Purification And Function Research Of Bioactive Peptides From Pig Blood Hydrolyzed By Trypsin

Bioactive peptides are some kind of biologically active peptides. Theseshort length peptides are containing up to 2 amino acid residues intheir primary sequence. Bioactive peptides are usually modified, andhave been shown to play physiological roles in cell metabolism andfunction. In recent years, bioactive peptides have been a drug researchhotspot.Various bioactive peptides have been reported in both the scientificliterature and in issued patents. Peptides historically have been isolatedfrom natural sources, and have recently been the subject ofstructure-function relationship studies. Additionally, natural peptideshave served as starting points for the design of synthetic peptideanalogs. Additionally, natural peptides have served as starting pointsfor the design of synthetic peptide analogs.Hidden or inactive in the amino-acid sequence of dairy proteins, theycan be released or activated in vivo during gastrointestinal digestion,or upstream during food processing via specific, enzyme-mediatedproteolysis. Enzyme-mediated proteolysis is the common use in recentyears. The natural sources are important sources of those bioactivepeptides. The development and exploitation of bioactive peptidesderived from pig's blood red cell have already been a hot researchtopic at home and abroad. We found the enzymolysis product of pigblood by trypsin hydrolysis is a mixture of some single amino acids,compound amino acids and some peptides, that may obvious enhancethe immunologic activities of immunologic system. As a matter of fact,it has been a long time that animal blood is applied to scienceresearches to get some useful biologic materials. Among these animalbloods, the source pig blood is very cheap and rich. The moreimportant reason of the abundant protein content in pig blood, makesthem become an important research object. Among these researches ofpig blood, using biological purification ways to get some useful singleamino acids, compound amino acids or some valuable peptides is themain aspect.In our research, we used trypsin to hydrolyze fresh pig blood and gotthe mixture that had ability to enhance organism immunologic activity,and this mixture is the raw material of our research. During thepurification progress, we use Sephadex G-25 chromatography,ion-changechromatograhpy, ultrafiltration to isolate and purify the aimproduct, and use HPLC to determine the purity of this peptide. In orderto identify the existence of our aim peptide, we conduct T lymphocytetransformation test and E rosette forming test to the product of theprogress. The result indicates that our aim product is a kind of whiteand easily absorbing water substance. In order to understand theenhancement immunologic activity of our aim peptide, we conductsome important immunology and pharmacology experiments in vitroand in vivo. In vitro experiments, we mainly research the effect of theaim peptide on T lymphocyte, so we do T lymphocyte transformationtest and E rosette forming test. But because in vivo experiments aremore important than in vitro experiments in immunology andpharmacology, and the results of in vivo experiments are morevaluable. In vivo experiments, to three main kind of immunologic cells:T lymphocyte, B lymphocyte, NK natural killer cell, we do Tlymphocyte transformation test, B lymphocyte agglutinating titer andNK cell cytotoxic activity test.From all these results, we have a basic understand to the peptide andget five important conclusions about it's enhancement immunologicactivity.1. Whether in vitro or in vivo immunologic experiments, this peptideall show much higher immunologic enhancement activity than control.It indicates that our aim peptide is a positive cellular immunityadjuster.2. From three tests in vivo related T lymphocyte, B lymphocyte, NKnatural killer cell, although this peptide can enhance all three kindscells immunologic activities, we found it affected the activity of Blymphocyte is much greater than T lymphocyte and NK natureal killercell. The result of the agglutination titer of B lymphocyte (5.0mg/kg .d) is 5.6 (injection) and (enema). Times as much as control. Theactivity enhanced is all greatly higher than T lymphocyte, NK naturalkiller cell.3. To injection groups and enema groups in vitro, althoughenhancement activity difference between them is not as much as thatbetween T lymphocyte, B lymphocyte, and NK natural killer cell. Inall three tests in vitro, injection groups show greater enhancementactivity than enema groups, no matter how much dosage level iscompared. The reason probaly is, when rats are delivered by enema,their digestive system, especially their stomachs and bowels will bestimulated. This can result in theirabsorbance ability becoming worse, so the peptide can't fully affecttheir functions. In the test we also found most rats of enema groups arethinner and with a poorer appetite, it is a also evidence of ourjudgement.4. To different dosage of groups, whether injection or enema, the levelof enhancement of immunologic activity decreased along withenhancement of dosage. When dosage is 5.0mg/kg . d, the activity ofenhancement is much greater than that of other dosage, and whendosage is 10.0mg/kg . d and 20.0mg/kg . d, the enhancement becamesmaller, when 40.0mg/kg . d, the peptide even didn't enhance theimmunologic activity, but suppressed their activity.5. In NK natural killer cell cytotoxic test, we performed the test ondifferent ratio of effector cell: target cell (E: T=20:1, 10:1, and 5:1),results indicated when E:T=20:1, the enhancement is the biggest, andwith the ratio decreased, the NK natural killer cell cytotoxic alsodecreased obviously.Because our research is in the basic stage now, there are manyunsolved questions, such as its mechanism to the immunologic activity,and its other effects to immunologic system. But for the aim peptidewith high enhancement immunologic activity, it has high values forapplication and research on improving immunologic conditions andenhancing immunologic defence. Our research improves the peptide isvery valuable in clinical trial, and because the source of pig blood ischeap and rich, method of purification is simple, purity of finalproduct is high and its effect is efficient and innocuous, it will arisegreat research interest.