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Pharmacologia
Year: 2013  |  Volume: 4  |  Issue: 2  |  Page No.: 89 - 94

Ameliorative Potential of Aqueous Root Extract of Withania somnifera Against Paracetamol Induced Liver Damage in Mice

Tabarak Malik, Devendra Kumar Pandey and Nitu Dogra    

Abstract: Paracetamol (also known as Acetaminophen) is a cheap but effective analgesic with few side-effects. However, outside of the therapeutic window, the liver’s ability to process the toxic metabolites of paracetamol is exceeded and hepatotoxicity may result in the process. The present study was undertaken exhaustively to investigate hepatoprotective activity of Withania somnifera root extract against paracetamol induced hepatotoxicity, in mice. Activities of liver markers enzymes Glutamate-oxaloacetate Transaminase (AST), Glutamate-pyruvate Transaminase (ALT), Alkaline Phosphatase (ALP), Bilirubin and total protein were estimated in serum, whereas, lipid peroxidation and antioxidant status (Superoxide dismutase, Catalase, Glutathione Peroxidase and Reduced glutathione content) were estimated in liver homogenate. Paracetamol (500 mg kg-1 b.wt.) induced hepatotoxicity was established by a significant increase in serum AST, ALT, ALP activity and bilirubin level accompanied by significant decrease in total serum protein level. Paracetamol hepatotoxicity was evident by an increase in lipid peroxidation, reduction of reduced glutathione content, Catalase, Glutathione reductase and Glutathione peroxidase activity in liver. Treatment of Withania somnifera root extract (500 mg kg-1 b.wt.) notably decreased AST, ALT, ALP, Bilirubin and Total Protein content in PCM compared to control. In addition Withania somnifera root extract significantly reduced hepatic lipid peroxidation, enhanced antioxidant enzyme activities and GSH levels in the liver of mice.

1,2, that can protect from oxidative stress, thus playing an important role in chemoprevention of diseases. Withania somnifera (L.) Dunal, also known as Ashwagandha, Indian ginseng, Winter cherry, Ajagandha Kanaje in Hindi and Amukkara in Tamil is a plant in the Solanaceae or nightshade family3. Withania somnifera has been used for centuries in Ayurvedic medicine. Current research supports its polypharmaceutical use, confirming antioxidant, anti-inflammatory, immune-modulating and antistress properties in the whole plant extract and several separate constituents4. Preliminary studies have found various constituents of ashwagandha exhibit a variety of therapeutic effects with little or no associated toxicity. Withania somnifera root has been considered to be the most active for therapeutic purposes. Root contains maximun amount of Alkaloides viz., Nicotine, Sominine, Somniferin, Somniferinine, Withanine, Withanonine, Pseudo withanine, Tropin, Withanoloides etc.5. Antihepatotoxic effect of Withania somnifera extracts on carbon tetrachloride-induced hepatotoxicity has been established6,7.

However, despite the observation of diverse medicinal activities attributed to this plant, no biochemical studies have been carried out to explore the role of W. somnifera on paracetamol induced hepatotoxicity in mice. An attempt has been made in the present study to elucidate the effects of oral feeding of aqueous root extract of Withania somnifera on paracetamol (PCM) induced liver damage in mice.

MATERIALS AND METHODS

Plant material, extraction and HPTLC quantification of Withania somnifera: The whole plant material of Withania somnifera was collected from Lovely Professional University campus, Punjab, India. The authenticity of the species was confirmed by a taxonomist. After collection the plant material was carefully washed and separated into root, stem and leaf and dried at 50°C.

Processing and extraction of withanolides (withaferine and withanolide A): The root part of the Withania somnifera was shade-dried and powdered in a grinder. To extract authentic withaferine A and withanolide A, the powdered root material (1.0 g) of Withania somnifera and 20 mL distilled water were added to a 50 mL conical flask. The contents were rapidly heated to reflux for one hour. After cooling, the aqueous extract was transferred to 50 mL conical flask. Aqueous extract was filtered and evaporated to dryness with the help of rotsavapour.

HPTLC quantification of withanolides: The content of withaferine A and withanolide A were determined by the modified method developed by Srivastava8. Standard Withanolide A and Withaferine A and the root samples were spotted on precoated silica gel 60 F254 TLC plates (10x10 cm; with 0.25 mm layer thickness: Merck KGaA; 1.05554. 0007) as bands 6 mm wide, with 10 mm distance between tracks, by means of Linomat-5 automatic sample applicator (with nitrogen flow) equipped with a 100 μL Hamilton syringe. HPTLC of W. somnifera aqueous root extracts was performed by using solvent system with dichloromethane-methanol-acetone-hexane (7.5:0.5:0.5:0.5 v/v) as a mobile phase. Upon separation, quantitative evaluation of these withanolides was performed in the absorption reflection mode at 235 nm. The method was validated for precision, reproducibility and accuracy. On the basis of RF values of 0.58 and 0.68 for withaferine A and withanolide A, respectively, were identified. On the basis of linear calibration curves for all withanolides in the range of 0.2-1.2 μg, an average recovery of withaferine A and withanolide A was 98 and 98%, respectively. The method is very simple, precise, specific, sensitive, accurate and economical for rapidly validating the aqueous root extract of W. somnifera (Ashwagandha).

Experimental animals: Female Swiss Albino mice weighing approximately 25±5 g were procured from the animal house of NIPER, Mohali, Punjab, India. The study was approved from Institutional Animal Ethical Committee (IAEC) (Registration no 954/ac/06). They were housed in air-conditioned room with temperature maintained at 25±3°C and fed with commercial pelleted feed from Hindustan Lever Ltd. and water was made freely available.

Experimental protocol: Animals were divided into three groups of six animals each.

Group I: Control: The animals were given 1.00 mL of saline (0.9% NaCl) through oral gavages once in a day for 6 days/week for 28 days
Group II: PCM: The animals were intoxicated with PCM (500 mg kg-1 b.wt.) through oral gavages once in a day for 6 days/week for 28 days
Group III: PCM and root extract of Withania somnifera: This group were given PCM (as in Group II) and after 60 min of the dose, animals were given root extract (500 mg kg-1 b.wt.) for 6 days/week for 28 days

Collection of serum and vital organs: After 28 days, the animals were weighed and sacrificed by cervical dislocation under light anesthesia. Blood samples were collected from each animal in clear sterile centrifuge tube without anticoagulant for obtaining serum for determination of ALT, AST, ALP, Bilirubin and serum total protein. The tubes were left in a big beaker to facilitate coagulation at room temperature. After 1 h the tubes were centrifuged at 2500 rpm for 10 min to pellet the coagulant mass. Supernatant which is serum, was siphoned off carefully in micro centrifuge tubes with the help of pipettes and was stored at 4°C until assayed. Liver was quickly excised from the mice and whole liver was washed with normal cold saline (pH 7.4). The washed liver was then kept in ice cold conditions. The liver was removed and cleansed from blood and extraneous tissue. Part of the liver was fixed with 10% buffered formal saline for 1 week and further processed for histopathological studies. Tissues were weighed for 10% homogenate and individually chopped into pieces with the help of forceps and homogenized using homogenizer in ice cold 0.1 M Phosphate buffer (pH 7.4) containing 0.15 M KCl for the estimation of different enzymes and biomolecules. Aliquot of homogenate was centrifuged at 9000 rpm to obtain supernatant (S9 fraction) for the estimation of various enzymes.

Biochemical assays: The activities of alanine and aspartate aminotransferases in serum were estimated by Reitman9 and the activity of alkaline phosphtase was measured according to the method of Bower10. Bilirubin level in serum was measured according to Jendrassik11. Lipid peroxidation was measured by the method of Utley12. Malondialdehyde, formed as an end product served as an index of oxidative stress. Catalase activity was measured according to the method of Sinha13. 0.1 mL of liver homogenate was mixed with 1.0 mL of 0.01 M phosphate buffer (pH 7.4) where one unit represents 1.0 μmole of H2O2 consumed/min/mg protein. Hepatic reduced glutathione level was determined by the method of Ellman14. The GSH contents were calculated using GSH as standard and expressed as mg g-1 tissue. Glutathione peroxidase (GPx) was assayed by the method of Rotruck15. Based on the reaction between glutathione remaining after the action of GPx and 5, 5′-dithiobis-(2-nitrobenzoic acid) to form a complex that absorbs maximally at 420 nm. Glutathione Reductase (GR) that utilizes NADPH to convert oxidized glutathione (GSSG) to the reduced form was assayed by the method of Tietze16. Protein content was determined by the method of Lowry17, using bovine serum albumin as standard.

Statistical analysis: Resulting data were represented as Mean±SD Statistical data was analyzed by student’s t-test. The p<0.05 was considered statistically significant.

RESULTS

Administration of paracetamol to mice by oral route caused liver damage as indicated by a significant increase in serum enzyme ALT, AST, ALP activity and bilirubin level compared to control mice (Table 1). Elevated levels of these enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver. Co-administration of mice with aqueous root extract of Withania somnifera remarkably (p<0.001) restored paracetamol induced elevated serum levels of ALT, ALP, AST and bilirubin towards normal value, respectively. The obtained results indicated a high degree of protection against the hepatotoxic effect of paracetamol. The serum total protein level was significantly (p<0.001) declined in paracetamol treated group. Treatment with the aqueous root extract of Withania somnifera could significantly (p<0.001) restored serum total protein level towards their control value.

The levels of TBARS as an index of lipid peroxidation, a degradative process of membranous lipids, in liver tissue of paracetamol treated mice were significantly (p<0.001) elevated when compared to control animals (Table 2). Lipid peroxidation level was restored towards their normal value by treatment with Withania somnifera root extract on paracetamol induced toxicity. Reduced glutathione level was depleted significantly (p<0.001) in the liver tissue of paracetamol treated mice compared to control group. Treatment with the aqueous root extract of W.somnifera had significantly improved level of GSH in liver tissue. Likewise, the decreased activities of catalase, glutathione reductase glutathione peroxidase and SOD (data not shown) as a result of the treatment with paracetamol were also restored by the aqueous root extract of Withania somnifera (p<0.001).

The results obtained from histopathological studies were in agreement with the biochemical studies. Exposure of paracetamol produced extensive vascular degenerative changes and centrolobular necrosis in hepatocytes (Fig. 1, 2). Treatment with PCM and Withania somnifera root extract shows normal architecture with mild hepatocyte degeneration comparable to control (Fig. 3). All these results indicate a hepatoprotective potential of the extract against paracetamol induced liver damage through its antioxidant potential, however, further studies are required to elucidate the molecular mechanisms involved in order to support the clinical use of the plant extract.



Fig. 1:
Liver from control mice showing normal hepatic architecture around the central vein (x400, H&E)

Fig. 2:
Liver from PCM exposed mice showing early degenerated changes in centrolobular areas and slight progression towards mid zonal area (x400, H&E)

Fig. 3:
Photomicrograph of liver from PCM and Withania somnifera coexposed mice showing normal architecture with mild hepatocyte degeneration comparable to control (x400, H&E)

DISCUSSION

The liver damage is largely assessed by rise of serum enzyme levels such as ALT and AST . The observed rise in the serum levels of alanine transaminase, aspartate transaminase and alkaline phosphatase in paracetamol treated mice has been ascribed to the damaged structural integrity of the liver18. The administration of root extract of Withania somnifera has diminished the increased serum marker enzymes AST, ALT, ALP and bilirubin level. Reduction of serum albumin in paracetamol treated mice may be due to formation of protein adduct. Hydrogen peroxide a harmful by-product of many normal metabolic processes is quickly converted in to H2O and O2 by Chelikani19.

The reduction in the activity of this enzyme may result in number of harmful effect due to accumulation of highly toxic metabolites and hydrogen peroxide in paracetamol administered mice. In addition, a metabolite of paracetamol NAPQI can increase the formation of reactive oxygen and nitrogen species such as superoxide anion, hydrogen radical, hydrogen peroxide, nitric oxide and peroxynitrite, respectively. Excess levels of reactive oxygen and nitrogen species can attack biological molecules which can lead to lipid peroxidation, nitration of tyrosine and depletion of the antioxidant enzymes resulting in oxidative stress Hinson20. The present study also demonstrates that paracetamol overdose resulted in a decrease in the SOD and CAT activities, when compared with control group. It could be due to inactivation of the antioxidant enzymes. Malondialdehyde (MDA), one of the end products in the lipid peroxidation process21, increases with oxidative stress. The increase in MDA level observed in liver suggests enhanced lipid peroxidation and tissue damage. A massive decrease in lipid peroxidation in liver tissue of root extract treated groups indicate that Withania somnifera possess antioxidative properties. Glutathione (GSH) is known to be an endogenous antioxidant, preventing damage to important cellular components caused by reactive oxygen species22. The principal toxic metabolite of paracetamol NAPQI23 is initially detoxified by conjugation with reduced glutathione (GSH) to form mercapturic acid24. However, when the rate of NAPQI formation exceeds the rate of detoxification by GSH (reduced glutathione), it oxidize tissue macromolecules such as lipid or -SH group of proteins. In this present study, the decreased level of glutathione has been observed in PCM treated mice, whereas, its level was significantly increased in Withania somnifera root extract treated PCM induced mice. Catalase, glutathione peroxidase and glutathione reductase are thought to be the fundamental antioxidant enzymes, for the direct elimination of reactive oxygen species. SOD is known to convert O2- to H2O2 and the resulting H2O2 is removed by Catalase and glutathione peroxidase25. Glutathione reductase reduces glutathione disulfide (GSSG) to the reduced form of glutathione (GSH) which is an important cellular antioxidant26. The activity of glutathione reductase is used as indicator for oxidative stress. In the present study, Withania somnifera root extract treatment was observed to exhibit hepatoprotective effect as demonstrated by enhanced activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) and significant increase in reduced glutathione content and decreased lipid peroxidation.

CONCLUSION

This study signifies that Withania somnifera root extract shows hepatoprotective effect through its antioxidant potential on Paracetamol induced liver damage in mice. However, further studies are required to elucidate the molecular mechanisms involved in order to support the clinical use of the plant extract.

ACKNOWLEDGMENTS

Authors are thankful to the Department of Medicinal Chemistry, Institute of Medical Sciences (IMS), BHU, Varanasi for their kind support for HPTLC analysis of bioactive compounds. Acknowlegdement is due to Dr. Kewal Lal, Inhalation Toxicology Division, IITR Lucknow for his help in histopathological studies.

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