CN114773430A - 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 - Google Patents
三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 Download PDFInfo
- Publication number
- CN114773430A CN114773430A CN202210390612.0A CN202210390612A CN114773430A CN 114773430 A CN114773430 A CN 114773430A CN 202210390612 A CN202210390612 A CN 202210390612A CN 114773430 A CN114773430 A CN 114773430A
- Authority
- CN
- China
- Prior art keywords
- cholesterol
- peptide
- cholesterol esterase
- lowering
- inhibitory activity
- 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 54
- 108010055297 Sterol Esterase Proteins 0.000 title claims abstract description 38
- 102000000019 Sterol Esterase Human genes 0.000 title claims abstract description 38
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 11
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 22
- 230000000694 effects Effects 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 14
- 235000013305 food Nutrition 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 9
- 230000007246 mechanism Effects 0.000 claims abstract description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 53
- 235000012000 cholesterol Nutrition 0.000 claims description 24
- 230000005764 inhibitory process Effects 0.000 claims description 13
- 240000001417 Vigna umbellata Species 0.000 claims description 9
- 235000011453 Vigna umbellata Nutrition 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 238000003032 molecular docking Methods 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 8
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000003531 protein hydrolysate Substances 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 238000011156 evaluation Methods 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 4
- 229940088598 enzyme Drugs 0.000 claims description 4
- 230000007062 hydrolysis Effects 0.000 claims description 4
- 238000006460 hydrolysis reaction Methods 0.000 claims description 4
- 229940055695 pancreatin Drugs 0.000 claims description 4
- 230000001603 reducing effect Effects 0.000 claims description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- 208000008589 Obesity Diseases 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims description 3
- 208000029078 coronary artery disease Diseases 0.000 claims description 3
- 206010012601 diabetes mellitus Diseases 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 235000020824 obesity Nutrition 0.000 claims description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 3
- 231100000419 toxicity Toxicity 0.000 claims description 3
- 230000001988 toxicity Effects 0.000 claims description 3
- 229940119495 Cholesterol esterase inhibitor Drugs 0.000 claims description 2
- 108010028921 Lipopeptides Proteins 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims description 2
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000001819 mass spectrum Methods 0.000 claims description 2
- 239000002417 nutraceutical Substances 0.000 claims description 2
- 235000021436 nutraceutical agent Nutrition 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims description 2
- 238000003041 virtual screening Methods 0.000 claims description 2
- 230000031891 intestinal absorption Effects 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 231100000956 nontoxicity Toxicity 0.000 abstract description 2
- 239000000825 pharmaceutical preparation Substances 0.000 abstract description 2
- 230000003993 interaction Effects 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 9
- 230000002209 hydrophobic effect Effects 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000002329 esterase inhibitor Substances 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 3
- 150000001840 cholesterol esters Chemical class 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- HVYWMOMLDIMFJA-UHFFFAOYSA-N 3-cholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 HVYWMOMLDIMFJA-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- CLPQUWHBWXFJOX-BQBZGAKWSA-N Gln-Gly-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O CLPQUWHBWXFJOX-BQBZGAKWSA-N 0.000 description 1
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 1
- OJNZVYSGVYLQIN-BQBZGAKWSA-N Gly-Met-Asp Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O OJNZVYSGVYLQIN-BQBZGAKWSA-N 0.000 description 1
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- QLRMMMQNCWBNPQ-QXEWZRGKSA-N Ile-Arg-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)O)N QLRMMMQNCWBNPQ-QXEWZRGKSA-N 0.000 description 1
- MGUTVMBNOMJLKC-VKOGCVSHSA-N Ile-Trp-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](C(C)C)C(=O)O)N MGUTVMBNOMJLKC-VKOGCVSHSA-N 0.000 description 1
- WRDTXMBPHMBGIB-STECZYCISA-N Ile-Tyr-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=C(O)C=C1 WRDTXMBPHMBGIB-STECZYCISA-N 0.000 description 1
- 108010028554 LDL Cholesterol Proteins 0.000 description 1
- 208000023137 Myotoxicity Diseases 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- HTKNPQZCMLBOTQ-XVSYOHENSA-N Phe-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N)O HTKNPQZCMLBOTQ-XVSYOHENSA-N 0.000 description 1
- GTMSCDVFQLNEOY-BZSNNMDCSA-N Phe-Tyr-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N GTMSCDVFQLNEOY-BZSNNMDCSA-N 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- OFGUOWQVEGTVNU-DCAQKATOSA-N Pro-Lys-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OFGUOWQVEGTVNU-DCAQKATOSA-N 0.000 description 1
- RYMZZMVNJRMUDD-UHFFFAOYSA-N SJ000286063 Natural products C12C(OC(=O)C(C)(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 RYMZZMVNJRMUDD-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000001906 cholesterol absorption Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000816 effect on animals Effects 0.000 description 1
- 229940125753 fibrate Drugs 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 230000009916 joint effect Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 150000002814 niacins Chemical class 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 238000013116 obese mouse model Methods 0.000 description 1
- DVDUMIQZEUTAGK-UHFFFAOYSA-N p-nitrophenyl butyrate Chemical compound CCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 DVDUMIQZEUTAGK-UHFFFAOYSA-N 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229960002855 simvastatin Drugs 0.000 description 1
- RYMZZMVNJRMUDD-HGQWONQESA-N simvastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)C(C)(C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 RYMZZMVNJRMUDD-HGQWONQESA-N 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000012795 verification 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
- 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
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- 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)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Obesity (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Diabetes (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Mycology (AREA)
Abstract
本申请提供了具有胆固醇酯酶抑制活性的肽及其应用和筛选方法，所述肽的序列选自SEQ ID NO.1‑3。本申请的肽具有良好的降胆固醇作用和人体肠道吸收性，稳定性高，无毒，并且其作用机理清楚、靶点明确，符合药物制剂开发的要求，同时也易作为功能因子添加到各类保健食品中，在医药和食品行业具有良好的市场前景。
Description
技术领域
本申请属于蛋白领域，具体地，本申请提供了三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用和筛选方法。
背景技术
由于血浆胆固醇水平与冠状动脉疾病、癌症、肥胖症和糖尿病等多种疾病有关，因此对胆固醇水平的控制备受人们关注。在过去的几十年中，胆固醇酯酶已被广泛研究为控制胆固醇水平的潜在目标。胆固醇酯酶是一种主要存在于小肠腔内的α/β水解酶，并从胰腺分泌到肠道中。正常情况下，饮食中的胆固醇酯类物质很难在肠道中直接吸收。胆固醇酯酶可以水解胆固醇酯类物质，从而产生胆固醇和游离脂肪酸，然后这些未酯化的胆固醇和游离脂肪酸很容易被肠道吸收到血液中，促进高胆固醇血症的发展。因此，对胆固醇酯酶的抑制被认为是限制和延迟膳食胆固醇吸收的关键。
目前市面上的降胆固醇药物主要有他汀类、贝特类、烟酸类等，其中他汀类药物疗效显著，但价格昂贵，且具有肝毒性、肌毒性等潜在的副作用。由于高胆固醇对人类健康危害极大，医药业人工合成的降胆固醇药物又不可避免地会给人体带来副作用，因此寻找天然、安全可靠的高胆固醇治疗药物便具有重要的现实意义。从可持续性和食品安全的角度来看，当前开展植物蛋白及其衍生的生物活性肽功能研究正变得越来越重要。已有很多的报道证明食物源蛋白质的水解产物对动物血清胆固醇水平有调节作用。先前的研究发现红小豆蛋白水解物可以显著降低肥胖小鼠的总胆固醇、低密度脂蛋白胆固醇以及抑制胆固醇酯酶的活性。基于此，我们通过超滤、质谱测序、分子对接等技术筛选出红小豆蛋白水解物中具有胆固醇酯酶抑制活性的降胆固醇肽并进行功能验证，为开发降胆固醇药物的安全替代品提供技术支持。
发明内容
针对上述问题，一方面，本申请提供了具有胆固醇酯酶抑制活性的降胆固醇肽，所述肽的序列选自SEQ ID NO.1-3。
进一步地，所述肽的序列为SEQ ID NO.2或SEQ ID NO.3。
另一方面，本申请提供了组合物，所述组合物包含上述肽以及药学、食品或保健品上可接受的辅料。
另一方面，本申请提供了上述肽或者组合物在制备胆固醇酯酶抑制剂中的用途。
另一方面，本申请提供了上述肽或者组合物在制备降低高胆固醇的药物中的应用。
另一方面，本申请提供了上述肽或者组合物在制备适用于高胆固醇人群的食品或保健品中的应用。
进一步地，所述高胆固醇情况为冠状动脉疾病、癌症、肥胖症或糖尿病中的高胆固醇情况。
另一方面，本申请提供了筛选上述肽的方法，所述方法包括：
(1)体外模拟消化：使用酶法对红小豆蛋白进行水解以得到蛋白水解物；
(2)降胆固醇肽的筛选：以胆固醇酯酶抑制活性为评价指标，在红小豆蛋白水解物的不同超滤级分中筛选出具有最佳降胆固醇活性的部分。然后通过质谱测序和虚拟筛选技术找出与胆固醇酯酶对接效果较好的肽段；
(3)抑制效果及机理分析：以胆固醇酯酶活性抑制率为评价指标，对采用Fmoc固相合成方法制备的降脂肽进行抑制效果评价，并进一步通过分子对接阐明其抑制作用机理。为了解降脂肽更多的特性，最后基于计算机软件评估其毒性、稳定性、等电点、空间位阻、总平均亲水性和人体肠道吸收性。
进一步地，所述水解使用胃蛋白酶和胰酶。
进一步地，所述超滤级分为＜3kDa、3-10kDa和＞10kDa级分。
有益效果：
本发明所述从红小豆蛋白中发现的三种功能性多肽(IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG)是纯天然的、无毒无害的植物源物质，并且具备显著的降胆固醇功效。胆固醇酯酶在水解膳食性胆固醇酯和转运游离胆固醇到肠细胞方面起着重要作用，常常作为控制胆固醇水平的靶标而被广泛研究。IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG是通过抑制胆固醇酯酶的活性从而达到降胆固醇的作用，并且这也得到体外酶活性抑制实验和分子对接的证实。因此，本发明所述IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG作为降胆固醇活性成分，具有良好的降胆固醇作用和人体肠道吸收性，稳定性高，无毒，并且其作用机理清楚、靶点明确，符合药物制剂开发的要求，同时也易作为功能因子添加到各类保健食品中，在医药和食品行业具有良好的市场前景。
附图说明
图1为不同超滤级分对胆固醇酯酶活性的抑制结果；
图2为降胆固醇肽对胆固醇酯酶活性的抑制结果；
图3为降胆固醇肽与胆固醇酯酶在活性位点处对接后的最佳位置概述和细节(A:IYVIRGQGQ；B:IWVGGSGMDM；C:FNTGSSFYNPKAG)。胆固醇酯酶残基用黄棒模型表示。蓝色、灰色、黄色和绿色虚线分别表示氢键、疏水相互作用、盐桥和π-π堆积。
具体实施方式
实施例1制备红小豆蛋白
脱脂红小豆粉和蒸馏水以1:10(w/v)的比例混合，并将溶液pH值调至8.5。在40℃连续搅拌1h后，收集上清液，并在上清液pH值调至4.5后，于室温静置1h以促使蛋白质沉淀。收集沉淀，用蒸馏水洗涤沉淀3次，并将蛋白质的pH值调至7.0。最后，冷冻干燥并保存在-20℃。
实施例2制备蛋白酶解液
配制5％的红小豆蛋白溶液以进行胃蛋白酶和胰酶两阶段酶解。首先加入4％(w/w)胃蛋白酶，酶解pH为2.0，酶解温度为37℃，酶解时间为2h；胃蛋白酶水解完成后，先将酶解液pH值调至5.3，再将pH值维持在7.5，随后加入4％胰酶(w/w)，酶解温度为37℃，酶解时间为2h。酶解结束后，沸水加热灭活酶，酶解液离心取上清。
实施例3制备降胆固醇粗肽
通过使用10kDa和3kD超滤装置对步骤(2)收集的上清液进行超滤。简而言之，12mL蛋白酶解上清液转移至离心超滤管后，5000×g离心30min即得超滤液。随后测定不同级分(>10kDa、3-10kDa和<3kDa)在4mg/mL浓度下对胆固醇酯酶活性的影响，并冷冻干燥不同级分样品。结果表明<3kDa级分对胆固醇酯酶活性抑制效果最好(见图1)。胆固醇酯酶活性抑制实验具体过程为：
在96孔酶标板中，将50μL的样品、50μL的25μg/mL胆固醇酯酶溶液和50μL的10mM对硝基苯丁酸酯作为底物，在pH 7.0的磷酸缓冲液(含100mM NaCl，5.16mM牛磺胆酸钠)中25℃孵育5min。酶标仪在405nm处记录吸光度。使用辛伐他汀作为阳性对照，并根据公式(1)进行计算。
式(1)中：A：对照的吸光度；B：对照空白的吸光度；C：样品的吸光度；D：样品空白的吸光度。
实施例4降胆固醇肽的筛选
采用液相色谱-串联质谱分析<3kDa级分的氨基酸序列，并利用Dock 6.9对得到的肽序列进行虚拟筛选，根据肽段的对接打分(<-130kcal/mol)筛选得到与胆固醇酯酶对接效果较好的肽段IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG(见表1)。
表1.肽段与胆固醇酯酶的对接打分
实施例5功能评价
根据实施例4的氨基酸序列分析结果，利用Fmoc固相合成法制备降胆固醇肽，并通过高效液相色谱和质谱分析，确定每种肽纯度均大于95％。经胆固醇酯酶活性抑制实验发现，在4mg/mL的浓度下，IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG的胆固醇酯酶抑制率分别是29.05％、34.08％和30.10％(见图2)。
从RCSB Protein Data Bank数据库(http://www.rcsb.org/)获取胆固醇酯酶(PDB编号：1F6W)的晶体结构，采用Dock 6.9分别将IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG与胆固醇酯酶进行半柔性对接，确定其与胆固醇酯酶作用的关键氨基酸残基及相互作用力(见图3)。由图3可知，IYVIRGQGQ肽可与10个氨基酸残基相互作用，并且相互作用包括氢键、疏水相互作用和盐桥。IYVIRGQGQ肽能与底物结合残基(Ala108)和产物释放通道(Val285)形成疏水相互作用(见图3A)；IWVGGSGMDM肽可与12个氨基酸残基相互作用，并且相互作用包括氢键、疏水相互作用和盐桥。IWVGGSGMDM肽能与催化残基(His435)、底物结合残基(Ala108)和产物释放通道(Val285)形成疏水相互作用(见图3B)；FNTGSSFYNPKAG肽可与13个氨基酸残基相互作用，并且相互作用包括氢键、疏水相互作用、盐桥和π-π堆积。FNTGSSFYNPKAG肽能与底物结合残基(Ala108)和产物释放通道(Val285)形成疏水相互作用，与催化残基(Ser194)形成氢键(见图3C)。总之，降胆固醇肽主要通过占据催化位点或底物结合位点以及阻碍产物释放通道来抑制胆固醇酯酶的活性。
计算机软件被用来对IYVIRGQGQ、IWVGGSGMDM、FNTGSSFYNPKAG进行功能预测，其中毒性和空间位阻通过ToxinPred(https://webs.iiitd.edu.in/raghava/toxinpred/index.html)，总平均亲水性和不稳定指数通过ExPasy(https://web.expasy.org/protparam/)，人体肠道吸收通过admetSAR(http://lmmd.ecust.edu.cn/admetsar1/home/)，等电点通过Pepdraw(http://www.tulane.edu/～biochem/WW/PepDraw/)评估。如表2所示，三种降胆固醇肽均是无毒的，且都具有良好的肠道吸收性和稳定性。IWVGGSGMDM的等电点小于7，表明它呈酸性，而IYVIRGQGQ和FNTGSSFYNPKAG呈碱性。总平均亲水性可以用来表征蛋白质的亲疏水性，其中正值越大表明疏水性越强，负值越大表明亲水性越强，故IYVIRGQGQ和FNTGSSFYNPKAG具有更好的亲水性，而IWVGGSGMDM具有更好的疏水性。3个降胆固醇肽的空间位阻值在0.63-0.69之间，其中FNTGSSFYNPKAG具有最低的空间位阻值，这有利于增强对胆固醇酯酶的抑制。
表2.降胆固醇肽的功能预测
注：不稳定指数小于40表示能够稳定存在。
SEQUENCE LISTING
<110> 中国农业大学
<120> 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> artificail
<400> 1
Ile Tyr Val Ile Arg Gly Gln Gly Gln
1 5
<210> 2
<211> 10
<212> PRT
<213> artificail
<400> 2
Ile Trp Val Gly Gly Ser Gly Met Asp Met
1 5 10
<210> 3
<211> 13
<212> PRT
<213> artificail
<400> 3
Phe Asn Thr Gly Ser Ser Phe Tyr Asn Pro Lys Ala Gly
1 5 10
Claims (10)
1.具有胆固醇酯酶抑制活性的肽，其特征在于，所述肽的序列选自SEQ ID NO.1-3。
2.根据权利要求1所述的肽，其中所述肽的序列为SEQ ID NO.2或SEQ ID NO.3。
3.组合物，其特征在于，所述组合物包含根据权利要求1或2所述的肽以及药学、食品或保健品上可接受的辅料。
4.根据权利要求1或2所述的肽或者根据权利要求3所述的组合物在制备胆固醇酯酶抑制剂中的用途。
5.根据权利要求1或2所述的肽或者根据权利要求3所述的组合物在制备降低高胆固醇的药物中的应用。
6.根据权利要求1或2所述的肽或者根据权利要求3所述的组合物在制备适用于高胆固醇人群的食品或保健品中的应用。
7.根据权利要求5或者6所述的应用，其中述高胆固醇情况为冠状动脉疾病、癌症、肥胖症或糖尿病中的高胆固醇情况。
8.筛选根据权利要求1或2所述的肽的方法，其特征在于，所述方法包括：
(1)体外模拟消化：使用酶法对红小豆蛋白进行水解以得到蛋白水解物；
(2)降胆固醇肽的筛选：以胆固醇酯酶抑制活性为评价指标，在红小豆蛋白水解物的不同超滤级分中筛选出具有最佳降胆固醇活性的部分。然后通过质谱测序和虚拟筛选技术找出与胆固醇酯酶对接效果较好的肽段；
(3)抑制效果及机理分析：以胆固醇酯酶活性抑制率为评价指标，对采用Fmoc固相合成方法制备的降脂肽进行抑制效果评价，并进一步通过分子对接阐明其抑制作用机理。为了解降脂肽更多的特性，最后基于计算机软件评估其毒性、稳定性、等电点、空间位阻、总平均亲水性和人体肠道吸收性。
9.根据权利要求8所述的方法，其中所述水解使用胃蛋白酶和胰酶。
10.根据权利要求8或9所述的方法，所述超滤级分为＜3kDa、3-10kDa和＞10kDa级分。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210390612.0A CN114773430B (zh) | 2022-04-14 | 2022-04-14 | 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210390612.0A CN114773430B (zh) | 2022-04-14 | 2022-04-14 | 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114773430A true CN114773430A (zh) | 2022-07-22 |
| CN114773430B CN114773430B (zh) | 2022-12-16 |
Family
ID=82429499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210390612.0A Active CN114773430B (zh) | 2022-04-14 | 2022-04-14 | 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114773430B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115838400A (zh) * | 2022-12-27 | 2023-03-24 | 中国农业大学 | 两种靶向预防或治疗代谢综合征的红小豆肽 |
| CN115925854A (zh) * | 2022-12-26 | 2023-04-07 | 中国农业大学 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
| CN117106020A (zh) * | 2022-12-07 | 2023-11-24 | 中国农业大学 | 具有消化酶抑制活性的红小豆来源的寡肽及其应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105831570A (zh) * | 2016-04-13 | 2016-08-10 | 华北电力大学（保定） | 辅助降胆固醇的八宝粥 |
-
2022
- 2022-04-14 CN CN202210390612.0A patent/CN114773430B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105831570A (zh) * | 2016-04-13 | 2016-08-10 | 华北电力大学（保定） | 辅助降胆固醇的八宝粥 |
Non-Patent Citations (3)
| Title |
|---|
| 张英蕾等: "红小豆的品质特性及加工利用研究概况", 《黑龙江农业科学》 * |
| 李家磊等: "红小豆保健价值研究进展", 《粮食与油脂》 * |
| 赵建京等: "红小豆保健功能研究进展", 《中国农业科技导报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117106020A (zh) * | 2022-12-07 | 2023-11-24 | 中国农业大学 | 具有消化酶抑制活性的红小豆来源的寡肽及其应用 |
| CN117106020B (zh) * | 2022-12-07 | 2024-05-28 | 中国农业大学 | 具有消化酶抑制活性的红小豆来源的寡肽及其应用 |
| CN115925854A (zh) * | 2022-12-26 | 2023-04-07 | 中国农业大学 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
| CN115925854B (zh) * | 2022-12-26 | 2023-08-25 | 中国农业大学 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
| CN115838400A (zh) * | 2022-12-27 | 2023-03-24 | 中国农业大学 | 两种靶向预防或治疗代谢综合征的红小豆肽 |
| CN115838400B (zh) * | 2022-12-27 | 2023-07-07 | 中国农业大学 | 两种靶向预防或治疗代谢综合征的红小豆肽 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114773430B (zh) | 2022-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114773430B (zh) | 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 | |
| CN114805483B (zh) | 五种来源于红小豆蛋白的胰脂肪酶抑制剂及其应用 | |
| Yu et al. | Identification and molecular docking study of a novel angiotensin-I converting enzyme inhibitory peptide derived from enzymatic hydrolysates of Cyclina sinensis | |
| CN114634552B (zh) | 一种抗肥胖十三肽及其应用 | |
| JPH0648956A (ja) | ヒト免疫不全ウィルス感染・増殖抑制剤 | |
| Wang et al. | Novel bioactive peptides from ginkgo biloba seed protein and evaluation of their α-glucosidase inhibition activity | |
| Cian et al. | Identification and in silico study of a novel dipeptidyl peptidase IV inhibitory peptide derived from green seaweed Ulva spp. hydrolysates | |
| Lee et al. | Purification and characterization of a novel angiotensin I-converting enzyme inhibitory peptide derived from an enzymatic hydrolysate of duck skin byproducts | |
| Zhao et al. | Exploration, sequence optimization and mechanism analysis of novel xanthine oxidase inhibitory peptide from Ostrea rivularis Gould | |
| CN114106128B (zh) | 具有α-淀粉酶抑制活性的小米醇溶蛋白肽 | |
| Zhao et al. | Separation, identification and docking analysis of xanthine oxidase inhibitory peptides from pacific cod bone-flesh mixture | |
| Hou et al. | Novel potential XOD inhibitory peptides derived from Trachinotus ovatus: Isolation, identification and structure-function analysis | |
| Ding et al. | Identification and characterization of dipeptidyl peptidase IV inhibitory peptides from wheat gluten proteins | |
| CN117003845B (zh) | 小米醇溶蛋白肽及其在治疗高脂血症中的用途 | |
| JPH0873499A (ja) | 新規ペプチドおよび免疫賦活剤 | |
| US20210309984A1 (en) | ChiA Enzyme | |
| CN112442108B (zh) | 一种枸杞ace、dpp-iv抑制肽及衍生多肽和应用、混合物 | |
| CN112457373A (zh) | 具有血管紧张素转化酶抑制活性的单环刺螠多肽及其应用 | |
| CN112679582A (zh) | 来源于淋巴细胞具有降血压和降血糖功效的生物活性肽及其应用 | |
| CN115838400B (zh) | 两种靶向预防或治疗代谢综合征的红小豆肽 | |
| Zheng et al. | In silico screening and characterization, inhibition mechanism on ACE, and stability of antihypertensive peptides with Zn-chelating capacity identified from millet bran albumin hydrolysates | |
| US7241735B2 (en) | Pharmacologically active polypeptide glycoconjugates | |
| Li et al. | Identification of novel renin inhibitory peptides from tartary buckwheat albumin hydrolysates by molecular docking | |
| CN115925799B (zh) | 一种具有降脂活性的小米寡肽 | |
| CN117820431B (zh) | 一种具有降尿酸功效的青稞酒糟肽及其制备方法与应用 |
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 |