2.6. In Vitro Antioxidant Activity

Free radical scavenging of MELHS was conducted according to the method described by Barca et al. [25]. About 3 mL of 0.004% DPPH solution was added with various concentrations (15.625 to 500 µg/mL) of crude extract, while a methanol plus DPPH solution was used as a negative control. The mixed solutions were then incubated for half an hour at a 30 °C temperature in a darkened room. A UV spectrophotometer was used to measure the absorbance at 517 nm. The reduction of absorbance with a high concentration implies effective radical scavenging activity. As a reference standard, ascorbic acid was applied [26]. The inhibition percentage of free radicals was measured by the following Equation (1):

where Ac is the absorbance of the control and As is the absorbance of the sample.

Reduction of the power capacity of MELHS was evaluated by following the method of Oyaizu [27]. About 1 mL of serially diluted concentration (31.25 to 500 µg/mL) was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and potassium ferricyanide (1% w/v). The mixed solution was incubated for 20 min at a 50 °C temperature to complete the reaction. After incubation, 2.5 mL of trichloroacetic acid (10%) was added and the whole mixture was centrifuged for 10 min at 3000 RPM. After that, the supernatant solution was dispelled and 0.5 mL of ferric chloride (0.1% w/v) was added to the solution with 2.5 mL of distilled water gradually mixed in. Therefore, the absorbance of the mixer was investigated on a UV spectrophotometer at 700 nm. A phosphate buffer was used as a blank solution, while ascorbic acid was used as a reference standard.

The total phenolic content (TPC) of MELHS was investigated by the method of Singleton et al. as an oxidizing agent, and Folin–Ciocalteu reagent (FCR) was applied [28]. Two and a half milliliters of sodium carbonate (20%) was mixed with 2.5 mL of FCR, which was diluted 10 times with water. Then, a 500 µg/mL extract was combined with the mixture. After that, distilled water was added to the mixed solution to make up a 10 mL solution and kept in incubation for 20 min at a 25 °C temperature to react effectively. The absorbance of the sample was taken at 760 nm on the UV spectrophotometer. TPC was calculated from a calibration curve using a standard gallic acid solution of several concentrations, whereas the absorbance value plotted against concentrations and the results was assessed in mg gallic acid equivalent concentrations.

The total flavonoid content of MELHS was determined by the colorimetric method described by Chang et al., whereas quercetin was used as a reference standard [29]. About 100 μL of aluminum chloride (10%) and 1.5 mL of methanol were mixed with a 500 µg/mL extract solution. Afterward, 100 μL of potassium acetate (1 M) and 2.8 mL of distilled water were mixed into the solution. The mixed solution was incubated for half an hour at room temperature (37 °C) so that the mixture reaction could be completed. A UV spectrophotometer was used to measure the absorbance of the mixed solution at 415 nm against the blank solution, which contained all the reagents except the extract. The calibration curve was developed by employing several quercetin concentrations and the total flavonoid content was assessed in mg/g of quercetin equivalent.