Hepatoprotective and Antioxidant Activities of Tribulus Terrestris

Tribulus terrestris L. has been used in folk medicine throughout history. The present study examined the acute toxicity of the total ethanolic extract of T. Terrestris followed by investigation of the hepatoprotective activity of the total ethanolic extract and different fractions of the aerial parts of the plant compared to silymarin against carbon tetrachlorideinduced hepatic damage in rats. In addition, in vivo antioxidant activity was examined and linked to the previous in vitro DPPH free radical scavenging activity investigation. This study established the plant’s safety and the hepatoprotective effect of the total ethanolic extract of the aerial parts of the plant and its different fractions due to significant decrease in CCl4induced rise in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin in rats. Treatment with the ethyl acetate fraction significantly reduced oxidative stress in CCl4intoxicated rats, as evident by a decrease inmalondialdehyde (MDA) content associated with elevation of hepatic reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Hence, this hepatoprotective effect could be due to the antioxidant activity of the plant which is mainly imparted by the two major di-p-coumaroylquinic acid derivatives isolated from the ethyl acetate fraction.


Introduction
Liver plays a vital role in transforming and clearing chemicals, so it is susceptible to the toxicity induced by these agents (Kashaw et al., 2011).Studies indicate that, oxidative stress might be a key originating factor in the pathogenesis of liver diseases including drug-induced hepatic damage.Over production of free radicals is toxic to hepatocytes and triggers reactive oxygen species (ROS) mediated cascade causing hepatocyte death (Kaplowitz, 2000;Bissel et al., 2001;Jaeschke et al., 2002).Accordingly, the need for reliable hepatoprotective agents is crucial.Nature has afforded some plants with the ability to prevent and cure hepatic disturbances.Thus, medicinal plants especially those rich in antioxidants may serve as a vital source for the development of effective therapy to combat liver problems (Kashaw et al., 2011).
Tribulus terrestris L. is a flowering plant native to warm temperate and tropical regions.It has been used in folk medicine throughout history for a variety of conditions such as hypertension, inflammation and sexual dysfunction (Vesilada et al., 1995).In ethno-botanical claims, the fruits of T. terrestris have been used in China for liver and eye diseases (Li et al., 1998), as well as, the hot water extract of the dried seeds which have been taken orally for liver diseases (Ross, 2005).Also, T. terrestris has been reported in Indian traditional medicine to be effective in treating jaundice (Pushpangadan and Atal, 1984).
The aim of the present study is to establish the ethno-botanical claims by evaluating the hepatoprotective activity of the total ethanolic extract and different fractions of the aerial parts of the plant against carbon tetrachloride-induced hepatic damage in rats.Also, we attempt to understand the cause of the hepatoprotective activity by evaluating the in vivo antioxidant activity and linking the results to the previously examined in vitro DPPH free radical scavenging activity (Hammoda, et al., 2013) in order to determine the main compound(s) responsible for the antioxidant and hepatoprotective effects of the plant.Moreover, acute toxicity study was carried out to ensure the safety of the plant.

Plant Material and Extraction
Tribulus terrestris L. was collected in August and September 2013, growing wildly on train railway and along the Mahmudiya Canal, Alexandria, Egypt.The plant material was identified by Prof. Dr. Salama Mohamed El-Dareer (Department of Botany and Microbiology, Faculty of Science, Alexandria University, Egypt).A voucher specimen (TT206) has been deposited in the Pharmacognosy Department, Faculty of Pharmacy, Alexandria University, Egypt.
Air dried powdered aerial parts (4 kg) of T. terrestris were extracted twice at room temperature (each for 7 days using 10 L 95% ethanol).The solvent was distilled off under reduced pressure to yield 400 g of total dry ethanolic extract.Three hundred and fifty gram of this dry extract was added to a mixture of water and ethyl alcohol (8:2).The hydroalcoholic phase (600 ml) was partitioned successively with chloroform, ethyl acetate and nbutanol to yield 18 g, 10 g and 15 g, respectively, which were separately dissolved in 0.3% sodium carboxymethylcellulose (CMC-Na) for oral administration to experimental animals.
Extraction yield (%) = (weight of the final dried extract x 100) / (weight of the original sample).

Animals
Male Wister rats weighing between 180 and 200 g were used in this study.Rats were kept at a controlled temperature of 23±2 o C, relative humidity of 60-70% and a light regime of 12 hr light: 12 hr dark (lights on at 6:00 a.m.).Animals were quarantined and acclimatized for 7 days prior to the initiation of the study; animals were kept in groups of six and were given a well-balanced pellet diet and tap water ad libitum.The use of animals in our study is conformed to the guidelines and bioethics of the Egyptian Scientific Research Academy that coincide well with the U.S. Department of Agriculture through the Animal Welfare Act (7USC 2131) 1985 and Animal Welfare Standards incorporated in 9 CFR Part 3, 1991.

Acute Toxicity Study
Thirty-six male Wister rats were randomly assigned to six groups of six rats each.Five groups then received orally one of following doses; 100, 200, 300, 500 or 800 mg/kg body weight of the total ethanolic extract dissolved in 0.3% sodium carboxymethylcellulose (CMC-Na).The control group (group 6) received 0.3% CMC-Na in distilled water (1ml/kg body weight, orally.).The groups were returned to their home cages, and provided with food and water ad libitum.Animals were observed for signs of toxicity and mortality for 72 hr (Litchfield and Wilcoxon, 1949).

Experimental Design for Hepatoprotective and in Vivo Antioxidant Study
Seventy-two male Wister rats were randomly assigned to twelve experimental groups (n=6).Group 1 (negative control) rats were given 0.3% CMC-Na in distilled water (1.0 ml/kg body weight) orally for 14 consecutive days.On days 11 and 12 the animals were injected with corn oil (1.0 ml/kg body weight, intraperitoneal -Jain, et al., 2008).Group 2 (positive control) rats were given 0.3% CMC-Na in distilled water (1.0 ml/kg body weight) orally for 14 consecutive days.On days 11 and 12 the animals were injected with fresh mixture of equal volumes of carbon tetrachloride and corn oil (2.0 ml/kg body weight, intraperitoneal- Huang et al., 2010).Groups 3 and 4 rats received silymarin dissolved in 0.3% CMC-Na at a dose of 200 mg/kg orally as a reference treatment for 14 consecutive days (Huang, et al., 2010).Groups 5 and 6 rats received the total ethanolic extract of T. terrestris dissolved in 0.3% CMC-Na at a dose of 500 mg/kg orally for 14 consecutive days.Groups 7 and 8 rats received the chloroform fraction of T. terrestris dissolved in 0.3% CMC-Na at a dose of 250 mg/kg orally for 14 consecutive days.Groups 9 and 10 rats received the ethyl acetate fraction of T. terrestris dissolved in 0.3% CMC-Na at a dose of 250 mg/kg orally for 14 consecutive days.Groups 11 and 12 rats received the n-butanol fraction of T. terrestris dissolved in 0.3% CMC-Na at a dose of 250 mg/kg orally for 14 consecutive days.On days 11 and 12 the animals in groups 4, 6, 8, 10 and 12 were injected with fresh mixture of equal volumes of carbon tetrachloride and corn oil (2.0 ml/kg body, intraperitoneal).Twenty-four hours after the last animal group treatment (day 15), the rats were anaesthetized by the mixture (alcohol: chloroform: ether, 1: 2: 3).Blood samples were collected from each rat in all groups by cardiac puncture (using 20 gauge needle and 5 ml syringe) in heparinized tubes.The plasma was separated and stored at -20 ºC for screening of plasma liver markers.

Biochemical Analysis of Plasma
The plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined according to the method described by Reitman and Frankel, (1957), total bilirubin by the method of Malloy and Evelyn (1937) and Tietz (1976), total protein by the method of Gornall et al., (1949) and albumin by the method of Dumas et al., (1997).Kits for determination of total proteins, albumin and transaminases activity were purchased from Bio Diagnostic Research office (Dokii, Giza, Egypt).The kit for determination of total bilirubin was bought from Diamond Diagnostics, El Gomhoria Company, Egypt.

Hepatoprotection Percentage
The hepatoprotection percentages of silymarin, total ethanolic extract of T. terrestris and its fractions (% H) were calculated for each biochemical parameter separately using the following equation (Wakchaure, et al., 2011).Moreover, total hepatoprotection percentage was compared to silymarin as follows: {(sum of % H of the biochemical parameters of each extract or fraction / sum of % H of the biochemical parameters of silymarin) × 100}.

Estimation of Hepatic Antioxidant Activity
After collection of blood samples, animals of groups 1, 2, 6 and 10 were sacrificed; livers were rapidly isolated and rinsed with ice cold saline.Tissue was then trimmed, washed in cold saline, blotted dry and immediately held on ice.Portions of the tissue were weighed (1.0g) and homogenized with 9 volumes of ice cold 0.05M phosphate buffer at pH 7.4, using electrical tissue homogenizer.Cell debris was removed by centrifugation at 12,000 r.p.m. (4°C) for 20 min.to collect supernatants for determination of malondialdehyde (MDA) concentration (Draper and Hadley, 1990), reduced glutathione (GSH) content (Chanarin, 1989) and superoxide dismutase (SOD) activity (Giannopolitis and Ries, 1977).

Statistical Analysis
Results are expressed as mean ± standard deviation (SD).Differences between means in different groups were tested for significance using a one-way analysis of variance (ANOVA) followed by Duncan's test and P value of 0.05 or less was considered significant using the statistical analysis system (SPSS, 1996).

Acute Toxicity Study
Oral administration of the total ethanolic extract of T. terrestris in doses up to 800 mg/Kg body weight showed no signs of toxicity or mortality in rats.

Effects of Total Ethanolic Extract and Fractions on AST, ALT, Total Bilirubin, Albumin and Total Protein
The tested total ethanolic extract and different fractions of T. terrestris showed no effect on serum biochemical parameters in normal rats (Table 1).On the other hand, the CCl 4 intoxication significantly elevated the bilirubin content and transaminases (AST and ALT) activity as compared to the negative control group.Course treatment with T. Terrestris ethanolic extract and its fractions significantly decreased serum AST, ALT and bilirubin in rats with CCl 4 -induced hepatotoxicity, moreover, the total ethanolic extract significantly elevated the level of serum albumin as shown in Table 1.

Hepatoprotection Percentage
Hepatoprotection percentage (% H) indicated that the total ethanolic extract of T. terrestris at a dose of 500 mg/kg has the highest hepatoprotective effect (99.07%) compared to silymarin followed closely by the ethyl acetate fraction at a dose of 250 mg/kg (97.90%) as shown in Table 2.

Effects of total ethanolic extract and Ethyl Acetate Fraction on MDA, GSH and SOD
CCl 4 administration increased MDA concentration and decreased GSH content and SOD activity markedly compared with the negative control rats, suggesting that lipid peroxidation was a result of CCl 4 poisoning.Treatment with the total ethanolic extract and ethyl acetate fraction of T. terrestris decreased MDA concentration and restored SOD activity and GSH content effectively relative to CCl 4 treated rats as shown in Table 3.
The ethyl acetate fraction significantly decreased MDA concentration to 0.43±0.039nmol/mg nearly restoring it to its original concentration in the negative control group (0.36±0.031 nmol/mg), while the total ethanolic extract was less effective (0.55±0.027 nmol/mg).Both the total ethanolic extract and the ethyl acetate fraction restored the concentration of GSH content and even elevated its concentration compared to the negative control group.Also, both fractions partially improved SOD activity but could not restore it to its original state.

Discussion
In the present experiment, acute toxicity study proved the safety of the plant extract in doses up to 800 mg/kg.The total ethanolic extract and different fractions of the aerial parts of T. terrestris were evaluated for the hepatoprotective activity using CCl 4 induced hepatotoxicity model in rats to find out the therapeutically more efficacious extract or fraction.In addition, an attempt was made to find out the correlation between antioxidant and hepatoprotective activities in order to determine the secondary metabolites responsible for these activities.The first part of the study was designed to evaluate the competency of the total ethanolic extract and different fractions of the aerial parts of T. terrestris in preventing CCl 4 -induced hepatic damage.Liver damage induced by CCl 4 is a commonly used model for the screening of hepatoprotective drugs.The rise in serum levels of AST and ALT has been attributed to the damaged structural integrity of the liver, because they are cytoplasmic in location and released into the circulation subsequent to cellular damage.The rise in the level of serum bilirubin is the most sensitive and confirms the intensity of jaundice (Krishna Mohan et al., 2007;Atta et al., 2010).
Hence, treatment of rats in this study with hepatotoxic CCl 4 resulted in severe damage of hepatocytes as indicated by the significant increase of serum transaminases (AST and ALT).Severe jaundice and hepatic cell injury were indicated by increased level of serum bilirubin.
Pretreatment of rats with the total ethanolic extract of the aerial parts of T. terrestris at a dose of 500 mg/kg induced a statistically significant decrease in the levels of AST, ALT and bilirubin, as well as, a significant increase in the level of serum albumin where stimulation of protein synthesis has been reported as a contributory hepatoprotective mechanism as it accelerates the regeneration process and the production of liver cells (Krishna Mohan et al., 2007;Atta et al., 2010).
The hepatoprotective activities of the different fractions of the ethanolic extract at a dose of 250 mg/Kg were also studied.The results revealed that the chloroform, ethyl acetate and n-butanol fractions induced a statistically significant decrease in the level of serum liver biochemical parameters to different extents.
The hepatoprotection percentage of the total ethanolic extract and different fractions was calculated compared to silymarin as shown in Table 2, aiming to identify which sample has the highest hepatoprotective effect.The results revealed that the total ethanolic extract, chloroform, ethyl acetate and n-butanol fractions caused 99.07, 86.08, 97.90 and 77.99 % protection, respectively.The total ethanolic extract at a dose of 500 mg/kg showed the highest hepatoprotective activity, closely followed by the ethyl acetate fraction at a dose of 250 mg/kg.
It has been reported in literature that one of the main causes of CCl 4 -induced liver injury is lipid peroxidation induced and accelerated by oxidative stress due to free radical derivatives of CCl 4 .CCl 4 is metabolically activated by cytochrome P450 dependent monooxygenase leading to the production of trichloromethyl radical (CCl . 3 ).The CCl .
3 radicals attack polyunsaturated fatty acids in the presence of oxygen to produce lipid peroxides contributing to hepatic damage.The lipid peroxidation is accelerated when free radicals are formed as the results of losing a hydrogen atom from the double bond in the structure of unsaturated fatty acids.Scavenging of free radicals is one of the major antioxidation mechanisms known to inhibit the chain reaction of lipid peroxidation (Ashok Shenoy et al., 2001;Jain et al., 2008;Huang et al., 2010;Desai et al., 2012).
Accordingly, the second part of the study was carried out as an attempt to elucidate the probable cause of the hepatoprotective activity and the role of the plant in reducing liver damage caused by oxidative stress.The in vivo antioxidant activity was investigated where the total ethanolic extract and ethyl acetate fraction were chosen as good candidates for this part of the study owing to their highest hepatoprotective activity.The in vivo antioxidant activity was assessed according to the effect of the candidates on superoxide dismutase (SOD) enzyme activity, malondialdehyde (MDA) and reduced glutathione (GSH) concentrations.
SOD is a metalloprotein and is the first enzyme involved in the antioxidant defense by lowering the steady state level of O 2 -.CCl 4 administration significantly reduced SOD activity compared to the negative control group.This reduction may be due to enhanced reactive oxygen radicals (superoxide and hydrogen peroxide) generation, which in turn overwhelmed the activity of the enzyme (Akinloye and Olaniyi, 2011).The first evidence of the antioxidant activity of the total ethanolic extract and ethyl acetate fraction was the partial improvement of SOD activity.
GSH, a pervasive tripeptide, is a major thiol in living organisms.It is present at a large concentration in the liver and plays a major role in the elimination and detoxification of many exogenous toxicants.Also, it has a central role in the non-enzymatic antioxidant defense system where GSH metabolism is important in quenching the reactive intermediates and radical species generated during oxidative stress so coordinating the body's antioxidant defense processes.Thus, disruption of GSH status of a biological system can lead to serious consequences (Lee et al., 2000;Mohammad et al., 2011).In this study, reduction of GSH stores of liver tissue in CCl 4 intoxicated rats suggested that oxidative stress due to free radical damage is one of the possible mechanisms in the pathophysiology of CCl 4- induced hepatopathy.On administration of the total ethanolic extract and ethyl acetate fraction of the plant, GSH levels increased markedly.The ethyl acetate fraction showed higher increase in GSH content (9.43±0.19µMol/mg) compared to the total ethanolic extract (6.650±0.17µMol/mg).
MDA is the last product of lipid breakdown caused by oxidative stress.The increase in MDA levels in liver suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals (Ashok Shenoy et al., 2001;Wu et al., 2007).CCl 4 intoxication increased the level of MDA compared to the negative control group.Pretreatment with the total ethanolic extract and the ethyl acetate fraction significantly reversed this increase.Hence, it is possible that the hepatoprotection of T. terrestris is due to its antioxidant effect.Moreover, it is apparent that the ethyl acetate fraction showed more potent effect than the total ethanolic extract.
Results of the in vivo antioxidant investigation were correlated to the former study of in vitro DPPH free radical scavenging activity, where both revealed that the ethyl acetate fraction possess the highest antioxidant effect.These findings prompted the phytochemical investigation of the most active fraction (ethyl acetate) leading to the isolation of two major phenolic compounds (4,5-di-p-ciscoumaroylquinic acid and 4,5-di-p-transcoumaroylquinic acid) showing IC 50 very close to that of ascorbic acid proving their responsibility for the antioxidant effect (Hammoda et al., 2013).
In conclusion, the data of this study provided convincing evidence that the ethyl acetate fraction of the aerial parts of T. terrestris has a significant hepatoprotective effect on acute liver injuries induced by CCl 4 .It can be postulated that this hepatoprotective effect may be due to its antioxidant activity proved by the in vivo and in vitro studies.Two major phenolic compounds (4, 5di-p-cis-coumaroylquinic acid and 4, 5-di-p-transcoumaroylquinic acid) isolated from the bioactive ethyl acetate fraction can be identified as the secondary metabolites responsible for the antioxidant effect and consequently the hepatoprotective activity of the plant.
% H = {1-[(T-N)/(C-N)]}× 100 T = mean value of treatment group.C = mean value of the positive control group.N= mean value of the negative control group.

Table 1 :
Effects of silymarin, total ethanolic extract and different fractions of T. terrestris on liver function markers in normal and CCl 4 -intoxicated rats.
a Significantly different compared to the negative control group (P<0.05).b Significantly different compared to the positive control group (P<0.05).

Table 2 :
Hepatoprotection percentages of the total ethanolic extract and different fractions of T. terrestris compared to silymarin following CCl 4 -induced hepatotoxicity in rats.

Table 3 :
Effects of the total ethanolic extract and ethyl acetate fraction of T. terrestris on MDA, GSH and SOD following CCl 4 -induced hepatotoxicity in rats.
bSignificantly different compared to the positive control group (P<0.05).