Patent GB-637309-A (GB)
Title: Improvements in or relating to the production of substances which reduce the bleeding time of animals
IPC: A61K35/28
Filed: 1947-05-13
Abstract: A preparation which reduces the bleeding time of animals is obtained by forming an aqueous extract from spleen material or soya bean flour, precipitating the enzymes therefrom, incubating the enzymes with an ascorbic acid substance having vitamin C activity, and recovering the product from the solution.  The product is designated Splenin A and has the empirical formula C18H40O5 or C18H36O5.  The precipitation of the enzymes from the aqueous extract may be effected by the addition of acetone or alcohol.  The precipitate may then be added to a solution of l-ascorbic acid in a phosphate or borate buffer solution at pH 8, the mixture being incubated at 37-38 DEG  C. for about 20 days.  The incubated material is then extracted with ether or chloroform at a temperature not greater than 50 DEG  C., the aqueous residue being subjected to further incubation and the non-aqueous phase being shaken twice with aqueous 2.5 per cent sodium bicarbonate.  The non-aqueous phase is then shaken with 0.5 N-sodium bicarbonate, the aqueous phase now containing the Splenin A being acidified to pH 4.7 by sulphuric acid.  The acid solution is extracted with chloroform, and the extract is dried by means of anhydrous sodium sulphate and is then evaporated to dryness.  The residue is taken up with heavy petroleum ether, the solution is shaken with methanol, and the petroleum ether solution is evaporated to dryness.  The residue is re-dissolved in boiling acetone and the acetone is allowed to come to room temperature, whereupon the Splenin A is precipitated.  The incubation step is activated by the presence of small quantities of cysteine or sodium cyanide and in this step dehydroascorbic acid, diketogluconic and or the sodium, calcium, or other salts of ascorbic acid may be used instead of l-ascorbic acid. The chloroform purification step prior to the bicarbonate treatment referred to above may be replaced by adsorption of the Splenin A on charcoal followed by treatment with hot methanol to recover the material.ALSO:A preparation which reduces the bleeding time of animals is obtained by forming an aqueous extract from spleen material or soya bean flour, precipitating the enzymes therefrom, incubating the enzymes with an ascorbic acid substance having vitamin C activity, and recovering the product from the solution.  The product is designated Splenin A and has the empirical formula C18H40O5 or C18H36O5.  The precipitation of the enzymes from the aqueous extract may be effected by the addition of acetone or alcohol.  The precipitate may then be added to a solution of l-ascorbic acid in a phosphate or borate buffer solution at pH 8, the mixture being incubated at 37-38 DEG C for about 20 days.  The incubated material is then extracted with ether or chloroform at a temperature not greater than 50 DEG C, the aqueous residue being subjected to further incubation and the non-aqueous phase being shaken twice with aqueous 2.5 per cent sodium bicarbonate.  The non-aqueous phase is then shaken with 0.5 N sodium bicarbonate, the aqueous phase now containing the Splenin A being acidified to pH 4.7 by sulphuric acid.  The acid solution is extracted with chloroform, and the extract is dried by means of anhydrous sodium sulphate and is then evaporated to dryness.  The residue is taken up with heavy petroleum ether, the solution is shaken with methanol, and the petroleum ether solution is evaporated to dryness.  The residue is re-dissolved in boiling acetone and the acetone is allowed to come to room temperature, whereupon the Splenin A is precipitated.  The incubation step is activated by the presence of small quantities of cysteine or sodium cyanide and in this step dehydroascorbic acid, diketogluconic and or the sodium, calcium, or other salts of ascorbic acid may be used instead of l-ascorbic acid.  The chloroform purification step prior to the bicarbonate treatment referred to above may be replaced by adsorption of the Splenin A on charcoal followed by treatment with hot methanol to recover the material.