CN102676621A - Antihypertensive rapeseed peptide and preparation method and application of antihypertensive rapeseed peptide - Google Patents
Antihypertensive rapeseed peptide and preparation method and application of antihypertensive rapeseed peptide Download PDFInfo
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Abstract
The invention provides an antihypertensive rapeseed peptide and a preparation method and an application of the antihypertensive rapeseed peptide. The method for preparing the antihypertensive rapeseed peptide includes using protease to hydrolyze a rapeseed protein water solution, then performing refining to obtain the antihypertensive rapeseed peptide. When the method for preparing the antihypertensive rapeseed peptide is compared with methods for preparing the antihypertensive rapeseed peptide in prior art, rapeseed meals or rapeseed isolated proteins are directly used as raw materials to obtain the antihypertensive rapeseed peptide by means of enzymatic hydrolysis, the sources of raw materials are wide, the production process is simple, the cost is low, the waste can be changed into the valuable, and environment protection is facilitated. The antihypertensive rapeseed peptide can be used for not only inhibiting angiotensin converting enzyme (ACE) activity, but also inhibiting renin activity to block an alternative pathway of a renin angiotensin system (RAS), the alternative pathway of the RAS is activated due to the fact that hypotensor of ACE inhibitor is taken for a long time, and the antihypertensive rapeseed peptide has a great developing prospect in decreasing blood pressure.
Description
Technical field
The present invention relates to relate to biological technical field, be specifically related to hypotensive rapeseed peptide (rsp).
Background technology
China is the maximum country of Semen Brassicae campestris output in the world, nearly 1,200 ten thousand tons of YO, and rapeseed meal is the main by product of rapeseed oil processing, accounts for 65% of vegetable seed total amount, wherein contains albumen 35% ~ 42%.Rape seed protein amino acid is formed rationally, and the content of Methionin, Gelucystine and methionine(Met) is higher, is a kind of good vegetable-protein.Development and use vegetable seed protein resource improves China people's trophic level to effectively utilizing the plant protein resource of China, has important social meaning and good economic benefit.
At present, China has 200,000,000 hyperpietics approximately, and still increase 1,000 ten thousand people every year newly, and development of new blood pressure lowering agent and nutrient substance help solving this increasingly serious social common problem.On the one hand, existing Altace Ramipril is taken for a long time and can be caused a lot of side reactions to health, especially gi system is had very big injury; In addition on the one hand; The target of present most of blood pressure lowering peptide researchs all is angiotensin transferase (ACE), still, takes the Altace Ramipril ACE inhibitor for a long time; Can activate the alternative pathway of hypertensin system (RAS), thereby influence hypertensive clinical treatment.And renin inhibitor can be blocked the RAS system from the source, has than the more potential benefit of ACE suppressor factor, still less untoward reaction and better tolerance, and in the middle of large-scale, demonstrates the effect with target-organ protection in the research of terminal point incident.But, less based on the Altace Ramipril of renin inhibitor.
Summary of the invention
The objective of the invention is to: provide the preparation of a kind of enzyme process to have feritin and suppress the method that active and ACE suppresses active hypotensive rapeseed peptide (rsp); This method is environmentally friendly; Reaction conditions is gentle; Can give full play to proteolytic enzyme to vegetable seed egg hydrolytic action, be the method that a kind of low cost, suitable industrial mass prepare the vegetable seed blood pressure lowering peptide.
Another object of the present invention is to provide hypotensive rapeseed peptide (rsp); Not only can suppress angiotensin transferase (ACE); And can suppress feritin, can block owing to take the alternative pathway of Altace Ramipril ACE inhibitor activated hypertensin system (RAS) for a long time.
Another object of the present invention is to provide the application of hypotensive rapeseed peptide (rsp) aspect preparation treatment Altace Ramipril.
The present invention provides a kind of method for preparing hypotensive rapeseed peptide (rsp), comprises the steps:
(1) be raw material with the rapeseed meal after pulverizing, said raw material is mixed with water, regulate pH value to 6.5～9,35 ℃～50 ℃ of temperature, dispersed with stirring 0.5～2 h obtains the rape seed protein aqueous solution;
(2) in the said rape seed protein aqueous solution of step (1), add proteolytic enzyme, at 35 ℃～50 ℃ following hydrolysis 1～4 h, deactivation proteolytic enzyme wherein, the centrifuging and taking supernatant gets hypotensive rapeseed peptide (rsp) crude extract;
(3) the refining back of the said hypotensive rapeseed peptide (rsp) crude extract of step (2) is obtained hypotensive rapeseed peptide (rsp).
The raw material of the said rapeseed meal of step (1) is conventional oil vegetable seed or double-low rapeseed seed, and the particle diameter of the rapeseed meal after the pulverizing is 10～200 μ m.
The raw material of the said rape seed protein aqueous solution of step (1) is an isolating protein from the vegetable seeds dregs of rice.
The said proteolytic enzyme of step (2) is Proteinase K, thermolysin, compound protease or papoid, preferential Proteinase K.
Proteinic mass ratio is 1～10:100 in the proteolytic enzyme of the said adding of step (2) and the rape seed protein aqueous solution.
The said process for purification of step (3) is: hypotensive rapeseed peptide (rsp) crude extract is the ultra-filtration membrane ultrafiltration of 3kDa through molecular weight cut-off, gets through the liquid dialysis desalting, and lyophilize gets hypotensive rapeseed peptide (rsp).
The said process for purification of step (3) also can for: hypotensive rapeseed peptide (rsp) crude extract is separated through RPLC; Getting the organic solvent volumetric concentration respectively is 10% ~ 15%; 35% ~ 40% and 55% ~ 60% three part elutriants; With the lyophilize respectively of said three part elutriants, promptly get hypotensive rapeseed peptide (rsp); The isolating condition of said RPLC is: chromatographic column is carbon 12 or carbon 18 silicagel columns; Moving phase is water and organic solvent, and said organic solvent is methyl alcohol or acetonitrile, and all containing mass concentration in said water and the organic solvent is 0.05～0.10% trifluoroacetic acid; Elution requirement is: linear elution, the organic solvent volumetric concentration in the moving phase changes to 80% from 5%.
The present invention also provides the hypotensive rapeseed peptide (rsp) that adopts method for preparing.
The present invention also provides and adopts the application of said hypotensive rapeseed peptide (rsp) aspect the preparation Altace Ramipril.
Beneficial effect of the present invention:
(1) the present invention prepares hypotensive rapeseed peptide (rsp) method compared with prior art, is raw material with rapeseed meal or vegetable seed separating protein directly, through enzymatic hydrolysis; Obtain hypotensive rapeseed peptide (rsp), raw material sources are extensive, and production technique is simple, cost is low; And turn waste into wealth, help environment protection.
(2) the hypotensive rapeseed peptide (rsp) that adopts two kinds of process for purification to obtain; Not only can suppress angiotensin transferase (ACE); And can suppress feritin; Can block owing to take the alternative pathway of Altace Ramipril ACE inhibitor activated hypertensin system (RAS) for a long time, aspect hypotensive, very big DEVELOPMENT PROSPECT arranged.
(3) the hypotensive rapeseed peptide (rsp) of the present invention comes from the blood pressure lowering peptide of natural animal and plant and mikrobe; Except having antihypertensive function; Anti-oxidant in addition, immunological enhancement, anticoagulation, be prone to digest and assimilate, to functions such as target cell toxicological harmless effects, be preferred raw materials for Altace Ramipril and nutrient substance.
Embodiment
The Proteinase K that uses among the present invention (Proteinase K) is purchased the company in Fisher, article No. 39450-01-6; Thermolysin (Thermolysin) is purchased the company in sigma, article No. 232-973-4, PIN P1512; Compound protease (Protamex) is purchased in sigma, PIN P0029; Papoid (Papain) is purchased in sigma, and article No. is 232-627-2, PIN P4762.
Embodiment 1 adopts Proteinase K to prepare hypotensive rapeseed peptide (rsp)
(1) rapeseed meal is pulverized,, processed the vegetable seeds dregs of rice after the pulverizing through 100 mesh sieves.Rapeseed meal with after pulverizing is a raw material, and said raw material by solid-to-liquid ratio 1:20, will be mixed with water, regulates pH value to 7.5,37 ℃ of temperature, and dispersed with stirring 1h obtains the rape seed protein aqueous solution.The raw material of said rapeseed meal is conventional oil vegetable seed or double-low rapeseed seed, and the particle diameter of the rapeseed meal after the pulverizing is 10～200 μ m.The raw material of the said rape seed protein aqueous solution can also be isolating protein from the vegetable seeds dregs of rice.
(2) be 5:100 by proteinic mass ratio in the proteolytic enzyme and the rape seed protein aqueous solution, in the said rape seed protein aqueous solution of step (1), add Proteinase K, controlled temperature and pH value are constant, hydrolysis 1～3 h.Reaction is heated to 90～100 ℃ with hydrolyzed solution after finishing, and keeps 10 min, and deactivation Proteinase K wherein is cooled to room temperature, and centrifugal 1 h of 10000 g cf-gets supernatant, gets hypotensive rapeseed peptide (rsp) crude extract.
(3) hypotensive rapeseed peptide (rsp) crude extract is separated through RPLC (RP-HPLC); During wash-out, get the organic solvent volumetric concentration respectively and be 10% ~ 15%, 35% ~ 40% and 55% ~ 60% three part elutriants; With the lyophilize respectively of said three part elutriants, promptly get hypotensive rapeseed peptide (rsp); The isolating condition of said RPLC is: chromatographic column is carbon 12 or carbon 18 silicagel columns; Moving phase is water and organic solvent, and said organic solvent is methyl alcohol or acetonitrile, and all containing mass concentration in said water and the organic solvent is 0.05～0.10% TFA (trifluoroacetic acid); Elution requirement is: linear elution, the organic solvent volumetric concentration in the moving phase changes to 80% from 5%.
When RPLC (RP-HPLC) separates, adopt UV-detector (detecting wavelength 215 nm).Wherein, component 2 (during wash-out, organic solvent body agent concentration is 35% ~ 40%) ACE suppresses activity and reaches 91.81%, and feritin suppresses activity and reaches 49.40%; Component 5 (during wash-out, organic this solvent body agent concentration is 45% ~ 50%) ACE suppresses activity and reaches 87.89%, and feritin suppresses activity and reaches 54.97%; Component 9 (during wash-out, organic solvent body agent concentration is 55% ~ 60%) ACE suppresses activity and reaches 86.89%, and feritin suppresses activity and reaches 58.19%.Can find out that the component 2,5 and 9 that this method of employing is separated to all has very high ACE inhibition activity and feritin inhibition activity, has good application prospects aspect hypotensive.
It is following that ACE suppresses activity test method: in cuvette, add 1 mL, 0.5 mM FAPGG (FAPGG full name N-[3-(2-Furyl) acryloyl]-L-phenylalanyl-glycyl-glycine; Be dissolved in that to contain 0.3 M NaCl concentration be the Tris-HCl damping fluid of 50 mM; PH 7.5), 20 μ L ACE (1U/mL, final vigor 20 mU); (protein concentration is 1mg/mL to the hypotensive rapeseed peptide (rsp) solution of 200 μ L; Solvent is 50 mM Tris-HCl damping fluids, contains 0.3 M NaCl, and pH 7.5); Under the mixing room temperature, measure the changing down of light absorption value under 345 nm in 2 min. blank with Tris – HCl damping fluid replacement sample. calculate ACE according to following formula and suppress active:
ACE suppresses active (%)=1-[△ A.min
-1 (sample)/ △ A.min
-1 (blank)] * 100%,
, wherein, △ A.min
-1 (sample)With △ A.min
-1 (blank)Be respectively the active speed that suppresses of expression sample and barren ACE.
It is following that feritin suppresses active detection method: hypotensive rapeseed peptide (rsp) suppresses the active external test of II Angiotensin II and (buys the Chemical in Cayman according to the RA test kit; Numbering 10006270) method is provided; Measure according to the following steps: add (1) blank in the 96-orifice plate successively: 20 μ L substrates; Damping fluid, 10 μ L pure water are used in 160 μ L test; (2) blank: 20 μ L substrates, damping fluid and 10 μ L pure water are used in 150 μ L test; (3) sample: 20 μ L substrates, damping fluid and 10 μ L samples are used in 150 μ L test.Add 10 μ L feritins afterwards respectively in each hole in (except that blank) and start reaction.Concussion 10 s under the 96-orifice plate room temperature are 37
0Preheating 15 min under the C, the fluorescence intensity of working sample in 10min, excitation wavelength 340nm, emission wavelength 490nm, active according to the feritin inhibition of following formula calculation sample:
Embodiment 2 adopts thermolysin to prepare hypotensive rapeseed peptide (rsp)
(1) rapeseed meal is pulverized,, processed the vegetable seeds dregs of rice after the pulverizing through 100 mesh sieves.Rapeseed meal with after pulverizing is a raw material, and said raw material by solid-to-liquid ratio 1:20, is mixed with water, regulates pH value to 9.0,50 ℃ of temperature, and dispersed with stirring 1h obtains the rape seed protein aqueous solution.The raw material of said rapeseed meal is conventional oil vegetable seed or double-low rapeseed seed, and the particle diameter of the rapeseed meal after the pulverizing is 10～200 μ m.The raw material of the said rape seed protein aqueous solution can also be isolating protein from the vegetable seeds dregs of rice.
(2) be 1:100 by proteinic mass ratio in the proteolytic enzyme and the rape seed protein aqueous solution, in the said rape seed protein aqueous solution of step (1), add thermolysin, controlled temperature and pH value are constant, hydrolysis 4 h.Reaction is heated to 95 ℃ with hydrolyzed solution after finishing, and keeps 10 min, and deactivation proteolytic enzyme wherein is cooled to room temperature, and centrifugal 1 h of 10000 g cf-gets supernatant, gets hypotensive rapeseed peptide (rsp) crude extract.
The ultra-filtration membrane ultrafiltration that this crude extract molecular weight cut-off is 3kDa; See through liquid through dialysis desalting; Lyophilize gets hypotensive rapeseed peptide (rsp), and this hypotensive rapeseed peptide (rsp) has feritin simultaneously and suppresses active and ACE inhibition activity, and wherein feritin suppresses active 53.93%; ACE suppresses activity 84.41%, aspect hypotensive, good application prospects is arranged.
Embodiment 3 adopts compound protease to prepare hypotensive rapeseed peptide (rsp) crude extract
(1) rapeseed meal is pulverized,, processed the vegetable seeds dregs of rice after the pulverizing through 100 mesh sieves.Rapeseed meal with after pulverizing is a raw material, and said raw material by solid-to-liquid ratio 1:20, is mixed with water, regulates pH value to 9.0,50 ℃ of temperature, and dispersed with stirring 1h obtains the rape seed protein aqueous solution.The raw material of said rapeseed meal is conventional oil vegetable seed or double-low rapeseed seed, and the particle diameter of the rapeseed meal after the pulverizing is 10～200 μ m.The raw material of the said rape seed protein aqueous solution can also be isolating protein from the vegetable seeds dregs of rice.
(2) be 4:100 by proteinic mass ratio in the proteolytic enzyme and the rape seed protein aqueous solution, in the said rape seed protein aqueous solution of step (1), add thermolysin, controlled temperature and pH value are constant, hydrolysis 4 h.Reaction is heated to 95 ℃ with hydrolyzed solution after finishing, and keeps 10 min, and deactivation proteolytic enzyme wherein is cooled to room temperature, and centrifugal 1 h of 10000 g cf-gets supernatant, gets hypotensive rapeseed peptide (rsp) crude extract.
(3) this crude extract is the ultra-filtration membrane ultrafiltration of 3kDa through molecular weight cut-off; See through liquid through dialysis desalting; Lyophilize gets hypotensive rapeseed peptide (rsp), and this hypotensive rapeseed peptide (rsp) has the active and ACE inhibition activity of feritin inhibition, and wherein feritin suppresses activity 87.41%, ACE suppresses activity 55.84%; Therefore, this hypotensive rapeseed peptide (rsp) has good application prospects aspect hypotensive.
Embodiment 4 adopts papoid to prepare hypotensive rapeseed peptide (rsp) crude extract
(1) rapeseed meal is pulverized,, processed the vegetable seeds dregs of rice after the pulverizing through 100 mesh sieves.Rapeseed meal with after pulverizing is a raw material, and said raw material by solid-to-liquid ratio 1:20, is mixed with water, regulates pH value to 6.5,40 ℃ of temperature, and dispersed with stirring 1h obtains the rape seed protein aqueous solution.The raw material of said rapeseed meal is conventional oil vegetable seed or double-low rapeseed seed, and the particle diameter of the rapeseed meal after the pulverizing is 10～200 μ m.The raw material of the said rape seed protein aqueous solution can also be isolating protein from the vegetable seeds dregs of rice.
(2) be 5:100 by proteinic mass ratio in the proteolytic enzyme and the rape seed protein aqueous solution, in the said rape seed protein aqueous solution of step (1), add papoid, controlled temperature and pH value are constant, hydrolysis 4 h.Reaction is heated to 95 ℃ with hydrolyzed solution after finishing, and keeps 10 min, and deactivation proteolytic enzyme wherein is cooled to room temperature, and centrifugal 1 h of 10000 g cf-gets supernatant, gets hypotensive rapeseed peptide (rsp) crude extract.
(3) hypotensive rapeseed peptide (rsp) crude extract is the ultra-filtration membrane ultrafiltration of 3kDa through molecular weight cut-off; Get and see through the liquid dialysis desalting; Lyophilize gets hypotensive rapeseed peptide (rsp), and its feritin suppresses activity 30.85%, and ACE suppresses active 48.23%; Therefore, this hypotensive rapeseed peptide (rsp) has good application prospects aspect hypotensive.
In addition, said hypotensive rapeseed peptide (rsp) crude extract can be the ultra-filtration membrane ultrafiltration of 1kDa through molecular weight cut-off also, sees through liquid through dialysis desalting, and lyophilize must have feritin inhibition activity and ACE suppresses active hypotensive rapeseed peptide (rsp).Hypotensive rapeseed peptide (rsp) feritin suppresses activity, ACE inhibition activity is respectively 41.85% and 60.19%, aspect hypotensive, has good application prospects.
Claims (10)
1. a method for preparing hypotensive rapeseed peptide (rsp) comprises the steps:
(1) be raw material with the rapeseed meal after pulverizing, said raw material is mixed with water, regulate pH value to 6.5～9,35 ℃～50 ℃ of temperature, dispersed with stirring 0.5～2 h obtains the rape seed protein aqueous solution;
(2) in the said rape seed protein aqueous solution of step (1), add proteolytic enzyme, at 35 ℃～50 ℃ following hydrolysis 1～4 h, deactivation proteolytic enzyme wherein, the centrifuging and taking supernatant gets hypotensive rapeseed peptide (rsp) crude extract;
(3) the refining back of the said hypotensive rapeseed peptide (rsp) crude extract of step (2) is obtained hypotensive rapeseed peptide (rsp).
2. according to the method for the hypotensive rapeseed peptide (rsp) of the said preparation of claim 1, the raw material that it is characterized in that the said rapeseed meal of step (1) is conventional oil vegetable seed or double-low rapeseed seed, and the particle diameter of the rapeseed meal after the pulverizing is 10～200 μ m.
3. according to the method for the hypotensive vegetable seed active peptide of the said preparation of claim 2, the raw material that it is characterized in that the said rape seed protein aqueous solution of step (1) is an isolating protein from the vegetable seeds dregs of rice.
4. according to the hypotensive rapeseed peptide (rsp) method of the said preparation of claim 2, it is characterized in that the said proteolytic enzyme of step (2) is Proteinase K, thermolysin, compound protease or papoid.
5. according to the hypotensive rapeseed peptide (rsp) method of the said preparation of claim 4, it is characterized in that the said proteolytic enzyme of step (2) is Proteinase K.
6. according to the method for claim 4 or the hypotensive rapeseed peptide (rsp) of 5 said preparations, it is characterized in that proteinic mass ratio is 1～10:100 in proteolytic enzyme and the rape seed protein aqueous solution of the said adding of step (2).
7. according to the hypotensive rapeseed peptide (rsp) method of the said preparation of claim 6; It is characterized in that the said process for purification of step (3) is: hypotensive rapeseed peptide (rsp) crude extract is the ultra-filtration membrane ultrafiltration of 3kDa through molecular weight cut-off; Get and see through the liquid dialysis desalting, lyophilize gets hypotensive rapeseed peptide (rsp).
8. according to the method for the hypotensive rapeseed peptide (rsp) of the said preparation of claim 6; It is characterized in that the said process for purification of step (3) is: hypotensive rapeseed peptide (rsp) crude extract is separated through RPLC; Getting the organic solvent volumetric concentration respectively is 10% ~ 15%; 35% ~ 40% and 55% ~ 60% three part elutriants with the lyophilize respectively of said three part elutriants, promptly get hypotensive rapeseed peptide (rsp); The isolating condition of said RPLC is: chromatographic column is carbon 12 or carbon 18 silicagel columns; Moving phase is water and organic solvent, and said organic solvent is methyl alcohol or acetonitrile, and all containing mass concentration in said water and the organic solvent is 0.05～0.10% trifluoroacetic acid; Elution requirement is: linear elution, the organic solvent volumetric concentration in the moving phase changes to 80% from 5%.
9. hypotensive rapeseed peptide (rsp) that adopts one of claim 8 method preparation.
10. the application of the said hypotensive rapeseed peptide (rsp) of claim 9 aspect the preparation Altace Ramipril.
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| CN107488697A (en) * | 2017-09-29 | 2017-12-19 | 南京仙草堂生物科技有限公司 | A kind of extraction of centipede anticoagulant peptide and purifying process |
| CN107574216A (en) * | 2017-09-29 | 2018-01-12 | 南京仙草堂生物科技有限公司 | A kind of extraction of scorpion toxin and purifying process |
| CN114409729A (en) * | 2021-11-11 | 2022-04-29 | 南京财经大学 | Rapeseed peptide and application thereof in preparation of drug nano-carrier |
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