Preparation Of Silkworm Pupa Peptide And Its Anti-hypertensive And Hypoglycemic Activity In Vivo

Silkworm pupa is a developmental stage in the life cycle of silkworm. Silkworm pupa production is very considerable in China and it is the main byproduct of the reeling industrial. Because of lacking of deep-processing technology, a large number of silkworm pupa basically used as feed and fertilizer. Protein content in dry silkworm pupa is as high as50%, the fat content is20%to30%. It also contains a variety of vitamins, trace elements, as well as chitin, antimicrobial peptides, polysaccharides, etc. Silkworm pupa is approved by the Ministry of Health as the only insect food in the list called new food resource. Therefore, comprehensive development and utilization of the silkworm resources is of great significance."Three high diseases" refers to hypertension, diabetes and hyperlipidemia. They are called "rich men’s disease" in modern society. They can exist alone, or interconnected. Anyone of these three disorders can cause the other easily. The risk of hypertension and diabetes can not be ignored. The clinical drugs that anti-hypertension have significant effect, but long-term using can lead side effects such as rash, urinary system diseases. Also the chemical drugs for diabetes treatment have big side effects. Based on the requirement of safe, convenient and effective drugs that anti-hypertension and hypoglycemic are urgent needed. Scientists and researchers turn to natural anti-hypertensive and hypoglycemic substances.Our research work is mainly focused on the preparation of active peptide in silkworm pupa protein and its activity in vivo. First choose appropriate protease to hydrolyze the silkworm pupa protein, using single factor test and design of response surface optimization test to reach the optimical condition. Then active peptides were isolated and purified by macroporous resin, Sephadex and semi-preparative HPLC. Lastly, the active component was tested in animals to evaluate the activity in vivo. The active components are to be further developped to a kind of functional health care products, avoiding waste of silkworm pupa resources. This method can promote deep development and utilization of pupa protein resources. Main parts of the research are as follows: (1) Optimization of enzymatic hydrolysis experimentThe enzymes used in this experiment are neutral protease, papain protease, alkaline protease, compound protease. The method we used was double enzyme digestion. DH and ACE inhibitory rate are used as the marker.Enzymatic time, pH, substrate concentration, enzyme dosage and temperature are researched in single factor test. We obtained a better value and then we conduct response surface test. The best enzyme technology parameters were got, hydrolysis time is3.5h, substrate concentration is10.12%, temperature is50.22℃, pH is8.5, enzyme dosage is7.03%.(2) The separation and purification experiment of silkworm pupa peptidesThe macroporous resin DA201-C was selected to conduct the desalination treatment. The sample flow rate is0.5ml/min.75%ethanol was used as elution and flow rate was2.0ml/min.Analytic solution after macroporous resin separation was separated by Seph-adex and five peaks were obtained. The ACE inhibition rates of every peak were:P1for24.82%, P2for22.37%, P3for32.64%, P4for34.82%, P5for44.37%. So choose the P5for further test.The solution of P5was performed in the semi-preparative RP-HPLC analysis and several components were separated preferably in the chromatogram. Its ACE inhibitory rate was measured, and its molecular weight was measured by SDS-PAGE electrophoresis.(3) Anti-hypertensive test of active polypeptide in SHRSpontaneously hypertensive rats were used as model for the animal test. Captopril was taken as control drug. Purified polypeptide after Sephadex was freeze dried and used in one-time and long-term administration in SHR. The results showed that the pupa polypeptide had anti-hypertensive activity.(4) The hypoglycemic activity of pupa polypeptide in vivoHyperglycemia model mice induced by Alloxan were used for animal test. Metformin hydrochloride was taken as control drug. Purified polypeptide after Sephadex was freeze dried and used in one-time and long-term administration in model mice. The results showed that, the pupa polypeptide had hypoglycemic activity.