냉초의 antioxidant response element 유도 이리도이드 성분

Abstract

냉초(Veronicastrum sibiricum (L.) Pennell)는 현삼과에 속하는 다년초 식물로 우리나라 강원도 이북 산지의 습한 곳에 자생하는 다년초 식물이다. 본초서 등에 기록되어 있지는 않으나 오랫동안 민간에서 어린 순을 숨위나물이라 하여 식용하였고, 그 외 감기나 부인병, 방광염, 간 손상 등을 치료하는 약이나 이담제 등으로 사용하였다고 알려져 있다 (Lee et al., 1987). Antioxidant response element (ARE) 유도 활성을 보인 냉초의 지상부로부터 화합물을 분리 정제하여, 그 구조를 동정하고자 본 실험에 착수하였다. 냉초의 지상부를 methanol로 초음파 추출한 다음, 용매 극성에 따른 분획을 통해 n-hexane 층 (13.89 g), ethyl acetate 층 (12.57 g), n-butanol 층 (51.68 g)을 얻었다. 이를 이용하여 ARE luciferase 활성 평가를 수행한 결과, ethyl acetate 층에서 가장 효과가 있음을 확인하였다. 활성 화합물을 분리 정제하고자 Diaion HP-20 open column chromatography를 수행하여 100% water부터 100% methanol까지 11개의 소분획물을 얻었다. 그 중 ethyl acetate 층의 major peaks가 있는 80%부터 100% methanol까지의 분획물로부터 preparative HPLC를 이용하여 총 5개의 단일 화합물을 정제하였다. 분리 정제한 화합물들의 구조는 1H-NMR, 13C-NMR, 1H-1H COSY, HSQC, 그리고 HMBC를 이용하여 각각 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl-5,7-bisdeoxycynanchoside (1), 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl -5-deoxycynanchoside (2), 2'',4''-dicinnamoyl-6-O-rhamnopyranosylcatalpol (3), 3'',4''-dicinnamoyl-6-O-rhamnopyranosylaucubin (4), 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl catalpol (5) 임을 확인 하였다. 화합물 (1)-(4)는 천연에서 처음 분리 보고 되었으며, 화합물 (5)는 냉초에서 처음 분리 보고되는 화합물이다. 추가적으로 이 5개의 단일 화합물을 이용하여 free radical 소거능 평가와 세포 독성 평가, ARE 유도 활성 평가 등을 수행하였다. DPPH assay와 ABTS assay를 통해 분리된 모든 화합물들의 라디칼 소거 활성은 미비하였지만, ethyl acetate 층의 가장 큰 peak 이었던 3'',4''-dicinnamoyl-6-O-rhamnopyranosylcatalpol (5)이 ARE 유도 활성이 가장 높음을 확인하였다.| Veronicastrum sibiricum (L.) Pennell is a perennial plant belonging to Scrophulariaceae, and is a native plant to humid regions in the north region of Gangwon-do in Korea. Although it is not recorded in the oriental old book, it used as a Korean folk medicines for treating a common cold, leucorrhea, cystitis and liver damage and as cholagogue (Lee et al.1987). The experiment was carried out to isolate and purify the compounds from the aerial part of the V. sibiricum showing the antioxidant response element (ARE) inducing activity and to identify its structure. The aerial part of the V. sibiricum was extracted with methanol. Through the fractionation according to the solvent polarity, n-hexane (13.89 g) fraction, ethyl acetate (12.57 g) fraction, n-butanol (51.68 g) fraction were obtained. As a result of ARE luciferase activity, it was founded that ethyl acetate fraction was evaluated as an active fraction. In order to separate and purify active compounds, 11 sub-fractions were obtained from 100% water to 100% methanol by performing Diaion HP-20 open column chromatography. Among them, a five single compounds were purified from the sub-fractions ranging from 80% to 100% methanol with major peaks of ethyl acetate fraction using preparative HPLC. The structures of the isolated compounds were identified by using 1H-NMR, 13C-NMR, 1H-1H COSY, HSQC and HMBC. The structure were named 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl-5,7-bisdeoxycynanchoside (1), 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl -5-deoxycynanchoside (2), 2'',4''-dicinnamoyl-6-O-rhamnopyranosylcatalpol (3), 3'',4''-dicinnamoyl-6-O-rhamnopyranosylaucubin (4), 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl catalpol (5), respectively. In addition, this compounds were used to evaluate free radical scavenging ability, cytotoxicity and ARE induction activity. As a result of identifying free radical scavenging activity using DPPH assay and ABTS assay showed that all purified compounds increased significantly in concentration dependent manner but were not effective as radical scavenging ability. And 3'',4''-dicinnamoyl-6-O-rhamnopyranosylcatalpol (5), the largest peak of ethyl acetate fraction, was found to have the highest ARE inducing activity.; Veronicastrum sibiricum (L.) Pennell is a perennial plant belonging to Scrophulariaceae, and is a native plant to humid regions in the north region of Gangwon-do in Korea. Although it is not recorded in the oriental old book, it used as a Korean folk medicines for treating a common cold, leucorrhea, cystitis and liver damage and as cholagogue (Lee et al.1987). The experiment was carried out to isolate and purify the compounds from the aerial part of the V. sibiricum showing the antioxidant response element (ARE) inducing activity and to identify its structure. The aerial part of the V. sibiricum was extracted with methanol. Through the fractionation according to the solvent polarity, n-hexane (13.89 g) fraction, ethyl acetate (12.57 g) fraction, n-butanol (51.68 g) fraction were obtained. As a result of ARE luciferase activity, it was founded that ethyl acetate fraction was evaluated as an active fraction. In order to separate and purify active compounds, 11 sub-fractions were obtained from 100% water to 100% methanol by performing Diaion HP-20 open column chromatography. Among them, a five single compounds were purified from the sub-fractions ranging from 80% to 100% methanol with major peaks of ethyl acetate fraction using preparative HPLC. The structures of the isolated compounds were identified by using 1H-NMR, 13C-NMR, 1H-1H COSY, HSQC and HMBC. The structure were named 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl-5,7-bisdeoxycynanchoside (1), 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl -5-deoxycynanchoside (2), 2'',4''-dicinnamoyl-6-O-rhamnopyranosylcatalpol (3), 3'',4''-dicinnamoyl-6-O-rhamnopyranosylaucubin (4), 3'',4''-dicinnamoyl-6-O-rhamnopyranosyl catalpol (5), respectively. In addition, this compounds were used to evaluate free radical scavenging ability, cytotoxicity and ARE induction activity. As a result of identifying free radical scavenging activity using DPPH assay and ABTS assay showed that all purified compounds increased significantly in concentration dependent manner but were not effective as radical scavenging ability. And 3'',4''-dicinnamoyl-6-O-rhamnopyranosylcatalpol (5), the largest peak of ethyl acetate fraction, was found to have the highest ARE inducing activity.