Use of jatamansin in preparation of medicine for treating insomnia

Abstract

This invention provides the application of swertisin in the preparation of drugs for treating insomnia. Through Western blotting experiments, this invention found that ERK phosphorylation can be dose-dependently activated by Swertisin, demonstrating that Swertisin has an agonistic effect on EP4 and can bind to the EP4 protein, thereby protecting EP4 from denaturation and precipitation under organic reagent treatment. By constructing a zebrafish insomnia model, the therapeutic effect of Swertisin on insomnia was studied. The results showed that Swertisin exhibited a concentration-dependent inhibitory effect on PTZ-induced insomnia-like activity; as the concentration increased, the zebrafish's movement trajectory gradually became sparser, showing a sedative-hypnotic potential similar to melatonin. Therefore, swertisin can be used to prepare an agonist of the prostaglandin receptor EP4, thereby effectively treating insomnia.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of swertiamarin in the preparation of drugs for treating insomnia. Background Technology

[0002] Insomnia is a common sleep disorder characterized by difficulty falling asleep, difficulty maintaining sleep, and early awakening. It is often accompanied by symptoms such as daytime fatigue, poor concentration, and mood swings. Long-term and recurrent episodes can lead to cognitive decline, weakened immunity, and even induce mental illnesses such as anxiety and depression, seriously affecting the patient's physical and mental health and quality of life.

[0003] Currently, benzodiazepines, non-benzodiazepines, and melatonin receptor agonists are commonly used medications for the clinical treatment of insomnia. However, these medications all have significant limitations in clinical application: long-term use of benzodiazepines easily leads to tolerance, dependence, and withdrawal symptoms, and may also cause adverse reactions such as dizziness, drowsiness, and memory loss; overdose can even lead to respiratory depression. While non-benzodiazepines have relatively lower dependence, they may still cause discomfort such as headaches, dizziness, and bitter taste in the mouth, and long-term use may also affect sleep structure. Melatonin receptor agonists have a slow onset of action, limited efficacy for some patients with severe insomnia, and may cause adverse reactions such as nausea and fatigue. Since the treatment of insomnia often requires long-term intervention, the adverse reactions and potential risks of existing medications impose a continuous burden on patients. Therefore, finding safe, effective, and non-addictive new therapeutic drugs and methods has become an important research direction in the field of insomnia treatment.

[0004] The significant advantage of traditional Chinese medicine (TCM) in treating insomnia lies in its good safety and tolerability. Modern research has confirmed that many active ingredients in TCM exert their anti-insomnia effects by regulating neurotransmitters and inflammatory pathways. TCM treatment of insomnia is based on the core principles of "holistic view" and "treatment according to syndrome differentiation," rooted in the self-healing concepts of "yin-yang harmony" and "mental tranquility." It is not limited to simply suppressing arousal function, but focuses on the fundamental pathogenesis of yin-yang imbalance and organ dysfunction in the body.

[0005] G protein-coupled receptors (GPCRs) are one of the largest families of membrane receptor proteins on the cell surface. They recognize a wide variety of extracellular signals and, through a series of precise steps including receptor conformational changes, G protein activation, and the initiation of downstream signaling cascades, convert extracellular signals into specific intracellular physiological responses. They are one of the most important drug targets in modern drug development. Prostaglandins (PGs) are potent lipid mediators derived from arachidonic acid, and their synthesis is rate-limited by cyclooxygenases (COX-1 and COX-2). Prostaglandin receptors (PGRs) are a class of GPCRs that specifically recognize prostaglandin mediators. Upon binding of a ligand to a PGR, a conformational change is induced, which in turn activates the coupled heterotrimeric G protein, initiating a complex downstream signal transduction network. PGRs are mainly classified into four classes based on ligand selectivity and signaling mechanisms: DP, EP, FP, and IP. EP receptors recognize and bind to prostaglandin PGE2, playing an important role in various physiological and pathological processes, such as inflammatory responses, pain perception, and reproductive function regulation. The EP receptor is further subdivided into four subtypes: EP1, EP2, EP3, and EP4. As a member of the GPCR family, EP4's ligand can activate the ERK1 / 2 signaling pathway to regulate a wide range of physiological and pathological functions. Once activated, ERK1 / 2 undergoes phosphorylation and then translocates to the nucleus to regulate gene transcription, ultimately achieving its broad regulation of cell-specific biological effects. Summary of the Invention

[0006] This invention provides a novel use for swertisin, namely its application in the preparation of drugs for treating insomnia.

[0007] The chemical structural formula of swertiamarin is as follows:

[0008] The purpose of this invention, using swertiamarin, is to prepare a drug for treating insomnia. Specifically, swertiamarin is used as the active ingredient, or in combination with other active ingredients, to exert its effect in the preparation of a drug for treating insomnia. In this application, one or more pharmaceutically acceptable excipients may be added, including conventional pharmaceutical fillers, diluents, binders, excipients, absorption enhancers, surfactants, stabilizers, etc., to improve absorption or facilitate administration, such as in the form of capsules, pills, powders, tablets, granules, oral liquids, etc.

[0009] The beneficial effects of this invention are: This invention, through Western blotting experiments, discovered that ERK phosphorylation can be dose-dependently activated by Swertisin, demonstrating that Swertisin has an agonistic effect on EP4. Furthermore, Swertisin can bind to the EP4 protein, thereby protecting EP4 from denaturation and precipitation under organic reagent treatment. By constructing a zebrafish insomnia model, the therapeutic effect of Swertisin on insomnia was studied. The results showed that Swertisin exhibited a concentration-dependent inhibitory effect on PTZ-induced insomnia-like activity; as the concentration increased, the zebrafish's movement patterns gradually became sparser, demonstrating a sedative-hypnotic potential similar to melatonin. Therefore, Swertisin can be used to prepare an agonist of the prostaglandin receptor EP4, thereby effectively treating insomnia. Attached Figure Description

[0010] Figure 1 A schematic diagram of the AutoDock Vina (docking) of Swertisin and EP4 molecules; Figure 2 This is a schematic diagram showing the phosphorylation level and expression level of ERK1 / 2 downstream of EP4 after treating Vector and EP4 overexpressing cells with Swertisin, PGE2, and CJ-42794 in Example 2. The upper figure is a Western Blot result, and the lower figure is a bar chart of WB grayscale scan values, where + represents the addition of the agonist PGE2 / EP4 receptor antagonist CJ-42794 / Swertisin. Figure 3 This is a schematic diagram showing the phosphorylation level and expression level of ERK1 / 2 downstream of EP4 after treating EP4-overexpressing cells with different concentrations of Swertisin in Example 2; the upper figure is the Western Blot result, and the lower figure is a bar chart of WB grayscale scan values. Figure 4 This is a schematic diagram showing the possible physical binding of EP4 and Swertisin using SIP in Example 3. The upper figure is the Western Blot band, and the lower figure is the thermostability curve of the protein to be tested. Figure 5 A diagram showing the behavioral movement trajectory of zebrafish; Figure 6 A bar chart quantifying the distance traveled by zebrafish (left) and their speed (right). Detailed Implementation

[0011] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. However, the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the methods in the embodiments are conventional methods or detection methods, and unless otherwise specified, the reagents used are conventional reagents or reagents prepared according to conventional methods. Example 1: Predicting the binding of swertiamarin to the EP4 receptor using the AutoDockvina molecular docking method 1. Preparation of ligand small molecules (1) Obtain the PubChem CID number of the target blood-entering component from the online database Drugbank, and download and save its molecular structure file in sdf format from the online database PubChem; (2) A small molecule library was built in the software MOE (Molecular Operating Environment), all small molecule structures were imported, and the small molecule library was "washed" and 3Dized to obtain the optimized ligand molecule (i.e. Swertisin). 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 non-protein components such as water molecules and organic solvents, and retain only the protein and the original ligand; 3. Molecular docking The optimized ligand from step 1 and the protein from step 2 were docked. During docking, the docking activity pocket was selected as LigandAtoms, and 30 conformations were generated and 3 docking results were output as references. The remaining parameters were kept at their default values. 4. Visual analysis of docking results The results were visualized using PyMOL software in a 3D model of the protein-ligand complex. The docking results are as follows: Figure 1 As shown; from Figure 1 It can be seen that Swertisin binds to the EP4 receptor.

[0012] Example 2: Verification of Swertisin's EP4 agonistic effect based on the ERK1 / 2 signaling pathway using Western blotting. 1. Cell Culture (1) Preparation: Irradiate the biosafety cabinet and necessary experimental supplies with ultraviolet light for 30 min to ensure a clean and sterile operating environment; turn on the 37℃ constant temperature water bath to preheat the culture medium, 1×PBS buffer and 0.25% trypsin to ensure that the reagent temperature is consistent with the in vivo environment and to provide a suitable growth environment for cells. (2) Washing: Discard the old culture medium in the culture dish, slowly add 1 mL of 1×PBS buffer along the wall of the culture dish, gently rinse the cell surface, then aspirate and discard, repeat 2 times; (3) Digestion: Add 0.4 mL of 0.25% trypsin to digest the cells. Shake gently until the cells flow down in a sand-like manner. Then add 0.8 mL of culture medium to stop the digestion. Use a pipette to gently blow the cells to completely detach them and obtain a cell suspension. (4) Centrifugation: Transfer the cell suspension 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 and DMEM medium containing 10% FBS was added to a total volume of 5 mL. The dish was then placed in a cell culture incubator at 37°C and 5% CO2 for cell culture until the cell density reached 80%. 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, wash cells with PBS, add 5 mL of preheated Opti-MEM medium to the cells, place in a 37°C, 5% CO2 cell culture incubator, and culture 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 (same as step (2) to (4) in Example 2) to obtain the treated cells. During the digestion process, the amount of trypsin added 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 cell well plate in a cross shape to mix. (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 -6 10 -7 10 -8 10 -9 10 - 10 EP4 receptor antagonist CJ-42794 (CJ) solution and concentration 10 mol / L -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 After starving cells for 12 hours with Swertisin solution at mol / L, the DMEM medium without FBS was replaced with 600 μL of CJ-42794 (CJ) solution or Swertisin solution, and incubated for 30 min. (2) After incubation, add EP4 receptor agonist PGE2 (10) -5 Activate with 1 mol / L for 5 min; then add an appropriate volume of strong RIPA lysis buffer containing the protease inhibitor Cocktail 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 4 °C and 15000 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: Figure 2 , 3 As shown; from Figure 2 , 3 As can be seen, the VSV tag was successfully expressed, indicating that the EP4 receptor was effectively overexpressed in the cells; the phosphorylation level of ERK could be significantly inhibited in a dose-dependent manner by the EP4 receptor-specific antagonist CJ-42794, proving that the model was successfully established; the phosphorylation of ERK could be activated by Swertisin in a dose-dependent manner, proving that Swertisin has an agonistic effect on EP4.

[0013] Example 3: Verifying the protective effect of Swertisin on EP4 using the SIP method via Western blotting. 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 30 min to obtain lysed cells; (3) Transfer the lysed cells to a 1.5 mL EP tube, centrifuge at 15000 rpm for 15 min, discard the precipitate, and keep the supernatant; (4) Transfer the supernatant to a clean 1.5 mL EP tube and add Swertisin; use sterile water as a blank control (NC). Dimethyl sulfoxide (DMSO) needs to be added to the sterile water to ensure that the concentration of DMSO in the Swertisin group and the NC group is consistent. Incubate in a shaker at 25°C for 3-5 h to obtain the incubated sample. (5) Mix 80 μL of the incubated sample with 16 μL of sterile water to prepare a 0% AEA mixed solution; mix 80 μL of the incubated sample with 4 μL of AEA organic solvent mixture (acetone: anhydrous ethanol: glacial acetic acid 50:50:0.1) and 12 μL of sterile water to prepare a 5% AEA mixed solution; mix 80 μL of the incubated sample with 8 μL of AEA organic solvent mixture and 8 μL of sterile water to prepare a 10% AEA mixed solution; mix 80 μL of the incubated sample with 12 μL of AEA organic solvent mixture and 4 μL of sterile water to prepare a 15% AEA mixed solution; mix 80 μL of the incubated sample with 16 μL of AEA organic solvent mixture to prepare a 20% AEA mixed solution. (6) Equilibrate the mixture at 25℃ and 60rpm for 20min, then centrifuge at 4℃ and 15000rpm for 20min and collect the supernatant. (7) Add 1 / 4 volume of 5×SDS Loading Buffer to the supernatant and vortex to mix to obtain the protein sample; (8) Western blotting was used to detect the protein samples, and the results are as follows: Figure 4 As shown; from Figure 4 As can be seen, Swertisin can bind to the EP4 protein, thereby protecting EP4 from denaturation and precipitation under organic reagent treatment, providing direct molecular binding evidence for Swertisin as an EP4 receptor agonist.

[0014] Example 4: Constructing a zebrafish insomnia model to study the therapeutic effect of Swertisin on insomnia. 1. Preparation of 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 using an analytical balance, and dilute to 2L with UP water to prepare a 60× stock solution. Dilute 60 times before use and adjust the pH to 7.2.

[0015] (2) Preparation of pentylenetetrazol stock solution 1,5-Pentamethylenetetrazole (PTZ), CAS No. 54-95-5, molecular formula C6H 10 N4, with a molecular weight of 138.17, is a well-proven chemical inducer that can mimic epileptic seizures. Weigh 276.34 mg of PTZ and dissolve it in 50 mL of E3 medium to prepare a 40 mM PTZ solution. Store at -20°C. Dilute with E3 medium to different concentrations before use.

[0016] Melatonin stock solution preparation Melatonin (N-Acetyl-5-methoxytryptamine, Melatonin), CAS No. 73-31-4, molecular formula C 13 H 16 N2O2, with a molecular weight of 232.28, is a first-line treatment for insomnia and can inhibit PTZ-induced insomnia-like activity in zebrafish. Therefore, melatonin was selected as a positive control in this experiment. 11.614 mg of melatonin was weighed and prepared into a 100 mM solution with 500 µL DMSO. This solution was then diluted to 400 µM to prepare the melatonin stock solution, which was stored at 4°C until use. It was diluted with E-culture medium before use.

[0017] Swertisin formulation Swertiacin, molecular formula C 22 H 22010 With a molecular weight of 446.4, it is an active ingredient in jujube seed. Weigh 1 mg of swertiamarin and prepare a 100 mM stock solution with 22.4 µL DMSO. Store at -20℃ until use. Dilute with E3 culture medium before use.

[0018] 2. Establishment of Zebrafish PTZ Model To investigate the PTZ-induced insomnia model in zebrafish, we immersed zebrafish completely in different concentrations of PTZ and recorded the total distance (in millimeters) of their swimming over 50 minutes to analyze their kinematic patterns. Six groups were established. Five-day-old post-fertilization zebrafish larvae (5-dpf) were placed in 48-well plates, one fish per well, with nine fish in each experimental group. The control group (Blank, E3 water only) and the PTZ-induced group (PTZ-2.5mM) each received 400µL of solution per well. Immediately after adding the solution, the larvae were placed in a zebrafish behavior tracker for acclimatization for 10 minutes. We then began recording the distance the larvae swam at a speed greater than 20 mm / s within one hour.

[0019] 3. Determination of Swertisin Concentration and Activity Testing To test the optimal concentration of swertiamarin for promoting sleep in zebrafish, zebrafish were pre-treated with swertiamarin solutions of varying concentrations. After 4 hours, PTZ was added, and the total distance swam by the zebrafish over 30 minutes was recorded for analysis. This experiment consisted of 7 groups. Zebrafish juveniles (5-dpf) were placed in 48-well plates, one fish per well, with 3 fish per group: a blank control group (Blank, E3 water only), a pentylenetetrazol group (PTZ-2.5mM), and swertiamarin gradient concentration groups (100, 200, 400, 600µM). For the swertiamarin gradient concentration groups, 300µL of the corresponding concentration of the drug solution was added for 4 hours, followed by 100µL of PTZ (except for the blank group) to bring the final concentration to 2mM, 400µL per well. The fish were then immediately placed in a zebrafish behavior tracker for acclimatization for 10 minutes, and the movement speed and distance swam at speeds greater than 20mm / s were recorded. Measurement results as follows Figures 5-6 As shown.

[0020] from Figure 5 The zebrafish movement trajectory diagram shows that PTZ (2mM) can successfully induce insomnia-like hyperactivity in zebrafish, while the positive control drug melatonin (400μM) can effectively inhibit this behavior. Swertisin showed a concentration-dependent inhibitory effect on PTZ-induced insomnia-like activity. As the concentration increased, the zebrafish movement trajectory gradually became sparse, showing a sedative-hypnotic potential similar to melatonin.

[0021] from Figure 6 As can be seen, compared with the control group, PTZ (2.5 mM) significantly increased the movement distance and average speed of zebrafish, successfully inducing insomnia-like activity; the positive control melatonin (400 μM) significantly inhibited this effect. Swertisin showed a concentration-dependent inhibitory effect on PTZ-induced hyperactivity; with increasing concentration, the movement distance and speed gradient of zebrafish decreased, showing a sedative-hypnotic potential similar to melatonin.

[0022] As can be seen from the above embodiments, swertisin has a sedative and hypnotic effect. Swertisin has an agonistic effect on EP4 and can be prepared as an agonist of the prostaglandin receptor EP4, which can effectively relieve insomnia.

Claims

1. Application of swertiamarin in the preparation of drugs for treating insomnia.

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