Hplc grade

The aflatoxins were extracted as per manufactures protocol (Mobile Assay Inc; Neogen Corporation, USA). Briefly, different samples were bring to laboratory and grind or paste so at least 75 % of material passes through a 20 mesh sieve, about the particle size of fine instant coffee. Aflatoxins were extracted by mixing 1 part of sample to 5 parts of 65 % ethanol (HPLC grade, HI-Media Laboratories Ltd. Mumbai, India) and were vigorously vortex for 3 min. The samples were allowed to settled and then filter with syringe filter and finally utilized for quantification of aflatoxins by using Reveal Q+ aflatoxin test strip (Neogen Corporation, USA).

The scavenging activity of cationic ABTS radical was determined using the method of Re et al., [28 ]. Briefly, 7 mM of ABTS stock solution was mixed with 2.45mM potassium persulfate solutions prepared in deionized water. The reaction mixture after sixteen hours of reaction in dark generates ABTS radical cation. The ABTS ^{● +} solution was suitably diluted with ethanol to yield an absorbance of 0.70 (± 0.2) at 734nm and equilibrated at 25°C to be used for the antioxidant assay. The assay was performed by adding 1ml methanolic extracts to be tested at different amounts to 3ml of ABTS^●+ radical cation solution and the mixture was shaken gently, incubated for six minutes at 37°C. The reduction of ABTS^●+ radical cation absorbance by adding compounds that contain antioxidants was measured by the change of absorbance of ABTS^●+ radical cation at 734nm using methanol as a blank, on UV- visible spectrophotometer (Thermo Fisher Scientific). A standard solution of ascorbic acid was also prepared and tested at a range of 2 to 10mg/ml in methanol (HPLC grade, HiMedia). The percentage inhibition was calculated using the formula:
$$\mathit{\text{Scavenging activity}}\left(\%\right)=\left[\mathit{Ac}-\mathit{As}\right)/\mathit{Ac}]100$$
Where, Ac and As is the absorbance of control and sample, respectively. The result was compared with control which was prepared by adding 1.0ml of methanol in place of the sample.