Optimization of the molybdenum blue method for estimating the antioxidant activity of natural products

Context: The present experimental conditions of the molybdenum blue spectrophotometric method, used for antioxidant activity estimation, promote the degradation of flavonoids with potential interferences in the above determination. Aims: To evaluate the effects of physicochemical factors on the formation of the complex for optimizing the total antioxidant activity method to better estimate the antioxidant activity of natural products. Methods: A 34-1 fractional experimental design was applied. Independent variables were temperature, color development time, type of acid and acidity, and the dependent variable was the absorbance of the complex. The concentration of ammonium molybdate tetrahydrate and sodium hydrogen phosphate remained constant throughout the study. The effects of the reducing agent and its concentration were studied independently. The effect of acidity in a wide range of values, the color development time considering temperature, the influence of co-solvents, and the antioxidant activity of various natural metabolites were also evaluated. Results: Acid concentration and temperature greatly influenced the complex formation, making the type of acid and incubation time less significant. Ascorbic acid showed a shorter color development time than reference metabolites. Ethanol negatively influenced the amount of complex formed. The proposed conditions for developing this method were: type of acid, HCl or H2SO4; acid concentration 0.01 N; incubation temperature 65°C; and incubation time 40 min. Under these experimental conditions, the ranking order of antioxidant activity was pyrogallol>quercetin>ascorbic acid>gallic acid>rutin. Conclusions: The new experimental conditions for the molybdenum complex assay give a more reliable determination of the antioxidant activity of natural products.