1.3.4. Fractionation
The hydro ethanolic crude extract (190g) was fractioned in hexane, ethyl acetate, n-butanol in increasing order of polarity. The hydro ethanolic extract was initially mixed with 500 ml of distilled water and 450 ml of hexane then transferred into a separating funnel. After shaking and decantation, the separation of two phases was obtained. The upper organic phase which represents the hexane fraction was transferred in to an erlenmeyer. The lower organic phase was extracted many times by adding fresh solvent (hexane) until a clear phase was obtained. The hexane fractions was then pooled and stored as such. The lower phase was mixed with subsequent solvents (ethyl acetate and then n-butanol) and they followed same treatment as hexane fraction. At the end of the fractionation the residue was called residual fraction. The four fractions were concentrated and dried.