Extraction, Isolation And Characteristic Of Active Substance Of Antler Base

Antler base is the ossified and rudimental antler on the pedicle of male sika deer(Cervus nippon) after sawing off the velvet antler, which then falls off by itself everyspring when the new velvet antler begins to germinate. It looks like a plate, so it istherefore named ‘antler base’. Antler base has high medicinal value according to theShen Nong Ben Cao Jing and is believed to tonify the liver and kidneys, andinvigorate the circulation of blood and detumescence. Over years, many researchershave focused on the antler base pharmacological value, which less than the researchof antler base protein. The range of research was narrow, that seriously hindered thedevelopment of antler base at industry. In this paper, antler base as the object of study,a broader and more in-depth study of antler base protein were carried out, to promotethe development of industry, and bring more economic and medicinal value.Through three channels to research the antler base protein: extraction, isolationand characteristic of antler base PRGPs; preparation and purification of antler baseantioxidant peptide; extraction and characteristic of antler base collagen. The researchcontents are as follows:(1) CnPGRP1is a novel antimicrobial protein from antler base of the sika deerCervus nipp, expressed at high levels to protect the wound of velvet antler frombacterial infections after removal. The samples were ground with10volumes ofprecooled solvent A (5mM sodium acetate, pH4.5) by using a colloidal mill. Theimpurity was removed from peptide by ethanol precipitation. The supernatant wasthen removed ethanol using a rotary evaporator at40℃, and lyophilized. The antlerbase antimicrobial proteins (AAP) were subjected to consecutive chromatographicmethods connected to Sephadex G-25gel filtration column,(CM) anion exchangecolumn, and RP-HPLC. The molecular weight of cnPGRP1was17.2kDa underSDS-PAGE, and peptide mass fingerprint analysis by MALDI-TOF-MS aspeptidoglycan recognition protein1matched to dasypus novemcinctus. The matchedamino acids sequences were RLYEIIQKWPHYRA.In a standard liquid assay in vitro, both Gram-positive bacteria andGram-negative bacteria are killed by cnPGRP-S, at a dose of50-250μg/mL withrange at4hours of incubation. Our results indicate that cnPGRP binds toGram-positive bacteria (S. aureus and B. subtilis), Gram-negative bacteria (E. coli andS. dysenteriae), and fungus (S. cerevisia). The requirement for Zn2+or Ca2+for microbicidal activity may explain why previously murine and human recombinantPGRP-S was only bacteriostatic, and not microbicidal. Our isolated cnPGRP from theantler base of the sika deer, a native protein, was the same as bovine PRGP-S, and theprobable retained Zn2+that was likely bound to them in vivo.(2) The protein of antler base was enzymatic hydrolyzed by alkaline protease,neutral protease, compound protease, pineapple protease and trypsin, and topreparated antioxidant peptides. The enzyme hydrolysis ability and antioxidantactivity of peptide were discussed, the hydrolysis active of alkaline protease and theantioxidant active of the peptide which were hydrolyzed by alkaline protease werehigher than other proteases, so the alkaline protease was chosen to hydrolyze antlerbase.According to the results of single factor experiment, enzyme concentration (X1),reaction temperature (X2) and pH (X3) were chosen to optimize the conditions ofenzymatic hydrolysis of antler base protein by three factors five levels of responsesurface analysis, and the clearance ratio of DPPH free radical by peptides as theresponse value (Y). The mathematical model was established based on the test result,as follow: Y=73.45+3.49X1-1.18X2-1.21X1X2-2.47X1X3-3.89X21-4.03X22-2.57X23, forthe significant level α<0.05. The optimal conditions of enzymatic hydrolysis as follow:the substrate concentration15%, enzyme concentration6%, temperature53℃, pH8.5,the clearance ratio of DPPH free radical by peptides was74%.(3) The dispose of antioxidant peptides mainly reflected in two aspects ofdecoloring and desalination. The peptide pH, reaction temperature, reaction time andactive carbon concentration were studied about the factors of decolorizing of effectson the decoloring of active carbon. The best conditions of active carbon decoloringwere determined as follow: the peptide pH5, reaction temperature50℃, reaction time3h and active carbon concentration4%, the decoloring rate of antler base peptides byactive carbon was79.86±0.26%.The pH, reaction temperature, peptide concentration and the ratio of peptide tomaterial were studied about the factors of adsorption of effects on the adsorptioncapacity of macroporous resin DA201-C. The best conditions of static adsorption ofmacroporous resin DA201-C were determined as follow: the peptide pH4, reactiontemperature25℃, peptides concentration10mg/mL the ratio of peptide to material5:1and adsorption time24h. Under this optimal condition, the adsorption rate ofantler base peptides by macroporous resin DA201-C was78.52±1.05%.According to the dynamic adsorption and elution experiment of resin DA201-Cof desalination processing of antler base peptides, the desalination rate of peptideswas98.64±2.45%, and the recovery of peptides was86.31±0.97%. The antioxidant activity of peptides which were desalted by resin DA201-C, still had high antioxidantactivity. So the resin DA201-C was suitable for desalting and purification of antlerbase peptides, and has a good industrial prospect.(4) The effects of enzyme concentration, electric field intensity and pulse numberon extraction of antler base collagen were studied, with the collagen concentration astest indexes, and jointed the collagen banding in the SDS-PAGE. The optimalconditions of extraction of collagen were confirmed as follow: electric filed intensity20kV/cm, pulse number8and pepsin concentration2%, under this optimal conditionof extraction, the extraction rate of antler base collagen was73.41±1.07%, and thebandings of collagen were more pure, there was not peptide banding under thecollagen bandings.The denaturation temperature and in vitro fibrillogenesis test of antler base collagen werestudied, the test showed that the denaturation temperature of collagen was36℃, the in vitrofibrillogenesis was good. The infrared spectrum showed that the extraction wascollagen.