Biological estimation of oestrogenic activity in red clover (Trifolium pratense): relative potencies of parts of plant and changes with storage

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Abstract
Summary: Immature ovariectomized mice were used to compare the oestrogenic activities of different parts of the same clover plants; to estimate the relative potencies of biochanin A, genistein and diethylstilboestrol; and to compare the effects of different methods of storage on the oestrogenic potency of red clover leaf and petiole. Test materials were incorporated in the diet fed to the mice and the uterine weight response was used to measure oestrogenic activity.With material harvested at four different times, the leaf or leaf and petiole had the highest activity; small and large stem fractions of the plant were both relatively less active, and blossom and seed head were inactive.Comparison of successive estimates of the relative potency of isoflavones and diethylstilboestrol indicated that the relative responsiveness to the two types of oestrogen did not remain constant. Thus comparisons of estimates of oestrogenic activity of plant material, obtained in terms of diethylstilboestrol in different experiments, could be invalid.The potencies of alcohol, acetone and freeze-dried preparations were compared after storage for 18, 115, 212 and 230 days. Of the procedures tested, the most satisfactory was that in which fresh material was placed in 95% alcohol and the liquor and residue dried and stored at room temperature over concentrated sulphuric acid at reduced pressure.