Solvent Fractionation of W. somnifera Extract

The solvent fractions were obtained using chloroform, n-butanol, and distilled water in increasing order of their polarity. The fractionation was done according to the method described by Mengie et al. [39 (link)]. Eighty gram (80 g) of 80% methanol crude extract of W. somnifera was dissolved in 400 ml of distilled water in a separator funnel. An equal volume of chloroform was added to the aqueous solution, and they were mixed well by gentle agitation. Then, the mixture was allowed to form a distinct layer (the chloroform at the bottom and the aqueous solution at the top). After 24 h, the chloroform fraction was isolated from the mixture, and the process was repeated twice with fresh chloroform. All the chloroform fractions were collected together and subjected to evaporation via a Rotary Evaporator (Yamato, Japan), which was set at 40°C to obtain the chloroform fraction. The remaining aqueous residue was further fractionated with 400 ml of n-butanol in the same manner as chloroform. The remaining aqueous residue after removal of n-butanol was frozen in a deep freezer overnight. Then, the frozen residue was dried with a lyophilizer (Operon, Korea Vacuum Limited, Korea). The three fractions were labeled and kept in the deep freezer with airtight containers in the refrigerator at −20°C until they were used in the antidiarrheal test.