Patent GB-430571-A (GB)
Title: Process for the production of a yellow oxidation ferment and a coloured component derived therefrom
IPC: A61K38/43
Filed: 1933-12-15
Abstract: A yellow oxidation enzyme is obtained by forming a substantially neutral aqueous extract of animal or vegetable cell material such as yeast at a low temperature, adding organic solvents miscible with water such as acetone, methanol or ethanol so as to precipitate impurities, and then adding more of the same solvents to precipitate the enzyme.  The precipitate is dissolved in water and precipitated by adding more solvent while saturating with carbon dioxide and reprecipitating from aqueous solution by more of the solvents.  The enzyme can be decomposed by adding solvents such as methanol, ethanol or acetone alone or admixed with water, at sufficient temperature, an insoluble protein and a soluble coloured component being produced. The coloured component may be obtained in crystalline form by rendering its aqueous solution alkaline and exposing to irradiation by a metal-filament lamp, or by allowing the alkaline solution to stand or by heating it, whereby in each case the coloured component is modified so that it can be extracted with chloroform or other water-immiscible solvent and crystallized therefrom.  Alternatively the same coloured component may be obtained direct from the animal or vegetable cell material by treating with the water-miscible solvents under such conditions that the ferment is decomposed.  In p  this modification acids or acid salts may be added to the extracting agent, and when these are added, the extracting agent may be water. The enzyme and the coloured component derived from it are useful as antineuritic vitamins.  In examples: (1) an aqueous solution obtained from bottom yeast and purified by adding lead acetate, octyl alcohol and soluble phosphate is treated with half its volume of acetone and kept at 0 DEG  C. for 24 hours; the solution is filtered and more acetone is added to the filtrate, the precipitated enzyme being treated as described above; (2) the enzyme is treated with a mixture of 1 volume water and 3 volumes methanol at 38 DEG  C., the precipitated protein is removed and the coloured component obtained by evaporation; (3) the coloured component is dissolved in \baN/2\be soda and the solution irradiated as described above, the coloured component being then extracted with chloroform and crystallized; (4) a solution of the coloured component obtained in example 2 is made alkaline so as to be equal to \baN/2\be to N soda and is allowed to stand for 48 hours when it is acidified, extracted with chloroform and crystallized; (5) bottom yeast is shaken for 72 hours at 38 DEG C. with a mixture of 1 volume water and 3 volumes methanol, undesired constituents being removed from the extract by chloroform; the solution is then made alkaline, irradiated and then acidified, extracted with chloroform, and the extract crystallized; the initial extraction of the yeast may be facilitated by adding an acid such as hydrochloric, sulphuric or phosphoric acid or an acid salt such as sodium bisulphate so as to give an acidity of about 5 per cent free acid; instead of aqueous methanol, aqueous ethanol or acetone may be used, or aqueous ethanol containing a small quantity of a water-immiscible solvent such as ether or ethyl acetate; (6) yeast is treated with 10 per cent hydrochloric acid at 30--60 DEG  C. and (7) with trichloracetic acid at room temperature and in each case the coloured component is obtained from the solution as described above. Specification 337,405, [Class 2 (iii)], is referred to.