Isolation And Efficacy Study Of The Hypotensive Octapeptide BUCM__L01 Derived From Coix Seed Glute

Hypertension is one of the major risk factors for cardiovascular disease which can lead to various complications such as heart attacks,stroke and renal failure.Hypertension is classified into the category of "headache or dizziness" in Traditional Chinese medicine clinic.And phlegm-dampness is one of major common types.The invigorating spleen to remove dampness is the principal treatment standard.Coix seed is dry and mature seed of Coix larchryma-jobi L.var.ma-yuen Stapf It is sweet,light and cold,and belongs to spleen and stomach meridian,with the function of inducing diuresis to remove dampness,strengthening the spleen to stop diarrhea,clearing heat and draining pus.It is a commonly used drug to treat phlegm-dampness hypertension.There are only reports of coixol and coix seed oil about its related pharmacodynamic substances.Our previous research demonstrated that adlay glutelin hydrolysates can significantly inhibite angiotensin-converting enzyme activity.Thus,it is vital to study the composition,structural and efficacy of Coix polypeptides.In this thesis,glutelin was obtained by sequential extraction,and hydrolyzed by pepsin for 48h with substrate concentration of 2%,[E]/[S]ratio of 1:10.Then,ion-exchange chromatography(IEC)and re versed-phase high performance liquid chromatography(RP-HPLC)were used for separation and purification.The ≤3KD fraction separated by ultra filtration(molecular weight cut-off 3KD)from glutelin hydrolysates showed the ACE inhibition rate of 16.9±1.29%at the final concentration of 0.01mg/mL.Seven faretions(A1～A7)were then separated by weak anion exchange chromatography(DEAE Sepharose Fast Flow).The ACE inhibitory rates of them were 30.39±0.49%,50.48±1.93,36.45±0.07%,27.39±1.19%,40.14±0.09%,23±0.52%and 1.49±0.27%,respectively.Among them,six ones components performanced a higher ACE inhibitory activity than ≤3KD fraction(p<0.05),and the fraction A2 ranked first with the rate of 50.48±1.93%(p<0.05).It was about 33.58%higher than that of ≤3KD fraction.RP-HPLC(TIAN-1825 Kromasil C18 column(250mm×4.6mm))was used for the separation of fraction A2.A total of 11 fractions were collected and named as B1 to B11.The ACE inhibitory activity of them was 61.87±1.48%,73.41±1.05%,55.74±1.03%,65.94±0.49%,62.98±1.09%,45.02±1.05%,60.88±1.54%,34.55±2.05%,34.85±1.01%,49.78±0.88%and 63.26±0.99%,respectively,at the final concentration of 0.01 mg/mL.Among them,activities of seven ons were significantly higher than fraction A2(p<0.05).Fraction B2 showed the highest activity,which was significantly different from the other fractions(p<0.05).The inhibition rate of B2 increased by 22.93%compared with that of A2 in the previous step,indicating that the activity gradually concentrated.Peptide sequences of fraction B2 were identified by HPLC-ESI-MS/MS.A total of 21,379 mass spectrums were obtained.Then Masote 2.3.02 software was used to search the Uniprot database and a Coix protein database established by our group.Finally 25 peptides were identified.The number of amino acids of the peptide was from 5 to 20.The 25 peptides were matched and screened by ACE inhibitor pharmacophore mode.There were 5 peptides with FitValue higher than 0.7,named BUCM_L01(0.963),BUCM_L02(0.960),BUCM_L03(0.893),BUCM_L04(0.834)and BUCM_L05(0.720),respectively.Then,the five peptides were tested by molecular docking.The crystal structures of the receptors were 1086,4CA5,and 4BZR,and their original ligands were lisinopril,phosphopeptides FI and K-26,with the-CDOCKER ENERGY of 106.29,102.32,and 156.91,respectively.The-CDOCKER_ENERGY of BUCM_L01 with 1086,4CA5,and 4BZR were 158.276,118.839,and 116.846,respectively,which showed that ithad a higher potential ACEI activity.BUCM_L01 was synthesized by Fmoc method and its IC50 was determined as 109.57μM.Antihypertensive effect of BUCM_L01 was evaluated in vivo with spontaneously hypertensive rat(SHR).In the single dose experiment,SHRs were randomly divided into 5 groups(n=6),including blank group(distilled water),positive drug group(captopril,15 mg/kg),high-dose group(BUCM_L01,30 mg/kg),middle-dose group(BUCM_L01,15 mg/kg)and low-dose group(BUCM_L01,7.5 mg/kg).After administration,the systolic blood pressure(SBP)of each group SHRs was measured by tail-cuff method.The data showed that the antihypertensive effect of BUCM_L01 was in a dose-dependent pattern.In the high-dose group,the blood pressure dropped to a maximum of 35.4±3.65 mmHg 6 h post administration.The decreasing trend of blood pressure in the middle-dose group and the positive-dose group was similar,with the maximum decrease of 23.6±3.01 mmHg and 22.0±1.14 mmHg,respectively.Compared with the blank group,the difference was statistically significant(P<0.01).For the low-dose group,blood pressure dropped maximumly of 9.25±2.63 mmHg 4 h post administration.The blood pressure of all groups restored to the initial level 24 h later.In the long-term administration experiment,SHRs were randomly divided into 3 groups(n=6),including blank group(distilled water),positive drug group(captopril,15 mg/kg)and peptide groqu(BUCM_L01,15 mg/kg).SHRs were daily administrated once for successive 5 weeks,and the SBP were weekly measured.The results showed that the blood pressure of the blank group increased gradually,with the data of 202±3.79 mmHg in the fifth week.Compared with blank group,the decrease of blood pressure was significant detected in the peptide group and positive drug group.During the first two weeks,SBP of the peptide group and the positive drug group decreased by 18.8±2.19 mmHg and 18.57±2.64 mmHg,respectively.And then,blood pressure increased slightly.At the end of fifth week,the blood pressure for the blank control group,peptide group,and positive drug group was 202±3.79 mmHg,176±5.33 mmHg,and 174.83±2.48 mmHg,respectively.The blood pressure in the peptide group and the positive drug group was much lower than that of the blank group(P<0.01).The results showed that BUCM_L01 derived from Coix seed had high antihypertensive effects in vivo and in vitro,and could provide antihypertensive peptide candidate drugs from traditional Chinese medicine(TCM).In this thesis,components and monomers with high antihypertensive activity were obtained from traditional Chinese medicine coix seed’s glutelin,which provides new experimental data for interpretation of the substance basis of Coix seed,and provides new scientific experimental data and theoretical basis for establishment of Coix seed quality standards based on efficacy and active ingredients.