Isolation And Identification Of Neurotrophic Factors From Carya Cathayensis Sarg.

This study has utilized a neuroblastoma(SH-SY5Y)cell line as an in vitro neuronal model and fresh hickory nut(Carya cathayensis Sarg.)extracts of lipsoluble and water-soluble constituents as test materials to verify neurotrophic activity of hickory nut extracts by the measurement of the cell neurite outgrowth and analysis the expression of correlated genes.By analysis of HPLC and HPLC-MS assays,neurotrophic factors from hickory nut extracts of lipsoluble and water-soluble constituents were isolated and identified.The main results are as follows:(1)In SH-SY5 Y cells treated with hickory lipsoluble and water-soluble constituents,the nuerite outgrowth was significantly promoted in a certain concentration range.The expression of NF160,NPY and NGF genes was significantly induced by hickory liposoluble constituents.However,hickory water-soluble constituents only significantly induced NF160 expression.The results showed that both liposoluble and water-soluble constituents of hickory had neurotrophic activity.(2)The analysis of HPLC and HPLC-MS displayed that the liposoluble constituent of hickory nuts had three specific peaks with molecular weights of 278,280,and 282 daltons,respectively,compared to control.They were identified as linolenic acid(LNA),linoleic acid(LA),and oleic acid(OA),respectively,by comparison of molecular weight and standard substance.A mixture of these three fatty acids based on their ratios in the hickory liposoluble constiuents was used to treat with SH-SY5 Y cells.The same results were observed as in the cells treated with hickory liposoluble constiuents,suggesting that neurotrophic activity factors the hickory liposoluble constituents is one or several of the three fatty acids.(3)SH-SY5 Y cells were treated with the mixture of three fatty acids based on the ratio in hickory liposoluble constituents at various concentrations.Data showed that the total concentration of 3.3?mol/L mixture had a highest activity.Alternatively,individual fatty acid and alternative ratios of three fatty acids at 3.3?mol /L did not display good activities as like the mixture.Data showed that the best neurotrophic activity came from a mixture of 1:8:16(linolenic acid : linoleic acid : oleic acid)ratio.These results suggested that the neurotrophic factors of hickory liposoluble constiuents is a certain ratio of linolenic acid,linoleic acid and oleic acid mixture.(4)The comparison of Hickory water-soluble constiuents by HPLC and HPLC-MS analysis to standard substances indicated that the the constiuents contain cytidine,uridine,guanosine,adenosine and adenine.However,these compounds had no neurotrophic activity under the same test conditions.