CN102600374A - Medicine for preventing parkinsonism and application of medicine - Google Patents
Medicine for preventing parkinsonism and application of medicine Download PDFInfo
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Abstract
The invention relates to a traditional Chinese medicine and application in terms of preparation of the medicine for preventing parkinsonism. The medicine is prepared by following bulk drug including, by weight, from 5 to 10 parts of gastrodia elata, from 5 to 10 parts of astragalus mongholicus, from 5 to 10 parts of prepared rehmannia roots, from 5 to 15 parts of radix paeoniae alba, from 5 to 10 parts of Chinese angelica, from 5 to 10 parts of uncaria, from 2 to 10 parts of bombyx batryticatus and from 2 to 10 parts of rhizoma arisaematis. The traditional Chinese medicine and the application thereof have the advantages that compatibility of the traditional Chinese medicine meets a traditional Chinese medicine 'assistant and guide' principle, medicine types are fewer, the traditional Chinese medicine is high in curative effect, free of toxic and side effects, cheap in price and the like, adopts rules including a rule of tonifying qi to promote blood circulation, a rule of nourishing yin for suppressing hyperactive yang, a rule of calming liver to stop endogenous wind, a rule of massaging muscles to stop endogenous wind, a rule of promoting blood circulation to remove blood stasis, a rule of dredging collaterals to stop endogenous wind and the like, aims to prevent pathogenic foundation, suits the remedy to the case so as to realize a prevention effect, is simple in preparation method and low in cost, is environment-friendly, is suitable for being orally taken for a long time, is a traditional Chinese medicine composition which safely and effectively prevents the parkinsonism and parkinsonism motion complications, and has a fine application prospect.
Description
Technical field
The present invention relates to a kind of Chinese medicine medicine, specifically, is Parkinsonian Chinese medicine composition medicine of a kind of prevention and application thereof.
Background technology
Parkinson disease (Parkinson ' s disease, PD) be the common nervous system degeneration illness of middle-aged and elderly people, mainly reach the nigrostriatum path dopamine mediator that causes thus and reduce relevant with black substance dense area dopaminergic neuron degeneration disappearance.Through clinical practice for many years, it is generally acknowledged that levodopa is still the most effectively medicine.But symptom fluctuation, the dyskinesia and mental symptom can appear in most of patient after the prolonged application.In addition laboratory find the high concentration dopamine (dopamine, DA) with levodopa because autoxidation produces free radical, can cause the neurocyte degeneration necrosis.Therefore, the associating other drug prevents PD and PD motor complication very urgent jointly.
In addition, discover that the development of striatum GRKs and Arrestins change of Expression and PD is in close relations.In recent years research shows; The generation of parkinson disease motor complication is activated in close relations with the direct path and the signal transduction pathways such as downstream cAMP deopendent protein kinases (PKA), ERK thereof of expressing the D1 receptor; Phosphorylated protein-32 (the dopamine and cAMP-regulated phosphoprotein of Mr 32000 that its downstream signal transducin dopamine and adenosine cyclophosphate are regulated; The change of albumen Thr75 site Expression of phosphorylated DARPP-32) possibly participated in unusual fluctuation disease (1evodopa-induced dyskinesia, morbidity LID).
Think at present and use levodopa controlled release agent, dopamine-receptor stimulant, monoamine oxidase B (monoamine oxidase-B type; MAO-B) inhibitor and catechol-oxygen position-methyltransferase inhibitors (eateehol-O-methyl transferase inhibitor, but the COMTI) appearance of delayed motion complication.But these medicines often side effect are bigger, form drug resistance easily, should not take for a long time.China is accumulating rich experiences aspect the Chinese medicine prevention PD, and summing up its prevention principle is the benefiting QI for activating blood circulation method, the nourishing YIN for attracting YANG method, and the suppressing the hyperactive liver to relieve the wind syndrome method is rubbed the muscle method that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., activating blood and removing stasis Method and collateral dredging rule such as relieve dizziness, high fever, infantile convulsions, epilepsy, etc.
Chinese patent document CN100409867C discloses a kind of capsule that only quivers that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., and the herbal mixture of being made up of the Radix Paeoniae Alba, Ramulus Uncariae Cum Uncis etc. is provided, and prevention PD improves tremble symptom, myotonia, akinesia shape.Li Min etc. have proposed invigorating kidney, promoting blood circulation Chinese medicine can improve PD patient moving complication symptom, and curative effect with the prevention time lengthening increase, this invigorating kidney, promoting blood circulation Chinese medicine is by Fructus Corni 10g; Radix Polygoni Multiflori 15g; Radix Salviae Miltiorrhizae 15g, Chinese medicines such as Hirudo 6g form, but not disclose other Chinese medicine ingredients (see for details: Li Min etc. the invigorating kidney, promoting blood circulation granule prevents the clinical research of parkinson disease motor complication. Chinese Chinese medicine magazine; 2011,26 (6): 1296-1299.).But also do not appear in the newspapers at present about Parkinsonian Chinese medicine composition medicine of a kind of prevention and application thereof.
Summary of the invention
The objective of the invention is to deficiency of the prior art, the application of a kind of Chinese medicine medicine in the Parkinsonian disease medicament of preparation prevention is provided.
One purpose more of the present invention is to provide a kind of prevention Parkinsonian Chinese medicine medicine.
For realizing above-mentioned purpose; The technical scheme that the present invention takes is: the application of a kind of Chinese medicine medicine in the Parkinsonian disease medicament of preparation prevention, described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 5-10 part, Radix Astragali 5-10 part, Radix Rehmanniae Preparata 5-10 part, Radix Paeoniae Alba 5-15 part, Radix Angelicae Sinensis 5-10 part, Ramulus Uncariae Cum Uncis 5-10 part, Bombyx Batryticatus 2-10 part, Rhizoma Arisaematis 2-10 part.
Described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 6-8 part, Radix Astragali 6-8 part, Radix Rehmanniae Preparata 6-8 part, Radix Paeoniae Alba 8-13 part, Radix Angelicae Sinensis 6-8 part, Ramulus Uncariae Cum Uncis 6-8 part, Bombyx Batryticatus 4-6 part, Rhizoma Arisaematis 4-6 part.
Described medicine is processed by following bulk drugs: 8 parts in Rhizoma Gastrodiae, 8 parts of the Radixs Astragali, 8 parts of Radix Rehmanniae Preparata, 10 parts of Radix Paeoniae Alba, 8 parts of Radix Angelicae Sinensis, 8 parts of Ramulus Uncariae Cum Uncis, 5 parts of Bombyx Batryticatus, 5 parts of Rhizoma Arisaematiss.
The application of described medicine in the disease medicament of preparation prevention parkinson disease motor complication.
For realizing above-mentioned second purpose; The technical scheme that the present invention takes is: the Parkinsonian Chinese medicine medicine of a kind of prevention, described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 5-10 part, Radix Astragali 5-10 part, Radix Rehmanniae Preparata 5-10 part, Radix Paeoniae Alba 5-15 part, Radix Angelicae Sinensis 5-10 part, Ramulus Uncariae Cum Uncis 5-10 part, Bombyx Batryticatus 2-10 part, Rhizoma Arisaematis 2-10 part.
Described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 6-8 part, Radix Astragali 6-8 part, Radix Rehmanniae Preparata 6-8 part, Radix Paeoniae Alba 8-13 part, Radix Angelicae Sinensis 6-8 part, Ramulus Uncariae Cum Uncis 6-8 part, Bombyx Batryticatus 4-6 part, Rhizoma Arisaematis 4-6 part.
Described medicine is processed by following bulk drugs: 8 parts in Rhizoma Gastrodiae, 8 parts of the Radixs Astragali, 8 parts of Radix Rehmanniae Preparata, 10 parts of Radix Paeoniae Alba, 8 parts of Radix Angelicae Sinensis, 8 parts of Ramulus Uncariae Cum Uncis, 5 parts of Bombyx Batryticatus, 5 parts of Rhizoma Arisaematiss.
The application of described medicine in the disease medicament of preparation prevention parkinson disease motor complication.
The dosage form of described medicine is tablet, capsule, granule, mixture, oral liquid or syrup.
The invention has the advantages that:
1, Chinese medicine composition medicine of the present invention, its compatibility meet Chinese medicine " monarch " principle, and the flavour of a drug number is less; Advantages such as having the curative effect height, have no side effect, price is low adopts the benefiting QI for activating blood circulation method, the nourishing YIN for attracting YANG method; The suppressing the hyperactive liver to relieve the wind syndrome method is rubbed the muscle method that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., activating blood and removing stasis Method and collateral dredging rule such as relieve dizziness, high fever, infantile convulsions, epilepsy, etc.; Start with from cause of disease at all, suit the remedy to the case, to reach preventive effect;
2, method for preparing is simple, and cost is low, and is environmentally friendly, is suitable for taking for a long time, is a kind of Chinese medicine composition that prevents parkinson disease and parkinson disease motor complication safely and effectively, has a good application prospect.
Description of drawings
Accompanying drawing 2 is the influences to PD rat model agent peak number of revolutions of various dose Chinese medicine composition medicine, and # Western medicine group and small dose group compare, all P < 0.05; * middle dose groups and small dose group compare, all P < 0.01; + heavy dose of group compares with middle dose groups, all P < 0.05.
Accompanying drawing 3 is Chinese medicine composition medicine various dose SABC results to the influence of PD rat model striatum district GRK6, and 1 is sham operated rats, and 2 is the PD matched group, and 3 is Western medicine group, and 4 is the TCM-small dose group, and 5 is dose groups among the TCM-, and 6 is the heavy dose of group of TCM-; U is not for damaging side, and L is the damage side; * PD matched group and sham operated rats relatively P 0.01, and # Western medicine group and PD matched group relatively P 0.05, and+TCM group and Western medicine group relatively, equal P < 0.05.
Accompanying drawing 4 is Chinese medicine composition medicine various dose Western results to the influence of PD rat model striatum district GRK6, and 1,2 is sham operated rats, and 3,4 is the PD matched group; 5,6 is Western medicine group; 7,8 is the TCM-small dose group, and 9,10 is dose groups among the TCM-, and 11,12 is the heavy dose of group of TCM-; 1,3,5,7,9,11 for not damaging side, and 2,4,6,8,10,12 are the damage side; * compare with sham operated rats, p < 0.01; # and PD matched group compare, and p < 0.05; + TCM and Western medicine group compare, and p < 0.01.
Accompanying drawing 5 is Chinese medicine various dose SABC results to the influence of PD motor complication rat model striatum district β-arrestin1, and 1 is sham operated rats, and 2 is the PD matched group; 3 is Western medicine group; 4 is the TCM-small dose group, and 5 is dose groups among the TCM-, and 6 is the heavy dose of group of TCM-; U is not for damaging side, and L is the damage side.
Accompanying drawing 6 is Chinese medicine various dose Western results to the influence of PD motor complication rat model striatum district β-arrestin1; 1,2 is sham operated rats; 3,4 is the PD matched group, and 5,6 is Western medicine group, and 7,8 is the TCM-small dose group; 9,10 is dose groups among the TCM-, and 11,12 is the heavy dose of group of TCM-; 1,3,5,7,9,11 for not damaging side, and 2,4,6,8,10,12 are the damage side.
Accompanying drawing 7 is Chinese medicine various dose SABC results to the influence of PD motor complication rat model striatum district phosphorylation DARPP-32 (Thr75) expression, the A sham operated rats, and B is the PD matched group; C is a Western medicine group; D is the TCM-small dose group, and E is a dose groups among the TCM-, and F is the heavy dose of group of TCM-.
Accompanying drawing 8 is Chinese medicine various dose Western results to the influence of PD motor complication rat model striatum district phosphorylation DARPP-32 (Thr75) expression; 1 is sham operated rats; 2 is the LID matched group, and 3 is Western medicine group, and 4 is the TCM-small dose group; 5 is dose groups among the TCM-, and 6 is the heavy dose of group of TCM-.
Accompanying drawing 9 is Chinese medicine various dose SABC results to the influence of PD motor complication rat model striatum district phosphorylation ERK1/2 expression, the A sham operated rats, and B is the PD matched group; C is a Western medicine group; D is the TCM-small dose group, and E is a dose groups among the TCM-, and F is the heavy dose of group of TCM-.
Accompanying drawing 10 is Chinese medicine various dose Western results to the influence of PD motor complication rat model striatum district phosphorylation ERK1/2 expression, and 1 is sham operated rats, and 2 is the PD matched group, and 3 is the TCM-small dose group, and 4 is dose groups among the TCM-, and 5 is the heavy dose of group of TCM-.
The specific embodiment
Below in conjunction with accompanying drawing the specific embodiment provided by the invention is elaborated.
The present invention provides a kind of prevention Parkinsonian Chinese medicine medicine, and described Chinese medicine medicine is processed by following bulk drugs: Rhizoma Gastrodiae 5-10 part, Radix Astragali 5-10 part, Radix Rehmanniae Preparata 5-10 part, Radix Paeoniae Alba 5-15 part, Radix Angelicae Sinensis 5-10 part, Ramulus Uncariae Cum Uncis 5-10 part, Bombyx Batryticatus 2-10 part, Rhizoma Arisaematis 2-10 part.The application of described Chinese medicine medicine in the Parkinsonian disease medicament of preparation prevention.
The preparation (one) of embodiment 1 Chinese medicine composition medicine
5 parts in Rhizoma Gastrodiae, 10 parts of the Radixs Astragali, 5 parts of Radix Rehmanniae Preparata, 15 parts of Radix Paeoniae Alba, 5 parts of Radix Angelicae Sinensis, 10 parts of Ramulus Uncariae Cum Uncis, 2 parts of Bombyx Batryticatus, 10 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (two) of embodiment 2 Chinese medicine composition medicines
6 parts in Rhizoma Gastrodiae, 9 parts of the Radixs Astragali, 6 parts of Radix Rehmanniae Preparata, 6 parts of Radix Paeoniae Alba, 6 parts of Radix Angelicae Sinensis, 9 parts of Ramulus Uncariae Cum Uncis, 6 parts of Bombyx Batryticatus, 8 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (three) of embodiment 3 Chinese medicine composition medicines
7 parts in Rhizoma Gastrodiae, 7 parts of the Radixs Astragali, 7 parts of Radix Rehmanniae Preparata, 12 parts of Radix Paeoniae Alba, 7 parts of Radix Angelicae Sinensis, 7 parts of Ramulus Uncariae Cum Uncis, 4 parts of Bombyx Batryticatus, 4 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (four) of embodiment 4 Chinese medicine composition medicines
8 parts in Rhizoma Gastrodiae, 8 parts of the Radixs Astragali, 8 parts of Radix Rehmanniae Preparata, 10 parts of Radix Paeoniae Alba, 8 parts of Radix Angelicae Sinensis, 8 parts of Ramulus Uncariae Cum Uncis, 5 parts of Bombyx Batryticatus, 5 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (five) of embodiment 5 Chinese medicine composition medicines
9 parts in Rhizoma Gastrodiae, 6 parts of the Radixs Astragali, 9 parts of Radix Rehmanniae Preparata, 8 parts of Radix Paeoniae Alba, 9 parts of Radix Angelicae Sinensis, 6 parts of Ramulus Uncariae Cum Uncis, 8 parts of Bombyx Batryticatus, 6 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (six) of embodiment 6 Chinese medicine composition medicines
10 parts in Rhizoma Gastrodiae, 5 parts of the Radixs Astragali, 10 parts of Radix Rehmanniae Preparata, 5 parts of Radix Paeoniae Alba, 10 parts of Radix Angelicae Sinensis, 5 parts of Ramulus Uncariae Cum Uncis, 10 parts of Bombyx Batryticatus, 2 parts of Rhizoma Arisaematiss, conventional method decocts.
Need to prove that it is the conventional manufacture method of Chinese medicine decoction that the described conventional method of embodiment 1-6 decocts, and is about to described crude drug decocte with water and becomes decoction.
The preparation of embodiment 7 Chinese medicine composition medicinal tablet/capsules
Get the arbitrary described Chinese medicine composition medicine of embodiment 1-6, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice.Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice, merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night.Get supernatant, be concentrated into thick extractum, or be concentrated into thick extract dry and be prepared into granule; Add pharmaceutical aids, vacuum drying is pulverized and is granulated, and is pressed into tablet or fills encapsulated.
The preparation of embodiment 8 Chinese medicine composition medicinal granules
Get the arbitrary described Chinese medicine composition medicine of embodiment 1-6, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice.Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice, merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night.Get supernatant, be concentrated into thick extractum, or be concentrated into thick extract dry and be prepared into granule; Add suitable pharmaceutical aids, granulate, drying, granulate gets granule, packing.
The preparation of embodiment 9 Chinese medicine composition drug mixture/oral liquid/syrups
Get the arbitrary described Chinese medicine composition medicine of embodiment 1-6, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice.Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice, merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night.Get supernatant, be concentrated into thick extractum, or be concentrated into thick extract dry and be prepared into granule; Add suitable pharmaceutical aids, process mixture, oral liquid or syrup.
The preclinical test of embodiment 10 Chinese medicine composition medicines of the present invention
One, experimental technique
1.PD rat model preparation:Prepare the PD rat model according to methods such as Ba Maowen.Get 65 rats and get into experiment, wherein 5 is sham operated rats, in MES (MFB) injecting normal saline.Lumbar injection 3% pentobarbital sodium anesthetized rat; Strict tack cranium position fixedly rat is shown the rat brain stereotaxic atlas in stereo brain orienting instrument with reference to the bag new people, confirms right side medial forebrain bundle (right medical forebrain; MFB) coordinate: 1. 3.7 mm behind the bregma; Sagittal suture right side 1.7 mm, 7.8 mm under the cranial periosteum, front tooth line 2.4 mm; 2. 4.4 mm behind the bregma, sagittal suture right side 1.2 mm, 7.8 mm under the cranial periosteum, front tooth line 2.4 mm.By the boring of above-mentioned definite injection site, extract 6-OHDA6 μ l (containing 0.2% ascorbic normal saline configuration, concentration 4 μ g/ μ l) with the microsyringe of 10 μ l, the withdraw of the needle behind every some injection 3 μ l, let the acupuncture needle remain at a certain point 10 min, stitching wound surface.After 3 weeks, rats by intraperitoneal injection apomorphine (0.5 mg/kg), average speed>7 times/min is successful PD model.
The model preparation is measured with behavioristics:The PD model of 25 successes is divided into 3 groups greatly at random.(1) PD matched group: PD rat model lumbar injection 0.2% vitamin C liquid 29 days; (2) Western medicine processed group: lumbar injection LDME+benserazide (50 mg/kg LDMEs and 25 mg/ kg benserazides, be dissolved in contain in the 0.2% ascorbic sterilization normal saline), continues 29d at 9 at 2 times/d at (in the morning with point in afternoons 5); (3) dose groups in Chinese medicine composition medicine small dose group (TCM-small dose group), the Chinese medicine composition medicine (the heavy dose of group of TCM-), the heavy dose of group of Chinese medicine composition medicine (the heavy dose of group of TCM-): give to add respectively with Chinese medicine composition medicine of the present invention on the basis of LDME/benserazide; 9 ml/kg; 1 filling every day stomach, continuous 4 weeks.Dose groups prepares the Chinese medicine composition medicine with reference to embodiment 4 described methods in the Chinese medicine composition medicine; The heavy dose of group of Chinese medicine composition medicine respectively increases by 20% according to each raw material of Chinese medicine medicine of middle dose groups; Chinese medicine composition medicine small dose group respectively reduces 20% according to each raw material of Chinese medicine medicine of middle dose groups.In therapeutic process, after medication in the morning in the 2nd, 8,15,22,29 day, carry out rat behavior and learn observation and scoring.AIM evaluation: AIM is divided into 4 components (upper limb AIM, the AIM of actinal surface portion, axle property AIM and rotation AIM) to be evaluated, and every part has or not with the order of severity according to it again and is divided into 5 grades (0-4): 0 does not have; 1 persistent period is less than 30s; 2 persistent period are greater than 30s, less than 60s; 3 persistent period, environmental stimuli can make it to stop greater than 60s; 4 persistent period, environmental stimuli can not make it to stop greater than 60s.Every 20min assessment is once observed 1min at every turn after the medication.The AIM total points is added up by the meansigma methods of total mark in observing time, and each component AIM maximum scores is 4 minutes behind 1 rat a drug in theory, and total AIM scoring is 16 minutes.Agent peak number of revolutions: behind the injection levodopa, every 5min record number of revolutions, maximum numbers of revolutions is an agent peak number of revolutions.
SABC shows the variation of striatum β-arrestin1, GRK6, DARPP-32 (Thr75) and ERK1/2:Finish injection back 12h, the anesthesia of 3% pentobarbital sodium, left ventricle is poured into 4% paraformaldehyde and is fixed, and broken end is got behind the brain in the same fixed liquid fixedly 8h of back, and piece, gradient alcohol dehydration, xylene are transparent through repairing, use FFPE behind the waxdip.The row paraffin section, slice thickness is 5 μ m.Paraffin section de-waxing is to water; 3% hydrogen peroxide room temperature lucifuge is hatched 5min, to eliminate the endogenous catalase activity; 1nmol/l ethylenediaminetetraacetic acid-Tris-hydrogen chloride of PH7.7 (repair by the EDTA-Tris-HCl microwave heating; 1% bovine serum albumin room temperature envelope 20min; Incubated overnight in the anti-Mus GRK6 of rabbit antibody, the anti-Mus β of rabbit-arrestin1 polyclonal antibody, the anti-Mus DARPP-32 of rabbit antibody, anti-phosphorylation DARPP-32 (Thr75) antibody, the total ERKl/2 of rabbit Chinese People's Anti-Japanese Military and Political College Mus and 4 ℃ of wet boxes of the Mus phosphorylation ERKl/2 of rabbit Chinese People's Anti-Japanese Military and Political College monoclonal antibody; The biotin labeling two that drips dilution is anti-, hatches 20min for 37 ℃; Drip the horseradish peroxidase-labeled strepto-avidin of dilution, hatch 20min for 37 ℃; Benzidine (3,3 ˊ-diaminobenzidine tetrahydrochloride, DAB) chromogenic reagent, tap water flushing, dehydration, transparent, mounting afterwards.All use the abundant rinsing of 0.01mol/l TBS between each step, TBS replaces one to resist as negative control.Examine under a microscope the SABC section; Each is observed the district and gets 5 not overlapping visuals field at random; Observe down in high power lens (10 * 40); Adopt OLYMPUS-IX50 to become phase system to take, Image-Pro Plus 5.1 imgae processing softwares carry out semi-quantitative analysis, calculate positive cell index (IOD)=positive cell area * correction OD value (measurement zone optical density-background indensity).
Method detects the expression of β-arrestin1, GRK6, DARPP-32 (Thr75) and ERK1/2:Last injection back 3h, the anesthesia of 3% pentobarbital sodium, broken end is got brain rapidly, peels off the bilateral striatum on ice, and ultrasonic degradation extracts total protein.After measuring protein concentration, put-80 ℃ frozen subsequent use.40 μ g egg door samples separate through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), change the film appearance with electricity and are transferred on polyvinylidene fluoride (PVDF) film.Confining liquid (5% defatted milk powder) room temperature sealing 2h; Dilute anti-GRK6 antibody (1: 500), anti-β-arrestin1 antibody (1:500), anti-total DARPP-32 antibody (1:1000) or anti-phosphorylation DARPP-32 (Thr75) antibody (1:1000), anti-total ERKl/2 antibody (1:1000) or anti-phosphorylation ERKl/2 antibody (1:500) or anti-β-actin (l:1000), 4 ℃ of jog incubated overnight according to respective concentration.Add two next day and resist, shaking table is hatched 1 h under the room temperature; Drip ECL colour developing mixed liquor, the exposure of Bio-Rad gel imaging appearance, video picture.Western blot image adopts the SmartView2000 image analysis software to calculate the product of each sample destination protein band OD value and area value, thereby it is quantitative to carry out protein band density.
Statistical analysis technique
Experimental data adopts SPSS 13.0 statistical softwares to analyze.The gained measurement data is with (
± s) expression of mean ± standard deviation; The relatively employing one factor analysis of variance of a plurality of sample averages relatively adopts the t check in twos.< 0.05 is that difference has statistical significance with P.
Two, result
1. the Chinese medicine composition medicine is to the influence of PD rat model behavior
1.1 the Chinese medicine composition medicine is to the influence of each component AIM scoring of PD rat model
With regard to the AIM of actinal surface portion scoring, average does not have significant difference between the 2nd day each group.8th, 15,22,29 days each the group between the average significant difference (
F=4.98,
F=22.70,
F=30.96,
F=23.27,
P<0.01).Wherein small dose group reduces than the Western medicine processed group the AIM of actinal surface portion scoring in the 22nd day, and difference has statistical significance (t=2.50, P<0.05), all the other several days equal not statistically significants of difference; In the 8th, 15,22,29 days AIM of actinal surface portion of dose groups scoring smaller dose group obviously reduce, difference have statistical significance (t=2.86, t=3.80, t=4.38, t=6.54, all
P<0.05); In dose groups and heavy dose of group relatively, the only middle dose groups actinal surface AIM of the portion scoring of big dose groups reduction in the 22nd day, difference have statistical significance (
t=2.50,
P<0.05).
With regard to upper limb AIM scoring, the 2nd, 8 day each class mean no significant difference, between the 15th, 22,29 day each group average difference have statistical significance (
F=15.12,
F=32.86,
F=35.80,
P<0.01).Medication the 2nd day and small dose group upper limb AIM scoring in the 8th day and Western medicine processed group be there was no significant difference relatively, is lower than Western medicine group to medication small dose group upper limb AIM scoring in the 15th, 22,29 days, difference have statistical significance (
t=4.45,
t=4.47, all
P<0.01).15th, dose groups obviously reduces than small dose group upper limb AIM scoring in 22,29 days, difference have statistical significance (
t=3.40,
t=4.04,
t=3.95, all
P<0.01).Compare the difference no difference of science of statistics between heavy dose of group and the middle dose groups.
With regard to axial AIM scoring, between the 15th, 22,29 day each group average difference have statistical significance (
F=3.30,
F=25.06,
F=36.41,
P<0.01).22,29 days axial AIM values of small dose group medication to the reduce than Western medicine processed group, difference have statistical significance (
t=3.93,
t=3.04, all
P<0.05); In dose groups than small dose group relatively, only in the 29th day difference have statistical significance (
t=3.05,
P<0.05); Heavy dose of group has obviously reduced axial AIM value, but compares with middle dose groups, only variant in the 29th day (
t=2.83,
P<0.05), put the difference no difference of science of statistics At All Other Times.
With regard to rotation AIM scoring, average significant difference between the 8th, 15,22,29 day each group (
F=10.50,
F=11.62,
F=18.92,
F=61.26,
P<0.01).The difference not statistically significant is compared in small dose group and Western medicine processed group rotation AIM scoring.In dose groups medication to the 15,22,29 days, rotation AIM scoring is than the obvious reduction of Western medicine group, difference have statistical significance (
t=3.43,
t=5.11,
t=7.63, all
P<0.01); Heavy dose of group and middle dose groups relatively, the 8th, 15,29 day obvious difference (
t=3.81,
t=2.52,
t=3.58, all
P<0.05).
The comparison of table 1 Chinese drug-treated group and Western medicine group AIM scoring (
± S divides)
| Time | Group | Mus number (only) | Actinal surface portion | Upper | Axially | Rotation | |
| 2d | The heavy dose of group of TCM- | 5 | 1.42±0.24 | 1.62±0.26 | 2.38±0.15 | 2.62±0.23 | |
| ? | Dose groups among the TCM- | 5 | 1.40±0.19 | 1.56±0.28 | 2.51±0.22 | 2.71±0.34 | |
| ? | The TCM- |
5 | 1.51±0.17 | 1.58±0.19 | 2.42±0.16 | 2.62±0.23 | |
| ? | The Western medicine processed |
5 | 1.44±0.18 | 1.51±0.22 | 2.49±0.17 | 2.67±0.28 | |
| 8d | The heavy dose of group of TCM- | 5 | 1.51±0.22 | 1.67±0.22 | 2.42±0.24 | 2.60±0.06 | |
| ? | Dose groups among the TCM- | 5 | 1.56±0.18 | 1.76±0.16 | 2.58±0.29 | 2.93±0.19 | |
| ? | The TCM- |
5 | 1.89±0.21 | 1.91±0.19 | 2.76±0.41 | 3.16±0.20 | |
| ? | The Western medicine processed |
5 | 1.82±0.17 | 1.93±0.20 | 2.71±0.41 | 3.20±0.25 | |
| 15d | The heavy dose of group of TCM- | 5 | 1.33±0.11 | 1.51±0.22 | 2.38±0.20 | 2.58±0.15 | |
| ? | Dose groups among the TCM- | 5 | 1.47±0.14 | 1.71±0.22 | 2.69±0.24 | 2.84±0.19 | |
| ? | The TCM- |
5 | 1.84±0.17 | 2.09±0.12 | 2.71±0.37 | 3.09±0.24 | |
| ? | The Western medicine processed |
5 | 2.11±0.22 | 2.18±0.15 | 2.93±0.28 | 3.36±0.28 | |
| 22d | The heavy dose of group of TCM- | 5 | 1.29±0.13 | 1.49±0.23 | 2.24±0.18 | 2.53±0.15 | |
| ? | Dose groups among the TCM- | 5 | 1.49±0.13 | 1.69±0.20 | 2.29±0.13 | 2.67±0.11 | |
| ? | The TCM- |
5 | 1.96±0.20 | 2.13±0.14 | 2.62±0.28 | 3.11±0.21 | |
| ? | The Western medicine processed |
5 | 2.40±0.37 | 2.56±0.16 | 3.18±0.15 | 3.38±0.29 | |
| 29d | The heavy dose of group of TCM- | 5 | 1.20±0.09 | 1.53±0.21 | 2.11±0.78 | 2.29±0.10 | |
| ? | Dose groups among the TCM- | 5 | 1.33±0.21 | 1.67±0.18 | 2.33±0.16 | 2.56±0.14 | |
| ? | The TCM- |
5 | 2.04±0.13 | 2.13±0.20 | 2.76±0.26 | 3.24±0.14 | |
| ? | The Western medicine processed |
5 | 2.36±0.36 | 2.60±0.13 | 3.16±0.13 | 3.47±0.23 |
1.2 various dose group Chinese medicine is to the influence of PD rat model AIM total points
Medication was marked in the 8th, 15,22,29 days; Find low dose group medication to the 15,22,29 days; The AIM total points is that (9.73 ± 0.52), (9.82 ± 0.37) and (10.18 ± 0.32) are divided, than Western medicine group (10.58 ± 0.40), (11.51 ± 0.17) divide and (11.58 ± 0.24) divide obviously and reduce (
t=2.88,
t=9.25,
t=7.77, all
p<0.05).And middle dose groups AIM total points is respectively (8.82 ± 0.36), (8.71 ± 0.47), (8.13 ± 0.37) and (7.89 ± 0.57) and divides and all be lower than the small dose group total points, difference have statistical significance (
t=3.32,
t=3.19,
t=7.56,
t=7.85, all
p<0.05); In the 8th, 15,22 day, heavy dose of group AIM total points was respectively (8.20 ± 0.27), (7.80 ± 0.34), (7.55 ± 0.38) are divided, and middle dose groups is low, difference have statistical significance (
t=3.12,
t=3.44,
t=2.57, all
p<0.05), and the 29th day big dose groups (6.71 ± 1.13) branch compares with middle dose groups, difference not statistically significant (see figure 1).
Various dose Chinese medicine composition medicine is to the influence of PD rat model agent peak number of revolutions
Carried out agent peak number of revolutions in the 8th, 15,22,29 day and detect, find that low dose group scoring (123.6 ± 11.91), (132 ± 10.07), (151 ± 15.73) and (155.8 ± 7.40) are inferior, little with the Western medicine group difference; And middle dose groups and small dose group are relatively, and middle dose groups agent peak number of revolutions is respectively (87.4 ± 15.60), (83.0 ± 21.76), (88 ± 10.20) and (73 ± 15.18), all is lower than small dose group, difference have statistical significance (
t=4.13,
t=4.57,
t=7.51,
t=10.96, all
p<0.05); Heavy dose of group and middle dose groups relatively, the inferior middle dose groups of big dose groups agent peak number of revolutions (51.2 ± 6.76) and (53.8 ± 7.79) was low in the 22nd, 29 day, difference have statistical significance (
t=6.73,
t=2.52, all
p<0.05) (see figure 2).
Various dose Chinese medicine composition medicine is to the influence of PD motor complication rat striatum district signal protein
2.1 Chinese medicine composition medicine various dose is to the influence of PD rat model striatum district GRK6
Showed by immune group result GRK6 expresses on cell membrane.Each group is not damaged the side average relatively, the difference not statistically significant (
F=0.17,
P>0.05), and each group damage side average significant difference (
F=4.48,
P<0.05).PD injury in rats side GRK6 expression is (4.53 ± 0.85) * 10
3, (6.10 ± 0.55) * 10 of normal rat
3Obviously reduce (
t=9.99,
P<0.01); GRK6 expresses and further is reduced to (3.52 ± 0.71) * 10 after the life-time service levodopa treatment
3, with the PD rat relatively, difference have statistical significance (
t=2.99,
P<0.05); And adding rat with Chinese herb on the prevention, striatal damage side GRK6 expresses and the PD rat is compared, and does not all have further to reduce, and compares small dose group (4.17 ± 1.20) * 10 with the rat of using levodopa
3With the Western medicine group zero difference, the expression of middle dose groups, heavy dose of group damage side GRK6 is respectively (4.21 ± 0.95) * 10
3, (4.78 ± 1.24) * 10
3, with Western medicine group relatively, the GRK6 expression obviously more (
t=3.79,
t=4.84, all
P<0.05) (see figure 3).
Western result and SABC basically identical.With regard to every rat, testing result is with damage side/do not damage the ratio value representation of side, and β-actin is as confidential reference items.Compare with (100.78 ± 5.57) % of sham operated rats, PD rat GRK6 expression is reduced to (81.32 ± 5.94) %, significant difference (
t=7.37,
P<0.01); The levodopa long-term treatment has further reduced the GRK6 expression to (72.66 ± 3.43) %, difference have statistical significance (
t=3.11,
P<0.05); Compare the difference not statistically significant and add with the rat striatum GRK6 expression and the PD rat of Chinese herb on the prevention.With the rat of life-time service levodopa relatively, % is obviously higher for middle dose groups striatum GRK6 expression (83.78 ± 4.50), difference have statistical significance (
t=7.26,
P<0.01), small dose group (79.02 ± 3.15) % and heavy dose of group (80.46 ± 4.74) % and its compare, the remarkable (see figure 4) of difference.
The Chinese medicine various dose is to the influence of PD motor complication rat model striatum district β-arrestin1
SABC shows that β-arrestin1 is expressed in cell membrane, each group damage side average significant difference (F=5.76, P 0.05).The proteic positive cell index of PD rat β-arrestin1 is (3.20 ± 0.75) * 10
4Be lower than (4.50 ± 0.63) * 10 of sham operated rats rat
4, but the difference not statistically significant.After the levodopa long-term treatment, β-arrestin1 expresses further and reduces, and the positive cell index is (2.50 ± 0.49) * 10
4, comparing with the PD rat, difference has statistical significance (t=3.90, P<0.05).Further decline does not appear in the rat β-arrestin1 positive cell index that adds with Chinese medicine, and particularly middle dose groups rat β-arrestin1 positive cell index is (3.67 ± 0.51) * 10
4, increase (t=6.80, P than PD rat<0.01).Large and small dose groups β-arrestin1 positive cell index is respectively (3.20 ± 0.68) * 10
4(3.10 ± 0.59) * 10
4Between relatively, with the PD group relatively, no significant difference (see figure 5).
Western result and SABC basically identical.Every rat Western result is with damage side/do not damage the ratio value representation of side, and β-actin is as confidential reference items.PD rat β-arrestin1 expressing quantity is (76.41 ± 5.12) %, than sham operated rats rat (100.86 ± 6.79) % obviously reduce (
t=11.48,
P<0.01).The levodopa long-term treatment has further reduced β-arrestin1 expression, is (65.04 ± 6.68) %, with PD group rat relatively, difference have statistical significance (
t=8.31,
P<0.01).Add with behind the Chinese herb on the prevention, small dose group β-arrestin1 expression is (69.34 ± 3.36) %, than PD rat group reduce (
t=3.42,
P<0.05), demonstrate the trend similar with Western medicine group; And middle dose groups is respectively (73.42 ± 3.82) % with heavy dose of group and further decline does not appear in (72.36 ± 6.66) %.Simultaneously, middle dose groups and the rat of heavy dose of group striatum β-arrestin1 expression, difference apparently higher than the life-time service levodopa have statistical significance (
t=3.11,
t=3.70,
P<0.05).Compare difference not statistically significant (see figure 6) between middle dose groups and the heavy dose of group.
The influence that the Chinese medicine various dose is expressed PD motor complication rat model striatum district phosphorylation DARPP-32 (Thr75)
Each group damage side average significant difference of SABC demonstration (
F=472.10,
P<0.01).Sham operated rats rat phosphorylation DARPP-32 (Thr75) expression is (7.01 ± 0.57) * 10
6, the PD group is increased to (11.50 ± 0.16) * 10
6, difference have statistical significance (
t=14.07,
P<0.01).After the levodopa long-term treatment, phosphorylation DARPP-32 (Thr75) is expressed as (3.85 ± 0.30) * 10
6, obviously reduce than PD group, difference have statistical significance (
t=38.99,
P<0.01).Add the rat with Chinese medicine, large, medium and small dose groups phosphorylation DARPP-32 (Thr75) expresses and is respectively (12.34 ± 0.19) * 10
6, (11.89 ± 0.47) * 10
6(7.20 ± 0.27) * 10
6, middle dose groups does not all occur significantly reducing with heavy dose of group, and only small dose group reduces.Compare difference not statistically significant (see figure 7) between heavy dose of group and the middle dose groups.
Western result and SABC basically identical, the gray value that sham operated rats rat phosphorylation DARPP-32 (THr75) expresses is (2.06 ± 0.24) * 10
6, PD group rat is (3.04 ± 0.20) * 10
6, two groups of comparing differences have statistical significance (
t=28.95,
P<0.01).Add with not playing preventive effect behind the low dose of Chinese medicine, phosphorylation DARPP-32 (THr75) expression is (1.85 ± 0.15) * 10
6, than the PD rat reduce (
t=19.77,
P<0.01); Rat striatum phosphorylation DARPP-32 (THr75) expression that adds dosage Chinese herb on the prevention in the usefulness is (3.10 ± 0.15) * 10
6, compare with the PD rat, the difference not statistically significant (
P>0.05).Heavy dose of group phosphorylation DARPP-32 (THr75) expression is (3.73 ± 0.17) * 10
6,, than the PD rat have slightly and increase (t=38.57,
P<0.01), simultaneously, dose groups increasing expression in heavy dose of group ratio (
t=21.78,
P<0.01) (see figure 8).
The influence that the Chinese medicine various dose is expressed PD motor complication rat model striatum district phosphorylation ERK1/2
Each group damage side average significant difference of SABC demonstration (
F=117.49,
P<0.01).PD rat phosphorylation ERK12 expression is (4.30 ± 0.23) * 10
4, be lower than (5.20 ± 0.34) * 10 of sham operated rats rat
4, difference have statistical significance (
t=15.32,
P<0.01).After the levodopa long-term treatment, phosphorylation ERK12 expresses and is increased to (8.08 ± 0.37) * 10
4, with PD group relatively, difference have statistical significance (
t=34.13,
P<0.01).Add the rat with Chinese medicine, small dose group phosphorylation ERK12 is expressed as (6.59 ± 0.36) * 10
4, in rising trend, middle dose groups is (4.49 ± 0.34) * 10 with heavy dose of group expression
4(4.26 ± 0.25) * 10
4, all do not occur significantly rising.Relatively, difference is not obvious between heavy dose of group and the middle dose groups.(see figure 9).
Western result and SABC basically identical, the gray value that PD rat phosphorylation ERK1/2 expresses is (2.83 ± 0.17) * 10
6, than (3.50 ± 0.12) * 10 of sham operated rats rat
6Reduce (
t=20.77,
P<0.01); Add the gray value of expressing and be respectively (3.23 ± 0.19) * 10 with the rat striatum phosphorylation ERK12 of large, medium and small dosage Chinese herb on the prevention
6, (3.15 ± 0.16) * 10
6(4.40 ± 0.16) * 10
6, with the PD rat rising is in various degree arranged more all, difference all have statistical significance (
t=13.81,
t=3.41,
t=45.12,
P<0.01).Wherein, middle dose groups phosphorylation ERK12 expression rising amplitude is minimum, the maximum (see figure 10) of small dose group rising amplitude.
In a word; Middle dosage Chinese medicine composition medicine has reversed the carrying out property rising of each component AIM scoring of PD rat, has reduced PD rat agent peak number of revolutions, through increasing GRK6, β-arrestin1 expressing quantity; The reduction and the phosphorylation ERK1/2 that reverse phosphorylation DARPP-32 (Thr75) expression express significantly rising; Improved the function of anomalous signals transduction molecule in the LID born of the same parents, reduced the generation of PD motor complication, prevented PD and delaying to have great using value aspect the disease progression.
Three, conclusion
Chinese medicine composition medicine of the present invention, its compatibility meet Chinese medicine " monarch " principle, and the flavour of a drug number is less; Advantages such as having the curative effect height, have no side effect, price is low adopts the benefiting QI for activating blood circulation method, the nourishing YIN for attracting YANG method; The suppressing the hyperactive liver to relieve the wind syndrome method is rubbed the muscle method that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., activating blood and removing stasis Method and collateral dredging rule such as relieve dizziness, high fever, infantile convulsions, epilepsy, etc.; Start with from cause of disease at all, suit the remedy to the case, to reach preventive effect; Method for preparing is simple, and cost is low, and is environmentally friendly, is suitable for taking for a long time, is a kind of Chinese medicine composition medicine that prevents parkinson disease and parkinson disease motor complication safely and effectively, has a good application prospect.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; Can also make some improvement and replenish, these improvement and replenish and also should be regarded as protection scope of the present invention.
Claims (9)
1. the application of Chinese medicine medicine in the Parkinsonian disease medicament of preparation prevention; It is characterized in that described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 5-10 part, Radix Astragali 5-10 part, Radix Rehmanniae Preparata 5-10 part, Radix Paeoniae Alba 5-15 part, Radix Angelicae Sinensis 5-10 part, Ramulus Uncariae Cum Uncis 5-10 part, Bombyx Batryticatus 2-10 part, Rhizoma Arisaematis 2-10 part.
2. application according to claim 1; It is characterized in that described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 6-8 part, Radix Astragali 6-8 part, Radix Rehmanniae Preparata 6-8 part, Radix Paeoniae Alba 8-13 part, Radix Angelicae Sinensis 6-8 part, Ramulus Uncariae Cum Uncis 6-8 part, Bombyx Batryticatus 4-6 part, Rhizoma Arisaematis 4-6 part.
3. application according to claim 1 is characterized in that, described medicine is processed by following bulk drugs: 8 parts in Rhizoma Gastrodiae, 8 parts of the Radixs Astragali, 8 parts of Radix Rehmanniae Preparata, 10 parts of Radix Paeoniae Alba, 8 parts of Radix Angelicae Sinensis, 8 parts of Ramulus Uncariae Cum Uncis, 5 parts of Bombyx Batryticatus, 5 parts of Rhizoma Arisaematiss.
4. according to the arbitrary described application of claim 1-3, it is characterized in that the application of described medicine in the disease medicament of preparation prevention parkinson disease motor complication.
5. one kind is prevented Parkinsonian Chinese medicine medicine; It is characterized in that described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 5-10 part, Radix Astragali 5-10 part, Radix Rehmanniae Preparata 5-10 part, Radix Paeoniae Alba 5-15 part, Radix Angelicae Sinensis 5-10 part, Ramulus Uncariae Cum Uncis 5-10 part, Bombyx Batryticatus 2-10 part, Rhizoma Arisaematis 2-10 part.
6. medicine according to claim 1; It is characterized in that described medicine is processed by following bulk drugs: Rhizoma Gastrodiae 6-8 part, Radix Astragali 6-8 part, Radix Rehmanniae Preparata 6-8 part, Radix Paeoniae Alba 8-13 part, Radix Angelicae Sinensis 6-8 part, Ramulus Uncariae Cum Uncis 6-8 part, Bombyx Batryticatus 4-6 part, Rhizoma Arisaematis 4-6 part.
7. medicine according to claim 1 is characterized in that, described medicine is processed by following bulk drugs: 8 parts in Rhizoma Gastrodiae, 8 parts of the Radixs Astragali, 8 parts of Radix Rehmanniae Preparata, 10 parts of Radix Paeoniae Alba, 8 parts of Radix Angelicae Sinensis, 8 parts of Ramulus Uncariae Cum Uncis, 5 parts of Bombyx Batryticatus, 5 parts of Rhizoma Arisaematiss.
8. according to the arbitrary described medicine of claim 5-7, it is characterized in that the application of described medicine in the disease medicament of preparation prevention parkinson disease motor complication.
9. according to the arbitrary described medicine of claim 5-7, it is characterized in that the dosage form of described medicine is tablet, capsule, granule, mixture, oral liquid or syrup.
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