VITAMIN B (5i) 29 



helping to restore to normal the degenerated nerve tissues the actual 

 rebuilding of which, he emphasized, must of necessity call for other 

 materials also. His work contributed little so far as methods of isolation 

 are concerned as he used the mercuric chloride precipitation which, as 

 in the case of Funk's early work, left the active substance scattered 

 in three or four fractions. 



In July 1912, Suzuki, Shimamura and Odake published a paper 

 representing the results of four years of chemical investigation of the 

 antineuritic substance of rice bran. 



The method of these Japanese investigators resembled Funk's original procedure 

 through the precipitation of the active substance by phosphotungstic acid. This 

 precipitate was decomposed by trituration with an excess of a solution of barium 

 hydroxide until the thick paste reacted strongly alkaline, or was dissolved in 

 acetone and water and enough of the barium hydroxide added to give a strongly 

 alkaline reaction. After freeing the filtrate from barium by sulfuric acid and 

 evaporating under reduced pressure there remained a slightly acid light brown 

 sirup which, on drying over sulfuric acid, formed a resinous mass to which the 

 name Crude Oryzanine I was given. The yield at this point was 0.4 per cent of 

 the original bran (1.2 grams from 300 grams of bran). 



Crude Oryzanine I proved to be ten times as active as the alcoholic extract. 

 From 0.03 to 0.04 gram, dissolved in a little water and administered either by 

 mouth or subcutaneously, cured polyneuritic pigeons in a day, while half the dose 

 prolonged the life of the pigeon but did not effect a complete cure. It was readily 

 soluble in water and dilute alcohol, forming a slightly acid solution which gave no 

 biuret reaction, a deep red color with Millon's reagent, and a flocculent precipitate 

 with phosphotungstic or phosphomolybdic acid in acid solution, and in concentrated 

 solution was precipitated by lead acetate and ammonia and partially precipitated 

 by mercuric chloride, acetate, or nitrate or by tannic acid. On hydrolysis by dilute 

 acid two acids described as a- and /?- acids were obtained as well as nicotinic acid, 

 and glucose. To the a- acid was ascribed the formula CioHsNOi and to the y8- acid 



CisHeN^Os. 



Precipitation with tannic acid was made the basis of further concentration of 

 the active material. Four grams of Crude Oryzanine I was dissolved in 100 cubic 

 centimeters of water and treated with 20 per cent aqueous tannic acid until a 

 whitish brown flocculent precipitate appeared. This on decomposition and concen- 

 tration, as in the case of the phosphotungstate, yielded a small amount of a light 

 brown sirup "Crude Oryzanine II." The yield was only about 6 per cent of the 

 weight of the Crude Oryzanine I taken for purification, but the antineuritic potency 

 was about three times as high, 0.01 gram proving sufficient to cure a polyneuritic 

 pigeon. Several modifications of this method are described as yielding similar 

 products. 



Picric acid was used in the next and final step of the concentration. After 

 repeated trials to prevent the simultaneous precipitation of nicotinic acid, traces of 

 which still remained in Oryzanine II, it was found that by adding small amounts of 

 the picric acid to Oryzanine II and triturating in the cold, the oryzanine picrate 

 appeared as a yellowish-brown, flocculent precipitate becoming crystalline on 

 standing in the cold, while the nicotinic acid remained in solution. On recrystalliza- 

 tion from acetone solution the picrate was obtained as yellowish brown, small 

 microscopic needles generally in clusters. These were insoluble in ether or 

 petroleum ether, rather difficultly soluble in cold water, and readily soluble in hot 

 water or alcohol. Unfortunately the yield of picrate was so small that attempts to 

 obtain free oryzanine were unsuccessful. 



For protection from polyneuritis, pigeons required 3 grams of bran daily, or 

 0.3 gram of alcoholic extract, or 0.03 gram of Crude Oryzanine I, or 0.01 gram 

 of Crude Oryzanine II, or about 0.005 gram of "Pure Oryzanine." This would 

 imply that the purification process had concentrated the active substance at least 



