VITAMIN B (5i) 27 



duction of the term vitamin and helped to crystalHze the conception 

 for which the term stands. 



The method of extraction first adopted by Funk (1911) was a modification of 

 that used by Fraser and Stanton, based on the customary procedure for isolating 

 simple natural bases. 



On account of the small amount of curative substance present, 54 kilograms of 

 rice polishings was used. This was extracted in separate portions of 1500 grams 

 each with 4 liters of alcohol containing gaseous hydrochloric acid to the extent 

 of from two to five per cent, the extraction being conducted in a shaking machine 

 and the liquid afterwards filtered on a Biichner funnel, the residue pressed out_ in 

 an hydraulic press and the liquid added to the original filtrate. On evaporation 

 in vacuo at 38° C. a fat-like residue was obtained which melted at 50° C. This 

 was heated with water on a water bath, and filtered at 38° -40° C. The filtrate 

 formed two layers which were separated and the aqueous layer extracted three 

 times with ether to remove fatty substances. The resulting aqueous solution, 

 which is described as containing no proteins and showing no reaction for artiino 

 acids when subjected to the Millon, glyoxylic acid, bromine, xanthoproteic, diazo 

 and diacetyl tests, was found to be eflfective in curing polyneuritic pigeons in 

 doses corresponding to 20 grams of the original rice. On adding to such an 

 aqueous extract sulfuric acid to five per cent concentration, followed by phospho- 

 tungstic acid, the active substance came down in the precipitate while the filtrate, 

 although containing nitrogen, proved inactive. The precipitate was washed with 

 5 per cent sulfuric acid, ground with barium hydroxide in a mortar, water 

 added, and the mixture shaken for three hours, then filtered and the precipitated 

 barium phosphotungstate washed with water. The filtrate, after removal of 

 ammonia by aeration and of excess barium by precipitation with sulfuric acid 

 and filtration, was neutralized with hydrochloric acid and evaporated^ in vacuo at 

 room temperature. The alcoholic extract of this residue in doses equivalent to 40 

 grams of the rice polishings was an eflfective cure for polyneuritic pigeons. 



On treating this extract with alcoholic mercuric chloride and allowing the 

 solution to stand in the cold for some time a crystalline precipitate (I) was 

 obtained which was soluble in water and recrystallized from the water solution in 

 needle-like crystals consisting largely of choline but containing some of the active 

 substance. At this point the active substance was also found in the alcoholic filtrate 

 from the mercuric chloride precipitate (III), and in the aqueous filtrate after 

 recrystallization (II). These filtrates were treated separately as follows: The 

 mercury was removed from the aqueous filtrate (II) by hydrogen sulfide, the 

 filtrate evaporated, the residue taken up by alcohol, the choline removed by 

 precipitation with platinic chloride and filtration, and the filtrate finally treated 

 with phosphotungstic acid. A crystalline precipitate was obtained which showed 

 activity on decomposition of the phosphotungstate with barium hydroxide and 

 carbon dioxide. The alcoholic filtrate (III) was evaporated, the residue taken up 

 in water, the mercury removed by hydrogen sulfide and the filtrate freed from 

 chloride by silver sulfate, the silver by hydrogen sulfide, and the sulfuric acid by 

 barium hydroxide. The filtrate thus purified was then acidified with nitric acid 

 and treated with silver nitrate and barium hydroxide according to the methods 

 used in isolating histidine and like bases as described, e.g., by Barger (1914), 

 the silver nitrate being added until a drop of the solution no longer gave a white 

 precipitate with ammoniacal silver nitrate. The precipitate thus obtained was 

 washed thoroughly with nitric acid, decomposed with hydrogen sulfide, freed from 

 the last traces of barium hydroxide with very dilute sulfuric acid and concen- 

 trated in vacuo. On evaporating with alcohol in a desiccator the active extract 

 thus prepared, a small yield (0.4 gram from 50 kilograms of the rice polishings) 

 of microscopic needle-like crystals was obtained which in doses of about 0.02 gram 

 eflfected a rapid cure in polyneuritic pigeons. These crystals melted at 233° C, 

 were diflficultly soluble in cold water and alcohol, more soluble in hot water, and 

 were free from chlorine, ash. and sulfuric acid. A single analysis of the substance 

 gave the percentage composition : C, 55.63 ; H, 5.29 ; N, 7.68 per cent, figures which 

 were thought to correspond best to the formula C17H20O7N2. 



