| The seed of adlay(Coix lachryma-jobi L.var.ma-yuen Stapf),one of effective herbs inducing diuresis to remove dampness,can reduce hypertension effectively.In our previous research,there was 14.17%proteins in coix seed and the highest content for prolamin that accounting for 44.74%of total protein.The hydrolysate of prolamin showed potential angiotensin converting enzyme(ACE)inhibitory activity in vitro.Therefore,in this thesis,the prolamin of coix seed was chosen to obtain the antihypertensive peptide with high ACE inhibitory activity and clear structure by enzymolysis method.The expression regulation effect of the peptide on local renin-angiotensin system(RAS)of human vascular endothelial cell(VEC)was also evaluated.Coix prolamin was hydrolyzed under the condition of simulated in vitro digestion by alone or in combined with pepsin,trypsin and alpha-chymotrypsin.The optimized hydrolysis condition was confirmed by determinations of ACE inhibitory activity and degree of hydrolysis.The optimal hydrolysis condition was substrate concentration of 1%(w/v),enzyme-to-substrate ratio of 1:5(w/w),and hydrolysis duration for 48 h at 37 ℃.Small molecular coix prolamin peptide(CPP)was obtained by ultrafiltration(MWCO=3 KDa).The antihypertensive activity in vivo of CPP was further evaluated by once-oral administration experiments with spontaneously hypertensive rat(SHR).Systolic blood pressure(SBP)of SHR was respectively decreased 12.5 mmHg,16.0 mmHg,and 28.8 mmHg at 125 mg/kg,250 mg/kg,and 500 mg/kg CPP with a dose-dependent manner.A transcriptome database was built by RNA-seq for enhancing the accuracy of peptide identification based upon mass spectrometry.The raw reads totaled 6.56 Gb.After being assembled and redundancy removed,76164 unigenes were rsulted.All unigene were annotated by compared unigene to seven functional databases including NR,NT,Swissprot,COG,KEGG,GO and Interpro.Totally 49092,56121,32748,20670,30941,27407 and 29789 unigenes were separately annotated to each database.Totally,48449 coding sequence(CDS)were obtained according to the gene annotation results except for 2711 more CDS from ESTScan.The protein data based of coix was successfully constructed.The CPP was further purified by ion exchange chromatography,gel filtration chromatography,and reversed-phasedhigh performance liquid chromatography(RP-HPLC).After further purified by DEAE-Sepharose anion exchange column,four fractions were collected:A,B,C and D.At the final concentration of 0.01 mg/mL,the ACE inhibition rates of these four fractions were 41.55±0.80%,7.26±0.46%,40.85±0.57%and 12.39±0.44%,respectively.The fraction C,which had higher content than fraction D,was then further separated into four fractions(C1-C4)using a Sephadex G-10 gel filtration chromatography.Among these,fraction C1 had the highest ACE inhibitory activity(53.05±2.19%at the final dose of 0.01 mg/mL),which increased by 12.20%compared with C fraction,and was further isolated through RP-HPLC.Thirteen fractions(C1-1～C1-13)were obtained and fraction C1-12 displayed the strongest ACE inhibitory activity(65.06±2.21%at 0.01 mg/mL).The C1-12 fraction was analyzed using high-performance iquid chromatography electrospray ionization tandem mass spectrometry(HPLC-ESI-MS/MS),and 21 peptides were then identified based on the protein database of Coix(Trans database)and Uniprot databases.All the peptides were screened by pharmacophore and molecular docking technology.According to the pharmacophore mapping results,7 peptides had the ACE inhibitory potency,while the molecular docking simulation indicated that none of them were successfully and wholly docked.Therefore,in silico proteolysis was implemented to hydrolyze these 7 peptides.Wherein,pepsin,trypsin and chymotrypsin,typical enzymes in the gastrointestinal digestion tract,were utilized.The resulted peptides were compared with data of BIOPEP,and 25 generated peptides were obtained finally.Among these peptides,5 peptides(CPPT02-1,CPPT03-3,CPPU05-1,CPPU10-2 and CPPU10-4)that might display high ACE inhibitory potential were selected,and the IC50 value of CPPT03-3 was 382.28±14.43 μM.An endothelial cell injury model was established by treating human umbilical vein endothelial cells(HUVEC-12)with Ang Ⅱ to explore the influence of CPPT03-3 on cell viability,the extracellular NO level and the key gene expression of RAS in injured cells.The groups were divided as follows:blank control group,Ang Ⅱ injury model group(1×10-6 mol/L Ang Ⅱ),positive control group(1×10-5 mol/L Captopril+1×10-6 mol/L Ang Ⅱ),low-dose CPPT03-3 group(1×10-4 mol/L CPPT03-3+1×10-6 mol/L Ang Ⅱ),middle-dose CPPT03-3 group(2 ×10-4 mol/L CPPT03-3+1×10-6 mol/L Ang Ⅱ),high-dose CPPT03-3 group(4×10-4 mol/L CPPT03-3+1× 10-6mol/L Ang Ⅱ).The data showed that the viability of HUVEC-12 was inhibited after being treated by Ang Ⅱ(1×10-6 mol/L)for 48 h.Captopril and CPPT03-3 significantly alleviated Ang Ⅱ-induced injury of HUVEC-12.The extracellular NO content significantly decreased after incubation with Ang Ⅱ and obviously increased in a dose-dependent manner by the simultaneous incubation with CPPT03-3.The effects of CPPT03-3 on the expressions of local RAS key genes in VEC were as follows.Compared with the blank control,the gene expression of ACE,AT1R in damage model group both increased,and that of ACE2 obviously decreased,while the mRNA expression level of Mas was not influenced significantly by Ang II.The up-regulation of ACE gene was antagonized by CPPT03-3 and Captopril.The decreasing expression of AT1R gene in the damage model group was detected after treated with middle-dose and high-dose CPPT03-3,while no remarkable changes in low-dose CPPT03-3 group and positive group.The ACE2 gene expression level in low-dose and middle-dose CPPT03-3 group were all lower than model group,and restored normally for high dose CPPT03-3 group.The ACE2 gene expression level of damage cells increased after being treated with Captopril.The Mas gene expression was enhanced by CPPT03-3 in a dose-dependent manner.In addition,compared with model group,the gene expression level of Mas had no significantly change in positive control group.This study showed that small molecule peptide CPP with antihypertensive activity in vitro and in vivo was produced by enzymolysis simulating gastrointestinal digestion.The active fraction C1-12 was further isolated and then was identified by mass spectrometry.The peptide CPPT03-3 with potent ACE inhibitory activity in vitro was finally obtained by virtual screening.The peptide CPPT03-3 played vital role on the regulation of VEC local RAS.This research lays a solid foundation for the development and mechanism research of coix seed antihypertensive peptide,and also provides theoretical guidance for the clinical application of coix. |
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