Application of 6-methoxysalicylic acid in preparation of medicine for relieving convulsion and soothing nerves

Abstract

The invention discloses a new application of 6-methoxysalicylic acid, namely application of 6-methoxysalicylic acid in preparation of a medicine for relieving convulsion and soothing nerves, through systematic pharmacological experiment research, it is found for the first time that 6-methoxysalicylic acid has EP4 receptor agonistic activity and can exert the effects of relieving convulsion and soothing nerves by specifically activating an EP4 receptor, and the 6-methoxysalicylic acid can be applied to preparation of medicines for relieving convulsion and soothing nerves. The symptoms such as palpitation and restlessness, sleep disorder and the like are effectively improved, and a new way is provided for treating diseases such as insomnia and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of 6-methoxysalicylic acid (6-SMA) in calming and tranquilizing. BACKGROUND

[0002] With the acceleration of modern social life pace, the increase of work pressure and the influence of environmental factors, the incidence of mental and psychological related diseases such as anxiety, insomnia and neurasthenia continues to rise. Such diseases are often accompanied by core symptoms such as palpitation, restlessness, irritability and sleep disorders, which seriously affect the physical and mental health and quality of life of patients.

[0003] Precise regulation of sleep-wake cycle is the key to maintaining normal physiological functions. Related studies have confirmed that prostaglandin E2 (PGE2) as an important bioactive substance in the mammalian brain, its bidirectional regulation of sleep-wake is closely related to the specific distribution and activation of EP receptor subtypes. Among them, the activation of EP1 / EP2 receptors can mediate the wake-promoting effect, while EP4 receptor as a key sleep-promoting target, its specific activation in the PGD2-SZ (PGD2-sensitive sleep zone) of the ventral surface area of the rostral basal forebrain and the nearby area of the subarachnoid space can significantly increase slow wave sleep (SWS) and paradoxical sleep (PS), and has fast onset and stable effect, which provides a clear target direction for the research and development of calming and tranquilizing drugs.

[0004] Chuanxiong is a traditional Chinese medicinal material in China, which was first recorded in Shennong's Herbal Classic and listed as the top grade. It has the effects of activating blood and promoting qi, dispelling wind and relieving pain, etc. Its active ingredients are rich, including alkaloids, phenolic acids, volatile oils, etc. It has shown good application potential in the treatment of cardiovascular system and nervous system diseases. 6-methoxysalicylic acid is a natural phenolic acid compound isolated and purified from Chuanxiong. Existing studies have confirmed that it has pharmacological activities such as anti-inflammatory, antioxidant and protection of nerve cells, but there is no related report that it acts as an EP4 receptor agonist and exerts calming and tranquilizing effect by activating EP4 receptor. There is also no technical scheme for targeting regulation of EP4 receptor by the compound to improve symptoms such as palpitation, sleep disorders, etc.

[0005] Based on the defects of existing calming and tranquilizing drugs, the clear mechanism of EP4 receptor as a sleep-promoting target and the development potential of natural active ingredients, it is of important practical significance and application value to develop a calming and tranquilizing drug with EP4 receptor as the target, high safety, definite curative effect and small side effects, to enrich the clinical treatment means and meet the needs of patients for medication. SUMMARY

[0006] This invention provides a novel use for 6-methoxysalicylic acid, namely its application in the preparation of sedative and tranquilizing drugs. Through systematic pharmacological experiments, this application has for the first time discovered that 6-methoxysalicylic acid has EP4 receptor agonist activity, which can exert sedative and tranquilizing effects by specifically activating EP4 receptors, effectively improving symptoms such as palpitations, anxiety, and sleep disorders.

[0007] The chemical structural formula of the 6-methoxysalicylic acid is as follows: .

[0008] The purpose of this invention is to use 6-methoxysalicylic acid in the preparation of sedative and tranquilizing drugs. Specifically, 6-methoxysalicylic acid is used as the active ingredient, or in combination with other active ingredients, to exert its effect. In the preparation of sedative and tranquilizing drugs, one or more pharmaceutically acceptable excipients may be added. These excipients include conventional pharmaceutical fillers, diluents, binders, excipients, absorption enhancers, surfactants, stabilizers, etc., to improve the absorption of the agonist or facilitate administration, such as in the form of capsules, pills, powders, tablets, granules, oral liquids, etc.

[0009] Preferably, the dosage form of the drug includes capsules, powders, tablets, granules, oral liquids, or injections.

[0010] The beneficial effects of this invention are: This invention provides the use of 6-methoxysalicylic acid, which can effectively stimulate the activity of prostaglandin receptor EP4. Therefore, it can be used to prepare agonists of prostaglandin receptor EP4. By stimulating the activity of prostaglandin receptor EP4, 6-methoxysalicylic acid can significantly increase slow-wave sleep (SWS) and REM sleep (PS), thereby effectively exerting a calming and sedative effect. Attached Figure Description

[0011] Figure 1 This is a schematic diagram showing the docking results of 6-methoxysalicylic acid with EP4 molecules. Figure 2 This is a schematic diagram of Western blot detection used in Example 2 to verify the activation of ERK phosphorylation by 6-MSA through the EP4 receptor using the EP4 receptor agonist PGE2. In this diagram, A represents the Western blot bands and B represents the band quantification results. Figure 3 This is a schematic diagram of the Western Blot detection results of 6-MSA on EP4 receptor-mediated p-ERK protein expression in Example 2; where A is the Western Blot result and B is the band quantification result. Figure 4This is a schematic diagram showing the detection of EP4 protein (VSVG marker) expression by 6-MSA at different temperatures, where A is the Western blot result and B is the band quantification diagram; Figure 5 This is a schematic diagram showing the detection of EP4 protein (VSVG label) expression by 6-MSA at different organic solvent (AEA) concentrations, where A is a schematic diagram of Western blot detection and B is a band quantification diagram; Figure 6 A diagram showing the distance traveled by zebrafish; Figure 7 A graph showing the speed of movement of zebrafish; Figure 8 This is a line graph showing the behavioral trajectory of zebrafish. Detailed Implementation

[0012] The methods described in this invention are further described below through examples, but the scope of protection of this invention is not limited by the examples. Unless otherwise specified, the reagents used in this embodiment are all commercially available reagents or reagents prepared by conventional methods, and the methods used are all conventional methods unless otherwise specified. Example 1: Predicting the binding of 6-methoxysalicylic acid to the EP4 receptor using molecular docking. 1. Preparation of ligand small molecules (1) Search for the PubChemCID number of the target blood-entering component in the Drugbank online database, and download and save its sdf format structure from the PubChem online database; (2) A small molecule library was created in the software AutoDockvina. All small molecules were imported, and the small molecule library was "Washed" and 3Dized to obtain the optimized ligand (i.e. 6-methoxysalicylic acid). 2. Protein receptor preparation (1) Download and save the 3D structure of the target receptor protein (i.e., the EP4 receptor) from the RCSB PDB (RCSB Protein Data Bank) online database; (2) Import the structure of the target receptor protein into the software MOE, remove water molecules and organic solvents, and retain only the protein and the original ligand; 3. Docking (1) Dock the optimized ligand in step 1 and the protein in step 2. When docking, select the docking active pocket as LigandAtoms, set to generate 30 conformations and output 3 docking results as reference, and keep the other parameters as default. (2) Waiting for docking: The evaluation standard is first to look at S, that is, the docking binding energy score (unit: kcal / mol). The smaller the value (usually negative), the lower the energy conformation. The smaller the conformation, the more tightly the small molecule binds to the ligand. 4. Visual analysis of docking results The results were visualized and analyzed using PyMOL in 3D mode, and the results were visualized and analyzed using MOE in 2D mode. The docking results are as follows: Figure 1 As shown; from Figure 1 It can be seen that 6-methoxysalicylic acid chuanxiong can bind to the EP4 receptor.

[0013] Example 2: Western blotting was used to verify the effect of 6-methoxysalicylic acid on EP4 based on the ERK1 / 2 signaling pathway.

[0014] 1. Culture of 293T cells overexpressing EP4 (1) Preparation: Disinfect the biosafety cabinet and necessary supplies with ultraviolet lamp for 30 minutes to ensure the cleanliness and sterility of the operating area; turn on the constant temperature water bath (37℃) and put the culture medium, 1×PBS buffer and 0.25% trypsin into it to preheat, so as to ensure that the temperature matches the in vivo environment and provide a suitable growth environment for the cells. (2) Washing: Slowly add 2 mL of 1×PBS buffer along the wall of the culture dish, rinse the cell surface, and then aspirate and discard. Repeat this process twice. (3) Digestion: Add 0.5 mL of 0.25% trypsin to digest the cells, place them in an incubator and let them digest statically. After the cells appear as quicksand, use complete culture medium to stop the digestion, mix the cells, and obtain a cell suspension. (4) Centrifugation: Gently disperse the cell suspension to distribute it evenly, transfer it to a clean and sterile 1.5mL EP tube, centrifuge at 800rpm for 3min, discard the supernatant after centrifugation, and retain the cell pellet. (5) Culture: The cell pellet was seeded into a 100 mm dish, 5 mL of DMEM medium containing 10% FBS was added, and the dish was placed in a cell culture incubator at 37°C and 5% CO2 for cell culture until the cell density reached 75%. 2. Cell transfection (1) Preparation: Preheat Opti-MEM medium and PBS in a 37°C water bath; remove cells from the cell culture incubator, discard the old medium by pipetting, wash the cells with preheated PBS, add 5 mL of preheated Opti-MEM medium to the cells, and incubate in a 37°C, 5% CO2 cell culture incubator for 1 h to obtain cells to be transfected. (2) Prepare solution A: 200 μL Opti-MEM medium + 18 μL lipofectamine 2000 / well, incubate at 37℃ for 5 min; Solution B: 200 μL Opti-MEM medium + 9 μL plasmid (pcDNA5 / FRT / TO-VSV-GluR5-EP4) (purchased from Beijing Qingke Biotechnology Co., Ltd.), incubate at 37℃ for 5 min; Mix solution A and solution B, gently pipette, and continue incubating at 37℃ for 20 min; obtain the mixture. (3) Add the cells to be transfected to the mixture and place them in a 37°C constant temperature incubator for transfection. After 6 hours of transfection, replace the medium with fresh DMEM medium containing 10% FBS and transfect for another 12 hours to obtain the transfected cells. 3. Cell plating (1) The transfected cells were washed, digested and centrifuged (refer to steps 1(2) to (4) of Example 2) to obtain the treated cells, wherein the amount of trypsin added during digestion was increased to 1 mL; (2) Open the cell well plate in the biological cabinet, wet each well with poly-L-lysine and aspirate it, and place it in the cabinet to air dry; (3) Add 1 mL of 10% FBS DMEM medium to the treated cells, mix by pipetting, and then add an equal amount evenly to the cell well plate coated with poly-L-lysine. Shake the well plate in a cross shape. (4) After 4 hours of plating, the cells were replaced with 600 μL of FBS-free DMEM medium to starve them. 4. Cell protein extraction (1) Prepare 10% concentration DMEM medium without FBS using DMEM medium. -5 10 mol / L of EP4 receptor antagonist CJ-42794 (CJ) solution and 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 10 -11 10 -12 After starving cells for 12 h, mol / L 6-methoxysalicylic acid (6-SMA) solution was used to replace the FBS-free DMEM medium with 600 μL 6-methoxysalicylic acid solution and incubated for 40 min. (2) After incubation, add EP4 receptor agonist PGE2 to activate for 5 min; then add an appropriate amount of strong RIPA lysis buffer containing protease inhibitor Cocktail and sodium orthostalate to each well, and lyse on ice for 20 min to obtain lysed cells. (3) Transfer the lysed cells to a 1.5 mL EP tube, centrifuge at 16000 rpm for 15 min, discard the precipitate and keep the supernatant; (4) Add 1 / 4 volume of 5×SDS Loading Buffer to the supernatant and vortex to mix to obtain the protein sample; (5) Western blotting was used to detect the protein samples, and the results are as follows: Figures 2-3 As shown; As can be seen from the figure, VSV tag expression indicates successful overexpression of EP4; ERK phosphorylation can be dose-dependently activated by the EP4 receptor agonist PGE2, proving the successful establishment of the model; ERK phosphorylation can be dose-dependently activated by 6-methoxysalicylic acid, proving that 6-methoxysalicylic acid has an agonistic effect on EP4, rather than an antagonistic effect.

[0015] Example 3: Verification of the physical binding of 6-SMA to the EP4 receptor using Western blotting based on the SIP method. According to the experimental principle of SIP, the binding of drugs to proteins can protect them from denaturation by organic solvents. The protective effect of drugs on protein receptors decreases with increasing organic solvent concentration. When the effective concentration reaches the limit of drug protection, the protein receptors almost completely disappear. 1. Cell culture and transfection: Same as the cell culture and transfection steps in Example 2; 2. Cell collection (1) After transfection, discard the original culture medium and wash the transfected cells twice with pre-cooled 1×PBS Buffer to obtain the washed transfected cells. (2) Add 1 mL of cell lysis buffer (lysis buffer formula: 200 μL CellLysisBuffer (10×) + 1800 μL ddH2O + 20 μL PMSF Solution + 20 μL protein inhibitor Cocktail) to the washed transfected cells, shake and lyse for 20 min to obtain lysed cells; (3) Transfer the lysed cells to a 1.5 mL EP tube, centrifuge at 16000 rpm for 15 min, discard the precipitate, and keep the supernatant; (4) Transfer the supernatant and evenly distribute it into three clean 1.5 mL EP tubes, 460 μL in each tube, and add the agonist PGE2 (concentration of 10) to each tube. -5 M), 6-methoxysalicylic acid (concentration of 10) -5M); Sterile water was used as a blank control (NC). Dimethyl sulfoxide (DMSO) was added to the sterile water to ensure that the concentration of DMSO in the PGE2 group, 6-methoxysalicylic acid group and NC group was consistent. The samples were incubated in a shaker at 25°C for 40 min to obtain the incubated samples. 3. Gradient-based deformation processing (1) Temperature gradient processing Take 80 μL of each incubated sample and add 20 μL of sterile water to prepare a 100 μL organic solvent-free mixture. Incubate the mixture precisely in metal baths at 37℃, 42℃, 47℃, 52℃, 57℃, and 62℃ for 15 min, respectively, for heat denaturation. Immediately transfer to 4℃ for pre-cooling, then centrifuge at 4℃ and 16,000 rpm for 20 min, and collect the supernatant. (2) Organic solvent (AEA) gradient treatment Take 80 μL of each incubated sample and prepare organic solvent mixtures of different concentrations according to the following proportions: No organic solvent group: 80 μL incubated sample + 20 μL sterile water; 5% organic solvent group: 80 μL incubated sample + 5 μL organic solvent mixture + 15 μL sterile water; 10% organic solvent group: 80 μL incubated sample + 10 μL organic solvent mixture + 10 μL sterile water; 15% organic solvent group: 80 μL incubated sample + 15 μL organic solvent mixture + 5 μL sterile water; 20% organic solvent group: 80 μL incubated sample + 20 μL organic solvent mixture. Then, equilibrate at 25°C and 60 rpm for 30 min to allow the organic solvent to fully react with the protein. Finally, centrifuge at 4°C and 16000 rpm for 20 min and collect the supernatant.

[0016] The protein samples were analyzed using Western blotting, and the results are as follows: Figure 4 , 5 As shown; from Figure 4 , 5 As can be seen, 6-MSA can significantly enhance the thermal stability of EP4 protein by specifically binding to it, while effectively antagonizing the inhibitory effect of organic solvent mixtures on EP4 protein. 6-MSA is a key small molecule ligand for regulating the stability and function of EP4 protein.

[0017] Example 4: Constructing a zebrafish insomnia model to study the sedative and tranquilizing effects of 6-methoxysalicylic acid from Ligusticum chuanxiong. 1. Preparation of the main experimental solutions (1) Preparation of E3 culture medium: Accurately weigh 34.8g NaCl, 1.6g KCl, 5.8g CaCl2·2H2O and 9.78g MgCl2·6H2O with an analytical balance and make up to 2L with ultrapure water to prepare a 60× stock solution. Dilute 60 times before use and adjust the pH to 7.2.

[0018] (2) Preparation of 15-Pentamethylenetetrazole (PTZ) stock solution: 15-Pentamethylenetetrazole (PTZ), CAS No. 54-95-5, is a well-proven chemical inducer that can simulate epileptic seizures. Prepare a 2.5 mM PTZ solution and store it at -20°C; (3) Preparation of 6-methoxysalicylic acid: 6-methoxysalicylic acid, molecular formula C8H8O3, molecular weight 152.15. Prepare a 100mM stock solution and store it at -20℃ for later use. Dilute with E3 culture medium before use. Dilute to 100μM, 200μM, and 400μM working solutions (DMSO final concentration 1‰), filter, and store at 4℃ protected from light.

[0019] 2. Randomly divide the zebrafish fry into 6 groups of 5 fish each. The specific grouping and treatment methods are as follows: (1) Drug administration and incubation phase (6-well plate operation) The first group was the control group, with 1 mL of E3 culture medium added to the corresponding wells of a sterile 6-well plate; the second group was the PTZ model group, with 1 mL of E3 culture medium added to the corresponding wells of a 6-well plate; the third group was the melatonin group, with 1 mL of 400 μM melatonin working solution added to the corresponding wells of a 6-well plate; the fourth group was the low-dose 6-MSA group, with 1 mL of 100 μM 6-MSA working solution added to the corresponding wells of a 6-well plate; and the fifth group was the medium-dose 6-MSA group, with 1 mL of 200 μM 6-MSA working solution added to the corresponding wells of a 6-well plate. The sixth group was the high-dose 6-MSA group. 1 mL of 400 μM 6-MSA working solution was added to the corresponding well of the 6-well plate. After gently placing 5 zebrafish juveniles into each well of the 6-well plate, the 6-well plate was placed in a constant temperature incubator. The melatonin group and each dose of 6-MSA were first incubated for 4 hours for pre-protection. After the pre-protection was completed, except for the control group, all other groups were supplemented with 2.5mM PTZ working solution and incubated for another 10 minutes to complete the modeling.

[0020] (2) Behavioral testing phase (100-well whiteboard operation) After incubation, gently aspirate each zebrafish fry from the 6-well plate using a Pasteur pipette and transfer them to individual wells of a sterile 100-well white plate (one fry per well). Add E3 culture medium to each well of the white plate to an appropriate volume to ensure that the zebrafish can swim freely and do not escape.

[0021] 3. Data Acquisition and Processing: (1) Data collection: Start the ZebraLab zebrafish behavior analysis system, set the shooting frame rate to 25 fps and the detection time to 10 min, and collect the movement trajectory, total movement distance (unit: cm) and average speed (unit: cm / s) of each zebrafish simultaneously. Save the raw data as a CSV file.

[0022] (2) Processing: A purple trajectory overlay map with a resolution of 300dpi was generated using the system's supporting software. The background was set to white and the hole boundary was set to yellow dashed lines. GraphPad Prism 9.0 software was used to perform statistical analysis on the quantitative data. The data were expressed as "mean ± standard deviation (Mean ± SD)". The differences between groups were analyzed by one-way ANOVA combined with Dunnett's multiple comparison test. The significance of the differences was marked as *P<0.05, P<0.01, P<0.001, and ***P<0.0001. A bar chart of total movement distance and average speed was drawn.

[0023] from Figure 6 , 7 It can be seen that the total movement distance and average speed of the PTZ model group were significantly higher than those of the control group; the total movement distance and average speed of the low, medium and high dose 6-MSA groups were significantly lower than those of the PTZ model group, and showed a concentration-dependent effect; the regulatory effect of the 400μM 6-MSA group was not significantly different from that of the melatonin group. Figure 8 It can be seen that the movement trajectory of zebrafish in the control group is diffuse and widely distributed, while the movement trajectory of zebrafish in the PTZ model group is densely distributed in the central area of ​​the hole. The degree of diffusion of the movement trajectory of zebrafish in each 6-MSA dose group gradually approaches that of the control group as the concentration of 6-MSA increases. Among them, the trajectory distribution of the 400μM 6-MSA group is not significantly different from that of the control group.

[0024] In summary, 6-MSA can significantly improve PTZ-induced behavioral abnormalities in zebrafish and has a significant therapeutic effect in this insomnia model.

Claims

Application of 1,6-methoxysalicylic acid in the preparation of sedative and tranquilizing drugs.

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