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논문 기본 정보
- 자료유형
- 학위논문
- 저자정보
- 지도교수
- 서영완
- 발행연도
- 2013
- 저작권
- 한국해양대학교 논문은 저작권에 의해 보호받습니다.
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As the human lifespan has been extended due to great advances in medical science, the incidence of various adult diseases has rapidly increased - cancer, cardiac disorder, diabetes, dementia etc. In recent years, there has been growing interest in the use of natural products, especially those derived from terrestrial plants or seaweeds. Plant-based medicine has definitely played an expanding role in the treatment of adult diseases.
As a part of our search for bioactive compounds from marine organisms, we collected Zostera asiatica, one of the marine flowering plants, from the southern coastal area of Korea. Dried samples were extracted twice: with acetone/methylene chloride (A+M) and with methanol (MeOH), respectively. The combined crude extracts were evaporated in vacuo, and then the residue was partitioned between water and methylene chloride. The aqueous layer was fractionated into H2O and n-butanol, and then the organic layer was also fractionated into 85% aq. MeOH and n-hexane, successively.
The crude extracts and their solvent fractions were evaluated for antioxidant, antiproliferative, and antiinflammatory effects, respectively. In antioxidant bioassay, n-BuOH and 85% aq. MeOH fractions showed a strong scavenging effect on DPPH radical and peroxynitrites. In a cellular system using 2'',7''-dichlorofluorescin diacetate (DCF-DA) as fluorescence probe in HT-1080 cells, all extracts and solvent fractions except the H2O fraction significantly decreased the level of intracellular reactive oxygen species(ROS). On the basis of the above, two flavonoids were obtained from n-BuOH fraction by bactivity-guided separation.
On the other hand, in cytotoxicity bioassay system using the MTT reduction method, the 85% aq. MeOH fraction among samples tested showed the most inhibitory effect on growth of human cancer cells AGS, HT-29, and MCF-7 cells. However, all samples didn’t exhibit the significant inhibitory effect on lipopolysaccharide(LPS)-stimulated nitric oxide(NO) production. Therefore, these results suggest that Z. asiatica may be useful as a potential candidate for both natural antioxidant to oxidative damage, and chemopreventive agent for cancer.
As a part of our search for bioactive compounds from marine organisms, we collected Zostera asiatica, one of the marine flowering plants, from the southern coastal area of Korea. Dried samples were extracted twice: with acetone/methylene chloride (A+M) and with methanol (MeOH), respectively. The combined crude extracts were evaporated in vacuo, and then the residue was partitioned between water and methylene chloride. The aqueous layer was fractionated into H2O and n-butanol, and then the organic layer was also fractionated into 85% aq. MeOH and n-hexane, successively.
The crude extracts and their solvent fractions were evaluated for antioxidant, antiproliferative, and antiinflammatory effects, respectively. In antioxidant bioassay, n-BuOH and 85% aq. MeOH fractions showed a strong scavenging effect on DPPH radical and peroxynitrites. In a cellular system using 2'',7''-dichlorofluorescin diacetate (DCF-DA) as fluorescence probe in HT-1080 cells, all extracts and solvent fractions except the H2O fraction significantly decreased the level of intracellular reactive oxygen species(ROS). On the basis of the above, two flavonoids were obtained from n-BuOH fraction by bactivity-guided separation.
On the other hand, in cytotoxicity bioassay system using the MTT reduction method, the 85% aq. MeOH fraction among samples tested showed the most inhibitory effect on growth of human cancer cells AGS, HT-29, and MCF-7 cells. However, all samples didn’t exhibit the significant inhibitory effect on lipopolysaccharide(LPS)-stimulated nitric oxide(NO) production. Therefore, these results suggest that Z. asiatica may be useful as a potential candidate for both natural antioxidant to oxidative damage, and chemopreventive agent for cancer.
목차
목 차List of schemes -------------------------------------------------------ⅰList of table ---------------------------------------------------------ⅱList of figures -------------------------------------------------------ⅲList of abbreviations -------------------------------------------------ⅵAbstract --------------------------------------------------------------11 서론 ----------------------------------------------------------------32 재료 및 방법 --------------------------------------------------------62-1. 재료 -----------------------------------------------------------62-2. 시약 -----------------------------------------------------------6(1) 추출, 분획 및 분리 --------------------------------------------6(2) 활성 ----------------------------------------------------------6(3) 기기 ----------------------------------------------------------7(4) 세포배양 ------------------------------------------------------72-3. 추출 및 분리 ---------------------------------------------------9(1) 왕거머리말의 추출 및 분획 -------------------------------------9(2) 왕거머리말의 활성 성분 분리 -----------------------------------112-4. 총 polyphenol 함량 측정 ----------------------------------------132-5. 항산화 활성 실험 -----------------------------------------------14(1) DPPH radical 소거 활성 ----------------------------------------14(2) Peroxynitrite 소거 활성 ---------------------------------------17(3) 세포 독성 측정 -------------------------------------------------20(4) 세포내 활성 산소종(ROS) 측정 -----------------------------------22(5) GSH 함량 측정 -------------------------------------------------24(6) Genomic DNA 추출 및 Genomic DNA 산화 생성물 측정 -------------- 252-5 암세포 증식 억제 실험 -------------------------------------------26(1) MTT assay를 이용한 세포 생존율 측정 ---------------------------26(2) 역전사 중합 연쇄 반응(RT-PCR) 분석 ----------------------------272-6 항염증 활성 실험 ------------------------------------------------29(1) NO 생성 억제 효과 ---------------------------------------------293. 결과 및 고찰 -------------------------------------------------------313-1 총 polyphenol 함량 측정 -----------------------------------------313-2 왕거머리말로부터 분리한 물질의 구조 결정 ------------------------333-3 In vitro 항산화 활성 ---------------------------------------------39(1) DPPH radical 소거 활성 ----------------------------------------39(2) Peroxynitrite 소거 활성 ---------------------------------------413-4 세포 수준에서의 항산화 활성 -------------------------------------44(1) HT-1080 세포에 대한 조추출물과 용매분획물의 독성 효과 ---------44(2) 세포내 활성 산소종(ROS) 소거 활성 -----------------------------46(3) 세포내 GSH 함량 측정 ------------------------------------------50(4) Genomic DNA의 산화 생성물 측정 --------------------------------533-3 인체 유래 암세포에 대한 세포증식 억제 효과 ----------------------55(1) HT-1080 세포 증식 억제 효과 -----------------------------------55(2) AGS 세포 증식 억제 효과 ---------------------------------------57(3) HT-29 세포 증식 억제 효과 -------------------------------------59(4) MCF-7 세포 증식 억제 효과 -------------------------------------61(5)용매 분획물이 apoptosis 관련 mRNA 발현에 미치는 효과 -----------633-4 NO 생성 억제 효과 -----------------------------------------------664. 요약 및 결론 -------------------------------------------------------695. 참고문헌 -----------------------------------------------------------716. 부록 ---------------------------------------------------------------75
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