Anti-hepatitis B activities of ganoderic acid from Ganoderma lucidum.

Abstract

Ganoderic acid, from Ganoderma lucidum, at 8 microg/ml inhibited replication of hepatitis B virus (HBV) in HepG2215 cells over 8 days. Production of HBV surface antigen and HBV e antigen were 20 and 44% of controls without ganoderic acid. Male KM mice were significantly protected from liver injury, induced with carbon tetrachloride, by treatment with ganoderic acid at 10 mg and 30 mg/kg x d (by intravenous injection) 7 days. Ganoderic acid at the same dosage also significantly protected the mice from liver injury induced by M. bovis BCG plus lipopolysaccharide (from Escherichia coli 0127:B8).

Full text

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Abstract
lucidum, at 8 lg/ml inhibited replication of hepa-
titis B virus (HBV) in HepG2215 cells over 8 days.
Production of HBV surface antigen and HBV e
antigen were 20 and 44% of controls without gan-
oderic acid. Male KM mice were significantly
protected from liver injury, induced with carbon
tetrachloride, by treatment with ganoderic acid at
10 mg and 30 mg/kgÆd (by intravenous injection)
7 days. Ganoderic acid at the same dosage also
significantly protected the mice from liver injury
induced by M. bovis BCG plus lipopolysaccharide
(from Escherichia coli 0127:B8).
Ganodericacid,from Ganoderma
Keywords
lucidum Æ Hepatitis Æ Hepatoprotection Æ
Virus
Ganoderic acid Æ Ganoderma
Introduction
Ganoderma lucidum (Fr.) Krast (Polyporaceae),
known as Lingzhi in China and Reisi in Japan, is a
traditional Chinese medicine used to prevent and
treat several diseases, including hepatitis B. It has
immunity regulation properties (Zhang et al.
2002) and also has anti-tumor (Wang et al. 1997),
anti-viral (Sahar et al. 1998; Kim et al. 2000) and
hepato-protective activities (Back et al. 1999).
The main bioactive substances in G. lucidum in-
clude sterols, triterpenes and polysaccharides
(Min et al. 1998, 2000, 2001; Oh et al. 2000).
Triterpenes of G. lucidum, known as ganoderic
acids (GA), have anti-HIV-1 activities (Sahar
et al. 1998), anti-thrombosis activity (Su et al.
1999), neuroprotective activity (Zhao et al. 2005),
anti-tumor activities (Li et al. 2005). GA can
selectively inhibit eukaryotic DNA polymerase
activities (Mizushina et al. 1999). In this study, we
have extracted GA from G. lucidum grown under
submerged culture and investigated its anti-hep-
atitis B activity.
Materials and methods
Materials
Lipopolysaccharide (LPS, from Escherichia coli
0127:B8) was purchased from Sigma. Bacille
Calmette-Gue ´rin (BCG, Mycobactarium bovis,
D2PB320) was purchased from Shanghai Institute
of Bioproducts.
Y.-Q. Li (&)
College of Life Science, South China Normal
University, Guangzhou 510631, P.R. China
e-mail: liyq9168@hotmail.com
S.-F. Wang
Jiangxi Academy of Science, Nanchang 330029,
P.R. China
Biotechnol Lett (2006) 28:837–841
DOI 10.1007/s10529-006-9007-9
123
ORIGINAL PAPER
Anti-hepatitis B activities of ganoderic acid
from Ganoderma lucidum
Yan-Qun Li Æ Æ Shun-Fa Wang
Received: 28 September 2005/Accepted: 20 February 2006/Published online: 31 May 2006
? Springer Science+Business Media B.V. 2006

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Cultivation of Ganoderma lucidum
G. lucidum CCGMC 5.616 (China General
Microbiological Fermentation Collection Center,
Beijing, China) was precultured in 500 ml flasks
for 7 days at 30?C on a rotary shaker (150 rpm).
This was inoculated at 10% (v/v) into a 5 l stirred-
tank bioreactors holding 3.5 l medium (40 g glu-
cose/l, 4 g peptone/l, 2 g yeast extract/l, 1.5 g
KH2PO4/l, 0.75 g MgSO4Æ7H2O/l and 0.01 g vita-
min B1/l) and the culture was grow at 30?C for
96 h. The culture broth was obtained by removal
of the cells by centrifugation.
Extraction of ganoderic acid (GA)
GA was extracted as described by Tsujikura et al.
(1992). The cell-free broth was concentrated to
one fourth of the initial volume under reduced
pressure, and then 3 · vol ethanol were added
gradually, and the mixture was stirred for 3 days
at 4?C. After removal of the precipitate by cen-
trifugation, the supernatant was concentrated at
45?C and lyophilized. The residue was suspended
in water and extracted with chloroform. The
chloroform phase was extracted with 5% (w/v)
NaHCO3. The pH of NaHCO3 phase was ad-
justed to 2–3 with HCl. GA was then extracted
again with chloroform. After removal of chloro-
form, GA was dissolved in dimethylsulfoxide
(DMSO) for bioactivity assay.
Cell line and antiviral assay
Cell line HepG2215 (HepG2 cell transferred with
HBV DNA, Sells et al. 1987) was obtained from
302 Hospital of the Chinese People’s Liberation
Army. Cells were cultured in 24-well plates at 105
cells/well in MEM medium containing 10% (v/v)
fetal bovine serum, 100 units streptomycin/ml and
100 units penicillin/ml, 1 mM glutamine/ml and
200 lg Geneticin G418/ml. After 3 days, the
medium was replaced with fresh medium con-
taining GA from 0.5 lg/ml to 8 lg/ml (the control
group contained no GA). The concentrations of
fetal bovine serum and Geneticin G418 in the
fresh medium were amended to 2% (v/v) and
380 lg/ml, respectively. After 4 days, the cultured
medium was replaced again with fresh medium
containing GA. After 8 days, the cultured med-
ium was replaced with PBS solution containing
0.5 mg MTT/ml. The cultured medium was used
to detect HBV surface antigen (HBsAg) and
HBV e antigen (HBeAg) using the radioimmu-
noassay kits (purchased from Institute of Atomic
Energy of China, Beijing, China). After 4 h
incubation, PBS solution was removed and 1 ml
DMSO was added into each well. The absorbancy
at 490 nm was measured with a microplate
reader.
Animals
Male KM mice weighing 18–22 g were reared at
23 – 3?C in a 12 h light/dark cycle. Animals were
fed with a standard laboratory chow and water ad
libitum. The animal experiments were carried out
in accordance with the Regulation of the Care
and Use of Medical and Laboratory Animals,
Ministry of Health, P.R. China.
Assay of protection from carbon
tetrachloride-induced liver injury of mice
Mice were randomly divided into five groups of
ten animals each. The groups included normal
control, model, standard reference and treatment
with GA at two concentrations. The normal
control group was given no drugs and the model
group was given carbon tetrachloride (CCl4).
Standard reference group was given biphenyldi-
carboxylate at 20 mg/kgÆd in saline (i.v.) and the
GA groups were given GA at 10 mg or 30 mg/
kgÆd (i.v.) for seven consecutive days. On the
eighth day, all groups were given olive oil with
CCl4[0.1% (v/v), 10 ml/kg, i.p.] except that the
normal control group was given 10 ml olive oil/kg
only (i.p.). No food was provided immediately
after CCl4 administration but tap water was
available ad libitum. Sixteen hours after CCl4
administration, blood was withdrawn from the
eye socket. All blood samples were centrifuged at
1200 · g and 4?C for 10 min to obtain the serum.
The activities of alanine aminotransferase (ALT)
and aspartate aminotransferase
measured with Venusleon-180 Eos bravo bio-
chemistry measuring system.
(AST)were
838Biotechnol Lett (2006) 28:837–841
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Assay of protection from BCG/LPS-induced
liver injury of mice
Mice were randomly divided into five groups of
ten animals each. The groups included normal
control, model, standard reference and treatment
with GA at two concentrations. Each animal,
except the animals in the normal control group,
was treated with 0.2 ml BCG (containing 5 · 106
bacteria, i.v) on the first day. The standard ref-
erence group was given 20 mg cyclophospha-
mide/kgÆd (i.v.) and GA groups were given 10 or
30 mg GA/kgÆd (i.v.) from the first to ninth day.
After 10 days, each animal (except normal con-
trol) was given 7.5 lg LPS (i.v.). Blood was
withdrawn from the eye socket 16 h post the
LPS-treatment. All blood samples were centri-
fuged at 1200 · g and 4?C for 10 min to obtain
the serum. The activities of ALT and AST were
measured as above.
Results and discussion
Antiviral activity of GA
The results in Table 1 indicate that GA inhibited
the HBsAgand HBeAg secretionfrom
HepG2215 cells, and the inhibition was GA dos-
age dependent. The HepG2215 cell line origi-
natedfromanindependent
HepG2 cells with HBV DNA. The cell line can
stably produce HBV virions (Sells et al. 1987).
The HBsAg and HBeAg, expressed by the cells,
are the structural proteins of HBV virions. Inhi-
bition of HBsAg and HBeAg secretion into the
medium by GA probably reflected inhibition ef-
fects of GA on the replication of HBV in the
cells, or on the expression of HBsAg and HBeAg.
GA was nontoxic to HepG2215 cells at the tested
dosages.
transfection of
Protection from CCl4-induced liver injur\stop
CCl4induced a significant increase in activities of
alanine and aspartate transaminases (ALT and
AST) in serum of the model group, as compared
to the control (Table 2). Significantly (P < 0.01)
lower ALT and AST levels were observed in GA-
treated groups than the model group, indicating
that GA offered a protection from CCl4-induced
liver injury in the animal of the treated groups.
Interestingly, the GA at 10 and 30 mg/kg was
even more effective for AST activity than the
standardreference biphenyldicarboxylate
20 mg/kg.
at
Table 1 The inhibition effects of ganoderic acid on hepatitis virus antigen and its cytotoxicity to HepG2215 cells
Dose (lg/ml) Inhibition ratio on HBsAgInhibition ratio on HBeAgCytotoxinic rate
4 days 8 days 4 days8 days
0.5
1
2
4
8
19
42
67
74
87
4 22
46
60
74
89
49
43
47
48
56
0
0
0
0
28
39
59
80 13
The HepG2215 cells (a hepatitis B virus DNA transferred HepG2 cell line) were cultured on 24-well plates in MEM
medium containing ganoderic acid (no ganoderic acid for normal control group). On the 4th day and 8th day after ganoderic
acid administration, the content of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in the cultured
medium were measured with radioimmunoassay. Each sample was tested four times. The content of HBsAg and HBeAg in
the cultured medium of normal control were 2.23 lg/ml and 0.56 lg/ml on the 4th day, and 2.86 lg/ml and 0.71 lg/ml on the
8th day, respectively. The inhibition ratio was calculated with the formula as:
Inhibition ratio ¼antigen content of control?antigen content of ganoderic acid group
antigen content of control
On the 8th day, the cytotoxicity of ganoderic acid to the cells was tested with MTT assay. The absorbance at 490 nm was
measured with an EL · 800 Microplate Reader and the cytotoxity was expressed as:
? 100%
Cytotoxitic rate ¼absorbance of control?absorbance of ganoderic acid group
absorbance of control
? 100%
Biotechnol Lett (2006) 28:837–841 839
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Protection from BCG/LPS-induced liver
injury
The levels of ALT and AST were significantly
(P < 0.01) higher in the model group than in the
normal control group (Table 3). This indicated
that BCG + LPS induced liver injury. Cyclo-
phosphamide could suppress the activities of ALT
and AST as expected. GA reduced liver injury
because the ALT and AST levels in the GA
treated groups were significantly (P < 0.01)
lower than those in the model group. However,
GA at the treated dosages appeared to be not
more effective than cyclophosphamide for ALT.
The models of CCl4-induced hepatotoxicity
and BCG + LPS-induced immunity liver injury
models are conventionally used to investigate
novel liver protective agents. Administration of
CCl4and BCG + LPS caused a rapid increase in
ALT and AST levels, which reflect liver injury.
Serum transaminase elevation has been reported
to be associated with a number of inflammatory
disorders (Hoder and Wilkinson 1980) and
hepatocellular damages (Sinha and Saran 1972).
Leakage of large quantities of enzymes into the
blood stream is often associated with massive
necrosis of liver (Rees and Spector 1961). CCl4-
induced acute liver injury is similar to the damage
of acute hepatitis (Recknagel 1967). The present
study indicated that GA could reduce ALT and
AST levels in the treated mice, implying that GA
had a hepatoprotective effect on CCl4- and
BCG + LPS-induced liver injuries.
The GA roles in antiviral activity and reduc-
tion of liver injury illustrated in this study
suggested that GA had potential to be a anti-
hepatitis medicine, though further investigation
on the mechanism of GA actions is necessary.
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