Study Of Separation Of Silkworm Moth Insulin-like Substance And Its Insulin-like Effect

According to the statistics of the World Health Organization,the global incidence of diabetes is increasing year by year,which has posed a serious threat to human health.At present,insulin is the most effective clinical drug for the treatment of diabetes,but long-term injection administration brings a lot of inconvenience and pain to patients.Therefore,the research and development of oral insulin preparations has become a hot spot in the field of diabetes treatment research.In traditional Chinese medicine treatment,silk moth is the main medicine for the treatment of diabetes.It has been found that silk moth has a pharmacodynamic effect on diabetes.In recent years,relevant researchers have used silkworm moth powder and silkworm moth crude extract to conduct anti-diabetic experiments and clinical studies.The research results show that they all have hypoglycemic effects,but the main medicinal components in silkworm moths that have hypoglycemic effects have not yet been revealed.After long-term preliminary research,the research team isolated silkworm moth insulin-like active substance(SMILAS)from silkworm moth,and preliminarily confirmed that it has insulin-like effect in mammalian cells.On the basis of many years of previous research by the team,through further transformation and optimization of the extraction process,the bioavailability and insulin-like biological activity of oral administration were improved,and the pharmacodynamic effect of anti-diabetes and comorbidities was further evaluated.For the first time,the antidiabetic effect of SMILAS was studied,and its insulin-like effect was verified through cell and animal experiments.Through the analysis of the natural properties of SMILAS to withstand strong acid environments and resist gastrointestinal digestive enzymes,and the optimization of the extraction process,high-efficiency oral insulin was obtained.The establishment of key technologies for similar drugs lays a solid foundation for the first development of oral insulin drugs at home and abroad.The diabetes drug market is huge,and the research results of this project have broad market application prospects and significant social and economic benefits.This study is divided into 3 parts:1.Optimization of SMILAS extraction process route.Three SMILAS extraction process routes were used to extract the head tissue of silkworm moth,and the extraction efficiency and yield of various methods was compared,and treatment processes such as high temperature and strong acid environment were set in the extraction process to explore the physical and chemical properties of SMILAS.2.Study on the insulin-like effect of SMILAS on mammalian cells.SMILAS was co-cultured with 3 target cells of insulin(preadipocytes 3T3-L1 cells,hepatoma cells Hep G2 cells and myoblast C2C12 cells)to study its effects on glucose metabolism,glycogen synthesis,cell proliferation and cell proliferation in mammalian cells.differentiation,etc.3.The effect of oral administration of SMILAS on blood glucose in diabetic mice.First,the diabetic model of male C57 BL mice was induced by multiple injections of different doses of streptozotocin.After successful induction,different components of SMILAS was orally administered to observe its effects on the body weight and blood sugar of diabetic mice,and to study oral administration of SMILAS Insulin-like effects in mammals.This study draws the following conclusions:1.Propose an optimized SMILAS extraction process route.This project uses a combination of chemical methods and physical methods to adopt three different extraction process routes.The three process routes are evaluated from the aspects of SMILAS yield,purity,extraction simplicity,etc.,and the optimized SMILAS extraction process is finally determined.route.This process route can obtain 3.24 g of mixture from the first step of chemical method from 5000 silkworm moth heads(weighing about 1600g),the protein is determined to be 1.96 g,and the purity is about60%.After the protein is further separated by physical method,SMILAS can be obtained.(Protein molecular weight is between 1KD-10KD)The yield is 0.231 g.2.The physical and chemical properties of SMILAS were clarified for the first time.We included high temperature denaturation(100°C),strong acid isoelectric point precipitation and different molecular weight cut-off in the SMILAS extraction process,indicating that the SMILAS substance is a peptide mixture,and confirmed that the extraction mixture has high temperature resistance and strong acid resistance.It is the key technology to obtain high-efficiency oral insulin drugs,which will lay a solid foundation for the first development of oral insulin drugs at home and abroad.3.To illustrate the insulin-like effect of SMILAS in mammals by in vitro experiments.SMILAS was applied to 3 target cells of insulin-Hep G2 cells,3T3L1 cells,and C2C12 cells.It was verified at the cellular level that SMILAS had the functions of promoting cell growth,promoting cellular glucose absorption and glycogen synthesis,showing varying degrees of insulin-like Function characteristics.4.To illustrate the insulin-like effect of SMILAS in mammals by in vivo experiments.The different molecular weight components of SMILAS were orally administered to diabetic mice.The experimental results showed that the different components of SMILAS had hypoglycemic effects,but the hypoglycemic effect of the1KD-10 KD components was significantly better than that of the components above10 KD.SMILAS is considered to have a lowering effect on blood glucose levels in mammals when orally administered.This study optimizes the process of efficient separation of SMILAS,improves the acquisition rate of SMILAS with innovative technology with independent intellectual property rights,and confirms the insulin-like effect of SMILAS through cell and animal experiments,especially the first time to prove the oral administration of insulin in mammals.biological effects.To this end,we will carry out related experiments such as protein mass spectrometry analysis of SMILAS components in the next step to explore the composition of SMILAS components,and further study the molecular mechanism and signaling pathway of its insulin-like effect through in vivo and in vitro experiments,so as to take the lead in research and development based on original innovation at home and abroad.The technical oral insulin-like drug should have broad market application prospects and significant social and economic benefits.