PHYTOCHEMICAL SCREENING AND PROXIMATE ANALYSIS OF CASTANOPSIS INDICA A DISSERTATION SUBMITTED FOR THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE MASTERS DEGREE OF SCIENCE IN CHEMISTRY BY Prakash Shrestha Exam Symbol No.: Chem.462/072 T.U. Regd. No.: 5-2-0022-0174-2011 CENTRAL DEPARTMENT OF CHEMISTRY INSTITUTE OF SCIENCE AND TECHNOLOGY TRIBHUVAN UNIVERSITY, KIRTIPUR KIRTIPUR, KATHMANDU NEPAL JUNE, 2019 1 BOARD OF EXAMINER AND CERTIFICATE OF APPROVAL This dissertation entitled “PHYTOCHEMICAL SCREENING AND PROXIMATE ANALYSIS OF Castanopsis indica” by Prakash Shrestha, under the supervision of Prof. Dr. Ram Chandra Basnyat Central Department of Chemistry, Tribhuvan University, Nepal, is hereby submitted for the partial fulfillment of the Master of Science (M.Sc.) Degree in Chemistry. This dissertation has not been submitted in any other university or institution previously for the award of a degree. Supervisior Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan University Kritipur, Kathmandu, Nepal Internal Examiner External Examiner Asst. Prof. Surya Kant Kalauni, PhD Prof. Daman Raj Gautam, PhD Central Department of Chemistry Amrit Science College Tribhuvan University Tribhuvan University Kritipur, Kathmandu, Nepal Lainchaur, Kathmandu Head of the Department Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan University Kritipur, Kathmandu, Nepal ii RECOMMENDATION LETTER This is to certify that the dissertation work entitled “PHYTOCHEMICAL SCREENING AND PROXIMATE ANALYSIS OF Castanopsis indica” has been carried out by Prakash Shrestha as a partial fulfillment for the requirement of Master Degree in Chemistry under my Supervision. To the best of my knowledge, this work has not been submitted to any other degree in this institute. ________________________________ Supervisor Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan, University Kirtipur, Kathmandu, Nepal iii DECLARATION I, Prakash Shrestha, hereby declare that the work presented herein is genuine work done originally by me and has not been published or submitted elsewhere for the requirement of a degree program. Any literature, data or works done by others, presented in this dissertation are cited, has been given due acknowledgement and listed in the reference section. ________________________________ Prakash Shrestha Central Department of Chemistry Tribhuvan University Kirtipur, Kathmandu, Nepal iv ACKNOWLEDGEMENTS The successful completion of this study was possible with the support, assistance, and advice of various people. I would like to express my gratitude and appreciation to the following: I would like to express my deepest gratitude and sincere appreciation to my supervisor and Head of the Central Department of Chemistry Prof. Ram Chandra Basnyat, PhD, Central Department of Chemistry, for his constant support, inspirable guidance, invaluable suggestions, and regular feedback at all stages of my dissertation work. My deepest appreciation goes to all my respected teachers of CDC. I would also like to convey my thanks to all the administration staff as well as laboratory staffs of the department for their continuous assistance throughout the dissertation work. I would especially like to thank National Herbarium and Plant Laboratories (KATH), for the identification of plants. I am thankful to the authors of books and articles which I have used as reference in this work. I would also like to thank Department of Food Technology and Quality Control for determination of proximate analysis. I am thankful to Pushpa Bhattarai for her cooperation. My special thanks go to all my colleagues whom I have been surrounded and enjoyed, especially for their co-operation, support and encouragement during the work. I would also like to convey my thanks to my seniors and my juniors for their help and suggestion during this work. Last but not least, my sincere obligation goes to my dearest family. Thank you so much your love, encouragement, inspiration and cooperation that have made this dissertation possible. Thank you all. Prakash Shrestha Email: [email protected] v LIST OF TABLES Table 1: Chemical composition of sea water .......................................................... 24 Table 2: Sample collection and identification ......................................................... 33 Table 3: Table showing % yield of methanol and hexane extract .......................... 33 Table 4: Phytochemical analysis of leaves and bark of Castanopsis indica ............ 34 Table 5: Total phenolic content of Castanopsis indica extracts ............................. 38 Table 6: Total Flavonoid content of Castanopsis extracts ...................................... 39 Table 7: Antimicrobial activity of methanol extract of bark and leaves ................. 40 Table 8: Antifungal activity of methanol extract of Castanopsis indica ................. 41 Table 9: Calculation of LC50 value of methanol extract of leaves and bark of Castanopsis indica ..................................................................................... 42 Table 10: Percentage composition of nutritional parameters in sample of Castanopsis indica in nut ........................................................................... 43 Table 11: Antioxidant activity of Ascorbic acid ........................................................ 55 Table 12: Antioxidant activity of methanol extract of bark ...................................... 55 Table 13: Antioxidant activity of methanol extract of leaf ....................................... 55 Table 14: Antioxidant activity of hexane extract of bark .......................................... 56 Table 15: Antioxidant activity of hexane extract of leaf ........................................... 56 Table 16: Total phenolic content in methanol extract of bark ................................... 56 Table 17: Total phenolic content in methanol extract of leaf .................................... 57 Table 18: Total phenolic content in hexane extract of bark ...................................... 57 Table 19: Total phenolic content in hexane extract of leaf ....................................... 57 Table 20: Total flavonoid content in methanol extract of bark ................................. 58 Table 21: Total flavonoid content in hexane extract of leaf ...................................... 58 Table 22: Total flavonoid content in methanol extract of leaf .................................. 58 Table 23: Total flavonoid content in hexane extract of lark ...................................... 59 Table 24: Determination of moisture ........................................................................ 59 Table 25: Determination of total ash ......................................................................... 59 Table 26: Determination of total nitrogen ................................................................. 59 Table 27: Determination of crude fiber ..................................................................... 60 Table 28: Determination of crude fat ........................................................................ 60 vi LIST OF FIGURES Figure 1: Castanopsis indica plant ........................................................................... 3 Figure 2: Castanopsis indica nuts ............................................................................. 3 Figure 3: Mechanism of DPPH free radical scavenging ........................................... 6 Figure 4: Mechanism of DPPH free radical scavenging by ascorbic acid ................ 6 Figure 5: Structure of compound found in Castanopsis indica .............................. 15 Figure 6: Scheme for extraction, fractionation and analysis of bark and ……………leaves of Castanopsis indica ................................................................... 17 Figure 7: Antioxidant activity of ascorbic acid ...................................................... 35 Figure 8: Antioxidant activity of methanol extract of Castanopsis indica leaf ...... 35 Figure 9: Antioxidant activity of methanol extract of Castanopsis indica bark ..... 36 Figure 10: Antioxidant activity of hexane extract of Castanopsis indica leaf .......... 36 Figure 11: Antioxidant activity of hexane extract of Castanopsis indica bark ......... 37 Figure 12: Absorbance vs concentration curve for gallic acid ................................. 38 Figure 13: Absorbance vs concentration curve for Quercetin .................................. 39 Figure 14: Percentage composition of nutritional parameters in nuts of Castanopsis indica........................................................................................................ 44 vii LIST OF ACRONYMS AND ABBREVIATIONS C. indica : Castanopsis indica DMSO : Dimethyl Sulphoxide DPPH : 1, 1-diphenyl-2-picryl hydrazyl FCR : Folin-Ciocalteau Reagent GAE : Gallic Acid Equivalent IC50 : Inhibitory Concentration for 50% Inhibition IR : Infra-Red LC50 : Lethal Concentration for 50% Mortality MHA : Mueller Hinton Agar MHB : Mueller Hinton Broth ppm : Part per million QE : Quercetin Equivalent ROS : Reactive Oxygen Species TFC : Total Flavonoid Content TPC : Total Phenolic Content UV : Ultraviolet ZOI : Zone of Inhibition viii ABSTRACT Castanopsis indica also known as Katus was selected as the plant for the dissertation work. Methanol and hexane extracts of leaves and bark of C. indica was prepared by Soxhlet extraction method. Phytochemical screening results showed the presence of glycosides, polyphenols, and tannins