목차

Ⅰ. 서 론 ··························································································· 1
Ⅱ. 재료 및 방법 ············································································· 8
1. 재료 ······································································································ 8
가. 실험재료 ··························································································· 8
나. 시약 ··································································································· 8
다. 세포 및 배지 ··················································································· 9
2. 실험방법 ···························································································· 10
가. 상황버섯, 영지버섯, 표고버섯의 성분분석 ···························· 10
1) 일반성분 분석 ·············································································· 10
2) 유리당 분석 ·················································································· 11
3) 아미노산 분석 ·············································································· 13
가) 구성아미노산 분석 ·································································· 13
나) 유리아미노산 분석 ·································································· 13
4) Vitamin 분석 ··············································································· 15
가) β-carotene 분석 ···································································· 15
나) Vitamin D2 분석 ····································································· 18
다) Ergosterol 분석 ······································································ 18
라) Vitamin E 분석 ······································································· 21
5) Ergothioneine 분석 ··································································· 21
6) β-glucan 분석 ············································································ 24
나. 상황버섯, 영지버섯, 표고버섯의 생리활성 ···························· 25
1) Total Polyphenols 함량 측정 ················································ 25
2) 항산화활성 측정 ·········································································· 26
가) 시료 추출 ·················································································· 26
(1) 물 추출 ···················································································· 26
(2) Ethanol 추출 ·········································································· 26
나) Electron donating ability ···················································· 27
다) Superoxide dismutase(SOD) 유사활성 ···························· 28
라) ABTS scavenging activity ················································· 29
마) Superoxide anion scavenging acitivity ························· 30
3) In-vitro test를 통한 항염증 활성 ········································· 31
가) 세포배양 ···················································································· 31
나) 세포독성 측정 ·········································································· 32
다) Nitric oxide (NO) 생성량 측정 ·········································· 33
다. 상황버섯 추출물 생리활성 물질 분리 및 활성 측정 ·········· 34
1) Ethanol 추출 ··············································································· 34
2) 용매 분획 ······················································································ 34
가) 항산화활성 측정 ······································································ 36
(1) Electron donating ability ·················································· 36
(2) Superoxide dismutase(SOD) 유사활성 ·························· 36
(3) ABTS scavenging activity ··············································· 36
(4) Superoxide anion scavenging acitivity ······················· 36
나) In-vitro test를 통한 항염증 활성 ······································ 36
(1) 세포배양 ·················································································· 36
(2) 세포독성 측정 ········································································ 36
(3) Nitric oxide(NO) 생성량 측정 ·········································· 36
3) Vacuum Liquid Chromatography(VLC)를 이용한
ethyl acetate 분획물의 분리 및 생리 활성 ························ 37
가) VLC에 의한 분획 ···································································· 37
나) In-vitro test를 통한 항염증 활성 ······································ 39
(1) 세포배양 ·················································································· 39
(2) 세포독성 측정 ········································································ 39
(3) Nitric oxide(NO) 생성량 측정 ·········································· 39
4) Preparative Liquid Chromatography(prep-LC)를 이용한
ethyl acetate 9번 fraction의 분리 및 생리 활성 ············· 40
가) Prep-LC를 이용한 ethyl acetate층 분리 ························ 40
나) In-vitro test를 통한 항염증 활성 ······································ 43
(1) 세포배양 ·················································································· 43
(2) 세포독성 측정 ········································································ 43
(3) Nitric oxide(NO) 생성량 측정 ·········································· 43
(4) Prostaglandin E2(PGE2) 생성량 측정 ····························· 44
(5) Western blot analysis ······················································· 45
4) High-performance liquid chromatography(HPLC)를
이용한 ethyl acetate 9-9번 fraction 순수 분리 및
생리 활성 ······················································································· 47
가) HPLC에 의한 순수 분리 ······················································· 47
나) In-vitro test를 통한 항염증 활성 ······································ 50
(1) 세포배양 ·················································································· 50
(2) 세포독성 측정 ········································································ 50
(3) Nitric oxide(NO) 생성량 측정 ·········································· 50
라. 생리활성 물질 동정 ···································································· 51
1) LC Mass에 의한 물질 동정 ······················································· 51
2) 1H-nuclear magnetic resonance(NMR)에 의한 물질 동정 ····· 53
Ⅲ. 결과 및 고찰 ··········································································· 54
1. 상황버섯, 영지버섯, 표고버섯의 성분분석 ································ 54
가. 일반성분 함량 ·············································································· 54
나. 유리당 함량 ·················································································· 57
다. 아미노산 함량 ·············································································· 59
1) 구성아미노산 함량 ······································································ 59
2) 유리아미노산 함량 ······································································ 62
라. Vitamin 함량 ················································································ 64
1) β-Carotene 함량 ······································································· 64
2) Vitamin D2 함량 ········································································ 66
3) Ergosterol 함량 ········································································· 68
4) Vitamin E 함량 ·········································································· 70
마. Ergothioneine 함량 ···································································· 72
바. β-Glucan 함량 ············································································ 74
2. 상황버섯, 영지버섯, 표고버섯의 생리활성 비교 ······················ 76
가. Total Polyphenol 함량 ····························································· 76
나. 항산화활성 ···················································································· 78
1) Electron donating ability ······················································· 78
가) Ethanol 추출물 ········································································ 78
나) 열수 추출물 ·············································································· 80
2) Superoxide dismutase(SOD) 유사활성 ······························· 82
가) Ethanol 추출물 ········································································ 82
나) 열수 추출물 ·············································································· 84
3) ABTS scavenging activity ····················································· 86
가) Ethanol 추출물 ········································································ 86
나) 열수 추출물 ·············································································· 88
4) Superoxide anion scavenging acitivity ····························· 90
가) Ethanol 추출물 ········································································ 90
나) 열수 추출물 ·············································································· 92
다. In-vitro test를 통한 면역력 증강 효과 ································ 94
1) LPS 처리 농도에 대한 세포독성 및 염증유발 ·················· 94
2) 세포독성 측정 ············································································ 96
3) Nitric oxide(NO) 생성량 측정 ············································· 98
3. 상황버섯 추출물 생리활성 물질의 분리 및 활성 측정 ······ 100
1) 용매 분획 ··················································································· 100
가) 항산화활성 ·············································································· 100
(1) Electron donating ability ··············································· 100
(2) Superoxide dismutase(SOD) 유사활성 ······················· 102
(3) ABTS scavenging activity ············································· 104
(4) Superoxide anion radical scavenging acitivity ······ 106
나) In-vitro test를 통한 항염증 활성 ··································· 108
(1) 분획물 세포독성 ·································································· 108
(2) 분획물 nitric oxide(NO) 생성 억제 ······························ 110
2) VLC를 이용해 분리한 ethyl acetate 분획물의 fraction별
항염증효과 ·················································································· 113
가) 세포독성 ·················································································· 113
나) Nitric oxide(NO) 생성 억제 ············································· 115
3) Prep-LC를 이용해 분리한 9번 fraction의 항염증효과 ··· 117
가) 세포독성 ·················································································· 117
나) Nitric oxide(NO) 생성 억제 ············································· 119
다) Prostaglandin E2 (PGE2) 분비능 억제 ·························· 121
라) 9-9번 fraction 처리를 통한 염증관련 단백질 발현 ···· 124
4) HPLC를 이용해 순수분리한 9-9번 fraction의 항염증효과 ··· 126
가) 세포독성 ·················································································· 126
나) Nitric oxide(NO) 생성 억제 ············································· 128
라. 생리활성 물질 동정 ·································································· 130
1) LC Mass에 의한 물질 동정 ····················································· 130
2) 1H-NMR에 의한 생리활성 물질 동정 ································· 132
Ⅳ. 요 약 ························································································ 134
Ⅴ. 참고문헌 ·················································································· 137