CN106518971A - Anti-obesity decapeptide CANPHELPNK - Google Patents
Anti-obesity decapeptide CANPHELPNK Download PDFInfo
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- CN106518971A CN106518971A CN201611103465.5A CN201611103465A CN106518971A CN 106518971 A CN106518971 A CN 106518971A CN 201611103465 A CN201611103465 A CN 201611103465A CN 106518971 A CN106518971 A CN 106518971A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The invention discloses anti-obesity decapeptide CANPHELPNK. The amino acid sequence of the decapeptide is shown as follows: Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys and is abbreviated to CANPHELPNK, the molecular weight of the decapeptide is 1122.27, and the purity is 97.55%. The decapeptide is synthesized by a polypeptide synthesizer with a solid-phase synthesis method. In-vitro mouse pre-adipocyte 3T3-L1 proliferation inhibition activity detection indicates that the decapeptide has a certain inhibiting effect on 3T3-L1 in the range of 0.125-2 mg/mL. The in-vitro proliferation inhibiting rate for 3T3-L1 is 59.51% when the concentration is 2 mg/mL. The synthetic decapeptide with potential in-vitro anti-obesity activity can be used for the field of biopharmacy.
Description
Technical field
The invention belongs to field of biological pharmacy, and in particular to a kind of anti-fat decapeptide and its application.
Background technology
Biologically active peptide is that the function on body or state have positive role and finally affect the special egg of body health
White matter fragment.For protein, the superiority of small-molecular peptides fragment is mainly reflected in：It is more easy to the utilization that is absorbed by the body；
It is active high, its distinctive physiological action can be played under less concentration；Molecular weight is little, it is easy to modification and transformation, can pass through
Artificial chemistry synthesis etc..And compared to single amino acid for, small-molecular peptides in addition to special physiologically active, inhale
Receive.Confirmation is studied, human small intestine exists special
Oligopeptide absorbing path, human body intake protein through the hydrolysis of various digestive ferments, mainly absorbed in the form of low peptide.
Many studies have shown that, the biologically active peptide in various sources has various works such as anti-oxidant, antitumor, antibacterial, step-down, hypoglycemic
With, become biological medicine and health products exploitation focus.Fat increase atherosclerotic, coronary heart disease, hypertension, diabetes,
Gout, the initiation potential of fatty liver diseases.Therefore, opening to the biologically active peptide safe and harmless with fat-reducing and antihyperglycemic
Research is sent out, becomes particularly important.At present, the polypeptide with fat-reducing and antihyperglycemic includes Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], perch active peptide, silkworm chrysalis peptide
Deng.
The content of the invention
It is research object that the present invention chooses murine preadipocyte cell 3T3-L1, determines the external suppression of synthetic peptide using mtt assay
System activity.It is an object of the invention to provide a kind of synthesis polypeptide with external anti-obesity activity, can be applicable to bio-pharmaceuticals neck
Domain.
The anti-fat decapeptide of present invention synthesis is abbreviated as CANPHELPNK, molecular weight 1122.27, and sequence is：Cys-Ala-
Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.Wherein,
Cys represents English name for Cysteine, corresponding residue of the Chinese for the amino acid of cysteine;
Ala represents English name for Alanine, corresponding residue of the Chinese for the amino acid of alanine;
Asn represents English name for Asparagine, corresponding residue of the Chinese for the amino acid of asparagine acid;
Pro represents English name for Proline, corresponding residue of the Chinese for the amino acid of proline；
His represents English name for Histidine, corresponding residue of the Chinese for the amino acid of histidine;
Glu represents English name for Glutamic acid, corresponding residue of the Chinese for the amino acid of glutamic acid;
Leu represents that English name is Leucine, and Chinese is the corresponding residue of leucic amino acid;
Pro represents English name for Proline, corresponding residue of the Chinese for the amino acid of proline；
Asn represents English name for Asparagine, corresponding residue of the Chinese for the amino acid of asparagine acid;
Lys represents English name for Lysine, corresponding residue of the Chinese for the amino acid of lysine.
Amino acid sequence of the present invention adopts standard Fmoc scheme, and by the screening of resin, rational polypeptide is closed
Into method.The C- end carboxyls of target polypeptides are connected with an insoluble macromolecule resin with covalent bond form, then with this
The amino of individual amino acid acts on forming peptide bond with the carboxyl of another molecule amino acid as starting point.Constantly repeat this process, i.e.,
Target polypeptides product can be obtained.After the completion of synthetic reaction, protection group is removed, peptide chain is separated with resin, that is, obtain target product
Thing.Peptide systhesis be one repetition add amino acid process, synthesis in solid state order from C-terminal to N ends synthesize.
The synthesis polypeptide of final concentration of 0.125-2 mg/mL is mixed by the present invention with 3T3-L1, after 48 h of incubation, Jing MTT
Method detects, reaches 32.63%-59.51% to adipocyte inhibiting rate, can apply in biomedicine field.
The decapeptide CANPHELPNK concentration be 2 mg/mL when, to the in-vitro multiplication inhibiting rate of 3T3-L1 be
59.51%。
Compared with prior art, the invention has the advantages that and technique effect：
The present invention has synthesized the peptide first, and the external lipocyte proliferation that have detected synthesis polypeptide using MTT methods suppresses
Activity, the synthesis polypeptide have certain adipocyte rejection ability.
Description of the drawings
Fig. 1 a are schemed for the HPLC of synthesis polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.
Fig. 1 b are schemed for the MS of synthesis polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.
Fig. 2 is synthesis polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys to adipocyte 3T3-L1
Inhibitory activity block diagram.
Specific embodiment
Below in conjunction with instantiation, the invention will be further described, but the present invention enforcement and protection domain be not limited to
This.
Solid-phase synthesis peptides
From macromolecule resin（Chinese Peptide Co., Ltd.）, according to amino acid sequence Cys-Ala-Asn-Pro-His-Glu-
The carboxyl of Lys is first connected in the form of covalent bond, then the ammonia of Asn by the feature of Leu-Pro-Asn-Lys with a resin
The carboxyl of base and Lys shrinks and reacts, and after process, then adds Pro, the carboxyl reaction of the amino and Asn of Pro, successively from right to left
Addition amino acid, plus after good last Cys amino acid, then cut off resin and obtain target polypeptides.Using high performance liquid chromatography
Purified, chromatographic column model Phenomenex C18, size 4.6*150mm, mobile phase A:Containing 0.1% trifluoroacetic acid
（TFA）Water；Mobile phase B:Solution containing 0.09%TFA (+20% water of 80% acetonitrile)；In 20 min, B phases are risen by 14.0%
To 24.0%, 1.0 mL/min of flow velocity, Detection wavelength 220nm.Liquid nitrogen flash freezer, freeze-drying obtain last product, it is desirable to pure
Degree reaches more than 98.06%, and Jing MS identification structures（As shown in Fig. 1）.
The external inhibitory activity of synthesis polypeptide
By growth inhibition effect of the MTT colorimetrically analysings peptide composition to 3T3-L1.Concrete operation step is as follows：
1）Take the logarithm the cell in growth period, Jing 0.25%（Volume）The digestion of trypsase-EDTA digestive juices after, add corresponding
Complete medium terminates digestion re-suspended cell, after blood cell plate count, adjusts the concentration of cell suspension to 5 × 104Individual/mL,
Add in 96 orifice plates, per 100 L of hole, in 37 DEG C of constant temperature CO2Cultivate in incubator；
2）Cell attachment after 24 h is cultivated, waste and old nutrient solution is suctioned out, adds final volume to be the to be measured containing variable concentrations of 200 L
The fresh complete medium of sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO2Cultivate in incubator；
3）Liquid is suctioned out after 48 h, with PBS board-washings 2 times, 20 l of MTT solution and fresh complete medium of 5 mg/mL is added
180 µL；Continue culture in 37 DEG C of constant temperature CO2 incubators；
4）After 4 h, the nutrient solution containing MTT is discarded, adds 150 l DMSO that 15 min are vibrated on microoscillator,
OD value is determined at 490nm wavelength and inhibiting rate is calculated:
Adipocytic cell growth inhibiting rate (%)=((control group OD- blank groups OD)-(administration group OD- blank group OD))/((control group
OD- blank groups OD)) × 100.
Application Example 1
100 L cell suspensions (5 × 10 of adipocyte 3T3-L14Individual/mL), add in 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Cultivate in foster case, cell attachment after 24 h, suction out waste and old nutrient solution, add the polypeptide of 125 g/mL that final volume is 100 L
The fresh complete medium of sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO2Cultivate in incubator, 48 h
After suction out liquid, with PBS board-washings 2 times, add 180 L of 20 l of MTT solution and fresh complete medium of 5 mg/mL；In 37
DEG C constant temperature CO2Continue culture in incubator；After 4 h, the nutrient solution containing MTT is discarded, add 150 l DMSO to shake after miniature
Swing, OD value is determined at 490nm wavelength and inhibiting rate is calculated.As shown in Figure 2,125 g/mL
Polypeptide is 32.63% to the inhibiting rate of adipocyte 3T3-L1.
Application Example 2
100 L cell suspensions (5 × 10 of adipocyte 3T3-L14Individual/mL), add in 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Cultivate in foster case, cell attachment after 24 h, suction out waste and old nutrient solution, add the polypeptide of 250 g/mL that final volume is 100 L
The fresh complete medium of sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO2Cultivate in incubator, 48
Liquid is suctioned out after h, with PBS board-washings 2 times, 180 L of 20 l of MTT solution and fresh complete medium of 5 mg/mL is added；In
37 DEG C of constant temperature CO2Continue culture in incubator；After 4 h, the nutrient solution containing MTT is discarded, 150 l DMSO are added after micro-
15 min are vibrated on type oscillator, OD value is determined at 490nm wavelength and inhibiting rate is calculated.As shown in Figure 2,250 g/
The polypeptide of mL is 42.28% to the inhibiting rate of adipocyte 3T3-L1.
Application Example 3
100 L cell suspensions (5 × 10 of adipocyte 3T3-L14Individual/mL), add in 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Cultivate in foster case, cell attachment after 24 h, suction out waste and old nutrient solution, add the polypeptide of 500 g/mL that final volume is 100 L
The fresh complete medium of sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO2Cultivate in incubator, 48 h
After suction out liquid, with PBS board-washings 2 times, add 20 l of MTT solution and fresh 180 L of base complete medium of 5 mg/mL；In
37 DEG C of constant temperature CO2Continue culture in incubator；After 4 h, the nutrient solution containing MTT is discarded, 150 l DMSO are added after micro-
15 min are vibrated on type oscillator, OD value is determined at 490nm wavelength and inhibiting rate is calculated.As shown in Figure 2,500 g/
The polypeptide of mL is 46.1% to the inhibiting rate of adipocyte 3T3-L1.
Application Example 4
100 L cell suspensions (5 × 10 of adipocyte 3T3-L14Individual/mL), add in 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Cultivate in foster case, cell attachment after 24 h, suction out waste and old nutrient solution, addition final volume is many of 1000 g/mL of 100 L
The fresh complete medium of peptide sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO2Cultivate in incubator, 48
Liquid is suctioned out after h, with PBS board-washings 2 times, 180 L of 20 l of MTT solution and fresh complete medium of 5 mg/mL is added；In
Continue culture in 37 DEG C of constant temperature CO2 incubators；After 4 h, the nutrient solution containing MTT is discarded, 150 l DMSO are added after micro-
15 min are vibrated on type oscillator, OD value is determined at 490nm wavelength and inhibiting rate is calculated.As shown in Figure 2,1000 g/
The polypeptide of mL is 48.36% to the inhibiting rate of adipocyte 3T3-L1.
Application Example 5
100 L cell suspensions (5 × 10 of adipocyte 3T3-L14Individual/mL), add in 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Cultivate in foster case, cell attachment after 24 h, suction out waste and old nutrient solution, addition final volume is many of 2000 g/mL of 100 L
The fresh complete medium of peptide sample, and with complete medium as negative control, cultivate in 37 DEG C of constant temperature CO2 incubators,
Liquid is suctioned out after 48 h, with PBS board-washings 2 times, 180 L of 20 l of MTT solution and fresh complete medium of 5 mg/mL is added；
In 37 DEG C of constant temperature CO2Continue culture in incubator；After 4 h, discard the nutrient solution containing MTT, add 150 l DMSO after
15 min are vibrated on microoscillator, OD value is determined at 490nm wavelength and inhibiting rate is calculated.As shown in Figure 2,2000
The polypeptide of g/mL is 59.51% to the inhibiting rate of adipocyte 3T3-L1.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University
<120>A kind of anti-fat decapeptide CANPHELPNK
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213>Artificial sequence
<400> 1
Cys Ala Asn Pro His Glu Leu Pro Asn Lys
1 5 10
Claims (3)
1. a kind of anti-fat decapeptide CANPHELPNK, is characterized in that the amino acid sequence of decapeptide CANPHELPNK is Cys-Ala-
Asn-Pro-His-Glu-Leu-Pro-Asn-Lys。
2. a kind of anti-fat decapeptide CANPHELPNK as described in claim 1, it is characterised in that the decapeptide CANPHELPNK
When concentration is 0.125-2 mg/mL, it is 32.63%-59.51% to the in-vitro multiplication inhibiting rate of PECTORAL LIMB SKELETON 3T3-L1.
3. a kind of anti-fat decapeptide CANPHELPNK according to claim 2, it is characterised in that the decapeptide
CANPHELPNK, when concentration is 2 mg/mL, is 59.51% to the in-vitro multiplication inhibiting rate of 3T3-L1.
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| CN201611103465.5A CN106518971B (en) | 2016-12-05 | 2016-12-05 | Anti-obesity decapeptide CANPHELPNK |
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| CN201611103465.5A CN106518971B (en) | 2016-12-05 | 2016-12-05 | Anti-obesity decapeptide CANPHELPNK |
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| CN106518971A true CN106518971A (en) | 2017-03-22 |
| CN106518971B CN106518971B (en) | 2019-12-10 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676073A (en) * | 2018-06-07 | 2018-10-19 | 华南理工大学 | The anti-obesity decapeptide LLVVYPWTQR of one kind and its application |
| CN109081862A (en) * | 2018-06-07 | 2018-12-25 | 华南理工大学 | The anti-obesity tetrapeptide PQTR of one kind and its application |
| CN114315965A (en) * | 2021-12-22 | 2022-04-12 | 华南理工大学 | Anti-obesity peptides and uses thereof |
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| JP2005220074A (en) * | 2004-02-05 | 2005-08-18 | Fancl Corp | Inhibitor of fat-cell differentiation |
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| US5525705A (en) * | 1995-01-31 | 1996-06-11 | Eli Lilly And Company | Anti-obesity proteins |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676073A (en) * | 2018-06-07 | 2018-10-19 | 华南理工大学 | The anti-obesity decapeptide LLVVYPWTQR of one kind and its application |
| CN109081862A (en) * | 2018-06-07 | 2018-12-25 | 华南理工大学 | The anti-obesity tetrapeptide PQTR of one kind and its application |
| CN108676073B (en) * | 2018-06-07 | 2021-10-26 | 华南理工大学 | Anti-obesity decapeptide LLVVYPWTQR and application thereof |
| CN114315965A (en) * | 2021-12-22 | 2022-04-12 | 华南理工大学 | Anti-obesity peptides and uses thereof |
| CN114315965B (en) * | 2021-12-22 | 2023-05-12 | 华南理工大学 | Anti-obesity peptides and uses thereof |
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| Publication number | Publication date |
|---|---|
| CN106518971B (en) | 2019-12-10 |
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