Application of N-acetyl serotonin in prevention and treatment of diabetes

Abstract

The invention relates to the technical field of biological medicines, in particular to application of N-acetyl serotonin in prevention and treatment of diabetes mellitus, and tests show that NAS has an obvious improvement effect on symptoms of weight loss, water intake increase and diet increase of T2DM mice; the NAS is used for improving the fasting blood glucose and the glucose tolerance level of the T2DM mouse; the NAS reduces the glycated serum protein and insulin resistance level of the T2DM mouse; the pancreatic beta cell function and insulin sensitivity of the T2DM mouse are improved. Therefore, the medicine N-acetyl serotonin has a good clinical application prospect in treating the type 2 diabetes mellitus and has important significance in developing a novel medicine composition for treating the type 2 diabetes mellitus.

Description

[Technical field]

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of N-acetyl serotonin in preventing and treating diabetes. [Background technology]

[0002] Diabetes is a common systemic chronic metabolic disease characterized by insulin resistance and chronic hyperglycemia, which has become a major public health problem worldwide. The most common type of diabetes is type 2 diabetes, which accounts for more than 90% of all diabetes cases worldwide.

[0003] According to an epidemiological survey report released by China in 2022, the prevalence of diabetes among Chinese adults is 12.8%, and the prevalence of type 2 diabetes continues to increase and tends to be younger. In patients with poor diabetes control, their persistent hyperglycemia and insulin resistance can cause diabetic complications. Controlling blood sugar and improving insulin resistance are the main means to prevent 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, the main constituent raw material of which is N-acetyl serotonin, which has good clinical application prospects in the treatment of type 2 diabetes.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A drug N-acetyl serotonin (NAS), source: Sigma-Aldrich; CAS number: 1210-83-9; molecular formula: C 12 H 14 N2O2; molecular weight: 218.25;

[0008] Its structural formula is as follows:

[0009]

[0010] The above-mentioned drug N-acetyl serotonin is used for preventing and treating diabetes.

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

[0012] In the present invention, further, the application includes preparing a drug for preventing and treating diabetes using N-acetyl serotonin as a raw material.

[0013] The present invention also provides a pharmaceutical composition for preventing and treating diabetes, which comprises an oral preparation or an injection preparation made of N-acetyl serotonin and a pharmaceutically acceptable excipient.

[0014] In the present invention, further, the pharmaceutical composition is specifically an oral preparation.

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

[0016] In the present invention, further, the preparation method of the pharmaceutical composition comprises the following steps: placing N-acetyl serotonin powder at the bottom of a centrifuge tube sterilized by high pressure, using a pipette to absorb dimethyl sulfoxide, and placing it at the bottom of the centrifuge tube, slowly shaking the N-acetyl serotonin powder and dimethyl sulfoxide to fully dissolve them to a transparent state without powder, and finally adding pure water to make up the volume, shaking the centrifuge tube up and down, and placing it on a vortex mixer to fully shake to make the solution concentration more uniform, so as to obtain the pharmaceutical composition.

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

[0018] The present invention proposes for the first time to use NAS as an active ingredient to prepare a drug for treating diabetes, specifically type 2 diabetes. In order to verify the effect, the applicant used db / db mice as a type 2 diabetes mouse model in the experiment, and used the drug N-acetyl serotonin to intervene in the mice by gavage. The mice were monitored for weight, weight gain, food intake, water intake, fasting blood sugar, glucose tolerance test, serum glycosylated serum protein, insulin detection, and insulin resistance index (HOMA-IR), pancreatic β cell function index (HOMA-β), and insulin sensitivity index (natural logarithm, ISILN) were calculated to confirm the new use of the drug N-acetyl serotonin in the treatment of type 2 diabetes. Specific performance: NAS has a significant improvement effect on the symptoms of weight loss, increased water intake, and increased food intake in T2DM mice; NAS improves the fasting blood sugar and glucose tolerance levels of T2DM mice; NAS reduces the glycosylated serum protein and insulin resistance levels of T2DM mice; and improves the pancreatic β cell function and insulin sensitivity of T2DM mice. This indicates that the drug N-acetyl serotonin has good clinical application prospects in the treatment of type 2 diabetes and is of great significance for the development of new drug compositions for type 2 diabetes.

Brief Description of the Drawings

[0019] Figure 1The drug NAS intervention improved the drinking water and diet status of type 2 diabetic mice;

[0020] Figure 2 The drug NAS intervention improved the weight changes and weight gain of type 2 diabetic mice;

[0021] Figure 3 The drug NAS intervention improved the fasting blood glucose and area under the glucose tolerance curve (AUC) of type 2 diabetic mice;

[0022] Figure 4 Drug NAS intervention improved serum glycosylated serum protein, insulin, insulin resistance index and insulin sensitivity in type 2 diabetic mice. [Specific implementation method]

[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0024] This embodiment specifically tests the improvement effect of the drug N-acetyl serotonin on type 2 diabetes, and the test method includes the following steps:

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

[0026] Fifty 8-week-old male type 2 diabetes model C57BL / KsJ-db / db (db / db) mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. with an experimental animal production license (license number: SCXK(Su)2016-0010), Jiangsu Province Laboratory Animal Quality Certificate (No. 202369340) and Animal Quarantine Certificate (Animal A, No. 3203395244). All mice were housed in the Animal Center of Guangxi Medical University (breeding environment: room temperature 22±2℃, relative humidity 50%±20%, indoor light-dark cycle 12 hours), and were housed in independently ventilated IVC cages (5 mice / cage). Each mouse was marked with an ear tag and fed with rat and mouse maintenance feed provided by the school (Beijing Ke'ao Xieli Feed Co., Ltd., protein 23.07%, fat 11.85%, carbohydrate 65.08%) and high-temperature sterilized water. They were allowed to eat and drink freely. The bedding, feed and water were changed three times a week. The food intake and water intake were recorded three times a week, and the body weight was recorded once a week.

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

[0028] (2) Animal grouping and treatment:

[0029] All mice were divided into 5 groups according to their body weight, fasting blood glucose and OGTT results using a random number table. Each group had 10 db / db mice, which were housed in 2 separate cages, with 5 mice in each cage.

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

[0031] (3) Intervention drugs:

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

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

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

[0035] 4) Metformin hydrochloride group solution (the oral administration dose is 150 mg / kg, and the oral administration standard is 0.05 ml / 10 g).

[0036] The solution is prepared as follows:

[0037] Preparation of NAS solution: Before preparing the solution, take out the N-acetyl serotonin (NAS) drug that is sealed and stored in a -20 degree refrigerator away from light and place it on ice. In a quiet and windless environment, use an electronic balance (accuracy of 0.0001g) and sterile weighing paper. After adjusting to the peeled state, use a sterile weighing spoon to take out an appropriate amount of NAS powder and place it on the weighing paper. Accurately weigh 0.004g of NAS powder and place it at the bottom of a 5ml centrifuge tube sterilized by high pressure (do not let the powder stick to the wall). Use a 200μL pipette to absorb 50μL of dimethyl sulfoxide (DMSO ), all placed at the bottom of the above centrifuge tube, slowly shake the powder and DMSO to fully dissolve them until they are transparent and free of powder, and finally add pure water to make the volume up to the 5mL mark. After shaking the centrifuge tube up and down, place it on a vortex mixer and fully oscillate to make the solution concentration more uniform. Finally, a high-dose NAS solution of 0.0008g / mL was prepared. The H-NAS group was marked on the centrifuge tube. The solution was prepared and used immediately, and a medium-dose NAS solution of 0.0002g / mL and a low-dose NAS solution of 0.00005g / mL were prepared in turn.

[0038] Preparation of metformin hydrochloride group solution: Before preparing the solution, the metformin drug is sealed and stored in the dark at room temperature. In a quiet and windless environment, use an electronic balance (accuracy of 0.0001g) and sterile weighing paper. After adjusting to the peeled state, use a sterile weighing spoon to scoop out an appropriate amount of metformin powder and place it on the weighing paper. Accurately weigh 0.2g of metformin powder and place it at the bottom of a 5ml centrifuge tube sterilized by high pressure (the powder should not stick to the wall). Add pure water to make up to the 5mL mark. After shaking the centrifuge tube up and down, place it on a vortex mixer and shake it thoroughly to make the solution concentration more uniform to a transparent state without powder. Finally, prepare a 0.04g / mL metformin solution. Mark the MET group on the centrifuge tube. The solution is 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, Qilin Bell Instrument Manufacturing Co., Ltd., Haimen City, Jiangsu Province); pure water / ultrapure water all-in-one machine (DirectQ8UV, Tongdi Scientific Instruments (Shanghai) Co., Ltd.); ultra-low temperature refrigerator (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)); microplate reader (Epoch2, Berton Instrument Co., Ltd., USA); high-speed centrifuge; ophthalmic scissors; cotton swabs; paper towels; sterile centrifuge tubes, etc.

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

[0042] After 23 weeks of intervention, after the mice were fasted overnight but not water for 12 hours, the T2DM mice were anesthetized with isoflurane for 10-15 seconds, and the blood of the mice was collected by enucleation. The collected blood samples were allowed to stand for 3 hours and then centrifuged (3000 rpm / min, 4°C, 10 min). The serum was collected by centrifugation, dispensed into 8-tube strips, and stored in a -80°C ultra-low temperature refrigerator for the detection of biochemical indicators. Jiangsu Jingmei Elisa kit (ELISA) was used to detect serum insulin (INS) and glycosylated serum protein (GSP); fasting blood glucose (FPG) and insulin (FINS) were used to calculate: HOMA-IR = fasting blood glucose level (FPG, mmol / L) × fasting insulin level (FINS, μU / mL) / 22.5; ISI = 1 / (FPG×FINS), and its natural logarithm, i.e., In[1 / (fasting blood glucose×FINS), was actually used for statistical analysis; HOMA-β = 20×fasting insulin level (FINS, μU / mL) / (fasting blood glucose level (FPG, mmol / L)-3.5)(%).

[0043] (6) Statistical analysis:

[0044] All experimental data were analyzed using SPSS Statistics 26.0. The results of each group were expressed as mean ± standard deviation (Mean ± SD). All graphics were completed using Graphpad Pism 9.5 and R 3.6.0 software.

[0045] (7) Test results:

[0046] The effect of N-acetyl serotonin on improving type 2 diabetes Figure 1-4 shown.

[0047] Results 1. NAS intervention improved the diabetic symptoms of type 2 diabetic mice:

[0048] Compared with the T2DM group, the NAS group had no significant differences in average water intake and average food intake before intervention ( Figure 1 A, D, P>0.05). After 23 weeks of intervention, the average water intake 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 among the MET, L-NAS and H-NAS groups were statistically significant ( Figure 1 BC, P<0.05); the average food intake and total food intake of mice in the L-NAS group and H-NAS group were significantly lower than those in the T2DM group ( Figure 1EF, P<0.05); during the whole experiment, the body weights of mice in the L-NAS group, M-NAS group, and H-NAS group were higher than those in the T2DM group and lower than those in the MET group, and the differences were not statistically significant (e.g. Figure 2 A, P<0.05). Compared with T2DM, the weight gain 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 from the 2nd to the 23rd week. Only the MET and M-NAS groups showed statistical significance (e.g. Figure 2 B, P<0.05). Therefore, NAS intervention improved the diabetic symptoms of type 2 diabetic mice.

[0049] Result 2. NAS intervention improved the fasting blood glucose level and glucose tolerance level of type 2 diabetic mice:

[0050] Compared with the T2DM group, there was no significant difference 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 group, L-NAS group, M-NAS group and H-NAS group were significantly lower than those in the T2DM group, among which the differences among the MET group, M-NAS group and H-NAS group 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 were significantly lower than that in the T2DM group, among which the difference between the MET group and the H-NAS group was statistically significant ( Figure 3 CD, P<0.05);

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

[0052] In summary, NAS has a significant improvement effect on the symptoms of weight loss, increased water intake, and increased food intake in T2DM mice; NAS improves fasting blood glucose and glucose tolerance levels in T2DM mice; NAS reduces the levels of glycosylated serum protein and insulin resistance in T2DM mice; and improves pancreatic β-cell function and insulin sensitivity in T2DM mice. This shows that the drug N-acetyl serotonin has a good clinical application prospect in the treatment of type 2 diabetes, and is of great significance for the development of new drug compositions for type 2 diabetes.

[0053] The above-mentioned embodiments merely express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention.

Claims

1. Application of N-acetyl serotonin in the prevention and treatment of diabetes.

2. The use of N-acetyl serotonin in preventing and treating diabetes according to claim 1, characterized in that: The diabetes is specifically type 2 diabetes.

3. The use of N-acetyl serotonin in preventing and treating diabetes according to claim 1, characterized in that: The application includes preparing medicine for preventing and treating diabetes by using N-acetyl serotonin as a raw material.

4. The pharmaceutical composition for preventing and treating diabetes according to any one of claims 1 to 3, characterized in that: The pharmaceutical composition comprises an oral preparation or an injection preparation made of N-acetyl serotonin and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition for preventing and treating diabetes according to claim 4, characterized in that: The pharmaceutical composition is specifically an oral preparation.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutically acceptable excipient is selected from one or any combination of solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents and antioxidants.

7. The pharmaceutical composition for preventing and treating diabetes according to claim 5, characterized in that: The preparation method of the pharmaceutical composition comprises the following steps: placing N-acetyl serotonin powder at the bottom of a centrifuge tube sterilized by high pressure, using a pipette to absorb dimethyl sulfoxide and placing it at the bottom of the centrifuge tube, slowly shaking the N-acetyl serotonin powder and dimethyl sulfoxide to fully dissolve them into a transparent state without powder, and finally adding pure water to make up the volume, shaking the centrifuge tube up and down, and placing it on a vortex mixer to fully shake it to make the solution concentration more uniform, so as to obtain the pharmaceutical composition.

Images