Lipopolysaccharide로 유도된 Raw 264.7 세포에서 마늘대 추출물(Allium sativum L. Stems)의 염증성 사이토카인 및 iNOS, COX-2 발현에 대한 효과 검증
Effect of garlic (Allium sativum L.) stems on inflammatory cytokines, iNOS and COX-2 expressions in Raw 264.7 cells induced by lipopolysaccharide
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초록
본 연구에서는 마늘의 부산물로 발생하는 마늘대의 항산화 및 항염증 효과를 알아보기 위하여 LPS로 염증을 유도한 Raw 264.7 세포에 대한 열수추출물(ASSW)과 70% 에탄올추출물(ASSE)의 효과를 살펴보았다. ASSW의 폴리페놀 함량은 37.08±1.51 mg(TAE)/g, ASSE의 폴리페놀 함량은 44.7±1.32 mg(TAE)/g 이 함유되어있음을 확인하였다. DPPH 실험과 ABTS+ 실험에서 ASSW, ASSE 모두 농도 의존적으로 증가하였으며, DPPH의 경우 1,000 μg/mL에서 대조군인 Vit.C의 50 μg/mL의 항산화능이 있다는 것이 확인되었고, ABTS+의 경우 500 μg/mL 이상부터 대조군인 Vit.C와 비슷한 효과를 나타냄으로서 ASSW, ASSE 모두 항산화능이 있다는 것을 확인하였다. MTT측정으로 인해 세포 독성을 가지지 않았던 농도대(5, 10, 25, 50, 100 μg/mL)에서 염증 억제 효과를 살펴보기 위해 NO를 측정한 결과ASSW, ASSE 모두 25 μg/mL에서부터 유의적으로 분비량이 감소함을 확인하였으며 특히 100 μg/mL의 농도에서 약 18%, 23%의 억제 효과를 보였다. 또한 대식세포의 염증성cytokine인 IL-6, TNF-α, IL-1β 및 PGE2의 분비량을 첨가 농도 의존적으로 억제함을 확인하였다. 특히 PGE2에 대해ASSW, ASSE 100 μg/mL의 농도에서 약 55%, 60%의 감소 효과를 보였다. ASSW의 iNOS, COX-2의 발현 저해효과는 나타내지 못하였지만, ASSE는 100 μg/mL의 농도에서 iNOS의 발현량이 현저하게 억제됨을 확인하였고, COX-2의 경우 농도 의존적으로 저하되어 특히 50 μg/mL, 100 μg/mL의 구간에서 단백질 발현 저해효과가 있음을 확인하였다. 이를 통해 ASSW, ASSE 모두 항산화 효과와 항염증 효과가 있음을 확인하였으며, ASSW 보다 ASSE에서 활성산소종(reactive oxygen species, ROS) 및 ROS에 의해 유발되는 염증을 억제시켜주는 소재가 될수있을 것이라 예상된다.
Abstract
In this study, the anti-oxidant and anti-inflammatory activities of water extract (ASSW) and 70% ethanol extract (ASSE) of Allium sativum L. stems were investigated using Raw 264.7 cells induced by lipopolysaccharide (LPS). ABTS radical scavenging activities of ASSW and ASSE at 1000 μg/mL concentration were 96.9% and 97.8%, respectively. In order to investigate the potential anti-inflammatory effects of ASSW and ASSE, nitric oxide (NO), pro-inflammatory cytokines, interleukin-6 (IL-6), and tumor necrosis factor including α (TNF-α), interleukin-1β (IL-1β), and prostaglandin-E2 (PGE2) were measured. ASSW and ASSE at 100 μg/mL concentration showed inhibitory effects against NO production by 18% and 23%, respectively. Production of IL-1β and IL-6 after treatment with ASSW and ASSE at 100 μg/mL decreased by approximately 28% and 15% for ASSW and 17% and 12% for ASSE, respectively. In addition, production of TNF-α after treatment of 100 μg/mL of ASSW and ASSE decreased by 24% and 23%, respectively. In addition, the treatment of 100 μg/mL of ASSW and ASSE showed inhibitory expressions against PGE2 by 45.47% and 33.87%, respectively. These results suggested that ASSE showed greater inhibitory activity than that of the ASSW by the suppression of inflammatory mediators, including NO, IL-6, TNF-α and PGE2 production, and the expressions of iNOS and COX-2 in macrophages. In conclusion, ASSW and ASSE may have some ancillary effects on inflammatory factors as potential anti-inflammatory agents.
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