CN112442109A - Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture - Google Patents
Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture Download PDFInfo
- Publication number
- CN112442109A CN112442109A CN201910806983.0A CN201910806983A CN112442109A CN 112442109 A CN112442109 A CN 112442109A CN 201910806983 A CN201910806983 A CN 201910806983A CN 112442109 A CN112442109 A CN 112442109A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- ace
- isnqppikf
- blood pressure
- inhibitor
- 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.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 40
- 244000241838 Lycium barbarum Species 0.000 title claims description 8
- 235000015459 Lycium barbarum Nutrition 0.000 title claims description 8
- 101000984728 Chiropsoides quadrigatus Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 title claims description 7
- 235000015468 Lycium chinense Nutrition 0.000 title claims description 4
- 239000000203 mixture Substances 0.000 title claims description 4
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 claims abstract description 36
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 claims abstract description 36
- 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 35
- 230000036772 blood pressure Effects 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 11
- 235000013376 functional food Nutrition 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 claims description 8
- 239000005541 ACE inhibitor Substances 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims 3
- 239000003937 drug carrier Substances 0.000 claims 3
- 230000004531 blood pressure lowering effect Effects 0.000 claims 2
- 239000003112 inhibitor Substances 0.000 claims 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims 1
- 101710129690 Angiotensin-converting enzyme inhibitor Proteins 0.000 claims 1
- 101710086378 Bradykinin-potentiating and C-type natriuretic peptides Proteins 0.000 claims 1
- 239000002671 adjuvant Substances 0.000 claims 1
- 230000003276 anti-hypertensive effect Effects 0.000 claims 1
- 238000000338 in vitro Methods 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 27
- 102000004169 proteins and genes Human genes 0.000 abstract description 23
- 108090000623 proteins and genes Proteins 0.000 abstract description 23
- 235000017784 Mespilus germanica Nutrition 0.000 abstract description 10
- 235000000560 Mimusops elengi Nutrition 0.000 abstract description 10
- 235000007837 Vangueria infausta Nutrition 0.000 abstract description 10
- 238000000855 fermentation Methods 0.000 abstract description 5
- 230000004151 fermentation Effects 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 4
- 230000001105 regulatory effect Effects 0.000 abstract description 4
- 240000002624 Mespilus germanica Species 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 23
- 239000000243 solution Substances 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 10
- 244000182216 Mimusops elengi Species 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 108010033276 Peptide Fragments Proteins 0.000 description 5
- 102000007079 Peptide Fragments Human genes 0.000 description 5
- 230000000975 bioactive effect Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 206010020772 Hypertension Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000005556 structure-activity relationship Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- ZDLZKMDMBBMJLI-FDMDGMSGSA-N 2-[[2-[[(2s)-2-[[(e)-3-(furan-2-yl)prop-2-enoyl]amino]-3-phenylpropanoyl]amino]acetyl]amino]acetic acid Chemical compound C([C@@H](C(=O)NCC(=O)NCC(=O)O)NC(=O)\C=C\C=1OC=CC=1)C1=CC=CC=C1 ZDLZKMDMBBMJLI-FDMDGMSGSA-N 0.000 description 2
- 108010048632 2-furanacryloyl-phenylalanyl-glycyl-glycine Proteins 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 102000013370 fibrillin Human genes 0.000 description 2
- 108060002895 fibrillin Proteins 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- WLRYGVYQFXRJDA-DCAQKATOSA-N Gln-Pro-Pro Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 WLRYGVYQFXRJDA-DCAQKATOSA-N 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- IDMNOFVUXYYZPF-DKIMLUQUSA-N Ile-Lys-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N IDMNOFVUXYYZPF-DKIMLUQUSA-N 0.000 description 1
- JODPUDMBQBIWCK-GHCJXIJMSA-N Ile-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O JODPUDMBQBIWCK-GHCJXIJMSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 244000241872 Lycium chinense Species 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- GUCCBOQKNLHXEA-KCWXNJEJSA-N O1C(=CC=C1)C(=O)C=CC(=O)[C@](N(CCCCC)C1=CC=CC=C1)(CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O Chemical compound O1C(=CC=C1)C(=O)C=CC(=O)[C@](N(CCCCC)C1=CC=CC=C1)(CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O GUCCBOQKNLHXEA-KCWXNJEJSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 208000007271 Substance Withdrawal Syndrome Diseases 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 159000000032 aromatic acids Chemical class 0.000 description 1
- -1 aromatic amino acid Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000005686 electrostatic field Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 208000037806 kidney injury Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108010020432 prolyl-prolylisoleucine Proteins 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 208000023516 stroke disease Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- 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
-
- 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
Abstract
The invention relates to a polypeptide for preparing angiotensin-converting enzyme (ACE) inhibitory activity by utilizing medlar protein through a fermentation method. The amino acid sequence of the polypeptide ISNQPPIKF is Ile-Ser-Asn-Gln-Pro-Pro-Ile-Lys-Phe, and the molecular weight is 1043.23 Da. The product has good ACE inhibitory activity and good application prospect in development of functional food or medicine for regulating blood pressure.
Description
Technical Field
The invention relates to a polypeptide ISNQPPIKF with angiotensin-converting enzyme (ACE) inhibitory activity prepared from medlar protein by a fermentation method, and application thereof in functional foods and medicines for regulating blood pressure.
Background
Hypertension is one of the most common cardiovascular and cerebrovascular diseases, and is the most important risk factor for cardiovascular diseases such as stroke and coronary heart disease. With the rapid development of social economy and the change of the living dietary structure of people, the incidence of hypertension also increases year by year, and the body health of residents in China is seriously influenced. The prevention and treatment of hypertension are major problems facing the present society.
ACE is widely present in the human body as a multifunctional enzyme, and functions to regulate blood pressure by acting on the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) in the body. Therefore, ACE inhibitors are widely used as a blood pressure lowering drug. However, the chemically synthesized western medicine antihypertensive drug has toxic and side effects on human bodies, particularly great burden is caused on liver and kidney tissues due to drug absorption, liver and kidney injury is caused, symptoms are repeated after drug withdrawal, and the curative effect after healing is not very ideal.
Bioactive peptides are a focus of research in recent years. The natural protein can release peptides with different structures under the action of protease and microorganisms, and researches show that the bioactive peptides show various biological activities such as blood pressure reduction, blood sugar reduction, immunity improvement, antibiosis, antioxidation and the like. The ACE inhibitor prepared by utilizing natural protein has the characteristics of definite function and high safety, so that the ACE inhibitor has wide prospect as functional food or medicine.
The invention relates to an ACE inhibitory active peptide prepared by fermenting medlar protein.
Disclosure of Invention
The object of the present invention is about the ACE inhibitory activity of polypeptide ISNQPPIKF; it has the sequence table of SEQ ID NO: 1, amino acid sequence; has good ACE inhibitory activity and has the function of lowering blood pressure by inhibiting the ACE activity. And has good application prospect in developing functional food or medicine for regulating blood pressure.
In order to achieve the purpose, the invention takes the product polypeptide ISNQPPIKF as a main component on the basis of fully utilizing the medlar protein, inhibits the activity of ACE and regulates blood pressure. On the basis, the polypeptide ISNQPPIKF can be used as the main component of ACE inhibitors, blood pressure regulating medicines and related functional products.
The polypeptide ISNQPPIKF has ACE inhibiting activity and blood pressure lowering activity, has the amino acid sequence of Ile-Ser-Asn-Gln-Pro-Pro-Ile-Lys-Phe, has single-chain linear structure, is white powder, is easily soluble in water, and has molecular weight of 1043.23 Da; has strong inhibiting effect on ACE activity, and half inhibitory concentration (IC50) is 105.77 μ M.
The three-dimensional structure of the Protein ACE (PDB code:1o8a) was derived from Protein Data Bank (https:// www.rcsb.org /), predicted for the binding site of polypeptide ISNQPPIKF to ACE using bioinformatics software Peptide (http:// pepsite2. russellab. org.). The presence or absence of interaction between the polypeptide and protein is indicated by the resulting P value and binding site, where a smaller P value indicates a greater probability of binding between the polypeptide and protein, and where a P value >0.5 indicates theoretically no probability of binding between the polypeptide and protein. Binding site means a binding site of a polypeptide to a protein, and if the polypeptide binds to the active site of the protein, it inhibits the protein from interacting with other proteins, thereby inhibiting the activity of the protein.
Polypeptide ISNQPPIKF has a p value of 0.00002537 and a binding site of Gln281#,His353#,Ala354#,His383#,Glu384#,His387#,Glu411#,Phe457,Phe460,Lys511#,His513#,Tyr520#,Tyr523#Phe527 therein having#Marked are important active sites and binding sites of ACE reported in the literature, and the polypeptide ISNQPPIKF has good ACE inhibitory activity.
In addition, the structure-activity relationship of bioactive peptides is related to the ACE inhibitory activity, the polypeptide ISNQPPIKF has the characteristics required by an ACE inhibitor, and the C-terminal tripeptide of the polypeptide has a strong influence on the ACE inhibitory activity. ACE inhibiting peptides are more prone to bioactive peptides where the C-terminal tripeptides all contain hydrophobic amino acids, such as aromatic and branched chain amino acids. The peptide segment with aromatic amino acid (Trp, Tyr, Phe) or Pro at the C terminal has stronger ACE inhibitory activity. The C-terminal amino acid of the polypeptide ISNQPPIKF is Phe, which meets the requirement.
The invention has the following advantages:
the invention obtains ACE inhibitory peptide from fermented medlar protein, the polypeptide ISNQPPIKF has good ACE inhibitory activity, and the half inhibitory concentration (IC50) is 105.77 mu M. Provides more diversified sources for ACE inhibitory peptides and provides more research references for the ACE inhibitory peptides.
Detailed Description
EXAMPLE 1 identification of polypeptide ISNQPPIKF
The medlar protein is subjected to bacillus subtilis fermentation, acid precipitation, ethanol precipitation and LC-MS/MS analysis, and bioactive peptides with ACE inhibitory activity are screened through bioinformatics and structure-activity relationship.
The specific method comprises the following steps:
(1) preparation of matrimony vine protein extract
Taking 10g of dry medlar as a raw material, weighing medlar, adding 100mL of deionized water, soaking for 2 hours, then crushing, ultrasonically extracting for 60min, centrifuging at 5000rpm for 10min, and using 9.6g of precipitate for extracting medlar protein.
Freeze-drying the precipitate, pulverizing again to obtain fructus Lycii powder, adding 192mL of organic solvent extract (n-hexane: ethanol: 2.6:1 (v/v)), stirring at 50 deg.C and 150rpm, extracting for 1 hr, filtering, extracting the filter cake with the same organic solvent extract once, filtering after two times, and freeze-drying to obtain 7.5g of fructus Lycii protein.
(2) Preparation of active peptide of medlar
Adding fructus Lycii protein into 112mL ionized water, adjusting pH to 7.5 with 1M sodium hydroxide solution, inoculating microorganism strain (Bacillus subtilis) with bacteria content of 2 x 108CFU/mL, fermenting at 37 ℃ for 48 hours; after fermentation was completed, centrifugation was carried out at 13000rpm for 15min, and the supernatant was freeze-dried to obtain 1g of crude fermentation extract.
Adding 5mL of 0.01M hydrochloric acid into the fermented crude extract, homogenizing at 2 deg.C for 8min, centrifuging at 12000rpm for 20min, collecting supernatant, adding 3 times of anhydrous ethanol, standing at 4 deg.C for 24 hr, centrifuging at 12000rpm for 20min, collecting supernatant, and lyophilizing to obtain 0.383g of fructus Lycii polypeptide.
Performing mass spectrometry on the lycium barbarum polypeptide by using LTQ Orbitrap Velos: desalting the Lycium barbarum polypeptide, C18The SPE pretreats the column, activated with 2mL acetonitrile, and then washed off with 2mL of 0.1% (v/v) TFA solution. The sample was loaded onto the column after adjusting p H to 2 with 50% (v/v) TFA solution. After desalting with 2mL of 0.1% (v/v) TFA, the eluate was eluted 3 times with 1mL of 80% (v/v) acetonitrile/0.1% (v/v) TFA, and the eluate was freeze-dried and stored at-20 ℃ for mass spectrometry. Adding appropriate amount of 0.1% (v/v) formic acid into lyophilized sample for redissolving to obtain 0.5 μ g/μ L solution, and usingA linear ion trap electrostatic field orbit trap combined mass spectrometer (LTQ Orbitrap Velos) performs mass spectrometry on a sample, and the temperature of an ion transmission capillary is set to be 250 ℃, the electrospray voltage is set to be 2.2kV, and the normalized collision energy is set to be 35.0%. Both MS and MS/MS were mapped using a data-dependent mode. The mass spectrometry scan conditions were set as: and selecting 10 highest-abundance ion peaks from the full scan with each m/z of 400-2000 to perform MS/MS scanning. Samples are analyzed in parallel for three times, and common peptide fragments identified by the three times of analysis are taken for statistics. The collected raw file data was converted to mgf format using Thermo protein discover Daemon (v1.4) and retrieved with Mascot (version 2.3.0, Matrix Science, London, UK) spectrogram in solanaceous plant protein library solanaceous (https:// www.uniprot.org /). The three analyses collectively identify 75 common peptide fragments derived from 14 different proteins, 41.33 percent of the peptide fragments are derived from the protein Fibrillin, the number of amino acids in the peptide fragments is between 8 and 27, the calculated molecular mass is 920.4491 to 2742.4664(Da), and the table I is the mass spectrum identification result of part of the lycium barbarum polypeptides.
table-Mass Spectrometry results for Lycium barbarum Polypeptides
According to the explanation of structure-activity relationship in the invention content, the identified common peptide fragments are screened to obtain the polypeptide with the sequence of ISNQPPIKF derived from the Fibrillin protein.
Example 2 detection of ACE inhibitory Activity of polypeptide ISNQPPIKF
The polypeptide ISNQPPIKF is synthesized by Nanjing Jie peptide Biotech Co., Ltd, has the amino acid sequence of Ile-Ser-Asn-Gln-Pro-Pro-Ile-Lys-Phe, has a single-chain linear structure, is white powder, is easy to dissolve in water, and has the molecular weight of 1043.23 Da.
Information of SEQ ID No.1
(a) Sequence characterization
Length: 9 amino acid
Type: amino acids
Chain type: single strand
(b) Molecular type: protein
Description of the sequence:
SEQ ID No.1
ISNQPPIKF
n- (3- (2-furoyl) acryloyl-phenylamyl-glutamyl-glutamic acid (fagg,. lamda.max. about.340 nm. epsilon. about.2270M)-1cm-1Molecular weight 399.40) can be enzymatically hydrolyzed by ACE to N- [3- (furan) propenoyl]-2-phenylalanine (FAP,. lamda.max. 340nm,. epsilon. 1512M-1cm-1) And glycylglycine (GG, no absorption at 340 nm) and thus can act as a mimetic substrate for ACE. The absorbance value of 1mM FAPGG completely converted to FAP and GG was 0.758, so that the inhibition rate could be calculated from the change value of absorbance at 340 nm.
Reaction system
(1) Buffer solution: 0.1M PBS buffer (pH 8.2, containing 300mM NaCl)
(2) Substrate solution: FAPGG solution was prepared at a concentration of 1.6mM using the above buffer.
(3) Enzyme solution: ACE was formulated as a 0.2U/mL solution using the above buffer solution.
(4) Sample solution: the polypeptide ISNQPPIKF was prepared as a 1.0, 0.5, 0.1mg/mL solution with the above buffers as required for the experiment.
Experiments were performed in 96-well plates. Adding the sample solution, the ACE solution and the substrate solution in sequence according to the second table, mixing uniformly, immediately measuring absorbance at 340nm by using an enzyme-labeling instrument, and recording as OD0After incubation at 37 ℃ for 30min, the absorbance at 340nm was again determined and recorded as OD1. The absorbance of each well was measured, and Δ OD was defined as OD0-OD1. Each sample was assayed in 3 replicates.
Sample adding method for epibiace inhibitory activity
"-" represents the addition of an equal volume of PBS buffer to the column
ACE inhibition ratio calculation formula:
I=[(ΔODControl-ΔODSample)/(ΔODControl-ΔODblank)]*100%
ΔODcontrolrepresenting the change of absorbance after the sample solution is replaced by the same amount of buffer solution in the reaction; delta ODSampleRepresenting the change in absorbance of the sample solution in the reaction; and Δ ODblankThe change in absorbance of the reaction mixture after the sample solution and the enzyme solution were replaced with the same amount of buffer solution
Absorbance of the assay for inhibitory Activity of Epsantel ACE
TABLE four inhibitory Activity of ISNQPPIKF on ACE at various concentrations
The ACE inhibitory activity was measured at different concentrations of polypeptide ISNQPPIKF as described above. Table three is the absorbance of experiment for ACE inhibitory activity at different concentrations of ISNQPPIKF, and the inhibitory activity of ISNQPPIKF against ACE at different concentrations of table four was calculated, thereby calculating the half inhibitory concentration (IC50) of ISNQPPIKF as 105.77 ± 72.80 μ M.
Sequence listing
<110> institute of chemistry and physics, large connection of Chinese academy of sciences
<120> Chinese wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ile Ser Asn Gln Pro Pro Ile Lys Phe
1 5
Claims (7)
1. An ACE inhibitory peptide of wolfberry, which is characterized in that: the polypeptide ISNQPPIKF has a sequence table SEQ ID NO: 1, amino acid sequence; the amino acid sequence of the polypeptide is specifically Ile-Ser-Asn-Gln-Pro-Pro-Ile-Lys-Phe.
2. A derivative polypeptide of ISNQPPIKF wherein the polypeptide of claim 1 is the polypeptide set forth in SEQ ID NO: 1 by substituting, deleting and/or adding one or more than 2 amino acids of the amino acid sequence shown in the claim 1, and the derivative polypeptide has the same function with the small peptide of the claim 1.
3. Use of a peptide according to claim 1 or 2 for the preparation of an angiotensin-converting enzyme (ACE) inhibitor and/or a blood pressure lowering medicament.
4. Use according to claim 3 of angiotensin converting enzyme
The (ACE) inhibitor and/or the blood pressure lowering drug takes poly ISNQPPIKF as an active ingredient, and can be added with pharmaceutically acceptable carriers or auxiliary materials.
5. Use of the polypeptide of claim 1 or 2 as an active ingredient in an in vitro angiotensin-converting enzyme (ACE) inhibitor, or as an active ingredient in a functional food having blood pressure lowering effect.
6. The use according to claim 5, wherein the inhibitor or functional food further comprises a pharmaceutically acceptable carrier or excipient.
7. A mixture as angiotensin converting enzyme inhibitor, antihypertensive medicine or functional food with blood pressure lowering effect is prepared from ISNQPPIKF powder as active component, and optionally pharmaceutically acceptable carrier or adjuvant.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910806983.0A CN112442109B (en) | 2019-08-29 | 2019-08-29 | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910806983.0A CN112442109B (en) | 2019-08-29 | 2019-08-29 | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112442109A true CN112442109A (en) | 2021-03-05 |
CN112442109B CN112442109B (en) | 2021-11-30 |
Family
ID=74740737
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910806983.0A Active CN112442109B (en) | 2019-08-29 | 2019-08-29 | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112442109B (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011064402A2 (en) * | 2009-11-30 | 2011-06-03 | Laboratoires Expanscience | Acacia macrostachya seed extract and compositions containing same |
CN102475884A (en) * | 2010-11-29 | 2012-05-30 | 中国科学院大连化学物理研究所 | Application of four polypeptides in preparation ACE inhibitor and antihypertensive drug |
CN104561205A (en) * | 2014-12-27 | 2015-04-29 | 宁夏医科大学 | Method for preparing wolfberry ACE inhibitory peptide |
CN106868081A (en) * | 2016-12-13 | 2017-06-20 | 国肽生物科技(北京)有限公司 | The extracting method of lbp peptide |
CN108949886A (en) * | 2018-08-23 | 2018-12-07 | 王召利 | A kind of hypoglycemic plant active peptides and preparation method thereof |
-
2019
- 2019-08-29 CN CN201910806983.0A patent/CN112442109B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011064402A2 (en) * | 2009-11-30 | 2011-06-03 | Laboratoires Expanscience | Acacia macrostachya seed extract and compositions containing same |
CN102475884A (en) * | 2010-11-29 | 2012-05-30 | 中国科学院大连化学物理研究所 | Application of four polypeptides in preparation ACE inhibitor and antihypertensive drug |
CN104561205A (en) * | 2014-12-27 | 2015-04-29 | 宁夏医科大学 | Method for preparing wolfberry ACE inhibitory peptide |
CN106868081A (en) * | 2016-12-13 | 2017-06-20 | 国肽生物科技(北京)有限公司 | The extracting method of lbp peptide |
CN108949886A (en) * | 2018-08-23 | 2018-12-07 | 王召利 | A kind of hypoglycemic plant active peptides and preparation method thereof |
Non-Patent Citations (3)
Title |
---|
CEREN DASKAYA-DIKMEN ET AL.: ""Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptides from Plants"", 《NUTRIENTS》 * |
庄晶玲 等: ""利用LC-MS/MS筛选发酵法制备的枸杞生物活性肽"", 《中国食品科学技术学会第十六届年会暨第十届中美食品业高层论坛论文摘要集》 * |
范彦娜 等: ""枸杞ACE抑制肽成分提取及测定研究"", 《中国卫生标准管理》 * |
Also Published As
Publication number | Publication date |
---|---|
CN112442109B (en) | 2021-11-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Li et al. | Moderation of hyperuricemia in rats via consuming walnut protein hydrolysate diet and identification of new antihyperuricemic peptides | |
Priyanto et al. | Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins | |
CN107163129B (en) | Preparation and application of kappa-casein-derived bioactive peptide | |
Mirzapour et al. | Identification of potent ACE inhibitory peptides from wild almond proteins | |
Zheng et al. | ACE-inhibitory and antioxidant peptides from coconut cake albumin hydrolysates: purification, identification and synthesis | |
Saputri et al. | Inhibition of angiotensin converting enzyme (ACE) activity by some Indonesia edible plants | |
Liu et al. | Novel peptides with α-glucosidase inhibitory activity from Changii Radix hydrolysates | |
Madadlou | Food proteins are a potential resource for mining cathepsin L inhibitory drugs to combat SARS-CoV-2 | |
Ding et al. | Identification and characterization of dipeptidyl peptidase IV inhibitory peptides from wheat gluten proteins | |
CN112442108B (en) | ACE and DPP-IV inhibitory peptide of medlar, derivative polypeptide, application and mixture | |
CN110105431B (en) | Sesame polypeptide, extraction method thereof and application of sesame polypeptide in preparation of anti-oxidation and/or blood pressure lowering medicines | |
Yu et al. | Anxiolytic effects of ACE inhibitory peptides on the behavior of rats in an elevated plus-maze | |
CN112442109B (en) | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture | |
CN112442111B (en) | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture | |
CN112500449B (en) | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture | |
CN112442110B (en) | Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture | |
Bhadkaria et al. | Peptide fraction from moth bean (Vigna aconitifolia (Jacq.)) seed protein hydrolysate demonstrates multifunctional characteristics | |
CN112442104B (en) | Fermentation method for preparing lycium barbarum bioactive peptide, method and application thereof, inhibitor and functional food | |
CN112125952B (en) | Pig source ACE inhibitory activity polypeptide, pharmaceutical composition or food and application | |
Guang et al. | Purification, activity and sequence of angiotensin I converting enzyme inhibitory peptide from alcalase hydrolysate of peanut flour | |
CN109912688B (en) | PTP1B polypeptide inhibitors and preparation method and application thereof | |
CN113801192B (en) | Tetrapeptides for inhibiting dipeptidyl peptidase IV and application thereof | |
Li et al. | Isolation and identification of angiotensin‐converting enzyme inhibitory peptides from Tartary buckwheat albumin | |
Du et al. | ACE inhibitory peptides from enzymatic hydrolysate of fermented black sesame seed: Random forest-based optimization, screening, and molecular docking analysis | |
CN110498833B (en) | Tripeptide with ACE (angiotensin converting enzyme) inhibition effect and application thereof |
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 |