CN117003845B - 小米醇溶蛋白肽及其在治疗高脂血症中的用途 - Google Patents
小米醇溶蛋白肽及其在治疗高脂血症中的用途 Download PDFInfo
- Publication number
- CN117003845B CN117003845B CN202311088956.7A CN202311088956A CN117003845B CN 117003845 B CN117003845 B CN 117003845B CN 202311088956 A CN202311088956 A CN 202311088956A CN 117003845 B CN117003845 B CN 117003845B
- Authority
- CN
- China
- Prior art keywords
- peptide
- millet
- prolamin
- amino acid
- millet prolamin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 88
- 244000062793 Sorghum vulgare Species 0.000 title claims abstract description 55
- 235000019713 millet Nutrition 0.000 title claims abstract description 55
- 108060006613 prolamin Proteins 0.000 title claims abstract description 47
- 102000000019 Sterol Esterase Human genes 0.000 claims abstract description 35
- 108050006759 Pancreatic lipases Proteins 0.000 claims abstract description 28
- 102000019280 Pancreatic lipases Human genes 0.000 claims abstract description 28
- 229940116369 pancreatic lipase Drugs 0.000 claims abstract description 28
- 108010087173 bile salt-stimulated lipase Proteins 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 239000002329 esterase inhibitor Substances 0.000 claims abstract description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 150000001413 amino acids Chemical class 0.000 claims description 14
- 125000006239 protecting group Chemical group 0.000 claims description 10
- 239000011347 resin Substances 0.000 claims description 10
- 229920005989 resin Polymers 0.000 claims description 10
- 238000004949 mass spectrometry Methods 0.000 claims description 7
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 7
- 239000003875 Wang resin Substances 0.000 claims description 3
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 239000002808 molecular sieve Substances 0.000 claims description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 3
- 230000008961 swelling Effects 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 39
- 230000000694 effects Effects 0.000 abstract description 22
- 208000031226 Hyperlipidaemia Diseases 0.000 abstract description 18
- 235000013305 food Nutrition 0.000 abstract description 15
- 102000038379 digestive enzymes Human genes 0.000 abstract description 14
- 108091007734 digestive enzymes Proteins 0.000 abstract description 14
- 150000002632 lipids Chemical class 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 13
- 230000036541 health Effects 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 8
- 239000002532 enzyme inhibitor Substances 0.000 abstract description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 231100001081 no carcinogenicity Toxicity 0.000 abstract description 3
- 231100000956 nontoxicity Toxicity 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000029087 digestion Effects 0.000 abstract description 2
- 229940125532 enzyme inhibitor Drugs 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 30
- 230000000975 bioactive effect Effects 0.000 description 29
- 230000005764 inhibitory process Effects 0.000 description 28
- 108010055297 Sterol Esterase Proteins 0.000 description 25
- 238000003032 molecular docking Methods 0.000 description 22
- 238000002835 absorbance Methods 0.000 description 20
- 230000003993 interaction Effects 0.000 description 18
- 125000000539 amino acid group Chemical group 0.000 description 16
- 229910052739 hydrogen Inorganic materials 0.000 description 16
- 239000001257 hydrogen Substances 0.000 description 14
- 230000002209 hydrophobic effect Effects 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 108010067035 Pancrelipase Proteins 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 239000000413 hydrolysate Substances 0.000 description 8
- 229940045258 pancrelipase Drugs 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 235000019626 lipase activity Nutrition 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- XRHVZWWRFMCBAZ-UHFFFAOYSA-L Endothal-disodium Chemical compound [Na+].[Na+].C1CC2C(C([O-])=O)C(C(=O)[O-])C1O2 XRHVZWWRFMCBAZ-UHFFFAOYSA-L 0.000 description 6
- 238000005411 Van der Waals force Methods 0.000 description 6
- 238000003556 assay Methods 0.000 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 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 108010009736 Protein Hydrolysates Proteins 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 208000032928 Dyslipidaemia Diseases 0.000 description 4
- 208000017170 Lipid metabolism disease Diseases 0.000 description 4
- 108090000284 Pepsin A Proteins 0.000 description 4
- 102000057297 Pepsin A Human genes 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 235000012000 cholesterol Nutrition 0.000 description 4
- 230000007071 enzymatic hydrolysis Effects 0.000 description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- DVDUMIQZEUTAGK-UHFFFAOYSA-N p-nitrophenyl butyrate Chemical compound CCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 DVDUMIQZEUTAGK-UHFFFAOYSA-N 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- 239000003531 protein hydrolysate Substances 0.000 description 4
- 238000004088 simulation Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 230000000678 effect on lipid Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 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 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 208000006011 Stroke Diseases 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 201000006549 dyspepsia Diseases 0.000 description 2
- 230000006862 enzymatic digestion Effects 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 231100001223 noncarcinogenic Toxicity 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 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 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical class C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- 102100029077 3-hydroxy-3-methylglutaryl-coenzyme A reductase Human genes 0.000 description 1
- 101710158485 3-hydroxy-3-methylglutaryl-coenzyme A reductase Proteins 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- OTLNPYWUJOZPPA-UHFFFAOYSA-M 4-nitrobenzoate Chemical compound [O-]C(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-M 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010014935 Enzyme abnormality Diseases 0.000 description 1
- 241000565391 Fraxinus mandshurica Species 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010023302 HDL Cholesterol Proteins 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010028554 LDL Cholesterol Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 230000007211 cardiovascular event Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002173 cutting fluid Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013367 dietary fats Nutrition 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940125753 fibrate Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000005095 gastrointestinal system Anatomy 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 231100000483 muscle toxicity Toxicity 0.000 description 1
- 150000002814 niacins Chemical class 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 210000004798 organs belonging to the digestive system Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 235000021395 porridge Nutrition 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- A61P3/04—Anorexiants; Antiobesity agents
-
- 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Diabetes (AREA)
- Genetics & Genomics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Polymers & Plastics (AREA)
- Child & Adolescent Psychology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本申请提供了小米醇溶蛋白肽WQHMMP及其在治疗高脂血症中的用途。本申请的小米醇溶蛋白肽具有天然、无毒、无致癌性、分子量小、不被胃肠道消化的特点,胰脂肪酶和胆固醇酯酶抑制活性高,制备方法简单,易于工业化生产,可大规模制备;可作为新型脂质消化酶抑制剂,亦可作为医药、食品、保健品等领域的优质原料进一步开发利用,有助于预防、缓解和治疗高脂血症,具有较高的经济价值和良好的市场前景。
Description
技术领域
本申请属于生物活性肽领域和代谢类疾病治疗领域,具体地,本申请提供了两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽及其在治疗高脂血症中的用途。
背景技术
高脂血症(又称血脂异常)通常指血清中甘油三酯、胆固醇、低密度脂蛋白胆固醇水平升高,高密度脂蛋白胆固醇水平降低,为临床常见、多发的一种全身性疾病,其主要与先天脂质代谢缺陷、不良生活习性、环境因素等有关。目前,中国成人血脂异常总体患病率高达40.4%,且逐年增加。此外,血脂异常也出现“低龄化”发展趋势,儿童青少年血脂异常检出率不断提高。高脂血症是心血管疾病、动脉粥样硬化、血栓形成、中风和肝损伤的重要危险因素,并能升高血尿酸水平,提高胰腺炎的发病率,增加罹患肿瘤的风险。有研究指出人群血脂水平的持续异常将导致2010-2030年期间中国心血管病事件增加920万,这预示未来中国成人高脂血症患病及相关疾病负担将继续加重。当前常规的降脂药物种类繁多如他汀类、贝特类、烟酸类等,其中最普遍应用的他汀类药物虽然耐受性良好,但也可能引起肌肉毒性、头痛、呼吸道感染、肝酶异常以及出血性脑卒中等不良反应。此外,他汀类药物的治疗靶点单一,主要限制胆固醇合成限速酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶,对于其他原因导致的脂代谢异常疗效不明显。随着高脂血症发病率的急剧升高,研发安全、有效的高脂血症预防与治疗药物、食品、保健品已成为国内外相关领域研发热点。
胰脂肪酶是一种由哺乳动物胰腺分泌后释放到胃肠体系,与肝脏分泌的胆汁酸盐协作将脂肪降解为甘油单酯和游离脂肪酸的重要消化道内源酶。胆固醇酯酶是一种从胰腺分泌,主要存在于小肠腔内的α/β水解酶,能催化膳食胆固醇酯中胆固醇和游离脂肪酸的释放。近年来,越来越多的研究表明抑制胰脂肪酶和胆固醇酯酶可减少消化器官中膳食脂肪的分解和吸收,进而改善高脂血症等代谢性疾病。因此,研发新型脂质消化酶(胰脂肪酶和胆固醇酯酶)抑制剂已受到高脂血症防治领域的高度关注。
与常规化学药物相比,生物活性肽因具有不良反应少、安全性高、多靶点起效、能达到增效减毒的治疗效果等优势,已成为脂质消化酶抑制剂研究的重点。然而,生物活性肽通常来源于蛋白水解释放,由于蛋白水解物所含成分复杂,若逐一展开研究则具有盲目性,且存在研究周期长及资金成本大等问题,因此,基于生物信息学技术的生物活性肽筛选对研发脂质消化酶抑制剂具有重要应用价值。
小米,原名粟,在中国和印度等地区种植广泛,是全球消费和工业使用的主要农产品。小米含有丰富的蛋白质(7%-12%),是一种良好的植物蛋白来源。小米蛋白主要包括清蛋白、球蛋白、醇溶蛋白和碱溶蛋白,其中醇溶蛋白含量最多,占总蛋白的50%以上。研究表明,以小米醇溶蛋白为原料,经酶解可制得具有抗氧化、抗炎症、降血压、降血糖等活性的小米醇溶蛋白水解物。虽然已有文献证明经常食用小米有助于缓解高脂血症,然而目前还没有从小米醇溶蛋白水解物中发现防治高脂血症的生物活性肽,这促使我们进一步挖掘小米醇溶蛋白水解物,以寻找多靶点、高效、低毒的能防治高脂血症的新型活性肽。
发明内容
本发明首先使用酶解法水解不同来源的小米醇溶蛋白,再基于对脂质消化酶(胰脂肪酶和胆固醇酯酶)的抑制作用筛选出具有最高酶抑制活性的小米醇溶蛋白水解物,进一步通过超滤、肽测序、生物信息学等技术筛选出生物活性肽,然后利用Fmoc固相合成方法制备生物活性肽并验证其对脂质消化酶的抑制效果,最后通过分子对接技术阐明抑制机理。
一方面,本申请提供了两种小米醇溶蛋白肽,其氨基酸序列为SEQ ID NO.1或SEQID NO.2。
另一方面,本申请提供了上述小米醇溶蛋白肽在制备胰脂肪酶和/或胆固醇酯酶抑制剂中的应用。
另一方面,本申请提供了上述小米醇溶蛋白肽在制备治疗高脂血症的药物或保健品中的应用。
另一方面,本申请提供了上述小米醇溶蛋白肽在制备减肥的药物或保健品中的应用。
另一方面,本申请提供了上述小米醇溶蛋白肽在制备食品中的应用,所述食品适用于肥胖人群。
一种药物、保健品或食品,其包含上述小米醇溶蛋白肽。
进一步地,上述药物、保健品或食品中还包含辅料、食品原料。
另一方面,本申请提供了制备上述小米醇溶蛋白肽的方法,所述方法为酶解方法或者Fmoc固相合成方法。
进一步地,所述酶解方法包括使用胃蛋白酶和胰酶酶解小米醇溶蛋白的步骤和分离上述小米醇溶蛋白肽的步骤。
进一步地,所述Fmoc固相合成方法包括(1)使用分子筛浸泡N,N-二甲基甲酰胺和甲醇以除杂;(2)N,N-二甲基甲酰胺溶胀活化Wang树脂;(3)接第一个氨基酸;(4)Fmoc保护基脱除;(5)接第二个氨基酸并脱除Fmoc保护基;(6)重复步骤(5)多次,直到合成到最后一个氨基酸并脱除Fmoc保护基;(7)树脂的脱落,通过液相和/或质谱方法进行检测。
本领域技术人员可以根据纯度等要求选择适合的方法从酶解物中分离所需肽,这些方法包括但不限于层析、沉淀等。
Fmoc固相合成的方法本领域公知,本领域技术人员可以根据仪器、树脂、安全性等要求选择不同的试剂和参数。
本申请所述的食品可以为本领域中已知的食品种类,包括但不限于点心、主食、膨化食品、液体或者冲饮形式的粥、糖、糊、饮料等。
上述辅料本领域技术人员可以根据寡肽的稳定性、溶解性等特性,结合药剂学、食品学中一般认识和工具书进行设计和选择。
本申请中的液相和质谱检测方法可以使用本领域市售的仪器和耗材,适合的参数和试剂本领域技术人员可以根据该领域的一般认识和适当的初步实验确定。
本发明的两种小米源亲水性/碱性醇溶蛋白肽,色氨酸-谷氨酰胺-组氨酸-谷氨酰胺-酪氨酸(Trp-Gln-His-Gln-Tyr,WQHQY),色氨酸-谷氨酰胺-组氨酸-蛋氨酸-蛋氨酸-脯氨酸(Trp-Gln-His-Met-Met-Pro,WQHMMP),是发明人通过大量工作筛选得到的,具有天然、无毒、无致癌性、分子量小、不被胃肠道消化的特点,胰脂肪酶和胆固醇酯酶抑制活性高,制备方法简单,易于工业化生产,可大规模制备。并且进一步发现它们通过疏水相互作用、盐桥、氢键、π-π堆积、π-阳离子相互作用与脂质消化酶的氨基酸残基结合实现酶抑制活性的发挥。总之,两种小米醇溶蛋白肽作为小米深加工产品,是新型脂质消化酶抑制剂,亦可作为医药、食品、保健品等领域的优质原料进一步开发利用,有助于预防、缓解和治疗高脂血症,具有较高的经济价值和良好的市场前景。
附图说明
图1为三种蛋白水解物的脂质消化酶活性抑制率;
图2为生物活性肽WQHQY(A)和WQHMMP(B)的高效液相色谱结果图;
图3为生物活性肽WQHQY(A)和WQHMMP(B)的质谱结果图;
图4为两种生物活性肽的脂质消化酶活性抑制率;
图5为生物活性肽WQHQY(A)和WQHMMP(B)的胰脂肪酶活性抑制机理;
图6为生物活性肽WQHQY(A)和WQHMMP(B)的胆固醇酯酶活性抑制机理。
具体实施方式
实施例1不同来源的小米醇溶蛋白的提取
将中谷2号、昭农21号及红苗压破车小米粉分别以1:5(w/v)的比例分散在正己烷中,37℃水浴震荡4h并静置1h。明显分层后,倒出上层正己烷,收集下层沉淀物,并在通风橱中干燥12h以完全除去正己烷残留。风干后的小米脱脂粉过60目筛备用。
将中谷2号、昭农21号及红苗压破车脱脂小米粉分别按1:7(w/v)的比例分散在70%乙醇中,37℃水浴震荡4h,随后在8000rpm下离心15min并收集上清液。用透析袋对上清液透析36h,期间更换蒸馏水4-5次。透析结束后,将透析液在7000rpm下离心5min,收集沉淀进行冻干,即得小米醇溶蛋白。
实施例2酶解法水解不同来源的小米醇溶蛋白
将中谷2号、昭农21号及红苗压破车小米醇溶蛋白分别按照5%(w/v)的比例在蒸馏水中混合均匀,使用1mol/L HCl调节蛋白溶液pH值到2.0。然后加入4%胃蛋白酶(w/w,250U/mg),混匀后置于摇床中振荡酶解,摇床转速为300rpm,酶解时间2h,酶解温度37℃。胃蛋白酶酶解结束后,先使用0.9mol/L NaHCO3将溶液pH值调节到5.3,再用1mol/L NaOH将pH值维持在7.5,最后加入4%胰酶(w/w,8×USP),并在37℃下酶解2h。胰酶酶解结束后,酶解液沸水浴10min,灭活残留的酶。酶解液经室温冷却后,在4℃,7000×g下离心20min,收集上清液即得小米醇溶蛋白水解物。
实施例3小米醇溶蛋白水解物的筛选
基于对脂质消化酶(胰脂肪酶和胆固醇酯酶)的抑制作用,筛选出具有最高酶抑制活性的小米醇溶蛋白水解物。
(1)胰脂肪酶活性抑制实验
使用pH 7.3的磷酸缓冲液配制2.5mg/mL胰脂肪酶溶液,然后以5500rpm离心5min取上清液。使用pH 7.3的磷酸缓冲液稀释对硝基苯基丁酸酯至10mM。
将50μL 5mg/mL中谷2号/昭农21号/红苗压破车小米醇溶蛋白水解物溶液(等量蒸馏水作为对照组)、40μL胰脂肪酶溶液和20μL对硝基苯丁酸酯溶液在37℃孵育30min后,酶标仪在405nm处记录吸光度。胰脂肪酶活性抑制率的计算公式如下:
式(1)中:A:对照吸光度;B:对照空白吸光度;C:样品吸光度;D:样品空白吸光度。(2)胆固醇酯酶活性抑制实验
使用pH 7.0的磷酸缓冲液(含100mM NaCl,5.16mM牛磺胆酸钠)分别配制25μg/mL胆固醇酯酶溶液和10mM对硝基苯基丁酸酯溶液。
将50μL 8mg/mL中谷2号/昭农21号/红苗压破车小米醇溶蛋白水解物溶液(等量蒸馏水作为对照组)、50μL胆固醇酯酶溶液和50μL对硝基苯丁酸酯溶液在25℃孵育5min后,酶标仪在405nm处记录吸光度。胆固醇酯酶活性抑制率的计算公式如下:
式(2)中:A:对照吸光度;B:对照空白吸光度;C:样品吸光度;D:样品空白吸光度。
体外试验结果显示,中谷2号、昭农21号和红苗压破车小米醇溶蛋白水解物的胰脂肪酶活性抑制率分别是31.68%、36.30%和41.15%,胆固醇酯酶活性抑制率分别是21.97%、26.87%和33.66%(图1)。这表明三种不同来源的小米醇溶蛋白水解物对脂质消化酶均有抑制效果,其中红苗压破车小米醇溶蛋白水解物具有最好的抑制效果。基于此,发明人以红苗压破车小米醇溶蛋白水解物为研究对象,从其中筛选出符合开发要求的生物活性肽。
小米醇溶蛋白水解物的超滤
12mL小米醇溶蛋白水解物溶液转移至3kDa离心超滤管,4℃,5000×g离心30min后即得分子量<3kDa级分,冷冻干燥并在-20℃保存。
实施例4<3kDa级分的肽序列鉴定
<3kDa级分样品用C18除盐柱除盐后,经由配备分析柱Acclaim PepMap C18,75μm×25cm和在线纳喷离子源的LC-MS/MS分析。进样量:3μL,柱流量:300nL/min,柱温:40℃,电喷雾电压:2kV。流动相A相:0.1%甲酸水溶液,B相:含0.1%甲酸的80%的ACN溶液。梯度从4%的B相起始,平衡1min,在53min 40sec以非线性梯度升高到50%,40s内升高到95%,维持5min 40sec。
质谱仪在数据依赖采集模式下运行,自动在MS和MS/MS采集间切换。质谱参数:(1)MS:扫描范围(m/z):350-1550,AGC target:8e5,分辨率:120000,最大注入时间:100ms;(2)HCD-MS/MS:分辨率:30000,AGC target:1e5,动态排除时间:30s,最大注入时间:54ms。串联质谱图经过PEAKS Studio version 10.6分析。PEAKSDB对uniprot-Setaria_italica(version202204,35905entries)数据库搜库,肽段卡值为:-10lgP≥20;在数据库中未检索到的肽段,通过设置ALC(%)≥80得出,部分典型结果见表1。
实施例5生物活性肽的筛选
作为功能成分用于和高脂血症有关的食品、保健品以及药品的生物活性肽,其无毒、无致癌性和高潜在生物活性是筛选的基本前提。生物活性肽发挥健康作用的重要考虑之一是确保肽的生物活性不受消化系统的影响,而由2-6个氨基酸组成,分子量集中在1000Da以下的活性肽可以不被胃肠道消化。故基于生物信息学技术对<3kDa级分中的肽段,依据分子量小(<1000Da)、无毒、无致癌性、高潜在生物活性和胃肠道不消化为标准,筛选出符合开发要求的生物活性肽。
采用ToxinPrep(https://webs.iiitd.edu.in/raghava/toxinpred/index.html)平台,基于SVM(Swiss-Port)算法,对肽进行毒性预测。利用admetSAR(http://lmmd.ecust.edu.cn/admetsar1/home/)对肽进行致癌性预测。通过PeptideRanker在线平台(http://distilldeep.ucd.ie/PeptideRanker/)分析肽的潜在生物学活性,其中阈值大于0.5则被认为具有生物活性。采用PeptideCutter(https://web.expasy.org/peptide_cutter/)对肽基于胃蛋白酶和胰蛋白酶进行胃肠道消化性预测。依据分子量小(<1000Da)、无毒、无致癌性、高生物活性(>0.5)和胃肠道不消化为标准,首次从小米醇溶蛋白水解物中筛选得到未经报道的生物活性肽WQHQY和WQHMMP(见表1)。
进一步运用计算机软件对肽WQHQY和WQHMMP的物理特性进行预测,其中总平均亲水性通过ExPasy(https://web.expasy.org/protparam/),等电点通过Pepdraw(http://www.tulane.edu/~biochem/WW/PepDraw/)评估。如表1所示,总平均亲水性可以用来表征蛋白质的亲疏水性,其中负值越大表明亲水性越强,结果表明肽WQHQY和WQHMMP具有较好的亲水性。肽WQHQY和WQHMMP的等电点大于7,表明它们均呈碱性。
表1基于生物信息学的小米醇溶蛋白肽性质预测
实施例6生物活性肽的人工合成
采用Fmoc固相合成法制备生物活性肽WQHQY和WQHMMP,具体如下:
(1)溶剂处理
N,N-二甲基甲酰胺(DMF)、甲醇在使用前用G3孔的分子筛浸泡过夜除杂质和水。
(2)树脂充分溶胀
称取2.0g空白Wang树脂于洁净干燥的反应管中,加入15mL DMF,室温活化30min。
(3)接第一个氨基酸
室温下,通过沙芯抽滤掉上步溶剂,加入1mmol 5倍摩尔过量的C端第一个氨基酸,5倍摩尔过量的DMAP,5倍摩尔过量的N,N-二异丙基碳二亚胺,DMF做溶剂室温反应3h。反应完毕用DMF洗4~6次,每次5-6mL。再加入体积比为1:1的吡啶和乙酸酐,反应30min。反应完毕用DMF洗4~6次,每次5-6mL。
(4)Fmoc保护基的离去
抽滤去掉③的溶剂,将10mL 20%的哌啶DMF溶液加入到树脂中,N2搅拌10min后滤出溶液,再加入10mL 20%的哌啶DMF溶液,N2吹动搅拌5min再滤去溶液,反复重复此操作两次后,用DMF洗4次,甲醇洗2次,每次5-6mL。
(5)茚三酮检测脱除效果
取出少量树脂,用甲醇洗三次,加入茚三酮、KCN、苯酚溶液各一滴,105℃-110℃加热5min,变深蓝色为阳性反应,说明脱除完全,即可进行下步反应;若呈无色,说明保护基没有脱除完全,则需要重复以上脱保护操作。
(6)接第二个氨基酸及Fmoc保护基脱除
称取3倍摩尔过量的C端第二个氨基酸,3倍摩尔过量的HBTU和3倍摩尔过量的1-羟基苯并三氮唑于反应管中,加入适量DMF溶液使其完全溶解后,再加入10倍摩尔过量的N,N-二异丙基乙胺,室温反应40min,用DMF洗4~6次,每次5-6mL。取少量树脂用茚三酮检测试剂检测,显无色,然后加入10mL 20%的哌啶DMF溶液脱Fmoc,进行两次,分别为10min、5min,之后用DMF洗4次,甲醇洗2次,每次5-6mL。取出少量树脂用茚三酮检测试剂检测,检测为蓝色,即可进行下一步反应。
(7)以此类推,重复(6)的步骤,直到合成到N端最后一个氨基酸,去掉Fmoc保护基,然后抽干。
(8)树脂的脱落及纯品分离检测
最后用三氟乙酸切割液(95%三氟乙酸:2%三异丙基硅烷:2%乙二硫醇:1%H2O)切割2h,抽滤反应液,得肽的三氟乙酸溶液,将裂解液用氮气吹干,再用乙醚沉淀、离心,然后用乙醚洗3~5次,得白色固体,用纯水溶解后,经HPLC脱盐提纯,冻干后析出晶体。
(9)肽的质量检测
取少量样品在超声溶解后,置于分析型高效液相色谱仪中检测。HPLC参数为:色谱柱:4.6×250mm,Sinochrom ODS-BP 5μm;流动相A:100%乙腈加0.1%三氟乙酸;流动相B:100%水加0.1%三氟乙酸;流速:1mL/min;进样量:5μL,检测波长:220nm。
对于WQHQY而言,梯度程序如下表:
对于WQHMMP而言,梯度程序如下表:
Agilent-6125B质谱参数:离子源为电喷雾离子化源(ESI源),雾化气流速:1.5L/min,CDL:-20.0V,CDL温度:250℃,加热块温度:200℃,离子源电压:+4.5kV,检测器电压:1.5kV,流动相流速:0.2mL/min,流动相比例:50%H2O/50%ACN。
最后通过高效液相色谱和质谱分析,确定肽WQHQY和WQHMMP的纯度均大于95%,具体的色谱和质谱结果分别见图2和图3。
实施例7生物活性肽的脂质消化酶抑制效果评价
(1)胰脂肪酶活性抑制实验
使用pH 7.3的磷酸缓冲液配制2.5mg/mL胰脂肪酶溶液,然后以5500rpm离心5min取上清液。使用pH 7.3的磷酸缓冲液稀释对硝基苯基丁酸酯至10mM。
将50μL 5mg/mL WQHQY/WQHMMP溶液(等量蒸馏水作为对照组)、40μL胰脂肪酶溶液和20μL对硝基苯丁酸酯溶液在37℃孵育30min后,酶标仪在405nm处记录吸光度。胰脂肪酶活性抑制率的计算公式如下:
式(1)中:A:对照吸光度;B:对照空白吸光度;C:样品吸光度;D:样品空白吸光度。(2)胆固醇酯酶活性抑制实验
使用pH 7.0的磷酸缓冲液(含100mM NaCl,5.16mM牛磺胆酸钠)分别配制25μg/mL胆固醇酯酶溶液和10mM对硝基苯基丁酸酯溶液。
将50μL 8mg/mL WQHQY/WQHMMP溶液(等量蒸馏水作为对照组)、50μL胆固醇酯酶溶液和50μL对硝基苯丁酸酯溶液在25℃孵育5min后,酶标仪在405nm处记录吸光度。胆固醇酯酶活性抑制率的计算公式如下:
式(2)中:A:对照吸光度;B:对照空白吸光度;C:样品吸光度;D:样品空白吸光度。
体外试验结果显示,生物活性肽WQHQY和WQHMMP的胰脂肪酶活性抑制率分别是66.77%和61.58%,胆固醇酯酶活性抑制率分别是45.75%和38.33%(图4)。这表明两个生物活性肽对胰脂肪酶和胆固醇酯酶均有较好的酶抑制活性。总之,这两个小米醇溶蛋白肽可有效预防、缓解和治疗高脂血症,并有望作为功能成分广泛应用于和高脂血症有关的食品、保健品以及药品中,具有良好的市场前景。
实施例8生物活性肽的脂质消化酶抑制机理分析
以筛选出的生物活性肽为配体,脂质消化酶(胰脂肪酶和胆固醇酯酶)为受体,通过分子对接技术明确生物活性肽与脂质消化酶之间的作用位点和相互作用力,进一步阐明抑制作用机理。
(1)肽WQHQY和WQHMMP进行胰脂肪酶分子对接试验
从RCSB蛋白数据库(http://www.rcsb.org/)获取胰脂肪酶(PDB编号:1ETH)的晶体结构,采用Dock 6.9将肽WQHQY和WQHMMP与胰脂肪酶进行半柔性对接,基于Grid打分函数进行能量评价。在分子对接前保留胰脂肪酶分子的链A(包含448个氨基酸残基)用于对接分析,同时去除共结晶分子和其他多肽链。分子对接以胰脂肪酶活性位点(Ser153、Asp177和His264)为中心,即坐标为X:64.152,Y:39.278,Z:127.241。对接打分是配体与大分子结合的近似势能,较低的得分值表明目的大分子与配体之间具有强亲和力。范德华力贡献指的是π-π堆积、疏水相互作用等非极性作用。静电力贡献则表现为盐桥、氢键等极性作用。对接打分是范德华力贡献和静电力贡献的总和。表2显示了肽WQHQY与胰脂肪酶的对接打分是-90.1657kcal/mol,范德华力贡献是-88.583kcal/mol,静电力贡献是-1.5827kcal/mol,内部排斥能是21.5718kcal/mol;肽WQHMMP与胰脂肪酶的对接打分是-99.0293kcal/mol,范德华力贡献是-98.6513kcal/mol,静电力贡献是-0.378kcal/mol,内部排斥能是25.9525kcal/mol。通常小于-50kcal/mol的对接打分值表示较好的结合力。内部排斥能数值越大,排斥力较大,表明该构象越不稳定,而内部排斥能小于30kcal/mol则表明构象较为稳定。两个生物活性肽与胰脂肪酶的对接打分均低于-50kcal/mol,内部排斥能也均低于30kcal/mol,这表明两者都能与胰脂肪酶形成稳定的结合状态。
分子对接模拟图5A展示了肽WQHQY与胰脂肪酶氨基酸残基的结合主要依赖于疏水相互作用、π-π堆积和氢键。具体地说,肽WQHQY与氨基酸残基(Phe216、Phe78、Leu265、Ile79、Val260、Trp253)形成疏水相互作用,与氨基酸残基(His264)形成π-π堆积,与氨基酸残基(Ser153、Asp80、Thr113)形成氢键。
分子对接模拟图5B展示了肽WQHMMP与胰脂肪酶氨基酸残基的结合主要依赖于疏水相互作用、盐桥和氢键。具体地说,肽WQHMMP与氨基酸残基(Phe216、Asn263)形成疏水相互作用,与氨基酸残基(Arg257、His152)形成盐桥,与氨基酸残基(Leu214)形成氢键。
胰脂肪酶N末端结构域含有酰基酶催化三联体Ser153、Asp177、His264以及底物结合残基Phe78、His152、Phe216的活性位点。故肽WQHQY通过π-π堆积和氢键占据催化位点,通过疏水相互作用占据底物结合位点来抑制胰脂肪酶的活性;肽WQHMMP通过疏水相互作用和氢键占据底物结合位点来抑制胰脂肪酶的活性。
(2)肽WQHQY和WQHMMP进行胆固醇酯酶分子对接试验
从RCSB蛋白数据库(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将肽WQHQY和WQHMMP与胆固醇酯酶进行半柔性对接,基于Grid打分函数进行能量评价。在分子对接前去除多余的水分子,同时分子对接以胆固醇酯酶活性位点(Ser194、Asp320和His435)为中心,即坐标为X:8.039,Y:-1.404,Z:21.544。表2显示了肽WQHQY与胆固醇酯酶的对接打分是-104.3077kcal/mol,范德华力贡献是-101.3097kcal/mol,静电力贡献是-2.9981kcal/mol,内部排斥能是19.4904kcal/mol;肽WQHMMP胆固醇酯酶的对接打分是-107.7835kcal/mol,范德华力贡献是-103.2768kcal/mol,静电力贡献是-4.5067kcal/mol,内部排斥能是28.2383kcal/mol。两个生物活性肽与胆固醇酯酶的对接打分均低于-50kcal/mol,内部排斥能也均低于30kcal/mol,这表明两者都能与胆固醇酯酶形成稳定的结合状态。
分子对接模拟图6A展示了肽WQHQY与胆固醇酯酶氨基酸残基的结合主要依赖于疏水相互作用、π-阳离子相互作用和氢键。具体地说,肽WQHQY与氨基酸残基(Ile439、Ala436、Ala108、Val285、Met281、Leu392、Phe324)形成疏水相互作用,与氨基酸残基(Lys445)形成π-阳离子相互作用,与氨基酸残基(Tyr453、Gly107)形成氢键。
分子对接模拟图6B展示了肽WQHMMP与胆固醇酯酶氨基酸残基的结合主要依赖于盐桥、疏水相互作用和氢键。具体地说,肽WQHMMP与氨基酸残基(Lys445)形成盐桥,与氨基酸残基(Ile439、Phe119、Ala108、Val285、Phe324、Met281)形成疏水相互作用,与氨基酸残基(Tyr453、Ser194)形成氢键。
胆固醇酯酶具有N端催化结构域,其中包含催化三联体Ser194、Asp320和His435。底物结合位点由与Ser194相邻的Gly107、Ala108和Ala195形成。肽WQHQY通过疏水相互作用和氢键占据底物结合位点;肽WQHMMP通过氢键占据催化位点,通过疏水相互作用占据底物结合位点来抑制胆固醇酯酶的活性。
表2两种生物活性肽的分子对接结果
/>
Claims (4)
1.小米醇溶蛋白肽,其特征在于,所述小米醇溶蛋白肽的氨基酸序列为SEQ ID NO.2。
2.根据权利要求1所述的小米醇溶蛋白肽在制备胰脂肪酶和/或胆固醇酯酶抑制剂中的应用。
3.制备根据权利要求1所述的小米醇溶蛋白肽的方法,其特征在于,所述方法为Fmoc固相合成方法。
4.根据权利要求3所述的方法,其中所述Fmoc固相合成方法包括(1)使用分子筛浸泡N,N-二甲基甲酰胺和甲醇以除杂;(2)N,N-二甲基甲酰胺溶胀活化Wang树脂;(3)接第一个氨基酸;(4)Fmoc保护基脱除;(5)接第二个氨基酸并脱除Fmoc保护基;(6)重复步骤(5)多次,直到合成到最后一个氨基酸并脱除Fmoc保护基;(7)树脂的脱落,通过液相和/或质谱方法进行检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311088956.7A CN117003845B (zh) | 2022-12-26 | 2022-12-26 | 小米醇溶蛋白肽及其在治疗高脂血症中的用途 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311088956.7A CN117003845B (zh) | 2022-12-26 | 2022-12-26 | 小米醇溶蛋白肽及其在治疗高脂血症中的用途 |
CN202211672882.7A CN115925854B (zh) | 2022-12-26 | 2022-12-26 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211672882.7A Division CN115925854B (zh) | 2022-12-26 | 2022-12-26 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117003845A CN117003845A (zh) | 2023-11-07 |
CN117003845B true CN117003845B (zh) | 2024-03-29 |
Family
ID=86553953
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311088956.7A Active CN117003845B (zh) | 2022-12-26 | 2022-12-26 | 小米醇溶蛋白肽及其在治疗高脂血症中的用途 |
CN202211672882.7A Active CN115925854B (zh) | 2022-12-26 | 2022-12-26 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211672882.7A Active CN115925854B (zh) | 2022-12-26 | 2022-12-26 | 两种抑制胰脂肪酶和胆固醇酯酶活性的小米醇溶蛋白肽 |
Country Status (1)
Country | Link |
---|---|
CN (2) | CN117003845B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117003845B (zh) * | 2022-12-26 | 2024-03-29 | 中国农业大学 | 小米醇溶蛋白肽及其在治疗高脂血症中的用途 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05339168A (ja) * | 1992-06-08 | 1993-12-21 | Suisanchiyou Chokan | 膵リパーゼおよびコレステロールエステラーゼ阻害剤 |
CN114634552A (zh) * | 2022-04-14 | 2022-06-17 | 中国农业大学 | 一种抗肥胖十三肽及其应用 |
CN115806589A (zh) * | 2022-12-09 | 2023-03-17 | 中国农业大学 | 两种小米源寡肽及其在治疗代谢综合征中的应用 |
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 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2239455B (en) * | 1989-12-25 | 1993-12-15 | Nisshin Flour Milling Co | Formulations of lipolytic enzyme inhibitors and use of said inhibitors |
JP2960947B2 (ja) * | 1989-12-25 | 1999-10-12 | 日清製粉株式会社 | 脂質分解酵素阻害剤 |
US20140356394A1 (en) * | 2011-12-09 | 2014-12-04 | Postech Academy-Industry Foundation | Composition for treatment or prevention of obesity, containing water extracts of fomitella fraxinea |
CN106573072A (zh) * | 2014-06-02 | 2017-04-19 | 阿尔莫生物科技股份有限公司 | 降低血清胆固醇的方法 |
CN114805483B (zh) * | 2022-04-02 | 2022-12-13 | 中国农业大学 | 五种来源于红小豆蛋白的胰脂肪酶抑制剂及其应用 |
CN114773430B (zh) * | 2022-04-14 | 2022-12-16 | 中国农业大学 | 三种具有胆固醇酯酶抑制活性的降胆固醇肽及其应用 |
-
2022
- 2022-12-26 CN CN202311088956.7A patent/CN117003845B/zh active Active
- 2022-12-26 CN CN202211672882.7A patent/CN115925854B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05339168A (ja) * | 1992-06-08 | 1993-12-21 | Suisanchiyou Chokan | 膵リパーゼおよびコレステロールエステラーゼ阻害剤 |
CN114634552A (zh) * | 2022-04-14 | 2022-06-17 | 中国农业大学 | 一种抗肥胖十三肽及其应用 |
CN115806589A (zh) * | 2022-12-09 | 2023-03-17 | 中国农业大学 | 两种小米源寡肽及其在治疗代谢综合征中的应用 |
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 |
Non-Patent Citations (2)
Title |
---|
Identification and characterization of cholesterol esterase and lipase inhibitory peptides from amaranth protein hydrolysates;Ajayi等;Food Chemistry: X;20211230;第12卷(第01期);1-11 * |
凡纳滨对虾蛋白体外降脂作用分析;龚受基等;食品工业科技;20200601;第41卷(第11期);316-321 * |
Also Published As
Publication number | Publication date |
---|---|
CN115925854A (zh) | 2023-04-07 |
CN115925854B (zh) | 2023-08-25 |
CN117003845A (zh) | 2023-11-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Gong et al. | Prospects of cereal protein-derived bioactive peptides: Sources, bioactivities diversity, and production | |
CN114805483B (zh) | 五种来源于红小豆蛋白的胰脂肪酶抑制剂及其应用 | |
CN117003845B (zh) | 小米醇溶蛋白肽及其在治疗高脂血症中的用途 | |
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 | |
CN111269290B (zh) | 一种鲟鱼抗炎肽的制备方法 | |
CN114716524B (zh) | 抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽 | |
CN114634552A (zh) | 一种抗肥胖十三肽及其应用 | |
CN116479077A (zh) | 高活性苦荞清蛋白降血压肽的制备方法 | |
CN115806589B (zh) | 两种小米源寡肽及其在治疗代谢综合征中的应用 | |
Chen et al. | Novel ACE inhibitory peptides derived from bighead carp (Aristichthys nobilis) hydrolysates: Screening, inhibition mechanisms and the bioconjugation effect with graphene oxide | |
Shao et al. | Peptides isolated from black soybean synergistically inhibit the activity of angiotensin converting enzyme (ACE) | |
CN114716523B (zh) | 具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽 | |
CN115838400B (zh) | 两种靶向预防或治疗代谢综合征的红小豆肽 | |
CN114891065A (zh) | 一种具有α-淀粉酶抑制活性的降血糖海参肽及其制备方法和应用 | |
CN115925799B (zh) | 一种具有降脂活性的小米寡肽 | |
Xiang et al. | Two novel angiotensin I-converting enzyme inhibitory peptides from garlic protein: In silico screening, stability, antihypertensive effects in vivo and underlying mechanisms | |
Liu et al. | Isolation and characterization of oligopeptides with vascular disease suppression effects derived from wheat gluten | |
CN116239652B (zh) | 三种红小豆来源的寡肽及其在控制肥胖和糖尿病中的应用 | |
Du et al. | Preparation, identification, and inhibitory mechanism of dipeptidyl peptidase IV inhibitory peptides from goat milk whey protein | |
JPS61171431A (ja) | アミラ−ゼ阻害物質およびその製造法 | |
JPH07224093A (ja) | ペプチド | |
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 | |
CN115677826B (zh) | 核桃乙酰胆碱酯酶抑制肽及其应用 | |
CN115010785B (zh) | 一种具有二肽基肽酶-4抑制活性的四肽及其应用 | |
CN117820431A (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 |