The isolation of a-oestradiol and oestrone from horse testes.

ZONDEK [1934] reported the preparation of crude alcoholic eXtracts of horse testes which possessed a marked oestrogenic potency. Extracts obtained in this way were three hundred times as active oestrogenically as those prepared from a similar weight of mares' ovaries. The high oestrogenic potency of horse testes extracts has been confirmed by later workers. The present investigation was undertaken in order to isolate and identify the oestrogens responsible. A crude alcoholic extract from 28 kg. of horse testes was generously placed at the disposal of the author by Dr A. S. Parkes. This extract was concentrated, in vacuo, the acetone-insoluble material was removed and the acetonesoluble fraction was saponified by refluxing with alcoholic NaOH. The greater part of the oestrogenic activity of the inmitial concentrate appeared in the alkalisoluble, saponifiable material. That part carried over into the non-saponifiable fraction was recovered by solvent partition. Following the partitioning of the saponifiable fraction between suitable solvents, the concentrate obtained was dissolved in NaOH and -the pH was adjusted to 9 0. Ether extraction gave a "phenolic" fraction, which on further solvent partition gave 112 mg. of "phenols" possessing practically all the oestrogenic activity of the original "saps". These "phenols" were separated into ketones and non-ketones with Girard's reagent-T. Oestrone was isolated from the ketonic material as the 3:5-dinitrobenzoate (M.P. 188-191° alone or 188-192' when mixed with authentic oestrone 3:5dinitrobenzoate of M.P. 193-1940). The parent substance (M.P. 238-245°) had the same order of biological activity as authentic oestrone. x-Oestradiol was isolated from the non-ketones, following digitonin precipitation-, as its di-oc-naphthoate which melted at 1940 alone or when mixed with authentic a-oestradiol di-a-naphthoate (M.P. 1940).