2.2. Flavonoid Extraction

Flavonoids were extracted using Soxhlet extractor (Sigma-Aldrich, St Louis, MO, USA) from B. lycium leaves in 80% methanol for 24 h. Methanol was subsequently evaporated by vacuum. The aqueous fraction was extracted with petroleum ether (40–60 °C), ethyl acetate, and diethyl ether [42] (Figure S1). Free flavonoids (FF) are aglycones, while in naturally occurring conjugated flavonoids (CF), most commonly as glycosylated and methylated derivatives, the fatty acid carbon chain is linked onto the primary –OH group on the glucose moiety of the flavonoids. Flavonoids screening was performed using Shinoda’s Test optimized by Mir et al. (2013) and Inalegwu and Sodipo (2013) [43,44]. FF and CF were dried using a rotary evaporator (Buchi Rotavapor R-200 system, Marshal Scientific, Cambridge, US).