Journal of Ethnopharmacology 106 (2006) 179–186

The anti-tumor effects of alkaloids from the seeds of Strychnos

nux-vomica on HepG2 cells and its possible mechanism
Xu-Kun Deng a,1 , Wu Yin b,1 , Wei-Dong Li a , Fang-Zhou Yin a , Xiao-Yu Lu a ,
Xiao-Chun Zhang a , Zi-Chun Hua b , Bao-Chang Cai a,∗
a College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029, China
b State Key Lab of Pharmaceutic Biotechnology, Nanjing University, Nanjing 210093, China
Received 6 April 2005; received in revised form 12 December 2005; accepted 15 December 2005
Available online 26 January 2006

Abstract
To screen the anti-tumor effects of the four alkaloids: brucine, strychnine, brucine N-oxide and isostrychnine from the seed of Strychnos nux-
vomica, MTT assay was used to examine the growth inhibitory effects of these alkaloids on human hepatoma cell line (HepG2). Brucine, strychnine
and isostrychnine revealed significant inhibitory effects against HepG2 cell proliferation, whereas brucine N-oxide didn’t have such an effect. In
addition, brucine caused HepG2 cell shrinkage, membrane blebbing, apoptotic body formation, all of which are typical characteristics of apoptotic
programmed cell death. The results of flow cytometric analysis demonstrated that brucine caused dose-dependent apoptosis of HepG2 cells through
cell cycle arrest at G0/G1 phase, thus preventing cells entering S or G2/M phase. Immunoblot results revealed that brucine significantly decreased
the protein expression level of cyclooxygenase-2, whereas increased the expression caspase-3 as well as the caspase-3-like protease activity in
HepG2 cells, suggesting the involvement of cyclooxygenase-2 and caspase-3 in the pro-apoptotic effects exerted by brucine. Therefore, this paper
indicate that the major alkaloids present in the seed of Strychnos nux-vomica are effective against HepG2 cells proliferation, among which brucine
proceed HepG2 cells death via apoptosis, probably through the participation of caspase-3 and cyclooxygenase-2.
© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Strychnos nux-vomica; Brucine; HepG2; Apoptosis; Cyclooxygenase-2; Caspase-3

1. Introduction tablet in liver cancer therapy. In addition to its anti-tumor effects,

Hepatocellular carcinoma is one of the most common malig-
nant tumors worldwide. In last decades, surgical resection, the
commonly used technique is frequently challenged in view of
metastasis and other pathological changes. Therefore, the devel-
opment of new agents for hepatocellular cancer is important to
reduce the mortality caused by this disease.
Nux vomica, the dried seed of Strychnos nux-vomica L.
(Loganiaceae), has been effectively used in Chinese folk
medicine for the treatment of liver cancer and associated patho-
logical abnormalities for a long history. As a major ingredient, it
has been frequently prescribed in some classic herbal remedies
such as “Ping-xiao” capsule, “Ci-dan” capsule, “Ma-qian-zi”
∗ processing, the intrinsic alkaloids such as brucine and strychnine
Corresponding author. Tel.: +86 25 86798281; fax: +86 25 86798281.
E-mail address: baochangcai@yahoo.com (B.-C. Cai).
1 These authors contribute equally to this work.
nux vomica is also claimed to have an ability of improving cir-
culatory system and relieving pains in patients with rheumatic
disorders (Guizhi, 1996). However, the mechanism underly-
ing the anti-tumor property of the nux vomica remains largely
unknown.
Alkaloids are the main bioactive chemicals in nux vomica
(Bisset and Phillipson, 1971), responsible for the pharmacolog-
ical and toxic effects exerted by nux vomica to a great extent. In
our previous study, 16 alkaloids have been separated and iden-
tified from the crude nux vomica, among which strychnine and
brucine take up 80% (Cai et al., 1994). Based on the principles
of Chinese medicine, nux vomica is commonly prescribed in
clinical practice after sand processing, the purpose of which is
not only to clean its fine hairs which causes throat irritation (Wu
et al., 1994), but also reduce its toxic side effects because after
transform into their isoforms or nitrogen oxidative derivatives
with less toxicity (Fig. 1) (Cai et al., 1990, 1993). However, the

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.12.021
180 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186
Fig. 1. Chemical structures of brucine, brucine N-oxide, strychnine, isostrychnine, major present alkaloids in nux vomica and the transformations between them after
hot sand processing. (1) Strychnine transformed into its isoform, isostrychnine after sand processing. (2) Brucine transformed into its nitrogen derivatives, brucine
N-oxide after sand processing.
alterations in chemical structures also result in different phar-
macological effects possessed by these alkaloids. For example,
brucine, in crude nux vomica, was claimed to be a morphine-like
analgesic substance, whereas its derivatives brucine N-oxide, in
sand processed nux vomica, act as a more NSAIDs-like com-
pound (Yin et al., 2003a).
In our previous study, the total alkaloid fractions from nux
vomica were shown to remarkably suppress the HeLa and K562
cells growth (Cai et al., 1995, 1998), the results of which pro-
vided the possibility that the traditional application of nux vom-
ica in the therapy of liver cancer is also due to the direct cytotoxic
effects on liver tumor cells elicited by these alkaloids. Therefore,
in order to test this hypothesis and identify the alkaloid which
is mainly responsible for the anti-tumor effect of nux vomica
as well as its mechanisms, we planned to screen the cytotoxic
effects of strychnine, brucine, isostrychnine and brucine N-oxide
as described above on HepG2 cells, and choose a representative
compound to further investigate its anti-tumor mechanisms. In
these four alkaloids, strychnine and brucine were selected to rep-
resent the alkaloids stemmed from crude nux vomica, whereas
isostrychnine and brucine N-oxide stood for the alkaloids from
processed nux vomica. The results of this investigation might
provide a scientific explanation for the traditional application of
this herbal medicine in liver cancer therapy.
2. Materials and methods
2.1. Materials
Strychnine, Hoechst 33258, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenylterazolium bromide (MTT) were purchased from
Sigma Chemical Co. (St. Louis, MO, USA); brucine and brucine
N-oxide were supplied by Carl Roth GmbH+ Co. (German);
isostrychnine was obtained from nux vomica based on the pre-
viously described method (Cai et al., 1994). In brief, after
extraction from nux vomica, isostrychnine was confirmed by
comparing the physical and chemical properties such as melting
point and thin layer chromatography with standard compounds
and 1 H NMR spectra. Its purity was more than 99%. The seeds
of Strychnos nux-vomica L. were collected and identified by
professor Bao-Chang Cai and De-Kang Wu from Nanjing Uni-
versity of Chinese Medicine. A voucher sample (Wu 200052)
was deposited in the College of Pharmacy, Nanjing University
of Chinese Medicine. All water soluble compounds were dis-
solved in RPMI 1640 medium (Gibco, USA), whereas insoluble
chemicals were dissolved into DMSO, the final concentration
of DMSO in each sample were less than 0.1%. Adjustment
of the solutions to pH 7.2–7.4 was made with HCl (0.5 M) or
NaOH (0.5 M) if necessary. All solutions were passed through
a 0.22 ␮m filter (GVMP 01230, Millipore) and stored at 4 ◦ C
until use.
2.2. Cell culture
Human hepatoma cell lines (HepG2) was purchased from
cell bank of Shanghai Institute of Cell Biology (Shanghai,
China), maintained in RPMI 1640 culture medium plus 10%
calf serum and 100 U/ml penicillin, 75 U/ml streptomycin, in
a 37 ◦ C incubator supplied with 95% room air and 5% CO2 .
After 60–80% confluency, the cells were trypsinized with 0.25%
trypsin (AMRESCO, dissolved in PBS, pH 7.4), counted and
placed down at needed density for treatment.
 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186
2.3. MTT-colorimetric assay
Assessment of in vitro neoplastic activity was determined
based on the previously described method (Fatope et al., 1996).
Briefly, the HepG2 cells in exponential growth were placed down
at a final concentration of 6 × 104 cells/ml in 96-well plate.
After 40% confluency, the cells were treated with strychnine,
brucine, brucine N-oxide or isostrychnine at varying concen-
trations, 100 ␮l MTT (1 mg/ml) was then added at 24, 48, 72 h
after treatments. For MTT assay, the supernatant was discarded
and 200 ␮l DMSO was added, the 96 wells plate was vibrated
on micro-vibrator for additional 10 min, the optical density of
each well was measured at λ492nm by Enzyme-immunoassay
instrument (Nanjing, China). The growth inhibitory ratio of each
alkaloid was calculated based on the following formula:
(average O.D. of control group − average O.D. of treated group)
Inhibitory ratio of growth (%) =
average O.D. of control group
2.4. Apoptosis analysis by Hoechst 33258 staining
The Hoechst 33258 staining was carried out as previously
described (Ramonede and Tomas, 2002). Briefly, the HepG2
cells in exponential growth were placed down at a final con-
centration of 1 × 105 /well in 12-well culture plate. A cover slip
was placed on the bottom of each well to allow cells to grow on
its surface as a monolayer. After 40–60% confluency, the cells
were exposed to brucine 0.25 mM for 36 h. For Hoechst 33258
staining, the cells were fixed with ice-cold methanol, acetic acid
(3:1) at room temperature for 5 min after washing twice with
ice-cold PBS, the re-washed with ice-cold PBS buffer twice,
loaded with Hoechst 33258 for additional 20 min, the changes
in the nuclei of cells after Hoechst 33258 staining were observed
under a fluorescence microscope (Olympus, BX-60, Japan) in
less than 15 min.
2.5. DNA content and cell cycle analyzed by flow cytometry
The DNA content and the cell cycle of HepG2 cells
were determined by flow cytometry based on previously
described method (Yin et al., 2003). Briefly, the HepG2 cells
were placed down at a density of 1 × 106 cells/well in a 6-
well plate. After 40% confluency, the cells were exposed to
brucine (0.25, 0.5, 1.0 mM) for additional 36 h. Cells were
collected, washed twice with ice-cold PBS buffer (pH 7.4),
fixed with 70% alcohol and stained with propidium iodide
(PI) (1 mg/ml) in the presence of 1% RNAase A atleast for
30 min before analysis by flow cytometry (Becton Dickinson,
USA). The data were analyzed with MODFIT and CellQuest
software.
2.6. Immunoblot analysis
The immunoblot analysis of caspase-3 and cyclooxygenase-
2 were performed as previously described (Yin et al., 2003b).
Briefly, the HepG2 cells were placed down at a final density
of 8 × 105 cells/well in a 12-well plate. After treatments, the
181
confluent cells were lysed and collected, resolved by a 12% SDS-
PAGE gel, the protein was then electrophoretically blotted onto
polyvinylidene fluoride (PVDF) membranes. The membranes
were first hybridized with primary antibodies and then with
horseradish peroxidase-conjugated anti-mouse or anti-rabbit
IgG secondary antibody (Sigma, St. Louis, MO, USA). The
antibody against the human cyclooxygenase-2 was purchased
from Santa Cruz Biotech (Santa Cruz, CA, USA), antibody
against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
were purchased from Upstate Biotechnology (Lake Placid, NY,
USA). The immune complexes were detected using chemilumi-
nescence (ECL) system (Amersham Pharmacia Biotech, Buck-
inghamshire, UK).
× 100
2.7. Measurement of caspase-3-like activity
The activity of caspase-3-like proteases was mea-
sured as increases in hydrolysis of fluorogenic tetrapeptide
substrate, Ac-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-
AMC), according to the manufacturer’s instructions (BIOMOL
Research Laboratories, Inc., Plymouth Meeting, PA, USA).
Because caspase 7 also cleaves the substrate, the activity
obtained here represents as caspase-3-like proteases activity.
Briefly, the HepG2 cells were exposed to brucine (0.125, 0.25,
0.5, 1.0 mM) for 36 h, after treatment, the cells were lysed, the
supernatant was taken for measurement of hydrolysis of Ac-
DEVD-AMC as a function of time at 22 ◦ C.
2.8. Statistical analysis
The results were expressed as mean ± S.D. The statistical
analysis involving two groups was performed by means of Stu-
dent’s t-test, whereas analysis of variance (ANOVA) followed by
Dunnett’s multiple comparison test were used in order to com-
pare more than two groups. All data were processed with SPSS
software. P < 0.05 was considered significant, and P < 0.01 was
considered more significant. Regression analysis was used to
calculate IC50 of tested chemicals.
3. Results
3.1. Growth inhibitory effects of strychnine, brucine,
brucine N-oxide, isostrychnine on HepG2 cells by MTT
assay
MTT assay was used as an indirect measure to determine
the viability of HepG2 exposed to the alkaloids. As shown
in Fig. 2(1)–(3), both brucine and isostrychnine caused the
cell death in a concentration, time-dependent manner. Brucine
(1 mM) showed the strongest cytotoxic effect on HepG2 at 72 h
after treatment (96.3%), whereas the maximal growth inhibitory
182 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186

Fig. 2. The growth inhibitory effects of strychnine, isostrychnine, brucine, brucine N-oxide on HepG2 cells at different time after treatment. The HepG2
(6 × 104 cells/ml) was incubated with the indicated four alkaloids at 0.125, 0.25, 0.5, 1 mM for 24 h (panel 1), 48 h (panel 2) and 72 h (panel 3). The cell via-
bility was measured in three plates in triplicate using MTT assay. * P < 0.05, ** P < 0.01 as compared to medium alone control.

rate for isostrychnine was 72.7%. As an analogue to isostrych- more effective against HepG2 cell proliferation than the latter.
nine, strychnine demonstrated the milder cytotoxic effect as The IC50 value of brucine N-oxide at any given time exceeded
compared to that of isostrychnine. Only after incubation with 1.0, further revealed its ineffectiveness on the HepG2 cells pro-
strychnine for 72 h, the HepG2 cells died in a dose-dependent liferation.
manner. However, its maximal cytotoxic effect (89%) was very
close to that of brucine, even slightly larger than isostrychnine. 3.2. Morphological changes of HepG2 cells induced by
In contrast to strychnine, brucine N-oxide didn’t present any brucine under fluorescent microscope
cytotoxic effect at any given time or concentrations. The cyto-
toxic effects of the four alkaloids were also revealed from the Hoechst staining was applied to investigate whether HepG2
results in Table 1, aside from brucine N-oxide and isostrychnine underwent cell death via apoptosis or necrosis. As shown
at 24 h after treatment, the IC50 of the cytotoxic effects of strych- in Fig. 3, after Hoechst staining, the HepG2 cells exposed
nine, isostrychnine or brucine at 72 h was below 1.0, the value of to brucine (0.25 mM) revealed marked nuclear condensation,
which was considered to be effective enough to inhibit the cell membrane blebbing, nuclear fragmentation and apoptotic bod-
growth. Brucine had a much smaller IC50 value as compared to ies (as illustrated by arrows), all of which are the characteristics
that of strychnine or isostrychnine, suggesting the former was of apoptotic programmed cell death. However, the control cells
didn’t display any of the given characteristics.
Table 1
IC50 (mM) effects of strychnine, brucine, brucine N-oxide, isostrychnine on the 3.3. Cell cycle analysis by flow cytometry
HepG2 cell growth at different time after treatment
Strychnine Brucine Brucine N-oxide Isostrychnine
The results presented in Fig. 4(1)–(4) showed that after
treatment for 36 h, brucine dose-dependently increased the sub
24 h 1.00 0.65 1.00 >1.00 G0/G1 population, which indicated the increased cell apopto-
48 h 1.00 0.32 1.00 0.98
72 h 0.52 0.10 1.00 0.61
sis. Also from the results of cell cycle distribution in Table 2,
HepG2 cells were arrested at G0/G1 phase, thus were prevented
 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186 183

Fig. 3. Morphological alterations of HepG2 cells following exposure to brucine (0.25 mM) for 36 h. The HepG2 cells were grown on the cover slip as a monolayer,
after treatment, the control cells (panel 1), brucine-treated cells (panel 2) were loaded with Hoechst 33258 and observed under the fluorescence microscope. Arrows
A (panel 2): nuclear shrinking; B: membrane blebbing; C: apoptosis body. Each experiment was performed in triplicate (magnification 100×).

Fig. 4. Flow cytometric analysis of HepG2 cells following exposed to brucine. Cells were treated with no brucine (panel 1), brucine 0.125 mM (panel 2), brucine
0.25 mM (panel 3), brucine 0.5 mM (panel 4) for 36 h, stained with PI and analyzed on a FACScalibur flow cytometer. The apoptotic rate and cell cycle distribution
of each group were stated in Table 2. Each experiment was performed in triplicate.
184 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186

Table 2
Cell cycle distributions of HepG2 after treatment with brucine for 36 h
Group Apoptotic Cell cycle distributions of HepG2 (%)
rate (%)
G0 /G1 G2 /M S

Control 4.52 47.55 12.02 40.43

Brucine 0.125 mM 28.15 53.61 6.89 39.50
Brucine 0.25 mM 36.76 58.76 7.15 34.10
Brucine 0.5 mM 48.49 60.80 11.05 28.15

from entering S phase since the population in G2/M or S phase

declined, whereas in G0/G1 phase increased.

3.4. Effects of brucine on the expression of caspase-3 and

cyclooxygenase-2 protein

To further explore the apoptotic mechanism of HepG2 cells Fig. 6. Effect of brucine on the caspase-3-like activity. The hydrolysis of Ac-
DEVD-AMC was considered as the indicator for the caspase-3-like activity.
induced by brucine, western blot was performed to examine Cells were treated with brucine at 0.125, 0.25, 0.5, 0.1 mM for 36 h. After treat-
the protein expression level of caspase-3 and cyclooxygenase- ment, the cells were lysed, the caspase-3-like activity of the supernatant was
2 in brucine-treated cells. As shown in Fig. 5, brucine measured. n = 3, ** P < 0.01, compared with control.
induced concentration-dependent increase of caspase-3-protein
abundance associated with the corresponding decrease of
activity. The strongest hydrolysis effect was exerted by brucine at
cyclooxygenase-2 expression. Sodium diclofenac (100 ␮g/ml),
0.5 mM after incubation for 36 h (72.41%). The highest concen-
a commonly used NSAIDs, served as a control, showed the sim-
tration of brucine (1.0 mM), however, decreased the hydrolysis
ilar inhibitory effect on the expression of cyclooxygenase-2 as
of Ac-DEVD-AMC as compared to other lower concentrations
that of brucine, but didn’t reveal any significant effect on the
(63.76%), perhaps due to the larger cell death rate.
level of caspase-3.

3.5. Effect of brucine on the caspase-3-like activity 4. Discussion and conclusions

Since brucine induced the over-expression of caspase-3
in HepG2 cells, Ac-DEVE-AMC, the normally used peptide
substrate to measure the caspase-3-like protease activity, was
employed to determine the caspase-3-like activity in HepG2
cells after treatment with brucine, the results in Fig. 6 demon-
strated that brucine dose-dependently increased the hydrolysis
of Ac-DEVD-AMC, an indicator of the caspase-3-like protease
Fig. 5. Immunoblot analysis of the protein abundance of caspase-3 and
cyclooxygenase-2 of HepG2 cells exposed to brucine. The HepG2 cells were
treated with 0.125 mM brucine (B), 0.25 mM (C), 0.5 mM (D) for 36 h, Sodium
diclofenac (100 ␮g/ml) (E) served as a positive control, cells with culture
medium alone (A) were included as a negative control. GAPDH was used as
an internal standard to normalize loadings. Each experiment was performed in
triplicate.
In the present study, we investigated the cytotoxic effects
of four alkaloids originated from crude or processed nux vom-
ica on HepG2 cells and the possible mechanisms. We found
that brucine, among the selected alkaloids, exhibited a strongest
inhibitory effect on HepG2 cells proliferation. In contrast to
brucine, brucine N-oxide didn’t reveal any anti-proliferation
activity. Strychnine, isostrychnine also demonstrated signifi-
cant inhibitory effects on HepG2 cells growth, although their
activities were less potent than that of brucine. Furthermore,
brucine was found to cause HepG2 cells via caspase-3 and
cyclooxygenase-2-mediated cells apoptosis.
In our previous study, brucine N-oxide was found to pos-
sess a more potent inhibitory effect on the ADP-induced platelet
aggregation than that of brucine (Jianyin et al., 1998), also the
anti-inflammatory effect of brucine N-oxide was stronger than
that of brucine in animal test (Yin et al., 2003a). However,
brucine N-oxide in this study didn’t show any inhibitory effect
on HepG2 cells proliferation. This phenomenon implied that the
pharmacological effects of brucine and brucine N-oxide were
strikingly different despite that only minor variations existed
in their chemical structures. Interestingly, this large difference
in structure–activity relationships of brucine didn’t applied to
strychnine, since both strychnine and isostrychnine demon-
strated significant inhibitory effects on HepG2 cells prolifera-
tion, although their effects were less potent than that of brucine.
Besides, based on the above results, it seemed that the crude nux
vomica was more advantageous over the processed one in treat-
 X.-K. Deng et al. / Journal of Ethnopharmacology 106 (2006) 179–186
ing liver cancer. However, in our recent study, we found that the
processed nux vomica was more effective against HepG2 cells
proliferation than that of crude nux vomica (unpublished data).
In detail, the IC50 effect of the total alkaloids from crude nux
vomica on HepG2 cells proliferation after treatment for 72 h was
53.1 mg/l, whereas the alkaloids from processed nux vomica was
37.8 mg/l. This data suggested that brucine may not fully rep-
resent the anti-proliferation effect of alkaloids in nux vomica,
some other alkaloids such as isobrucine may also make a con-
tribution to the anti-proliferation effect of nux vomica, although
they are present in nux vomica in small quantities. In addition,
we couldn’t formally exclude other mechanisms such as anti-
angiogenic or immunostimulant effects were also involved in
the anti-tumor property of alkaloids in nux vomica. Thus, more
experiments needs to be done to further identify the mechanisms
of nux vomica in live cancer therapy. In fact, there still existed
another advantage for processed nux vomica in treating liver can-
cer associated with inflammation, because the abundant brucine
N-oxide in processed nux vomica may inhibit the inflammatory
reactions in tumor-bearing patients as previously described (Yin
et al., 2003a).
During the past decades, the killing of tumors through the
induction of apoptosis has been recognized as a novel strategy
for the identification of anticancer drugs (Panchal, 1998; Smets,
1994; Watson, 1995). The apoptosis itself also plays an impor-
tant role in the development of various diseases including cancer
(Fisher, 1994; McConkey et al., 1996). In the present study,
brucine exhibited a caspase-3-dependent HepG2 cells apopto-
sis since the protein abundance of caspase-3 and caspase-3-like
protease activity in HepG2 cells were dramatically up-regulated
by brucine. Also, brucine was shown to cause HepG2 cells
cycle arrest at G0/G1 phase, thus preventing cells from enter-
ing S or G2/M phase and finally apoptosis occurred. Therefore,
whether the hindrance of HepG2 cells cycle progression caused
by brucine is related to caspase-3 needs more experiments to
clarify.
In the present study, brucine was also shown to reduce the
expression of cyclooxygenase-2 in HepG2 cells. Cyclooxyge-
nase is a rate-limiting enzyme in prostanoids (PGs) biosynthesis.
It consists of two isozymes, the constitutive cyclooxygenase-
1 and the inducible cyclooxygenase-2. The cyclooxygenase-2
gene has been characterized as an immediate early gene and
associated with inflammation, pain and cellular proliferation
as well as apoptosis. Recently, lines of evidences suggest that
the over-expression of cyclooxygenase-2 might be one of the
leading factors in hepatic carcinogenesis (Koga et al., 1999).
Non-steroid anti-inflammatory drugs (NSAIDs) are commonly
used drugs to specially inhibit the cyclooxygenase-2 activity.
NSAIDs can inhibit the tumor cell proliferation, induce apop-
tosis through down-regulation of bcl-2 expression and levels
of prostanoids (Li et al., 2002; Sheng et al., 1997). A case in
point is nimesulide, which suppress tumor growth and induce
apoptosis in implanted hepatoma mice by inhibiting the expres-
sion of cyclooxygenase-2 and prostaglandin E2 (Li et al., 2003;
Tsujii and DuBois, 1995). In our previous study, brucine was
shown to relieve pain, reduce the release of inflammatory factors
such as prostaglandin E2 , thromboxane B2 and 6-keto-PGF1a in
185
adjuvant-induced arthritis, the action mode of which is similar to
the common NSAIDs (Yin et al., 2003a). Given the above facts,
brucine could be a promising NSAID, which was effective in the
treatment of liver cancer associated with pain or inflammation.
Finally, we have to confess that the concentration brucine
used in this study was a little bit higher than its physiological
concentration, which may not be directly translated into clinical
practices. However, the effect of brucine, atleast in this study
has been proven to be specific because brucine at 0.5 mM or
higher didn’t cause significant human embryonic kidney cells
(HEK293) or other mammalian cells death (data not shown).
Taken together, we have successfully established an in vitro
model to evaluate the possible mechanism of alkaloids in nux
vomica in the therapy of liver cancer. The final goal we are will-
ing to achieve is to find a more effective anti-tumor drug by
properly modifying the structure of brucine, since many alka-
loids such as harringtonine, vincaleukoblastine and vincristine
which have strong anti-tumor effects share the indole ring in
their chemical structures with brucine. Thus, the experimental
model established in this paper will surely accelerate the under-
standing of the structure–activity relationships in brucine and
pave the way for discovering new drug.
Acknowledgements
This work was supported by National Nature Science Fund of
China (90209052, 30470644), PhD Candidate Fund of Jiangsu
Province (2005098). The authors express thanks for the assis-
tance from vice professor Wang Tian-Shan and Di Liu-Qing
from the College of Pharmacy, Nanjing University of Chinese
Medicine for the generation of the original research proposal,
also critical comments on the direction of this project and
great technical assistance from Xu Dong-Qing, Wang Ming-
Yan, Xiang Xiao-Ren, Sheng Yu-Qing.
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