Rice Polishings Extraction of the Antineuritic Substance
CHARLES A. COOK AND RICHARD H. CARROLL Experimental Research Laboratories, Burroughs Wellcome and Company, Tuckahoe, N. Y.
T
maximal yield and potency of vitamin from the dilute extracts. When assayed by the rat growth method, the preparations, employed as a substandard, were equivalent to the International Standard material and contained 100 I. U. of vitamin per gram. Such adsorption products contained a large proportion (75 to 80 per cent) of insoluble ash. The nature of the vitamin-silicate combination, as influenced by the conditions for adsorption, determined the efficiency of the subsequent elutions. The precise amount of adsorbent required for the clear vitamin extract (5 per cent extractives and solution a t pH 4.5) controlled the adsorption obtained by stirring for 24 hours (22" C.) or by percolation. ALKALINE ELUATES of the adsorption products were made following the older technics of Seidell (IO). Sodium hydroxide (0.5 M ) was used to extract a quantity (20,000 I. U.) of the standardized adsorbate at 20" C., and, after centrifuging, the clear solution was neutralized with hydrochloric acid. The extract represented a 60 per cent yield of the vitamin formerly combined with the silicate. When fed to rats, the dry eluate contained 220 I. U. per gram but such products consisted of 85 to 90 per cent ash. Lithium hydroxide was employed for similar elutions, and the lithium chloride, formed in neutralizing the alkali, was removed by extraction with butyl alcohol. Lithium hydroxide (0.25 M ) extracted the vitamin from a quantity of the adsorption product (40,000 I. U.) a t 10" C., and the clarified solution from the centrifuge was acidified to pH 4 with hydrochloric acid. On evaporation a t 30" C., a dry but hygroscopic preparation was obtained. This powder was extracted with anhydrous butyl alcohol; after centrifuging, the precipitate Experimental Procedure (vitamin fraction) was treated with pentane or ethyl ether to remove the residual alcohol. Losses of the vitamin in the ADSORPTION PRODUCTS were prepared, according to the butyl alcohol were negligible and within the limits of biological method for the International Standard of vitamin BI, from assay even on repeated extractions and with a fifteen fold 50 to 100 kg. of rice polishings.' The absorbates, on comvolume of the anhydrous parative b i o a s s a y , consolvent. The final product tained up to 98 per cent contained less than one-fifth of the a n t i n e u r i t i c sub(19.02 per cent sulfated ash, stance in the p o l i s h i n g s . Methods which obtained efficient con1 ) of the original ash and Xumerous p r e p a r a t i o n s centration of the vitamin in rice polishestablished that 7.5 mg. of supplied 165 I. U. of vitaf u l l e r ' s earth per Intermin B1 in one gram of the ings were investigated. The comparan a t i o n a l Unit (I. U.) of light brown p o w d e r . A tive potencies and yields of vitamin in vitamin B1 in the extracts low yield (18.3 per cent) of these different concentrates were estabwas the optimal quantity vitamin was obtained in the lished through biological assays. Pyridine of a d s o r b e n t to bind the preparation. O n f u r t h e r solutions and also aliphatic acids were trial, the method obtained 1 Rice polishings (commercial) a soluble preparation (13.15 found useful for the extraction and conf r o m C a l i f o r n i a , Texas, and per cent sulfated ash) with Louisiana were used for the excentration of the antineuritic vitamin. tracts. The vitamin BI potency 135 I. U. of vitamin in one These types of extraction may be apa p p e a f e d t o p a r a l l e l the oil gram and a low yield (19.8 (germ) oontent in the fresh polishplicable to other nitrogenous substances per cent) of the vitamin in ings and varied from 3 t o 7 Intersuch as amino acids. the adsorbate. - national Units per gram.
HIS paper summarizes an investigation on the concentration of the antineuritic vitamin in rice polishings. Of several chemieal procedures, adsorption has proved to be the most selective treatment for segregation of the active substance in dilute extracts. The extraction of the vitamin (B1) from an adsorption product with alcoholic pyridine solutions was found to yield a preparation of high biological potency and avoided the bothersome ash which resulted from the use of metallic alkalies. Similar pyridine mixtures were employed by Kuhn (6)and by Karrer (6) for extraction of the flavins in whey and malt adsorbates. When lithium hydroxide was employed for the extraction of an adsorption product, as by Levene ( 7 ) ,it was convenient to remove the lithium from the concentrated extract by extraction of its chloride with anhydrous butyl alcohol. The vitamin, in the form of a free organic base, was extracted with ether by Block and Cowgill ( 2 ) ; Cowgill (4) also suggested that it may exist in a chemical form which is soluble in fats. Myers and Voegtlin (9) succeeded in extracting the antineuritic substance directly from the autolyzed yeast filtrates with olive oil and oleic acid. The amino group of the vitamin molecule should form salts with organic acids and these salts probably exist in nature. Oleic and caproic acids formed such salts which were soluble in an excess of the respective acids. These aliphatic acids (more than four carbon atoms) were used to extract the vitamin from aqueous solutions.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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EXTRACTS OF ANTINEURITIC VITAMIN TABLEI. PYRIDINE I . U. in Extract
hlg. perI. L .
Per Cent Yield of Vitamin
Per Cent Sulfated .4sh in Extract
3732 7366
3 0 3.0
466 46 0
7 02 8 28
4624
3.9
38.5
2 10
3372
3.5
42 2
9 20
3460
3.0
54.1
5.65
2650
3.9
41.4
9.19
4500
2.0
63.0
7.73
5640
1.1
77.8
12.93
9120
1.9
57.1
11.28
150.836
1.8
75.4
8.15
I. U.a in Fraction R-4044
Adsorbate
R-4054 B-4064
16,000 12,000
B-4094
8,000
B-4144
6,400
B-4154
6,400
B-4046
7,136
B-4056
7.240
B-4134
16,000
B-4418
200,000
8,000
Extraction Mixtuieb 25% pyridine, 25% ethyl a h . . 5 0 % water (25’ C.)
ethyl ale. mixture (2) Aqueous extractc (1) 25% pyridineethyl alc. mixture (2) Abs. methyl alc. ert,rarrt ...... .. (1) 25% pyridineethyl ale. mixture (2) Aqueous extract (1) 25% wridineethyl a h mixture (2) Aqueous extract
a One International Unit = 10 mg. of adsorbate. b The adsorbates were extracted first with a seven-fold vplume of the solvent mixture, centrifuged a t high speed, and then washed thoroughly with a fivefold volume of the alcoholic pyridine. C The alcoholic pyridine extract was converted to a water-soluble fraction.
TABLE 11. OLEICAND CAPROIC ACIDEXTRACTS OF VITAMIN BI Expt. 1 5 7 35-c
I. U. ,in Mater,ala Extractloll Solvent 51,000 47,000 180,000 4,000
42-Cb
28,000
B-3764
25,000
1.0 kg. oleic acid 3.0 kg. oleic acid 27.2 kg. oleic acid satd. with HzS 0.65 kg. caproic acid 1.36 kg. caproic acid 1.50. kg. oleic acid - vitamin hydrolyzate
Per Cent
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