Measurement of Antioxidant Capacity of Vegetables Using Oxygen Radical Absorbance Capacity（ORAC）and Folin-Ciocalteu Reagent Reducing Capacity（FCR）Assay
Date Issued
2011
Date
2011
Author(s)
Cheng, Yu-Hsuan
Abstract
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.
Subjects
Antioxidant Capacity
ORAC
FCR
Type
thesis
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