표제지
국문초록
목차
List of Abbreviations 11
Ⅰ. 서론 18
Ⅱ. 실험 재료 및 방법 31
1. 식물재료 31
1-1. 순비기나무 31
2. 시약 및 기기 32
2-1. 시약 32
2-2. 기기 33
3. 순비기나무 부위별 추출 및 분획 34
3-1. 순비기나무 잎의 추출 및 분획 34
3-2. 순비기나무 열매의 추출 및 분획 35
3-3. 순비기나무 잔가지의 추출 및 분획 36
3-4. 순비기나무 뿌리의 추출 및 분획 37
3-5. 순비기나무 꽃의 추출 및 분획 38
4. 순비기나무 잎 유래 지표성분의 분리 39
4-1. 순비기나무 잎 추출물의 분획물로부터 화합물 분리 39
4-2. 순비기나무 잎 유래 지표성분의 분석 54
5. 순비기나무 유래 iridoid 계열 화합물의 분리 55
5-1. 순비기나무 잎으로부터 iridoid 계열 화합물 분리 55
5-2. 순비기나무 잔가지로부터 iridoid 계열 화합물 분리 61
6. 항산화 활성 검색 80
6-1. ABTS 라디칼 소거능 측정 80
6-2. DPPH 라디칼 소거능 측정 80
6-3. DPPH-HPLC 81
7. 항염증 활성 검색 82
7-1. 세포주 배양 82
7-2. 염증 유도 및 시료 처리 82
7-3. 세포 생존율 측정 83
7-4. NO 생성 측정 83
7-5. IL-8 생성 측정 84
7-6. 웨스턴 블롯 85
7-7. 통계처리 86
8. 함량 평가 87
8-1. 표준물질 87
8-2. 시료 전처리 87
8-3. 장비 및 분석 조건 88
8-4. 정량평가 88
Ⅲ. 결과 및 고찰 90
1. 순비기나무 지표성분 분리 분석 90
1-1. 화합물 1의 구조 분석 90
1-2. 화합물 2의 구조 분석 92
1-3. 화합물 3의 구조 분석 94
1-4. 화합물 4의 구조 분석 96
1-5. 화합물 5의 구조 분석 98
1-6. 화합물 6의 구조 분석 100
1-7. 화합물 7의 구조 분석 102
1-8. 순비기나무 잎 지표성분의 분석 104
2. 순비기나무 유래 iridoid 분리 109
2-1. 화합물 I1의 구조 분석 109
2-1. 화합물 I2의 구조 분석 118
2-3. 화합물 I3의 구조 분석 126
2-4. 화합물 I4의 구조 분석 128
2-5. 화합물 I5의 구조 분석 137
2-6. 화합물 I6의 구조 분석 139
2-7. 화합물 I7의 구조 분석 141
2-8. 화합물 I8의 구조 분석 143
2-9. 화합물 I9의 구조 분석 145
2-10. 화합물 I10의 구조 분석 147
2-11. 화합물 I11의 구조 분석 149
2-12. 화합물 I12의 구조 분석 151
3. 항산화 활성 평가 154
3-1. 순비기나무 부위별 추출물의 항산화 활성 평가 154
3-2. 순비기나무 부위별 분획물의 항산화 활성 평가 156
3-3. 순비기나무 잎 추출물의 DPPH-HPLC 라디칼 소거능 평가 158
4. 순비기나무의 지표성분 함량 평가 160
4-1. 순비기나무 잎 지표성분의 검량선 작성 160
4-2. 순비기나무 부위별 지표성분 함량 평가 162
4-3. 순비기나무 지역별 지표성분 함량 평가 164
5. RAW264.7 세포주에서의 염증 저해 활성 평가 168
5-1. 순비기나무 부위별 추출물 및 분획물의 세포 생존율 168
5-2. 순비기나무 부위별 추출물 및 분획물의 NO 생성 저해 활성 171
5-3. 순비기나무 유래 iridoid 화합물의 세포 생존율 173
5-4. 순비기나무 유래 iridoid 화합물의 NO 생성 저해 활성 174
5-5. 순비기나무 유래 iridoid 화합물의 iNOS 및 COX-2 발현 저해 활성 175
6. HT-29 세포주에서의 염증 저해 활성 평가 177
6-1. 순비기나무 유래 iridoid 화합물의 세포 생존율 177
6-2. 순비기나무 유래 iridoid 화합물의 IL-8 생성 저해 활성 178
Ⅳ. 결론 179
Ⅴ. 참고문헌 182
Table 1. Pharmacological studies reported in Vitex rotundifolia L. f. 25
Table 2. ¹H-NMR spectrum data of compounds 1-4 51
Table 3. ¹H-NMR spectrum data of compounds 5-7 52
Table 4. ¹³C-NMR spectrum data of compounds 1-7 53
Table 5. ¹H-NMR spectrum data of compounds I1-I3 74
Table 6. ¹H-NMR spectrum data of compounds I4-I6 75
Table 7. ¹H-NMR spectrum data of compounds I7-I9 76
Table 8. ¹H-NMR spectrum data of compounds I10-I12 77
Table 9. ¹³C-NMR spectrum data of compounds I1-I6 78
Table 10. ¹³C-NMR spectrum data of compounds I7-I12 79
Table 11. The mass data of seven marker compounds from V. rotundifolia leaves 107
Table 12. Antioxidant properties of active fractions derived from five organs of V. rotundifolia 157
Table 13. DPPH-HPLC of marker compounds of V. rotundifolia leaves 159
Table 14. Analysis of seven marker compounds of V. rotundifolia leaves 161
Table 15. Quantification of marker compounds content in the organs of V. rotundifolia 163
Table 16. Quantification of marker compounds content in the organs of V. rotundifolia located at different locations 166
Table 17. Anti-inflammatory activity of organs extracts and their fractions in LPS stimulated RAW264.7 cells 172
Figure 1. Three structure forms of iridoid 20
Figure 2. Origin of ROS and RNS and oxidative stress in bronchial asthma (BA) 21
Figure 3. The schematic of the anti-inflammatory activity 23
Figure 4. Structures of phenolic compounds known in V. rotundifolia 28
Figure 5. Structures of flavonoids known in V. rotundifolia 29
Figure 6. Structures of terpenes known in V. rotundifolia 30
Figure 7. V. rotundifolia herbs (A) and its organs; Leaves (B), Fruits (C), Twigs (D), Roots (E), Flowers (F) 31
Figure 8. ¹H- and ¹³C-NMR spectra of compound 1 91
Figure 9. ¹H- and ¹³C-NMR spectra of compound 2 93
Figure 10. ¹H- and ¹³C-NMR spectra of compound 3 95
Figure 11. ¹H- and ¹³C-NMR spectra of compound 4 97
Figure 12. ¹H- and ¹³C-NMR spectra of compound 5 99
Figure 13. ¹H- and ¹³C-NMR spectra of compound 6 101
Figure 14. ¹H- and ¹³C-NMR spectra of compound 7 103
Figure 15. TIC (negative ion mode) of 80% EtOH extract of V. rotundifolia leaves 104
Figure 16. Mass spectrum of compound 1 104
Figure 17. Mass spectrum of compound 2 105
Figure 18. Mass spectrum of compound 3 105
Figure 19. Mass spectrum of compound 4 105
Figure 20. Mass spectrum of compound 5 106
Figure 21. Mass spectrum of compound 6 106
Figure 22. Mass spectrum of compound 7 106
Figure 23. Structures of marker compounds isolated from V. rotundifolia leaves 108
Figure 24. ¹H- and ¹³C-NMR spectra of compound I1 112
Figure 25. HMQC spectrum of compound I1 113
Figure 26. COSY spectrum of compound I1 114
Figure 27. HMBC spectrum of compound I1 115
Figure 28. 2D-NOESY spectrum of compound I1 116
Figure 29. 1D-NOE spectrum of compound I1 117
Figure 30. ¹H- and ¹³C-NMR spectra of compound I2 121
Figure 31. HMQC spectrum of compound I2 122
Figure 32. HMBC spectrum of compound I2 123
Figure 33. 2D-NOESY spectrum of compound I2 124
Figure 34. 1D-NOE spectrum of compound I2 125
Figure 35. ¹H- and ¹³C-NMR spectra of compound I3 127
Figure 36. ¹H- and ¹³C-NMR spectrum of compound I4 131
Figure 37. HMQC spectrum of compound I4 132
Figure 38. COSY spectrum of compound I4 133
Figure 39. HMBC spectrum of compound I4 134
Figure 40. 2D-NOESY spectrum of compound I4 135
Figure 41. 1D-NOE spectrum of compound I4 136
Figure 42. ¹H- and ¹³C-NMR spectra of compound I5 138
Figure 43. ¹H-NMR spectrum of compound I6 140
Figure 44. ¹H- and ¹³C-NMR spectra of compound I7 142
Figure 45. ¹H- and ¹³C-NMR spectra of compound I8 144
Figure 46. ¹H- and ¹³C-NMR spectra of compound I9 146
Figure 47. ¹H- and ¹³C-NMR spectra of compound I10 148
Figure 48. ¹H- and ¹³C-NMR spectra of compound I11 150
Figure 49. ¹H- and ¹³C-NMR spectra of compound I12 152
Figure 50. Structures of iridoids isolated from V. rotundifolia 153
Figure 51. Antioxidant capacities (DPPH, ABTS) of five organs extracts. The data are expressed as the mean ± SD (n = 3) of three individual experiments. The data are... 155
Figure 52. DPPH-HPLC chromatograms of marker compounds of V. rotundifolia leaves 158
Figure 53. Chromatogram of seven marker compounds of V. rotundifolia leaves 160
Figure 54. Chromatograms detected at 254 nm of five organs 162
Figure 55. Cell viability of crude extracts on RAW264.7 cells. RAW 264.7 cells were treated with crude extracts for 2 h and stimulated with LPS (1 μg/mL) for 24 h. Cell... 169
Figure 56. Cell viability of crude extracts and fractions on RAW264.7 cells. RAW 264.7 cells were treated with crude extracts and fractions for 2 h and stimulated with... 170
Figure 57. Effects of iridoid compounds on cell viability. RAW 264.7 cells were treated with compound I1-I12 (100 μM) for 2 h and stimulated with LPS (1 μg/mL)... 173
Figure 58. Effects of iridoid compounds NO production. RAW 264.7 cells were treated with compound I1-I12 (100 μM) for 2 h and stimulated with LPS (1 μg/mL)... 174
Figure 59. Effects of iridoid compounds on iNOS and COX-2 expression. RAW 264.7 cells were incubated in the presence of compound I4, I6, I7 and I9 for two hour and... 176
Figure 60. Effects of iridoid compounds on the LPS-induced cell viability in HT-29 cells. HT-29 cells were treated with compounds I1-I12 (100 μM) for 2 h and... 177
Figure 61. Effects of iridoid compounds on the LPS-induced expression of IL-8 in HT-29 cells. HT-29 cells were treated with compounds I1-I12 (100 μM) for 2 h and... 178
Scheme 1. Extraction and fractionation of V. rotundifolia leaves 34
Scheme 2. Extraction and fractionation of V. rotundifolia fruits 35
Scheme 3. Extraction and fractionation of V. rotundifolia twigs 36
Scheme 4. Extraction and fractionation of V. rotundifolia roots 37
Scheme 5. Extraction and fractionation of V. rotundifolia flowers 38
Scheme 6. Isolation of compound from n-hexane fraction of V. rotundifolia leaves A: RP HPLC (YMC-Triart C₁₈, S-5 μm, 250 x 10 mm; 0.3% formic acid in water:... 41
Scheme 7. Isolation of compounds from the EtOAc fraction of V. rotundifolia leaves A: silica gel open column chromatography (DCM: methanol: water = 15: 1: 0.001 →... 42
Scheme 8. Isolation of compound from the aqueous fraction of V. rotundifolia leaves A: RP HPLC (YMC-Triart C₁₈, S-5 μm, 250 x 10 mm; 0.3% formic acid in water:... 43
Scheme 9. Isolation of compounds from EtOAc fraction of V. rotundifolia leaves A: Silica gel open column chromatography (DCM: methanol: water = 15: 1: 0.001 →... 56
Scheme 10. Isolation of compound from the aqueous fraction of V. rotundifolia leaves... 57
Scheme 11. Isolation of compounds from n-BuOH fraction of V. rotundifolia twigs A: silica gel open column chromatography (DCM: methanol: water = 50: 1: 0.001 →... 63