PHYTOCHEMICAL AND PHARMACOLOGICAL INVESTIGATION ON THE STEM OF Piper chaba Hunter. A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTERS OF PHILOSOPHY (M. PHIL.) IN CHEMISTRY SUBMITTED BY ANATH CHANDRA ROY STUDENT NO. : 0413033106 P REGISTRATION NO. : 0413033106 SESSON: APRIL-2013 ORGANIC RESEARCH LABORATORY DEPARTMENT OF CHEMISTRY BANGLADESH UNIVERSITY OF ENGINEERING AND TECHNOLOGY (BUET), DHAKA-1000, BANGLADESH FEBRUARY, 2017 DEDICATED TO MY PARENTS LATE Dr. SURENDRA NATH ROY And LATE GOURI RANI ROY DECLARATION This thesis work has been done by the candidate himself and does not contain any material extracted from elsewhere or from a work published by anybody else. The work for this thesis has not been presented elsewhere by the author for any degree or diploma. ____________________ Anath Chandra Roy M. Phil Student Roll No. 0413033106P Department of Chemistry BUET, Dhaka Bangladesh -1000. ACKNOWLEDGEMENT At first, I would like to express my gratitude to almighty Allah, who blessed me and given me the capability of completing the research successfully as well as to complete my M. Phil Degree. I am filling proud to express my sense of gratitude and sincere appreciation to Dr. Shakila Rahman, Professor, Department of Chemistry, BUET, my honorable supervisor and my ideal. She is the one who opened the door of research, freedom of thinking and shown the high way of organic chemistry for me. Without her unquenched trust of research, scholastic supervision, continuous encouragement and constructive suggestion, this achievement would not be possible. It is my great pleasure and I feel so proud to express my depth of gratitude and respect, indebtness and appreciation to Dr. Nasim Sultana, Principal Scientific Officer, Institute of National Analytical Research and Service (INARS), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka, my well wisher, for her constant supervision, affectionate inspiration, encouragement throughout the entire period of my research work. Her affectionate feelings and caring and an endless support during the entire course of this work inspired me to complete this work. I have been fortunate to learn a great deal of Chemistry from her. I extend my sincere thanks to all my honorable teachers of the Chemistry department of BUET, Dhaka for their help and co-operation during the course of this work. Specially, I am highly obliged to Prof. Dr. Abdur Rasid, Prof. Dr. Md. Wahab Khan, Prof. Dr. Md. Shakowat Hossain Feroze, Dr. Chanchal kumar Roy, Department of Chemistry, BUET and Dr. Abul Hashem, Professor, Department of Chemistry, JU for their helpful cooperation and continuous embodiment. I would like to thank Mr. Munsur Al- Neyam, Mr. Shamim Hossain, Mr. Ahedul Akbor, Mr. Mazedul Haque, Senior Sciencetific Officer, BCSIR, Mr. Mazharol Haque, Mr. Kamal Hossain, Associate Professor and Mr. Moinul Haque, Assistant Professor DCC, Dhaka for their friendly behavior and sharing experience. I wish to render my thank to all of my well wishers, specially to Chapol Roy, Mukunda Paul, Masbah Uddin Ahmed, Tapash Mujomdar, Altaf khan, Farjana Yesmain, Jobayet Tanvir and Nur- E-Jannat for their amicable contribution. I am thankful to all the staff members of the Chemistry Department of BUET, specially to Mr. Kobir, Mr. Mamun, Mr. Hakim, Mr. Alamgir and Mr. Rasul for their helps during the period of my university life. I am grateful to the Committee for Advanceed Studies and Research (CASR), BUET, Dhaka, for providing me with financial support in caring out this research project. Finally, my cordial thanks belong to my beloved wife Ashalata Roy, Monindra Nath Roy, Probitra Roy, Sraboni Das, Jibon Bikash Roy, Nittyajit Mondal, Pankoj Roy, Bithika Roy, Dhruba and Dhriti Roy for their continuous encouragement. Author ANATH CHANDRA ROY Contents PART-1 Page No. PHYTOCHEMICAL INVESTIGATION OF Piper chaba Hunter. ABSTRACT CHAPTER 1 Introduction 1.10 Aim of the project 29 1.11 Present study protocol 30 CHAPTER 2 Experimental 2.0 General methods 31 2.1 Solvents and chemicals 31 2.2 Distillation of solvents 31 2.3 Evaporation 32 2.4 Preparation of the reagents 32 2.5 Chromatographic techniques 33 2.6 Re-crystallization 37 2.7 Spectroscopic techniques 38 2.8 Investigation of Piper chaba Hunter. 38 2.8.9 Extraction and isolation of the compounds from the stem of 47 Piper chaba Hunter. 2.8.9.1 Investigation of n-hexane extract 41 2.8.6.2 Investigation of the ethyl acetate extract 44 2.9.1 Properties of compound -1 48 2.9.2 Properties of compound -2 50 2.9.3 Properties of compound -3 52 2.9.4 Properties of compound -4 54 CHAPTER 3 Results and Discussion 3.1 Preliminary investigation of the plant material 55 3.2 Characterization of isolated compounds from Piper chaba Hunter. 55 3.2.1 Characterization of compound-1 as stigmasterol 55 3.2.2 Characterization of compound-2 as Piperine 64 3.2.3 Characterization of compound-3 as 4ʹ, 5ʹ-dimethyl -3ʹ-hydroxy 77 isoflavan 3.2.4 Characterization of compound-4 as 3ʹ, 5ʹ-dimethyl -4ʹ-hydroxy 87 isoflavan PART-2 PHARMACOLOGICAL INVESTIGATION OF Piper chaba Hunter. CHAPTER 1 Antioxidant Activity 4.1 Introduction 99 4.2 Materials and methods 101 4.3 Results and discussion 107 CHAPTER 2 Antimicrobial Screening 5.1 Introduction 108 5.2 Principle of disc diffusion method 108 5.3 Experimental 110 5.4 Results and discussion of in vitro antimicrobial screening of Piper chaab 119 Hunter. CONCLUSION 120 REFERENCES 121 List of Tables Serial No. Tropics Page No. Table 1.1 Important drugs/chemicals from plant source and their actions. 10 Table 2.1 Fractions collected from solid-liquid column chromatography of 44 n-hexane extract (2.28g) using different solvent system. Table 2.2 Fractions collected from solid-liquid column chromatography of 47 n-hexane extract (4.32g) using different solvent system. Table 2.3 Isolation and purification of compound-2 by pipette column from 51 the fractions 73-85 (967mg). Table 2.4 Isolation and purification of compound-3 by pipette column from 53 the fractions 91-97 (624mg). Table 2.5 Isolation and purification of compound-4 by pipette column from 55 the fractions 103-115,127 (913mg). 13 Table 3.1 C-NMR data of compound-1 (PCH-1) compared with published 58 data of stigmasterol. 13 Table 3.2 C-NMR data of compound-2 (PCE-1) compared with published 66 data of Piperine 13 Table 3.3 C-NMR data of compound-3 (PCE-2) compared with published 79 data of 4ʹ,5ʹ- dimethyl-3ʹ-hydroxy isoflavan. 13 Table 3.4 C-NMR data of compound-4 (PCE-3) compared with published 89 data of 3ʹ,5ʹ- dimethyl-4ʹ-hydroxy isoflavan. Table 4.1 For DPPH radical scavenging activity of ethyl acetate extract. 104 Table 4.2 For DPPH radical scavenging activity of pure compound 105 Piperine. Table 4.3 For DPPH radical scavenging activity of Ascorbic acid 106 (standard). Table 5.1 List of the test bacteria and fungi. 110 Table 5.2 Antimicrobial activity of crude n-hexane extract, ethylacetate 117 extract, methanol extract and ethanol extract of Piper chaba. Table 5.3 Antimicrobial activity of the four pure compounds ( PCH-1, PCE- 118 1, PCE-2, PCE-3) of n-hexane and ethyl acetate extract of Piper chaba. List of the figures Serial No. Tropics Page No. Figure 1.1 Vincristine 3 Figure 1.2 Calanolide A 4 Figure 1.3 Artemisinin 4 Figure 1.4 Artemether 4 Figure 1.5 Huperzine 5 Figure 1.6 Galathmine 5 Figure 1.7 Acarbose 6 Figure 1.8 Colforsin daproate 6 Figure 1.9 Triptolide 6 Figure 1.10 Ginkgolide B 7 Figure 1.11 Gomisin A 7 Figure 1.12 Dextromethorphan 8 Figure 1.13 Camptothecin and Toptican 8 Figure 1.14 Quinine 9 Figure 1.15 Piper chaba plant with fruits 13 Figure 1.16 Whole plant of piper chaba 13 Figure 1.17 Piper chaba plant 14 Figure 1.18 Stem of piper chaba 15 Figure 1.19 5-hydroxy-7,8-dimethoxyflavone 17 Figure 1.20 Piperine 17 Figure 1.21 Pipernoaline 17 Figure 1.22 Guineensine 18 Figure 1.23 isobutylamide 18 Figure 2.1 Distillation process 31 Figure 2.2 Rotary vacuum evaporator 32 Figure 2.3 Spray reagent 32 Figure 2.4 Process of spoting 33 Figure 2.5 Developing of TLC plate 34 Figure 2.6 TLC plates under UV lamp 35 Figure 2.7 Calculation of Rf value 35 Figure 2.8 A plate for the calculation of Rf value 36 Figure 2.9 Various part of a column 37 Figure 2.10 Various stages in micro scale column 38 Figure 3.1 Structure of stigmasterol 56 Figure 3.2 IR spectrum of compound-1 59 Figure 3.3 1H NMR spectrum of compound-1 60 Figure 3.4 Expanded 1H NMR spectrum of compound-1 61 Figure 3.5 13C NMR spectrum of compound-1 62 Figure 3.6 Expanded 13C NMR spectrum of compound-1 63 Figure 3.7 Structure of Piperine 64 Figure 3.8 UV spectrum of compound-2 67 Figure 3.9 IR spectrum of compound-2 68 Figure 3.10 1H NMR spectrum of compound-2 69 Figure 3.11 Expanded 1H NMR spectrum of compound-2 70 Figure 3.12 Expanded 1H NMR spectrum of compound-2 71 Figure 3.13 13C NMR spectrum of compound-2 72 Figure 3.14 Expanded 13C NMR spectrum of compound-2 73 Figure 3.15 DEPT-135 spectrum of compound-2 74 Figure 3.16 Cosy spectrumof compound-2 75 Figure 3.17 Cosy spectrum of compound-2 76 Figure 3.18 Structure of the compound 4ʹ,5ʹ-dimethyl-3ʹ-hydroxy 77 isoflavan Figure 3.19 UV spectrum of compound-3 80 Figure 3.20 IR spectrum of compound-3 81 Figure 3.21 1H NMR spectrum of compound-3 82 Figure 3.22 Expanded 1H NMR spectrum of compound-3 83 Figure 3.23 13C NMR spectrum of compound-3 84 Figure 3.24 Expanded 13C NMR spectrum of compound-3 85 Figure 3.25 DEPT-135 spectrum of compound-3 86 Figure 3.26 Structure of the compound 3ʹ,5ʹ-dimethyl-4ʹ-hydroxy 87 isoflavan Figure 3.27 UV spectrum of compound-4 90 Figure 3.28 IR spectrum of compound-4 91 Figure 3.29 1H NMR spectrum of compound-4 92 Figure 3.30 Expanded 1H NMR spectrum of compound-4 93 Figure 3.31 Expanded 1H NMR spectrum of compound-4 94 Figure 3.32 13C NMR spectrum of compound-4 95 Figure 3.33 Expanded 13C NMR spectrum of compound-4 96 Figure 3.34 Expanded 13C NMR spectrum of compound-4 97 Figure 3.33 DEPT-135 spectrum of compound-4 98 Figure 4.1 How the antioxidant works in our immune system 100 Figure 4.2 Reaction involved in DPPH method 102 Figure 4.3 Plotting Concentration vs.