International Journal of Pharmaceutical Studies and Research E-ISSN

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1 ABSTRACT: Research Article "ANTIOXIDANT AND HEPATOPROTECTIVE ACTIVITY OF TRICHOSANTHES DIOICA ROXB. ON PARACETAMOL INDUCED TOXICITY" Mukesh Tanwar*, Ashok Sharma, Kedar Prasad Swarnkar, Monit Singhal, Kailash Yadav. Address for Correspondence Regional College of Pharmacy, Sitapura, Jaipur, Rajasthan. In present study, the hepatoprotective activity of ethanolic and aqueous extracts of Trichosanthes dioica Roxb. were evaluated against paracetamol induced hepatic damage in rats. The extracts at doses of 100, 200 and 400 mg/kg were administered orally once daily. The substantially elevated serum levels of glutamate oxaloacetate transaminases (AST), glutamate pyruvate transaminases (ALT), alkaline phosphatase (ALP), total protein and total bilirubin were significantly restored by the test extracts. Silymarin was used as standard reference which exhibited significant hepatoprotective activity in this model. The Histopathological studies further confirmed the hepatoprotective effect of the test extract. The results of this study indicate that Trichosanthes dioica Roxb. has hepatoprotective activity against paracetamol induced hepatic damage in rats. Aqueous extract was found to be more potent than ethanolic extract. In vitro antioxidant hydrogen peroxide (H 2 O 2 ) and DPPH free radical scavenging activities were also screened which were positive for both ethanolic and aqueous extracts. The possible mechanism of this activity may be due to free radical-scavenging and antioxidant activities which may be due to the presence of saponins, tannins, vitamin C and carotene in the extracts. Hence it can be concluded that Trichosanthes dioica Roxb. has significant hepatoprotective activity. KEYWORDS: Trichosanthes dioica Roxb., Paracetamol, Hepatoprotective activity, Antioxidant activity, DPPH, H 2 O 2. INTRODUCTION: have great potential in ameliorating these Liver diseases remain one of the serious health problems. In the absence of reliable liver protective drugs in allopathic medical practices, herbs play a role in the management of various liver disorders in ethanomedical practices as well as in traditional systems of medicine in India. More than 15 of these plants are evaluated for their hepatoprotective activity in light of modern science. However, we do not have satisfactory remedy for serious liver disease: most of the herbal drugs speed up the natural healing processes of liver. So the search for effective hepatoprotective drug continues (1). Free radicals have been implicated in the causation of several diseases such as liver cirrhosis, atherosclerosis, cancer, diabetes, etc. and compounds that can scavenge free radicals disease processes. Antioxidants thus play an important role to protect the human body against damage by reactive oxygen species (2). Many unknown and lesser known plants are used in folk and tribal medical practices in India. The medicinal values of these plants are not known to the scientific world. One such plant is Trichosanthes dioica Roxb. Trichosanthes dioica Roxb. (TD) commonly known as Kadu-padvala, is used in liver affections and jaundice (3). TD extract was found to possess anti-inflammatory (4), cholesterol-lowering (5, 6), blood sugar, serum cholesterol, high density lipoprotein, phospholipids and triglyceride levels (7, 8). The various chemical constituents present in TD are vitamin A, vitamin C, tannins, saponins, and

2 trichosanthin (9, 10). Phytochemical evaluations of aqueous and ethanolic extracts have showed the presence of saponins, tannins and a nonnitrogenous bitter glycoside trichosanthin (11). Being very rich in protein and vitamin A, Trichosanthes dioica has certain medicinal properties. The fruits are easily digestible and diuretic in nature. They are also known to have antiulcerous effects. The fruits and seeds have some prospects in the control of some cancer like conditions and haemagglutinating activities (6). According to ayurveda the plant is used for bronchitis, biliousness, cancer, jaundice, liver affections (Enlargement), cough, and blood diseases. It is also used as antipyretic diuretic, cardiotonic, laxative (3). MATERIALS AND METHODS: Plant material: The plant material was purchased from local market of Pune. It was authenticated by Agharkar Research Institute, Pune, with voucher specimen no Drugs and chemicals: Silymarin was obtained from Micro Lab. Ltd., India. Paracetamol was a gift sample from Indoco Ltd., India. Experimental animal: Albino rats of Wistar Strain weighing g were used for study and were kept in animal house at 26 ± 2 C with relative humidity % along with light and dark cycles of 12 h respectively. Institutional Animal Ethics Committee approved the experimental protocol. The animals were fed ad libitum with standard pellet diet and had free access to water. Preparation of extracts: 1) Preparation of ethanolic extract: Ethanolic extract of Trichosanthes dioica Roxb. was prepared by maceration method. Powdered plant material was defatted with petroleum ether and then macerated for 72 h in 95 % ethanol with occasional shaking. It was then filtered. Filtrate was then concentrated and the solvent was evaporated under vacuum. Yield of ethanolic extract of Trichosanthes dioica Roxb. was 2.8 % w/w. 2) Preparation of aqueous extract: Aqueous extract of Trichosanthes dioica Roxb. was prepared by maceration method. Powdered plant material was macerated for 72 h with occasional shaking in distilled water. It was then filtered. The solvent was evaporated under vacuum. Yield of aqueous extract of Trichosanthes dioica Roxb. was 4.2 % w/w. Preparation of drug solutions: TD extracts, Paracetamol and Silymarin were suspended in 1% CMC solution and were used. Acute oral toxicity: Dose selection was done according to OECD guideline. Wistar albino rat, fasting, for 24 h was administered ethanolic and aqueous extracts of TD at 2000 mg/kg, p.o. The animal was observed for 24 h. the animal survived and therefore 4 more animals were dosed at 2000 mg/kg, p.o. and were observed for 24 h. All five animals survived. Therefore 2000 mg/kg dose was considered safe and 1/10 th of the dose was selected for further evaluation (12). Antioxidant assays: 1) 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH ) free radical scavenging activity: The free radical scavenging activity of Trichosanthes dioica Roxb. was estimated by the method described by Blois (13), wherein the

3 bleaching rate of the stable free radical, DPPH is monitored at a characteristic wavelength in the presence of the sample. In its radical form, DPPH absorbs at 517 nm, but upon reduction by an antioxidant or a radical species its absorbance decreases. Briefly, 0.1 mm solution of DPPH in ethanol was prepared and 1ml of this solution was added to 3 ml of Trichosanthes dioica Roxb. solution in water at different concentration ( µg/ml). Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. IC 50 value in the test compound is concentration required to scavenge 50% DPPH free radicals. The DPPH radical scavenging activity was calculated according to the following equation. DPPH radical scavenging activity (%) = [{Ao - A 1 /Ao}] 100. Where, Ao is the absorbance of the DPPH, A 1 is the absorbance of the presence of the extract in DPPH solution. 2) Hydrogen peroxide (H 2 O 2 ) scavenging assays: The hydrogen peroxide scavenging ability of Trichosanthes dioica Roxb was determined according to the method of Ruch (14). A solution of H 2 O 2 (40 mm) was prepared in phosphate buffer (ph 7.4). Different concentrations of Trichosanthes dioica Roxb. (10-50 µg/ml) in phosphate buffer was added to a H 2 O 2 solution (0.6 ml, 40 mm). The absorbance value of the reaction mixture was recorded at 230 nm. Blank solution was containing phosphate buffer without H 2 O 2. The percentage of H 2 O 2 scavenging of Trichosanthes dioica Roxb. and standard compound was calculated as H 2 O 2 radical scavenging activity (%) = [{Ao - A 1 /Ao}] 100. Where, Ao is the absorbance of the H 2 O 2, A 1 is the absorbance of the presence of the extract in H 2 O 2 solution. Paracetamol Induced Hepatotoxicity: Treatment schedule: Animals were divided in nine groups (n=5) and treated in the following way (15). Group I (Normal control): Distilled water (1 ml/kg, p.o.) Group II (Intoxicated): Paracetamol (1 g/kg, p.o.) till 7 th day. Group III (Silymarin): Paracetamol (1 g/kg, p.o.) till 7 th day and silymarin (100 mg/kg, p.o.) from day 4 to day 12. Group IV (100e): Paracetamol (1 g/kg, p.o.) till 7 th day and ethanolic extract of TD (100 mg/kg, p.o.) from day 4 to day 12. Group V (200e): Paracetamol (1 g/kg, p.o.) till 7 th day and ethanolic extract of TD (200 mg/kg, p.o.) from day 4 to day 12. Group VI (400e): Paracetamol (1 g/kg, p.o.) till 7 th day and ethanolic extract of TD (400 mg/kg, p.o.) from day 4 to day 12. Group VII (100a): Paracetamol (1 g/kg, p.o.) till 7 th day and aqueous extract of TD (100 mg/kg, p.o.) from day 4 to day 12. Group VIII (200a): Paracetamol (1 g/kg, p.o.) till 7 th day and aqueous extract of TD (200 mg/kg, p.o.) from day 4 to day 12. Group IX (400a): Paracetamol (1 g/kg, p.o.) till 7 th day and aqueous extract of TD (400 mg/kg, p.o.) from day 4 to day 12.

4 Biochemical estimation: On 4 th and 13 th day all the animals were anesthetized with anesthetic ether and blood was collected from retro-orbital plexus using fine glass capillary and collected in plain sterile centrifuge tubes and allowed to clot. Serum was separated by centrifugation at 7000 rpm for 15 min. at 5 0 C. The separated serum was used for estimation of Alanine Transaminase (ALT), Aspartate Transaminase (AST), Alkaline phosphatase (ALP), Total protein (TP) and Total Bilirubin (TB). Histopathological investigations: On 13 th day animals were sacrificed after blood withdrawal and abdomen was cut open and the liver was dissected out. The liver was processed for the histopathological investigations. Statistical analysis: The results were expressed as mean ± SEM and statistically analyzed by ANOVA followed by Dunnett test, with level of significance set at p<0.05. RESULTS: Antioxidant assay: Several concentrations ranging from µg/ml of the aqueous and ethanolic extract of Trichosanthes dioica Roxb. were tested for their antioxidant activity in different in vitro models. It was observed that free radicals were scavenged by the test extract in a concentration dependent manner in both the models. The IC 50 value for aqueous and ethanolic extract in DPPH free radical scavenging assay was found to be and µg/ml respectively. And the IC 50 value for aqueous and ethanolic extract in H 2 O 2 method was found to be 46.2 and 48.8 µg/ml respectively. Paracetamol induced hepatotoxicity: In the model of Paracetamol induced hepatotoxicity, on the 4 th day serum levels of AST, ALT, ALP, TP and TB were analyzed (Table No. 1). When the serum levels in all the groups were compared with the normal control it was observed that there was significant (p<0.01) increase in the levels of AST, ALT, ALP, TB and decrease in TP. On the 13 th day, the serum levels of AST, ALT, ALP, TB and TP in the intoxicated group were compared with the normal control and it was observed that there was significant (p<0.01) increase in the levels of AST, ALT, ALP, TB and decrease in TP as compared to the normal control (Table No.2). Silymarin treated group showed significant (p<0.01) decrease in the levels of AST, ALT, ALP, TB and increase in TP as compared to intoxicated group. 100e and 100a treated group did not show any significant change in AST, ALT, ALP, TB and TP levels as compared to intoxicated group. 200e caused reduction in AST, ALT, TB and TP (p<0.05) as compared to intoxicated group. 200a caused a significant reduction of AST, ALT (p<0.01) ALP, TB (p<0.05) levels and increase in TP levels (p<0.01). There was also reduction in the levels of as compared to intoxicated group. 400e and 400a caused a significant (p<0.01) reduction of AST, ALT, ALP, TB and increase in TP levels.

5 Table 1:Effect of Trichosanthes dioica Roxb. in Paracetamol induced hepatotoxicity on day 4 Sr. no. Serum Biochemical Parameters 1. AST (U/ml) 2. ALT (U/ml) 3. ALP (KA units/ml) 4. TB (mg/dl) 5. TP (gm/dl) Normal control ± ± ± ± ±0.34 Intoxi- cated n=5. Values are expressed as Mean ± S.E.M. ##p < 0.01, when compared with normal control Silymarin 100e Groups (n=5) 200e 400e 100a 200a 400a ±1.31 ## ±1.01 ## ±1.23 ## ±1.55 ## ±1.55 ## ±1.42 ## ±1.62 ## ±1.31 ## ±1.93 ## ±1.89 ## ±1.85 ## ±1.39 ## ±1.17 ## ±0.89 ## ±0.84 ## ±1.22 ## ±0.89 ## ±0.71 ## ±0.62 ## ±1.03 ## ±0.91 ## ±0.43 ## ±0.58 ## ±0.37 ## ±0.13 ## ±0.10 ## ±0.12 ## ±0.08 ## ±0.09 ## ±0.15 ## ±0.11 ## ±0.14 ## ±0.29 ## ±0.44 ## ±0.22 ## ±0.43 ## ±0.53 ## ±0.51 ## ±0.43 ## ±0.65 ## Table 2: Effect of Trichosanthes dioica Roxb. in Paracetamol induced hepatotoxicity on day 13. Sr. no Serum Biochemical Parameters ALT (U/ml) AST (U/ml) ALP (KA units/dl) TB (mg/dl) 5. TP (gm/dl) n=5. Values are expressed as Mean ± S.E.M. ns: non-significant Normal control ± ± ± ± ±0.24 Intoxicated * p < 0.05, ** p < 0.01 when compared with intoxicated group ## p < 0.01, when compared with normal control Silymarin 100e Groups (n=5) 200e 400e 100a 200a 400a ±1.16 ## ±1.72 ** ±1.13 ns ±1.09 ** ±0.93 ** ±1.08 ns ±1.20 ** ±1.27 ** ±1.58 ## ±1.14 ** ±1.69 ns ±1.28 * ±1.22 ** ±1.06 ns ±1.43 ** ±1.08 ** ±0.41 ## ±0.68 ** ±0.58 ns ±0.65 ns ±0.45 ** ±0.47 ns ±0.55 * ±0.54 ** ±0.15 ## ±0.06 ** ±0.12 ns ±0.10 ns ±0.06 ** ±0.12 ns ±0.08 * ±0.04 ** ±0.13 ## ±0.23 ** ±0.07 ns ±0.15 ** ±0.30 ** ±0.14 ns ±0.20 ** ±0.12 **

6 Table 3: Free radical scavenging activity of Trichosanthes dioica Roxb. by DPPH radical inhibition. Concentration (µg/ml) TD Aqueous extract TD Ethanolic extract Ascorbic acid % IC50 % IC50 % IC50 Inhibition (µg/ml ) Inhibition (µg/ml) Inhibition (µg/ml ) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.41 TD: Trichosanthes dioica Roxb. Values are expressed in triplicate as Mean ± SEM Table 4: Hydrogen peroxide scavenging activity of Trichosanthes dioica Roxb. Concentration (µg/ml) % Inhibition TD: Trichosanthes dioica Roxb. Values are expressed in triplicate as Mean ± SEM. Histopathological examination of the rat liver in paracetamol induced hepatotoxicity: TD Aqueous extract TD Ethanolic extract Ascorbic acid IC 50 (µg/ml ) In the histopathological examination the normal control showed normal hepatic cells and normal hepatocytes (Fig.1) where as the intoxicated showed hydrophobic lesions with congestion and signs of necrosis (Fig.2). Treatment with silymarin showed normal histological appearance with no signs of necrosis (Fig.3). 100e treated showed hydrophobic lesions with congestion and signs of necrosis (Fig.4). % Inhibition 200e treated showed slight congestion and no evidence of necrosis. 400e treated group showed normal hepatocytes with regenerative changes. 100a showed focal areas of necrosis and the architecture was partly distorted. 200a treated group showed occasional swelling and cloudy appearance. 400a treated group showed regenerating hepatocytes and there was no evidence of necrosis. IC 50 (µg/ml) % Inhibition ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.49 IC 50 (µg/ml ) 29.5

7 Figure 1: Photomicrograph of rat liver of normal control showing normal hepatic cells. Figure 2: Photomicrograph of rat liver intoxicated showing hydrophobic lesions and signs of necrosis. Figure 3: Photomicrograph of rat liver silymarin group showing normal histological appearance with no evidence of necrosis.

8 Figure 4: Photomicrograph of rat liver 100e group showing hydrophobic lesion with congestion and signs of necrosis Figure 5: Photomicrograph of rat liver 200e group showing slight congestion and no evidence of necrosis. Figure 6: Photomicrograph of rat liver 400e group showing normal hepatocytes with regenerative changes.

9 Figure 7: Photomicrograph of rat liver 100a showing focal areas of necrosis. Architecture is partly distorted. Figure 8: Photomicrograph of rat liver of 200a showing regenerative changes with occasional swelling and cloudy appearance. Figure 9: Photomicrograph of rat liver of 400a showing regenerating hepatocytes with occasional mitotic activity. There is no evidence of necrosis

10 DISCUSSION: Paracetamol is mainly metabolized by glucoronide and sulphate conjugation. A small amount of paracetamol is metabolized by cytochrome p-450 to a highly reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and is initially detoxified by conjugation with reduced glutathione (GSH) to form mercapturic acid (15). The cells are normally protected from injury by conjugation of this toxic metabolite with glutathione. As the dose of paracetamol increases, the glutathione content of hepatocytes gets exhausted and the hepatocytes become vulnerable to the noxious effects of the metabolite resulting in liver cell necrosis (16). GSH is one of the most abundant tripeptide present in the liver and its functions are removal of free radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals, and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase and glutathione-s-transferase. This GSH protects hepatocytes by combining with reactive metabolite of paracetamol thus preventing their covalent binding to liver proteins (17). NAPQI binds covalently to tissue macromolecules which lead to mitochondrial dysfunction followed by acute hepatic necrosis through lipid peroxidation induced by decreasing GSH in the liver. Damage to the structural integrity of liver is reflected by an increase in the levels of serum transaminases AST, ALT and ALP, because they are cytoplasmic in location and are released into the circulation after cellular damage. Elevation of serum levels of these enzymes is considered as an index of liver damage (18). In the present study, it was observed that the animals treated with paracetamol for 7 days resulted in increase in the levels of serum marker enzymes like AST, ALT and ALP reflecting the hepatic injury. AST and ALT play a vital role in the conversion of amino acids to keto acids. ALT catalyses the conversion of alanine to pyruvate and glutamate. Therefore ALT is more specific to liver and is thus better parameter for detecting liver damage (15). Histopathology of liver samples revealed that the necrosis was reduced to few inflammatory cells. Thus the histopathological studies also support the hepatoprotective activity of Trichosanthes dioica Roxb. Based on results of present study it can be concluded that Trichosanthes dioica Roxb. suppress the paracetamol induced cell damage. Trichosanthes dioica Roxb. is rich in protein and vitamin A (6). It also contains vitamin C, carotene, tannins and saponins which possess antioxidant activity (19, 20). Beta carotene and other carotenes have antioxidant properties in vitro and in vivo models (21). Ascorbic acid may have counteracted the free radicals through effective scavenging and blocking the conjugation of reactive metabolite to GSH (22). The levels of vitamin C and E were significantly depleted in paracetamol intoxication which may be due to excessive utilization of quenching the enormous free radicals produced during paracetamol intoxication (23). The antioxidant potential of TD was assessed by DPPH assay and H 2 O 2 method. The DPPH assay is based on the measurement of scavenging ability of antioxidants towards the stable radical DPPH. Antioxidants reduce the radicals to the corresponding hydrazine when it

11 reacts with the hydrogen donor in the antioxidant principles. DPPH radicals react with suitable reducing agent, the electrons become paired off and the solution losses colour stoichiometrically depending on the number of electrons taken up (24). Hydrogen peroxide concentration is decreased by scavenger compounds and therefore absorbance value also decreases. Antioxidant shows good scavenging activity against H 2 O 2 and DPPH free radicals. TD extracts show reduction in absorbance value in DPPH assay and H 2 O 2 method, which might be due to radical scavenging activity of vitamin C and carotene (21, 26). Aqueous and ethanolic extracts of Trichosanthes dioica Roxb. effectively reduced the AST, ALT, ALP and TB levels and increased the TP levels in the paracetamol induced hepatotoxicity. The histopathological studies also substantiate the hepatoprotective activity of the Trichosanthes dioica Roxb. Hence it can be concluded that the possible mechanism of hepatoprotective activity of Trichosanthes dioica Roxb. may be due to its free radical scavenging activity and its ability to reduce elevated levels of serum marker enzymes which may be due to presence of vitamin C, carotene, saponins and tannins. REFERENCES: 1. Vishnu J. (2001) Herbal preparation as a source of hepatoprotective agents. Drugs New Prospect 14 (6), Sabu MC, Kuttan R. (2002) Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J. Ethnopharmacol 81, Kirtikar KR, Basu BD. (1996) Indian Medicinal Plants, pp , Jayyed Press, Allahabad. 4. Fulzule SV, Satturwar PM, Joshi SB. (2001) Studies on anti-inflammatory activity of a poly herbal formulation - Jatydi Ghrita. Indian drugs 39(1), Sharma G, Pant MC. (1988) Effect of raw deseeded fruit powder of Trichosanthes dioica (Roxb) on blood sugar, serum cholesterol, high density lipoprotein, phospholipids and triglyceride levels in the normal albino rabbits. Ind. J. Physiol. Pharmacol. 32(2), Sharmila BG, Kumar G, Rajasekhar PM. (2007) Cholesterol-lowering activity of the aqueous fruit extract of Trichosanthes dioica Roxb. in normal and streptozotocin diabetic rats. J. of clinical and diagnostic res. 1(4): Sharma, G, Pant MC. (1988) Effects of feeding Trichosanthes dioica (parval) on blood glucose, serum triglyceride, phospholipids, cholesterol, and high density lipoprotein-cholesterol levels in the normal albino rabbit. Current Science 57, Chandrasekhar B, Mukherjee B, Mukherjee SK. (1988) Blood sugar lowering effect of Trichosanthes dioica Roxb. in experimental rat models. Int. J. Crude Drug Res. 26, Raw materials. (2003). The Wealth of India, pp Chopra RN, Nayar SL, Chopra IC. (1956) Glossary of Indian Medicinal plants, pp. 256, CSIR, New Delhi. 11. Khandelwal KR. (2005) Practical Pharmacognosy, pp Nirali Prakashan, Pune. 12. OECD guidelines 425, Blois MS. (1958) Antioxidant determination by the use of a stable free radical. Nature; 26: Ruch RJ, Cheng SJ, Klaunig JE. (1989) Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10, Pimple BP, Kadam PV, Badgujar NS, Bafna AR, Patil MJ. (2007) Protective effect of Tamarindus indica linn. against paracetamol-induced hepatotoxicity in rats. Ind. J. of pharm. Sci Rao GM, Rao G, Ramnarayan K, Shrinivasan KK. (1992) Effect of hepatoguard on paracetamol induced liver injury in male albino rats. Indian drugs 30(1),

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