[논문]RAW 264.7 세포에서 조팝나무 에탄올 분획물의 항염 활성

서진형; 이한솔; 김수환; 이성진; 배은영; 이선영

doi:10.4163/jnh.2022.55.1.59

RAW 264.7 세포에서 조팝나무 에탄올 분획물의 항염 활성
Anti-inflammatory effects of the ethanol fraction of Spiraea prunifolia var. simpliciflora in RAW 264.7 cells 원문보기

Journal of nutrition and health, v.55 no.1, 2022년, pp.59 - 69

서진형 (충남대학교 식품영양학과) , 이한솔 (충남대학교 식품영양학과) , 김수환 ((주)차메디텍) , 이성진 ((주)차메디텍) , 배은영 (충남대학교 식품영양학과) , 이선영 (충남대학교 식품영양학과)

초록
AI-Helper

본 연구실의 선행 연구에서 항산화 활성이 가장 높았던 조팝나무 H₂O-EtOH (40:60) 분획물(SP60)의 항염 효능을 확인하여 기능성 소재로의 개발 가능성을 알아보고자 하였다. 항염활성 측정을 위해 RAW 264.7 세포에서 먼저 WST assay를 실시하여 SP60이 125 ㎍/mL 농도까지 세포 생존율에 영향을 미치지 않는 것을 확인하였다. Raw264.7 세포에 LPS와 SP60을 동시에 처리하여 NO, TNF-α, IL-6, IL-1β 생성과 iNOS, COX-2, NF-κB 발현을 측정하였다. SP60은 NO, TNF-α, IL-6, IL-1β 생성량을 모두 유의하게 억제시켰으며 western blot으로 iNOS, COX-2의 발현과 NF-κB의 활성을 측정한 결과 SP60을 처리한 군에서 농도 의존적으로 그 발현량과 활성을 억제하였다. 따라서 조팝나무 분획물은 항염 효능을 가진 것으로 판단되며 항염 관련 기능성 식품 및 소재로의 개발 가능성을 확인하였다.

Abstract ▼ AI-Helper

Purpose: Natural medicinal plant extracts have recently attracted attention as health beneficial foods and potential therapeutic agents for prevention of various diseases. This study was undertaken to measure the anti-inflammatory effect of the ethanol-water fraction obtained from the above-ground portion of Spiraea prunifolia var. simpliciflora, a wild-growing plant in Korea. The final fraction used in this study was the H2O-EtOH (40:60) fraction (SP60), which had the highest antioxidant activity, as determined in previous studies. Methods: The amounts of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β production were measured in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells exposed to SP60. Western blot was performed to measure the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and the activation of nuclear factor (NF)-κB. Results: SP60 exerted no cytotoxicity up to concentrations of 125 ㎍/mL. The levels of inflammatory cytokines, such as NO, TNF-α, IL-6, and IL-1β, were significantly decreased in LPS-stimulated RAW264.7 cells exposed to SP60. In addition, the expression levels of iNOS, COX-2, and phosphorylated p65 showed a concentration-dependent decrease subsequent to SP60 treatment. These results indicate that SP60 inhibits the LPS-induced production of inflammatory cytokines, iNOS, and COX-2, by inhibiting the activation of NF-κB, which is responsible for the expression of inflammatory mediators. Conclusion: The results presented in this study indicate that the H2O-EtOH (40:60) fraction (SP60) extracted from the above-ground portion of Spiraea prunifolia var. simpliciflora has the potential to be developed as a medicine or healthcare food and functional material possessing anti-inflammatory effects. However, it is necessary to first confirm the anti-inflammatory effects of SP60 in in vivo models.

주제어

표/그림 (6)

$Fig. 1. Effect of the SP60 on cell viability of LPS-induced RAW 264.7 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL) then with or without LPS (1 μg/mL) for 24 hours. Each bar represents the mean ± SD of three replicate determinations (n = 3). SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; LPS, lipopolysaccharide. Compared with control group: ***p < 0.001.$ 그림 Fig. 1. Effect of the SP60 on cell viability of LPS-induced RAW 264.7 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL) then with or without LPS (1 μg/mL) for 24 hours. Each bar represents the mean ± SD of three replicate determinations (n = 3). SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; LPS, lipopolysaccharide. Compared with control group: ***p < 0.001.
$Table 1. Yield of 70% ethanol extract and 60% ethanol fraction (SP60) of Spiraea prunifolia var. simplicifora$ 표 Table 1. Yield of 70% ethanol extract and 60% ethanol fraction (SP60) of Spiraea prunifolia var. simplicifora
$Fig. 2. Effect of SP60 on the production of NO in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL) then with or without LPS (1 μg/mL) for 24 hours. The culture supernatant of the treated cells were used to measure NO level. Levels of NO were determined by Griess reagent. Each bar represents the mean ± SD of three replicate determinations (n = 3). SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; NO, nitric oxide; LPS, lipopolysaccharide. Compared with control group: **p < 0.01, ***p < 0.001.$ 그림 Fig. 2. Effect of SP60 on the production of NO in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL) then with or without LPS (1 μg/mL) for 24 hours. The culture supernatant of the treated cells were used to measure NO level. Levels of NO were determined by Griess reagent. Each bar represents the mean ± SD of three replicate determinations (n = 3). SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; NO, nitric oxide; LPS, lipopolysaccharide. Compared with control group: **p < 0.01, ***p < 0.001.
$Fig. 3. Effect of SP60 on the production of TNF-α (A), IL-6 (B), IL-1β (C) in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL) then with or without LPS (1 μg/mL) for 24 hours. The levels of pro-inflammatory cytokines in the cell culture supernatant were determined by ELISA. Each bar represents the mean ± SD of three replicate determinations (n = 3). SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide. Compared with control group: *p < 0.05, **p < 0.01, ***p < 0.001.$ 그림 Fig. 3. Effect of SP60 on the production of TNF-α (A), IL-6 (B), IL-1β (C) in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL) then with or without LPS (1 μg/mL) for 24 hours. The levels of pro-inflammatory cytokines in the cell culture supernatant were determined by ELISA. Each bar represents the mean ± SD of three replicate determinations (n = 3). SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide. Compared with control group: *p < 0.05, **p < 0.01, ***p < 0.001.
$Fig. 4. Effect of SP60 on iNOS and COX-2 protein expression in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL), then with or without LPS (1 μg/mL) for 24 hours. Cell lysates were used for western blot analysis. The levels of protein expression in iNOS and COX-2 were normalized to the β-actin signals. The relative band intensities are indicated on the band of each treatment. SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase; LPS, lipopolyssacharide.$ 그림 Fig. 4. Effect of SP60 on iNOS and COX-2 protein expression in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL), then with or without LPS (1 μg/mL) for 24 hours. Cell lysates were used for western blot analysis. The levels of protein expression in iNOS and COX-2 were normalized to the β-actin signals. The relative band intensities are indicated on the band of each treatment. SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase; LPS, lipopolyssacharide.
$Fig. 5. Effect of SP60 on P65 protein expression in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL), then with or without LPS (1 μg/mL) for 24 hours. Cell lysates were used for western blot analysis. The levels of protein expression in p65 was normalized to the pp65 signal. The relative band intensities are indicated above each band. SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; LPS, lipopolyssacharide.$ 그림 Fig. 5. Effect of SP60 on P65 protein expression in LPS-induced RAW 267.4 cells. Cells were treated with SP60 (15.625, 31.25, 62.5, 125 μg/mL), then with or without LPS (1 μg/mL) for 24 hours. Cell lysates were used for western blot analysis. The levels of protein expression in p65 was normalized to the pp65 signal. The relative band intensities are indicated above each band. SP60, Spiraea prunifolia var. simpliciflora 60% ethanol fraction; LPS, lipopolyssacharide.

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