하수오(Polygonum multiflorum Thunberg) 건조방법에 따른 일반성분 및 생리활성

다국어 초록 (Multilingual Abstract)

This study was carried out to characterize the chemical components of Polygonum multiflorum root to evaluate its antioxidant activity and to identify the active compound for providing the basic data necessary for developing functional food and medicine. The results was as followings:
1. The results of proximate compositions in P. multiflorum root based on various dry methods were as followings; The highest contents of moisture(8.25%) and fat(1.11%) showed shade-dried P. multiflorum root among the dried P. multiflorum roots. The highest content of crude protein, crude ash and nitrogen free extract were highest in freeze-dried P. multiflorum root among the samples.
2. As the results of free sugars analysis, fructose, glucose, and sucrose were detected in dried P. multiflorum roots by various dry methods. The content of total free sugars were the highest in freeze dried P. multiflorum root.
Four kind organic acids were found in dried P.multiflorum roots. The contents of tartaric acid, malic acid, and malonic acid were highest in freeze dried P multiflorum root. But the highest content of oxalic acid was found in shade dried P. multiflorum root.
3. As the results of amino acids analysis sixteen kind amino acids were detected in dried P. multiflorum root by different dried methods. The major amino acid was determined arginine, and the highest content of arginine was found in freeze dried P. multiflorum root. The essential amino acids proportions(EAA/TAA) of shade dried P. multiflorum root, hot air dried P. multiflorum root, and freeze dried P. multiflorum root were 34.27%, 33.70%, and 31.91%, respectively.
4. Four kind minerals were found in dried P. multiflorum roots, and the potassium was the major minerals in all samples. The highest content of potassium was found in freeze dried P. multiflorum root (1,515.01 mg%).
5. The content of fatty acids in Polygonum multiflorum root based on various dry methods were high in the orders of linoleic acid, oleic acid and palmitic acid. The content of linoleic acid in freeze dried P. multiflorum root, shade dried P. multiflorum root, and hot air dried P. multiflorum root were 84.5 area%, 66.5 area%, 54.5 area%, respectively.
6. The content of total polyphenol in ethanol extract from dried P. multiflorum roots were higher than hot-water extract from dried P. multiflorum roots. The highest content of total polyphenols were found in ethanol extract from freeze dried P. multiflorum root (856.89 mg QE/g). The total flavonoid content of extracts from hot-air dried P. multiflorum were higher than the other samples.
7. The hot-water extracts of dried P. multiflorum roots showed higher activity for superoxide anion radical inhibition activity and DPPH radical scavenging activity than ethanol extract of dried P. multiflorum roots. The ethanol extract from dried P. multiflorum root showed higher ABTS radical scavenging activity than hot-water extract.
8. The cell viability of extract from dried P. multiflorum root was no cell toxic was observed in below the concentration 500 ug/mL of extract from dried P. multiflorum root application. The inhibition activity of nitric oxide production were not shows in all sample extracts.
9. As the result of anthraquinolic compounds analysis of P. multiflorum, 2,3,5,4`-tetrahydroxystilbene-2-O-β -D-glucoside(THSG), emodin, physcion were detected by HPLC.
10. Ethyl acetate fraction of ethanol extract from dried P multiflorum root was showed the highest total polyphenol content, DPPH radical scavenging activity and ABTS radical scavenging activity than the other extract fractions.
11. Fifteen fractions were obtained from ethylacetate fraction of P. multiflorum root ethanol extract by VLC. Number eleven and twelve were showed strong DPPH radical scavenging activity and ABTS radical scavenging activity among the VLC fractions.
12. The purified fifteen subfractions were obtained from subfraction number 12-4 of P. multiflorum root ethyl acetate fractions by HPLC. The purified subfraction 12-4-3 was identified the 2,3,5,4`-tetrahydroxystilbene–

목차 (Table of Contents)

• Ⅰ. 서 론 ····························································································· 1
• Ⅱ. 재료 및 방법 ··············································································· 6
• 1. 재료 ········································································································ 6
• 가. 실험재료 ····························································································· 6
• 나. 시료 건조방법 ··················································································· 6
• 다. 시약 ····································································································· 7
• 라. 세포 및 배지 ····················································································· 8
• 2. 실험방법 ································································································ 9
• 가. 건조방법에 따른 하수오의 성분분석 ··········································· 9
• 1) 일반성분 분석 ·················································································· 9
• 2) 유리당 분석 ···················································································· 10
• 3) 유기산 분석 ···················································································· 12
• 4) 아미노산 분석 ················································································ 14
• 가) 구성아미노산 분석 ···································································· 14
• 나) 유리아미노산 분석 ···································································· 15
• 5) 무기성분 분석 ················································································ 17
• 6) 지방산 분석 ···················································································· 19
• 나. 건조방법에 따른 하수오의 생리활성 ········································ 21
• 1) 시료 추출 ························································································ 21
• 가) Ethanol 추출 ·············································································· 21
• 나) 열수추출 ······················································································ 21
• 2) 항산화활성 측정 ············································································ 23
• 가) Total flavonoid content(TFC) ············································· 23
• 나) Total polyphenol content(TPC) ········································· 24
• 다) DPPH radical scavenging activity ···································· 25
• 라) ABTS cation radical scavenging activity ······················· 26
• 마) Superoxide anion scavenging activity ···························· 27
• 3) In-vitro test를 통한 항염증 활성 ··········································· 28
• 가) 세포배양 ······················································································ 28
• 나) 세포독성 측정 ············································································ 29
• 다) 대식세포주의 Nitric oxide(NO) 생성량 측정 ···················· 30
• 4) 하수오추출물의 rhein, emodin, chrysophanol, physcion 및
• 2,3,5,4`-tetrahydroxystilbene–2-O-β-D-glucoside 함유 검
• 증 ········································································································ 31
• 다. 하수오추출물 생리활성 물질의 분리 및 활성 측정 ·············· 33
• 1) 활성추출물의 용매 분획 ······························································ 33
• 2) 항산화활성 측정 ············································································ 35
• 가) Total flavonoid content(TFC) ············································· 35
• 나) Total polyphenol content(TPC) ········································· 35
• 다) DPPH radical scavenging activity ···································· 35
• 라) ABTS cation radical scavenging activity ······················· 35
• 마) Superoxide anion scavenging activity ···························· 35
• 라. Vacuum liquid chromatography(VLC)를 이용한
• ethyl acetate 분획물의 분리 및 생리 활성 ·························· 36
• 1) VLC에 의한 분획 ········································································· 36
• 2) 항산화 활성 검증 ·········································································· 38
• 가) DPPH radical scavenging activity ···································· 38
• 나) ABTS cation radical scavenging activity ······················· 38
• 마. 활성fraction에서의 물질 분리정제 및 생리 활성 검증 ········ 39
• 1) Preparative liquid chromatography(prep-LC)를 이용한
• 분리 정제 ······················································································· 39
• 2) High-performance liquid chromatography(HPLC)를
• 이용한 순수 분리 및 정제 ······················································ 42
• 가) HPLC에 의한 순수 분리 ························································· 42
• 바. 생리활성 물질 동정 ······································································ 45
• 1) LC Mass에 의한 물질 동정 ························································· 45
• 2) 1H-nuclear magnetic resonance(NMR) 및 1H-1H COSY
• spectrum에 의한 물질 동정 ··························································· 47
• 사. 건조방법에 따른 하수오의 발효 방법 및 성분분석 ·············· 48
• 1) 하수오 발효 방법 ·········································································· 48
• 가) 배지조성에 따른 균사의 성장 측정 ······································ 48
• 2) 일반성분 분석 ················································································ 50
• 3) 유리당 분석 ···················································································· 50
• 4) 유기산 분석 ···················································································· 50
• 5) 아미노산 분석 ················································································ 50
• 가) 구성아미노산 분석 ···································································· 50
• 나) 유리아미노산 분석 ···································································· 50
• 6) 무기성분 분석 ················································································ 50
• 아. 하수오 발효 조건 탐색 ································································ 51
• 1) 재료 및 방법 ·················································································· 51
• 가) 실험재료 ························································································ 51
• 나) 방법 ································································································ 51
• (1) 입도크기에 따른 균사의 생장 측정 ······································ 51
• (2) pH에 따른 하수오에서 따른 균사의 생장 측정 ················· 51
• (3) 탄소원에 따른 균사의 생장 측정 ·········································· 52
• (4) 질소원에 따른 균사의 생장 측정 ·········································· 52
• (6) 곡류배지에서의 대량 배양 ······················································ 53
• Ⅲ. 결과 및 고찰 ············································································· 55
• 1. 건조방법에 따른 하수오의 성분분석 ············································ 55
• 가. 일반성분 함량 ················································································ 55
• 나. 유리당 함량 ···················································································· 57
• 다. 유기산 함량 ···················································································· 59
• 라. 아미노산 함량 ················································································ 61
• 1) 구성아미노산 함량 ········································································ 61
• 2) 유리아미노산 함량 ········································································ 64
• 마. 무기성분 함량 ················································································ 66
• 바. 지방산 함량 ···················································································· 68
• 2. 건조방법에 따른 하수오의 생리활성 ············································ 70
• 가. 항산화활성 ······················································································ 70
• 1) Total flavonoid content(TFC) ················································ 70
• 2) Total polyphenol content(TPC) ············································· 72
• 3) DPPH radical scavenging activity ········································ 74
• 가) Ethanol 추출물 ········································································ 74
• 나) 열수 추출물 ·············································································· 76
• 4) ABTS cation radical scavenging activity ·························· 78
• 가) Ethanol 추출물 ········································································ 78
• 나) 열수 추출물 ·············································································· 80
• 5) Superoxide anion scavenging activity ································ 82
• 가) Ethanol 추출물 ········································································ 82
• 나) 열수 추출물 ·············································································· 84
• 나. In-vitro test 항염증 활성 ·························································· 86
• 1) 세포독성 및 nitric oxide(NO) 억제효과 ································ 86
• 다. 하수오추출물의 rhein, emodin, chrysophanol, physcion
• 및 2,3,5,4`-tetrahydroxystilbene–2-O-β-D-glucoside 함유
• 검증 ···································································································· 89
• 3. 하수오 추출물 생리활성 물질의 분리 및 활성 ·························· 96
• 가. 항산화활성 ······················································································ 96
• 1) Total flavonoid content(TFC) ················································ 96
• 2) Total polyphenol content(TPC) ············································· 98
• 3) DPPH radical scavenging activity ····································· 100
• 4) ABTS cation radical scavenging activity ························ 102
• 5) Superoxide anion scavenging activity ······························ 104
• 4. Vacuum liquid chromatography(VLC)를 이용한 ethyl
• acetate 분획물의 분리 및 생리 활성 ···································· 106
• 가. Ethyl acetate fraction 분리 ··················································· 106
• 나. 항산화 활성 검증 ········································································ 108
• 1) DPPH radical scavenging activity ····································· 108
• 2) ABTS cation radical scavenging activity ························ 110
• 5. 생리활성 물질 동정 ········································································ 112
• 가. LC Mass에 의한 물질 동정 ························································ 112
• 나. 1H-nuclear magnetic resonance(NMR) 및 1H-1H COSY
• spectrum에 의한 생리활성 물질 동정 ····································· 113
• 6. 건조방법에 따른 하수오 발효물의 성분 ···································· 115
• 가. 일반성분 함량 ·············································································· 115
• 나. 유리당 함량 ·················································································· 117
• 다. 유기산 함량 ·················································································· 119
• 라. 아미노산 함량 ·············································································· 121
• 1) 구성아미노산 함량 ····································································· 121
• 2) 유리아미노산 함량 ····································································· 123
• 마. 무기성분 함량 ·············································································· 125
• 7. 하수오 발효 조건 탐색 ·································································· 127
• 가. 하수오 발효 최적 입도크기와 온도 ········································ 127
• 나. 하수오 발효 최적 pH ······························································· 131
• 다. 하수오 발효 최적 탄소원 및 질소원 ······································ 134
• Ⅳ. 요 약 ························································································ 139
• Ⅴ. 참고문헌 ·················································································· 143