주목열매 추출물 구조분석

Abstract

주목열매에서 분리한 과육과 씨를 95% EtOH 용액을 사용하여 각각 1.15 kg과 0.94 kg을 유리 용기에 침지하여 추출하고, 농축된 추출용액을 용매의 극성에 따라 n-hexane, dichloromethane, ethyl acetate 및 수용성으로 순차 분획한 후, 동결건조하였다. 혼합물로부터 순수한 화합물을 분리하기 위하여 ethyl acetate 및 수용성 분획에 대하여 Sephadex-LH 20 칼럼크로마토그래피를 실시하였다. 단리된 화합물들의 구조는 flavan 화합물의 경우 <TEX>$^1H$</TEX>-NMR, <TEX>$^{13}C$</TEX>-NMR 및 EI-MS 스펙트럼을 측정하였으며, 탄수화물은 보다 정확한 구조를 결정하기 위해 COSY, HSQC와 같은 2-D NMR과 LC/MS 스펙트럼을 이용하여 구조를 결정하였다. 또한 구성당 분석을 위해 acid hydrolysis, permethylation을 실시하여 구성당의 성분과 결합위치를 확인하였다. 위의 실험을 통하여 (+)-catechin (1), (-)-epicatechin (2), (+)-gallocatechin (3), (-)-epigallocatechin(4) 및 <TEX>${\beta}$</TEX>-D-fructofuranose-(<TEX>$2{\rightarrow}4$</TEX>)-O-<TEX>${\beta}$</TEX>-D-glucopyranose(<TEX>$1{\rightarrow}4$</TEX>)-O-<TEX>${\alpha}$</TEX>-D-glucopyranose-(<TEX>$1{\rightarrow}2$</TEX>)-O-<TEX>${\beta}$</TEX>-D-fructofuranose (5)의 구조를 결정하였다. The fruits of Taxus cuspidata were collected, divided into seeds and fruits, and extracted with 95% EtOH. The extracts were evaporated under the reduced vacuum pressure, concentrated, then successively fractionated with a series of n-hexane, dichloromethane, ethyl acetate and water on a separatory funnel to get some freeze dried samples. A portion of the EtOAc (arils:1.65 g, seeds:1.04 g) and <TEX>$H_2O$</TEX> (arils:7 g, seeds:10 g) soluble samples were chromatographed on a Sephadex column using MeOH-<TEX>$H_2O$</TEX> (1:1, 1:3, 1:5, v/v), EtOH-hexane (3:1, v/v) mixture and 100% <TEX>$H_2O$</TEX> as eluting solvents to isolate pure compounds from the fractions. The isolates were developed by cellulose TLC using t-BuOH-HOAc-<TEX>$H_2O$</TEX> (TBA; 3:1:1, v/v/v) and 6% aqueous HOAc. Visualization was done under ultraviolet light and by spraying the vanillin-HCl-EtOH reagent (4.8:12:480, v/v/v). followed by heating. The structures of the isolates were characterized by <TEX>$^1H$</TEX>- and <TEX>$^{13}C$</TEX>-NMR, DEPT, 2D-NMR, LC/MS and EI-MS spectra. In addition to the NMR and MS spectra, acid hydrolysis and permethylation were used to determine the correct structure of the isolated sugar compound. Their structures were elucidated as (+)-catechin (1), (-)-epicatechin (2), (+)-gallocatechin (3), (-)-epigallocatechin (4) and <TEX>${\beta}$</TEX>-D-fructofuranose-(<TEX>$2{\rightarrow}4$</TEX>)-O-<TEX>${\beta}$</TEX>-D-glucopyranose(<TEX>$1{\rightarrow}4$</TEX>)-O-<TEX>${\alpha}$</TEX>-D-glucopyranose (<TEX>$1{\rightarrow}2$</TEX>)-O-<TEX>${\beta}$</TEX>-D-fructofuranose (5) on the basis of the above experimental evidences.