Epilepsy mixture as well as preparation method and application thereof

Abstract

The invention relates to an epilepsy mixture as well as a preparation method and application thereof. The mixture is prepared by crushing, decocting and concentrating active pharmaceutical ingredients including endive, fennel root bark, endive root, fennel (slightly fried), rose petals, pimpinella diversifolia fruit (slightly fried) and Chinese chive root into a concentrated solution, and then adding cane sugar. Tests show that the epilepsy mixture has the application in drugs for protecting neuronal cell injury and treating epilepsy.

Description

Technical Field

[0001] The invention relates to an epilepsy mixture and a preparation method and application thereof, and an application thereof in a medicine for protecting neuron cell damage and treating epilepsy. Background Art

[0002] Epilepsy is a chronic brain disease caused by multiple diseases. It is a common neurological disease characterized by abnormal discharges of brain neurons leading to sudden, recurrent, and transient epileptic seizures. It can cause neuronal damage, which in turn leads to learning, memory, and cognitive dysfunction. Every time epileptic symptoms occur, nerve cells will release excessive current, which will also cause damage to the patient's brain nerves and damage nerve cells. Pediatric epilepsy is a common chronic neurological disease in children. Related studies have shown that 70%-80% of epilepsy patients have varying degrees of cognitive dysfunction, and the impact of epilepsy on children's cognitive function is more obvious than that of adults. The Chinese Expert Consensus on the Diagnosis and Treatment of Status Epilepticus in Children (2022) points out the treatment principles: ① Early treatment, early and rapid termination of clinical convulsive seizures and persistent electroencephalographic epileptic discharges; ② Appropriate drug selection, sufficient dosage, appropriate usage, and continuous treatment procedures; ③ Comprehensive treatment, including life support, anticonvulsant treatment, etiology treatment, and treatment of complications; ④ Neurological function evaluation and rehabilitation treatment: Neurological function evaluation as soon as possible, and timely rehabilitation treatment. There are many treatments for epilepsy at present. In recent years, there have been many advances in drug treatment and neuromodulation. Common treatment methods can be divided into: (1) drug treatment of epilepsy; (2) surgical treatment of epilepsy (including neuromodulation therapy); (3) ketogenic diet. The World Health Organization (WHO) lists epilepsy as a neurological and psychiatric disease that requires key prevention and control. Epilepsy has become a serious public health and social problem that has received increasing attention in my country and around the world. In China, epilepsy is a common neurological disease second only to stroke, and is a chronic disease that seriously affects the physical and mental health and quality of life of patients. As an important part of the international anti-epilepsy cause, my country's anti-epilepsy cause is also constantly developing and progressing.

[0003] Epilepsy in modern medicine belongs to epilepsy in traditional Chinese medicine, also known as epilepsy syndrome or epilepsy. Due to its diverse causes and clinical manifestations, ancient doctors have never accurately named epilepsy. The earliest record was in the Yellow Emperor's Classic of Internal Medicine. Traditional Chinese medicine attaches great importance to the concept of holism, believing that a person is an organic whole, and the various components that constitute the human body are inseparable in structure, coordinated and complementary in function, and affect each other in pathology. Therefore, traditional Chinese medicine has a very rich understanding of the etiology and pathogenesis of epilepsy, which are mostly attributed to the six exogenous evils, internal injuries, congenital deficiency, improper diet, overwork or fright, phlegm and turbidity, obstruction of the mind, obstruction of the meridians and epilepsy. For the treatment of this disease, Western medicine mainly uses drug intervention and surgery. However, drug intervention can only control the disease but not eradicate it. Surgical resection of epileptic lesions can control most epileptic seizures and solve the fundamental problem of some focal epilepsy, but anti-epileptic drugs are still needed after surgery to prevent or control postoperative complications. Traditional Chinese medicine has a variety of methods for treating epilepsy, with significant efficacy and few side effects. It can relieve the onset of different types of epilepsy and effectively improve the quality of life of patients. In recent years, clinical research and research on the mechanism of action of traditional Chinese medicine in treating epilepsy have been increasing.

[0004] The epilepsy mixture provided by the present invention is a compound preparation formulated according to the theory of traditional Chinese medicine, and is composed of seven medicinal materials: chicory, fennel root bark, chicory root, fennel (slightly fried), rose petals, anise fruit (slightly fried), and leek root. It has the use of protecting neuronal cell damage and in medicines for treating epilepsy. Summary of the invention

[0005] The present invention aims to provide an epilepsy mixture and its preparation method and use. The mixture is prepared by crushing, decocting, concentrating to a concentrated solution, and then adding sucrose to the raw materials chicory, fennel root bark, chicory root, fennel (slightly fried), rose petals, anise fruit (slightly fried), and leek root. The experiment shows that the epilepsy mixture has the function of protecting neuronal cell damage and its use in medicine for treating epilepsy.

[0006] The epilepsy mixture of the present invention is prepared from raw materials chicory, fennel root bark, chicory root, fennel, rose petals, anise fruit, and leek root, and the specific operation is carried out according to the following steps:

[0007] a. The content of each component per 1 portion is: 7-22 grams of chicory, 8-24 grams of fennel root peel, 5-20 grams of chicory root, 6-21 grams of fennel, 4-17 grams of rose petals, 5-18 grams of anise fruit, and 6-18 grams of leek root are crushed according to a conventional method and mixed to obtain a mixture;

[0008] b. Place the mixture obtained in step a in an extraction tank, add 5-12 times the amount of water and boil for 2-3 times, boiling for 0.5-1.5 hours each time, filter, and combine the filtrate;

[0009] c. The filtrate obtained in step b is concentrated under reduced pressure of -0.01-0.10 MPa at a temperature of 40-80°C to a concentrated solution with a ratio of 0.2-1:2-8 of medicinal material: medicinal solution, and set aside;

[0010] d. Add 60-120 g of sucrose to 300-500 ml of the concentrated solution obtained in step c and mix evenly, and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0011] A method for preparing an epilepsy mixture is carried out according to the following steps:

[0012] a. According to the content of each component in each portion, 7-22 g of chicory, 8-24 g of fennel root peel, 5-20 g of chicory root, 6-21 g of fennel, 4-17 g of rose petals, 5-18 g of anise fruit, and 6-18 g of leek root are crushed and mixed using a crusher in a conventional manner to obtain a mixture;

[0013] b. Place the mixture obtained in step a in an extraction tank, add 5-12 times the amount of water and boil for 2 times, boiling for 0.5-1.5 hours each time, filter, and combine the filtrate;

[0014] c. The filtrate obtained in step b is concentrated under reduced pressure of -0.01-0.10 MPa at a temperature of 40-80°C to a concentrated solution with a ratio of 0.2-1:2-8 of medicinal material: medicinal solution, and set aside;

[0015] d. Add 60-120 g of sucrose to 300-500 ml of the concentrated solution obtained in step c and mix evenly, and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0016] The epilepsy mixture is used in preparing medicine for preventing neuronal cell damage.

[0017] The epilepsy mixture is used in preparing medicine for treating epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of the epilepsy mixture of the present invention on Morris water maze of KA-induced epileptic rats;

[0019] Figure 2 The effect of the epilepsy mixture of the present invention on the electroencephalogram of KA-induced epileptic rats;

[0020] Figure 3 The effect of the epilepsy mixture of the present invention on the pathological changes of hippocampal tissue in rats with epilepsy induced by KA;

[0021] Figure 4 The effect of the epilepsy mixture of the present invention on neurotransmitters in KA-induced epileptic rats;

[0022] Figure 5 The effect of the epilepsy mixture of the present invention on the cell viability of SH-SY5Y neuronal cells;

[0023] Figure 6-8 The effect of the epilepsy mixture of the present invention on the antioxidant effect in L-Glu-induced SH-SY5Y neuronal cells;

[0024] Fig. 9 The present invention shows the effect of the epilepsy mixture on the apoptosis of SH-SY5Y neuron cells induced by L-Glu. DETAILED DESCRIPTION

[0025] Example 1 (content of each component per 1 portion)

[0026] a. 12 g of chicory, 8 g of fennel root peel, 10 g of chicory root, 8 g of fennel (slightly fried), 11 g of rose petals, 12 g of anise fruit (slightly fried), and 7 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0027] b. Place the mixture obtained in step a in an extraction tank, add 6 times the amount of water and boil for 3 times, boiling for 0.5 hours each time, filter, and combine the filtrate;

[0028] c. The filtrate obtained in step b is decompressed to -0.01 MPa and concentrated at 40°C to a concentrate with a ratio of 0.3-1:2-7 of medicinal material: medicinal solution, and set aside;

[0029] d. Add 60 g of sucrose to 300 ml of the concentrated solution obtained in step c to prepare an epilepsy mixture.

[0030] Example 2 (content of each component per 1 portion)

[0031] a. 11 g of chicory, 13 g of fennel root peel, 9 g of chicory root, 14 g of fennel (slightly fried), 12 g of rose petals, 18 g of anise fruit (slightly fried), and 7 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0032] b. Place the mixture obtained in step a in an extraction tank, add 7 times the amount of water and boil twice, each time for 1.0 hour, filter, and combine the filtrate;

[0033] c. The filtrate obtained in step b was decompressed to -0.02MPa and concentrated to 50°C to a ratio of 0.3-1:3-7 of medicinal material: medicinal solution, and set aside;

[0034] d. Add 70 g of sucrose to 500 ml of the concentrated solution obtained in step c and mix well. Prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0035] Example 3 (content of each component per 1 portion)

[0036] a. 15 g of chicory, 14 g of fennel root peel, 9 g of chicory root, 10 g of fennel (slightly fried), 7 g of rose petals, 8 g of anise fruit (slightly fried), and 7 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0037] b. Place the mixture obtained in step a in an extraction tank, add 8 times the amount of water and boil for 3 times, boiling for 1.5 hours each time, filter, and combine the filtrate;

[0038] c. The filtrate obtained in step b is decompressed to -0.07MPa and concentrated to 80°C to a concentrate with a ratio of 0.3-1:3-6 of medicinal material: medicinal solution, and set aside;

[0039] d. Add 90 g of sucrose to 400 ml of the concentrated solution obtained in step c and mix evenly; and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0040] Example 4 (content of each component per 1 portion)

[0041] a. 18 g of chicory, 16 g of fennel root peel, 11 g of chicory root, 9 g of fennel (slightly fried), 8 g of rose petals, 9 g of anise fruit (slightly fried), and 9 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0042] b. Place the mixture obtained in step a in an extraction tank, add 9 times the amount of water and boil for 3 times, boiling for 1.5 hours each time, filter, and combine the filtrate;

[0043] c. The filtrate obtained in step b is decompressed to -0.05 MPa and concentrated at 70°C to a concentrated solution with a ratio of 0.1-2:3-5 of medicinal material: medicinal solution, and set aside;

[0044] d. Add 100 g of sucrose to 350 ml of the concentrated solution obtained in step c, mix well, and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0045] Example 5 (content of each component per 1 portion)

[0046] a. 12 g of chicory, 18 g of fennel root peel, 10 g of chicory root, 9 g of fennel (slightly fried), 10 g of rose petals, 7 g of anise fruit (slightly fried), and 12 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0047] b. Place the mixture obtained in step a in an extraction tank, add 9 times the amount of water and boil once, boil for 1 hour each time, filter, and combine the filtrate;

[0048] c. The filtrate obtained in step b is decompressed to -0.07 MPa and concentrated to 80°C to a concentrate with a ratio of 0.1-3:1-6 of medicinal material: medicinal solution, and set aside;

[0049] d. Add 80 g of sucrose to 450 ml of the concentrated solution obtained in step c, mix well, and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0050] Example 6 (content of each component per 1 portion)

[0051] a. 13 g of chicory, 15 g of fennel root peel, 11 g of chicory root, 9 g of fennel (slightly fried), 8 g of rose petals, 7 g of anise fruit (slightly fried), and 6 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0052] b. Place the mixture obtained in step a in an extraction tank, add 6 times the amount of water and boil twice, boiling for 1.5 hours each time, filter, and combine the filtrate;

[0053] c. The filtrate obtained in step b is decompressed to -0.09 MPa and concentrated to a concentrated solution with a ratio of 0.2-3:1-7 of medicinal material to medicinal solution, and set aside;

[0054] d. Add 70 g of sucrose to 500 ml of the concentrated solution obtained in step c and mix well. Prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0055] Example 7 (content of each component per 1 portion)

[0056] a. 16 g of chicory, 15 g of fennel root peel, 12 g of chicory root, 12 g of fennel (slightly fried), 9 g of rose petals, 6 g of anise fruit (slightly fried), and 6 g of leek root were crushed and mixed in a grinder according to a conventional method to obtain a mixture;

[0057] b. Place the mixture obtained in step a in an extraction tank, add 8 times the amount of water and boil twice, boiling for 1 hour each time, filter, and combine the filtrate;

[0058] c. The filtrate obtained in step b is decompressed to -0.10 MPa and concentrated at 4°C to a concentrate with a ratio of 0.3-1:2-7 of medicinal material: medicinal solution, and set aside;

[0059] d. Add 60 g of sucrose to 300 ml of the concentrated solution obtained in step c and mix well. Prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

[0060] Example 8

[0061] The obtained epilepsy mixture was subjected to experimental studies on the maximum drug tolerance, neuronal cell protection and chronic epilepsy treatment effects, to verify the neuronal cell protection and chronic epilepsy treatment effects and mechanisms of the epilepsy mixture;

[0062] 1. Effect of epilepsy mixture on chronic epilepsy induced by KA in rats:

[0063] 1.1 Experimental Materials:

[0064] 1.1.1 Drugs and reagents:

[0065] The epilepsy mixture of the present invention, KA (Sigma, batch number: 4051783), isoflurane (Jiangsu Hengfengqiang Biotechnology Co., Ltd., batch number: 20221101), 3% hydrogen peroxide (Shandong Xiekang Medical Technology Co., Ltd., batch number:), physiological sodium chloride solution (Sichuan Kelun Pharmaceutical Co., Ltd., batch number: L23091705), carbamazepine (Shanghai Fudan Fuhua Pharmaceutical Co., Ltd., batch number: 34K04051, national medicine standard: H31021366);

[0066] 1.1.2 Main instruments:

[0067] Brain stereotaxic instrument (Shenzhen Ruiwode Life Science Co., Ltd.), micro-injector (Shanghai Gaoge Industry and Trade Co., Ltd.), small animal intelligent anesthesia machine (model: ZS-MV-IDV, Beijing Zhongshi Di Chuang Technology Development Co., Ltd.), Morris water maze experimental equipment, programmable rat dark avoidance box (model: DBA-2, Institute of Materia Medica, Chinese Academy of Medical Sciences), BL420s biofunction experimental system (Chengdu Taimeng Software Co., Ltd.), open field box (Institute of Materia Medica, Chinese Academy of Medical Sciences);

[0068] 1.1.3 Animals:

[0069] 100 SPF SD rats (male), weighing 150±20g, were purchased from Spaif (Arctic) Biotechnology Co., Ltd., with the experimental animal license number of SCXK (Beijing) 2024-0001. The experimental animals were adapted to the environment for 3 days before the experiment, and had free access to food and water every day. The lighting was continuous for 12 hours every day. The temperature of the breeding environment was maintained at 22-25℃ and the relative humidity was 55-65%. The breeding and experimental procedures of the experimental animals were in accordance with the guidelines of the Animal Ethics Committee of the Chinese Academy of Medical Sciences.

[0070] 1.2 Experimental methods:

[0071] 100 SD rats were adaptively fed for 3 days and anesthetized by inhalation until the corneal reflex of the rats disappeared. They were fixed on a brain stereotaxic instrument, and the local skin was disinfected with 75% ethanol. KA was injected into the CA3 area of ​​the right hippocampus. An incision of about 1 cm was made on the midline to expose the skull and bregma. The bone surface was peeled off and the serum wound was stopped. 2.5 μL KA (0.4 μg / μL, pH=7.4) was drawn with a microsyringe and injected into the CA3 area of ​​the right hippocampus of the rats. The needle entered vertically and was slowly pushed at a speed of 0.1 μL / min. The needle was retained for 5 minutes after the injection. After the operation, 10-15 mL of normal saline was given subcutaneously, and penicillin 4×10 4 U / kg to prevent infection;

[0072] 1.2.1 Grouping and dosage:

[0073] During the modeling process, due to individual differences, 20 mice continued to have seizures until death, and were randomly divided into control group (sham), 12 mice, model group (KA), 14 mice, low-dose epilepsy mixture group (EC L), 13 mice, medium-dose epilepsy mixture group (EC M), 14 mice, high-dose epilepsy mixture group (EC H), 14 mice, and carbamazepine group (CBZ), 12 mice;

[0074] Table 1 Grouping and dosage of epilepsy mixture

[0075]

[0076] 1.2.2 Medication and observation:

[0077] Each group was given drugs by gavage once a day for 8 weeks, and the control group was given an equal volume of water;

[0078] 1.3 Effects of epilepsy mixture on behavior and cognitive impairment in KA epileptic rats:

[0079] 1.3.1 Morris water maze test

[0080] Before testing, rats were allowed to adapt to the new environment for at least 30 minutes. At the beginning of each subsequent trial, the rats were gently placed into the water from different platform locations, facing the edge of the pool, to collect the escape latency of each rat. If the rat failed to find the platform within 60 seconds, it was guided to the platform. Finding the platform was defined as staying on the platform for at least 5 seconds. The training of finding the platform was carried out for 4 consecutive days. After the last training session, the time it took the rat to find the platform within 60 seconds was recorded;

[0081] 1.3.2 Passive avoidance experiment:

[0082] The rat was placed in the light room with its back facing the dark room, and the time it took for the rat to enter the dark room was recorded as the latency period. The small door was closed and an electrical stimulus (50 Hz, 20 V, 5 s) was given to the rat. After 24 hours, the rat was placed in the light room again, and the time it took for the rat to enter the dark room was recorded as the latency period after stimulation.

[0083] 1.3.3 New object recognition experiment:

[0084] Preparation phase: rats were placed in the test environment 30 min in advance for familiarization. The first phase: adaptation to the experimental device, rats were allowed to move freely in the experimental device for 10 min; the second phase: familiarization period, two identical objects (AB, objects without any odor, firmly attached with double-sided tape to prevent rats from pushing) were placed in the device, the two objects were placed 10 cm away from the two side walls, the rats were placed in the device with their backs facing the objects from an equal distance from the objects, the exploration time of the rats on each object was recorded (the exploration of the object was counted as the mouth or nose touching the object and the object approaching within about 2-3 cm), and the time the animals explored each object was measured within 5 min; the third phase, the test phase, 24 h later, one of the two identical objects was replaced with a different object (AC) and placed in the device, and the rats were placed in the device with their backs facing the objects from an equal distance from the objects, the exploration time of A and C was recorded for 5 min, and the cognitive index RI = C / (A+C)×100% was calculated;

[0085] 1.3.4 Statistical methods:

[0086] The data were expressed as mean ± standard deviation SPSS27.0 data analysis software was used for statistical analysis, and GraphPad Prism 10.1.2 was used for drawing graphs. The experimental indicators of each group were analyzed by one-way ANOVA with random design, and P < 0.05 indicated that the difference was statistically significant;

[0087] 1.3.5 Experimental results:

[0088] 1.3.5.1 The effects of epilepsy mixture on Morris water maze of KA epileptic rats are shown in Table 2:

[0089] Table 2 Effect of epilepsy mixture on the time it takes KA epileptic rats to find the platform within 60 seconds:

[0090]

[0091] Note: Compared with the sham operation group, # p<0.05, compared with KA group, *p<0.05, **p<0.01;

[0092] Compared with the KA group, the three doses of epilepsy mixture and the positive control group can reduce the time it takes for rats to find the platform. The statistical differences between the medium and high doses of epilepsy mixture and the positive control group are statistically significant (EC M group & CBZ group, p<0.01; EC H group, p<0.05). Among the three doses of epilepsy mixture, the medium dose group took the shortest time to find the platform. The results show that epilepsy mixture has a certain effect on improving the behavior and cognition of rats (see Figure 1 );

[0093] 1.3.5.2 The effect of epilepsy mixture on passive avoidance in KA epileptic rats is shown in Tables 3 and 4:

[0094] Table 3 Effect of epilepsy mixture on the latency of KA epileptic rats to enter the dark room:

[0095]

[0096] Note: Compared with the sham operation group, ## p<0.01, compared with KA group, *p<0.05, **p<0.01;

[0097] Compared with the KA group, the latency of the three dose groups of epilepsy mixture and the positive control group increased, and the statistical difference of the high dose group of epilepsy mixture was statistically significant (EC H group, p<0.05); among the three doses of epilepsy mixture, the latency of the high dose group was the shortest, and the results showed that epilepsy mixture had a certain effect on improving the behavior and cognition of rats;

[0098] Table 4 Effect of epilepsy mixture on the number of shuttles into the dark room in KA epileptic rats

[0099]

[0100] Note: Compared with the sham operation group, ##p<0.01, compared with the KA group, *p<0.05, **p<0.01;

[0101] Compared with the KA group, the number of shuttles in the three dose groups of epilepsy mixture and the positive control group decreased, and the statistical difference between the high dose group of epilepsy mixture and the positive control group was statistically significant (ECH group, p<0.01; CBZ group, p<0.05); among the three doses of epilepsy mixture, the high dose group had the least number of shuttles, and the results showed that epilepsy mixture had a certain effect on improving the behavior and cognition of rats;

[0102] 1.3.5.1 The effect of epilepsy mixture on novel object recognition in KA epileptic rats is shown in Table 5:

[0103] Table 5 Effect of epilepsy mixture on novel object recognition index of KA epileptic rats

[0104]

[0105] Note: Compared with the sham operation group, ## p<0.01, compared with KA group, *p<0.05, **p<0.01;

[0106] Compared with the KA group, the recognition index of the three dose groups of epilepsy mixture and the positive control group was improved, among which the statistical results of the medium and high dose groups of pediatric epilepsy mixture and the positive control group were statistically significant (all p<0.01); among the three doses of epilepsy mixture, the recognition index of the medium dose group was improved most significantly, and the results showed that epilepsy mixture had a certain effect on improving the memory of rats;

[0107] The above behavioral experiments, Morris water maze, novel object recognition, and passive shuttle test results all indicate that the epilepsy mixture has a certain effect in improving the behavioral cognition and memory of KA rats.

[0108] Example 9

[0109] 1.4 Effects of epilepsy mixture on discharges in KA epileptic rats:

[0110] 1.4.1 BL420s electrophysiological function system monitors the discharge of rats:

[0111] Rats were anesthetized with isoflurane. Then, two stainless steel spiral electrodes with a diameter of 1.2 mm were implanted in the bilateral temporal cortex of the rats, and a third stainless steel electrode was implanted in the right frontal cortex as a reference electrode. The EEG electrodes were implanted in the rat cerebral cortex and fixed with dental cement. The three scattered electrodes were connected to the converter and connected to the amplifier. The rats were housed in a single cage and tested while awake. The rats were allowed to adapt for one week, and the discharge of the rats was monitored after one week, and the detection was continued for 7 days (see Figure 2 );

[0112] 1.4.2 Statistical methods:

[0113] The data were expressed as mean ± standard deviation SPSS27.0 data analysis software was used for statistical analysis, and GraphPad Prism 10.1.2 was used for drawing graphs. The experimental indicators of each group were analyzed by one-way ANOVA with random design, and P < 0.05 indicated that the difference was statistically significant;

[0114] 1.4.3 Experimental results:

[0115] 1.4.3.1 The effects of epilepsy mixture on seizure latency in KA rats are shown in Table 6;

[0116] Table 6 Effect of epilepsy mixture on seizure latency in KA epileptic rats

[0117]

[0118] Note: Compared with the KA group, **p<0.01;

[0119] Compared with the KA group, the high-dose epilepsy mixture had a longer onset latency, and the statistical results showed that the difference was statistically significant (all p<0.01); the results showed that the epilepsy mixture had a certain effect in improving epileptic seizures in rats;

[0120] 1.4.3.2 The effect of epilepsy mixture on the seizure frequency of KA rats is shown in Table 7;

[0121] Table 7 Effect of epilepsy mixture on seizure frequency in KA epileptic rats

[0122]

[0123] Note: Compared with KA group, *p<0.05;

[0124] Compared with the KA group, the high-dose epilepsy mixture had a shorter seizure frequency, and the statistical differences were statistically significant (all p<0.05); the results showed that the epilepsy mixture had a certain effect in improving epileptic seizures in rats;.

[0125] 1.5 Effect of epilepsy mixture on pathological changes of hippocampus tissue in KA epileptic rats

[0126] The rat brain tissues in each group were fixed by cardiac perfusion, embedded in conventional paraffin, sliced, and the coronal sections of the brain were Nissl stained, examined under a microscope, and imaged and analyzed;

[0127] The results are as follows Figure 3 As shown in the results of Nissl, the neurons in the hippocampus of the sham group rats were arranged regularly. However, the KA group rats showed disordered neurons, and a large number of neurons in the CA1 and CA3 regions were lost. Compared with the KA group, the epilepsy mixture administration group alleviated these phenomena.

[0128] Example 10

[0129] 1.6 Effect of epilepsy mixture on neurotransmitter content in KA epileptic rats:

[0130] 1.6.1 Effect of epilepsy mixture on GABA and Glu content in hippocampus of KA epileptic rats;

[0131] According to the kit, weigh an appropriate amount of tissue and use the corresponding kit to detect the content of GABA and Glu in rat hippocampal tissue.

[0132] 1.6.2 Effect of epilepsy mixture on the levels of DA and NE in the serum of KA epileptic rats:

[0133] The rats were anesthetized, and blood was collected from the abdominal aorta. The blood was kept at room temperature for 2 h, and then the supernatant was collected and tested using the corresponding kit.

[0134] 1.6.3 Statistical methods:

[0135] The data were expressed as mean ± standard deviation SPSS27.0 data analysis software was used for statistical analysis, and GraphPad Prism 10.1.2 was used for drawing graphs. The experimental indicators of each group were analyzed by one-way ANOVA with random design, and P < 0.05 indicated that the difference was statistically significant;

[0136] 1.6.4 Experimental results:

[0137] 1.6.4.1 Effect of epilepsy mixture on GABA and Glu content in hippocampus of KA epileptic rats;

[0138] The results showed that Figure 4 Compared with the sham group, the inhibitory neurotransmitter GABA content in the hippocampus tissue of the rats in the KA group was significantly reduced, and the excitatory neurotransmitter Glu content was significantly increased, and the statistical results showed statistically significant differences (P<0.01). After drug intervention, the GABA content of the rats in each drug group increased significantly, and the Glu content decreased significantly, and the statistical results showed statistically significant differences (P<0.01 or p<0.05);

[0139] 1.6.4.2 Effect of epilepsy mixture on the levels of DA and NE in the serum of KA epileptic rats;

[0140] The results showed that Figure 3 Compared with the sham group, the levels of dopamine (DA) and norepinephrine (NE) in the serum of the rats in the KA group were significantly reduced, and the statistical results showed significant differences (P<0.01). After drug intervention, the levels of DA and NE in the rats of each drug group increased significantly, and the statistical results showed significant differences (P<0.01 or p<0.05).

[0141] The above experimental results show that the epilepsy mixture has a certain improvement effect on neurotransmitters in KA-induced chronic epileptic seizures in rats.

[0142] Embodiment 11

[0143] 2. Effect of epilepsy mixture on L-Glu-induced SH-SY5Y neuronal cell damage:

[0144] 2.1 Experimental Materials:

[0145] 2.1.1 Drugs and reagents:

[0146] The epilepsy mixture of the present invention, L-Glutamic acid (batch number: WXBD0270V, Sigma), DMEM high-glucose culture medium (batch number: WHO1241131, Wuhan Punosai Life Science Technology Co., Ltd.), fetal bovine serum FBS (batch number: 10099-141, Gibco, USA), phosphate buffered saline PBS (batch number: SH30256.01, Hyclone, USA), 0.25% trypsin-EDTA (batch number: FG301-01, Beijing Quanshijin Biotechnology Co., Ltd.), active oxygen detection kit (batch number: CA1410, Beijing Solebow Technology Co., Ltd.), mitochondrial membrane potential detection kit (batch number: M8650, Beijing Solebow Technology Co., Ltd.), cell proliferation and cytotoxicity detection CCK-8 (Boster Biotechnology Co., Ltd.);

[0147] 2.1.2 Main instruments:

[0148] Optical microscope (OLYMPUS, CX21, Japan); cell culture incubator (HERAcell 150i, Themo, USA); flow cytometer (Beckman, Coulter EPICS XL, USA); multifunctional microplate detector (TECAN SPARK, Switzerland); multifunctional imaging system (VILBER, France, FUSION FX6); desktop high-speed refrigerated centrifuge (TGL-16K, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.);

[0149] 2.1.3 Cell lines:

[0150] Human neuroblastoma cells (SH-SY5Y) were purchased from Wuhan Punosai;

[0151] 2.2 Cell culture:

[0152] SH-SY5Y cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C and 5% CO2. When the cell density reached about 80%, 1:3 subculture was performed, and subculture was performed every other day.

[0153] 2.3 Grouping and Dosage:

[0154] SH-SY5Y cells were seeded in 6-well plates at 1×105 cells per well, and when the cells were completely attached, they were divided into control group, L-Glu group, curcumin group (2.5μmol / L), epilepsy mixture low-dose group (500μg / ml), and epilepsy mixture high-dose group (1000μg / ml). Epilepsy mixture and curcumin were pre-administered and cultured for 24 hours, and then L-Glu (9mmol / L) was given for 24 hours;

[0155] 2.4 Effect of epilepsy mixture on the viability of SH-SY5Y neuronal cells;

[0156] 2.4.1 CCK-8 method was used to detect the effect of epilepsy mixture on the activity of SH-SY5Y neuron cells;

[0157] SH-SY5Y cells were cultured at 1×10 5 / mL density was inoculated in the central 60 wells of a 96-well plate, 100μL of cell suspension was added to each well, and the remaining wells were filled with an equal amount of PBS. Six replicate wells were set for each drug concentration, 10μL of CCK-8 and 100μL of serum-free DMEM medium were added to each well, and the plate was placed in an incubator at 37°C for incubation. The treated culture plate was placed in an enzyme-linked immunosorbent assay at a wavelength of 450nm to detect the OD value. The cell survival rate was calculated;

[0158] 2.4.2CCK-8 method was used to detect the cell viability of SH-SY5Y neuron cells damaged by L-Glu induced by epilepsy mixture;

[0159] SH-SY5Y cells were inoculated in the central 60 wells of a 96-well plate at a density of 1×105 / mL, with 100μL of cell suspension in each well, and the remaining wells were filled with an equal amount of PBS. Six replicate wells were set for each drug concentration. After the cells were completely attached to the wall, they were divided into a control group, an L-Glu group, a curcumin group, a low-dose group of epilepsy mixture (500μg / ml), a high-dose group of epilepsy mixture (1000μg / ml), and a group of epilepsy mixture and curcumin pre-administered for 24 hours, and then L-Glu (9mmol / L) was given for 24 hours. 10μL CCK-8 and 100μL serum-free DMEM culture medium were added to each well, and the plates were placed in an incubator at 37°C for incubation. The treated culture plates were placed in an enzyme-linked immunosorbent assay at a wavelength of 450nm to detect the OD value and calculate the cell survival rate.

[0160] 2.4.3 Statistical methods:

[0161] The data were expressed as mean ± standard deviation SPSS27.0 data analysis software was used for statistical analysis, and GraphPad Prism 10.1.2 was used for drawing graphs. The experimental indicators of each group were analyzed by one-way ANOVA with random design, and P < 0.05 indicated that the difference was statistically significant;

[0162] 2.4.4 Experimental results

[0163] 2.4.4.1 Effect of epilepsy mixture on the viability of SH-SY5Y neuronal cells;

[0164] The results showed that Figure 5Compared with the control group, the cell proliferation inhibition rate of SH-SY5Y cells treated with different concentrations of epilepsy mixture for 24 hours did not change significantly, indicating that the epilepsy mixture had no obvious cytotoxicity to SH-SY5Y neuronal cells;

[0165] 2.4.4.2 Effect of epilepsy mixture on cell viability of L-Glu-induced SH-SY5Y neuronal cell damage;

[0166] The results showed that Figure 5 Compared with the control group, the cell viability of the L-Glu group was significantly reduced, and the statistical results showed significant differences (p<0.01). Compared with the L-Glu group, the cell viability of each drug-treated group was significantly increased, and the statistical results showed significant differences (EC L group & Cur group, p<0.05; EC H group, p<0.01). This shows that the epilepsy mixture has a certain protective effect on L-Glu-induced SH-SY5Y neuronal cells.

[0167] 2.5 Effect of epilepsy mixture on the antioxidant effect of L-Glu-induced SH-SY5Y neuronal cells;

[0168] 2.5.1 Determination of reactive oxygen species (ROS) content in each group of cells detected by DCFH-DA fluorescent probe

[0169] SH-SY5Y cells were plated at 1×10 5 The cells were inoculated in 6-well plates and divided into control group, L-Glu group, curcumin group, low-dose epilepsy mixture group (500μg / ml), and high-dose epilepsy mixture group (1000μg / ml) after the cells were completely attached to the wall. EC was pre-administered and cultured for 24 hours, and then L-Glu (9mmol / L) was given for 24 hours. The culture supernatant was discarded, and the cells were washed 3 times with PBS. 1ml DCFH-DA probe was added to each well and incubated at 37℃ for 20min. The cells were washed 3 times with serum-free medium to fully remove the DCFH-DA probe that did not enter the cells, and the fluorescence was captured under an inverted fluorescence microscope. The cell number was adjusted to be consistent and the cells were collected and scanned with an ELISA reader.

[0170] Example 12

[0171] 2.5.2 JC-1 fluorescent probe was used to detect the changes of MMP in each group of cells:

[0172] SH-SY5Y cells were seeded in 6-well plates at 1×105 cells per well. After the cells were completely attached to the wall, they were divided into control group, L-Glu group, curcumin group, low-dose epilepsy mixture group (500μg / ml), and high-dose epilepsy mixture group (1000μg / ml). EC was pre-administered and cultured for 24 hours, and then L-Glu (9mmol / L) was given for 24 hours. The culture supernatant was discarded, the cells were washed twice with PBS solution, and then 1ml 20μmol·L -1 The JC-1 probe was added and mixed thoroughly. The cells were incubated at 37°C for 20 min, the supernatant was removed, and the cells were washed twice with JC-1 staining buffer (1×). 2 ml of cell culture medium was added, and fluorescence was photographed under an inverted microscope. The data were processed using Image J software.

[0173] 2.5.3 Effects of epilepsy mixture on the contents of MDA, SOD, CAT, and GSH-Px in L-Glu-induced SH-SY5Y cells;

[0174] SH-SY5Y cells were seeded in 6-well plates at 1×105 cells per well. After the cells were completely attached to the wall, they were divided into control group, L-Glu group, curcumin group, low-dose epilepsy mixture group (500μg / ml), and high-dose epilepsy mixture group (1000μg / ml). EC was pre-administered and cultured for 24h, and then L-Glu (9mmol / L) was given for 24h. Total protein was extracted with RIPA lysis buffer, and protein was quantified by BCA method. The corresponding kits were used to detect the content of MDA, SOD, CAT, and GSH-Px in L-Glu-induced SH-SY5Y cells;

[0175] 2.5.4 Statistical methods:

[0176] The data were expressed as mean ± standard deviation SPSS27.0 data analysis software was used for statistical analysis, and GraphPad Prism 10.1.2 was used for drawing graphs. The experimental indicators of each group were analyzed by one-way ANOVA with random design, and P < 0.05 indicated that the difference was statistically significant;

[0177] 2.5.5 Experimental results

[0178] 2.5.5.1 Effect of epilepsy mixture on ROS in L-Glu-induced SH-SY5Y neuronal cells:

[0179] The results showed that Figure 6Compared with the control group, the content of ROS in the L-Glu group was significantly increased, and the statistical results showed that the difference was statistically significant (p<0.01). Compared with the L-Glu group, the content of ROS in the cells of each drug-treated group was significantly decreased, and the statistical results showed that the difference was statistically significant (Cur group, p<0.05; ECH group, p<0.01).

[0180] 2.5.5.2 Effect of epilepsy mixture on MMP induced by L-Glu in SH-SY5Y neuronal cells

[0181] The results showed that Figure 7 Compared with the control group, the mitochondrial membrane potential of the cells in the L-Glu group was significantly decreased, and the statistical results showed significant differences (p<0.01). Compared with the L-Glu group, the mitochondrial membrane potential of the cells in each drug-treated group was significantly increased, and the statistical results showed significant differences (Cur group, p<0.05; ECH group, p<0.01).

[0182] 2.5.5.3 Effects of epilepsy mixture on MDA, SOD, CAT and GSH-Px in L-Glu-induced SH-SY5Y neuronal cells

[0183] The results showed that Figure 8 Compared with the control group, the MDA content in the L-Glu group was significantly increased, and the SOD, CAT, and GSH-Px contents were significantly decreased, and the statistical results showed significant differences (P<0.01). The MDA content in the cells of each drug-treated group was significantly decreased, and the SOD, CAT, and GSH-Px contents were significantly increased, and the statistical results showed significant differences (P<0.01 or p<0.05).

[0184] The above results indicate that epilepsy mixture has a certain antioxidant effect on L-Glu-induced neuronal cell damage.

[0185] Example 13

[0186] 2.6 Effect of epilepsy mixture on L-Glu-induced apoptosis of SH-SY5Y neurons;

[0187] 2.6.1 Annexin V-FITC / PI method to detect cell apoptosis:

[0188] SH-SY5Y cells were seeded in 6-well plates at 1×105 cells per well. After the cells were completely attached to the wall, they were divided into control group, L-Glu group, curcumin group, low-dose epilepsy mixture group (500μg / ml), and high-dose epilepsy mixture group (1000μg / ml). EC was pre-administered and cultured for 24 hours, and then L-Glu (9mmol / L) was given for 24 hours. The culture supernatant was discarded, and the cells were washed twice with PBS. The cells were digested with EDTA-free trypsin at room temperature for 2 minutes, and the cell culture medium was added to terminate the digestion. The cells were collected by centrifugation at 2000rpm for 5 minutes; PBS was pre-cooled at 4℃, and the cells were washed twice (2000rpm×5min), and 5×10 5 . Centrifuge and discard the supernatant, add 500 μL of binding buffer and resuspend the cells by pipetting. Add 5 μL each of Annexin V-FITC and PI, pipette and mix, and incubate at room temperature in the dark for 15 minutes. Detect cell apoptosis by flow cytometry;

[0189] 2.6.2 Statistical methods:

[0190] The data were expressed as mean ± standard deviation SPSS27.0 data analysis software was used for statistical analysis, and GraphPad Prism 10.1.2 was used for drawing graphs. The experimental indicators of each group were analyzed by one-way ANOVA with random design, and P < 0.05 indicated that the difference was statistically significant;

[0191] 2.6.3 Experimental results:

[0192] The results showed that Fig. 9 Compared with the control group, the number of apoptotic cells in the L-Glu group increased significantly, and the statistical results showed significant differences (p<0.01). Compared with the L-Glu group, the cell apoptosis in each drug-treated group was reduced, among which the high-dose group of pediatric epilepsy mixture had a significant protective effect on L-Glu-induced SH-SY5Y cells, and the number of apoptotic cells was significantly reduced, and the statistical results showed significant differences (EC H group, p<0.01).

Claims

1. An epilepsy mixture, characterized in that The mixture is made from raw materials chicory, fennel root bark, chicory root, fennel, rose petals, anise fruit, and leek root. The specific operation is carried out according to the following steps: a. The content of each component per 1 portion is: 7-22 grams of chicory, 8-24 grams of fennel root peel, 5-20 grams of chicory root, 6-21 grams of fennel, 4-17 grams of rose petals, 5-18 grams of anise fruit, and 6-18 grams of leek root are crushed according to a conventional method and mixed to obtain a mixture; b. Place the mixture obtained in step a in an extraction tank, add 5-12 times the amount of water and boil for 2-3 times, boiling for 0.5-1.5 hours each time, filter, and combine the filtrate; c. The filtrate obtained in step b is concentrated under reduced pressure of -0.01-0.10 MPa at a temperature of 40-80°C to a concentrated solution with a ratio of 0.2-1:2-8 of medicinal material: medicinal solution, and set aside; d. Add 60-120 g of sucrose to 300-500 ml of the concentrated solution obtained in step c and mix evenly, and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

2. A method for preparing an epilepsy mixture, characterized in that Follow these steps: a. According to the content of each component in each portion, 7-22 g of chicory, 8-24 g of fennel root peel, 5-20 g of chicory root, 6-21 g of fennel, 4-17 g of rose petals, 5-18 g of anise fruit, and 6-18 g of leek root are crushed and mixed using a crusher in a conventional manner to obtain a mixture; b. Place the mixture obtained in step a in an extraction tank, add 5-12 times the amount of water and boil for 2 times, boiling for 0.5-1.5 hours each time, filter, and combine the filtrate; c. The filtrate obtained in step b is concentrated under reduced pressure of -0.01-0.10 MPa at a temperature of 40-80°C to a concentrated solution with a ratio of 0.2-1:2-8 of medicinal material: medicinal solution, and set aside; d. Add 60-120 g of sucrose to 300-500 ml of the concentrated solution obtained in step c and mix evenly, and prepare an epilepsy mixture according to a conventional method for preparing a pharmaceutical mixture.

3. Use of the epilepsy mixture according to claim 1 or 2 in the preparation of a drug for preventing neuronal cell damage.

4. Use of the epilepsy mixture according to claim 1 or 2 in the preparation of drugs for treating epilepsy.

Images