당근 지상부 추출물 유래 항염 및 항균 활성 성분

Anti-inflammatory and Anti-bacterial Constituents from the Extracts of Daucus carota var. sativa Aerial Parts

Abstract

본 연구에서는 당근 지상부 추출물 및 용매 분획물의 항염, 항균 활성을 확인하고 유효성분을 분리하여 화학구조를 동정하였다. 당근 지상부 에탄올 추출물 및 용매 분획물의 항염 활성을 측정하기 위해 LPS로 자극된 RAW 264.7 세포를 이용하여 NO 생성 억제 활성을 확인하였다. 그 중 에틸아세테이트 분획물이 NO의 생성을 농도 의존적으로 감소시키는 효과가 나타났고, 추가적인 항염 기전 연구를 위해 에틸아세테이트 분획물에 대해 $PGE_2$, 전염증성 사이토카인의 생성량 및 iNOS, COX-2 단백질의 발현량을 측정하였다. 그 결과, 에틸아세테이트 분획물이 $PGE_2$, IL-$1{\beta}$, IL-6의 생성을 감소시키고, iNOS, COX-2 단백질의 발현도 억제 시키는 효과가 있음을 확인하였다. 또한 표피포도상구균과 여드름균을 이용한 활성 실험 결과, 헥산 및 에틸아세테이트 분획물에서 항균 활성이 나타났다. 이와 같은 결과를 바탕으로 에틸아세테이트 분획물에 대해 컬럼 크로마토그래피를 수행하여 3개의 화합물을 분리하였으며, $^1H$ 및 $^{13}C$ NMR 데이터 분석과 문헌을 통하여 화학구조를 동정하였다; diosmetin (1), disomin (2), cynaroside (3). 분리된 화합물 1-3에 대해 항염 및 항균 활성을 측정하였으며, 그 결과 화합물 1-3 모두 NO의 생성을 저해시키는 효과가 있음을 확인하였다. 또한 화합물 3은 여드름균에 대해 항균 활성이 우수하였다. 이상의 연구 결과를 바탕으로 당근 지상부 추출물을 이용한 항염 및 항균 효과를 갖는 천연 화장품 소재로의 개발이 가능할 것이라 사료된다.

In this study, we investigated anti-inflammatory and anti-bacterial constituents from Daucus carota var. sativa (carrot) areal parts. For the extract and solvent fractions, the anti-inflammatory activities were examined by measuring the nitric oxide (NO) production using LPS-stimulated RAW 264.7 cells. Among them, the ethyl acetate (EtOAc) fraction decreased the NO level in a dose-dependent manner. To elucidate further anti-inflammatory mechanisms, EtOAc fraction was evaluated by estimating their effects on the production of prostaglandin $E_2$ and pro-inflammatory cytokines as well as on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). As a result, the EtOAc fraction was determined to inhibit the production of $PGE_2$, IL-$1{\beta}$, IL-6 and reduce the iNOS, COX-2 protein expression. Upon the anti-bacterial tests using Staphylococcus epidermidis and Propionibacterium acnes, n-hexane (Hex) and EtOAc fractions showed the most potent activities. Three phytochemicals were isolated form the EtOAc fraction; diosmetin (1), diosmin (2), cynaroside (3). The chemical structures of the isolated compounds were elucidated based on the spectroscopic data including $^1H$ and $^{13}C$ NMR spectra, as well as comparison of the data to the literatures. Anti-inflammatory and anti-bacterial effects were studied for the isolates. All of the compounds (1 - 3) decreased the NO production, effectively. Also, compound 3 showed anti-bacterial activity on P. acnes. Based on these results, D. carota var. sativa extract could be potentially applicable as anti-inflammatory and anti-bacterial ingredients in cosmetic formulations.

Figure 1. Effects of extract, solvent fractions (A) and EtOAc fraction (B) from D. carota var. sativa on NO production in LPS-induced RAW 264.7 cells. The cells were stimulated with 1 μg/mL of LPS only, or with LPS plus D. carota var. sativa extract and solvent fractions for 24 h. NO production was determined by the Griess reagent method. Cell viability was determined after 24 h culture of cells stimulated with LPS (1 μg/mL) in the presence of D. carota var. sativa. The data represent the mean ± SD of triplicate experiments (*p < 0.05, **p < 0.01).

Figure 2. Effects of EtOAc fraction from D. carota var. sativa on production of PGE₂, TNF-α, IL-1β and IL-6 in LPS-induced RAW 264.7 cells. The cells were stimulated with 1 μg/mL of LPS only, or with LPS plus EtOAc fraction for 24 h. PGE₂, TNF-α, IL-1β and IL-6 produced and released into the culture medium was assayed using the ELISA method. The data represent the mean ±SD of triplicate experiments (*p < 0.05, **p < 0.01).

Figure 3. Effects of EtOAc fraction from D. carota var. sativa on levels of iNOS and COX-2 protein in LPS-induced RAW 264.7 cells. The cells were stimulated with 1 μg/mL of LPS only, or with LPS plus EtOAc fraction for 24 h. Whole cell lysates (20 μg) were prepared and the protein was subjected to 10% SDS-PAGE; expression of iNOS, COX-2 and β-actin were determined by western blotting. β-action served as a loading control.

Figure 4. Isolated compounds 1-3 from D. carota var. sativa aerial parts.

Figure 5. Effects of isolated compounds 1-3 from D. carota var. sativa on NO production in LPS-induced RAW 264.7 cells. The cells were stimulated with 1 μg/mL of LPS only, or with LPS plus isolated compounds 1-3 for 24 h. NO production was determined by the Griess reagent method. Cell viability was determined after 24 h culture of cells stimulated with LPS (1 μg/mL) in the presence of isolated compounds 1-3. The data represent the mean ± SD of triplicate experiments (*p < 0.05, **p < 0.01).

Table 1. Anti-bacterial Activities of D. carota var. sativa

Table 2. MIC and MBC Values of D. carota var. sativa

Table 3. MIC and MBC Values of Isolated Compounds 1-3 from D. carota var. sativa