DPPH Radical Scavenging Assay of Curcuminoids

The antioxidant assay was carried out by the DPPH method. The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay, based on electron transfer that produces a violet solution in ethanol. This free radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving rise to a yellow-colored product, diphenylpicryl hydrazine in ethanol solution. The DPPH assay is used to determine antioxidant activities by the mechanism in which antioxidants act to inhibit lipid oxidation, scavenging of DPPH radical, and therefore determine free radical scavenging capacity. Free radical scavenging abilities of curcumin, TurmiZn, and THC were tested by the DPPH radical DPPH antioxidant assay kit (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Briefly, for three replicates of each analyte, a solution of 10 mL DPPH in ethanol was prepared, and 100 μL of this solution was mixed with 20 μL of the curcuminoids, THC or TurmiZn complex solubilized in ethanol at different concentrations (0.125–0.50 mg/mL). The reaction mixture was mixed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Trolox was used as a reference. The percentage DPPH radical scavenging activity was then calculated per manufacturing methodology and reported as mean values ± standard error of mean.