[보고서]Array 기법을 이용한 한약의 칠정관계 규명

하혜경

[국가R&D연구보고서] Array 기법을 이용한 한약의 칠정관계 규명
Evaluation of herb-herb interactions by array methods 원문보기

보고서 정보
주관연구기관	한국한의학연구원 Korea Institute of Oriental Medicine
연구책임자	하혜경
참여연구자	신현규 , 이미영 , 서창섭 , 정수진 , 김온순 , 김정훈 , 신인식 , 임혜선 , 진성은 , 김예지 , 전우영 , 신나라 , 김성실
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2013-02
주관부처	미래창조과학부 KA
연구관리전문기관	한국한의학연구원 Korea Institute of Oriental Medicine
등록번호	TRKO201300031244
DB 구축일자	2014-01-13
키워드	한약의 칠정,상수,상외,상오herb-herb interaction,mutual reinforcement,mutual restraint,mutual inhibition

초록

효능이 유사한 두 가지 이상의 약물들이 배합되어 원래 가지고 있는 치료효과를 현저히 증가시키는 상수 배합 1종, 한 가지 약물의 독성이나 부작용이 같이 배합되는 다른 약물에 의해 감소되거나 무력화되어 원래의 유효한 약효만 이용하는 상외 배합 1종, 두 가지 약물이 서로 배합되었을 때 상호 견제하여 원래의 성질과 효능이 감소되거나 상실되는 상오 배합 3종을 선정. 총 5종 배합의 배합 전·후 이화학적 성분 및 효능 변화 검증.

Abstract ▼

Multi-herb formulas have frequently been used in traditional medicine aimed at collectively exerting therapeutic actions and modulating the pharmacological effects of the chemical ingredients of the constituent herbs. In formulating these traditional medicine formulas, special herb-pairs for mutual enhancement, assistance, restraint, suppression, inhibition or antagonism. Mutual restraint and inhibition can be achieved by using an herb with ingredients capable of moderation of harshness and toxicity of the ingredients of the principal herb. In ancient formula books and according to ancient physicians, it was said that Raphani Semen might reduce the tonic activity of Ginseng Radix Alba (mutual inhibition) and Zingiberis Rhizoma Crudus might reduce the toxicity of Pinelliae Tuber (mutual restraint). In this study, interactions of herb-pairs were evaluated to detect chemical ingredient contents and efficacies of Anemarrhenae Rhizoma/Phellodendri Cortex, Pinelliae Tuber/Zingiberis Rhizoma Crudus, Ginseng Radix Alba/Raphani Semen, Syzygii Flos/Curcumae longae Radix and Scutellariae Radix/Zingiberis Rhizoma Crudus.
We established simultaneous determination to analyze several marker compounds in herbal mdeicines (EBM-82, -83, -85, -86, -88, -89, -91 and -92) and their mixtures (EBM-84, -87, -90 and –93) using the HPLC-PDA. Additionally, we were performed research on simultaneous analysis of herbal medicines (EBM-79 and –80) and the mixtrue (EBM-81) by LC-MS/MS. For HPLC or LC-MS/MS determination, we were obtained 15 water extracts (EBM-79 ∼ EBM-93) by reflux method and their yields were 3.88 ∼ 47.84%. As a result of HPLC or LC-MS/MS analysis, we identify ginsenoside Re, Rg1, Rb1, Rg2(S), Rg2(R), Rh1(S), Rh1(R), Ro, Rb2, Rb3, F1, Rd, C-O, Rg3(S), Rg3(R) and sinapic acid in EBM-79 and EBM-80, respectively. Contents of 16 constituents were 0.00 ∼ 0.66 mg/g. In EBM-81, the mixture of EBM-79 and EBM-80, 15 components were detected excluding ginsenoside F1. Content of 15 compounds were 0.00 ∼ 0.36 mg/g. We confirmed homogentisic acid and 6-gingerol in EBM82 and EBM-83, respectively. In EBM-84, the mixture of EBM-82 and EBM-83, while the 6-gingerol was detected, homogentisic acid not detected. We identify mangiferin and berberine in EBM-85 and EBM-86, respectively. Their contents were 8.21 mg/g and 11.40 mg/g, respectively. In EBM-87, the mixture of EBM-85 and EBM-86, contents of 2 components were 1.89 mg/g and 0.01 mg/g, respectively. We were applied to simultaneous analysis of gallic acid, ellagic acid, eugenol, bisdemethoxycurcumin, demetoxycurcurmin curcumin and turmerone in EBM-88 and EBM-89. However, only 3 compounds, gallic acid, ellagic and eugenol were detected. Contents of these components were 4.01 mg/g, 4.99 mg/g and 10.12 mg/g, respectively. In EBM-90, the mixture of EBM-88 and EBM-89, contents of 3 components were 1.61 mg/g, 1.08 mg/g and 5.90 mg/g, respectively. We identify baicalin and wogonoside in EBM-91 and 6-gingerol in EBM-92, respectively. Their contents were 25.13 mg/g, 11.36 mg/g and 0.38 mg/g, respectively. In EBM-93, the mixture of EBM-91 and EBM-92, contents of 3 components were 15.02 mg/g, 8.59 mg/g and 0.08 mg/g, respectively.

목차 Contents

표지 ... 1
제출문 ... 3
보고서 초록 ... 5
요약문 ... 7
SUMMARY ... 13
Contents ... 19
목차 ... 20
표목차 ... 23
그림 목차 ... 25
제1장 연구개발의 목적 및 필요성 ... 31
I. 연구개발의 목적 ... 33
II. 연구개발의 필요성 ... 34
1. 추진 배경 및 과학기술적 의의 ... 34
2. 국내외 연구동향 ... 36
3. 사업추진의 타당성 ... 37
4. 연구개발 추진 전략 ... 40
5. 기대효과 ... 41
제2장 연구개발의 내용 및 범위 ... 43
I. 한약재 배합 전·후의 이화학적 분석 ... 45
1. 추출물 제조 ... 45
2. LC-MS/MS를 이용한 EBM-79, EBM-80 및 EBM-81의 분석법 설정 ... 45
3. HPLC-PDA를 이용한 EBM-82, EBM-83 및 EBM-84의 분석법 설정 ... 49
4. HPLC-PDA를 이용한 EBM-85, EBM-86 및 EBM-87의 분석법 설정 ... 50
5. HPLC-PDA를 이용한 EBM-88, EBM-89 및 EBM-90의 분석법 설정 ... 52
6. HPLC-PDA를 이용한 EBM-91, EBM-92 및 EBM-93의 분석법 설정 ... 55
II. Array 법을 이용한 한약 배합의 효능 평가 ... 58
1. 칠정 이론에 따른 한약 배합 및 관련 효능 ... 58
2. 연구 내용 ... 59
III. 배합 전·후 대사 지표 변화 ... 67
1. 연구 내용 ... 67
2. 연구 방법 ... 67
제3장 연구개발 결과 ... 69
I. 한약재 배합 전·후의 이화학적 분석 ... 71
1. 추출물 제조 ... 71
2. 배합 전·후 추출물의 성분 분석법 설정 ... 71
II. Array 법을 이용한 한약 배합의 효능 평가 ... 89
1. EBM-79 + EBM-80 → EBM-81 : 상오 배합의 면역관련 활성 ... 89
2. EBM-82 + EBM-83 → EBM-84 : 상외 배합의 항천식, 항염증 활성 평가 ... 96
3. EBM-85 + EBM-86 → EBM-87 : 상수 배합의 항염증, 신장염 효능 평가 ... 106
4. EBM-88 + EBM-89 → EBM-90 : 상오 배합의 항염증, 알콜성 위염 효능 평가 ... 113
5. EBM-91 + EBM-92 → EBM-93 : 상오 배합의 항염증 활성 평가 ... 120
III. 배합 전·후 대사 지표 변화 ... 124
1. EBM79 + EBM80 → EBM81 : 상오 배합 ... 124
2. EBM82 + EBM83 → EBM84 : 상외 배합 ... 125
3. EBM85 + EBM86 → EBM87 : 상수 배합 ... 126
4. EBM88 + EBM89 → EBM90 : 상오 배합 ... 127
5. EBM91 + EBM92 → EBM93 : 상오 배합 ... 128
IV. 결 론 ... 129
1. 지모+황백의 상수 배합 ... 129
2. 반하+생강의 상외 배합 ... 129
3. 인삼+나복자의 상오 배합 ... 130
4. 정향+울금의 상오 배합 ... 131
5. 황금+생강의 상오 배합 ... 132
제4장 연구결과의 활용계획 ... 133
I. 과학기술적 성과 ... 135
II. 성과활용·확산계획 및 실적 ... 136
참고문헌 ... 137

표/그림 (64)

표 Fig. 1. Chemical structures of major constituents of EBM-79, EBM-80 and EBM-81
표 Fig. 1. Chemical structures of major constituents of EBM-79, EBM-80 and EBM-81
표 Fig. 2. Chemical structures of major constituents of EBM-82, EBM-83 and EBM-84
표 Fig. 3. Chemical structures of major constituents of EBM-85, EBM-86 and EBM-87
표 Fig. 4. Chemical structures of major constituents of EBM-88, EBM-89 and EBM-90
표 Fig. 5. Chemical structures of major constituents of EBM-91, EBM-92 and EBM-93
표 Fig. 6. TIC chromatogram of standard mixture (A and B) and EBM-79 (C), EBM-80 (D) and EBM-81 (E; saponins, F; sinapic acid)
표 Fig. 6. TIC chromatogram of standard mixture (A and B) and EBM-79 (C), EBM-80 (D) and EBM-81 (E; saponins, F; sinapic acid)
표 Fig. 7. Analytical results (mg/g) of EBM-79, EBM-80 and EBM-81
표 Fig. 8. HPLC chromatogram of standard mixture at 280 nm (A), EBM-82 extract (B), EBM-83 extract (C) and EBM-84 extract (D)
표 Fig. 9. HPLC chromatogram of standard mixture (A), EBM-85 (B), EBM-86 (C), and EBM-87 extract (D) at 260 nm
표 Fig. 10. Analytical results (mg/g) of EBM-85, EBM-86 and EBM-87
표 Fig. 11. HPLC chromatogram of standard mixture (A; 254 nm, B; 280 nm, C; 420 nm), EBM-88 (D; 254 nm, E; 280 nm), EBM-89 (F; 254 nm, G; 420 nm), and EBM-90 (H; 254 nm, I; 280 nm, J; 420 nm)
표 Fig. 11. HPLC chromatogram of standard mixture (A; 254 nm, B; 280 nm, C; 420 nm), EBM-88 (D; 254 nm, E; 280 nm), EBM-89 (F; 254 nm, G; 420 nm), and EBM-90 (H; 254 nm, I; 280 nm, J; 420 nm)
표 Fig. 12. Analytical results (mg/g) of EBM-88, EBM-89 and EBM-90.
표 Fig. 13. HPLC chromatogram of standard mixture (A), EBM91 (B), EBM92 (C), and EBM93 (D) at 280 nm
표 Fig. 14. Analytical results (mg/g) of EBM-91, EBM-92 and EBM-93.
표 Fig. 15. The cell proliferation of RAW 264.7 cells treated with herbal extracts (EBM-79, -80 and ?81)
표 Fig. 16. Effect of herbal extracts (EBM-79, -80 and -81) on nitric oxide (NO) production in RAW 264.7 cells
표 Fig. 17. The proliferation of mouse splenocytes treated with herbal extracts
표 Fig. 18. Flow cytometric analysis on EBM79-, 80- and 81-treated mouse splenocytes
표 Fig. 19. Effects of EBM79, 80 and 81 on IL-2 production in mouse splenocytes
표 Fig. 20. Changes on body weight during 3-week administration of herbal extracts (EBM79, 80 and 81) in OVA-immunized mice
표 Fig. 21. Effect of herbal extracts (EBM-79, -80 and ?81) on OVA-, LPSand Con A-stimulated splenocyte proliferation in vivo
표 Fig. 22. The plasma levels of total IgG, IgG1, IgM and OVA-specific IgG, IgG1 and IgG2a on OVA-immunized mice
표 Fig. 23. Seven bead populations with distinct fluorescence intensities (A) and standard curves from the mouse Th1/Th2/Th17 cytokine standards (B) using the cytokine bead array
표 Fig. 24. Effects of herbal extracts (EBM-82, -83 and ?84) on viability of RAW 264.7 cells
표 Fig. 25. Effects of herbal extracts (EBM-82, -83 and ?84) on LPS-stimulated NO production in RAW264.7 cells
표 Fig. 26. Effects of herbal extracts (EBM-82, -83 and ?84) on LPS-stimulated PGE2 production in RAW264.7 cells.
표 Fig. 27. BALF levels of IL-4 and IL-13 on EBM-82, -83 and ?84 administrated mice
표 Fig. 28. BALF levels of eotaxin and TNF-α on EBM-82, -83 and ?84 administrated mice
표 Fig. 29. Levels of total IgE and OVA-specific IgE on EBM-82, -83 and ?84 administrated mice
표 Fig. 29. Levels of total IgE and OVA-specific IgE on EBM-82, -83 and ?84 administrated mice
표 Fig. 30. Inflammatory cells in lung tissue on EBM-82, -83 and ?84 administrated mice
표 Fig. 31. Mucus production and goblet cell hyperplasia in lung tissue on EBM-82, -83 and ?84 administrated mice
표 Fig. 32. Cytokine array in plasma on EBM-82, -83 and ?84 administrated mice
표 Fig. 33. The proliferation of mouse splenocytes treated with EBM-82~84 extracts
표 Fig. 34. Flow cytometric analysis on EBM82-, 83- and 84-treated mouse splenocytes
표 Fig. 35. Effects of EBM-82~84 on IL-2 production in mouse splenocytes
표 Fig. 36. Effects of herbal extracts (EBM-85, -86 and -87) on viability of RAW 264.7 cells
표 Fig. 37. Effects of herbal extracts (EBM-85, -86 and -87) on LPS-stimulated NO and PGE2 production in RAW 264.7 cells
표 Fig. 38. Effects of EBM-85~87 on cytotoxicity in porcine renal epithelial cells
표 Fig. 39. Effects of EBM-85~87 on cisplatin-induced cytotoxicity in porcine renal epithelial cells (MTT assay)
표 Fig. 40. Effects of EBM-85~87 on cisplatin-induced cytotoxicity in porcine renal epithelial cells (crystal violet assay).
표 Fig. 41. Effects of EBM-85~87 on ROS generations in cisplatin-treated porcine renal epithelial cells.
표 Fig. 42. Effects of EBM-85~87 on GSH contents in cisplatin-treated porcine renal epithelial cells.
표 Fig. 43. Effects of EBM-85~87 on GSSG contents in cisplatin-treated porcine renal epithelial cells.
표 Fig. 44. Effects of EBM-85~87 on GSH:GSSG ratio in cisplatin-treated porcine renal epithelial cells
표 Fig. 45. Effects of EBM-85~87 on total GSH contents in cisplatin-treated porcine renal epithelial cells
표 Fig. 46. The proliferation of mouse splenocytes treated with EBM-85~87 extracts
표 Fig. 47. Effects of herbal extracts (EBM-88, -89 and -90) on viability of RAW 264.7 cells
표 Fig. 48. Effects of herbal extracts (EBM-88, -89 and -90) on LPS-stimulated NO and PGE2 production in RAW 264.7 cells
표 Fig. 49. Changes on body and organ weights during 3-day administration of herbal extracts (EBM-88, -89 or ?90) in mice with ethanol induced gastric injury
표 Fig. 50. Effect of herbal extracts (EBM-88, -89 and ?90) on MDA and GSH contents in mice with ethanol induced gastric injury
표 Fig. 51. Effect of herbal extracts (EBM-88, -89 and ?90) on activities of antioxidant enzymes in mice with ethanol induced gastric injury.
표 Fig. 52. Effect of herbal extracts (EBM-88, -89 and ?90) on PGE2 production in mice with ethanol induced gastric injury
표 Fig. 53. Effect of herbal extracts (EBM-88, -89 and -90) on gastric inflammation (H&E staining).
표 Fig. 54. Effect of herbal extracts (EBM-88, -89 and -90) on gastric mucus secretion (PAS staining).
표 Fig. 55. The proliferation of mouse splenocytes treated with EBM-88~90 extracts
표 Fig. 56. Effects of herbal extracts (EBM-91, -92 and -93) on viability of RAW 264.7 cells
표 Fig. 57. Effects of herbal extracts (EBM-91, -92 and -93) on LPS-stimulated NO and PGE2 production in RAW 264.7 cells
표 Fig. 58. Effects of herbal extracts (EBM-91, -92 and -93) on LPS-stimulated iNOS protein expression in RAW 264.7 cells
표 Fig. 59. The proliferation of mouse splenocytes treated with EBM-91~93 extracts
표 Fig. 60. Effects of EBM-79 on CYP450 isozymes

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[국가R&D연구보고서] Array 기법을 이용한 한약의 칠정관계 규명
Evaluation of herb-herb interactions by array methods 원문보기