Determination of Total Antioxidant Capacity Using Thiamine as a Natural Fluorescent Probe

This work proposes a spectrofluorometric method for the determination of total antioxidant capacity (CAO) in beverage samples, based on inhibition of thiochrome formation (λex = 370 nm, λem = 440 nm); a product of thiamine (vitamin B1) oxidation from K3Fe(CN)6. In the development of the method, gallic acid (GA) was used as a reference, and inhibition of thiochrome formation (in percentage) was used as the analytical response. The selectivity of the method was evaluated using seven different compounds (gallic acid, ascorbic acid, quercetin, butylhydroxytoluene (BHT), 6-hydroxy-2,5,7,8-tetramethylchroman-2 (Trolox), cysteine, and glucose). As proof of concept, the proposed method was applied in the determination of CAO in different samples, such as teas and infusions, red wines, and white wines. The results were compared with the Folin-Ciocalteu (FC), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+), and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) methods using the linear correlation between the methods (at 95% confidence). We observed excellent agreement between the proposed method and FC (correlation coefficient (r) = 0.9768) and ABTS•+ (r = 0.9842), compared with DPPH• method (r = 0.8502). For the determination of total CAO in beverages, the proposed method developed proved to be fast, sensitive, simple, and the results were in agreement with established assays.本研究提出了一种荧光分光光度法（spectrofluorometric method），用于测定饮料样品中的总抗氧化能力（total antioxidant capacity, CAO），该方法基于对硫色素（thiochrome）生成的抑制作用，而硫色素是硫胺素（thiamine，维生素B1）经铁氰化钾（K3Fe(CN)6）氧化后的产物，其激发波长（λex）为370 nm，发射波长（λem）为440 nm。方法开发过程中，以没食子酸（gallic acid, GA）作为参比物质，以硫色素生成抑制率（以百分比计）作为分析响应信号（analytical response）。本方法的选择性（selectivity）通过七种不同化合物进行评估，分别为没食子酸（GA）、抗坏血酸（ascorbic acid）、槲皮素（quercetin）、丁基羟基甲苯（butylhydroxytoluene, BHT）、6-羟基-2,5,7,8-四甲基色满-2（Trolox）、半胱氨酸（cysteine）以及葡萄糖（glucose）。作为概念验证（proof of concept），本研究将所提出的方法应用于多种样品的总抗氧化能力测定，包括茶及浸提液、红葡萄酒与白葡萄酒。将本方法的测定结果与福林-酚法（Folin-Ciocalteu, FC）、2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)自由基（2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate), ABTS•+）法以及2,2-二苯基-1-苦基肼基自由基（2,2-diphenyl-1-picrylhydrazyl, DPPH•）法进行对比，采用方法间的线性相关性进行分析（置信度为95%）。结果显示，本方法与FC法（相关系数（correlation coefficient, r）=0.9768）及ABTS•+法（r=0.9842）具有极佳的一致性，而与DPPH•法的相关系数仅为0.8502。综上，所开发的方法用于测定饮料中总抗氧化能力时，具备快速、灵敏、简便的优势，且测定结果与现有标准测定方法具有良好的一致性。