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 PDF

Info

Publication number
CN107260790A
CN107260790A CN201710688327.6A CN201710688327A CN107260790A CN 107260790 A CN107260790 A CN 107260790A CN 201710688327 A CN201710688327 A CN 201710688327A CN 107260790 A CN107260790 A CN 107260790A
Authority
CN
China
Prior art keywords
honegsukle flower
group
cerebral ischemia
honeysuckle
brain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710688327.6A
Other languages
Chinese (zh)
Inventor
苗明三
冯素香
白明
李瑞奇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan University of Traditional Chinese Medicine HUTCM
Original Assignee
Henan University of Traditional Chinese Medicine HUTCM
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan University of Traditional Chinese Medicine HUTCM filed Critical Henan University of Traditional Chinese Medicine HUTCM
Priority to CN201710688327.6A priority Critical patent/CN107260790A/en
Publication of CN107260790A publication Critical patent/CN107260790A/en
Pending legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/35Caprifoliaceae (Honeysuckle family)
    • A61K36/355Lonicera (honeysuckle)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10Preparation or pretreatment of starting material
    • A61K2236/15Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/39Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/51Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/55Liquid-liquid separation; Phase separation

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)

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

A kind of Honegsukle flower P.E is preparing treatment cerebral ischemia and focal cerebral ischemia medicine repeatedly Application in thing
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.
CN201710688327.6A 2017-08-12 2017-08-12 A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly Pending CN107260790A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710688327.6A CN107260790A (en) 2017-08-12 2017-08-12 A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710688327.6A CN107260790A (en) 2017-08-12 2017-08-12 A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly

Publications (1)

Publication Number Publication Date
CN107260790A true CN107260790A (en) 2017-10-20

Family

ID=60080296

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710688327.6A Pending CN107260790A (en) 2017-08-12 2017-08-12 A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly

Country Status (1)

Country Link
CN (1) CN107260790A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
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》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Similar Documents

Publication Publication Date Title
CN101444611B (en) Traditional Chinese medicine composition for treating lumbocrural pain and lumbar intervertebral disc protrusion and preparation method thereof
CN104225524A (en) Application of trichosanthes kirilowii maxim and cassia twig decoction in preparing medicament for treating or/and preventing cognitive impairment
CN105998276A (en) Application of total flavones of oldenlandia diffusa willd in preparing drugs for treating meningeal microcirculation disturbance and cerebral ischemia by serving as only active site
CN110327427A (en) It is a kind of to treat morning, the Chinese medicine composition of mid-term high blood pressure and preparation method
CN103655997A (en) Liver-soothing, brain-strengthening and depression-regulating tablet for treating post-stroke depression
CN102114170B (en) Traditional Chinese medicine composition for preventing and treating myocardial ischemia reperfusion injury and preparation method thereof
CN107260790A (en) A kind of Honegsukle flower P.E is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly
CN105521363A (en) Preparation method and quality control method of traditional Chinese medicine capsule for treating weakness of spleen and stomach
CN107912773A (en) A kind of preparation for being used to alleviate physical fatigue
CN102861159B (en) Medicine composition for treating dysmenorrheal as well as preparation method and application thereof
CN107441135A (en) Field thistle general flavone is preparing the application in treating cerebral ischemia and focal brain ischemia medicament repeatedly
CN105233089A (en) Traditional Chinese medicine preparation treating heart-kidney imbalance type insomnia
CN101829198B (en) Medicament for treating ischemic cranial vascular diseases
CN107243037A (en) Application of the field pennycress general flavone in treatment focal cerebral ischemia medicine is prepared
CN101297827B (en) Medicament for curing coronary disease and coronary artery coating bracket coated therewith
CN104189356B (en) A kind of pharmaceutical composition for the treatment of or auxiliary treatment infertility and its production and use
CN109663100B (en) Traditional Chinese medicine composition for treating optic neuritis and preparation method and application thereof
CN103830584B (en) Medicine for the treatment of Vertebral Artery Ischemic Disease and preparation method thereof
CN105833184A (en) Traditional Chinese medicine composition, fermented medicine liquor prepared from traditional Chinese medicine composition, preparation method and application
CN107441152A (en) A kind of flower of kudzuvine general flavone is preparing the application in treating brain ischemia medicament repeatedly
CN105412838A (en) Traditional Chinese medicine for treating diabetes and nephropathy
CN116870063B (en) Traditional Chinese medicine composition with blood circulation activating and nerve soothing functions and preparation method thereof
CN116898892B (en) Traditional Chinese medicine composition for improving or treating cerebral infarction and cerebral hemorrhage sequelae
CN101979084B (en) Chinese medicinal composition for warming kidney and tonifying yang and preparation method thereof
CN1453029A (en) Chinese medicine composition for treating senile dementia

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20171020

WD01 Invention patent application deemed withdrawn after publication