Chemical Composition and Anti-Proliferative Properties of Bursera graveolens Essential Oil

Abstract

Bursera graveolens is a wild tree of commercial importance native to the Neotropics, which has been widely used in folk medicine. In the present study, the chemical composition and anti-proliferative properties of the essential oil from B. graveolens were assayed. The chemical composition of the essential oil, determined by GC-MS, was complex and dominated by limonene (26.5%). Bursera oil inhibited the growth of MCF-7 breast tumor cells as well as amastigotes of L. amazonensis, with IC50 values of 48.9 +/- 4.3 and 36.7 +/- 4.7 microg/mL, respectively. In addition, the cytotoxicity of the oil was 103.9 +/- 7.2 microg/mL against peritoneal macrophages from BALB/c mice. These results demonstrate that the essential oil from B. graveolens is a promissory antiproliferative product.

Full text

Chemical Composition and Anti-proliferative Properties of Bursera
graveolens Essential Oil
Lianet Monzotea,*, Gabrielle M. Hillb, Armando Cuellarc, Ramón Scullc and William N. Setzerb,*
aInstitute of Medicine Tropical “Pedro Kourí”, Havana City, Cuba
bDepartment of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
cInstitute of Pharmacy and Food, Havana City, Cuba
monzote@ipk.sld.cu;wsetzer@chemistry.uah.edu
Received: September 23rd, 2012; Accepted: September 30th, 2012
Bursera graveolens is a wild tree of commercial importance native to the Neotropics, which has been widely used in folk medicine. In the present study, the
chemical composition and anti-proliferative properties of the essential oil from B. graveolens were assayed. The chemical composition of the essential oil,
determined by GC-MS, was complex and dominated by limonene (26.5%). Bursera oil inhibited the growth of MCF-7 breast tumor cells as well as
amastigotes of L. amazonensis, with IC50 values of 48.9 ± 4.3 and 36.7 ± 4.7 g/mL, respectively. In addition, the cytotoxicity of the oil was 103.9 ± 7.2
g/mL against peritoneal macrophages from BALB/c mice. These results demonstrate that the essential oil from B. graveolens is a promissory anti-
proliferative product.
Keywords: Bursera graveolens, Leishmania amazonensis, Tumor cell, Essential oil, Cytotoxicity, Chemical composition.
Burseraceae is a moderate-sized family composed by 17-18 genera
and about 540 species of flowering plants. The actual numbers
differ according to the time period in which a given source is
written describing this family. Burseraceae is also known as the
torchwood family, the frankincense and myrrh family, or simply the
incense tree family. The family includes both trees and shrubs, and
is native to tropical regions of Africa, Asia and the Americas [1].
Bursera graveolens Triana & Planch is one species of the family
with commercial importance. The plant is a wild tree, native to
tropical America (from Mexico south into Peru) and it is widely
used in folk medicine for stomach ache, as sudorific, and as
liniment for rheumatism [1a].
In general, Burseraceae trees or shrubs are chemically characterized
by resins, which contain triterpenoids and ethereal oils. Therefore, a
complex mixture of compounds can be present in the plant, which
impart characteristic aromas and medicinal properties. Herein was
studied the activity of the oil of B. graveolens against a cancer cell
line (MCF-7) and against a protozoal parasite (Leishmania
amazonensis) as models to evaluate the anti-proliferative properties
of the Bursera oil.
The composition of the essential oil was determined by GC-MS and
the major components were identified (Table 1). The Bursera oil
was dominated by limonene (26.5%), β-elemene (14.1%), (E)-β-
ocimene (13.0%), and menthofuran (5.1%). The EO inhibited the
growth of both breast tumor cells and amastigotes of L.
amazonensis (Table 2). In addition, murine macrophages were also
treated to determine safety and selectivity. The CC50 showed higher
values compared with IC50 of the tumor cell line and Leishmania
parasite.
Cancer is one of the most dreaded diseases worldwide and the
incidence is on the rise in both developing and developed countries.
Approved therapies include chemotherapy, radiotherapy, and
surgery. In spite of advances in therapeutic strategies, cancer still
remains a major cause of death. Research is going forward in order
to obtain better drugs, more effective and with fewer side effects
[2].
On the other hand, leishmaniasis, caused by parasites of Leishmania
genus, currently affects more than 12 million people in 88 countries
[3]. The control of leishmaniasis remains a serious problem. As a
zoonotic infection, transmission is difficult to interrupt, although
some attempts to reduce vector and mammalian reservoir
populations have been successful. There are currently no vaccines
for leishmaniasis prevention. The drugs available for leishmaniasis
therapy are toxic, expensive, and frequently ineffective [4]. The
problems previously mentioned, emphasize the importance of the
development of new drugs against leishmaniasis [5].
Currently, natural products have played a significant role in drug
discovery and development especially for agents against cancer and
infectious diseases. Natural compounds possess highly diverse and
complex molecular structures compared to small-molecule synthetic
drugs and often provide highly specific biological activities derived
from the rigidity and high number of chiral carbons [6]. In
particular, essential oils from plants have showed anti-tumor [7] and
anti-leishmanial [8] activity. In this sense, the anti-proliferative
potential of essential oil from B. graveolens has been explored in
our laboratories as a potential chemotherapeutic agent.
Essential oils are composed by a complex mixture of compounds. A
previous investigation of B. graveolens has revealed the essential oil
from the stems to contain limonene, α-terpineol, menthofuran,
carvone, germacrene, γ-muurolene, carveol and pulegone as major
constituents [9], whereas another stem sample was composed
largely of viridiflorol (70.8%) with a smaller amount of limonene
(4.8%) [10]. A previous examination of the leaf oil showed it to be
composed largely of limonene (30.7%), (E)-β-ocimene (20.8%),
and β-elemene (11.3%) [11]. Thus, the composition of the oil
evaluated in this current investigation is similar to this previous
report, with limonene as the main compound. B. simaruba leaf oil
NPC Natural Product Communications 2012
Vol. 7
No. 11
1531 - 1534

1532 Natural Product Communications Vol. 7 (11) 2012 Monzote et al.
Table 1: Chemical composition of essential oil from Bursera graveolens.
RIa Com
p
ound
b
%RIaCom
p
ound %
856 (3Z)-Hexenol 0.5 1425 2,5-Dimethoxy-p-cymene 0.1
867 (2Z)-Hexenol 0.1 1429 β-Copaene 0.2
941 α-Pinene 0.1 1439 α-Guaiene 0.1
976 Sabinene 0.3 1445 unidentifiede, m/z= 95, 81, 55 0.5
978 β-Pinene 0.2 1454 α-Humulene 0.3
993 Myrcene 0.7 1459 (E)-β-Farnesene 0.1
1005 α-Phellandrene 0.1 1464
cis-Cadina-1(6),4-diene 0.1
1030 Limonene 26.5
1466 cis-Muurola-4(14),5-diene 0.2
1042 (Z)-β-Ocimene 0.9 1470 4,5-di-epi-Aristolochene 0.2
1051 (E)-β-Ocimene 13.0 1477 β-Chamigrene 0.9
1061 γ-Terpinene trc 1482 Germacrene D 0.4
1089 p-Cymenene tr 1488 β-Selinene 1.4
1100 α-Pinene oxide 0.4 1498 α-Selinene 2.4
1105 (E)-6-Methyl-3,5-heptadien-2-oned 0.1 1506 trans-β-Guaiene 1.8
1120 trans-p-Mentha-2,8-dien-1-ol 0.1 1511 Germacrene A 3.9
1133 (Z)-Epoxyocimene 0.2 1517 γ-Cadinene 0.4
1135 cis-p-Mentha-2,8-dien-1-ol tr 1519 7-epi-α-Selinene 0.1
1143 (E)-Epoxyocimene 0.4 1526 δ-Cadinene 1.2
1154 Menthone 0.3 1534 trans-Cadina-1,4-diene 0.1
1165 Menthofurand 5.1 1539 α-Cadinene tr
1176 cis/trans-Isopulegoned 0.7 1544 α-Calacorene tr
1190 α-Terpineol 0.3 1578 Spathulenol 0.2
1194 neo-Dihydrocarveol 0.1 1593 Viridiflorol 0.2
1196 trans-Dihydrocarvone 0.1 1605 unidentified, m/z= 222, 204, 161, 109, 95 0.5
1210 unidentified,
m/z= 154, 137, 119, 109 1.1 1628 1-epi-Cubenol 0.3
1218 trans-Carveol 0.3 1645 τ-Muurolol 1.2
1230 cis-Carveol 0.1 1648 α-Muurolol (= Torreyol) 0.3
1238 Pulegone 1.7 1654 Cedr-8(15)-en-9α-ol 1.1
1241 Carvone 0.1 1658 α-Cadinol 2.4
1253 Piperitone 0.1 1658 unidentified,
m/z= 220, 205, 187, 121 0.5
1275 unidentified,
m/z= 97, 95, 72 0.7 1664 unidentified, m/z= 220, 205, 164, 93 0.7
1289 Limonen-10-ol 0.1 1669 unidentified,
m/z= 220, 205, 187, 157 0.5
1292 Thymol tr 1674 unidentified,
m/z= 220, 205, 93 0.5
1297 Perilla alcohol 0.1 1711 unidentified,
m/z= 220, 187, 159, 145, 133 1.8
1340 Piperitenone 0.1 1719 unidentified,
m/z= 220, 205, 187, 159 0.7
1372 Isoledene 0.1 1725 unidentified,
m/z= 220, 205, 187, 177, 159 2.0
1377 α-Copaene 2.3 1740 Cedr-8(15)-en-9α-ol acetate 0.3
1385 β-Bourbonene 0.9 1762 Benzyl benzoate 0.1
1393 β-Elemene 14.1 1765 Phenanthrene tr
1411 α-Gurjunene 0.2 1957 Palmitic acid 0.2
1420 β-Ylangene 0.2 Total Identified 90.5
a RI = retention index based on a homologous series of alkanes. b Unless indicated otherwise, all components identified based on RI and MS. c tr = trace (< 0.05%). d Identification
based on MS only. e Unidentified components with > 0.5 % are listed.
Table 2: Antileishmanial activity and cytotoxicity of essential oil from B. graveolens.
Products IC50a  SDb (g/mL) CC50c  SDb (g/mL)
MCF-7 Cells Leishmania
B. graveolens oil 48.9 ± 4.3 36.7 ± 4.7 103.9 ± 7.2
Positive controls 16.8 ± 1.7d 0.03  0.003e 5.8  0.5e
a IC50 = Concentration of drug that inhibited 50% of growth. b SD = Standard deviation.
c CC50 = Concentration of drug that caused 50% cell mortality.
d Positive control: Tingenone. e Positive control: Amphotericin B
has also been shown to be rich in limonene [12]. Limonene, a
natural cyclic monoterpene, is one of the most common constituent
of several citrus oils, which have been used especially in
aromatherapy [13]. Germacrene D, but not limonene, has been
found to be an abundant component in the leaf oils of many Bursera
species [14], but is a relatively minor component of B. graveolens
in this current study.
B. graveolens oil showed in-vitro cytotoxic activity against the
MCF-7 cancer cell line as well as Leishmania, with IC50 values less
than 50 µg/mL; one-third as high as the IC50 for normal
macrophages, which demonstrated the selectivity degree of the oil.
These properties of B. graveolens have not, to our knowledge, been
previously reported. However, it has been found that limonene, the
main component of B. graveolens leaf oil, has exhibited in-vitro anti
proliferative action on BW 5147 murine lymphoma cells [15], but
was inactive on MCF-7 human adenocarcinoma cells [16].
Limonene has shown also antiparasitic activity against Leishmania
amazonensis both in vitro and in vivo [17]. The mechanism of
action ascribed to limonene have been associated with the apoptosis
induction and increase of NO levels in the cell culture [18], which
could explain the activity shown against Leishmania. Notably, the
other major components in B. graveolens leaf oil, β-elemene and
β-ocimene, have also shown cytotoxic activity. β-Elemene has
demonstrated cytotoxic activity against murine P388 lymphocytic
leukemia [19], human A2780 ovarian carcinoma [20], and human
HEp-2 laryngeal carcinoma cells, while β-ocimene has shown
cytotoxic activity against human MDA-MB-231 and Hs 578T breast
carcinoma cell lines [21]. The activity of B. graveolens oil is a
complex mixture (Table 1) and the combination of components may
be acting synergistically or antagonistically [16], and therefore the
activities of the oil are not likely due to only the major components.
In-vitro studies have shown amphotericin B (IC50 = 0.14 μg/mL) to
be more active against Leishmania mexicana promastigotes than B.
graveolens oil, but Bursera oil has comparable IC50 values to
commonly used antileishmanial drugs such as pentostam (IC50 =
10,000 μg/mL) and aminosidine (IC50 = 54 μg/mL) [22]. In
addition, B. graveolens oil was more active (IC50 < 50 μg/mL) than
other essential oils studied recently, such as Ocimum gratissimum
(IC50 = 75 μg/mL) and Lippia sidoides (IC50 = 89 μg/mL) against
Leishmania spp. [23].
Against MCF-7 breast tumor cells, the oil showed lower activity in
comparison with conventional drugs, such as tamoxifen (IC50 = 19.7
μg/mL) [24] or doxorubicin (IC50 = 6.6 μg/mL) [25]. However,

Chemical and anti-proliferative properties of Bursera graveolens Natural Product Communications Vol. 7 (11) 2012 1533
other essential oils studied including Satureja thymbra, Sideritis
perfoliata, Laurus nobilis, Pistacia palestina and Salvia officinalis
did not showed appreciable activities against this cell line at
concentrations as high as 400 µg/mL [26]. Other essential oils like
Schinus molle and Schinus terebinthifolius [27], however, have
shown comparable cytotoxic activities on MCF-7 cells (IC50 = 47
and 54 μg/mL, respectively) to Bursera oil.
In conclusion, this study demonstrates the utility of essential oils as
promissory alternative antiproliferative products. Further
evaluation of essential oil from B. graveolens against experimental
models in animals are in progress in our laboratories.
Experimental
Plant Material: B. graveolens was collected in Lisa, La Habana,
Cuba, during September 2010. A voucher specimen (No. 42675)
was deposited in the National Ecology and Systematic Institute,
Havana, Cuba. The essential oil was obtained by hydrodistillation
of the aerial parts of the plant, using Clevenger type equipment.
Gas Chromatographic – Mass Spectral Analysis: The B.
graveolens essential oil was analyzed by GC-MS using an Agilent
6890 GC with Agilent 5973 mass selective detector [MSD, operated
in the EI mode (electron energy = 70 eV), scan range = 45-400 amu,
and scan rate = 3.99 scans/sec], and an Agilent ChemStation data
system. The GC column was an HP-5ms fused silica capillary with
a (5% phenyl)-polymethylsiloxane stationary phase, film thickness
of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm.
The carrier gas was helium with a column head pressure of 48.7 kPa
and a flow rate of 1.0 mL/min. Inlet temperature was 200°C and
interface temperature was 280°C. The GC oven temperature
program was used as follows: 40°C initial temperature, hold for 10
min; increased at 3°C/min to 200°C; increased 2°/min to 220°C. A
1 % w/v solution of the sample in dichloromethane was prepared
and 1 μL was injected using a splitless injection technique.
Identification of the oil components was based on their retention
indices determined by reference to a homologous series of n-
alkanes, and by comparison of their mass spectral fragmentation
patterns with those reported in the literature [28] and stored on the
MS library [NIST database (G1036A, revision
D.01.00)/ChemStation data system (G1701CA, version
C.00.01.080]. The percentages of each component are reported as
raw percentages based on total ion current without standardization.
Cell and Parasite Cultures: Human MCF-7 adenocarcinoma cells
(ATCC No. HTB-22) were grown in 25 cm2 tissue culture flasks
with feeding medium consisting RPMI 1640 medium with L-
glutamine and 7.5% NaHCO3, 100,000 units of penicillin and 10.0
mg streptomycin per liter, 15 mM Hepes at a pH of 7.35, and
supplemented with 10% fetal bovine serum at 37°C in a 5% CO2
incubator. Medium was replaced every two days to ensure optimum
growth conditions.
L. amazonensis (MHOM/77BR/LTB0016) was kindly provided by
the Department of Immunology, Oswaldo Cruz Foundation
(FIOCRUZ), Brazil. Parasites were routinely isolated from mouse
lesions and maintained as promastigotes at 26ºC in Schneider’s
medium (SIGMA, St. Louis, MO, USA) containing 10% heat-
inactivated fetal bovine serum (HFBS) (SIGMA, St. Louis, MO,
USA), 100 g of streptomycin/mL, and 100 U of penicillin/mL. The
parasites were used after three in-vitro passages.
Anti-Tumoral Cell Assay: In-vitro cytotoxic activity of the essential
oil on MCF-7 cells was carried out using the MTT assay [29]. Cells
were plated in 96-well cell culture plates at a concentration of 1.0 
105 cells per well for a final volume of 100 µL. Plates were allowed
to incubate for 48 hours in a 5% CO2 environment at 37˚C. After
48 hours, the medium was removed by suction from each well, and
100 µL of fresh medium containing 1.0 μL or 0.5 μL of DMSO
solution of the essential oil (1% w/w in DMSO) to give final
concentrations of 100 µg/mL and 50 μg/mL, respectively, of
essential oil. All tests were done in quadruplicate. Medium and
DMSO control wells were also included as negative controls;
tingenone [30] was used as a positive control. After 48 hours, the
medium and compound mixture was removed by suction and 100
µL of a 0.83 mg/mL MTT solution in medium (2 mL of 0.05 g
MTT in 10 mL of 10 mM phosphate buffered saline, pH 7.36, and
10 mL of medium) was added to each well and allowed to incubate
for 4 hours. After 4 hours, the MTT mixture and medium were
removed by suction, and 100 µL of DMSO was added to each well
to dissolve the formazan crystals. The plates were then incubated
for 15 minutes at 37ºC and the absorbance in each well was
determined with a microplate reader (Molecular Devices
SpectraMax Plus384) at 570 nm. Average absorbances, standard
deviations, and percent kill ratios (%killcmpd/%killDMSO) were
calculated. Median inhibitory concentration (IC50) values were
determined using the Reed-Muench method [31].
Anti-Amastigote Activity: Macrophages were harvested from
peritoneal cavities of normal BALB/c mice in RPMI medium
(Sigma). A volume of 1 mL of cells were plated at 106/mL in and
incubated at 37C under an atmosphere of 5% CO2 for 2 h. Non-
adherent cells were removed and stationary-phase L. amazonensis
promastigotes were added at a 4:1 parasite/macrophage ratio. The
cultures were incubated for 4 h, washed to remove free parasites
and 10 µL of different essential oil concentrations were added
between 6.25 and 50 µg/mL, two times, for 48 h. Control cultures
were included, which were treated with 10 µL of distilled water.
The cultures were then fixed with absolute methanol, stained with
Giemsa, and examined under light microscopy [32]. The number of
intracellular amastigotes and the percentage of infected
macrophages were determined. The infection rates were obtained by
multiplying the percentage of infected macrophages by the number
of amastigotes per infected macrophage. The results were expressed
as percent of reduction of the infection rate (%IR) in comparison to
that of the controls [33].
Macrophage Cytotoxicity Assay: Resident macrophages were col-
lected from peritoneal cavities of normal BALB/c mice in ice-cold
RPMI 1640 medium (SIGMA, St. Louis, Mo, USA), supplemented
with antibiotics, seeded at 30,000 cells/well and allowed to adhere
for 2 h at 37C in 5% CO2. After non-adherent cells were removed
by washing with PBS, dilutions of the essential oil, in 1 μL of
DMSO, were added in 200 μL of medium with 10% HFBS and
antibiotics. The macrophages were treated with six concentrations
of the product and cultures treated with 1 μL of DMSO were
included as controls. The cytotoxicity was determined after 3 days
of incubation using the colorimetric assay with 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
(SIGMA, St. Louis, MO, USA). MTT solutions were prepared at 5
mg/mL in PBS, filtered and sterilized at the time of use and 15 μL
of each solution were added to each well. After incubation for an
additional 4 h, the formazan crystals were dissolved by addition of
100 μL of DMSO. The optical density was determined using an
EMS Reader MF Version 2.4-0 at a test wavelength of 560 nm and
a reference wavelength of 630 nm [34]. The 50% cytotoxic
concentration (CC50) was obtained from dose-response curves fit to
data by means of the equation for the sigmoidal Emax model.
Selectivity indices (SI) were then calculated by dividing the CC50
for peritoneal macrophages of BALB/c mice by the IC50 for
Leishmania amastigotes [35].

1534 Natural Product Communications Vol. 7 (11) 2012 Monzote et al.
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