CN111233972B - 一种抗炎三肽及其提取分离方法和在改善记忆中的应用 - Google Patents
一种抗炎三肽及其提取分离方法和在改善记忆中的应用 Download PDFInfo
- Publication number
- CN111233972B CN111233972B CN202010060298.0A CN202010060298A CN111233972B CN 111233972 B CN111233972 B CN 111233972B CN 202010060298 A CN202010060298 A CN 202010060298A CN 111233972 B CN111233972 B CN 111233972B
- Authority
- CN
- China
- Prior art keywords
- inflammatory
- tripeptide
- lps
- walnut protein
- walnut
- 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
- 230000003110 anti-inflammatory effect Effects 0.000 title claims abstract description 31
- 230000006993 memory improvement Effects 0.000 title claims description 4
- 238000000926 separation method Methods 0.000 title abstract description 8
- 238000000605 extraction Methods 0.000 title abstract description 6
- XXXXOVFBXRERQL-ULQDDVLXSA-N Leu-Pro-Phe Chemical group CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XXXXOVFBXRERQL-ULQDDVLXSA-N 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 4
- 208000036110 Neuroinflammatory disease Diseases 0.000 claims description 6
- 230000003959 neuroinflammation Effects 0.000 claims description 6
- 230000036541 health Effects 0.000 claims description 4
- 210000004556 brain Anatomy 0.000 claims description 2
- 235000009496 Juglans regia Nutrition 0.000 abstract description 31
- 241000758789 Juglans Species 0.000 abstract description 30
- 235000020234 walnut Nutrition 0.000 abstract description 30
- 108090000623 proteins and genes Proteins 0.000 abstract description 17
- 102000004169 proteins and genes Human genes 0.000 abstract description 16
- 206010061218 Inflammation Diseases 0.000 abstract description 14
- 230000004054 inflammatory process Effects 0.000 abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 10
- 108010009736 Protein Hydrolysates Proteins 0.000 abstract description 6
- 239000008367 deionised water Substances 0.000 abstract description 6
- 229910021641 deionized water Inorganic materials 0.000 abstract description 6
- 238000010828 elution Methods 0.000 abstract description 6
- 235000012054 meals Nutrition 0.000 abstract description 6
- 239000003531 protein hydrolysate Substances 0.000 abstract description 6
- 239000006228 supernatant Substances 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 5
- 239000000047 product Substances 0.000 abstract description 5
- 238000001819 mass spectrum Methods 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 238000000108 ultra-filtration Methods 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 238000002523 gelfiltration Methods 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 3
- 239000012466 permeate Substances 0.000 abstract description 3
- 239000002778 food additive Substances 0.000 abstract description 2
- 235000013373 food additive Nutrition 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 42
- 239000002158 endotoxin Substances 0.000 description 40
- 229920006008 lipopolysaccharide Polymers 0.000 description 38
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 30
- 230000000770 proinflammatory effect Effects 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 239000003642 reactive oxygen metabolite Substances 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 11
- 230000028709 inflammatory response Effects 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 102000004127 Cytokines Human genes 0.000 description 8
- 108090000695 Cytokines Proteins 0.000 description 8
- 230000003834 intracellular effect Effects 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 102000003777 Interleukin-1 beta Human genes 0.000 description 7
- 108090000193 Interleukin-1 beta Proteins 0.000 description 7
- 102000004889 Interleukin-6 Human genes 0.000 description 7
- 108090001005 Interleukin-6 Proteins 0.000 description 7
- 229940100601 interleukin-6 Drugs 0.000 description 7
- 210000000274 microglia Anatomy 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 5
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 5
- 210000001700 mitochondrial membrane Anatomy 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000604 Hydrolases Proteins 0.000 description 4
- 102000004157 Hydrolases Human genes 0.000 description 4
- 108010019160 Pancreatin Proteins 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 230000004770 neurodegeneration Effects 0.000 description 4
- 230000036542 oxidative stress Effects 0.000 description 4
- 229940055695 pancreatin Drugs 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 210000001642 activated microglia Anatomy 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 210000003169 central nervous system Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 208000015122 neurodegenerative disease Diseases 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 2
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 238000003916 acid precipitation Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000008809 cell oxidative stress Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 125000003963 dichloro group Chemical group Cl* 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 230000017306 interleukin-6 production Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000006433 tumor necrosis factor production Effects 0.000 description 2
- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000010266 Sephadex chromatography Methods 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001781 electrospray-ionisation quadrupole time-of-flight tandem mass spectrometry Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000019189 interleukin-1 beta production Effects 0.000 description 1
- 230000008810 intracellular oxidative stress Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007074 memory dysfunction Effects 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000003961 neuronal insult Effects 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
-
- 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
- A61P25/00—Drugs for disorders of the nervous system
-
- 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)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种抗炎三肽及其提取分离方法和在改善记忆中的应用,该抗炎三肽的氨基酸序列为Leu‑Pro‑Phe,其提取分离方法包括以下步骤:碱溶酸沉从核桃粕中提取核桃蛋白,取核桃蛋白,加去离子水混合,酶解,取上清液,得到核桃蛋白酶解液;使用3kDa分子量超滤膜收集透过液;核桃蛋白酶解液的低分子量组分上样凝胶过滤色谱柱,用去离子水洗脱,检测波长为220nm,收集合并成多个洗脱峰组分,选择抗炎活性较强的组分做质谱检测,证实含有本发明的抗炎三肽。本发明的抗炎三肽对LPS刺激的BV‑2细胞炎症反应具有较好的抑制活性,可用于制备改善记忆类药物或改善记忆类保健品,也可与其他保健品或食品添加剂复配使用。
Description
技术领域
本发明属于活性肽领域,具体涉及一种抗炎三肽及其提取分离方法和在改善记忆中的应用。
背景技术
炎症是机体对外界刺激的一种防御反应,但过度的炎症反应会导致一些与细胞因子大量产生及组织破坏等相关的免疫系统疾病。
神经炎症可引发多种神经退行性疾病,包括阿尔茨海默症(AD),帕金森病(PD)和多发性硬化症等。小胶质细胞是巨噬细胞样细胞,在中枢神经系统(CNS)的炎症反应过程中发挥关键作用。通常,活化的小胶质细胞伴随形态变化而迁移到特定部位以清除入侵碎片和病原体。另一方面,当小胶质细胞过度活化时,它会释放各种促炎介质,包括一氧化氮(NO),前列腺素E2(PGE2)和促炎细胞因子,例如白介素-1β(IL-1β),白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)以及其他一些具有潜在神经毒性的化合物。这些因子在中枢神经系统中的过度产生会触发炎症反应,并最终导致神经变性。重要的是,持续进行性神经炎症会加速脑组织损伤和行为病理学的发展。另外,氧化应激和炎症反应总是相互联系的。活性氧(ROS)通过激活NF-κB途径诱导促炎基因的表达。激活的促炎性转录因子可增强细胞因子和趋化因子的形成,从而引起炎症反应的产生和发展。反之,该过程又会产生更多的ROS。这种恶性循环通过介导线粒体功能障碍,加剧细胞损伤并最终对神经元产生有害影响。这表明氧化应激在神经炎症中的重要作用。
BV-2是一种永生化的小鼠小胶质细胞,由于其激活后可持续、稳定和过量地产生炎性因子,因此被广泛应用于研究炎症相关的神经退行性疾病。
脂多糖(LPS)是革兰氏阴性菌外膜的主要成分,可通过介导多种细胞反应促进炎症因子的释放,通常用于在体内外诱导炎症的发生。
研究表明可通过在小鼠腹膜内注射LPS从而促进炎症反应进程来造成学习和记忆功能障碍模型。
此外,越来越多的研究报道了氧化应激在炎症反应发展中的作用。ROS的产生会增加BV-2小胶质细胞中炎症介质的表达,从而加速神经元损伤和死亡。据报道,小胶质细胞中ROS的过量产生可引起炎症并导致各种类型的神经系统损害。作为细胞中ROS的主要来源,线粒体的功能变化也参与其中。
发明内容
本发明的首要目的在于提供一种抗炎三肽。
本发明的另一目的在于提供上述抗炎三肽的提取分离方法。
本发明的再一目的在于提供上述抗炎三肽的用途。
本发明的目的通过下述技术方案实现:
一种抗炎三肽,其氨基酸序列为Leu-Pro-Phe(LPF)。
上述的抗炎三肽可以采用现有技术化学合成。例如通过固相合成仪合成本发明的三肽,将二氯树脂溶胀、洗涤,脱除Fmoc保护基后加入氨基酸进行缩合反应,重复脱除-保护-缩合的过程直至所有氨基酸连接完毕。
此外本发明的抗炎三肽也可采用超滤和葡聚糖凝胶色谱等分离手段从核桃蛋白酶解液中分离获得,具体地包括以下步骤:
(1)采用碱溶酸沉的方法从核桃粕中提取核桃蛋白,具体流程如下,核桃粕打粉后加水溶解(物料比为1:12),溶液调pH值至8.0-9.0,搅拌2-3h,离心(8000-10000rpm,10-15min,4℃),取上清液调pH值至4.0-5.0,再搅拌2-3h以上,再次离心(8000-10000rpm,10-15min,4℃),将沉淀溶解,透析脱盐2d(渣与水1:5)以上,真空干燥得到核桃蛋白粉;
(2)取核桃蛋白粉,加去离子水混合,使用复合植物水解酶和胰酶在50-60℃酶解12-16h,复合植物水解酶和胰酶的加入量均为0.5-1.0%(w/w,以核桃蛋白粉的质量计);然后灭酶,离心,取上清液,得到核桃蛋白酶解液;使用3kDa分子量超滤膜收集透过液以获得核桃蛋白酶解液的低分子量组分(MW<3kDa);
所述的灭酶是95℃下处理10-15min;
(3)核桃蛋白酶解液的低分子量组分上样凝胶过滤色谱柱Sephadex G-15,用去离子水以1.0-2.0mL/min的流速进行洗脱,检测波长为220nm,收集合并成多个洗脱峰组分,选择抗炎活性较强的组分做质谱检测,证实含有本发明的抗炎三肽。
本发明的抗炎三肽LPF可抑制LPS诱导的细胞炎症反应,同时缓解LPS诱导的细胞氧化应激反应,从而具有潜在的缓解脑部神经炎症的功能,可用于改善记忆类药物和保健品中。
所述的抑制LPS诱导的细胞炎症反应,包括降低LPS诱导的细胞中促炎介质和促炎细胞因子的生成量;
所述的促炎介质包括NO和PGE2;
所述的促炎细胞因子包括IL-6、IL-1β和TNF-α。
所述的缓解LPS诱导的细胞氧化应激反应是指降低LPS诱导的细胞内活性氧的生成量,以及逆转LPS诱导的线粒体膜电位的下降。
所述的细胞是小胶质细胞;
所述的细胞是BV-2细胞。
本发明相对于现有技术具有如下的优点及效果:
1、本发明的抗炎三肽结构清晰明了,可采用固相化学合成方法制得,也可通过对核桃蛋白酶解物进行分离纯化获得。
2、本发明的抗炎三肽对LPS刺激的BV-2细胞炎症反应具有较好的抑制活性,具有潜在的缓解脑部神经炎症的功效,可用于制备改善记忆类药物或改善记忆类保健品,也可与其他保健品或食品添加剂复配使用。
附图说明
图1是Sephadex G-15凝胶过滤色谱的洗脱曲线。
图2是三肽Leu-Pro-Phe的二级质谱图。
图3是LPF对LPS诱导的BV-2细胞内ROS和MMP的影响;其中,#代表与正常对照组比较,p<0.05;*表示与模型组比较,p<0.05。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
BV-2细胞培养
向DMEM培养基中加入胎牛血清(FBS),加入量为10%(v/v),然后加入由100U/mL青霉素和100μg/mL链霉素组成的混合双抗。细胞培养箱为BV-2细胞的生长提供了所需的环境,箱内条件设定为37℃且含有5%的CO2。根据细胞生长情况及时进行换液和传代。
实施例2
固相合成法合成Leu-Pro-Phe多肽
将1g二氯树脂溶胀、洗涤,脱除Fmoc保护基后加入氨基酸进行缩合反应,重复脱除-保护-缩合的过程直至所有氨基酸连接完毕。切割树脂,得到多肽Leu-Pro-Phe粗品,用反相高效液相色谱法纯化,得到多肽纯品(>95%)。
实施例3
Leu-Pro-Phe多肽的分离纯化方法,包括以下步骤:
(1)核桃蛋白粉的制备:采用碱溶酸沉的方法从核桃粕中提取核桃蛋白。具体流程如下,核桃粕打粉后加水溶解(物料比为1:12)→溶液调pH至8.5→搅拌2h→离心(8000rpm,15min,4℃)→取上清液调pH至4.5→搅拌2h→离心(8000rpm,15min,4℃)→沉淀溶解→透析脱盐2d(渣与水1:5)→干燥→核桃蛋白粉。
(2)核桃蛋白酶解物的制备:按重量取核桃蛋白粉1份和去离子水8份混合均匀,使用两种蛋白酶(复合植物水解酶和胰酶)在55℃酶解12小时,复合植物水解酶和胰酶的加酶量分别为1.0%和1.0%(w/w)。随后95℃下灭酶15min,离心,取上清液,得到核桃蛋白酶解液;使用3kDa分子量超滤膜收集透过液以获得核桃蛋白酶解液的低分子量组分。收集核桃蛋白酶解液低分子量组分(MW<3kDa),冷冻干燥,并在-20℃下储存备用。
(3)抗炎肽的分离纯化:使用凝胶过滤色谱柱Sephadex G-15进行分离纯化,用去离子水以1.5mL/min的流速进行洗脱,检测波长为220nm,洗脱曲线如图1所示,收集合并成多个洗脱峰组分,选择抗炎活性较强的组分G3和G6进行收集,然后冷冻干燥成粉末。得到目标多肽。目标多肽的二级质谱图如图2所示,序列是Leu-Pro-Phe。
表1 各洗脱峰组分对LPS诱导的BV-2细胞内NO和PGE2产生的影响
#代表与正常对照组比较,p<0.05,*表示与模型组比较,p<0.05。
LPS激活的小胶质细胞可通过过量产生促炎介质(NO和PGE2)加速炎症反应进程,最终导致细胞死亡。因此,NO和PGE2的产生量被用来评估样品对LPS刺激的BV-2细胞炎症的抑制作用。
通过Sephadex G-15凝胶过滤色谱将核桃蛋白酶解液低分子量组分(MW<3kDa)分成6个主要组分(G1-G6)。
测定结果如表1所示,LPS处理后细胞培养基中NO和PGE2含量显著增加,分别为101.73±7.98μM和205.58±23.58pg/mL(即模型组)。但是,G3和G6处理可以显著抑制LPS所致的BV-2细胞中NO和PGE2的产生(p<0.05)。因此,通过UPLC-ESI-Q-TOF-MS/MS分析G4和G6中包含的特定氨基酸序列抗炎肽,随后得到本发明抗炎三肽LPF。
实施例4
三肽LPF对LPS诱导的BV-2细胞促炎介质和促炎细胞因子水平的影响
本研究采用在BV-2细胞中同时添加诱导剂LPS和抗炎三肽LPF,即共培养细胞模型来评估LPF对LPS诱导的BV-2小胶质细胞炎症损伤的保护作用。
将细胞以2×105个/mL的密度接种于12孔板中贴壁生长24小时,随后加入LPF和LPS(0.1μg/mL)处理BV-2细胞24小时。正常对照组中不加LPF和LPS,仅加入相同量的DMEM培养基。
待细胞处理结束时,收集上清液并根据试剂盒使用说明书测定其中一氧化氮(NO)、前列腺素E2(PGE2)、白细胞介素-1β(IL-1β),白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)含量,如表2和表3所示:
表2 LPF对LPS诱导的BV-2细胞内NO和PGE2产生的影响
#代表与正常对照组比较,p<0.05,*表示与模型组比较,p<0.05。
LPS激活的小胶质细胞可通过过量产生促炎介质(NO和PGE2)加速炎症反应进程,最终导致细胞死亡。因此,NO和PGE2的产生量被用来评估LPF对LPS刺激的BV-2细胞炎症的抑制作用。
测定结果如表2所示,LPS处理后细胞培养基中NO和PGE2含量显著增加,分别为84.41±7.98μM和235.46±23.56pg/mL(模型组)。但是,LPF处理可以显著抑制LPS所致的BV-2细胞中NO和PGE2的产生(p<0.05),使其分别降低到48.74±3.75μM和125.82±13.29pg/mL。
表3 LPF对LPS诱导的BV-2细胞内IL-1β,IL-6和TNF-α产生的影响
#代表与正常对照组比较,p<0.05,*表示与模型组比较,p<0.05。
过度激活的小胶质细胞中促炎细胞因子的产生会引发急性和慢性炎症,从而引起神经退行性疾病。在本研究中,通过检测IL-6,IL-1β和TNF-α的产生水平以评估LPF对LPS刺激的BV-2细胞促炎性细胞因子暴增的抑制作用。
测定结果如表3所示,在LPS处理的BV-2细胞中观察到IL-6,IL-1β和TNF-α的含量突增(模型组)。然而,加入LPF保护的BV-2细胞中TNF-α产生从4000.53±357.75pg/mL显著降低至2200.41±249.65。同时,与LPS组相比,使用LPF可显著减少模型组细胞中IL-1β和IL-6的产生(p<0.05)。
实施例5
三肽LPF对LPS诱导的BV-2细胞内活性氧和线粒体膜电位的影响
细胞内的ROS水平使用荧光探针法(DCFH-DA)来测定:将细胞以2×105个/mL的密度接种于96孔板中贴壁生长24小时,随后加入LPF和LPS处理BV-2细胞24小时。待BV-2细胞样品处理完成后,将BV-2细胞与10μM DCFH-DA探针在培养箱中放置30分钟,然后用PBS清洗。在存在ROS的情况下,DCFH可以转化为具有强绿色荧光的二氯荧光素(DCF)。使用多模板读数器测量荧光强度(Ex=488nm,Em=525nm)。
细胞内线粒体膜电位(MMP)使用JC-1荧光探针法来测定:JC-1荧光探针法是一种对线粒体膜电位变化十分敏感的测定方法,当细胞具有较高的线粒体膜电位时,荧光探针在线粒体基质中形成有红色荧光的聚集体(J-aggregate)。反之,则JC-1以单体(J-monomer)的形式存在,本身具有绿色荧光。
将细胞以2×105个/mL的密度接种于96孔板中贴壁生长24小时,随后加入LPF和LPS处理BV-2细胞24小时。待BV-2细胞进行样品处理培养后,将细胞与JC-1工作溶液在37℃共孵育30分钟。然后收集细胞样品并用PBS洗涤两次,随后重新悬浮并用荧光分光光度计分析。计算ΔΨm单体和聚集体荧光强度比率的变化。
越来越多的证据表明氧化应激可促进炎症反应的进程,因此对细胞内氧化应激的抑制将有助于抗炎。
测定结果如图3所示,LPF可缓解LPS刺激引起的ROS生成。同时,LPF也显著逆转了BV-2细胞中LPS诱导的MMP损失。BV-2细胞经LPS处理后,细胞内ROS含量相比于对照组提高了2.24倍,LPF可将细胞内ROS的产生降低至对照组的167.97%±6.47%(p<0.05)。此外,LPS刺激引起BV-2细胞中MMP的损失(68.48%±3.34%),LPF可显著逆转细胞内因LPS刺激导致的MMP的降低(89.17%±2.79%)。
综上所述,本发明的三肽LPF可显著抑制LPS诱导所致的BV-2细胞内的炎症反应,同时LPF对氧化应激的缓解也有助于提高其抗炎活性。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
广州现代产业技术研究院
<120> 一种抗炎三肽及其提取分离方法和在改善记忆中的应用
<140> 2020100602980
<141> 2020-01-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> PRT
<213> 核桃(Juglans regia L.)
<400> 1
Leu Pro Phe
1
Claims (1)
1.一种抗炎三肽在制备改善记忆类的药物和保健品中的应用,其特征在于:
所述抗炎三肽的氨基酸序列为Leu-Pro-Phe;
所述的改善记忆是指缓解脑部神经炎症。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010060298.0A CN111233972B (zh) | 2020-01-19 | 2020-01-19 | 一种抗炎三肽及其提取分离方法和在改善记忆中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010060298.0A CN111233972B (zh) | 2020-01-19 | 2020-01-19 | 一种抗炎三肽及其提取分离方法和在改善记忆中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111233972A CN111233972A (zh) | 2020-06-05 |
CN111233972B true CN111233972B (zh) | 2021-08-10 |
Family
ID=70878050
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010060298.0A Active CN111233972B (zh) | 2020-01-19 | 2020-01-19 | 一种抗炎三肽及其提取分离方法和在改善记忆中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111233972B (zh) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113173970B (zh) * | 2021-06-15 | 2022-11-15 | 北京林业大学 | 一种核桃粕抗炎肽fpl及其应用 |
CN113307843B (zh) * | 2021-06-15 | 2022-09-23 | 北京林业大学 | 一种核桃粕抗炎肽wsl及其应用 |
CN113201047B (zh) * | 2021-06-15 | 2022-11-29 | 北京林业大学 | 一种核桃粕抗炎肽wpl及其应用 |
CN113402583B (zh) * | 2021-06-19 | 2023-03-21 | 江西农业大学 | Qgk三肽及其应用 |
CN114133430B (zh) * | 2021-11-30 | 2024-04-05 | 南开大学 | 一种与酶共生增强炎症治疗的多肽自组装药物递送载体的制备方法 |
WO2023143522A1 (zh) * | 2022-01-28 | 2023-08-03 | 北京达尔文细胞生物科技有限公司 | 一种神经修复蛋白组合物及其制备方法和其应用 |
CN114763370B (zh) * | 2022-03-14 | 2023-06-27 | 中国科学院华南植物园 | 一种抗氧化十肽及其制备方法和应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103497985A (zh) * | 2013-09-06 | 2014-01-08 | 华南理工大学 | 核桃粕抗氧化肽及其制备方法 |
CN105111282A (zh) * | 2015-09-11 | 2015-12-02 | 哈尔滨工业大学 | 一种具有ace抑制活性的核桃肽及其制备方法 |
CN107624118A (zh) * | 2015-04-24 | 2018-01-23 | 伊玛提克斯生物技术有限公司 | 用于肺癌(包括非小细胞肺癌和其他癌症)免疫治疗的新型肽和肽组合物 |
WO2019200298A1 (en) * | 2018-04-13 | 2019-10-17 | Northwestern University | Nanomolecules for the treatment of inflammatory bowel diseases |
-
2020
- 2020-01-19 CN CN202010060298.0A patent/CN111233972B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103497985A (zh) * | 2013-09-06 | 2014-01-08 | 华南理工大学 | 核桃粕抗氧化肽及其制备方法 |
CN107624118A (zh) * | 2015-04-24 | 2018-01-23 | 伊玛提克斯生物技术有限公司 | 用于肺癌(包括非小细胞肺癌和其他癌症)免疫治疗的新型肽和肽组合物 |
CN105111282A (zh) * | 2015-09-11 | 2015-12-02 | 哈尔滨工业大学 | 一种具有ace抑制活性的核桃肽及其制备方法 |
WO2019200298A1 (en) * | 2018-04-13 | 2019-10-17 | Northwestern University | Nanomolecules for the treatment of inflammatory bowel diseases |
Non-Patent Citations (1)
Title |
---|
核桃肽改善睡眠剥夺诱导大鼠记忆障碍及其对PC12细胞神经保护作用机制研究;王曙光;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20200115(第1期);摘要,第11页第1段、第1.5.1节,第25页第2.4.1节,第34-35页第3.3.1-3.3.3节,第37页第3.4.1节,第39-40页第3.4.2节,第40页表3-1 * |
Also Published As
Publication number | Publication date |
---|---|
CN111233972A (zh) | 2020-06-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111233972B (zh) | 一种抗炎三肽及其提取分离方法和在改善记忆中的应用 | |
CN111153963B (zh) | 抗炎五肽及其提取分离方法和在改善记忆中的应用 | |
CN111253466B (zh) | 抗炎四肽及其提取分离方法和在制备改善记忆类药物中的应用 | |
Xia et al. | Optimization and identification of antioxidant peptide from underutilized Dunaliella salina protein: Extraction, in vitro gastrointestinal digestion, and fractionation | |
KR20060069349A (ko) | 마데카소시드 및 터미놀로시드가 풍부한 센텔라 아시아티카추출물의 제조 방법 | |
CN110655556B (zh) | 一种免疫调节肽的制备及方法 | |
CN113845566B (zh) | 一种用于预防阿尔兹海默症的核桃多肽及其应用 | |
WO2020108629A1 (zh) | 一种多肽rdp1及其提纯方法与应用 | |
CN107828842B (zh) | 一种具有抗氧化和dpp-iv抑制功能的核桃蛋白肽 | |
CN107325155B (zh) | 一种抗氧化超短肽Mapin-WH2、其制备方法及应用 | |
JPH06256387A (ja) | 新規なペプチド、その製法およびそれを有効成分とする 血圧降下剤 | |
CN114907445B (zh) | 一种高抗氧化活性富硒肽及其应用 | |
CN104892730B (zh) | 一种带鱼肝脏抗菌肽 | |
CN113480597B (zh) | 一种来源于紫苏籽粕的ace抑制肽及其制备方法与应用 | |
CN104894200B (zh) | 路氏双髻鲨软骨血管生成抑制因子的制备方法 | |
CN116082443A (zh) | 一种金枪鱼鱼鳞寡肽及其制备方法和应用 | |
CN115109117A (zh) | 一种多管藻藻红蛋白血管紧张素转化酶抑制肽及其制备方法与应用 | |
CN108358998B (zh) | 一种星虫肽及其在制备妊娠期高血压治疗药物中的应用 | |
KR20120049047A (ko) | 굴 가수분해물을 유효성분으로 함유하는 항염증 조성물 | |
CN113201047A (zh) | 一种核桃粕抗炎肽wpl及其应用 | |
CN104892729B (zh) | 一种路氏双髻鲨软骨血管生成抑制因子 | |
JPH09157292A (ja) | 新規なペプチドおよび活性化酸素阻害剤 | |
CN102311484A (zh) | 一种抑制血管紧张素转移酶的六肽及其制备方法 | |
CN104762357B (zh) | 马面鲀鱼皮锌螯合肽制备方法 | |
JPH09157291A (ja) | 新規なペプチドおよび活性化酸素阻害剤 |
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 | ||
CB02 | Change of applicant information |
Address after: Nansha District Avenue South Ring of 511458 cities in Guangdong province Guangzhou City, No. 25 Hua Da Guangzhou production and Research Institute Applicant after: SOUTH CHINA University OF TECHNOLOGY Applicant after: GUANGZHOU INSTITUTE OF MODERN INDUSTRIAL TECHNOLOGY Address before: 510640 Tianhe District, Guangdong, No. five road, No. 381, Applicant before: SOUTH CHINA University OF TECHNOLOGY Applicant before: GUANGZHOU INSTITUTE OF MODERN INDUSTRIAL TECHNOLOGY |
|
CB02 | Change of applicant information | ||
GR01 | Patent grant | ||
GR01 | Patent grant |