Application of N-acetyl serotonin in preparation of medicine for treating type 2 diabetes

Abstract

The invention discloses application of N-acetyl serotonin in preparation of a medicine for treating type 2 diabetes mellitus, and belongs to the field of biological medicine. In-vivo and in-vitro experiments prove that the N-acetyl serotonin can promote secretion of GLP-1, has the effects of obviously reducing blood sugar, being good in sugar tolerance and insulin tolerance, and is obviously improved aiming at high fasting blood glucose, high postprandial blood glucose, poor glucose tolerance and reduced insulin tolerance; the GLP-1 secretion insufficiency is obviously improved. The invention is of great significance in maintaining blood sugar stability and improving glucose and insulin tolerance of diabetic patients, and provides a new solution for treatment of type 2 diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of N-acetyl serotonin in the preparation of medicines for treating type 2 diabetes. Background Art

[0002] Type 2 diabetes (T2D) is a chronic metabolic disease characterized by persistently elevated blood sugar and impaired insulin function caused by the interaction of genetics and the environment. With the rapid development of the economy, the pace of life has accelerated, and the sedentary lifestyle and unhealthy eating patterns have led to a rising trend of obesity worldwide, and the incidence of type 2 diabetes has also increased year by year. The prevention and treatment of diabetes has become a major challenge worldwide. Its current treatment status mainly relies on drug therapy, which aims to control blood sugar levels, reduce complications, and improve the quality of life of patients. At present, drug treatments for type 2 diabetes include biguanides, sulfonylureas, thiazolidinediones (TZDs), dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-glucose cotransporter 2 (SGLT-2) inhibitors, glucagon-like peptide-1 (GLP-1) analogs, and other drugs. Metformin has become the first choice for patients with type 2 diabetes because of its effects of reducing hepatic glucose output, enhancing peripheral tissue sensitivity, and stimulating GLP-1 secretion. However, despite the growing number of treatment options, existing diabetes treatments still face some bottlenecks, such as no single therapy that can effectively maintain long-term blood sugar control, insignificant blood sugar reduction in treatment effects, poor glucose tolerance, poor insulin tolerance, and high treatment costs.

[0003] N-Acetylserotonin is an acetylated derivative of serotonin (5-hydroxytryptamine) and a precursor of melatonin. It can indirectly participate in sleep regulation. Because it has a certain antioxidant effect, it can scavenge free radicals and reduce oxidative stress. At the same time, N-acetylserotonin has a neuroprotective effect and can reduce neuronal damage, especially in ischemia-reperfusion injury and neurodegenerative diseases. At present, there are no reports on the role of N-acetylserotonin in regulating blood sugar. Summary of the invention

[0004] The purpose of the present invention is to provide an application of N-acetyl serotonin in the preparation of a drug for treating type 2 diabetes, so as to solve the problems existing in the above-mentioned prior art. The present invention confirms through in vivo and in vitro experiments that N-acetyl serotonin has the effects of significantly lowering blood sugar, improving glucose tolerance and insulin tolerance, which is of great significance for diabetic patients to maintain blood sugar stability and improve glucose and insulin tolerance, and provides a new solution for the treatment of type 2 diabetes.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of N-acetyl serotonin as an effective component in preparing a medicine for treating type 2 diabetes.

[0007] Optionally, the type 2 diabetes includes type 2 diabetes caused by genetic factors, obesity factors and high-fat diet.

[0008] Optionally, the drug further includes pharmaceutically acceptable carriers and excipients.

[0009] Optionally, the dosage form of the drug includes tablets, granules, capsules and mixtures.

[0010] Furthermore, the N-acetyl serotonin achieves the effect of treating type 2 diabetes by lowering fasting blood sugar levels, improving glucose tolerance and improving insulin tolerance.

[0011] Furthermore, the N-acetyl serotonin reduces fasting blood sugar levels, improves glucose tolerance and improves insulin tolerance by promoting the secretion of GLP-1.

[0012] Optionally, the dosage of N-acetylserotonin is 5 mg / kg-50 mg / kg.

[0013] Further optionally, the dosage of N-acetylserotonin is 25 mg / kg-50 mg / kg.

[0014] The present invention discloses the following technical effects:

[0015] The present invention confirms the therapeutic effect of N-acetyl serotonin on type 2 diabetes through in vitro and in vivo experiments. Animal experiments have shown that N-acetyl serotonin can treat type 2 diabetes by lowering fasting blood sugar levels, improving glucose tolerance and improving insulin tolerance; cell experiments have shown that N-acetyl serotonin can lower fasting blood sugar levels, improve glucose tolerance and improve insulin tolerance by promoting the secretion of GLP-1.

[0016] N-acetylserotonin is widely available and easy to prepare, which reduces the cost of treating type 2 diabetes. It also has the effects of significantly lowering blood sugar, improving glucose tolerance, and improving insulin tolerance. It is of great significance for diabetic patients to maintain blood sugar stability and improve glucose and insulin tolerance, and provides a new solution for the treatment of type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 is a flow chart of Example 1;

[0019] Figure 2 The fasting blood glucose test results of mice in each group after 4 weeks of administration, *P<0.05;

[0020] Figure 3 OGTT curves (A) and area under the curves (B) of mice in each group at different time points after oral administration of glucose, **P<0.01, ****P<0.0001;

[0021] Figure 4 The ITT curves (A) and area under the curves (B) of each group of mice at different time points after intraperitoneal injection of insulin. One-way RMANOVA was used for inter-group comparison. The abscissa represents time, and the ordinate represents blood glucose level. *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 5 Fasting insulin levels (A) and insulin index (B) of mice in each group after 4 weeks of administration, *P<0.05, **P<0.01;

[0023] Figure 6 The GLP-1 secretion levels in mice of each group after 4 weeks of administration, **P<0.01, ***P<0.001;

[0024] Figure 7 are the GLP-1 secretion levels of NCI-H716 cells in different treatment groups. “25-” indicates a glucose concentration of 25 mM; “50-” indicates a glucose concentration of 50 mM; 0, 5, 25, 50 indicate that NAS concentrations were 0 mM, 5 mM, 25 mM, and 50 mM, respectively. *P<0.05, **P<0.01, ****P<0.0001. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The present invention provides application of N-acetyl serotonin in treating type 2 diabetes.

[0031] Optionally, N-acetyl serotonin is used as an active ingredient in the preparation of a glucose-lowering drug for treating type 2 diabetes.

[0032] Optionally, the drug further includes pharmaceutically acceptable carriers and excipients.

[0033] Specifically, "pharmaceutically acceptable carrier" refers to a carrier that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound; "excipient" refers to a solvent, diluent or other excipient, dispersant, surfactant.

[0034] Optionally, the medicine includes an oral N-acetylserotonin preparation and an N-acetylserotonin injection.

[0035] Optionally, the oral preparation of N-acetylserotonin includes N-acetylserotonin hydrochloride tablets, N-acetylserotonin sustained-release tablets, N-acetylserotonin sustained-release capsules, N-acetylserotonin enteric-coated tablets, N-acetylserotonin enteric-coated capsules, and N-acetylserotonin compound preparations.

[0036] Optionally, the oral N-acetylserotonin preparation further comprises N-acetylserotonin granules.

[0037] Optionally, the administration of the drug includes oral administration, intramuscular injection, fat layer injection, intravenous injection or subcutaneous injection.

[0038] Specifically, the N-acetyl serotonin achieves the effect of treating type 2 diabetes by lowering fasting blood sugar, improving glucose tolerance, and improving insulin tolerance in diabetic patients.

[0039] Optionally, chronic administration of N-acetyl serotonin can significantly reduce fasting blood glucose, fasting blood sugar, improve glucose tolerance, and improve insulin tolerance in diabetic patients to achieve the effect of treating type 2 diabetes.

[0040] More specifically, the N-acetyl serotonin promotes the secretion of GLP-1 in diabetic patients to reduce fasting blood sugar, improve glucose tolerance, and improve insulin tolerance, thereby achieving the effect of treating type 2 diabetes.

[0041] Optionally, chronic administration of N-acetyl serotonin significantly promotes the secretion of GLP-1 in diabetic patients to lower fasting blood sugar, improve glucose tolerance, and improve insulin tolerance, thereby achieving the effect of treating type 2 diabetes.

[0042] Optionally, the dosage of N-acetyl serotonin for regulating blood sugar is 5 mg / kg-50 mg / kg.

[0043] Optionally, the dosage of N-acetyl serotonin regulating insulin is 5 mg / kg-50 mg / kg.

[0044] In a specific embodiment, the dosage of N-acetyl serotonin for increasing the secretion of GLP-1 in mouse serum is 5 mg / kg-50 mg / kg.

[0045] The molecular formula of N-acetyl serotonin of the present invention is: 12 H 14 N 2 O 2 , molecular weight: 218.25, CAS number: 1210-83-9, molecular structure formula is as follows:

[0046]

[0047] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0048] Example 1 N-acetyl serotonin significantly reduces fasting blood sugar and improves glucose tolerance

[0049] 1. Experimental Methods

[0050] 1. Modeling method

[0051] After 1 week of adaptive feeding, C57BL / 6J mice were randomly divided into a normal diet group and a high-fat diet group. The normal diet group was fed with 10% control feed (Research diets, 12450J), and the high-fat diet group (HFD) was fed with 60% high-fat feed (Research diets, D12492). The feed was changed twice a week and weighed once. Feeding continued for 12 weeks. At the end of the 12th week, after fasting for 6 hours, the fasting blood glucose test and OGTT experiment were used to evaluate whether the model was successful. The standard for successful modeling was fasting blood glucose ≥11.1mmol / L and the presence of obvious impaired glucose tolerance.

[0052] 2. Experimental groups and treatments (6 mice per group)

[0053] Healthy control group (NC): each group was given 10% control feed + phosphate buffered saline (PBS);

[0054] Diabetes group (HFD): 60% high-fat diet + PBS;

[0055] N-acetyl serotonin experimental group (NAS): 60% high-fat diet + 5mg / kg N-acetyl serum. Oral gavage was used for administration. Specific experimental groups and treatment methods are shown in Table 1 and Figure 1 .

[0056] Table 1 Experimental groups and treatments

[0057]

[0058] 3. Fasting blood glucose test

[0059] Mice that had received oral administration of N-acetyl serotonin for 4 weeks were tested after fasting for 6 hours. Blood was collected by pricking the tail vein with a sterile needle, and blood glucose concentration was measured using a Roche blood glucose meter.

[0060] 4. Glucose tolerance test

[0061] Mice that had received oral administration of N-acetyl serotonin for 4 weeks were tested after fasting for 6 hours. Fasting blood glucose levels were first measured, followed by gavage with 20% glucose solution, and blood glucose changes were measured through the tail vein at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.

[0062] 2. Experimental Results

[0063] Fasting blood sugar test results Figure 2 As shown, the blue dots in the figure represent the fasting blood glucose values ​​of normal mice after four weeks of oral gavage with PBS. The purple dots represent the fasting blood glucose values ​​of diabetic mice after four weeks of oral gavage with PBS, and the red triangles represent the fasting blood glucose values ​​of diabetic mice after four weeks of oral gavage with 5 mg / kg N-acetyl serotonin. Compared with the diabetic group, oral gavage with 5 mg / kg N-acetyl serotonin significantly reduced the fasting blood glucose levels of mice.

[0064] Oral glucose tolerance test showed Figure 3 ), compared with the diabetic group, continuous oral gavage of 5 mg / kg N-acetyl serotonin for 4 weeks significantly reduced the fasting blood glucose level of diabetic mice and improved the impaired glucose tolerance of mice.

[0065] Conclusion: Compared with the diabetic group, oral administration of N-acetyl serotonin has a better hypoglycemic effect. Oral administration of 5 mg / kg N-acetyl serotonin significantly reduced the fasting blood glucose level of mice and improved the blood glucose clearance ability after oral administration of glucose.

[0066] Example 2 N-acetyl serotonin significantly reduces fasting insulin and insulin sensitivity

[0067] 1. Experimental Methods

[0068] 1. Modeling method

[0069] The diabetes modeling method and related detection scheme used in this example are the same as those in Example 1, and the diabetic mice were intervened when the disease course was 3 months.

[0070] 2. Experimental groups and treatments (6 mice per group)

[0071] Healthy control group (NC): each group was given 10% control feed + PBS;

[0072] Diabetes group (HFD): 60% high-fat diet + PBS;

[0073] N-acetyl serotonin experimental group (NAS): 60% high-fat diet + 5 mg / kg N-acetyl serotonin.

[0074] 3. Insulin sensitivity test

[0075] After four weeks of oral administration of N-acetyl serotonin, mice were tested after fasting for 4 hours. Fasting blood glucose levels were measured first, followed by intraperitoneal injection of 0.75 IU / kg insulin, and blood glucose changes were measured through the tail vein at 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 90 minutes.

[0076] 4. Detection of insulin levels in fasting serum samples of mice

[0077] 1) Take out the insulin test kit from the 4°C refrigerator 30 minutes in advance and return it to room temperature. Calculate the required half number of test strips in advance and store the rest in the refrigerator.

[0078] 2) Reagent preparation: Dilute Enzyme Conjugate 11× to Enzyme Conjugate 1× solution with Enzyme Conjugate Buffer for later use; 2 O Dilute Wash Buffer 21× to Wash Buffer 1× solution for later use; standard solution Calibrators 0, 1, 2, 3, 4, 5 for later use.

[0079] 3) Set up blank wells (Control), standard wells (Calibrator) and sample wells (Samples) respectively. The blank well is Calibrator 0, the standard wells are calibrators 1, 2, 3, 4, 5, and the sample wells are serum samples.

[0080] 4) Take 10 μL of Calibrator 0, calibrators 1, 2, 3, 4, 5 or plasma sample and add them to the plate, then add 100 μL of Enzyme Conjugate 1× solution prepared in advance and cover with sealing film.

[0081] 5) Place the plate on a shaker and incubate at 700-900 rpm for 2 h at room temperature (18-25°C).

[0082] 6) Discard the reaction solution in the well, turn the tube upside down and tap it on a dust-free paper to absorb the liquid.

[0083] 7) Add 350 μL Wash Buffer 1× to each reaction well for washing, discard the washing solution in the well, dry the liquid on a dust-free paper, and repeat the washing 6 times. Do not soak the reaction plate in Wash Buffer 1× for a long time during the washing process.

[0084] 8) Add 200 μL Substrate TMB to each well, seal the membrane, and incubate at room temperature (18-25°C) for 15 min in the dark.

[0085] 9) Add 50 μL Stop Solution to each well to terminate the reaction and gently shake for 5 seconds.

[0086] 5. Calculation of insulin resistance index (HOME-IR)

[0087] The formula is: HOMA-IR = fasting blood glucose (mmol / L) × fasting insulin (IU / mL) / 22.5.

[0088] 2. Experimental Results

[0089] Insulin sensitivity test results Figure 4 As shown, compared with the diabetic group, N-acetyl serotonin treatment significantly reduced the blood glucose levels of diabetic mice after insulin injection, and the corresponding area under the curve was significantly reduced.

[0090] Fasting serum insulin test showed ( Figure 5 ), compared with the diabetic group, 4-week N-acetyl serotonin treatment significantly reduced the fasting serum insulin levels of mice fed a high-fat diet, significantly restored the insulin index value, and significantly increased insulin sensitivity.

[0091] Conclusion: Compared with the diabetic group, oral administration of N-acetyl serotonin has a better insulin regulatory effect. Oral administration of 5 mg / kg N-acetyl serotonin significantly reduced the fasting insulin level of mice, reduced insulin resistance, and increased insulin sensitivity.

[0092] Example 3 N-acetyl serotonin significantly increases GLP-1 secretion levels

[0093] 1. Experimental Methods

[0094] 1. Modeling method

[0095] The diabetes modeling method and related detection scheme used in this example are the same as those in Example 1, and the diabetic mice were intervened when the disease course was 3 months.

[0096] 2. Experimental groups and treatments (6 mice per group)

[0097] Healthy control group (NC): each group was given 10% control feed + PBS;

[0098] Diabetes group (HFD): 60% high-fat diet + PBS;

[0099] N-acetyl serotonin experimental group (NAS): 60% high-fat diet + 5 mg / kg N-acetyl serotonin.

[0100] 3. Detection of GLP-1 in mouse serum

[0101] Mice that had received oral gavage of N-acetyl serotonin for 4 weeks were tested after fasting for 6 hours. 15 minutes after oral gavage of fasting mice, blood was collected from the canthus into an EP tube containing 1 μM DDP4 inhibitor, centrifuged at 3000 rpm, 4°C for 20 minutes, and the supernatant was tested using a mouse GLP-1 kit.

[0102] 4. ELISA kit to detect GLP-1 level

[0103] 1) Dilution of the standard: dilute the 8 pmol / L standard solution provided in the kit with the standard diluent according to the doubling dilution method to 4 pmol / L, 2 pmol / L, 1 pmol / L, 0.5 pmol / L, and 0.25 pmol / L standard solutions for later use.

[0104] 2) Sample addition: Set up blank wells, standard wells, and sample wells. Do not add enzyme labeling reagents and samples to the blank wells, and keep the rest the same. Add 50μL of standard solution to the wells of the ELISA plate, and dilute the sample to be tested five times with the sample diluent, that is, first add 40μL of sample diluent, and then add 10μL of the sample to be tested. Try to add the sample to the bottom of the well of the ELISA plate without touching the well wall, and shake gently to mix.

[0105] 3) Incubation: Seal the plate with a sealing film and place in a 37°C constant temperature incubator for 30 min.

[0106] 4) Solution preparation: Dissolve the concentrated washing solution in the kit with distilled water (ddH 2 O) Dilute 30 times before use.

[0107] 5) Washing: Remove the sealing film, pour out the liquid in the well plate and spin dry, fill the well plate with washing solution, let it stand for 30 seconds and then pour out the washing solution, repeat the washing 5 times, and pat dry on clean filter paper.

[0108] 6) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well except the blank well, incubate according to step 3) and wash according to step 5).

[0109] 7) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop the color in a 37°C incubator for 10 minutes in the dark.

[0110] 8) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0111] 9) Determination: Set the blank well to zero and measure the OD value of each well at a wavelength of 450 nm. The determination must be completed within 15 minutes after adding the stop solution.

[0112] 2. Experimental Results

[0113] GLP-1 secretion results Figure 6 As shown, N-acetyl serotonin treatment significantly increased the secretion level of GLP-1 after glucose stimulation in mice compared with the diabetic group.

[0114] Conclusion: High-fat diet leads to insufficient secretion of GLP-1 in mice, and oral administration of N-acetyl serotonin can significantly improve the phenomenon of insufficient secretion of GLP-1.

[0115] Example 4 In vitro experiment on increasing the secretion level of GLP-1 in cells under high glucose conditions by N-acetyl serotonin

[0116] 1. Experimental Materials

[0117] NCI-H716 cells were purchased from the National Certified Cell Culture Collection and were identified to exclude contamination by mycoplasma or chlamydia and were used within ten generations.

[0118] 2. Experimental steps

[0119] NCI-H716 cells were cultured at 5×10 5 The cells were inoculated into 96-well plates at a density of 100 cells / well and incubated for 48 hours. Then, 25mM or 50mM glucose and different concentrations of N-acetyl serotonin prepared by buffer were added to the cells, and the groups were named 25-0, 25-5, 25-25, 25-50, 50-0, 50-5, 50-25 and 50-50. In the group names, "25-" means that the glucose concentration is 25mM; "50-" means that the glucose concentration is 50mM; 0, 5, 25, 50 means that the NAS concentrations are 0mM, 5mM, 25mM, and 50mM, respectively.

[0120] After incubation for 2 hours, the supernatant of each group was collected and the release of GLP-1 was detected using a human GLP-1 ELISA kit (FANKEL Industrial Co., Ltd, Shanghai, China, F0203-A, 23090549N). The detection method was the same as in Example 3.

[0121] The results are as follows Figure 7 As shown, N-acetyl serotonin promotes the release of GLP-1 from glucose-stimulated NCI-H716 cells in a concentration-increasing manner.

[0122] The above examples demonstrate that N-acetyl serotonin can significantly improve high fasting blood sugar, high postprandial blood sugar, glucose intolerance, and decreased insulin tolerance; it can also significantly improve insufficient GLP-1 secretion.

[0123] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of N-acetyl serotonin as an active ingredient in the preparation of drugs for the treatment of type 2 diabetes.

2. The use according to claim 1, characterized in that: The type 2 diabetes includes type 2 diabetes caused by genetic factors, obesity factors and high-fat diet.

3. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable carriers and excipients.

4. The use according to claim 1, characterized in that: The dosage forms of the medicine include tablets, granules, capsules and mixtures.

5. The use according to claim 1, characterized in that: The N-acetyl serotonin achieves the effect of treating type 2 diabetes by lowering fasting blood sugar level, improving glucose tolerance and improving insulin tolerance.

6. The use according to claim 1, characterized in that: The N-acetyl serotonin reduces fasting blood sugar levels, improves glucose tolerance, and improves insulin tolerance by promoting the secretion of GLP-1.

7. The use according to claim 1, characterized in that: The dosage of N-acetyl serotonin is 5 mg / kg-50 mg / kg.

8. The use according to claim 7, characterized in that: The dosage of N-acetyl serotonin is 25 mg / kg-50 mg / kg.

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