Studies On Preparation, Purification Of The Sika Deer Placenta Oligopeptide

Sika deer placenta is the traditional rare herbs and senior nutrition,the study found deer placenta protein content of more than 70 percent,amino acids are rich,complete kinds,ratio reasonable,oligonucleotide peptide containing bioactive substances can increase the body's immune function;regulate the endocrine,maintain the normal function;anti-aging,anti-cancer effect such as。This study for raw materials Sika placenta, use of the traditional repeated freezing and thawing method combined with ultrasonic cell crushing technology extraction of sika deer placenta, using ultrafiltration method to the active oligonucleotide sika placenta peptide undertakes depart,finally, using the anion exchange column, gel chromatography column combined with reversed-phase high performance liquid chromatography oligonucleotide peptide purification of Sika placenta, and the molecular weight determination of active oligonucleotide peptides,analyze its biological activities,the main conclusions are as follows:1.Use of the traditional repeated freezing and thawing method combined with ultrasonic cell crushing technology extraction of sika deer placenta, consider freeze-thaw number,saline join multiples,ultrasonic broken frequency four factors on active oligonucleotide peptide extraction effect。Test shows, the optimum technological conditions for extraction:under 20 degrees below freezing on 24 hours,room temperature melting repeatedly three times,add 4 times saline,on 200w next ultrasonic crushing 18 seconds,repeated operation five times,every time interval 25s。In these conditions obtain sika deer oligonucleotide peptide coarse extractings from placenta concentration for the 247.85mg/ml。2.ultrafiltration method to oligonucleotide activity in sika placenta peptide undertakes depart,examines the concentration of liquid materials,pressure,temperature,velocity,enrichment multiples and operating time factors on the separation effect,determine the optimal separation conditions for:material liquid concentration 1.5%, pressure 0.06MPa,temperature 30℃, velocity of flow 2.0mL/s, concentrating multiple 2.5,operating time 3min,separation process to the removal rate of miscellaneous protein 43.78%。The membrane is soaked for 2 hour with 0.2%NaOH solution after being polluted, circulation time is 1 hours, oak 1h with HCl,circulation time is 0.5 hours,then washing to neutrality by water,flow recovery rate value is the largest for 0.834.3.Utilize the DEAE52 ion exchange column,Sephadex G-25 zeolites column to purify the oligonucleotide activity in sika placenta peptide in the test.Test result showes that the oligonucleotide activity in sika placenta peptide solution is separated by DEAE52 column, buffer with NaCl of 0.2mol/L,2 component are gained. It is verified that the biological activity of the first component is relatively high by the garland test of E-rosette.Its E-rosette rate of human T lymphocyte after off the receptor get back to 52.5%. High active component that isolated by Sephadex G-25column,buffer with NaCl of 0.15mol/L solution elute,get 2 components.It is verified that the biological activity of the second component is relatively high by the garland test of E-rosette.Its E-rosette rate of human T lymphocyte after off the receptor get back to 60%. Its characteristic absorbing peak locates at 223nm.4.Utilizing the reversed-phase high performance liquid chromatogram to carry out further purification to the oligonucleotide activity in sika placenta peptide,inspects TFA addition amount,the initial concentration of organic phase and velocity and temperature on the purification effect. the best purifying condition of separation is:Chromatogram column Hypersil ODS C18 (250x4.6mm,5μm), A liquid 0.10% TFA/ACN, B liquid 0.10% TFA/ water, in 30minl0%A～50% A and 90% B～50% B convex gradient elutes, velocity of flow 0.2mL/min, temperature 30℃, detecting wavelength 223nm. Under these conditions four separate components were gained,it is verified that the biological activity of the third component is relatively high through the test of E-rosette among three components.Its E-rosette rate of human T lymphocyte after off the receptor get back to 72.5%.Using high gel filtering chromatography determination polypeptide sequence is:0.385KDa.Through the amino acid analyzer determine activity oligonucleotide peptide homocysteine, aspartic acid tyrosine three kinds of amino acids, and the highest levels.