Study On The Technology Of Efficient Preparation Of Corn Protein Cyclo (His-pro) And Its Anti-hyperglycemic Activity

Cyclo(His-Pro)(CHP)is a kind of cyclic dipeptide derived from the cross-link between two amino acid residues via a pair of complementary amide bonds.Pharmacological studies showed that CHP could attenuate ethanol-induced anesthesia,defeat toxic effect of central depressive drugs,and have neuroprotective,anti-hyperglycemic and antioxidant actions.Besides,it has strong stability towards proteolysis and higher bioavailability.All these properties make CHP a short-peptide agent for the therapy of drug abuse,alcoholomania and diabetes.Diabetes mellitus is one of the major chronic metabolic diseases and 450 million people is tortured by diabetes and its syndromes.The anti-diabetic medicine in clinic,at present,is mainly western drugs,which could regulate blood glucose level to varying degree.However,long-terming taking of such western drugs has some side effects and becomes inefficient gradually.What's more,it is invalid to diabetic syndromes.Therefore,finding an effective anti-diabetic medicine with low side effect is urgent in the clinic treatment of diabetes.Natural product that has anti-hyperglycemic activity is becoming a hot point in research.In this study,the corn gluten meal was used as raw material to prepare CHP with controlled enzymatic hydrolysis,high temperature/pressure technology and microwave technology.Separation and purification of the hydrolysate was performed using a series of advanced isolation techniques and the structural identification of the obtained target peptide was performed by liquid chromatograph/mass spectrometer,Fourier transform infrared spectrum and nuclear magnetic resonance(NMR)spectrum.At last,the effect of corn CHP on the blood glucose regulation of diabetic mice models was investigated and the anti-hyperglycemic mechanism was preliminarily explored.Compared with the chemical synthesis,such preparation method of CHP has many advantages including short reaction path,cheap and easy-to-get raw material,simple post-treatment,short production period,environmental friendly,etc.,and is promising for industrial production.Corn gluten meal is a huge resource in China,which is not utilized sufficiently.If the achievement of this study is for practical application and Industrial transformation,it could increase the value of corn protein,solve pollution problems caused by the waste from corn starch processing effectively and promote research and promote the development of CHP anti-diabetic medicine and health product,which is significant for exploiting new approach and new resource for prevention and treatment of diabetes and syndromes.The main contents and results of study are as follows:1.The major protein fractions obtained from corn gluten meal were zein,G1-glutelin and G2-glutelin using continuous progressive extraction method,which account for 51.33%,14.65% and19.74% of the total protein respectively.Results of amino acid analysis showed that the content of histidine and proline of G1-glutelin and G2-glutelin was the highest among the three proteins.Results of electrophoretic analysis showed that the three extracted proteins was not single component and were composed of many subunits.The major subunit bands of the three proteins was cut from the gel,hydrolyzed in gel and identified by nanoscale liquid chromatography coupled to tandem mass spectrometry(Nano LC-MS/MS).The results showed that there are more His-Pro and Pro-His sequences in G1-glutelin and G2-glutelin,and therefore the two proteins are ideal substrates for enzymatic hydrolysis for preparation of CHP precursors.2.Corn glutelin was hydrolyzed by four common commercial proteases.By comparison of degree of hydrolysis and the yield of CHP precursors of each hydrolysates,the most appropriate proteases for preparation of CHP precursors were aclalase and flavourzyme.The conditions for the alcalase-flavourzyme hydrolysis were optimized by orthogonal experiment,and the optimum hydrolysis conditions were as follows: alcalase hydrolysis was performed with E/S ratio of 11000 U/g and substrate concentration of 40mg/mL for 7h,followed by flavourzyme hydrolysis with E/S ratio of 17000U/g and pH of 7.5 at 55 °C for 6 h.The CHP precursors with a yield of 7.38mg/g can be readily obtained under the optimum conditions.3.CHP precursors were isolated and purified from corn protein hydrolysate using alcohol precipitation,anion-exchange chromatography,gel filtration chromatography and two-step reverse phase high performance liquid chromatography(RP-HPLC)on the basis of quantitative trialing.And the content of CHP precursors of the final purified fraction Q-A2-a-I was 98.15%.Results of amino acid analysis showed that only histidine and proline was dectected in the Q-A2-a-I fraction,and the mole ratio of histidine to proline was 1: 1.18.Through identification by ultra high performance liquid chromatography coupled to tandem mass spectrometry(UPLC-MS/MS)and manual calculate,the amino acid sequences of the target peptides of Q-A2-a-I fraction was His-Pro and Pro-His,and the molecular weight were 252.6Da and 252.7Da,respectively.The structure of Q-A2-a-I fraction was identified by 1H-NMR and 13C-NMR,and the results showed that Q-A2-a-I fraction has the target structure.4.High pressure/temperature synthetic technique and microwave synthetic technique have been applied into the synthesis of corn CHP respectively,Using CHP precursor and corn protein hydrolysate as starting material,cyclization was performed in water by high pressure/temperature and microwave technique,respectively to prepare corn CHP.Through optimization of an orthogonal test,the optimum conditions of CHP precursor using high pressure/temperature cyclization were determined as follows: reaction pressure 0.20 MPa,reaction temperature 120?,reaction time 6h,solution pH 8.0,and substrate concentration 20mg/mL.When CHP precursor was cyclized under such conditions,the reaction yield was 93.25%.Using corn protein hydrolysate as starting material,the optimum conditions of high pressure/temperature cyclization were a reaction pressure of 0.20 MPa,a reaction temperature of 120?,a reaction time of 6.5h,solution pH of 8.0,and a substrate concentration of 30mg/mL.Under such conditions,the His-Pro and Pro-His linear dipeptides could be cyclized directly,and the reaction yield was 91.87%.The optimum conditions of CHP precursor using microwave cyclization were a microwave power density of 9W/mL,a reaction time of 45 min,solution pH of 8.0,and a substrate concentration of 25mg/mL.Under such conditions the reaction yield was 76.24%.The optimum conditions of corn protein hydrolysate using microwave cyclization were a microwave power density of 9W/mL,a reaction time of 55 min,solution pH of 8.0,and a substrate concentration of 30mg/mL.Under such conditions the reaction yield was 73.59%.By comparison of high pressure/temperature cyclization,microwave cyclization and ethanol reflux method,it can be seen that one-step synthesis of CHP using high pressure/temperature cyclization has the highest reaction yield and shorter reaction time.What's more,the procedure is simple,low-cost,environmental friendly,and is suitable for mass production.5.Using corn protein hydrolysate after high pressure/temperature cyclization as raw material,CHP were isolated and purified from the hydrolysate using anion-exchange chromatography,gel filtration chromatography and half-prepared RP-HPLC.A systematic separation and purification procedure of corn CHP was established successfully.The content of corn CHP of the final fraction A2-b was 97.36%,of which the purity increased by 45.1 times.Identification of the final purified fraction was performed by UPLC-MS/MS,and the results showed that the amino acid sequences of the target peptides of A2-b fraction was cyclo(His-Pro),and the molecular weight was 235.7Da.NMR and Fourier transform infrared spectra showed that fraction A2-b was the target compound.In vitro anti-hyperglycemic and antioxidant tests indicated that the corn CHP purified by this study exhibited the best ?-glucosidase inhibitory activity,DPPH scavenging activity and reducing capacity.Results of reaction kinetics and molecular docking showed that corn CHP could suppress?-glucosidase competitively,with IC50 value and inhibition constant being 1.3mg/mL and 1.02mg/mL respectively.CHP could enter into the subtrate-binging site and interact with the active center and surrounded amino acid residues via intermolecular forces such as van de waals' force and hydrogen bond.This provide basis and evidence for the follow-up anti-hyperglycemic and antioxidant tests in vivo as well as further development of CHP.6.Using alloxan-induced diabetic mice as model,the purified corn CHP was given to the animals by long-term intragastric administration.The results showed that corn CHP could relieve polyuria and emaciation symptom of diabetic mice,decrease water intake,food intake,and fast blood glucose level,improve oral glucose tolerance and promote secretion of insulin to some degree.What's more,corn CHP could reduce blood lipid level of diabetic mice,and improve lipid metabolism disorder of diabetic mice.Besides,it can been seen that corn CHP could reduce the serum nitric oxide(NO)and malondialdehyde(MDA)level of diabetic mice,which indicated that corn CHP could eliminate NO and oxygen radicals generated by alloxan and therefore relieve the damage of oxidative stress on body.Results of histopathology showed that corn CHP could improve damage of pancreas and liver caused by alloxan-induced diabetes.This indicated that the hypoglycemic mechanism of corn CHP in vivo was promoting the antioxidant function of whole body by eliminating NO and oxygen radicals generated by alloxan,suppressing damage of pancreatic ? cell and liver cell caused by oxidative stress,protecting and repairing damage of pancreatic ? cell and liver cell,which therefore promote secretion of insulin and suppress diabetic complications.