American Journal of Chinese Medicine, Vol. 29, Nos. 3-4, pp.

501-507
2001 Institute for Advanced Research in Asian Science and Medicine

Evaluation of the Hepatic and

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Renal-protective Effects of
Am. J. Chin. Med. 2001.29:501-507. Downloaded from www.worldscientific.com

Ganoderma lucidum in Mice

Ying-Hua Shieh1, Chi-Feng Liu2, Yao-Kuan Huang1, Jen-Yuann Yang2,
I-Lin Wu3, Chia-Hsien Lin4 and Song-Chow Lin5*

1
Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan
2
National Taipei College ofNursing, Taipei, Taiwan
3
National Taipei College ofNursing Hospital, Taipei, Taiwan
''Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
1
Department of Pharmacology, Taipei Medical University, Taipei, Taiwan
Corresponding author

(Accepted for publication March 1, 2001)

Abstract: The antioxidative effect of hot water extract of the mushroom Ganoderma lucidum on
ethanol-induced free radical generation had been studied. In order to further investigate the hepatic
and renal protective mechanism of Ganoderma lucidum, rates of lipid peroxidation were determined.
The hot water extract of Ganoderma lucidum dose-dependently exhibited antioxidative effect on
mouse liver and kidney lipid peroxidation; our results indicated that hepatic and renal homogenates
have a higher malonic dialdehyde level in an ethanol administered group than in the Ganoderma lu-
cidum treated group. It was concluded that the hepatic and renal protective mechanism of Ganoderma
lucidum, might be due at least in part to its prominent superoxide scavenging effect. Ganoderma ex-
tract could protect the liver and kidney from superoxide induced hepatic and renal damages.

1 he mechanism contributing to alcohol-induced hepatic and renal damage remains

unclear. Increasing evidence indicates that alcohol toxicity is associated with increased oxi-
dative stress and free radical-associated injury (Nanji et al, 1994; Manso, 1997). Several in-
vestigations indicated that the generation of oxygen metabolites, such as superoxide (02~),
hydrogen peroxide (H 2 0 2 ) and hydroxyl radical (-OH), is believed to be important in the
pathogenesis of alcoholic liver (Nordmann et al, 1996) and renal injury (Kera et al, 1985;
PallereJa/., 1988).

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 Y-H. SHIEH etal.

Ganoderma lucidum is a white rot basidiomycete widely distributed worldwide; its

fruit bodies have been used for the prevention and treatment of various diseases in Asia. Its
antitumor, immune enhancing and non-cytotoxic properties raise the possibility that it
could be effective in preventing oxidative damage related disease (Kim, et al., 1999).
In China, Ganoderma lucidum has been prescribed in Chinese Medicine for hundreds
of years as a tonic and sedative, and has been used for treatment of hypertension (Kabir, et
al, 1988), carcinoma (Kim, et al, 1999), inflammation and liver disease (Lin, et al, 1993).
Ganoderma lucidum has also been reported to have excellent antioxidative activities in a
dose dependent manner, and its terpene fraction was found to possess the most effective
constituents. Chemical isolation of the terpene fraction resulted in the detection of ganoder-
mic acids A, B, C and D, lucidenic acid B and ganoderma notriol as major ingredients (Zhu.
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M. etal., 1999).
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The aims of the present study were to investigate the hepatic and renal protective ef-
fect and antioxidative effect of this crude drug. The active oxygen scavenging activity of
Ganoderma lucidum was also examined by lipid peroxidation. TBA-MDA adduct was used
as a free radical index.

Materials and Methods

Animals and Grouping

Male ICR mice weighing about 20-25g were purchased from the Animal Center, College of
Medicine, National Yang-Ming University. They were kept at least one week on commer-
cial diets (Fu-So Co., Taipei) under environmentally controlled conditions (25 1C,
55 5% humidity) with free access to food and water. A 12-hr light/dark schedule was
maintained and hard wood chips were used as bedding.
ICR mice were divided into eight groups with 10 animals each. Group 1 (control, un-
treated mice) received saline (10 ml/kg). Group 2 received 95% ethanol (0.1 ml, p.o.).
Group 3-5 received the hot water extract of Ganoderma lucidum at the doses of 10,25, and
50 mg/kg p.o. respectively. Groups 6-8 received the hot water extract of Ganoderma lu-
cidum at the doses of 10,25 and 50 mg/kg p.o. respectively, 30 min before oral administra-
tion of 0.1 ml 95% ethanol. The animals were killed 1 hr after 0.1 ml 95% ethanol
administration. The above procedure was performed according to the method described by
Zhang et al. (1995).

Drugs and Chemicals

95% ethanol, thiobarbiruric acid (TBA), sodium dodecyl sulfate, ferric chloride, -butanol
were all purchased from Sigma Chemical Company (St. Louis, MO 63178, USA). Acetic
acid was obtained from a local company in Taipei, Taiwan. Ganoderma lucidum was a gift
of Kuun Yung, Co., Ltd., Taipei, Taiwan.

Preparation of Crude Extract

Ganoderma lucidum crude extract was prepared as follows: 100 gm of the dried, crushed
Ganoderma lucidum was decocted in a Chinese herb boiler with one liter of distilled water.
After filtration, the residues were decocted again in the same manner. Two fractions of fil-

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 HEPATIC AND RENAL-PROTECTIVE EFFECTS OF GANODERMA LUCIDUM
trate were combined and then concentrated into 1 gm/ml (w/v, based on the weight of the
dried crude drug). In order to obtain the exact dosage of crude extract, this concentrated so-
lution was freeze-dried into powder by a freeze-drier (Tokyo Rikakikai Co., Ltd.), then
stored in an auto-drying container until use.

Preparation of Liver and Kidney Homogenate

After sacrificing the animals, the liver and kidney were removed and cut into slices respec-
tively. The liver or kidney slices were homogenized with 1.15% KC1 solution according to
the method described by Mihara et al. (1978).
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Determination of Lipid Peroxidation by Measuring Thiobarbituric Acid Reactive

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Substance
The protective effect of the hot water extract of Ganoderma lucidum on the liver and kidney
lipid peroxidation was determined by the MDA-TBA adduct according to the modified
method described by Yuda et al. (1991). Briefly, 0.5 ml of prepared liver or kidney ho-
mogenate were placed in an incubator with 0.1 ml of Tris-HCl buffer (pH 7.2) for 1 hr at 37
C. After incubation, 9 ml of distilled water and 2 ml of 0.6% Thiobarbituric Acid (TBA)
were added to 0.5 ml of the incubation solution and were shaken vigorously. The mixture
was heated for 30 min in a boiling water bath. After cooling naturally at room temperature, 5
ml of n-BuOH was added and the mixture was again shaken vigorously. The n-BuOH layer
was separated by centrifugation at 1000 gr. for 10 min, and the malonic dialdehyde (MDA)
production amount was measured at 532 nm (Wong et al. 1987).

Statistical Analysis
All data were shown as mean S.E. (n=10). Statistical significance was assessed by one-
way analysis of variance coupled with Dunnett's test. The level of significance was chosen
as p<0.05.

Results

Ganoderma lucidum Effect on Mice Without Ethanol Treatment

Ganoderma lucidum (GL) (10,25 and 50 mg/kg) significantly improved the normal hepatic
and renal function of groups 3,4, 5 mice. Ganoderma lucidum dose-dependently inhibited
the lipid peroxidation-induced MDA formation, the end product of lipid peroxidation (Ta-
bles 1, 3, respectively) in mice hepatic and renal homogenate.

Free Radical Scavenging Assessment of Ganoderma lucidum on Ethanol-induced Acute

Toxicity
As shown in Tables 2 and 4, 95% ethanol treatment increased lipid peroxidation in the kid-
ney and liver homogenates, respectively. Various concentrations (10,25 and 50 mg/kg) of
Ganoderma lucidum dose-dependently inhibited the ethanol-induced lipid peroxidation in
the mice renal homogenate (Table 2) and liver homogenate (Table 4).

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 Y-H. SHIEH etal.

Table 1. Effect of Hot Water Extract of Ganoderma lucidum (GL) on Lipid Peroxidation
in the Mice (Without Ethanol Treatment) Kidney Homogenates
Groups MDA Inhibition rate
(nmole/mg protein) (%)
Control(Saline) 0.162 0.006 -
GL(10mg/kg) 0.129 0.02 20.37
GL (25 mg/kg) 0.125 0.0005* 22.84
GL (50 mg/kg) 0.121 0.008* 25.31
Each value represents mean S.E. (n=10)
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: p < 0.001, significantly different from normal control group

#
: p < 0.05, significantly different from normal control group
Am. J. Chin. Med. 2001.29:501-507. Downloaded from www.worldscientific.com

One-way analysis of variance coupled with Dunnett's test.

P value less than 0.05 was taken as significant.

Table 2. Inhibitory Effect of Hot Water Extract of Ganoderma lucidum (GL)

on Ethanol-induced Lipid Peroxidation in the Mice Kidney Homogenate
Groups MDA Inhibition rate
(nmole/mg protein) (%)
Control (Saline) 0.046 0.01
95% ethanol (0.1 ml) 0.095 0.01* -
95% ethanol (0.1 ml) 0.058 0.03 38.9
+ GL (10 mg/kg)
95% ethanol (0.1 ml) 0.048 0.02 49.5
+ GL (25 mg/kg)
95% ethanol (0.1 ml) 0.045 0.01* 52.6
+ GL (50 mg/kg)
Each value represents mean S.E. (n=10)
: p < 0.05, significantly different from normal control group
*: p < 0.05, significantly different from ethanol group
One-way analysis of variance coupled with Dunnett's test.
P value less than 0.05 was taken as significant.

Discussion

The aim of this study was to investigate the antioxidative effect of Ganoderma lucidum on
ethanol-induced hepatic and renal injury. It has been hypothesized that one of the principal
causes of ethanol-induced hepatic and renal injury is due to free radical induced lipid per-
oxidation, whereas free radicals could be generated largely by a long period of alcohol con-
sumption (Meagher et al, 1999; Bautista et al., 1999; Jaya et al, 1993; Rodrigo et al,
1998). In recent years, the biological and pharmacological properties of Ganoderma lu-
cidum have become a promising and important research. It was confirmed that Ganoderma

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Table 3. Effect of Hot Water Extract of Ganoderma lucidum (GL) on

Lipid Peroxidation in the Mice (Without Ethanol Treatment)
Liver Homogenates
Groups MDA Inhibition rate
(nmole/mg protein) (%)
Control (Saline) 0.213 0.007 _
GL(10mg/kg) 0.178 0.005* 16.43
GL (25 mg/kg) 0.170 0.002** 20.19
GL (50 mg/kg) 0.165 0.006* 22.54
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Each value represents mean S.E. (n=10).

: p < 0.05, significantly different from normal control group
Am. J. Chin. Med. 2001.29:501-507. Downloaded from www.worldscientific.com

"*: p < 0.001, significantly different from normal control group

One-way analysis of variance coupled with Dunnett's test.
P value less than 0.05 was taken as significant.

Table 4. Inhibitory Effect of Hot Water Extract of Ganoderma lucidum (GL) on

Ethanol-induced Lipid Peroxidation in the Mice Liver Homogenate
Groups MDA Inhibition rate
(nmole/mg protein) (%)
Control (Saline) 0.08 0.019 _
95% ethanol (0.1 ml) 0.199 0.02* _
95% ethanol (0.1 ml) 0.113 0.01* 43.2
+ GL (10 mg/kg)
95% ethanol (0.1 ml) 0.107 0.008* 46.2
+ GL (25 mg/kg)
95% ethanol (0.1 ml) 0.05 0.009* 74.9
+ GL (50 mg/kg)
Each value represents mean S.E. (n=10)
: p < 0.05, significantly different from normal control group
*: p < 0.05, significantly different from ethanol group
One-way analysis of variance coupled with Dunnett's test.
P value less than 0.05 was taken as significant.

lucidum could exhibit a broad spectrum of activities, including hypotensive action (Lee et
al., 1990), anti-tumor activity (Wang et al., 1997), antinociceptive effect (Koyama et al.,
1997), anti-platelet aggregation (Tao et al., 1990), hepatoprotective and anti-inflammatory
activities (Kim et al., 1999).
In the present study, the protective effect of Ganoderma lucidum and its action
mechanism on ethanol-induced hepatic and renal injury were investigated in ICR mice.
Superoxide scavenging activity was used as an indicator of hepatic and renal protective ef-
fect.
Results of this study showed that Ganoderma lucidum could inhibit lipid peroxida-
tion, hence decreasing significantly the malonic dialdehyde (MDA) formation in the control

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 Y-H. SHIEH et al.

mice (without ethanol treatment) kidney and liver homogenates (See Tables 1,3). It was re-
ported that ethanol could stimulate lipid peroxidation in the kidney (Kera et al, 1985) and
liver (Baustista et al, Meagher et al, 1999). Our results confirmed these findings (see Ta-
bles 2,4). We also found that Ganoderma lucidum had dose-dependently inhibited 9 5 %
ethanol-induced lipid peroxidation. These findings indicated that the preventive effect of
GL on hepatic and renal injuries induced by ethanol is due, at least in part, to the decrease in
MDA formation.
In conclusion, results of the present study suggested that production of free radicals
may be involved in the pathogenesis of hepatic and renal injuries induced by ethanol, and
that Ganoderma lucidum extract dose-dependently inhibited the ethanol-induced hepatic
and renal injury significantly. These effects may be due, in part, to its inhibitory activity on
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membrane lipid peroxidation and free radical formation, or free radical scavenging ability.
Am. J. Chin. Med. 2001.29:501-507. Downloaded from www.worldscientific.com

Clinical application of Ganoderma lucidum to treat ethanol-induced hepatic and renal dam-
age warrants further study.

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