Purification of some active ingredients of alcoholic extract of anise seed (Pimpinella anisum) and assessment their estrogenic effect on some physiological traits of immature female mice

Abstract

This experiment was conducted in order to purify some active ingredients of 70% alcoholic extract of aniseed by adsorption chromatography on silica gel and gel filtration on sepharose CL-6B column, using phosphate buffer (0.1M, pH 7.0) as eluent, as well as gel filtration on sephadex LH-20 where chloroform : methanol (3:1) was used as an eluent. Thin layer chromatography on silica gel was used as a criterion for the identification of purified fractions. Ultra violet transilluminator as well as vanillin-H2SO4 reagent were used to identify and develop of separated fractions, respectively. However, the fractions which purified on sephadex LH-20, showed an Rf value (0.71) which corresponded to the Rf values of anethole. To assess the estrogenic activity of purified fractions a dose of 150 mg/kg of body weight of collected fractions were given to immature female mice at three weeks age and administered orally for four weeks period. Twenty immature female mice were divided randomly into five groups. First group received crude extract of aniseed, while the second, third, and fourth groups received fractions purified on silica gel, sepharose CL-6B and sephadex LH-20 columns, respectively. Group five served as control, such treatments were continued until maturity, and then the animals were sacrified for studying weight changes in ovaries and uterus. The results of this study revealed that there is a significant increment (P<0.01) in the weights of ovaries in the treated groups as compared to the control, while fractions obtained from sepharose CL-6B had no significant effect (P>0.01). Also there is a significant increment (P<0.01) in the weights of uterus in the treated group with respect to fractions obtained from sephadex LH- 20 as compared to other treated groups and control.