Tài liệu Study on chemical constituents and biological activities of the lichen parmotrema praesorediosum (nyl.) hale (parmeliaceae)

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: 1. Except where due acknowledgement has been made, the work is that of the author alone; 2. The work has not been submitted previously, in whole or in part, to qualify for any other academic award; 3. 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; 4. 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 ..................................................................................................... 1 CHAPTER 1: LITERATURE REVIEW ................................................................ 3 1.1. GENERIC DESCRIPTION ............................................................................... 3 1.1.1. The lichen .......................................................................................................... 3 1.1.2. Parmotrema praesorediosum (Nyl.) Hale ......................................................... 4 1.2. CHEMICAL STUDIES ON THE LICHEN GENUS PARMOTREMA ........... 6 1.2.1. Lichen secondary metabolites ........................................................................... 6 1.2.2. Chemical studies on the lichen genus Parmotrema .......................................... 8 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 ix LIST OF ABBREVIATIONS 1D One dimensional 2D Two dimensional Ac Acetone AcOH Acetic acid br Broad C Chloroform calcd Calculated CC Column chromatography CD Circular dichroism COSY Homonuclear shift correlation spectroscopy CPCM Conductor-like polarized continuum model d Doublet dd Doublet of doublets DEPT Distortionless enhancement by polarisation transfer DMSO Dimethyl sulfoxide EA Ethyl acetate EI-MS Electron-impact ionization mass spectrum EtOH Ethanol H n-Hexane HMBC Heteronuclear multiple bond correlation spectroscopy HPLC High performance liquid chromatography HR-EIMS High resolution electron-impact ionization mass spectrum HR-ESIMS High resolution electrospray ionization mass spectrum x HSQC Heteronuclear single quantum correlation spectroscopy IR Infrared spectrophotometry m Multiplet M Methanol MeOH Methanol min Minutes MS Mass spectrum NMR Nuclear magnetic resonance NOESY Nuclear overhauser enhancement spectroscopy P Petroleum ether ppm Parts per million (chemical shift value) pre TLC Preparative thin-layer chromatography q Quartet quint Quintet ROESY Rotating-frame overhauser enhancement spectroscopy s Singlet sext Sextet t Triplet TD-DFT Time dependent density functional theory TLC Thin-layer chromatography TMS Tetramethylsilane UV Ultraviolet xi LIST OF TABLES Table 1.1. In vitro biological activities of the lichen genus Parmotrema 17 Table 3.1. Isolated compounds from Parmotrema praesorediosum 32 Table 3.2. 1 38 Table 3.3. 13 Table 3.4. NMR data of PRAES-T1, PRAES-E1, PRAES-T2 47 Table 3.5. NMR data of PRAES-E11, PRAES-T4, PRAES-T6, PRAES-E2 52 Table 3.6. NMR data of PRAES-C22, PRAES-C23, PRAES-C24 58 Table 3.7. NMR data of PRAES-C25, PRAES-C25M, PRAES-C26 65 Table 3.8. NMR data of PRAES-T3, PRAES-C7, PRAES-C9, PRAES-E18 68 Table 3.9. NMR data of PRAES-C14 and PRAES-C12 72 Table 3.10. 1 H NMR data of PRAES-C5 and Lecanorol 76 Table 3.11. 1 H NMR data of PRAES-C15, PRAES-C16, PRAES-C20, H NMR of aliphatic compounds C NMR of aliphatic compounds PRAES-C18, PRAES-C3 and PRAES-C4 Table 3.12 13 39 82 C NMR data of PRAES-C15, PRAES-C16, PRAES-C20, PRAES-C18, PRAES-C3 and PRAES-C4 83 Table 3.13. NMR data of PRAES-C21 96 Table 3.14. NMR data of PRAES-E5 and PRAES-E3 (CDCl3) 98 Table 3.15. NMR data of PRAES-C8, PRAES-E5 and Usimine A 102 Table 3.16. NMR data of PRAES-C27, Blennolide G, Blennolide B and Chromone lactone (CDCl3) Table 3.17. NMR data of PRAES-C27 and PRAES-C28 (CDCl3) 106 110 xii Table 3.18. NMR data of PRAES-E17, PRAES-E6, PRAES-E13 and 1β,3βDiacetoxyhopan-22-ol Table 3.19. NMR data of PRAES-E15 115 120 Table 3.20. % Inhibition of cytotoxic activity against three cancer cell lines of isolated compounds 122 Table 3.21. IC50 value of cytotoxic activity against three cancer cell lines of isolated compounds 122 Table 3.22. Acetylcholinesterase inhibition of some extracts and isolated compounds 123 xiii LIST OF FIGURES Figure 1.1. Types of the lichen 3 Figure 1.2. Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae) 5 Figure 1.3. Biosynthetic pathways of the major groups of lichen substances 7 Figure 2.1: Isolation of compounds from the prepicitate and petroleum ether extracts of Parmotrema praesorediosum (Nyl.) Hale 26 Figure 2.2: Isolation of compounds from the chloroform extract of Parmotrema praesorediosum (Nyl.) Hale 27 Figure 3.1. HMBC correlations of PRAES-C1 and PRAES-E14 36 Figure 3.2. HMBC correlations of PRAES-C10 37 Figure 3.3. HMBC correlations of PRAES-E19 41 Figure 3.4. HMBC correlations of PRAES-C2 42 Figure 3.5. Comparison of experimental CD spectrum of PRAES-C2Me and theoretical calculated one. 44 Figure 3.6. CD spectra of isolated aliphatic compounds 45 Figure 3.7. HMBC correlations of PRAES-E1 and PRAES-T2 48 Figure 3.8. HMBC correlations of PRAES-E11, PRAES-T4 and PRAES-E2 52 Figure 3.9. HMBC and NOESY correlations of PRAES-C22 54 Figure 3.10. HMBC and NOESY correlations of PRAES-C23 56 Figure 3.11. HMBC and NOESY correlations of PRAES-C24 57 Figure 3.12. COSY, HMBC and NOESY correlations of PRAES-C25M 61 Figure 3.13. Mechanism for the methylation of PRAES-C25 62 Figure 3.14. HMBC and NOESY correlations of PRAES-C25 and PRAES-C26 63 xiv Figure 3.15. HMBC correlations of PRAES-C9 and PRAES-C7 67 Figure 3.16. COSY and HMBC correlations of PRAES-E18 70 Figure 3.17. HMBC correlations of PRAES-C12 73 Figure 3.18. HMBC correlations of PRAES-C5 75 Figure 3.19. HMBC and NOESY correlations of PRAES-C15 78 Figure 3.20. HMBC and ROESY correlations of PRAES-C16 80 Figure 3.21. HMBC and ROESY correlations of PRAES-C20 85 Figure 3.22. 1H NMR data of PRAES-C18 and diphenyl ether 87 Figure 3.23. HMBC and ROESY correlations of PRAES-C18 88 Figure 3.24. HMBC correlations of PRAES-C3 90 Figure 3.25. HMBC correlations of PRAES-C4 93 Figure 3.26. HMBC correlations of PRAES-C21 94 Figure 3.27. ROESY correlations of PRAES-C21 95 Figure 3.28. HMBC correlations of PRAES-E5 97 Figure 3.29. HMBC correlations of PRAES-E3 99 Figure 3.30. HMBC correlations of PRAES-C8 101 Figure 3.31. The structure of Usimine A 102 Figure 3.32. COSY, HMBC and ROESY correlations of PRAES-C27 104 Figure 3.33. ROESY correlations of PRAES-C28 109 Figure 3.34. COSY and HMBC correlations of PRAES-C28 111 Figure 3.35. HMBC correlations of PRAES-E17 112 Figure 3.36. HMBC correlations of PRAES-E13 114 Figure 3.37. COSY and HMBC correlations of PRAES-E15 118 xv LIST OF APPENDICES Appendices 1-7: IR, MS and NMR spectra of PRAES-C1 146 Appendices 8-15: IR, MS and NMR spectra of PRAES-E14 149 Appendices 16-21: MS and NMR spectra of PRAES-C10 153 Appendices 22-26: MS and NMR spectra of PRAES-C11 156 Appendices 27-33: IR, MS and NMR spectra of PRAES-E19 159 Appendices 34-40: IR, MS and NMR spectra of PRAES-C2 162 Appendices 41-44: NMR spectra of PRAES-T1 166 Appendices 45-49: MS and NMR spectra of PRAES-E1 168 Appendices 50-54: NMR spectra of PRAES-T2 170 Appendices 55-58: NMR spectra of PRAES-E11 173 Appendices 59-62: NMR spectra of PRAES-T4 175 Appendices 63-66: MS and NMR spectra of PRAES-T6 177 Appendices 67-70: NMR spectra of PRAES-E2 179 Appendices 71-78: IR, MS and NMR spectra of PRAES-C22 181 Appendices 79-86: IR, MS and NMR spectra of PRAES-C23 185 Appendices 87-94: IR, MS and NMR spectra of PRAES-C24 189 Appendices 95-97: MS and NMR spectra of PRAES-C25 193 Appendices 98-106: IR, MS and NMR spectra of PRAES-C25M 194 Appendices 107-114: IR, MS and NMR spectra of PRAES-C26 199 Appendices 115-119: NMR spectra of PRAES-T3 203 Appendices 120-124: NMR spectra of PRAES-C7 205 xvi Appendices 125-131: MS and NMR spectra of PRAES-E18 208 Appendices 132-136: MS and NMR spectra of PRAES-C14 211 Appendices 137-141: NMR spectra of PRAES-C12 214 Appendices 142-147: MS and NMR spectra of PRAES-C5 216 Appendices 148-155: IR, MS and NMR spectra of PRAES-C15 219 Appendices 156-163: IR, MS and NMR spectra of PRAES-C16 223 Appendices 164-172: IR, MS and NMR spectra of PRAES-C20 227 Appendices 173-180: IR, MS and NMR spectra of PRAES-C18 232 Appendices 181-186: MS and NMR spectra of PRAES-C3 236 Appendices 187-192: MS and NMR spectra of PRAES-C4 239 Appendices 193-200: IR, MS and NMR spectra of PRAES-C21 242 Appendices 201-204: NMR spectra of PRAES-E5 246 Appendices 205-207: NMR spectra of PRAES-E3 248 Appendices 208-213: MS and NMR spectra of PRAES-C8 249 Appendices 214-222: IR, MS and NMR spectra of PRAES-C27 252 Appendices 223-231: IR, MS and NMR spectra of PRAES-C28 256 Appendices 232-235: NMR spectra of PRAES-E17 261 Appendices 236-237: NMR spectra of PRAES-E6 263 Appendices 238-244: MS and NMR spectra of PRAES-E13 264 Appendices 245-259: MS and NMR spectra of PRAES-E15 268 INTRODUCTION Lichens are by definition symbiotic organisms composed of a fungal partner (mycobiont) and one or more photosynthetic partners (photobiont/s). The photobiont can be either a green alga or a cyanobacterium. Morphologically lichens can be classified into three major groups. They are foliose, fruticose and crustose. Growing rates of lichens are extremely slow. More than twenty thousand species of lichens have been found. They can tolerate very drastic weather conditions and are resistant to insects and other microbial attacks. Lichens produce a variety of secondary compounds. They play an important role in protection and maintenance of the symbiotic relationship [1]. Many lichen secondary metabolites exhibited antibiotic, antitumour, antimutagenic, allergenic, antifungal, antiviral, enzyme inhibitory and plant growth inhibitory properties [5, 12]. In 2007, Balaji. P. et al. [3] indicated that dichloromethane, ethyl acetate and acetone methanol extracts of Parmotrema praesorediosum showed antimicrobial activity against ten bacterial (Gram + and -) (Bacillus cereus, Corynebacterium diptheriae, Proteus mirabilis, Proteus vulgari, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexnerii, Staphylococcus aureus, Streptococcus pyogenes and Vibrio cholera) and one fungal Candida albicans by using standard dics diffusion method. This lichen could therefore be a potential source in the search for pharmaceutical useful chemicals. The primary goal of the present work was to isolate secondary metabolites on the lichen Parmotrema praesorediosum (Nyl.) Hale. The chemical structure of isolated compounds was characterized by spectroscopic methods (1D-, 2D-NMR, HRMS, CD). Finally, the purified substances from this source were assayed for the cytotoxic activities against three cell lines: MCF-7 (breast cancer cell line), HeLa (cervical cancer cell line) and NCI-H460 (human lung cancer cell line) by 1 sulforhodamine B colorimetric assay method (SRB assay) [56] and the inhibition against acetylcholinesterase in vitro. Based on spectroscopic evidence and their physical properties, the chemical structures were attributed for be forty compounds, including six aliphatic acids, twelve mononuclear phenolic acids, three depsides, two depsidones, eight diphenyl ethers, three dibenzofurans, two xanthones, three triterpenoids and a macrocyclic compound. The latter twenty two compounds appeared to be new and among eighteen known compounds, twelve compounds were known for the first time from the genus Parmotrema. These results pointed out that the Vietnamese lichens could be new sources of bioactive compounds with novel skeletons 2