Hydroxyl radical scavenging assay of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (CUPRAC) method using catalase for hydrogen peroxide degradation

• Published in: Analytica chimica acta Vol. 616; no. 2; pp. 196 - 206
• Main Authors: Özyürek, Mustafa, Bektaşoğlu, Burcu, Güçlü, Kubilay, Apak, Reşat
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 02-06-2008; Elsevier
• Subjects: Analytical chemistry; Antioxidants - chemistry; Catalase - chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid - methods; Copper - chemistry; Cupric reducing antioxidant capacity (CUPRAC) assay; Edetic Acid - chemistry; Electrochemical methods; Exact sciences and technology; Flavonoids; Flavonoids - chemistry; Free Radical Scavengers - chemistry; Hydrogen Peroxide - chemistry; Hydroxyl Radical - analysis; Hydroxyl radical detection; Iron - chemistry; Kinetics; Molecular Structure; Other chromatographic methods; Oxidation-Reduction; Phenanthrolines - chemistry; Phenolics; Phenols - chemistry; Radical scavenging; Reproducibility of Results; Salicylates - chemistry; Sensitivity and Specificity; Spectrometric and optical methods; Time Factors; Flavonoids; Phenolics; Hydroxyl radical detection; Radical scavenging; Cupric reducing antioxidant capacity (CUPRAC) assay; Copper II; Second order; Ascorbic acid; Chemical analysis; Hydrogen peroxide; Electrochemical method; HPLC chromatography; Glucose; Flavonoid; Tissue; Catalase; Hydroxyl radical detection;Radical scavenging;Cupric reducing antioxidant capacity (CUPRAC) assay;Phenolics;Flavonoids; Reaction kinetics; Hydroxyl radicals; Inhibition; Rate constant; Chromophore; Human; Gallic acid; Enzyme; Color reaction; Chlorogenic acid; Antioxidant; EDTA; Radical scavenger; Absorption spectrometry; Peroxidases; Phenols; Oxidoreductases
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2008.04.033

Hydroxyl radicals (
OH) generated in the human body may play an important role in tissue injury at sites of inflammation in oxidative stress-originated diseases. As a more convenient, efficient, and less costly alternative to HPLC/electrochemical detection techniques and to the nonspecific, low-yield deoxyribose (TBARS) test, we used a salicylate probe for detecting
OH generated by the reaction of iron(II)–EDTA complex with H
2O
2. The produced hydroxyl radicals attack both the salicylate probe and the hydroxyl radical scavengers that are incubated in solution for 10
min. Added radical scavengers compete with salicylate for the
OH produced, and diminish chromophore formation from Cu(II)–neocuproine. At the end of the incubation period, the reaction was stopped by adding catalase. With the aid of this reaction, a kinetic approach was adopted to assess the hydroxyl radical scavenging properties of polyphenolics, flavonoids and other compounds (e.g., ascorbic acid, glucose, mannitol). A second-order rate constant for the reaction of the scavenger with
OH could be deduced from the inhibition of colour formation due to the salicylate probe. In addition to phenolics and flavonoids, five kinds of herbs were evaluated for their
OH scavenging activity using the developed method. The modified CUPRAC (cupric ion reducing antioxidant capacity) assay proved to be efficient for ascorbic acid, gallic acid and chlorogenic acid, for which the deoxyribose assay test is basically nonresponsive. An important contribution of this developed assay is the inhibition of the Fenton reaction with catalase degradation of hydrogen peroxide so that the remaining H
2O
2 would neither give a CUPRAC absorbance nor involve in redox cycling of phenolic antioxidants, enabling the rapid assay of polyphenolics.