Inhibitory effect of Koreinsis chinensis leaves extract on proinflammatory responses in lipopolysaccharide-induced Raw 264.7 cells

Lipopolysaccharide로 유도된 Raw 264.7 cell에서 잣 잎(Koreinsis chinensis L.) 추출물의 Pro-inflammatory 억제 효과

Hyaluronidase inhibitory activity as inflammatory factor of Koreinsis chinensis leaf ethanol extract was showed higher inhibitory activity than water extract. 29.5% inhibitory activity was shown at concentration of $200{\mu}g/mL$ phenolics. Lipopolysaccharide (LPS)-stimulated Raw 264.7 cells were treated with different concentrations ($5-25{\mu}g/mL$) of Koreinsis chinensis leaf extract and the amount of nitric oxide (NO) was determined; LPS-treated cells produced 3 times more NO than non-LPS treated cells. Moreover, the NO production in cells treated with Koreinsis chinensis leaf extract showed inhibitory effect in a concentration-dependent manner. Due to the stimulant-induced NO production is regulated by the inducible nitric oxide synthase (iNOS), we determined the iNOS protein level to elucidate the mechanism by which the NO production was inhibited. It was reduced by 40% with a Koreinsis chinensis leaf extract concentration of $25{\mu}g/mL$ and identified iNOS inhibition in dose-dependent manner. The prostaglandin $E_2$ production in cells treated with Koreinsis chinensis leaf extract was reduced by 26.2% at concentration of $25{\mu}g/mL$. The protein expression of cyclooxygenase-2 in LPS-treated Raw 264.7 cells was inhibited by 64% at $25{\mu}g/mL$ of Koreinsis chinensis leaf extract. Koreinsis chinensis leaf extract had a concentration-dependent inhibitory effect on the production of tumor necrosis factor-${\alpha}$ and interleukin-6 as pro-inflammatory cytokine in LPS-treated Raw 264.7 cells at $25{\mu}g/mL$ of Koreinsis chinensis leaf extract. Their levels were decreased by 61.7 and 62% respectively.

잣나무 잎 추출물에 대해 염증인자인 hyaluronidase 저해효과를 측정한 결과 ethanol 추출물에서 물추출물에 비해 상대적으로 더 높은 HAase 저해효과를 보여주었으며, $50-200{\mu}g/mL$ phenolic 농도에서 13.2-29.5%의 저해효과를 나타내었다. 잣나무잎 추출물을 Raw 264.7 cell에 농도별로 처리하여 생성되는 NO량을 측정한 결과 LPS처리군은 LPS 무처리군에 비하여 3배에 가까운 NO발현을 나타내었으며, 농도 의존적으로 NO발현을 억제하였다. LPS에 의해서 생성된 $PGE_2$의 생성억제 효과를 측정한 결과 $25{\mu}g/mL$의 처리농도에서 ethanol 추출물이 26.2%의 저해율을 나타내었다. NO 생성 억제기작에 관한 iNOS 단백질의 발현량을 측정한 결과 $25{\mu}g/mL$의 농도에서 40%의 높은 억제효과를 나타내었으며, 농도 의존적으로 감소하는 경향을 관찰 할 수 있었다. LPS 처리시 또 다른 염증인자인 COX-2의 단백질 발현을 측정한 결과 $25{\mu}g/mL$의 처리 농도에서 64%의 저해효과를 나타내었다. 잣나무잎 추출물이 Raw 264.7 세포에서 LPS에 의해서 생성되는 $TNF-{\cdot}$, IL-6의 생성억제 효과를 측정한 결과 $25{\mu}g/mL$의 처리농도에서 각각 61.7, 62%의 저해 효과를 나타내었다. 따라서 잣나무잎 추출물은 염증억제를 위한 천연 기능성 소재로 활용이 가능할 것으로 판단되었다.