CN106309637A - Traditional Chinese medicine composition for treating Parkinson disease and preparation method thereof - Google Patents
Traditional Chinese medicine composition for treating Parkinson disease and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition for treating Parkinson disease. The traditional Chinese medicine composition comprises the following bulk drugs in parts by weight: 1-10 parts of corydalis tuber, 1-10 parts of uncaria, 1-6 parts of fleece-flower root, and 1-6 parts of Ligusticum wallichii. The invention also discloses a preparation method of the traditional Chinese medicine composition. The pharmacological research confirms that the traditional Chinese medicine composition has the effects for mitigating limb tremble, alleviating muscular rigidity, protecting dopaminergic neuron, and delaying the condition development, and can be used for treating Parkinson disease.
Description
Technical field
The present invention relates to a kind of Chinese medicine composition, particularly relate to one and treat Parkinsonian Chinese medicine composition.
Background technology
Parkinson disease (Parkinson ' s disease, PD) it is a kind of incremental neurodegenerative disease being apt to occur in middle-aged and elderly people, more than the 60 years old crowd of the most about 1% is ill.Principle pathological mechanism is substantia nigra compacta striatal dopamine (dopamine, DA) neuronal degeneration, causes DA content in striatum to reduce.Clinically with static tremor, splinting, hypokinesia and posture insufficiency of accommodation as principal character.
Doctor trained in Western medicine for a long time as the medicine madopar of first-line treatment PD, is made up of with benserazide levodopa (L-DOPA) clinically, and it mainly treats PD, determined curative effect by the deficiency of DA in striatum in exogenous supplementary brain.Clinical and experimentation shows, this type of medicine of long-term taking can make curative effect reduce, and the serious side reaction such as " on-off phenomenon ", " agent end phenomenon " occurs, and can increase free radical generation in brain, damages dopamine neuron.The dopamine-receptor stimulant class such as bromocriptine effect protected to neuron that Equations of The Second Kind is conventional; onset is very fast; relatively L-DOPA length action time in striatum; alternative acts on DA receptor; but when being used alone curative effect not as L-DOPA obvious; there is individual variation in the reaction to medicine, the side effect such as mental symptom, postural hypotension easily occurs.
PD belongs to the traditional Chinese medical science " electric shock " " tremor " category, is apt to occur in person in middle and old age, and caused by liver and kidney deficiency, deficiency of marrow-reservoir are its origins of falling ill, the traditional Chinese medical science to the description of tremor syndrome first appeared in "Nei Jing"." Plain Questions-the most pure virginity to be discussed greatly " proposes " all wind trembling and dizziness come under liver ", wherein " falls " and i.e. refers to chatter, and this opinion is to for institute ancestor later age.Finding the PD clinical application research of Chinese medicine (including acupuncture) treatment during the nearly last ten years by consulting literatures, Chinese traditional treatment PD determined curative effect, untoward reaction is little.Its dialectical treatmert of clinical studies show is still its characteristic and elite, how relieves dizziness, high fever, infantile convulsions, epilepsy, etc. with the soft muscle of benefiting QI and nourishing blood, blood circulation promoting and blood stasis dispelling, spasmolytic and only quivers and nourishing the liver and kidney essence and blood etc. carries out the preventing and treating of PD, especially with nourishing the liver and kidney for main treatment PD for the rule for the treatment of.
Li Rukui etc. disclose a kind of traditional Chinese medicine composition for treating PD being made up of Radix Astragali Preparata 30g, Radix Paeoniae Alba 30g, Ramulus Uncariae Cum Uncis 15g, Herba Leonuri 30g, Radix et Rhizoma Rhei (processed) 30g, Rhizoma Cimicifugae 10g, result confirms that the flat soup rat circling behavior on 6-OHDA modeling that quivers, without impact, can not increase its Striatal Dopamine Content；Flat continuous gavage 3d of soup 144g/kg of quivering, there is obvious anti-shudder effect, the flat soup that quivers of prompting does not has the effect of dopamine receptor function in excited brain, can not increase the content of neurotransmitter, but have certain to anti-shudder effect [Li Rukui, Zhao Hong, Tu Yingmei, Deng. put down and quiver soup to the impact [J] of DOPAMINE CONTENT IN RABBIT in Parkinson disease model animal behavior and brain. Chinese medicine study, 2000,16 (2): 40-41].
Wang Hui is new etc. discloses one and only quivers decoction thing composition: Rhizoma Anemarrhenae 9g, Radix Paeoniae Alba 30g, Ramulus Uncariae Cum Uncis 18g, Radix Astragali 20g, Radix Salviae Miltiorrhizae 20g, Rhizoma Cimicifugae 9g, Radix Et Rhizoma Rhei 9g, 60 example PD patients is divided into Chinese medicine and only quivers soup treatment group (Chinese drug-treated group) and Western medicine pergolide treatment group (Western medicine group).Two groups of patient's Drug therapys, after 3 months, use the motion in PD unified score scale (UPDRS) to check scale score, and decline degree of marking before and after comparison therapy assesses its therapeutic effect.Result: after Chinese drug-treated group treatment, UPDRS scoring declines 4.55 points, Western medicine group declines 4.49 points, after two groups of treatments, UPDRS substantially reduces before the most relatively treating, difference statistically significant (P < 0.01), and Chinese drug-treated group total effective rate is 83.33%, it is better than 53.33% (P < 0.05) of Western medicine group.Without obvious adverse reaction in observation.Conclusion: Chinese medicine only quiver soup treatment PD have more definite clinical efficacy [Wang Huixin, Liu Yi, Li Rukui. the soup that only quivers associating madopar tablet treatment parkinson disease 30 example clinical research [J]. Journal of Traditional Chinese Medicine, 2010,51 (1): 146-147].
Li Rukui discloses a kind of traditional Chinese medicine composition for treating PD being made up of Rhizoma Anemarrhenae 8-16 part, Radix Angelicae Sinensis 6-12 part, Radix Paeoniae Alba 12-18 part, Ramulus Uncariae Cum Uncis 30-40 part, Radix Astragali 12-18 part, think and have an advantage in that the flat Tang Hezhi of quivering that therapeutic effect is better than in above-mentioned report quivers soup, and have preparation technology advantage simple, with low cost [Li Rukui. one treats Parkinsonian Chinese medicine composition and application thereof. number of patent application 201210415109.2].
Chen Mengyun etc. disclose a kind of by traditional Chinese medicine composition for treating PD such as Radix Astragali, Ramulus Uncariae Cum Uncis, Radix Polygoni Multiflori Preparata, Radix Paeoniae Alba (parched), the Rhizoma Anemarrhenaes, 120 example cases are randomly divided into treatment group and matched group, often organize 60 examples；Treatment group is given the granule that only quivers and is added conventional western medicine treatment, and matched group gives traditional Chinese medicine placebo and adds conventional western medicine treatment.Two groups of courses for the treatment of are 12 weeks, observe UPDRS scale integration, the situation of Chinese medicine syndrome integral before and after comparison therapy, and evaluate drug safety.Result: be 1. finally completed test case 108 example, treatment group 57 example, matched group 51 example.2. compare in group before and after treatment, treatment group UPDRS scale total score and subitem III (motion checks) integration difference statistically significant (P < 0.05)；Compare after treatment between group, UPDRS scale total score and subitem III (motion checks) integration difference statistically significant (P < 0.05).3. compare in group before and after treatment, treatment group tcm syndrome total score and cardinal symptom integration difference statistically significant (P < 0.05)；Compare after treatment between group, tcm syndrome total score and cardinal symptom integration difference statistically significant (P < 0.05).4. during test, untoward reaction 2 example, wherein treatment group 1 example, matched group 1 example occur altogether；Before and after treatment, experimenter's blood, urine, stool routine, liver function (ALT, AST), renal function (BUN, Cr), Electrocardioscopy does not all have found that it is likely that the abnormal change the most relevant with test.Conclusion: the granule that only quivers associating conventional western medicine treatment deficiency of the liver and kindey, the satisfactory effect of QIXUELIANGXU type PD, safety is preferable, the clinical symptoms of patient can be significantly improved, improve its quality of life [Chen Mengyun, Liu Yi, Ruan Kefeng etc. Parkinsonian random, the double blinding of the granule that only quivers associating conventional western medicine treatment deficiency of the liver and kindey, QIXUELIANGXU type, placebo controlled clinical research [J]. journal of shanghai Chinese medicine, 2014,48 (10): 27-30].
It is an object of the invention to pathogenesis pneumatic from PD blood stasis, deficiency of the liver and kindey start with, it is provided that the Chinese medicine composition of a kind of new treatment PD being different from above-mentioned known Chinese medicine composition.
Summary of the invention
The technical problem to be solved in the present invention is to provide one and treats Parkinsonian Chinese medicine composition.The Chinese medicines such as this Chinese medicine composition rhizoma corydalis, Ramulus Uncariae Cum Uncis, Rhizoma Chuanxiong, Radix Polygoni Multiflori are that primary raw material is made.Pharmacological research confirms, the water extract oral administration of each prescription of Chinese medicine compound of the present invention has and alleviates limb tremor, alleviates muscular rigidity, protects dopamine neuron, delays the effect of PD, and treatment parkinson disease are evident in efficacy.
The present invention combines with neural Research foundation, subordinate act, immunohistochemistry aspects has investigated the effect of Rhizoma Corydalis (having another name called: Rhizoma Corydalis, corydalis tuber, rhizoma corydalis, Rhizoma Corydalis etc.), Ramulus Uncariae Cum Uncis, Rhizoma Chuanxiong, Radix Polygoni Multiflori and the Chinese medicine compound of compatibility therewith, provides the experimental basis of science for the exploitation of further patent medicine and clinical practice.
Chinese medicine composition of the present invention mainly with Rhizoma Corydalis, Ramulus Uncariae Cum Uncis, Rhizoma Chuanxiong, Radix Polygoni Multiflori four Chinese medicine material composition, the PD pathogenesis of Liver and kidney pneumatic based on blood stasis, Rhizoma Corydalis, Rhizoma Chuanxiong can blood circulation promoting and blood stasis dispelling, Ramulus Uncariae Cum Uncis can suppressing the hyperactive liver to relieve the wind syndrome, Radix Polygoni Multiflori nourishing the blood and yin.Full side compatibility is precise and appropriate, complements each other, plays the merit of blood circulation promoting and blood stasis dispelling, nourishing the liver and kidney altogether.
The present invention carries out the following studies:
1, research is compared and is filtered out parkinson disease rationally effective compound recipe；
2, the optimum preparation technology of compound preparation is studied；
3, being studied by pharmacology pharmacodynamic, checking compound recipe is to alleviating limb tremor, alleviating muscular rigidity, protect dopamine neuron, delay the aspects such as PD to have effect.
In one aspect of the invention, it is provided that one treats Parkinsonian Chinese medicine composition, is mainly made up of the crude drug of following weight portion: Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, Rhizoma Chuanxiong 1-6 part.
The optimum ratio of above-mentioned raw materials medicine is: Rhizoma Corydalis 1-8 part, Ramulus Uncariae Cum Uncis 1-8 part, Radix Polygoni Multiflori 1-4 part, Rhizoma Chuanxiong 1-4 part.
Rhizoma Corydalis, Ramulus Uncariae Cum Uncis, Rhizoma Chuanxiong, Radix Polygoni Multiflori four taste Chinese medicine can be used according to above proportioning.
Other medicines can also be added on the basis of above-mentioned Rhizoma Corydalis, Ramulus Uncariae Cum Uncis, Rhizoma Chuanxiong, Radix Polygoni Multiflori four taste Chinese medicine, be combined into multiple prescription, play effect in various degree.
As currently preferred technical scheme, described crude drug is except Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, beyond Rhizoma Chuanxiong 1-6 part, also include one or more in agents: Radix Codonopsis 1-10 part, Radix Astragali 1-10 part, Radix Paeoniae Alba 1-8 part, Radix Paeoniae Rubra 1-8 part, Rhizoma Anemarrhenae 1-6 part, Radix Rehmanniae 1-6 part.
As currently preferred technical scheme, described crude drug is except Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, beyond Rhizoma Chuanxiong 1-6 part, also includes QI invigorating class medicine, this QI invigorating class medicine is selected from one or more of following medicine: Radix Codonopsis 1-10 part, Radix Astragali 1-10 part.
As currently preferred technical scheme, described crude drug is except Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, beyond Rhizoma Chuanxiong 1-6 part, also includes nourishing blood to suppress the hyperactive liver class medicine, this nourishing blood to suppress the hyperactive liver class medicine is selected from one or more of following medicine: Radix Paeoniae Alba 1-8 part, Radix Paeoniae Rubra 1-8 part.
As currently preferred technical scheme, described crude drug is except Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, beyond Rhizoma Chuanxiong 1-6 part, also includes nourishing YIN to lower pathogenic fire class medicine, this nourishing YIN to lower pathogenic fire class medicine is selected from one or more of following medicine: Rhizoma Anemarrhenae 1-6 part, Radix Rehmanniae 1-6 part.
As currently preferred technical scheme, described crude drug is except Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, beyond Rhizoma Chuanxiong 1-6 part, also includes the following: Radix Astragali 1-10 part, Rhizoma Anemarrhenae 1-6 part, Radix Paeoniae Alba 1-8 part.
The Chinese medicine composition of the present invention can use the conventional method of Chinese medicine preparation to be prepared as oral medicinal herb preparation, such as material medicine is carried out decocting, or extract with alcohol-water mixture, then concentrate drying decocting liquid or extracting solution, make powdered extract powder, extract powder is added suitable adjuvant with medicine, by the conventional pharmaceutical methods of those skilled in the art, it is prepared as oral medicinal herb preparation, including tablet, granule, oral liquid etc..
In another aspect of this invention, it is provided that the preparation method of a kind of described Chinese medicine composition, comprise the steps: by described weight portion weighting raw materials, add 6-12 times of soak by water, micro-boiling 30min-90min, filter, medicinal residues add 4-10 times of water, micro-boiling 30min-60min, filter；Merging twice decoction liquor, concentrate, extractum or dried extract powder add appropriate amount of auxiliary materials, prepare traditional Chinese medicine particle preparation.
Compared with prior art, the beneficial effects of the present invention is: prescription formula is precise and appropriate, and flavour of a drug are few；Confirm that the Chinese medicine composition of the present invention has through pharmacological research to alleviate limb tremor, alleviate muscular rigidity, protect dopamine neuron, delay the effect of PD, can be used for treating parkinson disease.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
The preparation of embodiment 1 pharmaceutical preparation of the present invention
Rhizoma Corydalis 10 parts (500g), Ramulus Uncariae Cum Uncis 6 parts (300g), Radix Polygoni Multiflori 1 part (50g), Rhizoma Chuanxiong 6 parts (300g).
Weighing pharmaceutical decocting piece by said ratio, add 10 times of soak by water, micro-boiling 90min, filter, medicinal residues add 6 times of water, micro-boiling 1h, filter.Merging twice decoction liquor, concentrating under reduced pressure, be dried, extract powder adds appropriate lactose, dextrin, magnesium stearate, mix homogeneously, pelletizes, makes 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 2 pharmaceutical preparation of the present invention
Rhizoma Corydalis 6 parts (300g), Ramulus Uncariae Cum Uncis 1 part (50g), Radix Polygoni Multiflori 1 part (50g), Rhizoma Chuanxiong 1 part (50g).
Weighing pharmaceutical decocting piece by said ratio, add 10 times of soak by water, micro-boiling 30min, filter, medicinal residues add 8 times of water, micro-boiling 30min, filter.Merging twice decoction liquor, concentrating under reduced pressure, be dried, extract powder adds appropriate lactose, dextrin, magnesium stearate, mix homogeneously, pelletizes, makes 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 3 pharmaceutical preparation of the present invention
Rhizoma Corydalis 1 part (50g), Ramulus Uncariae Cum Uncis 10 parts (500g), Radix Polygoni Multiflori 4 parts (200g), Rhizoma Chuanxiong 4 parts (200g).
Weighing pharmaceutical decocting piece by said ratio, add 10 times of soak by water, micro-boiling 60min, filter, medicinal residues add 8 times of water, micro-boiling 60min, filter.Merging twice decoction liquor, concentrating under reduced pressure, be dried, extract powder adds appropriate lactose, dextrin, magnesium stearate, mix homogeneously, pelletizes, makes 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 4 pharmaceutical preparation of the present invention
Rhizoma Corydalis 1 part (50g), Ramulus Uncariae Cum Uncis 10 parts (500g), Radix Polygoni Multiflori 6 parts (300g), Rhizoma Chuanxiong 3 parts (150g).
Weighing pharmaceutical decocting piece by said ratio, add 12 times of soak by water, micro-boiling 90min, filter, medicinal residues add 8 times of water, micro-boiling 60min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 5 pharmaceutical preparation of the present invention
Rhizoma Corydalis 8 parts (400g), Ramulus Uncariae Cum Uncis 5 parts (250g), Radix Polygoni Multiflori 1 part (50g), Rhizoma Chuanxiong 3 parts (150g), Radix Codonopsis 6 parts (300g).
Weighing pharmaceutical decocting piece by said ratio, add 8 times of soak by water, micro-boiling 60min, filter, medicinal residues add 8 times of water, micro-boiling 45min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 6 pharmaceutical preparation of the present invention
Rhizoma Corydalis 10 parts (500g), Ramulus Uncariae Cum Uncis 8 parts (400g), Radix Polygoni Multiflori 4 parts (200g), Rhizoma Chuanxiong 6 parts (300g), 4 parts of the Radix Rehmanniae (200g).
Weighing pharmaceutical decocting piece by said ratio, add 6 times of soak by water, micro-boiling 60min, filter, medicinal residues add 4 times of water, micro-boiling 45min, filter.Merge twice decoction liquor, be evaporated to relative density 1.25-1.30, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 7 pharmaceutical preparation of the present invention
Rhizoma Corydalis 1 part (50g), Ramulus Uncariae Cum Uncis 10 parts (500g), Radix Polygoni Multiflori 1 part (50g), Rhizoma Chuanxiong 1 part (50g), the Rhizoma Anemarrhenae 6 parts (300g), the Radix Paeoniae Alba 8 parts (400g).
Weighing pharmaceutical decocting piece by said ratio, add 12 times of soak by water, micro-boiling 90min, filter, medicinal residues add 10 times of water, micro-boiling 60min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 8 pharmaceutical preparation of the present invention
Rhizoma Corydalis 5 parts (250g), Ramulus Uncariae Cum Uncis 4 parts (200g), Radix Polygoni Multiflori 6 parts (300g), Rhizoma Chuanxiong 4 parts (200g), 1 part of the Radix Rehmanniae (50g), Radix Codonopsis 10 parts (500g).
Weighing pharmaceutical decocting piece by said ratio, add 10 times of soak by water, micro-boiling 60min, filter, medicinal residues add 6 times of water, micro-boiling 60min, filter.Merge twice decoction liquor, concentrating under reduced pressure, mix with appropriate dextrin, be dried, dry granulation, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 9 pharmaceutical preparation of the present invention
Rhizoma Corydalis 8 parts (400g), Ramulus Uncariae Cum Uncis 7 parts (350g), Radix Polygoni Multiflori 4 parts (200g), Rhizoma Chuanxiong 3 parts (150g), the Radix Astragali 1 part (50g), Radix Paeoniae Rubra 1 part (50g).
Weighing pharmaceutical decocting piece by said ratio, add 12 times of soak by water, micro-boiling 90min, filter, medicinal residues add 10 times of water, micro-boiling 45min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 10 pharmaceutical preparation of the present invention
Rhizoma Corydalis 6 parts (300g), Ramulus Uncariae Cum Uncis 8 parts (400g), Radix Polygoni Multiflori 1 part (50g), Rhizoma Chuanxiong 1 part (50g), the Radix Astragali 6 parts (300g), the Rhizoma Anemarrhenae 1 part (50g), the Radix Paeoniae Alba 6 parts (300g).
Weighing pharmaceutical decocting piece by said ratio, add 12 times of soak by water, micro-boiling 90min, filter, medicinal residues add 8 times of water, micro-boiling 45min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 11 pharmaceutical preparation of the present invention
Rhizoma Corydalis 1 part (50g), Ramulus Uncariae Cum Uncis 1 part (50g), Radix Polygoni Multiflori 4 parts (200g), Rhizoma Chuanxiong 4 parts (200g), the Radix Astragali 1 part (50g), the Rhizoma Anemarrhenae 4 parts (200g), the Radix Paeoniae Alba 1 part (50g).
Weighing pharmaceutical decocting piece by said ratio, add 12 times of soak by water, micro-boiling 90min, filter, medicinal residues add 8 times of water, micro-boiling 45min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
The preparation of embodiment 12 pharmaceutical preparation of the present invention
Rhizoma Corydalis 10 parts (500g), Ramulus Uncariae Cum Uncis 10 parts (500g), Radix Polygoni Multiflori 6 parts (300g), Rhizoma Chuanxiong 6 parts (300g), the Radix Astragali 10 parts (500g), the Rhizoma Anemarrhenae 6 parts (300g), the Radix Paeoniae Alba 8 parts (400g).
Weighing pharmaceutical decocting piece by said ratio, add 12 times of soak by water, micro-boiling 90min, filter, medicinal residues add 8 times of water, micro-boiling 45min, filter.Merge twice decoction liquor, concentrating under reduced pressure, with appropriate dextrin as bed material, spray into concentrated solution, fluid-bed marumerization, make 1000g, packaging, often bag 20 grams, every day 2 times, each 1 bag.
Below by way of test example, beneficial effects of the present invention is further elaborated:
Pharmacological research test example 1
(1) experiment material
Animal: male C57 mice, body weight 24 ± 1g, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..Cleaning grade, the quality certification number: SCXK (capital) 2009-0007.Rearing conditions: barrier level Animal House is raised, credit number: SYXK (capital) 2009-0004, raises in feeding standard box, 5, every box.
Test medicine:
The Chinese medicine composition (correspondence is administered 1-12 group respectively) of embodiment of the present invention 1-12.
Chinese medicine composition disclosed in the Rhizoma Anemarrhenae 3 parts (300g), Radix Angelicae Sinensis 1 part (100g), Radix Paeoniae Alba 3 parts (300g), Ramulus Uncariae Cum Uncis 5 parts (500g), the Radix Astragali 3 parts (300g), i.e. number of patent application 201210415109.2 (corresponding administration 13 groups).
Chinese medicine composition disclosed in 18 parts of Rhizoma Gastrodiae (180g), the Radix Astragali 18 parts (180g), Radix Rehmanniae Preparata 18 parts (180g), Radix Paeoniae Alba 22 parts (220g), Radix Angelicae Sinensis 16 parts (160g), Ramulus Uncariae Cum Uncis 20 parts (200g), i.e. number of patent application 201210062467.X (corresponding administration 14 groups).
L-DOPA: madopar (Benserazide sheet), Shanghai company limited of Roche Group, traditional Chinese medicines quasi-word H10930198, lot number: SH0895 (corresponding positive controls).
MPTP:Sigma Products, lot number: 128k1549.
DA and IP is purchased from Sigma company.
Remaining reagent is purchased from Beijing chemical reagents corporation, and purity is analytical pure.
(2) experimental technique
(1) model is set up
Mice employing turns rod method (Rotarod test) and trains 3 days in advance, and inharmonic mice that will move is rejected, random packet, often group 15.Mice lumbar injection every day MPTP (being dissolved in normal saline) 20mg/kg once, continuous 10 days.
(2) packet is administered
It is divided into Normal group, MPTP model group, positive controls, is administered 1-14 group.Being administered 1-14 group, be practiced formula extraction at example 1-14 respectively, dosage is converted according to each recipe quantity.Tested Chinese medicine is dissolved in aseptic double-distilled water, and madopar tablet (L-DOPA) is suspended in 0.5%CMC-Na.30min lumbar injection MPTP20mg/kg after tested Chinese medicine, L-DOPA administration, once a day, continuous 10 days.MPTP continues to give mice Chinese medicine by reagent, L-DOPA once a day after stopping injection, continuous 7 days.Normal group is administered orally the aseptic double-distilled water with lumbar injection same dose and normal saline, and MPTP model group is orally administered to the aseptic double-distilled water of same dose.
(3) behavioristics's detection
Employing turns rod method and pole-climbing method (Pole test) evaluates the sports coordination ability of mice.
1. rod method is turned: turning rod instrument (institute of Materia Medica,Chinese Academy of Medical Sciences development) is diameter 3cm, long about 50cm, it is divided into the horizon bar of 5 sections with dividing plate, it is ensured that animal is the most unaffected.Turn rod instrument rotating speed and be set to 14 turns/min of constant speed.Mice is placed on bar, opens switch, start timing, record and turn the time that rod falls down to mice from bar from starting, be designated as incubation period (time dropped i.e. for the first time), represent its sports coordination ability with this.Every mice is tested 3 times, and every minor tick 1h averages.Mice, in the 5th day of experiment, turns rod test for 9 days and 16 days.
2. pole-climbing method: pole-climbing method is with a diameter 13mm, high 50cm, the smooth rod (institute of Materia Medica,Chinese Academy of Medical Sciences development) of the wooden shot of one diameter 3cm is arranged at top, it is disposed vertically, mice head is placed on downwards on the top ball of bar, allow it naturally climb down along bar, observe animal behavior during climbing down.Behavior during mice climbs down is scored by standard, and standards of grading are as follows: 5 points: operated by arms and legs, coordinates step by step to creep downwards；4 points: creep downwards but have hind leg concurrently and slide behavior；3 points: get over a half-distance backward under slide, but bar can be held tightly；2 points: do not get over a half-distance and the behavior of sliding i.e. occurs；1 point: bar can not be grabbed after getting over a half-distance and drop from bar；0 point: do not get over a half-distance and i.e. can not grab bar, drop from bar.Before experiment, every mice is trained twice.In experiment pole-climbing in the 6th, 10,17 day test, every mice is tested 2 times, and every minor tick 1h marks according to above-mentioned standard, averages.
(4) striatal dopamine levels detection
High performance liquid chromatograph: Shimadzu LC-10AT vp Plus, detector: BASi CC-5e, LC-4C
1. chromatographic condition: flowing phase: sodium acetate-citrate buffer solution, containing citric acid 85mM, anhydrous sodium acetate 100mM, EDTA Na20.2mM, is first made into 850ml, adds 150ml methanol afterwards, tri-distilled water is settled to 1L, regulation pH is 3.68, adds appropriate SOS (first adding 90mg, depending on separation situation), positive dibutyl amine (first adding 15 μ L) makes peak be kept completely separate after sucking filtration.
2. instrument parameter: flow rate pump 1.2ml/min, detector detection sensitivity 10nA, glass carbon working electrode, Ag/AgCl reference electrode, detects current potential 0.76V, column temperature 25 DEG C.
3. standard substance preparation: weigh DA, IP and be dissolved in 0.1M HCLO respectively4In, it is configured to standard substance storing solution, takes storing solution and quantitatively mix, use 0.1M HCLO4Dilution makes each concentration of component be 0.125 μ g/ml, and IP is 0.25 μ g/ml.
4. the preparation of tissue homogenate: add IP in the perchloric acid solution of A liquid: 0.6mol/L, make the final concentration of 0.375 μ g/ml of IP, 4 DEG C of preservations.B liquid: containing potassium citrate 20mM, dipotassium hydrogen phosphate 300mM, EDTA Na22mM, 4 DEG C of preservations.
5. sample treatment: tissue sample processing procedure is all carried out under condition of ice bath, it then follows the quick principle of low temperature.Adding A liquid, be homogenized, 4 DEG C, 20000g is centrifuged 20 minutes, draws a certain amount of supernatant, adds the B liquid of half volume, ice bath 30min, mixing, stands, 20000g, and 4 DEG C are centrifuged 20 minutes, draws supernatant, 20000g, and 4 DEG C are centrifuged 20 minutes, draw supernatant, and 4 DEG C of preservations are to be measured.
(5) SABC detection nigral dopaminergic neuron
After Behaviors survey, often organize and take 5 mices at random, take brain after perfusion fixation, for Immunohistochemical detection.
The preparation of solution:
1. 4% paraformaldehyde perfusion fixative (pH7.4, containing 3% sucrose)
By 40g paraformaldehyde, 28.64g Na2HPO4·12H2O is dissolved in 800mL deionized water, heating for dissolving.Add 30g sucrose and 3.12g NaH2PO4·12H2O, adjusts pH value to 7.4 with 1N NaOH, adds distilled water and be settled to 1000mL, uses in filtering rear 4 DEG C of preservations, one week.
2. fixative after
4% paraformaldehyde perfusion fixative (pH7.4) adds 270g sucrose, dissolves, is settled to 1L.
3. 0.01mol/L phosphate buffer (PBS) (pH7.4)
9g NaCl is dissolved in 800mL distilled water, adds 0.1mol/L Na2HPO480mL, 0.1mol/L NaH2PO4120mL, adjusts pH to 7.4.
4. 0.01mol/L phosphate-Triton washing liquid (PBST)
0.01mol/L PBS 1000mL is placed in 37 DEG C of water-baths, is slowly added dropwise Triton X-100 (final concentration 0.3%) while stirring so that it is fully dissolve.
5. DAB nitrite ion
Each 1 of A, B, C liquid during order adds DAB colour reagent box in 1mL distilled water, mixing uses after filtering, matching while using.
6. bonding die liquid
The beaker filling a small amount of distilled water is put into 60-70 DEG C of water bath, adds 5g gelatin, after dissolving, put into 0.5g chromic potassium sulfate, to be dissolved after be settled to 1000mL, filter paper filtering, bonding die under water bath with thermostatic control, the slide glued is put in baking box 60 DEG C and is dried.
The perfusion fixation of cerebral tissue:
Mice, with after 0.5% pentobarbital sodium intraperitoneal injection of anesthesia, is lain on the back on platform, stretches fixing limbs, opens chest and abdomen and fully expose heart and liver.With No. 7 syringe needles from apex slightly biased left from inserting needle insert left ventricle, adjust needle point to ascending aorta direction, fixing, cut off right auricle simultaneously.After first quick filling warm saline to liver bleaches completely, right auricle flows out supernatant liquid, use 4% paraformaldehyde of 4 DEG C of pre-coolings again instead (containing 3% sucrose, prepare with the PBS of pH7.4) perfusion, first quick and back slow, treat that mouse liver is hardening, limb rigidity i.e. completes to fix.Broken end, takes brain, is placed in 4% paraformaldehyde solution, 4 DEG C overnight after, proceed in the rear fixative containing 30% sucrose 4% paraformaldehyde, the volume ratio of specimen and rear fixative is about 1:20.Change after mouse brain sinks to container bottom the newest after fixed solution, 4 DEG C of preservations.
The preparation of frozen section:
Cerebral tissue specimen is taken out from rear fixative, blots the residual liquid on surface with filter paper.Section is repaired so that it is smooth, in order to be fixed in the specimen torr of freezing stage with knife blade.The specimen fixed is placed in the cryovial filling a small amount of isopentane, puts into liquid nitrogen flash freezer about 10 seconds.Tissue fixed station adds a little frozen water, the specimen after quick-freezing is perpendicular to rapidly table top and puts on the freezing stage of the pre-freezing microtome being cooled to-25 DEG C.Frozen water is successively dripped so that it is be embedded in specimen torr around being organized into an icicle shape, make its major axis vertical with icicle plane, it is simple to completely to switch to the transverse section of whole tissue as far as possible around tissue contacts with ice face.After specimen embeds completely, cut into slices in row continuous coronal face, black substance position, thickness 20 μm, it is sequentially placed into equipped with in 24 orifice plates of PBST, cleans the fixative of residual with PBST, 5 minutes × 3 times.
Immunohistochemical staining:
3%H2O2 incubated at room 10 minutes, removes endogenous peroxidase activity, sucking-off H2O2, and distillation washing, 5min × 3 time, to non-foam；PBST washes, 5min × 3 time；30min is closed, to reduce non-specific adsorption by sheep blood serum stock solution room temperature；Sucking-off close serum, add dilute one anti-respectively with PBST and nonimmune normal serum replace one anti-foundation blank and negative control；It is placed in incubated at room temperature 2h in horizontal shaker, 4 DEG C of overnight incubation.Sucking-off one resists；PBST washes, 5min × 3 time；Add the anti-goat antibody of biotin labeled rabbit (two resist, and dilution factor is 1:300), incubated at room 2h；Sucking-off two resists；PBST washes, 5min × 3 time；Add horseradish peroxidase (HRP) labelling streptavidin (three resist, and dilution factor is 1:300), incubated at room 2h；Sucking-off three resists, and PBST washes, 5min × 3 time.With DAB working solution colour developing about 2min；Controlling dye levels under microscope, tap water color development stopping is reacted；Brain section after colour developing is placed in 0.01mol/L PBST.Brain section is fixed on the microscope slide processed with bonding die liquid, naturally dries.75%, 80%, 90%, 95% graded ethanol is dehydrated successively, each 10min；100% dehydration of alcohol 2 times, each 10min；Transparent 2 times of dimethylbenzene, each 5min；Resinene mounting.
Interpretation of result:
The change of light Microscopic observation mice TH neuron.Take pictures under 4 times of object lens, count substantia nigra compacta TH positive neuron number, the brain sheet of every mice is averaged into this mice TH positive neuron number last.
(6) statistical analysis
Statistical analysis is carried out with SPSS13.0.Change variance analysis between different groups, then checks with LSD-SNK.P < 0.05 is for having significant difference.
(3) experimental result
(1) the mice subacute PD model that MPTP is induced by Chinese medicine composition of the present invention turns the ethological improvement result of rod
Turning rod result to show, 5d, 9d and 16d MPTP model mice in experiment turns rod and drops incubation period significantly lower than normal control mice.Each administration group all can significantly improve mice at 5d, 9d and 16d and turn rod incubation period, has significant difference compared with model group；Compared with administration group 13,14, administration group 1-12 demonstrates and preferably turns rod behavior improvement result (table 1).
The impact of MPTP is caused by table 1. Chinese medicine composition of the present invention subacute PD model mice Zhuan Gun behavioristics damage
| Group | Day5 | Day9 | Day16 |
| Normal group | 100.2±8.13 | 101.37±6.09 | 108.27±4.67 |
| MPTP model group | 55.43±7.19** | 61.97±5.75** | 70.00±7.01** |
| It is administered 1 group | 79.07±8.35# | 98.30±7.69## | 101.47±8.64## |
| It is administered 2 groups | 79.17±9.25# | 92.60±7.57## | 96.25±9.62# |
| It is administered 3 groups | 78.80±8.14# | 89.79±7.83## | 94.21±8.91# |
| It is administered 4 groups | 83.93±9.11# | 91.37±7.93## | 96.83±9.13# |
| It is administered 5 groups | 81.08±7.95# | 94.28±6.98## | 102.54±7.94## |
| It is administered 6 groups | 80.26±9.86# | 95.30±6.87## | 96.76±9.58# |
| It is administered 7 groups | 78.23±6.74# | 93.79±8.43## | 94.21±5.65# |
| It is administered 8 groups | 82.43±7.42# | 92.76±9.13## | 95.78±8.37# |
| It is administered 9 groups | 85.10±7.93# | 88.83±8.68## | 91.43±7.92# |
| It is administered 10 groups | 90.25±8.12# | 99.31±7.25## | 102.28±5.93## |
| It is administered 11 groups | 92.34±8.67# | 98.65±8.05## | 103.24±6.45## |
| It is administered 12 groups | 9187±8.74# | 101.01±7.34## | 105.38±7.54## |
| It is administered 13 groups | 76.43±8.91# | 89.75±8.78## | 93.17±8.76# |
| It is administered 14 groups | 75.46±7.68# | 86.24±6.78## | 94.54±8.49# |
| Positive controls | 92.60±6.88# | 100.13±5.96## | 104.07±7.08## |
**P < 0.01vs.Control mice,#P<0.05,##P < 0.01vs.MPTP model mice.
(2) the mice subacute PD ethological improvement result of model pole-climbing that MPTP is induced by Chinese medicine composition of the present invention
Grasping clubglass test result shows, at 6d, 10d and the 17d of experiment, the pole-climbing scoring compared with normal mouse of MPTP model mice substantially reduces.Experiment 6d, the 10th day and 17 days, each administration group all can significantly improve mice pole-climbing scoring, there is compared with model group significant difference (table 2).
The impact of MPTP is caused by table 2. Chinese medicine composition of the present invention subacute PD model mice pole-climbing behavioristics damage
| Group | Day6 | Day10 | Day17 |
| Normal group | 4.77±0.08 | 4.83±0.08 | 4.80±0.08 |
| MPTP model group | 3.70±0.14** | 3.80±0.14** | 3.80±0.13** |
| It is administered 1 group | 4.13±0.13# | 4.67±0.14## | 4.37±0.13## |
| It is administered 2 groups | 4.03±0.11 | 4.43±0.15## | 4.46±0.15## |
| It is administered 3 groups | 4.08±0.12 | 4.61±0.14## | 4.50±0.14## |
[0129] [0129]
| It is administered 4 groups | 4.15±0.14# | 4.77±0.13## | 4.47±0.14## |
| It is administered 5 groups | 4.16±0.14# | 4.83±0.14## | 4.47±0.13## |
| It is administered 6 groups | 4.01±0.12 | 4.47±0.14## | 4.65±0.14## |
| It is administered 7 groups | 4.08±0.13 | 4.71±0.14## | 4.60±0.14## |
| It is administered 8 groups | 3.97±0.13 | 4.61±0.12## | 4.60±0.15## |
| It is administered 9 groups | 4.03±0.13 | 4.50±0.15## | 4.30±0.16# |
| It is administered 10 groups | 4.20±0.12# | 4.93±0.17## | 4.77±0.16## |
| It is administered 11 groups | 4.18±0.13# | 4.95±0.15## | 4.64±0.15## |
| It is administered 12 groups | 4.21±0.12# | 4.96±0.19## | 4.87±0.16## |
| It is administered 13 groups | 4.06±0.15 | 4.57±0.16## | 4.53±0.14## |
| It is administered 14 groups | 4.02±0.14 | 4.43±0.17## | 4.34±0.13# |
| Positive controls | 4.37±0.11## | 4.57±0.13## | 4.60±0.43## |
**P < 0.01vs.Control mice,#P<0.05,##P < 0.01vs.MPTP model mice
(3) DA and the impact of mice nigral DA neurons in the subacute PD model mice striatum that MPTP is caused by Chinese medicine composition of the present invention
DA is the regulation sensorimotor important neurotransmitter of human body, and it is to cause the handicapped immediate cause of PD patient moving that DA level reduces.HPLC-ED testing result shows that MPTP model group mouse striaturn DA content is significantly lower than Normal group.Administration group 1-12 all can improve MPTP model group mouse striaturn DA content, has significant difference compared with model group；Administration group 13-14 numerically can improve MPTP model group mouse striaturn DA content, but compared with model group, no difference of science of statistics (table 3).
Showed by immune group result, MPTP model group mice substantia nigra compacta TH positive neuron number compared with normal mice quantity substantially reduces.Positive controls to mice TH neuronal quantity without significantly improving effect；Administration group 1-12 is all remarkably improved mice TH neuronal quantity, has significant difference compared with model group；Administration group 13-14 numerically can improve mice TH neuronal quantity, but compared with model group, no difference of science of statistics (table 3).
DA and the impact of mice nigral dopaminergic neuron in the subacute PD model mice striatum that MPTP is caused by table 3. Chinese medicine composition of the present invention
| Group | DA (μ g/g wet tissue) | DA positive cell number |
| Normal group | 4.24±0.89 | 66.17±8.15 |
| MPTP model group | 1.52±0.11** | 35.47±4.72** |
| It is administered 1 group | 2.92±0.61# | 48.47±13.90# |
[0136] [0136]
| It is administered 2 groups | 2.94±0.25# | 47.53±5.15# |
| It is administered 3 groups | 3.03±0.10# | 46.56±12.30# |
| It is administered 4 groups | 3.24±0.04# | 49.63±6.18 |
| It is administered 5 groups | 2.98±0.28# | 46.67±7.09# |
| It is administered 6 groups | 3.06±0.26# | 47.80±7.24# |
| It is administered 7 groups | 3.14±0.06# | 46.38±12.26# |
| It is administered 8 groups | 2.98±0.25# | 40.03±6.27# |
| It is administered 9 groups | 3.08±0.18# | 48.60±5.87# |
| It is administered 10 groups | 3.18±0.19# | 49.27±6.45# |
| It is administered 11 groups | 3.23±0.17# | 49.56±6.48# |
| It is administered 12 groups | 3.27±0.16# | 49.39±6.39# |
| It is administered 13 groups | 2.38±0.54 | 40.47±7.57 |
| It is administered 14 groups | 2.31±0.64 | 41.54±8.14 |
| Positive controls | 4.06±0.26## | 35.53±4.56 |
**P < 0.01vs.Control mice,#P < 0.05vs.MPTP model mice
Pharmacological research test example 2
(1) experiment material
Cell:
BV-2 cell is purchased from cell institute of the Chinese Academy of Sciences.
Test medicine:
The Chinese medicine composition (correspondence is administered 1-12 group respectively) of embodiment of the present invention 1-12.
Chinese medicine composition disclosed in the Rhizoma Anemarrhenae 3 parts (300g), Radix Angelicae Sinensis 1 part (100g), Radix Paeoniae Alba 3 parts (300g), Ramulus Uncariae Cum Uncis 5 parts (500g), the Radix Astragali 3 parts (300g), i.e. number of patent application 201210415109.2 (corresponding administration 13 groups).
Chinese medicine composition disclosed in 18 parts of Rhizoma Gastrodiae (180g), the Radix Astragali 18 parts (180g), Radix Rehmanniae Preparata 18 parts (180g), Radix Paeoniae Alba 22 parts (220g), Radix Angelicae Sinensis 16 parts (160g), Ramulus Uncariae Cum Uncis 20 parts (200g), i.e. number of patent application 201210062467.X (corresponding administration 14 groups).
Reagent:
PDTC (Ammonium pyrrolidinedithiocarbamate) and MUP (4-Methylumbelliferyl phosphate) is purchased from sigma company, cell culture fluid and serum purchased from Gibco company.
(2) experimental technique
Amaxa gene electroporation is utilized to proceed in BV-2 cell, by 3 × 10 by combining promoter containing NF-κ B with Secreted alkaline phosphatase SEAP translation composition sequence plasmid downstream5/ ml kind, in 48 orifice plates, is placed in incubator and hatches, and after 12 hours, carries out modelling and experiment packet (1) blank group: BV-2 cell adds equal-volume LPS solvent, is placed in 37 DEG C and continues to cultivate 24 hours；(2) model control group: BV-2 cell adds the LPS of final concentration of 1mg/mL, is placed in 37 DEG C and continues to cultivate 24 hours；(3) PDTC positive compound and test-compound group: each compound is made into mother solution with DMSO respectively, culture fluid dilution 1000X becomes use liquid before use.BV-2 cell is hatched through the culture fluid containing PDTC (50uM) or 400mg/mL Chinese medicine test-compound and is cultivated 6 hours in 37 DEG C, adds the LPS of final concentration of 1mg/mL, continues to hatch 24 hours.Every culture hole takes culture fluid 100 μ l, is transferred to 96 orifice plates.Add MUP (72 μMs) 100 μ l immediately, after hatching 20 minutes, utilize the fluorescence intensity of Novostar microplate reader detection culture fluid；Represent the activity of NF-κ B with fluorescence intensity level, fluorescence intensity level is the lowest, illustrates that compound is the highest to the suppression ratio of NF-κ B, and action effect is the most obvious；Otherwise otherwise then.
Computational methods:
Suppression ratio=(to be measured group of fluorescence intensity-matched group fluorescence intensity)/matched group fluorescence intensity × 100%
Statistical analysis
Statistical analysis is carried out with SPSS13.0.Utilize one factor analysis of variance, then check with LSD-SNK.P < 0.05 is for having significant difference.
(3) experimental result
Chinese medicine composition of the present invention is to the inhibition by lipopolysaccharide-induced microglia BV-2NF-κ B Inflammatory Pathway
NF-κ B path is one of important path in inflammatory reaction, relates to multiple inflammatory factor and the translation of related inflammation albumen (such as COX-2, iNOS etc.) downstream.Under normal physiological condition, NF-κ B and I κ B exists with composite form.And in inflammatory reaction, IKK response stream signal is also activated, and by I κ B phosphorylation, the latter is degraded immediately, discharges and starts transcribing and translating of inflammation-related factor and albumen after NF-κ B, NF-κ B enters core.Microglia is one of inflammatory reaction cell important in central nervous system, is all that blood monocytes differentiates with the macrophage of periphery.It is to include that NF-κ B combines promoter and translates composition sequence with Secreted alkaline phosphatase SEAP downstream that the transfection used in this model builds plasmid, is proceeded to microglia system BV-2 cell, for screening compound.Process the BV-2 cell proceeding to related plasmids with lipopolysaccharide, in cell, NF-κ B signal pathway activity raises, and NF-κ B discharges via NF-κ B/I κ B complex, and combines the specificity promoter built on plasmid, starts synthesis and the secretion of SEAP.Weigh the activity height screening of NF-κ B path by content and the activity of SEAP in detection training liquid and evaluate compound.Test result indicate that, be administered 1,2,5,6,8-13 group to by lipopolysaccharide-induced BV-2 cell-stimulating NF-κ B path, there is significant impact, and administration group 14 is on not had significant difference compared with model control group by the impact of lipopolysaccharide-induced BV-2 cell-stimulating NF-κ B path, it is shown in Table 4.
Table 4. Chinese medicine composition of the present invention is on the impact by lipopolysaccharide-induced BV-2 cell-stimulating NF-κ B path
| Group | Fluorescence intensity |
| Blank group | 10252.16±1078.15 |
| Model control group | 64368.47±7823.51** |
| It is administered 1 group | 56328.45±5463.85# |
| It is administered 2 groups | 59621.25±7541.81# |
| It is administered 3 groups | 60234.54±5632.57 |
| It is administered 4 groups | 58995.75±5234.21 |
| It is administered 5 groups | 57826.65±5412.65# |
| It is administered 6 groups | 57245.85±5974.35# |
| It is administered 7 groups | 59875.27±5666.02 |
| It is administered 8 groups | 57235.44±5411.06# |
| It is administered 9 groups | 58027.05±6014.52# |
| It is administered 10 groups | 54258.39±6257.32## |
| It is administered 11 groups | 55643.36±6389.34## |
| It is administered 12 groups | 54743.33±6346.76## |
| It is administered 13 groups | 57574.49±7512.85# |
| It is administered 14 groups | 59257.95±6254.32 |
| PDTC positive controls | 51865.57±3695.27## |
**P < 0.01vs. blank fluorescent value,#P<0.05、#P < 0.01vs. model comparison fluorescent value；
Pharmacological research test example 3
(1) experiment material
Cell:
Undifferentiated PC12 cell is purchased from cell institute of the Chinese Academy of Sciences.
Test medicine:
The Chinese medicine composition (correspondence is administered 1-12 group respectively) of embodiment of the present invention 1-12.
Chinese medicine composition disclosed in the Rhizoma Anemarrhenae 3 parts (300g), Radix Angelicae Sinensis 1 part (100g), Radix Paeoniae Alba 3 parts (300g), Ramulus Uncariae Cum Uncis 5 parts (500g), the Radix Astragali 3 parts (300g), i.e. number of patent application 201210415109.2 (corresponding administration 13 groups).
Chinese medicine composition disclosed in 18 parts of Rhizoma Gastrodiae (180g), the Radix Astragali 18 parts (180g), Radix Rehmanniae Preparata 18 parts (180g), Radix Paeoniae Alba 22 parts (220g), Radix Angelicae Sinensis 16 parts (160g), Ramulus Uncariae Cum Uncis 20 parts (200g), i.e. number of patent application 201210062467.X (corresponding administration 14 groups).
Reagent:
Compound and reagent: H2O2Purchased from traditional Chinese medicines group；MTS reagent is purchased from Promega company；DMEM/LOW GLUCOSE culture fluid, hyclone and horse serum are purchased from Gibco company.
(2) experimental technique
Take undifferentiated PC12 cell, with every hole 5000c/w kind 96 orifice plate, after 37 DEG C of incubators continue to cultivate 16h, carry out modelling and experiment packet, (1) blank group: add equal-volume solvent, be not added with H2O2Rear continuation is cultivated；(2) model control group: add equal-volume solvent, every hole adds 300 μMs of H of final concentration2O2Rear continuation is cultivated；(3) Chinese medicine administration group: formula extraction at Chinese medicine embodiment 1-14, is made into mother solution with DMSO, dilutes 1000x with culture fluid before use, and after making final concentration reach 400mg/mL sample-adding, after continuing to cultivate 24h, every hole adds 300 μMs of H of final concentration2O2After 24h is cultivated in rear continuation, every hole adds 20uL MTs and continues to cultivate 4h, uses the OD value utilizing Novostar microplate reader detection culture fluid, and OD value is the biggest represents that the cell number of survival is the most, and vice versa.
Computational methods:
Survival rate=to be measured group of OD intensity/blank group OD intensity × 100%
Statistical analysis
Statistical analysis is carried out with SPSS13.0.Utilize one factor analysis of variance, then check with LSD-SNK.P < 0.05 is for having significant difference.
(3) experimental result
Chinese medicine composition of the present invention is to 300 μMs of H2O2The protective effect of induction PC12 cell model damage
The main pathological characters of parkinson disease is that the dopaminergic neuron of substantia nigra compacta significantly lacks, and Louis body occurs in the neuron remained.Recent domestic numerous studies show that oxidative stress causes the death of nigral dopaminergic neuron by all means, and oxidative stress is one of important pathogenesis of parkinson disease, and autopsy findings also indicates that and there is oxidative stress in parkinson disease.
Disturbances in patients with Parkinson disease dopamine oxidative metabolic processes produces a large amount of H2O2 and superoxide anion, under being catalyzed at black substance position Fe2+, generate the hydroxy radical that toxicity is bigger further, and the composite I activity decrease of now black substance mitochondrial respiratory chain, polyphenoils (particularly glutathion) disappears, it is impossible to remove free radical, therefore, free radical is by oxidation neurolemma lipoid, destruction DA neuron membrane function or directly destroys cell DNA, ultimately results in neuronal degeneration.
This experiment acts on Adrenal Pheochromocytoma PC12 simulation parkinson symptom for evaluating the anti-oxidation protection effect of different Chinese medicine composition by adding H2O2.Test result indicate that, be administered 1,3-8,10-12,14 groups of protective effects with significant PC12 cell, there is compared with model control group significant difference；Administration group 13 has the protective effect of certain PC12 cell, no difference of science of statistics compared with model control group.
Table 5. Chinese medicine composition of the present invention is to by H2O2The protective effect of induction PC12 cell
| Group | Light absorption value | Survival rate (%) |
| Blank group | 0.788±0.38 | / |
| Model control group | 0.382±0.49** | 48.50 |
| It is administered 1 group | 0.474±0.51# | 60.12 |
| It is administered 2 groups | 0.420±0.46 | 53.34 |
| It is administered 3 groups | 0.453±0.47# | 57.54 |
| It is administered 4 groups | 0.443±0.39# | 56.28 |
| It is administered 5 groups | 0.462±0.58# | 58.65 |
| It is administered 6 groups | 0.469±0.58# | 59.62 |
| It is administered 7 groups | 0.489±0.62# | 62.12 |
| It is administered 8 groups | 0.490±0.37# | 62.22 |
| It is administered 9 groups | 0.415±0.54 | 52.65 |
| It is administered 10 groups | 0.545±0.69# | 69.10 |
| It is administered 11 groups | 0.528±0.63# | 67.00 |
| It is administered 12 groups | 0.539±0.68# | 68.40 |
| It is administered 13 groups | 0.419±0.33 | 53.22 |
| It is administered 14 groups | 0.451±0.21# | 57.28 |
**P < 0.01vs. blank OD value,#P < 0.05vs. model comparison OD value.
Claims (9)
1. treat Parkinsonian Chinese medicine composition for one kind, it is characterised in that be mainly made up of the crude drug of following weight portion: prolong
Rhizoma Corydalis 1-10 part, Ramulus Uncariae Cum Uncis 1-10 part, Radix Polygoni Multiflori 1-6 part, Rhizoma Chuanxiong 1-6 part.
2. Chinese medicine composition as claimed in claim 1, it is characterised in that the weight portion of described crude drug is: Rhizoma Corydalis 1-8
Part, Ramulus Uncariae Cum Uncis 1-8 part, Radix Polygoni Multiflori 1-4 part, Rhizoma Chuanxiong 1-4 part.
3. Chinese medicine composition as claimed in claim 1, it is characterised in that described crude drug also includes the one in agents
Or several: Radix Codonopsis 1-10 part, Radix Astragali 1-10 part, Radix Paeoniae Alba 1-8 part, Radix Paeoniae Rubra 1-8 part, Rhizoma Anemarrhenae 1-6 part, Radix Rehmanniae 1-6 part.
4. Chinese medicine composition as claimed in claim 3, it is characterised in that described crude drug also includes QI invigorating class medicine, this benefit
Gas class medicine is selected from one or more of following medicine: Radix Codonopsis 1-10 part, Radix Astragali 1-10 part.
5. Chinese medicine composition as claimed in claim 3, it is characterised in that described crude drug also includes nourishing blood to suppress the hyperactive liver class medicine,
This nourishing blood to suppress the hyperactive liver class medicine is selected from one or more of following medicine: Radix Paeoniae Alba 1-8 part, Radix Paeoniae Rubra 1-8 part.
6. Chinese medicine composition as claimed in claim 3, it is characterised in that described crude drug also includes nourishing YIN to lower pathogenic fire class medicine,
This nourishing YIN to lower pathogenic fire class medicine is selected from one or more of following medicine: Rhizoma Anemarrhenae 1-6 part, Radix Rehmanniae 1-6 part.
7. Chinese medicine composition as claimed in claim 3, it is characterised in that described crude drug also includes the following: Radix Astragali 1-10
Part, Rhizoma Anemarrhenae 1-6 part, Radix Paeoniae Alba 1-8 part.
8. the Chinese medicine composition as described in any one of claim 1-7, it is characterised in that described Chinese medicine composition makes Chinese medicine
Oral preparations.
9. the preparation method of the Chinese medicine composition as described in any one of claim 1-8, it is characterised in that comprise the steps:
By described weight portion weighting raw materials, adding 6-12 times of soak by water, micro-boiling 30min-90min, filter, medicinal residues add 4-10 times of water,
Micro-boiling 30min-60min, filters；Merging twice decoction liquor, concentrate, extractum or dried extract powder add appropriate amount of auxiliary materials, system
Obtain traditional Chinese medicine particle preparation.
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| CN101167965A (en) * | 2007-10-25 | 2008-04-30 | 张玉红 | Traditional Chinese medicinal composition for treating senile parkinsonian syndrome |
| CN102366470A (en) * | 2011-09-30 | 2012-03-07 | 张丙焕 | Traditional Chinese medicinal preparation for treating Parkinsonism |
| CN103083623A (en) * | 2011-10-28 | 2013-05-08 | 上海中药制药技术有限公司 | Application of compound traditional Chinese medicine preparation in Parkinson disease treatment drug preparation |
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2015
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085062A (en) * | 2007-06-16 | 2007-12-12 | 梁子君 | Medicine for curing parkinson's disease and its preparing method |
| CN101167965A (en) * | 2007-10-25 | 2008-04-30 | 张玉红 | Traditional Chinese medicinal composition for treating senile parkinsonian syndrome |
| CN102366470A (en) * | 2011-09-30 | 2012-03-07 | 张丙焕 | Traditional Chinese medicinal preparation for treating Parkinsonism |
| CN103083623A (en) * | 2011-10-28 | 2013-05-08 | 上海中药制药技术有限公司 | Application of compound traditional Chinese medicine preparation in Parkinson disease treatment drug preparation |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109847044A (en) * | 2017-11-30 | 2019-06-07 | 兰州大学 | Four refreshing aproll extracts are preparing the application in antiparkinsonism drug |
| CN109847044B (en) * | 2017-11-30 | 2021-11-30 | 兰州大学 | Application of Sishen water extract in preparation of drug for resisting Parkinson's disease |
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| CN106309637B (en) | 2020-07-14 |
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