Am. J. Chin. Med. 2001.29:501-507. Downloaded from www.worldscientific.com by UNIVERSITY OF CALIFORNIA @ SAN DIEGO on 08/25/15. For personal use only.
American Journal of Chinese Medicine, Vol. 29, Nos. 3-4, pp. 501-507 Š 2001 Institute for Advanced Research in Asian Science and Medicine
Evaluation of the Hepatic and Renal-protective Effects of 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 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. 1 he 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 (•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 ganodermic acids A, B, C and D, lucidenic acid B and ganoderma notriol as major ingredients (Zhu. M. etal., 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 ± 1°C, 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, 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, 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 fil502
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 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
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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% KC1 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 °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 ¹ S.E. (n=10). Statistical significance was assessed by oneway 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). 503
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
Control(Saline)
0.162 ±0.006
Inhibition rate (%) -
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
Groups
MDA (nmole/mg protein)
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Each value represents mean ± 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
Control (Saline)
0.046 ±0.01
Inhibition rate (%) —
95% ethanol (0.1 ml)
0.095 ±0.01*
-
95% ethanol (0.1 ml) + GL (10 mg/kg)
0.058 ± 0.03
38.9
95% ethanol (0.1 ml) + GL (25 mg/kg)
0.048 ± 0.02
49.5
95% ethanol (0.1 ml) + GL (50 mg/kg)
0.045 ±0.01*
52.6
Groups
MDA (nmole/mg protein)
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 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 504
HEPATIC AND RENAL-PROTECTIVE EFFECTS OF GANODERMA LUCIDUM Table 3. Effect of Hot Water Extract of Ganoderma lucidum (GL) on Lipid Peroxidation in the Mice (Without Ethanol Treatment) Liver Homogenates
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Groups
MDA (nmole/mg protein)
Inhibition rate (%) _
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
Each value represents mean ± 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
Control (Saline)
0.08 ±0.019
Inhibition rate (%) _
95% ethanol (0.1 ml)
0.199 ±0.02*
_
95% ethanol (0.1 ml) + GL (10 mg/kg)
0.113 ±0.01*
43.2
95% ethanol (0.1 ml) + GL (25 mg/kg)
0.107 ±0.008*
46.2
95% ethanol (0.1 ml) + GL (50 mg/kg)
0.05 ±0.009*
74.9
Groups
MDA (nmole/mg protein)
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 effect. Results of this study showed that Ganoderma lucidum could inhibit lipid peroxidation, hence decreasing significantly the malonic dialdehyde (MDA) formation in the control 505
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Y-H. SHIEH et al. mice (without ethanol treatment) kidney and liver homogenates (See Tables 1,3). It was reported 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 Tables 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 membrane lipid peroxidation and free radical formation, or free radical scavenging ability. Clinical application of Ganoderma lucidum to treat ethanol-induced hepatic and renal damage warrants further study. Reference 1. Bautista, A.P. and J.J. Spitzer. Role of kupffer cells in the ethanol-induced oxidative stress in the liver. Front, in Bios. 4: 589-595,1999. 2. Jaya, D.S., J. Augstine and V.P. Menon. Role of lipid peroxides, glutathione and antiperoxidative enzymes in alcohol and drug toxicity. Ind. J. Exp. Biol. 31 (5): 453^59,1993. 3. Kabir, Y., S. Kimura and T. Tamura. Dietary effect of Ganoderma lucidum mushroom on blood pressure and lipid levels in spontaneously hypertensive rats (SHR). J. Nut. Sci. Vitam. 34(4): 433-438,1988. 4. Kera, Y., S. Komura, Y. Ohbora, T. Kiriyama and K. Inoue. Ethanol induced changes in lipid peroxidation and nonprotein sulfhydryl content. Different sensitivities in rat liver and kidney. Res. Com. Chem. Pathol. Pharmac. 47(2): 203-209, 1985. 5. Kim, D.H., S.B. Shim, N.J. Kim and I.S. Jang. Beta-glucuronidase-inhibitoroy activity and hepatoprotective effect of Ganoderma lucidum. Biol. Pharmaceut. Bull. 22(2): 162-164, 1999. 6. Kim, H.S., S. Kacew and B.M. Lee. In vitro chemopreventive effects of plant polysaccharides {Aloe barbadensis Miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor). Carcinogenesis.!^): 1637-1640, 1999. 7. Kim, K.C. and I.G. Kim. Ganoderma lucidum extract protects DNA from strand breakage caused by hydroxyl radical and UV irradiation. Int. J. Mol. Med. 4(3): 273-277, 1999. 8. Koyama, K, T. Imaizumi, M. Akiba, K. Kinoshita, K. Takahashi, A. Suzuki, S. Yano, S. Horie, K. Watanabe and Y. Naoi. Antinociceptive components of Ganoderma lucidum. Plant. Med. 63(3): 224-227, 1997. 9. Lee, S.Y. and H.M. Rhee. Cardiovascular effects of mycelium extract of Ganoderma lucidum: inhibition of sympathetic outflow as a mechanism of its hypotensive action. Chem. Pharmaceut. Bull. 38(5): 1359-1364, 1990. 10. Lin, J.M., C.C. Lin, H.F. Chiu, J.J. Yang and S.G. Lee. Evaluation of the anti-inflammatory and liver-protective effects of Anoectochilus formosanus, Ganoderma lucidum and Gynostemma pentaphyllum in rats. AJCM2\(\): 59-69, 1993. 11. Manso, C.F. Alcohol and free radicals. Various consequences: protein synthesis, endocrine disorders, immunity. Role of stress. Acta Med. Portug. 10(11): 809-817, 1997. 12. Meagher, E.A., O.P. Barry, A. Burke, M.R. Lucey, J A Lawson, J. Rokach and G.A. FitzGerald. Alcohol-induced generation of lipid peroxidation products in human. J. Clin. Investig. 104(6): 805-813, 1999. 506
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