| Summary: | 碩士 === 國立臺灣大學 === 食品科技研究所 === 94 === 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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