CN106518971A - Anti-obesity decapeptide CANPHELPNK - Google Patents

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

An anti-obesity decapeptide CANPHELPNK

technical field

The invention belongs to the field of biopharmaceuticals, and in particular relates to an anti-obesity decapeptide and its application.

Background technique

Bioactive peptides are special protein fragments that have a positive effect on the function or state of the body and ultimately affect the health of the body. Compared with proteins, the advantages of small-molecule peptide fragments are mainly reflected in: easier to be absorbed and utilized by the human body; high activity, which can exert its unique physiological effects at a small concentration; small molecular weight, easy to modify and transform, can By artificial chemical synthesis etc. Compared with single amino acids, small molecule peptides not only have special physiological activities, but also have incomparable advantages of amino acids in absorption channels and absorption speed. Studies have confirmed that there are special oligopeptide absorption channels in the small intestine of the human body, and the protein ingested by the human body is hydrolyzed by various digestive enzymes, and is mainly absorbed in the form of low peptides. Many studies have shown that bioactive peptides from various sources have various functions such as anti-oxidation, anti-tumor, antibacterial, antihypertensive, and hypoglycemic, and have become a hot spot in the development of biomedicine 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-loss and fat-lowering effects has become particularly important. Currently, peptides with weight-loss and lipid-lowering effects include liraglutide, perch active peptide, silkworm chrysalis peptide, etc.

Contents of the invention

The present invention selects mouse preadipocyte 3T3-L1 as the research object, and uses the MTT method to measure the in vitro inhibitory activity of the synthetic peptide. The purpose of the present invention is to provide a synthetic polypeptide with anti-obesity activity in vitro, which can be applied in the field of biopharmaceuticals.

The anti-obesity decapeptide synthesized by the present invention is abbreviated as CANPHELPNK, the molecular weight is 1122.27, and the sequence is: Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys. in,

Cys represents the corresponding residue of an amino acid whose English name is Cysteine and whose Chinese name is cysteine;

Ala represents the corresponding residue of the amino acid whose English name is Alanine and whose Chinese name is alanine;

Asn means the corresponding residue of the amino acid whose English name is Asparagine and whose Chinese name is Asparagine;

Pro means the corresponding residue of the amino acid whose English name is Proline and whose Chinese name is proline;

His represents the corresponding residue of the amino acid whose English name is Histidine and whose Chinese name is histidine;

Glu means the corresponding residue of the amino acid whose English name is Glutamic acid and whose Chinese name is glutamic acid;

Leu means the corresponding residue of the amino acid whose English name is Leucine and Chinese name is leucine;

Pro means the corresponding residue of the amino acid whose English name is Proline and whose Chinese name is proline;

Asn means the corresponding residue of the amino acid whose English name is Asparagine and whose Chinese name is Asparagine;

Lys represents the corresponding residue of the amino acid whose English name is Lysine and Chinese name is lysine.

The amino acid sequence described in the present invention adopts the standard Fmoc scheme, through resin screening, and a reasonable polypeptide synthesis method. Link the C-terminal carboxyl group of the target polypeptide to an insoluble polymer resin in the form of a covalent bond, and then use the amino group of this amino acid as the starting point to form a peptide bond with the carboxyl group of another molecule of amino acid. By repeating this process continuously, the target polypeptide product can be obtained. After the synthesis reaction is completed, the protecting group is removed, and the peptide chain is separated from the resin to obtain the target product. Peptide 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.

In the present invention, the synthetic polypeptide with a final concentration of 0.125-2 mg/mL is mixed with 3T3-L1, and after incubation for 48 hours, the inhibitory rate on adipocytes reaches 32.63%-59.51% by MTT method, which can be used in the field of biomedicine application.

When the concentration of the decapeptide CANPHELPNK is 2 mg/mL, the in vitro proliferation inhibition rate of 3T3-L1 is 59.51%.

Compared with the prior art, the present invention has the following advantages and technical effects:

The present invention synthesizes the peptide for the first time, and uses the MTT method to detect the in vitro adipocyte proliferation inhibitory activity of the synthetic polypeptide, and the synthetic polypeptide has a certain adipocyte inhibitory ability.

Description of drawings

Figure 1a is the HPLC chart of the synthetic polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.

Figure 1b is the MS image of the synthetic polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys.

Fig. 2 is a bar graph showing the inhibitory activity of the synthetic polypeptide Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys on adipocyte 3T3-L1.

detailed description

The present invention will be further described below in conjunction with specific examples, but the implementation and protection scope of the present invention are not limited thereto.

Peptide Solid Phase Synthesis

Select high molecular resin (China Peptide Biochemical Co., Ltd.), according to the characteristics of the amino acid sequence Cys-Ala-Asn-Pro-His-Glu-Leu-Pro-Asn-Lys, the carboxyl group of Lys is covalently bonded with a The resin is connected, and then the amino group of Asn shrinks with the carboxyl group of Lys. After treatment, Pro is added, and the amino group of Pro reacts with the carboxyl group of Asn. Amino acids are added in turn from right to left. After the last Cys amino acid is added, the resin is cut off. Get the target polypeptide. Purify by high performance liquid chromatography, the chromatographic column model is Phenomenex C ₁₈ , size 4.6*150mm, mobile phase A: water containing 0.1% trifluoroacetic acid (TFA); mobile phase B: solution containing 0.09% TFA (80% Acetonitrile + 20% water); Phase B increased from 14.0% to 24.0% within 20 min, flow rate 1.0 mL/min, detection wavelength 220nm. Quick-frozen in liquid nitrogen and freeze-dried to obtain the final product, which requires a purity of over 98.06%, and its structure was identified by MS (as shown in Figure 1).

In vitro inhibitory activity of synthetic peptides

The growth inhibitory effect of peptide fractions on 3T3-L1 was analyzed by MTT colorimetry. The specific operation steps are as follows:

1) Take the cells in the logarithmic growth phase, digest them with 0.25% (volume) trypsin-EDTA digestion solution, add the corresponding complete medium to stop the digestion and resuspend the cells, and adjust the concentration of the cell suspension to Add 5×10 ⁴ cells/mL to a 96-well plate, 100 µL per well, and culture in a constant temperature CO ₂ incubator at 37°C;

2) After 24 hours of culture, the cells adhered to the wall, sucked out the waste culture medium, and added a final volume of 200 µL of fresh complete medium containing different concentrations of the samples to be tested, and used the complete medium as a negative control, and cultured at a constant temperature of 37 °C in CO ₂ Cultivate in the box;

3) After 48 hours, suck out the drug solution, wash the plate twice with PBS, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO2 incubator at 37 °C;

4) After 4 h, discard the culture solution containing MTT, add 150 µl DMSO, shake on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490 nm and calculate the inhibition rate:

Adipocyte growth inhibition rate (%)=((OD of control group-OD of blank group)-(OD of drug administration group-OD of blank group))/((OD of control group-OD of blank group))×100.

Application Example 1

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a constant temperature CO ₂ incubator at 37 °C. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 125 μg/mL polypeptide sample with a final volume of 100 μL, and use the complete medium as a negative control, incubate in a constant temperature _CO2 incubator at 37 °C, suck out the drug solution after 48 h, and wash with PBS Plate twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37°C; after 4 h, discard the culture solution containing MTT and add 150 µl DMSO After oscillating on a micro-oscillator for 15 min, measure the optical density value at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 125 µg/mL polypeptide on adipocyte 3T3-L1 is 32.63%.

Application Example 2

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a 37°C constant temperature CO ₂ incubator. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 250 μg/mL peptide sample with a final volume of 100 μL, and use the complete medium as a negative control, incubate in a constant temperature CO ₂ incubator at 37 °C, suck out the drug solution after 48 hours, and wash the plate with PBS Twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37 °C; after 4 h, discard the culture solution containing MTT, add 150 µl DMSO Oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 250 µg/mL polypeptide on adipocyte 3T3-L1 is 42.28%.

Application Example 3

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate, and cultured in a constant temperature CO ₂ incubator at 37 °C. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 500 μg/mL polypeptide sample with a final volume of 100 μL, and use the complete medium as a negative control, culture in a constant temperature _CO2 incubator at 37 °C, suck out the drug solution after 48 h, and wash with PBS Plate twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh base complete medium; continue culturing in a constant temperature CO ₂ incubator at 37 ℃; after 4 h, discard the culture solution containing MTT and add 150 µl After DMSO, oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490 nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 500 µg/mL polypeptide on adipocyte 3T3-L1 is 46.1%.

Application Example 4

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate and cultured in a 37°C constant temperature CO ₂ incubator. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 1000 μg/mL peptide sample with a final volume of 100 µL, and use the complete medium as a negative control, incubate in a constant temperature CO ₂ incubator at 37 °C, suck out the drug solution after 48 hours, and wash the plate with PBS Twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO2 incubator at 37 °C; after 4 h, discard the culture solution containing MTT, add 150 µl DMSO and place in Oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 1000 µg/mL polypeptide on adipocyte 3T3-L1 is 48.36%.

Application Example 5

Adipocyte 3T3-L1 100 µL cell suspension (5×10 ⁴ cells/mL) was added to a 96-well plate, and cultured in a constant temperature CO ₂ incubator at 37 °C. After 24 h, the cells adhered to the wall, and the waste culture medium was sucked out. Add fresh complete medium of 2000 μg/mL polypeptide sample with a final volume of 100 μL, and use the complete medium as a negative control, incubate in a constant temperature CO2 incubator at 37 °C, suck out the drug solution after 48 h, and wash the plate with PBS Twice, add 20 µl of 5 mg/mL MTT solution and 180 µL of fresh complete medium; continue culturing in a constant temperature CO ₂ incubator at 37 °C; after 4 h, discard the culture solution containing MTT, add 150 µl DMSO Oscillate on a micro-oscillator for 15 min, measure the optical density at a wavelength of 490nm and calculate the inhibition rate. It can be seen from Figure 2 that the inhibitory rate of 2000 µg/mL polypeptide on adipocyte 3T3-L1 is 59.51%.

SEQUENCE LISTING

<110> South China University of Technology

<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 (3)

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. 2. A kind of anti-obesity decapeptide CANPHELPNK as claimed in claim 1, it is characterized in that when described decapeptide CANPHELPNK is at a concentration of 0.125-2 mg/mL, the in vitro proliferation inhibition rate to preadipocyte 3T3-L1 is 32.63 %-59.51%. 3. A kind of anti-obesity decapeptide CANPHELPNK according to claim 2, characterized in that when the decapeptide CANPHELPNK is at a concentration of 2 mg/mL, the in vitro proliferation inhibition rate of 3T3-L1 is 59.51%.