CN102512508A - Application of rheum emodi wall extract to preparation of medicines for resisting ischemic cerebral vascular diseases - Google Patents
Application of rheum emodi wall extract to preparation of medicines for resisting ischemic cerebral vascular diseases Download PDFInfo
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Abstract
The invention discloses new application of a rheum emodi wall extract, and particularly relates to application of the rheum emodi wall extract to the preparation of medicines for preventing and treating ischemic cerebral vascular diseases. Animal experiments prove that an extract which is prepared from rheum emodi wall, is percolated, leached and eluted by using ethanol and 3,5,3',4'-tetrahydroxystilbene-4'-O-beta-D-glucopyranoside can relieve the neuromotor dysfunction of rats subjected to middle cerebral artery occlusion, reduce the range of cerebral infarction, have an effect of relieving the ischemic cerebral vascular diseases, and can achieve the protective effect on the ischemic cerebral tissues by lowering the level of tumor necrosis factors and the expression level of fatty acid synthase (Fas) protein. Therefore, the rheum emodi wall extract has the obvious treatment effect on the ischemic cerebral vascular diseases of experimental animals, which are caused by multiple reasons, and can be applied to the preparation of the medicines for treating or preventing the ischemic cerebral vascular diseases.
Description
Technical field
The present invention relates to the application of Radix Rhei emodi extract, particularly relate to the application of Radix Rhei emodi extract in preparation ischemia resisting property cerebrovascular disease medicament, belong to the therapeutic activity field of chemical compound or pharmaceutical preparation.
Background technology
Radix Rhei emodi (Rheum emodi Wall.) is root and the rhizome of Polygonaceae Rheum ripple leaf group plant, mainly is distributed in ground such as Tibet, Qinghai, Sichuan and Yunnan, all is wild.Radix Rhei emodi sour in the mouth, hardship, cold in nature, the Tibetan medicine claims bent prick (Quza), is traditional medical material of Tibetan medicine.Application number be 201110124226.9 Chinese invention patent applications that are called a kind of Radix Rhei emodi extract and preparation method thereof disclose a kind of Radix Rhei emodi through the ethanol percolation extract again through the extract of ethanol elution.This extract contains the monomeric compound of structural formula suc as formula (I):
R wherein
1Be
Or H.Contain 30%～99% structural formula monomeric compound in this extract suc as formula (II):
Structural formula can be through above-mentioned Radix Rhei emodi extract through further extraction, eluting prepare suc as formula the monomeric compound of (II); And through the NMR Analysis and Identification be 3,5,3 '; 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) (3; 5,3 ', 4 '-tetrahydroxystilbene 4 '-O-β-D-glucopyranoside).Based on the compare of analysis of existing chemical compound, tentatively confirm 3,5; 3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside has and Radix Polygoni Multiflori composition 2,3; 5; 4 '-tetrahydroxystilbene-2-O-β-similar biological activity of D-glucoside, comprise oxidation and removing free radicals, antitumor, cholesterol reducing, inhibition atherosclerosis, the liver protecting, vasodilator, control senile dementia, improving memory etc., can be applied to the exploitation of related drugs.
Reference material:
Tibet Golden Khada Medical Co., Ltd.. a kind of Radix Rhei emodi extract and preparation method thereof [P] .CN201110124226.9
Lv Lishuan. the preparation of stilbene glucoside and antioxidation mechanism research [D] in the Radix Polygoni Multiflori. Wuxi: Southern Yangtze University, 2006.
Summary of the invention
The object of the present invention is to provide the Radix Rhei emodi extract to be used to prepare the application of ischemia resisting property cerebrovascular disease medicament, and the purposes of pharmaceutical composition in preparation ischemia resisting property cerebrovascular disease medicament that contains the Radix Rhei emodi extract.
The present invention also aims to provide 3; 5; 3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) is used for preparing the application of ischemia resisting property cerebrovascular disease medicament, and the purposes of pharmaceutical composition in preparation ischemia resisting property cerebrovascular disease medicament that contains PDG.
Ischemic cerebrovascular (Ischemic cerebral vascular disease ICVD) is meant that the main feeding artery of brain takes place narrow or inaccessible; Cause corresponding site cerebral tissue disturbance of blood circulation; Cause blood supply district cerebral cell ischemia, anoxia even necrosis; Cause part or full cranial nerve function obstacle persistent period greater than 24h even death, its sick key of sending out interrupts failing in time to form effective side Zhi Xunhuan for vascular occlusion causes blood flow.ICVD accounts for about 80% of cerebrovascular, is to cause one of human three dead big principal diseases, is only second to heart disease and cancer.ICVD belongs to modern medicine " transient ischemic attack ", " cerebral infarction ", " cerebral embolism ", " cerebral infarction " etc., and Chinese medicine belongs to " apoplexy ", " apoplexy ", " hemiplegia " category.Traditional ischemic cerebrovascular comprises transient ischemic attack, and (Transient ischemic attack is TIA) with cerebral infarction (Ischemic stroke).The latter claims that again (Cerebral infarction CT), accounts for about 70% of cerebrovascular sum to cerebral infarction, comprises that modal clinically type has cerebral thrombosis, lacunar infarction, cerebral embolism.
ICVD is because the of short duration or persistent minimizing of blood flow of the main tremulous pulse of brain causes; Pathogenesis comprises many-side, mainly contains the effect, excitatory amino acid (EAA) toxic action, inflammatory cytokine infringement, ischemia half blanking bar dysfunction of the overload of activation, calcium ion, nitric oxide (NO) and the nitric oxide synthetase (NOS) of oxygen free radical injury, apoptosis regulation gene etc.The ICVD morbidity is many-sided and quite complicated process, and the cerebral ischemia infringement also is a progressive process, and delayed ischemic neurological deficits increases the weight of with the prolongation of Ischemia Time.
Technical scheme provided by the invention, its principle are based on the following drug effect principle of the Radix Rhei emodi extract that discloses through animal experiment:
(1) the Radix Rhei emodi extract can alleviate the rat nerves dyskinesia after middle cerebral artery is blocked, and ICVD is shielded.
Animal experiment of the present invention proves; The Radix Rhei emodi extract can significant prolongation the breathing time of acute cerebral ischemia mice, have in various degree improvement to show that it alleviates the drug effect of the rat nerves dyskinesia after the middle cerebral artery blocking-up to the neuromotor dysfunction of focal rats with cerebral ischemia.
(2) the Radix Rhei emodi extract can dwindle the cerebral infarction scope, and ICVD is shielded.
Animal experiment of the present invention shows that the Radix Rhei emodi extract can significantly can reduce the degree of reperfusion injury after the cerebral ischemia, dwindles focal cerebral ischemia rat infarct size, embodies the protective effect to ICVD.
(3) 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) plays a protective role to ischemic rat brain through downward modulation tumor necrosis factor (TNF) level.
Tumor necrosis factor (tumor necrosis factor; TNF) be one type of cytokine that can directly cause death of neoplastic cells; Be divided into two kinds of TNF-α and TNF-β according to its source and structure, the former is produced by mononuclear phagocyte, claims cachectin (cachectin) again; The latter is produced by the activated T cell, has another name called lymphotoxin (lymphotoxin).The inflammatory reaction of cerebral ischemia is one of important damage mechanism of ischemic cerebrovascular.The inflammatory reaction of cerebral ischemia is by cytokine mediated.TNF is a kind of in the cytokine, can mediate the generation of multiple inflammatory mediator, participates in the pathophysiological process of body.TNF can quicken activated lymphocytes and get in the brain, inflammatory reaction in the aggravation brain through raising the expression of the adhesion factor (ICAM-1) on vascular endothelial cell and the leukocyte; And leukocyte and the adhesive attraction between the vascular endothelial cell in the blood are strengthened; Mechanically stop up the microcirculation passage; The blood supply of influence tissue (particularly ischemic penumbra), and activatory leukocyte can discharge a large amount of toxicity oxygen-derived free radicals proteolytic enzymes, the damage local vascular; Cause vascular permeability to increase, cause cerebral edema.
Animal experiment of the present invention proves that PDG plays a protective role to the animal pattern cerebral tissue through downward modulation TNF level.
(4) 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) can play a protective role to ischemic rat brain through downward modulation Fas proteic expression.
Cerebral infarction has damaged and can cause cellular energy exhaustion, and the various enzymes of activation activate its downstream factor, finally cause apoptosis and necrosis.Think that at present Fas is one of most important short apoptosis gene.Fas combines with Fas part (FasL), starts the caspase cascade reaction, directly causes apoptotic cell to disintegrate, thereby causes apoptosis, and this approach is called death receptor mediated Apoptosis approach.Fas albumen is as a kind of important promotion regulation of apoptosis gene; Belong to tumor necrosis factor/trk C family; Its expression product has the effect of transmitting apoptotic signal, but combines cell death inducing as the Fas albumen of death receptor with the Fas protein ligands.Cerebral ischemia reperfusion stream back apoptosis-related genes Fas and FasL can especially express in neuron in brain, and they promote apoptosis amount to take place.
The present invention shows that through the animal experiment example PDG possibly limit it and combine with FasL through suppressing the proteic expression of Fas, suppresses apoptotic generation through suppressing the death receptor approach thus, finally brings into play the cerebral protection of its ischemia resisting hypoxic damage.
Based on the Radix Rhei emodi extract drug effect principle of PDG ischemia resisting property cerebrovascular particularly, it is the pharmaceutical composition of active ingredient that the present invention provides with Radix Rhei emodi extract and PDG.This drug regimen with the Radix Rhei emodi extract particularly PDG be active ingredient, add pharmaceutically acceptable pharmaceutic adjuvant and be prepared from according to the conventional method of this area.Prepared drug regimen can further prepare becomes pharmaceutical preparation.Pharmaceutical technology based on existing plant extract; Particularly existing pharmaceutical technology is to 2; 3; 5,4 '-application of tetrahydroxystilbene-2-O-β-D-glucoside, the pharmaceutical preparation of the present invention's preparation can be: the dosage form of oral administration such as tablet, capsule (comprising hard capsule, soft capsule, enteric coated capsule and microcapsule), powder, granule and syrup; The dosage form of non-oral administration such as injection, suppository, pill, gel and patch.In addition, can also oral fast release solid formulation (for example tablet, granule etc.) and the slow releasing preparation (tablet, granule, fine granular, pill, capsule, syrup, Emulsion, suspension, solution) that is used for oral or non-oral administration be used for the present invention.Above-mentioned preparation can be the coating or the form of coating not, depends on the needs.
The present invention proves that through experimental study the Radix Rhei emodi extract has remarkable reverse to turn usefulness into to rat ischemia property cerebrovascular disease pathological changes, can be applied to the preparation of ischemia resisting property cerebrovascular disease medicament.Radix Rhei emodi has reliable medicinal safety as traditional Tibetan medicine, and the toxicity trial of PDG proves its safety too.
The specific embodiment
Below in conjunction with Test Example technical scheme of the present invention is further described.
Test Example one
The protective effect of the cerebral infarction that the Radix Rhei emodi extract causes chmice acute cerebral ischemia and focal cerebral ischemia in rats.
1, materials and methods
1.1 medicine and reagent Radix Rhei emodi extract specifically are Radix Rhei emodis through the ethanol percolation extract again through the extract of ethanol elution, extract from the Radix Rhei emodi medical material according to the open method of CN2011101722069.These article contain 52%3,5,3 approximately ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG), be chocolate brown powder, be mixed with 700mgml with distilled water during experiment
-1Aqueous solution is through using behind the 0.22 μ m aperture filter membrane EK, to call the Radix Rhei emodi solution of extract in the following text; Nimodipine (Tianjin Central Pharmaceutical Co., Ltd); Chloral hydrate, AR (5-linked chemical plant, Shanghai); Triphenyltetrazolium chloride (TTC), AR (Shanghai reagent three factories); Sodium dihydrogen phosphate, sodium hydrogen phosphate (granary chemical industry two factories).
1.2 animal health ICR mice, 18～22g; Wistar rat 250～300g, male and female have both (Nanjing University of Traditional Chinese Medicine's Experimental Animal Center provides).
1.3 experiment grouping chmice acute cerebral ischemia test divides 5 groups: model group, give 0.9% sodium chloride injection, the positive drug group gives 0.02% nimodipine solution, basic, normal, high 3 dose groups of Radix Rhei emodi extract (in crude drug content).Each is organized dosage and sees table 1, and dosage is 20ml/kg.The focal cerebral ischemia in rats test divides 6 groups: sham operated rats, model group, give 0.9% sodium chloride injection, and the positive drug group gives 0.02% nimodipine solution, and basic, normal, high 3 dose groups of Radix Rhei emodi extract (in crude drug content).Each is organized dosage and sees table 2, and the administration volume is 20ml/kg.
1.4 the ICR mice is got in chmice acute cerebral ischemia experiment, is divided into 5 groups at random, oral administration 4d, and behind last administration 0.5hr, dorsal position is fixed, and separates left and right sides common carotid artery and merges vagus nerve and ligation; The breathing of record mice is held time.
1.5 rat focal ischemia experiment
1.5.1 with a long 40cm, an end of the nylon wire of diameter 0.2mm is heated to be slick sphere with the preparation of nylon embolus in experiment.Clean and with wipes of alcohol in 18.5mm place labelling apart from the pommel, place 0.9% normal saline subsequent use.
1.5.2 body weight 240～270g rat is got in the making of cerebral ischemic reperfusion in rats (IR) model, is divided into 6 groups at random by body weight, oral administration 4d is behind last administration 0.5hr, with 10% chloral hydrate anesthesia (300mg/kg).Rat is lain on the back on operating-table, and neck medisection is separated right carotid (CCA); External carotid artery (ECA), internal carotid artery (ICA), the branch of pricking and cutting off ECA with No. 0 toe-in; One section on ligation (twice are beneficial to cut off) and free ECA trunk separate downwards along ICA.Clamp CCA and ICA with bulldog clamp.Cut an osculum at ECA, the nylon wire for preparing is inserted ECA, go into cranium to anterior cerebral artery (ACA) through the CCA crotch through ICA, the nylon wire insertion depth is about 18mm.Draw nylon wire to make its pommel be back to the blood supply that can recover middle cerebral artery in the ECA when pouring into again outward.Sham operated rats is not inserted nylon wire, and all the other steps are with other experimental grouies.
1.5.3 the scoring of the laggard capable behavioristics of neuroethology scoring operation: Mus tail built on stilts is carried in (1), and observes the forelimb situation.Normal rat two forelimbs stretch to ground symmetrically.Wrist elbow flexing appears in operation offside forelimb, and the shoulder inward turning is received in the left fore, is close to breast arm meter 4 minutes, otherwise counts 0 fen.(2) with on the sliding ground of Mus horizontalization, push away left and right shoulder respectively, check the resistance that opposing promotes to side shifting.Normal rat bilateral resistance is symmetry obviously.When the modeling rat is moved when right shoulder to the left, can find the resistance descender,, be chosen as 1～3 fen according to the difference of decline degree; (3) animal two forelimbs are put on the wire netting, observed the muscular tension of two forelimbs, normal rat two muscle of anterior limb tension force are symmetry obviously.The obvious descender of left fore muscular tension takes place in the modeling rat, and the degree according to descending is chosen as 1～3 fen.Mark according to above standard, full marks are 10 minutes, and mark is high more, explain that the behavior disorder of animal is serious more.Single blind method is adopted in scoring.
1.5.4TTc behind the dyeing measure cerebral infarct size operation 24hr rat broken end is got brain, brain is placed ice-cold normal saline 10min, behind removal olfactory bulb, XIAONAO and the low brain stem,, be cut into 5 with crown 4 cuttves of cutting of remainder.The 1st cutter is before brain in the middle of the utmost point and the optic chiasma line; The 2nd cutter is at the optic chiasma position; The 3rd cutter is at the infundibular stalk position; The 4th cutter is between the infundibular stalk and the posterior lobe tail utmost point.Place 5ml to contain the phosphate buffer of 1%TTC the brain sheet rapidly then, the lucifuge temperature is incubated 30min, wherein whenever stirs 1 time at a distance from 7～8min, dyed after, normal structure is rose, and blocking tissue is white in color, and boundary is clearly demarcated.After temperature is incubated and finished brain section is taken a picture, cut red white colour district and weigh.According to " weight area method ", calculate 5 brain sheets area of totally 10 planar gross areas and infarct area respectively, obtain the percentage ratio that the infarct area accounts for a side cerebral hemisphere gross area, i.e. infarction size.
1.6 statistical procedures t check.
2 results
2.1 the Radix Rhei emodi extract is to the influence of chmice acute cerebral ischemia and the comparison of model group; One-tenth live time (P＜0.05 of Radix Rhei emodi extract ability significant prolongation acute cerebral ischemia mice; P＜0.01); And the effect of Radix Rhei emodi extract increases with dosage, explains that the Radix Rhei emodi extract has anti-GBI effect.See table 1.
Table 1 Radix Rhei emodi extract is breathed the influence
of holding time to the chmice acute cerebral ischemia
Annotate: compare * P＜0.05, * * P＜0.01N=20 with model group
2.2 the Radix Rhei emodi extract is to the influence of the focal cerebral infarction of rat
2.2.1 the Radix Rhei emodi extract shows that to the influence experiment of focal rats with cerebral ischemia behavioral deficiency degree after the middle cerebral artery blocking-up, the nerve dyskinesia all appears in all animals.The scoring of 24hr inner model group behavioristics relatively has significant difference with sham operated rats, explains that model is successful.3 dose groups of Radix Rhei emodi extract make all that the neuromotor dysfunction of focal cerebral ischemia rat has improvement in various degree behind operation 10hr and the 24hr; Wherein, Middle and high two dose groups are compared difference with model group have highly significant meaning (P＜0.05, P＜0.01), sees table 2.
Table 2 Radix Rhei emodi extract is to the influence of focal rats with cerebral ischemia behavioristics defect level
Annotate: compare * * P＜0.01 with sham operated rats; Compare △ △ P＜0.01, △ P＜0.05 with model group
2.2.2 the Radix Rhei emodi extract influences the model group rat brain coronal section overwhelming majority it is thus clear that bigger pale districts are more obvious with the normal structure interface to focal rats with cerebral ischemia cerebral infarction scope.The middle and high dose groups of Radix Rhei emodi extract all can significantly be dwindled focal cerebral ischemia rat infarction size (P＜0.05, P＜0.01), compares with model group, has reduced by 72.7% and 87.5% respectively, sees table 3.
Table 3 Radix Rhei emodi extract is to the influence
of focal rats with cerebral ischemia cerebral infarction scope
Above-mentioned animal experiment example adopts chmice acute cerebral ischemia and focal cerebral ischemia in rats to irritate model again, observes the protective effect of Radix Rhei emodi extract to experimental cerebral ischemia.Test with vagus nerve and bilateral common carotid arteries ligation in the lump, in blocking-up cerebral blood flow supply, has stimulated vagus nerve when causing the chmice acute cerebral ischemic model, and causes and blood pressure drops cause cerebral ischemia.The pharmaceutical intervention test shows; The breathing time of Radix Rhei emodi extract ability significant prolongation acute cerebral ischemia mice; Neuromotor dysfunction to focal rats with cerebral ischemia has improvement in various degree, demonstrates it and alleviates the rat nerves dyskinesia after middle cerebral artery is blocked; The Radix Rhei emodi extract also can reduce the degree of reperfusion injury after the cerebral ischemia, dwindles the cerebral infarction scope.
Test Example two
This Test Example shows 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside plays a protective role to cerebral tissue through regulating tumor necrosis factor (TNF) level.
1 material
1.1 animal health male Wistar rat (animal origin is with Test Example one).
1.2 reagent and medicine 3,5,3 '; 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG); Extract from the Radix Rhei emodi medical material according to the open method of CN2011101722069, purity 92.80% adds normal saline and is mixed with PDG solution (hereinafter to be referred as the PDG aqueous solution); TNF reagent kit (Science and Technology Development Center of Chinese People's Liberation Army General Hospital puts and exempts from institute, lot number 20010218); Dexamethasone injection (Anyang Yi Kang Pharmacy stock Co., Ltd, lot number 0204021).
1.3 instrument SN-682 type radioimmunity gamma counter (Shanghai nuclear good fortune photoelectric instrument company limited); DY89-1 electric driven glass refiner (Ningbo Xin Zhike device institute).
2 methods
2.1 40 of model preparation and administration Wistar rats are divided sham operated rats, model group, Dexamethasone group and PDG group, 10 every group at random.Adopt two blood vessel blocking methods, 100mg/kg ketamine ip anesthetized rat, dorsal position is following admittedly, throat portion preserved skin; The surgery sterilization, the throat median incision separates bilateral common carotid arteries (CCA), threading; Ligation bilateral CCA, skin suture, sham operated rats animal threading only wherein, not ligation.3d tail iv normal saline (sham operated rats, model group) or medicine (Dexamethasone group dexamethasone pin 1.04mg/ (kgd), PDG20.83mg/ (kgd)) before the modeling, every day 1 time, and prick and close preceding 2h filling 1 time.
2.2 the index determining abdominal aortic blood, preparation serum; Other gets the right side cerebral tissue, in the rearmounted homogenizer of weighing, adds normal saline and fully grinds and process 20% homogenate, and 4 ℃ of centrifugal 15min of 3500r/min get supernatant.Adopt serum measured by radioimmunoassay and cerebral tissue TNF level.
2.3 statistical procedures: The data SPSS 10.0 software processes adopt variance analysis.
3 results
Rat blood serum and cerebral tissue TNF level influenced model group serum and cerebral tissue TNF level apparently higher than sham operated rats (P＜0.01); Compare with model group, Dexamethasone group serum and PDG group serum and cerebral tissue TNF level significantly reduce (P＜0.01); Compare with Dexamethasone group, PDG group serum TNF level reduces obviously (P＜0.01), and the result sees table 1.
Table 1 rat blood serum and cerebral tissue, and the variation of TNF and TGF level (x ± s, n=9)
Compare with model group: * P＜0.05**P＜0.01; Compare with Dexamethasone group: △ P＜0.01
Above-mentioned animal experiment example shows that TNF shows that apparently higher than sham operated rats TNF plays an important role in model group cerebral tissue and the serum in cerebral ischemia.Under PDG medication intervention, rats in test groups serum TNF level obviously reduces, and shows that PDG plays a protective role to cerebral tissue through downward modulation TNF level.
Test Example three
This Test Example explanation 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside is to the regulating and controlling effect of rat brain sheet Fas protein expression.
1 data and method
1.1 laboratory animal is divided into groups and 30 of medication healthy SD rats, the body constitution amount is 250～300g (source is with Test Example one); PDG (source is with Test Example two); Nimotop vial (the inferior precious Pharmaceutical in Shanxi); The Mus Fas of rabbit Chinese People's Anti-Japanese Military and Political College monoclonal antibody (SANTA CRUZ company).Animal is divided into 6 groups at random: sham operated rats (be called for short sham group), cerebral infarction group (being called for short the Model group), PDG administration group (10,20,40mg/kg) (hereinafter to be referred as T1, T2, T3 group), nimodipine group (1mg/kg) (being called for short the T4 group), every group of 5 rats.T1～T4 organizes respectively before the Rhizoma Atractylodis Macrocephalae 5d continuous lumbar injection ursodeoxycholic acid of 1h, nimodipine before draw materials, and all the other each groups are injected equivalent etc. respectively and oozed normal saline, 1 time/d.
1.2 rat right side cerebral infarction (MCAO) Preparation of model and checking adopt improvement line bolt legal system to be equipped with rat right side cerebral infarction model.The line bolt adopts the smooth boiling hot diameter of a circle 0.23mm fishing line of head end, slowly inserts (18.5 ± 0.5) mm approximately to internal carotid artery intracranial direction by the right carotid furcation, makes the end of a thread pass through the middle cerebral artery section start, and the sham operated rats insertion depth is less than 15mm.Behind the thromboembolism 2h, subcutaneous fascia and skin are extracted and sewed up to fishing line gently.Carry out function of nervous system's scoring in MCAO postoperative 24h with reference to 5 fens systems such as Longa standards of grading, 1～4 minute rat is the modeling success, for ineligible rat in advance to reject.For reject in the experiment and dead rat all by strict experiment condition unifiedly replenish, grouping.
1.3 SABC detects and graphical analysis MCAO postoperative 24h, with each group rat anesthesia, after the 4% paraformaldehyde perfusion fixation, broken end is got brain, gradient alcohol dehydration, directed FFPE, 5 μ m crown position section.The dyeing of SABC Envision method.The Fas positive expression is to be pale brown color in the endochylema.Every section is selected 5 high power fields at random at ischemia side cerebral cortex, detects the cell counting of average each visual field Fas positive expression through image analysis system.
1.4 statistical analysis is used the SPSS10.0 software analysis, and all data are all used
expression.Many group differences significance test adopts one factor analysis of variance.
2 experimental results
2.1 respectively organize rat nerves function scoring MCAO postoperative 24h, the scoring of Sham group function of nervous system is 0; The Model group all has obvious neurological deficit, and after PDG, nimodipine administration, T1, T2, T3, T4 group neurological deficit degree significantly alleviate than the Model group, but still be significantly higher than Sham group (P＜0.05); T1 group neurological deficit degree is significantly higher than T4 group (P＜0.05), and T2, T3 group and T4 group comparing difference not statistically significant (seeing table 1).
2.2 respectively organizing the comparison of rat cerebral cortex Fas protein expression content compares with the Sham group; Model group Fas positive cell counting showed increased, after PDG, nimodipine administration, T1, T2, T3, T4 group then obviously reduce; Difference has significance (P＜0.05), sees table 1.
Table 1 is respectively organized rat cerebral cortex Fas Caspase-3 protein expression positive cell counting
Annotate: compare △ P＜0.05 with the Model group; Compare #P＜0.05 with the Sham group; Compare ▲ P＜0.05 with the T4 group.
Above-mentioned zooperal immunohistochemical experiment result shows, MCAO postoperative 24h, and the Fas positive expression is positioned in the Cytoplasm of ischemic neuron, neurogliocyte, endotheliocyte etc.Sham operated rats Fas positive expression is more rare; Cerebral infarction group Fas positive expression amount showed increased.After giving the PDG treatment, each is organized the Fas positive expression and obviously reduces the minimizing of prompting apoptosis.Show that PDG maybe be through suppressing the Fas protein expression, resist itself and the combining of FasL, suppress apoptotic generation through inhibition death receptor approach thus, finally bring into play the cerebral protection of its ischemia resisting hypoxic damage.
Test Example four
3,5,3 ', 4 '-tetrahydroxystilbene-4 '-test of the toxicity of compound of O-β-D-glucoside.
1, experiment material
The ICR mice, male, body weight 18g～22g provides the (quality certification number: 1037764) by Sichuan University's West China Experimental Animal Center; The PDG aqueous solution is with Test Example two.
2, test method and result
The compounds of this invention is carried out acute toxicity test as test specimen to mouse gavaging.The result shows: the maximum tolerated dose of mouse gavaging The compounds of this invention is 20gkg
-124hr
-1, be about 2,3,5,4 '-(the 60kg body weight becomes human oral consumption per day 1.2gkg for the taking dose standard of tetrahydroxystilbene-2-O-β-D-glucoside medicine
-124hr
-1) 1000 times; The LD50 of the disposable filling clothes of mice is 15.2gkg
-1Inferior
-1, 95% confidence limit is 22.60～34.23gkg
-1
Long term toxicity test shows, rats gavaged The compounds of this invention 0.4,0.8,1.6gkg
-1D
-1(be equivalent to 2; 3; 5; 4 '-20,40,80 times of tetrahydroxystilbene-2-O-β-D-glucoside clinical drug amount), hematology, blood biochemical are learned relatively all no significant differences of index, organ coefficient and matched group behind the 24wk, and pathological examination results does not also see that the pathologic that health food causes changes.Stop using behind the invention chemical compound 2wk, each item index of each administration group and blank group compare, and all no significant difference points out this chemical compound not have obvious retardance toxicity.Explain that it also is safe that The compounds of this invention is taken for a long time.
Claims (9)
1. the application of Radix Rhei emodi extract in preparation control ischemic cerebrovascular medicine, said Radix Rhei emodi extract contains the monomeric compound of structural formula suc as formula (I):
R wherein
1Be
Or H; Said R
1Be
Monomeric compound be 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside, its structural formula is suc as formula (II):
2. the pharmaceutical composition that contains the Radix Rhei emodi extract is used to prepare the application of control ischemic cerebrovascular medicine, and said Radix Rhei emodi extract contains the monomeric compound of structural formula suc as formula (I):
R wherein
1Be
Or H; Said R
1Be
Monomeric compound be 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside, its structural formula is suc as formula (II):
3. application according to claim 2 is characterized in that: said pharmaceutical composition contains Radix Rhei emodi extract and pharmaceutically acceptable pharmaceutic adjuvant.
4. according to claim 1 or 2 or 3 described application, it is characterized in that: said medicine is that the tumor necrosis factor-alpha factor suppresses medicine.
5. according to claim 1 or 2 or 3 described application, it is characterized in that: said medicine is a downward modulation Fas protein expression level medicine.
6. according to claim 1 or 2 or 3 described application, it is characterized in that: said medicine is the neuromotor dysfunction medicine that alleviates after middle cerebral artery is blocked.
7. according to claim 1 or 2 or 3 described application, it is characterized in that: said medicine is to dwindle the cerebral infarct size medicine.
8. according to claim 1 or 2 or 3 described application, it is characterized in that: said ischemic cerebrovascular is meant focal cerebral infarction, or cerebral infarction, or cerebral infarction.
9. application according to claim 8; It is characterized in that: said medicine is that the tumor necrosis factor-alpha factor suppresses medicine; Or downward modulation Fas protein expression level medicine; Or alleviate the neuromotor dysfunction medicine after the middle cerebral artery blocking-up, or dwindle the cerebral infarct size medicine.
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| CN2012100098829A Pending CN102512508A (en) | 2012-01-12 | 2012-01-12 | Application of rheum emodi wall extract to preparation of medicines for resisting ischemic cerebral vascular diseases |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108472332A (en) * | 2015-11-13 | 2018-08-31 | 汉阳大学校产学协力团 | Composition for treating apoplexy by nasal administration |
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| CN1347698A (en) * | 2001-10-30 | 2002-05-08 | 首都医科大学宣武医院 | Use of stilbene glycoside in preparing medicine for ischemic cerebrovascular diseases |
| CN102058679A (en) * | 2010-12-10 | 2011-05-18 | 成都华西天然药物有限公司 | Medicine or health-care food composition for treating atherosclerosis |
| CN102228519A (en) * | 2011-06-24 | 2011-11-02 | 西藏金哈达药业有限公司 | Application of extract of Rheum emodi Wall. in preparing medicine for controlling ischemic heart disease (IHD) |
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| CN1347698A (en) * | 2001-10-30 | 2002-05-08 | 首都医科大学宣武医院 | Use of stilbene glycoside in preparing medicine for ischemic cerebrovascular diseases |
| CN102058679A (en) * | 2010-12-10 | 2011-05-18 | 成都华西天然药物有限公司 | Medicine or health-care food composition for treating atherosclerosis |
| CN102228519A (en) * | 2011-06-24 | 2011-11-02 | 西藏金哈达药业有限公司 | Application of extract of Rheum emodi Wall. in preparing medicine for controlling ischemic heart disease (IHD) |
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| CN108472332A (en) * | 2015-11-13 | 2018-08-31 | 汉阳大学校产学协力团 | Composition for treating apoplexy by nasal administration |
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Application publication date: 20120627 |