LC-MS 기반 대추나무 뿌리의 cyclopeptide alkaloids 및 triterpenoids 성분의 표적 분리 및 dereplication 방법 개발

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과 생약학전공, 2016. 2. 성상현.Ziziphus jujuba Mill. is the most economically important species in the family Rhamnaceae. Among hundreds of previously reported constituents, cyclopeptide alkaloids and ceanothane-type triterpenoids have been regarded as chemotaxonomic marker compounds of the Rhamnaceae species, and they have been focused as new drug candidates for their characteristic structures and bioactive potentials. The goal of this study was the development of targeted isolation strategy for bioactive cyclopeptide alkaloids and triterpenoids from Z. jujuba. UHPLC-qTOF-MS based dereplication-guided fractionation was selected as a main platform for this purpose. The analytical methods were optimized for LC-MS profiling of cyclopeptide alkaloids and triterpenic acids in MeOH extracts of Z. jujuba roots, twigs, leaves, and fruits. Based on UV, MS, and MS/MS spectral data, the dereplication methods were developed for cyclopeptide alkaloids and triterpenoids. Cyclopeptide alkaloids were hard to be fully predicted, but many triterpenoids could be tentatively identified with their spectra data. Based on these dereplication results, LC-MS profiles of extracts were compared for their potential as the isolation target. The root extract was selected to be a target for further separation, because of its abundance in both target compound classes. MS-guided fractionation resulted in the isolation of 9 cyclopeptide alkaloids (1-9), 37 triterpenoids (10-46), and 6 phenolic compounds (47-52) from the root extract. Among these 52 compounds, 5 cyclopeptide alkaloids (1-5) and 21 triterpenoids (16-20, 23, 24, 26, 27, 30, 33, and 35-43) were isolated from nature for the first time. In-house LC-MS library was built with the retention time and spectral data of isolated compounds, and it was confirmed that tentatively predicted structures of triterpenoids were correctely matched to their real structures. In addition, it was suggested that dereplication method for cyclopeptide alkaloids could be developed in the further study. Bioactivity of isolated compounds were tested, and as a result, antiviral activity of cyclopeptide alkaloid compounds 2, 3, and 6 against PEDV (EC50 4.49-3.41 μM) and cytotoxicity of triterpenic compounds 10, 22, 28-30, 33, 35, 36, and 41 against HepG2 cell line (IC50 1.93-9.24 μM) were evaluated. In conclusion, it was demonstrated that developed UHPLC-qTOF-MS based derelication method was an efficient strategy for the discovery of bioactive cyclopeptide alkaloids and triterpenoids from Z. jujuba.Chapter 1. Introduction 1 1.1. Study background 1 1.1.1. The family Rhamnaceae and the species Ziziphus jujuba 1 1.1.2. Cyclopeptide alkaloids 2 1.1.3. Ceanothane-type triterpenoids 4 1.1.4. Dereplication of natural products 9 1.2. Purpose of research 10 Chapter 2. Isolation of Cyclopeptide Alkaloids 12 2.1. LC-MS analysis of cyclopeptide alkaloids in extracts of Z. jujuba plant parts 12 2.2. Isolation of cyclopeptide alkaloids and development of dereplication method 17 2.3. Structural elucidation of cyclopeptide alkaloids 20 2.3.1. Compound 1 20 2.3.2. Compounds 2 and 3 25 2.3.3. Compounds 4 and 5 31 2.3.4. Compound 6 37 2.3.5. Compound 7 38 2.3.6. Compound 8 39 2.3.7. Compound 9 40 Chapter 3. Isolation of Triterpenoids 41 3.1. LC-MS analysis of triterpenic acids in extracts of Z. jujuba plant parts 41 3.2. MS-guided dereplication and isolation of triterpenoids 50 3.3. Structural elucidation of isolated triterpenoids 58 3.3.1. Compounds 10, 11, and 12 58 3.3.2. Compound 13 61 3.3.3. Compounds 14 and 15 62 3.3.4. Compound 16 64 3.3.5. Compounds 17 and 18 65 3.3.6. Compound 19 71 3.3.7. Compound 20 72 3.3.8. Compound 21 75 3.3.9. Compounds 22 and 23 76 3.3.10. Compound 24 80 3.3.11. Compounds 25, 26, and 27 83 3.3.12. Compounds 28 and 29 88 3.3.13. Compounds 30 and 31 90 3.3.14. Compounds 32 and 33 93 3.3.15. Compounds 34, 35 and 36 96 3.3.16. Compounds 37 and 38 100 3.3.17. Compound 39 103 3.3.18. Compound 40 106 3.3.19. Compound 41 107 3.3.20. Compound 42 110 3.3.21. Compounds 43 and 44 116 3.3.22. Compounds 45 and 46 121 3.4. Structural identification of isolated phenolic compounds 123 3.4.1. Compounds 47 and 48 123 3.4.2. Compound 49 125 3.4.3. Compound 50 126 3.4.4. Compounds 51 and 52 127 Chapter 4. Bioactivity of Isolated Compounds 128 4.1. Antiviral activity of isolated cyclopeptide alkaloids 128 4.2. Cytotoxicity of isolated triterpenoids against human liver hepatocellular carcinoma HepG2 cell line 130 Chapter 5. Experimental Section 132 5.1. Materials 132 5.1.1. Plant materials 132 5.1.2. Reagents 132 5.1.3. Equipment 133 5.2. UHPLC-qTOF-MS analysis for cyclopeptide alkaloids 135 5.3. Isolation of cyclopeptide alkaloids 136 5.4. Acid hydrolysis of compounds 1-5 138 5.4.1. Determining absolute configurations of the amino acids in compounds 1-5 by the advanced Marfeys method 138 5.4.2. Determining absolute configurations of the amino acids in compounds 2-5 by GITC analysis 139 5.4.3. Preparing L- and D-N,N-dimethylalanine 139 5.4.4. Determining absolute configurations of N,N-dimethylalanine in compounds 4 and 5 by PGME derivatization 139 5.5. UHPLC-qTOF-MS analysis for triterpenic acids 141 5.6. Isolation of triterpenoids and phenolic compounds 142 5.7. Preparation of (S)-MTPA ester and (R)-MTPA ester of 42 147 5.8. Spectral data of isolated compounds 148 5.8.1. Jubanine F (1) 148 5.8.2. Jubanine G (2) 148 5.8.3. Jubanine H (3) 149 5.8.4. Jubanine I (4) 149 5.8.5. Jubanine J (5) 149 5.8.6. Nummularine B (6) 153 5.8.7. Daechuine-S3(7) 153 5.8.8. Mucronine K (8) 153 5.8.9. Adouetine X (9) 155 5.8.10. Betulinic acid (10) 156 5.8.11. Alphitolic acid (11) 156 5.8.12. Betulin (12) 156 5.8.13. 3β-hydroxy-28-norlup-20(29)-en-17β-hydroperoxide (13) 157 5.8.14. Ceanothic acid (14) 160 5.8.15. Epiceanothic acid (15) 160 5.8.16. Epiceanothic acid 2-methyl ester (16) 160 5.8.17. 3-dehydroxy ceanothetric acid (17) 161 5.8.18. 3-dehydroxy ceanothetric acid 2-methyl ester (18) 161 5.8.19. Ceanothetric acid 2-methyl ester (19) 161 5.8.20. 3-dehydroxy-ceanotha-27α-carboxy-28β,19β-olide (20) 165 5.8.21. 24-hydroxy ceanothic acid (21) 165 5.8.22. Zizyberenalic acid (22) 165 5.8.23. 3-O-methyl-zizyberanalic acid (23) 166 5.8.24. 1,28-dinor-24-hydroxy-lup-2,17(22)-diene-27-oic acid (24) 166 5.8.25. 2-O-protocatechuoyl alphitolic acid (25) 169 5.8.26. 2-O-vanilloyl alphitolic acid (26) 169 5.8.27. 3-O-protocatechuoyl alphitolic acid (27) 169 5.8.28. 2-O-trans-p-coumaroyl alphitolic acid (28) 170 5.8.29. 2-O-cis-p-coumaroyl alphitolic acid (29) 170 5.8.30. 2-O-p-hydroxybenzoyl alphitolic acid (30) 171 5.8.31. 2-O-benzoyl alphitolic acid (31) 171 5.8.32. 3-O-protocatechuoyl ceanothic acid (32) 175 5.8.33. 3-O-protocatechuoyl ceanothic acid 2-methyl ester (33) 175 5.8.34. 3-O-vanilloyl ceanothic acid (34) 175 5.8.35. 3-O-vanilloyl epiceanothic acid (35) 176 5.8.36. 3-O-vanilloyl ceanothic acid 2-methyl ester (36) 176 5.8.37. 3-O-p-hydroxybenzoyl ceanothic acid (37) 177 5.8.38. 3-O-p-hydroxybenzoyl epiceanothic acid (38) 177 5.8.39. 2-O-protocatechuoyl isoceanothanolic acid (39) 177 5.8.40. 3-O-protocatechuoyl-ceanotha-28β,19β-olide (40) 178 5.8.41. 7β-O-vanilloyl-3-dehydroxy ceanothetric acid 2-methyl ester (41) 178 5.8.42. Epicatechinoceanothic acid A (42) 183 5.8.43. Epicatechinoceanothic acid B (43) 183 5.8.44. Epicatechinoceanothic acid C (44) 184 5.8.45. Maslinic acid (45) 187 5.8.46. Euscaphic acid (46) 187 5.8.47. (-)-epicatechin (47) 188 5.8.48. (+)-catechin (48) 189 5.8.49. Vanillic acid (49) 189 5.8.50. 6-O-vanilloylisotachioside (50) 190 5.8.51. Epiphyllocoumarin (51) 190 5.8.52. Isoepiphyllocoumarin (52) 191 5.9. Evaluation of antiviral effects of cyclopeptide alkaloids against pocrine epidemic diarrhea virus 192 5.9.1. Cell culture and virus stock 192 5.9.2. Cytotoxicity assay 192 5.9.3. Cytopathic effect (CPE) inhibition assay 193 5.10. Evaluation of cytotoxicity of triterpenoids 194 5.10.1. Cell culture 194 5.10.2. Cytotoxicity assay 194 Chapter 6. Conclusions 195 References 197 국문초록 210Docto