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Optimization and Validation of Two High-Throughput Methods Indicating Antiradical Activity

[ Vol. 13 , Issue. 6 ]

Author(s):

Graciela Granados-Guzman, Ricardo Salazar-Aranda*, Marsela Garza-Tapia, Rocio Castro-Rios and Noemi Waksman de Torres   Pages 499 - 507 ( 9 )

Abstract:


Background: The search for new natural or synthetic products with antioxidant activity is commonly based on methods that involve reduction of either 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 2-2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). However, the reported values of the effective concentrations are highly variable, even in controls. Herein, we optimize and validate both methods of determining antiradical activity.

Methods: Optimization was carried out using both a fractionated factorial design and a basic sequential simplex method, by monitoring the reduction percentage. Quercetin or Trolox were used as positive control. Furthermore, for each method, linearity, precision, accuracy, robustness, plate uniformity, signal variability, and Z factor, were established.

Results: The optimized conditions for the DPPH method were: DPPH 280 µM in ethanol and 15 min of reaction time in the dark. The linear range was between 7 and 140 µM with an R2 value of 0.9987. The optimized conditions for the ABTS method were: ABTS adjusted to 0.7 absorbance units, 70% concentration in ethanol, and a reaction time of 6 min in the dark. The linear range was found to be between 1 and 70% with an R2 = 0.9991. For both methods, the accuracy and precision were within limits and the Z factor value was higher than 0.89. The applicability of each method was assessed by analyzing eight plant extracts.

Conclusion: The DPPH and ABTS reduction methods were optimized and validated on a microscale and could be expected to be implemented in any laboratory.

Keywords:

ABTS, antiradical activity, DPPH, high-throughput screening methods, natural products, reduction.

Affiliation:

Universidad Autonoma de Nuevo Leon, Facultad de Medicina, Departamento de Quimica Analitica. Monterrey, N. L, Universidad Autonoma de Nuevo Leon, Facultad de Medicina, Departamento de Quimica Analitica. Monterrey, N. L, Universidad Autonoma de Nuevo Leon, Facultad de Medicina, Departamento de Quimica Analitica. Monterrey, N. L, Universidad Autonoma de Nuevo Leon, Facultad de Medicina, Departamento de Quimica Analitica. Monterrey, N. L, Universidad Autonoma de Nuevo Leon, Facultad de Medicina, Departamento de Quimica Analitica. Monterrey, N. L

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