CN105821102A - Method for preparing angiotensin-converting enzyme inhibitory peptide from Sinonovacula constricta - Google Patents
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Abstract
The invention discloses method for preparing angiotensin-converting enzyme inhibitory peptide from Sinonovacula constricta. The preparation method comprises the following steps: (1) pretreatment of Sinonovacula constricta; (2) enzymatic hydrolysis; (3) ultrafiltration and separation; (4) separation and purification via a rapid protein purifying system; and (5) separation and purification via a high-performance liquid chromatographic system. According to the invention, Sinonovacula constricta is used as a raw material and subjected to enzymatic hydrolysis via neutral protease, and ultrafiltration technology, the rapid protein purifying system and the high-performance liquid chromatographic system are employed for separation and purification of enzymatic hydrolysis liquid so as to obtain the novel angiotensin-converting enzyme inhibitory peptide (ACE inhibitory peptide); and the method is reasonable in extraction and separation process and strong in operability, and provides experimental basis for preparation of high-activity ACE inhibitory peptide from Sinonovacula constricta.
Description
Technical field
The present invention relates to biological pharmacy technical field, especially relate to a kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method.
Background technology
Hypertension is a kind of cardiovascular disease that puzzlement is current, may result in serious complication, very big to human health risk.Angiotensin converting enzyme (Angiotensin-converting enzyme, ACE) play an important role during body blood pressure regulating, on the one hand inactive angiotensinⅠ is converted into and has strong contraction whole body arteriole by ACE, makes the angiotensinⅡ that blood pressure raises；On the other hand ACE can promote that the Kallidin I having hypotensive activity decomposes, and causes elevation of the blood pressure.
The ACE inhibitor such as the captopril of synthetic, enalapril and lisinopril now, these medicines have a strongest hypotensive activity, but long-term taking can produce dizziness, feel sick, the acutely side effect such as cough, parageusia, renal function injury.Many studies have shown that the small peptide coming from food tool has efficacy in lowering high blood pressure, Dai-Hung Ngo etc., with Pacific Ocean cod skin as raw material, carries out enzymolysis with papain, separated obtains blood pressure lowering peptide: Thr-Cys-Ser-Pro after purification；Hasan F etc. utilizes pepsin enzymolysis Skipjack meat, separated obtains high activity blood pressure lowering peptide (IKYGD) after purification；Come from the biologically active peptide in food and have safe and free of toxic and side effects, and be prone to the features such as body absorption.Therefore, from food source, preparative separation goes out highly active ace inhibitory peptide and is still the focus of research.
Sinonovacula constricta (Sinonovacula ) it is the common shellfish of China coast, belong to high-quality protein source.Sinonovacula constricta extract has the effects such as antioxidation, antitumor and raising immunity of organism effect.But have not yet to see the research report relevant to Sinonovacula constricta blood pressure lowering peptide.
Summary of the invention
It is reasonable that the goal of the invention of the present invention is to provide for a kind of extraction and separation process, the Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method that workable, product stability is good, purity is high.
To achieve these goals, the present invention is by the following technical solutions:
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method, comprises the following steps:
(1) Sinonovacula constricta pretreatment: after Sinonovacula constricta is shelled meat except shell, with clear water, clam meat is cleaned, after tissue pulper homogenate at a high speed, soaks Sinonovacula constricta meat 3 ~ 5h with isopropanol, and filtered filtration residue to no isopropanol abnormal flavour, is dehydrated to obtain Sinonovacula constricta meat slag by purified water foam washing, standby.Sinonovacula constricta pretreatment is mainly removing impurities albumen and removes lipid.
(2) enzymolysis: the Sinonovacula constricta meat slag in step (1) is added neutral protease and carries out enzymolysis, enzymatic hydrolysis condition is: solid-liquid ratio 1:3 ~ 4, enzyme concentration 1500 ~ 2000U/g, pH value 6.5 ~ 7.5, hydrolysis temperature 50 ~ 60 DEG C, enzymolysis time 9 ~ 12 h, enzymolysis inactivates with boiling water bath after completing, it is centrifuged after pre-cooling, obtains supernatant.
(3) ultra-filtration and separation: by supernatant after 0.45 μm filter membrane sucking filtration, carry out ultrafiltration with the ultrafilter membrane that molecular cut off is 3 ku, obtain ultrafiltrate.
(4) FPLC purification system is isolated and purified: carry out isolated and purified by the ultrafiltrate in step (3) through FPLC purification system, obtain two active components, the angiotensin-converting enzyme inhibitory activity of these two active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component A.
(5) highly effective liquid phase chromatographic system is isolated and purified: component A is isolated and purified through highly effective liquid phase chromatographic system (HPLC), obtain three active components, the angiotensin-converting enzyme inhibitory activity of these three active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component B, component B is after purity detecting, and obtaining aminoacid sequence is: Pro-Trp-Gly-Met-Phe(PTGMP) angiotensin converting enzyme-inhibiting peptide.
As preferably, in step (1), Sinonovacula constricta is 1:3 ~ 5 with the mass ratio of isopropanol.
As preferably, in step (2), it is stirred continuously during enzymolysis.
As preferably, in step (2), it is cooled to 4 ~ 6 DEG C in advance.
As preferably, in step (2), centrifuging process condition is: rotating speed 5000 ~ 10000rpm/min, centrifugation time 10 ~ 30min.
As preferably, in step (4), FPLC purification system is isolated and purified method particularly includes: use DEAE-SepharoseFF ion exchange column (packing material size 10 μm, 10 × 300 mm), gel is filled post, after Tris-Hcl balance (pH is 7.4), ultrafiltrate is configured to the solution of 200mg/ml, applied sample amount is 3mL every time, be 7.4 with Tris-Hcl(pH respectively), the NaCl solution of 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L and 1mol/L carry out stepwise elution, elution speed is 2.2ml/min；Detection wavelength 280nm；Collect each peak.
As preferably, in step (5), highly effective liquid phase chromatographic system is isolated and purified method particularly includes: use sephadex G-25 chromatographic column (size: 2.6 × 80cm；Post material: Sephadex G-25), component B is configured to the solution of 200mg/ml, applied sample amount: 2.0mL；Flowing phase: ultra-pure water；Eluent flow rate: 1.0 mL/min；Detection wavelength: 280nm；Automatically gathering speed: 4.0min/ pipe；Collect each peak.
As preferably, in step (5), component B method for detecting purity is: use ZORBAX SB-C18 analytical type chromatographic column (packing material size: 5 μm, 4.6 × 150 mm)；Mobile phase A is ultra-pure water (containing 0.05%TFA), and Mobile phase B is acetonitrile (containing 0.05%TFA), employing isocratic elution mode: 80%A, 20%B；Flow velocity: 1.0 mL/min；Column temperature: 25 DEG C；Auto injection, sample size: 20 μ L；Detection wavelength: 280 nm.
Therefore, there is advantages that with Sinonovacula constricta as raw material, by neutral protease enzymolysis Sinonovacula constricta, and utilize hyperfiltration technique, FPLC purification system and highly effective liquid phase chromatographic system enzymolysis solution is carried out isolated and purified after obtain a kind of new angiotensin converting enzyme-inhibiting peptide (ACE peptide for inhibiting), extraction and separation process is reasonable, workable, provide experimental basis for preparing highly active ace inhibitory peptide from Sinonovacula constricta.
Detailed description of the invention
Below by detailed description of the invention, the present invention will be further described.
Embodiment 1
(1) Sinonovacula constricta pretreatment: after Sinonovacula constricta is shelled meat except shell, with clear water, clam meat is cleaned, after tissue pulper homogenate at a high speed, soaking Sinonovacula constricta meat 5h with isopropanol, the mass ratio of Sinonovacula constricta and isopropanol is 1:5, filtered filtration residue by purified water foam washing to no isopropanol abnormal flavour, it is dehydrated to obtain Sinonovacula constricta meat slag, standby.
(2) enzymolysis: the Sinonovacula constricta meat slag in step (1) is added neutral protease and carries out enzymolysis, enzymatic hydrolysis condition is: solid-liquid ratio 1:4, enzyme concentration 2000U/g, pH value 7.5, hydrolysis temperature 60 DEG C, enzymolysis time 12 h, it is stirred continuously during enzymolysis, enzymolysis inactivates with boiling water bath after completing, and is centrifuged 30min with rotating speed 10000rpm/min, obtains supernatant after being cooled to 6 DEG C in advance.
(3) ultra-filtration and separation: by supernatant after 0.45 μm filter membrane sucking filtration, carry out ultrafiltration with the ultrafilter membrane that molecular cut off is 3 ku, obtain ultrafiltrate.
null(4) FPLC purification system is isolated and purified: by the ultrafiltrate in step (3) through FPLC purification system (Apurifier UPC-100 type FPLC purification instrument，GE Life Sciences) carry out isolated and purified，Obtain two active components，The angiotensin-converting enzyme inhibitory activity of these two active components of separately sampled mensuration，The component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component A，FPLC purification system is isolated and purified method particularly includes: use DEAE-SepharoseFF ion exchange column (packing material size 10 μm，10 × 300 mm)，Gel is filled post，After Tris-Hcl balance (pH is 7.4)，Ultrafiltrate is configured to the solution of 200mg/ml，Applied sample amount is 3mL every time，It is 7.4 with Tris-Hcl(pH respectively)、0.1mol/L、0.3mol/L、0.5mol/L、The NaCl solution of 0.8mol/L and 1mol/L carries out stepwise elution，Elution speed is 2.2ml/min；Detection wavelength 280nm；Collect each peak.
(5) highly effective liquid phase chromatographic system is isolated and purified: component A is isolated and purified through highly effective liquid phase chromatographic system (HPLC), obtain three active components, the angiotensin-converting enzyme inhibitory activity of these three active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component B, component B is after purity detecting, obtaining aminoacid sequence is: Pro-Trp-Gly-Met-Phe(PTGMP) angiotensin converting enzyme-inhibiting peptide, highly effective liquid phase chromatographic system is isolated and purified method particularly includes: use sephadex G-25 chromatographic column (size: 2.6 × 80cm；Post material: Sephadex G-25), component B is configured to the solution of 200mg/ml, applied sample amount: 2.0mL；Flowing phase: ultra-pure water；Eluent flow rate: 1.0 mL/min；Detection wavelength: 280nm；Automatically gathering speed: 4.0min/ pipe；Collect each peak；Component B method for detecting purity is: use ZORBAX SB-C18 analytical type chromatographic column (packing material size: 5 μm, 4.6 × 150 mm)；Mobile phase A is ultra-pure water (containing 0.05%TFA), and Mobile phase B is acetonitrile (containing 0.05%TFA), employing isocratic elution mode: 80%A, 20%B；Flow velocity: 1.0 mL/min；Column temperature: 25 DEG C；Auto injection, sample size: 20 μ L；Detection wavelength: 280 nm.
Embodiment 2
(1) Sinonovacula constricta pretreatment: after Sinonovacula constricta is shelled meat except shell, with clear water, clam meat is cleaned, after tissue pulper homogenate at a high speed, soaking Sinonovacula constricta meat 3h with isopropanol, the mass ratio of Sinonovacula constricta and isopropanol is 1:3, filtered filtration residue by purified water foam washing to no isopropanol abnormal flavour, it is dehydrated to obtain Sinonovacula constricta meat slag, standby.
(2) enzymolysis: the Sinonovacula constricta meat slag in step (1) is added neutral protease and carries out enzymolysis, enzymatic hydrolysis condition is: solid-liquid ratio 1:3, enzyme concentration 1500U/g, pH value 6.5, hydrolysis temperature 50 DEG C, enzymolysis time 9h, it is stirred continuously during enzymolysis, enzymolysis inactivates with boiling water bath after completing, and is centrifuged 10min with rotating speed 5000rpm/min, obtains supernatant after being cooled to 4 DEG C in advance.
(3) ultra-filtration and separation: by supernatant after 0.45 μm filter membrane sucking filtration, carry out ultrafiltration with the ultrafilter membrane that molecular cut off is 3 ku, obtain ultrafiltrate.
null(4) FPLC purification system is isolated and purified: carry out isolated and purified by the ultrafiltrate in step (3) through FPLC purification system，Obtain two active components，The angiotensin-converting enzyme inhibitory activity of these two active components of separately sampled mensuration，The component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component A，FPLC purification system is isolated and purified method particularly includes: use DEAE-SepharoseFF ion exchange column (packing material size 10 μm，10 × 300 mm)，Gel is filled post，After Tris-Hcl balance (pH is 7.4)，Ultrafiltrate is configured to the solution of 200mg/ml，Applied sample amount is 3mL every time，It is 7.4 with Tris-Hcl(pH respectively)、0.1mol/L、0.3mol/L、0.5mol/L、The NaCl solution of 0.8mol/L and 1mol/L carries out stepwise elution，Elution speed is 2.2ml/min；Detection wavelength 280nm；Collect each peak.
(5) highly effective liquid phase chromatographic system is isolated and purified: component A is isolated and purified through highly effective liquid phase chromatographic system (HPLC), obtain three active components, the angiotensin-converting enzyme inhibitory activity of these three active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component B, component B is after purity detecting, obtaining aminoacid sequence is: Pro-Trp-Gly-Met-Phe(PTGMP) angiotensin converting enzyme-inhibiting peptide, highly effective liquid phase chromatographic system is isolated and purified method particularly includes: use sephadex G-25 chromatographic column (size: 2.6 × 80cm；Post material: Sephadex G-25), component B is configured to the solution of 200mg/ml, applied sample amount: 2.0mL；Flowing phase: ultra-pure water；Eluent flow rate: 1.0 mL/min；Detection wavelength: 280nm；Automatically gathering speed: 4.0min/ pipe；Collect each peak；Component B method for detecting purity is: use ZORBAX SB-C18 analytical type chromatographic column (packing material size: 5 μm, 4.6 × 150 mm)；Mobile phase A is ultra-pure water (containing 0.05%TFA), and Mobile phase B is acetonitrile (containing 0.05%TFA), employing isocratic elution mode: 80%A, 20%B；Flow velocity: 1.0 mL/min；Column temperature: 25 DEG C；Auto injection, sample size: 20 μ L；Detection wavelength: 280 nm.
Embodiment 3
(1) Sinonovacula constricta pretreatment: after Sinonovacula constricta is shelled meat except shell, with clear water, clam meat is cleaned, after tissue pulper homogenate at a high speed, soaking Sinonovacula constricta meat 4h with isopropanol, the mass ratio of Sinonovacula constricta and isopropanol is 1:4, filtered filtration residue by purified water foam washing to no isopropanol abnormal flavour, it is dehydrated to obtain Sinonovacula constricta meat slag, standby.
(2) enzymolysis: the Sinonovacula constricta meat slag in step (1) is added neutral protease and carries out enzymolysis, enzymatic hydrolysis condition is: solid-liquid ratio 1:3.5, enzyme concentration 1800U/g, pH value 7, hydrolysis temperature 55 DEG C, enzymolysis time 10 h, it is stirred continuously during enzymolysis, enzymolysis inactivates with boiling water bath after completing, and is centrifuged 20min with rotating speed 8000rpm/min, obtains supernatant after being cooled to 5 DEG C in advance.
(3) ultra-filtration and separation: by supernatant after 0.45 μm filter membrane sucking filtration, carry out ultrafiltration with the ultrafilter membrane that molecular cut off is 3 ku, obtain ultrafiltrate.
null(4) FPLC purification system is isolated and purified: carry out isolated and purified by the ultrafiltrate in step (3) through FPLC purification system，Obtain two active components，The angiotensin-converting enzyme inhibitory activity of these two active components of separately sampled mensuration，The component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component A，FPLC purification system is isolated and purified method particularly includes: use DEAE-SepharoseFF ion exchange column (packing material size 10 μm，10 × 300 mm)，Gel is filled post，After Tris-Hcl balance (pH is 7.4)，Ultrafiltrate is configured to the solution of 200mg/ml，Applied sample amount is 3mL every time，It is 7.4 with Tris-Hcl(pH respectively)、0.1mol/L、0.3mol/L、0.5mol/L、The NaCl solution of 0.8mol/L and 1mol/L carries out stepwise elution，Elution speed is 2.2ml/min；Detection wavelength 280nm；Collect each peak.
(5) highly effective liquid phase chromatographic system is isolated and purified: component A is isolated and purified through highly effective liquid phase chromatographic system (HPLC), obtain three active components, the angiotensin-converting enzyme inhibitory activity of these three active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component B, component B is after purity detecting, obtaining aminoacid sequence is: Pro-Trp-Gly-Met-Phe(PTGMP) angiotensin converting enzyme-inhibiting peptide, highly effective liquid phase chromatographic system is isolated and purified method particularly includes: use sephadex G-25 chromatographic column (size: 2.6 × 80cm；Post material: Sephadex G-25), component B is configured to the solution of 200mg/ml, applied sample amount: 2.0mL；Flowing phase: ultra-pure water；Eluent flow rate: 1.0 mL/min；Detection wavelength: 280nm；Automatically gathering speed: 4.0min/ pipe；Collect each peak；Component B method for detecting purity is: use ZORBAX SB-C18 analytical type chromatographic column (packing material size: 5 μm, 4.6 × 150 mm)；Mobile phase A is ultra-pure water (containing 0.05%TFA), and Mobile phase B is acetonitrile (containing 0.05%TFA), employing isocratic elution mode: 80%A, 20%B；Flow velocity: 1.0 mL/min；Column temperature: 25 DEG C；Auto injection, sample size: 20 μ L；Detection wavelength: 280 nm.
Extraction and separation process of the present invention is reasonable, workable, with Sinonovacula constricta as raw material, isolated and purified a kind of new angiotensin converting enzyme-inhibiting peptide (ACE peptide for inhibiting is obtained by utilizing hyperfiltration technique, FPLC purification system and highly effective liquid phase chromatographic system that enzymolysis solution is carried out after neutral protease enzymolysis, purity reaches more than 99%), it carries out the degraded order-checking of N end through Protein Sequencer, obtain pentapeptide, its aminoacid sequence is: Pro-Trp-Gly-Met-Phe(PTGMP), its relative molecular mass is 636.78 Da, the IC of TPMGP50It is 0.15 mg/ml, there is higher ACE inhibitory activity (ACE suppression ratio is more than 96.1%), provide experimental basis for preparing highly active ace inhibitory peptide from Sinonovacula constricta.
Embodiment described above is the one preferably scheme of the present invention, and the present invention not makees any pro forma restriction, also has other variant and remodeling on the premise of without departing from the technical scheme described in claim.
Claims (8)
1. a Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method, it is characterised in that comprise the following steps:
(1) Sinonovacula constricta pretreatment: after Sinonovacula constricta is shelled meat except shell, with clear water, clam meat is cleaned, after tissue pulper homogenate at a high speed, soaks Sinonovacula constricta meat 3 ~ 5h with isopropanol, and filtered filtration residue to no isopropanol abnormal flavour, is dehydrated to obtain Sinonovacula constricta meat slag by purified water foam washing, standby；
(2) enzymolysis: the Sinonovacula constricta meat slag in step (1) is added neutral protease and carries out enzymolysis, enzymatic hydrolysis condition is: solid-liquid ratio 1:3 ~ 4, enzyme concentration 1500 ~ 2000U/g, pH value 6.5 ~ 7.5, hydrolysis temperature 50 ~ 60 DEG C, enzymolysis time 9 ~ 12 h, enzymolysis inactivates with boiling water bath after completing, it is centrifuged after pre-cooling, obtains supernatant；
(3) ultra-filtration and separation: by supernatant after 0.45 μm filter membrane sucking filtration, carry out ultrafiltration with the ultrafilter membrane that molecular cut off is 3 ku, obtain ultrafiltrate；
(4) FPLC purification system is isolated and purified: carry out isolated and purified by the ultrafiltrate in step (3) through FPLC purification system, obtain two active components, the angiotensin-converting enzyme inhibitory activity of these two active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component A；
(5) highly effective liquid phase chromatographic system is isolated and purified: component A is isolated and purified through highly effective liquid phase chromatographic system (HPLC), obtain three active components, the angiotensin-converting enzyme inhibitory activity of these three active components of separately sampled mensuration, the component taking angiotensin-converting enzyme inhibitory activity the highest is designated as component B, component B is after purity detecting, and obtaining aminoacid sequence is: Pro-Trp-Gly-Met-Phe(PTGMP) angiotensin converting enzyme-inhibiting peptide.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterised in that in step (1), Sinonovacula constricta is 1:3 ~ 5 with the mass ratio of isopropanol.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterised in that in step (2), be stirred continuously during enzymolysis.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterised in that in step (2), be cooled to 4 ~ 6 DEG C in advance.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterised in that in step (2), centrifuging process condition is: rotating speed 5000 ~ 10000rpm/min, centrifugation time 10 ~ 30min.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterized in that, in step (4), FPLC purification system is isolated and purified method particularly includes: use DEAE-SepharoseFF ion exchange column (packing material size 10 μm, 10 × 300 mm), gel is filled post, after Tris-Hcl balance (pH is 7.4), ultrafiltrate is configured to the solution of 200mg/ml, applied sample amount is 3mL every time, it is 7.4 with Tris-Hcl(pH respectively), 0.1mol/L, 0.3mol/L, 0.5mol/L, the NaCl solution of 0.8mol/L and 1mol/L carries out stepwise elution, elution speed is 2.2ml/min；Detection wavelength 280nm；Collect each peak.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterized in that, in step (5), highly effective liquid phase chromatographic system is isolated and purified method particularly includes: use sephadex G-25 chromatographic column (size: 2.6 × 80cm；Post material: Sephadex G-25), component B is configured to the solution of 200mg/ml, applied sample amount: 2.0mL；Flowing phase: ultra-pure water；Eluent flow rate: 1.0 mL/min；Detection wavelength: 280nm；Automatically gathering speed: 4.0min/ pipe；Collect each peak.
A kind of Sinonovacula constricta angiotensin converting enzyme-inhibiting peptide preparation method the most according to claim 1, it is characterized in that, in step (5), component B method for detecting purity is: use ZORBAX SB-C18 analytical type chromatographic column (packing material size: 5 μm, 4.6 × 150 mm)；Mobile phase A is ultra-pure water (containing 0.05%TFA), and Mobile phase B is acetonitrile (containing 0.05%TFA), employing isocratic elution mode: 80%A, 20%B；Flow velocity: 1.0 mL/min；Column temperature: 25 DEG C；Auto injection, sample size: 20 μ L；Detection wavelength: 280 nm.
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| CN106084013A (en) * | 2016-08-26 | 2016-11-09 | 南京中医药大学 | Inhibiting peptide of tonin and its preparation method and application |
| CN106636279A (en) * | 2016-12-30 | 2017-05-10 | 浙江海洋大学 | Preparation method of sinonovacula constricta anti-hypertension peptide |
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| CN103242430A (en) * | 2013-05-31 | 2013-08-14 | 南京中医药大学 | Angiotensin-converting enzyme inhibitory peptide, and preparation method and application thereof |
| CN104829689A (en) * | 2015-04-30 | 2015-08-12 | 上海市奉贤区中心医院 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
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| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN103242430A (en) * | 2013-05-31 | 2013-08-14 | 南京中医药大学 | Angiotensin-converting enzyme inhibitory peptide, and preparation method and application thereof |
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| CN106636279A (en) * | 2016-12-30 | 2017-05-10 | 浙江海洋大学 | Preparation method of sinonovacula constricta anti-hypertension peptide |
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