Preparation And Activity Of Hemp Seed Antioxidant Peptide

Along with the improvement of standard of living and demand for nourishment, interest in utilizing natural antioxidant peptide which has potential application in food and medicine industry has increased substantially. In the processing of hemp fibres, its protein becomes an interesting byproduct. This paper main studies the preparation, antioxidant activity and isolation and purification of hemp enzymatic hydrolysates (HPHs). At the end, the effect on hydrogen peroxide-induced apoptosis in PC12 cells was studied.It was found that Alcalase2.4L alkaline protease had the best effect of scavenging to DPPH·. By response surface regression analysis, the optimum enzyme hydrolysis conditions of Alcalase 2.4L alkaline protease were achieved as substrate ([S])50 mg/mL, time 2h, temperature (T) 50℃, enzyme: substrate ratio ([E]/[S])2.2% (w/w), and pH 9.4. The resulted degree of hydrolylsis (DH) was about 19%. Furthermore, HPHs prepared on this condition had strong antioxidative activity, with the DPPH radical scavenging rate 82.65 %( 10 mg/mL).Because a lot of salts were introduced into HPHs during the process of hemp hydrolysis, it was necessary to make HPHs desalted. Macroporous adsorption resin DA201-C was used for removing the salts from HPHs in dynamic states. It was showed that the effect of desalination was very well. With 75% ethanol as desorption agent, the desalination rate was 93.03%, and the best reclaim rate was 98.45%. The color of desalted product became light and leaned to white; content of hydrophobic amino acid increased from 26.3g/100g to 34.47g/100g. Studying on the the molecular weight distribution of HPHs, found it distributed mainly between 310~2600. Reducing power and free radical-scavenging activities were enhanced. Before desalination the IC50 of DPPH·, O2-·and·OH were 3.75 mg/mL, 3.50 mg/mL, 8.25 mg/mL; after desalination they decreased to 2.75 mg/mL, 3.00 mg/mL, 7.70 mg/mL.Moreover, after isolated by gel filtration chromatography, semi-preparative RP-HPLC and RP-HPLC, two compenents G-25-1-F-a and G-25-1-F-b were achieved. Identified by MALDI-TOF- TOF, the amino acid sequence of m/z 441.02 and m/z 924.49 of G-25-1-F-a were NHAV and HVRETALV separately; m/z 1199.54 of G-25-1-F-b maybe LECNKVDTMF and LKRDEAFCAF.In this study, the effect of G-25-1-F on hydrogen peroxide-induced apoptosis in PC12 cells was studied. Exposure of cells to 0.15mmol/L H2O2 for 2h may cause significant viability loss and apoptotic rate increase. Pretreatment with different concentrations of G-25- 1-F(concentration 10μg/mL) for 2h increased the survival rate of PC12 cells significantly,inhibited apoptosis induced by H2O2. The mechanism of it may be the ability of scavenging reactive oxygen species.