A polypeptide and its application in preparing α-glucosidase inhibitor

Abstract

The present invention provides a polypeptide and its application in the preparation of α-glucosidase inhibitors. The present invention finds that the polypeptide having 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, thereby effectively preventing and / or treating diabetes and / or obesity, not only enriching the polypeptide resource library with α-glucosidase inhibitory and / or antioxidant effects, but also providing 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.

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] During digestion, carbohydrates are broken down into disaccharides like maltose, which are then broken down by α-glucosidase into monosaccharides like glucose, which are then absorbed into the bloodstream. Inhibiting α-glucosidase during this process can effectively delay the breakdown of disaccharides into absorbable monosaccharides, thereby controlling the body's absorption of carbohydrates. Therefore, α-glucosidase inhibitors can significantly suppress postprandial hyperglycemia, effectively preventing and / or treating diabetes, obesity, and other conditions.

[0003] Currently, the main α-glucosidase inhibitors on the market include acarbose, metformin hydrochloride, and miglitol. However, long-term use of these drugs may cause side effects such as gastrointestinal reactions and liver damage. Therefore, a growing number of pharmacologists and nutritionists are inclined to develop safe and side-effect-free products with α-glucosidase inhibitory activity.

[0004] Peptides are compounds composed of multiple α-amino acids linked together in peptide chains. They possess diverse biological activities and are safe and free of side effects. However, there are still relatively few reported peptides with α-glucosidase inhibitory activity, hindering the development of clinical drugs. Summary of the Invention

[0005] In response to 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. This not only enriches the resource library of polypeptides with α-glucosidase inhibitory effect, but also provides 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 biomaterials 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 biological materials related to 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 object of the present invention is to provide the use of biological materials related to 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 biological materials related to 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 has found that a polypeptide having 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 having 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 having 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) an α-glucosidase inhibitor, (2) an antioxidant product, (3) a product for preventing and / or treating diabetes, and (4) a product 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 pharmaceutical product.

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

[0022] More preferably, the excipients are 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, flavorings, tableting agents, stabilizers, antioxidants, pyrogen-free water, isotonic saline solution, and phosphate buffer. The type of excipient can be selected based on the need to improve 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] The present invention has the following beneficial effects:

[0025] The present invention discovered that a polypeptide having 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, thereby effectively preventing and / or treating diabetes and / or obesity. This not only enriches the resource library of polypeptides having α-glucosidase inhibitory and / or antioxidant effects, but also provides 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

[0026] Figure 1 The purity analysis results of the polypeptide obtained in Example 1 are shown in FIG.

[0027] Figure 2 The mass spectrometry results of the polypeptide obtained in Example 1 are shown.

[0028] Figure 3 The results of the α-glucosidase inhibitory activity test of the polypeptide obtained in Example 1 are shown.

[0029] Figure 4 The following are the results of the DPPH free radical scavenging activity test of the polypeptide obtained in Example 1. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the examples 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 present disclosure 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 one embodiment can be combined in another embodiment in a suitable manner to produce new embodiments. In addition, in the description below, a large amount of specific details are given in order to provide a more complete 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 stated, the reagents, methods and equipment used in the examples of the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the examples of the present invention are all commercially available.

[0031] The mass or volume of the relevant components mentioned in the embodiments of the present invention may not only refer to the specific amount of each component, but also represent the proportional relationship between the amounts of the components. Therefore, as long as the amount of the relevant components is proportionally increased or decreased according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. For example, the mass described in the description of the embodiments of the present invention may be units known in the biochemical field such as μg, mg, g, kg, etc., and the volume described may be units known in the biochemical field such as μL, mL, L, etc.

[0032] The temperature parameters in the present invention, unless otherwise specified, allow both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range of the instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted. In the present invention, when referring to the unit of a data range, if only the right endpoint is followed by the unit, it means that the units of the left and right endpoints 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).

[0033] In the examples of the present invention, each set of data was measured three times, and the results were expressed as mean ± standard deviation. The obtained data were analyzed using SPSS software, and the differences between samples were analyzed by one-way analysis of variance. P < 0.05 indicated a significant difference.

[0034] Example 1 Synthesis and identification of polypeptides

[0035] 1. Synthesis of Peptides

[0036] 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. SEQ ID NO: 1: KLNSTTEKLEE.

[0037] 2. Analysis and identification of peptides

[0038] (1) Purity analysis method

[0039] The purity of the peptide was analyzed by RP-HPLC. The analytical conditions of RP-HPLC were as follows: 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.

[0040] (2) Mass spectrometry identification method

[0041] Peptides were identified using ESI-MS, with the molecular weight and theoretical value of the mass spectra used to determine if they were the target product. ESI-MS identification conditions were: ESI source, capillary voltage of 2.5–3.5 kV, desolvation temperature of 450°C, desolvation gas flow rate of 800 L / hr, cone voltage of 10–30 V, and run time of 1 min.

[0042] (3) Analysis and identification results

[0043] 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, with only a few impurity peaks appearing, 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 its purity meets the requirements of subsequent experiments.

[0044] Example 2 Determination of α-glucosidase inhibitory activity of polypeptides

[0045] Sample solutions: 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.

[0046] The experiment was divided into four groups: blank wells, blank control wells, sample reaction wells, and sample background wells. The reagent types and volumes added to each well are shown in Table 1. 110 μL of PBS (pH 6.8, 0.2 M), 20 μL of sample solution, and / or 20 μL of α-glucosidase (3.3 U / mL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were added, and the volume was made up to 150 μL with distilled water. Each well was incubated at 37°C for 10 min, followed by the addition of 20 μL of pNPG (1.25 mmol / L, purchased from Shanghai Yuanye Biotechnology Co., Ltd.). The reaction was then incubated at 37°C for 20 min. The reaction was terminated by the addition of 80 μL of Na₂CO₃ solution (0.1 mol / L), and the absorbance at 405 nm was measured. Acarbose solutions at concentrations of 0.001, 0.01, 0.1, 1, and 10 mg / mL were used as positive controls. Three replicate wells were set up for each group, and the results were averaged.

[0047] Table 1 Types and volumes of reagents added to the four groups

[0048]

[0049] According to the formula "W α =[1-(A3-A4)] / (A1-A2)×100%” to calculate the α-glucosidase inhibition rate of the polypeptide obtained in Example 1, wherein A1 is the absorbance value of the blank well; A2 is the absorbance value of the blank control well; A3 is the absorbance value of the sample reaction well; and A4 is the absorbance value of the sample background well.

[0050] 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. 50 The value was (709.41±4.12) μM), indicating that the polypeptide of the present invention has excellent α-glucosidase inhibitory activity and is suitable for the preparation of α-glucosidase inhibitors, thereby effectively preventing and / or treating 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.

[0051] Example 3 Determination of DPPH free radical scavenging activity of polypeptides

[0052] 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 radical scavenging rates of the sample solutions were determined and calculated according to the instructions of the DPPH radical scavenging ability assay kit from Nanjing Jiancheng Bioengineering Institute.

[0053] The results are as follows Figure 4 As shown, sample solutions at concentrations of 2.0, 4.0, and 8.0 mg / mL all exhibited high DPPH radical scavenging rates of 52.49% ± 1.08%, 64.43% ± 2.34%, and 81.00% ± 1.43%, respectively. This indicates that the polypeptide of the present invention possesses excellent antioxidant activity and is suitable for use in the preparation of antioxidant products, thereby effectively preventing and / or treating diabetes. This not only enriches the resource library of polypeptides with antioxidant effects, but also provides more raw material options for antioxidant products, products for preventing and / or treating diabetes, and products for preventing and / or treating obesity.

[0054] In summary, the present invention discovered that the polypeptide having 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.

[0055] 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 considered as equivalent replacement methods and are included in the scope of protection 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 antioxidant products.

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

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

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

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

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

8. The use according to any one of claims 2 to 7, characterized in that: The product described is a pharmaceutical product.

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