Structural modification of ginsenoside Rh2 by fatty acid esterification and its detoxification property in antitumor

Article abstract of DOI:10.1016/j.bmcl.2011.11.104

Ginsenoside Rh₂, one of the most important ginsenosides with anticancer properties in red ginseng, has been developed as principal antitumor ingredient for clinical use. However, the cytotoxicity test in human hepatocyte cell line QSG-7701 (IC₅₀ 37.3 μM) indicated that Rh₂ might show strong cytotoxic side-effect on the normal liver cells. For blunting the toxicity, Rh₂ was structurally modified by reacting with octanoyl chloride to give a dioctanoyl ester of Rh₂ (D-Rh₂) in the present study. MTT assay in QSG-7701 cell line in vitro showed that the cytotoxicity of D-Rh₂ on human hepatocyte cells (IC₅₀ 80.5 μM) was significantly lower than that of Rh₂. While antitumor xenograft assay in mice bearing H22 liver cancer cells in vivo showed that the antitumor activity of D-Rh₂ retained to be strong as that of Rh ₂. According to previous pharmacokinetic studies, the fatty acid esterification of Rh₂ might be of detoxification reaction to cells. Additionally, D-Rh₂ showed significant enhancement on increasing thymus index at the dose of 10 mg/kg compared with vehicle treated control group. Thus, D-Rh₂ might indirectly affect tumor growth by stimulating lymphocytes to become cytotoxic to tumor cells. Finally, our findings suggested that D-Rh₂, the fatty acid ester of Rh ₂, might attenuate the side-effect by detoxification to human normal cell and could be a more potential candidate for developing as an antitumor drug.

Full text of DOI:10.1016/j.bmcl.2011.11.104

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1082–1085
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural modification of ginsenoside Rh2 by fatty acid esterification
and its detoxification property in antitumor
Gong-Qing Wei a, Yi-Nan Zheng a, Wei Li a, Wen-Cong Liu a, Ting Lin b, Wei-Yun Zhang c, Hai-Feng Chen b,
Jin-Zhang Zeng b, Xiao-Kun Zhang b, Quan-Cheng Chen b,
⇑
a College of Chinese Medicinal Material, Jilin Agricultural University, Changchun, Jilin 130118, China
b School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian 361005, China
c College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ginsenoside Rh2, one of the most important ginsenosides with anticancer properties in red ginseng, has
been developed as principal antitumor ingredient for clinical use. However, the cytotoxicity test in
human hepatocyte cell line QSG-7701 (IC50 37.3 lM) indicated that Rh2 might show strong cytotoxic
side-effect on the normal liver cells. For blunting the toxicity, Rh2 was structurally modified by reacting
with octanoyl chloride to give a dioctanoyl ester of Rh2 (D-Rh2) in the present study. MTT assay in QSG-
Received 16 October 2011
Revised 23 November 2011
Accepted 28 November 2011
Available online 7 December 2011
7701 cell line in vitro showed that the cytotoxicity of D-Rh2 on human hepatocyte cells (IC50 80.5 lM)
Keywords:
was significantly lower than that of Rh2. While antitumor xenograft assay in mice bearing H22 liver can-
cer cells in vivo showed that the antitumor activity of D-Rh2 retained to be strong as that of Rh2. Accord-
ing to previous pharmacokinetic studies, the fatty acid esterification of Rh2 might be of detoxification
reaction to cells. Additionally, D-Rh2 showed significant enhancement on increasing thymus index at
the dose of 10 mg/kg compared with vehicle treated control group. Thus, D-Rh2 might indirectly affect
tumor growth by stimulating lymphocytes to become cytotoxic to tumor cells. Finally, our findings sug-
gested that D-Rh2, the fatty acid ester of Rh2, might attenuate the side-effect by detoxification to human
normal cell and could be a more potential candidate for developing as an antitumor drug.
Ó 2011 Elsevier Ltd. All rights reserved.
12,60-Dioctanoyl ginsenoside Rh2
Antitumor
Structural modification
Detoxification
Ginsenoside Rh2 (Rh2) is a protopanaxadiol type saponin firstly
isolated from red ginseng which has been used as traditional med-
icine and natural tonic in oriental countries for thousands of years.1
This metabolite shows various biological effects such as reducing
blood glucose,2 ameliorating ischemic brain injury,3 and inhibiting
allergy.4 In particular, Rh2 exhibits beneficial impacts on antican-
cer. It has been previously reported to have a growth suppressive
effect on various cancer cells such as lung adenocarcinoma, neuro-
blastoma, breast cancer, prostate cancer, colorectal cancer, glioma,
malignant melanoma, and hepatocarcinoma cells.5–19 In addition,
Rh2 inhibits tumor growth in mice bearing human cancer cells,20,21
and exhibits some synergetic effects with chemotherapeutic
agents both in vitro and in vivo studies.22,23
tion, previous studies showed that little Rh2 could be absorbed into
plasma from the rat gastrointestinal tract.24 Collective evidences
indicated that the oral drug bioavailability of Rh2 is low.25,26 Thus,
it is essential to design and optimize the chemical structure of Rh2
by structural modification.
Previous pharmacokinetic studies have demonstrated that oral
main ginsenosides accounting for 90% (w/w) of the total saponins
in ginseng were cleaved the terminal sugar stepwise by colonic
bacteria to afford the major protopanaxadiol monoglucoside (M1
or Rh2) and protopanaxatriol (M4) metabolites. These metabolites
were then further esterified by fatty acids as ginsenoside fatty acid
esters which could be sustained longer in the body.27–29 Pharmaco-
kinetic studies also revealed that ginsenoside fatty acid esters
might be the real antitumor active species in vivo.30,31 More impor-
tantly, the fatty acid esterification of ginsenoside possibly repre-
sented a detoxification reaction in the body.28 Accordingly, fatty
acid esterified ginsenoside could be pharmaceutically active and
might be excellent potential drug candidate. In our previous re-
search, several fatty acid esters of ginsenoside M1 and Rh1 had
been chemically synthesized for improving their anticancer prop-
erties.32,33 In the present research, we aimed to optimize Rh2 by
structural modification with esterification for lowering its possible
side-effect.
Currently, Rh2 has been developed as a principal antitumor
ingredient of ‘Jinxing capsule’ (Authentication Code: GSJZ-
G20060157) for clinical use in China. However, Rh2 might exhibit
high toxicity to normal cells since being cytotoxic to the human
hepatocyte cell line QSG-7701 with an IC50 value 37.3 lM. In addi-
⇑
Corresponding author. Tel.: +86 592 218 7225; fax: +86 592 218 1879.
E-mail address: chenqc@xmu.edu.cn (Q.-C. Chen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.104

G.-Q. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1082–1085
1083
Table 1
Esterification of Rh2 was carried out using triethylamine-cata-
13C NMR spectroscopic data of compound 1 (13C NMR, 400 MHz, C5D5N)
lysed synthesis of ester, which is a typical esterification procedure.
An octanoyl chloride reagent was chosen because of being suffi-
cient in industrial supply and cheap. The esterification process is
quite simple (Scheme 1). Briefly, Rh2 (2 g) was dissolved in
500 mL of CHCl3 and was slowly added the octanoyl chloride
(1.2 mL) and Et3N (1.2 mL). The mixture was reacted under stirring
at room temperature for 15 min. Then the reactant was washed by
1.0 L distilled water and concentrated in vacuo. The concentrate
No.
Rh2
D-Rh2(1)
No.
Rh2
D-Rh2(1)
dC
17.87
dC
dC
dC
1
2
3
4
5
6
7
8
39.25
39.24
27.16
89.25
40.04
56.53
18.53
37.13
39.78
50.42
37.54
31.99
75.21
45.82
52.70
31.57
26.76
53.39
16.82
16.35
73.55
26.94
34.94
23.23
126.45
130.87
25.96
27
28
29
30
10
17.79
27.22
88.89
40.13
56.49
18.57
36.01
39.81
50.51
37.09
32.20
71.11
48.71
51.83
31.46
26.85
54.95
16.91
16.48
73.05
26.97
35.27
23.12
126.45
130.86
25.92
28.28
15.95
17.15
107.11
75.94
78.90
72.01
78.52
63.21
28.21
15.79
17.70
107.14
75.73
78.65
71.78
75.21
64.71
173.03
35.24
25.29
29.38a
29.51a
30.08a
22.92b
14.36c
173.79
34.59
25.39
29.38a
29.08a
29.70a
22.98b
14.29c
20
was dissolved in MeOH, and filtered through a 0.22 lm membrane.
30
Finally, the filtrate was condensed and subjected to silica gel col-
umn chromatography, eluted with CHCl3–MeOH = 15:1 to give a
pure compound 1 (1.38 g). The purification of 1 analyzed by HPLC
was more than 98%. The percentage yield of esterification reaction
from Rh2 to 1 was 48.1%.
40
9
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
60
100
200
300
400
500
600
700
800
1000
2000
3000
4000
5000
6000
7000
8000
The chemical structure of 1 was characterized by 1D and 2D
NMR analysis. At first, the 1H and 13C NMR data of 1 were similar
to those of Rh2, except for the obvious differences due to the pres-
ence of two additional fatty acyl signals (Table 1 and Supplemen-
tary data). In the 13C NMR spectra, carbon signals of dC 173.03,
35.24, 25.29, 29.38, 29.51, 30.08, 22.92, 14.36 (C-100ꢀ800), and dC
173.79, 34.59, 25.39, 29.38, 29.08, 29.70, 22.98, 14.29 (C-1000ꢀ8000)
could be identified as two separate octanoyl groups. In addition,
an upfield shift dC 45.82 and a downfield shift signal dC 75.21 com-
pared with dC 48.71 (C-13) and dC 71.11 (C-12) in Rh2 were ob-
served in the 13C NMR spectra. The observation indicated that
one of the octanoyl groups might combine to the hydroxyl group
at C-12 of Rh2 and change the original chemical surroundings of
C-12 and C-13 in Rh2. The conclusion could be confirmed by the
analysis of HMQC and HMBC spectra (Fig. 1). Firstly, dH 2.08 (dd)
and dH 5.13 (ddd) respectively correlated to dC 45.82 and dC
75.21 could be observed in HMQC spectra of 1. HMBC correlations
between dH 2.08 and dC 73.55, 75.21, 52.70, 53.39 indicated that dC
45.82 belongs to C-13, while dC 75.21 belongs to C-12. Secondly, dH
5.13 (H-12) showed correlationship with dC 173.03, as well as C-11
and C-13. Thus, one octanoyl group should be connected to the hy-
droxyl group of C-12 in Rh2. By 2D NMR spectra, another octanoyl
group could be deduced to connect with the hydroxyl group on C-
60 of the glucose moiety due to the correlations between H-60 (dH
4.74 and 4.95) and dC 173.79. Thus, 1 was finally elucidated as
12, 60-dioctanoyl ginsenoside Rh2 (D-Rh2).
a,b,c
Values may be interchangeable in each column.
O
21
24
27
22
HO
20
17
25
26
23
3"
1"
5"
7"
O
12
13
11
16
19
18
1
9
O
14
30
2
3
15
10
8
6'
7"'
4
1"'
5"'
3"'
O
7
5
O
6
5'
O
28
29
OH
1'
3'
H
HMBC
C
OH
OH
Figure 1. Selected HMBC correlations of 1.
D-Rh2 was firstly tested for its cytotoxicity against human hepa-
tocyte cell line QSG-7701 to evaluate the detoxification effect on
human normal cells comparing with Rh2. For the purpose, MTT as-
say for cell viability as previously described was used.34 QSG-7701
The supernate was removed and formed formazan crystals were
dissolved with 150 L DMSO. Each plate was shaken for 20 min
and the OD values were read at 570 nm (450 nm as a reference)
on the microplate reader within 30 min. The IC50 value was defined
as the concentration of sample needed to reduce 50% of absorbance
relative to the vehicle-treated control.
l
cells were seeded at 5 ꢁ 103 cells/mL in 96 well plates (180
lL per
well). The test samples were dissolved in DMSO and adjusted to
the final test concentration range by diluting with RPMI 1640 med-
ium. The final DMSO concentration was adjusted to <0.1%. Each
sample was prepared to triplicate, and added 20
lL to each well.
As shown in Fig. 2, Rh2 (50
while treating with D-Rh2 (50
Rh2 and D-Rh2 for inhibiting cell growth were 37.3 and 80.5
l
l
M) inhibited 83% of the cell growth,
M) inhibited 18%. The IC50 values of
M,
These cells were incubated for 48 h. Then 20 L MTT (Amresco,
l
OH, USA, 5 mg/mL in PBS) were added to each well and incubated
l
for 2 h. Microplates were centrifuged for 5 min (25 °C, 1500 rpm).
respectively. D-Rh2 might largely reduce the cytotoxicity for hu-
HO
OR
HO
OH
H
H
H
H
H
H
CH2OR
O
OH
CH2OH
RCl + Et3N
R=octanoyl
O
H
O
H
O
OH
OH
OH
OH
OH
Rh2
D-Rh2
Scheme 1. Fatty acid esterified reaction of ginsenoside Rh2.

1084
G.-Q. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1082–1085
100
90
80
70
60
50
40
30
20
10
11.4
16
l
mol/kg was observed almost as strong as that of Rh2 at dose
Rh2
l
mol/kg or CTX at dose 76.6 mol/kg. In addition, the average
l
body weights of normal mice and CTX, Rh2 (dose 20 mg/kg), and
D-Rh2 (dose 20 mg/kg) treated mice changed from 23.0 1.8 to
24.6 2.2 g, 25.3 1.5 to 24.0 1.3 g, 23.4 2.8 to 23.2 1.2 g,
and 24.8 1.1 to 27.1 1.5 g, respectively, after 10 days of treat-
ment. D-Rh2 (dose 20 mg/kg) showed increasing the mice body
weight similar to those of control group which grew up normally;
while Rh2 (dose 20 mg/kg) and CTX could not increase or even
block the increase of body weight.
Intriguingly, the above results indicated that D-Rh2 could signif-
icantly reduce the toxicity to human hepatocyte cell line QSG-7701
in vitro without blunting the antitumor activity in vivo. According
to the previous pharmacokinetic study,31 it is possible that part of
oral Rh2 were esterified by some fatty acids in the body after
digesting. The transformed Rh2 fatty acid esters might be sustained
longer in the body and served as antitumor species. However, the
remained Rh2 could be harmful to the human normal tissue due
to its considerable cytotoxicity. After oral intake, the absorbed D-
Rh2 might possess similar antitumor function in vivo to those of
transformed Rh2 fatty acid esters in the body. Compared with
Rh2, D-Rh2 which showed lower cytotoxicity would largely attenu-
ate the injury for normal tissue in the body. Thus, our findings sug-
gested that D-Rh2, the fatty acid ester of Rh2, might depress the
side-effect by detoxification to human normal cell and could be
an attractive candidate for antitumor. However, the molecular act-
ing mechanism and pharmacokinetics of D-Rh2 need to be further
studied.
Ginseng has been treasured in Asian countries, especially in Chi-
na, Korea and Japan due to its tonic property. There were many evi-
dences that ginseng and its active component ginsenosides
enhance immune activity.35,36 Jinxing Ginseng Rh2 Capsule, which
was declared to improve immune function and increase resistance,
was marketed as a health food for the patients in the treatment and
rehabilitation period with cancer. We therefore compared the ef-
fects of spleen and thymus indexes in the tumor bearing mice with
control and D-Rh2 treatment. Mice were inoculated tumor cells
and divided into groups (10 mice per group). Additionally, 10 nor-
mal mice were grouped as blank control. After 7 days of the tumor
cell implantation, mice were orally administrated once a day with
D-Rh2, Rh2, positive control CTX and negative control normal saline
(NS) for 10 days following above Xenograft assay. At the termina-
tion of the experiment, thymus gland, and spleen were harvested
and weighted, respectively.
As showed in Table 2, the CTX group significantly decreased
spleen and thymus indexes (P <0.05) compared with NS group.
D-Rh2 at the dose of 10 mg/kg significantly increased the thymus
index (P <0.05). No significant difference of spleen and thymus in-
dex was observed at 5 mg/ kg dose of the D-Rh2 while compared
with negative control. The above results indicated that cytoxan
could significantly (P <0.05) lead to atrophy of the spleen and thy-
D-Rh2
0
12.5
25
50
100
Concentration (µM)
Figure 2. The effects of Rh2 and D-Rh2 on inhibiting QSG-7701 cells growth.
man normal cells. The results indicated that the fatty acid esterifi-
cation of Rh2 might represent a detoxification reaction. This was
well consistent with the previous speculation by Hasegawa et al.28
We then examined D-Rh2 for its antitumor activity on Kunming
mice bearing mouse hepatoma (H22 cell line) using Xenograft as-
say. Kunming mice were provided by the Laboratory Animal Center
of Jilin University (Changchun, China) and housed at 23 0.5 °C,
60% humidity in a 12 h light–dark cycle. All animals were accli-
mated for seven days before the experiment began. Then the mice
were injected subcutaneously on right flanks above the hind limb
with 0.1 mL H22 cells (1 ꢁ 106) and divided into groups of 10 mice
at random. After 7 days of the tumor cell implantation, mice were
gavaged orally with cytoxan (CTX) (20 mg/kg), Rh2 (10 mg/kg), Rh2
(5 mg/kg), D-Rh2 (10 mg/kg), and D-Rh2 (5 mg/kg) in 0.5 mL nor-
mal saline solution with 1% Tween-80 once per day for 10 days.
The control mice were gavaged orally with 0.5 mL vehicle with a
similar dosing schedule. Body weights of mice were recorded
throughout the experiment. At the termination of the experiment,
the tumor tissues were harvested.
As shown in Fig. 3, CTX, Rh2, and D-Rh2 treated groups were all
significantly inhibited the tumor growth compared with the nega-
tive control group. Among them, CTX reduced ꢀ72% of tumor
growth in mice at oral treatment dose 20 mg/kg (body weight of
mouse). D-Rh2 exhibited ꢀ52–73% of tumor inhibition at dose 5
and 10 mg/kg (5.7 and 11.4 lmol/kg), while the parental Rh2
showed ꢀ53–75% inhibitory rate at dose 5 and 10 mg/kg (8.0 and
16.0
l
mol/kg). The tumor inhibitory activity of D-Rh2 at dose
1.2
1
0.8
0.6
0.4
0.2
0
*
*
*
*
Table 2
*
Effects of D-Rh2 on spleen and thymus index in Kunming mice
Group
Dose (mg/kg)
Spleen index (mg/g)
Thymus index (mg/g)
Blank
NS
CTX
Rh2
Rh2
—
—
20
5
10
5
10
6.64 1.04
6.61 1.84
4.58 1.11*,#
5.48 1.32
5.35 0.83
5.27 1.21
6.31 1.44
3.35 0.89
1.83 0.85*
0.74 0.23*,#
2.25 0.61*
2.09 0.78*
2.15 1.10*
3.42 0.82#
0
20
5
10
5
10 mg/kg
NS
CTX
Rh2
D-Rh2
Figure 3. Inhibition of tumor growth in animals. Tumors were dissected and
measured weights. Normal saline (NS) treated control group was considered as no
tumor inhibition. Data were presented as the means standard deviation (SD).
Statistical analysis was performed using the SPSS 13.0 statistical software (SPSS ins.,
Chicago, IL, USA). ANOVA procedures followed by the Dunnett’s Multiple Compar-
ison test were used to analyze variance. Differences with p-value less than 0.05
were considered to be statistically significant.
D-Rh2
D-Rh2
The data were presented as means SD (n = 10).
P <0.05 significantly different from the blank group.
*
#
P <0.05, significantly different from the NS treated group.

G.-Q. Wei et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1082–1085
1085
5. Cheng, C. C.; Yang, S. M.; Huang, C. Y.; Chen, J. C.; Chang, W. M.; Hsu, S. L. Cancer
Chemother. Pharmacol. 2005, 55, 531.
6. Popovich, D. G.; Kitts, D. D. Can. J. Physiol. Pharmacol. 2004, 82, 183.
7. Kim, Y. S.; Jin, S. H. Arch. Pharm. Res. 2004, 27, 834.
8. Kim, H. S.; Lee, E. H.; Ko, S. R.; Choi, K. J.; Park, J. H.; Im, D. S. Arch. Pharm. Res.
2004, 27, 429.
mus in tumor-bearing mice (Table 2), which were in a good agree-
ment with the previous data on immunostimulatory activities.37
While in D-Rh2 treated groups, there was significant enhancement
of thymus index at the dose of 10 mg/kg. The results indicated that
the antitumor effect of D-Rh2 might in part attribute to the immu-
nostimulatory function.
9. Fei, X. F.; Zheng, K. Y.; Wang, B. X.; Tashiro, S.; Ikejima, T. Chem. Res. Chin. Univ.
2003, 19, 49.
10. Fei, X. F.; Wang, B. X.; Tashiro, S.; Li, T. J.; Ma, J. S.; Ikejima, T. Acta Pharmacol.
Sin. 2002, 23, 315.
11. Oh, M.; Choi, Y. H.; Choi, S. H.; Chung, Y. H.; Kim, K. W.; Kim, S. I.; Kim, D. K.;
Kim, N. D. Int. J. Oncol. 1999, 14, 869.
12. Kim, Y. S.; Jin, S. H.; Lee, Y. H.; Kim, S. I.; Park, J. D. Arch. Pharm. Res. 1999, 22,
448.
13. Kim, H. E.; Oh, J. H.; Lee, S. K.; Oh, Y. J. Life Sci. 1999, 65, PI33.
14. Kim, H. E.; Oh, Y. J. J. Neurochem. 1998, 70, S15.
15. Zeng, X. L.; Tu, Z. G. Pharmacol. Toxicol. 2003, 93, 275.
16. Lee, K. Y.; Park, J. A.; Chung, E.; Lee, Y. H.; Kim, S. I.; Lee, S. K. Cancer Lett. 1996,
110, 193.
17. Li, B. H.; Zhao, J. O.; Wang, C. Z.; Searle, J.; He, T. C.; Yuan, C. S.; Du, W. Cancer
Lett. 2011, 301, 185.
18. Wu, N.; Wu, G. C.; Hu, R.; Li, M.; Feng, H. Acta Pharmacol. Sin. 2011, 32, 345.
19. Kim, Y. S.; Kim, D. S.; Kim, S. I. Int. J. Biochem. Cell Biol. 1998, 30, 327.
20. Nakata, H.; Kikuchi, Y.; Tode, T.; Hirata, J.; Kita, T.; Ishii, K.; Kudoh, K.; Nagata,
I.; Shinomiya, N. Cancer Sci. 1998, 89, 733.
In summary, for the purpose of reducing side-effect, Rh2 was
structurally modified as a dioctanoyl ester of Rh2 (D-Rh2) by re-
acted with octanoyl chloride. Compared with the parental Rh2, D-
Rh2 could significantly decrease toxicity to the human hepatocyte
cell line QSG-7701 in vitro but not attenuate the antitumor activity
in vivo. In addition, the enhanced effects of D-Rh2 on thymus index
indicated that D-Rh2 might indirectly affect tumor growth by stim-
ulating lymphocytes to become cytotoxic to tumor cells.30 D-Rh2
could be a more potential candidate for using as an antitumor drug
due to the possible lower side-effect. The results obtained in the
study will provide basic data for further development and utiliza-
tion of D-Rh2 in tumor prevention and therapy.
21. Choi, S.; Oh, J. Y.; Kim, S. J. J. Cell. Biochem. 2011, 112, 330.
22. Xie, X. W.; Eberding, A.; Madera, C.; Fazli, L.; Jia, W.; Goldenberg, L.; Gleave, M.;
Guns, E. S. J. Urol. 2006, 175, 1926.
23. Musende, A. G.; Eberding, A.; Jia, W.; Ramsay, E.; Bally, M. B.; Guns, E. T.
Prostate 2010, 70, 1437.
24. Oian, T. X.; Cai, Z. W.; Wong, R. N. S.; Jiang, Z. H. Rapid Commun. Mass Spectrom.
2005, 19, 3549.
25. Gu, Y.; Wang, G. J.; Sun, J. G.; Jia, Y. W.; Wang, W.; Xu, M. J.; Lv, T.; Zheng, Y. T.;
Sai, Y. Food Chem. Toxicol. 2009, 47, 2257.
Acknowledgments
This research was financially supported by the Science and
Technology Development Program from Jilin Provincial Science &
Technology Department (yyzx 201135), and in part by Grant
2010J05085 from Fujian Provincial Department of Science & Tech-
nology. We are grateful to the Laboratory Animal Center of Jilin
University (Changchun, China) for providing experimental animals.
26. Gu, Y.; Wang, G. J.; Wu, X. L.; Zheng, Y. T.; Zhang, J. W.; Ai, H.; Sun, J. G.; Jia, Y.
W. Xenobiotica 2010, 40, 602.
27. Bae, E. A.; Han, M. J.; Kim, E. J.; Kim, D. H. Arch. Pharm. Res. 2004, 27, 61.
28. Hasegawa, H.; Lee, K. S.; Nagaoka, T.; Tezuka, Y.; Uchiyama, M.; Kadota, S.;
Saiki, I. Biol. Pharm. Bull. 2000, 23, 298.
Supplementary data
29. Wakabayashi, C.; Hasegawa, H.; Murata, J.; Saiki, I. Oncol. Res. 1997, 9, 411.
30. Hasegawa, H.; Suzuki, R.; Nagaoka, T.; Tezuka, Y.; Kadota, S.; Saiki, I. Biol.
Pharm. Bull. 2002, 25, 861.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.104.
31. Hasegawa, H. J. Pharmacol. Sci. 2004, 95, 153.
32. Han, M.; Hou, J. G.; Dong, C. M.; Li, W.; Yu, H. L.; Zheng, Y. N.; Chen, L. Molecules
2010, 15, 399.
References and notes
33. Lei, J.; Li, X.; Gong, X. J.; Zheng, Y. N. Molecules 2007, 12, 2140.
34. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
35. Kim, J. Y.; Germolec, D. R.; Luster, M. I. Immunopharmacol. Immunotoxicol. 1990,
12, 257.
36. Shin, H. R.; Kim, J. Y.; Yun, T. K.; Morgan, G.; Vainio, H. Cancer Causes Control
2000, 11, 565.
1. Kitagawa, I.; Yoshikawa, M.; Yoshihara, M.; Hayashi, T.; Taniyama, T. Yakugaku
zasshi 1983, 103, 612.
2. Lai, D. M.; Tu, Y. K.; Liu, I. M.; Chen, P. F.; Cheng, J. T. Planta Med. 2006, 72, 9.
3. Park, E. K.; Choo, M. K.; Oh, J. K.; Ryu, J. H.; Kim, D. H. Biol. Pharm. Bull. 2004, 27,
433.
4. Park, E. K.; Choo, M. K.; Kim, E. J.; Han, M. J.; Kim, D. H. Biol. Pharm. Bull. 2003,
26, 1581.
37. Zhao, L. Q.; Chen, Y. L.; Ren, S.; Han, Y.; Cheng, H. B. Carbohydr. Res. 2010, 345,
637.