An active peptide and its use in the preparation of a medicament for treating diabetes

Abstract

The application provides an active peptide and application thereof in preparation of a medicine for treating diabetes. The application finds that the active peptide with an amino acid sequence shown in SEQ ID NO:1 can not only effectively remove DPPH free radicals, but also effectively inhibit alpha-glucosidase, which indicates that the active peptide can be used for preparing an antioxidant product, an alpha-glucosidase inhibitor, and simultaneously indicates that the active peptide can effectively treat obesity and diabetes, i.e., can be used for preparing a product for treating diabetes and a product for treating obesity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to an active peptide and its application in the preparation of a drug for treating diabetes. Background Technology

[0002] Diabetes is a chronic metabolic disease. Long-term hyperglycemia can damage various organs and systems in the body, leading to a variety of complications, including: (1) Cardiovascular disease: Hyperglycemia may damage vascular endothelial cells, promote the formation of atherosclerosis, and increase the risk of cardiovascular diseases such as heart disease, stroke, and lower extremity arterial occlusion; (2) Neuropathy: Long-term hyperglycemia may cause nerve damage, causing symptoms such as abnormal sensation in the extremities, pain, and numbness. In severe cases, it may lead to consequences such as lower extremity ulcers and amputation; (3) Kidney disease: Hyperglycemia may damage the glomeruli and renal tubules, leading to proteinuria and a gradual decline in kidney function, which may eventually develop into uremia; (4) Eye disease: Hyperglycemia may cause cataracts. (5) Diabetic autonomic neuropathy: including cardiovascular symptoms (such as tachycardia or bradycardia, blood pressure fluctuations, etc.), digestive symptoms (such as nausea, vomiting, abdominal distension, diarrhea or constipation, etc.), genitourinary symptoms (such as urinary incontinence, sexual dysfunction, etc.), and other symptoms (such as pupil abnormalities, abnormal sweating, etc.); (6) Decreased blood glucose self-regulation ability: leading to large blood glucose fluctuations, which may result in diabetic ketoacidosis, hyperosmolar hyperglycemia, etc.; (7) Other complications: diabetes can also reduce immunity, making patients more susceptible to various diseases, such as oral infections, skin infections, urinary tract infections, tuberculosis, etc.

[0003] Currently, there are three main treatment methods for diabetes, but each method has its own limitations: (1) Drug treatment: Metformin, glibenclamide, glipizide, nateglinide, insulin and other drugs are used, but these drugs may cause side effects such as hypoglycemia and weight gain; (2) Surgical treatment: Metabolic weight loss, pancreas transplantation, islet cell transplantation and other surgeries are performed, but the surgical risk is high and not all patients are suitable for surgical treatment; (3) Dietary regulation: A light diet, eat more high-fiber foods such as kelp and konjac, and eat less animal oil, as well as less foods with high sugar and cholesterol such as crab roe, shrimp roe, condensed milk, and candied fruit. However, dietary regulation can only be used as an auxiliary means and has limited effect on patients with severe conditions.

[0004] Bioactive peptides have received considerable attention in the treatment of diseases such as tumors, thrombosis, and hypertension due to their advantages such as safety, non-toxicity, and suitability for targeted therapy. However, the existing bioactive peptide resources available for the treatment of diabetes are still relatively scarce. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing an active peptide with an amino acid sequence as shown in SEQ ID NO:1, which can effectively scavenge DPPH free radicals and inhibit α-glucosidase, thereby playing a role in the prevention and treatment of diabetes and enriching the resource library of active peptides that can be used to treat diabetes.

[0006] The primary objective of this invention is to provide an active peptide.

[0007] Another object of the present invention is to provide a biomaterial related to an active peptide.

[0008] Another object of the present invention is to provide the use of the above-mentioned active peptides or related biomaterials in the preparation of products for treating diabetes.

[0009] Another object of the present invention is to provide a product for treating diabetes.

[0010] Another object of the present invention is to provide the use of the above-mentioned active peptides or related biomaterials in the preparation of products for treating obesity.

[0011] Another object of the present invention is to provide a product for treating obesity.

[0012] Another object of the present invention is to provide the application of the above-mentioned active peptides or related biomaterials in the preparation of antioxidant products.

[0013] Another object of the present invention is to provide an antioxidant product.

[0014] Another object of the present invention is to provide the application of the above-mentioned active peptides or related biological materials in the preparation of α-glucosidase inhibitors.

[0015] Another object of the present invention is to provide an α-glucosidase inhibitor.

[0016] The above-mentioned objective of this invention is achieved through the following technical solution:

[0017] This invention discovers that the active peptide with the amino acid sequence shown in SEQ ID NO:1 can not only effectively scavenge DPPH free radicals but also effectively inhibit α-glucosidase. This indicates that the active peptide of this invention can be used to prepare antioxidant products and α-glucosidase inhibitors. Furthermore, it also indicates that the active peptide of this invention can effectively treat obesity and diabetes, i.e., it can be used to prepare products for treating diabetes and obesity. These findings are of crucial significance in the field of obesity and diabetes treatment. Therefore, the active peptide with the amino acid sequence shown in SEQ ID NO:1, and its related biological materials, should be within the scope of protection of this invention. The related biological materials are nucleic acid molecules capable of expressing the above-mentioned active peptides, or recombinant bacteria, recombinant plasmids, recombinant cells, or expression cassettes containing the nucleic acid molecules.

[0018] Based on this, the present invention also provides (1) the application of the above-mentioned active peptide or the above-mentioned related biological material in the preparation of a product for treating diabetes, (2) a product for treating diabetes, (3) the application of the above-mentioned active peptide or the above-mentioned related biological material in the preparation of a product for treating obesity, (4) a product for treating obesity, (5) the application of the above-mentioned active peptide or the above-mentioned related biological material in the preparation of an antioxidant product, (6) an antioxidant product, (7) the application of the above-mentioned active peptide or the above-mentioned related biological material in the preparation of an α-glucosidase inhibitor, and (8) an α-glucosidase inhibitor.

[0019] Preferably, the diabetes is type 2 diabetes.

[0020] Preferably, the product is a drug.

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

[0022] More preferably, the excipients are one or more of the following: sugars (such as lactose, glucose and / or sucrose), starches (such as corn starch and / or potato starch), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and / or methyl cellulose), tragacanth powder, malt, gelatin, and talc.

[0023] More preferably, the drug formulation is one of the following: oral liquid, tincture, tincture, aerosol, powder inhaler, injection, sterile powder for injection, or suppository. The above formulation types can be understood according to the relevant definitions in *Pharmaceutics* (6th Edition, People's Medical Publishing House, Cui Fude), and the preparation of the above formulations can be carried out according to the relevant formulation methods in *Pharmaceutics* (6th Edition, People's Medical Publishing House, Cui Fude).

[0024] The present invention has the following beneficial effects:

[0025] The present invention has discovered that the active peptide with an amino acid sequence as shown in SEQ ID NO:1 can not only effectively scavenge DPPH free radicals, but also effectively inhibit α-glucosidase. This indicates that the active peptide of the present invention can be used to prepare antioxidant products and α-glucosidase inhibitors. Furthermore, it also indicates that the active peptide of the present invention can effectively treat obesity and diabetes, that is, it can be used to prepare products for treating diabetes and products for treating obesity. Attached Figure Description

[0026] Figure 1 The results are from mass spectrometry identification of bioactive peptides.

[0027] Figure 2 The results are from the purity analysis of the bioactive peptides.

[0028] Figure 3The results show the DPPH free radical scavenging activity of the bioactive peptides.

[0029] Figure 4 The results are the assay results for the α-glucosidase inhibitory activity of the active peptide. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0031] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features. In this invention, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The "numerical interval" can broadly include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0032] In this invention, where the method involves multiple steps, unless otherwise explicitly stated herein, the execution of these steps is not strictly limited in order and can be performed in any order other than that described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0033] In the embodiments described in this invention, each set of data was measured three times, and the results are expressed as mean ± standard deviation. The obtained data were analyzed using SPSS software, and the significance of differences between samples was determined by one-way ANOVA; p < 0.05 indicated a significant difference.

[0034] Example 1: Synthesis and Identification of Active Peptides

[0035] I. Synthesis of bioactive peptides

[0036] With the support of Qiangyao Biotechnology Co., Ltd., this embodiment uses solid-phase synthesis to prepare an active peptide with the amino acid sequence shown in SEQ ID NO:1. SEQ ID NO:1: TDADHKF.

[0037] II. Analysis and Identification of Bioactive Peptides

[0038] (1) Mass spectrometry identification method

[0039] Active peptides are identified using ESI-MS mass spectrometry, and their molecular weight and theoretical value are used to determine whether they are the target products.

[0040] The identification conditions for ESI-MS mass spectrometry are as follows:

[0041] ESI ion source

[0042] Capillary voltage: 2.5–3.5 kV

[0043] Desolventization temperature: 450 ℃

[0044] Desolvation gas flow rate: 800 L / hr

[0045] Tapered hole voltage: 10~30 V,

[0046] Running time: 1 min.

[0047] (2) Purity analysis method

[0048] The purity of the bioactive peptides was analyzed using RP-HPLC.

[0049] The analytical conditions for RP-HPLC are as follows:

[0050] Column: Kromasil C18 (100-5C18, 4.6×250 mm, 5 μm, column temperature 30 °C);

[0051] Mobile phase A: Acetonitrile (containing 0.1% ( v / v (trifluoroacetic acid);

[0052] Mobile phase B: Water (containing 0.1% ( v / v (trifluoroacetic acid);

[0053] Elution gradient: 0 → 15 min, 15% ( v / v A, 15→20 min, 40% ( v / v )A, 20→20.1 min, 100% ( v / v A;

[0054] Flow rate: 1 mL / min;

[0055] Detection wavelength: 220 nm;

[0056] Sample loading volume: 10 μL.

[0057] (3) Analysis and identification results

[0058] Mass spectrometry identification results of bioactive peptides are as follows Figure 1 As shown, the purity analysis results are as follows: Figure 2 As shown, the molecular ion peak m / z of the active peptide of this invention is 417.53, carrying two charges, consistent with its theoretical molecular weight. Furthermore, in the purity analysis results, the main peak accounts for the majority, with only a few impurity peaks appearing, indicating a purity as high as 98 wt%. This demonstrates that the active peptide synthesized in this embodiment is the active peptide with the amino acid sequence shown in SEQ ID NO:1, and has high purity.

[0059] Example 2: Determination of DPPH free radical scavenging activity of active peptides

[0060] The active peptide obtained in Example 1 was dissolved in distilled water to obtain sample solutions with concentrations of 1.0, 2.0, and 4.0 mg / mL, respectively. The DPPH free radical scavenging rate of the sample solutions was determined and calculated according to the instructions of the DPPH free radical scavenging ability assay kit of Nanjing Jiancheng Bioengineering Institute.

[0061] The results are as follows Figure 3 As shown, sample solutions with concentrations of 1.0, 2.0, and 4.0 mg / mL all exhibited high DPPH free radical scavenging rates of 28.50% ± 1.53%, 66.51% ± 3.13%, and 92.58% ± 2.94%, respectively. This indicates that the active peptide of the present invention possesses excellent antioxidant activity and is suitable for preparing antioxidant products. It also indicates that the active peptide of the present invention can effectively treat diabetes, i.e., it can be used to prepare products for treating diabetes.

[0062] Example 3: Assay of α-glucosidase inhibitory activity of active peptides

[0063] First, the active peptide obtained in Example 1 was dissolved in distilled water to obtain sample solutions with final concentrations of 0.0022, 0.0275, 0.055, 0.11, and 0.22 mg / mL (with acarbose solutions of 0.001, 0.01, 0.1, 1, and 10 mg / mL as positive controls). The experiment was then divided into four groups (each group had three parallel wells, and the results were averaged): sample reaction group, sample background group, blank group, and blank control group, as detailed below:

[0064] (1) Sample reaction group: 110 μL PBS (pH 6.8, 0.2 M), 20 μL sample solution, and 20 μL α-glucosidase (3.3 U / mL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were added to a 96-well plate.

[0065] (2) Sample background group: Add 110 μL PBS (pH 6.8, 0.2 M), 20 μL sample solution and 20 μL distilled water to a 96-well plate.

[0066] (3) Blank group: Add 110 μL PBS (pH 6.8, 0.2 M), 20 μL α-glucosidase (3.3 U / mL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and 20 μL distilled water to a 96-well plate.

[0067] (4) Blank control group: 110 μL of PBS (pH 6.8, 0.2 M) and 40 μL of distilled water were added to a 96-well plate.

[0068] After each group was placed at 37 °C for 10 min, 20 μL of pNPG (p-nitrophenyl-α-D-glucoside, 1.25 mmol / L, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added, and then the reaction was carried out at 37 °C for 20 min. The reaction was terminated by adding 80 μL of Na2CO3 solution (0.1 mol / L). Finally, the absorbance of the system at 405 nm was measured.

[0069] According to the formula "W" α =[1 (A1 A2)] / (A3 The α-glucosidase inhibition rate of the active peptide obtained in Example 1 was calculated by A4)×100%”, where A1 is the absorbance value of the sample reaction group; A2 is the absorbance value of the sample background group; A3 is the absorbance value of the blank group; and A4 is the absorbance value of the blank control group.

[0070] The results are as follows Figure 4 As shown. It can be seen that the active peptide obtained in Example 1 inhibits the IC50 of α-glucosidase. 50 The value was (136.96±2.12) μM, significantly lower than the IC50 value of acarbose for inhibiting α-glucosidase in the positive control. 50 The value ((709.41±4.12) μM) indicates that the active peptide of the present invention has excellent α-glucosidase inhibitory activity and is suitable for preparing α-glucosidase inhibitors. It also indicates that the active peptide of the present invention can effectively treat obesity and diabetes, that is, it can be used to prepare products for treating diabetes and products for treating obesity.

[0071] In summary, the present invention has discovered that the active peptide with the amino acid sequence shown in SEQ ID NO:1 can not only effectively scavenge DPPH free radicals, but also effectively inhibit α-glucosidase. This indicates that the active peptide of the present invention can be used to prepare antioxidant products and α-glucosidase inhibitors. Furthermore, it also indicates that the active peptide of the present invention can effectively treat obesity and diabetes, that is, it can be used to prepare products for treating diabetes and products for treating obesity.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An active peptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A biomaterial related to an active peptide, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:1; the related biological material is a nucleic acid molecule capable of expressing the active peptide of claim 1, or a recombinant bacterium, recombinant plasmid, recombinant cell or expression cassette containing the nucleic acid molecule.

3. The application of the active peptide of claim 1 or the related biomaterial of claim 2 in the preparation of products for treating diabetes, characterized in that, The product in question is a medicine.

4. A product for treating diabetes, characterized in that, Contains the active peptide of claim 1 or the related biomaterial of claim 2.

5. The use of the active peptide of claim 1 or the related biomaterial of claim 2 in the preparation of products for treating obesity, characterized in that, The product in question is a medicine.

6. A product for treating obesity, characterized in that, Contains the active peptide of claim 1 or the related biomaterial of claim 2.

7. The application of the active peptide of claim 1 or the related biomaterial of claim 2 in the preparation of antioxidant products, characterized in that, The product is a medicine.

8. An antioxidant product, characterized in that, Contains the active peptide of claim 1 or the related biomaterial of claim 2.

9. An α-glucosidase inhibitor, characterized in that, Contains the active peptide of claim 1 or the related biomaterial of claim 2.

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