以氧自由基吸收能力法(ORAC)測定園產品之 抗氧化能力

Abstract

摘要 氧自由基吸收能力(oxygen radical absorbance capacity, 簡稱ORAC)分析法是目前被認為具代表性之蔬果中總水溶性抗氧化力之分析方法。它利用螢光探針被AAPH [2,2-Azobis(2-amidinopropane) dihydrochloride]產生之過氧自由基(peroxyl radicals)氧化失去螢光之反應作為偵測反應。目前使用在ORAC分析法之螢光探針有β-Phycoerythrin (β-PE)及Fluorescein(FL)二種，前者為藻類螢光蛋白而後者為有機分子。本報告針對二種螢光探針就其螢光穩定度、反應是否受溶劑影響以及對不同抗氧化物標準品之反應曲線有無差異進行比較。結果顯示，在激發光照射下β-PE之螢光強度會隨時間而減弱，FL則維持穩定。以磷酸緩衝液及甲醇作為溶劑均不會影響空白反應之螢光變化，而以丙酮及乙醇作為溶劑則會干擾自由基作用。以β-PE及FL探針分析水溶性維生素E類似物-trolox之抗氧化力，在trolox濃度0~10 μM間呈直線性相關；對其他抗氧化物包括穀胱苷肽、抗壞血酸、鈉鹽-抗壞血酸、尿酸、芸香素、兒茶素與兒茶酚等之靈敏度亦在μM範圍內，且在濃度與抗氧化力之間皆能得到線性相關。以trolox為對照，比較各抗氧化物用二種探針做ORAC分析之相對抗氧化力，以β-PE為探針對穀胱苷肽得到之抗氧化力與FL相近；對抗壞血酸、鈉鹽-抗壞血酸及尿酸等分子之抗氧化力較FL為低；因此自由基與抗氧化物及螢光探針間之互動反應會依抗氧化物之種類而不同。由於酚類物質之相對抗氧化力均遠高於trolox，表示酚類物質對過氧自由基具有很好的抗氧化效果。 總氧自由基清除能力(total oxyradical scavenging capacity,簡稱TOSC)分析法為另一種分析總水溶性抗氧化力之方法；它是以2,2’-azobis-amidinopropane (ABAP)氧化α-keto-γ-methylthiobutyric acid，並以後者所分解產生的乙烯為定量的依據。以TOSC分析數種抗氧化物標準品包括trolox、抗壞血酸、穀胱苷肽、芸香素、兒茶素與兒茶酚，在濃度與抗氧化力之間均能得到線性相關。以trolox當量值來比較，抗壞血酸與穀胱苷肽之TOSC抗氧化力分析結果與ORACFL接近，而酚類物質芸香素、兒茶素與兒茶酚之TOSC抗氧化力則較ORACFL分析結果低。以ORACFL與TOSC兩種分析方法測試14種園藝作物之抗氧化力，二者之結果間僅有低度相關。對二種以上之抗氧化物標準品之混合物，ORACFL與TOSC分析法均能在實測抗氧化力值與估算值之間得到相關性。但ORACFL之相關性較佳，考慮分析方法之準確性及結果之代表性，仍然以ORACFL分析法為較佳之選擇。 試驗最後以ORACFL分析24種蔬菜作物及5種水果之總水溶性抗氧化力。結果顯示，所有分析之作物抗氧化力分佈範圍介於2.08 ± 1.11至58.19 ± 1.48 μmole Trolox eq./g F.W.之間，其中以‘台灣種’刈葉萵苣、‘夏峰1號’‘初秋’‘夏山’甘藍菜等屬於高抗氧化力種類(＞40 μmole Trolox equiv. /g)。相同作物之不同品種間，抗氧化力有明顯之差異。不同栽培地點及栽培方式亦是影響抗氧化力之因素。而分析相同農戶在不同栽培時期種植作物，抗氧化力則無顯著差異。由此可知，蔬果抗氧化力高低，除了先天之品種因素外，栽培時外界環境氣候及栽種方式也能對抗氧化力造成影響。

Abstract Oxygen radical absorbance capacity (ORAC) assay is regarded as the recommended method for representing the total hydrophilic antioxidant capacity in fruits and vegetables. This method measures the decay of fluorescence as the fluorescent probe is oxidized by the peroxy radicals generated from AAPH (2,2-Azobis(2-amidinopropane) dihydrochloride). At present, there are two fluorescent probes being used in the ORAC assay, the β-phycoerythrin (β-PE), an fluorescent algae protein; and fluorescein (FL), an organic fluorescent molecule. The object of this paper is to compare the fluorescence stability, influence of solvent on free radical reaction, and the responses of different antioxidants in the ORAC assay by using these two probes. Results showed that under excitation irradiation, FL remained stable during the assay period, while β-PE lost its fluorescence intensity with time. Using phosphate buffer or methanol as solvent did not influence the fluorescence change of the free radical reaction, but using acetone or ethanol as solvent would affect the reaction. Both β-PE and FL probes have shown linear correlation at the concentration range of 0~10 μΜ for the water-soluble vitamin E analog trolox. Similar linearity and sensitivity were obtained with other antioxidants, including glutathione, ascorbic acid, sodium ascorbate, uric acid, rutin, catechin and catechol. The relative antioxidant capacity (with respect to trolox) of various antioxidants were further compared, the antioxidant value of glutathione obtained with β-PE was similar to that of FL. For ascorbic acid, Sodium ascorbate and uric acid, the antioxidant values obtained with β-PE was higher than that of the FL. While for phenolic compounds including rutin, catechin and catechol, the antioxidant values obtained with β-PE was lower than that of FL. These results indicated that interactions between free radicals, antioxidants and fluorescence probes were different among various antioxidants. Since the relative antioxidant capacity of all the phenolic compounds was much higher than trolox in both assays, it is a good indication that phenolic compounds possess high antioxidation capacity against peroxy radicals. The total oxyradical scavenging capacity (TOSC) assay is another method for total hydrophilic antioxidant capacity, which is based on the oxidation of α-keto-γ-methylthiobutyric acid by 2,2’-azobis-amidino -propane (ABAP) with the evolution of ethylene as the quantifiable end product. Linear correlations were observed between TOSC antioxidant capacity and concentration of various antioxidants. The antioxidants being tested include trolox, ascorbic acid, glutathione, rutin, catechin and catechol. The antioxidant capacity of ascorbic acid and glutathione were similar to the ORACFL assay, and antioxidant capacity of all the phenolics, rutin, catechin and catechol are lower than that of the ORACFL assay. Fourteen horticulture crops were analyzed for their antioxidant capacity by both the TOSC assay and the ORACFL assay. The results of ORACFL and TOSC assay had rather poor correlation, indicating the difficulty of comparing results of two different assays. When using mixtures of two or more antioxidant standards, both ORACFL and TOSC assays gave linear regression curve between measured and calculated values, but the ORACFL had better correlation coefficients. Based on the accuracy and reliability of the assay methods, the ORAC assay was considered the better method to be chosen. In the last part, the antioxidant capacity of 24 vegetables and 5 fruits grown in Taiwan was measured by the ORACFL assay. The total hydrophilic antioxidant capacity of all the fruits and vegetables analyzed ranged between 2.08 ± 1.11 to 58.19 ± 1.48 μmole Trolox equiv./g. Among them, ‘Taiwan’ cutting lettuce, ‘Kaiya’, ‘C×Y cross’ and ‘Siashan’ cabbages all had relative high amount of antioxidant capacity, that is, higher than 40 μmole Trolox equiv./g. There are significant differences in antioxidant capacity among cultivars of the same crop, and there are differences among vegetables grown in different locations and by different growers. But there were no difference between vegetables grown by the same grower at the same location but harvested at different time. It is concluded that in addition to the differences among cultivars, antioxidant capacity of crops may be influenced by environmental factors as well as cultivation styles.