VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY UNIVERSITY OF SCIENCE  HUYNH BUI LINH CHI STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF THE LICHEN PARMOTREMA PRAESOREDIOSUM (NYL.) HALE (PARMELIACEAE) DOCTORAL THESIS IN CHEMISTRY Ho Chi Minh City, 2014 VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY UNIVERSITY OF SCIENCE  HUYNH BUI LINH CHI STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF THE LICHEN PARMOTREMA PRAESOREDIOSUM (NYL.) HALE (PARMELIACEAE) Subject: Organic Chemistry Code number: 62 44 27 01 Examination Board: Prof Dr Nguyen Minh Duc (1st Reviewer) Assoc Prof Dr Tran Cong Luan (2nd Reviewer) Assoc Prof Dr Pham Dinh Hung (3rd Reviewer) Assoc Prof Dr Le Thi Hong Nhan (1st Independent Reviewer) Dr Le Tien Dung (2nd Independent Reviewer) SUPERVISORS: PROF DR NGUYEN KIM PHI PHUNG PROF DR TAKAO TANAHASHI Ho Chi Minh City, 2014 i SOCIALIST REPUBLIC OF VIETNAM INDEPENDENCE-FREEDOM-HAPPINESS DECLARATION The work presented in this thesis was completed in the period of November 2009 to November 2013 under the co-supervision of Professor Nguyen Kim Phi Phung of the University of Science, Vietnam National University, Ho Chi Minh City, Vietnam and Professor Takao Tanahashi of the Kobe Pharmaceutical University, Japan In compliance with the university regulations, I declare that: Except where due acknowledgement has been made, the work is that of the author alone; The work has not been submitted previously, in whole or in part, to qualify for any other academic award; The content of the thesis is the result of the work which has been carried out since the official commencement date of the approved doctoral research program; Ethics procedures and guidelines have been followed Ho Chi Minh City, Sept 30, 2014 PhD student HUYNH BUI LINH CHI ii ACKNOWLEDGEMENTS There are many individuals without whom the work described in this thesis might not have been possible, and to whom I am greatly indebted Firstly, I wish to thank my supervisor, Prof Dr Nguyen Kim Phi Phung for her knowledge, support, and guidance, hundreds of meetings/emails and for always keeping me on my toes, from the very beginning to the very end of my PhD I would also like to acknowledge my second supervisor, Prof Dr Takao Tanahashi for his guidance, patience and who has taught me the true spirit of research I am deeply indebted to Dr Yukiko Takenaka at Kobe Pharmaceutical University, Japan for her teachings, kindness, helpful suggestion and valuable advice in this research I would also like to express my sincere thanks to PhD Vo Thi Phi Giao at University of Science, Vietnam National University, Ho Chi Minh City and Dr Harrie J M Sipman, Botanic Garden and Botany Museum Berlin-Dahlem, Freie University, Berlin, Germany for his expertise in the identification of lichen I am very grateful to thank Prof Dr Shigeki Yamamoto, Prof Dr Hitoshi Watarai at Osaka University, Japan and PhD Do Thi My Lien for giving up their precious time to help me with CD spectra, sample preparation and proof reading of some isolated compounds of the thesis A special thanks to Dr Le Hoang Duy for his helpful assistance and friendship during my work at Kobe Pharmaceutical University, Japan I would like to acknowledge the encouragement, insightful comments of the rest of examination board: Prof Dr Nguyen Cong Hao, Prof Dr Nguyen Minh Duc, Assoc Prof Dr Tran Cong Luan, Assoc Prof Dr Pham Dinh Hung, Assoc Prof Dr Nguyen Trung Nhan, Dr Pham Nguyen Kim Tuyen and Dr Le Tien Dung iii Similarly, I would also like to thank my teachers, friends and students in the Department of Organic Chemistry, Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City Most importantly, I would like to thank my husband, for being the most patient and supportive witness to my academic journey over the past four years Without his support, love and encouragement, this study would not have been possible Finally, I would like to thank my parents for believing in me and for being proud of me Their unconditional love and support has given me the strength and courage while I am far from home THANK YOU iv TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv LIST OF ABBREVIATIONS vi LIST OF TABLES xi LIST OF FIGURES xiii LIST OF APPENDICES xv INTRODUCTION CHAPTER 1: LITERATURE REVIEW 1.1 GENERIC DESCRIPTION 1.1.1 The lichen 1.1.2 Parmotrema praesorediosum (Nyl.) Hale 1.2 CHEMICAL STUDIES ON THE LICHEN GENUS PARMOTREMA 1.2.1 Lichen secondary metabolites 1.2.2 Chemical studies on the lichen genus Parmotrema 1.3 BIOLOGICAL ACTIVITIES 14 1.3.1 The biological significance of lichen metabolites 14 1.3.2 The biological significance of the lichen Parmotrema 15 CHAPTER 2: EXPERIMENTAL 20 2.1 MATERIALS AND ANALYSIS METHODS 20 2.2 LICHEN MATERIALS 22 v 2.3 EXTRACTION AND ISOLATION PROCEDURES 22 2.3.1 Isolating compounds from the methanol precipitate 22 2.3.2 Isolating compounds from the petroleum ether E1 extract 23 2.3.3 Isolating compounds from the petroleum ether E2 extract 23 2.3.4 Isolating compounds from the chloroform extract 24 2.4 PREPARATION OF SOME DERIVATIVES 28 2.4.1 Esterification of PRAES-C2 28 2.4.2 Methylation of PRAES-C25 29 2.5 BIOLOGICAL ASSAY 29 2.5.1 Cytotoxicity 29 2.5.2 In vitro acetylcholinesterase (AChE) inhibition assay 30 CHAPTER 3: RESULTS AND DISSCUSSION 32 3.1 CHEMICAL STRUCTURE ELUCIDATION 32 3.1.1 Chemical structure of aliphatic acids 33 3.1.1.1 Structure elucidation of compound PRAES-C1 33 3.1.1.2 Structure elucidation of compound PRAES-E14 34 3.1.1.3 Structure elucidation of compound PRAES-C10 35 3.1.1.4 Structure elucidation of compound PRAES-C11 39 3.1.1.5 Structure elucidation of compound PRAES-E19 40 3.1.1.6 Structure elucidation of compound PRAES-C2 41 3.1.2 Chemical structure of mononuclear phenolic compounds 45 3.1.2.1 Structure elucidation of compound PRAES-T1 45 3.1.2.2 Structure elucidation of compound PRAES-E1 46 vi 3.1.2.3 Structure elucidation of compound PRAES-T2 48 3.1.2.4 Structure elucidation of compound PRAES-E11 49 3.1.2.5 Structure elucidation of compound PRAES-T4 50 3.1.2.6 Structure elucidation of compound PRAES-T6 50 3.1.2.7 Structure elucidation of compound PRAES-E2 51 3.1.2.8 Structure elucidation of compound PRAES-C22 53 3.1.2.9 Structure elucidation of compound PRAES-C23 55 3.1.2.10 Structure elucidation of compound PRAES-C24 56 3.1.2.11 Structure elucidation of compound PRAES-C25 59 3.1.2.12 Structure elucidation of compound PRAES-C26 63 3.1.3 Chemical structure of depsides 66 3.1.3.1 Structure elucidation of compound PRAES-T3 66 3.1.3.2 Structure elucidation of compound PRAES-C7 67 3.1.3.3 Structure elucidation of compound PRAES-E18 69 3.1.4 Chemical structure of depsidones 70 3.1.4.1 Structure elucidation of compound PRAES-C14 70 3.1.4.2 Structure elucidation of compound PRAES-C12 73 3.1.5 Chemical structure of diphenyl ethers 74 3.1.5.1 Structure elucidation of compound PRAES-C5 74 3.1.5.2 Structure elucidation of compound PRAES-C15 77 3.1.5.3 Structure elucidation of compound PRAES-C16 79 3.1.5.4 Structure elucidation of compound PRAES-C20 84 3.1.5.5 Structure elucidation of compound PRAES-C18 86 vii 3.1.5.6 Structure elucidation of compound PRAES-C3 89 3.1.5.7 Structure elucidation of compound PRAES-C4 91 3.1.5.8 Structure elucidation of compound PRAES-C21 93 3.1.6 Chemical structure of dibenzofurans 97 3.1.6.1 Structure elucidation of compound PRAES-E5 97 3.1.6.2 Structure elucidation of compound PRAES-E3 99 3.1.6.3 Structure elucidation of compound PRAES-C8 100 3.1.7 Chemical structure of xanthones 103 3.1.7.1 Structure elucidation of compound PRAES-C27 103 3.1.7.2 Structure elucidation of compound PRAES-C28 108 3.1.8 Chemical structure of triterpenoids 111 3.1.8.1 Structure elucidation of compound PRAES-E17 111 3.1.8.2 Structure elucidation of compound PRAES-E6 112 3.1.8.3 Structure elucidation of compound PRAES-E13 113 3.1.9 Chemical structure of a macrocylic compound 117 3.1.9.1 Structure elucidation of compound PRAES-E15 117 3.2 BIOLOGICAL ASSAY 120 3.2.1 Cytotoxicity activitivy 120 3.2.2 Acetylcholinesterase inhibitory activity 121 CHAPTER 4: CONCLUSION 124 4.1 CONSTITUENTS OF PARMOTREMA PRAESOREDIOSUM 124 4.2 BIOLOGICAL ASSAY 132 FUTURE OUTLOOK 133 viii LIST OF PUBLICATIONS 134 REFERENCES 135 APPENDICES 145 Appendix 231 ROESY spectrum for PRAES-C28 Appendix 232 1H NMR spectrum for PRAES-E17 261 Appendix 233 13C NMR spectrum for PRAES-E17 Appendix 234 HSQC spectrum for PRAES-E17 262 Appendix 235 HMBC spectrum for PRAES-E17 Appendix 236 1H NMR spectrum for PRAES-E6 263 Appendix 237 13C NMR spectrum for PRAES-E6 [M+Na]+ Appendix 238 MS spectrum for PRAES-E13 264 Appendix 239 1H NMR spectrum for PRAES-E13 Appendix 240.13C NMR spectrum for PRAES-E13 265 Appendix 241 DEPT spectrum for PRAES-E13 Appendix 242 COSY spectrum for PRAES-E13 266 Appendix 243 HSQC spectrum for PRAES-E13 Appendix 244 HMBC spectrum for PRAES-E13 267 [M+Na]+ Appendix 245 MS spectrum for PRAES-E15 Appendix 246 1H NMR spectrum for PRAES-E15 268 Appendix 247 13C NMR spectrum for PRAES-E15 Appendix 248 DEPT spectrum for PRAES-E15 269 Appendix 249 COSY spectrum for PRAES-E15 Appendix 250 HSQC spectrum for PRAES-E15 270 Appendix 251 HMBC spectrum for PRAES-E15 Appendix 252 1H NMR spectrum for PRAES-C2Me 271 Appendix 253 13C NMR spectrum for PRAES-C2Me Appendix 254 1H NMR spectrum for PRAES-C2Et 272 Appendix 255 13C NMR spectrum for PRAES-C2Et Appendix 256 1H NMR spectrum for PRAES-C2Pro 273 Appendix 257 13C NMR spectrum for PRAES-C2Pro Appendix 258 HSQC spectrum for PRAES-C2Pro 274 Appendix 259 HMBC spectrum for PRAES-C2Pro 275 ... compared to the control (non-treated cells) Evaluation of the result based on the I% (at the concentration of 100 g/mL): 0–49% (inactive), 50–70% (active), 70–90% (strong), 90–100% (very strong) [78]... (9.0 mg) 0) (25.0 mg) PRAES-E14 0) (7.0 mg) PRAES-T2 0) (30.0 mg) PRAES-E15 0) (50.0 g) PRAES-E17 P:EA 0) (15.0 mg) PRAES-E18 (9: 1) 0)( 250.0 mg) PRAES-E19 0) 0) (5.0 mg) PRAES-T3 0)( 45.0 mg) PRAES-T4... (2.8 g) E2.5 (4.5 g) P:EA (95: 5) P:EA (9: 1) 0) E2.6 (3.7 g) P:EA (9: 1) 0) 0) (1.0 g) PRAES-E5 0) Acetone (45.0 g) E2.7 (2.5 g) Figure 2.1 Isolation of compounds from the precipitate and 0) petroleum