The Study Of Velvet Antler Protein Extration Establishment And Its Neuroprotective Effect

In this study, we compared two kinds of extraction condition of velvet antler protein(VAPs) in order to develop a VAPs extraction method with high yield, high protein content and active pharmaceutical effect.Two kinds of VAPs extract methods, homogenization and extra-micronization, were used to compare with the protein yield, protein content and pharmacological activity; Bradford assay was used for protein concentration measurement; Molecular weight of velvet antler protein was detected by SDS-PAGE; The morphology VAPs particle was visualized by scanning electron microscope; High Performance Liquid Chromatography(HPLC) and Mass Spectrometry were used to detect the molecular weight and ingredients of VAPs; The protective effect of VAPs against MPP+-induced cytotoxicity in SH-SY5 Y cell was measured using MTT assay; HE staining was used to evaluate the protective effect of VAPs on 6-OHDA induced cell death in Parkinson disease(PD) model in rat; Immunohistochemistry was used to detect the TH positive neuronal cell in substantia nigra; HPLC-MS was used to detect dopamine(DA), 3,4-Dihydroxyphenylacetic acid(DOPAC), homovani Hicacid(HVA), 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA) and glutamate(Glu) levels in the striatum and nigracerebrospinal fluid(CSF); The amino acid level in brain tissue and CSF was measured by HPLC-FLD; The VAPs influx through blood vessels was detected by HPLC.Compared to homogenization, VAPs extracted from extra-micronization extraction exerted characters of high-yield and rich-protein content and containing much more proteins with lower molecule weight. Scanning electron microscope observation showed that VAPs extracted using extra-micronization exerted much smaller particle than that of using homogenization. Next, the VAPs form extra-micronization also exerted much higher protective effect against MPP+-induced cell death than VAPs from homogenization in SH-SY5 Y cell. The protein content of VAPs from extra-micronization was 14.892%, molecular weight of VAPs ranged from 10 kD-250 kD, mainly in the range of 25 kD-70 kD. Mass Spectrometry analysis showed 157 protein components existed in the VAPs. Among them, 16 kinds of proteins exerted cell proliferation stimuli activity. In the 6-OHDA induced PD model of rat, VAPs inhibited the 6-OHDA induced neuronal death and TH positive cell decrease in substantia nigra. 6-OHDA also decreased the level of DA, DOPAC, HVA, 5-HT levels and DOPAC/DA, HVA/DA values in CSF, increased the level of Glu, GABA in CSF. VAPs intervention ameliorated these effects mentioned above. Fluorescent signals were detected form blood samples of rats received FITC conjufated VAPs treatment, but no fluorescent signals were detected from CSF samples.VAPs extracted by extra-micronization exerted characters of high-yield, high-protein content and higher neuroprotective effect, which contains protein content of 14.892%. 157 protein components of VAPs were collected using extra-micronization. VAPs also exerts protective effect against MPP+ induced SH-SY5 Y cell death and inhibits toxic insult of 6-OHDAin PD rat model.