Active peptide and application thereof in preparation of medicine for treating diabetes

Abstract

The invention provides an active peptide and application thereof in preparation of a medicine for treating diabetes mellitus. It is found that the active peptide with the amino acid sequence shown as SEQ ID NO: 1 can effectively remove DPPH free radicals and effectively inhibit alpha-glucosidase, it is indicated that the active peptide can be used for preparing antioxidant products and alpha-glucosidase inhibitors, and it is also indicated that the active peptide can effectively treat obesity and diabetes mellitus, that is, the active peptide can be used for preparing anti-oxidation products and alpha-glucosidase inhibitors. The traditional Chinese medicine composition can be used for preparing products for treating diabetes and obesity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to an active peptide and its application in preparing a medicine for treating diabetes. Background Art

[0002] Diabetes is a chronic metabolic disease. Long-term high blood sugar levels can cause damage to various organs and systems in the body, leading to a variety of complications, including: (1) Cardiovascular disease: High blood sugar levels may damage vascular endothelial cells, promote the formation of atherosclerosis, and increase the risk of cardiovascular diseases such as heart disease, stroke, and lower limb artery occlusion; (2) Neuropathy: Long-term high blood sugar levels may cause nerve damage, leading to symptoms such as paresthesia, pain, and numbness in the extremities. In severe cases, it may lead to lower limb ulcers, amputation, and other consequences; (3) Kidney disease: High blood sugar levels may damage the glomeruli and renal tubules, leading to proteinuria and a gradual decline in renal function, which may eventually develop into uremia; (4) Eye disease: High blood sugar levels may cause white spots in the eye. It can also affect the microcirculation of the retina and cause retinopathy, which may lead to blindness in severe cases; (5) Diabetic autonomic neuropathy: including cardiovascular system symptoms (such as tachycardia or bradycardia, blood pressure fluctuations, etc.), digestive system symptoms (such as nausea, vomiting, abdominal distension, diarrhea or constipation, etc.), genitourinary system symptoms (such as urinary incontinence, sexual dysfunction, etc.), and other symptoms (such as abnormal pupils, abnormal sweating, etc.); (6) Decreased blood sugar self-regulation ability: leading to large fluctuations in blood sugar, which may cause diabetic ketoacidosis, hyperosmolar hyperglycemic syndrome, 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 treatments for diabetes, but each has its own limitations: (1) Drug therapy: use of metformin, glibenclamide, glipizide, nateglinide, insulin and other drugs, but these drugs may cause side effects such as hypoglycemia and weight gain; (2) Surgical treatment: metabolic weight loss, pancreatic transplantation, islet cell transplantation and other surgeries, but the surgical risks are high and not all patients are suitable for surgical treatment; (3) Dietary conditioning: eat a light diet, eat more high-fiber foods such as kelp and konjac, and eat less animal oil, crab roe, shrimp eggs, condensed milk, candied fruit and other foods with high sugar and cholesterol content. However, dietary conditioning can only be used as an auxiliary means and has limited effect on patients with severe conditions.

[0004] Active peptides have received considerable attention in the treatment of diseases such as tumors, thrombosis, and hypertension due to their safety, non-toxicity, and suitability for targeted therapy. However, the existing active peptide resources that can be used to treat diabetes are still relatively scarce. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide an active peptide with an amino acid sequence as shown in SEQ ID NO: 1, which can effectively scavenge DPPH free radicals and effectively inhibit α-glucosidase, thereby playing a preventive and therapeutic role on diabetes, and enriching the active peptide resource library that can be used to treat diabetes.

[0006] The primary purpose of the present invention is to provide an active peptide.

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

[0008] Another object of the present invention is to provide the use of the above active peptides or the above 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 active peptides or the above 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 use of the above active peptides or the above related biological materials 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 use of the above active peptide or the above related biological material 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 purpose of the present invention is achieved through the following technical solutions:

[0017] The present invention finds that the active peptide with an amino acid sequence as shown in SEQ ID NO: 1 can not only effectively remove DPPH free radicals, but also effectively inhibit α-glucosidase, indicating that the active peptide of the present invention can be used to prepare antioxidant products and α-glucosidase inhibitors, and at the same time, it also shows 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. All of these are of vital importance to the field of obesity and diabetes treatment. Therefore, the active peptide with an amino acid sequence as shown in SEQ ID NO: 1, and its related biological materials should be within the scope of protection of the present invention. Among them, 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 use of the above-mentioned active peptide or the above-mentioned related biomaterial in the preparation of a product for treating diabetes, (2) a product for treating diabetes, (3) the use of the above-mentioned active peptide or the above-mentioned related biomaterial in the preparation of a product for treating obesity, (4) a product for treating obesity, (5) the use of the above-mentioned active peptide or the above-mentioned related biomaterial in the preparation of an antioxidant product, (6) an antioxidant product, (7) the use of the above-mentioned active peptide or the above-mentioned related biomaterial 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 medicine or a food.

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

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

[0023] Further preferably, the preparation of the drug is one of oral liquid, spirit, tincture, aerosol, powder spray, injection, sterile powder for injection, and suppository. The above preparation types can be understood according to the relevant definitions in Pharmaceutics (Sixth Edition, People's Medical Publishing House, Cuifude), and the preparation of the above preparation can be prepared according to the methods of relevant preparations in Pharmaceutics (Sixth Edition, People's Medical Publishing House, Cuifude).

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

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

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

[0027] The present invention found 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, indicating that the active peptide of the present invention can be used to prepare antioxidant products and α-glucosidase inhibitors, and also shows 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of mass spectrometry identification of active peptides.

[0029] Figure 2 The purity analysis results of active peptides.

[0030] Figure 3 The results are for the determination of DPPH free radical scavenging activity of active peptides.

[0031] Figure 4 The results are for the determination of α-glucosidase inhibitory activity of active peptides. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features. In the present invention, it is related to a numerical interval (i.e., a numerical range). Unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only points to the integers in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each 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 range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0034] In the present invention, if there are multiple steps involved in the method flow, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[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 active peptides

[0037] 1. Synthesis of active peptides

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

[0039] 2. Analysis and identification of active peptides

[0040] (1) Mass spectrometry identification method

[0041] ESI-MS mass spectrometry was used to identify the active peptides, and whether they were the target products was determined based on the molecular weight and theoretical value on the mass spectrum.

[0042] Among them, the identification conditions of ESI-MS mass spectrometry are:

[0043] ESI ion source,

[0044] Capillary voltage: 2.5~3.5kV,

[0045] Desolventization temperature: 450℃,

[0046] Desolvation gas flow rate: 800L / hr,

[0047] Cone voltage: 10~30V,

[0048] Running time: 1min.

[0049] (2) Purity analysis method

[0050] The purity of the active peptide was analyzed by RP-HPLC.

[0051] Among them, the analysis conditions of RP-HPLC are:

[0052] Chromatographic column: Kromasil C18 (100-5C18, 4.6×250 mm, 5 μm, column temperature 30°C);

[0053] Mobile phase A: acetonitrile (containing 0.1% (v / v) trifluoroacetic acid);

[0054] Mobile phase B: water (containing 0.1% (v / v) trifluoroacetic acid);

[0055] Elution gradient: 0→15 min, 15% (v / v) A, 15→20 min, 40% (v / v) A, 20→20.1 min, 100% (v / v) A;

[0056] Flow rate: 1 mL / min;

[0057] Detection wavelength: 220nm;

[0058] Sample volume: 10 μL.

[0059] (3) Analysis and identification results

[0060] The results of mass spectrometry identification of active peptides are as follows Figure 1 The purity analysis results are shown in Figure 2 As shown. It can be seen that the molecular ion peak m / z of the active peptide of the present invention is 417.53, with two charges, which is consistent with its theoretical molecular weight value, and in the purity analysis results, the main peak accounts for the main proportion, only very few impurity peaks appear, and the purity is as high as 98wt%. It shows that the active peptide synthesized in this example is the active peptide with an amino acid sequence as shown in SEQ ID NO: 1, and has a high purity.

[0061] Example 2 Determination of DPPH free radical scavenging activity of active peptides

[0062] 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 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.

[0063] The results are as follows Figure 3 As shown, the sample solutions with concentrations of 1.0, 2.0, and 4.0 mg / mL all have high DPPH free radical scavenging rates, which are 28.50%±1.53%, 66.51%±3.13%, and 92.58%±2.94%, respectively. This indicates that the active peptide of the present invention has excellent antioxidant activity and is suitable for preparing antioxidant products, indicating that the active peptide of the present invention can effectively treat diabetes, that is, can be used to prepare products for treating diabetes.

[0064] Example 3 Determination of α-glucosidase inhibitory activity of active peptides

[0065] First, the active peptide obtained in Example 1 was dissolved in distilled water to a final concentration of 0.0022, 0.0275, 0.055, 0.11, and 0.22 mg / mL, respectively, to obtain a sample solution (and 0.001, 0.01, 0.1, 1, and 10 mg / mL acarbose solutions were used as positive controls). The experiment was then divided into four groups (three parallel wells were set in each group, and the results were averaged), namely, a sample reaction group, a sample background group, a blank group, and a blank control group, as follows:

[0066] (1) Sample reaction group: In a 96-well plate, add 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.).

[0067] (2) Sample background group: In a 96-well plate, add 110 μL PBS (pH 6.8, 0.2 M), 20 μL sample solution, and 20 μL distilled water.

[0068] (3) Blank group: In a 96-well plate, 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 were added.

[0069] (4) Blank control group: 110 μL PBS (pH 6.8, 0.2 M) and 40 μL distilled water were added to a 96-well plate.

[0070] Each group was placed at 37°C for 10 min, and then 20 μL pNPG (p-nitrophenol-β-D-galactoside, 1.25 mmol / L, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added. The mixture was 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 was measured at 405 nm.

[0071] According to the formula "W α =[1-(A 1 -A 2 )] / (A 3 -A 4 )×100%” to calculate the α-glucosidase inhibition rate of the active peptide obtained in Example 1, where A 1 is the absorbance value of the sample reaction group; A 2 is the absorbance value of the sample background group; A 3 is the absorbance value of the blank group; A 4 is the absorbance value of the blank control group.

[0072] The results are as follows Figure 4 As shown. It can be seen that the IC of the active peptide obtained in Example 1 to inhibit α-glucosidase 50 The value was (136.96±2.12)μM, which was significantly lower than the IC of the positive control acarbose for inhibiting α-glucosidase 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 the preparation of α-glucosidase inhibitors, indicating that the active peptide of the present invention can effectively treat obesity and diabetes, that is, it can be used to prepare products for the treatment of diabetes and products for the treatment of obesity.

[0073] In summary, the present invention found 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, indicating that the active peptide of the present invention can be used to prepare antioxidant products and α-glucosidase inhibitors, and at the same time also shows 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.

[0074] 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. 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 according to claim 1, or a recombinant bacterium, recombinant plasmid, recombinant cell or expression cassette containing the nucleic acid molecule.

3. Use of the active peptide according to claim 1 or the related biomaterial according to claim 2 in the preparation of products for treating diabetes.

4. A product for treating diabetes, characterized in that: Containing the active peptide according to claim 1 or the related biological material according to claim 2.

5. Use of the active peptide according to claim 1 or the related biomaterial according to claim 2 in the preparation of products for treating obesity.

6. A product for treating obesity, characterized in that: Containing the active peptide according to claim 1 or the related biological material according to claim 2.

7. Use of the active peptide according to claim 1 or the related biological material according to claim 2 in the preparation of antioxidant products.

8. An antioxidant product, characterized in that: Containing the active peptide according to claim 1 or the related biological material according to claim 2.

9. Use of the active peptide according to claim 1 or the related biomaterial according to claim 2 in the preparation of α-glucosidase inhibitors.

10. An α-glucosidase inhibitor, characterized in that Containing the active peptide according to claim 1 or the related biological material according to claim 2.

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