Patent GB-810204-A (GB)
Title: Improvements in or relating to a method of extracting and purifying a pancreatic hormone acting on the respiratory centres
IPC: A61K35/38
Filed: 1955-06-15
Abstract: A method of extracting and purifying a pancreatic hormone which acts on the respiratory centres, comprises three steps, the first being the extraction of the hormone, insulin and vagotonin from ox p pancreas, the second, the elimination of vagotonin and a part of the insulin, and the third, the elimination of the remainder of the insulin and other glycohemiant substances to yield the required hormone.  The first step may be performed by one of three methods: (i) mincing and steeping fresh pancreas in 1.5 litres of 95 per cent ethyl alcohol acidified with hydrochloric acid to pH 3 to 4 per kilogramme pancreas, adding 1 to 4 c.c. 50 per cent sodium acetate solution per litre of the extract at pH 5, filtering the flakey precipitate formed and concentrating in a vacuum at a temperature of less than 20 DEG  C. to one-fifteenth of the original volume prior to cooling to 0 DEG  C. and addition of one-tenth volume of chloroform. The upper layer of the liquid is syphoned off, saturated with sodium chloride at pH 2.5 and the separated precipitate dehydrated in absolute alcohol and dried in a vacuum; (ii) powdered pancreas, removal of fats using trichlorethylene, is stabilized with acetate and steeped in water at 2 DEG  C. and pH 5.  A 50 per cent solution of sodium acetate at pH 5 is added to the separated liquid and the flakey precipitated separated.  The liquid is saturated with sodium chloride at pH 2.5 and the precipitate is separated, dehydrated with absolute alcohol and dried in vacuum; (iii) the mother liquor after the separation of insulin at its isoelectric point from a pancreatic extract, is saturated with sodium chloride at pH 2.5, the precipitate separated, dehydrated in absolute alcohol and dried in a vacuum. Step two consists of adjusting a solution of the crude extract obtained by step one, to pH about 6 by addition of sodium hydroxide or hydrochloric acid, dializing the solution, and adding 20 per cent zinc chloride solution, at 15 DEG  C. to precipitate a vagotonin-zinc complex without loss of the required hormone.  The separated solution is adjusted to pH 2.6, saturated with sodium chloride and the separated precipitate dissolved in water at pH 6 prior to dialysis at 15 DEG  C.  The dialysate is adjusted to pH 2.6, prior to dialysis at 15 DEG  C.  The dialysate is adjusted to pH 2.6, any clouding or flaking at pH 4.7 to 5 being removed, prior to addition of half volumes of 50 per cent lithium chloride solution.  The separated precipitate is dissolved in 70 per cent alcohol at pH 6 and poured into absolute alcohol at 0 DEG  C. The precipitate is dried in vacuum.  The third step consists of dialyzing an aqueous solution of the precipitate at pH 6, careful addition of ethyl alcohol to a concentration of 15 to 20 per cent at pH 4.7 and - 5 DEG  C.  The separated liquid, at pH 7, is poured into absolute alcohol at - 5 DEG C. and the precipitate dehydrated in absolute alcohol and dried in a vacuum.  A 50 per cent lithium chloride solution is added to an aqueous solution of the precipitate at pH 3.45 and 0 DEG  C. and the separated liquid adjusted to pH 2.7, the precipitate dissolved in 60 per cent alcohol and absolute alcohol added.  The dried precipitate obtained is dissolved in dilute potassium ferrocyanide solution at pH 3.8 to 4, the separated solution, at 0 DEG  C., is adjusted to pH 2.8 and any precipitate separated.  A 50 per cent solution of lithium chloride is added and the precipitate dissolved in 60 per cent alcohol at pH 7, the solution poured into absolute alcohol and the precipitate dehydrated by absolute alcohol and dried in a vacuum.