ANTIOXIDANT PROPERTIES OF Salvia absconditiflora EXTRACTS AND THEIR EFFECTS ON PHASE I AND PHASE II GENE EXPRESSIONS IN HEPG2 CELL LINE A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY DENİZ İRTEM KARTAL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY JULY 2015 Approval of the Thesis: ANTIOXIDANT PROPERTIES OF Salvia absconditiflora EXTRACTS AND THEIR EFFECTS ON PHASE I AND PHASE II GENE EXPRESSIONS IN HEPG2 CELL LINE submitted by DENİZ İRTEM KARTAL in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry Department, Middle East Technical University by, Prof. Dr. Gülbin Dural Ünver ______________ Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Orhan Adalı ______________ Head of Department, Biochemistry Prof. Dr. N. Tülin Güray ______________ Supervisor, Biological Sciences Dept., METU Assist. Prof. Dr. Gökhan Sadi ______________ Co-Supervisor, Biology Dept., Karamanoğlu Mehmetbey University Examining Committee Members: Prof. Dr. Meral Yücel _________________ Biological Sciences Dept., METU Prof. Dr. N.Tülin Güray _________________ Biological Sciences Dept., METU Prof. Dr Orhan Adalı _________________ Biological Sciences Dept., METU Prof. Dr. Benay Can Eke _________________ Pharmacy, Ankara University Prof. Dr. Özlem Yıldırım _________________ Biology Dept., Ankara University Date: 13/07/2015 I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last Name: Deniz İRTEM KARTAL Signature: iv ABSTRACT ANTIOXIDANT PROPERTIES OF Salvia absconditiflora EXTRACTS AND THEIR EFFECTS ON PHASE I AND PHASE II GENE EXPRESSIONS IN HEPG2 CELL LINE IRTEM KARTAL, Deniz PhD, Department of Biochemistry Supervisor: Prof. Dr. N. Tülin GÜRAY Co-Supervisor: Assistant Prof. Dr. Gökhan SADİ July 2015, 144 pages S.absconditiflora is one of the endemic Salvia species grown in Turkey, which is consumed as a herbal tea. Because of the presence of high amounts of vesicles on their leaves, it is very rich in bioactive compounds. S.absconditiflora water and methanol extracts were studied for their antioxidant capacity by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ABTS free radical scavenging assay. Total phenolic and total flavonoid contents were quantified by spectrophotometric methods, HPLC and LC-MS/MS analyses and the results revealed the presence of several compounds and their quantities were calculated by using 10 different standards. June water extracts showed the highest % Radical Scavenging Activity against DPPH radical. Total flavonoid content was found as one third of the total phenolic content. April methanol extracts had the highest TPC (180.77 mgGAE/ g of dried extract) and June methanol extracts had highest TFC (21.8 mg QE/ g of dried extract). Cytotoxic effects of S.absconditiflora extracts and two standards, as main phenolic components, were examined in human hepatocyte carcinoma cell line, HepG2, via XTT colorimetric and Trypan Dye Exclusion cell viability v assays. IC values in HepG2 cell line, after 48 and 72 hour incubations 50 were determined. Effects of S.absconditiflora water and methanol extracts on the gene expressions of several Phase I and II enzymes were further investigated. Among CYP450 enzymes, only five accounts for major drug metabolism, namely CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2 and water extracts induce the gene expression of all these CYP enzymes. Only CYP1A2, CYP2D6 and CYP2E1 gene expressions were induced with methanol extract. However, CYP2C9, CYP2C19 and CYP3A4 gene expressions were found to be inhibited with methanol extracts. CYP3A4 is the most important enzyme as its involved in almost all CYP450 mediated reactions. This inhibitory potential of S.absconditiflora methanol extract toward CYP3A4 could have potential herb-drug interaction in liver and might increase the bioavailability of co-adminestered drug and lead to toxicity. Inhibitory effect of methanol extract but not the water extract suggested that flavonoids, which exist abundantly in the methanol extract of S.absconditiflora may be responsible for this inhibition. Gene expression of all GST Mu enzyme (M1-M5) was inhibited by 3-18 fold with methanol extracts whereas water extracts inhibited M1 and M2 by 1.5 fold and induce M3 and M4 by again 1.5 fold. Both water and methanol extracts decreased the gene expression of GSTP1. According to our panel results we conclude that, the methanol extract of S.absconditiflora shows the most potent effects on CYP enzymes. On the other hand, the aqueous, water extract shows negligible effects on CYP activities. Since most individuals consume the leaves of S.absconditiflora either raw or boiled with water, the findings of this study suggest that the water extract may be safe considering the herb-drug interactions. Keywords: S.absconditiflora, HepG2 cell line, Phase I and Phase II gene expression vi ÖZ Salvia absconditiflora EKSTRELERİNİN ANTİOKSİDAN İÇERİKLERİ VE HEPG2 HÜCRELERİNDE FAZ I VE FAZ II ENZİMLERİNİN GEN EKSPRESYONLARI ÜZERİNE ETKİLERİ İRTEM KARTAL, Deniz Doktora, Biyokimya Bölümü Tez Yöneticisi: Prof. Dr. N. Tülin GÜRAY Ortak Tez Yöneticisi: Yrd. Doç.Dr. Gökhan SADİ Temmuz 2015, 144 sayfa S.absconditiflora Türkiye de yetişen ve bitki çayı olarak da tüketilen endemik Salvia türlerinden bir tanesidir. Yaprakları üzerindeki yüksek miktardaki damarlardan dolayı S.absconditiflora çok aktif bileşiklere sahiptir. S.absconditiflora’nın su ve metanol ekstrelerinin antioksidan kapasitelerine DPPH ve ABTS metodu ile bakıldı. Toplam fenolik ve flavanoid içerikleri spektroskopik teknik ile belirlendi. HPLC ve LC-MS/MS analizleri ile S.absconditiflora’nın içerisindeki bazı fenolik bileşiklerin varlığı 10 farklı standart kullanılarak belirlendi. Haziran su ekstresi en yüksek radikal yakalama kapasitesi gösterdi. Toplam flavanoid içeriği toplam fenolik içeriğinin 1/3’ü olarak belirlendi. Nisan metanol ekstresinin 108.77 mg GAE/g ekstre ile en yüksek toplam fenolik içeriğe sahip olduğu bulundu. S.absconditiflora ‘nın ve 2 ana aktif bileşeninin sitotoksik etkileri HepG2 hücre hattı üzerinde XTT kolorimetrik ve tripan mavisi boyası ile incelendi. IC değeri 48 ve 72 saat uygulamaları ile tripan mavisi sayımı ile belirlendi. 50 vii CYP enzimleri arasında, ilaç metabolizmasında rol alan en önemli beş enzim, CYP3A4, CYP2D6, CYP2C9, CYP2C19, ve CYP1A2 olarak bilinir ve bu enzimlerin gen ifadelerine bakıldığında Salvia su ekstresinin bu enzimlerin gen ifadelerinin hepsini arttırdığı görüldü. Sadece CYP1A2, CYP2D6 ve CYP2E1 gen ifadelerinin metanol ekstresi ile uyarıldığı, bununla birlikte, CYP2C9, CYP2C19 ve CYP3A4 gen ifadelerinin, metanol ekstresi uygulandığında inhibe edildiği bulundu. CYP3A4 enzimi tüm CYP enzim reaksiyonları içinde en önemli enzimdir. S.absconditiflora metanol ekstresinin CYP3A4 üzerindeki bu inhibe edici özelliği, karaciğerde, olası bitki-ilaç etkileşimine neden olabilir ve birlikte kullanılan ilacın biyoyarılanımını arttırarak toksik etkiye sebep olabilir. S.absconditiflora metanol ekstresinin inhibe edici etkisinin, içerisinde bol miktarda bulunan flavonoidler tarafından sağlanabileceği düşünülmektedir. GST Mu enzimlerinin hepsinin (M1-M5) gen ifadelerinin metanol ekstresi ile 3 ile 18 kat düştüğü görüldü. GSTM1 ve GSTM2 gen ifadeleri su ekstresi ile 1.5 kat düşerken, GSTM3 ve GSTM4 gen ifadelerinin su ekstresi ile 1.5 kat arttığı görüldü. Su ve metanol ekstrelerinin her ikiside de GST P1 gen ifadesini düştüğü gözlemlendi. Bu çalışmada kullanılan panel sonuçlarına göre, CYP enzimleri üzerine en güçlü etkiye S.absconditiflora metanol ekstresinin sahip olduğu bulundu. Diğer yandan, su ekstrelerinin CYP aktiviteleri üzerinde daha az etkisi olduğu görüldü. Çoğu kişi, S.absconditiflora yapraklarını çiğ ya da haşlanmış olarak tüketmektedir. Bu çalışmanın bulgularına bakıldığında su ekstrelerinin bitki- ilaç etkileşimleri dikkate alındığında güvenli olabileceğini düşünülmektedir. Anahtar kelimeler: S.absconditiflora, HepG2 hücre hattı, Faz I ve Faz II gen ifadeleri viii To My son, Arda Kaan ix ACKNOWLEDGEMENTS I would like to express my deep reverence to my supervisor Prof. Dr. N. Tülin Güray for her encouraging guidance, incredible patience and supervision throughout this study. I am glad to achieve the best in my research by her support and great personality which is absolutely not restricted with scientific area. I am also deeply grateful to my co-advisor Assistant Prof. Dr. Gökhan Sadi for his invaluable and precise commanding and guidance both in theoretical background and in experimental design of this research thesis. I am also thankful to my other PhD Examining Committee Members Prof. Dr. A. Meral Yücel, Prof. Dr. Orhan Adalı, Prof. Dr. Benay Can Eke and Özlem Yıldırım for their advices, suggestions and constructive criticisms. I wish to thank also to my friend Ahmet Altay specially supporting me all the time during this thesis study. I thank also to my friend Yeşim Kümbet and Tuba Çulcu for their sincere friendship and supports. I would like to thank to special study students Hülya Çöpoğlu and Başak Ezgi Saraç for their sincere friendship and supports and providing me a friendly and relaxed atmosphere in the lab. I also thank to Ceren Biler and Ürün Duru in Central Laboratory of METU for LC-MS/MS experiments. I am also thankful to the Scientific and Technological Research Council of Turkey (TUBITAK) for their support by providing scholarship during my PhD study. I would like to express my great appreciation to my family members my mother Mediha İrtem, my father Haydar İrtem and my sister Filiz Bozkurt for their physical and moral support and encouragement in my life. x