CN106749533A - A kind of anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR - Google Patents
A kind of anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR Download PDFInfo
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- CN106749533A CN106749533A CN201611103461.7A CN201611103461A CN106749533A CN 106749533 A CN106749533 A CN 106749533A CN 201611103461 A CN201611103461 A CN 201611103461A CN 106749533 A CN106749533 A CN 106749533A
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- lnnpsvcdcdcmmkaar
- polypeptide
- fat
- amino acid
- cys
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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
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Abstract
The invention discloses the anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR of one kind, the amino acid sequence of the synthesis polypeptide is as follows:Leu Asn Asn Pro Ser Val Cys Asp Cys Asp Cys Met Met Lys Ala Ala Arg, are abbreviated as LNNPSVCDCDCMMKAAR, molecular weight 1871.24, purity 98.06%.Polypeptide of the invention uses Peptide synthesizer, is synthesized using solid-phase synthesis.Detect that in the range of 0.125 2 mg/mL, polypeptide of the invention presents certain inhibition to 3T3 L1 by external murine preadipocyte cell 3T3 L1 proliferation inhibition activities.In 2 mg/mL, the in-vitro multiplication inhibiting rate to 3T3 L1 is 33.96%.The present invention provides a kind of synthesis polypeptide with potential external anti-obesity activity, can be applied to field of biological pharmacy.
Description
Technical field
The invention belongs to field of biological pharmacy, and in particular to a kind of anti-fat ten heptapeptide and its application.
Background technology
Biologically active peptide is the special egg for having positive role on the function or state of body and finally influenceing 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 smaller concentration;Molecular weight is small, 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
Receiving also has the unrivaled superiority of amino acid in passage and infiltration rate.Confirmation is studied, human small intestine exists special
Oligopeptide absorbing path, human body intake protein by 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 as the focus that biological medicine and health products are developed.Obesity increase atherosclerosis, 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
Hair research, becomes particularly important.At present, the polypeptide with fat-reducing and antihyperglycemic includes Liraglutide, 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, and the external suppression of synthetic peptide is determined using mtt assay
System activity.It is an object of the invention to provide a kind of synthesis polypeptide with external anti-obesity activity, bio-pharmaceuticals neck is can be applied to
Domain.
Anti- fat ten heptapeptide of present invention synthesis is abbreviated as LNNPSVCDCDCMMKAAR, molecular weight 1871.24, and sequence is:
Leu-Asn-Asn-Pro-Ser-Val-Cys-Asp-Cys-Asp-Cys-Met-Met-Lys-Ala-Ala-Arg.Wherein,
Leu represents English name for Leucine, and Chinese is the corresponding residue of the amino acid of leucine;
Asn represents English name for Asparagine, and Chinese is the corresponding residue of the amino acid of asparagine acid;
Asn represents English name for Asparagine, and Chinese is the corresponding residue of the amino acid of asparagine acid;
Pro represents English name for Proline, and Chinese is the corresponding residue of the amino acid of proline;
Ser represents English name for Serine, and Chinese is the corresponding residue of the amino acid of serine;
Val represents English name for Valine, and Chinese is the corresponding residue of the amino acid of valine;
Cys represents English name for Cysteine, and Chinese is the corresponding residue of the amino acid of cysteine;
Asp represents English name for Aspartic acid, and Chinese is the corresponding residue of the amino acid of aspartic acid;
Cys represents English name for Cysteine, and Chinese is the corresponding residue of the amino acid of cysteine;
Asp represents English name for Aspartic acid, and Chinese is the corresponding residue of the amino acid of aspartic acid;
Cys represents English name for Cysteine, and Chinese is the corresponding residue of the amino acid of cysteine;
Met represents English name for Methionine, and Chinese is the corresponding residue of the amino acid of methionine;
Met represents English name for Methionine, and Chinese is the corresponding residue of the amino acid of methionine;
Lys represents English name for Lysine, and Chinese is the corresponding residue of the amino acid of lysine;
Ala represents English name for Alanine, and Chinese is the corresponding residue of the amino acid of alanine;
Ala represents English name for Alanine, and Chinese is the corresponding residue of the amino acid of alanine;
Arg represents English name for Arginine, and Chinese is the corresponding residue of arginic amino acid.
Amino acid sequence of the present invention uses standard Fmoc schemes, and by the screening of resin, rational polypeptide is closed
Into method.The C- end carboxyls of target polypeptides are connected in covalent bond form with an insoluble macromolecule resin, then with this
The amino of individual amino acid acts on forming peptide bond as starting point, the carboxyl with another molecule amino acid.This process is constantly repeated, 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 are the processes that amino acid is added in a repetition, and synthesis in solid state order synthesizes from C-terminal to N ends.
The present invention mixes the synthesis polypeptide of final concentration of 0.125-2 mg/mL with 3T3-L1, after being incubated 48 h, through MTT
Method detection, 9.77%-33.96% is reached to fat cell inhibiting rate, can be applied in biomedicine field.
Compared with prior art, the invention has the advantages that and technique effect:
The present invention has synthesized the peptide first, and is suppressed using the external lipocyte proliferation that MTT methods have detected synthesis polypeptide
Activity, the synthesis polypeptide has certain fat cell rejection ability.
Brief description of the drawings
Fig. 1 a are synthesis polypeptide Leu-Asn-Asn-Pro-Ser-Val-Cys-Asp-Cys-Asp-Cys-Met-Met-
The HPLC figures of Lys-Ala-Ala-Arg.
Fig. 1 b are synthesis polypeptide Leu-Asn-Asn-Pro-Ser-Val-Cys-Asp-Cys-Asp-Cys-Met-Met-
The MS figures of Lys-Ala-Ala-Arg.
Fig. 2 is synthesis polypeptide Leu-Asn-Asn-Pro-Ser-Val-Cys-Asp-Cys-Asp-Cys-Met-Met-Lys-
Inhibitory activity block diagrams of the Ala-Ala-Arg to fat cell 3T3-L1.
Specific embodiment
Below in conjunction with instantiation, the invention will be further described, but implementation of the invention and protection domain are not limited to
This.
Solid-phase synthesis peptides
From macromolecule resin(Chinese Peptide Co., Ltd.), according to amino acid sequence Leu-Asn-Asn-Pro-Ser-Val-
The feature of Cys-Asp-Cys-Asp-Cys-Met-Met-Lys-Ala-Ala-Arg, first by the carboxyl of Arg in the form of covalent bond
Be connected with a resin, then the amino of Ala and Arg carboxyl shrink reaction, after treatment, then add Ala, the amino of Ala and
The carboxyl reaction of Ala, adds amino acid from right to left successively, plus after good last Leu amino acid, then cuts off resin and obtain
Target polypeptides.Purified using high performance liquid chromatography, chromatographic column model Phenomenex C18, size 4.6*150mm, stream
Dynamic phase A:Contain 0.1% trifluoroacetic acid(TFA)Water;Mobile phase B:Solution (water of 80% acetonitrile+20%) containing 0.09%TFA;
B phases rise to 24.0%, flow velocity 1.0 mL/min, Detection wavelength 220nm by 14.0% in 20 min.Liquid nitrogen flash freezer, freeze-drying,
Obtain last product, it is desirable to which purity reaches more than 98.06%, and identifies structure through MS(As shown in Fig. 1).
The external inhibitory activity of synthesis polypeptide
By MTT colorimetrically analysings peptide composition to the growth inhibition effect of 3T3-L1.Concrete operation step is as follows:
1)Take the logarithm the cell in growth period, through 0.25%(Volume)The digestion of trypsase-EDTA digestive juices after, add corresponding
Complete medium terminates digestion and re-suspended cell, after blood cell plate count, adjusts the concentration of cell suspension to 5 × 104Individual/mL,
In adding to 96 orifice plates, per the μ L of hole 100, in 37 DEG C of constant temperature CO2Cultivated in incubator;
2)Cell attachment after 24 h is cultivated, waste and old nutrient solution is suctioned out, it is the to be measured containing various concentrations of 200 μ L to add final volume
The fresh complete medium of sample, and with complete medium as negative control, cultivated in 37 DEG C of constant temperature CO2 incubators;
3)Liquid is suctioned out after 48 h, with PBS board-washings 2 times, the μ l of MTT solution 20 and fresh complete medium of 5 mg/mL is added
180 µL;Continue to cultivate in 37 DEG C of constant temperature CO2 incubators;
4)After 4 h, the nutrient solution containing MTT is discarded, add 150 μ l DMSO after vibrating 15 min on microoscillator,
OD value is determined at 490nm wavelength and inhibiting rate is calculated:
Adipocytic cell growth inhibiting rate (%)=((control group OD- blank group OD)-(administration group OD- blank group OD))/((control group
OD- blank group OD)) × 100
Application Example 1
The μ L cell suspensions (5 × 10 of fat cell 3T3-L1 1004Individual/mL), add in 96 orifice plates, trained in 37 DEG C of constant temperature CO2
Culture in case is supported, cell attachment after 24 h suctions out waste and old nutrient solution, adds the polypeptide of the 125 μ g/mL that final volume is 100 μ L
The fresh complete medium of sample, and with complete medium as negative control, cultivated in 37 DEG C of constant temperature CO2 incubators, 48
Liquid is suctioned out after h, with PBS board-washings 2 times, the μ l of MTT solution 20 and the μ L of fresh complete medium 180 of 5 mg/mL is added;In
Continue to cultivate in 37 DEG C of constant temperature CO2 incubators;After 4 h, the nutrient solution containing MTT is discarded, add 150 μ l DMSO 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 2,125 μ g/
The polypeptide of mL is 9.77% to the inhibiting rate of fat cell 3T3-L1.
Application Example 2
The μ L cell suspensions (5 × 10 of fat cell 3T3-L1 1004Individual/mL), in adding to 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Cell attachment after the h of 24 is cultivated in foster case, waste and old nutrient solution is suctioned out, the polypeptide of the 250 μ g/mL that final volume is 100 μ L is added
The fresh complete medium of sample, and with complete medium as negative control, cultivated in 37 DEG C of constant temperature CO2 incubators, 48
Liquid is suctioned out after h, with PBS board-washings 2 times, the μ l of MTT solution 20 and the μ L of fresh complete medium 180 of 5 mg/mL is added;In
37 DEG C of constant temperature CO2Continue to cultivate in incubator;After 4 h, the nutrient solution containing MTT is discarded, add 150 μ l DMSO 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 μ g/
The polypeptide of mL is 24.26% to the inhibiting rate of fat cell 3T3-L1.
Application Example 3
The μ L cell suspensions (5 × 10 of fat cell 3T3-L1 1004Individual/mL), in adding to 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Culture in case is supported, cell attachment after 24 h suctions out waste and old nutrient solution, adds the polypeptide of the 500 μ g/mL that final volume is 100 μ L
The fresh complete medium of sample, and with complete medium as negative control, cultivated in 37 DEG C of constant temperature CO2 incubators, 48
Liquid is suctioned out after h, with PBS board-washings 2 times, the μ l of MTT solution 20 and the μ L of fresh base complete medium 180 of 5 mg/mL is added;
Continue to cultivate in 37 DEG C of constant temperature CO2 incubators;After 4 h, discard the nutrient solution containing MTT, add 150 μ 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 2,500 μ
The polypeptide of g/mL is 28.24% to the inhibiting rate of fat cell 3T3-L1.
Application Example 4
The μ L cell suspensions (5 × 10 of fat cell 3T3-L1 1004Individual/mL), in adding to 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Culture in case is supported, cell attachment after 24 h suctions out waste and old nutrient solution, and addition final volume is many of the 1000 μ g/mL of 100 μ L
The fresh complete medium of peptide sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO2Cultivated in incubator, 48
Liquid is suctioned out after h, with PBS board-washings 2 times, the μ l of MTT solution 20 and the μ L of fresh complete medium 180 of 5 mg/mL is added;In
Continue to cultivate in 37 DEG C of constant temperature CO2 incubators;After 4 h, the nutrient solution containing MTT is discarded, add 150 μ l DMSO 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 μ g/
The polypeptide of mL is 27.93% to the inhibiting rate of fat cell 3T3-L1.
Application Example 5
The μ L cell suspensions (5 × 10 of fat cell 3T3-L1 1004Individual/mL), in adding to 96 orifice plates, in 37 DEG C of constant temperature CO2Training
Culture in case is supported, cell attachment after 24 h suctions out waste and old nutrient solution, adds the polypeptide of the 2000 μ g/mL that final volume is 100 μ L
The fresh complete medium of sample, and with complete medium as negative control, in 37 DEG C of constant temperature CO248 is cultivated in incubator
Liquid is suctioned out after h, with PBS board-washings 2 times, the μ l of MTT solution 20 and the μ L of fresh complete medium 180 of 5 mg/mL is added;In
Continue to cultivate in 37 DEG C of constant temperature CO2 incubators;After 4 h, the nutrient solution containing MTT is discarded, add 150 μ l DMSO 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 2,2000 μ g/
The polypeptide of mL is 33.96% to the inhibiting rate of fat cell 3T3-L1.
SEQUENCE LISTING
<110>South China Science & Engineering University
<120>A kind of anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> PRT
<213>Artificial sequence
<400> 1
Leu Asn Asn Pro Ser Val Cys Asp Cys Asp Cys Met Met Lys Ala Ala
1 5 10 15
Arg
Claims (3)
1. a kind of anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR, it is characterized in that the ammonia of ten heptapeptide LNNPSVCDCDCMMKAAR
Base acid sequence is Leu-Asn-Asn-Pro-Ser-Val-Cys-Asp-Cys-Asp-Cys-Met-Met-Lys- Ala-Ala-
Arg。
2. a kind of anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR as described in claim 1, it is characterised in that described 17
When concentration is 0.125-2 mg/mL, the in-vitro multiplication to PECTORAL LIMB SKELETON 3T3-L1 suppresses peptide LNNPSVCDCDCMMKAAR
Rate is 9.77%-33.96%.
3. a kind of anti-fat ten heptapeptides LNNPSVCDCDCMMKAAR according to claim 2, it is characterised in that described ten
When concentration is 2 mg/mL, the in-vitro multiplication inhibiting rate to 3T3-L1 is 33.96% to heptapeptide LNNPSVCDCDCMMKAAR.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114292311A (en) * | 2021-12-22 | 2022-04-08 | 华南理工大学 | Pancreatic lipase inhibitory peptide and application thereof |
Citations (2)
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CN104840943A (en) * | 2015-05-05 | 2015-08-19 | 中国农业科学院作物科学研究所 | Application of Lunasin polypeptide in aspect of preparing substance with weight-reducing activity |
WO2016175362A1 (en) * | 2015-04-28 | 2016-11-03 | (주)케어젠 | Peptide having anti-diabetic and anti-obesity effects, and use thereof |
-
2016
- 2016-12-05 CN CN201611103461.7A patent/CN106749533B/en active Active
Patent Citations (2)
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---|---|---|---|---|
WO2016175362A1 (en) * | 2015-04-28 | 2016-11-03 | (주)케어젠 | Peptide having anti-diabetic and anti-obesity effects, and use thereof |
CN104840943A (en) * | 2015-05-05 | 2015-08-19 | 中国农业科学院作物科学研究所 | Application of Lunasin polypeptide in aspect of preparing substance with weight-reducing activity |
Non-Patent Citations (1)
Title |
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霍兴华等: "obestatin对3T3-L1前脂肪细胞增殖和凋亡的研究", 《菏泽医学专科学校学报》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114292311A (en) * | 2021-12-22 | 2022-04-08 | 华南理工大学 | Pancreatic lipase inhibitory peptide and application thereof |
CN114292311B (en) * | 2021-12-22 | 2023-05-12 | 华南理工大学 | Pancreatic lipase inhibitory peptide and application thereof |
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