Preparation And Properties Of Hypoglycemic Peptides From Red Deer (Cervus Elaphus) Antlers

Antler is the young unossified hairy antler of male deer,which is mainly composed of amino acids,phospholipids,fatty acids,saccharides,hormone-like substances,chondroitin sulfate,polyamines,peptides,vitamins,enzymes and bases.The more than half of total composition are Amino acids.Proteins in the antler exist mainly in the forms of keratin,collagen,insulin-like growth factor and epidermal growth factor,which have the physiological functions of enhancing immune system,resisting oxidation,promoting tissue injury healing and lowering blood sugar.In order to reasearch on the physiological activity of hypoglycemic polypeptides and the relationship between structure and effect,to create conditions for the development and usage of antler.In this paper red deer(Cervus elaphus)antler was used as the raw material.The hypoglycemic peptides from red deer(Cervus elaphus)antler were isolated and purified,then the isolated components were subjected to in-vitro hypoglycemic effect verification.Finally,the structure of polypeptides were analyzed.The research contents and experimental results are as follows:(l)Preparation of hypoglycemic peptides from red deer(Cervus elaphus)antler:Hypoglycemic peptides were prepared from red deer(Cervus elaphus)antler by bi-enzyme hydrolysis process.Alcalase and Flavourzyme,two enzymes with higher a-glucosidase inhibition,were screened from Alcalase,Flavourzyme,Neutral Protease and Trypsin,and the sequence of action was confirmed to be Alcalase-Flavourzyme according to in-vitro hypoglycemic effect.The optimal process conditions for bi-enzyme hydrolysis were determined by the optimal process and orthogonal experiment as follows:hydrolysis with Alcalase at pH 8.0,60-C,12%substrate concentration and 5000 U/g enzyme dosage for 3 h,followed by hydrolysis with Flavourzyme at pH 6.5,45 ?,5%substrate concentration and 6000 U/g enzyme dosage for 1 h.The a-glucosidase inhibition rate of two-step double-enzyme hydrolysate was influenced by mass concentration.At a mass concentration of 3 mg/mL,the a-glucosidase inhibition rate was up to 94.09%,and the IC50 value 1.82 mg/mL.(2)Isolation and purification of red deer(Cervus elaphus)antler hypoglycemic peptides:Ultrafiltration technique was used to enrich the peptides,and the collected small molecular polypeptides were isolated and purified by Sephadex LH-20 gel chromatography.Under the conditions of 1.5 mL injection volume,55 mg/mL injection concentration and 0.25mL/min elution flow rate,four components could be isolated from red deer(Cervus elaphus)antler hypoglycemic peptides,which were P ?,P?,P? and P?,respectively.(3)In-vitro hypoglycemic function of red deer(Cervus elaphus)antler hypoglycemic peptides:Insulin resistance model of HepG2 cells was established,and the optimal insulin concentration was determined to be 10"7 mmol/L.The effects of purified components of red deer(Cervus elaphus)antler hypoglycemic peptides on HepG2 cells were studied.Component P?(0.05 mg/mL)exhibited the highest glucose consumption,and its effect on cell viability was greater than the positive control.To more comprehensive study the hypoglycemic function of the purified components of red deer(Cervus elaphus)antler hypoglycemic peptides,rat pancreatic ? cell line RIN-m5F was used to verify the hypoglycemic function in vitro.High-dose component P? exhibited a cell proliferative effect similar to the positive control(Exendin-4).Its cell proliferation rate for STZ-damaged pancreatic ? cells was higher than the other components(P I,PII,PIV),while close to the positive control group.(4)Molecular weight determination and amino acid sequencing of red deer(Cervus elaphus)antler hypoglycemic peptides:The ultrafiltrated red deer(Cervus elaphus)antler hypoglycemic peptides were analyzed by Tricine-SDS-PAGE gel electrophoresis.The results showed that there were four clear bands with molecular weight distribution ranging from 3.3k Da to 7.8 kDa.Nano-HPLC-MS/MS analysis revealed that the purified component P?contained a total of 308 polypeptide bands,of which the polypeptide with confidence greater than 90%have 15 polypeptide bands.The molecular weight were ranged from 420.46Da to 749.4Da.