CN115160408A - 降压肽或中国蛤蜊降压肽提取物及其应用 - Google Patents
降压肽或中国蛤蜊降压肽提取物及其应用 Download PDFInfo
- Publication number
- CN115160408A CN115160408A CN202210759639.2A CN202210759639A CN115160408A CN 115160408 A CN115160408 A CN 115160408A CN 202210759639 A CN202210759639 A CN 202210759639A CN 115160408 A CN115160408 A CN 115160408A
- Authority
- CN
- China
- Prior art keywords
- antihypertensive peptide
- antihypertensive
- clam
- chinensis
- ace
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 82
- 230000003276 anti-hypertensive effect Effects 0.000 title claims abstract description 45
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 claims abstract description 52
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 claims abstract description 52
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 claims abstract description 51
- 206010020772 Hypertension Diseases 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 9
- 229940079593 drug Drugs 0.000 claims abstract description 8
- 235000013305 food Nutrition 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 33
- 229920001184 polypeptide Polymers 0.000 claims description 28
- 241000417531 Mactra chinensis Species 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 108010067770 Endopeptidase K Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 239000003112 inhibitor Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 5
- 238000001728 nano-filtration Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- 238000011033 desalting Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000001694 spray drying Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 235000013376 functional food Nutrition 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 25
- 230000002496 gastric effect Effects 0.000 abstract description 12
- 238000011282 treatment Methods 0.000 abstract description 9
- 238000000338 in vitro Methods 0.000 abstract description 7
- 230000015556 catabolic process Effects 0.000 abstract description 6
- 238000006731 degradation reaction Methods 0.000 abstract description 6
- 241000700159 Rattus Species 0.000 abstract description 5
- 230000036541 health Effects 0.000 abstract description 5
- 230000002269 spontaneous effect Effects 0.000 abstract description 4
- 239000002220 antihypertensive agent Substances 0.000 abstract description 3
- 229940127088 antihypertensive drug Drugs 0.000 abstract description 3
- 230000001631 hypertensive effect Effects 0.000 abstract description 3
- 230000029087 digestion Effects 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 235000020639 clam Nutrition 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 230000036772 blood pressure Effects 0.000 description 11
- 239000012530 fluid Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 6
- 230000000415 inactivating effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 4
- 102000057297 Pepsin A Human genes 0.000 description 4
- 229960000830 captopril Drugs 0.000 description 4
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 4
- 238000003304 gavage Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000011699 spontaneously hypertensive rat Methods 0.000 description 3
- AAXWBCKQYLBQKY-IRXDYDNUSA-N (2s)-2-[[(2s)-2-[(2-benzamidoacetyl)amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-4-methylpentanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)C=1C=CC=CC=1)C1=CN=CN1 AAXWBCKQYLBQKY-IRXDYDNUSA-N 0.000 description 2
- BIDNLKIUORFRQP-XYGFDPSESA-N (2s,4s)-4-cyclohexyl-1-[2-[[(1s)-2-methyl-1-propanoyloxypropoxy]-(4-phenylbutyl)phosphoryl]acetyl]pyrrolidine-2-carboxylic acid Chemical compound C([P@@](=O)(O[C@H](OC(=O)CC)C(C)C)CC(=O)N1[C@@H](C[C@H](C1)C1CCCCC1)C(O)=O)CCCC1=CC=CC=C1 BIDNLKIUORFRQP-XYGFDPSESA-N 0.000 description 2
- 102000005367 Carboxypeptidases Human genes 0.000 description 2
- 108010006303 Carboxypeptidases Proteins 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 108010061435 Enalapril Proteins 0.000 description 2
- 108010007859 Lisinopril Proteins 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 230000004531 blood pressure lowering effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229960000873 enalapril Drugs 0.000 description 2
- GBXSMTUPTTWBMN-XIRDDKMYSA-N enalapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)CC1=CC=CC=C1 GBXSMTUPTTWBMN-XIRDDKMYSA-N 0.000 description 2
- 229960002490 fosinopril Drugs 0.000 description 2
- 108010016268 hippuryl-histidyl-leucine Proteins 0.000 description 2
- 230000001077 hypotensive effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 229960002394 lisinopril Drugs 0.000 description 2
- RLAWWYSOJDYHDC-BZSNNMDCSA-N lisinopril Chemical compound C([C@H](N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(O)=O)C(O)=O)CC1=CC=CC=C1 RLAWWYSOJDYHDC-BZSNNMDCSA-N 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000036454 renin-angiotensin system Effects 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 108010064733 Angiotensins Proteins 0.000 description 1
- 102000015427 Angiotensins Human genes 0.000 description 1
- 241000237519 Bivalvia Species 0.000 description 1
- 101000984728 Chiropsoides quadrigatus Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 241000514745 Mactra Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 241000237503 Pectinidae Species 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000035487 diastolic blood pressure Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- KVMLCRQYXDYXDX-UHFFFAOYSA-M potassium;chloride;hydrochloride Chemical compound Cl.[Cl-].[K+] KVMLCRQYXDYXDX-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 201000001474 proteinuria Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 235000020637 scallop Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 235000000891 standard diet Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了中国蛤蜊降压肽及其在制备降血压药物和保健食品中的应用。中国蛤蜊降压提取物经酶解获得，对该提取物进一步分离纯化得到一特定序列的多肽，该降压肽的氨基酸序列为Lys‑Asp‑Glu‑Tyr‑Lys‑His‑Leu‑Asn‑Phe‑Gly‑Glu‑Val(KDEYKHLNFGEV)。本发明公开的中国蛤蜊降压肽具有显著的血管紧张素转化酶(angiotensin converting enzyme，ACE)抑制活性。在体外表现为温度、pH、模拟胃肠消化和血管紧张素转化酶(ACE)降解稳定性。在体外和非自发性高血压大鼠(SHRs)体内都显示出良好的降压效果，可用于高血压治疗相关的保健品和药品。
Description
技术领域
本发明涉及生物活性肽技术领域，涉及中国蛤蜊的降压肽提取物和具有特定序列的降压肽及其在降血压药品和保健品方面的应用。
背景技术
高血压以人体循环动脉血压升高为主要特征(高压≥140mmHg，低压≥ 90mmHg)，高血压与心血管疾病和糖尿病的死亡率增加有关。世界卫生组织(WHO)报道，每年有超过1750万人死于心血管疾病。高血压已经变成了全球的主要死亡原因。肾素-血管紧张素系统(renin-angiotensin system,RAS)和激肽-缓激肽系统(kallikreo-bradykinin system,KKS)可在血压调节中发挥重要作用。血管紧张素转化酶(ACE)是普遍存在于哺乳动物组织中的能够调控肾素-血管紧张素系统(RAS)和激肽-缓激肽系统 (KKS)的一种Zn2+依赖性的羧肽酶。所以，ACE被认为是治疗高血压的重要靶点。
市场上普遍的ACE抑制药物多为“普利”类，像卡托普利(captopril)、赖诺普利(lisinopril)、依那普利(enalapril)、福辛普利(fosinopril)等，尽管这些药物能发挥好的降压效果，但是，他们也会产生一系列的副作用，像咳嗽、皮疹、恶心、急性肾功能衰竭、蛋白尿等。这些副作用会降低患者的依从性，增加额外医疗费用，因此越来越多的研究开始集中于对低毒性的天然ACE抑制肽的开发来作为抗高血压药物的辅助和替代品。食源性的多肽不仅能发挥同样的抗压作用还因为其天然来源具有安全性和低毒性，所以从天然食品中开发降压肽具有意义和前景。
中国蛤蜊，俗名“黄蚬子”、“黄蛤”、“飞蛤”，学名Mactra chinensis，隶属于软体动物门、瓣鳃纲、异柱目、蛤蜊科、蛤蜊属，主要分布在我国辽宁、山东的黄、渤海沿岸。人工养殖时常年均可采捕，尤以深秋和春季采捕效果为佳。中国蛤蜊适于生活在潮间带的细砂滩至水深60m的浅海区，是一种营养丰富、肉质鲜美的实用经济滩涂贝类。目前已经有多种从海洋贝类中筛选出具有ACE抑制作用的多肽，像牡蛎、扇贝、菲律宾蛤仔等。但很少有从中国蛤蜊中分离ACE抑制肽的报道。
发明内容
鉴于此，本发明的目的是选择中国蛤蜊作为原料，通过蛋白酶K酶解，液质连用技术，鉴定出氨基酸序列为 Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val(KDEYKHLNFGEV)的 ACE抑制肽。
本发明的中国蛤蜊降压肽：
Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val(KDEYKHLNFGEV)在体外显示较高的血管紧张素转化酶(ACE)抑制作用，在自发性高血压大鼠(SHRs)体内发挥良好的降压效果。
本发明的另个一个目的是提供一种中国蛤蜊降压肽提取物，其按照以下方法制备：将中国蛤蜊粉碎匀浆，加入中国蛤蜊重量0.5-20倍的纯化水，加入蛋白酶K进行酶解，离心过滤之后取上清液，经截留分子量为180道尔顿的纳滤脱盐，浓缩，将浓缩液冻干或喷雾干燥,获得中国蛤蜊酶解降压肽提取物。其中蛋白酶K添加量为中国蛤蜊重量的0.2％-5％，酶解温度控制在35℃-55℃，酶解pH控制在7.0-9.0，酶解时间为2h-5h，酶解结束后沸水10-60min灭酶。
本发明中的中国蛤蜊降压肽提取物及中国蛤蜊降压肽在血管紧张素抑制剂，降压药物及保健品中有广泛的应用。
本发明公开的中国蛤蜊降压肽具有显著的血管紧张素转化酶 (angiotensinconverting enzyme，ACE)抑制活性。在体外表现为温度、pH、模拟胃肠消化和血管紧张素转化酶(ACE)降解稳定性。在体外和非自发性高血压大鼠(SHRs)体内都显示出良好的降压效果，可用于高血压治疗相关的保健品和药品。
附图说明
图1为降压肽KDEYKHLNFGEV的液相色谱图(A)和质谱鉴定图(B)。
图2多肽KDEYKHLNFGEV(0.5mg/ml)在不同温度处理下的ACE抑制活性。
图3多肽KDEYKHLNFGEV(0.5mg/ml)在不同pH处理下的ACE抑制活性。
图4多肽KDEYKHLNFGEV(0.1mg/ml和0.5mg/ml)在模拟胃肠液中处理之后的ACE抑制活性。GC无胃蛋白酶的模拟胃液处理；G含胃蛋白酶的模拟胃液处理；G+IC无胃蛋白酶和胰酶的模拟胃肠液；G+I含有胃蛋白酶和胰酶的模拟胃肠液。
图5多肽KDEYKHLNFGEV(0.1mg/ml)与ACE结合稳定性研究。
图6多肽KDEYKHLNFGEV对自发性高血压大鼠(SHRs)高压的降低作用；灌胃剂量10mg/kg，A：收缩压；B：舒张压，●对照组，▉卡托普利，◆多肽KDEYKHLNFGEV
具体实施方式
通过以下具体实施例对本发明方案作进一步的具体说明：
实施例1
中国蛤蜊降压肽提取物的制备方法，包括以下步骤：
(1)中国蛤蜊鲜肉500g匀浆，加入其重量1倍(500g)的纯化水，混匀
(2)加入500mg蛋白酶K酶解，酶解温度42℃，酶解pH控制(采用1M氢氧化钠调节)在8.0，酶解时间为3h，酶解完成之后100℃灭酶10min，离心除杂。
(3)上清液采用截留分子量180道尔顿的纳滤膜脱盐，浓缩至100mL，冻干，制得降压肽提取物。
(4)使用下述高效液相色谱法(HPLC)对中国蛤蜊降压肽提取物的ACE 抑制活性进行评价，其抑制ACE的IC50值为1.02±0.31mg/ml。
实施例2
中国蛤蜊降压肽提取物的制备方法，包括以下步骤：
(1)中国蛤蜊肉烘干粉碎，取干粉20g加入400ml蒸馏水搅拌，并在室温下溶胀4h。
(2)加入400mg蛋白酶K搅拌酶解，酶解温度控制在42℃，酶解pH 控制(采用1M氢氧化钠调节)在7.8，酶解时间为2h，酶解完成后100℃沸水灭酶10min，过滤除杂。
(3)上清液采用截留分子量180道尔顿的纳滤膜脱盐，浓缩至20mL，喷雾干燥，制得中国蛤蜊降压肽提取物。
(4)使用下述高效液相色谱法(HPLC)对中国蛤蜊降压肽提取物的 ACE抑制活性进行评价，该降压肽抑制ACE的IC50值为0.93±0.24mg/ml。
实施例3
取实施例2制得的中国蛤蜊降压肽提取物，采用 UPLC-ESI-Q-Exactive Focus-MS/MS进行液相色谱-质谱分析(如图1所示)，所得质谱数据与中国蛤蜊转录组数据库比对，得到特定序列的多肽 KDEYKHLNFGEV。将该多肽采用固相合成法进行合成，得到序列为KDEYKHLNFGEV，纯度≥98％的多肽。
SEQ ID No.1的信息
(a)序列特征
*长度：12氨基酸残基
*类型：氨基酸
*链型：单链
*拓扑结构：线性
(b)分子类型：蛋白
序列描述：SEQ ID No.1
(一)Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val高效液相色谱法
(HPLC)法测定多肽体外ACE抑制活性
本实施例所用的药品及仪器无特殊说明均可从商业获得。
高效液相色谱法(HPLC)测定ACEI活性的原理为：马尿酰-组氨酰- 亮氨酸(Hip-His-Leu，HHL，美国Sigma公司)可被ACE(美国Sigma公司) 酶解成为马尿酸和二肽产物，马尿酸在紫外波长228nm处有特征的吸收峰，当抑制剂与血管紧张素转化酶(ACE)作用时，马尿酸的生成量减少，通过测定加入抑制剂前后生成马尿酸的量可以测定抑制剂对ACE的抑制率。计算公式如下：
上式中A空白为不添加抑制肽时马尿酸的峰面积，A抑制剂为添加抑制肽时马尿酸的峰面积；IC50表示能抑制ACE一半活性时抑制肽的浓度。
结果中国蛤蜊降压肽KDEYKHLNFGEV的IC50值为7.59±0.36μM。参考文献：Feng-Jun Wang,Xiao-Yan Yin,Joe M Regenstein,Jian-Zhong Wang.Separation andpurification of angiotensin-I-converting enzyme(ACE) inhibitory peptides fromwalnuts(Juglans regia L.)meal.Eur Food Res Technol (2016)242:911–918
(二)多肽的理化性质
多肽KDEYKHLNFGEV的分子量为1479.60Da，无毒。
(三)热稳定性和pH稳定性评价
多肽(0.5mg/ml)在温度20℃-70℃的温度范围内处理2h，处理完成后分别取20μL的多肽溶液按上述方法测定其ACE抑制活性。
分别用盐酸和氢氧化钠配置pH2-12的水溶液，多肽(0.5mg/ml)用 pH2-12范围内的溶液溶解，在此条件下处理2h后用1M的HCL和1M的 NaOH中和多肽溶液，取多肽溶液20μL按上述方法测定其ACE抑制活性。
附图2和附图3显示该中国蛤蜊降压肽KDEYKHLNFGEV在温度 20-70℃以及pH2-12范围内具有温度和pH稳定性。
(四)模拟胃肠液降解稳定性评价
体外模拟胃肠液降解来评价该多肽在模拟胃肠液中的稳定性。多肽(KDEYKHLNFGEV)溶解到0.1M氯化钾-盐酸(KCl-HCl)(pH 2.0)缓冲液中至终浓度分别为0.1mg/mL和0.5mg/ml。胃蛋白酶(≥250U/mg)添加到多肽溶液中至终浓度为0.8mg/mL，该溶液在37℃中孵育4h，之后体系在100℃沸水中灭酶10min，采用1M氢氧化钠调节溶液的pH至7.0， 12000rpm离心5min，取体系中上清液20μL来按上述高效液相色谱法测定 ACE抑制活性；剩余的上清液添加胰蛋白酶(8x USP)至终浓度10mg/mL，该溶液在37℃中继续孵育4h，100℃沸水浴10min灭酶，12000rpm离心 5min，取上清液20μL按上述高效液相色谱法测定ACE抑制活性。
附图4显示中国蛤蜊降压肽KDEYKHLNFGEV能够在抵抗模拟胃肠液的降解，在模拟胃肠液中保持其结构及ACE抑制活性稳定性。
(五)多肽与ACE结合稳定性考察
ACE是具有广泛的特异底物的羧肽酶，它能够产生降解降压肽的功能。 300μL的多肽(0.1mg/mL)与300μL 0.1U/mL ACE溶液混合在37℃下孵育4-24h，然后100℃沸水10min灭活ACE，取20μL多肽液、按上述高效液相色谱法测定ACE的抑制活性。附图5显示中国蛤蜊降压肽 KDEYKHLNFGEV在与ACE孵育24h之内仍保持ACE抑制活性。
(六)自发性高血压大鼠(SHRs)单次给药后降压效果评价
自发性高血压大鼠SHRs(雄性，10周龄，体重250-300g，无特异性病原体)购买自北京维通利华实验动物有限公司，SHRs在22±2℃下12h 光照/12h黑暗的日夜循环，每天定时喂养纯水和标准的饮食(即标准日粮)。 SHRs被分为分为三组(空白对照组、阳性对照组、实验组)，每组有6只，在高压高于180mmHg时，可以开始灌胃，KDEYKHLNFGEV灌胃剂量为 10mg/kg。
生理盐水用作空白对照，卡托普利作为阳性对照，灌胃剂量为10mg/kg，鼠尾无创血压测定系统CODA测定灌胃后0、1、2、4、6、8h的高压和低压。每次测量重复3次。
附图6说明KDEYKHLNFGEV在灌胃之后1h至2h能够显著的降低 SHRs的高压(p<0.05)，在2h，高压最多能从204mmHg降低至170mmHg，最多能使高压降低34mmHg，然后血压开始缓慢的恢复，直到灌胃后8h，血压恢复至之前的水平。KDEYKHLNFGEV在2h使SHRs的血压由145 mmHg降低至118mmHg(p<0.05)，然后血压开始缓慢恢复，8h恢复至原始的水平。总的来说，KDEYKHLNFGEV能在SHRs体内产生良好的降压效果。
以上结果表明：中国蛤蜊降压肽KDEYKHLNFGEV对ACE有较强的抑制活性；该多肽能在0℃-100℃和pH2-12的范围内都能保持活性，此外该多肽还能抵抗模拟胃肠液和ACE的降解；酶动力学显示该多肽为ACE 的非竞争性抑制剂；在自发性高血压大鼠(SHRs)体内也能显示出显著的降压活性。中国蛤蜊的降压肽和具有特定序列的降压肽KDEYKHLNFGEV 将广泛应用于高血压治疗相关的保健品和药物。
以上所述的实施例对本发明的技术方案进行了详细的说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明，凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种降压肽，其特征在于:具有序列表SEQ ID No.1所示的氨基酸序列。
2.按照权利要求1所述降压肽，其特征在于：多肽序列为Lys-Asp-Glu-Tyr-Lys-His-Leu-Asn-Phe-Gly-Glu-Val(KDEYKHLNFGEV)。
3.一种中国蛤蜊降压肽提取物，其特征在于：所述的中国蛤蜊降压肽提取物的制备方法包括：将中国蛤蜊肉粉碎或匀浆，加入中国蛤蜊肉重量0.5-20倍的纯化水，加入蛋白酶K进行酶解，离心过滤之后取上清液，经截留分子量为180-200道尔顿的纳滤脱盐，浓缩，将浓缩液冻干或喷雾干燥，获得中国蛤蜊降压肽提取物。
4.根据权利要求3所述的中国蛤蜊降压肽提取物，其特征在于：蛋白酶K添加量为中国蛤蜊重量的0.2％-5％，酶解温度控制在35℃-55℃，酶解pH控制在7.0-9.0，酶解时间为2h-5h，酶解结束后97℃-100℃沸水10-60min灭酶。
5.一种权利要求1或2所述降压肽、或3或4所述的中国蛤蜊降压肽提取物的应用，其特征在于：所述的降压肽或所述的中国蛤蜊降压肽提取物中的一种或二种以上在制备血管紧张素转化酶(ACE)抑制剂、高血压预防和/或治疗疾病的药品、保健食品、特医食品或功能食品中的应用。
6.按权利要求5所述的应用，其特征在于：以权利要求1或2所述降压肽、或3或4所述的中国蛤蜊降压肽提取物中的一种或二种以上配以任何符合食品(食品学)或药品(药物学)生产允许或可接受的辅料或载体制成。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210759639.2A CN115160408A (zh) | 2022-06-29 | 2022-06-29 | 降压肽或中国蛤蜊降压肽提取物及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210759639.2A CN115160408A (zh) | 2022-06-29 | 2022-06-29 | 降压肽或中国蛤蜊降压肽提取物及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115160408A true CN115160408A (zh) | 2022-10-11 |
Family
ID=83488491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210759639.2A Pending CN115160408A (zh) | 2022-06-29 | 2022-06-29 | 降压肽或中国蛤蜊降压肽提取物及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115160408A (zh) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100637415B1 (ko) * | 2005-05-02 | 2006-10-20 | 이무하 | Ａｃｅ 억제활성을 갖는 폴리펩타이드들을 유효성분으로함유하는 고혈압의 예방 및 치료용 조성물 |
| EP1818395A1 (en) * | 2006-02-08 | 2007-08-15 | Diatos | Compositions and methods for treating lysosomal storage diseases |
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN104862364A (zh) * | 2015-04-28 | 2015-08-26 | 辽宁天增祥生物科技有限公司 | 油菜蜂花粉寡肽组合物及其制备方法 |
| CN108892710A (zh) * | 2018-07-24 | 2018-11-27 | 中国科学院海洋研究所 | 龙须菜降压肽提取物和龙须菜降压肽及其应用 |
| CN113329758A (zh) * | 2018-12-14 | 2021-08-31 | 弗莱德哈钦森癌症研究中心 | 转铁蛋白受体靶向肽 |
| CN114195857A (zh) * | 2021-10-14 | 2022-03-18 | 中国科学院海洋研究所 | 一种降压肽及其制备方法和应用 |
| CN116925175A (zh) * | 2023-04-10 | 2023-10-24 | 浙江工业大学 | 一种珍珠多肽提取物、多肽及在制备酪氨酸酶抑制剂中的应用 |
-
2022
- 2022-06-29 CN CN202210759639.2A patent/CN115160408A/zh active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100637415B1 (ko) * | 2005-05-02 | 2006-10-20 | 이무하 | Ａｃｅ 억제활성을 갖는 폴리펩타이드들을 유효성분으로함유하는 고혈압의 예방 및 치료용 조성물 |
| EP1818395A1 (en) * | 2006-02-08 | 2007-08-15 | Diatos | Compositions and methods for treating lysosomal storage diseases |
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN104862364A (zh) * | 2015-04-28 | 2015-08-26 | 辽宁天增祥生物科技有限公司 | 油菜蜂花粉寡肽组合物及其制备方法 |
| CN108892710A (zh) * | 2018-07-24 | 2018-11-27 | 中国科学院海洋研究所 | 龙须菜降压肽提取物和龙须菜降压肽及其应用 |
| CN113329758A (zh) * | 2018-12-14 | 2021-08-31 | 弗莱德哈钦森癌症研究中心 | 转铁蛋白受体靶向肽 |
| CN114195857A (zh) * | 2021-10-14 | 2022-03-18 | 中国科学院海洋研究所 | 一种降压肽及其制备方法和应用 |
| CN116925175A (zh) * | 2023-04-10 | 2023-10-24 | 浙江工业大学 | 一种珍珠多肽提取物、多肽及在制备酪氨酸酶抑制剂中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| QUANBIN ZHANG等: "Isolation, identification and in vivo antihypertensive effect of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from Spirulina protein hydrolysate", 《FOOD & FUNCTION》, vol. 13, 25 July 2022 (2022-07-25), pages 9108 - 9118 * |
| 陈应运等: "发酵法制备蛤蜊ACE抑制肽的菌种筛选", 《核农学报》, no. 10, 31 December 2017 (2017-12-31), pages 1986 - 1992 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lin et al. | Angiotensin-I-converting enzyme (ACE)-inhibitory and antihypertensive properties of squid skin gelatin hydrolysates | |
| CN108892710B (zh) | 龙须菜降压肽提取物和龙须菜降压肽及其应用 | |
| JP2014500005A (ja) | トウモロコシ降圧活性ペプチドを調製するための工業的方法 | |
| CA2567582C (en) | Anti-hypertensive dietary supplement derived from salmo or oncorhynchus protein hydrolysates | |
| KR101533308B1 (ko) | 안지오텐신-i 전환 효소 저해능을 나타내는 펩타이드를 유효성분으로 포함하는 심혈관계 질환 예방 또는 치료용 약학적 조성물 | |
| TWI516280B (zh) | 紅藜萃取物用於製備促進膠原蛋白生成及抗皮膚老化之組合物之用途 | |
| Kumar et al. | Pharmacological review on natural ACE inhibitors | |
| CN111893157A (zh) | 一种降血糖藜麦肽及其制备方法和应用 | |
| US6217879B1 (en) | Enzyme-decomposed materials of laver and uses thereof | |
| JPWO2007108554A1 (ja) | 血圧上昇抑制作用を有するペプチド | |
| Kim et al. | A novel bioactive peptide derived from enzymatic hydrolysis of Ruditapes philippinarum: Purification and investigation of its free-radical quenching potential | |
| Harnedy et al. | Bioactive proteins and peptides from macroalgae, fish, shellfish and marine processing waste | |
| CN102336806A (zh) | 一种血管紧张素转化酶(ace)抑制物 | |
| CN108840909B (zh) | 一种紫菜降压肽和紫菜降压肽提取物及应用 | |
| CN114106092A (zh) | 一种具有ace抑制作用的活性多肽及其应用 | |
| CN115160408A (zh) | 降压肽或中国蛤蜊降压肽提取物及其应用 | |
| KR102693765B1 (ko) | 몰로키아 추출물을 유효성분으로 포함하는 알코올성 간 손상 개선, 예방 또는 치료용 조성물 | |
| CN111944009B (zh) | 一种海蓬子降血压肽的制备方法及应用 | |
| JP4934369B2 (ja) | 血圧低下作用を有するペプチド | |
| KR20140066317A (ko) | 헛개나무 추출물을 첨가한 표고버섯 균사체 흑미 배양액을 유효성분으로 함유하는 간기능 개선용 식품 | |
| CN113087773A (zh) | 一种具有降血糖和抗氧化功能的牦牛骨肽及其制备方法 | |
| CA2119660C (en) | Amylase inhibitors | |
| CN112521446A (zh) | 一种ace抑制肽及其用途 | |
| JP5312780B2 (ja) | 血中アンモニア濃度を低下させる飲食物および医薬組成物 | |
| KR100787949B1 (ko) | 대두 발효 조성물의 제조방법 및 이 조성물로부터 유래된펩타이드 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |