CN105906709A - Alaska Pollock fish skin active oligopeptides as well as synthesis method and application thereof - Google Patents
Alaska Pollock fish skin active oligopeptides as well as synthesis method and application thereof Download PDFInfo
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- CN105906709A CN105906709A CN201610327636.6A CN201610327636A CN105906709A CN 105906709 A CN105906709 A CN 105906709A CN 201610327636 A CN201610327636 A CN 201610327636A CN 105906709 A CN105906709 A CN 105906709A
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- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 31
- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 31
- 241001313700 Gadus chalcogrammus Species 0.000 title abstract 2
- 238000001308 synthesis method Methods 0.000 title abstract 2
- ONPXCLZMBSJLSP-CSMHCCOUSA-N Pro-Hyp Chemical group C1[C@H](O)C[C@@H](C(O)=O)N1C(=O)[C@H]1NCCC1 ONPXCLZMBSJLSP-CSMHCCOUSA-N 0.000 claims abstract description 36
- 108010017349 glycyl-prolyl-hydroxyproline Proteins 0.000 claims abstract description 31
- HVIBGVJOBJJPFB-OFQRNFBNSA-N Gly-Pro-Hyp Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)C(O)CC1 HVIBGVJOBJJPFB-OFQRNFBNSA-N 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000002347 injection Methods 0.000 claims abstract description 6
- 239000007924 injection Substances 0.000 claims abstract description 6
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 208000001132 Osteoporosis Diseases 0.000 claims abstract 3
- 206010003246 arthritis Diseases 0.000 claims abstract 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 60
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 59
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 49
- 239000011347 resin Substances 0.000 claims description 35
- 229920005989 resin Polymers 0.000 claims description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- 241001098054 Pollachius pollachius Species 0.000 claims description 32
- 239000012071 phase Substances 0.000 claims description 23
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 22
- 229920001184 polypeptide Polymers 0.000 claims description 20
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 15
- 238000007689 inspection Methods 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 11
- 239000003875 Wang resin Substances 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 11
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 11
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 230000008961 swelling Effects 0.000 claims description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- 150000003053 piperidines Chemical class 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 230000002265 prevention Effects 0.000 claims description 9
- GOUUPUICWUFXPM-XIKOKIGWSA-N (2s,4r)-1-(9h-fluoren-9-ylmethoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid Chemical compound C1[C@H](O)C[C@@H](C(O)=O)N1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 GOUUPUICWUFXPM-XIKOKIGWSA-N 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 8
- 238000010511 deprotection reaction Methods 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 150000002469 indenes Chemical class 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- NIPYQLPZPLBOLF-UHFFFAOYSA-N 3'-hydroxy-6'-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound OC1C(O)C(O)C(CO)OC1OC1=CC=C2C3(C4=CC=CC=C4C(=O)O3)C3=CC=C(O)C=C3OC2=C1 NIPYQLPZPLBOLF-UHFFFAOYSA-N 0.000 claims description 7
- 238000010189 synthetic method Methods 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000009833 condensation Methods 0.000 claims description 6
- 230000005494 condensation Effects 0.000 claims description 6
- 238000006482 condensation reaction Methods 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000011282 treatment Methods 0.000 claims description 4
- 150000002576 ketones Chemical class 0.000 claims description 2
- 230000020477 pH reduction Effects 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- 102000008186 Collagen Human genes 0.000 description 18
- 108010035532 Collagen Proteins 0.000 description 18
- 229920001436 collagen Polymers 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 9
- 229960003957 dexamethasone Drugs 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 102100031475 Osteocalcin Human genes 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000251468 Actinopterygii Species 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 230000008485 antagonism Effects 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 210000000963 osteoblast Anatomy 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 239000012925 reference material Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000004067 Osteocalcin Human genes 0.000 description 2
- 108090000573 Osteocalcin Proteins 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000001582 osteoblastic effect Effects 0.000 description 2
- 210000002997 osteoclast Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- ZPGDWQNBZYOZTI-UHFFFAOYSA-N 1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 108010048734 sclerotin Proteins 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses Alaska Pollock fish skin active oligopeptides as well as a synthesis method and an application thereof. The amino acid sequence of the oligopeptide is Pro-Hyp or Gly-Pro-Hyp, and the oligopeptide is synthesized by using a solid-phase synthesis method. The oligopeptide can be prepared into an injection liquid or an oral liquid for preventing and treating osteoporosis and arthritis.
Description
Technical field
The invention belongs to biologically active peptide field, be specifically related to a kind of wall pollack fish skin active oligopeptide and synthesis side thereof
Method and application.
Background technology
The gene of organism are stored on polynucleotide chain, and gene encode execution biology
The protein of function.Protein in organism is varied, and they are exercised various biological function and maintain
Vital movement.Although the kind of protein is unequal to lift more, but they are essentially all and are existed by 20 natures
Natural amino acid composition.Owing to amino acid whose composition and the difference that puts in order result in the thousand poor of protein
Ten thousand is other.In general, be referred to as protein containing more than 50 amino acid whose molecules, as more than 10 ammonia
The peptide chain of base acid is referred to as polypeptide, is referred to as oligopeptide less than 10 amino acid whose peptide chains.Have now been found that minimum
The little peptide of function only has 2 aminoacid, the most commonly little peptide of the function of more than 4.
Along with the development of China's sea fishery, processing of aquatic products process can produce substantial amounts of fish skin, fishbone, interior
Dirty the like waste, wall pollack fish is a kind of low form Fish living in North Pacific Region, China wall pollack fish year
Processing capacity about 500,000 tons, therefore can produce a large amount of wall pollack fish skin the like waste, and dry fish skin contains 70% collagen protein,
Collagen polypeptide is with collagen tissue as raw material.In recent years, the research of aquatic products collagen protein becomes domestic exterior-heat
Point, but the research to wall pollack collagen polypeptide rarely has report.Fan Jie etc. 2013 are in Affiliated Hospital of Qingdao University
Disclosing wall pollack fishskin collagen polypeptide physicochemical property research in journal, it is mainly studied 5 kinds of preparation technologies and obtains
The physicochemical property of wall pollack fishskin collagen polypeptide.But, for wall pollack fishskin polypeptide in prophylactic treatment disease etc.
The research of invention has no report, and lacks the artificial synthesis reporting such polypeptide.
Summary of the invention
For solving the problem that the existing concrete applied research of wall pollack fishskin collagen polypeptide lacks, the present invention proposes one
Wall pollack fish skin active oligopeptide, this active oligopeptide not only sequence understands, and has good medical efficacy.
The technical scheme is that and be achieved in that:
A kind of wall pollack fish skin active oligopeptide, the aminoacid sequence of described oligopeptide be Pro-Hyp or
Gly-Pro-Hyp。
It is a further object to provide a kind of wall pollack fish skin active oligopeptide at preparation prevention and treatment sclerotin
Application in terms of loose medicine.
Further, described medicine is injection or oral liquid.
It is a further object to provide a kind of wall pollack fish skin active oligopeptide in preparation prevention and treatment joint
Application in terms of scorching medicine.
Further, described medicine is injection or oral liquid.
The synthetic method of wall pollack fish skin active oligopeptide, the synthesis of described Pro-Hyp uses solid-phase synthesis, bag
Include following steps:
1) resin pretreatment: Fmoc-Hyp (tBu)-Wang resin is stirred in DCM so that it is fully
Swelling；
2) removing Fmoc protection group: add piperidines and DMF mixed solution in swelling resin；
3) connexon condensation: by Fmoc-Pro-COOH, HBTU, HOBT, DIEA of equimolar ratio
Be dissolved completely in DMF, after mixing, add step 2) resin of removing Fmoc radical protection is carried out anti-
Should；
4) indenes inspection: by step 3) solvent after condensation reaction terminates drains, washs with DMF, with indenes three
Ketone inspection is colourless, according to step 2) method removing Fmoc protection group, obtain the peptide resin of deprotection；
5) peptide chain cuts from resin: is acidified resin with 95wt%TFA aqueous solution, reacts 2-3 hour,
It is filtrated to get filtrate, in filtrate, adds ether separate out, obtain polypeptide crude product.
Further, described step 5) also include that polypeptide crude product is carried out high performance liquid chromatography is purified afterwards.
The synthetic method of wall pollack fish skin active oligopeptide, the synthesis of described Gly-Pro-Hyp uses solid-phase synthesis,
Comprise the following steps:
1) resin pretreatment: Fmoc-Hyp (tBu)-Wang resin is stirred in DCM so that it is fully
Swelling；
2) removing Fmoc protection group: add piperidines and DMF mixed solution in swelling resin；
3) connexon Pro condensation: by the Fmoc-Pro-COOH of equimolar ratio, HBTU, HOBT,
DIEA is dissolved completely in DMF, after mixing add step 2) removing Fmoc radical protection resin in enter
Row reaction；
4) indenes inspection for the first time: by step 3) solvent after condensation reaction terminates drains, washs with DMF,
Colourless, according to step 2 with 1,2,3-indantrione monohydrate inspection) method removing Fmoc protection group, obtain the Pro-Hyp of deprotection
Peptide resin；
5) connexon Gly condensation: by the Fmoc-Gly-COOH of equimolar ratio, HBTU, HOBT,
DIEA is dissolved completely in DMF, after mixing add step 4) deprotection Pro-Hyp peptide resin in
React；
6) indenes inspection for the second time: by step 5) solvent after condensation reaction terminates drains, washs with DMF,
Colourless, according to step 2 with 1,2,3-indantrione monohydrate inspection) method removing Fmoc protection group, obtain deprotection
Gly-Pro-Hyp peptide resin；
7) peptide chain cuts from resin: by 95wt%TFA aqueous solution acidification step 6) peptide resin, reaction
2-3 hour, it is filtrated to get filtrate, in filtrate, adds ether separate out, obtain polypeptide crude product.
Further, described step 7) also include that polypeptide crude product is carried out high performance liquid chromatography is purified afterwards.
It is also another object of the present invention to provide the separation method of a kind of wall pollack fish skin active oligopeptide, described
Pro-Hyp with Gly-Pro-Hyp uses Gradient Elution method to separate, and actual conditions is such as
Under: chromatographic column: venusic ASB-C18 (250mm × 4.6mm, 5um), column temperature: 40 DEG C, mobile phase A
For the acetonitrile solution of 0.1wt% trifluoroacetic acid, Mobile phase B is the aqueous solution of 0.1wt% trifluoroacetic acid, flow velocity:
1.0ml/min；Detection wavelength: 220nm；The time of gradient elution first stage is 0-20min, mobile phase A
Ratio be 2%-22%, the ratio of Mobile phase B is 78%-98%；The time of gradient elution second stage is
20.1-25min, the ratio of mobile phase A is 100%；The time of gradient elution phase III is 25.1-35min,
The ratio of mobile phase A is 2%, and the ratio of Mobile phase B is 98%.
The Chinese of the HBTU in the present invention is O-BTA-tetramethylurea hexafluorophosphoric acid ester,
The Chinese of HOBT is I-hydroxybenzotriazole, and the Chinese of DIEA is DIPEA,
DMF Chinese is dimethylformamide.
The method have the benefit that
1, to dexamethasone damaged bone cell OCN protein expression quantity research, wall pollack fish skin active oligopeptide egg
White expression is significantly raised compared with model group, and in dose dependent, prompting active oligopeptide can be filled on effectively antagonism ground
Rice pine causes osteoblastic damage, promotes it ripe and differentiation.
2, in statistical analysis model group blood supernatant, the content compared with normal group of Pro-Hyp significantly reduces
(p < 0.05), in prevention administration group, the content relatively model group of Pro-Hyp significantly raises (p < 0.01), prompting
The damage that Pro-Hyp possible antagonism dexamethasone causes has certain self-regeneration effect.
3, Pro-Hyp with Gly-Pro-Hyp uses Gradient Elution method to separate, point
From effective, the response rate is high.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to enforcement
In example or description of the prior art, the required accompanying drawing used is briefly described, it should be apparent that, describe below
In accompanying drawing be only some embodiments of the present invention, for those of ordinary skill in the art, do not paying
On the premise of going out creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is OCN protein expression result figure in Western Blot detection composition osteocyte；
Fig. 2 is the thick product of Gly-Pro-Hyp high-efficient liquid phase chromatogram spectrum for the first time；
Fig. 3 is Gly-Pro-Hyp thick product second time high-efficient liquid phase chromatogram spectrum；
Fig. 4 is the thick product of Pro-Hyp high-efficient liquid phase chromatogram spectrum for the first time；
Fig. 5 is Pro-Hyp thick product second time high-efficient liquid phase chromatogram spectrum.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clearly
Chu, be fully described by, it is clear that described embodiment be only a part of embodiment of the present invention rather than
Whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creation
The every other embodiment obtained under property work premise, broadly falls into the scope of protection of the invention.
Embodiment 1
The research that dexamethasone damage osteoblast is repaired by wall pollack fish skin active oligopeptide
Through 10-5After mol/L dexamethasone damage osteoblast 48h, its cell survival rate compared with normal group significantly drops
Low, only the 50.67% of normal group, (P < 0.05)；By 10 μm ol/L dexamethasone antagonists (RU-486)
2h preincubate osteoblast in advance, this group cell survival rate relatively model group significance raises, for
86.82% (P < 0.01)；Wall pollack collagen protein of fish skin peptide all can improve cell survival rate under test dose
(P<0.01), but group difference is without significance (P>0.05)；Small-molecular peptides group improves osteoclast survival rate up to
139.47%, far above three dosage groups of collagen peptide (P < 0.01), think the activity of collagen peptide
Molecule is tripeptides Gly-Pro-Hyp, the results are shown in Table 1.
Table 1 collagen peptide is to osteoclast survival rate effect
With normal group ratio, * P < 0.05；Respectively with model group ratio, * * P < 0.01；Compare with tripeptides group respectively,
##P<0.01。
Western Blot is utilized to detect OCN protein expression, dexamethasone model group osteoblast OCN egg
White expression compared with normal group significantly reduces (P < 0.05), after collagen peptide (three dosage) protection 48h,
This expressing quantity significantly raised compared with model group (P < 0.05), and in dose dependent, point out collagen peptide
Effectively can cause osteoblastic damage by antagonism dexamethasone, promote it ripe and differentiation；Gly-Pro-Hyp is pre-
Protection 48h group, OCN expressing quantity relatively collagen peptide group significantly raises (P < 0.01), sees Fig. 1.
With normal group ratio in Fig. 1, * P < 0.05；Respectively with dexamethasone model group ratio, #P < 0.05, ##P < 0.01；
Compare with tripeptides group respectively, * * P < 0.01.Conversion quantity: with wall pollack collagen protein of fish skin peptide molecular weight
3000Da counts, and Gly-Pro-Hyp molecular weight is 289.Clinic takes collagen peptide 5-10g, according to efficiently
Liquid chromatography testing result, converting as small active peptides amount is 2-4g.Here the full name of OCN is
Osteocalcin, Chinese is Bone Gla protein.
Embodiment 2
Blood-serum P ro-Hyp and the measurement result of Gly-Pro-Hyp content
In statistical analysis model group blood supernatant, the content compared with normal group of Pro-Hyp significantly reduces (p < 0.05),
In prevention administration group, the content relatively model group of Pro-Hyp significantly raises (p < 0.01), and prompting Pro-Hyp may
The damage that antagonism dexamethasone causes has certain self-regeneration effect；Additionally in prevention administration group
The content of Gly-Pro-Hyp the most relatively model group significance raises (p < 0.01), and model group compared with normal group significantly drops
Low (p < 0.05), prompting wall pollack collagen protein of fish skin peptide may be by being converted into the form quilt of Gly-Pro-Hyp
Absorb, refer to table 2.
Table 2 rat respectively organizes Pro-Hyp content in serum
With normal group ratio, #P < 0.05, and model group ratio, * * P < 0.01.
Embodiment 3
Gly-Pro-Hyp synthesis technique:
1), by swelling for Fmoc-Hyp (the tBu)-wang resin 0.7mmol/g 2g DCM that bought
Stand-by
2) the Fmoc group of Fmoc-Hyp (tBu)-wang resin, with 20% piperidines/DMF solution is sloughed,
Wash six times with DMF.
3), weigh 4.8mmol Fmoc-Pro-COOH, 4.8mmol HBTU, 4.8mmol HOBT,
4.8mmolDIEA 20ml DMF dissolves and is added to resin reaction 30min completely.
4), reaction 30min after pump reactant liquor, with DMF wash 6 times, with 1,2,3-indantrione monohydrate detection colourless, weight
Multiple 3rd step and the 4th step.
5) Fmoc of Fmoc-pro-Hyp (tBu)-wang resin, is sloughed with 20% piperidines/DMF solution
Group, washs 6 times with DMF.
6), weigh 4.8mmol Fmoc-Gly-COOH, 4.8mmol HBTU, 4.8mmol HOBT,
4.8mmol DIEA 20ml DMF dissolves and is added to resin reaction 30min completely.
7), reaction 30min after pump reactant liquor, with DMF wash 6 times, with 1,2,3-indantrione monohydrate detection colourless, weight
Multiple 6th step and the 7th step.
8) Fmoc of Fmoc-Gly-Pro-Hyp (tBu)-wang resin, is sloughed with 20% piperidines/DMF solution
Group, washs 6 times with DMF, and methanol washs 3 times, dry adsorbent
9), by TFA (trifluoroacetic acid) and water mixed solution (95%TFA), acidifying resin 2 hours, mistake
Filter off except resin, add 10 times of volume low temperature ether and polypeptide is separated out in TFA.Precipitation polypeptide is filtered and drains,
Carry out isolated and purified.
Purifying process:
Take 1mg thick product 1ml pure water ultrasonic dissolution, carry out thick product analysis, bar by high performance liquid chromatography
Part is: A (acetonitrile+0.1%TFA) B (pure water+0.1%TFA), A phase 0-25min (10-100%), inspection
Survey wavelength: 220nm, flow velocity 1ml min, sample size 20 microlitre, analyze collection of illustrative plates and see Fig. 2.
Take 400mg thick product water dissolution, converse preparative hplc condition by the retention time drawn: condition
For: A (acetonitrile) B (pure water+0.1%TFA), A phase 0-30min (2-12%), detect wavelength: 220nm,
Flow velocity 30ml min, sample size 400mg, analyze collection of illustrative plates and see Fig. 3.
Send Mass Spectrometric Identification molecular weight the fraction collected, liquid correct for mass spectrum is analyzed Liquid Detection purity.
Testing conditions is as follows: A (acetonitrile+0.1%TFA) B (pure water+0.1%TFA), A phase 0-25min (2-27%),
Detection wavelength: 220nm, flow velocity 1ml min, sample size 20 microlitre.
Embodiment 4
Pro-Hyp synthesis technique:
1), by swelling for Fmoc-Hyp (the tBu)-wang resin 0.7mmol/g 2g DCM that bought
Stand-by；
2) the Fmoc group of Fmoc-Hyp (tBu)-wang resin, with 20% piperidines/DMF solution is sloughed,
Wash six times with DMF；
3), weigh 4.8mmol Fmoc-Pro-COOH, 4.8mmol HBTU, 4.8mmol HOBT,
4.8mmol DIEA 20mlDMF dissolves and is added to resin reaction 30min completely.
4), reaction 30min after pump reactant liquor, with DMF wash 6 times, with 1,2,3-indantrione monohydrate detection colourless, weight
Multiple 3rd step and the 4th step.
5) Fmoc of Fmoc-Pro-Hyp (tBu)-wang resin, is sloughed with 20% piperidines/DMF solution
Group, washs 6 times with DMF, and methanol washs 3 times, and dry adsorbent is repeated once again.
6), with TFA and water mixed solution (95%TFA), acidifying resin 2 hours, filter and remove resin,
Add 10 times of volume ether to be separated out in TFA by polypeptide.Precipitation polypeptide is filtered and drains, carry out isolated and purified.
Purifying process:
Take 1mg thick product 1ml pure water ultrasonic dissolution.Thick product analysis, bar is carried out by high performance liquid chromatography
Part is: A (acetonitrile+0.1%TFA) B (pure water+0.1%TFA), A phase 0-25min (10-100%), inspection
Survey wavelength: 220nm, flow velocity 1 min, sample size 20 microlitre, analyze collection of illustrative plates and see Fig. 4.
Take 400mg thick product water dissolution, converse preparative hplc condition by the retention time drawn: condition
For: A (acetonitrile) B (pure water+0.1%TFA), A phase 0-30min (2-7%), detect wavelength: 220nm,
Flow velocity 30ml min, sample size 400mg, analyze collection of illustrative plates and see Fig. 5.
Send Mass Spectrometric Identification molecular weight the fraction collected, liquid correct for mass spectrum is analyzed Liquid Detection purity.
Testing conditions is as follows: A (acetonitrile+0.1%TFA), B (pure water+0.1%TFA), A phase 0-25min (2-27%),
Detection wavelength: 220nm, flow velocity 1ml min, sample size 20 microlitre.
Sample rotary evaporator qualified for detection is concentrated, and concentrated solution is upper freeze dryer after cryogenic refrigerator pre-freeze
Vacuum lyophilization.Weigh, quality inspection.
Embodiment 5
Pro-Hyp with Gly-Pro-Hyp separates
1, chromatographic condition
Chromatographic column: venusic ASB-C18 (250mm × 4.6mm, 5um)；Column temperature: 40 DEG C；Flowing phase:
Gradient elution (0-20min: acetonitrile (0.1% trifluoroacetic acid) A:2%-22%, pure water (0.1% trifluoroacetic acid)
B:98%-78%；20.1-25min: acetonitrile (0.1% trifluoroacetic acid) A:100%, pure water (0.1% trifluoro
Acetic acid) B:0%；25.1-35min: acetonitrile (0.1% trifluoroacetic acid) A:2%, pure water (0.1% trifluoro
Acetic acid) B:98%)；Flow velocity: 1.0ml/min；Detection wavelength: 220nm.
2, detection method
Take said method obtain ultrafiltrate according to each 50 μ l of above-mentioned chromatographic condition auto injection, according to peak area
Integrated value calculates the concentration of test sample.
3, linear relationship the result
The preparation of Pro-Hyp standard reference material solution: precision weighs 100mg Pro-Hyp (purity ＞ 99%)
Reference substance is dissolved in 1ml distilled water, successively with distilled water be diluted to concentration be respectively 0.5mg/ml, 0.25
Mg/ml, 0.1mg/ml, 0.05mg/ml, 0.01mg/ml, 0.005mg/ml gradient solution.
The preparation of Gly-Pro-Hyp standard reference material solution: precision weighs 100mg Pro-Hyp (purity ＞
99%) during reference substance is dissolved in 1ml distilled water, successively with distilled water be diluted to concentration be respectively 1mg/ml, 0.5
Mg/ml, 0.25mg/ml, 0.1mg/ml, 0.05mg/ml, 0.025mg/ml gradient solution.
Above-mentioned Pro-Hyp and Gly-Pro-Hyp standard reference material gradient solution is entered automatically according to this chromatographic condition
The each 50 μ l of sample, calculate each integrating peak areas value, with standard concentration as abscissa (X), and integrating peak areas value
For vertical coordinate (Y).Obtain Pro-Hyp regression equation: y=7E+06x, R2=0.9999, result shows: Pro-Hyp
Concentration in 0.005mg/ml～0.5mg/ml and its integrating peak areas value have good linear relationship；Obtain
Gly-Pro-Hyp regression equation: y=2E+07x, R2=0.9998, result shows: the concentration of Gly-Pro-Hyp
Good linear relationship is had with its integrating peak areas value in 0.01mg/ml～1mg/ml.
4, average recovery the result
(Pro-Hyp and Gly-Pro-Hyp concentration is respectively to take each 100 μ l of blood supernatant test sample 9 parts
0.045mg/ml and 0.075mg/ml), each addition 45 μ l Pro-Hyp and 75 μ l in low concentration group
Gly-Pro-Hyp；Each addition 90 μ l Pro-Hyp and 150 μ l Gly-Pro-Hyp in middle concentration group；High concentration group
In each add 135 μ l Pro-Hyp and 225 μ l Gly-Pro-Hyp.The average recovery rate of Pro-Hyp variable concentrations
Being respectively 99.26%, 98.77 and 99.07, RSD is respectively 1.71,2.63 and 2.33；Gly-Pro-Hyp
The average recovery rate of variable concentrations is respectively 98.89%, 98.22% and 99.57%, and RSD is respectively 1.70,
1.20 and 0.84；The results are shown in Table 3 and table 4.Show that the average recovery of Pro-Hyp and Gly-Pro-Hyp is good
Good.
Table 3 Pro-Hyp average recovery result
Table 4 Gly-Pro-Hyp average recovery result
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all at this
Within bright spirit and principle, any modification, equivalent substitution and improvement etc. made, should be included in this
Within bright protection domain.
Claims (10)
1. a wall pollack fish skin active oligopeptide, it is characterised in that the aminoacid sequence of described oligopeptide is Pro-Hyp
Or Gly-Pro-Hyp.
2. the wall pollack fish skin active oligopeptide described in a claim 1 is at preparation prevention and treatment osteoporosis drug
Application in terms of thing.
Wall pollack fish skin active oligopeptide the most according to claim 2 is at preparation prevention and treatment osteoporosis drug
Application in terms of thing, it is characterised in that described medicine is injection or oral liquid.
4. the wall pollack fish skin active oligopeptide described in a claim 1 is at preparation prevention and medicament for treating arthritis
The application of aspect.
Wall pollack fish skin active oligopeptide the most according to claim 4 is at preparation prevention and medicament for treating arthritis
The application of aspect, it is characterised in that described medicine is injection or oral liquid.
6. the synthetic method of wall pollack fish skin active oligopeptide as claimed in claim 1, it is characterised in that described
The synthesis of Pro-Hyp uses solid-phase synthesis, comprises the following steps:
1) resin pretreatment: Fmoc-Hyp (tBu)-Wang resin is stirred in DCM so that it is fully
Swelling；
2) removing Fmoc protection group: add piperidines and DMF mixed solution in swelling resin；
3) connexon condensation: by Fmoc-Pro-COOH, HBTU, HOBT, DIEA of equimolar ratio
Be dissolved completely in DMF, after mixing, add step 2) resin of removing Fmoc radical protection is carried out anti-
Should；
4) indenes inspection: by step 3) solvent after condensation reaction terminates drains, washs with DMF, with indenes three
Ketone inspection is colourless, according to step 2) method removing Fmoc protection group, obtain the peptide resin of deprotection；
5) peptide chain cuts from resin: is acidified resin with 95wt%TFA aqueous solution, reacts 2-3 hour,
It is filtrated to get filtrate, in filtrate, adds ether separate out, obtain polypeptide crude product.
The synthetic method of wall pollack fish skin active oligopeptide the most according to claim 6, it is characterised in that institute
State step 5) also include that polypeptide crude product is carried out high performance liquid chromatography is purified afterwards.
8. the synthetic method of wall pollack fish skin active oligopeptide as claimed in claim 1, it is characterised in that described
The synthesis of Gly-Pro-Hyp uses solid-phase synthesis, comprises the following steps:
1) resin pretreatment: Fmoc-Hyp (tBu)-Wang resin is stirred in DCM so that it is fully
Swelling；
2) removing Fmoc protection group: add piperidines and DMF mixed solution in swelling resin；
3) connexon Pro condensation: by the Fmoc-Pro-COOH of equimolar ratio, HBTU, HOBT,
DIEA is dissolved completely in DMF, after mixing add step 2) removing Fmoc radical protection resin in enter
Row reaction；
4) indenes inspection for the first time: by step 3) solvent after condensation reaction terminates drains, washs with DMF,
Colourless, according to step 2 with 1,2,3-indantrione monohydrate inspection) method removing Fmoc protection group, obtain the Pro-Hyp of deprotection
Peptide resin；
5) connexon Gly condensation: by the Fmoc-Gly-COOH of equimolar ratio, HBTU, HOBT,
DIEA is dissolved completely in DMF, after mixing add step 4) deprotection Pro-Hyp peptide resin in
React；
6) indenes inspection for the second time: by step 5) solvent after condensation reaction terminates drains, washs with DMF,
Colourless, according to step 2 with 1,2,3-indantrione monohydrate inspection) method removing Fmoc protection group, obtain deprotection
Gly-Pro-Hyp peptide resin；
7) peptide chain cuts from resin: by 95wt%TFA aqueous solution acidification step 6) peptide resin, reaction
2-3 hour, it is filtrated to get filtrate, in filtrate, adds ether separate out, obtain polypeptide crude product.
The synthetic method of wall pollack fish skin active oligopeptide the most according to claim 8, it is characterised in that institute
State step 7) also include that polypeptide crude product is carried out high performance liquid chromatography is purified afterwards.
10. the separation method of wall pollack fish skin active oligopeptide as claimed in claim 1, it is characterised in that institute
Stating Pro-Hyp with Gly-Pro-Hyp uses Gradient Elution method to separate, actual conditions
As follows: chromatographic column: venusic ASB-C18 (250mm × 4.6mm, 5um), column temperature: 40 DEG C, flow phase
A is the acetonitrile solution of 0.1wt% trifluoroacetic acid, and Mobile phase B is the aqueous solution of 0.1wt% trifluoroacetic acid, flow velocity:
1.0ml/min；Detection wavelength: 220nm；The time of gradient elution first stage is 0-20min, mobile phase A
Ratio be 2%-22%, the ratio of Mobile phase B is 78%-98%；The time of gradient elution second stage is
20.1-25min, the ratio of mobile phase A is 100%；The time of gradient elution phase III is 25.1-35min,
The ratio of mobile phase A is 2%, and the ratio of Mobile phase B is 98%.
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| CN109776652A (en) * | 2019-01-30 | 2019-05-21 | 浙江省医学科学院 | Cod skin oligopeptides and its isolation and purification method and preparing the application in ɑ-glucosidase inhibitor and type II diabetes resisting drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106929488A (en) * | 2017-03-29 | 2017-07-07 | 中南民族大学 | A kind of COX with bioactivity52‑69Solid phase synthesis process of polypeptide and application thereof |
| CN107759660A (en) * | 2017-12-05 | 2018-03-06 | 陕西慧康生物科技有限责任公司 | A kind of liquid-solid phase synthetic method of tripeptides 29 |
| CN108864249A (en) * | 2018-06-19 | 2018-11-23 | 南京肽业生物科技有限公司 | A kind of purification process of hydrophobic peptides |
| CN109355344A (en) * | 2019-01-08 | 2019-02-19 | 中国科学院烟台海岸带研究所 | A kind of preparation method of antihypertensive activity peptide |
| CN109776652A (en) * | 2019-01-30 | 2019-05-21 | 浙江省医学科学院 | Cod skin oligopeptides and its isolation and purification method and preparing the application in ɑ-glucosidase inhibitor and type II diabetes resisting drug |
| CN109776652B (en) * | 2019-01-30 | 2020-08-18 | 浙江省医学科学院 | Codfish skin oligopeptide, separation and purification method thereof, and application of codfish skin oligopeptide in preparation of alpha-glucosidase inhibitor and anti-type II diabetes drug |
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