Antioxidant Activity of Graptopetalum paraguayense E. Walther Leaf Extract Counteracts Oxidative Stress Induced by Ethanol and Carbon Tetrachloride Co-Induced Hepatotoxicity in Rats

(1) Background: Graptopetalum paraguayense E. Walther is a traditional Chinese herbal medicine. In our previous study, 50% ethanolic G. paraguayense extracts (GE50) demonstrated good antioxidant activity. (2) Methods: To investigate the hepatoprotective effects of GE50 on ethanol and carbon tetrachloride (CCl4) co-induced hepatic damage in rats, Sprague–Dawley rats were randomly divided into five groups (Control group; GE50 group, 0.25 g/100 g BW; EC group: Ethanol + CCl4, 1.25 mL 50% ethanol and 0.1 mL 20% CCl4/100 g BW; EC + GE50 group: Ethanol + CCl4 + GE50; EC + silymarin group: ethanol + CCl4 + silymarin, 20 mg/100 g BW) for six consecutive weeks. (3) Results: Compared with the control group, EC group significantly elevated the serum aspartate aminotransferase (AST), alanine aminitransferase (ALT), and lactate dehydrogenase (LDH). However, GE50 or silymarin treatment effectively reversed these changes. GE50 had a significant protective effect against ethanol + CCl4 induced lipid peroxidation and increased the levels of glutathione (GSH), vitamin C, E, total antioxidant status (TAS), and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione S-transferases (GST). Furthermore, in EC focal group, slight fat droplet infiltration was observed in the livers, while in the GE50 or silymarin treatment groups, decreased fat droplet infiltration. HPLC phytochemical profile of GE50 revealed the presence of gallic acid, flavone, genistin, daidzin, and quercetin. (4) Conclusions: The hepatoprotective activity of GE50 is proposed to occur through the synergic effects of its chemical component, namely, gallic acid, flavone, genistin, daidzin, and quercetin. Hence, G. paraguayense can be used as a complementary and alternative therapy in the prevention of alcohol + CCl4-induced liver injury.


Introduction
The liver, being a dynamic and vital organ, actively participates in multi-metabolic functions of foods, drugs, chemicals, biologicals, and xenobiotics, as well as detoxification of viral and bacterial products. These models, induced by toxins such as carbon tetrachloride (CCl 4 ), dimethylnitrosamine (DMN), acetaminophen, or thioacetamide, can represent chronic or acute/fulminant hepatitis. Experimentally induced cirrhotic response in rat by CCl 4 is shown to be similar to liver cirrhosis in the humans. Hepatotoxicity of CCl 4 is largely due to its degraded metabolites trichloromethyl (CCl 3 ) and trichloromethyl peroxyl (CCl 3 O 2 ) formed by hepatic microsomal enzyme [1,2]. The hepatotoxicity

Animal Treatment
Fifty male weanling Sprague-Dawley (SD) rats were obtained and fed commercial chow diets (Fwusow Industry Co., LTD, Taiwan). They were randomly divided into five groups, each containing ten animals. The control group was gavaged with 1.25 mL of normal saline daily for six weeks. The GE50 group was gavaged with GE50, dissolved in 1.25 mL normal saline, at a dose of 0.25 g/100 g BW for six weeks. The ethanol + CCl 4 (EC) group was gavaged with 50% ethanol 1.25 mL 50% ethanol/100 g BW (equal to 0.5 g ethanol/100 g BW) and 0.1 mL of 20% CCl 4 in olive oil twice a week and administered 1.25 mL of normal saline daily for six weeks. The ethanol + CCl 4 + GE50 (EC + GE50) group and ethanol + CCl 4 + silymarin (EC + silymarin) group were gavaged with GE50 (0.25 g/100 g BW) and silymarin (20 mg/100 g BW), respectively, daily for six weeks and received ethanol/CCl 4 in the same manner as EC group. The control and GE50 groups, which are not administered CCl 4 , received 0.1 mL of olive oil/100 g BW at the same time points. The animals were kept under standard laboratory conditions of light/dark cycle, a temperature of 22 ± 2 • C and humidity of 50 ± 10%. This animal research and all the procedures were reviewed and approved by the Animal Research Ethics Committee at Providence University, Taichung, Taiwan (Approval No: 20071210-A05).

Serum and Liver Tissue Preparation
After six weeks of feeding, the blood was collected. The serum was analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and total antioxidant status (TAS). The livers were homogenized in an ice-cold phosphate buffer (0.05 M, pH 7.4) using a Potter-Elvehjem-type homogenizer with a Teflon pestle. One portion of this tissue homogenate (0.3 g/mL) was used for assaying the levels of malondialdehyde (MDA), vitamin C, vitamin E, and reduced glutathione (GSH). After centrifuged at 12,000× g and 4 • C for 10 min. The resulting supernatant was used to determine the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), CAT, and glutathione S-transferase (GST).

Determination of AST, ALT, LDH, and TAS Serum Levels in Rats
The levels of AST and ALT in the serum samples were determined by enzymatic methods using an automatic analyzer at a commercial analytical service center (Lian-Ming Co., Taiwan, ROC). The levels of LDH and TAS were determined using commercial kits from Randox Laboratories Ltd. (Antrim, UK).

Measurement of MDA and GSH Levels, and GPx, SOD, and CAT, and GST Activities
To measure activities in the liver, MDA, GSH, GPx, SOD, and CAT were performed in accordance with our previously reported procedures. Tissue MDA levels were used to spectrophotometrically estimate thiobarbituric acid-reactive substances (TBARS) at 535 nm. GSH contents were measured by HPLC. SOD activity was determined spectrophotometrically at 325 nm. One unit of SOD activity was defined as half the rate of reduction of pyrogallol autoxidation over a 1-min period at 15-s intervals. GPx activity was determined by an enzyme coupled method with glutathione reductase (GR) using cumene hydroperoxide as the substrate at 30 • C. The rate of decrease in the NADPH concentration was observed at 340 nm over a 3-min period at 30-s intervals. One unit of GPx activity was defined as the amount of enzyme that catalyzed the oxidation of 1 µmol of NADPH/min/mL. CAT activity was determined using H 2 O 2 as the substrate. The rate of H 2 O 2 dismutated to H 2 O and O 2 was proportional to the CAT activity. The decrease in the amount of H 2 O 2 was observed at 240 nm over a 1-min period at 15-s intervals. One unit of CAT activity was defined as 1 mmole H 2 O 2 remaining per minute [22]. GST activity was determined at 340 nm by an enzyme-coupled method with glutathione-1-chloro-2,4-dinitrobenzene (CDNB) as the substrate at 25 • C [23]. One unit of GST activity was defined as the amount of enzyme that catalyzed the formation of glutathione-CDNB/min/ml. The protein content of the tissue cytosols was determined based on the Biuret reaction using a BCA kit. The specific activity of the enzyme was expressed as unit/mg protein.

Measurement of Antioxidants
The liver vitamin C content was stabilized by MPA and cysteine solution and determined by HPLC [24]. The vitamin E standard and the tissue cytosols were diluted in methanol solution containing 0.25% BHT and 0.2% ascorbate before HPLC analysis. The tissue vitamin E and GSH contents were measured by HPLC [25,26]. The tissue GSH was reduced by dithiothrietol and the monobromobimane derivative was produced before the HPLC analysis. As the eluent, 30 mM TBA in methanol solution was used at a flow rate of 1 mL/min.

Histopathology
The liver tissue of rats was immobilized in 10% formalin solution for 24 h. Then, the liver tissue was dehydrated, made transparent, waxed, embedded, and sectioned. Liver tissues were trimmed to 2-mm thickness. Then, the liver tissue was stained with hematoxylin and eosin (H&E).

Characterization of Phenolic Compounds in GE50 Extracts
Determine the polyphenolic compounds in GE50 extracts, HPLC analysis was performed in accordance with our previously described method with modifications [16,27]. GE50 was dissolved in 50% ethanol, filtered, and analyzed by HPLC. Peak areas and concentrations were determined. The identification of each compound was based on a combination of retention time and spectral matching by comparison with those of known standards.

Statistical Analysis
Data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using SPSS (v.16.0; SPSS, USA). One-way ANOVA and Scheffe's method were used to analyze the differences between the means. Differences with p < 0.05 were considered statistically significant. Table 1 shows the daily body weight gain, feed efficiency, and liver index of the rats in each group. The effects of GE50 on the body weight were evaluated. Silymarin, a polyphenolic flavonoid, was used in this study as a reference drug. Significant weight loss was observed in ethanol + CCl 4 (EC)-treated group. The change in body weight was highest in the EC-treated group (3.56 ± 0.66 g/day/rat) compared with the control (5.58 ± 0.66 g/day/rat), followed by the GE50 (5.92 ± 0.81 g/day/rat), EC + GE50 (4.68 ± 0.59 g/day/rat), and EC + silymarin (3.56 ± 0.74 g/day/rat) groups. The change in feed efficiency was the highest in EC-treated group compared with the other groups. The relative liver weight in the EC-treated group was the highest compared with the other groups. The administration of GE50 or silymarin over six weeks significantly reversed the ethanol + CCl 4 effects, inducing body weight gain and improving feed efficiency.

Effects of GE50 on Ethanol + CCl 4 -Induced Hepatotoxicity
To evaluate possible hepatocellular damage caused by ethanol and CCl 4 , the activities of AST and ALT were assessed. Ethanol and CCl 4 co-treatment significantly increased the activity of these enzymes in plasma, indicating intense hepatic damage. As shown in Table 2, the serum AST, ALT, LDH, and TAS levels were measured. A significantly higher serum levels of AST, ALT, and LDH were observed in the EC group than in the control group (AST: 184.40 ± 25.5 vs. 103.98 ± 14.0 U/L; ALT: 66.89 ± 4.9 vs. 44.10 ± 5.4 U/L; LDH: 563.04 ± 103.7 vs. 273.82 ± 94.9 U/L). However, EC + GE50 treatment efficiently decreased the AST level to 110.95 ± 14.0 U/L, ALT level to 50.01 ± 4.2, and LDH level to 403.20 ± 79.4 U/L. Similar results were obtained when the rats were treated with silymarin, which is a known hepatoprotective chemical. Table 2. Effects of oral administration of GE50 over six consecutive weeks on serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels, and total antioxidant status (TAS) in rats treated with ethanol + CCl 4 . The data are presented as mean ± S.D. of 10 rats. One-way ANOVA and Scheffe's method were used to analyze the differences between the means. a-c Mean values with different letters in the same row are significantly different (p < 0.05) according to Duncan's multiple-range test. Control group; GE50 group; EC group: Ethanol + CCl 4 ; EC + GE50 group: Ethanol + CCl 4 + GE50; EC + silymarin group: ethanol + CCl 4 + silymarin.

Groups AST (U/L) ALT (U/L) LDH (U/L) TAS (nmole/L)
The serum TAS level in rats showed a significantly decreased in the EC group compared to the control group (0.15 ± 0.1 vs. 0.39 ± 0.1 nmole/L). The EC + GE50 treatment reversed the TAS level to 0.28 ± 0.1 nmole/L. GE50 treatment alone did not affect the serum AST and ALT levels.

Histological Analyses
The hepatoprotective effects were confirmed by histological examinations. Hepatic steatosis represents an excess accumulation of fat in hepatocytes. To assess the degree of fatty liver, we examined the accumulation of hepatic triglyceride by H&E staining of the rat liver. Ethanol administration caused degenerative morphological changes, which were exhibited by fat droplets in the liver sections.
The results of H&E staining indicated that ethanol and CCl 4 co-administration presented extensive changes in the liver morphology, including marked enlarged areas of hepatocellular degeneration and infiltration inflammatory cells. No histological abnormality was observed in the control group. As illustrated in Figure 1, ethanol + CCl 4 (EC)-induced injury included increased vacuole formation, neutrophil infiltration, and necrosis. Liver section from the EC-treated group showed highly deformed liver architecture with fatty lesion due to intensive fatty infiltration (FI) and signs of necrosis (N). EC + GE50 group and EC + silymarin group demonstrated improved hepatocellular architecture with intact nucleus (IN) and normal sinusoids (NS). administration caused degenerative morphological changes, which were exhibited by fat droplets in the liver sections. The results of H&E staining indicated that ethanol and CCl4 co-administration presented extensive changes in the liver morphology, including marked enlarged areas of hepatocellular degeneration and infiltration inflammatory cells. No histological abnormality was observed in the control group. As illustrated in Figure 1, ethanol + CCl4 (EC)-induced injury included increased vacuole formation, neutrophil infiltration, and necrosis. Liver section from the EC-treated group showed highly deformed liver architecture with fatty lesion due to intensive fatty infiltration (FI) and signs of necrosis (N). EC + GE50 group and EC + silymarin group demonstrated improved hepatocellular architecture with intact nucleus (IN) and normal sinusoids (NS).

Effects of GE50 on Hepatic MDA, Vitamin C, Vitamin E, and GSH Levels in Ethanol + CCl 4 -Treated Group
In our study, the hepatic MDA levels were significantly elevated in the ethanol + CCl 4 (EC) group (2.40 ± 0.13 nmol/mg protein) compared with that in the control (1.28 ± 0.21 nmol/mg protein) and GE50 (1.19 ± 0.21 nmol/mg protein) groups (p < 0.05). In contrast, in the EC + GE50 group and EC + silymarin group, hepatic MDA levels were markedly decreased compared with the EC group ( Figure 2). This observation indicated that the plant extracts may provide phytochemicals that inhibit lipid peroxidation in the rat liver.
In our study, the hepatic MDA levels were significantly elevated in the ethanol + CCl4 (EC) group (2.40 ± 0.13 nmol/mg protein) compared with that in the control (1.28 ± 0.21 nmol/mg protein) and GE50 (1.19 ± 0.21 nmol/mg protein) groups (p < 0.05). In contrast, in the EC + GE50 group and EC + silymarin group, hepatic MDA levels were markedly decreased compared with the EC group ( Figure 2). This observation indicated that the plant extracts may provide phytochemicals that inhibit lipid peroxidation in the rat liver.
Vitamin C level was significantly decreased in the EC group (6.37 ± 0.87 nmol/mg protein) compared with the control group (8.97 ± 1.26 nmol/mg protein). Vitamin E level was also significantly decreased in the EC group (0.22 ± 0.03 nmol/mg protein) compared with the control group (0.36 ± 0.02 nmol/mg protein). GSH level was significantly decreased in the EC group (22.30 ± 3.62 nmol/mg protein) compared with the control group (32.78 ± 2.55 nmol/mg protein). However, EC + GE50 treatment reversed the vitamin C level to 8.55 ± 0.68 nmol/mg protein, vitamin E level to 0.33 ± 0.04 nmol nmol/mg protein, and GSH level to 27.41 ± 3.45 nmol/mg protein. The decline of GSH level in the EC group might be due to its excessively generated quantity of free radicals leading to hepatic injury.  Vitamin C level was significantly decreased in the EC group (6.37 ± 0.87 nmol/mg protein) compared with the control group (8.97 ± 1.26 nmol/mg protein). Vitamin E level was also significantly decreased in the EC group (0.22 ± 0.03 nmol/mg protein) compared with the control group (0.36 ± 0.02 nmol/mg protein). GSH level was significantly decreased in the EC group (22.30 ± 3.62 nmol/mg protein) compared with the control group (32.78 ± 2.55 nmol/mg protein). However, EC + GE50 treatment reversed the vitamin C level to 8.55 ± 0.68 nmol/mg protein, vitamin E level to 0.33 ± 0.04 nmol nmol/mg protein, and GSH level to 27.41 ± 3.45 nmol/mg protein. The decline of GSH level in the EC group might be due to its excessively generated quantity of free radicals leading to hepatic injury.

Effects of GE50 on Antioxidant Enzymatic Activities in Ethanol + CCl 4 -Treated Group
The activities of SOD, GPx, CAT, and GST were measured to evaluate the antioxidant effects of GE50 (Figure 3). The SOD activity significantly decreased in the ethanol + CCl 4 (EC) group (3.27 ± 0.11 unit/mg protein) compared with the control (3.95 ± 0.11 unit/mg protein) and GE50 (3.91 ± 0.19 unit/mg protein) groups (p < 0.05). The GPx activity significantly decreased in the EC group (627.25 ± 79.43 unit/mg protein) compared with the control (711.73 ± 37.97 unit/mg protein) and GE50 (702.96 ± 37.71 unit/mg protein) groups. The CAT activity significantly decreased in the EC group (19.61 ± 1.11 unit/mg protein) compared with the control (24.11 ± 1.44 unit/mg protein) and GE50 (23.87 ± 0.91 unit/mg protein) groups. The GST activity significantly decreased in the EC group (908.03 ± 88.92 unit/mg protein) compared with the control (1079.68 ± 41.13 unit/mg protein) and GE50 (1045.72 ± 52.27 unit/mg protein) groups (p < 0.05). GE50 treatment successfully recovered these enzymes levels to almost normal levels. The effect of GE50 was similar to silymarin, which has been previously shown to have a significant protective effect in rats.

Effects of GE50 on Antioxidant Enzymatic Activities in Ethanol + CCl4-Treated Group
The activities of SOD, GPx, CAT, and GST were measured to evaluate the antioxidant effects of GE50 (Figure 3). The SOD activity significantly decreased in the ethanol + CCl4 (EC) group (3.27 ± 0.11 unit/mg protein) compared with the control (3.95 ± 0.11 unit/mg protein) and GE50 (3.91 ± 0.19 unit/mg protein) groups (p < 0.05). The GPx activity significantly decreased in the EC group (627.25 ± 79.43 unit/mg protein) compared with the control (711.73 ± 37.97 unit/mg protein) and GE50 (702.96 ± 37.71 unit/mg protein) groups. The CAT activity significantly decreased in the EC group (19.61 ± 1.11 unit/mg protein) compared with the control (24.11 ± 1.44 unit/mg protein) and GE50 (23.87 ± 0.91 unit/mg protein) groups. The GST activity significantly decreased in the EC group (908.03 ± 88.92 unit/mg protein) compared with the control (1079.68 ± 41.13 unit/mg protein) and GE50 (1045.72 ± 52.27 unit/mg protein) groups (p < 0.05). GE50 treatment successfully recovered these enzymes levels to almost normal levels. The effect of GE50 was similar to silymarin, which has been previously shown to have a significant protective effect in rats. . The data are presented as mean ± S.D. of 10 rats. One-way ANOVA and Scheffe's method were used to analyze the differences between the means. a-c Mean values with different letters in the same row are significantly different (p < 0.05) according to Duncan's multiple-range test. Control group; GE50 group; EC group: Ethanol + CCl4; EC + GE50 group: Ethanol + CCl4 + GE50; EC + silymarin group: ethanol + CCl4 + silymarin.

Polyphenolic Profile in GE50
The HPLC chromatogram showed that gallic acid, quercetin, genistin, and daidzin were the major components among organic molecules found in GE50, which had maximum absorbance at 270 nm. G. paraguayense E. Walther mainly contained flavonoids that were identified in our study

Polyphenolic Profile in GE50
The HPLC chromatogram showed that gallic acid, quercetin, genistin, and daidzin were the major components among organic molecules found in GE50, which had maximum absorbance at 270 nm. G. paraguayense E. Walther mainly contained flavonoids that were identified in our study (Figure 4). It has also been reported that G. paraguayense E. Walther contains various antioxidants, such as gallic acid, quercetin, genistin, and daidzin [16,17]. The protective effects of GE50 may be attributed to the presence of polyphenolic compounds such as gallic acid, flavone, genistin, daidzin, and quercetin. The pharmacological fundamental constituents of the plant are flavonoids.  Figure 4). It has also been reported that G. paraguayense E. Walther contains various antioxidants, such as gallic acid, quercetin, genistin, and daidzin [16,17]. The protective effects of GE50 may be attributed to the presence of polyphenolic compounds such as gallic acid, flavone, genistin, daidzin, and quercetin. The pharmacological fundamental constituents of the plant are flavonoids.

Discussion
CCl4 is a typical hepatotoxic substance, and its mechanism of action is complex. CCl4-mediated hepatotoxicity was chosen as the experimental model. Due to ethanol and CCl4 toxicity, relative liver weight in the ethanol + CCl4 (EC)-treated group was higher than that in the control group (Table 1). Liver weight is known to increase due to hepatic damage inflicted by trichloromethyl radical, as well as consequent liver fibrosis and hypertrophy. Changes in body and liver weight after ethanol and CCl4 intoxication provides direct evidence of the overall hepatic damage [28,29]. The liver, the largest and the most metabolically complex organ in the body, is involved in the storage and biosynthesis metabolism. It is also responsible for detoxification and metabolic homeostasis. Ethanol and CCl4 resulted in loss of body weight, which is considered a putative indicator of health. We demonstrated that GE50 markedly ameliorated ethanol + CCl4 (EC)-induced chronic hepatitis in test rats, accompanied by reduced relative liver weight. Similar results were obtained when the rats were treated with silymarin, a commercial hepatoprotective agent. Silymarin was used as the positive control in this study.
AST and ALT are aminotransferases linked with liver parenchymal cells. If the hepatocellular plasma membrane is damaged, these will leak out from the cytosol into the bloodstream. Serum AST and ALT levels markedly increased in the ethanol + CCl4 (EC)-treated group, indicating altered permeability of membranes and hepatotoxicity. The results revealed that the serum AST and ALT levels significantly decreased after treatment with GE50. These results demonstrate the hepatoprotective effect of GE50 against ethanol + CCl4-induced liver injury in rats. Damage to the liver cells results in elevations of the both ALT and AST, which have been widely accepted as major biomarkers to assess the hepatic injury [30].

Discussion
CCl 4 is a typical hepatotoxic substance, and its mechanism of action is complex. CCl 4 -mediated hepatotoxicity was chosen as the experimental model. Due to ethanol and CCl 4 toxicity, relative liver weight in the ethanol + CCl 4 (EC)-treated group was higher than that in the control group (Table 1). Liver weight is known to increase due to hepatic damage inflicted by trichloromethyl radical, as well as consequent liver fibrosis and hypertrophy. Changes in body and liver weight after ethanol and CCl 4 intoxication provides direct evidence of the overall hepatic damage [28,29]. The liver, the largest and the most metabolically complex organ in the body, is involved in the storage and biosynthesis metabolism. It is also responsible for detoxification and metabolic homeostasis. Ethanol and CCl 4 resulted in loss of body weight, which is considered a putative indicator of health. We demonstrated that GE50 markedly ameliorated ethanol + CCl 4 (EC)-induced chronic hepatitis in test rats, accompanied by reduced relative liver weight. Similar results were obtained when the rats were treated with silymarin, a commercial hepatoprotective agent. Silymarin was used as the positive control in this study.
AST and ALT are aminotransferases linked with liver parenchymal cells. If the hepatocellular plasma membrane is damaged, these will leak out from the cytosol into the bloodstream. Serum AST and ALT levels markedly increased in the ethanol + CCl 4 (EC)-treated group, indicating altered permeability of membranes and hepatotoxicity. The results revealed that the serum AST and ALT levels significantly decreased after treatment with GE50. These results demonstrate the hepatoprotective effect of GE50 against ethanol + CCl 4 -induced liver injury in rats. Damage to the liver cells results in elevations of the both ALT and AST, which have been widely accepted as major biomarkers to assess the hepatic injury [30].
Alcoholic hepatitis should at least include inflammation, steatosis, fibrosis, and cell damage. The activity of alcohol dehydrogenase and aldehyde dehydrogenase causes a reduction in NAD + /NADH ratio, which is the process that causes a reduced mitochondrial capacity to metabolize lipids [11,31]. Otherwise, CCl 4 -induced liver injury is characterized by oxidative stress and activated hepatic macrophage, leading to hepatocyte damage and death [32]. Chronic alcohol consumption increases cellular nicotinamide adenine dinucleotide hydrate concentration and acetaldehyde dehydrogenase activity, which leads to severe free fatty acid overload, triglyceride accumulation, and subsequent hepatic steatosis [9]. Liver injury can lead to the transfer of fatty acids to the liver, resulting in an increase in the TG content in the liver. These results demonstrate that GE50 treatment significantly alleviated the degree of liver injury.
Lipid peroxidation is one of the major characteristics of CCl 4 -induced hepatotoxicity [33]. MDA, the end product of lipid peroxidation, is widely used as a marker of lipid peroxidation injury. Antioxidants, such as N-acetyl-cysteine, α-tocopherol, phenols, selenium, and vitamin C and E, have been proposed and used as therapeutic agents in liver damage [34]. Vitamin E is believed to be the most important lipophilic antioxidant in biological tissues. Cheeseman et al. demonstrated that an increased vitamin E level in liver protects from acute CCl 4 -induced damage by preventing lipid peroxidation [35].
Astrocytes contain one of the highest cytosolic concentrations of GSH amongst mammalian cells. GSH is the major non-protein thiol that plays a vital role in maintaining the antioxidant defense mechanism in the body. GSH levels in the liver dropped in CCl 4 -treated mice. The depletion of GSH may also be a consequence of liver damage. Ethanol inhibits the synthesis of reduced GSH. Moreover, acetaldehyde promotes GSH depletion, free radical-mediated toxicity, and lipid peroxidation [36]. The hepatoprotective effects of some compounds, such as silymarin, may be attributable to its ability to increase cellular GSH [37]. The increase in hepatic GSH by GE50 may be one of the ways in which G. paraguayense protects the liver against ethanol and CCl 4 co-induced hepatotoxicity in rats.
CCl 4 initiates lipid peroxidation, as well as reduces tissue GPx, GR, CAT, and SOD activities. In experimental animals, the induction of an SOD-CAT-insensitive free radical species by diet and alcohol facilitates liver damage [38]. The GST family represents one of the main detoxification systems in the hepatocytes. GST regulates apoptosis by influencing the lipid peroxidation pathway [39]. GPx and GSH are well-known reductants that metabolize toxic chemicals, drugs, and xenobiotics. In general, the liver can combat the free radical damage by biotransformation of these toxic agents in less reactive compounds through cytochrome P-450 and GPx [40]. CAT plays a role in the metabolism of ethanol. In addition to alcohol dehydrogenase and CYP2E1, CAT is involved in the metabolism of ethanol in the body.
The treatment of SD rats with alcohol and CCl 4 caused the development of significant hepatocellular damage, as was evident from a marked increase in the serum activities of AST, ALT, and LDH compared with the control rats (Table 2). We observed a large number of inflammatory cell infiltration in the ethanol + CCl 4 (EC) co-treatment group (Figure 1). Rats treated with the GE50 had lower concentration of MDA in the liver cells. Alcohol + CCl 4 (EC) co-treatment also caused a considerable increase (p < 0.05) in hepatic MDA formation ( Figure 2) and simultaneously induced a marked depletion (p < 0.05) in vitamin C, vitamin E, GSH, and SOD, GPx, CAT, and GST levels in the liver ( Figure 3) compared with the control rats. Our studies showed a decrease in food intake and increase in oxidative stress in rats co-treated ethanol and CCl 4 . Our results demonstrated that GE50 significantly enhanced the GSH, SOD, CAT, and GR levels, and may contribute to the important mechanisms underlying the liver preventive effects.
Plant flavonoid compounds are a gifted class of so-called "nutraceuticals" that include the ability to protect hepatic damage [41][42][43]. Phytochemicals are naturally occurring chemicals in plant that promote the prevention and treatment of various diseases. Plants are a good source of useful hepatoprotective agents that can modulate the activities of free radicals [41]. A significant decrease in lipid peroxides in liver tissues following co-treatment with CCl 4 and antioxidants indicated the protective effects of polysaccharide from Angelica and Astragalus [44] and Fagonia schweinfurthii [45] through the scavenging of free radicals produced by CCl 4 . Several reports have shown that Antrodia camphorate, also known as Antrodia cinnamomea (Niuchangchih), is a precious fungus grown in Taiwan. It has been reported to prevent ethanol-, CCl 4 -, and cytokine-induced liver injury, ameliorate fatty liver and liver fibrosis, and inhibit hepatoma cells [46][47][48]. In our study, GE50 and Niuchangchih were identical to the positive drug Silymarin.
The structural characteristics of plant polyphenols provide them with strong antioxidant and free radical scavenging abilities. Hepatoprotection using edible phytochemicals is a novel strategy for the treatment of various hepatic dysfunctions. Gallic acid (3,4,5-trihydroxybenzoic acid), a phenolic acid with strong antioxidant effect, is abundant in tea leaves, as well as white, red, and black mulberry, blackberry, raspberry, strawberry, dragon fruit, guava, mangosteen, papaya, and other plants. Gallic acid downregulated CYP2E1 expression in liver tissues while increasing SOD activities. These results support its ability to regulate the antioxidant enzymes activities and inhibit lipid peroxidation. Many studies have demonstrated its hepatoprotective effects [49][50][51].
Isoflavones are dietary phytoestrogens occurring naturally in legumes such as soybeans. Two major isoflavones found in soybean are daidzin and genistin, respectively. In soy and soy products, 95-99% of genistein exists in the conjugated form genistin (glycoside). Many studies demonstrated that daidzein and genistein alleviate hepatic damage [52,53].
It is, therefore, reasonable to assume that the hepatoprotective activities of GE50 is attributed to its gallic acid, flavone, genistin, daidzin, and quercetin components that most possibly act synergistically. G. paraguayense is an edible vegetable that has also been used in traditional Taiwanese folk medicine for protection against liver injury.

Conclusions
In conclusion, the results of our study indicate that GE50 enhances hepatic antioxidant enzyme activities, as well as inhibits lipid peroxidation in ethanol and CCl 4 (EC) co-induced liver injury, and its effect is similar to that of silymarin. The hepatoprotective activity of GE50 is proposed to occur through the synergic effects of its chemical component, namely gallic acid, flavone, genistin, daidzin, and quercetin. These results confirm that the in vivo hepatoprotective activity of GE50 may be associated with the phenolic phytochemicals present in the extract, which are known for their antioxidant potential. GE50 can be used as a functional food or even a pharmacological agent for the prevention of liver diseases.