| Adlay seed is the dried ripe seeds from Coix(soft-shelled Job's tears,Coix lachryma-jobi L.var.ma-yuen Stapf)and mainly produced in Fujian,Hebei and Liaoning Provinces of China.Adlay seed has the function of inducing diuresis to remove dampness,strengthening the spleen to stop diarrhea,clearing heat and draining pus.It is widely used in clinic in the treatment of edema,dysuria,diarrhea due to spleen deficiency,lung abscess,intestinal abscess,and muscular spasm or stiffness of tendons in the Bi Syndrome(arthritis)of the dampness type.Coix contains numerous biochemical components and has a wide range of pharmacological activities.Even though it is rich in protein(14.17%),little attention is paid on the function of Coix protein and derived bioactive peptides.Hypertension is a serious cardiovascular disease that threaten people's health worldwide.Although chemical drugs can lower down blood pressure quickly,they also bring about severe side effects at the same time,such as tussiculation,skin rash,pruritus and so on.Therefore,to develop an alternative antihypertensive drug that is safe and reliable is critical to public health.In this study,glutelin of Coix was selected as the research object.The bioactive peptides from glutelin were obtained by enzyme hydrolysis and various techniques were applied for separation and purification of them,then the antihypertensive activity in vivo of sma,ll molecular weight peptides(?3 KD)was further confirmed by animal experiments.At first,four kinds of protein,albumins,globulins,prolamins and glutelins,were sequentially extracted and then quantified,which accounted for 1.43,6.20,44.74,37.38 percent of total protein content,respectively.The contents of amino acids were measured after acid hydrolysis of Coix and 15 kinds of amino acids were detected including 9 kinds of essential or semi-essential amino acids.Then five different kinds of protease were employed to hydrolyze glutelin for 48:h with substrate concentration of 2%,[E]/[S]ratio of 1:10.The ACE inhibitory(ACEI)activity of the resulted peptides was determined by RP-HPLC method and pepsin was selected as the optimal protease in hydrolyzing glutelin.The optimization of hydrolysis conditions was conducted by single factor and orthogonal experiments with degree of hydrolysis(OPA method)as indicator.The optimum hydrolysis condition of glutelin was[E]/[S]ratio of 1:5,substrate concentration of 2%and hydrolysis duration for 48 h.For separation and purification of the glutelin derived peptides,ultrafiltration(molecular weight cut off 3 KD)was first used,and permeates were collected and freeze dried.ACEI activity of the small peptides was determined and the IC50 value was 201.2± 12.43 ?gmL.Three fractions(designated as A,B,C)were obtained after separating with Sephadex G-10 and the ACEI activity were 72.17±3.82%,69.75± 1.69%,25.2±2.18%,respectively,at the concentration of 1 mg/mL.The IC50 of fraction A and B were determined to be 91.65±6.41?g/mL and 98.78±13.8?g/mL.Ion exchange chromatography was performed to further separate A and B to obtain high ACEI sub-fractions A2(83.2±2.87%at 0.5 mg/mL)and B2(73.1±1.89%at 0.5 mg/mL).A2 and B2 were finally separated with semipreparative HPLC method and four fractions of A2-2,A2-8,B2-2,B24 with improved ACEI activity were obtained The ACEI activity of the four ones were 93.66±0.22%,73.23±3.86%,91.96±3.30%,89.70±2.40%,respectively'at the concentration of 0.1 mg/mL,which was elevated by more than five times compared vith A and B at the same concentration(18.62±1.26%,10.28±0.92%,respectively).SHR was taken as model of animal experiments in vivo to evaluate the pharmacodynamic effects of glutelin derived small peptides.The once-oral administration experiments showed that the blood pressure of SHR dropped to a minimum value 4 hours post-administration by 13.2 mmHg,21.5 mmHg,32.5 mmHg at a peptides dosage of 100 mg/kg,200 mg/kg,400 mg/kg,respectively.While the blood pressure of SHR treated with 20 mg/kg Captopril dropped to the minimum(161.8±3.3 mmHg)6 hours post-administration.The blood pressure of administration groups reduced significantly compared to the blank control(185.3±2.3 mmHg)and the antihypertensive effect of glutelin peptide groups showed a dose-dependent manner.The results of long-term oral administration with peptides in two different doses(300 mg/kg3 450 mg/kg)showed that the blood pressure of SHR declined to 154.5±3.2,149.0±3.4 unHg,respectively,comparing to the Captopril group of 153.9±2.8 mmHg at the dosage of 20 mg/kg,but the peptide did not affect the blood pressure of Wistar rats(118.1±4.5 mmHg)signif cantly during the whole experiment.The Ang ? level of SHR administrated with peptides at doses of 300 mg/kg,450 mg/kg and Captopril(20 mg/kg)were 819.6±346.5,513.4±199.6,388.7± 145.3 pg/mL,respectively,and there was no significant difference compared with the control group(542.5±295.9 pg/nL).The level of plasma renin activity(PRA)were 59.66±48.16,29.03±29.29,27.01± 14.46 ng/mL·br and only the Captopril group showed significantly higher than the control group(31.23±18.82 ng/mL·hr).Our study showed that Coix glutelin can release antihypertensive peptides by being hydrolyzed by pepsin in vivo.Therefore,long-term intake of Coix can exert antihypertensive effect to a certain extent and Coix is a potential resource for health food with antihypertensive peptides. |
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