CN1785223A - Medicine for treating muscular dystrophy and myasthenia gravis, and its prepn. method - Google Patents
Medicine for treating muscular dystrophy and myasthenia gravis, and its prepn. method Download PDFInfo
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Abstract
A Chinese medicine for treating the myophagism and myasthenia gravis caused by motor neuron diseases, prograssive myodystrophy and congenital myopathy is prepared from ginseng and epimedium. Its preparing process is also disclosed.
Description
Technical field
The present invention relates to a kind of medicine that is used for the treatment of amyotrophy and myasthenia gravis and preparation method thereof, belong to the Chinese herbal and crude drugs preparations technical field.Be mainly used in diseases such as amyotrophy due to the diseases such as motor neuron disease (amyotrophic lateral sclerosis, progressive spinal myodystrophia, progressive bulbar palsy, primary lateral sclerosis), progressive muscular dystrophy, congenital myopathy and myasthenia gravis clinically.
Background technology
Motor neuron disease (motor neuron disease, MND) comprise amyotrophic lateral sclerosis (amyotrophiclateral sclerosis, ALS), progressive spinal myodystrophia, progressive bulbar palsy, primary lateral sclerosis etc., the extensive degeneration of neuron causes because the central nervous system moves up and down, its symptom has that muscular strength goes down, amyotrophy, and develop into gradually diet choke cough, dysphagia, dyspnea, up to death.ALS is the chronic progressive external degenerative disease that the not bright selectivity of a kind of cause of disease is invaded ventricornu cell, brain stem nervus motorius nuclear and tractus pyramidalis, clinical manifestation is the impaired sign of neuron merging that moves up and down, and is modal type in the chronic exercise neuronal disease.Doctor trained in Western medicine thinks under the situation that the pathogenesis of ALS is not understood fully, not have special therapeutic method at present, mainly be based on supporting treatment to the ill, guarantee enough nutrition, improve General Symptoms, with the psychology support, make it establish the courage and resolution of struggling with disease for a long time to patient.
The traditional Chinese medical science thinks that the muscular atrophy diseases that ALS etc. cause belongs to the myopathy category of the traditional Chinese medical science, and ancients once proposed the viewpoint of " treating flaccidity syndromes have to treat first YANG MING ".Professor Li Renxian thinks that primary disease is this with the deficiency of spleen and stomach, and there is the not smooth pathological factor of blood vessels in stirring-up of pathogenic wind in the interior resulting from deficiency for mark, should serve as the treatment rule with strengthening spleen, tonifying kidney, relieving spasm by subduing liver-wind, promoting blood circulation to remove obstruction in the collateral, faces card from intending strengthening spleen, tonifying kidney side's plus-minus, has obtained certain curative effect; Professor Deng Tietao thinks that the ALS card belongs to the deficiency of spleen-YANG and kidneyYANG folder expectorant folder stasis of blood, for oral administration with the BUZHONG YIQI TANG plus-minus, and quiet and acupoint injection therapy Radix Astragali injection are with strengthening spleen, tonifying kidney, washout side's fumigation and wash method upper limb is with eleminating phlegm and freeing channels, coloclysis amasss just to arrange, be aided with multiple therapies such as acupuncture, massage, diet, feelings will again, also obtained certain curative effect, but all not fully up to expectations.
Summary of the invention
The object of the present invention is to provide a kind of treatment amyotrophy evident in efficacy and medicine of myasthenia gravis and preparation method thereof.
This drug invention people professor Wu Yiling finds in practice, only adopts the Therapeutic Method of function of spleen and stomach regulating, and clinical effectiveness is unsatisfactory.Therefore, inventor professor Wu Yiling at first walks around the inherent pattern of controlling from Treatise on the spleen and stomach, and the feeler of thinking is stretched to meridians, and foothold is placed on the governor vessel of eight extra-channel the most at last.He is that amyotrophic pathogenesis is inquired in the breach with the eight extra-channel, and the treatment rule of the neodoxy that proposition eight extra-channel opinion is controlled and " hold up first Conclusion Decreasing, support flourish granulation promoting " focuses on Wen Liqiyang, grows and fills out very essence, warm logical eight arteries and veins.And from a large amount of Chinese medicine, choose tens flavor replenishing vital QI with drugs of warm nature governor vessels, rouse oneself neural medicine, scientific formula, development cost invention medicine.Drug test of the present invention proves: can resist the inhibitory action of amyotrophic lateral sclerosis patients serum to the teleneuron regeneration capacity; promote the regeneration and the reparation of injured nerve tip; obviously improve the laboratory animal swimming endurance; obvious muscle strength reinforcing effect is arranged; and can make active rising of muscle fatigue mice serum phosphagen creatase be subjected to obvious suppression, the function of skeletal muscle there is significant protective effect.
Medicine of the present invention is to be made by the crude drug of following weight portion ratio:
Radix Ginseng 2-10 part, Herba Epimedii 2-8 part
The weight portion ratio of above-mentioned raw materials medicine is preferred:
5 parts of Radix Ginsengs, 5 parts of Herba Epimedii and:
5 parts of Radix Ginsengs, 2 parts of Herba Epimedii and:
9 parts of Radix Ginsengs, 7 parts of Herba Epimedii and:
3 parts of Radix Ginsengs, 1 part of Herba Epimedii
Medicine of the present invention can be made the acceptable any conventional dosage form of pharmaceutics, for example injection, capsule, pill, tablet, oral liquid etc. by preparation process routinely.
The preparation method of medicine of the present invention may further comprise the steps:
1), get above-mentioned Chinese crude drug, choosing is clean respectively, pulverizes, and presses the weighing of prescription amount;
2), get the ginseng crude drug, with 60-90% ethanol extraction 1-3 time, add medical material 8-12 the 1st time doubly to measure solvent extraction 1-2 hour, add 6-10 the 2nd time doubly to measure solvent extraction 1-2 hour, add 6-10 and doubly measured solvent extraction 0.5-1 hour for the 3rd time; Merge said extracted liquid, filter while hot, decompression recycling ethanol, and concentrate, put into stainless steel cask, add pure water and stir, cold preservation 24-36h; Cold preservation liquid is taken out, put to room temperature, plate filter filters, and washs sheet frame with low amounts of water, and filtrate adds the pure water dilution, and is standby; Get the AB-8 type macroporous adsorptive resins of Radix Ginseng concentrated medicament, and use ammonia, 20% ethanol, 80% ethanol elution of pH value 8-9 respectively, collect 80% ethanol elution by handling well.80% eluent is added pure water, be made into 50% alcoholic solution,, collect eluent by the neutral alumina post, a small amount of 50% washing with alcohol pillar of reuse, it is standby to merge eluent; Taking liquid adds active carbon, and reflux boils 20min, and the sheet frame sucking filtration is collected filtrate while hot, concentrates, and cold preservation is spent the night; The clarification plate filters, and drying under reduced pressure promptly gets Radix Ginseng extract.
3), get epimedium herb, decoct with water and extract 2-5 time, amount of water be 10-30 doubly, the extraction time first time is decided to be 0.5-2h, later 3 times is 0.5-1h; Merge said extracted liquid, filter while hot, concentrating under reduced pressure is put into stainless steel cask, and adding ethanol to medicinal liquid, to contain determining alcohol be 70%, cold preservation; Filter, filtrate recycling ethanol also concentrates, and adds water and stirs, and cold preservation is filtered, and is standby; Get the AB-8 type macroporous adsorptive resins of Herba Epimedii medicinal liquid, and use pure water, 20% ethanol, 50% ethanol elution respectively, collect 50% ethanol elution by handling well; Eluent is admixed an amount of kieselguhr mixing, uses dehydrated alcohol extraction 2-5 time, and extracting solution merges, reclaim ethanol and concentrated, concentrated solution adds acetone, and stirs, cold preservation, filter, precipitation adds acetone again with pre-treatment 2 times, merges 3 times filtrate, reclaim solvent, and concentrate, drying under reduced pressure promptly gets Herba Epimedii extract.
4), get 2) and 3) in the extract of preparation, add 1,2 propylene glycol, sodium dihydrogen phosphate, sodium hydrogen phosphate additives, and adding injection water standardize solution to 10-20ml, microporous filter membrane, ultra-filtration filters are used in heat treatment cold preservation successively, fill, sterilization promptly gets injection of the present invention.
Perhaps
With 2) and 3) the middle extract for preparing, formulation method is made capsule, pill, tablet or oral liquid routinely.
The specific embodiment
Example below in conjunction with the preparation of drug injection of the present invention, capsule, tablet and pill illustrates the specific embodiment of the present invention.
Embodiment 1:
5 parts of Radix Ginsengs, 2 parts of Herba Epimedii
Preparation method:
1), get above-mentioned Chinese crude drug, choosing is clean respectively, pulverizes, and presses the weighing of prescription amount;
2), get the ginseng crude drug, with 70% ethanol extraction 3 times, add 10 times of amounts of medical material solvent extraction 1 hour the 1st time, add 8 times of amounts solvent extraction 1 hour the 2nd time, add 8 times of amounts solvent extraction 0.5 hour the 3rd time; Merge said extracted liquid, filter while hot, concentrating under reduced pressure is put into stainless steel cask, adds pure water and stirs, cold preservation 24h; Cold preservation liquid is taken out, put to room temperature, plate filter filters, and washs sheet frame with low amounts of water, and filtrate adds the pure water dilution, and is standby; Get the AB-8 type macroporous adsorptive resins of Radix Ginseng concentrated medicament, and use ammonia, 20% ethanol, 80% ethanol elution of pH value 8-9 respectively, collect 80% ethanol elution by handling well.80% eluent is added pure water, be made into 50% alcoholic solution,, collect eluent by the neutral alumina post, a small amount of 50% washing with alcohol pillar of reuse, it is standby to merge eluent; Taking liquid adds active carbon, and reflux boils 20min, and the sheet frame sucking filtration is collected filtrate while hot, concentrates, and cold preservation is spent the night; The clarification plate filters, drying under reduced pressure, promptly.
3), get epimedium herb, decoct with water and extract 4 times, amount of water is 20 times, extraction time is decided to be 1h for the first time, later 3 times is 0.5h; Merge said extracted liquid, filter while hot, concentrating under reduced pressure is put into stainless steel cask, and adding ethanol to medicinal liquid, to contain determining alcohol be 70%, cold preservation; Filter, filtrate recycling ethanol also concentrates, and adds water and stirs, and cold preservation is filtered, and is standby; Get the AB-8 type macroporous adsorptive resins of Herba Epimedii medicinal liquid, and use pure water, 20% ethanol, 50% ethanol elution respectively, collect 50% ethanol elution by handling well; Eluent is admixed an amount of kieselguhr mixing, uses dehydrated alcohol extraction 3 times, and extracting solution merges, and reclaims ethanol and concentrates concentrated solution adding acetone, and stir, cold preservation is filtered, and precipitation adds acetone again with pre-treatment 2 times, merges 3 times filtrate, reclaim solvent, and concentrate, drying under reduced pressure, promptly.
4), get 2), 3) in the semi-finished product of preparation, add additives such as 1,2 propylene glycol, sodium dihydrogen phosphate, sodium hydrogen phosphate, and adding injection water standardize solution to 16ml, microporous filter membrane, ultra-filtration filters are used in heat treatment cold preservation successively, fill, sterilization promptly gets injection of the present invention.
Embodiment 2:
5 parts of Radix Ginsengs, 5 parts of Herba Epimedii
Preparation method: with among the embodiment 1 1), 2) 3) step and make capsule according to a conventional method.
Embodiment 3:
9 parts of Radix Ginsengs, 7 parts of Herba Epimedii
Preparation method: with among the embodiment 1 1), 2) 3) step and make tablet according to a conventional method.
Medicine of the present invention, be according to traditional Chinese medicine theory, adopting " holding up first Conclusion Decreasing; support flourish granulation promoting " is the Therapeutic Principle, develop in conjunction with clinical application experience for many years, being intended to provides a kind of determined curative effect, takes safe new product of Chinese medicine for amyotrophy and myasthenia gravis patient due to diseases such as motor neuron disease (amyotrophic lateral sclerosis, progressive spinal myodystrophia, progressive bulbar palsy, primary lateral sclerosis), progressive muscular dystrophy, congenital myopathy.
Take a broad view of drug prescription of the present invention, in sweet flat Radix Ginseng, be equipped with sweet using warming therapy Herba Epimedii in a small amount, make full side on the flat basis of property, both helped nourishing the essence and strengthening QI, nourish Yuanyang, do not make its too warm-dryness syndrome again.Though medicine is two flavors only, clear efficacy, compatibility is rigorous.
Its injection of pharmacological action medicine of the present invention has carried out following animal experiment to prove its curative effect:
The flesh protective effect of clever injection of withering to the In vitro culture motor neuron
[summary] purpose: observe the flesh protective effect of clever injection (JWL) of withering to the In vitro culture motor neuron.Method: use Density Gradient Centrifugation and separate Mus embryo dynamoneure and carry out formerly being commissioned to train fosterly, add glutamic acid and set up the toxicity of excitatory amino acid damage model, estimate the protective effect of clever injection of withering of variable concentrations flesh to motor neuron.Mtt assay detects cell viability, and nervous process trunk length is measured in the row image analysis of going forward side by side of NF-200 immunohistochemical staining, and biochemistry analyzer detects the lactic acid dehydrogenase (LDH) in the culture supernatant, TUNEL positive neuron counting observation of cell apoptosis.The result: (1) flesh clever injection that withers can obviously promote the vigor of In vitro culture motor neuron, promotes the growth of nervous process; (2) the flesh clever injection that withers can reduce spilling of toxicity of excitatory amino acid damage motor neuron LDH; (3) the flesh clever injection that withers can significantly reduce the motor neuron apoptosis of glutamate induction.Conclusion: the flesh clever injection that withers all has protective effect to normal motor neuron and toxicity of excitatory amino acid damage motor neuron.
The reparation of central nervous system injury and protection are the difficult problems of neural area research, still lack effective treatment means and medicine at present.Especially to cause the absolute minimizing of quantity be irreversible for the damage of motor neuron or degeneration death, makes the function corresponding forfeiture, has a strong impact on patient moving function and life quality.The proper motion neuron that remains of protection, it is significant to the treatment central nervous system injury to alleviate the damage that the degeneration neuron is subjected to.The flesh clever injection that withers is the new product of Chinese medicine of the treatment neuromuscular system disease of professor Wu Yiling development, obtains tangible curative effect in diseases such as clinical treatment motor neurons.The cell in vitro culture technique is used in this research, observes flesh and withers clever injection to the effect of motor neuron, is intended to seek effective medicine for the clinical treatment central nervous system injury.
1. material and method
1.1 experiment material
The clever injection 1.1.1 medicine flesh withers is produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd, and 8ml/ props up (every ml contains crude drug 0.9g), preparation number: stone is defended medicament word (98) 501-00296 that goes out.This medical instrument holds up unit to play flaccidity, supports the effect of flourish granulation promoting.Rilutek
RRiluzole Tablet is produced by Aventis company, imported medicine registration certificate number: H200020006, and lot number: 28097, dissolve through 0.9%NaCl-0.01NHCl during application.
1.1.2 reagent L15 culture medium, butanediamine, trypsin 1: 250), poly-D-lysine, the anti-NF-200 antibody of rabbit, insulin, DAB developer, MTT, DMSO purchase the company in Sigma, horse serum and Laminin are provided by Gibco BRL company, hyclone is the Hyclone product, the TUNEL test kit is a Roche molecular Biochemical product, biotin labeling two resists and resists with horseradish peroxidase-labeled three is Beijing Zhong Shan biological product company limited product, and surplus reagent is homemade analytical pure.
1.1.3 the female pregnant Mus of animal cleaning level SD, pregnancy period 10～14d is provided by big level ground hospital of Chongqing Third Military Medical University aseptic sursery institute Experimental Animal Center.
1.1.4 instrument microplate reader (DG3022A type, Shanghai), image analysis system (Image Pro Plus4.5 type, the U.S.), automatic clinical chemistry analyzer (BechMan CX7 type, the U.S.).
1.2 method
1.2.1 tire Mus dynamoneure is former be commissioned to train foster
Get the tire Mus about pregnant 14d, below the brain stem plane, take out spinal cord, be cut into 1～2mm after peelling off spinal meninges and blood vessel
3Fritter, trypsinization 30min with 0.125%, 37 ℃, cross 200 order steel meshes, filtered solution moves into and presets in the centrifuge tube of 1ml 4%BSA (bovine serum albumin), the centrifugal 10min of 300g removes supernatant, L15 culture medium dissolution precipitation, add and preset in the 5ml centrifuge tube of 1ml 6.8%Metrizamide, the centrifugal 15min of 500g, light carrying liqs between sucking-off two liquid levels, the centrifugal 10min of 300g, remove supernatant, the L15 planted medium is dissolved, and promptly gets the single cell suspension of ventricornu neurocyte, and adjusting concentration is 4.5 * 10
5/ ml plants in 24 well culture plates and 96 well culture plates, puts CO
2Cultivate in the incubator, change behind the 24h and keep culture medium, add 12.5 μ g/ml 5-Fu effect 24h sucking-offs behind the 48h, renew the bright culture medium of keeping.
1.2.2 experiment condition control:
After motor neuron is cultivated 72h, it is 0.5%, 1%, 5% the flesh clever injection that withers that experimental group adds with fresh culture dilution respectively, the Rilutek group adds riluzole (final concentration 10 μ mol/L), matched group adds equivalent culture medium co-cultivation 24h, and the toxicity of excitatory amino acid damage adds the laggard row index of glutamic acid (final concentration is 0.5mmol/L) effect 24h and detects.
Mtt assay is measured cell viability:
With the careful culture fluid that goes in 96 orifice plates of inhaling of suction pipe, every hole adds MTT solution (concentration 5mg/mL) 10 μ L, 37 ℃, 5% humidifying CO
2Behind the reaction 4h, remove supernatant, add solvent dimethyl sulfoxide (DMSO) 100 μ L/ holes, place on the miniature oscillator under the room temperature and shake 5min, the full-automatic microplate reader in quality product dissolving back is measured wavelength X=570nm, and reference wavelength λ=630nm measures the OD value.
The NF-200 immunohistochemical staining:
After cultivating the 6d cell routine and fixing, seal, add the anti-NF-200 monoclonal antibody of rabbit (1: 1000), 24h (4 ℃), add biotin labeled two anti-IgG (1: 300), 60min (37 ℃) adds the strepto-avidin (1: 300) of horseradish peroxidase-labeled, 60min ((37 ℃) again.More than all use 0.01mol/LPBS rinsing 5min totally 3 times between each step.DAB solution colour developing 5min, gradient alcohol dehydration, dimethylbenzene is transparent, the neutral gum mounting.Negative control group replaces the NF-200 antiserum with PBS and carries out immunohistochemical reaction.Get each 2 of coverslipes respectively, every cover plate is got 50 NF-200 positive neurons at random, and the Quantimet-a image analysis system is measured its projection trunk length
LDH detects:
Take out 24 orifice plate cultured cells, carefully draw culture supernatant, packet numbering.Detect lactic dehydrogenase enzyme activity in the culture supernatant respectively with automatic clinical chemistry analyzer.
TUNEL detects:
After the cultured cell routine is fixing, 0.3%H
2O
2/ methanol sealing 30min, add the 0.1%Triton-0.1% liquor sodii citratis and place 2min on ice, add end deoxyribonucleic acid transferase (TdT) and fluorescein-labeled dUTP, hatch 2h for 37 ℃, the anti-fluorescein antibody of horseradish peroxidase is hatched 2h for 37 ℃, the NBT/BCIP colour developing, more than all use 0.01M PBS rinsing 3 times, each 5min, dehydration between each step, transparent, mounting.Negative control group does not add the TdT enzyme.Each experimental group repeats 3 times, counts the TUNEL stained positive neuron in 5 visuals field at random.
Statistical analysis:
(x ± s) expression, relatively with the t check, the group difference significance detects and adopts variance analysis data between mean with mean ± standard deviation.
2. result
2.1 dynamoneure morphological observation
Beginning is adherent behind the neuron inoculation 4h, and cell is single circle or ellipse, and plantation 24h has seen that the componental movement neuron grows projection, and 1%JWL group motor neuron projection is obviously longer.Adherent cell is obviously grown up after cultivating 48～72h, engenders particulate material in the cell space, and cell week is dizzy clear, and projection increases prolongation, and the whole visual field inner process is crisscross after the 96h.The cell of motor neuron growth 5～6d is the most plentiful, and refractivity is strong, and karyon is positioned at cell central authorities or relatively on one side, and karyon is shallow brightly to be separated easily with perikaryon, and karyon and kernel are high-visible.The motor neuron that matched group is cultivated 5d adds glutamic acid 24h rearward projection retraction, and 1% flesh withers, and to cultivate neure growth good for clever injection, and density is moderate, and visible projection is thick and intermesh.More apoptotic body appears in the TUNEL visible glutamic acid matched group motor neuron apoptosis that dyes, and 1% flesh withers, and apoptotic body obviously reduces in the clever injection group.
Clever injection is to the influence (table 1) of normal motor neuron growth 2.2 flesh withers
0.5%, 1%, 5%JWL group motor neuron cell viability has significant difference (P＜0.05) with matched group, wherein 1%JWL organizes cell viability and obviously is better than Rilutek group (P＜0.05); 0.5% and 1%JWL group dynamoneure enation obviously be better than matched group (P＜0.05), and significant difference (P＜0.05) is arranged with the Rilutek group.
Table 1 flesh withers clever injection to the influence of normal motor neuron vigor and projection (x ± s)
Tab 1 The effect of JWL injection on vigor and outgrowth of normal motor neuron
| Grouping Group | Concentration Density | OD value OD value | Projection (L, μ m) Outgrowth |
| Matched group Rilutek group 0.5%JWL group 1%JWL group 5%JWL group | 10umol/L 4.5μg/L 9μg/L 45μg/L | 0.230±0.130 0.266±0.141 0.317±0.054 1 0.396±0.087 1，2 0.329±0.097 1 | 159.71±48.95 192.08±89.07 315.96±32.32 1，2 373.46±80.24 1，2 151.46±69.97 |
1. compare P＜0.05 with matched group; 2.P＜0.05 with the Rilutek group relatively
1.P＜0.05 vs control group；2.P＜0.05 vs riluzole group
The influence (table 2) that clever injection spills toxicity of excitatory amino acid damage motor neuron LDH 2.3 flesh withers
Testing result O.5%JWL with Rilutek group culture supernatant in LDH all be lower than matched group (p＜0.05), there was no significant difference (p＞0.05) between the two illustrates that both can reduce glutamic acid damage motor neuron and cause spilling of LDH in the cell.
The table 2 flesh influence that clever injection spills the toxicity of excitatory amino acid damage motor neuron LDH (x ± s) that withers
Table 2 The effect of JWL injection on LDH leakage of neurotoxicity motor neurons
| Grouping Group | Concentration Density | LDH Lactic dehydrogenase |
| Glutamic acid matched group Rilutek+glutamic acid group 0.5%JWL+ glutamic acid group 1%JWL+ glutamic acid group 5%JWL+ glutamic acid group | 10umol/L 4.5μg/L 9μg/L 45μg/L | 67.75±5.12 58.50±4.20 1 59.00±4.97 1 61.50±3.11 69.00±5.35 2 |
1. compare P＜0.05 with matched group; 2.P＜0.05 with the Rilutek group relatively
1.P＜0.05 vs control group；2.P＜0.05 vs riluzole group
Clever injection is to the toxicity of excitatory amino acid damage apoptotic influence of motor neuron (table 3) 2.4 flesh withers
1%JWL group and Rilutek group TUNEL positive cell are less than matched group (p＜0.05).0.5% and 5%JWL group TUNEL positive cell reduce but with matched group there was no significant difference (p＞0.05), with Rilutek group variant (p＜0.05).
Table 3 flesh withers clever injection to the apoptotic influence of toxicity of excitatory amino acid damage motor neuron (x ± s)
Table 2 The effect of JWL injection on apoptosis of neurotoxicity motor neurons
| Grouping Group | Concentration Density | TUNEL positive cell number Cell number of TUNEL positive |
| Glutamic acid matched group Rilutek+glutamic acid group 0.5%JWL+ glutamic acid group 1%JWL+ glutamic acid group 5%JWL+ glutamic acid group | 10umol/L 4.5μg/L 9μg/L 45μg/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. compare P＜0.05 with matched group; 2.P＜0.05 with the Rilutek group relatively
1.P＜0.05 vs control group；2.P＜0.05 vs riluzole group
3. discuss
Chinese medicine has certain advantage in central nervous system's repair process.The flesh clever injection that withers is the new product of Chinese medicine of the treatment neuromuscular system disease that is developed under traditional Chinese medical science eight extra-channel theoretical direction of Wu Yiling professor, is made up of medicines such as Radix Ginsengs, has and holds up unit to play flaccidity, the effect of foster flourish granulation promoting.Experimental studies have found that the flesh clever injection that withers can obviously improve the motor function of experimental autoimmune motor neuron (EAMND) model mice, the anterior horn motor neurons of damaged is had protection and improvement effect.
Glutamic acid plays important physiological action as excitatory neurotransmitter in the central nervous system, but in cerebral infarction, amyotrophic lateral sclerosis diseases such as (ALS), glutamic acid can the nervous pathway abnormal activation and produce excitatory toxicity, is the key factor that causes neuronal damage.The development of glutamic acid inhibitor has been opened up new approach for the clinical treatment nervous system disease.Rilutek is the new drug of the treatment ALS that passes through of U.S. FDA, and its main pharmacological is the antagonism toxicity of excitatory amino acid, the clinical research advancing of disease of can slowing down, but cost an arm and a leg and can not cure ALS and obviously improve patient's symptom.This research will be used density In vitro culture motor neuron; add glutamic acid and set up the toxicity of excitatory amino acid damage model; with the contrast medicine of Rilutek as anti-toxicity of excitatory amino acid, research flesh withers clever injection to protective effect normal and toxicity of excitatory amino acid damage motor neuron.
3.1 the motor neuron In vitro culture is to observe medicine acts on curative effect to the central nervous system important means
Because central nervous system's particularity, clinical and experimentation can not be observed motor neuron intuitively.It is to use more a kind of experimental technique and means in recent years that cell in vitro is cultivated, and can observe the comparative drug curative effect effectively, and further inquire into mechanism of action.This research is used density and is separated Mus embryo dynamoneure and carry out formerly being commissioned to train fosterly, has set up and has observed the external model of medicine to the motor neuron effect.In order to obtain the motor neuron of certain purity, take two measures: the one, the centrifuge tube of built-in 6.8%Metrizamide has formed a complete density gradient system, the motor neuron that volume is bigger after centrifugal is big because of buoyancy, on float on the top of Metrizamide; The 2nd, after cultivating 24h, add 5-Fu, use the hypertrophy that mitotic inhibitor reduces neurogliocyte.The motor neuron purity of using above method acquisition can reach 85%, and growth activity is uninfluenced, and growth is stable, helps pharmaceutically-active observation.
Clever injection promotes the neuronic growth of proper motion 3.2 flesh withers
Discover that when clinical symptoms appearred in patients with motor neuron disease, its motor neuron reduced more than 50%, even reached 80%, and its minimizing speed approximately is to reduce half in per 6 months.Because motor neuron in case damage can not repair, therefore, the proper motion neuron that remains keep the patient moving function and effectively compensatory in play an important role.This result of study shows, the flesh clever injection that withers can significantly strengthen normal cultivation motor neuron vigor, and promotes dynamoneure enation (P＜0.05).Because the main mechanism of action of Rilutek is the antagonism toxicity of excitatory amino acid, and normal motor neuron is not seen the effect of increase cell viability, this result of study is consistent with document.Wither clever injection of 0.5% and 1% flesh promotes the neurite-outgrowth effect obviously to be better than Rilutek (P＜0.05).Wither clever injection of this explanation flesh promotes the neuronic vigor of proper motion, and it promotes that the effect of neurite-outgrowth is significant to the functional compensation of central nervous system injury.
Clever injection alleviates the damage of toxicity of excitatory amino acid to motor neuron 3.3 flesh withers
Wither clever injection to the protective effect of toxicity of excitatory amino acid damage motor neuron in order to study flesh, at first handle and cultivate motor neuron, add glutamic acid then and set up the toxicity of excitatory amino acid damage model with the flesh clever injection that withers.LDH is intracellular a kind of marker enzyme, burst size is few under the normal condition, the corresponding increase of LDH burst size behind neuronal damage, therefore the LDH content of measuring in the culture fluid can the neuronic extent of damage of quantitative assessment, and the flesh that the found that doses clever injection of withering can alleviate spilling of cell lactic acid dehydrogenase.But in In vitro culture, when drug dose was too high, pair cell may have certain damaging action on the contrary.Think at present, neuron under the effect of low dosage excitatory amino acid mainly with the death of apoptosis mode, and when high dose mainly with downright bad mode death.Originally after discovering adding glutamic acid, occurring the TUNEL positive cell in the cultured cell, is the sign of glutamate induction motor neuron apoptosis.The spirit of withering of 1% flesh can reduce the TUNEL positive cell number, illustrates that the flesh spirit of withering can resist the neuronal cell apoptosis of glutamate induction.
Originally studies show that; the flesh clever injection that withers both can strengthen proper motion neuron vigor; promote neurite-outgrowth; can protect toxicity of excitatory amino acid damage motor neuron again; reduce apoptosis; keeping central nervous system function, delay PD, improve patient's life quality aspect and have important clinical application value.
Claims (9)
1, a kind of medicine for the treatment of amyotrophy and myasthenia gravis is characterized in that being being made by the crude drug of following weight portion ratio:
Radix Ginseng 1-10 part, Herba Epimedii 1-10 part.
2, medicine according to claim 1, the weight portion ratio of its crude drug is:
5 parts of Radix Ginsengs, 5 parts of Herba Epimedii.
3, medicine according to claim 1, the weight portion ratio of its crude drug is:
5 parts of Radix Ginsengs, 2 parts of Herba Epimedii.
4, medicine according to claim 1, the weight portion ratio of its crude drug is:
9 parts of Radix Ginsengs, 7 parts of Herba Epimedii.
5, medicine according to claim 1, the weight portion ratio of its crude drug is:
3 parts of Radix Ginsengs, 1 part of Herba Epimedii.
6,, it is characterized in that this medicine is injection, capsule, pill, tablet or oral liquid according to the arbitrary described medicine of claim 1-5.
7, medicine according to claim 6 is characterized in that this medicine is an injection.
8, the preparation method of the described medicine of claim 7 is characterized in that may further comprise the steps:
1), get above-mentioned Chinese crude drug, choosing is clean respectively, pulverizes, and presses the weighing of prescription amount;
2), get the ginseng crude drug, with 60-90% ethanol extraction 1-3 time, add medical material 8-12 the 1st time doubly to measure solvent extraction 1-2 hour, add 6-10 the 2nd time doubly to measure solvent extraction 1-2 hour, add 6-10 and doubly measured solvent extraction 0.5-1 hour for the 3rd time; Merge said extracted liquid, filter while hot, decompression recycling ethanol, and concentrate, put into stainless steel cask, add pure water and stir, cold preservation 24-36h; Cold preservation liquid is taken out, put to room temperature, plate filter filters, and washs sheet frame with low amounts of water, and filtrate adds the pure water dilution, and is standby; Get the AB-8 type macroporous adsorptive resins of Radix Ginseng concentrated medicament, and use ammonia, 20% ethanol, 80% ethanol elution of pH value 8-9 respectively, collect 80% ethanol elution by handling well.80% eluent is added pure water, be made into 50% alcoholic solution,, collect eluent by the neutral alumina post, a small amount of 50% washing with alcohol pillar of reuse, it is standby to merge eluent; Taking liquid adds active carbon, and reflux boils 20min, and the sheet frame sucking filtration is collected filtrate while hot, concentrates, and cold preservation is spent the night; The clarification plate filters, and drying under reduced pressure promptly gets Radix Ginseng extract.
3), get epimedium herb, decoct with water and extract 2-5 time, amount of water be 10-30 doubly, the extraction time first time is decided to be 0.5-2h, later 3 times is 0.5-1h; Merge said extracted liquid, filter while hot, concentrating under reduced pressure is put into stainless steel cask, and adding ethanol to medicinal liquid, to contain determining alcohol be 70%, cold preservation; Filter, filtrate recycling ethanol also concentrates, and adds water and stirs, and cold preservation is filtered, and is standby; Get the AB-8 type macroporous adsorptive resins of Herba Epimedii medicinal liquid, and use pure water, 20% ethanol, 50% ethanol elution respectively, collect 50% ethanol elution by handling well; Eluent is admixed an amount of kieselguhr mixing, uses dehydrated alcohol extraction 2-5 time, and extracting solution merges, reclaim ethanol and concentrated, concentrated solution adds acetone, and stirs, cold preservation, filter, precipitation adds acetone again with pre-treatment 2 times, merges 3 times filtrate, reclaim solvent, and concentrate, drying under reduced pressure promptly gets Herba Epimedii extract.
4), get 2) and 3) in the extract of preparation, add 1,2 propylene glycol, sodium dihydrogen phosphate, sodium hydrogen phosphate additives, and adding injection water standardize solution to 10-20ml, microporous filter membrane, ultra-filtration filters are used in heat treatment cold preservation successively, fill, sterilization promptly gets injection of the present invention.
9, the preparation method of the described medicine of claim 6 is characterized in that may further comprise the steps:
With step 2 in the claim 8) and 3) preparation extract, formulation method is made capsule, pill, tablet or oral liquid routinely.
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| CN102288686A (en) * | 2010-06-17 | 2011-12-21 | 河北以岭医药研究院有限公司 | Method for measuring residual amount of organic solvent in traditional Chinese medicine freeze-drying injection |
| CN102451209A (en) * | 2010-10-29 | 2012-05-16 | 河北以岭医药研究院有限公司 | Application of Chinese medicine composition to preparation of medicines for neuroprotection after spinal cord injury |
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| CN103301178A (en) * | 2013-06-08 | 2013-09-18 | 中国人民解放军第四军医大学 | Fatigue syndrome prevention and treatment oral drug |
| CN102091114B (en) * | 2009-12-15 | 2014-11-26 | 河北以岭医药研究院有限公司 | Traditional Chinese medicament freeze-drying injection and preparation method thereof |
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| CN1253011A (en) * | 1998-11-04 | 2000-05-17 | 刘相章 | Chinese medicine for treating myasthenia and its preparing process |
| CN1199656C (en) * | 2000-09-08 | 2005-05-04 | 姜东海 | Medicine for treating myashthenia gravis |
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| CN102288686A (en) * | 2010-06-17 | 2011-12-21 | 河北以岭医药研究院有限公司 | Method for measuring residual amount of organic solvent in traditional Chinese medicine freeze-drying injection |
| CN102451209A (en) * | 2010-10-29 | 2012-05-16 | 河北以岭医药研究院有限公司 | Application of Chinese medicine composition to preparation of medicines for neuroprotection after spinal cord injury |
| CN102451209B (en) * | 2010-10-29 | 2015-04-15 | 河北以岭医药研究院有限公司 | Application of Chinese medicine composition to preparation of medicines for neuroprotection after spinal cord injury |
| WO2012100408A1 (en) * | 2011-01-25 | 2012-08-02 | 河北以岭医药研究院有限公司 | Dextrin inclusion complex, preparation method thereof and pharmaceutical formulation comprising said inclusion complex |
| CN103301178A (en) * | 2013-06-08 | 2013-09-18 | 中国人民解放军第四军医大学 | Fatigue syndrome prevention and treatment oral drug |
| CN103301178B (en) * | 2013-06-08 | 2016-12-28 | 中国人民解放军第四军医大学 | Prevention and the oral drugs for the treatment of fatigue syndrome |
| CN106177629A (en) * | 2016-08-22 | 2016-12-07 | 王延林 | A kind of Chinese and Western compound medicine treating progressive spinal muscular atrophy and preparation method |
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| CN100353951C (en) | 2007-12-12 |
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