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Chemical and Biological Study of Essential Oils from Eugenia pruniformis

Cambess., an Endemic Species from Brazilian Atlantic Forest

Article  in  LATIN AMERICAN JOURNAL OF PHARMACY · August 2012

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 Latin American Journal of Pharmacy Regular Article
(formerly Acta Farmacéutica Bonaerense) Received: June 19, 2012
Revised version: July 15, 2012
Lat. Am. J. Pharm. 31 (6): 830-4 (2012) Accepted: July 16, 2012

Chemical and Biological Study of Essential Oils from Eugenia

pruniformis Cambess., an Endemic Species from Brazilian Atlantic Forest
Ricardo D.D.G. ALBUQUERQUE 1, Luis A.C. TIETBOHL 1, Caio P. FERNANDES 1,3,
Pedro P. COUTEIRO 2, Débora N. EIRIZ 3, Marcelo G. SANTOS 4, Moacélio V. SILVA-FILHO 5,
Gutemberg G. ALVES 6,7, Róber BACHINSKI 6,7 & Leandro ROCHA 1,2,3*

1 Laboratório de Tecnologia de Produtos Naturais, 2 Programa de Pós-Graduação em

Ciências Aplicadas a Produtos para Saúde, Departamento de Farmácia e Administração Farmacêutica,

3 Departamento de Tecnologia Farmacêutica, 5 Departamento de Farmácia e Administração Farmacêutica,

Faculdade de Farmácia, Universidade Federal Fluminense,

Rua Doutor Mário Viana 523, CEP 24241-000, Niterói, RJ, Brazil
4 Departamento de Ciências, Faculdade de Formação de Professores, Universidade do

Estado do Rio de Janeiro, Dr. Francisco Portela 1470, CEP 24435-000, São Gonçalo, RJ, Brazil
6 Unidade de Pesquisa Clínica, Hospital Universitário Antônio Pedro, Universidade Federal Fluminense,

Rua Marques de Paraná, 303/emergência - 4º andar, Centro, CEP: 24033-900, Niterói, RJ, Brazil
7 Programa de Pós-graduação em Biologia das Interações (PPBI)/UFF Universidade Federal Fluminense,

Morro do Valonguinho S/N0, CEP 24001-970, Niterói, RJ, Brazil

SUMMARY. Eugenia pruniformis Cambess. is an endemic species from Brazilian Atlantic Forest. Essential
oils from leaves and fruits from this species were obtained by hydrodistillation and analyzed by GC-
MS/CG-FID. In all, 25 compounds were identified, with predominance of sesquiterpene hydrocarbons in
both plant parts. The major compounds were β-caryophyllene, bicyclogermacrene, germacrene D, δ-
cadinene and α-copaene. Antioxidant activity was performed for essential oil from leaves using ORAC
method, showing value of 0.30 ± 0.06 mmol TE/g. Anticholinesterasic evaluation was also performed for
this oil, indicating that it inhibited acetylcholinesterase, showing an IC50 of 1798 μg/mL. These results in-
dicate that this essential oil may be considered as a potential source of substances for Alzheimer’s disease
Treatment. To our knowledge, these are the first contributions to biological and phytochemical characteri-
zation of E. pruniformis, an almost unexplored species from Brazilian Atlantic Forest.

INTRODUCTION
Restinga is a type of habitat originated from
quaternary marine deposits and represented by
usually arbustive-herbaceous vegetation cover-
ing typically sandy soils 1. It is characterized by
large sandy coastal plains of sedimentary origin
that are rippled by rows of dunes isolating la-
goons, lakes, ponds, bogs and marshes. Such a
diversity of physical conditions gives rise to a
great diversity of habitats that are colonized by
a great variety of vegetal communities 2. Restin-
ga contains many species in common with the
Atlantic forest, but presents diverse physiologi-
cal responses to a drier habitat 3.
On this context, Restinga de Jurubatiba Na-
tional Park is an area for permanent preserva-
tion of restinga habitats (Brazilian sandy coastal
plain vegetation) on Rio de Janeiro State, Brazil.
This area (22° to 22°23’S and 41°15’ to 41°45’
W) comprises the municipalities of Macaé, Cara-
pebus and Quissamã 2.
One of the most representative families in
Restinga de Jurubatiba National Park is Myr-
taceae. This family is represented in Brazil by 24
genera and 927 species, of which 707 are en-
demic 4. Species from this family are frequently
used in folk medicine, being especially impor-
tant in the country and also one of the domi-
nant woody families in the Atlantic Forest. One

KEY WORDS: Acetylcholinesterase, β-caryophyllene, Essential oil, Eugenia pruniformis, Myrtaceae, restinga.
* Author to whom correspondence should be addressed. E-mail: lean@vm.uff.br

830 ISSN 0326-2383


of the synapomorphies of this family is the
leaves with spherical secretory cavities contain-
ing terpenoids and other aromatic, spicy-
resinous compounds 4,5.
Essential oils have been assayed for various
biological activities. Studies indicated that essen-
tial oils from several species are able to inhibit
the growth of microorganisms related to dental
caries and food spoilage, including Gram-nega-
tive and Gram-positive bacteria 6. They are also
cited by many authors as being responsible for
medicinal properties, such as antifungal, anti-
rheumatic, diuretic and anti-inflammatory activi-
ties 7,8.
The genus Eugenia belongs to family Myr-
taceae and is described as a significant source of
terpenoids, essential oils and showed antimicro-
bial, larvicidal, nociceptive and hypothermic ac-
tivities 9-11. Essential oils from this genus also
demonstrated anticholinesterasic and antioxidant
activities 12,13. Eugenia pruniformis Cambess. is
mainly found in Atlantic Forest of Southeastern
of Brazil 5. To our knowledge, there are no pre-
vious studies about its chemical composition or
biological activities.
Neurodegenerative diseases involving im-
pairment of cognitive functions, such as the
Alzheimer’s disease (AD), are the most common
health problems in elderly populations 14. There
is currently no cure and treatment provides only
relief of the symptoms in some patients 15.
Thus, the aim of this study was to identify
substances from essential oils of leaves and
fruits. It was also evaluated anticholinesterasic
and antioxidant activities of essential oil from
leaves of E. pruniformis.
MATERIALS AND METHODS
Plant material
Leaves and fruits of Eugenia pruniformis
were collected from four specimens in Restinga
de Jurubatiba National Park, Rio de Janeiro
State, Brazil, in open Clusia scrub vegetation (S
22°12’40.85’’ – W 41°35’14.61’’; S 22°12’40.85’’ –
W 41°35’14.61’’; S 22°12’36.36’’ – W
41°35’20.18’’; S 22° 12’34.90’’ – W 41°35’21.04’’).
This species was identified by the botanist Dr.
Marcelo Guerra and a voucher specimen (M.G.
Santos 2206) was deposited at the herbarium of
the Faculdade de Formação de Professores (Uni-
versidade Estadual do Rio de Janeiro, Brazil).
Extraction of the essential oil
Leaves and fruits were obtained from four
specimens of E. pruniformis. Then, fresh leaves
(3250 g) and fresh fruits (160 g) were individu-
Latin American Journal of Pharmacy - 31 (6) - 2012
ally ground with distilled water using an auto-
matic blender (Ética Equipamentos Científicos
S.A, Brazil). Hydrodistillation method was em-
ployed using Clevenger apparatus and each
plant material was placed in a 5 L flask. At the
end of extraction, essential oils were collected,
dried over anhydrous sodium sulphate and
stored at 4 °C for further analyses.
Chemical analysis of the essential oils
Essential oils were analyzed by a GCMS-
QP2010 (Shimadzu) gas chromatograph
equipped with a mass spectrometer using elec-
tron ionization. The gas chromatographic (GC)
conditions were as follows: injector temperature,
260 °C; detector temperature, 290 °C; carrier gas
(Helium), flow rate 1 mL/min and split injection
with split ratio 1:40. Oven temperature was ini-
tially 60 °C and then raised to 290 °C at a rate of
3 °C/min. One microliter of each sample, dis-
solved in CH2Cl2 (1:100 mg/µL), was injected at
RTX-5 column (i.d. = 0.25 mm, length 30 m, film
thickness = 0.25 µm). The mass spectrometry
(MS) conditions were voltage, 70 eV and scan
rate; 1 scan/s. The retention indices (AI) were
calculated by interpolation of retention times of
the substances to the retention times of a mix-
ture of aliphatic hydrocarbons (C7-C40) (Sigma)
analyzed in the same conditions 16. The identifi-
cation of substances was performed by compari-
son of their retention indices and mass spectra
with those reported in literature 17. The MS frag-
mentation pattern of compounds was also
checked with NIST mass spectra libraries. Quan-
titative analysis of the chemical constituents was
performed by flame ionization gas chromatogra-
phy (CG/FID), under same conditions of GC/MS
analysis and percentages obtained by FID peak-
area normalization method.
Anticholinesterasic activity
Anticholinesterasic activity was performed
according to method described by Ellman with
some modifications 18,19, using a 96-well mi-
croplate. A total volume of 200 µL of test media
was composed by 65 µL of Phosphate buffered
saline (PBS), 60 µL of 5,5’-dithiobis-(2-nitroben-
zoic acid) (DTNB) 1,5 mM, 25 µL of electric eel
acetylcholinesterase (Sigma) (AchE) 0.2 U/mL,
25 µL of essential oil from leaves and 25 µL of
acetylthiocholine iodide (ASCh). Different con-
centrations of essential oil from leaves and posi-
tive control (eserine), dissolved in methanol,
were tested for a dose-response curve and posi-
tive control. The negative control, used to calcu-
late the percentage of inhibition, was measure
831
ALBUQUERQUE R.D.D.G., TIETBOHL L.A.C., FERNANDES C.P., COUTEIRO P.P., EIRIZ D.N., SANTOS M.G. et al.
using only methanol sample, without inhibitor
or chemical test. The spontaneous hydrolysis of
substrate was calculated replacing the enzyme
solution by PBS, with methanol sample. The ab-
sorbance was measured kinetically 17 times in
13 min at 412 nm.
Antioxidant activity
The antioxidant evaluation was performed
using oxygen radical absorbing capacity assay
(ORAC) 20 using a 96-well microplate reader
(Fluostar Optima – Fluoroluminometer, BMG
Labtech). The antioxidant activity of essential oil
from leaves was measured by fluorescence de-
cay of fluorescein (Sigma), induced by 2,2’-azo-
bis(2-amidinopropane) dihydrochloride (AAPH).
Measurements were done by following time
course of the fluorescence decay, in order to es-
timate antioxidant activity. Using Trolox stan-
dard, a calibration curve was generated using
the net area under the curve AUC (AUCTrolox –
AUCblank). ORAC values were calculated using
the linear regression and expressed as Trolox
Equivalent (TE). TE = slope regression curve
(sample) / slope regression curve (Trolox). The
experiments were realized in triplicate.
RESULTS AND DISCUSSION
After extraction, the essential oil obtained
from fresh leaves presented a bright green color
and yielded 0.160 %. Essential oil obtained from
fresh fruits showed a greenish-blue colour and
yielded 0.003 %.
In all, 25 components were identified, mainly
constituted by sesquiterpene hydrocarbons (80.2
% in leaves and 89.9 % in fruits). β-caryophyl-
lene was the major substance found in both oils,
corresponding to 46.9 % and 27.8 % of total rel-
ative composition of leaves and fruits, respec-
tively. The compounds bicyclogermacrene (14.9
% in leaves, and 7.6 % in fruits), germacrene-D
(14.2 % in fruits), δ-cadinene (3.4 % in leaves
832
and 12.4 % in fruits) and α-copaene (5.5 % in
leaves and 11.4 % in fruits) were also represen-
tative on these essential oils. The constituents
and relative amounts of essential oils obtained
from leaves and fruits of E. pruniformis are indi-
cated in Table 1.
The assay performed for anticholinesterasic
substances indicated that essential oil from
leaves of E. pruniformis was able to inhibit the
acetylcholinesterasic with an IC50 of 1798
µg/mL (R2 = 0.8420). The positive control (eser-
ine) showed an IC50 of 15 µg / mL (R 2 =
0.9752) (Fig. 1).
A variety of plants and essential oils has
been reported to show anticholinesterasic activi-
ty 21. Moreover, some Myrtaceae plants from
Restinga de Jurubatiba National Park, including
β-caryophyllene rich essential oil from leaves of
Eugenia sulcata 12, exhibited anticholinesterasic
activity 22-24. These results may indicate a possi-
ble relationship between the presence of β-
caryophyllene with the existence of anti-
cholinesterase activity. On this context, sub-
stances with the ability to inhibit this enzyme
may be relevant to treatment of neurodegenera-
tive disorders such as Alzheimer’s disease (AD),
since the rationale for the current major thera-
peutic approach to this disease is directed to the
inhibition of acetylcholinesterase 25.
Several studies have demonstrated that phyto-
chemicals and complex natural mixtures, such as
essential oils, may contain numerous antioxidant
molecules 26,27. Essential oils with antioxidant ca-
pacity have been invoked to promote diverse ef-
fects, such as preservation of food and cosmetic
formulations 8. The antioxidant activity of essen-
tial oil from leaves of Eugenia pruniformis
showed a value of 0.30 ± 0.06 mmol TE/g. It is
especially interesting from a therapeutic ap-
proach that substances with antioxidant effects,
such as the essential oil from leaves of E. pruni-
formis has potential aplication to protect humans
Figure 1. Linear regression
between AchE activity (%)
x natural logarithm of ef-
fective concentration of es-
sential oil from leaves of E.
pruniformis and positive
control (eserine).
 Latin American Journal of Pharmacy - 31 (6) - 2012

Nº Compound Leaves (%) Fruits (%) RI leaves RI fruits

1 α-copaene 5.5 11.4 1374 1377

2 β-caryophyllene 46.9 27.8 1417 1422
3 Aromadendrene 1.6 - 1439 -
4 α-humulene 4.4 4.2 1452 1456
5 9-epi-β-caryophyllene 2.0 1.5 1464 1463
6 Bicyclogermacrene 14.9 7.6 1500 1499
7 δ-cadinene 3.4 12.4 1522 1526
8 Maaliol 1.5 - 1566 -
9 Spathulenol 1.6 - 1577 -
10 Globulol 8.9 - 1590 -
11 Viridiflorol 3.3 - 1592 -
12 cubeban-11-ol 1.4 - 1595 -
13 Rosifoliol 2.2 - 1600 -
14 α-cubebene - 2.5 - 1351
15 β-cubebene - 2.4 - 1392
16 α-bergamotene - 2.2 - 1437
17 β-Z-farnesene - 1.0 - 1444
18 trans-cadina-1(6),4-dieno - 0.5 - 1476
19 germacrene-D - 14.2 - 1483
20 α-muurolene - 0.9 - 1502
21 β-bisabolene - 1.3 - 1509
22 caryophyllene oxide - 1.7 - 1586
23 1-epi-cubenol - 1.0 - 1631
24 Cubenol - 1.5 - 1645
25 α-cadinol - 0.7 - 1658

Monoterpene hydrocarbons - -
Oxygenated monoterpenes - -
Sesquiterpene hydrocarbons 80.2 89.9
Oxygenated sesquiterpenes 17.4 4.9
Total identified (leaves) 97.6 -
Total identified (fruits) - 94.8

Table 1. Relative abundance of essential oil constituents from leaves and fruits of Eugenia pruniformis.

from oxidative stress damage and may slow the
progression of Alzheimer’s disease by minimizing
neuronal degeneration based on it ability to scav-
enge oxidants and free radicals 15,28.
CONCLUSIONS
The drugs approved and licensed by the U.S.
Food and Drug Administration (FDA) for treat-
ment of cognitive dysfunction and memory loss
associated with Alzheimer’s disease (AD) have
been generally acetylcholinesterase inhibitors,
such as tacrine, donepezil, rivastigmine and
galantamine. However, none of these are com-
pletely effective to cure AD patients and often
present adverse effects. Thus, there is a continu-
ous need to discover new anticholinesterasic
agents from natural resources 21,29. The present
study describes the chemical composition, anti-
cholinesterasic activity and antioxidant proper-
ties of essential oil from an almost unexplored
species Eugenia pruniformis. This multi target
approach is especially interesting regarding to
Alzheimer’s disease treatment, based on the pro-
posal of different mechanisms of action with the
same purpose. On this context, essential oil
with enhanced activities may be helpful in the
treatment of this neurodegenerative disease
based on the hypotheses that it results from a
deficit of cholinergic function or oxidative stress
in the brain.
As part of our ongoing studies to discovering
anticholinesterasic and antioxidant substances
with potential therapeutic effects for Alzheimer’s
disease treatment, further investigations will be
carried out for essential oils from Myrtaceae
species from Restinga de Jurubatiba National
Park with the aforementioned assays.
Acknowledgements. The authors thank CAPES and
FAPERJ for their financial support.

833
 ALBUQUERQUE R.D.D.G., TIETBOHL L.A.C., FERNANDES C.P., COUTEIRO P.P., EIRIZ D.N., SANTOS M.G. et al.
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