β-Carotene bleaching assay

The antioxidant capacity of M. latifolia fractions and isolated constituents by β-carotene bleaching was performed according to a method described by Kassim et al. (2019). In brief, 210 µL of β-carotene solution (1 mg/mL in chloroform) was added to Tween 20 (42 µL) and linoleic acid (5 µL) in a round bottom flask. The chloroform was removed by rotary evaporation prior to the addition of 10 µL of distilled water and shaken to form an emulsion. The emulsion (200 µL) was then transferred into each well of a 96-well microplate that contains 50 µL of samples (1 mg/mL). The mixture was incubated at 50 °C in the dark for 2 h, and its absorbance was measured at 470 nm at the initial time (t = 0) and every subsequent 30 min for 2 h (t = 2). Ascorbic acid, α-tocopherol, and BHT were used as positive controls. Methanol was used to replace the test samples as a negative control. The antioxidant activity (AA) was calculated according to the formula (5):

whereby A_t=0 and A_t=2 are the absorbance of the samples measured at 0 and 2 h, respectively; A_c=0 and A_c=2 are the absorbance of negative control measured at 0 and 2 h, respectively.