CN104127485A - Application of Rabdosia rubescens extract in preparation of cerebral ischemia tolerance improving medicines - Google Patents
Application of Rabdosia rubescens extract in preparation of cerebral ischemia tolerance improving medicines Download PDFInfo
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Abstract
The invention relates to an application of a Rabdosia rubescens extract in the preparation of cerebral ischemia tolerance improving medicines, and effectively solves the problems of the preparation of the Rabdosia rubescens extract and the application of the extract in the preparation of the cerebral ischemia tolerance improving medicines. The Rabdosia rubescens extract is prepared through the following steps: crushing Rabdosia rubescens to form crude powder, carrying out reflux extraction by using petroleum ether two times, volatilizing petroleum ether from the obtained extract liquid until the crude Rabdosia rubescens powder is dry, adding ethanol, immersing for 0.5h, carrying out reflux extraction two times, carrying out reduced pressure concentration on the obtained ethanol extract liquid until no ethanol smell, dispersing with distilled water until the crude Rabdosia rubescens extract concentration is 0.4g/ml, adding the obtained dispersion to an AB-8 resin as a loading medicine solution in a flow manner, allowing the dispersion and the resin to stand for 12h for full adsorption, sequentially eluting with distilled water and ethanol, and volatizing until dryness in order to obtain powder which is the Rabdosia rubescens extract. Repeated tests of the Rabdosia rubescens extract prove that the Rabdosia rubescens extract can improve the cerebral ischemia tolerance, can be effectively used to prepare cerebral ischemia resistant medicines, and has substantial economic and social benefits.
Description
Technical field
The present invention relates to medicine, particularly a kind of Rabdosia rubescens extract improves the application in cerebral ischemic tolerance medicine in preparation.
Background technology
Cerebral ischemic tolerance is actually environmental stimuli and activates the endogenous protection mechanism in body; thereby give body to the resistivity of major injury next time; although being proved, physical property preprocessing means can induce cerebral ischemic tolerance; but be a kind of damage to body; although some chemicalses can improve tolerance, its mechanism that tolerance is formed also imperfectly understands; side effect after the taking of chemicals long-term taking, has certain restriction to the treatment of Imaging in Patients with Cerebral Ischemia Disease.Chinese medicine action temperature and lasting; there is the effect of Comprehensive Treatment; can delay the generation of complication; the feature of its multicomponent, many target spots is having great advantage aspect control cerebrovascular disease; by the mode of " preventive treatment of disease "; when induction Ischemic Tolerance, the multiple endogenous protection mechanism of body, alleviates ischemic brain injury, becomes preprocessing means safely and effectively.It is the crack rice dry aerial parts of fork Rabdosia rubescenss (Hemsl.) Hara of labiate that 2010 editions pharmacopeia are recorded Rabdosia rubescens, bitter in the mouth, glycosides, be slightly cold, return lung, stomach, Liver Channel, have heat-clearing and toxic substances removing, the merit of promoting blood circulation and stopping pain, for laryngopharynx swelling and pain mass in the abdomen mass in the abdomen, worm venom.Main chemical compositions has the terpenoids such as monoterpene, diterpene, triterpene, also contains volatile oil, steroidal, flavone, alkaloid, aminoacid, organic acid, monosaccharide composition etc.Modern study shows that Rabdosia rubescens has good antitumor, and antimicrobial antiphlogistic strengthens immunity, antioxidation, blood pressure lowering, the effects such as mutation.Clinical experiment shows, it has good therapeutical effect as esophagocardial carcinoma, colon cancer, hepatocarcinoma etc. to certain cancers, in addition, Rabdosia rubescens also has good curative effect as treatment suppurative tonsillitis to some inflammation, acute and chronic pharyngitis and chronic tracheitis etc., rubescensine A in Rabdosia rubescens composition is that a kind of hypotoxicity, anti-tumor activity are good, is called as " paclitaxel second ".But at present, also do not find its any report to cerebral ischemic tolerance.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the present invention's object is just to provide a kind of Rabdosia rubescens extract and improves the application in cerebral ischemic tolerance medicine in preparation, can effectively solve the preparation of Rabdosia rubescens extract, and this extract improves the application problem in cerebral ischemic tolerance medicine in preparation.
The technical scheme that the present invention solves is, Rabdosia rubescens extract improves the application in cerebral ischemic tolerance medicine in preparation, this Rabdosia rubescens extract is that Rabdosia rubescens is pulverized as coarse powder, add the petroleum ether reflux extraction 2 times of 10 times of weight at every turn, each extraction 1h, merge extract twice, fling to petroleum ether dry to Rabdosia rubescens coarse powder, add the mass concentration 50% soak with ethanol 0.5h of 10 times of weight at every turn, reflux, extract, 2 times, each 1h, merge alcohol extract twice, be evaporated to without alcohol taste, extraction ratio is 18-22%, be separated into and be equivalent to containing crude drug concentration 0.4g/ml with distilled water, as loading drug solns, cross AB-8 resin, the weight ratio of crude drug in whole and AB-8 resin is 1: 8, loading flows, standing 12h is after fully adsorbing, first with 2 times of column volume distillation washings, discard water liquid, the ethanol remove impurity that is 10% by the mass concentration of 3 times of column volumes again, the ethanol elution that is finally 80% by the mass concentration of 5 times of column volumes, collect eluent, volatilize to obtain powder, obtain Rabdosia rubescens extract of the present invention, extraction ratio 6-8%, wherein Rabdosia rubescens general flavone content is 55-60% (note: solvent quality used is different because of Rabdosia rubescens quality and in extracting, therefore, can affect the content of total flavones in extract, but through repeated tests, general flavone content is substantially at 55-60%, average out to 58%).
Extract of the present invention is through repetition test, prove that Rabdosia rubescens extract has the cerebral ischemic tolerance of raising ability, be effective to prepare anti-cerebral ischemia drugs, thereby realize Rabdosia rubescens extract and improve the application in cerebral ischemic tolerance medicine in preparation, new purposes and the medical value of Rabdosia rubescens are opened up, having significant economic and social benefit, is the innovation in Rabdosia rubescens medicinal application.
Brief description of the drawings
Fig. 1 is the affect figure of the present invention on cerebral ischemic tolerance rat model mortality rate.
Fig. 2 is the affect figure of the present invention on the scoring of cerebral ischemic tolerance rat model neurological deficit.
Fig. 3 is the affect figure of the present invention on NSE level in cerebral ischemic tolerance rat model serum.
Fig. 4 is the affect figure of the present invention on cerebral ischemic tolerance rat model cerebral infarction rate.
Fig. 5 is the affect figure of the present invention on TNF-α, IL-1 β, IL-8 content in cerebral ischemic tolerance rat model brain homogenate.
Fig. 6 is the affect figure of the present invention on BAX content in cerebral ischemic tolerance rat model brain homogenate.
Fig. 7 is the affect figure of the present invention on Bcl-2 content and Bcl-2/Bax ratio in cerebral ischemic tolerance rat model brain homogenate.
Fig. 8 is the affect figure of the present invention on Caspase-3 content in cerebral ischemic tolerance rat model brain homogenate.
Fig. 9 is the affect figure of the present invention on the Hippocampus CA1He CA3 district GDNF of rat cerebral tissue positive cell expression rate.
Figure 10 is the affect figure of the present invention on the Hippocampus CA1He CA3 district BDNF of rat cerebral tissue positive cell expression rate.
Figure 11 is the affect figure of the present invention on Hippocampus CA1He CA3 district of rat cerebral tissue NF κ Bp65 positive cell expression rate.
Detailed description of the invention
Below in conjunction with embodiment, the specific embodiment of the present invention is elaborated.
The present invention, in concrete enforcement, can be provided by following examples.
Embodiment 1
Rabdosia rubescens extract improves the application in cerebral ischemic tolerance medicine in preparation, this Rabdosia rubescens extract is, Rabdosia rubescens 1kg is pulverized as coarse powder, add 10kg petroleum ether reflux extraction 2 times at every turn, each 1h, merge extract twice, fling to petroleum ether dry to Rabdosia rubescens coarse powder, add 10kg mass concentration 50% soak with ethanol 0.5h at every turn, reflux, extract, 2 times, each 1h, merges twice alcohol extract, is evaporated to without alcohol taste, extraction ratio is 19%, be separated into and be equivalent to containing crude drug concentration 0.4g/ml with distilled water, as loading drug solns, cross AB-8 resin; The weight ratio of crude drug in whole and AB-8 resin is 1: 8, mobile loading, standing 12h is after fully adsorbing, first with 2 times of column volumes distillation washings, discard water liquid, the ethanol remove impurity that is 10% by the mass concentration of 3 times of column volumes again, the ethanol elution that is finally 80% by the mass concentration of 5 times of column volumes, collects eluent, volatilize to obtain powder, obtain Rabdosia rubescens extract of the present invention, yield is 7%, and wherein Rabdosia rubescens general flavone content is 58%.
Embodiment 2
Rabdosia rubescens extract improves the application in cerebral ischemic tolerance medicine in preparation, this Rabdosia rubescens extract is, Rabdosia rubescens 1.5kg is pulverized as coarse powder, add 15kg petroleum ether reflux extraction 2 times at every turn, each 1h, merge extract twice, fling to petroleum ether dry to Rabdosia rubescens coarse powder, add 15kg mass concentration 50% soak with ethanol 0.5h at every turn, reflux, extract, 2 times, each 1h, merges twice alcohol extract, is evaporated to without alcohol taste, extraction ratio is 22%, be separated into and be equivalent to containing crude drug concentration 0.4g/ml with distilled water, as loading drug solns, cross AB-8 resin; The weight ratio of crude drug in whole and AB-8 resin is 1: 8, mobile loading, standing 12h is after fully adsorbing, first with 2 times of column volumes distillation washings, discard water liquid, the ethanol remove impurity that is 10% by the mass concentration of 3 times of column volumes again, the ethanol elution that is finally 80% by the mass concentration of 5 times of column volumes, collects eluent, volatilize to obtain powder, obtain Rabdosia rubescens extract of the present invention, yield is 8%, and wherein Rabdosia rubescens general flavone content is 60%.
Embodiment 3
Rabdosia rubescens extract improves the application in cerebral ischemic tolerance medicine in preparation, this Rabdosia rubescens extract is, Rabdosia rubescens 0.5kg is pulverized as coarse powder, add 5kg petroleum ether reflux extraction 2 times at every turn, each 1h, merge extract twice, fling to petroleum ether dry to Rabdosia rubescens coarse powder, add 5kg mass concentration 50% soak with ethanol 0.5h at every turn, reflux, extract, 2 times, each 1h, merges twice alcohol extract, is evaporated to without alcohol taste, extraction ratio is 18%, be separated into and be equivalent to containing crude drug concentration 0.4g/ml with distilled water, as loading drug solns, cross AB-8 resin; The weight ratio of crude drug in whole and AB-8 resin is 1: 8, mobile loading, standing 12h is after fully adsorbing, first with 2 times of column volumes distillation washings, discard water liquid, the ethanol remove impurity that is 10% by the mass concentration of 3 times of column volumes again, the ethanol elution that is finally 80% by the mass concentration of 5 times of column volumes, collects eluent, volatilize to obtain powder, obtain Rabdosia rubescens extract of the present invention, yield is 6%, and wherein Rabdosia rubescens general flavone content is 55%.
Above-mentioned Rabdosia rubescens extract, through scientific experimentation repeatedly, proves that Rabdosia rubescens extract (comprising Rabdosia rubescens total flavones) can improve large and small Mus neurological deficit scoring, reduces infarct size, alleviates ischemic injuries, improves the pathological changes of cerebral cortex and hippocampus; Alleviate because of the too high damage to cerebral tissue of acidity, reduce because of the damage of energy metabolism impairment to cerebral tissue; Remove free radical, suppress lipid peroxidation, thereby alleviated cerebral ischemia reperfusion injury degree; Inflammatory reaction after inhibition ischemia, and improve a series of cascade reaction media and the cytokine that inflammation causes, cerebral ischemia reperfusion injury is played a protective role.
Study and also show, ischemia pretreatment time and intensity are two principal elements that produce cerebral ischemic tolerance.Cerebral ischemia intensity is too weak, the time is too shortly not enough to produce tolerance, and intensity is too strong, overlong time can cause the irreversible ischemic injuries of cerebral tissue.Carry out the research of cerebral ischemic tolerance mechanism, the most important thing is to set up a good animal model, find out best ischemia pretreatment time dosage, induction time window, could further carry out the research of its mechanism.Cerebral tissue neurocyte is one of easily vulnerable position of ischemic animal; the time windows producing by observation different ischemic pretreatment induction cerebral ischemic tolerance is on the morphologic impact of mouse tissue; find that the little mousetrap closes bilateral common carotid arteries blocking blood flow 10min and does ischemia pretreatment; taking 120h as induction time window; folder closes bilateral common carotid arteries 30min again; cerebral tissue edema degree alleviates, and pathological change is light, presents protective effect.By to rat cerebral ischemia Tolerance Model (2VO+MCAO) pretreatment time dosage, induction time window experimentation; find that rat adopts of short duration blocking-up bilateral common carotid arteries 10min; taking 72h as induction time window; adopt again transience middle cerebral artery occlusion (MCAO) model; as the blocking-up of ischemic injuries model again medium-sized artery blood flow 2h, then pour into after 22h, cerebral tissue edema degree alleviates; infarcted region scope is dwindled, and protective effect is strong.
The application by copying above-mentioned mouse brain Ischemic Tolerance model, rat cerebral ischemia Tolerance Model is object of study; observe the effect of Rabdosia rubescens extract to cerebral ischemic tolerance animal pattern, the variation of its endogenous protection effect of further investigated and relevant cell factor and neurotrophic factor.Intervening and treat ischemia apoplexy for clinical practice Rabdosia rubescens provides experimental basis, and related tests data is as follows:
1 experiment material
1.1 Experimental agents
Rabdosia rubescens extract of the present invention (comprising Rabdosia rubescens total flavones).
Nimodipine tablet, main component: nimodipine.The cerebral vasospasm and the convalescent blood circulation of acute cerebrovascular disease that are applicable to after a variety of causes subarachnoid hemorrhage improve.Lot number: 130660, producer: Shanxi Yabao Pharmaceutical Group Corp..
NAOLUOTONG JIAONANG, main component: Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, the Radix Astragali, hesperidin methyl, tolperisone hydrochloride, vitamin B6.Function cures mainly: dredge the meridian passage, and the lively atmosphere of enriching blood, has blood vessel dilating, increases cerebral blood flow effect, for cerebral arteriosclerosis, cerebral thrombosis, the various cerebrovascular disease such as apoplexy sequela, the headache that syndrome of blood stasis due to qi deficiency causes, dizzy, hemiplegia, the disease such as limbs are numb, spiritlessness and weakness.Lot number: 130901, producer: Jilin Jinbao Pharmaceutical Co., Ltd..
1.2 experiment reagent
Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd, lot number: C1206807;
Sodium chloride injection, Henan Shuan Hehuali pharmaceutcal corporation, Ltd, lot number: 130629;
Chloral hydrate, Tianjin recovery fine chemistry industry institute, lot number: 20120827;
Formaldehyde (analytical pure), Yantai City is Chemical Co., Ltd. in pairs, lot number: 20130702;
Sodium carboxymethyl cellulose, Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd, lot number: 20120418;
75% medical alcohol, big disinfectant preparation company limited of Xinxiang City three, lot number: 20131020;
Red tetrazolium: upper seamount Pu Chemical Co., Ltd., lot number: 20120508;
Sodium hydrogen phosphate, Tianjin Zhi Yuan chemical reagent company limited, lot number: 20130402;
Sodium dihydrogen phosphate, Tianjin chemical reagent three factories, lot number: 20051028;
Rat NSE detection kit, producer: R & D, lot number: 20131202B;
Rat TNF-α ELISA detection kit, producer: R & D, lot number: 20131202B;
Rat IL-8ELISA detection kit, producer: R & D, lot number: 20131202B;
Rat IL-1 β ELISA detection kit, producer: R & D, lot number: 20131202B;
Rat BAX ELISA detection kit, producer: R & D, lot number: 20131202B;
Rat Bcl-2ELISA detection kit, producer: R & D, lot number: 20131202B;
Rat Casp-3ELISA detection kit, producer: R & D, lot number: 20131202B;
1.3 experimental apparatus
MCAO line bolt, Shadong Biological Technology Co., Ltd., Beijing, production number: 2838-A4;
SHHW21600S type electric heating constant temperature three use water-baths, Shanghai leap Medical Devices Co., Ltd. produces;
FA2204B electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd produces;
KDC-160HR High speed refrigerated centrifuge, Zhong Jia branch company of Keda Innovation Co., Ltd;
BIORAD-680 microplate reader, 680Microplate Reader, Bio-Rad Laboratories;
Adjustable pipette, Sai Mo flies generation that (Shanghai) Instrument Ltd.;
Specialty image analysis system, NIS-Elements AR4.10.01.
1.4 laboratory animal
Wistar rat, male, SPF level, body weight 260-280g, Lukang Medical Co., Ltd., Shandong, the quality certification number: 0017228.Laboratory quality certification SYXK (Henan) 2010-001.
2 experimental techniques
2.1 grouping and administrations: get 128 of the male rats of about body weight 280～300g, be evenly divided into immediately 8 groups: sham operated rats, ischemical reperfusion injury group, BIT model group, nimodipine group, NAOLUOTONG JIAONANG group, large, medium and small dosage Rabdosia rubescens extract group, 16 every group.Nimodipine group (positive control drug is nimodipine tablet, and dosage is 20mg/kg, is made into drug level 2mg/ml before use with 0.5%CMC, 1ml/100g, 10 times of quantity); NAOLUOTONG JIAONANG group (positive control drug is NAOLUOTONG JIAONANG, and dosage is 500mg/kg, is made into drug level 50mg/ml before use with 0.5%CMC, 1ml/100g, 10 times of quantity); Large, medium and small dosage Rabdosia rubescens extract group (200mg/kg, 100mg/kg, 50mg/kg are made into drug level 20mg/ml, 10mg/ml, 5mg/ml, 1ml/100g with 0.5%CMC before use); Sham operated rats, ischemical reperfusion injury group, BIT model group (gavaging same volume 0.5%CMC).
2.2 modeling methods:
2.2.1 the pretreated model of ischemic injuries method-cerebral ischemia for the first time
All rats are through 10% chloral hydrate 0.3ml/100g intraperitoneal anesthesia, lie on the back on fixing operation platform, the sterilization of ethanol cervical region, then makes cervical region median incision with scalpel, blunt separation bilateral common carotid arteries, except sham operated rats, ischemical reperfusion injury group, all the other each group rats close bilateral common carotid arteries with noinvasive arteriole folder folder, and blocking blood flow 10min, then recovers perfusion respectively, sham operated rats, ischemical reperfusion injury group only expose bilateral common carotid arteries, but blocking blood flow not.After animal revives, give respectively relative medicine, every day 1 time, successive administration 3 days, after ischemia pretreatment 72h, i.e. after the 4th administration, 1h carries out MCAO art.
2.2.2 ischemic injuries method-middle cerebral artery occlusion Ischemia-Reperfusion Injury Model again
Rat chloral hydrate anesthesia, lie on the back fixing, separate left carotid (CCA), internal carotid artery (ICA) and external carotid artery (ECA), except rats in sham-operated group, all the other each group rat ligation ECA and CCA, close after internal carotid artery distal end with bulldog clamp folder, make a kerf in external carotid artery and internal carotid artery crotch, insert line bolt from incision, insertion depth 20mm left and right, ligation porch, line bolt stays 1～2mm, skin suture outward.After 2h, lift gently extremely slightly resistance of stayed the end of a thread, realize and pouring into again.Rats in sham-operated group only exposes tremulous pulse, but blocking blood flow not.In operation process, keep 23～25 DEG C of room temperatures.
2.3 nervous symptoms scorings
According to Zea Longa5 level point system, evaluate 0 point and the person of remaining unconscious or died rejecting in postoperative 24h (pouring into again 22h).Standards of grading are without obvious nervous symptoms, count 0 point; Can not full extension left side fore paw, count 1 point; Rotation, counts 2 points to the left; When walking, topple over to the left, count 3 points; Can not walk voluntarily, count 4 points; Death, counts 5 points.
2.4 serum preparations
Pour into after 22h, eyeball is got blood again, separation of serum, and-20 DEG C of Refrigerator stores, for the mensuration of NSE.
2.5 cerebral tissue are drawn materials and are processed
Get after blood the complete brain of getting of broken end rapidly, put into immediately-20 DEG C of cryogenic refrigerators, after cooling 15min, the remainder removing after olfactory bulb, cerebellum and low brain stem is cut into 3 parts along coronalplane, (the first cutter 1mm place before optic chiasma coronalplane is that Part I is 1mm place before best optic chiasma coronalplane before brain, the second cutter 1mm place after optic chiasma coronalplane is that Part II is 1mm place before and after optic chiasma coronalplane, and Part II is about the thin slice of 2mm; Part III be after optic chiasma coronalplane 1mm place to the cerebral tissue of tail end).
2.6 brain infarction areas are measured
Get the 2nd brain section and put into rapidly the 1%TTC phosphate solution with the configuration of pH=7.2 phosphate buffer, put into subsequently calorstat, 37 DEG C of lucifuges are hatched 15min, stir gently therebetween, in order to abundant dyeing.Take out brain sheet as for the 24h that keeps in Dark Place in 10% formalin, under blue background, take pictures, normal cerebral tissue is rose, and infarction position is white.Use scanner scanning image, input picture analytical system, calculates the area of infarcted region, obtains infarcted region area and account for the percentage ratio of the gross area.
The preparation of 2.7 brain tissue homogenates
Part I cerebral tissue, sagittal cuts left side cerebral tissue (plug wire bolt side cerebral tissue), weigh, prepare 10% brain homogenate with normal saline, brain homogenate, through the centrifugal 10min of 3000r/min, is got supernatant, subpackage, to-20 DEG C of Refrigerator stores, for the assay of brain homogenate TNF-α, IL-8, IL-1 β, Bcl-2, BAX, Casp-3.
2.8HE dyeing and immunohistochemical staining
Part III cerebral tissue, routine paraffin wax embedding, carries out conventional H E dyeing by step, adopts SP immunohistochemistry staining method to carry out BDNF, GDNF and NF κ Bp
65dyeing.
2.8.1 image processing
With reference to Kato stage division, under optical microscope, Histological change of ischemia side cortical areas is carried out to classification, standard is as follows: I level, cortical neuron cellular edema, distortion, impassivity unit is dead; II level, the death of minority neuronal cell, infarct size is less than 1/3 of the ischemia side cortex gross area; III level, neuronal death in flakes, infarct size is greater than 1/3 of the left cortical gross area and is less than 2/3 of the gross area; IV level, large stretch of neuronal death, infarct size is greater than 2/3 of the left cortical gross area.
2.8.2 SABC graphical analysis
In the section of Olympus optical microphotograph Microscopic observation immunohistochemical staining, Olympus digital micro-analysis photographing unit gathers image, respectively chooses two visuals field at each treated animal ischemia side (left side) cortex, hippocampus, meter BDNF, GDNF and NF κ Bp
65positive expression cell number, calculates positive cell expression rate.
NSE in 2.9 serum, the mensuration of TNF-α, IL-8, IL-1 β, BAX, Casp-3, Bcl-2 content in brain homogenate
Detect principle: test kit adopts double antibody one step sandwich assay elisa (ELISA).In the coated micropore of coated refreshing NSE in advance (or TNF-α, BAX, Casp-3, Bcl-2) antibody, add successively the detection antibody of specimen, standard substance, HRP labelling, through hatching and thoroughly washing.With substrate TMP colour developing, TMP changes into blueness under the catalysis of peroxidase, and changes into final yellow under sour effect.NSE in the depth and the sample of color (or TNF-α, IL-8, IL-1 β, BAX, Casp-3, Bcl-2) is proportionate.Under 450nm wavelength, measure (OD) value by microplate reader, calculation sample concentration.
Operating procedure: take out required lath from the aluminium foil bag equilibrium at room temperature 20min, residue lath is put back to 4 DEG C of preservations with valve bag sealing.Standard substance hole and sample aperture are set, and standard substance hole adds the standard substance 50 μ l of various variable concentrations.Sample aperture first adds sample to be tested 10 μ l, then adds sample diluent 40 μ l; Blank well does not add.Except blank well, in standard substance hole and sample aperture, every hole adds the detection antibody 100 μ l of horseradish peroxidase (HPR) labelling, seals reacting hole with shrouding film, 37 DEG C of water-baths or calorstat incubation 60min.Discard liquid, in absorbent paper, pat dry, cleaning mixture is filled it up with in every hole, leaves standstill 1min, gets rid of cleaning mixture, in absorbent paper, pats dry, and so repeats to wash plate 5 times (also can with washing plate machine washing plate).Every hole adds substrate A, the each 50 μ l of B, and 37 DEG C of lucifuges are hatched 15min.Every hole adds stop buffer 50 μ l, in 15min, measures the OD value in each hole at 450nm wavelength place.
Drawing standard curve: in Excel worksheet, make abscissa with standard substance concentration, corresponding OD value is made vertical coordinate, draws out standard substance linear regression, calculates each sample concentration value by curvilinear equation.
3 statistical procedures methods
Use SPSS17.0for windows to carry out the statistical analysis of data information, measurement data adopts average ± standard deviation
represent, between group, relatively adopt one factor analysis of variance (One-way ANOVA) analysis, the neat person of variance test uses least significant difference (LSD) method, heterogeneity of variance person checks by Games-Howell method, ranked data adopt Ridit inspection, have statistical significance taking P<0.05 as difference.
4 experimental results
4.1 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) on the impact of cerebral ischemic tolerance rat model neurological deficit scoring, mortality rate the results are shown in Table 1, Fig. 1, Fig. 2
The impact of table 1 on the scoring of cerebral ischemic tolerance rat model neurological deficit, mortality rate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
After rat MCAO, occur certain mortality rate and neurologic impairment symptom in various degree, showing as right side fore paw can not full extension, rotates to the right or topple over when walking, even can not walk.Compared with ischemical reperfusion injury group, BIT model group and each administration group mortality rate obviously reduce and can significantly improve rat model neural disappearance symptom (P<0.01), compared with BIT model group, nimodipine group, dosage Rabdosia rubescens extract big or middle all can significantly improve rat neural disappearance symptom (P<0.01), and NAOLUOTONG JIAONANG group can obviously be improved rat neural disappearance symptom (P<0.05).
4.2 Rabdosia rubescens extracts on the impact of NSE level, brain area infarction rate in cerebral ischemic tolerance rat model serum the results are shown in Table 2, Fig. 3, Fig. 4
The impact of table 2 on NSE level, brain area infarction rate in cerebral ischemic tolerance rat model serum
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
From Fig. 3, Fig. 4 and upper table, with sham operated rats comparison, ischemical reperfusion injury group serum level of NSE significantly increases (P<0.01); With the comparison of ischemical reperfusion injury group, BIT model group serum level of NSE significantly reduces (P<0.01); With the comparison of BIT model group, nimodipine group, NAOLUOTONG JIAONANG group, big or middle dosage Rabdosia rubescens extract group serum level of NSE all significantly reduces (P<0.01).With the comparison of ischemical reperfusion injury group, BIT model group and low dose of Rabdosia rubescens extract group cerebral infarction percentage ratio obviously reduce (P<0.05), nimodipine group, NAOLUOTONG JIAONANG group, big or middle dosage Rabdosia rubescens extract group brain infarction area percentage ratio significantly reduces (P<0.01); With the comparison of BIT model group, heavy dose of Rabdosia rubescens extract group brain infarction area percentage ratio obviously reduces (P<0.05), and NAOLUOTONG JIAONANG group brain infarction area percentage ratio significantly reduces (P<0.01).
4.3 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) on the impact of TNF-α, IL-1 β, IL-8 content in cerebral ischemic tolerance rat model brain homogenate the results are shown in Table 3, Fig. 5
The impact of table 3 on TNF-α, IL-1 β, IL-8 content in cerebral ischemic tolerance rat model brain homogenate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
From upper table, Fig. 5, with sham operated rats comparison, TNF-α, IL-1 β content significantly raise (P<0.01) in ischemical reperfusion injury group brain homogenate, with the comparison of ischemical reperfusion injury group, all significantly risings (P<0.01) of TNF-α, IL-1 β content in BIT model group and each administration group brain homogenate, with the comparison of BIT model group, TNF-alpha content obviously raise (P<0.05) in nimodipine group brain homogenate, IL-1 β content significantly raise (P<0.01), IL-1 β content obviously raise (P<0.05) in NAOLUOTONG JIAONANG group brain homogenate, TNF-alpha content obviously raise (P<0.05) in heavy dose of Rabdosia rubescens extract group brain homogenate, IL-1 β content significantly raise (P<0.01), in, TNF-α in low dose of Rabdosia rubescens extract group brain homogenate, IL-1 β content is without significant change (P>0.05).With sham operated rats comparison, IL-8 content significantly raise (P<0.01) in ischemical reperfusion injury group brain homogenate; With the comparison of ischemical reperfusion injury group, in BIT model group and each administration group brain homogenate, IL-8 content all significantly reduces (P<0.01); With the comparison of BIT model group, nimodipine group can significantly reduce IL-8 content (P<0.01) in brain homogenate, in big-and-middle dosage Rabdosia rubescens extract group brain homogenate, IL-8 content obviously reduces (P<0.05), and in low dose of Rabdosia rubescens extract group brain homogenate, IL-8 content is without significant change (P>0.05).
4.4 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) on the impact of Bcl-2, BAX content in cerebral ischemic tolerance rat model brain homogenate the results are shown in Table 4, Fig. 6, Fig. 7
The impact of table 4 on BAX, Bcl-2 content in cerebral ischemic tolerance rat model brain homogenate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
By upper table and Fig. 6, Fig. 7 is known, with the comparison of ischemical reperfusion injury group, Bcl-2 content obviously raise (P<0.05) in BIT model group brain homogenate, Bax content significantly reduces (P<0.01), nimodipine group, NAOLUOTONG JIAONANG group and large, Bcl-2 content significantly raise (P<0.01) in middle low dose of Rabdosia rubescens extract group brain homogenate, Bax content significantly reduces (P<0.01), the ratio of BIT model group and each administration group Bcl-2/BAX significantly raise (P<0.01), with the comparison of BIT model group, in nimodipine group, NAOLUOTONG JIAONANG group and the homogenate of big or middle dosage Rabdosia rubescens extract group, Bcl-2 content significantly raises (P<0.01), BAX content significantly reduces (P<0.01), Bcl-2/BAX ratio significantly reduces (P<0.01), in low dose of Rabdosia rubescens extract group brain homogenate, BAX content obviously reduces (P<0.05), Bcl-2 content and Bcl-2/BAX rate of change not obvious (P>0.05).
4.4 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) on the impact of Caspase-3 content in cerebral ischemic tolerance rat model brain homogenate the results are shown in Table 5, Fig. 8
The impact of table 5 on Caspase-3 content in cerebral ischemic tolerance rat model brain homogenate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
From upper table, Fig. 8, with sham operated rats comparison, Caspase-3 content significantly raise (P<0.01) in ischemia-reperfusion group brain homogenate; With the comparison of ischemical reperfusion injury group, in BIT model group and each administration group brain homogenate, Caspase-3 content significantly reduces (P<0.01); With the comparison of BIT model group, nimodipine group, heavy dose of Rabdosia rubescens extract group Caspase-3 content all can significantly reduce (P<0.01), in NAOLUOTONG JIAONANG group, middle dosage Rabdosia rubescens extract group brain homogenate, Caspase-3 content obviously reduces (P<0.05), and in low dose of Rabdosia rubescens extract group brain homogenate, Caspase-3 content is without significant change (P>0.05).
The impact of 4.5 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) on cerebral ischemic tolerance rat model histopathology morphological change
4.5.1 cerebral tissue cosmetic variation
Rats in sham-operated group both sides cerebral hemisphere symmetry, ditch, return clear, have no edema.There is obvious ischemic necrosis kitchen range the visible ischemia side of ischemical reperfusion injury group rat brain coronal section (left side) middle cerebral artery blood supply center, and simultaneously visible ischemia tricorn edema expands.
4.5.2 brain section HE dyeing
4.5.2.1 the impact of Rabdosia rubescens extract (comprising Rabdosia rubescens total flavones) on rat cerebral ischemia Tolerance Model cerebral tissue cortical areas pathological change
Rabdosia rubescens extract is to cerebral tissue cortical areas pathological observation following (annex 1 is shown in by photo): sham operated rats: under light microscopic, rat cerebral cortex district organizational structure is complete, and neuron is intensive, queueing discipline, and endochylema is abundant, and karyon is obvious; Neurogliocyte is many, and karyon is clear, minimum with neuron, blood capillary peripheral clearance, without obvious edema.Ischemical reperfusion injury group: the softening kitchen range of the visible significantly oval sieve shape in cerebral cortex district, in kitchen range, neuronal cell disappears or sees that a small amount of pyknotic nucleus is residual; Transition region has obvious edema, and not exclusively dissolving appears in nerve fiber.BIT model group: the most neurons slabbing necrosis of cerebral cortex district, the softening kitchen range of visible a small amount of sieve shape, in kitchen range, neuronal cell disappears or sees that a small amount of pyknotic nucleus is residual; Transition region neuronal cell edema, not exclusively dissolving appears in nerve fiber.Nimodipine group and NAOLUOTONG JIAONANG group: cerebral cortex district infarction size dwindles, minority neuronal necrosis.Neuronal cell edema degree obviously alleviates.Rabdosia rubescens extract medication group: cerebral cortex district infarction size obviously dwindles, neuronal cell edema degree significantly alleviates, wherein, low dose of Rabdosia rubescens extract group occurs that only a few sample neuron is downright bad in the form of sheets.Concrete pathological grading sees table:
The impact of table 6 rat cerebral ischemia Tolerance Model cerebral tissue cortical areas pathological change
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
"-" cerebral cortex and neurocyte are normal; " I " cerebral cortex neurons edema, degeneration, impassivity unit is downright bad; II cerebral cortex edema, minority neuronal necrosis, infarct size account for cortex 1/3 in; " III " cerebral cortex edema, in flakes neuronal necrosis, infarct size account for cortex 1/3～2/3 in; " IV " cerebral cortex edema, large stretch of neuronal necrosis, infarct size accounts for the more than 2/3 of cortex.
Through Ridit inspection, compared with sham operated rats, ischemical reperfusion injury group has significant statistical significance (P<0.01), and modeling success is described.With the comparison of ischemical reperfusion injury group, cortical areas of nimodipine group rat cerebral tissue pathology damage is significantly improved (P<0.01), and large, medium and small dosage Rabdosia rubescens extract group and cortical areas of NAOLUOTONG JIAONANG group rat cerebral tissue pathology damage are obviously improved (P<0.05).With the comparison of BIT model group, nimodipine group, cortical areas of heavy dose of Rabdosia rubescens extract group rat cerebral tissue pathology damage are obviously improved (P<0.05).
4.5.2.1 the impact of Rabdosia rubescens extract (comprising Rabdosia rubescens total flavones) on rat cerebral ischemia Tolerance Model cerebral tissue hippocampus pathological change
Rabdosia rubescens extract is to following (annex 1 is shown in by the photo) sham operated rats of cerebral tissue hippocampus pathological observation: under light microscopic, rat hippocampus district organizational structure is complete, and neuron is intensive, queueing discipline; Endochylema is abundant, and kernel is obvious.Ischemical reperfusion injury group: hippocampus neuronal cell is sparse, irregular arrangement, the obvious edema of most cells, endochylema is light to be dyed, karyon shrinkage, major part is downright bad in the form of sheets.BIT model group: hippocampus neuronal cell is sparse, irregular arrangement, cellular edema endochylema is light to be dyed, and karyon shrinkage, is spotty necrosis.Nimodipine group: most of hippocampus neuronal cell queueing discipline, edema appears in a few cell, a few sample hippocampus clear in structure normal.NAOLUOTONG JIAONANG group: most of hippocampus neuronal cell queueing discipline, cellular edema, minority neuronal necrosis, indivedual sample hippocampus clear in structure normals.Rabdosia rubescens extract medication group: most of hippocampus neuronal cell queueing discipline, cellular edema, minority neuronal necrosis, indivedual sample hippocampus clear in structure normals.Concrete pathological grading sees table:
The impact of table 7 rat cerebral ischemia Tolerance Model cerebral tissue hippocampus pathological change
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
"-" hippocampus structure is normal, and structure of neurons is clear; " I " cerebral tissue hippocampus edema, neuron edema, endochylema is light to be dyed; " II " cerebral tissue hippocampus edema, neuron edema, endochylema is light to be dyed, minority neuronal necrosis; " III " cerebral tissue hippocampus edema, in flakes neuronal necrosis, infarct size account for Hippocampus 1/3 in; " IV " cerebral tissue hippocampus edema, neuronal necrosis in flakes, infarct size accounts for the more than 1/3 of Hippocampus.
Through Ridit inspection, compared with sham operated rats, ischemical reperfusion injury group has significant statistical significance (P<0.01), and modeling success is described.With the comparison of ischemical reperfusion injury group, nimodipine group rat cerebral tissue hippocampus pathology damage is significantly improved (P<0.01), and big or middle dosage Rabdosia rubescens extract group and NAOLUOTONG JIAONANG group rat cerebral tissue hippocampus pathology damage are obviously improved (P<0.05).With the comparison of BIT model group, nimodipine group rat cerebral tissue hippocampus pathology damage is obviously improved (P<0.05).
4.6 the impact that Rabdosia rubescens extract is expressed GDNF in cerebral ischemic tolerance rat model cerebral tissue the results are shown in Table 8, Fig. 9
The comparison of the Hippocampus CA1He CA3 district GDNF of table 8 Ge Zu rat cerebral tissue positive cell expression rate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
Immunohistochemical staining demonstration, sham operated rats and ischemia are respectively organized HeCA3 district, CA 1 of Hippocampus all the expression of GDNF, and positive cell product is sundown, is arranged in neuronal cell endochylema.From upper table, Fig. 9, with ischemical reperfusion injury comparison, nimodipine group, NAOLUOTONG JIAONANG group Hippocampus CA1, the CA3 district GDNF positive cell expression rate (P<0.01 that obviously raises, P<0.05), large, medium and small dosage Rabdosia rubescens extract group Hippocampus CA1, CA3 district GDNF positive cell expression rate significantly raise (P<0.01); With the comparison of BIT model group, heavy dose of Rabdosia rubescens extract group Hippocampal CA 1 GDNF positive cell expression rate obviously raises (P<0.05), big or middle dosage Rabdosia rubescens extract group Hippocampus CA 3 Region GDNF positive cell expression rate significantly raise (P<0.01).
The impact that 4.7 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) are expressed BDNF in cerebral ischemic tolerance rat model cerebral tissue the results are shown in Table 9, Figure 10
The comparison of the Hippocampus CA1He CA3 district BDNF of table 9 Ge Zu rat cerebral tissue positive cell expression rate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
Immunohistochemical staining demonstration, sham operated rats and ischemia are respectively organized HeCA3 district, CA 1 of Hippocampus all the expression of BDNF, and positive cell product is sundown, is arranged in neuronal cell endochylema.From upper table, Figure 10, with sham operated rats comparison, ischemical reperfusion injury group Hippocampus CA1, CA3 district BDNF positive cell expression rate obviously raise (P<0.01, P<0.05); With ischemical reperfusion injury comparison, nimodipine group, NAOLUOTONG JIAONANG group Hippocampus CA1, the CA3 district BDNF positive cell expression rate (P<0.01 that obviously raises, P<0.05), large, medium and small dosage Rabdosia rubescens extract group Hippocampus CA1, CA3 district BDNF positive cell expression rate obviously raise (P<0.01, P<0.05); With the comparison of BIT model group, brain ruton brain network Capsules group Hippocampal CA 1 BDNF positive cell expression rate significantly raise (P<0.01), dosage Rabdosia rubescens extract group big or middle Hippocampal CA 1 HeCA3 district BDNF positive cell expression rate significantly raises (P<0.01), low dose of Rabdosia rubescens extract group Hippocampus CA 3 Region BDNF positive cell expression rate obviously raise (P<0.05).
The impact that 4.8 Rabdosia rubescens extracts (comprising Rabdosia rubescens total flavones) are expressed NF κ Bp65 in cerebral ischemic tolerance rat model cerebral tissue the results are shown in Table 10, Figure 11
The comparison of Hippocampus CA1He CA3 district of Biao10Ge Zu rat cerebral tissue NF κ Bp65 positive cell expression rate
Note: with the comparison of ischemical reperfusion injury group,
△p<0.05,
△ △p<0.01; With the comparison of BIT model group, * P<0.05, * * P<0.01
Immunohistochemical staining demonstration, sham operated rats and ischemia are respectively organized HeCA3 district, CA 1 of Hippocampus all the expression of NF κ Bp65, and positive cell product is sundown, is mainly positioned at karyon, and separately having part is that endochylema is expressed.From Figure 11, upper table, with sham operated rats comparison, ischemical reperfusion injury group Hippocampus CA1, CA3 district NF κ Bp65 positive cell expression rate obviously raise (P<0.01, P<0.05); With ischemical reperfusion injury comparison, HeCA3 district, dosage Rabdosia rubescens extract group big or middle Hippocampal CA 1 NF κ Bp65 positive cell expression rate obviously reduces (P<0.01, P<0.05), nimodipine group Hippocampal CA 1 NF κ Bp65 positive cell expression rate obviously reduces (P<0.05).With the comparison of BIT model group, nimodipine group, middle dosage Rabdosia rubescens extract group Hippocampal CA 1 NF κ Bp65 positive cell expression rate obviously reduce (P<0.05), and heavy dose of Rabdosia rubescens extract group Hippocampal CA 1 NF κ Bp65 positive cell expression rate significantly reduces (P<0.01).
5 conclusions
By observing the neurological deficit of rat cerebral ischemia Tolerance Model, mortality rate, infarct size percentage ratio; Detect NSE level in serum, TNF-a, IL-1 β, IL-8, Bcl-2, Bax, Caspase-3 content in rat cerebral even slurry; Observe the pathological change of cerebral tissue cortical areas and hippocampus neuronal cell; Immunohistochemical Method is observed GDNF, BDNF, NF κ Bp65 positive cell expression rate.Known Rabdosia rubescens extract can improve the scoring of rat model function of nervous system, reduces mortality rate, lowers infarct size percentage ratio, improves the pathological changes situation of cerebral tissue; By reducing NSE level in serum, the ratio of endogenous anti-apoptosis factor Bcl-2 protein content and Bcl-2/Bax in raising brain homogenate, reduces Bcl-2 associated protein BAX content, reducing NF κ Bp65 expresses, reduce caspase-3 content, thus inhibited apoptosis, neuroprotective unit cell; Increase neurotrophic factor GDNF, BDNF and express, and then the effect of enhancing body endogenous protection; Reduce IL-8 content in brain homogenate, appropriate TNF-α, the IL-1 β of generation can stimulate body to produce endogenic protection mechanism, suppresses the excessive release of caused inflammatory cytokines.
Eventually the above; Rabdosia rubescens extract (comprising Rabdosia rubescens total flavones) can suppress the excessive release of inflammatory factor due to ischemic injuries; stimulate body to produce endogenous protection effect; inhibited apoptosis; neuroprotective unit cell; further improve the protection of ischemia pretreatment to brain injury; improve cerebral ischemic tolerance; be effective to preparation and improve cerebral ischemic tolerance medicine; improve the application in cerebral ischemic tolerance medicine thereby realize Rabdosia rubescens extract in preparation, opened up new purposes and the medical value of Rabdosia rubescens.
Claims (1)
1. a Rabdosia rubescens extract improves the application in cerebral ischemic tolerance medicine in preparation, this Rabdosia rubescens extract is that Rabdosia rubescens is pulverized as coarse powder, add the petroleum ether reflux extraction 2 times of 10 times of weight at every turn, each extraction 1h, merge extract twice, fling to petroleum ether dry to Rabdosia rubescens coarse powder, add the mass concentration 50% soak with ethanol 0.5h of 10 times of weight at every turn, reflux, extract, 2 times, each 1h, merge alcohol extract twice, be evaporated to without alcohol taste, extraction ratio is 18-22%, be separated into and be equivalent to containing crude drug concentration 0.4g/ml with distilled water, as loading drug solns, cross AB-8 resin, the weight ratio of crude drug in whole and AB-8 resin is 1: 8, mobile loading, standing 12h is after fully adsorbing, first with 2 times of column volumes distillation washings, discard water liquid, the ethanol remove impurity that is 10% by the mass concentration of 3 times of column volumes again, the ethanol elution that is finally 80% by the mass concentration of 5 times of column volumes, collects eluent, volatilize to obtain powder, obtain Rabdosia rubescens extract of the present invention, extraction ratio 6-8%, wherein Rabdosia rubescens general flavone content is 55-60%.
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| CN101978987A (en) * | 2010-11-18 | 2011-02-23 | 河南中医学院 | Application of herba rabdosiae rubescentis extract to preparation of medicament for treating and resisting cerebral ischemia |
| CN103432240A (en) * | 2013-09-14 | 2013-12-11 | 河南中医学院 | Method for extracting anti-cerebral ischemia substance from China rose and application of extracted anti-cerebral ischemia substance |
| CN103463244A (en) * | 2013-09-26 | 2013-12-25 | 河南中医学院 | Method for extracting blood sugar lowering substance from China roses and application of blood sugar lowering substance |
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| CN101978987A (en) * | 2010-11-18 | 2011-02-23 | 河南中医学院 | Application of herba rabdosiae rubescentis extract to preparation of medicament for treating and resisting cerebral ischemia |
| CN103432240A (en) * | 2013-09-14 | 2013-12-11 | 河南中医学院 | Method for extracting anti-cerebral ischemia substance from China rose and application of extracted anti-cerebral ischemia substance |
| CN103463244A (en) * | 2013-09-26 | 2013-12-25 | 河南中医学院 | Method for extracting blood sugar lowering substance from China roses and application of blood sugar lowering substance |
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