CN1785223A - Medicine for treating muscular dystrophy and myasthenia gravis, and its prepn. method - Google Patents

Abstract

The present invention is based on the traditional Chinese medicine theory of "supporting the vitality and atrophy, nourishing the vitality and promoting the muscle" as the treatment principle, and provides a kind of treatment for motor neuron diseases (amyotrophic lateral sclerosis, progressive spinal muscular atrophy, progressive medulla oblongata) Paralysis, primary lateral sclerosis), progressive muscular dystrophy, congenital myopathy and other diseases caused by muscle atrophy and myasthenia gravis and its preparation method, to achieve both symptoms and root causes. The medicine is prepared from raw materials ginseng and epimedium. The medicine of the present invention has the following effects: anti-amyotrophic lateral sclerosis patient serum inhibits nerve ending regeneration ability, promotes the regeneration and repair of damaged nerve endings, obviously improves the swimming endurance of experimental animals, has the effect of obviously enhancing muscle strength, and It can significantly inhibit the increase of serum creatine phosphokinase activity in muscle fatigue mice, and has a significant protective effect on the function of skeletal muscle.

Description

A kind of medicine for treating muscle atrophy and myasthenia gravis and preparation method thereof

technical field

The invention relates to a medicine for treating muscle atrophy and myasthenia gravis and a preparation method thereof, belonging to the technical field of Chinese herbal medicine preparations. Clinically, it is mainly used for motor neuron diseases (amyotrophic lateral sclerosis, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis), progressive muscular dystrophy, congenital myopathy and other diseases. Muscle atrophy and myasthenia gravis.

Background technique

Motor neuron disease (motor neuron disease, MND) includes amyotrophic lateral sclerosis (ALS), progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, etc. It is caused by extensive degeneration of neurons, and its symptoms include muscle weakness and muscle atrophy, and gradually develop into eating and drinking cough, dysphagia, dyspnea, and even death. ALS is a chronic progressive degenerative disease of unknown etiology that selectively invades the anterior horn cells of the spinal cord, brainstem motor nuclei and pyramidal tracts. The most common type in . Western medicine believes that when the pathogenesis of ALS is not fully understood, there is currently no special treatment. It is mainly based on symptomatic and supportive therapy to ensure adequate nutrition, improve systemic symptoms, and provide psychological support to patients so that they can establish a long-term struggle against the disease. courage and determination.

Chinese medicine believes that muscular atrophy diseases caused by ALS and others belong to the category of impotence syndrome in traditional Chinese medicine. The ancients once put forward the view that "the treatment of impotence depends on Yangming". Professor Li Renxian believes that the disease is based on the deficiency of the spleen and kidney, the internal movement of the deficiency wind is the standard, and there are pathological factors of poor blood vessels. Modified Spleen and Kidney Recipe has achieved certain curative effect; Professor Deng Tietao believes that ALS syndrome is spleen and kidney yang deficiency with phlegm and blood stasis, so he uses Buzhong Yiqi Decoction for oral administration, and intravenous infusion and acupoint injection of Astragalus Injection to strengthen the spleen and kidney , the external washing recipe fumigation and washing of the upper limbs to remove phlegm and dredging collaterals, enema to expel accumulated stools, supplemented by acupuncture, massage, diet, emotion and other treatments, also achieved certain curative effects, but they were not satisfactory.

Contents of the invention

The object of the present invention is to provide a medicine for treating muscular atrophy and myasthenia gravis with significant curative effect and a preparation method thereof.

Professor Wu Yiling, the inventor of the drug, found in practice that the clinical effect is not satisfactory only by regulating the spleen and stomach. Therefore, the inventor, Professor Wu Yiling, first bypassed the inherent model of treating from the spleen and stomach, and extended the tentacles of thinking to the meridians, and finally put the foothold on the Governor Vessel of the Eight Extraordinary Meridians. He explored the pathogenesis of muscular atrophy with the eight extra meridians as a breakthrough point, and put forward a new view on the treatment of extra meridians and the treatment principle of "supporting the yuan to atrophy, nourishing the body and muscle", focusing on warming and regulating Qiyang, nourishing and filling the essence, and warming the body. Through the eight veins. And from a large number of traditional Chinese medicines, select dozens of medicines that warm and nourish the governor vessel and stimulate nerves, scientifically formulate them, and develop the medicines of this invention. The drug test of the present invention proves that it can resist the inhibitory effect of the serum of patients with amyotrophic lateral sclerosis on the regeneration ability of nerve endings, promote the regeneration and repair of damaged nerve endings, significantly improve the swimming endurance of experimental animals, and have the effect of obviously enhancing muscle strength, and It can significantly inhibit the increase of serum creatine phosphokinase activity in muscle fatigue mice, and has a significant protective effect on the function of skeletal muscle.

The medicine of the present invention is made from the crude drug in the following parts by weight ratio:

2-10 parts of ginseng, 2-8 parts of epimedium

The weight ratio of above-mentioned crude drug is preferred:

Ginseng 5 parts, Epimedium 5 parts and:

5 parts ginseng, 2 parts epimedium and:

9 parts ginseng, 7 parts epimedium and:

3 parts ginseng, 1 part epimedium

The medicine of the present invention can be made into any conventional pharmaceutically acceptable dosage form according to the conventional preparation process, such as injection, capsule, pill, tablet, oral liquid and the like.

The preparation method of medicine of the present invention comprises the following steps:

1), take the above-mentioned Chinese herbal medicines, select and clean respectively, pulverize, and weigh according to the prescription amount;

2), take the ginseng medicinal material, extract it with 60-90% ethanol for 1-3 times, add 8-12 times the amount of solvent extraction of the medicinal material for 1-2 hours for the first time, and add 6-10 times the amount of solvent extraction for 1-2 hours for the second time hour, add 6-10 times the amount of solvent for the third time and extract for 0.5-1 hour; combine the above extracts, filter while hot, recover ethanol under reduced pressure, concentrate, put it in a stainless steel bucket, add pure water to stir evenly, and refrigerate for 24 hours -36h; take out the refrigerated liquid, put it at room temperature, filter the plate and frame filter, wash the plate and frame with a small amount of water, dilute the filtrate with pure water, and set aside; take the concentrated ginseng liquid and pass it through the processed AB-8 macroporous adsorption resin The column was eluted with ammonia water with a pH value of 8-9, 20% ethanol, and 80% ethanol, and the 80% ethanol eluate was collected. Add 80% of the eluate into pure water to form a 50% alcohol solution, pass it through a neutral alumina column, collect the eluate, wash the column with a small amount of 50% ethanol, and combine the eluent for later use; take the liquid and add activated carbon, Heating under reflux, boiling for 20 minutes, suction filtration while hot, collecting the filtrate, concentrating, and refrigerating overnight; filtering with a clarification plate, drying under reduced pressure to obtain the ginseng extract.

3), take Epimedium medicinal material, add water and decoct and extract 2-5 times, the amount of water added is 10-30 times, the first extraction time is set at 0.5-2h, and the next three times are 0.5-1h; combine the above extracts, Filtrate while it is hot, concentrate under reduced pressure, put it into a stainless steel bucket, add ethanol until the alcohol concentration of the liquid is 70%, refrigerate; filter, recover ethanol from the filtrate and concentrate, add water and stir evenly, refrigerate, filter, and set aside; take epimedium Huoyao liquid passes through the treated AB-8 macroporous adsorption resin column, and is eluted with pure water, 20% ethanol, and 50% ethanol respectively, and the 50% ethanol eluate is collected; the eluent is mixed with an appropriate amount of diatomaceous earth Mix well, extract 2-5 times with absolute ethanol, combine the extracts, recover the ethanol and concentrate, add acetone to the concentrated solution, and stir evenly, refrigerate, filter, add acetone to the precipitate and then treat it twice as before, combine the filtrates 3 times, The solvent is recovered, concentrated, and dried under reduced pressure to obtain the epimedium extract.

4), take the extracts prepared in 2) and 3), add 1,2 propylene glycol, sodium dihydrogen phosphate, disodium hydrogen phosphate additives, and add water for injection to make up to 10-20ml, heat-treat and refrigerate, and use micro pore filter membrane, ultrafiltration, filling, and sterilizing to obtain the injection of the present invention.

or

The extracts prepared in 2) and 3) are made into capsules, pills, tablets or oral liquids according to conventional preparation methods.

Detailed ways

The specific embodiment of the present invention will be described below in conjunction with the examples of the preparation of the pharmaceutical injection, capsule, tablet and pill of the present invention.

Example 1:

5 parts ginseng, 2 parts epimedium

Preparation:

1), take the above-mentioned Chinese herbal medicines, select and clean respectively, pulverize, and weigh according to the prescription amount;

2), take ginseng medicinal material, extract 3 times with 70% ethanol, add 10 times the amount of solvent extraction of the medicinal material for 1 hour for the first time, add 8 times the amount of solvent extraction for 1 hour for the second time, add 8 times the amount of solvent extraction for 0.5 times for the third time hours; combine the above extracts, filter while hot, concentrate under reduced pressure, put them in a stainless steel bucket, add pure water to stir evenly, and refrigerate for 24 hours; take out the refrigerated solution, put it at room temperature, filter it with a plate and frame filter, and wash with a small amount of water Plate and frame, dilute the filtrate with pure water, and set aside; take the concentrated ginseng liquid and pass it through the treated AB-8 macroporous adsorption resin column, and elute with ammonia water with a pH value of 8-9, 20% ethanol, and 80% ethanol , and collect the 80% ethanol eluate. Add 80% of the eluate into pure water to form a 50% alcohol solution, pass it through a neutral alumina column, collect the eluate, wash the column with a small amount of 50% ethanol, and combine the eluent for later use; take the liquid and add activated carbon, Heating under reflux, boiling for 20 minutes, suction filtration while hot, collecting the filtrate, concentrating, and refrigerating overnight; clarifying plate filtration, drying under reduced pressure to obtain the product.

3), take Epimedium medicinal material, add water to decoct and extract 4 times, the amount of water added is 20 times, the first extraction time is set to 1h, and the next 3 times are 0.5h; combine the above extracts, filter while hot, and decompress Concentrate, put it into a stainless steel bucket, add ethanol until the alcohol concentration of the liquid is 70%, refrigerate; filter, recover the ethanol from the filtrate and concentrate, add water and stir evenly, refrigerate, filter, and set aside; take the Epimedium liquid through the processed AB-8 macroporous adsorption resin column, and eluted with pure water, 20% ethanol, and 50% ethanol respectively, and collected the 50% ethanol eluate; Extract 3 times, combine the extracts, recover ethanol and concentrate, add acetone to the concentrated solution, stir evenly, refrigerate, filter, add acetone to the precipitate and treat 2 times as before, combine the filtrates 3 times, recover the solvent, concentrate, and dry under reduced pressure , that is.

4), take the semi-finished product prepared in 2) and 3), add 1,2 propylene glycol, sodium dihydrogen phosphate, disodium hydrogen phosphate and other additives, and add water for injection to make the volume to 16ml, heat treatment and refrigeration, and then use microporous filter membrane, ultrafiltration, filling, and sterilization to obtain the injection of the present invention.

Example 2:

5 parts ginseng, 5 parts epimedium

Preparation method: Same as 1), 2) and 3) steps in Example 1 and make capsules according to conventional methods.

Example 3:

9 parts of ginseng, 7 parts of epimedium

Preparation method: Same as 1), 2) and 3) steps in Example 1 and make tablets according to conventional methods.

The medicine of the present invention is based on the theory of traditional Chinese medicine, adopts the principle of "supporting the vitality and atrophy, nourishing the vitality and promoting muscle growth" as the treatment principle, combined with many years of clinical drug experience, and is designed to treat the disease caused by motor neuron disease (amyotrophic lateral sclerosis) , progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis), progressive muscular dystrophy, congenital myopathy, etc. new preparations of traditional Chinese medicine.

Taking a comprehensive view of the drug prescription of the present invention, a small amount of sweet and warm Epimedium is added to the sweet and flat ginseng, so that the whole prescription is beneficial to nourishing essence and nourishing Yuanyang without making it too warm on the basis of flat nature. dry. Although the medicine has only two flavors, its efficacy is clear and its compatibility is strict.

Pharmacological action Its injection of medicine of the present invention has carried out following animal experiment to prove its curative effect:

Protective effect of Jiweiling injection on cultured motor neurons in vitro

【Abstract】Objective: To observe the protective effect of Jiweiling Injection (JWL) on the cultured motor neurons in vitro. METHODS: Density centrifugation was used to isolate mouse embryonic spinal motor neurons for primary culture, and glutamic acid was added to establish a model of excitatory amino acid toxicity injury, and the protective effect of different concentrations of Jiweiling injection on motor neurons was evaluated. MTT method was used to detect cell viability, NF-200 immunohistochemical staining and image analysis were performed to measure the length of the neurite trunk, a biochemical analyzer was used to detect lactate dehydrogenase (LDH) in the culture supernatant, and TUNEL positive neurons were counted to observe cell apoptosis. Death. Results: (1) Jiweiling injection can significantly promote the activity of cultured motor neurons in vitro and promote the growth of neurite; (2) Jiweiling injection can reduce the leakage of LDH in motor neurons injured by excitatory amino acid toxicity; ( 3) Jiweiling injection can significantly reduce glutamate-induced motor neuron apoptosis. Conclusion: Jiweiling injection has protective effect on both normal motor neurons and motor neurons injured by excitatory amino acid toxicity.

The repair and protection of central nervous system injury is a difficult problem in the field of neuroscience, and there is still a lack of effective treatment methods and drugs. In particular, the absolute reduction in the number of motor neurons caused by injury or degeneration and death is irreversible, resulting in the loss of corresponding functions and seriously affecting the patient's motor function and quality of life. Protecting the surviving normal motor neurons and reducing the damage of degenerated neurons is of great significance for the treatment of central nervous system injuries. Jiweiling Injection is a new traditional Chinese medicine preparation developed by Professor Wu Yiling for the treatment of neuromuscular system diseases. It has achieved obvious curative effects in the clinical treatment of motor neuron disease and other diseases. In this study, the in vitro cell culture technique was used to observe the effect of Jiweiling injection on motor neurons, aiming to find effective therapeutic drugs for the clinical treatment of central nervous system injuries.

1. Materials and methods

1.1 Experimental materials

1.1.1 Drugs Jiweiling injection, produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd., 8ml/bottle (each ml contains 0.9g of crude drug), preparation number: Shiwei Pharmaceutical Zi (98) Mie 501-00296. This medicine has the effect of supporting Yuan to relieve flaccidity, nourishing vitality and promoting muscle growth. ^Riluzole Tablets, produced by Aventis Company, import drug registration certificate number: H200020006, batch number: 28097, dissolved in 0.9% NaCl-0.01NHCl during application.

1.1.2 Reagents L15 medium, butylenediamine, trypsin (1:250), polylysine, rabbit anti-NF-200 antibody, insulin, DAB chromogenic reagent, MTT, DMSO were purchased from Sigma, horse serum and Laminin are provided by Gibco BRL, fetal bovine serum is Hyclone product, TUNEL kit is product of Roche molecular Biochemical, biotin-labeled secondary antibody and horseradish peroxidase-labeled third antibody are products of Beijing Zhongshan Biological Products Co., Ltd., other reagents It is domestic analytically pure.

1.1.3 Animals Clean-grade SD female pregnant mice, 10-14 days pregnant, were provided by the Experimental Animal Center of the Field Surgery Research Institute, Daping Hospital, Third Military Medical University, Chongqing.

1.1.4 Instrument Microplate reader (DG3022A, Shanghai), image analysis system (Image Pro Plus4.5, USA), automatic biochemical analyzer (BechMan CX7, USA).

1.2 Method

1.2.1 Primary culture of fetal mouse spinal motor neurons

Fetal mice around 14 days pregnant were taken, and the spinal cord was taken out below the brainstem level, the meninges and blood vessels were peeled off, and then cut into ^small pieces of 1-2 mm, digested with 0.125% trypsin for 30 minutes, passed through a 200-mesh steel mesh at 37°C, Transfer the filtrate into a centrifuge tube with 1ml of 4% BSA (bovine serum albumin), centrifuge at 300g for 10min, remove the supernatant, dissolve the precipitate in L15 medium, add 1ml of 6.8% Metrizamide into a 5ml centrifuge tube, centrifuge at 500g for 15min , suck out the light-colored liquid between the two liquid surfaces, centrifuge at 300g for 10min, remove the supernatant, dissolve in L15 planting medium, and obtain the single-cell suspension of spinal cord anterior horn neurons, adjust the concentration to 4.5×10 ⁵ /ml, and plant in 24-well culture plate and 96-well culture plate were cultured in a CO ₂ incubator. After 24 hours, the maintenance medium was replaced. After 48 hours, 12.5 μg/ml 5-Fu was added and sucked out for 24 hours, and the fresh maintenance medium was replaced.

1.2.2 Experimental condition control:

After the motoneurons were cultured for 72 hours, the experimental group was added with 0.5%, 1%, and 5% Jiweiling injection diluted with fresh medium respectively, the Lirutai group was added with riluzole (final concentration 10 μmol/L), and the control group was added with riluzole. The same amount of medium was added for co-cultivation for 24 hours, and glutamic acid (final concentration: 0.5mmol/L) was added for 24 hours after excitotoxicity damage, and then index detection was performed.

MTT assay for cell viability:

Use a pipette to carefully suck off the culture medium in the 96-well plate, add 10 μL of MTT solution (concentration 5 mg/mL) to each well, react at 37 °C and 5% humidified CO _for 4 hours, remove the supernatant, and add the solvent dimethyl sulfoxide (DMSO) 100 μL/well, shake on a micro-oscillator at room temperature for 5 minutes, and use an automatic microplate reader to measure the wavelength λ=570nm and reference wavelength λ=630nm to measure the OD value after dissolving the colored product.

NF-200 immunohistochemical staining:

On the 6th day of culture, after the cells were routinely fixed and blocked, the rabbit anti-NF-200 monoclonal antibody (1:1000) was added for 24 hours (4°C), and the biotin-labeled secondary antibody IgG (1:300) was added for 60 minutes (37°C) , then horseradish peroxidase-labeled streptavidin (1:300), 60min ((37°C). The above steps were rinsed with 0.01mol/LPBS for 5min for a total of 3 times. DAB solution developed color for 5min, Gradient alcohol dehydration, transparent xylene, and neutral gum sealing. Negative control group, replace NF-200 antiserum with PBS for immunohistochemical reaction. Take 2 coverslips respectively, and randomly take 50 NF for each cover slip - 200 positive neurons, Quantimet-a image analysis system to measure the length of the trunk

LDH detection:

Remove the cultured cells from the 24-well plate, carefully aspirate the culture supernatant, and number the groups. The lactate dehydrogenase activity in the culture supernatant was detected with an automatic biochemical analyzer.

TUNEL detection:

After routine fixation of cultured cells, block with 0.3% H ₂ O ₂ /methanol for 30 min, add 0.1% Triton-0.1% sodium citrate solution and place on ice for 2 min, add terminal deoxyribonucleic acid transferase (TdT) and fluorescein-labeled dUTP , incubate at 37°C for 2h, incubate with horseradish peroxidase-coupled anti-fluorescein antibody at 37°C for 2h, develop color with NBT/BCIP, rinse with 0.01M PBS for 3 times, each time for 5min, dehydrate and become transparent , cover film. In the negative control group, no TdT enzyme was added. Each experiment group was repeated 3 times, and the TUNEL-positive neurons in 5 visual fields were randomly counted.

Statistical analysis:

Data are expressed as mean ± standard deviation ( x ± s) means, the t test was used for the comparison between the means, and the analysis of variance was used for the significant difference between groups.

2. Results

2.1 Morphological observation of spinal motor neurons

The neurons began to adhere to the wall 4 hours after inoculation, and the cells were single round or oval. After 24 hours of inoculation, some motor neurons had grown protrusions, and the protrusions of the motor neurons in the 1% JWL group were significantly longer. After culturing for 48-72 hours, the adherent cells grew up obviously, granular substances gradually appeared in the cell body, the halo around the cells was clear, and the protrusions increased and extended. After 96 hours, the protrusions criss-crossed in the entire visual field. Motor neurons grown for 5 to 6 days are the most plump cells with strong refraction. The nucleus is located in the center of the cell or on one side. The nucleus is light and easily separated from the perinuclear substance. The motor neurons in the control group cultured on the 5th day after adding glutamic acid for 24 hours had their protrusions retracted, and the neurons cultured with 1% mustrophin injection grew well, with moderate density, and the protrusions were thick and interlaced. TUNEL staining showed that the motor neurons in the glutamate control group were apoptotic, and many apoptotic bodies appeared, and the apoptotic bodies in the 1% Jiweiling injection group were significantly reduced.

2.2 Effect of Muswelling Injection on the Growth of Normal Motor Neurons (Table 1)

0.5%, 1%, 5% JWL group motor neuron cell viability was statistically different from the control group (P<0.05), and the cell viability of 1% JWL group was significantly better than that of Riruta group (P<0.05); 0.5% The outgrowth of spinal motoneuron process in the 1% JWL and 1% JWL groups was significantly better than that in the control group (P<0.05), and there was a significant difference from the Riruta group (P<0.05).

Table 1 The effect of Jiweiling injection on normal motoneuron vigor and process ( x±s)

Tab 1 The effect of JWL injection on vigor and outgrowth of normal motor neuron Grouping Group Density OD valueOD value Protrusion (L, μm) Outgrowth Control group Riruta group 0.5% JWL group 1% JWL group 5% JWL group 10umol/L4.5μg/L9μg/L45μg/L 0.230±0.1300.266±0.1410.317±0.054 ¹ 0.396±0.087 ^{1, 2} 0.329±0.097 ¹ 159.71±48.95192.08±89.07315.96±32.32 ^1,2 373.46±80.24 ^1,2 151.46±69.97

1. P<0.05 compared with the control group; 2. P<0.05 compared with the Rirutai group

1.P＜0.05 vs control group; 2.P＜0.05 vs riluzole group

2.3 The effect of Jiweiling injection on LDH leakage of motor neurons injured by excitatory amino acid toxicity (Table 2)

Test results LDH in the culture supernatant of O.5% JWL and Riruta groups were all lower than those of the control group (p<0.05), and there was no significant difference between the two (p>0.05), indicating that the two can reduce glutamic acid Injury to motor neurons results in leakage of intracellular LDH.

Table 2 The effect of Jiweiling injection on LDH leakage of motor neurons injured by excitatory amino acid toxicity ( x±s)

Table 2 The effect of JWL injection on LDH leakage of neurotoxicity motor neurons Grouping Group Density LDHLactic dehydrogenase Glutamic acid control group Riruta+glutamic acid group 0.5% JWL+glutamic acid group 1% JWL+glutamic acid group 5% JWL+glutamic acid group 10umol/L4.5μg/L9μg/L45μg/L 67.75±5.1258.50±4.20 ¹ 59.00±4.97 ¹ 61.50±3.1169.00±5.35 ²

1. P<0.05 compared with the control group; 2. P<0.05 compared with the Rirutai group

1.P＜0.05 vs control group; 2.P＜0.05 vs riluzole group

2.4 Effect of Jiweiling Injection on Apoptosis of Motor Neurons Injured by Excitatory Amino Acid Toxicity (Table 3)

TUNEL-positive cells in the 1% JWL group and Riruta group were less than those in the control group (p<0.05). TUNEL positive cells decreased in 0.5% and 5% JWL groups but had no significant difference with the control group (p>0.05), and there was a difference with Rirutai group (p<0.05).

Table 3 The effect of Jiweiling injection on the apoptosis of motor neurons injured by excitatory amino acid toxicity ( x±s)

Table 2 The effect of JWL injection on apoptosis of neurotoxicity motor neurons Grouping Group Density Cell number of TUNEL positive Glutamic acid control group Riruta+glutamic acid group 0.5% JWL+glutamic acid group 1% JWL+glutamic acid group 5% JWL+glutamic acid group 10umol/L4.5μg/L9μg/L45μg/L 19.8±5.63 11.2±2.84 ¹ 16.2±2.08 ² 9.8±4.34 ¹ 16.4±3.65 ²

1. P<0.05 compared with the control group; 2. P<0.05 compared with the Rirutai group

1.P＜0.05 vs control group; 2.P＜0.05 vs riluzole group

3 Discussion

Chinese medicine has certain advantages in the process of repairing the central nervous system. Jiweiling Injection is a new traditional Chinese medicine preparation for the treatment of neuromuscular system diseases developed by Professor Wu Yiling under the guidance of the Theory of Extraordinary Classics of Traditional Chinese Medicine. Experimental studies have found that Jiweiling injection can significantly improve the motor function of experimental autoimmune motor neuron disease (EAMND) model mice, and can protect and improve the injured spinal cord anterior horn motor neurons.

As an excitatory neurotransmitter, glutamate plays an important physiological role in the central nervous system, but in diseases such as ischemic stroke and amyotrophic lateral sclerosis (ALS), the abnormal activation of glutamatergic neural pathways produces excitability Sexual toxicity is an important factor causing neuronal damage. The development of glutamate inhibitors has opened up a new way for the clinical treatment of neurological diseases. Rirutai is a new drug for the treatment of ALS approved by the US FDA. Its main pharmacological effect is to resist the toxicity of excitatory amino acids. Clinical research can slow down the development of the disease, but it is expensive and cannot cure ALS and significantly improve the symptoms of patients. In this study, density gradient centrifugation will be used to culture motor neurons in vitro, and glutamic acid will be added to establish an excitatory amino acid toxicity injury model. Protective effect on motoneuron injury caused by sexual amino acid toxicity.

3.1 In vitro culture of motor neurons is an important means to observe the effect of drugs on the central nervous system

Due to the particularity of the central nervous system, clinical and experimental studies cannot directly observe motor neurons. In vitro cell culture is an experimental method and method that has been widely used in recent years. It can effectively observe and compare the efficacy of drugs, and further explore the mechanism of action. In this study, density gradient centrifugation was used to isolate mouse embryonic spinal motor neurons for primary culture, and an in vitro model for observing the effect of drugs on motor neurons was established. In order to obtain a certain purity of motor neurons, two measures are taken: one is that the centrifuge tube with built-in 6.8% Metrizamide forms a complete density gradient system. Top; second, 5-Fu was added after 24 h of culture, and mitotic inhibitors were applied to reduce the proliferation of glial cells. The purity of the motor neuron obtained by the above method can reach 85%, and the growth activity is not affected, and the growth is stable, which is beneficial to the observation of the drug effect.

3.2 Jiweiling injection promotes the growth of normal motor neurons

Studies have found that when patients with motor neuron disease have clinical symptoms, their motor neurons have been reduced by more than 50%, or even 80%, and the reduction rate is about half every 6 months. Since motor neurons cannot be repaired once they are damaged, the surviving normal motor neurons play an important role in maintaining the motor function and effective compensation of patients. The results of this study showed that Jiweiling injection could significantly enhance the activity of normal cultured motor neurons and promote the growth of spinal cord motor neurons (P<0.05). Since the main mechanism of action of Rirutai is to counteract the toxicity of excitatory amino acids, but it has no effect on increasing cell viability in normal motor neurons, the results of this study are consistent with the literature. The effect of 0.5% and 1% Jiweiling injection on promoting the growth of neurite was significantly better than that of Lirutai (P<0.05). This shows that Jiweiling injection can promote the vitality of normal motor neurons, and its role in promoting the growth of neurite is of great significance to the functional compensation of central nervous system injury.

3.3 Jiweiling Injection Reduces the Damage of Excitatory Amino Acid Toxicity to Motor Neurons

In order to study the protective effect of Jiweiling injection on motor neurons injured by excitatory amino acid toxicity, the cultured motor neurons were first treated with Jiweiling injection, and then glutamic acid was added to establish the model of excitatory amino acid toxicity injury. LDH is a marker enzyme in cells, and the release amount is very small under normal circumstances. When the neurons are damaged, the release amount of LDH increases accordingly. Therefore, the determination of the LDH content in the culture medium can quantitatively evaluate the damage degree of neurons. It was found that A certain dose of mustrophin injection can reduce the leakage of cellular lactate dehydrogenase. However, in in vitro culture, when the drug dose is too high, it may have a certain damage to the cells. At present, it is believed that neurons mainly die by apoptosis under the action of low doses of excitatory amino acids, and mainly die by necrosis at high doses. This study found that after adding glutamate, TUNEL-positive cells appeared in the cultured cells, which was a sign of glutamate-induced motor neuron apoptosis. 1% myotrophin can reduce the number of TUNEL-positive cells, indicating that myotrophin can resist glutamate-induced neuronal apoptosis.

This study shows that Jiweiling injection can not only enhance the vitality of normal motor neurons, promote the growth of neurites, but also protect motor neurons from excitatory amino acid toxicity, reduce cell apoptosis, maintain the function of the central nervous system, and delay the progression of the disease. , It has important clinical application value in improving the quality of life of patients.

Claims (9)

1. A medicine for the treatment of muscular atrophy and myasthenia gravis, characterized in that it is made of the crude drug in the following parts by weight: 1-10 parts of ginseng, 1-10 parts of epimedium. 2. The medicine according to claim 1, the proportion by weight of the crude drug is: 5 parts of ginseng, 5 parts of epimedium. 3. The medicine according to claim 1, wherein the proportion by weight of the crude drug is: 5 parts of ginseng, 2 parts of epimedium. 4. The medicine according to claim 1, wherein the proportion by weight of the crude drug is: 9 parts of ginseng, 7 parts of epimedium. 5. The medicine according to claim 1, wherein the proportion by weight of the crude drug is: 3 parts of ginseng, 1 part of epimedium. 6. The medicine according to any one of claims 1-5, characterized in that the medicine is injection, capsule, pill, tablet or oral liquid. 7. The medicine according to claim 6, characterized in that the medicine is an injection. 8. The preparation method of the medicine according to claim 7, characterized in that it comprises the following steps: 1), take the above-mentioned Chinese herbal medicines, select and clean respectively, pulverize, and weigh according to the prescription amount; 2), take the ginseng medicinal material, extract it with 60-90% ethanol for 1-3 times, add 8-12 times the amount of solvent extraction of the medicinal material for 1-2 hours for the first time, and add 6-10 times the amount of solvent extraction for 1-2 hours for the second time hour, add 6-10 times the amount of solvent for the third time and extract for 0.5-1 hour; combine the above extracts, filter while hot, recover ethanol under reduced pressure, concentrate, put it in a stainless steel bucket, add pure water to stir evenly, and refrigerate for 24 hours -36h; take out the refrigerated liquid, put it at room temperature, filter the plate and frame filter, wash the plate and frame with a small amount of water, dilute the filtrate with pure water, and set aside; take the concentrated ginseng liquid and pass it through the processed AB-8 macroporous adsorption resin The column was eluted with ammonia water with a pH value of 8-9, 20% ethanol, and 80% ethanol, and the 80% ethanol eluate was collected. Add 80% of the eluate into pure water to form a 50% alcohol solution, pass it through a neutral alumina column, collect the eluate, wash the column with a small amount of 50% ethanol, and combine the eluent for later use; take the liquid and add activated carbon, Heating under reflux, boiling for 20 minutes, suction filtration while hot, collecting the filtrate, concentrating, and refrigerating overnight; filtering with a clarification plate, drying under reduced pressure to obtain the ginseng extract. 3), take Epimedium medicinal material, add water and decoct and extract 2-5 times, the amount of water added is 10-30 times, the first extraction time is set at 0.5-2h, and the next three times are 0.5-1h; combine the above extracts, Filtrate while it is hot, concentrate under reduced pressure, put it into a stainless steel bucket, add ethanol until the alcohol concentration of the liquid is 70%, refrigerate; filter, recover ethanol from the filtrate and concentrate, add water and stir evenly, refrigerate, filter, and set aside; take epimedium Huoyao liquid passes through the treated AB-8 macroporous adsorption resin column, and is eluted with pure water, 20% ethanol, and 50% ethanol respectively, and the 50% ethanol eluate is collected; the eluent is mixed with an appropriate amount of diatomaceous earth Mix well, extract with absolute ethanol for 2-5 times, combine the extracts, recover the ethanol and concentrate, add acetone to the concentrated solution, and stir evenly, refrigerate, filter, add acetone to the precipitate and treat it twice as before, combine the filtrates three times, The solvent is recovered, concentrated, and dried under reduced pressure to obtain the epimedium extract. 4), take the extracts prepared in 2) and 3), add 1,2 propylene glycol, sodium dihydrogen phosphate, disodium hydrogen phosphate additives, and add water for injection to make up to 10-20ml, heat treatment and refrigeration, and then use micro pore filter membrane, ultrafiltration, filling, and sterilizing to obtain the injection of the present invention. 9. The preparation method of the medicine as claimed in claim 6, characterized in that it comprises the following steps: The extract prepared by steps 2) and 3) in claim 8 is made into capsules, pills, tablets or oral liquids according to conventional preparation methods.