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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Natural Product Chemistry

Background:

• Antioxidant activity screening commonly employs 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2-2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays.
• Variability in reported effective concentrations, even for controls, necessitates method optimization and validation.
• This study focuses on refining and validating DPPH and ABTS assays for accurate antiradical activity determination.

Purpose of the Study:

• To optimize and validate microscale methods for determining antiradical activity using DPPH and ABTS assays.
• To establish reliable and reproducible conditions for antioxidant capacity measurements.
• To ensure the applicability of the optimized methods for analyzing various plant extracts.

Main Methods:

• Optimization employed a fractionated factorial design and a sequential simplex method.
• Assays monitored the reduction percentage of DPPH and ABTS radicals.
• Method validation included linearity, precision, accuracy, robustness, and Z factor assessment, using Quercetin or Trolox as positive controls.

Main Results:

• Optimized DPPH conditions: 280 μM in ethanol, 15 min reaction time; linear range 7–140 μM (R²=0.9987).
• Optimized ABTS conditions: 0.7 absorbance units, 70% in ethanol, 6 min reaction time; linear range 1–70% (R²=0.9991).
• Both methods demonstrated excellent accuracy, precision, and a Z factor > 0.89, with successful application to eight plant extracts.

Conclusions:

• The optimized and validated DPPH and ABTS reduction assays provide reliable microscale methods.
• These refined assays are readily implementable in any laboratory setting.
• The validated methods enhance the accuracy and reproducibility of antioxidant activity assessments.