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Protective Effect of Hypericum triquetrifolium Turra. on Cyclophosphamide-Induced Hepatotoxicity in Rat Asst.Prof. Songul CETIK Mardin Artuklu University, TURKEY İntroduction Cyclophosphamide (CP) is the most widely used alkylating agent in chemotherapy with a high therapeutic index and broad spectrum of activity against a variety of cancers since the late-1950s 1. The antitumor effect of CP is in proportion to the dose of CP administered, which often results in hepatotoxicity, immunosuppression and cytotoxic effects 2 . Side Effect of CP Therapeutic effectiveness of anticancer drugs is associated with severe side effects due to their toxicity. Liver involvement during CP therapy may represent treatment failure, disease progression or CP-induced hepatotoxicity. Thus, strategies to minimize the side effects of chemotherapeutic agents while preserving their chemotherapeutic efficacy are needed. CP Metabolits CP produces two active metabolites such as phosphoramide mustard (PAM) and acrolein (ACR). Lipid peroxidation (LPO) is one of the principal causes of CPinduced toxicity and is mediated by the production of ACR, a metabolite for much of its toxicity 3, 4.5. Acrolein interferes with the tissue antioxidant defense system, produces highly ROS, and interacts with protein amino acids causing structural and functional changes in enzymes 6,7. Experimental evidence suggests that oxidative stress is responsible for cyclophosphamide hepatotoxicity8,9. In CP treatment, toxicity is a vital dose limiting factor. Plasma antioxidant concentration has shown a decrease of patients who had a high dose chemotheraphy. As also suggested by previous studies, in order to benefit from CP at higher doses, there is a need for protective agents that would eliminate the toxic side effects of CP. We need new agents to let CP's higher dose useges by preventing the CP's toxic effects, without tumor preserving and tumor growth stimulating properties, to protect normal tissues from chemotherapy induced toxicity. By preventing dose-limiting antineoplastic drugs toxic effects, several methods have been developed to let their more effective doses in many conducted experimental and clinical studies, in aspect of importance of role of some the natural phenolic compounds on body. Hypericum Antioxidants that can eliminate toxic side effects of chemotherapy can provide the use of higher and more effective doses of the anticancer drugs. The genus Hypericum (family Hypericaceae) have been ethnomedically used in different parts of the world for its sedative, antiseptic, antioxidant, antimicrobial and cytotoxic properties. Hypericum triquetrifolium TURRA. Hypericum triquetrifolium Turra (HT) belongs to the Hypericaceae family and it is native to eastern Europe and the Mediterranean area. The methanolic extract of H.triquetrifolium contain chlorogenic acid, rutin, hyperoside, quercitrin, quercetin, kaempferol, phenolic - flavonoid compounds and other water-soluble components that possess a wide array of biological properties. HT is a phenolic component with antioxidative, analgesic and anticarcinogenic properties. The methanolic extract of H. triquetrifolium aerial parts, possesses a significant antioxidant activity. PURPOSE This study aimed to investigate the possible protective effect of HT on CPinduced hepatotoxicity. In order to determine the protective effect of HT upon the liver, the levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) as well as the total levels of total oxidant capacity (TOC) and total antioxidant capacity (TAC) were determined. Also, oxidative stres index (OSI) was measured. Furthermore, the liver tissues were analysed histologically. Plant Extraction Wild-growing plants (H. triquetrifolium seeds) were harvested at seeding stages from August to September, 2015 from wild populations located in Zınar, Mardin, Turkey. A total of 20 g of each dried material were ground in a grinder with a 2-mmdiameter mesh and then incubated into a glass flask with 200 mL (99%) methanol for 3 days under magnetic stirrer. After it was filtrated, the methanol was removed on rotary evaporator. Approximately, 2 g of the crude methanol extracts of H. triquetrifolium seed was obtained and kept in dark and airtight glass bottles at 4ºC until it was used. Then when used for injetions solved with % 0.2 DMSO. Experimental Groups Albino rats (Wistar, 3-4 months old, male, weight 220 ± 20 g healthy) were randomly divided in 9 groups, each including seven animals: Group 1 (control) treated with saline; Group 2 treated with 150 mg/kg CP, respectively; Groups 3, 4 and 5 treated with 25, 50 and 100 mg/kg HT; Groups 6, 7 and 8 treated with 25, 50 or 100 mg/kg HT+CP, Group 9 treated with 0.5ml - %0.2 DMSO. All injections i.p. CP and HT Administered to Groups Days ---> Given Matter v 1 2 3 CP+HT Control & DMSO 5 6 + CP (150mg/kg) HT(25, 50,100) 4 7 Sacrifice + + + + + + Sacrifice HT HT HT CP + HT HT HT Sacrifice SF SF SF SF SF SF Sacrifice DMSO DMSO DMSO DMSO DMSO DMSO Statistical Analysis The results were expressed as means ± S.E.M. Statistical analysis was performed by using One Way Analysis of Variance and Kruskal-Wallis One Way Analysis of Variance on Ranks Test and p<0.05 accepted as considering statistical significance. Each experiment was repeated at least three times. Histological Analysis Histological: Liver were fixed with 10% formaldehyde solution. Through routine histological preperations, samples were embedded in paraffin and 5.0 micron thick serial sections were made, which were stained with HematoxylinEosin and histopathologic features were evaluated. The results was analyzed by One Way Analysis of Variance and Kruskal-Wallis One Way Analysis of Variance on Ranks Test. Results Our study demonstrated that in line with the rise in the dose of 150 mg/kg CP, we determined an increase the levels of serum ALT, AST, ALP, LDH, TOS and OSI, besides of moderate degeneration in the liver tissue. With 25, 50 and 100 mg/kg HT, there was an important decrease with respect to CP toxicity. In the groups given both CP plus HT, there was a rise in serum TAS levels, while the levels of AST, ALT, ALP, LDH, TOS and OSI showed a remarkable decrease. DMSO(Dimethyl Sulfoxide) When the obtained results are evaluated, the control values close to the %0.2 DMSO, which we used for solving the extracts. This result showed that DMSO doesn’t affect on the potential of plants and the test results. ALT (U/L) ALT 30.00 ALT (U/L) 25.00 20.00 15.00 10.00 5.00 0.00 DOSES (mg/kg) AST (U/L) AST 120.00 AST (U/L) 100.00 80.00 60.00 40.00 20.00 0.00 DOSES (mg/kg) ALP (U/L) ALP (U/L) ALP 100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 DOSES (mg/kg) LDH (U/L) LDH 600.00 LDH (U/L) 500.00 400.00 300.00 200.00 100.00 0.00 DOSES (mg/kg) TOC ( μmol H2O2 equivalent/L ) TOC (μmol H2O2 equivalent/L) TOC 3.00 2.50 2.00 1.50 1.00 0.50 0.00 DOSES (mg/kg) TAC ( μmolTroloxequivalent/L ) (TAC, μmolTroloxequivalent/L) TAC 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 DOSES mg/kg OSI( TOC/TAC X 100 ) OSI OSI ( TOC/TAC x 100 ) 0.30 0.25 0.20 0.15 0.10 0.05 0.00 DOSES (mg/kg) HISTOLOGICAL RESULTS Fig. 1- CP grubu A: H-E, B: kaspaz-3, C: Bcl-2, D: Bax. Fig. 2- 100 HP grubu A: H-E, B: kaspaz-3, C: Bcl-2, D: Bax. Fig. 3- CP+100 HP group A: H-E, B: kaspaz-3, C: Bcl-2, D: Bax. Conclusion Results showed for the first time that the methanolic extract of H. triquetrifolium seed extract, possesses a significant antioxidant activity. The signs of recovery in the serum biochemical levels were also true fort the histological findings of the liver. Conclusion CP induced oxidative stress in the liver as evident from the increased lipid peroxidation (LPO), reactive oxygen species (ROS). Our data suggest that HT is a highly effective antioxidant substance with a cell-protecting effect on LPO and ROS. Therefore, HT could serve as effective agent that could lessen the adverse effects of anticancer drugs in the course of chemotherapy protocols. REFERENCES 1. Pass, G.J., Carrie, D., Boylan, N., Lorimore, S., Wright, E., Houston, B., Henderson, C.J., Wolf, C.R., 2005. 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Drug Interact. 19, 67–82. THANKS for your attention.