CN102258530A

CN102258530A - Application of myricitrin in preparation of drugs for treating cardiovascular diseases

Info

Publication number: CN102258530A
Application number: CN 201110187042
Authority: CN
Inventors: 孙晓波; 孙桂波; 潘瑞乐; 秦蒙; 李宗阳; 李明
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2011-07-05
Filing date: 2011-07-05
Publication date: 2011-11-30

Abstract

The invention discloses an application of myricitrin in the preparation of drugs for treating cardiovascular diseases, and belongs to the technical field of medicines. The cardiovascular diseases indicated by the invention comprise acute myocardial ischemic coronary heart disease, heart ischemia-reperfusion injury, hyperlipidemia, and vascular endothelial cell injury. The medicament for treating the cardiovascular diseases can be prepared into a pharmaceutical composition by the use of myricitrin and one or a plurality of pharmaceutically acceptable carriers or excipients.

Description

Myricetrin is used for the treatment of application in the cardiovascular disease medicine in preparation

Technical field

The present invention relates to the application of a kind of medicinal active ingredient in the preparation medicine, relate in particular to the application in the preparation medicine of myricetrin and derivant thereof, belong to medical technical field.

Technical background

Myricetrin, shown in (I) structure, its chemistry is called 3 ', 4 ', 5 ', 5,7-pentahydroxyflavone-3-O-alpha-L-rhamnoside.Myricetrin is the natural polyhydroxy flavone compound, the bark and the leaf that extensively are present in Myruca ceas plant Fructus Myricae rubrae Myrica rubra Sieb.et Zucc., the branch and leaf of leguminous plant Cortes seu Caulis Caraganae jubatae Caragana jubata Poir. are in the aerial parts of polygonaceae plant Herba Polygoni Avicularis Polygonum aviculare L.

Myricetrin has very strong oxidation resistance, can be used as antioxidant and adds in food, medicine and the cosmetics.In addition, myricetrin also have vasoconstrictive, blood sugar lowering, protect the liver, multiple effect such as function of gallbladder promoting, antiinflammatory, mutation, antitumor.

Other purposes of research and development myricetrin with and related derivatives can give full play to the use potentiality of myricetrin, improve its effectiveness.

Summary of the invention

The inventor has found the activity of myricetrin and derivatives for treatment cardiovascular disease thereof, and the treatment cardiovascular disease is had better curative effect.

The invention provides myricetrin and derivant thereof and be used for the treatment of application in the cardiovascular disease medicine in preparation.

Described myricetrin derivant has following structure:

Wherein:

R independently is selected from following group, and this group comprises and replacing or unsubstituted alkyl, replaces or unsubstituted thiazolinyl, replace or unsubstituted cycloalkyl, replace or unsubstituted aryl, replace or unsubstituted amino group, acetoxyl group, hydroxyl, acyl group, alkyl sulphonyl, alkyl sulphinyl, mercapto, alkyl sulfide alcohol radical, and heterocyclic radical group;

R1 is selected from following group, this group comprises and replacing or unsubstituted alkyl, replaces or unsubstituted thiazolinyl, replaces or unsubstituted cycloalkyl, replace or unsubstituted aryl, replace or unsubstituted amino group, and acyl group, alkoxyl, alkyl sulphinyl, alkyl sulphonyl, alkyl sulfide alcohol radical, hydroxyl;

R2 is selected from acyl group, hydrogen, alkyl group.

Described myricetrin derivant is 2-(3,4-dihydroxy, 5-methoxyphenyl)-3,5-dihydroxy-7-ethyoxyl chromone.

Cardiovascular disease of the present invention comprises acute myocardial ischemia coronary heart disease, heart ischemia reperfusion damage, hyperlipemia, the inductive vascular endothelial cell damage of oxidative stress etc.

The invention provides myricetrin and be used for the treatment of application in the cardiovascular disease medicine in preparation, the described medicine that is used for the treatment of cardiovascular disease is made pharmaceutical composition by myricetrin and one or more pharmaceutically acceptable carriers or excipient.Also can make pharmaceutical composition by myricetrin and one or more other active component, one or more pharmaceutically acceptable carriers or excipient.

The amount of the contained active component myricetrin of pharmaceutical composition minimum unit of the present invention is 1.25-10mg.More preferably: 2.5-5mg; Most preferably be 2.5mg.

The treatment cardiovascular disease medicine of myricetrin preparation of the present invention comprises any clinically acceptable forms form.Various dosage forms as oral and parenteral form.Being used for when oral, can be tablet, capsule, soft capsule, oral liquid, syrup, granule, drop pill, oral cavity disintegration tablet, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule etc.; When being used for the parenteral approach, can be liquid drugs injection, freeze-dried powder, aseptic powder injection, transfusion etc.Pharmaceutical composition preferred tablet of the present invention and liquid drugs injection dosage form.

Pharmaceutical composition provided by the invention, described pharmaceutically acceptable carrier or the optional self application of excipient comprise filler, binding agent, lubricant, disintegrating agent, cosolvent, surfactant, absorption carrier etc. in the pharmaceutical excipient of oral formulations.

Pharmaceutical composition provided by the invention, described pharmaceutically acceptable carrier or the optional self application of excipient comprise solvent, antioxidant, cosolvent, adsorbent, osmotic pressure regulator, PH regulator in the pharmaceutical excipient of injection.

The pharmaceutical composition minimum unit is meant a slice, a capsule, one a bag of granule or an injection etc.

Any method that dosage form of the present invention can use pharmaceutical preparation technology to learn the known usual use of those skilled in the art produces and this is not particularly limited.

For example, tablet of the present invention can be by using suitable method granulation, drying well known in the art and sieving main medicament and excipient, binding agent or the like, adds lubricant or the like and mix then and form tablet in the gained mixture.Pelletize can be undertaken by any suitable method well known in the art, for example wet granulation, non-slurry pelletizing or heating pelletize.Suitable limiting examples comprises uses high-speed stirred comminutor, fluidized granulation drying machine, Squeezinggranulator or cylinder compactor to carry out these prilling process.In addition, method for example dry and screening can be carried out according to the needs that carry out pelletize.The mixture of main medicament, excipient, binding agent, lubricant or the like also can be formed directly in tablet.

Film coating can use any film coating device known in the art if desired, and as film coating substrate, suitable example comprises sugar-coat base, hydrophilic film coating base, enteric film coating base and sustained release film coat base.

Description of drawings

Fig. 1 variable concentrations H ₂O ₂Influence to ECV-304 effect different time pair cell vigor.

Fig. 2 myricetrin (12.5,25,50 μ g/ml) is to the influence of effect separately of ECV-304 cell

Fig. 3 myricetrin (12.5,25,50 μ g/ml) is to ECV-304 cell pretreatment H after 12 hours ₂O ₂(100 μ M) damages modeling

The influence of Fig. 4 myricetrin pair cell LDH burst size

The influence of Fig. 5 myricetrin pair cell NO burst size

The influence of Fig. 6 myricetrin pair cell MDA content

The influence of Fig. 7 myricetrin pair cell MDA content

Fig. 8 myricetrin is to H ₂O ₂The apoptotic influence of the ECV-304 that causes

Fig. 9 myricetrin is to H ₂O ₂The influence that the ECV-304 cell mitochondrial transmembrane potential that causes (Δ Ψ m) descends

The specific embodiment

Below in conjunction with the specific embodiment the present invention is described in further details.

Embodiment one: myricetrin is to the protective effect of acute myocardial ischemia coronary heart disease.

Materials and methods:

1, animal; The SD rat, male, body weight 18～21g is available from Beijing Vital River Experimental Animals Technology Co., Ltd..

2, test kit: the LDH test kit, Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd. produces, lot number: 100341.201008; The CK test kit, Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd. produces, lot number: 080511.200811; The SOD test kit, Nanjing is built up bio-engineering research and is produced, lot number: 20101206; MDA testing cassete, Nanjing are built the poplar bio-engineering research and are produced, lot number: 20101206; Coomassie brilliant blue, Nanjing are built the poplar bio-engineering research and are produced, lot number: 20101206.

3, instrument: the LD5-2A centrifuge, Beijing Medical Centrifugal Machine Factory produces; GF-D800 type semi-automatic biochemical analyzer, Shandong Gaomi Caihong Analytical Instrument Co., Ltd. produces.

4, method: get 40 of rats, be divided into the blank group at random, model group, SHENGMAI ZHUSHEYE 4.5mg/kg, the myricetrin group, 5mg/kg, 2.5mg/kg administration group, every day, intravenously administrable was 1 time, successive administration 7 days, will be respectively at giving the 7th day subcutaneous injection isoprenaline 15mg/kg, behind the last injection isoprenaline 24 hours, 1 hour mensuration rat electrocardiogram after the last administration, then with the rat sacrificed by decapitation, get blood system and survey LDH by kit method from serum, the CK activity, the active and MDA content of coring by the SOD in the kit method thought-read muscular tissue.And the mensuration myocardial infarction area, The data SPSS software is organized a t check.

The result:

Myricetrin causes the protective effect of Acute Myocardial Ischemia in Rats to isoproterenol, the results are shown in Table 1,2,3.

Table 1 myricetrin causes the electrocardiogram of Acute Myocardial Ischemia in Rats, the influence of LDH, CK to isoproterenol

Compare with the blank group: ^#P＜0.05, ^##P＜0.01; Compare with model group: ^*P＜0.05, ^*P＜0.01

Table 2 myricetrin causes the influence of the SOD of Acute Myocardial Ischemia in Rats cardiac muscular tissue, MDA content to isoproterenol

Table 3 myricetrin causes the influence of Acute Myocardial Ischemia in Rats myocardial infarction area to isoproterenol

The result shows, normal health rat J point shows as more to be lifted on slight, with the blank group relatively, behind model group and the myricetrin high and low dose group injection ISO during 20min J point all significantly move down (P＜0.05); Compare active obviously raise (P＜0.01) of model group and myricetrin high and low dose group rat blood serum CK, LDH with the blank group.Compare active significantly reduce (P＜0.01) of myricetrin high and low dose group rat blood serum CK, LDH with model group; This experiment is by the percentage measuring myocardial necrosis in cardiac muscular tissue's section and partly account for whole myocardial area reflecting myocardium degree of necrosis recently.Measurement result shows: after giving ISO, compare with the blank group, model group and myricetrin high and low dose group have obvious myocardial necrosis, and difference has significance meaning (P＜0.05).Compare with model group, myricetrin high and low dose group all can significantly reduce rat heart muscle necrosis area (P＜0.01).Compare with the blank group, SOD is active in the model group cardiac muscular tissue significantly descends (P＜0.01), and MDA content significantly increases (P＜0.01).Compare with model group, myricetrin high and low dose group SOD is active significantly to be increased, and is dose dependent; MDA content significantly reduces, and is dose dependent.The prompting myricetrin can significantly improve SOD activity in the ischemia-reperfusion cardiac muscular tissue, reduce MDA content, strengthens the oxidation resistance of cardiac muscle.

Embodiment two: myricetrin is to the protective effect of isolated rat heart ischemical reperfusion injury

1. materials and methods

1.1 animal: SPF level SD rat,

Body weight 300～350g is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..The animal quality certification number is: SCXK (capital) 2006-0009.

1.2 medicine and reagent and instrument: myricetrin: provide by China Medical Sciences Academy Medical Plants Institute; Verapamil hydrochloride injection: Shanghai Hefeng Pharmaceutical Co., Ltd.; All the other reagent are homemade analytical reagent.U.S. Radnoti isolated perfusion system; BL-420/820 four road physiology monitors; Tianjin Ao Teensi AP-01P type vacuum pump; Prunus mume (sieb.) sieb.et zucc. Teller PH meter; DY89-∏ type electric driven glass refiner; MQX200 type microplate reader; Hitachi's 7060 type automatic clinical chemistry analyzers; TGL-16 high speed tabletop refrigerated centrifuge.

1.3 method

1.3.1 isolated heart Langendorff perfusion model preparation

The SD rat after 20% urethane (lumbar injection) anesthesia, sublingual vein injecting heparin sodium (25mgkg-1) row heparinization.Open breast rapidly, it is dirty to core, hang on the Langendorff perfusion device, with constant temperature (37 ℃), fill binary oxygen (95%O2,5%CO2), K-H liquid continues the adverse current perfusion, flow velocity is 10～15mlmin-1.K-H liquid composition following (g/L): NaCl 6.8912, NaHCO3 2.09, KCl 0.35015, KH2PO4 0.1606, GLU 1.998, MgSO47H2O 0.2908, CaCl2 0.283 PH7.4.Adopt water pocket transmission piezometry intraventricular pressure in the left ventricle, write down left chamber shrinkage curve by BL-420 bio signal acquisition system, calculate the every parameters of left ventricular function of left ventricle, comprise left ventricular systolic pressure (LVSP), left ventricular end diastolic presssure (LVEDP), the maximum climbing speed of left indoor pressure (+dp/dtmax), the maximum fall off rate of left indoor pressure (dp/dtmax) etc.

1.3.2 ischemia/reperfusion experimental technique

Isolated heart with K-H liquid perfusion 15min balance after, can carry out modeling.This experiment is poured into 30min after adopting global ischemia reperfusion injury model promptly to stop to irritate 25min again.Experimental specimen is divided into 4 groups, and every group 8 example is respectively the normal control group: continous perfusion 80min; Ischemia-reperfusion (I/R) group; Myricetrin high and low dose group (3mgL-1,1.5mgL-1); Each administration group is stopping irritating preceding 10min, adds the pastille infusion liquid of variable concentrations respectively, and continues whole refilling process, and except that the blank group, all isolated hearts all stop to irritate 25min, pour into 30min again and cause the I/R model.

1.4 detection index: the mensuration of hemodynamic index

Cut an osculum at the left auricle root, insert sacculus (connecing the BL/420 signal acquiring system) from here to the left ventricular recording hemodynamic index, real time record hemodynamic index: LVSP, LVEDP ,+dp/dtmax.

2. result: see Table 4.

With the blank group relatively, model group is when pouring into 30min again, LVSP obviously reduces, LVEDP obviously increases, ± dp/dtmax obviously descend (p＜0.05).Compare with model group, myricetrin high and low dose group These parameters all has clear improvement.The prompting myricetrin can obviously improve the systolic and diastolic function of damaged myocardium, significantly reduces the inhibitory action of I/R damage to myocardium systolic and diastolic function.

Table 4 myricetrin to isolated rat heart pour into again the hemodynamic influence of 30min ( N=8)

Embodiment three: myricetrin is to the experimentation of hyperlipemia model mice effect for reducing blood fat

1, materials and methods

1.1 trial drug

Myricetrin: provide by China Medical Sciences Academy Medical Plants Institute; All the other reagent are homemade analytical reagent.

1.2 test apparatus

Tianjin Ao Teensi AP-01P type vacuum pump; Prunus mume (sieb.) sieb.et zucc. Teller PH meter; DY89-∏ type electric driven glass refiner; Hitachi's 7060 type automatic clinical chemistry analyzers; TGL-16 high speed tabletop refrigerated centrifuge.

1.3 animal

The ICR mice, male and female half and half, body weight 18-22g, is provided by Beijing Vital River Experimental Animals Technology Co., Ltd. by 60.

1.4 reagent:

High lipid food: 75% egg yolk Emulsion, adopt the fresh egg yolk of 30ml to add 10ml normal saline vortex mixer mixing, become emulsus, 4 ℃ of storages are standby.T-CHOL (TC), triglyceride (TG) test kit build up the biological engineering company limited by Nanjing.

1.5 method:

Mice gives the normal diet adaptability and feeds after 3 days, is divided into normal control group, model control group, myricetrin 7.0,3.5mg/kg dosage group at random, 12 every group.Each group gives normal feedstuff simultaneously, and experimental session organizes respectively respectively that lumbar injection gives relative medicine, the 0.1ml/10g body weight, once a day, successive administration 7 days.Normal control group and model control group give the distilled water of respective volume.Behind the last administration 2h, except that the normal control group, all the other each groups are injected fresh egg yolk Emulsion by 0.1ml/10g dosage abdominal cavity.Pluck eyeball behind the 20h and get blood (water is can't help in the 12h fasting before getting blood), the centrifugal 10min of 3000rpm gets serum, detects serum total cholesterol (TC), triglyceride (TG) in accordance with the law.

2, result sees Table 5.

Result of study shows, compares with the normal control group, and model control group mice serum TC, TG significantly increase, and compares with model control group, and myricetrin high and low dose group all can significantly reduce mice serum TC, TG, has significant difference.

Table 5, myricetrin are to the influence of hyperlipemia in mice serum TG, TC

Compare with the blank group: ^#P＜0.05, ^##P＜0.01; Compare with model group: ^*P＜0.05, ^*P＜0.01.

Embodiment four: myricetrin is to the protective effect of the inductive vascular endothelial cell damage of H2O2

1, experiment material

1.1 medicine myricetrin monomer is by China Medical Sciences Academy Medical Plants Institute's naturalization thing chemistry chamber separation and Extraction (identifying purity＞98% through professor Pan Ruile); Quercetin (is gone up Hiroad standing grain medical sci-tech Development Co., Ltd (purity＞99%)

1.2 being ECV-304, the cell Human umbilical vein endothelial cells purchases in China typical culture collection center (collecting center of Wuhan University)

1.3 reagent D MEM culture medium, hyclone (U.S. Gibco company), DMSO, H ₂O ₂, MTT, JC-1 (U.S. Sigma company), the two transfection reagent boxes (American I nvitrogen company) of AnnexinV/PI apoptosis, NO, LDH, MDA, SOD test kit (Nanjing of China is built up bio-engineering research institute)

1.4 instrument is just being put fluorescence microscope (German Leika), flow cytometer (U.S. BD), enzyme-linked immunosorbent assay instrument (U.S. Bio-Tek), CO ₂Incubator (U.S. Harris)

2, experimental technique

2.1 cell culture

The ECV-304 cell culture is in containing 10% hyclone, 2mM L-glutaminate, and the 100U/ml penicillin in the DMEM culture fluid of 100 μ g/ml streptomycins, places 37 ℃, contains 5%CO ₂Cell culture incubator in cultivate, went down to posterity with 0.25% trypsinization in 2-3 days.

2.2 experiment grouping

2.2.1 the blank group is abandoned culture medium and is handled with serum-free DMEM

2.2.2 H ₂O ₂It is the H of 100 μ M that model group serum-free DMEM adds final concentration ₂O ₂Hatch 24h

2.2.3 myricetrin+H ₂O ₂Myricetrin (12.5,25, the 50 μ g/ml) preincubate of group variable concentrations adds final concentration after 12 hours be the H of 100 μ M ₂O ₂Hatch 24h

2.2.4 Quercetin+H ₂O ₂Group Quercetin (20 μ g/ml) preincubate adds final concentration after 12 hours be the H of 100 μ M ₂O ₂Hatch 24h)

2.2.5 the myricetrin of myricetrin group different depth (12.5,25,50 μ g/ml) preincubate 12 hours

2.3 tetramethyl azo azoles salt (mtt assay) detects cell viability

Take the logarithm the cell of trophophase with 1 * 10 ⁴The density in individual hole is inoculated in 96 orifice plates, and 37 ℃, 5%CO ₂Cultivated 24 hours under the condition, carry out different disposal by the grouping requirement.After processing finished, every hole added 5mg/mlMTT solution 20 μ l, hatches after 4 hours for 37 ℃ and removes culture fluid, every hole adds 150 μ lDMSO, microwell plate oscillator concussion 10min, the purple result is fully dissolved after, adopt microwell plate scanning microplate reader to survey 570nm place's light absorption value (OD value).Cell survival rate (%)=experimental group OD value/matched group OD value * 100%

2.4 LDH, NO, SOD, MDA assay

Take the logarithm the cell of trophophase with 1 * 10 ⁵The density in individual hole is inoculated in 6 orifice plates, and 37 ℃, 5%CO ₂Cultivated 24 hours under the condition, carry out different disposal by the grouping requirement.After processing finishes, collecting cell culture fluid and cell, explanation detects according to test kit

2.5 Flow Cytometry detects apoptosis

Take the logarithm the cell of trophophase with 1 * 10 ⁵The density in individual hole is inoculated in 6 orifice plates, and 37 ℃, 5%CO ₂Cultivated 24 hours under the condition, carry out different disposal by the grouping requirement.Adopt the two labelling methods that dye of flow cytometry Annex in V-FITC/PI, modeling after pharmaceutical intervention 12h is respectively organized in experiment is removed supernatant, 0.25% trypsinization collecting cell, 1000rmin ^-15min is centrifugal, PBS washed cell 2 times, with cell with 1 * 10 ⁵106/mL is resuspended among 1 * Binding Buffer, shift again in the streaming pipe of 100..L to 5mL after blowing and beating mixing gently, the mass concentration that adds 5..L Annex in V-FITC dyeing liquor and 5 μ L is the propidium iodide (PI) of 20 μ g/mL, mixing, lucifuge is hatched 15min under the room temperature, goes up machine testing apoptotic cell percentage ratio in the 1h.Excitation wavelength Ex=488nm; Emission wavelength Em=530nm.

2.6 the detection of cell mitochondrial transmembrane potential (Δ Ψ m)

Take the logarithm the cell of trophophase with 5 * 10 ⁴The density in individual hole is inoculated in 24 orifice plates.37 ℃, 5%CO ₂Cultivate after 24 hours under the condition and manage outward by different grouping.With the cell in 0.25% enzymic digestion, 24 orifice plates, collect the E ffindoff pipe of putting into 1.5ml then, add 1mg/ml JC-15 μ l, bullet is even gently, lucifuge, 37 hatch 30min (every 10min jolting 1 time).The PBS washing, the centrifugal 5min of 2000r/min discards dye liquor, and flow cytometer detects, and excitation wavelength is 488nm, wavelength of transmitted light 575nm, each sample collects 1 * 10 at least ⁴Individual cell, nm), determination data carries out date processing by the software that flow cytometer disposed.The level relatively of representing the myocardial cell transmembrane potential with the average fluorescent strength of positive cell.

2.7. data analysis

All experiments repeat more than three times, adopt SPSS 12.0 softwares to carry out date processing, and (x ± s) expression adopts one factor analysis of variance (ANOVA) to experimental data, and P＜0.05 has statistical significance for difference with mean ± standard deviation.

3, result

3.1 the active influence of myricetrin pair cell

3.1.1 H ₂O ₂Establishment to ECV-304 damage model condition

Adopt the H of variable concentrations (12.5-400 μ M) ₂O ₂Hatch 4,8,24 with the ECV-304 cell respectively, 48h, begin cell viability from 8h and significantly reduce, be dosage-time dependence, experimental result is seen Fig. 1.Wherein 100 μ M handled 24 hours, and cell viability drops to 48.2%, and this concentration and time point are as the unified modeling condition of testing later.

3.1.2 myricetrin is to the independent effect of ECV-304 cell

Adopt the myricetrin and the ECV-304 cell of variable concentrations (12.5,25,50 μ g/ml) to hatch 12 hours, the MTT cell viability is measured demonstration myricetrin pair cell avirulence in the administration concentration scope does not have proliferation function yet, the results are shown in Figure 2.

3.1.2 myricetrin is to H ₂O ₂The ECV-304 cell injury protective effect that causes

The myricetrin of variable concentrations (12.5,25,50 μ g/ml) and ECV-304 cell preincubate be after 12 hours, 100 μ M H ₂O ₂Act on 24 hours, measure cell viability.The Quercetin of 20 μ g/ml is as positive controls.The myricetrin H that significantly gos up ₂O ₂The cell viability that causes descends, and onset concentration is 12.5 μ g/ml, and 50 μ g/ml effects are the most remarkable, and cell viability rises to 70.9% from 59.9%, illustrates that myricetrin is to H ₂O ₂The ECV-304 cell injury that causes has protective effect, the results are shown in Figure 3.

3.2 the influence of LDH and NO burst size in the myricetrin pair cell liquid

Compare H with the blank group ₂O ₂Model group LDH burst size significantly raises, and 25,50 μ g/ml myricetrin pretreatment are after 12 hours, and the increase of LDH burst size reduction the results are shown in Figure 4, ^##P＜0.01: compared significant difference with the blank group; * P＜0.05, significant difference has been compared with model group in * * P＜0.01.

Compare H with the blank group ₂O ₂Model group NO content descends, and 25, the 50 μ g/ml myricetrin pretreatment NO content that can significantly go up in 12 hours the results are shown in Figure 5, ^#P＜0.05: compared significant difference with the blank group; * P＜0.05: compared significant difference with model group.

3.3 the influence of MDA content and SOD vigor in the myricetrin pair cell

Compare H with the blank group ₂O ₂Model group MDA content significantly raises, and the SOD vigor obviously descends, and 25,50 μ g/ml myricetrin pretreatment are after 12 hours, and MDA content descends in the cell, the results are shown in Figure 6, ^##P＜0.01: compared significant difference with the blank group; ^*P＜0.05, ^*Significant difference has been compared with model group in P＜0.01.The SOD vigor raises, and the results are shown in Figure 7, ^##P＜0.01: compared significant difference with the blank group; ^*Significant difference has been compared with model group in P＜0.01.

3.4 myricetrin is to H ₂O ₂The apoptotic effect that causes

ECV-30 is through 100 μ M H ₂O ₂Handle 24h after flow cytometer detects the two apoptosis that dye of AnnexinV/PI, apoptosis rate is elevated to (16.68 ± 0.13) % from (3.86 ± 0.09) %, the myricetrin pretreatment of 50 μ g/m significantly reduced apoptosis rate (7.85 ± 0.15) % after the modeling in 12 hours, the results are shown in Figure 8 ^#P＜0.05: compared significant difference with the blank group; * significant difference has been compared with model group in P＜0.05.

3.4 myricetrin is to H ₂O ₂The influence that the cell mitochondrial transmembrane potential that causes (Δ Ψ m) descends

ECV-304 is through 100 μ M H ₂O ₂Handle 24h after fluorized marking poststaining and flow cytometer detection display line mitochondrial membrane potential obviously descend, red fluorescence becomes green, the myricetrin pretreatment of 50 μ g/ml showed the painted green fluorescence amount of minimizing JC-1 in 12 hours, recover mitochondrial membrane potential, the result is through the streaming detection validation, the results are shown in Figure 9 ^#P＜0.05: compared significant difference with the blank group; * significant difference has been compared with model group in P＜0.05.

Embodiment five myricetrin specifications are the preparation of 10mg/ sheet

Prescription:

Technology:

1, former, that the adjuvant pulverize separately is crossed 80 mesh sieves is standby;

2, getting 2%HPMC, to add concentration be that 30～95% medicinal alcohols are made 5～10% solution, promptly;

3, get myricetrin, microcrystalline Cellulose, amylum pregelatinisatum, carboxymethyl starch sodium mix homogeneously, add 2%HPMC alcoholic solution system soft material, 16 mesh sieves are granulated, 60 ℃ of dryings;

4,16 mesh sieve granulate add magnesium stearate, Pulvis Talci mixing 10 minutes, make evenly, and tabletting promptly.

Embodiment six myricetrin specifications are the preparation of 5mg/ sheet

Prescription:

Technology:

Embodiment seven myricetrin specifications are the preparation of 1.25mg/ sheet

Prescription:

Technology:

Embodiment eight myricetrin specifications are the preparation of 2.5mg/ sheet

Prescription:

Technology:

4,16 mesh sieve granulate add magnesium stearate, Pulvis Talci mixing 10 minutes, make evenly, and tabletting promptly.The stable contrast test of embodiment nine myricetrin tablets under hot conditions

Get each 10 in the tablet of embodiment five, six, seven, eight or four kind of specification, put respectively in the sealing clean container, under 60 ℃ of conditions, placed 10 days; Respectively at the 0th day, the 5th day and sampling in the 10th day, detect, the result is as follows:

Place situation of change 60 ℃ of hot conditionss

Time	0 day: outward appearance	5 days: outward appearance	10 days: outward appearance
				Myricetrin 10mg/ sheet	No change	No change	No change
Myricetrin 5mg/ sheet	No change	No change	No change
				Myricetrin 2.5mg/ sheet	No change	No change	No change
Myricetrin 1.25mg/ sheet	No change	No change	No change

As seen from the above table, after placing 10 days under 60 ℃ of conditions of high temperature, the myricetrin tablet appearance changes little,

Fine to pyritous stability.

Stable contrast test under the embodiment nine myricetrin tablet super-humid conditions.

Get each 10 in the tablet of embodiment five, six, seven, eight or four kind of specification, put in the constant humidity hermetic container, under 92.5% relative humidity condition, placed 10 days; Respectively at the 0th day, the 5th day and sampling in the 10th day, detect, the result is as follows:

Under 92.5% relative humidity super-humid conditions, place situation of change

Time	0 day: outward appearance	5 days: outward appearance	10 days: outward appearance
				Myricetrin 10mg/ sheet	No change	No change	No change
Myricetrin 5mg/ sheet	No change	No change	No change
				Myricetrin 2.5mg/ sheet	No change	No change	No change
Myricetrin 1.25mg/ sheet	No change	No change	Wherein 2 to 5 pittings appear in two surfaces

As seen from the above table, after placing 10 days under the high humidity 92.5% relative humidity condition, 2 to 5 pittings appear in the tablet appearance no change of 10mg/ sheet and 8mg/ sheet, the tablet of 1.25mg/ sheet wherein two surfaces, outward appearance changes slightly, shows that the myricetrin tablet is stable to high humidity.

The stable contrast test of embodiment ten myricetrin tablets under the strong illumination condition.

Get each 10 in the tablet of embodiment five, six, seven, eight or four kind of specification, put lighting box, placed under illumination 5000Lx condition 10 days, respectively at the 0th day the, 5 days and sampling in the 10th day, detect, the result is as follows:

Under illumination 5000Lx condition, place situation of change

As seen from the above table, after placing 10 days under the illumination 5000Lx condition, the myricetrin tablet is stable fine to illumination.

Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be qualification to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give exhaustive to all embodiments.And these belong to conspicuous variation or the change that spirit of the present invention extended out and still are among protection scope of the present invention.

Claims

1. myricetrin and derivant thereof are used for the treatment of application in the cardiovascular disease medicine in preparation.

2. application according to claim 1 is characterized in that, described derivant has following structure: described myricetrin derivant has following structure:

Wherein:

R2 is selected from acyl group, hydrogen, alkyl group.

3. application according to claim 2 is characterized in that, described myricetrin derivant is 2-(3,4-dihydroxy, 5-methoxyphenyl)-3,5-dihydroxy-7-ethyoxyl chromone.

4. application according to claim 1 is characterized in that, described cardiovascular disease medicine is the acute myocardial ischemia medicaments for coronary disease.

5. application according to claim 1 is characterized in that, described cardiovascular disease medicine is the heart ischemia reperfusion damage medicine.

6. application according to claim 1 is characterized in that, described cardiovascular disease medicine is a hyperlipidemia.

7. application according to claim 1 is characterized in that, described cardiovascular disease medicine is protection vascular endothelial cell damage medicine.

8. according to the described application of arbitrary claim among the claim 1-7, it is characterized in that described medicine is made pharmaceutical composition by myricetrin or derivatives thereof and one or more pharmaceutically acceptable carriers or excipient.

9. application according to claim 8 is characterized in that, the amount of the contained active component myricetrin of described pharmaceutical composition minimum unit is 1.25-10mg.

10. application according to claim 9 is characterized in that, the amount of the contained active component myricetrin of described pharmaceutical composition minimum unit is 2.5-5mg.

11. application according to claim 10 is characterized in that, the amount of the contained active component myricetrin of described pharmaceutical composition minimum unit is 2.5mg.

12. application according to claim 8 is characterized in that, described medicine composition dosage form is tablet, capsule, soft capsule, oral liquid, syrup, granule, drop pill, oral cavity disintegration tablet, slow releasing tablet, slow releasing capsule, controlled release tablet or controlled release capsule.

13. application according to claim 8 is characterized in that, described medicine composition dosage form is liquid drugs injection, freeze-dried powder, aseptic powder injection, transfusion.

14. application according to claim 8 is characterized in that, described pharmaceutically acceptable carrier or excipient comprise filler, binding agent, lubricant, disintegrating agent, cosolvent, surfactant, absorption carrier.

15. application according to claim 8 is characterized in that, described pharmaceutically acceptable carrier or excipient are selected from solvent, antioxidant, cosolvent, adsorbent, osmotic pressure regulator or the PH regulator that is applicable to injection.