CN106518971B - Anti-obesity decapeptide CANPHELPNK - Google Patents
Anti-obesity decapeptide CANPHELPNK Download PDFInfo
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- CN106518971B CN106518971B CN201611103465.5A CN201611103465A CN106518971B CN 106518971 B CN106518971 B CN 106518971B CN 201611103465 A CN201611103465 A CN 201611103465A CN 106518971 B CN106518971 B CN 106518971B
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- 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 an anti-obesity decapeptide CANPHELPNK, the amino acid sequence of the polypeptide is shown as follows: Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys, abbreviated CANPHELPNK, molecular weight 1122.27, purity 97.55%. The polypeptide of the invention is synthesized by a solid phase synthesis method by using a polypeptide synthesizer. Through in vitro mouse preadipocyte 3T3-L1 proliferation inhibition activity detection, the polypeptide of the invention has certain inhibition effect on 3T3-L1 within the range of 0.125-2 mg/mL. The in vitro proliferation inhibition rate of 3T3-L1 at 2 mg/mL was 59.51%. The invention provides a synthetic polypeptide with potential in vitro anti-obesity activity, which can be applied to the field of biological pharmacy.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to anti-obesity decapeptide and application thereof.
Background
Bioactive peptides are specific protein fragments that have a positive effect on the function or state of the body and ultimately affect the health of the body. The superiority of small molecule peptide fragments over proteins is mainly reflected in: is easier to be absorbed and utilized by human body; the activity is high, and the special physiological effect can be exerted under a smaller concentration; small molecular weight, easy modification and reconstruction, and can be synthesized by artificial chemistry, etc. Compared with single amino acid, the small molecule peptide has the advantages of no comparable amino acid in absorption channel and absorption speed besides special physiological activity. It has been proved that the small intestine of human body has special oligopeptide absorption channel, and the protein ingested by human body is hydrolyzed by various digestive enzymes and absorbed mainly in the form of oligopeptide. Many researches show that bioactive peptides from various sources have multiple functions of resisting oxidation, resisting tumors, inhibiting bacteria, reducing blood pressure, reducing blood sugar and the like, and become hot spots for developing biological medicines and health care products. Obesity increases the risk of atherosclerosis, coronary heart disease, hypertension, diabetes, gout, fatty liver, and other diseases. Therefore, the development and research of safe and harmless bioactive peptides with weight-reducing and lipid-lowering effects become important. At present, the polypeptide with the functions of losing weight and reducing fat comprises liraglutide, weever active peptide, silkworm pupa peptide and the like.
Disclosure of Invention
The invention selects mouse preadipocyte 3T3-L1 as a research object, and uses MTT method to determine the in vitro inhibitory activity of synthetic peptide. The invention aims to provide a synthetic polypeptide with in vitro anti-obesity activity, which can be applied to the field of biological pharmacy.
The synthesized anti-obesity decapeptide is abbreviated as CANPHELPNK, has a molecular weight of 1122.27, and has a sequence as follows: Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys. Wherein the content of the first and second substances,
Cys represents the corresponding residue of an amino acid with the English name Cystein and the Chinese name Cysteine;
Ala represents the corresponding residue of the amino acid with the English name Alanine and the Chinese name Alanine;
Asn represents the corresponding residue of an amino acid named asaragine in english and Asparagine in chinese;
pro represents the corresponding residue of an amino acid having the English name Proline and the Chinese name Proline;
His represents the corresponding residue of an amino acid known by the English name Histidine and the Chinese name Histidine;
glu represents the corresponding residue of the amino acid known by the English name Glutamic acid and the Chinese name Glutamic acid;
Leu represents the corresponding residue of the amino acid named Leucine in England and Leucine in Chinese;
Pro represents the corresponding residue of an amino acid having the English name Proline and the Chinese name Proline;
Asn represents the corresponding residue of an amino acid named asaragine in english and Asparagine in chinese;
Lys represents the corresponding residue of an amino acid with the English name Lysine and the Chinese name Lysine.
The amino acid sequence of the invention adopts a standard Fmoc scheme, and a reasonable polypeptide synthesis method is realized by screening resin. The C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymeric resin, and then the amino group of the amino acid is used as a starting point to react with the carboxyl group of another molecule of amino acid to form a peptide bond. The process is repeated continuously to obtain the target polypeptide product. And after the synthesis reaction is finished, removing the protecting group, and separating the peptide chain from the resin to obtain the target product. Polypeptide synthesis is a process of repeated addition of amino acids, and the solid phase synthesis sequence is synthesized from the C-terminus to the N-terminus.
the invention uniformly mixes the synthetic polypeptide with the final concentration of 0.125-2 mg/mL and 3T3-L1, incubates for 48 h, and detects by an MTT method that the inhibition rate of the fat cells reaches 32.63% -59.51%, thus the invention can be applied in the field of biological medicine.
When the concentration of the decapeptide CANPHELPNK is 2 mg/mL, the in vitro proliferation inhibition rate of the decapeptide CANPHELPNK to 3T3-L1 is 59.51%.
Compared with the prior art, the invention has the following advantages and technical effects:
the peptide is synthesized for the first time, and the in vitro adipocyte proliferation inhibition activity of the synthesized polypeptide is detected by adopting an MTT method, wherein the synthesized polypeptide has certain adipocyte inhibition capacity.
Drawings
FIG. 1a is an HPLC plot of the synthetic polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.
FIG. 1b is an MS picture of the synthetic polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.
FIG. 2 is a bar graph of the inhibitory activity of the synthetic polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys on adipocytes 3T 3-L1.
Detailed Description
The present invention is further illustrated by the following specific examples, but the scope of the invention is not limited thereto.
Solid phase synthesis of polypeptides
18Selecting high molecular resin (Zhongji Biochemical Co., Ltd.), connecting the carboxyl of Lys with a resin in a covalent bond mode according to the characteristics of an amino acid sequence Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys, then carrying out a shrinkage reaction on the amino of Asn and the carboxyl of Lys, adding the amino of Pro, Pro and the carboxyl of Asn for reaction, sequentially adding amino acid from right to left, adding the last Cys amino acid, and cutting off the resin to obtain the target polypeptide.
In vitro inhibitory Activity of synthetic Polypeptides
The growth inhibitory effect of the peptide fraction on 3T3-L1 was analyzed by MTT colorimetry. The specific operation steps are as follows:
1) taking cells in logarithmic growth phase, digesting with 0.25% (volume) trypsin-EDTA digestive juice, adding a corresponding complete culture medium to stop digestion and resuspending the cells, counting a blood cell plate, adjusting the concentration of cell suspension to 5 × 10 4/mL, adding the cell suspension into a 96-well plate, adding 100 μ L of each well, and culturing in a constant-temperature CO 2 incubator at 37 ℃;
2) after culturing for 24 hours, adhering the cells to the wall, sucking out waste culture solution, adding a fresh complete culture medium with a final volume of 200 mu L and containing samples to be detected with different concentrations, and culturing in a constant-temperature CO 2 incubator at 37 ℃ by taking the complete culture medium as a negative control;
3) Sucking out the liquid medicine after 48 hours, washing the plate for 2 times by using PBS, and adding 20 mul of 5 mg/mL MTT solution and 180 mul of fresh complete culture medium; continuously culturing in a CO2 incubator at the constant temperature of 37 ℃;
4) after 4 h, discarding the culture solution containing MTT, adding 150 mul DMSO, oscillating for 15 min on a micro oscillator, measuring the optical density value at the 490nm wavelength and calculating the inhibition rate:
The adipocyte growth inhibition ratio (%) = ((control OD-blank OD) - (administration OD-blank OD))/((control OD-blank OD)) × 100.
Application example 1
Adding adipocyte 3T 3-L1100 mu L cell suspension (5 multiplied by 10 4/mL) into a 96-well plate, culturing in a CO 2 incubator at constant temperature of 37 ℃, adhering the cells after 24 hours, sucking out waste culture solution, adding fresh complete culture medium of a polypeptide sample of 125 mu g/mL with the final volume of 100 mu L, culturing in a CO 2 incubator at constant temperature of 37 ℃ by taking the complete culture medium as a negative control, sucking out liquid medicine after 48 hours, washing the plate with PBS for 2 times, adding 20 mu L of 5 mg/mL MTT solution and 180 mu L of fresh complete culture medium, continuously culturing in a CO 2 incubator at constant temperature of 37 ℃, discarding culture solution containing MTT density value after 4 hours, adding 150 mu L DMSO, oscillating for 15 min on a micro-oscillator, measuring light at 490nm wavelength and calculating the inhibition rate, wherein the inhibition rate of polypeptide of 125 mu g/mL to adipocyte 3-1 is 32.63%.
Application example 2
Adding adipocyte 3T 3-L1100 [ mu ] L cell suspension (5 multiplied by 10 4/mL) into a 96-well plate, culturing in a CO 2 incubator at constant temperature of 37 ℃, adhering the cells after 24 hours, sucking out waste culture solution, adding a fresh complete culture medium of a polypeptide sample of 250 [ mu ] g/mL with the final volume of 100 [ mu ] L, culturing in a CO 2 incubator at constant temperature of 37 ℃ by taking the complete culture medium as a negative control, sucking out liquid medicine after 48 hours, washing the plate with PBS for 2 times, adding 20 [ mu ] L of 5 mg/mL MTT solution and 180 [ mu ] L of the fresh complete culture medium, continuously culturing in a CO 2 incubator at constant temperature of 37 ℃, discarding culture solution containing MTT density value after 4 hours, adding 150 [ mu ] L DMSO, oscillating for 15 min on a micro-oscillator, measuring light at 490nm wavelength and calculating the inhibition rate, wherein the inhibition rate of the polypeptide of 250 [ mu ] g/mL to adipocyte 3-1 is 42.28%.
Application example 3
Adding adipocyte 3T 3-L1100 [ mu ] L cell suspension (5 multiplied by 10 4/mL) into a 96-well plate, culturing in a CO 2 incubator at constant temperature of 37 ℃, adhering the cells after 24 hours, sucking out waste culture solution, adding a fresh complete culture medium of a polypeptide sample of 500 [ mu ] g/mL with the final volume of 100 [ mu ] L, culturing in a CO 2 incubator at constant temperature of 37 ℃ by taking the complete culture medium as a negative control, sucking out liquid medicine after 48 hours, washing the plate with PBS for 2 times, adding 20 [ mu ] L of 5 mg/mL MTT solution and 180 [ mu ] L of the fresh complete culture medium, continuously culturing in a CO 2 incubator at constant temperature of 37 ℃, discarding culture solution containing MTT after 4 hours, adding 150 [ mu ] L, oscillating DMSO for 15 min on a micro-oscillator, measuring light at the wavelength of 490nm and calculating the inhibition rate, wherein the inhibition rate of the polypeptide of 500 [ mu ] g/mL on adipocyte 3-L1 is 46.1%.
application example 4
Adding adipocyte 3T 3-L1100 [ mu ] L cell suspension (5 multiplied by 10 4/mL) into a 96-well plate, culturing in a CO 2 incubator at constant temperature of 37 ℃, adhering the cells after 24 hours, sucking out waste culture solution, adding a fresh complete culture medium of a polypeptide sample of 1000 [ mu ] g/mL with the final volume of 100 [ mu ] L, culturing in a CO 2 incubator at constant temperature of 37 ℃ by taking the complete culture medium as a negative control, sucking out liquid medicine after 48 hours, washing the plate with PBS for 2 times, adding 20 [ mu ] L of 5 mg/mL MTT solution and 180 [ mu ] L of the fresh complete culture medium, continuously culturing in a CO2 incubator at constant temperature of 37 ℃, discarding culture solution containing MTT density value after 4 hours, adding 150 [ mu ] L DMSO, oscillating for 15 min on a micro-oscillator, measuring light at 490nm wavelength and calculating the inhibition rate, wherein the inhibition rate of the polypeptide of 1000 [ mu ] g/mL to adipocyte 3-1 is 48.36%.
application example 5
Adding adipocyte 3T 3-L1100 [ mu ] L cell suspension (5 multiplied by 10 4/mL) into a 96-well plate, culturing in a CO 2 incubator at constant temperature of 37 ℃, adhering the cells after 24 hours, sucking out waste culture solution, adding a fresh complete culture medium of a polypeptide sample with the final volume of 100 [ mu ] L of 2000 [ mu ] g/mL, culturing in a CO2 incubator at constant temperature of 37 ℃ by taking the complete culture medium as a negative control, sucking out liquid medicine after 48 hours, washing the plate for 2 times by PBS, adding 20 [ mu ] L of 5 mg/mL MTT solution and 180 [ mu ] L of the fresh complete culture medium, continuously culturing in a CO 2 incubator at constant temperature of 37 ℃, discarding culture solution containing MTT density value after 4 hours, adding 150 [ mu ] L DMSO, oscillating for 15 min on a micro-oscillator, measuring light at 490nm wavelength and calculating the inhibition rate, wherein the inhibition rate of the polypeptide with the density of 2000 [ mu ] g/mL to adipocyte 3-1 is 59.51%.
SEQUENCE LISTING
<110> university of southern China's science
<120> an anti-obesity 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 (2)
1. an anti-obesity decapeptide CANPHELPNK, characterized in that the amino acid sequence of the decapeptide CANPHELPNK is Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys; when the concentration of the decapeptide CANPHELPNK is 0.125-2 mg/mL, the in vitro proliferation inhibition rate of the preadipocyte 3T3-L1 is 32.63% -59.51%.
2. The anti-obesity decapeptide CANPHELPNK according to claim 1, wherein the in vitro proliferation inhibition rate of the decapeptide CANPHELPNK to 3T3-L1 at a concentration of 2 mg/mL is 59.51%.
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| CN108676073B (en) * | 2018-06-07 | 2021-10-26 | 华南理工大学 | Anti-obesity decapeptide LLVVYPWTQR and application thereof |
| 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 |
| CN114315965B (en) * | 2021-12-22 | 2023-05-12 | 华南理工大学 | Anti-obesity peptides and uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5525705A (en) * | 1995-01-31 | 1996-06-11 | Eli Lilly And Company | Anti-obesity proteins |
| US5532336A (en) * | 1995-01-31 | 1996-07-02 | Eli Lilly And Company | Anti-obesity proteins |
| JP2005220074A (en) * | 2004-02-05 | 2005-08-18 | Fancl Corp | Inhibitor of fat-cell differentiation |
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| KR101669140B1 (en) * | 2015-04-28 | 2016-10-26 | (주)케어젠 | Peptides having Anti-obesity and Anti-Diabetes 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 |
| CN104829705B (en) * | 2015-05-06 | 2017-11-14 | 广东省生物资源应用研究所 | The leptin activity peptide of one C spiral region mutation and its encoding gene and application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5525705A (en) * | 1995-01-31 | 1996-06-11 | Eli Lilly And Company | Anti-obesity proteins |
| US5532336A (en) * | 1995-01-31 | 1996-07-02 | Eli Lilly And Company | Anti-obesity proteins |
| JP2005220074A (en) * | 2004-02-05 | 2005-08-18 | Fancl Corp | Inhibitor of fat-cell differentiation |
Non-Patent Citations (1)
| Title |
|---|
| 藻蓝蛋白酶解条件优化及产物抑制肥胖相关酶活性的研究;周丽丽;《中国优秀硕士学位论文全文数据库》;20150215(第2期);1-73 * |
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