Measurement of Antioxidant Capacity of Vegetables Using Oxygen Radical Absorbance Capacity（ORAC）and Folin-Ciocalteu Reagent Reducing Capacity（FCR）Assay

蔬果內含豐富的抗氧化物質如胡蘿蔔素、類黃酮及酚類物質等，提供人類膳食中豐富的抗氧化力。抗氧化力之分析方法眾多且原理不同，本試驗以目前最主要之二種抗氧化力分析方法：氧自由基吸收能力方法（Oxygen radical absorbance capacity assay, ORAC）與FC試劑還原能力法（Folin-Ciocalteo reagent reducing capacity assay, FCR）為對象，探討影響分析反應之因子並比較二種方法對於不同抗氧化物質標準品之相對抗氧化力之差異性，探討植物樣品之萃取技術，並以所建立之分析方法分析十六種國內蔬菜之抗氧化力，進而分析抗氧化物質與抗氧化力分析方法間之相互關係。 氧自由基吸收能力（ORAC）方法是以Fluorescein為螢光探針，其最適激發波長為490 nm，散射波長為510 nm；分析時於恆溫37℃避光五分鐘後，加入自由基產生物AAPH進行反應，以螢光強度衰減曲線下面積計算抗氧化力值。ORAC系統中含有5%甲醇或2%丙酮，不影響螢光強度衰減；但含有0.5%乙醇即會干擾自由基作用，影響螢光強度衰減。以ORAC分析抗壞血酸、綠原酸、沒食子酸、兒茶素、木犀草素、楊梅酮、芸香苷、山萘酚、芹菜素、槲皮素等十種抗氧化物質，其濃度與淨螢光衰減曲線下面積皆能得到良好的線性相關；依照螢光衰減曲線不同，可分為三種類型。因此以ORAC方法分析不同類型抗氧化物質皆能得到一致性的結果。 FC試劑還原能力（FCR）方法以Folin-Ciocalteu試劑作為反應液，於鹼性環境與酚類及其他具有還原力之物質進行氧化反應。以沒食子酸與Trolox作為標準品均可得良好相關的標準曲線，因此可作為抗氧化力分析之方法。用Trolox 為共同的參考物質，比較十種抗氧化物質用FCR、ORAC及DPPH三種方法分析結果的一致性。結果以ORAC方法最適作為抗氧化力之分析，可廣泛分析不同類型抗氧化物質，減少系統中的偏差；FCR方法整體相對抗氧化力數值接近ORAC方法，然而較偏好水溶性抗氧化物質，但也為良好分析方法。DPPH 方法則與前二方法之差異較大且比較欠缺區別不同抗氧化力的能力。 洋蔥粉末樣品在分析前以含1.2 N HCl之甲醇加熱萃取兩小時，萃取物可分別在ORAC、FCR、DPPH三個抗氧化力測定方法中得到較高之抗氧化值，為最佳萃取條件。以此條件製備十六種國內常見蔬菜之萃取液，以DPPH、FCR、ORAC方法分析其抗氧化力。以DPPH方法分析所有蔬菜樣品，皆可得95%以上清除自由基能力，無法區別蔬菜種類間之差異。FCR及ORAC二種方法均可區別不同蔬菜間抗氧化力之差異。以FCR方法得到具有最高抗氧化能力的蔬菜為秋葵、甘薯葉及芫荽；ORAC方法則以甘薯葉、芫荽、葉萵苣具最高抗氧化能力。十六種蔬菜以FCR測定之抗氧化能力值介於300~1500 μmol TE･g-1，以ORAC測定則介於100~800 μmol TE･g-1。 將三種方法得到之抗氧化力與抗壞血酸含量以及類黃酮含量，進行相關性與多元迴歸曲線分析。三種方法以ORAC與FCR之間有0.77的顯著相關性，為較能真實反映樣品抗氧化能力的方法。抗壞血酸含量與ORAC 以及FCR抗氧化力值的相關係數分別為0.73與0.62，顯示兩者為顯著相關。以多元迴歸方法分析抗氧化能力與抗氧化物質含量的關係，其中ORAC抗氧化力值的最佳多元迴歸方程式以抗壞血酸為主；FCR多元迴歸方程式則包含抗壞血酸與槲皮素以及芹菜素，初步釐清十六種蔬菜的抗氧化力主要與抗壞血酸有關。本研究的結果顯示，ORAC方法與FCR方法皆能正確區別不同抗氧化物之抗氧化力，可作為日後分析園產品之抗氧化能力之理想方法。Fruits and vegetables are rich in compounds that possess antioxidative capacity, such as carotenoids, flavonoids and phenolics, and these compounds act as the major source of antixidants in our daily diet. A number of methods, that were based on different principles, had been developed to determine the antioxidant capacity in our food. In this research, two of the major methods for antioxidant capacity assay: the Oxygen radical absorbance capacity assay（ORAC）and the Folin-Ciocalteo reagent reducing capacity assay（FCR）were studied in detail. First, standard assay procedures were established for these two assays and the relative antioxidant capacity of ten phytochemicals representing antioxidants commonly found in vegetables were obtained and the differences between these two assays were compared. Then, the most effective way of extracting antioxidant components from vegetable tissue was studied and the antioxidant capacities of sixteen vegetables sold in local market were assayed and the correlation between the antioxidant capacity and the antioxidant compounds were further analyzed. The ORAC assay utilizes Fluorescein as the fluorescent probe, its optimum excitation and emission wavelengths were 490 nm and 510 nm respectively. During the assay, the sample and the probe were mixed first and incubated at 37℃for 5 min in darkness, then the free radical generating compound AAPH was added to initiate the reaction. The changes in fluorescence with time were monitored with a spectro- fluorometer until the fluorescence dropped to less than 1% of the initial value. The area under the fluorescence curve（AUC）was then used to calculate the antioxidant capacity of the sample. The ORAC assay system was not affected by the presence of 5% methanol or 2% acetone; however, the decline of fluorescence was strongly interfered by the presence of 0.5% ethanol in the system. Ten phytochemicals commonly found in vegetables, including ascorbic acid, chlorogenic acid, gallic acid, catechin, luteolin, myricetin, rutin, kaempferol, apigenin, and quecertin were assayed by the ORAC method and all of them gave good linear correlations between concentration and AUC, indicating the validity of the ORAC assay. Based on the curves of fluorescence decline obtained from these ten compounds, three types of pattern could be distinguished, probably representing different mode of antioxidation reaction by these compounds. The FCR assay utilize the Folin-Ciocalteu reagent as reactant, it undergo oxidation reaction with phenolics or other compounds having reducing capacities under alkaline condition. Both gallic acid and Trolox gave good linear correlations by the FCR assay, indicating the method was suitable for antioxidant assay. By using Trolox as the mutual reference compound, the ten phytochemicals were assayed by three antioxidant capacity assays: the FCR, the ORAC and the DPPH assay; and the resulting relative antioxidant capacities were compared for consistency. The ORAC assay was found to be able to react with different types of antioxidant, and the results were highly representative of the antioxidant capacity of the system. The FCR assay showed preference for water soluble compounds, but also gave comparable results with the ORAC assay. The DPPH assay was less consistent with the other two assays and was less responsive to compounds with higher antioxidant capacity. Onion powders extracted with methanol containing 1.2 N HCl at 90℃for two hours gave the highest antioxidant capacity by all three assay methods. This extraction condition was used to obtain extracts from 16 vegetables commonly found in local market, and they were assayed for antioxidant capacity by the ORAC, the FCR and the DPPH assay. In the DPPH assay, over 95% free radical scavenging capacity was obtained for all vegetable samples, suggesting the inability of this assay in distinguishing the antioxidant capacities among different vegetables. On the other hand, both the FCR and the ORAC assay gave different results for different vegetables. Vegetables that showed the highest antioxidant capacity were okra, sweetpotato leaf and Chinese parsley by the FCR assay; and sweetpotato, Chinese parsley and leaf lettuce by the ORAC assay. The absolute antioxdant capacity were between 300~1500 μmol TE･g-1，by the FCR assay and between 100~800 μmol TE･g-1 by the ORAC assay. Statistical analyses were carried out with data from the results of antioxidant capacity assay of 16 vegetables and the contents of ascorbic acid and 6 individual flavonoids. The result showed that there was a significant correlation, r=0.77, between the ORAC assay and the FCR assay, indicating the consistency of these two assays in measuring the antioxidant capacity of the sample. The correlation coefficient between ascorbic acid content and the ORAC assay and the FCR assay were 0.73 and 0.62 respectively, indicating the dominant role of ascorbic acid played in the antioxidant capacity of vegetables. The relationship between antioxidant capacity and antioxidant compounds were further analyzed with multiple linear regression. The best multiple linear regression equation for the ORAC assay showed that ascorbic acid was the major component, and the best multiple linear regression equation for the FCR assay showed that ascorbic acid, quecertin and apigenin were the important factors. The result also showed that ascorbic acid was involved in the antioxidant capacity in all 16 vegetable studied. Results of this study indicated that both the ORAC assay and the FCR assay could distinguish the antioxidant capacity of different vegetable correctly and therefore were considered as the recommended method for antioxidant capacity assay for horticultural products in the future