Antihepatotoxic and antioxidant defense potential of Mimosapudica

Reactive Oxygen species (ROS) are believed to be responsible for pathogenesis of various diseases affecting tissues and the main organ, the Liver. Hence, in the present study, the extent of Lipid Peroxidation (LPO) and ROS elimination and its defense mechanisms by the enzymic & non enzymic antioxidants in liver & serum was investigated. Hepatoxicity was manifested by significantly decreased (p<0.05) levels in the activities of the enzymic antioxidants such as Superoxide dismutase (SOD) Catalase (CAT), Glutathione peroxidase and the non enzymic antioxidants such as glutathione & Vitamin C in rats induced hepatic damage by ethanol Simultaneous administration of the leaf extract Mimosa pudica along with the toxin ethanol in rats showed a considerable protection against the toxin induced oxidative stress and liver damage as evidence by a significant increase (p<0.05) in antioxidant activities. The study reveals that the co administration of Mimosa pudica aqueous extract significantly lowered the level of lipid peroxidation in alcohol fed mice. Key wordsMimosa pudica, Hepatoxicity, antioxidants Introduction Liver damage due to consumption of alcohol may be caused by oxygen radicals such as superoxide and hydroxyl radicals generated during the metabolism of ethanol by the microsomal oxidising system [1]. Antioxidants are likely to provide beneficial effects on hepatocytes desensitization against oxidant stress while inhibiting primary mechanism for expression of pro-inflammatory and cytotoxic mediators [2]. In spite of tremendous scientific advancement in the field of hepatology in recent years, liver problems are on the rise. [3]. Ingested alcohol produces striking metabolic imbalances in the liver and leads to the formation of reactive oxygen species [4] and free radial increase [5]. The free radical produced during the metabolism of the drug is considered to be responsible for alteration induced in lipid peroxidation, enzymic & non enzymic antioxidants [6]. Antioxidants stop the free radical from forming in the first place or interrupt an oxidizing chain reaction to minimize the damage caused by free radicals [7]. Medicinal plants are considered to be an important source of antioxidant compounds and the therapeutic benefit of many medicinal plants in often attributed to their antioxidant properties [8]. Mimosa pudica is an annual shrub found through out India. Stem and rachis are clothed with prickles arising from bulbous bases. Previous studies have shown that Mimosa pudica cures congestion with flem, biliousness, leucoderma and diseases of blood [9]. The present study investigates Mimosa pudica used in herbal medicine for their potential to scavenge free radicals as consequence may be considered as effective sources for combating oxidative hepatic damage [10]. Although a number of reports described the usefulness of this herb against various disorders, no studies have been published to date describing its beneficial role against ethanol induced hepatotoxicity in rats. In this study, the extent of liver damage was assessed by determining the hepatic antioxidant status by measuring the hepatic content of SOD, GSH, CAT, Reduced glutathione Vitamin C and TBARS expressed as malanoldialdehyde equivalents. Materials and Methods Animals Male albino Wister rats (150 gm) were housed in large spacious cages and were fed on standard pellet diet (M/s Hindustan Lever Ltd.,) and water and libitum. The experimental procedures were carried out in strict compliance with the Animal Ethics committee’s rules and regulations of this institute. Plant Material The plant material was taxonomically identified by Prof. Parthiban, Associate, Prof. (Forestry), TNAU, Tamil Nadu, India.


Introduction
Liver damage due to consumption of alcohol may be caused by oxygen radicals such as superoxide and hydroxyl radicals generated during the metabolism of ethanol by the microsomal oxidising system [1]. Antioxidants are likely to provide beneficial effects on hepatocytes desensitization against oxidant stress while inhibiting primary mechanism for expression of pro-inflammatory and cytotoxic mediators [2]. In spite of tremendous scientific advancement in the field of hepatology in recent years, liver problems are on the rise. [3]. Ingested alcohol produces striking metabolic imbalances in the liver and leads to the formation of reactive oxygen species [4] and free radial increase [5]. The free radical produced during the metabolism of the drug is considered to be responsible for alteration induced in lipid peroxidation, enzymic & non enzymic antioxidants [6]. Antioxidants stop the free radical from forming in the first place or interrupt an oxidizing chain reaction to minimize the damage caused by free radicals [7]. Medicinal plants are considered to be an important source of antioxidant compounds and the therapeutic benefit of many medicinal plants in often attributed to their antioxidant properties [8]. Mimosa pudica is an annual shrub found through out India. Stem and rachis are clothed with prickles arising from bulbous bases. Previous studies have shown that Mimosa pudica cures congestion with flem, biliousness, leucoderma and diseases of blood [9]. The present study investigates Mimosa pudica used in herbal medicine for their potential to scavenge free radicals as consequence may be considered as effective sources for combating oxidative hepatic damage [10]. Although a number of reports described the usefulness of this herb against various disorders, no studies have been published to date describing its beneficial role against ethanol induced hepatotoxicity in rats. In this study, the extent of liver damage was assessed by determining the hepatic antioxidant status by measuring the hepatic content of SOD, GSH, CAT, Reduced glutathione Vitamin C and TBARS expressed as malanoldialdehyde equivalents.

Materials and Methods Animals
Male albino Wister rats (150 gm) were housed in large spacious cages and were fed on standard pellet diet (M/s Hindustan Lever Ltd.,) and water and libitum. The experimental procedures were carried out in strict compliance with the Animal Ethics committee's rules and regulations of this institute.

Experimental Design
The rats were divided into four groups (n = 4). The ethanol dose for rats was calculated on the basis of their body weight Group I: Control -Normal Healthy Rats with control vehicle i.e., distilled water. Group II: Alcohol Control -Rats administered with 40% alcohol orally. Group III: Simultaneous Induction and Treatment -(Alcohol + Aqueous extract) Simultaneous administration of 40 % alcohol and aqueous plant (Mimosa pudica) extract (1 gm /kg body wt.) was given orally.
Group IV: Aqueous Extract Control -Rats administered with aqueous extract (1gm/kg body wt.) of the plant Mimosa pudica orally.

Preparation of Sample for Antioxidant Studies
After the end of the experimental period of 30 days the rats were deprived of food over night and sacrificed by cervical dislocation. Blood was collected and kept for 30 minutes without disturbing. The blood clots were then centrifuged for 15 minutes at 2000 rpm to separate the serum and it is used for antioxidant analysis. The liver was washed with ice cold tris buffered saline, blotted dry and10% homogenate was prepared using tris buffered saline (pH 7.4) to follow lipid peroxidation rate. 15% homogenate were prepared using phosphate buffered saline (pH 7.0) in cold condition for the estimation of antioxidants and 20% liver homogenate was prepared using 5% trichloroacetic acid to estimate reduced glutathione. The homogenate was then centrifuged at 2000 rpm for 10 minutes and the supernatant was used for the experimental analysis.

Results and Discussion Antioxidant levels in Mimosa pudica Enzymic and non-enzymic antioxidants
The activity of the enzymic antioxidants such as superoxide dismutase, peroxidase, catechol oxidase and laccase were found to be present in the plant extract of Mimosa pudica. Superoxide dismutase activity was found to be maximum in the leaf sample followed by polyphenol oxidase. Peroxidase activity was found to be less when compared to superoxide dismutase. Polyphenol was found to be 5.3 X 10 Lipid peroxidation is a free radical mediated process and acts as a potential ¯³ units as catechol oxidase and laccase respectively (Table I). The presence of non-enzymic antioxidants in Mimosa pudica is illustrated in Table II and III; the levels of nonenzymic antioxidants (total phenols, flavonoids, GSH, vitamin C and vitamin E are shown (Tables  II and III).

In vitro and in vivo inhibition of lipid peroxidation
Free radical scavenging activity of different extracts of Mimosa pudica induced lipid peroxidation is depicted in Table IV. When different extracts of Mimosa pudica were compared for free radical, scavenging activity aqueous extract showed higher percentage of inhibition (89.73 %) over ethanol, methanol, chloroform and petroleum ether extract. Based on the above results, water extract of Mimosa pudica was selected for its free radical scavenging activity. Lipid peroxidation is a free radical mediated process and acts as a potential marker of susceptibility of early and irreversible tissue damage. The hepatoprotective effect of Mimosa pudica against alcohol toxicity is presented in Table V. The levels of TBARS in liver were elevated in alcoholic group when compared with control group. Our study reveals that co-administration of Mimosapudica aqueous extract significantly lowered the level of lipid peroxidation in alcohol fed mice.
The first line of cellular defense against free radicals consists of catalase, superoxide dismutase and peroxidase [11]. These enzymes react directly with oxygen radicals to yield non -radical products [12]. A significant reduction (P<0.05) in the activity of antioxidant enzyme like SOD and catalase were observed in rats intoxicated with alcohol (Group II). But Co-administration with Mimosa pudica extract increases its activity. The enzyme antioxidant levels were increased when alcohol fed rats were treated with Mimosa pudica. Inhibition of SOD and catalase activities could be due to exhaustion or inactivation as a result of oxidative stress [13]. The increase in the SOD and Catalase activity by treatment with aqueous extract of Mimosa pudica effectively eliminates the super oxides and peroxides by alcohol intoxication. Table IV depicts the activity of  Glutathione-S-transferase and Glutathione peroxidase. Recent studies have indicated that the cytosolic liver enzyme -glutathione Stransferase ( GST) functions as an early and sensitive indicator of hepatocyte damage caused by various adverse conditions, including hemorrhagic shock [14] ischemia and reperfusion [15] liver transplant rejection [16] and acetaminophen overdose [17]. Liver failure can be manifested by the increased level of GST or when GST remains persistently elevated [18]. Hepatocytes in severe alcoholic liver disease co expressed both alpha and pi class glutathione Stransferase [19]. The activity of Glutathione -Stransferase was considerably elevated in alcohol group than in other groups. Severe liver damage was observed on exposure to alcohol. On administration of Mimosa pudica extract the rat liver cells were protected from oxidative damage. GPx and GR activities in haemolysate decreased after 12 weeks of exposure to chronic ethanol treatment in mice [20]. The activity of GPx was significantly decreased in the liver of rats treated with alcohol. The decreased level of GPx can result in increased level of free radical [21] which induced lipid peroxidation [22]. Co-administration of Mimosa pudica showed the adaptive nature of the system against the damage of super oxide and peroxide radical. This agrees with the result of Omotuyi and Oluyemi who reported a decrease in Glutathione peroxidase which requires selenium for its optimum activity [23]. Selenium is decreased during increased free radical formation [24]. GSH antioxidant system consists of array of enzymic and non enzymic pathway involved in neutralisation of reactive free radical species [25]. Under conditions of increased oxidative stress to cells, levels of GSH are usually reduced [26]. The hepatoprotection was associated with the significant increase in hepatic GSH status, as indicated by substantial increase in tissue GSH levels in Mimosa pudica treated rats. From our finding it was observed that the activity of Vitamin C and reduced Glutathione decreased significantly in the liver tissues of animals administered with alcohol when compared to the control. Vitamins C and E are antioxidants that scavenge for free radicals [27]. The level of vitamin C and reduced glutathione was near normal when Mimosa pudica extract was fed with alcohol in rats. GSH, Vitamin E and C exist in their inter convertible forms and participate in the detoxification of the toxic reactive oxygen species [28]. So the present results indicate the protective potential offered by the Mimosa pudica extract against hepatic dysfunction caused by alcohol.

List of abbreviations
CAT -catalase GPx -glutathione peroxidase GR -glutathione reductase GSH -reduced glutathione GST -glutathione-S-transferase SOD -superoxide dismutase TBARS -thiobarbituric acid reactive substances  (Values are mean ± S.D. from 6 animals in each group) Statistical Comparison a b=significant(P< 0.05) when Group III is compared with Group II significant(P< 0.05) when Group II is compared with Group I significant(P< 0.05) when Group II is compared with Group I b=significant(P< 0.05) when Group III is compared with Group II a=significant(P< 0.05) when Group II is compared with Group I b=significant(P< 0.05) when Group III is compared with Group II SOD-Units : 50% inhibition of nitrite/min/mg protein Catalase-Units : n moles of H2O2 decomposed / min/mg protein GPx-Units : n moles of GSH / min/mg protein GST-Units : n moles of CDNB conjugated / min/mg protein