野牡丹花部抗酪胺酸酶成分之研究

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题名:	野牡丹花部抗酪胺酸酶成分之研究
作者:	陳余峰
贡献者:	生物科技研究所
关键词:	野牡丹 Melastoma candidum 抗酪胺酸酶 anti-tyrosinase
日期:	2012
上传时间:	2012-10-16 10:06:22 (UTC+8)
摘要:	野牡丹為野牡丹科之多年生常綠灌木，分佈於台灣、大陸以及東南亞地區。野牡丹葉及根莖部之生物活性報導包括抗氧化、抗發炎、抗脂質過氧化、抗潰瘍、抗肝毒性及抗酪胺酸酶等活性。酪胺酸酶是一種含銅離子酵素，具有催化酪胺酸、多巴及多巴醌氧化的功能，其活性與銅離子密切相關。抗酪胺酸酶研究中酪胺酸酶常見的濃度為 1375 unit/mL、1000 unit/mL 及 138 unit/mL。本研究目的為探討分離自野牡丹花部的黃酮化合物，對文獻中常出現高低二種濃度下抗酪胺酸酶活性之差異。野牡丹花部之乙醇萃取物依極性分割方式得到正己烷、乙酸乙酯、正丁醇以及水萃取物，接著進行抗高及低濃度酪胺酸酶及清除 ABTS 自由基活性分析。抗酪胺酸酶活性結果顯示正丁醇萃取物 (200 μg/mL) 的抑制活性比其他三種萃取物具有潛力，其抑制率為 10.6%。正丁醇萃取物 (10 μg/mL) 在清除 ABTS 自由基活性測試中亦具有最佳活性，清除率為 98.1%。在核磁共振氫譜中發現乙酸乙酯萃取物有許多黃酮化合物之氫訊號。根據抗酪胺酸酶活性實驗結果，將正丁醇及乙酸乙酯萃取物以 DIAION HP20、Sephadex LH-20、silica gel、C18 以及高效能液相層析儀進一步純化，並以核磁共振儀解析化合物結構。本研究共得到 6 個黃酮化合物，包括 naringenin (MCF-1)、kaempferol (MCF-2)、afzelin (MCF-3)、astragalin (MCF-4)、herbacetin 3-O-β-glucoside (MCF-5) 及 kaempferol 6-O-rhamnoside (MCF-6)。其中化合物 MCF-5 及 MCF-6 為新化合物。抗酪胺酸酶活性結果顯示在高濃度 (1375 unit/mL) 的酪胺酸酶條件下，化合物 MCF-1~4 (200 μg/mL) 的抗酪胺酸酶活性抑制率分別為 3.2%、25.9%、3.1% 及 7.8%；低濃度 (137.5 unit/mL) 時，化合物 MCF-1~6 (200 μg/mL) 的抗酪胺酸酶活性抑制率分別為 4.2%、27.4%、2.2%、4.2%、1.3% 及 1.8%。結果顯示黃酮類化合物結構中糖基的存在與否對活性有顯著的影響。例如 kaempferol (MCF-2) 與 afzelin (MCF-3) 比較，3-OH 接鼠李糖基團後，抗酪胺酸酶活性降低 9.5 倍；astragalin (MCF-4) 與 herbacetin 3-O-β-glucoside (MCF-5) 比較，8-OH 對於抗酪胺酸酶的活性無影響；kaempferol (MCF-2) 與 kaempferol 6-O-rhamnoside (MCF-6) 比較，當 6-OH 存在時接糖基，抗酪胺酸酶活性變差 14.2 倍。　Melastoma candidum is a perennial evergreen shrub of Melastomataceae family distributed in Taiwan, China and Southeast Asia. Bioactivity report of the leaf, root and stem parts of M. candidum include anti-oxidative, anti-inflammatory, anti-lipid peroxidative, anti-ulcer, anti-hepatotoxic and anti-tyrosinase activities. Tyrosinase is a copper-containing enzyme that catalyzes the oxidation of tyrosine, DOPA and dopaquinone, and its activity is related to copper. Common concentrations of tyrosinase used in anti-tyrosinase studies are 1375, 1000 and 138 unit/mL. This research aims to explore anti-tyrosinase activity of the flavones from the flower part of M. candidum. The ethanolic extract of the flower part of M. candidum was partitioned with hexane, EtOAc, BuOH and H2O subsequently to give the corresponding fractions, and then further assayed on anti-tyrosinase and ABTS radical scavenging assays. The anti-tyrosinase activity results revealed BuOH extracts (200 μg/mL) demonstrated the highest potency than the other three extracts, with an inhibitory activity of 10.6%. BuOH extracts (10 μg/mL) also have the best scavenging activity in ABTS free radical scavenging activity test, with an inhibitory rate of 98.1%. The EtOAc extract was found many proton signals of flavonoid in 1H NMR spectra. According to the experimental result of anti-tyrosinase activity, the EtOAc and BuOH fractions were further purified by DIAION HP20, Sephadex LH-20, silica gel, C18 and HPLC, and the structures of these compounds were determined by nuclear magnetic resonance. Six flavonoids were isolated in this study, including naringenin (MCF-1), kaempferol (MCF-2), afzelin (MCF-3), astragalin (MCF-4), herbacetin 3-O-β-glucoside (MCF-5) and kaempferol 6-O-rhamnoside (MCF-6). Among these, MCF-5 and MCF-6 were confirmed to be new compounds. The anti-tyrosinase activity results revealed that at high tyrosinase concentration (1375 unit/mL), the anti-tyrosinase activity of MCF-1~4 (200 μg/mL) were 3.2%, 25.9%, 3.1% and 7.8%, respectively; at low tyrosinase concentration, the anti-tyrosinase activity of MCF-1~6 (200 μg/mL) were 4.2%, 27.4%, 2.2%, 4.2%, 1.3% and 1.8%, respectively. The results revealed that the presence and absence of glycosyl groups on flavonoids play key roles in their activity. For example, comparing the anti-tyrosinase activity of kaempferol (MCF-2) with that of afzelin (MCF-3), when a rhamnose moiety was existed on 3-OH, the anti-tyrosinase activity was 9.5-fold reduced; comparing the anti-tyrosinase activity of astragalin (MCF-4) with that of herbacetin 3-O-β-glucoside (MCF-5), the 8-OH did not affect the anti-tyrosinase activity; comparing the anti-tyrosinase activity of kaempferol (MCF-2) with that of kaempferol 6-O-rhamnoside (MCF-6), when the presence a rhamnosyl group on 6-OH of MCF-6, the anti-tyrosinase activity was 14.2-fold reduced.
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