Application of peganum harmala dichloromethane extract as antiepileptic drug
By extracting dichloromethane substances from camel boars as anti-epileptic drugs, the drug resistance and side effects of existing drugs were solved, the behavioral and cognitive functions of epilepsy rats were significantly improved, and the damage to hippocampal neurons was reduced, and the development of new anti-epileptic drugs was achieved.
Patent Information
- Application Number
- CN202510683271.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-07-15
AI Technical Summary
Existing anti-epileptic drugs such as carbamazepine and sodium valproate have strong resistance and great side effects. It is urgent to develop new anti-epileptic drugs. Moreover, the substance basis and mechanism of anti-epileptic drug efficacy of larchopone are not clear, which limits its in-depth development and clinical application.
The alkaloids were extracted from the herbicides by specific methods and prepared into clinically suitable drug dosage forms, such as tablets, oral liquids, pills, capsules, granules, injections and slow-release agents, for use in the Li-Pilo-induced epilepsy model for behavioral experiments to evaluate its neuroprotective effect.
Camel-pop dichloromethane extract significantly improved the anxiety, autonomous motor ability, cognitive memory ability of epilepsy rats, and reduced hippocampal neuron damage, which had significant neuroprotective effects.
Smart Images
Figure CN120305298A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of traditional Chinese medicine drug applications, and specifically relates to the application of a dichloromethane extract of Peganum harmala as an anti-epileptic drug. Background Art
[0002] Epilepsy is a chronic neurological disease caused by abnormal discharges of brain neurons. Its pathogenesis is complex and involves multiple factors such as neurotransmitter imbalance, ion channel dysfunction, oxidative stress, and neuroinflammation. At present, although clinical commonly used anti-epileptic drugs such as carbamazepine and sodium valproate have certain curative effects, they have problems such as strong drug resistance and large side effects, and there is an urgent need to develop new anti-epileptic drugs.
[0003] Traditional ethnic drugs have unique advantages in the treatment of neurological diseases. Peganum harmala L. has the effects of dispelling wind and relieving pain, relieving cough and asthma, and opening orifices and strengthening the brain. "Chinese Ethnic Medicine" further points out that Peganum harmala can be used to treat mental depression, paralysis, amnesia, and epilepsy, suggesting its potential neuroprotective effect. Modern research shows that Peganum harmala is rich in active ingredients such as alkaloids, flavonoids, and triterpenes. Its extract can play an anti-Alzheimer's disease role by inhibiting acetylcholinesterase, but the pharmacodynamic material basis and mechanism of its anti-epileptic effect have not been clarified, which severely restricts the further development and clinical application of this drug. Summary of the Invention
[0004] In view of this, the present invention provides the application of a dichloromethane extract of Peganum harmala as an anti-epileptic drug to solve the above technical problems.
[0005] To achieve the above object, the present application adopts the following scheme:
[0006] The application of a dichloromethane extract of Peganum harmala as an anti-epileptic drug.
[0007] Preferably, the preparation method of the dichloromethane extract of Peganum harmala includes the following steps:
[0008] S10. Extraction step: Select Peganum harmala medicinal materials, place the Peganum harmala medicinal materials in an ethanol aqueous solution with a mass concentration of 95%, carry out soaking extraction at room temperature, the soaking time is 5 - 7 days, and the material-liquid ratio of the Peganum harmala medicinal materials to the ethanol aqueous solution is 1:20 (g / mL); repeat the extraction operation three times according to this soaking extraction method. After each extraction is completed, separate the extract from the medicinal material residue, collect the extracts obtained from the three extractions, and remove the solvent ethanol by conventional methods such as vacuum concentration to prepare a dry extract of the ethanol extract of Peganum harmala medicinal materials;
[0009] S20. Extraction step: Place the dry extract of ethanol extract in an appropriate amount of water, stir well to dissolve it to obtain an aqueous solution of the dry extract; slowly add 2% sulfuric acid solution to the obtained aqueous solution of the dry extract, adjust the pH to 2 - 3 to obtain an acidified aqueous solution, so that the alkaloids existing in the form of organic salts in Peganum harmala L. are converted into small molecule inorganic salts, increasing their solubility in water; add ethyl acetate to the acidified aqueous solution for extraction, fully shake and mix, then let it stand for stratification, separate the ethyl acetate layer, repeat the above extraction operation at least once to remove impurities such as lipids, and obtain an acid aqueous solution. The volume ratio of the acidified aqueous solution to ethyl acetate is 1:1. Slowly add 1% sodium hydroxide solution to the acid aqueous solution, adjust the pH to 10 - 11 to free the alkaloids in the aqueous solution, and obtain an alkalized aqueous solution. Add dichloromethane to the alkalized aqueous solution for extraction according to a volume ratio of 1:1, fully shake and mix, then let it stand for stratification, separate the dichloromethane layer, repeat the above extraction operation with dichloromethane three times to enrich the alkaloids, collect the extraction solutions obtained from the three dichloromethane extractions, and remove the solvent dichloromethane by conventional methods such as vacuum concentration to obtain the dichloromethane extract of Peganum harmala L.
[0010] An antiepileptic drug, comprising the above-mentioned dichloromethane extract of Peganum harmala L.
[0011] Preferably, the dosage of the dichloromethane extract of Peganum harmala L. is 10 mg / kg - 40 mg / kg.
[0012] Preferably, the dosage of the dichloromethane extract of Peganum harmala L. is 20 mg / kg.
[0013] Preferably, it includes being prepared into a drug dosage form suitable for clinical use, including any one of tablets, oral liquids, pills, granules, capsules, injections and sustained-release or controlled-release preparations.
[0014] In the application of the above-mentioned dichloromethane extract of Peganum harmala L. as an anti-epileptic drug, a Li-Pilo-induced epilepsy model was used, and behavioral experiments were conducted on the induced epileptic rats through open field experiments, novel object recognition experiments, and Morris water maze experiments. After the dichloromethane extract of Peganum harmala L. was administered in the open field experiment, the anxiety of epileptic rats was alleviated and the spontaneous motor ability of the rats was enhanced; after the dichloromethane extract of Peganum harmala L. was administered in the novel object recognition experiment, the novel object recognition index of epileptic rats increased significantly, thus significantly improving the cognitive memory ability of epileptic rats; after the dichloromethane of Peganum harmala L. was administered in the Morris water maze experiment, the results showed that the dichloromethane extract of Peganum harmala L. could significantly shorten the escape latency of rats, thus significantly improving the spatial memory ability of epileptic rats, indicating that the dichloromethane extract of Peganum harmala L. has a neuroprotective effect on Li-Pilo-induced epileptic rats. The results of H&E staining and Nissl staining showed that the dichloromethane extract of Peganum harmala L. significantly reduced hippocampal neuron damage and neuronal necrosis, making the neuronal arrangement relatively regular and increasing the number of intracellular Nissl bodies, further indicating that the dichloromethane extract of Peganum harmala L. can be used in the preparation of anti-epileptic drugs. Description of the Drawings
[0015] Figure 1 It is a statistical chart of the effect of the dichloromethane extract of Peganum harmala L. on the open field behavior of rats.
[0016] Figure 2 It is a statistical chart of the effect of the dichloromethane extract of Peganum harmala L. on the novel object recognition of rats.
[0017] Figure 3 It is a statistical chart of the effect of the dichloromethane extract of Peganum harmala L. on the water maze behavior of rats.
[0018] Figure 4 It is an H&E micrograph of the effect of the dichloromethane extract of Peganum harmala L. on the neuronal damage in the CA3 region of the hippocampus of epileptic rats.
[0019] Figure 5 It is a Nissl micrograph of the effect of the dichloromethane extract of Peganum harmala L. on the neuronal damage in the CA3 region of the hippocampus of epileptic rats.
[0020] Figure 1 In , A is a schematic diagram of the movement trajectory of rats, B is the activity distance in the central area (within the blue area), C is the total movement distance of rats within 5 min, and D is the average speed of rats' movement; Figure 2 In , A is a schematic diagram of the movement trajectory of rats, and B is the novel object recognition index; Figure 3 In , A is the time for rats to reach the platform, B is the number of times rats cross the platform area, C is the percentage of time rats stay in the quadrant where the platform is located, and D is a schematic diagram of the movement trajectory of rats; Figure 4 and Figure 5A, B, C, D, E, and F in it respectively represent the normal group, the model group, the sodium valproate group, the low-dose harmel group, the medium-dose harmel group, and the high-dose harmel group. Detailed implementation manners
[0021] To make the purpose and technical solutions of the present invention clearer, the content of the present invention will be further described in detail through experiments. However, it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following experiments only. Any corresponding substitution or modification made according to the common general knowledge and customary means in the art without departing from the above-mentioned technical solutions of the present invention is included in the present invention.
[0022] Epilepsy is a type of chronic recurrent neurological disorder, and its core pathological feature is the sudden abnormal synchronous discharge of the cortical network. The clinical impact of this disease goes beyond the seizures themselves, presenting multi-dimensional pathophysiological effects, especially the progressive cognitive decline as a significant neuropsychological sequela. Temporal lobe epilepsy, as the most common type of epilepsy, usually causes memory defects and severe neuronal damage. The Li-Pilo-induced epilepsy model is one of the "gold standard" models for studying temporal lobe epilepsy. By simulating the pathological process of human temporal lobe epilepsy, it is widely used in mechanism exploration and drug research and development.
[0023] The following uses the Li-Pilo-induced epilepsy model and, through behavioral experiments, including the Morris water maze, novel object recognition, and open field experiments, evaluates the improvement effect of harmel dichloromethane on epilepsy-induced anxiety and depression-like behaviors and cognitive memory impairment. The neuroprotective effect of harmel dichloromethane on hippocampal CA3 region nerve damage induced by Li-Pilo-induced epilepsy is evaluated by H&E staining and Nissl staining. It should be noted that the inventors have carried out relevant pharmacodynamic experimental studies to prove the efficacy of the harmel dichloromethane extract of the present invention as an anti-epileptic drug. It should be noted that the following experimental studies are all carried out on the basis of proving the drug safety, and the dosing doses in the experimental studies are all within the safe dose range. The drugs selected in the following pharmacodynamic experiments are the drugs obtained by the preparation method in the present invention; the inventors have also carried out pharmacodynamic experiments on other drugs, and the experimental results show the same or similar effects. However, due to space limitations, they are not listed one by one here.
[0024] 1. Establishment of the Li-Pilo-induced epilepsy rat model
[0025] 1.1 Experimental animals
[0026] Thirty-six SPF-grade male rats, weighing 180-220 g, were provided by the Experimental Animal Center of Ningxia Medical University. They were freely given water and fed with a fixed amount of rat feed in an animal room with ventilation, dryness, a room temperature of 22-25 °C, and a humidity of 50%-70%.
[0027] 1.2. Experimental drugs
[0028] Preparation of Peganum harmala dichloromethane extract: Select Peganum harmala medicinal materials, place the Peganum harmala medicinal materials in an ethanol aqueous solution with a mass concentration of 95%, and carry out immersion extraction at room temperature for 5 - 7 days. The material - liquid ratio of the Peganum harmala medicinal materials to the ethanol aqueous solution is 1:20 (g / mL); repeat the extraction operation three times according to this immersion extraction method. After each extraction, separate the extract from the medicinal residue, collect the extracts obtained from the three extractions, and remove the solvent ethanol by conventional methods such as vacuum concentration to prepare a dry extract of the ethanol extract of Peganum harmala medicinal materials; place the dry extract of the ethanol extract in an appropriate amount of water, stir well to dissolve it to obtain an aqueous solution of the dry extract; slowly add a 2% sulfuric acid solution to the obtained aqueous solution of the dry extract, adjust the pH to 2 - 3 to obtain an acidified aqueous solution, so that the alkaloids existing in the form of organic salts in Peganum harmala are converted into small - molecule inorganic salts, increasing their solubility in water; add ethyl acetate to the acidified aqueous solution for extraction, fully oscillate and mix, then let it stand for layering, separate the ethyl acetate layer, and repeat the above extraction operation at least once to remove impurities such as lipids to obtain an acid aqueous solution. The volume ratio of the acidified aqueous solution to ethyl acetate is 1:1. Slowly add a 1% sodium hydroxide solution to the acid aqueous solution, adjust the pH to 10 - 11 to free the alkaloids in the aqueous solution to obtain an alkalized aqueous solution. Add dichloromethane to the alkalized aqueous solution according to a volume ratio of 1:1 for extraction, fully oscillate and mix, then let it stand for layering, separate the dichloromethane layer, and repeat the above extraction operation with dichloromethane three times to enrich the alkaloids. Collect the extracts obtained from the three dichloromethane extractions, and remove the solvent dichloromethane by conventional methods such as vacuum concentration to obtain the Peganum harmala dichloromethane extract.
[0029] Peganum harmala dichloromethane extract high - dose (40 mg / kg), medium - dose (20 mg / kg), low - dose (10 mg / kg) solutions: Prepare Peganum harmala dichloromethane extract solutions with concentrations of 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL by respectively mixing an appropriate amount of dry extract of Peganum harmala dichloromethane with a 0.5% CMC - Na solution.
[0030] Sodium valproate solution (200 mg / kg): Prepare a 5 mg / mL sodium valproate solution by mixing an appropriate amount of sodium valproate with physiological saline.
[0031] 1.3. Model grouping and establishment
[0032] After 7 days of environmental adaptation, 6 out of 36 SD rats were randomly selected as the control group, and the remaining 30 rats were intraperitoneally injected with lithium chloride (127 mg / kg, dissolved in normal saline). Pilocarpine (30 mg / kg, prepared with normal saline) was injected at an interval of 20 h. Scopolamine methylbromide (1 mg / kg, diluted with normal saline) was pre-injected 30 min before pilocarpine administration to relieve peripheral side effects. After administration, the behavioral changes of the animals were continuously monitored, and the degree of epileptic seizures was evaluated according to the Racine grading standard (Table 1). If the seizure reached grade IV or above and the behavior of the rats did not return to normal between two seizures and lasted for more than 15 min, the model was considered successfully established. If the rats did not show epileptic seizures of grade IV or above 30 minutes after administration, pilocarpine was supplemented at 1 / 3 of the initial dose, and at most two supplements were made. If epileptic seizures of grade IV or above still could not be induced after three cumulative administrations (pilocarpine), the model construction was determined to fail and the rats were excluded from the subsequent experiments. At the same time, the control group was not modeled and was given intraperitoneal injection of normal saline.
[0033] Among them, 28 rats reached grade 4 or above seizures. During the acute phase (within 24 h after modeling), 1 EP model rat died due to frequent convulsions caused by EP. No rats died during the latent period (within two weeks after modeling), and no EP rats died during the chronic phase (two to three weeks after modeling).
[0034] Table 1 Racine grading standard for epileptic behavior in rats
[0035]
[0036]
[0037] 1.4 Experimental grouping and drug administration
[0038] The 6 rats that were not modeled were used as the normal group. 24 rats were randomly selected from the 27 successfully modeled rats and divided into a model group, a valproate group, a low-dose Peganum harmala group, a medium-dose Peganum harmala group, and a high-dose Peganum harmala group, with 6 rats in each group. The 6 groups of rats were administered drugs once a day for 4 weeks. The specific drug administration situations were as follows:
[0039] Valproate group (VAS): Intragastric administration of valproate solution at 200 mg / kg;
[0040] Low-dose Peganum harmala group (PhDE-L): Administration of PhDE solution at 10 mg / kg dissolved in 0.5% CMC-Na;
[0041] Medium-dose Peganum harmala group (PhDE-M): Administration of PhDE solution at 20 mg / kg dissolved in 0.5% CMC-Na;
[0042] High-dose Peganum harmala group (PhDE-H): Administration of PhDE solution at 40 mg / kg dissolved in 0.5% CMC-Na;
[0043] Normal group (Natural) and model group (Model): Equal amounts of blank CMC-Na solution were administered.
[0044] 2. Experimental methods related to the antiepileptic effect of Peganum harmala dichloromethane
[0045] 2.1 Open field test (OFT)
[0046] 2.1.1 Experimental apparatus
[0047] A black cubic box with dimensions of 100 cm × 100 cm × 30 cm. After dividing the base surface into 16 equal square grids, the four adjacent grids at the geometric center are the central area.
[0048] 2.2.2 Experimental method
[0049] After the administration ended, the experimental rats were subjected to OFT to evaluate their spontaneous locomotor ability: The rats were placed with their backs to the experimenter at the same position into the above-mentioned apparatus, and their free exploration in the box (open field) within 5 minutes was observed. Record and analyze the total movement distance, movement distance in the central area, and average movement speed within 5 minutes in the open field. After each rat was tested, the feces and urine in the field were cleaned, and alcohol was sprayed in the field to remove the residual odor to avoid interfering with the subsequent tests of rats.
[0050] 2.2 Novel object recognition test (NOR)
[0051] 2.2.1 Experimental apparatus
[0052] Two identical square items (A) with no special odor, and a cylindrical object (B) with a similar size but different color. The NOR experiment was carried out in the same black cubic box as the OTF.
[0053] 2.2.2 Test method
[0054] Examine the learning and memory abilities of rats in each group, including the old object exploration stage (10 minutes) and the new object exploration stage (10 minutes).
[0055] In the old object exploration stage, the two A objects were fixed in the field, and the objects were required to be symmetrically distributed at 2 / 3 of the side length of the field. After the odor was eliminated with alcohol, the rats were placed with their backs to the A objects from the other side of the field and allowed to explore autonomously for 10 minutes.
[0056] In the new object exploration stage, one of the A objects in the box was replaced with a cylindrical object B. After spraying and drying with alcohol, the experimental rats were placed in the field with their backs to both A and B objects. The exploration behaviors (including smelling and touching) of the rats towards two objects with different geometric features (color and shape) were observed within 10 minutes. The time for the rats to explore each object was recorded. The object exploration time and movement trajectory of the rats were synchronously collected through the Smart3.0 video tracking system. The recognition index RI of each rat towards object B (the new object) was calculated (TimeA: the time to explore object A; Time B: the time to explore object B):
[0057]
[0058] 2.3 Morris water maze experiment (MWM)
[0059] 2.3.1 Experimental apparatus
[0060] A cylindrical black pool with a diameter of 160 cm and a height of 50 cm was equally divided into four areas: SE, SW, NW, and NE. A hidden escape platform (Φ10 cm) was fixed in the SW area, 15 cm away from the pool wall and kept 1 cm below the liquid level. Warm water was injected into the pool in advance, and 2 - 4 heating rods were added to maintain the water temperature at a constant 23 - 25 °C. Since the experimental animals used were SD rats, an appropriate amount of ink was added to the pool so that the hidden platform in the water could not be seen. The pool was covered with a curtain around and on the top to make the data collection more sensitive and reduce the influence of external factors on the experimental results.
[0061] 2.3.2 Test method
[0062] Place navigation stage: The rats were placed into the water facing the pool wall in the order of SE, SW, NW, and NE, and the escape latency (the time from entering the water to reaching the platform) was recorded. If the rats reached the platform successfully within 60 s, the time spent was the escape latency; if they were still in the water within the specified time (60 s) and could not find the platform, items such as a glass plate could be used to guide the rats to swim to the platform, and the escape latency was recorded as 60 s at this time. And let the rats familiarize themselves with the environment on the platform for 60 s. After each rat was fished out of the water, the moisture on its body surface needed to be dried and its hair was blown dry with a hair dryer to prevent the rats from inducing EP due to cold and affecting their vital signs. There was a 0.5 - h interval between the tests of each rat in every two areas. The average value of the escape latencies in the four areas was the escape latency data of the rats on that day. This stage of the experiment needed to be carried out continuously for 4 days at the same time period.
[0063] Space exploration phase: Conduct space exploration experiments at the same time period on the 5th day. Remove the submerged platform underwater, keep other experimental conditions unchanged, place the rats facing the pool wall in the area far from the quadrant where the platform is located, that is, the NE area. Start timing when putting the rats into the water and let the rats freely explore in the water for 60 s. The camera records the movement trajectories of the rats, and analyzes the residence time of the rats in the SW area and the number of times they cross the area demarcated by the platform.
[0064] 2.4 Tissue sampling
[0065] 2.4.1 Sampling of whole brain tissue
[0066] The rats were anesthetized by inhaling isoflurane. After fixing their four limbs, open the chest cavity and free the heart tissue. Connect a peristaltic pump to a reagent bottle containing normal saline or paraformaldehyde and an injection needle. Turn on the peristaltic pump to ensure smooth flow in the pipeline. Insert the needle at the apex of the rat's heart, and quickly cut open the right auricle with surgical scissors. It can be seen that blood flows out from the notch until the outflowing liquid becomes transparent, and the rat's liver changes from dark red-brown to gray-yellow. Immediately replace the normal saline with 4% paraformaldehyde solution and perfuse until the rat's tail becomes stiff. Each rat was perfused with approximately 300 mL of liquid. During this period, hemostatic forceps can be used to clamp the apex of the heart and the needle to play a fixing role. At the same time, perfusion requires operating in a fume hood because paraformaldehyde is toxic. After perfusion, turn off the peristaltic pump, properly place the injection needle, and decapitate the rats to remove the brain. Place the brain tissue in 4% paraformaldehyde solution for further fixation.
[0067] 2.4.2 Sampling of hippocampal tissue
[0068] After the rats were anesthetized, they were perfused with normal saline through the heart until the liver turned gray-yellow. Free the bilateral hippocampi in the brain tissue with forceps on the ice surface, put them into cryotubes, and store them at -80 °C.
[0069] 2.5 Histopathological examination
[0070] 2.5.1 H&E staining
[0071] Pre-treatment before sectioning: After the brain tissue was fixed for 24 h, transfer it to a 30% sucrose-PBS solution and gradient sediment until the tissue completely sinks.
[0072] Deparaffinization and rehydration of paraffin sections: Immediately after resection of the cerebellum with a scalpel, place it on a specimen holder and embed it in paraffin. Place the specimen holder on the quick-freezing stage of a cryostat at -40°C for quick freezing. Refer to the Paxinos rat brain stereotaxic atlas and trim the block in the coronal plane at -20°C in a cryostat. Retain the brain tissue in the interval of 2.3 - 4.5 mm posterior to the bregma, and use a cryostat to prepare 10-μm serial sections. Focus on retaining the sections containing the complete hippocampal structure and attach them to adhesive slides. The sections are deparaffinized with gradient xylene and rehydrated with gradient ethanol (100% → 95% → 85% → 70% for 3 min each), and finally washed with deionized water.
[0073] Staining: Stain the sections of each group with hematoxylin staining solution for 3 - 5 min. After differentiation and blue return, perform eosin staining for 5 min.
[0074] Dehydration, mounting and scanning: Dehydrate with gradient alcohol, permeabilize, mount, and then observe the sections under a microscope.
[0075] 2.5.2 Nissl staining
[0076] Similar to the steps of H&E staining, prepare sections by paraffin embedding. After deparaffinization and rehydration of the sections, add Nissl staining solution at 57°C and stain for 20 min. After staining, dehydrate with gradient alcohol and clear with xylene, and finally mount and dry completely.
[0077] 2.6 Statistical analysis
[0078] All data in this study were processed using SPSS 26.0, and the results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the statistical differences among multiple groups of data. A P value < 0.05 was considered statistically significant. The results were visualized using GraphPad Prism 8 software.
[0079] 3 Experimental results
[0080] 3.1 Results of open field test
[0081] From Figure 1 The results showed that compared with the normal group, the total movement distance (P < 0.001), the movement distance in the central area (P < 0.05), and the average speed (P < 0.001) of rats in the Li-Pilo-induced epilepsy model group were significantly reduced. Compared with the model group, the valproate group and each dichloromethane administration group of Peganum harmala L. could significantly increase the total movement distance and the movement distance in the central area of rats (P < 0.01). The results indicated that Li-Pilo-induced epilepsy could lead to the inhibition of the motor ability of rats and the generation of anxiety. After administration of dichloromethane of Peganum harmala L., the anxiety of epileptic rats was alleviated and the spontaneous motor ability of rats was enhanced.
[0082] 3.2 NOR Results
[0083] It can be seen from Figure 2 that the exploration time of normal rats for new objects is longer than that for old objects. In contrast, the relative exploration time of new objects in the model group of rats was significantly decreased (P<0.001). After administration of sodium valproate and dichloromethane extract of Peganum harmala L., the novel object recognition index of rats was significantly increased compared with that of the model group. The results showed that dichloromethane extract of Peganum harmala L. could significantly improve the cognitive memory ability of epileptic rats, and the medium-dose group had the best effect (P<0.01).
[0084] 3.3 MWM Results
[0085] It can be seen from Figure 3 A that after four days of training with water entry in four quadrants in turn (place navigation experiment), the average time (escape latency) for normal rats to find the platform was significantly shortened compared with the first day (P<0.01), while there was no significant change in the escape latency of the model group. Comparing the escape latencies of each group on the fourth day, the results showed that dichloromethane extract of Peganum harmala L. could significantly shorten the escape latency of rats.
[0086] In the spatial exploration experiment ( Figure 3 B-D), the residence time of epileptic rats in the SW quadrant was significantly reduced compared with the normal group (P<0.05), and the number of times of crossing the platform range was also significantly reduced (P<0.001). After administration of dichloromethane extract of Peganum harmala L., the residence time of rats in the SW quadrant and the number of times of crossing the platform range were significantly increased compared with the model group.
[0087] The above results showed that dichloromethane extract of Peganum harmala L. could significantly improve the spatial memory ability of epileptic rats, and the medium-dose group had the best effect.
[0088] 3.2 H&E Staining Results
[0089] Histopathological examination showed ( Figure 4 ) that the nerve cells in the hippocampal region of the normal group were arranged in regular layers, the cell membrane boundaries were clear, the nuclear cytoplasm was uniform and translucent, and no obvious pathological changes were seen. The model group showed typical neurodegenerative lesion characteristics: the layered structure of neurons was disordered, and karyolysis was seen in local areas. The structural integrity of nerve cells in the drug intervention group was significantly improved. Among them, the arrangement of neurons in the medium-dose group of Peganum harmala L. was the most regular, and only apoptotic bodies were occasionally seen. The degree of pathological improvement was significantly better than that of other drug administration groups.
[0090] 3.4 Nissl Staining Results
[0091] The Nissl results showed ( Figure 5) In the normal group, the hippocampal tissue structure was intact, neurons were arranged in an orderly palisade pattern, and Nissl bodies were densely distributed in the cytoplasm, with staining characteristics consistent with the physiological state. The model group presented typical nerve injury characteristics: the neuron density was significantly reduced, the arrangement direction of the remaining cells was disordered, and significant loss of Nissl bodies occurred. Among the drug intervention groups, the medium-dose Peganum harmala group showed the best neuroprotective effect, with the neuron density restored and the number of Nissl bodies increased compared with the model group, and its therapeutic effect was significantly better than that of other dosing groups.
[0092] The above results indicate that dichloromethane extract of Peganum harmala has a neuroprotective effect on Li-Pilo-induced epileptic rats. The behavioral results show that dichloromethane extract of Peganum harmala can significantly improve the manifestations of reduced spontaneous activity ability, cognitive and memory ability impairment in rats caused by epilepsy. The results of H&E staining and Nissl staining show that dichloromethane extract of Peganum harmala significantly reduces hippocampal neuron injury and neuronal necrosis, making the neuron arrangement relatively neat and increasing the number of intracellular Nissl bodies. The medium dose (20 mg / kg) of Peganum harmala has the best effect. This further indicates that the dichloromethane fraction of the ethanol extract of Peganum harmala can be used for the preparation of anti-epileptic drugs.
[0093] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should be covered within the protection scope of the present invention.
Claims
1. Use of the dichloromethane extract of Peganum harmala L. as an anti-epileptic drug.
2. Use of the dichloromethane extract of Peganum harmala L. as claimed in claim 1 as an anti-epileptic drug, characterized in that, The preparation method of the dichloromethane extract of Peganum harmala L. comprises the following steps: S10. Extraction step: Select the medicinal materials of Peganum harmala L., place the medicinal materials of Peganum harmala L. in an ethanol aqueous solution with a mass concentration of 95%, carry out immersion extraction at room temperature, the immersion time is 5 - 7 days, and the material-liquid ratio of the medicinal materials of Peganum harmala L. to the ethanol aqueous solution is 1:20 (g / mL); repeat the extraction operation three times according to this immersion extraction method. After each extraction, separate the extract from the medicinal residues, collect the extracts obtained from the three extractions, and remove the solvent ethanol by conventional methods such as vacuum concentration to prepare a dry extract of the ethanol extract of the medicinal materials of Peganum harmala L. S20. Extraction step: Place the dry extract of the ethanol extract in an appropriate amount of water, stir well to dissolve it to obtain an aqueous solution of the dry extract; slowly add a 2% sulfuric acid solution to the obtained aqueous solution of the dry extract to adjust the pH to 2 - 3 to obtain an acidified aqueous solution, so that the alkaloids existing in the form of organic salts in Peganum harmala L. are converted into small molecule inorganic salts, increasing their solubility in water; add ethyl acetate to the acidified aqueous solution for extraction, fully oscillate and mix, then let it stand for layering, separate the ethyl acetate layer, repeat the above extraction operation at least once to remove impurities such as lipids to obtain an acid aqueous solution. The volume ratio of the acidified aqueous solution to ethyl acetate is 1:
1. Slowly add a 1% sodium hydroxide solution to the acid aqueous solution to adjust the pH to 10 - 11 to free the alkaloids in the aqueous solution to obtain an alkalized aqueous solution. Add dichloromethane to the alkalized aqueous solution according to a volume ratio of 1:1 for extraction, fully oscillate and mix, then let it stand for layering, separate the dichloromethane layer, repeat the above extraction operation with dichloromethane three times to enrich the alkaloids, collect the extracts obtained from the three dichloromethane extractions, and remove the solvent dichloromethane by conventional methods such as vacuum concentration to obtain the dichloromethane extract of Peganum harmala L.
3. An antiepileptic drug, characterized in that, It includes the dichloromethane extract of Peganum harmala L. as described in claim 1 or 2.
4. The anti-epileptic drug according to claim 3, characterized in that, The dosage of the dichloromethane extract of Peganum harmala L. is 10 mg / kg - 40 mg / kg.
5. The antiepileptic drug according to claim 3, characterized in that, The dosage of the dichloromethane extract of Peganum harmala L. is 20 mg / kg.
6. The antiepileptic drug according to claim 3, characterized in that, It includes being prepared into a drug dosage form suitable for clinical use, including any one of tablets, oral liquids, pills, granules, capsules, injections and sustained-release and controlled-release preparations.