Studies on the Angiotensin Ⅰ-converting Enzyme Inhibitory Peptide from Pumpkin Seed Protein
|School||Hunan Agricultural University|
|Course||The horticultural products of Postharvest Science and Technology|
|Keywords||pumpkin seeds angiotensin-coverting enzyme ACE Inhibitory peptide human umbilical vein endothelial cells Real-time fluorescence quantitative PCR Protein two dimensional electrophoresis|
This paper studied the collection, isolation, purification of the pumpkin seeds meal peptides (PSMPS) and its cardiovascular functional effects and mechanisms using pumpkin seed meal (PSM) as raw materials. The meal was hydrolyzed by six proteases and those productions inhibiting ability of angiotensin-coverting enzyme (ACE) were analyzed, some proteases PSMPs were screened for making ACEI and their process condition was optimized by Response Surface Analysis. The PSMPs’ were collected, isolated, purified and analyzed through many methods including Gel column chromatography, RP-HPLC and UHPLC-Q-TOF technology. The PSMPs’ effects on proliferation of human umbilical vein endothelial cells (HUVECs) and expression of some related genes’mRNA were analyzed by Real time fluorescence quantitative PCR technology so as to analyze the PSMPs’cardiovascular functional and mechanisms. The main results are showed as follows:1. The protein content, isoelectric point and molecular weight of oil removed PSMs were tested by two dimensional electrophoresis technology, the result showed their protein content was75.63%, its isoelectric point was pH5-7and Molecular weight were22KDa-42Kda.The amino acid composition analysis of enzymatic hydrolysate of PSM’s neutral protease by automatic amino acid analyzer showed there were34.40g hydrophobic amino acid,11.49g aromatic amino acid,6.68g Proline and12.92g branched chain amino acid in100g protein, its average hydrophobicity was4.189kJ/mol. These results indicated that the protein of PSM and its enzymatic hydrolysate had great amino acid positive related to ACEI activity, its composition was good for producing antihypertensive peptides through enzyme method.2.Six Protease including Trypsin, Hydrolysis protease, Papain, Neutral protease, Protease N and Protease S were screened for producing ACEI Peptides according to their ACE Inhibiting ability. The Neutral protease was the best and its hydrolysis reaction factors were optimized using Response Surface Analysis. The results showed its best reaction condition were45℃and pH7.0, the protease content was4.8%, Substrate content was4.0%, Hydrolysis time was320min, the ACE inhibiting percentage could be80.56±0.23%under the condition.3. four compositions were isolated from enzymatic hydrolysates of PSM using Sephadex G-75Gel chromatography, they were Z1(MW>14220Da), Z2(MW14200 -8400Da), Z3(MW8400-4000Da), Z4(MW<4000Da), respectively, the Z4had80.23±0.21%inhibitory percentage. The Z4was isolated by Sephadex G-15Gel chromatography,3compositions were isolated and named Z4-1, Z4-2和Z4-3, the Z4-2had81.56±0.41%inhibitory percentage so it was isolated and purified by RP-HPLC method.7compositions were isolated from Z4-2and named R、R2、R3、 R4、R5、R6and R7, respectively, R4had the highest inhibitory percentage(79.63±0.32%). R4was tested to be Single component through RP-HPLC method again, its Relative molecular weight was905.67Da and Amino acid sequence was L(/I)L(/I)L(/I)SHDL(/I)V[Leu (/Ile)-Leu (/Ile)-Leu (/Ile)-Ser-His-Asp-Leu (/Ile)-Val]. R4was a novel ACEI peptide.4. PSMPs’effects on HUVEC experiment indicated the effects were Proportional to the PSMPs’concentration and reaction time, the PSMP might have similar mechanism with Captopril, it inhibited cell’s proliferation through blocking the cell at S stage. The ACEI could lower the blood pressure through reduce the ET-l’s concentration of cell’s supernatant, but which need enough content.The mRNA expression of ET-1, ACE and ACE2genes were tested by Real-time fluorescence quantitative PCR. The results showed both the PSMP and Captopril could reduce the3genes’ expressions, the PSMPs’ concentration higher, the expressions reduce more. The results also indicated the Generation of Angll were influenced by expression of ACE/ACE2mRNA. The termination agent method showed PSMP could clear02-at some degree, which eases endothelial cell’s injury so as to reduce blood pressure.The above results indicated the PSMP had determined reduce blood pressure function, its mechanism might be associated with inhibiting the HUVEC’s Proliferation, clearing the O2-, reducing the activity of ACE and expression of mRNA of ACE and ET-1genes, Promoting expression of mRNA of ACE2. all those factors could reduce the content of Angll and ET-1. PSMP is Worthy of research and development as reduce blood pressure natural products.