The Preparation Of Cholesterol-lowering Silkworm Pupa Protein Peptides And Research On Its Quantitative Structure- Activity Relationship

Hyperlipidemia belongs to the diseases of human body lipid metabolic abnormalities. High blood lipid levels will induce atherosclerosis,myocardial infarction,coronary heart disease and other diseases of heart head blood-vessel.These diseases have become a serious threat to human health.Therefore,peptide screening which has the hypolipidemic and cholesterol-lowering effect have attracted more attentions of scientists.This study used silkworm pupa protein as raw material, established a HPLC method of detection the activity of cholesterol-lowering in vitro and optimized the hydrolysis conditions of cholesterol-lowering peptides of silkworm pupa protein. We separated and purified the cholesterol-lowering peptides of silkworm pupa protein.The pharmacophore model of structure-activity relationship was setted up.On this basis,we identificated the primary structure of the cholesterol-lowering peptides of silkworm pupa protein.The main results were as follows:1. A HPLC method was established for the determination of HMG-CoA reductase inhibitor activity in vitro by measuring the concentration of NADPH in the systerm.The chromatographic conditions were as follows Symmetry (?) C18 column (5μm,4.6 mmx250 mm), mobile phase:V (K2HPO4-KH2PO4):V (methanol)= 85:15, pH 7.2, the flow rate of 1 ml/min, Detection wavelength of 337 nm, Sample quantity of 20 mu L, Column temperature of 25℃. In this condition, the NADPH linear range was 0.6-600μmol/L, the standard addition recovery was 94.48%-101.81%, relative standard deviation (n= 5), less than 4.49%, the lowest detection limit was 0.1μmol/L.2. Optimization of the hydrolysis conditions of cholesterol-lowering silkworm pupa protein peptides by response surface methodology was performed. The optimized conditions were as follows:enzyme dosage 5.12%, hydrolyzing 5 hours at the enzymolysis temperature 52℃ and pH=7.0 with a ratio of substrate/water (w/v) 3.91%,the HMGR inhibitory rate of cholesterol-lowering silkworm pupa protein peptide reached 45.24%. A response surface optimized model based on the cholesterol-lowering silkworm pupa protein peptide extraction conditions was established. The verified test results showed the model with a 97.31% accurate rate.3.The molecular weight of peptides that less than 5000Da and 3000Da had been separated by G-15 gel column chromatography. Then the HMGR inhibitory activities of each peak were compared, the results showed that the molecular weight of less than 3000Da peptides isolated from the peak 4 has the best inhibitory activity. The fouth peak were separated second time,and get its fifth peak with high activity,its inhibitory activity was 32.92%. This peak was the target peak for primary structure identification of cholesterol-lowering peptide of silkworm pupa protein.At the same time,we collected 20 peptides which had released data structure and activity of HMGR inhibitory as training set to generate pharmacophore models DDAN for HMGR inhibitory peptides by MOE molecular software.The pharmacophore models were 2 hydrogen bond donors,1 hydrogen bond receptor,1 carboxyl donor.4. We measured amino acid composition of the fifth peak, this silkworm pupa protein mainly contain 16 kinds of amino acid compositions. The contents of aspartic acid (Asp), glutamic acid (Glu) and histidine (His) with higher content.The percentage were respectively 20.07%,16.00% and 9.66%. Through the determination of molecular weight of the fifth peak, its molecular weight range was 125-503 Da. According to the results of molecular weight range and determination of amino acid composition, we judged the silkworm pupa protein peptide should give priority to with three peptides,the peptide sequence may be DEH, DHE, HDE.These three peptides were synthesised,its molecular weight were 400Da,the purity were more than 98%.The HMGR inhibitory activities IC50 of these three peptides were 0.18mg/mL,0.06mg/mL,0.004mg/mL.