1048

PHYTOTHERAPY RESEARCH
Phytother. Res. 17, 10481053 (2003)
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/ptr.1295
S. R. M. LIMA ET AL.

In vivo and in vitro Studies on the Anticancer

Activity of Copaifera multijuga Hayne and
its Fractions
Sylvia R. M. Lima1, Valdir F. Veiga Junior2, Herick B. Christo2, Angelo C. Pinto2 and
Patricia D. Fernandes1,*
1
2

Departamento de Farmacologia Bsica e Clnica, ICB

Instituto de Qumica, Centro de Tecnologia, Bloco A. Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

Copaiba oil resin (COR) obtained from Copaifera multijuga Hayne has been used in popular medicine as an
antinammatory and for the treatment of bronchitis, ulcers and cancer. The aim of this study was to evaluate
the action of COR and its fractions on the inhibition of lung metastasis and tumour growth induced by B16F10
melanoma cells in mice and cytotoxicity in vitro using Trypan Blue exclusion method and MTT conversion.
Mice which have received subcutaneously B16F10 cells developed a solid tumour that reached a peak at
17 days. Together with the increase in tumour growth we also observed an increase in the number of lung
nodules. There was a positive correlation between the in vitro cytotoxic assay and in vivo antitumour activity.
The oral administration of COR (at 2 g/Kg in the days 3, 5, 7, 10, 12 and 14 after inoculation of tumoral cells)
reduced tumour growth by 58% and tumour weight by 76%. At the same dose COR reduced the number of
lung nodules by 47.1%. In vitro experiments showed that COR incubated with the melanoma cell line reduced
cell viability in a concentration and time-dependent manner. Diterpenic and sesquiterpenic fractions or reconstituted oil induced cytotoxicity. Our results shows that COR and its fractions have tumouricidal activity in the
melanoma cell line in both models in vivo and in vitro. Copyright 2003 John Wiley & Sons, Ltd.
Keywords: Copaifera multijuga Hayne; copaiba; melanoma; cytotoxicity.

INTRODUCTION
Indians from the North and Northeast of Brazil have
been using copaiba oil resins since the 19th century.
Botanically classied as Leguminosae, genus Copaifera,
Copaiba oil is popularly known as copaiba, copaiva,
pau-de-oleo (Veiga Jr and Pinto, 2001). The most
popular species are Copaifera ofcinalis L., Copaifera
langsdori Desf., Copaifera reticulata Ducke and
Copaifera multijuga Hayne (Pio Correa, 1984).
Recent studies have demonstrated the effects of
copaiba oil resin in antinammatory processes and
ulcers (Basile et al., 1988; Veiga Jr. et al., 2001). Fractions obtained from the oil resin were tested in several
models and demonstrated antioedematogenic activity
in rat paw edema induced by carrageennan (Shimizu et
al., 1990), bactericidal activity in S. mutans and P. acnes
(Kang et al., 1992).
The extracts of several plants which have been used
in folk remedies have been tested on a variety of experimental tumours. In 1997, Moraes et al. described
that Copaifera langsdori Desf. inhibited Walker sarcoma 256 growth in rats. Copaifera multijuga which has
* Correspondence to: Dr P. D. Fernandes, Department of Pharmacology,
Universidade Federal do Rio de Janeiro. PO Box 68016, 21941-970. Rio
de Janeiro, RJ, Brazil.
E-mail: patfern@farmaco.ufrj.br
Contract/grant sponsor: CNPq.
Contract/grant sponsor: FAPERJ.
Contract/grant sponsor: PRONEX-FINEP; contract/grant number: 400296, 0888-96.
Copyright 2003 John Wiley & Sons, Ltd.
Copyright 2003 John Wiley & Sons, Ltd.

been used as an antinammatory, recently showed antitumour activity in mice (Ohsaki et al., 1994). The lack
of success in cancer treatment is often due to the growth
of secondary, metastatic lesions in distant organs. The
majority of patients die in consequence of metastasis.
In this work we studied the effects of orally administered Copaifera multijuga Haynes oil resin (COR) on
C57/black6 mice injected intravenously or subcutaneously with melanoma cell line (B16F10). In vitro assays
were conducted in order to evaluate the cytotoxic effects of COR by Trypan blue exclusion method and by
MTT conversion. The effects of diterpenic (DF) and
sesquiterpenic (SF) fractions as well as reconstituted
oil (RO) were also tested against B16F10 cell line. Our
results show that COR displays cytotoxic and antitumour activity in vitro and in vivo.

MATERIALS AND METHODS

Plant Material. The copaiba oil exuded directly from
the trunk of the tree was collected from Copaifera
multijuga Hayne tree at the Ducke Reserve from the
Instituto Nacional de Pesquisas da Amaznia (INPA),
Manaus, Brazil, in February 1993.
Chromatographic analyses. High resolution gas chromatography (HRGC) analyses of the oils and fractions was carried out after prior derivatization with
freshly prepared diazomethane. A Hewlett-Packard
(HP) model 5890 gas chromatography instrument was
12 February(2003)
2002
Phytother. Received
Res. 17, 10481053
Accepted 14 October 2002

COPAIFERA MULTIJUGA HAYNE ANTICANCER ACTIVITY

used equipped with FID and an SE-54 glass column:

25 m 0.25 mm i.d., lm thickness 0.25 m; ow rate:
2 mL/min H2. Samples were injected using the split
mode (1:20) with the injector temperature 270 C and
the detector at 300 C.
High resolution gas chromatography-mass spectrometry (HRGC-MS) analyses were performed by using
a Hewlett-Packard model 5880 (quadrupole analyser),
operated in the electron impact mode (70 eV) coupled
to a Hewlett-Packard model 5897A mass spectrometer.
High purity hydrogen was used as carrier gas at a
linear velocity of 2 mL/min. The oven temperature was
programmed from 110 C (2 min hold) to 130 C at
3 C/min and (no hold) to 290 C at 8.5 C/min.
Retention indices (RI) were calculated using cochromatographed standard hydrocarbons. Identications were done by comparison with MS literature data,
computer matching with the Wiley library and by comparison of their RI values with those of pure standards
and conrmed with the aid of RI from published sources
whenever necessary.
Animals. Experiments were performed on male
C57/black 6 mice, 78 weeks old, purchased from Instituto Nacional do Cncer (INCa), Rio de Janeiro,
Brazil. Mice were housed in a temperature-controlled
room (22 1 C) with 12:12 lightdark cycle with free
access to standard certied rodent diet and tap water.
All experiments were conducted in a quiet room at
constant temperature to avoid animal stress. Copaiba
oil resins were administered orally in different doses.
Tumour cell line. B16F10 melanoma cells were obtained
from Ludwig Cancer Research Institute (So Paulo,
Brazil) and maintained in RPMI-1640 medium, supplemented with 2 mM L-glutamine, 100 g/mL of streptomycin sulphate, 60 g/mL of penicillin, 2 mg/mL of
sodium bicarbonate and 10% fetal calf serum. On
the day of the experiments, cells were harvested by
trypsinization, counted and left to adhere in 96 well
plates at a density of 105 cells/well, in RPMI for 2 h.
Determination of tumour growth in mice. Animals
received subcutaneously 2 106 B16F10 cells (day zero).
The tumour volume was estimated on alternate days,
from two-dimensional tumour measurements performed
with a slide clipper following the formula and according to Kato et al., 1994: Tumoural vol. (mm3) = 0.5
A B2, where A is the longest diameter and B the
shortest diameter of the tumour. The median tumour
volume of the treated group was compared with that
of the control group and the results were expressed
as T/C, where T/C% = (tumour mean in treated
group/tumour mean in control group) 100.
Determination of metastasis formation in mice. Mice
were injected intravenously, via the lateral tail vein,
with 2 106 B16F10 melanoma cells/mL, in a nal
volume of 0.1 mL (Poste and Nicolson, 1980). Day of
inoculation was considered as day zero. After 17
days mice were sacriced, lungs were removed and
total tumour nodules on the surface were counted
under a dissecting microscope.
Treatment with Copaifera multijuga Oil resin (COR).
Animals were divided into two groups. The control
Copyright 2003 John Wiley & Sons, Ltd.

1049

group received PBS and the treated group received

Copaifera multijuga oil resin (2 g/Kg of body weight) in
a nal volume of 0.1 mL. Oral treatment of animals
with COR or PBS was done in the days 3, 5, 7, 10, 12
and 14 after inoculation of tumoral cells. In the 17th
day, mice were sacriced, tumour and lungs were
removed in order to quantify tumour volume and lung
metastasis as cited above.
Cytotoxicity assay by Trypan Blue exclusion method.
Melanoma cell line B16F10 cells were incubated with
0.5 and 1.0 mg/mL of Copaifera multijuga oil resin for 1
or 3 h. Cell viability was quantied in a haemocytometer
using Trypan blue exclusion method and were expressed
as percentage of viable cells when compared with control groups.
Cytotoxicity assay in vitro by MTT conversion method.
B16F10 cells (at 0.1 106/mL in 96 well plates), were
incubated in a nal volume of 0.1 mL with COR or its
fractions, for different times. After the incubation
period, 10 L of MTT solution (5 mg/mL) was added and
further incubated for 2 h. The supernatant was discarded
and 100 L of dimethylsulfoxide (DMSO) was added.
Optical density was read at 570 nm in a Spectramax
Plus Elisa reader. The control group was cells without
any addition and is referred to as 100% of viable cells.
Statistical analysis. Results are presented as means SD
of three experiments (n = 5 animals) of at least triplicates. All data were analysed statistically by Students
t-test for unpaired data. A p value <0.05 was considered
to be indicative of signicance.

RESULTS
Effects of Copaifera multijuga oil resin on tumour
growth in vivo. The subcutaneous injection of 2 106
B16F10 cells resulted in a tumoural growth that reached
a peak at 17 days. The injection of COR at 2 g/Kg,
signicantly reduced tumour volume at 10, 12, 14 and
17 days after inoculation (Fig. 1). Growth inhibition of
solid tumour was about 58% and the reduction in
tumour weight was 76% (Table 1).
Effects of Copaifera multijuga oil resin on lung metastasis. The number of lung nodules was evaluated 17
days after intravenous administration of 2 105 B16F10
melanoma cells. When mice were injected orally with
2 g/Kg of COR, a signicant reduction in the number
of nodules was observed in comparison with the control group (animals injected with saline) (Table 1).
Determination of cytotoxicity of Copaifera multijuga
oil resin in vitro. In order to elucidate if COR in vivo
effect could be reproducible using in vitro experiments,
we incubated B16F10 cells with COR at 0.5 and
1 mg/mL for 1 h and 3 h respectively, and evaluated the
results by Trypan blue exclusion and MTT conversion
methods. Together with the increase in incubation time,
we observed reduction in the number of viable cells
observed by the Trypan blue exclusion method. Differences also could be noted between the two doses used.
At 1 h incubation, 0.5 and 1 mg/mL reduced the number
Phytother. Res. 17, 10481053 (2003)

1050

S. R. M. LIMA ET AL.

Table 1. Effect of Copaifera multijuga Haynes on tumour volume and weight and lung nodule formation
Group

Dose (g/Kg)

Tumour Volume

% Inhibition

Tumour Weight

% Inhibition

Nodules/Animal

% Inhibition

Control
COR

0
2

1,500 144
6,296 118

58.0

1.7 0.07
0.41 0.1

75.9

210 70.4
111 11.0

47.1

The values are the mean SD from two separate experiments, with five animals/group. COR or PBS was administered orally at days
3, 5, 7, 10, 12 and 14 after inoculation of tumoral cells. The animals were sacrificed on the 17th day, tumour volume (in mm3) and
weight (in grams) and lung nodules counted.

Figure 1. Effect of Copaifera multijuga Hayne oil resin on

tumour volume. B16F10 cells (0.2 106) were injected subcutaneously in C57B/6 mice. Tumour volume was estimated
3 days a week. COR was administered orally an alternating days
at 2 g/Kg of body weight. After 17 days mice were sacrified,
tumours and lungs removed, and tumour volumes quantified.
* p < 0.005 (Kruskal-Wallis Test).

of viable cells from 89.0% 16.8% (control group) to

49.4% 6.1% and 30.9% 10.7%, respectively (Fig. 2A).
At 3 h incubation viable cells in the control group
were 110.9% 16.7% and this number was reduced to
34.0% 9.1% and 16.8% 7.9% when 0.5 and 1 mg/mL
of Copaifera multijuga oil resin was used, respectively (Fig. 2B). In order to prove the results obtained
with Trypan blue we used the MTT conversion method.
Again differences with incubation time and doses used
could be observed. As we increased the time of incubation, the percentage of viable cells reduced drastically
(Fig. 3).
Effects of Copaifera multijuga oil resin fractions on
cell viability. In addition to the cytotoxic effects of COR,
we decided to test DF and SF fractions obtained from
the original oil. We also tested RO obtained by addition of DF and SF in the same proportions of that
obtained from the original oil. In an experiment with
an incubation time of 1 h and at 1 mg/mL, the results
were 43.4% 7.9% to DF, 47.0% 18.6% to SF and
35.0% 9.8% to RO (Fig. 4).
Phytochemical analyses. The sample of Copaifera
multijuga oil resin (COR) was fractionated on a silica
gel column impregnated with a 5% aqueous solution
of KOH (Pinto et al., 1997; Pinto et al., 2000). The
chromatographic column was eluted with hexanedichloromethane and methanol. The methanol fraction
Copyright 2003 John Wiley & Sons, Ltd.

Figure 2. Effect of Copaifera multijuga Hayne oil resin on cell

viability. COR at 0.5 and 1 mg/mL was incubated for 1 and 3 h
respectivily, with B16F10 cells. Cell viability was measured by
Trypan Blue exclusion method. * p < 0.005 (Kruskal-Wallis Test).

was acidied to pH4 and extracted with dichloromethane. Aliquots from these two fractions and the crude
oil were methylated with diazomethane and analysed
by high resolution gas chromatography (HRGC) and
high resolution gas chromatography-mass spectrometry
(HRGC-MS). Chromatographic analyses of the crude
methylated Copaifera multijuga oil showed sesquiterpenes (hydrocarbons and alcohols) and diterpenic
carboxylic acid methyl esters. The analyses of the two
fractions showed a perfect separation of the sesquiterpenes (hydrocarbons and alcohols) from the diterpenic
carboxylic acids, named sesquiterpenic fraction (SF)
and diterpenic fraction (DF), respectively. The two
fractions were added to recover the original oil and
analyse the possibility of degrading reactions from the
separation process. This new recovered oil (RO) was
re-analysed and the same proportion of the original
compounds was found.
Phytother. Res. 17, 10481053 (2003)

1051

COPAIFERA MULTIJUGA HAYNE ANTICANCER ACTIVITY

Table 2. Chemical composition of the studied Copaifera multijuga

oil resin
Compounds

Figure 3. Effect of Copaifera multijuga Haynes oil resin on cell

viability. COR at 0.5 and 1 mg/mL was incubated for 1 and 3 h
respectivily, with B16F10 cells. Cell viability was measured by
MTT conversion method. * p < 0.005 (Kruskal-Wallis Test).

-elemene
-cubebene
-copaene
-cedrene
Calarene
Longifolene
-caryophyllene
-bergamotene
-sesquiphellandrene
aromadendrene
-humulene
-amorphene
germacrene D
germacrene B
-bisabolene
-cadinene
-cadinene
-cadinene
-vetivenene
-caryophyllenol
Ledol
caryophyllene oxide
Guaiol
Cedrol
NI
Cadalene
NI
-bisabolene oxide
-bisabolol
acetoxy-caryophyllene
methyl eperuate
methyl copalate
dimethyl pinifolate
NI
dimethyl agathate
methyl 3-hydroxy-copalate
methyl 3-acetoxy-copalate

RI

1344
1352
1382
1400
1417
1423
1426
1436
1442
1447
1457
1478
1483
1499
1509
1515
1524
1531
1542
1554
1565
1582
1595
1616
1625
1637
1642
1655
1666
1700

0.34
0.30
2.51
1.12
0.30
0.11
57.46
2.58
0.10
0.15
8.28
1.88
2.42
0.98
0.33
0.58
1.67
0.24
0.12
0.74
0.24
0.54
0.19
0.37
0.16
0.36
0.73
0.42
0.09
0.23
0.41
6.16
0.16
1.29
2.10
0.63
3.35

NINot identified

Table 2 shows the composition of the methylated

Copaifera multijuga oil resin (COR) with the retention
indices used to identify sesquiterpenes.

DISCUSSION

Figure 4. Effect of Copaifera multijuga Haynes fractions on cell

viability. Diterpenic (DF), sesquiterpenic (SF) or reconstituted
oil (RO) at 0.5 and 1 mg/mL was incubated for 1 and 3 h respectivily, with B16F10 cells. Cell viability was measured by MTT
conversion method. * p < 0.005 (Kruskal-Wallis Test).

Copyright 2003 John Wiley & Sons, Ltd.

The results obtained by HRGC and HRGC-MS agree

with the previous work about Copaifera species composition. The sesquiterpenes are the most common components of the copaiba oils and have been found to be
almost the same in several different copaiba oils (Veiga
et al., 2001), changing the quantitative but not most of
the qualitative composition. The composition of the oil
analysed in this work is quite similar to that analysed
by Gilbert (Gilbert and Cascon, 2000), with almost
60% consisting of the sesquiterpene -caryophyllene,
but less caryophyllene oxide (less than 1%), possibly
due to better storage conditions. This sesquiterpene,
-caryophyllene, has been described as the main
component from several active essential oils, showing
antiallergic (Tanaka et al., 1996), antianaemic (Kudo,
Phytother. Res. 17, 10481053 (2003)

1052

S. R. M. LIMA ET AL.

1953), antiinammatory (Martin et al., 1993) and

anticarcinogenic activities (Zheng et al., 1992).
The diterpenic fraction, on the other hand, is quite
different among copaiba oils. The composition of the
Copaifera multijuga resin oil analysed here, rich in labdanic diterpenes such as copalic, eperuic, pinifolic and
agathic acids, is very different from that of Copaifera
langsdori oil resin. Analysed by Ohsaki (Ohsaki et al.,
1994), the oil from Copaifera langsdori is made up of
labdane and clerodane diterpenes but only the clerodane
diterpenes showed antitumoral activity.
Several reports describe other activities from copaiba
oils, obtained from different Copaifera species, such
as bactericidal (Maruzzela and Sicurella, 1960), antihelmintic (Pellegrino, 1967; Gilbert et al., 1972), analgesic (Fernandes and Pereira, 1989), antiinammatory
(Basile et al., 1988; Fernandes et al., 1992), gastroprotective (Paiva et al., 1998) and trypanocidal (Cascon et al.,
1998).
Based on these results, we analysed not only the
Copaifera multijuga oil resin, but also the fractions containing sesquiterpenes or diterpenes, separated by column chromatography. As the separation methodology
could produce artefacts, the corresponding percentage
of each fraction of the oil were remixed, regenerating
the original oil, which was pharmacologically and
chromatographically analysed too. This reconstituted
oil (RO) showed the same chemical composition and
pharmacological activity (statistically) as the original
Copaifera multijuga resin oil.
The results of the present study demonstrate that the
Copaifera multijuga oil resin (COR), when administered
orally to C57black/6 mice reduces the growth of subcutaneously injected melanoma cell line B16F10, as well
as the number of lung metastases. When incubated at
different doses and with different times COR reduced
the number of viable cells evaluated by the technique
of Trypan blue exclusion as well as by MTT conversion
method.

Our results corroborate with other reports on the

inhibitory action of Copaifera oil resin on tumoural
growth using different experimental designs. The
anticancer nature of Copaifera langsdori Desf oil resin
was reported in Walker sarcoma in rats (Moraes et al.,
1997) and in CD1 mice against IMC carcinoma. The
antitumour activity was evaluated by an increase in life
span (Ohsaki et al., 1994). Copaifera multijuga Hayne
demonstrated a signicant antiproliferative effect in
Sp2/0 murine mieloma (Henriques, personal communication). However, the antitumour activity was not investigated in detail. Our results with cell culture were
complemented with that obtained with mice and clearly
shows an important tumouricidal activity reducing drastically the number of viable cells.
Cancer treatment is made by radiotherapy and/or
intravenous chemotherapy. One of the problems in
patients with melanoma cancer is due to metastatic
nodules that appear after to the principle focus. In a
great number of cases, patients die due to the nodules
found in brain and/or in lungs. The most interesting
nding presented in this study is the effect of COR
(given orally) in vivo. In the two doses tested, COR
reduced the formation of nodules in the lung consequently reduncing death in animals. Considering that
COR administered orally are inuenced by pharmacokinetics parameters such as absorption in gastrointestinal
tract, rst passage metabolism in liver and plasma
biodisponibility we could say that not all from an initial
dose is avaiable to develop its pharmacological actitivy.
The results presented suggest that COR may be safe
and useful as an antitumoural and antineoplasmic agent
and could be used in oral chemotherapy.

Acknowledgements
Antonio Vicente C. Leite for technical assistance, CNPq, FAPERJ
and PRONEX-FINEP 4002-96 and 0888-96, for nancial support.

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