Akademik Veri Yönetim Sistemi
Tezin Türü: Doktora
Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye
Tezin Onay Tarihi: 2013
Öğrenci: SERDAR KARAKURT
Danışman: ORHAN ADALI
Özet:The genus Epilobium sp. (Onagraceae) is widely distributed all over the world and consists of nearly 200 species. Epilobium hirsutum L. (EH) one of the members of this genus is known as its analgesic, anti-microbial and anti-proliferative activity and it is used in our country as an alternative medicine. The pharmacological effect of EH could be explained by the presence of polyphenolics including steroids, tannins and flavonoids in the aerial parts. Cytochrome P450s (CYPs) known as phase I enzymes have important function on the metabolism of xenobiotics including drugs and other chemicals. Several procarcinogens require metabolic activation by CYP enzymes in order to show their toxic and carcinogenic effects. This toxic effect can be eliminated by phase II enzymes known as conjugation or antioxidant enzymes. In the first part of the thesis study, EH was collected, by using aerial parts of the plant extract was prepared and lyophilised then active substances were identified and quantitated by LC-MS MS. In the second part of the study, the water extract of EH was dissolved in ddH2O and 37 mg extract/kg body weight/day was intraperitoneally (i.p.) injected to 4 months old male Wistar Albino rats for a period of nine days to investigate in vivo therapeutic/toxic effects of EH on rat liver enzymes. To our knowledge, this is the first study which investigates the effects of EH on liver phase I, phase II and antioxidant enzymes activities, as well as mRNA and protein levels of those in rats. Spectrophotometric analysis of lactate dehydrogenase activity was shown that EH does not cause any cytotoxicity on rat liver (1.01-fold). EH has an inhibitory effect on rat liver cytochrome P450 dependent enzyme activities; CYP1A1 catalyzed ethoxyresorufin-o-demethylase (1.46-fold) p<0.0003, CYP2B1 dependent benzphetamine N-demethylase (2.12-fold) p<0.0001 and ethylmorphine N-demethylase (8.68-fold) p<0.0001 and CYP2E1 dependent aniline 4-hydroxylase (4.93-fold) p<0.0001 and NDMA N-demethylase (1.2-fold) p<0.05. Similar to cytochrome P450s, total GST and its isoforms GST mu and GST theta were also inhibited significantly (p<0.0001) when rats were injected with EH . On the other hand, phase II enzyme NQO1 and antioxidant enzymes SOD and GPx activities significantly increased (p<0.0001) upon treatment of animals with EH. In order to investigate the causes of the alteration of enzyme activities, protein and mRNA expressions of those enzymes were determined by western blot and qRT-PCR, respectively. Western blotting analysis have shown that CYP1A1, CYP2B1, CYP2E1 and CYP3A1 and GST-mu protein expressions significantly decreased (p<0.0001) in EH treated rats as compared to control animals. In contrast to these findings, treatment of rats with EH caused induction of the protein expressions of NQO1 and GPx as 2.97-fold and 1.93-fold, respectively. Analysis of mRNA expressions by qRT-PCR demonstrated that mRNA expressions of CYP1A1, CYP2B1, CYP2E1, CYP3A1 GST-mu and GSTT1 were significantly decreased (p<0.0001) In contrast to these, EH treatment increased the GPx and NQO1 mRNA expressions as 4.97-fold and 3.5-fold (p<0.0001) compared to control, respectively. Cross analyses of enzyme activity versus protein and mRNA expressions showed that there is a correlation between mRNA and protein expression levels and corresponding enzyme activities in EH treated samples Inhibition of CYP enzymes may affect the metabolism of many chemicals and drug molecules. Since procarcinogens require metabolic activation by CYP enzymes in order to show their toxic and carcinogenic effects, inhibition of the enzyme system prevents the formation of hazardous metabolites. Pro-drugs or active drugs’ metabolism may be modulated by EH which resulted in toxicity or enhanced response. The presented evidences point out that EH alters the activity and expression of enzymes involved in xenobiotics activation/detoxification pathways. Therefore, modulatory effect of EH on these enzymes suggests an inherent chemopreventive action against a group of chemicals including carcinogens and drugs. However, necessary precautions such as medical advice should be taken regarding the usage of this plant in replacement treatments since it reveals possible interactions with drugs and dietary foods.