CN113801195B - 一种金枪鱼鱼卵降血压肽及其应用 - Google Patents
一种金枪鱼鱼卵降血压肽及其应用 Download PDFInfo
- Publication number
- CN113801195B CN113801195B CN202111203878.1A CN202111203878A CN113801195B CN 113801195 B CN113801195 B CN 113801195B CN 202111203878 A CN202111203878 A CN 202111203878A CN 113801195 B CN113801195 B CN 113801195B
- Authority
- CN
- China
- Prior art keywords
- tuna
- roe
- antihypertensive peptide
- ace
- sth
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 28
- 230000003276 anti-hypertensive effect Effects 0.000 title claims abstract description 26
- 239000003814 drug Substances 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 230000036772 blood pressure Effects 0.000 claims description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 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 abstract description 39
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 abstract description 39
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 abstract description 39
- 230000005764 inhibitory process Effects 0.000 abstract description 12
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 5
- 206010020772 Hypertension Diseases 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 2
- 238000000108 ultra-filtration Methods 0.000 description 14
- 238000004007 reversed phase HPLC Methods 0.000 description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000005227 gel permeation chromatography Methods 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 229920005654 Sephadex Polymers 0.000 description 7
- 239000012507 Sephadex™ Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- 108091005507 Neutral proteases Proteins 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 239000007986 glycine-NaOH buffer Substances 0.000 description 3
- 239000000413 hydrolysate Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 2
- 239000005541 ACE inhibitor Substances 0.000 description 2
- 102400000344 Angiotensin-1 Human genes 0.000 description 2
- 101800000734 Angiotensin-1 Proteins 0.000 description 2
- 102400000345 Angiotensin-2 Human genes 0.000 description 2
- 101800000733 Angiotensin-2 Proteins 0.000 description 2
- 102400000967 Bradykinin Human genes 0.000 description 2
- 101800004538 Bradykinin Proteins 0.000 description 2
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 2
- 241000269819 Katsuwonus pelamis Species 0.000 description 2
- 241000269851 Sarda sarda Species 0.000 description 2
- 229960002478 aldosterone Drugs 0.000 description 2
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 2
- 229950006323 angiotensin ii Drugs 0.000 description 2
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000001077 hypotensive effect Effects 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 1
- 101100493735 Arabidopsis thaliana BBX25 gene Proteins 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 206010010164 Hypertension complications Diseases 0.000 description 1
- 102000001399 Kallikrein Human genes 0.000 description 1
- 108060005987 Kallikrein Proteins 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 101100311254 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) STH1 gene Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 101100277996 Symbiobacterium thermophilum (strain T / IAM 14863) dnaA gene Proteins 0.000 description 1
- 208000009205 Tinnitus Diseases 0.000 description 1
- 206010047139 Vasoconstriction Diseases 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000886 tinnitus Toxicity 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000025033 vasoconstriction Effects 0.000 description 1
- 238000005303 weighing Methods 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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种具有血管紧张素转化酶(ACE)抑制活性的金枪鱼鱼卵降压肽及其制备方法和用途，该降压肽的氨基酸序列为Trp‑Pro‑Phe‑Ala‑Cys‑Ser(WPFACS)，ESI/MS检测分子量为709.8Da。本发明制备的金枪鱼鱼卵降压肽具有显著的血管紧张素转化酶(ACE)抑制活性，可应用于治疗高血压相关的药物或保健食品。本发明以金枪鱼鱼卵为原料，利用酶解工艺和色谱制备技术制备得到了一种具有ACE抑制的降血压肽，为金枪鱼鱼卵高值化利用提供技术支持。
Description
技术领域
本发明涉及多肽技术领域，具体涉及一种金枪鱼鱼卵的降血压肽及其应用。
背景技术
高血压是一种常见病和多发病。此病一般起病缓慢，患者早期常无症状，或仅有头晕、头痛、心悸、耳鸣等症状，表面上看是一种独立的疾病，实际上是引发心、脑血管和肾病变的一个重要的危险因素，如果治疗不当就会病变成为较严重的脑中风、心肌梗死和肾功能衰竭等这些常见高血压并发症。血管紧张素转化酶(ACE)又称为肽基-羧基肽酶或者激肽酶Ⅱ，分子量为150,000Da，属血管内皮细胞膜结合酶。ACE可以将十肽的血管紧张素Ⅰ水解成八肽的血管紧张素Ⅱ，促使血管进一步收缩，导致血压升高。同时，ACE也可通过作用于肾上腺皮质而促进醛固酮的分泌。因此，ACE是肾素-血管紧张素-醛固酮(RAS)的重要成分。另外，ACE还可以催化具有降压作用的缓激肽水解而使其失去活性。因此，寻找合适的抑制体内ACE活性的物质，就可以达到降低或者控制血压的目的。血管紧张素转化酶抑制剂(ACEI)是一类发展迅速的抗高血压药物，其主要通过抑制ACE活性，致使血管紧张素Ⅰ不能转换为血管紧张素Ⅱ，同时抑制ACE对缓激肽的灭活作用，从而产生降压效应。
发明内容
本发明以金枪鱼鱼卵为原料，利用酶解工艺和色谱制备技术制备得到了一种具有ACE抑制的降血压肽，为金枪鱼鱼卵高值化利用提供技术支持。
一方面，本发明提供一种金枪鱼鱼卵降血压肽，其氨基酸序列为：Trp-Pro-Phe-Ala-Cys-Ser，ESI-MS测定其分子量为709.8Da。
另一方面，本发明提供了一种金枪鱼鱼卵降血压肽的制备方法，其包括以下步骤：将金枪鱼鱼卵微波和超声预处理、酶解、超滤、凝胶色谱分离和反相高效液相色谱制(RP-HPLC)纯化，得到降压肽Trp-Pro-Phe-Ala-Cys-Ser。
具体地，一种金枪鱼鱼卵降血压肽包括以下步骤：
1)金枪鱼鱼卵的预处理：将金枪鱼鱼卵解冻、除杂，然后加入双蒸水混合、匀浆，放置于电脑微波超声组合成/萃取仪中，在微波功率500W条件下，处理5min，取出冻干；冻干后的粉末加入乙酸乙酯混合，于室温3000W超声处理25min，离心出去上清液，得金枪鱼鱼卵粉末；
2)金枪鱼鱼卵酶解液的制备：将上述金枪鱼鱼卵粉末与甘氨酸-NaOH缓冲液混合，调节混合液pH值至7.0，调温度至50℃，加入中性蛋白酶(5.0×104U/g)，酶解3.5h；酶解液放于沸水种10min后，灭酶活，得金枪鱼鱼卵酶解液(STH)；
3)金枪鱼鱼卵酶解液的超滤分级：将金枪鱼鱼卵酶解液(STH)采用1kDa，3.5kDa和5kDa超滤膜进行超滤处理，收集分子量小于1kDa(STH1)，得超滤酶解物；
4)金枪鱼鱼卵降压肽的制备：将超滤酶解物(STH1)依次经Sephadex G-15凝胶色谱和反相高效液相色谱(RP-HPLC)纯化，得金枪鱼鱼卵降压肽。
作为优选，所述Sephadex G-15凝胶色谱分离步骤如下：将超滤酶解物(STH1)配成50μg/mL溶液，经过葡聚糖凝胶G-15柱层析分离，用双蒸水进行洗脱，根据220nm下的吸光度曲线收集洗脱组分，测定各组分的血管紧张素转化酶(ACE)抑制活性，具有最高ACE抑制活性的组分即为凝胶层析酶解物。
作为优选，所述RP-HPLC纯化步骤如下：将凝胶层析酶解物配成45μg/mL溶液，利用RP-HPLC(条件为：进样量为10μL；色谱柱为Zorbax C18(250×4.6mm，5μm)；流动相为50％乙腈；洗脱速度为0.5mL/min；紫外检测波长为220nm)进行纯化，测定各分离组分的血管紧张素转化酶(ACE)半数抑制率(IC50)，选择最优的组分，测定其氨基酸序列和分子量，得氨基酸序列为Trp-Pro-Phe-Ala-Cys-Ser(WPFACS)，ESI/MS检测分子量为709.8Da。
作为优选，所述的步骤1)中的金枪鱼为鲣鱼Katsuwonus pelamis。
作为优选，所述步骤1)中解冻除杂后的金枪鱼与蒸馏水的重量体积比g/ml为1:5。
作为优选，所述步骤1)中冻干后的粉末与乙酸乙酯的重量体积比g/ml为1:4。
作为优选，所述步骤2)中中性蛋白酶的添加量为金枪鱼鱼卵粉末重量的0.8％～1.5％。
作为优选，所述步骤2)中金枪鱼鱼卵粉末与甘氨酸-NaOH缓冲液的重量体积比g/ml为1:8。
另一方面，本发明提供了一种金枪鱼鱼卵降血压肽用于制备降血压相关疾病的药物中的用途。
本发明公开了一种具有血管紧张素转化酶(ACE)抑制活性的金枪鱼鱼卵降压肽及其制备方法和用途，该降压肽的氨基酸序列为Trp-Pro-Phe-Ala-Cys-Ser(WPFACS)，ESI/MS检测分子量为709.8Da。本发明采用的是一种可控和对环境友好的生物酶法，易通过对酶解过程的监控，使金枪鱼鱼卵降压肽最大程度地释放出来，提高了原料的利用率；同时所制备的降压肽安全、无毒副作用；本发明制备的降压肽具有显著的血管紧张素转化酶(ACE)抑制活性，可应用于治疗高血压相关的药物或保健食品。
附图说明
图1实施例中金枪鱼鱼卵酶解物(STH)及其超滤分级组分(STH1～STH5)在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)。
图2为实施例中葡聚糖凝胶G-15柱层析分离后各组分的吸光度曲线。
图3为实施例中金枪鱼鱼卵超滤酶解物(STH1)及其凝胶色谱分离组分(STH1A～STH1D)在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)。
图4是本发明实施例用Zorbax C18(250×4.6mm，5μm)柱进行纯化时所获得的色谱图。
图5是本发明实施例用RP-HPLC制备的各组分对ACE的半数抑制率(IC50)。
图6是本发明实施例金枪鱼鱼卵降压肽Trp-Pro-Phe-Ala-Cys-Ser的结构图。
图7是本发明实施例金枪鱼鱼卵降压肽Trp-Pro-Phe-Ala-Cys-Ser的质谱图。
具体实施方式
下列实施例用于进一步解释说明本发明，但是，它们并不构成对本发明范围的限制或限定。本发明实验所用的金枪鱼为鲣鱼(Katsuwonus pelamis)，由宁波今日食品有限公司提供。
实施例
1)金枪鱼鱼卵的预处理：将金枪鱼鱼卵解冻、除杂，称取1000g，然后与双蒸水按照1g：5mL的重量体积比混合、匀浆，放置于电脑微波超声组合成/萃取仪中，在微波功率500W条件下，处理5min，取出冻干；冻干粉末与乙酸乙酯按照1g：4mL的重量体积比混合，于室温3000W超声处理25min，离心出去上清液，得金枪鱼鱼卵粉末；
2)金枪鱼鱼卵酶解液的制备：将上述金枪鱼鱼卵粉末与甘氨酸-NaOH缓冲液按照1g：8mL的重量体积比混合，调节混合液pH值至7.0，调温度至50℃，按照金枪鱼鱼卵粉末质量的1.5％加入中性蛋白酶(5.0×104U/g)，酶解3.5h；酶解液放于沸水种10min后，灭酶活，得金枪鱼鱼卵酶解液(STH)；
3)金枪鱼鱼卵酶解液的超滤分级：将金枪鱼鱼卵酶解液(STH)采用1kDa，3.5kDa和5kDa超滤膜进行超滤处理，分别收集分子量小于1kDa(STH1)、1-3.5kDa(STH2)、5-5kDa(STH3)、3.5-10kDa(STH4)、大于10kD(STH5)的组分，测定各组分在5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)(见图1)，STH1具有最高ACE抑制活性，即为超滤酶解物；
4)金枪鱼鱼卵降压肽的制备：将超滤酶解物(STH1)依次经Sephadex G-15凝胶色谱和反相高效液相色谱(RP-HPLC)纯化，得金枪鱼鱼卵降压肽；
Sephadex G-15凝胶色谱纯化步骤：将超滤酶解物(STH1)配成50μg/mL溶液，经过葡聚糖凝胶G-15柱层析分离，用双蒸水进行洗脱，根据220nm下的吸光度曲线收集洗脱组分(见图2)，测定个组分(STH1A～STH1D)5mg/mL浓度下对血管紧张素转化酶(ACE)抑制率(％)(见图3)，其中，STH1D具有最高ACE抑制活性，即为凝胶层析酶解物。
RP-HPLC纯化步骤：将凝胶层析酶解物(STH1D)配成45μg/mL溶液，利用RP-HPLC(条件为：进样量为10μL；色谱柱为Zorbax C18(250×4.6mm，5μm)；流动相为50％乙腈；洗脱速度为0.5mL/min；紫外检测波长为220nm)进行纯化(见图4)，测定各分离组分(STP1～STP6)的血管紧张素转化酶(ACE)半数抑制率(IC50)(见图5)，STP5具有最低的ACE半数抑制率(IC50)，测定STP5的氨基酸序列和分子量。
结构检测：收集ACE半数抑制率(IC50)最低的组分，，最低组分(STP5)的降压肽经检测为单一峰，利用蛋白/多肽序列分析仪测定氨基酸序列为Trp-Pro-Phe-Ala-Cys-Ser(WPFACS)(见图6)，ESI/MS检测分子量为709.8Da(见图7)。
Claims (2)
1. 一种金枪鱼鱼卵降血压肽，其特征在于所述降压肽的氨基酸序列为：Trp-Pro-Phe-Ala-Cys-Ser，ESI-MS测定其分子量为709.8 Da。
2.如权利要求1所述一种金枪鱼鱼卵降血压肽用于制备降血压相关疾病的药物中的用途。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111203878.1A CN113801195B (zh) | 2021-10-15 | 2021-10-15 | 一种金枪鱼鱼卵降血压肽及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111203878.1A CN113801195B (zh) | 2021-10-15 | 2021-10-15 | 一种金枪鱼鱼卵降血压肽及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113801195A CN113801195A (zh) | 2021-12-17 |
| CN113801195B true CN113801195B (zh) | 2023-06-27 |
Family
ID=78937767
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111203878.1A Active CN113801195B (zh) | 2021-10-15 | 2021-10-15 | 一种金枪鱼鱼卵降血压肽及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113801195B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5238921A (en) * | 1990-02-27 | 1993-08-24 | Agency Of Industrial Science And Technology | Oligopeptide, angiotensin converting enzyme inhibitors, hypotensive agent, and method for treatment of hypertension |
| JPH1129594A (ja) * | 1997-02-03 | 1999-02-02 | Hagoromo Foods Kk | アンジオテンシン変換酵素阻害物質、その製造方法およびそれを用いた血圧降下剤 |
| JP2006096747A (ja) * | 2004-08-31 | 2006-04-13 | Nichiro Corp | サケ由来アンジオテンシンｉ変換酵素阻害ペプチド化合物またはそれを含有するペプチド組成物とそれらの製造方法 |
| CN104774899A (zh) * | 2015-04-24 | 2015-07-15 | 浙江海洋学院 | 金枪鱼暗色肉蛋白抗宫颈癌多肽的制备方法 |
| CN110724178A (zh) * | 2019-10-14 | 2020-01-24 | 浙江海洋大学 | 一种金枪鱼白肉ace抑制肽及其制备方法 |
| CN110759965A (zh) * | 2019-10-14 | 2020-02-07 | 浙江海洋大学 | 一种金枪鱼红肉ace抑制肽及其制备方法 |
-
2021
- 2021-10-15 CN CN202111203878.1A patent/CN113801195B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5238921A (en) * | 1990-02-27 | 1993-08-24 | Agency Of Industrial Science And Technology | Oligopeptide, angiotensin converting enzyme inhibitors, hypotensive agent, and method for treatment of hypertension |
| JPH1129594A (ja) * | 1997-02-03 | 1999-02-02 | Hagoromo Foods Kk | アンジオテンシン変換酵素阻害物質、その製造方法およびそれを用いた血圧降下剤 |
| JP2006096747A (ja) * | 2004-08-31 | 2006-04-13 | Nichiro Corp | サケ由来アンジオテンシンｉ変換酵素阻害ペプチド化合物またはそれを含有するペプチド組成物とそれらの製造方法 |
| CN104774899A (zh) * | 2015-04-24 | 2015-07-15 | 浙江海洋学院 | 金枪鱼暗色肉蛋白抗宫颈癌多肽的制备方法 |
| CN110724178A (zh) * | 2019-10-14 | 2020-01-24 | 浙江海洋大学 | 一种金枪鱼白肉ace抑制肽及其制备方法 |
| CN110759965A (zh) * | 2019-10-14 | 2020-02-07 | 浙江海洋大学 | 一种金枪鱼红肉ace抑制肽及其制备方法 |
Non-Patent Citations (3)
| Title |
|---|
| 血管紧张素转化酶抑制肽研究进展;于志鹏;赵文竹;刘博群;刘静波;;食品科学(11);第315-318页 * |
| 超声预处理对金枪鱼皮胶原ACE抑制肽消化稳定性的影响及消化产物的分离纯化、鉴定;陈雪珂;马良;黄丹丹;夏文银;李晓艺;张宇昊;;食品工业科技(22);第76-80页 * |
| 酶法制备阿拉斯加鳕鱼降压肽的工艺优化及其产物的结构鉴定;杨贵兰;秦松;王晓艳;李文军;;食品工业科技(05);第169-174页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113801195A (zh) | 2021-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103980347B (zh) | 一种大黄鱼鱼鳔降压肽及其制备方法和用途 | |
| CN107141336B (zh) | 具有dpp-iv抑制活性的牦牛骨蛋白肽及制备方法 | |
| CN103992385B (zh) | 一种大黄鱼鱼鳔抗氧化胶原肽及其制备方法和用途 | |
| US8940685B2 (en) | Method for preparing active peptides from corn germ proteins | |
| CN113215212B (zh) | 一种具有抗氧化和ace抑制功能的大豆蛋白肽及其制备方法 | |
| CN105111282B (zh) | 一种具有ace抑制活性的核桃肽及其制备方法 | |
| CN104762358B (zh) | 贻贝蛋白降压肽的快速制备方法 | |
| CN103204904B (zh) | 一种抗前列腺癌赤魟软骨蛋白多肽及其制备方法和用途 | |
| CN112442112B (zh) | 一种银杏蛋白源ace抑制肽组合物及其制备方法和应用 | |
| CN103539831B (zh) | 山杏α-葡萄糖苷酶抑制肽及其制备方法和用途 | |
| CN103539833B (zh) | 高活性α-葡萄糖苷酶抑制肽及其制备方法和用途 | |
| CN104877007B (zh) | 牦牛乳乳清蛋白源ace抑制肽及其制备方法 | |
| CN113801195B (zh) | 一种金枪鱼鱼卵降血压肽及其应用 | |
| CN117965668A (zh) | 一种具有抗氧化和抗衰老功能的牛骨胶原蛋白肽及其制备方法与应用 | |
| CN116789748A (zh) | 一种鲟鱼鱼鳔抗肺癌寡肽及其应用 | |
| CN104945471B (zh) | 贻贝蛋白降压肽 | |
| CN113072621B (zh) | 一种牦牛骨降血压肽及其制备方法与应用 | |
| CN111825754B (zh) | 一种从芝麻蛋白中获得的具有降血压与降血糖活性的多肽 | |
| KR101497506B1 (ko) | 안지오텐신 전환효소 저해용 약학 조성물 및 건강기능식품 | |
| CN102787154A (zh) | 一种乌鸡低聚肽的制备及其活性肽段分离、鉴定方法 | |
| CN117567562B (zh) | 南极磷虾ace抑制肽及其制备方法和应用 | |
| CN118812644A (zh) | 一种源于乌贼皮的降血压寡肽及应用 | |
| JPH04275298A (ja) | ぺプチド及びこれを有効成分とするアンジオテンシン変換酵素阻害剤 | |
| CN112480210B (zh) | 一种抗凝血活性肽及其用途 | |
| CN116082442A (zh) | 一种麒麟菜降血压六肽、制备方法及其应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240126 Address after: 710000 No. B49, Xinda Zhongchuang space, 26th Street, block C, No. 2 Trading Plaza, South China City, international port district, Xi'an, Shaanxi Province Patentee after: Xi'an Huaqi Zhongxin Technology Development Co.,Ltd. Country or region after: China Address before: Plot C2-10, Putuo Zhanmao Xiaohui Industrial Zone, Putuo marine science and Technology Industrial Park, Zhoushan City, Zhejiang Province Patentee before: Zhejiang Ocean University Country or region before: China |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240602 Address after: 075800 Unit 101, Building 36, Lijing New City, Xicheng Town, Yangyuan County, Zhangjiakou City, Hebei Province Patentee after: Guoxian Min'an Pharmaceutical Hebei Co.,Ltd. Country or region after: China Address before: 710000 No. B49, Xinda Zhongchuang space, 26th Street, block C, No. 2 Trading Plaza, South China City, international port district, Xi'an, Shaanxi Province Patentee before: Xi'an Huaqi Zhongxin Technology Development Co.,Ltd. Country or region before: China |
|
| TR01 | Transfer of patent right |