Anti-inflammatory and Antioxidant Effects of Hot Water Extracts from Kaempferia Galanga L

삼내자 열수추출물의 항산화 및 항염 효과

Abstract

In this study, we investigated the possibility of Kaempferia Galanga(KG) hot water extract on the antioxidant, cytotoxic and anti-inflammatory efficacy as a cosmetic ingredient. Antioxidant effects were evaluated based on DPPH and ABTS radical scavenging activity, FRAP assay, and total polyphenol contents. The MTT assay was used to confirm the cell toxicity in mouse macrophage RAW264.7 cells. Anti-inflammatory effects were also investigated in LPS-induced RAW264.7 cells by measuring secretion of NO, $TNF-{\alpha}$ and iNOS, $TNF-{\alpha}$ mRNA expression level. As a result, DPPH and ABTS radical scavenging activities were increased in a concentration-dependent manner. The ferric reducing antioxidant power(FRAP) was the highest at 5 mg/mL as 24.5 uM. The measurements of total polyphenol content was $1.28{\pm}0.064mg\;GAE/g$. The cytotoxicity of the KG extract results showed no cytotoxicity at concentration of 0.625 to 2.5 mg/mL. In addition, the extract of KG significantly suppressed the LPS-induced nitrite, $TNF-{\alpha}$ secretion and the mRNA expression of iNOS, $TNF-{\alpha}$ in RAW264.7 cells. Taken together, these data suggest that the KG hot water extracts can be used as a safe and functional cosmetic raw material.

본 연구의 목적은 삼내자(Kaempferia Galanga) 열수추출물의 항산화, 세포독성, 항염 효능을 조사하여 화장품 성분으로서 활용 가능성을 알아보고자 하였다. 항산화 활성 평가를 위해 DPPH, ABTS 라디컬 소거능 활성, FRAP 환원력, 총폴리페놀 함량을 측정하였다. 쥐의 대식세포인 RAW264.7 세포를 대상으로 MTT assay를 수행하여 세포독성을 확인하였다. 항염 효능을 알아보기 위해 LPS로 유도된 RAW264.7 세포에서 nitric oxide(NO), $TNF-{\alpha}$ 분비 및 iNOS, $TNF-{\alpha}$ mRNA 발현 수준을 조사하였다. 그 결과 DPPH와 ABTS 라디칼 소거능은 농도 의존적으로 라디칼 소거 활성이 증가하였다. FRAP 값은 5 mg/mL에서 24.5 uM로 가장 높게 나타났으며, 총폴리페놀 함량은 $1.28{\pm}0.064mg\;GAE/g$로 나타났다. KG 추출물 농도 0.625~2.5 mg/mL에서 세포독성은 보이지 않았다. 또한 RAW264.7 세포에서 LPS 유도에 의한 NO, $TNF-{\alpha}$ 분비 그리고 iNOS, $TNF-{\alpha}$의 mRNA 발현이 KG 추출물에 의해 유의하게 감소하였다. 이러한 결과는 삼내자 열수추출물이 안전하고 효과적인 화장품 원료로서의 활용 가능성이 있음을 보여준다.

Fig. 1. Effect of KG extracts on the DPPH radical scavenging activity. Results are the mean±S.D. from three independent experiments. *p<0.05 compared to the negative control.

Fig. 2. Effect of KG extracts on the ABTS radical scavenging activity. Results are the mean±S.D. from three independent experiments. *p<0.05; **p<0.001 compared to the negative control.

Fig. 3. Ferric reducing antioxidant power assay of KG extracts. Results are the mean±S.D. from three independent experiments. *p<0.05 compared to the negative control.

Fig. 4. Effect of KG extracts on RAW264.7 cell viability. Cells were treated with various concentration of KG for 24 h. Cell viability was evaluated using the MTT assay. Results are the mean±S.D. from three independent experiments. *p<0.05; **p<0.001 compared to the negative control.

Fig. 5. Effect of KG extracts on the NO production and iNOS protein expression in LPS-induced RAW264.7 cells. Cells were incubated with LPS in the presence or absence of KG for 24 h. The NO concentration in medium was determined by Griess reagent assay(a). The whole cell lysates were then subjected to Western blot analysis(b). Results are the mean±S.D. from three independent experiments. *p<0.05 compared to the negative control.

Fig. 6. Effect of KG extracts on TNF-α secretion in LPS-induced RAW264.7 cells. Cells were incubated with LPS in the presence or absence of KG for 24 h. Results are the mean±S.D. from three independent experiments. *p<0.05 compared to the negative control.

Fig. 7. Effect of KG extracts on iNOS and TNF-α gene expression in LPS-induced RAW264.7 cells. Cells were incubated with LPS in the presence or absence of KG for 12 h. iNOS(a) and TNF-α (b) mRNAlevels were determined using real-time PCR. Results are the mean±S.D. from three independent experiments. *p<0.05; **p<0.001 compared to the negative control.

Table 1. TNF-α, iNOS and GAPDH primer

Table 2. Total polyphenol contents (TPC) of KG extracts.