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The Journal of Essential Oil Research

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Volume 23, Number 5
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Aims
The Journal of Essential Oil Research (JEOR) is a scientific journal devoted entirely to all facets of pure and applied studies on essential
oils or plant volatiles, excluding those of a purely agricultural or horticultural nature. The main areas of emphasis of the journal are:
Analytical Chemistry
Chemical Composition
Microbiological Activity
Biological Activity
Chemical Synthesis
Plant Biochemistry/Biosynthesis
Biotechnology
Chemosystematics
Toxicology

The Journal of Essential Oil Research is international in scope, as can be seen by the composition of the Editorial Board. The goal of
this Board is the timely publication of papers of a high standard of technical merit and scientific quality. All manuscripts submitted
for publication in the JEOR will be formally reviewed by no less than two members of the scientific community who are regarded
as authorities in that field. To be considered as a subject for publication, the manuscript must contain information on the aromatic
principles of a plant or its isolate, or must be directed toward furthering our knowledge of the aromatic plant and animal kingdoms.
This journal will serve as a forum for the publication of formally refereed manuscripts devoted to the field of essential oils and plant
volatiles. Consequently, concise contributions on the experimental or theoretical investigations of some facet of essential oils, aromatic
plants, or plant and animal interactions are invited for publication.
The Journal of Essential Oil Research is reviewed by Chemical Abstracts Service for referencing in CAS abstract literature.
The Journal of Essential Oil Research is published as January/February, March/April, May/June, July/August, September/October, and November/December issues by
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i
Calendar of Events
September 4959th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research; Antalya, Turkey; contact: The Society for Medicinal Plant
and Natural Product Research; www.ga2011.org
September 682011 PAM (Aromatic and Medicinal Plants) Congress and 30th International
Days of Essential Oils & Extracts; Digne-les-Baines, France; www.pole-pass.fr
September 111442nd International Symposium of Essential Oils; Antalya, Turkey; www.
iseo2011.org
October 31November 2IFSCC Conference; Bangkok, Thailand; contact: International Federation of Cosmetic Chemists; tel: 44-0-1582-726661; enquiries@ifscc.org; www.ifscc.org
November 3RIFMs 2011 Annual Meeting; contact: The Research Institute for Fragrance Materials; www.rifm.org
November 610IFEAT 2011; Barcelona, Spain; contact: International Federation of Essential
Oils and Aroma Trades; www.ifeat.org
2/Journal of Essential Oil Research

ii
Vol. 22, September/October 2010

(ISSN 1041-2905)
Volume 23, Number 5
www.JEORonline.com
Contents
Leaf Secretory Structure and Volatile Compounds of Eugenia

copacabanensis Kiaersk. (Myrtaceae)
R. do Carmo de O. Arruda and C.P. Victrio.......................1
Anti-inflammatory Activity of Some Essential Oils

S.Prez G., M. Zavala S., L. Garca A.
and M. Ramos L..................................................................38
Chemical Composition and Cytotoxic Activity of Essential Oil

from Myrcia laruotteana Fruits
M..A. Stefanello, D. Riva, E.L. Simionatto,
J.E. de Carvalho, A.L.T. Gis Ruiz and M.J. Salvador........7
Chemical Composition and Antibacterial Activity of Origanum

majorana L. Essential Oil from the Venezuelan Andes
S. Ramos, L.B. Rojas, M. Eugenia Lucena,
G. Meccia and Alfredo Usubillaga......................................45
Chemotaxonomic Importance of Sesquiterpenes and Flavonoids

in Five Argentinian Species of Polygonum Genus
M. Derita and S. Zacchino..................................................11
Anti-inflammatory and Antioxidant Activity of a Methanolic

Extract of Phyllanthus orbicularis and its Derived Flavonols
Y.I. Gutirrez Gaitn, M.M. Martnez, A.B. Alarcn,
M.M.Vzquez, J.L.F. Hernndez, L.D. Roche
and L. Rastrelli....................................................................50
Seasonal Evaluation and Chemical Composition of Volatile

Fractions from Piper claussenianum by Hydrodistillation and
SPME
A.M. Marques and M.A.C. Kaplan.....................................15
Analysis of the Chemical Composition and Antimicrobial
Activity of the Essential Oil from Lippia triplinervis Gardner
(Verbenaceae)
S.G. Leito, J.P.L. Damasceno, M.G. Martini,
S.N. Miranda, P.M. Neufeld, F. Regina Salimena
and H.R. Bizzo.....................................................................20
Activity against Streptococcus pneumoniae of the Essential Oil
and d-Cadinene Isolated from Schinus molle Fruit
A. Prez-Lpez, A.T. Cirio, V.M. Rivas-Galindo,
R.S. Aranda and N.W. de Torres.........................................25
Chemical Composition of Leaf Essential Oils of Calyptranthes
microphylla B. Holts & M.L., Myrcia aff fosteri Croat and
Eugenia octopleura Krug & Urb from Panama
A.I.S. Tenorio, D. Vargas, A. Espinosa, A. Daz and
M.P. Gupta...........................................................................29
Essential Oil from the Leaves of Campomanesia guaviroba
(DC.) Kiaersk. (Myrtaceae): Chemical Composition, Antioxidant
and Cytotoxic Activity
A.C.R.F. Pascoal, C.C. Loureno, L. Sodek, J.Y. Tamashiro,
G.C. Franchi, Jr., A.E. Nowill, M..A. Stefanello and
M. Jos Salvador..................................................................34
Chemical Composition of Essential Oils from Ripe and Unripe

Fruits of Piper amalago L. var. medium (Jacq.) Yunck and Piper
hispidum Sw.
M.L.F. Simeone, S. Bos Mikich, L.C. Ccco,
F.A. Hansel and G.V. Bianconi............................................54
Chemical Composition and Larvicidal Effects of Essential Oil
from Bauhinia acuruana (Moric) against Aedes aegypti
R.W. da Silva Gois, L.M. de Sousa, T.L.G. Lemos,
A.M.C. Arriaga and M. Andrade-Neto, G.M.P. Santiago,
Y.S. Ferreira, P.B. Alves and H.C.R. de Jesus.....................59
Chemical Composition and Biological Properties of the Leaf
Essential Oil of Tagetes lucida Cav. from Cuba
E.L. Regalado and M.D. Fernndez, J.A. Pino,
J. Mendiola and O.A. Echemendia . ...................................63
Analytical Characterization of Industrial Essential Oils from
Fruits and Leaves of C. aurantifolia Tan. and C. latifolia Swing
I. Bonaccorsi, P. Dugo, L. Mondello, D. Sciarrone,
G. Dugo and L. Haro-Guzman...........................................68
Appendix: Submission Guidelines
iii
Journal of Essential Oil Research
The Italo-Latin American Society of Ethnomedicine (SILAE, www.silae.it) is an international nonprofit 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 in 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
has aimed to individualize new ways of collaboration between its member countries and other European nations, as well
as Asiatic countries, to sign accords with intergovernmental organizations. SILAE proposes to establish contacts with
scientific communities, universities, and research centers for the pursuit of medicinal and food plants knowledge and to
encourage the exchange and mobility of professors, researchers, and PhD students. Moreover SILAE_live, the oneto-one live chat and messenger at www.silae.it, is the first scientific chat on the Web and is a tool developed to engage
the interest and imagination of the public and for helping non-scientists to understand and enjoy scientific discoveries
and 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.
In Latin America, aromatic plants are an essential part of traditional health care systems. This has recently attracted
the attention of many scientists and encouraged them to screen plants to study the biological activities of their oils
from chemical and pharmacological investigations to therapeutic aspects. Essential oils are valuable natural products
used as raw materials in many fields, including perfumes, cosmetics, aromatherapy, phytotherapy, spices and nutrition.
Although essential oils have been used therapeutically for centuries, there is little published research on many of them.
Most of the chemical constituents of plant essential oils belong to terpenoid compounds, including monoterpenes,
sesquiterpenes, and their oxygenated derivatives. These low molecular weight (most below 300 g/mol) compounds easily
diffuse across cell membranes to induce biological reactions. In recent years, there has been a tendency for applied
studies of essential oils to focus on antimicrobial and the mosquito larvicidal activities as well as anti-inflammatory
bioactivity.
This special issue focused on Latin American and Caribbean Aromatic Plants, which you now hold, is comprised of 15
research articles related to different areas of aromatic plants: chemical composition and biological properties of the
essential oil, novel techniques for analysis and characterization of secondary metabolites, and several of these papers are
collaborative works between two or more countries.
Many papers present the compositions of essential oils from different aromatic plants using analytical techniques such
as GC, GC/MS, esGC, and MDGC. Various authors report the in vitro activity of an essential oil against bacteria, yeasts,
plasmodia and HHV 1/HHV 2 strains, leukemic cells lines, different human cancer cells and Aedes aegypti. Four papers
present the compositions of essential oils from different Myrtaceae species; one paper covers reports published in the
last five years on the anti-inflammatory activities of several essential oils isolated from 43 plants. Two papers deal with
the GC and GC/MS analysis of more polar compounds in aromatic plant extracts as sesquiterpenes and flavonoids.
The editors would like to thank the contributors who gave so generously their time and experience and who made this
publication a valuable tool for scientists in the field of essential oil and aromatic plants chemistry, analysis 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.
The editors are also very grateful to the Editorial Board of JEOR for embracing this project with interest and
enthusiasm, and for the opportunity to publish this special issue. We hope that this will be the first in a long series in this
attractive and interesting journal.
Luca Rastrelli
Guest Editor
Dipartimento di Scienze Farmaceutiche e Biomediche
University of Salerno, Italy
Luigi Mondello
Editor in Chief, JEOR
iv
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J. Essent. Oil Res., 23 (September/October 2011)
Leaf Secretory Structure and Volatile Compounds of

Eugenia copacabanensis Kiaersk. (Myrtaceae)
Rosani do Carmo de O. Arruda
Centro de Cincias Biolgicas e da Sade, Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS,
Brasil
Cristiane P. Victrio*
Centro Universitrio Estadual da Zona Oeste (UEZO), Rio de Janeiro, Rio de Janeiro, 23070-200, Brasil
Abstract
Eugenia copacabanensis Kiaersk. (Myrtaceae) is restricted to salt marshes or restinga areas along the southeastern
coast of Brazil. This work analyzes the leaf secretory structures and volatile compounds produced in E. copacabanensis plants collected in the Marambaia Restinga of Rio de Janeiro City. Simultaneous-distillation extraction (SDE),
when combined with GC-FID and GC/MS analyses, revealed a-pinene (20.2%) and b-pinene (50.4%) as the major
volatiles of this plant, proving that it is rich in monoterpenes. trans-Caryophyllene (10.3%) was the only sesquiterpene
identified. Using light microscopy, E. copacabanensis leaves presented numerous randomly distributed oil secretory
cavities in the mesophyll, while histochemical tests showed the presence of terpenoids and lipophilic substances accumulated in secretory cavities and parenchyma cells.
Key Word Index
Eugenia copacabanensis Kiaersk., Myrtaceae, Atlantic Rainforest, a-pinene, b-pinene, caryophyllene, restinga,
secretory activity, simultaneous-distillation extraction, volatile composition.
Introduction
Restingas comprise one of Brazils prime ecosystems.
Restingas are characterized by areas of open vegetation within
the coastal plains, and because of their abundant endemic
species, both known and unknown, restingas are protected
(1). Under the influence of the Atlantic Ocean, these ecosystems occur in areas between the inner dunes and the lowland
altitudinal zone of the Atlantic Rainforest. Families such as
Bromeliaceae, Leguminosae, Myrtaceae and Clusiaceae are
among the most represented in this biome (2). The diversity
of genera and endemic species provides both food and shelter
to forest fauna.
Myrtaceae is an ecologically important family in Brazils
Atlantic Rainforest, and it represents the largest number of
species in the Brazilian restinga (3). This monophyletic family
is characterized by the presence of entire leaves containing oil
glands and internal phloem (3, 4). Secreted oil is accumulated
in a spherical internal cavity bounded by a secretory epithelium.
Against the light, the secretory cavities in leaves are visible to
the naked eye as translucent dots.
Considered the largest of the New World Myrtaceae
genera, Eugenia L. is currently estimated to contain 500 to
2,000 species, and their distribution ranges from southern

Mexico to Cuba and the Antilles and then south to Uruguay
and Argentina. A few species are found in Africa (5). Eugenia
includes species with fleshy fruits used as food, and some are
cultivated for ornamental purposes.
Eugenia copacabanensis Kiaersk. (subtribe Eugeniinae
O. Berg, Myrtoideae) is known as cambu amarelo, or yellow cambu, by the yellow color of its fruits (Figure 1A). Its
specific name most likely derives from the Copacabana district
of Rio de Janeiro City. The distribution of E. copacabanensis
occurs sporadically in shoals surrounding Rio de Janeiro City.
This plants natural habitat is restricted to salt marshes or
restinga areas in Rio de Janeiro State (specifically, in the South
Beach Reserve, Marambaia Restinga, Jacarepagu, and state
ecological reserves in Maric Municipalities and Cabo Frio).
Consequently, this species is considered endemic and rare (6).
In the Marambaia Restinga, E. copacabanensis occurs in open
and unflooded areas, where it is exposed to intense wind and
light. The reduction in the number of individuals of this species
is directly related to human occupation of these natural salt
marshes, thus posing a threat to the entire tropical ecosystem.
It is hoped that the dual goals of creating conservation units
*Address for correspondence: cris.pvictor@gmail.com
Rec: May 2011

Acc: May 2011
1041-2905/11/0001-05$14.00/0 2011 Allured Business Media

Journal of Essential Oil Research/1
Arruda et al.
Table I. The main leaf volatiles (%) of Eugenia copacabanensis Kiaersk. collected in the Marambaia Restinga, Rio de Janeiro.
Constituent
a-pinene
b-pinene
RI lit.a
RI calculated
939
936
979
980
1,8 cineole
1031
1034
geijerene (n.d.)*
1150
1146
menth-1,5-dien-8-ol (n.d.)*
1170
1168
-ylangene (n.d.)*
1375
1373
trans-caryophyllene
1419
1414
Monoterpenes total
Sesquiterpenes total
Total identified
Relative area (%)b

20.27.5
50.414.8
2.40.5
1.70.6
1.00.2
1.20.3
10.32.8
74.0
13.2
87.2
a
RI= Retention Index (see Adams, 2007). bResults are given as the mean of triplicate extractions; n.d. = not detected by GC/MS analysis by low concentration. *Probably
terpenes obtained by comparison with RI analysis of Nakamura et al. (27).
Figure 1. Light microscopy images showing secretory structures of Eugenia copacabanensis Kiaersk. A. Leaves and
fruit of E. copacabanensis. B-C. Paradermic sections of leaf mesophyll showing the distribution of secretory cavities.
Histochemical tests: (B) Sudan IV and (C-D, F) Red oil O. D. Cross-section of leaves: reaction of Red oil O showed
terpenoids in secretory cavities, parenchyma and a thick cuticle; arrows indicate druses. E. Paradermic section, in detail,
overlying cells Nile blue test. F, H and I. Petiole. F. Red oil O test; arrows indicate the thick cuticle on epidermis. G-H.
KOH (potassium hydroxide) test for flavonoids (phenolic compounds). I. Ferric chloride test for phenolic compounds.
*Secretory cavity.
E. copacabanensis
in the State of Rio de Janeiro and protecting arbustive plants

where this species grows will dramatically reduce the risk of
extinction.
Leaf anatomy of many Eugenia species has been studied
in order to identify diagnostic characters, morpho-anatomy
and the histochemistry of secretory cavities. Since this genus
is edible and has medicinal uses, research has also provided
chemical identification of its volatile compounds (7). In the
Myrtaceae family, Eugenia is the fourth most important genera
in the production of essential oils, after Eucalyptus, Melaleuca
and Psidium. The chemical composition of essential oils in
the Eugenia species reveals the presence of various types of
terpenoids that can be used in the pharmaceutical, cosmetic
and agrochemical industries. Particularly, the boiling of E.
uniflora and E. dysenterica leaves produces a liquid yielding
anti-inflammatory, antimicrobial and antihypertensive effects
(8, 9). Anti-rheumatic effect has been attributed to the leaves
of E. brasiliensis (10), and hypoglycemic activity has been
reported for E. punicifolia (11).
Despite the large number of Brazilian species of Eugenia and
their wide distribution in several biomes, relatively few studies
have addressed either the anatomical structure of their leaves
or the chemical composition of their essential oils. Therefore,
this study aimed to identify the composition of volatile secretions, characterize the leaf anatomy, as well as localize the leaf
volatile secretory structures, of E. copacabanensis.
Experimental
Plant material: Leaves of Eugenia copacabanensis were
collected between 10 and 11 h from three individual plants
growing in open shrub formation in July 2010, in the Marambaia Restinga (Municipality of Mangaratiba; 230275S,
433568W), altitude 7 m, Rio de Janeiro City, Brazil. At the
collection site, the following environmental conditions are
typically observed. The climate is tropical, with a wet season
having an average monthly rainfall between 40 and 50 mm or
less between July and August and a dry winter season with
temperatures averaging 20.9C. The average annual rainfall
is 1,239.7 mm, and the average annual temperature is 23.7C
(Instituto Nacional de Meteorologia-INMET, Brazil).
Marcelo da Costa Souza (Rio de Janeiro Botanical Garden,
Brazil) undertook the taxonomic identification of E. copacabanensis. A voucher specimen is deposited at the Herbarium
of Rio de Janeiro Botanical Garden under accession number
RB 415728.
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 (12) and preserved in 70% ethanol. The leaves
were sectioned in longitudinal and transverse planes by the

free-hand method or by Ranvier microtome. The leaf sections were clarified in sodium hypochlorite and rinsed in 1%
acetic acid and distilled water. Afterwards, the samples were
stained with Alcian Blue 1% and fucsin 0.1% and rinsed in
distilled water. All samples were mounted in 50% glycerin on
slides with cover slips. The epidermis was described using
leaf segments separated by maceration in solution of acetic

acid and hydrogen peroxide 1:1 for 12 h, rinsed in distilled
water, stained with 1% Alcian Blue in 50% ethanol, and

mounted in 50% glycerin.
Histochemical tests were performed on fresh leaf blades
and petioles, which were sectioned by the free-hand method
and then submitted to the following staining reagents: Sudan
III, Sudan IV (11) and Sudan black B for total lipids (13); Red
oil O for terpenoid compounds (14); Nile blue for acid and
neutral lipophilic compounds (15); aqueous solution containing 5% KOH (potassium hydroxide) for flavonoids; and ferric
chloride for phenolic compounds (12). Control procedures for
histochemical tests were carried out. 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 using an Olympus (BX-41).
Volatile extraction and analyses: Fresh leaves (5 g) of
E. copacabanensis were cut and submitted to simultaneous
distillation-extraction (SDE) for 90 min using 2 mL of dichloromethane as an organic collecting solvent (16). 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. The vapors
were condensed as a result of the circulation of cooling water
pumped to the apparatus. SDE samples were introduced to
GC-FID and GC/MS for analysis. Table I shows averages of
three extractions collected in the same period, July 2010.
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 m film thickness) and
equipped with flame ionization detection (FID). Temperature
was programmed from 60-240C at 3C/min. The injection
consisted of 1 L of samples. Hydrogen was used as the carrier
gas at a flow rate of 1 mL/min. The injector temperature was
260C, with interface of 200C. 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 m film thickness). GC/
MS conditions were the same as above, except for 1) He 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. Quantification was performed from GC-FID
profiles using relative areas (%). Injections were carried out
in triplicate from three volatile extractions, and standard deviations were considered. 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 05) library, as well as
comparison between mass fragmentation patterns and those
reported in the literature (17).
Results and Discussion

E. copacabanensis can be classified as a shrub or tree that
grows up to 6 m in height. The trunk has a laminated outer
skin, exfoliating in papyraceous blades. The leaves are elliptical, ovate or lanceolate and glabrous, with yellowish petiole. In
addition, the leaves are shiny with midrib prominent adaxially,
densely dotted; revolute leaf margin with yellowish thickening
(3) (Figure 1A).
Arruda et al.
In frontal view, anatomical analyses showed that the epidermal cells of E. copacabanensis present straight and thick
walls covered by a shiny, smooth and thick cuticle (Figure
1D-E). Numerous stomata occur only on the abaxial surface.
According to Fontenelle et al. (18), the stomata are anomocytic,
and the entire leaf surface is covered by a layer of wax flakes.
Both the thick, shiny cuticle and the wax reduce the invasion
of pathogens, aid in water conservation, and reflect incident
light, thereby cooling the leaf (19). The cuticular layer showed
an intense reaction to the presence of lipophilic substances and
terpenoids, as detected by staining with Sudans and Red oil,
indicating a possible relationship of this class of metabolites
with protection against radiation and high air evaporative demand. Environments like restingas are subject to fluctuations
in water availability; therefore, plants exhibit many attributes
associated with resource conservation (20). On drier, nutrientpoor sites, many species have a strongly thickened cuticular
layer and cutinization with protective function, reducing the
loss of nutrients by leaching (21).
On both sides of the leaf cells, either isolated or pairs of
polygonal cells overlap the internal secretory cavities (termed
overlying cells) (Figure 1E). The overlying cells have low affinity for dyes, and in fresh leaves of E. copacabanensis, they
are translucent. The number of overlying cells among Eugenia
species varies and, as such, represents a taxonomic feature (18,
22). Although optical microscopy does not show visible pores,
it is possible that these cells are related to the elimination of
volatile substances accumulated in the secretory cavities whose
odor pervades the site collection areas of the plants analyzed.
More detailed studies could clarify the function of these cells
for the species of Eugenia.
In cross section, the leaf of E. copacabanensis presents a
one-layered epidermis. The mesophyll is isobilateral (Figure
1D, G), a pattern very different from most Eugenia species
(4, 23), but similar to the description of Fontenelle et al. (18)
for E. copacabanensis found in the City of Maric. Two layers
of palisade parenchyma with many chloroplasts on each side
of the epidermis can be observed. The spongy parenchyma
is composed of six bulky layers and a few chloroplasts, constituting a watery tissue (Figure 1G).In E. copacabanensis,
exposure to the intense light is reflected in the development
of the palisade under both sides of the leaf. A positive reaction
to tests for phenolics and flavonoids was found throughout the
mesophyll and in cells of the epidermis. These two important
substances reduce the absorption of wavelengths harmful
to the sub-cellular structure (24). In restinga environments,
plants are exposed to intense light, and, as such, the production of flavonoids shows an evolutionary correlation with the
protection of plants against high levels of ultraviolet radiation
found in these locations. Abundant calcium oxalate druses are
often found in the ground parenchyma of E. copacabanensis
(Figure 1D, G, H) (18).
The essential oil cavities appear to be randomly distributed
in mesophyll, and they accumulate a yellowish-green secretion
directed toward both sides of the leaf (Figure 1B-D). In the
leaf blade of E. copacabanensis, secretory structures are located
adjacent to the leaf epidermis, and in the petiole, they more

deeply embedded (Figure 1F). The location of the secretory
cavities in the leaf seems to vary between species of Eugenia.
In E. umbelliflora and E. brasiliensis, the secretory structures
may be the deepest on the adaxial side and near the periphery
on the abaxial surface, connecting with the epidermis through
a set of cells that form a neck (25).
The secretory cavities are spherical, large and formed by
a secretory epithelium that delimits a space which retains the
material secreted (Figure 1B-C, G). Secretory cells have thin
walls and react positively to tests for phenolic compounds. The
histochemical tests showed that secretions, which accumulated
in the cavities of E. copacabanensis, showed positive reaction
to tests for the recognition of acidic and neutral lipophilic substances, terpenoids and essential oils (Figure 1B-D). According
to Metcalfe and Chalk (4), the presence of secretory cavities
containing oil terpenoids and other aromatic compounds is a
hallmark of Myrtaceae.
The number of secretory cavities can vary within the same
species, depending on environmental factors. Accordingly,
Donato and Morretes (26) found that individuals of E. brasiliensis grown under conditions of high brightness, as well as
high salinity and temperature, had a greater number of cavities
compared to plants of the same species grown under low light
conditions and more humidity.
The vascular system of E. copacabanensis consists of vascular
bundles associated with fibers surrounded by a parenchymatous
endoderm with chloroplasts. In the petiole and midrib region,
the vascular system is organized in the form of an open arc, and
the phloem completely surrounds the xylem characterizing the
amphicrival bundles (Figure 1H) common to some Myrtaceae
(4) and observed in some Myrtales. Pericyclic fibers bypass the
vascular system of the petiole. Peripheral layers of collenchyma
and internal parenchyma cells are observed to fill the cortical
region of the petiole and midrib. Secretory cavities are located
deep, and flavonoids and phenolic substances were identified
in this region of the leaf (Figure 1G, I).
Morpho-physiological changes in plants in response to different environmental and survival conditions are, collectively,
an important component of tropical biodiversity. In this regard,
the distribution of E. copacabanensis in both open and shaded
sandbanks suggests the plasticity of this species in adapting to
the environment. In fact, environmental conditions account for
many phenotypic differences in the morpho-anatomical and
chemical features seen in the Eugenia species.
Through histochemical testing, we verified the correlations
among volatile composition, production and accumulation,
especially in secretory cavities. The content of the cavities
is composed of terpenoids, essential oils and lipophilic substances, as confirmed by gas chromatography analysis. The
main volatile components identified were a-pinene (20.22%),
b-pinene (50.41%), 1,8-cineole (2.38%) monoterpenes, and
trans-caryophyllene (10.34%). Such substances are also found
in other species of Eugenia (26), being intensively volatilized
at times of increased solar intensity.
Using SDE, this study revealed a higher concentration of
E. copacabanensis
a-pinene and b-pinene in E. copacabanensis when compared
to data provided in other studies for E. candolleana DC., E.

punicifolia (HBK) DC, or E. copacabanensis collected in January using hydrodistillation (27). A high content of a-pinene
and b-pinene was also found in E. rotundifolia collected in the
Grumari Restinga, Rio de Janeiro (28). On the basis of other
studies, many variations in volatile composition have been
shown among Eugenia species as a consequence of ecosystem
type, seasonal changes, including rainfall, and the intensity
of sunlight (29, 30). As determined by SDE, caryophyllene,
among all volatiles of E. copacabanensis, was the unique sesquiterpene found in this study. Caryophyllene is one of the
most recognizable sesquiterpenes in chemical profiles of the
essential oils of Eugenia spp, as shown in the present study
(27). It should be noted that detection by GC/MS may have
been prevented by the low concentration of other sesquiterpenes in the volatile dichloromethane extract, particularly in
view of the contradictory findings of Nakamura et al. (27), who
reported the occurrence of about 81.15% of sesquiterpenes
against 13.37% monoterpenes in leaf oils of E. copacabanensis
using hydrodistillation. Differences in the composition of E.
copacabanensis volatiles may be associated with differences
in the ecosystem of origin. We collected this species in its
natural habitat, the restinga ecosystem, where it is under
strong environmental pressure caused by high solar intensity,
temperature and salinity when compared with the specimen
used by Nakamura et al. (27). Their specimen was cultivated in
a canopied area with low light intensity and low temperature.
Other factors can also influence volatile composition, such as
seasonality and the method of volatile extraction.
Numerous secretory cavities with spherical lumen were
observed to be randomly distributed in mesophyll. The use of
histochemical reagents, such as Sudans, Red oil and Nile blue,
showed that these cavities are responsible for the production and
accumulation of lipophilic substances, terpenoids and essential
oils. This finding agrees with volatile composition obtained by gas
chromatography, in which it was found that the main compounds
were a- and b-pinenes and 1,8-cineole, representing more than
70%, and trans-caryophyllene, consisting of 10.34%, of volatiles.
Overall, the anatomical and phytochemical results of this study
could help researchers engaged in species identification and
could be applied to phylogenetic approaches. Moreover, the
volatile composition of E. copacabanensis, as demonstrated
in this work, supports the pharmacological effects indicated
for the essential oils of some Eugenia species (8, 9). Finally,
in addition to medicinal uses, research has demonstrated that
the essential oils of some species of neotropical Myrtaceae
can be used as natural insecticides, suggesting that the volatile
substances in E. copacabanensis, as determined in this study,
might also serve as safe insect control agents.
Anestor Mezzomo and Marco Lacerda, who lent us their photos of

E. copacabanensis habits, and David Martin who edited the English
version.
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M. laruotteana
Chemical Composition and Cytotoxic Activity of

Essential Oil from Myrcia laruotteana Fruits
Maria lida A. Stefanello and Dilamara Riva
Departamento de Qumica, Universidade Federal do Paran, Caixa Postal 19081, 81531-900, Curitiba, PR, Brazil
Edsio L. Simionatto
Instituto de Pesquisas Tecnolgicas de Blumenau, Universidade Regional de Blumenau, 89030-080, Blumenau, SC, Brazil
Joo E. de Carvalho and Ana Lucia T. Gis Ruiz

Diviso de Farmacologia e Toxicologia, CPQBA, UNICAMP, Caixa Postal 6109, 13083-970, Campinas, SP, Brazil
Marcos J. Salvador*
Instituto de Biologia, Departamento de Biologia Vegetal,Curso de Farmcia, UNICAMP, Caixa Postal 6109, 13083-970,
Campinas, SP, Brazil
Abstract
The essential oil isolated by hydrodistillation from unripe fruits of Myrcia laruotteana Camb. (Myrtaceae) was
analyzed by GC and CG/MS. Forty-four components were identified, representing around 83% of total oil. The major
components were a-bisabolol (23.6%) and a-bisabolol oxide B (11.5%). The cytotoxicity of the oil and of a fraction
rich in a-bisabolol was tested in vitro against U251 (glioma), UACC-62 (melanoma), MCF-7 (breast), NC1-ADR/RES
(ovarian-resistant), 786.0 (kidney), NCI-H460 (lung), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon) and K562
(leukemia) human cancer cells and against VERO (no cancer cell). The oil exhibited antiproliferative activity against
all cell lines (TGI < 100 mg/mL), with exception of NCI-H460 cell line (TGI > 125 mg/mL). The highest activity of
the oil was observed against U251 (TGI 20.46 mg/mL), 786.0 (TGI 20.74 mg/mL), UACC-62 (TGI 26.98 mg/mL) and
PC-3 (TGI 27.63 mg/mL) cell lines. The fraction rich in a-bisabol showed a similar activity profile. It was most active
against OVCAR-3 (TGI 8.58 mg/mL) and 786.0 (TGI 8.74 mg/mL).
Key Word Index

Myrcia laruotteana, Myrtaceae, essential oil composition, a-bisabolol, a-bisabolol oxide B, antiproliferative
activity.
Introduction
Experimental
Myrcia DC (Myrtaceae) is a large genus represented

in Brazil by more than 300 species (1). Myrcia laruotteana
Camb., known as cambui is a shrub or treelet, growing wild
in the Central, Southeastern and Southern Brazil, Paraguay
and Northern Argentine. Its leaves are oblong (6 cm long and
2 cm wide), with a weak aroma. The flowers are white and, in
contrast with leaves, strongly aromatic. The unripe fruits are
bright-green, containing essential oil that decrease during the
ripening, giving edible deep purple small berries (5-10 mm),
almost without smell (2). The chemical composition of essential oils of leaves and flowers was previously reported (3). The
present work describes the essential oil composition of unripe
fruits and its antiproliferative activity against cell lines.
Plant material: Unripe fruits of M. laruotteana (2-5 mm

long) were collected in November 2009 in Curitiba, Paran
State, Brazil (S 25o 25 48, W 49o 16 15, 934 m). The plant
was identified by Dr. Armando C. Cervi (Departamento de
Botnica, UFPR) and a voucher was deposited in the Herbarium of Universidade Federal do Paran (UPCB) under
code number 53303.
Fresh unripe fruits were submitted to hydrodistillation in
a Clevenger-type apparatus for 4 h. The oil was recovered with
diethyl ether and dried over anhydrous Na2SO4. The solvent
was removed under vacuum. The oil was kept under refrigeration for further analysis.
Analysis of the essential oils: Oil sample analyses were
performed on a Shimadzu GC-17A gas chromatograph (FID)
*Address for correspondence: marcosjs@unicamp.br
Rec: Feb 2011

Acc: June 2011

Stefanello et al.
equipped with a DB-5 fused silica capillary column (30 m

0.25 mm, 0.25 mm film thickness), temperature programmed as
follows: 50C for 3 min, and then programmed from 50-240C at
3C/min, after which it was kept isothermal at 240C for 5 min.
The carrier gas was He at a flow of 1.2 mL/min; injector port
and detector temperature were 240C and 250C, respectively.
Samples were injected by splitting and the split ratio was 1:20.
The relative percentage of components was based on the peak
areas obtained by electronic integration without FID response

factor correction. The results are average of three analyses.
GC/MS analysis was performed on Varian Saturn 2000
apparatus using a CP-Sil-8CB fused silica capillary column
(30 m x 0.25 mm; 0.25 mm film thickness) in the same conditions described above, with MS operating by electron impact
ionization at 70 eV with scan mass range of 40-400 m/z at a
sampling rate of 1.0 scan/s. Compounds were identified by
Table I. Chemical composition (%) of essential oil from M. laruotteana unripe fruits.
Number
Componenta
R.I.b
R.I.c
1
perillene
1102
1092
2
endo-fenchol
1114
1112
3
4-hydroxycryptone
1315
1306
4
ethyl nerolate
1354
1359
1414
1418
5
E-b-damascone
6
dehydrosesquicineole
1471
1466
7
trans-cadina-1(6),4-diene
1476
1473
a-muurolene
1500
1497
8
d-amorphene
1512
1512
9
g-cadinene
1513
1517
10
11
trans-calamenene
1522
1521
a-calacorene
1545
1541
12
13
selina-3,7(11)-diene
1546
1550
14
trans-dauca-4(11),7-diene
1557
1556
15
germacrene B
1561
1563
16
maaliol
1567
1569
17
spathulenol
1578
1579
18
caryophyllene oxide
1583
1581
19
globulol
1590
1587
20
viridiflorol
1592
1595
21
guaiol
1600
1605
22
rosifoliol
1600
1608
23
,10-di epi-cubenol
1619
1615
1623
1622
24
10-epi-g-eudesmol
25
1-epi-cubenol
1628
1630
1630
1631
26
muurola-4,10(14)-dien-1b-ol
27
cis-cadin-4-en-7-ol
1636
1638
1640
1644
28
epi-a-cadinol
1642
1647
29
epi-a-muurolol
a-muurolol
1646
1650
30
a-bisabolol oxide B
1658
1657
31
a-cadinol
1654
1659
32
33
khusinol
1680
1681
a-bisabolol
1685
1690
34
35
10-nor-calamenen-10-one
1702
1705
36
oplopanone
1740
1737
37
E, E-farnesol
1741
1739
a-bisabolol oxide A
1749
1758
38
b-bisabolen-12-ol
1760
1765
39
40
2E, 6E-methylfarnesoate
1784
1781
b-bisabolenol
1789
1789
41
1808
1810
42
eudesm-11-en-4a,6a-diol
43
iso-acorone
1811
1815
44
2Z, 6E-farnesyl acetate
1822
1820

Total identified
Monoterpenes
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
%
0.4 0.1
tr
0.6 0.1
0.2 0.0
tr
0.6 0.1
0.6 0.1
0.6 0.1
0.4 0.1
0.4 0.1
0.1 0.0
0.4 0.0
0.6 0.1
tr
2.1 0.1
tr
5.4 0.1
tr
6.3 0.2
4.6 0.1
0.6 0.2
0.6 0.2
tr
tr
0.9 0.1
0.9 0.1
tr
2.4 0.0
2.2 0.0
tr
11.5 0.5
5.8 0.5
tr
23.6 0.5
tr
tr
1.4 0.1
0.6 0.0
1.3 0.1
5.8 0.6
tr
0.5 0.1
tr
1.4 0.1
82.8
1.2
5.8
75.8
Compounds are listed in order of their elution from a CP-Sil-8CB column; tr = trace < 0.09%;
a Identification based on mass spectra and RI published (5) and computer matching of the mass spectra with NIST 1998 library (quality level more than 90%); b retention
index published (5); c retention index experimental on a CP-Sil-8CB column.
M. laruotteana
computer search using digital libraries of mass spectral data

(4) and by comparison of their retention indices and authentic mass spectra (5), relative to C8-C32 n-alkane series (6) in a
temperature-programmed run.
Isolation of a fraction rich in a-bisabolol: A sample of
oil (80 mg) was submitted to silica gel column chromatography
eluted with pentane, dichlorometane and diethyl ether, giving
13 fractions of 3 mL each one. Fraction 6 (8.5 mg) was rich in
a-bisabolol (78.4%). This fraction was analyzed by 1H and 13C

NMR (Brucker AC200, 200 MHz, CDCl3). The spectra data
were compatible with a-bisabolol (7).
Cytotoxicity assay: It was used the U251 (glioma, CNS),
UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES
(ovarian-resistent), 786-0 (kidney), NCI-H460 (lung, no small
cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon),
K562 (leukemia) and VERO cell lines. The assay was done as
Table II. Antiproliferative activity of essential oil of Myrcia laruotteana and a fraction rich in a-bisabolol
Cell lines

TGI (mg/mL) a
Essential Oil
EOF b
Doxorubicin
U251
UACC-62
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-3
HT-29
K562
20.46
26.98
88.31
>125
20.74
79.83
27.63
52.36
59.29
-
16.89
28.00
20.84
>50
8.74
33.50
-
8.58
19.16
25.48
6.24
0.30
>25
>25
25
>25
6.32
3.30
>25
0.15
VERO
36.45
7.91
>25
-: No tested; TGI Total Growth Inhibition concentration that inhibited cell growth by 100%. The coefficients of variation obtained in these analyses were below to 5%;
b
fraction of the essential oil containing a-bisabolol (78.4 2.0%), a-bisabolol oxide B (7.8 0.6%), viridiflorol (3.4 0.5%), epi-a-muurolol (2.5 0.2%) and 1-epi-cubenol
(2.0 0.1%) as determined by GC-FID.
a
Figure 1. GC chromatogram of the essential oil from Myrcia laruotteana fruits showing the major components: spathulenol
(17), globulol (19), viridiflorol (20), a-bisabolol oxide B (31), a-cadinol (32), a-bisabolol (34) and, 2E, 6E-methylfarnesoate
(40).
Stefanello et al.
described previously (8). Briefly, the cells were distributed in

96-well plates (100 mL cells/well) and exposed to various concentrations of essential oil (0.25, 2.5, 25.0 and 250.0 mg/mL)
in DMSO (0.1%) at 37oC, with 5% of CO2, for 48 h. The final
concentration of DMSO did not affect the cell viability. A 50%
trichloroacetic acid solution was added and after incubation for
30 min at 4oC, the cells were washed and dried. Cell proliferation was determined by spectrophotometric quantification (at
540 nm) of the cellular protein content using sulforhodamine
B. The experiments were carried, at least, in triplicate and the
concentration necessary to total growth inhibition (TGI) was
calculated in mg/mL. Doxorubicin was used as positive control.
The data were analyzed using ANOVA and the F-test used to
determine any difference among the groups.

The hydrodistillation of M. laruotteana unripe fruits furnished colorless oil, with yielding of 0.3%, which was higher
than previously reported yields from leaves (0.05%) and flowers (0.07%) (3). The components of the oils, the percentage
of each constituent and the retention indices are summarized
in the Table I and the GC chromatogram is shown in the
Figure 1. The oil was characterized by high content of oxygenated sesquiterpenes, mainly of bisabolane group. The major
components were a-bisabolol (23.6%) and a-bisabolol oxide
B (11.5%). This composition is very similar to those of leaves
and flowers, except for presence of a-bisabolol oxide (3). The
predominance of sesquiterpenes, mainly type bisabolane, in
the essential oil has been reported for several species of Myrcia
(9-11). The essential oil of Myrcia splendens contains almost
80% of a-bisabolene (10) while a-bisabolol is almost half of
the oil of M. bracteata (11).
The oil exhibited antiproliferative activity for almost all
cell lines evaluated, with TGI varying of 20.46-88.31 mg/mL,
with exception of NCI-ADR/RES cell line, for which the TGI
was higher than 125 mg/mL. The most significant activity was
observed against U251 (glioma), 786-0 (kidney), UACC-62
(melanoma) and PC-3 (prostate), all with TGI lower than 30
mg/mL. The activity of the fraction rich in a-bisabolol was
similar, with TGI varying of 8.58-33.50 mg/mL (Table II).
These results suggest that the activity of oil is related to presence of a-bisabolol.
The sesquiterpene a-bisabolol has been used in pharmaceutical products as drug permeation, anti-inflammatory,
antispasmodic, anti-allergic and vermifuge. Besides, antimicrobial, antiplasmodial, antioxidant and anticancer activities
also have been reported for pure a-bisabolol or oil rich in
this compound (12, 13). The antitumor activity was previously observed against glioma and pancreatic carcinoma cells
(13, 14). The present study show that, in addition to glioma,
a-bisabolol is also effective against ovarian (OVCAR-3) and
kidney (786.0) carcinoma cells, with TGI of 8.58 and 8.74 mg/
mL, respectively. However, both oil and the fraction rich in
a- bisabolol exhibited toxicity against VERO cell (no cancer
cell) with TGI of 36.45 and 7.91 mg/mL, respectively. This

result with VERO cell was unexpected because a-bisabolol is
considered a nontoxic compound (14). Thus, further investigations are necessary to confirm the potential of the essential oil
of M. laruotteana fruits as a citotoxic agent useful for in vivo
applications in cancer treatment.
Acknowledgements
The authors thank the FAPESP for financial support. MJS and
JEC are grateful to CNPq for research scholarships. D. Riva is grateful
to CAPES for scholarship.
References
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Essential oil composition of Myrcia laruotteana Camb..J. Essent. Oil
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Commerce, Gaithersburg, MD (1998).
H. van Den Dool, P.D.J.A. Kratz, Generalization of the retention index
system including linear temperature programmed gas-liquid partition
chromatography. J. Chromatogr., 11, 463-471 (1963).
M. Miyazawa, H. Nankai and H. Kameoka, Biotransformation of
(-)-a-bisabolol by plant pathogenic fungus, Glomerella cingulata.
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properties. Chem. Biodiv., 8, 73-94 (2011).
M. J. Nakamura, S. S. Monteiro, C. H. B. Bizarri, A. C. Siani, Essential
oils of four Myrtaceae species from the Brazilian Southeast. Biochem.
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R. A. Pereira, M. G. B. Zoghbi and M. N. C. Bastos, Essential oils
of twelve species of Myrtaceae growing wild in the sandbank of the
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V. Popovic, S. Petrovic, M. Pavlovic, M. Milenkovic, M. Couladis, O.
Tzakou, S. Duraki and M. Niketic, Essential oil from the underground
parts of Laserpitium zernyi: potential source of a-bisabolol and its
antimicrobial activity. Nat. Prod. Comm., 5, 307-310 (2010).
G. P. P. Kamatou and A. M. Viljoen, A review of the application and
pharmacological properties of a-bisabolol and a-bisabolol-rich oils.

J. Am. Oil Chem. Soc., 87, 1-7 (2010).
14. E. Cavalieri, S. Mariotto, C. Fabrizi, A. C. Prati, R. Gottardo, S. Leone,
L. V. Berra, G. M. Lauro, A. R. Ciampa and H. Suzuki, a-bisabolol,
a nontoxic natural compound, strongly induces apoptosis in glioma
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Polygonum
Chemotaxonomic Importance of Sesquiterpenes and

Flavonoids in Five Argentinian Species of Polygonum
Genus
M. Derita* and S. Zacchino
Pharmacognosy Area, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario,
Rosario, Argentina
Abstract
Leaves of five species of Polygonum genus belonging to Persicaria section were collected from the northeast and
central lowlands of Argentina and their dichloromethane extracts were analyzed by GC/EM. They were investigated
for the presence of four drimane-type sesquiterpenes: polygodial (1), isopolygodial (2), drimenol (3) and confertifolin
(4), previously isolated from P. acuminatum; and the presence of three flavonoids: pinostrobin (5), flavokawin B (6)
and cardamonin (7), previously isolated from P. persicaria. Results showed that among the five species of Persicaria
section studied, two species contained sesquiterpenes 1-4 but not flavonoids 5-7, other two species contained flavonoids 5-7 but not sesquiterpenes 1-4, and only one species contained compounds 1-7. These results add evidences
to a previous proposal to sub-classify the Persicaria section of Polygonum genus from a chemotaxonomic point of
view. [Editors note: This papers references are notated within the main body of the text in superscript, so as to avoid
reader confusion with the discussed sesquiterpenes and flavonoids.]
Key Word Index
Polygonum genus, Persicaria section, sesquiterpenes, flavonoids, chemotaxonomic importance.
Introduction
The Polygonum genus (Polygonaceae) is well known for producing a variety of secondary metabolites including flavonoids, 1
triterpenoids,2 anthraquinones,3 coumarins,4 phenylpropanoids,5
lignans,6 stilbenoids,7 tannins8 and sesquiterpenoids.9 It is represented in Argentina by 21 species, which are divided into
five sections: Echinocaulon, Amblygonum, Persicaria, Tiniaria
and Polygonum.10
Polygonum punctatum Elliot, P. persicaria L., P. acuminatum Kunth., P. lapathifolium L. and P. hydropiperoides
Michux var. hydropiperoides, are five out of the 11 perennial
herbs, belonging to the Persicaria section, which grow in the
northeast and central lowlands of Argentina.
In a previous work, and considering that Gattuso11 and
Cialdella10 suggested a delimitation of the Persicaria section
to those species of Polygonum genus containing some kind of
irritant valves, we suggested that the presence of the sesquiterpene polygodial (1) could be also of diagnostic value for the
delimitation of the Persicaria section. Following this point of
view, the inclusion of P. hydropiperoides var. hydropiperoides
and P. lapathifolium (which do possess neither polygodial
nor valvate glands) within the Persicaria section could be the
subject of a further revision.12

In this work we add evidence to that proposal, investigating
the presence of three sesquiterpenes (in addition to polygodial) isolated from P. acuminatum [isopolygodial (2), drimenol
(3) and confertifolin (4)], and three flavonoids isolated from
P. persicaria [pinostrobin (5), flavokawin B (6) and cardamonin (7)], in DMC extracts of the species aforementioned.
(Figure 1).
Experimental
Plant material: Polygonum hydropiperoides Michux var.
hydropiperoides was collected during the flowering season
(March 2005) in San Luis province, Merlo district (3235S Lat.,
6503O Long. and 850 m elevation), identified by Elisa Petenatti
and deposited at the Herbarium of the National University of
San Luis (UNSL # 9256). P. punctatum Elliot, P. persicaria L.,
P. acuminatum Kunth. and P. lapathifolium L. were harvested
in March 2005 in Santa Fe province, Puerto Gaboto district
(3227S Lat., 6048O Long. and 25 m elevation), identified
by Susana Gattuso and deposited at the Herbarium of the
National University of Rosario, Argentina (UNR Gattuso, S.
97, 108, 94, and 115, respectively).
*Address for correspondence: mgderita@hotmail.com
Rec: Jan 2011

Acc: April 2011

Derita et al.
Figure 1. A) Sesquiterpenes isolated from P.

acuminatum: polygodial (1), isopolygodial (2),
drimenol (3), confertifolin (4). B) Flavonoids isolated
from P. persicaria: pinostrobin (5), flavokawin B (6),
cardamonin (7).
Dried and powdered leaves of the species mentioned above

(30 g each) were macerated with dichloromethane (DCM) for
24 h (3X). The solvent was evaporated under reduced pressure to yield 0.86, 1.14, 1.05, 0.80 and 0.50 g of DCM soluble
extracts, respectively.
Compounds isolation: Fractionation by column chromatography of P. acuminatum leaves DCM extract allowed us to
isolate 70, 53, 35 and 27 mg of compounds 1-4 respectively.
Fractionation by column chromatography of P. persicaria
leaves DCM extract allowed us to isolate 40, 36 and 25 mg of
compounds 5-7 respectively. Compounds 1-4 were previously
isolated from Drymis spp.,13,14 P. punctatum15 and P. acuminatum,9 while compounds 5-7 were previously isolated from
Boesenbergia pandurata, Myrica pensilvanica, P. ferrugineum
and Piper spp.1,16,17 All spectrums of the isolated compounds
in this work, were compared with the literature cited to assure
the identity of each structure.
Analysis of the DCM extracts: All the extracts were
submitted to GC-MS using a Turbo Mass Perkin Elmer chromatograph, equipped with a fused silica column (SE-30 25
m x 0.22 mm ID) with He as a carrier gas, coupled to a mass
selective detector, film 0.25 m, ionization energy 70 eV with
a temperature programme of 70-200C at 10C/min; total time
30 min. Compounds 1-7 were identified by comparison of
their retention time and their MS spectrum with the authentic
samples obtained from our previous works. Chromatograms
are shown in Figure 2.
Figure 2: Gas chromatograms of dichloromethane extracts of P. acuminatum (A), P. persicaria (B), P. punctatum (C), P.
lapathifolium (D) and P. hydropiperoides var. hydropiperoides (E). Peaks at 14.74/14.36 min in (A), (B) and (C) belong to
drimenol (3). Peaks at 15.59/15.38 min in (A), (B) and (C) belong to confertifolin (4). Peaks at 16.16/16.38 min in (A),
(B) and (C) belong to isopolygodial (2). Peaks at 17.41/17.40 min in (A), (B) and (C) belong to polygodial (1). Peaks at
20.81, 20.93, 20.91 min in (B), (D) and (E) belong to pinostrobin (5). Peaks at 22.69, 22.83, 22.80 min in (B), (D) and
(E) belong to flavokawin B (6). Peaks at 23.51, 23.19, 23.14 min in (B), (D) and (E) belong to cardamonin (7).
Polygonum
Compound descriptions:
Polygodial (1) MP: 48C. [a]D: -27 (c 1.00, CHCl3). IR

(KBr): 2927, 2850, 2726, 1722, 1680, 1642, cm-1. 1H NMR
(300 MHz, CDCl3): 9.48 (1H, d, J= 4.2 Hz, H-11); 9.42 (1H, s,
H-12); 7.11 (1H, m, H-7); 2.78 (1H, dddd, J= 6.2, 2.1, 2.1, 2.1
Hz, H-9); 2.55-2.40 (1H, m, H-6a); 2.35-2.20 (1H, m, H-6b);
1.82 (1H, m, H-1b); 1.54-1.43 (3H, m, H-2a, 2b, 3b); 1.34 (1H,
td, J= 4.0 and 13.4 Hz, H-1a); 1.26-1.16 (2H, m, H-3a, H-5);
0.92; (3H, s, Me-15); 0.91 and 0.89 (6H, 2s, Me-14 and Me-15).
13
C NMR (75 MHz, CDCl3): 201.9 (HC=O); 193.2 (HC=O);
154.4 (=CH); 138.1 (=C); 60.2 (CH); 48,8 (CH); 41.7 (CH2);
39.5 (CH2); 36.8 (C); 33.0 (C); 33.0 (CH3); 25.1 (CH2); 21.9
(CH3); 17.9 (CH2); 15.2 (CH3). MS (EI, 70 eV): m/z (%) = 234
[M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me].
Isopolygodial (2) [a]D: +30 (c 1.00, CHCl3). IR (KBr):
2927, 2850, 2726, 1722, 1680, 1642, cm-1. 1H NMR (300 MHz,
CDCl3): 9.86 (1H, d, J= 2.7 Hz, H-11); 9.41 (1H, s, H-12); 7.11
(1H, dd, J= 2.7 and 4.9 Hz, H-7); 3.26 (1H, bm, H-9); 2.57 (1H,
dt, J= 5.0, 5.0, 20.6 Hz, H-6a); 2.22 (1H, dddd, J= 1.8, 2.6,
11.6 and 20.6 Hz, H-6b); 1.80 (1H, m, H-1b); 1.62-1.52 (5H,
m, H-1a, 2a, 2b, 3b, 5); 1.19 (1H, ddd, J= 0.62, 4.1 and 12.0
Hz, H-3a); 0.97 (3H, s, Me-15); 0.94 and 0.92 (6H, 2s, Me-13
and Me-14). 13C NMR (75 MHz, CDCl3): 202.2 (HC=O); 192.8
(HC=O); 153.5 (=CH); 137.3 (=C); 58.5 (CH); 44.2 (CH); 42.0
(CH2); 37.6 (CH2); 37.1 (C); 32.9 (C); 32.7 (CH3); 25.5 (CH2);
21.9 (CH3); 21.5 (CH2); 18.4 (CH3). MS (EI, 70 eV): m/z (%) =
234 [M+], 216 [M+ - H2O], 206 [M+ - CO], 191 [206 - Me].
Drimenol (3) MP: 98C. [a]D: -15 (c 1.00, CHCl3). IR
(KBr): 3405, 2922, 1620 cm-1. 1H NMR (300 MHz, CDCl3):
5.54 (1H, m, H-7); 3.85 (1H, dd, J= 3.3, 11.3 Hz, H-11B);
3.73 (1H, dd, J= 3.3, 11.3 Hz, H-11A); 1.97 (1H, m, H-6a);
1.95 (1H, m, H-1b); 1.92 (1H, m, H-6b); 1.86 (1H, m, H-9);
1.79 (3H, s, Me-12); 1.48 (2H, m, H-2a and 2b); 1.41 (1H,
m, H-3b); 1.20 (2H, m, H-3a and H-5); 1.07 (1H, m, H-1a);
0.89, 0.87 and 0.85 (9H, 3s, Me-13, 14 and 15). 13C NMR (75
MHz, CDCl3): 132.9 (=C); 124.0 (=CH); 60.9 (H2C-OH); 57.3
(CH); 49.9 (CH); 42.1 (CH2); 39.8 (CH2); 36.0 (C); 33.3 (CH3);
32.9 (C); 23.6 (CH2); 22.0 (CH3); 21.9 (CH3); 21.9 (CH3); 18.8
(CH2); 14.9 (CH3). MS (EI, 70 eV): m/z (%) = 234 [M+], 216
[M+ - H2O], 206 [M+ - CO], 191 [206 - Me].
Confertifolin (4) MP: 153C. [a]D: +70 (c 1.00, CHCl3).
IR (KBr): 1769, 1677 cm-1. 1H NMR (300 MHz, CDCl3): 4.72
(1H, ddd, J= 2.8, 2.8, 16.9 Hz, H-1a); 4.62 (1H, ddd, J= 1.7,
3.5, 16.9 Hz, H-1b); 2.54-2.05 (2H, m, H-4a and b); 1.92-1.30
(9H, m, H-5a and b, H-5a, H-7a and b, H-8a and b, H-9a and
b); 1.15 (3H, s, Me-9a); 0.93 and 0.89 (6H, 2s, Me-6a and b).
13
C NMR (75 MHz, CDCl3): 174.5 (C=O); 170.8 (=C); 123.4
(=C); 68.2 (CH2); 51.2 (CH); 41.6 (CH2); 36.6 (CH2); 36.0 (C);
33.2 (C); 33.2 (CH3); 21.4 (CH2); 21.4 (CH3); 20.9 (CH3); 18.3
(CH2); 18.0 (CH2). MS (EI, 70 eV): m/z (%) = 234 [M+], 216
[M+ - H2O], 206 [M+ - CO], 191 [206 - Me].
Pinostrobin (5) MP: 87C. [a]D: -51 (c 1.00, CHCl3). IR
(KBr): 3450, 2950, 1647, cm-1. 1H NMR (300 MHz, CDCl3):
12.04 (1H, s, OH); 7.49-7.40 (5H, m, H-2-6); 6.10 (1H, d,
J= 2.3 Hz, H-8); 6.09 (1H, d, J= 2.3 Hz, H-6); 5.44 (1H, dd,
J= 3.1 and 12.9 Hz, H-2); 3.83 (3H, s, O-Me); 3.11 (1H, dd,
J= 12.9 and 17.2 Hz, H-3a); 2.84 (1H, dd, J= 3.1 and 17.2
Hz, H-3b). 13C NMR (75 MHz, CDCl3): 195.8 (C=O); 168.0
(=C); 164.1 (=C); 162.8 (=C); 138.4 (=C); 128.9 (=CH); 128.9
(=CH); 128.9 (=CH); 126.2 (=CH); 126.2 (=CH); 103.1 (=C);
95.1 (=CH); 94.2 (=CH); 79.2 (CH); 55.7 (OCH3); 43.4 (CH2).
MS (EI, 70 eV): m/z (%) = 270 [M+].
Flavokawin B (6) MP: 95C. IR (KBr): 3450, 2940, 1630,
cm-1. 1H NMR (300 MHz, CDCl3): 12.04 (1H, s, -OH); 7.92
(1H, d, J= 15.4 Hz, H-b); 7.79 (1H, d, J= 15.4 Hz, H-a); 7.647.28 (5H, m, H2-6); 6.12 (1H, d, J= 2.3 Hz, H-5); 5.98 (1H,
d, J= 2.3 Hz, H-3); 3.93 and 3.85 (6H, 2s, 2 O-Me). 13C NMR
(75 MHz, CDCl3): 192.6 (C=O); 168.4 (=C); 166.2 (=C); 162.5
(=C); 142.3 (=CH); 135.6 (=C); 130.0 (=CH); 128.9 (=CH);
128.9 (=CH); 128.4 (=CH); 128.4 (=CH); 127.5 (=CH); 106.3
(=C); 93.8 (=CH); 91.3 (=CH); 55.9 (O-CH3); 55.6 (O-CH3).
MS (EI, 70 eV): m/z (%) = 284 [M+].
Cardamonin (7) MP: 98C. IR (KBr): 3400, 2924, 1638,
cm-1. 1H NMR (300 MHz, DMSO-d6): 13.7 (1H, s, -OH); 7.83
(1H, d, J= 15.6 Hz, H-b); 7.73-7.70 (2H, m, H-2 and H-6); 7.68
(1H, d, J= 15.6 Hz, H-a); 7.46-7.44 (3H, m, H-3, 4 and 5); 6.01
(1H, d, J= 2.2 Hz, H-5); 5.92 (1H, d, J= 2.2 Hz, H-3); 3.88
(3H, s, O-Me). 13C NMR (75 MHz, DMSO-d6): 192.2 (C=O);
166.7 (=C); 165.6 (=C); 163.1 (=C); 142.3 (=CH); 135.4 (=C);
130.8 (=CH); 129.5 (=CH); 128.8 (=CH); 128.8 (=CH); 128.0
(=CH); 128.0 (=CH); 105.5 (=C); 96.3 (=CH); 92.2 (=CH);
55.5 (O-CH3). MS (EI, 70 eV): m/z (%) = 270 [M+].

The analysis of the GC spectra clearly showed that P.
hydropiperoides var. hydropiperoides and P. lapathifolium
do not possess sesquiterpenes 1-4 but contain flavonoids 5-7;
meanwhile P. punctatum and P. acuminatum do not possess
flavonoids 5-7 but contain sesquiterpenes 1-4. Surprisingly, P.
persicaria (the specie that gives the name to the whole section)
possesses compounds 1-7.
Hereby, taking into account our previous work12and these
new results, we can propose that within the Persicaria section
of Polygonum genus there are two kinds of species that could
be sub-classified by a chemotaxonomic point of view: those that
produce the sesquiterpenes (1-4) but not the flavonoids (5-7),
and those that biosynthesize the flavonoids (5-7) but not the
sesquiterpenes (1-4). To assure this proposal, we must go on
studying other species that belong to the Persicaria section of
Polygonum genus in terms of their content in sesquiterpenes
and flavonoids.
Acknowledgements
The authors wish to acknowledge CONICET, ANPCyT, ERASMUS

MUNDUS lot 18 ARBOPEUE, UNR.
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of Polygonum genus belonging to Persicaria section. Biochem. Syst.
Ecol., 36, 55-58 (2008).
13. V. Cechinel Filho, V. Schlemper, A. Santos, T. Pinheiro, R. Yunes, G.
Mendes, J. Calixto, F. Delle Monache, Isolation and identification of
active compounds from Drymis winteri barks. J. Ethnopharmacol.,
62, 223-227 (1998).
14. D. Muoz-Concha, H. Vogel, R. Yunes, I. Razmilic, L. Bresciani, A.
Malheiros, Presence of polygodial and drimenol in Drymis populations
from Chile. Biochem. Syst. Ecol., 35, 434-438 (2007).
15. T. De Almeida Alves, F. Lacerda Ribeiro, H. Kloos, C. Zani,
Polygodial, the fungitoxic component from the Brazilian medicinal
plant Polygonum punctatum. Mem. do Inst. Oswaldo Cruz, 96,
831-833 (2001).
16. K. Hodgetts, Approaches to 2-substituted chroman-4-ones: synthesis
of (-)-pinostrobin. Tetrahedron Let., 42, 3763-3766 (2001).
17. B. Burke, M. Nair, Phenylpropene, benzoic acid and flavonoids
derivatives from fruits of Jamaican Piper species. Phytochemistry,
25, 1427-1430 (1986).
P. claussenianum
Seasonal Evaluation and Chemical Composition

of Volatile Fractions from Piper claussenianum by
Hydrodistillation and SPME
Andr Mesquita Marques* and Maria Auxiliadora Coelho Kaplan
Ncleo de Pesquisas de Produtos Naturais (NPPN), Centro de Cincias da Sade, Bloco H . Universidade Federal do Rio
de Janeiro (UFRJ), Brasil. CEP: 21941-590
Abstract
The essential oils from Piper species are chemically diverse, with several biological activities as well as commercial
and pharmacological values reported. This work aims to compare the volatile constituents from leaves and inflorescences obtained by two different extraction techniques and show the seasonal variation of nerolidol in the essential
oil from leaves of Piper claussenianum. The P. claussenianum volatile components were obtained by headspace
solid-phase microextraction (HS-SPME) and hydrodistillation, coupled to gas chromatography/mass spectrometry
(GC/MS) analysis. Leaf and inflorescence volatile fractions were analyzed in fresh and dried conditions. In total, 40
compounds were identified, accounting for 88.897.7% of the constituents. The high percentage of nerolidol in the
leaves and linalool in the inflorescences were remarkable in this species, accounting for up to 77.0% and up to 50.0%,
respectively, collected monthly. Results of (E)-nerolidol seasonal variation study carried out in the growth period of
January/December 2009, suggest the plant leaves which provide an essential oil with high content of (E)-nerolidol
over 88.0% are primarily observed in the September/December collection period.
Key Word Index
Piper claussenianum, nerolidol, essential oil, SPME, hydrodistillation.
Introduction
The essential oils (EOs) from aromatic herbs traditionally obtained by hydrodistillation are of increasing use in aromatherapy
due to the popular interest for natural compounds with curative
properties and economical value, such as scents in perfumes,
cosmetics and cleaning agents. Various novel techniques have
been developed for EO extraction from plants. Among these,
headspace solid phase microextraction (HS-SPME) allows the
rapid fingerprinting of a plant headspace (1). Over the last
several years, solid-phase microextraction (SPME) has gained
acceptance in many fields as an accurate, rapid, sensitive and
solvent-free sampling method. Many researchers report its application as a useful approach in sample preparation of volatile
compounds from complex matrices (2). Chemically diversified
essential oils from Piper species have noteworthy biological
activities as well as commercial and pharmacological values.
Terpenes such as linalool and nerolidol have been used for
scent composition bouquet in perfumes and as antimicrobial
products (3-7). In a previous work, we reported, for the first
time, the chemical compositions of essential oils from the leaves
and flowers of Piper claussenianum, in addition to their antiparasitic activity against a strain of Leishamania amazonensis.
The high percentage of the sesquiterpene (E)-nerolidol in the

leaves and the monoterpene linalool in the inflorescences was
remarkable to this species. Due to this fact and previous reports
concerning about leishmanicidal properties of terpenes we
were encouraged to carry out the investigation of these oils on
this parasite. Both assayed EOs extracted by hydrodistillation
have inhibited the growth of parasites. MIC and IC50 values
of the leaf EO were respectively 57.6 mg/mL and 30.4 mg/
mL while the EO from inflorescences were 664.0 mg/mL and
1328.0 mg/mL. These results suggest the higher percentage of
nerolidol in leaf EO to be the responsible compound for the
more effective growth inhibition (3).
This work aims to investigate the chemical composition of
these piperaceous essential oils extracted using hydrodistillation (HD) from separated organs (leaves and flowers) as well
as the volatile fractions extracted using HS-SPME from P.
claussenianum. Seasonal monitoring of nerolidol in the leaf
essential oils was also performed. In both procedures, analyses were carried out using gas chromatography (GC) and gas
chromatography/mass spectrometry (GC/MS). SPME constitutes a suitable alternative to extract volatile and semivolatile
chemicals in a wide range of matrices. A major advantage
*Address for correspondence: andrefarmaciarj@yahoo.com.br
Rec: Feb 2011

Acc: June 2011

Marques et al.
very critically since transformation processes of genuine

aroma-active compounds due to the influence of heat, steam,
and pH may occur (11). On the other hand, highly volatile
components as well as water-soluble components can get lost
during hydrodistillation. As with hydrodistillation, non-volatile
substances are not detected with SPME, and thus do not affect
the evaluation of the essential oils and related compounds.
Similarly hydrodistillation is a very time-consuming method
and therefore not useful, especially for the screening of very
large quantities of plant samples for their aromatic composition. Solid-phase microextraction (SPME) is a comparatively
new, very simple and efficient method for routine laboratory
analysis of organic compounds (12-15). Furthermore, SPME
is the most time-saving sample preparation method for the
subsequent gas chromatographic determination of the composition of aroma-active compounds.
includes the capability to combine volatile fraction extraction and preconcentration in a single step (8). Some physical
factors such as sample agitation, headspace equilibration
temperature, extraction time, and analyte diffusion rate from
the vapour phase to the fibre surface also contribute to high
SPME efficacy. (9). The evaluation of aromatic fractions from
natural compounds is important in agricultural science and
food chemistry. The determination of essential oil composition is necessary for quality control of breeding parameters,
cultivation, and production of aromatic plants. Quality control
of raw material, intermediates, and end products is furthermore required in the food industry for production of spices,
essential oils, or flavored food (10). The most usual method for
the determination of the essential oil content is by hydrodistillation. However, its use for the subsequent determination of
the aroma compound compositions has often been discussed
Table I. Identified Compounds in the Essential Oil from Leaves of P. claussenianum.

Compounds
RI
FRESH HD %
DRY HD %
SPME %
Identification
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
a-pinene
b-pinene
b-myrcene
limonene
(Z)-b-ocimene
(E)-b-ocimene
cis Linalool oxide
linalool
g-elemene
b-elemene
(E)-caryophyllene
(Z)-b-farnesene
a-humulene
g-muurolene
a-selinene
(Z)-a-bisabolene
d-cadinene
(E)-g-bisabolene
a-cadinene
germacrene B
(E)-nerolidol
caryophyllene oxide
gleenol
a-eudesmol
939
979
991
1029
1037
1050
1087
1097
1338
1391
1419
1443
1455
1480
1498
1507
1523
1531
1539
1561
1563
1583
1587
1654
936
976
987
1027
1033
1043
1083
1101
1340
1387
1415
1439
1451
1477
1497
1508
1520
1531
1545
1558
1563
1577
1586
1654
0.2
0.2
-
-
0.6
0.9
0.6
5.2
0.1
0.5
0.6
-
0.6
1.1
0.4
-
0.4
-
-
-
81.4
-
-
0.5
93.3
-
0.1
0.2
-
-
-
-
2.2
1.8
0.8
1.4
-
1.1
3.2
-
-
0.8
-
-
-
83.2
-
-
2.9
97.7
0.6
0.6
-
0.3
3.4
4.1
-
4.6
5.4
-
-
6.9
1.3
15.9
-
0.3
4.1
1.2
0.6
0.5
42.1
1.1
1.0
-
94.0
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS, STD
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS, STD
RI, GCMS
RI, GCMS
RI, GCMS
RILit
% PEAK SUM OF IDENTIFIED COMPOUNDS:

a
RI Lit: Literature Retention Indices16; b RI: Experimental Retention Indices; H.D: Hydrodistillation;
Table II. Monoterpene and Sesquiterpene fractions of the analyzed Essential Oils.

Monoterpene
Sesquiterpene
Total
Fresh HD %
Dry HD %
SPME %
Leaves
Inflorescences
Leaves
Inflorescences
Leaves
Inflorescences
7.6
85.7
93.3
52.9
35.9
88.8
2.5
95.2
97.7
56.5
35.5
92.0
13.0
81.0
94.0
61.9
31.2
93.1
P. claussenianum
Experimental
Plant material and essential oil extraction: Leaves
(100 g) of Piper claussenianum were collected in Castelo, ES
in January 2010. The botanical vouchers were identified by Dr.
Elsie Franklin Guimares and kept at the Herbarium (HB) of
the Rio de Janeiro Botanical Garden (JBRJ), registered under
number RB 489043. The fresh and dried plant materials were
submitted for hydrodistillation for 2 h in a modified Clevengertype apparatus. The obtained essential oils (EO) were dried
over anhydrous sodium sulphate, yielding 1.0% w/w in both
studied specimens.
GC-FID analysis: The gas chromatography analyses
were carried out on a GC 2010 Shimadzu with a ZB-1MS
fused silica capillary column (30 m x 0.25 mm x 0.25 mm film
thickness). The operating temperatures used were as follows:
injector 260C, detector 290C and column oven 60C up to
290C (10C/min). Hydrogen at 1.0 mL min-1 was used as a
carrier gas. The percentages of the compounds were obtained
by GC-FID analysis.
GC/MS analysis: Qualitative analyses were carried out
on a GC-QP2010 PLUS Shimadzu with a ZB-5MS fused silica
capillary column (30 m x 0.25 mm x 0.25 mm film thickness)
under the experimental conditions reported for GC-FID
analysis. The essential oil components were identified by
comparing their retention indices and mass spectra to pub-
lished data and computer matching with WILEY 275 and the
National Institute of Standards and Technology (NIST 3.0)
libraries provided by a computer-controlled GC/MS system.
The results were also confirmed by comparing the compounds
elution order with their relative retention indices reported in
the literature (16). The retention indices were calculated for
all the volatile constituents using the retention data of linear
n-alkanes C8C24.
Nuclear magnetic resonance spectroscopy: The crude
EO obtained from leaves of P. claussenianum was analyzed by
1
H and 13C NMR and recorded on a Brker DRX 400 spectrometer in order to confirm the presence of the sesquiterpene
and to show the 13C NMR spectra value on characterization
of terpenes. The chemical shifts were determined in CDCl3,
using TMS as the internal standard. The spectrometric data
for the 1H NMR spectra are organized in agreement with the
convention: chemical shift (number of protons and multiplicity),
and the data for 13C NMR spectra are organized by chemical
shift. The signals of the NMR analyses were compared to the
literature data (3, 16).
Head-space/Solid phase microextraction (HSSPME): Fresh leaves and inflorescences of P. claussenianum

C.DC. (500 mg) were reduced to small pieces and powdered
one by one in separated way. Four headspaces with these
materials were separately extracted by HS-SPME using
Figure 1. Seasonal evaluation of Nerolidol in EO of P. claussenianum leaves monthly harvested in 2009. The percentages
of the nerolidol remains always over 77.0% (April) achieving the maximum content level in October (94.0%) during the
Brazilian spring.
Marques et al.
a carboxen-divinylbenzene 75 mm fiber (CAR-DVB) at

80C, and sample/ headspace equilibration time for 15
min. The extracted materials were immediately desorbed
and analyzed by GC/MS.
sesquiterpene (E)-nerolidol in the leaves and the monoterpene

linalool in the inflorescences was remarkable to this species.
The oil from leaves of P. claussenianum was characterized by its
high content in (E)-nerolidol (81.4%, 83.3%, 42.1%) as well as in
linalool (5.2%, 2.2%, 4.6%), g-muurolene (1.1%, 3.2%, 15.9%),
(E)-caryophyllene (0.6%, 1.4%, - ) and g-elemene (0.5%, 0.8%,
5.4%) in fresh HD, dry HD and SPME analyses, respectively
(Table I). (E)-nerolidol, the main volatile compound from the
leaf EOs, represents (81.0%) of the volatile fractions in the
hydroditillation of fresh and (83.0%) in dried samples while
SPME analysis has shown only a half percent of this compound
of fresh leaf EO composition. As was expected, the EO from
fresh leaf HD displays a higher content of monoterpenes: 6
(7.6%), than dried material: 3 (2.5%). On the other hand the
sesquiterpene percentage in the EO from dried leaves showed
the highest value, accounting for 95.2% while in the fresh EO
it was found to be 85.7%. (Table II).
Although the dried material displays a higher percentage of
sesquiterpenes in its composition the content did not differ that
much from that of the fresh material. The relative composition
of HS-SPME volatile fractions obtained from leaves shows a

Analyses of the essential oils from leaves and inflorescences collected in Castelo, ES, Brazil, led to the identification of 40 components, corresponding to 89.0-97.7% of the
constituents.
The identification of the compounds was performed by
comparing their EI-MS and retention indices with those reported in the literature libraries. The structure of nerolidol was
confirmed by 1H NMR and 13C NMR spectra obtained by the
crude leaf EOs. Both oils, from leaves and inflorescences showed
rich monoterpene and sesquiterpene fractions. Sesquiterpenes
were identified as the main volatile fraction of the leaves EOs
while monoterpenes were found in great amount in the inflorescences. The essential oils obtained by hydrodistillation from
fresh and dried material of P. claussenianum aerial parts yielded
about 1.0% (w/v) for both cases. The high percentage of the
Table III. Identified Compounds in the Essential Oils from Inflorescences of P. claussenianum.

Compounds
RI
FRESH HD %
DRY HD %
SPME %
Identification
1
2
3
4
5 l
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
a-pinene
b-pinene
b-myrcene
a-phellandrene
imonene
b-phellandrene
cineole
(Z)-b-ocimene
(E)-b-ocimene
a-pinene oxide
linalool
a-terpineol
piperitone
g-elemene
a-copaene
b-cubebene
(E)-caryophyllene
(Z)-b-farnesene
a-humulene
g-muurolene
germacrene D
b-selinene
a-selinene
trans guaiene
(Z)-a-bisabolene
a-amorphene
d-cadinene
(E)-nerolidol
t-cadinol
eudesmol <7-epi-alpha>
eudesm-7(11)-en-4-ol
939
979
991
1003
1029
1030
1031
1037
1050
1099
1097
1189
1253
1338
1377
1388
1419
1443
1455
1480
1485
1490
1498
1503
1507
1512
1523
1563
1640
1664
1700
936
976
987
1006
1027
1029
1030
1033
1043
1101
1102
1195
1257
1340
1380
1385
1414
1439
1451
1476
1483
1484
1497
1501
1508
1514
1527
1564
1635
1666
1707
0.2
0.2
0.1
0.1
0.3
0.3
0.3
-
0.3
-
50.2
0.3
0.6
-
0.1
0.1
2.0
-
2.6
0.3
-
0.6
-
0.7
-
0.3
0.9
22.7
0.3
5.0
0.3
-
-
-
-
0.3
-
0.3
-
0.5
0.5
54.5
-
0.5
0.3
-
-
2.0
-
2.6
0.4
-
4.0
0.4
-
-
0.2
0.9
24.3
-
-
0.5
0.6
0.6
0.3
0.5
1.5
-
-
2.6
5.3
-
50.6
-
-
1.3
-
-
-
4.4
4.9
-
0.5
0.3
-
-
1.7
1.0
2.4
14.6
-
-
-
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS, STD.
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS
RI, GCMS, STD.
RI, GCMS
RI, GCMS
RI, GCMS
88.8
92.2
93.1
RILit
% PEAK SUM OF IDENTIFIED COMPOUNDS:

a
RI Lit: Literature Retention Indices16; b RI: Experimental Retention Indices; H.D: Hydrodistillation;
P. claussenianum
higher diversity and/or quantity of monoterpenes (13.0%) and

sesquiterpenes (81.0%) on the essential oil profile comparing
to hydrodistillation. These results suggest most of the highly
volatile components absent in fresh and dried hydrodistillation
EOs were lost due to lengthy times at a high temperature during the extraction process. In the inflorescences, the consituent
found in the highest concentration was identified as linalool.
It was present in concentrations of up to 50.0% in the
volatile fractions produced by each extraction technique. The
highest concentration of the major terpenes (linalool and
nerolidol) were found in the dried HD essential oil. Linalool
was found as the main constituent (50.2%, 54.5%, 50.6%), followed by (E)-nerolidol (22.7%, 24.3%, 14.6%), a-humulene
(2.6%, 2.6%, 4.9%), (E)-caryophyllene (2.0%, 2.0%, - ) and
b-selinene (0.6%, 4.0%, 0.3%) in fresh HD, dried HD and
SPME analysis, respectively (Table III).
The HS-SPME was a useful tool in qualitative analysis of
highly volatile fractions, mainly the fresh material due the presence of a higher quantity of monoterpenes. In these analyses, a
low percentage of monoterpenes was obtained from the leaves,
but higher when compared to HD for fresh or dried material.
However, HS-SPME analysis of inflorescence showed a high
percentage (61.9%) of the monoterpenes, mainly linalool content (50.6%). This was expected since HS-SPME is suitable
for volatile compounds. The sesquiterpene (E)-nerolidol was
identified in lower amounts in the HS-SPME of leaves and
inflorescences. In fact, this sesquiterpene was identified in
approximately half of headspaces when compared to the hydrodistillation of fresh and dried leaves. Once again, this is also
expected, since HS-SPME is not a suitable technique for the
analysis of semi-volatile compounds. As a result the nerolidol
quantification was performed with the crude EO extracted by
hydrodistillation. In HS-SPME procedure the samples remained
at 80C during a time span of 15 min, which was not sufficient
for the release of all the nerolidol content present in the leaves.
Similarly, HS-SPME was more sensible for the determination
and analysis of the presence of linalool in the inflorescences
compared with nerolidol in the leaves. The seasonal monitoring of the EO from leaves of P. claussenianum was performed
in order to determine the optimal time of harvest. In order
to choose the appropriate harvest period for the specimen,
about 100 g of fresh plant aerial parts were collected monthly.
The EOs extracted by hydrodistillation were analyzed and no
variation in the chromatographic profile was found. Thoughout
the entire year, the leaf specimens displayed a marked higher
essential oil rich in nerolidol, always up to 77.0%.
The highest nerolidol content was observed during the
Brazilian spring collection period: Sept/Oct/Nov (87.0%, 94.0%,
92.0% respectively). The minimum level recorded was found
during the autumn collection period Mar/Apr/May (78.0%;
77.0%; 80.0%) respectively (Figure 1). We have enhanced
the importance of SPME as a useful technique to analyze the
endangered species from the Brazilian Tropical Rain Forest,
due to its simplicity, speed, low cost and solventless operation,
needing just a small quantity of material for the analysis to pro-
duce high quality of data. Furthermore, this study emphasizes

the importance of choosing the appropriate harvest period in
order to obtain the richest nerolidol content EO from leaves
of P. claussenianum.
Acknowledgements
The authors thank Conselho Nacional de Desenvolvimento Cientfico Tecnolgico (CNPq) for financial support and Mark English for
English correction.
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Faculdade de Farmcia, Universidade Federal do Rio de Janeiro, CCS, Bloco A, 2o andar, Ilha do Fundo, Rio de Janeiro,
RJ, 21941-590 Brazil
Ftima Regina Salimena

Instituto de Cincias Biolgicas, Universidade Federal de Juiz de Fora, Juiz de Fora-MG, Brazil
Humberto R. Bizzo
Embrapa Agroindustria de Alimentos, Avenida das Amricas 29501, Rio de Janeiro, RJ, 23020-470 Brazil
Abstract
The chemical composition of the essential oil from Lippia triplinervis (Verbenaceae), as well as its antimicrobial
activity against Candida albicans and other clinical isolates, was analyzed. The major compounds identified in the oil
obtained in April 2010 were myrcene (2.6%), ipsenone (11.4%), myrcenone (57.7%), (Z)-ocimenone (1.3%) and (E)calamenene (4.8%). The essential oil of this same plant collected in September 2010, showed a quite similar chemical
composition, but the relative percentage of myrcenone was reduced from 57.7% to 13.5%, whereas limonene (8.0%),
(E)-tagetone (2.0%) and (Z)-tagetone (19.4%) were now major constituents of this oil. The minimum inhibitory concentration (MIC) activity against C. albicans as well as other clinical isolates (C. glabrata, C. krusei, C. parapsilosis and
C. tropicalis) ranged from 156 to 1,250 mg/mL. This is the first report of the chemical composition and antimicrobial
activity of L. triplinervis essential oil.
Key Word Index
Lippia triplinervis, Verbenaceae, myrcene, myrcenone, limonene, ipsenone, (E)-tagetone, myrcenone, (Z)tagetone.
Presented in part at the 41st International Symposium on Essential Oils, Wroclaw, Poland.
Introduction
The genus Lippia (Verbenaceae) comprises about 200
species occurring mainly in Central and South America, as
well as in some areas of Tropical Africa (1). One of the main
diversity centers of the genus Lippia is located at the Cadeia
do Espinhao Mountains, in the State of Minas Gerais, Brazil
(2). During one of our field trips for plant collecting in the
mountains of Aiuruoca (Minas Gerais State), Brazil, we found
a strongly aromatic shrub, growing above the height of 1800 m.
This plant was identified as L. triplinervis Gardner (syn. Lippia iodophylla Schauer), a species with no previous chemical
investigation reports and thus, was collected for investigation.
This species is a shrub to 1.5 m tall, with coriaceous leaves, and
the flowers are fragrant, rose or violet. The first specimen of this
rarity, endemic of the campos de altitude species, has been
collected between stones at an altitude of 2000 meters, in Serra

dos rgos, Itatiaia and Mantiqueira (Rio de Janeiro, Minas
Gerais and So Paulo States) flowering in January, February,
April and May (3). As of 1998, it has been included in the list
of endangered species of the State of So Paulo, Brazil (4).
Due to the scarce data about this plant in the literature, we
decided to analyze the chemical composition of its essential oil,
as well as to evaluate the antimicrobial activity against Candida
albicans and other clinical isolates: C. glabrata, C. krusei, C.
parapsilosis and C. tropicalis.
Experimental
Plant material and essential oil extraction: Lippia
triplinervis Gardner was collected in flower above 1800 m,
*Address for correspondence: sgleitao@pharma.ufrj.br
Rec: Feb 2011

Acc: July 2011

L. triplinervis
Table I. Chemical composition (relative % peak area) of the essential oils from Lippia triplinervis (leaves) collected
in April and September 2010.
No.
Constituents
RI lit 5,6
RIcalc
April %
September %
1
2
3
4
5
6
7

8
9
10

11
12
13
14
15

16

17
18
19

20
21
22
23
24
25

26
27

28

29

camphene
myrcene
n.i.
limonene
1,8 cineole
dihydro-tagetone
ipsenone
n.i.
linalool
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
(E)-tagetone
myrcenone
(Z)-tagetone
borneol
terpinen-4-ol
n.i.
a-terpineol
n.i.
n.i.
(Z)-ocimenone
(E)-ocimenone
2-phenylethyl acetate
n.i.
bornyl acetate
(Z)-methyl cinnamate
piperitenone
a-cubene
a-copaene
(E)-methyl cinnamate
b-cubebene
a-gurjunene
(E)-caryophyllene
n.i.
a-humulene
9-epi-(E)-caryophyllene
g-muurolene
(E)-muurola4(14),5diene
n.i.
n.i.
g-cadinene
(E)-calamenene
(E)-cadina1,4-diene
a-cadinene
n.i.
germacrene B
caryophyllene oxide
n.i.
n.i.
guaiol
humulene epoxide II
1,10-di-epi-cubenol
n.i.
epi-a-cadinol
(Z)-calamenen-10-ol
(E)-calamenen-10-ol
n.i.
954
991
-
1029
1031
1053
10836
-
1091
-
-
-
-
-
-
1144
1150
1152
1169
1177
-
1189
-
-
1229
1238
1258
-
1289
1300
1343
1351
1377
1378
1388
1410
1419
-
1455
1466
1480
1494
-
-
1514
1529
1535
1539
-
1561
1583
-
-
1601
1608
1619
-
1640
1661
1669
-
944
992
1020
1031
1034
1055
1088
1093
1100
1102
1115
1123
1132
1138
1141
1148
1156
1157
1168
1178
1182
1191
1211
1218
1233
1242
1259
1273
1285
1306
1342
1350
1374
1386
1388
1406
1416
1447
1451
1458
1478
1488
1491
1500
1511
1521
1530
1535
1541
1553
1579
1584
1591
1598
1604
1611
1617
1639
1657
1665
1675
-
2.6
0.3
0.1
0.1
0.1
11.4
0.1
0.4
0.4
0.2
-
-
-
-
0.2
57.7
0.1
2.1
0.2
0.2
1.4
-
-
1.1
1.3
0.5
-
0.6
t
-
0.2
0.5
0.2
0.3
0.2
0.2
0.6
0.4
0.3
0.1
0.4
0.1
0.1
0.5
4.7
1.1
0.2
0.2
0.8
0.2
0.6
0.3
0.4
0.2
1.1
0.3
0.2
0.2
0.2
0.4
0.2
2.4
0.3
8.0
0.1
0.7
17.4
0.2
0.3
0.4
0.1
0.2
0.1
0.2
0.1
2.0
13.5
19.4
2.0
0.3
0.9
0.2
0.3
1.8
5.6
0.4
0.2
0.8
0.5
3.6
0.7
1.5
0.2
0.4
0.4
0.4
0.4
5.5
0.8
0.2
0.7
0.3
1.5
-
Leito et al.
Table I. Continued
No.
Constituents
RI
RI
April %
September %

n.i.
-
1764

n.i.
-
1795

n.i.
-
1804

n.i.
-
1829

n.i.
-
1908
Number of Identified Compounds
0.9
0.8
0.4
0.5
1.0
55
0.2
0.5
0.4
46
Total Identified Compounds %
92.5
96.3
lit 5,6
calc
n. i. not identified , t trace (< 0.1%).
Table II. Minimum inhibitory concentration (MIC, SD) of the essential oila of Lippia triplinervis against Candida clinical strains.
Sample
C. albicans
C. parapsilosis
C. krusei
C. tropicalis
C. glabata
MIC (mg. mL-1)
L. triplinervis

1,250.0
(721.6878)
1,250.0
(0.0)
312.5
(180.422)
625.0
(0.0)
156.25
(0.0)
Fluconazoleb
0.5
0.5
16.0
0.25
1.0
September, only one assay.
at Aiuruoca, Minas Gerais, Brazil, in a place called Campo

dos Poejos in April and September 2010. Plant material was
identified by Dr. Fatima Salimena, from Universidade Federal
de Juiz de Fora, Juiz de Fora, MG, Brazil, where a voucher
specimen is deposited. The volatile oils from fresh leaves of L.
triplinervis were obtained by hydrodistillation in a Clevenger
type apparatus for 2 h, yielding 1.1% (April) and 1.0% (September) of yellow essential oils, respectively.
Analysis of the essential oils: The oils were analyzed
in an Agilent (Palo Alto, USA) 6890N gas chromatograph
fitted with a 5% phenyl/95% methylsilicone (HP5, 25 m x
0.32 mm x 0.25 mm) fused silica capillary column. The oven
temperature was programmed from 60C to 240C at 3C/min,
and H2 was used as carrier gas (1.4 mL/min). It was injected
1.0 mL of a 1% solution of the oil in dichloromethane, in split
mode (1:100). Injector was kept at 250C and detector (FID)
at 280C. Mass spectra were obtained in an Agilent 5973N
system operating in electronic ionization mode (EI) at 70 eV,
with scan mass range of 40-500 m/z. Sampling rate was 3.15
scan/s. Ion source was kept at 230C, mass analyzer at 150C
and transfer line at 260C. The mass detector was coupled to
an Agilent 6890 gas chromatograph fitted with a low bleeding
5% phenyl/95% methylsilicone (HP-5 MS, 30 m x 0.25 mm x
0.25 mm) fused silica capillary column. Injection procedure and
oven temperature program were the same as above. Helium was
the carrier gas, at 1.0 mL/min. Linear retention indices (LRI)
were measured by injection of a series of n-alkanes (C7-C26)
in the same column and conditions as above for GC analyses.
Identification of the oil components was based on computer
search using the Wiley 6th ed. library of mass spectral data
and by comparison of their calculated linear retention indices
with literature data (5,6).
Antimicrobial assay: Minimum inhibitory concentrations
(MIC) were determined by broth microdilution method according to the document M27-A3 (Candida spp) of the Clinical and
Laboratory Standard Institute (7), using resazurin as indicator
for cell viability (8). All determinations were performed in
triplicate and two independent experiments lead to concordant
results. Positive (medium plus inoculum without essential
oil) and negative (medium without inoculum or essential oil)
growth controls were included in all assays. Fluconazole was
used as reference antibiotic.

Fifty-five compounds were identified in the essential oil
from the leaves of Lippia triplinervis collected in April 2010, of
which the major components were myrcene (2.6%), ipsenone
(11.4%), myrcenone (57.7%), (Z)-ocimenone (1.3%) and (E)calamenene (4.8%) (Table I). The essential oil of this same
plant collected in September 2010, showed a quite similar
chemical composition, with 46 identified compounds (Figure
1). However, in this oil the relative percentage of myrcenone
was drastically reduced from 57.7% to 13.5%, whereas limonene
(8.0%), (E)-tagetone (2.0%) and (Z)-tagetone (19.4%) now
figure as major constituents.
Myrcenone, one the major components of these essential
oils, has been described as an important constituent of some
L. triplinervis
Figure 1. Gas Chromatograms (GC-FID) of the essential oils of Lippia triplinervis collected in April and September 2010.
Leito et al.
other Lippia species such as L. lacunosa (64.2%), L. adoensis (0.8114.89%); L. alba (38% to over 50%), L. multiflora
(54.6%), L. juneliana (17.2%), L. javanica and L. asperifolia
(1). Ipsenone, a rarer monoterpene, has been described as
one of the components of the volatile pheromone from Ips
cembrae, the large larch bark beetle (9). It has been reported
for the first time in the plant kingdom in the essential oil of
Lippia multiflora from Congo (10). Later, it was also found to
be the major component of an ipsenone rich-type (4261%) of
Lippia javanica (Burm. F.) Spreng. indigenous to South Africa
(11), as well as in L. adoensis Hoechst ex Walp. var. adoensis
(12). The presence of high yields of (Z)-tagetone (19.4%) in
the essential oil of L. triplinervis collected in September is
noteworthy, since this substance has been reported to occur
in high amounts (11.3% (Z)-tagetone; 30.2% (E)-tagetone) in
the essential oil of another chemotype of L. multiflora (13).
Apart from the presence of ipsenone, the chemical composition
of these oils closely resembles that of L. lacunosa previously
studied by our group, which has a strong mango-like aroma,
due to the presence of myrcenone (1). In the same way, the
essential oil from L. triplinervis displayed similar sensory
characteristics, which was one of the features that guided the
plant collection in the field.
The antimicrobial activity of the essential oil of L. triplinervis collected in September was evaluated by broth dilution
method and the minimum inhibitory concentration (MIC)
was determined to confirm the antimicrobial activity agaisnt
Candida clinical strains. The MIC data are summarized in
Table II. Candida glabrata was the most susceptible to the
essential oil, followed by C. krusei and C. tropicalis, with MIC
values below 625 mg/mL.
This is the first report of the chemical composition and
antimicrobial activity of L. triplinervis essential oil.
Acknowledgements
The authors thank CNPq (Edital Universal) and FAPERJ for

financial support.
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Y. Plissier, C. Marion, J. Cassadebaig, M. Milhau, D. Kone, G.
Loukou, Y. Nanga and J.M. Bessire, A Chemical, Bacteriological,
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S. pneumoniae
Activity against Streptococcus pneumoniae of the

Essential Oil and d-Cadinene Isolated from Schinus
molle Fruit
Alejandro Prez-Lpez*, Anabel Torres Cirio, Vernica M. Rivas-Galindo,
Ricardo Salazar Aranda and Noem Waksman de Torres
Departamento de Qumica Analtica, Facultad de Medicina, Universidad Autnoma de Nuevo Len, Monterrey, Nuevo
Len, Mxico., P.O. Box 2316 Sucursal Tecnolgico, C.P. 64841 Monterrey, N.L; Mxico
Abstract
Streptococcus pneumoniae is a major cause of respiratory infections. This study aims to test the activity of the
essential oil obtained from the fruit of Schinus molle against S. pneumoniae resistant to conventional antibiotics and
to identify the compounds responsible for the activity. A fraction showing antimicrobial activity (MIC 125 mg/mL) was
obtained. The principal components were identified as: b-myrcene (39.7%), p-cymene (19.5%), d-cadinene (7.8%),
a-phellandrene (7.1%) and limonene (4.1%). Bioassay-guided fractionation led to the identification of d-cadinene
as the principal active constituent (MIC of 31.25 mg/mL). The findings reported here highlight and justify the global
spread of the use of S. molle for the treatment of respiratory infections.
Key Word Index
Schinus molle, essential oil, antimicrobial, d-cadinene, Streptococcus pneumoniae.
Introduction
Streptococcus pneumoniae is the causal agent of infections
in the respiratory tract such as pneumonia and sinusitis; this
bacteria causes meningitis, septicemia, and otitis media in the
general population as well as in hospital patients. Pneumonia
has a high rate of annual mortality worldwide, mainly in children (1). Studies in Latin American countries show that most
serious clinical cases of pneumonia are associated with bacterial infection with predominance of S. pneumoniae followed
by Haemophilus influenzae type b (2,3). Multiple microbial
resistances acquired over time is alarming; many species of
infection-causing bacteria, which once seemed under control,
are now difficult to treat (4-6). Furthermore, the number of
strains of S. pneumoniae resistant to penicillin and other blactamics and to vancomycin is increasing (2, 7).
An alternative approach to fight these antibiotic-resistant
strains of microorganisms is to use antimicrobially active ingredients obtained from plants, in particular from those used
in traditional medicine (8).
In Mxico, as in other developing countries, traditional
medicine is an important source of products for treating common infections. About 25% of the Mexican population depends
exclusively on the use of medicinal plants (9).
It is well known that plants containing essential oils, which
serve to protect and prolong the life of the plant because they
often constitute a means of defense against predators, can
act as insect repellents (10,11). The essential oils are used in
natural therapies and alternative medicine as remedies for
many infectious diseases, and the antimicrobial properties
of essential oils have been long recognized and traditionally
used for respiratory tract infections and as ethnic medicines
for colds. In the medicinal field, inhalation therapy with essential oils has been used to treat acute and chronic bronchitis
and acute sinusitis (12). Several studies have confirmed that
essential oils have highly significant antimicrobial properties
against bacteria, yeast, and fungi (13-15).
Schinus molle is a plant belonging to the Anacardiaceae
family and is native to South America; however, it is found
worldwide (16). In traditional medicine, the plant is used
against coughs, colds, tuberculosis, bronchitis, and fever (9,
17). The antimicrobial efficacy of S. molle and related species
has been previously reported (18-21).
In a previous work, we reported the antimicrobial activity of a hexane extract obtained from S. molle fruit against S.
pneumoniae (9). Following from these antecedents, the present work aimed to determine the chemical composition of the
essential oil by GC/MS analysis, evaluate its activity against S.
pneumoniae, and isolate the main active component.
*Address for correspondence: lualejandro@hotmail.com
Rec: Feb 2011

Acc: July 2011

Prez-Lpez et al.
Experimental
Chemicals: The following chemicals and other materials
were used in the study: hexane, ethyl acetate, and methanol
(Fermont, Productos Qumicos Monterrey, Monterrey, N.L.
Mexico); dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis,
MO, USA); alkane standard solution C8C20 (Fluka, SigmaAldrich, Switzerland); oxacillin and vancomycin (Sigma-Aldrich);
silica gel 60, 0.20.5 mm (Merck, Darmstadt, Germany); thin
layer chromatography (TLC) silica gel 60 aluminum sheets 20
x 20 cm (Merck); agar supplemented with bovine blood (BBL,
Becton Dickinson of Mexico, Estado de Mxico, Mexico); and
cation-adjusted Mueller-Hinton broth supplemented with 5%
lysed horse blood (CAMHB-LHB; BBL, Becton Dickinson,
Sparks, MD, USA).
Bacterial culture: Two isolates of S. pneumoniae resistant
to oxacillin but sensitive to vancomycin (InDRE 24-CCpn-02;
InDRE 49619) were obtained from the Instituto Nacional de
Diagnstico y Referencia Epidemiolgicos (InDRE, Mxico.
D.F., Mexico). The microorganisms were maintained on agar
supplemented with bovine blood (BBL, Becton Dickinson de
Mxico) until use.
Plant material: S. molle fruit was collected in Arteaga,
Coahuila, Mexico, in November 2008. A voucher specimen (UNL
024166) was deposited in the herbarium of the Facultad de
Ciencias Biolgicas, Universidad Autnoma de Nuevo Len.
Isolation of essential oil: The oil was isolated by hydrodistillation of ground fresh fruit (100 g/1 L water) for 4 h, using
a Clevenger-type apparatus. The oil obtained was collected in
two fractions (according to as they distilled), one colorless and
the other yellow, and conserved at -4C until use.
Antimicrobial activity: S. pneumoniae strains resistant to
b-lactamic antimicrobials were tested with microdilution assays
according to the National Committee for Clinical Laboratory
Standards (9, 22). In order to prepare the inocula, S. pneumoniae strains were cultured in Petri dishes containing blood agar
(Bacto, Becton Dickinson). Plates were incubated overnight at
37C 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 MuellerHinton broth supplemented with 5%
lysed horse blood (CAMHB-LHB; BBL, Becton Dickinson)
to make the working suspensions of S. 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
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 mg/mL. Oxacillin and vancomycin
were used as antimicrobial drug controls (644 mg/mL). The
final concentration of microorganisms was 1 x 104 UFC. Plates
were incubated at 37C 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.
GC analysis: GC analysis was carried out on a GC Perkin
Elmer Autosystem XL equipped with a flame ionization detector and a HP-5MS column (30 m x 0.25 mm i.d., 0.25 mm film
thickness). Helium (99.999%) was used as carrier gas at a flow
rate of 0.5 mL/min. Injector and detector temperature were
set at 220C and 290C respectively. Oven temperature was

programmed to 35C for 9 min, then from 35C to 150C at
3C/min and held for 10 min, then at 10C/min to 250C, and
finally at 3C/min to 270C and held for 10 min. The samples
were injected using the splitless mode. The injection volume
was 2 L. Percentage composition was calculated using peak
normalization method assuming equal detector response for
all the compounds.
GC/MS analysis: GC/MS was performed using an Agilent
Technologies 6890N gas chromatograph equipped with an HP5MS column (30 m x 0.25 mm i.d., 0.25 mm film thickness) and
a 5973 INERT selective mass spectrometer. The carrier gas was
He (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 230C, quadrupole temperature was 150C,
and the injector temperature was 220C. Oven temperature
was programmed to 35C for 9 min, then from 35C to 150C
at 3C/min and held for 10 min, then at 10C/min to 250C,
and finally at 3C/min to 270C 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 relative to C8C20 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 (23).
Activity guided fractionation: Yellow oil (8 g) obtained
from the S. molle hydrodistillation was subjected to column
chromatography over silica gel. Elution was started with hexane,
and then with eluents of increasing polarity including EtOAc
and MeOH to give four fractions (F1 to F4). On the basis of
the antimicrobial activity, F1 (1.1 g) was further fractioned
with 97:3 hexane:EtOAc and MeOH to give 14 fractions (F11 to F1-14). According to its antimicrobial activity, fraction
F1-2 was further analyzed by GC/MS. Fraction F2 (0.32 g)
was further subjected to column chromatography over silica
gel, eluted with 97:3 hexane:EtOAc; fractions with similar Rf
were combined to give, finally, seven fractions (F2-1 to F2-7).
F3 (0.22 g) was further purified by preparative TLC on silica
gel eluted with 10:2 hexane:AcOEt to yield three fractions
(F3-1 to F3-3), which were analyzed by GC/MS. Fraction 4
(0.5 g) was subjected to column chromatography over silica
gel, eluted with 97:3 hexane:EtOAc to give 10 fractions (F4-1
to F4-10). Fraction F4-6 was further separated by preparative TLC on silica gel and eluted with 10:3 hexane:EtOAc to
give three fractions (F4-6-1 to F4-6-3). Fraction F4-6-1 was
analyzed by GC/MS. All GC/MS analyses were conducted as
previously described.

Several papers reporting the potential antimicrobial activity
of the essential oil obtained from S. molle have been published;
Gundiza showed the activity of the essential oil obtained from
leaves against Klebsiella pneumoniae, Pseudomonas aeruginosa,
and Escherichia coli, among other bacteria (24). Fuselli et al.
demonstrated the activity of aerial parts (fruits and leaves)
against Paenibacillus larvae larvae, a casual agent of American
foulbrood in honey bees (20). Dikshit et al. found that the aerial
parts of the plant inhibited 100% of the growth of mycelia of
S. pneumoniae
Microsporum gypseum, Trichophyton mentagrophytes, and

Trichophyton rubrum, pathogenic fungi of several animals;
they also demonstrated inhibition (although to a lesser degree) of the storage fungi Alternaria alternata, Aspergillus
flavus, and Penicillium italicum (18). Padin et al. found that
the ethanolic extract obtained from the fruit of S. molle, was
active against Listeria monocytogenes, Staphylococcus aureus,
Bacillus cereus, E. coli, Salmonella enteritidis, P. aeruginosa,
and K. pneumoniae, with MICs between 1 and 5 mg/mL (21).
Belhamel et al. reported that an extract obtained from the
aerial parts was active against S. aureus, S. pyogenes, E coli,
and Vibrio vulnificus (25). Our research group demonstrated
the activity of a hexane extract obtained from the S. molle fruit
against S. pneumoniae, H. influenzae, and Mycobacterium
tuberculosis (9). Considering these findings and taking into
account the importance of S. pneumoniae as a causal agent
of respiratory tract diseases, in the present contribution we
investigated whether the essential oil was responsible for the
reported activity.
During the hydrodistillation process, colorless oil was first
obtained (2.3% w/w) and subsequently yellowish oil could be
distilled (1.0% w/w). The fractions were assayed for antimicrobial activity. The yellow fraction was found to be more active
against both antibiotic resistant S. pneumoniae strains with a
MIC of 125 mg/mL; the colorless fraction showed a MIC of
250 mg/mL (Table I).
In this way, the yellow fraction was further analyzed by GC/
MS. The results demonstrate that this essential oil is very rich
in terpenes and 24 major components were found, of which 22
were identified (97% from the total area). Unidentified compounds are minor components and none of them represents
more than 3% of the total area (Table II). The oil was abundant
in monoterpenes (76.8% from the total area), predominantly
b-myrcene (39.7%), p-cymene (19.5%), a-phellandrene (7.1%),
limonene (4.1%) and a-pinene (3.2%). d-cadinene (7.8%) and
a-cadinol (3.1%) were the main sesquiterpene components.
This composition is consistent with that of the essential oil
obtained from the fruits of S. molle collected in Peru (26) and
Tunisia (27).
Following a bioassay-guided fractionation, column chromatography of the yellow essential oil obtained from S. molle
fruit over silica gel, yielded four fractions (F1 to F4), and all
fractions showed antimicrobial activity against both S. pneumoniae strains; however, F1 showed the best activity, with an
MIC of 31.25 mg/mL (Table I). Purification of F1 yielded
14 fractions (F1-1 to F1-14), with fraction F1-2 being the
most abundant and the most active against S. pneumoniae, as
its MIC was 31.25 mg/mL for both strains. This fraction was
identified through GC/MS as d-cadinene by matching the mass
spectrum with the NIST and Adams libraries and its retention
index was consistent with that reported for d-cadinene in the
literature (23).
F2 yielded seven fractions upon purification (F2-1 to F27). From F3, three fractions were obtained (F3-1 to F3-3);
MIC values for F3-1 to F3-3 against S. pneumoniae were 500
mg/mL for both resistant strains. The principal component
in these fractions was identified as a-cadinene. On the other
hand, F4 yielded 10 fractions (F4-1 to F4-10). Fraction F4-6
was further separated to afford three fractions (F4-6-1 to F4Vol. 23, September/October 2011
Table I. Minimal Inhibitory Concentration (MIC) of essential

oil obtained from Schinus molle fruit and fractions against
Streptococcus pneumoniae strains.
Essential oil from
Schinus molle
oil fruit (fraction)
MIC (mg/mL)
Strain ATCC 49619
Strain 24-ccpn-02
Colorless
Yellow
F1
F2
F3
F4
F1-2 (d-cadinene)
F2-5 to F2-7
(a-cadinene)
F3-1
F3-2
F3-3
F4-6-1 (t-muurolol)
Cephalotin
250
125
31.25
250
62.5
62.5
31.25
250
125
31.25
125
62.5
62.5
31.25
500
Not active
Not active
Not active
Not active
1
500
Not active
Not active
Not active
Not active
4
Oxacillin
Streptococcus pneumoniae
Table II. Composition of the yellow fraction from the essential

oil of the fruit of Schinus molle
Name
Percentage
RIa
RIb
a-pinene
b-myrcene
a-phellandrene
p-cymene
limonene
methyl octanoate
unknown 1 FW 152
verbenyl acetate <trans>
unknown 2 FW152
b-elemene
a-gurjunene
b-caryophyllene
a-humulene
g-muurolene
germacrene D
a-muurolene
g-cadinene
d-cadinene
elemol
gleenol
viridiflorol
ledol
t-muurolol
3.2
39.7
7.1
19.5
4.1
2.3
0.9
1.33
2.0
0.2
1.2
0.8
0.7
0.5
0.7
1.6
1.6
7.8
0.3
0.6
0.4
0.21
0.2
930
992
1002
1023
1028
1126
1200
1291
1317
1390
1408
1422
1452
1475
1480
1499
1511
1520
1543
1589
1591
1601
1636
939
990
1002
1024
1029
1127
1291
1389
1409
1417
1452
1478
1485
1500
1513
1522
1548
1586
1592
1602
1642
a-cadinol
3.1
1650
1652
IR= Retention indices relative to n-alkanes on HP-5MS column.

b
IR= Retention indices from literature
a
6-3). t-Muurolol was identified as the principal component

in fraction F4-6-1; however, no activity against either strain
of S. pneumoniae could be found. The results of the activity found strongly suggest that d-cadinene is the principal
Prez-Lpez et al.
compound responsible for the antimicrobial activity against

S. pneumoniae. The fractions named as F3 and F4 obtained
from the yellow fraction also showed good activity (MICs 62.5
mg/mL); however, this antimicrobial activity was lost during
fractionation (Table I). These findings were also reported by
other authors investigating other plants, which suggests that
the compounds must remain together to be active, perhaps
through a synergic mechanism, or that the presence of minor
components enhances the activity (28).
The antimicrobial activity displayed by S. molle essential oil
against S. pneumoniae supports the traditional use of this plant
for the treatment of infectious diseases. 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.
Acknowledgements
The authors are grateful to the biologists Marco Antonio Guzmn

Lucio and M.C. Mara del Consuelo Gonzlez de la Rosa for definitive
taxonomic identification of species reported here. We thank Ivonne
Carrera for her technical assistance in the extraction procedures and
acknowledge grants 103.5/08/3125 and 103.5/09/4913 from PROMEPMexico and U.A.N.L. (PAICYT) SA 233-09.
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C. microphylla
Chemical Composition of Leaf Essential Oils of

Calyptranthes microphylla B. Holts & M.L., Myrcia
aff fosteri Croat and Eugenia octopleura Krug & Urb
from Panama*
Ana I. Santana Tenorio
Centro de Investigaciones Farmacognosticas de la Flora Panamea (CIFLORPAN),Departamento de Quimica Organica,
Universidad de Panama, Panama, Republicade Panama
Deisy Vargas and Alex Espinosa

Centro de Investigaciones Farmacognsticas de la Flora Panamea (CIFLORPAN), Universidad de Panam, Panama,
Republica de Panama
Albano Daz
Instituto de Investigaciones Cientficas y Servicios de Alta Tecnologa (INDICASAT-AIP), Panama, Republica de Panama
Mahabir P. Gupta**
Centro de Investigaciones Farmacognsticas de la Flora Panamea (CIFLORPAN), Universidad de Panam, Panama,
Republica de Panama
Abstract
The chemical compositions of essential oils obtained by hydrodistilation from leaves of Calyptranthes microphylla
B. Holts & M.L., Myrcia aff fosteri Croat and Eugenia octopleura Krug & Urb, belonging to family Myrtaceae were
studied by GC and GC/MS. Forty-three compounds, representing 93.5% of the total, were identified in C. microphylla,
b-pinene (48.4%) and b-bisabolene (12-0%) being the major components, while the essential oils of M. aff fosteri
contained 38 compounds, a-bisabolol (19.2%) and b-bisabolol oxide (19.2%) being the principal components. From
the essential oil of E. octopleura 40 components, consisting principally of a-pinene (43.0%) and limonene (23.6%)
were identified. The oil from Myrcia aff fosteri showed activity against Staphylococcus aureus and Bacillus subtilis,
which was comparable to chloramphenicol.
Key Word Index
Myrtaceae, Calyptranthes microphylla, Myrcia aff fosteri, Eugenia octopleura, a-pinene, b-bisabolene, b-bisabolol
oxide, a-bisabolol, limonene.
*Presented in XIII National Congress of Science and Technology, Panamanian Association for the Advancement of Science,
City of Knowledge, Panama, 6-9 October 2010.
Introduction
The family Myrtaceae consists of 129 genera and 4620
species distributed throughout the world (1). In Panama, 72
species pertaining to 17 genera have been reported (2). This
family is rich in essential oils. In a previous study on Plinia
cerrocampanensis Barrier, an endemic species of Panama,
an oxygenated sesquiterpene a-bisabolol, a highly valuable
constituent in the pharmaceutical and cosmetic industries,
has been reported in very high yields (3).
In our ongoing research on the aromatic flora of Panama,

we herein report the chemical composition of essential oils
of three species Calyptranthes microphylla B. Holst & M.L.
Kawas, Eugenia octopleura Krug & Urb and Myrcia aff Fosteri
Croat, of which the latter is endemic to Panama. No chemical
data on essential oil compositions of these essential oils has been
published previously. However, the infusion of the leaves from
a related species Calyptranthes bipennis O. Berg has been used
as a stimulant and analgesic (4). Eugenia uniflora, known as
*Address for correspondence: mahabirpgupta@gmail.com
Rec: Jan 2011

Acc: July 2011

Tenorio et al.
Brazilian cherry tree, has been investigated and determined to

be effective in treatment against digestive disorders and commonly used as an antifebrile, ant-irheumatic, anti-inflammatory,
diuretic, to lower blood glucose levels (5, 6), and has been
used in the cosmetic industry. Oil from this species contains a
mixture of atractylone and 3-furanoesdesmene (7). Essential
oil composition of seven species of Eugenia from Costa Rica
has been investigated. E. austin-smithii and E. cartagensis
contain trans-2-hexenal, while a-pinene from E. haberi and
E. zuchowskiae, linalool from E. monteverdensi and 1,8-cineol
and zingiberene from Eugenia sp have been reported (8).
Experimental
Plant material: Leaves from wild plants were collected
from two national parks (Cerro Jefe, Chagres National Park
and National Park of Altos de Campana) with the permission
of the National Environment Authority of Panama. Taxonomical identification of plants was established by Alex Espinosa
Taxonomist CIFLORPAN. The voucher specimens are deposited in the Herbarium the University of Panama (PMA) under
number FLORPAN 8543 for C. microphylla (N:09o13.444;
W:79o22.327; altitude: 863.0 m), FLORPAN 8548 for E.
octopleura (N: 08o40.994; W: 779o55.588; altitude: 845.0
m) and FLORPAN 8544 for M. aff fosteri (N: 09o13.532; W:
79o22.320; attitude: 860.0 m). Leaves from the three species
were hydrodistilled with a Clevenger-type apparatus to obtain
essential oils in yields of 0.55, 0.75 and 0.74 for C. microphylla,
M. aff fosteri (endemic) and E. octopleura, respectively.
Chemicals: Numerous authentic standards were used
to build the homemade MS library and to determine the RI
as described in the experimental section on polar and apolar
column (9). All chemicals were purchased from Fluka - Sigma
Aldrich (Quifar International SA Panama City, Panama).
Analysis of the essential oils: Oils were analyzed by GC
and GC/MS. The quantification of the chemical components
was carried out by using Agilent Technologies, model 6890N Gas
Chromatograph (FID) with HP-5 capillary column with a (5%
phenyl)-methylpolysiloxane as a stationary phase (30 m x 0.32
mm i.d.; 0.25 m film thickness). Carrier gas: high purity N2,
flow rate 1.0 mL/min, temperature 250oC, oven temperature
program from 50oC to 220oC at 4oC/min, for 5 min and then at
6oC/min to 250oC with detector temperature of 280oC. Samples
were injected by splitting and the split ratio was 1:20.
GC/MS analysis was done using an Agilent System model
6890N Gas Chromatograph, a model 5973 mass selective detector (MSD), and an Agilent Chem Station data system. The
GC column was an HP-5 MS fused silica capillary with a (5%
phenyl)-methylpolysiloxane stationary phase (30 m x 0.25 mm
id.. x 0.25 m film thickness). Helium was the carrier gas with
a flow rate of 1.0 mL/min. GC temperature program were the
same as GC-FID. The inlet temperature was 250oC and the
oven temperature program was as follows: 50oC to 220oC at
4oC/min, for 5 min and then at 6oC/min to 250oC with the interphase temperature of 280oC. The split injection mode (1:142),
detector temperature 270oC. 0.1L of the pure essential oil
was injected. MS interface temperature 270C, Ms mode, E.I.
detector voltage 1300 V; mass range 40-400 u, 1 scan/s.
Identification of components was achieved based on their
retention indices, (determined with reference to a homologous series of fatty acids methyl esters) as previously reported
(9). Table I shows the values of RI experimentally obtained
compared with references RI values either calculated within
this research or available in our homemade library built with
authentic standards or with naturally occurring components
identified in previous studies by 13C NMR and analyzed on
SE-30 capillary column. The identification was considered
reliable with a difference between experimental and standard
RI below 5 units. The MS spectra were also compared to the
spectral fragmentation patterns available from literature [Adams
(10) and MS library (NIST database, Adams, Wiley9, Wiley6,
Wiley275) Chen Station Data System (11)].
Determination of antimicrobial activity: Antimicrobial
activity was carried out according to the disc diffusion assay,
Rondon et al. (12).
The bacterial strains used were Staphylococcus aureus,
Bacillus subtilis, Pseudomonas aeroginosa y Klebsiella sp.
The fresh cultures were seeded on TSA media and incubated at 37oC for 24 h. The bacterial inoculum was diluted in
sterile 0.85% saline to obtain a turbidity visually comparable
to a McFarland No. 5 standard (106-8 CFU/mL). The bacterial suspension was inoculated on LB agar plates and later 5
mm filter paper disc impregnated with the known quantity of
chloramphenicol was placed on the plates. This served as a
standard. The disc impregnated with 5 g/mL of essential oil was
used to test the activity. A disc impregnated with 0.85% sterile
saline served as a negative control. The plates were incubated
at 37oC for 24 h and the diameter of the zones of inhibition
was read. Each experiment was done in duplicate.

The qualitative and quantitative analytical results of three
essential oils are shown in Table I.
In the essential oil form leaves E. octopleura, forty components, corresponding to 92.5% of the total, were identified.
These were mainly sesquiterpene hydrocarbons, oxygenated
monoterpenes, and monoterpene hydrocarbons. a-Pinene
(43.1%) was the principal component, followed by limonene
(23.6%), b-ocimene (5.5%), viridiflorol (3.6%), linalool (3.0%),
trans-caryophyllene (1.5%) and a-terpineol (1.5%). Essential
oil from a related species Eugenia speciosa also contained apinene (47.3%) and limonene (23.0%) in similar proportions,
while the oil form E. cuprea was constituted principally by
sesquiterpenes spathulenol, b-caryophyllene and caryophyllene oxide; the oil from E. octopleura contained higher amount
monoterpenes. (13).
The oil from the leaves of an endemic species Myrcia aff
fosteri, had an intense yellow color. In it, 38 compounds corresponding to the 76.7% of total were identified. These were
mainly sesquiterpene hydrocarbons and oxygenated sesquiterpenes, represented by a-bisabolol (19.2%), b-bisabolene
oxide (19.3%), b-bisabolol oxide B (7.0%), caryophyllene oxide
(3.5%), trans-6,11-dimethyl-3,8-oxoethane-bicyclo-(6,3,0)undeca-4,6-diene (3.1%), 1-epicubenol (3.4%), grosonol
(3.2%), trans-6,11-dimethy-3,8-oxomethane-bicyclo-(6,3,0)
undeca-4,6-diene (2.3%), a-calacorene (2.1%), a-cadinene
C. microphylla
Table I.
RIref
COMPONENTS
RIexp

hexanal
2-hexenal
3-hexen-1-ol
cyclohexanol
a-pinene
camphene
b-pinene
6-methyl-5-hepten-2-one
b-myrcene
p-cymene
limonene
1,8-cineol
b-ocimene*
endo-fenchol
p-cymenene
terpinolene
linalol
6-methyl-3,5-heptadien-2-one
acamphonelal
4-acetyl-1-metylcyclohexane
cis-pinocarveol
pinocarvone
endo-borneol
terpinen-4-ol
acetophenone
aterpineol
verbenone
trans-carveol
cis-p-mentha-1(7),8-dien-2-ol
geraniol
carvone
pinocarvyl acetate
bornyl acetate
trans-carvyl acetate
acopaene
bbourbononene
acedrene
agurjunene
cis-a-bergamotene
asantalene
trans-caryophyllene
germacrene D
bcopaene
bgurjunene
trans-a-bergamotene
ahumulene
santalene stereoisomer*
aromadendrene
allo-aromadendrene
gcurcumene
trans-cadine-1-(6)-4-diene
aamorphene
ar-curcumene
trans-b-farnesene
viridiflorene
amuurolene
bbisabolene
bcurcumene
b-amorphene
d-cadinene
d-amorfene
cadine-1,4-diene
C. microph. B. Holst
& M.L. Kawas
M. aff fosteri
Croat
nd
nd
t b
nd
nd
0.1 b
nd
nd
0.3 b
nd
nd
t b
a,b
126
127
48.4
139
138
142
144
4.5 a,b
155
157
0.4 a,b
160
165
201
204
0.1 a,b
211
213
1.1 a,b
t a,b
213
214
0.8 a,b
228
231
b
245

301
299
0.1 a,b
292
293
308
311
309
313
0.2 a,b
a,b
321
322
0.7
323
323
0.3 a,b
330
330
3.7 a,b
339
340
1.6 a,b
340
341
0.3 a,b
345
345
348
349
1.0 a,b
351
353
1.1 a,b
359
357
t a,b
362
362
0.4 a,b
366
365
376
374
0.2 a,b
384
384
t a,b
388
390
t a,b
392
394
404
402
t a,b
431
431
0.4 a,b
0.3 a,b
436
440
0.1 a,b
446
444
t a,b
449
448
451
451
0.6 a,b
454
454
0.2 a,b
a,b
455
453
3.4
458
457
0.1 a,b
a,b
459
460
0.1
461
461
461
463
0.2 a,b
0.8 a,b
471
470
1.0 a,b
473
473
0.7 a,b
464
465
b
474
474
0.3 a,b
474
474
0.1 a,b
481
480
0.3 a,b
482
482
0.6 a,b
0.5 a,b
483
484
3.0 a,b
a,b
485
485
t
0.2 a,b
489
490
491
492
0.3 a,b
0.5 a,b
495
495
12.0 a,b
498
498
0.4 a,b
502
501
503
503
2.0 a,b
a,b
503
504
0.8
507
509
0.5 a,b
E. Octopl.
Krug & Urb
43.0 b
0.2 a,b
0.8 a,b
0.2 a,b
0.8 a,b
0.8 a,b
23.6 a,b
5.1 a,b
0.2 a,b
0.8 a,b
-b
3.0 a,b
0.1 a,b
0.2 a,b
0.4 a,b
1.5 a,b
0.1 a,b
0.2 a,b
0.2 a,b
0.6 a,b
0.2 a,b
0.2 a,b
1.5 a,b
0.2 a,b
0.3 a,b
0.5 a,b
0.4 a,b
0.2 a,b
0.1 a,b
0.4 a,b
0.1 a,b
0.5 a,b
Tenorio et al.
Table I. Continued
RIref
COMPONENTS
RIexp

g-bisabolene
a-cadinene
a-calacorene
cis-a-bisabolene
507
512
516
514
525
528
536
537
537
540
546
549
C. microph. B. Holst
& M.L. Kawas
M. aff fosteri
Croat
E. Octopl.
Krug & Urb
510
1.0 a,b
512
0.2 a,b
517
2.1 a,b
514
t a,b
526
0.5 a,b
528
0.6 a,b
536
0.5 a,b
3.5 a,b
537
1.4 a,b
538
540
546
549
0.1 a,b
nerolidol
-calacorene
ar-tumerol
caryophyllene oxide
viridiflorol
viridiflorol isomer*
t-cadinol
iso-longifolene
trans-6,11-dimethyl-3,
8-oxomethane-bicyclo-(6,3,0)
undeca-4.6-diene
553
554
5.4 a,b
1-epi-cubenol
561
562
3.4 a,b
gossonorol
564
563
3.2 a,b
cedreanol
567
566
1.4 a,b
tmuurolol
565
566
1.0 a,b
acadinol
574
574
1.4 a,b
-bisabolol oxide
577
578
19.2 a,b
bisabolol oxide B
578
578
7.0 a,b
iso-italacene
580
580
cadalene
580
581
1.7 a,b
a-bisabolol
590
592
1.6 a,b
19.2 a,b
melaleucol
594
596
t a,b
cryptomerione
608
609
0.8 a,b
phytol
793
793
0.3 a,b
Total
93.5%
76.7%
Monoterpene Hydrocarbons
54.0%
0.2%
Oxygenated monterpenes
8.8%
2.0%
Sesquterpene hydrocarbons
24.5%
8.2%
5.9%
65.9%
Other
0.3%
0.4%
0.3 a,b
Number of compounds identified
40
43
3.6 a,b
0.5 a,b
0.5 a,b
0.2 a,b
0.8 a,b
0.2 a,b
92.5%
75.1%
6.1%
5.4%
5.9%
0.0%
38
RI: average retention rate calculated experimentally

a
: identified by GC-FID; b: identified by GC/MS; t: trace amount, standard deviation= 0.1%; *correct isomeric form not identified
(2.0%), cadelene (1.7%) and a-cubebene (1.4%).

In the oil from the leaves of C. microphylla 43 compounds
corresponding to 93.5% of the total were identified. It was
constituted principally of a-pinene (48.4%), b-bisabolene
(12.0%), b-pinene (4.5%), trans-caryophyllene (3.4%), cispinocarveol (3.7%), Ar-curcumene (2.9%), a-cadinol (1.8%),
caryophyllene oxide (1.4%), limonene (1.1%); endo-fenchol
and aromadendrene were identified by GC/MS, but in GCFID their amounts could not be quantified. It is noteworthy,
that a- and b-pinene in this oil are lost easily, resulting in an
increase in the amount of bisabolene.
Of the three essential oils tested, only the oil form Myrcia
aff fosteri showed good antimicrobial activity against S. aureus
and B. subtilis. This did not show activity against Gram negative
bacteria P. aeroginosa and Klebsiella sp. Chloramphenicol was
used as a positive control. Candino et al. (14) have also reported
antimicrobial activity in essential oil form Myrcia ovata against
Escherichia faecalis, E. coli, Salmonela cholorasies, Streptococcus pneumoniae and Candida parapsilosis.
Results show that Myrtaceae is an important aromatic

family, and its other species warrant further studies.
Acknowledgements
Thanks are due to the National Secretariat for Science, Technology, and Innovation of Panama (SENACYT), and Organization of
American States for financial support. Thanks are due to Raineldo
Urriola of Smithsoniam Tropical Research Institute for permintting
the use of GC/MS.
References
1.
2.
3.
D.J. Mabberley, The Plant-book. Cambridge University Press,

Cambridge, UK. (1997).
M. Correa, M. Staff, C. Galdames, Catlogo de Plantas Vasculares.
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Technology, 10 (7): 25102514 (2010).
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Bioresource
C. microphylla
4.
J. Sanz-Biset, J. Campos de la Cruz, M.A. Epiquin-Rivera, S.

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(2000).
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essential oil and isolated terpenoids form Eugenia uniflora L. (Brazilian
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Oils of Seven Species of Eugenia from Monteverde, Costa Rica.
Biochemical Systematics and Ecology 35, 877-886 (2009).
9. R. Vila, F. Tomi, M. Mundina, A.I. Santana, P.N. Solis, J.B. Lopez
Arce et al. Unusual composition of the essential oils from the leaves
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10. R.P. Adams, Identification of Essential Oil Components by Gas

11. National Institute of Standards and Technology, PC version of
the NIST/EPA/NIH Mass Spectral Database. U.S. Department of
Commerce, Gaithersburg, MD (1998).
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Rojas, J. Carmona, T. Daz, Chemical composition and antibacterial
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Pascoal et al.
Essential Oil from the Leaves of Campomanesia

guaviroba (DC.) Kiaersk. (Myrtaceae): Chemical
Composition, Antioxidant and Cytotoxic Activity
Aislan C. R. F. Pascoal, Caroline C. Loureno, Ladaslav Sodek and Jorge Y. Tamashiro
Departament of Plant Biology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, 13083-970,
Brazil
Gilberto C. Franchi, Jr. and Alexandre E. Nowill

Onco-Hematological Child Research Center, Faculty of Medical Sciences, State University of Campinas (UNICAMP),
13083-970, Campinas, SP, Brazil
Maria lida A. Stefanello

Department of Chemistry, University of State of Parana, 81530-900, Curitiba, PR, Brazil
Marcos Jos Salvador*

Pharmacy Course, Department of Plant Biology, Institute of Biology, State University of Campinas (UNICAMP),
Campinas, SP, cp6109, 13083-970, Brazil
Abstract
The essential oil from the leaves of Campomanesia guaviroba (DC.) Kiaersk., obtained by hydrodistillation, was
analyzed by GC and GC/MS. Sixteen compounds could be identified, representing around 94% of the total oil. The
major components were myrtenal (27.0%), myrtenol (24.7%) and trans-pinocarveol (15.7%). The essential oil was
also evaluated for DPPH radical-scavenging activity by TLC autographic assay, antioxidant capacity by ORAC-FL
assay and antiproliferative activity against leukemic cells lines.
Key Word Index
Campomanesia guaviroba, Myrtaceae, essential oil composition, myrtenal, myrtenol, trans-pinocarveol, antioxidant, cytotoxic.
Introduction
The genus Campomanesia (Myrtaceae) comprises around
30 species of shrubs or small trees, aromatic, distributed mainly
in tropical and subtropical South America (1). Most of the
species produce edible fruits that are widely used to make
liqueurs, juices and sweets (2). Several species are considered
medicinal and have been used in folk medicine against digestive problems, fever, cough, flu, diabetes, bladder, heart and
liver diseases (3). Campomanesia guaviroba is a tree up to
11 m high, belonging to the family Myrtaceae, distributed in
Minas Gerais to Santa Catarina from the coastal region to the
eastern highlands (4).
The essential oils are used as alternative remedies for the
treatment of many infectious diseases or the preservation of
food from the toxic effects of oxidants (5). The antioxidants
block the free radicals formation in different ways and establish
important control functions in some oxidative stress (6) and in
food conservation (7). Then, new natural antioxidants, mainly

those isolated from medicinal plants, acquire great pharmacological importance and the search for these compounds has been
developed much in the last several years (8, 9). Besides these
facts, essential oils have shown antiproliferative activity and
induction of apoptosis in various tumor cell lines (10-12).
Previous studies have shown that Campomanesia species
produce oils rich in terpenes. The oils of C. aurea (12), C.
guazumifolia (12), C. rhombea (12), C. xanthocarpa (12-15),
C. phaea (16) and C. sessiliflora (14, 17) are characterized by
predominance of sesquiterpenes, along with variable amounts
of monoterpenes. In C. pubescens the oils from fruits and
flowers also are rich in sesquiterpenes (18, 19), while in the
leaf oil monoterpenes are predominant (20). C. adamantium,
the most studied species, exhibited great chemical variability,
with some samples producing mainly sesquiterpenes and
others accumulating monoterpenes (13, 21-24). The present
*Address for correspondence: marcosjs@unicamp.br
Rec: Jan 2011

Acc: June 2011

C. guaviroba
work reports, for the first time, the chemical composition and
biological activity of the essential oil of C. guaviroba.
Table I. Chemical composition (%) of leaf essential oil of

Campomanesia guaviroba
Number Compounda
RIb
RIc
1
trans-pinocarveol
1133
1139
2
pinocarvone
1156
1164
3
myrtenal
1189
1195
4
myrtenol
1192
1195
d-elemene
1266
1335d
5
a-copaene
1285
1374d
6
b-bourbonene
1389
1387
7
b-elemene
1393
1389
8
9
iso-caryophyllene
1412
1408
a-muurolene
1494
1500
10
d-cadinene
1508
1513
11
12
selina-3,7(11)-diene
1547
1545
13
spathulenol
1572
1576
14
caryophyllene oxide
1576
1582
15
allo-aromadendrene oxide 1633
1639
a-cadinol
1651
1654
16

Total identified
Monoterpenes
%
15.7
3.7
27.0
24.7
0.5
0.2
0.6
0.5
0.3
0.5
0.4
0.8
5.4
5.0
7.5
1.2
94.0
71.1
3.7
19.2
Compounds are listed in order of their elution from a DB-5 column; the coefficients
of variation obtained in these analyses were below 5%.
a
Identification based on mass spectra and RI published (25) and computer
matching of the mass spectra with NIST 1998 library (quality level more than 90%);
b
retention index published (5); c retention index experimental on a DB-5 column;
d retention index experimental calculated was not similar to that described in
literature, but the fragmentation confirmed these compounds.
Table II. Concentration of the essential oil of Campomanesia

guaviroba leaves able to inhibit 50% of the proliferative activity
of leukemic cell lines
Cell line
IC50 g/mL oila
IC50 g/mL Vincristinea
K562
HL60
NB 4
RAMOS
RAJI
Jurkat
CEM
MOLT 4
NALM 16
NALM 16
B15
63.14
19.31
22.65
21.21
57.94
19.64
19.63
80.00
20.48
33.28
26.80
0.006
0.008
0.003
1.887
0.042
0.008
0.004
0.014
>100
>100
0.009
RS4
38.09
>100
The coefficients of variation obtained in these analyses were below 5%.a

*Leukemic cell lines: K562, HL60, NB4 human Myeloid Leukemia cell line,
RAMOS, RAJI human lymphoma cell line Burkit, Jurkat, CEM, MOLT4 Lymphoid
human leukemia cell line T, NALM6, NALM16, B15, RS4 B human leukemia cell
line lymphoid.
Experimental
Plant material: Leaves from C. guaviroba were collected
in June 2010 in the state of So Paulo, Brazil, in the city of
Campinas. Specimens were identified by one of the authors
(J.Y.T.) and vouchers were deposited in the Herbarium of the
University of Campinas (Unicamp). Fresh leaves were submitted to hydrodistillation in a Clevenger-type apparatus for 4 h.
At the end of each distillation the oils were collected, dried
with anhydrous Na2SO4, measured, and transferred to glass
flasks that were filled to the top and kept at a temperature of
18C for further analysis.
Analysis of the essential oil: The GC/EIMS (70 eV) analysis
was performed on a Shimadzu GC/MS spectrometer equipped
A Durabond-DB5 capillary column (30 m x 0.32 mm, 0.25 m
film thickness; J&W Scientific) was operated at 60C for 3 min,
and then programmed from 60-220C at 5C/min, after which
it was kept isothermal at 220C for 5 min. The carrier gas was
He (99.99 g%; 1 mL/min) and the injector temperature was
250C. The analyses were performed in split mode, with a split
ratio of 1:20. The essential oil components were identified by
comparison of their retention indices (relative to n-alkanes)
and mass spectra with those found in the literature (25, 26)
and stored on the spectrometer database (NIST 1998). The
results are average of three analyses.
Evaluation of antioxidant properties ORACFL kinetic assay: The antioxidant capacities of the essential oil of C. guavirova
was assessed through the oxygen radical absorbance capacity
(ORAC) assay. The ORAC assay is based upon the inhibition of
the peroxylradical-induced oxidation initiated by decomposition
of a biological relevant peroxyl radical (2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), Aldrich, Milwaukee, WI),
using fluorescein (Aldrich, Milwaukee, WI) as the fluorescent
probe (27). The ORAC assays were carried out on a Synergy
2 (Biotek, Winooski, VT) multidetection microplate reader
system. The temperature of the incubator was set at 37C. The
procedure was carried out according to the method established
by Ou et al. (28) with modifications (29). The data are expressed
as mol of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid, Aldrich, Milwaukee, WI) equivalents (TE)
per gram of oil on a dry basis (mol of TE/g). In these tests,
quercetin, isoquercitrin, and caffeic acid were used as positive
controls. The analyses were performed in triplicate.
TLC autographic assay for DPPH Radical-Scavenging:
Ten microlitres of a 1:250 dilution of the essential oil in methanol were applied to TLC plates (silica gel 60 GF254, Fluka,
AG, Switzerland). The TLC plates were sprayed with a 0.2%
2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, St Louis,
MO) solution in MeOH and left at room temperature. Plates
were observed 30 min after spraying. Active compounds are
observed as yellow spots against a purple background. Relative
radical-scavenging activity was assigned as strong (samples
that produced an intense bright yellow zone), medium
(samples that produced a clear yellow spot), weak (samples
that produce only a weakly visible yellow spot), or not active
(samples that produced no sign of any yellow spot) (30).
Antiproliferative assay: This test aims to evaluate the cell
viability of 12 different types of leukemias and they are: K562,
HL60, NB4 RAMOS, RAJI Burkit, Jurkat, CEM, MOLT4,
Pascoal et al.
NALM6, NALM16, B15, RS4 B. Cellular viability was determined by the MTT reduction assay using a tetrazolium salt (3[4,5 - dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide,
MTT (31). Briefly, the cells were distributed in 96-well plates
(100 L cells/well) and exposed to various concentrations of
essential oil (100, 10, 0.1, 0.01, 0.001, 0.0001 and 0.00001
g/mL) in DMSO (0.1%) at 37C, with 5% of CO2, for 48 h.
The final concentration of DMSO did not affect the cell viability. The selected method for assessing cell viability is the
MTT (Sigma M2128) and the optical density was measured
by spectrophotometry at 570 nm (Bio-Tek Power Wave XS).
The experiments were carried out, at least, in triplicate and
the median inhibitory concentration (IC50) was calculated
in mg/mL as the concentration of the sample that decreased
50% of the viable cells compared with that of the control using OD values of viable cells. Vincristine Sulfate was used as
positive control.

The hydrodistillation of the leaves of C. guaviroba provided
oil slightly yellowish, with pleasant and persistent aroma and
yielding 0.016% (m/m). GC analyses showed a few prominent
peaks (Figure 1). Sixteen compounds could be identified,
representing around 94% of the total oil (Table I). The oil was
dominated by monoterpenes (71.1%), followed by sesquiterpenes. The major components were myrtenal (27.0%), myrtenol
(24.7%) and trans-pinocarveol (15.7%). The sesquiterpenes
alloaromadendrene oxide, spathulenol and caryophyllene
oxide were identified as minor constituents. A similar profile
was previously reported for Campomanesia pubescens (20)
and C. adamantium (23). The oils of these species also were
characterized by predominance of monoterpenes along with
a large number of minor constituents, including myrtenol.
Monoterpenes with pinane skeleton, such as a-pinene and b-
pinene were present in significant amounts. The sesquiterpene

spathulenol was identified in all analyzed Campomanesia oils,
while caryophyllene oxide was a major component in the oils
of C. guazumifolia (12) and C. phaea (16).
The antioxidant activity was evaluated by two methods. In
the ORAC-FL assay, the essential oil result was 511 mol TE/g,
while TLC produced a yellow spot where the essential oil was
applied, due to DPPH reduction. The essential oil was able to
capture the radicals but the antioxidant activity by DPPH assay
was lower in essential oil. Similar results were obtained with
essential oil from leaves of C. adamantium that showed low
activity (9.91% reduction of DPPH with 2270 g/mL) compared
to the flavonoid quercetin (90% reduction of DPPH with 20
g/mL) (24). ORAC-FL and DPPH assays show 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 chainbreaking antioxidant capacity (28, 29). The essential oil of C.
guaviroba is rich in monoterpenes and with or without minor
compounds with donor groups of the electron in ortho position
in relation to phenolic hydroxyl (32, 33).
Cytotoxic activity of essential oil of C. guaviroba was
investigated on a series of leukemia human cells by the MTT
assay. The results are shown in Table II. The cytotoxic effect
on JURKART, HL60, NB4, CEM, RAMOS B15 and cell lines
was significantly stronger than that on the other six cell lines
(K562, RS4, RAJI, MOLT4, NALM6 and NALM16). Of all
leukemia lines, HL 60 was the most vulnerable to the essential
oil with an IC50 of 19.31 g/mL. Cytotoxic activities have been
reported for several essential oils and their components (34).
In particular, myrtenal and caryophyllene oxide showed activ-
Figure 1. GC chromatogram of the essential oil from the leaves of Campomanesia guaviroba showing the major
components: trans-pinocarveol (1), myrtenal (3), and myrtenol (4).
C. guaviroba
ity against human leukaemia cells (HL-60), with IC50 values of

114.85 g/mL and 37.88 g/mL, respectively (35). Therefore,
the presence of these compounds in the essential oil of C.
guaviroba may be responsible, at least in part, for observed
antileukaemic activity.
Acknowledgments
The authors thank FAPESP, CNPq and FAEPEX-UNICAMP for financial support. MJS is grateful to CNPq for research scholarships.
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Prez et al.

Salud Prez G., Miguel Zavala S., Lucina Garca A. and Miguel Ramos L.*
Departamento de Sistemas Biolgicos, Universidad Autnoma Metropolitana-Xochimilco, Calzada del Hueso 1100, Col.
Villa Quietud, Del. Coyoacn, C.P. 04960 Mxico D.F.
Abstract
There are many diseases that are associated with inflammation, such as infections by bacteria, virus and protozoa,
autoimmune diseases such as arthritis and diabetes, Alzheimers disease, and cancer. There are many medications
available to prevent or minimize the progression of the inflammation; they include non-steroidal anti-inflammatory
drugs (NSAIDs) and corticosteroids, but they have some secondary effects. Traditional medicine has been used to
address the health demands of the population and nowadays it presents many opportunities in health care. Essential
oils are use in this medicine to treat many diseases.
In a review of the last five years it was found that several essential oils with anti-inflammatory activity were isolated
from 43 plants. In some cases, oils of the same genus but different species have this activity, such as the essential
oils obtained from three species of genus Origanum, as well as three oils from three species of the Citrus genus, and
three from the Pimpinella genus. In many cases the essential oil composition obtained has been determined, and in
some cases the anti-inflammatory activity of the main compounds of these essential oils has been evaluated, such as
carvacrol and isoeugenol, which showed an important anti-inflammatory activity. On the basis of this review, we can
say that some essential oils could be an important source for the treatment of inflammatory diseases.
Key Word Index
Anti-inflammatory activity, essential oils, inflammatory diseases, review.
Introduction
Inflammation is a physiological response to a variety of
agents including infectious microorganisms, toxic chemical
compounds and physical injury. There are many diseases that
are associated with the inflammation process, such as skin
inflammation (1, 2), autoimmune diseases such as arthritis and
diabetes, Alzheimers disease and cancer.
Many medications are available to prevent or minimize the
progression of inflammation, includuing non-steroidal antiinflammatory drugs (NSAIDs) and corticosteroids. NSAIDs
such as acetyl salicylic acid, ibuprofen, diclofenac and their
new related compounds are mainly selective COX-2 inhibitors;
cyclooxygenase-2 is involved in the inflammation pathway. The
regular use of NSAIDs can cause a number of side effects, some
of which may be very serious. The most common are increases
in the development of ulcers in the stomach and duodenum,
as well as inhibition of uterine motility and hypersensitivity
reaction (3), nausea, vomiting, indigestion, diarrhea, heartburn,
headache, dizziness, rapid weight gain and breathing problems
(4). The lengthy use of corticosteroids could produce the suppression of the function of pituitary-adrenal, hyperglycemia
and increase susceptibility to infections (5).
The biological activities of many plants have been long
known in ethnomedicine to treat inflammatory diseases. These
biological properties are often due to essential oils contained in

plants which are used as herbal remedy in traditional medicine.
It has been found that these essential oils possess different
activities such anti-inflammatory and antiradical properties
(6-8). For this reason, we did a review of the last five years
and in this period we found that several essential oils with antiinflammatory activity were isolated from 43 plants.
Essential oils with anti-inflammatory properties

Afromomum danielli (Hook f.) Schum and A. melegueta Schum (Zingiberaceae): The analysis of the chemical
composition of A. melengueta seed essential oil indicated that
it is rich in sesquiterpenes. The other samples were rich in
monoterpenes like limonene, 1,8-cineole, a- and b-pinenes,
linalool and (E)-b-ocimene as the major components. The
anti-inflammatory activity of A. daniellii seed essential oil was
measured and gave an inhibition concentration 50 (IC50) of 237
ppm against 0.7 ppm for nordihydroguiaretic acid (NDGA).
The results achieved highlight the potential of essential oils to
be developed against inflammatory disorders (9).
Ageratum fastigiatum R. M. King et H. Rob. (Asteraceae):
This plant is used in folk medicine as an anti-inflammatory,
analgesic and antimicrobial. The main compounds found
*Address for correspondence: agromyke@yahoo.com
Rec: Feb 2011

Acc: May 2011

in the essential oil were germacrene D, a-humulene and bcedrene. The oil, with LD50 of 2.50 g/kg, inhibited the acetic
acid-induced writhing at the dose of 200 mg/kg in the formalin
test. In the hot plate test, after 30 min and 60 min of treatment,
doses of 100 and 200 mg/kg increased the reaction time. The
anti-edematogenic effect, reduction on the exudate volume
and leukocyte mobilization were observed at doses of 100
and 200 mg/kg. A. fastigiatum possessed analgesic and antiinflammatory properties (10).
Aucoumea klaineana Pierre (Burseraceae): a-Pinene,
a-phelandrene, p-cymene and 1,8-cineole were the major
components of the essential oil. The anti-inflammatory activity was carried out by lipoxygenase method and the oil was
not active (11).
Canarium scheinfurthii Engl. (Burseaceae): This plant
grows in Cameroon; the main components of the essential oil
obtained by hydrodistillation were p-cymene, limonene and
a-terpineol. The oil had anti-inflammatory activity in lipoxygenase method with an IC50 of 62.6 ppm (11).
Calycorectes sellowianus O. Berg (Myrtaceae): It is
endemic to Brazil. The major constituents of 37 compounds
of the leaves essential oil (GC/MS) were guaiol (13.1%) and
b-caryophyllene (8.6%). The anti-inflammatory activity of this
oil was investigated in vitro and in vivo. It reduced the treated
neutrophils chemotaxis with 91% inhibition and had no effect
on the carrageenan-induced paw edema (12).
Cinnamomum insularimontanum Hayata (Lauraceae):
It has a strong fragrance and has been used as a folk medicine
in Taiwan for a long time. The fruit essential oil was analyzed
by GC/MS; the main constituents were a-pinene (9.45%),
camphene (1.70%), b-pinene (4.30%), limonene (1.76%), citronellal (24.64%), citronellol (16.78%) and citral (35.89%). The
results obtained from nitric oxide (NO) inhibitory activity assay,
essential oil and its dominant compound (citral) presented the
significant NO production inhibitory activity, IC50 of essential
oil and citral were 18.68 and 13.18 g/mL, respectively. Moreover, based on the results obtained from the protein expression
assay, the expression of IKK, iNOS, and nuclear NF-kB was
decreased and I Ba was increased in dose dependent manners. It proved that the anti-inflammatory mechanism of citral
was blocked via the NFjB pathway, but it could not efficiently
suppress the activity on COX-2. In addition, citral exhibited a
potent anti-inflammatory activity on croton oil-induced mice
ear edema, at doses of 0.1 and 0.3 mg per ear. The inhibition
was 22% and 83%, respectively. The results presented that
the fruit essential oil of C. insularimontanum and citral have
anti-inflammatory effect. (13).
Cinnamomum osmophloeum Kaneh (Lauraceae): It is an
endemic tree that grows in natural hardwood forest of Taiwan.
The leaf essential oil components showed inhibitory effects
as anti-bacterial, anti-termite, anti-mites, anti-mildew, antimosquito larvae, and anti-fungal. The chemical constituents
of the essential oil were analyzed by GC/MS and they were
found to be L-bornyl acetate (15.89%), caryophyllene oxide
(12.98%), g-eudesmol (8.03%), b-caryophyllene (6.60%),
T-cadinol (5.49%), -cadinene (4.79%), trans-b-elemenone
(4.25%), cadalene (4.19%), and trans-cinnamaldehyde (4.07%).
The effects of essential oil on oxide NO and prostaglandin E2
production in lipopolysaccharide (LPS)-activated RAW 264.7
macrophages were also examined. Results of nitric oxide tests

indicated that the essential oil and its major constituents such
as trans-cinnamaldehyde, caryophyllene oxide, L-borneol,
L-bornyl acetate, eugenol, b-caryophyllene, E-nerolidol, and
cinnamyl acetate have anti-inflammatory activity (14).
Citrus aurantium L. var bergamia (Rutaceae): The essential oil is extracted from the peel of the fruit, whose main
components are limonene (40%), linalool (8%) and linalyl
acetate (28%) (15). The anti-inflammatory activity of essential
oil of Bergamot (BO) was tested on carrageenan-induced rat
paw edema at different doses: 0.025, 0.05 and 0.1 mL/kg; the
reduction in paw edema was 27.56%, 30.77% and 63.93%
respectively, and indomethacin used as a reference produced
an inhibition of 95.7%. These results showed that BO possesses
anti-inflammatory effect (16).
Citrus sinensis L. (Rutaceae): Orange essential oil can
be attributed to its properties like anti-inflammatory, antidepressant, anti-spasmodic, antiseptic, aphrodisiac, carminative,
diuretic, tonic, sedative and cholagogue. The anti-inflammatory
activity of the oil was tested using the lipoxygenase enzymatic
method; the IC50 was 20.3 mg/L (17).
Citrus sunki (Hayata) Tanaka (Rutaceae): C. sunki is used
in traditional medicine for digestion, cold, and fever. The analysis
of the essential oil of this plant by GC/MS showed that the
major components were dl-limonene (68.18%) and b-myrcene
(4.36%). The oil reduced the LPS-induced secretion of NO in
RAW 264.7 cells. This result suggests that the essential oil has
anti-inflammatory activity (18).
Cleistocalyx operculatus Roxb. (Myrtaceae): In folk
medicine in China, Vietnam and some other tropical countries,
it is widely used for the treatment of gastric ailments and as
an antiseptic agent. The anti-inflammatory activity of the essential oil of C. operculatus buds inhibited lipopolysaccharide
induced secretion of pro-inflammatory cytokines, including
tumor necrosis factor-a (TNF-a) and interleukin-1 b (IL-1b)
in RAW 264.7 cells, a mouse macrophage-like cell line. Also
the mRNA expression of TNF-a and IL-1b was suppressed.
Moreover, reporter gene analysis revealed that the oil blocked
LPS-induced transcriptional activation of NF-kB in RAW
264.7 cells. Besides, the essential oil inhibited the ear edema
induced by TPA (19).
Cordia verbenacea DC (Boraginaceae): This plant is a
medicinal plant popularly used in Brazil as anti-inflammatory,
antiulcer and anti-rheumatic agent without detailed pharmacological and toxicological studies (20). a-Humulene and transcaryophyllene were identified in C. verbenacea essential oil
and the anti-inflammatory activity of the both compounds was
evaluated in a model of acute inflammation in rat paw, induced
by LPS. The treatment with a-humulene or trans-caryophyllene
inhibited the LPS-induced NF-kB activation and neutrophil
migration; however, only a-humulene prevented the production
of cytokines TNF-a and IL-1b and the in vivo up-regulation of
kinin B1 receptors, so that both sesquiterpenes might be used
as agents to treat inflammatory diseases (21).
Cyperus esculentus L. and C. rotundus Linn. (Cyperaceae): Anti-inflammatory, anti-arthritic, analgesic and anticonvulsant activities of the oils of both plants were study.
Phytochemical tests of the oil are positive for flavonoids, triterpenoids, carbohydrates and proteins. The effects of the oils
Prez et al.
were evaluated in the models of carrageenan-induced edema,

formaldehyde induced arthritis, formalin induced writhing
and MES induced convulsion. It was found that both essential
oils posses good anti-inflammatory, anti-arthitic, analgesic and
anticonvulsant activities (22).
Chenopodium album L. (Chenopodiaceae): This plant
is commonly known as pigweed and in folk medicine is used
as laxative, antihelmitic, against round and hook worms, and
as a blood purifier. Also it is used for the treatment of hepatic
disorders, spleen enlargement, intestinal ulcers and burns (23).
The analysis of the essential oil of C. album leaves revealed
that the main constituents were p-cymene (40.9%), ascaridole
(15.5%), pinane-2-ol (9.9%), a-pinene (7.0%), b-pinene (6.2%)
and a-terpineol. The oil had strong anti-inflammatory activity
against TPA-induced ear edema in mice (24).
Dennettia tripetala G. Baker (Annonaceae): The fruit,
bark, leaves and roots are used as spices and condiments. The
leaves are used in combination with other medicinal plants to
treat fever, typhoid, cough, worm infestation, vomiting and
stomach upset (25). Atinociceptive and anti-inflammatory
activity of the essential oil were tested in mice using the hot
plate, acetic acid-induced writhings and formalin tests, while
carrageenan-induced paw edema as anti-inflammatory model.
The anti-inflammatory activity of the oil was comparable to
dexamethasone (1 mg/kg) (26).
Drimys brasiliensis Miers (Winteraceae): This species has
been used in folk medicine as analgesic and anti-inflammatory.
The essential oils from leaves and stem barks were characterized by GC-FID and GC/MS. The main components were
monoterpenes (leaves 4.31% and stem barks 90.02%) and
sesquiterpenes (leaves 52.31% and stem barks 6.35%). The
evaluation of antinociceptive and anti-inflammatory potential
of the essential oils and the sesquiterpene polygodial were
evaluated in paw edema induced by carrageenan and formalin
test in mice. The essential oil obtained from the stem barks
significantly reduced the edema induced by carrageenan. The
anti-inflammatory effect of stem barks oil (at 200 mg kg-1) was
similar to that observed with indomethacin (at 10 mg kg-1) 30
and 60 min after the administration of essential oils. The effect
of polygodial (at 200 mg kg-1) was lower than the oils (27).
Fortunella japonica (Thunb.) Swingle (Rutaceae): F.
japonica is also known as round kumquat or Marumi kumquat.
The fruit is rich in vitamins A and C. The main components
of the essential oil are dl-limonene (61.58%) and carvone
(6.36%). The oil significantly reduced LPS-induced NO release in RAW 264.7 cells. This fact indicates that this oil has
anti-inflammatory effect (18).
Hedychium coronarium Koen. (Zingiberaceae): It is
commonly known as butterfly ginger, cinnamon jasmine,
gargland flower and ginger lily. The rhizome has been used for
the treatment of headache, diabetes, contusion inflammation
and sharp pain due to rheumatism. Twenty-nine components
were identified in the flowers essential oil and the main components were b-trans-ocimenone (28.05%), linalool (18.52%),
1,8-cineole (11.35%), a-terpineol (7.11%), 10-epi-g-eudesmol
(6.06%), sabinene (4.59%) and terpinen-4-ol (3.17%). At doses
of 100 mg/kg p.o. the oil produced significant inhibition of
carrageenan-induced hind paw edema in rats (28).
Illicium anisatum Hayata (Illiciaceae): It is widely used
for treatment of some skin problems in traditional Chinese

medicine. The fruit is an important source of essential and
volatile oil. Fifty-two components were identified in the essential oil and the main components were eucalyptol (21.8%),
sabinene (5.3%), a-terpinenyl acetate (4.9%), kaurene (4.5%),
isopimaradiene (3.2%), safrol (2.7%), b-linalool (2.6%), gcadinene (2.2%), a-cadinol (2.2%) and terpinen-4-ol (1.9%).
The mechanism of the anti-inflammatory activities of I. anisatum
essential oil (IAE) was evaluated whether it could modulate the
production of nitric oxide (NO) and prostaglandin E2 (PGE2)
by activated macrophages. The results indicate that IAE is an
effective inhibitor of LPS-induced NO and PGE2 production
in RAW 264.7 cells. These inhibitory activities were accompanied by dose-dependent decreases in the expression of iNOS
and COX-2 proteins and iNOS and COX-2 mRNA. Also was
evaluated the cytotoxic effects of the oil, It was found that IAE
exhibited low cytotoxicity at 100 mg mL1 (29).
Lippia sidoides Cham. (Verbenaceae): It is mainly used
as an antiseptic (30). It was found that the topical application
of leaf essential oil at doses of 1 and 10 mg/ear, respectively,
reduced 45.93% and 32.26% the acute ear edema induced by
12-O-tetradecanoylforbol 13-acetate (TPA).
Melaleuca alternifolia Maiden et Betche (Myrtaceae): It
has well established traditional and folk uses in Australia, specially
as an antiseptic. The major constituent from the essential oil
was terpinen-4-ol. This compound is considered, together with
a-terpinene, g-terpinene, and a-terpineol, the main responsible
for the anti-inflammatory activity from this essential oil. The
oil showed anti-inflammatory activity on edema-induced by
histamine in mice. Several clinical studies and observations,
endorse the clinical external use of the oil for the treatment
of vulvovaginitis, mainly candidiasic cases (31).
Mezoneuron benthamianum Baill. (Caesalpinoideae):
This plant is used for the treatment of dermal infection,
healing of refractory sores, blood disorders, as a laxative, for
stomach troubles, eye treatments, genital stimulants/depressants, hemorrhoids, pain-killers, pulmonary troubles and as a
chewing stick. The oil contained 15 compounds and the main
components were 3-carene, pinene (11.8%), trans-nerolidol
(13.5%), farnesene (11.6%) and thujene (6.7%). The essential oil
was tested at different concentrations for its anti-inflammatory
activity evaluated as inhibition of TPA induced ear edema in
mice. The oil at 5.0 mg and 2.5 mg dose levels exhibited a
significant anti-inflammatory activity with percentage edema
reduction of 92.3% and 76.9%, respectively (32).
Myrciaria tenella (DC.) Berg. (Myrtaceae): It is known as
Cambu. The GC/MS analysis revealed that the main constituents of the leaf essential oil were b-caryophyllene (25.1%) and
spathulenol (9.7%). The oil reduced significantly the treated
neutrophil chemotaxis with 93% inhibition, and in the systemic
treatment at doses of 50 mg/kg p.o. reduced the carrageenaninduced paw edema with a similar effect for indomethacin (10
mg/kg), the positive control (33).
Nepeta cataria L. var. citriodora (Becker) (Lamiaceae):
It is used as anti-tussive, expectorant and antiathmatic (34).
The essential oil was analyzed by gas chromatography-flame
ionization detector (GC-FID), four major components were
identified trans,trans-nepetalactone, cis,trans-nepetalactone,
trans,cis-nepetalactone and nepetalactol. At doses of 0.0005
mL/kg the oil presented peripheric anti-inflammatory properties by reducing the induced edema after carrageenan injection (35).
Ocotea quixos Lam. (Lauraceae): The main components of
the essential oil were trans-cinnamaldehyde (27.9%) and methyl
cinnamate (21.6%) (36). The anti-inflammatory activity of the
essential oil and these two compounds were investigated in in
vitro and in vivo models. The oil and trans-cinnamaldehyde,
but not methyl cinnamate, significantly reduced LPS-induced
NO release from J774 macrophages, inhibited LPS-induced
COX-2 expression, and increased forskolin-induced cAMP
production. At doses of 30-100 mg/kg of essential oil and 10
mg/kg of trans-cinnamaldehyde showed anti-inflammatory
activity against paw edema in rats carrageenan-induced without
damaging gastric mucosa (37).
Olea europea L. (Oleaceae): In Tunisian folk medicine
this plant is used in the treatment of inflammatory diseases and
bacterial infections. The analysis of the essential oil resulted
in the identification of 32 compounds and the major compounds were a-pinene (52.7%), 2,6-dimethyloctane (16.57%)
and 2-methoxy-3-isopropylpyrazine (6.01%). Intraperitoneal
administration of O. europea essential oil at doses of 100,
200 and 300 mg/kg reduced acetic acid-induced abdominal
constrictions and paw edema (38).
Origanum ehrenbergii Boiss and O. syriacum L. (Lamiaceae): In the essential oil of O. ehrenbergii was found 37
components of which thymol (19%) and p-cymene (16.1%) were
the main abundant compounds. Thirty-six components were
found in the O. syriacum essential oil and the main compounds
were thymol (24%) and carvacrol (17.6%). O. ehrenbergii oil
inhibited NO production in the murine monocytic macrophage
cell line RAW 264.7 with an IC50 value of 66.4 g/mL (39).
Origanum vulgare L. (Labiatae): It is an aromatic plant of
the Mediterranean flora that has been commonly used to treat
diarrhea and pain. Identified in the essential oil were transsabinene hydrate, thymol and carvacrol. THP-1 macrophages
were used as cellular model of atherogenesis and the release/
secretion of cytokines (TNT-a, IL-1b, IL-6 and IL-10) and their
respective mRNA expressions were quantified both in presence or absence of supercritical oregano extracts. The results
showed a decrease in pro-inflammatory TNF-a, IL-1b and IL-6
cytokines synthesis, as well as an increase in the production
of anti-inflammatory cytokine IL-10. These results may suggest an anti-inflammatory effect of oregano extracts and their
compounds in a cellular model of atherosclerosis (40).
Pelargonium graveolens LHr (Geraniaceae): This plant
is commonly known as geranium. For many years in traditional
medicine it has been used as an anti-asthmatic, anti-allergic,
antioxidant, anti-diarrheic, antihepatotoxic, diuretic, tonic,
haemostatic, stomachic and anti-diabetic (41). The main components of the essential oil were citronellol (26%), citronellyl
formate (16%), linalool (10%), geraniol (8%), isomenthone (6%)
and menthone (4%). It was found that this essential oil could
inhibit the LPS-elicited expression of the induced proinflammatory enzymes COX-2 and iNOS, as well as the NO produced
by LPS-activated microglial cells. This inhibition did not result
from a cytotoxic effect of the oil. Although high concentrations
of citronellol could inhibit NO production from the cells, when
administered at their natural relative concentrations in the oil,
neither citronellol nor the other constituents of the oil were

effective at inhibiting NO production (42).
Pimpinella corymbosa Boiss, P. tragium Vill. and P.
rhodanta Bois (Apiaceae): Pimpinella species have been
used as animal feed to increase milk secretion (43), also the
estrogenic activity of some isolated compounds and essential
oils of different Pimpinella species were reported. The oils of
these three species were effective in inhibiting NF-kB mediated transcription. The roots showed notably potent activities
with IC50 values of 2, 3 and 6 g/mL, respectively (44).
Rosmarinus officinalis L. (Labiatae): It is known as a
common herb and household plant broadly used all around the
world for different medicinal purposes, being a component of
various established anti-inflammatory plant drug preparations,
and having a long tradition of use for treating headaches, colds
and colic, as well as other diseases (45). The effect of R. officinalis essential oil dietary administration at concentrations
of 1250, 2500 and 5000 ppm in carrageenan paw edema and
trinitrobenzene sulfonic acid (TNBS) colitis was studied (46).
Dietary supplementation with 5000 ppm of the oil initially
increased after 2 h, but after 24 h suppressed the extent of
paw edema, and in the TNBS model exhibited protective effects on colonic mucosa and decreased macroscopic scores for
colonic inflammation.
Sabina virginiana L. Antoine (Cupressaceae): It is commonly known as eastern west cedar and has been used in the
treatment of psoriasis, dermatitis, hemorrhoids and varicose
veins. The leaves are found to exert effects on emmenagogue,
as a stimulant, and as a diaphoretic in rheumatism (47). The
leaves essential oil was analyzed by GC/MS; 31 compounds
were identified, and the major constituents were limonene
(32.9%), safrole (23.0%), asarone (15.9%) and a-pinene (5.2%).
The essential oil was tested at different concentrations (0.075,
1.25, 2.5 and 5.0 mg/ear) for its anti-inflammatory assay evaluated as inhibition of TPA induced ear edema in mice. At doses
of 5.0 mg/ear the inhibition was 66.7%. This effect was similar
to that obtained with indomethacin (57.7%) (48).
Thymus vulgaris L. (Labiatae): Thyme has been used
for respiratory ailments for its infection-fighting and cough
suppressive qualities. Thyme tea is an old time favorite cough
and cold remedy. The essential oils of thyme are grouped into
three main types: thyme oil, which contains 4260% phenols
and is mainly thymol; origanum oil, which contains 6374%
phenols and is mainly carvacrol; and lemon thyme oil, which
contains citral. The dietary addition of thyme essential oils to
the diet at 3 concentrations (5000, 2500 and 1250 ppm) and
fed to Balb/c mice. The extent of ear swelling in DTH/CHS
reaction, paw edema induced by carrageenan administration and
TNBS-induced colitis were evaluated. Dietary supplementation
with 5000 ppm of oil decreased paw edema and ear swelling
and the microscopic and macroscopic scores of colitis (49).
Zanthoxylum piperitum AP DC (Rutaceae): The major
constituents of the essential oil were limonene and geranyl
acetate. The oil decreased approximately 38% of nitrite
production, as compared to LPS-induced nitrite production.
However, the essential oil and its components did not suppress
NO chemically in a cell-free system and inhibited iNOS mRNA
transcription. The inhibition of E-selectin gene transcription
by the oil caused the suppression of cellular adhesion. These
Prez et al.
results suggest that the essential oil of this plant might have
anti-immunological anti-inflammatory activity (50).
Zanthoxylum schnifolium Sieb. et Zucc.(Rutaceae): This
plant has been used in traditional medicine for treatment of
the common cold, stomachache and diarrhea (51, 52). The
chemical composition of the essential oil was found by GC/MS
and 55 compounds were detected. The main constituents were
b-phellandrene (22.54%), citronellal (16.48%), and geranyl
acetate (11.39%). The oil and its constituents (b-phellandrene,
citronellal and geranyl acetate) significantly suppressed gene
transcription of iNOS, the COX-2 gene, and biosynthesis of
IL-1b by LPS-stimulated macrophage cells. This result suggests
that the essential oil may be useful to relief and retardation of
immunological inflammatory responses (53).
Zingiber officinale Roscoe (Zingiberaceae): This plant
is commonly known as ginger. It is used in folk medicine
to treat pain, inflammation, arthritis, urinary infections, and
gastrointestinal disorders. The ginger essential oil at doses of
50, 100 and 200 mg/kg, p.o. significantly suppressed the acetic
acid-induced writhing response in a dose-dependent manner.
Maximum inhibition of the oil was observed at 200 mg/kg.
GEO was found to contain monoterpenes and sesquiterpenes
as principal compounds, suggesting that the anti-inflammatory
and analgesic effects could be correlated to these essential oil
constituents (54).
Zingiber zerumbet (L) Sm. (Zingiberaceae): It is locally
known as lempoyang or wild ginger. In traditional medicine
is used to cure swelling and loss of appetite. The juice of the
boiled rhizomes has also been used as a medicine for worm
and ascaris in children (55). The rhizomes essential oil was
evaluated in acute and chronic inflammatory models, using
carrageenan-induced paw edema and cotton pellet-induced
granuloma, respectively; non-inflammatory-mediated pain was
also assessed using a formalin test. The oil exhibited significant
anti-inflammatory activity both in acute and chronic inflammation, and also had anti-nociceptive activity (56).
Zizyphus jujube Miller (Rhamanaceae): In traditional
medicine it is used in the treatment of diabetes and anti-fertility
(57, 58), diarrhea and insomnia. The anti-inflammatory activity
of the essential oil obtained from the seeds of Z .jujube was
evaluated on ear edema induced with TPA in mice. The treatment with 1% and 10% of the essential oil caused significant
decreased in ear thicknesses. Furthermore, histological analysis
confirmed that this oil inhibited the inflammatory responses
of skin inflammation in mice (59).
Discussion
Inflammatory diseases are generally treated with steroidal
and non-steroidal anti-inflammatory drugs (60). However, both
of them have significant negative side effects, reducing their
use in certain segments of the population (61). Hence, there
is a need to develop new drugs with novel modes of action
and fewer side effects. The use of herbal therapy or alternative medicine constituents is an attractive approach for the
treatment of several inflammatory disorders (62). Essential
oils are plant secondary metabolites that are used extensively
in aromatherapy and various traditional medicinal systems and
many of these oils possesses different pharmacological proper42/Journal of Essential Oil Research
ties, one of which is the anti-inflammatory effects on several

different models of inflammation as shown in this review. A
large number of the essential oils contain various bioactive
compounds, some of which have potent anti-inflammatory
effect including carvacrol, limonene, citronellal, and cinnamaldehyde, among others.
The results presented in this report suggest the applications of essential oils or their components as anti-inflammatory
agents and might accelerate the development of new drugs for
different inflammatory diseases.
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O. majorana
Chemical Composition and Antibacterial Activity

of Origanum majorana L. Essential Oil from the
Venezuelan Andes
Sulymar Ramos1, Luis B. Rojas1, Maria Eugenia Lucena2,
Gina Meccia1* and Alfredo Usubillaga1
Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mrida, Venezuela
Bioanalysis Clinic Department, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mrida, Venezuela
1
Abstract
Origanum majorana L. (Lamiaceae) is a plant that is used in gastronomy and natural medicine. The plant material used in this study was collected at San Isidro de Apartaderos, Mrida State. A yield of 0.6% of essential oil was
obtained by hydro-distillation using a Clevenger trap. The main constituents found were: cis-sabinene hydrate (30.2%),
terpinen-4-ol (28.8%), g-terpinene (7.2%), a-terpineol (6.9%), trans-sabinene hydrate (4.4%), linalyl acetate (3.8%),
and a-terpinene (3.6%). The essential oil was also fractionated over a silica gel dry column. Two main fractions were
isolated, the first containing 67.6% of cis-sabinene hydrate and the second 72.8% of terpinen-4-ol. The antibacterial
activity of the oil was determined using the agar diffusion method and it was found that it was active against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae. Antibacterial activity of the fractions
obtained by dry column chromatography was also tested. It was found that the fraction rich in cis-sabinene hydrate
was more active than the one rich in terpinen-4-ol. It was concluded that in the essential oil of Origanum majorana,
cis-sabinene hydrate is the more important compound responsible for inhibition of bacterial growth.
Key Word Index
Origanum majorana L., Lamiaceae, essential oil composition, antibacterial activity, cis-sabinene hydrate, terpinen4-ol.
Introduction
The Lamiaceae family has about 3,000 species, most of
them aromatic plants. They are distributed around the world,
but they are especially abundant in the Mediterranean region
and Eastern Asiatic countries. In Venezuela there are around
90 species that belong to 19 genuses (1). The genus Origanum
has about 20 species which are aromatic herbs. Origanum
majorana L., popularly known as sweet marjoram, is an aromatic herbaceous plant with small leaves and white or pink
flowers native to the Middle East. It is used to treat digestive
disorders, as well as appetizer, carminative, aphrodisiac, diaphoretic hypotensor, expectorant, and sudorific (1-2). Its active
constituents are used in the manufacture of anti-rheumatic
ointments. In the food industry it is used to season meats and
sausages (1). Its essential oil and alcoholic extracts are used in
pharmaceuticals, perfumes and cosmetics (3).
The essential oil obtained by steam distillation contains
mainly terpinen-4-ol, which along with cis-sabinene hydrate is

responsible for the characteristic flavor and fragrance of marjoram oil. In addition to these compounds, a- and g-terpinene
and terpinolene are the other major components (4-14); only
the oil from Turkey (15) contains carvacrol as a main constituent. Antioxidant activity of marjoram essential oil (4, 14) has
been reported. Its volatile oil possesses antimicrobial properties
against foodborne bacteria and mycotoxigenic fungi (4, 6, 8,
16). Previous studies from Brazil, Hungary and Tunisia (1719) reported antibacterial activity against Gram-positive and
Gram-negative microorganisms. In Venezuela, activity against
Escherichia coli, Staphylococcus aureus y Pseudomonas sp.
(20) has been reported.
In the present report, the essential oil of O. majorana,
collected at San Isidro de Apartaderos, was analyzed and its
antibacterial activity was determined. The oil was fractionated
in an attempt to identify the components that were responsible
for such antibacterial activity.
*Address for correspondence: gmeccia@ula.ve
Rec: Feb 2011

Acc: June 2011

Ramos et al.
Experimental
Plant collection and oil extraction: Origanum majorana
was collected at San Isidro de Apartaderos, a town located
3,340 meters above sea level at Mrida State, Venezuela. It
was identified by Ing. Juan Carmona, and voucher specimen
No. LR062 was deposited at the Merf Herbarium. Fresh leaves
(500 g) were hydrodistilled for 3 h to obtain 3.1 mL of oil which
was stored at 4C in the dark.
Essential oil fractionation: The oil (2 g) was treated
over a silica dry column. A plastic 1.0 inch wide column was
filled with silica gel (Merck, 230-400 mesh). Hexane was added
until the solvent reached the lower part of the column which
was then cut lengthwise in five equal parts. The pieces were
labeled from the top to the bottom (A-E). The silica of each
portion was extracted twice with 10 mL of diethyl ether and
filtered. Each portion was then treated for 12 h with 20 mL of
ethyl acetate. All fractions were then concentrated to a volume
of 1.0 mL and stored for later analysis.
Analysis of the essential oil: GC-FID analysis was performed on a Perkin-Elmer Auto System gas chromatograph
equipped with a 5% phenylmethylpolysiloxane fused-silica
capillary column (AT-5, Alltech Associates Inc., Deerfield,
IL, 60 m x 0.25 mm, film thickness 0.25 mm). The initial oven
temperature was 60C, it was then heated to 260C at 4C/
min, and the final temperature kept for 20 min. The column
injector and detector temperatures were 200C and 250C,
respectively, and the carrier gas was He at 1.0 mL/min. A 1.0
mL sample was injected using a split ratio of 1:10. Retention
indices were calculated relative to C8-C24 n-alkanes, and com-
pared with values reported in the literature (21).

The GC/MS analysis was done on a Hewlett Packard GCMS system, Model 5973, fitted with a 30 m long, cross-linked
5% phenylmethylpolisiloxane (HP-5MS, Hewlett Packard, USA)
fused-silica column (0.25 mm, film thickness 0.25 mm). The
oven temperature conditions were the same used for GC-FID
analysis. Source temperature 230C; quadrupole temperature,
150C; carrier gas He adjusted to a linear velocity of 34 cm/s;
ionization energy, 70 eV; scan range, 40-500 amu; 3.9 scans/s.
The injected volume was 1.0 mL of 2% solution of oil in nheptane. A Hewlett-Packard ALS injector was used with split
ratio 1:100. The identification of the oil components was based
on a Wiley MS Data Library (sixth ed.), followed by comparison
of MS data with published literature (21, 22).
Antibacterial activity: The essential oil and its fractions
were assayed for their antibacterial activity. The microorganisms
used were Staphylococcus aureus (ATCC 25923), Enterococcus
faecalis (ATCC 19433), Escherichia coli (ATCC 25992) and
Klebsiella pneumoniae (ATCC 23357).
The antibacterial activity was carried out according to the
disc diffusion assay described by Rondn et al. (23). The strains
were maintained in agar at room temperature. 0.5 mL of every
bacteria inoculum was diluted in sterile 0.85% saline solution
to obtain a turbidity visually comparable of a McFarland N
0.5 standard (106-8 CFU/mL). Every inoculum was spread over
plates containing Mueller-Hinton agar and a paper filter disc
(4 mm) saturated with both 10 L of essential oil and the two
fractions. The plates were left for 30 min at room temperature
and then incubated at 37C for 24 h. The inhibitory zone around
Table I. Percentage composition of the essential oil of Origanum majorana L.

Peak
Constituents
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Butyl acetate
a-Thujene
Sabinene
Myrcene
a-Terpinene
p-Cymene
b-Phellandrene
g-Terpinene
trans-Sabinene hydrate
a-Terpinolene
cis-Sabinene hydrate
cis-para-Menth-2-en-1-ol
trans-para-Menth-2-en-1-ol
endo-Borneol
Terpinen-4-ol
a-Terpineol
cis-Piperitol
trans-Piperitol
Geraniol
Linalool acetate
Eugenol
Neryl acetate
Geranyl acetate
trans-Caryophyllene
Bicyclogermacrene
% Oil
Fr. A
Fr. C
t
1.2
0.2
1.4
0.5
3.6
2.4
1.5
7.2
4.4
5.1
3.2
2.0
30.2
67.6
9.9
2.0
2.1
1.7
1.1
2.0
0.4
28.8
9.5
72.8
6.9
11.4
2.6
0.5
0.5
t
1.1
3.9
8.2
0.3
0.3
0.6
1.6
0.6
1.0
RI
RIlit
805
928
974
989
1017
1025
1030
1060
1069
1089
1100
1125
1144
1172
1185
1196
1200
1213
1260
1262
1362
1369
1387
1427
1508
812
931
976
991
1018
1026
1031
1062
1065
1088
1098
1121
1140
1165
1177
1189
1193
1205
1255
1257
1356
1365
1383
1418
1494
RI: Retention Indices were determined by GC on a HP-5 column.

t: traces (< de 0,1 %)
O. majorana
the disc was measured and expressed in mm. A positive control

was also assayed to check the sensitivity of the tested organisms using the following antibiotics: Ampicillin, Piperaciline
and Amikacine. 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 twice.

Origanum majorana leaves produced 3.1 mL of oil which
was equivalent to 0.6% yield. It was possible to identify 25
components. Table I shows the percentage composition of
the essential oil. The major constituents of the oil were: cissabinene hydrate (30.2%), terpinen-4-ol (28.8%), g-terpinene
(7.2%), a-terpineol (6.9%), trans-sabinene hydrate (4.4%),
linalool acetate (3.8%) and a-terpinene (3.6%). The essential
oil from Germany was reported to contain cis-sabinene hydrate,
linalool, sabinene and b-caryophyllene as main constituents.
French and Italian studies reported similar results (5), but
the oil from Turkey (15) was reported to have a completely
different composition, because O. majorana from Turkey
contained 78% carvacrol. On the other hand, essential oils
from Cuba (12), Brazil (17), Hungary (18), and Tunisia (19)
were reported to have terpinen-4-ol, g-terpinene and linalool
as main components.
Figure 1. Chromatogram of Marjoram essential oil obtained by hydrodistillation.
Figure 2. Chromatogram of Marjoram essential oil (fraction A).

Ramos et al.
Figure 3. Chromatogram of Marjoram essential oil (fraction C).
Table II. Antibacterial activity of the essential oil of Origanum majorana L.

Microorganisms
Inhibition zone (mm)
Oil
1:10
Fr. A
Staphylococcus aureus ATCC 25923

Enterococcus faecalis ATCC 19433
Escherichia coli
ATCC 25992
Klebsiella pneumoniae ATCC 23357
16
12
15
13
-
-
9
-
13
23
13
28
Fr. C
Controls
AN
AM
PRL
-
34
13
30
10
32
-
32
DMSO
-
Inhibition zone, diameter measured in mm, disc diameter 4 mm. average of two consecutive trial.
AN: Ampicillin (10 g); AM: Amikacine (30 g); PRL: Piperaciline (8 g)
A previous study performed on O. majorana collected at

the Medicinal Plants Garden at the Faculty of Pharmacy (20)
reported that the oil contained 3-ciclohexen-1-ol (41.7%) and
cis-sabinene-hydrate (14.8%) as major components. Different
results obtained in this study could be attributed to climatic
differences since the Medicinal Plant Garden is located at an
altitude of 1400 m above sea level.
The oil fractionation on the silica gel dry column permitted to isolate cis-sabinene-hydrate (67.6%) on fraction A (Fr.
A) and terpinen-4-ol (72.8%) on fraction C (Fr. C) (Figures
1, 2, and 3).
Antibacterial activity was measured using the agar diffusion method. ATCC Gram positive and Gram negative strains
were used. These results are shown on Table II. Our results
confirm the activity found on previous studies. The antibacterial
activity of fractions A and C were also assayed.
Fraction A and the total oil were found to be active against
all assayed bacteria, but fraction A was found to be twice as active against E. faecalis ATCC 19433 and K. pneumoniae ATCC
23357. On the other hand fraction C was found to be active
only against E. faecalis ATCC 19433 and E. coli ATCC 25992
and in general it was less active than the total oil. Fraction
A, which was enriched chromatographycally in cis-sabinene
hydrate, is more active possibly because the OH group is next
to a methyl while terpinen-4-ol, which is the main component
of fraction C, has an isopropyl group next to the OH. Since
fraction A showed the largest activity, it was concluded that
cis-sabinene-hydrate was the substance responsible for such
antibacterial activity.
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Gaitn et al.
Anti-inflammatory and Antioxidant Activity of a

Methanolic Extract of Phyllanthus orbicularis and its
Derived Flavonols
Yamilet Irene Gutirrez Gaitn*1, Migdalia Miranda Martnez1, Adonis Bello Alarcn1, Mariano
Martnez Vzquez2, Jose Luis Figueroa Hernndez3, Livn Delgado Roche1 and Luca Rastrelli4
Pharmacy and Foods Institute, University of Havana, 222 and 23 Ave., La Coronela, La Lisa, Cod, 13600, Havana, Cuba
2
Institute of Chemical, Nacional Autonomous University of Mexico, Coyoacan, Cod, 04510, Mexico. D.F.
3
Faculty of Medicine, Nacional Autonomous University of Mexico, Coyoacan, Cod, 04510, Mexico. D.F.
4
Departimento di Scienze Farmaceutiche e Biomediche, University of Salerno, Via Ponte Don Melillo, 84084, FiscianoSalerno, Italy
Abstract
In order to validate the use of Phyllanthus orbicularis (Phyllantaceae) in the traditional medicine of Cuba as an
anti-inflammatory remedy, the methanolic (MeOH) extract has been evaluated in vivo for anti-inflammatory activity
on 12-O-tetradecanoyl-13-acetate phrobol (TPA) assay in mouse and in vitro for the antioxidant activity on Ferric
Reduction Antioxidant Power assay. This extract exerted in vivo a significant anti-inflammatory activity. Subsequent
fractionation and analysis of the extract has led to the isolation and characterization as major constituents of apigenin
(1), rutin (quercetin 3-O--L-rhamnopyranosyl-(1->6)--D-glucopyranoside) (2) and quercetin (3) and of rutin decaacetate (4) and quercetin pentaacetate (5) from acetylated methanolic extract. Their structures were elucidated by
spectral methods. The bioassay-directed analysis of flavonols 1-3 indicated that rutin (2) and quercetin (3) were the
most active compounds, whereas apigenin showed no significant activity. The acetylation process increased the antiinflammatory activity but decreased the antioxidant activity. The MeOH extract and all of flavonoids tested did not
show in vitro significant cytotoxic effect in J774.A1 macrophage cell line. [Editors note: The five compounds under
discussion are denoted throughout this paper by bolded number (1, 2, etc.). This should not be confused with the
referenced ancillary texts, denoted in non-bolded font and within parentheses.]
Key Word Index
Phyllanthus orbicularis leaf and steam extracts, flavonoids, anti-inflammatory activity, antioxidant activity.
Introduction
Phyllanthus orbicularis (Phyllantaceae) is an herbaceous
plant growing in Cuba. Its leaves have been used as a remedy
in local folk medicine for the treatment of pathological processes such as ulcers and rheumatism, and as a febrifuge (1).
In previous papers, the aqueous extract has been reported to
show antiviral activity against human hepatitis B virus, herpes
simplex virus type 1 and 2, bovine herpes (2, 3) as well as shown
anti-mutagenic and antioxidant properties (4, 5).
Flavonoids are reported to affect the inflammatory process
of the mammalian system and possess anti-inflammatory activity
in vitro and in vivo (6). Prostaglandin biosynthesis and nitric
oxide production have been implicated in the process of inflammation and NO, produced by inducible and constitutive nitric
oxide synthase (cNOS), is one of the inflammatory mediators
that plays an important role in inflammation (6).
In vitro studies have confirmed that the flavonoid quercetin

inhibits nitric oxide production in IL-1-stimulated hepatocytes
through the inhibition of iNOS expression (7). In addition to
its anti-inflammatory activity, quercetin also has a gastric ulcer
protective effect, by the reduction in lipid peroxidation and
an increase in the activity of antioxidant enzymes (8). These
properties make quercetin an anti-inflammatory agent with no
gastrointestinal side effect. However, quercetin has not been
used widely in therapeutic medicine because it is practically
insoluble in water or oil, and many other reasons such as low of
oral absorption and bioavailability (9). It is therefore important
for the molecular modification to enhance the solubility and
bioavailability of quercetin. Some acetic acid esters of quercetin were reported enhanced the anti-inflammatory activity
of quercetin (10). We reported that quercetin pentaacetate
*Address for correspondence: ygutierrez@infomed.sld.cu
Rec: Mar 2011

Acc: July 2011

P. orbicularis
Table I. Inhibitory effects of extracts and compounds 25 from

P. orbicularis leaves and steams on TPA-induced inflammation
in mice
Samples
(mg/ear)
Indomethacin
0.36
MeOH extract
1
Ac-MeOH extract
1
rutin
1
rutin decaacetate
1
quercetin
1
quercetina pentaacetate
1

Edema (mg)
Inhibition (%)
2.880.73
8.570.49
6.832,53
12.930.57
6.104.51
5.470.56
11.272.24
78.76*
50.00*
59.80*
24.51*
64.12*
33.73*
66.80*
*p< 0.05 by Studentst-test as compared to control group; the results were

analyzed by means of a test of t- Student. Different letters mean that differences
exist statistically significant for 95% of confidence.
Table II. Antioxidant activity of extracts and compounds 25

from P. orbicularis leaves and stems
Samples (1 mg/mL)
FRAP (mol/g)a
l-ascorbic acid
MeOH extract
Ac-MeOH extract
rutin
rutin decaacetate
quercetin
quercetin pentaacetate
404.0 9.2b
1067.2 22.7
103.1 4.1
467.4 4.3
82.3 3.2
1559.7 120.5
90.3 6.9
FRAP, relative activities of the individual antioxidants to the reaction of Fe+2. For
protocols used, see Experimental section. bMean SD of three determinations
showed stronger inhibitory activity on TPA-induced inflammatory assay than quercetin.

In the context of our research on medicinal plants from
Cuba, the present paper reports on the composition and
anti-inflammatory activity of the methanolic and acetylated
methanolic extracts of P. orbicularis and on the involvement
of its major constituents, flavonols and flavonol glycosides, in
mediating this activity.
Experimental
Apparatus: A Bruker DRX-600 spectrometer operating at
599.2 MHz for 1H and 150.9 MHz for 13C using the UXNMR
software package was used for NMR measurements with CD3OD
solutions. DEPT, 1H-1H DQF-COSY, 1D TOCSY and HMBC
spectra were obtained by employing the conventional pulse
sequences. Optical rotations were measured on a Perkin-Elmer
141 polarimeter using a sodium lamp operating at 589 nm in
1% w/v solutions in MeOH. Electrospray ionization mass spectrometry (ESIMS) was performed using a Finnigan LCQ Deca
instrument from Thermo Electron (San Jose, CA) equipped
with Xcalibur software. Full mass and collision-induced dissociation (CID) MS/MS spectra were acquired both in positive
and negative mode. HPLC separations were performed with
a Waters model 6000A pump equipped with a U6K injector
and a Model 401 refractive index detector.
Plant material: Phyllanthus orbicularis HBK was identified and collected by Agronomist Engineer Rafael Carbonel
Paneque in August 2007 during flowering, in the zone of
Cajalbana, Pinar del Rio Province (Cuba). A specimen was
deposited in the herbarium of the Nacional Botanical Garden
of Cuba (HFC-85589-HAJA).
Extraction and isolation: The dried, powered stem
and leaves were mixed (500 g) and partitioned successively
(7 days) with n-hexane (1500 mL x 3), ethyl acetate (EtOAc)
(1500 mL x 3) and methanol (MeOH) (1500 mL x 3) to yield
three extracts. The methanolic extract was used in this work.
A portion of methanolic extract was acetylated with acetic
anhydride/pyridine (2 mL, 1:1) to 70C by 6h. The methanolic and acetylated methanolic extracts were fractionated on a
column packed with silica gel 60 GF254 (Merck) and eluted
with hexane, EtOAc, MeOH and mixtures of them. Fraction
containing flavonoids was subjected to reversed-phase HPLC
separation on Bondapak C18 column (30 cm x 7.8 mm i.d. flow
rate 1.5 mL/min) with MeOH- H2O (1:1) as solvent system.
This procedure gave three pure compounds identified by their
observed NMR, FABMS spectra and optical rotation data in
comparison with the literature values as apigenin, quercetin
and rutin from MeOH extract and as rutin decaacetate and
quercetin pentaacetate from acetylated methanolic extract.
Animals: Male CD1/c mice were housed in an environment with controlled temperature, (21-24C), lighting (12:12
light: darkness cycle), standard laboratory chow and drinking
water ad libitum for a period of 7 days before any experimental
manipulation. Their body weights ranged 20-25 g. All experiments were conducted according to guidelines established by
the Animal Care Committee.
Assay of TPA-induced inflammation ear edema in mice: Ear
edema was induced according to the method of De Young et al.
(11). The right ear of each mouse received TPA (0.125 mg/mL
acetone solution) as a topical application (10 mL for each side
of the ear). The methanolic and acetylated methanolic extracts,
rutin (2), quercetin (3), rutin decaacetate (4) and quercetin
pentaacetate (5) from Phyllanthus orbicularis (dissolved in
acetone), were applied topically immediately after TPA at
doses of 1.0 mg/ear. The left ear, used as a control, received
the vehicle. Indomethacin (0.36 mg/ear/20 L) was used as a
reference compound. Four hours after TPA administration,
the animals were sacrificed and disks of 6 mm diameter were
removed from each ear and their weights determined. Swelling
was measured as the difference in weight between the punches
from right and left ears, and the percent inhibition of edema
was calculated in comparison with control animals.
Determination of reducing power: The total antioxidant
potential of samples was determined using the ferric reducing antioxidant power (FRAP) assay of Benzie and Strain
(12). A solution of 10 mM TPTZ in 40 mM HCl and 12 mM
ferric chloride was diluted in 300 mM sodium acetate buffer
(pH 3.6) at a ratio of 1:1:10. Solutions of the MeOH and AcMeOH extracts and pure compound 2-5 (60 mL) were added
to 3 mL of the FRAP solution, and the absorbance at 593 nm
was determined every 10 min, for 90 min. Aqueous solutions
of known Fe(II) concentration (FeSO47H2O) were used
for calibration of the FRAP assay and antioxidant power was
expressed as mmol/g FRAP. l-ascorbic acid was used as referJournal of Essential Oil Research/51
Gaitn et al.
ence compound. All extracts and compounds were diluted and

analyzed in triplicate.
Cell culture: The macrophage cell line J774.A1 was
cultured in Dulbeccos modified Eagles medium (DMEM)
supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL
penicillin and 100 g/mL streptomycin at 37C under 5% CO2
humidified air.
MTT assay for cell viability: Cytotoxicity studies were
performed in a 96 well-plate. J774.A1 cells were mechanically
scraped and plated at a seeding density of 35.000 J774.A1
cells/well to a final volume of 150 L. After 2 h of incubation in
DMEM 5% FCS, cells were treated with LPS 1 g/mL alone or
in combination with P. orbicularis MeOH extract (1-100 g/mL)
or derived flavonoids 1-3 (0.5-50 g/mL) dissolved in DMSO.
This DMSO percentage allows the optimal solubilization of
flavonoids in aqueous solution. Control and LPS wells received
the same amount of DMSO. After 22 h of incubation at 37C,
25 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT, 5 mg/mL) were added in each well and 3 h
later the cells were lysed with 100 L of lysis buffer (20% SDS
and 50% DMF, pH 4.7). After an incubation of 18 h at 37C,
the optical densities (OD620) for the serial dilutions of the
methanolic extract of P. orbicularis and its derived flavonols
were compared to the OD of the control or LPS-stimulated
wells to assess the cytotoxicity (6).
Statistical Analysis
Experimental results are expressed as the means SEM of
measurements of at least six different mice. Data were assessed
by the method of analysis of variance (ANOVA). If this analysis
indicated significant differences among the group means, then
each group was compared with those for controls by the Students t test, and p values of less than 0.05 were considered to
be statistically significant. For antioxidant activity analysis data
are reported as mean standard deviation (SD) of triplicate
determinations. The statistical analysis was carried out using
the Microsoft Excel software package (Microsoft Corp.).

Chemical studies: The TLC analysis of the MeOH extract
of P. orbicularis leaves and steams revealed the presence of
flavonoids as major constituents. A silicagel column of the
MeOH extract (15 g) gave a flavonoid enriched fraction C that
was chromatographed by reversed-phase HPLC to yield three
pure compounds 1 (0.008%), 2 (0.533%) and 3 (0.035 % of
MeOH extract). They were identified as apigenin (1), rutin (2)
and quercetin (3) respectively (13), by ESIMS and extensive
NMR analysis. In particular the sugar moiety was determined
to be 3-O--L-rhamnopyranosyl-(1->6)--D-glucopyranoside
linked at C3 of the aglycone in compound 2 by a combination
of 1D TOCSY, 2D DQF-COSY and HMQC 2-D NMR experiments. The position of the interglycosidic linkage and the site
of glycosylation on the aglycone were deduced by long-range
C-H correlations in the HMBC spectra. The chemical shift,
multiplicity, absolute value of the proton coupling constants,
as well as the resonances of C2, C3 and C5, indicated the
configuration of D-glucopyranose and L-rhamnopyranose. The
acetylated methanolic extract was fractionated by CC obtaining
160 fractions. The fractions 40-61 eluted with hexane:EtOAc

(60:40) gave 27.5 mg of a crystalline powder cream color (4)
and fractions 98-109 eluted with hexane:EtOAc (10:90) were
gave 33.2 mg of a yellow crystalline product (5). 4 and 5 were
identified as quercetin pentaacetate (14) and rutin decaacetate
(15) respectively by comparison of their physical and spectroscopic data with reported values.
Inhibition of TPA-induced inflammation in ear mice:
TPA can act as an inducer of epidermal hyperplasia, a tumor
promoter, and an activator of various biological systems. TPAinduced ear edema is an in vivo model of acute inflammation;
it offers a simple and useful assay for screening the efficacy of
topical anti-inflammatory capacities of plant extracts, in this case
blackberry preparations. The inhibitory effects of MeOH and
Ac-MeOh extracts of and their isolated compounds 2-5 were
evaluated on TPA-induced inflammation in mice together with
those of a commercially available anti-inflammatory drugs, indomethacin. The results are shown in Table I. All of the extracts
and compounds tested showed inhibitory effects comparable or
more active than indomethacin. Rutin and quercetin were less
potent compared to the extract; conversely rutin decaacetate
and quercetina pentaacetate showed an increase of edema
percentages inhibition (64.12% and 66.80% respectively) and
were more active when compared to the acetylated extract.
Apigenin present in small amounts in the extract was not
tested for its anti-inflammatory activity. Quercetin pentacetate
exhibited a strong inhibitory effect that was almost the same
order of potency as that of indomethacin (Table I). Our results
suggest that chemical transformation by means of a reaction of
acetylation of the methanolic extract can improve the activity
and the results are in agree with those reported by Chen et al.
(16) and Gusdinar et al. (17).
These flavonoids therefore contribute to the anti-inflammatory activity of the MeOH extract of the leaves and steams
of P. orbicularis. The inhibitory effect against TPA-induced
inflammation has been demonstrated to closely parallel that of
the inhibition of tumor promotion in two stage carcinogenesis
initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and
TPA, a well-known promoter, in a mouse skin model. Thus,
compounds 2-5 may have anti-tumor-promoting activity in
this animal model.
Antioxidant activity: The antioxidant potential of extracts
and pure compounds from P. orbicularis were evaluated in
the antioxidant (FRAP) assays. l-Ascorbic acid was used as
the reference antioxidant. The results (Table II) showed that
flavonoids 2 and 3 exhibited free-radical scavenging activity
at potency levels comparative to the reference antioxidant
compound, while 4 and 5 had more moderate activities.
In this case, the acetylation process decreases the antioxidant activity. It is well known that the free radical scavenging
and antioxidant activities of phenolics are dependent upon the
arrangement of functional groups about the nuclear structure.
Both the number and configuration of H-donating hydroxyl
groups are the main structural features influencing the antioxidant capacity of phenolics.
Citoxicity: As previously indicated the MeOH extract of
P. orbicularis and its derived flavonoids (1-3) have been also
P. orbicularis
tested for their in vitro cytotoxic activity on J774.A1 cell line,

but we did not observe any significant cytotoxic effect here
(data not shown).
6.
7.
Conclusion
The flavonoids have been considered as the active principles of many anti-inflammatory plants. It has been speculated
that anti-inflammatory properties are a consequence of their
inhibitory actions on arachidonic acid metabolism as demonstrated in vitro and in vivo (6). Furthermore, flavonoids have
been reported to possess free radical scavenging or antioxidant
properties, which can be related with the inhibition exerted in
the metabolism of arachidonic acid via lipoxygenase activity
(18). On the basis of our results, we can hypothesize that the
anti-inflammatory activity of the extracts and compounds of
P. orbicularis may be due to the presence of a combination of
flavonoids and flavonoid glycosides.
Acknowledgements
The authors give thanks to the Institute of Chemical of Nacional

Autonomous University of Mexico for the provision of necessary
facilities.
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Simeone et al.
Chemical Composition of Essential Oils from Ripe

and Unripe Fruits of Piper amalago L. var. medium
(Jacq.) Yunck and Piper hispidum Sw.
Maria Lcia Ferreira Simeone*
Embrapa Milho e Sorgo, Caixa Postal 151, Sete Lagoas-MG, 35700-297, Brazil
Sandra Bos Mikich

Embrapa Florestas, Caixa Postal 319, Colombo-PR, 83411-000, Brazil
Llian Cristina Ccco

Centro Politcnico, Usinas Piloto A, Universidade Federal do Paran, Caixa Postal 19.024, Curitiba-PR, 81530-990, Brazil
Fabrcio Augusto Hansel

Embrapa Florestas, Caixa Postal 319, Colombo-PR, 83411-000, Brazil
Gledson Vigiano Bianconi

Instituto Neotropical: Pesquisa e Conservao, Caixa-Postal 19009, Curitiba-PR, 81531-980, Brazil
Abstract
The chemical composition of essential oils from unripe and ripe fruits of Piper amalago L. var. medium (Jacq.)
Yunck and Piper hispidum Sw. was examined using GC/MS analysis. The analysis of oils from P. amalago revealed
a predominance of oxygenated sesquiterpenes and 65 compounds were identified; their main constituents are: (E)nerolidol (14.2% and 19.9%), germacrene-D-4-ol (10.3% and 12.7%), a-cadinol (11.1% and 8.2%) in 99.6% and 98.7%
of the compounds for unripe and ripe fruits, respectively. Piper hispidum revealed a predominance of sesquiterpene
hydrocarbons, from which we identified 53 compounds including: a-copaene (28.7% and 36.2%), a-pinene (13.9%
and 7.1%), b-pinene (13.3% and 7.5%), and (E)-nerolidol (2.9% and 7.0%) which represented 97.8% and 98.1% of
the compound constituents for unripe and ripe fruits, respectively. The essential oils of fruits of P. amalago and P.
hispidum are reported for the first time.
Key Word Index

Piperaceae, Piper amalago L. var. medium (Jacq.) Yunck, Piper hispidum Sw., essential oil composition, a-pinene,
b-pinene, a-copaene, (E)-nerolidol, germacrene d-4-ol, a-cadinol.
Introduction
The genus Piper (Piperaceae) has been recently revised,
and includes approximately 700 species, represented by herbs,
shrubs and trees (1). The genus is widely distributed in tropical
and subtropical regions of both hemispheres. Several plants of
this genus are widely used in folk medicine in several parts of
the world and have been reported to produce compounds with
diverse biological and pharmacological properties (2). Many
Piper species are aromatics and as a consequence the chemi-
cal composition of the essential oils from several species has

been studied in detail. These studies revealed a diverse range
of oil components, including monoterpenes, sesquiterpenes,
arylpropanoids, aldehydes, ketones and long chain alcohols (35). The oil components from fruit parts of Piperaceae species
have been reported in a number of investigations and consist
of variable mixtures with a predominance of monoterpenes
(C10) and sesquiterpenes (C15) (6).
Previous results suggest that Piperaceae are very important
*Address for correspondence: malu@cnpms.embrapa.br
Rec: Nov 2010

Acc: Mar 2011

P. amalago and P. hispidum
Table I. Chemical composition of unripe and ripe fruits P. amalago and P. hispidum.
Oil components
RIb
RIc
Relative Area%
Piper amalago
Unripe
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
heptanal
tricyclene
a-pinene
canfene
sabinene
b-pinene
myrcene
n-decane
a-phellandrene
para-cymene
limonene
b-phellandrene
1,8-cineole
cis-b-ocimene
trans-b-ocimene
g-terpinene
cis-sabinene hydrate
para-mentha-2,4(8)-diene
linalool
trans-hydrate sabinene
perilene
perilene isomer
4-terpineol
exo-fenchol
cis-pinene hydrate
cis-b-terpineol
trans-b-terpineol
borneol
cryptone
a-terpineol
n-decanal
undec-9-en-1-al
g-elemene isomer
g-elemene
a-cubebene
ciclosativene
isoledene
a-copaene
b-bourbonene
b-cubebene
b-elemene
b-isocomene
a-gurjunene
trans-caryophyllene
b-gurjunene
aromandrene
geranyl acetone
a-humulene
trans-b-farnesene
seichelene
g-gurjunene
g-himachalene
g-muurolene
germacrene D
b-selinene
valencene
b-cis-guaiene
bicyclogermacrene
a-muurolene
germacrene A
g-cadinene
cubebol
d-cadinene
cis-calamenene
904
925
933
951
973
980
988
1004
1008
1025
1030
1032
1034
1034
1045
1058
1072
1087
1101
1104
1113
1113
1183
1122
1127
1145
1158
1175
1191
1198
1207
1309
1332
1335
1347
1370
1369
1376
1384
1388
1390
1392
1407
1420
1431
1439
1448
1456
1453
1460
1475
1472
1479
1481
1489
1491
1496
1496
1498
1507
1513
1516
1518
1521
Piper hispidum
Ripe
Unripe
899
-
-
<0.1
926
-
0,1
-
939
0.7
3.6
13.9
953
-
0.2
0.3
976
1.3
3.0
-
980
0.2
0.6
13.3
991
1.8
2.6
0.6
999
-
-
0.3
1005
0.7 -
-
1026
0.7
2.2
0.6
1031
1.0
1.5
0.9
1031
8.2
7.3
-
1033
0.3
0.1
-
1040
-
-
-
1040
0.1
<0.1
0.4
1062
-
<0.1
-
1068
-
<0.1
-
1086
-
<0.1
-
1098
2.0
1.4
-
1097
-
-
-
1099
-
-
1.6
1099
0.3
-
-
1177
0.3
0.2
0.2
1117
- -
0.6
1121
0.06
-
-
1144
0.04 -
1163 -
0.3
1165
0.1
-
1.4
1185
0.1
0.9
-
1189
0.2
0.5
1.5
1201
-
-
0.2
1308
-
-
<0.1
1339
0.1
-
-
1339
0.4
0.5
-
1351
0.2
0.1
0.2
1368
0.1
<0.1
-
1373
-
-
1.3
1376
3.0
0.7
28.7
1384
0.2
0.2
-
1390
3.3
2.5
-
1391
-
0.5
-
1403
-
-
0.2
1409
-
-
0.1
1418
2.6
2.7
1.7
1432
-
0.2
-
1439
0.1
<0.1
2.6
1453
-
-
0.9
1454
1.0
0.8
3.3
1458
-
<0.1
-
1460
-
<0.1
0.3
1473
-
0.5
0.8
1476
-
2.3
-
1477
2.1
-
-
1480
2.0
1,0
-
1485
-
-
-
1491
0.3
-
0.3
1490
-
0.2
1494
9.1 3.0
-
1499
1.5
<0.1
0.6
1503
0.9
0.7
-
1513
0.9
0.9
0.4
1514
-
-
-
1524
6.6
2.3
3.4
1521
-
<0.1
1.7
Ripe
7.1
0.1
7.5
0.7
0.3
0.4
0.9
<0.1
<0.1
0.2
1.9
0.4
0.1
0.1
1.3
36.2
0.2
<0.1
4.9
0.2
0.8
3.0
4.8
0.3
0.9
<0.1
<0.1
0.2
0.2
0.8
0.4
<0.1
2.3
2.2
Simeone et al.
Table I. Continued. Chemical composition of unripe and ripe fruits P. amalago and P. hispidum.
Oil components
RIb
RIc
Relative Area%
Piper amalago
Unripe
65
a-colacorene
1542
1542
66
(E)-nerolidol
1561
1564
67
ledol
1571
1565
68
germacrene d-4-ol
1578
1576
69
spathulenol
1578
1576
70
caryophyllene oxide
1583
1581
71
globulol
1588
1583
72
humulene epoxide II
1612
1606
73
1-epi-cubenol
1629
1627
74
a-acorenol
1631
1630
b-acorenol
1635
1634
75
76
epi-a-cadinol
1644
1640
77
epi-a-muurolol
1646
1641
78
a-muurolol
1649
1645
79
a-cadinol
1658
1653
80
9-methoxicalamelene
1667
d
81
cadalene
1674
1674
a-bisabolol
1682
1683
82
83
n-heptadecane
1699
1700
84
oplopanone
1737
1733
85
khusinol acetate
1829
1816
Terpenoid Classes
Monoterpene hydrocarbons
Oxygenated monoterpenes
Total
Piper hispidum
Ripe
Unripe
Ripe
-
<0.1
14.2
19.9
- -
10.3
12.7
-
-
-
0.7
-
0.8
-
-
-
<0.1
1.2
2.1
-
<0.1
6.1
4.9
1.5
-
2.6
2.1
11.1
8.2
-
<0.1
-
-
-
<0.1
- -
0.1
3.9
-
<0.1
1.1
2.9
-
-
4,16
1.82
0.94
0.97
0.30
-
-
-
-
0.2
-
-
1.7
-
0.9
-
-
1.3
7.0
0.1
3,67
1.39
1.27
0.84
0.44
0.4
0.2
0.2
1.9
0.7
-
14.9
3.3
34.5
46.9
99.6
30.4
5.8
48.6
13.0
97.8
17.2
2.4
59.3
19.2
98.1
21.2
3.0
19.1
55.4
98.7
Not detected.
a
-Compounds are listed in order of their elution from a CPSIL 8 CB column;
b
- RI = retention indices relative to C8 C26 n-alkanes;
c
- RI = retention indices from literature (DB-5 column);10
d
- mass spectrum agreed with NIST mass spectral database (tentative identification);
Obs: DB-5 and CPSIL 8 CB columns are similar in dimensions and chemical composition.
for the fruit-eating bat genus Carollia. Fruit-eating bats are

amongst the main seed dispersers for a variety of species and
improving this ecological role may have application in forest
restoration projects since the essential oils isolated from mature
chiropterochoric fruits are able to attract frugivorous bats both
inside forest remnants and in order to promote tropical forest
restoration (7-8).
The objective of this study was to evaluate the oil constituents in both unripe and ripe fruits of two Piper species
(Piper hispidum Sw. and Piper amalago L. var. medium (Jacq.)
Yunck) for use in future research to attract bats, encouraging
seed dispersal and subsequent forest regeneration.
Experimental
Plant material: Unripe and ripe fruits of P. amalago and
P. hispidum were harvested at the Parque Estadual Vila Rica
do Esprito Santo, in the town of Fnix (Paran State, Brazil;
2355S, 5157W), in 2006. Specimens were identified by Dr.
Sandro M. Silva and vouchers were deposited in the Herbarium
of Universidade Federal do Paran, Curitiba, Brazil, under code
numbers UPCB 32345, 32346 for P. amalago L. var. medium
(Jacq.) Yunck and UPCB 32337, 32339 for P. hispidum Sw.
Isolation of the essential oils: Unripe and ripe fruits of

P. amalago and P. hispidum species (200 g) were subjected to
hydrodistillation for 4 h in a Clevenger-type apparatus. The
oil layers obtained were dried over anhydrous sodium sulfate
after extraction with ethyl ether and, after filtration, evaporated
under nitrogen flux and maintained under refrigeration (-10C)
before analysis. Yields were calculated from the weight of fresh
material. The yields were averaged over three experiments
and calculated through the relation of the oil weight from the
Clevenger-type equipment to the mass of fruit material used
in the extraction.
performed using a Varian CP-3800 gas chromatograph equipped
with an auto sampler Varian 8200 and a flame ionization detector using a CP-SIL 8 CB (30 m x 0.25 mm id, 0.25 m film
thickness) capillary column. The injector and detector temperature were maintained at 250C and 300C, respectively.
The samples (1.0 L), dissolved in ethyl acetate, were injected
in split mode (1:200), using He as the carrier gas, flow rate 1
mL/min-1. The oven temperature was programmed as follows:
60C (1 min), heating to 240C at 3C/min-1 and holding for 5
min. Peak areas were measured by electronic integration. The
relative amounts of individual components were determined
P. amalago and P. hispidum
on the basis of their GC peak areas, without corrections for

FID response factors. GC/MS analysis was carried out on
a Varian ion trap Model CP 3800/Saturn 2000. GC/MS, EI
electron impact ion source, 70 eV using a CP-SIL 8 CB fused
silica capillary column (30 m x 0.25 mm i.d. x 0.25 m film
thickness); helium as the carrier gas, with a flow rate of 1 mL/
min-1 and a split ratio of 1:100. The injector temperature and
ion trap temperature was 250C and 150C, respectively. The
oven temperature was programmed as follows: 60C (1 min),
heating to 240C at 3C.min-1 and holding for 5 min, following
the same conditions from literature (11). The transfer line and
manifold temperatures were 200C and 100C, respectively.
Mass range was used from 35 to 500 m/z and scan time was
set in 0.4 s/scan-1.
Oil components were identified by comparison of their mass
spectra with those obtained from the spectrometer data base,
held at the National Institute for Standard TechnologyNIST
library using retention indices as a pre-selection routine (9-10).
The linear retention indices were calculated by co-injection
with a standard saturated n-alkanes homologous series. The
identifications were confirmed by comparison of the fragmentation pattern and corresponding linear retention indices with
those reported in the literature (10).

Average essential oils yields were for unripe and ripe P.
amalago, 0.09% and 0.03%, and for P. hispidum, 0.01% and
0.04%, respectively.
In total, there were identified 85 oil constituents in the fruits
of the two Piper species (Table I). The 32 monoterpenes and
53 sesquiterpenes identified are shown in order of elution on a
CPSIL 8 CB column, corresponding to essential oils from the
unripe and ripe fruits of P. amalago and P. hispidum.
Although P. hispidum showed a different chemical composition of essential oils compared with those identified from
P. amalago, in both species there was a predominance of sesquiterpene compounds in both ripe and unripe fruit.
Chemical analysis using GC/MS (Table I) of essential
oil from fruits of P. amalago revealed a predominance of oxygenated sesquiterpenes and 65 compounds were identified;
the main constituents are: (E)-nerolidol (14.2% and 19.9%),
germacrene-D-4-ol (10.3% and 12.7%), a-cadinol (11.1%
and 8.2%) and b-phellandrene (8.2% and 7.3%) in 99.6% and
98.7% of the compounds obtained from unripe and ripe fruit
oils, respectively.
In the analysis of essential oils from fruits of P. hispidum
there was a predominance of sesquiterpene hydrocarbons, and
were identified 53 compounds including: a-copaene (28.7% and
36.2%), a-pinene (13.9% and 7.1%), b-pinene (13.3% and 7.5%),
(E)-nerolidol (2.9% and 7.0%) and trans-caryophyllene (1.7%
and 4.9%) the majorities constituents. The whole indentified
compounds represented 97.8% and 98.1% of the constituents
of unripe and ripe fruit oils, respectively.
As expected, there are some differences in the proportions of the oil compounds in the unripe and ripe fruits of
the two Piper species. Table I showed that sesquiterpenes
hydrocarbons decreased and sesquiterpenes oxygenated in-
creased from unripe to ripe fruit oil of P. amalago. As for P.

hispidum, the monoterpenes decreased and sesquiterpenes
increased from unripe to ripe fruit oil. These results confirm
that the formation of ripe compounds in fruit is a dynamic
process, during which concentrations of constituents change
both qualitatively and quantitatively, which can cause changes
in the oil composition.
Mesquita et al, 2005 (11) analyzed the composition of volatile oil from leaves of P. amalago and P. hispidum harvested in
the state of Minas Gerais (Brazil). Piper amalago had a yield
of 0.6% and showed a predominance of sesquiterpenoids
containing the following major constituents: caryophyllene
oxide (18.0%), E-caryophyllene (17.8%), bicyclogermacrene
(16.4%), germacrene D (10.9%), a-pinene (9.3%). Piper
hispidum had a yield of 0.2% in which the predominant compound was monoterpenoid b-pinene (14.0%), followed by
sesquiterpenoid spathulenol (7.0%), germacrene D (6.9%),
caryophyllene oxide (6.4%).
The chemical composition of the essential oils of P. hispidum, from Cerrado (Brazillian savannah) was determined and
compared with the composition of oils from the same species
collected in the Atlantic Rain Forest. The distillation of leaves
of P. hispidum (12) had a yield of 0.3% and 26 compounds
were identified in the essential oil. The main constituents
were: b-pinene (19.7%), a-pinene (9.0%), d-3-carene (7.4%),
a-cadinol (6.9%) and spathulenol (6.2%). The identification
of chemical constituents of essential oils from the roots of P.
hispidum were 99.9% and 92.2%, represented by phenylpropanoids, corresponding to three major components: dillapiole
(57.5%), elemicine (24.5%) and apiole (10.2%) (13).
The essential oil from the fruits of P. tuberculatum were
identified 90.4% from the major constituent compounds (E)caryophyllene with 12.3% and caryophyllene oxide 26.6%. This
finding contrasts with previous studies (14) that revealed the
prevalence of monoterpenes found in fruits of the other Piperaceae species.The sesquiterpenes are frequent in the composition of the oils from the leaves, roots and fruits of these species,
and are also commonly recorded in the essential oils obtained
from other Piper species. This is the first report of the chemical
composition of essential oils from the fruits of P. amalago and P.
hispidum, and the observed chemical profile is different from
those obtained from other plant parts (11-13).
In summary, the study of essential oil components of Piper
species collected in Paran state showed a predominance of
oxygenated sesquiterpenes in P. amalago and sesquiterpene
hydrocarbons were mostly detected in P. hispidum. The essential oils of these chiropterochoric fruits can be used to
improve autoecological studies of fruit-eating bats and to
promote tropical forest restoration through the attraction of
frugivorous bats to degraded areas.
Acknowledgements
This work was funded by the Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico - CNPq (Process 476253/2007-1). The
authors are grateful to Dr. Carlos Itsuo Yamamoto (Laboratrio de
Anlise de Combustveis Automotivos LACAUT UFPR) for recording the GC and GC/MS spectra. S.B. Mikich is grateful to CNPq for
the Productivity Fellowship (Process 308419/2008-1).
Simeone et al.
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B. acuruana
Chemical Composition and Larvicidal Effects of

Essential Oil from Bauhinia acuruana (Moric) against
Aedes aegypti
Roberto W. da Silva Gois, Lencio M. de Sousa, Telma L. G. Lemos, Angela M. C. Arriaga and
Manoel Andrade-Neto
Departamento de Qumica Orgnica e Inorgnica, Universidade Federal do Cear, CEP 60451-970, Fortaleza, CE, Brazil
Gilvandete M. P. Santiago* and Yana S. Ferreira

Departamento de Farmcia, Universidade Federal do Cear, Rua Capito Francisco Pedro 1210, CEP 60430-370,
Fortaleza, CE, Brazil
Pricles B. Alves and Hugo C. R. de Jesus

Departamento de Qumica, Universidade Federal de Sergipe, CEP 49100-00, So Cristovo, SE, Brazil
Abstract
The essential oil from leaves of Bauhinia acuruana Moric was obtained by hydrodistillation and analyzed by gas
chromatography (GC-FID) and gas chromatography/mass spectrometry (GC/MS). In total, thirty compounds comprising 91.4% of the total peak area were identified. The main constituents of the essential oil were the sesquiterpenes
spathulenol (23.4 0.08%), epi-a-cadinol (20.7 0.12%) and caryophyllene oxide (16.4 0.04%). The essential oil
was tested against Aedes aegypti larvae and showed LC50 value of 56.2 0.4 mg/mL.
Key Word Index
Bauhinia acuruana, Caesalpinioideae, essential oil composition, sesquiterpenes, epi-a-cadinol, spathulenol,
Aedes aegypti.
Introduction
Bauhinia (family: Leguminosae, subfamily: Caesalpinioideae) is a genus of shrubs or trees, very rarely climbers,
distributed throughout the tropical regions of the world (1).
This genus comprises 300 species and is popularly known in
the northeast Brazil as pata-de-vaca due to their leaf format
(2). Species of this genus have been frequently used in folk
medicine to treat diabetes (3-5).
There are reports in the literature on the chemical composition of some species of the genus Bauhinia. Thus, different
classes of compounds such as lactones, flavonoids. terpenoids,
steroids, tannins and quinones were isolated and identified from
these species (3). No previous work on the chemical composition of Bauhinia acuruana has been reported.
Mosquitoes play a predominant role in the transmission
of several diseases which are today among the greatest health
problems in the world (6). Aedes aegypti is the principal vector
for the arboviruses responsible for yellow fever and dengue,
including the hemorrhagic form. The incidence of dengue has
grown significantly around the world in recent years. Today,
dengue is the most important mosquito-borne viral disease

affecting humans (7). Due to the lack of vaccines for dengue,
vector control is the main strategy to prevent or contain disease
outbreaks. Many synthetic insecticides such as organochlorides,
organophosphates and carbamates have been used to control
A. aegypti, but the toxicity problem, together with the growing
incidence of insect resistance (8,9), has called attention to the
necessity for novel insecticides.
The demand for new natural larvicides has increased and
plant essentials oils are a source of potential larvicides (6,10-16)
because they are, in some cases, highly active, economically
viable, and biodegradable (16).
As part of our program to evaluate essential oils from
northeastern Brazilian flora, this work reports the composition
and larvicidal activity of the essential oil from the leaves of B.
acuruana against Aedes aegypti.
Experimental
Plant material: Leaves of B. acuruana were collected in
May 2008 in Tiangu County, State of Cear, northeast Brazil.
Rec: Nov 2010
*Address for correspondence: gil@ufc.br
Acc: June 2011

da Silva Gois et al.
under the following conditions: J&W Scientific DB-5MS fused

silica capillary column (30 m x 0.25 mm i.d., x 0.25 mm film
thickness, composed of 5% phenyl/95% methylpolysiloxane)
operating in EI mode at 70 eV. Helium (99.999%) was used as
the carrier gas at a constant flow of 1.2 mL/min. The injection
volume was 0.5 L (split ratio of 1:100), the injector temperature 250oC and the ion-source temperature 280oC. The oven
temperature was programmed from 50oC (isothermal for 1.5
min), with an increase of 4oC/min to 200oC, then 10oC/min to
300oC, ending with a 10 min isothermal period at 300oC. Mass
spectra were taken at 70 eV with a scan interval of 0.5 s and
fragments from 40 to 500 Da.
A voucher specimem (#EAC42405) has been deposited at the

Herbrio Prisco Bezerra, Departamento de Biologia, Universidade Federal do Cear, Cear, Brazil.
The fresh leaves of B. acuruana were subjected to hydrodistillation in a Clevenger-type apparatus for 2 h to afford a
pale yellow oil. The isolated oil, after drying over anhydrous
sodium sulfate and filtration, was stored in sealed glass vials and
maintained under refrigeration until further analysis. The yield
(w/w) was calculated based on the fresh weight of the leaves.
Analysis of the essential oils: The essential oil was analyzed
by GC/MS on a Shimadzu QP5050A (Shimadzu Corporation,
Kyoto, Japan) system equipped with a AOC-20i autosampler
Table I. Chemical composition (%) of essential oil from the leaves of Bauhinia acuruana.
Peak
Compound
1
d-Elemene
a-Copaene
2
b-Elemene
3
4
cis-a-Bergamotene
b-Caryophyllene
5
b-Copaene
6
7
trans-a-Bergamotene
8
(E)-b-Farnesene
a-Humulene
9
10
allo-Aromadendrene
11
Germacrene D
b-Selinene
12
14
Bicyclogermacrene
g-Cadinene
15
b-Sesquiphellandrene
16
17
Elemol
18
Germacrene B
20
(E)-Nerolidol
22
Spathulenol
24
Caryophyllene oxide
26
Globulol
27
Viridiflorol
29
M 220
31
Humulene epoxide II
32
M 220
33
g-Eudesmol
34
epi-a-Cadinol
a-Muurolol
35
37
Valerianol
38
M 220
39
trans-Calamenen-10-ol
40
14-Hydroxy-9-epi(E)-caryophyllene
41
M 222
42
2,3-Dihydrofarnesol
43
Unidentified
44
M 222
45
Unidentified
46
Unidentified
Total
RI1
RI Lit (18)
1334
1338
1375
1376
1388
1390
1412
1412
1418
1419
1429
1432
1432
1434
1452
1456
1454
1454
1458
1460
1480
1485
1487
1490
1493
1500
1511
1513
1523
1522
1547
1549
1557
1561
1560
1563
1576
1578
1581
1583
1585
1590
1593
1592
1603
-
1609
1608
1627
-
1631
1632
1641
1640
1646
1646
1654
1658
1658
-
1664
1669
1668
1668
1674
-
1686
1689
1739
-
1749
-
1844
-
2057
-
-
-
Percentage
Identification
0.3 0.01
0.4 0.01
2.3 0.03
0.3 0.01
1.5 0.01
0.2 0.01
0.8 0.01
0.4 0.03
0.6 0.00
0.7 0.00
0.7 0.02
0.4 0.05
0.7 0.01
0.8 0.00
1.8 0.02
0.1 0.01
0.7 0.01
2.4 0.03
23.4 0.08
16.4 0.04
2.4 0.02
1.6 0.01
1.0 0.01
2.2 0.02
1.2 0.00
1.0 0.01
20.7 0.12
0.4 0.04
5.7 0.09
1.4 0.07
0.5 0.03
0.6 0.00
0.4 0.01
1.4 0.01
0.4 0.08
1.1 0.21
0.5 0.02
1.2 0.03
12.6
78.8
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
-
91.4
RI: Relative retention index calculated against n-alkanes applying the Van den Dool & Kratz (1963) equation. RI (17).
m/z (rel. int.): RI=1603, 220[M+] (5), 204(28), 189(24), 177(12), 161(49), 147(40), 133(36), 119(42), 107(76), 105(85), 91(74), 79(49), 69(41), 55(52), 41(100); RI =1627, m/z (rel.
int.): 220[M+] (1), 202(36), 187(25), 183(20), 159(100), 145(40), 131(56), 119(49), 117(34), 105(46), 91(43), 41(60); RI=1658, m/z (rel. int.): 220[M+] (7), 204(44), 189(60), 175(16),
159(65), 147(35), 133(46), 119(40), 105(66), 93(62), 81(66), 67(42), 55(61), 41(100); RI=1674, m/z (rel. int.): 222[M+] (10), 202(24), 187(26), 159(50), 145(38), 131(32), 125(43),
119(37), 107(49), 105(49), 93(43), 91(55), 79(41), 67(40), 55(41), 41(100); RI=1749, m/z (rel. int.): 222[M+] (5), 202(17), 200(18), 185(13), 177(24), 159(76), 143(27), 131(26),
117(22), 105(23), 91(47), 79(17), 65(13), 55(18), 43(100).
B. acuruana
Quantitative analysis of the chemical constituents was performed by flame ionization gas chromatography (FID), using
a Shimadzu GC-17A (Shimadzu Corporation, Kyoto, Japan)
instrument, under the following operational conditions: capillary
ZB-5M5 column (5% phenyl-arylene/95% methylpolysiloxane
fused silica capillary column 30 m x 0.25 mm i.d. x 0.25 mm film
thickness), under the same conditions reported for the GC/
MS. Quantification of each constituent was estimated by area
normalization (%). Compound concentrations were calculated
from the GC peak areas and they were arranged in order of GC
elution. Three independent analyses were carried out, and data
on average value and standard deviation were calculated.
Identification of individual components of the essential
oils was performed by computerized matching of the acquired
MS with those stored in NIST21 and NIST22 mass spectral
library of the GC/MS data system. Retention indices (RI) for
all compounds were determined according to literature (17)
for each constituent, as previously described (18).
Larvicidal bioassay: Aliquots of the essential oils tested
(12.5500 mg/mL) were placed in a beaker (50 mL) and dissolved in DMSO/H2O 1.5% (20 mL). Fifty instar III larvae
of Aedes aegypti were delivered to each beaker. After 24 h at
room temperature, the number of dead larvae was counted
and the lethal percentage calculated. A control using DMSO/
H2O 1.5% was carried out in parallel. For each sample, three
independent experiments were run (19).

The essential oil yield was 0.01%. A total of 30 compounds
were identified, all of which were sesquiterpenes, representing
91.4% of the essential oil. Its chemical composition, including retention index (RI) values listed in order of elution from
the DB-5MS column, and the percentage relative to each
constituent, are presented in Table I. The GC chromatogram
with peak identification of essential oil from the leaves of B.
acuruana is presented in Figure 1.
The major components of this essential oil were spathulenol
(23.4 0.08%), epi-a-cadinol (20.7 0.12%) and caryophyllene
oxide (16.4 0.04%). Other minor constituents were found
to be valerianol (5.7 0.09%), (E)-nerolidol (2.4 0.03%),
globulol (2.4 0.02%), b-elemene (2.3 0.03%) and humulene
epoxide II (2.2 0.02%).
The large abundance of sesquiterpenoid compounds in the
essential oil from leaves of B. acuruana is in accordance with
findings about the chemical composition of the essential oils
from leaves of B. aculeata (20), B. brevipes (20), B. longifolia
(20), B. pentandra (20), B. rufa (20), B. variegata (20), B.
forficata (20, 21) and B. ungulata (22). Thus, it seems that the
occurrence of sesquiterpenes as the predominant constituents
in the essential oil from leaves of Bauhinia species is a chemical
characteristic of this genus (20).
Furthermore, the larvicidal potential of the essential was
evaluated against A. aegypti larvae and exhibited an LC50 value
of 56.2 0.4 mg/mL. Several studies have shown that sesquiterpenoid compounds possess significant larvicidal activities (15,
23, 24), and that essential oils containing sphathulenol exhibit
properties against the larvae of Aedes aegypti (15, 25).
This is the first report on the chemical composition and
larvicidal activity against A. aegypti of essential oil from B.
Figure 1. GC chromatogram of essential oil from the leaves of Bauhinia acuruana.
da Silva Gois et al.
acuruana and the findings of the present study indicate this

essential oil as a potential natural larvicide, effective in the
control of the A. aegypti.
Acknowledgements
The authors thank the Brazilian agencies CNPq, CAPES, FUNCAP,

PRONEX for fellowships and financial support, and Laboratrio de
Entomologia, Ncleo de Endemias da Secretaria de Sade do Estado
do Cear, Brazil, where the bioassays were performed.
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T. lucida
Chemical Composition and Biological Properties of

the Leaf Essential Oil of Tagetes lucida Cav. from
Cuba
Erik L. Regalado and Miguel D. Fernndez
Center of Marine Bioproducts, Loma y 37, Havana, C.P. 10400, Cuba
Jorge A. Pino*
Food Industry Research Institute, Carretera al Guatao km 3, Havana, C.P. 19200, Cuba
Judith Mendiola
Institute of Tropical Medicine Pedro Kour, P.O. Box 601, Havana, Cuba
Olga A. Echemendia
Institute Finlay. Ave 27 319805, Havana, C.P.11600, Cuba
Abstract
The leaf essential oil of Tagetes lucida Cav. (Asteraceae) from Cuba has been obtained by hydrodistillation and
analyzed by GC-FID and GC/MS. Forty volatile compounds were identified, of which estragole (96.8%) was the
major constituent. The antioxidant capacity of this essential oil was measured by two different in vitro assays (DPPH
and TBARS) and significant activities were evidenced. The preliminary screening of its antiplasmodial, antibacterial,
antifungal and antiviral activities was carried out against Plasmodium berghei, Staphylococcus aureus, Pseudomonas
aeruginosa, Escherichia coli, Candida albicans, Acinetobacter lwoffi, Enterobacter aerogenes and against strains HHV
1 and HHV 2. The results showed a moderate activity against P. berghei and E. coli.
Key Word Index
Tagetes lucida, Asteraceae, essential oil composition, estragole, antioxidant capacity, antiplasmodial activity, antimicrobial activity, antiviral activity.
Introduction
Tagetes lucida Cav. (syn. T. florida Sweet, T. schiedeana
Less.), commonly called Pericn, hierbans, ans, santa Mara,
Mexican mint marigold, Mexican tarragon, Spanish tarragon,
or Texas tarragon, is a perennial herb that grows in dry rocky
slopes and woods native to Central America and South America
and naturalized elsewhere in the tropics and subtropics (1,
2). It is cultivated commercially in Costa Rica as a spice herb;
it contains an essential oil having an anise-like odor, and the
fresh aerial parts of this plant are sold in the supermarket as a
substitute of tarragon (3), which have been very used as spice
and to preserve meat (4). This species has been cultured in
Cuba in gardens due to the beauty of its foliage, but it is not
common its exploitation for medicinal aims.
Tagetes lucida has been referred in Mexican traditional
medicine for different therapeutic applications. The infusion
of leaves and flowers is drunk to combat diarrhea, rheumatism,
asthma, and cold (5, 6). The decoction of the aerial parts is
employed in the treatment of amoebic dysentery, giardiasis,

ascaridiasis and other infections caused by helminthes (6).
Moreover, a bibliographic survey of plants for malaria in Latin
America (2) reported the use of the dried powdered plant or
the plant decoction for the treatment of malaria in Mexico.
Essential oils from aromatic and medicinal plants have
been known to possess important biological properties, notably antibacterial, antifungal and antioxidant activities. Their
biological potential depends on their chemical composition
determined by genotype and influenced by environmental and
agronomic conditions (7). The chemical composition of T. lucida
volatile oil has been the subject of previous studies. The major
constituents of this volatile oil were determined to be methyl
eugenol (80%) and estragole (12%) in Mxico (8); estragole
(45%) and methyl eugenol (20%) in Hungary (9); anethole
(23.8%), eugenol (24.3%) and estragole (33.9%) in Guatemala
(10) or estragole (95-97%) in Costa Rica (3). T. lucida extracts
have reported to act on bacteria and phytopathogenic fungi (5)
and also possesses antidepressant-like properties in rats (11).
*Address for correspondence: jpino@iiia.edu.cu
Rec: Dec 2010

Acc: Mar 2011

Regalado et al.
Moreover, considering both chemical composition and biomass

yields, T. lucida appeared to be a promising species, with high
potential for use as biocidal crops for the implementation of
pest control practices (12).
In spite of the worldwide use of T. lucida in the folk
medicine, the biological properties of its essential oil based
on experimental models have remained largely unexplored.
In this context, the present work describes a detail chemical
composition and examines the antiplasmodial, antibacterial,
antifungal and antiviral activities of the essential oil isolated
from the leaves of Tagetes lucida Cav. from Cuba.
Experimental
Plant material: Leaves of T. lucida were collected in
February 2010, in the medicinal plants field of the Food
Industry Research Institute in Havana, Cuba. The plant was
identified by Dr. Pedro Herrera of the Institute of Ecology
and Systematic (IES) and a voucher specimen was deposited
at the Herbarium of IES (HAC 44100). Leaves (200 g) were
submitted to hydrodistillation in a Clevenger-type apparatus
for 2 h. At the end of each distillation the oils were collected,
dried with anhydrous Na2SO4, measured, and transferred to
glass flasks that were filled to the top and kept at a temperature
of 18C for further analysis. Analyses were made by duplicate.
Yields were calculated according to the weights of oils and plant
material before distillation.
performed on A Konik 4000A instrument (Barcelona, Spain)
equipped with a HP-5ms fused silica column (25 m x 0.25 mm
i.d., film thickness 0.25 m), split injection 1:10, and flame
ionization detection. Injector and detector temperatures were
at 220C and 250C. The oven temperature was held at 70C
for 2 min and then raised to 250C at 4C/min and held for
10 min. The carrier gas was H2 at 1 mL/min. Samples were
injected by splitting and the split ratio was 1:20. The lineal
retention indices (RI) were obtained from GC by logarithmic
interpolation between bracketing a homologous series of
n-alkanes used as standards. Peak areas were measured by
electronic integration using the EZChrom Chromatography
Data System 6.07 program (Scientific Software, Inc., FL).
The relative amount of the individual components was based
on the peak areas.
GC/MS analysis was performed on a Shimadzu 17A (Tokyo,
Japan) gas chromatograph coupled to a Shimadzu QP-5000
high performance quadrupole mass selective detector was
used. The GC was fitted with a HP-5ms fused silica column (25
m x 0.25 mm i.d., film thickness 0.25 m). The GC operating
conditions were identical with those described above except
that He was used as carrier gas. The MS operating conditions
were: ionization potential 70 eV with scan mass range of 35-400
m/z and ion source temperature at 250C. Compounds were
identified by computer search using digital libraries of mass
spectral data (NIST 02, Wiley 275, Adams 2001, Palisade 600,
and Flavorlib homemade library) and by comparison of their
retention indices of either reference substances or literature
values (13), relative to C8-C32 n-alkane series in a temperatureprogrammed run.
2,2-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scav64/Journal of Essential Oil Research
enging Assay: The antioxidant activity of the essential oil was

measured in terms of free-radical scavenging ability according
to DPPH reported method (14) with minor modifications.
Basically, a 60-M methanolic solution of DPPH (980 L)
[Sigma-Aldrich Co. (St. Louis, MO)], prepared daily, was placed
in a spectrophotometer cuvette, and eight concentrations of
the essential oil of 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 and 3.0 mg/
mL or ascorbic acid (standard) (0.16, 0.26, 0.6, 1.0 and 1.30
mg/mL) in methanol (v/v) solution (20 L) were added. The
decrease in absorbance at 515 nm was determined in a UV1201 spectrophotometer, until the reaction plateau step was
reached. Methanol was used to zero the spectrophotometer.
EC50 values were determined from the plotted graph of scavenging activity against sample concentrations, which is defined
as the total antioxidant necessary to decrease the initial DPPH
radical concentration by 50%. Triplicate measurements were
carried out, and their scavenging effect was calculated based
on the percentage of DPPH scavenged.
TBARS (thiobarbituric acid reactive species) assay: The lipid peroxidation assay as TBARS was carried out
according to a modified method (15). The reaction mixture
containing, in a final volume of 1.1 mL, 100 mL of cerebral
tissue (whole brain) and 1 mL (0.05M) of KH2PO4/K2HPO4
buffer, pH 7.4 in NaCl (0.9%), and seven concentrations of the
essential oil (20, 50, 100, 150, 200, 250 and 500 g/mL) was
incubated at 37C for 1 h. Then, 1 ml of thiobarbituric acid
(0.5%) and 1 mL of acetic acid (20%) were added to the test
tubes and were incubated at 100C for 60 min. After cooling,
absorbance was measured at 532 nm against control and buffer,
BHT being used as reference compound. All the experiments
were performed in triplicate, and the results were averaged.
The inhibition percentage was determined by comparison of
the results between the samples and control.
Antimalarial assay: In vitro drug susceptibility was determined in the standard short-term cultures of Plasmodium
berghei ANKA blood stages as described before (16). Briefly,
erythrocytes infected with parasites of P. berghei ring forms/
young trophozoites are incubated at 2% parasitemia at a final cell
concentration of 1% in complete culture medium (RPMI 1640
with 20% Fetal Calf Serum, Sigma, St. Louis, MO) containing
serial dilutions of essential oil maximal concentration tested,
from 200 g/mL to 12.5 g/mL, each in duplicate wells of 96well culture plates. These plates are incubated for a period of
24 h at 37C under standardized in vitro culture conditions. The
antimalarial activity was expressed as inhibitory concentration
50 (IC50), defined as the concentration of the volatile oil that
induces 50% reduction of production of schizonts, which was
calculated according to reported methodologies (17). IC50 was
expressed as mean standard deviation of tests performed in
two different assays. Chloroquine diphosphate and artemisinin
(Sigma, St. Louis, MO) were used as references.
Antibacterial activity: The antibacterial activity was carried out by the diffusion method according to the National Committee for Clinical Laboratory Standard Guideliness (NCCLS)
(18) and evaluated against several bacterial reference strains:
Staphylococcus aureus (ATCC 6538), Pseudomona aeruginosa
(ATCC 9027), Escherichia coli (10576), and Candida albicans
(ATCC10231) and strains from clinical samples: Pseudomona
aeruginosa, E. coli, Acinetobacter lwoffi, and Enterobacter
T. lucida
aerogenes. All the suspensions of microorganism were adjusted

0.5 MacFarland and six concentrations of the essential oil were
tested. The plates were incubated to 37C in a humidified
atmosphere, containing 5% CO2 for 24 h.
Cytotoxicity assay: Vero line was grown (37C, 5% CO2)
in 96-well culture plates, in 199 medium, supplemented with
10% fetal calf serum. Confluent monolayers were incubated
3 days with each dilution of the essential oil. All experiments
were performed in triplicate. The effect of samples on cell
viability was measured using the Naftol Blue Black (NBB)
method (19).
Antiviral activity: The antiviral activity was evaluated
against strains HHV 1 and HHV 2 and performed in 96-well
flat bottom tissue culture plates. Different dilutions of the
essential oil were added to confluent monolayer of Vero cell.
After 1 hour of incubation at 37C in a 5% CO2, the virus
suspensions were added. All plates were further incubated at
37C in a 5% CO2 atmosphere for 3 days.

A yield of 0.79% (v/m) of leaf essential oil was obtained by
hydrodistillation of the fresh leaves of T. lucida.
A gas chromatogram of the volatile compounds of the
leaf oil is shown in Figure 1, indicating the presence of 40
components, all of them were identified by comparing and
matching the mass spectra and GC retention index of the
unknown compounds with those of reference.
Table I lists the volatile components identified in the
leaf oil. Sixteen compounds are reported for the first time.
Estragole (96.8%) dominated the leaf oil composition. The
pleasant sweet odor of T. lucida leaves is assumed to be caused
by the anise-like smelling estragole. Among all molecules
identified, the other quantified components included myrcene,
germacrene D, (E)-b-ocimene, linalool, -caryophyllene and
(E,E)-a-farnesene.
Previous studies on leaf oils classified T. lucida into different

chemotypes (3, 9, 10) on the basis of the main constituents. The
leaf oil of T. lucida from Cuba could therefore be classified as
the estragole high content type, similar to the species reported
in Costa Rica (3). A minor difference with the Costa Rican leaf
oil is that other phenylpropanoids such as (E)-anethole and
eugenol were found in trace in the Cuban leaf oil and they
were not detected in Costa Rican leaf oil.
To establish the antioxidant activity of this essential oil, we
used two well-established in vitro assays. The first is based on
the free-radical-scavenging capacity of the stable DPPH radical and the second concerns the spectrophotometric detection
of TBARS, namely being malonaldehyde (MDA), one of the
secondary lipid peroxidation products, whose quantification
gives a measure of the extent of lipid degradation.
For the first assay, solutions with eight volatile-oil concentrations of 0.13 mg/mL, and different doses of ascorbic acid
(positive control) were prepared to evaluate the DPPH radicalscavenging capacity. The respective scavenging capacities ranged
from 10.2 0.3% to 85.4 1.1% with an EC50 value of 1.4
0.3 mg/mL for the essential oil and 0.025 0.004 mg/mL for
ascorbic acid. For the second test, seven different concentration
of volatile oil (20500 g/mL) and BHT as positive control,
also showed antioxidant activities in a dose dependent manner
and had 14.04 0.09% to 96.26 0.05% inhibition on lipid
peroxidation. The IC50 value was found to be 0.23 0.03 mg/
mL for the essential oil and 0.18 0.04 g/mL for BHT.
These results were in agreement with published data, which
demonstrated that estragole is a weak DPPH radical scavenger
(IC50 > 400 M) (20). However, several investigations have
demonstrated that some essential oils (estragole chemotype)
exhibit antioxidant potential (15, 16). At the same time, the
other significant component of this volatile oil, myrcene (2.3%)
and a minor one (linalool, 0.1%) have been previously found
to possess substantial protective effect against oxidant induced
genotoxicity, which is predominately mediated by their radi-
Figure 1. Total ion chromatogram of Tagetes lucida leaf essential oil.

Regalado et al.
Table I. Chemical composition of Tagetes lucida leaf essential oil
Peak Nr.
Compound
RIE1
RIR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
ethyl 2-methylbutanoate
(Z)-3-hexenol
myrcene
(Z)-3-hexenyl acetate
(Z)-b-ocimene
(E)-b-ocimene
linalool
estragole
carvone*
chavicol
(E)-anethole
a-cubebene
eugenol*
b-bourbonene*
b-elemene
b-caryophyllene
b-copaene*
trans-a-bergamotene
aromadendrene*
(E)-b-farnesene
germacrene D
(Z,E)-a-farnesene*
bicyclogermacrene
a-muurolene
(E,E)-a-farnesene
d-cadinene
elemol
1,10-di-epi-cubenol*
epi-a-muurolol*
a-muurolol*
a-cadinol
14-hydroxy-9-epi-(E)-caryophyllene*
n-octadecane*
hexahydrofarnesylacetone*
n-nonadecane*
n-eicosane*
n-heneicosane
phytol
n-docosane*
851
859
991
1005
1037
1050
1097
1194
1243
1250
1285
1350
1359
1385
1391
1419
1432
1435
1441
1456
1481
1490
1500
1502
1505
1523
1550
1619
1642
1646
1654
1670
1800
1840
1900
2000
2100
2112
2200
852
860
992
1007
1038
1048
1097
1196
1243
1252
1287
1351
1359
1388
1393
1419
1432
1435
1441
1457
1485
1491
1499
1500
1505
1526
1550
1619
1642
1646
1656
1670
1800
1844
1900
2000
2100
2115
2200
tr
tr
2.3 0.04
tr
tr
0.2 0.01
0.1 0.01
96.8 1.1
tr
tr
tr
tr
tr
tr
tr
0.1 0.01
tr
tr
tr
tr
0.3 0.02
tr
tr
tr
0.1 0.01
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
40
n-tricosane*
2300
2300
tr
*Reported for the first time in this essential oil; RIE and RIR: Experimental and reference retention index; Trace constituent (< 0.1%)
1
cal scavenging activity (21). In this context, the weak (DPPH)

and moderate (TBARS) antioxidant effects of T. lucida essential oil could be attributed in a great part to these volatile
metabolites.
T. lucida essential oil exhibited a moderate antimalarial
activity, with IC50 value equal to 72 3.62 g/mL, which was
evaluated following recommended endpoint criteria for natural
complex mixtures (22). IC50 values for chloroquine (30.92 ng/
mL) and artemisinin (18.3 4.48 ng/mL) were significantly
lower than this obtained for the oil. In addition, they closely
corresponded with previously reported IC50 values of P. berghei
ANKA (23). Previous studies have underlined the potential
biological activities of various essential oils against malaria
parasites. At earliest research, eight essential oils were tested
in vitro against P. falciparum and displayed IC50 values ranging
1491000 g/mL (24), while more recently, other five essential

oils (Xilopia phloiodora, Pachypodanthium confine, Antidesma
laciniatum, Xylopia aethiopica, Hexalobus crispiflorus) (25) with
IC50 values ranging 230 g/mL were considered very active.
Essential oils from fresh leaves of Cymbopogon citratus and
Ocimum gratissimum growing in Cameroon showed significant
antimalarial activities in the four-day suppressive in vivo test
in mice infected with Plasmodium berghei, at concentrations
of 200, 300 and 500 mg/kg of mouse weight per day (26). In
these reports, no clear conclusions can be derived from the
chemical composition of the tested essential oils in association
with their antimalarial activity.
The antioxidant activity of T. lucida essential oil is an interesting biological property. Endothelial cell injury by adherent
parasitized red blood cells is ameliorated by superoxide disVol. 23, September/October 2011
T. lucida
mutase, ascorbic acid, or tocopherol in vitro, highlighting the

potential therapeutic benefit of antioxidants for severe malaria.
At present, it is difficult to predict what will be their effect on
malaria disease, but future strategies might specifically target
antioxidants to the endothelium while keeping or enhancing
oxidative stress in infected RBC (27). It will depend on the
distribution of the essential oil in the cells (28).
To the best of our knowledge, only extracts of flowers of T.
erecta in ethanol/water and methanol/water solvent mixtures
were evaluated in the in vitro antimalarial screening and against
P. berghei in mice, which gave negative results (29), so the
moderate antimalarial activity exhibited by T. lucida essential
oil should be further explored.
Additionally, T. lucida essential oil only showed a moderate
antibacterial activity against E. coli (10576) using the diffusion
method (18). This essential oil was not cytotoxic from 103
dilutions and did not show antiviral activity with strains HHV
1 and HHV 2. Estragol-rich essential oils are reputed for their
antifungal properties (30), but have also exhibited significant
activity against bacterial strains (31), including against E. coli
(32). The antibacterial properties of T. lucida extracts have
been previously demonstrated where some isolated coumarins
were the most effective compounds against Gram-negative and
Gram-positive bacteria (5). However, we report for the first
time the activity of its essential oil against E. coli.
Acknowledgements
The authors thank Pedro Herrera for the identification of the

species.
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27. H. C. Ackerman, S. D. Beaudry and R. M. Fairhurst, Antioxidant
therapy: Reducing malaria severity? Crit. Care Med., 37, 758-760
(2009).
28. F. Bakkali, S. Averbeck, D. Averbeck and M. Idaomar, Biological
effects of essential oils A review. Food Chem. Toxicol., 46, 446475 (2008).
29. M. Misra, R. E. Rodriguez, S. L. North and K. S. Kasprzak,
Nickel-induced renal lipid peroxidation in different strains of mice:
concurrence with nickel effect on antioxidant defense systems.
Toxicol. Lett., 58, 121-133 (1991).
30. S. Shin and C. A. Kang, Antifungal activity of the essential oil of
Agastache rugosa Kuntze and its synergism with ketoconazole. Lett.
Appl. Microbiol., 36, 111-115 (2003).
31. K. Carovi-Stanko, S. Orli, O. Politeo, F. Striki, I. Kolak, M. Milos
and Z. Satovic, Composition and antibacterial activities of essential
oils of seven Ocimum taxa. Food Chem., 119, 196-201 (2010).
32. A. Guerrini, G. Sacchetti, M. Muzzoli, G. Moreno Rueda, A. Medici,
E. Besco and R. Bruni, Composition of the volatile fraction of Ocotea
bofo Kunth (Lauraceae) Calyces by GC-MS and NMR fingerprinting
and its antimicrobial and antioxidant activity. J. Agric. Food Chem.,
54, 7778-7788 (2006).
Bonaccorsi et al.
Analytical Characterization of Industrial Essential

Oils from Fruits and Leaves of C. aurantifolia Tan.
and C. latifolia Swing.
Ivana Bonaccorsi*, Paola Dugo, Luigi Mondello, Danilo Sciarrone and Giovanni Dugo
Dipartimento Farmaco-chimico, University of Messina, V.le annunziata -98168-Messina, Italy
Luis Haro-Guzman
Jos Vasconcelos, 105, Col. Jard. Vista Hermosa, 28010 Colima Col., Mexico
Abstract
The physicochemical indices, the composition of the volatile fraction, the enantiomeric ratios of some volatile
components and the oxygen heterocyclic fraction of cold-pressed Key lime oils (types A and B), Persian lime oils,
and petitgrain lime oils are reported. The volatile fraction of cold-pressed Persian lime oil is characterized by a
higher content of limonene, g-terpinene and esters and a lower content of b-pinene, sesquiterpene hydrocarbons,
alcohols and aldehydes than cold-pressed Key lime oils. In petitgrain oils the oxygenated compounds are present
at levels higher than the peel oils. Oxypeucedanin, probably due to the extraction technology, was almost absent in
cold-pressed Key lime type A, while it is present in cold-pressed Key lime type B and in Persian lime oil. The enseparation was performed by direct enantioselective GC (esGC) and by multidimensional GC (MDGC) to obtain the
most appropriate antiomeric separation of all the components analyzed. The enantiomeric excess of S-(-)-a-pinene,
1S,4R-(-)-camphene, S-(-)-b-pinene, S-(-)-sabinene, and R-(-)-b-phellandrene are lower in cold-pressed Persian lime
oil than in Key lime oils.
Key Word Index
C. aurantifolia Tan., C. latifolia Swing., peel and leaf lime oils, volatiles, non-volatiles, enantiomeric ratios, esGC,
GC/MS, MDGC, HPLC.
Introduction
Lime essential oil is known as one of the most complex
citrus essential oils, difficult to characterize for a series of
variables (variety, extraction method, geographic origin) and
it is extremely appreciated in the food and beverage market.
Usually Key lime (C. aurantifolia Tanaka), also known as acid
lime or Mexican lime, is the most appreciated, although Persian
lime (C. latifolia Swingle) is commonly used in transformation
industry.
From the Key lime tree it is possible to obtain the following
products: Key lime oil type A (cold-pressed by screw press from
the whole fruits, then separated by centrifuge); distilled lime
oil (obtained by steam distillation of the juice/oil emulsion);
Key lime type B (cold-extracted by common extractor/roller
machines which rasp the peel to release the oil. The oil is never
in contact with the acid juice); Key lime petitgrain (obtained
from the young green parts of the tree by distillation).
According to the world map showing production centers
of essential oils created by Treat PLC, attached to the very
recent volume edited by Baser and Buckbauer (1) the main

producer countries of lime oils are Mexico, Per, Brazil, Haiti,
Cuba, Guatemala, Ivory Coast, Ghana, Gambia, Madagascar,
India and Indonesia.
This study reports results on the analytical characterization of the different types of lime oils, of different geographic
origin as described in Table I to complete and update the
knowledge on these products which is often obsolete and scant
in recent literature.
The composition of the volatile fraction of Key lime oils
type A and B, and of Persian lime oil was reviewed in 1979
by Shaw (2) and successively, for the articles published until
1999, by Dugo et al. (3). These authors also published in the
same year a review on the composition of petitgrain lime oils
(4). Also in 2002 Haro-Guzman (5) revised the composition
of distilled lime oils and Mondello et al. (6) reported on the
enantiomeric distribution of some volatile components of these
oils. The composition of the oxygen heterocyclic compounds
was extensively revised by Dugo and McHale (7). Information
on the composition of these oils can be found in periodical
*Address for correspondence: bonaccor@pharma.unime.it
Rec: Dec 2010

Acc: Jan 2011

C. aurantifolia and C. latifolia
The results relative to the oxygen heterocyclic fraction of lime

oils are even fewer (16-19). More data is available in literature
relative to commercial oils of uncertain origin, some probably
polluted.
Recent literature is unavailable concerning the composition of lime petitgrain, and the enantiomeric distribution of
any lime oil industrially produced.
More information on oils extracted in laboratory, published
in the last decade, is available. The results relative to the volatile fraction of peel oils (20-29) were published between 1998
reviews by Lawrence (8). In the above mentioned reviews are

included samples obtained by industrial processes of secure
origin, and results relative to commercial samples and oils
extracted in laboratory by different techniques.
Few are the articles published on industrial genuine oils
after those revised in the above mentioned reviews in 2002.
Those relative to the volatile fraction of cold-pressed lime
oils found in literature in the new millennium are quite scant
(9-15). These are unfortunately relative to a single sample or
focused on particular aspects of the composition of the oils.
Table I. Analyzed samples of lime essential oils and their physicochemical indices.

Key lime oils
Persian lime oils
Cold-pressed
Type A
(1)a
(2)a
(3)a
(4)a
(5)a
(6)a
(7)b
(8)a
(9)a
(10)
(11)
Refractive index
1.484
1.484
1.486
1.484
1.483
1.480
1.484
1.482
1.482
1.479
n.d.
CD
5.05
6.76
5.55
7.00
5.50
7.06
7.06
3.47
Petigrain
Cold-pressed
Type B
Commercial
Crude Colorlessc
a) Mexican; b) Egyptian; c) obtained by distillation of sample 10
Figure 1. GC-FID chromatogram of cold pressed Key lime Type B. Peak identification: 1 tricyclene; 2 a-thujene; 3
a-pinene; 4 a-fenchene; 5 camphene; 6 thuja-2,4(10)-diene; 7 sabinene; 8 b-pinene; 9 6-methyl-5-hepten-2-one; 10
myrcene; 11 decane; 12 octanal; 13 a-phellandrene; 14 d-3-carene; 15 a-terpinene; 16 p-cymene; 17 limonene; 18 (Z)b-ocimene; 19 (E)-b-ocimene; 20 g-terpinene; 21 cis-sabinene hydrate; 22 terpinolene; 23 p-cymenene; 24 linalool; 25
trans-sabinene hydrate + nonanal; 26 fenchol*; 27 trans-p-mentha-2,8-dien-1-ol; 28 cis-p-menth-2-en-1-ol; 29 allocimene;
30 cis-limonene oxide; 31 trans-limonene oxide; 32 trans-pinocarveol; 33 citronellal; 34 (Z)-isocitral; 35 borneol; 36
isogeranial + terpinen-4-ol; 37 p-cymen-8-ol; 38 a-terpineol; 39 decanal; 40 citronellol; 41 nerol; 42 neral; 43 carvone; 44
geraniol; 45 geranial; 46 perilla aldehyde; 47 bornyl acetate; 48 trans-pinocarvyl acetate; 49 tridecane; 50 undecanal; 51
methyl geranate; 52 d-elemene; 53 citronellyl acetate; 54 neryl acetate; 55 geranyl acetate; 56 b-elemene; 57 dodecanal;
58 cis-a-bergamotene; 59 b-caryophyllene; 60 g-elemene; 61 trans-a-bergamotene; 62 (E)-b-farnesene; 63 a-humulene;
64 b-santalene; 65 g-curcumene; 66 germacrene D; 67 trans-b-bergamotene; 68 b-selinene; 69 a-selinene; 70 (Z)-abisabolene; 71 (E,E)-a-farnesene; 72 b-bisabolene; 73 (E)-g-bisabolene; 74 (E)-a-bisabolene; 75 germacrene B; 76
caryophyllene oxide*; 77 dodecyl acetate; 78 tetradecanal; 79 a-bisabolol; 80 (E,E)-farnesal.
Bonaccorsi et al.
and 2008. Five articles are relative to laboratory extracted leaf

oils (30-34). The enantiomeric distribution of some volatile
components were the object of two articles (35,36).
Experimental
Plant material: The present study was carried out on eleven
samples of industrially produced oils described in Table I.
Physicochemical indices: The analyses of CD were carried
out on a Hitachi U-2000 spectrophotometer, UV absorbance
was measured in the range of 200-400 nm. The CD values
were determined in accordance with the method of Sale (37),
25 mg of lime essential oil accurately weighed was diluted in
100 mL of ethanol. The analyses of refractive index were carried out on an Optec refractometer at 21C with a Carlo Erba
Kryo-Thermo stat WK5.
GC-Flame ionization detector (GC-FID): A Shimadzu
GC2010 gas chromatograph, equipped with an AOC-20i series
autoinjector, was used in all applications (Shimadzu, Kyoto,
Japan).
All the samples were injected and analyzed in triplicates
under the following conditions: column, SLB-5MS (silphenylene
polymer, virtually equivalent in polarity to 5% diphenyl/95%
methylpolysiloxane) 30 m x 0.25 mm i.d. x 0.25 mm df (Supelco,
Milan, Italy); temperature program: 50C to 250C at 3.0C
min; split/splitless injector (250C); injection mode: split, ratio:1:100; injection volume: 1.0 mL; inlet pressure: 99.5 kPa;
carrier gas: He; constant gas linear velocity: 30.0 cm/s. Data
handling by means of GCsolution software.
GC/Mass spectroscopy (GC/MS): Samples were analyzed
by GC/MS (EI) on a GC/MS-QP2010 system coupled to FFNSC ver. 1.3 database (38), created in the authors laboratory
now commercially available (Shimadzu, Japan, Wiley, USA)
and to Adams library (39); GC conditions: fused silica capillary column SLB-5MS 30 m x 0.25 mm i.d., 0.25 mm df film
thickness; column temperature, 50250C (10 min) at 3C/min;
carrier gas, He delivered at a constant pressure of 30.6 KPa
(30.1 cm/s); 1.0 mL of solution (1/10, v/v, essential oil/hexane)
injected on a split-splitless injector; injector temperature,
250C; injection mode, split; ratio: 1:50. MS scan conditions:
source temperature, 200C; interface temperature, 250C; E
energy 70eV; mass scan range, 40-400 amu. Data were handled
through the use of GC/MSsolution software.
Direct enantioselective GC (esGC): All the samples were
analyzed in triplicate under the following conditions: column,
Megadex DETTBS-b (diethyl-tert-butil-silyl b-cyclodextrin) 25
m x 0.25 mm i.d. x 0.25 mm df (Mega, Legnano, Italy); temperature program: 50C to 200C at 2.0C min; split/splitless
injector (220C); injection mode: split, ratio: 1:10; injection
volume: 1.0 mL (1/100, v/v, essential oil/hexane); inlet pressure:
Figure 2. GC-FID chromatogram of Persian lime oil. Peak identification: 1 tricyclene; 2 a-thujene; 3 a-pinene; 4
camphene; 5 sabinene; 6 b-pinene; 7 6-methyl-5-hepten-2-one; 8 myrcene; 9 octanal; 10 a-phellandrene; 11 d-3-carene;
12 a-terpinene; 13 p-cymene; 14 limonene + 1,8-cineole + (Z)-b-ocimene; 15 (E)- b-ocimene; 16 g-terpinene; 17 cissabinene hydrate; 18 terpinolene; 19 linalool; 20 nonanal; 21 cis-limonene oxide; 22 trans-limonene oxide; 23 citronellal;
24 borneol; 25 terpinen-4-ol; 26 a-terpineol; 27 decanal; 28 nerol + citronellol; 29 neral; 30 geraniol; 31 geranial; 32 perilla
aldehyde; 33 bornyl acetate; 34 undecanal; 35 d-elemene; 36 citronellyl acetate; 37 neryl acetate; 38 geranyl acetate; 39
cis- b-elemene; 40 dodecanal; 41 cis-a-bergamotene; 42 b-caryophyllene; 43 g-elemene; 44 trans-a-bergamotene; 45
(Z)- b-farnesene; 46 (E)- b-farnesene; 47 a-humulene; 48 b-santalene; 49 g-curcumene; 50 b-selinene; 51 (Z)-a-bisabolene;
52 (E,E)-a farnesene; 53 b-bisabolene; 54 (Z)-g-bisabolene; 55 (E)-g-bisabolene; 56 E)-a-bisabolene; 57 germacrene B;
58 caryophyllene oxide*; 59 tetradecanal; 60 2,3-dimethyl-3-(4-methyl-3-penten-1-yl)-2-norbornanol; 61 campherenol; 62
a-bisabolol
96.6 kPa; carrier gas: He; constant gas linear velocity: 35.0 cm/s.
Detector: FID (220C); H2: 50.0 mL/min; air: 400 mL/min;
makeup (N2): 40.0 mL/min; sampling rate: 80 msec. Data were
collected by the GCSolution software (Shimadzu).
Multidimensional enantio-GC (MDGC): The MDGC
system consisted of two GC2010 (defined as GC1 and GC2) gas
chromatographs, equipped with a Deans type switch transfer
device, an MS-QP2010 quadrupole mass spectrometer, and an
AOC-20i autosampler (Shimadzu). GC1 was equipped with a
split/splitless injector and a flame ionization detector (FID1).
The MDGC switching element, located inside the oven, was
connected to an advanced pressure control (APC) system which
supplied carrier gas (He) at constant pressure. GC1 column
was an SLB-5MS 30 m x 0.25 mm i.d. x 0.25 mm df . All samples
were analyzed in triplicate. The operational conditions were as
follows: constant inlet pressure 220 kPa (300C), split mode
1:20 (gas carrier, He); injected volume, 1.5 mL; initial linear
velocity, 30 cm/sec. Temperature program: 50280C at 3C/
min. The FID (300C) was connected, via a stainless steel
retention gap, to the transfer system; sampling rate: 80 ms.
APC constant pressure: 130 kPa. GC2 was equipped with a
split/splitless injector and a flame ionization detector (both not
used in the present research). Transfer line between GC1 and
GC2: 180C. The chiral column was a Megadex DETTBS-b
(diethyl-tert-butil-silyl b-cyclodextrin) 25 m x 0.25 mm i.d. x

0.25 mm df (Mega, Legnano, Italy). Temperature program:
40100C (20 min) at 1C/min, to 160C at 3C/min.
Reversed phase liquid chromatography (RP-HPLC/
PDA): Samples were analyzed in triplicates by HPLC using a
Shimadzu instrument equipped with two LC 10 AD Vp pumps,
an SPD-M10 Avp UV detector, a SCL-10 Avp controller, a
CTO-20AC column oven at 40C and a DGU-14A degasser.
Sample: ca. 100 mg of oil accurately weighed were diluted in 6
mL of ethanol. Before HPLC analysis, 50 mL of I.S. coumarin
(53.1 mg in 50 mL of ethanol) were added to 1.0 mL of this
solution. The injection volume was 2.0 mL. The column used was
a partially porous Ascentis Express C18, 150 x 4.6 mm i.d. with
particle size of 2.7 mm (Supelco, Bellefonte, PA). Two mobile
phases were used: eluent A (Water:Methanol:THF, 85:10:5)
and eluent B (Methanol:THF, 95:5). The HPLC analyses of
lime oil were performed according to the following program:
0-5 min, 0% B, 5-25 min, 0-40% B, 25-45 min, 40-90% B, 4555 min, 90% B, 55-60 min, 0% B. The flow-rate was 1.0 mL/
min, the pressure was 225 bar. Detection was performed by a
photodiode array (PDA) detector in the range 190-370 nm and
the chromatograms were extracted at 315 nm. Time constant
was 0.64 s and sample frequency 1.5625 Hz. Data was handled
by Shimadzu LCsolution software Ver. 3.3.
Figure 3. GC chromatogram of the essential oil of Petitgrain key lime from Egypt. Peak identification: i.s.: internal
standard; 1 a-thujene; 2 a-pinene; 3 camphene; 4 sabinene; 5 b-pinene; 6 6-methyl-5-hepten-2-one; 7 myrcene; 8 octanal;
9 d-3-carene; 10 p-cymene; 11 limonene; 12 (Z)-b-ocimene; 13 (E)-b-ocimene; 14 bergamal; 15 cis-linalool oxide; 16
trans-linalool oxide; 17 a-pinene oxide; 18 linalool; 19 6-methyl-3,5-heptadien-2-one; 20 4,8-dimethyl-1,3,7-nonatriene
(3E)-; 21 trans-p-mentha-2,8-dien-1-ol; 22 cis-limonene oxide; 23 trans-limonene oxide; 24 isopulegol; 25 citronellal;
26 (Z)-isocitral; 27 rose furan oxide; 28 nonanol; 29 isogeranial; 30 terpinen-4-ol; 31 trans-isocarveol; 32 a-terpineol; 33
g-terpineol; 34 decanal; 35 octyl acetate; 36 nerol; 37 citronellol; 38 cis-p-mentha-1(7),8-dien-2-ol; 39 neral; 40 carvone; 41
linalyl acetate; 42 geraniol; 43 geranial; 44 neryl formate; 45 limonen-10-ol; 46 geranyl formate; 47 undecanal; 48 methyl
geranoate; 49 d-elemene; 50 citronellyl acetate; 51 neryl acetate; 52 geranyl acetate; 53 b-bourbonene; 54 b-elemene; 55
tetradecane; 56 dodecanal; 57 cis-a-bergamotene; 58 b-caryophyllene; 59 trans-a-bergamotene; 60 aromadendrene; 61
geranyl acetone; 62 a-humulene; 63 neryl isobutyrate; 64 g-muurolene; 65 b-selinene; 66 a-selinene; 67 (E,E)-a-farnesene;
68 b-bisabolene; 69 g-cadinene; 70 7-epi-a-selinene; 71 (Z)-nerolidol; 72 cis-sesquisabinene hydrate; 73 germacrene B; 74
caryophyllene oxide; 75 humulene epoxide II; 76 spathulenol; 77 a-cadinol; 78 selin-11-en-4-a-ol; 79 a-bisabolol.
Bonaccorsi et al.

Physicochemical indices: In Table I for each of the
samples analyzed are reported the physicochemical indices.
CD values are in agreement with those previously reported
(40) for cold-extracted peel oils of secure origin; the sample of
crude commercial Persian oil has a low CD value.
Volatile fraction: Table II reports the composition of
the volatile fraction in single components (average values
determined out of triplicates) and in class of substances of
the samples analyzed. In the same table are reported the
LRI experimentally determined compared to those reported
in literature. The coefficient of variation determined for the

peak area % of each component out of triplicates was never
above 5.9%. In general, the repeatability of the method was
excellent.
GC/MS identified 137 components. The composition is
expressed as relative percent of peak area determined by
GC-FID without considering the response factors nor the
non-volatile residue. The gas chromatograms in Figures 1-3,
respectively show Key lime oil cold-pressed, Persian lime oil
cold-pressed and Key lime petitgrain oil. The components
identified (Table II) in peel oils represent about 98-99% of
Figure 4. A) TIC chromatogram obtained by MDGC of a cold-pressed Key lime Type A. B) enlargement of the direct esGC
separation showing the separation of the enantiomers of a-pinene and of camphene; C) enlargement of the direct esGC
separation of (-)/(+) a-terpineol separation. Peak identification: 1 S-(+)-a-thujene; 2 R-(-)-a-thujene; 3 R-(+)-a-pinene; 4
S-(-)-a-pinene; 5 1S,4R-(-)-camphene; 6 1R,4S-(+)-camphene; 7 R-(+)-b-pinene; 8 S-(-)-b-pinene; 9 R-(+)-sabinene; 10
S-(-)-sabinene; 11 R-(-)-a-phellandrene; 12 S-(+)-a-phellandrene; 13 R-(-)-b-phellandrene; 14 S-(-)-limonene; 15 S-(+)b-phellandrene; 16 R-(+)-limonene; 17 R-(-)-linalool; 18 S-(-)-citronellal; 19 R-(+)-citronellal; 20 S-(+)-linalool; 21 S-(+)terpinen-4-ol; 22 R-(-)-terpinen-4-ol; 23 S-(-)-a-terpineol; 24 R-(+)-a-terpineol.
Table II. Composition of the volatile fraction (relative percentage of uncorrected peak areas).

Key Lime oils
Cold-pressed

Compound
Type A
LRI
exp
Tricyclene
923
a-Thujene
925
a-Pinene
933
a-Fenchene
947
Camphene
953
Thuja-2,4(10)-diene
954
Sabinene
972
b-Pinene
978
6-Methyl-5-hepten-2-one 983
Myrcene
988
6-Methyl-5-hepten-2-ol
994
Decane
1000
Octanal
1004
a-Phellandrene
1006
d-3-Carene
1010
a-Terpinene
1017
p-Cymene
1024
Limonene*
1030
1,8-Cineole
1033
(Z)-b-Ocimene
1035
(E)-b-Ocimene
1045
Bergamal
1053
g-Terpinene
1058
cis-Sabinene hydrate
1068
Octanol
1070
cis-Linalool oxide
1069
Terpinolene
1086
p-Cymenene
1095
a-Pinene oxide
1097
Linalool
1099
trans-Sabinene hydrate
1100
6-Methyl-3,5-heptadien2-one
1102
n-Nonanal
1105
4,8-Dimethyl-1,3,7nonatriene, (3E)-
1113
1,3,8-p-Menthatriene
1110
Fenchyl alcohol
1122
trans-p-Mentha-2,8-dien1-ol
1122
cis-p-Menth-2-en-1-ol
1125
(4E,6Z)-Allocimene
1128
cis-p-Mentha-2,8-dien-1-ol 1132
cis-Limonene oxide
1134
trans-Limonene oxide
1138
trans-Pinocarveol
1140
Isopulegol
1142
Citronellal
1152
1161
(Z)-Isocitral
Pinocarvone
1159
Rose furan oxide
1169
Borneol
1170
cis-Pinocamphone
1173
Nonanol
1176
Isogeranial
1180
Terpinen-4-ol
1182
p-Cymen-8-ol
1188
trans-Isocarveol
1189
a-Terpineol
1197
g-Terpineol
1200
Decanal
1206
trans-Piperitol
1208
Octyl acetate
1214
LRI
Persian Lime oils

Petitgrain
Commercial
Type B
(1)
(2)
(3)
(4)
(5)
923
927
933
948
953
953
972
978
986
991
995
1000
1006
1007
1009
1007
1025
1030
1032
1026
1046
1053
1058
1069
1073
1072
1086
1093
1099
1101
1099
0.03
0.33
2.09
tr
0.07
tr
2.45
20.17
0.03
1.01
tr
tr
0.03
0.03
tr
0.20
0.24
51.14
-
0.09
0.22
-
9.29
0.04
-
-
0.38
tr
-
0.15
0.03
0.01
0.27
1.96
-
0.08
-
2.43
21.16
tr
1.05
-
-
0.05
0.03
-
0.19
0.11
50.29
-
0.12
0.29
-
7.66
-
-
-
0.33
-
-
0.17
-
0.01
0.31
1.92
0.01
0.09
tr
2.31
18.25
0.03
1.07
-
-
0.04
0.05
tr
0.23
0.33
50.96
-
0.13
0.30
-
9.79
0.05
tr
-
0.52
0.01
-
0.24
tr
0.01
0.29
1.91
-
0.07
-
3.02
20.10
0.10
1.04
-
-
0.03
0.03
-
0.13
0.10
49.60
-
0.12
0.28
-
8.68
-
0.06
-
0.33
-
-
0.22
-
0.01
0.31
2.03
0.01
0.09
tr
2.40
18.01
0.03
1.13
-
0.01
0.04
0.05
0.01
0.22
0.31
49.25
-
0.13
0.30
-
9.87
0.05
-
-
0.53
0.01
-
0.24
0.02
1103
1107
-
0.02
-
0.03
-
0.03
1115
1108
1123
tr
-
tr
-
-
-
-
0.01
0.04
-
-
-
1125
1124
1132
1133
1137
1140
1141
1145
1152
1160
1164
1170
1173
1176
1178
1179
1180
1189
1190
1195
1200
1208
1209
1210
tr
tr
tr
tr
-
-
tr
-
0.01
-
tr
-
tr
0.01
-
0.30
0.06
-
-
0.27
-
0.17
tr
-
-
-
-
-
tr
tr
-
-
tr
-
-
-
-
tr
-
0.41
0.01
-
-
0.22
-
0.24
-
-
0.01
tr
-
-
0.01
tr
-
-
0.03
0.02
-
-
0.04
0.02
-
0.33
0.07
0.02
-
0.80
-
0.18
-
tr
-
-
-
-
-
tr
-
-
0.03
-
-
-
-
tr
-
-
0.08
-
-
0.31
-
0.22
-
tr
lit
Cold-pressed
-
-
0.03
(6)
(7)
(8)
(9)
(10)
-
-
0.03
0.01
0.01
0.05 0.01
0.48
0.61
0.57
0.30 0.11
1.99
2.24
2.25
-
-
tr
-
tr
tr
0.01
0.04
0.06
0.06
-
-
-
-
-
0.89 0.14
1.70
1.95
2.10
0.37 0.32
10.52 11.39
12.16
0.87 0.34
tr
0.01
0.02
0.78 0.45
1.26
1.46
1.38
-
-
-
-
-
-
-
-
-
-
0.07 0.01
0.01
0.01
0.01
-
0.04
0.05
0.04
-
0.04 0.02
tr
0.01
0.01
0.03
-
0.23
0.28
0.12
0.25 0.14
0.08
0.18
1.07
45.22 45.29
58.15 54.77
53.61
0.14
-
0.45 0.23
0.18
0.22
2.33 0.50
0.08
0.08
0.12
-
-
-
-
0.03
1.09
-
14.61 13.90
12.38
-
-
0.04
0.05
0.04
0.03
-
-
-
-
-
0.01
-
-
-
0.10
-
0.49
0.63
0.45
-
-
-
-
-
-
0.04
-
-
-
1.20 0.61
0.13
0.18
0.18
-
-
0.05
0.07
0.05
(11)
0.03
0.51
2.05
tr
0.06
1.92
11.82
0.01
1.30
0.01
0.04
tr
0.20
0.30
54.20
0.11
12.84
0.04
0.51
0.14
-
-
0.04
0.04
-
-
0.01
-
-
-
0.01
0.01
-
0.01
0.05
-
-
-
0.02
-
-
-
-
-
-
tr
-
-
tr
0.01
0.01
0.01
0.01
-
0.01
0.01
0.01
tr
0.03
0.02
-
-
0.02
-
-
0.32
0.05
0.02
-
0.80
-
0.17
-
-
0.01
-
0.02
-
-
0.03
-
-
0.74
0.24
-
-
-
-
-
0.47
0.05
-
-
0.16
-
0.28
-
-
0.05
-
-
-
-
-
-
-
0.37
0.25
-
-
0.04
-
1.64
0.03
0.04
-
-
-
0.10
-
-
0.01
-
-
0.01
-
0.16
0.01
-
-
-
-
0.01
tr
-
-
0.04
0.01
-
-
0.02
tr
-
0.02
tr
0.01
-
-
0.04
0.03
-
-
0.06
tr
-
-
0.02
tr
-
0.04
0.01
0.01
0.06
0.01
-
-
0.04
0.18
0.08
0.18
-
0.02
tr
-
0.32
-
0.06
-
-
0.03
-
0.26
-
0.06
-
-
0.21
0.06
-
-
-
0.25
-
0.05
-
-
Bonaccorsi et al.
Table II. Continued

Key Lime oils
Cold-pressed

Compound
Type A
LRIexp
Nerol
1225
Citronellol
1227
cis-p-Mentha-1(7),8-dien-2-ol 1230
Neral
1239
Carvone
1246
Linalyl acetate
1249
Geraniol
1251
Piperitone
1263
Geranial
1269
Decanol
1276
Perillaldehyde
1276
Neryl formate
1276
Bornyl acetate
1283
Isobornyl acetate
1284
Geranyl formate
1298
2,3-Benzopyrrole
1299
trans-Pinocarvyl acetate 1300
Tridecane
1300
Undecanal
1308
Geranic acid methyl ester 1321
d-Elemene
1337
a-Terpinyl acetate
1347
Citronellyl acetate
1349
Neryl acetate
1358
Geranyl acetate
1377
b-Bourbonene
1382
b-Elemene
1391
Tetradecane
1400
Dodecanal
1409
Decyl acetate
1410
cis-a-Bergamotene
1415
b-Caryophyllene
1423
a-Santalene
1416
g-Elemene
1432
trans-a-Bergamotene
1435
Aromadendrene
1438
(Z)-b-Farnesene
1140
b-Sesquisabinene
1450
(E)-b-Farnesene
1451
Geranyl acetone
1450
a-Humulene
1459
b-Santalene
1460
g-Curcumene
1479
Germacrene D
1481
trans-b-Bergamotene
1484
Valencene
1489
Neryl isobutyrate
1485
g-Muurolene
1485
b-Selinene
1492
Bicyclogermacrene
1499
a-Selinene
1501
(Z)-a-Bisabolene
1502
(E,E)-a-Farnesene
1504
b-Bisabolene
1509
(Z)-g-Bisabolene
1510
b-Sesquiphellandrene
1521
(E)-g-Bisabolene
1530
(E)-a-Bisabolene
1539
g-Cadinene
1512
7-epi-a-Selinene
1520
(Z)-Nerolidol
1531
cis-Sesquisabinene hydrate 1544
Persian Lime oils

Petitgrain
Cold-pressed
Commercial
Type B
LRIlit
(1)
(2)
1229
1232
1233
1238
1244
1243
1255
1267
1268
1278
1278
1280
1285
1287
1298
1297
1296
1300
1309
1320
1335
1349
1353
1361
1380
1381
1391
1400
1410
1412
1416
1424
1418
1431
1434
1439
1439
1455
1452
1449
1454
1459
1482
1480
1483
1492
1482
1485
1490
1497
1500
1503
1501
1508
1511
1523
1528
1540
1510
1520
1530
1545
0.04
0.01
-
1.12
-
-
0.07
tr
1.90
0.01
0.02
-
tr
-
-
-
-
0.01
0.02
-
0.02
-
0.01
0.16
0.18
-
0.16
-
0.10
-
0.07
0.79
-
0.03
1.12
-
-
-
0.10
-
0.09
0.04
0.03
0.26
0.07
-
-
-
0.04
-
0.06
0.14
1.00
1.85
-
-
-
0.03
-
-
-
0.01
-
0.01
-
1.36
-
-
tr
-
2.29
-
0.01
-
-
-
-
-
-
-
0.02
-
0.56
tr
tr
0.07
0.19
-
0.29
-
-
0.13
-
0.96
-
-
1.14
-
-
-
0.11
-
0.11
0.04
-
0.07
-
0.07
-
-
-
0.15
-
-
1.27
1.83
-
0.01
0.04
-
-
-
-
-
(3)
(4)
0.01
-
-
-
-
-
1.61
1.82
tr
-
-
-
0.07
tr
tr
-
2.63
2.88
-
-
0.02
0.02
-
-
-
-
0.01
-
-
-
-
0.01
0.01
-
0.01
-
0.02
0.02
tr
tr
0.39
0.46
-
-
0.01
-
0.14
0.12
0.25
0.19
-
-
0.19
0.27
-
-
0.09
-
tr
0.12
0.06
-
0.73
0.97
-
-
0.06
-
0.86
0.93
-
-
0.03
0.09
0.01
-
0.06
-
-
-
0.10
0.11
0.03
0.03
0.02
-
0.27
0.06
-
-
0.01
0.06
-
-
-
-
0.02
tr
0.12
0.07
-
0.12
-
1.06
1.20
1.35
1.50
tr
-
-
-
0.02
0.03
0.03
-
-
-
0.01
-
-
-
0.01
tr
(5)
0.04
0.01
-
1.65
tr
-
0.09
-
2.67
-
0.02
-
0.01
-
-
-
0.01
tr
0.01
tr
0.38
-
0.01
0.13
0.24
-
0.18
-
0.09
-
0.05
0.72
-
0.06
0.86
0.03
-
-
0.08
-
0.09
0.04
0.02
0.26
0.06
-
-
-
0.03
-
0.07
0.11
1.09
1.34
0.01
-
tr
0.03
-
0.03
-
0.01
(6)
(7)
(8)
-
1.12
0.16
3.27 0.59
-
0.06
-
10.38 7.45
1.09
-
0.10
-
-
0.12
-
3.91 1.82
0.04
-
-
-
13.96 10.75
1.79
-
-
-
-
-
0.02
0.03 0.05
-
-
-
-
-
-
-
0.05 0.11
-
-
-
-
-
-
-
-
-
-
0.05 0.27
0.01
0.01 0.17
-
0.57 0.58
0.03
-
-
-
0.06 0.53
0.01
0.45 2.11
1.11
1.31 6.22
0.14
0.01 0.09
-
0.81 0.67
0.04
-
0.08
-
0.11 0.15
0.01
-
-
-
0.02 0.04
0.08
2.72 2.63
0.29
-
-
0.20
-
0.01
0.28 0.25
0.67
0.01 0.05
-
-
-
-
-
-
-
-
0.10
-
-
0.34
-
0.39 0.02
0.03
-
-
0.01
-
-
0.01
-
-
0.05
-
-
0.05
-
-
-
-
0.09
-
0.29 0.11
-
-
0.15
0.02
-
-
-
0.06 0.16
0.02
-
-
0.18
0.99 0.48
0.21
0.58 0.48
1.67
-
-
-
-
-
-
-
-
-
-
-
-
tr
tr
-
-
0.03
-
-
0.29
-
-
0.03
tr
(9)
(10)
(11)
0.19
0.03
-
1.25
-
0.01
0.04
0.01
2.02
-
0.03
-
-
0.01
-
-
-
-
0.01
-
0.06
-
0.01
1.21
0.19
-
0.05
tr
0.04
-
0.08
0.59
-
0.01
1.11
-
0.05
0.01
0.12
-
0.05
0.04
0.03
0.06
0.07
-
-
-
0.01
-
0.03
0.15
0.27
1.70
0.02
-
0.01
0.04
-
-
-
0.01
0.08
0.06
-
1.46
0.01
0.01
0.03
0.01
2.42
-
0.04
-
-
0.02
-
-
-
-
0.03
-
0.04
-
0.03
1.31
0.28
-
0.07
0.01
0.04
-
0.08
0.68
-
0.01
1.18
-
0.05
0.01
0.12
-
0.06
0.05
0.02
0.06
0.08
-
-
-
0.02
-
0.04
0.15
0.25
1.79
0.02
-
0.01
0.04
-
0.01
-
0.02
0.09
1.35
0.03
2.20
0.03
0.02
0.07
0.01
1.08
0.23
0.06
0.04
0.06
0.61
0.01
0.98
0.01
0.10
0.05
0.04
0.02
0.02
0.13
0.23
1.53
0.02
0.01
0.03
tr
Table II. Continued

Key Lime oils
Cold-pressed

Compound
Type A
LRIexp
LRIlit
Germacrene B
1564
1557
trans-Sesquisabinene hydrate 1573 1577
Spathulenol
1576
1577
Caryophyllene oxide
1587
1587
Dodecyl acetate
1609
1610
Humulene epoxide II
1612
1613
Tetradecanal
1617
1614
a-Cadinol
1652
1652
Selin-11-en-4-a-ol
1655
1658
Norbornanol**
1662
-
cis-Nerolidyl acetate
1669
1665
Campherenol
1675
-
a-Bisabolol
1688
1685
(E,E)-Farnesal
1739
1737
HYDROCARBONS
Monoterpene
Sesquiterpene
Aliphatic
ALDEHYDES
Monoterpene
Sesquiterpene
Aliphatic
KETONES
Monoterpene
Aliphatic
ALCOHOLS
Monoterpene
Sesquiterpene
Aliphatic
ESTERS
Monoterpene
Sesquiterpene
Aliphatic
OXIDES and ETHERS
OTHERS
ALL
Persian Lime oils

Petitgrain
Cold-pressed
Commercial
Type B
(1)
(2)
0.54
-
-
tr
-
-
0.04
-
-
-
-
-
0.08
0.01
94.19
87.74
6.44
0.01
3.74
3.35
0.01
0.38
0.04
0.01
0.03
0.71
0.61
0.09
0.01
0.35
0.35
-
-
-
0.07
99.11
0.55
0.01
-
0.01
-
-
-
-
-
0.04
-
0.02
0.07
-
93.18
85.98
7.20
-
4.41
4.07
-
0.34
-
-
-
0.54
0.40
0.14
-
0.39
0.26
-
0.13
0.01
-
98.53
(3)
(4)
0.52
0.45
tr
0.02
-
-
0.01
-
0.01
-
-
-
0.03
-
-
-
-
-
tr
0.04
0.01
tr
0.02
0.06
0.06
0.02
tr
92.33
91.99
86.30
85.71
6.02
6.28
0.01
-
4.72
5.05
4.31
4.75
0.02
-
0.39
0.30
0.05
0.01
0.02
-
0.03
0.10
1.68
0.79
1.61
0.59
0.07
0.14
-
0.06
0.44
0.43
0.42
0.31
0.01
-
0.01
0.12
0.02
-
0.01
-
99.25
98.36
(5)
(6)
(7)
(8)
(9)
(10)
(11)
0.51
tr
-
0.01
0.01
-
0.03
-
-
-
-
-
0.06
0.01
90.75
84.69
6.05
0.01
5.06
4.71
0.01
0.34
0.03
-
0.03
1.44
1.37
0.07
-
0.41
0.40
-
0.01
0.03
0.21
97.93
1.33
-
-
0.15
-
-
-
-
-
-
-
-
-
-
60.18
51.92
8.26
-
25.87
25.32
-
0.55
0.87
-
0.87
9.05
9.02
-
0.03
1.91
1.91
-
-
0.32
-
98.20
0.31
-
0.58
2.09
-
0.28
-
0.09
0.17
-
-
-
0.09
-
53.29
47.22
6.05
0.02
20.68
20.07
-
0.61
0.82
0.44
0.38
5.86
4.6
1.25
0.01
9.42
9.40
-
0.02
2.86
0.28
93.21
0.09
-
-
-
-
-
-
-
-
-
-
-
-
-
93.26
89.70
3.56
-
3.03
2.94
-
0.09
-
-
-
0.75
0.75
-
-
1.26
1.26
-
-
-
-
98.30
0.12
-
-
tr
tr
-
0.02
-
-
-
0.01
-
0.09
0.02
92.46
87.78
4.67
0.01
3.53
3.37
0.02
0.14
0.02
0.01
0.01
1.09
0.98
0.11
-
1.45
1.44
0.01
-
0.01
-
98.56
0.11
-
-
0.05
0.01
-
0.03
-
-
-
0.01
-
0.10
0.02
91.52
86.56
4.95
0.01
4.23
4.03
0.02
0.18
0.04
0.02
0.02
0.95
0.83
0.12
-
1.68
1.66
0.02
-
0.12
-
98.54
0.12
0.01
0.02
0.05
0.05
0.08
89.99
85.89
4.10
3.80
3.64
0.16
0.01
0.01
0.75
0.57
0.18
1.32
1.32
0.03
95.90
* coeluted with -phellandrene; **2,3-dimethyl-3-(4-methyl-3-pentenyl)-2-norbornanol; a: crude; b: colorless; LRIexp : LRI measured on SLB-5MS column; LRIlit : FFNSC
1.3 GC/MS library, Wiley, USA, 2008; Adams RP, Identification of essential oil components by gas chromatography/massspectrometry, 4th Edn,: Allured Pub Corp; 2007;
Hochmuth, D.H., Joulain, D., Knig, W.A., 2002, MassFinder Software and Data Bank, University of Hamburg.
the volatile fraction; those determined in the two samples of

petitgrain represent 93% in the Egyptian sample and 98% in
the Mexican one; in the sample of distilled colorless Persian
lime the components identified represent about 96%. According to what previously determined (40), the main components
determined in Key lime oil type A and B, are in decreasing
amount order limonene (49-51%), b-pinene (18-21%) and gterpinene (8-10%); in Persian lime oils the main components
are the same, but limonene riches the value of 54-58% while,
with an opposite trend compared to Key lime oils, g-terpinene
is higher (~14%) than b-pinene (~12%). The total content of
sesquiterpene hydrocarbons is about 6-7% in Key lime oils,
while in Persian lime decreases to about 2/3: this is due to the
behavior of many single sesquiterpene hydrocarbons such as
b- and d-elemene, b-caryophyllene, germacrenes B and D, and
mainly (E,E)-a-farnesene; this behavior was also determined
by Dugo et al. (40) with the exception of d-elemene in Key lime
oil type B, which presented, in the single sample analyzed in

1997, a value significantly lower than what presently determined
(0.07% vs. 0.4%). Total oxygenated components are higher in
Key lime type B (~7%) compared to type A (~5%); in Persian
lime the content is about the same as Key lime type A. This is
probably due to the behavior of total aldehydes and alcohols. The
values determined for these two classes of substances slightly
differ from what previously determined (40), mainly for Key
lime type B and Persian lime. These differences are probably
due to the conditions used for the extraction of the oil.
In the two samples of petitgrain, as it happens for all the
citrus leaf oils, hydrocarbons are present at lower levels and
oxygenated compounds at higher levels, if compared to peel
oils. In the two oils, among oxygenated compounds, monoterpene aldehydes are predominant. Among the two samples
some important quantitative differences are however noticed.
These can be only explained by the different geographic
Bonaccorsi et al.
Figure 5. A) RP-HPLC Chromatotram of Key lime essential oil type A. B) RP-HPLC Chromatotram of key lime essential oil
type B. C) RP-HPLC Chromatotram of Persian lime essential oil. For peak identification see Table IV.
Table III. Enantiomeric distribution of some volatile components of the oils analyzed.
Key lime oils Persian lime oils
Type A
(1)a
Cold-pressed
Commercial
(2)a
(3)a
Type B
(4)a
(5)a
Petigrain
(6)a
(7)b
(8)a
(9)a
(10)
(11)
a-thujene

a-pinene*

camphene

b-pinene

sabinene

a-phellandrene

b-phellandrene

limonene

linalool

citronellal

terpinen-4-ol

a-terpineol*
S-(+)
1.00
R-(-)
99.00
R-(+)
21.83
S-(-)
78.17
1S,4R-(-) 92.80
1R,4S-(+) 7.20
R-(+)
3.58
S-(-)
96.42
R-(+)
15.23
S-(-)
84.77
R-(-)
50.86
S-(+)
49.14
R-(-)
64.94
S-(+)
35.06
S-(-)
2.82
R-(+)
97.18
R-(-)
72.70
S-(+)
27.30
S-(-)
-
R-(+)
-
S-(+)
29.22
R-(-)
70.78
S-(-)
84.08
1.06
98.94
22.55
77.45
92.89
7.11
4.47
95.53
15.44
84.56
53.30
46.70
65.59
34.41
2.93
97.07
68.69
31.31
77.04
22.96
29.15
70.85
85.37
0.62
99.38
22.27
77.73
93.85
6.15
3.71
96.29
15.37
84.63
52.19
47.81
73.70
26.30
2.91
97.09
65.23
34.77
81.04
18.96
20.78
79.22
79.89
1.15
98.85
22.72
77.28
94.60
5.40
4.45
95.55
14.83
85.17
54.53
45.47
68.54
31.46
2.86
97.14
66.11
33.89
73.41
26.59
29.13
70.87
78.49
0.48
99.52
23.34
76.66
91.78
8.22
4.24
95.76
15.18
84.82
58.35
41.65
69.09
30.91
2.91
97.09
66.01
33.99
72.79
27.21
29.14
70.86
83.77
12.62
87.38
78.08
21.92
28.52
71.48
31.05
68.95
85.68
14.32
16.66
83.34
30.05
69.95
2.43
97.57
58.80
41.20
53.43
46.57
95.87
4.13
77.62
12.45
87.55
77.80
22.20
27.81
72.19
5.89
94.11
31.32
68.68
15.45
84.55
26.67
70.33
0.75
99.25
70.71
29.29
89.25
10.75
88.29
11.71
80.40
0.50
99.50
29.96
70.04
89.27
11.73
10.34
89.66
18.60
81.40
55.16
44.84
55.08
44.92
2.66
97.34
63.45
36.55
78.68
21.32
19.34
80.66
77.36
0.74
99.26
28.57
71.43
88.37
11.63
10.10
89.90
19.28
80.72
55.82
44.18
54.48
45.52
2.69
97.31
63.94
36.06
80.50
19.50
20.41
79.59
78.04
1.28
98.72
28.61
71.39
88.96
11.04
9.20
90.80
18.61
81.39
56.15
43.85
30.45
69.55
2.59
97.41
68.01
31.99
80.49
19.51
23.64
76.36
79.79
1.56
98.44
32.56
67.44
87.80
12.20
9.50
90.50
20.35
79.65
49.06
50.94
27.89
72.11
4.07
95.93
52.34
47.66
29.52
70.48
77.26
R-(+)
14.63
20.11
21.51
16.23
22.38
19.60
22.64
21.96
20.21
22.74
15.92
* values determined by direct esGC.
Table IV. Composition of the oxygen heterocyclic fraction of the analyzed oils (ppm).

Key lime oils
Persian lime oils
N Group Compound Cold-pressed
Petigrain
Cold-pressed
Comm.
(6)
(8)
(9)
(10)
1 CUM Herniarin
1460 2970
3880
4670
3350
120
170
7860
2 PSO
Oxypeucedanin hydrate
780
1160
1690
1710
1620
-
-
500
3 CUM Citropten
7350 11740
10950
9230
5940
40
80
8860
4 PSO
Isopimpinellin
5670 10210
7300
6540
3010
60
60
3830
5 PSO
Bergapten
2000 3450
3000
3920
2160
140
120
3620
6 PSO
Byacangelicol
90
-
1020
460
80
-
-
220
7 PSO
Oxypeucedanin
260
-
10720
6660
7560
-
140
10540
8 PSO
Isoimperatorin
370
390
70
210
410
2010 1160 40
9 PSO
Imperatorin
830
900
380
430
660
2780 1720 120
10 CUM 5-Isopentenyloxy-7-methoxy-coumarin 4170 4830
2790
2670
2100
-
-
580
11 PSO
5-Isopentenyloxy-8-(2,3
epoxyisopentenyloxy)-psoralen
n.a.*
n.a.*
n.a.*
n.a.*
n.a.*
-
-
-
12 PSO
Cnidicin
340
90
250
110
70
-
-
-
13 PSO
8-Geranyloxy-psoralen
6520 8100
4470
4540
3800
-
-
1880
14 PSO
5-Geranyloxy-8-methoxy-psoralen
n.a.*
n.a.*
n.a.*
n.a.*
n.a.*
-
-
-
15 PSO
Bergamottin
37300 56130
36400
41590 25320 460
760
48830
16 CUM 5-Geranyloxy-7-methoxy-coumarin
41550 63320
43140
45350 27770 400
240
53040

Coumarins
54530 82860
60750
61920 39160 560
500
70340
Psoralens
54160 80410
65300
66160 44700 5450 3960 69580
4132
174
6415
2014
1977
49
5543
58
153
322
2220
190
1650
1140
560
70
4090
310
510
370
n.a.*
24
1164
n.a.*
33012
30049
n.a.*
40
990
n.a.*
26430
8120
40918
44168
12360
34320
85086
46680
Type A
(1)
ALL
Type B
(2)
108690 163270
(3)
126050
(4)
(5)
128080 83860
6010
(7)
4460
139920
*n.a.: standard was not available in sufficient amount for quantitative calculation
CUM = coumarin
PSO = psoralen
Bonaccorsi et al.
origin, since one sample is from Egypt, the other from Mexico.
In fact, further comments cannot be given, due to the lack of
information available in literature on industrial lime petitgrain
oils. The data available on laboratory petitgrain oils, relative to
samples of different geographic origin, obtained by different
techniques, provide a very large range of variability of their
composition. In all these oils, as confirmed in this study, are
present high amounts of neral and geranial.
Enantiomeric ratios (MDGC): Table III reports the
enantiomeric ratios of the volatiles determined in all samples
analyzed by MDGC and, when necessary, by direct esGC.
The CV% values determined from triplicates ranged from 0 to
9.78. The highest values were determined for (+)-camphene
and (-)-a-thujene (9.78 and 5.52 respectively). The elution
order of the a-thujene enantiomers was established based on
Casabianca et al. (41). The enatiomeric excesses determined
by the two techniques were in excellent agreement. Figure
4 shows the TIC chiral chromatogram obtained by MDGC
of a cold-pressed Key lime oil type A. In the same figure are
reported two enlargements of the enantio-selective separation
obtained by direct GC. In the first enlargement (B) it can be
observed the separation achieved for (+)/(-)-a-pinene. The
enatio separation of the two a-pinene enantiomers cannot
be accomplished by MDGC, since the diethyl-tert-butil-silyl
b-cyclodextrin column shows poor selectivity for these two
enantiomers, providing sufficient resolution by direct esGC,
while under the conditions applied in MDGC the resolution
of this critical pair is completely lost (42). For this reason the
peak of a-pinene is not transferred during MDGC analysis.
The enlargement (C) relative to a-terpineol shows the excellent separation by direct esGC, used for quantitative analysis
of the two enantiomers. It must be however underlined that
these enantiomers are usually well determined by MDGC for
all citrus oils (43), but in lime essential oil the separation is
compromised due to a probable coelution of the (+) isomer
with an unknown compound.
The MDGC system allowed to determine the enantiomeric
distribution of camphene, a- and b-phellandrene in lime essential
for the first time. The results determined in the five samples
of Key lime oils are in good agreement among each other; the
only exception is given by a-thujene determined in sample 4,
with a percentage of the (+)-a-thujene about double than what
determined in the other tow samples of Key lime Type B. The
results determined in Persian lime are also in good agreement.
Some differences are noticed between Key and Persian coldpressed lime oils: the enantiomeric excess of S-(-)-a-pinene
and of S-(-)-b-pinene is slightly lower in Persian lime oils; the
enantiomeric excess of R-(-)-b-phellandrene is higher in Key
lime. The enantiomeric distribution of b-phellandrene in the
two commercial samples (10,11) differs from that determined
in the cold-pressed oils of secure origin. As predicted, a slight
tendency to racemization is noticed for many components
analyzed in the colorless sample (sample 11) obtained by
distillation from the cold-pressed oil (sample 10).
The values here determined for the samples of cold-pressed
lime oils are in very good agreement with those previously
determined by Mondello et al. and by Dugo et al., at least for
the components investigated in these studies (42,44).
The enantiomeric ratios determined in the Egyptian and
Mexican petitgrain oils are similar for a-thujene, a-pinene,

camphene, a- and b-phellandrene, and a-terpineol; those
determined for all the other components analyzed differ
more or less evidently, and for sabinene it can be observed
the inversion of the enantiomeric excess. It is impossible to
provide further comments on these two oils, due to the lack
of information in literature.
Oxygen heterocyclic components (RP-HPLC/PDA):
To determine this fraction an Ascentis Express column was
employed, which is packed with partially porous particles of
2.7 mm based on Fused-Core technology. This consists of 1.7
mm solid core and a 0.5 mm porous shell, with the advantage
of a small diffusion path, compared to a totally porous particle,
which reduces axial dispersion of solutes and minimized peak
broadening allowing for higher resolving power (19). Table IV
reports the qualitative and quantitative composition (expressed
as mg/100 g of oil) of the oxygen heterocyclic components.
The quantitative analysis was tested for repeatability with
an average CV% value of 6.63%. Figure 5A-5C shows the
HPLC separations of the three samples of cold-pressed lime
oils. The total content of this class of components ranges from
8% to 16% in Key lime oils; in the sample of Persian lime of
secure origin the amount of these fractions ranges from 8%
to 14%; in commercial Persian lime the oxygen heterocyclic
components are less than 5% as confirmed by the CD values.
Thus, there are rising doubts on the genuineness of this oil,
which could have been diluted by the addition of distilled products, although this hypothesis cannot be proven by the other
analytical results. Sixteen components were identified in this
fraction, 4 coumarins and 12 psoralens. The main components,
as reported in literature, are bergamottin and 5-geranyloxy-7methoxycoumarin. In Key lime type A oxypeucedanin is absent
or present at levels lower than in Key lime type B and in the
Persian lime oil of secure origin. In fact, as previously observed
by Radford and Olansky (45), and by Dugo et al (40,46) respectively in lemon, bitter orange and lime oils, oxypeucedanin
is destroyed by contact with the water and juice phase during
extraction. When these conditions occur, the epoxy ring is
opened by hydrolysis, forming oxypeucedanin hydrate, which
is completely water-soluble.
Although some qualitative and quantitative differences are
noticed, the results here obtained are in agreement with those
previously determined (19, 40). The qualitative differences
are due to the presence of imperatonin and byacangelicol,
not identified in the article published in 1997 by Dugo et al.
The quantitative differences are mainly relative to the values
of herniarin, citropten and bergapten. It is possible to assume,
from the present results, that the sample used by Dugo et al.
(19), of unknown origin, could have been Key lime type A, due
to its content of oxypeucedanin and oxypeucedanin hydrate.
As predictable, oxygen heterocyclic components are absent
in Persian lime oil obtained by distillation (sample 11). The
CD value for this oil was almost zero.
The two samples of petitgrain (samples 6 and 7) have very
low amounts of oxygen heterocyclic compounds, among which
imperatorin and isoimperatorin are the most abundant.
Acknowledgements
The authors thank Shimadzu for its continuous support.

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Appendix
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where authors wish to place Figures and Tables.
Manuscripts should be e-mailed to: Prof. Luigi Mondello at jeorsubmission@yahoo.com.
In order to speed up processing times, authors are strongly encouraged to prepare their manuscript using the JEOR
Word Template.
Authors are encouraged to supply the names, addresses, and email addresses of 3-4 potential referees. Authors may
also mention persons who they would prefer not to review their paper.
Submission Checklist

Letter including statement of justification for publication

A single Word file containing text, tables, and graphic materials
Names of preferred referees
Supporting materials as appropriate
Completed Copyright Transfer Form
Preparation of Manuscript
Manuscripts must be written in English using American spelling. Manuscripts written in ambiguous English or
otherwise incomprehensible, in the opinion of the Editor, will be returned to the authors with the request to
resubmit once the language has been improved.
In order to achieve uniform presentation and to avoid unnecessary delays because of further inquiries, all
authors are invited to observe these guidelines.
JEOR Word Template

The use of the JEOR Word Template allows authors to view their paper in a style close to the final printed form.
All manuscripts will be fully typeset from the authors electronic files. It should be noted that due to defined
typesetting standards and the complex requirements of electronic publishing, the Publisher will not always be
able to exactly match the layout the author has submitted. The use of journal templates is preferable and its
adoption will speed the publication process.
The elements of the template are:
Title
The Title should be concise and specific, describing the nature of the paper. If the paper has been previously
reported in whole or in part at a scientific meeting, this should be stated as a footnote on the introductory page.
Authors Names
The Authors Names should have forename in full and middle initial(s) (e.g., Hubert H. Smith). Give full
addresses for each author, and connect each author with an address. Authors with the same address must be
given together. Do not list all of the authors and then all of their affiliations separately. Indicate with an asterisk
the author to whom all correspondence concerning the manuscript should be sent, and indicate email address
for this author. Specify very clearly the affiliation of each author.
Abstract
The Abstract is an important summary of the work because it is often the only portion of the paper read by
abstracting journals. As a consequence, the abstract should be written so that it can be used verbatim in an
abstracting journal. It should be a concise summary giving essential information, data, etc., and be intelligible
without reference to the paper itself. No references to cited publications are permitted in the Abstract, nor
should there be any numerical reference to a structure found in the text.
Key Word Index
The Key Word Index should provide relevant key words for both indexing and abstracting. For a specific plant,
list species name and plant family name, and also common name if well known. For reports on the analysis of an
essential oil, list essential oil composition and all the components found in amounts greater than 10%.
Introduction
The Introduction should present the object or reason for the investigation, and clearly states what is new in the
paper submitted. A summary of the pertinent literature should be included; however, only the relevant work
should be described in a brief, concise manner.
Experimental
The Experimental section should describe clearly and in sufficient detail the materials and methods used so that
the experiment could be reproduced by others. Only new techniques need to be described fully, while known
methods must have adequate references.
The Results should be presented in a clear, concise manner using tables and illustrations for clarity. Do not
list tabular data in the text. Do not list significant data figures for which the level of experimentation error is
unacceptable; e.g., for capillary GC data obtained from electronic integration, 1.4%, not 1.35 or 1.349% unless
this is a mean of a large number of analyses. Following presentation of the results they should be discussed and
interpreted where possible. The results of other studies of a similar or related nature should be compared with
only the most pertinent data and can be listed in tabular form for comparative purposes.
References
The References must be numbered consecutively in the text (one reference per number) and should be typed
in order on a separate page. Within the text, the reference should appear as follows: as described by Lincoln
et al. (5). A maximum of 30 references is suggested unless the manuscript is a review article. Recent review
articles can be used as a substitute for all but the most pertinent original articles. All references must be typed in
full, including all editors names for book citations, using the following style:
1.
2.
3.
4.
5.
6.
7.
B. M. Lawrence, Essential Oils 19881991. Allured Publ. Corp., Carol Stream, IL (1993).
B.M. Lawrence, A Study of the Monoterpene Interrelationships in the Genus Mentha with Special Reference to the Origin of Pulegone and Menthofuran.
Ph.D. Thesis, Rijksuniversiteit, Groningen (1978).
B.M. Lawrence and J.K. Morton, Cytological and Chemical Variation in Mentha. Paper No. AG/b-01, Vth International Essential Oil Congress, Sao
Paulo, Brazil (1971).
B.M. Lawrence, A further examination of the variation of Ocimum basilicum L. In: Flavors and Fragrances: A World Perspective. Edits., B.M. Lawrence,
B.D. Mookerjee and B.J. Willis, pp. 161170, Elsevier Sci. Publ. B.V., Amsterdam (1988).
D.E. Lincoln, M.J. Murray and B.M. Lawrence, Chemical composition and genetic basis for the isopinocamphone chemotype of Mentha citrata
hybrids. Phytochemistry, 8, 18571863 (1986).
L. Mondello, R. Shellie, A. Casilli, P.Q. Tranchida, P. Marriott, G. Dugo, Ultra-fast essential oil characterization by capillary GC on 50 m ID column.
J. Sep. Sci., 27, 699-702 (2004).
The International Fragrance Association Website. http://www.ifraorg.org/ (Feb 10, 2010)
Acknowledgments
The number of acknowledgments should be kept to a bare minimum.

Footnotes
Footnotes should be kept to a minimum. They should be indicated by a superscript number.
GC Data
All reported GC analyses must contain a description of the analytical procedures used including the make
and model number of the equipment, the column type and dimensions (e.g. OV-101 30 m x 0.22 mm fused
silica capillary column, film thickness 0.25 m; or 20 ft. x 3/4 inch stainless steel packed column, coated with
10% Carbowax 20 m on 80/100 mesh Chromosorb W NAW). The temperature programming conditions used
along with carrier gas flow rate must be described. It is no longer sufficient to refer to a previous publication
for a description of analytical conditions. A statement as to how the quantitative data was obtained should be
included.
In summary:
Column: dimensions (length, internal diameter), manufacturer and location, type of column (packed, capillary,
etc.), support material, film thickness.
Carrier gas: type, purity, flow-rate/linear velocity, inlet pressure and/or pressure programmes.
Temperatures: temperatures of injector, detector, oven (and temperature programmes)
e.g.: GC-FID analyses were carried out on a Shimadzu GC-2010 gas chromatograph operated with a split/
splitless injector and a Shimadzu AOC-20i autoinjector (Shimadzu, Kyoto, Japan). Column: SLB-5MS
(silphenylene polymer) 30 m x 0.25 mm x 0.25 m film thickness (Supelco, Bellefonte, IL, USA). Temperature
program: from 50250C (10 min) at 3C/min. Injection temperature: 250C. Injection volume: 1.0 L. Inlet
pressure: 100 kPa. Carrier gas: He, linear velocity (u): 30 cm/sec. Injection mode: split (50:1). FID (250C):
H2 flow: 50 mL/min; air flow: 400 mL/min; make up flow (N2/Air): 50 mL/min. Sampling rate: 40 msec. Data
handling was carried out by means of GCsolution 2.3 (Shimadzu).
GC/MS Data
In addition to compliance with the above GC instructions, add EI mode operating at 70 eV, scan time and
acquisition mass range. Add modifier if operating in CI mode. Specify type of mass analyzer, Scan mode.
Detection conditions, ions monitored in SIM and dwell time.

e.g.: GC/MS analyses were performed with a Shimadzu GCMS-QP2010 model gas chromatograph-mass
spectrometer equipped with an AOC-20i autoinjector. Column: SLB-5MS, 30 m x 0.25 mm ID x 0.25 m film
thickness. Temperature program: from 50C (2 min) to 250C (10 min.) at 3C/min. Injection temperature: 250C.
Injection volume: 1.0 L. Inlet pressure: 37.1 kPa. Carrier gas: He, linear velocity (u): 32.4 cm/sec. Injection mode:
split (10:1). MS interface temp.: 250C; MS mode: EI; detector voltage: 0.9 kV; mass range: 40-400 u; scan speed:
769 u/s; interval: 0.50 s (2 Hz). Data handling was made through GCMSsolution 2.5 (Shimadzu).
HPLC Data
All reported HPLC analyses must contain a description of the analytical procedures used including the make
and model number of the equipment, the column type and dimension (e.g. RP-18 150 x 2.1 mm HPLC column
packed with particles of 5 m). The solvents used, column temperature, flow rate and gradient program/isocratic
conditions must be described. Type of detector used, and specific conditions used. It is no longer sufficient to
refer to a previous publication for a description of analytical conditions.
e.g.: HPLC separation was carried out on a Shimadzu system equipped with two LC 10 AD Vp pumps, an
SPD-M10 Avp UV detector, a SCL-10-Avp controller, a CTO-20AC column oven thermostated at 30C and a
degasser DGU-14A, data were acquired and processed by LC-solution ver 3.3 software. The column used was an
Ascentis Express C18 150 mm x 4.6 mm i.d. with particle size of 2.7 m (Supelco, Bellefonte, PA). The injection
volume was 2 l, mobile phase consisted of Water, Acetonitrile, THF (85:10:5) (solvent A) and Acetonitrile,
Methanol, THF (65:30:5) (solvent B), the linear gradient profile was as follow: 0-5 min, 0% B, 5-25 min, 0-40%
B, 25-45 min, 40-90% B, 45-55 min, 90% B, 55-60 min, 0% B. Flow-rate was 1.0 mL/min, data were acquired
using a photodiode array detector in the range 190-370 nm and the chromatograms were extracted at 315 nm.
Time constant was 0.64 s and sample frequency 1.5625 Hz. Data acquisition was performed by Shimadzu LC
solution software ver 3.3.
HPLC/MS Data
In addition to compliance with the above HPLC instructions, specify inlet system, source (vaporizer and
capillary temperature, nebulising, auxiliary or ionizing gases, source voltage, CID voltage), mass analyzer (scan
mode, resolution and mass range), detection.
Spectra
The inclusion of MS, IR or NMR spectra of uncommon or newly characterized compounds is encouraged. If no
adequate MS or IR spectrum of a compound can be found in the readily accessible literature, then its inclusion
is also encouraged.
Chromatograms
The inclusion of a chromatogram adds substantial value to the paper, the incorporation of chromatographic
profiles is strongly encouraged.
Tables
Tables should be double-space typed in the same form as the manuscript and should be numbered using Roman
numerals; however, they should not be formatted within the text but should be included as an attachment, at
the end of the article. Each table should be on a separate page. The inclusion of retention indices in tables of
components identified is encouraged whereas the inclusion of retention times is not. Unless the quantitative data
is an average of more than 5 analyses, only one decimal place is acceptable. Averages of more than 5 analyses
can be presented in two decimal places. Tables should be typed as word documents only, using tabs, and not
the space bar. Tables should be typed using Microsoft Word program only. Do not use programs like Excel to
create tables, because eventually when you send the article to us on disk, we are not able to read anything that is
created with cells.
Keep the number of columns in a table as few as possible and keep the titles or headings concise. Essential
details in the title can be added as a footnote to the table. The compounds must be listed in the table in elution
order from the GC column. Compounds that can exist in isomeric form that have not been fully characterized
should have an asterisk and footnote stating correct isomeric form not identified. Unknowns, tentatively
identified compounds or those partially characterized as, for example, sesquiterpene hydrocarbon, can only be
included in the table if the MS data is included as a footnote to the table.
Figures
Figures can be high quality photographic prints or camera-ready original diagrams, graphs or drawings. Spectra
or chromatograms should be presented as high quality photographic prints or camera-ready computer drawn
representations. All figures should be accompanied by a descriptive phrase or sentence typed on a separate page.
All figure captions can be listed on the same page. The size of figures submitted should not exceed 6 x 10 inches.
Chemical Nomenclature
Use generally accepted chemical nomenclature. For example, the use of trivial terpene names is recommended:
a-pinene, spathulenol, b-bourbonene, etc. For IUPAC names, the author is requested to determine whether
there is a trivial name for the compound; if there is, it should be used.
Species Names
All experimental plants listed must be given their correct taxonomic classification, including the author citation;
e.g., Micromeria teneriffae Benth. Once cited in the text, the following reference to the species can be written
as M. teneriffae. Depositing a voucher specimen of each plant species in the herbarium of a reputable university
or institution is mandatory for all plants collected from the wild state. Also, if other Micromeria species are
mentioned after the genus has been introduced, then they may be cited as follows: M. benthamii: Webb. &
Berthel., M. biflora Benth., etc. When a sentence commences with a species name, the species should be written
in full, i.e. Micromeria biflora, not M. biflora.
Structural Formulae
All structural formulae should be drawn with the aid of a graphics software package, dry transfers, a template, or
some accurate structural design facsimile. Under each structure should be a boldface number. This is the same
number that should appear in the text in bold (within square brackets) after the compound has been named; e.g.,
4-ketoisophorone [8]. The inclusion of structural formulae of known compounds is discouraged unless they are
considered to be essential for a better understanding of the text.
NMR Data
NMR Data must be specified as either 1H-NMR or 13C-NMR. It is necessary to state the frequency of the
instrument, the solvent used, and the internal standard. Chemical shifts should be noted in d (ppm) values
relative to TMS. The type of signal should also be noted; e.g., singlet s, doublet d, triplet t, multiplet m, etc. Two
examples of NMR data presentation are:
1. 1H-NMR (250 CDCl3/TMS): d 0.87(d, CH3), d 0.89(d, CH3), d 1.28(s, CH3OH), d 1.93(bm, CH2OH), d
5.63(bs, HC=CH).
2. 13C-NMR (25.15 MHz CDC13): d 67.9(C-1), d 134.0(C-2), d 133.7(C-3), d 42.6(C-4), d 22.7(C-5), d
37.7(C-6), d 32.3(C-7), d 32.1(C-8), d 20.1(C-9), d 19.1(C-10)a decoupled experiment. If coupled
experiments are performed, then the type of signal should be included.
MS Data
Presentation of data should indicate the method used; e.g., MS (this is for EIMS), CIMS, GC/MS, and the
ionizing energy. An example of data presentation can be seen as follows: MS, 70 eV, 210C, m/z(rel. int.):
154[M]+(6), 139(28), 136(20), 121(22), 111(27), 93(100), 84(30), 79(50), 77(48), 71(45), 69(35), 55(22), 43(37).
Qualitative Analysis/Identification of Compounds

Compound identification must be supported by at least two methods of analysis. They can be mass spectrometry
and gas chromatography with calculation of retention indices. Retention Indices should be included in the
tabular data and compared to literature data obtained on the same stationary phase.
Some available databases of LRI are suggested here:

http://www.alluredbooks.com/idofesoilbyg.html
http://www.odour.org.uk/index.html
http://www.shimadzu.com/products/lab/ms/oh80jt00000059kf.html
http://www.wiley.com/WileyCDA/WileyTitle/productCd-0470425210.html
Quantitative Analysis/Composition of the Sample

Authors may report simple GC percentages obtained using FID or TCD as detector, assuming response factors
equal to unity for all the components. This procedure is not possible if mass spectrometer and NPD are used
as detectors. Authors are strongly encouraged to provide a true quantification at least of major components
(greater than 10% of the whole oil) of the analyzed sample. If standards are not commercially available, for the
measurement of response factors, compounds can be grouped into chemical classes (hydrocarbons, aldehydes,
etc.) and subclasses (monoterpenes, sesquiterpenes, etc.) and semi-quantification can be carried out using one
(or more) pure standard for each class of similar components. Refer to Results and Discussion paragraph for
an appropriate number of significant figures to be reported.
Abbreviations
Standard abbreviations should be used throughout the manuscript, particularly in the experimental section.
Some examples of common abbreviations are:
C, IR, GC, GC/MS, HPLC, TLC, FTIR, 1H-NMR, 13C-NMR, CD, l max (solvent), [a]D Temp, nm, cm-1, L,
C/min, m, kg/ha, mL, L, mg, min, eV, ppm, FID, TC, EC, etc. (Note: No periods are needed).
Copyright
The copyright of all papers remains the property of the author unless transferred to JEOR, but the Journal has
the sole right of publication for a period of six months from the date of publication. Papers appearing in JEOR
may be published elsewhere after the six-month grace period has elapsed, provided that acknowledgment of the
original publication is given.
Manuscript Checklist
Manuscript prepared according to the journal guidelines
Experimental part includes the description of all the techniques and conditions used, so that methods can
be reproduced by others.
Correct taxonomic classification of all plant material has been given
A voucher specimen of each plant specie collected from the wild state has been deposited in the
herbarium of a reputable university or institution
Identification of components has been carried out using at least two methods (e.g. MS and GC data
correlated with LRI)
LRI compared with those reported in referred literature or reputable databases
Proofs
Prior to publication, galley proofs will be sent to the contact author for checking. Corrections should be
restricted to typographical or similar errors. Modifications to the original text should be avoided at all costs,
otherwise the publication of the article will be seriously delayed. Galley proofs should be returned to the
publisher within three days of receipt. Please provide e-mail address to receive proof.
Reprints
The contact author will be e-mailed a PDF file of the paper to use for a limited number of reprints. To order
additional reprints of the article, please check with the publisher regarding the prices.
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The Journal of Essential Oil Research

Volume 23, Number 5

Editor in Chief Luigi Mondello

Dipartimento Farmaco-chimico, Facolt di Farmacia

Founding Editor Brian M. Lawrence

Karl Heinz Kubeczka

2/Journal of Essential Oil Research

Vol. 22, September/October 2010

The Journal of Essential Oil Research

Volume 23, Number 5

Leaf Secretory Structure and Volatile Compounds of Eugenia

Anti-inflammatory Activity of Some Essential Oils

Chemical Composition and Cytotoxic Activity of Essential Oil

Chemical Composition and Antibacterial Activity of Origanum

Chemotaxonomic Importance of Sesquiterpenes and Flavonoids

Anti-inflammatory and Antioxidant Activity of a Methanolic

Seasonal Evaluation and Chemical Composition of Volatile

Chemical Composition of Essential Oils from Ripe and Unripe

Appendix: Submission Guidelines

Journal of Essential Oil Research

Vol. 22, September/October 2010

Now available in PRINT

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The Journal of Essential Oil Research

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J. Essent. Oil Res., 23 (September/October 2011)

Leaf Secretory Structure and Volatile Compounds of

2,000 species, and their distribution ranges from southern

*Address for correspondence: cris.pvictor@gmail.com

Rec: May 2011

1041-2905/11/0001-05$14.00/0 2011 Allured Business Media

Journal of Essential Oil Research/1

Relative area (%)b

Vol. 23, September/October 2011

in the State of Rio de Janeiro and protecting arbustive plants

two to three individuals were taken from the third or fourth

were sectioned in longitudinal and transverse planes by the

leaf segments separated by maceration in solution of acetic

water, stained with 1% Alcian Blue in 50% ethanol, and

Results and Discussion

adjacent to the leaf epidermis, and in the petiole, they more

a-pinene and b-pinene in E. copacabanensis when compared

to data provided in other studies for E. candolleana DC., E.

Anestor Mezzomo and Marco Lacerda, who lent us their photos of

We especially acknowledge the FAPERJ for financial support, the

F.R. Scarano, Marginal plants: functional ecology at the Atlantic

Journal of Essential Oil Research/5

23. S.M. Gomes, N.S.D.N. Somavilla, K.M. Gomes-Bezerra, S.C.

6/Journal of Essential Oil Research

Vol. 23, September/October 2011