Use of n-acetylserotonin for the preparation of a medicament for the treatment of type 2 diabetes

Abstract

The application discloses application of N-acetylserotonin in preparation of a medicine for treating type 2 diabetes, and belongs to the field of biological medicines. The application proves through in-vivo and in-vitro experiments that N-acetylserotonin can promote the secretion of GLP-1, has obvious effects of lowering blood sugar, good glucose tolerance and good insulin tolerance, and has obvious improvement on high fasting blood sugar, high postprandial blood sugar, poor glucose tolerance and decreased insulin tolerance. The application has obvious improvement on insufficient GLP-1 secretion. The application has important significance for maintaining stable blood sugar and improving glucose and insulin tolerance of diabetes patients, and provides a new solution for type 2 diabetes treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of N-acetylserotonin in the preparation of a drug for treating type 2 diabetes. BACKGROUND

[0002] Type 2 diabetes (T2D) is a chronic metabolic disease characterized by persistent hyperglycemia and impaired insulin function, which is caused by the interaction of genetics and environment. With the rapid development of economy, the pace of life is accelerating, and the sedentary lifestyle and unhealthy dietary patterns have led to a rising global obesity trend, and the incidence of type 2 diabetes has also increased year by year. The prevention and treatment of diabetes has become a major challenge in the world, and its current treatment mainly relies on drug therapy, aiming to control blood glucose levels, reduce complications, and improve the quality of life of patients. At present, the drug treatment of type 2 diabetes includes biguanides, sulfonylureas, thiazolidinediones (TZDs), dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-dependent glucose transporters 2 (SGLT-2) inhibitors, glucagon-like peptide-1 (GLP-1) analogs and other drugs. Metformin is the first choice for patients with type 2 diabetes due to 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 the inability of a single therapy to maintain good long-term glycemic control, the lack of significant blood glucose-lowering effects, poor glucose tolerance, poor insulin tolerance, and high treatment costs.

[0003] N-acetylserotonin is an acetylated derivative of serotonin (5-hydroxytryptamine), a precursor of melatonin, which can indirectly participate in sleep regulation. It has certain antioxidant effects, can scavenge free radicals, and reduce oxidative stress. At the same time, N-acetylserotonin has neuroprotective effects, can reduce neuronal damage, especially in ischemia-reperfusion injury and neurodegenerative diseases. Currently, there is no related report on the regulation of blood glucose by N-acetylserotonin. SUMMARY

[0004] The application aims to provide application of N-acetylserotonin in preparation of a medicine for treating type 2 diabetes, so as to solve the problems in the prior art. The application proves that N-acetylserotonin has the effects of obviously reducing blood sugar, good glucose tolerance and good insulin tolerance, and is of great significance for maintaining blood sugar stability and improving glucose and insulin tolerance of diabetes patients, and provides a new solution for type 2 diabetes treatment.

[0005] To achieve the above object, the application provides the following scheme.

[0006] The application provides application of N-acetylserotonin as an effective component in preparation of 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 medicine further includes a pharmaceutically acceptable carrier and excipient.

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

[0010] Further, the N-acetylserotonin achieves the effect of treating type 2 diabetes by reducing fasting blood sugar level, improving glucose tolerance and improving insulin tolerance.

[0011] Further, the N-acetylserotonin reduces fasting blood sugar level, improves glucose tolerance and improves insulin tolerance by promoting secretion of GLP-1.

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

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

[0014] The application discloses the following technical effects:

[0015] The application proves the effect of N-acetylserotonin on treatment of type 2 diabetes through in-vivo and in-vitro experiments. Animal experiments prove that N-acetylserotonin achieves the effect of treating type 2 diabetes by reducing fasting blood sugar level, improving glucose tolerance and improving insulin tolerance; cell experiments prove that N-acetylserotonin reduces fasting blood sugar level, improves glucose tolerance and improves insulin tolerance by promoting secretion of GLP-1.

[0016] N-acetylserotonin is widely sourced and easy to prepare, which reduces the treatment cost of type 2 diabetes, and has obvious effects of reducing blood sugar, good glucose tolerance and insulin tolerance, is of great significance to maintain blood sugar stability and improve glucose and insulin tolerance for diabetes patients, and provides a new solution for type 2 diabetes treatment. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

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

[0019] Figure 2 is the fasting blood glucose detection result of each group of mice after 4 weeks of administration, *P<0.05;

[0020] Figure 3 is the OGTT curve (A) and the area under the curve (B) of each group of mice at each time point after oral glucose, **P<0.01, ****P<0.0001;

[0021] Figure 4 is the ITT curve (A) and the area under the curve (B) of each group of mice at each time point after intraperitoneal injection of insulin, the comparison between groups uses One-way RMANOVA, the abscissa represents time, and the ordinate represents blood glucose value, *P<0.05, **P<0.01, ***P<0.001;

[0022] Figure 5 is the fasting insulin level (A) and insulin index (B) of each group of mice after 4 weeks of administration, *P<0.05, **P<0.01;

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

[0024] Figure 7 is the GLP-1 secretion level of NCI-H716 cells in different treatment groups, "25-" represents a glucose concentration of 25mM; "50-" represents a glucose concentration of 50mM; 0, 5, 25, 50 represent NAS concentrations of 0mM, 5mM, 25mM, 50mM respectively, *P<0.05, **P<0.01, ****P<0.0001. DETAILED DESCRIPTION

[0025] The following detailed description of various exemplary embodiments of the application should not be considered to be limiting of the application, but rather a description of certain aspects, features and embodiments of the application.

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range is inclusive of its end points.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to the extent allowed by law. In the case of conflict between the description herein and the incorporated material, the description herein shall control.

[0028] In the description of the application specific embodiments, numerous specific details are set forth in order to provide a thorough understanding of the application. However, those of ordinary skill in the art will realize that the application can be practiced without many of these details. In other instances, well-known methods, procedures and components have not been described in detail so as not to unnecessarily obscure aspects of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from a review of the description of the application and practice of the application.

[0029] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" or the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0030] The present application provides the use of N-acetylserotonin in the treatment of type 2 diabetes.

[0031] Optionally, the use of N-acetylserotonin as an active ingredient in the preparation of a hypoglycemic drug for treating type 2 diabetes.

[0032] Optionally, the drug further comprises a pharmaceutically acceptable carrier and excipient.

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

[0034] Optionally, the drug comprises an oral preparation of N-acetylserotonin and an injection of N-acetylserotonin.

[0035] Optionally, the N-acetylserotonin oral preparation 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, N-acetylserotonin compound preparation.

[0036] Optionally, the N-acetylserotonin oral preparation also includes N-acetylserotonin granules.

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

[0038] Specifically, the N-acetylserotonin can reduce the fasting blood glucose of diabetic patients, improve glucose tolerance and insulin tolerance to achieve the effect of treating type 2 diabetes.

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

[0040] More specifically, the N-acetylserotonin can promote the secretion of GLP-1 in diabetic patients to reduce fasting blood glucose, improve glucose tolerance and insulin tolerance to achieve the effect of treating type 2 diabetes.

[0041] Optionally, chronic administration of N-acetylserotonin can significantly promote the secretion of GLP-1 in diabetic patients to reduce fasting blood glucose, improve glucose tolerance and insulin tolerance to achieve the effect of treating type 2 diabetes.

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

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

[0044] In one specific embodiment, the dosage of N-acetylserotonin for increasing the secretion of serum GLP-1 in mice is 5mg / kg-50mg / kg.

[0045] The molecular formula of the N-acetylserotonin is: C 12 H 14 N2O2, the molecular weight is: 218.25, the CAS number is 1210-83-9, and the molecular structure formula is as follows:

[0046]

[0047] The test methods used in the embodiments of the present application are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0048] Example 1 N-acetylserotonin significantly reduces fasting blood glucose and improves glucose tolerance

[0049] I. Experimental Methods

[0050] 1. Modeling Method

[0051] After C57BL / 6J mice were adapted to feeding for 1 week, they were randomly divided into a normal diet group and a high-fat diet group. The normal diet group was fed 10% control feed (Research diets, 12450J), and the high-fat diet group (HFD) was fed 60% high-fat feed (Research diets, D12492). During this period, the feed was replaced twice a week and weighed once a week. The feeding was continued for 12 weeks, and at the end of the 12th week, after fasting for 6 hours, the mice were evaluated for modeling success by fasting blood glucose detection and OGTT experiment. The standard for modeling success was fasting blood glucose ≥ 11.1 mmol / L and obvious glucose tolerance impairment.

[0052] 2. Experimental grouping and treatment (6 mice per group)

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

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

[0055] N-acetylserotonin experimental group (NAS): 60% high-fat diet + 5 mg / kg N-acetylserotonin. Oral gavage administration was used. The specific experimental grouping and treatment methods are shown in Table 1 and Figure 1 .

[0056] Table 1 Experimental grouping and treatment methods

[0057]

[0058] 3. Fasting blood glucose detection experiment

[0059] The mice that received N-acetylserotonin oral gavage administration for 4 weeks were tested after fasting for 6 hours. Blood was taken from the tail vein with a sterile needle, and the blood glucose concentration was measured with a Roche blood glucose meter.

[0060] 4. Glucose tolerance test

[0061] The mice were tested after 4 weeks of oral gavage of N-acetylserotonin after fasting for 6 hours. The fasting blood glucose level was first determined, followed by gavage of 20% glucose solution, and the blood glucose change was detected through the tail vein at 15 min, 30 min, 60 min, 90 min, and 120 min.

[0062] II. Experimental Results

[0063] The fasting blood glucose test results are shown in Figure 2 , where the blue dots in the figure are the fasting blood glucose values of the normal mice after gavage of PBS for four weeks. The purple square points represent the fasting blood glucose values of the diabetic mice after gavage of PBS for four weeks, and the red triangles represent the fasting blood glucose values of the diabetic mice after oral gavage of 5 mg / kg N-acetylserotonin for four weeks. Compared with the diabetic group, oral gavage of 5 mg / kg N-acetylserotonin significantly reduced the fasting blood glucose level of the mice.

[0064] The oral glucose tolerance test shows Figure 3 that compared with the diabetic group, continuous oral gavage of 5 mg / kg N-acetylserotonin for four weeks significantly reduced the fasting blood glucose level of the diabetic mice and improved the impaired glucose tolerance of the mice.

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

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

[0067] I. Experimental Methods

[0068] 1. Modeling method

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

[0070] 2. Experimental grouping and treatment (6 mice per group)

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

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

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

[0074] 3. Insulin sensitivity experiment

[0075] The mice were tested after 4 hours of fasting after receiving N-acetylserotonin orally by gavage for four weeks. The fasting blood glucose level was first determined, then insulin was injected intraperitoneally at 0.75 IU / kg, and the blood glucose change was detected through the tail vein at 15 min, 30 min, 45 min, 60 min, and 90 min.

[0076] 4. Detection of insulin level in mouse fasting serum samples

[0077] 1) Take the insulin detection kit out of the 4°C refrigerator 30 min in advance, and let it recover to room temperature. Calculate the required number of half strips in advance, and put the rest back in the refrigerator for storage.

[0078] 2) Reagent preparation: dilute Enzyme Conjugate 11x to Enzyme Conjugate 1x solution with Enzyme Conjugate Buffer; dilute Wash Buffer 21x to Wash Buffer 1x solution with ddH2O; standard solution Calibrators 0, 1, 2, 3, 4, 5 are ready.

[0079] 3) Set up blank wells (Control), standard wells (Calibrator), and sample wells (Samples) respectively. The blank wells are 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 samples and add them to the plate, then add 100 μL of Enzyme Conjugate 1x solution prepared in advance, and cover with a sealing film.

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

[0082] 6) Discard the reaction solution in the wells, invert and pat on a dust-free paper to dry the liquid.

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

[0084] 8) Add 200 μL of Substrate TMB to each well, seal the film, 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, tap to shake 5 s.

[0086] 5. Insulin resistance index (HOMA-IR) calculation

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

[0088] II. Experimental results

[0089] The results of the insulin sensitivity experiment are shown in Figure 4 As shown in the table, compared with the diabetes group, N-acetylserotonin treatment significantly reduced the blood glucose value of diabetic mice after insulin injection, and the corresponding area under the curve was significantly reduced.

[0090] Detection of fasting serum insulin showed Figure 5 Compared with the diabetes group, 4-week N-acetylserotonin treatment significantly reduced the fasting serum insulin level of high-fat diet mice, significantly restored the insulin index value, and significantly increased the insulin sensitivity.

[0091] Conclusion: Compared with the diabetes group, N-acetylserotonin has good insulin regulation effect. Oral gavage of 5 mg / kg N-acetylserotonin significantly reduced the fasting insulin level of mice, reduced insulin tolerance, and increased insulin sensitivity.

[0092] Example 3 N-acetylserotonin significantly increases GLP-1 secretion level

[0093] I. Experimental method

[0094] 1. Modeling method

[0095] The diabetes modeling method and related detection scheme used in this embodiment are the same as in Example 1, and intervention is performed when the diabetes mice are 3 months old.

[0096] 2. Experimental grouping and treatment (6 mice per group)

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

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

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

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

[0101] The mice after receiving N-acetylserotonin oral gavage for 4 weeks were tested after fasting for 6 hours. After fasting, the mice were orally gavaged 15 min, and blood was collected from the canthus into an EP tube containing a DDP4 inhibitor at a concentration of 1 μM, centrifuged at 3000 rpm for 20 min at 4°C, and the supernatant was used for detection with a mouse GLP-1 kit.

[0102] 4. GLP-1 level detection by ELISA kit

[0103] 1) Standard dilution: The 8 pmol / L standard solution provided in the kit was diluted to 4 pmol / L, 2 pmol / L, 1 pmol / L, 0.5 pmol / L, and 0.25 pmol / L standard solutions by the standard dilution solution according to the dilution method.

[0104] 2) Sample addition: Blank wells, standard wells, and sample wells were set up, and the blank wells did not contain enzyme-labeled reagents and samples, and the rest were the same. 50 μL of the standard solution was added to the enzyme-labeled plate wells, and the sample was diluted five times with the sample dilution solution, i.e., 40 μL of the sample dilution solution was added first, and then 10 μL of the sample to be tested was added. The sample was added to the bottom of the enzyme-labeled plate well as much as possible without touching the well wall, and was mixed gently.

[0105] 3) Incubation: After sealing the plate with a sealing film, it was placed in a 37°C incubator for 30 min.

[0106] 4) Solution preparation: The concentrated washing solution in the kit was diluted 30 times with distilled water (ddH2O) for standby.

[0107] 5) Washing: The sealing film was removed, the liquid in the plate was poured out and dried, the washing solution was added to the plate, and after standing for 30 seconds, the washing solution was poured out, and the washing was repeated 5 times, and the plate was dried on a clean filter paper.

[0108] 6) Enzyme addition: 50 μL of enzyme-labeled reagent was added to each well except the blank wells, and after incubation according to operation 3), the washing was performed according to operation 5).

[0109] 7) Color development: 50 μL of color developing agent A was added to each well, followed by 50 μL of color developing agent B, and the mixture was mixed gently, and after avoiding light, color development was performed in a 37°C incubator for 10 min.

[0110] 8) Termination: 50 μL of termination solution was added to each well to terminate the reaction.

[0111] 9) Measurement: The blank well was zeroed, and the OD values of each well were measured at 450 nm. The measurement needed to be completed within 15 min after the addition of the termination solution.

[0112] II. Experimental results

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

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

[0115] Example 4 In vitro experiment of N-acetylserotonin increasing GLP-1 secretion level of cells in high glucose state

[0116] 1. Experimental materials

[0117] NCI-H716 cells were purchased from the National Accredited Cell Culture Preservation Center, and were identified to exclude contamination of mycoplasma or chlamydia, and were used within ten generations.

[0118] 2. Experimental steps

[0119] NCI-H716 cells were inoculated into 96-well plates at a density of 5 x 10 5 cells / well, and incubated for 48 hours. Then 25 mM or 50 mM glucose and N-acetylserotonin with different concentrations prepared from buffer were added to the cells, respectively, and the groups were recorded as 25-0, 25-5, 25-25, 25-50, 50-0, 50-5, 50-25 and 50-50, respectively. In the group designation, “25–” indicates that the glucose concentration is 25 mM; “50–” indicates that the glucose concentration is 50 mM; 0, 5, 25, 50 indicate that the NAS concentration is 0 mM, 5 mM, 25 mM, 50 mM, respectively.

[0120] After 2 hours of incubation, the supernatant 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), and the detection method was the same as in Example 3.

[0121] As shown in the results, Figure 7 N-acetylserotonin promoted the release of GLP-1 from glucose-stimulated NCI-H716 cells in a concentration-dependent manner.

[0122] The above examples demonstrate that N-acetylserotonin has obvious improvement on high fasting blood glucose, high postprandial blood glucose, glucose intolerance, and decreased insulin tolerance; and has obvious improvement on insufficient GLP-1 secretion.

[0123] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. The application of N-acetylserotonin as an active ingredient in the preparation of drugs for treating type 2 diabetes.

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

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers and excipients.

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

5. The application according to claim 1, characterized in that, The N-acetylserotonin is used to treat type 2 diabetes by lowering fasting blood glucose levels, improving glucose tolerance, and improving insulin tolerance.

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

7. The application according to claim 1, characterized in that, The dosage of N-acetylserotonin used is 5 mg / kg to 50 mg / kg.

8. The application according to claim 7, characterized in that, The dosage of N-acetylserotonin used is 25 mg / kg to 50 mg / kg.

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