Cockscomb hydrolysate was found to have neurite outgrowth-promoting activity in PC12 cells. To investigate the neurite outgrowth-promoting compounds derived from cockscomb hydrolysate, bioassay-guided purification was carried out. Purified active fractions were obtained by liquid-liquid partition, followed by column chromatography. High-performance liquid chromatography and proton nuclear magnetic resonance analyses of the purified active fractions clarified that the main compounds are threonine, alanine, valine, and methionine. By screening for 20 kinds of amino acids, it was shown that valine and methionine, but not threonine and alanine, have neurite outgrowth-promoting activity. The results of activity evaluation of the mixture of amino acids indicated that alanine enhanced the activity of valine and that the mixture of valine and methionine showed a higher ratio of neurite formation than did each of them alone. On the other hand, dipeptides formed by valine and methionine showed weak neurite outgrowth-promoting activity. A mixture of threonine, alanine, valine, and methionine at the same concentrations as those in cockscomb hydrolysate showed neurite outgrowth-promoting activity comparable to that of cockscomb hydrolysate although threonine, alanine, valine, and methionine alone did not show activity at their concentrations in cockscomb hydrolysate. Therefore, the strong neurite outgrowth-promoting activity of cockscomb hydrolysate was considered to be due to the synergistic effect of threonine, alanine, valine, and methionine.
Keywords: NGF; PC12 cells; amino acid; cockscomb hydrolysate; dibutyryl cyclic AMP.