A STUDY ON BIOCHEMICAL ANALYSIS OF SOME TRADITIONALLY USED HEPATOPROTECTIVE MEDICINAL PLANTS & IN-VIVO ASSAY IN RODENT MODEL A THESIS SUBMITTED TO BODOLAND UNIVERSITY, KOKRAJHAR, ASSAM FOR THE AWARD OF THE DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF BIOTECHNOLOGY, UNDER THE FACULTY OF SCIENCE AND TECHNOLOGY. SUBMITTED BY HANKHRAY BORO ENROLLMENT NO: PHDBT13003 PH. D REGISTRATION NO: FINAL/03BIO0003 OF 2013- 2014 SUPERVISED BY DR. SANDEEP DAS DEPARTMENT OF BIOTECHNOLOGY, BODOLAND UNIVERSITY, KOKRAJHAR, ASSAM, INDIA, 783370. JUNE, 2019. Certificate This is to certify that the Ph.D thesis entitled “A study on biochemical analysis of some traditionally used hepatoprotective medicinal plants & in-vivo assay in rodent model” is a bonafide record of Ph.D work done by Mr. Hankhray Boro under my supervision during the year 2013 to 2019. He has been registered under Bodoland University vide registration no: 001915 of 2013-14 and the Ph.D enrollment no: PHDBT13003 along with the final registration no: FINAL/03BIO0003 of 2013-2014. The thesis has not been previously submitted for the award of any degree, diploma or other research title. Signature of Supervisor (Dr. SANDEEP DAS) Department of Biotechnology Bodoland University Kokrajhar, Assam, India, 783370. Declaration I Hankhray Boro declare that the thesis entitled “A study on biochemical analysis of some traditionally used hepatoprotective medicinal plants & in-vivo assay in rodent model” is a bonafide Ph.D work done by me under the supervision of Dr. Sandeep Das (Associate Professor, Dept. of Biotechnology, Bodoland University). This thesis is submitted to the Department of Biotechnology, Bodoland University for the award of degree for Doctor of Philosophy bearing registration no: 001915 of 2013-14 and the Ph.D enrollment no: PHDBT13003 and the final registration no: FINAL/03BIO0003 of 2013-2014. The thesis has not been previously submitted for the award of any degree, diploma or other research title. (Hankhray Boro) Place: Kokrajhar Department of Biotechnology Dated: 14th Nov, 2019. Bodoland University i CONTENTS Title Pages Acknowledgment v Abstract vii List of Tables ix List of Figures x List of Appendices xi CHAPTER-I Introduction 1-4 1.1 Introduction 1 1.2 Necessity of the research 3 1.3 Research problem 4 1.4 Objectives 4 CHAPTER- II Literature Review 5-10 2.1 Survey review 5 2.2 Plant phytochemicals and antioxidant review 6 2.3 Hepatoprotective review 7 2.4 GC-MS analysis review 9 2.5 Docking review 9 CHAPTER- III Description of Plants 11-12 3.1 Sample selection 11 3.2 Morus indica L. 11 3.3 Averrhoa carambola L. 11 3.4 Phlogacanthus thyrsiflorus Nees. 12 CHAPTER- IV Materials and Methods 13-24 4.1 Area of study 13 4.2 Topography 13 4.3 Collection and Identification 13 ii 4.4 Extract preparation of sample 14 4.5 Phytochemical screening 14 4.6 In-vitro antioxidant tests 16 4.6.1 Total phenolic contents 16 4.6.2 Total flavonoid contents 16 4.6.3 Total reducing power assay 16 4.6.4 Total antioxidant capacity 16 4.6.5 DPPH radical scavenging assay 17 4.6.6 ABTS radical cation scavenging assay 17 4.6.7 Iron chelating capacity 17 4.6.8 H2O2 radical scavenging assay 18 4.6.9 Ferric reducing antioxidant assay 18 4.7 Gas chromatography coupled with mass-spectrometry analysis 18 4.8 In-vivo animal model experiment 19 4.8.1 Experimental animal 19 4.8.2 Acute toxicity study 19 4.8.3 Experimental design 19 4.8.4 Assessment of serum biochemical assays 20 4.8.5 Assessment of levels of antioxidant enzymes 20 4.8.5.1 Superoxide dismutase (SOD) assay 21 4.8.5.2 Catalase assay 21 4.8.5.3 Gluthathione peroxidase 21 4.8.5.4 Gluthathione assay 21 4.8.5.5 Lipid-peroxidation 22 4.8.5.6 Total protein assay 22 4.9 Histopathological study 22 iii 4.10 Molecular docking with hepatoprotective bio-target 22 4.10.1 Ligand selection 22 4.10.2 Selection of target protein 23 4.10.3 Ligand molecules 23 4.10.4 Ligand preparation 23 4.10.5 Preparation of receptor 23 4.10.6 Molecular docking 24 4.10.7 ∆G binding affinity 24 4.10.8 ADME property of ligands 24 4.10.9 Statistical analysis 24 CHAPTER- V Results 25-59 5.1 Hepatoprotective medicinal plants 25 5.2 Some herbal formulation 29 5.3 Extract yield 30 5.4 Phytochemical screening 30 5.5 Total phenolic & flavonoid content 31 5.6 Total reducing power assay 33 5.7 Total antioxidant capacity 34 5.8 DPPH radical scavenging assay 35 5.9 ABTS radical scavenging assay 37 5.10 Iron chelating capacity 38 5.11 H2O2 radical scavenging activity 40 5.12 Ferric reducing antioxidant property 41 5.13 GC-MS analysis 42 iv 5.14 In-vivo hepatoprotective study 45 5.14.1 Acute toxicity study 45 5.14.2 Effect of RoMi interventions on biochemical parameters 45 5.14.3 Effect of RoMi on parameters of oxidative stress in liver 47 5.15 Effect of interventions on histopathological changes in liver 50 5.16 Effect of interventions on histopathological changes in kidney 52 5.17 Docking analysis of NFκB protein 53 5.17.1 Receptor sitemap & site score 53 5.17.2 Ligand docking 54 5.18 Docking analysis of COX-2 protein 56 5.19 ADME property of ligands 58 CHAPTER- VI Discussion 60-72 CHAPTER-VII Conclusion 73-74 CHAPTER- VIII Summary 75-77 CHAPTER- IX Salient Features of the Study 78 CHAPTER- X Bibliography 79-97 Appendix-A Questionnaires used 98-99 Appendix-B GC-MS chromatogram 100-101 Appendix-C Chemicals used 102-103 Appendix-D Instruments used 104 Appendix-E Paper publication 105 Appendix-F Seminar presentation 106 Appendix-G Photos of medicinal plants 107-113 Appendix-H Abbreviations used 114-115 v Acknowledgment The present compilation is the result of comprehensive work of my Ph.D thesis from the Department of Biotechnology, Bodoland University, Kokrajhar, Assam, during a period of 2013-2019. The whole period was one of the most intensive stage for me, which was not possible without the collective supports, inspiration and valuable suggestions from a number of individuals. At the very beginning, I owe my thanks and gratitude to the kindness, confidence and patience showered by my supervisor Dr. Sandeep Das, Department of Biotechnology, Bodoland University, Kokrajhar, for his worthless supervision, suggestion and incessant encouragement all through my research work. This research work was possible only because of his well planning and arrangement as well as his advice and focus to achieve the goal. Sir, my heartfelt thanks goes to you for your kind companion throughout the research work. I would like to extend my sincere gratitude to Dr. Jatin Sharma (Associate professor), Dr. Sharmistha Brahma Kaur (Assistant professor) and Mrs. Jonali Owary (Assistant professor), Department of Biotechnology, Bodoland University, for giving me valuable suggestion as well as constant encouragement throughout the research work. I utilize this opportunity to extend my sincere, gratitude and gratefulness to former Hon’ble Vice-Chancellor, Prof. Hemanta Kumar Baruah, former registrar, Dr. Sekhar Brahma and all the administrative personnel of Bodoland University for their authoritative approval and necessary help in the period of my research work. With deepest affection, I attempt to extend my thanks to NFHE, UGC, Govt. of India, for providing fellowship. I must express my hearty gratitude to the Director of MLACW and Dr. M.B. Nagaveni (HOD), Department of Biotechnology and Biochemistry, Maharani Lakshmi Amman College for Women, Malleswaram, Bengaluru, for allowing me an authoritative approval to pursue the experimental research works in their institution. I express my heartfelt thanks to Dr. Sushil Kumar Middha and Smt. Usha Talambedu, Department of Biotechnology and Biochemistry, Maharani Lakshmi Ammani College for Women, Bengaluru for allowing and steering me to execute the animal model experimentation in their organised laboratory (Life Science Research Centre) and spending their valuable time for completion of my experimental research work. vi I express my whole hearted thanks to Dr. A. A. Mao, Scientist- ‘F’, Joint Director, Dr. Ranjit Daimary and Ms. Chaya Deori, BSI, Eastern Regional Centre, Shillong for their significant resourcefulness for identification and authentication of my research specimen. With deepest affection, I attempt to extend my thanks to all the supporting non- teaching staff of the Department of Biotechnology and the J.R.F of Biotech-hub as well as Mushroom Spawn Unit for their imperative endorsement, necessary and continuous help throughout my research. I wish to express my profound sense of gratitude to Dr. Chawngthanliana, Director of RIPANS, Mizoram, Mr. Vinod Devaraji, Application Scientist of Schrodinger and Dr. M. Ravikumar, Senior Application Scientist, Schrodinger for their significant resourceful assist in carrying out the docking work smoothly for my research. Words evade me to express a great debt of thankfulness to my mother Smt. Amila Gayary, father Mr. Mukunda Ram Boro and all my family members for their constant inspirations with moral support and endless caring with courage in my academic life. I express my heartfelt debt of gratitude with special appreciation to my beloved wife Mrs. Phungja Brahma, without whom the survey work would have been very tough. Her love, caring, moral and constant co-operative motivation shouldered me to achieve this milestone. Besides, I would like to offer my sincere thanks to all associate scholar friends for uphold of excellent spirit of research environment with kind co-operation. Indeed the words at my command are just not sufficient to express my sincere thanks and deep sense of profound respect to my native villagers of Purana Titaguri and the people/ herbal medicine practitioner who shared lot of information and everyone who directly or indirectly helped me to achieve this research successfully.