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Preventive effect of Ganoderma amboinense on acetaminophen-induced acute liver injury.

Abstract

In vivo preventive effects of Gunoderma amboinense against acetaminophen-induced hepatotoxicity in Balb/cA mice were studied. G. amboinense powder at 1% and 2% was mixed with standard diet and supplied to mice for 6 weeks, and followed by acetaminophen (350 mg/kg body weight) intraperitoneal injection. In normal mice (without acetaminophen treatment), the consumption of G. amboinense significantly increased hepatie glutathione (GSH) level. Acetaminophen treatment significantly elevated both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities; however, the pre-intake of G. amboinense significantly and dose-dependently protected liver against the subsequent acctaminophen-intake of G. amboinense significantly and dose-dependently protected liver against the subsequent acctaminophen-induced clevation of ALT and AST activities. Acetaminophen treatment also caused significant GSH depletion, malondialdehyde (MDA) and reactive oxygen species (ROS) increase, and activity reduction of glutathione peroxidase (GPX) and catalase. However, the pre-intake of G. amboinense significantly diminshed the subsequent acetaminophen-induced GSH depletion, MDA and ROS increase, and retarded the loss of catalase and GPX activities, in which the effect of G. ambionense on GPX activity, and preventive agent for acute liver injury.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Ganoderma amboinense: Hepatotoxicity; Glutathione: Polyphenols

Introduction

Ganoderma amboinense is a fruit body of ganoderma fungi and has been used as folk medicine for thousands of years in Asia. Folk medicine implies the Ganoderma family has anti-cancer and immunomodulatory activities, and is often used for tonics and remedies for ailments such as cough, asthma, bronchitis and hepatitis (Yan et al., 1999). So far, many ganoderma triterpenes has been identified from G. amboinense, and it is reported that some of them could arrest cell growth, and consequently induce senescence or apoptosis in human hepatoma HepG2 cells and HuH-7 cells (Li et al., 2005; Chang et al., 2006). Although these previous cell-line studies already suggested that this herb had hepato-protective capability, the information regarding in vivo hepatic protection of this herb is lacking. On the other hand, the anti-oxidative activity of G. lucidum, another member in the Ganoderma family, has been indicated (Thyagarajan et al., 2006); however, it is unknown whether G. amboinense also has anti-oxidative activity. In order to further understand the hepato-protective function of G. amboinense, and examine whether this herb could provide anti-oxidative protection, a study regarding G. amboinense against acetaminophen-induced hepatotoxicity in animal was designed.

Acetaminophen is an analgesic drug and metabolized by the cytochrome P450 system, which leads to the formation of n-acety1-p-benzoquinoneimine (NAPQI) (Song et al., 2004; Masubuchi et al., 2005). A large does of this drug causes depletion of the cellular glutathione (GSH) level in liver because NAPQI reacts rapidly with glutathione (Nelson, 1990; Oz et al., 2004; Masubuchi et al., 2005), with consequently exacerbates oxidative stress in conjunction with mitochondrial dysfunction. Thus, the GSH depletion, especially occurring in acute hepatotoxicity, affects liver functions and leads to massive hepatocyte necrosis, liver failure or death. Since oxidative stress and GSH depletion contributed to acetaminophen-induced liver injury; the agent(s) with antioxidant property and/or GSH reserving ability may provide preventive effect against the progression of lipid peroxidation and hepatocellular injury.

The purpose of this animal study was to examine the preventive effects of G. amboinense on acetaminophen-induced acute hepatic oxidative injury. The content of polyphenols and flavonoids in this herb was measured. The influence of G. amboinense upon liver GSH level and activity of antioxidant enzymes were also evaluated.

Materials and methods

Chemicals

Ganoderma amboinense obtained from Rich & Health Bio-Tech. Co. (Taichung City, Taiwan) was freeze-dried and ground into powder. Acetaminophen was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Determination of total polyphenols and total flavonoids

Total polyphenols of G. ambinense were determined by a spectrophotometric method (Taga et al., 1984). The absorbance was measured at 750 n, compared with the prepared blank. Total polyphenols were expressed on a fresh weight basis as mg gallic acid equivalent (GAE)/ 100g. Total flavonoids of G. amboinense were measured using a colorimetric assay developed by Zhishen et al. (1999). The absorbance of the pink mixture was measured at 510 nm compared with the prepared blank. Total flavonoids were expressed on a fresh weight basis as mg catechin equivalent (CE)/100g. All analyses were carried out 5 times.

Animals

Five- to six-week-old male Balb/cA mice were obtained from National Laboratory Animal Center (National Science Council, Taipei City, Taiwan). Mice were housed on a 12-h light-12-h dark schedule, and fed with water and mouse standard diet (PMI Nutrition International LLC, Brentwood, MO, USA) ad libitum. Use of the mice was reviewed and approved by Chung Shan Medical University animal care committee.

Experimental design

G. amboinense powder at 1 or 2 g was mixed with 99 or 98 g standard powder diet. Each mouse was given a 5g diet with or without G. amboinense supplement per day. Body weight, feed intake and water intake were recorded weekly. After 6 weeks of supplementation, mice were treated by acetaminophen intraperitoneally (ip 350 mg/kg body weight), and killed with carbon dioxide after 24h. Liver from each mouse was collected and weighted. Blood was also collected, and plasma was separated from erythrocyte immediately. Liver at 0.2g was homogenized on ice in 2ml phosphate buffer (pH 7.2) and the filtrate was collected. The protein concentration of plasma and liver filtrate was determined by the method of Lowry et al. (1951) using bovine serine albumin as a standard. In all experiments, sample was diluted to a final concentration of 1 g protein/1.

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) analyses

Plasma activities of ALT and AST were determined by using commerical assay kits (Randox Laboratories Ltd., Crumlin, United Kingdom).

Glucose, triglyceride (TG) and total cholesterol (TC) measurements in plasma

Plasma glucose level (mmol/1) was measured by a glucose HK kit (Sigma Chemical, St. Louis, MO, USA). TG and TC levels (mmol/l) in plasma were determined by triglycerides/GB kit and cholesterol/HP kit (Boehringer Mannheim, Mannheim, Germany), respectively.

Glutathione (GSH) and oxidized glutathione (GSSG) levels, catalase and glutathione peroxidase (GPX) activities assay

GSH and GSSG concentrations (nmol/mg protein) in liver were determined by commerical colorimetric GSH and GSSG assay kits (OxisResearch, Portland, OR, USA). Catalase and GPX activities (U/mg protein) in liver were determined by catalase and glutathione peroxidase assay kits (Calbiochem, EMD Bioscience, Inc., San Diego, CA).

Determination of lipid oxidation and reactive oxygen species (ROS)

Lipid oxidation was determined by measuring the level of malondialdehyde (MDA, [mu]mol/1) via an HPLC method (Hsu et al., 2006) in liver. The method described in Gupta et al. (2007) was used to measure the amount of ROS in liver. Briefly, 10 mg liver was homogenized in 1 mi of ice cold 40 mM Tris-HCI buffer (pH 7.4), and further diluted to 0.25% with the same buffer. Then, samples were divided into two equal fractions. In one fraction, 40[micro] 1 1.25 mM 2',7' -dichlorofluorescin diacetate in methanol was added for ROS estimation. Another fraction, in which 40[micro]l methanol was added, served as a control for auto fluorescence. After incubating for 15min at 37[degrees] C, fluorescence was determined at 488 nm excitation and 525 nm emission using a fluorescene plate reader.

Statistical analyses

The effect of each treatment was analyzed from 10 mice (n = 10) in each group. Data were subjected to analysis of variance (ANOVA) and computed using the SAS General Linear Model procedure (SAS Institute, 1999). Difference with (p.< 0.05) were considered to be significant

Results

The content of total polyphenols and total flavonoids in G. amboinense is 193.8 [+ or -] 11.9 mg GAE/100g and 241.5 [+ or -] 9.3 mg CE/100 g, respectively. The intake of G. amboinense at 1% or 2% did not affect final body weight, feed intake and water intake (p > 0.05), data not shown). Without acetaminophen treatment, the consumption of G. amboinense did not affect ALT and AST activities (Table 1, (p > 0.05). Acetaminophen treatment caused significant elevation of both ALT and AST activities (p < 0.05); however, the pre-intake of G. amboinense significantly and dose-dependently protected liver against the subsequent acetaminophen-induced elevation of ALT and AST activities in plasma (p < 0.05). The effect of G. amboinense intake and acetaminophen treatment upon glucose level, TG and TC contents in plasma is presented in Table 2. Without acetaminophen treatment, the consumption of 2% G. amboinense significantly decreased plasma TC level (p < 0.05). Acetaminophen treatment resulted in significantly lower plasma glucose level (p < 0.05). The pre-intake of G. amboinense significantly alleviated plasma glucose reduction (p < 0.05). Without acetaminophen treatment, the consumption of G. amboinense significantly and dose-dependently increased hepatic GSH level (Table 3, (p < 0.05). but did not affect GPX and catalase activities (p > 0.05). Acetaminophen treatment caused significant GSH depletion, GSSG, ROS and MDA increase and activity reduction of GPX and catalase (p < 0.05). However, the pre-intake of G. amboinense significantly diminished the subsequent acetaminophen-induced MDA formation, GSH depletion, GSSG and ROS production, and retarded the loss of catalase and GPX activities (p < 0.05), in which the effect of G. amboinense on GPX activity and the formation of MDA and ROS was dose-dependent (p < 0.05).
Table 1. ALT and AST levels in plasma from mice treated with 1% or 2%
Ganoderma amboinense (GA) alone or plus acetaminophen

 ALT (U/1) AST (U/1)

Control 54 [+ or -] 4 (a) 77 [+ or -] 9 (a)
1% GA 48 [+ or -] 8 (a) 81 [+ or -] 5 (a)
2% GA 56 [+ or -] 5 (a) 73 [+ or -] 7 (a)
Acetaminophen 505 [+ or -] 36 (d) 326 [+ or -] 20 (d)
1% GA + acetaminophen 423 [+ or -] 20 (c) 251 [+ or -] 13 (c)
2% GA + acetaminophen 293 [+ or -] 18 (b) 203 [+ or -] 10 (b)

Data are mean [+ or -] SD (n = 10)
(a-d) Least-squares means with a common superscript within a column are
not different at the 5% level.

Table 2. The content of glucose, TG and TC in plasma from mice
treated with 1% or 2% Ganoderma amboinense (GA) alone or plus
acetaminophen

 Glucose (mmol/l) TG (mmol/l) TC (mmol/l)

Control 6.8 [+ or -] 0.2 (b) 0.23 [+ or -] 1.59 [+ or -]
 0.08 (a) 0.09 (b)

1% GA 6.4 [+ or -] 0.3(b) 0.31 [+ or -] 1.50 [+ or -]
 0.06 (a) 0.11 (b)

2% GA 6.7 [+ or -] 0.4(b) 0.25 [+ or -] 1.22 [+ or -]
 0.05 (a) 0.07 (a)

Acetaminophen 5.5 [+ or -] 0.3 (a) 0.27 [+ or -] 1.62 [+ or -]
 0.08 (a) 0.12 (b)

1% GA + 6.4 [+ or -] 0.4 (b) 0.28 [+ or -] 1.55 [+ or -]
acetaminophen 0.09 (a) 0.08 (b)

2% GA + 6.5 [+or -] 0.5 (b) 0.03 [+ or -] 1.50 [+ or -]
acetaminophen 0.04 (a) 0.13 (b)

Data are mean [+ or -] SD (n = 10).
(a-b) Least-squares means with a common superscript within a column are
not different at the 5% level

Table 3. The content of MDM, ROS, GSH, GSSG and activity of GPX and
catalase in liver from mice treated with 1% or 2% Ganoderma amboinense
(GA) alone or plus acetaminophen

 MDA ([mu] mol/1) ROS (nmol/ mg GSH (nmol/ mg
 protein) protein)

Control 0.65 [+ or -] 0.29 [+ or -] 23.1 [+ or -] 1.8
 0.10 (a) 0.10 (a) (e)

1% GA 0.67 [+ or -] 0.31 [+ or -] 25.9 [+ or -] 1.4
 0.12 (a) 0.09 (a) (d)

2% GA 0.63 [+ or -] 0.26 [ -or -] 28.3 [+ ] 1.5
 0.08 (a) 0.11 (a) (e)

Acetaminophen 1.52 [+ or -] 1.37 [+ or -] 10.1 [+ or -] 1.2
 0.32 (d) 0.17 (c) (a)

1% GA + 1.24 [+ or -] 0.72 + 0.15 (c) 15.3 [+ or -]1.3
acetaminophen 0.22 (c) (b)

2% GA + 0.96 [+ or -] 0.55 [+ or -] 14.7 [+ or -] 1.1
acetaminophen 0.10 (b) 0.08 (b) (b)

 GSSG (nmol/ mg GPX (U/mg Catalase (U/ mg
 protein) protein) protein)

Control 0.34 [+ or -] 30.9 [+ or -]2.6 21.3 [+ or -]
 0.09 (a) (d) 2.1 (c)

1% GA 0.33 [+ or -] 31.2 [+ or -] 20.8 [+ or -]
 0.07 (a) 2.0 (a) 2.4 (e)

2% GA 0.32 [+ or -] 31.6 [+ or -] 21.6 [+ or -]
 0.06 (a) 2.2 (d) 1.8 (e)

Acetaminophen 1.10 [+ or -] 16.8 [+ or -]1.7 11.5 [+ or -]
 0.14 (e) (a) 1.2 (a)

1% GA + 0.89 [+ or -] 19.3 = 1.5 (b) 13.9 [+ or -]
acetaminophen 0.10 (b) 0.9 (b)

2% GA + 0.81 [+ or -] 23.0 [+ or -] 14.2 = 1.1 (b)
acetaminophen 0.07 (b) 2.0 (e)

Data are mean [+ or -] SD (n = 10).
(a-e) least-squares means with a common superscript within a column are
not different at the 5% level.


Discussion

It has been indicated that G. amboinense is rich in polysaccharides, glycoproteins and triterpenes (Shiao et al., 1994). Our present study further found that this herb also contains polyphenols and flavonoids. Polyphenols and flavonoids could act as radical scavengers (Zou et al., 2004; Correia et al., 2007); thus, these components might contribute to the antioxidant protection from this herb because the intake of this herb effectively reduced liver ROS level. It has been reported that some of ganoderma triterpenes could inhibit cholesterol synthesis (Kimura et al., 2002; Shaio, 2003). In our present study, the consumption of G. amboinense at 2% markedly decreased total cholesterol content in plasma in normal mice. This finding agreed that this herb might possess cholesterol-lowering effects, and could be applied for attenuating hypercholesterolemia. Further study is necessary to examine the effect and mechanism of this herb upon prevention and/or alleviation of hypercholesterolemia.

As reported by others (Gamal et al., 2003; Song et al., 2004), acetaminophen ip injection increased serum ALT and AST activities, and exacerbated oxidative injury. The result from our present study agreed with those previous studies, and supported that oxidative damage was an important factor responsible for acute liver injury induced by acetaminophen. Furthermore, our present study found that the intake of G. amboinense markedly alleviated acetaminophen-induced oxidative stress and improved liver function. This finding implied that this herb could provide anti-oxidative protection against acute hepatic oxidative injury.

Reduced glutathione is a substrate for glutathionerelated enzymes, and a regenerator for alpha-tocopherol; therefore, it plays an important role in the amtioxidant defense system (Vatassery et al., 1989; Meister, 1991). It is well known that a large dose of acetaminophen causes hepatic GSH depletion because NAPQI reacts rapidly with glutathione (Nelson, 1990; Oz et al., 2004 Masubuchi et al., 2005), which consequently exacerbates oxidative stress in conjuction with mitochondrial dysfunction. Our present study found that intake of Ganoderma amboinense could increase hepatic GSH level in mice without acetaminophen treatment, and could restore hepatic GSH level in acetaminophen-treated mice. These findings suggested that this herb might be rich in GSH, or the component (s) of this herb could act as GSH precursor (s). Therefore, the observed antioxidant protection from G. amboinense against acetaminophen-induced hepatotoxicity might be partially due to the increased GSH. Further study is necessary to examine whether G. amboinense contains GSH, or GSH precursor (s) such as cysteine, or other compounds by which the enzymes involved in GSH biosynthetic pathway are up-regulated. On the other hand, G. amboinense supplement enhanced hepatic activity of GPX and catalase in acetaminophentreated mine. this finding revealed that this herb could provide anti-oxidative protection via sparing or elevating the activity of enzymatic antioxidants. thus, the observed diminished oxidative stress from G. amboinense supplement could be partially ascribed to the enhanced activity of these antioxidant enzymes. G. amboinense has been used as folk medicine for thousands of years in China and Asia. Thus, it should be safe for people to consume. Since this herb could provide anti-oxidative protection via increasing GSH level and enhancing GPX activity, this herb could be also applied to prevent and/ or alleviate other diseases with oxidative stress.

In conclusion, G. amboinense contained polyphenols and flavonoids; the pre-intake of this herb could restore hepatic GSH content and enhance catalase and GPX activities, which effectively protected the liver against subsequent acetaminophen-induced oxidative injury These results support that G. amboinense may be considered as a preventive agent for acute liver injury.

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Cheng-chin Hus (a), Ko-yen Lin (a), Zhi-hong Wang (a), Wea-lung Lin (b), Mei-chin Yin (c), *

(a) Department of Nutritional Science, Chung Shan Medical University, Taichung City, Taiwan

(b) Department of Pathology, Chung Shan Medical University, Taichung City, Taiwan

(c) Department of Nutrition, China Medical University, Taichung City, Taiwan

* Corresponding author. Tel: + 88642205 3366x7510;

fax: + 88622062891.

E-mail address: mcyin@mail.cmu.edu.tw (M.-C. Yin).

0944-7113/$-see front matter [C] 2008 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2008.04.011
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Author:Hus, Cheng-chin; Lin, Ko-yen; Wang, Zhi-hong; Lin, Wea-lung; Yin, Mei-chin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9TAIW
Date:Nov 1, 2008
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