Effect of n-hexane extract of Tinospora crispa on reduced glutathione and malondialdehyde level in paracetamol induced hepatotoxicity in rats

The present study demonstrates that, treatment with the nhexane extract of Tinospora crispa was able to ameliorate chronic hepatotoxicity due to oxidative stress e.g. Paracetamol induced hepatotoxicity. Considering the biochemical and histological parameters together, it was observed that hepatic reduced glutathione (GSH) concentrations of the n-hexane extract post treated group was increased to significant level and hepatic malondialdehyde (MDA) concentration was reduced to significantly lower level compared to the paracetamol treated groups. This study suggests near normal alleviation of paracetamol induced chronic hepatotoxicity in rats.

In the past, researchers studied the mechanism of toxic action of paracetamol (Acetaminophen) focused on metabolic activation of the drug, depletion of glutathione and covalent binding of the reactive metabolite n-acetyl-p-benzoquinone imine ( NAPBQI ) to cellular protein as the main cause of hepatic cell death 7 . In addition to covalent binding of the metabolite, oxidative stress appeared as another mechanism in the development acetaminophen toxicity 6 . Under conditions of NAPBQI overproduction, reduced glutathione ( GSH ) concentrations may be very low in the centrilobular cells and the major peroxide detoxification enzyme GSH peroxidase becomes inhibited 8 . Nake 9 reported that the administration of encapsulated superoxide dismutase decreased the

Article info.
Website: https://www.banglajol.info/index.php/UpDCJ paracetamol toxicity in rat. Jaeschke and Bajt 10 also demonstrated that oxidant stress causes mitochondrial membrane permeability trasition pore, loss of membrane potentials of mitochondria, depletion of ATP might be responsible for the typical nuclear DNA fragmentation of paracetamol induced cell death.
Metabolism of various endogenous and exogenous compounds generate reactive oxygen and nitrogen species ( ROS and RNS ) which are involved in the pathogenesis of liver disease 11,12,13 . ROS rapidly react with a variety of cellular molecules and thereby interfere with cellular function 14 .
Infection with HCV is associated with increased levels of ROS/RNS and decreased antioxidant levels in patients. Patients infected with HCV show increase in lipid peroxidation levels in liver samples, serum, and peripheral blood mononuclear cells 2 .
Adachi and Ishii 15 reported that long term ethanol fed rats showed an increase ROS production, hepatocytes damage and apoptosis; this reaction was prevented by antioxidants. Most hepatocellular carcinoma occur in cirrhotic livers, and the common mechanism for carcinogenesis is chronic inflammation associated with oxidative stress 16 . Amimoto 17 reported that treatment of fasted mice with paracetamol lead to hepatic injury. Paracetamol caused an increased amount of Thiobarbituric acid reactive substance ( TBARS ), which was accompanied by a loss of reduced form of coenzyme Q9 and coenzyme Q10 functioning as antioxidants. Paracetamol also markedly decreased hepatic glutathione levels. These results suggested that oxidative stress followed by lipid peroxidation might play a role in the pathogenesis of paracetamol induced hepatic injury.
Cellular GSH ( reduced glutathione ) concentration is decreased markedly in response to protein malnutrition, oxidative stress and many pathological conditions 18 . Levels of GSH are regarded as an index of toxicity, since its depletion in parenchymal organs leads to binding of toxic chemicals to cellular structure and eventually cell death 19 .
The traditionally available remedies for hepatic disorders from natural sources also are worth mentioning at this point. The herbal agents are cheap, easily obtainable, environment friendly and expected to lack organ-specific significant adverse effects. In this subcontinent, stem, leaves and whole plant of Tinospora crispa known in Bengal as 'guloncha' has been used widely for the treatment of bodyache, rheumatic pain, jaundice, pyrexia, tetanus, leprosy, diabetes, malaria, syphilis, sprain, eczema, sedative, loss of appetite, cold for a long period apparently without any scientific basis by traditional medical practioners 20 . Tinospora crispa belongs to the Menispermaceae family. Several plants belonging to this family have also been scientifically recognized as to contain phytochemical constituents having significant pharmacological activities 20 . The Tinospora crispa stem extract possess high antioxidative properties and the activities is comparable to the established antioxidants, such as BHT and vitamin C 21 . The flavonoids detected in Tinospora crispa are Catechin, Luteolin, Morin, and Rutin 21 . Catechin could prevent cardiovascular disease by protecting LDL from oxidative damage through its free radical quenching and metal chelating abilities 22 . Luteolin has strong scavenging properties for superoxide radicals 23 . The present study decided to explore the curative role of the n-hexane extract of Tinospora crispa (Tc) in chronic hepatotoxicity induced by paracetamol in the rat model.

MATERIALS AND METHODS
The study was carried out in the laboratory of the Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University ( BSMMU ), Dhaka, during the period from July 2010 to March 2011. 48 adult rats of the Long-Evans Norwegian strain aged between 3-4 months ( weighing between 160-180 g ) were obtained from the animal house of BSMMU. The rats were housed in standard sized metallic cages (3-4 rats / cage) in a well ventilated room. They were allowed to live at room temperature, fed normal rat diet.

Procedure for obtaining n-hexane extract of Tinospora crispa stem:
About 7 Kg of the herb (Tinospora crispa ) was collected from suburbs of Dhaka, Tongi and Gazipur. The stems of the herb were cleaned, cut into small pieces and shed dried for several days. They were grinded by clean sterile grinding machine. About 1500 g powder of Tinospora crispa stem were obtained. The dried powdered herb was suspended in 6 litre of ethanol for 3 days. The whole was then filtered with filter paper. The filtrate was concentrated by rotary vaccum evaporator at 40-50 o C and reduced pressure. About 600 ml of crude ethanol extract of Tinospora crispa was obtained. An equal amount of n-hexane was added to the crude ethanol extract and left for 24 hours. 2 layers were formed : n-hexane soluble nonpolar upper portion and ethanol soluble aqueous lower portion. The n-hexane soluble layer was separated by a separator funnel and was concentrated with rotary vaccum evaporator at 40 o C. About 113.28 ml ( 14.5 g ) light green n-hexane extract was obtained which was preserved in an amber coloured air tight glass bottle at 0-4 0 C until preparation of suspension.

Description of groups treated with drugs and extracts:
The experiment was designed to demonstrate the effect of post-treatment of the n-hexane extracts of Tinospora crispa on hepatic GSH, MDA concentrations as well as liver histology in paracetamol induced hepatotoxicity. For this purpose 48 rats were taken and grouped as follows-Group C (control group) -6 rats received normal rat diet and water ad libitum for 21 days and were sacrificed on the 22 nd day.
Website: https://www.banglajol.info/index.php/UpDCJ Group P22 (paracetamol control group) -6 rats received daily a single dose of paracetamol at a dose of 150 mg/Kg body weight orally by ryle's tube from day 1 to day 21 and were sacrificed on the 22 nd day.
Group P36 (paracetamol treated group) -6 rats received daily a single dose of paracetamol at a dose of 150 mg/Kg body weight orally by ryle's tube from day 1 to day 21 and were sacrificed on the 36 th day.
Group PH36 (paracetamol + n-hexane extract treated group) -6 rats received daily a single dose of paracetamol at a dose of 150 mg/Kg body weight orally by ryle's tube from day 1 to day 21 and n-hexane extract solution of Tinospora crispa at a dose of 200 mg/Kg body weight orally by ryle's tube from day 22 to day 35 & were sacrificed on the 36 th day.
Group P43 (paracetamol treated group) -6 rats received daily a single dose of paracetamol at a dose of 150 mg/Kg body weight orally by ryle's tube from day 1 to day 21 and were sacrificed on the 43 rd day.
Group PH43 (paracetamol + n-hexane extract treated group) -6 rats received daily a single dose of paracetamol at a dose of 150 mg/Kg body weight orally by ryle's tube from day 1 to day 21 and n-hexane extract solution of Tinospora crispa at a dose of 200 mg/Kg body weight orally by ryle's tube from day 22 to day 42 & were sacrificed on the 43 rd day.

Sacrifice of animals and collection of samples:
After inducing chloroform anaesthesia on the animal, The liver was dissected out after opening the abdomen by median incision. A small part (approximately 500 mg) of liver tissue was immersed immediately into Tyrode's solution contained in a separate beaker placed on an ice bath, weighed (after being blotted on filter paper) and kept in deep freeze until homogenized. The rest of the portion was preserved in 10% formalin for subsequent histological processing.

Estimation of hepatic reduced glutathione (GSH) concentrations
2-nitrobenzoic acid is reduced by SH group to form 2-nitro-5mercaptobenzoic acid which has an intense yellow colour & can be used to measure SH groups by spectrophotometer.

Estimation of hepatic malondialdehyde (MDA) concentrations
Malondialdehyde (MDA) is formed as a result of lipid peroxidation. It reacts with thiobarbituric acid (TBA). The reaction yields a pink MDA-TBA adduct. The coloured complex can be measured by spectrophotometer.

Statistical analysis:
Data obtained from the findings of the above experiments have been expressed as mean ± Standard devitation (mean ± SD). The results were presented by tables. Significance of difference between groups were assessed by using 'One way Analysis of Variance' (ANOVA) followed by Bonferroni 't' test. The difference between groups were considered highly significant at P < 0.001, moderately significant at P < 0.01, and significant at P < 0.5. Table I. Effect of Tinospora crispa extract on hepatic reduced glutathione(GSH) level ( mg/ g of tissue ) in paracetamol induced hepatotoxicity in rats.

Group
GSH (mg/g) (mean ± SD)  Histological examination of the liver sections post treated with extracts of Tinospora crispa showed remarkable reduction in necrosis and degenerative changes against paracetamol induced toxicity.

DISCUSSION
The reputation of Tinospora crispa ( guloncha ) to the rural physians regarding alleviation of hepatitis encouraged the present researchers in their attempts to evaluate the curative effect of its n-hexane extract in chronic hepatotoxicity. This study was designed to observe the post treatment effect of the n-hexane extract of

CONCLUSION
Tinospora crispa stem are rich in flavonoid compounds like Catechin, Luteolin, Morin, and Rutin which all have antioxidant activities 21 . These findings are in accordance with the findings of the present study where the n-hexane extract of Tinospora crispa stem administration suggest less lipid peroxidation or less oxidative stress.