Patent GB-435830-A (GB)
Title: A process for purifying the hormones of the corpus luteum
IPC: A61K31/56
Filed: 1934-09-05
Abstract: The hormones of the corpus luteum are obtained in crystalline form by causing crude preparations thereof to react with reagents for the carbonyl group, separating the reaction products, and decomposing them to liberate the hormones.  Reagents specified are bisulphite, hydroxylamine or hydrazine or derivatives thereof, such as semi-carbazide and phenyl-hydrazine sulphonic acid. According to examples; (1) an extract of corpus luteum prepared according to Specification 426,166, [Group VI], is dissolved in alcohol and treated with an aqueous solution of semi-carbazide hydrochloride and sodium acetate, the crude precipitate is washed with alcohol, dissolved in glacial acetic acid, and heated with dilute sulphuric acid, and the reaction mixture extracted with ether to yield the hormone; (2) a corpus luteum preparation is reacted in alcoholic solution with hydroxylamine hydrochloride and sodium acetate, and the oil which separates is washed with water, dissolved in hot methyl alcohol, and water is added to precipitate the oxime which is then boiled with hydrochloric acid to liberate the hormone; (3) an ether solution of a hormone preparation is shaken with an aqueous solution of sodium bisulphite and the aqueous layer is separated and heated with sulphuric acid and the released hormone extracted with ether; (4) a preparation of corpus luteum is dissolved in ethyl acetate, the active constituents are adsorbed on animal charcoal from this solution, extracted from the adsorbate with a mixture of methyl alcohol and chloroform, and boiled with an alcoholic solution of phenyl-hydrazine-4-sulphonic acid; the reaction mixture is distilled to remove the alcohol, extracted with ether to remove inactive products, and the aqueous portion treated with sulphuric acid and extracted with ether to obtain the liberated hormone. Specification 391,686, [Group VI], also is referred to.  The Specification as open to inspection under Sect. 91 states that the crystalline hormones are diketones and may be further purified by recrystallization, by further reaction with a carbonyl reagent such as hydroxylamine, or by sublimation in a high vacuum; refers to recrystallization of the oxime obtained according to example 1, whereby a dioxime of m.p. 240 DEG  C. is obtained which can be separated by further recrystallization into two dioximes of higher melting points leaving a mother liquor which on saponification yields an oxyketone; and includes an additional example according to which an extract of corpus luteum is dissolved in methyl alcohol and boiled with phenyl hydrazine or para-nitrophenyl hydrazine and glacial acetic acid, and the reactive product diluted with water to precipitate a crude hydrazone which is recrystallized from ethyl-or methyl-alcohol and then decomposed with dilute acid.  This subject matter does not appear in the Specification as accepted.ALSO:A preparation of corpus luteum hormone dissolved in ethyl acetate is treated with animal charcoal to adsorb the active constituents which are then extracted from the adsorbate by means of a mixture of methyl alcohol and chloroform.