CN101461939B - Angiotensin converting enzyme inhibitor and preparation thereof - Google Patents
Angiotensin converting enzyme inhibitor and preparation thereof Download PDFInfo
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- CN101461939B CN101461939B CN2007101590294A CN200710159029A CN101461939B CN 101461939 B CN101461939 B CN 101461939B CN 2007101590294 A CN2007101590294 A CN 2007101590294A CN 200710159029 A CN200710159029 A CN 200710159029A CN 101461939 B CN101461939 B CN 101461939B
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Abstract
The invention relates to a deer blood active peptide, in particular to an angiotensin converting enzyme inhibitor and a method for preparing the same. The inhibitor takes the deer blood active peptide as an active component, and the deer blood active peptide has the activities of inhibiting the angiotensin converting enzyme and improving the impunity. Because the deer blood active peptide is prepared by enzymolysis, the method has the advantages of simple preparation process, mild condition and good application prospect.
Description
Technical field
The present invention relates to active peptide of deer blood; Specifically a kind of angiotensin converting enzyme (angiotensin-converting enzyme; ACE) inhibitor and preparation thereof, the active peptide of deer blood of the present invention's preparation has angiotensin converting enzyme inhibition activity and enhancing immunity is active.
Background technology
Protein content accounts for more than 13% in the Sanguis cervi, is the main component in the Sanguis cervi.Protein is made up of peptide; Peptides with physiological action is called Functional Polypeptides or biologically active peptide; Bioactive peptide has antioxidation, blood pressure lowering, raising immunity, antitumor, promotion mineral absorption isoreactivity; The bioactive peptide that the protein preparation of separate sources has various functions has become this hot research fields (list of references 1:Hannu Korhonen, Anne Pihlanto; Bioactive peptides:Production andfunctionality, International Dairy Journal 16 (2006) 945-960).Have that rich in protein is an ideal activity peptide raw material in the Sanguis cervi of health care.
Hypertension is the very high common cardiovascular disease of China's sickness rate, is to cause various complication such as the heart, brain, kidney and eye blood vessel and cause apoplexy, promote the significant risk factor of atherosclerosis, coronary heart disease.Hypertension does not generally have malaise symptoms in early days, until clinical picture-heart morbidity, rupture of blood vessel in brain take place, therefore has family history of hypertension and light symptoms person's early stage health care just most important.
Angiotensin converting enzyme (angiotensin-converting enzyme; ACE) inhibitor is main medicine (list of references 2:YANJUN YANG, EWAD.MARCZAK, the MEGUMI YOKOO that treats hypertension and heart failure now; HACHIRO USUI, AND MASAAKIYOSHIKAWA; Isolation and Antihypertensive Effect of AngiotensinI-Converting Enzyme (ACE) Inhibitory Peptides from Spinach Rubisco; J. Agric.Food Chem.2003; 51,4897-4902 4897).Angiotensin converting enzyme can be converted into the octapeptide Angiotensin II with strong vasoconstrictive effect with the active decapeptide angiotensin I of tool not; Cause hypertension, so ACE inhibitor can effectively be controlled hypertension and in treatment of diseases such as heart failure, plays a significant role.Peptides is one type of important ACE inhibitor, and peptide class ACE inhibitor has hypotensive effect and the normotensive is not had effect the hyperpietic, is ideal potential hyperpietic's health product therefore.
The enzymolysis of native protein is that preparation has a main method that ACE suppresses active peptide, and gentle enzymatic hydrolysis condition can not destroy its inherent activity and can produce new bioactive peptide chemical compound for the known Sanguis cervi that health-care effect arranged.
Summary of the invention
The purpose of this invention is to provide a kind of angiotensin-convertion enzyme inhibitor and preparation thereof; It is active ingredient with the active peptide of deer blood, and active peptide of deer blood has angiotensin converting enzyme inhibition activity and enhancing immunity is active.The present invention prepares active peptide of deer blood through the method for enzymolysis, and preparation technology is simple, mild condition, has a good application prospect
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
A kind of angiotensin-convertion enzyme inhibitor, it is active ingredient with the active peptide of deer blood, active peptide of deer blood has angiotensin converting enzyme inhibition activity and enhancing immunity is active.
The method for preparing of said angiotensin-convertion enzyme inhibitor is a raw material with fresh Sanguis cervi, and Sanguis cervi is after centrifugalize; Get supernatant, supernatant is plasma protein, the protein lyophilization of plasma protein after chemistry or thermal denaturation, degeneration; Be dissolved into the solution of weight concentration 1～50% with the phosphate buffer of pH2～9; Add proteolytic enzyme, the addition of enzyme is an enzyme: protein wt is than=1: 10～500, and enzymolysis is 0.5～24 hour under 20～80 ℃ of temperature; Lyophilization obtains the Sanguis cervi polypeptide again, and bioactive peptide detects has angiotensin converting enzyme inhibition activity and immune-enhancing activity.
Said centrifugal separation processes does, is raw material with fresh Sanguis cervi, gets 1～2 liter of Sanguis cervi, 4 ℃, Sanguis cervi under 3000～15000 rev/mins of rotating speeds centrifugal 0.1～2 hour, is precipitated as hemocyte, and supernatant is used to prepare bioactive peptide for the Sanguis cervi plasma protein.
Said chemical modification process is: in the Sanguis cervi plasma protein, adding carbamide to final concentration is 8-10mol/L, stirs 10-20 minute; Said thermal denaturation process is: with the Sanguis cervi plasma protein be heated to 60-100 ℃ 10-60 minute.
Proteolytic enzyme is pepsin, trypsin, flavor protease, papain, alkaline protease or neutral protease.
The present invention can obtain protein content, purity plasma protein preferably with Sanguis cervi through centrifugal, has omitted loaded down with trivial details purification procedures; Plasma protein has an immunologic function Sanguis cervi component by what peptide, immune protein and many trace element were formed, and the method for enzymolysis has kept and itself has active function.
Characteristics of the present invention are following:
1. the physiologically active of bioactive peptide is remarkable.Adopt preparation technology of the present invention, from Sanguis cervi, obtain to have the peptide matters of angiotensin converting enzyme inhibition activity and immune-enhancing activity first.
2. reaction condition is gentle, method is simple and feasible.The preparation of bioactive peptide of the present invention is the method through enzymolysis, and reaction condition is gentle, not only can prepare bioactive peptide but also keep original active component in the Sanguis cervi, reaction method simple possible.
3. have a good application prospect.Angiotensin-convertion enzyme inhibitor can reduce hyperpietic's blood pressure, and the bioactive peptide for preparing through the present invention might become the function that the antihypertensive healthcare article have raising immunity simultaneously again, therefore has a good application prospect.
Description of drawings
Fig. 1 is the active peptide of deer blood graph of molecular weight distribution of the embodiment of the invention 1 preparation.
The specific embodiment
Embodiment 1
With the fresh deer blood is raw material, according to following prepared Sanguis cervi polypeptide:
Get 1 liter of fresh deer blood, under 4 ℃, 3000 rev/mins rotating speeds centrifugal 1 hour, get supernatant 100 ℃ of degeneration 4 hours; It is 1% solution that protein lyophilization after the degeneration, exsiccant protein powder use the phosphate buffer of pH=2 to be dissolved into concentration, adds pepsin; The addition of enzyme is an enzyme: protein=1: 100; Hydrolysis is 0.5 hour under 20 ℃ of temperature, and reaction finishes postlyophilization, obtains Powdered active peptide of deer blood.
The molecular weight distribution of the bioactive peptide that the embodiment of the invention of being measured through substance assistant laser desorpted ionized mass spectrum (MALDI-MS) 1 prepares is as shown in Figure 1; The peptide molecular weight of enzymolysis gained mainly is distributed in below 2000 dalton, explains that through this technology can be the less peptides of molecular weight with the proteolysis of macromolecule.
The angiotensin converting enzyme inhibition activity of active peptide of deer blood is IC
50When=0.4mg/ml, the inductive mouse spleen lymphocyte transformation experiment of the ConA of mice were illustrated in concentration and are 10ug/ml, immune-enhancing activity was positive.
ACE suppresses activity and the enhance immunity active process is:
The bioactive peptide of method for preparing is carried out the active and immune-enhancing activity detection of ACE inhibition, and the result is following:
1) ACE suppresses activity test method: adopt document (B.A.Murray; D.J.Walsh, R.J.Fitzgerald, Modification of the furanacryloyl-L-phenylalanyglycylglycine assayfor determination of angiotensin-I-converting enzyme inhibiotory activity; Journal of biochemical and biophysical methods; 2004,59:127-137) institute's reported method, it is active that detection of active peptide ACE suppresses; With peptide FAGG is substrate; ACE is converted into FA and two peptides of GG with FAGG, obtains the activity of ACE in the variation of 340nm ultraviolet absorptivity, and the ACE activity is compared the ACE suppression ratio that can obtain bioactive peptide when adding bioactive peptide with bioactive peptide not.The ACE suppression ratio of the bioactive peptide through the preparation of this method is 90%, and the ACE suppression ratio of the plasma protein of enzymolysis is not 10%.
2) the active detection of enhance immunity:, detect with mtt assay through the inductive mouse spleen lymphocyte transformation experiment of ConA.When T lymphocyte receptor ConA stimulates back generation blast transformation, living cells particularly proliferative cell is decomposed into blue purple crystal through the mitochondrion hydrolytic enzyme with MTT (a kind of flaxen azoles nitrogen salt) and develops the color, and its OD value can reflect the propagation situation of cell.The result adopts variance analysis, adds the experimental mice splenocyte increment remarkable (P＜0.05) of bioactive peptide, explains that bioactive peptide has the enhance immunity activity.
Embodiment 2
Get 1 liter of fresh deer blood, under 4 ℃, 15000 rev/mins rotating speeds centrifugal 0.1 hour, get supernatant and add carbamide 480 grams; The protein lyophilization is taken out in degeneration after 1 hour, it is 50% solution that exsiccant protein powder uses the phosphate buffer of pH8 to be dissolved into concentration, adds neutral protease; The addition of enzyme is an enzyme: protein=1: 500; Hydrolysis is 24 hours under 80 ℃ of temperature, and reaction finishes postlyophilization, obtains Powdered active peptide of deer blood.
The active testing result of bioactive peptide is identical with embodiment 1.
Claims (5)
1. angiotensin-convertion enzyme inhibitor, it is characterized in that: it is active ingredient with the active peptide of deer blood, active peptide of deer blood has angiotensin converting enzyme inhibition activity and enhancing immunity is active;
It is following that active peptide of deer blood prepares process: with fresh Sanguis cervi is raw material, and Sanguis cervi is got supernatant after centrifugalize; Supernatant is plasma protein; The protein lyophilization of plasma protein after chemistry or thermal denaturation, degeneration is dissolved into the solution of weight concentration 1～50% with the phosphate buffer of pH2～9, adds proteolytic enzyme; The addition of enzyme is an enzyme: protein wt is than=1: 10～500; Enzymolysis is 0.5～24 hour under 20～80 ℃ of temperature, and lyophilization obtains the Sanguis cervi polypeptide again, and bioactive peptide detects has angiotensin converting enzyme inhibition activity and immune-enhancing activity.
2. the method for preparing of the said angiotensin-convertion enzyme inhibitor of claim 1, it is characterized in that: with fresh Sanguis cervi is raw material, and Sanguis cervi is after centrifugalize; Get supernatant, supernatant is plasma protein, the protein lyophilization of plasma protein after chemistry or thermal denaturation, degeneration; Be dissolved into the solution of weight concentration 1～50% with the phosphate buffer of pH2～9; Add proteolytic enzyme, the addition of enzyme is an enzyme: protein wt is than=1: 10～500, and enzymolysis is 0.5～24 hour under 20～80 ℃ of temperature; Lyophilization obtains the Sanguis cervi polypeptide again, and bioactive peptide detects has angiotensin converting enzyme inhibition activity and immune-enhancing activity.
3. according to the described method for preparing of claim 2, it is characterized in that: said centrifugal separation processes does,
4 ℃, Sanguis cervi under 3000～15000 rev/mins of rotating speeds centrifugal 0.1～2 hour, be precipitated as hemocyte, supernatant is used to prepare bioactive peptide.
4. according to the described method for preparing of claim 2, it is characterized in that:
Said chemical modification process is: in the Sanguis cervi supernatant, adding carbamide to final concentration is 8-10mol/L, stirs 10-20 minute; Said thermal denaturation process is: the Sanguis cervi supernatant is heated to 60-100 ℃, 10-60 minute.
5. according to the described method for preparing of claim 2, it is characterized in that: proteolytic enzyme is pepsin, trypsin, flavor protease, papain, alkaline protease or neutral protease.
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| CN2007101590294A CN101461939B (en) | 2007-12-19 | 2007-12-19 | Angiotensin converting enzyme inhibitor and preparation thereof |
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| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102199648A (en) * | 2011-03-25 | 2011-09-28 | 伊通满族自治县吉云鹿业发展有限公司 | Method for acquiring bioactive peptide by sequentially hydrolyzing deer haemproteins with two enzymes |
| CN102640927A (en) * | 2012-05-15 | 2012-08-22 | 华东理工大学 | Application of metapenaeus ensis in preparation of angiotensin converting enzyme (ACE) inhibitors |
| CN104621551A (en) * | 2013-11-07 | 2015-05-20 | 中国科学院兰州化学物理研究所 | Deer blood polypeptide effervescent tablet and preparation method thereof |
| CN106552255A (en) * | 2015-09-25 | 2017-04-05 | 中国科学院大连化学物理研究所 | A kind of preparation method of Sanguis cervi peptidoliposome |
| CN107217082A (en) * | 2017-06-05 | 2017-09-29 | 深圳知本康业有限公司 | A kind of deer hemalbumin polypeptide and application |
| CA3078625C (en) | 2017-10-09 | 2023-01-17 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CN112516166A (en) * | 2020-12-03 | 2021-03-19 | 青岛大学附属医院 | Method for freeze drying and powdering deer plasma protein peptide powder |
| CN112662724B (en) * | 2021-01-20 | 2023-04-28 | 重庆市药物种植研究所 | Deer blood polypeptide and extraction method and application thereof |
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| CN1552891A (en) * | 2003-11-10 | 2004-12-08 | 东北农业大学 | Preparation of dairy ACE inhibitory peptide |
| CN1680580A (en) * | 2005-01-04 | 2005-10-12 | 华南理工大学 | Preparation of inhibiting peptide of tonin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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Effective date of registration: 20230531 Address after: 116023 No. 457-41 Zhongshan Road, Shahekou District, Dalian City, Liaoning Province Patentee after: DALIAN INSTITUTE OF CHEMICAL PHYSICS, CHINESE ACADEMY OF SCIENCES Patentee after: Jiang Bo Patentee after: Jin Yan Address before: 116023 No. 457, Zhongshan Road, Liaoning, Dalian Patentee before: DALIAN INSTITUTE OF CHEMICAL PHYSICS, CHINESE ACADEMY OF SCIENCES Patentee before: Fushun Jiujiu Deer Industry Co.,Ltd. |