2.6.2. Metal Chelating Activity

The metal chelating activity was examined by measuring the ability of the compound to compete with ferrozine for Fe2+, complex of which can be quantified spectrophotometrically. Metal chelating activity was measured by the method previously described by Adjimani and Asare with minor modifications [20]. Assay measures the reduction in the color intensity as a result of disruption of ferrous ion and ferrozine complexes. Briefly, varying concentration of extract was added to 0.15 mL of 2 mM FeCl2. The reaction was initiated with the addition of 5 mM ferrozine (Sigma), followed by the incubation at the room temperature for 10 min. The absorbance was measured at 562 nm. The percentage of inhibition was calculated using the following equation:

Metal chelating activity (%) = [(Ao − A1)/Ao] × 100,

where Ao is the absorbance of control and A1 is the absorbance of the sample. EDTA was used as a positive control.

#### 2.6.3. 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Radical Scavenging Activity (ABTS)

ABTS (Sigma) scavenging activity is based upon the reduction of ABTS\* radicals by compounds having lower redox potential than that of ABTS. The 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay was carried out according to the method developed by Ser et al. [21]. Initially, ABTS stock solution (7 mM) was mixed with potassium persulfate (2.45 Mm, Himedia) to form ABTS cation complex for 12 h. The ABTS complex solution was added to varying concentrations of the extract preloaded in a 96-well microplate. The reaction was kept for incubation at room temperature for 20 min and the absorbance was measured at 734 nm. The percentage scavenging activity was calculated using the following formula:

$$\text{ABTS radical scanning activity (\%)} = \left[ (\text{A}\_{\text{o}} - \text{A}\_{\text{l}}) / \text{A}\_{\text{o}} \right] \times 100\_{\text{c}}$$

#### *Microorganisms* **2018**, *6*, 72

where Ao is the absorbance of control and A1 is the absorbance of the sample. Trolox (Sigma) was used as a positive control.
