Tez özetleri Astronomi ve Uzay Bilimleri Anabilim Dalı

Application of Spectrofluorometric Analysis Methods to Total Antioxidant Capacity Assays

Flavonoids are health beneficial compounds in regard to their antioxidant, anticancer and anti-inflammatory activity, and therefore their analysis is important. Flavonols are the most widely found flavonoid class in the plant kingdom, and their leading representatives are quercetin, myricetin, fisetin, and kaempferol. The most frequently used method in literature for total flavonoid assay is the spectrophotometric aluminium chloride/potassium acetate (AlCl₃/KAc) method, and this work proposes an alternative spectrofluorometric method for flavonoid assay using the same reagent. For this purpose, the developed AlCl₃/KAc method was applied to flavonoids dissolved in ethanol and in acetone-methyl-β-cyclodextrin (M-β-CD) solution (previously developed for the assay of lipophilic antioxidants), and the results were compared to those of both the AlCl₃/KAc the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric assays. Compared to the 10^-7 M detection limits (LOD values) of the AlCl₃/KAc spectrophotometric method, the LOD of the spectrofluorometric method was reduced down to 10^-8 M levels for most flavonoids, except for rutin, galangin, luteolin, and quercitrin, which gave poorer linear calibration curves. The lower linear correlation coefficients for the latter flavonoids in spectrofluorometry may arise from the fact that spectrophotometry measures the color of a unique chromophore while a number of complexes with different stoichiometries may emerge in spectrofluorometry each having different fluorescence decay lifetimes. The major chromophore in AlCl₃/KAc spectrophotometry is the stable Al-chelate formed with the C-4 keto and either C-3 or C-5 hydroxyl groups of flavons and flavonols, and the flavon, flavonol, and isoflavon sub-classes of flavonoids respond to this spectrophotometric method. As mutual analytical wavelengths of flavonoids in the developed fluorometric method, fluorescence intensities with excitation at 470 nm and emission at 525 nm were measured. Spectrofluorometry was more dependent on pH than spectrophotometry, and the optimal pH of fluorescence measurement was 7. The strong dependence on pH of spectrofluorometry may be attributed to changes in fluorescence properties of Al-chelates of flavonoids arising from intense changes in the excited-state pK_a values of certain –OH groups that have not chelate-bonded to Al(III). The quercetin-equivalent flavonoid concentrations (QREFC values) of individual flavonoids were found from the calibration curves drawn in ethanol and in acetone/ M-β-CD media. In both solvent media, binary and ternary mixtures of flavonols were prepared, and the fluorescence intensities of mixture constituents were found to have additive property (i.e., signal obtained from the mixture was approximately equal to the sum of the signals due to individual mixture constituents). This additive property is essential for precise capacity measurements of antioxidants and flavonoids. The total QREFC flavonoid capacities of a number of real samples such as red wine, onion, and green tea were measured with both the developed spectrofluorometric and the two spectrophotometric (i.e., AlCl₃/KAc and CUPRAC) methods carried out in two different solvent media. In spectrofluorometry, the QREFC coefficients of certain flavonoids (which gave relatively poor calibration curve linearities) were quite lower than found in spectrophotometry, which may pose a problem in the representative weights of these flavonoids in the observed total flavonoid capacities of mixtures. The order of QREFC flavonoid capacities of real samples measured with four different methods were generally in accordance, with red wine showing the highest QREFC value in each method.