Solanum trilobatum L. Ameliorate Thioacetamide-Induced Oxidative Stress and Hepatic Damage in Albino Rats

Solanum trilobatum L. (Solanaceae) has been well known as nightshade, commonly used by diverse populations to heal several disorders. Earlier studies in Solanum trilobatum were focused on different pharmacological activities and a few were concerned with antioxidant and hepatoprotective effects. Thus, the current study was focused to evaluate the antioxidant potential and hepatoprotective effects of S. trilobatum L. on thioacetamide (TAA) intoxication in Wistar albino rats. The rats were kept into four groups and six animals each. Group A was normal control. Group B was the TAA treated control. Groups C and D were pretreated with the aqueous extract from the leaves of S. trilobatum (100 mg, 200 mg/kg bw p.o.) once daily for 10 consecutive days administration followed by a single dose infusion of TAA (100 mg/kg s.c.). After 10 days, blood and livers were collected. The biochemical assay was carried out in the GSH (reduced glutathione), TBARS(thiobarbituric acid reactive substances), Na+-K+-ATPase, and antioxidant enzymes viz., SOD (superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase), GST (glutathione-S-transferase), and GR (glutathione reductase) were analyzed in samples of blood and liver. Treatment with S. trilobatum reduced blood and liver TBARS, and Na+ K+ ATPase activity in TAA (thioacetamide)-induced hepatotoxicity rats. Furthermore, the above antioxidant enzymes were increased in the pretreatment of S. trilobatum in TAA intoxicated rats. Finally, we concluded that S. Trilobatum displayed potent antioxidant properties and alleviate oxidative stress induced hepatotoxic effects and possible engross mechanisms related to free radical scavenging properties.


Introduction
Currently, oxidative stress associated free-radical biology have become niche research attention. Free radicals play a crucial function in the progress of tissue damage and pathological events in living beings [1]. In a cell, lipid peroxidation is restricted by several cellular defense mechanisms consisted of enzymatic and non-enzymatic scavenging systems [2]. Recent studies have also specified that there is a contrary connection between the intake of food rich in antioxidants and the occurrence of human illness [3]. Thus the investigations of new synthetic or natural antioxidants are urgently important.
The properties of medicinal plants have been investigated in the recent scientific progress due to its effective antioxidant potentials and ability in healing various ailments. Normally, antioxidants inhibit oxidative stress related tissue damage caused by free radicals; either obstruct oxidation mechanisms or scavenge the oxygen [4,5]. Potentially reactive oxygen derivatives are known to be recognized as reactive oxygen species (ROS), which is consistently produced by various biochemical mechanisms in and Center for Molecular Systematics in Management and Science University, Malaysia. Voucher specimens are maintained in the institution.

Preparation of Plant Extract
Fresh powdered leaves (500 g) were subjected to 2 L of distilled water and extraction was kept in a cold room with constant stirring overnight. The extraction was filtered using cheesecloth, and Whatmann filter paper followed by centrifugation (1200× g for 10 min). The supernatant was evaporated under reduced pressure using a vacuum rotary evaporator and residues were kept under refrigeration until used (yield: 210 w/w).

Experimental Design
Animals were kept into four groups and six rats each. Group A was normal control. Group B was TAA treated control. Group C and D were pretreated with the aqueous extract from the leaves of S. trilobatum (100 mg, 200 mg/kg bw p.o.) once daily for 10 consecutive days administration followed by a single dose infusion of TAA (100 mg/kg s.c.) as a 2% W/V solution in distilled water. The control group received the vehicle as distilled water. The method of acute hepatotoxicity induction was followed according to the method of Kumar et al. [46]. After 24 h of toxin administration, all rats were sacrificed by cervical dislocation; blood was collected and used for the measurement of antioxidant enzymes. Livers were dissected immediately and homogenized. The homogenates were then centrifuged at 3200× g for 20 min at 4 • C and supernatant obtained was used for the assay of various enzymes.

Assay of Antioxidant Enzymes
2.6.1. Reduced Glutathione (GSH) Assay GSH was estimated using DTNB by the method of Sedlak and Lindsay [47]. 1 mL sample of 10% homogenate was precipitated with 1 mL of (4%) sulfosalicylic acid. The samples were maintained at 4 • C for 1 h and then centrifuged at 1200× g for 20 min at 4 • C. Added 0.1 mL filtered aliquot, 0.2 mL of 100 mM DTNB, 2.7 mL phosphate buffer (0.1 M, pH 7.4) in the cuvette. The yellow color of the mixture was developed, read immediately at 412 nm on the spectrophotometer and expressed as µM GSH/g tissue.

Superoxide Dismutase (SOD) Assay
SOD activity was estimated by the method of Kakkar et al. [48]. Reaction mixture of this method contained 0.1 mL of phenazine methosulphate (186 µM), 1.2 mL of sodium pyrophosphate buffer (0.05 mM, pH 7.0), 0.3 mL of supernatant after centrifugation (1500× g, 10 min followed by 10,000× g, 15 min) of 10% homogenate was added to the reaction mixture. The enzyme reaction was initiated by adding 0.2 mL of NADH (780 µM) and stopped after 1 min by adding 1 mL of glacial acetic acid. The changes in the absorbance were measured at 560 nm using the spectrophotometer. Results are expressed in units/mg protein.

Catalase Activity (CAT)
CAT activity was measured by following decomposition of H 2 O 2 according to the method of Aebi and Vergmeyer [49]. The reaction solution contained 0.1 mL enzyme extract, 2.5 mL of 50 mM phosphate buffer (pH 5.0), and 0.4 mL of 6 mM H 2 O 2 . After one min, the absorbance of the reaction solution was read at 240 nm. One unit of CAT activity was defined as an absorbance change of as 0.01 units/min.

Glutathione-S-Transferase (GST) Assay
GST activity was measured using CDNB as substrate according to Habig et al. [51]. The reaction mixture consisted of 1.475 mL phosphate buffer (0.1 M, pH 6.5), 0.025 mL CDNB (1 mM), 0.2 mL reduced GSH (1 mM), and 0.3 mL of 10% homogenate in a total volume of 2.0 mL. The absorbance was measured at 340 nm using a spectrophotometer and GST activity was determined as nM CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 10 3 /M cm.
2.6.6. Glutathione Reductase (GSR) Assay GSR activity was determined by the method of James et al. [52]. The reaction solution composed of 1.65 mL phosphate buffer: (0.1 M, pH 7.6), 0.1 mL EDTA (0.5 mM), 0.1 mL NADPH (0.1 mM), 0.05 mL oxidized glutathione (1 mM), and 0.1 mL 10% homogenate in a total volume of 2 mL. Enzyme activity was estimated at 25 • C by measuring the disappearance of NADPH at 340 nm and was calculated as nM NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 10 3 /M cm.

Estimation of Lipid Peroxidation (TBARS) Assay
The TBARS assay for lipid peroxidation was carried out by following the method of Ohkawa et al. [53]. The reaction mixture in a total volume of 1 mL contained 0.58 mL phosphate buffer (0.1 M, pH 7.4), 0.2 mL homogeneous samples, 0.2 mL ascorbic acid (100 mM), and 0.02 mL ferric chloride (100 mM). The composite was kept at 37 • C for 1 h in a water bath. The reaction was stopped by the addition of 1.0 mL 10% TCA. After addition of 1.0 mL 0.67% TBA, all the tubes were boiled in a water bath for 20 min and then shifted to an ice-bath before centrifuging at 2500× g for 10 min. The quantity of TBARS was analyzed by determining the optical density of the supernatant at 535 nm using a spectrophotometer. The results were expressed as nM TBARS/min/mg tissue at 37 • C using a molar extinction coefficient of 1.56 × 10 5 /M cm. The concentration of TBARS was expressed as nmol of malondialdehyde per mg of protein using 1,1,3,3-tetraethoxypropane as the standard.
2.6.8. Measurement of Na + K + ATPase Activity Na + K + ATPase activity was measured by Bonting method [54]. This method is based on the measurement of orthophosphate released from ATP during incubation of membranes with a medium containing: 1 mM ATP, 10 mM MgCl 2 , 100 mM Tris-HCl buffer (pH 7.4), and 0.1 mM ouabain, which is added to block the Na + K + ATPase. Samples were incubated for 30 min at 37 • C and of 0 • C. After the incubation, 0.6 M TCA was added. The concentration of the orthophosphate was determined in the supernatant by the Veldhoven and Mannaers' method.

Statistical Analysis
All data obtained in the study were expressed as Mean ± S.D., using the Statistical Package for Social Sciences (SPSS) software made for Windows Version 20.0 and statistically assessed by one-way analysis of variance (ANOVA). The difference between test animals and controls were evaluated by Student's t-test [55].

Results
The level of glutathione (GSH) in the blood and in liver homogenate, liver Na + K + ATPase and liver TBARS in TAA intoxicated rats were given in Table 1. Animals treated with TAA elevated blood and liver GSH, and Na + K + ATPase level. Pretreatment of rats with 100 mg, 200 mg/kg bw of aqueous leaf extracts of S. trilobatum showed a significant reclamation in the reduction of blood (75%) and liver GSH (70%), liver Na + K + ATPase level (75.7%). While TBARS of TAA treated animals were significantly higher than the control animals. Administration of aqueous extract of leaves of S. trilobatum markedly decreased the level of TBARS (85.6%). The protection was maximal with the treatment at a higher dose of the extract. Table 1. Effect of aqueous extract of Solanum trilobatum on GSH (blood & liver), liver Na + K + ATPase, and liver TBARS in rats subjected to TAA toxicity.

Treatment Blood GSH (mg%) Liver GSH (µmol/g of Liver)
Liver Na + K + ATPase (U/g prOtein)  The effect of aqueous extract of leaves of S. trilobatum on GSR, GST, GP X , SOD, and CAT in TAA intoxicated rats were shown in Table 2. Levels of these antioxidant enzymes were decreased significantly (p < 0.001) in TAA-induced rats when compared with those of control animals. Treatment of rats with aqueous extract from leaves of S. trilobatum (100 mg, 200 mg/kg bw p.o.) showed an elevated level of those antioxidant enzymes such as GSR (69.6%), GST (67.7%), GPx (87.5%), SOD (75.7%), and CAT (88.7%) in TAA intoxicated rats. The hepatoprotection was higher with the treatment at a higher dose of the plant extracts.

Discussion
Generally, glutathione plays a noteworthy cellular antioxidant defense mechanism that scavenges singlet oxygen, superoxide, and hydroxyl radicals [1,2]. The degradation of the GSH-dependent antioxidant defense mechanism enhances the intracellular flux of oxygen radicals [3] which creates cellular oxidative stress and promoting apoptosis [56]. The protective functions of GSH on cellular lipid peroxidation have been well documented [5]. An increase hepatic lipid peroxidation was evident by lifting MDA in liver homogenate and its reduction to nearly normal levels by S. trilobatum administration.
Thioacetamide was the well-known hepatotoxin and causes hepatocellular carcinoma in rodents [46]. The studies further suggested that the acute or chronic exposure of TAA caused liver cirrhosis, encephalopathy, and fibrosis in rats [13][14][15][16]. In the present findings, TAA produced depletion of blood and liver GSH and elevation of hepatic TBARS. Pretreatment of rats with S. trilobatum (100, 200 mg/kg bw) significantly abridged the TBARS levels and increased the concentration of GSH. These findings demonstrated that a high concentration of GSH in blood and liver could provide a strong tissue defense system against oxidative stress, and thus contribute to attenuating the hepatotoxicity effect of TAA. In addition, detoxification of TAA in the liver could be triggered by GST-catalyzed conjugation with GSH [57], the elevated levels of GST in the liver persuaded by the administration of crude extracts of S. trilobatum, as a result, diminish the acute hepatotoxicity effect of TAA. The GSR is a member of cytosolic enzymes, mainly involved in the detoxification of xenobiotic compounds by their GSH conjugation [58]. It has been reported that TAA produces an increased blood GSR activity, which occurs at a maximum of 24 h after administration. Pre-treatment of S. trilabatum significantly reduced GSR in TAA administered rats.
The enzyme activities of Na + K + ATPase, SOD, and CAT were significantly decreased (p < 0.001), the experiment was positively correlated with previous studies associated with hepatotoxin-treated animals [57,59]. The administration of plant extract showed the reversal of these enzyme activities and could be helpful to reduce the hepatotoxin-induced liver damage. These findings suggested that pre-treatment of leaf extract of S. trilabatum elevated the activity of SOD and CAT, and thereby inhibited the generation of lipid peroxides and reduced hepatic damage. In addition, these elevated levels of enzymes in S. trilobatum treated animals has prominent free radical scavenging activity, which may exert a beneficial effect against pathological alterations, and protect the membrane damage in liver caused by ROS. Antioxidants may be molecules/substances that can neutralize free radicals by accepting or donating electron(s) to eliminate the unpaired condition of the radical [7,8,59]. S. trilobatum possesses enormous bioactive substances, acts as antioxidant molecules, which may directly react with the reactive radicals and destroy or neutralize them [60]. Hence, the present findings strongly suggest that the hepatoprotective action of the plant extract might be due to its antioxidant potential. However, the potential of antioxidant agents and active principles can only be realized after further comprehensive pharmacological and molecular mechanisms.

Conclusions
In conclusion, the current finding highlights that S. trilobatum is known to be possess potent antioxidants and ameliorate the effect of TAA-induced oxidative stress and liver toxicity in rats appears to be associated with the inhibition of lipid peroxides and elevation the antioxidant enzymes in the blood and liver.