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M.S Latha et al. / International Journal of Advances in Pharmaceutical Research liver (Fig.2B) is morphologically different from normal liver. In NDEA treated groups rat liver become very large in size and a large number of hepatic nodules were observed in the liver. In MEWF 200mg/kg treated group and in MEWF alone treated groups liver morphology is very similar to normal rats. 3.3 Tissue biochemical analysis 3.3.1 Estimation of Glutathione - S - transferase (GST) The GST activity of liver tissues were significantly (p ≤ 0.05) reduced in NDEA intoxicated rats of pretreatment groups compared to normal control. The MEWF dose dependently increased (p ≤ 0.05) the activity of GST in hepatic tissues (Table 1). Treatment with 200 mg/kg methanolic extract exhibited prominently increased i.e., 89.9% GST levels. In addition, Silymarin treated rats also prevented the NDEA induced decrease in GST activity by 69.9% in hepatic tissue. 3.3.2 Estimation of Glutathione Reductase (GR) GR activity was significantly decreased (p ≤ 0.05) in NDEA treated animals when compared to control. A significant increase (p ≤ 0.05) in the level of GR was observed in MEWF (100 and 200 mg/kg) and Silymarin (100 mg/kg) treated rats intoxicated with NDEA (Table 1). The percentage of protection in liver tissue was 83.8% for 200 mg/kg of MEWF. Silymarin restored the GR activity up to 62.6% in rat liver. 3.3.3 Estimation of Glutathione Peroxidase (GPx) Activities of hepatic GPx was significantly (p ≤ 0.05) lowered in NDEA treated rats (Table 1). MEWF dose dependently prevented the lowering of GPx compared to NDEA alone treated groups. In liver, 200 mg/kg of methanolic extract showed a protection of 94.5%. Silymarin-treated rats also prevented the lowering of GPx by 73.3% in hepatic tissues. 3.4 Immunohistochemical analysis Fig. 3 and 4 shows the immunohistochemical analysis of PCNA and Cyclin D1 respectively. Here normal rat tissue showed regularly stained nucleus. NDEA administered rat liver tissue showed overexpression of Proliferating Cell Nuclear Antigen (PCNA) and Cyclin D1. Administration of MEWF showed a reduction in the expression of PCNA and Cyclin D1. PCNA is a common index for proliferation of hepatocytes at late G1 stage and early S stage. Fig. 3B and 4B (HCC bearing) groups stained positively for PCNA and Cyclin D1 indicating active cell proliferation when compared with normal control. The plant extract given at a concentration of 200mg/kg showed a remarkable lowering of the expression of PCNA and cyclin D1 and was comparable to the effect rendered by silymarin at a concentration of 100mg/kg. 4. DISCUSSION Abnormal proliferation of cells is the main feature of carcinogenesis, and there for exploration of drugs that can affect malignant proliferation of liver cells is of primary importance in chemical prevention of liver cancer. PCNA present in cell nucleus is directly involved in DNA replication. It has been found that the positive expression of PCNA were a common index for proliferation of hepatocytes at late G1 stage and early S stage (20). The positive expression of proteins was mainly found in the precancerous proliferation focus and cancerous liver tissue during NDEA induced hepatocarcinogenesis. The high expression of PCNA in the present studies suggested that the ability of cell proliferation become stronger, and this was closely related to malignant cell proliferation and carcinogenesis (21). The results indicated that the administration of MEWF could remarkably decrease the PCNA in HCC bearing tissue, suggesting that MEWF had the ability to suppress malignant proliferation of hepatocytes in experimental liver cancer and inhibited tumor growth through inhibition of DNA synthesis. Cyclin D1 is another important cancer marker has been associated with a variety of cancers including HCC. Cyclin D1 is the regulatory subunit of a holoenzyme that promotes progression through the G1-S phase of cell cycle. Amplification or overexpression of Cyclin D1 plays important roles in the development of a subset of human tumorigenesis and cellular metastasis (22). Amino transferases (AST, ALT) and GGT are the important cytoplasmic enzymes present in the liver. During chemical intoxication the membrane integrity is lost due to necrosis and these enzymes move into the circulatory system followed by elevated levels of enzymes in the serum (23). Increased levels of serum AST, ALT and GGT was detected in NDEA treated rats, effectively down- regulated by the treatment with MEWF indicating the protective role of Woodfordia fruticosa in NDEA induced HCC. The intra cellular antioxidant system comprises of different free radical scavenging antioxidant enzymes such as GST, GR and GPx constitute the first line of cellular antioxidant defense system. When excess free radicals are produced, the equilibrium is lost and consequently oxidative insult is established (24). GST offers protection against lipid peroxidation by promoting the conjugation of toxic electrophiles with GSH (25). GR is also essential for the maintenance of IJAPR / Dec. 2012/ Vol. 3 /Issue. 12 / 1322 – 1330 1325
M.S Latha et al. / International Journal of Advances in Pharmaceutical Research GSH levels in vivo. The significant (p ≤ 0.05) restoration of GPx activity in MEWF and silymarin might be due to the antioxidant activity by detoxifying the endogenous metabolic peroxides generated after NDEA intoxication in hepatic tissues. These biochemical restorations may be due to the inhibitory effects of MEWF in the process of cellular damage in the liver. Free radical scavenging activity of Woodfordia fruticosa methanolic extract is well established and that effectively protects the liver against carbon tetrachloride [13], diclofenac (26) thioacetamide (27) and phenytoin (28) induced hepatotoxicity in rats. Preliminary phytochemical analysis identified the major class of chemical components present in methanolic extract are saponins (steroids and terpenes), phenolics, alkaloids, flavanoids, tannins etc. The phytochemicals identified are more potent in inhibiting free radicals and this could be protective against the progression of NDEA induced HCC. The compounds reported from the dried flowers of Woodfordia fruticosa are β-sitosterol, kaempferol, ellagic acid, octacosanol, meso-inositol, quercetin, woodfordins A, B, C, oenothein B, woodfordin D and oenothein A (29). Ellagic acid is a compound reported to have chemopreventive effect and is useful in inflammatory disorders like colorectal cancer (30). Quercetin is a plant derived Flavanoid, used in cancer prevention and treatment. Antiinflammatory effect of quercetin in hepatoma cell lines (Hep G2) was reported (31). Woodfordin C and oenothein B, a class of macrocyclic hydrolysable tannins exhibited potent host-mediated antitumor activity against sarcoma 180 in mice (32-33). Woodfordin C shows remarkable inhibition of DNA topoisomerase II (34). Woodfordin D and Oenothein A, trimeric hydrolysable tannins also have antitumor activity (35). Thus protection yielded by this extract may be due to the combined effects of these compounds or fractions rather than any single component. AND FIGURES Table 1: Effect of MEWF on GST, GR and GPx in the liver tissue of different experimental groups Treatment groups Normal control NDEA control (0.02%) Silymarin(100mg/kg)+ NDEA MEWF(100mg/kg)+ NDEA MEWF(200mg/kg)+ NDEA MEWF alone GST 1 72.5±0.70 33.1±0.43 * 58.3±0.52 ** 53.9±0.78 ** 68.5±0.51 ** 70.2±0.53 ** 1: μmol CDNB-GSH conjugate formed/min/mg protein ; 2 : nmol of GSSG utilized/min/mg protein; 3: nmol of GSH oxidized/min/mg protein Values are mean± S.D., n=6 * p≤ 0.05 versus normal control ** p≤ 0.05 versus NDEA control GR 2 21.4±0.77 7.2±0.38 * 16.16±0.43 ** 12.3±0.45 ** 19.15±0.83 ** 21.7±0.61 ** GPx 3 283.1±2.85 162.4±3.09 * 250.8±3.05 ** 217.5±2.51 ** 276.4±3.81 ** 281.8±3.0 ** IJAPR / Dec. 2012/ Vol. 3 /Issue. 12 / 1322 – 1330 1326
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