CN1695608A - Application of pinocembrin in preparing medication for treating disease relevant to hurt of nerve cell - Google Patents

Abstract

An application of pinocembrin in preparing the medicines for preventing and treating cerebral ischemia and its sequelae, and the diseases associated with the injury and function variation of neurocyte is disclosed.

Description

Give birth to the plain application in preparation control neural cell injury relevant disease medicine of pine

Technical field

The present invention relates to give birth to Song Suhe and contain the application in relevant disease medicines such as preparation prevention and treatment cerebral ischemia, cerebral ischemia sequela, neural cell injury and changing function of giving birth to the plain compositions of pine.

Background technology

Along with expanding economy, the raising of health care level, aged crowd significantly increases, and cerebrovascular disease such as apoplexy have become one of major disease of serious harm human health, and its sickness rate, disability rate and mortality rate are all very high; Patient more back quality of life relatively poor, take medicine throughout one's life, make troubles and heavier financial burden for patient's life.As: in 2000, U.S.'s patients with cerebral apoplexy reached 4,000,000 person-times, and annual medical total expenses reaches 4,000,000,000 dollars, on average 10,000 U.S. dollars for each person every year.

Brain is an organ extremely responsive to ischemia, the dysfunction that energy shortage causes in case ischemia promptly occurs.If the situation of ischemia fails to improve, then dysfunction continues to spread, and causes cerebral infarction, i.e. the cascade reaction of cerebral ischemia.Present brain injury protection medicine has calcium channel blocker, has been widely used in the treatment of ischemic cerebrovascular as nimodipine; Free radical scavenger (tocopherol, SOD); Neurotrophic factor (nerve growth factor, neurotrophic factor); Nmda antagonist; Ganglioside and excitatory amino acid receptor antagonists etc.

To the treatment of acute ischemic cerebral apoplexy, still lack medicine preferably both at home and abroad.The medicine of present clinical use can't effectively be corrected the symptom of cerebral ischemia, improve after the cerebral ischemia more after.U.S. FDA was ratified first and is treated this sick thrombolytic medicine t-PA in 1996, because the high untoward reaction of intracranial hemorrhage is arranged, and still disputable to its curative effect.Result of study to all kinds cerebral protective agent shows that a lot of clinical drug curative effects are good inadequately, or have than serious side effects.Several medicines are arranged still in the clinical verification stage.Pharmacy worker is always in the medicine effort that can fundamentally treat cardiovascular disease for searching.Therefore the protection medicine of apoplexy is effectively treated in exploitation, and market prospect is wide.

Give birth to loose element and belong to flavone compound, be widespread in nature, chemical structural formula is as follows:

Pharmacological evaluation in the past shows that it has stronger antibacterial, antiviral, antifungal activity.Give birth to the pine element as just being rich in the traditional health food Mel of China, often edible Mel does not only have obstruction to tooth; can also in the oral cavity, play the effect of sterilizing; can alleviate oral ulcer, and accelerating wound healing, but it does not see as yet that to the cerebral ischemia protection someone reports.

Summary of the invention

The invention provides the loose element of giving birth to shown in the formula (I) and protect the application in the medicine at the protection of preparation cerebral ischemia brain injury, treatment cerebral ischemia sequela, neural cell injury.

The present invention is by having carried out a series ofly experimental results show that it is the active drug of cerebral ischemia, cerebral ischemia sequela and neural cell injury protection to giving birth to loose element.

The rat cerebral ischemia experimentation proves, it is plain that pine is given birth in the injection of animal brain ischemia posterior vein, the dyskinesia that cerebral ischemia is drawn, ability of learning and memory goes down and behavioristics's change all improves significantly, improve rat behavior and learn index, the significant prolongation rat is in the time of staying of hang plate, helps promoting the recovery of the various symptoms that cause behind the animal brain ischemia.

The later on visible tangible ischemic necrosis of cerebral tissue of animal brain ischemia; ischemic tissue's volume and degree of ischemia have substantial connection; it is plain to give birth to pine behind the ischemia; the ischemic tissue of brain degree of injury is alleviated; the cerebral ischemia area dwindles, and giving birth to the plain Action Specification that obviously reduces cerebral infarct size of pine has significant protective effect to ischemic tissue of brain.

Experimental result proves that it is plain to give birth to pine behind the animal brain ischemia, can suppress ischemic tissue of brain LDH and discharge; suppress the active rising of Calpain in the ischemic tissue of brain; suppress c reactive protein level rising in the serum, therefore, giving birth to loose element is a valuable cerebral ischemia protection medicine.

Neuronal cell cultures result shows that it is plain for extrinsic factor such as H to give birth to pine ₂O ₂The neural cell injury that processing causes has significant protective effect; can improve cell survival rate; reducing the release of the lactic acid dehydrogenase that cell causes because of damage, can reduce the cell free calcium ion concentration, is the main mechanism of giving birth to the effect of the plain performance of pine neurocyte protection.

C-reactive protein (CRP) is a kind of acute phase protein.CRP is a kind of very ancient protein on system takes place, and in the evolution of long period of time, CRP is retained, and shows that it has important biological significance.It is generally acknowledged that CRP is a member in the body defending system.Detect CRP clinically and be mainly used in and diagnose the illness, estimate curative effect; It has multiple effects such as activating complement, adjusting immunity.Content is atomic in the normal serum, and in the inflammation acute stage, among the patients serums such as rheumatic fever, rheumatoid arthritis, malignant tumor, wound, CRP content can thousand times of increases.Because it is fast, responsive, objective that CRP reacts, and often CRP is applied to declaring of disease condition as an index clinically and estimates, and understands course of disease process and direction of medication usage.CRP increases the danger that can increase cerebral infarction ^[11]Result of study shows, CRP raise with apoplexy after release, cardiovascular complication, infection, the malignant tumor of inflammatory mediators relevant with deep venous thrombosis ^[12]The rising of operation group CRP, and due to the unprovoked operation wound, and derive from the reaction of infarction lesion itself, the CRP variable quantity is observed in this experiment simultaneously and neurological's symptom is proportionate, prompting CRP can be used as the early signal of the instantaneous damage of cerebral infarction, and promptly CRP raises and can be used as one of sign of MCAO model success ^[13], the result of this and wes' ischemia model group is very approaching, and discovers: pre-adaptation can be improved the CRP that ischemia causes and raise.CRP is by a large amount of acute reaction albumen that the excretory cytokine of activated macrophage stimulates and inducing hepatocyte produces, and obviously increases suppressor T cell and enhancing T miscellaneous function when inflammation or disorganization; Promote leukocyte motion and phagocytic function.It is by combining with lipoprotein, and producing in a large number eventually by classical pathway activating complement system, last albumen and complex cause the tunica intima damage.This may be that pre-adaptation has alleviated the inflammation initial reaction so caused the CRP level to reduce.This provides a frontier to the pre-adaptation Mechanism Study.We find simultaneously: giving birth to loose plain high dose has inhibitory action to c reactive protein level rising in the serum.This prompting: living loose element may react by inflammation-inhibiting brings into play the cerebral ischemia protective effect.

In recent years, the relevant effect of Calpain in cerebral ischemia becomes a new research focus.Calpain is a kind of activatory neutral protease of calcium ion that needs, and is distributed widely in the different tissues that comprises cerebral tissue ^[14]In the quiescent stage cell, the major part of Calpain exists with the zymogen forms of non-activity.Because the temporary transient or partial calcium summation of cell, fraction is activated, and certain target substrate molecule is produced limited proteolysis.The cerebral ischemia Calpain activity that can raise, and cause following damage cascade reaction: short ionizing glutamate receptor overactivity on (1) postsynaptic membrane makes Na ⁺And Ca ²⁺Continue to flow into cell; (2) Na ⁺The film depolarization is induced in inflow, opens by voltage-controlled Ca thereupon ²⁺Passage, and then increase Ca ²⁺Flow into; (3) Ca in the neuron ²⁺Continue to be increased to high concentration; (4) depend on Ca ²⁺System (comprise calmodulin, CaM, Protein kinase C, lecithinase C and A ₂, depend on kinases II, nitricoxide synthase and the Calpain of calmodulin, CaM by overactivity; (5) these processes some or all neuron has been produced irreversible damage finally causes cell death ^[15]Albuminolysis due to reports such as Bartus, Calpain activate is a potential pathological process of global brain ischemia, and the inductive albuminolysis of Calpain is very fast behind ischemic injuries promptly to be occurred, far away early than can detected infarction on the pathology.Because Calpain is a kind of calcium-activated protease, its activation degree and cytosolic free calcium level are proportionate.So infer: loose plain high dose reduction Calpain activity is handled, given birth to pre-adaptation may be relevant with reduction cytosolic free calcium level.Kitagawa etc. ^[16]Studies show that: behind rat or the mouse brain ischemia, Calpain is easy to be activated, and causes Proteolytic enzyme to increase, and this is likely one of reason of neuron rapid wear.

Nitric oxide under the physiological status (NO) plays many-sided important function as a kind of neurotransmitter or courier's material of reversibility disperse in central nervous system (CNS), for example blood vessel dilating, inhibition platelet and leukocyte recruitment.NO also is a kind of free radical and cytotoxic substance simultaneously, can pass through NO-iron complexes, sulfydryl protein oxidation formation superoxide anion generation neurotoxicity.Can improve antiotasis because loose element is given birth in our previous work prompting, and NO can vasodilator, improve the blood supply of cerebral tissue, so we has studied the plain influence to rats with cerebral ischemia homogenate NO level of living pine.NO role in the ischemic cerebral lesion is the emphasis of research always.Although still there is not clear conclusions so far, there is any to affirm, promptly the effect of NO is dual in the brain ^[17], its brain protection mechanism can realize by following approach: (1) regulates rCBF; (2) antiplatelet and leukocytic adhesion; (3) downward modulation nmda receptor; (4) the regulation and control mediator discharges; (5) to the feedback regulation of self NO synthase (NOS).Its neurotoxicity mechanism has: (1) mediation toxicity of excitatory amino acid; (2) suppress mitochondrial function; (3) free radical generates and suppresses by free radical the reuptake of excitatory amino acid; (4) generation of peroxynitrite; (5) suppress dna replication dna and reparation.For illustrating the effect of NO, in recent years, many scholars use different no inhibitor treatment ischemic cerebral lesion, but the result of gained is not quite similar, or conflicting ^[18], this may illustrate present we the rule that produces in the double action mechanism of NO and the ischemia process and change is also lacked enough understanding.This research finds that by homogenate behind the rat cerebral ischemia is detected the NO level cerebral ischemia can promote NO to discharge.This may be because intracellular calcium concentration increase behind the ischemia activates cNOS by calmodulin, CaM and causes NO generation increase.But in experiment, do not find the influence of pre-adaptation processing or medicine, give birth to plain other blood vessel dilating mechanism of passing through of pine probably, increase tissue perfusion, the damage of minimizing apoplexy NO.

Lactic acidosis is the common trait of ischemic tissue of brain.The hydrogen exchange body of sodium is activated when hanging down pH.Present evidence shows: the hydrogen exchange body inhibitor of sodium also has the better protect effect to cerebral ischemia: significantly reduce infarct size; improve degree of cerebral edema, suppress arachidonic release, motor function is strengthened; excitatory amino acid discharges and reduces, and the neuron loss number reduces ^[7-11]The hydrogen exchange body of sodium can be regulated the pH of glial cell, improves more remarkable to the pH under the pathologic condition.When anoxia, use the hydrogen exchange body inhibitor of sodium can improve the energy supply of glial cell ^[12]The hydrogen exchange body of sodium is higher at astrocyte film surface content ^[13-15]And when In vitro culture, astrocyte can continuous passage.Therefore select for use astrocyte to set up the high flux screening model of the hydrogen exchange body inhibitor of sodium as the donor of the hydrogen exchange body of sodium.

The feature of brain tolerance is that multiple special gene is expressed (referring to gene as c-fos, c-jun, zinc) in the inducing cell, grow out of nothing synthetic or increase relevant stress protein (as Fos albumen, heat shock protein HSP), normal protein matter synthetic then is suppressed or stops.(heat shockproteins is a kind of of stress protein HSP) to heat shock protein, and molecular weight is between 7～200KD.HSP has multiple again, for example HSP27, HSP47, HSP70, HSP90 etc.In most of biologies, HSP70 is abundant, the most conservative stress protein of content and molecular chaperones ^[19,20]In eukaryote, multiple HSP70 associated protein is arranged, wherein some is can constitutive expression under the normal growth condition, and is that normal growth is requisite, remaining then can only stress the time just can induce synthetic ^[21,22]Under the mammalian cell stress state, HSP70 mRNA is (heat shocktranscription factor, activation HSF) and changing because the heat shock conversion factor.Its mechanism is that stressors is induced formation HSF, and the heat shock combination of elements in HSF and the HSP70 gene promoter begins to change, and HSP70mRNA is converted to the synthetic HSP70 albumen of endochylema, and combines to keep protein from steady with denatured protein in endochylema ^[23]HSP70 enters karyon and combines with ribosomal precursor and other nuclear protein, prevents that degeneration from causing the disorder of cell life-information.Suppressed otherwise HSP70 albumen is synthetic, the change of the release of excitatory neurotransmitter and DNA self sequence finally causes the necrosis of neuronic tardy property in addition ^[24]Heat tolerance phenomenon has also clearly demonstrated the endogenous Neuroprotective Mechanisms of HSP.When injecting the HSP antibody of anti-70kD in the individual cells, cell loses the heat tolerance; When HSP gene expression was by competitive inhibition in the cell, cell lost the heat tolerance equally.

In the experimental cerebral ischemia, find that by immunocytochemistry these HSP mainly are distributed in neuron, some expression are also arranged at glial cell and endotheliocyte.Focal ischemia handles can cause the proteic expression of cortex Hippocampus CA1 district neuron HSP70, but its weak strength.Studies show that HSP70 albumen expresses in ischemia 5d, expression decreased so that disappear in the 7d-14d in ischemia cortex ^[25]In recent years studies show that almost detect in the normal brain activity less than HSP70 mRNA or HSP70, behind transient cerebral ischemia, the Denatured protein of damaged cell can be induced HSP70 gene expression, also obtain an of short duration opposing simultaneously damaging subsequently.This result of study shows, same rat own control finds in the ischemia side cerebral tissue that HSP70 expresses, do not see in the non-ischemia side cerebral tissue that HSP70 expresses, show that cerebral ischemia can induce HSP70 to express, it is the impaired index of reliable cerebral ischemia that HSP70 expresses, and this is consistent with relevant report.

Kitagawa etc. studies show that the synthetic of HSP70 is that the ischemia tolerance is essential, and the expression persistent period of HSP70 and the persistent period basically identical that tolerates effect ^[26-28]The proteic expression of HSP is consistent with the susceptibility of selective brain ischemia, at first at CA1, secondly at cortex and thalamus, afterwards in the stronger zones of resistance such as dentate nucleus granulocytes.The HSP that is mainly 70kD continues to raise in ischemia tolerance brain, and expression pattern is consistent with " dose response " and the time window of ischemia tolerance.Because it is especially responsive that Hippocampus CA1 district neuron stimulates ischemia, damage easily behind the ischemia, therefore more to the research of Hippocampus CA1 district neuron ischemia tolerance phenomenon ^[29]After pre-adaptation was handled, cerebral cortex and the proteic expression of Hippocampus CA1 district neuron HSP70 significantly increase, and be consistent with cerebral infarct size and behavioristics's index, and prompting HSP70 protein expression and the effect of its organization protection are closely related.

Experiment in vitro proves: HSP70 and HSP27 increase can protect cell ^[30]The possible mechanism of action: suppress proteic synthetic in the heat shock process, proteic amount not folding in the eukaryotic cell is reduced, thereby weaken the destruction of pair cell.The synthetic need of eukaryotic protein forms initial medicated cap complex eLF4F, and HSP27 can combine with the constituent eLF4G of eLF4F, stops the translation of mRNA, and pair cell shields ^[31]HSP27 can prevent the destruction of Actin, safeguards the stable of cytoskeleton, increases the toleration to heat ^[32]Stimulate RNA and proteinic synthetic after heat shock, helper cell was repaired the damage that heat shock causes before death, make cell recover normal physiological function as early as possible.We test discovery: Heat shock protein 27 HSP27 belt leather layer behind ischemia is expressed to some extent and is risen after the pretreatment.Report: Heat shock protein 27 HSP27 is expressed at astrocyte to be increased, and may be the reaction of glial cell to neuronal damage, helps contiguous neuronic survival ^[33]

Former study finds to use sympatheticomimetic thing amphetamine can induce HSP27, and HSP70 and HSP89 are expected to be used for inducing of pre-adaptation in the expression of heart, lung, liver, kidney and brain ^[34]The thrombin of intracerebral injection low dosage can be induced the great expression of HSP27, and the performance protective effect ^[35]Our result of study is found: give birth to the plain high dose of pine and can induce cerebral cortex and the proteic expression of Hippocampus CA1 district neuron HSP70 significantly to increase, HE coloration result showed cell form and number all are protected simultaneously; Point out living pine to have and to bring into play protective effect by inducing the HSP70 protein expression.Its mechanism may be to give birth to the plain ischemia cell HSP70 gene that quickens of pine in the expression of transcribing with translation skill, and HSP70 is synthetic to be increased thereby make, and makes neurocyte produce tolerance to ischemia, plays protecting neuron from acute.Give birth to mechanism that loose element induces endogenous protection mechanism to start and remain further research, will help understanding to the research of the temporal mode/spatial model of HSP70 protein expression simultaneously its protection mechanism.

Calcium ion is the cell internal information material that extensively exists in the body, effectively effluxes with the enclose inside system by one and keeps the stable of intracellular Ca2+ environment.Since the eighties, the research of the cell function of calcium causes people's common concern, and people recognize Ca gradually ²⁺Almost influence the physiological activity of cell each side.Ca ²⁺Trigger many important cellular activities as courier in the cell, as motion, division, pinocytosis, the exocytosis process of cell, the synthetic and decomposition of cyclic adenosine monophosphate and synthetic and release of neurotransmitter and some hormone or the like.

The function of cell depends on the inside and outside calcium ion concentration of cell and maintains a proper proportion.What experimental results show that calcium ion strides stream and intracellular calcium in the film unusually at the time distributes and the time transports the unusual final cell death that causes.Therefore the concentration that detects intracellular calcium not only helps understanding, the diagnosis of disease, the treatment of pathophysiological mechanism in the pair cell damage process, also for exploring the new way that effective medicine provides.Illustrate mechanism of drug action and link.The therapeutical effect that has now proved many medicines is realized by influencing intracellular Ca2+.

In sum, living loose element can improve cerebral ischemia and cause the neurocyte cells injury, and may pass through Calpain, NO, CRP and cross expression of heat shock in the startup brain, and performance is to ischemic tissue of brain, protecting neuron from acute.

Therefore the present invention also relates to and containing as the The compounds of this invention of active ingredient and the pharmaceutical composition of conventional medicine excipient or adjuvant.Usually pharmaceutical composition of the present invention contains the The compounds of this invention of 0.1-95 weight %.

The pharmaceutical composition of The compounds of this invention can be according to method preparation well known in the art.When being used for this purpose, if desired, The compounds of this invention and one or more solids or liquid medicine excipient and/or adjuvant can be combined, make and can be used as suitable administration form or the dosage form that people's medicine or veterinary drug use.

The compounds of this invention or contain its pharmaceutical composition can the unit dosage form administration, route of administration can be intestinal or non-intestinal, as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum etc.

The compounds of this invention or the route of administration that contains its pharmaceutical composition can be drug administration by injection.Injection comprises intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection therapy etc.

Form of administration can be liquid dosage form, solid dosage forms.As liquid dosage form can be true solution class, colloidal type, particulate formulations, emulsion dosage form, mixed suspension form.Other dosage forms are tablet, capsule, drop pill, aerosol, pill, powder, solution, suspensoid, Emulsion, granule, suppository, lyophilized injectable powder etc. for example.

The compounds of this invention can be made ordinary preparation, also can be slow releasing preparation, controlled release preparation, targeting preparation and various particulate delivery system.

For the unit form of administration is made tablet, can be extensive use of various carrier well known in the art.Example about carrier is, for example diluent and absorbent are as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, carbamide, calcium carbonate, kaolin, microcrystalline Cellulose, aluminium silicate etc.; Wetting agent and binding agent are as water, glycerol, Polyethylene Glycol, ethanol, propanol, starch slurry, dextrin, syrup, Mel, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethyl cellulose, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.; Disintegrating agent, for example dry starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.; Disintegrate inhibitor, for example sucrose, glyceryl tristearate, cocoa butter, hydrogenation wet goods; Absorption enhancer, for example quaternary ammonium salt, sodium lauryl sulphate etc.; Lubricant, for example Pulvis Talci, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, Polyethylene Glycol etc.Tablet further can also be made coated tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablet and multilayer tablet.

For example, can be extensive use of various carrier well known in the art for pill is made in the administration unit.Example about carrier is, for example diluent and absorbent are as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Kaolin, Pulvis Talci etc.; Binding agent is as arabic gum, Tragacanth, gelatin, ethanol, Mel, liquid sugar, rice paste or batter etc.; Disintegrating agent is as agar powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.

For example for capsule is made in the administration unit, the effective ingredient The compounds of this invention is mixed with above-mentioned various carriers, and the mixture that will obtain thus places hard gelatine capsule or soft capsule.Also the effective ingredient The compounds of this invention can be made microcapsule, be suspended in and form suspensoid in the aqueous medium, in the hard capsule of also can packing into or make injection and use.

For example, The compounds of this invention is made injection preparation, as solution, suspensoid solution, Emulsion, lyophilized injectable powder, this preparation can be moisture or non-water, can contain acceptable carrier, diluent, binding agent, lubricant, antiseptic, surfactant or dispersant on a kind of and/or multiple pharmacodynamics.Can be selected from water, ethanol, Polyethylene Glycol, 1 as diluent, the isooctadecanol of ammediol, ethoxylation, the isooctadecanol of polyoxyization, Polyoxyethylene Sorbitol Fatty Acid Esters etc.In addition, ooze injection, can in injection preparation, add proper amount of sodium chloride, glucose or glycerol, in addition, can also add conventional cosolvent, buffer agent, pH regulator agent etc. in order to prepare etc.These adjuvants are that this area is commonly used

In addition, as needs, also can in pharmaceutical preparation, add coloring agent, antiseptic, spice, correctives, sweeting agent or other material.

For reaching the medication purpose, strengthen therapeutic effect, medicine of the present invention or pharmaceutical composition can be with any known medication administrations.

The dosage of The compounds of this invention pharmaceutical composition depends on many factors, for example to prevent or treat the character and the order of severity of disease, the sex of patient or animal, age, body weight, personality and individual reaction, route of administration, administration number of times, therapeutic purposes, therefore therapeutic dose of the present invention can have large-scale variation.In general, the using dosage of Chinese materia medica composition of the present invention is well known to a person skilled in the art.Can be according to the actual drug quantity that is contained in the preparation last in the The compounds of this invention compositions, in addition suitable adjustment to reach the requirement of its treatment effective dose, is finished prevention of the present invention or therapeutic purposes.The suitable dose scope of the every day of The compounds of this invention is preferably the 0.1-100mg/kg body weight, more preferably 1-20mg/kg body weight, more preferably 0.1-100mg/ days/people.Above-mentioned dosage can the single dose form or be divided into several, for example two, three or four dosage form administrations this be subject to administration doctor's clinical experience and comprise the dosage regimen of using other treatment means.

Each treats that required accumulated dose can be divided into repeatedly or by the dose administration.Chemical compound of the present invention or compositions can be taken separately, or merge use and adjust dosage with the other treatment medicine.

Description of drawings

Fig. 1. the shock index of each group in the nervous symptoms scoring experiment.( ^{* *}P＜0.001vs. infraction group)

Fig. 2. each group time of staying on hang plate in the hang plate experiment.( ^{* *}P＜0.001, ^*P＜0.01vs. infraction group)

Fig. 3. give birth to the plain influence of pine to ischemia rat cerebral infarction area.

Fig. 4. it is plain to the active influence of LDH in the ischemia rat cerebral tissue to give birth to pine.( ^*P＜0.05vs. infraction group)

Fig. 5. give birth to pine plain to the active influence of Calpain in the ischemia rat cerebral tissue ( ^*P＜0.05vs. infraction group)

Fig. 6. give birth to the plain influence of pine to the CRP content in the ischemia rat blood serum.( ^*P＜0.05vs. infraction group)

Fig. 7. give birth to the plain inhibitory action of pine to smooth muscle cell NHE.

Fig. 8. give birth to the plain inhibitory action of pine to astrocyte NHE.

Fig. 9. ischemic region cortex HE (400 *) (A: false processed group that dyes; B: solvent control group; C: pretreated group; D: middle cerebral artery infraction group; E: give birth to loose plain low dose group; F: give birth to loose plain high dose group)

Figure 10. ischemic region Hippocampus CA1 district HE (200 *) (A: false processed group that dyes; B: solvent control group; C: pretreated group; D: middle cerebral artery infraction group; E: give birth to loose plain low dose group; F: give birth to loose plain high dose group)

Figure 11. give birth to expression (200 *) (A: false processed group of loose plain HSP70 at the ischemia cortex; B: solvent control group; C: pretreated group; D: middle cerebral artery infraction group; E: give birth to loose plain low dose group; F: give birth to loose plain high dose group)

Figure 12. give birth to expression (200 *) (A: false processed group of loose plain HSP70 in Hippocampus CA1 district; B: solvent control group; C: pretreated group; D: middle cerebral artery infraction group; E: give birth to loose plain low dose group; F: give birth to loose plain high dose group)

Figure 13. give birth to expression (200 *) (A: false processed group of loose plain HSP27 at the ischemia cortex; B: solvent control group; C: pretreated group; D: middle cerebral artery infraction group; E: give birth to loose plain low dose group; F: give birth to loose plain high dose group)

Figure 14, living pine element are to neuronal cell line PC12 cell H ₂O ₂Handle the influence of back cell survival.

Figure 15, living pine element are to neuronal cell line PC12 cell H ₂O ₂Handle the influence that the back discharges lactic acid dehydrogenase

Figure 16. give birth to pine plain to the PC12 neurocyte through H ₂O ₂Handle the influence of back endocellular liberation calcium concentration

The specific embodiment

Embodiment 1, the plain rat cerebral ischemia protective effect of living pine to rat medium-sized artery infraction

The foundation of model: adopt suture method to prepare the middle cerebral artery cerebral infarction models.The SD rat is along cervical region medisection, and separation right side neck is total, neck interior and external carotid artery., internal carotid artery place total in neck closes with the bulldog clamp folder, external carotid artery proximal part and distal end ligation, and cut off the centre.Be pulled to internal carotid artery the external carotid artery free-end in alignment, nylon wire is inserted by external carotid artery, insert back with No. 0 silk thread ligation, hemorrhage to prevent, open internal carotid artery place bulldog clamp, nylon wire is inserted internal carotid artery, continue to be inserted into intracranial, stop when slight resistance, insertion depth is about 2cm.

Grouping and administration: be divided into 5 groups, 10 every group, be respectively: 1) false processed group, 2) the solvent control group; 3) infraction group; 4) low dose group is given birth to loose plain 0.3mgkg ^-1Tail vein injection; 5) high dose group is given birth to loose plain 3mgkg ^-1Tail vein injection.After experiment finished, rat carried out nervous symptoms scoring and hang plate experiment; The animal broken end is got brain immediately subsequently, detects ischemic areas with the cardinal principle staining.

Neural behavior scoring method and standard are as follows, and mark is high more, illustrate that the behavior disorder of animal is serious more.

Method	Symptom	Classification	Mark
					1. put animal and put animal in smooth flat, push the side respectively with hands in its walking 2. of ground observation.3. carry afterbody and make about the liftoff chi of rat, normal rat forelimb symmetry is stretched downwards.1+2+3	Animal reduces to sideway swivel damage back offside muscular strength, easily topples over to offside.It is negative that medial rotation is received 1,2,3 tests in the offside forelimb of damage back	3 grades 2 grades 1 grade 0 grade	6310

In nervous symptoms scoring experiment, the nervous symptoms index of false processed group is 0, and the infraction group is 8.8, tangible neurological handicap occurs, gives birth to pine element 3,0.3mgkg ^-1Iv.Can significantly improve the nervous symptoms of postoperative, its shock index reduces to 2.40 ± 1.14, and 4.40 ± 1.14 (P＜0.001vs. infraction group) (Fig. 1).

In the hang plate experiment, rats with cerebral ischemia time of staying on hang plate is significantly shorter than the sham operated rats animal, and gives birth to pine element 3,0.3mgkg ^-1Iv can obviously prolong animal time of staying on hang plate, and its time of staying is respectively 236.00 ± 73.95,105.33 ± 66.83 (P＜0.001, P＜0.01vs. infraction group).Above presentation of results is given birth to the plain high low dose group of pine and can obviously be improved the behavioristics unusual (Fig. 2) that is caused by cerebral ischemia.

Behind the cerebral ischemia 6h, rat brain can be observed the right side ischemic region and large-area infraction necrosis occurs after TTC dyeing, presents white.The sham operated rats animal brain presents uniform rose, shows ischemic necrosis not occur.The infarct size that high dose group can make cerebral ischemia cause is dwindled (Fig. 3).

Embodiment 2, give birth to the influence of the plain rat cerebral even slurry biochemical indicator to rat medium-sized artery infraction of pine

Adopt suture method to prepare middle cerebral artery cerebral infarction models (the same), get the ischemic region cerebral tissue and make 1: 10 (W/V) homogenate with ice-cold normal saline ,-20 ℃ of packing are preserved, and carry out biochemistry detection.

Compare with sham operated rats, LDH is active in the ischemia rat cerebral tissue obviously increases.Give birth to loose plain low dose group and can significantly suppress to organize interior LDH activity (P＜0.05vs. infraction group), (Fig. 4).

Compare with sham operated rats, Calpain is active in the ischemia rat cerebral tissue obviously increases.Give birth to loose plain high dose group and can significantly suppress to organize interior Calpain activity (P＜0.05vs. infraction group), suppression ratio is 22.38% (Fig. 5).

Compare with sham operated rats, the CRP content in the ischemia rat blood serum significantly increases.Give birth to loose plain high dose group and significantly suppress CRP content in the serum (P＜0.05vs. infraction group) (Fig. 6).

Embodiment 3, the plain inhibitory action of living pine to smooth muscle cell sodium hydrogen exchange (NHE)

Under aseptic condition, get thoracic aorta, in Hank ' s liquid, wash blood stains, remove fatty tissue, adventitia and inner membrance, middle film is placed the small beaker that contains culture fluid, be cut into 1mm ²Organize fritter, be inoculated in the culture bottle, arrange by equidistant, add the culture medium that contains 10% hyclone, the jam-pack bottleneck makes piece of tissue be positioned at the culture bottle top, places 37 ℃, 5%CO ₂In the incubator, the culture bottle that overturns gently behind the 5h immerses in the culture fluid piece of tissue, leave standstill cultivation then, every 3d changes liquid once, generally grows from the piece of tissue edge at the 4-7d visible cell, grow fine and close cellular layer after 2 weeks, get the cell of cultivating 10-20 generation and be used for experimentation.

Use and give birth to the plain different concentration (10 of pine ^-8, 10 ^-7, 10 ^-6With 10 ^-5Mol/L) hatch with rat aorta smooth muscle cell, it is plain 10 wherein to give birth to pine ^-7Mol/L is the strongest to the inhibitory action of NHE, is 80.79%, (n=5).The result as shown in Figure 7.

Embodiment 4, the plain inhibitory action of living pine to astrocyte sodium hydrogen exchange (NHE)

Fluorescence indicator BCECF/AM can the interior hydrionic variation of reacting cells.BCECF/AM (no fluorescence) facilitates penetration of cell membrane, is become BCECF (fluorescence is arranged) by the esterase hydrolyzed in the born of the same parents.The fluorescence of BCECF is that pH relies on, and the different fluorescence intensities of pH value are also different.Under certain pH, F ₄₉₀/ F ₄₅₀Linear with pH ^[16-18]

Behind the cell loading BCECF/AM, by using NH ₄Cl stimulates acidify in the inducing cell, and uses high sodium buffer to induce pH to recover.Because no HCO in the cell ₃ ^-Ion is so the recovery of pH is only by the hydrogen exchange body mediation of sodium.Measure under the different condition change in fluorescence of BCECF in the cell.Because acting on of the hydrogen exchange body of sodium recovers to be in the 2min linear behind the cell acidify, so select for use following formula to calculate the hydrogen exchange body suppression ratio of sodium.

PH recovery rate=(R _Recover-R _Acidify)/R _Acidify* 100% (R=F ₄₉₀/ F ₄₅₀)

NHE suppression ratio=medicine pH recovery rate/normal control group pH recovery rate * 100%

Newborn 1d rat brachycephaly in super-clean bench is got brain, puts ice-cold D-hank ' s liquid (g/L KCl0.4 KH ₂PO ₄0.06 NaCl 8.0 NaHCO ₃0.35 Na ₂HPO ₄7H ₂O0.06 phenol red 0.02) in divide the bark fetching layer, shred digestion 15min, ice-cold complete medium stops digestion, crosses 200 order cells sieve, 1500rpm is centrifugal, plants in the culture bottle that is covered with 100 μ g/ml poly-D-lysines in advance 5%CO ₂, 37 ℃ of CO ₂Incubator is cultivated, and changes liquid once in per 3 days.In Day10 culture bottle is put into the isothermal vibration water-bath, 200rpm concussion 18hr is to remove neuron and microglia.Numerous heteroproteose cells that suspending in the culture medium this moment with the culture medium flush away, change with fresh complete medium and cultivate.The cell seeding of purification is gone into 24 holes or 96 well culture plates, cultivates 48hr, carries out GFAP cytochemistry and identifies and test processing.

Test the serum of removing in the astrocyte culture fluid in preceding 24 hours, after the PBS washing, change with serum-free medium.37 ℃ of preheatings of all reagent.In culture medium, add BCECF/AM1.5 μ g/ml 10 μ L, hatch 15min for 37 ℃.Continue to add 50mM NH ₄Cl stimulates 10min. not have Na ⁺(mM choline chloride 140, KCl 3, MgCl for HEPES ₂1, glucose 10, Hepes20) liquid washes the outer BCECF/AM with the removal born of the same parents, balance 5min, 450nm/490nm excites, and fluorescence is surveyed in the 530nm emission, calculating R=F490/450, the R value is high more, and pH value is high more.(mM NaCl 140, KCl 3, MgCl for high sodium HEPES liquid ₂1, glucose 10, Hepes 20) flushing is to induce the pH recovery of sodium ion dependence, mensuration fluorescence calculates R=F ₄₉₀/ F ₄₅₀With nigericin calibration pH value.

Use and give birth to the plain different concentration (10 of pine ^-8, 10 ^-7, 10 ^-6With 10 ^-5Mol/L) hatch with Rat Astroglia.Give birth to the plain inhibitory action of pine and have good dose-effect relationship, its IC NHE ₅₀Be 2.95 * 10 ^-10Mol/L is far below the IC of DMA ₅₀(n=5), be a more potential NHE inhibitor.As shown in Figure 8.

Embodiment 5, living pine element are to ischemia rat layer and the painted influence of Hippocampus CA1 district HE

Obvious damage is not seen in false processed group cortex HE dyeing, and cellularity is complete.Solvent control group and middle cerebral artery infraction group cortical cell are dispersed in, and intercellular substance enlarges, neuron swelling.Pretreated group and medicine high dose group all can significantly be improved the ischemic neuron damage.The result as shown in Figure 9.

False processed group Hippocampus CA1 district HE staining cell level is more, is evenly distributed,, arrange closely, it is clear even to dye, and cellular morphology is complete, no abnormal change.Solvent control group and middle cerebral artery infraction group cell disappearance are obvious, and cell is arranged loose, rarely seen indivedual neurocyte endochylema engrains, karyon pyknosis.The medicine high dose group all can significantly be improved the ischemic neuron damage, and neuron density increases, and it is more even to dye, and cellular morphology is more complete.Result such as Figure 10, table 1.

It is plain to cortex, the painted influence of Hippocampus granule after the cerebral ischemia that table .1. gives birth to pine.

Group	Cortex	Hippocampus
			False processed group solvent control group MCAO IP+MCAO 0.3mg/kg 3mg/kg	??104.67±8.50 **??63.00±11.27 ??55.00±6.00 ??110.33±11.50 **??60.00±7.00 ??107.67±13.32 **	??50.33±5.13 ***??9.33±3.51 ??7.33±2.52 ??42.00±4.36 ***??33.33±9.07 **??50.00±8.00 ***

^*P＜0.01, ^{* *}P＜0.001vs.MCAO group, and n=3. (meansigma methods ± S.D)

Embodiment 6, the plain influence of living pine to rats with cerebral ischemia cortex and Hippocampus CA1 district HSP70 expression

The false processed group of heat shock protein (HSP) 70 dyeing is not seen cortex dyeing.The expression that is positive of solvent control group, middle cerebral artery infraction group ischemia cortex marginal zone neuron; The dyeing of pretreated group cortex increases the endochylema engrain.Give birth to the loose plain high dose group positive cell number that also can raise and reaching color depth.As shown in figure 11

The HSP70 false processed group that dyes is not seen hippocampus dyeing.The solvent control group, the dyeing of the visible Hippocampus CA1 of the painted pretreated group of middle cerebral artery infraction group positive cell district increases, and brown yellow granule is more common in the endochylema of neuronic cyton and aixs cylinder.Giving birth to loose element can increase positive cell number, and tinctorial strength increases.As Figure 12, shown in the table 2

Table .2. gives birth to the plain influence to rats with cerebral ischemia cortex and Hippocampus CA1 district HSP70 expression of pine

Group	Cortex	Hippocampus
			False processed group solvent control group MCAO IP+MCAO 0.3mg/kg 3mg/kg	??------ ??0.72±0.05 ??0.66±0.07 ??0.84±0.09 *??0.50±0.10 ??0.82±0.07 *	??------- ??0.48±0.08 ??0.46±0.05 ??0.69±0.09 *??0.57±0.05 *??0.61±0.07 *

With the dummy of false processed group as average transmittance mensuration. ^*P＜0.05vs.MCAO group, and n=3. (meansigma methods ± S.D)

Embodiment 7, living pine element are expressed ischemia rat ischemia cortex, hippocampus HSP27

False processed group is not seen obvious dyeing at whole brain, and middle cerebral artery infraction group HSP27 dyeing strengthens, and pretreated group has dyeing more by force at neuron cell body, and cytochrome significantly increases.It is suitable with middle cerebral artery infraction group number to give birth to the plain high low dose group staining cell of pine.As Figure 13, shown in the table 3.

It is plain to ischemia rat ischemia cortex, hippocampus HSP27 expression that table .3. gives birth to pine.

Group	Cortex
		False processed group solvent control group MCAO IP+MCAO 0.3mg/kg 3mg/kg	??------ ??0.15±0.04 ??0.20±0.02 ??0.26±0.03 *??0.16±0.05 ??0.22±0.04

With the dummy of false processed group as average transmittance mensuration. ^*P＜0.05vs.MCAO group, and n=3. (meansigma methods ± S.D)

Embodiment 8, the plain protective effect that hydrogen peroxide is caused the PC12 neural cell injury of living pine

At 37 ℃, 5%CO ₂In the constant incubator, cultivate the PC12 cell with 1640 culture medium (containing 10% hyclone, penicillin 100U/ml, streptomycin 100ug/ml).After cell is paved into monolayer, absorb former culture medium, add the H of final concentration 200uM ₂O ₂100ul experimentizes behind the 24h.Adopt mtt assay to decide cell viability; Adopt LDH to measure LDH Determination on content in the kit measurement culture fluid.The result shows 10 ^-5Mol/L gives birth to pine element, 10 ^-6The living loose element of mol/L can obviously suppress the decline of the PC12 cell viability due to the hydrogen peroxide, 10 ^-5The living loose element of mol/L can reduce the release of the LDH due to the hydrogen peroxide, and hydrogen peroxide is caused the PC12 cell injury protective effect.The results are shown in Figure 14, Figure 15.

Embodiment 9, the plain influence of living pine to free calcium concentration in the neurocyte

Add PC12 cell suspension 90 μ l holes in 96 orifice plates, with 5 μ mol.L ^-1Fura-2/AM is at 37 ℃ of isothermal vibration 35min.After load finishes, respectively to contain 0.2% BSA Hanks, not contain Mg ²⁺0.2% BSA Hanks and D-Hanks liquid wash twice, transferring cell suspension is 2 * 10 ⁶Individual/ml, add 10 in advance ^-5After the living Song Suwen of mol/L is incubated 5 minutes, measure the quiescent condition intracellular Ca2+.The maximum excitation wavelength of Fura-2 is 380nm, with Ca ²⁺In conjunction with after the maximum excitation wavelength be 340nm, emission wavelength is 500nm.Carry out fluoroscopic examination with the FLUO-STAR exometer.Add Triton X-100 broken cell membrane, measure fluorescence behind the concussion mixing, be Ca this moment ²⁺Fluorescence intensity when saturated.Add EGTA complexation intracellular Ca2+, measure fluorescence behind the concussion mixing, be zero Ca this moment ²⁺The time fluorescence intensity.

Be calculated as follows Ca ²⁺Concentration:

[Ca ²⁺]＝KDa*(R-Rmin)/(Rmax-R)*(F _f2/F _b2)?????(R＝F ₃₄₀/F ₃₈₀)

Drug treating: cell loading adds 10 after finishing in advance ^-5After the living Song Suwen of mol/L is incubated 5 minutes, add stimulant KCl respectively, glutamic acid (Glu) and norepinephrine (NE) are measured change in fluorescence immediately.

Experimental result shows 10 ^-5It is [the Ca of D-Hanks that mol/L gives birth to the outer liquid of loose plain pair cell ²⁺] not influence of i, when extracellular fluid is Hanks liquid, 10 ^-5The living pine of mol/L have the effect that reduces the quiescent condition intracellular Ca2+, sees Table 1.10 ^-5Mol/L gives birth to that stream has inhibitory action in the plain calcium that KCL, NE are caused of pine, and Figure 16 is seen in not influence of stream in the calcium that glutamic acid is caused.

Table 4. is given birth to the plain influence to the quiescent condition intracellular Ca2+ of pine

Handle	???????????????????[Ca 2+]i(nmol/L)
				??Hanks	??Hanks(Mg 2+free)	??D-Hanks
	Contrast	??114.4±17.1	??158.3±18.7				??93.2±14.6 *
Give birth to pine plain 10 -5mol/L				??91.7±12.7	??132.8±11.1	??91.0±10.9 *

Claims (4)

1, the loose plain chemical compound of the life shown in general formula (I) is in the application of the medicine of relevant diseases such as preparation prevention or treatment cerebral ischemia, cerebral ischemia sequela, neural cell injury and changing function.

2, according to the application of claim 1, the dosage of wherein said medicine is the loose plain chemical compound of life of 0.1-100mg/kg body weight every day.

3. a pharmaceutical composition that prevents or treat relevant diseases such as cerebral ischemia, cerebral ischemia sequela, neural cell injury and changing function is characterized in that, contains the loose plain chemical compound of the life shown in general formula (I) and the pharmaceutically suitable carrier for the treatment of effective dose.

According to the pharmaceutical composition of claim 2, it is characterized in that 4, described pharmaceutical composition is made into the form of tablet, capsule, pill, injection, slow releasing preparation, controlled release preparation and various particulate delivery systems.

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