송악(常春藤)의 생리활성과 성분연구 : Studies on components and biological activities of hedera rhombea Bean

Abstract

송악(常春藤) Hedera rhombea Bean은 담장나무라고도 불리우며 오가피나무과(Araliaceae)에 속하는 常綠活葉蔓木으로 平肝 解毒의 效能이 있으며 肝炎, 目眩, 打撲傷, 癰疽腫毒을 치료하고 민간약으로는 黃疸, 結石症, 動脈硬化症 등을 치료하는데 효과가 있다고 알려져 있다. 그리하여 본 연구는 송악에서 미량으로 抗癌 活性物質을 檢索할 수 있는 FPTase 저해활성 검색에서 강한 활성을 나타내는 물질의 단리와 간 보호작용, 간기능 촉진 작용을 가지는 페놀성 물질을 단리하고 구조와 활성 관계를 규명하고자 송악잎의 MeOH extract의 EtOAc분획, n-BuOH분획을 silica gel column chromatography, reverse phase column chromatography, HPLC를 실시하여 EtOAc분획에서 compoundⅠ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ을 분리하였고, n-BuOH분획에서 Ⅶ,Ⅷ,Ⅸ,Ⅹ,xi,xii을 분리하였다. 단리된 화합물은 각종 이화학적 분석 및 분광학적 분석(UV, IR, FAB-MS, EI-MS, 1H-NMR, 1H-1H COSY, 13C-NMR, DEPT, HMBC, HMQC) 등을 통하여 그 구조를 해석하였다. 그 결과, compound Ⅰ과 Ⅱ는 천연에서 처음으로 단리된 carbon 27위의 methyl기가 없어진 dammarane 型의 nortriterpenoid이다. compound Ⅰ의 구조는 27-demethyl -20(S)-dammar-23-ene-6α,20-diol-3,25-dione으로 구조를 밝혔으며, rhombenone으로 명명하였다. compound Ⅱ는 27-demethyl-20(S)-dammar -23-ene-3β,6α,20-triol-25-one으로 해석하고, dihydrorhombenone으로 명명하였다. compound Ⅲ,Ⅳ는 dammarane 型 kizuta saponin K7, kizuta saponin K7A, compound Ⅶ,Ⅷ은 hederagenin 型 kizuta saponin K8, kizuta saponin K11로 동정하였다. 또한, compound Ⅴ,Ⅵ은 methyl 3,5-di-O-caffeoyl quinate, methyl 3,4-di-O-caffeoyl quinate, compound Ⅸ는 chlorogenic acid, compound Ⅹ, xi, xii는 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid로 동정하였다. 분리된 물질에 대한 FPTase 저해 활성 측정 결과 compound Ⅰ=rhombenone (IC50=2.5μM), compound Ⅱ=dihydrorhombenone (IC50=1.2μM)으로 강한 저해 .활성을 나타내었다. 또한 사염화탄소 유발 간세포 독성 실험에서는 compound Ⅴ, Ⅵ Ⅹ, xi, xii에서 glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) 유리를 농도 의존적으로 억제하였고, 사염화탄소에 의해 상승된 GOT값에 대하여 대조군과 비교시 compound X(66.4%), compound XII(87.5%), compound V,VI(83.2%)로 유의성 있는 억제 효과를 나타내었고, GPT값은 compound X(77.7%), compound XI(68.5%), compound XII(68.5%), compound V,VI(71%)로 억제 효과가 나타났다. 또한, HIV에 대한 저해 활성 측정 결과 caffeic acid, compound Ⅸ, compound Ⅹ에서 약한 활성이 나타났고 HSV-2에 대해서는 caffeic acid만 미약한 저해 활성을 나타내었다. ; Hedera rhombea Bean (Araliaceae) has been used to treat hemorrage, chronic catarrh, jaundice, lithiasis and convulsion as a folk medicine. On this study Hedera rhombea was subjected to phytochemical analysis to investigate biological active constituents. The EtOAc and n-BuOH extracts of Hedera rhombea were subjected to silica gel column chromatography, reverse phase chromatography, HPLC, preparative TLC. Two new compounds of 27-demethyl dammarane type (compound I, II), two dammarane type triterpeneglycoside (compound III,IV), two hederagenin triterpeneglycoside (compound VII,VIII) and two methyl caffeoyl quinate and four caffeoyl quinic acid (compound V,VI,IX,X, XI,XII) were isolated. The structures of isolated compounds were identified by spectroscopic data of UV, IR, EI-MS, FAB-MS, 1H-NMR, 1H-1H COSY, 13C-NMR, DEPT, HMBC and HMQC. By spectural analysis, structure of compound I was elucidated as (27-demethyl-20(S)-dammar-23-ene-6α,20diol-3,25-dione) and named as rhombenone. Rhombenone was the first 27-demethyl nortriterpene of dammarane type isolated from natural sources and compound II was elucidated as (27-demethyl-20(S)-dammar-23-ene-3β,6α,20 triol-25-one) and named 3-dihydrorhombenone. Compound III,IV were identified as kizutasaponin K7 (20(S)- dammar-24-ene-3β,6α,26triol-26-O-β-D-glucopyranoside), kizutasa -ponin K7A (3-oxo-20(S)-dammar-24-ene-6α,20,21,26tetraol-26-O-β-D glycopyranoside). Compound VII,VIII were identified as kizutasaponin K8 (3-O-α-L-arabinopyranosyl-hederagenin-28-O-α-L-rhamnopyranosyl-(1→4)-6-O-acetyl-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-ester), kizutasaponin K11 (3-O-2-L-rhamnopyranosyl-(1→2)-α-L-arabino pyranosyl-hederagenin-28-O-α-L-rhamnopyranosyl-(1→4)-6-O-acetyl-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosylester). Compound V,VI,IX,X,XI,XII were identified as methyl-3,5-di-O- caffeoyl quinate, methyl-3,4-di-O-caffeoyl quinate, 3-caffeoyl quinic acid, 3,5-dicaffeoyl quinic acid, 4,5-dicaffeoyl quinic acid, 3,4-dicaffeoyl quinic acid. Ras Farnesyl Protein Transferase (FPTase) is an enzyme which catalyzes the transfer of the farnesyl group from farnesyl pyrophosphate (FPP) onto cysteine 186 at the C-terminal of the Ras protein. This is a mandatory process before anchoring to plasma membrane which is critical for triggering ras oncogene toward tumor formation. Therefore, the inhibitor of FPTase have the potential to be used as antimutagenic agents. Compound I,II were subjected to farnesyl protein Transferase (FPTase) inhibitory assay. As a result, compound I and II showed strong inhibitory activity, against FPTase (IC50=2.5 M, IC50=1.2 M). Phenolic compounds were subjected to protective effects on CCl4 -induced hepototoxicity in vivo. At the dose of compound X 100-300mg/kg, CCl4-induced marked evalution of GOT,GPT activity in the serum was significantly suppressed to 45.0%-66.4% respectively. Compound XI had no effect on evaluation of GOT, but GPT activity was significantly suppressed to (100mg/kg:68.5%). Compound XII suppressed to GOT activity (100mg/kg:52.4%, 300mg/kg:87.5%), GPT activity (300mg/kg:68.5%) and compound V,VI suppressed to GOT activity (100mg/kg:54%, 300mg/kg:83.2%), GPT activity (100mg/kg:46%, 200mg/kg:71%) respectively. Phenolic compounds were subjected to HIV inhibitory assay. Compound IX, X caffeic acid showed mild inhibitory activity against HIV and also caffeic acid showed mild inhibitory activity against HBV-2. These result suggest that nortriterpenes and phenolic compounds would be used as potent antimutagenic agents, and liver protective agents.

Key Words: Hedera rhombea Bean, FPTase, IC50, nortriterpene, rhombenone, 3-dihydrorhombenone, Kizutasaponin K7, K7A, Kizutasaponin K8, K11, methyl-3,4-di-O-dicaffeoyl quinate, 3-5 dicaffeoyl quinic acid, 3,4-di-O-caffeoyl quinic acid, 3,5-di-O-caffeoyl quinic acid, 4,5-di-O-caffeoyl quinic acid, GPT, GOT