Evaluation of the hepatic and renal-

protective effects of ganoderma

lucidum in mice
American Journal of Chinese Medicine, Summer-Fall, 2001 by Ying-Hua Shieh, Chi-
Feng Liu, Yao-Kuan Huang, Jen-Yuann Yang, I-Lin Wu, Chia-Hsien Lin, Song-Chow
Lin

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 lucidum 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 extract could protect the liver and kidney from superoxide induced hepatic
and renal damages.

The mechanism contributing to alcohol-induced hepatic and renal damage remains

unclear. Increasing evidence indicates that alcohol toxicity is associated with increased
oxidative stress and free radical-associated injury (Nanji et al., 1994; Manso, 1997).
Several investigations indicated that the generation of oxygen metabolites, such as
superoxide (*[O.sub.2.sup.-]), hydrogen peroxide ([H.sub.2][O.sub.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; Paller et al., 1988).

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 ganodermic acids A, B, C and D, lucidenic acid B and ganoderma notriol as
major ingredients (Zhu. M. et al., 1999).
The aims of the present study were to investigate the hepatic and renal protective effect
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
commercial diets (Fu-So Co., Taipei) under environmentally controlled conditions (25 [+
or -] 1 [degrees] C, 55 [+ or -] 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,
untreated 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
lucidum at the doses of 10, 25 and 50 mg/kg p.o. respectively, 30 min before oral
administration 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, thiobarbituric acid (TBA), sodium dodecyl sulfate, ferric chloride, n-
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 filtrate 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 solution 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
respectively. The liver or kidney slices were homogenized with 1.15% KCl solution
according to the method described by Mihara et al. (1978).
Determination of Lipid Peroxidation by Measuring Thiobarbituric Acid Reactive
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 homogenate were placed in an incubator with 0.1 ml of Tris-HCl buffer (pH 7.2)
for 1 hr at 37 [degrees] 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 [+ or -] 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 (Tables 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
kidney 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).

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 peroxidation, whereas free radicals could be generated largely by a long period of
alcohol consumption (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 lucidum have become a promising and important research. It was confirmed
that Ganoderma 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
effect.

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 [+ or -] 0.006 --

GL (10 mg/kg) 0.129 [+ or -] 0.02 20.37
GL (25 mg/kg) 0.125 [+ or -] 0.0005 * 22.84
GL (50 mg/kg) 0.121 [+ or -] 0.008 (#) 25.31

Each value represents mean [+ or -] S.E. (n=10)

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

(#): p < 0.05, 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 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 [+ or -] 0.01 --

95% ethanol (0.1 ml) 0.095 [+ or -] 0.01 * --
95% ethanol (0.1 ml) 0.058 [+ or -] 0.03 38.9
+ GL (10 mg/kg)
95% ethanol (0.1 ml) 0.048 [+ or -] 0.02 49.5
+ GL (25 mg/kg)
95% ethanol (0.1 ml) 0.045 [+ or -] 0.01 (#) 52.6
+ GL (50 mg/kg)

Each value represents mean [+ or -] 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.

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 [+ or -] 0.007 --

GL (10 mg/kg) 0.178 [+ or -] 0.005 * 16.43
GL (25 mg/kg) 0.170 [+ or -] 0.002 (##) 20.19
GL (50 mg/kg) 0.165 [+ or -] 0.006 * 22.54

Each value represents mean [+ or -] S.E. (n=10).

*: p < 0.05, significantly different from normal control group

(##): 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 [+ or -] 0.019 --

95% ethanol (0.1 ml) 0.199 [+ or -] 0.02 * --
95% ethanol (0.1 ml) 0.113 [+ or -] 0.01 (#) 43.2
+ GL (10 mg/kg)
95% ethanol (0.1 ml) 0.107 [+ or -] 0.008 (#) 46.2
+ GL (25 mg/kg)
95% ethanol (0.1 ml) 0.05 [+ or -] 0.009 (#) 74.9
+ GL (50 mg/kg)

Each value represents mean [+ or -] 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.

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Ying-Hua Shieh (1), Chi-Feng Liu (2), Yao-Kuan Huang (1), Jen-Yuann Yang (2), I-Lin
Wu (3), Chia-Hsien Lin (4) and Song-Chow Lin (5) *

(1) Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan

(2) National Taipei College of Nursing, Taipei, Taiwan

(3) National Taipei College of Nursing Hospital, Taipei, Taiwan

(4) pharmacological Institute, College of Medicine, National Taiwan University, Taipei,

Taiwan

(5) Department of Pharmacology, Taipei Medical University, Taipei, Taiwan

* Corresponding author
(Accepted for publication March 1, 2001)

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