CN110183517B - Blood sugar reducing undecapeptide - Google Patents

Abstract

The invention discloses an undecapeptide. The undecapeptide amino acid sequence is as follows: Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys, abbreviated as VVDLVFFAAAK, molecular weight 1179.44Da, purity 97.71%. The present invention uses a polypeptide synthesizer and adopts a solid-phase synthesis method to synthesize. In vitro α-amylase and α-glucosidase inhibitory activity assays showed that both enzymes had good inhibitory effects, and the 50% inhibitory concentration (IC50) for α-amylase was 1.44 mg/mL (1.70 μmol/L ), the 50% inhibitory concentration (IC50) of α-glucosidase was 0.0435 mg/mL (0.0513 μmol/L). The invention provides a synthetic polypeptide with potential in vitro hypoglycemic activity, which can be applied to the field of biopharmaceuticals.

Description

A hypoglycemic undecapeptide

technical field

The invention belongs to the field of biopharmaceuticals, and particularly relates to a hypoglycemic undecapeptide.

Background technique

Diabetes mellitus is a chronic disease, which is a disorder of protein, fat and carbohydrate metabolism caused by insufficient insulin in the body, and is mainly characterized by chronic hyperglycemia. Studies have found that there are many natural anti-diabetic active ingredients, such as: Ginkgo biloba extract, plant polysaccharides and so on. There are few studies on the hypoglycemic aspects of bioactive peptides. Some studies have shown that bioactive peptides can effectively improve the effect of diabetes. For example, in the study of Wang Junbo et al., marine collagen peptides can alleviate the structural damage of islet β cells in hyperinsulinemic rats, increase the secretion of granules, reduce the formation of lipid droplets, and significantly improve the biological activity of insulin; Fasting insulin levels, fasting blood glucose and oral glucose tolerance also have a certain improvement effect. In the research of Huang Fengjie et al., shark liver active peptide S-8300 has antioxidant effect, protects islet beta cells by scavenging free radicals, regulates glucose and lipid metabolism, delays islet beta cell failure, and can treat diabetes to a certain extent.

The digestion and absorption of starch and other carbohydrates in the human body depends on two key enzymes, α-glucosidase and α-amylase. Therefore, inhibiting the activities of these two key enzymes can slow down the degradation of carbohydrates into monosaccharides, so as to achieve the purpose of regulating the rapid rise of postprandial blood sugar.

Therefore, the present invention provides a hypoglycemic undecapeptide, the synthetic polypeptide has hypoglycemic ability.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a hypoglycemic undecapeptide and its application.

The present invention selects α-amylase and α-glucosidase as research objects, and determines the in vitro inhibitory activity of the synthetic peptide. The purpose of the present invention is to provide a synthetic polypeptide with in vitro hypoglycemic activity, which can be used in the field of biopharmaceuticals.

The invention provides a hypoglycemic undecapeptide, the amino acid sequence of the undecapeptide is Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys, abbreviated as VVDLVFFAAAK.

Further, described a kind of hypoglycemic undecapeptide, described undecapeptide VVDLVFFAAAK has inhibitory activity to alpha-amylase, and the 50% inhibitory concentration (IC50) value of alpha-amylase is 1.44 mg/mL (1.70 μmol/L).

Further, the undecapeptide VVDLVFFAAAK has inhibitory activity on α-glucosidase, and the 50% inhibitory concentration (IC50) value of α-glucosidase is 0.0435 mg/mL (0.0513 μmol/L).

Further, the undecapeptide VVDLVFFAAAK has a molecular weight of 1179.44 Da and a purity of 97.71%.

The application of the above-mentioned one kind of hypoglycemic undecapeptide.

The synthetic polypeptide of the present invention is abbreviated as VVDLVFFAAAK, the molecular weight is 1179.44 Da, the purity is 97.71%, and the sequence is: Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys. in,

Val represents the corresponding residue of the amino acid whose English name is Threonine and Chinese name is threonine;

Val represents the corresponding residue of the amino acid whose English name is Threonine and Chinese name is threonine;

Asp represents the corresponding residue of the amino acid whose English name is Aspartic acid and Chinese name is aspartic acid;

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

Val represents the corresponding residue of the amino acid whose English name is Threonine and Chinese name is threonine;

Phe represents the corresponding residue of the amino acid whose English name is Phenylalanine and Chinese name is phenylalanine;

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

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

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

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. The C-terminal carboxyl group of the target polypeptide is connected to an insoluble polymer resin in the form of a covalent bond, and then the amino group of this amino acid is used 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. Polypeptide synthesis is a process of repeatedly adding amino acids, and the solid-phase synthesis sequence is synthesized from the C-terminus to the N-terminus.

The present invention evaluates its hypoglycemic effect by studying the inhibitory effect of synthetic peptide on α-amylase and α-glucosidase.

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

This peptide is synthesized for the first time in the present invention, and the inhibitory activity of the synthetic polypeptide on α-amylase and α-glucosidase is detected, and the synthetic polypeptide has certain hypoglycemic ability.

Description of drawings

Figure 1a is the HPLC chart of the synthetic polypeptide Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys.

Figure 1b is the MS image of the synthesized polypeptide Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys.

Figure 2a is a line graph showing the inhibitory activity of synthetic polypeptide Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys on α-amylase.

Figure 2b is a line graph showing the inhibitory activity of synthetic polypeptide Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys on α-glucosidase.

Detailed ways

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

Peptide Solid Phase Synthesis

Select macromolecule resin (China Peptide Biochemical Co., Ltd.), according to the characteristics of the amino acid sequence Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys, first the carboxyl group of Val in the form of a covalent bond It is connected with a resin, and then the amino group of Val and the carboxyl group of another Val are shrunk and reacted. After processing, Asp is added, the amino group of Asp and the carboxyl group of Leu are reacted, and amino acids are added from right to left in turn. After adding the last Lys amino acid, The resin is then excised to obtain the target polypeptide. Purified by high performance liquid chromatography, the column model is Phenomenex C18, size 4.6*150mm, mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid (TFA); mobile phase B: water containing 0.1% TFA; B within 25min The phase decreased from 95.0% to 30.0%, the flow rate was 1.0 mL/min, and the detection wavelength was 214 nm. Liquid nitrogen is quick-frozen and freeze-dried to obtain the final product, which requires a purity of more than 95%, and the structure is identified by MS (as shown in Figure 1a and Figure 1b).

In vitro inhibitory activity of synthetic peptides against α-amylase

1 Preparation of reagents

1) 0.2M phosphate buffer: Weigh 2.84 g of Na ₂ HPO ₄ and 2.72 g of KH ₂ PO ₄ and dissolve them in 100 mL of distilled water respectively, take appropriate amounts of the two solutions and mix them to pH=6.9 under the action of a magnetic stirrer, and stir. The process measures real-time pH with a pH meter.

2) 1 U/mL alpha-amylase solution.

3) 1% starch solution: take 1 g of soluble starch and dissolve it in 99 mL of buffer.

4) Sample solution: Take a certain quality of sample and configure it into sample solutions of different doses (0-10 mg/mL), and the solvent is water.

5) DNS termination reaction solution: weigh 1 g of DNS and 12 g of sodium potassium tartrate into a conical flask, and add 87 mL of 0.4M Na ₂ CO ₃ solution.

6) Acarbose solution: for positive control, weigh a certain amount of acarbose to prepare solutions with different concentration gradients (0-10mg/mL)

2 Experimental steps

1) 1% starch solution was denatured by pretreatment in a water bath at 95°C for 8 minutes.

2) The experimental group was mixed with 20 μL of inhibitor (0-10 mg/mL) and 10 μL of α-amylase solution in a test tube with a pipette, and 20 μL of buffer solution in the control group was mixed with 10 μL of α-amylase solution. 20 μL of carbose (0-10 mg/mL) was mixed with 10 μL of α-amylase solution, and the reaction was shaken at 37° C. for 15 min.

3) Add 500 μL of the pretreated starch solution, and react at 37° C. for 5 min on a shaker table.

4) Add 600 μL of DNS solution, and take a water bath at 100° C. for 15 minutes.

5) After the reaction, 200 μL of the reaction solution was drawn with a pipette, and the absorbance was measured at 540 nm. The absorbances of the experimental group and the control group were expressed as the _{experimental group} A and the _{control group} A, respectively.

6) Drawing of inhibition rate-concentration curve: OriginPro 9.1 software was used for nonlinear fitting of the obtained data, Logistic function within the range of Origin Basic Function was selected, 95% confidence interval was selected, and Find Yfrom X was used for output data. The IC50 value can be obtained by making the inhibition rate-concentration curve.

The logistic function formula is as follows:

A1 is the minimum value of y, A2 is the maximum value of y, P=3, X0 is the value of x at y=50%.

In vitro inhibitory activity of synthetic peptides against α-glucosidase

1 Preparation of reagents

1) 0.2M phosphate buffer: Weigh 2.84 g of Na ₂ HPO ₄ and 2.72 g of KH ₂ PO ₄ and dissolve them in 100 mL of distilled water respectively, take appropriate amounts of the two solutions and mix them to pH=6.9 under the action of a magnetic stirrer, and stir. The process measures real-time pH with a pH meter.

2) P-NPG solution: for the substrate solution, 0.03765 g of p-NPG was weighed and dissolved in 25 mL of distilled water.

3) 0.2U/mL α-glucosidase solution: draw 5 μL of the aliquoted α-glucosidase solution (200U/ml), and make 5 mL with distilled water.

4) Sample solution: Take a certain quality of sample and configure it into sample solutions of different concentrations (0-10 mg/mL), and the solvent is water.

5) 0.2M Na ₂ CO ₃ : Weigh 0.848 g of Na ₂ CO ₃ and dissolve in 40 mL of distilled water.

2 Experimental steps

1) Reaction in a 96-well plate, the experimental group, background group, control group, and positive control group were added with reagents as shown in Table 1, and the reaction was shaken at 37°C for 20 min.

Table 1 Amount of sample added

2) Add 50 μL of buffer solution and 40 μL of substrate solution to each well, react at 37° C. for 20 min after shaking, and then add 140 μL of Na ₂ CO ₃ solution to stop the reaction.

3) The absorbance was measured at 405 nm, and the absorbance of the experimental group and the control group were expressed as the _{experimental group} A and the _{control group} A, respectively. .

4) Drawing of inhibition rate-concentration curve: OriginPro 9.1 software was used for nonlinear fitting of the obtained data, Logistic function within the range of Origin Basic Function was selected, 95% confidence interval was selected, and Find Yfrom X was used for output data. The IC50 value can be obtained by making the inhibition rate-concentration curve.

The logistic function formula is as follows:

A1 is the minimum value of y, A2 is the maximum value of y, P=3, X0 is the value of x at y=50%.

Application Example 1

From the peak area percentage shown in Figure 1a, the purity of the undecapeptide is 97.71%, which meets the purity requirements of synthetic peptides. Take a 1% starch solution in a water bath at 95°C for 8 minutes, and pretreat it to denature it. In the experimental group, 20 μL of undecapeptide (10 mg/mL) was mixed with 10 μL of α-amylase enzyme solution in a test tube with a pipette, and 20 μL of buffer solution in the control group was mixed with 10 μL of α-amylase enzyme solution. 20 μL of carbose (10 mg/mL) was mixed with 10 μL of α-amylase enzyme solution, and the reaction was shaken at 37° C. for 15 min. 500 μL of the above pretreated starch solution was added, and the reaction was carried out at 37° C. for 5 min. Add 600 μL of DNS solution, 100 ℃ water bath for 15 min. After the reaction, 200 μL of the reaction solution was drawn with a pipette, and the absorbance was measured at 540 nm to calculate the inhibition rate. It can be seen from Figure 2a that the inhibition rate of undecapeptide to α-amylase is 84.75%, which is slightly higher than that of acarbose (81.87%).

Application Example 2

From the peak area percentage shown in Figure 1a, the purity of the undecapeptide is 97.71%, which meets the purity requirements of synthetic peptides. Take a 1% starch solution in a water bath at 95°C for 8 minutes, and pretreat it to denature it. In the experimental group, 20 μL of undecapeptide (4 mg/mL) was mixed with 10 μL of α-amylase enzyme solution in a test tube with a pipette, and 20 μL of buffer solution in the control group was mixed with 10 μL of α-amylase enzyme solution. 20 μL of carbose (4 mg/mL) was mixed with 10 μL of α-amylase enzyme solution, and the reaction was shaken at 37° C. for 15 min. 500 μL of pretreated starch solution was added, and the reaction was shaken at 37° C. for 5 min. Add 600 μL of DNS solution, 100 ℃ water bath for 15 min. After the reaction, 200 μL of the reaction solution was drawn with a pipette, and the absorbance was measured at 540 nm to calculate the inhibition rate. It can be seen from Figure 2a that the inhibition rate of undecapeptide to α-amylase is 65.97%, and the inhibition rate of acarbose at the same concentration is 77.15%.

Application Example 3

From the peak area percentage shown in Figure 1a, the purity of the undecapeptide is 97.71%, which meets the purity requirements of synthetic peptides. Take a 1% starch solution in a water bath at 95°C for 8 minutes, and pretreat it to denature it. In the experimental group, 20 μL of undecapeptide (1.5 mg/mL) was mixed with 10 μL of α-amylase enzyme solution in a test tube with a pipette, and 20 μL of buffer solution in the control group was mixed with 10 μL of α-amylase enzyme solution. 20 μL of carbose (1.5 mg/mL) was mixed with 10 μL of α-amylase enzyme solution, and the reaction was shaken at 37° C. for 15 min. 500 μL of pretreated starch solution was added, and the reaction was shaken at 37° C. for 5 min. Add 600 μL of DNS solution, 100 ℃ water bath for 15 min. After the reaction, 200 μL of the reaction solution was drawn with a pipette, and the absorbance was measured at 540 nm to calculate the inhibition rate. It can be seen from Figure 2a that the inhibition rate of undecapeptide to α-amylase is 51.12%, and the inhibition rate of acarbose is 68.14% at the same concentration.

Application Example 4

From the peak area percentage shown in Figure 1a, the purity of the undecapeptide is 97.71%, which meets the purity requirements of synthetic peptides. The experimental group (undecapeptide (4mg/mL) 20 μL and α-glucosidase enzyme solution 10 μL), background group (undecapeptide (4 mg/mL) 20 μL and buffer 10 μL), control group ( Buffer 10 μL and α-glucosidase enzyme solution 10 μL), positive control group (acarbose solution (4 mg/mL) 20 μL and α-glucosidase enzyme solution 10 μL), react at 37°C with shaking for 20 min. 50 μL of buffer solution and 40 μL of substrate solution were added to each well, and the reaction was removed after shaking at 37° C. for 20 min, and 140 μL of Na ₂ CO ₃ solution was added to stop the reaction. The absorbance was measured at 405 nm and the inhibition rate was calculated. It can be seen from Figure 2b that the inhibition rate of undecapeptide to α-glucosidase is 98.26%, which is close to the inhibition rate of acarbose (99.5%).

Application Example 5

From the peak area percentage shown in Figure 1a, the purity of the undecapeptide is 97.71%, which meets the purity requirements of synthetic peptides. The experimental group (undecapeptide (1 mg/mL) 20 μL and α-glucosidase enzyme solution 10 μL), the background group (undecapeptide (1 mg/mL) 20 μL and buffer 10 μL), the control group ( 10 μL of buffer solution and 10 μL of α-glucosidase enzyme solution), positive control group (20 μL of acarbose solution (1 mg/mL) and 10 μL of α-glucosidase enzyme solution), were reacted at 37°C on a shaking table for 20 min. 50 μL of buffer solution and 40 μL of substrate solution were added to each well, and the reaction was removed after shaking at 37° C. for 20 min, and 140 μL of Na ₂ CO ₃ solution was added to stop the reaction. The absorbance was measured at 405 nm and the inhibition rate was calculated. It can be seen from Figure 2b that the inhibition rate of undecapeptide on α-glucosidase is 95.16%, which is 2.4 times that of acarbose (40.22%).

Application Example 6

From the peak area percentage shown in Figure 1a, the purity of the undecapeptide is 97.71%, which meets the purity requirements of synthetic peptides. Add the experimental group (undecapeptide (0.5mg/mL) 20 μL and α-glucosidase enzyme solution 10 μL), background group (undecapeptide (0.5 mg/mL) 20 μL and buffer 10 μL), control group to 96-well plate Group (10 μL of buffer solution and 10 μL of α-glucosidase enzyme solution), positive control group (20 μL of acarbose solution (0.5 mg/mL) and 10 μL of α-glucosidase enzyme solution), reacted at 37°C for 20 min on a shaking table . 50 μL of buffer solution and 40 μL of substrate solution were added to each well, and the reaction was removed after shaking at 37° C. for 20 min, and 140 μL of Na ₂ CO ₃ solution was added to stop the reaction. The absorbance was measured at 405 nm and the inhibition rate was calculated. It can be seen from Figure 2b that the inhibition rate of undecapeptide on α-glucosidase is 94.26%, which is 4.7 times that of acarbose (20.19%).

The above examples are only preferred embodiments of the present invention, and are only used to explain the present invention, but not to limit the present invention. Changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention shall belong to the present invention. the scope of protection of the invention.

sequence listing

<110> South China University of Technology

<120> A hypoglycemic undecapeptide

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> Artificial sequences (artificial synthesis)

<400> 1

Val Val Asp Leu Val Phe Phe Ala Ala Ala Lys

1 5 10

Claims (4)

1. An undecapeptide for lowering blood sugar, characterized in that the amino acid sequence of the undecapeptide is Val-Val-Asp-Leu-Val-Phe-Phe-Ala-Ala-Ala-Lys, abbreviated as VVDLVFFAAAK.

2. The use of a hypoglycemic undecapeptide in the preparation of a biopharmaceutical having hypoglycemic activity in vitro of claim 1, wherein said undecapeptide VVDLVFFAAAK has inhibitory activity on α -amylase and an IC50 value of 1.44 mg/mL.

3. The use of a hypoglycemic undecapeptide in the preparation of a biopharmaceutical for its in vitro hypoglycemic activity of claim 1, wherein said undecapeptide VVDLVFFAAAK has inhibitory activity on α -glucosidase with an IC50 value of 0.0435 mg/mL.

4. Use of a hypoglycemic undecapeptide in the preparation of biopharmaceuticals having hypoglycemic activity in vitro according to claim 1, wherein said undecapeptide VVDLVFFAAAK has a molecular weight of 1179.44Da and a purity of 97.71%.

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