Studies On The Ejiao Low Mol. Wt. Peptide And Its Preparation

In the article, the biological enzyme is used to extract the traditional Chinese medicine Ejiao. First by pharmacodynamics experiment, the effective and safe MWt. Range of Ejiao Low MWt. Peptide for Oral use and for injection and the dose-effect relationship are established. Then single factor experiment and orthogonal test are designed to optimize the extraction craft with the index of peptide content. Next, the affected ingredients in Ejiao are purified by the method of Membrane Separation Technology, optimized the craft through inspecting the loss percentage, Ejiao Low MWt. Peptide for oral use and the gelsiccation powder of Ejiao Low MWt. Peptide for injection are prepared. Then by inspecting the method of Sephadex, HPCE, HPLC, we have penetratingly separated the gelsiccation powder solution, researched the Spectrum-Pharmacodynamic action relationship by SDS-PAGE and Cell culture, researched the structure by HPLC-MS preliminarily. Next, we checked the safety and researched the pharmacology. Finally the quality specification is researched.Use sensitization as index, the experiments determined the effective MWt. range of peptide for oral use is <5kD, the effective and safe MWt. range of peptide for injection is 1kD～3kD.<5kD Ejiao low MWt. peptide has positively correlation of doses and efficacy in the context of 5mg·kg-1～15mg·kg-1,1kD～3kD Ejiao low MWt. peptide has positively correlation of doses and efficacy in the context of 1mg·kg-1～20mg·kg-1.Process for the enzymatic hydrolysis (for oral use) is:temperature 42℃, pepsin digestion pH 2.0, the amount of enzyme pepsin 100kU·g-1, pepsin digestion time is 1.5 hours, trypsin digestion pH 8.0, the amount of enzyme trypsin 5k U·g-1, trypsin digestion time is 2.5 hours. Process for the enzymatic hydrolysis (for injection) is:temperature 42℃, pepsin digestion pH 2.0, the amount of enzyme pepsin 100kU·g-1, pepsin digestion time is 2 hours, trypsin digestion pH 8.0, the amount of enzyme trypsin 5k U·g-1, trypsin digestion time is 3 hours. Process for the ultrafiltration is:Make powder of Ejiao 50g to low-peptide hydrolyzate, diluted to 1000mL, water-bath preheated to 40℃, the column pressure 0.15±0.01MPa, respectively through the MWCO 30kD,5kD(for oral use),3kD(for injection) of the ultrafiltration membrane, and washed once.Ejiao Low MWt. Peptide for oral use had no abnormal toxicity, is the actual non-toxic substances; Ejiao Low MWt. Peptide for injection had significant low-allergenic, non-toxic side effects, no pyrogen reaction. Use four kinds of common anemia model experiments (equivalent to clinical long-term poisoning, blood loss, chemotherapy and radiotherapy) confirmed that Ejiao Low MWt. peptides for different types of anemia has a different degree of improvement.Load swimming test in mice, serum urea nitrogen and liver glycogen tests showed that Ejiao Low MWt. peptides have a certain trend of anti-fatigue effects, mouse immune-enhancing experimental results show that Ejiao Low MWt. peptides can increase the level of circulating antibodies serum hemolysin and enhance DNCB-induced DTH responses in mice to play a role in the humoral and cell immunity.Macrophages of non-specific immune system also have a certain role in promoting.Clarity, pH, density and purity determination can test the quality of Ejiao Low MWt. Peptide for oral use.By examining the visible foreign matter, insoluble particles, pH, molecular weight range, purity (or low-peptide concentration), and promoting blood titer can test the quality of Ejiao Low MWt. Peptide for injection. Through Tricine-SDS-PAGE study for Ejiao Low MWt. Peptide and its components, the qualitative identification method is determined:the MWt. Of the preparation is<2kD. Stability of the experimental results show that stored in the Xilin bottle of Ejiao Low MWt. Peptide for injection freeze-dried powder can store at 4℃for 2 years.Based on the preparation of Ejiao Low MWt. Peptide, we studied the composition and structure. First separated 3 components by Zeolite, then purified them and did cell culture, meantime, studied the HPLC condition of No.2 and No.3 peak which had high content of Low MWt. Peptide, and concluded No.2 peak is effective component. Finally studied the HPLC-MS of No.2 peak, and searched the sequence of possible peptide segment by SEQUEST and Mascot search engine.