꾸지뽕나무 열매 isoflavonoid 화합물에 대한 정량분석법 개발

Abstract

본 연구는 꾸지뽕나무 열매에서 분리 및 정제한 isoflavonoid 성분에 대하여 Ultra Performance Liquid Chromatography(UPLC)을 이용하여 분석법을 개발하고 검증하고자 하였다. 꾸지뽕나무 열매를 methanol로 추출하여 n-hexane, ethyl acetate, 물 순으로 분액 후, ethyl acetate 추출물을 silica gel 컬럼과 sephadex LH-20 컬럼을 사용하여 4종의 isoflavonoid들을 분리하였다. 분리한 4종의 isoflavonoid들은 기기분석(UV-Vis spectroscopy, ESI-MS, <TEX>$^1H\;NMR$</TEX>, <TEX>$^{13}C\;NMR$</TEX>)을 통하여 alpinumisoflavone, 6,8-diprenyl orobol, 6,8-diprenyl genistein, 4'-O-methylalpinumisoflavone으로 확인 및 동정하였다. 이 성분들을 2% acetic acid 용액(용매 A)과 2% acetic acid가 함유된 MeOH 용액(용매 B)을 기울기 이동상으로 하여 <TEX>$C_{18}$</TEX> 컬럼이 장착된 UPLC로 분석하였다. 분석 조건은 ICH (International Conference on Harmonisation) 가이드라인에 기술된 선택성, 직선성, 정량한계, 검출한계, 정확성 및 정밀성을 측정하여 분석법의 타당성을 검증하였다. 또한 검증한 분석방법을 이용하여 꾸지뽕나무 열매의 채취시기별 함량을 조사하였다. 그 결과, 미숙과는 7월과 8월에 각 성분의 함량이 증가하였다가 9월에는 감소하였고, 성숙과의 경우 9월 미숙과 보다 각 성분들이 전반적으로 많이 함유되어 있었다. In this study, an analytical method to evaluate the quality of isoflavonoid compounds purified and isolated from the fruits of Cudrania tricuspidata was developed and validated using Ultra Performance Liquid Chromatography (UPLC). The fruits of C. tricuspidata were extracted with methanol and further fractioned with n-hexane, ethyl acetate and water. The resulting ethyl acetate extract separated into four isoflavonoid compounds by a combination of silica gel and sephadex LH-20 column chromatography. The structures of the compounds were elucidated as alpinumisoflavone, 6,8-diprenyl genistein, 6,8-diprenyl orobol, 4'-O-methylalpinumisoflavone by various techniques such as UV-Vis, ESI-MS, <TEX>$^1H\;NMR$</TEX> and <TEX>$^{13}C\;NMR$</TEX> spectroscopy. Finally, a method to characterize the compounds was developed by using the UPLC equipped with a <TEX>$C_{18}$</TEX> column and a gradient mobile phase system consisting of 2% acetic acid in water (solvent A) and 2% acetic acid in methanol (solvent B). The developed method was validated with the parameters such as selectivity, linearity, limit of detection, limit of quantitation, accuracy, and precision, which are defined by the ICH (International Conference on Harmonization). Using the validated method, the compounds in the fruits harvested in different months were also quantitatively analyzed. We propose this approach this approach can readily be utilized as an efficient evaluation method to quantify the extracts of C. tricuspidata.