Polypeptide and application thereof in preparation of alpha-glucosidase inhibitor

Abstract

The invention provides a polypeptide and application thereof in preparation of an alpha-glucosidase inhibitor. The invention finds that the polypeptide with the amino acid sequence as shown in SEQ ID NO: 1 has excellent alpha-glucosidase inhibitory activity and DPPH free radical scavenging activity, that is, the polypeptide is suitable for preparing an alpha-glucosidase inhibitor and an antioxidant product, so that diabetes and / or obesity can be effectively prevented and / or treated, and the application prospect is broad. According to the present invention, the polypeptide resource library having the alpha-glucosidase inhibition effect and / or the anti-oxidation effect can be enriched, and more raw material choices can be provided for the alpha-glucosidase inhibitor, the anti-oxidation product, the diabetes prevention and / or treatment product, and the obesity prevention and / or treatment product.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a polypeptide and its application in the preparation of α-glucosidase inhibitors. Background Art

[0002] Carbohydrates are broken down into disaccharides such as maltose during digestion in the body, and then broken down into monosaccharides such as glucose by α-glucosidase, which are then absorbed into the blood vessels. In this process, if the action of α-glucosidase can be inhibited, the cleavage of disaccharides into absorbable monosaccharides can be effectively delayed, thereby controlling the body's absorption of carbohydrates. Therefore, α-glucosidase inhibitors can significantly inhibit postprandial hyperglycemia, thereby effectively preventing and / or treating diabetes, obesity, etc.

[0003] Currently, the α-glucosidase inhibitors that have been marketed mainly include acarbose, metformin hydrochloride and miglitol, but the long-term use of these drugs may cause gastrointestinal reactions, liver damage and other side effects. Therefore, more and more pharmacologists and nutritionists prefer to develop safe and side-effect-free products with α-glucosidase inhibitory activity.

[0004] Peptides are compounds formed by connecting multiple α-amino acids with peptide chains. They have multiple biological activities and are safe and have no side effects. However, there are still few reported peptides with α-glucosidase inhibitory activity, which is not conducive to the development of clinical drugs. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, which has excellent α-glucosidase inhibitory activity, which can not only enrich the polypeptide resource library with α-glucosidase inhibitory effect, but also provide more raw material options for α-glucosidase inhibitors.

[0006] The first object of the present invention is to provide a polypeptide.

[0007] The second object of the present invention is to provide the use of the above polypeptide in the preparation of α-glucosidase inhibitors.

[0008] The third object of the present invention is to provide the use of the related biological materials of the above polypeptide in the preparation of α-glucosidase inhibitors.

[0009] The fourth object of the present invention is to provide the use of the above polypeptide in the preparation of antioxidant products.

[0010] The fifth object of the present invention is to provide the use of the related biological materials of the above polypeptide in the preparation of antioxidant products.

[0011] The sixth object of the present invention is to provide the use of the above polypeptide in preparing products for preventing and / or treating diabetes.

[0012] The seventh objective of the present invention is to provide the use of the related biological materials of the above polypeptide in the preparation of products for preventing and / or treating diabetes.

[0013] The eighth object of the present invention is to provide the use of the above polypeptide in the preparation of products for preventing and / or treating obesity.

[0014] The ninth objective of the present invention is to provide the use of the related biological materials of the above polypeptide in the preparation of products for preventing and / or treating obesity.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] The present invention finds that the polypeptide with an amino acid sequence as shown in SEQ ID NO:1 has excellent α-glucosidase inhibitory activity and DPPH free radical scavenging activity, that is, the polypeptide is suitable for preparing α-glucosidase inhibitors and antioxidant products, and can effectively prevent and / or treat diabetes and / or obesity. It can not only enrich the polypeptide resource library with α-glucosidase inhibitory and / or antioxidant effects, but also provide more raw material options for α-glucosidase inhibitors, antioxidant products, products for preventing and / or treating diabetes, and products for preventing and / or treating obesity. Therefore, the present invention provides a polypeptide with an amino acid sequence as shown in SEQ ID NO:1, and the use of the polypeptide in preparing any of the following products: (1) α-glucosidase inhibitors, (2) antioxidant products, (3) products for preventing and / or treating diabetes, and (4) products for preventing and / or treating obesity.

[0017] Based on this, the present invention also provides the use of related biological materials of the above-mentioned polypeptide in the preparation of any of the following products: (1) α-glucosidase inhibitors, (2) antioxidant products, (3) products for preventing and / or treating diabetes, and (4) products for preventing and / or treating obesity; wherein the related biological materials are nucleic acid molecules capable of expressing the above-mentioned polypeptide, or expression cassettes, recombinant cells, recombinant plasmids or recombinant bacteria containing the nucleic acid molecules.

[0018] Preferably, the diabetes is type 2 diabetes.

[0019] In addition, the present invention also provides a product containing the above polypeptide or its related biological material, wherein the related biological material is a nucleic acid molecule capable of expressing the above polypeptide, or an expression cassette, recombinant cell, recombinant plasmid or recombinant bacteria containing the nucleic acid molecule.

[0020] Preferably, the product is a medicine or a food.

[0021] More preferably, the medicine also contains pharmaceutically acceptable excipients.

[0022] More preferably, the auxiliary material is one or more of solid lubricants (such as stearic acid and / or magnesium stearate), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and / or cocoa butter), polyols (such as propylene glycol, glycerol, sorbitol, mannitol and / or polyethylene glycol), alginic acid, emulsifiers (such as Tween and / or polyoxyethylene castor oil), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, tablets, stabilizers, antioxidants, pyrogen-free water, isotonic saline solution, and phosphate buffer. The type of the auxiliary material can be selected according to the needs of improving drug stability, activity and / or bioavailability.

[0023] More preferably, the pharmaceutical preparation is one of tablets, capsules, powders, granules, syrups, solutions and oral liquids.

[0024] More preferably, the food further contains a food-acceptable auxiliary material.

[0025] More preferably, the food is a functional food.

[0026] The present invention has the following beneficial effects:

[0027] The present invention finds that the polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 has excellent α-glucosidase inhibitory activity and DPPH free radical scavenging activity, that is, the polypeptide is suitable for preparing α-glucosidase inhibitors and antioxidant products, and can effectively prevent and / or treat diabetes and / or obesity. It can not only enrich the resource library of polypeptides with α-glucosidase inhibitory and / or antioxidant effects, but also provide more raw material options for α-glucosidase inhibitors, antioxidant products, products for preventing and / or treating diabetes, and products for preventing and / or treating obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The purity analysis results of the polypeptide obtained in Example 1 are shown in FIG.

[0029] Figure 2 This is the mass spectrometry identification result of the polypeptide obtained in Example 1.

[0030] Figure 3 The results of the α-glucosidase inhibitory activity test of the polypeptide obtained in Example 1 are shown in FIG.

[0031] Figure 4 The following are the results of the DPPH free radical scavenging activity test of the polypeptide obtained in Example 1. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification, but the embodiments do not limit the present invention in any form. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. For example, the features described or described as part of an embodiment can be combined in another embodiment in a suitable manner to produce a new embodiment. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details. Unless otherwise specified, the reagents, methods and equipment used in the embodiments of the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the embodiments of the present invention are commercially available.

[0033] The mass or volume of the relevant components mentioned in the embodiments of the present invention description can not only refer to the specific amount of each component, but also represent the proportional relationship between the amounts of each component. Therefore, as long as the amount of the relevant components is proportionally enlarged or reduced according to the embodiment of the present invention description, it is within the scope disclosed in the embodiment of the present invention description. For example, the mass described in the embodiment of the present invention description can be units known in the biochemical field such as μg, mg, g, kg, etc., and the volume can be units known in the biochemical field such as μL, mL, L, etc.

[0034] The temperature parameters in the present invention, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed. In the present invention, if the units of the data range are only followed by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0035] In the examples of the present invention, each set of data was measured three times, and the results were expressed as mean ± standard deviation. SPSS software was used to analyze the obtained data, and the significance of the difference between samples was analyzed by one-way ANOVA, and p < 0.05 indicated that there was a significant difference.

[0036] Example 1 Synthesis and identification of polypeptides

[0037] 1. Synthesis of Peptides

[0038] With the support of Qiangyao Biotechnology Co., Ltd., this example uses solid phase synthesis to prepare a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1. Wherein, SEQ ID NO: 1: KLNSTTEKLEE.

[0039] 2. Analysis and identification of peptides

[0040] (1) Purity analysis method

[0041] The purity of the peptide was analyzed by RP-HPLC. The analysis conditions of RP-HPLC were as follows: chromatographic column: Kromasil C18 (100-5C18, 4.6×250 mm, 5 μm, column temperature 30°C); mobile phase A: acetonitrile (containing 0.1% (v / v) trifluoroacetic acid); mobile phase B: water (containing 0.1% (v / v) trifluoroacetic acid); elution gradient: 0→15 min, 15% (v / v) A, 15→20 min, 40% (v / v) A, 20→20.1 min, 100% (v / v) A; flow rate: 1 mL / min; detection wavelength: 220 nm; sample volume: 10 μL.

[0042] (2) Mass spectrometry identification method

[0043] The peptide was identified by ESI-MS mass spectrometry, and whether it was the target product was determined based on the molecular weight and theoretical value on the mass spectrum. The identification conditions of ESI-MS mass spectrometry were: ESI ion source, capillary voltage of 2.5-3.5 kV, desolvation temperature of 450°C, desolvation gas flow of 800 L / hr, cone voltage of 10-30 V, and running time of 1 min.

[0044] (3) Analysis and identification results

[0045] The results of peptide purity analysis are as follows Figure 1 The mass spectrometry identification results are shown in Figure 2 As shown. Figure 1 It can be seen that in the polypeptide of the present invention, the main peak accounts for the majority, only a few impurity peaks appear, and the purity is as high as 98wt%. Figure 2 It can be seen that the molecular ion peak m / z of the polypeptide of the present invention is 646.83, with two charges, which is consistent with its theoretical molecular weight value. This shows that the polypeptide synthesized in this example is the polypeptide with the amino acid sequence shown in SEQ ID NO: 1, and the purity meets the requirements of subsequent experiments.

[0046] Example 2 Determination of α-glucosidase inhibitory activity of polypeptides

[0047] Sample solution: The polypeptide obtained in Example 1 was dissolved in distilled water to a final concentration of 5, 62.5, 125, 250, and 500 μg / mL, respectively.

[0048] The experiment was divided into four groups, namely blank well, blank control well, sample reaction well, and sample background well. The types and volumes of reagents added to each well are shown in Table 1. 110 μL PBS (pH 6.8, 0.2 M), 20 μL sample solution and / or 20 μL α-glucosidase (3.3 U / mL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were added, and distilled water was used to make up to 150 μL. After each well was placed at 37°C for 10 min, 20 μL pNPG (1.25 mmol / L, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added, and then reacted at 37°C for 20 min, and 80 μL Na 2 CO 3 The reaction was terminated by adding 0.1 mol / L solution, and the absorbance of the system at 405 nm was finally measured. At the same time, 0.001, 0.01, 0.1, 1, and 10 mg / mL acarbose solutions were used as positive controls. Three parallel wells were set for each group, and the results were averaged.

[0049] Table 1 Types and volumes of reagents added to the four groups

[0050] Reagents Blank / μL Blank control / μL Sample reaction / μL Sample background / μL PBS 110 110 110 110 Sample solution -- -- 20 20 α-Glucosidase 20 -- 20 -- Distilled water 20 40 -- 20 pNPG 20 20 20 20 <![CDATA[Na 2 CO 3 Solution]]> 80 80 80 80

[0051] According to the formula "W α =[1-(A 3 -A 4 )] / (A 1 -A 2 )×100%” to calculate the α-glucosidase inhibition rate of the polypeptide obtained in Example 1, where A 1 is the absorbance value of the blank well; A 2 is the absorbance value of the blank control well; A 3 is the absorbance value of the sample reaction well; A 4 is the absorbance value of the sample background well.

[0052] The results are as follows Figure 3 As shown. It can be seen that the IC of the polypeptide obtained in Example 1 for inhibiting α-glucosidase is 50 The value was (144.89±3.04) μM, which was significantly lower than the IC of the positive control acarbose for inhibiting α-glucosidase. 50The value of (709.41±4.12) μM indicates that the polypeptide of the present invention has excellent α-glucosidase inhibitory activity, is suitable for the preparation of α-glucosidase inhibitors, and can effectively prevent and / or treat diabetes and / or obesity. It can not only enrich the resource library of polypeptides with α-glucosidase inhibitory effect, but also provide more raw material options for α-glucosidase inhibitors, products for preventing and / or treating diabetes, and products for preventing and / or treating obesity.

[0053] Example 3 Determination of DPPH free radical scavenging activity of polypeptides

[0054] The polypeptide obtained in Example 1 was dissolved in distilled water to obtain sample solutions with concentrations of 2.0, 4.0, and 8.0 mg / mL, respectively. The DPPH free radical scavenging rates of the sample solutions were determined and calculated according to the instructions of the DPPH free radical scavenging ability assay kit of Nanjing Jiancheng Bioengineering Institute.

[0055] The results are as follows Figure 4 As shown, the sample solutions with concentrations of 2.0, 4.0, and 8.0 mg / mL all have high DPPH free radical scavenging rates, which are 52.49%±1.08%, 64.43%±2.34%, and 81.00%±1.43%, respectively. This indicates that the polypeptide of the present invention has excellent antioxidant activity, is suitable for preparing antioxidant products, and can effectively prevent and / or treat diabetes. It can not only enrich the resource library of polypeptides with antioxidant effects, but also provide more raw material options for antioxidant products, products for preventing and / or treating diabetes, and products for preventing and / or treating obesity.

[0056] In summary, the present invention finds that the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 has excellent α-glucosidase inhibitory activity and DPPH free radical scavenging activity, that is, the polypeptide is suitable for preparing α-glucosidase inhibitors and antioxidant products, and can effectively prevent and / or treat diabetes and / or obesity. It can not only enrich the resource library of polypeptides with α-glucosidase inhibitory and / or antioxidant effects, but also provide more raw material options for α-glucosidase inhibitors, antioxidant products, products for preventing and / or treating diabetes, and products for preventing and / or treating obesity.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in the preparation of α-glucosidase inhibitors.

3. Use of the related biological material of the polypeptide according to claim 1 in the preparation of α-glucosidase inhibitors, characterized in that: The related biological material is a nucleic acid molecule capable of expressing the polypeptide according to claim 1, or an expression cassette, recombinant cell, recombinant plasmid or recombinant bacteria containing the nucleic acid molecule.

4. Use of the polypeptide according to claim 1 in the preparation of antioxidant products.

5. Use of the related biological materials of the polypeptide according to claim 1 in the preparation of antioxidant products, characterized in that: The related biological material is a nucleic acid molecule capable of expressing the polypeptide according to claim 1, or an expression cassette, recombinant cell, recombinant plasmid or recombinant bacteria containing the nucleic acid molecule.

6. Use of the polypeptide according to claim 1 in preparing a product for preventing and / or treating diabetes.

7. Use of the related biological materials of the polypeptide according to claim 1 in preparing products for preventing and / or treating diabetes, characterized in that: The related biological material is a nucleic acid molecule capable of expressing the polypeptide according to claim 1, or an expression cassette, recombinant cell, recombinant plasmid or recombinant bacteria containing the nucleic acid molecule.

8. Use of the polypeptide according to claim 1 in preparing a product for preventing and / or treating obesity.

9. Use of the related biological materials of the polypeptide according to claim 1 in preparing products for preventing and / or treating obesity, characterized in that: The related biological material is a nucleic acid molecule capable of expressing the polypeptide according to claim 1, or an expression cassette, recombinant cell, recombinant plasmid or recombinant bacteria containing the nucleic acid molecule.

10. The use according to any one of claims 4 to 9, characterized in that: The product is a medicine or a food.

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