CUPRAC total antioxidant capacity assay of lipophilic antioxidants in combination with hydrophilic antioxidants using the macrocyclic oligosaccharide methyl β-cyclodextrin as the solubility enhancer

摘要

Antioxidants are important health beneficial compounds in regard to their ability to quench reactive O,N-species under 'oxidative stress' conditions. We recently reported a simple, low-cost, and widely applicable total antioxidant capacity assay for dietary polyphenols, vitamins C and E, and plasma antioxidants in alcoholic aqueous media utilizing the copper(Ⅱ)-neocuproine (Nc) reagent as the chromogenic oxidant, which we named the CUPRAC method. Although this novel method was applied to synthetic mixtures of complex nature, plant extracts, human serum, and hydroxyl radical scavengers, lipophilic antioxidants were normally treated separately from hydrophilic ones. With oil-soluble antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), rert-butylhydroquinone (TBHQ), propyl gallate (PG), lauryl gallate (LG), and vitamin E (α-tocopherol), it was necessary to apply the CUPRAC method with dichloromethane extraction, which is not useful for hydrophilic antioxidants. Thus, as an improvement to classical CUPRAC methodology, this work reports the assay both lipophilic and hydrophilic antioxidants simultaneously, by making use of their 'host-guest' complexes with methyl β-cyclodextrin (M-β-CD), a cyclic oligosaccharide, in aqueous medium. The turbidity limit for a-tocopherol, BHT, PG, and LG were 1:3 (v/v) alcohol-water solutions, but when these suspensions were mixed with equal volumes of 7% M-β-CD aqueous solution, clear solutions were obtained in which the CUPRAC assay could be directly performed. In addition to solubility enhancement, molecular spectroscopic evidence was also provided for the formation of (M-β-CD + antioxidant) inclusion complexes. The calibration curves of individual (lipophilic and hydrophilic) antioxidants were constructed in such media, with their molar absorptivities and linear concentration ranges determined. Testing of synthetic ternary and quaternary mixtures of lipophilic and hydrophilic antioxidants in M-β-CD-containing media yielded theoretically expected CUPRAC antioxidant capacities, considering the additivity of absorbances of constituents obeying Beer's law. Thus M-β-CD served as the solubility enhancer host molecule to establish a modified CUPRAC method for the total antioxidant capacity assay of a mixture of diverse antioxidants of different lipophilicity levels in solutions rich in water content. The relative M-β-CD concentration used to form the inclusion complex could be restricted so as not to significantly decrease the antioxidant capacity exhibited by the tested compounds.