Application of small molecule peptide LKLKLL in preparation of hypoglycemic products

Abstract

The invention discloses application of a small molecule peptide LKLKLL in preparation of a hypoglycemic product and application of the small molecule peptide LKLKLL in preparation of the hypoglycemic product. Experiments prove that the small molecule peptide LKLKLL can effectively reduce the blood sugar level and remarkably improve insulin resistance, and a brand new target is provided for treatment of type 2 diabetes mellitus. The small molecule peptide has a unique action mechanism in the aspect of reducing blood sugar, helps a patient to better manage the blood sugar level, and provides a solution for diabetes treatment.

Description

Technical Field

[0001] The invention relates to the field of biomedicine or health food, and specifically to application of a small molecule peptide LKLKLL in the preparation of a blood sugar-lowering product. Background Art

[0002] At present, the glucose-lowering drugs widely used in the market, such as insulin and metformin, have significant effects in the short term, but long-term use may cause some adverse reactions, such as hypoglycemia, gastrointestinal discomfort and weight gain. Therefore, it is particularly important to explore new glucose-lowering therapies.

[0003] In recent years, studies have found that intestinal microorganisms play an important role in the regulation of host metabolism, especially certain probiotics, such as Akkermansia muciniphila (AKK). AKK bacteria can significantly improve the metabolic health of the host by regulating the intestinal environment, improving intestinal barrier function, and enhancing incretin secretion. For example, AKK's metabolites (such as short-chain fatty acids) can promote the secretion of GLP-1 (glucagon-like peptide-1); its membrane protein Amuc_1100 can enhance the integrity of the intestinal barrier and regulate the intestinal flora, thereby further promoting the secretion of GLP-1; in addition, AKK's secretory protein P9 can directly induce the secretion of GLP-1 and help improve metabolic disorders by enhancing the thermogenesis of brown adipose tissue. These studies have shown that intestinal microorganisms and their metabolites have important potential in improving metabolic diseases.

[0004] As a molecule with unique molecular structure and biological activity, small molecule peptides have been widely studied in the treatment of diabetes and metabolic diseases in recent years. Studies have shown that some small molecule peptides can not only promote insulin secretion, but also improve insulin sensitivity and regulate blood sugar balance. At the same time, peptide molecules can also improve the intestinal microecological environment, inhibit inflammatory response and oxidative stress, thereby having a positive impact on the overall metabolic health of diabetic patients. Based on their multiple biological effects, peptides are expected to become a key ingredient in hypoglycemic products. However, although the potential of peptides in hypoglycemic treatment has been preliminarily verified, related research and product development still face many challenges. For example, different small molecule peptides have different absorption, metabolic pathways and hypoglycemic effects in the body, and further research is urgently needed to optimize their preparation process, improve activity and ensure their stability. In addition, how to effectively integrate small molecule peptides into existing health food and drug systems to achieve their hypoglycemic effects and meet market demand is still the focus of current research. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of the small molecule peptide LKLKLL in the preparation of a blood sugar lowering product.

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

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

[0008] Preferably, the blood sugar lowering product is a blood sugar lowering oral health food or a blood sugar lowering oral medicine.

[0009] Advantages of the present invention:

[0010] Experiments have shown that the small molecule peptide LKLKLL significantly improves insulin sensitivity and glucose tolerance by inhibiting the increase in blood sugar induced by a high-fat diet. The experiment randomly divided 6-week-old male C57BL / 6 male mice into groups and fed them with a high-fat diet combined with a low-dose STZ-induced type 2 diabetes model. The study found that LKLKLL can effectively reduce the blood sugar levels of mice in the experimental group and improve insulin resistance. Further OGTT tests showed that compared with the control group, the blood sugar concentration and area under the curve of mice treated with LKLKLL after glucose load were significantly reduced, indicating that LKLKLL has significant effects on lowering blood sugar and improving metabolic health, providing a new target for the treatment of type 2 diabetes. This small molecule peptide has a unique mechanism of action in lowering blood sugar, helping patients better manage blood sugar levels and providing a solution for the treatment of diabetes. The small molecule peptide LKLKLL of the present invention can be used as a candidate ingredient for improving diabetes and lowering blood sugar, and is widely used in hypoglycemic oral health foods or hypoglycemic oral medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The small molecule peptide LKLKLL improves the increase in blood sugar induced by a high-fat diet;

[0012] Figure 2 The small molecule peptide LKLKLL improves the blood glucose level of type 2 diabetic mice;

[0013] Figure 3 The small molecule peptide LKLKLL improves insulin resistance (A: OGTT; B: AUC (area under the glucose curve)).

[0014] Figure 4 Targeted metabolomics and tolerability evaluation of the small molecule peptide LKLKLL, including:

[0015] A: Targeted metabolomics;

[0016] B: Artificial gastric juice (referred to as gastric juice) tolerance test;

[0017] C: Artificial intestinal fluid (abbreviated as intestinal fluid) tolerance test. DETAILED DESCRIPTION

[0018] 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.

[0019] The reagents or formulations used in the following examples are:

[0020] 1. Preparation of LKLKLL solution: After Akkermansia muciniphila ATCC BAA-835 was cultured anaerobically for 2 hours in complete cell culture medium, non-targeted metabolomics analysis was performed, and a small molecule peptide (the amino acid sequence of the small molecule peptide is: leucine-lysine-leucine-lysine-leucine-leucine, referred to as LKLKLL) content was significantly increased. Thereafter, 3.75 mg of LKLKLL was synthesized by chemical synthesis (by Beijing Baipeptide Biotechnology Co., Ltd.) and taken out from a -20℃ refrigerator and restored to activity at room temperature for 1 hour. Physiological saline was added to prepare 2 mL of solution, mixed, ultrasonicated for 5 minutes to assist melting, and centrifuged for use.

[0021] Preparation of STZ solution: In an ice bath, add 100 mg of STZ powder directly into 18 ml of ice-cold 0.1 mol / L sodium citrate buffer and stir rapidly to dissolve it completely. STZ should be used immediately after preparation and should be used as soon as possible under ice-cold conditions (within 30 minutes after preparation) and away from direct sunlight.

[0022] 60% high-fat feed was purchased from Sino Bio; Zhihang blood glucose meter and blood glucose test strips were purchased from Roche JD flagship store; STZ was purchased from Sigma, USA; 0.1 mol / L sodium citrate buffer (pH 4.5) was purchased from Beijing Solebow Biotechnology Co., Ltd.

[0023] Example 1

[0024] Small molecule peptide LKLKLL improves high blood sugar levels

[0025] 1. Test methods

[0026] (1) Six-week-old male C57BL / 6 mice were selected and, after one week of adaptive feeding, randomly divided into three groups:

[0027] Normal control group (normal diet, n = 6), given standard feed;

[0028] The high-fat diet group (HFD, High-fat diet, n = 6) was given a high-fat diet with 60% fat content;

[0029] The experimental group (high-fat diet + LKLKLL, n = 6) was fed the same diet as the high-fat diet group, and LKLKLL (12.5 mg / kg body weight) was orally administered daily.

[0030] (2) Type 2 diabetic mouse modeling method: After the mice were fed a high-fat diet for 8 weeks, they were fasted for 16 hours and weighed. STZ dissolved in 0.1 mol / L sodium citrate buffer was intraperitoneally injected at a dose of 35 mg / kg according to body weight for 5 consecutive days to establish a type 2 diabetic mouse model. The normal control group was intraperitoneally injected with sodium citrate buffer at a dose of 35 mg / kg.

[0031] After STZ injection, random blood glucose levels were measured every 3 days until the blood glucose level in the model group reached ≥16.7mmol / L, confirming that the diabetes model was successfully established. After the model was established, the high-fat diet was continued for 5 weeks, and blood glucose levels were tested weekly.

[0032] (3) Oral glucose tolerance test (OGTT): Four weeks after successful modeling, an OGTT test was performed to measure the glucose tolerance of mice. The mice were weighed after fasting for 16 hours, and a 30% glucose solution was prepared (3 g of glucose was dissolved in 10 mL of sterile PBS). After collecting fasting blood glucose, glucose was injected at 2 mg / g body weight. The tail blood glucose was measured at 15 minutes, 30 minutes, 60 minutes and 120 minutes after injection, and the blood glucose concentration was recorded to evaluate the glucose tolerance of the mice.

[0033] 2. Experimental results

[0034] (1) LKLKLL inhibits the increase in blood sugar induced by a high-fat diet: By monitoring the blood sugar levels of mice in the experiment, the blood sugar levels of mice in the high-fat diet group (HFD) were significantly higher than those in the normal control group; while the blood sugar levels of mice that received LKLKLL intervention (experimental group) were significantly lower than those in the high-fat diet group. This indicates that LKLKLL can effectively inhibit the increase in blood sugar induced by a high-fat diet ( Figure 1 ).

[0035] (2) LKLKLL prevents and improves elevated blood sugar in type 2 diabetic mice: In a type 2 diabetes model established by inducing mice with a high-fat diet combined with a low dose of STZ, it was found that the blood sugar level of mice in the high-fat diet group was significantly higher than that in the normal diet group. The blood sugar 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 in preventing and improving elevated blood sugar in type 2 diabetic mice ( Figure 2 ).

[0036] (3) LKLKLL can improve insulin resistance in type 2 diabetic mice: OGTT test was performed 4 weeks after successful modeling. The blood glucose and glucose area under the curve of mice in the high-fat diet group were significantly higher than those in the normal diet group at each time point after glucose loading. The blood glucose levels of mice in the LKLKLL intervention group (experimental group) were significantly lower than those in the high-fat diet group at each time point, and the glucose area under the 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 ).

[0037] Example 2 Targeted metabolomics and tolerability evaluation of a small molecule peptide LKLKLL

[0038] 1. Targeted metabolomics of LKLKLL

[0039] (1) Experimental method: The present invention uses triple quadrupole mass spectrometry (Triple Quadrupole MS / MS) as a 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-sugar complete medium, and collecting and filtering after culturing for 2 hours and 6 hours, respectively. Then, add a 20% acetonitrile methanol solution extract (1.5mL), vortex mix for 3 minutes, and then centrifuge at 12000r / min for 10 minutes at 4°C. After centrifugation, the supernatant (500μL) is aspirated and transferred to an injection bottle. The chromatographic conditions are to use a Waters Atlantis T3 Column column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, the column temperature is set to 40°C, the flow rate is 0.4mL / min, and the injection volume is 3μL. The separated components were detected by triple quadrupole mass spectrometry, with the first two quadrupoles selecting specific parent ions and daughter ions to enhance the selectivity and sensitivity of the signal. Finally, the mass spectrometry data was analyzed to quantify the abundance of small molecule peptides and compared with the control group to verify the detection results.

[0040] (2) Test results

[0041] The present invention successfully detected the content of the small molecule peptide LKLKLL in the supernatant of AKK bacteria using triple quadrupole mass spectrometry. The results showed that the abundance of LKLKLL in the supernatant of AKK bacteria was significantly higher than that in the control group, and the content at 6h was higher than that at 2h. Figure 4 A.

[0042] 2. Evaluation of LKLKLL artificial gastric juice tolerance

[0043] (1) Test method

[0044] LKLKLL was dissolved in 1 mL of artificial gastric juice with pH = 2 and 1 mL of ddH2O to prepare a 1000 ng / mL solution, and the solution was thoroughly mixed and placed in an environment of 37°C for 1.5 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0045]

[0046] Wherein A0 is the content of LKLKLL dissolved in ddH2O, and An is the content of LKLKLL dissolved in artificial gastric juice.

[0047] (2) Test results

[0048] Degradation of LKLKLL in artificial gastric fluid Figure 4 B. In the artificial gastric fluid environment of pH=2, the content of the small molecule peptide LKLKLL was almost unaffected, and the degradation rate was 2%.

[0049] 3. Evaluation of tolerance to LKLKLL artificial intestinal fluid

[0050] (1) Test method

[0051] LKLKLL was dissolved in 1 mL of artificial intestinal fluid and 1 mL of ddH2O to prepare a 1000 ng / mL solution, and the solution was thoroughly mixed and placed at 37°C for 4 h. The content of LKLKLL was immediately detected by triple quadrupole mass spectrometry, and the degradation rate was calculated.

[0052]

[0053] (2) Test results

[0054] Degradation of LKLKLL in artificial intestinal fluid Figure 4 C. In the artificial intestinal fluid environment, the content of the small molecule peptide LKLKLL was almost unaffected, and the degradation rate was 0.2%.

[0055] In summary, the small molecule peptide LKLKLL provided by the present invention can significantly inhibit the increase in blood sugar induced by a high-fat diet, improve insulin resistance, and effectively reduce the blood sugar level of type 2 diabetes model mice, showing its application prospect in hypoglycemic effect.

[0056] Based on the biological functions of LKLKLL, relevant health foods or medicines are prepared to provide new options for the treatment of type 2 diabetes and metabolic diseases.

[0057] The health food is prepared into convenient oral dosage forms such as tablets, capsules, solutions or granules by conventional technical means.

[0058] The drug is prepared into convenient oral dosage forms such as tablets, capsules, solutions or granules by conventional techniques.

[0059] A composition with the above-mentioned small molecule peptide as the only active ingredient.

[0060] Application of the above composition in the preparation of hypoglycemic products.

[0061] The above product is preferably a health food or a medicine.

[0062] The above products are oral health foods or oral medicines.

[0063] The dosage form of the above-mentioned oral health food is tablet, capsule, solution or granule.

[0064] The above-mentioned oral medicine is in the form of tablets, capsules, solutions or granules.

[0065] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

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

2. The use according to claim 1, characterized in that The blood sugar lowering product is a blood sugar lowering oral health food or a blood sugar lowering oral medicine.

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