| Hypertension,one cardiovascular syndrome,is mainly characterized by constant increasing arterial blood pressure.It is the major risk factor that leads to cardiovascular and cerebrovascular diseases,and has a high incidence,disability and death rates.In recent years,hypertensive individuals are increasing year by year in China,furthermore,their life quality has been badly influenced.Traditional Chinese medicine has obvious advantages and characteristics for the treatment of hypertension.The invigorating spleen to remove dampness is the principal treatment standard.Adlay seeds,the dried ripe seeds from Coix lachryma-jobi L.var.ma-yuen Stapf,are mainly produced in Fujian,Hebei and Liaoning Province of China.Adlay seed has the effects of strengthening the spleen to stop diarrhea,inducing diuresis to remove dampness,draining pus and clearing heat.It is widely used for the treatment of dysuria,edema,diarrhea due to spleen deficiency,intestinal abscess,lung abscess,and muscular spasm or stiffness of tendons in the Bi Syndrome(arthritis)of the dampness type in clinic.Our previous researches showed that Coix seed is a typical of high ptotein content and rich in amino acid with preference for the ends of antihypertensive peptides.In this thesis,glutelin of Coix was selected as the research object.The angiotensin converting enzyme(ACE)inhibitory peptides from glutelin were obtained by pepsin hydrolysis.Then the peptides with molecular weight less than 3 kDa(designated as the ACE inhibitory peptides)obtained by ultralitration(UF).The inhibitory activity of peptides obtained under different pressures was investigated.The results showed that the inhibitory rate of the peptides obtained under 0.1 Mpa UF pressure was significantly higher than those of other pressure(P<0.05),and the IC50 of it was further determined to be 46.70±1.34?g/mL.Therefore,0.1 Mpa was considered as the optimal UF pressure.The solubility and stability of the ACE inhibitory peptides from glutelin of Coix were then investigated.The results showed that the solubility of the ACE inhibitory peptides derived from Adlay glutelin was above 70%and not affected by pH.Data of hermal stability manifeasted that the ACE inhibitory peptides could remain high activity(62%)after being incubated at the temperature of 0-80?,which suggested that the peptides had satisfying thermal stability.Furthermore,at pH range of 2.0 to 12.0,the maximum ACE inhibitory rate was 66.37%at pH 6.0.The ACE inhibitory rates were reduced to 59.25%and 57.60%,respectively,at pH 2.0 and 12.0,eventhough still with high inhibitory activity.Furthermore,after being digested by the mixture of pepsin,?-chymotrypsin and trypsin,the inhibitory rate of the peptides was significantly improved to 78.98%vs 59.92%for the blank control which provided some guidance for the following research.The ACE inhibitory peptides were seperated by gel filtration chromatography(Sephadex G-10)and five fractions(designated as F1-F5)were obtained.The inhibitory rates were 68.62±0.21%,76.90±1.09%,70.53±1.12%,75.41±0.37%,84.75±0.42%,respectively,at the concentration of 0.02 mg/mL.The inhibitory rate of F5 was significantly higher than that of other fractions(P<0.05)and the IC50 was 14.6±0.58 ?g/mL.Semi-preparative RP-HPLC was performed to further purify F5 and nine fractions(F5-1?F5-9)were obtained.The ACE inhibitory rates of the corresponding fractions were 42.14±0.48%,50.42 ± 1.81%,60.06 ±0.72%,3.39 ± 2.07%,31.22±0.71%,30.18±2.01%,14.91 ± 2.73%,41.10±1.26%,9.61±3.21%,respectively,at the concentration of 0.01 mg/mL.Fraction F5-3 showed the highest inhibitory activity and IC50 was 9.80±0.02 ?g/mL.The activity of F5-3 was further improved than the former fraction F5.F5-3 was then analyzed by ESI-MS/MS and six peptides(BUCM001-BUCM006)were identified by search engine against NCBI non-redundant and Uniprot protein database.The peptides consisted of 6 to 18 amino acids with molecular weight rangeing from 756.40 to 1966.95.The ACE pharmacophore model and molecular docking(three different human's ACE protein receptor models:1086,4BZR,4CA5)were employed for predicting the ACE inhibitory activity of the six identified peptides.The results showed that BUCM003 and BUCM004 were well matched with the pharmacophore,and their Fitvalue were 0.97 and 0.96,seperately.Then,BUCM003 and BUCM004 were tested by molecular docking,and the results demonstrated that BUCM004 had a better the interaction with receptor proteins,and successfully bonded with 1086 and 4CA5,with their-CDOCKER ENERGY were 180.40 and 184.83,respectively.Our previous results showed that the ACE inhibitory activity of peptides increased significantly after the digestion by proteases,thus BUCM004 were further simulated gastrointestinal hydrolysis and structure optimization with the assistance of BIOPEP and PeptideCutter software.Finally,compound named BUCM004-3-1 was well matched with the pharmacophore with the Fitvalue of 0.96.It successfully bonded with 1086,4CA5 and 4BZR,and their-CDOCKER ENERGY were 163.62,147.68 and 117.09,respectively.Moreover,the IC50 value of this synthetic compound was 14.19?M.The systolic blood pressure(SBP)of spontaneously hypertensive rats(SHR)was measured by tail-cuff method to estimate the antihypertensive effects in vivo of Coix powder,Coix glutelin,the ACE inhibitory peptides from Coix glutelin,trapped fluid after UF,fraction F5,F5-3 and BUCM004-3-1.The once-oral administration results showed that SBP decreased of 9.17 mmHg after administration of Coix at the middle dose of 2.70 g/kg body weight(BW),which has significant difference(P<0.05)from the negative control group,suggesting that the Coix has specific antihypertensive effect;The Coix glutelin at the middle dose of 200 mg/kg BW(calculated as 1.35 g/kg Coix powder)decreased the SBP of 9.83 mmHg,which mean the antihypertensive effect was significantly higher than isodose original Coix herb;The maximal decrease of 20.50 mmHg post administration of the ACE inhibitory activity peptides of Coix glutelin(200 mg/kg BW)suggested that the antihypertensive effect was further improved;The SBP of SHR dropped of 20.33 mmHg after administration of the fraction F5 at dosage of 50 mg/kg BW,which indicated that active components were more concentrated than the former fraction.For fraction F5-3 at the middle dose of 100 mg/kg BW,SBP decreased of 20.83 mmHg.However,the BUCM004-3-1 at the dosage of 15.0 mg/kg significantly and maximally decreased the SBP of 27.50 mmHg,which was consistent with that treated with 17.5 mg/kg Captopril,and showed a dose-dependent manner.Our research showed that peptides derived from Coix glutelin had distinct antihypertensive effects,as well as high solubility and stability.The results confirmed that BUCM004-3-1 derived from Coix exerted significant antihypertensive activities,which was expected to be a candidate for pharmaceuticals against hypertension.Meanwhile,it may provide further theoretical and experimental guidance for rclinical antihypertensive application of Coix. |
PDF Full Text Request