Study On The Preparation And Inhibition Mechanism Of Dual Renin And ACE Inhibitory Peptide Derived From Rapeseed Protein

Hypertension is one of the primary risk factors associated with cardiovascular diseaseevents including myocardial infarction, stroke, heart failure and chronic kidney disease. Theprimary tasks for prevention and treatment of hypertension are building hypertensionprevention and control system engineering, and developing new blood pressure loweringagents and nutraceuticals. Renin-angiotensin system (RAS) is a major regulator of bloodpressure and fluid homeostasis, and the renin and angiotensin-converting enzyme-I (ACE)inhibitors could reduce the blood pressure resulting from the deregulation of the RAS system.Food-derived peptides have been used for development of functional foods and healthproducts related to lower blood pressure due to their safety and multi-functionality. In thisstudy, rapeseed protein is going to be hydrolyzed by proteinase to release the dual renin andACE-inhibitory peptides, which might provide a experimental evidence for the relatedresearch and development of dual inhibitory peptides.Firstly, rapeseed protein hydrolysates (RPHs) were produced using various commercialproteases such as Alcalae, proteinase k, Thermolysin, Pepsin and Pancreatin (P+P), andFlavourzyme, and then separated into different peptide fractions (<1kDa,1-3kDa,3-5kDa,and5-10kDa) by membrane ultrafiltration. The antioxidant activities,, renin and ACE-inhibitory and hypotensive properties were discussed. The results showed that Alcalasehydrolysis significantly (p <0.05) produced the highest yield of protein hydrolysate whileFlavourzyme produced the least. The <1kDa fraction was the most abundant after themembrane ultrafiltration of the protein hydrolysates, which indicates that the proteases wereefficient at reducing the native rapeseed proteins into low molec ular weight peptides. TheAlcalase and Proteinase K fractions had less ability to reduce ferric ion but better capacity toscavenge superoxide radicals and inhibit linoleic acid oxidation, while the opposite resultswere observed from P+P and Flavourzyme fractions. The <1kDa peptide fractions weregenerally the most effective scavengers of free radicals and ferric reducing power but hadweaker iron chelating ability when compared to peptide fractions with sizes>3kDa. In vitroinhibition of the ACE was significantly higher (p <0.05) for the Thermolysin, Proteinase Kand Alcalase RPHs when compared to the P+P and Flavourzyme RPHs. The Alcalase RPHhad significantly higher (p <0.05) renin inhibition among the RPHs. Oral administration (100mg/kg body weight) of the RPHs to spontaneously hypertensive rats showed the AlcalaseRPH to be the most effective in blood pressure (BP) reduction (~24mmHg) while ProteinaseK RPH was the least effective (~5mmHg) after8h. However, the P+P RPH had the mostprolonged effect with BP reduction of~20mmHg after24h of oral administration. Weconclude that Alcalase and P+P (especially its <3kDa fraction) hydrolysates can beconsidered potential alternative peptides ingredients for applications in the hypotensive foodand nutrition industry, and are selected for the further purification to find the dual inhibitorypeptides.Secondly, The Alcalase hydrolyzate was separatied by2times preparative and analyticalreversed-phase column, three peptides Thr-Phe (TF), Leu-Tyr (LY) and Arg-Ala-Leu-Pro (RALP) were purified and amino acid sequence determined by tandem mass spectrometry. LY(IC50,0.11mmol/L) was the most potent (p <0.05) against ACE activity when compared toTF (IC50,0.65mmol/L) and RALP (IC50,0.81mmol/L). However, RALP (IC50,0.97mmol/L)was the most potent (p <0.05) against renin activity when compared to LY (IC50,1.87mmol/L) and TF (IC50,3.1mmol/L). Single oral administration (30mg/kg body weight) tospontaneously hypertensive rats (SHRs) showed LY and RALP to be the more effectivehypotensive agents with maximum blood pressure reduction of~26and~16mmHg,respectively when compared to TF (~12mmHg). The results suggest that the higher numberof hydrophobic amino acid residues LY and RALP contributed to their higher in vitro and invivo activities when compared to TF. In addition, A novel antihypertensive peptide Gly-His-Ser (GHS) with dual inhibition of angiotensin I-converting enzyme (ACE) and renin activitieswas isolated from the <3kDa ultrafiltration fraction of a pepsin+pancreatin rapeseed proteindigest. The IC50values of GHS were1.74mmol/Land1.07mmol/Lfor ACE and renininhibitions, respectively. Oral administration (30mg/kg body weight) to spontaneouslyhypertensive rats showed GHS to be an effective hypotensive agent with maximum bloodpressure reduction of~17mmHg. It was a similar hypotensive effect with the RALP obtainedfrom Aclalase hydrolyzate.Thirdly, On the basis of rapeseed dual inhibitory peptide, the interaction between TF, LY,RALP with ACE and renin was investigated by the enzyme inhibition kinetics, fluorescencespectroscopy and circular dichroism spectroscopy and molecular docking. The ACE inhibitionpatterns were uncompetitive for TF, and competitive for LY and RALP, while TF exhibited tobe a non-competitive mode of the renin inhibition, and LY and RALP were like that ofmixed-type. The fluorescence intensity of ACE was mainly quenched by TF by collision withthe ACE molecular, while LY and RALP were quenching the fluorescence intensity by staticbinding to ACE molecular. The quenching mechanism between TF and renin was staticquenching by binding to the two active sites, while LY and RALP quenched the fluorescenceintensity by the combination of binding to renin and forming renin-peptide complex andcolliding renin molecular. The observed structural changes of ACE and renin were distinctlydependent on the concentration and variety of the added peptides. For example, Theinteraction of LY with ACE principally led to the increase of unordered structure (38.03±1.21%) and the decreases of α-helix and β-strand at low concentration of LY, while theadditional LY (25μg/mL) further reduced α-helix(1.41±0.17%) and β-turnto form β-strandstructure (42.80±0.17%). RALPresulted in the transformation of ordered β-strand and β-turnto α-helix (10.93±1.15%) at12.5μg/mLwhile high concentration of RALP totally exposedthe renin structure with the83.05±0.07%unordered structure and small amount of α-helixand β-sheet formation. LY possessed better docking and affinity effects with ACE moleculeswhen compared with that of TF and RALP; though molecular weight of RALP was greaterthan that of TF and LY, hydrogen bonds formed between RALP and active center of reninmolecular were much more than that of TF and LY.Finally, the electrodialysis with ultrafltrationsystem(EDUF) was employed to separatedAlcalase hydrolyzate (ORPH). The results showed that the hydrolysate was mainly separatedinto three fractions a cationic peptide, an anionic peptide and the separation residual (FRPH) of the hydrolyzate by EDUF. their IC50values of ACE inhibitory activities were0.0932±0.0037、0.1439±0.0044、0.3146±0.0101and0.1034±0.0024mg/mL, and0.68±0.12,0.47±0.05,0.89±0.17and0.56±0.07mg/mL for renin inhibitory activity (IC50).Single oral administration (100mg/kg body weight) to SHRs showed ORPH and FRPH to bethe more effective hypotensive agents with maximum blood pressure reduction of~47and~51mmHg, respectively, which were better than that of Captopril (10mg/kg bw). The higherrenin inhibition might contribute to hypotensive effect of FRPH when compared to ORPH. Inaddition, hypotensive effect of cationic peptide was similar to that of Captopril within24h.Overall, hypotensive effect of ORPH obtained by a direct hydrolysis rapeseed meal withAlcalase was improved after electrodialysis with ultrafltration separation, evidenced with theobtained FRPH and cationic peptides.