| Angiotensin ? converting enzyme inhibitor(ACEI)as one of first-line antihypertensive drugs,can exert antihypertensive effect by reducing the generation of angiotensin ?(Ang ?)and weakening the degradation of bradykinin(BK).However,these synthetic drugs may also cause obvious side effects such as cough,taste disorder,rash and leucopenia.Food-derived ACE inhibitory peptide has drawn considerable attention since its gentle,high safety and the characteristic of no influence on the normal blood pressure.Male black soybean(Glycine max(L.)Merr.),a variety type of soybean with black seed coat,is often used to treat dizziness,vertigo,restlessness and thirst due to yin deficiency,and edema with scanty urine.Vinegar soaked black soybean is made of black soybean by soaking in the vinegar.It is widely used as a antihypertensive prescription in the folk.Black soybean has protein content as high as 36-40%,and its peptides have ACE inhibitory activity with some characteristics such as easy digestion and absorption and high solubility.Therefore,we speculate that the peptides of black soybean may be one of the important effective constituent for its antihypertensive effect.However,it is uncompletely clear that the effect of vinegar soaking procession on black soybean protein content,its fractions ratio,and its ACE inhibitory peptides activity.In this thesis,with male black soybean as the research object,the effect of vinegar soaking on black soybean protein content and its peptides' ACE activity was analyzed to selecte the optimal protein fracton.ACE inhibitory peptides were obtained by the enzymatic hydrolysis.Then the ultrafiltration(UF),ion exchange chromatography(IEC)and reversed phase high performance liquid chromatography(RP-HPLC)were employed to separate and purify the peptides.The liquid mass spectrometry(HPLC/ESI-MS/MS)and molecular simulation technology were used to identify the structure and virtual screening,respectively.Finally,highly active ACE inhibitory peptide was obtained and then its in vitro and in vivo antihypertensive activity were evaluated.Research on the major component of black soybean.The contents of protein,crude fat,starch,water and ash were dermined by Kjeldahl,Soxhlet extraction,Polarimetry,Drying and Muffle furnace method,respectively.The results showed that:black soybean had the highest protein content,accounting for 46.74 ± 0.22%of fresh weight.Crude fat,starch,water and ash accounted for 19.70 ± 0.08%?15.96 ± 0.83%?6.67 ± 0.01%and 4.45± 0.01%,respectively.Therefore,black soybean is rech in protein,and its biological peptides obtained by enzymatic hydrolysis may have great research value.Research on the effect of vinegar soaking on black soybean protein content and its fractions ratio.Black soybean was soaked in vinegar for 7 d and 14 d,respectively.Albumin,globulin,prolamin and glutelin were sequentially extracted and then quantified.The results showed that the total protein content of black soybean was changing within 0.5%(46.73 ± 0.22%(0 d),47.22± 0.62%(7 d),46.41 ± 0.23%(14 d))after soaking in vinegar.While the contents of four protein fractions had a large change.Overall change trend was that after soaking albumin content reduced,however,globulin,prolamin and glutelin content increased.The glutelin content of 14 d vinegar soaked black soybean accounted for 59.45%of total protein.Among them,albumin content reduced from 25.76%(0 d)to 4.46%(14 d).Glutelin content were 9.87%(0 d),27.56%(7 d)and 27.59%(14 d),respectively.Research on the effect of vinegar soaking procession on peptides' ACE inhibitory activity.Four protein fractions were hydrolyzed using pepsin and the hydrolysates were fractionated by ultrafiltration(MWCO= 3 KD)to prepare low weight peptides(<3 KD).The data showed that after soaking in vinegar for 7 d,peptides from albumin and prolamin had no significant activity change.However,after 14 d,albumin peptides activity reduced from 21.09%(7 d)to 6.11%(14 d),while prolamin peptides activity increased from 30.05%(7 d)to 35.85%(14 d).Globulin peptides activity had no significant change during soaking in vinegar.Glutelin peptides activity increased with the extension of vinegar soaked time,from 18.18%(0 d)to 37.58%(14 d).Therefore,glutelin from 14 d vinegar soaked black soybea has been selected as the optimal protein to obtain ACE inhibitory peptides.Research on preparation parameters of ACE inhibitory peptides from glutelin.Glutelin was hydrolyzed using pepsin,trypsin,chymotrypsin and alcalase,respectively,and the hydrolysates were then fractionated by ultrafiltration(MWCO=3 KD)to prepare low weight peptides(? 3 KD).The result showed that the peptides from alcalase hydrolysates had the highest activity with ACE inhibition ratio of 47.47%,at the final concentration of 0.01 mg/mL.With the degree of hydrolysis and ACE inhibitory activity as selection index,the glutelin hydrolysis conditions for alcalase was[S]= 2%,[S]/[E]= 1:10 at 60 ? for 4 h.The separation and purification of ACE inhibitory peptides.The vinegar soaked black soybean glutelin hydrolysates were large-scale fractionated using ultrafiltration membrane(MWCO= 3 KD)to obtain:<3 KD fraction.The ACE inhibition ratio was 44.16%at the final concentration of 0.01 mg/mL.The<3 KD fraction was then separated into four fractions L1,L2,L3 and L4 on anion exchange chromatography.Their inhibition ratio were 68.81 ± 1.51%,27.50 ± 0.73%,30.57 ± 0.99%and 14.50 ±0.32%,respectively.Fraction L1 was then further separated using RP-HPLC,and seven fractions were obtained(L1-1-L1-7).Among those fractions,fraction Ll-2 had the lowest activity with inhibition ratio of 15.37± 0.73%.However,fraction L1-4 displayed the strongest activity with inhibition ratio of 83.41 ± 0.91%.The amino acid sequence of fraction L1-4 was analyzed by electrospray ionization tandem mass spectrometry(ESI-MS/MS)based on a LTQ Orbitrap Velos system.In total,five peptide sequences(CD001-CD005)were identified by searching the UniProt database.The activity prediction and structural optimization of the identified peptides.ACE pharmacophore model and molecular docking(three human's ACE protein receptor models:1086,4BZR,4CA5)were performed to explore the ACE inhibitory activity of five identified peptides.Among them,four sequences CD002?CD005 matched with pharmacophore model with FitValue 0.96,0.96,0.92 and 0.95,respectively.CD002 docked with three receptors 1086,4CA5 and 4BZR,while CD001 and CD003 just only docked with 4BZR.Based on above two results,we speculate that CD002 and CD003 may have great activity potentail.CD002 and CD003 were further studied by simulating gastrointestinal hydrolysis with the Peptide Cutter database.Finally,A peptide CD003-1 with high ACE inhibitory potential was obtained with pharmacophore FitValue of 0.96.Moreover,it was successfully matched with 1086,4BZR and 4CA5 with-CDOCKER-ENERGY 134.79,121.88 and 97.20,respectively.It was chemically synthesized,and its IC50 value was 117.11?M.Research on in vivo antihypertensive effect of the peptide CD003-1.Spontaneously hypertensive rats were devided into the blank control group(distilled water),the positive control group(15 mg/kg captopril),low dose group of CD003-1(10 mg/kg),medium dose group of CD003-1(20 mg/kg)and high dose group of CD003-1(40 mg/kg)to evaluate in vivo antihypertensive effect of CD003-1.The result showed that the maximum reduction of SBP of low dose,medium dose group and high dose group were 10.41 mmHg,21.95 mmHg and 33.58 mmHg,respectively.The antihypertensive effect of medium dose group was similar to that of captopril group(23.00 mmHg),and this effect could continue for about 8 h.It mean that peptide CD003-1 had significant antihypertensive effect.This thesis showed that CD003-1,which was obtained from vinegar soaked black soybean,had significant in vitro and in vivo antihypertensive activity.Peptide CD003-1 could be a good ingredient for pharmaceuticals against hypertension.Meanwhile,our research also provides new idea that from the perspective of peptides,the antihypertensive effect of vinegar soaked black soybean could be revealed. |
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