INVESTIGATION OF ANTIOXIDANT PROPERTIES AND ANTICARCINOGENIC EFFECTS OF ETHANOLIC EXTRACT FROM BARK OF SALIX AEGYPTIACA AND ITS FRACTIONS A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY MÜŞERREF ŞEYMA CEYHAN IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGY FEBRUARY 2014 Approval of the thesis: INVESTIGATION OF ANTIOXIDANT PROPERTIES AND ANTICARCINOGENIC EFFECTS OF ETHANOLIC EXTRACT FROM BARK OF SALIX AEGYPTIACA AND ITS FRACTIONS Submitted by MÜŞERREF ŞEYMA CEYHAN in partial fulfillment of the requirements for the degree of Master of Science in Biology Department, Middle East Technical University by, Prof. Dr. Canan Özgen _____________________ Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Gülay Özcengiz _____________________ Head of the Department, Biology Assoc. Prof. Dr. Sreeparna Banerjee _____________________ Supervisor, Biology Dept., METU Examining Committee Members: Assoc. Prof. Dr. A. Elif Erson-Bensan _____________________ Biology Dept., METU Assoc. Prof. Dr. Sreeparna Banerjee _____________________ Biology Dept., METU Prof. Dr. Arif Ahmet Başaran _____________________ Pharmacognosy Dept, Hacettepe University Assoc. Prof. Dr. Mesut Muyan _____________________ Biology Dept., METU Assist. Prof. Dr. Erhan Astarcı _____________________ Crop and Animal Production Dept., AIBU Date : 06.02.2014 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: Müşerref Şeyma Ceyhan Signature : iv ABSTRACT INVESTIGATION OF ANTIOXIDANT PROPERTIES AND ANTICARCINOGENIC EFFECTS OF ETHANOLIC EXTRACT FROM BARK OF SALIX AEGYPTIACA AND ITS FRACTIONS Ceyhan, Müşerref Şeyma MSc., Department of Biology Supervisor: Assoc. Prof. Dr. Sreeparna Banerjee February 2014, 97 pages Cancer is a multi-factorial disease that may arise as a consequence of different factors such as age, diet, and environmental factors as well as genetic makeup. Moreover, cancerous cells can develop certain strategies to evade immune system and develop resistance against chemotherapy drugs. Therefore, multi-targeted therapeutics have gained importance for both prevention of cancer and better prognosis. Extracts from Salix aegyptiaca includes several active phytochemicals; phenolic compounds and flavonoids and therefore have the potential for use as herbal remedies. The aim of this study was to investigate antioxidant properties and anticarcinogenic effects of ethanolic extract from bark (EEB) of Salix aegyptiaca and its fractions in colon cancer. EEB was fractionated into different fractions by solvent solvent partitioning. The antioxidant property of EEB and its fractions were investigated by 1, 1-diphenyl -2-picryl hydrazyl (DPPH) free radical scavenging activity, total phenolic and flavonoid content assays. Effect of EEB and its active v fractions on cancer cell proliferation were evaluated by MTT and BrdU incorporation assays. The ethyl acetate fraction showed the highest free radical scavenging activity (11 ± 1 μg/ml) and total phenolic content (29.6 ± 2 mg GAE/100 g dried weight). EEB had the highest total flavonoid content (47.5 ± 2 mg of EGCG equivalent/100 g dried weight) compared to its fractions. EEB, ethyl acetate and aqueous fractions decreased proliferation of cancer cells. Catechin, catechol and salicin were detected in high quantity in the ethyl acetate and aqueous fractions by tandem mass spectrometry (MS). Treatment of cancer cells with a combination of catechin and catechol had higher efficacy in reduction of proliferation than the individual pure compounds. EEB led to significant decrease in phosphorylation of ERK1/2, JNK1/2 and p38, but its active fractions were only functional in reducing the phosphorylation of ERK1/2. To conclude, our data suggest that EEB and its active fractions could be thought of as a potent antioxidant and anticarcinogenic agent that may be used in complementary and alternative medicine. Key words: Salix aegyptiaca, Colon cancer, Antioxidant, MAPK pathways vi ÖZ SALIX AEGYPTIACA KABUĞUNDAN ELDE EDİLEN ETANOLİK ÖZÜTÜN VE ONUN FRAKSİYONLARININ ANTİOKSİDAN ÖZELLİĞİNİN VE ANTİKARSİNOJENİK ETKİLERİNİN İNCELENMESİ Ceyhan, Müşerref Şeyma Yüksek Lisans, Biyoloji Bölümü Tez Yöneticisi: Doç. Dr. Sreeparna Banerjee Şubat 2014, 97 sayfa Kanser genetik yapının yanısıra yaş, diyet, ve çevresel etmenler gibi birçok farklı faktörün sonucunda ortaya çıkan multi-faktöriyel bir hastalıktır. Ayrıca, kanserli hücreler bağışıklık sisteminden kaçmak ve kemoterapi ilaçlarına karşı direnç göstermek için belirli stratejiler geliştirmişlerdir. Böylece çok hedefli terapötikler hem kanserden korunma hem de daha iyi prognoz için önem kazanmıştır. Salix aegyptiaca özütü fenolik bileşikler ve flavonoidler gibi birçok aktif fitokimyasalları içerir ve bu yüzden bitkisel ilaç olarak kullanılma potansiyeli vardır. Bu çalışmanın amacı Salix aegyptiaca kabuğundan elde edilen etanolik özütün (EEB) ve onun fraksiyonlarının antioksidan özelliğinin ve MAPK yolağıyla antikarsinojenik etkilerinin kolon kanserinde incelenmesidir. EEB çekme yöntemi ile farklı fraksiyonlara ayrılmıştır. EEB ve fraksiyonlarının antioksidan özelliği DPPH serbest radikalleri uzaklaştırma aktivite, total flovanoid ve total fenolik içerik deneyleri ile araştırılmıştır. EEB ve onun aktif fraksiyonlarının kanserli hücre vii çoğalmasının üzerindeki etkisi MTT ve BrdU katılım deneyleri ile değerlendirilmiştir. En yüksek serbest radikalleri uzaklaştırma aktivitesi (11 ± 1 μg/ml) ve total fenolik içeriği (29.6 ± 2 mg gallik asit eşleniği / 100 gram kurutulmuş ekstrakt) etil asetat fraksiyonu göstermiştir. Fraksiyonlarına kıyasla en yüksek total flavonoid içeriğe (47.5 ± 2 mg EGCG eşleniği / 100 gram kurutulmuş ekstrakt) EEB sahiptir. EEB, etil asetat ve su fazı fraksiyonları kanser hücrelerinin çoğalmasını azaltmıştır. Ardışık kütle spektrometre yöntemi ile etil asetat ve sulu faz fraksiyonlarında yüksek miktarda katekin, katekol ve salisin belirlenmiştir. Katekin ve katekol kombinasyonunun kanserli hücrelere uygulanması saf bileşiklere kıyasla hücre çoğalımının azalmasında daha yüksek bir etkiye sahiptir. ERK1/2, JNK1/2 ve p38 fosforilasyonunda önemli azalma EEB ile sağlanmıştır, fakat aktif fraksiyonları sadece ERK1/2 fosforilasyonunda azalmaya neden olmuştur. Sonuç olarak, verilerimiz gösteriyor ki EEB ve onun fraksiyonları tamamlayıcı ve alternatif tıpta güçlü bir antioksidan ve antikarsinojenik ajan olarak kullanılabilir. Anahtar kelimeler: Salix aegyptiaca, Antioksidan, Kolon kanseri, MAPK yolakları viii To My Family ix ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor Assoc. Prof. Dr. Sreeparna Banerjee for her guidance, encouragement, enthusiasm, immense knowledge and comments throughout my research. Her advices helped me in all the time of research and writing of this thesis. I would like to thank all my jury members; Assoc. Prof. Dr. Mesut Muyan, Assoc. Prof. Dr. A.Elif Erson Bensan, Prof. Dr. Arif Ahmet Başaran and Assist. Prof. Dr. Erhan Astarcı for their invaluable suggestions, comments and contributions to my thesis. I would like to extend my thanks to my lab mates, Aslı Sade Memişoğlu, Seda Tuncay Çağatay and Mümine Küçükdemir who made the lab a friendly environment for working with their care, helpful advices, support and encouragement, Richardas Rachkauskas, Doğukan Hazar Ülger and Yanuar Limasale. Moreover, I would like to special thank to Shabnam Enayat Berber for introducing me to the topic as well for the support on the way. I am deeply thankful to my friends Feride Kaşıkcı and Ayşe Senem Haser for their friendship, encouragement, moral support and suggestions throughout this study. My special thanks to Merve Öyken for her friendship, patience, and motivation throughout my undergraduate and graduate studies. I would like to express my deepest gratitude to my mother and father who experienced all of the ups and downs of my research for always believing in me, their continuous love and their supports in my decisions. Also I am deeply grateful to my brother and sister for their care, absolute patience, understanding and encouragements with their best wishes. I will be grateful forever for your love. This study was supported by METU Internal Funds. x