CN107137410B - It is a kind of for treating the Compound Chinese Herbal Monomer Recipe compatibility agent and preparation method of cerebral ischemia - Google Patents
It is a kind of for treating the Compound Chinese Herbal Monomer Recipe compatibility agent and preparation method of cerebral ischemia Download PDFInfo
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Abstract
It is a kind of for treating the Compound Chinese Herbal Monomer Recipe compatibility agent of cerebral ischemia, it is characterised in that: its active pharmaceutical ingredient is mixed by tanshinone IIA and lamp-dish flower acetic；The tanshinone IIA structural formula are as follows:The structural formula of the lamp-dish flower acetic are as follows:The present invention can be used for preparing after ischemic cerebral disease, cerebral hemorrhage rehabilitation after rehabilitation, cerebral thrombosis and alleviate myocardial ischemia, brain ischemia medicament.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition compound compatibility preparations and preparation method and the pharmaceutical composition to prepare
Treat the purposes in brain ischemia medicament.
Background technique
Headstroke is since cerebral vessels rupture suddenly or block and cause blood circulation disorder and cause brain tissue impairment
One group of disease, also known as cerebral apoplexy or cerebrovascular accident have high case fatality rate and disability rate.In China, 200 are had more than every year
Ten thousand people's kainogenesis cerebrovascular diseases have 1,500,000 people to die of the disease, and about 3/4 people loses labour to some extent in survivor
Ability, there are about 1/4-1/3 to recur in 2 to 5 years.Principal pathogenetic object is 60 years old or more crowd, and headstroke has become sternly
The principal disease of middle-aged and the old's health is threatened again.The patient survived after apoplexy has different degrees of deformity there are about 60%-80%,
The most common consequence is exactly that patient can generate " three partially ", disfluency, aphetite disorder, cognitive disorder, daily activities obstacle
And stool and urine obstacle." three partially ": hemiplegia, side disorder of limb's activity；Hemidysesthesia is exactly that side limbs are not felt
Feel；Hemianopsia refers to a certain segmental defect in the visual field.These can generate very serious consequence, and bring to countless patients and family
Huge pain and heavy burden.The high fact of Stroke In Aged disease incidence causes the common pass of medical worker and society
Note.The relationship of age and apoplexy is very close, and the apoplexy rate of 70 years old or more people is 20 times of the right side of fifty, and it is exactly dynamic for tracing it to its cause
Arteries and veins hardening.
The common method of clinical treatment apoplexy sequelae has at present: Chinese medicinal recovery treatment, acupuncture-moxibustion recovery method, scientific fortune
Dynamic functional training etc., and the Western medicine for treating this illness is very few.The Chinese medicine of common treatment apoplexy sequelae is handed down from one's ancestors with China
Secret recipe is in the majority, such as the glad safe Xueshuan xinmaining Tablet in huatuo zaizao pill, ginseng restorative pill, dahuoluo pills, day.In these prescriptions
In medicine, for ginseng restorative pill by 56 taste Chinese medicinal compositions, dahuoluo pills then contain 50 taste Chinese medicines.Effective component in big prescription is complicated,
And so in big prescription, be on earth any a few herbs or which composition play vital effect but must not and
Know.
Radix Salviae Miltiorrhizae has the effects that promoting blood circulation for regulating menstruation, stasis-dispelling and pain-killing, cool blood to disappear carbuncle, relieving restlessness and restlessness, nourishing blood and tranquilization.It is its bitter, micro-
It is pungent, cold nature.It commonly uses it and treats coronary disease and angina pectoris, atherosclerosis, bronchial asthma, palpitation and restlessness, vexed insomnia etc.
Disease.Fleabane flower is cold in nature, slight bitter, Gan Wenxin, with slightly cold removing toxic substances, dispelling wind and eliminating dampness, activating microcirculation and removing stasis medicinal, clearing and activating the channels and collaterals, anti-inflammatory analgetic
Effect.
Fleabane injection is clinically mainly used for diseases of cardiovascular and cerebrovascular systems, such as hypertensive cerebral hemorrhage, cerebral thrombosis shape
At, cerebral embolism etc..
Since above two Chinese medicine all has the function of expanding coronary artery and antithrombotic, we will be in this two taste
Medicine extracts wherein effective ingredient, to enhance therapeutic effect and further decrease the adverse reaction of drug as medicine pair.
Domestic and international present Research: headstroke is broadly divided into hemorrhagic cerebral apoplexy (cerebral hemorrhage or subarachnoid hemorrhage) and lacks
Hemorrhagic headstroke (cerebral infarction, cerebral thrombosis) two major classes, it is most commonly seen with cerebral infarction.Headstroke morbidity is anxious, and it is the world that case fatality rate is high
One of upper most important fatal disease.High blood pressure and atherosclerosis are the main and common causes of disease of headstroke.It is right
It in headstroke, is only capable of through illness such as control hypertension, diabetes, and keeps diet light and the progress such as maintenance is emotionally stable
Prevention, and once occur and survive by sheer good luck, most patients are but therefore disabled.Can treat cerebral apoplexy sequela method and
Drug is very limited, and has the characteristics that administration time length, poor prognosis.
Conventional Chinese medicine for treating apoplexy sequelae has:
1) dahuoluo pills: there are the multiple efficacies such as relaxing tendons and activating collaterals dredging collateral, warming channel and expelling cold analgesic, dispelling wind and eliminating dampness slit phlegm.It is usually used in
Paralysis caused by wind phlegm addiction, glossolalia, sufficient wither numbness pain, muscle arteries and veins contraction, lumbocrural pain and traumatic injury, walking in treatment
Inconvenience etc..Show that dahuoluo pills have effects that anticoagulation, anti-cerebral thrombosis and promoting blood circulationization addiction by clinical observation, having improves disease
The effect for becoming position blood circulation and limbs nutrition can also play prevention cerebral thrombosis again while treating ischemic hemiplegia
The effect of secondary formation.But the adverse reaction for thering are a few patients mouth parched and tongue scorched and constipation occur.
2) huatuo zaizao pill: major function is promoting blood circulationization addiction, eleminating phlegm and freeing channels, promoting qi circulation and relieving pain.Apoplexy for phlegm addiction resistance network
Hemiplegia caused by convalescence and sequelae, contraction, facial paralysis, glossolalia etc..Pharmacological research shows Hua Tuozai
Brain blood circulation can be enhanced by making ball, reduce blood viscosity, promoted to remove intracranial hematoma block, inhibited platelet aggregation and thrombus shape
At keeping blood circulation smooth；Brain cell more importantly can be directly protected, normal brain cells function is activated, inherently restores
Nervous function is effectively treated the apoplexy sequelaes such as numb in every limb, facial paralysis, paralysis.But patient will appear insomnia
Dreaminess, dysphoria, constipation and dry throat with a bitter taste etc..
3) ginseng restorative pill: having the benefits of inrigorating qi and promoting blood circulation, promoting menstruation dredging collateral, strengthening the muscles and bones, for treatment arthralgia and myalgia, four limbs fiber crops
The diseases such as wood, hemiplegia, facial paralysis, slurred speech, brothers' muscular constricture and set.Studies have shown that ginseng restorative pill can promote blood
Circulation, accelerates blood flow, improves immunity of organisms and repair function, has good treatment and adjustment effect to ishemic stroke.
But this product contains cinnabar and aristolochiaceae plant asarum, should not take for a long time, taking should inspect periodically, and should check blood, mercury in urine
Ion concentration checks Liver and kidney function, deactivates immediately more than prescribed limit person.In addition, old man should not generally use.
4) Xiao Huoluo Dan: there is the effect of warming channel and expelling cold, by addiction dredging collateral.It is usually used in treating cerebrovascular accident sequelae, especially
There is good action for numb in every limb, stretching, extension is unfavorable, limbs are not warm.Not with adverse cardiac, hypertension and hepatic and renal function
Full patient, which avoids, takes this medicine.
5) Xie Yudan: having the benefits of dispelling wind and removing obstruction in the meridians, dissipating phlegm for resuscitation, be suitable for the facial paralysis of apoplexy sequelae, spasm of muscles and vessels,
Numb crymodynia, glossolalia, salivation are not understood, swallow inconvenience etc..
6) it BUCHANG NAOXINTONG JIAONANG: becomes silted up for blood stagnancy due to deficiency of QI, train of thought and hinders caused apoplexy symptoms include hemiplegia, limbs fiber crops
Wood, facial paralysis, tongue are talked about by force stuttering.But having the patient of stomach trouble to should be noted after meal takes medicine.
Apoplexy pathomechanism is complicated, and Chinese medicine thinks that stagnation of blood stasis is its pathology key.Radix Salviae Miltiorrhizae bitter is slightly cold, removing blood stasis for promoting tissue regeneration,
Effect is distinguished；Fleabane flower bitter is warm-natured, blood-activating analgetic, expelling wind and clearing away cold.The two mutually must be used, and the function of stagnation resolvation more wins, and two medicines respectively take
Its active component, power are specially imitated macro.Treatment suitable for apoplexy sequelae.
Radix Salviae Miltiorrhizae also known as red ginseng, radix salviae miltiorrhizae, red root etc..It is born in by patana woods by-pass or on sparse woods dry ground.For Shuangzi
Leaf plant Labiatae, dry root and rhizome.Main product is in Anhui, Henan, Shaanxi and other places.It is sturdy with root item, it dries, color is purplish red, nothing
Reed head and fibrous root person are preferred.Radix Salviae Miltiorrhizae is the conventional Chinese medicine of qi and activate blood circulation and treatment cardiovascular and cerebrovascular disease, its effect mainly has:
1) effect of cardiovascular system:
A. heart tonifying reinforces myocardial contractive power, improves cardiac function, does not increase myocardial oxygen consumption；
B. coronary artery is expanded to blood vessel function, increases myocardial blood flow；Peripheral blood vessel is expanded, blood flow increases；Under cerebral blood flow (CBF)
Drop；
C. antithrombus formation improves plasmin activity；Extend bleeding and cotting time；Platelet aggregation is inhibited (to improve blood platelet
Interior cAMP level inhibits TXA2 synthesis)；Improve hemorheological properties (blood viscosity reduces, erythrocyte electrophoretic time shortens)；
D. improve microcirculation.
2) promote the reparation and palingenesis of tissue:
A. the reparation of tissue and regeneration necrotic myocardium is promoted to remove fast；Fibroblast differentiation, collagenous fibres form brighter
It is aobvious；Granulation forms comparative maturity.Local extravasated blood mitigates, blood circulation improves, and healing time shortens；
B. hyperplasia is inhibited to have inhibiting effect to the fibroblast of hyperplasia.
3) liver protection improves liver microcirculation.
4) contain Cryptotanshinone, dihydrotanshinone in antibacterial red sage formulation, to external staphylococcus, Escherichia coli, change
Property bacillus has inhibiting effect.
Radix Salviae Miltiorrhizae contains plurality of active ingredients, but can be summarized as liposoluble constituent and water soluble ingredient.The former is with tanshinone
Main, the latter is based on phenolic acid.There are three types of liposoluble constituent in Radix Salviae Miltiorrhizae is most important: Tanshinone I, tanshinone IIA and hidden Radix Salviae Miltiorrhizae
Ketone, wherein tanshinone IIA content is relatively high, about the 0.1~0.9% of medicinal material, controls as the quality of effective component always
Index.Tanshinone separation is generally extracted using organic solvent such as methanol, ethyl alcohol, methylene chloride etc., and traditional separation method is to use
95% alcohol heat reflux extracts.But since tanshinone is unstable when heated, thus ultrasonic method can effectively be kept away as non-thermal extraction
Exempt from loss caused by heating, improves its content.Means of supercritical extraction is recent development new method, is especially suitable for the extraction point of tanshinone
From purity and yield have been greatly improved, and are a kind of methods being worthy to be popularized.Tanshin Water-soluble Ingredient be protocatechualdehyde,
Danshensu and danshinolic acid.Deeply with research, it has been found that the most effective ingredient of salvia-soluble not instead of the former two, red phenol
Acid, wherein salviandic acid A activity is most strong.The content of salviandic acid A is about the 0.01~0.06% of red rooted salvia, and tanshin polyphenolic acid B contains
Amount is higher, up to 2~8%.Therefore tanshin polyphenolic acid B is most important active constituent in red sage root water soluble ingredient.Traditional water solubility
Phenolic acid separation is heated using water extract-alcohol precipitation, including thermal extraction, thermal concentration, the dry drop for causing danshinolic acid
Solution, therefore shortening heated time is the effective ways for improving its content, such as is concentrated under reduced pressure, spray drying.Macroreticular resin extracts
It is the method that developed recently gets up for separating salvia-soluble phenolic acid, high using this method separative efficiency, phenolic content is high, loss
It is small.Laboratory separation has been obtained for a variety of effective component in red sage, but still less with the drug that this is developed, such as danshen injections
With the Danshen Root dropping ball preparation based on water soluble ingredient and liposoluble constituent respectively.
Fleabane flower is the drying herb also known as erigeron breviscapus, eastern chrysanthemum of compositae plant Erigeron breviscapus.It is born in hillside fields on the sunny side, it is main
It is distributed in Southwestern China area, it is especially more with Yunnan.Hypertension, cerebral hemorrhage, cerebral thrombosis shape clinically mainly used for treating
At, cerebral embolism, polyneuritis, the paralysis sequelae such as chronic archnoiditis, in addition, to diabetes, nephrosis, cervical vertigo,
Geriatric disease also has a better effect.
Breviscapinun is the flavonoids effective constituent extracted from fleabane flower, and principal component is that (4 '-hydroxyls are yellow for lamp-dish flower acetic
A kind of reed mentioned in ancient books element -7-o- glucuronide aldehydic acid glycosides), another (apiolin -7-o- glucuronide) containing a small amount of oil lamp A prime.Fleabane flower
Element has the function of expansion of cerebral vascular, can reduce cerebral vascular resistance, increases cerebral blood flow (CBF), improves microcirculation, and has confrontation blood small
Plate aggtegation.Therefore can be used for treating ischemic cerebrovascular disease, it paralyses as caused by cerebral thrombosis, cerebral embolism, cerebral hemorrhage etc.
Sequelae patient has good therapeutic effect, and to curative effect of the course of disease within 6 months, than 6 months or more persons are good.
However, Radix Salviae Miltiorrhizae and fleabane flower also have respective side effect.It was verified that Radix Salviae Miltiorrhizae is to the irritating effect of stomach and intestine, it is such as long
Phase takes Radix Salviae Miltiorrhizae, can cause the diseases such as different degrees of indigestion and loss of appetite, pantothenic acid.Radix Salviae Miltiorrhizae can not be taken together with aspirin.Fleabane flower class
The application effect of drug clinically is significant, and civil utilization is with a long history, has extensive social base, in addition toxic side effect
It is small, it receives praises from customers, is listed in the herbal variety and Chinese traditional treatment cardiovascular and cerebrovascular disease of state key development, but in use
There are fash, out of strength, the side effects such as dry.Therefore, proper use of Chinese medicine, using its effective component, removal causes adverse reaction
Other compositions, realize the modernization of Chinese medicine, be the key that Chinese medicine development and move towards the world.
Summary of the invention
The object of the present invention is to provide a kind of compound Chinese medicinal preparation being made of Radix Salviae Miltiorrhizae and fleabane flower active pharmaceutical ingredient and
Preparation method is used to prepare the medical application for the treatment of brain ischemia medicament.
Purpose of the invention be achieved in that it is a kind of for treating the Compound Chinese Herbal Monomer Recipe compatibility agent of cerebral ischemia,
It is characterized by: its active pharmaceutical ingredient is mixed by tanshinone IIA and lamp-dish flower acetic；
The tanshinone IIA structural formula are as follows:
The structural formula of the lamp-dish flower acetic are as follows:
The purpose of the present invention can be realized with following manner: the matter of active pharmaceutical ingredient tanshinone IIA and lamp-dish flower acetic
Amount is than being 3 ︰ 5.
Active pharmaceutical ingredient tanshinone IIA and lamp-dish flower acetic are according to human body weight reckoning dosage ratio range are as follows: red
Join ketone IIA:0.3mg/kg-7.5mg/kg, lamp-dish flower acetic 0.5mg/kg-12.5mg/kg.
The Compound Chinese Herbal Monomer Recipe compatibility agent for treating cerebral ischemia is oral preparation or injection.
The oral preparation is tablet or capsule.
The injection is intravenous drip preparations.
The present invention is used to treat the preparation method of the Compound Chinese Herbal Monomer Recipe compatibility agent of cerebral ischemia, it is characterised in that: drug
Active constituent tanshinone IIA and lamp-dish flower acetic are bought by market or raw medicinal material is extracted and obtained, and are then 3 ︰ 5 according to mass ratio
Ratio mixing, be prepared into tablet or capsule or injection according to conventional plus auxiliary material；Usage and dosage: tablet, capsule, specification are
30mg/ piece (grain), adult 1 to 2 tablets once, 3 times a day；Injection: intravenous drip, daily overall control 100mg-200mg it
Between.
The present invention is used to treat the Compound Chinese Herbal Monomer Recipe compatibility agent of the cerebral ischemia health after preparing ischemic cerebral disease, cerebral hemorrhage
It can be applied in rehabilitation and alleviation myocardial ischemia, brain ischemia medicament after multiple, cerebral thrombosis.
The invention discloses tanshinone IIAs and lamp-dish flower acetic composition and medicine compatibility method to treat brain ischemia medicament in preparation
In purposes, compatibility significantly can protect focal cerebral ischemia to damage in proportion for tanshinone IIA and lamp-dish flower acetic composition in the present invention
Wound.With artery ligation model, Primary cortical neurons Nervous toxicity and model of oxidative in mouse brain, Morris water is utilized
Maze experiment, the experiment of NMDA excititoxic and hydrogen peroxide oxidation injury experiment, find tanshinone IIA and lamp-dish flower acetic
Composition is remarkably improved the spatial memory capacity of cerebral middle artery occlusion model mice, improves motor function, and reduce brain group
Damaged area is knitted, neuronal function is promoted to restore, reduces oxidative damage；Cell in vitro model is it has furthermore been found that tanshinone IIA
The effect of excititoxic caused by oxidativestress damage and NMDA can significantly be mitigated with lamp-dish flower acetic compatibility.Show benefit
With tanshinone IIA and lamp-dish flower acetic composition ratio compatibility, be conducive to the pleiotropism for playing traditional Chinese medicine monomer prescription, that is, meet
Medical knowledge opinion, and the mechanism of action of Chinese medicine multiple location, multiple target point is embodied, effect is obvious, safe.The present invention has treatment ischemic
The features such as cerebral injury curative effect is high, dosing is small, using facilitating.
Detailed description of the invention
Fig. 1 primary neuronal cell viability experiment
(A) primary neuronal culture NMDA excititoxic model；(B) primary neuronal culture hydrogen peroxide (H2O2)
Model of oxidative.Tanshinone I (Tanshinone-I), tanshinone IIA (Tanshinone-IIA), danshinolic acid
(Salvianolic acid), lamp-dish flower acetic (Scutellarin), oil lamp A prime apigenin-7-O-glucronide.n
=6 culture dishes/group, mean+SD, * * P < 0.01vs Control,$P<0.05,$$P < 0.01vs NMDA or H2O2Mould
Type.
Inhibiting effect of Fig. 2 monomer compatibility to cerebral injury
(A) TTC dyeing display each group cerebral infarction dead zone, red are normal cerebral tissue, and white is infarcted cerebral constitution.(B) each
Group infarcted cerebral constitution volume (brain volume %) is quantitative.(C) groups of animals neurological score is quantitative.Tanshinone IIA
(Tanshinone-IIA), lamp-dish flower acetic (Scutellarin) n=6, mean+SD,#P < 0.05,##P < 0.01 with
MCAO model group compares.
Influence of Fig. 3 monomer compatibility to learning and memory and motor function
(A) mouse finds platform incubation period in Morris water maze training.(B) each group mouse is when platform quadrant stops
Between.(C) groups of animals residence time (total time 3min) on roller.Tanshinone IIA (Tanshinone-IIA), fleabane flower
B prime (Scutellarin).N=6, mean+SD, P < 0.05 * P < 0.05, * *, compared with Sham；#P < 0.05,##P<
0.01 compared with MCAO model group.
The influence that Fig. 4 monomer compatibility expresses cerebral injury marginal zone glutamate NMDA receptor
(A) Western-blot band example.(B) each group NR2A subtype expression is horizontal.(C) each group NR2B subtype expression water
It is flat.(D) each group NR2B Ser1303 site phosphorylation is horizontal.(E) each group NR2B Tyr1472 site phosphorylation is horizontal.Radix Salviae Miltiorrhizae
Ketone IIA (Tanshinone-IIA), lamp-dish flower acetic (Scutellarin).N=6, mean+SD, * P < 0.05, * * P
< 0.05, compared with Sham；#P < 0.05,##P < 0.01 is compared with MCAO model group.
Specific embodiment
The effect analysis and main component function analysis that the present invention is based on Radix Salviae Miltiorrhizaes and fleabane flower in compound Chinese medicinal preparation, really
Vertical monomer compound composition and proportion compatibility.
(1) experimental method and material
1. animal: adult mice
Pregnant 14~15 days C57BL/6 mouse, cleaning grade, healthy male C57BL/6 mouse are tested by The Fourth Military Medical University
Animal center provides.It is raised in The Fourth Military Medical University's pharmacology teaching and research room animal house, 23 ± 2 DEG C of temperature, humidity 50% ± 10%,
12h daily cycle, animal ad lib, drinking-water.Mouse need to adapt to environment one week before testing, and all Behaviors surveys are arranged in white
It carries out (morning 9:00-12:00 and afternoon 14:00-18:00).All experiments relevant to animal are by The Fourth Military Medical University
Ethics Committee's audit passes through.
2. primary neuronal culture
The culture of Cerebral Cortex neuron is referring to this laboratory front-end process and appropriately adjusts [43], and pregnant 13~15
Its C57BL/6 mouse cervical dislocation is put to death, and takes out embryo under aseptic condition, is put into the plate equipped with pre-cooling D-Hank ' s balanced salt solution
In, endocranium is removed under the microscope and takes out brain, is rejected tunica vasculose, is separated and take out cerebral cortex, is added final concentration of
0.25% tryptic digestive juice digests 10 minutes, and cortical tissue then is sucked out with connector bend dropping tube, is transferred to equipped with pre-cooling
In the centrifuge tube of DMEM culture solution, rinsing three times, terminates digestion, and finally gently piping and druming cell prepares Single cell suspensions for several times,
200 mesh net filtrations simultaneously count.Adjustment cell density presses 1 × 10 respectively6/ hole kind enters in 6 orifice plates, and 5 × 104/ hole kind enters 96 orifice plates
It is interior, 2 × 105/ hole kind enters to be covered in 24 orifice plate of slide in advance, and all culture plates are coated with final concentration of 50 μ g/ml's in advance
Poly-D-Lys (PLL).Full dose is changed to Neurobasal culture solution (Neurobasal+2%B27+ after cultivating in incubator for 24 hours
1% is dual anti-).Change the liquid once in half every three days later, change before liquid should by culture solution in 37 DEG C of environment abundant rewarming, reduce cold
Stimulation.It cultivates to 7~10 days cells for testing.
3.MTT experiment
Under aseptic condition, culture medium is removed, with no Mg2+Extracellular fluid (ECS) wash twice, first treated with medicaments neuron
15min, adding NMDA (200 μM) and hydrogen peroxide, ((100 μM), drug and NMDA and hydrogen peroxide act on cell simultaneously at this time.
Each hole is washed twice with ECS after 30min, and every hole adds 90 μ l original fluids to continue culture 24 hours, and then final concentration is added in every hole
For the 10 μ l of MTT solution of 5mg/ml, continue to be incubated for 4-6 hours.Careful inhale abandons culture solution, and 150 μ l dimethyl sulfoxides are added in every hole
(DMSO), optical density (OD) value value is tested with automatic microplate reader (wavelength 490nm) rapidly.
4. arterial embolism (MCAO) is tested in mouse brain
For animal to receive MCAO preoperative, continuous gavage is administered 3 days (2 times a day, in each stomach-filling one of AM9:00 and PM5:00
It is secondary), 1 hour after last time was administered in the morning on the 4th, model group and sesamin group mouse are used into arteria cerebri media embolism
(MCAO) models of cerebral ischemia-reperfusion injury is made in method.Intraperitoneal injection fiber crops are carried out to mouse with chloraldurate (300mg/kg)
Liquor-saturated, dorsal position is fixed, neck disinfection, under surgical operation microscope, along neck median line notch, successively separates subcutaneous and muscle groups
It knits, sufficiently after exposure arteria carotis communis, carefully free arteria carotis communis is to crotch, by external carotid artery (ECA) and arteria carotis communis (CCA)
Ligation closes internal carotid (ICA) with artery clamp folder.A kerf is cut in arteria carotis communis proximal part with eye scissors, by line bolt (diameter
0.2mm, length 20mm) through notch it is softly inserted into internal carotid about 10mm or so along arteria carotis communis, encounter light resistance
Stop, showing that line bolt head end is located exactly at middle artery starting point, by ipsilateral cerebral Middle cerebral artery occlusion.Stay 5mm the end of a thread in body
Table sutures muscle and skin, animal is put back in rearging cage, and operation neutralization is postoperative to be kept the temperature with electric baking lamp.Reperfu- sion is 2 after ischemic
Hour carries out, and line bolt is gently pulled to internal carotid section start, and arteria cerebri media blood supply is restored at this time.Sham-operated control group operation and
MCAO model group is essentially identical, does not need insertion line bolt only.It is postoperative to continue administration 2 times.
5. Neuroscore and cerebral infarction volume measurement
Neuroscore carries out after Reperfu- sion 24 hours, divides standards of grading processed with reference to Longa method 5: having no neural damage
Hurting defect is 0 point；Forelimb bending, cannot stretch completely is 1 point；Turn-take to the left when walking is 2 points；Astasia, to the left
Topple over is 3 points；Being unable to independent ambulation is simultaneously 4 points with conscious disabilities.0 point and 4 points of persons are excluded, it is impossible to be used in real
It tests.After Neuroscore, Some Animals (every group 6) take out rapidly mouse brain and are placed in -20 DEG C of ice with ice normal saline flushing
Case 20 minutes, coronal-plane is taken uniformly to be cut into 2mm slab 6 in brain slot, is put into the 2,3 of the 2% of 37 DEG C of preheatings rapidly,
It is dyed 30 minutes in 5-triphenyltetrazolium chlorides (TTC) solution.Stir a brain piece within every 5 minutes.Then with 10% formal
Woods solution is fixed overnight, is taken pictures with digital camera.Normal district's groups are woven to red, and cerebral infarction district's groups are woven to white.Use image procossing
Software photoshop CS3 calculates Infarction volume, and the sum of each infarct size is total infarct body multiplied by brain piece thickness (2mm)
Product.
6.Morris water maze
After Neuroscore, Some Animals (every group 10) continue administration 7 days, then stop administration, restore 3 weeks, in
Space and ability of learning and memory of the month after operation using Morris water maze assessment animal.Water maze used in this experiment is one
The water vat of a round plating, pool inner water depth 45cm, pool wall painted black.Because mouse is black, milk powder, which is added, in water makes it
Become milky.Light in whole experiment process room, temperature, in pond temperature all keep constant it is constant.Pond is divided into four
Quadrant, it is assumed that target quadrant is first quartile, a white platform is placed at pool wall 35cm in target quadrant, diameter is
8cm is about 2cm apart from the water surface.During the experiment, the position of platform is constant.Different colours and the marker of shape are attached to pond
In wall.Video camera is mounted on the top in labyrinth.The motion profile of mouse can be recorded by real-time synchronization.Screen analysis in experiment
The system developed with processing using Jiliang Software Sci-Tech Co., Ltd., Shanghai.The system can provide appearing on the stage for animal
Incubation period wears multiple indexs such as platform number, swimming distance, swimming rate and target quadrant activity time percentage.Specific experiment step
It is rapid as follows, it is broadly divided into two steps:
1. space learning is tested: before experiment, first mouse being placed on platform, it is allowed to adapt to 20s.Then one is selected to remove
Mouse is slowly put into water by the quadrant except target quadrant along pool wall, and the position of place of entry is at a distance from platform each time
Want almost the same.The mouse 5s that goes up on the stage calculates success of going up on the stage, and stops recording.The record time is set as 60s every time.If small after 1min
Mouse climbs up platform not yet, equally stops recording, and then guides it to go up on the stage and it is allowed to stop 20s.After experiment, animal is wiped
Mouse cage is put into after clean.It repeats the above steps, the interval that every mouse is tested every time at least 30min, to guarantee that mouse has abundance
Time regain one's strength, reduce swimming bring stress.By under software records mouse swim total distance, speed, appear on the stage it is latent
The indexs such as phase, for evaluating its Spatial learning ability.Same experiment, continuously repeats test 5 days.
2. spatial memory is tested: interval two days the 8th day and the 9th day, removes platform, any quadrant is by mouse along pool wall
It is slowly put into water, records the swimming track of 90s mouse, equally mouse is dried and is put back in cage.By recording track curve,
Come measure mouse target quadrant activity time percentage and wear platform number, to assess the spatial memory capacity of mouse.
The experiment of 7.Rotarod idler wheel
After Morris water maze assesses space and the ability of learning and memory Neuroscore of animal, using Rotarod
Roller devices, the progress turn-club test training in the 40th day after cerebral ischemia.Mouse is by 8 turns/min revolving speed training 3 times, every time
5min, twice train between interval 20min as the fatigue recovery time.Postoperative 43rd day, all mouse by 16 turns of revolving speed/
Min is tested, and the time of mouse continuous walking in transfer rod instrument is recorded.
8.Western-blot
After Neuroscore, Some Animals (every group 6) take out rapidly mouse brain, take Penumbra zone surrouding brain tissue PBS
After rinsing, the RIPA lysate (containing appropriate protease inhibitors cocktail and 1mM PMSF) of pre-cooling is added, on ice with super
Sound wave cell disruptor is homogenized 5 times (25Hz, 3s/ time, interval 5s), and final liquid is to limpid.4 DEG C, 12000rpm centrifugation
20min collects supernatant in new 1.5ml centrifuge tube；A small amount of supernatant BCA method protein quantification is taken, draws standard curve, really
Determine loading volume；5 × sample-loading buffer boiling water boiling 8min of 1/4 volume is added in remaining supernatant, makes albuminous degeneration.Using SDS-
PAGE protein isolate successively prepares 9% separation gel and 3% concentration glue, after gel, 500ml electrophoretic buffer is added and (contains
1.51g Tris, 7.4g Glycine, 0.5g SDS)；Take each 30 μ g of protein sample through well loading, first 80V electrophoresis 0.5h,
Then it is adjusted to 120V and continues electrophoresis about 1.5h, when bromophenol blue reaches separation gel bottom, that is, stop electrophoresis；Pvdf membrane methanol is pre-
2min is handled, is added 1L transfering buffering liquid (methanol containing 200ml, 14.4g Glycine, 3.03g Tris), by gel and PVDF
Film is placed with the direction of " black glue tunica albuginea ", and constant pressure 100V shifts 2h.After transferring film, needed for being cut according to albumen marker
Destination protein band is placed in 5% skimmed milk power and closes, and room temperature slowly shakes 2-4h；After PBST rinses extra milk powder, by band
In enclosed polybag, it is added and uses the diluted primary antibody of PBST, be placed in 4 DEG C of overnight incubations；Next day takes out polybag, room temperature rewarming
0.5h, then PBST is washed film 3 times, each 10min, then band is enclosed in the diluted secondary antibody of PBST, is incubated at room temperature 2h；PBST
Rinsing 3 times after, then with PBS rinse 10min, by ECL luminescent solution A and B by 1:1 mix after, drop evenly on film, be immediately placed in
Exposure in light-emitting appearance, photograph.Antibody NR1 and NR2B antibody is purchased from U.S. Millipore company；NR2A,NR2B,NR2B-P-
Ser1303, NR2B-P-Tyr1472 antibody are purchased from Britain Abcam company.
9. oxidative damage parameters measure
Specific experiment step please refers to kit specification.The homogenate of side ischemia margin zone brain tissue tissue is damaged,
12000rpm is centrifuged 20min, supernatant according to kit, by spectrophotometer chemical colorimetry measure malonaldehyde (MDA),
Superoxide dismutase (SOD), glutathione (GSH) are horizontal.
(2) result
1. the establishment of main active in Radix Salviae Miltiorrhizae
According to the literature, effective component in red sage mainly includes Tanshinone I, tanshinone IIA, Cryptotanshinone and danshinolic acid
Deng.Wherein tanshinone IIA content is relatively high, about the 0.1~0.9% of medicinal material, controls as the quality of effective component always
Index.Danshen injections and Danshen Root dropping ball are respectively based on water soluble ingredient and liposoluble constituent.Foundation tanshinone expansion blood vessel,
Improve many-sided effect such as microcirculation, anti-oxidant, the selection primary neuronal culture NMDA excitability of our screening studies early period
Neurotoxicity model and hydrogen peroxide (H2O2) model of oxidative, we are respectively to Tanshinone I, tanshinone IIA and danshinolic acid three
Monomer component is studied.Find above-mentioned three kinds of monomers to excitement caused by NMDA in NMDA excititoxic model
Nerve toxicity has certain inhibiting effect.Neuron viability significantly reduces after NMDA processing, and saline control group neuron is living
Power is only 46.2%, and positive control medicine nmda receptor NR2B blocking agent Ro25-6981 (0.3 μM), which can significantly inhibit NMDA, to be caused
Neure damage (neuron viability increases to 96.3%).Neuron viability increases in various degree after drug-treated, specifically
Are as follows: three concentration Tanshinone Is make neuron viability rise to 52.3%, 63.2% and 66.5%；Three concentration tanshinone IIAs make mind
66.6%, 74.5% and 81.1% is risen to through first vigor；Three concentration danshinolic acids make neuron viability be upgraded to 50.2%, 60.4%
With 68.5% (Figure 1A).It can be seen that three Tanshinone I, tanshinone IIA and danshinolic acid monomer component confrontation NMDA excitatory neuron poison
Property acted on tanshinone IIA when acting on comparable sodium it is most strong.
In addition, primary neuronal culture hydrogen peroxide (H2O2) in model of oxidative, neuron viability is aobvious after dioxygen water process
Writing reduces, and saline control group neuron viability is only 38.4%, and positive control medicine Catechin (5 μM) can significantly inhibit dioxygen
Neure damage caused by water (neuron viability increases to 64.3%).Neuron viability increases in various degree after drug-treated
Add, specifically: three concentration Tanshinone Is make neuron viability rise to 42.4%, 48.5%, 52.8%；Three concentration tanshinones
IIA makes neuron viability rise to 46.5%, 56.7% and 67.5%；Three concentration danshinolic acids are upgraded to neuron viability
46.0%, 52.1% and 57.1 (Figure 1B).It can be seen that three Tanshinone I, tanshinone IIA and danshinolic acid monomer components fight hydrogen peroxide
Oxidative injury is acted in comparable sodium with tanshinone IIA most strong.
Tanshinone IIA: cherry red acicular crystal is insoluble or poorly soluble in water, is soluble in dimethyl sulfoxide, ethyl alcohol, acetone, second
The organic solvents such as ether and benzene.Structural formula are as follows:
2. the establishment of main component in fleabane flower
Breviscapinun is the important component that fleabane flower puts in overprotection effect, mainly includes that (4 '-hydroxyls are yellow for lamp-dish flower acetic
A kind of reed mentioned in ancient books element -7-o- glucuronide aldehydic acid glycosides) and oil lamp A prime (apiolin -7-o- glucuronide).It is clinically main to use
Completeness and incomplete paralysis caused by treatment cerebral thrombosis and brain bolt are waited indefinitely, since its antioxidation is stronger,
We still select primary neuronal culture NMDA excititoxic model and hydrogen peroxide (H2O2) model of oxidative evaluation lamp
The neuroprotection of small cup flower main component.In NMDA excititoxic model, three concentration lamp-dish flower acetics make nerve
First vigor rises to 58.5%, 68.5% and 73.3%；Three concentration oil lamp A primes make neuron viability rise to 50.2%, 58.7%
With 62.3% (Figure 1A).It can be seen that work when lamp-dish flower acetic monomer component confrontation NMDA excititoxic acts on comparable sodium
With most by force.
In primary neuronal culture hydrogen peroxide oxidation damage model, three concentration lamp-dish flower acetics make neuron viability liter
To 45.1%, 64.5%, 72.5%；Three concentration oil lamp A primes make neuron viability rise to 44.1%, 48.4% and 52.5%
(Figure 1B).It can be seen that two monomer component confrontation hydrogen peroxide oxidation damaging actions of lamp-dish flower acetic and oil lamp A prime are in comparable sodium
It is acted on lamp-dish flower acetic most strong.
Lamp-dish flower acetic: to be faint yellow to yellow powder, there is certain hygroscopicity；Odorless, tasteless or taste is micro- salty.Methanol,
It is dissolved in pyridine, dilute alkaline soln, it is slightly molten in hot water, ethyl alcohol, ethyl acetate, have in water, ether, chloroform, benzene, acetone etc.
It is almost insoluble in solvent.Structural formula are as follows:
3. cytology orthogonal experiment establishes monomer compound composition
According to above-mentioned experimental result, select the best experimental concentration of respective drug respectively (see Fig. 2).By tanshinone in salvia miltiorrhiza bunge
I, three kinds of monomers of tanshinone IIA and danshinolic acid are as factor 1, two kinds of monomer conducts of lamp-dish flower acetic and oil lamp A prime in fleabane flower
Factor 2 carries out the compatibility of two kinds of ingredients using Factorial Design principle respectively；And two kinds of cytology damage models of utilization, that is, primary
Neuronal culture models NMDA excititoxic model and primary neuron hydrogen peroxide oxidation damage model, to evaluate monomer
The optimum effect of compound compatibility.It is tested according to above-mentioned concentration gradient, determines the best use concentration of each monomeric substance.
(1) first with the synergistic effect of NMDA excititoxic model evaluation monomer compatibility.(Radix Salviae Miltiorrhizae has 2 factors
Imitate ingredient, fleabane flower effective component) factorial test design (complete intersection group experiment design) discovery, it is effective that Radix Salviae Miltiorrhizae is used alone
Ingredient Tanshinone I, tanshinone IIA and danshinolic acid, three monomer components can significantly inhibit neuronal death, increase cell survival
Rate, respectively 52.3%, 74.5% and 68.4%, there were significant differences compared with saline control group model group (42.5%).Equally,
Lamp-dish flower acetic and oil lamp A prime is used alone and also significantly increases neuron survival rate to 68.5% and 62.3%.Factorial test list
Variable the results of analysis of variance shows: effective component in red sage or fleabane flower effective component single factor test main effect are significant (p < 0.05),
Show that effective component in red sage or fleabane flower effective component is used alone, there is certain neuroprotection；Single factor test main effect
Comparison discovery, it is (square: 0.604vs 0.308) that effective component in red sage protective effect is greater than fleabane flower effective component.It finds simultaneously,
Significant interaction (p < 0.05) between two factors shows that effective component in red sage and fleabane flower effective component are used in combination to have and assists
Same effect.Optimal dose group is combined into tanshinone IIA (5 μM) and lamp-dish flower acetic (5 μM), and neuron survival rate reaches 89.7%.
Inhibiting effect (cell survival rate %, mean+SD) of 1 monomer compatibility of table to NMDA excitatory neuron poison
It is handled using SPSS statistical software, the statistical method of comparison among groups one-way ANOVA variance analysis；It is more
Group compares between seeing two-by-two with Post-Hoc-Tests LSD method.* P < 0.01 P < 0.05, * * is compared with physiological saline group；$P<
0.05,$$The compatibility of P < 0.01 group is compared with wherein any one component.
(2) synergistic effect of primary neuron hydrogen peroxide oxidation damage model evaluation monomer compatibility is secondly utilized.Compatibility is same
Upper table.Effective component in red sage Tanshinone I, tanshinone IIA and danshinolic acid, three monomer components, which are used alone, can significantly inhibit mind
Through first dead, raising cell survival rate, respectively 48.5%, 56.7% and 57.2%, with saline control group model group
(37.9%) compared to there were significant differences.Equally, lamp-dish flower acetic and oil lamp A prime is used alone and also significantly increases neuronal survival
Rate is to 65.5% and 53.2%.The factorial test univariate analysis of variance the result shows that: effective component in red sage or fleabane flower effectively at
Divide single factor test main effect significant (p < 0.05), shows that effective component in red sage or fleabane flower effective component is used alone, there is one
Fixed neuroprotection；The comparison discovery of single factor test main effect, effective component in red sage protective effect and fleabane flower effective component phase
When (square: 0.487vs0.517).Find simultaneously, significant interaction (p < 0.05) between two factors, show Radix Salviae Miltiorrhizae effectively at
Divide and fleabane flower effective component is used in combination with synergistic effect.Optimal dose group is combined into tanshinone IIA (5 μM) and fleabane flower second
Plain (5 μM), neuron survival rate reaches 78.9%.
2 monomer compatibility of table causes inhibiting effect (cell survival rate %, the average value ± mark of neuron oxidative damage to hydrogen peroxide
It is quasi- poor)
It is handled using SPSS statistical software, the statistical method of comparison among groups one-way ANOVA variance analysis；It is more
Group compares between seeing two-by-two with Post-Hoc-Tests LSD method.* P < 0.01 P < 0.05, * * is compared with physiological saline group；$P<
0.05,$$The compatibility of P < 0.01 group is compared with wherein any one component.
4. tanshinone IIA and lamp-dish flower acetic compatibility improve cerebral ischemia
(1) tanshinone IIA and lamp-dish flower acetic compatibility improve neurological deficit and cerebral injury
Arteria cerebri media embolism (MCAO) is a generally accepted brain damage model.Cerebral ischemia re-pouring injured generation
It is a many factors and the complicated pathophysiological process that mechanism participates in jointly.During acute brain injury, oxygen radical
Caused oxidative damage includes the damage of many molecules and cell, such as protein, liposome, DNA, mitochondria and cell membrane
Structure and consequential Apoptosis and necrosis.Tanshinone IIA (5 μM) and lamp-dish flower acetic are combined into according to optimal dose group
(5 μM) and molecular weight tanshinone IIA (molecular weight 294.34) and lamp-dish flower acetic (molecular weight 462.36) calculate that animal is real
It tests compatibility dosage and is respectively set as tanshinone IIA 1.5mg/kg and lamp-dish flower acetic 2.5mg/kg.Experiment is set as 5 groups, that is, does evil through another person
Art control group, MCAO model group, tanshinone IIA 1.5mg/kg group, lamp-dish flower acetic 2.5mg/kg group, compatibility group (tanshinone
IIA 1.5mg/kg and lamp-dish flower acetic 2.5mg/kg).The experimental results showed that Reperfu- sion 24 was as a child, it is real in addition to sham-operation group
It tests animal and different degrees of neurological deficit and cerebral infarction occurs, compared with sham-operation group, MCAO significantly increases small
The cerebral infarction volume (39.64 ± 0.78%) and Neuroscore (3.4 ± 0.09) of mouse, tanshinone IIA 1.5mg/kg group,
Lamp-dish flower acetic 2.5mg/kg group and the pretreatment of compatibility group can reduce cerebral infarction volume caused by MCAO performs the operation and increase (Fig. 2A, 2B)
With neurological deficit (Fig. 2 C).It is wherein best with the two compatibility curative effect.
(2) tanshinone IIA and lamp-dish flower acetic compatibility improve learning and memory and motor function
After mouse MCAO art Neuroscore, Some Animals (every group 10) continue administration 7 days, then stop administration,
Restore 3 weeks, assesses space and the ability of learning and memory of animal using Morris water maze in month after operation.Experimental result table
Bright, MCAO mouse shows space learning and is substantially reduced with memory capability, tanshinone IIA 1.5mg/kg group, lamp-dish flower acetic 2.5mg/
Kg group and the treatment of compatibility group can reduce learning and Memory damage (Fig. 3 A, 3B) caused by MCAO performs the operation, and restore its locomitivity
(Fig. 3 C), wherein best with the two compatibility curative effect.
5. pharmacological mechanism is studied
The molecular mechanism of collaboration neuroprotection is played further to inquire into tanshinone IIA and lamp-dish flower acetic compatibility,
Both Western-blot detections compatibility combination is respectively adopted first to the ionotropic glutamate for mediating excititoxic effect
The expression of type nmda receptor and the influence of phosphorylation.As shown in figure 4, Reperfu- sion 24 was as a child, infarct border area brain tissue is taken to carry out
Western-blot, discovery ischemic injuries dramatically increase the expression of nmda receptor NR2B hypotype, and NR2A subtype expression is without significant
Variation；Further study show that ischemic injuries are also significantly enhanced NR2B receptor phosphorylation.Tanshinone IIA 1.5mg/kg
Group, lamp-dish flower acetic 2.5mg/kg group and the treatment of compatibility group can reduce expression and its phosphorylation level of nmda receptor NR2B hypotype
(Fig. 4).It is wherein most powerful with the two compatibility effect.
Obvious oxidative damage occurs for ischemic tissue of brain, tanshinone IIA 1.5mg/kg group, lamp-dish flower acetic 2.5mg/kg group and
The treatment of compatibility group can substantially reduce Oxidation Damage Products malonaldehyde (MDA) content in damage brain tissue, increase superoxide dismutase
Enzyme (SOD), glutathione (GSH) are horizontal (table 3).It is wherein most powerful with the two compatibility effect.
Influence (mean+SD) of 3. monomer compatibility of table to brain tissue Antioxidant Indexes
It is handled using SPSS statistical software, the statistics of comparison among groups one-way ANOVA variance analysis
Method；Multiple groups compare between seeing two-by-two with Post-Hoc-Tests LSD method.* P < 0.01 P < 0.05, * * and vacation
Operation group compares；$P < 0.05,$$P < 0.01 is compared with MCAO group.
(3) compatibility agent and medication
1. proportion compatibility and recommended dose
According to mouse test results tanshinone IIA 1.5mg/kg and lamp-dish flower acetic 2.5mg/kg proportion compatibility, it may be assumed that Radix Salviae Miltiorrhizae
Ketone IIA/ lamp-dish flower acetic is 3/5, which is the ratio between mass parts.If being scaled molar ratio, for 1:1.The effective model of ratio
It encloses: tanshinone IIA: 0.3mg/kg-7.5mg/kg, lamp-dish flower acetic 0.5mg/kg-12.5mg/kg.According to mouse/people (70
Kilogram) body surface area conversion, recommendation prescription clinic people's dosage is that 30mg/70kg (is calculated, wherein tanshinone IIA according to 3/5 ratio
11.25mg/70kg and lamp-dish flower acetic 18.75mg/70kg).
2. preparation type
(1) oral preparation: can be tablet, capsule, and specification is 30mg/ piece, it is proposed that 1 to 2 tablets once, 3 times a day.
(2) injection: intravenous drip, daily overall control is between 100mg-200mg.
(4) safety is investigated
Acute toxicity: acute toxicity is detected using Bliss method intragastric administration on mice, does not measure LD50.It is calculated according to effective dose,
Any toxic side effect is had no at 400mg/kg (for 100 times of mouse effective dose).
(5) conclusion
The present invention utilizes cytology and whole animal research, using effective component in Factorial Design discovery Radix Salviae Miltiorrhizae and fleabane flower
Tanshinone IIA and the combination of lamp-dish flower acetic compatibility, have significant synergistic effect, and utmostly play neuroprotection,
Have no obvious toxic-side effects.Its mechanism of action is related with excititoxic effect and anti-oxidative damage is inhibited.
Claims (1)
1. a kind of for treating the Compound Chinese Herbal Monomer Recipe compatibility agent of cerebral ischemia, active pharmaceutical ingredient is by tanshinone IIA and lamp
Small cup flower B prime mixes；
The tanshinone IIA structural formula are as follows:
The structural formula of the lamp-dish flower acetic are as follows:
It is characterized by: the mass ratio of active pharmaceutical ingredient tanshinone IIA and lamp-dish flower acetic is 3 ︰ 5；
Preparation method: active pharmaceutical ingredient tanshinone IIA and lamp-dish flower acetic are extracted by market purchase or raw medicinal material and are obtained, so
It is mixed afterwards according to the ratio that mass ratio is 3 ︰ 5, is prepared into tablet or capsule or injection according to conventional plus auxiliary material；Usage and dosage: piece
Agent, capsule, specification are 30mg/ piece (grain), and adult 1 to 2 tablets once, 3 times a day；Injection: intravenous drip, daily overall control
Between 100mg-200mg.
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