Eggshell membrane polypeptide with DPP-IV enzyme inhibitory activity and application of eggshell membrane polypeptide in blood glucose regulation

Abstract

The invention discloses eggshell membrane polypeptide with DPP-IV enzyme inhibitory activity and application of the eggshell membrane polypeptide in blood glucose regulation, and belongs to the technical field of biology. The amino acid sequence of the eggshell membrane polypeptide is as shown in SEQ ID NO. 1. According to the invention, a high-activity polypeptide Ala-Pro-Ser-Pro-Trp-Glu is separated and identified from an eggshell membrane, the polypeptide is proved to have certain DPP-IV inhibition ability both in cells and outside cells, the stable DPP-IV inhibition activity is maintained in an in-vitro simulated gastrointestinal fluid environment, and a theoretical basis is provided for research and development of novel diabetes drugs.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an eggshell membrane polypeptide with DPP-IV enzyme inhibitory activity and application thereof in blood sugar regulation. Background Art

[0002] In recent years, the number of patients with diabetes has continued to increase worldwide, and it has become an important public health issue. Among them, about 90-95% of patients suffer from type 2 diabetes, which is characterized by incretin dysregulation, leading to insulin resistance. Glucagon-like peptide 1 (GLP-1) is a hormone that is secreted by intestinal L cells after food stimulation and can stimulate insulin secretion. GLP-1 can promote insulin secretion and reduce glucagon secretion, thereby lowering blood sugar. However, more than 95% of GLP-1 will be cleaved and inactivated under the action of the serine protease DPP-IV in the human body, making it difficult to play a role. Therefore, DPP-IV inhibitors have become the main means of treating type 2 diabetes in recent years. Although the current drugs for the treatment of type 2 diabetes are effective, they may also cause adverse side effects such as liver dysfunction and high blood pressure. Therefore, food-derived bioactive peptides have received considerable attention due to their potential as natural alternatives or supplements to DPP-IV inhibitory drugs.

[0003] As a byproduct of egg processing, eggshell membrane contains a variety of proteins such as collagen and keratin, as well as bioactive substances such as hyaluronic acid and chondroitin sulfate, and is a potential source of active peptides. As a serine protease, DPP-IV enzyme can specifically hydrolyze the peptide containing the second Pro or Ala residue at the NH2 end, and the enzymatic hydrolysate in eggshell membrane is rich in Pro, which has the potential to prepare DPP-IV inhibitory peptides. At present, the waste eggshell membrane resources are seriously wasted, causing certain environmental pressure. Therefore, the development of DPP-IV inhibitors in eggshell membrane is of great significance. Summary of the invention

[0004] The purpose of the present invention is to provide an eggshell membrane polypeptide with DPP-IV enzyme inhibitory activity and its application in blood sugar regulation to solve the problems existing in the above-mentioned prior art. The present invention separates and identifies a highly active polypeptide Ala-Pro-Ser-Pro-Trp-Glu from eggshell membrane, confirms that the polypeptide has a certain DPP-IV inhibitory ability both inside and outside the cell, and maintains a stable DPP-IV inhibitory activity in an in vitro simulated gastrointestinal fluid environment, and the activity is more stable, which provides a theoretical basis for the research and development of new diabetes drugs.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an eggshell membrane polypeptide having DPP-IV inhibitory activity, wherein the amino acid sequence of the eggshell membrane polypeptide is shown in SEQ ID NO. 1, specifically: Ala-Pro-Ser-Pro-Trp-Glu.

[0007] The present invention also provides the use of the eggshell membrane polypeptide in preparing a drug for treating diabetes.

[0008] Preferably, the diabetes is type 2 diabetes.

[0009] The present invention also provides a medicine for treating diabetes, wherein the active ingredient of the medicine is the eggshell membrane polypeptide.

[0010] Preferably, the medicine also includes pharmaceutically acceptable excipients.

[0011] Preferably, the diabetes is type 2 diabetes.

[0012] The present invention also provides the use of the eggshell membrane polypeptide in the preparation of a DPP-IV inhibitor.

[0013] The present invention also provides the above-mentioned DPP-IV inhibitor, wherein the active ingredient of the DPP-IV inhibitor is the above-mentioned eggshell membrane polypeptide.

[0014] Preferably, the DPP-IV inhibitor further comprises a pharmaceutically acceptable excipient.

[0015] The present invention discloses the following technical effects:

[0016] The present invention identifies and prepares the polypeptide Ala-Pro-Ser-Pro-Trp-Glu derived from eggshell membrane for the first time, and uses an in vitro DPP-IV inhibitory activity evaluation method to prove that the polypeptide has a certain DPP-IV inhibitory ability both inside and outside the cell, and maintains a stable DPP-IV inhibitory activity in an in vitro simulated gastrointestinal fluid environment. In subsequent drug development, the use of excipients can be reduced, the drug utilization rate is higher, and a new treatment option is provided for patients with type II diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 is the secondary mass spectrum of Ala-Pro-Ser-Pro-Trp-Glu polypeptide;

[0019] Figure 2 This is the molecular simulation docking result diagram of Ala-Pro-Ser-Pro-Trp-Glu peptide and DPP-IV receptor;

[0020] Figure 3 is a high performance liquid chromatogram of Ala-Pro-Ser-Pro-Trp-Glu polypeptide;

[0021] Figure 4 The results of the DPP-IV inhibition rate determination of different concentrations of Ala-Pro-Ser-Pro-Trp-Glu polypeptide;

[0022] Figure 5 The results of the DPP-IV inhibition rate determination of Ala-Pro-Ser-Pro-Trp-Glu peptide before and after gastrointestinal digestion;

[0023] Figure 6 The effect of different concentrations of Ala-Pro-Ser-Pro-Trp-Glu peptide on the proliferation activity of Caco-2 cells;

[0024] Figure 7 The inhibitory activity of different concentrations of Ala-Pro-Ser-Pro-Trp-Glu polypeptide on intracellular DPP-IV enzyme. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solution of the present invention is as follows:

[0031] Eggshell membrane was used as raw material, and eggshell membrane hydrolysate was prepared by enzymatic hydrolysis. The polypeptide components with molecular weight less than 3K Da in the eggshell membrane hydrolysate were separated by ultrafiltration and other technologies. Then, a highly active polypeptide Ala-Pro-Ser-Pro-Trp-Glu was identified by HPLC-MS / MS identification, bioinformatics analysis and AutoDock Vina molecular simulation docking. The polypeptide was subjected to in vitro DPP-IV inhibitory activity verification experiments and intracellular DPP-IV inhibitory activity verification experiments to detect its inhibitory effect on DPP-IV.

[0032] The preparation process of the eggshell membrane hydrolysate is as follows:

[0033] 1. Take dried and pure eggshell membranes, add purified water according to the material-liquid ratio of 100 g: (700-1000) mL, add 10-50 mmol / L of one or more of arginine, proline, serine, and cysteine ​​and mix with the eggshell membranes to obtain an eggshell membrane suspension.

[0034] 2. Transfer the eggshell membrane suspension to an enzymolysis tank, and add 3000U / g keratinase and 4000U / g collagenase to the eggshell membrane substrate, and perform a segmented or synchronous composite enzymolysis reaction. The reaction temperature of keratinase is 30-60℃, pH 6-10, and the reaction time is 1-5h; the reaction temperature of collagenase is 30-60℃, pH 6-10, and the reaction time is 1-4h. After the reaction is completed, the temperature is raised to 80-90℃ for 10-30min to inactivate the enzymes and obtain the eggshell membrane hydrolyzate.

[0035] 3. Centrifuge the eggshell membrane hydrolyzate and dry it to obtain a powdered eggshell membrane hydrolyzate.

[0036] Example 1 Preparation of eggshell membrane polypeptides

[0037] 1. Take dried and pure eggshell membranes, add purified water according to the solid-liquid ratio of 100 g: 800 mL, add 40 mmol / L proline and mix with the eggshell membranes to obtain an eggshell membrane suspension.

[0038] 2. Transfer the eggshell membrane suspension to an enzymolysis tank, and add 3000U / g keratinase and 4000U / g collagenase to the eggshell membrane substrate in sequence, and perform a segmented or synchronous composite enzymolysis reaction. The reaction temperature of the keratinase enzymolysis process is 50°C, pH 7.0, and the reaction time is 4h; the reaction temperature of the collagenase enzymolysis process is 50°C, pH 7.0, and the reaction time is 3h. After the reaction is completed, the temperature is raised to 90°C for 30 minutes to inactivate the enzymes and obtain the eggshell membrane hydrolyzate.

[0039] 3. Centrifuge the eggshell membrane hydrolysate, dry it, and extract the powdered eggshell membrane polypeptide.

[0040] 4. Screening and identification of target peptides: The peptide components with molecular weight less than 3K Da in the hydrolyzate were prepared into peptide solutions for HPLC-MS / MS identification and bioinformatics analysis, that is, the peptide property calculator online tool was used to predict the function of the peptides, and then AutoDock Vina was used for molecular simulation docking to identify a highly active peptide Ala-Pro-Ser-Pro-Trp-Glu (SEQ ID NO.1). The secondary mass spectrum of the peptide is shown in Figure 1 The molecular simulation docking results are shown in Figure 2 shown.

[0041] 5. The peptide APSPWE was tested by high performance liquid chromatography under the following conditions: chromatographic column: Agilent 300SB-C18250×4.6mml.dS-5um, 12nm; mobile phase A was 0.1% trifluoroacetic acid dissolved in water, and mobile phase B was 0.1% trifluoroacetic acid dissolved in acetonitrile; elution procedure was: flow rate 1mL / min, wavelength 214nm, 0.01-25min: 15%B, 85%A; 25-30min: 40%B, 60%A; the experiment was stopped after 30min. The results are as follows Figure 3 As shown, it can be seen that the peptide APSPWE peaks at around 13 minutes and the purity reaches 97.585%.

[0042] Example 2 Efficacy verification of eggshell membrane polypeptides

[0043] The polypeptide Ala-Pro-Ser-Pro-Trp-Glu obtained by screening in Example 1 was synthesized by solid phase synthesis, and the following experiment was performed:

[0044] 1. In vitro simulated digestion experiment

[0045] Gastric phase simulated digestion: 10 mL of 10 mg / mL polypeptide solution was mixed with 7.5 mL of SGF electrolyte stock solution (purchased from Solebao Bioreagent Co., Ltd.) and 1.6 mL of 25000 U / mL porcine pepsin solution. Then 5 μL of 0.3 mol / LCaCl2, 0.695 mL of deionized water and 0.2 mL of 1 mol / L HCl were added, the pH value was monitored to keep it at about 3.0, and magnetic stirring was continued in a 37°C water bath for 120 min to obtain a gastric phase product.

[0046] Intestinal phase simulated digestion: 10 mL of gastric phase product was mixed with 5.5 mL of SIF stock solution (purchased from Solebao Bioreagent Co., Ltd.), 2.5 mL of 800 U / mL trypsin solution, 1.25 mL of 160 mmol / L bile salt, 20 μL of 0.3 mol / L CaCl2, 0.655 mL of deionized water, and 0.075 mL of 1 mol / L NaOH. The intestinal phase digestion process was the same as the gastric phase digestion process, lasting 120 min, and the pH was monitored at around 7.0 to obtain the intestinal phase product.

[0047] All prepared samples were freeze-dried for further testing.

[0048] 2. DPP-IV In vitro Inhibitory Activity Experiment

[0049] The DPP-IV inhibition screening kit (fluorescence method) (purchased from Cayman Reagent Company) was used to determine the inhibition rate of APSPWE polypeptide samples (1, 0.5, 0.1, 0.05, 0.01 mg / mL), gastric phase product samples, and intestinal phase product samples on DPP-IV. The steps are as follows:

[0050] Before the assay, the sample lyophilized powder was dissolved in a buffer solution (20mM Tris-HCl, pH 8.0, containing 100mM NaCl, 1mM EDTA) and diluted to prepare a sample solution. The sample solution (10μL) and DPP-IV solution (50μL) were added to a 96-well plate and mixed, incubated at 37°C for 10 minutes, and then 25μL of substrate solution was added to all wells and incubated at 37°C for 15 minutes. After that, fluorescence detection was performed using an ELISA reader at an excitation wavelength of 360nm and an emission wavelength of 460nm and the results were recorded. All steps were kept in a dark environment to ensure that the substrate was not decomposed. The DPP-IV inhibitory activity was calculated according to the following formula.

[0051] DPP-IV inhibition rate = (F sample - F control) / F control × 100%

[0052] The results of DPP-IV inhibition rate determination of APSPWE peptide at different concentrations are as follows: Figure 4 As shown. It can be seen that APSPWE polypeptide has significant DPP-IV inhibitory activity and is concentration-dependent; when the concentration is 1 mg / mL, the DPP-IV inhibition rate of APSPWE polypeptide can reach about 90%. It was determined that the half-inhibitory concentration of APSPWE polypeptide is 0.192 mg / mL, so this concentration was used to conduct the DPP-IV inhibitory activity experiment of in vitro simulated digestion products.

[0053] The gastric phase product samples and intestinal phase product samples were prepared into sample solutions with a concentration of 0.192 mg / mL according to the above steps, and their DPP-IV inhibition rates were detected and compared with the APSPWE polypeptide sample solutions at the half-inhibitory concentration. Figure 5 As shown. It can be seen that the DPP-IV inhibition rate of APSPWE polypeptide before gastrointestinal digestion can reach about 50%, the DPP-IV inhibition rate of APSPWE polypeptide after gastric juice digestion can still reach about 38.5%, and the DPP-IV inhibition rate of APSPWE polypeptide after gastrointestinal digestion can reach about 35.79%. This shows that the activity of APSPWE polypeptide is relatively stable, the protease in the gastrointestinal tract has little effect on its activity, and its application prospects are more extensive.

[0054] 3. Cytotoxicity Verification Experiment

[0055] Caco-2 cells (purchased from the Cell Resource Center of the Chinese Academy of Sciences) were seeded in 96-well plates at a density of approximately 2×10 5 / mL, 100 μL was inoculated in each well, and the cells were cultured with different concentrations of APSPWE polypeptide solution (50, 100, 200, 400, 800 μg / mL) for 24 hours, and the cell proliferation activity was tested using the CCK8 kit (purchased from Beyotime Biotechnology Co., Ltd.).

[0056] The results of the effects of different concentrations of APSPWE peptide on the proliferation activity of Caco-2 cells are shown in Figure 6 As shown, it can be seen that 50-400 μg / mL of APSPWE polypeptide has a significant promoting effect on the proliferation of Caco-2 cells, and APSPWE polypeptide has no toxic effect on cells and is safe and reliable.

[0057] 4. Intracellular activity verification experiment

[0058] Caco-2 cells were seeded in black 96-well plates (with transparent bottom) at a density of approximately 10 6 Each well was inoculated with 100 μL of APSPWE peptide solution at different concentrations (50, 100, 200, 400, 800 μg / mL) and the cells were cultured for 24 hours. 2+ / Mg2+ ) of Gly-Pro-AMC, 100 μL of Gly-Pro-AMC substrate and cell lysate were added to each well. After incubation for 5 minutes, the fluorescence signal (excitation wavelength / emission wavelength is 360 nm / 460 nm) was measured using a microplate reader and the results were recorded.

[0059] The results are as follows Figure 7 As shown. It can be seen that the inhibition rate of APSPWE polypeptide on intracellular DPP-IV can reach more than 10%. When the concentration is 800μg / mL, the DPP-IV inhibition rate of APSPWE polypeptide can reach 50%. This shows that APSPWE polypeptide also has stable DPP-IV inhibition activity in cells and can be used in the actual treatment of type 2 diabetes.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An eggshell membrane polypeptide having DPP-IV inhibitory activity, characterized in that The amino acid sequence of the eggshell membrane polypeptide is shown in SEQ ID NO.

1.

2. Use of the eggshell membrane polypeptide according to claim 1 in the preparation of a drug for treating diabetes.

3. The use according to claim 2, characterized in that: The diabetes is type 2 diabetes.

4. A drug for treating diabetes, characterized in that: The active ingredient of the medicine is the eggshell membrane polypeptide according to claim 1.

5. The drug according to claim 4, characterized in that The medicine also includes pharmaceutically acceptable excipients.

6. The drug according to claim 4 or 5, characterized in that The diabetes is type 2 diabetes.

7. Use of the eggshell membrane polypeptide according to claim 1 in the preparation of a DPP-IV inhibitor.

8. A DPP-IV inhibitor, characterized in that The active ingredient of the DPP-IV inhibitor is the eggshell membrane polypeptide according to claim 1.

9. The DPP-IV inhibitor according to claim 8, characterized in that The DPP-IV inhibitor also includes pharmaceutically acceptable excipients.

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