Protective effect of ethanolic extract of Actinoscirpus grossus tubers against ethanol induced liver toxicity in albino rats

1 Introduction

Majority of xenobiotics gets metabolised in liver and thus liver plays a important role in the maintenance of homeostasis. Despite of its great capacity to handle toxicants and high regenerative power, liver exhibit symptoms after extensive damage. Recently the liver disease related complications are increasing to a greater extent due to overwhelming use of drugs and alcohol. The acute hepatitis with or without cirrhosis, liver steatosis, cirrhosis are results of chronic alcoholism and can also leads to hepatocellular carcinoma (Liu, 2014). Clinical alcoholic patients may remain asymptomatic for years and when the symptoms developed the lesions may be quite advanced. There exist only few universal treatment options for common liver diseases such as fatty liver, cirrhosis and chronic hepatitis (Mello et al., 2008; Moghe et al., 2011).

Herbal treatments have been used to alleviate the liver disorders from centuries and are well documented in traditional system of medicines and it becomes a favourable therapy for most of the liver dysfunction worldwide (Neetu et al., 2011). There are number of plants and polyhedral formulations claimed to have hepatoprotective activities, however only a handful of drugs has been studied thoroughly for their therapeutic uses (Sabayan et al., 2007). Thus increasing use of herbal medicines shows their perceived effectiveness and prevention of liver diseases and believed to be safe because of its natural origin (Lee et al., 2004).

Actinoscirpus grossus (A. grossus, family: Cyperaceae) is a perennial plant with long rhizomes ending in small tubers and popularly known as ‘Kasheruka’ in Ayurveda. It is distributed throughout India and neighbouring Asian countries (Sharma and Sharma, 2006; Nisteshwar, 2007). In Ayurveda the decoction prepared from A. grossus tubers has been indicated for liver, digestive disorders and as anti-emetic agent (Kirtikar et al., 2007). It has been reported that the ethanolic extract of tubers possess biphasic hypotensive effect in anesthetized cats (Sharma and Sharma, 2006). The ethanolic extract of tubers of A. grossus (EAGT) has revealed the presence of flavonoids, carbohydrate, coumarins, steroids, tannins, and terpenoids (Savin et al., 2017). The acute oral toxicity test indicated that LD 50 of EAGT exceeded 2000 mg/kg in rats (Savin et al., 2018). Despite of its folklore claim as a liver tonic there is no experimental data available for its hepatoprotective activity. Thus the present study was undertaken to evaluate the hepatoprotective potentials of EAGT in ethanol induced liver toxicity in albino rats.

2 Materials and methods

3 Results

3.6

3.6 Effect of EAGT on antioxidant parameters

The antioxidant activity of EAGT was evaluated in the liver homogenate of rats administered with repeated dose of ethanol. The activity of endogenous antioxidant enzymes such as catalase and glutathione peroxidase in ethanol intoxicated group significantly reduced as compared to normal control. The lipid peroxidation was significantly increased after ethanol intoxication (p < 0.01). Treatment with EAGT at both dose level significantly restored the catalase and glutathione peroxidase to normal level (p < 0.01) and significantly attenuated ethanol induced elevated lipid peroxidation (p < 0.05) (Fig. 1).

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Fig. 1

Graphical representation of the effect of EAGT on antioxidant parameters.

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3.7

3.7 Effect of EAGT on liver histopathological examination

Repeated administration of ethanol caused extensive changes in the liver histology (Fig. 2A & B). The changes observed were micro and macro fatty changes, hepatocyte necrosis, cell infiltration, appearance of balloon cells and sinusoidal dilatation. Liver sections from reference standard drugs especially the Silymarin exhibited good protection against ethanol induced liver toxicity. We could observe mild to moderate fatty infiltration and regenerative changes and almost normal cytoarchitecture (Fig. 2C &D). The test drug EAGT administered group rats exhibited good protection against ethanol toxicity. We could observe only mild to moderate fatty changes without perisinusoidal fibrosis, Lobular infiltration and necrosis. Test drug EAGT exhibited almost normal cytoarchitecture in sections in both dose level (Fig. 2E & F).

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Fig. 2

Photomicrographs of representative liver sections. Normal control shows normal Cytoarchitecture of liver (A). Liver sections from ethanol administered group shows extensive fatty changes with necrosis and appearance of balloon cells (B). Liver sections from ethanol and Liv 52 administered rats (C) and ethanol + silymarin administered rats (D). Photomicrographs of representative liver sections from ethanol + EAGT (200& 400 mg/kg) rats shows mild fatty changes and mild cell infiltration with normal cytoarchitecture (E and F). HC– Hepatic cell; KC- Kupffer cell; Sn- Sinusoid; PT- Portal triad; CI- Cell infiltration; Fc- Fatty changes; CV- Central vein.

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4 Discussion

Liver is the primary site of ethanol metabolism and hence more prone to ethanol intoxication. Thus, repeated and or acute high dose of ethanol has high chances of developing alcoholic liver diseases (Baraona et al., 1977; Hasumura et al., 1975). Ethanol undergoes oxidation and produce highly toxic substances like acetaldehyde by alcohol dehydrogenase. Along with alcohol dehydrogenase the enzymes like Cytochrome P450 isoenzyme CYP2E1 and catalase also involved in the metabolism. In the ethanol metabolism molecular oxygen (O₂) is recipient of the electrons from ethanol via microsomal enzyme system. At last the catalase reduces hydrogen peroxide into water and produce acetaldehyde from ethanol. Acetaldehyde dehydrogenate quickly oxidises acetaldehyde into less toxic acetate. Thus, rapid removal of ethanol by the liver is clearly offering significant protection from its toxic metabolites (Baraona et al., 1977). During the oxidative process there will be release of reactive oxygen species, which inter causes severe oxidative stress followed by release of inflammatory cytokines, which intern causes inflammatory process and leads to liver cirrhosis (Gallagher, 1962).

Normally hepatocytes have both enzymatic and non-enzymatic defensive antioxidant molecules to combat the reactive oxygen species. Among non-enzymatic antioxidant molecules glutathione, thioredoxine and thiol containing molecules are well recognized antioxidant molecules. The endogenous enzymes like superoxide dismutase, catalase and glutathione peroxidase are enzymatic antioxidants. Superoxide dismutase (SOD) enzyme is the first enzyme which defends the reactive oxygen species; superoxide’s and converts superoxide anions to hydrogen peroxide and oxygen. Thus generated hydrogen peroxide is neutralized by catalase (CAT) and glutathione peroxidase (GSH-Px) enzymes. There by it cause protective action in hepatocytes against free hydroxyl radicals (Comporti et al., 2010). The catalase and glutathione peroxidase activity were measured to evaluate the antioxidant effect of EAGT in ethanol induced oxidative stress in liver tissue. Repeated administration of ethanol caused significant decrease in the CAT and GSH-Px as compared to the control rats. EAGT at 200 mg/kg and 400 mg/kg significantly attenuated the ethanol induced decrease in the CAT and GSH-Px level in a dose dependant manner. Depletion of defensive antioxidant molecules in liver cells results in mitochondrial dysfunctions and increased lipid peroxidation, which leads to the liver necrosis. The level of MDA in the ethanol group were increased significantly (p < 0.01). However the MDA level was reduced significantly with the treatment of EAGT. Thus the degenerative hepatic cells releases enzymes such as SGOT and SGPT into the circulation and it can be measured as a marker for liver abnormalities. The measurement of SGPT is considered as essential marker for mitochondrial distortion in the centrilobular zone in the liver tissues.

Ethanol 5 g/kg 20%v/v dose administration for 30 consecutive days caused decrease in the body weight of rats, however the treatment with EAGT mild to moderate increase in the body weight has been observed as compared to ethanol intoxicated group rats. Ethanol intoxication significantly increased serum SGOT, SGPT, ALP, total bilirubin, HDL, total protein and albumin as compared to the normal control. The histopathological results supports the evidence of reduced number of viable cells with massive necrotic cells around the centrilobular zone extending to the parenchymal zone, which is characterized by increased glycogen depletion, condensation of nuclei, pyknotic, karyolitic, lymphatic infiltrations and endothelial disruptions.

Interestingly, treatment with EAGT showed capability to increase the body weight and simultaneously caused significant reduction in the liver enzymes such as SGOT, SGPT and ALP levels in blood dose dependently. It is further supported by significant attenuation of ethanol intoxicated decrease in the CAT and GSH-Px antioxidant enzymes and decreased lipid peroxidation level in the liver homogenate. The biochemical parameters such as total bilirubin, cholesterol, total protein and albumin were significantly attenuated as compared to the ethanol intoxicated rats. Further the histopathological investigation shows the normal cytoarchitecture and presence of normal healthy hepatocytes. This liver regenerative process was almost comparable with that of reference standards like Silymarin and Liv 52 treated groups. Whereas increased viable hepatocytes, decreased hepatic enzymes and increased endogenous anti-oxidant enzymes such as CAT and GSH-Px and decreased lipid peroxidation could be observed.

Ethanol induced hepatotoxicity is mainly attributed to the ethanol active metabolite acetaldehyde induced free radical generation subsequent oxidative stress and inflammatory process in liver tissues. Thus, it is clear that the extract possesses free radical scavenging activity and prevented the process of initiation and progression of hepatocellular damage. Interestingly our present findings did demonstrate the EAGT ability of scavenging free radicals and exerting antioxidant activity (Pari and Prasath, 2008).

The plant derived phytochemicals such as flavonoids, polyphenols, unsaturated fatty acids, glycosides, tannins, saponins and alkaloids having wide range of biological activities and found useful in the treatment of liver disorders (Mundugaru et al., 2014; Xu et al., 2007). It is a valuable source for future research in the therapeutics. Some of the potent flavonoids like Silymarin, which is derived from seeds of the milk thistle, Silybum marianum is used in the treatment of hepatitis, liver cirrhosis and toxins induced liver dysfunctions (Sy-Cordero et al., 2010). Many chemicals like hydroxy citric acid, curcumin, garcinol and ginkjo alkaloids were derived from plant sources were found to be effective in liver diseases (Abdel-Wahab, 2013; Liu et al., 2013; Yang et al., 2011). The phytochemical analysis demonstrated that EAGT are rich in flavonoids, tannins, terpenoids and steroids. Flavonoids are important phenolic contents of the plants and serves as secondary metabolites having potent antioxidant and anti-inflammatory activities. Thus we could infer that the presence of flavonoids, tannins, terpenoids and steroids might have possessed free radical, anti-oxidant and anti-inflammatory and hepatoprotective activities.

5 Conclusion

Herbs are the natural products considered to be the rich source of structurally diverse molecules and exhibit multiple therapeutic functions. It is very important in the treatment of chronic degenerative disorders like liver diseases. We have reported for the first time that the ethanolic extract of tubers of Actinoscirpus grossus showed protective effects in ethanol induced hepatotoxicity. Thus, the plant tuber can be used as potent source of phytoconstituents from which we could further develop efficient hepatoprotective agents.