CN104523676A - Application of genipin in prevention or treatment of ischemic brain injury - Google Patents

Abstract

The invention relates to application of genipin in prevention or treatment of ischemic brain injury. Brain injury is particularly related to injury of microglial cells or human umbilical vein endothelial cells. The invention further relates to a medicine composition containing genipin for preventing or treating ischemic brain injury.

Description

Genipin is for preventing or treat the purposes of ischemic brain injury

Technical field

The pharmaceutical composition that the present invention relates to genipin and contain it is preparing the purposes prevented and/or treated in the medicine of ischemic brain injury.

Background technology

Cerebrovascular causes one of human death's three large diseases, wherein ischemic cerebrovascular (i.e. ischemic brain injury) is clinical common critical illness, it refers to degeneration that local brain tissue comprises the cell such as neurocyte, glial cell and occur due to blood supply disorder, necrosis or transient afunction, account for about 80% of cerebrovascular, become serious threat human life and one of healthy principal disease with its high incidence, high mortality, high disability rate, high relapse rate.Ischemic brain injury is very harmful to patient health, often causes irreversible brain to lose.Because of the interruption that cerebral arterial blood flow and oxygen are supplied, remarkable infringement brain physiologically active, thus result in the pathophysiological process of series of complex, comprise Energy Metabolism of Brain Tissue disorder, free radical loss, toxicity of excitatory amino acid, apoptosis, inflammatory reaction etc.Ischemic brain injury can cause apoplexy, cerebral infarction, cerebral palsy etc.

The mechanism of cerebral ischemia is a complicated pathophysiological process, cerebral blood flow interrupts causing local brain tissue hypoxic-ischemic, Energy Metabolism of Brain Tissue obstacle, there is again the Reperfu-sion of blood flow to cerebral tissue simultaneously, these can make brain tissue cell produce Cascade of Injury, at least relate to several different mechanism, and they cause by the Reperfu-sion of hypoxic-ischemic and blood flow, occur in different time points, overlap each other and connect each other.

Control cerebral ischemia becomes one of key for the treatment of cerebral ischemia brain injury, has the following aspects: 1. protect blood vessel endothelium, alleviate cerebral edema the machine-processed main manifestations of cerebral ischemia therapeutical effect; Regulate vasomotoricity, improve brain microcirculation; Improve blood flow rheology, improve cerebral circulation, increase cerebral blood flow, alleviate cerebral ischemia; 2. the morphology loss of neurocyte is alleviated, neuroprotective cell; 3. brain cell energy metabolism is improved; 4. antioxidation; 5. antagonism neurotoxicity; 6. reduce inflammation reaction; 7. promote differentiation and proliferation of neural stem cells, suppress neuronal apoptosis; 8. angiogenesis is promoted; 9. neurotransmitter or neuropeptide disorder etc. is regulated.

Microglia (microglia) is the one of neurogliocyte, is equivalent to the macrophage in brain and spinal cord, is that the first in central nervous system (CNS) is also topmost immune defence line together.Microglia accounts for greatly 20% of the neurogliocyte in brain.Microglia ceaselessly removes the nerve, speckle and the infectious substance that damage in central nervous system.After brain tissue ischemia, microglia is activated, and produces a large amount of cytokines and chemokine.Cytokine makes cerebrovascular endothelial cell expression of adhesion molecule raise, in circulation, leukocyte is under the effect of adhesion molecule and chemotactic factor, stick and migrate to the brain essence of damage, the cytokine such as a large amount of il-1s (IL-1) and tumor necrosis factor-alpha (TNF-α) is discharged together with glial cell, thus reinforcement leukocyte infiltration, simultaneously the inflammatory mediator such as arachidonic acid metabolite rolls up, increase the weight of local damage, finally cause neuronal apoptosis, necrosis.

Vascular endothelial cell is often referred to the simple squamous epithelium being lining in the heart, blood vessel and intralymphatic surface, also claim endotheliocyte, have and engulf foreign body, antibacterial, necrosis and old and feeble tissue, participate in the effect of collective's Immunization Activities, as vasoconstriction and vasodilation, thus control blood pressure; Blood coagulation (thrombosis and fibrinolysis); Arteriosclerosis; Angiogenesis; Inflammation and swelling etc.Wherein ICAM-1 (ICAM-1) is the critical mediator strengthening adhesive attraction between leukocyte and endotheliocyte, expresses the strongest at vascular endothelial cell.During cerebral ischemia, ICAM-1 obviously raises, and suppresses its unconventionality expression, can adhesion process effectively between blocking leukocyte and vascular endothelial cell, thus alleviates brain tissue impairment degree.Nitric oxide (NO) is messenger molecule important in body and effector molecule; for the endogenous mediator of neurotransmission, vasodilation, function of nervous system; with neural physiology and pathological process in close relations; the nitricoxide synthase eNOS secreted by blood vessel endothelium and the NO synthesized; expansion of cerebral vascular is had in local; increase cerebral blood flow, protective effect is played to cerebral tissue.

International curing apoplexy preclinical study guide (STAIR) is pointed out, in brain ischemia medicament research, multiple indexes overall merit is very important, histology and ethological result all should be evaluated, except measurement Infarction volume, the result of function score is also very important, and it is a crucial pharmacodynamics index in anti-cerebral ischemia drugs research.

Genipin and glucosides thereof have the various biological characteristics such as hepatic cholagogic, anti-inflammatory and antalgic, antitumor, blood sugar lowering, anti-senile dementia, suppression neurotoxicity, antidepressant, antithrombotic.But the effect of genipin to ischemic brain injury so far there is no report.By specific experiment, the present inventor finds, genipin is in protection microglia and Human umbilical vein endothelial cells and then improve in ischemic brain injury and have outstanding effect, for the treatment of ischemic brain injury and relevant disease thereof provides new selection.

Summary of the invention

The present inventor found through experiments, genipin can resist the loss that lipoprotein causes mice microglia (BV2 cell), increase the survival of BV2 cell, regulate tumor necrosis factor-alpha (TNF-α), IL-1β (IL1-β) active; The loss that opposing hydrogen peroxide causes human umbilical vein epithelial cell (HUVEC), increase the survival of HUVEC cell, regulate nitric oxide (NO), eNOS (eNOS), ICAM-1 (ICAM1) active; And observed by further experiment, find that genipin can improve the function of nervous system of permanent cerebral ischemia rat, reduce cerebral infarction rate.

Therefore, one object of the present invention is preparing for providing genipin the purposes prevented and/or treated in the medicine of ischemic brain injury.Wherein, described ischemic brain injury can be cerebral infarction/cerebral infarction, ischemic cerebrovascular, ischemic cerebral thrombosis, cerebral infarction, cerebral ischemia attack etc.

The present invention further provides genipin and prepare the purposes prevented and/or treated in the medicine of the disease relevant to microglia or damage of human umbilical vein endothelial.The wherein said disease relevant to microglia or damage of human umbilical vein endothelial can be Alzheimer, epilepsy, parkinsonism, cerebral ischemia etc.

Another aspect of the present invention provides a kind of pharmaceutical composition for preventing and/or treating ischemic brain injury, and it contains genipin as active component and pharmaceutically acceptable carrier.

The present invention further provides a kind of pharmaceutical composition for preventing and/or treating with microglia or damage of human umbilical vein endothelial relevant disease, it contains genipin as active component and pharmaceutically acceptable carrier.

Those skilled in the art can understand, pharmaceutical composition of the present invention can be formulated into various dosage form well known in the art according to concrete method of application, such as peroral dosage form (powder, tablet, capsule, soft capsule, liquid medicine, syrup, wine made of broomcorn millet ball, powder, wafer, granule), or topical formulation (emulsifiable paste, ointment, lotion, gel, face cream, plaster, paste, spray, aerosol etc.), or ejection preparation (solution, suspending agent, Emulsion).

Pharmaceutically acceptable carrier, adjuvant or diluent can be comprised according to pharmaceutical composition of the present invention, such as, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythroglucin, maltose alcohol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicates, cellulose, methylcellulose, polyvinylpyrrolidone, water, hydroxymethyl-benzoic acid, hydroxypropyl benzoic acid, Pulvis Talci, magnesium stearate and mineral oil.These preparations can comprise filler, anticoagulant, lubricant, wetting agent, flavoring agent, emulsifying agent, antiseptic etc. in addition.Compositions of the present invention can be made by utilizing any known method in this area, can provide active component that is quick, lasting or slow releasing after making patient's medication.

Such as, compositions of the present invention can be dissolved in oil, propyleneglycoles or other be generally used for preparing in the solvent of injection, the Examples of carriers be applicable to comprises normal saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate etc., but is not limited to them.

Genipin can adopt the form of its pharmaceutically acceptable salt in described pharmaceutical dosage form, and it can be used alone, also can with other pharmacy activity component conbined usage.

The dosage of inventive compound genipin can be different from the difference of the order of severity of the situation of individuality and weight, the state of an illness, medicament forms, route of administration and dosage period, and it also can be selected by those skilled in the art.But in order to obtain the effect of needs, suggestion effective dose every day is 0.2-5.0mg/kg, more preferably 0.03-3.00mg/kg usually.Dosage can be single-dose or the every day of administration several times every day.

Pharmaceutical composition of the present invention is administered to individual animals by all means as mammal (rat, mice, domesticate animals or the mankind), all administering modes are all expections, such as, administration can be oral, rectally or in vein, intramuscular, subcutaneous, Intradermal, sheath, epidural or intracerebral ventricle injection.

Illustrate the present invention further below with reference to the drawings and specific embodiments, but should to understand it be only illustrational effect, and limit the scope of the invention never in any form.

Accompanying drawing explanation

Fig. 1 show inverted phase contrast microscope observe metamorphosis after LPS induction of BV2 cell and genipin it is intervened after metamorphosis, it is Figure 1A respectively: blank group, Figure 1B: LPS intervention group, Fig. 1 C-Fig. 1 F: genipin 2.75mM group, 5.50mM group, 11.00mM group, 22.00mM group.

Fig. 2 shows inverted phase contrast microscope and observes HUVEC cell through H ₂o ₂metamorphosis after induction and genipin, to the metamorphosis after its intervention, are Fig. 2 A: blank group respectively, Fig. 2 B:H ₂o ₂intervention group, Fig. 2 C-Fig. 2 F: genipin 2.75mM group, 5.50mM group, 11.00mM group, 22.00mM group.

Fig. 3 shows various dose genipin to the Neuroscore after Cerebral Ischemia.

Fig. 4 shows various dose genipin to MCAO operation 12h cerebral Ischemia rat brain slice cerebral ischemia area TTC coloration result.

Fig. 5 shows various dose genipin to the cerebral infarction rate after Cerebral Ischemia.

Detailed description of the invention

Experimental example 1 genipin causes the protective effect of BV2 cell injury to lipopolysaccharide (LPS)

1. material

1.1 cell derived

BV2 cell is purchased from consonance cellular resources center, Beijing.

1.2 medicines and reagent

Genipin is purchased from Guangxi Shanyun Biochemistry Technology Co., Ltd.; DMEM culture medium, glutamine, dual anti-all purchased from GIBCO company; CCK-8 test kit is rich biological purchased from upper sea cowry; Tumor necrosis factor-alpha, IL-1β ELISA kit all purchased from Cloud clone corp, the U.S..

1.3 test apparatus

Microplate reader (the vigorous MK3 type of Finland's thunder, Finland), inverted microscope (Olympus IX2-SLP, Olympus, Japan), hypervelocity refrigerated centrifuge (Thermo, the U.S.), CO2 gas incubator (STIK, the U.S.), superclean bench (HFsafe-1500 Biohazard Safety Equipment, Haier, China), digital camera (Lycra DC300 type, Germany), ultraviolet spectrophotometer (Japanese Shimadzu UV-2450).

2 methods

The preparation of 2.1 culture medium

By volume, 90%DMEM culture medium, 10% hyclone (gibco, the U.S.), simultaneously also containing 0.1% glutamine and penicillin and streptomycin (Thermo, the U.S.) each 100,000 u/L.

The cultivation of 2.2BV2 cell

The method that BV2 cell cultivates collection warehousing according to Beijing consonance cellular resources center mode is cultivated, and namely cell and culture medium are placed in 37 DEG C, 5%CO ₂hatch in incubator, within 2 days, change liquid, within 2-3 days, go down to posterity.

2.3 grouping and administrations

Blank group is divided into (namely not have LPS to intervene, there is no pharmaceutical intervention yet), LPS intervention group (be only LPS intervene), (genipin 10mg medicine is dissolved in 1mL tri-distilled water and is made into 44mM storing solution genipin administration group,-20 DEG C frozen, take out after melting during experiment, variable concentrations is mixed with, i.e. 22mM, 11mM, 5.5mM, 2.75mM) with PBS doubling dilution.Choose the BV2 cell being in exponential phase, the medical preconditioning cell 4h of administration group variable concentrations, then LPS intervention group and administration group are all by LPS 1 μ g/mL process cell, be placed in after cultivating 24h in incubator, carry out CCK-8 detection, the ELISA of morphological observation and TNF-a and IL1-β detects.

2.4CCK-8 detect

Get the BV2 cell after intervening 24h, after using pancreatin/EDTA Digestive system (Trypsin/EDTA Solution, ScienCell, the U.S.) peptic cell of 0.25%, by culture medium, concentration of cell suspension is adjusted to 2.5 × 10 ⁴individual/ml, is inoculated in 96 orifice plates, every hole 100ul.Be placed in CO ₂preculture in incubator, stablizes and spends the night (>5h), add 10ul CCK-8 solution therewith, at CO to every hole ₂hatch 2h in incubator, microplate reader (450nm) reads OD value.

2.5TNF-α and IL1-'beta ' activity measure

The mensuration of TNF-α and IL1-'beta ' activity is carried out according to corresponding ELISA kit description.

2.6 statistical method

Experimental data with mean+SD ( ) represent, adopt SPSS19.0 software to do statistical analysis, the comparison between multiple sample average adopts one factor analysis of variance, and relatively row SNK method inspection between two between group, P<0.05 is that difference has statistical significance.

3 results

3.1 variable concentrations genipin are to the morphologic observation of the BV2 impact cell of LPS process

Fig. 1 show inverted phase contrast microscope observe BV2 cell through LPS induction after metamorphosis and genipin on its intervene after metamorphosis impact.After the BV2 cell of four concentration genipin and LPS process is hatched altogether as can be seen from this figure, BV2 cell viability increases.

3.2 variable concentrations genipin are to the Activity determination of the BV2 impact cell of LPS process

The BV2 cytoactive impact of genipin on LPS process is shown in following table 1.As shown in Table 1, after the genipin of four concentration and the impaired BV2 cell of LPS process are hatched altogether, the survival rate of BV2 cell significantly increases.

Table 1 variable concentrations genipin is on the impact of the BV2 cytoactive of LPS process

Note: compare with blank group, ^*p<0.05, ^*p<0.01; Compare with LPS intervention group, ^Δp<0.05, ^{Δ Δ}p<0.01

3.3 variable concentrations genipin promotes on the BV2 cell of LPS process the impact that TNF-α and IL1-β discharges

Table 2 shows variable concentrations genipin and promotes on the BV2 cell of LPS process the impact that TNF-α and IL1-β discharges.As can be seen from Table 2, BV2 cell is after LPS process, and TNF-α, IL1-β release significantly raises (P<0.05); Compared with LPS intervention group, genipin 2.75mM group TNF-α significantly lowers (P<0.01), and genipin 5.50mM group, 11.00mM, 22.00mM group TNF-α, IL1-β all significantly lower (P<0.01).

Table 2 variable concentrations genipin promotes on the BV2 cell of LPS process the impact that TNF-α and IL1-β discharges

Note: compare with blank group, ^*p<0.05, ^*p<0.01; Compare with LPS intervention group, ^Δp<0.05, ^{Δ Δ}p<0.01

In summary, through the BV2 cell of LPS process, when giving after variable concentrations genipin intervenes, the survival rate of BV2 cell increases, and the release of TNF-α, IL1-β reduces.Genipin has been pointed out to have the effect protecting impaired cerebrovascular function cell BV2 cell.

Experimental example 2 genipin is to H ₂o ₂cause the protective effect that Human umbilical vein endothelial cells (HUVEC) is damaged

1. material

1.1 cell derived

HUVEC cell is purchased from consonance cellular resources center, Beijing.

1.2 medicines and reagent

Genipin is purchased from Guangxi Shanyun Biochemistry Technology Co., Ltd.; DMEM culture medium, glutamine, dual anti-all purchased from GIBCO company; CCK-8 test kit is rich biological purchased from upper sea cowry; Nitric oxide (NO) test kit builds up Bioengineering Research Institute purchased from Nanjing; ENOS (eNOS) test kit, intercellular adhesion molecular1 (ICAM-1) test kit all purchased from Cloudclone corp, the U.S..

1.3 test apparatus

Microplate reader (the vigorous MK3 type of Finland's thunder, Finland), inverted microscope (Olympus IX2-SLP, Olympus, Japan), hypervelocity refrigerated centrifuge (Thermo, the U.S.), CO2 gas incubator (STIK, the U.S.), superclean bench (HFsafe-1500 Biohazard Safety Equipment, Haier, China), digital camera (Lycra DC300 type, Germany), ultraviolet spectrophotometer (Japanese Shimadzu UV-2450).

2 methods

The preparation of 2.1 culture medium

By volume, 90%DMEM culture medium, 10% hyclone (gibco, the U.S.), simultaneously also containing 0.1% glutamine and penicillin and streptomycin (Thermo, the U.S.) each 100,000 u/L.

The cultivation of 2.2HUVEC cell

Human umbilical vein endothelial cells (HUVEC) at 37 DEG C, 95% air/5%CO ₂condition under cultivate.When cell grows to 90% degrees of fusion, cell is through 0.125% pancreatin/EDTA Digestive system (Trypsin/EDTA Solution, ScienCell, the U.S.) digest 3 minutes, add the low sugar DMEM containing 10% hyclone, the centrifugal (1600 × g of Digestive system hypervelocity refrigerated centrifuge, 5 minutes), discard culture medium, cell blows even with culture medium, with 1 × 10 ⁴the cell density of/mL is seeded to 96 orifice plates.Continue to cultivate 12h, administration.

2.3 grouping and administrations

Be divided into blank group (namely do not have LPS to intervene, also do not have pharmaceutical intervention), H ₂o ₂intervention group (is only H ₂o ₂intervene), (genipin 10mg medicine is dissolved in 1mL tri-distilled water and is made into 44mM storing solution genipin administration group,-20 DEG C frozen, take out during experiment after melting, be mixed with variable concentrations with PBS doubling dilution, i.e. 22mM, 11mM, 5.5mM, 2.75mM).Choose the HUVEC cell being in exponential phase, the medical preconditioning 24h of administration group variable concentrations, then H ₂o ₂intervention group and administration group are all by H ₂o ₂400uM process cell, is placed in after cultivating 12h in incubator, carries out CCK-8 detection, get cell conditioned medium and carry out NO detection after centrifugal, and the ELISA carrying out e-NOS and ICAM-1 detects.

2.4CCK-8 detect

To the HUVEC cell after 12h be intervened, after using pancreatin/EDTA Digestive system (Trypsin/EDTA Solution, ScienCell, the U.S.) peptic cell of 0.25%, by culture medium, concentration of cell suspension is adjusted to 2.5 × 10 ⁴individual/ml, is inoculated in 96 orifice plates, every hole 100ul.Be placed in CO ₂preculture in incubator, stablizes and spends the night (>5h), add 10ul CCK-8 solution therewith, at CO to every hole ₂hatch 2h in incubator, microplate reader (450nm) reads OD value.

2.5NO, e-NOS and ICAM-1 activity determination method

NO, e-NOS and ICAM-1 determination of activity is all carried out according to the operating instruction of corresponding reagent box.

2.6 statistical method

Experimental data with mean+SD ( ) represent, adopt SPSS19.0 software to do statistical analysis, the comparison between multiple sample average adopts one factor analysis of variance, and relatively row SNK method inspection between two between group, P<0.05 is that difference has statistical significance.

3 results

3.1 variable concentrations genipin are to H ₂o ₂the morphologic observation of the HUVEC impact cell of process

Fig. 2 shows inverted phase contrast microscope and observes HUVEC cell through H ₂o ₂metamorphosis after induction and genipin are on the metamorphosis impact after its intervention.As can be seen from this figure, four concentration genipin and H ₂o ₂after the HUVEC cell processed is hatched altogether, HUVEC cell viability increases.

3.2 variable concentrations genipin are to H ₂o ₂the Activity determination of the HUVEC impact cell of process

Genipin is to H ₂o ₂the impact of the HUVEC cytoactive of process is shown in following table 3.As shown in Table 3, four concentration genipin and H ₂o ₂after the HUVEC cell processed is hatched altogether, the survival rate of HUVEC cell increases.

Table 3 variable concentrations genipin is to H ₂o ₂the impact of the HUVEC cytoactive of process

Note: compare with blank group, ^*p<0.05, ^*p<0.01; With H ₂o ₂intervention group compares, ^Δp<0.05, ^{Δ Δ}p<0.01

3.3 variable concentrations genipin are to H ₂o ₂the impact of the release of HUVEC cell NO, e-NOS and ICAM-1 of process

Table 4 shows variable concentrations genipin to H ₂o ₂the impact of the release of HUVEC cell NO, e-NOS and ICAM-1 of process.As can be seen from Table 4, H ₂o ₂nO, eNOS discharge minimizing, ICAM-1 expresses and significantly raises (P<0.01) to stimulate HUVEC cell to cause; With H ₂o ₂intervention group is compared, and genipin 2.75mM group ICAM-1 reduces (P<0.01); Genipin 5.50mM group eNOS raises (P<0.05), and ICAM-1 lowers (P<0.01); Genipin 11.00mM, 22.00mM group NO, eNOS raise (P<0.05), and ICAM-1 lowers (P<0.01).

Table 4 variable concentrations genipin is to H ₂o ₂the impact of the release of HUVEC cell NO, e-NOS and ICAM-1 of process

Note: compare with blank group, ^*p<0.05, ^*p<0.01; With H ₂o ₂intervention group compares, ^Δp<0.05, ^{Δ Δ}p<0.01

In summary, through H ₂o ₂the HUVEC cell of process, when giving after variable concentrations genipin intervenes, the survival rate of HUVEC cell increases, and NO, e-NOS discharges increase, and the release minimizing of ICAM-1, has pointed out genipin to have the effect protecting impaired cerebrovascular function cell HUVEC.

Experimental example 3 genipin anti-cerebral ischemia experimentation

Adopting muroid to make cerebral ischemic model is the means that anti-cerebral ischemia research is commonly used the most, common model has global brain ischemia, permanent Focal Cerebral Ischemia and focal cerebral ischemia reperfusion, they respectively have feature, simulate the pathogenesis of acute cerebral ischemic stroke from different perspectives.The present invention has used rat permanent cerebral ischemia model (pMCAO) to evaluate the curative effect of genipin anti-cerebral ischemia drugs,

1. material

1.1 laboratory animals and source

Healthy male Sprague-Dawley (SD) pathogen-free domestic (SPF level) rat, 7 months ages of Mus, body weight 250-270g, purchased from Laboratory Animal Science portion of Department Of Medicine, Peking University (quality certification number: SCXK (capital) 2011-0012), raise in Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences's Experimental Animal Center.

1.2 medicines and reagent

Genipin is purchased from Guangxi Shanyun Biochemistry Technology Co., Ltd., Semen Ginkgo extrac EGB761 is purchased from Tianjin Taiyang Pharmaceutical Co., Ltd. (lot number: H20130206), TTC (chlorination 2,3,5-triphenyltetrazolium chloride) is purchased from the green symphysis Technology Co., Ltd. in Nanjing product.

2 methods

2.1 animal grouping and administrations

Rat is placed in SPF environment (Specific pathogen free) i.e. barrier system (barriersystem) and raises.In experimentation, animal freely ingests and drinks water, room temperature 20 DEG C-25 DEG C, and relative humidity is 60%-70%, and periodicity of illumination is (7:00-19:00 illumination in 12 hours; 19:00-7:00 is dark).Suitable the supporting of SD rat is divided into sham operated rats in 3 days afterwards at random, cerebral ischemic model group (model group), 50mg/kg Semen Ginkgo extrac treatment group (positive drug group), 0.2mg/kg genipin treatment group (genipin small dose group), 1mg/kg genipin treatment group (in genipin dosage group), 5mg/kg genipin treatment group (the heavy dose of group of genipin), often organizes 10.Experiment was started the 4th day morning.Each group before anesthesia 1h gavage once, and to be again administered once after Post operation 6h, and after modeling 12h, get brain, section, dyeing.Sham operated rats, model group distilled water gavage (8ml/kg), Folium Ginkgo treatment group and each treatment group of genipin give the medicine gavage of equal volume.

The foundation of 2.2 cerebral ischemic models

Rat weight, 10% chloral hydrate (0.35ml/kg) intraperitoneal injection of anesthesia, is fixed on dissecting operation table by its dorsal position, with left side middle cerebral artery for operation is surveyed, cervical region preserved skin, iodophor disinfection.Median incision before row cervical region, cuts off skin, is separated subcutaneous tissue, exposes bubbling gland and pushes it against both sides, blunt separation tissue, exposes common carotid sheath.Blunt separation common carotid artery (common carotid artery; CCA), internal carotid artery (internal carotidartery; ICA), external carotid artery (external carotid artery; ECA) and the Main Branches tremulous pulse, occipital artery, superior thyroid artery etc. of ECA, protection peripheral nerve is as injury-free in vagus nerve.The Main Branches tremulous pulse of first ligation ECA, ligation CCA proximal part, puts a silk thread for subsequent use at distal end and slightly does ligation, give over to static line bolt use, put a silk thread for subsequent use at ICA proximal part, gives over to hemostasis and uses.CCA cuts a v-notch between two ligatures, the line bolt got ready is inserted in CCA, slightly tighten up distal end ligature, crotch line bolt being crossed ECA and ICA enters ICA tube chamber, slow feeding intracranial, arrive the initial part (mean depth of insertion for inside and outside neck aortic bifurcation place 18 ± 0.5mm) of middle cerebral artery (middle cerebral artery, MCA) when running into light resistance, tighten up and ligature on ligation CCA.Confirm that wound is without oozing of blood, layer-by-layer suture muscle and skin, iodophor disinfection operative incision, line bolt ends exposed outside skin, and by its labelling.The nursing of Postoperative insulation and otch, operated cohort is single cage raising after reviving, and keeps mouse cage dry cleansing, supply liquid, close observation.

Sham operated rats model is prepared the same, but inserts line bolt only about 10mm through ECA sidewall, and namely line bolt does not insert ICA intracranial segment.

2.3MACO model inclusion criteria

With reference to Bederson scoring, after rat operation anesthesia is clear-headed, carry out four Performance Level scorings:

0 point: impassivity injury symptoms;

1 point: outstanding tail test can not full extension offside fore paw;

2 points: forelimb opposing offside thrust capacity declines;

3 points: turn-take to offside.

Scoring enters group the 2-3 person of dividing.

The exclusion standard of 2.4MACO model

According to Bederson point system, Neuroscore is lower than 2 points of persons;

Find when getting brain and the person that finds subarachnoid hemorrhage;

The TTC dyeing person that has no ischemic focus.

Because above-mentioned factor causes each test group of animals number deficiency person, polishing number of animals also modeling again at any time.

3 statistical method

Experimental data with mean+SD ( ) represent, adopt SPSS19.0 software to do statistical analysis, the comparison between multiple sample average adopts one factor analysis of variance, and relatively row SNK method inspection between two between group, P<0.05 is that difference has statistical significance.

4 results

4.1 neurological deficits score

After last administration, 12h adopts blind to carry out Neuroscore, and carry out Bederson scoring, Bederson is divided into four Performance Levels, 0 point: impassivity injury symptoms; 1 point: outstanding tail test can not full extension offside fore paw; 2 points: forelimb opposing offside thrust capacity declines; 3 points: turn-take to offside.

The evaluation of 4.2 rat cerebral infarction volumes

Application TTC dyeing is evaluated cerebral infarction volume.

After MCAO rat ischemia 12h, get brain with the excessive anesthesia of 10% chloral hydrate, remove olfactory bulb, cerebellum and low brain stem, take out after freezing 20 minutes in-20 DEG C of refrigerators, from forebrain antinion, cut out the Coronal brain section that 5 thickness are about 2mm backward continuously.Brain sheet is put into the 2%TTC solution prepared in advance, 37 DEG C of lucifuges hatch about 15 minutes, and then brain sheet is placed in freshly prepared 4% paraformaldehyde solution and fixes 24h, observe and take pictures, normal cerebral tissue is cerise, do not dye and be pale asphyxia in necrotic area.

Analyze: application IPWIN 32 image processing software calculates the percentage ratio that Infarction volume accounts for homonymy brain volume.

Infarction rate=(Vc-Vl)/(Vc × 2) × 100%

Vc=d × ∑ Ac (Ac-normal side half brain sheet area)

Vl=d × ∑ Al (the red area of Al-Ipsilateral half brain sheet).

Table 5 shows various dose genipin to the Neuroscore after Cerebral Ischemia and cerebral infarction rate.

As shown in Table 5, with model group ratio, genipin small dose group (0.2mg/kg), in genipin, the Neuroscore of the heavy dose of group (5mg/kg) of dosage group (1mg/kg) and genipin and cerebral infarction volume all decline (P<0.05, see Fig. 3 ~ Fig. 5); Further, the Neuroscore of genipin heavy dose group is similar even lower to positive drug group (50mg/kg Semen Ginkgo extrac group) with cerebral infarction rate.

Table 5 various dose genipin is to the Neuroscore after Cerebral Ischemia and cerebral infarction rate

Note: compare with sham operated rats, ^*p<0.05, ^*p<0.01, ^{* *}p<0.001; Compare with model group, ^△p<0.05, ^{△ △}p<0.01, ^{△ △ △}p<0.001

Above result shows, gives permanent cerebral ischemia rat genipin, can improve the neurological deficit after cerebral ischemia, reduces cerebral infarction volume, realize the cerebral protection after ischemia, and its protective effect for cerebral ischemia becomes dose dependent to increase.

Embodiment 1: capsule

Genipin 50g is mixed homogeneously with 280g starch, with starch slurry (get starch 220g water and make starch slurry) granule processed, sieves, dry, encapsulated.

Embodiment 2: tablet 1

Genipin 80g is mixed homogeneously with starch 340g, with starch slurry (get starch 210g water and make starch slurry) granule processed, sieves, dry, adds 6% magnesium stearate, mixing, tabletted, film coating.

Embodiment 3: tablet 2

Genipin 100g, starch 100g and cellulose sieve in right amount, and fully mix, appropriate polyvinylpyrrolidonesolution solution is mixed with above-mentioned powder, sieve, obtain wet granular in 60 DEG C of dryings, by hydroxymethyl starch sodium salt, 6% magnesium stearate and Pulvis Talci sieve in advance, then join tabletting in above-mentioned granule.

Above by instantiation to invention has been detailed description, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.

Claims (7)

1. genipin is preparing the purposes prevented and/or treated in the medicine of ischemic brain injury.

2. purposes according to claim 1, wherein said ischemic brain injury comprises cerebral infarction/cerebral infarction, ischemic cerebrovascular, ischemic cerebral thrombosis, cerebral infarction, cerebral ischemia attack.

3. genipin is preparing the purposes prevented and/or treated in the medicine of the disease relevant to microglia or damage of human umbilical vein endothelial.

4. purposes according to claim 3, the wherein said disease relevant to microglia or damage of human umbilical vein endothelial comprises Alzheimer, epilepsy, parkinsonism, cerebral ischemia.

5. one kind for preventing and/or treating the pharmaceutical composition of ischemic brain injury, it contains genipin as active component and pharmaceutically acceptable carrier, and wherein said ischemic brain injury is cerebral infarction/cerebral infarction, ischemic cerebrovascular, ischemic cerebral thrombosis, cerebral infarction or cerebral ischemia attack particularly.

6. one kind for preventing and/or treating the pharmaceutical composition with microglia or damage of human umbilical vein endothelial relevant disease, it contains genipin as active component and pharmaceutically acceptable carrier, particularly Alzheimer, epilepsy, parkinsonism, the cerebral ischemia of the wherein said disease relevant to microglia or damage of human umbilical vein endothelial.

7. the pharmaceutical composition according to claim 5 or 6, it can be prepared to peroral dosage form as powder, tablet, capsule, granule, or ejection preparation is as solution, suspending agent, Emulsion, or topical formulation is as emulsifiable paste, ointment, spray, aerosol.