CN107260790A - A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly - Google Patents
A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly Download PDFInfo
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Abstract
The application in treating cerebral ischemia and focal brain ischemia medicament repeatedly is being prepared the present invention relates to Honegsukle flower P.E, effectively solve to extract Honegsukle flower P.E from honeysuckle, and the pharmaceutical problems of cerebral ischemia and focal cerebral ischemia are treated repeatedly in preparation as sole active agent, described Honegsukle flower P.E is, honeysuckle powder is broken into coarse powder, aether backflow extracts degreasing, the dregs of a decoction volatilize ether, plus the immersion of the ethanol of mass concentration 70%, refluxing extraction, filtering, decompression filtrate recycling ethanol is to without alcohol taste, plus distilled water is dispersed to the dispersion liquid equivalent to the 0.3g/mL containing crude drug, use second acid for adjusting pH value, HPD 600 or the type macroporous absorbent resins of AB 8 on dispersion liquid, use 30% ethanol elution, eluent is concentrated, freeze-drying, crush, obtain Honegsukle flower P.E, the present invention can effectively solve to extract Honegsukle flower P.E from honeysuckle, and effective for treating cerebral ischemia and focal cerebral ischemia repeatedly, the new application and medical value of honeysuckle are opened up, there is significant economic and social benefit.
Description
Technical field
The present invention relates to medicine, preparing treatment, cerebral ischemia and focal brain lack particularly a kind of Honegsukle flower P.E repeatedly
Application in blood medicine.
Background technology
Cerebral ischemia diseases are one group causes brain, cerebellum or brain stem part or multiple location blood supply insufficiency by many reasons,
So as to cause the disease of corresponding neurological symptom.Including cerebral ischemia repeatedly and focal cerebral ischemia, cause of disease complexity is formed, is given people
It is healthy bring very major injury, therefore be asking of being concerned about very much of people to the treatment of cerebral ischemia repeatedly and focal cerebral ischemia
Topic, though having multi-medicament at present, for various reasons, its curative effect is simultaneously unsatisfactory, therefore, and developing new treatment, brain lacks repeatedly
The medicine of blood and focal cerebral ischemia is the desired technical problem solved of people.
Honeysuckle, also known as honeysuckle (scientific name：Lonicera japonica), it is cold in nature, it is sweet, enter lung, the heart, stomach, honeysuckle
Clearing heat and detoxicating good medicine is described as from ancient times, has effects that clearing heat and detoxicating, anti-inflammatory, qi-restoratives treat wind, the sweet cold air fragrance of its property, Gan Han
Heat-clearing is without injuring one's stomach, and fragrance reaches thoroughly again can eliminating evil.Honeysuckle can dispelling wind-heat, be also apt to removing summer-heat blood poison, for various febrile diseases,
Such as body heat, dermexanthesis, hair spot, heat toxin sore carbuncle, abscess of throat disease, equal effect significantly, cure mainly disease under turgor, warm disease heating, heat toxin
The disease such as large carbuncle and tumour.It is for dizzy dizziness, thirst, hidrosis unhappiness, enteritis, bacillary dysentery, measles, pneumonia, encephalitis, stream
The diseases such as brain, acute mastitis, septicemia, appendicitis, skin infection, ulcer furunculosis, erysipelas, parotitis, suppurative tonsillitis
Disease has certain curative effect.But so far there are no Honegsukle flower P.E prepares treatment cerebral ischemia and focal brain ischemia medicament repeatedly
Open report.
The content of the invention
For above-mentioned situation, to overcome the defect of prior art, the purpose of the present invention is just to provide a kind of honeysuckle and extracted
Thing is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly, can effectively solve to extract gold and silver from honeysuckle
Flower extract, and preparing the pharmaceutical problems for the treatment of cerebral ischemia and focal cerebral ischemia repeatedly as sole active agent.
The technical scheme that the present invention is solved is that a kind of Honegsukle flower P.E is preparing treatment cerebral ischemia and focal brain repeatedly
Application in ischemia drugs, described Honegsukle flower P.E is that honeysuckle powder is broken into coarse powder, and aether backflow is extracted degreasing, abandoned
Remove ether；The honeysuckle dregs of a decoction volatilize ether, plus the immersion of the ethanol of mass concentration 70%, and refluxing extraction, filtering, filtrate decompression reclaims second
Alcohol is used on second acid for adjusting pH value, dispersion liquid to the dispersion liquid equivalent to the 0.3g/mL containing crude drug is dispersed to without alcohol taste, plus distilled water
HPD-600 or AB-8 type macroporous absorbent resins, with the ethanol elution of mass concentration 30%, collect eluent, concentrate, freeze-drying,
Crush, obtain Honegsukle flower P.E.
The present invention can effectively solve to extract Honegsukle flower P.E from honeysuckle, and effective for treatment repeatedly cerebral ischemia and
Focal cerebral ischemia, has opened up the new application and medical value of honeysuckle, is the big innovation on medicine, have it is significant economical and
Social benefit.
Embodiment
Embodiment below in conjunction with concrete condition for the present invention elaborates.
The present invention is in specific implementation, and a kind of Honegsukle flower P.E is preparing treatment cerebral ischemia and focal cerebral ischemia repeatedly
Application in medicine, described Honegsukle flower P.E is that honeysuckle powder was broken into the coarse powder of 20-30 mesh sieves, first adds 10 times of weights
The petroleum ether refluxing extraction 1h of amount, degreasing discards petroleum ether；The honeysuckle dregs of a decoction volatilize petroleum ether, plus 10 times of weight of honeysuckle,
The ethanol of mass concentration 70% soaks 0.5h, 80 DEG C of refluxing extraction 1h, and filtering obtains first time filtrate；The dregs of a decoction add 10 times of honeysuckle again
Weight, the ethanol of mass concentration 70%, 80 DEG C of refluxing extraction 1h, filtering obtain second of filtrate, merge filtrate twice, depressurize back
Receive ethanol and be dispersed to the dispersion liquid equivalent to the 0.3g/mL containing crude drug to without alcohol taste, plus distilled water, plus acetic acid adjusts pH to 2.4-4.2,
Quality of Flos Lonicerae/macroreticular resin mass ratio is 1 ︰ 6-8 in HPD-600 or AB-8 types macroporous absorbent resin, dispersion liquid on dispersion liquid,
With the ethanol elution of mass concentration 30%, eluted with 2.3BV/h flow velocity, eluent consumption is 8-12 times of macroreticular resin body
Product, collects eluent, concentrates, and freeze-drying obtains Honegsukle flower P.E.
The effect of Honegsukle flower P.E prepared by the present invention has the multiple cerebral ischemia of anti-reflective and focal cerebral ischemia through experiment,
The medicine of cerebral ischemia and focal cerebral ischemia is treated repeatedly effective for preparing, is to treat cerebral ischemia repeatedly and focal cerebral ischemia
Innovation on medicine, and extraordinary advantageous effects are achieved through experiment, relevant experimental data is as follows：
Test influence experiment of the Honegsukle flower P.E to mouse repeated cerebral ischemia-reperfusion model
1 material
1.1 animal
KM kind mouse, SPF grades, male, 96,25~30g of body weight is provided, the quality certification by experimental animal center of henan province
Number：41003100000258；Laboratory quality certification number：SYXK (Henan) 2010-001.
1.2 medicines and reagent
Honegsukle flower P.E of the present invention；Nimodipine tablet, Shanxi Yabao Pharmaceutical Group Corp.；Sodium chloride injection,
Henan Shuan Hehuali pharmaceutcal corporation, Ltds；Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd；Formalin (analysis
It is pure), Chemical Co., Ltd. in pairs of Yantai City；Chloraldurate, Tianjin recovery fine chemistry industry research institute；Sodium carboxymethylcellulose
(CMC), Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd；Medical ethanol (alcohol), the limited public affairs of big disinfectant preparation of Xinxiang City three
Department；ICAM-1 (ICAM-1) ELISA detection kit, R＆D companies；Tumor necrosis factor α (TNF-α) ELISA is examined
Test agent box, R＆D companies；Neuronspecific enolase (NSE) ELISA detection kit, R＆D companies；Myeloperoxidase
Enzyme (MPO) testing cassete, Bioengineering Research Institute is built up in Nanjing.
1.3 instrument
Electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd；Electronic scale, Shanghai Precision Scientific Apparatus Co., Ltd；
Table-type low-speed autobalance centrifuge, Changsha Xiang Zhi centrifuges Instrument Ltd.；Electric-heated thermostatic water bath, Shanghai
One permanent scientific instrument Co., Ltd；Ultraviolet-uisible spectrophotometer, Shanghai Techcomp Instrument Ltd.；ELIASA, the U.S.
BIO-RAD companies；Adjustable pipette, Shanghai Lei Bo Analytical Instrument Co., Ltd；Ultrasonic cleaner, Shanghai section leads Ultrasound Instrument
Device Co., Ltd；Glass homogenizer, Zhengzhou Glassware Factory.
2 methods
2.1 modelings and administration
Healthy mice 96, male, 25~30g of body weight is normal to raise 3 days, weighs, and is randomly divided into 6 groups, every group 16,
Respectively do evil through another person Shu Zu ﹑ model groups, Nimodipine group, large, medium and small dosage Honegsukle flower P.E group.By 0.1ml/10g gavage Buddhist nuns
(positive control drug, dosage is not 30mg/kg to Horizon suspension, equivalent to 15 times of clinical dosage, before use with 0.5%
CMC is made into drug concentration 3mg/ml), large, medium and small dosage Honegsukle flower P.E suspension (dosage is respectively 100mg/kg,
50mg/kg, 25mg/kg, are made into drug concentration 10mg/ml, 5mg/ml, 2.5mg/ml with 0.5%CMC before use), sham-operation
Zu ﹑ model groups give same volume 0.5%CMC gavages, are administered once daily, successive administration 7d.
Started fasting in batches in the 6th day at 8 points in evening to can't help after water, 12h, batch weighing administration, after 1h, with 10% hydration chlorine
Aldehyde (0.03ml/10g) intraperitoneal injection of anesthesia (righting reflex loss of mouse), is sterilized with alcohol neck, is then made with scalpel
Neck median incision, is gradually disengaged Surrounding muscles, and week is peeled off until seeing bilateral common carotid arteries (CCA), then with the accurate tweezer of elbow
Enclose nerve, separate CCA, threading is standby, the acupuncture needle that each arteria carotis communis is about 0.3mm with diameter hook come, make into about 90 degree,
Ischemic 10min, loosens acupuncture needle, recovers perfusion 10min, then ischemic 10min, recovers blood supply.Site of injury applies and spreads penicillin powder, stitches
Close neck wound.Sham-operation group is not in addition to blocking CCA, and other operations are with modeling group.
2.2 Testing index and detection method
2.2.1 Testing index
After all reper-fusion 24h, pluck eyeball and take blood, stand after half an hour, 3500r/min centrifugation 10min take blood
Clearly, NSE, MPO content in serum are surveyed.De- cervical vertebra is put to death, and brain is taken rapidly, sagittal cuts half and is put into 10% formalin solution,
One week is fixed, FFPE makees HE dyeing, and observation cerebral morphology changes；Second half is with the blood above normal saline flushing
Mark, then with the physiological saline blotted on filter paper above net, its weight of precise, according to the ice-cold life of this re-computation addition
Salt water is managed, with physiological saline (ml)：Brain tissue (g)=9: 1 ratio uses glass homogenizer in the large beaker for fill ice cube
10% brain homogenate is made, 4 DEG C, 3000r/min centrifugation 10min take supernatant in less than -20 DEG C Cord bloods, determined respectively
ICAM-1, TNF-α content in brain homogenate.
2.2.2 in serum NSE assay method
Cleaning Principle：Kit uses the step sandwich method EUSA (ELISA) of double antibody one.Toward coating in advance
In the coating micropore of Serum Neuron Specific Enolase (NSE) protein antibodies, sequentially add sample, standard items, HRP mark detection resist
Body, by incubating and thoroughly washing.Developed the color with substrate TMB, TMB converts au bleu under the catalysis of peroxidase, and in acid
In the presence of change into final yellow.Serum Neuron Specific Enolase (NSE) in the depth and sample of color is proportionate.Use enzyme
Mark instrument determines absorbance (OD values) under 450nm wavelength, calculates sample concentration.
Operating procedure：Lath needed for being taken out from the aluminium foil bag after equilibrium at room temperature 20min, remaining lath is sealed with valve bag
Put back to 4 DEG C of preservations.Setting standard sample wells and sample aperture, standard sample wells add the μ l of standard items 50 of various various concentrations；Sample aperture is first
Plus the μ l of sample to be tested 10, then add the μ l of Sample dilution 40；Blank well is not added with.It is every in standard sample wells and sample aperture in addition to blank well
Hole adds the μ l of detection antibody 100 of horseradish peroxidase (HPR) mark, seals reacting hole with shrouding film, 37 DEG C of water-baths or
Insulating box incubates 60min.Discard and patted dry on liquid, blotting paper, cleaning solution is filled it up with per hole, stand 1min, get rid of cleaning solution, absorb water
Patted dry on paper, so repeat board-washing 5 times (can also be machine-washed plate with board-washing).Substrate A, each 50 μ l of B are added per hole, 37 DEG C of lucifuges are incubated
15min.Added per hole in terminate liquid 50 μ l, 15min, the OD values in each hole are determined at 450nm wavelength.
Draw standard curve：In Excel worksheets, abscissa is made with standard concentration, correspondence OD values are made ordinate, painted
Standard items linear regression curves are made, each sample concentration value is calculated by curvilinear equation.
2.2.3 in serum MPO assay method
MPO determines sequence list in serum
Mix, 60 DEG C of water-baths 10 minutes, after taking-up immediately 460nm at, 1cm optical paths, distilled water zeroing, each pipe extinction of survey
Angle value.
Calculation formula：
MPO vigor (units per liter)=(determine OD values-compare OD values) ÷ (11.3 × sampling amount (liter))
2.2.4 in brain tissue ICAM-1 assay method
Cleaning Principle, operating procedure：Same Serum Neuron Specific Enolase (NSE).
2.2.5 in brain tissue TNF-α assay method
Cleaning Principle, operating procedure：Same Serum Neuron Specific Enolase (NSE).
3 statistical procedures methods
Data analysis carries out the statistical procedures of data information with the statistical packages of SPSS 17.0, and measurement data is with averagely
Number ± standard deviationRepresent, compare between each group and use one-way analysis of variance, the neat person's least significant difference of variance test
(LSD) method, heterogeneity of variance person is examined with Games-Howell methods, and ranked data are examined with Radit.
4 results
4.1 pairs of the repeated cerebral ischemia-reperfusion model mice death rates, serum NSE, influences (the results are shown in Table 1) of MPO levels
The Honegsukle flower P.E of table 1 to the repeated cerebral ischemia-reperfusion model mice death rate, serum NSE, MPO levels influence
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, the death rate highest of model group, Nimodipine group, large, medium and small dosage Honegsukle flower P.E
The death rate of group mouse decreases, and illustrates that the reduction repeated cerebral ischemia-reperfusion model that administration each group can be different degrees of is small
The mouse death rate, reduces brain tissue impairment, protects brain tissue.With sham-operation group ratio, model group mice serum NSE, MPO level show
Write rise (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group mice serum NSE, MPO level is substantially reduced
(P<0.05), heavy dose of Honegsukle flower P.E group mice serum NSE levels significantly reduce (P<0.01) in, low dose of honeysuckle
Extract group mice serum NSE levels have reduction trend (P ＞ 0.05), big, middle dosage Honegsukle flower P.E group mice serum MPO
Level significantly reduces (P<0.01), low dose of Honegsukle flower P.E group mice serum MPO levels substantially reduce (P<0.05), explanation
Administration each group has different degrees of protection repeated cerebral ischemia-reperfusion model mice neuron, reduction serum NSE, MPO levels
Effect.
4.2 pairs of repeated cerebral ischemia-reperfusion model mice brain tissue ICAM-1, influences (the results are shown in Table 2) of TNF-α level
The Honegsukle flower P.E of table 2 is to repeated cerebral ischemia-reperfusion model mice brain tissue ICAM-1, the influence of TNF-α level
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, with sham-operation group ratio, model group Mice brain tissues ICAM-1, TNF-α level significantly on
Rise (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group, heavy dose of Honegsukle flower P.E group Mice brain tissues
ICAM-1 levels significantly reduce (P<0.01), middle dosage Honegsukle flower P.E group Mice brain tissues ICAM-1 levels substantially reduce (P
<0.05), low dose of Honegsukle flower P.E group Mice brain tissues ICAM-1 levels have reduction trend (P ＞ 0.05), Nimodipine
Group, big, middle dosage Honegsukle flower P.E group Mice brain tissues TNF-α level significantly reduce (P<0.01), low dose of honeysuckle carries
Thing group Mice brain tissues TNF-α level is taken substantially to reduce (P<0.05), illustrate that administration each group has reduction cerebral ischemia reperfusion repeatedly
Injection molding type Mice brain tissues ICAM-1, TNF-α level, reduce inflammation effect of the reaction to brain tissue impairment.
The influence (the results are shown in Table 3) of 4.3 pairs of repeated cerebral ischemia-reperfusion model mice brain tissue cortical area pathological changes
It is as follows to repeated cerebral ischemia-reperfusion model mice brain tissue cortical area Histopathological Studies：Sham-operation group brain skin
Matter nerve cell is normal；Model group Neurons of Cerebral Cortex oedema, most neurons necrosis；Nimodipine group cerebral cortex
A small number of nerve cell oedema, a small number of neuronal degenerations, the light dye of endochylema, structural fuzzy, indivedual neuronal necrosis；Heavy dose of honeysuckle
Extract group cerebral cortex minority nerve cell oedema, are dispersed in, a small number of neuronal degenerations, the light dye of endochylema, structural fuzzy；Middle dosage
Honegsukle flower P.E group cerebral cortex partial nerve edema, is dispersed in, a small number of neuronal degeneration, the light dye of endochylema, structural fuzzy,
Indivedual neuronal necrosis；Low dose of Honegsukle flower P.E group Neurons of Cerebral Cortex oedema, neuronal degeneration in blocks, endochylema is light
Dye, structural fuzzy, indivedual neuronal necrosis.
Influence of the Honegsukle flower P.E of table 3 to repeated cerebral ischemia-reperfusion model mice brain tissue cortical area pathological change
Note：With model group ratio, * * P<0.01, * P<0.05
"-" nerve cell is normal；"+" Neurons of Cerebral Cortex oedema, is dispersed in, a small number of neuronal degeneration, and endochylema is light
Dye, structural fuzzy；" ++ " Neurons of Cerebral Cortex oedema, neuronal degeneration in blocks, the light dye of endochylema, structural fuzzy, indivedual god
Through first necrosis；" +++ " Neurons of Cerebral Cortex oedema, most neurons necrosis.
Examined through Ridit, with sham-operation group ratio, model group has significant statistical significance (P<0.01) model group, is illustrated
There is significant pathological change, modeling success in Mice brain tissues cortical area；With model group ratio, Nimodipine group, large, medium and small dose
Amount Honegsukle flower P.E group has significant statistical significance (P<0.01), illustrate that each administration group can significantly improve repeatedly cerebral ischemia again
The pathology damage degree of perfusion model Mice brain tissues cortical area, protects brain tissue.
The influence (the results are shown in Table 4) of 4.4 pairs of repeated cerebral ischemia-reperfusion model mice brain tissue hippocampus pathological changes
It is as follows to repeated cerebral ischemia-reperfusion model mice brain tissue hippocampus Histopathological Studies：The big brain of sham-operation group
Horse nerve cell is normal；Model group cerebral hippocampus nerve cell oedema, most neurons necrosis；Nimodipine group cerebral hippocampus
A small number of nerve cell oedema, a small number of neuronal degenerations, the light dye of endochylema, structural fuzzy, indivedual neuronal necrosis；Heavy dose of honeysuckle
Extract group cerebral hippocampus minority nerve cell oedema, are dispersed in, a small number of neuronal degenerations, the light dye of endochylema, structural fuzzy；Middle dosage
Honegsukle flower P.E group cerebral hippocampus partial nerve edema, is dispersed in, a small number of neuronal degeneration, the light dye of endochylema, structural fuzzy,
Indivedual neuronal necrosis；Low dose of Honegsukle flower P.E group cerebral hippocampus nerve cell oedema, neuronal degeneration in blocks, endochylema is light
Dye, structural fuzzy, indivedual neuronal necrosis.
Influence of the Honegsukle flower P.E of table 4 to repeated cerebral ischemia-reperfusion model mice brain tissue hippocampus pathological change
Note：With model group ratio, * * P<0.01, * P<0.05
"-" nerve cell is normal；"+" cerebral hippocampus nerve cell oedema, is dispersed in, a small number of neuronal degeneration, and endochylema is light
Dye, structural fuzzy；" ++ " cerebral hippocampus nerve cell oedema, neuronal degeneration in blocks, the light dye of endochylema, structural fuzzy, indivedual god
Through first necrosis；" +++ " cerebral hippocampus nerve cell oedema, most neurons necrosis.
Examined through Ridit, with sham-operation group ratio, model group has significant statistical significance (P<0.01) model group, is illustrated
There is significant pathological change, modeling success in Mice brain tissues hippocampus；With model group ratio, Nimodipine group, large, medium and small dose
Amount Honegsukle flower P.E group has significant statistical significance (P<0.01), illustrate that each administration group can significantly improve repeatedly cerebral ischemia again
The pathology damage degree of perfusion model Mice brain tissues hippocampus, protects brain tissue.
5 brief summaries
By observing Honegsukle flower P.E to the death rate of mouse repeated cerebral ischemia-reperfusion model, serum NSE, MPO water
ICAM-1, TNF-α level, the influence of cerebral tissue pathology injury's change, point out Honegsukle flower P.E to reduce dynamic in flat, brain tissue
The thing death rate, reduction serum NSE, MPO levels and brain tissue ICAM-1, TNF-α level, reduce inflammation damage of the reaction to brain tissue
Wound, protects neuron, mitigates damage of the cerebral ischemia re-pouring to brain tissue nerve cell, using heavy dose of Honegsukle flower P.E group as
Most preferably.
Two Honegsukle flower P.Es are tested to focal cerebral ischemic in mice re-perfusion model brain infarction area and serum S-100 β
The influence experiment of albumen
1 material
1.1 animal
KM kind mouse, SPF grades, male, 96,25~30g of body weight is provided by Lukang Medical Co., Ltd., Shandong,
Quality certification number：0017138；Laboratory quality certification number：SYXK (Henan) 2010-001.
1.2 medicines and reagent
Honegsukle flower P.E of the present invention；Sodium chloride injection, Henan Shuan Hehuali pharmaceutcal corporation, Ltds；Nimodipine tablet,
Shanxi Yabao Pharmaceutical Group Corp.；Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd；Formalin (analysis
It is pure), Chemical Co., Ltd. in pairs of Yantai City；Chloraldurate, Tianjin recovery fine chemistry industry research institute；Sodium carboxymethylcellulose
(CMC), Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd；Medical ethanol (alcohol), the limited public affairs of big disinfectant preparation of Xinxiang City three
Department；Red tetrazolium (TTC), upper seamount Pu Chemical Co., Ltd.；Disodium hydrogen phosphate, Tianjin Zhi Yuan chemical reagent Co., Ltd；
Sodium dihydrogen phosphate, the factory of Tianjin chemical reagent three；S-100 β protein ELISA detection kits, R＆D companies.
1.3 instrument
MCAO line bolts, Shadong Biological Technology Co., Ltd., Beijing；Electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd；
Electronic scale, Shanghai Precision Scientific Apparatus Co., Ltd；Table-type low-speed autobalance centrifuge, Changsha Hunan intelligence centrifuge instrument is limited
Company；Electric-heated thermostatic water bath, Shanghai Yiheng Scientific Instruments Co., Ltd；ELIASA, BIO-RAD companies of the U.S.；It is adjustable to move
Liquid device, Shanghai Lei Bo Analytical Instrument Co., Ltd；Specialized image analysis system, NIS-Elements AR 4.10.01；Ultrasonic wave
Washer, Shanghai High Kudos Science Instrument Co., Ltd.；Glass homogenizer, Zhengzhou Glassware Factory.
2 methods
2.1 modelings and administration
Healthy mice 96 is taken, male, 25~30g of body weight is normal to raise 3 days, weighs, random point 6 groups, every group 16,
Respectively do evil through another person Shu Zu ﹑ model groups, Nimodipine group, large, medium and small dosage Honegsukle flower P.E group.By 0.1ml/10g gavage Buddhist nuns
(positive control drug, dosage is not 30mg/kg to Horizon suspension, equivalent to 15 times of clinical dosage, before use with 0.5%
CMC is made into drug concentration 3mg/ml), large, medium and small dosage Honegsukle flower P.E suspension (dosage is respectively 100mg/kg,
50mg/kg, 25mg/kg, are made into drug concentration 10mg/ml, 5mg/ml, 2.5mg/ml with 0.5%CMC before use), sham-operation
Zu ﹑ model groups give same volume 0.5%CMC gavages, are administered once daily, successive administration 7d.
Started fasting in batches in the 6th day at 8 points in evening to can't help after water, the 7th day 8 a.m. batch weighing administration 1h, with 10%
After chloraldurate (0.03ml/10g) intraperitoneal injection of anesthesia mouse, mouse anesthesia, neck hits exactly otch to the left, successively separation exposure
Left common carotid (CCA), external carotid artery (ECA), internal carotid (ICA), ligation arteria carotis communis and external carotid artery, use artery clamp
Internal carotid is blocked, a wide osculum of about 0.2mm is cut at bifurcated 1mm in arteria carotis communis, line bolt is inserted, always moved through neck
Arteries and veins furcation enters internal carotid, upwards deeply to more than bifurcated 8~10mm, until there is resistance, that is, blocks arteria cerebri media to enter
Mouthful place, gently extracts bolt line out after ligaturing internal carotid otch and bolt line, 2h, realizes Reperfu- sion, makes middle cerebral artery occlusion-fill again
Note (MCAO) animal model.Only exposure left side blood vessel does not do plug wire processing to sham-operation group.
2.2 Testing index and detection method
2.2.1 Testing index
After all reper-fusion 22h, clinic nerve function assay is carried out to modeling mouse：Using Longa[13]Standard is entered
Row scoring.Standards of grading：0 point：Impassivity afunction symptom, activity is normal；1 point：It is unable to full extension offside fore paw；2 points：
Occur turn-taking to hemiplegia side when creeping；3 points：During walking, body rolls to hemiplegia；4 points：Spontaneous it can not walk, the loss of consciousness；
5 points：It is dead.Score is that 0 point and 5 points of persons reject；Pluck eyeball and take blood, stand after half an hour, 3500r/min centrifugation 10min take blood
Clearly, S-100 β protein contents in serum are surveyed.Mouse takes off cervical vertebra and put to death, and brain tissue is stripped rapidly, -20 DEG C of refrigerator freezings are placed in
15min, removes the remainder after cerebellum, olfactory bulb and low brain stem, and coronal-plane cuts four knives, brain homogenate is cut into five, so
It is rapid afterwards that brain piece is placed in the 1%TTC dye liquors configured with pH=7.2 phosphate buffers, incubated under the conditions of 37 DEG C of water-baths, lucifuges
Educate 10min.It is placed in after taking-up in 10% formalin solution and 24h is kept in dark place.Dyed rear non-ischemic region is rose, infarct
Area is white.Digital camera is taken pictures, and calculates the gross area and infarct of five brain pieces totally 10 planes respectively with image analysis software
The area in region, obtains the percentage that infarcted region area accounts for the gross area.
2.2.2 in serum S-100 β assay method
Cleaning Principle, operating procedure：With Serum Neuron Specific Enolase (NSE) in experiment one.
3 statistical procedures methods
Data analysis carries out the statistical procedures of data information with the statistical packages of SPSS 17.0, and measurement data is with averagely
Number ± standard deviationRepresent, compare between each group and use one-way analysis of variance, the neat person's least significant difference of variance test
(LSD) method, heterogeneity of variance person is examined with Games-Howell methods, and ranked data are examined with Radit.
4 results
(result is shown in for the influence of 4.1 pairs of Focal Cerebral Ischemia Reperfusion model mice death rates and clinic nerve function assay
Table 5)
The Honegsukle flower P.E of table 5 is to the Focal Cerebral Ischemia Reperfusion model mice death rate and clinic nerve function assay
Influence
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, the death rate highest of model group, Nimodipine group, large, medium and small dosage Honegsukle flower P.E
The death rate of group mouse decreases, and illustrates the reduction Focal Cerebral Ischemia Reperfusion model that each group can be different degrees of is administered
Mouse death rate, reduces brain tissue impairment, protects brain tissue.With sham-operation group ratio, the scoring of model group mouse Nerve afunction
Significantly rise (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group, heavy dose of Honegsukle flower P.E group mouse
Clinic nerve function assay significantly reduces (P<0.01), the scoring of middle dosage Honegsukle flower P.E group mouse Nerve afunction is obvious
Reduce (P<0.05), there is low dose of Honegsukle flower P.E group reduction Focal Cerebral Ischemia Reperfusion mouse Nerve afunction to comment
The trend (P ＞ 0.05) divided, illustrates that administration each group has different degrees of improvement Focal Cerebral Ischemia Reperfusion mouse cranial nerve
The effect of function.
The influence of 4.2 pairs of Focal Cerebral Ischemia Reperfusion model mice serum S-100 β levels and brain infarction area percentage
(the results are shown in Table 6)
The Honegsukle flower P.E of table 6 is to Focal Cerebral Ischemia Reperfusion model mice serum S-100 β levels and brain infarction area
The influence of percentage
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, with sham-operation group ratio, model group mice serum S-100 β levels, brain infarction area percentage
Significantly rise (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group, large, medium and small dosage Honegsukle flower P.E
Group mice serum S-100 β levels, brain infarction area percentage significantly reduce (P<0.01), illustrate that administration each group is respectively provided with not
With the protection Focal Cerebral Ischemia Reperfusion model mice brain cell and nervous function of degree, serum S-100 β levels are reduced
With the effect of brain infarction area.
5 conclusions
By observing the death rate, neurological deficit of the Honegsukle flower P.E to focal cerebral ischemic in mice re-perfusion model
Scoring, brain infarction area percentage, the influence of Nerve tissue protein S-100 level, point out Honegsukle flower P.E to reduce animal dead
Rate, clinic nerve function assay and brain infarction area percentage are died, Nerve tissue protein S-100 level is reduced, to brain tissue and god
There is certain protective effect through member, be optimal using heavy dose of Honegsukle flower P.E group.
Three Honegsukle flower P.Es are tested to focal cerebral ischemic in mice re-perfusion model brain homogenate biochemical indicator and brain tissue
The influence experiment of form
1 material
1.1 animal
KM kind mouse, SPF grades, male, 96,25~30g of body weight is provided by Lukang Medical Co., Ltd., Shandong,
Quality certification number：0017138；Laboratory quality certification number：SYXK (Henan) 2010-001.
1.2 medicines and reagent
Honegsukle flower P.E of the present invention；Sodium chloride injection, Henan Shuan Hehuali pharmaceutcal corporation, Ltds；Nimodipine tablet,
Shanxi Yabao Pharmaceutical Group Corp.；Benzylpenicillin sodium for injection, Huabei Pharmaceutic Co., Ltd；Formalin (analysis
It is pure), Chemical Co., Ltd. in pairs of Yantai City；Chloraldurate, Tianjin recovery fine chemistry industry research institute；Sodium carboxymethylcellulose
(CMC), Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd；Medical ethanol (alcohol), the limited public affairs of big disinfectant preparation of Xinxiang City three
Department；The B cell lymphoma factor 2 (Bcl-2) ELISA detection kit, R＆D companies；Bcl-2 correlations X protein (BAX) ELISA is examined
Test agent box, R＆D companies；Caspase-3 (Caspase-3) ELISA detection kit, R＆D companies；Coomassie brilliant blue
Protein quantification testing cassete, Bioengineering Research Institute is built up in Nanjing；Atriphos (ATP) enzyme testing cassete, biological work is built up in Nanjing
Journey research institute.
1.3 instrument
MCAO line bolts, Shadong Biological Technology Co., Ltd., Beijing；Electronic balance, Shanghai Precision Scientific Apparatus Co., Ltd；
Electronic scale, Shanghai Precision Scientific Apparatus Co., Ltd；Table-type low-speed autobalance centrifuge, Changsha Hunan intelligence centrifuge instrument is limited
Company；Electric-heated thermostatic water bath, Shanghai Yiheng Scientific Instruments Co., Ltd；Ultraviolet-uisible spectrophotometer, the U.S. science in Shanghai day
Instrument Ltd.；ELIASA, BIO-RAD companies of the U.S.；Adjustable pipette, Shanghai Lei Bo Analytical Instrument Co., Ltd；It is super
Sound wave washer, Shanghai High Kudos Science Instrument Co., Ltd.；Glass homogenizer, Zhengzhou Glassware Factory.
2 methods
2.1 modelings and administration
Healthy mice 96 is taken, male, 25~30g of body weight is normal to raise 3 days, weighs, random point 6 groups, every group 16,
Respectively do evil through another person Shu Zu ﹑ model groups, Nimodipine group, large, medium and small dosage Honegsukle flower P.E group.By 0.1ml/10g gavage Buddhist nuns
(positive control drug, dosage is not 30mg/kg to Horizon suspension, equivalent to 15 times of clinical dosage, before use with 0.5%
CMC is made into drug concentration 3mg/ml), large, medium and small dosage Honegsukle flower P.E suspension (dosage is respectively 100mg/kg,
50mg/kg, 25mg/kg, are made into drug concentration 10mg/ml, 5mg/ml, 2.5mg/ml with 0.5%CMC before use), sham-operation
Zu ﹑ model groups give same volume 0.5%CMC gavages, are administered once daily, successive administration 7d.
Started fasting in batches in the 6th day at 8 points in evening to can't help after water, the 7th day 8 a.m. batch weighing administration 1h, with 10%
After chloraldurate (0.03ml/10g) intraperitoneal injection of anesthesia mouse, mouse anesthesia, neck hits exactly otch to the left, successively separation exposure
Left common carotid (CCA), external carotid artery (ECA), internal carotid (ICA), ligation arteria carotis communis and external carotid artery, use artery clamp
Internal carotid is blocked, a wide osculum of about 0.2mm is cut at bifurcated 1mm in arteria carotis communis, bolt line is inserted, always moved through neck
Arteries and veins furcation enters internal carotid, upwards deeply to more than bifurcated 8~10mm, until there is resistance, that is, blocks arteria cerebri media to enter
Mouthful place, gently extracts bolt line out after ligaturing internal carotid otch and bolt line, 2h, realizes Reperfu- sion, makes middle cerebral artery occlusion-fill again
Note (MCAO) animal model.Only exposure left side blood vessel does not do plug wire processing to sham-operation group.
2.2 Testing index and detection method
2.2.1 Testing index
After all reper-fusion 22h, clinic nerve function assay is carried out to modeling mouse：Carried out using Longa standards
Scoring.Standards of grading：0 point：Impassivity afunction symptom, activity is normal；1 point：It is unable to full extension offside fore paw；2 points：Climb
Occur turn-taking to hemiplegia side during row；3 points：During walking, body rolls to hemiplegia；4 points：Spontaneous it can not walk, the loss of consciousness；5
Point：It is dead.Score is that 0 point and 5 points of persons reject；Mouse takes off cervical vertebra and put to death, and brain is taken rapidly, sagittal cuts left half brain, discards right half
Brain.The half of left half brain is put into 10% formalin solution, one week is fixed, FFPE makees HE dyeing observation brain tissue shapes
State changes；Second half is with the bloodstain above normal saline flushing, then with the physiological saline blotted on filter paper above net, accurate to claim
Its weight is measured, according to the ice-cold saline volume of this re-computation addition, with physiological saline (ml)：Brain tissue (g)=9: 1
10% brain homogenate is made in ratio in the large beaker for fill ice cube of glass homogenizer, and 4 DEG C, 3000r/min centrifuges 10min,
Take supernatant in less than -20 DEG C Cord bloods, Bax, Bcl-2, Caspase-3, ATP enzyme in brain homogenate are determined respectively.
2.2.2 in brain tissue Bax, Bcl-2, Caspase-3 assay method
Cleaning Principle, operating procedure：With Serum Neuron Specific Enolase (NSE) in experiment one.
2.2.3 the assay method of Coomassie brilliant blue protein quantification testing cassete
Coomassie brilliant blue protein determination Operations Sequence Chart
Mix, stand 10 minutes, at wavelength 595nm, each pipe absorbance is surveyed in optical path 1cm, distilled water zeroing.
Calculation formula：
2.2.4 the assay method of ATPase in Brain Tissue
Atpase assay Operations Sequence Chart in brain homogenate
The preparation of five kinds of mix reagents of A, B, C before test
Enzymatic reaction
Mix, 37 DEG C of water-baths 30 minutes are cooled to room temperature, 660nm at, 1cm optical paths, distilled water returns to zero colorimetric.
Note：A pipes are control tube；B pipes are Na+K+-ATP enzyme pipes；C pipes are Mg++-ATP enzyme pipes；
Calculation formula：
ATP vigor (μm olPi/mgprot/hour)=(determining pipe OD values-control tube OD values) ÷ standard pipes OD in tissue
Sample extension rate (2.5) × 6 ÷ homogenate proteins concentration in value × standard pipe concentration (0.5 μm of ol/ml) × reaction system
(mgprot/ml)
3 statistical procedures methods
Data analysis carries out the statistical procedures of data information with the statistical packages of SPSS 17.0, and measurement data is with averagely
Number ± standard deviationRepresent, compare between each group and use one-way analysis of variance, the neat person's least significant difference of variance test
(LSD) method, heterogeneity of variance person is examined with Games-Howell methods, and ranked data are examined with Radit.
4 results
(result is shown in for the influence of 4.1 pairs of Focal Cerebral Ischemia Reperfusion model mice death rates and clinic nerve function assay
Table 7)
The Honegsukle flower P.E of table 7 is to the Focal Cerebral Ischemia Reperfusion model mice death rate and clinic nerve function assay
Influence
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, the death rate highest of model group, Nimodipine group, large, medium and small dosage Honegsukle flower P.E
The death rate of group mouse decreases, and illustrates that administration each group can reduce Focal Cerebral Ischemia Reperfusion model in various degree small
The mouse death rate, reduces brain tissue impairment, protects brain tissue.With sham-operation group ratio, the scoring of model group mouse Nerve afunction shows
Write rise (P<0.01) modeling success, is illustrated；With model group ratio, heavy dose of Honegsukle flower P.E group mouse Nerve afunction is commented
Dividing significantly reduces (P<0.01), middle dosage Honegsukle flower P.E group mouse Nerve afunction scoring substantially reduces (P<0.05),
Nimodipine group, low dose of Honegsukle flower P.E group mouse Nerve afunction scoring have reduction trend (P ＞ 0.05), illustrate to
Medicine each group has the effect of different degrees of improvement Focal Cerebral Ischemia Reperfusion mouse cranial nerve function.
Influence (the knot of 4.2 pairs of Focal Cerebral Ischemia Reperfusion model mice brain tissue Bax, Bcl-2, Caspase-3 levels
8) fruit is shown in Table
The Honegsukle flower P.E of table 8 is to Focal Cerebral Ischemia Reperfusion model mice brain tissue Bax, Bcl-2, Caspase-3
The influence of level
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, with sham-operation group ratio, model group Mice brain tissues Bax, Caspase-3 level significantly rises
Height, illustrates to promote apoptosis factor increase in Mice brain tissues, modeling success；With model group ratio, it is Nimodipine group, large, medium and small
Dosage Honegsukle flower P.E group Mice brain tissues Bax, Caspase-3 level significantly reduces (P<0.01), Nimodipine group,
Large, medium and small dosage Honegsukle flower P.E group Mice brain tissues Bcl-2 levels significantly raise (P<0.01), illustrate each group is administered
Suppression apoptosis gene level in focal cerebral ischemia in rats Mice brain tissues can be increased in various degree, reduction promotees Apoptosis base
Because of level, suppress Apoptosis, so as to protect brain tissue, alleviate symptoms of cerebral ischemia.
4.3 couples of Focal Cerebral Ischemia Reperfusion model mice Na+K+- ATP enzyme vigor and Mg++The influence of-ATP enzyme vigor
(the results are shown in Table 9)
The Honegsukle flower P.E of table 9 is to Focal Cerebral Ischemia Reperfusion model mice Na+K+- ATP enzyme and Mg++- ATP enzyme vigor
Influence
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from the above table, with sham-operation group ratio, model group Mice brain tissues Na+-K+- ATP enzyme and Mg++- ATP enzyme is lived
Power significantly reduces (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group, big, middle dosage Honegsukle flower P.E
Group Mice brain tissues Na+-K+- ATP enzyme vigor significantly raises (P<0.01), low dose of Honegsukle flower P.E group Mice brain tissues
Na+-K+Significantly raised (the P of-ATP enzyme vigor<0.05), Nimodipine group, heavy dose of Honegsukle flower P.E group Mice brain tissues Mg++- ATP enzyme vigor significantly raises (P<0.01), middle dosage Honegsukle flower P.E group Mice brain tissues Mg++- ATP enzyme vigor is bright
Aobvious rise (P<0.05), low dose of Honegsukle flower P.E group Mice brain tissues Mg++- ATP enzyme vigor has rise trend (P ＞
0.05), illustrate that administration each group can raise Focal Cerebral Ischemia Reperfusion model mice brain tissue ATP enzyme vigor in various degree, delay
Solve Energy Metabolism of Brain Tissue obstacle.
The influence (the results are shown in Table 10) of 4.3 pairs of Focal Cerebral Ischemia Reperfusion model mice brain tissue cortical area pathological changes
It is as follows to Focal Cerebral Ischemia Reperfusion model mice brain tissue cortical area Histopathological Studies result：Sham-operation group
Cerebral cortex is without oedema, and nerve cell is normal；Model group Neurons of Cerebral Cortex oedema, large stretch of neuronal necrosis, infarct face
Product accounts for more than the 2/3 of the left side cortex gross area；Nimodipine group cerebral cortex partial nerve edema, a small number of neurons are bad
Extremely, infarct size is accounted within the 1/3 of the left side cortex gross area；Heavy dose of Honegsukle flower P.E group Neurons of Cerebral Cortex water
Swollen, a small number of neuronal necrosis, infarct size is accounted within the 1/3 of the left side cortex gross area；Middle dosage Honegsukle flower P.E group brain
Cortical neural cell oedema, a small number of neuronal necrosis, infarct size is accounted within the 1/3~2/3 of the left side cortex gross area；It is low dose of
Honegsukle flower P.E group Neurons of Cerebral Cortex oedema, neuronal necrosis in blocks, infarct size accounts for the left side cortex gross area
Within 1/3~2/3.
Shadow of the Honegsukle flower P.E of table 10 to Focal Cerebral Ischemia Reperfusion model mice brain tissue cortical area pathological change
Ring
Note：With model group ratio, * * P<0.01, * P<0.05
"-" cerebral cortex is without oedema, and nerve cell is normal；"+" Neurons of Cerebral Cortex oedema, a small number of neurons are bad
Extremely, infarct size is accounted within the 1/3 of the left side cortex gross area；" ++ " Neurons of Cerebral Cortex oedema, neuronal necrosis in blocks,
Infarct size is accounted within the 1/3~2/3 of the left side cortex gross area；" +++ " Neurons of Cerebral Cortex oedema, large stretch of neuron is bad
Extremely, infarct size accounts for more than the 2/3 of the left side cortex gross area.
Examined through Ridit, with sham-operation group ratio, model group has significant statistical significance (P<0.01) model group, is illustrated
There is significant pathological change, modeling success in Mice brain tissues cortical area；With model group ratio, Nimodipine group, heavy dose of gold and silver
Flower extract group has significant statistical significance (P<0.01), middle dosage Honegsukle flower P.E group has obvious statistical significance (P
<0.05), low dose of Honegsukle flower P.E group is not statistically significant (P ＞ 0.05), illustrates the improvement that each administration group can be different degrees of
The pathology damage degree of the office's property made cerebral ischemia re-pouring model mice brain tissue cortical area, protects brain tissue.
The influence (the results are shown in Table 11) of 4.4 pairs of Focal Cerebral Ischemia Reperfusion model mice brain tissue hippocampus pathological changes
It is as follows to Focal Cerebral Ischemia Reperfusion model mice brain tissue hippocampus Histopathological Studies：Sham-operation group brain
Hippocampal tissue is without oedema, and nerve cell is normal；Model group cerebral hippocampus tissue edema, large stretch of neuronal necrosis, infarct size is accounted for
More than the 2/3 of left hippocampus tissue；Nimodipine group cerebral hippocampus portion of tissue oedema, a small number of neuronal necrosis, infarct size
Within account for left hippocampus tissue 1/3；Heavy dose of Honegsukle flower P.E group cerebral hippocampus tissue edema, a small number of neuronal necrosis,
Infarct size is accounted within the 1/3 of left hippocampus tissue；Middle dosage Honegsukle flower P.E group cerebral hippocampus tissue edema, a small number of nerves
First necrosis, infarct size is accounted within the 1/3~2/3 of left hippocampus tissue；Low dose of Honegsukle flower P.E group cerebral hippocampus tissue
Oedema, neuronal necrosis in blocks, infarct size is accounted within the 1/3~2/3 of left hippocampus tissue.
Influence of the table 11 to Focal Cerebral Ischemia Reperfusion model mice brain tissue hippocampus pathological change
Note：With model group ratio, * * P<0.01, * P<0.05
"-" cerebral hippocampus tissue is without oedema, and nerve cell is normal；"+" cerebral hippocampus tissue edema, a small number of neurons are bad
Extremely, infarct size is accounted within the 1/3 of left hippocampus tissue；" ++ " cerebral hippocampus tissue edema, neuronal necrosis in blocks, infarct face
Product is accounted within the 1/3~2/3 of left hippocampus tissue；" +++ " cerebral hippocampus tissue edema, large stretch of neuronal necrosis, infarct size
Account for more than the 2/3 of left hippocampus tissue.
Examined through Ridit, with sham-operation group ratio, model group has significant statistical significance (P<0.01) model group, is illustrated
There is significant pathological change, modeling success in Mice brain tissues hippocampus；With model group ratio, Nimodipine group, big, middle dosage gold
Honeysuckle flower extract group has significant statistical significance (P<0.01), low dose of Honegsukle flower P.E group has obvious statistical significance
(P<0.05) the improvement office property the made cerebral ischemia re-pouring model mice brain tissue hippocampus that each administration group can be different degrees of, are illustrated
Pathology damage degree, protects brain tissue.
5 conclusions
By observing the death rate, neurological deficit of the Honegsukle flower P.E to focal cerebral ischemic in mice re-perfusion model
Bax, Bcl-2, Caspase-3, ATP enzyme level, the influence of cerebral tissue pathology injury's change, point out gold and silver in scoring, brain tissue
Flower extract can reduce mortality of animals, clinic nerve function assay, improve the degree of impairment of brain tissue, improve Bcl-2 water
It is flat, Bax, Caspase-3 level are reduced, suppresses nerve cell apoptosis, ATP enzyme level is raised, improved because of energy metabolism impairment pair
The damage of ischemic tissue of brain, is optimal with Honegsukle flower P.E heavy dose group.
6 clinical experimental datas
Clopidogrel adds treats transient cerebral ischemia (middle danger layer) 60 with Honegsukle flower P.E
Selection meets the transient cerebral ischemia diagnostic criteria that the 4th cerebrovascular disease academic conference in the whole nation is worked out:It is as of short duration
, reversible, local brain blood circulation disorder, can recurrent exerbation, few person 1~2 time, up to tens of times, breaking-out every time continues
Time, sings and symptoms should be wholly absent in 24h generally at several minutes to 1h or so.
ABCD2 standards of grading:Age ＞ 60 years old (1 point)；Blood pressure is systolic pressure ＞ 140mmHg or diastolic pressure ＞ 90mmHg (1
Point)；Clinical symptoms are single-sided weakness (2 points), not with powerless disfluency (1 point)；Symptom duration ＞ 60min (2 points),
10~59min (1 point)；With diabetes (1 point).Risk stratification:0~3 point is low danger group；4~5 points are middle danger group；6~7 points
For high-risk group.
Select ABCD2 standards of grading in the people of patient 60 of middle danger layer, be randomly divided into 2 groups, every group of 30 people, the oral chlorine of control group
Pyrrole Gray (trade name Plavix, 75mg/ pieces, match Norfin, Inc of France), each 75mg, 1d 1 time, first dose of 300mg.Treatment
(extract is made capsule to group, every 0.25g, every time in addition to clopidogrel is taken by control group method, plus with Honegsukle flower P.E
1,2 times a day).Two groups of the above treats conventional control blood pressure and blood glucose during 30d, treatment, and disabling other can influence to coagulate
The medicine of blood function.
Clopidogrel group adds the number of cases point terminated with TIA in Honegsukle flower P.E group 7d with clopidogrel
Wei not be 24,26, time when TIA is terminated in 7d is respectively (0.96 ± 1.11) d, (0.62 ± 1.18)
The number of cases for still having breaking-out after d, 7d is respectively 6,4, and the number of cases that transient cerebral ischemia is aggravated in 30d is respectively 2,0.Plus use
Honegsukle flower P.E treatment group general curative effect is better than conventional Effect of Clopidogrel in Treating group.
It should be apparent that the Honegsukle flower P.E that the present invention is extracted from honeysuckle has anti-brain from above-mentioned experiment
The effect of ischemic, the medicine of cerebral ischemia and focal cerebral ischemia is treated repeatedly effective for preparing, realizes that Honegsukle flower P.E exists
The application treated in cerebral ischemia and focal brain ischemia medicament repeatedly is prepared, its preparation method is simple, easy to operate, has opened up gold and silver
Hua Xin medical value and commercial value, is to treat the innovation in cerebral ischemia repeatedly and focal brain ischemia medicament, economy and society
Can remarkable benefit.
Claims (1)
1. a kind of Honegsukle flower P.E is preparing treatment cerebral ischemia and focal brain ischemia medicament repeatedly as sole active agent
In application, described Honegsukle flower P.E is that honeysuckle powder was broken into the coarse powder of 20-30 mesh sieves, first plus 10 times of weight
Petroleum ether refluxing extraction 1h, degreasing discards petroleum ether；The honeysuckle dregs of a decoction volatilize petroleum ether, plus honeysuckle 10 times of weight, quality
The ethanol of concentration 70% soaks 0.5h, 80 DEG C of refluxing extraction 1h, and filtering obtains first time filtrate；The dregs of a decoction add 10 times of weight of honeysuckle again
, the ethanol of mass concentration 70%, 80 DEG C of refluxing extraction 1h, filtering obtains second of filtrate, merges filtrate twice, ethanol is recovered under reduced pressure
The dispersion liquid equivalent to the 0.3g/mL containing crude drug is dispersed to without alcohol taste, plus distilled water, plus acetic acid adjusts pH to 2.4-4.2, dispersion liquid
Quality of Flos Lonicerae/macroreticular resin mass ratio is 1 ︰ 6-8 in upper HPD-600 or AB-8 types macroporous absorbent resin, dispersion liquid, uses quality
The ethanol elution of concentration 30%, is eluted with 2.3BV/h flow velocity, and eluent consumption is 8-12 times of macroreticular resin volume, and collection is washed
De- liquid, is concentrated, and freeze-drying obtains Honegsukle flower P.E.
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| CN117398422A (en) * | 2023-09-26 | 2024-01-16 | 河南中医药大学 | Japanese ardisia herb extract and preparation method and application thereof |
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2017
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| WAN032: ""绿原酸对大、小鼠脑缺血再灌注模型的干预作用研究"", 《WWW.DOCIN.COM/P-1803462955.HTML》 * |
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| CN117398422A (en) * | 2023-09-26 | 2024-01-16 | 河南中医药大学 | Japanese ardisia herb extract and preparation method and application thereof |
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Application publication date: 20171020 |
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