CN105327153B - Chinese medicine composition containing ganoderma lucidum and ginkgo leaf is used for the application of promotion rehabilitation after apoplexy - Google Patents
Chinese medicine composition containing ganoderma lucidum and ginkgo leaf is used for the application of promotion rehabilitation after apoplexy Download PDFInfo
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Abstract
The invention discloses a kind of Chinese medicine compositions containing ganoderma lucidum and ginkgo leaf to promote application in the drug of rehabilitation after preparing apoplexy, wherein, the composition is made of the following components: ganoderma lucidum 300-400 parts by weight, ginkgo leaf 300-400 parts by weight, Radix Achyranthis Bidentatae 200-290 parts by weight, radix scrophulariae 200-290 parts by weight, rhizoma acori graminei 150-190 parts by weight, Rhizoma Gastrodiae 100-140 parts by weight.The present invention is administered containing the traditional Chinese medicine composition for treating of ganoderma lucidum and ginkgo leaf has significant protective effect to intraluminal middle cerebral artery occlusion in rats occlusion reperfusion induced brain injury；Permanent cerebral ischemia caused by occluding for treatment intraluminal middle cerebral artery occlusion in rats has significant protective effect；There is significant inhibiting effect to PAF, ADP induction Platelet Aggregation in Rabbits；There is significant inhibiting effect to thrombosis；Mouse bleeding time and clotting time can significantly be extended, for promoting rehabilitation that there is remarkable result after apoplexy.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, and in particular to a kind of Chinese medicine composition containing ganoderma lucidum and ginkgo leaf is used for apoplexy
Promote the application of rehabilitation afterwards.
Background technique
Apoplexy, names of disease of tcm are divided into exogenous wind and interior wind, caused by exogenous wind is because of being invaded by exogenous pathogen,invasion of exogenous pathogen (ailment said due to cold or exposure), in treatise on Febrile Diseases name
Say apoplexy (also known as syndrome of decoction GUI ZHI)；Interior wind category diseases caused by internal damage, also known as apoplectic stroke, cerebral apoplexy, stroke, hemiplegia.Modern times commonly referred to as in
Wind refers to the apoplectic stroke of diseases caused by internal damage more, mostly because qi and blood is inverse disorderly, the resistance of brain vessle-Bi symptom-complex or blood are overflow caused by brain.Not with unexpected dusk servant, half body
Then, extremity numbness, tongue are not smoothgoing in silence, and dispute skew, hemianesthesia etc. is the brain disease mainly showed, and, change anxious with onset
Change fastly, the characteristics of change such as ailment said due to cold or exposure benefaction number.
Inventor promotes after experimental exploring finds that a kind of Chinese medicine composition containing ganoderma lucidum and ginkgo leaf is used to prepare apoplexy
The application of the drug of rehabilitation.
Summary of the invention
The object of the present invention is to provide a kind of Chinese medicine compositions containing ganoderma lucidum and ginkgo leaf to promote rehabilitation after preparing apoplexy
Drug in application.
The purpose of the present invention is what is be accomplished by the following way:
A kind of Chinese medicine composition containing ganoderma lucidum and ginkgo leaf promotes the application in the drug of rehabilitation after preparing apoplexy,
In, the composition is made of the component of following parts by weight:
Ganoderma lucidum 300-400 parts by weight ginkgo leaf 300-400 parts by weight Radix Achyranthis Bidentatae 200-290 parts by weight
Radix scrophulariae 200-290 parts by weight rhizoma acori graminei 150-190 parts by weight Rhizoma Gastrodiae 100-140 parts by weight
Above-mentioned composition is preferably made of the following components:
Ganoderma lucidum 300-350 parts by weight ginkgo leaf 300-350 parts by weight Radix Achyranthis Bidentatae 250-290 parts by weight
Radix scrophulariae 250-290 parts by weight rhizoma acori graminei 150-180 parts by weight Rhizoma Gastrodiae 110-140 parts by weight
Above-mentioned composition is most preferably made of the following components:
333 parts by weight ginkgo leaf of ganoderma lucidum, 333 parts by weight Radix Achyranthis Bidentatae, 278 parts by weight
278 parts by weight rhizoma acori graminei of radix scrophulariae, 167 parts by weight Rhizoma Gastrodiae, 125 parts by weight
Dosage form as defined in pharmacy can be made in above-mentioned Chinese medicine composition with the auxiliary material pharmaceutically allowed, and the dosage form is preferably
Capsule.
The preparation method of the composition the following steps are included:
A, rhizoma acori graminei is ground into particle；
B, rhizoma acori graminei particle is used into CO2Supercritical extract, extracting pressure 22-30Mpa, extraction kettle temperature are 45-60
DEG C, extraction time is 0.5-1.5 hours, obtains volatile oil and rhizoma acori graminei extract remainder；
C, it takes volatile oil to be dissolved with isometric ethyl alcohol, obtains volatile oil ethanol solution；Separately 4-8 times of volatile oil weight is taken to measure
Beta-cyclodextrin, the water measured with 3-5 times of beta-cyclodextrin grind well, pour into colloid mill, and it is molten that above-mentioned volatile oil ethyl alcohol is slowly continuously added dropwise
Liquid is milled and is included 20-40 minutes, pours out, and 0-4 DEG C of refrigeration stands 24 hours or more, filtration, in 40-80 DEG C of vacuum drying to get
Volatile oil clathrate compound；
D, ganoderma lucidum, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae and Rhizoma Gastrodiae and rhizoma acori graminei extract remainder are added total 6-10 times of medicinal material of prescription
Weight concentration is the ethyl alcohol of 70-90%, and heating and refluxing extraction 1-3 times is 1-2 hours each, and extracting solution merges, and is stood, and is filtered, filter
Ethyl alcohol is recovered under reduced pressure in liquid, the clear cream that relative density is 1.15~1.30 under the conditions of being concentrated into 60 DEG C；
E, clear cream is dried under reduced pressure at 60-80 DEG C, is got dry extract；
F, dry cream and volatile oil clathrate compound are uniformly mixed.
The preparation method of above-mentioned composition preferably includes following steps:
A, rhizoma acori graminei is ground into particle；
B, rhizoma acori graminei particle is used into CO2Supercritical extract, extracting pressure 22-30Mpa, extraction kettle temperature are 45-60
DEG C, extraction time is 0.5-1.5 hours, obtains volatile oil and rhizoma acori graminei extract remainder；
C, it takes volatile oil to be dissolved with isometric ethyl alcohol, obtains volatile oil ethanol solution；Separately 4-8 times of volatile oil weight is taken to measure
Beta-cyclodextrin, the water measured with 3-5 times of beta-cyclodextrin grind well, pour into colloid mill, and it is molten that above-mentioned volatile oil ethyl alcohol is slowly continuously added dropwise
Liquid is milled and is included 20-40 minutes, pours out, and 0-4 DEG C of refrigeration stands 24 hours or more, filtration, in 40-80 DEG C of vacuum drying to get
Volatile oil clathrate compound；
D, ganoderma lucidum, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae and Rhizoma Gastrodiae and rhizoma acori graminei extract remainder are added total 6-10 times of medicinal material of prescription
Weight concentration is the ethyl alcohol of 70-90%, and heating and refluxing extraction 1-3 times is 1-2 hours each, and extracting solution merges, and is stood, and is filtered, filter
Ethyl alcohol is recovered under reduced pressure in liquid, the clear cream that relative density is 1.15~1.30 under the conditions of being concentrated into 60 DEG C；
E, clear cream is mixed with the microcrystalline cellulose of clear cream weight 30-40%, is dried under reduced pressure, obtains under the conditions of setting 60 DEG C -80 DEG C
Dry cream；
F, dry cream and volatile oil clathrate compound are ground into fine powder, the microcrystalline cellulose and dry cream weight with clear cream weight 5-55%
0.5% magnesium stearate is uniformly mixed, and obtains fine powder.
The apoplexy sequelae shows as hemiplegia, and limb adynamia, dispute skew, speech is stuttering puckery, hypophrenia, mouth
Angular flux saliva.
The above-mentioned Chinese medicine composition containing ganoderma lucidum and ginkgo leaf is in the drug for preparing invigorating Qi, eleminating phlegm and freeing channels restoring consciouness
Middle application, wherein the composition is made of the component of following parts by weight:
Ganoderma lucidum 300-400 parts by weight ginkgo leaf 300-400 parts by weight Radix Achyranthis Bidentatae 200-290 parts by weight
Radix scrophulariae 200-290 parts by weight rhizoma acori graminei 150-190 parts by weight Rhizoma Gastrodiae 100-140 parts by weight.
Above-mentioned composition is preferably made of the following components:
Ganoderma lucidum 300-350 parts by weight ginkgo leaf 300-350 parts by weight Radix Achyranthis Bidentatae 250-290 parts by weight
Radix scrophulariae 250-290 parts by weight rhizoma acori graminei 150-180 parts by weight Rhizoma Gastrodiae 110-140 parts by weight.
Above-mentioned composition is most preferably made of the following components:
333 parts by weight ginkgo leaf of ganoderma lucidum, 333 parts by weight Radix Achyranthis Bidentatae, 278 parts by weight
278 parts by weight rhizoma acori graminei of radix scrophulariae, 167 parts by weight Rhizoma Gastrodiae, 125 parts by weight.
Beneficial effects of the present invention compared with the prior art:
The present invention is administered containing the traditional Chinese medicine composition for treating of ganoderma lucidum and ginkgo leaf to intraluminal middle cerebral artery occlusion in rats occlusion reperfusion institute
Cause cerebral injury that there is significant protective effect；Permanent cerebral ischemia caused by occluding for treatment intraluminal middle cerebral artery occlusion in rats has aobvious
The protective effect of work；There is significant inhibiting effect to PAF, ADP induction Platelet Aggregation in Rabbits；Have to thrombosis significant
Inhibiting effect；Mouse bleeding time and clotting time can significantly be extended.
In addition, the traditional chinese medicine composition of the invention, which can merge sodium dithionite (Na2S2O4), lacks the external cortex neural of sugar cause
First cell hypoxia sugar deficiency injury model plays a very good protection；Cortical neuron can be caused to platelet activating factor (PAF)
The protective effect of cellular oxidation stress damage model is obvious.The traditional chinese medicine composition of the invention Contained Serum therapeutic administratp is in vitro culture
Cortical neuron has significant protective effect.
Rehabilitation is promoted to be specifically described to after apoplexy below by way of test example.The test specimen abbreviation compound silver spirit of use
Open capsule is prepared by 1 method of embodiment.
One, test material
1, drug and reagent
Test drug title: compound silver Lingtong capsule: character: brown ceramic powder (is prepared) according to embodiment method, and people uses
Amount: adult takes raw medicinal herbs amount: 18.168g, 6.0g medicinal extract powder/people .d-1, every gram of medicinal extract powder is equivalent to crude drug 3.028g.
Preparation method: tested medicinal 0.5%CMC-Na grinding is configured to the homogenous suspension of required concentration.
Folium Ginkgo: Yangzijiang Pharmaceutical Group Co., Ltd, specification: every 19.2mg containing total flavonoids, terpene lactone
4.8mg, lot number: 12053121.Drug is prepared: being configured to the uniform suspension of required concentration with 0.5%CMC-Na grinding before use
Liquid.
Aspirin enteric coated tablet: Bayer HealthCare Co, specification: 100mg is ground with 0.5%CMC-Na before use
The homogenous suspension of required concentration is made in grinding-in.
Chloraldurate: Sinopharm Chemical Reagent Co., Ltd. is configured to concentration with 0.9% sodium chloride injection before use
For 3% solution.
Red tetrazolium (TTC), Chinese medicines group chemical reagents corporation product, weighs TTC powder before use, uses phosphate-buffered
Liquid dissolution is configured to 1% TTC solution.
Formalin: A.R, Xilong Chemical Co., Ltd's product, specification: 500ml/ bottles, lot number: 1005152.
Platelet activating factor (PAF): SIGMA Products.
It prepares: 1. taking PAF1mg, being placed in 2.6ml sodium chloride injection makes to dissolve, and is sub-packed in tool plug seal pipe
0.1ml/ branch, -20 DEG C of preservations are spare (concentration is 7.072 μm of ol/L) with 100 times of dilutions of sodium chloride injection before use.2. taking
PAF 5mg is formulated as mother liquor, -20 DEG C of preservations with DMSO dissolution.Required concentration, which is diluted to, with sterilizing PBS before use (acts on mind
Final concentration of 40 μm of ol/L through member).
5 ' ﹣ adenosine diphosphate disodium salts (ADP): SIGMA company, lot number: YY1124B056W.
It prepares: 1. taking 10mg ADP, set in 0.9% sodium chloride injection of 6.64ml, shake to being completely dissolved, be first made into
The preservation liquid of 3mmol/L is sub-packed in tool plug seal pipe, 0.1ml/ branch, -20 DEG C of preservations.Before use with 0.9% sodium chloride injection
10 times of dilution can (concentration be 300 μm of ol/L).2. taking 400mg ADP, the solution of 20mg/ml is made into physiological saline.
MEM culture medium: GIBCO company, the U.S., lot number: 1133365.It takes MEM powder to be dissolved in three steaming of 1000ml sterilizing, uses
To 7.0, filtration sterilization, packing, 4 DEG C of refrigerators save NaHCO3 tune pH value.Use 10% fetal calf serum of preceding addition.
Sugar-free EBSS (Earle ' s balanced salt solution) buffer: weigh NaCl 6.8g,
NaHCO32.2g、KCl 0.4g、CaCl2·2H2O 0.265g、MgSO40.09767g、NaH2PO40.122g is dissolved in 1000ml tri-
It steams in water, pH7.2-7.4, filtration sterilization, 4 DEG C of refrigerators save.
Containing sugared EBSS buffer: weighing NaCl 6.8g, NaHCO32.2g、KCl 0.4g、CaCl2·2H2O 0.265g、
MgSO40.09767g、NaH2PO40.122g, glucose 1g, are dissolved in 1000ml tri-distilled water, pH7.2-7.4, filtration sterilization, and 4 DEG C
Refrigerator saves.
Digestive juice: pancreatin, BIOSHARP Products；Pancreatin is assigned to 0.25% with PBS, filtration sterilization；EDTA is matched with PBS
To 0.02%, high pressure sterilization.Pancreatin, EDTA 1:1 mixing, 4 DEG C of refrigerators save.
β-actin, goat antirabbit (anti-mouse) IgG: Beijing Bo Aosen Bioisystech Co., Ltd product.
Syn antibody, NF- κ B p65 antibody, NF- κ B pp65 antibody: I κ B Alpha antibodies, pI κ B Alpha antibodies are Santa Cruz
Biotechnology Products.
2, experimental animal
Male cleaning grade SD rat, 250~300g of weight, Suzhou Industrial Park Ai Ermaite Science and Technology Ltd. provide,
Production licence number: SCXK (Soviet Union) 2009-0001；White rabbit, weight 1.8-2.0kg are moved by Jiangning county's Qinglongshan
Object breeding farm provides, animal productiong licensing number: SCXK (Soviet Union) 2012-0008.
Two, experimental method and result:
1, compound silver Lingtong capsule therapeutic administratp makees the protection of intraluminal middle cerebral artery occlusion in rats occlusion reperfusion induced brain injury
With (being administered 3 times within first day, be administered once a day, successive administration 14 days) later
The influence of 1.1 pairs of death rates, Neurological deficits, brain water content, infraction rate
Cleaning grade male SD rat 105, weight 250-300g, using line brush preparation intraluminal middle cerebral artery occlusion in rats occlusion
(MCAO) model[1].For pseudo- operation group rat in addition to being not inserted into line bolt, other operate same model group.Animal is divided into 7 groups after modeling,
Every group 15,3 dosage groups of compound silver Lingtong capsule are respectively as follows: 1.2g/kg (0.24g/ml), 0.6g/kg (0.12g/ml),
0.3g/kg(0.06g/ml).Positive control drug ginkgo biloba p.e group 4mg/kg (0.8mg/ml).Separately set pseudo- operation group and ischemic
Model control group (0.5%CMC-Na for giving isometric(al)).3h starts gastric infusion after multiple filling, is administered once a day, always
It is administered 15 days altogether, first administration dosage is 3 times of maintenance dose.
The death condition (symptom and death toll before dead) of animal is observed and recorded in experimentation.Record depositing for animal
Motility rate.
The preparation of fishing line: by a long 40mm, one end of the fishing line of diameter 0.26mm is marked with marking pen, apart from this label
A label is made again of marking pen at the 18mm of end, is placed in spare in physiological saline.
0.5h begins after being administered in first time, every other day referring to neurological deficit point system (18 minute marks quasi-) to each group rat into
Row scoring[2], standard is as follows:
Rat nerve neurological deficit score standard
After These parameters measurement, by rat anesthesia, brain temperature is surveyed, puts to death rat later.Brain is taken out, is immediately placed in -20 DEG C
Quick-frozen 10-15min, until when mouse brain is hardened, since antinion 2mm, do it is coronal cut five knives, rear to brain and hindbrain have a common boundary
Brain piece is placed in rapidly in the phosphate buffer solution of appropriate 1%TTC by place after being cut into 2mm thick six[3], it is protected from light temperature and incubates[4], often
It was stirred every 7~8 minutes once, observes brain piece color change frequently, take out brain piece (about 30min) when distinctness shows when red color area,
Computer is inputted after being taken pictures with digital camera, calculates infarct (white) and two halves brain volume, meter with Image pro plus later
It is as follows to calculate infarct percent:
Cerebral infarction volume percentage={ [total Infarction volume-(right half brain volume-half brain volume of a left side)]/left half brain volume } ×
100%.
Claim to obtain the brain tissue after dyeing to big brain wet weight, is subsequently placed in 110 DEG C of baking oven drying, compares big brain wet weight and find out brain
Water content is as follows[5]:
Brain water content (%)=(1- brain tissue dry weight/brain tissue weight in wet base) × 100%
It the results are shown in Table 1~table 2.
Influence of the table 1 to the Focal Cerebral Ischemia-Reperfusion in Rats death rate
The each group death rate is as shown above.It is increased with the well-informed dosage of silver, rats death rate has the tendency that gradually decreasing.
Influence of the table 2 to Focal Cerebral Ischemia-Reperfusion in Rats Neurological deficits
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；*P < 0.05,**P < 0.01vs. model control group；ap<
0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
As can be seen from Table 2, pseudo- operation group has no apparent neuroethology damage.Compared with pseudo- operation group, model group lacks certainly
Daystart after blood, the extremely significant property of Neurological deficits reduce (p < 0.01).Compared with model control group, the well-informed height of silver
Dosage group energy conspicuousness increases 8d rat nerve neurological deficit score (p < 0.05) after ischemia-reperfusion, and the extremely significant property of energy, which increases, to be lacked
10d, 12d, 14d rat nerve neurological deficit score (p < 0.01) after blood Reperfu- sion；The well-informed middle dose group energy conspicuousness of silver increases
12d rat nerve neurological deficit score (p < 0.05) after ischemia-reperfusion, the extremely significant property of energy increase 14d rat after ischemia-reperfusion
Neurological deficits (p < 0.01).Silver has quick access to information each dosage group compared with Folium Ginkgo group, the well-informed low dose group 14d nerve of silver
Neurological deficit score conspicuousness reduces (p < 0.05).
Influence of the table 3 to brain of rat with focal cerebral ischemia reperfusion temperature, infarct percent and brain water content
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；*P < 0.05,**P < 0.01vs. model control group；ap<
0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
Seen from table 3,14d after ischemia-reperfusion, each mild brain water content of modeling group brain equal nothing compared with pseudo- operation group
Significant difference (p > 0.05)；14d after ischemia-reperfusion, pseudo- operation group have no apparent infarcted region, model control group cerebral infarction
Percentage is 41.4 ± 9.8%, with model control group compared with the extremely significant property reduction of the well-informed high dose group cerebral infarction percentage of silver (p <
It 0.01), is 19.5 ± 6.7%；The well-informed middle dose group cerebral infarction percentage of silver is 29.9 ± 5.5%, compared with model control group
Conspicuousness reduces (p < 0.05)；Also extremely significant property reduces (p < 0.05) for Folium Ginkgo group and aspirin group cerebral infarction percentage,
Respectively 16.6 ± 4.1% and 14.3 ± 4.4%.
Compared with Folium Ginkgo group, during silver is well-informed, the extremely significant property of low dose group cerebral infarction percentage increase (p < 0.01)；With
Aspirin group is compared, during silver is well-informed, the extremely significant property of low dose group cerebral infarction percentage increase (p < 0.01).
The influence of 1.2 pairs of learning and memory functions, local cerebral blood flow and pathologyofbraintissue
Male SD rat 105 separately are taken, modeling and medication are same as above.After the 14th administration 0.5h (it is multiple fill after the
13d measures rat learning and memory function using Y type labyrinth[6]: tested rat is put into after adapting to 8min in labyrinth, gives electricity
Stimulation, random to change electric shock area and safety zone, otherwise it is that mistake is anti-that it is correct response that rat, which is escaped straight to safety zone, when electric shock
It answers[7].With in continuous 10 times electric shocks, accumulative number of shocks when correct response is up to 9 times indicates school grade；Learn into
After achievement measurement for 24 hours, it continuously shocks by electricity to rat 10 times, wherein correct response number is the Memory result of the rat.Then abdominal cavity
Inject 3% chloraldurate (300mg/kg) anesthetized rat.Rat head is fixed with rat stereotaxic apparatus, exposure skull,
The bone window (endocranium cannot be damaged) that a diameter is 2-3mm is bored at 2mm under the right 2mm/ of bregma with gear division sander, it will
TSD144 type laser Doppler probe is placed in the surface of thicker blood vessel in bone window, fixes probe with stereotaxic apparatus, connection
MP150 type 16 leads physiograph progress brain blood flow monitoring and records being averaged for steady section after curve steadily about 15min
Cerebral blood flow (CBF)[2].Then rat is put to death, takes out damage side brain and fixed in 10% formalin, by pathology professional's system
Piece, HE dyeing, and the diagosis under light microscopic, observe cerebral cortex hippocampus morphological changes of cell[8]。
Influence of the table 4 to Focal Cerebral Ischemia-Reperfusion in Rats ability of learning and memory
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
13d carries out learning ability measurement, by table 4 as it can be seen that compared with pseudo- operation group, model control group after ischemia-reperfusion
The rat study extremely significant property of number increases, and shows the 13d after ischemia-reperfusion, the learning ability of rat is still badly damaged.
Compared with model control group, the well-informed high dose group of silver, the well-informed middle dosage of silver, Folium Ginkgo group and aspirin group rat learn energy
The extremely significant property of power improves (p < 0.01), and the well-informed low dose group learning ability conspicuousness of silver improves (p < 0.05).Learning ability is surveyed
Memory capability measurement is carried out after fixed for 24 hours, the well-informed high dose group of silver and Folium Ginkgo group and aspirin group can be extremely significant
Property promote ischemia-reperfusion Rats With Memory ability (p < 0.01), during silver is well-informed, low dose group energy conspicuousness promote ischemia-reperfusion
Rats With Memory ability (p < 0.05).
Influence of the table 5 to Focal Cerebral Ischemia-Reperfusion in Rats local cerebral blood flow
#P < 0.05,##P < 0.01vs. puppet operation group；* p < 0.05, * * p < 0.01vs. model control group；aP < 0.05,aap<
The well-informed group vs. Folium Ginkgo group of 0.01 silver medal；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
By table 5 as it can be seen that compared with pseudo- operation group, there was no significant difference for Folium Ginkgo group local cerebral blood flow (p > 0.05),
The well-informed high dose group cerebral blood flow (CBF) conspicuousness of silver reduces (p < 0.05), the extremely significant property of local cerebral blood flow after other each groups 15d
It reduces (p < 0.01).Compared with model control group, local cerebral blood flow is extremely aobvious after other each groups 15d in addition to compound low dose group
Work property increases (p < 0.01).
Compared with Folium Ginkgo group, the well-informed middle dose group cerebral blood flow (CBF) conspicuousness of silver reduces (p < 0.05), and silver is had quick access to information low dose
The extremely significant property of amount group local cerebral blood flow reduces (p < 0.01)；Compared with aspirin group, the well-informed low dose group cerebral blood flow (CBF) of silver
Extremely significant property reduces (p < 0.01), and there was no significant difference for other each comparison among groups (p > 0.05).
Pathologic diagnosis sample is divided into 7 groups, and respectively pseudo- operation group, model group, compound silver Lingtong capsule is high, it is low to neutralize
Dosage group, 1 group of positive drug and 2 groups of positive drug, every group 8.Testing result is as described below:
Pseudo- operation group: cerebral cortex layer and hippocampus plot structure understand that completely, neurotagma is neat, and nuclear membrane is clear
Chu, kernel is obvious, and chromatin is abundant.
Model control group: right side cerebral cortex and hippocampus structure disturbance are shown in different degrees of infarcted region, infarcted region it is thermophilic she
Red light dye, infarcted region central nervous member, Deiter's cells and nerve fibre necrosis, structure are destroyed, and institutional framework is loose in sieve
Netted, there is inflammatory cell infiltration on a other infarct periphery.
Compound silver Lingtong capsule is high, neutralizes low dose group: high, neutralization low dose group part example brain and the big encephalopathy of model group
Reason feature is similar, right side cerebral cortex and/or hippocampus structure disturbance, sees that different degrees of infarcted region, the thermophilic Yihong in infarcted region are light
Dye, infarcted region central nervous member, Deiter's cells and nerve fibre necrosis, structure are destroyed, and loose institutional framework is in mesh-like,
There is inflammatory cell infiltration on a other infarct periphery.The infarcted region range and model group of low dose group are close, the stalk of middle dose group
Dead zone range is slightly less than model group, and the infarcted region range of high dose is significantly less than model group.
1 ginkgo biloba p.e group of positive drug and 2 aspirin group of positive drug
1 group of positive drug and 2 groups of brains of positive drug are similar to model group brain pathological characteristic, and only lesser extent, is shown in the right side
Side cerebral cortex and/or hippocampus structure disturbance are shown in the infarcted region of lesser extent, the light dye in the thermophilic Yihong in infarcted region, infarct district center
Neuron, Deiter's cells and nerve fibre necrosis, structure are destroyed, and institutional framework is loose, and there is inflammation on a other infarct periphery
Property cellular infiltration.
The influence of 1.3 pairs of brain tissues and Biochemical Indices In Serum
Male SD rat 105 are taken, modeling and medication are same as above.0.5h after the last administration, orbital venous plexus take blood system
Serum is separated out, rat is put to death immediately after taking blood, takes out brain, by the separation of damage side brain front, is sufficiently shredded and is mixed with tissue shear
Even, weighing to be fully ground in the physiological saline that 0.5g adds 4.5ml ice-cold makes tissue homogenization, and 4 DEG C 1500 turns are centrifuged 10 minutes, takes
Supernatant is 10% brain tissue homogenate's liquid, the measurement for biochemical indicator.By lactic acid in kit specification measurement rat blood serum
Dehydrogenase (LDH) vigor[9]And total antioxidant capacity (T-AOC) vigor, superoxide dismutase (SOD) are living in brain tissue
Power[10], malonaldehyde (MDA) content, Na+-K+ATP enzyme and Ca2+-Mg2+ATP enzyme, creatine kinase (CK) vigor, lactic acid (LD) contain
Amount, histone measurement BCA albuminometry.It the results are shown in Table 6~table 8.
Influence of the table 6 to Focal Cerebral Ischemia-Reperfusion in Rats brain tissue T-AOC, SOD, MDA
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
From table 6
For model control group compared with pseudo- operation group, total antioxidant capacity (T-AOC), SOD vigor and MDA vigor are extremely significant
Property reduce (p < 0.01)；
Compared with model control group, the well-informed high dose group of silver, the extremely significant property of aspirin group energy increase brain tissue T-AOC's
Vigor (p < 0.01), the well-informed middle dose group energy conspicuousness of Folium Ginkgo, silver improve the vigor (p < 0.05) of brain tissue T-AOC, silver
The well-informed high, middle dose group of apricot blade, aspirin, the silver extremely significant property of energy improves Brain Tissue SOD Vigor (p < 0.01), and silver is well-informed
Low dose group energy conspicuousness improves Brain Tissue SOD Vigor (p < 0.05), and the well-informed high dose group of Folium Ginkgo, silver can extremely significant property drop
Low brain tissue MDA vigor (p < 0.01), the well-informed middle dose group energy conspicuousness of aspirin, silver reduce the vigor of MDA in brain tissue
(p<0.05)。
Compared with Folium Ginkgo group, the well-informed extremely significant property of low dose group MDA vigor of silver increases (p < 0.01)；With aspirin
Group is compared, during silver is well-informed, low dose group T-AOC conspicuousness reduce (p < 0.05), the well-informed low dose group MDA vigor conspicuousness liter of silver
High (p < 0.05)；Silver has quick access to information other each dosage groups there are no significant compared with positive drug group difference (p > 0.05).
Table 7 is to Focal Cerebral Ischemia-Reperfusion in Rats brain tissue Na+-K+ATP enzyme/Ca2+-Mg2+The influence of ATP enzyme
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
By table 7 as it can be seen that model control group is compared with pseudo- operation group, model control group Na+-K+ATP enzyme vigor, Ca2+-Mg2+
The extremely significant property of ATP enzyme vigor reduces (p < 0.01)；
Compared with model control group, the well-informed high dose group of Folium Ginkgo, aspirin, silver can conspicuousness raising brain tissue
Na+-K+ATP enzyme vigor (p < 0.05), the well-informed high dose group energy conspicuousness of Folium Ginkgo, aspirin, silver increase in brain tissue
Ca2+-Mg2+ATP enzyme vigor (p<0.05), other each groups compare that there was no significant difference (p>0.05)；
Compared with Folium Ginkgo group, the well-informed low dose group Ca of silver2+-Mg2+ATP enzyme vigor conspicuousness reduces (p < 0.05)；Silver
Other each dosage groups of having quick access to information there are no significant compared with positive drug group difference (p > 0.05).
Influence of the table 8 to Focal Cerebral Ischemia-Reperfusion in Rats brain tissue CK, LD vigor and Serum LDH
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
By table 8 as it can be seen that model control group is compared with pseudo- operation group, CK vigor there are no significant difference between each group (p >
0.05), model control group brain tissue LD content and the extremely significant property of Serum LDH vigor increase (p < 0.01)；
Compared with model control group, silver well-informed height, middle dose group, Folium Ginkgo group, aspirin group brain tissue LD vigor
Conspicuousness reduces (p < 0.05), and the well-informed height of silver, middle dose group, Folium Ginkgo group, aspirin group Serum LDH vigor are extremely significant
Property reduce (p < 0.01)；
Compared with Folium Ginkgo group, the well-informed low dose group Serum LDH vigor conspicuousness of silver reduces (p < 0.05)；Other each groups
Between the difference (p > 0.05) that compares that there are no significant.
2, the experimental study (first of permanent cerebral ischemia caused by compound silver Lingtong capsule treatment intraluminal middle cerebral artery occlusion in rats occludes
Its administration 3 times, is administered once a day, successive administration 15 days later)
The influence of 2.1 pairs of death rates, Neurological deficits, brain water content, infraction rate
Cleaning grade male SD rat 105, weight 250-300g, with reference to the method for Longa et al., using internal carotid line
Bolt method prepares intraluminal middle cerebral artery occlusion in rats and occludes (MCAO) model[1].After rats in sham-operated group anesthesia, artery point inside and outside neck is only exposed
Fork, does not occlude MCA.Animal is divided into 7 groups after modeling, every group 15,3 dosage groups of compound silver Lingtong capsule are respectively as follows:
1.2g/kg (0.24g/ml), 0.6g/kg (0.12g/ml), 0.3g/kg (0.06g/ml).1 Folium Ginkgo group of positive control drug
4mg/kg(0.8mg/ml).2 aspirin group 30mg/kg (6mg/ml) of positive control drug.Separately set sham-operation group and ischemia model
Control group (0.5%CMC-Na for giving isometric(al)).3h starts to be administered after ischemic, and daily gastric infusion is primary, continuously gives
Medicine 15 days, first administration dosage was 3 times of maintenance dose, and administration volume is 0.5ml/100g weight.
The death condition (symptom and death toll before dead) of animal is observed and recorded in experimentation.
The preparation of fishing line: by a long 40mm, one end of the fishing line of diameter 0.26mm is marked with marking pen, apart from this label
A label is made again of marking pen at the 18mm of end, is placed in spare in physiological saline.
In 0.5h after the last administration, referring to neurological deficit point system, (18 minute marks are quasi-[2]) each group rat is commented
Point, standard such as test 1.1.After These parameters measurement, sacrificed by decapitation rat takes out full brain weighing.Be immediately placed in -20 DEG C it is quick-frozen
10-15min, until when mouse brain is hardened, since antinion 2mm, do it is coronal cut five knives, rear to brain and hindbrain intersection, cut
Contain in the phosphate buffer solution of 1%TTC at brain piece is set 5ml rapidly after six of 2mm thickness, is protected from light temperature and incubates 30 minutes, in temperature
It was stirred once during incubating every 7~8 minutes, temperature takes out brain piece after incubating 30 minutes, is taken pictures with digital camera and inputs computer, later
Pale area's (infarct) and non-pale area (normal area) volume are calculated with Image pro plus, calculates infarct percent such as
Under[3]:
Cerebral infarction volume percentage={ [total Infarction volume-(right half brain volume-half brain volume of a left side)]/left half brain volume } ×
100%.
Brain tissue after dyeing is placed in the drying of 110 DEG C of baking ovens, compareing big brain wet weight, to find out brain water content as follows:
Brain water content (%)=(1- brain tissue dry weight/brain tissue weight in wet base) × 100%
Influence of the table 9 to the property rats with cerebral ischemia death rate forever
The each group death rate is as shown above.It is increased with the well-informed dosage of silver, rats death rate is in gradually decrease trend.
Table 10 is to the behavior of permanent cerebral ischemia rat nerve, the influence of cerebral infarction rate and brain water content
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaP < 0.01vs. Folium Ginkgo group；bP < 0.05,bbP < 0.01vs. aspirin group
By table 10 as it can be seen that compared with pseudo- operation group, model group brain water content conspicuousness increases (p < 0.05), other
Each group brain water content there are no significant compared with pseudo- operation group difference (p > 0.05)；
Compared with model group, the well-informed high dose group Neurological deficits of Folium Ginkgo group, aspirin, compound silver are extremely aobvious
Work property increases (p < 0.01), and the well-informed middle dose group Neurological deficits conspicuousness of compound silver increases (p < 0.05), Folium Ginkgo,
The well-informed high, middle dose group of aspirin, the compound silver extremely significant property of energy reduces cerebral infarction percentage (p < 0.01), Folium Ginkgo
The well-informed high dose group energy conspicuousness of group, compound silver reduces brain water content (p < 0.05), other each groups are equal compared with model group
There was no significant difference (p > 0.05).
Compared with Folium Ginkgo group, the extremely significant property reduction of the well-informed high dose group cerebral infarction percentage of aspirin group, silver (p <
0.01)；Compared with aspirin group, silver have quick access to information high, medium and low dosage group cerebral infarction percentage extremely significant property increase (p <
0.01)；Silver has quick access to information other each dosage groups there are no significant compared with positive drug group difference (p > 0.05).
The influence of 2.2 pairs of death rates, learning and memory function, cerebral blood flow (CBF) and brain tissue
Male SD rat 105 separately are taken, modeling and medication are same as above.The death of animal is observed and recorded in experimentation
Situation (symptom and death toll before dead).Rat learning and memory function is measured using Y type labyrinth, specific method learns with 1.2
Achievement has 9 correct suffered accumulative number of shocks to indicate and (14d is administered) to reach continuous 10 electric shocks；(last after for 24 hours
1h after administration), continuous electric shock 10 times is then popped one's head in laser dopper flowmeter and is surveyed using wherein correct response number as Memory result
Determine local cerebral blood flow.
Then sacrificed by decapitation is taken out damage side brain and is fixed in 10% formalin, by pathology professional's film-making, HE dye
Color, and diagosis, observe cerebral cortex hippocampus morphological changes of cell, be denoted as respectively according to lesion degree: "-", "+", " ++ ",
“+++”、“++++”。
Influence of the table 11 to the property rats with cerebral ischemia death rate forever
The each group death rate is as shown above.It is increased with the well-informed dosage of silver, rats death rate is in gradually decrease trend.
Influence of the table 12 to permanent cerebral ischemia learning and memory function
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaThe silver medal vs. Folium Ginkgo group of p < 0.01；bP < 0.05,bbP < 0.01vs. aspirin group
By table 12 as it can be seen that compared with pseudo- operation group, model group adds up number of shocks and the extremely significant property of correct response number
Increase (p < 0.01), the well-informed low dose group of silver adds up the extremely significant property of number of shocks and increases (p < 0.01)；It is positive compared with model group
1 Folium Ginkgo of medicine, 2 aspirin of positive drug, the well-informed high dose of silver and middle dose group add up the equal conspicuousness of number of shocks reduce (p <
0.05), the well-informed middle dose group energy conspicuousness of Folium Ginkgo group, aspirin, silver increases correct response number (p < 0.05), silver spirit
Logical high dose group can extremely significant property increase correct response number (p<0.01), other each groups compare that there was no significant difference (p>
0.05).Silver has quick access to information each dosage group there are no significant compared with positive drug group difference (p > 0.05).
Influence of the table 13 to property rats with cerebral ischemia local cerebral blood flow forever
#P < 0.05,##The model control group vs. puppet operation group of p < 0.01；* p < 0.05, * * p < 0.01vs. model control group；ap
< 0.05,aaP < 0.01vs. Folium Ginkgo group；bP < 0.05,bbP < 0.01vs. aspirin group
By table 13 as it can be seen that model control group is compared with pseudo- operation group, the extremely significant property drop of model control group local cerebral blood flow
Low (p < 0.01).Compared with model control group, local cerebral blood flow pole after the well-informed high dose group 15d of aspirin, compound silver
Conspicuousness increases (p < 0.01)；Folium Ginkgo group and the well-informed middle dose group local cerebral blood flow conspicuousness of silver increase (p < 0.05)；
Other each groups no significant difference (p > 0.05) compared with model group.
Compared with aspirin group, the well-informed extremely significant property of low dose group local cerebral blood flow of silver reduces (p < 0.01)；Silver-colored spirit
Lead to other each dosage groups there are no significant compared with positive drug group difference (p > 0.05).
Influence to pathologyofbraintissue
Sample is divided into 7 groups, and respectively pseudo- operation group, model group, compound silver Lingtong capsule is high, neutralizes low dose group, the positive
1 group of medicine and 2 groups of positive drug, every group 8.Testing result is as described below:
Pseudo- operation group: most of example cerebral cortex layer and hippocampus plot structure understand that completely, neurotagma is neat, carefully
Karyon film understands that kernel is obvious, and chromatin is abundant, and No. 7 and No. 8 cerebral cortex shallow-layers are shown in focal slight infarct, infarct
The light dye in the thermophilic Yihong in area, cellular morphology are still normal.
Model group: different degrees of infarcted region, the light dye in the thermophilic Yihong in infarcted region, part are shown in right side Cerebral Cortex Structure disorder
Infarcted region central nervous member, Deiter's cells and nerve fibre necrosis, structure are destroyed, and loose institutional framework is in mesh-like, portion
Divide infarcted region cellular morphology still intact, loose institutional framework is in mesh-like, and there is inflammatory cell infiltration on a other infarct periphery.It is a
Other example ductus arteriosus wall necrosis, lumen are shown in thrombus, and Guan Zhoujian brain tissue oedema is loose, and has cell infiltration.
1 Folium Ginkgo group of positive drug:, most of example on the right side of cerebral cortex similar to model group brain pathological characteristic, 1 right side
Side hippocampus brain tissue structure disturbance is shown in different degrees of infarcted region, the light dye in the thermophilic Yihong in infarcted region, part infarcted region central nervous
Member, Deiter's cells and nerve fibre necrosis, structure are destroyed, and loose institutional framework is in mesh-like, part infarcted region cell shape
State is still intact, and loose institutional framework is in mesh-like, and there is inflammatory cell infiltration on a other infarct periphery.Part example ductus arteriosus wall goes out
Blood, necrosis and cell infiltration, lumen are shown in big thrombus, and Guan Zhoujian brain tissue oedema is loose, and has cell infiltration.
2 aspirin group of positive drug: 2 groups of brains of positive drug are similar to model group brain pathological characteristic, only lesser extent,
4 right side cerebral cortexes and 1 hippocampus are shown in the infarcted region of lesser extent, and the light dye in the thermophilic Yihong in infarcted region, institutional framework is loose, a
There is inflammatory cell infiltration on other infarct periphery.Part example arterial wall bleeding, necrosis, tube wall and pipe week cell infiltration, 2 pipes
Chamber is shown in thrombus.
Compound silver Lingtong capsule high dose group: high dose group brain is similar to model group brain pathological characteristic, and 6 right sides are big
Cerebral cortex and 2 hippocampus are shown in the infarcted region of lesser extent, and the light dye in the thermophilic Yihong in infarcted region, institutional framework is loose, a other infarct
There is inflammatory cell infiltration on stove periphery.Part example ductus arteriosus wall necrosis, Guan Zhoujian brain tissue oedema is loose, and has cell infiltration.
Compound silver Lingtong capsule middle dose group: low dose group brain is similar to model group brain pathological characteristic, most of example
Right side cerebral cortex and hippocampus are shown in the infarcted region of lesser extent, and the light dye in the thermophilic Yihong in infarcted region, institutional framework is loose, a other stalk
There is inflammatory cell infiltration on dead stove periphery.Part example arterial wall bleeding, necrosis, tube wall and pipe week cell infiltration.
Compound silver Lingtong capsule low dose group: different degrees of infarct is shown in Cerebral Cortex Structure disorder on the right side of most of example
Area, the light dye in the thermophilic Yihong in infarcted region, part infarcted region central nervous member, Deiter's cells and nerve fibre necrosis, structure are destroyed,
Loose institutional framework is in mesh-like, and part infarcted region cellular morphology is still intact, and loose institutional framework is in mesh-like, a other infarct
There is inflammatory cell infiltration on stove periphery.Individual example ductus arteriosus walls and Guan Zhoujian bleeding, cell infiltration.
For brain section cytological stains the results show that compared with pseudo- operation group, model group brain is shown in apparent infarcted region.Each group
Infarct lesion is divided equally as follows from high to low: model group > low dose group > Folium Ginkgo group > middle dose group > high dose group > Ah Si
Woods group > puppet operation group.
3, effect (experiment in vivo) of the compound silver Lingtong capsule to PAF, ADP induction Platelet Aggregation in Rabbits function[9]
3.1 groups and dosage
Male New Zealand rabbit 48,2~2.5kg of weight are taken, by being randomly divided into 6 groups, every group 8.Respectively compound silver
3 dosage groups of Lingtong capsule, are respectively as follows: 0.6g/kg (0.6g/ml), 0.3g/kg (0.3g/ml), 0.15g/kg (0.15g/
ml).1 Folium Ginkgo group 2mg/kg (2mg/ml) of positive control drug, 2 aspirin group 15mg/kg (15mg/ml) of positive control drug
The 0.5%CMC-Na of isometric(al) is given with negative control group, administration volume is 1ml/kg.Daily stomach-filling (ig) is administered once, even
Continuous administration 7 days, first administration dosage are 3 times of maintenance dose,
3.2 take blood
Rabbit dorsal position is fixed on operating table for rabbit and cuts off the neck rabbit hair by 1h after the last administration, and 1% salt is subcutaneously injected
Sour Procaine injection local infiltration anesthesia.Skin of neck about 3cm is cut off, carries out blunt separation, exposure branch gas with haemostatic clamp
Arteria carotis communis is isolated after pipe.Filament ligatures arteria carotis communis distal end, then closes proximal part with artery clamp folder, after carrying out vascular catheterization
It is fixed.Then artery clamp is opened, blood is put into the test tube containing 3.8% sodium citrate, blood and 3.8% sodium citrate are made
The ratio between volume is 9:1.
3.3 prepare platelet rich plasma (RPR) and platelet poor plasma (PPP)
After the anticoagulated blood of above-mentioned preparation is mixed, 500rpm draws upper plasma after being centrifuged 10min up to PRP；It will remain
Upper plasma is drawn after remaining blood 3500rpm centrifugation 10min up to PPP.
3.4 measurement platelet aggregation rates[11]
It first takes 300 μ l PPP to be added in test cup, is then placed in instrument connection, presses " PPP " key and calibrated.Then 270 μ are taken
L PRP is added in test cup, is put into instrument connection after preheating 3min in 37 DEG C of pre-temperature slots, induction is added immediately when pressing " beginning " key
30 μ l of agent (concentration of two kinds of inducers is respectively PAF7.072 μm of ol/L, ADP300mmol/L), to measure maximum aggregation rate.Often
4 channels of secondary measurement, in the hope of average maximum aggregation rate.
L-Arginine is calculated according to the following formula:
3.5 result
3.5.1 influence of the compound silver Lingtong capsule to PAF induction rabbit extracorporeal platelet aggregation rate
Influence in 14 body of table to compound silver Lingtong capsule to PAF induction rabbit extracorporeal platelet aggregation rate
*P < 0.05,**P < 0.01vs. blank control group；aP < 0.05,aaP < 0.01vs. Folium Ginkgo group；bP < 0.05,bbp<
0.01vs. aspirin group
By table 14 as it can be seen that compared with blank control group, Folium Ginkgo group energy conspicuousness inhibits rabbit extracorporeal platelet aggregation
(p < 0.05), silver-colored high, medium and low three dosage groups of having quick access to information have inhibiting effect to the PAF Platelet Aggregation in Rabbits induced, and with dosage
It increases, aggregation inhibiting rate is increased in dose dependent, wherein the well-informed high dose group max platelet rate of silver and blank pair
Comparing according to group has significant difference (p<0.05), and there was no significant difference compared with blank control group for aspirin group (p>0.05).
3.5.2 influence of the compound silver Lingtong capsule to ADP induction rabbit extracorporeal platelet aggregation rate
Influence in 15 body of table to compound silver Lingtong capsule to ADP induction rabbit extracorporeal platelet aggregation rate
* p < 0.05, * * p < 0.01vs. blank control group；aP < 0.05,aaP < 0.01vs. Folium Ginkgo group；bP < 0.05,bbp
< 0.01vs. aspirin group
By table 15 as it can be seen that compared with blank control group, Folium Ginkgo group and aspirin group energy conspicuousness inhibit ADP to lure
The rabbit extracorporeal platelet aggregation (p < 0.05) led, the Rabbit Blood Platelets that silver-colored high, medium and low three dosage groups of having quick access to information induce ADP
It is populated with inhibiting effect, and is increased with dosage, aggregation inhibiting rate is increased in dose dependent, wherein the well-informed high dose group of silver is most
Large platelet aggregation rate has extremely significant sex differernce (p < 0.01) compared with blank control group, the well-informed middle dose group of silver and blank control
Group, which is compared, significant difference (p < 0.05).
Compared with aspirin group, the well-informed high dose group energy conspicuousness of silver inhibits the external platelet aggregation of rabbit of ADP induction
Collect (p < 0.05)；Silver has quick access to information other each dosage groups there are no significant compared with positive drug group difference (p > 0.05).
4, inhibiting effect of the compound silver Lingtong capsule to thrombosis
The influence of thrombosis on 4.1 pairs of rat neck artery-vein bypass silk threads
Male SD rat 60,250~280g of weight are taken, animal is randomly divided into 6 groups, every group 10, compound silver is well-informed
3 dosage groups of capsule, are respectively as follows: 1.2g/kg (0.24g/ml), 0.6g/kg (0.12g/ml), 0.3g/kg (0.06g/ml).Sun
Property comparison medicine Folium Ginkgo group 4mg/kg (0.8mg/ml), 2 aspirin group 30mg/kg (6mg/ml) of positive control drug.It is negative
Control group (0.5%CMC-Na for giving isometric(al)).Successive administration 7 days, volume was administered in the daily gastric infusion of each group rat 1 time
For 0.5ml/100g.
1h after the last administration, rat are anaesthetized with 3% chloraldurate solution 1ml/100g ip, separate right common carotid artery and a left side
Vena jugularis externa.No. 4 operation silk threads of a root long about 6cm are put into three sections of polyethylene pipes.Physiological saline is full of polyethylene pipe
Chamber.After left vena jugularis externa is first inserted into one end of pipe, the other end of pipe is inserted into right common carotid artery.Open blood after the completion of operation
Stream, then blood flows through polyethylene pipe from right common carotid artery, returns to vena jugularis externa.Middle clinopodium polycephalum after open blood flow 15min.It takes rapidly
Band clot silk thread is weighed to obtain total weight out, and it is wet weight of thrombus that total weight subtracts silk thread again.70 DEG C of silk thread are dried, takes out and claims
Weight, it is thrombus dry weight that total weight subtracts silk thread again.Inhibiting rate is calculated according to the following formula[12,13]:
As a result as shown in the table.
Influence of the 16 silver medal Lingtong capsule of table to thrombosis on rat neck artery-vein bypass silk thread
* p < 0.05**p < 0.01 is compared with blank control group；aP < 0.05,aaP < 0.01vs. Folium Ginkgo group；bp<0.05
,bbP < 0.01vs. aspirin group
By table 16 as it can be seen that compared with negative control group, during Folium Ginkgo, silver are well-informed, low dose group energy conspicuousness reduce blood
Bolt weight in wet base (p < 0.05), the extremely significant property of the well-informed high dose group energy of aspirin group, silver reduce wet weight of thrombus (p < 0.01), and silver is well-informed
High, medium and low dosage is in the trend gradually decreased to wet weight of thrombus inhibiting rate；The well-informed high dose group of Folium Ginkgo, aspirin, silver
Can extremely significant property reduce thrombus dry weight (p < 0.01), during silver is well-informed, low dosage energy conspicuousness reduce thrombus dry weight (p < 0.05),
Silver high, medium and low dosage of having quick access to information is in the trend that gradually decreases to thrombus dry weight inhibiting rate.
Compared with aspirin group, during silver is well-informed, the equal conspicuousness of low dose group thrombus dry weight increase (p < 0.05)；Silver is well-informed
Other each dosage groups there are no significant compared with positive drug group difference (p > 0.05).
4.2 couples of ADP cause the influence of mouse lung thrombosis[14,15]
Healthy ICR mouse 60 is taken, half male and half female, 19~22g of weight, be divided into 5 groups, every group 10 at random: compound is silver-colored
3 dosage groups of Lingtong capsule, are respectively as follows: 2.4g/kg (0.12g/ml), 1.2g/kg (0.06g/ml), 0.6g/kg (0.03g/
ml).1 Folium Ginkgo group 8mg/kg (0.4mg/ml) of positive control drug, 2 aspirin group 60mg/kg (3mg/ of positive control drug
ml).Negative control group (0.5%CMC-Na for giving isometric(al)).The daily gastric infusion of each group 1 time, successive administration 7 days, administration
Volume is 0.4ml/20g.1h after the last administration forms acute lung thrombus (mouse breathing by 200mg/kg tail vein injection ADP
Syndrome characterized by dyspnea is unable to autonomic activities), record the time for restoring autonomic activities after injecting ADP to mouse.
17 silver medal Lingtong capsule of table induces ADP the influence of chmice acute lung thrombosis
* p < 0.05**p < 0.01 is compared with blank control group；aP < 0.05,aaThe well-informed group vs. Folium Ginkgo group of the silver medal of p < 0.01
；bP < 0.05,bbThe well-informed group vs. aspirin group of the silver medal of p < 0.01
By table 17 as it can be seen that compared with negative control group, the extremely significant property of Folium Ginkgo group energy shortens ADP and causes mouse pulmonary embolism extensive
Again autonomic activities time (p < 0.01), silver has quick access to information high, medium and low dosage energy conspicuousness shortening ADP cause mouse lung refilling certainly
Main activity time (p < 0.05).
Compared with Folium Ginkgo group, the well-informed high dose group of silver restores autonomic activities time conspicuousness and extends (p < 0.05), silver
In well-informed, low dose group restores autonomic activities time extremely significant property and extends (p < 0.01)；Silver is had quick access to information other each dosage groups and positive
Difference (p > 0.05) that medicine group compares that there are no significant.
5, influence of the compound silver Lingtong capsule to mouse bleeding time and clotting time
The influence in 5.1 pairs of mouse bleeding times
Healthy ICR mouse 60 is taken, half male and half female, 19~22g of weight, be divided into 6 groups, every group 10 at random: compound is silver-colored
3 dosage groups of Lingtong capsule, are respectively as follows: 2.4g/kg (0.12g/ml), 1.2g/kg (0.06g/ml), 0.6g/kg (0.03g/
ml).1 Folium Ginkgo group 8mg/kg (0.4mg/ml) of positive control drug, 2 aspirin group 60mg/kg (3mg/ of positive control drug
ml).Negative control group (0.5%CMC-Na for giving isometric(al)).The daily gastric infusion of each group rat 1 time, successive administration 7 days,
Administration volume is 0.4ml/20g.
After last dose 1h, mouse is fixed, rat-tail length is measured with milimeter scale and is marked, is then cut respectively with benefit small
It is cross-section at rat-tail point 3mm, it is voluntarily overflowed to blood, starts timing, sucked drop of blood 1 time every 30s with filter paper, until blood is natural
Stop until (without blood when filter paper is inhaled), as the bleeding time[16]。
The influence in bleeding time after 18 compound silver Lingtong capsule of table docks to mouse
* p < 0.05**p < 0.01vs. blank control group；aP < 0.05,aaP < 0.01vs. Folium Ginkgo group；bP < 0.05,bbp<
0.01vs. aspirin group
By table 18 as it can be seen that compared with negative control group, the bleeding time, extremely significant property extended after the docking of aspirin group mouse
(p < 0.01), Folium Ginkgo, the well-informed high dose of silver, middle dose group bleeding time conspicuousness extend (p < 0.05), other groups with
Negative control group is compared to there was no significant difference (p > 0.05).
Compared with aspirin group, during silver is well-informed, low dose group bleeding time conspicuousness shorten (p < 0.05)；Silver has quick access to information it
His each dosage group there are no significant compared with positive drug group difference (p > 0.05).
The influence of 5.2 pairs of clotting time of mice
Healthy ICR mouse 60 is taken, half male and half female, 19~22g of weight, be divided into 5 groups, every group 10 at random: compound is silver-colored
3 dosage groups of Lingtong capsule, are respectively as follows: 2.4g/kg (0.12g/ml), 1.2g/kg (0.06g/ml), 0.6g/kg (0.03g/
ml).1 Folium Ginkgo piece group 8mg/kg (0.4mg/ml) of positive control drug, 2 aspirin group 60mg/kg (3mg/ of positive control drug
ml).Negative control group (0.5%CMC-Na for giving isometric(al)).The daily gastric infusion of each group 1 time, successive administration 7 days, administration
Volume is 0.4ml/20g.
It after last dose 1h, is inserted into capillary glass-tube from the intraocular corner of the eyes, that is, has blood outflow, and drip on glass slide.Every
15s is gently stirred inwards with pin autoblood edge, and whether there is or not the traces of blood to provoke for observation.Stop since blood sampling to provoking the trace of blood, institute
With time, that is, clotting time[17,18]。
Influence of the 19 compound silver Lingtong capsule of table to clotting time of mice
* p < 0.05**p < 0.01vs. blank control group；aP < 0.05,aaP < 0.01vs. Folium Ginkgo group；bP < 0.05,bbp<
0.01vs. aspirin group
By table 19 as it can be seen that compared with negative control group, aspirin group can extremely significant property extend clotting time of mice (p <
0.01), the well-informed high dose group energy conspicuousness of silver extends clotting time of mice (p < 0.05), other each groups are compared with negative control group
Difference that there are no significant (p > 0.05).
Silver has quick access to information each dosage group there are no significant compared with positive drug group difference (p > 0.05).
6, compound silver Lingtong capsule Contained Serum prevention administration grinds protective effect-mechanism of in vitro culture cortical neuron
Study carefully
The preparation of 6.1 rat Contained Serums[19,20]
SD rat 24,250g-300g, 1 Folium Ginkgo group 26.7mg/kg of positive drug, positive drug 2 are randomly divided by weight
The well-informed group 4g/kg of aspirin group 200mg/kg, silver, blank control group, every group 6, gastric infusion, administered volume 1ml/
100g, blank control group are given isometric 0.5%CMC-Na, are taken twice daily, and successive administration 4 days.Last time is administered
1h afterwards, sterile arteria carotis communis take blood, are stored at room temperature 2h after taking blood, and 3000rpm is centrifuged 20min, takes supernatant, will mix with group serum
Close, 56 DEG C of inactivation 30min, super-clean bench filtration sterilization, dispense be stored in -80 DEG C it is spare[21]。
6.2 cortical neuronal cells originally cultures
It is improved slightly according to the method for Choi DW, carries out rat primary cortex neurons culture.It takes pregnancy 15-18 days
SD rat 1[22], chloral hydrate anesthesia.Female rat abdomen is sterilized with 75% ethyl alcohol, abdominal cavity is cut off, tire mouse is taken out and is first put into
Of short duration soaking disinfection, places into the PBS of pre-cooling in 75% ethyl alcohol.The head (Cong Erhou is cut) of tire mouse is cut in super-clean bench,
It is put into the PBS of pre-cooling, brain is all taken out and is put into the PBS of pre-cooling.Then, cerebral cortex is removed, removes meninx, it will
Cortex is transferred in centrifuge tube after shredding.Pancreatin is added to digest 10 minutes in 37 DEG C, carries out piping and druming mixing within every 5 minutes.Tire is added
Cow's serum terminates digestion, then proceedes to blow and beat, and adds 10%MEM culture medium and (blows in every 500mLMEM containing fetal calf serum 50mL)
It beats and is resuspended.Then it with 100 mesh net filtrations, then with 400 mesh net filtrations, shifts filtrate and is centrifuged into clean centrifuge tube
(1000rpm) 5 minutes.Liquid is discarded supernatant after centrifugation, and the piping and druming of 10% culture medium is added and is resuspended.It is counted under inverted microscope, according to
Cell density 1 × 106A/mL handles cell inoculation in overnight sterile culture plate in advance with cell adherence agent, is placed in 37
DEG C, CO2It is incubated for about 4h in the sterile culture case that concentration is 5%, in microscopically observation after cell is sufficiently adherent, by 10%MEM
Culture medium changes the dedicated maintenance culture medium of neuron (Neurobasal Media 49mL+B27-Supplement 1mL) into[23].To thin
The form that neuron is presented in born of the same parents can be used to test[24,25]。
6.3 experimental model preparation methods
6.3.1 sodium dithionite (Na2S2O4) merge scarce sugar cause cortical neuronal cells oxygen-glucose deprived injury model (in advance
Anti- administration)
Reference literature slightly improves[26], sub- two sodium sulphate of company of final concentration of 10mM are added in sugar-free EBSS solution
(Na2S2O4) dry powder sufficiently dissolves, use NaHCO3Adjusting pH is 7.2, as anoxic solution.This solution needs matching while using, in order to eliminate
Na2S2O4The influence of decomposition product in water separately sets a non-hypoxemia solution control group in addition to setting blank control group (control group 1)
(control group 2), the preparation of non-hypoxemia solution are the preparatory anoxic Na for preparing 100mM2S2O4Stock solution, the used time was by stock solution (3 days
More than) it is diluted to 10mM, as 10mM Na2S2O4Normal oxygen solution.
If blank control group (the blank control group serum that addition accounts for total volume 15%), non-hypoxemia solution control group (are added
Account for the blank control group serum of total volume 15%), model control group (the blank control group serum that addition accounts for total volume 15%), sun
Property control 1 Folium Ginkgo group (the Folium Ginkgo group Contained Serum that addition accounts for total volume 15%), 2 aspirin group of positive control
(the aspirin group Contained Serum that addition accounts for total volume 15%) and compound silver Lingtong capsule are high, and (addition accounts for the silver of total volume 15%
Well-informed Contained Serum), in (silver that addition accounts for total volume 10% have quick access to information+5% blank control group serum of Contained Serum), low (be added
The silver for accounting for total volume 5% is had quick access to information+10% blank control group serum of Contained Serum) dosage group, each dosage sets 3 multiple holes, tests
It is repeated 3 times (MTT sets 6 multiple holes, and experiment is repeated 3 times).It takes and is incubated at the well-grown cortical neuronal cells of 24 orifice plates and is used for
Each group serum is added in test, and effect for 24 hours, discards stoste, is gently swung and is washed twice with the EBSS solution containing sugar, changes into containing dense eventually
Degree is the Na of 10mM2S2O4Sugar-free EBSS hydroponics 1h (EBSS containing sugar is added in blank control group), while using medical proof fabric
Plate surrounding is sealed, changes anoxic culture solution after being incubated for 1h, is gently swung and is washed twice with the EBSS solution containing sugar, is changed into normally containing B-
The Neurobasal culture solution of 27supplement, sets 37 DEG C, 5%CO2Continue culture in incubator for 24 hours, forms anoxia _ reoxygenation
Damage model.
6.3.2 platelet activating factor (PAF) causes cortical neuronal cells damage model (prevention administration)[27]
By cortical neuronal cells with 1 × 106A/mL density is inoculated in 24 holes, sets 37 DEG C, 5%CO2It is incubated in incubator.
Every hole sets blank control group (the blank control group serum that addition accounts for total volume 15%), (it is total that Zhan is added in non-hypoxemia solution control group
The blank control group serum of volume 15%), it is model control group (the blank control group serum that addition accounts for total volume 15%), positive right
It (is added according to 1 Folium Ginkgo group (the Folium Ginkgo group Contained Serum that addition accounts for total volume 15%), 2 aspirin group of positive control
Account for the aspirin group Contained Serum of total volume 15%) and compound silver Lingtong capsule high (silver that addition accounts for total volume 15% is well-informed
Contained Serum), in (addition account for total volume 10% silver have quick access to information+5% blank control group serum of Contained Serum), it is low (be added Zhan it is total
Well-informed+10% blank control group serum of Contained Serum of the silver of volume 5%) dosage group, each dosage of 24 orifice plates sets 3 multiple holes, real
It tests and is repeated 3 times.It takes the cortical neuronal cells in culture third day for testing, discards original fluid, gently swung with PBS and wash two
It is secondary, after each group serum free culture system is added for 24 hours, original fluid is discarded, is gently swung and is washed twice with PBS, fresh medium is added and end is dense
Degree is 4 × 10-5The PAF of M sets 37 DEG C, 5%CO2Continue to cultivate 48h in incubator.
The measurement of 6.4 index observings
Morphological observation, mtt assay measure cell viability, and LDH release measures, LD assay, SOD vitality test,
MDA assay is conventionally.
6.5 experimental result
6.5.1 compound silver Lingtong capsule Contained Serum prevention administration is to sodium dithionite (Na2S2O4) merge scarce sugar cause skin
Layer mind
Influence through first cell hypoxia sugar deficiency injury model
Morphological observation: cortical neuronal cells are through Na2S2O4Processing about for 24 hours after, observed under inverted microscope: just
Normal cellular control unit form is elongated, transparency is high, gloss is preferable；Model group cellular damage is obvious.The cortical neuron of damage is thin
Born of the same parents are compared with normal cell, and cell process disappears, swelling, and diopter weakens born of the same parents, and the contracting of cytoplasm circle falls off.Be added Folium Ginkgo, Ah
Department woods, compound silver are had quick access to information, and then form is obviously improved the cell that Contained Serum is protected in advance compared with damaging cells, more thin
Born of the same parents retain raised structures, swelling Leukopenia, and cell is glossy.
Mtt assay measures cell viability
20 silver medal of table has quick access to information Contained Serum prevention administration to Na2S2O4The influence of cell survival rate after modeling
cp<0.05ccThe non-hypoxemia control group in p < 0.01 is compared with blank control group；▲p<0.05▲▲The model control group of p < 0.01
Compared with non-hypoxemia control group；#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 with
Model control group ratio
Compared with；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 20 as it can be seen that compared with model control group, the well-informed high, middle dose group of Folium Ginkgo group, aspirin group, silver contains
The medicine serum extremely significant property of energy improves cell survival rate (p < 0.01), and the well-informed Contained Serum of silver is to Na2S2O4Merge caused by lacking sugar
Cortical neuronal cells damage model OD570nmProtective effect enhance with the increase of Contained Serum concentration.
Silver has quick access to information each dosage group there are no significant compared with positive drug group difference (p > 0.05).
LDH leakage rate measures cellular damage degree
21 silver medal of table has quick access to information Contained Serum prevention administration to Na2S2O4The influence of cell LDH leakage rate after modeling
cp<0.05ccThe non-hypoxemia control group in p < 0.01 is compared with blank control group；▲p<0.05▲▲The model control group of p < 0.01
Compared with non-hypoxemia control group；#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 with
Model control group ratio
Compared with；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 21 as it can be seen that compared with model control group, the well-informed high dose group LDH leakage rate of Folium Ginkgo group, silver is extremely aobvious
Work property reduces (p < 0.01), and the well-informed equal conspicuousness of middle dose group LDH leakage rate of aspirin group, silver reduces (p < 0.05)；Other
There was no significant difference for each comparison among groups (p > 0.05).
LD assay
22 silver medal of table has quick access to information Contained Serum prevention administration to Na2S2O4The influence of cell LD content after modeling
cp<0.05ccThe non-hypoxemia control group in p < 0.01 is compared with blank control group；▲p<0.05▲▲The model control group of p < 0.01
Compared with non-hypoxemia control group；#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 with
Model control group compares；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 and aspirin group ratio
Compared with.
By table 22 as it can be seen that compared with model control group, Folium Ginkgo group and the well-informed extremely significant property of high dose group LD content of silver
It reduces (p < 0.01), aspirin group and the well-informed middle dose group LD content conspicuousness of silver reduce (p < 0.05)；With Folium Ginkgo group
Compare, during silver is well-informed, the extremely significant property of low dose group LD content increase (p < 0.01), aspirin group LD content conspicuousness increases (p
<0.05)；Compared with aspirin group, the well-informed extremely significant property of high dose group LD content of silver reduces (p < 0.01)；Compare between other each groups
Compared with there was no significant difference (p > 0.05).
SOD vitality test
23 silver medal of table has quick access to information Contained Serum prevention administration to Na2S2O4The influence of cell SOD vigor after modeling
cp<0.05ccThe non-hypoxemia control group in p < 0.01 is compared with blank control group；▲p<0.05▲▲The model control group of p < 0.01
Compared with non-hypoxemia control group；#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 with
Model control group compares；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 and aspirin group ratio
Compared with.
By table 23 as it can be seen that compared with model control group, the well-informed extremely significant property of high dose group SOD vigor of Folium Ginkgo group, silver
It increases (p < 0.01), the well-informed middle dose group SOD vigor conspicuousness of aspirin group, silver increases (p < 0.05)；With Folium Ginkgo group
Compare, during silver is well-informed, low dose group SOD vigor conspicuousness reduce (p < 0.05)；There was no significant difference for other each comparison among groups (p >
0.05)。
MDA assay
24 silver medal of table has quick access to information Contained Serum prevention administration to Na2S2O4The influence of cell MDA content after modeling
cp<0.05ccThe non-hypoxemia control group in p < 0.01 is compared with blank control group；▲p<0.05▲▲The model control group of p < 0.01
Compared with non-hypoxemia control group；#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 with
Model control group compares；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 and aspirin group ratio
Compared with.
As shown in Table 24, compared with model control group, the well-informed high dose group MDA vigor of Folium Ginkgo group, silver is extremely significant
Property reduce (p < 0.01), aspirin group, the equal conspicuousness of the well-informed middle dose group MDA vigor of silver reduce (p < 0.05)；With ginkgo leaf
Piece group is compared, and the well-informed extremely significant property of low dose group MDA vigor of silver increases (p < 0.01)；Compared with aspirin group, silver is had quick access to information low
Dosage group MDA vigor conspicuousness increases (p < 0.05)；There was no significant difference (p > 0.05) for other each comparison among groups.
6.5.2 compound silver Lingtong capsule Contained Serum prevention administration causes cortical neuron to platelet activating factor (PAF)
The influence of cellular oxidation stress damage model
Morphological observation
Cortical neuron is observed: Normal group cell after PAF handles about 48h under inverted microscope (10 × 10 times)
Form is elongated, transparency is high, gloss is preferable；Model group cellular damage is obvious.The cortical neuronal cells and normal cell of damage
It compares, cell process disappears, swelling, and diopter weakens born of the same parents, and the contracting of cytoplasm circle falls off.Folium Ginkgo, aspirin, compound is added
Then form is obviously improved the cell that the well-informed Contained Serum of silver is protected in advance compared with damaging cells, retains protrusion knot compared with many cells
Structure, swelling Leukopenia, and also cell is glossy.
Mtt assay measures cell viability
25 silver medal of table is had quick access to information influence of the Contained Serum prevention administration to cell survival rate after PAF modeling
#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 and model control group
Compare；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 25 as it can be seen that compared with model control group, the well-informed height of Folium Ginkgo group, silver, middle dose group cell survival rate are equal
Extremely significant property increases (p < 0.01), and aspirin group cell survival rate conspicuousness increases (p < 0.05)；Compared with Folium Ginkgo group,
The well-informed low dose group cell survival rate conspicuousness of silver reduces (p < 0.05)；There was no significant difference for other each comparison among groups (p >
0.05)。
LDH release measurement
26 silver medal of table is had quick access to information influence of the Contained Serum prevention administration to cell LDH leakage rate after PAF modeling
#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 and model control group
Compare；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 26 as it can be seen that compared with model control group, the extremely significant property reduction of the well-informed high dose group LDH leakage rate of silver (p <
0.01), the well-informed middle dose group LDH leakage rate conspicuousness of Folium Ginkgo group, silver reduces (p < 0.05)；Other each comparison among groups are without aobvious
It writes sex differernce (p > 0.05).
LD assay
27 silver medal of table is had quick access to information influence of the Contained Serum prevention administration to cell LD content after PAF modeling
#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 and model control group
Compare；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 27 as it can be seen that compared with model control group, the well-informed high dose group cell LD content of Folium Ginkgo group, silver is extremely significant
Property reduce (p < 0.01), the well-informed middle dosage of silver reduces (p < 0.05) in cell LD content conspicuousness；Other each comparison among groups are without aobvious
It writes sex differernce (p > 0.05).
SOD vitality test
28 silver medal of table is had quick access to information influence of the Contained Serum prevention administration to cell SOD vigor after PAF modeling
#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 and model control group
Compare；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 28 as it can be seen that compared with model group, the well-informed extremely significant property of high dose group SOD vigor of Folium Ginkgo group, silver is increased
(p < 0.01), the well-informed middle dose group SOD vigor conspicuousness of aspirin group, silver increase (p < 0.05)；Compared with Folium Ginkgo group,
The well-informed extremely significant property of low dose group SOD vigor of silver reduces (p < 0.01)；Compared with aspirin group, the well-informed low dose group SOD of silver
Vigor conspicuousness reduces (p < 0.05)；There was no significant difference (p > 0.05) for other each comparison among groups.
MDA assay
29 silver medal of table is had quick access to information influence of the Contained Serum prevention administration to cell MDA content after PAF modeling
#p<0.05##The model control group of p < 0.01 is compared with blank control group；* p < 0.05**p < 0.01 and model control group
Compare；aP < 0.05,aaP < 0.01 is compared with Folium Ginkgo group；bP < 0.05,bbP < 0.01 is compared with aspirin group.
By table 29 as it can be seen that compared with model control group, the well-informed high dose group MDA vigor conspicuousness drop of Folium Ginkgo group, silver
Low (p < 0.05)；There was no significant difference (p > 0.05) for other each comparison among groups.
7, compound silver Lingtong capsule Contained Serum therapeutic administratp grinds protective effect-mechanism of in vitro culture cortical neuron
Study carefully
The preparation of 7.1 rat Contained Serums
With 6.1.
7.2 cortical neuronal cells originally cultures
With 6.2.
7.3 experimental model preparation methods
7.3.1 sodium dithionite (Na2S2O4) merge lack sugar cause cortical neuronal cells oxygen-glucose deprived injury model (control
Treat administration)[26]
The company two sodium sulphate (Na of Asia of final concentration of 10mM are added in reference literature in sugar-free EBSS solution2S2O4) dry powder fills
Divide dissolution, uses NaHCO3Adjusting pH is 7.2, as anoxic solution.
It takes and is incubated at the well-grown cortical neuronal cells of 24 orifice plates for testing, original fluid is discarded, with containing sugar
EBSS solution, which is gently swung, to be washed twice, changes the Na containing final concentration of 10mM into2S2O4Sugar-free EBSS solution (blank control group adds
Enter the EBSS containing sugar), while being sealed plate surrounding with medical proof fabric, anoxic culture solution is changed after being incubated for 1h, with the EBSS containing sugar
Solution gently swings to wash changes the normally Neurobasal culture solution containing B-27 into twice, while each group serum is added, and sets 37 DEG C, 5%
CO2Continue culture in incubator for 24 hours.Blank control group serum, the mould for accounting for total volume 10% is added in blank control group (Control)
The blank control group serum for accounting for total volume 15%, (the silver spirit of compound silver Lingtong capsule high dose group is added in type control group (Vehicle)
Logical-H) addition accounts for the well-informed Contained Serum of silver of total volume 15%, the silver for accounting for total volume 10% is added in middle dose group (well-informed-M of silver)
The well-informed drug containing of silver for accounting for total volume 5% is added in well-informed+5% blank control group serum of Contained Serum, low dose group (well-informed-L of silver)
The ginkgo leaf for accounting for total volume 15% is added in+10% blank control group serum of serum, positive control Folium Ginkgo group (Folium Ginkgo)
The aspirin Contained Serum for accounting for total volume 15% is added in piece group Contained Serum, aspirin group (ASP), and each group sets 3
Multiple holes, experiment are repeated 3 times.
Observation index measurement:
Mtt assay measures cell viability, and LDH release and SOD, MDA vitality test are conventionally.
7.3.2 platelet activating factor (PAF) causes cortical neuronal cells damage model[27]
It takes and is incubated at the well-grown cortical neuronal cells of 24 orifice plates for testing, discard original fluid, gently with PBS
It swings and washes twice, fresh medium and final concentration of 4 × 10 is added-5The PAF of M sets 37 DEG C, continues to cultivate in 5%CO2 incubator.
Each group Contained Serum is added after 3h.If blank control group (the blank control group serum that addition accounts for total volume 15%), model comparison
(addition accounts for the silver spirit of total volume 15% for group (the blank control group serum that addition accounts for total volume 15%) and compound silver Lingtong capsule height
Logical Contained Serum), in (silver that addition accounts for total volume 10% have quick access to information+5% blank control group serum of Contained Serum), it is low that (addition accounts for
Well-informed+10% blank control group serum of Contained Serum of the silver of total volume 5%) dosage group, (addition accounts for positive control Folium Ginkgo group
The Folium Ginkgo group Contained Serum of total volume 15%), it sets 37 DEG C, continue culture for 24 hours in 5%CO2 incubator, each dosage sets 3
A multiple holes, experiment are repeated 3 times.
Index observing measurement:
Mtt assay measures cell viability: LDH release and SOD, MDA vitality test are conventionally.
Research to the extracorporeal neuron damage NF- κ B signal Signal Transduction Pathways of PAF induction:
Neuron culture is carried out after PAF modeling terminates to be added Contained Serum for 24 hours using 6 orifice plates to be swung with PBS and wash cell training
Feeding plate twice, is added appropriate 0.125% pancreatin and is blown and beaten with liquid-transfering gun to lower layer's cells float in digestion, micro tire ox blood is added
Clear terminate digests, and merges with group and is transferred in 1.5ml centrifuge tube, and 2000rpm is centrifuged 10min, and it is thin to outwell digestive juice reservation lower layer
Born of the same parents add appropriate RIPA lysate (1mM containing PMSF) to be vortexed and mix, 30min is then cracked in ice bath, are vortexed every 5min mixed
Even, 4 DEG C, 12000rpm is centrifuged 5min, isolates supernatant, and BCA method carries out protein quantification, measures supernatant volume, it is added 5 ×
Boiling water boils 5min after SDS sample-loading buffer, saves after packing in -80 DEG C.Prepare 4% concentration polyacrylamide concentration glue and
10% concentration polyacrylamide gel (PAGE) separation gel carries out electrophoresis, by the liquor capacity loading of 20 μ g albumen.To required mesh
Albumen at MARK be sufficiently separated after stop electrophoresis.It is indicated referring to the molecular weight of MARK, is cut into the PAGE glue containing destination protein,
" sandwich " shape is made into together with the pvdf membrane activated in advance with methanol, arrangement is successively are as follows: sponge/filter paper/glue/film/filter
Paper/sponge.Wet process transferring film carries out electricity under 100mA constant current and turns 2 hours.After transferring film, pvdf membrane, confining liquid closing are removed
1h is to eliminate non-specific background.TBST washes off confining liquid, 4 DEG C of primary antibody overnight incubations.It is swung and is washed three times at room temperature with TBST, every time
10min.After secondary antibody is incubated for 2h at room temperature, TBST, which is swung, to be washed three times, each 10min.Appropriate Ecl developing solution is added dropwise, uses gel imaging
Instrument is exposed film and takes pictures, with the net OD value of Quantity one image processing software analysis target stripe.Experiment weight
Again three times.
7.4 experimental result
7.4.1 compound silver Lingtong capsule Contained Serum therapeutic administratp is to sodium dithionite (Na2S2O4) merge scarce sugar cause skin
The influence of layer neuronal cell oxygen-glucose deprived injury model
30 silver medal of table is had quick access to information influence of the therapeutic administratp to cell survival rate in neure damage caused by sodium dithionite
(average value ± s.d., n=3)
The model comparison of #p < 0.05, ##p < 0.01 is compared with blank control group；* p < 0.01 p < 0.05, * * and model control group
Compare.
Cortical neuronal cells are through Na2S2O4Be added after processing Contained Serum about for 24 hours after, observed under inverted microscope: normal
Cellular control unit form is elongated, transparency is high, gloss is preferable；Model group cellular damage is obvious.The cortical neuronal cells of damage
Compared with normal cell, cell process disappears, swelling, and diopter weakens born of the same parents, and the contracting of cytoplasm circle falls off.Compound silver is added and has quick access to information and contains
Then form is obviously improved the cell of medicine serum compared with damaging cells, compared with many cells retain raised structures, swelling Leukopenia, and
And cell is glossy.
By table 30 as it can be seen that compared with model control group, the well-informed-H group of silver and Folium Ginkgo group can be external caused by OGD
Neure damage plays a very good protection (p < 0.01), and the well-informed-M of silver also can conspicuousness raising neuron depositing after OGD
Motility rate (p < 0.05).
The well-informed therapeutic administratp of 31 silver medal of table is living to LDH leakage rate, SOD and MDA in neure damage caused by sodium dithionite
The influence (average value ± s.d., n=3) of power
The model comparison of #p < 0.05, ##p < 0.01 is compared with blank control group；* p < 0.01 p < 0.05, * * and model control group
Compare.
By table 31 as it can be seen that compared with model control group, the extremely significant property of the well-informed high dose group energy of silver reduces cell after OGD modeling
LDH leakage rate (p < 0.01) in supernatant, the well-informed middle dose group energy conspicuousness of silver reduce LDH leakage rate (p < 0.05).Show silver
It is well-informed to mitigate cellular damage caused by OGD well.The extremely significant property of the well-informed high dose group energy of silver reduces external mind caused by OGD
Through increasing for first MDA vigor, lipid peroxidation injury is reduced.
After OGD modeling, the extremely significant property of SOD vigor reduces (p < 0.01), shows the antioxidant system of neuron by serious
It destroys, and silver increases SOD vigor (p < 0.05) after neuron OGD damage with having quick access to information high dose group energy conspicuousness, plays good
Anti oxidative damage.
The influence of cell survival rate is (average in the extracorporeal neuron damage that the well-informed therapeutic administratp of 32 silver medal of table induces PAF
Value ± s.d., n=3)
The model comparison of #p < 0.05, ##p < 0.01 is compared with blank control group；* p < 0.01 p < 0.05, * * and model control group
Compare.
After PAF modeling be added Contained Serum processing about for 24 hours after, observed under inverted microscope (10 × 10 times), normal control
Group cellular morphology is elongated, transparency is high, gloss is preferable；Model group cellular damage is obvious.The cortical neuronal cells of damage with just
Normal cell is compared, and cell process disappears, swelling, and diopter weakens born of the same parents, and the contracting of cytoplasm circle falls off.The well-informed drug containing blood of compound silver is added
Into the cell of protection, then form is obviously improved compared with damaging cells clearly, compared with many cells retain raised structures, swelling Leukopenia,
And cell is glossy.By MTT result as it can be seen that the well-informed extremely significant property of high dose group cell survival rate of silver increases (p < 0.01), silver
Well-informed middle dose group cell survival rate conspicuousness increases (p < 0.05).
LDH leakage rate, SOD and MDA are living in the extracorporeal neuron damage that the well-informed therapeutic administratp of 33 silver medal of table induces PAF
The influence (average value ± s.d., n=3) of power
The model comparison of #p < 0.05, ##p < 0.01 is compared with blank control group；* p < 0.01 p < 0.05, * * and model control group
Compare.
By table 33 as it can be seen that compared with model control group, in the well-informed high dose group cell conditioned medium of silver under LDH leakage rate conspicuousness
It drops (p < 0.05), the also conspicuousness decline (p < 0.05) of the well-informed middle dose group LDH leakage rate of silver.
By table 33 as it can be seen that compared with model control group, silver well-informed high dose group, middle dose group, Folium Ginkgo group MDA vigor
Conspicuousness reduces (p < 0.05), and the well-informed extremely significant property of high dose group SOD vigor of silver increases (p < 0.01), the well-informed middle dose group mind of silver
(p < 0.05) is increased through first SOD vigor conspicuousness.
Influence (the average value of NF- κ B signal Signal Transduction Pathways in the well-informed extracorporeal neuron damage induced PAF of 34 silver medal of table
± s.d., n=3)
The model comparison of #p < 0.05, ##p < 0.01 is compared with blank control group；* p < 0.01 p < 0.05, * * and model control group
Compare.
Such as table 34 as it can be seen that the raising of p65 and pp65 expression that the well-informed high dose group of silver can inhibit PAF to induce to extremely significant property
(p < 0.01), the well-informed middle dose group of silver can also play obvious action (p < 0.05).In addition, silver is well-informed can also to inhibit I κ B α phosphoric acid
Change the expression of albumen.It can be seen that the well-informed NF- κ B that PAF can be inhibited to induce by preventing the phosphorylation of p65 and I κ B α of silver believes
The activation of number access.
By table 34 as it can be seen that after PAF is damaged, the extremely significant property of the expression quantity of extracorporeal neuron cell Syn reduces (p < 0.01),
Show under this condition, neuronal cell is subject to damage very serious.And the well-informed shadow to extracorporeal neuron Syn expression quantity of silver
Sound is consistent with vivo results, compared with model control group, the well-informed high dose group of silver can extremely significant property increase Syn expression quantity (p <
0.01), the well-informed middle dose group of silver also can increase to extremely significant property the expression quantity (p < 0.01) of Syn, to the ex vivo nerve of PAF induction
Member damage plays a very good protection.
Specific embodiment
Embodiment 1
1, it is formulated:
Ganoderma lucidum 333g ginkgo leaf 333g Radix Achyranthis Bidentatae 278g
Radix scrophulariae 278g rhizoma acori graminei 167g Rhizoma Gastrodiae 125g
2, preparation method:
(1) it crushes: rhizoma acori graminei is ground into particle, it is spare.
(2) CO2Supercritical extract: take rhizoma acori graminei particle in extraction kettle, CO2Supercritical extract, wherein pressure be
30Mpa, extraction kettle temperature are 60 DEG C, and extraction time is 1.5 hours, obtains volatile oil and rhizoma acori graminei extract remainder, spare.
(3) inclusion essential oil: taking volatile oil to be dissolved with isometric ethyl alcohol, obtain volatile oil ethanol solution, spare；Separately take
The beta-cyclodextrin of 4 times of volatile oil weight amounts, the water measured with 4 times of beta-cyclodextrin grind well, pour into colloid mill, slowly continuous to be added dropwise
Volatile oil ethanol solution is stated, mills inclusion 20 minutes, pours out, 0-4 DEG C of refrigeration stands 24 hours or more, filtration, in 40 DEG C of vacuum
Drying is spare to get volatile oil clathrate compound；
(4) alcohol extracting: the total medicinal material of prescription is added in ganoderma lucidum, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae and Rhizoma Gastrodiae and rhizoma acori graminei extract remainder
The ethyl alcohol that 8 times of weight concentrations are 90%, twice, 1.5 hours every time, extracting solution merged heating and refluxing extraction, stood, and filtered, filter
Ethyl alcohol is recovered under reduced pressure in liquid, the clear cream that relative density is 1.15~1.30 under the conditions of being concentrated into 60 DEG C, measures dry cream rate, spare.
(5) combination drying: clear cream is mixed with the microcrystalline cellulose of clear cream weight 30-40%, sets 60 DEG C of items of vacuum oven
It is dried under reduced pressure, gets dry extract spare under part.
(6) it crushes, mix, capsule filling: dry cream, cyclodextrin inclusion compound being set and are ground into fine powder in Universalpulverizer, with
The magnesium stearate that remaining microcrystalline cellulose and dry cream weigh 0.5% is uniformly mixed, and obtains fine powder.Wherein, step 5 and step 6 share
The weight ratio of microcrystalline cellulose total amount and clear cream is 237.5:260.
(7) capsule is filled: taking No. 0 hard capsule filling, loading amount: 0.5g.
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Claims (9)
1. a kind of Chinese medicine composition containing ganoderma lucidum and ginkgo leaf promotes the application in the drug of rehabilitation after preparing apoplexy,
In, the composition is made of the component of following parts by weight:
Ganoderma lucidum 300-400 parts by weight ginkgo leaf 300-400 parts by weight Radix Achyranthis Bidentatae 200-290 parts by weight
Radix scrophulariae 200-290 parts by weight rhizoma acori graminei 150-190 parts by weight Rhizoma Gastrodiae 100-140 parts by weight；
The composition is made by following preparation method, comprising the following steps:
A, rhizoma acori graminei is ground into particle；
B, rhizoma acori graminei particle is used into CO2Supercritical extract, extracting pressure 22-30Mpa, extraction kettle temperature are 45-60 DEG C, extraction
Taking the time is 0.5-1.5 hours, obtains volatile oil and rhizoma acori graminei extract remainder；
C, it takes volatile oil to be dissolved with isometric ethyl alcohol, obtains volatile oil ethanol solution；Separately take β-ring that 4-8 times of volatile oil weight is measured
Dextrin, is ground well with the water of 3-5 times of weight of beta-cyclodextrin, is poured into colloid mill, and above-mentioned volatile oil ethanol solution is slowly continuously added dropwise,
It mills and includes 20-40 minutes, pour out, 0-4 DEG C of refrigeration stands 24 hours or more, filtration is dried in vacuo to get waving in 40-80 DEG C
Hair oil inclusion compound；
D, the total 6-10 times of weight of medicinal material of prescription is added in ganoderma lucidum, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae and Rhizoma Gastrodiae and rhizoma acori graminei extract remainder
Concentration is the ethyl alcohol of 70-90%, and heating and refluxing extraction 1-3 times is 1-2 hours each, and extracting solution merges, and is stood, and filtration, filtrate subtracts
Receipts ethyl alcohol is pushed back, the clear cream that relative density is 1.15 ~ 1.30 under the conditions of being concentrated into 60 DEG C；
E, clear cream is dried under reduced pressure at 60-80 DEG C, is got dry extract；
F, dry cream, volatile oil clathrate compound are uniformly mixed.
2. application according to claim 1, it is characterised in that the composition is made of the following components:
Ganoderma lucidum 300-350 parts by weight ginkgo leaf 300-350 parts by weight Radix Achyranthis Bidentatae 250-290 parts by weight
Radix scrophulariae 250-290 parts by weight rhizoma acori graminei 150-180 parts by weight Rhizoma Gastrodiae 110-140 parts by weight.
3. application according to claim 1, it is characterised in that the composition is made of the following components:
333 parts by weight ginkgo leaf of ganoderma lucidum, 333 parts by weight Radix Achyranthis Bidentatae, 278 parts by weight
278 parts by weight rhizoma acori graminei of radix scrophulariae, 167 parts by weight Rhizoma Gastrodiae, 125 parts by weight.
4. application according to claim 1,2 or 3, it is characterised in that the Chinese medicine composition and the auxiliary material system pharmaceutically allowed
At dosage form as defined in pharmacy.
5. application according to claim 4, it is characterised in that the dosage form is capsule.
6. application according to claim 1, it is characterised in that apoplexy sequelae shows as hemiplegia, limb adynamia, mouth
Tongue skew, speech is stuttering puckery, hypophrenia, slobbering.
7. a kind of Chinese medicine composition containing ganoderma lucidum and ginkgo leaf is in the drug for preparing invigorating Qi, eleminating phlegm and freeing channels restoring consciouness
Application, wherein the composition is made of the component of following parts by weight:
Ganoderma lucidum 300-400 parts by weight ginkgo leaf 300-400 parts by weight Radix Achyranthis Bidentatae 200-290 parts by weight
Radix scrophulariae 200-290 parts by weight rhizoma acori graminei 150-190 parts by weight Rhizoma Gastrodiae 100-140 parts by weight.
8. application according to claim 7, it is characterised in that the composition is made of the following components:
Ganoderma lucidum 300-350 parts by weight ginkgo leaf 300-350 parts by weight Radix Achyranthis Bidentatae 250-290 parts by weight
Radix scrophulariae 250-290 parts by weight rhizoma acori graminei 150-180 parts by weight Rhizoma Gastrodiae 110-140 parts by weight.
9. application according to claim 7, it is characterised in that the composition is made of the following components:
333 parts by weight ginkgo leaf of ganoderma lucidum, 333 parts by weight Radix Achyranthis Bidentatae, 278 parts by weight
278 parts by weight rhizoma acori graminei of radix scrophulariae, 167 parts by weight Rhizoma Gastrodiae, 125 parts by weight.
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| CN113384605B (en) * | 2021-06-28 | 2022-09-06 | 广东康盾制药有限公司 | Medicament for treating infantile autism caused by hypoxic-ischemic brain injury and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1224614A (en) * | 1998-01-24 | 1999-08-04 | 倪德贵 | Medicine for preventing and curing cardiac and cerebral vascular diseases and its preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1224614A (en) * | 1998-01-24 | 1999-08-04 | 倪德贵 | Medicine for preventing and curing cardiac and cerebral vascular diseases and its preparation |
Non-Patent Citations (1)
| Title |
|---|
| 中医辨证治疗脑梗塞480例;栗广明等;《中日友好医院学报》;19981231;第12卷;第220页 |
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