全 文 ：Inhibitory effect of total saponins isolated from Taraphochlamys affinis
on duck hepatitis B virus replication
LIN Xing1，HUANG Quanfang2，ZHANG Shijun 1，HUANG Jianchun 1，HUANG Renbin 1*
(1. Guangxi Medical University，Nanning 530021，China;
2. The First Affiliated Hospital of Guangxi Traditional Chinese Medicine University，Nanning 530023，China)
［Abstract］ It has been previously shown that Taraphochlamys affinis possessed anti-hepatitis B virus (HBV)activities． To
identify the active ingredients，the total saponins (TSTA)were isolated from T． affinis and the inhibitory effect of TSTA on HBV in the
duck HBV model was examined． The results showed that serum levels of DHBV-DNA decreased in all ducks treated with TSTA (1． 0
and 2． 0 g·kg－1·d －1)and lamivudine (3TC) (50 mg·kg －1·d －1)during treatment，but 7 days after the cessation of treatment
(p7)with 3TC，the viral replication level returned to the pretreatment baseline． Contrariwise in ducks treated with TSTA，the effect of
DHBV DNA inhibition lasted． Compared with model control group，the alanine aminotransferase (ALT) ，aspartate aminotransferase
(AST)and duck hepatitis B surface antigen (DHBsAg)values of 1． 0 and 2． 0 g·kg－1·d －1-dose TSTA groups were significantly
lower on 7，14 days after the treatment (d7，d14)and p7，and at p7，the ALT and DHBsAg levels of 2． 0 g·kg －1·d －1-dose TSTA
group was significantly lower than that of 3TC group． Furthermore，significant histological improvement was noted in ducklings of TSTA
treatment group 7 days after the withdrawal． The study results demonstrate that TSTA possesses potent anti-HBV activity．
［Key words］ total saponins of Taraphochlamys affinis(TSTA) ;anti-hepatitis B virus
［Manuscript number］ 20110603008
［Funding］ Science and technology research development of Guangxi prov-
ince (0322024-5E) ;The department of education of Guangxi province
(200710MS014，200911MS28)
［Corresponding author］ * Huang Renbin，Tel: (0771)5358272，
E-mail:huangrenbin@ 163. com
The hepatitis B virus (HBV)belongs to the fami-
ly of hepadnaviruses (hepatotropic DNA viruses).
HBV causes acute and chronic infections of the liver
and is responsible for 1. 2 million deaths annually［1］.
Approximately，80% of carriers have different levels of
hepatocyte destruction，which may develop into liver
cirrhosis and hepatocellular carcinoma (HCC)［2］.
Worldwide deaths from liver cancer caused by HBV in-
fection probably exceed 1 million per year ［3］. Despite
the availability of a safe and effective vaccine against
hepatitis B，chronic infection with HBV remains a ma-
jor health problem worldwide. Although anti-HBV
drugs now available have improved the quality of the
lives of HBV patients，the apparently inevitable devel-
opment of drug resistance has prompted the search for
new anti-HBV agents. Natural compounds，because of
their structural diversity，provide a large opportunity
for screening anti-HBV agents with novel structure and
mechanism of action.
Taraphochlamys affinis，the herb of T. affinis
(Giff) Bremekhu ［Strobilanthes affinis (Griff)
Y. C. Tang］，has been widely used in traditional Chi-
nese medicine since ancient times with an excellent
safety record and demonstrated efficacy in the improve-
ment of immune disorders and liver diseases. The total
saponins，one of the major active constituents of T. af-
finis，had been found to be capable of relieving hepatic
fibrosis［4］，immunological liver injury［5］，or lipid per-
oxidation［6］ independently. Furthermore， the direct
effect of TSTA on HBV replication in HepG2 2. 2. 15
cell line was examined. TSTA appeared to downregulate
the secretion of HBsAg and HBeAg［7］as well as the re-
lease of HBV DNA［8］ from HepG2. 2. 2. 15 in a dose-
and time-dependent manner. Unfortunately， unlike
IFN-α or antiviral nucleoside analogues，little is known
about the exact mechanism or target of the total sapo-
nins of T. affinis(TSTA)against HBV in vivo. In this
study，further to investigate TSTA for its anti-HBV ac-
tivity，DHBV-infected duck model to observe and eval-
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uate its effect on the DHBV replication in vivo was
used，and explored its possible antiviral target.
1 Materials
1. 1 Reagents Lamivudine (3TC)was purchased
from GlaxoSmithKline. L-glutamine was obtained from
Sigma (St Louis，MO，USA). HBsAg and HBeAg en-
zyme immunoassay (EIA)kits were purchased from
Nanjing Jianchen Bioengineering Institute (Nanjing，
China).
1. 2 Experimental animals Guangxi ducklings within
1d of hatching were obtained from an animal breeding
farm，The center of experimental animal，Guangxi
Medical University［SCXKG 2003-0003］.
2 Methods
2. 1 Preparation of the total saponins of Taraphochlamys
affinis(TSTA) Total saponins were prepared by the
method described previously with slight modification.
The dry and powdered sample (1 000 g)was extracted
three times with 80% ethanol (herb-ethanol 1∶ 10)un-
der reflux (85 ℃)for 90 min. The alcohol extract was
concentrated，suspended in distilled water and then
partitioned successively with chloroform (ratio 1∶ 5)
and n-butanol saturated with water (ratio 1∶ 3，3
times). The n-butanol extract was combined and evap-
orated using a rotary evaporator at 70 ℃ to give a pow-
der residue. The yield for the extract (TSTA)was
41. 28 g (4. 128%). TSTA was easily soluble in wa-
ter. Then TSTA 0. 1 mL (0. 106 9 g·mL －1)were
mixed with the vanillin (8%，0. 5 mL)and sulphuric
acid (72%，5 mL). The mixture was incubated at 60
℃ for 10 min，cooled in an ice water bath for 15 min
and the absorbance read at 538 nm. Oleanolic acid was
used as a reference standard and the content of total
saponins was expressed as oleanolic acid equivalents
(OAE mg·g －1 extract). The total saponins content
determination showed that TSTA content was 71. 8% .
It should be noted that the vanillin-sulphuric acid rea-
gent gives only an approximate estimate of the total
saponins content.
2. 2 Experimental infection of ducklings Sixty
one-day-old ducklings were intravenously infected with
about 6 × 106 viral DNA equivalent (VGE，1 VGE =
3. 3 × 106 pg)of duck hepatitis B virus (DHBV).
Seven days after infection，50 ducklings were divided
into 5 groups with each consisting of 10 ducks:the
control group (normal saline) ，the positive drug group
(3TC，50 × 10 －3 g· kg －1· d －1) ，and the TSTA
0. 5，1. 0，2. 0 g·kg －1·d －1 groups. Drugs were ad-
ministered orally，bid for 14 d. The serum samples
were obtained before treatment (d0) ，d7，and d14
during treatment，and p7 (d21)after the cessation of
treatment. The serums were stored at － 70 ℃ for fu-
ture analysis.
2. 3 Analysis of serum samples viral DNA Isolation
of DNA from each serum sample，300 μL sera was in-
cubated at 65 ℃ for 4 h in lysis buffer［20 mmol·L －1
Tris-HCl(pH 8. 0) ，10 mmol· L －1 EDTA，0. 1%
SDS，and 0. 8% proteinase K］. DNA was then extrac-
ted with phenol-chloroform and precipitated with etha-
nol. The pellet was dissolved in 50 μL of RNase- and
DNase-free ddH2O.
PCR was employed to exclude congenitally DH-
BV-infected ducklings. The serum samples were tested
by PCR for the presence of DHBV DNA. The PCR
mixture also contained 2. 5 μL of 10 × reaction buffer
(TaKaRa，Dalian，China) ，1. 5 μL of 25 mmol·L －1
MgCl2，1. 25 μL of 10 × dNTP，0. 2 μL Taq polymer-
ase (TaKaRa，Dalian，China) ，1 μL of both forward
and reverse primers，and 16. 55 μL ddH2O. The PCR
primers were designed according to the sequence in
GenBank (Accession No． M32990)and were as fol-
lows: 5-AACCATTGAAGCAATCACTAGAC-3 and
5-ATCTATGGTGGCTGCTCGAACTA-3 . The PCR
protocol was 10 min at 94 ℃，followed by 40 cycles of
30 s at 94 ℃，30 s at 55 ℃，and 45 s at 72 ℃. The
PCR product was detected by 1. 5% agarose gel elec-
trophoresis.
2. 4 Oligonucleotide primers and probe for Real-time
PCR Real-time PCR was performed using the BioRad
Lightcycler (BioRad，iCycler iQ，USA)on extracted
DHBV DNA samples and quantitated using fluores-
cence probe. Each 25 μL reaction mixture contained
50 ng DNA，2 μL probes (Genecore，Shanghai，Chi-
na) ，2. 5 μL MgCl2(at a final concentration of 2. 5
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mmol·L －1) ，0. 25 μL TaKaRa Ex Taq HS (5 U·
μL －1) (TaKaRa，Dalian，China) ，and 0. 5 μL of 10
μmol · L －1 primers. The probe sequence (5-
CGGGCTCCCCTCTCCCACG-3)was labeled with 6-
carboxy-fluorescein dye at the 5-end and with the
quencher dye 6-carboxy-tetramethyl-rhodamine at the
3-end. The sense primer sequence was 5-GAGC-
CCCTTCACCCCAAC-3 and antisense primer sequence
was 5-ATCTATGGTGGCTGCTCGAACT-3 . The
primers and probe were synthesized by Shanghai Ge-
necore (TaqMan minor groove binder，Shanghai，Chi-
na). The PCR protocol required initial incubation for
10 min at 95 ℃ and then 45 cycles of 10 s at 95 ℃，
20 s at 55 ℃，and 30 s at 72 ℃. Each sample was de-
tected in triplicate.
2. 5 Preparation of standards The 218 bp PCR prod-
uct was inserted into the vector of a pMD18-T(TaKa-
Ra，Dalian，China). It resulted in plasmid and named
pDHBV. Plasmid DNA was then isolated with the DNA
purification system and quantified by spectrophotome-
try. Concentrations were converted to copies /μL by
use of the Avogadro constant. Serial dilutions of plas-
mids were used for the construction of the standard
curve and for the validation of the real-time PCR as-
say. Plasmids were detected in triplicate. The correla-
tion between the quantification of DHBV and pDHBV
plasmids was evaluated by the regression analysis.
2. 6 Analysis of serum ALT，AST and DHBsAg Ser-
um samples were tested on the day of collection for lev-
els of alanine aminotransferase (ALT)and aspartate
aminotransferase (AST) ，using an automatic analyzer
(Abbott Laboratories Co. Ltd. ，Diagnostics Division，
USA). Serum DHBsAg was analyzed by quantitative
enzyme-linked immunosorbent assay (ELISA)accord-
ing to the manufacturers instructions.
2. 7 Histopathological examination of duck liver On
p7 (d21)after the cessation of treatment，each duck-
ling was laparotomized to obtain the liver immediately
after collecting blood from the leg vein. Fragments of
the ducklings liver were fixed in 10% formalin solu-
tion， embedded in paraffin， sectioned at 5 μm，
stained with hematoxylin and eosin，and examined by
light microscopy.
2. 8 Statistics The data were expressed as 珋x ± s，and
analyzed by one-way repeated-measure ANOVA and
t-test for comparisons between groups. P ＜ 0. 05 was
considered statistically significant.
3 Results
During the experiments，no obvious side effects
were observed in animals receiving antiviral therapy or
in control animals.
3. 1 Detection of DHBV-related avihepadnaviruses in
serum samples In total，7 of 60 ducklings detected
were found to be congenitally-infected birds by PCR
and were excluded from experiments (Fig. 1).
Lane 1. DHBV-positive control;lane 2. negative control;lanes 3-6. ser-
um samples;lane 7. 100 bp size markers.
Fig. 1 Detection of congenitally DHBV-infected ducklings by
PCR in serum samples
3. 2 Standard curve The concentration of plasmid
DNA was 183. 92 μg·L －1. Concentrations were con-
verted to 1. 0 × 107 copies /μL by use of the Avogadro
constant. The amplification lines of reaction templates
were 1. 0 × 107copies /μL diluting as 10 × grad (10 －1，
10 －2，10 －3，10 －4，10 －5，10 －6). The standard curve
was drawn by 45 cycles of amplification of the plasmid
samples and we obtained a linear quantification with a
slope of － 7. 146 and an R2 ＞ 0. 998 . According to
the standard curve，the sample copies were equal to lg
(80. 069 － Y)/7. 146，where Y is the Ct (cycle
threshold). The sensitivity limit was 5 copies /μL
(100% positive results).
3. 3 Detection of duck serum DHBV replication level
The effects of TSTA and 3TC，which were used for
comparison，on DHBV replication in vivo were deter-
mined by quantification of DHBV-DNA by real-time
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PCR. The levels of serum viral DNA were recorded in
the 5 groups before the experiment. During treatment，
serum levels of DHBV-DNA decreased in all 10 ducks
treated with TSTA 1. 0 and 2. 0 g·kg －1·d －1(P ＜
0. 05). But 7 days after the cessation of treatment with
3TC，the viral replication level returned to the pretreat-
ment baseline. In ducks treated with TSTA，the effect
of DHBV DNA inhibition lasted(P ＜ 0. 05). No signif-
icant decrease of serum DHBV-DNA was observed dur-
ing treatment with TSTA 0. 5 g·kg －1·d －1(Fig. 2 ).
Control. DHBV model control;3TC. 3TC(50 × 10 －3 g·kg －1·d －1) ;
TSTA 0. 5. TSTA (0. 5 g·kg －1·d －1) ;TSTA 1. 0. TSTA (1. 0 g·
kg －1·d －1) ;TSTA 2. 0. TSTA (2. 0 g·kg －1·d －1) ;1)P ＜ 0. 05，
2)P ＜ 0. 01 vs control;3)P ＜ 0. 05 vs 3TC group (the same to Fig. 3-5)
.
Fig. 2 Effect of treatment with TSTA on DHBV replication level
in duck serum(珋x ± s，n = 10)
3. 4 Serum ALT，AST and DHBsAg Compared with
DHBV model control group，the ALT，AST and DHB-
sAg values of 1. 0 and 2. 0 g·kg －1·d －1-dose TSTA
groups were significantly lower on d7，d14 and p7
(P ＜ 0. 05 or P ＜ 0. 01) ，and at p7，the ALT and DH-
BsAg levels of 2. 0 g·kg －1·d －1-dose TSTA group
was significantly lower than that of 3TC group (P ＜
0. 05) (Fig. 3，4，5).
3. 5 Histopathological examination of duck livers
Histopathological profiles of the liver from model con-
trol group ducklings revealed necrosis，steatosis，and
often swelling of the hepatic cytoplasm. Pathological
changes in the 3TC group was obviously improved. A
significant improvement of the hepatocellular architec-
ture and a considerable reduction in necrosis and vacu-
olation were found. The protective effect of TSTA
was confirmed by histopathological examinations. Ad-
ministration of TSTA to the experimental animals (2. 0
g·kg －1·d －1)showed a significant improvement of
the hepatocellular architecture over the model group，
as evident from a considerable reduction in necrosis
and vacuolation (Fig. 6).
4 Discussion and conclusion
In primary study，some kinds of traditional Chi-
nese medicine was selected，which have long been
used in the folk treatment of chronic hepatitis in
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A. control;B. 3TC group;C-E. TSTA-treated group at a dose of 0. 5，
1. 0，2. 0 g·kg －1·d －1，respectively.
Fig. 6 Histopathological examinations of liver in ducklings trea-
ted for 14 d (Hematoxylineosin staining;× 100)
China，for screening novel anti-HBV agents. Among
them， the water extract from Helicteres angustifolia
could effectively inhibited the HBV DNA duplication in
HepG2. 2. 15 cell culture［9］，and inhibit DHBV［10］ .
Similar results of the inhibitory effect of it on HBsAg
and HBeAg production in cultured 2. 2. 15 cells was al-
so observed［11］. However，what the active part of T.
affinis against HBV is still unknown. Previously，we
isolated the total saponins from T. affinis(TSTA) ，and
our studies showed that TSTA could markedly decrease
ALT，AST，MDA content，increase SOD，T-AOC ac-
tivity in serum and in liver［12］，and could significantly
increase percentage of CD3
+，CD4
+ cell and CD4
+ /
CD8
+，decrease the ratio of CD8
+［5］，which indicated
TSTA had a effect of anti-lipiperoxidation and a protec-
tive effect on immunological liver injury in mice.
In this paper，we attempted to demonstrate the ef-
ficient anti-HBV activity of TSTA in the duck HBV
model. This experiment with TSTA (0. 5，1. 0，2. 0 g
·kg －1·d －1)in ducklings pointed to a suppressive
action on DHBV replication in vivo. During treatment，
serum levels of DHBV-DNA significantly decreased in
all 10 ducks treated with TSTA 1. 0 and 2. 0 g·kg －1
·d －1. It was well known that most antivirus medicines
had the inevitable rebound effect after drug cessation.
This shortcoming had limited the therapy to those dis-
eases infected by viruses such as HB or AIDS. The
similar phenomena appeared in the positive control
drug 3TC in the present study. TSTA showed therapeu-
tic effects as well as 3TC，and no difference was ob-
served after cessation of TSTA therapy compared to
TSTA-treated animals. It suggested that TSTA could
maintain for a long time in treating viremia of HBV and
the effect of DHBV-DNA inhibition showed a concen-
tration-dependent response.
Compared with the control，the serum ALT，AST
levels were decreased 7days after the treatment and 7
days after the treatment withdrawal， indicating that
TSTA could improve the serum biochemistry and was as-
sociated significantly with high rates of virologic，bio-
chemical and histological improvement after treatment.
The presence of both DHBsAg and antisurface an-
tibodies may have caused some of the variability seen
in levels of DHBsAg and antisurface antibodies，due to
the varying amounts present in the form of antigen-anti-
body complexes. The presence of circulating antisur-
face antibodies is generally thought to be protective，
and in HBV it is recognized as a marker of immunity
and of the resolution of disease. However，complexes
of HBsAg and immunoglobulin have been detected in
acute HBV infection［13］ and in healthy carriers and in
patients with symptoms of chronic infection［14］ . The
presence of these complexes has been related to HBV
infection outcomes，with an appearance time of 1 to 27
weeks seen in acute infection and long-term persistence
of complexes in chronically infected individuals［15］ .
Our study showed that TSTA significantly inhibited the
secretion of HBsAg，with TSTA concentration increas-
ing，a dose-dependent response was observed.
Furthermore，significant histological improvement
was noted in ducklings of TSTA treatment group 7 days
after the withdrawal.
In summary，TSTA，a potent inhibitor of the hep-
adnaviral polymerases，prevents the development of in-
fection when administered in the early stages of DHBV
infection. It is a potent and rapid-acting suppressor of
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DHBV replication. The mechanism which allows DNA
to rebound withdrawal and the spontaneous viral ge-
nome variability leading to the emergence of drug-re-
sistant mutants requires further study. A long-term
study and the toxicity of TSTA also require further
study. It is likely that dosage form is important for
TSTA. This study has opened the way for further stud-
ies，including an optimal dosing regimen and effective
treatment approaches.
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六月青总皂苷对鸭 HBV病毒复制的抑制作用
林兴1，黄权芳2，张士军1，黄建春1，黄仁彬1*
(1. 广西医科大学，广西 南宁 530021;
2. 广西中医学院 第一附属医院，广西 南宁 530023)
［摘要］ 采用鸭乙型肝炎动物模型观察六月青总皂苷抗鸭乙型肝炎病毒与保护肝细胞的作用，实验结果表明，六月青总
皂苷(1. 0，2. 0 g·kg －1·d －1)和 3TC(50 mg·kg －1·d －1)能明显抑制 DHBV-DNA 复制，抑制 ALT，AST 和 DHBsAg 水平，改
善肝组织病理学损伤。不同于 3TC的是六月青总皂苷在停药后仍能显示有抑制病毒的作用，没有出现反跳现象，提示，六月
青总皂苷抗病毒的作用机制有别于 3TC。实验结果表明六月青总皂苷具有有效抑制 DHBV-DNA和保护肝细胞的作用。
［关键词］ 六月青总皂苷;抗乙肝病毒
doi:10． 4268 /cjcmm20120327
［责任编辑 吕冬梅］
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