Total phenolic content analysisThe total phenolic content of Shiikuwasha juice was examinedusing the Folin–Ciocalteu method.12 Briefly, various concentrationsof sample (20 μL), distilled water (60 μL), and Folin–Ciocalteureagent (15 μL, previously diluted twofold with distilled water)were transferred to a 96-well microplate (Nunc, Roskilde, Denmark)and mixed well. The mixture was allowed to react at roomtemperature for 5 min, and then 75 μL sodium carbonate (2%) wasadded to the well. The microplate was immediately placed in amicroplate reader (PowerWave XS2, BioTek, Winooski, VT, USA),agitated, and then allowed to stand for 15min until stable absorptionvalues were obtained. The absorbance was then measured at750 nm. Total phenolic content was calculated from a linear calibrationcurve of gallic acid and expressed as milligrams of gallicacid equivalents (GAE) per liter of juice. All assays were performedin triplicate.