相思樹葉子抗氧化活性成分之分析與鑑定

Abstract

本研究利用DPPH（1,1-Diphenyl-2-picrylhydrazyl）自由基清除活性、超氧自由基捕捉活性、抑制脂質過氧化、還原力與金屬螯合能力等抗氧化活性試驗，評估相思樹（Acacia confusa）葉子抽出物之抗氧化活性，測定其總抗氧化能力、總酚類及總黃酮類含量，並利用管柱層析及高效能液相層析等技術分離、純化相思樹葉子抽出物，所得到之抗氧化成分則進一步運用核磁共振光譜、質譜、紅外線光譜與紫外光-可見光光譜等分析技術鑑定化合物。相思樹葉子甲醇抽出物抗氧化試驗結果顯示，甲醇抽出物之粗抽出物、乙酸乙酯可溶部及正丁醇可溶部皆具有良好的總抗氧化能力，且其總酚類含量及總黃酮類含量很高，故能有效的清除DPPH自由基、清除超氧自由基、抑制脂質過氧化及還原過氧化物質。其中，又以乙酸乙酯可溶部總酚類含量（307.8 mg of GAE/g）及總黃酮類含量（108.0 mg of QE/g）最高，故具有最強的清除DPPH自由基之效用（EC50 = 7.0 μg/mL）、抑制脂質過氧化作用（EC50 = 110.0 μg/mL）及還原過氧化物質之能力，其超氧自由基抑制作用（EC50 = 2.5 μg/mL）甚至比已知之抗氧化劑兒茶素（EC50 = 7.0 μg/mL）優異。 續以管柱層析將乙酸乙酯可溶部分離成8個次分離部後，測定其DPPH自由基清除活性、超氧自由基捕捉活性、還原力與金屬螯合能力等抗氧化活性試驗，並測定其總酚類及總黃酮類含量。試驗結果顯示Et6、Et7及Et8次分離部具有良好的DPPH自由基清除能力及超氧自由基抑制作用，並含有很高的總酚類含量及總黃酮類含量，其中，又以Et8次分離部之抗氧化活性最強。 乙酸乙酯次分離部共分離出10種化合物，包括：Gallic acid gallate、Luteolin、Myricetin 3-O-(2’’-O-galloyl)-α-rhamnopyranoside 7-methyl ether、Myricetin 3-O-(3’’-O-galloyl)-α-rhamnopyranoside、Myricetin 3-O-(3’’-O-galloyl)-α- rhamnopyranoside 7-methyl ether、Myricetin-3-glucopyranoside、Myricetin-3-rhamnopyranoside、Myricetin 3-O-(2’’-O-galloyl)-α-rhamnopyranoside、Quercetin-3-rhamnopyranoside及Europetin-3-rhamnopyranoside，其中，除了Luteolin與Gallic acid methyl ester清除DPPH自由基之效果及總抗氧化能力較差外，其他化合物清除DPPH自由基之效果均較兒茶素優異。此外，相思樹葉子熱水抽出物亦具有良好的清除DPPH自由基之效果及總抗氧化能力，其總酚類含量及總黃酮類含量則分別為254.7 mg of gallic acid equivalent/g及35.8 mg of quercetin equivalent/g。综合試驗結果顯示，相思樹葉子抽出物頗具潛力開發為抗氧化之保健用品。

In this research, the antioxidant activity of extract from the leaves of Acacia confusa was evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical and superoxide radical scavenging assays, lipid peroxidation assay, reducing power and metal chelating ability on ferrous ions assays, and trolox equivalent antioxidant capacity (TEAC) assay. Total phenolic contents and total flavonoid contents were also evaluated. In addition, column chromatography and high performance liquid chromatography are used to separate and purify the antioxidant compounds. The chemical structure of active constituents from A. confusa leaves was identified by the analysis of nuclear magnetic resonance spectroscopy, mass spectroscopy, IR spectroscopy and UV/VIS spectroscopy. Antioxidant results of methanolic extract from A. confusa leaves revealed that the crude extract, EtOAc soluble fraction and BuOH soluble fraction have great effectiveness in TEAC assay, and they also possess high amounts of total phenolic contents and total flavonoid contents, therefore these extracts exhibited great effectiveness in scavenging DPPH free radical, scavenging superoxide radical, inhibition of lipid peroxidation and reducing peroxidated substrates. Among those extracts, EtOAc soluble fraction possesses the highest amount of total phenolic content (281.0 mg of GAE/g) and total flavonoid content (108.0 mg of QE/g), and demonstrated the great capacity of scavenging DPPH free radical (EC50 = 7.0 μg/mL), inhibition of lipid peroxidation (EC50 = 110.0 μg/mL) and reducing peroxidated substrates. Moreover, the scavenging of superoxide radical of EtOAc soluble fraction (EC50 = 2.5 μg/mL) is more effective than catechin (EC50 = 7.0 μg/mL). EtOAc soluble fraction was then separated into 8 subfractions which were assayed by DPPH free radical and superoxide radical assays, reducing power and metal chelating on ferrous ions assays, and also determined their total phenolic contents and total flavonoid contents. Results revealed that subfractions Et6, Et7, and Et8 possessed the high amounts of total phenolic contentand total flavonoid content, they also exhibited the best antioxidant activities in DPPH free radical and superoxide radical scavenging assays. Ten compounds were isolated from EtOAc subfractions, including gallic acid gallate, luteolin, myricetin 3-O-(2’’-O-galloyl)-α-rhamnopyranoside 7-methyl ether, myricetin 3-O-(3’’-O-galloyl)-α-rhamnopyranoside, myricetin 3-O-(3’’-O-galloyl)-α- rhamnopyranoside 7-methyl ether, myricetin-3-glucopyranoside, myricetin-3- rhamnopyranoside, myricetin 3-O-(2’’-O-galloyl)-α-rhamnopyranoside, quercetin-3 -rhamnopyranoside and europetin-3-rhamnopyranoside. Among them, all compounds exhibited more effective capacity in scavenging DPPH free radical and TEAC than catechin except for luteolin and gallic acid methyl ether. In addition, the hot-water extract of A. confusa also have great effectiveness in DPPH and TEAC assays, and it possess high amounts of total phenolic contents (254.7 mg of GAE/g) and total flavonoid contents (35.8 mg of QE/g). Results obtained above indicated that the leaves of A. confusa have the potential to be used as a source for natural health products such as antioxidants.