NPC Natural Product Communications

EDITOR-IN-CHIEF
HONORARY EDITOR
DR. PAWAN K AGRAWAL
PROFESSOR GERALD BLUNDEN
Natural Product Inc.
The School of Pharmacy & Biomedical Sciences,
7963, Anderson Park Lane,
University of Portsmouth,
Westerville, Ohio 43081, USA
Portsmouth, PO1 2DT U.K.
agrawal@naturalproduct.us
axuf64@dsl.pipex.com

EDITORS ADVISORY BOARD

PROFESSOR ALESSANDRA BRACA
Dipartimento di Chimica Bioorganicae Biofarmacia, Prof. Berhanu M. Abegaz Prof. Leopold Jirovetz
Universita di Pisa, Gaborone, Botswana Vienna, Austria
via Bonanno 33, 56126 Pisa, Italy
braca@farm.unipi.it Prof. Viqar Uddin Ahmad Prof. Karsten Krohn
Karachi, Pakistan Paderborn, Germany
PROFESSOR DEAN GUO Prof. Øyvind M. Andersen Prof. Hartmut Laatsch
State Key Laboratory of Natural and Biomimetic Drugs, Bergen, Norway Gottingen, Germany
School of Pharmaceutical Sciences,
Peking University, Prof. Giovanni Appendino Prof. Marie Lacaille-Dubois
Beijing 100083, China Novara, Italy Dijon, France
gda5958@163.com Prof. Yoshinori Asakawa Prof. Shoei-Sheng Lee
Tokushima, Japan Taipei, Taiwan
PROFESSOR YOSHIHIRO MIMAKI Prof. Lee Banting Prof. Francisco Macias
School of Pharmacy, Portsmouth, U.K.
Tokyo University of Pharmacy and Life Sciences, Cadiz, Spain
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan Prof. Julie Banerji Prof. Imre Mathe
mimakiy@ps.toyaku.ac.jp Kolkata, India Szeged, Hungary
Prof. Alejandro F. Barrero Prof. Joseph Michael
PROFESSOR STEPHEN G. PYNE Granada, Spain
Department of Chemistry Johannesburg, South Africa
University of Wollongong Prof. Anna R. Bilia Prof. Ermino Murano
Wollongong, New South Wales, 2522, Australia Florence, Italy Trieste, Italy
spyne@uow.edu.au Prof. Maurizio Bruno Prof. M. Soledade C. Pedras
Palermo, Italy Saskatoon, Cnada
PROFESSOR MANFRED G. REINECKE
Department of Chemistry, Prof. César A. N. Catalán Prof. Luc Pieters
Texas Christian University, Tucumán,Argentina Antwerp, Belgium
Forts Worth, TX 76129, USA Prof. Josep Coll Prof. Peter Proksch
m.reinecke@tcu.edu Barcelona, Spain Düsseldorf, Germany
PROFESSOR WILLIAM N. SETZER Prof. Geoffrey Cordell Prof. Phila Raharivelomanana
Department of Chemistry Chicago, IL, USA Tahiti, French Plynesia
The University of Alabama in Huntsville Prof. Cristina Gracia-Viguera Prof. Monique Simmonds
Huntsville, AL 35809, USA Murcia, Spain
wsetzer@chemistry.uah.edu
Richmond, UK
Prof. Duvvuru Gunasekar Prof. Valentin Stonik
PROFESSOR YASUHIRO TEZUKA Tirupati, India Vladivostok, Russia
Institute of Natural Medicine Prof. A.A. Leslie Gunatilaka Prof. Winston F. Tinto
Institute of Natural Medicine, University of Toyama, Tucson, AZ, USA
2630-Sugitani, Toyama 930-0194, Japan
Barbados, West Indies
tezuka@inm.u-toyama.ac.jp Prof. Kurt Hostettmann Prof. Karen Valant-Vetschera
Lausanne, Switzerland Vienna, Austria
PROFESSOR DAVID E. THURSTON Prof. Martin A. Iglesias Arteaga Prof. Peter G. Waterman
Department of Pharmaceutical and Biological Chemistry, Mexico, D. F, Mexico
The School of Pharmacy,
Lismore, Australia
University of London, 29-39 Brunswick Square, Prof. Jerzy Jaroszewski
London WC1N 1AX, UK Copenhagen, Denmark
david.thurston@pharmacy.ac.uk

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 Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

NPC-SILAE: Special Issue

I am very grateful to Professor Luca Rastrelli, Dipartimento di Scienze Farmaceutiche e Biomediche, University of Salerno,
84084 Fisciano (SA), Italy, for organizing this issue, originating from the XIX SILAE (Società Italo-Latinoamericana di
Etnomedicina) Congress, which was held at Cagliari, Italy, from September 6th-10th, 2010, and attended by a large number of
participants from Latin America and the European Union. The present issue highlights some significant aspects of
ethnomedicine. The editors join me in thanking Professor Rastrelli, the authors and the reviewers for their efforts that have
made this issue possible, and to the production department for putting it into print.

Pawan K. Agrawal
Editor-in-Chief
 Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Editorial
The Italo-Latin American Society of Ethnomedicine (SILAE, www.silae.it) is an international non-profit organization
dedicated to advancing science around the world by serving as an educator, leader, spokesperson and professional association.
The fundamental objective of SILAE is to promote research and development into the use of medicinal and food plants in
different countries of the World. SILAE welcomes and actively seeks opportunities to work cooperatively, activating and
intensifying scientific relations between countries and between SILAE members. Since SILAE was founded (1990) its
objective has been set to contribute to the close examination of the themes of great interest and actuality in the context of the
relationships between Latin America and the European Union. In addition to this, SILAE aimed to individualize new ways of
collaboration between its member countries and other European as well as Asiatic countries to sign accords with
intergovernmental organizations. SILAE proposes to establish contacts with Scientific Communities, Universities, and
Research Centres for the pursuit of medicinal and food plants knowledge. Moreover SILAE_live, the one-to-one live Chat and
Messenger on our website (www.silae.it), is the first scientific chat on the web and is a developed tool to engage the interest
and imagination of the public and for helping non-scientists to understand and enjoy scientific discoveries and the scientific
processes. In addition to organizing membership activities, SILAE publishes the SILAE Special Issues, as well as many
scientific newsletters, books and reports, and spearheads programs that raise the bar of understanding for science worldwide.

Natural Product Communications is publishing a special issue that contains a selection of papers that were presented at the
XIX SILAE Congress (Cagliari, Italy, September, 6-10, 2010). For the Conference, 292 papers from authors coming from 19
different countries were accepted and published in the Proceedings of the SILAE 2010 (Abstract book ISBN: 88-8160-218-0).
The most promising 60 submissions were proposed for publication in the special issue of Natural Product Communications in
October 2010, each of which was reviewed by at least two anonymous referees. Following the review, 31 papers from different
universities of Argentina (7), Brazil (8), Colombia (2), Cuba (1), Honduras (1), Ireland and Serbia (1), Italy (6), Mexico (2) and
Venezuela (3) were selected for publication in this Special Issue. They are original papers on all aspects of natural products
including isolation, characterization, spectroscopic properties, biological activities, synthesis, analytical methods and tissue
culture; several are collaborative works between two or more countries.

Ten papers present the compositions of essential oils from different aromatic plants using analytical techniques such as GC,
GC/MS, GC/MS-LRI, esGC, GC-C-IRMS (1, 2, 4, 7, 8, 11, 14, 16, 22, 28) and also NMR spectroscopy (22). Although
essential oils have been used therapeutically for centuries, there is little published research on many of them. Bonaccorsi et al.
(1) report in their article on samples of Egyptian nerolì oils, obtained from the flowers of bitter orange (Citrus aurantium,
Rutaceace). For all the samples the composition was determined by GC/FID and by GC/MS-LRI; the samples were also
analyzed by esGC to determine the enantiomeric distribution of twelve volatiles and by GC-C-IRMS for the determination of
the 13CVPDB values of some mono and sesquiterpene hydrocarbons, alcohols and esters. The analytical procedures allowed the
quantitative determination of 86 components (1). Radulović et al. (7) identify 109 constituents from an essential oil sample
obtained from dry leaves of Nepeta × faassenii Bergmans, a hybrid species produced by crossbreeding N. mussinii Spreng. with
N. nepetella L. The chemical composition of the oil was compared, using multivariate statistical analyses (MVA), with those of
the oils of other Nepeta taxa, in particular N. mussinii and N. nepetella. The authors also report the chemical composition
dissimilarity relationships of 36 Nepeta essential oil samples. Some authors report the in vitro activity of the essential oil
against bacteria (2, 11, 16, 28). Bruno et al. (2) record the results obtained with the oil from the aerial parts of Salvia verbenaca
(Labiatae) aerial parts against Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Streptococcus faecalis, Escherichia coli,
Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa; Perez et al. (11), the essential oil of Chrysactinia
mexicana (Astaraceae) roots against Streptococcus pneumonia; Rios Tesch et al. (16), Lantana camara var. moritziana
(Verbenaceae) leaf essential oil against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella
pneumoniae, Salmonella typhi, and Pseudomonas aeruginosa; and Mora et al. (28), Phthirusa adunca (Loranthaceae) aerial
parts essential oil against Salmonella typhi, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Klebsiella
pneumonia. Results confirm that many essential oils possess in vitro antimicrobial activity against pathogens when compared
with reference drugs. Martini et al. (8) report the chemical composition of two Lamiaceae, Ocimum selloi and Hesperozygis
myrtoides, widely used in Brazilian traditional medicine. The authors report, for the first time, the chemical composition of the
essential oil from H. myrtoides, a very aromatic small bush found in the region of Aiuruoca (Minas Gerais State, Brazil); this
plant is also used in the preparation of a drink with “cachaça”, the Brazilian sugar cane spirit, where the plant is soaked in the
bottle’s spirit and buried for one year before being consumed. The essential oils showed activity against Candida albicans C.
glabrata, C. krusei, C. parapsilosis and C. tropicalis. Oliva et al. (14) also report the activity against Candida yeasts (C.
albicans, C. dubliniensis, C. glabrata, C. krusei, C. guillermondii, C. parapsilosis and C. tropicalis) of an essential oil obtained
from Aloysia triphylla (Verbenaceae), a promising alternative from the Argentinean flora for the treatment of candidiasis.
Natural Product Communications Vol. 6 (7) 2011
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Veloza et al. report the application of dioxirane chemistry to essential oils in order to generate modified compounds with
potential uses in several areas of medicine and industry (22). Polyphenols are among the most widespread class of metabolites
in nature, and their distribution is almost ubiquitous.

Nine papers describe the isolation and structure elucidation, as well as the bioactivity, of phenolic compounds. Interesting
biological properties are reported, such as antioxidant (17, 18, 26, 29), antifungal (13), antimicrobial (17), antiplatelet (20),
antiangiogenic (25), chemopreventive and apoptotic activities (21). Arevalo et al. (3) report a new phenolic apiosyl derivative,
two uncommon apiosyl derivatives, and known phenyl propanoids and flavonoids from Martinella obovata (Bignoniaceae)
collected in Honduras, and used by indigenous peoples to treat various eye ailments, including inflammation and conjunctivitis.
It is well known that the consumption of polyphenol-rich products, mainly due to their antioxidant properties, is beneficial for
human health, Salvador et al. (18), as well as Aislan et al. (29), report the antioxidant capacity and phenolic organic acids and
flavonoids content of Myrtaceae plants of the south of Brazil. The article by Mendiondo et al. [17] presents the antioxidant and
antimicrobial activity of the methanolic extract of Chuquiraga straminea (Asteraceae). The extract was also active against ten
methicillin resistant and sensitive S. aureus strains isolated from nosocomial infection; kaempferol and quercetin glycoside
derivatives seem to be responsible for some of the observed biological activity of the extracts. Derita and Zacchino (13)
dedicate their article to the bio-guided fractionation of the active dichloromethane extract of Polygonum persicaria L.
(Polygonaceae). This genus is represented in Argentina by 21 species and some of them have been used in traditional medicine
of that country to treat complaints related to fungal infections, such as skin ailments and vaginal disease. Isolated sesquiterpene
dialdehydes and flavonoids showed activity against yeasts, Aspergillus spp. and dermatophytes with MICs between 3.9 - 250
µg/mL. The results validate the popular use of this plant.

Data from the literature show that some flavonoids and other phenolic substances have the property to interfere with the
platelet system. A diet rich in phenolic compounds may favorably contribute to reducing the risks of cardiovascular diseases
through several mechanisms. Douglas et al. (20) assessed the inhibitory activity toward clotting formation and platelet
aggregation of an aqueous extract of leaves from Petroselinum crispum, an aromatic herb from the Apiaceae family that has
been employed in the food, pharmaceutical, perfume and cosmetic industries. The active principles, cosmosiin (apigenin 7-O-
glucoside) and apigenin, showed in vitro antiplatelet aggregation activity. Angiogenesis is a crucial step in many pathological
conditions like cancer, inflammation and metastasis formation; on this basis, the search for antiangiogenic agents has widened.
In order to identify new compounds able to interfere with the Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1),
Lepore et al. (25) investigated the extract of Calycolpus moritzianus (Myrtaceae) leaves by a competitive ELISA-based assay.
Phytochemical and pharmacological investigation of the active fractions led to the isolation of flavonoids and terpenes. The
authors hypothesized that the inhibitory activity of PlGF and VEGF interaction with Flt-1 receptor by the C. moritzianus
CHCl3 extracts and fractions may be due to the presence of a combination of compounds acting synergistically or as vehicles
enhancing the biological activity. These results suggest that the use of C. moritzianus extract is preferable to that of a purified
single compound. Torrenegra et al. (21) evaluated the activity of 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-
methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone present in Chromolaena leivensis (Asteraceae) on cell viability, cell
cycle distribution, mitochondrial membrane depolarization and viability of peripheral blood mononuclear cells and fibroblasts.
3,5-Dihydroxy-7-methoxyflavone showed activity on mitochondrial membrane, whereas both 3,5-dihydroxy-7-methoxy-
flavanone and 3,5-dihydroxy-7-methoxyflavone slightly increased the proliferation of peripheral blood mononuclear cells
either with phytohemagglutinin or without it, and the proliferation of fibroblasts.

The chemical composition of naturally grown herbs may vary according to climatic conditions, harvest time, storage condition,
and so on. As such, the same type of herb can vary in its composition and concentrations of chemical constituents from batch to
batch. These variabilities can result in significant differences in pharmacological activity. Therefore, the identification and
extraction of active ingredients from a medicinal plant represent a new approach to the development of natural product based
drugs. Picerno et al. (26) report on the evaluation of polyphenol components and antioxidant properties of fresh bergamot juice
(Citrus bergamia, Rutaceae), as well as on the production and characterization of powders obtained by loading the fresh juice
onto maltodextrins as a carrier (BMP) for spray-drying. Moreover, a formulation study to develop tablets containing BMP for
oral administration has been performed. The characteristics of the tablets were evaluated in terms of disintegration time and the
release of the active compounds into water and simulated biological fluids.

Five papers deal with the evaluation of pharmacological activity of crude extracts from plants used in Mexican (6),
Argentinean (9, 10 and 31) and Cuban (12) traditional medicine. Pazos et al. (6) investigated the effect of Morinda citrifolia
(Rubiaceae) seed (noni oil) on serum lipid levels in normolipidemic and hyperlipidemic induced mice. They found that
administration of noni oil causes a reduction in total cholesterol and triglyceride levels in both models. GC-MS analysis of the
fatty acid methyl esters indicated the presence of five major fatty acids. The mean linoleic acid content of crude noni seed oil
was 67.8%; these results indicate that noni seeds may be a useful new source of vegetable oil. Few medicinal plants have been
 Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

scientifically evaluated for their safety, efficacy and potential benefits, despite the great public interest in these herbs. Sabini et
al. (10) evaluated the cytotoxic and genotoxic activities of a cold aqueous extract obtained from Achyrocline satureioides
(Asteraceae) using the Allium cepa test, whereas Escobar et al. (31) assessed the genotoxic and cytotoxic activities of a
methanolic extract of Verbascum thapsus (Scrophulariaceae) using a micronucleus test in mouse bone marrow. Numerous
investigations have reported bioactive properties for both medicinal plants and the results obtained in these present studies
allow the conclusion to be made that the aqueous extract of A. satureioides and the methanolic extract of V. thapsus do not
contain genotoxic and cytotoxic compounds. Cytotoxicity, antiviral and virucidal activities of aqueous extracts of Baccharis
articulata were also evaluated by Cristina Vanesa Torres et al. (9). Extracts exhibited more than 95% virucidal activity against
Herpes suis virus type 1. These findings support the potential application of these extracts as a disinfectant or antiseptic
consistent with ancient ethnopharmacological thinking. In Cuba, alcoholic extracts of propolis are popular as a homemade
remedy. Three main types of Cuban propolis directly related to their secondary metabolite classes were described: brown
Cuban propolis (BCP), rich in polyisoprenylated benzophenones, red Cuban propolis (RCP), containing isoflavonoids as the
main constituents, and yellow Cuban propolis (YCP) with a variety of triterpenoids as the major chemical components.
Monzote et al. (12) assessed the activity of Cuban propolis extracts (brown, red and yellow type) on Leishmania amazonensis
and Trichomonas vaginalis. All propolis samples caused inhibition of growth of the Leishmania parasite. RCP was the most
active and the most cytotoxic. Only five propolis extracts showed activity against T. vaginalis and in this case YCP samples
were the most active.

According to the World Health Organization (WHO), more than 80% of the world's people, mostly in poor and less-developed
countries, depend on traditional medicine for their primary healthcare requirements. They use medicinal plants not only for
themselves but also for their domestic animals. Traditionally, people collected the ingredients for their medicines from forests.
However, due to rapid and extensive deforestation, accompanied by uncontrolled over-exploitation, the wild populations of
medicinal plants are disappearing very fast.

Three papers deal with biodiversity and nature protection. The cerrado, a vast tropical savanna ecoregion of Brazil,
particularly in the states of Goiás and Minas Gerais, is characterized by an enormous range of plant and animal biodiversity.
The cerrado is one of the world's threatened biodiversity hotspots. About 60% of its vegetation has already been removed and
the remaining areas are isolated in forest fragments. Due to the devastation, many natural compounds with potential biological
activities have been lost. Soares et al. (23) compared the basal cytotoxicity of active compounds extracted from plants of the
Brazilian “cerrado”. Those with low toxicity were subjected to further anti-inflammatory assays as natural products with low
cytotoxicity constitute an excellent alternative source for complementary treatments for inflammatory disease. The viability
was assayed using the neutral red uptake assay in Mac Coy cells after 24 h of exposure. The dose evaluated was 50µg/µL. The
test substances were: cinnamic acid, p-coumaric acid, chlorogenic acid, syringic acid, vanilic acid, homogentisis acid,
scandenin, palustric acid, diosgenin, and cabraleone. From 1975 until the beginning of the 1980s, many governmental
programs have been launched with the intent of stimulating the development of the "cerrado" region, through subsidies for
agriculture. As a result, there has been a significant increase in agricultural and cattle production. On the other hand, urban
pressure and rapid establishment of agricultural activities in the region have rapidly reduced the biodiversity of the ecosystems.
Camargo et al. (30) tried to diagnose the current public programs focused on herbal medicines in Brazil from 1985 to 2006.

Sharry et al. (19) dedicated their article to the establishment of vegetative propagation systems for three native forest species
widely used in Argentinean folk medicine: Erythrina crista-galli (Fabaceae), Acacia caven (Mimosaceae) and Salix
humboldtiana (Salicaceae). In the last few years the use of in vitro culture techniques for trees has facilitated the cloning of
selected phenotypes, leading to the preservation and manipulation of vegetal material. The authors are able to support the
conservation of native forest resources for medicinal use by means of vegetative propagation techniques: macro and
micropropagation and somatic embryogenesis.

The last four papers are quite different from the others, each one dealing with its own subject of great importance. Marques
et al. (5) report for the first time the isolation of six aristolactams from Ottonia anisum (Piperaceae). Aristolactams belong to a
large and important group of naturally occurring alkaloids that possess a phenanthrene lactam skeleton with a
phenolic hydroxy function. They constitute an important alkaloid group due to their unique structural features and potent
biological activities, such as anti-inflammatory, anti-arthritis, anti-PAF, anti-mycobacterial, and neuro-protective.
Aristolactams have been reported from plants of the Annonaceae, Monimiaceae, Menispermaceae, Piperaceae and Saururaceae
families. Nicoletti (15) analyzed, first by HPTLC and later by isolation and analysis of spectroscopic data, the presence of a
sildenafil derivative (thiosildenafil) in herbal products. Quality assurance has enabled health professionals to prescribe safely
herbal medicines that the population has been taking for quite a long time.The presence of synthetic drugs in the formulation of
herbal products in order to improve the efficacy has been reported in several cases.
Natural Product Communications Vol. 6 (7) 2011
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Viegi et al. (24) revised the use of toxic or potentially toxic plants for the treatment of ailments in livestock and pets in
ethnoveterinary practice in Italy. More than 250 of the entities used (81% for curative purposes) can be toxic unless dosed
appropriately. The species belong to 71 families, among which the Fabaceae predominates. Drugs derived from natural
sources are usually produced by harvesting the natural source or through semi-synthetic methods: semisynthesis is usually used
when the precursor molecule is too structurally complex, too costly or too inefficient to be produced by total synthesis. It is
also possible that the semisynthetic derivative outperforms the original biomolecule itself with respect to potency, stability and
safety. Usubillaga et al. (27) undertook the isomerization of kaurenic acid to obtain ent-kaurenic acid, a tetracyclic diterpene
that has been reported to have antimicrobial, antiparasitic and cytotoxic activity. The occurrence of kaurenic acid, which has an
exocyclic double bond at Δ16, is widespread in the plant kingdom, while the occurrence of its isomer ent-kaur-15-en-19-oic
acid is rare.

The congresses of SILAE are international events whose organizations are submitted to an International Organizer Committee
composed of professors from Italian and Latin America Universities. The Italo-Latin American Congress of Ethnomedicine
arose from the necessity to evaluate the important potentialities of little known medicinal and alimentary plants, typical and
traditional plants of the Latin American continent and to provide connections between Italian and other European and Latin-
American researchers, with common objectives of research in the areas in which the projects will be articulated. Traditional
medicine is used by 85% of the World’s population and is of great importance in developing countries. In accordance with the
requirements of the World Health Organization, a scientific basis and proof for the use of medicinal plants is required and so
the organization of such a Congress provides an important exchange of such information and coordination of scientific activity.
This Natural Product Communications special issue provided an opportunity for publication of original, peer-reviewed, full-
length articles on new research on medicinal plants used in Latin America; this will serve to stimulate the studies in these areas
that are extremely important for academia and industry.

The Guest Editor would like to thank the contributors who gave so generously of their time and experience and who made this
publication a valuable tool for scientists in the field of natural products chemistry and biology. Thanks are also due to the
referees for their valuable comments and for the very detailed and accurate review of manuscripts; their comments certainly
helped to improve the papers. I am also grateful to my staff who lent their considerable talents to the project: Annalisa
Piccinelli, Florence Somma, Luca Campone and the webmaster of SILAE, Vincenzo Barbarulo. I thank all of them for their
commitment, continued support and friendship.

I am also very grateful to the Editorial Board of Natural Product Communications for embracing this project with interest and
enthusiasm, and for the opportunity to publish this Special Issue. I hope that this will be the first of a long series in this
attractive and interesting Journal. Finally, I would like to thank the Editor-in-Chief, Pawan Agrawal, for his valuable input and
for careful supervision. Thank you Pawan!
Luca Rastrelli
Dipartimento di Scienze Farmaceutiche e Biomediche,
University of Salerno,
Via Ponte don Melillo,
84084 Fisciano (SA), Italy
E-mail: rastrelli@unisa.it

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3. Arevalo C, Ruiz I, Piccinelli AL, Rastrelli L. (2011) Phenolic derivatives from the leaves of Martinella obovata (Bignoniaceae).
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Rio de Janeiro, Brazil. Natural Product Communications, 6, 1045-1050
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Natural Product Communications, 6, 1005-1008
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mussinii Spreng. x N. nepetella L.): a comparison study. Natural Product Communications, 6, 1015-1022
 Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

8. Martini MG, Bizzo HR, de L. Moreira D, Neufeld PM, Miranda SN, Alviano CS, Alviano DS, Leitão G. (2011) Analysis of the
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St.-Hil.) Epling (Lamiaceae) . Natural Product Communications, 6, 1027-1030
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Chrysactinia mexicana roots. Natural Product Communications, 6, 1035-1038
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13. Derita M, Zacchino S. (2011) Validation of the ethnopharmacological use of Polygonum persicaria for its antifungal properties.
Natural Product Communications, 6, 931-933
14. Oliva M, Carezzano E, Gallucci N, Casero C, Demo M. (2011) Antimycotic effect of the essential oil of Aloysia triphylla against
Candida species obtained from human pathologies. Natural Product Communications, 6, 1039-1043
15. Nicoletti M. (2011) Isolation and identification of thiosildenafil in a health supplement. Natural Product Communications, 6,
1003-1004
16. Rios Tesch N, Mora F, Rojas L, Díaz T, Velasco J, Yánez C, Rios N, Carmona J, Pasquale S. (2011) Chemical composition and
antibacterial activity of the essential oil of Lantana camara var. moritziana (Otto & Dietr.) López-Palacios. Natural Product
Communications, 6, 1031-1034
17. Mendiondo ME, Juárez BE, Zampini C, Isla MI, Ordoñez R. (2011) Bioactivies of Chuquiraga straminea Sandwith, subfamily
Barnadesioideae (Asteraceae) . Natural Product Communications, 6, 965-968
18. Salvador MJ, Andreazza NL, de Lourenço CC, Aislan Pascoal CRF, Alves Stefanello ME. (2011) Antioxidant capacity and
phenolic content of four Myrtaceae plants of the South of Brazil. Natural Product Communications, 6, 977-982
19. Sharry S, Adema M, Basiglio Cordal MA, Villarreal B, Nikoloff N, Briones V, Abedini W. (2011) Propagation and conservation of
native forest genetic resources of medicinal use by means of in vitro and ex vitro techniques. Natural Product Communications, 6,
985-988
20. Chaves DSA, Frattani F, Assafim M, de Almeida AP, Zingali RB, Costa SS. (2011) Phenolic chemical composition of
Petroselinum crispum extract and its effect on haemostasis. Natural Product Communications, 6, 961-964
21. Torrenegra RDG, Rodriguez AO. (2011) Chemical and biological activity of leaves extracts of Chromolaena leivensis. Natural
Product Communications, 6, 947-950
22. Veloza LA, Orozco LM, Sepúlveda-Arias JC. (2011) Use of dimethyldioxirane in the epoxidation of the main constituents of the
essential oils obtained from Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora. Natural Product
Communications, 6, 925-930
23. Soares VCG, Bonacorsi C, Andrela ALB, Bortoloti LV, Campos SC, Fagundes FHR, Piovani M, Cotrim CA, Vilegas W, Toyama
MH. (2011) Cytotoxicity of active ingredients extracted from plants of the Brazilian “Cerrado”. Natural Product Communications,
6, 983-984
24. Viegi L, Vangelisti R. (2011) Toxic plants used in ethnoveterinary medicine in Italy. Natural Product Communications, 6, 999-
1000
25. Lepore L, Gualtieri MJ, Malafronte N, Dal Piaz F, Ambrosio L, De Falco S, De Tommasi N. (2011) Anti-angiogenic activity
evaluation of secondary metabolites from Calycolpus moritzianus (O. Berg) Burret leaves. Natural Product Communications, 6,
943-946
26. Picerno P, Sansone F, Mencherini T, Prota L, Aquino RP, Rastrelli L, Lauro MR. (2011) Citrus bergamia juice: phytochemical and
technological studies Natural Product Communications, 6, 951-955
27. Rojas J, Aparicio R, Villasmil T, Peña A, Usubillaga A. (2011) On the isomerization of ent-kaurenic acid. Natural Product
Communications, 6, 935-938
28. Mora FD, Ríos N, Rojas LB, Díaz T, Velasco J, Carmona JA, Silva B. (2011) Chemical composition and in vitro antibacterial
activity of the essential oil of Phthirusa adunca (Meyer) from Venezuelan Andes Natural Product Communications, 6, 1051-1053
29. Pascoal ACRF, Ehrenfried CA, Eberlin MN, Alves Stefanello ME, Salvador MJ. (2011) Free-radical scavenging activity,
determination of phenolic compounds and HPLC-UV/DAD/ESI-MS profile of Campomanesia adamantium leaves. Natural
Product Communications, 6, 969-972
30. Saranz Camargo EE, Medeiros Bandeira MA, Gomes de Oliveira A. (2011) Diagnosis of public programs focused on herbal
medicines in Brazil. Natural Product Communications, 6, 1001-1002
31. Escobar FM, Sabini MC, Zanon SM, Cariddi LN, Tonn CE, Sabini LI. (2011) Genotoxic evaluation of a methanolic extract of
Verbascum thapsus using micronucleus test in mouse bone marrow Natural Product Communications, 6, 989-991
 Natural Product Communications
2011
Volume 6, Number 7
Contents
Original Paper Page

Use of Dimethyldioxirane in the Epoxidation of the Main Constituents of the Essential Oils Obtained from
Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora
Luz A. Veloza, Lina M. Orozco and Juan C. Sepúlveda-Arias 925
Validation of the Ethnopharmacological Use of Polygonum persicaria for its Antifungal Properties
Marcos Derita and Susana Zacchino 931
On the Isomerization of ent-Kaurenic Acid
Julio Rojas, Rosa Aparicio, Thayded Villasmil, Alexis Peña and Alfredo Usubillaga 935
Aristolactams from Roots of Ottonia anisum (Piperaceae)
André M. Marques, Leosvaldo S. M. Velozo, Davyson de L. Moreira, Elsie F. Guimarães and
Maria Auxiliadora C. Kaplan 939
Anti-angiogenic Activity Evaluation of Secondary Metabolites from Calycolpus moritzianus Leaves
Laura Lepore, Maria J. Gualtieri, Nicola Malafronte, Roberta Cotugno, Fabrizio Dal Piaz, Letizia Ambrosio,
Sandro De Falco and Nunziatina De Tommasi 943
Chemical and Biological Activity of Leaf Extracts of Chromolaena leivensis
Ruben D. Torrenegra G. and Oscar E. Rodríguez A. 947
Citrus bergamia Juice: Phytochemical and Technological Studies
Patrizia Picerno, Francesca Sansone, Teresa Mencherini, Lucia Prota, Rita Patrizia Aquino, Luca Rastrelli and
Maria Rosaria Lauro 951
Phenolic Derivatives from the Leaves of Martinella obovata (Bignoniaceae)
Carolina Arevalo, Ines Ruiz, Anna Lisa Piccinelli, Luca Campone and Luca Rastrelli 957
Phenolic Chemical Composition of Petroselinum crispum Extract and Its Effect on Haemostasis
Douglas S. A. Chaves, Flávia S. Frattani, Mariane Assafim, Ana Paula de Almeida, Russolina B. Zingali and
Sônia S. Costa 961
Bioactivities of Chuquiraga straminea Sandwith
María Elena Mendiondo, Berta E. Juárez, Catiana Zampini, María Inés Isla and Roxana Ordoñez 965
Free Radical Scavenging Activity, Determination of Phenolic Compounds and HPLC-DAD/ESI-MS Profile of
Campomanesia adamantium Leaves
Aislan C.R.F. Pascoal, Carlos Augusto Ehrenfried, Marcos N. Eberlin, Maria Élida Alves Stefanello and
Marcos José Salvador 969
Activity of Cuban Propolis Extracts on Leishmania amazonensis and Trichomonas vaginalis
Lianet Monzote Fidalgo, Idalia Sariego Ramos, Marley García Parra, Osmany Cuesta-Rubio, Ingrid Márquez Hernández,
Mercedes Campo Fernández, Anna Lisa Piccinelli and Luca Rastrelli 973
Antioxidant Capacity and Phenolic Content of four Myrtaceae Plants of the South of Brazil
Marcos José Salvador, Caroline C. de Lourenço, Nathalia Luiza Andreazza, Aislan C.R.F. Pascoal and
Maria Élida Alves Stefanello 977
Cytotoxicity of Active Ingredients Extracted from Plants of the Brazilian “Cerrado”
Veronica CG Soares, Cibele Bonacorsi, Alana LB Andrela, Lígia V Bortoloti, Stepheny C de Campos,
Fábio HR Fagundes, Márcio Piovani, Camila A Cotrim, Wagner Vilegas and Marcos H Toyama 983
Propagation and Conservation of Native Forest Genetic Resources of Medicinal Use by Means of in vitro and
ex vitro Techniques
Sandra Sharry, Marina Adema, María A. Basiglio Cordal, Blanca Villarreal, Noelia Nikoloff, Valentina Briones and
Walter Abedini 985
Genotoxic Evaluation of a Methanolic Extract of Verbascum thapsus using Micronucleus Test in Mouse
Bone Marrow
Franco Matías Escobar, María Carola Sabini, Silvia Matilde Zanon, Laura Noelia Cariddi, Carlos Eugenio Tonn and
Liliana Inés Sabini 989

Continued Overleaf
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Study of Antiviral and Virucidal Activities of Aqueous Extract of Baccharis articulata against Herpes suis virus
Cristina Vanesa Torres, María Julia Domínguez, José Luis Carbonari, María Carola Sabini, Liliana Inés Sabini and
Silvia Matilde Zanon 993
Evaluation of Cytogenotoxic Effects of Cold Aqueous Extract from Achyrocline satureioides by Allium cepa L test
María C. Sabini, Laura N. Cariddi, Franco M. Escobar, Romina A. Bachetti, Sonia B. Sutil, Marta S. Contigiani,
Silvia M. Zanon and Liliana I. Sabini 995
Toxic Plants Used in Ethnoveterinary Medicine in Italy
Lucia Viegi and Roberta Vangelisti 999
Diagnosis of Public Programs focused on Herbal Medicines in Brazil
Ely Eduardo Saranz Camargo, Mary Anne Medeiros Bandeira and Anselmo Gomes de Oliveira 1001
Identification of Thiosildenafil in a Health Supplement
Marcello Nicoletti 1003
Hypolipidemic Effect of Seed Oil of Noni (Morinda citrifolia)
Diana C. Pazos, Fabiola E. Jiménez, Leticia Garduño, V. Eric López and M. Carmen Cruz 1005
Composition of Egyptian Nerolì Oil
Ivana Bonaccorsi, Danilo Sciarrone, Luisa Schipilliti, Alessandra Trozzi, Hussein A. Fakhry and Giovanni Dugo 1009
Essential oil of Nepeta x faassenii Bergmans ex Stearn (N. mussinii Spreng. x N. nepetella L.): A Comparison Study
Niko Radulović, Polina D. Blagojević, Kevin Rabbitt and Fabio de Sousa Menezes 1015
Chemical Composition and Biological Activity of Salvia verbenaca Essential Oil
Marisa Canzoneri, Maurizio Bruno, Sergio Rosselli, Alessandra Russo, Venera Cardile, Carmen Formisano,
Daniela Rigano and Felice Senatore 1023
Chemical Composition and Antimicrobial Activities of the Essential Oils from Ocimum selloi and
Hesperozygis myrtoides
Márcia G. Martini, Humberto R. Bizzo, Davyson de L. Moreira, Paulo M. Neufeld, Simone N. Miranda,
Celuta S. Alviano, Daniela S. Alviano and Suzana G. Leitão 1027
Chemical Composition and Antibacterial Activity of the Essential Oil of Lantana camara var. moritziana
Nurby Rios Tesch, Flor Mora, Luis Rojas, Tulia Díaz, Judith Velasco, Carlos Yánez, Nahile Rios, Juan Carmona and
Sara Pasquale 1031
Activity against Streptococcus pneumoniae of the Essential Oil and 5-(3-Buten-1-ynyl)-2, 2'-bithienyl Isolated
from Chrysactinia mexicana Roots
Bárbara Missiam Mezari Guevara Campos, Anabel Torres Cirio, Verónica Mayela Rivas Galindo, Ricardo Salazar Aranda,
Noemí Waksman de Torres and Luis Alejandro Pérez-López 1035
Antimycotic Effect of the Essential Oil of Aloysia triphylla against Candida Species Obtained from
Human Pathologies
María de las Mercedes Oliva, María Evangelina Carezzano, Mauro Nicolás Gallucci and Mirta Susana Demo 1039
Secretory Cavities and Volatiles of Myrrhinium atropurpureum Schott var. atropurpureum (Myrtaceae): An
Endemic Species Collected in the Restingas of Rio de Janeiro, Brazil
Cristiane Pimentel Victório, Claudio B. Moreira, Marcelo da Costa Souza, Alice Sato and
Rosani do Carmo de Oliveira Arruda 1045
Chemical Composition and in vitro Antibacterial Activity of the Essential Oil of Phthirusa adunca from
Venezuelan Andes
Flor D. Mora, Nurby Ríos, Luis B. Rojas, Tulia Díaz, Judith Velasco, Juan Carmona A and Bladimiro Silva 1051

Manuscripts in Press 1054


LIST OF AUTHORS
Abedini, W ............................ 985
Adema, M .............................. 985
Almeida, AP .......................... 961
Alviano, CS ......................... 1027
Alviano, DS ......................... 1027
Ambrosio, L .......................... 943
Andreazza, NL ...................... 977
Andrela, ALB ........................ 983
Aparicio, R ............................ 935
Aquino, RP ............................ 951
Aranda, RS .......................... 1035
Arevalo, C ............................. 957
Arruda, ACO ....................... 1045
Assafim, M ............................ 961
Bachetti, RA .......................... 995
Bandeira, MAM .................. 1001
Bizzo, HR ............................ 1027
Blagojević, PD .................... 1015
Bonaccorsi, I........................ 1009
Bonacorsi, C .......................... 983
Bortoloti, LV ......................... 983
Briones, V.............................. 985
Bruno, M ............................. 1023
Camargo, EES ..................... 1001
Campone, L ........................... 957
Campos, BMMG ................. 1035
Canzoneri, M ....................... 1023
Carbonari, JL ......................... 993
Cardile, V ............................ 1023
Carezzano, ME .................... 1039
Cariddi, LN..................... 989,995
Carmona A,J ........................ 1051
Carmona, J ........................... 1031
Chaves, DSA ......................... 961
Cirio, AT ............................. 1035
Contigiani, MS ...................... 995
Cordal, MAB ......................... 985
Costa, SS ............................... 961
Cotrim, CA ............................ 983
Cotugno, R ............................ 943
Cruz, MC ............................. 1005
Cuesta-Rubio, O .................... 973
de Campos, SC .......................983
de Lourenço, CC ....................977
de Oliveira, AG ....................1001
Demo, MS ............................1039
Derita, M ................................931
Díaz, T ........................ 1031,1051
Domínguez, MJ ......................993
Dugo, G ................................1009
Eberlin, MN............................969
Ehrenfried, CA .......................969
Escobar, FM ................... 989,995
Fagundes, FHR.......................983
Fakhry, HA ...........................1009
Falco, SD ................................943
Fernández, MC .......................973
Fidalgo, LM............................973
Formisano, C ........................1023
Frattani, FS .............................961
Galindo, VMR ......................1035
Gallucci, MN ........................1039
Garduño, L ...........................1005
Gualtieri, M ............................943
Guimarães, EF ........................939
Hernández, IM........................973
Isla, MI ...................................965
Jiménez, FE ..........................1005
Juárez, BE ..............................965
Kaplan, MAC .........................939
Lauro, MR ..............................951
Leitão, SG ............................1027
Lepore, L ................................943
López, VE ............................1005
Malafronte, N .........................943
Marques, AM .........................939
Martini, MG .........................1027
Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)
Mencherini, T .........................951
Mendiondo, ME .....................965
Menezes, FS .........................1015
Miranda, SN .........................1027
Mora, F .................................1031
Mora, FD ..............................1051
Moreira, CB .........................1045
Moreira, DL ..................939,1027
Neufeld, PM .........................1027
Nicoletti, M ..........................1003
Nikoloff, N .............................985
Oliva, MM ............................1039
Ordoñez, R .............................965
Orozco, LM ............................925
Parra, MG ...............................973
Pascoal, ACRF ................969,977
Pasquale, S ...........................1031
Pazos, DC .............................1005
Peña, A ...................................935
Pérez-López, LA ..................1035
Piaz, FD ..................................943
Piccinelli, AL ..................957,973
Picerno, P ...............................951
Piovani, M ..............................983
Prota, L ...................................951
Rabbitt, K .............................1015
Radulović, N ........................1015
Ramos, IS ...............................973
Rastrelli, L ............... 951,957,973
Rigano, D .............................1023
Rios, N.........................1031,1051
Rodríguez A, OE ....................947
Rojas, J ..........................935,1031
Rojas, LB .............................1051
Rosselli, S.............................1023
Ruiz, I .....................................957
Russo, A ...............................1023
Sabini, LI ................. 989,993,995
Sabini, MC .............. 989,993,995
Salvador, MJ ................... 969,977
Sansone, F .............................. 951
Sato, A .................................. 1045
Schipilliti, L ......................... 1009
Sciarrone, D ......................... 1009
Senatore, F ........................... 1023
Sepúlveda-Arias, JC............... 925
Sharry, S ................................. 985
Silva, b ................................. 1051
Soares, VCG .......................... 983
Souza, MC ............................ 1045
Stefanello, MEA ............. 969,977
Sutil, SB ................................. 995
Tesch, NR............................. 1031
Tommasi, ND ......................... 943
Tonn, CE ................................ 989
Torrenegra G., RD ................. 947
Torres, CV.............................. 993
Torres, NW .......................... 1035
Toyama, MH .......................... 983
Trozzi, A .............................. 1009
Usubillaga, A ......................... 935
Vangelisti, R .......................... 999
Velasco, J ....................1031,1051
Veloza, LA ............................. 925
Velozo, LSM .......................... 939
Victório, CP ......................... 1045
Viegi, L .................................. 999
Vilegas, W.............................. 983
Villarreal, B ............................ 985
Villasmil, T ............................ 935
Yánez, C ............................... 1031
Zacchino, S ............................ 931
Zampini, C ............................. 965
Zanon, SM................989,993,995
Zingali, RB ............................. 961
 2011
NPC Natural Product Communications Vol. 6
No. 7
Use of Dimethyldioxirane in the Epoxidation of the 925 - 930

Main Constituents of the Essential Oils Obtained from

Tagetes lucida, Cymbopogon citratus, Lippia alba and
Eucalyptus citriodora
Luz A. Velozaa*, Lina M. Orozcoa and Juan C. Sepúlveda-Ariasb
a
Laboratorio de Fitoquímica, Escuela de Química, Universidad Tecnológica de Pereira, A.A. 097,
Vereda La Julita Pereira, Risaralda – Colombia
b
Laboratorio de Fisiología Celular e Inmunología, Facultad de Ciencias de la Salud, Universidad
Tecnológica de Pereira, A.A. 097, Vereda La Julita, Pereira, Risaralda – Colombia

lveloza@utp.edu.co

Received: December 10th, 2010; Accepted: March 16th, 2011

Dimethyldioxirane (DMDO), a widely used oxidant in organic synthesis is considered an environmentally friendly oxygen transfer reagent
because acetone is the only byproduct formed in its oxidation reactions. This work describes the isolation of the main constituents (terpenes)
in the essential oils obtained from Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora, their epoxidation with
DMDO in acetone solution and the characterization of the resulting epoxides by GC-MS (EI) and NMR. This is one of the first reports
involving the application of dioxirane chemistry to essential oils in order to generate modified compounds with potential uses in several areas
of medicine and industry.

Keywords: dimethyldioxirane, epoxidation, estragole, citral, carvone, citronellal.

The use of dimethyldioxirane (DMDO), a member of a
new class of three-membered ring peroxide containing
oxidants, has increased notably in recent years due to its
ability to do oxygen atom transfer reactions with a wide
range of substrates, including C=C bonds [1], C-H
insertions in hydrocarbons [2], as well as oxidations of
atoms containing lone pairs of electrons, such as
sulfides [3] and primary and secondary amines [4]. DMDO
is also considered to be an environmentally friendly
oxygen transfer reagent, and is attractive from the “green
chemistry” [5] point of view due to its lack of toxic or
harmful metals. DMDO’s reactions occur under extremely
mild and neutral conditions, and the only byproduct of its
reactions is acetone, which can be easily removed. Thus,
DMDO reactions do not require complicated purification
procedures to obtain pure products.
This electrophilic oxygen transfer reagent is the reagent of
choice for the epoxidation of both conjugated and
unconjugated double bonds containing other functional
groups, such as hydroxyls or carbonyls. Isolated double
bonds are selectively and easily oxidized under mild
conditions (0-25°C and neutral pH) [6]. On the other hand,
allylic alcohols are oxidized frequently with tert-butyl
hydroperoxide (TBHP) [7] in the presence of transition
metals, which are necessary to initiate the reaction.
Typically, enones are epoxidized with alkaline hydrogen
peroxide (H2O2) [8], but in some cases the hydrolytically-
sensitive epoxide products open via a side reaction to form
a diol. The three types of olefins mentioned above have
been successfully oxidized with DMDO in high regio- and
stereoselectivity.
Several methods for the generation of epoxides from
alkenes have been developed, such as the Sharpless [9] and
Jacobsen [10] catalytic epoxidations, the use of alternative
routes involving the formation of intermediate halohydrins
by using trichloroisocyanuric acid in aqueous acetone [11]
and the use of peracids to generate both mono- and
bisepoxides, as well as cleaved oxidation products. A
system involving a hydrogen peroxide-dinuclear
manganese (IV) complex and a carboxylic acid has been
successfully employed for the efficient epoxidation of
terpenes such as limonene, citral, carvone and linalool,
while other sterically hindered terpenes (i.e., citronellal, -
and-pinene) were epoxidized in low yields [12]. The
selective oxidation of monoterpenes (i.e., limonene,
terpinene, neryl acetate, geranyl acetate, citral and
geranyl nitrile) with H2O2 catalyzed by
peroxotungstophosphate under biphasic conditions
produced mono- and bisepoxides in good yields [13].
While alkene epoxidation has found widespread use in the
926 Natural Product Communications Vol. 6 (7) 2011
total synthesis of natural products, there are very few
reports in the literature of regioselective epoxidations of
natural products with DMDO. Therefore, in this work, we
have carried out the DMDO epoxidation of the main
terpene constituents in the essential oils extracted from
Tagetes lucida, Cymbopogon citratus, Lippia alba and
Eucalyptus citriodora, which makes this one of the first
reports where dioxirane chemistry has been applied to
isolated components of essential oils.
Tagetes lucida (Asteraceae) is an endemic plant that is
widely distributed in Central and South America, and is
known in Colombia as “Estragón de invierno” [14]. Its
extracts are reported to possess bactericidal [15],
antimycotic [16] and antioxidant [17] activities, but this
plant has not been well studied from the chemical point of
view. In Colombia, the essential oil extracted from this
plant contains a large amount (93.8%) of a
phenylpropanoid, estragole (1-allyl-4-methoxybenzene),
which is present in many vegetables as a flavoring [18].
Cymbopogon citratus (Poaceae) is known worldwide as
the herb lemongrass, and is widely used in folk medicine
due to its antimicrobial [19], anti-inflammatory,
antispasmodic [20] and antifungal [21] activities. The
essential oil from lemongrass has been widely used in the
perfume and cosmetics industries. It has also been used in
chemical synthesis, due to its high citral (3,7-dimethyl-2,6-
octadienal) content. Citral exists as a natural mixture of
two isomeric aldehydes, geranial and neral. Recently,
some citral derivatives with potential bactericidal activity
were generated using “green” thiol conjugate additions
[22]. Citral is a polyfunctional molecule that mediates a
wide number of chemical transformations, mainly
epoxidation reactions.
Lippia alba (Verbenaceae) is a shrub widely distributed
from Mexico to South America [23] and is known in
Colombia as “Pronto alivio”. L. alba essential oil and some
organic and polar extracts have shown analgesic, anti-
inflammatory [24], antifungal [25], and antibacterial [26]
properties. In Colombia the essential oil from this plant is
distinguished by its high content of S-carvone [2-methyl-5-
(1-methylethenyl)-2-cyclohexene-1-one] (40-57 %), a
substance with a high-value for the perfume and cosmetics
industries and as a starting material for fine organic
synthesis [27].
Eucalyptus citriodora (Myrtaceae) is an aromatic specie
known as “lemon scented gum”. Water extracts from dried
leaves of E. citriodora are traditionally used as
antipyretics, anti-inflammatory and analgesic [28]. E.
citriodora essential oil has been shown to contain high
concentration (70-80%) of citronellal (3,7-dimethyl-6-
octenal) [29], which is effective against bacterial and
fungal infections [30]. Besides, this compound is attractive
from the stereochemical point of view since it can be used
in an efficient way to introduce a new stereogenic center in
more complex structures [31].
Veloza et al.
As an important application of dioxirane chemistry to
essential oils, this work describes the epoxidation of the
main constituents of the essential oil from Tagetes lucida,
Cymbopogon citratus, Lippia alba and Eucalyptus
citriodora with acetone solutions of DMDO. In general,
the importance of the derivatives generated is due to their
polyfunctionality and the high reactivity of the oxirane
ring. Such terpene epoxides can be readily transformed
into alcohols, aldehydes, ketones and heterocycles, which
give these terpene epoxides a promising position in terpene
chemistry with future applications in industry and
medicine.
The chemical composition and identity of the main
constituents of the essential oils obtained from Tagetes
lucida, Cymbopogon citratus, Lippia alba and Eucalyptus
citriodora were determined by GC-MS (EI). The main
constituent of the oil from T. lucida was estragole, which
had a relative abundance of 93.8% and a retention time of
7.7 minutes. Citral was the main component of the oil from
C. citratus. It had a relative abundance of 74% and
retention times of 8.1 and 8.3 minutes for the isomers.
Carvone was the main constituent of the oil from L. alba,
with a relative abundance of 42% and a retention time of
7.9 minutes. Citronellal was the main constituent of the oil
from E. citriodora with a relative abundance of 74% and a
retention time of 6.7 minutes. All of the compounds were
isolated in high purity (95%), which was confirmed by
GC-MS.
The epoxidation of estragole, citral, carvone and
citronellal with DMDO in an acetone solution
afforded epoxyestragole 1, 6,7-epoxycitral 2a and 2b,
8,9-epoxycarvone 3 and 6,7-epoxycitronellal 4 (Figure 1),
with greater than 95% conversion (confirmed by GC-MS).
The resulting epoxides were stored at room temperature
and evaluated at several points during the course of one
year and found to be stable (data not shown).
O
6
CHO
O O 7
1 2a, 2b
9
O
10 O
7 8
7
6
CHO
3
O 9
9
3 4
Figure 1: Epoxides obtained from the DMDO reaction.
Estragole was chosen for DMDO epoxidation because it
possesses allyl and methoxy substituents. The isolated
double bond does not have electron-donating groups that
would promote epoxidation. In addition, the electron-
donating methoxy group (–OMe) bound to the aromatic
ring does not have a direct influence on the olefin’s
reactivity. Under the conditions employed for the reaction,
1 was obtained in a short period of time (0.5-1 h) in
85% yield (101 mg). When this substrate and a series of
other substrates, including unsubstituted styrenes and
Dimethyldioxirane epoxidation of essential oils
cycloalkenes, were epoxidized with aqueous H2O2 and a
polystyrene-supported triphenylarsine reagent, the
moderately unstable epoxide products were isolated in
poor yield [32].
Kim, et al. [33] synthesized trans-anethole oxide from
trans-anethole (an isomer of estragole) using an acetone
solution of DMDO as the oxidizing reagent. This oxide
was stable for one year, and reaction’s yield was >95%.
This yield was much better than that obtained by Mohan
and Whalen [34] and Greca et al [35], who used m-CPBA
to oxidize the same substrate, but obtained low yields
(38%) that were complicated by the presence of m-
CPBA’s acid byproduct. These results and estragole’s
structural characteristics provide evidence supporting the
high reactivity of DMDO. Therefore, it is clear that this
oxygen transfer reagent is the reagent of choice for the
epoxidation of double bonds, including electron deficient
olefins. The use of this oxidant offers a variety of
advantages; it is generated from readily available reagents,
it reacts to generate epoxides under mild conditions and its
only byproduct is acetone and prevents the need for work-
up conditions that can cause the opening of the oxirane
ring.
Another substrate chosen for DMDO epoxidation was
citral, which is widely distributed in nature as a mixture of
E/Z isomers. This substrate can undergo a wide range of
reactions due to its multiple functional groups, including
two trisubstituted double bonds, one of which is adjacent
to a carbonyl group. One benefit of using this substrate is
that the regioselectivity in the epoxidation of the 6,7
double bond versus the 2,3 double bond provides a
measure of the relative rate of reaction of the two olefins
that can be read from the ratio of the two possible
monoepoxides (i.e., the 6,7 and 2,3 epoxides) because this
is a simple intramolecular competition experiment.
Although the two double bonds are trisubstituted, the
inductive electron- withdrawing effect of the carbonyl
group lowers the nucleophilicity of the 2,3 double bond,
and if electronic properties are the decisive factor in the
reactivity, then preferential epoxidation of the 6,7 double
bond would be expected.
The 6,7-epoxycitral 2a, 2b was obtained as a mixture of
diastereoisomers with an E/Z ratio of 50:50 which
corresponds to the content in essential oil and in 87% yield
(92 mg). The preferential epoxidation of the 6,7 double
bond is probably due to its greater nucleophilicity, which
is a consequence of its electron-donating alkyl
substituents. Conversely, the reactivity of the 2,3 double
bond is drastically reduced due to the electron-
withdrawing effect of the conjugated carbonyl group,
which results in much lower nucleophilicity. This result
shows the electrophilic character of DMDO and its
preference for electron-rich double bonds. In addition, the
high conversion to the 6,7 monoepoxide indicates that the
reaction’s rate increases with the nucleophilic character of
Natural Product Communications Vol. 6 (7) 2011 927
citral’s terminal double bond due to the presence of its
electron-donating substituents. The exclusive formation of
the monoepoxide shows that the oxidation of the aldehyde
function to a carboxylic acid did not occur under the
reaction conditions employed, which indicates that DMDO
promotes the epoxidation of the double bonds in
preference to oxygen insertion.
Other studies have shown that under the same conditions
used in our epoxidation of citral, the oxidation of geraniol
(the allylic alcohol corresponding to the reduction of citral)
with DMDO produces the 2,3 and 6,7 epoxides, as well as
the bis-epoxide [36]. Clearly, the nature of the substrate is
an important factor in determining the regioselectivity of
the epoxidation reaction. In the case of geraniol, the
formation of the 2,3 epoxide has been explained in terms
of the stability gained from an intermolecular hydrogen
bond formed by the hydroxyl and the DMDO molecule in
the epoxidation transition state. The lack of 2,3 epoxycitral
formation can be explained by the inductive electron-
withdrawing effect of the carbonyl group, which lowers
the nucleophilicity of the adjacent double bond. The
resonance effect of the -unsaturated aldehyde also
contributes to the decreased reactivity of the double bond.
Finally, in the case of citral, the formation of a hydrogen
bond between the substrate and the DMDO molecule is not
possible.
The treatment of citral with H2O2 in an alkaline medium in
the presence of a phase-transfer catalyst gives rise to the
formation of the 2,3 epoxide and 6-methyl-5-hepten-2-one,
which forms as a result of the epoxide’s decomposition
[37]. Alternatively, treating citral with peracetic acid gives
the 6,7-epoxy derivative and the (E/Z)-2,6-dimethyl-5,6-
epoxy-1-heptenyl isomers resulting from a Baeyer-Villiger
side reaction [38]. The formation of the 6,7 derivative has
also been reported in moderate yield (43%) by Woitiski
[12] by reacting citral with a H2O2-dinuclear manganese
(IV) complex of oxalic acid.
Carvone is another substrate chosen for DMDO
epoxidation, which contains two electron-poor double
bonds, the exocyclic double bond due to the low number of
electron-donating alkyl substituents and the endocyclic
bond due to the bearing electron-withdrawing effect of the
carbonyl group. These carvone’s structural characteristics
allow to compare the reactivity of the two double bonds in
an intramolecular competition experiment with this cyclic
substrate. The results show the regioselectivity in the
reaction of carvone with DMDO and the formation of the
exocyclic monoepoxide 3 as the only product of the
reaction in 80% yield (102 mg) and 50:50
diastereoselectivity. The selective epoxidation of the
carvone exocyclic double bond corroborates that DMDO is
a powerful reagent for the epoxidation of electron-poor
double bonds. The lack of endocyclic oxirane formation
demonstrates the low nucleophilicity of the enone double
bond due to the carbonyl group electron-withdrawing
effect.
928 Natural Product Communications Vol. 6 (7) 2011
In the epoxidation of carvone under oxygen using
Nickel(II) acetylacetonate as catalyst, (R)-(-)-carvone
exhibited low reactivity due to long times reaction to
obtain the exocyclic monoepoxide [39]. When the
anhydrous hydrogen peroxide/alumina system is used, a
high regioselectivity of the exocyclic monoepoxide
(93.8%) and a low substrate conversion (8.7%) were
observed, however, yields decreased to 0.8% in the
absence of alumina catalyst [40].
Citronellal is an aliphatic olefin which contains an
aldehyde group and a terminal trisubstituted double bond.
This substrate can be used as a chemoselectivity probe for
DMDO epoxidation in terms of the aldehyde oxidation to
carboxylic acid versus the double bond epoxidation. The
results show the double bond oxidation leading to the
formation of the diastereomeric epoxides in 84% yield
(272 mg) and 50:50 ratio, confirming the high reactivity
and chemoselectivity of DMDO to nucleophilic olefins.
Similar results were obtained for the epoxidation of
Citronellal with tert-butyl hydroperoxide (TBHP) and
molybdenum catalyst in nonpolar and aprotic solvents,
giving a mixture of diastereomeric epoxides with a 50:50
ratio and 71% yield [41].
Essential oils are an important source of terpenes that can
be modified chemically to generate a wide variety of
polyfunctional compounds. From the chemical point of
view, epoxidation of the main constituents of Tagetes
lucida, Cymbopogon citratus, Lippia alba and Eucalyptus
citriodora with DMDO generates oxirane derivatives and
provides evidence for the high reactivity of this new
oxygen transfer reagent. Although a large number of
compounds have been epoxidized with DMDO, to our
knowledge, this is the first report of the synthesis of
estragole, citronellal and citral epoxides using DMDO.
Epoxide stability is an important characteristic when
carrying out tests of biological activity.
The high conversion of estragole and the good yield of
epoxyestragole indicate that unactivated olefins can be
efficiently oxidized with DMDO. The epoxidation of citral
proved to be regioselective, providing only 6,7 epoxide as
a result of the higher nucleophilicity of the 6,7 double
bond relative to the 2,3 double bond, which is electron
deficient due to the electron-withdrawing effect of the
conjugated carbonyl group. In addition, the use of citral
shows that DMDO oxidizes double bonds in preference to
oxidizing aldehydes to carboxylic acids. For carvone, the
nucleophilicity of the double bond adjacent to the carbonyl
group is quite low due to the electronic-withdrawing effect
of this group, which is an important reactivity factor. The
DMDO epoxidation of citronellal confirms the
chemoselectivity of this oxidant agent in terms of the
aldehyde oxidation to carboxylic acid versus the double
bond epoxidation. Our results indicate that it is important
to consider both the regio and chemoselectivity of the
DMDO in the epoxidation of natural compounds.
Veloza et al.
Experimental
General: Gas Chromatography-Mass Spectrometry (GC-
MS) analyses were performed using a Shimadzu GC-2010
gas-chromatograph coupled to a selective mass detector
(Shimadzu QP-2010) and equipped with an Rtx-5Sil-MS
column (30 m x 0.25 mm i.d., 0.25 m film thickness)
operating in electronic ionization mode at 70 eV. Helium
was used as the carrier gas. All data processing was done
on the Shimadzu Labsolutions software (GCMS Solution
version 2.5), which includes the Wiley Registry of Mass
Spectral Data, 7th Edition (Wiley Interscience, New York).
1
H NMR spectra were acquired on a Bruker Avance
DRX400 (400.13 MHz) spectrometer at 25 °C using
CDCl3 as the solvent and TMS as an internal standard.
Essential oils extraction: Essential oils were obtained
from plant leaves by microwave-assisted hydrodistillation
(MWHD) at CENIVAM (Centro Nacional de
Investigaciones para la Agroindustrialización de Especies
Vegetales Aromáticas y Medicinales Tropicales),
Universidad Industrial de Santander, Bucaramanga
(Colombia).
Terpenes isolation: The essential oils from T. lucida (800
mg) and L. alba (812 mg) were fractionated over a silica
gel column eluting with n-hexane-chloroform (90:10) and
increasing the polarity of the solvent to 50:50. Estragole
(119 mg) and carvone (128 mg) were obtained with a
purity of 95% as determined by GC-MS. The structure of
each compound was identified by comparison of their
mass spectrum (Wiley Registry of Mass Spectral Data) and
was further confirmed by comparison to 1H NMR data
reported in the literature.
The essential oils from C. citratus (3 mL) and E.
citriodora (3 mL) were distilled under reduced pressure
(60ºC/550 mm Hg and 90ºC/550 mm Hg, respectively).
Citral (106 mg) and citronellal (324 mg) were obtained
with a purity of 95% as determined by GC-MS. The
structure of citral and citronellal were identified by
comparison of their mass spectrum (Wiley Registry of
Mass Spectral Data) and was further confirmed by
comparison to 1H NMR data reported in the literature.
Preparation of DMDO in acetone solution: A
concentrated solution of DMDO in acetone (0.11 M) was
prepared using Oxone (Caroate, 2KHSO5.KHSO4.K2SO4),
acetone and NaHCO3 according to a previously reported
literature procedure [42]. The DMDO concentration was
determined by UV/Vis spectrophotometry.
General procedure for the epoxidation of terpenes with
DMDO: Estragole, citral, carvone and citronellal were
dissolved in acetone (1 mL) and 1.0-1.2 equiv. of DMDO
(0.11 M solution in acetone) was rapidly added at 25 °C.
The solution was stirred at this temperature and monitored
by TLC and GC-MS until the peroxide test (KI/HOAc)
was negative (total reaction time 0.5-1 h). The solvent was
Dimethyldioxirane epoxidation of essential oils Natural Product Communications Vol. 6 (7) 2011 929

removed under reduced pressure to give the respective 8,9-Epoxycarvone (3)

epoxides in high purity. The structure of the epoxides was
confirmed with spectral data and comparison with spectral
data reported in the literature of 1 [43], 2a,2b [44], 3[45]
and 4 [44].
Epoxyestragole (1)
H-NMR (400 MHz, CDCl3): 7.16 (2H, d, J=8.0 Hz,
1
H-5, H-9), 6.84-6.87 (2H, m, H-6, H-8), 3.79 (3H, s,
H-10), 3.09-3.11 (1H, m, H-2), 2.74-2.89 (3H, m, H-1,
H-3), 2.52 (1H, dd, J=2.8 Hz, J=5.0 Hz, H-3).
6,7-Epoxycitral
1
H-NMR (400 MHz, CDCl3) 2a epoxygeranial:  10.0
(1H, d, J=8.0 Hz, H-1), 5.91-5.93 (1H, m, H-2), 2.71-2.77
(3H, m, H-4, H-6), 2.01 (3H, s, H-9), 1.64-1.83 (2H, m,
H-5), 1.28 and 1.31 (6H, s, H-8, H-10).
2b epoxyneral:  9.97 (1H, d, J=8.0 Hz, H-1), 5.91-5.93
(1H, m, H-2), 2.71-2.77 (1H, m, H-6), 2.29-2.48 (2H, m,
H-4), 2.20 (3H, s, H-9), 1.64-1.83 (2H, m, H-5), 1.28 and
1.31 (6H, s, H-8, H-10).
1
H-NMR (400 MHz, CDCl3) Isomer A:  6.72-6.76 (1H,
m, H-3), 2.71 (1H, d, J=4.4 Hz, H-9), 2.03-2.57 (6H, m, H-
4, H-5, H-6, H-9), 1.77-1.80 (3H, m, H-7), 1.33 (3H, s, H-
10).
Isomer B:  6.72-6.76 (1H, m, H-3), 2.68 (1H, d, J=4.8 Hz,
H-9), 2.03-2.57 (6H, m, H-4, H-5, H-6, H-9), 1.77-1.80
(3H, m, H-7), 1.32 (3H, s, H-10).
6,7-Epoxycitronellal (4)
1
H-NMR (400 MHz, CDCl3):  9.77 (1H x 2, m, H-1),
2.68-2.71 (1H x 2, m, H-6), 2.39-2.46 (2H, m, H-2’), 2.24-
2.30 (2H, m, H-2), 2.08-2-18 (1H x 2, m, H-3), 1.52-1.57
(4H x 2, m, H-4, H-5), 1.27, 1.31 (6H x 2, s, H-8, H-10),
1.00 (3H x 2, d, J=6.8 Hz, H-9).
Supplementary data: 1H-NMR spectra for compounds 1,
2a, 2b, 3 and 4 are contained in the supplementary data.
Acknowledgments - The authors gratefully acknowledge
financial support from COLCIENCIAS-CENIVAM
(contrato RS-432-2004), Universidad Tecnológica de
Pereira and Red ALMA MATER.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Validation of the Ethnopharmacological Use of Polygonum 931 - 933

persicaria for its Antifungal Properties

Marcos Derita*and Susana Zacchino

Pharmacognosy Area, Faculty of Biochemical and Pharmaceutical Sciences, National University of

Rosario, Suipacha 531, Rosario, 2000, Argentina

mgderita@hotmail.com

Received: November 13th, 2010; Accepted: March 16th, 2011

Polygonum L. genus (Polygonaceae) is represented in Argentina by 21 species and some of them have been used in the traditional medicine
of our country to treat affections related with fungal infections, such as skin ailments and vaginal diseases. With the aim of contributing to the
correct ethnopharmacological use of this genus, in the present work we describe the antifungal properties of P. persicaria (species not studied
up to now) and the bio-guided isolation of the main active compounds. Results showed that dichloromethane extracts was the most active
with MICs (Minimun Inhibitory Concentrations) between 31.2 – 1000 µg/mL, validating the ethnopharmacological use of P. persicaria to
treat affections related with fungal infections in the Argentinean traditional medicine.

Keywords: Validation, Ethnopharmacological use, Polygonum, Antifungal activity.

Since the early 1980s, fungal infections have emerged as A

major causes of morbi-mortality, mainly among 11

immunocompromised patients. The majority of deaths

11
15 CHO
15 CHO 12
12 1
1 CHO
were associated with species of Candida, Aspergillus and CHO 9
9 2 10
2 8
10 8
5

Cryptococcus [1]. Instead, dermatophytes such as

5 3
3 4 7
4 7
6 6
H H

Trichophyton and Microsporum spp. produce superficial 13 14 (1) 13 14 (2)

B
infections (tineas) which are usually not threatening but 5

dramatically diminish the quality of life of human beings

6 4

B
H 5´ 1

[2]. Although it appears to be an array of antifungal agents

1 2´
8 3´ H3CO OCH3 3
H3CO O 4´ 
7
6´ 2
8a 2 1´ A
(polyenes, azoles, allylamines and the recent 6
4a
4
3
6´
4´
3´
1´
´


echinocandins) there are, in fact, few therapeutic options.

5 5´ 2´
H

OH O
H
(3) OH O (4)
Decreased susceptibilities of yeasts to the currently 6
5

available antifungal agents [3] added to the increase in the HO

5´
OCH3
1
3

number of reported cases of resistance [4], have led to a

4´ 
6´ 2

1´

general consensus that new efforts for detecting novel

3´ 
´
2´

antifungal entities remain a priority. In this context, the OH O (5)

study of plants with history of ethnopharmacological use Figure 1: A) Sesquiterpene [polygodial (1) and isopolygodial (2)] and B)
for ailments related to fungal infections, can serve two flavonoids [pinostrobin (3), flavokawin B (4) and cardamonin (5)] isolated
from P. persicaria DCM extract.
goals: validation of the use of traditional medicines and
finding new leads [5]. of two sesquiterpene dialdehydes: polygodial (1),
isopolygodial (2), and three flavonoids: pinostrobin (3),
Polygonum L. genus (Polygonaceae) is represented in
flavokawin B (4) and cardamonin (5) (Figure 1).
Argentina by 21 species and some of them have been used
Compounds 1 and 2 were previously isolated from Drymis
to treat affections related with fungal infections, such as
spp. [9,10], P. punctatum [7] and P. acuminatum [8], while
skin ailments and vaginal diseases [6]. Previous studies of
compounds 3-5 were previously isolated from
this genus reported that P. punctatum possessed antifungal
Boesenbergia pandurata, Myrica pensilvanica, P.
properties against yeasts and dermatophytes [7]. With the
ferrugineum and Piper spp [11-13].
aim of contributing to the correct ethnopharmacological
use of this genus, in a previous work we described
Compounds 1-5 were evaluated for their antifungal
the antifungal properties of P. acuminatum [8] and
activities with the microbroth dilution assay recommended
in this work we describe those of P. persicaria (species
by the Clinical and Laboratory Standards Institutes (CLSI)
not studied up to now) and the bio-guided isolation
932 Natural Product Communications Vol. 6 (7) 2011 Derita M. et al.

Table 1: Antifungal activity (MICs in µg/mL) of P. persicaria extracts.

Species Extract Antifungal activity (MICs in µg/mL)
Ca Sc Cn Afu Afl An Mg Tr Tm

P. persicaria Hex I I 1000 I I I 1000 500 1000

DCM 1000 500 500 1000 1000 1000 125 62.5 31.2
EtOAc 1000 1000 1000 I I I 1000 500 1000
MeOH I I I I I I I I I
Standards drugs Ketoconazole 0.50 0.50 0.25 0.12 0.50 0.25 0.04 0.02 0.02
Amphotericin 1.00 0.50 0.25 0.50 0.50 0.50 0.12 0.07 0.07
Terbinafine - - - - - - 0.04 0.01 0.04
Ca: Candida albicans ATCC 10231; Sc: Saccharomyces cerevisiae ATCC 9763; Cn: Cryptococcus neoformans ATCC 32264; Afu: Aspergillus fumigatus ATCC 26934;
Afl: Aspergillus flavus ATCC 9170; An: Aspergillus niger ATCC 9029; Mg: Microsporum gypseum C 115; Tr: Trichophyton rubrum C 113; Tm: Trichophyton
mentagrophytes ATCC 9972. I: Inactive = MIC > 1000 µg/mL.

Table 2: Antifungal activity (MICs in µg/mL) of compounds isolated from P. persicaria.

Compounds Antifungal activity (MICs in µg/mL)

Ca Sc Cn Afu Afl An Mg Tr Tm
1 3.90 15.6 7.8 250 250 250 62.5 7.8 7.8
2 250 125 125 250 250 250 62.5 62.5 31.2
3 250 250 250 125 125 250 62.5 62.5 62.5
4 I I 250 250 125 250 125 125 125
5 250 250 250 250 250 250 62.5 15.6 15.6
Standards drugs Ketoconazole 0.50 0.50 0.25 0.12 0.50 0.25 0.04 0.02 0.02
Amphotericin 1.00 0.50 0.25 0.50 0.50 0.50 0.12 0.07 0.07
Terbinafine - - - - - - 0.04 0.01 0.04
Ca: Candida albicans ATCC 10231; Sc: Saccharomyces cerevisiae ATCC 9763; Cn: Cryptococcus neoformans ATCC 32264; Afu: Aspergillus fumigatus ATCC 26934;
Afl: Aspergillus flavus ATCC 9170; An: Aspergillus niger ATCC 9029; Mg: Microsporum gypseum C 115; Tr: Trichophyton rubrum C 113; Tm: Trichophyton
mentagrophytes ATCC 9972. I: inactive = MIC > 250 µg/mL).

[14] and results are shown in Table 2. They were active useful in guiding the discovery of antifungal compounds
against yeasts, Aspergillus spp. and dermatophytes with against dermatophytes, as it was demonstrated in a recent
MICs between 3.90 - 250 µg/mL survey among seven Latinaoamerican countries [15].

As it can be observed in Table 2, the five compounds Experimental

isolated from P. persicaria, drimanes as well as
flavonoids, all showed antifungal activity. Among them,
polygodial (1) showed the best activity against yeasts and
dermatophytes with MICs between 3.9 to 62.5 µg/mL and
it was almost inactive against species of Aspergillus genus.
Its epimer, isopolygodial (2), showed a lower antifungal
activity (MICs between 31.2 to 250 µg/mL), suggesting
that the C-9 configuration plays an important role in the
antifungal activity, as we have been found in a previous
paper [8].
Regarding flavonoids 3-5, there is not a clear difference
among the antifungal activities of them against yeasts
and Aspergillus spp. Nevertheless, chalcone 5 showed
a high antifungal activity against T. rubrum and T.
mentagrophytes with MICs = 15.6 µg/mL, eight times
higher than the activity showed by chalcone 4 against the
same strains. This striking difference in activity against
Trichophyton spp. could be attributed to the phenolic OH
present in compound 5 which is absent in 4.
These results show that the antifungal activity of P.
persicaria could be attributed to polygodial but it is clear
that the rest of the isolated compounds could contribute to
the antifungal behavior of this traditional used species. In
addition, these results validate the ethnopharmacological
use of P. persicaria to treat affections related to fungal
infections in the Argentinean traditional medicine and add
a new evidence that the ethnopharmacological approach is
Extracts preparation and compounds isolation: Air-dried
aerial parts of each species (100 g) were powdered and
successively macerated (3×24 h each) with Hexane (Hex),
dichloromethane (DCM), ethyl acetate (EtOAc) and
methanol (MeOH) with mechanical stirring to obtain the
corresponding extracts, after filtration and evaporation.
Bioassay-guided fractionation of DCM extract allowed us
to isolate the compounds responsible for the antifungal
activity. 1.1 g of P. persicaria DCM extract were
submitted to column chromatography using mixtures of
Hex: AcOEt in increasing polarity as elution solvents. We
obtained 10 fractions; three of them were actives (fractions
6-8). From 150 mg of fraction 6, by repeated column
chromatography, we obtained 55 and 30 mg of compounds
1 and 2 respectively. From 170 mg of fraction 7, by
repeated column chromatography, we obtained 50, 46 and
25 mg of compounds 3, 4 and 5 respectively. Additionally,
from 70 mg of fraction 8, we obtained 10 mg of compound
5. All the compounds were characterized by UV-visible,
IR, 1H NMR and 13C NMR spectroscopy.
Antifungal assay: For the antifungal evaluation, strains
from the American Type Culture Collection (ATCC,
Rockville, MD, USA) and Centro de Referencia en
Micología, CEREMIC [C, Faculty of Biochemical and
Pharmaceutical Sciences, Suipacha 531 (2000)-Rosario,
Argentina] were used: Candida albicans (Ca) ATCC
10231, Saccharomyces cerevisiae (Sc) ATCC 9763,
Cryptococcus neoformans (Cn) ATCC 32264, Aspergillus
Ethnopharmacological use of Polygonum Natural Product Communications Vol. 6 (7) 2011 933

flavus (Afl) ATCC 9170, Aspergillus fumigatus (Afu)
ATTC 26934, Aspergillus niger (An) ATCC 9029,
Trichophyton rubrum (Tr) C 110, Trichophyton
mentagrophytes (Tm) ATCC 9972 and Microsporum
gypseum (Mg) C 115. Strains were grown on Sabouraud-
chloramphenicol agar slants for 48 h at 30 ºC, maintained
on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and
subcultured every 15 days to prevent pleomorphic
transformations. Inocula of cell or spore suspensions were
obtained and quantified following reported procedures
(CLSI).[14]
techniques according to the guidelines of CLSI for yeasts:
document M27-A2 and for filamentous fungi, M38A. For
the assay, stock solutions of extracts or pure compounds
(100 µL) were two-fold diluted with the culture medium.
A volumen of 100 µL of inoculum suspension [adjusted to
1–5 × 104 cells/spores as Colony Forming Units
(CFU/mL)] was added to each well with the exception of
the sterility control where sterile water was added to the
well instead. Ketoconazole (Sigma Chem. Co., St. Louis,
MO), Terbinafine (Novartis) and Amphotericin B (Sigma)
were used as positive controls.

Minimum Inhibitory Concentration (MIC) of each extract Acknowledgments - CONICET, ANPCyT, UNR,
or compound was determined by using broth microdilution ERASMUS MUNDUS, UNIBO.

References

[1] Pfaller M, Diekema D. (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clinical Microbiology
Reviews, 20, 133-163
[2] Weitzman I, Summerbell R. (1995) The dermatophytes. Clinical Microbiology Reviews, 8, 240-259
[3] Hsueh P, Lau Y, Chuang Y, Wan J, Huang W, Shyr J, Yan J, Yu K, Wu J, Ko W, Yang Y, Liu Y, Teng L, Liu Ch, Luh K. (2005)
Antifungal susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species from Taiwan:
Surveillance of multicenter antimicrobial resistance on Taiwan program data from 2003. Antimicrobial Agents and Chemotherapy,
49, 512-517
[4] White T, Holleman S, Dy F, Mirels L, Stevens D. (2002) Resistance mechanisms in clinical isolates of Candida albicans.
Antimicrobial Agents and Chemotherapy, 46, 1704-1713
[5] Verpoorte R. (2000) Pharmacognosy in the new millennium: leadfinding and biotechnology. Journal of Pharmacy and
Pharmacology, 52, 253-262
[6] Del Vitto L, Petenatti E, Petenatti M. (2003) Materia Medica Vegetal. Plantas medicinales nativas y exóticas empleadas en
fitomedicinas, homeopáticos, galénicos. Servicio Técnico Herbario UNSL, San Luis, pp. 1-63
[7] De Almeida Alves T, Lacerda Ribeiro F, Kloos H, Zani C. (2001) Polygodial, the fungitoxic component from the Brazilian
medicinal plant Polygonum punctatum. Memórias do Instituto Oswaldo Cruz, 96, 831-833
[8] Derita M, Leiva M, Zacchino S. (2009) Influence of plant part, season of collection and content of the main active constituent, on
the antifungal properties of Polygonum acuminatum Kunth.. Journal of Ethnopharmacology, 124, 377-383
[9] Cechinel Filho V, Schlemper V, Santos A, Pinheiro T, Yunes R, Mendes G, Calixto J, Delle Monache F. (1998) Isolation and
identification of active compounds from Drymis winteri barks. Journal of Ethnopharmacology, 62, 223-227
[10] Muñoz-Concha D, Vogel H, Yunes R, Razmilic I, Bresciani L, Malheiros A. (2007) Presence of polygodial and drimenol in Drymis
populations from Chile. Biochemical Systematics and Ecology, 35, 434-438
[11] Hodgetts K. (2001) Approaches to 2-substituted chroman-4-ones: synthesis of (-)-pinostrobin. Tetrahedron Letters, 42, 3763-3766.
[12] Burke B, Nair M. (1986) Phenylpropene, benzoic acid and flavonoids derivatives from fruits of Jamaican Piper species.
Phytochemistry, 25, 1427-1430
[13] López S, González Sierra M, Gattuso S, Furlán R, Zacchino S. (2006) An unusual homoisoflavanone and a structurally-related
dihydrochalcone from Polygonum ferrugineum. Phytochemistry, 67, 2152-2157
[14] CLSI (Clinical and Laboratory Standards Institute). (2002) Methods M 27-A2, Vol. 22 (15): 1-29 and M 38-A, Vol. 22 (16): 1-27.
Wayne Ed.
[15] Svetaz L, Zuljan F, Derita M, Petenatti E, Tamayo G, Cáceres A, Cechinel Filho V, Giménez A, Pinzón R, Zacchino S, Gupta M.
(2010) Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin
American countries. Journal of Ethnopharmacology, 127, 137-158
 2011
NPC Natural Product Communications Vol. 6
No. 7
On the Isomerization of ent-Kaurenic Acid 935 - 938

Julio Rojasa, Rosa Apariciob, Thayded Villasmilc, Alexis Peñac and Alfredo Usubillagab*
a
Postgrado de Quimica de Medicamento, Facultad de Farmacia y Bioanálisis,
Universidad de Los Andes, Mérida, Venezuela
b
Instituto de Investigaciones, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida,
Venezuela
c
Postgrado Interdisciplinario de Química, Facultad de Ciencias, Universidad de Los Andes, Mérida,
Venezuela

usubillaga@intercable.net.ve

Received: December 12th, 2010; Accepted: March 15th, 2011

Kaurenic acid (1a) is a tetracyclic diterpene that has an exocyclic double bond at  Isokaurenicacid (2a) has an endocyclic 15double
bondThis compound has been isolated from Espeletia tenore (Espeletinae), a resinous plant from the Venezuelan Andes, but its occurrence
is rare. In order to obtain a larger amount of 2a, the isomerization of 1a, which is easily obtained from other Espeletinae, was tried. Kaurenic
acid methyl ester (1b) was treated with dil. HCl in CH3Cl/EtOH, after 6 h under reflux a yield of 41.5% isokaurenic acid methyl ester (2b)
was obtained but 35.7% 16-ethoxy-kauran-19-oic acid methyl ester (3b) had formed as a byproduct. Treating 1b with CF3COOH in
refluxing CH2Cl2 permitted to obtain a yield of 66.6 % of 2b in 4 h and only traces of 16-hydroxy-kauran-19-oic acid methyl ester (3a) as
a byproduct. Both isomers were separated on a silica gel column impregnated with 20% AgNO3. Treating 2b with KOH in refluxing DMSO
yielded pure isokaurenic acid, no back isomerization was observed.

Keywords: Ent-kaur-16-en-19-oic acid, ent-kaur-15-en-19-oic acid, CF3COOH, methyl esters, silver nitrate chromatography.

Ent-kaurenic acid (1a, Figure 1) is a tetracyclic diterpene 20 11 13

17

that has been reported to have antimicrobial [1] and 1

15
16

8 R
antiparasitic activity [2]. It also shows cytotoxicity against 4

several cancer cell lines [3]. The occurrence of kaurenic 19

COOR
acid, which has an exocyclic double bond at Δ16, is COOR COOCH3

widespread in the plant kingdom, while the occurrence of
its isomer ent-kaur-15-en-19-oic acid (2a), also called
isokaurenic acid, is rare. This substance (2a) was first
obtained by Ekong and Ogan by lithium reduction of
Xylopic acid [4a], but 2a has been isolated as a natural
product from Espeletia tenore, a midget Espeletiinae from
the Venezuelan Andes [4b].
Since the quantity of isokaurenic acid that could be
obtained from E. tenore is small, isomerization of kaurenic
acid was studied in order to obtain it in sufficient quantity
to explore its biological properties. Double bond migration
in olefins could be base-catalyzed or acid-catalyzed
[5a,5b]. Base-catalyzed isomerization can be effected in
homogeneous solution or in the presence of basic
heterogeneous catalysts. Isomerization of 1b was tried
with sodium on alumina according to Shabtai and Gil-Av
[6], but no reaction was observed after 24 hours.
Isomerization was also attempted treating 1b with iodine
in benzene solution under reflux, according to Barnes and
MacMillan [7] but only 10.6% of 2b was obtained after 6
hr.
1a R= H 2a R= H 3a R= OH
1b R= CH3 2b R= CH3 3b R= OC2H
5
Figure 1
Since it had been observed that, during isolation of the
acidic fraction of some Espelettinae, treatment with HCl
produced traces of isokaurenic acid, isomerization of 1b
in CHCl3/EtOH solution was tried adding 5 drops of
HCl:H2O (10:1) at room temperature. The course of the
reaction was followed by gas chromatography. As it is
shown on Table 1, after one hr of reaction 35.4% of the
original amount of 1b had isomerized into 2b, while the
relative concentration of 1b had diminished to 55.6%, but
at the same time 9.0% of ent-16α-ethoxy-kauran-19-oic
acid (3b) had formed as a by-product. After 4 hr of
reaction a maximal yield of 2b was achieved (46.4%), but
after 6 hr the relative concentration of 2b had diminished
and settled at 41.4%. At the same time the by-product (3b)
represented 35.7% of the reaction mixture.
Since formation of 3b was undesirable, isomerization
was tried using CF3COOH in dry CH2Cl2.The course of
the reaction is shown on Table 2. After 1.0 hr of reaction
936 Natural Product Communications Vol. 6 (7) 2011
Table 1: Acid-isomerization of ent-kaurenic acid using HCl in
CHCl3/EtOH.
Time (hr) % 2b % 1b
0 -------- 100
1 35.4 55.6
2 46.4 35.3
4 41.8 24.7
6 41.5 22.8
54.4% of isomerization had been achieved. After 2.0 hr the
yield was 63.5%. After 4.0 hr the reaction had arrived to
equilibrium where relative concentration of 2b was 66.6%
and concentration of 1b was down to 33.3%. But after 4.0
hr it was observed that 0.1% of 3a had been formed,
probably caused by the presence of traces of water.
Table 2: Acid-isomerization of ent-kaurenic acid using CF3COOH.
Time (hr) % 2b % 1b
0 -------- 100
1 54.4 45.6
2 63.5 36.5
3 66.2 33.8
4 66.6 33.3
t : traces
Figure 2 shows the increase of 2b as a function of time and
the abatement of kaurenic acid methyl ester concentration
(1b) to end up with a constant concentration mixture made
up of 66.6% 2b and 33.3% 1b. Absolute exclusion of
water would hinder the formation of 16-hydroxy-kauran-
19-oic acid methyl ester (3a). But from a practical
point of view two hours of reaction is enough to obtain
a good yield (63.5%) of the desired product. Separation
of both isomers is accomplished over a silica gel
column impregnated with 20% silver nitrate. Taking into
consideration that silver nitrate chromatography is required
to separate both isomers, the methyl ester of kaurenic acid
(1b) was used instead of the free acid (1a). Isokaurenic
acid (2a) was recovered refluxing 2b with KOH in DMSO
solution.
According to Hubert and Reimlinger [5b] the acid
catalyzed isomerization takes place via carbonium ions. In
the case of kaurenic acid, presence of an acid causes the
formation of a carbonium ion at C-16. The catalyst acts as
a proton donor and acceptor. Migration of the double
bonds occurs because a proton is transferred to the C-17
exocyclic methylene moiety, which becomes a methyl
group, and at the same time, a proton is lost from C-15
generating a Δ15 double bond. But this process is
reversible and the reaction proceeds until it reaches a
thermodynamic equilibrium. In this case a Δ15 double
bond is thermodynamically more stable and therefore its
formation is enhanced leading to the migration of 2/3 of
the original exocyclic double bond to a Δ15 position.
The rate of formation of 3a increases with reaction time.
After 72 h under reflux the yield of 3a was 13%.
Rojas et al.
% 3b
------
9.0
18.2
33.5
35.7
Figure 2
Experimental
% 3b General procedures: Melting points were determined on a
------ Fisatom 430 D apparatus and are uncorrected. IR spectra
0 were measured on a Shimadzu Affinity instrument as KBr
0 discs. NMR spectra were recorded with a Bruker Avance
t 400 MHz instrument for solutions in CDCl3, 1H, 13C,
0.1 DEPT, H-H COSY, HMQC, and HMBC experiments
were performed. GC was made on a Perkin Elmer
Autosystem gas chromatograph equipped with FID
detector. A 5% phenylmethyl polysiloxane capillary
column was used (30 m, 0.25 mm i.d., film thickness 0.25
m). The oven temperature was programmed from 250°C
to 300°C at 10°C/min., and kept isothermal at the higher
temperature for 10 min. The injector and detector
temperatures were 200°C and 300°C respectively. The
carrier gas was helium at 0.9 mL/ min. The samples (1.0
L) were injected using a split ratio of 1:10. For mass
spectrometry an Agilent MSD 5973 instrument equipped
with a DB-5MS capillary column (30 m, 0.25 mm, 0.25
m film).The oven temperature program was the same
used for GC analysis. Injector temperature, carrier gas, and
sample injection conditions were also the same but a split
ratio of 1:50 was used. Analytical TLC was performed on
Merck aluminum-backed silica gel foils (F254). Flash
chromatography was performed on Merck silica gel Grade
9385 (230-400 mesh) by gradient elution with hexane-
EtOAc or hexane-diethyl ether mixtures. Kaurenic isomers
were separated on a silica gel column impregnated with
20% AgNO3.
Isolation of kaurenic acid (1a): Espeletia semiglobuta
was collected at Paramo of Piedras Blancas in February
2009. A voucher specimen (AU-30) was deposited at the
MERF Herbarium. The leaves (10 Kg) were air dried,
ground and extracted with a hexane-diethyl ether mixture
(3:1) at room temperature. The extract was shaken with
0.5 N NaOH solution. The aqueous layer was made acidic
by addition of dil. HCl and shaken with hexane to recover
the acid fraction. Kaurenic acid was purified by flash
chromatography over silica gel using hexane and hexane-
diethyl ether (9:1) as solvent. Chromatographic fractions
were inspected by TLC, fractions containing pure kaurenic
acid were combined and crystallized Pure kaurenic acid
Isomerization of Kaurenic acid
(13.5 g) was obtained, mp 175-178°C (lit [8] 179-181).
It was compared with an authentic sample (TLC,
1
H-NMR) [4b].
Kaurenic acid methyl ester (1b): A solution of kaurenic
acid (1a, 20 mmol) was dissolved in Et2O and mixed with
freshly distilled diazomethane (about 30 mmol) obtained
from nitrosomethylurea [9a,9b]. After 24 h at room
temperature the solvent and excess diazomethane were
distilled off at low pressure. Kaurenic acid methyl ester
(1b) crystallized from hexane, MP 75°C. GC retention
time 3.80 min. MS (EI, 70 eV): m/z (%) = 316.2 (51), 302
(35), 284 (62), 257 (199), 241 (79), 213 (33), 187 (2), 121
(45), 91 (47).
Attempted isomerization of 1b with sodium on alumina:
The catalyst was prepared according to Shabtai and Gil-Av
[6]. Alumina was heated at 300oC during 24 h . Pretreated
alumina (5.0 g) was mixed with 1.0 g of sodium at 140°C
with stirring under argon atm. The catalyst was cooled at
2-3°C and a solution of 1b (500 mg) in dry hexane was
added. Samples were taken after continuous stirring at 1.0
h, 6.0 h, and 24 h, and analyzed by GC-MS after 1.0 h, 6.0
h, and 24 h, but no isomerization occurred and only the
peak of 1b, with a retention time of 3.79 min., was
observed on the gas chromatogram.
Isomerization of 1b with iodine in benzene solution:
Kaurenic acid methyl ester (1b, 300 mg) was dissolved in
dry benzene containing 20 mg of iodine and it was heated
under reflux for 6 hr. The solution was cooled and shaken
twice with aqueous sodium thiosulphate. The organic layer
was taken to dryness. A 5 mg sample of the solid was
dissolved in diethyl ether and analyzed by GC. It was
observed that after six hours of reaction only 10.6% of 1b
had isomerized into 2b which had a retention time of 3.55
min.
Isomerization of 1b with diluted HCl in CHCl3/EtOH:
Kaurenic acid methyl ester (2.0 mmol) was dissolved in a
mixture 50 mL of CHCl3 and 10 mL of EtOH. After
addition of 5 drops of 10% HCl, the mixture was heated
under reflux. Aliquot samples (5 mL) were taken at 1h, 2h,
4h, and 6 h, washed with dil. NaHCO3, and with H2O. The
organic layer was dried over Na2SO4 and evaporated to
dryness. Samples (5 mg) were dissolved in Et2O and
analyzed by GC. Results are shown on table 1.
Isolation of ent-16-ethoxy-kauran-19-oic acid methyl
ester (3b): The rest of the solution (40 mL) of the previous
isomerization reaction was washed with dil. NaHCO3 and
H2O. The chloroform layer was treated with dry Na2SO4,
filtered, and mixed with 1 g of silica gel. CHCl3 was
evaporated under vacuum and the silica gel containing the
product of isomerization was added to the top of a flash
column charged with 12 g of silica gel. The column was
eluted with hexane which yielded a mixture of 1b and 2b.
Natural Product Communications Vol. 6 (7) 2011 937
When both isomers had eluted the column was treated with
hexane-EtOAc (10:1) to elute 3b.
MP: 140°C.
IR (cm-1): 2981, 2932, 2854, 1724, 1466, 1443, 1234,
1157, 1068, 987.
1
H NMR (400 MHz, CDCl3): 0.77 (1H, dt, J = 4; 14 Hz,
H-1a), 0.81 (3H, s, H-20), 0.96 (1H, m, H-9), 1.02 (1H,
dd, J= 2; 14 Hz, H-5), 1.10 (3H, t, J = 7 Hz, Me), 1.17
(3H, s, H-18), 1.26 (3H, s, H-17), 1.32 (1H, m, H-15a),
1.38 (1H, m, H-14a), 1.5 (2H, m, H-12), 1.51 (2H, m, H-
11), 1.52 (1H, m H-3a), 1.53 (2H, m, H-2), 1.75 (1H, m,
H-3b), 1.78 (2H, m, H-6), 1.84 (1H, m, H-1b), 2.02 (1H,
bs, H-13), 2.15 (2H, m, H-7), 3.31 (2H, q, J = 7 Hz,
OCH2), 3.63 (3H, s, OMe).
13
C NMR (100 MHz, CDCl3): 178.2 (COO), 83.9 (C-17),
57.1 (C-5), 56.8 (OCH2), 56.2 (C-9), 55.2 (C-15), 51.2
(OCH3), 44.9 (C-4), 43.9 (C-8), 42.3 (C-14), 44.0 (C-13),
40.9 (C-1), 39,6 (C-10), 38.3 (C-7), 37.2 (C-3), 28.9
(C-18), 26.8 (C-12), 22.3 (C-6), 19.3 (C-2), 19.2 (C-17),
18.2 (C-11), 16.7 (CH3), 15.5 (C-20).
MS (EI, 70 eV): m/z (%) = 362 (4), 347 (34), 316 (85), 274
(100), 257 (55), 217 (54), 121 (70).
Isomerization of 1b with CF3COOH in CH2Cl2: To a
solution of kaurenic acid methyl ester (5.0 mmol) in dry
CH2Cl2 (100 mL) 10 drops of trifluoroacetic acid were
added and the mixture was heated under reflux in an argon
atmosphere. Aliquot samples (5 mL) were taken at 1h, 2h,
and 4h. They were washed with H2O; the organic layer
was dried over Na2SO4 and evaporated to dryness.
Samples (5mg) were dissolved in Et2O and analyzed by
gas chromatography. Table 2 shows the results of this
reaction as a function of time. To recover the isokaurenic
acid methyl ester the rest of the reaction mixture was
washed with H2O, dried over Na2SO4, filtered, and the
solvent was evaporated to dryness. The reaction product
was dissolved in hexane and chromatographed on a
column of silica gel impregnated with 20% of AgNO3. The
column was eluted with hexane; 100 mL fractions were
taken and inspected by GC. Fractions 6-12 eluted pure 2b
(722 mg). MP 74-75°C. GC retention time 3.55 min. It was
identical to an authentic sample obtained by methylation of
2a isolated from Espeletia tenore [4b] (IR, 1H NMR, MS).
Fractions 13-18 eluted a mixture of 1b and 2b (330 mg),
and fractions 19-27 pure 1b (275 mg).To a solution of 500
mg of 2b in 50 mL of DMSO 300 mg of KOH were added
and the mixture heated under reflux for 2 h. The solution
was cooled; 50 mL of H2O was added, and taken to pH 3.0
by addition of dil. HCl. The solution was then shaken with
100 mL of hexane. The hexane layer was shaken twice
with 20 mL of H2O, dried over Na2SO4, and the solvent
distilled under vacuum. The isokaurenic acid (455 mg) was
crystallized from hexane, MP 169-171°C. Identical to 2a
isolated from Espeletia tenore (IR, 1H NMR )[4b]. A
sample (5 mg) was methylated and examined by GC. Only
one peak was observed at 3.55 min (100%).
938 Natural Product Communications Vol. 6 (7) 2011 Rojas et al.

Isolation of ent-16-hydroxy-kauran-19-oic acid methyl
ester (3a): To a solution of kaurenic acid methyl ester (2.0
mmol) in CH2Cl2 five drops of trifluoroacetic acid were
added and the mixture was heated under reflux. After 72 h.
the reaction mixture was cooled and shaken with H2O. The
organic layer was dried over Na2SO4, filtered, and
evaporated to dryness. A 5 mg sample was dissolved in
Et2O and inspected by GC. The gas chromatogram showed
peaks at 3.54 (2b, 60%), 3.79 (1b, 27%), and 4.94 min (3a,
13%). Flash chromatography over a silica gel column
afforded 42 mg of 3a.
MP: 159-160°C.
IR (KBr, cm-1): 3412, 2982, 2953, 2868, 1728, 1424, 1157,
925.
1
H NMR (400 MHz, CDCl3): 0.77 (1H, dt, J = 4; 14 Hz,
H-1a), 0.80 (3H, s, H-20), 0.96 (1H, m, H-9), 1.01 (1H, m,
H-5), 0.97 (1H, m, H-3a), 1.15 (3H, s, H-18), 1.35 (3H, s,
H-17), 1.40 (1H, m, H-2a), 1.42 (1H, m, H-14a), 1.5 (2H,
m, H-11), 1.53 (2H, m, H-15), 1.57 (1H, m, H-7a), 1.58
(1H, m, H-14b), 1.75 (2H, m, H-6), 1.80 (1H, bs, H-13),
1.82 (1H, m, H-2b), 1.84 (1H, m, H-1b), 1.89 (1H, m,
H-7b), 3.63 (3H, s, OCH3).
13
C NMR (100 MHz, CDCl3): 174.3 (COO), 79.7 (C-16),
58.1 (C-15), 57.3 (C-5), 56.3 (C-9), 51.4 (OCH3), 49.2
(C-13), 45.6 (C-8), 44.1 (C-4), 42.4 (C-7), 41.0 (C-1), 39.8
(C-10), 38.4 (C-3), 37.9 (C-14), 29.0 (C-18), 27.1 (C-12),
24.8 (C-17), 22.4 (C-6), 19.4 (C-2), 18.6 (C-11), 15.7
(C-20).
MS (EI, 70 eV): m/z (%) = 334 (11), 316 (100), 302 (26),
276 (67), 257 (73), 217 (30), 180 (32), 121 (85).
Acknowledgments – This work was possible thanks to the
financial support of Mision Ciencia (Grant 2007000881).
The authors are indebted to Dr. Andres Leon for IR spectra
and to Dr. Ali Bahsas and Dr. Sonia Koteich for NMR
analyses.

References
[1] Mitscher LA, Rao GS, Veysoglu T, Drake S, Haas T. (1983) Isolation and identification of trachyloban-19-oic and kaur-16-en-19-
oic acids as antimicrobial agents from the prairie sunflower, Heliantus annuus . Journal of Natural Products, 46, 745-746.
[2] Alves TMA, Chaves PPG, Santos LMST, Nagem TJ, Murta SMF, Cevarolo IP, Romanha AJ, Zani CI. (1995) A diterpene from
Mikania obtusata, active on Trypanosoma cruzi. Planta Medica, 61, 85-86.
[3] Fatope MO, Audo OT, Takeda O, Zeng L, Shi G, Shimada H, McLaughlin JL. (1996) Bioactive ent-kaurene diterpenoids from
Annona senegalensis. Journal of Natural Products, 59, 301-303.
[4] (a) Ekong EU, Ogan AU (1968) Chemistry of the constituents of Xylopia aethiopica. The .structure of xylopic acid, a new
diterpene acid. Journal of the Chemical Society (C), 311-312; (b) Usubillaga A, Morales Mendez A. (1970) Derivados del Kaureno
en la Espeletia tenore. Revista Latinoamericana de Química, 1, 128-131.
[5] (a) Hubert AJ, Reimlinger H. (1969) The isomerization of olefins. Part I. Base catalysed isomerization of olefins. Synthesis, 97-112.
(b) Hubert AJ, Reimlinger H. (1969) The isomerization of olefins. Part II. Thermal and catalytic isomerization of olefins using
acids, metals, metal complexes, or boron compounds as catalysts. Synthesis, 405-430.
[6] Shabtai J, Gil-Av E. (1963) A convenient method for the preparation of 1-methylcyclobutene. Journal of Organic Chemistry, 28,
2893-2894.
[7] Barnes MF, MacMillan J. (1967) The Garryfoline-Cuauchichicine rearrangement: A study of the mechanism in the (-)-kaurenols.
Journal of the Chemical Society C, 361-366.
[8] Henrick CA, Jefferies PR. (1964) The chemistry of the Euphorbiaceae. VII. The diterpenes of Ricinocarpus stylosus Diels.
Australian Journal of Chemistry, 17, 915-933.
[9] (a) Amstutz ED, Myers MM. (1957) Nitrosomethylurea from acetamide. In Organic Syntheses. Blatt AH (Ed). John Wiley and
Sons, Inc. Collective Vol. II, 462-463. New York; (b) Arndt F (1957) Diazomethane. In Organic Syntheses. Blatt AH (Ed). John
Wiley and Sons, Inc. Collective Vol. II, 165-166. New York
 2011
NPC Natural Product Communications Vol. 6
No. 7
Aristolactams from roots of Ottonia anisum (Piperaceae) 939 - 942

André M. Marquesa, Leosvaldo S. M. Velozoa, Davyson de L. Moreirab, Elsie F. Guimarãesc and

Maria Auxiliadora C. Kaplana
a
Núcleo de Pesquisas de Produtos Naturais (NPPN), Centro de Ciências da Saúde, Bloco H,
Universidade Federal do Rio de Janeiro (UFRJ). CEP: 21941-590 - Rio de Janeiro, RJ, Brazil
b
Departamento de Produtos Naturais, Far-Manguinhos, FIOCRUZ. CEP: 21041-250 - Rio de Janeiro,
RJ, Brazil
c
Instituto de Pesquisa Jardim Botânico do Rio de Janeiro. CEP: 22.460-030 - Rio de Janeiro, RJ,
Brazil

dmoreira@far.fiocruz.br

Received: December 15th, 2010; Accepted: March 14th, 2011

The Piperaceae species are known worldwide for its medicinal properties and its chemical compounds. In Brazil, many species of this family
are distributed mainly in Amazon Region and in the Atlantic Forest. The genus Ottonia is known as source of amides, flavonoids,
arylpropanoids and terpenes with record biological activities. Six aristolactams, including, aristolactam BII, piperolactam C, goniothalactam,
stigmalactam, aristolactam AII and aristolactam BIII were isolated from roots of this species. GC-MS, 1H NMR and NOESY techniques were
used to characterize these compounds. This is the first report about the occurrence of aristolactams in the Ottonia anisum Sprengel.

Keywords: Piperaceae, Ottonia anisum, aristolactams, alkaloids.

The family Piperaceae is composed approximately by 2000
species with wide tropical and subtropical distribution and
great representation in Central and South Americas,
occurring in Mexico, Panama, Peru, Costa Rica, Argentina
and from North to Southern Brazil. The Piper species are
mostly tropical plants of worldwide occurrence and are
represented by ca. 700 species. In Brazil, 260 species are
distributed mainly in Amazon Region and in the Atlantic
Forest of the country [1a-c]. The phytochemical
investigation of Piper has led to the isolation of a large
number of bioactive compounds such as alkaloids, amides,
propenylphenols, piperolides, flavonoids, chromenes,
prenylated benzoic acid derivatives and lignoids.
Furthermore, literature records registered the occurrence of
interesting secondary metabolites in species of the genus
Peperomia (benzoic acid derivatives and seconeolignans),
Ottonia (amides, flavonoids and arylpropanoids) and
Pothomorphe (catechol derivatives) [2a-e]. The chemistry
of Piperaceae from Rio de Janeiro State, Brazil, has been
addressed with great success. Previous phytochemical
investigations of Piperaceae species from Brazilian
Atlantic Forest performed by our research group led to the
isolation of several compounds, such as kaplanin,
lhotzchromene and blandachromenes I and II [3a-c]. In
order to continue with the phytochemical studies of native
Piperaceae species from Southeast Brazil, Ottonia anisum
have been successfully studied [4]. The O. anisum root
extracts were chemically investigated to afford six
aristolactams. This is the first work regarding the presence
of aristolactams in Ottonia species. Aristolactams belong
to a large and important group of naturally occurring
alkaloids that possess the phenanthrene lactam skeleton
[5]. The phenanthrene chromophore group is found in the
Aristolochiaceae together with the aristolochic acids and
4,5-dioxoaporphines alkaloids. Aristolactams have been
reported from plants of the Annonaceae, Monimiaceae,
Menispermaceae, Piperaceae and Saururaceae families
[6a,b]. The phenanthrene lactam core is frequently found
in biologically active natural products [7a-d]. Among
them, aristolactams and aporphines constitute an
important alkaloid group due to their unique structural
features and potent biological activities, such as anti-
inflammatory, to treat arthritis, gout, rheumatism, antiPAF,
antimycobacterial, and neuro-protective [8a-e].
Major secondary compounds from non polar root extracts
of O. anisum were isolated by chromatographic techniques
and identified as 3,4-dimethoxy-aristolactam (aristolactam
BII, 1), 2,3,4-trimethoxy-aristolactam (piperolactam C, 2),
6-hydroxy-3,4-dimethoxy-aristolactam (stigmalactam, 3,
6-hydroxy-2,3,4-trimethoxy-aristolactam (goniothalactam,
4), 3-hydroxy-4-methoxy-aristolactam (aristolactam AII,
5), 3,4,6-trimetoxy-aristolactam (aristolactam BIII, 6)
(Figure 1). These compounds 1-6 were identified by
comparison of physical and spectroscopic data (MS and
NMR) with literature records [9a-c]. Compound (1) was
obtained as a yellow needle crystalline solid. The
molecular formula was confirmed by GC-MS measurement
940 Natural Product Communications Vol. 6 (7) 2011
R1
O
R2
2
1 NOESY spectrum of compound (3) showed the methoxyl
3
NH
R3 4 (1) R1=R4=H; R2=R3=OCH3;
(2) R1=R2=R3=OCH3; R4=H;
5 9 (3) R1=H; R2=R3=OCH3; R4=OH;
(4) R1=R2=R3= OCH3; R4=OH;
6
8
(5) R1=R4=H; R2=OH; R3=OCH3;
R4
7
(6) R1=H; R2=R3=R4=OCH3;
Figure 1: Aristolactams isolated from O. anisum. Arrows show the
NOESY correlations.
m/z [M+] 279, suggesting the Cl7Hl3NO3. The 1H NMR
spectrum of compound (1) showed signals related to six
aromatic protons, a broad D2O-exchangeable proton at 
10.67 and protons of two methoxy groups attached to
aromatic ring at  4.06 (3H, s) and 4.04 (3H, s). The
signal registered at  9.17 (1H, dd, J = 6.0, 3.0 Hz) was
assigned to H-5, and the signals at  7.52 (2H, m) and
7.94 (1H, dd, J = 6.0, 3.0 Hz) corresponding to H-6, H-7
and H-8 respectively (Table 1). A singlet was identified at
 7.86 (1H, s) corresponding to H-2. The NOESY
spectrum of compound (1) showed correlations between
H-2/CH3O-3, CH3O-3/CH3O-4, CH3O-4/H-5, as well as
between H-8/H-9 (Figure 1). Significant correlations
between H-5 ( 9.17) and H-6 ( 7.58), H-7 ( 7.58) and
H-8 ( 7.94) were also observed. Considering these data as
well as literature records, the structure of compound (1)
was identified as aristolactam BII. The NMR analysis of
the compounds (2-6) led us to conclude that these
constituents have the same aristolactam skeleton of
compound (1). Compound (2) was isolated as a yellow
powder. The GC-MS established the molecular formula
that was confirmed by m/z [M+] 309. The 1H NMR
spectrum of compound (2) showed signals related to five
aromatic protons, a broad D2O-exchangeable proton at 
10.96 and protons of three methoxy groups attached to
aromatic ring at  3.92 (3H, s), 4.17 (3H, s) and  4.42
(3H, s). The signals at  9.12 (1H, dd, J = 6.0, 3.0 Hz) was
assigned to H-5, and the signals at  7.52 (2H, m)
corresponding to H-6, H-7 besides  7.94 (1H, dd, J = 6.0,
3.0 Hz) assigned to H-8. The NOESY spectrum of (2)
confirms the spatial correlation of the protons between
CH3O-2/CH3O-3, CH3O-3/CH3O-4, CH3O-4/H-5, as well
as between H-8/H-9. NOESY correlations between
H-5, H-6, H-7 and H-8 were also observed (Figure 1).
These data confirmed compound (2) as piperolactam C.
Compound (3) was isolated as yellow powder. GC-MS
analysis for compound (3) showed a molecular ion at m/z
[M+] 295. The 1H NMR spectrum of compound (3)
showed five aromatic protons, a broad D2O-exchangeable
proton at  10.64 and two methoxy groups attached to
aromatic ring at  4.02 (3H, s) and 4.04 (3H, s). Signal at
 8.57 (1H, d, J = 3 Hz) was assigned to H-5, and the
signals at  7.08 (1H, dd, J = 6.0, 3.0 Hz) and  7.76 (1H,
d, J = 6.0 Hz) were assigned to H-7 and H-8, respectively.
Moreira et al.
A singlet was at  7.86 (1H, s) corresponds to H-2. The
groups attached to C-3 and C-4 due to the spatial
proximity of H-2/CH3O-3, CH3O-3/CH3O-4, CH3O-4/H-5.
NOESY correlations between H-7 and H-8 were also
observed, as well as between H-8/H-9 (Figure 1)
confirming (3) as goniothalactam. Compound (4) was
isolated as brownish yellow needles. The mass spectrum
m/z [M+] 325 established the molecular formula
C18H15NO5. The 1H NMR spectrum of compound (4)
showed signals related to four aromatic protons, a broad
D2O-exchangeable proton at  10.79 and three methoxy
groups attached to aromatic ring at  3.93 (3H, s), 4.04
(3H, s) and  4.38 (3H, s). The signal at  8.58 (1H, d, J =
3 Hz) assigned to H-5, and the signals at  7.08 (1H, dd,
J = 6.0, 3.0 Hz) and 7.79 (1H, d, J = 6.0, Hz)
corresponding to H-7 and H-8 respectively. The NOESY
spectrum was quite similar to the compound (2), showing
the spatial correlation between the methoxyl groups at C-2,
C-3 and C-4. These data are in accordance with
stigmalactam, 4. The compound (5) was isolated as a pale
yellow powder. The same aristolactam skeleton was
observed by the MS and NMR analysis. The mass
spectrum m/z [M+] 265 established the molecular formula
C16H11NO3. The 1H NMR spectrum of compound (5)
showed signals related to six aromatic protons, a broad
D2O-exchangeable proton at  10.69 and a single methoxy
group attached to aromatic ring at  4.03 (3H, s). The
signal at  9.25 (1H, dd, J = 6.0, 3.0 Hz) assigned to H-5,
the signals at 7.96 (1H, dd, J = 6.0, 3.0 Hz)
corresponding to H-8 and the signals at  7.54 (2H, m)
corresponding to H-6 and H-7. Spatial correlations were
found between the hydroxyl group at C-3 and the H-2 and
CH3O-4. NOESY correlations between H-5, H-6, H-7 and
H-8 were also confirmed, as well as between H-8/H-9.
Analysis of GC-MS, 1H NMR and NOESY spectral data
showed chemical shifts very similar to the analogous
previous analized compounds and allowed to confirmed
(5) as aristolactam AII. Compound (6) was isolated as a
yellow green powder with m/z [M+] 265 obtained from
GC-MS. The 1H NMR spectrum of compound (6) showed
signals related to five aromatic protons, a broad D2O-
exchangeable proton at  7.67 and three methoxy groups
attached to aromatic ring at  3.96 (3H, s), 4.18 (3H, s)
and 4.45 (3H, s). The signals at  8.65 (1H, d, J = 3 Hz)
was assigned to H-5, and the signals at  7.15 (1H, dd,
J = 6.0, 3.0 Hz) and 7.77 (1H, d, J = 6.0 Hz)
corresponding to H-7and H-8, respectively. A singlet
observed at  7.80 was assigned to H-2. NOESY
correlations between the methoxyl group at  3.96 and H-5
and H-7 was confirmed. This is in agreement with the
aristolactam skeleton and suggested that the signal at
3.96 refers to the methoxyl group located at position C-6,
confirming compound (6) as aristolactam BIII.
Nowadays, many communities in the North of Brazil still
remain using O. anisum popularly in the treat toothache
Aristolactams from Ottonia anisum
Table 1: 1H NMR spectroscopic data of compounds 1-5 in DMSO-d6 and compound 6 in CDCl3, H J (Hz).
Position 1 2
5 9.17 (1H) dd (6.0, 3.0) 9.12 (1H) dd (6.0, 3.0)
7 7.58 (1H) m 7.52 (1H) m
8 7.94 (1H) dd, (6.0, 3.0) 7.94 (1H) dd, (6.0, 3.0)
9 7.13 (1H) s 7.12 (1H) s
R1 7.86 (1H) s 3.92 (3H) s
R2 4.06 (3H) s 4.42 (3H) s
R3 4.04 (3H) s 4.17 (3H) s
R4 7.58 (1H) m 7.52 (1H) m
N-H 10.67 (1H) s 10.96 (1H) s
and as local anesthetic, even if a survey of the literature
data shows that some of natural compounds have
carcinogenic and nephrotoxic properties. Aristolactams are
known to be nephrotoxic, carcinogenic and mutagenic
[8b,d] [10a-f]. However, naturally occurring aristolactams
such as cepharanone B (aristolactam BII), aristolactam
BIII, piperolactam A and goniothalactam have shown
potent inhibitory activity against human cancer cells. For
example, aristolactam BII inhibits T and B lymphocyte
proliferation as well as shows cytotoxic activity, while
aristolactam FI (piperolactam A,) displays inhibitory
effects on NO generation by RAW264 [5]. Several
synthetic aristolactam derivatives exhibited potent
antitumor activities against a broad array of cancer cell
lines with submicromolar range and some are equally
potent toward multidrug resistant cell lines compared to
the commercially available drug [8a,d]. It is noteworthy
that the methoxy-substituted compounds are more potent
than the hydroxyl-substituted ones. The tetra-methoxy
substituted phenanthrene lactam has highly potent
cytotoxic activity [11]. Although the cytotoxicity of
aristolactams is well known, structure-activity
relationships have not been explored mainly as a
consequence of the synthetic difficulties associated with
preparing a diverse array of aristolactam analogues.
Therefore, it is important the continuous knowledge about
the native Piperaceae species of Brazil since many of them
have been used in folk medicine to treat many conditions.
To the best of our knowledge this is the first report of
aristolactams in the genus Ottonia.
Experimental
Plant material: Branches and roots of Ottonia anisum
Sprengel were collected in Duque de Caxias, Rio de
Janeiro, RJ, Brazil in april of 2007. A Voucher specimen
was identified by Dr. Elsie Franklin Guimarães and is
deposited at the RB Herbarium of Jardim Botânico do Rio
de Janeiro, under the number RB393494.
Chromatographic materials: Sephadex LH-20
(Pharmacia) was used for column chromatographic
separation. Silica gel PF254 (Merck) was used for TLC
preparative/purification. All compounds were visualized
on analytical TLC under UV light (254 and 365 nm) and
by spraying with ceric sulfate solution followed by
heating.
Natural Product Communications Vol. 6 (7) 2011 941
3 4 5 6
8.57 (1H) d (3.0) 8.58 (1H) d (3.0) 9.25 (1H) dd (6.0, 3.0) 8.65 (1H) d (3.0)
7.08 (1H) dd (6.0, 3.0) 7.08 (1H) dd (6.0, 3.0) 7.54 (1H) m 7.15 (1H) dd (6.0, 3.0)
7.76 (1H) d (6.0) 7.79 (1H) d (6.0) 7.96 (1H) dd (6.0, 3.0) 7.77 (1H) d (6.0)
7.07 (1H) s 7.15 (1H) s 7.09 (1H) s 7.07 (1H) s
7.86 (1H) s 3.93 (3H) s 7.82 (1H) s 7.80 (1H) s
4.04 (3H) s 4.04 (3H) s - 4.45 (3H) s
4.02 (3H) s 4.38 (3H) s 4.03 (3H) s 4.18 (3H) s
- - 7.54 (1H) m 3.96 (3H) s
10.64 (1H) s 10.79 (1H) s 10.69 (1H) s 7.67( 1H) s
Extraction and Isolation: Air-dried and powdered roots
(650 g) were extracted with MeOH. The solvent was
evaporated to dryness under reduced pressure to give the
MeOH extract (15g) which was partitioned successively
between n-hexane, followed by dichloromethane, ethyl
acetate and n-butanol. Hexane and dichloromethane
fractions were combined providing about 2g that were
subjected to a column chromatography on Sephadex LH
20, eluted with a MeOH/CHCl3 (7:3) system furnishing 30
fractions. The last 10 fractions were combined furnishing
200 mg of a very strong UV fluorescence fraction. This
sample was subjected to preparative normal phase TLC
eluted with hexane/ethyl acetate (3:2) yielding six fractions
with different UV fluorescence color. Aristolactam 1 (17.0
mg), 2 (7.0 mg), 3 (4.0 mg), 4 (2.0 mg), 5 (4.5 mg) and 6
(3.0 mg) were isolated and analyzed by GC-FID, GC-MS
and 1H and NOESY NMR techniques.
GC-FID analysis: Qualitative analyses were carried out on
a GC 2010 Shimadzu apparatus with a DB-1MS fused
silica capillary column (30 m x 0.25 mm x 0.25 m film
thickness). The operating temperatures used were: injector
260oC, detector 290oC and column oven 60°C up to 290oC
(10oC min-1). Hydrogen at 1.0 mL min-1 was used as
carrier gas.
GC-MS analysis: Qualitative analysis was carried out on a
GC-MS QP 5000 Shimadzu machine with a ZB-5MS
fused silica capillary column (30 m x 0.25 mm x 0.25 m
film thickness) under the same experimental conditions
reported for GC-FID analysis. The aristolactams mass
spectra were matching with WILEY 275 and National
Institute of Standards and Technology (NIST 3.0) libraries
provided with the computer controlling the GC–MS
system. The results were also confirmed by comparison of
data of the isolated compounds with mass and
fragmentation reported in the literature [9a-c].
Nuclear Magnetic Resonance Spectroscopy: The pure six
constituents obtained were analyzed by 1H NMR and
NOESY recorded on a Varian VNMRS 500 spectrometer.
Chemical shifts were determined in DMSO-d6 and CDCl3,
using TMS as internal standard. The signals of NMR
analysis were compared with literature data [9a-c].
Acknowledgments – This work was supported by CNPq.
942 Natural Product Communications Vol. 6 (7) 2011 Moreira et al.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Anti-angiogenic Activity Evaluation of Secondary 943 - 946
Metabolites from Calycolpus moritzianus Leaves
Laura Leporea, Maria J. Gualtieria, Nicola Malafrontea, Roberta Cotugnoa, Fabrizio Dal Piaza,
Letizia Ambrosioa, Sandro De Falcob and Nunziatina De Tommasia*
a
Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo,
84084 Fisciano, Salerno, Italy
b
Angiogenesis Lab, Institute of Genetics and Biophysics ‘Adriano Buzzati-Traverso’, CNR,
Via Pietro Castellino 111, 80131 Napoli, Italy

detommasi@unisa.it

Received: November 13th, 2010; Accepted: March 25th, 2011

Angiogenesis is a crucial step in many pathological conditions like cancer, inflammation and metastasis formation; on these basis the search
for antiangiogenic agents has widened. In order to identify new compounds able to interfere in the Vascular Endothelial Growth Factor
Receptor-1 (VEGFR-1, also known as Flt-1) recognition by VEGFs family members, we screened Calycolpus moritzianus (O. Berg) Burret
leaves extracts by a competitive ELISA-based assay. MeOH and CHCl3 extracts and several their fractions demonstrated to be able to prevent
VEGF or PlGF interaction with Flt-1, with an inhibition about 50% at concentration of 100 g/mL. Phytochemical and pharmacological
investigation of the active fractions led to the isolation of flavonoids, and terpenes.

Keywords: Calycolpus moritzianus, Angiogenesis, VEGFR1/Flt-1, VEGF and PlGF, bioassay-oriented study.

In the last decade the inhibition of angiogenesis and

vascular targeting has been the focus of new treatment
strategies against the cancer. Among the long list of
growth factors involved in the angiogenic process, VEGF-
A has been considered for years the most important
mediator of tumor angiogenesis [1]. Consequently, several
strategies have been developed to inhibit the release of this
growth factor, or to interfere in its interaction with
receptors, VEGF receptor 1 (Flt-1) and VEGF receptor 2
(Flk-1 in mouse, KDR in human) [2]. Recent data support
the concept that tumor infiltration by bone marrow-derived
myeloid cells confers resistance to current antiangiogenic
drugs targeting primary VEGF-A and its receptors
(VEGF(R)s) [3]. For this reason, novel targets out of
VEGF-A have been studied to diversify antiangiogenic
treatments and to overcome resistance [4]. Genetic and
pharmacological studies have identified Flt-1 and Placental
Growth Factor (PlGF) as possible therapeutic targets for Figure 1: Inhibitory effect of C. moritzianus leaves extracts were assayed on
PlGF/Flt-1 (A) and VEGF/Flt-1 interaction (B). The extracts were used at 500-
anticancer therapy [5]. Furthermore, has been proven that a 100-20 µg/mL. As control a specific inhibiting peptide was used (CP). The
combination of lower amount of VEGF(R)s inhibitors and white bar refers to ELISA experiment carried out without inhibitors. Each
compounds able to block PlGF showed equal antitumor experiment was performed three times and average values ± SD were reported.
efficacy compared to the standard dose of VEGF(R)s [5].
Accordingly to these data, the research of new natural
These findings suggested that molecules able to inhibit the compounds which may inhibit both PlGF and VEGF-A
activity of both PlGF and VEGF-A driven angiogenesis activity, has been the target of the present study. We
may be an opportunity for patients with cancer who may carried out a screening of Calycolpus moritzianus
suffer excessive or prohibitive adverse effects from (O. Berg) Burret leaves extracts by a competitive ELISA-
VEGF(R)s inhibitors. based assay [6]. We aimed to identify natural molecules as
inhibitors of PlGF and VEGF-A recognition by Flt-1.
944 Natural Product Communications Vol. 6 (7) 2011
Figure 2: Inhibitory properties of plant fractions from MeOH extract were assayed on PlGF/Flt-1 interaction (A) and VEGF/Flt-1 interaction (B). The fractions were
tested at 500-100-20 µg/mL. As control a specific inhibiting peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each
experiment was performed three times and average values ± SD were reported.
Figure 3: Inhibitory activity of compounds isolated from active methanolic fractions assayed at concentration of 100 µg/ml on PlGF/Flt-1 (A) and VEGF/Flt-1 (B). As
control a specific inhibiting peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each experiment was performed three times
and average values ± SD were reported.
Several extracts from C. moritzianus leaves have been
tested at the doses of 0.5, 0.1, and 0.02 mg/mL.
Methanol and chloroform residues exhibited a good
activity in the inhibition on both PlGF/Flt-1 and VEGF-
A/Flt-1 interaction, with a binding reduction higher
than 60% at 20 µg/mL (Figure 1).Therefore these extracts
were submitted to a bioassay-oriented fractionation. C.
moritzianus MeOH extract was fractioned by sephadex
column chromatography giving 9 fractions (A-I), while
CHCl3 extract was separated using silica gel column
chromatography giving 7 fractions (AA-GG). The effect of
the obtained fractions was tested on both PlGF/Flt-1 and
VEGF-A/Flt-1, leading to the results reported in Figure
2-4.
C. moritzianus MeOH extract fractions were assayed in
dose-dependent experiments at concentration ranging
between 500 and 20 µg/mL on PlGF/Flt-1; the active frs.
D-F and H were then assayed on VEGF-A/Flt-1 at
concentration of 100 and 20 µg/mL. Among the MeOH
fractions, Fr. H revealed the highest dose-dependent
activity for PlGF/Flt-1 inhibition, provoking a reduction of
its Flt-1 binding to 20% at 500 µg/mL and to 60% at 100
µg/mL, while at dose 100 µg/mL a 25% reduction of
VEGF-A/Flt-1 interaction was observed.
Also D, E, F, I fractions exhibited a moderate inhibition
for PlGF/Flt-1 complex, even if only Fr. D revealed to be
able to inhibit also hVEGF-A / Flt-1 complex, leading to a
binding reduction of 60% at 100 µg/mL (Figure 2). The
active fractions were studied in order to identify the
compounds responsible for this inhibitory activity.
Chromatographic and spectroscopic analyses of active
Frs. D-F indicated the presence of flavonoidic derivatives
Lepore et al.
as main components, which were identified as quercetin
3-O-β-D-glucopyranoside 1 [7], kampferol-3-O-β-D-
glucopyranoside 2 [7], quercetin-7-O-β-D-glucopyranoside
3 [8], kaempferol-3-O-β-D-rhamnopyranoside 4 [9],
quercetin-3-O-β-D-rhamopyranoside 5 [9], and quercetin 6
[10].
The main compound identified in Fr. H was quercetin
(85% w/w abundance). All isolated compounds were
identified by means of 1D- and 2D-NMR spectroscopy,
ESI-MS analysis, and by comparison of their data with
those reported in the literature.
Among the pure compounds assayed, only quercetin
showed a moderate activity (60% inhibition of PlGF/Flt-1
interaction at 100 µg/mL), compounds 1, 3, 5 its
glycosides were inactive (Figure 3). These data suggest
that the glycosylation at C-3 and C-7 of quercetin core is
fatal for the activity. To better investigate a structure-
activity relationship we tested also kaempferol aglycon of
compounds 2, 4. Kaempferol was inactive in our test. The
structures of quercetin and kaempferol are very similar
except for the substituent at C-3’; the different activity
observed for these compounds indicated that the presence
of OH group at C-3’ influence the resultant activity.
Fractions obtained from C. moritzianus CHCl3 extract
were assayed on PlGF/Flt-1 and VEGF-A/Flt-1 interaction
by a competitive ELISA screening at concentration of 100
and 20 µg/mL.
Data in Figure 4 showed that the most active fraction was
Fr. AA which revealed a moderate activity for both the
growth factors causing a reduction of their Flt-1 binding to
40% for PlGF and 60% for VEGF at dose 100 µg/mL.
Antiangiogenic metabolites from Calycolpus moritzianus
Figure 4: Inhibitory properties of chloroformic extract fractions were assayed on PlGF/Flt-1 interaction (A) and VEGF/Flt-1 interaction (B). The fractions were used at
100-20 µg/mL. As control a specific inhibiting peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each experiment was
performed three times and average values ± SD were reported.
Figure 5: Inhibitory activity of compounds 7-12 assayed at concentration of 100-20 µg/mL on PlGF/Flt-1 (A) and VEGF/Flt-1 (B). As control a specific inhibiting
peptide was used (CP). The white bar refers to ELISA experiment carried out without inhibitors. Each experiment was performed three times and average values ± SD
were reported.
Nonetheless BB showed a moderate inhibition for both
PlGF / Flt-1 and VEGF / Flt-1 interaction with a binding
reduction of about 40% at dose 100 µg/mL.
Chromatographic separation of AA and BB fractions
allowed to obtain the pure components: rosifoliol 7 [11],
platanic acid 8 [12], oleanolic acid 9 [13], (-)-4,10-di-epi-
5β,11-dihydroxyeudesmane 10 [14], 4,5-dioxoseco-γ-
eudesmol 11 [15], and ursolic acid 12 [13], all identified
by means of 1D- and 2D-NMR spectroscopy, ESI-MS
analysis, and a comparison of their data with those
reported in the literature.
The isolated pure compounds were tested in dose
dependence manner on both PlGF/Flt-1 and hVEGF/Flt-1
systems (Figure 5). Only compound 8 was moderately able
to inhibit PlGF/Flt-1 recognition; anyway the inhibition
activity showed by this compound cannot explain by itself
the activity of the original fraction .
On the basis of our results, we could hypothesize that the
inhibition activity of PlGF and VEGF interaction with Flt-
1 receptor by the C. moritzianus CHCl3 extracts and
fractions may be due to the presence of a combination of
compounds acting synergistically or as vehicles enhancing
the biological activity. However, we cannot rule out that
the activity of the extracts and fractions could be due to a
very minor compound not isolated.
Experimental
General experimental procedures: The instrumentation
used in this work is described in our previous paper [13].
Plant material: The leaves of C. moritzianus were
collected in Venezuela in 2008 and identified by Ing. Juan
Carmona of Herbarium (MERF), Facultad de Farmacia y
Bioanalisis - Universidad de Los Andes, Merida; where a
voucher specimen n.761 is deposited.
Natural Product Communications Vol. 6 (7) 2011 945
Extraction and isolation: The air-dried powdered leaves
of C. moritzianus (890 g) were defatted with n-hexane and
extracted successively by exhaustive maceration (3 x 1 L,
for 48 h) with CHCl3, CHCl3-MeOH (9:1), and MeOH.
The MeOH extract (5 g) was chromatographed over a
sephadex LH-20 column (100 x 5 cm) with MeOH as the
eluent. A total of 110 fractions were collected (15 mL
each) and combined according to TLC analysis [silica 60
F254 gel-coated glass sheets with n-BuOH-AcOH-H2O
(60:15:25) and CHCl3-MeOH-H2O (40:9:1)] to give nine
pooled fractions (A-I). Fraction D (106 mg) was purified
by RP-HPLC with a C18 μ-Bondapak column (30 cm x 7.8
mm, flow rate 2 mL/min) using MeOH-H2O (35:65) to
obtain compound 1 (4.0 mg, tR = 20 min), and 2 (15.0 mg,
tR = 26 min).
Fraction E (95 mg) was purified by RP-HPLC with a
C18 μ-Bondapak column (30 cm x 7.8 mm, flow rate 2
mL/min) using MeOH-H2O (35:65) to obtain compound 2
(5.0 mg, tR = 26 min). Fractions F (90 mg) was separately
purified by RP-HPLC using MeOH-H2O (2:3) to give
compounds 3 (16 mg, tR = 10 min), 4 (6 mg, tR = 16 min),
and 5 (2 mg, tR = 20 min). Fraction H (20 mg) was
identified as quercetin.
The CHCl3 extract (5.0 g) was submitted to silica gel flash
column chromatography eluting with CHCl3 followed by
increasing concentrations of MeOH (between 1% and
70%). The following volumes of solvents were used: 4.2 L
of CHCl3, 1 L of CHCl3-MeOH (99:1), 4.3 L of CHCl3-
MeOH (49:1), 1 L of CHCl3-MeOH (95:5), 0.5 L of
CHCl3-MeOH (9:1), 0.5 L of CHCl3-MeOH (1:1), 0.5 L of
CHCl3-MeOH (3:7), and 0.3 L of MeOH. Fractions of 30
mL were collected and analyzed by TLC on silica 60 F254
gel-coated glass sheets eluting with CHCl3 or mixtures
CHCl3-MeOH, 99:1, 49:1, 95:5, 9:1, 4:1, and grouped into
seven fractions (AA-GG). Fraction AA (95 mg) was
subjected to RP-HPLC on a C18 μ-Bondapak column
946 Natural Product Communications Vol. 6 (7) 2011 Lepore et al.

(30 cm x 7.8 mm, flow rate 2.0 mL/min) with MeOH-H2O
(73:27) to yield compounds 7 (4 mg, tR = 10 min), 8 (10
mg, tR = 16 min) and 9 (2 mg, tR = 34 min). Fractions BB
(70 mg) and was purified by RP-HPLC with MeOH-H2O
(37:13) to give compounds 10 (3.5 mg, tR = 16 min), 11 (8
mg, tR = 4 min), 12 (1.5 mg, tR = 26 min).
ELISA-based assays: The ELISA based assay for plant
extract, fractions and pure compounds screening was
performed as described elsewhere [6].
Plant extracts, fractions and compounds 1-13 dissolved in
DMSO (Sigma) were properly diluted and added to the
wells pre-mixed with ligand. For dose-dependent
experiments, concentration ranging between 20 and 500
µg/mL were used.
Acknowledgements - Authors are grateful to Ing. Forestal
Juan Carmona of Herbarium (MERF), Facultad de
Farmacia y Bioanalisis - Universidad de Los Andes,
Merida for the help in collecting the plant material.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Chemical and Biological Activity of Leaf Extracts of 947 - 950

Chromolaena leivensis
Ruben D. Torrenegra G. and Oscar E. Rodríguez A.

Facultad de Ciencias y Tecnología, Universidad de Ciencias Aplicadadas y Ambientales U.D.C.A,

Bogota, Colombia

ruben.torrenegra@hotmail.com; rtorrenegra@udca.edu.co

Received: December 12th, 2010; Accepted: March 26th, 2011

The flavonoids 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone were isolated

from the leaves of C. leivensis. Preliminary observations in K562 cells (human erythroleukemia) using the trypan blue test, showed a 90%
viability at a concentration of 100 g/mL; however, further testing of the flavonoids at concentrations of 25, 50 and 100 g/mL showed
toxicity affecting the morphology of human erythroleukemia cells (K562) and human melanoma cells (A375). Induction of apoptosis was
produced by 3,5-dihydroxy-7-methoxyflavone at 72 hours after treatment with arrest in the G2 / M phase of the cell cycle. The A375 cells
treated with 50 µg/mL of 3,5-dihydroxy-7-methoxy-flavanone for 24, 48 and 72 hours, display effects on the behavior of the cell cycle. The
flavonoid 3,5-dihydroxy-7-methoxyflavone has activity on the mitochondrial membrane at concentrations of 25, 50 and 100 µg/mL, at time
intervals of 8 to 12 hours. The flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone at a concentration of
25 g/mL increased the expression of costimulatory molecules corresponding to the phenotype presented by mature dendritic cells with
differentiation markers CD40, CD83, CD86 and HLA-DR. The two flavonoids at concentrations between 0.39 and 100 g/mL slightly
increased the proliferation of peripheral blood mononuclear cells in the presence and in the absence of phytohemagglutinin. These flavonoids
at concentrations of 50 and 100 g/mL slightly increased the proliferation of fibroblasts.

Keywords: Chromolaena leivensis, flavonoids, cytotoxicity, apoptosis.

Chromolaena species are invasive and cosmopolitan with
morphological diversity given by adaptations to different
environments and are considered weeds. This genus is
constituted by 202 species of which only 13 have
undergone chemical studies, and only a few studies have
included biological activity. There are no known reports on
the species Chromolaena leivensis, C. perglabra, C.
tacotana, C. subscandens, C. opadoclinia, C. odorata, C.
arnottiana, C. morii, C. Collina, C. connivens, C.
glaberrima, C. pseudoinsignis and C. chasleae.
Compounds such as sesquiterpenes, diterpenes, triterpenes,
flavonoids, cyclic fatty acids, sesquiterpene lactones,
germacranolides, have been identified from Chromolaenas.
A sesquiterpene lactone was identified from the
dichloromethane extract of Chromolaena opadoclina [1].
5,3-dihydroxy-6, 7,4’-trimethoxyflavone, 5-hydroxy-6,7,
3’,4’-tetramethoxyflavone, 5-hydroxy,6,7,3',4’,5’-penta-
methoxyflavone and a common derived 3,4-dihydroxy-
acetophenone have been identified in Chromolaena
arnottiana [2]. The heliangolide 4’-dihydrochromolaenid
and a sesquiterpene lactone were found in Chromolaena
glaberrima [3]. The acid 7α-acetoxy-trans-communic was
identified In Chromolaena collina and in Chromolaena
morii, germacrane D, squalene, flavonols and a fatty acid
type prostaglandin were found [5b].
Many studies on biological activity have been conducted
in Chromolaena odorata and a few in C. hirsuta, C.
moritziana C. perglabra, C. bullata and C tacotana.
Among the tested biological activities the following are
highlighted: pesticide, insect repellent, antiprotozoal,
insecticidal, trypanocidal, antibacterial, antifungal,
cytotoxic, antioxidant, mutagenic, proliferative agent of
human keratinocytes and fibroblasts. Taleb et al. [6]
evaluated the antiprotozoal effect of total extracts and
purified flavonoids from Chromolaena hirsuta and
determined antiprotozoal activity against trypomastigotes
of Trypanosoma cruzi and amastigotes of Leishmania
amazonensis; the crude extracts significantly reduced
parasite viability and the flavonoids showed an
antiproliferative effect on these. Phan found that phenolic
compounds of Chromolaena odorata, p-hydroxybenzoic
acid and p-coumaric acid, flavones, flavanones and
chalcones protect skin cells from oxidative damage and
repair skin conditions [7]. Bouda observed the effect of
essential oils from leaves of Chromolaena odorata on the
mortality of Sitophilus Curculionidae (Coleoptera) with a
LD50 of 6.78%[8]. Thang evaluated the antioxidant effect
of extracts of Chromolaena odorata on human dermal
fibroblasts by measuring the protectant effect against
damage from hydrogen peroxide and hypoxanthine-
xanthine oxidase [9]. Phan showed that Eupolin extract
948 Natural Product Communications Vol. 6 (7) 2011 Torrenegra G. & Rodríguez A.

Table 1: Effect of the flavonoids on cell viability at 24h of treatment. Table 5: Percentage of viability of normal cells at several concentrations
of each flavonoid and control + Vincristine and DMSO.
CELL VIABILITY
Trypan blue MONONUCLEAR CELL 24H
FLAVONOID -g % TO 24 h 100 50 12,5 6,25
3,5-dihydroxy-7-methoxyflavanone-100 94.4 g/mL g/mL g/mL g/mL
3,5-dihydroxy-7-methoxyflavanone-50 100 Ethanol 23% 23% 22% 24%
3,5-dihydroxy-7-methoxyflavone-100 89 3,5-dihydroxy-7-methoxy
3,5-dihydroxy-7-methoxyflavone-50 100 flavanone 24% 28% 23% 23%
3,5-dihydroxy-7-methoxy
Table 2: Concentrations that affect cell morphology. flavone 13% 20% 21% 22%
Vincristine 26% 22% 24% 23%
EFFECT ON MORPHOLOGY DMSO 25% 23% 24% 25%
Direct microscopic observation
CELL-K562 CELL-K562 CELL-A375
[g/24 H] [g/48 H] [g/24H] Table 6: Percentage of viability of normal cells at several concentrations
3,5-dihydroxy-7- of each flavonoid and control + Vincristine and DMSO.
methoxy flavanone 25 25 100
3,5-dihydroxy-7- MONONUCLEAR CELL 24H
methoxy flavone 100 100 50 100 50 12,5 6,25
g/mL g/mL g/mL g/mL
Ethanol 23% 23% 22% 24%
Table 3: Percentage of cells in each phase of the cell cycle.
3,5-dihydroxy-7-methoxy
EFFECT ON CELL CYCLE, A375 CELLS flavanone 24% 28% 23% 23%
FLAVONOID - mg G0/G1 - S -% G2/M - SUB G1- 3,5-dihydroxy-7-methoxy
% % % flavone 13% 20% 21% 22%
3,5-dihydroxy-7-methoxy Vincristine 26% 22% 24% 23%
flavone-50 75.9 19.39 4.62 5.99 DMSO 25% 23% 24% 25%
CONTROL(-) DMSO 54.21 26.3 19.49 0.62
CONTROL (+) G2/M
VINC 8.63 16.27 75.10 2.29 Table 7: Percentage of viability of fibroblasts at several concentrations of
the flavonoids and control + Vincristine after 24h of treatment
Table 4: Percentage of depolarized cells at several concentrations of each FIBROBLASTS 24H
flavonoid after 8h and 12h of treatment. 100 25 12,5 6,25
g/mL g/mL g/mL g/mL
DEPOLARIZATION OF MEMBRANE MITOCHONDRIAL, JC-1 vincristine 26% 28% 26% 30%
FLAVONOID-g-time % DEPOLARIZED 3,5-dihydroxy-7-methoxy
3,5-dihydroxy-7-methoxyflavanone flavanone 40% 53% 45% 55%
25-8H 9.5 3,5-dihydroxy-7-methoxy
50-8H 10.1 flavone 41% 39% 40% 42%
100-8H 19.8
25-12H 9.5
50-12H 13.8 The species Chromolaena perglabra, C. tacotana, C.
100-12H 21.7 bullata, C. subscandens, C. leivensis and C. scabra are
3,5-dihydroxy-7-methoxy flavone
25-8H 18.4
found in the Cundiboyacense region of Colombia, and C.
50-8H 77.3 barranquillensis is found in the Atlantic coast., These
100-8H 95.4 species have not been studied at depth with respect to their
25-12H 32.1 biological activities, specifically as antiparasitic agents
50-12H 72.8
100-12H 97.4
against Chagas and Leishmania and their cytotoxic and
CONTROL(-) DMSO antitumor potential.
8H 19.6
12H 11.8 In this investigation we studied the production of
EtOH
8H 13.0
secondary metabolites in the leaves of C. leivensis and
12H 13.0 isolated the flavonoids 3,5-dihydroxy-7-methoxy-
flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-
adhesion complex and fibronectin in human keratinocytes trihydroxy-6-methoxyflavone, the first two compounds
[10]. They found increased expression of integrin b1 and were tested for their activity on cancer cell lines. Using the
b4 induced by the extract at concentrations of 0.1 and 1 trypan blue test, it was observed in K562 cells
(erythroleukemia) viability percentages of 90% using a
g/mL, but the expression was reduced at higher doses of
concentration of 100 g/mL, which indicates that
Eupolin (10 to 150 g/mL). Other researchers [11] have
flavonoids at these concentrations are not cytotoxic. The
studied the proliferation of fibroblasts and endothelial cells
flavonoids tested at concentrations of 25, 50 and 100
treated with hydroethanolic leaves extracts of
Chromolaena odorata (Eupolin). The greatest growth of g/mL showed toxicity affecting the morphology of
fibroblasts and endothelial cells was found at human erythroleukemia cells (K562) and human
melanoma cells (A375). Induction of apoptosis was
concentrations of 10 g/mL and 100 g/mL of Eupolin
produced by the flavonoid 3,5-dihydroxy-7-methoxy-
extract, but it was found to be toxic at concentrations
flavone at 72 hours of treatment with arrest in G2/ M. In
exceeding 250 g/mL.
A375 cells treated with 50 g/mL of the flavonoids for 24,
Secondary metabolites in the leaves of C. leivensis
48 and 72 hours, it was observed that the flavonoid 3,5-
dihydroxy-7-methoxy-flavanone influences the behavior of
the cell cycle. The flavonoid 3,5-dihydroxy-7-methoxy-
flavone have activity on mitochondrial membrane at
concentrations of 25, 50 and 100 g/mL, at time intervals
of 8 to 12 hours. It was also observed that the flavonoids
3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-
methoxyflavone at a concentration of 25 g/mL increased
the expression of costimulatory molecules corresponding
to the phenotype presented by mature dendritic cells with
differentiation markers CD40, CD83, CD86 and HLA-
DR. The two flavonoids at concentrations between 0.39
and 100 g/mL slightly increased the proliferation of
peripheral blood mononuclear cells in the presence and in
the absence of phytohemagglutinin. It was also determined
that fibroblast proliferation increased slightly at
concentrations of 50 and 100 g/mL of these flavonoids.
Experimental
Materials were collected in the outskirts of Bogota,
Colombia; a control sample was sent to the National
Herbarium of Colombia for identification and was
determined to be Chromolaena leivensis (Hieron). King &
H. Rob. with the number COL-535 219 Colombian
National Herbarium. The extraction was carried out in
Soxhlet with 95% ethanol (4L) with a yield of 31.04%.
200 g of extract was mixed with silica gel (1:2) and
extracted solid - liquid with petroleum ether (3L), toluene
(2L), dichloromethane (2L), ethyl acetate (2L) and
methanol (3L), successively, with yields of 2.66% for
petrol, 29.09% for Toluene, 15.68% for CH2Cl2, 14.66%
for AcOEt and 30.08% for MeOH. Five grams of the
toluene fraction were subjected to column chromatography
with silica gel (100 g silica gel Merck Kieselgel), eluting
with petroleum ether, toluene and methanol in various
proportions. By fractional crystallization three flavonoids
were isolated and identified as 3,5-dihydroxy-7-
methoxyflavanone, 3,5-dihydroxy-7-methoxyflavone [13]
and 3,5,7-trihydroxy-6-methoxyflavone.[14].
Cell viability: Cell viability was measured using trypan
blue, a negatively charged chromophore that interacts with
cell membranes whose integrity has been altered. Living
cells exclude the dye while dead cells allow entry and are
stained. The effect of flavonoids 3,5-dihydroxy-7-
methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone
on cell populations with concentrations less than or equal
to 100 g/mL were analysed at 24 and 48 hours. The data
are listed in Table 1.
Evaluation of cell cycle distribution: To demonstrate the
effect of flavonoids 3,5-dihydroxy-7-methoxy-flavanone
and 3,5-dihydroxy-7-methoxyflavone on the cell cycle in
the A375 tumor cell line, cells were synchronized in G1
phase by total withdrawal of fetal bovine serum for 3
days. Once synchronized, the cells were grown in twelve-
well plates at a density of 400,000 cells/mL and incubated
in the presence and absence of the test compounds. After
Natural Product Communications Vol. 6 (7) 2011 949
the incubation period, samples were analyzed in a flow
cytometer, using Cell Quest Pro program and subsequent
analysis was performed by the Modfit program V. 2.0.
(FACSCalibur, Beckton Dickinson). The calibration
parameters were established, and the linearity of the laser
was measured for further analysis with the program
Modfit. The data are shown in Table 3.
Mitochondrial membrane depolarization: The iodide
of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl
carbocyamine (JC-1) is a lipophilic cationic compound
sensitive to changes in mitochondrial membrane potential.
In healthy cells the tagged mitochondria emits red
fluorescence. The negative charge established by the intact
mitochondrial membrane allows the JC-1, which has
delocalized positive charge to enter the mitochondrial
matrix where it accumulates. When it reaches a critical
concentration it forms J-aggregates that emit red
fluorescence. In apoptotic cells, the membrane potential of
mitochondria declines, and the JC-1 cannot accumulate
within the organelle and remains in the cytosol as a
monomer emitting green fluorescence. The aggregate red
form has a maximum absorption / emission 585/590 nm,
while for the green monomer is 510/527 nm. The test was
performed using the cationic lipophilic fluorochrome JC-1,
at concentration of 10 g/mL, from a diluted stock solution
in DMSO and kept at 4°C. The K562 cells (1X106) were
treated with flavonoids 3,5-dihydroxy-7-methoxy-
flavanone and 3,5-dihydroxy-7-methoxyflavone in 24 well
plates and read at 4, 8 and 12 hours. The cells were then
incubated for 10 minutes at 37°C and read immediately
using a flow cytometer, using the Cell Quest Pro program
(FACSCalibur, Beckton Dickinson). The data obtained are
shown in Table 4.
Dendritic cells analysis: Peripheral Blood mononuclear
cells (PBMC) were obtained and washed in RPMI 1640
supplemented with 1% fetal bovine serum (FBS). The
viability and cell numbers were assessed by trypan blue
staining and Neubauer cell counting chamber, respectively.
The CD14+ cells were incubated in RPMI 1640 in the
presence of 35 g/mL of interleukin (IL-4) and 50 g/mL
growth factor granulocyte and monocyte GM-CSF (R&D
system) for 5 days of culture, verifying their morphology
by light microscopy. At day 5 of culture, DCs were treated
with different concentrations (12, 25 and 50 g/mL) of
flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-
dihydroxy-7-methoxyflavone, and 1 μg/mL of lipopoly-
saccharide (LPS) as a positive control for differentiation .
The expression of surface markers was assessed by flow
cytometry after 48 hours using antibodies against CD40,
CD83, CD86 and HLADR. The data obtained showed
proliferation of dendritic cells.
Evaluation of the effect of the flavonoids on the
viability of PBMNC and fibroblasts: The effect of
flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-
dihydroxy-7-methoxyflavone on cell viability of peripheral
950 Natural Product Communications Vol. 6 (7) 2011 Torrenegra G. & Rodríguez A.

blood mononuclear cells (PBMNC) cells was analyzed by
seeding the cells in 96-well plates at a density of 200,000
cells/well in 200 L of RPMI 1640 without phenol red
supplemented with 10% FBS and stimulated for 12 hours
with the mitogen PHA (250 L/100 mL) and then placed
in contact with different concentrations of each flavonoid.
Cells treated with DMSO (vehicle) were used as a control.
Fibroblasts were analyzed in a similar manner. Briefly,
cells were seeded at a density of 15,000 cells/well in
200 L of medium in a 96-well plate and allowed to
adhere for 24 hours; different concentrations of the
flavonoids were added and incubated for 24 hours (37°C,
5% CO2, 95% humidity). After incubation with the
flavonoids, 110 L of medium was added to 10 L of 12
mM MTT and incubated for 4 hours (37°C, 5% CO2, 95%
humidity) protected from light. After this time, the crystals
of formazan resulting from the metabolism of MTT by
mitochondria of viable cells were dissolved with 100 L of
SDS-HCl 0.01M. The plates were incubated again under
the same conditions for 4 hours. The color intensity was
read by absorbance at 540 nm in an ELISA reader
(Labsystems Multiskan MCC/3340).
Acknowledgments - This work was supported by funds
from Universidad de Ciencias Aplicadas y Ambientales,
UDCA. Thanks to Dr Fernando Echeverri,Universidad de
Antioquia by NMR spectra and Dr. Victoria Ramsauer,
East Tennessee State University .

References
[1] El-Sayed NH, Misk IM, Whittemore AT, Mabry TJ. (1988) Sesquiterpene lactones from Chromolaena opadoclinia.
Phytochemistry, 27, 3312-3314.
[2] De Gutiérrez AN, Catalán AN, Díaz JG, Herz W. (1995) Sesquiterpene lactones, a labdane and other constituents of Urolepis
hecatantha and Chromolaena arnottiana. Phytochemistry, 39, 795-800.
[3] Ahmed AA, Whittemore AT, Mabry TJ. (1985) A heliangolide from Chromolaena glaberrima. Phytochemistry, 24, 605-606.
[4] Bohlmann F, Zdero C, Fiedler L, Robinson H, King RM. (1981) A labdane derivative from Chromolaena collina and a
p-hydroxyacetophenone derivative from Stomatanthes corumbensis. Phytochemistry, 20, 1141-1143.
[5] Bohlmann F, Gupta R, King K, Robinson HM. (1981) Prostaglandin-like fatty acid derivative from Chromolaena morii.
Phytochemistry, 20, 1417-1418.
[6] Taleb SH, Salvador MJ, Balanco JM, Albuquerque S, De Oliveira DC. (2004) Antiprotozoal effect of crude extracts and flavonoids
isolated from Chromolaena hirsuta (Asteraceae). Phytotherapy Research, 18, 250-254.
[7] Phan TT, Hughes MA, Cherry GW. (2001) Effects of an aqueous extract from the leaves of Chromolaena odorata (Eupolin) on the
proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization. Wound Repair and
Regeneration, 9, 305-313.
[8] Bouda H, Tapondjou LA, Fontem DA, Gumedzoe M. (2001) Effect of essential oils from leaves of Ageratum conyzoides, Lantana
camara and Chromolaena odorata on the mortality of Sitophilus zeamais (Coleoptera, Curculionidae). Journal of Stored Products
Research, 37, 103-109.
[9] Thang PT, Patrick S, Teik LS,Yung CS. (2001) Anti-oxidant effects of the extracts from the leaves of Chromolaena odorata on
human dermal fibroblasts and epidermal keratinocytes against hydrogen peroxide and hypoxanthine-xanthine oxidase induced
damage. Burns, 27, 319-327.
[10] Phan TT, Allen J, Hughes MA, Cherry G, Wojnarowska F. (2000) Upregulation of adhesion complex proteins and fibronectin by
human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin). European Journal of
Dermatology, 10, 522-527.
[11] Phan TT, Hughes MA, Cherry GW. (1998) Enhanced proliferation of fibroblasts and endothelial cells treated with an extract of the
leaves of Chromolaena odorata (Eupolin), an herbal remedy for treating wounds, Plastic & Reconstructive Surgery, 101, 756-765.
[12] Mabry TJ, Mabry H. (1975) The Flavonoids. Edited by Harborne JB, Chapman and Hall, 1204 p.
[13] Goel RN, Seshardri TR. (1958) New synthesis of tamaraxetin, alpinone and izalpinin. Proceeding of the Indian Academy of
Sciences section A, 47, 191-195.
[14] Asakawa J, Genjida F, Suga T. (1971) Four new flavonoids isolated from Alnus Seboldina. Bulletin of the Chemical Society of
Japan, 44, 297.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Citrus bergamia Juice: Phytochemical and Technological 951 - 955

Studies
Patrizia Picerno, Francesca Sansone, Teresa Mencherini, Lucia Prota, Rita Patrizia Aquino,
Luca Rastrelli and Maria Rosaria Lauro*

Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano,
Salerno, Italy

lauro@unisa.it

Received: December 10th, 2010; Accepted: March 25th, 2011

Fresh juice from bergamot (Citrus bergamia Risso) has been studied to evaluate the polyphenolic composition by HPLC-DAD analysis and
total polyphenols content by UV method. The main constituent, Naringin, has been selected as analytical and biological marker of the juice.
Juice has been loaded onto maltodextrin matrix by spray-drying. The produced maltodextrin/juice powder (BMP) showed neither significant
change in total polyphenols content nor decrease in antioxidant properties with respect to fresh juice. Moreover, BMP displayed high in vitro
dissolution rate of the bioactive constituents in water and in simulated biological fluids. BMP appears as promising functional raw material
for food, nutraceutical and pharmaceutical products. With this aim, a formulation study to develop tablets (BMT) for oral administration has
been also performed. The produced solid oral dosage form preserved high polyphenols content, showed complete disaggregation in few
minutes and satisfying dissolution rate of the bioactive constituents in simulated biological fluids.

Keywords: Citrus bergamia Risso, fresh juice, polyphenols content, Naringin, maltodextrin/juice powder, tablets, in vitro disaggregation and
dissolution tests.

Bergamot (Citrus bergamia Risso) is a natural hybrid fruit
derived from bitter orange and lemon. The plant grows
almost exclusively in the Reggio Calabria region (South
Italy). Bergamot is used for production of essential oil
obtained from the peel, and its fruit juice is considered a
waste product of the industrial process [1a-1e]. Bergamot
juice has drawn attention for its polyphenolic, mainly
flavonoids content [2a-2c], being an attractive raw material
for the food and nutraceutical industry. It is well known
that the consumption of polyphenol-rich products, mainly
due to their antioxidant properties, is beneficial for human
health [3a,3b]. Flavonoids from citrus fruits have many
health benefits including anticancer, antiviral, and anti-
inflammatory activities, as well as effects on capillary
fragility, and inhibition activity on human platelet
aggregation [4a,4b]. In recent studies [5a,5b], bergamot
juice has been shown to be effective in the prevention of
diet-induced hyperlipidemia. Moreover, it is able to
enhance the antioxidant values of others industrial juices,
acting as synergistic compound for the synthetic additives
normally used [6].
Despite these beneficial effects, the unprocessed fresh
bergamot juice, showing penetrating smell and bitter taste,
involves practical difficulties for an industrial use.
Alterations of the functional and organoleptic properties of
polyphenols can take place during the storage period due
to constituents release and degradation/oxidative process
[7,8]. A convenient way to increase the shelf-life and to
improve the organoleptic characteristics of a plant
derivative is to transform it into a stable dry powder form
[9a,9b]. Spray-drying is a micro-encapsulation technique
appropriate for sensitive components such as polyphenols,
and commonly used in pharmaceutical and food industry
[10]. Food ingredients and additives in spray-dried powder
form have reduced bulk weight and size, long-last
biological stability, and are suitable for transportation and
handling. Common carriers for spray-drying process
include carbohydrates, gums, semisynthetic cellulose
derivatives and synthetic polymers [11]. Currently,
maltodextrins, water soluble modified starch derivatives,
are used alone or in combination with other materials in
food and drug processing of plant extracts, aromatic
additives, carotenoids and vitamins [12a-12c].
Maltodextrins have multifaceted functions including
bulking, caking resistance, film formation, binding of
flavour and fat as well as reduction of oxygen permeability
of wall matrix. Moreover, as one of the administration
problems of the bergamot juice is the bitter taste,
maltodextrins are also able to sweeten the final product.
This paper reports on the evaluation of polyphenol
components and antioxidant properties of fresh bergamot
juice, as well as on the production and characterization of
powders obtained loading the fresh juice onto malto-
dextrins as carrier (BMP) by spray-drying. Moreover, a
952 Natural Product Communications Vol. 6 (7) 2011
formulation study to develop tablets containing BMP for
oral administration has been performed. Characteristics of
the tablets (BMT) were evaluated in term of disintegration
time and active compounds release in water and simulated
biological fluids.
Hand-squeezed crude bergamot juice was analyzed by
HPLC-DAD method. As shown in the chromatogram
(Figure 1A) the eluted constituents were three flavanone
neohesperidosides, Neoeriocitrin (1), Naringin (2),
Neohesperedin (3), and a flavone neohesperidoside,
Neodiosmin (4) (Figure 1B). Each compound was
identified by comparison of retention time, MS and UV
spectra with those of standards. In agreement with
literature data [2a-2c], compounds (1-3), were found as the
most abundant flavonoids in the bergamot juice (0.6 ±
0.01, 0.6 ± 0.01, and 0.4 ± 0.01 mg/mL, respectively). To
produce the powder form, an aqueous liquid feed,
containing both maltodextrins and bergamot juice, was
prepared and processed by spray-drying technique, as
described in the experimental section. The production
yield of Bergamot-Maltodextrin Powder (BMP) was very
high (90%). The presence of maltodextrin, having high
water solubility, significantly reduced apparent viscosity of
the feed dispersion favouring the atomization and drying
of the liquid feed [12b]. Moreover, increasing temperature
during the spray-drying process, maltodextrins are able to
induce the rapid formation of a glassy surface which
allows air expansion inside particles, favouring the
increase of particles diameter [12b]. For this reason,
smallest and lightest particles which are normally lost with
the exhaust of the spray dryer are reduced, and the yield
increases. On the other hand, a low viscosity liquid feed
led to a low retention of core material because of the delay
in the formation of a semi-permeable layer by the internal
components during drying [12b,13].
Polyphenol content of both unprocessed juice (actual
polyphenol content, APCB) and BMP (APCBMP) was
determined by UV method (5.49 and 3.57%, respectively).
These values led to calculate the effectively loaded juice
(actual juice content, AJCBMP 32.7%) as described in the
experimental section. AJCBMP was reasonable with respect
to the theoretical juice content (TJC 50.0%).
Consequently, the loading efficiency (LE) value,
calculated as the ratio of AJC to TJC, was 65.4%.
The functional stability of juice, before and after the spray-
drying process, was evaluated as free-radical scavenging
activity using the DPPH test [14]. The antioxidant activity
of bergamot juice, expressed as EC50, (130 ± 5 and 140 ±
12 g/mL, respectively) was at the same level and
remained quite unaltered after the spray-drying process.
The process conditions used did not determine significant
loss of antioxidant activity. To evaluate the dissolution/
release profile of juice from the powder, its solubility in
each dissolution medium was previously detected as
described in the experimental section. Solubility of BMP
resulted 4.0 g/L in water, 3.4 g/L in simulated gastric fluid
Picerno et al.
(GF) and 4.0 g/L in simulated intestinal fluid (IF),
respectively. Sink conditions, which describe a dissolution
system sufficiently dilute so that the dissolution process is
not impeded by saturation of the solution, resulted 1.0 g/L.
The in vitro dissolution/release profiles of active
compounds from the BMP in each dissolution medium
(water, GF and IF) are reported in Figure 2. After 5
minutes a high amount (about 90%) of juice was
dissolved/released both in water and simulated intestinal
fluid; and about 60% in GF, in agreement with solubility
results previously reported. The complete dissolution
(about 100%) was achieved after 30 minutes in both water
and IF, and after 2 hours in GF (Figure 2). These results
are very interesting, because the high water solubility of
the powder, displaying high in vitro dissolution rate with
complete release of the active compounds in all dissolution
media. Formulation of BMP into tablets meets the
challenge to retain the original properties of powder during
compression. This was achieved by keeping low the
compression pressure and using the direct compression
procedure instead of wet granulation, thus avoiding
lengthy granulation steps and exposure to solvents used in
wet granulation [15]. BMP itself was used both as a
directly compressible binder and as diluent. Bergamot
juice is rich in sugars which can act as binder, and
maltodextrins, used as carrier, which may also act as
diluent in the tablets preparation [15]. Moreover, CMC
was used as disintegrant and directly compressible filler,
and magnesium stearate as a lubricant. The final
formulation of bergamot-maltodextrin tablets (BMT) is
reported in the experimental section. Each BMT resulted
8-mm tick (Figure 3) and their disintegration time (see
experimental section) was in about 15 minutes. Figure 3
shows the dissolution profiles of BMT in three different
dissolution media. In 5 minutes, about 20% and 30% of
juice was dissolved in water and IF, respectively. In the
same time only 5% was dissolved in GF. 90% of
dissolution was obtained in 90 minutes in IF, and in 150
minutes, a total release was observed in all dissolution
media. Release of juice from the BMT resulted slower than
from the BMP, probably due to the enhancement of
binding forces of sugars, contained in the juice, during the
compression. Any how, this effect was balanced by the
presence of CMC which promotes the disintegration of
tablets and enhancement of the active compounds
dissolution rate. In conclusion, the obtained water-soluble
powder BMP is able to preserve the polyphenols content
and antioxidant activity of unprocessed juice. Furthermore,
the spray-dried powder is suitable for the production of
oral dosage tablets (BMT) by directly compression as well
as for manufacturing raw material functional food, and for
pharmaceutical and food supplements products.
Experimental
Chemicals: HPLC grade methanol (MeOH), formic acid
(HCOOH) and the reagents used for the extractions were
purchased from Carlo Erba (Rodano, Italy). HPLC grade
water (18mΩ) was prepared using a Millipore Milli-Q
Polyphenol and antioxidant properties of fresh bergamot juice
Figure 1: 1A ) HPLC-DAD chromatogram of C. bergamia juice. In increasing retention order: Neoeriocitrin (1), Naringin (2), Neohesperidin (3), Neodiosmin
(4); detection wavelength: 284 nm; 1B) compounds isolated from bergamot juice.
in vitro BMP Dissolution test
wate r GF
100
80
% Dissolved
60
40
20
0
0 15 30 45 60 75
Time (min)
Figure 2: in vitro BMP dissolution profiles.
purification system (Millipore Corp., Bedford, MA).
Neoeriocitrin, Naringin, Neohesperidin, Neodiosmin, and
Folin-Ciocalteau’s phenol reagent were provided from
Sigma Chemical Co. (Milan, Italy). Maltodexstrins D.E.
16, magnesium stearate and microcrystalline cellulose
from Acef, Italy.
Instruments: HPLC analysis was carried out on an
Agilent 1100 series system equipped with a Model G-1312
pump, and Rheodyne Model G-1322A loop (20 μl), and a
DAD G-1315 A detector. Peaks area were calculated with
an Agilent integrator. ESI-MS was performed on a
Finnigan LC-Q Deca instruments (Thermoquest, San Jose,
CA) equipped with Xcalibur software. To produce the
juice-maltodexstrin powder a Mini Spray Dryer B-191
Büchi (Laboratoriums-Tecnik, Flawil, Switzerland) was
used. Dissolution test was carried out by SOTAX AT
Smart Apparatus (Basel, CH) on line with a
spectrophotometer (UV/Vis spectrometer Lambda 25,
Perkin Elmer Instruments, MA, USA). Balance Crystal
100 CAL – Gibertini (max 110 g d=0,1 mg;+ 15°C/30°C).
Mixer Galena Top (Ataena, Tecno-Pro srl, Italy).
Alternative compression apparatus GP1, Costamac srl,
Casatenovo (LC) Italy.
Plant material: Citrus bergamia fruits Risso were
collected in February 2009 from plants growing in Reggio
Calabria, Italy. Bergamot juice was prepared by hand
squeezing fresh fruits, immediately after collection. It was
filtered through steel sieves of 1 mm and stored at -20°C
until required for our study.
Sample preparation for HPLC and UV analyses:
Unprocessed bergamot juice and processed spray dried
Natural Product Communications Vol. 6 (7) 2011 953
in vitro BMT Dissolution Test
IF
water GF IF
100
80
% Dissolved
60
40
20
0
90 105 120 135 150
0 15 30 45 60 75 90 105 120 135 150
Time (min)
Figure 3: BMT picture and in vitro dissolution profiles.
juice (BMP) were extracted according to Pernice et al.
2009 [6] with slight modifications. 2 mL of bergamot juice
was extracted whit 10 mL methanol, agitated and sonicated
for 10 min. Juice was centrifuged for 10 min at 4000 rpm;
supernatant was collected, while the pellets was extracted a
second time using the same procedure. Supernatants were
combined and centrifuged for 10 min at 2000 rpm. The
concentration of solid material was 20.4 mg/mL.
HPLC-DAD analysis: A part of supernatant was
separated by HPLC using a 3.9 × 300 mm i.d. C18 μ-
Bondapack column. The mobile phase consisted in water
(solvent A) containing 0.1% formic acid, and methanol
(solvent B). The elution gradient was as follows: 0→5
min, 15→30% B; 5→10 min, 30→35% B, 10→20 min,
35→50% B, 20→30 min, 50→75% B; 30→35 min,
75→95% B; 35→40 min, 100% B. The flow rate was 1.0
mL min−1 with a DAD detector set at 284 nm. Elution
yielded four major compounds (Figure 1): Neoeriocitrin
(1, tR 12.6 min), Naringin (2, tR 15.1 min), Neohesperidin
(3, tR 16.6 min), and Neodiosmin (4, tR 18.4 min), in
according to data reported in literature [2c]. Identification
of constituents was carried out by comparison of their
retention times, UV and MS spectra data with those of
standard compounds, and confirmed by co-injections.
Bergamot-powder (BMP) production by Spray drying:
200 g of maltodextrins (16 D.E.) were dissolved in 200 mL
of fresh bergamot juice, with a 1:1 polymer/juice weight
ratio. The liquid feed was spray dried under the following
process conditions: inlet temperature 120°C; outlet
temperature 69-71°C; spray flow feed rate 5 mL/min;
nozzle diameter 0.5 mm; drying air flow 500 l/h, air
pressure 6 atm, aspirator 100%. In order to keep
954 Natural Product Communications Vol. 6 (7) 2011 Picerno et al.

homogeneity, while feed was pumping into the spray
dryer, the suspension was gently stirred using a magnetic
stirring. Each preparation was carried out in triplicate.
Spray-dried bergamot-maltodextrin powder (BMP) was
collected and stored under vacuum for 48h at room
temperature until the characterization.
Quantitative HPLC analysis: HPLC equipment and
conditions were the same used for the qualitative analysis.
Unprocessed juice and BMP were subjected to extraction
as reported in Sample preparation. Reference standard
solutions of Neoeriocitrin, Naringin and Neohesperedin
were prepared at three concentration levels in the range
0.25-1.0 mg/mL. Standard curves were analyzed using the
linear least-squares regression equation derived from the
peak area (R2>0.9999) corresponding to each compound.
Results were expressed as mg/mL of compound ± standard
deviation.
Total polyphenol content: Actual phenol content of
bergamot unprocessed juice (APCB) and BMP (APCBMP),
were determined by UV/Vis spectrometry at λ 284 nm.
Each analysis was made in triplicate. APC was expressed
in percentage as total Naringin (N) equivalents (mg N/100
mL juice).
Yield of the process and loading efficiency: Production
yield was gravimetrically determined and expressed as the
weight percentage of the final product compared to the
total amount of the materials sprayed.
Theoretical juice content (TJC) was calculated as
percentage of juice content compared to the initial total
content of all feed components before spray-drying. Actual
juice content (AJC), theoretical polyphenol content (TPC)
and loading efficiency (LE) were calculated as reported in
the following formula:
AJC% = APCBMP / APCB x 100
TPC% = APCB x loaded juice/ feed components
LE% = AJC/TJC x 100
BMP Solubility: Solubility of the powder was determined
in distilled water and in simulated biological fluids (gastric
fluid, pH 1.2, and intestinal fluid, pH 7.5 without enzymes)
prepared according to USP 31 (2008) [16] at the conditions
reported elsewhere [8]. Concentration of juice in the media
was determined by UV/Vis spectrometry at λ 284 nm and
expressed as Naringin equivalents. Each analysis was
made in triplicate. Naringin calibration curves in the same
solvents were previously worked out. Proportionality
between absorbance and concentration was verified in the
range 1 g/L to 5 g/L (R2>0.999).
Bleaching of the Free-radical 1,1-Diphenyl-2-
picrylhydrazyl (DPPH Test): Free radical scavenging
activity of unprocessed juice and BMP was determined
using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method
[14]. Results were expressed as amount (g/mL) of
antioxidant necessary to decrease the DPPH initial
concentration by 50% (EC50) ± standard deviation (SD).
Tocopherol (EC50 10.1±1.3g/mL) was used as a
positive control in the test.
Tablets products: Ingredients and their relative amounts
used for the formulation of Bergamot-maltodextrin-tablet
(BMT) were: BMP 1000 mg, magnesium stearate 10 mg,
and microcrystalline cellulose (CMC) 5 mg. The
ingredients were mixed until uniformity. The resultant
mixture was compressed using round double concave
punches of 10 mm diameter.
In vitro dissolution test: In vitro dissolution/release tests
were carried out according with the Farmacopea Ufficiale
Italiana (F.U.I. XII, 2009) [17]. Release profile of BMP
and BMT were determined in water, phosphate buffer at
pH 6.8 (simulated intestinal fluid without enzymes) and
hydrochloric acid buffer pH 1.2 (simulated gastric fluid
without enzymes). Samples were analyzed
spectrophotometrically at λmax 284 nm. Briefly, 1000 mg
of BMP or one BMT were placed in six dissolution vessels
containing 1000 mL of dissolution medium on a
dissolution test apparatus n.2: paddle, 100 rpm at
37°C±0.5°C. All the dissolution tests were made in
triplicate; only the mean values are reported (standard
deviations < 5%). Amount of juice dissolved was
measured as Naringin equivalents. Results are graphically
expressed as the dissolution rate (in percentage) with
respect to the time (in minutes).
In vitro disintegration time: Test was carried out
according with the Farmacopea Ufficiale Italiana (F.U.I.
XII, 2009) [18]. A modified dissolution apparatus (paddle
type) was used. The disintegration fluid was HCl pH 1.0
(1000 mL) at the temperature of 37±0.5°C with a stirring
of 100 rpm. Six tablets were placed individually in six
sinkers and disintegration time was determined as the point
at which the tablet disintegrated completely and passed
through the screen of the sinker.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Phenolic Derivatives from the Leaves of Martinella obovata 957 - 960

(Bignoniaceae)
Carolina Arevaloa*, Ines Ruiza, Anna Lisa Piccinellib, Luca Camponeb and Luca Rastrellib
a
Departamento de Control Químico, Facultad de Farmacia, Universidad Nacional Autonoma –
Tegucigalpa, Honduras
b
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte don Melillo,
84084 Fisciano (SA), Italy

anarevalohn23@yahoo.com

Received: December 15th, 2010; Accepted: March 16th, 2011

A new phenolic derivative, 4-methoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O--D-glucopyranoside (1), has been identified together with
uncommon 3,4-dimethoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O--D-glucopyranoside (2) and 3-hydroxy, 4-methoxyphenol 1-O-β-D-
apiofuranosyl-(1→6)-O--D-glucopyranoside (3) from the leaves of Martinella obovata (Kunth) Bureau & K. Schum., an Honduran species
used in folk medicine for the treatment of eyes diseases. Verbascoside, isoverbascoside, leucoceptoside A, vitexin, isovitexin, luteolin 8-C-β-
D-glucopiranoside and spireoside were also found. All structures were elucidated on the basis of mass spectrometry and 2D NMR techniques.

Keywords: Bignonaceae, Martinella obovata, Phenolic apiosides, 1D and 2D NMR.

The Bignoniaceae family includes about 120 genera and O O

R1

800 species, growing mainly in Africa, Central and South OH

O
OMe

America. Species of the Bignoniaceae are used for many OH

HO
O
purposes, such as horticulture, timber, dyes and medicine.
HO HO
OH

The best-known medicinal use of the Bignoniaceae is the 1; R1 = H

application of bark preparations of various species of

2; R1 = OMe
3; R1 = OH

Tabebuia as cancer cures [1]. However, members of the

Figure 1. Compounds 1-3 isolated from the leaves of Martinella obovata
family have been sparsely chemically investigated [2].

Martinella (Bignoniaceae) is a tropical genus consisting of
nine species. Root extracts of the Martinella
iquitosensis vine, found in Amazonian lowland rainforests,
are used by indigenous peoples to treat various eye
ailments, including inflammation and conjunctivitis
[3]. This medicinal use may be attributed, at least in part,
to the presence guanidine alkaloids which have been
demonstrated to be modest antibiotics and micromolar
binders of several G-protein coupled receptors [4]. A
predominance of information regarding M. obovata use
comes from Amazon Indian tribes of Peru, where this
liana, uniformly called “yuquilla”, is often cultivated as the
preferred treatment for eye diseases. In general, the thick
fleshy root bark, with the rough outside part scraped off, is
pounded and the resultant juice strained through cloth. One
or two drops of this juice placed into the eyes is said to
have an immediate effect on inflammation. However, there
are no data in the literature concerning the possible
pharmacological effects and the chemical constituents; this
is the first chemical investigation of M. obovata leading to
the isolation of 10 phenolic compounds.
The ESIMS in negative mode of compound 1 exhibited a
quasi-molecular ion peak at m/z 417 [M-H]- and a high
resolution measurement indicated the molecular formula,
C18H26O11, in accordance with 13C NMR data. Major
fragments at m/z 285 and 123 were assigned to the loss of
a pentose unit (132 amu) and the successive loss of an
hexose unit (162 amu). The 1H NMR spectrum of 1
exhibited a set of AAXX coupling system at  H 7.09
(2H, d, J=8.5, H-2, H-6), and  H 7.01 (2H, d, J=8.5,
H-3, H-5), a methoxy signal at  H 3.85 (3H, s, OMe-3),
and two anomeric proton signals at  5.01 (1H, d, J=2.5
Hz, H-1'') and 4.78 (1H,d, J=8.0 Hz, H-1'). The 13C-NMR
spectrum of 1 revealed 19 carbon signals, including one
benzene ring carbon signal, a set of hexose carbon signals,
a set of pentose carbon signals, and a methoxy signal.
NMR data were in agreement with a 1,4-disubstitution of
benzene ring. The nature of the terminal sugar unit as
β-D-apiofuranosyl was deduced by the following
evidence: the 1H NMR spectrum indicated an anomeric
signal at δ 5.01 (H-1'', d, J = 2.0 Hz); in the 1D TOCSY
experiment, selective excitation of the signal at δ 5.01 led
958 Natural Product Communications Vol. 6 (7) 2011 Arevalo et al.

Table 1: 1H and 13C NMR (600 MHz) Data for Compound 1in CD3ODa. spectra indicated the presence of 1,2,4-trisubstituted
1 aromatic ring, with one methoxyl group as well as a -D-
Position δ 1H (JHH in Hz) δ13C
1 ----- 149.6
apiofuranosyl-(1→6)-O--D-glucopyranosyl unit. The
2 7.09 (d, 8.5) 116.8 spectroscopic data of compound 3 suggested the same
3 7.01 (d, 8.5) 114.4 skeleton as compound 2, but lacking a methoxyl group.
4 ----- 154.6
5 7.01 (d, 8.5) 114.4
The complete assignment was established by the
6 7.09 (d, 8.5) 116.8 resonance of C-3 () shifted upfield by 3.6 ppm and
Glu of C-4 (144.7) and C-2 () shifted downfield by
1' 4.78 (d, 8.0) 102.8
2' 3.28 (dd, 7.5, 9.5) 74.3 ca. 2.4 and 1.6 ppm with respect to dimethoxylated model
3' 3.43 (t, 9.5, 9.5) 78.3 (2). The 3-hydroxy, 4-methoxy substitution was also
4' 3.34 (t, 9.5, 9.5) 70.9 confirmed by HMBC experiments, the correlation peaks
5' 3.30 (m) 77.3
6' 3.70 (dd, 12.0, 3.5) 69.0 of H-2/C-6, C-4; H-6/C-2, C-4, H-5/ C-1, C-3; CH3O–/
3.56 (dd, 12.0, 5.0) C-4; H-1'/C-1 and H-1''/C-6' indicated that benzene ring
Api was substituted at C-1 with the apiofuranosyl-(1→6)-
1'' 5.01 (d, 2.0) 110.3
2'' 4.03 (d, 2.0) 77.4 glucopyranosyl unit and the methoxyl group was located
3'' ----- 80.5 at the C-4. Therefore, the structure of 3 was determined
4'' 3.82 (d, 10.0) 75.4 as 3-hydroxy, 4-methoxyphenol 1-O-β-D-apiofuranosyl-
4.05 (d, 10.0)
5'' 3.61 (2H, s) 66.2 (1→6)-O--D-glucopyranoside, reported previously only
OCH3 3.85 56.4 in the Indonesian medicinal plant Fagara rhetza
a
Chemical shift values are in ppm from TMS, and values in Hz are (Rutaceae) [7].
presented in parentheses. All signals were assigned by DQFCOSY,
HSQC and HMBC experiments.
The structures and molecular formulas of compounds
4-10 were determined from their ESIMS spectra, as well
to the the enhancement only of H-2'' (δ 4.03, d, J = 2.0
as from 1D and 2D 1H and 13C NMR data and by
Hz); and the multiplicity of H-2'' may be derived only
comparison of their NMR data with those in the
from the presence of a quaternary carbon at C-3''
literature. Compound 4 was identified as verbascoside by
characteristic of an apiofuranosyl structure. The 13C NMR
HPLC comparison with authentic standard and according
spectrum gave 11 carbon signals for the sugar moiety, of
to its 1H, 13C NMR, and ESI-MS data [8]. The structure
which three methylenes were ascribable to C-4'' (δ 75.4)
of isoverbascoside (5) was confirmed using 1H and
and C-5'' (δ 66.2) of an apiofuranosyl unit and to C-6' 13
C NMR. The 1H NMR spectrum of isoverbascoside was
(δ 69.0) of a glucopyranosyl unit, respectively. Analysis
similar to that of verbascoside, except for differences in
of the correlated 13C NMR signals in the HSQC spectrum
and of the resonances of the quaternary carbon signal the chemical shifts of H-4' (verbascoside,  4.81;
(δ 80.5, C-3'') matched well with a terminal β-D- isoverbascoside,  3.41) and 2H-6 (verbascoside,  3.63
apiofuranosyl linked to an inner β-D-glucopyranosyl. C-6' and 3.84; isoverbascoside,  4.34 and 4.50) in their
of the glucopyranosyl unit was shifted downfield glucosyl moiety. The 13C NMR chemical shifts of
(β-effect) demonstrating the (1→6) linkage between the isoverbascoside were close to those of verbascoside,
apiosyl and glucosyl units. The interglycosidic linkage but slight differences were observed in the shifts at
was also confirmed unambiguously to be at C-6’’ based C-3', C-4', and C-6' (verbascoside,  81.66, 70.69, 62.49;
on the HMBC cross-peak, between H-1'' and C-6'. isoverbascoside,  84.45, 70.94, 65.20) [9]. Compound 6
Correlations due to long-range HMBC couplings were showed ESI-MS and 1H and 13C NMR data
also observed between H-1' and C-1.Therefore, the superimposable with those reported in the literature for
structure of 1 was determined as 4-methoxyphenol 1-O-β- leucoceptoside A [10]. Compounds 7-9 were identified as
D-apiofuranosyl-(1→6)-O--D-glucopyranoside (1). the flavones vitexin, isovitexin and luteolin 8-C-β-D-
glucopiranoside, compound 10 as the flavonol spireoside
Comparison with the NMR spectral showed that the on the basis of their spectroscopic data and specifically
NMR signals in 2 were similar to those of 1, except for by comparison of their NMR data with those in the
the presence of an extra O-methyl, suggesting that 2 was literature [11].
a 1,3,4- trisubstituted benzene with two methoxyl groups
and one apiofuranosyl(1→6)-glucopyranosyl group. The Experimental
location of these groups was verified by HMBC and NOE General Experimental Procedure: A Bruker DRX-600
spectra. Therefore, compound 2 was concluded to be NMR spectrometer, operating at 599.19 MHz for 1H and at
3,4-dimethoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O-- 150.86 MHz for 13C, was used for NMR experiments;
D-glucopyranoside reported previously only in two chemical shifts are expressed in (parts per million)
species Symplocos caudata (Symplocaceae) [5] and referring to the solvent peaks  H 3.34 and  C 49.0 for
Tabebuia impetiginosa (Bignoniaceae) [6]. CD3OD; coupling constants, J, are in Hertz. DEPT, 13C,
DQF-COSY, HSQC, HMBC and NOESY NMR
Compound 3 has the molecular formula C18H26O12, as experiments were carried out using the conventional pulse
deduced from ESIMS analysis. The 1H and 13C NMR sequences as described in the literature. Electrospray
Phenolic compounds from Martinella obovata
ionization mass spectrometry (ESIMS) was performed
using a Finnigan LCQ Deca instrument from Thermo
Electron (San Jose, CA) equipped with Xcalibur software.
Instrumental parameters were tuned for each investigated
compound: capillary voltage was set at 3 V, the spray
voltage at 5.10 kV and a capillary temperature of 220°C
and the tube lens offset at - 60 V was employed; specific
collision energies were chosen at each fragmentation step
for all the investigated compounds, and the value ranged
from 15-33% of the instrument maximum. Data were
acquired in the MS1 scanning mode (m/z 150-700). All
compounds were dissolved in MeOH : H2O (1:1) and
infused in the ESI source by using a syringe pump; the
flow rate was 5 L/min. Exact masses were measured by a
Q-TOF premier (Waters, Manifold, MA, USA) instrument.
Chromatography was performed over Sephadex LH-20
(Pharmacia, Uppsala, Sweden) employing MeOH as
solvent. Column chromatography was carried out
employing Silica gel RP18 (0.040–0.063 mm; Carlo Erba)
and MeOH:H2O gradients. HPLC separations were
performed on a Waters 590 series pumping system
equipped with a Waters R401 refractive index detector and
a Kromasil C18 column (250 x 10 mm i.d., 10m,
Phenomenex). HPLC-grade methanol was purchased from
Sigma Aldrich (Milano, Italy). HPLC-grade water (18
mΩ) was prepared by a Milli-Q50 purification system
(Millipore Corp., Bedford, MA). TLC analysis was
performed with Macherey-Nagel precoated silica gel 60
F254 plates.
Plant Material: The leaves of M. obovata (Kunth) Bureau
& K. Schum. were collected in Pico Bonito, Francisco
Morazan, Honduras, in August 2005. The plant was
identified by Dr. Cirilo Nelson. A voucher specimen was
deposited in the herbarium of the Botanical Department of
the Universidad Nacional Autonoma de Honduras,
Tegucigalpa, Honduras, (Voucher No. 314).
Extraction and Isolation Procedure of Compounds 1-
10: Dried and powdered leaves (1 kg) of M. obovata were
extracted for a week, three times, at room temperature
using solvents of increasing polarity; namely, petroleum
ether, chloroform, and methanol. Part (3 g) of MeOH
extract was chromatographed on a Sephadex LH-20
column (100 cm x 5.0 cm) using CH3OH as mobile phase
and a flow rate of 1 mL/min to furnish 6 fractions (I-VI).
Fraction II and III (258.4 mg) were purified by RP-HPLC
(40% CH3OH) to give 2 (9.5 mg) and 3 (4.9 mg) and 1
(9.2 mg). Fr. IV (184.1 mg) was purified by RP-HPLC
(35% CH3OH) to give 4 (16.8 mg), 5 (6.4 mg) and 6 (3.8
mg). Finally Fr. V and VI containing flavones were
purified with 70:30 MeOH-H2O to yield compounds 7
(15.1 mg), 8 (12.2 mg), 9 (6.1 mg) and 10 (7.2 mg).
References
[1] Gentry AH. (1992) A synopsis of Bignoniaceae ethnobotany and economic botany. Annals of the Missouri Botanical Garden, 79,
53-64.
Natural Product Communications Vol. 6 (7) 2011 959
4-methoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O--D-
glucopyranoside (1)
White amorphous solid.
[α]D: -58.9 (c 0.20, MeOH).
UV/Vis λmax (MeOH) nm (log ε): 202 (4.42), 223 (3.85),
279 (3.42).
1
H and 13C NMR (600 MHz, CD3OH): see Table 1
ESI-MS m/z 417 [M-H]-, m/z 285 [M-132]- and m/z 123
[M-132-162]-
HREIMS m/z 418.3560 (calcd for C18H26O11, 418.6570).
3,4-dimethoxyphenol 1-O-β-D-apiofuranosyl-(1→6)-O-
-D-glucopyranoside (2)
1
H and 13C NMR data were consistent with those
previously reported [4].
ESI-MS m/z 447 [M-H]-, m/z 315 [M-132]- and m/z 153
[M-132-162]-
HREIMS m/z 448.2560 (calcd for C19H28O12, 448.4430).
3-hydroxy, 4-methoxyphenol 1-O-β-D-apiofuranosyl-(1-
>6)-O--D-glucopyranoside (3)
1
H and 13C NMR data were consistent with those
previously reported [7]
ESI-MS m/z 433 [M-H]-, m/z 301 [M-132]- and m/z 139
[M-132-162]-
HREIMS m/z 434.3177 (calcd for C18H26O12, 434.7100).
Verbascoside (4)
1
H and 13C NMR data were consistent with those
previously reported [8]. ESI-MS m/z 621 [M-H]-.
Isoverbascoside (5)
1
H and 13C NMR data were consistent with those
previously reported [9]. ESI-MS m/z 621 [M-H]-.
Leucosceptoside A (6): 1H and 13C NMR data were
consistent with those previously reported [10]. ESI-MS
m/z 635 [M-H]-.
Vitexin (7)
1
H and 13C NMR data were consistent with those
previously reported [11]. ESI-MS m/z 431 [M-H]-.
Isovitexin (8)
1
H and 13C NMR data were consistent with those
previously reported [11]. ESI-MS m/z 431 [M-H]-.
Luteolin 8-C--D-glucopiranoside (9)
1
H and 13C NMR data were consistent with those
previously reported [11]. ESI-MS m/z 447 [M-H]-.
Spireoside (10)
1
H and 13C NMR data were consistent with those
previously reported [11]. ESI-MS m/z 463 [M-H]-.
960 Natural Product Communications Vol. 6 (7) 2011 Arevalo et al.

[2] (a) Von Poser GL, Schripsema J, Henriques AT, Jensen SR. (2000) The distribution of iridoids in Bignoniaceae. Biochemical
Systematics and Ecology, 28, 351-366; (b) Frederic M, Hay AE, Corno L, Gupta MP, Hostettmann K. (2007) Iridoid glycosides
from the stems of Pithecoctenium crucigerum. Phytochemistry, 68, 1307-1311.
[3] Gentry AH, Cook K. (1984) Martinella (Bignoniaceae): a widely used eye medicine of South America. Journal of
Ethnopharmacology, 11, 337−343.
[4] Witherup KM, Ransom RW, Graham AC, Bernard AM, Salvatore MJ, Lumma WC, Anderson PS, Pitzenberger SM, Varga SL.
(1995) Martinelline and Martinellic Acid, Novel G-Protein Linked Receptor Antagonists from the Tropical Plant Martinella
iquitosensis (Bignoniaceae). Journal of American Chemical Society, 117, 6682-6685.
[5] Jiang J, Feng Z, Wang Y, Zhang P. (2005) New Phenolics from the Roots of Symplocos caudata Wall. Chemical & Pharmaceutical
Bulletin, 53, 110-113
[6] Warashina T, Nagatani Y, Noro T. (2006) Constituents from the Bark of Tabebuia impetiginosa. Chemical & Pharmaceutical
Bulletin, 54, 14-20.
[7] Shibuya H, Takeda Y, Zhang RS, Tanitame A, Tsai YL, Kitagawa I. (1992) Indonesian medicinal plants. IV. On the constituents of
the bark of Fagara rhetza (Rutaceae). (2). Lignan glycosides and two apioglucosides. Chemical & Pharmaceutical Bulletin, 40,
2639-2646.
[8] Sticher O, Lahloub MF (1982) Phenolic glycosides of Paulownia tomentosa bark. Planta Medica, 46, 145-148.
[9] Kawada T, Asano R, Makino K, Sakuno T. (2002) Synthesis of isoacteoside, a dihydroxyphenylethyl glycoside. Journal of Wood
Science, 48, 512-515.
[10] Miyase T, Koizumi A, Ueno A, Noro T, Kuroyanagi M, Fukushima S, Akiyama Y, Takemoto T. (1982) Studies on the acyl
glycosides from Leucoseptrum japonicum (Miq.) Kitamura et Murata. Chemical & Pharmaceutical Bulletin, 30, 2732-2737.
[11] (a) Agrawal PK. (1989) Carbon-13 NMR of Flavonoids. Elsevier, London; (b) Harborne JB. (1994) The flavonoids: Advances in
Research since 1986. Chapman & Hall, New York.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Phenolic Chemical Composition of Petroselinum crispum 961 - 964
Extract and Its Effect on Haemostasis
Douglas S. A. Chavesa#, Flávia S. Frattanib, Mariane Assafimb, Ana Paula de Almeidac,d,e,
Russolina B. Zingalib and Sônia S. Costaa*
a
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21 941-902,
Rio de Janeiro, RJ, Brazil
b
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21 941-902, Rio de Janeiro,
RJ, Brazil
c
Departamento de Ciências Químicas, Laboratório de Química Orgânica e Farmacêutica,
Faculdade de Farmácia, Universidade do Porto, 4050-047, Porto, Portugal
d
Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), 4050-047, Porto, Portugal
e
Laboratório de Estudo Químico e Farmacológico de Produtos Naturais, Universidade Severino Sombra,
27 700-000, Vassouras, RJ, Brazil
# Present address: Instituto de Ciências Exatas, Departamento de Química, Universidade Federal
Rural do Rio de Janeiro, 23890-000, Seropédica, RJ, Brazil

sscbh@terra.com.br; sscosta@nppn.ufrj.br

Received: November 11th, 2010; Accepted: March 16th, 2011

From the aqueous extract (Pc) of Petroselinum crispum (Mill) flat leaves specimens were isolated and identified the flavonoids apigenin (1),
apigenin-7-O-glucoside or cosmosiin (2), apigenin-7-O-apiosyl-(1→2)-O-glucoside or apiin (3) and the coumarin 2’’,3’’-dihydroxy-
furanocoumarin or oxypeucedanin hydrate (4). The inhibitory activity toward clotting formation and platelet aggregation was assessed for Pc
flavonoids (1) and (2), and the coumarin (4). Pc showed no inhibition on clotting activity when compared with the control. On the other
hand, a strong antiplatelet aggregation activity was observed for Pc (IC50 = 1.81 mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin
(IC50 = 0.18 mg/mL). In all cases ADP was used as inductor of platelet aggregation. Our results showed that Pc, apigenin and cosmosiin
interfere on haemostasis inhibiting platelet aggregation. To the best of our knowledge this is the first report for the cosmosiin antiplatelet
aggregation in vitro activity.

Keywords: Petroselinum crispum, parsley, flavonoids, cosmosiin, cardiovascular disease, haemostasis, platelet aggregation.

The species Petroselinum crispum, known as parsley, is an
aromatic herb from Apiaceae family that has been
employed in food, pharmaceutical, perfume and cosmetic
industries [1]. Widespread in all continents, parsley may
be one of the oldest herbs used as condiment in food.
Previous studies on the chemical composition of parsley
have revealed the presence of flavonoids [2-5], coumarins
[6-8] and terpenes [9,10]. In popular medicine, parsley is
used to treat various illnesses such as Alzheimer’s disease,
thrombosis and strokes [11,12]. In Morocco and Brazil,
parsley is widely employed against cardiovascular diseases
[12-14].
The ethnopharmacological knowledge is useful to identify
potential therapeutic targets from medicinal plants.
Substances from vegetal kingdom, which have already
contributed with several compounds in prophylaxis and
treatment of a large variety of pathologies, have been
investigated for their potential as antithrombotic agents
[15,16].
Aspirin, an antiplatelet agent and warfarin, an oral
anticoagulant were developed from secondary metabolites.
However, aspirin consumption can increase the risk of
gastrointestinal bleeding and other adverse effects [16].
Antiplatelet and anticoagulant drugs are used to treat
cardiovascular diseases and strokes preventing or slowing
down blood clots formation and enlargement of existing
blood clots. [17].
According to World Health Organization (WHO), in the
next 20 years there will be 24 million deaths from
cardiovascular diseases [18]. The current spending with
antithrombotic treatment is extremely high [19]. Many
antithrombotic substances from synthetic or semi-synthetic
origin or derived from natural products are currently under
clinical evaluation (phases I, II, III or IV). About 450
substances are considered promising candidates as new
antithrombotic drugs in the Stroke Trials Registry [20].
962 Natural Product Communications Vol. 6 (7) 2011 Chaves et al.

OH OH

RO O O
A possible explanation for the difference in these results
OH should be due to the different extraction procedures
H employed in both studies. We prepared a decoction from
OH O O O O
fresh leaves at 10% (w/v), while Mekhfi et al. (2004)
1-3 4
prepared an infusion at 5.5% (w/v) without mention if
Compounds R Rt UV [M-H+]
fresh or dried aerial parts were used. The variety of P.
min λ max (nm) m/z crispum was not mentioned by those authors, while we
1 H 13.98 256; 336 269.18 used the flat leaf specimens. Furthermore, other factors
2 β-D-Glc 12.91 256; 337 433.26 such as cultivation conditions, sunlight exposure and
3 β-D-Api (1→2)-O-Glc 21.52 268; 339 563.30 season may lead to differences in the production of
4 ------- 33.78 249; 265; 315 305.20 bioactive secondary metabolites [25].
Figure 1: Compounds identified in the aqueous extract of Petroselinum
crispum (Pc) based on HPLC-DAD, ESI and NMR analyses.

This study led to the isolation of known 5,7,4’-trihydroxy-

flavone (apigenin), apigenin 7-O-glucoside (cosmosiin),
apigenin 7-O-apiosyl-(1→2)-O-glucoside (apiin)
and 2’’,3’’-dihydroxyfuranocoumarin (oxypeucedanin
hydrate) identified according to reported NMR data
[21,22]. These flavones and the coumarin derivative were
previously described for this species [4,6]. Two other Figure 2: Inhibitory effect of Petroselinum crispum (Pc) aqueous extract
on the platelet aggregation in vitro induced by ADP (5 µM) using human
flavones diosmetin apiosyl-glucoside and diosmetin platelet-rich plasma (n = 3).
apiosyl-glucoside isomer were identified with basis on
HPLC-DAD chromatogram and comparison with literature
data [4]. Apiin is the most abundant flavonoid in parsley,
while apigenin is the minor component as reported
previously in the literature [3].

In experiments assessing the intrinsic pathway (aPTT), and

the extrinsic pathway (PT) of coagulation, Pc extract,
apigenin, cosmosiin and oxypeucedanin hydrate did not
show any significant activity, since the clotting time was Figure 3: Inhibitory effect of apigenin on the platelet aggregation in vitro
not significantly increased. Figures 2, 3 and 4 show the induced by ADP (5 µM) using human platelet-rich plasma (n = 3).
antiplatelet aggregation effect observed for Pc, apigenin
and cosmosiin, respectively. A strong antiplatelet
aggregation activity was observed for Pc (IC50 = 1.81
mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin
(IC50 = 0.18 mg/mL).

Our results confirm the already known antiplatelet activity

reported for Pc and apigenin [12,23]. Cosmosiin exhibited
a significant antiplatelet activity, although less active than
apigenin. We can deduce from our findings that the
presence of glycosylation decreases the activity of
cosmosiin, since the apigenin skeleton is common to both
structures [24].
Studies on the medicinal species P. crispum (Pc) showed
that its aerial parts aqueous extract was able to inhibit
platelet activity induced by ADP [13]. In these studies, a
crude extract at 10 mg/mL inhibited the platelet
aggregation by 78%. In our study we observed that Pc
leaves extract significantly inhibited (IC50 = 1.81 mg/mL)
the platelet aggregation induced by ADP in human
platelet–rich plasma. Moreover, at concentrations 2.6 times
lower (3.80 mg/mL) than that used by Mekhfi et al. (2004)
we obtained a higher platelet aggregation inhibition
(94.4%) [13].
Figure 4: Inhibitory effect of cosmosiin on the platelet aggregation in
vitro induced by ADP (5 µM) using human platelet-rich plasma (n = 3).
Flavonoids and other phenolic substances are able to
interfere in the platelet system. Apigenin was shown to
block the inducer collagen and ADP in platelet-rich plasma
[24,26]. A diet rich in phenolic compounds may favorably
contribute for reducing risks of cardiovascular diseases
through several mechanisms. Several studies on the
cardiovascular protective effect of flavonoids have been
reported, suggesting that both apigenin and luteolin may
act as competitors with the receptor of thromboxane A2
(TXA2), an inducer of platelet aggregation [27].
We can observe that some flavonoids, particularly
flavones, may be related to the in vitro inhibitory effect of
Phenolic composition of P. crispum and haemostasis
platelet aggregation induced by ADP [12,13]. Our results
showed that Pc extract, apigenin and cosmosiin interfere
on haemostasis inhibiting platelet aggregation. To the best
of our knowledge this is the first report for the cosmosiin
in vitro-antiplatelet aggregation activity. The study of apiin
and the coumarin oxypeucedanin hydrate effect on the
coagulation process is undergoing in our laboratories.
Experimental
Chemical
General: Melting points were determined using a Koppler
melting point apparatus. Optical rotations were measured
on a Jasco P-2000 digital polarimeter. All 1D and 2D
experiments were performed on a Varian 400 MHz
spectrometer. The NMR spectra were recorded in DMSO-
d6. ESI-MS spectra were recorded on a tandem – triple
quadrupole m/z 30-3000. HPLC separation was performed
using a Shimadzu liquid chromatograph LC-10AD
equipped with an UV SPD-10A wavelength detector.
The reversed-phase column used was Merck C18 (5 µm,
250 mm, 2.5 mm) with mobile phase consisted of water
containing phosphoric acid 0.01% (eluent A) and methanol
(eluent B). The samples were run for 44 minutes at
1 mL/min and absorbance was monitored between
200 – 500 nm. The gradient used was 0 – 5 min (100 –
65% A), 5 – 15 min (65 – 55% A), 15 – 25 min (55 – 52%
A), 25 – 35 min (52 – 45% A), 35 – 40 min (45 – 20% A),
40 – 42 min (20 – 0% A) and 42 – 44 min (0 – 100% A).
Thin layer chromatography (TLC) was performed on silica
gel 60 F254 (Merck) eluted with n-butanol/acetic acid/water
(BAW) 8:1:1, visualized under UV light (254 and 365 nm)
and developed with ceric sulfate solution for flavonoids.
Coumarin was detected using 5% potassium hydroxide
solution in ethanol.
Plant material: Leaves from Petroselinum crispum (Mill.)
Nym.ex A.W. Hill (flat leaf specimens) were collected out
of blooming season from specimens grown in an
experimental garden at Severino Sombra University
(Vassouras, RJ, Brazil). A voucher specimen (RFA –
31241) was classified by Dr. Ricardo C. Vieira and
deposited in the herbarium of the Institute of Biology,
UFRJ, Brazil.
Extraction and isolation: Fresh leaves (160 g) were
triturated using a food processor and extracted with
distilled water (10% w/v) by decoction (10 minutes). After
the extract filtration, a spontaneous precipitation at room
temperature yielded a solid that was separated by
centrifugation. This precipitate (1.6 g) was re-suspended in
methanol and chromatographed over Sephadex LH-20
(30 x 1.5 cm; MeOH) yielding 2 fractions: a minor
methanol-soluble fraction (396.2 mg) and a methanol-
insoluble one (1.2 g). Each fraction was purified over
Sephadex LH-20 (23 x 0.7 cm; MeOH/H2O 1:1) affording
cosmosiin (2), for the soluble fraction, as a yellow powder
(23.4 mg) and apiin (3), for the insoluble fraction as a
white powder (676.8 mg). After the precipitate separation
Natural Product Communications Vol. 6 (7) 2011 963
the extract was filtered (Whatman filter paper Nr.1), frozen
at -20°C and lyophilized (1.8 g). After its re-suspension in
water, the resulting solution was partitioned successively
with ethyl acetate (3 x 300 mL) and n-butanol (3 x 300
mL). The ethyl acetate fraction (301.0 mg) was
chromatographed over Sephadex LH-20 (23 x 0.7 cm;
ethanol) affording three fractions. The second fraction
(64.8 mg) showed a yellow crystalline solid (22.3 mg) that
was separated by centrifugation and identified as apigenin
(1). The third fraction was purified on an RP-2 column (30
x 1.2 cm; H2O/EtOH), followed by a Sephadex LH-20
chromatography (23 x 0.7 cm; MeOH/H2O 1:1), affording
oxypeucedanin hydrate (4) as a brown powder (15.4 mg).
Biological
In vitro determination of activated partial thromboplastin
time (aPTT) and prothrombin time (PT): Blood samples
were centrifuged (2000 x g, 10 min), and the platelet-poor
plasma was stored at -20°C until use). aPTT and PT were
measured on an Amelung KC4A coagulometer as follows.
For aPTT tests, cephalin plus kaolin (aPTT reagent,
BioMériaux, RJ, Brazil) were incubated for 1 min with 50
µL of pre-warmed plasma (37°C) and P. crispum (Pc),
apigenin, cosmosiin and oxypeucedanin hydrate at various
concentrations (suspension in PBS buffer). The reaction
was started by addition of 100 µL of pre-warmed CaCl2
(25 mM).
For PT tests, 50 µL of pre-warmed plasma was incubated
with Pc extract, apigenin, cosmosiin and oxypeucedanin
hydrate at various concentrations (suspension in PBS
buffer) for 2 min (37°C) and reaction was started by
addition of 100 µL of pre-warmed thromboplastin with
calcium (PT reagent, BioMériaux, RJ, Brazil). Apiin was
not evaluated since it is insoluble in water.
Platelet aggregation assays: Human blood was collected
in EDTA 0.2 M (9:1 v/v). Platelet-rich plasma (PRP) was
prepared by centrifugation (500 x g, 10 min) at room
temperature. The platelet-poor plasma (PPP) was prepared
by centrifugation of the PRP (2000 x g, 10 min) at room
temperature). In some cases, experiments were performed
using washed platelets as described [28]. Platelet
aggregation was monitored by the turbidimetric method on
a Chrono-Log aggregometer. PRP (400 µL) was incubated
(37°C, 1 min) with continuous stirring at 900 rpm. Platelet
aggregation was induced by ADP (2-10 mM). P. crispum
extract (Pc), apigenin, cosmosiin and oxypeucedanin
hydrate at various concentrations (suspension in PBS
buffer) or vehicle (0.5% DMSO v/v) was added to PRP
samples 1 min before addition of the agonist.
Supplementary data: Compounds from Petroselinum
crispum (Mill.) Nym.ex A.W. Hill, an aromatic herb
popularly known as parsley.
Acknowledgments: Authors gratefully acknowledge
CNPq and CAPES for financial support. We also thank
964 Natural Product Communications Vol. 6 (7) 2011 Chaves et al.

Dr. R. C. Vieira (IB, UFRJ, Brazil) and Dr. Jean-Pierre thank CEQUIMED (FCT, I&D 4040) for allowing Ana
Férézou (ICMMO, Université Paris-Sud, France). We Paula de Almeida to collaborate in this study.

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[21] Beier RC, Ivie GW, Oertli EH. (1994) Linear furanocoumarins and graveolone from the common herb parsley. Phytochemistry, 36,
869-872.
[22] Yoshikawa M, Uemura T, Shimoda H, Kishi A, Kawahara Y, Matsuda H. (2000) Medicinal foodstuffs. XVIII. Phytoestrogens from
the aerial parts of Petroselinum crispum Mill. (parsley) and structures of 6”- Acetylapiin and a new monoterpene glycoside,
petroside. Chemical & Pharmaceutical Bulletin, 48, 1039-1044.
[23] Navarro-Núñez L, Lozano ML, Palomo M, Martínez C, Vicente V, Castillo J, Benavente-García O, Diaz-Ricart M, Escolar G,
Rivera J. (2008) Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the
arachidonic acid pathway. Journal of Agricultural and Food Chemistry, 56, 2970-2976.
[24] Mladěnka P, Zatloukalová L, Filipský T, Hrdina R. (2010) Cardiovascular effects of flavonoids are not caused only by direct
antioxidant activity. Free Radical Biology & Medicine, 49, 963-975.
[25] Muzitano MF, Bergonzi MC, De Melo GO, Lage CL, Bilia AR, Vincieri FF, Rossi-Bergmann B, Costa SS. (2011) Influence of
cultivation conditions, season of collection and extraction method on the content of antileishmanial flavonoids from Kalanchoe
pinnata. Journal of Ethnopharmacology, 133, 132-137.
[26] Janssen K, Mensink RP, Cox FJJ, Harryvan JL, Hovenier R, Hollman PCH, Katan MB. (1998) Effects of the flavonoids quercetin
and apigenin on hemostasis in healthy volunteers: results from an in vitro and a dietary supplement study. American Journal of
Clinical Nutrition, 67, 255-262.
[27] Guerrero JA, Navarro-Nunez L, Lozano ML, Martinez C, Vicente V, Gibbins JM, Rivera J. (2007) Flavonoids inhibit the platelet
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[28] Soslau G, Class R, Morgan DA, Foster C, Lord ST, Marchese P, Ruggeri ZM. (2001) Unique pathway of thrombin-induced platelet
aggregation mediated by glycoprotein-Ib. Journal of Biological Chemistry, 276, 21173-21183.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Bioactivities of Chuquiraga straminea Sandwith 965 - 968

María Elena Mendiondoa, Berta E. Juáreza, Catiana Zampinia,b, María Inés Islaa,b and
Roxana Ordoñeza,b,*
a
Facultad de Ciencias Naturales e Instituto Miguel Lillo.UNT. Fundación Miguel Lillo. CONICET
Miguel Lillo 205/251. (4000).San Miguel de Tucumán. Tucumán. Argentina
b
INQUINOA. CONICET. Facultad de Bioquímica, Química y Farmacia. UNT. Ayacucho 471.
San Miguel de Tucumán. Tucumán. Argentina

rmordoniez@fbqf.unt.edu.ar

Received: December 10th, 2010; Accepted: March 16th, 2011

Methanolic extracts of Chuquiraga straminea Sandwith, subfamily Barnadesioideae (Asteraceae) showed the presence of quercetin-3-O-
glucoside, quercetin-3-O-rutinoside, kaempferol, kaempferol-3-O-glucoside and kaempferol-3-O-rutinoside. Antioxidant and antimicrobial
activity was determined. The total extracts showed antioxidant activity by DPPH and ABTS method (SC50 14.5 to 34.9 µg/mL). A
significantly positive correlation was observed between the antioxidant activity and the total phenolics (R2>0.93). The extracts were active
against ten methicillin resistant and sensitive Staphylococcus aureus strains isolated from nosocomial infection (MIC values between 200 to
800 μg/mL). These preliminary studies are highly interesting as they open new ways for further applications in the treatment of infections by
methicillin resistant S. aureus.

Keywords: Flavonoids, Chuquiraga straminea, Antioxidant activity, Antimicrobial activity.

From an estimated 250,000 higher plants in the world only has long been considered a major problem of Public
5-15% have been studied for a potential therapeutic value. Health. The aim of this study was to investigate
The Argentinian flora offers great possibilities for the antioxidant and antimicrobial activities of C. straminea
discovery of new compounds with medicinal uses. The extracts obtained from aerial parts and flowers.
genus Chuquiraga is represented in Argentina by 15
species distributed in arid regions between the Andes and Extracts were analyzed for their contents of phenolic
Patagonia. Previous reports indicated that the flavonoids compounds (total phenols, flavonoids). A relationship
identified in the species of Chuquiraga genus are between antimicrobial activity, antioxidant activities and
identical, so the compounds are useful as phylogenetic the content of phenolic compounds was evaluated.
micromolecular markers in the genus [1]. Chuquiraga
straminea is a medium xerophytic shrub. Its distribution is The content of total phenols and flavonoids of flower and
in southern Argentina, from northwestern Chubut province aerial parts extracts from C. straminea are given in Table
to eastern Neuquen province. It inhabits in Patagonian 1. The amount of total phenolic compounds extracted
phytogeographic province between 600 and 1000 meters ranged from 1.56 to 2.74 mg gallic acid equivalent
above sea level (masl). This species has been employed as (GAE)/mL extract. The C. straminea aerial parts extracts
traditional medicine by native people, either as building contained the highest amounts of phenolic compounds.
and crafts material and forage [2].
Table 1: Phenolic compounds of C. straminea extracts.
It is reported that phenolic compounds from herbs are Alcoholic Phenolic compounds Flavonoids Compounds
active against many human pathogenic bacteria and fungi Extracts (mg GAE/mL) (mg QE /mL) isolated
quercetin-3-O-glucoside,
and have antioxidant activity [3-5]. Recently, there has Flowers 1.56±0.04 0.12±0.01 quercetin-3-O-rutinoside,
been considerable interest in the use of such antioxidants kaempferol, kaempferol-
and antimicrobial compounds from natural sources, not 3-O-glucoside and
Aerial 2.74±0.08 0.14±0.01 kaempferol-3-O-
only in pharmaceutical industry but also for the rutinoside
Parts
preservation of foods and improving the shelf life of food
products, for increasing the stability of fats and oils and to
control the plant diseases of microbial origin. Kaempferol, quercetin-3-O-glucoside, quercetin-3-O-
Staphylococcus aureus is a common pathogen associated rutinoside, kaempferol-3-O-glucoside and kaempferol-3-
with serious community and hospital acquired diseases and O-rutinoside were identified in the extracts. The flavonoid
966 Natural Product Communications Vol. 6 (7) 2011 Mendiondo et al.

A B

Figure 1: Residual scavenging activity (% RSA) of samples on DPPH (A) and ABTS (B) radicals at a phenolic compounds concentration until of 50 µgGAE/mL. (■) C.
straminea flower extract (SC50DPPH=34.9±1.2 and SC50ABTS= 31.0±1.1 µgGAE/mL) and (♦) C. straminea aerial parts extract (SC50 DPPH= 18.0±0.7µg and
SC50ABTS= 14.5±0.8µg GAE/mL).

glycosides are comparable with those previously isolated, Table 2: Antibacterial activities of C. straminea extracts against sensitive
and antibiotic resistant Staphylococcus aureus strains.
by us from other argentine species belonging to
Chuquiraga genus, [1,6]. C. straminea extracts
Strain Phenotype MIC/MBC (μgGAE/mL)
Antioxidant activity: Free radical species play a critical Flowers Aerial parts
role in cardiovascular and inflammatory diseases as well as MRSA F2 Metr Oxar Genr 400/400 800/>800
in neurodegenerative disorders, cancer and aging. The C. MRSA F7 Metr Oxar GenrVans 400/800 800/>800
straminea extracts were effective as ABTS [2,2`-azinobis MRSA F31 Metr Oxar GenrVans 800/>800 800/>800
(3-ethylbenzothiazoline-6 sulfonic acid) diammonium salt] MSSA F13 Mets Oxas GensVans 200/400 800/>800
MSSA F16 Mets Oxas GensVans 400/400 400/>800
and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-
MSSA F24 Mets Oxas GensVans 400/400 400/>800
scavengers. The results obtained are represented in Figure MRSCN F22 Metr Oxar Genr Vans 400/800 800/>800
1 (SC50 values denote the sample concentration required to MRSCN F27 Metr Oxar Genr Vans 800/>800 800/>800
scavenge 50% ABTS or DPPH free radicals). Aerial parts MSSCN F29 Mets Vans 400/800 400/>800
extracts were the most effective radical-scavengers with MSSCN F30 Mets Vans 400/800 800/>800
SC50 of 18.0±0.7 and 14.5±0.79 µg GAE/mL for DPPH ATCC 29213 Control strain 400/400 800/800
and ABTS respectively, flowers extracts result also active MRSA: methicillin resistant Staphylococcus aureus, MSSA: methicillin
with SC50 of 34.5±1.2 and 31.0±1.1 µg GAE/mL for DPPH sensitive Staphylococcus aureus, MRSCN: methicillin resistant
Staphylococcus coagulase negative, MSSCN: methicillin sensitive
and ABTS radicals, respectively. Staphylococcus coagulase negative, ATCC: American Type Culture
Collection. r and s, resistance or susceptibility to antibiotics. Met,
A significantly positive correlation was observed between methicillin; Oxa, oxacillin; Gen, gentamicin; Van, vancomycin. MIC was
defined as the lowest concentration of extract that had restricted growth to
the antioxidant potential determined by ABTS and DPPH a level <0.05 at 550nm. MBC (minimal bactericidal concentration) was
assays, and the total phenolics (R2>0.93).Contact defined as the lowest extract concentration at which 99.9% of the bacteria
autography, indicated that more than one compounds with have been killed.
antioxidant activity in all crude extracts. The flavonoids
could be the more active metabolites in this plant specie. differences between minimal inhibitory concentration
(MIC) values with respect to the control strain were not
Antimicrobial activity: The antibiotic resistant clinical S. significant. Similar behavior was observed for methicillin
aureus strains assayed in this work were isolated from sensitive and resistant Staphylococcus coagulase negative
human infections from a local hospital. (MSSCN and MRCN, respectively). All nosocomial
strains were sensitive to extracts and vancomycin, a
By means of bioautographic assay was qualitatively glycopeptidic antibiotic. In most cases the extracts showed
demonstrated that several phenolic compounds in the bactericidal effect.
methanolic extracts were active against methicillin
resistant and sensitive S. aureus strains. Table 2 show the The reported data in the present work for S. aureus was
antimicrobial activity of C. straminea methanolic extracts similar to those obtained for other Chuquiraga species that
against ten S. aureus antibiotic resistant and sensitive grow in arid regions of Argentine [5].
strains.
Up to the present, there are limited reports about the
The two methanolic extracts of C. straminea were active bioactivities of C. straminea. The ethnobotanical data
against all methicillin resistant and sensitive S. aureus indicated that this species was used by mapuches and
strains (MRSA and MSSA, respectively) and the observed tehuelches as the main therapeutic tool in traditional
Bioactivity of Chuquiraga straminea
medicine as antiinflammatory and antiseptic on skin
infection [2]. According with our results the C. straminea
extracts could serve as good candidates for the
development of new antimicrobial and antioxidant agents
and/or standardized phytomedicines.
Experimental
Plant material: The plant was collected in the province of
Neuquén, Department Collan Cura. Grow in patches on the
slope of Piñon Cerrito, on rocky ground. A voucher
specimens is deposited at the Fundación Miguel Lillo
Herbarium (LIL 605812).
Preparation of plant extracts: The aerial parts (flowers and
leaves) of C. straminea were dried and extracted with
methanol 80%. The solvent was evaporated at reduced
pressure and then, resuspended in DMSO
(dimethylsulfoxide) for avoid the extraction solvent
interference in the biological screening.
Determination of total phenolics compounds (TPC) and
flavonoids: TPC was determined using the Folin-
Ciocalteau method [7] and gallic acid was used as
standard. The results were expressed as mg GAE/mL
extract. Flavonoid content was determined according to
Woisky and Salatino [8] and quercetin was used as
standard. The results were expressed as mg QE/mL
extract.
Compounds identification: The extracts were
chromatographed bidimensionally according to Mabry [9]
using TBA (tert-butanol-acetic acid-water 3:1:1) and 15%
AcOH as development solvents. Eluted spots were
analyzed by paper and thin layer chromatography in
different solvent systems. The plates were observed under
ultraviolet light, in the absence and presence of ammonia
and natural product reagent. Spectral data with shift
reagents, NaOMe (sodium methoxide), AlCl3 (aluminum
trichloride), HCl (hydrochloric acid), NaOAc (sodium
acetate), H3BO3 (boric acid) were used.
Free radical scavenging activity
The DPPH method: The reduction capability of extracts
was measured by DPPH method according to Zampini et al
[10]. DPPH solution (1.5 mL of 300 μM in 96% ethanol)
was incubated with the samples (5-50 μg GAE). The
reaction mixture was shaken and incubated during 20 min.
at room temperature. Then, absorbance was measured at
515 nm.
The percentage (%) of radical scavenging activity (RSA)
was calculated using the following equation:
RSA % = [(A0 - As)/A0] x 100.
Where A0 is the absorbance of the control and As is the
absorbance of the samples at 515 nm. SC50 values denoted
the sample concentration required to scavenge 50% DPPH
free radicals.
Natural Product Communications Vol. 6 (7) 2011 967
The ABTS method: Antioxidant capacity assay was carried
out by the improved ABTS method as described Re [11].
ABTS•+ radical cation was generated by reacting 7 mM
ABTS and 2.45 mM potassium persulfate after incubation
at room temperature (23 ºC) in the dark for 16 h. ABTS•+
solution (1mL; absorbance of 0.7 ± 0.02 at 734 nm) was
added to 5-50 μg GAE of each tested sample and mixed
thoroughly. The reactive mixture was allowed to stand at
room temperature and the absorbance was recorded at 734
nm, 1 min. after initial mixing and up to 6 min. Results
were expressed in terms of percentage (%) of radical
scavenging activity (RSA) at 6 minute and SC50 values
denoted the sample concentration required to scavenge
50% ABTS free radicals.
Autographic assay: For rapid visualization of antiradical
activity, 5 μg of extracts were applied on silica gel 60 F254
TLC plates. Mixtures of chloroform-ethyl acetate (80:20;
v/v) was used as mobile phase. Then, the plates were dried
overnight and covered with 3 mL of soft medium (agar
0.9%) containing 1mL ABTS•+ (7 mM ABTS and 2.45
mM potassium persulfate) or DPPH (1mg/mL). Plates
were incubated at room temperature during 1 minute in the
dark. Active samples appeared as light spots against a
green-blue or purple background for ABTS or DPPH
assay, respectively [12].
Microorganism: The microorganisms used in this study
consisted of ten Staphylococcus aureus strains recovered
from clinical samples obtained from the Hospital Nicolás
Avellaneda, San Miguel de Tucumán, Tucumán,
Argentina: methicillin resistant S. aureus (MRSA) (n=3),
methicillin sensitive S. aureus (MSSA) (n=3), methicillin
resistant S. coagulase negative (MRSCN) (n=2) and
methicillin sensitive S. coagulase negative (MSSCN)
(n=2). A reference strain was included in the study: S.
aureus ATCC 29213.
Antimicrobial activity
Bioautographic assays: Extracts (25 μg) were seeded on
TLC plates and the components were separated using
chloroform-ethyl acetate (80:20; v/v) as development
solvents. Then, the plates were dried overnight in a sterile
room and were covered with 3 mL of soft medium (BHI
with 0.6% agar) containing 1 x 105 colony forming units
(CFU) of S. aureus (F7) incubated at 35C for 20 h. Next,
the plates were sprayed with a 2.5 mg/mL MTT solution
(3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) in
PBS (10 mM sodium phosphate buffer, pH 7, with 0.15 M
NaCl) to determine cellular viability. Plates were
incubated at 35C for 1h in the dark for color development
[13].
Broth microdilution susceptibility assay: This assay was
performed in sterile 96-well microplates. The extracts were
transferred to each microplate well in order to obtain two-
fold serial dilutions of the original extract (25 to 800
µg/mL). The inoculum (100 µL) containing 5×105 CFU
968 Natural Product Communications Vol. 6 (7) 2011 Mendiondo et al.

was added to each well. A number of wells were reserved
in each plate for sterility control (no inocula added),
inocula viability (no extract added), and solvent effect
(DMSO) [14].
Plates were aerobically incubated at 35°C. After
incubation for 16-20 h, bacterial growth was indicated
by the presence of turbidity and a pellet on the well
bottom. A cytotoxicity assay was also carried out.
After the broth microdilution susceptibility assay, 20 L
of methylthiazolyltetrazolium chloride solution (MTT)
(12 mg/mL in PBS) was added to the wells and incubated
for 1 h. Cellular viability was determined by absorbance at
550 nm.
MIC was defined as the lowest concentration of extract
that had restricted growth to a level <0.05 at 550nm (no
macroscopically visible growth).
To confirm MIC and to establish MBC, 10 µL of each
culture medium was removed from each well with no
visible growth and inoculated in Müller Hinton Agar
plates. After 16-20 h of aerobic incubation at 35°C, the
number of surviving organisms was determined.
MBC was defined as the lowest extract concentration at
which 99.9% of the bacteria have been killed.
MIC values were also determined for different commercial
antibiotics. Resistance was defined for each case:
methicillin (Met, MIC > 16 μg/mL), oxacillin (Oxa, MIC >
16 μg/mL), gentamycin (Gen, MIC > 100 μg/mL) and
vancomycin (Van, MIC > 6 μg/mL) for S aureus strains.
All experiments were carried out in triplicate.
Statistical analysis: Data are represented as mean ±
standard deviation. The statistical tests were carried out by
analysis of variance (one-way ANOVA) and the post-test
of Turkey, using a probability level of less than 5% (p <
0.05).
Acknowledgments - This research was partially supported
by Grants from Consejo de Investigación de la
Universidad Nacional de Tucumán (CIUNT, Tucumán,
Argentina) and Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET; Buenos Aires,
Argentina).

References
[1] Mendiondo ME, Juarez BE, Seeligmann P. (2000) Flavonoid profiles of some Argentine species of Chuquiraga (Asteraceae).
Biochemical Systematics and Ecology, 28, 283-285.
[2] González S, Morales S (2004) Plantas medicinales utilizadas en comunidades rurales del Chubut, Patagonia-Argentina. Boletín
Latinoamericano de Plantas Medicinales y Aromáticas, 3, 58-62.
[3] Arias ME, Gómez, JD, Vattuone MA, Isla MI. (2004) Antibacterial activity of ethanolic and aqueous extract of Acacia aroma Gill
ex Hook. Life Science, 75, 191-202.
[4] Zampini IC, Vattuone M, Isla MI. (2005) Antibacterial activity against antibiotic-resistant Gram negative human pathogenic
bacteria of hydroxychalcone isolated from Zuccagnia punctata Cav. Journal of Ethnopharmacology, 102, 450-456.
[5] Zampini IC, Cuello S, Alberto MR, Ordoñez RM, D’ Almeida R, Solorzano E, Isla MI. (2009) Antimicrobial activity of selected
plant species from “the Argentine Puna” against sensitive and multi-resistant bacteria. Journal of Ethnopharmacology, 124,
499–505.
[6] Juárez BE, Mendiondo ME. (2002) Flavonoid Chemistry of Chuquiraga (Asteraceae). Biochemical Systematics and Ecology, 30,
371-373.
[7] Singleton VL, Orthofer R, Lamuela-Raventos RM. (1999) Analysis of total phenols and other oxidation substrates and antioxidants
by means of Folin Ciocalteu reagent. Methods in Enzymology, 299, 152-178.
[8] Woisky R, Salatino A (1998) Analysis of propolis: some parameters and procedures for chemical quality control. Journal
Apiculture Research, 37, 99-105.
[9] Mabry TJ, Markham KR, Thomas MB. (1970) In The systematic identificaction of flavonoids. Springer-Verlag, New York, Chapter 5.
[10] Zampini IC, Meson Gana J, Ordoñez RM, Sayago JE, Nieva Moreno MI, Isla MI. (2008) Antioxidant and xanthine oxidase
inhibitory activities of plan species from the Argentine Puna (Antofagasta, Catamarca). Recent Progress in Medicinal Plants, 21,
95-110
[11] Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice Evans C. (1999) Antioxidant activity applying an improvement ABTS
radical cation decoloration assay. Free Radical Biology and Medicine, 26, 1231-1237.
[12] Zampini I, Ordoñez R, Isla MI. (2010) Autographic assay for the rapid detection of antioxidant capacity of liquid and semisolid
pharmaceutical formulations using ABTS●+immobilized by gel entrapment. AAPSPharm Sci Technology, 11, 1159-1163.
[13] Nieva Moreno MI, Isla MI, Cudmani NG, Vattuone MA, Sampietro AR. (1999) Screening of antibacterial activity of Amaicha del
Valle (Tucumán, Argentina) propolis. Journal of Ethnopharmacology, 68, 97-102.
[14] CLSI (Clinical and Laboratory Standards Institute, formerly National Committee for Clinical and Laboratory Standards, NCCLS).
(2006) Methods M27-A2 and M-38A, 2nd ed.; Wayne, PA. Ed.; Vol. 22 (15), 1-29; (16), 1-27.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Free Radical Scavenging Activity, Determination of 969 - 972
Phenolic Compounds and HPLC-DAD/ESI-MS Profile of
Campomanesia adamantium Leaves
Aislan C.R.F. Pascoala, Carlos Augusto Ehrenfriedb, Marcos N. Eberlinc,
Maria Élida Alves Stefanellob and Marcos José Salvadora,*
a
Pharmacy School, Department of Plant Biology, Institute of Biology,
State University of Campinas (UNICAMP), Campinas, SP, 13083-970, Brazil
b
Departament of Chemistry, Federal University of Paraná (UFPR), Curitiba, PR, 81531-990, Brazil
c
Thomson Mass Spectrometry Laboratory, Institute of Chemistry, State University of Campinas
(UNICAMP), Campinas, SP, 13083-970, Brazil

marcosjs@unicamp.br

Received: December 10th, 2010; Accepted: March 16th, 2011

Numerous diseases are induced by free radicals via lipid peroxidation, protein peroxidation and DNA damage. It has been known that a
variety of plant extracts have antioxidant activity to scavenge free radicals. Campomanesia adamantium (Myrtaceae) is a small tree with
edible fruit, commonly known as “guavira” or “guabiroba-branca” that has been used in popular medicine as depurative anti-diarrhoeic, anti-
inflammatory, anti-rheumatic and to liver diseases. In this study, the antiradical activities of ethanol crude extract of the leaves from C.
adamantium and the ethyl acetate and butanol fractions obtained by partition, were determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl
radical) and ORAC-FL (Oxygen Radical Absorbance Capacity) assays. The total phenol content in the samples was estimated by Folin
Ciocalteau method (FCR). In an initial evaluation the ethanolic extract and the fractions ethyl acetate and butanol have shown levels of
phenolic compounds between 15- 74 mg GAE/g in FCR assay, showed DPPH free-radical scavenging activity with SC50 in the range of 7.77-
13.35 µg/mL and demonstrated antioxidant capacity between 2648-3502 µmol TE/g of extract and fractions in the ORAC-FL assay. HPLC-
DAD and ESI-MS analysis revealed were that the extract of the leaves of C. adamantium studied appears to contain flavonoids as major
constituents, including isoquercetrin and quercetin that exhibit proven antioxidant activity.

Keywords: radical scavenger, DPPH, Myrtaceae, Campomanesia adamantium, HPLC- DAD/ESI-MS.

The organic and inorganic molecules and atoms that
contain one or more unpaired electrons, with independent
existence, can be classified as free radicals [1]. Free
radicals are responsible for lipid peroxydation occurred
during production and storage of nutrients [2], and are
directly involved in some cancers, cardiovascular
disorders, diabetes [3], Alzheimer's disease, atherosclerosis
and others human pathologies [4]. The radicals O2• and
their reduction products, H2O2, and especially the radical
OH•, are some of those responsible for cell damage by
promoting lipid peroxidation, with damage to
mitochondria, lysosomes and cell membrane itself, leading
to cell death. In animal, different biochemical routes
involve free radicals formation, but in these cases defense
mechanisms against the oxidative process propagation are
also involved. These mechanisms do not show a constant
efficacy [3]. However, exogenous antioxidant compounds
act as an auxiliary function in this defense processes.
Antioxidants block the free radicals formation through
different ways and establish important control function in
some oxidative stress diseases [4] and in food conservation
[5]. Thus, new natural antioxidants, mainly those isolated
from medicinal plants, acquire great pharmacological
importance and the research on these classes of
compounds has been increased in the last years [6-8].
The genus Campomanesia (Myrtaceae) comprises around
30 species of shrubs or small trees, aromatic, distributed
mainly in tropical and subtropical South America [9]. Most
species produce edible fruits that are widely used to make
liqueur, juices and jellies [10]. Several species are
considered medicinal and have been used in folk medicine
mainly against digestive problems and diarrhea [11].
Campomanesia adamantium Camb. is a small tree, known
as “guavira” or “guabiroba-branca”, largely spread in
Brazil. It can be found growing wild in the Midwest,
Southeast and South regions of Brazil, and frequently is
cultivated in home gardens for its fruits [12]. Its leaves and
fruits have been used against rheumatism, liver and urinary
diseases [13]. Previous phytochemical studies in
Campomanesia have reported the identification of
quercetin, myricetin and rutin in C. xanthocarpa [14] and
β-triketone type compounds, named champanones in C.
lineatifolia [15]. Recently it was reported the isolation of
970 Natural Product Communications Vol. 6 (7) 2011
flavonoids in C. adamantium [16,17] as well as the
antioxidant activity of extracts and fractions [17,18].
However, these studies were carried on specimens growing
in Midwest region that can have a chemical profile
different from those growing in other regions of country.
These facts prompted us to investigate the antioxidant
capacity of the ethanolic extract and fractions of C.
adamantium from South region of Brazil and, characterize
the major constituents responsible for antioxidant activity.
The samples analyzed in the present study showed a total
phenol content in the range of 15.78 – 74.83 mg GAE/g
extract (Table 1). Phenolic compounds are recognized as
one of most important class responsible for antioxidant
capacity in plants [19].
Ethanol extract, ethyl acetate and butanol fractions
exhibited antioxidant activity concentration-dependent in
DPPH assays, with SC50 varying from 7.77 to 13.35
µg/mL. The highest antioxidant activity was exhibited by
butanol fraction. In ORAC-FL kinetic assay, based on
hydrogen transfer mechanism, the extracts showed
antioxidant capacity between 2648 and 3502 µM of
TROLOX equivalent per gram of extract (Table 1) In
comparison with previous studies [16,18], our extracts and
fractions showed higher antioxidant activity and there were
chemical differences between C. adamantium leaves
analyzed in this study of South region of Brazil and C.
adamantium growing in Midwest region of Brazil [16-18].
The chalcones were previously reported in C. adamantium
growing in Midwest region of Brazil [16], while the
flavonols isoquercetrin, myricetin, quercitrin and quercetin
are being reported for the first time in this plant. Myricetin
had been already identified in C. xanthocarpa [14].
Table 1: Total phenol content and antioxidant capacity by the DPPH and
ORAC assays of ethanol extract of Campomanesia adamantium leaves
and its fractions ethyl acetate (EtOAc) and butanolic (BuOH).
Samples Phenol contenta DPPH assay, ORAC a
(mg of GAE/g)b SC50a (µg/mL)c (µmol TE/g) d
Ethanol extract 35.04(5.48) 13.00 (5.03) 2648 (1.77) d
EtOAc fraction 74.83(10.77) 13.35 (16.85) 3150 (6.66) d
BuOH fraction 15.78(15.29) 7.77 (5.00) 3502 (5.71) d
Quercetin* - 12.80 (2.00) 5.62 (0.89) e
Isoquercitrin* - - 5.21 (1.60) e
Trolox* - 2.55 (1.40) - in Curitiba, Paraná State, Brazil (25o25’48’’ S, 49o16’15’’
*Experimental positive controls.
-: not evaluated.
a
b
Mean (%RSD, relative standard deviation) of triplicate assays.
Total phenolics data expressed as milligrams of gallic acid equivalents per
gram (mg of GAE/g) of extract or fractions.
c
DPPH assay data expressed as SC50 (concentration that inhibited 50% of the
DPPH radical) in micrograms per milliliters (µg/mL).
d
ORAC data expressed as micromol of Trolox equivalents per gram (µmol of
TE/g) of extract or fractions.
e
ORAC data expressed as relative Trolox equivalent, mean (%RSD, relative
standard deviation) of triplicate assays
The ESI-MS technique has been applied in the analyze of
several complex matrix, such as wine, oil, beer and
extracts from natural sources. In this study, the samples
presented high content of phenolic compounds and were
Pascoal et al.
analyzed by ESI (-)-MS [20-21]. The analysis by ESI(-)-
MS showed that major constituents in the samples of
C. adamantium leaves, including the crude ethanol extract
and TLC yellow spot sample, were coincided with the
mass of the chalcones 2’,4’-dihydroxy-6’-methoxy-
chalcone, 2’,4’-dihydroxy-5’-methyl-6’- methoxychalcone
and, 2’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone
together with the flavonols isoquercitrin, quercitrin,
quercetin, and myricetin (Table 2, Figure 1). Structural
analysis of single ions in the mass spectra from extract and
fractions were performed by ESI-MS/MS. The compounds
were identified by comparison of their ESI-MS/MS
fragmentation spectra with fragmentation spectra of the
authentic standard samples (compounds 1, 5, 6 and 7) and
with literature data [21].
To confirm the presence of the flavonoid isoquercitrin
were made the HPLC-UV/DAD analysis of standard
isoquercitrin and of the crude extract. We noted the
formation of a peak with a retention time coincident with
the same pattern and absorption peaks. Furthermore, the
identity of major constituent isoquercitrin was also
confirmed through co-elution with authentic standard
sample. These results are enough to confirm that one of the
major constituents and the responsible for antioxidant
activity is the flavonoid isoquercitrin, since that the same
mass was also found in the analysis of TLC spot yellow
sample that was reveled with solution of DPPH.
Thus, the results of the present study suggest that the
antioxidant capacity of C. adamantium is correlated to the
content of flavonoids, including isoquercitrin, which is
present in the crude ethanolic extract and TLC spot yellow
sample. Moreover, this activity presents a positive
correlation with the total phenolic soluble content
measured by FCR assay. However, further investigations
are necessary to confirm if this plant and its constituents
represent a source of powerful antioxidant products useful
in vivo.
Experimental
Plant Material: The leaves of Campomanesia
adamantium were collected from wild specimen growing
W) at 934 m of altitude. The plant was identified by Dr.
Armando Carlos Cervi, which deposited a voucher
specimen at the herbarium of UFPR (UPCB 60503).
Extracts preparation: The powder was subjected to the
process of maceration with ethanol at a ratio of powder /
solvent of 1:5 (weight / volume). The ethanolic crude
extract was suspended in methanol/water (9:1, v/v) and
fractionated by liquid-liquid extraction with hexane and
ethyl acetate. The hydroalcoholic phase remaining was
partitioned with n-butanol and water to afford an n-
butanol-soluble portion. This procedure yielded the
fractions of hexane (Hex), ethyl acetate (EtOAc) and
butanol (BuOH).
Antioxidant activity and HPLC/ESI-MS profile of C. adamantium Natural Product Communications Vol. 6 (7) 2011 971
OH

OH
R3 CH3

HO O HO O
R1

OR2
R4
OH O
OH O

1 Isoquercitrin R1=H, R2=glucose

5 2’,4’-dihydroxy-6’-methoxychalcone R3=R4=H
2 Quercitrin R1=H, R2=rhamnose
6 2’,4’-dihydroxy-5’-methyl-6’-methoxychalcone R3=CH3, R4=H
3 Myricetin R1=OH, R2=H
7 2’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone R3=R4=CH3
4 Quercetin R1=R2=H

Figure 1: Structure of the compounds identified in ethanol extract from the leaves of Campomanesia adamantium of the South of Brazil and its fractions

Table 2: Compounds identified in ethanol extract from the leaves of Campomanesia adamantium and its fractions using ESI(-)-MS/MS.

ESI-MS ions (m/z)

Compounds Campomanesia adamantium samples Deprotonated ions [M-H]- m/z MS/MS ions m/z
Ethanol EtOAc BuOH TLC
extract fraction fraction yellow spot
1 + + + + 463 25 eV: 463→301, 255, 151
2 + + - + 447 25 eV: 447→301
3 + - - + 317 25 eV: 317→287
4 + + + - 301 25 eV: 301→271, 255
5 + + - + 269 25 eV: 269→253, 226, 198, 184, 177, 165, 150, 139,
122, 108, 97, 94, 65
6 + + - + 283 25 eV: 283→268, 240, 198, 179, 164, 136, 108, 79
7 + + - + 297 25 eV: 297→282, 254, 191, 178, 163, 150, 134, 122
+: detected; -: not detected

Radical scavenging activity by DPPH assay: The
antiradical activity of extract and fractions of EtOAc and
BuOH were determined using the stable 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH) [22]. Fifty microliters of a
250 μM DPPH solution in ethanol was added to a range of
solutions of different concentrations (seven serial 3-fold
dilutions to give a final range of 100 to 1.6 μg mL-1) of
extracts to be tested in ethanol (10μL). Absorbance at 517
nm was determined 20 min after the addition of each of the
compounds tested, and the percentage of activity was
calculated. Quercetin and Trolox were used as positive
controls. The antioxidant activity was expressed as the
SC50 value in μg mL-1. All samples were tested in triplicate.
extract or fraction (mg of GAE/g). The analyses were
performed in triplicate.
Separation and analysis of antioxidants on thin layer
chromatography: The sample was applied on TLC and
eluted with BAW (butanol: acetic acid: water), after that
we sprayed with a solution 500 µg/mL of DPPH in
ethanol. After solvent evaporation (about 5 minutes), the
potential anti-free radical was verified by the appearance
of yellow spots on violet background, according to the
described in the literature [25]. The yellow spot was
scraped, dissolved in methanol, filtered and this was called
TLC yellow spot sample.

Evaluation of antioxidant capacity by ORAC assay:
The antioxidant capacity of the ethanolic extract and
AcOEt and BuOH fractions were assessed through the
oxygen radical absorbance capacity (ORAC) [23]. In this
assay, measures the antioxidant scavenging activity against
peroxyl radicals using fluorescein as the fluorescent probe.
ORAC assays were carried out on a Synergy-2 multi-
detection microplate reader system. The temperature of the
incubator was set at 37 °C. The data were expressed as
micromoles of Trolox equivalents (TE) per gram of extract
or fraction on dry basis (μmol of TE/g) and as relative
Trolox equivalent for pure compounds. The analyses were
performed in triplicate.
Quantitative determination of total soluble phenols:
The dried ethanolic extract and its EtOAc and BuOH
fractions, dissolved in ethanol, were analyzed for their total
soluble phenolic content according to the Folin-Ciocalteau
colorimetric method [24]. The results were expressed as
milligrams of gallic acid equivalents (GAE) per gram of
HPLC-UV/DAD/ESI-MS profile: The TLC yellow spot
sample, crude extracts and fractions of C. adamantium
leaves were diluted in a solution containing 50% (v/v)
methanol (chromatographic grade), 50% (v/v) deionized
water and, 0.5% of ammonium hydroxide (Merck,
Darmstadt, Germany). In the fingerprinting ESI-MS
analysis, the general conditions were: source temperature
of 100 oC, capillary voltage of 3.0 kV and cone voltage of
30 V. For measurements were performed according to the
described in the literature [20,21]. Structural analysis of
single ions in the mass spectra from extract and fractions
were performed by ESI-MS/MS. The ion with the m/z of
interest was selected and submitted to 25 eV collisions
with argon in the collision quadrupole. The compounds
were identified by comparison of their ESI-MS/MS
fragmentation spectra.
HPLC analyses were conducted using a RP-18 column
(Lichrospher“, 5 μm, 225\4.6 mm, Merck). The mobile
phase consisted of a linear gradient combining solvent A
972 Natural Product Communications Vol. 6 (7) 2011 Pascoal et al.

(acetonitrile) and solvent B (water/acetic acid, 99:1, v/v,
pH 2.88) as follows: 15% A (15 min), 15-40% A (5 min),
40-60% A (5 min), 60-100% A (5 min), 100-15% A (5
min), 15% A (5 min). The analyses were carried out in
triplicate at a flow rate of 0.8 mL/ min and an injection
volume of 20 μL. UV-DAD detector was set to record
between 200 and 600 nm, and the UV chromatograms
were measured at 254 and 350 nm. The samples were
crude extract and isoquercitrin standard sample at
1mg/mL.
Statistical analysis: Data are reported as mean (%RSD,
relative standard deviation) of triplicate determinations.
The statistical analyses were carried out using the
Microsoft Excel 2002 software package (Microsoft Corp.,
Redmond, WA)
Acknowledgments - The authors are grateful to Dr.
Armando C. Cervi (UFPR) for plant identification and to
FAPESP, CNPq and FAEPEX-UNICAMP for financial
support.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Activity of Cuban Propolis Extracts on Leishmania 973 - 976

amazonensis and Trichomonas vaginalis

Lianet Monzote Fidalgo*, Idalia Sariego Ramosa, Marley García Parraa, Osmany Cuesta-Rubiob,
Ingrid Márquez Hernándezb, Mercedes Campo Fernándezb, Anna Lisa Piccinellic and Luca Rastrellic
a
Parasitology Department, Institute of Tropical Medicine “Pedro Kourí”, Havana City, Cuba
b
Department of Pharmacy, Institute of Pharmacy and Food, University of Havana, Cuba
c
Department of Pharmaceutical Sciences, Via Ponte Don Melillo 84135, Fisciano, Salerno, Italy

monzote@ipk.sld.cu

Received: December 10th, 2010; Accepted: March 16th, 2011

In this paper we analyzed the antiprotozoal effects of eighteen Cuban propolis extracts (brown, red and yellow type) collected in different
geographic areas, using Leishmania amazonensis (as a model of intracellular protozoa) and Trichomonas vaginalis (as a model of
extracellular protozoa). All evaluated propolis extracts caused inhibitory effect on intracellular amastigotes of L. amazonensis. However,
cytotoxicity on peritoneal macrophages from BALB/c mice was observed. Only five samples decreased the viability of T. vaginalis
trophozoites at concentrations lower than 10 g/mL. No correlation between the type of propolis and antiprotozoal activity was found. Cuban
propolis extracts demonstrated activity against both intracellular and extracellular protozoa model, as well as the potentialities of propolis as
a natural source to obtain new antiprotozoal agents.

Keywords: Propolis, antiprotozoal, Leishmania amazonensis, Trichomonas vaginalis.

Propolis is a resinous hive product that honey bees produce
employing parts of plants as buds, leaves, exudates or
resins, and beeswax. Its chemical composition is highly
variable and depends greatly according to the plants found
around the hive [1a]. Cuban propolis has shown significant
differences in its chemical composition with respect to
propolis from temperate zone. They also vary significantly
among themselves and can be clearly divided into three
groups: brown Cuban propolis type (BCP type) has shown
to be rich in polyisoprenylated benzophenones, red type
(RCP type), containing isoflavonoids as the main
constituents, and yellow type (YCP type) with a variety of
triterpenoids as the major chemical components [1b].
Diverse pharmacological activities of propolis have been
explored, such as: anti-inflammatory and anti-tumoral
effect [2a]. Up to now, antimicrobial properties have been
widely investigated; including antibacterial [2b], antiviral
[2c] and antifungal activity [2d]. Antiparasitic activities
have also been reported against Trypanosoma cruzi [2e]
and Giardia duodenalis [2f]. However, only a few studies
have been carried out for the antileishmanial [3a-3c] and
antitrichomonocidal activity [4a]. In Cuba, propolis has
displayed therapeutic potentialities as antipsoriatic, anti-
inflamatory, analgesic [4b], antibacterial [4c] and
antitumoral [4d]. Scarce reports can be found about its
antiparasitic activity. Thus, its biological potentiality has
not been explored totally.
In the present work, we analyzed the antiprotozoal effects
of brown, red and yellow Cuban propolis extracts collected
in different geographic areas, using Leishmania
amazonensis (as a model of intracellular protozoa) and
Trichomonas vaginalis (as a model of extracellular
protozoa. Leishmania are protozoa that cause leishmaniasis
[5a]. The disease is endemic in 88 countries throughout
Latin America, Africa, Asia and Southern Europe.
Approximately 350 million people are thought to be at risk
with a worldwide prevalence of 12 million and annual
incidence of 2 million new cases [5b].
All propolis samples caused inhibition growth against
L. amazonensis, with IC50 < 27 µg/mL; although a high
toxicity on peritoneal macrophage from BALB/c mice was
observed (Table 1). The better selectivity was showed by
BCP-1, a propolis sample that exhibited a high content of
nemorosone [1b]. However, other BCP samples containing
also high content of this compound exhibited higher values
of IC50. BCP-16 and RCP-29 showed unspecific action. In
general, RCP was the most active (IC50 average of 13.9
µg/mL) and the most cytotoxic (CC50 average of 37.2
µg/mL). There are a number of studies documenting the
antiprotozoal activity of flavonoids [6a-6e]. These
compounds have shown activity against Plasmodium
falciparum, Leishmania spp., Trypanosoma cruzi or
Giardia intestinalis. Four flavonoids detected in RCP
including biochanin A, 3,8-dihydroxy-9-methoxy-
pterocarpan, formononetin, and liquiritigenin have shown
974 Natural Product Communications Vol. 6 (7) 2011 Monzote et al.

Table 1: Antileishmanial activity and cytotoxicity of Cuban propolis extracts. Table 2: Activity of Cuban propolis extracts on T. vaginalis.
Propolis extracts IC50† CC50§ SI ƒ
Propolis extracts IC50† (µg/mL) ± SD‡
(µg/mL) ± SD‡ (µg/mL) ± SD‡ BCP-1 6.2 ± 0.3
BCP-1 7.8 ± 0.9 51.8 ± 4.5 7 BCP-4 > 200
BCP-4 23.4 ± 0.9 70.0 ± 4.4 3 BCP-5 > 200
BCP-5 21.2 ± 2.4 52.0 ± 0.3 2 BCP-16 > 200
BCP-16 33.9 ± 1.7 43.8 ± 0.7 1 BCP-17 > 200
BCP-17 22.5 ± 0.7 47.3 ± 0.8 2 RCP-9 > 200
RCP-9 12.5 ± 1.8 25.0 ± 1.5 2 RCP-29 8.9 ± 0.1
RCP-29 17.8 ± 1.5 23.8 ± 2.3 1 RCP-35 > 200
RCP-35 14.6 ± 0.7 50.1 ± 2.2 3 RCP-37 > 200
RCP-37 11.8 ± 1.5 47.7 ± 2.2 4 RCP-45 > 200
RCP-45 17.2 ± 2.7 51.2 ± 0.1 3 RCP-72 > 200
RCP-72 9.5 ± 1.2 25.6 ± 0.2 3 YCP-2 3.7 ± 0.7
YCP-2 13.3 ± 2.2 50.7 ± 1.5 4 YCP-18 9.1 ± 0.8
YCP-18 20.2 ± 0.3 31.7 ± 3.2 2 YCP-39 > 200
YCP-39 13.0 ± 0.5 31.8 ± 2.4 2 YCP-41 > 200
YCP-41 19.9 ± 2.2 52.1 ± 0.4 3 YCP-48 3.2 ± 0.1
YCP-48 15.4 ± 1.1 50.4 ± 0.4 3 YCP-50 > 200
YCP-50 20.7 ± 1.4 91.0 ± 0.6 4 YCP-60 > 200
YCP-60 26.4 ± 0.4 66.0 ± 4.1 3 Metronidazol 0.4  0.04
Pentamidine 1.3 ± 0.1 11.7 ± 1.7 9
†: IC50: Concentration of drug that caused 50 % of inhibition growth.
†: IC50: Concentration of drug that caused 50 % of inhibition growth. ‡: SD:
‡: SD: Standard deviation.
Standard deviation. §: CC50: Concentration of drug that caused 50 % of mortality.ƒ:
SI: Selectivity index = CC50 / IC50
contained also other prenylated benzophenone derivatives
inhibitory activity against parasites mentioned above. such as garcinielliptone I, hyperibone B and propolone B,
These observations correlate well with the in vitro studies C and D [8a]. RCP is composed by isoliquiritigenin,
obtained herein due to these flavonoids are among the liquiritigenin, biocanin A, formononetin, vestitol,
major components of RCP. neovestitol, isosativan, medicarpin, homopterocarpin and
vesticarpan, mainly [8b]; while YCP samples are rich
Previously, Ayres et al. reported the activity of Brazilian in triterpenoids including lanosterol, - and -amyrin,
propolis on L. amazonensis promastigotes and amastigote -amyrin acetate, -amyrone, germanicol and germanicol
form [3a]. In this sense, Machado et al. compared the acetate, lupeol and lupeol acetate, cycloartenol, lanosterol
antileishmanial activity of Brazilian and Bulgarian acetate and 24-methylene-9,19-cyclolanostan-3β-ol [8c].
propolis against four different species of Leishmania (L.
amazonensis, L. braziliensis and L. chagasi from New Although Cuban propolis has been characterized and
World and L. major from Old World). They also observed grouped into three different types according its main
significant differences in the leishmanicidal activities with chemical components, we could not correlate its
IC50 values that ranged from 2.8 to 229.3 µg/mL [3b]. antiparasitic activity against both protozoa with the
Duran et al., demonstrated that propolis samples from chemical composition. Promissory results were found in
Turkey also reduced the proliferation of L. tropica some samples of each type of propolis, but this activity has
promastigotes [3c]. It is very interesting to note that all not been shown by all samples belonging to a same group.
these results were obtained employing propolis samples This apparent contradiction can indicate that the
from different geographical origins containing very antiparasitic activity can be associated to compounds that
dissimilar chemical constituents. are present in a minor percentage in the sample or due to
interaction of different compounds that act as synergistic
On the other hand, T. vaginalis is a flagellated protist that and enhance the effects or interact as antagonist, which
causes trichomoniasis, a common but overlooked sexually provoke the lost of activity in some samples of the same
transmitted human infection, with approximately 170 type. This unresolved situation is very common in products
million cases occurring annually worldwide [7]. Only five that exist in the nature as complex mixture of substances,
propolis extracts showed activity against T. vaginalis; which can interact and change the expected
while thirteen were inactive (Table 2). A high contrast was pharmacological activity. Further experiments with the
observed since only some samples belonging to each compounds obtained from all types of propolis can be
propolis type caused an important inhibition growth of developed in order to elucidate the responsible of the high
trophozoites. In this sense, YCP samples were the most antiparasitic activity showed in some cases.
active, which should be corroborate using in vivo models
of infection. A previous report demonstrated that propolis In conclusion, some Cuban propolis extracts exhibited
possesses in vitro anti-trichomonas activity [4a]. activity against both intracellular and extracellular
protozoa model. Our results corroborated the high
In both protozoa, the reference drug caused better activity, potentiality of propolis as a natural source of new
which is logical since the pentamidine and metronidazol antiprotozoal agents with a wide spectrum of activity. This
are pure compounds and propolis extracts are complex study also confirms that both the chemical composition of
mixture of substances. Chemical studies revealed that propolis and its biological potentiality should be evaluated
BCP has shown nemorosone as the main component and during the quality control process of this natural product.
Activity of propolis on Leishmania and Trichomonas Natural Product Communications Vol. 6 (7) 2011 975

Experimental Activity of propolis samples on L. amazonensis

Propolis samples: Eighteen samples of Cuban propolis amastigotes: Peritoneal macrophages were harvested from
were provided by “La Estación Experimental Apícola”, normal BALB/c mice in RPMI medium (Sigma) and
Havana, Cuba, between October 2003 and December plated at 106/mL in Lab-Tek 16 chamber slides (Costar,
2004. Samples were collected in nine provinces of Cuba Naperville, US) and incubated at 37ºC and 5% CO2 for 2
including Eastern, Central and Western regions (Table 3). hours. Non-adherent cells were removed and stationary-
Propolis samples were extracted by maceration with phase L. amazonensis promastigotes were added at a 4:1
methanol (10 mL, 3 times) for 1 hour at room temperature parasite/macrophage ratio. The cultures were incubated for
employing agitation. Extracts were filtered on paper filters 4 hours and washed to remove free parasites. Propolis was
and the solvent was evaporated at 40ºC under reduced added at a concentration ranging from 100 to 12.5 g/mL
pressure to obtain dry extracts. Each sample was for 48 hours. The cultures were then fixed with absolute
characterized and classified using a combination of NMR, methanol, stained with Giemsa, and examined under light
HPLC-PDA and HPLC-ESI/MS techniques as previously microscopy [9b]. The number of intracellular amastigotes
was described [1b]. Cuban propolis samples used in this was determined by counting the amastigotes residents on
study, their origin and classification are reported in table 3. 100 macrophage per each sample, and the results were
The extracts were dissolved in dimethylsulphoxide expressed as percent of reduction of the infection rate
(DMSO, BDH, Poole, England) at 20 mg/mL and stored at (%IR) in comparison to that of the controls [%IR = 100 –
4ºC. (infection rate of the treated culture/infection rate of the
untreated culture x 100)]. The infection rates were
Table 3: Cuban propolis samples used in this study, classification and origin. obtained by multiplying the percentage of infected
Samples Province (Municipality) Samples Province (Municipality) macrophages by the number of amastigotes per infected
macrophages [9c].
BCP-1 Ciudad Habana (Jardín RCP-39 Pinar del Río
Botánico) (Candelaria)
BCP-4 Granma (Buey Arriba) RCP-41 Pinar del Río (Bahía Cytotoxicity of propolis samples on macrophage:
Honda)
BCP-5 Guantánamo (Imías) RCP-48 Matanzas (Unión de Peritoneal macrophages were collected from normal
Reyes) BALB/c mice in RPMI medium supplemented with
BCP-16 Las Tunas (Puerto Padre) RCP-50 Matanzas (Unión de
Reyes)
antibiotics, and seeded at 30000 cell/well. The cells were
BCP-17 Guantánamo (Salvador) RCP-60 Holguín (Báguanos) incubated for 2 hours at 37ºC in 5% CO2. Non-adherent
RCP-9 Pinar del Río (Cabo de YCP-2 Ciudad Habana (Jardín cells were removed and dilutions of propolis in 1 L
San Antonio) Botánico) DMSO were added to 200 L medium at 10% HFBS and
RCP-29 Villa Clara (Manicaragua) YCP-18 Ciudad Habana (Jardín
Botánico) antibiotics. The macrophages were treated by eight
RCP-35 Pinar del Río (La YCP-39 Pinar del Río concentrations of the product ranging from 200 to 1.7
Coloma) (Candelaria)
RCP-37 Pinar del Río (Güanes) YCP-41 Pinar del Río (Bahía g/mL for 48 hours. The viability was determined using
Honda) the colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-
RCP-45 Matanzas (Jagüey YCP-48 Matanzas (Unión de
Grande) Reyes)
2,5-diphenyltetrazolium bromide (MTT) (SIGMA, St.
RCP-72 Ciego de Ávila YCP-50 Matanzas (Unión de Louis, MO, USA). MTT solutions were prepared at
Reyes) 5 mg/mL, filtered and sterilized at the moment of use,
RCP-2 Ciudad Habana (Jardín YCP-60 Holguín (Báguanos)
Botánico) 15 L was added to each well. After incubation for 4 hours
RCP-18 Ciudad Habana (Jardín the formazan crystals were dissolved by addition of 100
Botánico)
L DMSO. The optical density was determined using an
EMS Reader MF Version 2.4-0, at a test wavelength of
Parasites culture: The strain of L. amazonensis
560 nm and a reference wavelength of 630 nm [9d].
(MHOM/77BR/LTB0016) was kindly provided by the
Department of Immunology, Oswaldo Cruz Foundation,
Activity of propolis samples on T. vaginalis trophozoites:
Brazil. The parasites were routinely isolated from mouse
The assay was performed in 96 well microtiter plates.
lesions and maintained as promastigotes at 26ºC in
First, 99 µL of TYI-S-33 medium were added, followed of
Schneider’s medium (Sigma Chem Co, St. Louis, Mo,
1µL of the dilution of the propolis in DMSO. The assayed
US), containing 10% heat-inactivated foetal bovine
concentrations of the propolis varied from 200 until 1.6
serum (Sigma), 200 U penicillin/mL and 200 g µg/mL. Finally 100 µL of the parasites suspension (25 x
streptomycin/mL (Sigma). The parasites were not used 104 trichomonas/mL) were seeded. Plates were incubated
after the fifth passage. For examining the effect of these
during 46 hours, in a candle jar at 37C. Each product
propolis samples on T. vaginalis, the isolate C173,
concentration and controls were tested in quadruplicate
axenized from a symptomatic woman suffering of
and the experiment was repeated three times. To determine
trichomoniasis, was used [9a]. Parasites were cultured in
the viability of the parasites the medium was aspirated and
TYI-S-33 supplemented with heat-inactivated bovine
replace by one lacking ascorbic acid and L-cysteine,
serum at final concentration of 10%, under anaerobic
followed of the addition of 20 µL of MTT to each well.
conditions, at 37C. MTT was prepared as explained above. After a period of
incubation of 2 hours, the plates were centrifuged (5 min,
976 Natural Product Communications Vol. 6 (7) 2011 Monzote et al.

800 x g), and the supernatant medium was aspirated from Statistical analysis: The 50 % inhibition concentration
the wells, as completely as possible, without disturbing the (IC50) value was determined from the lineal concentration-
formazan crystals or the cells on the plastic surface. response curves in each parasite evaluation. The 50 %
DMSO (100 µL) was added to each well to dissolve the cytotoxic concentration (CC50) was obtained from dose-
formazan crystals, the plates were shaked for 1 min and response curves fit to data by means of the equation for the
immediately the optical density was determined at 560 nm, sigmoidal Emax model [9e]. Selectivity indices (SI) were
using 630 nm as the reference wavelength. then calculated through of division the CC50 for host cells
by the IC50 for L. amazonensis [9f].
References
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3-15; (b) Cuesta-Rubio O, Piccinelli AL, Fernandez MC, Hernández IM, Rosado A, Rastrelli L. (2007) Chemical characterization
of Cuban propolis by HPLC-PDA, HPLC-MS, and NMR: the brown, red, and yellow Cuban varieties of propolis. Journal of
Agricultural and Food Chemistry, 55, 7502-7509.
[2] (a) De Castro SL. Propolis: biological and pharmacological activities. Therapeutic uses of this bee-product. (2001) Annal Review of
Biomedical Science, 3, 49-83; (b) Duran N, Koc A, Oksuz H, Tamer C, Akaydin Y, Kozlu T, Celik M. (2006) The protective role
of topical propolis on experimental keratitis via nitric oxide levels in rabbits. Molecular Cellular Biochemistry, 281, 153-61; (c)
Gekker G, Hu S, Spivak M, Lokensgard JR, Peterson PK. (2005) Anti-HIV-1 activity of propolis in CD4(+) lymphocyte and
microglial cell cultures. Journal of Ethnopharmacology, 102, 158-63; (d) Sforcin JM, Fernandez A, Lopes CA, Funari SR, Bankova
V. (2001) Seasonal effect of Brazilian propolis on Candida albicans and Candida tropicalis. Journal of Venomous Animal Toxins,
7, 139-144; (e) Dantas AP, Salomão K, Barbosa HS, De Castro SL. (2006) The effect of Bulgarian propolis against Trypanosoma
cruzi and during its interaction with host cells. Memorias do Instituto Oswaldo Cruz, 101, 207-211; (f) Freitas FS, Shinohara L,
Sforcin JM, Guimara S. (2006) In vitro effects of propolis on Giardia duodenalis trophozoites. Phytomedicine, 13, 170–175.
[3] (a) Ayres DC, Marcucci MC, Giorgio S. (2007) Effects of Brazilian propolis on Leishmania amazonensis. Memorias do Instituto
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different species of Leishmania. Memorias do Instituto Oswaldo Cruz; 102, 73-77; (c) Duran G, Duran N, Culha G, Ozcan B, Oztas
H, Ozer B. (2008) In vitro antileishmanial activity of Adana propolis samples on Leishmania tropica: a preliminary study.
Parasitology Research, 102, 1217-1225.
[4] (a) Xu BH, Shi MZ. (2006) An in vitro test of propolis against Trichomonas vaginalis. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng
Chong Bing Za Zhi, 24, 477-8; (b) Ledón N, Casacó A, González R, Merino N, González A, Tolón Z. (1996) Efectos
antipsoriásico, antiinflamatorio y analgésico del propóleo rojo colectado en Cuba. Revista Cubana Farmacia, 30, 28-32; (c) Cuesta
O, Cuellar A, Rojas N, Aquino R, Velez H, Rastrelli L. (1999) A polyisoprenylated benzophenone from Cuban propolis. Journal
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(2009) Antiproliferative activity of Brown Cuban Propolis extract on Human Breast Cancer Cells. Natural Product
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[5] (a) Bailey MS, Lockwood DNJ. (2007) Cutaneous leishmaniasis. Clinics in Dermatology, 25, 203-211; (b) Desjeux P. (2001) The
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Yamada H. (2008) In vitro antimalarial activity of prenylated flavonoids from Erythrina fusca. Journal Naturat Medicine, 62, 217-
220; (b) Sartorelli P, Carvalho CS, Reimão JQ, Ferreira MJ, Tempone AG. (2009) Antiparasitic activity of biochanin A, an isolated
isoflavone from fruits of Cassia fistula (Leguminosae). Parasitology Research, 104, 311-314; (c) Khan IA, Avery MA, Burandt
CL, Goins DK, Mikell JR, Nash TE, Azadegan A, Walker LA. (2000) Antigiardial activity of isoflavones from Dalbergia
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vitro leishmanicidal constituents of Millettia pendula. Chemical & Pharmaceutical Bulletin, 54, 915-917; (e) Kraft C, Jenett-Siems
K, Siems K, Gupta MP, Bienzle U, Eich E. (2000) Antiplasmodial activity of isoflavones from Andira inermis. Journal of
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de Simone F, Rastrelli L. (2005) Isoflavonoids isolated from Cuban propolis. Journal of Agricultural and Food Chemistry, 53,
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on the constituents of Yellow Cuban Propolis: GC-MS determination of triterpenoids and flavonoids. Journal of Agricultural and
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Selective effect of 2’,6’-dihydroxy-4’methoxychalcone isolated from Piper aduncum on Leishmania amazonensis. Antimicrobial
Agents and Chemotherapy, 43, 1234-1241; (c) Delorenzi JC, Attias M, Gattass CR, Andrade M, Rezende C, Da Cunha A,
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Antimicrobial Agents and Chemotherapy, 45, 1349-1354; (d) Sladowski D, Steer SJ, Clothier RH, Balls M. (1993) An improve
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parthenolide, a sesquiterpene lactone isolated from Tanacetum parthenium. Antimicrobial Agents and Chemotherapy, 49, 176-182.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Antioxidant Capacity and Phenolic Content of four 977 - 982
Myrtaceae Plants of the South of Brazil
Marcos José Salvador*a, Caroline C. de Lourençoa, Nathalia Luiza Andreazzaa,
Aislan C.R.F. Pascoala and Maria Élida Alves Stefanellob
a
Instituto de Biologia, Departamento de Biologia Vegetal, Curso de Farmácia,
Universidade Estadual de Campinas (UNICAMP), 13083-970, Campinas, SP, Brasil
b
Departamento de Química, Universidade Federal do Paraná (UFPR), 81531-990, Curitiba, PR, Brasil

marcosjs@unicamp.br

Received: December 11th, 2010; Accepted: March 26th, 2011

Antioxidant compounds can be useful to prevent several degenerative diseases or as preservative in food and toiletries. Species of the
Myrtaceae family are able to accumulate phenolic substances and those are closely related to the antioxidant activity due to their capacity to
scavenge free radicals, protect against lipid peroxidation and quench reactive oxygen species. These facts prompted us to investigate the
antioxidant capacity of the ethanolic extracts of the leaves of four Myrtaceae plants collected of the south of Brazil: Eugenia chlorophylla O.
Berg., Eugenia pyriformis Cambess, Myrcia laruotteana Cambess and Myrcia obtecta (Berg) Kiacrsk. The antioxidant potential was
performed using the DPPH (a single electron transfer reaction based assay) and ORAC (Oxygen Radical Absorbance Capacity, a hydrogen
atom transfer reaction based assay) assays. Moreover, the total soluble phenolic content was also measured using the Folin-Ciocalteu reagent.
A preliminary evaluation of the ethanolic extracts of these Myrtaceae plants revealed high levels of phenolic compounds (343.7-429.3 mg
GAE) as well as high antioxidant activity according to both methods (1338 a 3785 µmol of TE/g of extract in ORAC and SC50 in the range of
1.70 and 33.7 µg/mL in the DPPH). The highest antioxidant activity obtained by DPPH assay was exhibited by ethanol extract of the leaves
of E. pyriformis (1.70 g/mL), followed by extracts of M. laruotteana (3.38 g/mL) and M. obtecta (6.66 g/mL). In comparison
with controls, in the DPPH assay, the extract of E. pyriformis was more active than trolox (SC50 = 2.55 g/mL), while the extracts of
M. laruotteana and M. obtecta were more actives than quercetin (SC50 = 7.80 g/mL). In the ORAC assay, all species also show good
antioxidant capacity (1000 µmol of TE/g). Initial HPLC-UV/DAD and ESI-MS confirmed the presence of phenolic acids constituents in the
ethanol extracts. The results indicate the presence of compounds possessing promising antioxidant/free-radical scavenging activity in the
analyzed extracts of Myrcia and Eugenia plants of the south of Brazil.

Keywords: Myrtaceae, Myrcia, Eugenia, radical scavenger, DPPH, HPLC-UV/DAD, ESI-MS.

Antioxidant compounds can be useful to prevent several
degenerative diseases or as preservative in food and
toiletries products. Currently there is an increasing interest
in searching natural antioxidants to replace synthetic
compounds, which can be dangerous for human health.
Thus many plants have been screened for a possible source
of non-toxic and effective antioxidants [1-3]. Compounds
with antioxidant activity are generally phenolics and
the free radical-scavenging is a suitable method for
preliminary search of them in plants [4]. Myrtaceae is an
important family in Brazil, with more than 1000 species
over the country. Myrcia and Eugenia, with around 400
and 350 species, respectively, are the major genera [5].
Many species produce edible fruits and, some have an
essential ecological role in the conservation of Brazilian
biodiversity [6]. Screening for antioxidant activity of
Myrtaceae has been focused on its edible fruits [7-9], with
only one report on leaves [10]. The aim of this work was
evaluate the antioxidant capacity of leaves of Eugenia
chlorophylla O. Berg., Eugenia pyriformis Cambess,
Myrcia laruotteana and Myrcia obtecta (Berg) Kiacrsk.
The selected plants are trees, growing in Southern region
of Brazil [11]. Previous studies have reported the essential
oil composition of these plants (12-17], identification of
the flavonoids myricitrin, rutin and quercitrin in the leaves
of E. pyriformis [18] and the antimicrobial and antioxidant
activities of the fruits of E. pyriformis [7,19-20]. No
phytochemical reports were found on E. chlorophylla,
M. laruotteana and M obtecta.
In this study, the phytochemical screening showed the
presence of triterpenes/sterols, phenolic compounds,
tannins and saponins in all samples. Theses results are in
agreement with previous reports of flavonoids, triterpenes
and phenolic compounds in Eugenia and Myrcia [21-26].
The ethanol extracts showed a total phenol content in
the range of 343.7-429.3 mg GAE/ g extract (Table 1).
Phenolic compounds (including simple phenols, tannins
and flavonoids) are recognized as one of most important
class responsible for antioxidant capacity in plants [27].
978 Natural Product Communications Vol. 6 (7) 2011
All samples exhibited antioxidant activity concentration-
dependent in DPPH assays, with SC50 varying from 1.70 to
33.72 μg/mL. In DPPH assays the highest antioxidant
activity was exhibited by ethanol extract of the leaves of
E. pyriformis (1.70 μg/mL), followed by extracts of
M. laruotteana (3.38 μg/mL), M. obtecta (6.66 μg/mL) and
E. chlorophylla (33.72 μg/mL). In comparison with
controls, the extract of E. pyriformis was more active
than trolox (SC50 = 2.55 μg/mL), while the extracts of
M. laruotteana and M. obtecta were more actives than
quercetin (SC50 = 7.80 μg/mL) (Table 1). Moreover, in
ORAC-FL kinetic assay the extracts showed a good
antioxidant capacity with value between 1338.58 and
3785.70 µM of Trolox equivalent per gram of extract
(µM of TE/g). In accordance to literature data, samples
with values ≥1000.00 µM of TE/g can be considered
samples with good antioxidant capacity in this assay
[28,29]. In ORAC-FL assays, highest antioxidant activity
was exhibited by ethanol extract of the leaves of
M. laruotteana, followed by extracts of E. chlorophylla,
E. pyriformis and M. obtecta (Table 1).
Table 1: Total phenol content and antioxidant capacity by the DPPH and
ORAC assays of ethanol extracts of Myrtaceae plants.
Sample Phenol contenta (mg DPPH assay, SC50a ORAC assaya
Ethanol extract of GAE/g of extract (µg/mL)c
or fraction)b
Eugenia chlorophylla 429.3 (5.2) 33.72 (2.25)
Eugenia pyriformis 396.2 (4.5) 1.70 (1.19)
Myrcia laruotteana 401.4 (2.5) 3.38 (3.38)
Myrcia obtecta 343.7 (4.9) 6.66 (5.30)
Quercetin* - 7.80 (2.00)
Caffeic acid* - 10.80 (2.60)
Chlorogenic acid* - 12.15 (1.80)
Trolox* - 2.55 (1.40)
a
Mean value (%RSD, relative standard deviation) of triplicate assays.
b
Total phenolics data expressed as milligrams of gallic acid equivalents
per gram (mg of GAE/g).
c
DPPH assay data expressed as SC50 (concentration that inhibited 50% of
the DPPH radical) in micrograms per milliliters (µg/mL).
d
ORAC data expressed as micromol of Trolox equivalents per gram
(µmol of TE/g).
e
ORAC data expressed as relative Trolox equivalent, mean (%RSD,
relative standard deviation) of triplicate assays.
*Experimental positive controls.
-: not evaluated.
The differences in best antioxidant capacity of the species
studied among the two assays happens due to differences
of sensibility and on the basis of the chemical reactions
involved in each test: ORAC is a hydrogen atom transfer
reaction based assay (HAT) and DPPH is a single electron
transfer reaction based assay (ET). It is apparent that the
hydrogen atom transfer reaction is a key step in the radical
chain reaction. Therefore, the HAT based method is more
relevant to the radical chain-breaking antioxidant capacity.
Overall, there are a multitude of ET-based assays for
measuring the reducing capacity of antioxidants. The
assays are carried out at acidic (FRAP and DPPH), neutral
(TEAC and ORAC), or basic (total phenols assay by Folin-
Ciocalteau reagent, FCR assay) conditions. The pH values
have an important effect on the reducing capacity of
antioxidants. At acidic conditions, the reducing capacity
Salvador et al.
may be suppressed due to protonation on antioxidant
compounds, whereas in basic conditions, proton
dissociation of phenolic compounds would enhance a
sample’s reducing capacity [28,29].
Thus, the results documented in this study demonstrated
that all extracts analyzed showed antioxidant capacity
(measured by DPPH and ORAC assays) and this activity
present a positive correlation with the total phenolic
content (measured by FCR assay). Moreover the caffeic
and chlorogenic acids presented considerable antioxidant
(Table 1) capacity and were detected in some of the
extracts studied (Table 2).
Phenolic compounds has been presented as important
substances in combating free radical production mainly
due to its chemical structure and redox capacity, allowing
them to act as reducing agents, hydrogen donating,
neutralizing free radicals [30], chelating of transition
metals and inhibiting lipid peroxidation [31]. In biological
systems this capacity confers pharmacological properties
to this compounds that act preventively against diseases
related to oxidative stress.
The results for E. pyriformis corroborate previous work
(µmol of that reported the inhibition of xanthine oxidase, an enzyme
TE/g)d responsible by super-oxide anion production [32] and
2197.20 (1.12)
1456.50 (4.92) antioxidant activity of fruits [7,19].
3785.70 (2.67)
1338.58 (4.85) The ESI-MS fingerprints technique with direct infusion
5.62 (0.89)e
2.86 (2.02)e [33-35] was used to characterize the presence of
2.65 (1.50)e compounds with potent free-radical scavenging activity in
- this work. The extracts were analyzed by direct insertion
both in the negative and positive ion modes. However,
ESI(+)-MS fingerprints produce by far the most
characteristic mass spectra; hence only the ESI(-)-MS data
will be presented and discussed. This method in the
negative ion mode provides a sensitive and selective
method for the identification of polar organic compounds
with acidic sites, such as the phenolic organic acids.
Deprotonated forms of the compounds of interest were
then selected and dissociated and their ESI-MS/MS were
compared to those of standards.
Chlorogenic acids are esters of trans-cinnamic acids
(coumaric, caffeic, ferulic, and 3,4-dimethoxycinnamic)
with quinic acid. The trans-cinnamic acids can be
esterified at one or more of the hydroxyls at positions 1, 3,
4, and 5 of quinic acid, originating series of positional
isomers. In HPLC-UV/DAD and ESI-MS analysis the
Myrcia samples presented m/z 353 as base peaks (bp) in
negative ionization mode mass spectra and UV spectra
characteristics of caffeoylquinic derivatives (UV max:
≈298 and 325 nm) which, when taken together, suggest
positional isomers of a quinic acid (QA) esterified with a
single caffeoyl (CAF) unit. The product ion spectra
obtained by negative ion MS/MS for precursor ions m/z
353 were different from each other, and comparison with
Antioxidant capacity of four Myrtaceae plants Natural Product Communications Vol. 6 (7) 2011 979

Figure 1: ESI-MS fingerprints of ethanol extracts from the leaves of four Myrtaceae plants of the South of Brazil (a: Eugenia chlorophylla; b: Eugenia
pyriformis; c: Myrcia obtecta; d: Myrcia laruotteana).

Table 2: Compounds identified in ethanol extracts from the leaves of four Myrtaceae plants of the South of Brazil using ESI(-)-MS/MS.
ESI-MS ions (m/z)
Compound Myrtaceae plants Deprotonated ions [M-H]- m/z MS/MS ions m/z
ECE EPE MOE MLE
Caffeic acid - + + + 179 15 eV: 179→135
Quinic acid + + + + 191 25 eV: 191→173, 127, 111, 93, 85
Ferulic acid - + - - 195 15 eV: 193→178, 149, 134
Chlorogenic acid (5-O-(E)-caffeoylquinic acid) - - + + 353 15 eV: 353→191, 179, 173
+: detected; -: not detected. (ECE: Eugenia chlorophylla; EPE: Eugenia pyriformis; MOE: Myrcia obtecta; MLE: Myrcia laruotteana).

identification of peak as 5-O-(E)-caffeoylquinic acid (5-

Caffeic acid Ferrulic acid
Quinic acid Chlorogenic acid
Figure 2: Phenolic organic acids identified in antioxidant ethanol extracts
from the leaves of four Myrtaceae plants of the South of Brazil.
the caffeoylquinic acids (CQA) identification keys [36,37]
led to the individualization of three CQA positional
isomers. The product ion spectrum for m/z 353 showed m/z
191 (bp) and m/z 179 at 4% ri. The greater relative
intensity of m/z 179 in the product ion spectrum, led to the
CQA). Furthermore, the identity of this compound was
also confirmed through co-elution with a 5-CQA authentic
standard. In the ESI-MS fingerprints of the samples of
Myrtaceae extracts (Figure 1, Table 2) the following
components were identified in their deprotonated forms:
caffeic acid (m/z 179), quinic acid (m/z 191), ferulic acid
(m/z 193), and chlorogenic acid 5-CQA (m/z 353), figure
2. The content of phenolic compounds in the extracts,
possibly could explain the high antioxidant activity
verified for these extracts of Myrtaceae.
The investigation by direct infusion electrospray ionization
mass spectrometry (ESI-MS) provided important
information about bioactive components present in the
Myrtaceae extracts, that are widely reported as potent
antioxidants, probably explaining the antioxidant activity
of the studied extracts [35, 38-40].
980 Natural Product Communications Vol. 6 (7) 2011
Experimental
Plant Material: The leaves of Eugenia chlorophylla O.
Berg. (I), Eugenia pyriformis Cambess (II), Myrcia
laruotteana Cambess (III) and Myrcia obtecta (Berg)
Kiacrsk. (IV) were collected in Curitiba, Paraná State,
Brazil. Voucher specimens were deposited at the
herbarium of Universidade Federal do Paraná (UPCB
53304, 16741, 53303 and 60504, respectively).
Extracts preparation and chemical analysis: Dried and
powdered leaves of each plant (50 g) were extracted
with ethanol (3 X 300 mL) at room temperature. The
solvent was removed under reduced pressure to give the
crude extracts (E. chlorophylla 5.4%, E. pyriformis 5.7%,
M. laruottena 14.1% and M. obtecta 21.4%), which were
used in DPPH assays.
Phytochemical Analysis: Phytochemical tests for
sterols/triterpenes, phenolic compounds, tannins, saponins
and alkaloids were carried on according usual
methodology [41].
Quantitative determination of total soluble phenols:
The extracts, dissolved in methanol, were analyzed for
their total soluble phenolic content according to the Folin-
Ciocalteau colorimetric method [42-43], using gallic acid
as reference. The results were expressed as milligrams of
gallic acid equivalents (GAE) per gram of extract or
fraction (mg of GAE/g). The analyses were performed in
triplicate.
Radical scavenging activity using the DPPH method:
The antiradical activity of extracts was determined using
the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
[44]. The test was performed in 96-well microplates. Fifty
microliters of a 250 μM DPPH solution in MeOH was
added to a range of solutions of different concentrations
(seven serial 3-fold dilutions to give a final range of 100 to
1.6 μg mL-1) of extracts to be tested in MeOH (10μL).
Absorbance at 517 nm was determined 30 min after the
addition of each of the compounds tested, and the
percentage of activity was calculated. Quercetin and 6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) were used as positive controls. All samples were
tested in triplicate. The antioxidant activity of each sample
was expressed as the SC50 value, which is the
concentration in μg mL-1of each extract that scavenged
50% of the DPPH radicals. All of the results are expressed
as mean of three different trials.
Evaluation of antioxidant capacity by ORAC assay:
The antioxidant capacity of the ethanolic extract was
assessed through the oxygen radical absorbance capacity
(ORAC) assay. This assay measures antioxidant
scavenging activity against peroxyl radicals using
fluorescein as the fluorescent probe. ORAC assays were
carried out on a Synergy HT multi-detection microplate
reader system. The temperature of the incubator was set at
Salvador et al.
37°C. The procedure was carried out according to the
method established by Ou and co-workers [29] with
modifications [45]. The data were expressed as
micromoles of Trolox equivalents (TE) per gram of extract
on dry basis (μmol of TE/g) and as relative Trolox
equivalent for pure compounds. Quercetin and 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)
were used as positive controls. The analyses were
performed in triplicate.
HPLC analysis: HPLC analyses were conducted using a
RP-18 column (Lichrospher, 5 μm, 225\4.6 mm, Merck).
The mobile phase consisted of a linear gradient combining
solvent A (acetonitrile) and solvent B (water/acetic acid,
99:1, v/v, pH 2.88) as follows: 15% A (15 min), 15-40% A
(5 min), 40-60% A (5 min), 60-100% A (5 min), 100-15%
A (5 min), 15% A (5 min). The analyses were carried out
in triplicate at a flow rate of 0.8 mL/ min and an injection
volume of 20 μL. UV-DAD detector was set to record
between 200 and 600 nm, and the UV chromatograms
were measured at 254 and 330 nm. The samples were
analyzed at 1 mg/mL. The standard sample of quinic acid
and chlorogenic acid (5-O-(E) caffeoylquinic acid) were
also analyzed and then used for co-elution with authentic
standard.
Electrospray ionization mass spectrometry
fingerprinting: Crude extracts of Myrtaceae plants were
diluted in a solution containing 50% (v/v) chromatographic
grade methanol and 50% (v/v) deionized water and 0.5%
of ammonium hydroxide (Merck, Darmstadt, Germany). In
the fingerprinting ESI-MS analysis, the general conditions
were: source temperature of 100 oC, capillary voltage of
3.0 kV and cone voltage of 30 V. For measurements in the
negative ion mode, ESI(-)-MS, 10.0 L of concentrated
NH4OH were added to the sample mixture having a total
volume of 1000 L yielding 0.1% as final concentration.
For measurements in the positive ion mode ESI(+)-MS,
10.0 L of concentrated formic acid were added giving a
final concentration of 0.1%. ESI-MS was preformed by
direct infusion with a flow rate of 10 L min mL-1 using a
syringe pump (Harvard Apparatus). Structural analysis of
single ions in the mass spectra from extract was performed
by ESI-MS/MS. The ion with the m/z of interest was
selected and submitted to 15–45 eV collisions with argon
in the collision quadrupole. The collision gas pressure was
optimized to produce extensive fragmentation of the ion
under investigation. The compounds were identified by
comparison of their ESI-MS/MS fragmentation spectra
with literature data [33-35].
Statistical analysis: Data are reported as mean (%RSD,
relative standard deviation) of triplicate determinations.
The statistical analyses were carried out using the
Microsoft Excel 2002 software package (Microsoft Corp.,
Redmond, WA)
Antioxidant capacity of four Myrtaceae plants Natural Product Communications Vol. 6 (7) 2011 981

Acknowledgments - The authors are grateful to Dr. and to FAPESP, CNPq and FAEPEX-UNICAMP for
Armando C. Cervi, from Departamento de Botânica, financial support.
Universidade Federal do Paraná, for plant identification

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Cytotoxicity of Active Ingredients Extracted from Plants of 983 - 984
the Brazilian “Cerrado”
Veronica CG Soaresa, Cibele Bonacorsib, Alana LB Andrelad, Lígia V Bortolotie,
Stepheny C de Campose, Fábio HR Fagundesd,e, Márcio Piovanie, Camila A Cotrima,
Wagner Vilegasc and Marcos H Toyamaf
a
Institute of Biology, UNICAMP, Campinas, Sao Paulo, 13083-862. Brazil
b
Department of Microbiology, UNESP, Araraquara, Sao Paulo, 14801-902. Brazil
c
Institute of Chemistry,UNESP, Araraquara, Sao Paulo, 14801-970. Brazil
d
Department of Pharmacy, Unianchieta, Jundiai, Sao Paulo, 13207-270. Brazil
e
Department of Pharmacy, UNIP, Jundiai, Sao Paulo, 13214-525.Brazil
f
Institute of Biology, UNESP, Campus Litoral Paulista, Sao Vicente, Sao Paulo,11330-150. Brazil

vcgsoares@gmail.com

Received: December 10th, 2010; Accepted: March 16th, 2011

Cytotoxicity assays are needed for the screening of natural products with potential anti-inflammatory. The purpose of this study was to
compare the basal cytotoxicity of active ingredients extracted from plants of the Brazilian “cerrado”. The viability was assayed with the
neutral red uptake assay in Mac Coy cells after 24h of exposition. The dose evaluated was 50 µg/µL. The test substances were: cinnamic
acid, p-coumaric acid, chlorogenic acid, syringic acid, vannilic acid, homogentisic acid, scandenin, palustric acid, diosgenin, cabraleone.
Studies of cytotoxicity demonstrated that all active compounds evaluated have low toxicity in vitro. The substances showed cell viability
above 60% for the concentration used. However, the cinnamic acid, sacandenin and palustric acid showed highest toxicity with a 50%
reduction in cell viability for the dose of 50 µg/µL. Cytotoxic screening results are useful to estimate the best concentrations of those
compounds with potential anti-inflammatory without their cause cell death.

Keywords: Natural products, Cytotoxicity, Brazilian Cerrado.

The Cerrado is one of the world's threatened biodiversity Table 1: Viability was performed with the neutral red uptake assay in Mac
Coy cells after 24h of exposure to the test substances.
hotspots [1a]. About 60% of its vegetation has already
been removed [1b] and the remaining areas are isolated in Natural Products (50 µg/mL nm (±SD)* (%)
Control + 0.519 ± 0.010 100%
forest fragments [2a]. Due to the devastation many natural Cinnamic acid 0.155 ± 0,015 30%
compounds with potential biological activities were lost. Scandenin 0.207 ± 0.012 40%
Palustric acid 0.249 ± 0.016 48%
Screenings of natural compounds using cytotoxicity assays Homogentisic acid 0.259 ± 0.013 50%
Diosgenin 0.404 ± 0.014 78%
offer the advantage of evaluate several compounds p-coumaric acid 0.415 ± 0.013 30%
simultaneously. The comparative sensitivity of cells to Cabraleone 0.337 ± 0.012 65%
toxicity tests that evaluate direct-acting and indirect-acting Syringic acid 0.337 ± 0.015 65%
Vannilic acid 0.363 ± 0.016 70%
cytotoxicants may be important to explaining their mode Chlorogenic acid 0.415 ± 0.013 80%
of action [2b]. Natural products with low cytotoxicity * Values presented with standard deviation.
activities constitute an excellent alternative search for
complementary treatments for inflammatory disease. The compounds showed cell viability above 60% for the
concentration used. However, the cinnamic acid, scandenin
The values obtained from the cytotoxicity test performed and palustric acid showed highest toxicity with a 50%
on cells MacCoy can be used as parameters for doses used reduction in cell viability for the dose of 50 µg.
in trials of anti-inflammatory activity. The purpose of this
study was to compare the basal cytotoxicity of active The natural products obtained from the Brazilian cerrado
compounds extracted from plants of the Brazilian show large potential for pharmaceutical product
“cerrado” and find those whose toxicity is low to further development. Some researches have shown the use of
anti-inflammatory assays. Due to this purpose dose of these compounds as an alternative for the treatment of
50 µg was used. The percentage of cell viability was cancer [3]. In addition, the potential of these products
established for each of the compounds evaluated and the mighty be exploited to treat other inflammatory disorders
results are presented in Table 1. such as physiological dysfunctions. Several xanthones,
984 Natural Product Communications Vol. 6 (7) 2011 Soares et al.

coumarins, terpenoids and phenolic compounds were
isolated from plants Brazilian cerrado and with remarkable
activities including antiHIV [4], antibacterial [5],
trypanocidal [6], and anticancer against the cell lines
KM12 (colon adenocarcinoma), U251 (glioma), PC3
(prostate), and K562 and HL60 (leukemia) [7-9a]. Our
results agree with those previously reported. According to
the results, all the products have shown low cytotoxicity.
The differences between the chemical structures of the
substances evaluated, was the determining factor for
difference of cytotoxicity. Complex structures (diosgenin,
cabraleone) showed less toxic, possibly because these
compounds are not able to go into de cells. Structures
with coumarin ring were the most toxic (cinnamic acid,
p-coumaric acid) Glycoside radical (acid chlorogenic)
usually shows low toxicity due to the presence of
hydroxyls that make difficult the transport of the
compounds to into the cells. Natural compounds with low
toxicity and potential as anti-inflammatory drugs have
been a source of research, once the search for new drugs
with fewer side effects has increased significantly.
Culture Section of the Adolfo Lutz Institute, São Paulo,
Brazil) was maintained in Eagle medium with 7.5% fetal
bovine serum at 37ºC.
Cytotoxicity assay: After trypsinization, 0.2 mL aliquots
of Eagle, containing approximately 104-105 cells/mL were
transferred to 96-well microtiter tissue-culture plates and
incubated at 37°C. After 24 hr, the medium was removed
and the cells were covered in unmodified medium
(control) or in medium modified with various
concentrations of the test compound. After incubating for
another 24 hr, the medium was removed and the plates
were prepared for the neutral red (NR) assay [9b]. After
brief agitation, the plates were transferred to a microplate
reader (Spectra (Shell) & Rainbow (Shell) Reader, Tecan
Austria GMBH) and the optical density of each well at
620 nm was measured, using a 540 nm filter. All
experiments were performed at least four times, using
three wells for each concentration of chemical tested. The
cytotoxicity data was standardized by determining
absorbance and calculating the corresponding chemical
concentrations [9c].

Experimental Compounds: The test substances were obtained after

Chemical and culture media: Neutral red was purchased
from Sigma (St. Louis, MO, USA). Fetal bovine serum
(FBS) was obtained from Cultilab (Campinas, SP, Brazil).
Dulbecco´s Modified Eagle Medium (DMEM) was
obtained from Instituto Adolfo Lutz (São Paulo, SP
Brazil).
Cell Line: Mac Coy mouse fibroblast cell line (CCL1;
American Type Culture Collection, USA, from Cell
maceration of different plants species from Brazilian
cerrado, resulted in hexane, dichloromethane, ethanol and
hydro-ethanol extracts, depending on extraction procedures.
The substances, cinnamic acid, p-coumaric acid, chloro-
genic acid, syringic acid, vannilic acid, homogentisic acid,
scandenin, palustric acid, diosgenin, cabraleone were
obtained using methods described by dos Santos [9d].
Acknowledgments - Financial Support: FAPESP, CNPq.

References
[1] (a) Myers N, Mittermeier RA, Mittermeier CG, Fonseca GAB, Kent J. (2000) Biodiversity hotspots for conservation priorities.
Nature, 403, 853-858; (b) Machado RB, Ramos Neto MB, Pereira PGP, Caldas EF, Gonçalves DA, Santos NS, Tabor K, Steininger
M. (2004) Estimativas de perda da área do Cerrado brasileiro. Brasília: Conservação Internacional, 1-6.
[2] (a) Durigan G, Siqueira MF, Franco GADC. (2007) Threats to the Cerrado remnants of the State of São Paulo, Brazil. Scientia
Agricola, 64, 355-363; (b) Soares VCG, Varanda EA, Raddi MSG. (2006) In vitro basal and metabolism-mediated cytotoxicity of
flavonoids. Food and Chemical Toxicology, 44, 835-838.
[3] Mesquita ML, de Paula JE, Pessoa C, de Moraes MO, Costa Lotufo LV, Grougne R, Michel S. (2009) Cytotoxic activity of
Brazilian Cerrado plants used in traditional medicine against cancer cell lines. Journal of Ethnopharmacology, 123, 439-445.
[4] Huerta-Reyes M, Basualdo MdelC, Abe F, Jimenez-Estrada M, Soler C, Reyes-Chilpa R. (2004) HIV1 inhibitory compounds from
Calophyllum brasiliense leaves. Biological & Pharmaceutical Bulletin, 27, 1471–1475.
[5] Pretto JB, Cechinel-Filho V, Noldin VF, Sartori MRK, Isaias DEB, Cruz AB. (2004) Antimicrobial activity of fractions and
compounds from Calophyllum brasiliense (Clusiaceae/Guttiferae). A Journal of Biosciences Zeitschrift fur Naturforschung C, 59,
657–662.
[6] Abe F, Nagafuji S, Okawa M, Kinjo J, Akahane H, Ogura T, Martinez-Alfaro MA, Reyes-Chilpa R. (2005) Trypanocidal
constituents in plants 5. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in the seeds of
Persea americana. Biological & Pharmaceutical Bulletin, 28, 1314–1317.
[7] Reyes-Chilpa R, Estrada-Muniz E, Ramirez Apan T, Amekraz B, Aumelas A, Jankowski CK, Vazquez Torres M. (2004) Cytotoxic
effects of mammea type coumarins from Calophyllum brasiliense. Life Sciences, 75, 1635–1647.
[8] Ito C, Murata T, Itoigawa M, Nakao K, Kaneda N, Furukawa H. (2006) Apoptosis inducing activity of 4 substituted coumarins
from Calophyllum brasiliense in human leukaemia HL60 cells. Journal of Pharmacy and Pharmacology, 58, 975–980.
[9] (a) Suffredini IB, Paciência MLB, Varella AD, Younes RN. (2007) In vitro cytotoxic activity of Brazilian plant extracts against
human lung, colon and CNS solid cancers and leukemia. Fitoterapia, 78, 223–226; (b) Borenfreund E, Puerner JA. (1985) Toxicity
determined in vitro by morphological alterations and neutral red absorption. Toxicology Letters, 24, 119-124; (c) Barile FA. (1994)
In vitro cytotoxicology. New York: CRC Press, 96p; (d) dos Santos LC, da Silva MA, Rodrigues CM, Carbone V, Napolitano A,
Bassarello C, Mari A, Piacente S, Pizza C, Vilegas W. (2009) Characterization of flavonoid and naphthopyranone derivatives from
Eriocaulon ligulatum using liquid chromatography tandem mass spectrometry. Natural Product Communications, 4, 1651-1656.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Propagation and Conservation of Native Forest Genetic 985 - 988

Resources of Medicinal Use by Means of in vitro and

ex vitro Techniques
Sandra Sharry, Marina Adema, María A. Basiglio Cordal, Blanca Villarreal, Noelia Nikoloff,
Valentina Briones and Walter Abedini

Centro Experimental de Propagación Vegetativa (C.E.ProVe)Facultad de Ciencias Agrarias y

Forestales. Universidad Nacional de La Plata. CICPBA. Diagonal 113 N° 469 (1900) La Plata,
Buenos Aires, Argentina

ssharry@gmail.com

Received: December 13th, 2010; Accepted: March 10th, 2011

In Argentina, there are numerous native species which are an important source of natural products and which are traditionally used in
medicinal applications. Some of these species are going through an intense extraction process in their natural habitat which may affect their
genetic diversity. The aim of this study was to establish vegetative propagation systems for three native forestal species of medicinal interest.
This will allow the rapid obtainment of plants to preserve the germplasm. This study included the following species which are widely used in
folk medicine and its applications: Erythrina crista-galli or “seibo” (astringent, used for its cicatrizant properties and for bronchiolitic
problems); Acacia caven or “espinillo” (antirheumatic, digestive, diuretic and with cicatrizant properties) and Salix humboldtiana or “sauce
criollo” (antipyretic, sedative, antispasmodic, astringent). The methodology included the micropropagation of seibo, macro and
micropropagation of Salix humboldtiana and the somatic embryogenesis of Acacia caven. The protocol for seibo regeneration was adjusted
from nodal sections of seedlings which were obtained from seeds germinated in vitro. The macropropagation through rooted cuttings of
“sauce criollo” was achieved and complete plants of this same species were obtained through both direct and indirect organogenesis using in
vitro cultures. The somatic embryogenesis for Acacia caven was optimized and this led to obtain a high percentage of embryos in different
stages of development. We are able to support the conservation of native forest resources of medicinal use by means of vegetative
propagation techniques.

Keywords: Micropropagation, native forest species, macropropagation, somatic embryogenesis.

Medicinal plants have been used since ancient times as
new therapeutic agents and their uses have been
transmitted from generation to generation, either in oral or
written forms, up to the present, and this is known as the
“traditional therapeutic practice”, the use of extracts or
active principles of plants, which has been essential to take
care of people’s health in the first level of attention.
The developed countries as well as the developing ones
have increased the use of medicinal plants or their products
[1]. Medicinal plants have played a vital role in societies
including Argentina for centuries. Most of these plants are
wild plants which were available in some forest
ecosystems. With the intensity of development and
clearing of land many of these wild plants used for
medicinal purposes are no longer available in their natural
habitat. Now we can address this environmental situation
by ex situ conservation and propagation of medicinal
plants for its sustainable utilization using new and
traditional techniques.
In Buenos Aires province (Argentina), there are different
native forest species of medicinal interest and some of
them are Salix humboldtiana or “sauce criollo”, Erythrina
crista-galli or “seibo” and Acacia caven or “espinillo”.
Salix humboldtiana is distributed in river banks or islands,
sometimes in sandy places, too. The different parts of the
plant contain salicin. The bark is used in folk medicine as a
quinine substitute (it contains glucosides). The decoction
of this bark is used “against intermittent fever” (rubber).
The bark is bitter and has febrifugal, tonic, sedative and
spasmodic properties. It is also astringent [2]. Erythrina
crista-galli has several important pharmacological uses. It
is an astringent and a sedative to heal wounds (3% of bark
decoction). It is antihemorrhoidal and used for vaginal
lavage in candidiasis cases (bark). It is disinfectant and
deodorant, it has cicatrizant properties and it is
ahemostatic, emollient for colds, coughs, catarrh,
bronchitis and asthmatic pains (the leaves are smoked in a
pipe or rolled up like a cigar). It has narcotic, sedative and
hypnotic properties: this is attributed to the most inner part
986 Natural Product Communications Vol. 6 (7) 2011 Sharry et al.

of the bark when it is used in an infusion (it contains

several alkaloids). This plant is also used for muscular and
rheumatic pains (a balm prepared with its bark and flowers
in 70% of alcohol). The leaves are used as
antihemorrhoidal for external use and they are antiseptic
and astringent [2]. Acacia caven or “espinillo” is a
medicinal plant that lives in South America and it is not
tropical. It grows in the arid highlands in the centre and
north of the country as well as on the islands in the Paraná Figure 1 a-b: Macropropagation of Salix humboldtiana. Plants
River, in very humid areas. The parts of the plant which obtained by rooting of cuttings.
are used for medicinal purposes are its leaves, stems and
seeds. The leaves of this medicinal plant have cicatrizant
properties, and the seeds are used as a digestive. The
leaves and stems have antiphlogistic and sedative
properties (http//www.tusplantasmedicinales.com/).

Vegetative reproduction keeps the parental genotype and

its characteristics are preserved in its off springs. Thus, the
genotypes of selected trees which are propagated
vegetatively reproduce identically and form clones. Grafts,
cuttings and layering are the traditional methods of
vegetative propagation [3].

In the last few years the use of in vitro culture techniques

in trees has facilitated the clonation of select phenotypes,
the preservation and the manipulation of vegetal material.
These techniques allow the multiplication of clones in a
short period of time, in any season of the year and in a
limited space [4]. By using these techniques, the long time
it takes a plant to reach maturity, the low viability of seeds,
and the difficulties some species have to propagate by
traditional methods can be reduced [5]. Besides, hundreds
of clones from the same species can be reproduced in vitro.
Later, they are taken to a plant nursery and then, they are
cultivated in fields where they will develop and finally
become a product with a specific economic interest
(http://www.biologia.org/).
The aim of this study was to establish vegetative
propagation systems for three native trees of medicinal
interest: Salix humboldtiana (sauce criollo), Erythrina
crista-galli (seibo) and Acacia caven (espinillo). This will
help to obtain plants to conserve germplasm in a rapid way.
Macropropagation of Salix humboldtiana: The substrate
with the mixture of soil, perlite and vermiculite (6:3, 5:0, 5)
was adequate to place the Salix humboldtiana cuttings. The
cuttings rooted 15 days after they were placed in water.
90% of the cuttings which were treated with IBA rooted,
and 75% of them originated complete plants (Figure 1).
In vitro culture of Salix humboldtiana: The culture media
used for callus, shoot and root induction were adequate.
Callus with de novo shoots (indirect organogenesis) were
obtained approximately 35 days after the leaves were
cultured. The shoots formed roots 25 days later. In this
way, vitroplants were obtained and they became
acclimatized successfully.
Figure 2a-f: In vitro culture of Salix humboldtiana. a. In vitro seedlings
of Salix humboldtiana obtained from immature embryos. b. Calli obtained
from the seedling leaves. c. Shoots formed from calli. d. Shoots from
nodal sections. e. Induction of roots from microcuttings. f. Whole plants
under greenhouse conditions (acclimatization and hardening).
The percentage of contamination of the nodal sections
which grew from fully grown plants was of 80-90% when
they were treated with 50% of sodium hypochlorite during
25 minutes and of 60% when they were treated with 50%
of sodium hypochlorite during 45 minutes. The WPM
culture medium without growth regulators was the best
one to obtain preformed shoots and whole plantlets. The
first shoots were observed approximately 20 days later,
and the roots, 25-30 days after they were cultured in vitro.
The culture media with BAP promote the formation of
callus on the base of the microcuttings. This callus turned
out to be organogenic and the production of shoots was
induced by using cytokinins (Figure 2).
Micropropagation of Erythrina crista-galli : Growth of
the preformed shoots was induced in MS with 1 mg.L-1
of BAP and 0.5 mg.L-1 of NAA. These shoots were
subcultured in a WPM medium with 0.1 mg.L-1 of IBA
where they elongated. Whole plants were obtained in a
WPM rooting medium with 0.1 mg.L-1 of NAA. These
plants were acclimatized under controlled light and
temperature conditions (16 hours of light and 8 hours of
darkness, T 21°C +/-2°C) (Figure 3).
Somatic embryogenesis of Acacia caven: Direct
and indirect somatic embryogenesis were obtained on the
Vegetative propagation systems for three medicinal plants
Figure 3a-d: Micropropagation of Erythrina crista-galli. a. In vitro seedlings. b. Shoot elongation from stem section. c. Induction of roots. d.
Acclimatization of whole plants.
Figure 4a-e: Somatic embryogenesis of Acacia caven.a. Mature fruits of Acacia caven. b y c Seeds in chemical scarification. d. Somatic embryos in a
globular and heart stage formed fron cotyledons. e. Embryos in torpedo and cotiledonar stage.
culture medium and the PGR concentration (1 mg.L-1 of
2,4 D and 0.1 mg/L-1 of BAP) used. The somatic embryos
occur directly over the cotyledons, on their adaxial side, or
indirectly from the formation of the callus after 6 months
of culture. Somatic embryos were observed in different
stages of development, and this showed there is no
synchronicity in their maturation (Figure 4). The somatic
embryos in a globular stage were subcultured on MS
medium free from PGR, and they germinated there. 30%
of them were converted into complete plants.
Conclusion: When the vegetative propagation techniques
of the species mentioned before are adjusted, they
contribute to the preservation of the forest genetic
resources. Excellent opportunities for scientific research
are generated by installing germplasm banks with a wide
range of genetic material. This becomes very important if
we consider that these species have a great importance in
the medicinal use and among others.
These studies pretend to be preliminary stages to achieve
the production of native plants of medicinal interest,
without the degradation of the genetic base of this
resource, which is vitally important for the preservation of
the forest resources.
Experimental
This study has been developed at the Centro Experimental
de Propagación Vegetativa (C.E.Pro.Ve.) in the Facultad
de Ciencias Agrarias y Forestales at Universidad Nacional
de La Plata, Buenos Aires, Argentina. In order to reach the
objectives, we used vegetative propagation strategies for
each species.
Natural Product Communications Vol. 6 (7) 2011 987
For Salix humboldtiana or “sauce criollo”, we used micro
and macropropagation systems. Macropropagation was
done through the rooting of cuttings with the exogenous
application of growth regulators and the optimization of
their nutritional requirements. The micropropagation was
done through the use of in vitro plant tissue culture
techniques, following the direct and indirect organogenic
pathways.
For Erythrina crista-galli or “seibo” and Acacia caven or
“espinillo” we used micropropagation following the
pathway of organogenesis and somatic embryogenesis.
Mother plants which were in good sanitary conditions,
optimal growth, and adaptability to the particularities of
the local site were chosen as a donating source for the
different explants. The methodology included:
Macropropagation of Salix humboldtiana: Between 100
and 150 plant stem cuttings with single node each were
cut of approximately 30 cm long and from 0.8 to 1.5 cm
in diameter. They were treated superficially with 1000 mg.
L-1 of Bennomyl fungicide for 3 hours in order to avoid
the presence of fungi. After that, the bases of the cuttings
were dippedin 50 mg. L-1 of indole-3-butyric acid (IBA)
for 24 hours to induce rooting. The control was not treated
with IBA. Then, they were placed in running water for 15
days and they were planted in plastic flowerpots N° 14
(1500 cm3) with a mixture of soil, perlite and vermiculite
(6:3.5:0.5) as substrate [6]. The pots were arranged in
randomized block design and replicated three times.
In vitro culture of Salix humboldtiana: Explants were
nodal sections with internodes (microcuttings) from adult
plants and leaves from in vitro seedlings obtained from
988 Natural Product Communications Vol. 6 (7) 2011 Sharry et al.

immature embryos. The embryos were surface sterilized Micropropagation of Erythrina crista-galli: In order to
with 10% of commercial sodium hypochlorite (55% active induce the shoot proliferation nodal sections from in vitro
chlorine) for 5 minutes in order to place them in vitro, and germinated seedlings were used as source of explants.
later obtain the seedlings. The leaves from the seedlings Different culture media with different growth regulators
were placed on Murashige & Skoog (MS) [7] basal were tested. The explants were cultured in MS medium in
medium at full strength supplemented with 1 mg.L-1 of a complete, half, and a quarter concentrations, with the
benzyl amino purine (BAP) and 0.5 mg.L-1 of naftalen addition of different growth regulators: 1 and 2 mg.L-1 of
acetic acid (NAA), 2% sucrose and 7.5 g. L-1 agar. Calli BAP, 0.5 mg.L-1 of NAA, 1mg.L-1 of IBA, 2% sucrose and
were subcultivated on MS medium at full strength with 7.5 g.L-1 of agar, alone or combined. Cultures were
1mg/L-1 of BAP and 1 mg.L-1 of NAA. The nodal sections incubated at 21°C +/-2°C with a 16 hours photoperiod.
or microcuttings of adult “sauce” plant were washed with
running water for 5 minutes, and they were surface Somatic embryogenesis of Acacia caven: In this case,
disinfected with 2000 mg/ L-1 of Bennomyl fungicide for 3 cotyledons from mature seeds were used as explants.
hours and 50% of commercial sodium hypochlorite (55% These seeds were treated with 98% of sulfuric acid in
active chlorine) for 25 and 45 minutes. For shoot order to scarification for two hours and they were washed
induction, the following culture media were tested: Woody with running water for 10 minutes. Then, they were
Plant Medium (WPM) without growth regulators and disinfected with 70% of ethanol during 5 minutes and 20%
WPM supplemented with 0.1; 0.5; 1; 1.5; and 2 mg.L-1 of of sodium hypochlorite (55% active chlorine) for 30
the 6- benzilaminopurine (BAP). minutes. After that, they were washed 3 times with
distilled water under a laminar flow hood and they were
The cultures were maintained in the culture room under a put in sterile water during 7 days in order to soften the
regime of 16 h photoperiod (intensity - 40 µEcm- seed coat and obtain the cotyledons. The latter were sowed
2/min/sec) at 21°C +/-2°C. All experiments were in a Murashige & Skoog (MS) basal medium, at half
conducted at least three times with 15 replicates each. concentration of macro and micronutrients, supplemented
with 1 or 2 mg.L-1 of 2,4-Dichlorophenoxyacetic acid
Shoots from both explants were placed in a rooting (2.4-D) and 0.1 mg.L-1 of BAP, 3% of sucrose and 7.5 g.
medium with the macro and micronutrients of WPM [8] L-1 agar (Table 2). The cotyledons were placed with their
supplemented with 0.1 mg.L-1 of Indolebutyric acid (IBA) abaxial side in contact with the culture medium and were
(Table 1). maintained in the culture room under complete darkness at
21°C +/-2°C.
Table 1: Culture media used to the differents explants (leaves and nodal
sections) of Salix humboldtiana. Table 2: Culture media used in the somatic embryogenesis of Acacia
Basal media Growth mg.L -1
Type of explant caven.
regulator PGR
Explants Media 2.4D BAP Condition
MS complete BAP/ANA 1:0.5 Leaves
(mg.L-1) (mg.L-1)
MS complete BAP/ANA 1:1 Leaves
Cotyledon (Control) MS/2 0 0 25 +/- 2°C
WPM IBA 0,1 Shoots
Cotyledon MS/2 1 0.1 in the
WPM BAP 0.1; 0.5; 1; 1.5 y 2 Nodal sections
Cotyledon MS/2 2 0.1 darkness.
WPM - - Nodal sections

References
[1] García González M. (2000) Plantas medicinales científicamente validadas. II Congreso de Ciencias “Exploraciones dentro y fuera
del aula”. Fundación CIENTEC. INBioparque, Santo Domingo, Heredia, Costa Rica.
[2] Lahitte HB, Hurrell JA. (1994) Flora arbórea de la Isla Martín García nativa y naturalizada. Reserva Natural y Cultural. Provincia
de Buenos Aires. República Argentina. ISSN 0325-1225. Serie informe Nº 47. 27-229
[3] Hartmann HT, Kester DE. (1998) Propagación de plantas. Ed. CECSA. México, D.F. Parte II. 220, 221-760
[4] Mroginski L, Sansberro P. y Flaschland E. (2010) Herramientas básicas. En: Biotecnología y Mejoramiento Vegetal II. Ediciones
INTA-Eds: Gabriela Levitus, Viviana Echenique, Clara Rubinstein, Esteban Hopp, Luis Mroginski.
[5] Neumann K. (2009) Plant Cell and Tissue Culture - A Tool in Biotechnology: Basics and Application (Principles and Practice),
Springer.
[6] Abedini W, Adema M, Herrera J, Sharry S, Villarreal B, Nikoloff N. (2008) Recursos Forestales nativos de la provincia de Buenos
Aires: la Biotecnología como una estrategia de conservación. III Congreso Nacional de Conservación de la Biodiversidad. Facultad
de Ciencias Exactas y Naturales, UBA. Ciudad Autónoma de Buenos Aires. CD ROM
[7] Murashige T, Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia
Plantarum, 15, 473-497.
[8] Lloyd G, McCown B. (1980) Commercially feasible micropropagation of mountain laurel Kalmia latifolia, by use of shoot tip
culture. Combined Proceedings International Plant Propagation Society, 30, 421–427.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Genotoxic Evaluation of a Methanolic Extract of Verbascum 989 - 991

thapsus using Micronucleus Test in Mouse Bone Marrow

Franco Matías Escobara, María Carola Sabinia, Silvia Matilde Zanona, Laura Noelia Cariddia,
Carlos Eugenio Tonnb and Liliana Inés Sabinia
a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y
Naturales.Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba. Argentina
b
INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San
Luis, San Luis. Argentina

fescobar@exa.unrc.edu.ar

Received: November 15th, 2010; Accepted: March 25th, 2011

Verbascum thapsus L. is a medicinal plant and has been used to treat numerous pulmonary diseases, asthma, inflammatory disease,
spasmodic coughs and migraine headaches. Several studies have demonstrated that different extracts of V. thapsus present antimicrobial
activity. Thus, the goal of this study was to evaluate the genotoxic and cytotoxic activities of a methanolic extract of Verbascum thapsus,
using micronucleus test in mouse bone marrow. No toxicity in bone marrow was detected in the extract-treated groups. The methanolic
extract of V. thapsus at doses of 100, 300 and 500 mg / kg, did not produce a significant increase in the frequency of MNPCE in bone
marrow and neither altered the relationship PCE / NCE respect to negative control. These cytogenotoxic findings contribute the preclinical
knowledge of methanolic extract of V. thapsus and provide security in its use as herbal medicine.

Keywords: Verbascum thapsus L., methanolic extract, micronucleus, bone marrow, genotoxicity.

The use of medicinal plants in therapy or as dietary
supplements remounts centuries ago, but it has increased
substantially in the last decades [1a,1b]. The popularity of
herbal medicines is related to their easy access, therapeutic
efficacy, relatively low cost, and assumed absence of toxic
effects.
Widespread public opinion is that being a natural product,
herbal medicines are harmless and free from adverse
effects. However, the safety of their use has recently been
questioned due to the reports of illness and fatalities
[2a-2c]. Considering the complexity of herbals in general
and their inherent biological variation, it is now necessary
to evaluate their safety, efficacy and quality [1b]. Thus, an
assessment of their mutagenic and cytotoxic potential is
necessary to ensure the relatively safe use of plant-derived
medicines.
Scrophulariaceae is an important family of plants
comprising over 200 genera and about 2500 species. It
includes Mimulus, Penstemon, Digitalis, Veronica and
Verbascum [3a]. Different members have been valued for
their curative properties and are widely employed in
domestic and regular medicine. At least 250 species of
Verbascum are known. Among the species traditionally
used in medicine, the most important is Verbascum
thapsus L., commonly known as mullein, common
mullein, great mullein [3b]. V. thapsus is distributed
worldwide. In Argentina this species is abundant however;
it is considered an exotic plant. Many studies carried
out with plants collected in other countries, have shown
that different extracts have antimicrobial, antitumor
and cytotoxic activities [4a-4c]. Therefore, given the
abundance of the species in Córdoba province, Argentina,
cytotoxic and antiviral properties of methanolic extract of
V. thapsus were investigated. The results of these previous
studies have indicated that the extract markedly inhibits
Herpes suis virus type 1 at non cytotoxic concentrations
[5a,5b]. Since this information is hopeful, it is necessary to
define the cytogenotoxic potential to ensure the use of the
extract at safe levels.
The aim of this study was to determine the genotoxic and
cytotoxic activities of a methanolic extract of Verbascum
thapsus, using micronucleus test in mouse bone marrow.
Evaluation of micronucleus induction is the primary in
vivo test in a battery of genotoxicity tests and is
recommended by the regulatory worldwide agencies to be
conducted as part of product safety assessment.
The results of micronucleus (MN) test in BALB/c mice
treated with different doses of the extract are summarized
in Table 1. In all cases these results are expressed as mean
(± standard deviation).
990 Natural Product Communications Vol. 6 (7) 2011 Escobar et al.

Table 1: Mean of polychromatic erythrocytes with micronuclei (MNPCE) observed in bone marrow cell of female (F) and male (M) BALB/c mice treated with
a Verbascum thapsus methanolic extract, and respective controls.
Dose MNPCE PCE/NCE
Treatments Number of MNPCE per animal
mg/kg (mean ± SD) (mean ± SD)
F1 F2 F3 M1 M2 M3
Negative control (saline) 0 2 3 3 2 2 2 2.50 ± 0.55 1.69 ± 0.14
V. thapsus methanolic extract 100 1 2 5 1 3 1 2.17 ± 1.60 1.77 ± 0.12
V. thapsus methanolic extract 300 3 1 3 1 2 2 2.00 ± 0.89 1.79 ± 0.23
V. thapsus methanolic extract 500 5 1 1 2 1 2 2.00 ± 1.55 1.74 ± 0.15
Positive control (cyclophophamide) 20 11 14 11 13 9 12 11.7 ± 1.7* 1.69 ± 0.10
Thousand cells were analyzed per animal, for a total of 6000 cells per group. SD = Standard deviation. * p< 0.001, statistically significant difference from
saline group (ANOVA. Tukey`s test).

25 Male
Female
20

MNPCE / 1000 PCE

MNPCE 15

10

NCE
0

kg

kg

kg
(-)

)
(+
NCE PCE

g/

g/
g/
ol

l
ro
m

m
tr

t
on

on
10

30

50
C

Figure 1: A photomicrograph of mice whole bone-marrow smear
showing nucleated as well as enucleated cells (PCEs and NCEs). One the
polychromatic erythrocyte also contains micronuclei.
C
Figure 2: Frequency of Micronucleated Polychromatic Erythrocytes
(MNPCE) induced in bone-marrow cells of female (F) and male (M)
BALB/c mice treated with a V. thapsus methanolic extract: negative
control (saline solution), positive control (cyclophosphamide 20 mg/kg
body weight). Values are shown as mean ± SD.

Examples of polychromatic and normochromatic
erythrocytes unaltered, normal, and the presence of
micronucleated polychromatic erythrocytes are shown in
Figure 1.
The percentage frequency of MN in the groups treated
with 100, 300 and 500 mg / kg of methanolic extract,
which were 2.17 (±1.60), 2.00 (±0.89), 2.00 (±1.55)
respectively, showed no significant differences from the
saline-treated group: 2.5 (±0.55). However, there was a
significant increase in the frequency of micronucleus in
PCE from the positive control group treated with
cyclophosphamide (Figure 2).
Therefore, the results obtained in the present study allow
concluding that the methanolic extract of Verbascum
thapsus does not contain genotoxic and cytotoxic
compounds since its administration in mice at doses of
100, 300 and 500 mg/kg, showed no evidence of
genotoxicity or cytotoxicity in vivo. The extract did not
produce a significant increase in the frequency of MNPCE
in bone marrow and neither altered the relationship PCE /
NCE respect to negative control.
These cytogenotoxic findings contribute the preclinical
knowledge of methanolic extract of V. thapsus and provide
security in its use as herbal medicine.

No citotoxicity in bone marrow was detected in the Experimental

extract-treated groups. Statistical analysis of the proportion
PCE / NCE revealed no differences in any study group.
There were no sex-dependent changes in any treatment.
V. thapsus methanolic extract contains iridoid glycosides
(laterioside, harpagoside, ajugol, picroside IV), three
iridoid ((+)-genipin, α-gardiol and β-gardiol), one
phenylethyl glycoside (verbacoside), two sesquiterpenes
(buddlindeterpene A and buddlindeterpene B), one
diterpene (buddlindeterpene C), and one biflavonoid
(amentoflavone) [6].
Plant material and extraction: Aerial parts of Verbascum
thapsus L. were collected in San Luis province, Argentina.
The plant material was identified by Ing. Luis A. del Vitto.
A voucher specimen (N° #514) was preserved and
deposited in herbal library of the “Herbario de la
Universidad Nacional de San Luis, Argentina”. The leaves
were dried and chopped finely using a blender. Eight
hundred grams of dried material were successively
extracted with 3.5 L of the following solvents: n-hexane,
chloroform and methanol at room temperature for 48 h.
The evaporation of the extracts in vacuum at 40ºC yielded
Genotoxic evaluation of a methanolic extract of Verbascum thapsus Natural Product Communications Vol. 6 (7) 2011 991

the hexane, chloroform and methanol extracts. The
methanolic extract was dissolved in saline solution and
subsequently diluted to appropriate working
concentrations.
Animal’s treatments: Two months old male/female
BALBc mice weighing ca. 20 g were intraperitoneally
injected with a single dose of V. thapsus methanolic
extract (volume 0.2 ml). Three doses were selected (100,
300 and 500 mg/kg) considering previous citotoxicity data
obtained with Vero cells. Cyclophosphamide (Sigma) at 20
mg/kg and saline solution were used as positive and
negative controls respectively.
Mouse bone marrow micronuclei assay: Six mice per
dose were sacrificed at 24 h post-injection and femurs
were removed. Femurs were prepared for the boned-
marrow micronucleus test as previously described [7a].
Slides were stained with May-Grünwald and Giemsa
solutions [7b] which maximized the differentiation
between the polychromatic (PCE) and normochromatic
(NCE) erythrocytes. To determine index of genotoxicity
the number of micronucleated polychromatic erythrocytes
(MNPCE) was obtained at an average of 1000 PCE,
counted per animal per dose. In order to evaluate any
cytotoxic effect of extract, the ratio of PCE/NCE was
determined in the same sample. Statistical significance was
determined by analysis of variance (ANOVA), applying
software GraphPad Prism 5.0.
Acknowledgments - The authors are grateful to
CONICET, MinCyT of Córdoba, Universidad Nacional de
Río Cuarto and PICTOR program, BID 1728 /OC-AR for
financial support. We also would like to thank Ing. Luis A.
del Vitto for taxonomic determination of the plant
specimen.

References
[1] (a) Woods PW. (1999) Herbal healing. Essence, 30, 42-46; (b) WHO (World Health Organization). (2002) Drug Information Herbal
Medicines. Vol. 16. World Health Organization, Geneva.
[2] (a) Stewart MJ, Moar JJ, Steenkamp P, Kokot M. (1999) Findings in fatal cases of poisoning attributed to traditional remedies in
South Africa. Forensic Science International, 101, 177-183; (b) Ernst E. (2002) Toxic heavy metals and undeclared drugs in Asian
herbal medicines. Trends in Pharmacological Sciences, 23, 136-139; (c) Veiga-Junior VF, Pinto AC, Maciel MAM. (2005)
Medicinal plants: safe cure? Química Nova, 28, 519-528.
[3] (a) Grieve M. (1981) A Modern Herbal Vol 2. Dover Publications: New York, 562-566; (b) Turker AU, Gurel E. (2005) Common
Mullein (Verbascum thapsus L.): Recent Advances in Research. Phytotherapy Research, 19, 733-739.
[4] (a) McCutcheon AR, Ellis SM, Hancock REW, Towers GHN. (1992) Antibiotic screening of medicinal plants of the British
Columbian native peoples. Journal of Ethnopharmacology, 37, 213-223; (b) McCutcheon AR, Roberts TE, Gibbons E, Ellis SM,
Babiuk LA, Hancock REW, Towers GHN. (1995) Antiviral screening of British Columbian medicinal plants. Journal of
Ethnopharmacology, 49, 101-110; (c) Turker AU, Camper ND. (2002) Biological activity of common mullein, a medicinal plant
Journal of Ethnopharmacology, 82, 117-125.
[5] (a) Escobar F, Gallotti V, Salas M, Sabini C, Giordano O, Contigiani M, Sabini L. (2008) Comparative assays of cytotoxicity
induced by extracts of Verbascum thapsus. Biocell, 32, 108; (b) Escobar F., Konigheim BV, Aguilar J, Sabini C, Giordano O,
Contigiani M, Sabini L. (2008) Preliminary studies of antiherpetic activity exerted by extracts of Verbascum thapsus. Biocell, 32,
109.
[6] Hussain H, Aziz S, Miana GA, Ahmad VU, Anwar S, Ahmed I. (2009) Minor chemical constituents of Verbascum thapsus.
Biochemical Systematic and Ecology, 37, 124-126.
[7] (a) Schmid W. (1975) The micronucleus test. Mutation Research, 31, 9-15; (b) Cole RJ, Taylor NA, Cole J, Arlette CF. (1979)
Transplacental effect of chemical mutagens detected by micronucleus test. Nature, 277, 317-318.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Study of Antiviral and Virucidal Activities of Aqueous 993 - 994
Extract of Baccharis articulata against Herpes suis virus
Cristina Vanesa Torres, María Julia Domínguez, José Luis Carbonari, María Carola Sabini,
Liliana Inés Sabini and Silvia Matilde Zanon

Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Fisico-Químicas y

Naturales. Universidad Nacional de Río Cuarto. Río Cuarto, Córdoba. Argentina

ctorres@exa.unrc.edu.ar

Received: December 10th, 2010; Accepted: March 16th, 2011

Baccharis articulata is native of América and traditionally used for the treatment of digestive disorders and urinary infections. Cytotoxicity
of aqueous extracts of B. articulata was investigated in Vero cells. As the maximal non cytotoxic concentration has been established, this
concentration has been used to evaluate antiviral and virucidal activities against Herpes suis virus type 1, member of the same subfamily of
Herpes simplex virus. Aqueous extracts of B. articulata exhibited more than 95% of virucidal activity. These findings support their potential
application as a disinfectant or antiseptic with low toxicity and provide a valuable knowledge to ethnopharmacology properties of Baccharis
articulata.

Keywords: cytotoxicity, virucidal, antiviral activity, aqueous extract, Baccharis articulata, Herpes suis virus.

Baccharis articulata, commonly known as carqueja, Table 1: Cytotoxic effect and inhibition of viral replication of aqueous
extracts of Baccharis articulata.
frequently found in the hills region of Cordoba province of
Cytotoxicity Antiviral activity (%) Virucidal (%)
Argentina, exhibit antioxidant, antibacterial, anti-HIV and MNCC* A B C MNCC MNCC 2X
antifungal abilities, [1a-1c]. In treatment of herpetic CAE 1000 g/mL 25.6 33 0 31.5 97.8
infections one or more drugs currently available were used, HAE 600 g/mL 54 6.8 11 80 96
but both continuous use and self-medication promote the * Maximal non-cytotoxic concentration, A: viral adsorption, B: post-viral adsorption,
development of resistance and tolerance of these viruses C: pre-treatment cell.

[2]. This background encouraged the study of cytotoxic,

antiviral and virucidal activities of aqueous extracts of B. (54%) during the stage of viral adsorption and penetration.
articulata against Herpes suis type 1, virus closely related This value showed that Herpes suis virus were more
to Herpes simplex types 1 and 2. sensitive at HAE than CAE of B. articulata.

The aim of the present study was to determine
concentration of aqueous extracts that do not affect
monolayer cell and to be used in later assays. Therefore
cytotoxic effect on Vero cells of aqueous extracts was
evaluated by daily microscopic observation of treated cells
to determine the MNCC (Maximal Non Cytotoxic
Concentration). The MNCC values were 1000 µg/mL and
600 µg/mL for cold aqueous extract (CAE) and hot
aqueous extract (HAE), respectively. The cultures exposed
to extract concentrations lower than MNCC exhibited
morphology similar to control cultures. The cytotoxic
effect was characterized by retraction cell and
disruption of cell monolayer. Virucidal and antiviral
assays performed at different stages of virus replication
revealed percentages of inhibition shown in Table 1. The
CAE inhibited 25 and 33% viral replication when the
extract was added at 1000 µg/mL (MNCC) during the viral
adsorption and later that step, respectively. The HAE,
at 600 µg/mL, demonstrated to exert the inhibitory activity
Only the pre-treatment of Vero cells with HAE slightly
interfered Herpes suis type 1 adsorption to cellular
receptor. Therefore, both extracts would not induce
antiviral state in the cells and neither would interfere to the
mechanism of endocytosis used in the entry of virus into
the cell. The CAE and HAE demonstrated to exert strong
extracellular virus inactivation mostly when the assays
were carried out with extracts at double concentration of
MNCC (97.8 and 96% respectively). Results obtained in
this work allow concluding that the aqueous extracts of
B. articulata exert slight antiviral activities against Herpes
suis virus type 1.
It is known that plant aqueous extracts, among other
components, contain anthocyanins, saponins, polypeptides
and terpenes [3a]. Studies on the chemical composition of
B. articulata have reported presence of several terpenes
such as articulina, germacrene and ɑ-pinene [3b]. These
compounds in other plant species (Glyptopetalum
sclerocarpum, Thymus vulgaris) have exhibited antiviral
994 Natural Product Communications Vol. 6 (7) 2011 Torres et al.

activity [3a,3c]. As a consequence they could be
responsible of the antiviral activity demonstrate in this
work. Furthermore, additional studies are needed in order
to identify which compounds could be responsible for this
effect and how they exert antiviral action.
Experimental
Plant material: Baccharis articulata was collected in the
hills of Cordoba, Argentina. Taxonomic identification was
performed by Prof. Margarita Grosso of the Universidad
Nacional de Río Cuarto. A specimen of the plant was
deposited (Nº RCV 1810) in the Herbarium. Dried aerial
parts (branch and leaf) (15 g) were submitted to extraction
with 700 mL of cold water at 4°C for 2 days (cold aqueous
extract, CAE) and at 70°C for 2 days (hot aqueous extract,
HAE). The extracts were filtered and lyophilized.
Viruses and cells: Vero cells (African green monkey
kidney) were grown in Eagle´s minimum essential medium
(MEM) supplemented with 8% FCS, 1% gentamicin and
1% L-glutamine and maintained at 37ºC in 5% CO2
atmosphere. Herpes suis virus type 1 strain RC/79 was
isolated in Río Cuarto in 1979 [4a].
Cytotoxicity assays: Confluent cell monolayers cultivated
in 96-well culture plates were treated with different
concentrations of extracts and incubated at 37ºC for 72 h.
At this time, maximal non cytotoxic concentration
(MNCC) was determined by microscopic observation.
Antiviral assays: The antiviral activity of tested extracts
was evaluated at different stages of viral replication by
plaque reduction method. Virus titres were calculated by
plaque forming units per mL (PFU/mL) [4b]. The
percentage of inhibition was calculated as the ratio
between virus titres in treated cells and in untreated cells.
During adsorption and viral penetration: Monolayer
cells grown in 24-well culture plates were incubated for 90
min with Herpes suis virus type 1 (105 PFU/mL) in
combination or not with extract at MNCC. Then, residual
virus was discarded. The cells were overlaid with an
overlay medium containing 1% of methylcellulose. The
plates were further incubated at 37ºC for 72 h. Later cell
monolayer was fixed with 10% formalin. The virus
plaques formed on Vero cells were stained with 1% crystal
violet. Percentage of viral inhibition was determined.
Post-adsorption and penetration of virus: Confluent
monolayer of Vero cells grown in 24-well culture plates
were infected with Herpes suis type 1 (105 PFU/mL) and
incubated at 37ºC for 90 min. After residual virus was
removed, the cells were covered with the overlay medium
containing 1% of methylcellulose and extract at MNCC,
and incubated at 37ºC for 72 h. Percentage of viral
inhibition was determined.
Pretreatment: Monolayer cells grown in 24-well culture
plates were incubated for 2 h with extract at MNCC. After
the extract was discarded, culture cells were inoculated
with Herpes suis type 1 (105 PFU/mL) and incubated at
37ºC for 90 min. The remainder virus was discarded and
the cells were incubated with the overlay medium
containing 1% of methylcellulose at 37°C for 72 h.
Percentage of viral inhibition was determined.
Virucidal activity assay: Viral suspensions (105 PFU/mL)
were incubated with extract at MNCC and at double
concentration. After incubation of virus at 37°C for 2 h,
monolayer cells grown in 24-well culture plates were
infected with treated virus. The infected cells were
incubated at 37°C for 90 min. The remainder virus was
discarded and the cells were incubated with overlay
medium containing 1% of methylcellulose at 37°C for 72
h. Percentage of viral inactivation was determined.
Acknowledgments - The authors thank Universidad
Nacional de Río Cuarto and PICTOR program, BID 1728
/OC-AR for financial support. We are grateful to Prof.
Margarita Grosso for help in identification of the plant
specimen.

References
[1] (a) Palacios PS, Wilson EG, Debenedetti SL. (1999) HPLC analysis cafeilquínicos acid present in three species of Baccharis.
Dominguezia, 15, 39; (b) De Oliveira SQ, Dal-Pizzol F, Gosmann G, Guillaume D, Moreira JC, Schenkel E. (2003) Antioxidant
activity of Baccharis articulata extracts: isolation of new compound with antioxidant activity. Free Radical Research, 37,
555-559; (c) Vivot Lupi EP, Sanchez Brisuela CI, Casik Jeifetz F, Sequin Acosta CJ. (2009) Screening of antifungal activity of
extracts of plant species in Entre Ríos. Revista Cubana de Farmacia, 43, 74-84.
[2] Kimberlin DW, Whitley RJ. (1996) Antiviral resistance: Mechanisms, Clinical Significance and Future Implications. Journal of
Antimicrobial Chemotherapy, 37, 403-421
[3] Ambrogi A, Giraudo J, Busso J, Bianco O, Bagnat E, Segura de Aramburu M, Ramos B, Ceriatti F. (1981) Primer diagnóstico de la
enfermedad de Aujeszky en cerdos en la República Argentina. Gaceta Veterinaria. Buenos Aires, Tomo XLIII, 357, 58-64
[4] Dulbecco, R. (1962) Production of plaques in monolayer tissue culture by single particles of an animal virus. Proceedings of the
National Academy of Sciences, USA, 38, 747-752
 2011
NPC Natural Product Communications Vol. 6
No. 7
Evaluation of Cytogenotoxic Effects of Cold Aqueous 995 - 998

Extract from Achyrocline satureioides by Allium cepa L test

María C. Sabinia*, Laura N. Cariddia, Franco M. Escobara, Romina A. Bachettia, Sonia B. Sutila,
Marta S. Contigianib, Silvia M. Zanona and Liliana I. Sabinia
a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico-Químicas y
Naturales. Universidad Nacional de Río Cuarto. Río Cuarto, Córdoba, Argentina
b
Instituto de Virología “José María Vanella”. Facultad de Ciencias Médicas. Universidad Nacional
de Córdoba. Córdoba, Argentina

csabini@exa.unrc.edu.ar

Received: December 14th, 2010; Accepted: March 16th, 2011

Achyrocline satureioides (“marcela del campo”) is native to America. Numerous investigations have reported several bioactive properties
such as anti-inflammatory, hepatoprotective, immunomodulatory, antimicrobial and antiviral. Nowadays, few medicinal plants have been
scientifically evaluated to test its safety, efficacy and potential benefits, despite the great public interest in these herbs. The aim of this work
was to evaluate the cytotoxic and genotoxic activities of cold aqueous extract obtained from A. satureioides using Allium cepa L test. The
results demonstrated the absence of genotoxicity of the extract. Only higher concentrations induced cytotoxicity but interestingly this effect
was reversible and was not associated with mutagenicity. The contribution of this research provides assurance of safety in the application of
Achyrocline satureioides in treatment of microbial diseases and other pathologies helping to define selective toxicity.

Keywords: Achyrocline satureioides (Lam.) DC, Allium cepa L test, cytogenotoxicity, cold aqueous extract.

Asteraceae (Compositae) family includes some of the
oldest and most valued plants for medicinal purposes [1a].
It is known that certain genus of this family contain toxic
compounds such as tannic, cyanide, formic and malic acid
[1b]. Achyrocline satureioides (Lam.) DC. is a relevant
species that belongs to the family Asteraceae. This plant,
commonly known as “marcela del campo”, is native to
America and extends throughout the continent, as well as
Europe and Africa. In our country it is often found
in the hills of Córdoba, San Luis and Buenos Aires
[2]. Numerous investigations have reported several
bioactive properties, such as anti-inflammatory [3a],
sedative [3b], hepatoprotective [3c], antioxidant [3d,3e],
immunomodulatory and antimicrobial [3f], antitumoral
[3g], antiviral [3h,3i,3j] and photoprotective [3k].
Nowadays, few medicinal plants have been scientifically
evaluated to test its safety, efficacy and potential benefits,
despite the great public interest in these herbs [4].
Regulatory worldwide authorities require information on
the genotoxic potential of new drugs as part of the safety
evaluation process. Allium cepa L test allows assessment
of toxicity of substances in terms of macroscopic
parameters such as growth and form of roots and,
evaluation of genotoxicity from microscopic parameters
such as types and frequencies of chromosomal aberrations
and abnormal cell divisions.
For all previously described, it is of paramount importance
to know the cytotoxicity and genotoxicity of extracts from
A. satureioides. In order to evaluate the cytotoxic and
genotoxic activities of cold aqueous extract (CAE)
obtained from Achyrocline satureioides experiments were
performed using Allium cepa L test with modifications.
Table 1 summarizes the results of the effects of CAE of
A. satureioides on root of Allium cepa. Taking into account
the number of bulbs roots, the different concentrations of
CAE induced normal development, similar to control.
The analysis of length of roots treated with CAE for 5 days
showed statistically significant differences (p<0.05)
among all treatments vs. negative control and absence of
dose-response relationship. The inhibition of roots length
was ≥ 50% (p<0.05) for all tested concentrations.
Considering the bulbs treated for 2 days (with reversion),
there were no significant differences between treatments of
0.5 and 2 mg/mL vs. negative control. By the contrary,
there were significant differences for the remaining
concentrations of CAE compared to negative control
(Figure 1). The average root length for treatment of 2 days
with CAE (with reversion) was always higher than of 5
days, although there was no statistically significant
difference within the same concentration considered. The
concentration of 0.5 mg/mL was the exception because it
996 Natural Product Communications Vol. 6 (7) 2011 Sabini et al.

Table 1: Macroscopic parameters analyzed in roots of Allium cepa L after treatment with different concentrations of CAE of A. satureioides for 5 and 2 days.
TREATMENTS
(n = 4 bulbs)
PARAMETER
CAE (mg/ml) for 5 days CAE (mg/ml) for 2 days
C (-) C (+) C (-) C (+)
0.5 1 2 3 4 0.5 1 2 3 4
Mean root number 54 37 67 41 46.5 60.5 56 48.5 34.5 43.5 62.5 39 38 39.5
Mean root length 20.41 13.97 10.03 7.78 8.42 7.51 6.61 25.91 17.55 18.98 11.51 13.07 7.91 10.95
(mm) ± SEM ±2.00 ±0.65 ±0.62 ±0.56 ±0.72 ±0.60 ±0.45 ±2.63 ±0.94 ±1.48 ±0.64 ±1.03 ±0.66 ±0.86
Ha 8 32 11 0 2 10 3 10 20 7 5 10 6 4
Geb 0 0 0 10 14 16 25 0 0 0 15 6 15 37
Abnormalities
Nec 0 0 16 17 6 4 0 0 0 12 17 1 0 18
Tud 10 5 2 0 0 0 1 9 5 0 1 3 0 0

Ha: hook; Geb: gelling; Nec: necrosis; Tud: tumor.

2 days (with reversion) 10

30
MI 1 MI 3
5 days 8 MI 2 C+

Mitotic Index (%)

Roots length

20
6
(mm)

4
10

0
0
C- C+ 0.5 1 2 3 4 C- 0.5 1 2 3 4 C- 0.5 1 2 3 4 C- 0.5 1 2 3 4 C+
Concentration (mg/mL)
Treatment (mg/mL)
Figure 1: Roots length of bulbs of Allium cepa L treated with different Figure 2: Comparison of mitotic indices 1, 2 and 3 of the roots of the
concentration of CAE of A. satureioides for 5 days vs. 2 days (with Allium cepa bulbs treated with different concentrations of CAE of
reversion). Achyrocline satureioides.

showed a statistical difference (p<0.001), indicating the
recovery of the roots by mineral water action, after
treatment with the extract. This result suggests that bulbs
would have the ability to recover from damage induced by
this concentration of extract.
Respect to macroscopic abnormality, the study revealed
the presence of hooks and tumours in negative control with
low frequency. These spontaneous changes are normal and
concordant with other authors [5a,5b]. Positive control,
paracetamol (acetaminophen 0.3 mg/mL), showed a high
incidence of root hooks and also induced the appearance of
tumours. None of the roots treated with CAE induced the
development of hooks. The results of all treatments with
the extract were markedly different to the effect caused by
paracetamol.
Gelling and necrosis were present with varying frequency,
having the first one the highest incidence. Roots treated
with extract did not show significant values in the number
of tumours. Pigmentation was observed in most of roots
treated due to intense colour of extract.
inhibition of cell division. For the MI 2, the trend of the
curve revealed a drastic reduction for MI% at the
concentrations 2, 3 and 4 mg/mL. This behaviour could be
due to these concentrations are extremely toxic when they
are used for 5 days. The values of MI 3 (treatment with
reversion) did not show statistically significant differences
between treatments, neither between these treatments and
negative control, indicating the ability of roots to recovery
of the toxic action of CAE. The comparative analysis of
MI 1 vs. MI 3, for each concentration, showed significant
difference (p<0.5), indicating that the damage was
reversed (Figure 2).
Analysis of phases index for bulbs treated for 2 and 5 days
showed that cell division in roots treated with 0.5 and
1 mg/mL was similar to negative control. By the contrary,
cell cycle stages were modified in the bulbs treated with 2,
3 and 4 mg/mL. Prophase was the most observed phase,
demonstrating an arrest of cell division at this stage.
Application of CAE at these concentrations would be
affecting the formation of chromosomes and avoiding their
placement in equatorial plane of the cell.

Statistical analysis of mitotic index of bulbs treated with
CAE did not show significant difference between negative
control and treatments with 0.5 and 1 mg/mL, for 2 days
(MI 1) and 5 days (MI 2). So, these concentrations did
not exert toxicity. In contrast, there were significant
differences between negative control and treatments of
2, 3 and 4 mg/mL. These concentrations would exert an
Statistical analysis of the results obtained in treatment with
reversion did not show significant differences in phase
index of treated roots vs. negative control, indicating the
reversion of the changes induced by CAE of A.
satureioides. These results point out that mitosis was
normally developed as it confirms by the value of MI 3,
(Figure 3).
Cytogenotoxic evaluation of the extract of Achyrocline satureioides Natural Product Communications Vol. 6 (7) 2011 997

A
A A A A
Phase index (%)

100

50

B C D E
0
C- 0,5 1 2 3 4

Treatment
F F
B

100
Phase index (%)

Figure 4: Microphotographs of meristematic cells of root tips of Allium

50 cepa. Squash preparations, stained in acetocarmine and observed in light
microscope. (A) A typical view of different size, shape and basophility of
nuclei and interphase and mitotic phases; (B) cells with sticky
chromosomes; (C) cells with delayed chromosomes; (D) cells with
0
chromosomal bridges; (E) physiological aberrations: c-mitosis; (F)
C- C+ 0,5 1 2 3 figures similar to apoptotic bodies in interphase cells.

Treatment
Experimental
C Plant material: Healthy plants of A. satureioides species
were collected manually from Villa Jorcoricó, southern
Phase index (%)

100
50
0
C- 0,5 1 2
Treatment
Prophase index
Metaphase index
Figure 3: Phase index of cell division in roots treated with CAE of
Achyrocline satureioides (A) MI 1, (B) MI 2, and (C) MI 3.
Microscopic evaluation of cells showed physiological and
clastogenic aberrations as previously described [6]. The
physiological aberrations observed were c-mitosis and,
sticky and delayed chromosomes, while the other ones
presented chromosomal bridges. These alterations were
found more often in roots with normal cell division.
Similar figures to apoptotic bodies were observed in inter-
phase cells treated for 2 and 5 days with CAE, (Figure 4).
This study demonstrated the absence of genotoxicity of
CAE from A. satureioides. High concentrations of the
extract induced citotoxicity but this effect was reversible
and was not associated with mutagenicity. The
contribution of this research provides assurance of safety
in the application of Achyrocline satureioides in treatment
of microbial diseases and other pathologies helping to
define selective toxicity. Nowadays, studies referred to
chemical characterization of CAE from A. satureioides are
in progress.
Córdoba hills in 2009. The plant material was identified by
Dr. Luis Del Vitto, Facultad de Farmacia y Bioquímica,
Universidad de San Luis, San Luis, Argentina. A voucher
specimen was deposited in the Herbarium of the
University of San Luis (Nº 6362).
3 4 Obtention of cold aqueous extract: Dried aerial vegetal
parts (15 g) were submitted to extraction with 700 mL of
Anaphase index
cold water at 4°C for 2 days. The mixture was filtered and
lyophilized. This extract was identified as cold aqueous
Telophase index
extract (CAE).
Determination of genotoxic activity of cold aqueous
extract from A. satureioides by the Allium cepa test:
Allium test as described [7a,7b,7c] was developed with
some modifications. Qualitative and quantitative changes,
macro and microscopic, induced by treatment with CAE in
plant cells were assessed.
Onion root tips of Allium cepa L grown in mineral water,
in darkness, with aeration and constant temperature of 25 ±
0.5 °C were employed. CAE was assayed at 0.5, 1, 2, 3
and 4 mg/mL of mineral water. Positive (paracetamol 0.3
mg/mL) and negative (mineral water) controls were
included in the system. Extract concentrations were
applied for different times: 2 and 5 days, and 2 days
followed by 3 days with water (reversion). At the end of
each treatment, 2-3 root tips from these bulbs were cut and
fixed in a mixture of absolute alcohol:glacial acetic acid
(3:1, v/v). These roots were hydrolyzed in 1N HCL for 5
minutes after which they were washed in distilled water.
Two root tips were then squashed on each slide, stained
with acetocarmine for 10 min and cover slips carefully
998 Natural Product Communications Vol. 6 (7) 2011 Sabini et al.

lowered on to exclude air bubble. The cover slips were
sealed on the slides with clear fingernail polish as
suggested by [8]. These roots were reserved for evaluating
the cytogenetic abnormalities (microscopic). Six slides
were prepared for each concentration and controls (at 1000
cells per slide) were analyzed at ×1000 magnification for
induction of chromosomal aberration. The mitotic index
was calculated as the ratio between the number of cells in
division and 1000 observed cells. In addition, phases
indices were also calculated [7a,7c]. The percentage
frequency of aberrant cells was calculated based on the
number of aberrant cells per total cells scored at each
concentration of the extract, [5b,7c,9].
Previously, macroscopic changes of the roots in terms of
root number, length and presence of abnormalities, were
evaluated.
Statistical significance was determined by analysis of
variance (ANOVA) using GraphPad Prism 5.0 software.
Acknowledgments - The authors thank to CONICET,
MinCyT of Córdoba, Universidad Nacional de Río Cuarto
and the PICTOR programme, BID 1728 /OC-AR, for
providing financial support. The authors are also grateful
to Dr Luis Del Vitto for taxonomic classification of the
plant specimen.

References
[1] (a) Paulsen E. (2002) Contact sensitization from Compositae containing herbal remedies and cosmetics. Contact Dermatitis, 47,
189–198; (b) Duke JA. (2000) Toxins: their toxicity and distribution in plant genera. In: Handbook of medicinal herbs, pp.
525-568.
[2] Instituto Nacional de Investigación Agropecuaria (INIA). (2004) Estudios en domesticación y cultivos de especies medicinales y
aromáticas nativas. Estación experimental Las Brujas. Ruta 48 km 10, Rincón del Colorado, Canelones, Uruguay.
[3] (a) De Souza K, Bassani VL, Schapoval E. (2007) Influence of excipients and technological process on anti-inflammatory activity
of quercetin and Achyrocline satureioides (Lam.) D.C. extracts by oral route. Phytomedicine, 14, 102-108; (b) Hnatyszyn O,
Moscatelli V, Rondina R, Costa M, Arranz C, Balaszczuk A, Coussio J, Ferraro G. (2004) Flavonoids from Achyrocline
satureioides with relaxant effects on the smooth muscle of Guinea pig corpus cavernosum. Phytomedicine, 11, 366-369; (c)
Kadarian C, Broussalis AM, Miño J, Lopez P, Gorzalczany S, Ferraro G, Acevedo C. (2002) Hepatoprotective activity of
Achyrocline satureioides (LAM). Pharmacology Research, 45, 57-61; (d) Arredondo M, Blasina F, Echeverry C, Morquio A,
Ferreira M, Abin-Carriquiry J, Lafon L, Dajas F. (2004) Cytoprotection by Achyrocline satureioides (Lam.) D. C. and some of its
main flavonoids against oxidative stress. Journal of Ethnopharmacology, 91, 13-20; (e) Polydoro M, Souza KC, Andrades ME, Da
Silva EG., Bonatto F, Heydrich J, Dal-Pizzol F, Shapoval EE, Bassani VL, Moreir JC. (2004) Antioxidant, a pro-oxidant and
cytotoxic effects of Achyrocline satureioides extracts. Life Sciences, 74, 2815–2826; (f) Calvo D, Cariddi N, Grosso M, Demo M,
aldonado A. (2006) Achyrocline satureioides (LAM.) DC (Marcela): Actividad antimicrobiana sobre Staphylococcus spp. y efectos
inmunomoduladores sobre linfocitos humanos. Revista Latinoamericana de Microbiología, 48, 247-255; (g) Ruffa MJ, Ferraro G.,
Wagner ML, Calcagno ML, Campos RH, Cavallaro L. (2002) Cytotoxic effect of argentine medicinal plant extracts on human
hepatocellular carcinoma cell line. Journal of Ethnopharmacology, 79, 335-339; (h) Zanon SM, Ceriatti FS, Rovera M, Sabini LI,
Ramos BA. (1999) Search for antiviral activity of certain medicinal plants from Córdoba, Argentina. Revista Latinoamericana de
Microbiología, 41, 59-62; (i) Bettega JMR, Teixeira H, Bassani VL, Barardi CRM, Simões CM. (2004) Evaluation of the
antiherpetic activity of standardized extracts of Achyrocline satureioides. Phytotherapy Research, 18, 819-23; (j) Sabini MC,
Escobar FM, Tonn CE, Zanon SM, Contigiani MS, Sabini LI. (2010) Evaluation of antiviral activity of aqueous extracts from
Achyrocline satureioides against Western equine encephalitis virus. Natural Product Research, DOI:
10.1080/14786419.2010.490216; (k) Morquio A, Rivera-Megret F, Dajas F. (2005) Photoprotection by topical application of
Achyrocline satureioides (‘Marcela’). Phytotherapy Research, 19, 486-90.
[4] Calixto JB. (2000) Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic
agents). Brazilian Journal of Medical and Biological Research, 33, 179-189.
[5] (a) Fiskesjö G. (1994) Allium test II: Assessment of a chemical genotoxic potential by recording aberrations in chromosomes and
cells divisions in root tips of Allium cepa. Environmental Toxicology and Water Quality, 9, 235-241; (b) Bidau CJ, Amat AG., Yajia
M, Martí D, Riglos GA, Silvestroni A. (2004) Evaluation of the genotoxicity of aqueous extracts of Ilex paraguariensis St. Hil.
(Aquifoliaceae) using Allium Test. Cytologia, 69, 109-117.
[6] Rani G, Kaur K, Wadhwa R, Kaul SC, Nagpal A. (2005) Evaluation of the anti-genotoxicity of leaf extract of Ashwagandha. Food
and Chemical Toxicology, 43, 95-98.
[7] (a) Fiskesjö G. (1985) The Allium test as a standard in environmental monitoring. Hereditas, 102, 99-112; (b) Fiskesjö G, Levan A.
(1993) Evaluation of the first ten MEIC chemicals in the Allium test. ATLA, 21, 139-149; (c) Fiskesjö G. (1997) Allium test for
screening chemicals; evaluation of cytological parameters. Plants for Environmental Studies, CRC, Lewis Publishers, New York,
pp. 307-333.
[8] Grant WF, (1982) Chromosome aberrations assays in allium report of the USEPA gene tox program. Mutation Research, 99,
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[9] Bakare AA, Mosuro AA, Osibanjo O. (2000) Effect of simulated leachate on chromosomes and mitosis in roots of Allium cepa (L).
Journal of Environmental Biology, 21, 263-271.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Toxic Plants Used in Ethnoveterinary Medicine in Italy 999 - 1000

Lucia Viegi* and Roberta Vangelisti

Dipartimento di Biologia, Unità di Botanica generale e sistematica, Pisa University,Via L. Ghini 5,

56126 Pisa, Italy

lviegi@biologia.unipi.it

Received: November 10th, 2010; Accepted: March 16th, 2011

This study was conducted to document the use of toxic or potentially toxic plants for the treatment of ailments in livestock and pets in
ethnoveterinary practice in Italy. More than 250 of the entities used (81% for curative purposes) can be toxic unless dosed appropriately.
Many (55%) are dietary supplements. The list included 186 species (45%) for internal and 175 (55%) for external use, many used in places
where animals are kept. The species belong to 71 families, among which the Fabaceae predominate. The purpose of the study was to provide
information that can be validated and associated with correct determination, permitting even potentially dangerous plants to be used in
veterinary practice.

Keywords: Toxic plants, ethnoveterinary medicine, Italy.

The number of plants used in Italy to treat domestic
animals was previously reported to be 260 [1] and is now
more than 500 [2a-c]. These plants include fungi, ferns,
gymnosperms and angiosperms. Most are dietary
supplements, chosen for their positive effect on growth
and ease of administration. Many are used for prevention,
but more than 60% of all uses are curative. Some plants
are valued against parasites and as repellents, others for
their toxic effects on fish. Other plants are considered to
have magic properties. In this study we analyze use of
toxic and potentially toxic plants in ethnoveterinary
medicine in Italy.
Bibliographic and unpublished data in our database were
examined and screened for toxic and potentially toxic
plants. Shepherds and farmers generally avoid
administering such plants, though many were used
traditionally and considered relatively safe. More than
250 toxic or potentially toxic plants were identified, about
50% of all species used in ethnoveterinary medicine.
Most (81%) are used for curative purposes and can be
toxic if not appropriately dosed. A good number (55%)
are dietary supplements. 186 species (45%) are used
internally and 175 externally (55%) (Table 1). The active
ingredients, largely glycosides and alkaloids, are listed in
Table 2. The types of animals treated were cattle
(23.93%), sheep (10.73%), poultry (9.5%), horses
(7.83%), pigs (6.38 %), goats (5%), dogs and cats
(3.19%), rabbits (2.18%) and animals in general (27%).
Many plants were used in places where animals are kept,
such as stables, chicken houses, drinking troughs (3.92%)
(e.g. Alnus glutinosa, Artemisia absinthium, Datura
stramonium, Nerium oleander, Sambucus ebulus, S.
nigra).
Toxic plants belong to 71 families, 62 of which are
Angiosperms, one Gymnosperm, seven Pteridophytes,
one fungus (Amanitaceae). The most common families
were Fabaceae, followed by Asteraceae, Ranunculaceae,
Labiatae, Euphorbiaceae, Apiaceae and Liliaceae. This
differs slightly from the general statistics for
ethnoveterinary medicine, which indicate species of the
family Asteraceae to be the most numerous [3a,3b], as
found in the Mediterranean area in general [4].
The toxic or potentially toxic species identified had largely
curative uses. The main methods of administration were as
such, decoction, crushed and macerated. The main active
ingredients were glycosides and alkaloids, with saponins
and triterpenoids accounting for more than 13%, followed
by tannins, volatile oils, terpenoids and resins [5a-5d]. In
many species, the toxic substances are not distributed
throughout the plant: many are concentrated in certain
organs while the rest of the plant is innocuous; sometimes
substances are influenced by the vegetative period or age
of the plant. Toxic substances are often more abundant in
certain phases of the life cycle, usually in seeds and
juvenile plants, and in certain phases of the vegetative
cycle, usually spring. Sometimes plants can be toxic if not
appropriately dosed, if infected by fungi, if they
accumulate harmful substances or if combined with
conventional remedies [6a-6c].
Toxic plants were certainly used with caution in
ethnoveterinary traditions, because loss of an animal was a
serious event. Their use cannot be encouraged. The aim of
the present study was to provide information that can be
validated and associated with correct determination,
permitting even potentially dangerous plants to be used in
1000 Natural Product Communications Vol. 6 (7) 2011 Viegi & Vangelisti

veterinary practice. The study is part of a series concerned pharmacological research and for the conservation of
with the enormous heritage of empirical experience and native flora. Benefits may range from local to European
knowledge still traceable in Italy. Such documentation is community level.
useful to save animal lives, for phytochemical-
Table 1: Uses and examples of toxic or potentially toxic plants used in ethnoveterinary medicine in Italy.
Internal use Vermicides Ichthyotoxic Flea, mouse and mole repellents Pesticides and repellents
Agrostemma githago Allium sativum Achillea ligustica Aconitum napellus Colchicum autumnale,
Amanita muscaria Artemisia absinthium, Anthirrinum majus Alnus glutinosa Anemone hortensis
Anemone hortensis A. vulgaris Conium maculatum Artemisia absinthium Colchicum autumnale
Crocus neapolitanus Calamintha nepeta Cyclamen repandum Calamintha nepeta Cestrum parqui
Daphne mezereum Cucurbita pepo Daphne gnidium Conium maculatum Daphne laureola
Dryopteris filix-mas Dryopteris filix-mas Euonymus europaeus Delphinium consolida, Delphinium consolida
Euphorbia dendroides, Fraxinus ornus Euphorbia characias, D. staphysagria Euphorbia helioscopia
E. lathyris Glechoma hederacea E. dendroides, E. helioscopia, Ficus carica Helichrysum italicum
Eucalyptus resinifer Juglans regia E. lathyris, E. paralias, Laburnum alpinum Veratrum album
Helleborus sp.pl. Mercurialis annua E. pinea, E. pithyusa L. anagyroides
Ilex aquifolium Polypodium australe Juglans regia Lupinus albus
Ligustrum vulgare Ruta angustifolia, Marrubium vulgare Ruscus aculeatus
Glechoma hederacea R. chalepensis Oenanthe crocata Ruta graveolens
Mercurialis annua R. graveolens Pistacia lentiscus Tanacetum vulgare
Papaver rhoeas, Santolina insularis Plumbago europaea Veratrum album
P. somniferum Sempervivum tectorum Sambucus sp.
Polypodium australe, Verbascum thapsus Solanum nigrum
P. vulgare Teucrium chamaedrys
Polystichum setiferum Thapsia garganica
Rhamnus sp.pl. Urginea maritima
Ricinus communis Verbascum pulverulentum,
Veratrum album V. sinuatum, V. thapsus

Table 2: Active principles of toxic or potentially toxic plants used in ethnoveterinary medicine in Italy.
% no. of species Notes (active compounds and the no. of species that contain them)
Glycosides 25.82 110 coumarin and furocoumarin (11); saponinic glycosides (15)
Alkaloids 19.48 83
Saponins and triterpenoids 13.38 57 saponins (non saponinic glycosides) (51)
Tannins 8.92 38
Volatile oils, terpenoids, resins 8.22 35 resins (5)
Organic acids 6.10 26 oxalates (13)
Pigments (flavonoids) 4.23 18
Diterpenoids 3.29 14
Phytoestrogens 3.05 13 phytoestrogens, phytosterols
Nitrates and nitrites 2.11 9
Lignans and lignins 2.11 9
Toxic if infected with fungus or in cases of accumulation 1.88 8
Enzymes 1.41 6 thiaminase, urease, ficin

References
[1] Viegi L, Pieroni A, Guarrera PM, Vangelisti R. (2003) A review of plants used in folk veterinary medicine in Italy as basis for a
databank. Journal of Ethnopharmacology, 89, 221-244.
[2] (a) Viegi L, Camarda I, Piras G. (2005) Some aspects of ethnoveterinary medicine in Sardinia (Italy). Proceed. IV International
Congress of Ethnobotany (ICEB 2005), 21-26 August, Istanbul, Turkey, 135-136; (b) Bullitta S, Piluzza G, Viegi L. (2007) Plant
resources used for traditional ethnoveterinary phytotherapy in Sardinia (Italy). Genetic Resources and Crop Evolution, 54,
1447-1464; (c) Felicioli A, Giusti M, Vangelisti R, Viegi L. (2008) Plants used as antiparasitic in italian ethnoveterinary medicine.
Parassitologia, 50, Suppl. 1, 210.
[3] (a) Fossati F, Bianchi A, Favali MA. (1999) Farmacopea popolare del parmense: passato e presente. Informatore Botanico Italiano,
31, 171-176; (b) Barbini S, Tarascio M, Sacchetti G, Bruni A. (1999) Studio preliminare sull'etnofarmacologia delle comunità
ladino dolomitiche. Atti Colloquio S.B.I. "Botanica Farmaceutica ed etnobotanica alle soglie del duemila: passato e futuro a
confronto", Genova, 9-11 aprile 1999. Informatore Botanico Italiano, 31, 181-182.
[4] Pieroni A, Giusti ME, de Pasquale C, Lenzarini C, Censorii E, Gonzáles-Tejero MR, Sánchez-Rojas CP, Ramiro-Gutiérrez J,
Skoula M, Johnson C, Sarpaki A, Della A, Paraskeva-Hadijchambi D, Hadjichambis A, Hmamouchi M, El-Jorhi S, El-Demerdash
M, El-Zayat M, Al-Shahaby O, Houmani Z, Scherazed M. (2006) Circum-Mediterranean cultural heritage and medicinal plant uses
in traditional animal healthcare: a field survey in eight selected areas within the RUBIA project. Journal of Ethnobiology and
Ethnomedicine, 2, 16-28.
[5] (a) Debelmas AM, Delaveau P. (1978) Guide des Plantes Dangereuses. Ed. Maloine, Paris; (b) Maugini E. (1994) Manuale di
Botanica Farmaceutica, VII edizione, Piccin, Padova; (c) Lorgue G, Lechenet J, Rivière A. (1999) Tossicologia clinica
veterinaria. C. Giraldi Ed.; (d) Frohne D, Pfänder HJ. (2004) Poisonous plants. 2nd Edition. Manson Publishing Ltd, London
[6] (a) Bruneton J. (1999) Toxic Plants Dangerous to Humans and Animals. Lavoisier Publishing, Paris; (b) Frohne D, Pfänder HJ.
(2004) Poisonous plants. 2nd Edition. Manson Publishing Ltd, London; (c) Wynn SG, Fougère BJ. (2007) Veterinary Herbal
Medicine. Mosby, Elsevier.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Diagnosis of Public Programs focused on Herbal Medicines 1001 - 1002
in Brazil
Ely Eduardo Saranz Camargoa, Mary Anne Medeiros Bandeirab and
Anselmo Gomes de Oliveirac
a
Programa de Pós-graduação em Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas,
Universidade Estadual Paulista Julio de Mesquita Filho-Unesp, Rodovia Araraquara-Jaú, km 01,
14801-902, Araraquara, SP. Brazil
b
Universidade Federal do Ceará, Faculdade de Farmácia, Odontologia e Enfermagem,
Rua Alexandre Baraúna, 949, Fortaleza, CE

elycamargo@ipcs.com.br

Received: November 12th, 2010; Accepted: March 25th, 2011

The present study is aimed to diagnose the current public programs focused on herbal medicines in Brazil by means of in loco visits to 10
programs selected by means of questionnaires sent to 124 municipalities that count on herbal medicine services. The main purpose of the
implementation of program programs is related to the development of medicinal herbs. 70% of them are intended for the production of herbal
medicines and 50% are aimed to ensure the access of the population to medicinal plants and or herbal medicines. The initiative of the
implementation of these programs was related to the managers (60%). The difficulties in this implementation were due to the lack of funding
(100%) of the programs. In 60% of the programs, the physicians did not adhere to herbal medicine services due to the lack of knowledge of
the subject. Training courses were proposed (80%) to increase the adhesion of prescribers to the system. Some municipalities use information
obtained from patients to assess the therapeutic efficiency of medicinal plants and herbal medicines. Of the programs underway, cultivation
of medicinal plants was observed in 90% and 78% of them adopt quality control. In most programs, this control is not performed in
accordance with the legal requirements. The programs focused on medicinal plants and herbal medicines implemented in Brazil face some
chronic problems of infrastructure, management, operational capacity and self-sustainability, which can be directly related to the absence of a
national policy on medicinal plants and herbal medicines.

Keywords: Medicinal plants, herbal medicines, public health.

The use of medicinal plants dates back thousands of years.
However, the traditional allopathic medicine faces
increased competition from alternative treatments due to
the production of herbal medicines in accordance to the
standards recommended by the legislation these days [1a].
The development of quality assurance within the
pharmaceutical industry involves the concern with the
production of seeds, planting, harvesting, drying,
extraction, production practices and storage of drugs, and
all these processes must be carried out according to a strict
quality control, pre-clinical and clinical trials and data
record. Quality assurance has enabled the health
professionals to prescribe safely herbal medicines that the
population has been taking for quite a long time [1b].
The use of medicinal plants for therapeutic purposes (both
the traditional and popular usage of these plants and their
use based on scientific evidence) is a common practice
nowadays. The use of plants to mitigate symptoms or cure
diseases has been a very common practice for long,
especially because the resources used in the production of
drugs are not available according to the needs of the
population, or else, many of these drugs are currently
being developed. These historical aspects of the use of
medicinal plants clarify the importance of the interaction
between local communities and their natural environment
to the entire society in the present and in the future [1c]. At
the same time, there is the urgent need to investigate the
abundant biodiversity and, particularly, the medicinal
flora, its proper and rational use, in order to find out how
medicinal plants can be used by the population for
medicinal purposes in an efficient and safe way [2a].
Data generated from the questionnaires completed has
demonstrated the existence of well-structured programs,
and the activities carried out within these programs
involved pharmaceutical care, corroborating the expansion
and effectiveness of the National Policy of Integrative and
Complementary Practices at the SUS (Brazilian Public
Health System) [2b]. Thus, visits to 10 programs focused
on herbal medicines were scheduled and had excellent
results, as demonstrated in the previously described
research. [2c]. The visits were conducted from December
2008 to December 2009 and were aimed to collect
information on the activities developed within the scope
of the programs, as well as information on their
1002 Natural Product Communications Vol. 6 (7) 2011 Camargo et al.

implementation, difficulties faced and solutions
encountered to ensure the maintenance of the referred
programs. The programs selected for visit were carried out
in all the Brazilian regions as follows: 1 in the Southern
region, 5 in the Southeastern region, 2 in the Center-West
region, 1 in the Northeastern region and 1 in the Northern
region. The selection was based on the data generated from
the questionnaires sent to 124 Brazilian municipalities. It
was found that in all programs visited (10) the main
purpose of their implementation was the development of
medicinal plants. In 70% of them the purpose was the
production of herbal medicines, 50% were aimed to ensure
the access of the population to these medicinal drugs and
only one program (10%) was aimed to decrease the
healthcare costs incurred by the population. 60% of the
programs stemmed from the initiative of municipal
governments, 50% were suggested by experts, 20% by
users and 30% had different origins. The same results were
obtained when the initiatives were carried out by more
than one group. Regarding the inclusion of herbal
medicine in pharmaceutical care programs in the
municipalities, such inclusion was found to occur in 70%
of them and in 30% of them it did not occur. These
findings may have a direct influence on the maintenance of
the programs, since funding issues can compromise the
survival of those programs not included in the
pharmaceutical care programs of the municipalities.
The difficulties in the implementation of these programs
included: lack of funding (100%), poor adhesion by
prescribers (60%), lack of space (70%) lack of qualified
professionals (40%) and 1 (10%) mentioned the lack of
interest of the population. Still regarding the difficulties
encountered, it was found that 90% of the programs were
funded by the municipality and 40% were funded by the
state, but only 1 (10%) was exclusively funded by the
state. Regarding the cultivation of medicinal plants, only 1
(10%) did not count on a garden and raw material for
herbal medicines was obtained from suppliers. The
selection of the medicinal species cultivated was based on
literature research (100%), an in 80% of them it was also
based on popular knowledge and in 40% the local species
were used. Only 1 (10%) program did not count on a
herbal medicine workshop, being focused on the
distribution of medicinal plants. In all the programs that
involved herbal medicine handling a pharmacist was the
technical responsible person, according to the country’s
legislation. 7 out of these professionals (80%) are
registered with their respective regional board of pharmacy
and only 2 (20%) were not registered with their regulatory
bodies. The pharmaceutical forms handled in the programs
are distributed according to the number of quotations in the
herbal medicine workshops: 67% of the programs deliver
solutions, 22% suspensions, 55% capsules, 78% dyes, 11%
elixirs, 89% syrups, 89% creams, 55% ointments and 44%
liquid soaps. It has also been found that 78% of the programs
that handle herbal medicines performed quality control.
The distribution of medicinal plants and herbal medicines
is performed by the pharmacist in all the cases, and in
some of them, it is also performed by physicians (50%),
nurses (50%), technicians (40%), other professionals, such
as dentists, biologists and nutritionists (20%). Therefore, in
80% of the total programs visited there was some kind of
follow-up of patients taking medicinal plants or herbal
medicines, 20% did not have any kind of follow-up
service. However, in some of the programs that reported
follow-up services, the evaluation was not recorded.
Regarding efficiency assessment, 80% of the programs
performed this assessment and 20% of them did not
perform any efficiency assessment.
80% of the programs provide training courses to the
professionals involved in herbal medicines and 20% of
them do not count on any kind of training courses or
activities. However, it was found that all the programs
visited had educational activities targeted at the local
community. One issue that has been greatly discussed
with the coordinators of the programs visited was the
partnership with universities or other institutions, both
public and private, Only 3 (30%) of them have established
partnerships, mostly with universities, and 70% have no
kind of partnership. Nevertheless, most programs had tried
unsuccessfully to establish partnerships, and the main
reason for this was the lack of funding.
Finally, the results obtained in this study conclude that
herbal medicine is getting considerable attention and has
become a valuable asset that ensures the access of the
population to basic health care.

References

[1] (a) Brasil. (2010) Ministério da Saúde. Agencia Nacional de Vigilância Sanitária. Resolução de Diretoria Colegiada (RDC) no. 10,
de 09 de março de 2010. Dispõe sobre a Notificação de Drogas Vegetais Junto a Agencia Nacional de Vigilância Sanitária. Diário
Oficial de União, Brasília; (b) Goodman LS, Gilman AG, Hardman JG. (2003) As Bases Farmacológicas Da Terapêutica. 10a ed.
Rio de Janeiro: Mc GrawHill, 2003, 1436 p; (c) Brasil. (2006) Ministério da Saúde. Secretaria de Ciência, Tecnologia e Insumos
Estratégicos. Departamento de Assistência Farmacêutica. A fitoterapia no SUS e o programa de pesquisa de plantas medicinais da
central de medicamentos/ Ministério da Saúde, Secretaria de Ciência, Tecnologia e Insumos Estratégicos, Departamento de
Assistência Farmacêutica. – Brasília: Ministério da Saúde. 148p. – “serie B. textos básicos de saúde”.
[2] (a) Alzugaray D. (1988) Enciclopédia da plantas medicinais, Editora três Ltda São Paulo; (b) Brasil. (2006) Ministério da Saúde.
Secretaria de Atenção a Saúde. Departamento de Atenção Básica. Política nacional de práticas integrativas e complementares no
SUS – PNPIC-SUS/Ministério da Saúde, Secretaria de Atenção a Saúde, Departamento de Atenção Básica. – Brasília: Ministério
da Saúde, 92 p. – “serie B. texto básico de saúde”; (c) Camargo EES. (2010) Avaliação dos programas de plantas medicinais e
medicamentos fitoterápicos, visando subsidiar sua reorientação no sistema único de saúde. Tese (Doutorado em Ciências
Farmacêuticas) Universidade Estadual Paulista. Araraquara – SP, 230p
 2011
NPC Natural Product Communications Vol. 6
No. 7
Identification of Thiosildenafil in a Health Supplement 1003 - 1004

Marcello Nicoletti

Dep. Enviromental Ecology, University Sapienza, Rome, Italy

marcello.nicoletti@uniroma1.it

Received: December 11th, 2010; Accepted: March 16th, 2011

The presence of a sildenafil derivative, the thiosildenafil, in an herbal product has been evidenced first by HPTLC and later determined by
isolation and analysis of spectroscopic data. The analyzed product is nowadays marketed as dietary supplement containing herbal extracts
and claimed for male and female sexual improvement. This report is noteworthy since it is clear that adulterated materials can cause serious
health problems if they are consumed as herbal “natural” products, generally considered deprived of toxicity by the consumers. The use of a
simple and reliable method, based on HPTLC, to determine synthetic adulterations is reported in this paper.

Keywords: thiosildenafil, sildenafil, dietary supplement, HPTLC, NMR.

The presence of synthetic drugs in the formulation of

R
10

herbal products in order to improve the efficacy has been HN 6 5

4
N

N
reported in several cases. The case seems to be very 24
O2
S
7
8 9
1
N
16 15 N
important in adulteration of herbal products marked as N
25
28 17
18
14
19 20
21
11 12

27
“natural” in cases of erectile dysfunction, since an abuse 29
O 13

can be very dangerous for patients who unwittingly R = O sildenafil

consume a synthetic potent drug instead of a botanical.
In the recent years, in particular, several cases have been
reported about such herbal products produced in China
[1-3]. The reported monitoring aspects were essentially
based on the analytical analyses concerning the detection
of the adulterants that in these cases are synthetic selective
inhibitors of cyclic guanosine monophosphodiesterase-5
(PDE-5). Such active principles are the well known
registered drugs, but also a plethora of analogues obtained
by minor modifications to the basic structure of PDE-5
inhibitors was found. The reason of this proliferation is
mainly due to the intention to escape analytical controls.
Therefore, several efforts were focused on the best
analytical tool to catch the adulterant, since first HPLC
determination [4], followed by NMR [5], LC/MS and
LC/MS/MS [6], until the recent 2D and 3D DOSY 1H
NMR [7]. In this paper we report a further case of
adulteration and propose the use of HPTLC as simple,
direct and low cost method to detect such and other
adulterants, also in case of their presence in complex
herbal mixtures.
Whereas sildenafil citrate (Viagra®, manufactured by
Pfizer), vardenafil hydrochloride (manufactured by
Levitra) and tadalafil (Cialis®, manufactured by Lilly) are
well known compounds approved by the U.S. Food and
Drug Administration for the treatment of erectile
dysfunction, the analogues usually are not subjected to
any control. In any case whereas registered products are
R = S thiosildenafil
prescription drugs and must be used under medical
supervision, herbal products are self administered and
generally regarded as being harmless because of their
natural origin. Confusion between the two categories is
therefore very dangerous.
Recently, a survey of the analysis of the presence of
synthetic PDE-5 inhibitors in dietary supplements has been
reported [3]. Among the seventeen considered commercial
formulations of herbal drugs or dietary supplements
marketed for sexual dysfunction, eight resulted
adulterated, containing sildenafil, tadalafil, vardenafil,
hydroxyhomosildenafil and/or thiomethisosildenafil.
We were able to examine the content of an herbal
supplement heavily marketed in internet sites as Sensual
Tea or Jinshenkang, commercialized as able to rapidly
solve any sexual problem of females and males. The
product, also marketed in Italy, came from Spain, where
now the product has been removed from the market. First,
a HPTLC in dichloromethane: methanol (9:1, v/v) in a
horizontal chamber (Camag 20X10) after saturation with
the same mobile phase was performed. The plate showed a
great spot very strong at UV lamp at 250 nm; its position
and its intensity, in comparison with the other spots due to
components of the herbal extracts, were an evident clue of
the adulteration. Direct HPTLC comparison with
sildenafil, verdenafil and tadalafil excluded the identity
with these substances. Also densitometry analysis
confirmed the differences.
1004 Natural Product Communications Vol. 6 (7) 2011 Nicoletti

Easily, extraction with ethyl acetate afforded a complete
remove of the substance from the product and NMR
spectrum of the extract resulted in a highly pure compound
identified as thiosildenafil, by comparison with reported
data and analysis of 2D spectra. In particular, making a
1
H-NMR comparison with sildenafil [3], it is diagnostic
the upfield shift (δH = + 0.12) of the N-Me, due the
influence of the thiocarbonyl. The LC-MS analysis
confirmed the presence of thiosildenafil, as well as a little
quantity of sildenafil, probably as remaining product
of the conversion into the thioderivative. Quantitative
determination was obtained by the NMR method proposed
by Balyssac [1] and gave for each package a quantity equal
to that of a tablet of Viagra. Independently, HPLC analysis
was performed confirming the non identity with previous
compounds and affording similar quantitative results.
Herbal products spiked with synthetic drugs are dangerous
to consumers and noxious for future correct developing of
use of natural products. Controls must be based on simple,
viable and low cost analyses. Therefore, HPTLC is a
strong candidate to be used in the detection of anomalous
constituents in botanicals to obtain easy clues of the
adulteration.
Experimental
HPTLC in silica gel 60 in dichloromethane: methanol (9:1,
v/v) developed in a horizontal chamber (Camag 20X10).
Deposition with CAMAG Linomat IV, TLC scanner 3
WINCATS software. For the densitometric analysis a
Camag TLC scanner 3 linked to winCATS software was
used after multi-wavelength scanning between 250 and
400 nm. NMR by BRUKER AM400 at 400 MHz for 1H
NMR and 100 MHz for 13C NMR. Spectra were recorded
in CDCl3 using CHCl3 signal (7.23 and 77.0 ppm) as
internal reference. MS by hyphenated LC/MS LXQ
Thermo Electron.
Samples and extraction: The analyzed samples were
imported from Spain and sold as a dietary supplement in
package containing white granules. The reported content
of the granules was mainly sugar and a series of herbal
extracts. Sildenafil, tadalafil and vardenafil were obtained
from corresponding marketed products.
HPTLC analysis: The granules of a package (40 g) were
grounded and extracted with ethyl acetate (50 mL) for 4 h.
After filtration over 0.45 m filter, the filtrate was
evaporated and dissolved in 50 mL of ethyl acetate/H2O
1:1 (v/v). The content of the organic phase was directly
used for analysis, including HPTLC using sildenafil and
vardenafil as reference standards. At UV lamp at 350 nm
the Rf value of the unknown constituent (0.81) resulted
significantly higher than those of the two reference
compounds (0.79 and 0.74 for sildenafil and vardenafil,
respectively). The layers, treated with H2SO4 2N spray
reagent and subsequent worming at 110°C, showed other
minor spots at lower RF, probably due to the natural
products. For complete identification and improve the
quality of spectroscopic analyses, pure thiosildenafil was
obtained after silica gel CC in CHCl3:MeOH 40:1 of the
above organic phase.
Thiosildenafil: The ethyl acetate extract was evaporated
and the residue directly examined by NMR. 1H NMR (400
MHz, CDCl3) and ITMS data were in accordance with
reported ones [7].

References

[1] Balayssac S, Trefi S, Gilard V, Malet-Martino M, Martino R, Delsuc M-A. (2009) 2D and 3D DOSY 1H NMR, a useful tool for
analysis of complex mixtures: Application to herbal drugs or dietary supplements for erectile dysfunction. Journal of
Pharmaceutical and Biomedical Analysis, 50, 602-612.
[2] Blok-Tip L, Zomer B, Bakker F, Hartog KD, Hamzink M, ten Hove J, Vredenbregt M, de Kaste D. (2004) Structure elucidation of
sildenafil analogues in herbal products. Food Additives and Contamination, 21, 737-748.
[3] Singh S, Prasad B, Savaliya AA, Shah RP, Gohil VM, Kaur A (2009) Strategies for characterizing sildenafil, vardenafil, tadalafil
and their analogues in herbal dietary supplements, and detecting counterfeit products containing these drugs. Trends in Analytical
Chemistry, 28, 13-26.
[4] Daraghmeh N, Al-Omari M, Badwan AA, Jaber AMY. (2001) Determination of sildenafil citrate and related substances in the
commercial products and tablet dosage form using HPLC. Journal of Pharmaceutical and Biomedical Analysis, 25, 483-491.
[5] Wawer I, Pisklak M, Chilmonczyk Z. (2005) 1H, 13C, 15N NMR analysis of sildenafil base and citrate (Viagra) in solution, solid
state and pharmaceutical dosage forms. Journal of Pharmaceutical and Biomedical Analysis, 38, 865-870.
[6] Park HJ, Jeong HK, Chang MI, IM MH, Jeong JY, Choi DM, Park K, Hong MK, Youm J, Han SB, Kim DJ, Park JH, Kwon SW.
(2007) Structure determination of new analogues of verdenafil and sildenafil in dietary supplements. Food Additives and
Contaminations, 24, 122-129.
[7] Trefi S, Gilard V, Balayssac S, Malet-Martino M, Martino R. (2009) The usefulness of 2D DOSY and 3D DOSY-COSY 1H NMR
for mixture analysis: application to genuine and fake formulations of sildenafil (Viagra). Magnetic Resonance in Chemistry, 47,
S163-173.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Hypolipidemic Effect of Seed Oil of Noni (Morinda citrifolia) 1005 - 1008

Diana C. Pazosa, Fabiola E. Jiménezb, Leticia Garduñoa, V. Eric Lópezb and M. Carmen Cruzb,*
a
Departamento de Farmacia, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional,
CP 11340, México DF, México
b
Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, CP 90700,
Tepetitla, Tlaxcala, México

dianadelcpg@yahoo.com, ccruz25@hotmail.com

Received: November 14th, 2010; Accepted: March 16th, 2011

Morinda citrifolia, has been reported to posses different biological activities and almost all parts of this have been studied phytochemically.
However there are few studies on the seeds of fruit. The objective of present study was investigated the effect to Noni Seed Oil (NSO) on
serum lipid levels in normolipidemic and hyperlipidemic induced mice. We find that administration of noni oil causes a reduction in total
cholesterol and triglycerides levels in both models. However hypolipidemic effect is higher when hyperlipidemia is presented.

Keywords: Morinda citrifolia seed oil, Atherogenic Index, Linolenic acid, hypolipidemic effect, tyloxapol.

Morinda citrifolia L. (Rubiaceae) commonly known as Hyperlipidemic is defined as elevated lipid levels in
noni, is an evergreen tree may reach heights of 3 to 8 m plasma, and represent one of the factors associated with
tall. Its leaves range from 10 to 45 cm long and it bears cardiovascular diseases, which are a worldwide death
tubular white flowers and a green fruit. This fruit turns cause [11-13]. Treatment of dyslipidemia reduces
yellow and then white as it ripens, has a pungent odor and cardiovascular events. The modern pharmacological
contains seeds of about 3 mm in length. This plant is therapy for abnormal lipids is effective but is expensive
native to Asia, Australia and Polynesia [1]. The plant is and it is associated with side-effects leading to patient
used in the treatment of arthritis, headaches, digestive incompliance. For this reason, our work evaluates the
problems, diabetes mellitus, high blood pressure, and effect of NSO on lipid levels (total cholesterol (chol),
angina pectoris among others [2]. The Leaves, steam, root, triglyceride (Tg), high density lipoprotein (chol-HDL), in
fruit and seeds of noni are used in various forms such as normolipidemic and hyperlipidemic mice. Castelli’s
capsules, teas, juice and oil [2,3]. Atherogenic Index (AI) was calculated for determined risk
factor of cardiovascular disease with noni seed oil
Due to the great popularity of this plant many consumed.
phytochemical studies have been carried out in which have
reported compounds as iridoids, anthraquinones phenolics, Table 1: Gas chromatographic retention times (Rt) and molecular weights
of fatty methyl esters from NSO.
glycosides of fatty acids and alcohols, cumarins,
flavonoids, alkaloids and terpenes [4]. Regarding the Compound Rt (min) M+ (amu) %
biological activity of this specie, the antimicrobial effect Methyl palmitate 38.5 270 9.4
Methyl palmitoleate 39.2 268 0.7
was the first observed property [5], however other effects Methyl stearate 42.5 298 4.2
like antitubercular [6], hypoglycemic [7], anti- Methyl oleate 43.1 296 15.9
inflammatory [8], antitumor [9] and analgesic [10] have Methyl linoleate 44.2 294 67.8
been reported.
Analysis of NSO
Among these one evaluated the toxicity and nutritional The total yield of oil for two extractions of dried seeds was
value, as well as the determination of the fatty acid 12%. GC-MS analysis of the FAME (fatty acid methyl
composition to assess if it is usable as edible vegetable oil. esters) prepared by transesterification procedure indicated
Seeds constitute 2.5% of the whole fruit and are the presence of five fatty acids with the relative
considered a waste in the industrial process for making composition shown in Table 1. The FAME showed mass
juice [3]. Although anthraquinones and fatty acids such as spectra with molecular ions at m/z 270, 268, 298, 296 and
arachidonic and palmitoleic have been isolated from these 294, corresponding to the retention times of 38.5, 39.2,
seeds, it has not been established whether they show any 42.5, 43.1, 44.2 min. These times indicated the presence of
biological activity [1]. palmitic (C16), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1)
1006 Natural Product Communications Vol. 6 (7) 2011 Pazos et al.

Table 2: Hypolipidemic effect produced by NSO in normolipidemic mice.

Doses
Total-Chol (%) Tg (%) chol-HDL (%)
(mg/kg/day)
CONTROL 100±2.1 100±0.18 100±1.58
NSO 150 4.16±1.07 11.26±0.17 -13.91±1.49
NSO 300 -11.01±1.73 -12.2±0.22 -10.36±2.11
NSO 600 -19.09±2.69 -33.7±0.19 -22.42±3.2

Table 3: Hypolipidemic effect produced by NSO in hyperlipidemic mice

male.
Doses
Total-chol (%) Tg (%) chol-HDL (%)
(mg/kg/day)
CONTROL 100±6.6 100±1.08 100±0.95
NSO 150 -22.13±1.72 -74.2±0.09* 33.54±1.06
NSO 300 5.73±7.16 -64.97±0.31 39.54±1.49
NSO 600 -15.57±1.8 -31.79±0.24 42.85±1.29
* Represents significant difference when compared with the control when Figure 1: Castelli’s Atherogenic index for normolipidemic and
analyzed by ANOVA ± standard error. hyperlipidemic mice treated with different doses of NSO.

and linoleic (C18:2) fatty acids respectively, being the inhibition of cholesterol biosynthesis by inhibition of
principal component linoleic acid. However further studies HMG Co-A. However, the failure of NSO to cause
are necessary to determine the exact chemical complete inhibition indicates the involvement of additional
composition. mechanisms.

Biological assay: Data on the effect on lipid-lowering
activity of the three parameters evaluated are summarized
in Tables 2 and 3. All animals showed good health during
the period of administration. At necropsy no changes were
found in the organs of the treated animals.
Animals administered with 600 mg/Kg (Table 2) registered
the most important effect with a reduction of -19.09% and
-33.7% for total cholesterol and triglycerides respectively.
These results suggest a hypolipidemic activity of seed oil
and a relationship between dose and effect.
Treatment of ICR mice with Triton WR 1339 (tyloxapol)
resulted in a significant elevation in total serum
cholesterol, HDL-cholesterol and triglycerides respect to
control.
Tyloxapol is a non-ionic surfactant being widely used to
explore possible mechanism of lipid lowering drugs, it
causes drastic increase in serum triglycerides and
cholesterol levels due to increase in hepatic cholesterol
synthesis particularly by the increase in HMG Co-A
(3-hydroxy-3-methyl-glutaryl Co-A) activity and by the
inhibition of lipoprotein lipase responsible for hydrolysis
of plasma lipids [14].
The Table 3 shows data about the use of NSO in
hyperlipidemic mice. The lowest reduction values for
cholesterol (-22.15%) and triglycerides (-74.2%) were
obtained with dose of 150 mg/kg, exhibiting the best
hypolipidemic effect. Not relationship dose-effect in
cholesterol was observed. Nevertheless for triglycerides is
notorious inverse relationship dose-effect.
Significant inhibition of lipid levels increase by noni seed
oil of Morinda citrifolia in this model is indicative of the
Various indices have been used for the diagnosis and
prognosis of cardiovascular disease, one of them was
reported by Castelli [11]. Castelli’s Atherogenic index (AI)
is total cholesterol/HDL ratio and it is considered that a
value below than four units represents a low risk of
cardiovascular disease [11-13]. AIs were calculated for
the different groups, being that it is smaller for
normolipidemic group when administered NSO (Figure 1).
While for the hyperlipidemic group a slight decrease
regarding the control is observed only at dose of 300
mg/Kg. However the values for this group stay below the
one it limits.
We find that the administration of noni oil causes a
hypolipidemic effect, mainly evident when hyperlipidemia
occurs. The results from this study rationalize the
medicinal use of noni seed oil in dislipidemia. However
further studies are required to prove efficacy of noni seed
oil in dyslipidemia and to prove that it can be used as a
potential medicine for cardiovascular diseases.
The main constituent of noni seed oil is linoleic acid,
however hypolipidemic activity may be due to the
presence of other compounds in oil.
Experimental
Extraction seed oil (NSO): The noni fruit was obtained
from Córdoba Veracruz, México. The seeds were obtained
from fresh fruits. The batch of seeds were washed and
dried at room temperature. Dried seeds were ground and
extracted by maceration with food-grade hexane at 1:5
ratio at room temperature for 24 h. The extract was filtered
and the solvent removed by distillation under reduced
pressure. The crude oil was used in the bioassay.
Hypolipidemic effect of Morinda citrifolia seed oil Natural Product Communications Vol. 6 (7) 2011 1007

Oil analysis: Fatty acid composition was determined for
gas chromatography (GC) of methyl ester obtained after
transesterification of the crude oil [15] on a Hewlett-
Packard 5890 Series II Gas Chromatograph equipped with
a 5971A Mass Selective detector and using an HP-
Innowax capillary column (30 m x 0.2 mm x 0.25 μm thick
coating). Helium was used as the carrier gas at a flow rate
of 1 mL/min with the injector set to split mode at 250ºC.
The oven was programmed from 100 to 140°C at
1.5°C/min and then from 140 to 250°C at 5°C/min and
held at 250ºC for 10 min. The detector was operated at
70 eV in scanning mode over the range of 50–550 amu.
Mass spectra were compared with data in NIS library and
% of fatty acid was calculated for integration value for
chromatogram.
Hypolipidemic Evaluation: Hypolipidemic activity was
studied in (ICR) male mice weighing 25-30g (Birmex, S.
A., Mexico City). All animals were housed in hanging
metal cages and maintained at 24±2 ºC and 50±10%
relative humidity, with 12 h light/dark cycle. They were
fed on standard pellet diets (Rodent Diet 5001, PMI
Nutrition International, Inc. Brenwood, MO) and drinking
water was freely available. All animals appeared healthy
throughout the dosing period, maintaining normal food
intake and weight gain. At sacrifice, no gross
abnormalities were observed in any treated mice.
All animals were treated in accordance with ethical
principles and regulations specified by the Animal Care
and Use Committee of our institution and the standards of
the National Institutes of Health of Mexico.
The mice were randomly divided into groups of six
animals. Hyperlipidemia was induced in the mice by
administration of Triton WR 1339 (Tyloxapol) was
dissolved in water at 400 mg/Kg. The seed oil was
administered 1h before and 22 and 48 h after the tyloxapol
injection [16].
Mice were treated with the oil seed suspended in a
1:4.5:4.5 tween 80: mineral oil: saline solution and
administered orally by an incubation needle at doses of
150, 300 or 600 mg/Kg/day for 28 days. Animals receiving
the vehicle were used as the non-cholesterol control group.
For tyloxapol-treated mice blood samples were taken 48 h
after injection. On the other hand, animals receiving
treatment for 28 days were fasted for 12 h before sacrifice.
Blood samples were collected by periorbital plexus
bleeding and centrifuged at 3000 rpm for 15 min. Total
cholesterol (Col), high-density lipoprotein cholesterol (col-
HDL), and triglycerides (Tg) levels were determined in the
serum, using a Wiener lab, Selectra 2000 automatic
analyzer.
All data are expressed as the percentage of the cholesterol
group control (the mean± standard error) by using Student
9t test. P values less than 0.05 considered statistically
significant.
Castelli Atherogenic index (AI) were calculated using the
equation IA= Total cholesterol/HDL-chol [11].
Acknowledgments – We thank SIP/IPN by financial
support (20090806). D.C-P is grateful to PIFI-IPN for the
scholarships awarded. M.C-C, L-C., and V.E.-L are
fellows of the EDI/IPN and COFAA/IPN programs.

References

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Madaeva Rao US, Subramanian S. (2009) Biochemical evaluation antihyperglycemic and antioxidative effects of Morinda
citrifolia extract studied in streptozotocin-induced diabetic rats. Medicinal Chemistry Research, 18, 433-446.
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Seino K, Nakamoto K, Kuninaga H, Suzuki T, Kimura Y. (2007) Anti-inflammatory and potential cancer chemopreventive
constituents of the fruits of Morinda citrifolia (Noni). Journal of Natural Products, 70, 754-757.
[9] (a) Hirazumi A, Furusawa E. (1999) An immunomodulatory polysaccharide- rich substance from the fruit juice of Morinda
citrifolia (Noni) with antitumor activity. Phytotherapy Research, 13, 380–387; (b) Hirazumi A, Furusawa E, Chou S C, Hokama Y.
(1996) Immunomodulation contributes to the anticancer activity of Morinda citrifolia (noni) fruit juice. Proceedings of the Western
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[10] Younos C, Rolland A, Fleurentin J, Lanhers MC, Misslin R, Mortier F. (1990) Analgesic and behavioral effects of Morinda
citrifolia. Planta Medica, 56, 430–434.
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Nacional de Gestión sanitaria, 1-19.
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 2011
NPC Natural Product Communications Vol. 6
No. 7
Composition of Egyptian Nerolì Oil 1009 - 1014

Ivana Bonaccorsia*, Danilo Sciarronea, Luisa Schipillitia, Alessandra Trozzib,

Hussein A. Fakhryc and Giovanni Dugoa
a
Dipartimento Farmaco-chimico, Università di Messina, V.le Annunziata, 98168 Messina, Italy
b
Dipartimento Farmaco-biologico, Università di Messina, V.le Annunziata, 98168 Messina, Italy
c
A. Fakhry & Co. 1081 Cornich El-Nil Cairo 11451, Egypt

bonaccor@pharma.unime.it

Received: November 10th, 2010; Accepted: March 3rd, 2011

The bitter orange flower oil (or nerolì) is an essential product, largely used in perfumery. Nerolì is obtained by hydrodistillation or steam
distillation, from the flowers of bitter orange (Citrus aurantium L.). Since a long time nerolì production is limited and its cost on the market
is considerably high. The annual production in Tunisia and Morocco is ca. 1500 Kg, representing more than 90% of the worldwide
production. A small amount of nerolì is also produced in Egypt, Spain and Comorros (not exceeding 150 kg totally). Due to the high cost, the
producers and the users have tried to obtain less expensive products, with odor characters close to that of nerolì oil to be used as substitute
and sometimes as adulterants of the genuine oil. In this study are investigated five samples of Egyptian nerolì oils produced in 2008 and
2009, in the same industrial plant, declared genuine by the producer. For all the samples the composition was determined by GC/FID and by
GC/MS-LRI; the samples were also analyzed by esGC to determine the enantiomeric distribution of twelve volatiles and by GC-C-IRMS for
the determination of the 13CVPDB values of some mono and sesquiterpene hydrocarbons, alcohols and esters. The analytical procedures
allowed to quantitatively determining 86 components. In particular the variation of the composition seems to be dependent on the period of
production. In fact, the amount of linalool decreases from March to April while linalyl acetate presents an opposite trend, increasing in the
same period. The RSD determined for the 13CVPDB are very small (max. 3.89%), ensuring the authenticity of all samples. The results are
also discussed in function of the limits provided by the European Pharmacopoeia (EP) (2004), AFNOR (1995) and ISO (2002) regulations for
genuine nerolì oils.

Keywords: Nerolì oil, Citrus aurantium L., GC, GC/MS-LRI, GC-C-IRMS, es-GC.

The oil of nerolì is obtained by hydrodistillation or
by steam distillation of the flowers of bitter orange
(C. aurantium L.).
Nerolì, rose and jasmine are often cited as “the three pearls
of perfumery”. Nerolì is the classic ingredient of the most
famous and prestigious perfumes and eau de cologne. It is
also used as flavor ingredient in food and beverages. In
traditional Chinese medicine the extracts from bitter
orange flowers are used to treat digestive problems and
insomnia.
Nerolì is the product of a laborious work: the flowers,
which bloom between the end of April and the beginning
of June are collected manually during the first hours of the
day; one worker can collect about 20 Kg per day for a
period of 20 days; the flowers are hydro- or steam-distilled
with a yield ranging from 0.08% at the beginning of the
season to a maximum of 0.13% under the most favorable
conditions. The production in the European Mediterranean
Countries (mainly France) is subject to a strong decrease,
mainly for the specialized working cost necessary to
collect the flowers, and for the contraction of the cultivated
fields of bitter orange.
The annual world production of nerolì is today less than
2000 Kg; most of it is concentrated in Morocco and in
Tunisia. Small amounts of nerolì, about 150 Kg/year, are
produced in Egypt, Spain, and Comorros. The market
price of nerolì is considerably high and the organic product
can be sold at more than 4,500 USD/Kg. It is therefore
predictable that this oil can be subject to adulteration by
the addition of less valuable natural products, such as the
oils obtained from flowers of citrus different from C.
aurantium, or by addition of leaf oils or of synthetic
compounds. The adulteration of nerolì oil is not easily
identifiable, mainly because the reference data available in
literature, relative to oils produced industrially and
extracted in laboratory [1-3], ranges widely and is also
probably affected by the geographic origin of the trees.
Based on the rules AFNOR (Association Francaise de
Normalization) [4a], ISO (International Organization for
Standardization) [4b] and EP (European Pharmacopeia)
[5], some of the results available in literature should not
indicate genuine samples. Very few results are available in
literature on the enantiomeric distribution of volatiles
determined in nerolì oils [6-10]. In the authors knowledge
the IRMS analysis was never performed before on nerolì.
1010 Natural Product Communications Vol. 6 (7) 2011
Usually essential oils quality assessment is obtained by
traditional chromatographic techniques (GC-FID, GC-MS,
Es-GC, HPLC) as recently reported by our research group
[11], recognized for their validity in the quality control
field. Advanced chromatographic techniques have been
also exploited for different essential oils as fast GC/MS
[12] and multidimensional GC-GC for quantitative [13]
and enantiomeric ratio assessment [14,15]. Recently has
gained importance the Gas Chromatography-Combustion-
Isotope Ratio Mass Spectrometry (GC-C-IRMS) that,
determining small differences in the isotopic carbon
composition of the matrices, can be exploited to
discriminate between products of different origin [10,16-
18a]. In this regards GC-C-IRMS can be an useful tool in
the flavour and fragrance authenticity control, unveiling
illicit essential oils production methods, such as the oils
adulteration by the addition of synthetic or natural
compounds, different from the genuine ones [18b,19,20].
To our knowledge nerolì oil was never investigated by
IRMS. It is therefore particularly interesting to provide
information useful for the genuineness assessment of
nerolì, also in function of the geographic origin.
The present article reports the results relative to the
composition of five samples of Egyptian nerolì produced
in 2008 and 2009, to the enantiomeric distribution and the
isotopic ratio of selected components.
The samples analyzed are described below:
Sample Description
1 hydrodistilled from Egypt (2008)
2 steam-distilled March 23th 2009
3 steam-distilled March 28th 2009
4 steam-distilled April 7th 2009
5 steam-distilled April 9-11th 2009
Table 1 reports the composition determined by GC-FID, of
the samples analyzed. To facilitate comparison with
information already available in literature the results are
here reported as raw peak area %. The correction factors
(C.F.) for each class of substances determined by GC-FID
are however reported in Table 1 to provide complete
information to the reader. In the case of distilled oils, as
nerolì, the volatile fraction should represent the whole oil.
The quantitative results obtained from triplicates show
CV% values always below 5%. The 86 components
identified by GC/MS with the use of LRI as filters
interactively applied during the mass spectral identification
process [21] represent about 99% of the whole oils. In
comparison with literature information this study led to the
identification of numerous components (indicated by * in
Table 1), while the presence of numerous minor
components previously reported were not here confirmed.
Hydrocarbons range between 20-25%, oxygenated
compounds vary between 73-78%; among these alcohols
range from 58 to 70% and esters from 7 to 19%, while
aldehydes are present at small amounts (0.16-0.26%). The
main alcohol is linalool (44-53%), followed by -terpineol
Bonaccorsi et al.
(5-6%) and by geraniol (3-4%). The sesquiterpene alcohols
(E)-nerolidol and (E,E)-2,6-farnesol are also well
represented ranging together between 2-5%. The main
ester is linalyl acetate (2-15%) followed by geranyl
acetate (about 3%) and neryl acetate (about 1.5%).
The most abundant monoterpene hydrocarbon is limonene
(8-12%) followed by (E)--ocimene (3-5%), and by
-pinene (2-4%); the main sesquiterpene hydrocarbon is
-caryophyllene (0.6-0.9%).
The sample produced in 2008 by hydrodistillation,
compared to all the other oils obtained by steam
distillation, has the highest amount of linalool and of total
alcohols, and the lowest amount of linalyl acetate and of
total esters.
In the samples produced in 2009 the composition varies
gradually but significantly during the productive season.
The total monoterpene hydrocarbons and the single
components of this class of compounds, the total
monoterpene alcohols and the single components of this
class of compounds as well as the ratio linalool/linalyl
acetate decrease during the season. Total esters and linalyl
acetate present an opposite behavior, as well as the
sesquiterpene hydrocarbons and aldehydes. Neryl and
geranyl acetate remain constant during the whole season.
In Figure 1 are graphically described the seasonal variation
of class of substances and some single components. The
results confirm, as reported in literature, that the main
components of nerolì oil are linalool, linalyl acetate and
limonene. The amount of these components determined in
this study fall in the ranges hitherto determined for nerolì
oil. It should be however mentioned that in one Egyptian
oil [1a] it was determined the 30% of linalool content and
1% of linalyl acetate; the highest value (74%) of linalool
was reported for a Chinese oil [1a] which presented a very
unusual low amount of limonene (1%); in some Spanish
oils [3] were reported very low values of linalyl acetate
(0.6%).
The results determined in the present study also confirm
the presence of some Key compounds such as methyl
anthranilate, methyl N-methyl anthranilate, phenyl ethyl
alcohol, (E)-nerolidol, and some newly identified
components such as the (E,Z)- and (E,E)-2,6-farnesals,
useful for the characterization of this product.
Table 2 reports the enantiomeric distribution of some
components determined by es-GC. The values are
determined from triplicates with CV% never exceeding
5.5% with the exception of that relative to the (-)--
thujene isomer which is 8.9% due to the chromatographic
behavior of this component. Figure 2 shows the chiral
chromatogram of one of the samples analyzed.
The enantiomeric ratios of camphene, sabinene, - and
-phellandrene and citronellal were determined in
nerolì oils for the first time. The values of the enantiomeric
Composition of Nerolì oil Natural Product Communications Vol. 6 (7) 2011 1011

Table 1: Composition of the five samples analyzed (% of peak areas) 2,3-Dihydrofarnesol* 1688 1688 0.02 0.01 0.01 0.01 0.04
determined by GC-MS-LRI and GC-FID. β-Sinensal* 1695 1699 0.09 0.09 0.09 0.08 0.08
(E,Z)-2,6-Farnesal* 1711 1714 0.02 0.01 0.01 0.02 0.03
LRIa LRIb 1 2 3 4 5 (E,E)-2,6-Farnesol 1718 1716 2.03 1.17 1.29 1.59 1.66
-Thujene 925 927 0.01 0.02 0.02 0.02 0.02 (E,E)-2,6-Farnesal* 1739 1737 0.03 0.02 0.02 0.03 0.04
-Pinene 933 933 0.15 0.26 0.22 0.22 0.23 -Sinensal* 1752 1749 0.02 0.02 0.02 0.02 0.01
Camphene 950 953 0.01 0.01 0.01 0.01 0.01 Farnesyl acetate° 1833 1832 0.04 0.02 0.02 0.03 0.03
Sabinene 973 972 0.85 1.56 1.15 1.44 1.33 (E,E)-Geranyl linalol* 2024 2026 0.02 0.02 0.02 0.03 0.03
β-Pinene 979 978 1.89 3.70 3.44 3.29 3.04 C.F. Sum of uncorrected % of peak areas
6-Methyl-5-hepten-2-one 984 978 0.02 0.02 0.02 0.02 0.02 HYDROCARBONS 1.0 21.79 24.95 23.97 22.57 20.10
Myrcene 989 991 1.43 1.74 1.61 1.44 1.33 Monoterpene 20.67 24.16 22.76 20.99 18.30
cis-Dehydrolinalol oxide* 1006 1006 0.02 0.02 0.02 0.02 tr Sesquiterpene 1.09 0.74 1.17 1.52 1.77
-Phellandrene 1007 1002 0.01 0.02 0.01 0.01 0.02 Aliphatic 0.03 0.05 0.04 0.06 0.03
δ-3-Carene 1010 1009 0.09 0.06 0.05 0.06 0.03 ALDEHYDES 1.3 0.26 0.16 0.22 0.25 0.26
(E)-2-Hexenyl acetate 1014 1017 - 0.01 0.01 0.01 tr Monoterpene 0.10 0.05 0.08 0.10 0.10
-Terpinene 1018 1018 0.04 0.13 0.09 0.07 0.04 Sesquiterpene 0.16 0.14 0.14 0.15 0.16
o-Cymene* 1020 1022 tr tr tr tr tr KETONES 1.3 0.09 0.08 0.08 0.11 0.09
p-Cymene 1025 1025 0.13 0.09 0.06 0.13 0.13 Monoterpene 0.05 0.05 0.05 0.07 0.05
Limonene 1031 1030 11.89 10.14 10.10 9.18 7.87 Aliphatic 0.04 0.03 0.03 0.04 0.04
(Z)-β-Ocimene 1035 1026 0.48 0.61 0.58 0.50 0.47 ALCOHOLS 1.3 69.72 59.58 58.68 57.81 58.19
(E)-β-Ocimene 1046 1046 3.31 5.11 4.81 4.09 3.40 Monoterpene 65.21 57.17 56.14 54.07 53.17
γ-Terpinene 1058 1049 0.12 0.28 0.19 0.17 0.11 Sesquiterpene 4.49 2.39 2.52 3.71 4.99
cis-Linalool oxide 1071 1069 0.13 0.17 0.17 0.18 0.25 Aliphatic 0.02 0.02 0.02 0.03 0.03
Terpinolene 1087 1086 0.26 0.43 0.42 0.36 0.27 ESTERS 1.6 6.88 13.39 15.55 17.55 19.35
Linalool 1107 1101 53.33 45.58 45.31 43.80 43.69 Monoterpene 6.83 13.36 15.52 17.50 19.30
4,8-Dimethyl-1,3(E),7- Sesquiterpene 0.04 0.02 0.02 0.03 0.03
nonatriene* 1114 1113 0.03 0.04 0.04 0.05 0.03 Aliphatic 0.01 0.01 0.01 0.02 0.02
+ Phenylethyl alcohol* OXIDES + ETHERS 1.5 0.22 0.30 0.32 0.37 0.34
Fenchol° 1122 1123 tr - - - tr OTHERS 0.10 0.14 0.16 0.14 0.10
trans-p-Menth-2,8-dienol 1124 1122 tr - - - tr ALL 99.06 98.60 98.98 98.80 98.43
cis-p-Menth-2-en-1-ol 1127 1124 0.03 0.03 tr 0.02 0.02 Notes a: LRI measured on SLB-5MS column; b: Reference LRI reported in
trans-Limonene oxide* 1135 1142 0.01 0.09 0.09 0.10 0.02 literature (FFNSC 1.3GC-MS library, Shimadzu, Japan; or Adams RP.
trans-p-Menth-2-enol* 1144 1141 0.02 0.02 0.01 0.02 0.03 Identification of essential oil components by gas chromatography/mass
Terpinen-4-ol 1182 1177 0.44 0.79 0.52 0.57 0.59 spectrometry, 4th Edn. Carol Stream, IL, USA: Allured Publishing Corp;
p-Cymen-8-ol* 1189 1189 0.02 0.01 0.01 0.01 0.03 2007; or Hochmuth, D.H., Joulain, D., König, W.A., 2002. MassFinder
(Z)-3-Hexenyl butanoate* 1190 1184 0.01 tr tr 0.01 0.02 Software and Data Bank, University of Hamburg); tr: ≤ 0.005; C.F. Correction
-Terpineol 1199 1195 6.22 6.17 5.90 5.50 4.89 Factor (FID response) for class of compounds; * identified, in the authors
trans-Piperitol 1211 1209 0.01 0.02 0.01 0.01 0.02 knowledge, for the first time in nerolì oils; ° correct isomer not identified.
Nerol 1226 1229 1.28 1.12 1.06 1.01 0.95
cis-Carveol* 1234 1232 tr - - - -
Neral 1239 1238 0.03 tr 0.03 0.03 0.03
Carvone 1246 1246 0.01 0.02 0.02 0.03 0.01
Linalyl acetate 1250 1243 2.19 8.77 10.97 12.97 14.57 Table 2: Enantiomeric distribution of some volatile components in the
Geraniol 1253 1255 3.83 3.42 3.30 3.06 2.94 samples analyzed.
trans-Myrtanol* 1257 1261 tr 0.01 0.02 0.02 0.01
Geranial 1269 1264 0.07 0.05 0.05 0.07 0.07 1 2 3 4 5
Bornyl acetate* 1286 1287 tr 0.01 0.01 0.01 0.01 S-(+)--Thujene 59.02 48.80 33.53 56.53 30.98
Geranyl formate* 1298 1298 0.03 0.01 0.02 tr 0.04 R-(-)--Thujene 40.98 51.20 66.47 43.47 69.02
Methyl geranoate* 1321 1320 0.01 tr tr tr 0.02 R-(+)--Pinene 41.37 36.77 32.96 34.70 n.d.
Linalyl propanoate* 1331 1333 tr - - - 0.02 S-(-)--Pinene 58.63 63.23 67.04 65.30 n.d.
Bicycloelemene* 1334 1338 tr 0.01 0.01 0.01 tr 1S,4R-(-)-Camphene n.d. n.d. n.d. n.d. 90.85
δ-Elemene 1337 1335 0.03 0.05 0.06 0.06 0.06 1R,4S-(+)-Camphene n.d. n.d. n.d. n.d. 9.15
Methyl anthranilate 1342 1337 0.04 0.10 0.12 0.09 0.06 R-(+)-β-Pinene 2.05 2.04 1.72 1.76 2.08
-Terpinyl acetate 1348 1349 0.05 0.06 0.07 0.06 0.07 S-(-)-β-Pinene 97.95 97.96 98.28 98.24 97.92
Citronellyl acetate 1349 1353 0.02 - - - 0.02 R-(+)-Sabinene 80.89 81.40 76.72 81.93 81.14
Neryl acetate 1359 1361 1.45 1.45 1.43 1.44 1.45 S-(-)-Sabinene 19.11 18.60 23.28 18.07 18.86
Geranyl acetate 1379 1380 3.08 3.06 3.02 3.01 3.08
R-(-)--Phellandrene 44.55 27.42 n.d. 14.37 32.01
β-Elemene 1392 1391 0.09 0.11 0.09 0.12 0.18
(E)-Jasmone* 1395 1390 0.02 0.01 0.01 0.02 0.02 S-(+)--Phellandrene 55.45 72.58 n.d. 85.63 67.99
n-Tetradecane* 1400 1400 tr 0.01 tr 0.01 R-(-)-β-Phellandrene 39.17 27.75 53.74 36.50 49.31
0.01 S-(+)-β-Phellandrene 60.83 72.25 46.26 63.50 50.69
cis--Bergamotene* 1404 1411 0.01 tr tr 0.01
Methyl N-methyl anthranilate 1409 1405 0.04 0.02 0.02 0.02 0.01 S-(-)-Limonene 1.65 2.54 2.42 2.58 2.60
β-Caryophyllene 1424 1424 0.56 0.60 0.68 0.82 0.94 R-(+)-Limonene 98.35 97.46 97.58 97.42 97.40
Perillyl acetate* 1435 1435 - tr tr tr 0.01 S-(-)-Citronellal n.d. 47.14 45.09 n.d. n.d.
Aromadendrene 1443 1439 0.01 tr tr tr 0.01 R-(+)-Citronellal n.d. 52.86 54.91 n.d. n.d.
Geranyl acetone* 1448 1453 0.04 0.03 0.03 0.04 0.04 R-(-)-Linalyl acetate 98.95 99.39 99.47 99.41 99.28
(E)-β-Farnesene 1453 1452 0.09 0.13 0.13 0.18 0.22 S-(+)-Linalyl acetate 1.05 0.61 0.53 0.59 0.72
-Humulene 1460 1452 0.06 0.06 0.06 0.08 0.11 R-(-)-Linalol 78.25 78.51 78.54 78.48 78.55
9-epi-β-Caryophyllene* 1464 1464 0.01 tr tr 0.01 0.01 S-(+)-Linalol 21.75 21.49 21.46 21.52 21.45
Geranyl propanoate* 1469 1471 tr tr tr 0.01 0.01 S-(+)-Terpinen-4-ol 62.19 62.74 60.00 61.68 62.13
Germacrene D 1485 1479 0.03 0.07 0.07 0.08 0.08 R-(-)-Terpinen-4-ol 37.81 37.26 40.00 38.32 37.87
Bicyclogermacrene* 1500 1497 0.10 0.14 0.16 0.14 0.05 S-(-)--Terpineol 28.77 28.98 28.88 29.21 28.99
-Muurolene* 1502 1497 0.01 0.02 0.01 0.02 0.01
R-(+)--Terpineol 71.23 71.02 71.12 70.84 70.79
(E,E)--Farnesene 1505 1504 0.05 0.02 0.01 0.02 0.03
γ-Cadinene 1517 1513 tr tr tr tr 0.01 n.d.: not determined.
δ-Cadinene 1522 1518 0.03 0.02 0.03 0.03 0.04
β-Sesquiphellandrene* 1526 1523 0.01 0.01 0.01 0.01 0.01 distribution of limonene, linalol, terpinen-4-ol and
(E)-Nerolidol 1563 1561 2.35 1.15 1.16 2.04 3.21
Spathulenol 1582 1576 0.05 0.03 0.03 0.05 0.05 -terpineol here determined are in good agreement
Caryophyllene oxide° 1587 1587 0.04 0.02 0.02 0.04 0.04 with literature results relative to genuine nerolì oils
Globulol 1591 1587 0.02 0.01 0.01 0.01 0.02
Cadin-4-en-10-ol* 1660 1659 0.02 0.02 0.02 0.01 0.01 [6,7a]; however, if compared to literature, the enantiomeric
n-Tetradecanol* 1677 1680 0.02 0.02 0.02 0.03 0.03 excess of (-)--pinene is slightly lower, that of (-)--pinene
1012 Natural Product Communications Vol. 6 (7) 2011 Bonaccorsi et al.

Table 3: (-)13CVPDB values calculated for the samples analyzed, average uV(x10,000)
Chromatogram

and relative standard deviation% (RSD). 3.0

6
14 15 16 19 24

1 2 3 4 5 Ave RSD
-Pinene 25.24 25.75 25.16 25.24 25.48 25.37 0.95 2.5

Myrcene 24.77 26.26 25.05 24.25 24.30 24.92 3.28

Limonene 27.29 27.78 27.20 27.84 27.32 27.48 1.09 2.0
Linalol 25.64 25.85 25.80 26.08 26.06 25.89 0.72 7
Terpinen-4-ol 28.10 27.74 27.01 27.68 27.81 27.67 1.46 23
1.5
-Terpineol 26.84 27.14 26.57 26.93 27.22 26.94 0.96
Nerol 27.05 27.39 26.27 26.89 28.19 27.16 2.59
Neryl acetate 28.14 27.86 27.48 28.42 28.52 28.09 1.51 1.0
Geranyl acetate 27.42 28.06 27.75 28.54 28.41 28.03 1.65
Caryophyllene* 25.74 25.15 23.18 25.04 24.61 24.74 3.89 0.5 21
8
(E)-Nerolidol 30.60 30.43 30.12 30.79 30.13 30.41 0.96 4
12 22

Farnesol** 30.18 29.3 29.64 29.58 29.79 29.71 1.03 3

1 2
5
910
11
13
17 18
20

Correct isomer identification: *-; **(2E,6E)-

0.0
15.0 20.0 25.0 30.0 min

Table 4: Comparison between the limits reported in the regulations [4,5] and Figure 2: Chiral chromatogram of one sample of neroli oil. Peak identification: 1.
the results experimentally obtained for the five samples of Egyptian Nerolì (+)-α-thujene; 2. (-)-α-thujene; 3. (+)-α-pinene; 4. (-)-α-pinene; 5. (+)-β-pinene; 6. (-
(peak area %). )-β-pinene; 7. (+)-sabinene; 8. (-)-sabinene; 9. (-)-α-phellandrene; 10. (+)-α-
phellandrene; 11. (-)-β-phellandrene; 12. (-)-limonene; 13. (+)-β-phellandrene; 14.
AFNOR ISO EP 2006 Range
(+)-limonene; 15. (-)-linalol; 16. (+)-linalol; 17. (-)-citronellal; 18. (+)-citronellal;
1995 3517:2002
19. (-)-linalyl acetate; 20. (+)-linalyl acetate; 21. (+)-terpinen-4-ol; 22. (-)-terpinen-
Limonene 9-18 9-18 9.0-18.0% 7.87-11.89
4-ol; 23. (-)-α-terpineol; 24. (+)-α-terpineol.
Myrcene 1-4 1-4 - 1.33-1.74
(E)-β-Ocimene 3-8 3-8 - 3.31-5.11
α-Pinene max. 2 tr-2 - - -23

β-Pinene 7-17 7-17 7.0-17.0 1.89-3.70 -24

1
Sabinene - tr-3% - 0.85-1.44 -25
Linalool 28-44 28-44 28.0-44.0 43.31-53.33
-26
α-Terpineol 2-5.5 2-5.5 2.0-5.5 4.89-6.22
(E,E)-Farnesol 1-4 1-4 0.8-4.0 1.29-2.03 -27 2

(E)-Nerolidol 1-5 1-5 1.0-5.0 1.15-3.21 -28

13CVPDB
Linalyl acetate 3-15 3-15 2.0-15.0 2.19-14.57 -29
Geranyl acetate 1-5 1-5 1.0-5.0 3.0-3.08 3
-30
Neryl acetate max. 2.5 tr-2.5 max 2.5 1.43-1.45
Methyl anthranilate - - 0.1-1.0 0.04-0.12 -31
4
-32
Chiral purity

e
Te -ol

l
e

eo

ar l
yo tate
in ol
Li e

(+)-Linalol - - max 30 21.45-21.75

so
(E nyl ate
l

)-F do
m e

ry ero

en
en

en
n

in

ne
i
ce

Te nal

yll
t

ol
ph en-
in

e
ce
on

rp

)-C ac

er
-P

ph
yr

(+)-Linalyl acetate - - max 5 0.53-1.05

la

(2 )-N
M
ta

rp
Li

a-

5
be

6E
(E
ar
Ne

a
er

E,
al

G
70,00
Seasonal variation

60,00
Figure 3: Diagram obtained for 13CVPDB values for each components in
function of the period of production. For sample description see table in text.
50,00

by plotting the 13CVPDB values for each component

40,00 Linalyl acetate analysed in function of the period of production. The
relative standard deviation of the 13CVPDB values range
Linalol
%

Linalol + Linalyl acetate

for the samples analyzed between 3.52 ((E)--

30,00 Monoterp HYDROCARB
Alcohols

caryophyllene) and 0.96 (-terpineol). These low values

Esters
20,00

indicate very narrow ranges of variation, therefore it is

10,00
posible to assume that the 13CVPDB can be considered
0,00
characteristic of authenticity and of the geographic origin
1 2
sample #
3 4
of the samples.

Table 4 provides a comparison of the ranges determined

Figure 1: Seasonal variation of class of components, of linalool and linalyl
acetate in the four samples of nerolì produced during 2009.
extremely lower and that of (-)-linalyl acetate is higher.
With the exception of -pinene, the results here obtained
for the components analyzed are overall in good agreement
with the enantiomeric purity previously determined by
Mosandl [6] considered characteristic of genuine nerolì oils.
Table 3 reports the (-)13CVPDB values calculated for the
nerolì oils samples. This study reports for the first time the
GC-C-IRMS analysis of selected volatile components in
nerolì oil. It is impossible to compare these values with
literature information. Figure 3 shows the graph obtained
from the present results with the limits provided by the
AFNOR, ISO and EP regulations [4,5]. Some of the results
fall within these limits; others fall only slightly outside
them; in three of the samples analyzed linalol is present at
levels sensibly higher than the limits provided by the
aformentioned regulations; -pinene is always below the
minima reported for nerolì oils. These behavior could be
due to the geographic origin of the oils analyzed.
Considering the high commercial value of nerolì, its
limited production in different geographic areas and the
high possibility that this product can be subject to
adulteration, it is necessary to fix quality parameters in
Composition of Nerolì oil
consideration of its variability among different geographic
areas. To accomplish this, for a correct evaluation, not
only the indices given by these regulations should be taken
into account, but also the 13CVPDB of selected compounds
and the chiral purity of more compounds than the two
already indicated, thus providing adequate tools for quality
and genuineness assessment.
Experimental
Analysis of the essential oils: On the five samples
described in text the following analytical investigations
have been carried out: GC/FID, GC/MS of the volatile
fraction; direct enantio-GC for the determination of the
enantiomeric distribution of some volatiles. Each analysis
was performed in triplicates. Results are expressed as
average peak area %.
GC/FID: The volatile fraction was analyzed by
HRGC/FID as described. Gas chromatograph: Shimadzu
GC2010 equipped with a Flame Ionization Detector, a
split/splitless injector and an AOC-20i series auto-injector.
Capillary column: 30 m x 0.25 mm I.D. 0.25 m df coated
with SLB-5MS [silphenylene polymer, virtually equivalent
in polarity to poly (5% diphenyl/95% methyl)siloxane)]
(Supelco, Milan, Italy); column temperature, 50-250°C (10
min) at 3°C/min; injector temperature: 250°C; detector
temperature: 280°C; carrier gas, He at 99.5 kPa (30.0
cm/s); injection mode: split; split ratio, 1:100; injected
volume, 1.0 L of diluted oil. Data handling was made by
means of GCsolution software.
GC/MS Analysis: Samples were analyzed by GC/MS (EI)
on a GCMS-QP2010 system equipped with commercially
available libraries (see notes to Table 1) including the
commercial version of the FFNSC ver. 1.3 (Shimadzu,
Japan) database (created in the authors’ laboratory)
consisting of about 2000 reference standards and their
relative linear retention indices determined on apolar
column, interactively used as filters for the spectral
interpretation. GC conditions: capillary column and
temperature program as in GC/FID; carrier gas, He
delivered at a constant pressure of 30.6 kPa (30.1 cm/s);
1.0 L of solution (1/10, v/v, essential oil/hexane) injected
on a split/splitless injector; injector temperature, 250°C;
injection mode, split; split ratio, 1:50. MS scan conditions:
source temperature, 200°C; interface temperature, 250°C;
E energy 70eV; mass scan range, 40-400 amu. Data was
handled through the use of GCMSsolution software.
Enantio-GC: Shimadzu GC2010 gas chromatograph
equipped with a Flame Ionization Detector, a split/splitless
injector and an AOC-20i series autoinjector. Capillary
chiral column was a Megadex DETTBS-(diethyl-tert-
butil-silyl -cyclodextrin) 25 m x 0.25 mm I.D. x 0.25m
df (Mega, Legnano, Italy). Temperature program: 50°-
200°C at 2°C/min. Inlet pressure 96.6 kPa (220°C), split
mode 1:20 (gas carrier He); injected volume, 1.0 l; linear
velocity, 30 cm/sec (constant). Data handling was made by
means of GCsolution software
Natural Product Communications Vol. 6 (7) 2011 1013
GC-C-IRMS device and analyses: Trace GC Ultra
equipped with a TriPlus autosampler, retrofitted to the
combustion interface GC/CIII and hyphenated to the
isotope ratio mass spectrometer Delta V Advantage (all
purchased from Thermo Fisher Scientific, Milan, Italy).
GC: column: SLB-5ms (silphenylene polymer) 30 m x
0.25 mm i.d., 0.25 m df (Supelco, Milan, Italy.);
temperature program: 50°C to 230°C at 3°C/min;
split/splitless injector (250°C). Inlet pressure: 167 kPa;
column flow: 2.0 ml/min (constant flow mode); carrier
gas: He.
GC/C III: ox. reactor (Cu/Ni/Pt): 980°C; red. reactor:
640°C; He: 1 bar; O2: 0.8 bar; CO2: 0.5 bar.
IRMS: EI; electron voltage: 123.99 eV; electron current:
1.5 mA; 3 Faraday cup collectors at m/z 44, 45, and 46;
peak center pre-delay and post-delay: 15 s, cup 3;
reference: 60-80 s, 100-120 s, 140-160 s, 180-200 s; split:
open; evaluation type: CO2_SSH, ref. time: 155.90 s,
13C/12C -60.300‰; integration time 0.2 s.
GC-C-IRMS instrument achieves highly precise
measurement of carbon isotopic abundance, converting the
eluted volatile components, in CO2 and water into an
oxidation chamber. After removing water, just behind the
furnace, by a capillary-shaped phase separator, CO2
reaches an ionization chamber where it will be transformed
into three ion traces for the different isotopomers: 12C16O2,
13 16
C O2 and 12C18O16O, with their corresponding masses at
(m/z) 44, 45, 46.The three ion beams are registered
simultaneously by an Universal Faraday collector that
detects the different contributions of ionic fragments
obtained. Isotopic ratios, 45/44 and 46/44, are expressed in
‰ and are related to a certified standard (VPDB-standard)
of known value [20]. Exploiting the GC-Combustion
backflush, the most concentrated components were not
introduced into the combustion chamber.
The samples dilutions and the GC-Combustion conditions
were as follows: concentration 1:10 (v/v), 1 L split
injection, 1:100 split ratio, backflush: off, for the
determination of 13CVPDB of limonene and linalool.
Concentration 1:10 (v/v), 1 L split injection, 1:50 split
ratio, backflush open: 780-830 s and 970-1060 s, for the
determination of 13CVPDB of -pinene, myrcene, terpinen-
4-ol, -terpineol, nerol, neryl acetate, geranyl acetate, (E)-
caryophyllene, nerolidol, (2E,6E)-farnesol. Data are
collected in triplicate, by using the Isodat 2.5 software
(Thermo Fisher Scientific).
CO2 reference gas cylinder calibration: The attained
carbon isotope ratio of the unknown sample is compared to
that of a calibrated CO2 reference. The CO2 reference gas
was calibrated by injecting 1 L of a carbon stable isotope
ratio reference alkanes mixture comprising C16 to C30
(Indiana University, Bloomington, U.S.A.), calibrated
against VPDB standard with a defined 13C content. Isotope
ratios were expressed as  values (‰), versus a standard.
1014 Natural Product Communications Vol. 6 (7) 2011 Bonaccorsi et al.

(13C/12C)sample - (13C/12C)standard x 1000 Acknowledgements - Authors whish the acknowledge

13CVPDB = Shimadzu for the continuous support.
(13C/12C)sample

Tricosane (C23) was arbitrarily chosen as reference alkane.

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[17] (a) Kelly S, Parker I, Sharman M, Dennis J, Goodall I. (1997) Assessing the authenticity of single seed vegetable oils using fatty
acid stable carbon isotope ratios.(13C/12C). Food Chemistry, 59, 181-186; (b) Pupin AM, Parker MJ, Kelly S, Bigwood T, Toledo
MCF. (1998) Use of isotopic analysis to determine the authenticity of Brazilian orange juice.(Citrus sinensis). Journal of
Agricultural and Food Chemistry, 46, 1369-1373; (c) Juchelka D, Mosandl A. (1996) Authenticity profiles of bergamot oil.
Pharmazie, 51, 417-422; (d) Faulhaber S, Hener U, Mosandl A. (1997) GC-IRMS analysis of mandarin essential oils. 2. δ13CPBD
values of characteristic flavour components. Journal of Agricultural and Food Chemistry, 45, 4719-4725.
[18] (a) Schipilliti L, Tranchida PQ, Sciarrone D, Russo M, Dugo P, Dugo G, Mondello L. (2010) Genuineness assessment of mandarin
essential oils employing Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS). Journal of Separation
Science, 33, 1–6; (b) Schipilliti L, Dugo P, Dugo G, Santi L, Mondello, L. (2010)Authentication of bergamot essential oil by Gas-
Chromatography-Combustion-Isotope Ratio Mass Spectrometer (GC-C-IRMS). Journal of Essential Oil Research, 23, 60-71.
[19] Schipilliti L, Dugo P, Bonaccorsi I, Mondello L. (2011) Authenticity control on lemon essential oils employing Gas-
Chromatography-Combustion-Isotope Mass Spectrometry (GC-C-IRMS). Submitted to Food Chemistry.
[20] Russe K, Valkiers S, Taylor PDP. (2004) Synthetic isotope mixtures for the calibration of isotope amount ratio measurements of
carbon. International Journal of Mass Spectrometry, 235, 255-262.
[21] Mondello L, Dugo P, Basile A, Dugo G. (1995) Interactive use of linear retention indices, on polar and apolar columns, with a MS
library for reliable identification of complex mixtures. Journal of Microcolumn Separations, 7, 581-591.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Essential oil of Nepeta x faassenii Bergmans ex Stearn 1015 - 1022

(N. mussinii Spreng. x N. nepetella L.): A Comparison Study

Niko Radulovića, Polina D. Blagojevića, Kevin Rabbittb and Fabio de Sousa Menezesb,*
a
Department of Chemistry, Faculty of Science and Mathematics, University of Nis, Serbia
b
School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences,
Trinity College Dublin, Ireland

DESOUZAF@tcd.ie

Received: December 12th, 2010; Accepted: March 16th, 2011

Analysis (GC and GC/MS) of an essential oil sample obtained from dry leaves of Nepeta × faassenii Bergmans ex Stearn, a hybrid species
produced by crossbreeding N. mussinii Spreng. with N. nepetella L., led to the identification of 109 constituents that represented 95.9% of
the oil. The major constituents were 4aα,7α,7aα-nepetalactone (67.8%), 1,8-cineole (6.6%), germacrene D (4.8%), β-pinene (2.7%),
(E)-β-ocimene (2.6%), 4aα,7β,7aα-nepetalactone (2.3%) and (E)-β-farnesene (1.0%). Chemical composition of the oil was compared, using
multivariate statistical analyses (MVA) with those of the oils of other Nepeta taxa, in particular N. mussinii and N. nepetella. This was done
in order to explore the mode of inheritance of the monoterpene biosynthetic apparatus of N. faassenii. Chemical composition of the volatiles
of a Nepeta taxon (different populations) can be subject to variation due to environmental and geographical factors. To accommodate this fact
in the MVAs, along side with N. faassenii essential oil, additional 6 oils (3 different populations of N. nuda L. and N. cataria L. from Serbia)
were included in this study (isolated and analyzed (chemically and statistically)). The MVA analyses recognized N. faassenii as being closely
related to both N. mussinii and N. nepetella. If the relative content of oil constituents per plant and not per chromatogram were used as
variables in the MVA (this was done by simple multiplication of the yields and relative percentages of components) a higher degree of
mutual similarity (in respect to the monoterpene biosynthesis) of N. faassenii to N. mussinii, than to the other parent species, was observed.

Keywords: Nepeta × faassenii Bergmans ex Stearn, Lamiaceae, Nepeta nuda L., Nepeta cataria L., Nepeta mussinii Spreng, Nepeta
nepetella L., essential oil, nepetalactone, chemometrics.

The genus Nepeta L., Lamiaceae, includes over 200
species of perennial plants and some annuals [1]. The
members of this genus are known as catnip or catmint
because of the purported effect on cats, pleasantly
stimulating cats’ pheromonic receptors, typically resulting
in temporary euphoria [1]. The active substances of catnip
plants, some of which have a sedative effect on humans,
have been widely used in medicine, food industry and
perfumery. They are also used in folk medicine as
remedies for bronchitis, flu, cold and other illnesses.
Number of catnips have sudorific, diuretic and
bacteriostatic properties, reduce fever and help treat
stomach-aches [1,2]. Generally speaking, Nepeta species
are essential oil-rich taxa, and some of them contain more
than 1% of the essential oil [2,3]. Moreover, many of them
are cultivated as garden plants. In order to produce new,
interesting garden/ornamental plants, many Nepeta species
have been crossbred repeatedly. The aim of the breeding
processes is not only creating plants with an appalling
look, but also producing larger amounts of biologically
active compounds [2]. One of the outcomes of these
experiments is the sterile hybrid Nepeta × faassenii
Bergmans ex Stearn, which has been produced by
crossbreeding N. mussinii Spreng. with N. nepetella L. [4].
Nepeta × faassenii is cultivated in Europe, C Asia and
Persia, and is used as a spice and an ornamental plant [4].
There is only limited data on the composition of the
volatile oil of the mentioned hybrid taxon [3a]. For this
reason, the aim of this study was set to analyze in detail
(GC and GC/MS) the essential oil of N. faassenii and to
compare its chemical composition, using multivariate
statistical analyses (MVA: agglomerative hierarchical
cluster analysis (AHC) and principal component analysis
(PCA)) with those of the oils of other Nepeta taxa, in
particular N. mussinii and N. nepetella. Having in mind
that the monoterpene diversity and morphology could serve
as a mirror of the gene flow [5], multivariate analyses were
primarily done in order to provide an insight into the
inheritance of the chemical characters for this taxon.
The chemical composition of the essential oils of a Nepeta
taxon (different populations) can be subject to variation
due to environmental and geographical factors. To
accommodate this fact in the MVAs, along side with N.
faassenii essential oil, additional 6 oils (3 different
populations of N. nuda L. and N. cataria L. from Serbia)
were included in this study (isolated and analyzed
(chemically and statistically)).
1016 Natural Product Communications Vol. 6 (7) 2011 Radulović et al.

Table 1: Chemical composition of the essential oils extracted from Table 1 (contd.)
Nepeta faassenii (NF), N. cataria (NC) and N. nuda (NN) 1163 Lavandulol tr M
1165 Sabina ketone tr M
RIa Compound NF NCb NNb Class
1166 Pinocarvone tr 0.20.1 M
765 (Z)-2-Penten-1-ol trc,d tr O
1169 Lavandulol 0.10.0 tr M
778 3-Methyl-2-butenal tr tr O
Methyl 3- 1170 Rosefuran epoxide tr M
778
methylbutanoatee
tr tr tr O 1173 δ-Terpineole tr 1.60.5 MM
801 Hexanale tr tr O 1184 Terpinen-4-ole 0.1 tr 0.30.1 MM
837 Furfurale tr tr O 1193 Cryptone tr MM
845 (Z)-2-Hexenal tr 0.10.0 O 1198 α-Terpineole 0.1 tr 3.91.0 MM
851 (E)-2-Hexenal 0.1 O 1199 Methyl salicylatee tr 0.10.1 tr O
Ethyl 3- 1201 Myrtenale tr 0.30.2 M
852 tr O 1207 Decanale tr O
methylbutanoatee
855 (Z)-3-Hexen-1-ol tr O (E,E)-2,6-Dimethyl-
1208 0.1 M
886 2-Butylfuran tr O 3,5,7-octatrien-2-ol
897 2,6-Dimethypyridine tr O 1218 Verbenone tr M
900 Nonanee 0.10.1 O 1221 β-Cyclocitral tr tr tr O
900 Styrenee,f 0.8 tr tr O 1226 trans-Carveole tr MM
927 Diethyldisulfidee tr O (Z)-3-Hexenyl 2-
1238 tr O
932 α-Thujenee 0.1 tr tr M methyl butanoate
937 α-Pinenee 0.4 tr 1.10.3 M 1239 Nerale tr tr M
951 Allylbenzenee tr O (Z)-3-Hexenyl 3-
1242 0.30.3 O
955 Camphenee tr tr tr M methylbutanoate
963 Hexanoic acide tr O Hexyl 3-methyl
1248 tr O
970 Benzaldehydee tr tr tr O butanoatee
975 Sabinenee 0.8 0.20.1 2.00.5 M 1251 Cumin aldehyde tr MM
978 1-Octen-3-ol tr O 1251 Geraniole tr tr tr M
984 β-Pinenee 2.7 0.40.2 4.00.7 M 1256 Carvonee tr MM
987 3-Octanone tr 0.1 O 1269 Geraniale 0.1 tr M
Methyl 3-
989 Myrcenee 0.3 tr 1.10.1 M 1275 0.3 O
phenylpropanoatee
993 2-Pentyl furan tr tr O
1282 Lavandulyl acetate tr M
995 Dehydro-1,8-cineole tr 0.10.0 MM
1293 Dihydroedulan IA tr 0.10.0 O
997 3-Octanol 0.1 O
1324 (E)-3-Hexenyl tiglate tr O
999 (Z,E)-2,4-Heptadienal tr tr O
Ethyl 3-
1004 Octanale tr O 1350 tr O
phenylpropanoatee
1009 p-Mentha-1(7),8-diene tr tr MM
4aα,7α,7aα-
1014 (E,E)-2,4-Heptadienal tr O 1378 67.8 91.77.8 0.10.0 MI
Nepetalactonee
1021 α-Terpinene tr tr 0.10.1 MM
1381 α-Copaene 0.2 0.20.1 S
1028 p-Cymenee tr tr tr MM
1387 Geranyl acetatee tr M
Methyl 1-cyclohexenyl
1029 tr tr M (Z)-3-Hexenyl (Z)-3-
ketone 1389 0.40.4 tr O
hexenoate
1032 Limonenec 0.5 tr MM
1391 β-Bourbonene 0.2 0.90.6 S
1034 β-Phellandrene 0.1 0.10.0 MM
1392 β-Cubebene tr tr S
1037 1,8-Cineolee 6.6 tr 37.54.9 MM
1393 β-Elemene 0.2 0.40.0 S
1040 (Z)-β-Ocimene tr 0.10.0 M
1393 (E)-β-Damascenone tr O
1045 (E)-β-Ocimene 2.6 0.20.1 0.70.3 M 4aα,7α,7aβ-
1050 2-Phenylacetaldehydee 0.20.2 O 1396 0.3 0.40.3 MI
Nepetalactone
1060 γ-Terpinene tr tr 0.10.0 MM 1397 (E)-Jasmone tr O
1061 (E)-2-Octenal tr O 4aα,7β,7aα-
1062 Bergamal tr M 1404 2.3 1.00.7 12.92.3 MI
Nepetalactone
1070 1-Octen-3-ol tr O 1414 Isocaryophyllene tr S
(E)-3-Hexenyloxy- 1415 α-cis-Bergamotene 0.1 S
1070 tr O
acetaldehyde 1416 α-Gurjunene 0.10.1 S
1071 1-Octanole tr O 1423 β-Ylangene tr S
1073 cis-Sabinene hydrate 0.1 0.40.3 M 1424 Dihydronepetalactone tr MI
cis- Linalool oxide 1425 β-Caryophyllenee 0.9 2.30.8 4.62.5 S
1077 tr tr M
(furanoid)e 1434 β-Copaene 0.1 0.20.0 S
1089 Terpinolenee tr 0.10.0 MM 1441 (Z)-β-Farnesene tr S
1092 Rose furan tr M 1451 Isogermacrene D 0.10.0 S
1101 Linaloole tr tr tr M 1453 (E)-β-Farnesene 1.0 S
0.20.2 4.63.1
1102 Perillene tr MM
1460 α-Humulenee 0.1 0.20.2 tr S
1105 trans-Sabinene hydrate tr tr M
1463 2-Methyltetradecane tr tr O
1106 Nonanale tr tr tr O
1468 allo-Aromadendrene tr S
1107 α-Thujonee tr M
cis-Cadina-1(6),4-
(E)-6-Methyl-3,5- 1470 tr S
1109 tr tr O diene
heptadien-2-one
cis-Muurola-4(14),5-
1128 Dehydrosabina ketone tr M 1473 tr 0.10.0 S
diene
1128 allo-Ocimene tr M
1483 ar-Curcumene tr S
1132 α-Campholenal tr M
1487 γ-Muurolene 0.10.1 S
1139 (E)-Epoxy-ocimene tr M
1145 trans-Pinocarveol tr tr M 1486 Germacrene De 4.8 tr 12.64.4 S
0.20.2
1146 Nopinone tr tr M 1490 (E)-β-Iononee tr O
1147 trans-Sabinole tr M trans-β-Ionon-5,6-
1491 tr O
1151 Citronellal tr M epoxide
1152 trans-Verbenol M 1496 α-Zingiberene 0.1 0.10.1 S
0.10.1
1159 (E)-2-Nonenal tr O trans-Muurola-4(14),5-
1499 tr S
diene
Nepeta x faassenii essential oil Natural Product Communications Vol. 6 (7) 2011 1017

Table 1 (contd.) p-Menthane (MM) 7.4 0.1±0.0 47.3±1.7

1499 Bicyclogermacrene 0.1 0.7±0.6 S Sesquiterpenoids (S) 9.5 3.4±0.5 30.6±5.2
1500 α-Muurolene tr tr S Hydrocarbons 8.6 2.7±0.4 26.4±4.2
1504 (E,E)-α-Farnesene 0.1 tr tr S Oxygenated 0.9 0.7±0.4 26.5±3.1
1509 β-Bisabolene 0.3 0.9±0.2 S Others (O) 1.4 0.9±0.5 0.6±0.1
1512 Germacrene A 0.1 S a
Compounds listed in order of elution from a DB-5MS column with
1520 γ-Cadinene 0.1 tr S
retention indices (RI) determined experimentally by co-injection of a
1523 endo-1-Bourbonanol tr S
homologous series of C7-C28 n-alkanes; bPercentages given as average
1526 β-Sesquiphellandrene 0.2 0.8±0.2 S
1536 trans-γ-Bisabolene tr S values ± absolute deviations of the components’ relative abundances
1538 trans-Cadina-1,4-diene tr S (three oil samples); ctr-trace (<0.05%); dAll compounds present below
1543 α-Cadinene tr S 0.05% in at least one of the analyzed N. nuda and N. cataria oil samples
1550 α-Calacorene tr tr S are given as tr; eThe identity of the constituent was determined by
1561 (E)-Nerolidol tr S comparison of MS and RI matching and confirmed by co-injection of an
1568 1-nor-Bourbonanone 0.1±0.1 S authentic sample; fProbably a contaminant of the isolation procedure;
1569 Dendrolasin tr tr S syn.-synonym.
1572 Mint oxide tr S
1575 Palustrol 0.1±0.0 S
(Z)-3-Hexenyl
1575 tr O
benzoate
1581 Germacrene D-4-ol 0.1 S
1584 Spathulenole 0.2±0.0 S
1587 Caryophyllene oxidee 0.2 0.7±0.1 1.9±0.5 S
1593 β-Copaen-4α-ol tr S
1597 Salvial-4(14)-en-1-one tr tr S
1610 Ledol 0.1±0.0 S
1611 β-Oplopenone tr S
1617 Humulene epoxide II tr 0.4±0.3 S
1626 Junenol tr S
1633 Zingiberenol tr S
1635 1-epi-Cubenol tr S
allo-Aromadendrene
1643 0.1 S
epoxide
Caryophylla-
1644 tr tr S
4(12),8(13)-dien-5-ol
epi-α-Cadinol
1645 tr tr S
(τ-cadinol)
epi-α-Murrolol (syn.g
1648 0.3±0.2 S
τ-muurolol)
1650 3-iso-Thujopsanone tr S
α-Muurolol (syn.
1652 0.1±0.1 S
torreyol)
1659 α-Cadinol 0.1 0.6±0.6 S
14-Hydroxy-9-epi-
1678 0.1±0.0 S
caryophyllene
Germacra-
1688 4(15),5,10(14)-trien- 0.1 0.3±0.1 S
1α-ol
1690 2,3-Dihydrofarnesol tr S
Eudesma-4(15),7-dien-
1694 tr S
1β-ol
1697 (Z,Z)-2,6-Farnesol 0.1 S
1743 Mint sulfide tr S
1745 α-Oxobisabolene 0.1 S
1768 β-Costol tr S
1770 Benzyl benzoate tr O
14-Hydroxy-α-
1777 tr S Figure 1: Typical TIC chromatograms of the herein analyzed N. cataria,
muurolene
14-Hydroxy-δ- N. nuda and N. fasenii essential oil samples (for sample designation see
1797 0.1 S Table 2).
cadinene
Hexahydrofarnesyl
1845 tr 0.1±0.0 O
acetone Almost 200 different compounds, representing 95.9-99.1%
2067 Abietatriene tr O
2107 (E)-Phytol 0.1 tr O of the total oils, were identified in the 7 presently analyzed
2300 Tricosane tr tr tr O samples (Table 1). Typical TIC chromatograms of the
2364 (Z)-9-Octadecenamidef tr O analyzed samples are depicted in Figure 1. Chemical
2400 Tetracosanee tr O
2500 Pentacosanee tr tr O compositions of the analyzed N. nuda and N. cataria oils
2600 Hexacosanee tr tr O were in general agreement with the literature data [3a,g].
2800 Octacosanee tr O Major compounds identified in N. faassenii oil were
Total 95.9 98.5±1.1 98.0±1.5
Number of
4aα,7α,7aα-nepetalactone (67.8%), 1,8-cineole (6.6%) and
119 75±5 115±9 germacrene D (4.8%). Additional 4 compounds with the
components
Monoterpenoids (M) 85.0 94.2±7.1 66.8±5.1 relative amount higher than 1% of were: β-pinene (2.7%),
Hydrocarbons 7.5 1.0±0.3 9.4±3.1 (E)-β-ocimene (2.6%), 4aα,7β,7aα-nepetalactone (2.3%)
Oxygenated 77.5 93.8±4.3 57.4±5.5
Iridane (MI) 70.4 93.1±6.8 13.1±2.9
and (E)-β-farnesene (1.0%).
1018 Natural Product Communications Vol. 6 (7) 2011 Radulović et al.

Table 2: List of the essential oil samples used in the statistical analyses.
Sample Yielda
Dendrogram
Taxon Ref. 60000
designation (%)
N. faas1 p.s.b 0.2
N. faassenii Bergmans ex Stearn N. faas2 [3a] 0.1 50000
N. faas3 [3a] 0.2
N. muss1 [3b] -c
N. muss2 [3c] 0.2 40000
N. mussinii Spreng.
N. muss3 [3d] 0.2
(syn. N. racemosa Lam.)

Dissimilarity
N. muss4 [3e]d 0.7
N. muss5 [3e] 0.1 30000
N. nep1 [3c] -
N. nepetella L. N. nep2 [3a] 0.8
N. nep3 [3f] 1.2 20000
N. nuda1 p.s. 0.5
N. nuda2 p.s. 0.6
N. nuda L. N. nuda3 p.s. 0.6 10000
N. nuda4 [3a] 0.03
N. nuda5 [3a] 0.2
N. cat1 p.s. 0.5 0
N. cat2 p.s. 0.5

N. cadmea
N. cat2
N. cat1

N. muss1
N. rtanj
N. muss4
N. muss5
N. muss3

N. muss2
N.cat5
N.nep3
N.nep2
N. cat3
N. nep1
N.sib
N.cat6
N. faas1
N.fass2

N.nuda4
N.nuda5

N. cilicia

N.faas3

N. cat4
N. crispa
N. ispah
N. mahan

N. nuda2
N. nuda1
N. nuda3
N. ucr
N. erem
N. mac
N. sept
N. folio

N. rivul
N. oxy
N. cat3 p.s. 0.4
N. cataria L.
N. cat4 [3g] -
N. cat5 [3a] 0.1
N. cat6 [3a] 0.2 A
N. macrosiphon Boiss. N. mac [3h] 0.2
N. ucrainica L. ssp. kopetdaghensis N. ucr [3i] 0.04
N. oxydonata Boiss. N. oxy [3j] 0.1
N. foliosa Moris N. foli [3k] 0.1 Dendrogram
12000
N. septemcrenata Erenb N. sept [3l] 0.4
N. sibirica L. N.sib [3a] 1.0 11000
N. rtanjensis Diklic & Milojevic N. rtanj [3m] 1.0 10000
N. cadmea Boiss. N. cadmea [3n] 2.1
N. crispa Willd. N. crispa [3o] - 9000
N. mahanensis Jamzad & Simmonds N. mahan [3o] - 8000
N. ispahanica Boiss. N. ispah [3o] -
Dissimilarity

N. eremophila Hausskn. & Bornm. N. erem [3o] - 7000

N. rivularis Bornm. N. rivul [3o] - 6000
N. cilicia Boiss. N. cilicia [3p] -
a b 5000
Given as in the original references (%, w/w or v/w); p.s.-present study;
c
Yield not given in the original references; d Reference [3e] plus personal 4000
correspondence with K. H. C. Baser; syn.-synonym. 3000

2000
As already mentioned, monoterpene profiles of hybrids 1000
and their parent taxa could serve as a mirror of the gene
0
flow [5]. We wanted to verify this hypothesis in the case of
N. cadmea
N. cat2
N. cat1

N. muss1
N. rtanj
N. muss4
N. muss5
N. muss3

N. muss2
N.cat5
N.nep3
N.nep2
N. cat3
N. nep1
N.sib
N.cat6
N. faas1
N.fass2

N.nuda4
N.nuda5

N. cilicia

N.faas3
N. crispa
N. ispah
N. mahan
N. cat4
N. nuda2
N. nuda1
N. nuda3
N. ucr
N. erem
N. mac
N. sept
N. folio

N. rivul
N. faassenii, and decided to use essential oil composition N. oxy

as a reflection of the inheritance of the biosynthetic

apparatus of this hybrid taxon.
For that reason, chemical compositions of the herein
analyzed samples and 29 other oils originating from
different Nepeta taxa (Table 2), including the two
previously studied oils of N. faassenii and 8 oils of the
parent species N. nepetella and N. mussinii, were
compared using multivariate statistical analysis (MVA:
AHC and PCA), Figures 2-5. The exact identity of the
Nepeta taxa other than N. faassenii, N. nepetella and N.
mussinii was unimportant for the present study and these
were chosen randomly (either from the literature or
analyzed in parallel with N. faassenii) to provide a
representative data set, large enough to be suitable for
MVAs. Moreover, several different populations of N. nuda
and N. cataria were included as a sort of a “control group”,
to estimate the potential influence of environmental and
geographical factors, and the possibility of the existence of
different chemotypes of one taxon.
B
Figure 2: A-Dendrogram (AHC1 analysis; original variables)
representing chemical composition dissimilarity relationships of 36
Nepeta essential oil samples (observations) obtained by Euclidian
distance dissimilarity (dissimilarity within the interval [0, 50300], using
aggregation criterion-Ward's method). Three groups of samples (C1-C3)
were found (from left to right); B-Expanded section of dendrogram A,
showing the dissimilarity interval [0, 12000].
The dendrogram depicted in Figure 2, obtained as the
result of AHC analysis performed using original variables
(percentage composition of the oils, AHC1), delimitates
three statistically different classes of samples, C1-C3.
Class C1 (4aα,7α,7aα-nepetalactone class) groups all
samples with a high relative amount of 4aα,7α,7aα-
nepetalactone (more than 65%).
Oils obtained from N. nepetella (N. nep1-N. nep3), as well
as the two N. faassenii samples (N. faas1 and N. faas2)
were also grouped within this class. The mutual feature of
the oils, including two N. mussinii samples, clustered
within the C2 ((4aα,7α,7aβ-nepetalactone class)) was the
Nepeta x faassenii essential oil Natural Product Communications Vol. 6 (7) 2011 1019

Dendrogram Dendrogram
60000
35000

50000 30000

25000
40000
Dissimilarity

Dissimilarity
20000
30000

15000
20000
10000

10000
5000

0
0

N. ucr
N. muss2

N. muss1
N. muss4
N. muss3
N. rtanj

N. muss5
N.nuda4
N. faas1
N. cat2
N. cat1
N.cat5
N.cat6

N.fass2
N.nep2
N.sib
N. cat3
N. nep1
N. cadmea
N.nep3

N. mahan
N. crispa
N. ispah
N. cat4
N. nuda3
N. nuda1
N. nuda2
N.nuda5

N. cilicia

N.faas3
N. erem

N. sept

N. mac
N. rivul

N. folio

N. oxy

N. rtanj

N. ucr
N. muss4

N. muss5

N. muss2

N. muss3
N. nuda2
N. nuda1
N. nuda3

N.faas3

N.nuda4
N.faas1
N.cat6

N.cat5
N. faas2

N.nuda5
N. cadmea
N. cat3
N. cat1
N. cat2
N. nep2
N. nep3
N.sib
N. sept
N. mac

N. folio

N. oxy
A A

Dendrogram Dendrogram

10000
5000
9000

4000 8000

7000
Dissimilarity

Dissimilarity

3000 6000

5000

2000 4000

3000

1000 2000

1000

0 0
N. ucr
N. muss2

N. muss1
N. muss4
N. muss3
N. rtanj

N. muss5
N.nuda4
N. faas1
N. cat2
N. cat1
N.cat5
N.cat6

N.fass2
N.nep2
N.sib
N. cat3
N. nep1
N. cadmea
N.nep3
N. crispa
N. ispah
N. mahan
N. cat4
N. nuda3
N. nuda1
N. nuda2
N.nuda5

N. cilicia

N.faas3
N. erem

N. sept

N. mac
N. rivul

N. folio

N. oxy

N. rtanj

N. ucr
N. muss4

N. muss5

N. muss2

N. muss3
N. nuda2
N. nuda1
N. nuda3

N.faas3

N.nuda4
N.faas1
N.cat6

N.cat5
N. faas2

N.nuda5
N. cadmea
N. cat3
N. cat1
N. cat2
N. nep2
N. nep3
N.sib
N. sept
N. mac

N. folio

N. oxy

B B
Figure 3: A-Dendrogram (AHC2 analysis; transformed variables – Figure 4: A-Dendrogram (AHC3 analysis; transformed variables
summation of the nepetalactones) representing chemical composition (information about the oil yields included to give the per plant content))
dissimilarity relationships of 36 Nepeta essential oil samples representing chemical composition dissimilarity relationships of 27
(observations) obtained by Euclidian distance dissimilarity (dissimilarity Nepeta essential oil samples (observations) obtained by Euclidian
within the interval [0, 50000], using aggregation criterion-Ward's distance dissimilarity (dissimilarity within the interval [0, 30000], using
method). Three groups of samples (C4-C6) were found (from left to aggregation criterion-Ward's method). Three groups of samples (C7-C9)
right); B-Expanded section of dendrogram A, showing the dissimilarity were found (from left to right); B-Expanded section of dendrogram A,
interval [0, 5500]. showing the dissimilarity interval [0, 11000].

high relative amount of 4aα,7α,7aβ-nepetalactone (>70%).
The remaining class (C3) was much more heterogenic with
respect to the dominant components of its comprising
samples. One sample of N. faassenii (N. faas3) and 3
samples of the N. mussinii oil were placed within the same
subclass of C3.
Germacrene D, (E)-β-ocimene, 4aα,7α,7aβ-nepetalactone,
4aα,7α,7aα-nepetalactone, 4aα,7β,7aα-nepetalactone and
1,8-cineole were among the dominant components of these
N. faassenii and N. mussinii oils.
According to the results of AHC1, one could speculate that
in general, the hybrid N. faassenii is, as far as the
monoterpene biosynthesis is concerned, generally more
similar to N. nepetella than to its other parent species (C1).
The placement of one N. faassenii oil sample outside of the
C1 class might be possibly explained by the existence of
more than one chemotype of this hybrid.
It could be even possible that the crossbreeding might not
give identical N. nepetella x N. mussinii hybrids all of
the time. However, although N. faassenii, N. nepetella and
1020 Natural Product Communications Vol. 6 (7) 2011 Radulović et al.

N. mussinii oils didn’t share the same amounts of

O b s e r v a t io n s ( a x e s F 1 a n d F 2 : 2 4 .0 7 % )
(different) nepetalactones. For this reason, additional
MVA analyses, using transformed variables were 10 N . r iv u l
preformed to answer this situation. In the first transformed
set of variables (MVA2: AHC2 and PCA2) 4 N. nud a1,2
N . f o lio
N . c a t4
nepetalactone diastereoisomers were not used as individual 5
N. mahan
N. crispa
variables, but were replaced with one collective variable N. ispah N . m ac
N . c ilic ia
(the sum of the relative abundances of the 4

nepetalactones). The results of AHC 2 are given in Figure
3. The three observed statistically different groups of
samples (C4-C6) are not identical with those from AHC1,
but can be correlated to a certain level. Once again, the N.
faassenii oils were not placed within the same class of the
resulting dendrogram. The sample N. faas3 (collected from
the same botanical garden as N. faas2), characterized with
a high percentage of germacrene D, (E)-β-ocimene, was
again recognized as statistically different, as might be
expected. Except for this sample and N. muss5, all other N.
faassenii, N. nepetella and N. mussinii oils were not
recognized as statistically different (C4, AHC2).
It has been previously shown that not only the identity of
the dominant essential oil constituents is important for the
comparison and classification of plant species, but the oil
yield as well [6]. Therefore, an additional set of variables,
taking into account the yields of the compared oils, was
used for the MVAs (MVA3, for each sample, the relative
amount of the every original variable was multiplied with
the corresponding oil yield, to reflect the per plant and not
per chromatogram relative amount of the volatile
constituents). MVA3 were performed using a slightly
reduced set of observations, as the yields of all oils listed
in Table 2 were not given in the original references. The
resulting dendrogram of the AHC3 is given in Figure 4.
This time, all oil samples of N. faassenii were placed within
the same class (C7), together with N. mussinii oils. Contrary
to that, N. nepetella samples were recognized as statistically
different and were grouped within C9. As can be seen from
Table 2, the yields of N. faassenii and N. mussinii oils
were comparable and fall within the interval 0.1-0.2% (the
only exception was N. muss4), whereas the yields of N.
nepetella oils were significantly higher (0.8-1.2%).
Alongside with the AHCes, the samples listed in Table 2
were analyzed by PCA, using the same three sets of
variables (original and transformed). The results of PCA1-
PCA3 were almost identical. As can be seen from a typical
byplot of the PCA (PCA1, original variables), given in
Figure 5, values of F1 and F2 factors for all analyzed N.
faassenii, N. nepetella and N. mussinii oils were very
similar, suggesting a high level of likeness of the
mentioned samples.
It could be interesting to mention that all performed MVA
analyses (AHC1-AHC3 and PCA1-PCA3) generally
showed that oils of the “control” species (N. nuda and N.
cataria) were not statistically different (except for the N.
cat4) within the taxon.
F 2 ( 1 0 .8 3 % )
0
N . nuda3
N . sept
N . oxy
N. faas1 -3
N. mu ss1-5
-5
N. nep 1-3
N. cat1-3,5,6
N. nud a4,5 N . ucr
N. cadmea
N. rtanj
N. sib
-1 0
N. erem
-1 5 -1 0 -5 0 5 10 15
F 1 ( 1 3 .2 4 % )
Figure 5: Principal component analysis - ordination of 36 oil samples
(observations). Axes (F1 and F2 factors-the first and second principal
component) refer to the ordination scores obtained for the samples. Axis
F1 accounts for ca. 13% and axis F2 accounts for a further 11% of the
total variance.
Thus, one could conclude that the oil composition of these
Nepeta taxa were not susceptible, at least not significantly,
to external factors (environmental and geographical). For
this reason, it is not unreasonable to assume that the
statistically significant differences in the chemical
composition of the all herein analyzed Nepeta oils could,
at least partially, reflect the differences of corresponding
taxa on the biosynthetic/genetic level. Having all the
above stated in mind, some general conclusions regarding
the inheritance of the monoterpene biosynthetic apparatus
of N. faassenii could be drawn. Considering only the
chemical composition of the analyzed oils, both AHC and
PCA (Figures 2, 3 and 5) recognized, as expected, N.
faassenii as closely related to both N. mussinii and N.
nepetella. It could be even speculated that the hybrid can
unselectively inherit the monoterpene biosynthetic
apparatus from both of its parent species (AHC1 and
AHC2). Nevertheless, when the chemical composition of
the compared oils was not the only compositional data
taken into account, but also their corresponding yields
(AHC3), a higher degree of mutual similarity (with respect
to the monoterpene biosynthesis) of N. faassenii to N.
mussinii, than to its other parent species, was observed.
Experimental
Plant material: Above-ground parts of N. faassenii were
collected in the botanical gardens, Trinity College, Dartry,
Dublin 6. Nepeta nuda was collected from three different
locations in SE Serbia (slopes of the Suva planina
mountain (Donji Dušnik), the Ploče highland and the
vicinity of the city of Niš (village Knez selo)), in August,
2010. Above-ground parts of N. cataria were collected in
the vicinity of the city of Pirot, the slopes of the mountain
Rtanj and the vicinity of the city of Niš (village Knez
Nepeta x faassenii essential oil
selo), SE Serbia), also in August, 2010. Voucher
specimens were deposited in the Herbarium of the Faculty
of Science and Mathematics, University of Niš under the
acquisition number – 201035A-C and 201036A-C.
Isolation of the essential oil: Air-dried, to constant
weight, aboveground parts of Nepeta × faassenii, N.
cataria and N. nuda (batches of 25 – 150 g) were subjected
to hydrodistillation with ca. 1 l of distilled water for 2.5 h
using the original Clevenger-type apparatus [7]. Yields of
the obtained oils (w/w) are given in Table 2. The obtained
oils were separated by extraction with diethyl ether and
dried over anhydrous magnesium sulphate. The solvent
was evaporated under a gentle stream of nitrogen at room
temperature in order to exclude any loss of the essential oil
and immediately analyzed. When the oil yields were
determined, after the bulk of ether was removed under a
stream of N2, the residue was exposed to vacuum at room
temperature for a short period to eliminate the solvent
completely. The pure oil was then measured on an
analytical balance and multiple gravimetric measurements
were taken during 24 h to ensure that all of the solvent had
evaporated.
GC and GC/MS analyses: The GC/MS analyses were
repeated three times for each sample using a Hewlett-
Packard 6890N gas chromatograph. The gas
chromatograph was equipped with a fused silica capillary
column DB-5MS (5% phenylmethylsiloxane, 30 m  0.25
mm, film thickness 0.25 m, Agilent Technologies, USA)
and coupled with a 5975B mass selective detector from the
same company. The injector and interface were operated at
250° and 300°C, respectively. The oven temperature was
raised from 70° to 290°C at a heating rate of 5°C/min and
then isothermally held for 10 min. As a carrier gas helium
at 1.0 mL/min was used. The samples, 1 L of the oil
solutions in diethyl ether (1 : 100), were injected in a
pulsed split mode (the flow was 1.5 mL/min for the first
0.5 min and then set to 1.0 mL/min throughout the
remainder of the analysis; split ratio 40 : 1), which enabled
sufficient separation (narrower peaks due to a pulsed
injection mode despite starting the run at 70°C) and
positive identification of numerous components with RI
lower than 900. The identification of partially overlapping
peaks was also aided by NIST AMDIS (Automated Mass
Spectral Deconvolution and System) Software version 2.4,
supplied by National Institute of Standards and
Technology (NIST, USA). Мass selective detector was
operated at the ionization energy of 70 eV, in the 35–500
amu range with a scanning speed of 0.34 s. GC (FID)
analyses were carried out under the same experimental
References
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236-238.
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Herba Polonica, 54, 68-78.
Natural Product Communications Vol. 6 (7) 2011 1021
conditions using the same column as described for the
GC/MS. The percentage composition was computed from
the GC peak areas without the use of correction factors.
Qualitative analyses of the essential oil constituents wеrе
based on several factors. Firstly, the comparison of the
essential oils linear retention indices relative to the
retention times of C8-C28 n-alkanes on the DB-5MS
column [8] with those reported in the literature [9].
Secondly, by comparison of their mass spectra with those
of authentic standards, as well as those from Wiley 6,
NIST02, MassFinder 2.3. Also, a homemade MS library
with the spectra corresponding to pure substances and
components of known essential oils was used, and finally,
wherever possible, the identification was achieved by GC
coinjection with an authentic sample (see Table 1).
Relative standard deviation (RSD) of peak areas of the
repeated measurements (independent sample preparations
and GC-MS) was for all substances below 1%. The only
exceptions which had higher RSD were minor components
such as bicyclogermacrene, (E)-β-ocimene, (Z)-2-hexenal,
α-terpineol, β-caryophyllene, cis-sabinene hydrate and
β-sesquiphellandrene where RSD was 6, 3, 4, 9, 8, 2 and
13%, respectively. In repeated measurements, no
significant deviation of the component retention times was
observed.
Multivariate statistical analyses: Principal component
analysis (PCA) and agglomerative hierarchical clustering
(AHC) were performed using the Excel program plug-in
XLSTAT version 2010.03.5. Both methods were applied
utilizing three sets of variables: original variables
(percentages of individual oil constituents (only
constituents with the percentage higher than 1% in at least
one sample were taken into account)) and transformed
variables (a-original variables were transformed in such a
way that 4 nepetalactone isomers were summed into one
collective variable; b-for each sample, the relative amount
of the every original variable was multiplied by the
corresponding oil yield). AHC was determined using
Pearson dissimilarity where the aggregation criterion were
simple linkage, unweighted pair-group average and
complete linkage and Euclidean distance where the
aggregation criterion were weighted pair-group average,
unweighted pair-group average and Ward’s method. PCA
of the Pearson (n) type was performed.
Acknowledgments – The research has been supported by
the Trinity College Dublin, Ireland and the Ministry of
Science and Technological Development of the Republic
of Serbia (Project 172061).
1022 Natural Product Communications Vol. 6 (7) 2011 Radulović et al.

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Ozek T, Akgul A, Tumen G. (1993) Composition of the essential oil of Nepeta racemosa Lam. Journal of Essential Oil Research,
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mechanisms underlying the spasmolytic and bronchodilatory properties of the essential oil of Nepeta cataria L. Journal of
Ethnopharmacology, 121, 405-411; (h) Javidnia K, Miri R, Jafari A, Rezai H. (2004) Analysis of the volatile constituents of Nepeta
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H. (2005) Volatile constituents of the essential oil of Nepeta ucrainica L. ssp. kopetdaghensis from Iran. Flavour and Fragrance
Journal, 20, 219-221; (j) Sajjadi SE, Eskandari B. (2005) Chemical constituents of the essential oil of Nepeta oxyodonta. Chemistry
of Natural Compounds, 41, 175-177; (k) Giamperi L,Bucchini A, Cara P, Fraternale D, Ricci D, Genovese S, Curini M, Epifano F.
(2009) Composition and antioxidant activity of Nepeta foliosa essential oil from Sardinia (Italy). Chemistry of Natural Compounds,
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Products, 3, 103-111; (m) Ljaljević-Grbić M, Stupar M, Vukojević J, Soković M, Mišić D, Grubišić D, Ristić M. (2008)
Antifungal activity of Nepeta rtanjensis essential oil. Journal of the Serbian Chemical Society, 73, 961-965; (n) Celik A, Mercan
N, Arslan I, Davran H. (2008) Chemical composition and antimicrobial activity of essential oil from Nepeta cadmea. Chemistry of
Natural Compounds, 44, 119-120; (o) Sefidkon F, Jamzad Z, Mirza M. (2006) Chemical composition of the essential oil of five
Iranian Nepeta species (N. crispa, N. mahanensis, N. ispahanica, N. eremophila and N. rivularis). Flavour and Fragrance Journal,
21, 764-767; (p) Karaman S, Comlekciogolu N. (2007) Essential oil composition of Nepeta cilicia Boiss. Apud Bentham and
Phlomis viscosa Poiret from Turkey. International Journal of Botany, 3, 122-124.
[4] Seidemann J. (2005) World spice plants: economic usage, botany, taxonomy. Springer-Verlag, Berlin Heildeberg, 251.
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Distribution, Vol. 6. Recent Advances in Phytochemistry Series. Runeckles VC, Mabry TJ (Eds). Academic Press, New York,
187-214.
[6] (a) Radulović NS, Blagojević PD, Palić RM. (2009) Fatty acid derived compounds – the dominant volatile class of the essential oil
poor Sonchus arvensis subsp. uliginosus (Bieb.) Nyman. Natural Product Communications, 4, 405-410; (b) Radulović NS,
Blagojević PD. (2010) Essential oil yield-composition hypothesis: could the oil yield give the first insight into its chemical
composition? In 41st International Symposium on Essential Oils: Programe and book of abstracts. Lochynski S, Wawrzenczyk C.
(Eds). Wroclaw Univrsity of Envinronmental and Life Sciences, Wroclaw, Poland, OP-12.
[7] Clevenger, J. P. (1928) Content of essential oil in plants. American Perfumer and Essential Oil Review, 23, 467-503.
[8] Van den Dool H, Kratz PD. (1963) A generalization of the retention index system including linear temperature programmed gas-
liquid partition chromatography. Journal of Chromatography, 11, 463-471.
[9] Adams RP. (2007) Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing
Corporation, Carol Stream, IL.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Chemical Composition and Biological Activity of 1023 - 1026

Salvia verbenaca Essential Oil

Marisa Canzoneria, Maurizio Brunob,*, Sergio Rossellib, Alessandra Russoc, Venera Cardiled,
Carmen Formisanoe, Daniela Riganoe and Felice Senatoree
a
Ente Sviluppo Agricolo – Dipartimento Regionale Azienda Foreste Demaniali, Regione Siciliana,
Via Libertà 97, Palermo, Italia
b
Dipartimento di Chimica Organica, Università degli Studi di Palermo, Parco d’Orleans II,
I-90128 Palermo, Italia
c
Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università di Catania,
V.le A. Doria 6, 95125 Catania, Italia
d
Dipartimento di Scienze Fisiologiche, Università degli Studi di Catania, V.le A. Doria 6,
95125 Catania, Italia
e
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II,
via D. Montesano 49, Napoli, Italia

maurizio.bruno@unipa.it

Received: December 15th, 2010; Accepted: March 14th, 2011

Salvia verbenaca L. (syn. S. minore) is a perennial herb known in the traditional medicine of Sicily as “spaccapetri” and is used to resolve
cases of kidney stones, chewing the fresh leaves or in decoction. The chemical composition of the essential oil obtained from aerial parts of
S. verbenaca collected in Piano Battaglia (Sicily) on July 2009, was analyzed by GC and GC-MS. The oil was strongly characterized by fatty
acids (39.5%) and carbonylic compounds (21.2%), with hexadecanoic acid (23.1%), (Z)-9-octadecenoic acid (11.1%) and benzaldehyde
(7.3%) as the main constituents. The in vitro activity of the essential oil against some microorganisms in comparison with chloramphenicol
by the broth dilution method was determined. The oil exhibited a good activity as inhibitor of growth of Gram + bacteria.

Keywords: Salvia verbenaca, Lamiaceae, volatile components, hexadecanoic acid, (Z)-9-octadecenoic acid, benzaldehyde, β-phellandrene,
antibacterial activity.

The genus Salvia (sage) is one of the largest and the most
important aromatic and medicinal genus of the Lamiaceae
family, comprising about 900 species widespread
throughout the world [1]. Salvia species are used in folk
medicine all around the world for their antibacterial,
antitumor [2], antidiabetic [3], and antioxidant [4]
activities. Members of this genus produce many useful
secondary metabolites including terpenes and phenolics
and their derivatives that have been in the center of
pharmacopoeias of many countries [5].
Salvia verbenaca L. is known in Italy as Salvia minore and
it’s a tall herbaceous perennial plant, 20-50 cm high, with
bluish purple flowers of about 1 cm length arranged in
verticillasters (each of which generally contains six
flowers). The calyx (green, 4-8 mm long) encloses a 6-10
mm long corolla. Nutlet fruits contain 1-4 seeds. The
verticillasters are close together on the stern at flowering,
but move further apart by fruit set. Flowering commences
in mid-April and finishes towards the end of May [6]. The
species is distributed in the Mediterranean area and in Italy
is found frequently throughout the territory with the
exclusion of the Alps. In Sicily, the plant is spread in
scrublands and grasslands throughout all the island, from
sea level to 1.200 m. above sea level [7]. In the Sicilian
traditional medicine is known as “spaccapetri” and its
leaves and flowering aerial parts are used to resolve cases
of kidney stones, chewing the fresh leaves or in decoction.
The plant is also known as bactericide against respiratory
ailments, as healing in wounds and ulcers, and above all as
eyedrops, because fruits or seeds when applied on the eyes
remove impurities or dust particles.
As part of our extensive screening program of Salvia
species from Mediterranean Area [2,4,8,9], we report in
this paper the qualitative and quantitative analyses of the
essential oil of wild population of S. verbenaca collected
in Sicily and compare it with those previously reported.
A total of 76 constituents, representing 91.8% of the total
oil, have been identified from the essential oil extracted
from the aerial parts of S. verbenaca. In Table 1 the
1024 Natural Product Communications Vol. 6 (7) 2011 Canzoneri et al.

Table 1: Volatile components of aerial parts of Salvia verbenaca.

COMPONENT LRI a LRI b %c Identificationd
Hydrocarbons 4.7
Nonane 900 900 1.2 Ri, MS
α-Ionene 1208 0.2 Ri, MS
Tricosane 2300 2300 0.9 Ri, MS, Co-GC
Tetracosane 2400 2400 0.4 Ri, MS, Co-GC
Pentacosane 2500 2500 0.8 Ri, MS, Co-GC
Heptacosane 2700 2700 0.7 Ri, MS, Co-GC
Nonacosane 2900 2900 0.5 Ri, MS, Co-GC
Carbonylic compounds 21.2
(E)-2-Hexenal 854 1209 1.5 Ri, MS
Heptanal 903 1188 0.4 Ri, MS
Benzaldehyde 963 1543 7.3 Ri, MS, Co-GC
1-Octen-3-one 975 1308 0.3 Ri, MS
Phenyl acetaldehyde 1044 1663 1.5 Ri, MS, Co-GC
Acetophenone 1058 1657 0.2 Ri, MS, Co-GC
Nonanal 1102 1401 1.1 Ri, MS
(E,E)-2,4-Octadienal 1125 0.3 Ri, MS
(E,Z)-2,6-Nonadienal 1154 1572 0.5 Ri, MS
Decanal 1204 1508 0.3 Ri, MS
(E)-2-Decenal 1260 1655 0.5 Ri, MS
(E,E)-2,4-Decadienal 1315 1827 0.3 Ri, MS
β-Damascenone 1380 1835 0.8 Ri, MS
(E)-Geranylacetone 1453 1867 0.5 Ri, MS
(E)-β-Ionone 1484 1958 1.9 Ri, MS, Co-GC
Tetradecanal 1619 1934 0.2 Ri, MS
Hexahydrofarnesyl acetone 1845 2131 0.7 Ri, MS
9,12,15-Octadecatrienal 2111 2.9 Ri, MS, Co-GC
Terpenoids
Monoterpene hydrocarbons 11.5
α-Pinene 936 1075 0.5 Ri, MS, Co-GC
Sabinene 973 1132 0.5 Ri, MS, Co-GC
β-Pinene 978 1118 0.5 Ri, MS, Co-GC
α-Terpinene 1012 1189 0.9 Ri, MS, Co-GC
p-Cymene 1025 1278 0.4 Ri, MS, Co-GC
β-Phellandrene 1029 1218 5.9 Ri, MS, Co-GC
Limonene 1030 1203 2.0 Ri, MS, Co-GC
γ-Terpinene 1057 1256 0.8 Ri, MS, Co-GC
Sesquiterpene hydrocarbons 2.2
(E)-Caryophyllene 1418 1612 1.2 Ri, MS, Co-GC
(E)-β-Farnesene 1452 1673 0.3 Ri, MS
-Humulene 1455 1689 0.2 Ri, MS
allo-Aromadendrene 1463 1661 0.1 Ri, MS
α-Amorphene 1475 1679 t Ri, MS
β-Selinene 1475 1715 0.1 Ri, MS
Germacrene D 1477 1726 t Ri, MS
γ-Cadinene 1515 1776 0.3 Ri, MS
α-Calacorene 1542 1918 t Ri, MS
Oxygenated monoterpenes 3.3
1,8-Cineole 1034 1213 0.2 Ri, MS, Co-GC
Linalool 1098 1553 0.7 Ri, MS, Co-GC
Camphor 1143 1532 t Ri, MS, Co-GC
Pinocarvone 1154 1587 1.1 Ri, MS
Borneol 1167 1719 0.1 Ri, MS, Co-GC
Terpinen-4-ol 1176 1611 t Ri, MS, Co-GC
p-Cymen-8-ol 1185 1856 0.3 Ri, MS
α-Terpineol 1187 1706 0.5 Ri, MS, Co-GC
Safranal 1201 1618 0.2 Ri, MS
Carvone 1242 1750 0.2 Ri, MS
Oxygenated sesquiterpenes 3.9
Isocaryophyllene oxide 1527 2001 t Ri, MS
Germacrene D 4-ol 1575 2065 0.3 Ri, MS
Spathulenol 1577 2148 1.7 Ri, MS
Caryophyllene oxide 1581 2208 1.9 Ri, MS, Co-GC
Caryophylladienol I 1640 2316 t Ri, MS
Fatty acids and esters 39.5
Bornyl angelate 1566 t Ri, MS
Dodecanoic acid 1566 2503 0.4 Ri, MS, Co-GC
Tetradecanoic acid 1769 2713 0.9 Ri, MS, Co-GC
Pentadecanoic acid 1873 2740 0.5 Ri, MS, Co-GC
Methyl hexadecanoate 1925 2208 0.7 Ri, MS, Co-GC
Hexadecanoic acid 1972 2931 23.1 Ri, MS, Co-GC
Ethyl hexadecanoate 1994 2245 2.6 Ri, MS
(Z)-9-Octadecenoic acid 2117 3157 11.1 Ri, MS
(Z,Z)-9,12-Octadecadienoic acid ethyl ester 2162 2532 0.2 Ri, MS
Volatile components of Salvia verbenaca
Phenolic compounds
Thymol methyl ether 1239
Thymol 1290
Carvacrol 1297
Eugenol 1353
Others
1-Octen-3-ol 977
2-Pentylfuran 1002
2-Phenylethanol 1115
(Z)-Phytol 1949
(E)-Phytol 2132
Squalene 2828
Total amount of compounds
a
Linear Retention Index on a HP-5 MS column, bLinear Retention Index: retention index on an Innowax column, ct: trace, less than 0,05%, d Ri: retention index matches
with bibliography; MS: identification based on comparison of mass spectra; Co-GC: comparison of retention time of authentic compounds.
retention indices, percentage composition and
identification methods are given; the components, grouped
in class of substances, are listed in order of elution on a HP
5MS column. Carbonylic compounds (21.2%) and fatty
acids (39.5%) were the main fractions of the oil, while the
terpenoidic fraction of the oil amounted to 20.9%, with
monoterpenes accounting to 14.8% and sesquiterpenes to
6.1%. The most abundant compound was hexadecanoic
acid (23.1%), followed by (Z)-9-octadecenoic acid
(11.1%), benzaldehyde (7.3%) and the monoterpene
hydrocarbon β-phellandrene (5.9%).
The essential oil of S. verbenaca from Sicily presented
noticeably different qualitative and quantitative results
compared with the other studied oils. Pitarokili et al.,
(Greece) [10] detected as main compounds β-phellandrene
(30.3%) and (E)-caryophyllene (16.1%), whereas Al-
Howiriny (Saudi Arabia) [11] reported sabinene (16.0%),
cadinene (7.9%), 4-terpineol (7.4%) and pinene (7.3%) as
the dominating compounds. S. verbenaca essential oil from
Morocco presents terpineol (19.2%), camphor (6.6%) and
β-thujone (6.1%) as main compounds [12], while Ben
Taarit et al. [13,14] showed that S. verbenaca wild-
growing in different locations in Tunisia is particularly
rich in oxygenated sesquiterpenes and monoterpene
hydrocarbons, particularly viridiflorol (21.8%), tricyclene
(18.8%), (Z)-β-ocimene (29.5%) for the samples collected
in Sabelet Ben Ammar, Somaa and Sers respectively and
β-caryophyllene (23.1%) and caryophyllene oxide (15.9%)
for the samples collected in the northeast region of
Tunisia. Previous papers [15, 16] showed that many
factors affect the yield and the composition of essential
oils of Salvia species, including plant source, individual
plant chemotypes, time of harvest, the environmental
conditions and the proportion of plant parts distilled.
The in vitro antibacterial activity of the essential oil of S.
verbenaca against eight bacterial strains was evaluated by
determining the minimum inhibitory concentration (MIC)
and the minimum bactericidal concentration (MBC) using
the broth method. The oil appeared to be not active against
Gram – bacteria (MICs >100 μg/mL), while it showed
antibacterial activity against the Gram + bacteria tested. In
particular, Staphylococcus aureus and Streptococcus
faecalis were slightly sensitive to the action of the oil
Natural Product Communications Vol. 6 (7) 2011 1025
Table 1 (contd.)
2.3
1611 0.2 Ri, MS
2198 0.8 Ri, MS, Co-GC
2239 0.6 Ri, MS, Co-GC
2186 0.7 Ri, MS, Co-GC
3.2
1452 0.4 Ri, MS
1243 0.9 Ri, MS
1934 0.4 Ri, MS
2622 1.5 Ri, MS
2625 T Ri, MS
3408 t Ri, MS
91.8
(MIC = 100 μg/mL), while the oil exerted an appreciable
activity against Bacillus subtilis and Staphylococcus
epidermidis (MIC = 50 μg/mL for both)
Experimental
Plant material: Aerial parts of S. verbenaca were collected
at the full flowering stage on July 2009 from plants
growing in Piano Battaglia (Sicily). Voucher specimens
were deposited at the Herbarium of the Botanical Gardens
of Palermo (Italy) under the number PAL 09-876.
Isolation of the volatile components: The fresh samples
were cut into small pieces, then subjected to
hydrodistillation according to the standard procedure
described in the European Pharmacopoeia [17]. The yield
(w/w) was 0.18%. The oil was dried over anhydrous
sodium sulphate and then stored in sealed vials, at - 20°C,
ready for the GC and GC-MS analyses.
Gas chromatography: Analytical gas chromatography
was carried out on a Perkin-Elmer Sigma 115 gas
chromatograph fitted with a HP-5 MS capillary column
(30 m x 0.25 mm i.d.; 0.25 μm film thickness). Column
temperature was initially kept at 40°C for 5 min, then
gradually increased to 250°C at 2°C min-1, held for 15 min
and finally raised to 270°C at 10°C min-1. Diluted samples
(1/100 v/v, in n-pentane) of 1 μL were injected manually at
250°C, and in the splitless mode with a 1 minute purge-off
due to the small amount of oil partially utilized for
biological tests. Flame ionization detection (FID) was
performed at 280°C. Analysis was also run by using a
fused silica HP Innowax polyethylenglycol capillary
column (50 m x 0.20 mm i.d.; 0.20 μm film thickness).
Helium was the carrier gas (1 mL min-1) in both cases.
Gas chromatography - mass spectrometry: GC-MS
analysis was performed on an Agilent 6850 Ser. II
apparatus, fitted with a fused silica HP-1 capillary column
(30 m x 0.25 mm i.d.; 0.33 μm film thickness), coupled to
an Agilent Mass Selective Detector MSD 5973; ionization
energy voltage 70 eV; electron multiplier voltage energy
2000 V. Mass spectra were scanned in the range 35-450
amu, scan time 5 scans/s. Gas chromatographic conditions
were as reported above; transfer line temperature, 295°C.
1026 Natural Product Communications Vol. 6 (7) 2011 Canzoneri et al.

Identification of components: Most constituents were
identified by gas chromatography by comparison of their
retention indices (LRI) with either those of the literature
[18, 19] or with those of authentic compounds available in
our laboratories. The retention indices were determined
by GC-FID mode in relation to a homologous series of
n-alkanes (C8-C28) under the same operating conditions on
both columns. Further identification was made by
comparison of their mass spectra on both columns with
either those stored in NIST 02 and Wiley 275 libraries or
with mass spectra from the literature [19, 20] and our
home made library. Component relative concentrations
were calculated based on GC-FID peak areas without
using correction factors.
Antimicrobial activity: The antibacterial activity was
evaluated by determining the minimum inhibitory
concentration (MIC) and the minimum bactericidal
concentration (MBC) using the broth dilution method as
previously described [2]. Eight bacteria species, selected as
representative of the class of Gram positive and Gram
negative, were tested: Bacillus subtilis (ATCC 6633),
Staphylococcus aureus (ATCC 25923), Staphylococcus
epidermidis (ATCC 12228), Streptococcus faecalis (ATTC
29212), Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (ATCC 10031), Proteus vulgaris (ATCC
13315) and Pseudomonas aeruginosa (ATCC 27853).
Acknowledgments – The GC and GC-MS analyses were
performed at the "C.S.I.A.S." of the University "Federico
II" of Napoli. The assistance of the staff is gratefully
appreciated.

References
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radiation, and ecological specializations of Salvia and tribe Mentheae. American Journal of Botany, 91, 1115-1125.
[2] Cardile V, Russo A, Formisano C, Rigano D, Senatore F, Arnold NA, Piozzi F. (2009) Essential oils of Salvia bracteata and Salvia
rubifolia from Lebanon: Chemical composition, antimicrobial activity and inhibitory effect on human melanoma cells. Journal of
Ethnopharmacology, 126, 265-272.
[3] Kim EJ, Jung SN, Son KH, Kim SR, Ha TY, Park MG, Jo IG, Park JG, Choe W, Kim SS, Ha J. (2007) Antidiabetes and antiobesity
effect of cryptotanshinone via activation of AMP-activated protein kinase. Molecular Pharmacology, 72, 62-72.
[4] Tenore GC, Ciampaglia R, Arnold NA, Piozzi F, Napolitano F, Rigano D, Senatore F. Antimicrobial and antioxidant properties of
the essential oil of Salvia lanigera from Cyprus. Food and Chemical Toxicology, in press, doi:10.1016/j.fct.2010.10.022.
[5] Hedge IC. (1972) Salvia L. In Flora Europaea III; T. G., Tutin, V.H. Heywood, N.A. Burges, D.M. Moore, D.H. Valentine, S.M
Walters and D.A Webb Eds.; Cambridge University Press, Cambridge, UK; pp. 289-290.
[6] Navarro L. (1997) P1ant Systematics and Evolution, 207, 111-117.
[7] Pignatti S. (1982) Flora d’Italia.1-3. Edagricole Bologna.
[8] Formisano C, Senatore F, Apostolides AN, Piozzi F, Rosselli S. (2007) GC and GC/MS analysis of the essential oil of Salvia
hierosolymitana Boiss. growing wild in Lebanon. Natural Product Communications, 2, 181-184.
[9] Mancini E, Arnold NA, De Martino L, De Feo V, Formisano C, Rigano D, Senatore F. (2009) Chemical composition and
phytotoxic effects of essential oils of Salvia hierosolymitana Boiss. and Salvia multicaulis Vahl. var. simplicifolia Boiss. growing
wild in Lebanon. Molecules, 14, 4725-4736.
[10] Pitarokili D, Tzakou O, Loukis A. (2006) Essential oil composition of Salvia verticillata, S. verbenaca, S. glutinosa and S.
candidissima growing mild in Greece. Flavour and Fragrance Journal, 21, 670-673.
[11] Al-Howiriny TA, (2002) Chemical composition and antimicrobial activity of essential oil of Salvia verbenaca. Biotechnology, 1,
45–48.
[12] Holeman M, Berrada M, Bellakhdar J, Ilidrissi A, Pinel R. (1984) Comparative chemical study on essential oils from Salvia
officinalis, S. aucheri, S. verbenaca, S. phlomoides and S. argentea. Fitoterapia, 55, 143-148.
[13] Ben Taarit M, Msaada K, Hosni K, Chahed T, Marzouk B. (2010) Essential oil composition of Salvia verbenaca L. growing wild in
Tunisia. Journal of Food Biochemistry, 34, 142-151.
[14] Ben Taarit M, Msaada K, Hosni K, Ben Amor N, Marzouk B, Kchouk ME. (2010) Chemical composition of the essential oils
obtained from the leaves, fruits and stems of Salvia verbenaca L. from the northeast region of Tunisia. Journal of Essential Oil
Research, 22, 449-453.
[15] Perry NB, Anderson RA, Brennan NJ, Douglas MH, Heaney AJ, McGimpsey JA, Smallfield BM. (1999) Essential oils from
Dalmatian sage (Salvia officinalis L.): Variations among individuals, plant parts, seasons, and sites. Journal of Agricultural Food
and Chemistry, 47, 2048–2054.
[16] Piccaglia R, Marotti M, Della Cecca V. (1997) Effect of planting density and harvest date on yield and chemical composition of
sage oil. Journal of Essential Oil Research, 9, 187–191.
[17] European Pharmacopoeia 6.0, (2008) Determination of essential oils in herbal drugs, 2.8.12, pp. 251–252.
[18] Davies NW. (1990) Gas chromatographic retention indexes of monoterpenes and sesquiterpenes on methyl silicone and Carbowax
20M phases. Journal of Chromatography A, 503, 1-24.
[19] Jennings W, Shibamoto T. (1980) Qualitative Analysis of Flavour and Fragrance volatiles by Glass Capillary Gas
Chromatography. Academic Press, New York.
[20] Adams RP. (2007) Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, 4th Edn, Allured
Publishing Co. Carol Stream, Illinois.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Chemical Composition and Antimicrobial Activities of the 1027 - 1030

Essential Oils from Ocimum selloi and Hesperozygis myrtoides

Márcia G. Martinia, Humberto R. Bizzob, Davyson de L. Moreirac, Paulo M. Neufelda,
Simone N. Mirandaa, Celuta S. Alvianod, Daniela S. Alvianod and Suzana G. Leitãoa*
a
Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, CCS, Bloco A 2o andar,
Ilha do Fundão, CEP 21941-590, Rio de Janeiro, Brazil
b
Embrapa Agroindústria de Alimentos, Avenida das Américas 29501, CEP 23020-470, Rio de Janeiro,
RJ, Brazil
c
Departamento de Produtos Naturais, Far-Manguinhos, FIOCRUZ. CEP 21041-250 - Rio de Janeiro,
RJ, Brazil
d
Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Professor Paulo de Goes, CCS,
Bloco I, Ilha do Fundão, CEP 21941-590, Rio de Janeiro, Brazil

sgleitao@pharma.ufrj.br

Received: December 13th, 2010; Accepted: March 16th, 2011

Ocimum selloi, a traditional medicinal plant from Brazil, is sold in open-air markets at Rio de Janeiro State. Hesperozygis myrtoides is a very
aromatic small bush found in the State of Minas Gerais, Brazil, growing at an altitude of 1800m. The chemical composition of both essential
oils was analyzed as well as their antimicrobial activity against fungi and bacteria. For all specimens of Ocimum selloi obtained at open-air
markets, methylchavicol was major compound found (93.6% to 97.6%) in their essential oils. The major compounds identified in the oil of
H. myrtoides were pulegone (44.4%), isomenthone (32.7%), and limonene (3.5%). Both oils displayed antimicrobial activity against all tested
microorganisms but Candida albicans was the most susceptible one. Combinations of the two oils in different proportions were tested to
verify their antimicrobial effect against C. albicans, which, however, was not modified in any of the concentrations tested. The minimum
inhibitory concentration (MIC) was determined to confirm the antimicrobial activity against C. albicans as well as other clinical isolates
(C. glabrata, C. krusei, C. parapsilosis and C. tropicalis).

Keywords: Lamiaceae, Ocimum, Hesperozygis, essential oils, methylchavicol, pulegone, antimicrobial activity.

The family Lamiaceae is composed by 220 genera and
about 3500-4000 species, many of them used in folk
medicine and as aromatic herbs in the cosmetic, food and
perfumery industries [1]. The most abundant secondary
metabolites of Lamiaceae are terpenoids (mono, sesqui, di
and triterpenes) and flavonoids, but the medicinal use of
this family is primarily related to their essential oil content
[1]. Ocimum and Hesperozygis belong to the Lamiaceae
family and comprise about 160 and 6 species, respectively.
Ocimum selloi Benth. is widely used in traditional
medicine, including in the mountain region of Rio de
Janeiro State, where it is sold in open-air markets [2]. This
species has a pleasant fragrance, similar to that of anise
and fennel’s fruits. Hesperozygis myrtoides (A.St.-Hil.)
Epling is a very aromatic small bush found in the region of
Aiuruoca (Minas Gerais State, Brazil) where it grows at an
altitude of 1800m high. This plant, locally called “poejo”
(“pennyroyal”) because of its strong mint odor, is also
used for the treatment of respiratory disorders. Another
traditional use of this plant is in the preparation of a drink
with “cachaça”, the Brazilian sugar cane spirit, where the
plant is soaked into the bottle’s spirit and buried for one
year before it is consumed (personal communication to the
author by local people of Aiuruoca.). This work aimed to
investigate the chemical composition of the essential oils
obtained from these species as well as to evaluate their
antimicrobial activity.
The essential oils of O. selloi were obtained from fresh
leaves of individuals purchased at different open markets
in the State of Rio de Janeiro. Colorless oils, with a
characteristic odor of anise and with yields ranging
from 0.2% to 0.5% (Table 1) were obtained. From
chromatographic analysis by GC-FID and GC-MS a quite
similar chemical composition was observed among
different individuals of O. selloi. The propenylphenol
methylchavicol was identified in high concentration in all
analyzed samples (93.6% to 97.6%). The oil of O. selloi
from Rio de Janeiro can be considered a good new source
of this substance in a practically pure form. The compound
methylchavicol, also called estragol, is a propenylphenol
responsible for the anise flavor in some plant species and
 1028 Natural Product Communications Vol. 6 (7) 2011 Martini et al.

Table 1: Essential oil yields of Ocimum selloi and Hesperozygis Table 3: Identified compounds in the essential oil from fresh leaves of H.
myrtoides according to the collection sites. myrtoides.
Plants Collection Plant collection Essential oil Compounds RI RIlit Fresh
sites Month/Year yield (%)a Leaves (%)
O. selloi Itaguaí, (RJ) September/ 2009 0.5b -Pinene 937 939 0.2
O. selloi Petrópolis (RJ) November/ 2009 0.4 Sabinene 976 976 0.2
-Pinene 979 980 0.3
O. selloi Petrópolis (RJ) January/ 2010 0.5
Myrcene 992 991 0.4
O. selloi Itaguaí (RJ) February /2010 0.5 Limonene 1031 1031 3.5
O. selloi Madureira (RJ) March/ April/ 2010 0.5 cis-β-Ocimene 1040 1040 0.2
H. myrtoides Aiuruoca (MG) April/ 2010 1.6 Heptanyl acetate 1113 1113 0.2
a
Average of three extractions. Menthone 1156 1154 0.4
b Isomenthone 1167 1164 32.7
Only one extraction was done.
-Terpeneol 1189 1189 0.1
Pulegone 1242 1242 44.4
Table 2: Chemical composition (relative percentage %) of the essential Isomenthyl acetate 1307 1306 7.0
oils from O. selloi purchased at different open markets in Rio de Janeiro. Isopulegol acetate 1312 1308 0.7
Terpinyl acetate 1350 1350 0.4
Rio de Janeiro State
NI* 1.1
Madureira Valencene 1493 1491 0.2
Itaguaí Petrópolis
Substance RIcalc RIlit Fair Monoterpenes 91.8
Sept. Feb. March/April Nov. Jan. Sesquiterpenes 0.2
2009 2010 2010 2010 2010 Total of Identified Compounds % 92.0
Methylchavicol 1204 1195 95.9 94.9 94.7/ 97.6 93.6 94.6 *not identified.
Byciclogermacrene 1497 1494 0.8 0.7 0.8/ 0.4 1.7 1.3
-Caryophyllene 1420 1418 0.6 0.9 0.9/ 0.6 1.0 0.9
-Copaene 1377 1376 0.1 0.1 0.1/ 0.1 0.3 0.3 (collected in the Paraná State, Brazil) has lower levels of
Methyleugenol 1406 1401 0.1 0.2 0.2/ 0.1 0.3 0.3 this compound. Isomenthone, the other monoterpene
3-Octen-1-ol 980 978 0.2 0.4 0.2/ 0.1 0.4 0.6 present in relevant concentration in the oil of H. myrtoides
-Bisabolene 1510 1509 0.1 0.1 0.1/ 0.1 0.5 0.5
Total of
(32.7%), is absent in H. ringens and present only at low
Identified 97.8 97.3 97.0/ 99.0 97.8 98.2 concentration in the oil of H. rhododon (2.2%). On the
Compounds %
other hand, menthone, which was characterized in tiny
proportions in H. myrtoides (0.3%) and H. ringens (2.2%),
is often used in perfumery industry [3,4]. Methylchavicol was one of the major compounds in the essential oil of H.
is also responsible for various biological activities, rhododon (43.4%).
including insecticide and nematicide [4-6]. Interestingly, it
is the main component of essential oils of medicinal plants The antimicrobial activity of the essential oils of O. selloi
belonging to different plant families such as Apiaceae and H. myrtoides were evaluated for a series of
(Pimpinella anisum L., anise; Foeniculum vulgare Mill., microorganisms (Table 4). Both oils displayed
Fennel), Magnoliaceae (Illicium verum Hook F., star anise) antimicrobial activity against all tested microorganisms but
and Asteraceae (Artemisia dracuncullus L., tarragon or the major inhibition halos were shown for the resistant
dragon's-wort) [4]. strain of C. albicans (Table 4). Due to the interesting
results of these oils against C. albicans and the possibility
The occurrence of three different chemotypes for O. selloi to combine the odor of fresh mint of H. myrtoides with the
has been previously reported in literature [6-9] being one anise odor of O. selloi in a future pharmaceutical
chemotype rich in methylchavicol, the second one rich in formulation to treat oral candidiasis, we tested the
methyleugenol, and a third one, rich in methylchavicol and combination of the two oils in order to verify the effect in
trans-anethole. For all plants analyzed in this study, their antimicrobial activity. The proportions tested aimed
methylchavicol was major compound found in their to spare the oil of H. myrtoides in respect to that of
essential oils (Table 2). This suggests that maybe the same O. selloi, since H. myrtoides is native to regions of high
clone was introduced in the State, and has been cultivated altitudes, where it grows wild (making it hard to harvest),
by local plant producers for commercial purposes. and the species O. selloi can be easily grown [5,9]. The
proportions tested and the results obtained are shown in
H. myrtoides furnished a colorless essential oil with a Table 4. The combination of the two oils did not modify
pleasant mint odor and an average yield of 1.6% (Table 3). the activity against C. albicans strain, in any of the
The major compounds were identified as the oxygenated concentrations tested, but it is worth to note that the
monoterpenes pulegone (44.4%) and isomenthone inhibition zone (cm) remained quite the same even
(32.7%), besides limonene in small amount (3.5%). To the with a relatively low concentration of the essential oil of
best of our knowledge, this is the first report on the H. myrtoides.
chemical composition of H. myrtoides essential oil.
Comparison of the chemical composition of the essential Nowadays, opportunistic fungal infections that assault
oil of H. myrtoides with essential oils from other species of immunocompromised patients are frequently resistant to
this genus [10], showed that H. ringens (collected in the ordinary clinical drugs and, hospital-acquired infections
State of Rio Grande do Sul, Brazil) also has a high and antibiotic-resistant bacteria continue to be major health
concentration of pulegone (79.2%), while H. rhododon
Essential oils of Ocimum selloi and Hesperozygis myrtoides Natural Product Communications Vol. 6 (7) 2011 1029

Table 4: Antimicrobial activity of essential oils from O. selloi (A) and H. Fitoquimica e Farmacognosia, Faculdade de Farmácia,
myrtoides (B), and their association at different proportions (A + B):
UFRJ. Samples of Hesperozygis myrtoides (A.St.-Hil.)
Sample C. albicans S. aureus E. coli A. niger L. casei Epling were collected in Aiuruoca, Minas Gerais State. H.
Inhibition Zone (cm)
O. selloi (A) 1.2 0.3 0.5 0.7 0.5
myrtoides was identified by Dr. R. M. Harley and voucher
H. myrtoides (B) 1.5 1.2 0.8 --- 0.8 specimens are deposited at Universidade Estadual de Feira
A + B (4:1) 1.3 0.6 0.6 0.8 0.6 de Santana Herbarium (Feira de Santana, Bahia State,
A + B (3:1) 1.2 0.6 0.6 0.7 0.6
A + B (2:1) 1.2 0.8 0.6 0.7 0.7
Brazil) under the number 1333584.
A + B (1:1) 1.2 0.6 0.7 0.7 0.8
Essential oil extraction: The essential oils were extracted
concerns worldwide [11-13]. Therefore, there is a constant separately from fresh leaves (150 g, O. selloi, 3,802 g H.
search for new antifungal agents. The determination of the myrtoides) by hydrodistillation in a Clevenger-type
minimum inhibitory concentration (MIC) has apparatus for 2 h. Yields are reported in Table 1.
demonstrated that essential oils have a varied ability to
inhibit fungal growth and which justifies susceptibility GC and GC-MS analyses: Gas chromatographic analyses
studies [14]. Since the essential oils tested positive against were performed using an Agilent 7890A gas
C. albicans resistant strain ATCC 24433 in a preliminary chromatograph (Palo Alto, CA, USA) equipped with a
test (drop test) the minimum inhibitory concentration flame ionization detector (FID) and a HP-5 (5% phenyl/
(MIC) was determined to confirm the antimicrobial 95% dimethylpolysiloxane) fused silica capillary column
activity on Candida clinical strains. The MIC data are (30m x 0.32mm x 0.25μm). Hydrogen was the carrier gas
summarized in Table 5. (1.5 mL min-1). The injector temperature was kept at
250°C and the oven temperature programmed from 60° to
Table 5: MIC of the essential oils of O. selloi and H. myrtoides obtained 240°C at a rate of 3°C min-1. Detector (FID) was operated
for Candida clinical strains. at 280°C. One microliter of a 1% solution of the oil in
Sample C. albicans C. parapsilosis C. krusei C. tropicalis C. glabata dichloromethane was injected in split mode (1:100). GC-
MIC (g. mL-1) MS analyses were performed in an Agilent 5973N mass
O. selloi 312.5 2,500.0 2,500.0 2,500.0 78.1 selective detector coupled to an Agilent 6890 gas
H. myrtoides 1,250.0 625.0 625.0 625.0 19.5 chromatograph (Palo Alto, CA), equipped with a HP5-MS
capillary column (30m X 0.25mm X 0.25μm), operating in
C. glabrata was the most susceptible to both essential oils electron impact (EI) mode at 70eV, with transfer line
followed by C. albicans, which was the most susceptible to maintained at 260°C, while mass analyzer and ion source
the oil of O. selloi. On the other hand, the other Candida temperature were held at 150°C and 230°C respectively.
strains - C. krusei, C. parapsilosis and C. tropicalis were Helium (1.0 mL min-1) was used as carrier gas. Oven
more susceptible to the oil of H. myrtoides. The antifungal temperature program, injector temperature and split rate
activity of pulegone, the major compound present in the were the same as stated for GC analyses. A standard
essential oil of H. myrtoides, has already been described solution of n-alkanes (C7-C26), injected in the same column
for C. albicans [15,16] which leads to infer that the and conditions as above, was used to obtain the retention
activity of this oil is may be due to its high content of this indices [19]. Individual volatile components were
substance. As for what concerns the essential oils of O. identified by comparison of their mass spectra (MS) and
selloi, all analyzed samples bear more than 94% of retention indices (RI) with those reported in literature [20]
methylchavicol. Even if we cannot rule out the possible and also to the Wiley Registry of Mass Spectral Data, 6th
antimicrobial activity of minor compounds, it is reasonable Edition [21].
to suggest that methylchavicol may be the principal
antifungal agent in these oils. It has been suggested that the Antimicrobial Assay:
antimicrobial action of rich propenylphenol essential oils Drop Test: The antimicrobial activity of the essential oils
can arise from the complexation between the protein or was preliminarily evaluated by agar diffusion technique
other components of the cell membrane of the microbes (drop test) [22] against resistant strains of Candida
and the phenolic components [17]. Essential oils rich in albicans B type ATCC 36802, Staphylococcus aureus
methylchavicol generally show weak antimicrobial activity MRSA (BMB9393), Escherichia coli, Aspergillus niger
[18] but our results points out interesting results and Lactobacillus casei. The inhibition zone generated
demonstrated for Candida clinical strains. after application of 1 L of pure essential oil obtained
from each species or in combination (Table 4) was
Experimental measured in centimeters.
Plant material: Samples of Ocimum selloi Benth. were
purchased from producers at open markets at Petropolis Minimum inhibitory concentration (MIC): Minimum
and Itaguai cities, Rio de Janeiro State, as well as at the inhibitory concentrations were determined by broth
Madureira fair (“Mercadão de Madureira”), at Rio de microdilution method according to the document M27-A3
Janeiro city. Plants were identified by Dr. R. M. Harley (Candida albicans) of the Clinical and Laboratory
and voucher specimens are deposited at Laboratorio de Standard Institute (CLSI, 2008) [23], using resazurin as
indicator for cell viability [24]. All determinations were
1030 Natural Product Communications Vol. 6 (7) 2011 Martini et al.

performed in triplicate and two independent experiments Acknowledgments – This work was supported by CNPq.
lead to concordant results. Positive and negative growth We are grateful for Associação de Proteção e Educação
controls were included in all assays. Ambiental da Serra e do Vale dos Garcias (Aspasg) for
assisting with plant collection.

References
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composition of Lamiaceae species growing in Southern Brazil. Brazilian Archives of Biology and Technology, 52, 473-478.
[2] Leitão F, Fonseca-Kruel VS, Silva IM, Reinert F. (2009) Urban ethnobotany in Petrópolis and Nova Friburgo (Rio de Janeiro,
Brazil). Revista Brasileira de Farmacognosia, 19, 333-342.
[3] Vincenzi M, Silano M, Maialetti F, Scazzocchio B (2000) Safety data review. Constituents of aromatic plants: II. Estragole.
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VCH, Weinheim.
[5] David EFS, Pizzolato M, Morais R, Ferri AF, Marques MOM, Ming LC. (2006) Influência da temperatura de secagem no
rendimento e composição química do óleo essencial de Ocimum selloi Benth. Revista Brasileira de Plantas Medicinais, 8, 66-70.
[6] Paula JP, Farago PV, Ribas JLC, Spinardi GMS, Döll PM, Artoni RF, Zawadzki SF. (2007) In vivo evaluation of the mutagenic
potencial of estragole and eugenol chemotypes of Ocimum selloi Benth. essential oil. Latin American Journal of Pharmacy, 26,
846-851.
[7] Martins RE, Casali WDV, Barbosa CAL, Carazza F. (1997) Essential oil in the taxonomy of Ocimum selloi Benth. Journal of the
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[8] Moraes LAS, Facanali R, Marques MOM, Ming CML, Meirelles AMA. (2001) Phytochemical characterization of essential oil
from Ocimum selloi. Anais da Academia Brasileira de Ciencias, 74, 183-186.
[9] Costa LCB, Pinto JEBPP, Bertolucci SKVE, Evangelino TS. (2009) Variação no rendimento e composição química do óleo
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Plantas Medicinais, 11, 43-48.
[10] Poser GL, Menut C, Toffoli ME, Verin P, Sobral M, Bessiere JM, Lamaty G, Henriques AT. (1996) Essential oil composition and
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[11] Kanafani Z, Perfect J. (2008) Antimicrobial resistance: resistance to antifungal agents: mechanisms and clinical impact. Clinical
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[12] Niimi M, Firth NA., Cannon RD. (2010) Antifungal drug resistance of oral fungi. Odontology, 98, 15–25.
[13] Warnke PH, Becker ST, Podschun R, Sivananthan S, Springer IN, Russo PAJ, Wiltfang J, Fickenscher H, Sherry E. (2009) The
battle against multi-resistant strains: Renaissance of antimicrobial essential oils as a promising force to fight hospital-acquired
infections. Journal of Cranio-Maxillofacial Surgery, 37, 392-397.
[14] Duarte MCT, Figueira GM, Sartoratto A, Rehder VLG, Delarmelina C. (2005) Anti-Candida activity of Brazilian medicinal plants.
Journal of Ethnopharmacology, 97, 305–311.
[15] Duru ME, Oztark M, Uayur A, Ceylan O. (2004) The constituents of essential oil and in vitro antimicrobial activity of Micromeria
cilicica from Turkey. Journal of Ethnopharmacology, 94, 43-48.
[16] Arruda TA, Antunes RMP, Catão RMR, Lima EO, Sousa DP, Nunes XP, Pererira MSV, Barbosa-Filho JM, Cunha EVL. (2006)
Preliminary study of the antimicrobial activity of Mentha x villosa Hudson essential oil, rotundifolone and its analogues. Revista
Brasileira de Farmacognosia, 16, 307-311.
[17] Omidbeygi M, Barzegar M, Hamidi Z, Naghdibadi H. (2007) Antifungal activity of thyme, summer savory and clove essential oils
against Aspergillus flavus in liquid medium and tomato paste. Food Control, 18, 1518-1523.
[18] Bagamboula CF, Uyttendaele M, Debevere J. (2003) Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol,
linalool and p-cymene towards Shigella sonnei and S. flexneri. Food Microbiology, 21, 33–42.
[19] Van Den Dool H, Kratz PD. (1963) A generalization of the retention index system including linear temperature programmed gás
liquid partition chromatography. Journal of Chromatography A, 11, 463-471.
[20] Adams RP. (2007) Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. 4th Ed. Allured
Publishing Co, Carol Stream, Illinois.
[21] Wiley Registry of Mass Spectral Data (2000) 6th Edition, Wiley Interscience, New York.
[22] Hili P, Evans CS, Veness RG. (1997) Antimicrobial action of essential oils: the effect of dimethylsulphoxide on the activity of
cinnamon oil. Letters in Applied Microbiology, 24, 269–275.
[23] Clinical Laboratory Standard Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved
standard, M27-A3. Wayne, PA: Clinical Laboratory Standard Institute, 2008.
[24] Sarker SD, Nahar L, Kumarasamy Y. (2007) Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of
cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods, 42, 321–324.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Chemical Composition and Antibacterial Activity of the 1031 - 1034

Essential Oil of Lantana camara var. moritziana

Nurby Rios Tescha*, Flor Moraa, Luis Rojasb, Tulia Díazc, Judith Velascoc, Carlos Yánezd,
Nahile Riosd, Juan Carmonaa and Sara Pasqualee
a
Departamento de Farmacognosia y Medicamentos Orgánicos. bInstituto de Investigaciones.
c
Departamento de Microbiología. dDepartamento de Farmacología y Toxicología. eDepartamento de
Farmacia Galenica. Facultad de Farmacia y Bioanálisis. Universidad de Los Andes, Mérida,
Venezuela, 5101

nurbyrios@ula.ve

Received: December 14th , 2010; Accepted: March 16th, 2011

The essential oil obtained from the leaves of Lantana camara var. moritziana (Otto & Dietr.) López-Palacios collected at Rubio, Táchira
State, Venezuela, was obtained by hydrodistillation in a Clevenger trap (0.1% yield). The oil was analyzed by gas chromatography-mass
spectrometry (GC/MS) on HP GC-MS System, model 5973, identifying 33 compounds (97.1%) of which the major components were
germacrene D (31.0%), followed by β-caryophyllene (14.8%), α-phellandrene (6.7%), limonene (5.7%) and 1,8-cineole (5.2%). Evaluation of
the antibacterial activity by agar diffusion method with discs against international reference bacteria (Staphylococcus aureus, Enterococcus
faecalis, Escherichia coli, Klebsiella pneumoniae, Salmonella Typhi, Pseudomonas aeruginosa) showed growing inhibition of E. faecalis
and S. aureus at MIC of 350 mg/mL and 400 mg/mL, respectively.

Keywords: Lantana camara, Verbenaceae, essential oil, antibacterial activity.

Lantana camara Linn (Verbenaceae) is an ornamental
grass with aromatic leaves, orange to bright red flowers
and blue or black fruits (drupes). It is native to tropical
and warm regions worldwide [1-3]. These plants are
used in ethnomedicine to treat various diseases of the
gastrointestinal tract, respiratory tract, as well as
tranquilizers, anti-tumor, rheumatism, hypertension,
uterine bleeding, and applied externally as an antiseptic to
treat leprosy, scabies, tetanus, pustules [4-8].
In Venezuela we found 17 species of Lantana Linn among
which Lantana camara L. commonly known as red
cariaquito, is frequently found as three varieties L. camara
var aculeata (L.) Moldenke (L.), L. camara. var. mista
(L.) L H Bailey, and L. camara var. moritziana (Otto and
Dietr.) Lopez-Palacios [2,9-11]. The last one is a shrub
sometimes of unpleasant smell with yellow to orange or
red flowers which are small and fragrant [10,11].
Previous studies on the analysis of the chemical
composition of the essential oil of Lantana camara from
different parts of the world have been reported: studies of
the essential oil of Lantana camara growing in Brail
showed the presence of monoterpenes, sesquiterpenes and
bisabolone derivatives. Germacrene D is one of the mayor
component; even at different times of collection [12-14].
From Madagascar, Africa, the main constituents found
were β-caryophyllene (19%), an unknown sesquiterpene
(16%), and δ-3-carene (10%) [15]. From Calicut, India, the
major ones were β-caryophyllene (34.8%), geranyl acetate
(22.1%) terpinyl acetate (5.8%), bornyl acetate (4.1%) and
D-limonene (2.3%).
Lantana essential oil is used in traditional medicine, for
example, with insecticidal and nematicidal properties
[16,17]. Insecticidal activity of the essential oil of Lantana
camara was found against Tribolium castaneum (LC50
0.45mg/cm2) [18]. This oil showed antibacterial activity
against Pseudomonas aeruginosa MBC 10 μg/mL,
Staphylococcus aureus MBC 25 μg/mL, E. coli 1.25
μg/mL and antifungal Aspergillus niger, Fusarium solani,
Candida albicans [3,6,8,19,20].
In the present investigation, the study of the composition
and antibacterial activity of the essential oil of Lantana
camara var. moritziana is presented. Leaves of Lantana
camara var. moritziana were hydrodistilled yielding 0.1%
of the essential oil. Analysis of this by GC-MS allowed the
identification of thirty three compounds (97.1% of
whole sample), which are listed in Table 1. The three
major ones were Germacrene D (31.0%), β-caryophyllene
(14.8%) and α-phellandrene (6.7%). Germacrene D and
β-caryophyllene were also found as main constituents
in the composition of the essential oils of Lantana camara
1032 Natural Product Communications Vol. 6 (7) 2011 Tesch et al.

Table 1: Chemical composition of essential oil of Lantana camara var. from several countries [6,8,13,14,16,21-23]. Citral has
moritziana*.
been detected as the common major compound from five
Peak Compounds Area (%) LRI LRI [20] varieties of L. camara from Egypt [24].
1 (Z)-3-Hexenol 0.8 854 851
2 n-Hexanol 0.6 866 837
3 α-Pinene 1.9 938 936 Bacterial resistance is a growing phenomenon driven
4 Sabinene 1.4 975 973 primarily by indiscriminate and irrational use of
5 β-Pinene 1.9 979 978 antibiotics. Resistant Staphylococcus aureus and E.
6 Myrcene 0.7 990 991 faecalis has emerged as a serious public-health problem
7 α-Phellandrene 6.7 1005 1005 that demands increased vigilance in the diagnosis and
8 δ-3-Carene 2.2 1011 1011
investigation of new alternative treatments [25].
9 o-Cymene 2.3 1026 1022
10 Limonene 5.7 1031 1031
11 1,8-Cineole 5.2 1034 1033 The antibacterial activity of the essential oil of Lantana
12 (E)-β-Ocimene 0.8 1050 1041 camara var. moritziana was evaluated. The oil had a weak
13 Terpinolene 0.9 1091 1082 activity against S. aureus ATCC (25923) and E. faecalis
14 Linalool 1.0 1101 1086 ATCC (29212) with MIC values of 400 and 350μg/mL,
15 Camphor 0.5 1152 1143 respectively. The complete results are shown in Table 2.
16 α-copaene 0.5 1383 1379
17 β-Bourbonene 0.5 1391 1378
18 β-Cubebene 0.5 1396 1390
Experimental
19 β-Elememe 0.7 1397 1391 Plant material: The leaves of Lantana camara var.
20 β-Caryophyllene 14.8 1430 1446 moritziana were collected (April, 2010) at Rubio, Táchira
21 β-Copaene 0.4 1439 1430 State, Venezuela, located at 101 m.s.n.m 8°53'07"N
22 β-Guaiene 0.3 1449 1440
64°89'11"O. A voucher specimen Nº NR005, has been
23 α-Humulene 1.3 1466 1455
24 (E)-β-Farnesene 0.9 1468 1458 deposited at the Herbarium of the Faculty of Pharmacy and
25 Alloaromadendrene 1.4 1474 1461 Bioanalysis, University of the Andes (MERF herbarium).
26 t-Muurolene 0.6 1491 1480
27 Germacrene D 31.0 1497 1480 Isolation of the essential oil: Fresh leaves (1000g) were
28 α-Chamigrene 5.1 1511 1503 cut into small pieces and subjected to hydrodistillation for
29 α-Farnesene 1.6 1519 1516 6 h using a Clevenger-type apparatus. One mL of pale
30 δ-Cadinene 0.8 1535 1524
yellow essential oil (0.1% yield) was obtained. The oil was
31 Germacrene B 2.4 1566 1560 [13]
32 Spathulenol 0.8 1584 1576
kept at -4 ° C until used for biological tests.
33 Caryophyllene oxide 0.9 1589 1581
*
Compounds were identified by comparison of the mass spectrum of each Gas chromatography: The volatile components of
component with the Wiley GC/MS library data base and from logarithmic essential oil were analyzed by gas chromatography
retention index (LRI) data. Area % was determined by GC-FID.
using a Perkin-Elmer chromatograph. A 5% phenylmethyl
polysiloxane fused-silica column (AT-5, Alltech Associates
Table 2: Antimicrobial activity of the essential oil of Lantana camara Inc., Deerfield, IL), 60 m x 0.25 mm, film thickness 0.25
var. moritziana. m, was used. The initial oven temperature was 60°C; it
Inhibition zone (mm)* MIC was then heated to 260°C at 4°C/min, and the final
Microorganism
Oil E VA SAM AZT CIP CAZ μg/mL temperature maintained for 20 min. The injector and
Staphylococcus detector temperatures were 200°C and 250°C, respectively.
aureus 8* 35* 400 The carrier gas was helium at 1.0 mL/min. The sample (1
ATCC (25923)
μL) was injected using a Hewlett-Packard ALS injector
Enterococcus
faecalis 8* 21* 350 with a split ratio of 50:1. The identification of each of the
ATCC (29212) compounds was performed by GC-MS with a Hewlett
Escherichia Packard Model 5973, equipped with a HP-5MS column 30
coli NA 24* NT
ATCC (25922) m long x 0.25 diameter, film thickness 0.25 μm Hewlett-
Klebsiella Packard. The oven temperature program was the same as
pneumoniae NA 32* NT that used for the HP-5 column for GC analysis; the transfer
ATCC (23357)
Salmonella Typhi 40*
line temperature was programmed from 150ºC to 280ºC;
NA Injector temperature 230ºC, interphaase temperature
CDC57
Pseudomonas 150°C, helium carrier gas at a linear speed of 34 m/s,
aeruginosa NA 40* NT
ATCC (27853)
ionization energy 70 eV, scan range of 40-50 amu, 3.9
E: Erythromycin® 150μg, VA: Vancomycin® (30 μg ), SAM: Sulbactam - scan /s. Injection volume 1 μL of a solution diluted to 2%
Ampicillin® (10μg/10μg), AZT: Aztreonam® (30μg) , CIP: Ciprofloxacin® in diethyl ether. The identification of compounds was
(30μg), CAZ: Ceftazidime® (30 μg), NA: non active, NT: not tested. based on database Wiley and NIST MS Data Library 05,
*Inhibition zone, diameter measured in mm, disc diameter 6 mm, average logarithmic retention indices (LRI) were compared with
of two consecutive assays.
MIC: Minimal inhibitory concentration, concentration range 10-600 values available in the literature (6th edition) [26].
μg/mL.
Essential oil of Lantana camara var. moritziana Natural Product Communications Vol. 6 (7) 2011 1033

Microbiological analysis minimum inhibitory concentration (MIC) was determined

Bacterial strains: Staphylococcus aureus ATCC 25923,
Enterococcus fecalis ATCC 29212, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC 23357,
Salmonella Typhi CDC57 y Pseudomonas aeruginosa
ATCC 27853 were used in this study, provided by the
Department of Microbiology and Parasitology, Faculty of
Pharmacy and Bioanalysis, University of the Andes.
only against organisms that showed inhibition zone,
preparing dilutions of the oil with dimethylsulfoxide at
concentrations of 10 to 600 μg/mL. The reference
antibiotics employed were: Erythromycin® 150μg,
Vancomycin® 30 μg, Sulbactam-Ampicillin® 10 μg/10 μg,
Aztreonam® 30 μg, Ciprofloxacin® 30 μg, and Ceftaxidime®
30 μg. The tests were performed in duplicate.

Antibacterial method: Antibacterial activity was evaluated
according to the agar diffusion method with disks [27].
The strains were maintained in agar conservation at room
temperature. Every bacterial inoculum (2.5 mL) was
incubated in Mueller-Hinton broth at 37ºC for 18 h. The
Acknowledgments – The authors would like to thank
Consejo de Desarrollo Científico, Humanístico, Tecnoló-
gico y de las Artes (CDCHTA-Mérida-Venezuela) for
financial support of this study (Project FA-480-10-08-B).

References
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[2] Hokche O, Berry P, Huber O. (2008) Catálogo de la flora vascular venezolana. Fundación Instituto Botánico de Venezuela “Dr.
Tobbias Lasser”. Caracas-Venezuela. 654pp.
[3] Pattnaik S, Pattnaik, B. (2010) A study of Lantana camara linn aromatic oil as an antibacterial agent. International Journal of
Research in Pharmaceutical Sciences, 01, 32-34.
[4] Mahato S, Sahu N, Roy S, Sharma O. (1994) Potencial antitumor agents from Lantana camara: structures of flavonoid, and
phenylpropanoid glycosides. Tetrahedron, 50, 9439-9446.
[5] Ghisalberti E. (2000) Lantana camara L. (Verbenaceae). Fitoterapia, 71, 467-486.
[6] Deena M, Thoppil, J. (2000) Antimicrobial activity of the essential oil of Lantana camara. Fitoterapia, 71, 453-455.
[7] Silva G, Martins F, Matheus M, Leitão S, Fernández P. (2005) Investigation of anti-inflammatory and antinociceptive activities of
Lantana trifolia. Journal of Ethopharmacology, 100, 254-259.
[8] Sonibare O, Effiong I. (2008) Antibacterial activity and cytotoxicity of the essential oil of Lantana camara L. leaves from Nigeria.
African Journal of Biotechnology, 7, 2618-2620.
[9] Schnee L. (1960) Plantas comunes de Venezuela. Revista de la Facultad de Agronomía de la Universidad Central de Venezuela
(UCV), 3, 150.
[10] López S. (1977) Flora de Venezuela. Verbenaceae. Universidad de los Andes (ULA). Facultad de Farmacia, 327-328, 360-367.
[11] López P. (1974) Lantana camara var. moritziana (Otto & A. Dietr.). Revista de la Facultad de Farmacia, 14, 21.
[12] Weyerstahl P, Marschall H, Eckhardt A, Christians C. (1999) Constituents of commercial Brazilian Lantana oil. Flavour and
Fragrance Journal, 14, 15-28.
[13] Oliveira J, Neves I, Camara C. (2008) Essential oil composition of two Lantana species from Mountain Forests of Pernambuco
(Northeast of Brazil). Journal of Essential Oil Research, 20, 530-532.
[14] Sousa E, Colares A, Rodríguez F, Campos A, Lima S, Costa J. (2010) Effect of collection time on essential oil composition of
Lantana camara Linn (Verbenaceae) growing in Brazil Northeastern. Records of Natural Products, 4, 31-37.
[15] Möllenbeck S, König T, Shereier P, Schwab W, Rajaonarivony J, Ranarivelo L. (1997) Chemical composition and analysis of
enantiomers of essential oils from Madagascar. Flavour and Fragrance Journal, 12, 63-69.
[16] Love A, Naik D, Basak S, Pathak N, Babu C. (2009) Variability in foliar essential oils among different morphotypes of Lantana
species complexes, and its taxonomic and ecological signicance. Chemistry & Biodiversity, 6, 2263-2274.
[17] Padalia R, Verma R, Sundaresan V. (2010) Volatile constituents of three invasive weeds of Himalaya Region. Records Natural
Products, 4, 109-114.
[18] Mohamed M, Abdelgaleil S. (2008) Chemical composition and insecticidal potential of essential oils from egyptian plants against
Sitophilus oryzae (L.) (Coleóptera: Curculionidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Applied
Entomology and Zoology, 43, 599-607.
[19] Kurade N, Jaitak V, Kaul V, Sharma O. (2010) Chemical composition and antibacterial activity of essential oils of Lantana
camara, Ageratum houstonianum and Eupatorium adenophrum. Pharmaceutical Biology, 16, 216-218.
[20] Randrianalijaona J, Ramanoelina P, Rasoarahona J, Gaydou E. (2006) Chemical compositions of aerial part essential oils of
Lantana camara L. chemotypes from Madagascar. Journal of Essential Research, 18, 405-407.
[21] Khan M, Srivastava K, Syamasundar K, Singh M, Naqvi A. (2002) Chemical composition of leaf and flower essential oil of
Lantana camara from India. Flavour and Fragrance Journal, 17, 75-77.
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[24] Saleh M. (1974) Gas-Cromatrographic analysis of the essential oil of Lantana camara L. Varieties. Planta Médica, 25, 373-375.
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[26] Adams R. (2007) Identification of essential oil components by gas chromatograpy/mass spectroscopy, 4th Edition. Illinois USA:
Allured Publishing Corporation, Carol Stream, Ill, 804 pp.
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 2011
NPC Natural Product Communications Vol. 6
No. 7
Activity against Streptococcus pneumoniae of the Essential 1035 - 1038
Oil and 5-(3-Buten-1-ynyl)-2, 2'-bithienyl Isolated from
Chrysactinia mexicana Roots
Bárbara Missiam Mezari Guevara Camposa, Anabel Torres Ciriob, Verónica Mayela Rivas Galindob,
Ricardo Salazar Arandab, Noemí Waksman de Torresb and Luis Alejandro Pérez-Lópezb*
a
Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Química,
Av. Pedro de Alba y Manuel L. Barragán s/n, Cd. Universitaria, C.P. 66451, San Nicolás de los Garza,
Nuevo León, México
b
Universidad Autónoma de Nuevo León, Facultad de Medicina, Departamento de Química Analítica,
Av. Francisco I. Madero y Dr. Eduardo Aguirre Pequeño s/n, Col. Mitras Centro, C.P. 64460,
P.O. Box 2316 Sucursal Tecnológico, Monterrey, Nuevo León, México

lualejandro@hotmail.com

Received: December 14th, 2010; Accepted: March 16th, 2011

The essential oil of Chrysactinia mexicana retrieved from the root bark was characterized by gas chromatography coupled to a mass detector.
The compounds silphiperfol-5-ene, 7-epi- silphiperfol-5-ene, modheph-2-ene, -isocomene, -isocomene and methyl-linoleate were
identified. The principal compound (76.42%) could not be identified by the library and was further isolated through a reverse phase C-18
chromatography followed by silica gel chromatography and identified as 5-(3-buten-1-ynyl)-2,2'-bithienyl. Both the oil and the isolated
compound were tested for their antimicrobial activity against two strains of Streptococcus pneumoniae resistant to -lactam antibiotics. MICs
were 250 g/mL and 125 g/mL respectively. This is the first report about extraction of oil and compound 5-(3-buten-1-ynyl)-2, 2'-bithienyl
from roots of Chrysactinia mexicana as well as the determination of antimicrobial activity against S. pneumoniae.

Keywords: Chrysactinia mexicana, essential oil, 5-(3-buten-1-ynyl)-2, 2'-bithienyl, antimicrobial activity, Streptococcus pneumoniae.

Essential oils are used in alternative medicine as a remedy
for many infectious diseases, antimicrobial properties have
been long established and several studies have confirmed
that they have activity against bacteria, yeasts and fungi
[1-3]. Essential oils have been traditionally used for
respiratory tract infections [4].
Chrysactinia mexicana, is a fragrant plant that contains
essential oils in aerial part, commonly known as calanque,
damianita, hierba de San Nicolás or falsa damiana. It is
distributed throughout northern Mexico and Texas, USA.
It use is known for to treat respiratory illnesses and skin
infections. The leaves and stems are used as a diuretic, to
counteract stomach ailments or postpartum pain, using the
root as a home remedy [5]. The activity of an ether extract
of the roots of Chrysactinia mexicana against two strains
of Streptococcus pneumoniae resistant to penicillin have
been reported [6].
Streptococcus pneumoniae, is a major causative agents of
infectious diseases of the respiratory tract, causing a
significant number of deaths worldwide, in both hospital
and community, affecting mainly children and elderly.
This situation is exacerbated by the increasingly frequent
emergence of resistant strains. This infectious disease is
the leading cause of death in developing countries,
followed by heart disease, diarrhea, HIV/AIDS and stroke.
Pneumonia is the leading cause of death in children under
5 years and about two million deaths each year among this
group of people around the world, mainly in low resource
environments, poor nutrition and poor hygiene conditions
[7,8]. In the last decades has increased the incidence of
pneumococcal infections in adults, despite the wide
availability of effective antibiotics, continue to cause high
morbidity and mortality, reporting mortality rates of up to
20% in adult patients with bacterium [9,10]. Due to the
great problem posed by bacterial infections, was reviewed
the importance of using traditional medicine, mainly using
natural products of plant origin, to counteract these
diseases, including respiratory tract infections [11]. Our
aim was to obtain the essential oil from the root of
Chrysactinia mexicana, characterize their composition, to
isolate some key components and evaluate their
antimicrobial activity against two strains of Streptococcus
pneumoniae resistant to -lactam antibiotics.
Since there are reports of Chrysactinia mexicana use in
folk medicine for the treatment of respiratory diseases and
scientific studies of activity of both the shoot and root
against various microorganisms [5,6,12], our aim in this
1036 Natural Product Communications Vol. 6 (7) 2011 Campos et al.

work was, to characterize their composition, to isolate Table 1: Chemical composition from essential oil of the root bark of
Chrysactinia mexicana.
some key components and to evaluate their antimicrobial
activity against two strains of Streptococcus pneumoniae Compound tR Area % IK
Silphiperfol-5-ene 32.90 0.57 1329
resistant to -lactam antibiotics. 7-epi-silphiperfol-5-ene 33.64 1.59 1348
Modheph-2-ene 35.13 1.53 1384
In preliminary experiments we tested the activity of -isocomene 35.40 3.29 1388
essential oil from the root bark and root without bark of -isocomene 36.17 1.11 1407
Chrysactinia mexicana against two strains of 5-(3-buten-1-ynyl)-2,2'-bithienyl 59.85 76.42 -
Streptococcus pneumoniae resistant -lactam antibiotics No identified 61.43 4.01 -
(ATCC 49619 and 24-CCPN-02) and we found major No identified 61.75 0.68 -
No identified 62.14 0.48 -
activity in root bark (MIC of 250 g/mL against both
Methyl-linoleate 63.16 0.41 2096
strains), therefore we decided to work specifically with the No identified 64.19 1.57 -
root bark. We obtained a dark yellow essential oil with No identified 66.43 8.34 -
yield of 0.41%.
1
Cárdenas et al. reported the characterization of the H NMR spectrum showed three signals at  5.56 ppm (dd,
essential oil from the leaves of this plant showing that the J =11.20, 1.90 Hz),  5.74 ppm (dd, J = 17.50, 1.90 Hz)
main components are terpenes, including eucalyptol and  6.40 ppm (dd, J = 17.50, 11.20 Hz) for terminal
(41.3%) and piperitone (37.7%) [12]. So far, no studies are olefin. Signals between  7.04 ppm and  7.25 ppm were
available on the characterization of extract components or assigned to olefinic protons from heterocycles according to
essential oil from root of Chrysactinia mexicana. This J values (J = 3.60, 3.80, 5.09, 5.12 Hz) (Table 2. The 13C
work is the first report on the study of the root of this NMR spectra exhibited 12 signals, including one
plant, specifically of the essential oil obtained from the methylene, six methines and five quaternary carbons, as
bark. GC/MS analysis showed the presence of 12 seen determined by a DEPT experiment. Structure of 5-(3-
compounds. The main component had a retention time of buten-1-ynyl)-2,2'-bithienyl (figure 1) was established by
59.85 minutes and represented the 76.42%, other 2D NMR experiments: COSY, HMQC and HMBC. The
component with retention time of 66.43 minutes, compound has been reported in aerial part of Chrysactinia
represented the 8.34%. However, neither could be mexicana and roots of other plants [15-18].
identified using the NIST library. Only six of the 12 Table 2: NMR spectral data of 5-(3-buten-1-ynyl)-2,2'-bithienyl, in CDCl3.
compounds were identified from GC/MS spectra using the
Position Type Chemical shifts, (ppm) Coupling constants, Hz
NIST library and they represented the 8.50% of total area. 13C 1H JHH
Five of them were sesquiterpenes (silphiperfol-5-ene, 7- 2 C 139.01
epi-silphiperfol-5-ene, modheph-2-ene, ɑ-isocomene and 2’ C 136.70
4 CH 132.86 7.11 (d) 3.80
ß-isocomene) and one was the esther methyl-linoleate. The 4’ CH 127.97 7.02 (dd) 5.09, 3.60
sesquiterpenes have been reported in roots from Silphium C, terminal CH2 127.04 5.74 (dd) 17.50, 1.90
perfoliatum and Echinops giganteus [13,14]. Other three olefine 5.56 (dd) 11.20, 1.90
5’ CH 125.04 7.25 (d) 5.12
minor component representing 15.08% of the total area 3’ CH 124.27 7.18 (d) 3.60
were no identified (Table 1). Because the main component 3 CH 123.54 7.04 (d) 3.80
could not be identified by NIST library, we decided to 5 C 121.75
C, terminal CH 116.82 6.04 (dd) 17.52, 11.20
isolate it and identified by spectroscopic techniques. olefine
C, alkynes C 92.98
Essential oil obtained from the root bark of Chrysactinia C 83.26
mexicana was further purified by means of reverse phase 1'
3 4

low-pressure liquid chromatography, to afford the active S H

compound six fractions were obtained (F1 to F6). The
most important compound was found in fraction F5 and
the yield was 34%. A gravitational column chromatography
on silica gel was performed from F5 to give 4 fractions
(F5a to F5d) and, pure compound was obtained in F5b
representing a yield of 64.4% from the F5 initially placed
in this column.
Compound was subjected to structural analysis. CG/MS
analysis displayed one peak at 59.85 minutes with m/z 216
(M+). IR showed several important signals at  max 3000-
3104 cm-1 (C-H), 2192.53 cm-1 (triple bound C-C),
1636.14 cm-1 and 1602.86 cm-1 (terminal olefin) and 785 y
838 cm-1 (corresponded to derivatives 2,2'-bithienyl 5
substituted).
2 5 C C
5' 2' S C
1
4' 3'
C H
H
Figure 1: Structure of 5-(3-buten-1-ynyl)-2, 2'-bithienyl.
5-(3-buten-1-ynyl) -2, 2 '-bithienyl have been isolated from
several plants and it antimicrobial and nematicidal
activities [16] have been reported (antimicrobial activity
against the plants pathogens: Colletotrichum sp, Fusarium
oxysporum, Phomopsis viticola, and Peltula. obscurans
[19] or against the human pathogens: Candida albicans,
Escherichia coli and Sarcina lutea [20]). We studied the
activity of the pure compound 5-(3-buten-1-ynyl) -2,2'-
bithienyl against two resistant strains of Streptococcus
pneumoniae (ATCC 49619 and 24-CCPN-02) and the MIC
value was equal for both strains (Table 3). This is the first
Essential oil of Chrysactinia mexicana
Table 3: Minimal Inhibitory Concentration of essential oil and 5-(3-buten-1-
ynyl)-2,2'-bithienyl obtained from the root bark of Chrysactinia mexicana
against Streptococcus pneumoniae strains.
Sample MIC (g/mL)
Streptococcus pneumoniae Strain
ATCC 49619 24-ccpn-02
Essential oil 250 250
5-(3-buten-1-ynyl)-2,2'-bithienyl 125 125
Oxacillin 125 31.25
Cephalotin 31.25 31.25
Vancomycin <1.95 <1.95
report of the activity of 5-(3-buten-1-ynyl)-2, 2'-bithienyl
on the pathogen causing respiratory tract infectious
diseases in humans. The activities found for both, the
compound isolated and essential oil were similar, this
finding could be explained because the main component in
the essential oil is 5-(3-buten-1-ynyl)-2,2'-bithienyl that
representing 75% of the total area.
On the other hand, we did an analysis of the essential oil of
leaves but the compound 5-(3-buten-1-ynyl)-2, 2'-bithienyl
was not detected. Therefore, we decide to prepare again
extracts of the leaves, root bark, twigs and bark of the
trunk (side closest to the root) using the solvents hexane,
methylene chloride and methanol (1:1:1), agree with the
report by Domínguez [15]. Our results showed that the
compound was not present in the aerial part. The
difference in the composition of the oil could be attributed
to the date and place of collection.
The antimicrobial activity displayed by Chrysactinia
mexicana essential oil against Streptococcus pneumoniae
supports the traditional use of this plant for the treatment
of infectious diseases. The isolation biodirected afforded
the 5-(3-buten-1-ynyl)-2,2'-bithienyl as the principal
compound responsible of the antimicrobial activity shown
by this plant. The results presented here are important in
the search for new antimicrobial agents against bacteria
responsible for respiratory diseases, especially those
resistant to conventional antibiotics.
Experimental
Bacterial culture: Streptococcus pneumoniae (InDRE 24-
CCpn-02) and Streptococcus pneumoniae (InDRE 49619),
resistant to oxacillin and sensitive to vancomycin
respectively, were obtained from the “Instituto Nacional de
Diagnóstico y Referencia Epidemiológicos” (InDRE,
México. D.F., Mexico). Microorganisms were maintained
on agar supplemented with bovine blood (BBL, Becton
Dickinson de México) until use.
Plant material: Chrysactinia mexicana roots were
collected in Arteaga, Coahuila, Mexico, in November
2009. A voucher specimen (UNL 024102) was deposited
in the herbarium of the Facultad de Ciencias Biológicas,
Universidad Autónoma de Nuevo León.
Isolation of essential oil: The essential oil was obtained by
hydrodistillation of ground fresh root bark for 4 h, using
a Clevenger-type apparatus. Essential oil was conserved at
–4C until use.
Natural Product Communications Vol. 6 (7) 2011 1037
CG/MS analysis: Analysis of the essential oil was
performed using an Agilent Technologies 6890N gas
chromatograph equipped with an HP-5ms column (30 m 
0.25 mm i.d., 0.25 m film thickness) and a 5973 INERT
selective mass spectrometer. The carrier gas was helium
(99.999%) at a flow rate of 0.5 mL/min; ionization energy
was 70 eV. Data acquisition was scan mode. Ionization
source temperature was 230C, quadrupole temperature
was 150C, and the injector temperature was 220C. Oven
temperature was programmed to 35C for 9 min, then from
35C to 150C at 3C/min and held for 10 min, then at
10C/min to 250C, and finally at 3C/min to 270C and
held for 10 min. The samples were injected using the
splitless mode. The injection volume was 2 L.
Components were identified by comparison of their
retention indices (Kovats indices) relative to C8–C20
n-alkanes, and their mass spectra were compared with
mass spectra from the US National Institute of Standards
and Technology (NIST) library and reference data [21].
Relative percentages of components were calculated based
on GC peak areas without using correction factors.
Isolation of 5-(3-buten-1-ynyl)-2,2'-bithienyl: Essential
oil (17.3 mg) obtained from the Chrysactinia mexicana
root bark by hydrodistillation was subjected to inverse
phase column chromatography over C-18. Elution was
started with MeOH:H2O 90:10 to 0.5 mL/min. until the
principal compound was eluted and then with MeOH.
Finally 6 fractions were obtained (F1 to F6). On the basis
of the antimicrobial activity, F5 (5.9 mg) was further
fractioned in column chromatography over silica gel with
hexane and then with MeOH to give 4 fractions (F5a to
F5d). According to its antimicrobial activity, fraction F5b
(3.8 mg) was further analyzed by CG/MS, IR, 1HNMR,
13
C NMR and 2D NMR (COSY, HMQC and HMBC) and
identified as 5-(3-buten-1-ynyl)-2, 2'-bithienyl. NMR
Spectra were recorder on Bruker Avance DPX 400
equipment.
Antimicrobial activity: Both the oil and the isolated
compound were tested for their antimicrobial activity.
Streptococcus pneumoniae strains resistant to -lactamic
antimicrobials, were tested with microdilution assays
according to the National Committee for Clinical
Laboratory Standards [22]. In order to prepare the inocula,
Streptococcus pneumoniae strains were cultured in Petri
dishes containing blood agar (Bacto, Becton Dickinson).
Plates were incubated overnight at 37C and suspensions
were prepared by transferring colonies to 0.85% NaCl
solution until the turbidity of the 0.5 McFarland standard
was reached. The suspensions were diluted 1:50 with
cation-adjusted Mueller–Hinton broth supplemented with
5% lysed horse blood (CAMHB-LHB; BBL, Becton
Dickinson) to make the working suspensions of
Streptococcus pneumoniae. The essential oil was prepared
at a concentration of 2 mg/mL in 20% of DMSO in
CAMHB-LHB. The antimicrobial activity assay was
performed in flat-bottom 96-well polystyrene microplates
1038 Natural Product Communications Vol. 6 (7) 2011 Campos et al.

covered with a low evaporation lid. The culture medium
was CAMHB-LHB. The concentration of the essential oil
ranged from 500,000 to 15.625 g/mL. Oxacillin and
vancomycin were used as antimicrobial drug controls (64–
4 g/mL). The final concentration of microorganisms was
1  104 UFC. Plates were incubated at 37C for 24 h and
bacterial growth was examined. The MIC was defined as
the minimum concentration of essential oil that stops
growth. Every biological assay was conducted in duplicate.
Acknowledgements - The authors are grateful to the
biologists Marco Antonio Guzmán Lucio and M.C. María
del Consuelo González de la Rosa for definitive taxonomic
identification of species reported here. We thank Dr.
Adolfo Caballero Quintero for support in IR analysis and
Ivonne Carrera for her technical assistance in the
extraction procedures and acknowledge grants
103.5/08/3125 and 103.5/09/4913 from PROMEP-Mexico.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Antimycotic Effect of the Essential Oil of Aloysia triphylla 1039 - 1043
against Candida Species Obtained from Human Pathologies
María de las Mercedes Oliva, María Evangelina Carezzano, Mauro Nicolás Gallucci and
Mirta Susana Demo

Departmento de Microbiologia e Inmunología, Fac. de Ciencias Exactas, Fco-Qcas y Naturales.

Universidad Nacional de Río Cuarto. Córdoba, Argentina

moliva@exa.unrc.edu.ar

Received: December 10th, 2010; Accepted: March 14th, 2011

The research of alternative substances to treat infections caused by Candida species is a need. Aromatic plants have the ability to produce
secondary metabolites, such as essential oils (EO). The antimicrobial properties of Aloysia triphylla (L`Her.) Britton (cedrón) EO has been
previously described. The aims of this work were to determine the antimicrobial activity and the effect on the cell structure of the EO of
A. triphylla against Candida sp isolated from human illnesses. The EO was obtained by hydrodistillation of A. triphylla leaves.
The minimum inhibitory concentration (MIC) was performed with microdilution method and the minimum fungicidal concentration (MFC)
was determined. A. triphylla EO´s showed antifungal activity against all yeast: C. albicans, C. dubliniensis, C. glabrata, C. krusei,
C. guillermondii, C. parapsilosis and C. tropicalis which were resistant to fluconazol (150 mg/mL). The range of MIC values was from: 35
to 140 µg/mL and the MFC: 1842 to 2300µg/mL. The time of killing at the MFC against C. albicans (3 x 105 UFC/mL) was 140 min. The
dates of OD620 and OD260 suggest lysis and loss of absorbing material, respectively. The HROM shows distortion in morphology and shape
of the cell, with large vacuoles in the cytoplasm. These studies clearly show that A. triphylla EO is a promising alternative for the treatment
of candidiasis.

Keywords: candidiasis, Aloysia triphylla, essential oil, antimycotic activity.

Candida species are commonly part of the normal flora in
the digestive tract of healthy humans; however they have
been described as responsible for opportunistic infections,
particularly in neonates and inmunocompromised patients
[1]. These infections are difficult to treat with traditional
drugs because they have multiple side effects, high toxicity
and yeasts develop resistance against antifungal
chemotherapics. Thus, searching for alternative antifungal
compounds has been a major concern in recent years [2,3].
The investigation of alternative substances to treat these
infections is necessary to find a solution to these
problematic. Several medicinal plants have been
extensively studied in order to find more effective and less
toxic compounds [4].
Aromatic plants constitute an interesting group of
vegetables with ability to produce secondary metabolites,
such as essential oils (EO). Aloysia triphylla (L`Her.)
Britton, (Aloysia citriodora Palau,) popularly known as
“cedrón”, is a member of the Verbenaceae Family. It is
perennial and grows widely in North and South America
and also in northeast, northwest and central regions of
Argentine. It is cultivated from Mexico till the South
region of the continent. It is a bush with white flowers and
fruits, with an intense scent lemon-like, sweet, lightly
floral, and herbaceous [5,6]. This specie is used in
folk medicine to treat many digestive disorders, as
anti-inflammatory, analgesic, antipyretic, tonic and
stimulating. It shares an important place on the
international herbal market due to the sensory and
medicinal properties of it EO. These attributes determine
its use as a primary ingredient for infusions and
nonalcoholic beverages as well as aromatic ingredient for
the flavor and fragrance industries. The pharmaceutical
industry uses A. triphylla for its carminative,
antispasmodic and sedative properties [7,8]. EO are
constituted by a complex mixture of organic compounds
including monoterpenes, diterpenes, carbonylated
products and polyenes. There are many studies that
suggest the antibacterial and antifungal activity of these
compounds [9,10]. A. triphylla could be used to treat
infections produced by Candida species.
The aims of this work were to determine the antimicrobial
activity and the effect on the cell structure of the EO of A.
triphylla against Candida species isolated from human
illnesses.
The A. triphylla EO´s were analyzed with GC-MS. The
average yield obtained in the hydrodistillation process was
0.4% (w/v) and the main components identified were:
limonene (2.9%), neral (20%), geranial (29.2%),
spathulenol (8.9%) and caryophyllene oxide (7%), in
concordance with other authors that previously described
1040 Natural Product Communications Vol. 6 (7) 2011 Oliva et al.

all of them as the characteristic constituents of the EO of Table 2: OD620 lectures in C. albicans cells treated with A. triphylla EO
and not treated
A. triphylla [6,10-12].
OD 620nm
Treatment 0h Not centrifuged (18 h) Centrifuged (18 h)
The antifungal activity of A. triphylla EO was tested using 0.513 1.785 1.605
Control
a microdilution broth method. The EO presented With EO 0.513 0.010 0.074
antifungal activity against all yeast. The range of MIC CS+EO 0.081 0.009 0.073
values was from 35 µg/mL to 143 µg/mL (Table 1). It is
interesting to note low values of MIC necessary to inhibit
C. albicans and C. dubliniensis (MIC: 35 µg/mL). In
addition to this, the EO was able to cause the death of all
Candida species. The fungicidal effect of the EO against
the yeasts reached values of MFC from 230 µg/mL to
1842 µg/mL. (Table 1)

Table 1: Minimum Inhibitory Concentration (MIC) and Minimum

Fungicidal Concentration (MFC) of A. triphylla essential oil against
Candida species
Species MIC (µg/mL) MFC (µg/mL)
C. albicans 35 460
C. dubliniensis 35 921
C. glabrata 143 230
C. krusei 71 230
C. guillermondii 71 1842
C. parapsilopsis 71 1842 Figure 2: High Resolution Optic Microscopy of Candida albicans
C. tropicalis 143 230 untreated.

For more accurate evaluation of the antifungal activity of

the EO, time-kill assays were performed using the yeast C.
albicans. The killing time was tested at different cell
concentration and results are shown in Figure 1. For 3 x
105 CFU/mL, killing time was 140 min. For a
concentration of 3 x 106 CFU/mL the killing time was 300
min, for 3 x 108 CFU /mL was 480 min. and for 3 x 1012
CFU /mL was 1140 min. (Figure 1). These data shows that
it is necessary more time to kill a bigger cell population;
this means that killing time of the EO is directly
proportional to number of cell. In all cases the viability
control of the yeast presented a macroscopically visible
growth while treated cells did not show visible growth at
each killing time.

Figure 3: High Resolution Optic Microscopy of Candida albicans

treated at the MFC (460 µg/mL) of A. triphylla EO.
CFU/ml

detectable values; this last result could be explained by

Figure 1: Killing time of Aloysia triphylla EO (MFC=460µg/mL) at
different cell concentrations of Candida albicans (CFU /mL)
Table 2 shows the results obtained in the cellular lyses
assay with C. albicans cells treated at the MFC. The
control showed an increase in OD620 what means that
the yeast continued with its cellular growth. In contrast,
in treated cells the OD 620 diminished almost at not
the possibility that cellular lysis caused by the EO
occurred.
In the suspensions obtained from the centrifugation of C.
albicans inoculums treated with EO and not treated
(control), the OD260 of the treated samples increased
(OD260 = 0.270) compared to the control suspensions
(OD260 =0.008). This suggests that there is a lost of 260-
nm-absorbing material.
The HROM showed in control cells that the morphology
and the cytoplasmic density were characteristic of a
normal cell (Figure 2). After EO treatment at the MFC
Antimycotic activity of the essential oil of Aloysia triphylla
(460 µg/mL), morphology and shape of the cell was
distorted and a notable structural disorganization was seen
within the cytoplasm with the observation of large
vacuoles. In addition, the contents of some treated cells
appeared depleted and amorphous. Lost of cellular
material was also observed (Figure 3).
A. triphylla EO showed antifungal activity against all
Candida species, particularly in those that acquire
resistance to conventional chemotherapics, like C.
albicans and C. dubliniensis. The last one was recently
identified as an opportunistic pathogen associated with
oral candidiasis, particularly in individuals who are
positive for human immunodeficiency virus (HIV) and
immunocompromised patients [1,13]. C. albicans is the
most common fungal pathogen in humans, responsible for
skin, oral, esophagic, intestinal tract, vaginal and
circulatory diseases commonly affecting immunologically
compromised patients and those undergoing prolonged
antibiotic treatment [14].
The results presented in this work demonstrate that A.
triphylla EO had the potential to kill Candida cells
causing lysis. What is more, this study clearly
demonstrates that there was a direct relationship between
number of cells and the time that the EO needed to cause
killing effect.
C. albicans suspensions treated with A. triphylla EO lost
significant 260- nm-absorbing material, suggesting that
nucleic acids were released through a damaged
cytoplasmic membrane. These data are coincident to
experiences made with C. albicans treated with tea tree oil
[15]. Marked leakage of cytoplasmic material is
considered indicative of gross and irreversible damage to
the cytoplasmic membrane. Many antimicrobial
compounds that act on the bacterial cytoplasmic
membrane induce the loss of 260-nm absorbing material.
Some antimicrobial agents cause gross membrane damage
and provoke whole-cell lysis and this has been reported
previously for essential oils from oregano, rosewood, and
thyme [16].
EO components have the capability to alter cell
permeability by entering between the fatty acyl chains
making up membrane lipid bilayers and disrupt the lipid
packing. Due to this, the membrane properties like
permeability, fluidity and consequently its functions may
get changed [17]. This may also affect the regulation and
function of the membrane bound enzymes that alter the
synthesis of many cell wall polysaccharide components
and alter the cell growth morphogenesis [18].
The data obtained by Vataru Nakamura, et al. 2004. with
electronic microscopy showed that C. albicans as well as
C. tropicalis, C. parapsilosis, and C. krusei underwent
remarkable ultrastructural alterations which were visible
by electron microscopy, when treated with the essential oil
Natural Product Communications Vol. 6 (7) 2011 1041
of O. gratissimum [4]. This study is in concordance with
our experience in which changes in the morphology of C.
albicans cells were observed, as well as the formation of
large vacuoles in the cytoplasm when they were exposed
to the MFC of A. triphylla EO. Tyagy et al. 2010, worked
with C. albicans cells treated with lemongrass EO and
they observed similar results in experiences with different
techniques of electronic microscopy [18].
A. triphylla EO is a promising natural product for the
treatment of candidiasis. Therefore further studies on their
pharmacokinetics and toxicological behavior are
warranted. The results obtained represent a contribution to
the characterization of the anti-Candida activity of EO of
traditional medicinal plants from the Argentinean flora.
Experimental
Plant material and EO extaction: The EO was obtained
by hydrodistilation of samples of A. triphylla collected
from plants growing in farms (plantations) located in La
Paz, Córdoba Province (Argentina).
Gas Chromatography: The EO were analyzed with a
Shimadzu GC-R1A gas chromatograph equipped with a
fused silica column (30 m x 0.25 mm) coated with CBP-1.
The temperature of the column was programmed from
60°C to 240°C at 4°C/min. The injector and detector
temperatures were at 270°C. The gas carrier was He, at a
flow rate of 1 mL/min. Peak areas were measured by
electronic integration. The relative amounts of the
individual components are based on the peak areas
obtained, without FID response factor correction.
Programmed temperature retention index of the
compounds were determined relative to n-alkanes. GC
analysis was still performed using a column Supelcowax-
10 with the same conditions as described above [19].
Gas Chromatography-Mass Spectrometry: GC-MS
analyses were performed on a Perkin Elmer Q-910 using a
30 m x 0.25 mm capillary column coated with CBP-1. The
temperature of the column and the injector were the same
than those from GC. The carrier gas was He, at a flow rate
of 1mL/min. Mass spectra were recorded at 70 eV. The oil
components were identified by comparison of their
retention indices, mass spectra with those of authentic
samples, by peak enrichment, with published data, mass
spectra library of National Institute of Standards and
Technology (NIST 3.0) and our mass spectra library
which contains references mass spectra and retention
indices of volatile compounds. GC-MS analysis was still
performed using a column Supelcowax 10 with the same
conditions as describe above [20].
Microorganisms: The activity of the EO was tested
against the following yeasts: C. albicans, C. dubliniensis,
C. glabrata, C. krusei, C. guillermondii, C. parapsilosis
and C. tropicalis. These strains were resistant to
fluconazol (150 mg/mL). The strains were isolated in the
1042 Natural Product Communications Vol. 6 (7) 2011
Central Hospital of Rio Cuarto and identified in the
Mycology Area of Department of Microbiology and
Immunology of the National University of Rio Cuarto.
Antimicrobial activity: The minimum inhibitory
concentration (MIC) of the A. triphylla EO was evaluated
against yeast species with the broth microdilution method
described by Mann and Markham (1998) [21]. The
minimum fungicidal concentration (MFC) was determined
[23].
Culture methods: Tubes containing Sabouraud Broth
(SB) (Britania) with 0.1% (w/v) agar (SBA) were
prepared at pH 7 inoculated with each microorganism and
incubated overnight (18 h) at 37ºC. Optical densities were
measured at 620nm in a spectrometer and number of cells
was confirmed by the viable plate count on Sabouraud
Agar (SA) (Britania).
Firstly, the cell concentration necessary to cause reduction
of resazurin within 3.30 h was determined for each of the
test microorganisms. Serial 10 fold dilutions of the
overnight culture were prepared in SBA and aliquots (170
μL) from these dilutions were dispensed into microplates
containing 20 μL of diluent (Dimethylsulphoxide-distilled
water 1:1). The resazurin solution (10 μL) was added; then
they were incubated for 3.30 h at 37ºC. The appropriate
dilution to work was the last one unable to reduce
resazurin (blue), which was tested, by the plate count
method. Resazurin is a redox indicator that is blue in its
oxidized form and pink in its reduced form [21].
Determination of the Minimum Inhibitory
Concentration (MIC): Serial two fold dilutions of the
EO were prepared by vortexing it in the diluent at room-
temperature. The resazurin assay medium, SBA, was
inoculated with the test organism to yield a final cell
density ≈ 1 log cycle lower than the cell density required
to reduce resazurin (usually 106 cfu/mL). The inoculum
density was confirmed by plate count. A sterile 96-well
microtitre tray was set up with each of the tested Candida
sp as follows: column 1-10, 170 μL inoculum plus 20 μL
of the EO dilution; column 11, 170 μL inoculum plus 20
μL EO diluent (positive control); column 12, sterile
resazurin assay medium plus 20 μL of EO diluent
(negative control, respectively). Well contents were
thoroughly mixed and were incubated at 37ºC for 18h.
After incubation 10 μL of resazurin solution was added to
all except column 12, to which 10 μL of distilled water
was added. After a second incubation of 3 h at 37ºC, wells
were assessed visually for color change, with the highest
dilution remaining blue indicating the MIC. Each
experience was made by triplicate [21].
References
[1] Jewtuchowicz VM, Mujica MT, Brusca MI, Sordelli N, Malzone MC, Pola SJ, Iovannitti CA, Rosa AC. (2008) Phenotypic and
genotypic identification of Candida dubliniensis from subgingival sites in immunocompetent subjects in Argentina. Oral
Microbiology and Immunology, 23, 505–509.
Oliva et al.
Determination of the Minimum Fungicidal
Concentration (MFC): 100 µl of the dilution belonging
to the MIC and the previous dilutions were inoculated in
SA and incubated at 37ºC for 24 h. The MFC was
considered as the last dilution that did not show cell
growth [22].
Yeast killing assays: The time of killing of A. triphylla
EO against the yeast C. albicans were evaluated by
measuring the cellular viability at the MFC. The treatment
consisted on a suspension of cells (UFC/mL) in SBA plus
EO dilution (MFC) was incubated at 37ºC, 120 rpm. A
sample (0.1 mL) was removed at 30 min intervals and
plated on SA (viability control) and incubated overnight.
A suspension of cells (UFC/mL) in SBA without EO was
incubated at the same conditions (suspension control).
Cellular lysis: Suspensions of C. albicans (104 UFC/mL)
were prepared in SB (control) and SB supplemented with
EO at the MFC and incubated at 37ºC for 18 h. The
OD620nm was measured at 0 min and at 18 h. Then both of
them were centrifuged at 10000 rpm for 5 min. The pellet
was resuspended in PBS (Phosphate buffer saline) and
OD620nm was measured. Each experience was made by
triplicate.
Loss of 260-nm-absorbing material: The supernatant
from the suspensions of C. albicans prepared for cellular
lysis assay was used to measure the loss of OD 260-nm-
absorbing material.
High Resolution Optic Microscopy (HROM): Thin cuts
(± 0.25 m) obtained using a manual ultramicrotome
(Sorvall MT-1A, DuPont) of C. albicans were processed
for high resolution optic microscopy. They were placed on
a slide and stained with toluidine blue on a thermic
platine, allowing the income of the dye in the fungal cell.
Thin stained cuts were mounted in DPX (Merk) and
observed with an optic microscope Axiophot (Carl Zeiss,
Alemania). Images were obtained with a digital camera
Powershot G6, 7.1 megapixels (Canon INC, Japón) joined
to the optic microscope. Software AxioVision Release
4.6.3 (Carl Zeiss, Alemania) was used to process the
images [23].
Acknowledgments - María de las Mercedes Oliva, Mauro
Nicolás Gallucci are researchers from CONICET. We are
grateful to SECyT of Universidad Nacional de Río Cuarto
for financial support. We thank to Electronic Microscopy
Area and Mycology Area of Universidad Nacional de Río
Cuarto and Dr. Julio Alberto Zygadlo for GC-MS service
(UNC).
Antimycotic activity of the essential oil of Aloysia triphylla Natural Product Communications Vol. 6 (7) 2011 1043

[2] Brito Gamboa A, Mendoza M, Fernandez A, Diaz E. (2006) Detection of Candida dubliniensis in patients with candidiasis in
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[3] Neppelenbroek KH, Campanha NH, Splidorio DMP, Spolidorio LC, Séo RS, Pavarina AC. (2006) Molecular fingerprinting
methods for the discrimination between C. albicans and C. dubliniensis. Oral Diseases, 12, 242-253.
[4] Vataru Nakamura C, Ishida K, Faccin LC, Dias Filho BP, Garcia Cortez DA, Rozental S, de Souza W, Ueda-Nakamura T. (2004)
In vitro activity of essential oil from Ocimum gratissimum L. against four Candida species. Research in Microbiology, 155,
579–586.
[5] Barboza GE, Bonzani, N, Filipa, E, Lujan M, Morero R, Bugatti M, Decolatti N, Ariza-Espinar L. (2001) Atlas Histo-Morfológico
de plantas de interés medicinal de uso corriente en Argentina. Museo de Botánica. Fac. de Cs. Ex., Fcas. y Nat. y Fac. de Cs. Qcas.
de la Universidad Nacional de Córdoba.
[6] Gil A, Van Baren CM, Di Leo Lira PM, Bandoni AL. (2007) Identification of the Genotype from the Content and Composition of
the Essential Oil of Lemon Verbena (Aloysia citriodora Palau). Journal of Agricultural and Food Chemistry, 55, 8664–8669.
[7] Sartoratto A, Machado ALM, Delarmelina C, Figueira GM, Duarte MC, Rehder VLG. (2004) Composition and antimicrobial
activity of essential oil from aromatic plants used in Brazil. Brazilian Journal of Microbiology, 35, 275-280.
[8] Oskay, M, Usame Tamer A, Ay G, Sari D, Aktas K. (2005) Antimicrobial activity of the leaves of Lippia triphylla (L`Her) O.
Kuntze (Verbenaceae) against on bacteria and yeasts. Journal of Biological Sciences, 5, 620-622.
[9] Demo MS, Oliva Ma de las M, Lopez ML, Zunino MP; Zygadlo JA. (2005) Antimicrobial activity of essential oils obtained from
aromatic plants of Argentina. Pharmaceutical Biology, 43, 129-134.
[10] Oliva M. de las M, Beltramino E, Gallucci N, Casero C, Zygadlo J. Demo, M. (2010) Antimicrobial activity of the essential oils of
Aloysia triphylla (L`Her.) Britton from different regions of Argentina. BLACPMA, 9, 29-37.
[11] Pascual ME, Slowing K, Carretero E, Sanchez Mata D, Villar A. (2001) Lippia: traditional uses, chemistry and pharmacology: a
review. Journal of Ethnopharmacology, 76, 201-214.
[12] Stashenko EE, Jaramillo BE, Martinez JR. (2003) Comparación de la composición química y de la actividad antioxidante in vitro
de los metabolitos secundarios volátiles de plantas de la familia Verbenaceae. La Revista de la Academia Colombiana Ciencias, 27,
579-597.
[13] Sullivan DJ, Haynes K, Bille J. (1997) Widespread geographic distribution of oral Candida dubliniensis strains in human
immunodeficiency virus-infected individuals. Journal of Clinical Microbiology, 35, 960–964.
[14] Teixeira Duarte MC, Mara Figueira G, Sartoratto A, Garcia Rehder VL, Delarmelina C. (2005) Anti-Candida activity of Brazilian
medicinal plants. Journal of Ethnopharmacology, 97, 305–311
[15] Hammer KA, Carson CF, Riley TV. (2004) Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on
Candida albicans, Candida glabrata and Saccharomyces cerevisiae. Journal of Antimicrobial Chemotherapy, 53, 1081–1085
[16] Carson C, Mee B, Riley T. (2002) Mechanism of action of Melaleuca alternifolia (tea tree oil) on Staphylococcus aureus
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[17] Sikkema J, de Bont JAM, Poolman B. (1995) Mechanism of membrane toxicity of hydrocarbons. Microbiological Review, 59,
201-221.
[18] Tyagi AK, Malik S. (2010) In situ SEM, TEM and AFM studies of the antimicrobial activity of the lemon grass oil in liquid and
vapour phase against Candida albicans. Micron, 41, 797-805.
[19] Zunino MP, Newton MN, Maestri DM, Zygadlo JA. (1998) Essential oils of three Baccharis species. Planta Medica, 64, 86-87.
[20] Adams RP. (1995) Identification of essential oil components by gas chromatography and mass spectroscopy. Allured Publ. Corp.,
Carol Stream, IL. USA
[21] Mann CM, Markham JL. (1998) A new method for determining the minimum inhibitory concentration of essential oils. Journal
Applied Microbiology, 84, 538-544.
[22] Gallucci N, Oliva M, Casero C, Dambolena J, Luna A, Zygadlo J, Demo M. (2009) Antimicrobial combined action of terpenes
against the food borned microorganisms: Escherichia coli, Staphylococcus aureus and Bacillus cereus. Flavour and Fragrance
Journal, 24, 348-354.
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(2009) Cellular apoptosis in porcine placenta of different periods of gestation. Acta Microscopica, 1 (C), 691-692.
 2011
NPC Natural Product Communications Vol. 6
No. 7
Secretory Cavities and Volatiles of Myrrhinium atropurpureum 1045 - 1050
Schott var. atropurpureum (Myrtaceae): An Endemic Species
Collected in the Restingas of Rio de Janeiro, Brazil
Cristiane Pimentel Victórioa,*, Claudio B. Moreirab, Marcelo da Costa Souzac, Alice Satob and
Rosani do Carmo de Oliveira Arrudad
a
Colegiado de Ciências Biológicas e da Saúde, Centro Universitário Estadual da Zona Oeste (UEZO),
Rio de Janeiro, RJ, 23070-200, Brasil
b
Departamento de Botânica, Universidade Federal do Estado do Rio de Janeiro (UNIRIO),
Rio de janeiro, RJ, Brasil
c
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
d
Centro de Ciências da Saúde, Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande,
MS, Brasil

cristianevictorio@uezo.rj.gov.br

Received: December 15th, 2010; Accepted: March 16th, 2011

In this study, we investigated the leaf anatomy and the composition of volatiles in Myrrhinium atropurpureum var. atropurpureum endemic
to Rio de Janeiro restingas. Particularly, leaf secretory structures were described using light microscopy, and histochemical tests were
performed from fresh leaves to localize the secondary metabolites. To observe secretory cavities, fixed leaf samples were free-hand
sectioned. To evaluate lipophilic compounds and terpenoids the following reagents were employed: Sudans III and IV, Red oil O and Nile
blue. Leaf volatiles were characterized by gas chromatography after hydrodistillation (HD) or simultaneous distillation-extraction (SDE).
Leaf analysis showed several cavities in mesophyll that are the main sites of lipophilic and terpenoid production. Monoterpenes, which
represented more than 80% of the major volatiles, were characterized mainly by α- and β-pinene and 1,8-cineole. In order to provide tools for
M. atropurpureum identification, the following distinguishing characteristics were revealed by the following data: 1) adaxial face clear and
densely punctuated by the presence of round or ellipsoidal secretory cavities randomly distributed in the mesophyll; 2) the presence of cells
overlying the upper neck cells of secretory cavities; 3) the presence of numerous paracytic stomata distributed on the abaxial leaf surface, but
absent in vein regions and leaf margin; and 4) non-glandular trichomes on both leaf surfaces. Our study of the compounds produced by the
secretory cavities of M. atropurpureum led us to conclude that volatile terpenoid class are the main secretory compounds and that they
consist of a high concentration of monoterpenes, which may indicate the phytotherapeutic importance of this plant.

Keywords: Atlantic rain forest, histochemistry, volatile compounds, secretory cavity, restinga, leaf anatomy, Myrtaceae.

The Atlantic Rain Forest is among the world’s foremost
biodiversity hotspots [1a]. From evolutionary and
conservation view points, the Atlantic coastal vegetation of
Brazil, particularly in the case of the state of Rio de
Janeiro, should be treated as a mosaic comprising all forest
types and also the neighboring open vegetation [1b,1c].
Within this environment, a plant community represented
by usually arbustive-herbaceous vegetation, known as a
restinga, is located in part in Rio de Janeiro State. Restinga
ecosystems are areas of coastal plains covered by marine
deposits, which are the result of geological processes that
occurred during the Quaternary, around 5 thousand years
ago [2]. In this peculiar environment, plants are influenced
by the Atlantic Ocean and thus subjected to high salinity
and high temperatures. This ecosystem presents different
physiognomies and diverse flora where Myrtaceae is one
of the most important families. Plants in this area are under
high risk of extinction as a consequence of increasing land
development.
Myrrhinium atropurpureum Schott var. atropurpureum
(Myrtaceae), which is restricted to Rio de Janeiro
restingas, is a native source of medicinal compounds used
as astringents and antimicrobials. A different variety,
M. atropurpureum var. octandrum Bentham (syn. M.
loranthoides Hook. et Arn.), may be found in Brazil’s
southland to Uruguay and northwestern Argentina to
Ecuador [3]. M. atropurpureum var. atropurpureum
differentiates itself by having ovaries with 10-14 ovules
per locule, leaves 1.7-2.5 times longer than wide, with
thick blades and a leathery margin that is strongly revolute.
This species is a large evergreen shrub, or small tree,
reaching from 1.5 to 5.0 m high. In Brazil, M.
atropurpureum is known as “pau-ferro”, “carrapato” and
“murtilo”. It is an ornamental plant with aromatic leaves
and fleshy, sweet and swollen flower petals commonly
used as food for birds [4]. Similar to other Myrtaceae
species, M. atropurpureum presents large secretory
cavities in leaves, a peculiar structure that is a taxonomic
1046 Natural Product Communications Vol. 6 (7) 2011
Figure 1: Aspect of Marambaia restinga, showing arbustive habitat (up to 5 m) of a “Marambaia restinga” specimen - (→) Myrrhinium atropurpureum var.
atropurpureum. B-D. Aspect of M. atropurpureum morphology. B. Elliptic leaves, rounded, border revolute and leathery. C. Flowery branches. D. Details of
red reproductive axis showing flower buds and advanced stage of floral development from flower bud.
feature responsible for the accumulation of important
secondary metabolites involved in plant defense [5,6]. In
order to register a phytotherapeutic, it is necessary to
perform a macroscopic and microscopic characterization
of plant material to help identify a given species, as well as
define its phytochemical features. Therefore, the purpose
of the current study was to characterize the leaf anatomy
and localize the leaf volatile secretory structures of M.
atropurpureum, in addition to investigating the compo-
sition of volatiles produced in such secretory structures.
M. atropurpureum specimens are described as shrubs up to
5 m with opposite phyllotaxy (Figure 1A, B) Adult leaves
are elliptical, obtuse or rounded apex; base acute or
rounded; margin revolute, leathery and discolored, with the
adaxial face clear and densely punctuated by the presence
of secretory cavities. M. atropurpureum produces flowers
with petals elliptical to oval, 4-5 x 2-3 mm, red in floral
bud, becoming purple or pink, juicy and sweet in
full anthesis (Figures 1B-D) [7]. As discussed by Roitman
et al. [4], this floral type is very uncommon and seems to
be restricted to some genera of Myrtaceae and
Scrophulariaceae, both of which occur in South America.
In frontal view, both faces of M. atropurpureum leaves are
covered by an epidermis composed of common cells with
straight and thickened walls (Figure 2A). In cross section,
the epidermis is uniseriate and covered with a thick
cuticular layer, especially on adaxial surface, as revealed
by Sudan III and IV tests (Figure 2I-J). These features are
common to species occurring in dry places under high
temperature and intense light [8a], as well as leaves of
plants found in areas with saline soil near the sea
(halophytes and psamophilous plants) [8b,8c]. In
environments where the availability of nutrients in the
substrate is reduced, such as Brazilian salt marshes, or
restingas, the cuticular layer and dense wax deposits
observed in leaves of some Myrtaceae [9] can serve a
protective function by reducing the loss of phosphorus and
potassium by leaching [10].
While numerous paracytic stomata are randomly
distributed only on the abaxial leaf surface, they are absent
in the vein regions and leaf margin (Figures 2E-F). On
both surfaces of the epidermis, isolated cells, or groups
with two, three, and even four colorless cells, are observed,
separated from each other by a strongly cutinized wall
(Figure 2A-B). The cross section evidences that these cells
Victório et al.
are related to oil secretory cavities in mesophyll. Around
these sets, or groups of cells, the remaining epidermal cells
occur concentrically. These cells, called overlying cells
[9,12], can occur frequently, either in isolation or in pairs,
and they are distinguished by low affinity for histological
staining. In M. atropurpureum, the cell walls between
overlying cells are straight and strongly cutinized (Figure
2A). It is possible that these special cells are related to the
elimination of secretions, which are mainly composed of
volatile compounds accumulated in the secretory cavities,
by the intense reaction of cell wall thickening to reagents
for lipophilic substances. Similar anatomical results were
observed by Donato and Morretes [6] for Eugenia
brasiliensis. The authors cite the study of List et al. [12],
in which Melaleuca alternifolia (Myrtaceae) leaves, when
subjected to a vacuum, release drop of oil on the surface
through overlying cells. Judging from studies of other
Myrtaceae species, such as Myrcia sp. and Campomanesia
sp. [11] we suggest that overlying cells are involved in the
process of aroma elimination on the leaf surface, based on
the strong olfactory properties of Myrtaceae population in
natural habitats (personal observations).
Non-glandular trichomes are observed on both leaf
surfaces (Figure 3). A second trichome type was observed
and it was pluricellular, rounded-shape, and accumulate
phenolic substances (Figures 2D, G; 3). These trichomes
are composed of two or three cells in the basal region with
one apical enlarged cell (Figures 2E-F). Some of these
trichomes can occur near the overlying cells. Only
phenolic substances are detected in these epidermal
appendages. No mention of these types of trichomes is
found in the literature, but, given the saline environment in
which the studied plants live, these trichomes could be
related to the elimination of salts impregnated in plant
tissues, as proposed for Eucalyptus species living in
salinized soils [13].
The cross section shows that the secretory cavities are
round or ellipsoidal and outlined by slightly thickened cell
walls. Internally, the secreting cells have a thin wall, and
they show positive reaction to tests for lipophilic
compounds (Figures 2I-N). The cavities are randomly
distributed in the mesophyll, and they accumulate a
yellow-greenish secretion in fresh material (Figure 2A.).
Under the adaxial surface, the secretory cavities are
located more deeply, and they connect with the epidermal
Secretory cavities and volatiles of Myrrhinium atropurpureum Natural Product Communications Vol. 6 (7) 2011 1047

Figure 2: Cross sections of Myrrhinium atropurpureum var. atropurpureum leaves obtained by light microscopy. A. General aspect of leaf cross sections showing
dorsiventral organization of the mesophyll, single-layered epidermis, and oil cells distributed in mesophyll. B-C. Secretory cavities. D and G. Non-glandular trichomes
observed on both leaf surfaces. H. Vascular system of the leaf blade showing phloem (*) proportionally more abundant than xylem. E-F. Overlying cells (arrows). I-N.
Histochemical characterization of the contents in secretory cavities, as evidenced by the oil droplets after reaction with Sudan IV (I and J) or Nile blue (L-N). Transversal
sections, bar = 100 μm; epidermis, bar= 30 μm.

layer through a series of cells that form a "neck" (Figures
2B-C). As suggested previously, the set formed by the
neck cells is also possibly related to the elimination of
secretions that are mainly composed of volatile compounds
accumulated in the secretory cavities. On the abaxial face,
the cavities are near the epidermis, or separated from it by
a pair of cells. The histochemistry analysis confirmed that
the content of secretory cavities of M. atropurpureum is
lipophilic in nature, as revealed by the positive reaction
with Sudan IV and Nile blue tests (Figures 2I-N). The Nile
blue test showed that the lipids produced in the secretory
cavities have an acidic composition (Figures 2L-N), and
the positive reaction with Sudan confirms their volatile
property (Figures 2I-J).
The number of secretory cavities in the leaf may be
affected by environmental conditions. For instance, in
Eugenia brasiliensis, which inhabits two contrasting
environments in the Atlantic Rain Forest (Brazil), the
number of secretory cavities in both faces of foliar blade
was lower in individuals inhabiting moist shaded areas
than individuals collected in the mostly dry and brightly lit
areas at sea level, suggesting that the development of some
tropical plants in restinga areas may reflect a greater
production of essential oils and other compounds
associated with therapeutic effects, in contrast to plants
growing in shaded areas [6].
The palisade is formed by two layers of narrow, thin-
walled cells, and the spongy parenchyma has up to 12
layers of voluminous cells with thin walls, presenting, a
leaf with isolateral structure. Tiny druse-type crystals were
observed in the chlorenchyma cells. Phenolic reaction was
observed in all mesophyll. Such features are typically
found in xeromorphic plants growing in saline or dry
environments, where a reduction in nutrients and water is
common [8b]. The vascular system of the leaf blade is
composed of vascular bundles with phloem proportionally
more abundant than xylem (Figure 2H). Some morpho-
anatomical and phytochemical features of resistance to
1048 Natural Product Communications Vol. 6 (7) 2011 Victório et al.

restinga environment conditions are recognized in M.

atropurpureum: thick cuticle and walls in the epidermis,
secretory structures, thick leaves, possibly accumulating
water, and phenolic compounds. The lipophilic substances
that accumulate in the cavities may also be a chemical
defense that confer protection against herbivory.

The presence of lipophilic content observed in the

secretory cavities led us to a phytochemical analysis of the
composition of the volatiles, which is summarized in Table
1. The lipophilic nature of the secretion was detected after
reaction with Nile blue, which showed blue staining
indicative of lipid acids, in agreement with the presence of
1,8-cineole, which is weakly acid with a hydroxyl in its
chemical structure. Positive reaction to Sudan confirmed
the lipophilic character of the contents of secretory
cavities. The leaf volatiles were found to be very poor in
sesquiterpenes. Using both extraction methods, the Figure 3: Leaf surface of Myrrhinium atropurpureum imaged by scanning
components found in greater concentration among the electron microscopy. Non-glandular trichomes observed on both leaf
volatiles were α and β-pinene and 1,8-cineole. On the other surfaces. → The same pluricellular trichome showed in Fig. 2D.
hand, the oil composition of the var. atropurpurem from
Table 1: Volatile compounds (%) of Myrrhinium atropurpureum var.
southern Brazil presented limonene (35%), 1,8-cineole atropurpureum collected in Grumari and Marambaia restingas in Rio de
(23%) and α-pinene (12%) as the main compounds [14]. Janeiro City. HD– hydrodistillation and SDE– simultaneous distillation-
Based on our studies comparing volatiles from plants of extraction.
the Grumari and Marambaia restingas, we found the RI RI Relative Area (%)a
Constituents literatur*
concentration of α–pinene to be equally high. Similarly, calculated
Grumari Marambaia
α–pinene (75%) was found to be the main component in HDa SDEb SDEb
the volatile compounds of a different variety (M. α-Thujene 931 929 0.7 - -
α-Pinene 939 936 25.5 54.1 50.6
artopurpureum var. octandrum) from Argentina [15]. This β-Pinene 980 981 9.5 22.1 8.4
result may indicate that α–pinene is a plant marker p-Cymene 1026 1030 1.1 1.4 tr
1,8-Cineole 1033 1037 33.0 1.7 15.9
compound in this genus. γ-Terpinene 1062 1061 2.9 0.9 9.6
Terpinolene 1088 1088 0.4 tr tr
We compared volatiles from M. atropurpureum collected Terpinen-4-ol 1177 1184 1.1 tr tr
α-Terpineol 1189 1198 6.0 0.6 3.1
from two distinct restingas during the same period (July, Monoterpenes 80.2 80.8 87.6
2010). Using SDE, we showed variations in concentrations β-Caryophyllene 1418 1413 2.9 tr 0.2
Aromadendrene 1439 1433 0.6 tr tr
of the monoterpenes β-pinene, 1,8-cineole and γ–terpinene α-Humulene 1454 1450* tr tr tr
between Marambaia and Grumari restingas. In samples Epiglobulol - 1560* tr tr tr
Spathulenol 1576 1575 2.3 tr 0.3
from Marambaia, there was a significant increase in Caryophyllne
1,8-cineole and γ–terpinene concentrations, while in oxide 1581 1578 4.5 tr 2.6
Grumari, an increase in β-pinene was revealed. However, Globulol 1586 1583 2.3 tr tr
Viridiflorol 1590 1590 0.3 tr tr
no difference was found in α–pinene concentration, as Guiol 1595 1594 2.1 tr 0.2
noted above. These data demonstrate how the environment Ledol 1565 1599 0.4 tr tr
n.d. - 1604* 0.4 tr tr
affects the production of volatiles, even though both Epi-β-eudesmol 1649 1621 0.5 tr tr
restingas are in the same State, Rio de Janeiro. Tetracyclo[6.3.2.0
(2,5).0(1,8)]tridec
an-9-ol, 4,4-
According to Araújo [16], the restingas that occur in Rio dimethyl - 1636* 0.6 tr tr
de Janeiro can be divided on the basis on their flora and Sesquiterpenes 16.9 tr 3.3
the physiognomy of ten types of vegetation, as influenced tr – trace (less than 0.05%). n.i. – not identified. *Adams [20a] does not
by both topography and climate. The Marambaia restinga have these mass spectra. **Identified by similarity with Kovats indices of
the study of Myrrhinium from Argentina [15]. aFresh leaves were
is formed by a largely well-preserved extension of sand collected in April 2009. bFresh leaves were collected in July, 2010.
dunes approximately 40 km long. This area forms an east-
west peninsular-like extension of the continent into the The hydrodistillation method was used to obtain essential
Atlantic Ocean linked to Marambaia Island. In contrast, oil. Similar to SDE, HD revealed the following major
Grumari is one of the smallest remaining restinga components: pinenes (35.0%) and 1,8-cineole (33.0%). β-
fragments located within the metropolitan area of Rio de caryophyllene and its oxide, spathulenol, globulol and
Janeiro which has experienced increasing degradation by guiol were the main sesquiterpenes found, totaling 16.9%.
the expansion of farmland areas, contributing, in turn, to Therefore, considering the different methods of extraction,
the loss of this habitat. HD and SDE, for samples collected in April (HD) and July
(SDE) in the same restinga - Grumari, the main volatile
Secretory cavities and volatiles of Myrrhinium atropurpureum
constituents, α and β-pinene and 1,8-cineole, presented
significant differences in relative concentrations. That is,
by using HD, α-pinene (25.5%) and β-pinene (9.5%)
presented reduced concentrations, while the concentration
of 1,8-cineole (33.0%) was high when compared to
the SDE method where a reduction of only 1.7% for
1,8-cineole was found, but an increase in both α-pinene
(54.0%) and β-pinene (22.0%). These differences in
volatile composition may have resulted from the extraction
method used or the influence of relative humidity and
precipitation in accordance with seasonal variation in April
and in July.
Overall, therefore, phytochemical analyses of the volatiles
showed the presence of compounds with pharmacological
interest in that the lipophilic content of secretory cavities
and phenolics have previously been reported to
demonstrate antibacterial, antifungal, anti-inflammatory
and antioxidant activity [17,18a].
In conclusion, the leaf secretory system has numerous
cavities with circular lumen close to the interface between
the epidermis and the parenchymas. Histochemical
reagents, such as Nile blue and Sudan, showed that these
secretory cavities are responsible for the production of
essential oil and lipophilic compounds. This finding agrees
with volatile composition obtained by GC-FID and
GC-MS, in which it was found that the main compounds
were monoterpenes, such as pinenes and 1,8-cineole,
representing more than 50%. These morpho-anatomical
and phytochemical data based on our study of M.
atropurpureum var. atropurpureum from restinga areas
may help in species identification, and the volatile
composition of this plant, consisting of pinenes and
1,8-cineole, has specific implications in the context of
phytotherapeutics.
Experimental
Plant material: Leaves from Myrrhinium atropurpureum
var. atropurpureum were collected between 10-11 h in two
Brazilian restingas: Grumari, Municipality of Rio de
Janeiro (23º02’94’’S, 43º31’98’’W), which is located in
the Grumari Protection Environmental Area, and
Marambaia Restinga - “Linha 4”, 23º02’75’’S,
43º35’68’’W (Municipality of Mangaratiba), both in Rio
de Janeiro City, Brazil. The collection occurred in April,
2009 (HD), and in July, 2010 (SDE). A voucher specimen
is deposited at the Herbarium of Rio de Janeiro Botanical
Garden under accession number RB 415731. M. atropur-
pureum leaves were collected from three georeferenced
specimens growing in open shrub formation. Restingas are
at sea level and present the following environmental
conditions: temperature (± 24-23ºC), annual precipitation
(1172-1240 mm) and sandy soil.
Leaf anatomy and histochemistry: Mature leaves of two
to three individuals were taken from the third or fourth
nodes from the apex of the branch. The leaves were fixed
in FAA70 and preserved in 70% ethanol. The leaves were
Natural Product Communications Vol. 6 (7) 2011 1049
free-handed, or, by using a Ranvier microtome, sectioned
in longitudinal and transverse planes. The sections were
clarified in sodium hypochlorite and rinsed in 1% acetic
acid and distilled water. Afterwards, the samples were
stained with alcian blue and fucsin 0.1%. The epidermis
was described using leaf segments separated by the
solution of Franklin [18b], stained with 0.25% fuchsin in
50% ethanol. All samples were mounted in 50% glycerin
on slides with cover slips. For scanning electron
microscopy (SEM), fixed leaves were dehydrated in
graded ethanol series, submitted to critical point drying
with CO2 (Leica EM CPD-030), mounted on stubs, and
coated with a thin layer of gold (Denton vacuum Desk IV,
LLC). The samples were analyzed with a JEOL JSM-
6490LV scanning electron microscopy (JEOL, Tokyo,
Japan).
Histochemical tests were performed on fresh leaves from
the Marambaia restinga, sectioned by the free-hand
method, and submitted to Sudan III, Sudan IV [19a] and
Red oil O staining to determine lipophilic compounds
[19b], followed by the Nile blue test for acid and neutral
lipophilic compounds [19c]. Histochemical tests were
adapted and carried out following Victório et al. [19d].
Control procedures for histochemical tests were carried
out. All samples were rinsed with distilled water before
mounting in glycerin on slides with cover slips. To
confirm the nature of calcium oxalate crystals, tests were
carried out with acetic acid and hydrochloric acid [19a].
All samples were rinsed with distilled water and mounted
in glycerin on slides with cover slips. Observations were
carried out and captured on light microscopy Olympus®
(BX-41).
Volatile extraction and gas chromatography analyses:
Two extraction methods were used: hydrodistillation (HD)
and simultaneous distillation-extraction (SDE). For the HD
method, fresh leaves (50 g) of M. atropurpureum were cut
and submitted to a Clevenger-type apparatus for 2h,
yielding 0.4% v/w of the oil. For the second extraction
method, fresh leaves (5 g) of M. atropurpureum were
homogenized with 50 mL of distilled water and submitted
to SDE for 2 h [20a]. A 2 mL volume of dichloromethane
was used as an organic collecting solvent and placed in the
solvent flask. Boiling chips were added to both flasks.
Mineral oil bath under stirrer/heat plate was used to apply
heat to flasks. The heating temperatures for the sample and
solvent flasks were controlled to 110-130oC and 55-60oC,
respectively, so that boiling in sample flasks began, and
extraction was carried out for 2 h. The vapors were
condensed as a result of the circulation of cooling water
pumped to the apparatus. HD and SDE samples were
introduced to GC/FID and GC/MS for analysis.
Analytical GC (gas chromatography) was carried out on a
Varian Star 3400 gas chromatograph fitted with a DB-1-
MS column (30 m × 0.25 mm i.d., 0.25 μm film thickness)
and equipped with flame ionization detection (FID).
Temperature was programmed from 60ºC to 240ºC at
1050 Natural Product Communications Vol. 6 (7) 2011 Victório et al.

3°C/min. The injection consisted of 1 μL of distilled oil
diluted with dichloromethane obtained by SDE. And, the
essential oil obtained by HD was diluted in
dichloromethane (1:1). Hydrogen was used as the carrier
gas at a flow rate of 1 mL min-1. The injector temperature
was 260°C, with interface temperature of 200oC. Leaf
volatile samples were analyzed in splitless mode. GC-MS
analyses were carried out on a Shimadzu Model GC-MS-
QP 5000 fitted with a HP-5/MS fused silica capillary
column (30 m x 0.25 mm i.d., 0.25 μm film thickness).
GC-MS conditions were the same as above, except for 1)
helium which was used as the carrier gas at a flow rate of 1
mL/min and 2) the mass spectrometer which was operated
on electron impact mode at 70 eV, at a scan rate of 0.5
scans/s and fragments from 40 to 500 Da. Quantification
was performed from GC-FID profiles using relative areas
(%). Identification of components in the volatiles was
based on retention indices relative to n-alkanes (C8 - C19)
and computer matching with the National Institute of
Standards and Technology (NIST 98) library, as well as by
comparison between mass fragmentation patterns and
those reported in the literature [14, 20b].
Acknowledgements - We thank the Brazilian Army, in
particular those responsible for administration and care of
the Marambaia restinga area, for their kind hospitality and
access to the material used in this study. To Ms. Aline
Carvalho de Azevedo, Bruna Nunes de Luna and Mr.
Eliandro Joaci de Lima assisted with preparation of the
plant material and microscopy.

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 2011
NPC Natural Product Communications Vol. 6
No. 7
Chemical Composition and in vitro Antibacterial Activity of 1051 - 1053

the Essential Oil of Phthirusa adunca from Venezuelan Andes

Flor D. Moraa*, Nurby Ríosa, Luis B. Rojasb, Tulia Díazc, Judith Velascoc, Juan Carmona Aa
and Bladimiro Silvaa
a
Department of Pharmacognosy and Organic Medicaments, bResearch Institute, cGastrointestinal
and urinary syndromes laboratory " Lic. Luisa Vizcaya", Facultad de Farmacia y Bioanálisis,
University of Los Andes, Mérida, Venezuela, 5101

flormv@ula.ve

Received: December 14th, 2010; Accepted: March 25th, 2011

In this paper, preliminary studies on the chemical characterization of Phthirusa adunca Meyer essential oil, obtained by hydrodistillation, is
presented. The separation of the components was performed by GC-MS. Twenty-three compounds (94.5% of the sample) were identified of
which the three major ones (76% of the sample) were β-phellandrene (38.1%), germacrene D (26.8%) and β-pinene (11.5%). The essential
oil showed a broad spectrum of activity against Salmonella Typhi CDC 57 (100 g/mL), Staphylococcus aureus ATCC 25923 (200 g/mL),
Enterococcus faecalis ATCC 29212 (250 g/mL), Escherichia coli ATCC 25922 y Klebsiella pneumoniae ATCC 23357 (500 g/mL). This
is the first report on the composition and activity of the essential oil of this species.

Keywords: Phthirusa adunca (Meyer), Loranthaceae, essential oil, antibacterial activity, β-phellandrene.

Loranthaceae family comprises intertropical plants,
hemiparasite on different species. It is composed for more
than one thousand species. The main characteristic of this
family is the conexion with xilematic vases of the host
from which obtain water and nutrients but with a life cycle
independent of the host. They possess unisex flowers,
berry fruits and seeds protected for sticky substances [1,2].
Several references relate the ethnobotanical use of plants
of the genus Phthirusa. Among them, P. pyrifolia (HBK)
Eichl is used for stomach burning relief and bone fractures
[3-5]. From P. pyrifolia a leaf lectin (PpyLL) with
antimicrobial activity have been isolated [6]. The protector
effect on gastric lesions induced by ethanol at 1 g/Kg
weigh on rats have been reported [7] as well as its effect
on the inhibition of fatty acid synthase and reduction of
weight on rats [8].
Phthirusa adunca G. (Meyer) Maguire, known in
Venezuela as guatepajarito, grows on a variety of shrubs
and trees as well on rocky substrates [9]. Several of its
physiological characteristics structure of their haustorium
will depend on its host. It is used in Peru for delivery and
fracture bone treatment [10]. In the current report the
chemical composition of the essential oil of P. adunca
collected in Venezuela and its antibacterial activity by the
agar diffusion method on international reference bacteria is
shown. Leaves of P. adunca were hydrodistilled yielding
0.8% of essential oil. Analysis of this by GC-MS allowed
the identification of Twenty-three compounds which are
listed in Table 1 (94.5% of the sample). However, 76% of
the sample is represented by three compounds: β-
phellandrene (38.1%), germacrene D (26.8%) and β-pinene
(11.5%). These results are preliminary, and further study to
characterize this essential oil is necessary. The essential oil
showed a weak antimicrobial activity, as shown in table 2,
against Salmonella Typhi CDC 57 (100 g/mL),
Staphylococcus aureus (ATCC 25923) (200 g/mL),
Enterococcus faecalis (ATCC 29212) (250 g/mL),
Escherichia coli (ATCC 25922) Pseudomonas aeruginosa
(ATCC 27853) y Klebsiella pneumoniae (ATCC 23357)
(500 g/mL).
Even thought the presence of β-phellandrene as the major
compound of an essential oil has not been associated with
good antimicrobial activity [11], the presence of
germacrene D in the oils seems to be responsible for some
antimicrobial activity [12]. Therefore the mix of the
components in the oils seems to have a potential effect on
the activity.
Experimental
Plant material: The aerial parts of Phthirusa adunca were
collected (May 2008) at El Caucho, Mérida State,
Venezuela, located at 2000 m.a.s.l. A voucher specimen Nº
FMBS011, collected by Bladimiro Silva, has been
deposited at the Herbarium of the Facultad de Farmacia y
Bioanálisis, University of Los Andes (MERF herbarium).
1052 Natural Product Communications Vol. 6 (7) 2011 Mora et al.

Table 1: Chemical composition of the essential oil of Phthirusa adunca. (AT-5, Alltech Associates Inc., Deerfield, IL), 60 m x
0.25 mm, film thickness 0.25 m, was used. The initial
Compound
Peak Area
LRI
LRI oven temperature was 60°C; it was then heated to 260°C at
(%) Literature
14
4°C/min, and the final temperature maintained for 20 min.
α-Pinene 1.3 937 939 The injector and detector temperatures were 200°C and
Sabinene 5.0 976 976 250°C, respectively. The carrier gas was helium at 1.0
β-Pinene 11.5 980 980 mL/min. The sample (1μL) was injected using a Hewlett-
Myrcene 1.2 990 991 Packard ALS injector with a split ratio of 50:1. Retention
1-Phellandrene 1.2 1004 1005
indices were calculated relative to C8-C24 n-alkanes, and
α-Terpinene 0.2 1018 1018
β-Phellandrene 38.1 1036 1031 compared with values reported in the literature 14.
trans-β-Ocimene 0.3 1050 1050
-Terpinene 0.3 1061 1062 Gas chromatography-mass spectrometry: GC-MS
α-Terpinolene 0.2 1090 1088 analyses were carried out on a Model 5973 Hewlett-
1-Terpineol 0.1 1145 1144 Packard GC-MS system fitted with a HP-5MS fused silica
4-Terpineol 0.8 1183 1174
column (30 m x 0.25 mm i.d., film thickness 0.25 m,
α-Terpineol 0.1 1196 1189
1339
Hewlett-Packard). The oven temperature program was the
δ-Elemene 0.3 1344
α-Copaene 0.3 1382 1376 same as that used for the HP-5 column for GC analysis; the
β-Cubebene 0.1 1395 1390 transfer line temperature was programmed from 150ºC to
β-Caryophyllene 1.6 1428 1418 280ºC; source temperature, 230ºC; quadrupole
α-Humulene 0.9 1465 1454 temperature, 150ºC; carrier gas, helium, adjusted to a
Germacrene-D 26.8 1497 1480 linear velocity of 34 cm/s; ionization energy, 70 eV; scan
Bicyclogermacrene 1.8 1509 1494
range, 40:500 amu; 3.9 scans/s. The sample was diluted
δ-Cadinene 0.9 1534 1524
1645
with diethyl ether (20μL in 1 mL) and 1μL was injected
T-Muurolol 0.3 1629
Kaur-16-ene 1.2 2039 2042 using a Hewlett-Packard ALS injector with a split ratio of
Total 94.5 50:1. The identity of the oil components was established
from their GC retention indices, by comparison of their
Table 2: Antimicrobial activity of the essential oil of Phthirusa adunca. MS spectra with those of standard compounds available in
the laboratory, and by a library search (Nist, 05) 14-16.
Inhibition zone (mm)* MIC
Microorganism
μg/mL
Oil E VA SAM AZT CIP CAZ Microbiological analysis
Staphylococcus
aureus 17* 35* 200 Bacterial strains: Staphylococcus aureus (ATCC 25923),
(ATCC 25923) Enterococcus faecalis (ATCC 29212), Escherichia coli
Enterococcus
faecalis 10* 21* 250 (ATCC 25922), Klebsiella pneumoniae (ATCC 23357),
(ATCC 29212) Salmonella Typhi (CDC 57) and Pseudomonas aeruginosa
Escherichia
coli 10* 24* 500
(ATCC 27853) were used in this study.
(ATCC 25922)
Klebsiella Antimicrobial method: The antimicrobial activity was
pneumoniae 7* 32* 500
(ATCC 23357) tested according to the disc diffusion assay described by
Salmonella Typhi
12* 40* 100 Velasco et al. 17. The strains were maintained in agar
(CDC 57)
Pseudomonas
conservation at room temperature. Every bacterial
aeruginosa NA 35* NT inoculum (2.5 mL) was incubated in Mueller-Hinton broth
(ATCC 27853) at 37ºC for 18 h. The bacterial inoculum was diluted in
E: Erythromycin® (15 μg), VA: Vancomycin® (30 μg), SAM: Sulbactam -
Ampicillin® (10μg/10μg), AZT: Aztreonam® (30 μg), CIP: Ciprofloxacin®
sterile 0.85% saline to obtain a turbidity visually
(30 μg), CAZ: Ceftazidime® (30 μg), NA: non active, NT: not tested. comparable to that of a McFarland Nº 0.5 standard (106-8
*Inhibition zone, diameter measured in mm, disc diameter 6 mm, average of CFU/mL). Every inoculum was spread over plates
two consecutive assays. MIC: Minimal inhibitory concentration, concentration
range 10-600 μg/mL.
containing Mueller-Hinton agar and a paper filter disc (6
mm) saturated with 10 μL of essential oil. The plates were
Isolation of the essential oil: Fresh leaves (1000 g) were left for 30 min at room temperature and then incubated at
cut into small pieces and subjected to hydrodistillation for 3 37ºC for 24 h. The inhibitory zone around the disc was
h using a Clevenger-type apparatus. The oil (0.8% yield) measured and expressed in mm. A positive control was
was dried over anhydrous sodium sulfate and stored at 4ºC also assayed to check the sensitivity of the tested
organisms using the following antibiotics: Erythromycin®
13.
(15 μg), Vancomycin® (30 μg), Sulbactam-Ampicillin®
(10 μg/10 μg), Aztreonam® (30 μg), Ciprofloxacina®
Gas chromatography: GC analyses were performed using
(30 μg), Ceftazidime® (30 μg) (Table 2).
a Perkin-Elmer AutoSystem gas chromatograph equipped
with a flame ionization detector and data handling system.
The minimum inhibitory concentration (MIC) was
A 5% phenylmethyl polysiloxane fused-silica column
determined only with microorganisms that displayed
Essential oil of Phthirusa adunca Natural Product Communications Vol. 6 (7) 2011 1053

inhibitory zones. MIC was determined by dilution of the
essential oil in dimethylsulfoxide (DMSO), pipetting 10
μL of each dilution onto a filter paper disc. Dilutions of the
oil within a concentration range of 10-600 μg/mL were
also carried out. MIC was defined as the lowest
concentration that inhibited the visible bacterial growth
18. A negative control was also included in the test using
a filter paper disc saturated with DMSO to check possible
activity of this solvent against the bacteria assayed. The
experiments were repeated at least twice.
Acknowledgments - The authors would like to thank Dr.
Alfredo Usubillaga for collaboration with GC-MS
analysis. Consejo de Desarrollo Científico, Humanístico,
Tecnológico y de las Artes (CDCHTA–Mérida–
Venezuela) for partial financial support of this study
(project FA-236-99-08-C). ADG (Grupo de Bacteriología
Clínica) and Fondo Nacional de Ciencia y Tecnología
(FONACIT), Ministerio de Ciencia y Tecnología, Caracas,
Venezuela (project Nº F-2000001633).

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1054 Natural Product Communications Vol. 6 (7) 2011
Natural Product Communications
Manuscripts in Press
Volume 6, Number 7 (2011)
Chemical Composition and Antioxidant Activities of the
Essential Oil from Tornabenea bischoffii (Apiaceae)
Risoleta Ortet, Erik L. Regalado, Olivier P. Thomas, Jorge A. Pino
and Miguel D. Fernández
Insect Growth Regulatory Activity of Blechnum chilense
Carlos A. Hincapié L., Zulma Monsalve F., Katherine Parada,
Claudio Lamilla, Julio Alarcón and Carlos L. Céspedes A.
GC/MS Analysis of the Essential Oil of Senecio belgaumensis
Flowers
Rajesh K. Joshi
Neolitsea aciculata Essential Oil Inhibits Drug-Resistant Skin
Pathogen Growth and Propionibacterium acnes-Induced
Inflammatory Effects of Human Monocyte Leukemia
Sang Suk Kim, Jung Eun Kim, Chang-Gu Hyun and Nam Ho Lee
Two Compounds from the Endophytic Colletotrichum sp. of
Ginkgo biloba
Sheng-Liang Zhou, Song-Lin Zhou, Mei-Xia Wang and Shuang-
Lin Chen
Biflavonoids from the Roots of Wikstroemia indica
Xiaoli Zhang, Guocai Wang, Weihuan Huang, Wencai Ye and
Yaolan Li
New Virescenosides from the Marine-derived Fungus
Acremonium striatisporum
Shamil Sh. Afiyatullov, Anatoly I. Kalinovsky and Alexandr S.
Antonov
Drypetdimer A: A New Flavone Dimer from Drypetes gerrardii
Margaret Mwihaki Ng’ang’a, Hidayat Hussain, Sumesh Chhabra,
Caroline Langat-Thoruwa, Muhammad Riaz, and Karsten Krohn
Composition and Antimicrobial Activity of Seseli globiferum
Essential Oil
Peđa Janaćković, Marina Soković, Ljubodrag Vujisić, Vlatka Vajs,
Ivan Vučković, Zoran Krivošej and Petar D. Marin
Composition of the Essential oils from Anthriscus cerefolium
var. trichocarpa and A. caucalis Growing Wild in the Urban
Area of Vienna (Austria)
Remigius Chizzola
Additional Minor Diterpene Glycosides from Stevia rebaudiana
Venkata Sai Prakash Chaturvedula and Indra Prakash
Two New Alkylanacardic Acids, Ozorcardic A and B, from
Ozoroa pulcherrima
Tsague Dongmo Christelle, Hidayat Hussain, Etienne Dongo, Jatsa
-Megaptche Boukeng Hermine, Ishtiaq Ahmed and Karsten Krohn
Cordioxime: A New Dioxime γ-Lactam from Cordia platythyrsa
Tsague Dongmo Christelle, Hidayat Hussain, Etienne Dongo,
Oben Enyong Julius and Javid Hussain
Chemical Constituents of Cichorium intybus and their Inhibi-
tory Effects against Urease and -Chymotrypsin Enzymes
Sumayya Saied, Shazia Shah, Zulfiqar Ali, Ajmal Khan, Bishnu
P. Marasini and Muhammad Iqbal Choudhary
Artocarpus Plants as a Potential Source of Skin Whitening
Agents
Enos Tangke Arung, Kuniyoshi Shimizu and Ryuichiro Kondo
Epoxidation of Soybean Oil Catalyzed by [π-C5H5NC16H33]3
[PW4O16] with Hydrogen Peroxide and Ethyl Acetate as
Solvent
Shuang-Fei Cai and Li-Sheng Wang
Chemical Composition of the Essential Oil of Pituranthos
scoparius
Nadhir Gourine, Bahia Merrad, Mohamed Yousfi, Pierre Stocker
and Emile M. Gaydou
Prenylated Flavonoids from the Leaves of Derris malaccensis
and their Cytotoxicity
Daranee Chokchaichamnankit, Vorawan Kongjinda, Nisachon
Khunnawutmanotham, Nitirat Chimnoi, Somchai Pisutcharoen-
pong and Supanna Techasakul
Inhibition on HIV-1 Integrase Activity and Nitric Oxide
Production of Compounds from Ficus glomerata
Kingkan Bunluepuech, Teeratad Sudsai, Chatchai Wattanapirom-
sakul and Supinya Tewtrakul
3
Influence of Growth Phase on the Essential Oil Composition
and Antimicrobial Activities of Satureja hortensis
Mohammad Jamal Saharkhiz, Kamiar Zomorodian, Mohammad
Reza Rezaei, Farshid Saadat and Mohammad Javad Rahimi
Anthraquinone Profile, Antioxidant and Antimicrobial
Properties of Bark Extracts of Rhamnus catharticus and R.
orbiculatus
Marcello Locatelli, Francesco Epifano, Salvatore Genovese,
Giuseppe Carlucci, Marijana Zovko Končić, Ivan Kosalec and
Dario Kremer
Antifungal Activity of Essential Oil from Asteriscus graveolens
against Postharvest Phytopathogenic Fungi in Apples
Mohamed Znini, Gregory Cristofari, Lhou Majidi, Hamid
Mazouz, Pierre Tomi, Julien Paolini and Jean Costa
Inhibition of Gastric H+, K+-ATPase Activity by Compounds
from Medicinal Plants
Cristina Setim Freitas, Cristiane Hatsuko Baggio, Bárbara Mayer,
Ana Cristina dos Santos, André Twardowschy, Cid Aimbiré de
Moraes Santos and Maria Consuelo Andrade Marques
Aroma-therapeutic Effects of Massage Blended Essential Oils
on Humans
Tapanee Hongratanaworakit
Toxic Effects of Citrus aurantium and C. limon Essential Oils
on Spodoptera frugiperda (Lepidoptera: Noctuidae)
Emilio Villafañe, Diego Tolosa, Alicia Bardón and Adriana Neske
Biosynthesis, Characterization and Biological Evalutation of
Fe(III) and Cu(II) Complexes of Neoaspergillic Acid, a
Hydroxamate Siderophore Produced by Co-cultures of two
Marine-derived Mangrove Epiphytic Fungi
Feng Zhu, Jingshu Wu, Guangying Chen, Weihong Lu and Jiahui
Pan
 Natural Product Communications Vol. 6 (7) 2011
Published online (www.naturalproduct.us)

Study of Antiviral and Virucidal Activities of Aqueous Extract of Baccharis articulata against Herpes suis virus
Cristina Vanesa Torres, María Julia Domínguez, José Luis Carbonari, María Carola Sabini, Liliana Inés Sabini and
Silvia Matilde Zanon 993
Evaluation of Cytogenotoxic Effects of Cold Aqueous Extract from Achyrocline satureioides by Allium cepa L test
María C. Sabini, Laura N. Cariddi, Franco M. Escobar, Romina A. Bachetti, Sonia B. Sutil, Marta S. Contigiani,
Silvia M. Zanon and Liliana I. Sabini 995
Toxic Plants Used in Ethnoveterinary Medicine in Italy
Lucia Viegi and Roberta Vangelisti 999
Diagnosis of Public Programs focused on Herbal Medicines in Brazil
Ely Eduardo Saranz Camargo, Mary Anne Medeiros Bandeira and Anselmo Gomes de Oliveira 1001
Identification of Thiosildenafil in a Health Supplement
Marcello Nicoletti 1003
Hypolipidemic Effect of Seed Oil of Noni (Morinda citrifolia)
Diana C. Pazos, Fabiola E. Jiménez, Leticia Garduño, V. Eric López and M. Carmen Cruz 1005
Composition of Egyptian Nerolì Oil
Ivana Bonaccorsi, Danilo Sciarrone, Luisa Schipilliti, Alessandra Trozzi, Hussein A. Fakhry and Giovanni Dugo 1009
Essential oil of Nepeta x faassenii Bergmans ex Stearn (N. mussinii Spreng. x N. nepetella L.): A Comparison Study
Niko Radulović, Polina D. Blagojević, Kevin Rabbitt and Fabio de Sousa Menezes 1015
Chemical Composition and Biological Activity of Salvia verbenaca Essential Oil
Marisa Canzoneri, Maurizio Bruno, Sergio Rosselli, Alessandra Russo, Venera Cardile, Carmen Formisano,
Daniela Rigano and Felice Senatore 1023
Chemical Composition and Antimicrobial Activities of the Essential Oils from Ocimum selloi and
Hesperozygis myrtoides
Márcia G. Martini, Humberto R. Bizzo, Davyson de L. Moreira, Paulo M. Neufeld, Simone N. Miranda,
Celuta S. Alviano, Daniela S. Alviano and Suzana G. Leitão 1027
Chemical Composition and Antibacterial Activity of the Essential Oil of Lantana camara var. moritziana
Nurby Rios Tesch, Flor Mora, Luis Rojas, Tulia Díaz, Judith Velasco, Carlos Yánez, Nahile Rios, Juan Carmona and
Sara Pasquale 1031
Activity against Streptococcus pneumoniae of the Essential Oil and 5-(3-Buten-1-ynyl)-2, 2'-bithienyl Isolated
from Chrysactinia mexicana Roots
Bárbara Missiam Mezari Guevara Campos, Anabel Torres Cirio, Verónica Mayela Rivas Galindo, Ricardo Salazar Aranda,
Noemí Waksman de Torres and Luis Alejandro Pérez-López 1035
Antimycotic Effect of the Essential Oil of Aloysia triphylla against Candida Species Obtained from
Human Pathologies
María de las Mercedes Oliva, María Evangelina Carezzano, Mauro Nicolás Gallucci and Mirta Susana Demo 1039
Secretory Cavities and Volatiles of Myrrhinium atropurpureum Schott var. atropurpureum (Myrtaceae): An
Endemic Species Collected in the Restingas of Rio de Janeiro, Brazil
Cristiane Pimentel Victório, Claudio B. Moreira, Marcelo da Costa Souza, Alice Sato and
Rosani do Carmo de Oliveira Arruda 1045
Chemical Composition and in vitro Antibacterial Activity of the Essential Oil of Phthirusa adunca from
Venezuelan Andes
Flor D. Mora, Nurby Ríos, Luis B. Rojas, Tulia Díaz, Judith Velasco, Juan Carmona A and Bladimiro Silva 1051
 Natural Product Communications
2011
Volume 6, Number 7
Contents
Original Paper Page

Use of Dimethyldioxirane in the Epoxidation of the Main Constituents of the Essential Oils Obtained from
Tagetes lucida, Cymbopogon citratus, Lippia alba and Eucalyptus citriodora
Luz A. Veloza, Lina M. Orozco and Juan C. Sepúlveda-Arias 925
Validation of the Ethnopharmacological Use of Polygonum persicaria for its Antifungal Properties
Marcos Derita and Susana Zacchino 931
On the Isomerization of ent-Kaurenic Acid
Julio Rojas, Rosa Aparicio, Thayded Villasmil, Alexis Peña and Alfredo Usubillaga 935
Aristolactams from Roots of Ottonia anisum (Piperaceae)
André M. Marques, Leosvaldo S. M. Velozo, Davyson de L. Moreira, Elsie F. Guimarães and
Maria Auxiliadora C. Kaplan 939
Anti-angiogenic Activity Evaluation of Secondary Metabolites from Calycolpus moritzianus Leaves
Laura Lepore, Maria J. Gualtieri, Nicola Malafronte, Roberta Cotugno, Fabrizio Dal Piaz, Letizia Ambrosio,
Sandro De Falco and Nunziatina De Tommasi 943
Chemical and Biological Activity of Leaf Extracts of Chromolaena leivensis
Ruben D. Torrenegra G. and Oscar E. Rodríguez A. 947
Citrus bergamia Juice: Phytochemical and Technological Studies
Patrizia Picerno, Francesca Sansone, Teresa Mencherini, Lucia Prota, Rita Patrizia Aquino, Luca Rastrelli and
Maria Rosaria Lauro 951
Phenolic Derivatives from the Leaves of Martinella obovata (Bignoniaceae)
Carolina Arevalo, Ines Ruiz, Anna Lisa Piccinelli, Luca Campone and Luca Rastrelli 957
Phenolic Chemical Composition of Petroselinum crispum Extract and Its Effect on Haemostasis
Douglas S. A. Chaves, Flávia S. Frattani, Mariane Assafim, Ana Paula de Almeida, Russolina B. Zingali and
Sônia S. Costa 961
Bioactivities of Chuquiraga straminea Sandwith
María Elena Mendiondo, Berta E. Juárez, Catiana Zampini, María Inés Isla and Roxana Ordoñez 965
Free Radical Scavenging Activity, Determination of Phenolic Compounds and HPLC-DAD/ESI-MS Profile of
Campomanesia adamantium Leaves
Aislan C.R.F. Pascoal, Carlos Augusto Ehrenfried, Marcos N. Eberlin, Maria Élida Alves Stefanello and
Marcos José Salvador 969
Activity of Cuban Propolis Extracts on Leishmania amazonensis and Trichomonas vaginalis
Lianet Monzote Fidalgo, Idalia Sariego Ramos, Marley García Parra, Osmany Cuesta-Rubio, Ingrid Márquez Hernández,
Mercedes Campo Fernández, Anna Lisa Piccinelli and Luca Rastrelli 973
Antioxidant Capacity and Phenolic Content of four Myrtaceae Plants of the South of Brazil
Marcos José Salvador, Caroline C. de Lourenço, Nathalia Luiza Andreazza, Aislan C.R.F. Pascoal and
Maria Élida Alves Stefanello 977
Cytotoxicity of Active Ingredients Extracted from Plants of the Brazilian “Cerrado”
Veronica CG Soares, Cibele Bonacorsi, Alana LB Andrela, Lígia V Bortoloti, Stepheny C de Campos,
Fábio HR Fagundes, Márcio Piovani, Camila A Cotrim, Wagner Vilegas and Marcos H Toyama 983
Propagation and Conservation of Native Forest Genetic Resources of Medicinal Use by Means of in vitro and
ex vitro Techniques
Sandra Sharry, Marina Adema, María A. Basiglio Cordal, Blanca Villarreal, Noelia Nikoloff, Valentina Briones and
Walter Abedini 985
Genotoxic Evaluation of a Methanolic Extract of Verbascum thapsus using Micronucleus Test in Mouse
Bone Marrow
Franco Matías Escobar, María Carola Sabini, Silvia Matilde Zanon, Laura Noelia Cariddi, Carlos Eugenio Tonn and
Liliana Inés Sabini 989

Continued inside backcover