The American Journal of Chinese Medicine, Vol. 34, No.

2, 341–349
© 2006 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine

Anti-hepatitis Activities in the Broth of

Ganoderma lucidum Supplemented with a
Chinese Herbal Medicine
Yanqun Li
College of Life Science, South China Normal University
Guangzhou 510631, China

Yailong Yang
School of Life and Environment Science, Wenzhou University
Wenzhou 325000, China

Lu Fang
Jiangxi Institute of Medicine, Nanchang 330046, China

Zhibin Zhang
Wuxi Hospital of Chinese Traditional Medicine, Wuxi 214001, China

Jian Jin and Kechang Zhang

School of Biotechnology, Southern Yangtze University
Wuxi 214036, China

Abstract: The anti-hepatitis B virus activity and hepatoprotective activity of a liquid

fermentation broth of Ganoderma lucidum were investigated. The cultured broth was
supplemented with aqueous extract of Radix Sophorae flavescentis, a kind of Chinese herbal
medicine. Our results indicated that the cultured broth had effects of anti-hepatitis B virus
activity in vitro and protected mice from liver damage in vivo. Our results also indicated that
the co-fermentation broth of Ganoderma lucidum in the presence of aqueous extract of Radix
Sophorae flavescentis has better medicinal effects than simply mixing these two ingredients
together, suggesting a potential novel way to prepare Chinese herbal mixtures.

Keywords: Ganoderma lucidum; Radix Sophorae flavescentis; Anti-hepatitis B Virus;

Hepatoprotection; Submerged Culture.

Correspondence to: Dr. Yanqun Li, College of Life Science, South China Normal University, Guangzhou 510631,
China. Tel/Fax: (+86) 20-3170-8125, E-mail: liyq9168@hotmail.com

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 342 Y. LI et al.

Introduction

Ganoderma lucidum (Fr.) Krast (Polyporaceae), a member of the basidiomycete family, is

a traditional Chinese medicine generally used for invigorating healthy qi and strengthening
body resistance to diseases. Recent literatures showed that G. lucidum had the ability to
regulate and boost the immune system (Zhang et al., 2002), suppress tumor growth (Wang
et al., 1997), inhibit viral infection (Sahar et al., 1998; Kim et al., 2000), and enhance
hepatic functions (Back et al., 1999). The dried root of Sophora flavescens Ait. is another
traditional Chinese medicine named Radix Sophorae flavescentis (RSF). It is often used for
the treatment of skin and mucosal ulcers and sores, gastrointestinal hemorrhage, diarrhea,
inflammation, and arrhythmia. In recent years, the antiviral effects of RSF have attracted
considerable attention (Ma et al., 2002) and RSF has been used in prescriptions for the
treatment of chronic hepatitis B virus (Liu et al., 2003).
In traditional Chinese medicine practices, G. lucidum is usually used in conjunction
with other traditional Chinese medicines, such as RSF. A common practice of such clinical
approach is to allow RSF and the fruiting body of G. lucidum to be decocted together to
treat hepatitis. The fruiting body of G. lucidum grows slowly and it takes several months
before it can be harvested. In addition, the quality of G. lucidum varies from batch to
batch depending on the growth conditions. Therefore, a faster and more quality consistent
method is needed to grow G. lucidum. In several recent reports, it was demonstrated that
the liquid fermentation broth of G. lucidum can be produced and harvested in less than
a week (Yang and Liau, 1998; Fang and Zhong, 2002). It was further demonstrated that
the liquid fermentation broth of G. lucidum culture has the same medical effects as the
fruiting body.
In the present study, G. lucidum was cultured in a medium supplemented with an
aqueous extract of RSF. It is anticipated that the RSF extract will not only mix with the
cultured broth, but also participate in the fermentation process of G. lucidum. We found
that the fermentation broth of G. lucidum, when supplemented with the RSF extracts,
has higher anti-hepatitis B virus activity than the non-supplemented broth, as well as
the RSF extracts alone. Our results suggested that the co-fermentation process may bring
out some novel ingredients with desired medicinal effects. Our liquid co-fermentation
process provided a novel methodology to the preparation of Chinese herbal mixture for
medicinal usage.

Materials and Methods

Materials and Chemicals

3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) and lipopoly-

saccharide (LPS) were purchased from Sigma Chemical Co. (USA); Minimum Essential
Medium (MEM) for cell culture was purchased from GIBCO-BRL (USA). Carbon
tetrachloride (CCl4) was purchased from Merck (Germany). Radioimmunoassay (RIA)

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 G. LUCIDUM SUPPLEMENTED WITH A CHINESE HERBAL MEDICINE 343

kits for HBV surface antigen (HBsAg) and e antigen (HBeAg) test were purchased
from the Institute of Atomic Energy of China (Beijing, China). Cyclophosphamide (CP)
was purchased from Shanghai Hua Lian Pharmaceutical Co. Ltd. (Shanghai, China).
Biphenyldicarboxylate (DDB) was purchased from Jiangsu Huanghe Pharmaceutical
Co. Ltd. (Jiangsu, China). Bacille Calmette-Guérin (BCG) was purchased from Shanghai
Institute of Bio-products (Shanghai, China). RSF was purchased from SHANHE
Pharmaceutical Group (Wuxi, China). All the other chemicals used were at least
reagent grade.

Animals

Male KM mice weighing 18–22 g (seven weeks of age) were housed in a temperature
controlled room (23 ± 3°C) with lighting maintained at a 12-hour light/dark cycle. Animals
were fed with a standard laboratory chow and water ad libitum. Animals were randomly
divided into control, hepatotoxin-treated and medicine-treated groups (ten mice in
each group).

Preculture of G. lucidum

The strain of G. lucidum CCGMC 5.616 was purchased from China General Microbiological
Fermentation Collection Center (Beijing, China). Preculture medium contained (g l−1):
glucose, 40; peptone, 4; yeast extract, 2; KH2PO4·3H2O, 1.5; MgSO4·7H2O, 0.75; vitamin
B1, 0.01. G. lucidum was grown in a 250 ml flask containing 70 ml medium at 30˚C for
7 days with shaking at 150 rpm.

Preparation of RSF Extracts (RSFE)

After having soaked for 1 hour, 1 kg sliced RSF was extracted with 6 liters of boiling water
for 30 minutes. The residue was subjected to further extraction in 4 liters of boiling water.
The decoction from the two successive extractions was combined and filtered through a
filter paper (Xinhua No.1 filter paper). The filtrate was collected and lyophilized.

Preparation of Cultured Broth of G. lucidum (GL)

A broth of G. lucidum was prepared through cultivation in a basic medium that contained
(g l−1): glucose, 40; peptone, 4; yeast extract, 2; KH2PO4·3H2O, 1.5; MgSO4·7H2O, 0.75;
and vitamin B1, 0.01. 70 ml medium was added into a 250 ml flask and inoculated with
8 ml precultured broth. After 96 hours of cultivation at 30ºC on a rotary shaker (150 rpm),
the mycelia were removed from the cultured broth by centrifugation, and the supernatant
was concentrated and lyophilized.

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 344 Y. LI et al.

Broth of G. lucidum Cultured with the Extracts from RSF (BGR)

BGR was prepared by culturing G. lucidum in the medium described above except with the
addition of 10 g/l lyophilized RSF extract. The culture conditions and supernatant harvest
were performed as described above.

Mixture of RSF Extract and Broth of G. lucidum (Mix.)

A mixture was prepared with 10 g dried RSF extract and 1 liter G. lucidum broth which
was cultured in the basic medium as GL and the mycelia removed by centrifugation. After
thorough mixing, the mixture was concentrated at 50ºC and finally lyophilized.

Cell Culture

Hepatitis B virus (HBV) transfected human hepatoma HepG2-2.2.15 cells (Sells et al.,
1987) were maintained in MEM with 10% fetal bovine serum, 100 units/ml of streptomycin
and penicillin, 1 mM glutamine and 200 µg/ml G418. Cells were seeded into 24-well
plates at the density of 105 cells/well. After 3 days of incubation, the medium was replaced
with fresh medium containing test samples at various concentrations (the control group
contained no test samples). Fetal bovine serum and G418 in the fresh medium were
changed to 2% and 380 µg/ml, respectively. After 4 days of incubation, the cultured
medium was replaced again with fresh medium containing the same test samples. To
measure the effects of test samples on cell growth, culture medium at day 12 was replaced
with PBS solution containing 0.5 mg/ml MTT. After 4 hours of incubation, PBS solution
was removed and 1 ml dimethyl sulfoxide was added into each well. Absorbance (A) at
490 nm was measured and the cytotoxicity was calculated with the formula as rate of
A
living cell (RLC) = i � 100%, where A0 is the absorbance of control cultures and Ai is
A0
absorbance of cultures containing test samples. To measure the effects of test samples
on viral replication, HBV surface antigen (HBsAg) and e antigen (HBeAg) in the
cultured medium were tested by immunoassay kits. Inhibition ratio of antigen (IRA) was
C � Ci
calculated with a formula IRA = 0 � 100%, where C0 is the antigen content of
C0
control culture medium and Ci is the antigen content of culture medium with test samples.

CCl4-Induced Hepatotoxicity in Mice

Mice were randomly divided into 11 groups of ten animals each. Normal control
group received 10 ml/kg olive oil (i.p.) and 30 ml/kg·day saline (i.g.). The model group
was given olive oil with CCl4 (0.1%, 10 ml/kg, i.p.). The standard reference group received
CCl4/olive oil (0.1%, 10 ml/kg, i.p.) and DDB (200 mg/kg in saline, i.g.). The test groups
were treated with CCl4/olive oil (0.1%, 10 ml/kg, i.p.) and BGR, Mix, RSFE or GL
(100 mg or 300 mg/kg i.g.), respectively, prepared as mentioned above in saline solution.

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 G. LUCIDUM SUPPLEMENTED WITH A CHINESE HERBAL MEDICINE 345

Animals were treated for 7 consecutive days before CCl4/olive oil administration. No
food was provided after CCl4 administration, but tap water was made available ad libitum.
Blood was drawn from the eye socket 16 hours after CCl4 administration. Blood samples
were centrifuged at 3000 g and 4°C for 10 minutes to obtain the serum. The activities of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were
measured with VENUSLEON-180 Eos bravo biochemistry measuring system (Hospitex
Diagnostics Landao Laboratories LTD, Italy).

BCG/LPS-Induced Hepatotoxicity in Mice

Mice were randomly divided into 12 groups of ten animals each. Normal control group
was given 30 ml/kg·day saline (i.g.). In the other 11 groups, each animal was treated with
0.2 ml BCG (contained 5 × 106 bacteria, i.v) on the first day. Then, the model group
received 30 ml/kg·day saline (i.g.). The two standard reference groups were given either
200 mg/kg·day DDB (i.g.) or 20 mg/kg·day CP (i.g.). The treated groups were given
a prepared drug samples (BGR, Mix, RSFE or GL), which was suspended in saline at
a dose of 100 mg or 300 mg/kg (i.g.), respectively. Ten days later, each animal except
normal control was given 7.5 µg LPS (i.v.). 16 hours later, blood was drawn from the eye
socket. The blood samples treatment and the activities of ALT and AST measurements as
mentioned above.

Statistical Analysis

All values were presented as mean ± SD. Completely randomized design and t-test were
used to analyze the statistical difference between the treatment and the control groups. A
level of difference of p < 0.05 was considered significant.

Results

The results in Table 1 indicated that the fermentation broth of G. lucidum in the presence
of RSF extract (BGR) inhibited 2.2.15 cells to secret HBsAg and HBeAg. The extent of
inhibition seems to be dose effective. In addition, the inhibitory effects of BGR seem to be
higher than all the other test samples (Mix, RSFE and GL). The cytotoxicity of both Mix
and RSFE was higher than BGR. However, GL had low cytotoxicity on the 2.2.15 cells at
the tested dosages.
CCl4 induced a marked increase of ALT and AST levels in mice serum as shown in
Table 2. However, administration with BGR significantly (p < 0.01) decreased the ALT
and AST levels and the effects appeared to be dose-dependent. Our results also indicated
that BGR, Mix and RSFE, but not GL, had significant protective effects on hepatotoxicity
induced by CCl4.
The liver damage induced by BCG + LPS significantly increased the levels of ALT
and AST in mice serum of the model group, but the levels were significantly decreased
(p < 0.01) in BGR-, Mix- and GL-treated groups as shown in Table 3. Furthermore, the

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 346 Y. LI et al.

decrease of ALT and AST levels in BGR-treated groups was more significant than DDB
at 200 mg/kg. The levels in BGR groups were significantly (p < 0.01) lower than in Mix
groups and GL groups. The RSFE groups did not show a marked decrease in the ALT and
AST levels.

Table 1. The Anti-HBV Effects of BGR, Mix, RSFE and GL in Vitro (x̄ ± s, n = 4)
IRA of HBsAg/ (%) IRA of HBeAg/ (%)
Cont./ RLC/
Sample
(µg/ml) 4d 8d 12 d 4d 8d 12 d (%)
BGR 0.5 45.3 ± 3.8 37.4 ± 2.3 34.4 ± 3.7 63.8 ± 5.1 50.4 ± 4.8 49.5 ± 3.6 100
1 67.6 ± 2.1 70.2 ± 3.4 77.1 ± 5.1 73.2 ± 2.4 59.8 ± 3.3 57.8 ± 4.1 100
2 78.1 ± 2.7 79.2 ± 6.8 77.8 ± 3.5 82.4 ± 4.5 64.9 ± 4.8 63.9 ± 2.8 100
4 87.5 ± 3.5 81.3 ± 4.2 82.2 ± 4.3 89.7 ± 3.9 79.5 ± 3.8 77.6 ± 3.9 100
8 100 ± 0.0 97.6 ± 2.5 96.3 ± 3.1 91.0 ± 4.1 85.2 ± 2.8 92.1 ± 4.2 79.8

Mix 0.5 31.9 ± 3.6 25.4 ± 3.4 20.2 ± 4.8 33.6 ± 2.8 25.5 ± 4.1 23.0 ± 3.8 100
1 43.5 ± 5.8 49.1 ± 5.2 45.7 ± 3.8 43.2 ± 4.4 45.1 ± 4.8 31.5 ± 3.4 96.6
2 55.1 ± 3.7 59.3 ± 4.8 59.1 ± 5.1 52.3 ± 3.7 54.7 ± 5.2 63.0 ± 2.8 78.8
4 63.2 ± 5.1 75.7 ± 2.8 85.1 ± 3.2 60.6 ± 6.3 79.5 ± 3.1 83.8 ± 4.3 57.2
8 67.3 ± 4.3 88.8 ± 6.0 89.5 ± 3.5 65.9 ± 5.0 85.3 ± 5.8 92.2 ± 4.8 35.8

RSFE 0.5 34.7 ± 2.9 4.8 ± 1.8 3.3 ± 1.2 22.1 ± 3.0 9.94 ± 1.5 8.3 ± 1.8 100
1 43.0 ± 1.8 13.1 ± 2.2 10.7 ± 2.1 30.4 ± 2.8 45.2 ± 2.4 30.8 ± 3.2 95.9
2 54.1 ± 5.7 56.5 ± 3.6 40.4 ± 3.2 43.4 ± 2.3 55.2 ± 3.8 60.3 ± 2.8 87.3
4 59.9 ± 3.1 70.7 ± 3.5 80.5 ± 4.8 55.4 ± 3.1 79.1 ± 4.2 83.8 ± 3.1 69.2
8 63.6 ± 4.5 84.2 ± 4.5 86.7 ± 5.7 58.3 ± 2.4 88.3 ± 3.1 90.5 ± 3.5 38.5

GL 0.5 10.3 ± 1.5 11.7 ± 1.7 17.9 ± 3.8 9.2 ± 2.7 8.4 ± 1.9 16.7 ± 2.3 100
1 26.6 ± 2.2 29.8 ± 3.1 32.7 ± 3.0 25.4 ± 3.2 27.7 ± 3.5 26.8 ± 3.6 100
2 44.9 ± 2.3 49.0 ± 3.6 52.5 ± 1.9 48.3 ± 4.6 43.9 ± 4.8 48.3 ± 4.8 100
4 59.9 ± 3.4 65.8 ± 3.8 70.3 ± 2.8 62.7 ± 3.9 61.3 ± 5.3 65.9 ± 5.4 100
8 76.1 ± 3.1 80.6 ± 4.8 83.2 ± 4.2 82.0 ± 1.8 81.3 ± 3.5 79.4 ± 3.2 100

Table 2. The Protective Effects of BGR, Mix, RSFE and GL on Chemical Liver Injury Induced by CCl4
(x̄ ± s, n = 10)
Group Dosage (mg/kg·d) ALT (U/L) AST(U/L)
Control 60.33 ± 10.12 84.9 ± 11.0
Model 441.6 ± 61.3* 495.4 ± 71.2*
DDB 200 332.6 ± 69.1† 387.3 ± 73.9†
BGR 100 320.6 ± 69.5† 362.0 ± 78.7†
300 289.2 ± 67.4† 322.8 ± 63.7†
Mix 100 321.7 ± 75.1† 378.1 ± 60.7†
300 292.1 ± 69.9† 329.4 ± 66.3†
RSFE 100 314.5 ± 77.6† 348.4 ± 74.1†
300 284.7 ± 71.3† 317.0 ± 61.5†
GL 100 406.1 ± 58.8 431.7 ± 61.2
300 380.0 ± 68.6 396.0 ± 78.9‡
Notes:*Compared with control p < 0.01, †compared with model p < 0.01, ‡compared with model p < 0.05.

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 347 Y. LI et al.

Table 3. The Protective Effects of BGR, Mix, RSFE and GL on Mice Immunity Liver Injury Induced by
BCG + LPS (x̄ ± s, n = 10)
Group Dosage (mg/kg·d) ALT (U/L) AST(U/L)
Control 64.51 ± 37.42 81.1 ± 31.3
Model 264.8 ± 44.5* 301.3 ± 56.3*
CP 20 98.4 ± 28.4† 85.8 ± 36.5†
DDB 200 155.1 ± 54.2† 221 ± 59.23‡
BGR 100 150.0 ± 40.3† 1 81.6 ± 57.1†
300 110.1 ± 52.6† 131.1 ± 42.3†
Mix 100 233.0 ± 44.0 239.2 ± 45.3‡
300 207.4 ± 65.5‡ 216.2 ± 33.5†
RSFE 100 256.7 ± 44.5 296.4 ± 52.6
300 248.9 ± 47.8 288.8 ± 47.1
GL 100 210.7 ± 52.7‡ 234.6 ± 50.0‡
300 194.0 ± 55.4‡ 217.2 ± 45.1†
Notes:*Compared with control p < 0.01, †compared with model p < 0.01, ‡compared with model p < 0.05.

Discussion and Conclusions

Hepatitis B is a disease caused by the hepatitis B virus. Liver cell injury is its primary
characteristic. Therefore, it is very important that an anti-hepatitis B medicine has effects
of both anti-virus activity and hepatoprotection.
HepG2 2.2.15 cell line originated from an independent transfection of HepG2 cells
with HBV DNA (Sells et al., 1987) and it was established as an in vitro model system for
analyses of HBV replication (Sells et al., 1987; Sureau et al., 1986). The results presented
in this paper clearly demonstrated the anti-HBV activities of a liquid fermentation broth of
G. lucidum supplemented with RSF extract. The co-fermentation broth of G. lucidum with
RSF extracts has better antiviral and hepatoprotective activities than simply mixing the
cultured broth of G. lucidum with RSF extracts (Mix).
CCl4-induced hepatotoxicity and BCG + LPS-induced liver injury models are
conventionally used for the investigation of novel liver protective agents. Administration of
CCl4 and BCG + LPS caused a rapid increase in ALT and AST levels. Serum transaminase
elevation has been reported to be associated with a number of inflammatory disorders
(Hoder and Wilkinson, 1980) and hepatocellular damage (Sinha and Saran, 1972). Leakage
of large quantities of enzymes into the blood stream is often associated with massive
necrosis of the liver (Rees and Spector, 1961). CCl4-induced acute liver injury is similar to
the damage of acute hepatitis (Recknagel, 1967).
In this study, the anti-HBV effects of BGR in vitro were more significant than the other
three samples and the cytotoxicity of BGR was lower than Mix and RSFE. It suggested that
fermentation of RSF extracts with G. lucidum might have promoted its anti-HBV effects
and reduced its cytotoxicity.
The protective effects of BGR on chemical liver injury showed no significant difference
compared to Mix and RSFE, but were better than GL in this research. The fact that BGR
and Mix all contained RSFE suggested that the protective effects on chemical liver injury

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 348 Y. LI et al.

induced by CCl4 might mainly be contributed by RSFE and that the fermentation with
G. lucidum did not reduce the protective effects of RSFE.
However, the protective effect of BGR on autoimmune liver injury was significantly
better than the other three samples. These results suggest that the co-fermentation of
G. lucidum with RSFE has additive effects on the protection of autoimmune liver injury
induced by BCG + LPS.
In conclusion, our study demonstrated that the liquid fermentation of G. lucidum
is an effective way to process medicinal herbs. Addition of the RSF extract during the
fermentation process augmented the anti-HBV virus activity in vitro, as well as the
hepatoprotective effects in vivo.

Acknowledgments

The authors wish to thank Professor Y.L. Xiong (College of Agriculture Food Science
Section, Lexington, KY, USA) and Dr. Richard Zhang (Xcyte Therapies, Inc. Seattle, WA,
USA) for their advice on the writing of this article.

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