2.6. Antioxidant activity using DPPH free radical

Antioxidant activity of the selected cyanobacterial extract was measured with DPPH (2.2-diphenyl-1-picrylhydrazyl) scavenging assay using the method of Assuncao (Assunção et al., 2017). Concisely, 1.8 ml of 0.06 mM DPPH (CDH, India) dissolved in methanol was added to the 0.2 ml of the selected cyanobacterial extract with six different concentrations (1, 5, 10, 15, 20 and 30 mg/mL). A blank solution was prepared by adding 0.2 ml of DMSO to 1.8 ml DPPH solution. The absorbance of the reaction mixture, as well as the blank, was measured after 15 min at 515 nm using the UV–VIS double beam spectrophotometer (Systronics AU 2701, India). While the same amount of DPPH mixed with same volume of synthetic antioxidant 3, 5-di-tert-butyl-4-hydroxytoluene (BHT) solution was used as the positive control. The DPPH radical scavenging ability of the extract and the positive control was expressed in terms of inhibition percentage and calculated with the equation given below.

where A_DPPH is the absorbance of blank and A_sample is the absorbance of the reaction mixture containing cyanobacteria extract after 15 min.

A curve was plotted between the concentration of extract and corresponding inhibition percentage. The concentration (%, v/v) of the extract that reduces the initial absorbance of DPPH by 50% was calculated by logarithmic regression with R² value of 0.98 and considered as IC₅₀.