CN100369928C - Blood pressure lowering polypeptide of corn gluten powder, its separation method and application - Google Patents
Blood pressure lowering polypeptide of corn gluten powder, its separation method and application Download PDFInfo
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- CN100369928C CN100369928C CNB2005101047877A CN200510104787A CN100369928C CN 100369928 C CN100369928 C CN 100369928C CN B2005101047877 A CNB2005101047877 A CN B2005101047877A CN 200510104787 A CN200510104787 A CN 200510104787A CN 100369928 C CN100369928 C CN 100369928C
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Abstract
The present invention discloses a zein powder blood pressure reducing polypeptide, a separation method thereof and the application thereof. The present invention relates to a polypeptide and the preparation thereof, which provides a zein powder blood pressure reducing polypeptide, a separation method thereof and the application thereof to the preparation of blood pressure reducing medicines and foods. The zein powder blood pressure reducing polypeptide has an amino acid sequence of Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala. The present invention has the separation method that collected zein powder is pulverized, and the undersized powder is used as enzymolysis substrate; alkali protease is added for hydrolysis to obtain hydrolyzate on which ion exchange chromatography is carried out to obtain active constituents of highly blood pressure reducing peptide, and then gel chromatography is carried out to obtain the active constituents of highly blood pressure reducing peptide; reversed-phase high-performance liquid chromatography is carried out for two times, and the constituents with high inhibitory activity, which are filtered out, are target products. The present invention has the advantages of highly blood pressure inhibiting activity, no side effects and easy absorption because food protein is used as a source, and no effects on normal blood pressure. The present invention can be used for preparing blood pressure reducing medicines and foods.
Description
Technical field
The present invention relates to a peptide species and preparation thereof, especially relating to a kind of is raw material with the Zein powder, separate the highly active blood pressure lowering polypeptide of corn gluten powder that has that obtains by enzymolysis process, and blood pressure lowering polypeptide of corn gluten powder is in preparation Altace Ramipril and Application in Food.
Background technology
Blood pressure lowering peptide (Angiotensin-converting Enzyme Inhibitory Peptides, ACEIP) be the micromolecule polypeptide that a class can reduce human blood-pressure, it is angiotensin-converting enzyme (Angiotensin-convertingEnzyme, ACE) inhibitor can reach the purpose that brings high blood pressure down by the generation that suppresses the nervous plain II of human vas.Nineteen sixty-five Ferreira (Ferreira, S.H.Abradykinin-potentitiating factor pressing the venom ofBothropsjiararaca, Brit.Pharmacol, 1965,24:163-169) from the venom of South America pallas pit viper, extract a kind of peptide material, find that it has antihypertensive function.Research to blood pressure lowering peptide in the food starts from (G.Oshima such as Oshima, H.Shimabukuro, K.Nagasawa.Peptide inhibitors of angiotensin-convertingenzyme in digests of gelatin by bacterial collagenase.Biochimaica et BiophysicaActa, 1979, the 566:128-137) ace inhibitory peptide that from the gelatin enzymolysis solution, extracts.Because it is obvious that blood pressure lowering peptide has blood pressure lowering effect, normal arterial pressure there are not advantages such as influencing and has no side effect, become the focus of present research.Nineteen eighty-two Maruyamas etc. isolates the ACEI of 12 peptides from the caseic trypsin hydrolyzing thing of ox, begin from more protein source, to obtain the research of ACEI peptide subsequently, now successfully from numerous food proteins such as fish and shellfish, soybean, corn and vinasse, obtained ace inhibitory peptide.Discovery and the new blood pressure lowering peptide of isolation identification remain an important directions of blood pressure lowering peptide research from new raw material.Authorize publication number to provide the synthetic of a kind of cardiovascular active polypeptide and them and the application in medical science for the patent of invention of CN1055479C, said polypeptide is AA-Arg-Pro-Ala-Lys-OH and AA-Arg-Pro-Ala-lys-Arg-Gly-Asp-AA-OH, wherein AA and AA ' are the amino acid of L configuration, relate to their application aspect medical science, for example as expanding blood vessel, hypotensive and antithrombotic reagent use, with the pharmaceutical composition of this class polypeptide as activeconstituents.Publication number is that the application for a patent for invention of CN1552894A discloses a kind of low-molecular-weight function corn small peptide product, utilizes the zein in the direct degrading maize protein powder of enzyme process to prepare the method for this small peptide and the application of this small peptide product.This corn peptide product dry powder is a kind of flaxen powder, and molecular weight distribution is the small peptide mixture that contains the 2-9 amino-acid residue mainly below 1000, measures 4-5 amino acid of mean chain length with the TNBS method.The preparation method relates to steps such as raw materials pretreatment, enzymic hydrolysis, enzyme-deactivating, refining and dry powder process.
Summary of the invention
The object of the present invention is to provide a kind of blood pressure lowering polypeptide of corn gluten powder; Another object of the present invention is to provide a kind of method of utilizing enzymolysis process from Zein powder, to separate blood pressure lowering polypeptide of corn gluten powder; Another object of the present invention is to the application that blood pressure lowering polypeptide of corn gluten powder is used to prepare Altace Ramipril and food.
Its aminoacid sequence of blood pressure lowering polypeptide of corn gluten powder of the present invention is:
Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala，
Its molecular weight is 970.6855, and theoretical iso-electric point is 5.52, hypotensive activity IC
50Be 17.5 μ mol/L, be white powder, soluble in water.
IC
50Be defined as and suppress the needed inhibitor concentration of ACE enzymic activity one half under certain condition.Described blood pressure lowering polypeptide of corn gluten powder does not all come from zein, but comes from other albumen.
The separation method of blood pressure lowering polypeptide of corn gluten powder of the present invention the steps include:
1) the raw material Zein powder is collected undersized pale yellow powder after crushed as the enzymolysis substrate;
2) in Zein powder, add hydrolysis by novo and get hydrolyzed solution, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: (0.03～0.05AU), pH is 7～9, and hydrolysis temperature is 50～70 ℃, and hydrolysis time is 7～8h;
3) hydrolyzed solution separates obtaining high blood pressure lowering peptide active ingredient through ion exchange chromatography;
4) the high blood pressure lowering peptide active ingredient that separation is obtained gets high blood pressure lowering peptide active ingredient through gel chromatography;
5) the high blood pressure lowering peptide active ingredient that separation is obtained is through twice RPLC, filters out to have the high active component that suppresses, and through measuring, its sequence is Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala.
In step 1), the most well after crushed 200 mesh sieves of raw material Zein powder.
In step 2) in, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.042AU, and pH is 8.0, and hydrolysis temperature is 60 ℃, and hydrolysis time is 6h, Sumizyme MP is selected from Alacalase 2.4L.
Blood pressure lowering polypeptide of corn gluten powder of the present invention has the high blood pressure activity that suppresses, and can be used for preparing Altace Ramipril.
Blood pressure lowering polypeptide of corn gluten powder of the present invention has the high blood pressure activity that suppresses, and can be used for preparing blood pressure reducing food.
By being compared with the aminoacid sequence of the zein of having reported, the blood pressure lowering polypeptide of corn gluten powder of the present invention's acquisition shows, the polypeptide that the present invention obtains does not all come from zein, but come from other albumen, and this peptide sequence has not yet to see report.Can suppress the ACE activity and reduce the blood pressure of essential hypertension mouse when doses, its aminoacid sequence is different with the blood pressure lowering peptide sequence in the corn source of having reported, belongs to newfound blood pressure lowering peptide.The inside and outside activity experiment that provides from following examples will show that blood pressure lowering polypeptide of corn gluten powder of the present invention has tangible hypotensive activity.Because blood pressure lowering polypeptide of corn gluten powder of the present invention derives from food proteins, therefore have characteristics such as having no side effect, easily absorb, normal arterial pressure is not had effect, can be developed as functional medicine and food with hypotensive activity.
Description of drawings
The change curve of SHR systolic pressure in 24 hours after Fig. 1 administration.In Fig. 1, abscissa is fed the back time (h) for irritating, and ordinate zou is Δ SBP (mmHg).
Embodiment
Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.
Embodiment 1
1) with the zein is raw material, it is bigger brown platy shaped particle, enzyme-to-substrate can fully contact during for the ease of enzyme digestion reaction, raw material carries out pre-treatment as follows: the raw material Zein powder is crossed 200 mesh sieves after pulverizer is pulverized, collect undersized pale yellow powder as the enzymolysis substrate.
2) in Zein powder, add hydrolysis by novo, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.042AU, and pH is 8.0, and hydrolysis temperature is 60 ℃, hydrolysis time is 6h, and Sumizyme MP is selected from Alacalase 2.4L.
3) hydrolyzed solution is through ion exchange chromatography, ion-exchange packing is QAE-A25, processing step is: filler pre-treatment → dress post → last sample → gradient elution, processing condition are: (1) filler pre-treatment: take by weighing 7g QAE-A25 anionite-exchange resin powder in beaker, the elutriant A that adds certain volume, swelling 2h in boiling water bath, outwell buoyant fine particle on the liquid level after the cooling, clean repeatedly several times with elutriant A, be made into 75% suspension, it is standby to vacuumize the bubble of catching up with in the suspension then in vacuum chamber.(2) dress post: with chromatography column (C16/40, internal diameter 16mm, long 40cm) vertically is put on the iron stand after cleaning up with deionized water, adds elutriant A, open water outlet to drive the bubble of bottom away, when the damping fluid of column bottom has 2cm high approximately, close chromatography column bottom water outlet.With disposable the pouring in the chromatography column of swelling gel suspension good, that stir, connect peristaltic pump rapidly, open water outlet, with the flow velocity of 2mL/min elutriant A is pumped into and to make the rapid sedimentation of filler in the chromatography column, treat to use the flow velocity of 5mL/min to push filler again after the whole sedimentations of filler, treat that the post bed height no longer changes the back termination of pumping, connects Adaptor and compress rapidly, make bed a surperficial no redundant space and an elutriant, this moment, the bed volume height was about 13cm.With using behind 2 bed volumes of elutriant A balance.(3) go up sample: sample on the peristaltic pump, applied sample amount are 300mg, and last sample speed is 0.5mL/min.(4) gradient elution: elutriant A is 30mmol/LpH8.0Tris-HCl, and the ionic strength of elutriant B is 2mol/LNaCl, and ionic strength gradient (NaCl) is that 0.024mol/L.min, elution flow rate are 0.9mL/min.
4) separate the high blood pressure lowering peptide active ingredient obtain through gel chromatography by step 3), filler is Superdex 30p.g..Processing step is: filler pre-treatment → dress post → last sample → wash-out.Processing condition are: (1) filler pre-treatment: swelling is good in advance in 20% ethanol for filler, therefore only needs in use earlier ethanol is removed and can be used.With cleaning filler repeatedly,, put into vacuum drying oven and vacuumize and to adorn post until ethanol is removed fully through the filterable deionized water of 0.45 μ m filter membrane.(2) dress post: the chromatography column that the water washed with de-ionized water is clean (XK16/70, internal diameter 16mm, long 70cm) vertically is positioned on the iron stand.In post, add earlier elutriant, the bubble of post bottom is discharged, close column outlet then, and connect Revisior on the top of post.The filler of having handled well is transferred to the uniform suspension liquid of the about 475mL of volume with deionized water, pour in the chromatography column along glass stick is disposable then, rapidly peristaltic pump is connected, with elutriant with the speed of 1mL/min flushing pillar 2h, with the sedimentation of quickening filler and make the uniform filling sedimentation.Treat carefully to take off Revisior after filling settlement fully, connect Adaptor,, treat that the post height gets final product termination of pumping when no longer changing again with the speed punching press pillar of 4mL/min.Adaptor is a little firmly compressed downwards, make it not interspace with tight contact the in bed surface.Can use with two bed volumes of damping fluid balance again.With this method dress post good repeatability is arranged.(3) go up sample: sample on the peristaltic pump, applied sample amount are 2% of bed volume, and last sample speed is 0.5mL/min.(4) wash-out: elutriant is pH6.050mol/Lm acetate-ammonium acetate buffer solution, and elution flow rate is 0.9mL/min.
5) separated the high blood pressure lowering peptide active ingredient that obtains through twice RPLC by step 4), chromatographic column is Everest C18 reversed-phase column (4.6mm * 250mm, 238TP54), adopt gradient elution, mobile phase A is for being 5% acetonitrile+0.1% trifluoroacetic acid, and B is pure acetonitrile+0.1% trifluoroacetic acid mutually.Chromatographic separation condition is for seeing Table 1.Filter out and have the high active component that suppresses, through measuring, its sequence is Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala.
Table 1 chromatographic separation condition
| Time (min) | A(％) | B(％) | Flow velocity (mL/min) | Temperature (℃) |
| 0.00 2.00 35.00 | 75.0 70.0 55.0 | 25.0 30.0 45.0 | 0.800 0.800 0.800 | 30 30 30 |
Below provide principle and result that the external hypotensive activity of blood pressure lowering polypeptide of corn gluten powder detects.
Measuring principle: angiotensin-converting enzyme (ACE) can hydrolysis hippuryl histidyl-leucine (Hip-His-Leu under 37 ℃, pH8.3, HHL) generate urobenzoic acid (Hippuric acid), and blood pressure lowering peptide is by suppressing the output that the ACE enzymic activity reduces urobenzoic acid, urobenzoic acid has maximum absorption at the 228nm place, and the urobenzoic acid that reaction generates can extract with ethyl acetate is specific.Just can calculate blood pressure lowering peptide to ACE enzymic activity inhibiting rate by the amount that detects the urobenzoic acid that generates within a certain period of time.
The calculation formula of inhibiting rate is: inhibiting rate (%)=(E
c-E
s)/(E
c-E
b) * 100%, wherein, E
cFor not adding the optical density value that obtains under the inhibitor condition; E
sFor adding the optical density value that records behind the inhibitor; E
bStop reaction that the optical density value that records at last takes place for the preceding HCl of adding takes place in reaction.
The activity IC of blood pressure lowering peptide
50Represent IC
50Be defined as and suppress the needed inhibitor concentration of ACE enzymic activity one half under certain condition.
Record the IC of blood pressure lowering polypeptide of corn gluten powder as stated above
50Be 17.5 μ mol/L.
Below provide hypotensive activity detects in the blood pressure lowering polypeptide of corn gluten powder body principle and result.
Laboratory animal is original hypertensive rat (SHR), and 40 SHR are divided into control group, Captopril group (clinical application at present) and blood pressure lowering peptide group at random by body weight, and the blood pressure lowering peptide group comprises high dose group, middle dosage group and low dose group, 8 every group.Wherein high, medium and low dosage group gives blood pressure lowering peptide 1000,500,100mg/kg bw, Captopril group gives captopril 10mg/kg bw, control group gives distilled water 5mL, other gets 8 WKY and gives corn anti-hypertensive peptides 1000mg/kgbw, administering mode is fed for irritating, and blood pressure lowering peptide and captopril all are dissolved in the 5mL distilled water.All rats all begin formal experiment after one week of domestication before experiment.All rat sub-cage rearings, 2 in every cage, the feed of all freely drinking water, activity is not limit.Temperature is 23 ± 1 ℃, and humidity is 55 ± 5%, illumination and dark each 12h.Adopt the caudal artery volumetric method to measure the systolic pressure (SBP) of rat.Study after the administration blood pressure lowering peptide blood pressure lowering effects in 24, the results are shown in Figure 1.
As shown in Figure 1, after SHR irritate to feed corn anti-hypertensive peptides, systolic pressure descended, and antihypertensive effect is corresponding relation with filling hello dosage.Compare with control group, polypeptide group SHR feeds corn anti-hypertensive peptides 2h post shrinkage in filling and presses off beginning decline, high dose group SHR touches the bottom at the 10h systolic pressure, SBP has reduced 22mmHg, in, low dose group SHR respectively the 6th, the 12h systolic pressure touches the bottom, SBP has reduced by 12 respectively, 13mmHg.The step-down time length of polypeptide group reaches more than the 12h.The maximum reducing effect of high dose group (Δ MaxSBP=22mmHg) wants high by 30% with the maximum reducing effect (Δ MaxSBP=17mmHg) of Captopril group, both antihypertensive effects do not have the difference (P＞0.05) on the statistical significance in 2-8h, after the 8th hour, the antihypertensive effect of high dose group is better than Captopril group (P＜0.05).In addition, its step-down time length is than the step-down longer duration of captopril.Though in, the maximum reducing effect (Δ MaxSBP=12,13mmHg) of low dose group is lower by 30% and 25% than Captopril group, their step-down time length (＞12h) but be longer than Captopril group.Blood pressure lowering polypeptide of corn gluten powder is irritated hello SHR rat with 120mg/kg, 150mg/kg and 100mg/kg bw dosage respectively, and blood pressure reduces behind the 2h, and the maximum reducing effect is respectively 13mmHg, and the step-down time length reaches more than the 12h.
Embodiment 2
Similar to Example 1, its difference is that the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.05AU, and pH is 7～9, and hydrolysis temperature is 50 ℃, and hydrolysis time is 8h, and Sumizyme MP is selected from Alacalase2.4L.
Embodiment 3
Similar to Example 1, its difference is that the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.03AU, and pH is 7～9, and hydrolysis temperature is 70 ℃, and hydrolysis time is 7h, and Sumizyme MP is selected from Alacalase2.4L.
Claims (10)
1. blood pressure lowering polypeptide of corn gluten powder is characterized in that its aminoacid sequence is:
Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala，
Its molecular weight is 970.6855, and theoretical iso-electric point is 5.52, hypotensive activity IC
50Be 17.5 μ mol/L, be white powder, soluble in water.
2. the separation method of blood pressure lowering polypeptide of corn gluten powder is characterized in that the steps include:
1) the raw material Zein powder is collected undersized pale yellow powder after crushed as the enzymolysis substrate;
2) in Zein powder, add hydrolysis by novo and get hydrolyzed solution;
3) hydrolyzed solution separates obtaining high blood pressure lowering peptide active ingredient through ion exchange chromatography;
4) the high blood pressure lowering peptide active ingredient that separation is obtained gets high blood pressure lowering peptide active ingredient through gel chromatography;
5) the high blood pressure lowering peptide active ingredient that separation is obtained is through twice RPLC, filters out to have the high active component that suppresses, and through measuring, its sequence is Leu-Leu-Pro-Pro-Tyr-Leu-Ser-Pro-Ala.
3. the separation method of blood pressure lowering polypeptide of corn gluten powder as claimed in claim 2 is characterized in that in step 1), and the raw material Zein powder is crossed 200 mesh sieves after crushed.
4. the separation method of blood pressure lowering polypeptide of corn gluten powder as claimed in claim 2 is characterized in that in step 2) in, the content of Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.03～0.05AU, pH is 7～9.
5. the separation method of blood pressure lowering polypeptide of corn gluten powder as claimed in claim 4, the content that it is characterized in that Zein powder and Sumizyme MP is Zein powder: Sumizyme MP=1g: 0.042AU, pH is 8.0.
6. the separation method of blood pressure lowering polypeptide of corn gluten powder as claimed in claim 2 is characterized in that in step 2) in, in Zein powder, adding hydrolysis by novo, hydrolysis temperature is 50～70 ℃, hydrolysis time is 7～8h.
7. the separation method of blood pressure lowering polypeptide of corn gluten powder as claimed in claim 2 is characterized in that hydrolysis temperature is 60 ℃.
8. the separation method of blood pressure lowering polypeptide of corn gluten powder as claimed in claim 2 is characterized in that Sumizyme MP is selected from Alacalase 2.4L.
9. blood pressure lowering polypeptide of corn gluten powder as claimed in claim 1 is used to prepare the application of Altace Ramipril.
10. blood pressure lowering polypeptide of corn gluten powder as claimed in claim 1 is used to prepare the application of blood pressure reducing food.
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| CN101390564B (en) * | 2007-09-18 | 2011-01-26 | 中国食品发酵工业研究院 | Production method of corn separation protein |
| CN103052717B (en) * | 2011-05-17 | 2015-01-14 | 中国食品发酵工业研究院 | Industrial production method for producing antihypertensive bioactive peptide |
| CN107574205B (en) * | 2016-07-05 | 2021-01-15 | 陈栋梁 | Preparation method of homoarginine mixed peptide and application of homoarginine mixed peptide in treatment of gastric cancer |
| CN107868124B (en) * | 2017-11-13 | 2021-04-20 | 江苏大学 | Zein anti-inflammatory polypeptide and preparation method thereof |
| CN111105845B (en) * | 2020-01-13 | 2023-03-28 | 深圳职业技术学院 | Preparation of antihypertensive peptide based on protein folding fingerprint bar code design |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1526299A (en) * | 2003-09-23 | 2004-09-08 | 江苏大学 | Wheat plumule protein hydrolysate and its prepn process and use |
| CN1552894A (en) * | 2003-12-19 | 2004-12-08 | 吉林大学 | Corn peptide and its preparation and use |
| CN1563404A (en) * | 2004-04-01 | 2005-01-12 | 武汉工业学院 | Method for preparing decrease blood pressure peptide 'Angiotensin I-converting enzyme inhibitor' of rice bran protein |
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| CN1526299A (en) * | 2003-09-23 | 2004-09-08 | 江苏大学 | Wheat plumule protein hydrolysate and its prepn process and use |
| CN1552894A (en) * | 2003-12-19 | 2004-12-08 | 吉林大学 | Corn peptide and its preparation and use |
| CN1563404A (en) * | 2004-04-01 | 2005-01-12 | 武汉工业学院 | Method for preparing decrease blood pressure peptide 'Angiotensin I-converting enzyme inhibitor' of rice bran protein |
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