Isolation and chemical characterization of bound niacin (niacinogen) in cereal grains.

The existence of an alkali-labile bound form of niacin, different from other known derivatives of niacin, in cereal grains has already been established (l-6). The concentration of this substance was found to be the highest in the bran among the different parts of the grain (7). Krehl et al. (5, 6) concentrated it from wheat bran to a certain degree and considered from its solubility and dipolar ion behavior that it was a compound of niacin with the nicotinyl radical attached to a substituent, bearing terminal groups, which render the molecule acidic and watersoluble. Chaudhuri and Kodicek (8) obtained a concentrate from wheat bran, which showed a bluish white fluorescence in ultraviolet light changing to greenish yellow on treatment with alkali. A higher concentration of the material was obtained in this laboratory by Chaudhuri (9) from rice bran by solvent fractionation and chromatography on a Decalso column. However, a pure crystalline material was not obtained. In this paper a method has been described for the crystallization of the bound form of niacin, called niacinogen for convenience. The work of separation has been facilitated by the observation that on subjecting a niacinogen concentrate to paper chromatography, with phenol-water (80: 20) as the solvent system, two ninhydrin-sensitive spots moved with the solvent. The slower moving spot was colorless and gave a violet color with ninhydrin, whereas the faster moving one was brownish in color and gave a blackish violet coloration with the same reagent. Further, the faster moving spot gave an orange coloration on exposure to cyanogen bromide vapor followed by spraying with the p-aminobenzoic acid reagent (CNHr-PABA) according to the method of Kodicek and Reddi (10). This test would indicate the presence of either niacin or niacinogen or both. Das and Ghosh (11) had, however, observed that the colored material formed from niacinogen by the CNBr-PAPA reaction was insoluble in amyl alcohol, whereas that formed from niacin was soluble (12). It was now found that the colored material from the faster moving spot was insoluble in amyl alcohol, indicating that this spot was given by niacinogen, free from niacin. The same spot gave a bluish white fluorescence in ultraviolet light, which also indicated the presence of niacinogen (13). Since it also gave a ninhydrin reaction, the presence of an amino acid or peptide moiety in niacinogen was indicated. It has later been shown that niacinogen contains a peptide (16). In the present work crystallization of niacinogen has been effected from rice bran, wheat bran, and whole corn by solvent fractionation and chromatography on a cellulose column, in