1.4.2. Determination of total saponins
20 g of P. glandulosus leaves was taken in conical flask and 20% aqueous ethanol (100 ml) was added. The mixture was heated at about 55°C for 4 h over a hot water bath with continuous stirring. The mixture was filtered and the residue re-extracted with another 200 ml of 20% ethanol. The extracts were combined and reduced to 40 ml. The concentrate was transferred to a separating funnel and 20 ml of diethyl ether was added and shaken vigorously. Ether layer was discarded and aqueous layer was further purified. Concentrate was then fractionated with 60 ml n-butanol repeatedly for three times. Butanol fractions were combined and washed twice with 10 ml of 5% aqueous sodium chloride. Purified butanol fractions were evaporated to dryness in the oven to a constant weight. Saponin content was calculated as percentage (Aliet al., 2019 ; Obdoni et al .,2001).