Oestrogenic Activity of Subterranean Clover 2. THE ISOLATION OF GENISTEIN FROM SUBTERRANEAN CLOVER AND METHODS OF QUANTITATIVE ESTIMATION By D. H. CURNOW*

The isolation of small amounts of genistein (5:7:4'trihydroxy-isoflavone) from subterranean clover (Trifolium subterraneum var. Dwalganup) and the demonstration of its weakly oestrogenic properties (Bradbury & White, 1951, and Biggers & Curnow, 1954), together with the difficulties in the synthesis of this compound, made it important that a more efficient method be found for the isolation of this isoflavone from the clover. Also, the amount of genistein isolated by Bradbury & White (2 mg./ 100 g. of fresh clover) and its low oestrogenic activity (Biggers & Curnow, 1954) were not nearly sufficient to account for the activity of the fresh plant (Robinson, 1949) and left considerable doubt as to whether this compound was responsible for the oestrogenic activity of the clover. The present study has shown, however, that the clover contains much more genistein than found by these workers. The amount now found indicates that genistein could indeed be responsible for the activity of the plant and therefore for the infertility in sheep grazing on subterranean-clover pastures. This finding called for rapid and efficient methods of estimation of this compound in the plant. For the determination of the genistein content of the clover at various stages of growth, under various nutritional conditions, in different seasons and in various strains, an accurate method of estimation was required, whereas for the purposes of the plant geneticist a rapid method was desired for use with small amounts of the material (parts of a single plant), in which great accuracy is a secondary consideration. Methods fulfilling these two sets of requirements have been developed and are presented here.