Cytotoxic and Anti-inflammatory Activities of Lipids from the Nuruk (Rhizopus oryzae KSD-815)

누룩(Rhizopus oryzae KSD-815)으로부터 분리한 지질화합물의 세포독성 및 항염증 활성

Nuruk is the Korean traditional Koji that contains various microorganisms and has been used to make the traditional fermented foods including alcoholic beverages. Rhizopus oryzae KSD-815 was isolated from the alcohol-fermenting Nuruk used for manufacturing traditional alcohol. In this study, the authors reported the isolation and identification of four lipids from the Nuruk (Rhizopus oryzae KSD-815) that inoculated wheat with Rhizopus oryzae KSD-815. The dried and powdered Nuruk (Rhizopus oryzae KSD-815) were extracted three times at room temperature with 80% aqueous MeOH. The extracts were partitioned with EtOAc, n-BuOH, and water, successively. The EtOAc extract was suspended in 80% MeOH and partitioned repeatedly with n-hexane. From the n-hexane fraction, four lipids were isolated through the repeated silica gel and ODS column chromatographies. According to the results of physico-chemical data including NMR, GC and MS, the chemical structures of the compounds were determined as linolenic acid methyl ester (1), palmitic acid methyl ester (2), linoleic acid (3), palmitic acid (4). Cytotoxicity was evaluated in huamn breast cancer cells, MDA-MB-231 and human hepatocarcinoma, SK-HEP-1 cells using MTT assay. Exposure of compounds 1 and 3 led to a dose-dependent inhibition of cell viability in both cancer cell lines. In addition, treatment of RAW264.7 cells with compound 3 caused inhibition of lipopolysaccharide/interferon-${\gamma}$-induced nitric oxide production.

Rhizopus oryzae KSD-815를 밀에 접종하여 만든 누룩(Rhizopus oryzae KSD-815)에서 4종의 지질 화합물을 분리하여 화학구조를 동종하였다. 건조된 누룩을 실온에서 80% MeOH 수용액으로 추출하고 이추출물을 EtOAc 분획, n-BuOH 분획, $H_2O$ 분획으로 나누었다. EtOAc분획은 다시 80% MeOH 과 n-hexane 으로 분획하였다. n-Hexane 분획에 대해 silica gel 및 ODS column chromatography를 반복 실시하여 4종의 지질 화합물을 분리, 정제하였다. NMR, IR, GC/MS 등을 통하여 화합물 1(linolenic acid methyl ester), 화합물 2(palmitic acid methyl ester), 화합물 3(linoleic acid), 화합물 4(palmitic acid)의 구조를 결정하였다. 이 지방산 화합물의 세포독성을 평가하기 위해 인체 유래 유방암(MDA-MB-231)과 간암(SK-HEP-1)세포주에 대해 MTT assay를 수행하였다. 두 암 세포주에서 화합물 1(linolenic acid methyl ester)과 화합물 3(linoleic acid)은 농도의존적인 세포독성을 확인하였다. 또 염증반응 매개체의 일종인 nitric oxice(NO)의 생성 억제 활성을 Griess 방법으로 평가한 결과, 화합물 3(linoleic acid)은 LPS와 IFN-${\gamma}$에 의해 유도 된 NO 생성도 저해하는 것을 RAW264.7 세포에서 확인하였다.