Application of N-acetylserotonin in the prevention and treatment of diabetes

Abstract

This invention relates to the field of biomedical technology, and particularly to the application of N-acetylserotonin (NAS) in the prevention and treatment of diabetes. Experiments have shown that NAS significantly improves symptoms such as weight loss, increased water intake, and increased food intake in type 2 diabetes mellitus (T2DM) mice; NAS improves fasting blood glucose and glucose tolerance levels in T2DM mice; NAS reduces glycated serum protein and insulin resistance levels in T2DM mice; and it enhances pancreatic β-cell function and insulin sensitivity in T2DM mice. This indicates that N-acetylserotonin has promising clinical application prospects in the treatment of type 2 diabetes and is of great significance for the development of novel pharmaceutical compositions for type 2 diabetes.

Description

[Technical Field]

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of N-acetylserotonin in the prevention and treatment of diabetes. [Background Technology]

[0002] Diabetes mellitus is a common systemic chronic metabolic disease characterized by insulin resistance and chronic hyperglycemia, and has become a major public health problem worldwide. Type 2 diabetes is the most common type, accounting for over 90% of all diabetes cases globally.

[0003] According to an epidemiological survey report released in China in 2022, the prevalence of diabetes among Chinese adults was 12.8%, and the prevalence of type 2 diabetes continued to increase and showed a trend towards affecting younger people. In patients with poorly controlled diabetes, persistent hyperglycemia and insulin resistance can lead to diabetic complications. Controlling blood sugar and improving insulin resistance are the main means of preventing diabetic complications.

[0004] Therefore, developing drugs to prevent and treat diabetes has always been a hot topic in medical research. [Summary of the Invention]

[0005] In view of the above, it is necessary to provide a pharmaceutical composition whose main ingredient is N-acetylserotonin, which has good clinical application prospects in the treatment of type 2 diabetes.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A drug, N-acetylserotonin (NAS), source: Sigma-Aldrich; CAS No.: 1210-83-9; molecular formula: C 12 H 14 N2O2; molecular weight: 218.25;

[0008] Its structural formula is as follows:

[0009]

[0010] The aforementioned drug, N-acetylserotonin, is used for the prevention and treatment of diabetes.

[0011] In this invention, the diabetes is specifically type 2 diabetes.

[0012] Furthermore, in this invention, the application includes the preparation of drugs for the prevention and treatment of diabetes using N-acetylserotonin as a raw material.

[0013] The present invention also provides a pharmaceutical composition for the prevention and treatment of diabetes, the pharmaceutical composition comprising an oral or injectable formulation of N-acetylserotonin and pharmaceutically acceptable excipients.

[0014] In this invention, the pharmaceutical composition is specifically an oral formulation.

[0015] In this invention, the pharmaceutically acceptable excipient is further selected from one or any combination of solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants.

[0016] In this invention, the preparation method of the pharmaceutical composition further includes the following steps: placing N-acetylserotonin powder at the bottom of a centrifuge tube after autoclaving; using a pipette to draw dimethyl sulfoxide and place it at the bottom of the centrifuge tube; slowly shaking the N-acetylserotonin powder and dimethyl sulfoxide until fully dissolved and transparent with no powder; finally, adding pure water to make up to the final volume; shaking the centrifuge tube up and down; and then placing it on a vortex mixer to fully agitate it to make the solution concentration more uniform, thereby obtaining the pharmaceutical composition.

[0017] The present invention has the following beneficial effects:

[0018] This invention is the first to propose using NAS as an active ingredient to prepare a drug for treating diabetes, specifically type 2 diabetes. To verify the efficacy, the applicant used db / db mice as a type 2 diabetes mouse model in the experiment. The mice were treated with N-acetylserotonin via gavage. Monitoring of body weight, weight gain, food intake, water intake, fasting blood glucose, glucose tolerance, and serum glycated serum protein and insulin levels were conducted, and the insulin resistance index (HOMA-IR), pancreatic β-cell function index (HOMA-β), and insulin sensitivity index (natural logarithm, ISILN) were calculated to demonstrate the novel application of N-acetylserotonin in the treatment of type 2 diabetes. Specifically, NAS significantly improved symptoms of weight loss, increased water intake, and increased food intake in T2DM mice; NAS improved fasting blood glucose and glucose tolerance levels in T2DM mice; NAS reduced glycated serum protein and insulin resistance levels in T2DM mice; and NAS improved pancreatic β-cell function and insulin sensitivity in T2DM mice. This indicates that the drug N-acetylserotonin has promising clinical application prospects in the treatment of type 2 diabetes and is of great significance for the development of novel drug compositions for type 2 diabetes. [Attached Image Description]

[0019] Figure 1To improve the drinking and eating conditions of type 2 diabetic mice through NAS intervention;

[0020] Figure 2 To improve body weight changes and weight gain in type 2 diabetic mice through NAS intervention;

[0021] Figure 3 The drug NAS intervention was used to improve fasting blood glucose and area under the glucose tolerance curve (AUC) in type 2 diabetic mice.

[0022] Figure 4 The drug NAS intervention improved serum glycated serum protein, insulin, insulin resistance index and insulin sensitivity in type 2 diabetic mice.

Detailed Implementation Methods

[0023] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0024] This embodiment specifically tests the effect of the drug N-acetylserotonin on improving type 2 diabetes. The test method includes the following steps:

[0025] (1) Selection and feeding of type 2 diabetes mellitus (T2DM) mouse models:

[0026] Fifty 8-week-old male C57BL / KsJ-db / db(db / db) type 2 diabetes mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., and were certified with Laboratory Animal Production License (License No.: SCXK(Su)2016-0010), Jiangsu Province Laboratory Animal Quality Certificate (No. 202369340), and Animal Quarantine Certificate (Animal A, No. 3203395244). All mice were housed at the Animal Center of Guangxi Medical University (hospitalization environment: room temperature 22±2℃, relative humidity 50%±20%, indoor light and dark cycle 12 hours). They were housed in individual ventilated cages (IVC cages) with 5 mice per cage. Each mouse was tagged with an ear tag. They were fed a maintenance diet provided by the university (Beijing Keao Xieli Feed Co., Ltd., protein 23.07%, fat 11.85%, carbohydrates 65.08%) and sterilized water. They had free access to food and water. The bedding, food, and water were changed 3 times a week. Food intake and water intake were recorded 3 times a week, and body weight was recorded once a week.

[0027] All mouse cages were equipped with LED flexible light strips (5730 household waterproof 220V double-row flexible light strips), and a photometer (Delixi DLY-1801C) was used to measure the light intensity in the center of each cage to be 48-55 lx. That is, the lighting conditions of each cage were balanced and met the 12-hour light-dark cycle (8:00 am-8:00 pm). After the animals were acclimatized, an oral glucose tolerance test (OGTT) was performed for subsequent animal grouping.

[0028] (2) Animal grouping and treatment:

[0029] Using a random number table method, all mice were divided into 5 groups based on their body weight, fasting blood glucose, and OGTT results. Each group consisted of 10 db / db mice, which were housed in 2 separate cages with 5 mice per cage.

[0030] The five groups of mice were: (1) model group (T2DM group, T2DM + pure water 0.05ml / 10g BW / day, N=10); (2) metformin group (MET group, T2DM + MET 150mg / kg BW / day, N=10); (3) low-dose NAS group (L-NAS group, T2DM + NAS 0.25mg / kg BW / day, N=10); (4) medium-dose NAS group (M-NAS group, T2DM + NAS 1mg / kg BW / day, N=10); (5) high-dose NAS group (H-NAS group, T2DM + NAS 4mg / kg). BW / day, N=10); After grouping, the mice were intervened. Their weight and water intake were measured and recorded daily. Their food intake was measured every 3 days, and their growth and activity were observed. Fresh feces were collected from the mice before and after the intervention. Fasting blood glucose (FBG) was measured weekly, and an oral glucose tolerance test (OGTT) was performed every 4 weeks. The principle of blood glucose measurement was to exclude stress caused by temperature, sound, light, odor, and handling.

[0031] (3) Intervention drugs:

[0032] 1) Low-dose group NAS solution (gavage dose of 0.25 mg / kg, gavage standard of 0.05 ml / 10 g);

[0033] 2) Medium-dose group NAS solution (gavage dose of 1 mg / kg, gavage standard of 0.05 ml / 10 g);

[0034] 3) High-dose group NAS solution (gavage dose of 4 mg / kg, gavage standard of 0.05 ml / 10 g)

[0035] 4) Metformin hydrochloride solution (gavage dose of 150 mg / kg, gavage standard of 0.05 ml / 10 g).

[0036] The solution preparation method is as follows:

[0037] Preparation of NAS solution: Before preparing the solution, remove the N-acetylserotonin (NAS) drug, which is sealed and protected from light from storage at -20 degrees Celsius, and place it on ice. In a quiet and windless environment, use an electronic balance (accuracy 0.0001g) and sterile weighing paper. After adjusting to the tare setting, use a sterile weighing scoop to place an appropriate amount of NAS powder onto the weighing paper. Accurately weigh 0.004g of NAS powder and place it at the bottom of a 5ml autoclaved centrifuge tube (do not allow the powder to stick to the tube walls). Use a 200μL pipette to pipette 50μL of dimethyl sulfoxide (DMSO). All of the ingredients were placed at the bottom of the centrifuge tubes. The powder and DMSO were slowly shaken until fully dissolved and transparent with no powder residue. Finally, pure water was added to bring the volume to the 5 mL mark. The centrifuge tubes were shaken up and down and then placed on a vortex mixer to agitate the solution to ensure a more uniform concentration. A high-dose NAS solution of 0.0008 g / mL was prepared. The centrifuge tubes were labeled as H-NAS group. The solution was prepared fresh for each use. A medium-dose NAS solution of 0.0002 g / mL and a low-dose NAS solution of 0.00005 g / mL were prepared in sequence.

[0038] Preparation of metformin hydrochloride solution: Before preparing the solution, the metformin drug should be sealed and stored at room temperature in a light-proof environment. In a quiet and windless environment, use an electronic balance (accuracy 0.0001g) and sterile weighing paper. After adjusting to the tare setting, use a sterile weighing spoon to place an appropriate amount of metformin powder on the weighing paper. Accurately weigh 0.2g of metformin powder and place it at the bottom of a 5ml autoclaved centrifuge tube (do not let the powder stick to the wall). Add pure water to make up to the 5mL mark. Shake the centrifuge tube well and then place it on a vortex mixer to make the solution concentration more uniform until it is transparent and free of powder. The final solution is 0.04g / mL metformin. Label the centrifuge tube with the MET group. The solution should be prepared and used immediately.

[0039] (4) Experimental materials and instruments:

[0040] Electronic analytical balance (BSA124S, Sartorius Scientific Instruments (Beijing) Co., Ltd.), vortex mixer (Vortex Kylin-bell 5, Jiangsu Haimen Qilin-bell Instrument Manufacturing Co., Ltd.); pure water / ultrapure water system (DirectQ8UV, Tongdi Scientific Instruments (Shanghai) Co., Ltd.); ultra-low temperature freezer (995, Thermo Scientific, USA); blood glucose meter (RocheACCU-CHECK, Roche Diagnostics Products Co., Ltd. (Shanghai)); blood glucose test strips (RocheACCU-CHECK, Roche Diagnostics Products Co., Ltd. (Shanghai)); ELISA reader (Epoch2, Bertek Instruments, USA); high-speed centrifuge; ophthalmic scissors; cotton swabs; tissues; sterile centrifuge tubes, etc.

[0041] (5) Mouse sample collection, index detection and calculation:

[0042] After 23 weeks of intervention, mice were fasted overnight but allowed free water for 12 hours. T2DM mice were anesthetized with isoflurane for 10-15 seconds, and blood was collected by enucleation. After the blood samples were left to stand for 3 hours, they were centrifuged (3000 rpm / min, 4℃, 10 min). The serum was collected by centrifugation, aliquoted into 8-tube containers, and stored in an ultra-low temperature freezer at -80℃ for the detection of biochemical indicators. Serum insulin (INS) and glycated serum protein (GSP) were detected using the Jiangsu Jingmei ELISA kit. Fasting plasma glucose (FPG) and fasting insulin (FINS) were used to calculate: HOMA-IR = fasting plasma glucose level (FPG, mmol / L) × fasting insulin level (FINS, μU / mL) / 22.5; ISI = 1 / (FPG × FINS), and its natural logarithm, i.e., ln[1 / (fasting plasma glucose × FINS), was used for statistical analysis; HOMA-β = 20 × fasting insulin level (FINS, μU / mL) / (fasting plasma glucose level (FPG, mmol / L) - 3.5) (%).

[0043] (6) Statistical analysis:

[0044] All experimental data were analyzed using SPSS Statistics 26.0. Results for each group are expressed as mean ± standard deviation (Mean ± SD). All images were generated using Graphpad Pism 9.5 and R 3.6.0 software.

[0045] (7) Experimental results:

[0046] The effect of the drug N-acetylserotonin on improving type 2 diabetes, such as Figure 1-4 As shown.

[0047] Result 1: NAS intervention improved diabetes symptoms in type 2 diabetic mice.

[0048] Compared with the T2DM group, there was no significant difference in average water intake and average food intake in the NAS group before the intervention. Figure 1 (A, D, P>0.05) After 23 weeks of intervention, the average and total water intake of mice in the MET, L-NAS, M-NAS, and H-NAS groups were significantly lower than those in the T2DM group. The differences in the MET, L-NAS, and H-NAS groups were statistically significant. Figure 1 BC, P<0.05); the average and total food intake of mice in the L-NAS and H-NAS groups were significantly lower than those in the T2DM group (BC, P<0.05); Figure 1EF, P<0.05); Throughout the experiment, the body weight of mice in the L-NAS, M-NAS, and H-NAS groups was higher than that in the T2DM group and lower than that in the MET group, but the differences were not statistically significant (e.g., EF, P<0.05); Figure 2 A, P<0.05). Compared with T2DM, between weeks 2 and 23, the weight gain of mice in the L-NAS, M-NAS, and H-NAS groups was higher than that in the T2DM group but lower than that in the MET group, with only the MET and M-NAS groups showing statistical significance (e.g., ...). Figure 2 B, P<0.05). Therefore, NAS intervention improved the diabetes symptoms in type 2 diabetic mice.

[0049] Result 2: NAS intervention improved fasting blood glucose and glucose tolerance levels in type 2 diabetic mice.

[0050] Compared with the T2DM group, there were no significant differences in fasting blood glucose and glucose tolerance levels in the NAS group before intervention. After 23 weeks of intervention, the fasting blood glucose levels of mice in the MET, L-NAS, M-NAS, and H-NAS groups were significantly lower than those in the T2DM group. Among them, the differences in the MET, M-NAS, and H-NAS groups were statistically significant. Figure 3 AB, P<0.05); The AUC of mice in the MET group, M-NAS group, and H-NAS group after 4 weeks of intervention was significantly lower than that in the T2DM group, among which the differences in the MET group and H-NAS group were statistically significant (AB, P<0.05); Figure 3 CD, P<0.05);

[0051] Result 3: NAS intervention reduced glycated serum protein and insulin resistance in T2DM mice, and improved pancreatic β-cell function and insulin sensitivity in T2DM mice: Compared with the T2DM group, the glycated serum protein levels in the MET group, L-NAS group, M-NAS group, and H-NAS group were all lower than those in the T2DM group. Among them, the differences in the MET group, L-NAS group, and H-NAS group were statistically significant. Figure 4 A, P<0.05). Compared with the T2DM group, the serum insulin and insulin resistance index of the MET group, L-NAS group, M-NAS group and H-NAS group were significantly lower than those of the T2DM group. Figure 4 B, C, P<0.05), and all showed a decreasing trend with increasing NAS intervention dose; conversely, the insulin sensitivity of the MET group, L-NAS group, M-NAS group, and H-NAS group was higher than that of the T2DM group, and the differences between the MET group, M-NAS group, and H-NAS group were statistically significant. Figure 4EP<0.05), and showed a trend of increasing with increasing NAS intervention dose; the insulin β-cell index of the MET group, L-NAS group, M-NAS group and H-NAS group were all higher than that of the T2DM group, and only the difference in the MET group was statistically significant. Figure 4 D, P<0.05).

[0052] In summary, NAS significantly improved the symptoms of weight loss, increased water intake, and increased food intake in T2DM mice; NAS improved fasting blood glucose and glucose tolerance levels in T2DM mice; NAS reduced glycated serum protein and insulin resistance levels in T2DM mice; and NAS improved pancreatic β-cell function and insulin sensitivity in T2DM mice. This indicates that N-acetylserotonin has promising clinical application prospects in the treatment of type 2 diabetes and is of great significance for the development of novel drug compositions for type 2 diabetes.

[0053] The above embodiments are merely examples of several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. The use of N-acetylserotonin in the preparation of drugs for the prevention and / or treatment of type 2 diabetes.

Images