*2.8. Bioautography-Guided Fractionation*

The exocarp dichloromethane fraction (EDF) that exhibited strong antioxidant activities for most assays was further fractionated by column chromatography. A glass column (32 × 1000 mm) packed with silica gel 60 (70–230 mesh, 63–200 μm) was loaded with the fraction at a ratio of 130:1. The column was eluted with binary solvent systems of increasing polarity starting with 200 mL of DCM/hexane (8:2, *v*/*v*) up to 100% DCM at 5% increments to afford five subfractions (Di-1 to Di-5), then DCM/ethyl acetate (EA) (99:1, *v*/*v*) up to the volume ratio of 7:3 at 1%, 2% and 5% increments of EA successively to yield fifteen subfractions (Di-6 to Di-20). Next, DCM/acetone (95:5, *v*/*v* up to 8:2, *v*/*v*) at 5% increments of acetone, yielding two subfractions (Di-21 and Di-22). Finally, DCM/methanol (99:1, *v*/*v*) at 1% and 2% increments of methanol up to 8:2 and 9:1 ratios, respectively, giving four subfractions (Di-23 to Di-26). Antioxidant subfractions were monitored by TLC bioautographic screening using 0.4 mM DPPH in methanol as the spraying reagen<sup>t</sup> and the DPPH radical scavenging activity was evaluated.
