CN107184694A

CN107184694A - The new application of Cortex Dictamni extract

Info

Publication number: CN107184694A
Application number: CN201610144900.2A
Authority: CN
Inventors: 许扬
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2016-03-15
Filing date: 2016-03-15
Publication date: 2017-09-22

Abstract

The present invention relates to a kind of Chinese medical extract, after Dictamnus dasycarpus Turcz water extraction, impurity is removed through ethanol precipitation, is spray-dried and obtains.Chinese medical extract of the present invention has has significant protective effect to Myocytes Anoxia reoxygenation; and myocardial infarction area can be reduced; significantly reduce T sections of myocardial ischemia-reperfusion injury electrocardiogram S rise; improve superoxide dismutase (SOD) activity; reduce the content of MDA (MDA); the activity of creatine kinase (CK) is reduced, improves pathological lesion of the myocardial ischemia-reperfusion to cardiac muscle cell, so as to improve to myocardial ischemia in rats symptom.

Description

The new application of Cortex Dictamni extract

Technical field

The invention belongs to medicine field, and in particular to the new application of Cortex Dictamni extract.

Background technology

In recent years, the incidence of disease of domestic ischemic cardiomyopathy is constantly rising, and the sick pathogenic factor is mainly many reasons and causes coronary blood flow to lower, and then myocardial blood supply is obstructed, nutriment is insufficient and metabolite is removed and reduced, and causes myocardial cell injury even dead.Such as include the foundation and popularization and application of coronary arteriography, PTCA+ stentings, percutaneous balloon mitral commissurotomy, radio-frequency ablation procedure, pacemaker implantation, interventional therapy in congenital heart diseases, coronary artery intracavitary thrombolysis art method with the intervention operation of heart disease, blood reperfusion is retrieved after making heart tissue organ ischemia.In most cases, postischemic reperfusion can recover the normal configuration of damaged myocardium and improve cardiac function, but also there is experiment to show, some researchers are after the Reperfu- sion for making cardiac muscle obtain blood by above treatment means, the function of damaged myocardium is not restored, and degree of injury is aggravated on the contrary, or even the irreversible damages such as infarct size expansion occurs, this phenomenon is the ischemical reperfusion injury (Myocardial ischemia-reprefusion injury, MIRI) of cardiac muscle.

With the treatment technology means of myocardial ischemia raising and application it is extensive, the treatment of cardiomyopathy has been enter into Reperfu- sion period, explores preventing and treating MIRI medicine and turns into important research direction.

With deepening continuously for the study of incident mechanism to MIRI, associated treatment medicine such as Trimetazidine, lidocaine, rapamycin etc. are applied to clinic.But the research and application of Chinese medicine are also limited only to the red sage root and its extract tanshinone, Bao Yuan Tang etc., clinical treatment is high to curative effect, the Chinese medicine of Small side effects demand is huge.

Dictamnus dasycarpus Turcz is the dry root skin of Rutaceae shaggy-fruited dittany platymiscium shaggy-fruited dittany, is Chinese conventional Chinese medicine, also known as northern fresh hide, Zanthoxylum simulans root skin, smelly root skin etc..Modern pharmacology research shows that Dictamnus dasycarpus Turcz has the effects such as heat-clearing and damp-drying drug, dispelling wind and arresting itching and removing toxic substances, to treat the diseases such as dripping yellow water, eczema, rheumatic fever.Relevant report there is no to show that root bark of shaggy-fruited dittany water extract has the effect of the ischemical reperfusion injury for the treatment of cardiac muscle after ethanol extracts precipitation.

The content of the invention

Technical problem solved by the invention is to provide the new application of Cortex Dictamni extract.

Particular content is purposes of the Cortex Dictamni extract in the ischemical reperfusion injury medicine for preparing treatment cardiac muscle, wherein, Cortex Dictamni extract is prepared by following methods：

A, the root bark of shaggy-fruited dittany add water circumfluence distillation 3 times, 1 hour every time, filtration, merging filtrate, being concentrated under reduced pressure into 1: 1, (relative density is 1.05~1.10,70 DEG C), release, it is 50% to add ethanol regulation ethanol content, decompression filtration, filtrate is recycled to no alcohol taste, adds water and is adjusted to suitable concentration, obtains reserve liquid.

B, reserve liquid are by pre-processing the D101 macroreticular resins that finish, and adding water, it is shallow to be eluted to efflux color, merges water lotion, is concentrated under reduced pressure, is spray-dried, obtains Powder Extract.

Inventor passes through Myocytes Anoxia reoxygenation model and rat myocardial ischemia and reperfusion model; drug effect and pharmacological action to Cortex Dictamni extract have carried out system thinking; show that Cortex Dictamni extract has significant protective effect to Myocytes Anoxia reoxygenation; and myocardial infarction area can be reduced; improve superoxide dismutase (SOD) activity; reduce the content of MDA (MDA); reduce the activity of creatine kinase (CK); improve pathological lesion of the myocardial ischemia-reperfusion to cardiac muscle cell, so as to improve to myocardial ischemia in rats symptom.

The formulations such as the formulation that medicine of the present invention can be made into is, capsule, granule, tablet, pill, oral liquid, pill, soft capsule.

Auxiliary material can be used in the capsule, starch, magnesia, magnesium carbonate, calcium monohydrogen phosphate, calcium dihydrogen phosphate, starch slurry, syrup, water, ethanol, superfine silica gel powder, sodium carboxymethyl starch etc..

The auxiliary material that the granule is used can be starch, lactose, dextrin, Icing Sugar, calcium sulfate, sucrose, twenty long, rectangular bag ferment, microcrystalline cellulose, glucose.

The auxiliary material that the tablet is used can be that starch, dextrin, Icing Sugar, calcium carbonate, calcium phosphate, lactose, mannitol, microcrystalline cellulose are disorderly, sodium carboxymethyl starch, talcum powder, magnesium stearate etc..

Auxiliary material can be used in the pill, the poly- ferment class of second two, stearic acid, odium stearate, poloxamer, porphin glyceryl stearate etc..

The auxiliary material that the oral solutions is used can be honey, citric acid, poly yamanashi esters, sodium benzoate, benzoic acid and its salt, sorbic acid etc..

The auxiliary material that the pill is used can be water, honey, syrup, carboxymethyl cellulose, sodium carboxymethylcellulose, hydroxypropyl methyl cellulose etc..

The auxiliary material that the soft capsule is used can be vegetable oil, PEG400, beeswax, glycerine, gelatin, water etc..

The embodiment of form, is described in further detail again to the above of the invention by the following examples.But the scope that this should not be interpreted as to above-mentioned theme of the invention is only limitted to following example.All technologies realized based on the above of the present invention belong to the scope of the present invention.

Embodiment

Below by way of two aspects of pharmacodynamic action of the Cortex Dictamni extract to Myocytes Anoxia reoxygenation and the pharmacological action to myocardial ischemia-reperfusion injury, beneficial effects of the present invention are illustrated to the preclinical study of root bark of shaggy-fruited dittany extract for treating myocardial ischemia-reperfusion injury.

The pharmacodynamic experiment of 1 Cortex Dictamni extract

1.1 test materials and instrument

The root bark of shaggy-fruited dittany (is purchased from Anguo medicinal material market, this institute professor Chang Qi identifies), H9c2 cardiac muscle cell's strain (fundamental research institute of Chinese Academy of Medical Sciences cell resource center), DMEM high glucose mediums (Gibco companies of the U.S.), hyclone (Gibco companies of the U.S.), BSA (Amersco companies of the U.S.), injection sound mycin sodium (HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory), streptomycin sulphate for injection (North China pharmacy group), CO2 constant incubators (Thermo companies of the U.S.), super quiet workbench (AirClean companies of the U.S. of the U.S.), inverted microscope CKS31 (Japanese Olympus company), the types of enzyme-linked immunosorbent assay instrument Model 550 (Bio-Red companies of the U.S.).Rat cTn-IElisa detection kits (RD companies of the U.S.), lactic dehydrogenase (LDH) kit, superoxide dismutase (SOD) kit, MDA (MDA) kit (Bioengineering Research Institute is built up in Nanjing).Caspase-3 spectrophotometries kit (Nanjing KaiJi Biology Science Development Co., Ltd).JC-1, Hoechst33342, MTT, pancreatin (Sigma Co., USA).Mitochondrial cytochrome C detection kits (USCN companies of the U.S.), mitochondrial protein extracts kit (the green skies biotech company in Shanghai), Annexin V/PI anti-apoptotic detection kits (U.S. company BD).The antibody such as Bcl-2, Bax (CST companies of the U.S.), horseradish peroxidase Goat anti-mouse antibodies (Beijing CoWin Bioscience Co., Ltd.).

1.2 experimental method

1.2.1 cell culture

1.2.1.1 recovery

The DMEM high glucose mediums 10mL containing 12% hyclone (FBS) is prepared in advance in super-clean bench into 15mL centrifuge tubes.Hold cell cryopreservation tube taken out from liquid nitrogen container after, rapid be put into 37 DEG C of warm water is ceaselessly stirred, until after ice cube all melts, quickly cell is transferred in preprepared centrifuge tube, cell is set to be sufficiently mixed with culture medium, 1000 turns/min centrifugations 5min.Supernatant is abandoned in suction, adds the DMEM high glucose mediums of the hyclones of 4mL 12%, after gently mixing, all moves to blake bottle, and it is 5% to be put into CO2 contents, and temperature is is cultivated in 37 DEG C of cell culture incubator.After 8h, cell state is observed, liquid is changed once.The culture medium of 10% hyclone can be changed into according to the state of cell later.

1.2.1.2 passage

When Myocyte growth is fused to 80%~90%, you can take out and passed on from incubator.First, inhale and abandon original culture medium, be rinsed once with 4mL PBS.Exhausted completely after PBS with suction pipe, add 2mL 0.125% pancreatin, after rapid mixing, be put into cell culture incubator and digest 1min.Micro- Microscopic observation cell shape, when space between cells increase, cellular morphology shrinkage is rounded, during bright color, and pancreatin is abandoned in suction, is rapidly added the complete medium that 4mL contains 10% hyclone, terminates pancreatin digestion.Cell is gently blown and beaten repeatedly with suction pipe, is allowed to come off from bottle wall completely.After piping and druming is uniform, is moved into according to 1: 2 ratio sub-bottle in new blake bottle, be put into incubator and cultivated.

1.2.2 the foundation of Hypoxia-reoxygenation model

After H9C2 cardiac muscle cell's bed board, carried out in incubator normally cultivating 24h with complete medium.When cell enters logarithmic phase, orifice plate is covered with substantially, anoxic treatment is now carried out.Anoxic treatment method is：Complete medium is abandoned in suction, is added after anoxic liquid, is put into anaerobism gloves constant incubator.

After anoxic treatment certain time, reoxygenation processing is carried out.Reoxygenation processing method is：Cell is taken out from anaerobism gloves incubator, anoxic liquid is abandoned in suction, and addition is diluted to certain density medicine with complete medium, is put into normal incubator and is cultivated.Certain time is cultivated, after reoxygenation terminates, the experimental arrangement in downstream is carried out.

1.2.3 experiment packet

H9C2 cardiac muscle cell into experiment is randomly divided into blank control group (Con groups), hypoxia-reoxygenation group (H/R), the total water extract various dose treatment group of the root bark of shaggy-fruited dittany (H/R+Cortex Dictamni aqueous extract), and the specific processing mode of each group is as follows：

Blank control group：Whole experiment process uses complete medium, is positioned in cell culture incubator and normally cultivates.

Model group (H/R groups)：Handled according to above-mentioned hypoxia-reoxygenation condition, reoxygenation after anoxic 6h, in reoxygenation, culture 12h is carried out with the complete medium of not drug containing.

H/R+ drug-treated groups：Handled according to above-mentioned hypoxia-reoxygenation condition, reoxygenation after anoxic 6h, in reoxygenation, addition complete medium dilutes Cortex Dictamni extract to finite concentration, is put into normal incubator and cultivates 12h.

1.2.4 the establishment of anoxia/reoxygenation model

H9C2 cardiac muscle cell is with 1 × 10⁴Individual/hole is inoculated in 96 orifice plates, cultivate 24h to cell confluency be 80%~90% after, with anoxic liquid replace normal incubation medium after, be put into anaerobism gloves constant incubator, it is pure nitrogen gas and gaseous mixture (hydrogen 5%+ nitrogen 95%) to carry out gas in anoxic 6h, incubator.96 orifice plates are taken out from anaerobic box, anoxic liquid is exhausted, adds to be put into normal incubator after complete medium and is cultivated, as reoxygenation.The reoxygenation time is respectively 8h, 12h, 16h, 24h.After reoxygenation terminates, the vigor of cell is determined by MTT methods, so that it is determined that can guarantee that the Hypoxia-reoxygenation model successful optimal reoxygenation time.

1.2.5 the determination (MTT methods) of Cortex Dictamni extract the best use of concentration

After cell dissociation in blake bottle, it is resuspended and is counted with complete medium, equivalent (1 × 104/hole) is inoculated into 96 orifice plates, is 100 μ L per hole nutrient solution volume, puts 37 DEG C, 5%CO₂Incubator culture 48h reaches that cell 80~90% is merged.Former culture medium is abandoned, PBS is rinsed twice.The cell cultivated in each hole of culture plate is randomly divided into Con groups, H/R groups, H/R+Cortex Dictamni aqueous extract (0.4 μ g/mL, 1.56 μ g/mL, 6.25 μ g/mL, 25 μ g/mL, 100 μ g/mL).Blank well is seen as with acellular hole.After being handled respectively according to experiment group technology, at the end of reoxygenation, the 5mg/mL μ L of MTT 20 are added per hole into culture medium, 4h is cultivated in cell culture incubator, then inhale and abandon culture hole supernatant, the μ L/ holes of dimethyl alum (DMSO) 150 are added, shaking table is placed and slightly shakes after 20min, the OD values under 570nm wavelength are determined with ELIASA.The decline of absorbance is considered the decline of cell viability, and the cell viability of Normal group regards 100% as.Every group of 6 multiple holes, experiment is repeated 3 times.

1.2.6 LDH is determined in cell supernatant

When each experimental port cell reoxygenation is completed, cell supernatant is collected with pipettor, is operated according to assay method in LDH kits, as shown in following table Tab1.

Tab 1 Preparation procedure of solution for coenzyme I and substrate buffer

Calculation formula：

1.2.7 Troponin I is determined in culture cell supernatant

1.2.7.1 experimental procedure

(1) lath needed for being taken out from aluminium foil bag, equilibrium at room temperature 20min.

(2) standard sample wells and sample aperture are set, and standard sample wells respectively adds the μ L of standard items 50 of various concentrations.

(3) sample aperture first adds the μ L of sample to be tested 10, then adds the μ L of Sample dilution 40, and blank well is not added with.

(4) in addition to blank well, the μ L of detection antibody 100 of horseradish peroxidase (HRP) mark are added in standard sample wells and sample aperture per hole, reacting hole is sealed with shrouding film, 37 DEG C of insulating boxs incubate 60min.

(5) discard and patted dry on liquid, blotting paper, cleaning solution is filled it up with per hole, stand 1min, get rid of and patted dry on cleaning solution, blotting paper, so repeat board-washing 5 times.

(6) substrate A, each 50 μ L of B are added per hole, 37 DEG C of lucifuges are incubated 15min.

(7) added per hole in terminate liquid 50 μ L, 15min, the OD values in each hole are determined at 450nm wavelength.

1.2.7.2 calculate

Using standard concentration as abscissa, corresponding OD values are ordinate, draw out standard items linear regression curves, cTn-I concentration values in each sample are calculated according to curvilinear equation.

1.2.8 SOD determinations of activity in cell supernatant

1.2.8.1 reagent composition is with preparing

The preparation of substrate application liquid

Reagent one：Buffer solution, 4 DEG C of preservations.

Reagent two：Stock substrate, 4 DEG C of preservations.

Stock substrate and buffer solution are mixed in 1: 200 ratio, are made into substrate application liquid, and matching while using matches somebody with somebody how many, exhaustless 4 DEG C can preserve 7 days with how many.

The preparation of enzyme working solution

Reagent three：Enzyme stock solution, 4 DEG C of preservations.

Reagent four：Enzyme dilution, 4 DEG C of preservations.

Enzyme stock solution and enzyme dilution are mixed in 1: 10 ratio, are made into enzyme working solution, and matching while using matches somebody with somebody how many, exhaustless 4 DEG C can preserve 3 days with how many.

1.3.3.2 table is operated

The configuration process of enzyme working solution, substrate application liquid and enzyme dilution is as shown in Tab 2.

Tab 2 Preparation procedure of solutionfor enzyme and substrate

Note：Control, control blank, a collection of experiment of measure blank only need to respectively do 1-2 hole.

1.2.8.2 computational methods

1.2.9 MDA assays in cell supernatant

1.2.9.1 preparation of reagents

Reagent one：1 bottle of 20mL liquid, 4 DEG C of preservations.

Reagent two：1 bottle of 12mL liquid, the used time adds 340mL distilled waters to mix, and 4 DEG C of refrigerations (note：It should not encounter on skin).

Reagent three：Pulvis is added in 90 DEG C~100 DEG C of hot distilled water 60mL by 1, pulvis, the used time, can suitably be heated in course of dissolution, and 60mL acetic acid 60mL on the rocks again are fully complemented to distilled water after dissolving, are mixed, the reagent lucifuge room temperature preservation prepared.

Standard items：10nmol/mL tetraethoxypropanes 5mL, 4 DEG C of refrigerations.

1.2.9.2 operation

After reoxygenation terminates, cell supernatant is drawn, kit specification is determined according to MDA, the measure of MDA contents is carried out, refers to Tab 3.

Tab2 Operating procedure of the measurement of MDAcontent

Swirl mixing device is mixed, and test tube mouthful is tightened with antistaling film, and an aperture is pierced with syringe needle, 95 DEG C of water-baths (or uncapped with water-bath boil) 60min, and flowing water is cooled down after taking-up.Then at 532nm, the absorbance of each pipe is determined with ELIASA.

1.2.9.3 computational methods

1.2.10 mitochondrial membrane potential is determined

(1) after the completion of each group cell is handled as requested, the cell culture fluid volley of rifle fire is gently inhaled and abandoned, plus appropriate PBS is washed 2 times.

(2) 100 μ L, 2 μm of ol/L JC-1 dyestuffs are added per hole, incubator is put in and is incubated 30min.

(3) gently inhaled with the volley of rifle fire and abandon supernatant containing dyestuff and cell is slowly washed with PBS 3 times, added the μ g/mL Hoechst33342 dyestuffs of 100 μ L 10 and be put in incubator incubation 15min.

(4) it is incubated after 15min, gently discards supernatant containing dyestuff and washed with PBS 3 times.

(5) after the washing of each group cell is finished, 100 μ L PBS is added per hole and pass through machine testing in high intension.

(6) high each parameter of intension is adjusted, observes and takes pictures, picture is obtained.

1.2.11 in endochylema cromoci content measure

(1) mitochondria extracts kit is utilized, suppressor proteins are obtained according to kit operating procedure.- 20 DEG C of refrigerators preserve stand-by.

(2) illustrate according to cromoci detection kit, standard items gradient series are diluted, gauge orifice, blank well and sample well is determined.

(3) 100 μ L standard dilution, blank solution and sample are separately added into the hole positioned.Sealed with shrouding film, 37 DEG C of incubation 2h.

(4) inhale and abandon liquid in hole, each hole adds 100 μ L detection reagent A working solutions, is sealed with shrouding film, 37 DEG C of incubation 1h.

(5) inhale and abandon liquid in every hole, 350 μ L1 × cleaning solution is added per hole, stand 1~2min, get rid of and patted dry on cleaning solution, blotting paper.So it is repeated 3 times.Last time must eliminate moisture.

(6) 100 μ L detection reagent B working solutions are added, are sticked after shrouding film, 37 DEG C of incubation 30min.

(7) repeat (5), wash 5 times.

(8) 90 μ L matrix liquids are added, are sealed with shrouding film, 37 DEG C are incubated 15~25min (being no more than 30min).

(9) 50 μ L terminate liquids are added.Liquid now starts to become yellow.The another side of plank is gently patted, it is mixed.

(10) determine each hole absorbance at 450 nm with ELIASA, record and make standard curve.Using standard curve, to calculate the content of cromoci in each sample cytoplasm.

1.2.12 spectrophotometry Caspase-3 activity expression

(1) cell or tissue cracking supernatants of the 50 μ L containing 100~200 μ g albumen is drawn；If tissue is less than 50 μ L, the μ L of cumulative volume 50 are complemented to Lysis Buffer (each group is measured and compared using same protein content)；

(2) 2 × Reaction Buffer for adding 50 μ L (note：0.5 μ LDTT are added using preceding every 50 μ, 2 × Reaction of L Buffer)；

(3) 5 μ L Caspase-3 Substrate are added and 4h are incubated in 37 DEG C of lucifuges；

(4) its OD value is determined in wavelength 405nm with ELIASA.By calculating OD_Derivant/OD_{Negative control}Multiple determine inducer of apoptosis group Caspase-3 activation degree.

Note：Blank control is used as using Lysis Buffer and Reaction Buffer.

1.2.13 FITC-Annexin V/PI are double contaminates flow cytomery Apoptosis

(1) handled respectively after each group according to experimental method, cell is washed with PBS twice, after being digested to cell, neutralized with complete medium, collect cell in centrifuge tube, 1000 turns/min centrifugations 5min.

(2) after abandoning supernatant, plus appropriate PBS is resuspended cell and counted.105 cells are taken in centrifuge tube, after resuspension, 1000 turns/min centrifugation 5min, abandoning supernatant.

(3) cell is gently resuspended in the Binding Buffer for adding 100 μ L, adds 5 μ L FITC-Annexin V and 5 μ L PI, and room temperature lucifuge is incubated 15min.

(4) the Binding Buffer for adding 400 μ L on before machine are gently mixed, you can upper machine testing.

1.2.14 Western blotting detect correlative protein expression

H9C2 cardiac muscle cell's total protein extraction：

(1) each group cell is after processing terminates as requested, digested with tryptic digestive juice and collect each group cell with centrifuge tube.

(2) cell is collected by centrifugation, condition is 1000 turns/min, centrifuges 5min.

(3) the mammalian proteins extraction agent containing 1% protease inhibitors PMSF (phenylmethylsulfonyl fluoride) of appropriate (about 10 times of amounts) is added, after fully being mixed with cell, placement cracks 40min on ice.

(4) after the completion of cracking, centrifuged at 4 DEG C, centrifugal condition is 12000 turns/min, 20min.

(5) after the completion of centrifuging, take supernatant to dispense, preserve stand-by under the conditions of -20 DEG C.

BCA method protein quantifications：

(1) measure of BSA standard curves：Operation dilution BSA standard items are carried out by kit explanation.

(2) BCA working solutions are configured：According to the quantity of BSA standard items and testing sample, appropriate reagent A and reagent B are taken, is mixed according to 50: 1 volume stand-by.The BCA working solutions room temperature newly prepared can be stablized under confined conditions preserves 24h.

(3) micropore detection sample protein concentration：Each 25 μ L of the BSA standard items and sample that have diluted (stoste or dilution) are added separately in 96 labeled orifice plates.200 μ L BCA working solutions are added per hole, are fully mixed, lucifuge, 37 DEG C of incubation 30min.It is cooled to after room temperature, the light absorption value at wavelength 562nm is determined with ELIASA.According to BSA standard curves, the protein concentration (scope in sample is calculated：20-2000μg/mL).

(4) surveyed after protein concentration, it is applied sample amount to calculate the protein solution volume containing 20~40 μ g albumen (testing applied sample amount with batch identical).SDS-PAGE sample-loading buffers and protein sample are mixed by 1: 4,5~10min is boiled in boiling water bath, makes albuminous degeneration, ice bath 1min coolings, -20 DEG C of preservation samples are stand-by.Used time directly takes appropriate loading.

Polyacrylamide gel electrophoresis：

(1) glass plate, comb, neck etc. are cleaned and is dried stand-by.Glass plate after alignment is put into vertical card in folder and on the top of the shelf, after clamping, prepares encapsulating, preventing from cementing leakage.

(2) preparation of separation gel.Determine after resolving gel concentration that volume needed for being selected according to operating instruction mixes a certain proportion of 30%Acri-Bis (29: 1), separation gel buffer solution and distilled water in test tube according to molecular weight of albumen size.Add 10%APS (matching while using) to mix, add a certain amount of TEMED, poured into after rapid mixing in gel mold.Mould is filled with distilled water again, bubble is driven out of.At least 30min is stored at room temperature, is occurred between glue and distilled water to be separated behind clearly interface, shows that gel is aggregated good.The distilled water on separation gel upper strata is gently sucked with filter paper, is careful not to encounter glue surface.

(3) preparation of glue is concentrated.According to required volume, a certain proportion of 30%Acri-Bis (29: 1), concentration glue buffer solution and distilled water are mixed in test tube.Add 10%APS (matching while using) to mix, add a certain amount of TEMED, after rapid mixing, concentration glue is slowly added on separation gel from an angle, until filling it up with mould.During comb is gently inserted concentration glue from one end, flatten, bubble-free in comb holding level, concentration glue is made during Boxwood comb.

(4) loading.Electrophoresis liquid will be filled it up with the middle of two clotting offset plates, stripping fork is carefully extracted straight up.10 μ L samples are slowly added in gel pore with micropipettor.

(5) electrophoresis

Gel neck is put into electrophoresis tank and appropriate electrophoresis liquid is added in electrophoresis tank, is turned on the power, regulation voltage is between 70-80V, constant pressure electrophoresis concentration glue.When sample enters lower floor's separation gel, regulation voltage to 110V, constant pressure electrophoretic separation glue.After electrophoresis terminates, concentration glue is cut, stays separation gel to carry out transferring film.

(6) transferring film

A, standby film.After electrophoresis terminates, the sizableness of NC films and filter paper, NC films and glue is cut with scissors, but it is bigger, and the size of filter paper should be slightly less than NC films, and the NC films and filter paper that cut are placed in transferring film buffer solution and soak 15min.

B, dress film.By NC films, sponge pad, filter paper the saturation 15min in transferring film buffer solution in advance.The clip of transferring film is opened, stacked successively according to the order of clip black flour-filter paper of fiber mat-1-clip fine flour of one fiber mat of filter paper one of gel-NC films one 1, last clip clamping puts it into rapidly in transferring film groove, fills transferring film buffer solution, prevent bubble.

C, transferring film.Transferring film groove is placed in mixture of ice and water, 50~70min of 110V constant pressure transferring films is used according to molecular weight of albumen (molecular weight is bigger, and required time is longer).

(7) it is immunized and combines

A, closing.NC films are rinsed 3 times with TBS-T, NC films are put into the plate added with 5% skimmed milk power by each 15min after rinsing, it is slow on shaking table at room temperature to shake 2h.

B, primary antibody are incubated.NC films after closing are put into TBS-T, shaking table is rinsed 3 times, each 15min.Film is drained after washing as far as possible, is put into the hybridization bag containing primary antibody, hybridization bag is sealed with sealing machine, 4 DEG C are put into refrigerator overnight.

C, secondary antibody are incubated.The NC films being incubated by primary antibody are rinsed 3 times in TBS-T, then each 15min adds in the plate containing secondary antibody, be incubated 2h on shaking table at room temperature.

D, development.After secondary antibody incubation terminates, rinsed three times with TBS-T, each 15min.The ECL now matched somebody with somebody is lighted into working solution dropwise addition on film, room temperature lucifuge is incubated timing 5min, is scanned, made film with gel imaging system after colour developing.

1.2.15 statistical analysis

All enumeration datas are represented using mean ± standard deviation (mean ± SD), statistical analysis is carried out using statistic software SPSS 18, it is compared in one-way analysis of variance (One-way ANOVA) method, is to have statistically significant gender gap with P ＜ 0.05.

1.3 experimental result

1.3.1 the foundation of H9C2 Myocytes Anoxias reoxygenation model

In anoxic 6h, on reoxygenation different time point, the survival rate of cell is determined by MTT methods, as shown in figure 1, between 8~24h of reoxygenation, the change of cell survival rate have passed through two stages.First stage is the stage of reoxygenation, and cell survival rate is gradually reduced, and during to reoxygenation 12h, its survival rate has reached minimum under conditions of anoxic 6h, is 51.3%；Second stage is after reoxygenation 12h, cell is cultivated under normal operation, has gradually broken away from influence of the hypoxia-reoxygenation to it, the process that its growth conditions gradually takes a turn for the better.

Analyzed from result above, this experiment takes anoxic 6h, reoxygenation 12h to study cellular damage the most serious time point, cell survival rate now is 50% or so, the influence of other factors is eliminated, to can preferably show the curative effect of medicine.

1.3.2 to the influence of H9C2 cell survival rates

At the end of anoxic, the Cortex Dictamni extract of various dose is given, the cell survival rate of each dosage group and model group is determined by MTT methods.Compared with H/R groups, the survival rate of Cortex Dictamni extract various dose administration group cell has different degrees of increase.Wherein, the μ g/mL of small dose group 0.4 cell survival rate has increased trend, but statistical result is shown compared with H/R groups, and there was no significant difference.And equally compared with H/R groups, the cell survival rate of 1.56 μ g/mL groups has significant difference (P ＜ 0.05), and 6.25 μ g/mL, 25 μ g/mL, 100 μ g/mL, tri- dosage groups have more significant sex differernce (P ＞ 0.01).And be compared to each other between 6.25 μ g/mL, 25 μ g/mL, 100 μ g/mL, tri- dosage groups, then without significant difference (P ＞ 0.05) (Fig. 2).

According to the result of this experiment; show that each dosage group of Cortex Dictamni extract has certain protective effect to the H9C2 cardiac muscle cell of anoxia/reoxygenation; in follow-up experiment; it have selected 1.56 μ g/mL, 6.25 μ g/mL to be studied respectively as low dosage and high dose group, be expressed as H/R+L and H/R+H.

1.3.3 the measure of LDH contents

Tetra- groups of Con, H/R, H/R+L, H/R+H LDH burst sizes have been investigated respectively.From measurement result it can be seen that, compared with H/R groups, each dosage group can effectively reduce the LDH burst sizes (P ＜ 0.01) in culture cell supernatant, and with the increase of administration concentration, LDH burst sizes are gradually decreased (Fig. 3).

1.3.4 the measure of cardiac troponin (cTnI)

By the escaped quantity for determining cTnI in cell supernatant after anoxia/reoxygenation, measurement result shows, it is compared with H/R groups, H/R+L, H have conspicuousness to reduce (P ＜ 0.01 in Cortex Dictamni extract dosage group, P ＜ 0.01), i.e., each dosage group of Cortex Dictamni extract can significantly mitigate degree of injury (Fig. 4) of the hypoxia-reoxygenation condition to cardiac muscle cell.

1.3.5 to the influence of SOD vigor in cardiac muscle cell's supernatant of anoxia/reoxygenation

Compared with Con groups, the SOD vigor in H/R group cell supernatants has conspicuousness to reduce (P ＜ 0.01).Each dosage group H/R+L, H of Cortex Dictamni extract, the vigor (P ＜ 0.01) of SOD in H9C2 cardiac muscle cell's supernatant can be significantly improved, and with the increase of Cortex Dictamni extract dosage, SOD vigor gradually strengthens (Fig. 5) in supernatant.This explanation Cortex Dictamni extract can effectively raise the activity of SOD in cardiac muscle cell's supernatant of anoxia/reoxygenation, and with certain concentration dependent, dosage is higher, acts on more obvious.

1.3.6 to the influence of MDA contents in cardiac muscle cell's supernatant of anoxia/reoxygenation

H/R groups are compared with Con groups, and MDA content is significantly higher in H/R cell supernatants.And MDA contents have conspicuousness to decline (P ＜ 0.01, P ＜ 0.01) in H/R groups and each dose comparison of Cortex Dictamni extract, the cell supernatant of two administration groups, and in dose-dependent relationship (Fig. 6).Show that Cortex Dictamni extract can substantially reduce the MDA contents of supernatant in the cardiac muscle cell of anoxia/reoxygenation.

1.3.7 to the influence of cardiac muscle cell's mitochondrial membrane potential of anoxia/reoxygenation

Experiment detects the mitochondrial membrane potential of cell using JC-1 fluorescence probe methods, and the decline of mitochondrial membrane potential is the hallmark events of Apoptosis early stage.Pass through transformations of the JC-1 from red fluorescence to green fluorescence, the decline of cell membrane potential can be reliably detected, transformations of the JC-1 from red fluorescence to green fluorescence can also be used simultaneously, and as the Testing index of Apoptosis early stage, specific measurement result is as shown in Fig 8.Test result indicates that, H/R group mitochondrial membrane potentials are significantly reduced, and Apoptosis ratio is significantly raised.And each dosage administration group of the root bark of shaggy-fruited dittany can significantly inhibit the reduction of film potential caused by Myocytes Anoxia reoxygenation, Apoptosis ratio is significantly reduced, and with dose dependent (Fig. 7).

1.3.8 the expression quantity of cromoci

Pass through the burst size of cromoci in each group endochylema in EUSA (ELISA) determination experiment, measurement result shows, compared with CON groups, the burst size of cromoci is dramatically increased (P ＜ 0.01) in H/R group endochylemas；And compared with H/R groups, each dosage group of Cortex Dictamni extract (H/R+L and H/R+H) significantly reduces content (the P ＜ 0.01 of cromoci in endochylema；P ＜ 0.01), and with the increase of administration concentration, the content for the cromoci that mitochondria discharges into endochylema gradually lowers, and has certain concentration dependent (Fig. 8).

1.3.9 influence active the cardiac muscle cell Caspase-3 to anoxia/reoxygenation

Caspase-3 is that most important apoptosis performs albumen in Caspase families, is main apoptosis effect molecule in apoptotic process, and its activation marker Apoptosis and enters the irreversible stage.The testing result of Caspase-3 activity is as shown in Fig10, by the way that compared with Con groups, the activation levels of the Caspase-3 in H/R have the rise of highly significant (P ＜ 0.01)；Each dosage group is compared with H/R groups, and the Caspase-3 of activation has different degrees of reduction, and this effect has dose-dependent relationship (Fig. 9).Show that the activation that Anti-G value that Cortex Dictamni extract has may be with suppressing Caspase families cascade reaction has relation.

1.3.10 to the influence of the apoptosis rate of anoxia/reoxygenation

This experiment carries out the detection of apoptosis rate by flow cytometer, specific result of the test is as shown in Tab 4, Figure 10 using the method for the double dyes of FITC-Annexin V/PI.Compared with blank control group, model group apoptosis rate has reached 8.80 ± 1.09, and statistical analysis has significant difference.And compared with model group, the apoptosis rate of each administration group of Cortex Dictamni extract has a conspicuousness reduction, only 3.14 ± 0.57 and 2.79 ± 0.30, and apoptosis rate reduces with the increase of administration concentration.Illustrate that Cortex Dictamni extract is inhibited to the cardiac muscle cell apoptosis of anoxia/reoxygenation.

Tab 3 Effects of aqueous extract of Cortex Dictamni on H/R-induced H9C2 myocardial cells apoptosis.

Note：Datawere presented as means±SD from three independent experiments.

The vs H/R of *, P ＜ 0.01；The vs Con. of ##, P ＜ 0.01

1.3.11 to the influence of the cardiac muscle cell apoptosis expressing quantity of anoxia/reoxygenation

Pass through Western Blot experimental method, Cortex Dictamni extract be have studied to being related to the influence of expression of apoptosis protein amount, result of the test shows, compared with Con groups, the expression quantity of pro apoptotic protein Bax in H/R groups is significantly raised (P ＜ 0.01), and anti-apoptotic proteins Bcl-2 expression quantity is then remarkably decreased, so that Bcl-2/Bax ratio reduction, promotes Apoptosis.Compared with H/R groups, each dosage administration group of Cortex Dictamni extract can significantly inhibit the increase (P ＜ 0.05) of the downward of Bcl-2 expression or Bax expression caused by hypoxia-reoxygenation, the Bcl-2/Bax made ratio rise (P ＜ 0.05), it is suppressed that Apoptosis (Figure 11).

1.4 conclusion

Cortex Dictamni extract effectively improves the survival rate of the rat myocardial cell of anoxia/reoxygenation, reduction damaged cell LDH, cTn-I burst size, and has certain concentration dependent.

Cortex Dictamni extract significantly increases H9C2 cardiac muscle cell's antioxidase SOD of anoxia/reoxygenation activity, so as to reduce lipid within endothelial cells Peroxidation Product MDA content, the protective effect to hypoxia-reoxygenation cell may be played by Antioxidation Mechanism.

Cortex Dictamni extract have to the cardiac muscle cell of hypoxia-reoxygenation it is significant suppress apoptotic effect, and the mechanism of its Anti-G value is related to multiple links of the mitochondrial pathways, it may be possible to produce significant Anti-G value to cell by mitochondria pathway.

2. the pharmacological experiment of Cortex Dictamni extract

2.1 materials and methods

2.1.1 experiment material

Animal：

SPF grades of Wistar rats 48, male, body weight (130 ± 10) g ties up the magnificent experimental animal company of tonneau by Beijing and provides [SCXK (capital) 20124) 001].Raise in [SYXK (capital) 20084) 019] in China Medical Sciences Academy Medical Plants Institute SPF grades of Animal House.Keep 14h illuminations, circulation environment dark 10h and is ingested at free water.

Instrument：

The road physiological signal record system of MP150 types 16 (Biopac companies of the U.S.)；DM4000B types are just putting fluorescence microscope and imaging system (German Leica companies)；Heraeus Labofuge400R types high speed freezing centrifuge (German Thermo Scientific companies)；MQX200 types ELIASA (Bio-Tek companies of the U.S.)；CM1900 types freezing-microtome (German Leica companies)；ALC-V8S type toys lung ventilator (Shanghai Alcott bio tech ltd)；U725 types ultra low temperature freezer (NBS companies of the U.S.).

Reagent：

Creatine kinase (CK), superoxide dismutase (SOD), Serum MDA (MDA) detection kit (bio tech ltd is built up in Nanjing), red tetrazolium (TTC) (U.S. Amresc.Company), Diaoxinxue Kang (Diao Group Chengdu Pharmaceutical Co., Ltd., lot number：1109006).

2.2 experimental method

2.2.1 animal packet

48 rats are randomly divided into 6 groups, every group 6.Respectively

Sham-operation group (Sham Operation, SO), model group (model group, MG), root bark of shaggy-fruited dittany low dose group (CDL), middle dose group (CDM), high dose group (CDH), and positive drug group (positive drug control group, PC).

2.2.2 medication

Cortex Dictamni extract bacterium distilled water is dissolved, be made into basic, normal, high three dosage be respectively 0.128,0.64,1.28g/ (Kg*bw) dosage is administered], positive drug group gives Diaoxinxue Kang 0.054g/ (kg*bw) (by the conversion of adult's dosage), and sham-operation group presses the solvent gavage of same dose with model group.The daily gastric infusion of each group 1 time.Weigh weekly 1 time, dosage, successive administration 15 days are adjusted in time.

2.2.3 prepared by model

With 20% urethane [1000mgKkg*bw)] anesthetized rat, face upward position and fix, it is subcutaneous that fixed Acupuncture needle electrode inserts in four limbs, connects electrocardiograph monitoring standard limbs n lead electrocardiogram.Row endotracheal intubation, connects animal respirator positive pressure respiration (75 beats/min of frequency, tidal volume 5mL/min, inspiratory/expiratory 2: 1).The 3rd~4 intercostal opens chest on the left of breastbone, cuts off pericardium, exposure heart.Coronary artery left anterior descending branch is found out between pulmonary conus and left auricle of heart, and is placed under ramus descendens anterior arteriae coronariae sinistrae initial part with the circular noninvasive silk thread of sewing needle 5/0 at 2mm and ligatures.In addition to except sham-operation group, only threading is not ligatured, remaining each group is ligatured.After following coronary artery occlusion left anterior descending branch, alarmmed with electrocardiogram II lead T wave height merge with QRS wave, the broaden towel width that shakes of QRS wave is increased and indicated for ligation is successful.Ligature, Reperfu- sion 120min are unclamped after ligation 30min.

2.2.4 the measurement of ECG ST-T section

Rat anesthesia is faced upward after the fixation of position, and needle electrode connection MP150 type electrocardiographs are subcutaneously inserted in four limbs.Continuous cardioelectric monitor, and trace successively before ligation, ischemic 30min and distant lead electrocardiogram (chart speed 50mm/s, gain 1mV=10mm) during Reperfu- sion 120min.

2.2.5 rat blood serum CK, SOD activity and Content of MDA measure

After Reperfu- sion terminates, rat is put to death, immediately abdominal aortic blood, 3500r/min centrifugation 10min separation serum.Method determines indices as shown in kit specification.

2.2.6 after rat heart muscle TTC dyeing and myocardial infarction area measure Reperfu- sion terminate, win heart, hematocele in heart is cleaned with physiological saline, remove blood vessel, fat, right ventricle, to the apex of the heart below self-ligating line, uniformly five are cut into by heart is parallel, myocardium piece is inserted in 1%TTC solution, 37 DEG C of dyeing 10mm, coloration result is shot and stored, the data of storage are read in Image Pro plus (version 6.0, Media Cybernetics companies of the U.S.), the infarct size and the gross area of every piece of myocardium piece is calculated.According to myocardial infarction area/calculating myocardium infarct rates of total myocardial area x 100%.

2.2.7 rat heart muscle histopathology morphological observation

Core the cardiac muscular tissues of Zang Xia 1/3, the embedding of OCT fixers, makes frozen section.5 sections, HE dyeing, the metamorphosis of optical microphotograph Microscopic observation cardiac muscular tissue are taken from each group.

2.2.8 statistical analysis

In each group of data input computer, using SPSS statistical softwares (version 17.0), variance analysis is carried out according to data requirement.

2.3 result

2.3.1 myocardial ischemia-reperfusion injury myocardial infarction area

Rat left ventricle thin slice dyes the situation that observation infarct occurs through TTC, observe and show under stereomicroscope, model group infarcted region is larger, and the infarcted region after the basic, normal, high dosage group of Cortex Dictamni extract (Figure 12 D, Figure 12 E, Figure 12 F) administration after myocardial ischemia-reperfusion injury is less than model group (Figure 12 B).Infarcted region (white portion) area is counted with ImagePro Plus softwares, calculate the ratio of Infarct area and whole Myolaminar sheet area, as a result show, the ratio of the middle and high dosage group of Cortex Dictamni extract is significantly lower than model group (P ＜ 0.01), and low dose group is compared then no significant difference (P ＞ 0.05) with model group.The middle and high dosage group of Cortex Dictamni extract is compared with positive drug group (Figure 12 C), and myocardial infarction area ratio difference is without conspicuousness (P ＞ 0.05).Control rats cardiac muscle has no obvious infarcted region (Figure 12 A).

2.3.2 the change of myocardial ischemia-reperfusion injury ECG ST-T section

Rat anesthesia layback position is fixed, and records each group electrocardiogram before ligation, the then electrocardiogram after record ligation 30min with Reperfu- sion 2h, the millimeters that ST-T sections of measurement is raised, and calculate the millivolt number that ST-T is raised.As a result show, compared with sham-operation group, model group ischemic 30min and Reperfu- sion 2h ST-T sections greatly drive up (P ＜ 0.01) (Figure 13 B), show modeling successful surgery.

ECG data shows after each administration group administration, and Cortex Dictamni extract high dose group is in ischemic 30min and Reperfu- sion 2h, and ST-T sections of substantially reductions have very significant (P ＜ 0.01) with model group comparing difference.Cortex Dictamni extract middle dose group is in ischemic 30min and Reperfu- sion 2h, and ST-T sections of reductions have conspicuousness (P ＜ 0.05) with model group comparing difference.Cortex Dictamni extract low dose group is in ischemic 30min and Reperfu- sion 2h, though ST-T sections have reduction, with model group comparing difference without conspicuousness (P ＞ 0.05) (Figure 13 G).

2.3.3 after myocardial ischemia-reperfusion injury serum CK activity

Rat aorta blood is gathered at the end of experiment, serum is separated, each group CK activity is determined by kit specification requirement.As a result show, after Ischemia and Reperfusion in vivo in Rats operation terminates, the CK activity of the middle and high dosage group of Cortex Dictamni extract is compared with model group, and difference has conspicuousness (P ＜ 0.01), and Cortex Dictamni extract low dose group then changes (Figure 14) without obvious.

2.3.4 myocardial ischemia-reperfusion injury in rats activity of SOD in serum and MDA contents

At the end of experiment, the blood sampling of each group rat aorta separates serum, requires to determine activity of SOD in serum and MDA contents according to kit specification.As a result show, in model group rats serum SOD activity reduction, MDA contents substantially increase (with sham-operation group ratio, compared with P ＜ 0.01).Compared with model group, after the middle and high dosage group administration of Cortex Dictamni extract, SOD activity is significantly raised (P ＜ 0.05 and P ＜ 0.01) in serum, Cortex Dictamni extract low dose group then no significant difference (P ＞ 0.05)；After the basic, normal, high dosage group administration of Cortex Dictamni extract, MDA content is substantially reduced (P ＜ 0.05 and P ＜ 0.01) (Figure 15) in serum.

2.3.5 the pathological change of myocardial ischemia-reperfusion injury rear myocardium tissue

After each group cardiac muscular tissue HE dyeing, the morphological change of light Microscopic observation cardiac muscular tissue and cell.As a result show, Sham-operated control group cardiac muscle marshalling, structure understands that band is high-visible, core is oval to be located at cell center, and cardiac muscle cell is evenly distributed, myocardial vascular, and cardiac interstitium etc. is into normal configuration (Figure 16 A).Model group cardiac muscle cell's irregular arrangement, structure disturbance, band is fuzzy or disappears, and iuntercellular has inflammatory cell infiltration, local unclear transverse striation of muscle fiber or disappearance, and muscle fibre space is broadening.Focal areas has myolysis change, and visible stove shape liquefaction and necrosis (Figure 16 B).The basic, normal, high dosage group postoperative myocardial HE dyeing of Cortex Dictamni extract shows, the pathological change of cardiac muscle is presented by weight to light change of successively decreasing; lesion degree is substantially lighter than model group; and it is lighter with the middle and high dosage group lesion of Cortex Dictamni extract, cardiac morphology is substantially close to sham-operation group (Figure 16 after the administration of Cortex Dictamni extract water extract high dose group：C-E).

2.4 conclusion

Myocardial infarction area can be significantly reduced after the middle and high dosage group administration of the root bark of shaggy-fruited dittany, substantially during reduction ischemic 30min and Reperfu- sion 120min ST sections raise, and MDA contents in rat blood serum can be reduced, increased SOD activity reduces the myocardial histopathology caused by ischemic and damaged.

Above drug effect and pharmacological experiment show; Cortex Dictamni extract has has significant protective effect to Myocytes Anoxia reoxygenation; and myocardial infarction area can be reduced; significantly reduce the rise of myocardial ischemia-reperfusion injury electrocardiogram S-T segment; improve superoxide dismutase (SOD) activity; reduce the content of MDA (MDA); reduce the activity of creatine kinase (CK); improve pathological lesion of the myocardial ischemia-reperfusion to cardiac muscle cell, so as to improve to myocardial ischemia in rats symptom

Brief description of the drawings

Influence of Fig. 1 difference reoxygenation times to H9C2 cell viabilities

Effect of the Cortex Dictamni extract of Fig. 2 various doses to hypoxia-reoxygenation H9C2 cardiac muscle cell

Each administration group LDH assays of Fig. 3

The measure of each administration group cardiac troponins of Fig. 4 (cardiac troponin I (cTn-I))

Each administration group superoxide dismutase (SOD) determinations of activity of Fig. 5

The measure of each administration group MDA (MDA) contents of Fig. 6

The influence for H9C2 cardiac muscle cell's mitochondrial transmembrane potentials that Fig. 7 Cortex Dictamni extracts are induced hypoxia-reoxygenation

Influence of Fig. 8 Cortex Dictamni extracts to expression of cytochrome C amount

The influence for the H9C2 cardiac muscle cell Caspase-3 activity that Fig. 9 Cortex Dictamni extracts are induced hypoxia-reoxygenation

The influence for the H9C2 cardiac muscle cell apoptosis that Figure 10 Cortex Dictamni extracts are induced hypoxia-reoxygenation

H9C2 cardiac muscle cell Bcl-2, Bax that Figure 11 Cortex Dictamni extracts are induced hypoxia-reoxygenation influence

Influence of Figure 12 Cortex Dictamni extracts to myocardial infarction region and infarct size ratio after ischemia-reperfusion

Influence of Figure 13 Cortex Dictamni extracts to the electrocardiogram ST-T changes during Ischemia and Reperfusion in vivo in Rats

Influence of Figure 14 Cortex Dictamni extracts to activity of serum CK after myocardial ischemia-reperfusion injury

Influence of Figure 15 Cortex Dictamni extracts to activity of SOD in serum after ischemia-reperfusion and MDA contents

Protective effect of Figure 16 Cortex Dictamni extracts to myocardial ischemia-reperfusion injury cardiac muscular tissue.

Claims

1. purposes of the Cortex Dictamni extract in the medicine for preparing treatment myocardial ischemia-reperfusion injury, it is characterised in that the Cortex Dictamni extract It is to be prepared by following methods：

A, the root bark of shaggy-fruited dittany add water circumfluence distillation 3 times, 1 hour every time, filtration, merging filtrate, be concentrated under reduced pressure into 1: 1 (relative density is 1.05~ 1.10,70 DEG C), release, it is 50% to add ethanol regulation ethanol content, and decompression filtration, filtrate is recycled to no alcohol taste, adds water and be adjusted to suitable Concentration, obtains reserve liquid.

B, reserve liquid are by pre-processing the D101 macroreticular resins that finish, and adding water, it is shallow to be eluted to efflux color, merges water lotion, is concentrated under reduced pressure, sprays Dry, obtain Powder Extract.

2. purposes of the Cortex Dictamni extract in the medicine for preparing treatment myocardial ischemia-reperfusion injury according to claims 1, its feature It is：The condition of the heat that added water in step A backflow is the heat backflow 3 times, every time 1 hour that adds water.

3. purposes of the Cortex Dictamni extract in the medicine for preparing treatment myocardial ischemia-reperfusion injury according to claims 1, its feature It is：Powder Extract described in step B is added into pharmaceutically acceptable auxiliary material, the preparation being conventionally prepared into.

4. purposes of the Cortex Dictamni extract in the medicine for preparing treatment myocardial ischemia-reperfusion injury according to claims 1, its feature exists In：The preparation is capsule, granule, tablet, pill, oral liquid, pill, soft capsule.