부산 태종대 굴통머리 및 찬물개 구역 돌미역(Undaria pinnatifida)의 영양화학적 특성과 항산화 및 항암 효과 = Nutritional chemical characteristics and antioxidant and anticancer effects of sea mustards from Gultongmeori and Chanmulgae area, Taejongdae in Busan

다국어 초록 (Multilingual Abstract)

This study investigated to analyze flavonoid and phenol contents of sea mustards from Taejongdae in Busan and determined biological activities of fractions from sea mustards. Total flavonoid and phenol contents showed that n-Hexane fraction from sea mustard of Gultongmeori area had more phenol content, while 85% aqueous methanol (85% aq. MeOH) fraction from sea mustard of Chanmulgae area contained the highest flavonoid and phenol contents. In anticancer assay using the cell counting kit-80 (CCK-8) reduction method, treatments with fractions from sea mustard of Chanmulgae area significantly inhibited the growth of HT-29, MDA-MB-231, SNU-719 and AsPC-1 human cancer cell lines (p < 0.05). The fractions with the highest inhibitory effect on the proliferation of all cancer cells were 85% aq. MeOH and n-Hexane fractions. In addition, we conducted experiments related to matrix metalloproteinase (MMP), which is closely related to the occurrence of cancer. expressions of MMP-2 and MMP-9 in HT-1080 cells were measured by Western blot, respectively. The 85% aq. MeOH fraction from sea mustard of both Gultongmeori and Chanmulgae areas revealed the strong inhibitory effect against the expression of MMP-2 and MMP-9. In antioxidant activity of sea mustard, all tested fractions from sea mustard of both Gultongmeori and Chanmulgae areas 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 85% aq. MeOH fraction showed a higher inhibitory effect on cellular ROS production than that of other fractions at all concentration stested. In DPPH and ABTS radical scavenging assay, the 85% aq. MeOH fraction from seamustard of both Gultongmeori and Chanmulgae areas showed a scavenging effect greater than that of other fractions (p < 0.05). The 85% aq. MeOH and n-Hexane fractions from sea mustard of both Gultongmeori and Chanmulgae areas showed a higher inhibitory effect against DNA oxidation compared with control (p<0.05). In addition, fractions from sea mustard of both Gultongmeori and Chanmulgae areas tended to increase levels of intracellular glutathione (GSH) but the differences were not statistical. In anti-inflammatory activity, the production of nitric oxide (NO) assay showed that all fractions from sea mustard of both Gultongmeori and Chanmulgae areas significantly reduced NO production induced by lipopolysaccharide (LPS) (p < 0.05). Among fractions, the n-Hexane n and 85% aq. MeOH fractions from sea mustard of Gultongmeori area showed higher reduction of NO production by 72% and 54%, respectively. In addition, in case of sea mustard of Chanmulgae area, the n-Hexane and 85% aq. MeOH fractions showed higher decreased NO production by 72% and 71%, respectively (p < 0.05). These results suggested that n-Hexane and 85% aq. MeOH fractions of sea mustard from Gultongmeori and Chanmulgae areas inhibited cellular oxidation and growth of human cancer cells, which suggested that their activity may be associated with the contents of flavonoids and phenols. Therefore, this study proposes that processed food products supplemented with sea mustard can be developed as functional foods to promote health in the local population.

목차 (Table of Contents)

• 1. 서 론 4
• 2. 재료 및 방법 7
• 2.1. 재료 7
• 2.2. 돌미역의 추출 및 분획 7
• 2.4. 돌미역의 총 플라보노이드 함량 측정 11
• 2.5. 돌미역의 총 페놀 함량 측정 12
• 2.6. 돌미역의 생리활성 측정 13
• 2.6.1. 항암 실험 13
• ① 세포 배양 13
• ② Cell viabillity assay를 통한 항암 효과 측정 14
• ③ Western blot을 통한 Matrix metalloprotinase (MMP) 발현 측정 16
• 2.6.2. 항산화 실험 17
• ① 세포 배양 17
• ② 세포 내 활성산소종 (Reactive oxygen species) 측정 18
• ③ 1,1-diphenyl-2-picrylhydrazly (DPPH) 라디칼 소거활성 측정 20
• ④ 2.2'-azino-bis(3-ethybenzthiazoline-6-sulphonicacid) diammonium salt
• radicalcation (ABTS+) 라디칼 소거활성 측정 22
• ⑤ Genomic DNA 추출 및 DNA 산화 생성물 측정 24
• ⑥ 세포 내 Glutathione (GSH) 함량 측정 24
• 2.6.3. 항염증 실험 25
• ① 세포 내 Nitric oxide (NO) 생성 측정 25
• 2.7. 통계 분석 25
• 3. 결과 및 고찰 26
• 3.1. 돌미역의 총 플라보노이드 및 페놀 함량 26
• 3.1.1. 굴통머리 구역 돌미역의 총 플라보노이드 및 총 페놀 함량 26
• 3.1.2. 찬물개 구역 돌미역의 총 플라보노이드 및 총 페놀 함량 28
• 3.2. 돌미역 분획물의 항암 활성 30
• 3.2.1. 굴통머리 구역 돌미역 분획물의 항암 효과 30
• ① 인체 유래 암세포에 대한 세포 증식 억제 효과 30
• ② Western Blot을 통한 MMP-2과 MMP-9 level의 발현 36
• 3.2.2. 찬물개 구역 돌미역 분획물의 항암 효과 38
• ① 인체 유래 암세포에 대한 세포 증식 억제 효과 38
• ② Western Blot을 통한 MMP-2과 MMP-9 level의 발현 44
• 3.3. 돌미역 분획물의 항산화 활성 46
• 3.3.1. 굴통머리 구역 돌미역 분획물의 항산화 효과 46
• ① 세포 내 활성산소종 (Reactive oxygen species) 생성 억제 효과 46
• ② DPPH 라디칼 소거 활성 48
• ③ ABTS+ 라디칼 소거 활성 50
• ④ Genomic DNA 산화 억제 효과 52
• ⑤ Glutathione (GSH) 생성 상승 효과 54
• 3.3.2. 찬물개 구역 돌미역 분획물의 항산화 효과 56
• ① 세포 내 활성산소종 (Reactive oxygen species) 생성 억제 효과 56
• ② DPPH 라디칼 소거 활성 58
• ③ ABTS+ 라디칼 소거 활성 60
• ④ Genomic DNA 산화 억제 효과 62
• ⑤ Glutathione (GSH) 생성 상승 효과 64
• 3.4. 돌미역 분획물의 항염증 활성 66
• 3.4.1. 굴통머리 구역 돌미역 분획물의 항염증 효과 66
• ① 세포 내 Nitric oxide (NO) 생성 저해효과 66
• 3.4.2. 찬물개 구역 돌미역 분획물의 항염증 효과 71
• ① 세포 내 Nitric oxide (NO) 생성 저해효과 71
• 4. 요약 및 결론 76
• 참고문헌 79