This study analyzed flavonoid and phenol contents and fatty acid composition of Stachys sieboldii Miq. and investigated biological activities of crude extracts and fractions of S. sieboldii Miq. The flavonoid and phenol contents of the methanol (MeOH) extract were greater than those of the acetone+methylene chloride (A+M) extract. Among fractions, the flavonoid and phenol contents of the n-buthanol (n-BuOH) fraction were highest in root of S. sieboldii Miq., while the flavonoid and phenol contents of the 85% aqueous methanol (85% aq. MeOH) fraction were highest in leaves of S. sieboldii Miq. In fatty acid composition of root of S. sieboldii Miq., the percentages of linoleic acid (LA, 18:2n-6), linolenic acid (LNA, 18:3n-3) and oleic acid (OA, 18:1n-9) were 54.27%, 20.82% and 2.28%, respectively. Leaves of S. sieboldii Miq. showed that the percentages of LA, LNA and OA were 11.27%, 16.37% and 5.45%, respectively.
In anticancer activity, treatments of crude extracts and fractions from S. sieboldii Miq. significantly inhibited the growth of HT-29, AGS and HT-1080 human cancer cell lines (p<0.05). Both A+M and MeOH extracts from roots and leaves showed inhibitory effects on growth by more than 50% at the concentrations of 0.5 mg/mL in AGS and HT-1080 cancer cells. Among the fractions, the 85% aq. MeOH and n-Hexane fractions exhibited a higher inhibitory effect on proliferation of three types of cancer cells.
In antioxidant activity, all tested extracts and fractions of S. sieboldii Miq. dose-dependently decreased cellular reactive oxygen species (ROS) production induced by H202 in comparison with that produced by exposure to the extract-free control. The A+M extract of roots showed a higher inhibitory effect on cellular ROS producing than that of the MeOH extract at all concentrations tested. On the other hand, MeOH extract of leaves showed a higher inhibitory effect on cellular ROS producing than that of the A+M extract at all concentrations tested. Among the fractions, both BuOH fraction (0.1 mg/mL concentration) from roots and leaves showed a higher inhibitory effect on cellular ROS production. In DPPH and ABTS radical scavenging assay, the A+M extract from roots showed a greater scavenging effect than that of the MeOH extract of roots(p<0.05). Among extracts from leaves, MeOH extract showed a greater scavenging effect than that of the A+M extract (p<0.05). The n-BuOH fraction showed a stronger radical inhibitory effect than dibutyl hydroxy toluene (BHT). The A+M and MeOH extracts from S. sieboldii Miq. significantly inhibited genomic DNA oxidation (p<0.05). The 85% aq. MeOH fraction showed a higher inhibitory effect against DNA oxidation compared with control (p<0.05). In addition, the extracts and fractions from S. sieboldii Miq. increased levels of intracellular glutathione (GSH) in a dose manner.
In anti-inflammatory activity, the productions of nitric oxide (NO) assay showed that the extracts and fractions significantly reduced NO production induced by lipopolysaccharide (LPS) (p<0.05). The n-BuOH fraction from roots of S. sieboldii Miq. showed the highest reduction of NO production by 75.4% and then A+M and MeOH extracts showed reduced NO production by 67.9% and 78.4%. The n-BuOH fraction from leaves of S. sieboldii Miq. showed the highest reduction of NO production by 61.4% and then A+M and MeOH extracts showed reduced NO production by 57.7% and 61.1%. respectively (p<0.05).
These results suggested that 85% aq. MeOH and n-BuOH fractions from roots and leaves of S. sieboldii Miq. inhibited cellular oxidation and growth of human cancer cells, suggesting that their biological activity may be associated with the contents of flavonoids and phenols. Thus, S. sieboldii Miq. might be useful for providing valuable materials to the pharmaceutical, cosmetic nutraceutical and food industries.1. 서 론 4
2. 재료 및 방법 7
2.1. 재료 7
2.2. 초석잠 뿌리와 잎 추출 및 분획 7
2.3. 초석잠 뿌리와 잎의 활성 성분 분리 8
2.4. 초석잠 뿌리와 잎의 총 플라보노이드 함량 측정 10
2.5. 초석잠 뿌리와 잎의 총 페놀 함량 측정 11
2.6. 초석잠 뿌리와 잎의 지질 및 지방산 추출 12
2.7. Gas chromatography를 이용한 초석잠 뿌리와 잎의 지방산 분석 14
2.8. 초석잠 뿌리와 잎 생리활성 측정 15
2.8.1. 항암 실험 15
① 세포 배양 15
② MTT assay 16
2.8.2. 항산화 실험 18
① 세포 배양 18
② 세포 내 활성산소종 (Reactive oxygen species) 측정 19
③ 1,1-diphenyl-2-picrylhydrazly (DPPH) 라디칼 소거 활성 측정 21
④ 2.2'-azino-bis(3-ethybenzthiazoline-6-sulphonic acid) diammonium salt radical cation (ABTS+) 라디칼 소거활성 측정 23
⑤ Genomic DNA 추출 및 DNA 산화 생성물 측정 25
⑥ 세포 내 Glutathione (GSH) 함량 측정 25
2.8.3. 항염증 실험 25
① 세포 내 Nitric oxide (NO) 생성 측정 25
2.9. 통계 분석 26
3. 결과 및 고찰 27
3.1. 초석잠 뿌리와 잎의 총 플라보노이드 및 총 페놀 함량 27
3.1.1 초석잠 뿌리의 총 플라보노이드 및 총 페놀 함량 27
3.1.2 초석잠 잎의 총 플라보노이드 및 총 페놀 함량 29
3.2. 초석잠 뿌리와 잎의 지방산 조성 31
3.2.1 초석잠 뿌리의 지방산 조성 31
3.2.2 초석잠 잎의 지방산 조성 33
3.3. 초석잠 뿌리와 잎의 추출물 및 분획물의 생리활성 35
3.3.1. 초석잠 뿌리와 잎 추출물 및 분획물의 항암 효과 35
3.3.1.1 초석잠 뿌리 추출물 및 분획물의 항암 효과 35
3.3.1.2 초석잠 잎 추출물 및 분획물의 항암 효과 45
3.3.2. 초석잠 뿌리와 잎 추출물 및 분획물의 항산화 효과 55
3.3.2.1 초섬잠 뿌리 추출물 및 분획물의 항산화 효과 55
① 초석잠 뿌리가 세포 내 활성산소종 (Reactive oxygen species) 생성 억제효과 55
② 초석잠 뿌리의 DPPH 라디칼 소거활성 58
③ 초석잠 뿌리의 ABTS+ 라디칼 소거활성 60
④ 초석잠 뿌리의 Genomic DNA 산화 억제효과 62
⑤ 초석잠 뿌리가 Glutathione (GSH) 생성에 미치는 효과 64
3.3.2.2 초석잠 잎 추출물 및 분획물의 항산화 효과 67
① 초석잠 잎의 세포 내 활성산소종 (Reactive oxygen species) 생성 억제효과 67
② 초석잠 잎의 DPPH 라디칼 소거활성 70
③ 초석잠 잎의 ABTS+ 라디칼 소거활성 72
④ 초석잠 잎의 Genomic DNA 산화 억제효과 74
⑤ 초석잠 잎의 Glutathione (GSH) 생성에 미치는 효과 76
3.3.3.1 초석잠 뿌리 추출물 및 분획물의 항염증 효과 67
① 세포 내 Nitric oxide (NO) 생성 저해효과 67
3.3.3.2 초석잠 잎 추출물 및 분획물의 항염증 효과 67
① 세포 내 Nitric oxide (NO) 생성 저해효과 67
4. 요약 및 결론 93
참고문헌 96
감사의 글 103Maste
