Application of small molecule peptide LKLKLL in the preparation of hypoglycemic products

Abstract

This invention discloses the application of the small molecule peptide LKLKLL in the preparation of hypoglycemic products. Experiments have demonstrated that the small molecule peptide LKLKLL of this invention can effectively lower blood glucose levels, significantly improve insulin resistance, and provide a novel target for the treatment of type 2 diabetes. This small molecule peptide has a unique mechanism of action in lowering blood glucose, helping patients better manage their blood glucose levels and providing a solution for diabetes treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically the application of a small molecule peptide LKLKLL in the preparation of hypoglycemic drugs. Background Technology

[0002] Currently, widely used hypoglycemic drugs on the market, such as insulin and metformin, while showing significant effects in the short term, may cause adverse reactions with long-term use, such as hypoglycemia, gastrointestinal discomfort, and weight gain. Therefore, exploring new hypoglycemic therapies is particularly important.

[0003] Recent studies have revealed the crucial role of gut microbiota in host metabolic regulation, particularly certain probiotics such as Akkermansia muciniphila (AKK). AKK bacteria significantly improve host metabolic health by modulating the gut environment, enhancing intestinal barrier function, and boosting incretin secretion. For instance, AKK metabolites (such as short-chain fatty acids) promote GLP-1 (glucagon-like peptide-1) secretion; its membrane protein Amuc_1100 enhances intestinal barrier integrity and regulates gut microbiota, further promoting GLP-1 secretion; additionally, AKK's secretory protein P9 directly induces GLP-1 secretion and helps improve metabolic disorders by enhancing thermogenesis in brown adipose tissue. These studies demonstrate the significant potential of gut microbiota and their metabolites in improving metabolic diseases.

[0004] Small molecule peptides, with their unique molecular structure and biological activity, have been extensively studied in the treatment of diabetes and metabolic diseases in recent years. Studies have shown that certain small molecule peptides can not only promote insulin secretion but also improve insulin sensitivity and regulate blood glucose balance. Simultaneously, peptide molecules can improve the gut microecological environment, inhibit inflammatory responses and oxidative stress, thus positively impacting the overall metabolic health of diabetic patients. Based on their multiple biological functions, peptides are expected to become key components in hypoglycemic products. However, although the potential of peptides in lowering blood glucose has been preliminarily validated, related research and product development still face many challenges. For example, different small molecule peptides exhibit differences in absorption, metabolic pathways, and hypoglycemic effects in vivo, requiring further research to optimize their preparation processes, improve activity, and ensure stability. Furthermore, how to effectively integrate small molecule peptides into existing drug systems to achieve their hypoglycemic effects and meet market demands remains a key focus of current research. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the small molecule peptide LKLKLL in the preparation of hypoglycemic oral drugs.

[0006] The technical solution of this invention is summarized as follows:

[0007] Application of small molecule peptide LKLKLL in the preparation of oral hypoglycemic drugs.

[0008] Advantages of this invention:

[0009] Experiments have demonstrated that the small molecule peptide LKLKLL significantly improves insulin sensitivity and glucose tolerance by inhibiting high-fat diet-induced hyperglycemia. In a type 2 diabetes model induced by randomly grouping 6-week-old male C57BL / 6 mice and feeding them a high-fat diet combined with a low-dose STZ, the study found that LKLKLL effectively reduced blood glucose levels and improved insulin resistance in the experimental group mice. Further OGTT experiments showed that, compared with the control group, mice treated with LKLKLL had significantly lower blood glucose concentration and area under the curve after glucose loading, indicating that LKLKLL has significant hypoglycemic and metabolic health-improving effects, providing a novel target for the treatment of type 2 diabetes. This small molecule peptide has a unique mechanism of action in lowering blood glucose, helping patients better manage their blood glucose levels and providing a solution for diabetes treatment. The small molecule peptide LKLKLL of this invention can serve as a candidate ingredient for improving diabetes and lowering blood glucose, and can be widely used in oral hypoglycemic drugs. Attached Figure Description

[0010] Figure 1 The small molecule peptide LKLKLL improves the hyperglycemia induced by a high-fat diet;

[0011] Figure 2 The small molecule peptide LKLKLL improves blood glucose levels in type 2 diabetic mice;

[0012] Figure 3 The small molecule peptide LKLKLL improves insulin resistance (A: OGTT; B: AUC (area under the glucose curve)).

[0013] Figure 4 For the targeted metabolomics and tolerability evaluation of the small molecule peptide LKLKLL, including:

[0014] A: Targeted metabolomics;

[0015] B: Tolerance test for artificial gastric juice (referred to as gastric juice);

[0016] C: Tolerance test for artificial intestinal fluid (hereinafter referred to as intestinal fluid). Detailed Implementation

[0017] 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.

[0018] The reagents or formulations used in the following examples:

[0019] 1. Preparation of LKLKLL solution: After anaerobic culture of Akkermansia myxophilus ATCCBAA-835 in complete cell culture medium for 2 hours, non-targeted metabolomics analysis revealed a significant increase in the content of a small peptide (the amino acid sequence of the small peptide is: leucine-lysine-leucine-lysine-leucine-leucine, abbreviated as LKLKLL). Subsequently, LKLKLL was synthesized chemically (by Beijing Baitai Biotechnology Co., Ltd.). 3.75 mg of LKLKLL was removed from a -20℃ freezer and allowed to recover its activity at room temperature for 1 hour. A 2 mL solution was prepared by adding physiological saline, mixing thoroughly, and sonicating for 5 minutes to aid dissolution. The solution was then immediately aliquoted for use.

[0020] STZ solution preparation: In an ice bath, add 100 mg of STZ powder directly to 18 ml of ice-cold 0.1 mol / L sodium citrate buffer solution and stir rapidly until completely dissolved. STZ should be prepared and used immediately under ice-cold conditions (within 30 minutes of preparation) and protected from direct sunlight.

[0021] 60% high-fat feed was purchased from Sinobio; the Zhihang blood glucose meter and blood glucose test strips were purchased from Roche's JD.com flagship store; STZ was purchased from Sigma-Aldrich, Inc.; and 0.1 mol / L sodium citrate buffer (pH 4.5) was purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0022] Example 1

[0023] The small molecule peptide LKLKLL improves high blood sugar levels.

[0024] 1. Test Methods

[0025] (1) Six-week-old male C57BL / 6 mice were selected and, after one week of acclimatization, were randomly divided into three groups:

[0026] The normal control group (normal diet, n=6) was given standard feed;

[0027] The high-fat diet group (HFD, n=6) was given a high-fat diet with 60% fat content;

[0028] The experimental group (high-fat diet + LKLKLL, n=6) had the same diet as the high-fat diet group, but were orally fed LKLKLL (12.5 mg / kg body weight) daily.

[0029] (2) Method for establishing a type 2 diabetic mouse model: Mice were fed a high-fat diet for 8 weeks, then fasted for 16 hours and weighed. Based on body weight, STZ dissolved in 0.1 mol / L sodium citrate buffer was injected intraperitoneally at a dose of 35 mg / kg for 5 consecutive days to establish a type 2 diabetic mouse model. The normal control group was injected intraperitoneally with sodium citrate buffer at a dose of 35 mg / kg.

[0030] Random blood glucose levels were measured every 3 days after STZ injection until the blood glucose level in the model group reached ≥16.7 mmol / L, confirming successful establishment of the diabetes model. After model establishment, a high-fat diet was continued for 5 weeks, with blood glucose levels monitored weekly.

[0031] (3) Oral Glucose Tolerance Test (OGTT): Four weeks after successful modeling, an OGTT was performed to measure the glucose tolerance of mice. Mice were weighed after fasting for 16 hours, and a 30% glucose solution (3 g glucose dissolved in 10 mL sterile PBS) was prepared. After collecting fasting blood glucose, glucose was injected at a dose of 2 mg / g body weight. Blood glucose in the tail of the mice was measured at 15, 30, 60, and 120 minutes after injection, and the blood glucose concentration was recorded to assess the glucose tolerance of the mice.

[0032] 2. Experimental Results

[0033] (1) LKLKLL inhibits high-fat diet-induced hyperglycemia: Monitoring of blood glucose levels in mice during the experiment showed that the blood glucose level in the high-fat diet group (HFD) was significantly higher than that in the normal control group; while the blood glucose level in mice treated with LKLKLL (experimental group) was significantly lower than that in the high-fat diet group. This indicates that LKLKLL can effectively inhibit high-fat diet-induced hyperglycemia (HDL). Figure 1 ).

[0034] (2) LKLKLL prevents and improves hyperglycemia in type 2 diabetic mice: In a type 2 diabetes model induced by a high-fat diet combined with a low dose of STZ, the blood glucose level of mice in the high-fat diet group was significantly higher than that in the normal diet group. However, the blood glucose level of mice in the LKLKLL intervention group (experimental group) was significantly lower than that in the high-fat diet group, indicating that LKLKLL has a significant effect on preventing and improving hyperglycemia in type 2 diabetic mice (Figure 2).

[0035] (3) LKLKLL can improve insulin resistance in type 2 diabetic mice: An OGTT test was performed 4 weeks after successful modeling. The blood glucose and area under the glucose curve of mice in the high-fat diet group were significantly higher than those in the normal diet group at all time points after glucose loading. However, the blood glucose level of mice in the LKLKLL intervention group (experimental group) was significantly lower than that in the high-fat diet group at all time points, and the area under the glucose curve was significantly lower than that in the high-fat diet group, indicating that LKLKLL can significantly improve insulin resistance in type 2 diabetic mice (Figure 3).

[0036] Example 2: Targeted metabolomics and tolerability evaluation of a small molecule peptide LKLKLL

[0037] 1. Targeted metabolomics of LKLKLL

[0038] (1) Experimental Method: This invention uses triple quadrupole mass spectrometry (Triple Quadrupole MS / MS) as the detection method for small molecule peptide-targeted metabolomics, which has high sensitivity and high selectivity and is widely used in the qualitative and quantitative analysis of small molecule compounds in biological samples. The specific steps include: First, collecting the metabolic supernatant of Akkermansia muciniphila (AKK bacteria) cultured in high glucose complete medium, collecting and filtering after 2 hours and 6 hours of culture, respectively. Then, adding 1.5 mL of 20% acetonitrile methanol solution extraction buffer, vortexing for 3 minutes, and then centrifuging at 12000 r / min for 10 minutes at 4°C, and then aspirating the supernatant (500 μL) and transferring it to a sample vial. The chromatographic conditions are as follows: using a Waters Atlantis T3 Column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, column temperature is set at 40°C, flow rate is 0.4 mL / min, and injection volume is 3 μL. The separated components were detected using triple quadrupole mass spectrometry. The first two quadrupoles were used to select specific precursor and daughter ions to enhance signal selectivity and sensitivity. Finally, the mass spectrometry data were analyzed to quantify the abundance of small molecule peptides and compared with a control group to verify the detection results.

[0039] (2) Test results

[0040] This invention successfully detected the content of the small peptide LKLKLL in the supernatant of *A. akadina* bacteria using triple quadrupole mass spectrometry. The results showed that the abundance of LKLKLL in the *A. akadina* supernatant was significantly higher than that in the control group, and the content at 6 h was higher than that at 2 h. Figure 4 A.

[0041] 2. Evaluation of tolerance to LKLKLL artificial gastric juice

[0042] (1) Test methods

[0043] LKLKLL was dissolved in 1 mL of artificial gastric fluid at pH=2 and 1 mL of ddH2O to prepare a 1000 ng / mL solution. After thorough mixing, the solution was placed in an environment of 37℃ for 1.5 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0044]

[0045] Where A0 is the content of LKLKLL dissolved by ddH2O, and An is the content of LKLKLL dissolved by artificial gastric juice.

[0046] (2) Test results

[0047] The degradation of LKLKLL in artificial gastric fluid is as follows: Figure 4 B. In an artificial gastric fluid environment at pH=2, the content of the small molecule peptide LKLKLL was almost unaffected, with a degradation rate of 2%.

[0048] 3. Evaluation of tolerance to LKLKLL artificial intestinal fluid

[0049] (1) Test methods

[0050] LKLKLL was dissolved in 1 mL of artificial intestinal fluid and 1 mL of ddH2O to prepare a 1000 ng / mL solution. After thorough mixing, the solution was placed in an environment of 37℃ for 4 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0051]

[0052] (2) Test results

[0053] The degradation of LKLKLL in artificial intestinal fluid is as follows: Figure 4 C. Under artificial intestinal fluid conditions, the content of the small molecule peptide LKLKLL was almost unaffected, with a degradation rate of 0.2%.

[0054] In summary, the small molecule peptide LKLKLL provided by this invention can significantly inhibit the hyperglycemia induced by a high-fat diet, improve insulin resistance, and effectively reduce blood glucose levels in type 2 diabetic model mice, demonstrating its application prospects in hypoglycemia.

[0055] Based on the biological functions of LKLKLL, related drugs are prepared to provide new options for the treatment of type 2 diabetes and metabolic diseases.

[0056] The drug is made into a convenient oral dosage form, such as tablets, capsules, solutions or granules, using conventional technical methods.

[0057] A composition in which one of the above-mentioned small molecule peptides is the sole active ingredient.

[0058] The application of the above composition in the preparation of hypoglycemic drugs.

[0059] The above-mentioned drugs are oral medications.

[0060] The dosage forms of the above-mentioned oral medications are tablets, capsules, solutions, or granules.

[0061] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of small molecule peptide LKLKLL in the preparation of hypoglycemic oral drugs.

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