Application of oroxylin A in preparation of preparation for preventing or treating epilepsy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of oridonin in preparation of preparations for preventing or treating epilepsy. The application studies the anti-epilepsy effect of oridonin by giving zebra fish oridonin treatment, and the research content includes movement distance, behavior characterization and corresponding neurotoxicity detection. The application shows that oridonin is suitable for controlling the occurrence and development of epilepsy, and the zebra fish rapid movement distance is significantly reduced. In the application, it is detected that oridonin does not cause obvious nerve damage to zebra fish at a therapeutic dose, and has no neurotoxicity; specifically, the fluorescence intensity of central nerve apoptosis cells and the fluorescence intensity of brain nerve apoptosis cells of zebra fish are not obviously affected.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of hyoscyamine in the preparation of agents for the prevention or treatment of epilepsy. Background Technology

[0002] Epilepsy is a chronic neurological disorder caused by abnormal electrical activity in the brain, characterized by recurrent seizures. Currently, medication remains the primary treatment for epilepsy, but due to its complex pathogenesis, existing antiepileptic drugs can only control seizures in a portion of patients and do not alter long-term prognosis; approximately one-third of patients develop drug-resistant epilepsy. Therefore, current antiepileptic drugs are insufficient to meet the treatment needs of epilepsy patients. The development of new antiepileptic drugs is slow, and few new targeted drugs have emerged. Most commonly used clinical antiepileptic drugs primarily inhibit neuronal excitability, providing symptomatic treatment. However, there is a need for targeted improvements in neuronal protection and recovery after seizures. This is a significant reason why epilepsy patients experience memory, cognitive, and psychological impairments after seizures, and the inability to promptly recover from damage can also lead to recurrent seizures and various complications. Therefore, there is an urgent need to explore and discover new antiepileptic strategies that can effectively control seizures and provide neuronal protection and recovery after seizures. Summary of the Invention

[0003] To address the issue that existing antiepileptic drugs have a low protective and restorative effect on patients' nerve cells during treatment, this invention provides the application of diosgenin in the preparation of agents for the prevention or treatment of epilepsy.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides the application of thymol in the preparation of agents for the prevention or treatment of epilepsy.

[0006] Preferably, the epilepsy prevention or treatment preparation uses styrax ethylsin as the active ingredient, supplemented with antiepileptic drugs and pharmaceutically acceptable excipients.

[0007] Preferably, the antiepileptic drug comprises at least one of a sodium channel blocker, an enhancer that works by inhibiting γ-aminobutyric acid transaminase, and a calcium channel modulator;

[0008] The enhancers that work by inhibiting γ-aminobutyric acid transaminase include at least one of sodium valproate, gabapentin, and pregabalin.

[0009] Preferably, the sodium channel blocker includes at least one of phenytoin sodium, carbamazepine, and oxcarbazepine.

[0010] Preferably, the calcium channel modulator includes at least one of ethosuximide, vigabatrin, and topiramate.

[0011] Preferably, the pharmaceutically acceptable excipient is one or more of the following: diluent, disintegrant, precipitation inhibitor, flow aid, binder, dispersant, suspending agent, isotonic agent, thickener, emulsifier, preservative, antioxidant, and stabilizer.

[0012] Preferably, the diluent includes any one of starch, lactose, sucrose, and mannitol.

[0013] Preferably, the disintegrant comprises any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0014] Preferably, the precipitation inhibitor comprises any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

[0015] Preferably, the flow aid comprises any one of cationic polyacrylamide, polydiallyldimethylammonium chloride, and cationic starch.

[0016] Preferably, the adhesive comprises any one of starch paste, hydroxypropyl methylcellulose, and povidone.

[0017] Preferably, the dispersant comprises any one of sodium dodecyl sulfate, polyvinylpyrrolidone, and sodium carboxymethyl cellulose.

[0018] Preferably, the suspending agent includes any one of gum arabic, tragacanth gum, sodium carboxymethyl cellulose, and hydroxypropyl methylcellulose.

[0019] Preferably, the isotonic agent includes any one of sodium chloride, glucose, and mannitol.

[0020] Preferably, the thickener includes any one of gum arabic, xanthan gum, and sodium carboxymethyl cellulose.

[0021] Preferably, the emulsifier comprises any one of sodium dodecyl sulfate, benzalkonium chloride, and fatty acid sorbitan.

[0022] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methylparaben, and benzalkonium bromide.

[0023] Preferably, the stabilizer comprises any one of sodium sulfite, sodium bisulfite, tocopherol, and disodium EDTA.

[0024] Preferably, the antioxidant is tocopherol.

[0025] Preferably, the diluent is dimethyl sulfoxide.

[0026] Preferably, the acceptable dosage form of the epilepsy prevention or treatment preparation includes one of the following: tablets, capsules, granules, injections, pills, powders, ointments, and oral liquids.

[0027] Preferably, the acetaminophen exerts its anti-epileptic effect by upregulating gabral gene expression.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention provides the application of thiamethoxam in the preparation of agents for the prevention or treatment of epilepsy. This invention studies the anti-epileptic effect of thiamethoxam by treating zebrafish with thiamethoxam, including studies on movement distance, behavioral characteristics, and corresponding neurotoxicity detection. The results of this invention show that thiamethoxam is suitable for controlling the occurrence and development of epilepsy, manifested by a significant reduction in the rapid movement distance of zebrafish. This invention also detected that thiamethoxam at therapeutic doses did not cause significant neurological damage in zebrafish, exhibiting no neurotoxicity; specifically, it had no significant effect on the fluorescence intensity of apoptotic cells in the central nervous system and the brain of zebrafish. Attached Figure Description

[0030] Figure 1 This represents the distance a zebrafish can travel at high speed.

[0031] Figure 2 The fluorescence intensity of apoptotic cells in the central nervous system of zebrafish is shown.

[0032] Figure 3 The fluorescence intensity of apoptotic cells in the zebrafish brain.

[0033] Figure 4 Typical zebrafish behavior diagrams: A: Normal control group; B: Model control group; C: Carbamazepine; D: 25 ng / fish of ethanoic acid; E: 50 ng / fish of ethanoic acid; F: 100 ng / fish of ethanoic acid.

[0034] Figure 5 Typical fluorescence intensities of apoptotic cells in the central nervous system and brain of zebrafish.

[0035] Figure 6 To compare the relative expression levels of the gabra1 gene with the model control group, **p<0.01, ***p<0.001. Detailed Implementation

[0036] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0037] The inventive concept of this invention is as follows:

[0038] There are three main classes of existing antiepileptic drugs: sodium channel blockers, GABA enhancers, and calcium channel modulators. Sodium channel blockers include phenytoin sodium, carbamazepine, and oxcarbazepine; GABA enhancers include sodium valproate, gabapentin, and pregabalin; and calcium channel modulators include ethosuximide, vigabatrin, and topiramate. Currently, existing antiepileptic drugs can only control seizures in some patients and do not change the long-term prognosis; approximately one-third of patients develop drug-resistant epilepsy. Therefore, existing antiepileptic drugs are no longer sufficient to meet the treatment needs of epilepsy patients.

[0039] Based on this, the present invention provides the application of thymol in the preparation of agents for the prevention or treatment of epilepsy.

[0040] Febrifugine, also known as Changshan alkaloid, is a quinazoline alkaloid found in the Changshan plant. It is a crystalline solid with a melting point of 139℃~140℃ and is soluble in a variety of organic solvents, such as DMF, DMSO, and ethanol.

[0041] In traditional Chinese medicine, Changshan (Dichroa febrifuga) is associated with the concept of "phlegm." In TCM, Changshan is believed to have the effects of inducing vomiting of phlegm and treating malaria. Specifically, Changshan is bitter, pungent, and cold in nature, slightly toxic, and enters the lung, heart, and liver meridians. It has a strong emetic effect, promoting the expulsion of phlegm from the chest, effectively clearing phlegm stagnation. Its upward-moving nature allows it to induce vomiting of phlegm and fluid retention in the chest, making it suitable for phlegm accumulation and chest congestion. Through vomiting, it can remove old phlegm and fluid buildup from the body, thereby achieving a therapeutic effect.

[0042] In Traditional Chinese Medicine (TCM), phlegm and fluid retention are considered a significant contributing factor to epilepsy. TCM believes that epilepsy is related to "phlegm obstructing the heart orifices," meaning that phlegm and fluid retention in the heart orifices, including the heart and brain, leads to mental confusion and triggers seizures. The accumulation of phlegm and fluid can also obstruct the meridians, affecting the flow of qi and blood, and consequently impacting brain function, leading to seizures. Chest congestion can further impede qi circulation, exacerbating the accumulation of phlegm and fluid, thus affecting the mind and triggering epilepsy.

[0043] Therefore, in the treatment of epilepsy using Traditional Chinese Medicine (TCM), herbs that can eliminate phlegm, resolve blood stasis, and induce vomiting are often used, such as Changshan (Dichroa febrifuga), to eliminate phlegm accumulation and chest congestion, thereby achieving the goal of treating epilepsy. For example, herbs with specific effects are used, or formulas that regulate qi, resolve phlegm, and promote blood circulation are employed. This also provides the TCM theoretical basis for this invention.

[0044] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0045] The list of abbreviations for this invention is shown in Table 1.

[0046] Table 1 List of Abbreviations

[0047] Abbreviation Full name PTZ Pentylenetetrazole GABA γ-aminobutyric acid <![CDATA[GABA A ]]> Gamma-aminobutyric acid type A receptor DMSO Dimethyl sulfoxide

[0048] The acetylcholine used in this invention was purchased from a reagent company, brand: Maclean, purity: greater than 98%.

[0049] The technical means used in this invention is to induce a zebrafish epilepsy model using pentylenetetrazole (PTZ). Epilepsy is a chronic disease caused by sudden abnormal discharges of neurons in the brain, leading to transient brain dysfunction. Its main manifestations include convulsions, loss of consciousness for several seconds, myoclonus, loss of muscle tone, and prolonged muscle contractions. The zebrafish brain contains brain structures similar in function to mammals, such as the cerebral cortex and hippocampus, as well as the same neurotransmitters such as γ-aminobutyric acid (GABA), acetylcholine, and glutamate. PTZ is a central nervous system stimulant that can induce neuronal apoptosis in the hippocampus and inhibit GABA production. A Activity on receptors causes clonic convulsions in the body, presenting as an epileptic seizure. During an epileptic seizure, the distance of rapid movement in zebrafish is positively correlated with the severity of the epilepsy.

[0050] The antiepileptic activity experiment of this invention is as follows: Thirty wild-type AB strain zebrafish (6 dpf) were randomly selected and placed in 6-well plates. Experimental groups and dosages are shown in Table 2. The experimental groups received intravenous injections of different doses of thiamethoxam; the positive control group received carbamazepine (200 ng / fish); a normal control group was also set up, receiving only DMSO as the solvent; and a model control group was also included. Each well contained 3 mL. After treatment at 28℃ for 1 h, the zebrafish were transferred to 96-well plates, 200 μL / fish, 1 fish / well. Except for the normal control group, the other groups received PTZ in aqueous solution to establish a zebrafish epilepsy model. The rapid movement distance of the zebrafish over 1 h was detected using a behavioral analyzer, and the antiepileptic efficacy of the samples was evaluated based on the statistical analysis results of this index. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0051] Neurotoxicity assay: Wild-type AB strain zebrafish (1 dpf) were randomly selected and placed in 6-well plates. Different doses of thiamethoxam were injected into each well (see Table 3). A normal control group and a solvent control group were also included, with 3 mL per well. The normal control group was treated with DMSO. After treatment at 28℃ for one day, zebrafish in each group were stained with AO in the dark for 30 min, washed three times with standard dilution water, and then 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The fluorescence intensity of apoptotic cells in the central nervous system and brain of the zebrafish was analyzed, and the neurotoxicity of the samples was evaluated based on the statistical analysis results of the above indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0052] Table 2. Results of the antiepileptic efficacy evaluation experiment, sample size n=10

[0053]

[0054] Table 3. Results of neurotoxicity evaluation experiments on samples, sample size n=10

[0055]

[0056] In Tables 2 and 3, "-" indicates that this item is not present; "***" indicates that P < 0.001.

[0057] Example 1

[0058] The application of diosgenin in the preparation of agents for the prevention or treatment of epilepsy is as follows:

[0059] 1. Evaluation of the efficacy and neurotoxicity of diosgenin.

[0060] The efficacy evaluation results of chebulic ethylsin showed that it had antiepileptic efficacy at a dose of 100 ng / tail, and its effect was slightly better than that of the positive control drug carbamazepine. (See results below.) Figure 1 At a dose of 100 ng / tail, dimethicone had no significant effect on the fluorescence intensity of apoptotic cells in the central nervous system and the brain of zebrafish. Therefore, this indicates that it does not exhibit neurotoxicity at this dose. Results are shown below. Figure 2 and Figure 3 .

[0061] According to Table 2, Figure 1 and Figure 4The effects of different interventions on the hyperkinetic phenotype were assessed by rapid movement distance. Results showed that the rapid movement distance in the model control group (951±90.4 mm) was significantly higher than that in the normal control group (92.2±11.8 mm), indicating that the epilepsy model successfully induced the hyperkinetic phenotype (P<0.001). The rapid movement distance in the positive control group (219±37.8 mm) was 77% lower than that in the model control group, confirming its anti-hyperkinetic effect was consistent with clinical efficacy. Notably, the diosgenin intervention group exhibited a significant dose-dependent effect: the rapid movement distance of 857±111 mm in the 25 ng / tail dose group was close to that of the model control group, suggesting that low doses had no significant intervention effect; the rapid movement distance of 522±66 mm in the 50 ng / tail dose group was 45% lower than that in the model control group, partially inhibiting hyperkinesis; while the inhibitory effect of the 100 ng / tail dose group was 78.6%, comparable to carbamazepine (P<0.001), and the intragroup variability was smaller (SE: 28.8 vs 37.8), suggesting that high-dose diosgenin has better stability and efficacy. Furthermore, its mechanism of action may be related to the regulation of excitatory pathways in the central nervous system.

[0062] Table 2 Figure 2 , Figure 3 and Figure 5 In the normal control group, the fluorescence intensity of apoptosis in the central nervous system and brain nerves was 58831±2149 pixels and 56931±3429 pixels, respectively, indicating that the basal level of apoptosis was in a stable state. The fluorescence intensity of the solvent control group was not significantly different from that of the normal control group, with the central nervous system at 57423±1907 pixels and the brain nerves at 57691±1979 pixels, suggesting that the solvent had no effect on cell apoptosis.

[0063] Dose-response analysis of the diosgenin intervention group showed that the apoptosis fluorescence intensity in the central nervous system of the 100 ng / tail group was 61527 ± 1214 pixels, and in the brain nerves it was 60318 ± 1515 pixels. There was no statistically significant difference compared with the normal control group and the solvent control group (P > 0.05). Dose-dependent analysis indicated that diosgenin had a relatively small regulatory effect on apoptosis fluorescence intensity within the range of 25 ng / tail to 50 ng / tail, with a fluctuation range of ±5%. While the fluorescence intensity of the 100 ng / tail group was slightly higher than other groups, the values ​​were still within the normal physiological fluctuation range. For the central nervous system, the diosgenin concentration at 100 ng / tail was 61527 pixels, compared to 58831 pixels in the normal control group; for the brain nerves, the diosgenin concentration at 100 ng / tail was 60318 pixels, compared to 56931 pixels in the normal control group, with standard errors of ±1214 and ±1515, respectively, showing no significant deviation from baseline.

[0064] Therefore, at a dose of 100 ng / tail, thiamethoxam had no significant effect on the fluorescence intensity of apoptotic cells in the central nervous system and brain nerves of zebrafish, indicating that it does not have neurotoxicity at this dose.

[0065] 2. Expression level of the gabra1 gene.

[0066] The expression of β-actin and gabra1 genes in zebrafish treated with different doses of thiamethoxam was detected by q-PCR in normal control group, model control group, positive control group, and zebrafish treated with different doses of thiamethoxam. β-actin was used as an internal reference for gene expression, and the relative RNA expression level of the gabra1 gene was calculated. The group treated with thiamethoxam was designated as DZ-CS-1.

[0067] The gabra1 gene encodes the GABA receptor. GABA primarily functions as an inhibitory neurotransmitter in adults, playing a crucial role by inhibiting or suppressing excitatory signal transduction. Zebrafish brains possess brain structures similar in function to mammals, such as the cerebral cortex and hippocampus, and share the same neurotransmitters such as GABA, acetylcholine, and glutamate. PTZ is a central nervous system stimulant that induces neuronal apoptosis in the hippocampus and works by inhibiting GABAergic signal transduction. A Activity on the receptors causes clonic convulsions in the body, resembling an epileptic seizure. (See Table 4 of this invention.) Figure 6 The data leads to the conclusion that diosgenin has anti-epileptic effects, specifically by upregulating the expression of the gabra1 gene.

[0068] Table 4. Genetic experimental results evaluating the antiepileptic efficacy of thymol.

[0069]

[0070] In Table 4, compared with the model control group, **p<0.01, ***p<0.001. “-” indicates that this item was not present.

[0071] The RNA extraction results and primer sequence information for the q-PCR experiment are as follows:

[0072] At the experimental endpoint, total RNA was extracted from zebrafish, and the RNA concentration and A260 / A280 ratio were determined using a UV-Vis spectrophotometer (see Table 5). The A260 / A280 ratios were all between 1.8 and 2.2, indicating that the extracted total RNA from zebrafish was of good quality and suitable for subsequent q-PCR experiments. Primer sequences are shown in Table 6.

[0073] Table 5. Total RNA concentration and A260 / A280 ratio, n=3

[0074]

[0075] In Table 5, "-" indicates that this item is not present.

[0076] Table 6 Primer sequence information

[0077]

[0078] The present invention also provides a specific formulation of the aforementioned anti-epileptic or anti-epilepsy agent, as shown in Table 7.

[0079] Table 7 Formulations of the epilepsy prevention or treatment agents of the present invention

[0080]

[0081]

[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of hyoscyamine in the preparation of agents for the prevention or treatment of epilepsy.

2. The application as described in claim 1, characterized in that, The proposed epilepsy prevention or treatment preparation uses styrax ethylsin as the active ingredient, supplemented with antiepileptic drugs and pharmaceutically acceptable excipients.

3. The application as described in claim 2, characterized in that, The antiepileptic drug includes at least one of sodium channel blockers, enhancers that work by inhibiting γ-aminobutyric acid transaminase, and calcium channel modulators; The enhancers that work by inhibiting γ-aminobutyric acid transaminase include at least one of sodium valproate, gabapentin, and pregabalin.

4. The application as described in claim 3, characterized in that, The sodium channel blocker includes at least one of phenytoin sodium, carbamazepine, and oxcarbazepine.

5. The application as described in claim 3, characterized in that, The calcium channel modulator includes at least one of ethosuximide, vigabatrin, and topiramate.

6. The application as described in claim 2, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, antioxidants, solvents, and stabilizers. The precipitation inhibitor is any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone, and hydroxypropyl methylcellulose; The solvent is dimethyl sulfoxide.

7. The application as described in claim 1, characterized in that, The acceptable dosage forms of the proposed epilepsy prevention or treatment preparations include one of the following: tablets, capsules, granules, injections, pills, powders, ointments, and oral liquids.

8. The application as described in claim 1, characterized in that, The thiamethoxam is upregulated gabral Gene expression plays an anti-epileptic role.

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