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Investigation of the Anticancer Potential of Pasaltakaza, Helicia Nilagirica Bedd

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Investigation of the Anticancer Potential of Pasaltakaza, Helicia Nilagirica Bedd Investigation of the anticancer potential of Pasaltakaza, Helicia nilagirica Bedd. A thesis submitted to Mizoram University (A Central University) For the degree of Doctor of Philosophy Submitted by JENNIFER ZOREMSIAMI, M.Sc. Department of Zoology, Mizoram University Tanhril, Aizawl- 796004 (India), 2017 ACKNOWLEDGEMENT First and foremost I would like to thank God for his never-ending grace, mercy and provision during the course of my research. I would like to express my deep gratitude to Prof. Ganesh Chandra Jagetia, my guide and mentor, for his expert advice, encouragement and his brilliant support which allows me to complete my work throughout difficult times. I am also very thankful to him for sharing his precious knowledge and experiences which was very fruitful in carrying out my research work. I wish to express my sincere thanks to all the faculty members of the Department of Zoology, Mizoram University, Prof. G.S Solanki, Dean, School of Life Sciences, Prof. G. Gurusubramanian, Head of Department, Dr. Esther Lalhmingliani, Dr. H.T Lalremsanga, Dr. Vikas, Dr. Zothansiama and Dr. Amit for their valuable and timely support. I am also grateful to Dr. Rosy Lalnunsangi, Dr. Zonuntluangi Bawitlung, Lalduhsaki Ralte, Pu Lalhmangaiha, RD-a and all the other staffs, who have helped me in various ways in times of need. I am indebted to my colleagues, Dr. Longjam Shantabi, Dr. M. Vabeiryureilai, Dr. K. Lalrinzuali, K. Lalhminghlui, C. Lalrinengi, Hari Krishna Verma (L), N. Bonika Devi, B. Lalruatfela, Jeremy Malsawmhriatzuala, Sanjeev, Sunita Devi, Mr. Bipra Singh and all my fellow research scholars, for their valuable collaboration and timely help during the course of work. My sincere thanks go to Prof. Senthil Kumar, former Head, Department of Biotechnology, for providing the required facilities to carry out my work and all the other research scholars of the department for helping me out whenever I needed. I would like to express my gratitude to Prof. Amritesh Kumar Shukla, Dr. Awadesh and all the staff members of Department of Horticulture, Aromatic and Medicinal Plants for their support while carrying out my work. My sincere thanks also go to Prof. Diwakar Tiwari, Head, Department of Chemistry, Mizoram University, for allowing me to access the required facility, it would have been impossible to finish my work without their support. This work would not have been possible without the financial support of the Department of Biotechnology (DBT) and University Grant Commission (UGC), Government of India, New Delhi. Last but not the least I would like to express my heartfelt gratitude to my parents, Mr. Lalkima Ralte and Mrs. Lalsangzuali for their never ending love and guidance in the pursuit of this degree and without whom I cannot pursue anything in life, and to all my loving and supporting family members and friends. Aizawl Jennifer Zoremsiami CONTENTS Page No. List of tables List of figures Abbreviations CHAPTER I General introduction 1-24 Phytochemical profiling of Helicia nilagirica CHAPTER II 25-45 (Bedd.) Determination of free radical scavenging activity CHAPTER III 46-65 of Helicia nilagirica (Bedd.) in vitro Evaluation of the antineoplastic activity of CHAPTER IV Helicia nilagirica (Bedd.) in preclinical models in 66-88 vitro Investigation of the anticancer activity of Helicia CHAPTER V nilagirica (Bedd.) in the Swiss albino mice 89-118 transplanted with Dalton’s lymphoma tumor cells Summary and conclusions 119-125 List of tables Chapter II Table 1: Results of the Phytochemical analysis of Helicia nilagirica Table 2: Quantitative determination of the chemical constituent of Helicia nilagirica. Table 3: Percentage of loss on drying fresh bark of Helicia nilagirica Table 4:Physicochemical parameters of dried bark powder of Helicia nilagirica Table 5: Yield of various extracts of Helicia nilagirica. Table 6: TLC profile of the different extracts of Helicia nilagirica on pre- coated aluminium TLC plates. Table 1: Effect of different concentrations on the cytotoxic effects of aqueous Chapter IV extract of Helicia nilagirica in various cell lines by conventional MTT assay. Table 2: Effect of different exposure time on the cytotoxic effects of aqueous extract of Helicia nilagirica and DOX in various cell lines by MTT assay at different post treatment time. Table 3: Effect of different concentrations of the aqueous extract of Helicia nilagirica (HNA) and doxorubucin (DOX) treatment on the survival of HeLa cells. Table 4: Alterations in the Glutathione contents of HeLa cells induced by different concentrations of Helicia nilagirica (HNA) and doxorubicin. Table 5: Alterations in the GST activity of HeLa cells treated with different concentrations of Helicia nilagirica (HNA) and doxorubicin. Table 6: Alterations in the catalase activity of HeLa cells treated with different concentrations of Helicia nilagirica (HNA) and doxorubicin (DOX). Table 7: Alterations in the SOD activity of HeLa cells treated with different concentrations of Helicia nilagirica extract (HNA) and doxorubicin (DOX). Table 1: Acute toxxcity of different extracts of Helicia nilagirica in Swiss Chapter V albino mice after intraperitoneal administration. Table 2: Change in the body weight of Dalton’s lymphoma bearing Swiss albino mice administered with ethanol or aqueous extract of Helicia nilagirica. Table 3: Alteratiuon in the Survival of Dalton’s lymphomas ascites bearing mice treated with various doses of Helicia nilagirica intraperitoneally. Table 4: Alteration in the survival time of Dalton’s lymphoma ascites bearing mice treated with different extracts of Helicia nilagirica. Table 5: Frequency of micronuclei in Dalton’s lymphoma ascites bearing mice treated with 175 mg/kg b.wt. aqueous extract of Helicia nilagirica (HNA) or 0.5 mg/kg b. wt. doxorubucin (DOX) at different post treatment time. Table 6: Induction of apoptosis and necrosis in Dalton’s lymphoma ascites bearing mice treated with 175 mg/kg.b.wt. aqueous extract of Helicia nilagirica (HNA) or 0.5 mg/kg b.wt. doxorubucin (DOX) at different post treatment time. Table7: Alteration in the glutathione (GSH) contents of mice bearing Dalton’s lymphoma treated with 175 mg/kg.b.wt. aqueous Helicia nilagirica extract (HNA) or 0.5 mg/kg b.wt. doxorubicin (DOX). Table 8: Alteration in the glutathione-S-transferase (GST) activity of Mice bearing Dalton’s lymphoma treated with 175 mg/kg.b.wt. aqueous Helicia nilagirica extract (HNA) or 0.5 mg/kg b.wt. doxorubicin (DOX). Table 9: Alteration in the catalase activity of mice bearing Dalton’s lymphoma treated with 175 mg/kg.b.wt. aqueous Helicia nilagirica extract (HNA) or 0.5 mg/kg b.wt. doxorubicin (DOX). Table 10: Alteration in the superoxide dismutase (SOD) activity in mice bearing Dalton’s lymphoma treated with 175 mg/kg.b.wt. aqueous Helicia nilagirica extract (HNA) or 0.5 mg/kg b.wt. doxorubicin (DOX). Table 11: Alterations in the lipid peroxidation level in mice bearing Dalton’s lymphoma treated with 175 mg/kg.b.wt. aqueous Helicia nilagirica extract (HNA) or 0.5 mg/kg b.wt. doxorubicin (DOX). Table 12: Effect of 175 mg/kg.b.wt. aqueous Helicia nilagirica extract (HNA) or 0.5 mg/kg b.wt. doxorubicin (DOX) on the liver and kidney function tests of Dalton’s lymphoma bearing mice. List of figures Chapter II Figure 1: Phytochemical screening of H.nilagirica extracts. Figure 2: TLC profile of different extracts of H.nilagirica using different solvent systems observed under normal light to detect phytochemicals present in the extracts (aqueous, chloroform and ethanol). Figure 3: TLC profile of H.nilagirica on different solvent systems observed under UV 365 nm to detect phytochemicals present in the extracts (aqueous, chloroform and ethanol). Figure 4: TLC profile of H.nilagirica on different solvent systems observed under UV 254 nm to detect phytochemicals present in the extracts (aqueous, chloroform and ethanol). Chapter III Figures 1-8: Effect of different Helicia nilagirica extracts on the free radical scavenging activity. 1) DPPH; 2) Hydroxyl; 3) Superoxide anion 4) ABTS cation 5) Nitric oxide; 6) Reducing power; 7) Total phenol 8) Total flavonoid Chapter IV Figure 1: Cytotoxic effect of different concentrations of aqueous extract of Helicia nilagirica in V79 (a) and HeLa (b) cell lines by conventional MTT assay. Figure 2: Cytotoxic effect at different exposure time of the aqueous extract of Helicia nilagirica and DOX in V79 (a) and HeLa (b) cell lines by MTT assay. Figure 3: Effect of different concentrations of the aqueous extract of Helicia nilagirica (HNA) and Doxorubucin (DOX) treatment on the survival of HeLa cells. Figure 4: Alteration in the GSH content of HeLa cells induced by different concentrations of Helicia nilagirica and doxorubicin. Figure 5: Alteration in the GST activity of HeLa cells induced by different concentrations of Helicia nilagirica and doxorubicin. Figure 6: Alteration in the catalase activity of HeLa cells induced by different concentrations of Helicia nilagirica and doxorubicin. Figure 7: Alteration in the catalase activity of HeLa cells induced by different concentrations of Helicia nilagirica and doxorubicin. Chapter V Figure 1: Change in body weight of Dalton’s lymphoma bearing Swiss albino mice after treatment with different concentrations of the aqueous extract of Helicia nilagirica. Figure 2: Effect on the survivality of Dalton’s lymphoma ascites bearing mice after 9 days consecutive treatment with different doses of a) ethanol and b) aqueous extracts of Helicia nilagirica in vivo. Figure 3: Effect of extract of Helicia nilagirica in Dalton’s lymphoma ascites bearing mice on the tumor response assessment based on average survival time (AST), median survival time (MST), mean life span (% IMLS) and increase in average life span (% IALS) . Figure 4: Induction of micronuclei in Dalton’s lymphoma ascites bearing mice treated with 175mg/kg.b.wt. aqueous extract of Helicia nilagirica (HNA) and 0.5mg/kg.b.wt. doxorubucin (DOX) at different post treatment time. Figure 5: Induction of apoptosis and necrosis in Dalton’s lymphoma ascites bearing mice treated with 175 mg/kg.b.wt.
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