CHAPTER VII EFFECT OF H. ZEYLANICA, R. NASUTA AND S. CILIATA ON PARACETAMOL - INDUCED HEPATOTOXICITY IN WISTAR RATS
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1 CHAPTER VII EFFECT OF H. ZEYLANICA, R. NASUTA AND S. CILIATA ON PARACETAMOL - INDUCED HEPATOTOXICITY IN WISTAR RATS
2 7.1. Introduction Paracetamol is a remarkably safe drug at therapeutic doses, but it is also the drug most commonly consumed by the patients in gross therapeutic overdosage, which may be responsible for the development of acute liver failure (Grady, 1997 b). Considerable interest has been shown in the study of toxicity of paracetamol because of its clinical use as an antipyretic as well as an analgesic.in large doses, paracetamol is known to produce hepatotoxicity both in experimental animals and also in human beings (Davidson, 1960).lndiscriminate ingestion can lead to accidental poisoning and potentially lethal hepatotoxicity. Its mode of action in liver is by covalent binding of its toxic metabolite, n - acetyl - p- benzoquinone - amine to tissue macromolecules resulting in cell necrosis (Mitchell et al., 1973).Protection against paracetamol - induced toxicity has been used as a test for a potential hepatoprotective agent by several investigators. (Dwivedi et al., 1991; Shukla et al., 1991). Damage to the structural integrity of liver is reflected by an increase in the levels of serum transaminases. (Schimidt et al., 1975) because these are cytoplasmic in location and are released into circulation after cellular damage (Sallie et al., 1991). It has been reported that paracetamol toxicity is the consequence of its biotransformation by the microsomal drug metabolizing enzymes and is associated with depletion of GSH and covalent binding to protein and other cellular macromoleculs (Mitchell et al., 1973). The involvement of a radical mechanism and generation of superoxide have been suggested. In this chapter, the protective effect of the three plant extracts (HZ, RN, SC) on paracetamol- induced hepatotoxicity in Wistar rats is presented Materials and Methods Paracetamol, Silymarin and biochemical kits were obtained from various sources as mentioned in The plant extracts, H. zeylanica (HZ), R. nasuta (RN), S. ciliata (SC) were obtained as stated in Wistar albino rats (males) 12 weeks old weighing g were used. Groups of six rats were used for each set of experiments, control and experimental Paracetamol induced hepatotoxicity studies The procedure of paracetamol induced liver toxicity was stated in
3 7.2.2 Biochemical estimations Biochemical parameters like serum enzymes, serum aspartate aminotransferase (SGOT), serum alanine aminotransferase (SGPT) serum alkaline phosphatase (SAKP) and serum bilirubin (SB) were assayed according to standard methods (Reitman and Frankel, 1957; King and Armstrong 1980; Malloy and Evelyn 1937) Histopathological studies Histopathological Studies were conducted as mentioned in Results Administration of paracetamol caused significant increase in serum enzyme levels namely SGOT, SGPT, SAKP and SB in rats as compared to normal rats. Pre treatment with the plant extracts HZ, RN, SC (100 mg/kg and 200 mg/kg) caused significant reduction in the serum enzyme levels namely SGOT, SGPT and SAKP in a dose dependent manner and was almost comparable to silymarin treated groups (Figs. 5-7). Treatment with RN (200 mglkg) and HZ (200 mg/kg) significantly reduced SGOT, SGPT and SAKP levels. SC (200 mg/kg) was the best of the three in lowering SGOT, SGPT, SAKP and SB levels. In the case of serum bilirubin, both HZ and SC gave almost similar values for 200 mg/kg and it was significantly lower than RN (200mg/kg) (Fig. 8). Histopathological observations basically support the results obtained from serum enzyme assays. Histological architecture of paracetamol - treated liver sections showed fatty degeneration of hepatocytes, severe centrizonal hemorrhagic hepatonecrosis, ballooning with nuclear pycnosis and karyolysis by SC treatment. The histological architecture of liver of rats pretreated with all the three plant extracts showed normalization of the defects showing its potent hepatoprotective effects almost comparable to the normal control and silymarin groups.(figs 9-14). 7.4 Discussion In recent years, there has been many researchers in traditional medicines, attempting to develop new drugs for hepatitis (Liu, 1989). In the present study, an overdose of paracetamol was used to produce liver toxicity in Wistar rats, the experimental model to investigate the protective effect of the three plant drugs HZ, RN and SC on paracetamol induced liver toxicity. Paracetamol is a hepatotoxicant which is primarily metabolized by 104
4 Fig. 5. Effect of H. zeylanica I R. nasuta and S. ciliata extracts on SGOT levels in rats after paracetamol 250 -, , J ~ b C) en 50 o+-----j'-- H. zeylanica R. nasuta. S. ciliata o Normal control Paracetamol + plant extract (100 mg/kg) Paracetamol +silymarin (100 mg/kg) Paracetamol + plant extract (200 mg/kg) Values are mean ± SO, n=6, **p<0.01,***p<0.001 as compared to toxin control Fig. 6. Effect of H. zeylanica I R. nasuta and S. ciliata extracts on SGPT levels in rats after paracetamol 200 -, , :::; ~... :::: D.. ~ o H. zeylanica R. nasuta S. ciliata Normal control o Paracetamol + plant extract (100 mg/kg) Paracetamol + silymarin (100 mg/kg) Paracetamol + plant extract (200 mg/kg) Values are mean ± SO, n=6, **p<0.01,***p<o.001 as compared to toxin control
5 Fig.7. Effect of H. zey/anica, R. nasuta and S. ciliata extracts on SAKP levels in rats after paracetamol == , , E o ~ j--- ' ~ 60 -j-- ~ 30 «(/) 0 H. zey/anica R. nasuta Normal control o Paracetamol + plant extract (100 mg/kg) Paracetamol + silymarin (100 mg/kg) S. ciliata Paracetamol + plant extract (200 mg/kg) Values are mean ± SD, n=6 **p<o.01,***p<o.001 as compared to toxin control Fig. 8. Effect of H. zey/anica, R. nasuta and S. ciliata extracts on serum bilirubin levels in rats after paracetamol 1.2-, , - ~ s:: :c :I ~ :c E 0.3 :I "- CD en H. zey/anica R. nasuta S. ciliata o Normal control Paracetamol + plant extract (100 mg/kg) Paracetamol + silymarin (100 mg/kg) Paracetamol + plant extract (200 mg/kg) Values are mean ± SO, n=6, **p<o,01,***p<o,001 as compared to toxin control
6 ... Fig. 9. Section of rat liver (normal control) showing normal hepatic architecture with hepatic cells having prominent nuclei (t ) and cytoplasm (x 350). Fig. 10. Section ofparacetamol - treated (toxin group) rat liver showing broad infiltration of lymphocytes and Kupffer cells (t ), loss of cell boundaries, degeneration ofhepatic cells with centrilobular necrosis (t t), inflammation, extensive vacuolization (t t t), ballooning degeneration (t t t t) and sinusoidal congestion (x 350). Fig. 11. Section ofhz (200 mg/kg) + Paracetamol - treated rat liver showing mild inflammation (t ) and no necrosis when compared to Paracetamol toxin group (x 350). Fig. 12. Section ofrn (200 mg/kg) + Paracetamol- treated rat liver showing less vacuolization(t ), mild degeneration ofhepatocytes when compared to Paracetamol toxin group (x 350). Fig. 13. Section of SC (200 mg/kg) + Paracetamol treated rat liver showing almost normal picture with reduced sinusoidal dilation (t ) and mild hepatic degeneration (x 350). Fig. 14. Section of Silymarin (100 mg/kg) + Paracetamol treated rat liver showing almostnormal hepatic cells with mildhepatic degeneration only (x 350).
7 sutfation and glucuronidation to unreactive metabolites and then activated by tile cytochrome P 450 system to produce liver injury. (Moldeus, 1978b). It is established that paracetamol is bioactivated to a toxic electrophile, N- acetyl P benzoquinoineamine (NAPQI) which binds covalently to tissue macromolecules. (Mitchell et al.,1973) and probably oxidizes lipids (Pable et al." 1992), the critical sulfhydryl groups (protein thiols) and alters the homeostasis of calcium. However NAPQI is readily detoxified by conjugation with glutathione (GSH) to form mercapturic acid (Nelson, 19'90) rendering it inactive. Hepatic necrosis occurs only when the amount of NAPQl exceeds the binding capacity of GSH. Hepatic injury by paracetamol therefore is dependent on the adequacy of GSH stores. (Hymann and Willis, 1993). Paracetamol, an analgesic and antipyretic is assumed to be safe in recommended doses, overdoses however, produce hepatic necrosis. Small doses are eliminated by conjugation followed by excrenon, but wilen the conjugation enzymes are saturated, the drug is diverted to an alternative metaboli,c pathway resulting in the formation of a hydroxylamine derivative by cytochrome P<t51)i enzyme. The hydroxy:lamine derivative, a reactive electrophilic agent reacts non enzymatica.liy with gllutathione and detoxifies it. When the hepatic reserves of glutathione dep,letes, the hydmxyllamil1l,e reacts with macromolecu'les and disrupts their structure and f\menon. Extenslive Iliver damage by paracetamoi itself decreases its rate of metabolism. Induction of cytochrome P450 or depl,enon of hepatic gmathione is a prerequisite for paracetamol induced toxidt'f. The present study indicated that treatment with paracetamol produced severe hepatic necrosis and fatty changes. aalso increased significantly the serum hepatic enzyme levels. Compared to parncetamol group, it was found that paracetamol + plant extracts (HZ/RN/SC) treated groups represented the best hepatoprotective effects in serum enzyme level studies. Histopathologi,ca! damage caused by paracetamol was improved in rat liver treated with the three plant extracts. In conclusion, paracetamol is dependent on cytochrome P450 system to produce the injurious and toxic reactive metabolite NAPQI. Therefore the possible hepatoprotective action of the three plant extracts HZ, RN and SC may be due to the following factors.(1} inhibiting the cytochrome P450 activity (2 ) preventing the process of lipid peroxidation, (3) stabilizing the hepatocellular plasma membrane ( 4) enhancing protein synthesis (5 ) reducing generation of toxic metabolites and (6) free radical scavenging. 105
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