Free radical scavenging activity was estimated as percentage inhibition of radicals. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is an artificially stabilized free radical [29 (link)]. An aliquot of diluted extract or gallic acid standard solution (5 mg/mL; ≥98% purity) was added to 1.7 mL DPPH methanolic solution (0.06 mM), mixed thoroughly, and kept in the dark for 20 min at room temperature. The mixture’s absorbance was examined at 517 nm using an Ultrospec 4300 Pro UV–visible spectrophotometer (Amersham Biosciences Corp. San Francisco, CA, USA). Blank was prepared using aqueous acetone (80%) mixed with the DPPH solution. The gallic acid standard curve (at ≥98% purity) was prepared, and the linearity of the gallic acid standard curve (r2 = 0.98) was obtained in the range of 20–80 μg/mL. The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of fresh leaf (mg GAE/g fl). The following formula was used to calculate percentage inhibition [30 (link)]:
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