Studies on the Antioxidative Components of Longan (Dimocarpus longan Lour.) Flower
|Keywords:||抗氧化;龍眼花;epicatechin;proanthocyanidin A2;antioxidant;Longan flower;epicatechin;proanthocyanidin A2||Issue Date:||2006||Abstract:||
動脈粥狀硬化為造成心血管疾病之主要原因，而低密度脂蛋白（low density lipoprotein, LDL）膽固醇的氧化為啟動動脈粥狀硬化的重要步驟；抗氧化劑可以增加LDL對氧化傷害的抵抗力，因此攝取含有抗氧化成分之食品應有助於預防動脈粥狀硬化之發生。本研究室於前一年度研究顯示，龍眼（Dimocarpus longan Lour.）花具有良好之抗氧化效果，因此本研究以抗氧化活性為導向進行龍眼花活性成分之分離純化，並以「抑制銅離子誘導人類LDL之氧化」為主要抗氧化活性之檢測方法，進而鑑定出有效之抗氧化成分。
龍眼花甲醇萃取物以正己烷、乙酸乙酯、正丁醇及水四種溶劑進行分配萃取，其中以乙酸乙酯區分層之抗氧化效果最佳，其對於清除DPPH自由基之EC50為5.28 ± 1.14 μg/mL。在延緩LDL氧化方面，其效果為同濃度（1 μg/mL）下Trolox之1.14倍，並且具有最高之總酚（701.7 ± 29.3 mg gallic acid /g dry weight of sample）與總類黃酮（213.7 ± 14.8 mg catechin equivalent /g dry weight of sample）含量。
進一步將乙酸乙酯區分層以矽膠管柱層析法予以分離純化，得到20個次區分。由DPPH自由基清除能力試驗可知，中至高極性之次區分（F9~19）具有較好之抗氧化效果。ORAC試驗結果顯示F8~11之抗氧化效果十分優異，其中以F9（以ethyl acetate/n-hexane=60/40, v/v沖提）最佳，ORAC值為25.07 ± 4.08 Trolox equivalent。抑制銅離子誘導人類LDL氧化試驗結果亦顯示，F8~11可有效延緩LDL之氧化，其中以F10（以ethyl acetate/n-hexane=70/30, v/v沖提）之效果特別突出，為同濃度（1 μg/mL）下Trolox之1.72倍。分析各次區分之抗氧化物質含量，發現F9之總酚（970.4 ± 11.2 mg gallic acid /g dry weight of sample）與總類黃酮（732.9 ± 19.0 mg catechin /g dry weight of sample ）含量皆為最高；且F8~11之總類黃酮含量趨勢與抗氧化效果之趨勢相符，並皆顯著高於區分前之乙酸乙酯區分層。因此推測龍眼花之抗氧化性可能與酚類中類黃酮物質含量或組成具有高度相關性。
HPLC分析F9發現其僅含一個成分，該化合物以 IR、MS、UV-Vis與1H-NMR、13C-NMR、2D-NMR等光譜分析，鑑定出F9中主要抗氧化活性成分為(-)-epicatechin。以Sephadex LH-20分離F10中成分後，則鑑定出其中兩個主要抗氧化活性成分為(-)-epicatechin與proanthocyanidin A2，兩者皆具抑制LDL氧化效果，分別為同濃度（0.5 μg/mL）下Trolox之1.95與2.04倍。每克龍眼花中(-)-epicatechin與proanthocyanidin A2之含量經HPLC分析，各約為5.58 mg及1.70 mg。本實驗結果顯示龍眼花中具有優異抗氧化效果之成分，有潛力發展為具有保健功效之食材。
Atherosclerosis is the major cause of cardiovascular disease, and the oxidation of low density lipoprotein (LDL) cholesterol is the important step to initiate atherosclerosis. Antioxidants can increase the resistance against oxidative damage, so the supplementation of food with antioxidants may help prevent the incidence of atherosclerosis. Previous year study in our laboratory has shown that Longan (Dimocarpus longan Lour.) flower had good antioxidative activity. Therefore, the objective of this study is to conduct antioxidant activity-guided separation and purification of Longan flower by the major antioxidative assay, the inhibition of Cu2+-induced human LDL oxidation, and to identify the effective compounds.
After liquid-liquid partition of Longan flower methanol extract with n-hexane, ethyl acetate, n-butanol and water, the ethyl acetate fraction showed the best antioxidant activity. The EC50 value of the ethyl acetate fraction in scavenging DPPH radicals was 5.28 ± 1.14 μg/mL, and its effect of delaying LDL oxidation is 1.14 times better than Trolox at the same concentration level (1μg/mL). Besides, the ethyl acetate fraction had the highest contents of total polyphenol (701.7 ± 29.3 mg gallic acid /g dry weight of sample) and flavonoid (213.7 ± 14.8 mg catechin equivalent /g dry weight of sample).
Silica gel chromatography was employed to fractionate the ethyl acetate fraction of Longan flower methanol extract, and twenty sub-fractions were obtained. DPPH assay showed that the sub-fractions with medium to high polarity had better antioxidative activities. Result of ORAC assay revealed that F8~11 were the more effective sub-fractions and F9 (eluted by ethyl acetate/n-hexane=60/40, v/v) gave the highest ORAC value (25.07 ± 4.08 Trolox equivalent). As for the effect of Cu2+-induced oxidation of human LDL, F8~11 also showed better effect in delaying LDL oxidation. Among them, F10 (eluted by ethyl acetate/n-hexane=70/30, v/v), which had superior effect, was 1.72 times better than Trolox at the same concentration level (1μg/mL). Further analysis of these sub-fractions showed that F9 contained the hightest amounts of total polyphenol (970.4 ± 11.2 mg gallic acid /g dry weight of sample) and total flavonoid (732.9 ± 19.0 mg catechin /g dry weight of sample). The trends of total flavonoid contents and the antioxidative activities of the four sub-fractions (F8~11) were similar, we therefore supposed that the antioxidative activity of Longan flower was highly related to its total flavonoid content or composition.
There was only one major compound present in F9 by HPLC analysis and it was identified as (-)-epicatechin by spectrometric analysis of IR, MS, UV-Vis, 1H-NMR, 13C-NMR and 2D-NMR. After separating F10 by Sephadex LH-20, two major components were identified as (-)-epicatechin and proanthocyanidin A2. Both of them had superior effect in delaying LDL oxidation, and the lag time of each compound was 1.95 ( (-)-epicatechin ) and 2.04 ( proanthocyanidin A2 ) times better than Trolox at the same concentration level (0.5μg/mL). The contents of (-)-epicatechin and proanthocyanidin A2 in Longan flower were quantified by HPLC to be 5.58 and 1.70 mg/g dry weight, respectively. This study showed that Longan flower contained components with excellent activity, it thus has good potential to be developed as a functional food.
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