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Essential oil analysis and anticancer activity of

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 Journal of Ethnopharmacology 103 (2006) 99–102

Essential oil analysis and anticancer activity of leaf essential oil

of Croton flavens L. from Guadeloupe
Muriel Sylvestre a , André Pichette a , Angélique Longtin a , Francine Nagau b , Jean Legault a,∗
aLaboratoire d’analyse et de séparation des essences végétales, Département des Sciences fondamentales,
Université du Québec à Chicoutimi, Chicoutimi, Qué., Canada G7H 2B1
b Laboratoire de chimie des substances naturelles, Département de Chimie, Université des Antilles et de la Guyane,

B.P. 250 - 97157, Pointe-à-Pı̂tre Cedex, Guadeloupe

Received 22 October 2004; received in revised form 8 June 2005; accepted 26 July 2005
Available online 15 September 2005

Abstract
The leaf essential oil of Croton flavens L., a native plant from the Caribbean area used in traditional medicine, was extracted by hydrodistillation.
The composition of the volatile fraction of this essential oil was determined by GC and GC–MS analyses. We have identified 47 compounds,
of which viridiflorene (12.22%), germacrone (5.27%), (E)-␥-bisabolene (5.25%) and ␤-caryophyllene (4.95%) are the main components. The
anticancer activity of this extract was tested on human lung carcinoma cell line A-549 and human colon adenocarcinoma cell line DLD-1. Croton
flavens leaf essential oil was found to be very active against both tumor cell lines, with a GI50 of 27 ± 4 ␮g/ml for A-549 and 28 ± 3 ␮g/ml for
DLD-1. Three compounds identified in the leaf essential oil, ␣-cadinol (3.97%), ␤-elemene (1.53%) and ␣-humulene (1.06%) are cytotoxic against
tumor cell lines.
© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Croton flavens L.; Leaf essential oil; Anticancer activity; Viridiflorene; Germacrone; (E)-␥-bisabolene

1. Introduction as an abortificant. This plant is also used to contract the uterus

Croton flavens L. (Euphorbiaceae), commonly called ti
baume or copahu in French West Indies islands and yellow
croton in English Caribbean territories (USDA, 2002), is an
endemic plant of the Caribbean archipelago (Missouri Botanical
Garden, 2003). The plant is a very common aromatic shrub
of 1–3 m high, which grows on dried coastlines and chalky
grounds. Its leaves (2–10 cm long) are oval to closely oval
and young branches are covered with white-yellowish hairs
(Fournet, 1978). When leaves are crushed, they give off a scent
due to the presence of an essential oil. Croton flavens is widely
used in folk medicine in the West Indies (Morton, 1981), and
on the Netherlands Antilles island of Curaçao it is one of the
medicinal plants most frequently used by the Black and Creole
populations. Locally, fresh green leaves infusions are prepared
as bush tea, but other parts of the plant are also used in traditional
medicine. Its main applications are against menstrual pain and
during post-partum (Morton, 1971). In 2000, a study on essential
oils of different parts of the Croton flavens plant from Curaçao,
including the leaves, was reported by Woerdenbag (Woerdenbag
et al., 2000). However, the composition of Croton flavens leaf
essential oil from Guadeloupe has not been studied previously.
Moreover, the widespread use of Croton flavens in traditional
medicine prompted us to determine the anticancer potential of
the essential oil of this plant. So far, to the best of our knowl-
edge, no such study exists, but the anticancer activity of some
monoterpenes and sesquiterpenes was reported in the literature
(Ren and Gould, 1998; Legault et al., 2003). In this article, we
report the chemical composition of Croton flavens leaf essential
oil from Guadeloupe and the results of its anticancer testing.
2. Materials and methods
2.1. Plant material and essential oil

∗ Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545 5012. The leaves of Croton flavens were collected at Petit Canal,
E-mail address: jean legault@uqac.ca (J. Legault). Guadeloupe in July 2002. The specimen was identified by Dr.

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.07.011
100 M. Sylvestre et al. / Journal of Ethnopharmacology 103 (2006) 99–102
Félix Lurel (Département de biologie végétale, Université des
Antilles et de la Guyane). A voucher specimen of this plant
(Fournet 4180) has been deposited at the Guadeloupe INRA-
National Park herbarium.
2.2. Extraction of the essential oil
Essential oil was obtained from freshly harvested leaves
(441.1 g) by hydrodistillation for 2 h in a Clevenger apparatus
(Pharmacopée française, 1985). The oil was dried over anhy-
drous sodium sulfate and stored under nitrogen at 4 ◦ C.
2.3. Analysis conditions
The essential oil was analysed by GC on a gas chromato-
graph Hewlett-Packard 5890 (FID detector) equipped with a
polar Supelcowax 10 column and an apolar DB-5 column
(30 m × 0.25 mm × 0.25 ␮m). Analyses by GC–MS were per-
formed on a Hewlett-Packard mass spectrometer 5972 at 70 eV
coupled to an HP 5890 equipped with a DB-5 column (same
as above). The temperature program was 40 ◦ C for 2 min, then
2 ◦ C/min to 210 ◦ C and held constant for 33 min. For the injec-
tion (split injector), 5 ␮L of essential oil was diluted in 500 ␮L
of hexane, and 5 ␮L of this diluted solution was injected. Iden-
tification of volatile constituents was made on the basis of their
retention indices (Kovats, 1965) and their mass spectra, which
were compared with reference data (Adams, 2001).
2.4. Cell culture
Cell lines A-549 and DLD-1 (human lung carcinoma and
human colon adenocarcinoma, respectively) were obtained from
the American Type Culture Collection (ATCC). Cells were
maintained at 37 ◦ C in a 5% CO2 atmosphere. Both cell lines
were grown in minimum essential medium complete with
Earle’s salts and l-glutamine (Mediatech Cellgro, VA) and sup-
plemented with 10% fetal bovine serum (Hyclone), vitamins
(1×), penicillin (100 IU/ml) and streptomycin (100 ␮g/ml),
essential amino acids (1×) and sodium pyruvate (1×) (Medi-
atech Cellgro).
2.5. Cytotoxicity assay
Exponentially growing cells were plated in 96-well
microplates (Costar, Corning Inc.) at a density of 5 × 103 cells
per well in 100 ␮l of culture medium and were allowed to adhere
for 16 h before treatment. Increasing concentrations of essen-
tial oil in ethanol (Sigma–Aldrich) were then added (100 ␮l per
well). The final concentration of ethanol in the culture medium
was maintained at 0.5% (v/v) to avoid solvent toxicity. The cells
were incubated for 48 h in the presence or absence of essential
oil. Cytotoxicity was assessed using the resazurin reduction test
as described by O’Brien et al. (2000). Fluorescence was mea-
sured on an automated 96-well Fluoroskan Ascent FlTM plate
reader (Labsystems) using excitation and emission wavelengths
of 530 and 590 nm, respectively. The cell survival curves were
calculated from cells incubated in the presence of 0.5% ethanol.
Cytotoxicity is expressed as the concentration of drug inhibiting
cell growth by 50% (GI50 ).
3. Results and discussion
3.1. Chemical composition of the essential oil
Croton flavens leaf extraction produces a dark yellow and
viscous essential oil. The physico-chemical characteristics
of this essential oil are presented in Table 1. The chemical
composition of the leaf essential oil is listed in Table 2.
Chromatographic analysis of the essential oil of Croton
flavens collected in Guadeloupe shows 55 compounds, but
we could only identify 47 components. The volatile extract
contains 77.27% sesquiterpenes, including 28.58% oxygenated
sesquiterpenes. Viridiflorene (12.22%), germacrone (5.27%),
(E)-␥-bisabolene (5.25%) and ␤-caryophyllene (4.95%) are the
major constituents of the oil. Some sesquiterpenes, representing
13.62% of the total composition, are not identified. With regards
to their mass spectra, unidentified compounds D and E seem
to be isomers. Monoterpenes are weakly represented (2.27%)
with one oxygenated monoterpene: (E)-piperitol (0.31%). GC
analysis of Croton flavens leaf essential oil from Curaçao
(Woerdenbag et al., 2000) showed that the major components
of this oil were ␣-pinene (16.0%), isocaryophyllene (9.3%),
ar-curcumene (7.9%), unknown C15 H24 O (4.8%) and spathu-
lenol (4.5%). Surprisingly, none of these components were
found in Croton flavens leaf essential oil from Guadeloupe.
Indeed, of the 47 components identified in Croton flavens
essential oil from Guadeloupe, only 10 minor components
(8.9%) are also found in Croton flavens essential oil of from
Curaçao. The main identified constituents common to both
oils are germacrene D, ␤-elemene, allo-aromadendrene and
␣-humulene.
3.2. Anticancer activity of Croton flavens essential oil
The anticancer properties of Croton flavens leaf essential oil
were assessed against cell lines A-549 (human lung carcinoma)
and DLD-1 (human colon adenocarcinoma). Both cell lines
were submitted to growing concentrations of Croton flavens leaf
essential oil for 48 h. Fig. 1 shows the percentage of cell sur-
vival versus the concentration of essential oil. Concentrations
of oil for which each cell line’s growth was inhibited by 50%
(GI50 ) were calculated from the curve. GI50 values obtained were
27 ± 4 ␮g/ml for A-549 and 28 ± 3 ␮g/ml for DLD-1. These low
GI50 values confirm the strong anticancer properties of Croton
flavens leaf essential oil. Very few of the compounds found in
Croton flavens leaf essential oil have been tested for anticancer
Table 1
Physico-chemical characteristics of the essential oil of Croton flavens
Oil yield (w/w) (%) 0.18
Refractive index (at 20 ◦ C) 1.5065
Density (g/ml) 0.810
 M. Sylvestre et al. / Journal of Ethnopharmacology 103 (2006) 99–102 101

properties. However, it has been reported in literature that ␣- Table 2 (Continued )

cadinol (3.97%) shows selective toxicity against human colon Components RI DB-5a RI Spwaxb Percent
adenocarcinoma cell line HT-29 (He et al., 1997) and that ␤-
Germacrone 1693 2217 5.27
elemene (1.53%) is cytotoxic against tumor cells (Duh et al., 14-Hydroxy-␣-humulene 1718 2475 1.02
1999). Moreover, we found that ␣-humulene is active against Unidentified Gj 1732 – 2.81
A-549 and DLD-1 cell lines with GI50 values of 62 ± 2 and Unidentified Hk 1755 – 1.06
71 ± 2 ␮M, respectively (Legault et al., 2003). However, no such Sinensall 1781 2348 0.72
cytotoxicity assays have been performed for the other major Total 93.16
components of Croton flavens leaf essential oil (viridiflorene, a Retention indices on apolar DB-5 column.
germacrone and (E)-␥-bisabolene). b Retention indices on polar Supelcowax 10 column.
c m/z (relative intensity): 41(100), 43(81), 119(61), 55(53), 161(49), 91(48),

105(45), 79(43), 67(31), 179(27), 204(18).

Table 2 d m/z (relative intensity): 222(M+ , 8), 41(100), 43(87), 55(51), 79(50), 95(46),
Chemical composition (%) of the leaf essential oil of Croton flavens 69(38), 112(21), 109(20).
e m/z (relative intensity): 220(M+ , 4), 43(100), 41(95), 91(71), 59(65), 79(61),
Components RI DB-5a RI Spwaxb Percent
119(57), 161(54), 105(49), 204(18), 189(13).
␣-Thujene 933 1032 0.27 f Correct isomer not characterized.

␣-Sabinene 974 1128 0.22 g m/z (relative intensity): 220(M+ , 0.5), 43(100), 41(66), 79(64), 67(48),

␦-3-Carene 1007 1154 0.18 55(27), 91(25), 109(15), 107(15), 159(9), 162(5), 202(1).
h m/z (relative intensity): 220(M+ ), 43(100), 79(60), 41(45), 91(35), 67(31),
p-Cymene 1025 1284 0.75
␥-Terpinene 1066 1257 0.54 55(20), 106(13), 162(3), 202(0.5).
i m/z (relative intensity): 220(M+ , 2), 43(100), 41(79), 91(48), 79(45), 67(32),
(E)-Piperitol 1201 1713 0.31
␣-Copaene 1375 1496 0.50 55(34), 105(28), 119(18), 205(10), 145(9), 159(9), 177(5), 187(5).
␤-Bourbonene 1382 1521 0.46 j m/z (relative intensity): 220(M+ , 10), 41(100), 79(85), 119(71), 91(70),

␤-Elemene 1389 1589 1.53 55(59), 67(51), 105(50), 123(28), 159(22), 147(22), 133(21), 187(13), 177(12),
␤-Caryophyllene 1414 1589 4.95 202(11).
␤-Copaene 1428 1585 0.24 k m/z (relative intensity): 220(M+ , 1), 41(100), 67(46), 55(40), 81(35), 91(33),

␣-Guaiene 1438 1589 2.10 109(33), 123(28), 138(22), 145(9), 202(8), 159(8), 187(7).
l Correct isomer not characterized.
Dehydroaromadendrane 1450 1643 1.59
␣-Humulene 1452 1666 1.06
Allo-aromadendrene 1460 1639 1.21
␥-Muurolene 1479 1690 0.80
In conclusion, we have identified most of the volatile con-
Germacrene D 1482 1708 2.45
Valencene isomer 1492 1713 1.97 stituents of Croton flavens leaf essential oil and evaluated the
Valencene 1495 1718 1.24 oil’s anticancer activity. The chemical composition of Guade-
4-Epi-cubebol 1496 1956 0.77 loupe Croton flavens leaf essential oil is different from that of
Bicyclogermacrene 1496 – Curaçao Croton flavens oil since only 10 minor constituents
Viridiflorene 1505 1736 12.22
from the 47 identified components of Guadeloupe Croton flavens
␣-Muurolene 1505 1729 0.92
␦-Guaiene 1508 1723 0.58 oil are common to both oils. Our results clearly show that this
␤-Bisabolene 1512 1736 0.37 essential oil is active against both tumor cell lines tested (A-549
␥-Cadinene 1516 1758 2.16
7-Epi-␣-selinene 1516 1758
(Z)-␥-Bisabolene 1518 1736 0.79
␦-Cadinene 1526 1758 2.31
(E)-␥-Bisabolene 1535 1761 5.25
␣-Cadinene 1539 1787 0.35
Germacrene B 1555 1819 3.64
Germacrene D-4-ol 1572 2050 3.97
Caryophyllene oxide 1577 1971 3.55
Humulene epoxide I 1596 2050 1.82
Humulene epoxide II 1600 2027 0.44
␤-Oplopenone 1600 2064 0.60
Unidentified Ac 1608 – 0.60
Unidentified Bd 1619 – 0.97
Unidentified Ce 1623 – 1.65
Caryophylla-4(14),8(15)-dien-5-olf 1631 2294 0.60
␶-Cadinol 1638 2184 1.69
␶-Muurolol 1638 2170 1.27
Gossonorol 1638 2312 0.73
␣-Muurolol 1643 2196 0.70
Allo-aromadendrene epoxide 1646 2054 1.46
␣-Cadinol 1652 2229 3.97 Fig. 1. Cytotoxic activity of Croton flavens leaf essential oil. Anticancer proper-
Unidentified Dg 1661 2085 2.12 ties of essential oil against cell lines A-549 (human lung carcinoma) and DLD-1
Unidentified Eh 1666 2151 3.79 (human colon adenocarcinoma). Values represented are the means of three deter-
Unidentified Fi 1669 – 0.62 minations.
 102 M. Sylvestre et al. / Journal of Ethnopharmacology 103 (2006) 99–102
and DLD-1). These cytotoxic properties could be explained, in
part, by the presence of ␣-cadinol, ␤-elemene and ␣-humulene.
However, further studies aimed at determining the anticancer
properties of the other major constituents of Croton flavens
leaf essential oil, as well as identifying the oil’s unknown com-
pounds, are necessary to fully understand its bioactivity.
Acknowledgments
We express our gratitude to F.-I. Jean and H. Gagnon for
help and suggestions. We wish to thank Professor G.J. Collin
for comments. M. Sylvestre is grateful to Région Guadeloupe,
Conseil Général de la Guadeloupe and AFFDU France, for her
postdoctoral training scholarship.
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