CN114522190A

CN114522190A - Pharmaceutical composition for resisting myocardial ischemia injury and preparation method and application thereof

Info

Publication number: CN114522190A
Application number: CN202011307052.5A
Authority: CN
Inventors: 詹常森; 张建革; 周俊杰; 汪飞云; 姜鹏
Original assignee: Shanghai Hutchison Pharmaceuticals Ltd
Current assignee: Shanghai Hutchison Pharmaceuticals Ltd
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2022-05-24
Anticipated expiration: 2040-11-20
Also published as: CN114522190B

Abstract

The invention provides a pharmaceutical composition for resisting myocardial ischemia injury, which comprises the following components in parts by weight: 2-10 parts of musk, 11-25 parts of ginseng, 6-13 parts of storax, 2-10 parts of bezoar and 10-26 parts of cinnamon. The invention also provides a preparation method and application of the pharmaceutical composition.

Description

Pharmaceutical composition for resisting myocardial ischemia injury and preparation method and application thereof

Technical Field

The invention belongs to the field of medicine and pharmacy, and particularly relates to a pharmaceutical composition for resisting myocardial ischemia injury, and a preparation method and application thereof.

Background

Cardiovascular disease is the first cause of death worldwide: more people die annually from cardiovascular disease than from any other cause. Patients with hypertension, coronary heart disease, cerebrovascular disease, peripheral vascular disease, heart failure, congenital heart disease, cardiomyopathy and the like all cause myocardial ischemia to damage the shape, function and the like of myocardial cells, and the symptoms are chest distress, chest pain, palpitation and the like. Myocardial cell damage and even necrosis caused by restoring blood reperfusion after myocardial ischemia is called myocardial ischemia reperfusion injury, and myocardial cell hypoxia/reoxygenation injury is one of the main pathogenesis of the myocardial cell damage.

Musk, ginseng, storax, bezoar, cinnamon, toad venom, borneol and other medicinal materials are frequently used in the traditional Chinese medicine formula for treating cardiovascular diseases. The musk is warm and leads to resuscitation, promotes blood circulation and relieves pain; borneol and bezoar help musk to induce resuscitation; the toad venom and the storax have the effects of inducing resuscitation and refreshing mind, removing dirt and relieving pain; ginseng, radix Ginseng, acts to reinforce primordial qi and Rou Gui, warms yang and dispels cold. The medicines are compatible, and have the effects of promoting blood circulation, relieving pain, inducing resuscitation and removing filth.

Bufalin, bufotalin, resibufogenin, cinobufagin, 3-epi bufalin, and other toad-derived components derived from venenum bufonis are part of the blood-entering components of venenum bufonis that have been detected (Yan S, et al. chemical refining and quantifying analysis of proteins in expensing Baoxin beta chromatography with a crystallization and mass spectrometry detection [ J ]. J Anal Chem,2009,64(2): 149-. According to the Shore research, the venenum bufonis can improve ischemic myocardial metabolism, correct sugar and fatty acid metabolic disorder and prevent myocardial damage caused by free fatty acid accumulation; toad venom can also obviously increase the SOD activity of rabbit with myocardial ischemia reperfusion injury and reduce the content of M DA, suggesting that the toad venom can reduce the injury of oxygen free radicals to the myocardium by inhibiting the lipid peroxidation process and simultaneously improving the activity of endogenous antioxidase (Shao Ling, low temperature and the protective effect of toad venom on the myocardial ischemia reperfusion injury of anesthetized rabbit [ D ]. Wuhan: Wuhan university, 2005). However, the cardiotoxicity of the toad venom is a problem which cannot be ignored. Another study shows that borneol has a protective effect on acute myocardial ischemia injury caused by isoproterenol, and the possible mechanism is to relieve intracellular calcium overload and inhibit apoptosis (vitex flame. borneol has a protective effect on acute myocardial ischemia injury [ D ]. Sichuan: Sichuan university, 2003).

In 2017, deaths caused by cardiovascular diseases account for more than 40% of deaths caused by diseases of residents in China, are higher than those caused by malignant tumors and other diseases, and are in a continuously rising state (chengwei, et al, China cardiovascular disease report 2017 (summary [ J ]. J. China J. China circulation, 2018, 33(1): 1-8). Therefore, the clinical need for effective and safe drugs for cardiovascular diseases is still not met.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a novel pharmaceutical composition for resisting myocardial ischemia injury. The pharmaceutical composition has a protective effect on anoxic-reoxygenation myocardial cells, has the same effect as the medicines on the market, but has simpler and safer formula.

Therefore, the invention adopts the following technical scheme:

the pharmaceutical composition for resisting myocardial ischemia injury comprises the following components in parts by weight:

2-10 parts of musk, 11-25 parts of ginseng, 6-13 parts of storax, 2-10 parts of bezoar and 10-26 parts of cinnamon.

Preferably, the pharmaceutical composition for resisting myocardial ischemia injury comprises the following components in parts by weight:

3-10 parts of musk, 12-25 parts of ginseng, 7-13 parts of storax, 3-10 parts of bezoar and 11-26 parts of cinnamon.

More preferably, the pharmaceutical composition for resisting myocardial ischemia injury comprises the following components in parts by weight:

4-10 parts of musk, 13-25 parts of ginseng, 8-13 parts of storax, 4-10 parts of bezoar and 12-26 parts of cinnamon.

Preferably, the ginseng is selected from ginseng medicinal materials or ginseng extracts.

More preferably, the ginseng extract is prepared by the following method: pulverizing Ginseng radix into granules, reflux-extracting with 75% ethanol, filtering the extractive solution while it is hot, concentrating, and vacuum drying the extract.

Preferably, the musk is selected from natural musk or artificial musk.

More preferably, the musk is an artificial musk.

Preferably, the calculus bovis is selected from natural calculus bovis, in vitro cultured calculus bovis or artificial calculus bovis.

More preferably, the bezoar is an artificial bezoar.

The invention also aims to provide a preparation method of the pharmaceutical composition for resisting myocardial ischemia injury, which comprises the steps of preparing the components according to the parts by weight, mixing the components, adding or not adding pharmaceutically acceptable auxiliary materials, and preparing a clinically acceptable preparation according to a conventional method in the field.

In addition, the invention also provides application of the pharmaceutical composition in preparing a medicament for treating diseases related to myocardial ischemia injury.

Preferably, the diseases associated with myocardial ischemia injury are selected from one or more of coronary heart disease, angina pectoris and myocardial infarction.

In the present specification, the "parts by weight" of each component means a relative amount ratio between the components, not an actual absolute mass. As the case may be, 1 part by weight may be 1g, 10g, 50g, 100g, 500g, 1kg or any other mass number.

In the present specification, the "ginseng material" refers to dried roots and rhizomes of ginseng (Panax ginseng c.a.mey.) belonging to the family araliaceae, including wild "mountain ginseng", cultivated "round ginseng", and "mountain ginseng under forest" which is naturally grown in wild state of mountain forest.

The "natural musk" is dry secretion in mature male sachet of forest musk deer (Moschus berezovski Flerov), horse musk deer (Moschus sifanicus Przewalski) or original musk deer (Moschus moschefferus Linnaeus) belonging to Cervidae.

The artificial musk is a substitute of endangered animal medicinal material musk and is used as the same as natural musk.

The "calculus bovis" is dry gallstone of cattle (Bos taurus domesticus Gmelin) belonging to family Bovidae.

The bezoar cultured in vitro is prepared by taking fresh bile of cattle (Bos taurus domesticus Gmelin) of Bos family as mother liquor, and adding deoxycholic acid, cholic acid, compound calcium bilirubinate, etc., according to the records of Chinese pharmacopoeia (2015 or 2020 edition).

The artificial bezoar is prepared by processing bovine bile powder, cholic acid, hyodeoxycholic acid, taurine, cholesterol, trace elements and the like, and is subject to the records of Chinese pharmacopoeia (2015 or 2020 edition).

Drawings

The invention will be further explained with reference to the drawings.

Figure 1 is a bar graph showing the effect of musk heart-protecting pills at different concentrations on the survival rate of myocardial cells after hypoxic injury. In the figure, x: p <0.001 compared to model group.

FIG. 2 is a bar graph showing the effect of Musk BaoXin pill (SBP, 75mg/L) on LDH, MDA and NO release following hypoxic injury to cardiac myocytes. In the figure, x: p <0.001 compared to model group. Wherein:

a: the effect of musk heart protecting pill (SBP, 75mg/L) on LDH release after myocardial cell hypoxia injury;

b: the effect of musk heart protecting pill (SBP, 75mg/L) on MDA release after myocardial cell hypoxia injury;

c: moschus BAOXI pill (SBP, 75mg/L) has effect in releasing NO after myocardial cell anoxia injury.

FIG. 3 is a bar graph showing the effect of the pharmaceutical composition of example 2 of the present invention on the survival of myocardial cells after hypoxic injury at various concentrations. In the figure, x: p <0.01 compared to model group; ***: p <0.001 compared to model group.

Figure 4 is a bar graph showing the effect of the pharmaceutical composition of example 2 of the invention on ldh (a), mda (b) and no (c) release after hypoxic injury to cardiomyocytes at different concentrations. In the figure, x: p <0.01 compared to model group; ***: p <0.001 compared to model group. Wherein:

a: effect of the pharmaceutical composition of example 2 on LDH release following hypoxic injury to myocardial cells at different concentrations;

b: effect of the pharmaceutical composition of example 2 on MDA release following hypoxic injury to cardiac myocytes at different concentrations;

c: effect of the pharmaceutical composition of example 2 on NO release after hypoxic injury of myocardial cells at different concentrations.

The histogram of fig. 5 shows the effect of comparative 1, comparative 2, comparative 3, comparative 4, comparative 5 and comparative 6 on survival of myocardial cells after hypoxic injury at different concentrations.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified. Wherein, the purchase conditions of partial reagents and medicinal materials are as follows:

musk heart-protecting pill: shanghai and yellow pharmaceuticals, Inc., lot number 20190812;

artificial musk: beijing Bixin pharmaceutical Co., Ltd, lot number 20190504;

ginseng: liaoning xintianjian medicinal materials Co., Ltd., lot number 20190113;

storax: zhejiang forest rainbow medicine, Inc., lot number 20190328;

artificial bezoar: hunan Dibo pharmaceutical Co., Ltd, lot number 20190329;

cinnamon: shanghai Zhenrutang pharmaceutical Co., Ltd., lot number 20190512;

borneol: shanghai medicinal materials Co., Ltd., lot number 20190606;

and (3) toad venom: shandong Kangyuan Tang Chinese herbal pieces, Inc., lot number 20190603.

Example 1A pharmaceutical composition for treating myocardial ischemic injury

10 parts of artificial musk, 25 parts of ginseng, 8 parts of storax, 4 parts of bezoar and 12 parts of cinnamon; wherein 1 part by weight is 1 g.

Example 2A pharmaceutical composition for treating myocardial ischemic injury

8 parts of artificial musk, 13 parts of ginseng, 8 parts of storax, 10 parts of bezoar and 26 parts of cinnamon; wherein 1 part by weight is 1 g.

Example 3A pharmaceutical composition for treating myocardial ischemic injury

4 parts of artificial musk, 25 parts of ginseng, 8 parts of storax, 10 parts of bezoar and 20 parts of cinnamon; wherein 1 part by weight is 1 g.

Test example 1 protection of hypoxic-reoxygenation myocardium by the pharmaceutical composition of the present invention

1. Primary reagent material

2. Main instrument

3. Test sample

3.1 pharmaceutical compositions of the invention

Pharmaceutical composition of example 2

3.2 Musk Baoxin Wan:

3.4 comparative pharmaceutical compositions

Comparative pharmaceutical composition 1 (hereinafter referred to as "comparative 1"): 8g of artificial musk, 20g of ginseng and 6g of bezoar. Weighing 8g of artificial musk, 20g of ginseng and 6g of bezoar, crushing into fine powder and stirring uniformly to obtain the musk-containing Chinese medicinal composition.

Comparative pharmaceutical composition 2 (hereinafter referred to as "comparative 2"): 8g of artificial musk, 20g of ginseng and 1g of toad venom. Weighing 8g of artificial musk, 20g of ginseng and 1g of toad venom, crushing into fine powder and stirring uniformly to obtain the musk-containing compound powder.

Comparative pharmaceutical composition 3 (hereinafter referred to as "comparative 3"): 6g of artificial musk, 15g of ginseng and 15g of cinnamon. Weighing 6g of artificial musk, 15g of ginseng and 15g of cinnamon, crushing into fine powder, and uniformly stirring to obtain the musk-containing tea.

Comparative pharmaceutical composition 4 (hereinafter referred to as "comparative 4"): 4g of artificial musk, 13g of storax, 25g of ginseng and 2g of toad venom. Weighing 4g of artificial musk, 12g of storax, 25g of ginseng and 2g of toad venom, crushing into fine powder and uniformly stirring to obtain the musk-containing compound powder.

Comparative pharmaceutical composition 5 (hereinafter referred to as "comparative 5"): 4g of artificial musk, 25g of ginseng, 2g of toad venom and 26g of cinnamon. Weighing 4g of artificial musk, 25g of ginseng, 2g of toad venom and 26g of cinnamon, crushing into fine powder and stirring uniformly to obtain the musk-containing compound powder.

Comparative pharmaceutical composition 6 (hereinafter referred to as "comparative 6"): 4g of artificial musk, 13g of storax, 10g of bezoar and 26g of cinnamon. Weighing 4g of artificial musk, 25g of ginseng, 2g of toad venom and 26g of cinnamon, crushing into fine powder and stirring uniformly to obtain the musk-containing compound powder.

The medicine composition, the musk heart-protecting pill and each comparative medicine composition are all stored in a sealed way at the temperature of 20 ℃ below zero.

Weighing a test sample, and preparing a solution with the concentration of 30000mg/L by using DMSO and a complete culture solution (a complete culture solution prepared by DMEM medium, fetal bovine serum and penicillin-streptomycin) as a stock solution (the concentration of the musk heart-protecting pill is 20000mg/L), wherein the content of the DMSO is ten percent. The stock solution is diluted by 100 times to obtain 300mg/L solution, and the 300mg/L solution is further diluted by different times to obtain corresponding working solution.

4. Experimental methods

4.1 myocardial cell H9C2 hypoxia injury model

1) Plate paving: 96-well plates were plated with 6000H 9C2 cardiomyocytes per well using DMEM, 10% fetal bovine serum, 1% complete medium mixed with double antibody, 100. mu.L of cell suspension per well, and cultured overnight.

2) The next day, the liquid in the wells was discarded, and 5mmol/L Na was added₂S₂O₄The solution (200. mu.L, prepared with sugarless RPMI 1640) was placed in a cell culture chamber (37 ℃, 5% CO)₂)1.5h。

4.2 cell viability assay

1) After myocardial cells H9C2 are damaged by hypoxia for 1.5H, liquid in the wells is discarded, 200 mu L of complete culture solution is added into each well of a control group and a model group, 200 mu L of test sample solution is added into each well of an administration group, and the cells are placed in a cell culture box for 24H.

2) After 24h, the wells were discarded and 20. mu.L of 5mg/ml MTT was added to each well and placed in a cell incubator for 4 h.

3) After 4h, the wells were drained, 150. mu.L of DMSO was added to each well, the wells were placed on a shaker for 10min, and the absorbance of each well was measured at 570nm using a microplate reader.

4) Calculating the cell survival rate (%) ═ OD model group/OD control group 100; survival (%) ═ OD administration group/OD control group 100.

4.3 LDH Release assay

1) After myocardial cells H9C2 are damaged by hypoxia for 1.5H, liquid in the wells is discarded, 200 mu L of complete culture solution is added into each well of a control group and a model group, 200 mu L of compound liquid is added into each well of an administration group, and the cells are placed in a cell culture box for 24H.

2) After 24h, the cell culture plate was centrifuged at 400g for 5min, and 120. mu.L of the supernatant was aspirated into each well of a new 96-well plate, followed by sample assay.

3) Preparing an LDH detection working solution: LDH detection working solutions with different volumes are prepared according to the number of samples to be detected, and the following table is provided.

4) 60 μ L of LDH detection working solution was added to each well.

5) Mixing, wrapping with aluminum foil, slowly shaking on horizontal shaker for 30min, and measuring absorbance at 490 nm.

4.4 MDA assay

1) Plate paving: petri dishes were plated with 60 ten thousand H9C2 cardiomyocytes per dish using DMEM, 10% fetal bovine serum, 1% double antibody mixed complete medium. The control group, model group and administration group were set up, and 2mL of cell suspension was added to each dish and cultured overnight.

2) The next day, the fluid in the dish was discarded, 2mL of DMEM was added to each dish for the control group, and 2mL of 5mmol/L Na was added to each dish for the model group and the compound group₂S₂O₄The solution was put into a cell culture chamber (37 ℃, 5% CO)₂)1.5h。

3) After 1.5h, the liquid in the dish is discarded, 2mL of DMEM complete culture solution is added to each dish of the control group and the model group, 2mL of test sample solution with different concentrations (the compound is prepared by biological-grade DMSO and DMEM complete culture solution, the DMSO content is not more than one thousandth) is added to each dish of the drug group, and the mixture is placed in a cell culture box for 24 h.

4) After 24h the dish was discarded, 1mL of DPBS was added to each dish, the cells were scraped off with a cell scraper, and the cell suspension was transferred to a 1.5mL EP tube.

5) Centrifuging at 1000 rpm for 10min, discarding supernatant, adding 130 μ L of reagent five extract in MDA kit, mixing for 2min, and sampling 100 μ L in 1.5mL EP tube.

6) According to the reagent I: and a second reagent: preparing working solution according to the ratio of 0.2:3:1 of the reagent III, adding 1ml of the working solution into each sample, mixing the solution evenly by vortex, pricking a small hole on a tube cover by using a syringe needle, and heating for 40min at 100 ℃.

7) Taking out, cooling, centrifuging at 4000 rpm for 10min, sucking 130 μ L of liquid into 96-well plate, and measuring absorbance at 530 nm.

8) The sample protein concentration was determined with the BCA kit.

9) MDA content (nmol/mg prot) ═ OD_{Measurement of}-OD_{Blank space})/(OD_{Standard of merit}-OD_{Blank space}) Concentration of standard substance/concentration of protein in sample

4.5 determination of NO

1) Plate paving: petri dishes were plated with 50 ten thousand H9C2 cardiomyocytes per dish using DMEM, 10% fetal bovine serum, 1% double antibody mixed complete medium. The control group, model group and administration group were set up, and 2mL of cell suspension was added to each dish and cultured overnight.

2) The next day, the fluid in the dish was discarded, 1mL of DMEM was added to each dish for the control group, and 1mL of 5mmol/L Na was added to each dish for the model group and the compound group₂S₂O₄The solution was placed in a cell culture chamber (37 ℃, 5% CO)₂)1.5h。

3) After 1.5h, the liquid in the dish is discarded, 1mL of DMEM complete culture solution is added to each dish of the control group and the model group, 1mL of test sample solution with different concentrations is added to each dish of the administration group (the compound is prepared by biological-grade DMSO and DMEM complete culture solution, the DMSO content is not more than one thousandth), and the mixture is placed in a cell culture box for 24 h.

5) Centrifuging at 1000 rpm for 10min, discarding supernatant, adding 90 μ L tissue lysate, and mixing by vortexing to lyse cells.

6) The liquids were added as in the table below.

NADPH(2mM)(μL)	5
		FAD(μL)	10
Nitrate Reductase(μL)	5

7) Vortex the mix and incubate at 37 ℃ for 30 min.

8) The following liquids were added to each tube, vortexed and mixed, and incubated at 37 ℃ for 30 min.

LDH Buffer(μL)	10
		LDH(μL)	10

9) The following liquids were added to each tube, vortexed and mixed, incubated at room temperature (20-30 ℃) for 10min, and absorbance was measured at 540 nm.

Griess ReagentⅠ(μL)	50
		Griess ReagentⅡ(μL)	50

10) The sample protein concentration was determined with the BCA kit.

5. Data processing: data are shown as mean ± SEM, single-sided T-test performed.

6. The experimental results are as follows:

6.1 protective action of Musk Baoxin pill on myocardial cell hypoxia injury

6.1.1 Effect of Musk Baoxin pill on survival rate of myocardial cells after hypoxia injury at different concentrations

Compared with a model group, the musk heart-protecting pill has the protection effect on myocardial cell hypoxia injury under the concentration of 5mg/L, 25mg/L, 50mg/L, 75mg/L, 100mg/L, 150mg/L and 200mg/L, and SBP can promote the survival of myocardial cells with hypoxia injury at 75mg/L, and has significant difference. As shown in detail in fig. 1. And determining 75mg/L as the working concentration of the heart-protecting musk pill according to the test results, and carrying out subsequent tests.

6.1.2 action of Musk Baoxin Wan (75mg/L) on LDH, MDA and NO release after myocardial cell hypoxia injury

Compared with a normal control group, the release of LDH, MDA and NO of the myocardial cells of the hypoxia group is obviously increased; compared with the hypoxia group, the musk heart-protecting pill can reduce the release of LDH, MDA and NO at 75mg/L, and has significant difference. The results are shown in FIG. 2, panels A, B and C, respectively.

6.2 example 2 protective Effect on myocardial cell hypoxic injury

6.2.1 Effect of example 2 of the invention on survival of myocardial cells following hypoxic injury

Compared with a model group, the embodiment of the invention can promote the survival of hypoxic myocardial cells under the concentration of 100mg/L and 300mg/L, has significant difference, and has no significant difference compared with a musk heart-protecting pill group. See in particular fig. 3.

6.2.2 Effect of embodiments of the invention on LDH, MDA and NO release following myocardial cell hypoxic injury

Compared with a normal control group, the release of LDH of the myocardial cells of the hypoxia group is obviously increased; compared with the hypoxia group, the embodiment 2 can reduce the release of LDH at the concentration of 100mg/L and 300mg/L, and has significant difference; example 2 had a slightly weaker effect in reducing LDH release compared to the musk heartburn pill group. See in particular fig. 4A.

Compared with a normal control group, the release of the myocardial cell MDA of the hypoxia group is obviously increased; compared with the anoxia group, the example 2 can obviously reduce the release of MDA at 100mg/L and 300mg/L, and has no obvious difference compared with the musk heart-protecting pill group. See in particular fig. 4B.

Compared with a normal control group, the release of the NO of the myocardial cells of the hypoxia group is obviously increased; compared with the hypoxia group, the example 2 can obviously reduce the release of NO at 100mg/L and 300mg/L, and has NO obvious difference compared with the musk heart-protecting pill group. See in particular fig. 4C.

6.3 Effect of comparative pharmaceutical composition on survival Rate of myocardial cells after hypoxic injury

Comparative 1, comparative 2, comparative 3, comparative 4, comparative 5 and comparative 6 had no promoting effect on survival after hypoxic injury to cardiac myocytes at concentrations of 100mg/L and 300 mg/L. See in particular fig. 5.

7. Conclusion

The pharmaceutical composition provided by the embodiment of the invention has an obvious protection effect on myocardial cell ischemic injury, and the effect of the pharmaceutical composition is basically equivalent to that of a musk heart-protecting pill. Each comparative pharmaceutical composition has no effect of resisting myocardial cell ischemic injury.

Claims

1. A pharmaceutical composition for resisting myocardial ischemia injury comprises the following components in parts by weight:

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition for resisting myocardial ischemia injury comprises the following components in parts by weight:

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition for resisting myocardial ischemia injury comprises the following components in parts by weight:

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the ginseng is selected from ginseng or a ginseng extract.

5. The pharmaceutical composition of claim 4, wherein the ginseng extract is prepared by the method comprising: pulverizing Ginseng radix into granules, reflux-extracting with 75% ethanol, filtering the extractive solution while it is hot, concentrating, and vacuum drying the extract.

6. A pharmaceutical composition according to any one of claims 1 to 3 wherein the musk is selected from natural musk or artificial musk;

more preferably, the musk is an artificial musk.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the bezoar is selected from the group consisting of natural bezoar, in vitro cultured bezoar or artificial bezoar;

more preferably, the bezoar is an artificial bezoar.

8. A process for preparing a pharmaceutical composition as claimed in any one of claims 1 to 7, which comprises preparing the components in the said parts by weight, mixing the components, with or without pharmaceutically acceptable excipients, and preparing into clinically acceptable preparations according to conventional methods in the art.

9. Use of a pharmaceutical composition according to any one of claims 1 to 7 or a pharmaceutical composition prepared by the process according to claim 8 for the preparation of a medicament for the treatment of a disease associated with myocardial ischemic injury.

10. The use according to claim 9, wherein the diseases associated with myocardial ischemic injury are selected from one or more of coronary heart disease, angina pectoris and myocardial infarction.