[논문]식물추출물 항산화효능 기전의 일부로서의 활성산소 발생 억제 효과

송선범; 정구준; 정희진; 장정윤; 정해영; 김남득; 이지현; 민경진; 박순영; 곽충실; 황은성

doi:10.9721/kjfst.2021.53.6.706

식물추출물 항산화효능 기전의 일부로서의 활성산소 발생 억제 효과
Suppression of reactive oxygen species generation as a part of antioxidative effect of plant extracts 원문보기

한국식품과학회지 = Korean journal of food science and technology, v.53 no.6, 2021년, pp.706 - 714

송선범 (서울시립대학교 생명과학과) , 정구준 (서울시립대학교 생명과학과) , 정희진 (부산대학교 약학대학) , 장정윤 (부산대학교 약학대학) , 정해영 (부산대학교 약학대학) , 김남득 (부산대학교 약학대학) , 이지현 (인하대학교 생명과학과) , 민경진 (인하대학교 생명과학과) , 박순영 (다인내추럴) , 곽충실 (서울대학교 의과대학 노화고령사회연구소) , 황은성 (서울시립대학교 생명과학과)

초록
AI-Helper

세포 내에서의 활성산소(ROS) 생성을 억제하는 일은 항산화제의 작동방식으로 기존에 알려진 ROS의 화학적 소거보다 조직의 산화적 손상을 줄이는 방법으로서 효과가 뛰어날 것으로 기대되지만, 이러한 전략은 지금까지의 연구들에서 거의 검토되지 않고 있다. 본 연구에서는 항산화 효능이 알려진 식물시료들이 실제로 세포에서 ROS 생성을 제어하여 항산화 효과를 발휘하는지를 쇠뜨기, 가죽나무잎, 달맞이순, 그리고 토마토의 에탄올 추출물을 가지고 조사하였다. 이 네 가지 식물 시료들은 모두 비슷하게 세포내 ROS의 수준을 감소시켰다. 그러나, 가죽나무잎, 달맞이순, 그리고 토마토 시료들만이 미토콘드리아 질을 개선하여 미토콘드리아로부터의 ROS 생성을 감소시켰는데, 이들은 또한 세포와 조직내 lipofuscin과 malondialdehyde의 축적을 줄여서 뚜렷한 산화적 손상을 억제하는 효과를 보였다. 이들은 나아가서 초파리의 수명을 연장시키는 효과도 보였다. 미토콘드리아 질 향상 효과가 거의 없었던 쇠뜨기 시료는 산화적 손상물과 초파리 수명에 거의 영향을 미치지 못하였다. 이러한 결과는 식물 시료들의 항산화 효과가 ROS의 화학적 소거와 미토콘드리아 질의 향상을 통해 발현될 수 있는데, 후자의 효과가 실제로 체내에서 산화적 손상을 억제하는데 중요하게 작용할 가능성을 시사해 준다. 향후, 식물시료들의 항산화 효능에 대해서 이러한 ROS 발생억제 기전을 대상으로 조사하는 일은 그 유용성을 판단하는데 있어서 큰 도움이 될 것으로 기대된다.

Abstract ▼ AI-Helper

Chemical scavenging of reactive oxygen species (ROS) is considered a major mechanism of antioxidant effects, but preventing ROS generation can be more efficient in attenuating oxidative damage. In this study, the extracts of plants, Solanum lycopersicum, Ailanthus altissima, Equisetum arvense, and Oenothera biennis, were tested to determine whether their antioxidative effects are driven by the prevention of superoxide generation from mitochondria, a major ROS generator. While all the extracts efficiently attenuated the elevation of ROS levels in human fibroblasts and inflammation-induced mice, those of S. lycopersicum, A. altissima, and O. laciniata only suppressed mitochondrial ROS generation and reduced levels of lipofuscin and lipid peroxidation. Furthermore, the extracts of A. altissima and O. laciniata extended the lifespan of fruit flies. Our results suggest that plant extracts with anti-oxidative effects differ in their ability to prevent ROS generation, which may be associated with the attenuation of oxidative damage in cells and animal tissues.

주제어

표/그림 (4)

그림 Fig. 1. Effect of plant extracts on the levels of free radicals in human cells and mouse kidney. (A) Human fibroblasts cultivated in wells of 6-well plates were treated with 0.25 μM Adriamycin for 4 h and further incubated in mock-treated medium (Ad) or in the presence of 0.2, 1, or 5 mg/mL extracts of tomato (Ad+Sol), Ailanthus (Ad+Ail), Equisetum (Ad+Equ), or Oenothera (Ad+Oen). After 72 h, cells were collected, and applied for flow cytometry to determine cellular superoxide levels. Mean values of the measurements from 3 wells were normalized by that of the cells not treated with adriamycin (C), and the relative numbers are plotted. The values of the treatments of adriamycin alone ( −) and adriamycin+extracts (Ad+Sol, Ad+Ail, Ad+Equ), and those of the treatments of 0.2 mg/mL and either of 1 or 5 mg/mL of all the extracts except for the cases of Oenothera are significantly different at p<0.01 (**) by Anova test. (B) C57BL6/N mice (male, n=6) were given either distilled water ( −) or the extracts through oral gavage at a dose of 70 mg/kg per day for 3 days, and 6 h hours before sacrifice, were given intraperitoneally 5 mg/kg lipopolysaccharide (LPS). Homogenates of isolated kidneys were applied to fluorometry to quantitate the levels of ROS. Mean values of the measurements from 6 mice were normalized by that of the non-LPS-treated mice (C), and the relative numbers are plotted. The differences of the treatments of adriamycin alone (−) and adriamycin+tomato (Sol) or Oenothera (Oen) are significant at p<0.05 (*), and that of the treatment of adriamycin alone and adriamycin+Ailanthus (Ail) is different at p<0.01 (**) by Anova test.
그림 Fig. 2. Effect of plant extracts on mitochondrial ROS, content, membrane potential as well as cellular autophagy activity. (A) Human fibroblasts in wells of 6-well plates were pulsed with 0.25 μM Adriamycin and chased in mock-treated medium (Ad) or in the presence of 0.2, 1, or 5 mg/mL extracts of Equisetum (Ad+Equ), Ailanthus (Ad+Ail), tomato (Ad+Sol), or Oenothera (Ad+Oen). After 72 h, cells were collected, and applied to determination of mitochondrial ROS (A), content (B), and membrane potential (D). Or the collected cells were applied to western blotting analysis for the levels LC3 type I and type II, or p62SQSTM1 (C, top). To quantitatively compare the changes in the ratios of LC3 type II/type I, band intensities were quantitated through densitometry and normalized by that of β-actin. Measured vales for the cel ls from three different wells were averaged, and normalized by that of control cells (not senescence-induced) (C), and plotted. The differences between the treatments of adriamycin alone (−) and adriamycin+extracts are significant at p<0.01 (**) by ANOVA.
그림 Fig. 3. Effect of plant extracts on cellular lipofuscin accumulation and MDA levels in cells and mouse kidneys. (A) Human fibroblasts were treated with adriamycin as in Fig. 1, and treated with 0.2, 1, 5 mg/mL extracts of Equisetum (Ad+Equ), Ailanthus (Ad+Ail), tomato (Ad+Sol), or Oenothera (Ad+Oen) or mock treated. After 72 h, cells were collected and applied to fluorometry for auto-fluorescence quantitation (A) or determination of mitochondrial ROS (B). Or, the collected cells were lysed to determine levels of malondialdehyde (C). Mean values of cells from three different wells were normalized to those of control cells (C), and relative values were plotted. The differences between the treatments of adriamycin alone (−) and adriamycin+extracts are significant at either p<0.1 (*) or p<0.01 (**) by ANOVA. In (D), kidney homogenates from mice untreated (C) or treated either with LPS alone (−), Ailanthus +LPS (Ail), or Oenothera+LPS (Oen) were applied to determination of malondialdehyde levels. Mean values of the measurements from 6 mice treated were normalized by those of the mice not treated with LPS, and the relative numbers are plotted. The differences between LPS alone (−) and extract+LPS in the case of female mouse are significant at p<0.05 (*) by ANOVA.
그림 Fig. 4. Effect of plant extracts on longevity of fruit flies. Flies (n=300 for male and female per experiment) were fed with either 0, 25, 100, 500, 1000 μg/mL of ethanol extracts of Ailanthus (A), Oenothera (B), tomato (C), or Equisetum (D) in SY medium. In every 2 days, the extract-fortified medium was changed anew. At this time, dead flies were counted, and the survival curve was plotted, and applied to Log-rank test. For each extract, the effects on male (♂, left) and female (♀, right) are presented.

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