A pharmaceutical composition and its application

Abstract

The present invention provides a pharmaceutical composition and its application. Specifically, the pharmaceutical composition comprises: salvianolic acid B as a first active ingredient; and ginsenoside Rg1 as a second active ingredient, and the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5). The pharmaceutical composition of the present invention has a better active ingredient ratio than the prior art, has a better therapeutic effect in preventing and / or treating ischemic diseases and ischemic reperfusion tissue and organ damage, and the pharmaceutical composition of the present invention can be used to treat ischemia and / or ischemia reperfusion injury of different tissues and organs.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a pharmaceutical composition and its application in preventing and / or treating ischemic diseases and / or ischemic reperfusion injury and the like. Background Art

[0002] Blood vessels are responsible for supplying blood to tissues and organs throughout the body. Once blood vessels are blocked, tissues and organs will develop diseases due to insufficient blood supply, especially the heart, brain and other tissues and organs that have a high demand for blood oxygen supply. With the establishment and promotion of methods such as unblocking microcirculation during shock, relief of coronary artery spasm, arterial bypass surgery, thrombolytic therapy, percutaneous transluminal coronary angioplasty, extracorporeal circulation in cardiac surgery, cardiopulmonary and cerebral resuscitation, replantation of severed limbs and organ transplantation, many tissues and organs can resume blood supply (i.e., reperfusion) after ischemia. However, this reperfusion after ischemia sometimes not only fails to restore the function of tissues and organs, but also aggravates the dysfunction and structural damage of tissues and organs. This phenomenon in which tissue damage is aggravated after blood flow is restored on the basis of ischemia, and even irreversible damage occurs, is called ischemia-reperfusion injury.

[0003] The Chinese application with application number CN2011102229806 discloses a pharmaceutical composition containing compounds salvianolic acid B and ginsenoside Rg1, which has therapeutic effects on cardiac ischemia-reperfusion injury. However, the research on the compatibility of the two is still insufficient, and it is necessary to carry out further in-depth research on the formulation and compatibility to provide a pharmaceutical composition with therapeutic effects on ischemia-reperfusion injury of different tissues and organs. Summary of the invention

[0004] The purpose of the present invention is to provide a pharmaceutical composition for preventing and / or treating diseases such as tissue and organ ischemia and ischemia-reperfusion injury.

[0005] In a first aspect, the present invention provides a pharmaceutical composition comprising:

[0006] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid B, or a combination thereof;

[0007] (b) a second active ingredient selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof; and

[0008] (c) a pharmaceutically acceptable carrier;

[0009] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5), wherein the weight ratio is calculated based on salvianolic acid B and ginsenoside Rg1.

[0010] In another preferred embodiment, the first active ingredient includes a purified product of salvianolic acid B or a pharmaceutically acceptable salt thereof.

[0011] In another preferred embodiment, the purity of the purified product, calculated as salvianolic acid B, is ≥90%, preferably ≥95%, more preferably ≥98% or 99%, based on the total weight of the purified product.

[0012] In another preferred embodiment, the first active ingredient comprises a salvianolic acid extract having a salvianolic acid B content C1 ≥ 30 wt %, wherein the content C1 is calculated by weight of salvianolic acid.

[0013] In another preferred embodiment, in the extract, the content C1 of salvianolic acid B is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0014] In another preferred embodiment, the second active ingredient comprises a total saponin extract having a ginsenoside Rg1 content C2 ≥ 30 wt %, wherein the content C2 is based on the weight of the total saponins.

[0015] In another preferred embodiment, in the extract, the content C2 of ginsenoside Rg1 is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0016] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.0), preferably 5:(1.2-3.8), and more preferably 5:(1.5-3.5).

[0017] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1.8-3.2), preferably, 5:(1.9-3.1), more preferably 5:(2-3), and most preferably 5:2.

[0018] In another preferred embodiment, the first active ingredient is salvianolic acid B, and the second active ingredient is ginsenoside Rg1.

[0019] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as freeze-dried preparations), gaseous dosage forms, and semisolid dosage forms.

[0020] In another preferred embodiment, the dosage form is selected from the following group: injection (such as injection solution or powder injection), oral preparation (such as capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), sublingual preparation, respiratory tract administration preparation, skin administration preparation, mucosal administration preparation, more preferably, the dosage form is an injection.

[0021] In a second aspect, the present invention provides a combination of active ingredients, the combination of active ingredients comprising:

[0022] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid B, or a combination thereof;

[0023] (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof;

[0024] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5), wherein the weight ratio is calculated based on salvianolic acid B and ginsenoside Rg1.

[0025] In another preferred embodiment, the combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.

[0026] In a third aspect, the present invention provides a medicine kit, comprising:

[0027] A first pharmaceutical composition, comprising: (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid B, or a combination thereof; and a pharmaceutically acceptable carrier;

[0028] A second pharmaceutical composition, comprising: (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof; and a pharmaceutically acceptable carrier;

[0029] Furthermore, the first pharmaceutical composition and the second pharmaceutical composition are administered in combination, wherein the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5), wherein the weight ratio is calculated based on salvianolic acid B and ginsenoside Rg1.

[0030] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are different (or independent) pharmaceutical compounds, or are the same pharmaceutical composition.

[0031] In a fourth aspect, the present invention provides the use of the pharmaceutical composition as described in the first aspect of the present invention, the combination of active ingredients as described in the second aspect of the present invention, or the medicine kit as described in the third aspect of the present invention, for preparing a drug or medicine kit, wherein the drug or medicine kit is used for (i) preventing and / or treating ischemic diseases; (ii) preventing and / or treating ischemia-reperfusion injury; (iii) inhibiting lactate dehydrogenase.

[0032] In another preferred embodiment, the drug or drug kit is used for (i) preventing and / or treating ischemic heart disease; (ii) preventing and / or treating ischemic reperfusion injury; (iii) inhibiting lactate dehydrogenase and / or (iv) preventing and / or treating ischemic diseases.

[0033] In another preferred embodiment, the ischemic disease is selected from the following group: tissue and organ damage caused by acute ischemia and / or tissue and organ damage caused by chronic ischemia.

[0034] In another preferred embodiment, the ischemic disease is selected from the following group: ischemic damage to tissues and organs due to primary tissue and vascular lesions, and / or ischemic lesions caused by secondary causes, such as vascular severance caused by trauma, vascular occlusion caused by inflammation, and vascular compression caused by tumors.

[0035] In another preferred embodiment, the ischemic disease is selected from the group consisting of ischemic heart disease, ischemic stroke (such as acute cerebral infarction), ischemic liver damage, pulmonary embolism, ischemic kidney damage, ischemic nerve damage, or a combination thereof.

[0036] In another preferred embodiment, the ischemic heart disease includes: coronary heart disease, myocardial infarction, angina pectoris, myocardial fibrosis, heart failure, or a combination thereof.

[0037] In another preferred embodiment, the ischemia-reperfusion injury is tissue and organ damage caused by reperfusion.

[0038] In another preferred embodiment, the tissues and organs are selected from the group consisting of heart, brain, liver, spleen, lung, kidney, muscle, nerve, or a combination thereof. In another preferred embodiment, the tissues and organs are selected from the group consisting of liver, spleen, lung, kidney, brain, nerve, or a combination thereof.

[0039] In another preferred embodiment, the tissue organ is selected from the group consisting of heart, brain, or a combination thereof.

[0040] In another preferred embodiment, the drug or drug kit is also used to improve myocardial hypertrophy induced by pulmonary embolism. In another preferred embodiment, the drug or drug kit is also used to improve the diastolic function of the heart with reperfusion injury, preferably, the diastolic function of the heart includes the diastolic rate of the heart.

[0041] In another preferred embodiment, the drug or drug kit is also used to improve the contractile function of the heart (such as the contraction rate of the heart).

[0042] In another preferred embodiment, the drug or drug kit is used to improve renal reperfusion injury, preferably, including improving renal structure.

[0043] In another preferred embodiment, the tissue and organ damage is post-operative reperfusion injury. Preferably, the surgery is selected from the following group: arterial bypass surgery, thrombectomy or thrombolytic therapy, percutaneous transluminal coronary angioplasty, heart surgery under extracorporeal circulation, heart, lung and / or brain resuscitation after cardiac arrest, limb replantation or organ transplantation, or reperfusion injury caused by other major surgeries.

[0044] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The figure shows the effect of salvianolic acid B / ginsenoside Rg1 on reducing the area of ​​cardiac infarction in an animal model of myocardial infarction; (A) Representative images of TTC staining of cardiac sections; (B) quantification of infarct area (the percentage of the infarct area in the entire heart area).

[0046] Figure 2 The effect of salvianolic acid B / ginsenoside Rg1 on the lactate dehydrogenase level in the blood in an animal model of myocardial infarction is shown.

[0047] Figure 3 The results showed that salvianolic acid B / ginsenoside Rg1 has a protective effect on cardiac tissue structure in an animal model of myocardial infarction.

[0048] Figure 4 The results show that in the animal model of myocardial ischemia-reperfusion injury, the salvianolic acid B / ginsenoside Rg1 (5:2) group significantly reduced myocardial infarction area and improved cardiac structure compared with the salvianolic acid B / ginsenoside Rg1 (2:5) group; (A) Representative images of TTC staining of cardiac sections; (B) quantification of infarct area (the percentage of the infarct area in the entire heart area); (C) Representative images of HE staining of cardiac tissue.

[0049] Figure 5 The results of hemodynamics (maximum relaxation rate and maximum relaxation rate) of rats in the salvianolic acid B / ginsenoside Rg1 (2:5) group and the salvianolic acid B / ginsenoside Rg1 (5:2) group in an animal model of myocardial ischemia-reperfusion injury are shown. "*" indicates P < 0.05 compared with the salvianolic acid B / ginsenoside Rg1 (2:5) group.

[0050] Figure 6 The graph shows the hemodynamic (end diastolic pressure and mean arterial pressure) test results of rats in the salvianolic acid B / ginsenoside Rg1 (2:5) group and the salvianolic acid B / ginsenoside Rg1 (5:2) group in an animal model of myocardial ischemia-reperfusion injury.

[0051] Figure 7 The results of HE staining of rat kidneys in the sham operation group, renal ischemia-reperfusion model group, salvianolic acid B / ginsenoside Rg1 (2:5) group, and salvianolic acid B / ginsenoside Rg1 (5:2) group in the renal ischemia-reperfusion injury model are shown.

[0052] Figure 8 The results of periodic acid Schiff staining (PAS) of rats in the sham operation group, renal ischemia-reperfusion model group, salvianolic acid B / ginsenoside Rg1 (2:5) group, and salvianolic acid B / ginsenoside Rg1 (5:2) group in the renal ischemia-reperfusion injury model are shown.

[0053] Fig. 9 The figure shows the therapeutic effect of salvianolic acid B / Rg1 (5:2) on pulmonary embolism in the pulmonary embolism model; (A) left lung index; (B) right lung index; (C) representative image of lung HE staining, (D) quantitative results of lung interstitial area (E) representative image of heart HE staining, (F) average optical density of lung neutrophils.

[0054] Fig.10 It is shown that in the pulmonary embolism model, salvianolic acid B / Rg1 (5:2) reduces the occurrence of pulmonary embolism-induced myocardial hypertrophy, (A) representative image of cardiac HE staining, (B) quantitative image of myocardial cell cross-sectional area.

[0055] Fig.11 It shows that in the acute cerebral infarction model, salvianolic acid B / Rg1 (5:2) significantly reduced the infarct area, (A) representative image of brain TTC staining, (B) quantitative results of infarct area.

[0056] Fig.12 The results show that in the acute cerebral infarction model, salvianolic acid B / Rg1 (5:2) improved the behavioral scores of rats after cerebral infarction.

[0057] Fig.13 The protective effect of salvianolic acid B / Rg1 (5:2) on cerebral cortical neurons in an acute cerebral infarction model is shown. (A) is a representative image of HE staining of the cerebral cortex; (B) is the quantitative result of the number of neurons stained by HE; (C) is a representative image of Nissl staining of the cerebral cortex; (D) is the quantitative result of the number of Nissl bodies in the cerebral cortex.

[0058] Fig.14Representative images of HE staining of CA1, CA2, and CA3 in the hippocampus in an acute cerebral infarction model are shown.

[0059] Fig.15 Shown are representative Nissl staining images of CA1, CA2, and CA3 of the hippocampus and their quantitative results in an acute cerebral infarction model.

[0060] Fig.16 It shows that in the cerebral ischemia-reperfusion injury model, salvianolic acid B / Rg1 (5:2) reduces the cerebral infarction area of ​​reperfusion injury, (A) representative image of TTC staining of brain tissue, (B) quantitative results of infarction area.

[0061] Fig.17 The results showed that in the cerebral ischemia-reperfusion injury model, salvianolic acid B / Rg1 (5:2) improved the behavioral scores of rats after cerebral ischemia-reperfusion.

[0062] Fig.18 The figure shows the protective effect of salvianolic acid B / Rg1 on cerebral cortical neurons in the cerebral ischemia-reperfusion injury model. A is HE staining of the cerebral cortex; B is Nissl staining of the cerebral cortex.

[0063] Fig.19 Representative images of HE staining of CA1, CA2, and CA3 in the hippocampus in a rat cerebral ischemia-reperfusion injury model are shown.

[0064] Fig. 20 The figure shows representative images of Nissl staining of CA1, CA2, and CA3 in the hippocampus in rat cerebral ischemia-reperfusion injury model.

[0065] Fig.21 Representative images of HE staining of liver tissue in a rat liver ischemia-reperfusion injury model are shown. DETAILED DESCRIPTION

[0066] After extensive and in-depth research, a large number of screenings and tests, the inventors have provided a pharmaceutical composition comprising salvianolic acid B and ginsenoside Rg1 as active ingredients. Compared with the prior art, the pharmaceutical composition of the present invention shows a more excellent improvement and therapeutic effect on ischemic diseases and ischemic reperfusion injuries of tissues and organs; and surprisingly, the composition of the present invention has therapeutic effects not only on ischemic reperfusion injuries of organs such as the heart, brain, and liver, but also on ischemic reperfusion injuries of organs such as the kidney, and can be used for ischemic reperfusion injuries of various tissues and organs. The present invention was completed on this basis.

[0067] the term

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0070] As used herein, the term "stereoisomer" is intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the active ingredients of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformers) are all within the scope of the present invention.

[0071] The active ingredient of the present invention may be in an amorphous form, a crystalline form or a mixture thereof.

[0072] As used herein, "pharmaceutically acceptable salt" refers to a salt formed by a compound of the active ingredient of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the active ingredient of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, and organic acids such as aspartic acid and glutamic acid. A preferred class of salts is a salt formed by a compound of the active ingredient of the present invention and a base. Suitable bases for forming salts include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, and organic bases such as ammonia water, triethylamine, and diethylamine. Another preferred salt is a salt formed by the active ingredient of the present invention and a metal ion, including but not limited to magnesium salt, sodium salt, calcium salt, potassium salt and the like.

[0073] As used herein, "pharmaceutically acceptable ester" refers to an ester suitable for use as a drug formed by a compound of the active ingredient of the present invention and an acid or an alcohol. A preferred class of esters are esters formed between one or more hydroxyl groups of the active ingredient of the present invention and an acid. Acids suitable for forming esters include but are not limited to: phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, etc.; another preferred class of esters are esters formed between the carboxyl group of the active ingredient of the present invention and an alcohol. Alcohols suitable for forming esters include but are not limited to: C1-C6 alkyl-OH, such as methanol, ethanol, n-propanol, isopropanol, etc.

[0074] Unless otherwise specified, in the pharmaceutical composition, the weight ratio is calculated based on the original compound forms of salvianolic acid B and ginsenoside Rg1.

[0075] The "prevention" and "treatment" described in the present invention include delaying and stopping the progression of the disease, or eliminating the disease, and do not require 100% inhibition, elimination and reversal. In some embodiments, compared with the level observed in the absence of the composition or pharmaceutical composition of the present invention, the composition or pharmaceutical composition of the present invention prevents, alleviates, inhibits and / or reverses ischemia-reperfusion injury by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0076] As used in the present invention, the term "SalB" and "salvianolic acid B" can be used interchangeably; the term "Rg1" and "ginsenoside Rg1" can be used interchangeably.

[0077] First active ingredient

[0078] In the present invention, the first active ingredient is selected from the following group: salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, extracts containing salvianolic acid B, or combinations thereof.

[0079]

[0080] In another preferred embodiment, the first active ingredient includes a purified product of salvianolic acid B or a pharmaceutically acceptable salt thereof.

[0081] In another preferred embodiment, the purity of the purified product, calculated as salvianolic acid B, is ≥90%, preferably ≥95%, more preferably ≥98% or 99%, based on the total weight of the purified product.

[0082] In another preferred embodiment, the first active ingredient comprises a salvianolic acid extract having a salvianolic acid B content C1 ≥ 30 wt %, wherein the content C1 is calculated by weight of salvianolic acid.

[0083] In another preferred embodiment, in the extract, the content C1 of salvianolic acid B is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0084] Second active ingredient

[0085] In the present invention, the second active ingredient is selected from the following group: ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof.

[0086]

[0087] In another preferred embodiment, the second active ingredient comprises a total saponin extract having a ginsenoside Rg1 content C2 ≥ 30 wt %, wherein the content C2 is based on the weight of the total saponins.

[0088] In another preferred embodiment, in the extract, the content C2 of ginsenoside Rg1 is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0089] Pharmaceutical composition, combination of active ingredients, and kit

[0090] The present invention provides a pharmaceutical composition comprising:

[0091] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid B, or a combination thereof;

[0092] (b) a second active ingredient selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof; and

[0093] (c) a pharmaceutically acceptable carrier;

[0094] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5), wherein the weight ratio is calculated based on salvianolic acid B and ginsenoside Rg1.

[0095] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.0), preferably 5:(1.2-3.8), and more preferably 5:(1.5-3.5).

[0096] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1.8-3.2), preferably, 5:(1.9-3.1), more preferably 5:(2-3), and most preferably 5:2.

[0097] In another preferred embodiment, the first active ingredient is salvianolic acid B, and the second active ingredient is ginsenoside Rg1.

[0098] The dosage form of the pharmaceutical composition is selected from the following group: liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as lyophilized preparations).

[0099] In another preferred embodiment, the dosage form is selected from the following group: injection (such as injection solution or powder injection), oral preparation (such as capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), more preferably, preferably, the dosage form is injection.

[0100] In the pharmaceutical composition of the present invention, the first active ingredient and the second active ingredient can be prepared into preparations separately or mixed together into a preparation.

[0101] The pharmaceutical composition of the present invention comprises a first active ingredient and / or a second active ingredient within a safe and effective amount. Wherein "safe and effective amount" means: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the active ingredient of the present invention / dose, more preferably, contains 10-500 mg of the active ingredient of the present invention / dose. Preferably, the "one dose" is a capsule, tablet, injection, etc.

[0102] In the present invention, "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that each component in the composition can be mixed with the first active ingredient and / or the second active ingredient without significantly reducing the efficacy of the first active ingredient and / or the second active ingredient. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0103] The present invention also provides a combination of active ingredients, the combination of active ingredients comprising:

[0104] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid B, or a combination thereof;

[0105] (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof;

[0106] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5), wherein the weight ratio is calculated based on salvianolic acid B and ginsenoside Rg1.

[0107] In another preferred embodiment, the combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.

[0108] In the active ingredient combination, the first active ingredient and the second active ingredient may be independent of each other, or they may be combined together to form an active ingredient composition.

[0109] The present invention also provides a medicine box, which comprises:

[0110] A first pharmaceutical composition, comprising: (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid B, or a combination thereof; and a pharmaceutically acceptable carrier;

[0111] A second pharmaceutical composition, comprising: (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing ginsenoside Rg1, or a combination thereof; and a pharmaceutically acceptable carrier;

[0112] Furthermore, the first pharmaceutical composition and the second pharmaceutical composition are administered in combination, wherein the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.5), wherein the weight ratio is calculated based on salvianolic acid B and ginsenoside Rg1.

[0113] In another preferred embodiment, the medicine kit further includes instructions.

[0114] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are different (or independent) pharmaceutical compounds, or are the same pharmaceutical composition.

[0115] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously, separately or sequentially.

[0116] The pharmaceutical composition, active ingredient combination and medicine kit of the present invention can be prepared by conventional methods and equipment.

[0117] Use and method of application

[0118] The present invention provides a use of the pharmaceutical composition, combination of active ingredients or kit described herein for preparing a medicament or kit for (i) preventing and / or treating ischemic diseases; (ii) preventing and / or treating ischemia-reperfusion injury; and / or (iii) inhibiting lactate dehydrogenase.

[0119] In the present invention, the ischemic disease refers to damage or pathology caused by ischemia in tissues or organs. The "ischemia" refers to a decrease in the blood supply to a tissue or organ compared to a normal value, especially when the blood supplied to a tissue or organ cannot meet the metabolic needs of the tissue or organ.

[0120] The active ingredients of the present invention have obvious therapeutic effects on ischemic diseases. Common ischemic diseases include but are not limited to: ischemic heart disease, ischemic stroke, ischemic liver injury, ischemic lung injury, ischemic kidney injury, or a combination thereof.

[0121] In the present invention, the ischemic heart disease is a heart disease caused by myocardial ischemia and hypoxia due to changes in coronary circulation. Common ischemic heart diseases include the following groups (but not limited to): coronary heart disease, myocardial infarction, myocardial fibrosis, angina pectoris, or a combination thereof.

[0122] In the present invention, the ischemia-reperfusion injury includes tissue and organ damage caused by reperfusion. The tissue and organ include the following group (but not limited to): heart, liver, spleen, lung, kidney, brain, muscle, nerve, or a combination thereof. The tissue and organ damage also includes reperfusion injury after surgery, and the surgery includes the following group (but not limited to): arterial bypass surgery, thrombolytic therapy, percutaneous transluminal coronary angioplasty, heart surgery under extracorporeal circulation, heart, lung and / or brain resuscitation after cardiac arrest, limb replantation or organ transplantation. The reperfusion injury also includes: reperfusion injury after microcirculation dredging after shock, and reperfusion injury after relief of coronary artery spasm.

[0123] In the present invention, the applications of preventing and / or treating ischemic diseases, preventing and / or treating ischemic reperfusion injury, etc. include preventive applications and subsequent ameliorative applications. For example, for reperfusion injury, the pharmaceutical composition, active ingredient composition or kit of the present invention is applied before, during, and / or after reperfusion to improve or enhance the protection, repair or function of tissues and organs after reperfusion injury.

[0124] In the pharmaceutical composition, combination of active ingredients or medicine kit of the present invention, the first active ingredient and the second active ingredient can also be co-administered with other pharmaceutically acceptable compounds, including (but not limited to): antihypertensive drugs, lipid-lowering drugs, glucose-lowering drugs, antiplatelet aggregation drugs, etc.

[0125] The pharmaceutical composition, combination of active ingredients or kit of the present invention can also be used to inhibit lactate dehydrogenase. Lactate dehydrogenase (LDH) is an enzyme required in the process of converting sugar into cellular energy, and is present in various organs and tissues throughout the body, such as liver, heart, pancreas, kidney, skeletal muscle, lymphatic tissue and blood cells. Lactate dehydrogenase is involved in the final step of glycolysis in which pyruvate is converted into lactic acid. Although normal tissues usually use glycolysis only when oxygen supply is insufficient, cancer tissues rely heavily on aerobic glycolysis, regardless of oxygen supply level. LDH inhibitors are used for pathologies involving metabolic conversion from oxidative phosphorylation to glycolysis, for example, can be used for (but not limited to): treatment of patients with cancer, fibrosis or other conditions in which metabolic conversion from oxidative phosphorylation to glycolysis occurs. At the same time, lactate dehydrogenase is responsible for the enzyme that converts glyoxylate into oxalate in the mitochondrial / peroxisomal glycine metabolic pathway of the liver and pancreas, and inhibition of LDH can be used to treat chronic kidney disease, such as hyperoxaluria. The pharmaceutical composition of the present invention can reduce the LDH concentration in the blood and / or inhibit the activity of LDH, and can be used as an LDH inhibitor.

[0126] In the pharmaceutical composition, combination of active ingredients or kit of the present invention, the first active ingredient and the second active ingredient may be administered simultaneously, separately or sequentially.

[0127] In the pharmaceutical composition, combination of active ingredients or medicine kit of the present invention, there is no particular limitation on the mode of administration of the first active ingredient and the second active ingredient. Representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0128] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0129] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient can also be formed into microcapsules with one or more of the above-mentioned excipients.

[0130] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0131] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0132] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0133] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0134] The dosage forms of the active ingredient of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

[0135] In the pharmaceutical composition, combination of active ingredients or kit of the present invention, the general range of the therapeutically effective dose of the active ingredient will be: about 1-2000 mg / day, about 10-about 1000 mg / day, about 10-about 500 mg / day, about 10-about 250 mg / day, about 10-about 100 mg / day, or about 10-about 80 mg / day, based on the total amount of salvianolic acid B and ginsenoside Rg1. The therapeutically effective dose will be administered in one or more doses. However, it should be understood that the specific dose of the active ingredient of the present invention for any particular patient will depend on a variety of factors, such as the age, sex, weight, general health condition, diet, individual response of the patient to be treated, the time of administration, the severity of the disease to be treated, the dosage form, the mode of application and the concomitant medication. The therapeutically effective amount for a given situation can be determined by routine experiments and is within the ability and judgment of the clinician or physician. In any case, the active ingredient will be administered in multiple doses based on the individual situation of the patient and in a manner that allows the delivery of a therapeutically effective amount.

[0136] The main advantages of the present invention include:

[0137] 1. Compared with the prior art, the pharmaceutical composition of the present invention has a more optimal active ingredient ratio and has a better therapeutic effect on the treatment of ischemic diseases and ischemia-reperfusion injury (such as excellent effects of reducing infarct area and improving tissue and organ function).

[0138] 2. The pharmaceutical composition of the present invention has a therapeutic effect not only on the heart, but also on reperfusion injuries of different organs such as the brain, liver, lungs and kidneys, and can be widely used in ischemia-reperfusion injuries of various tissues and organs.

[0139] 3. The pharmaceutical composition of the present invention also has the effect of inhibiting lactate dehydrogenase, can quickly reduce the lactate dehydrogenase content and / or activity in the blood, and can be used as a lactate dehydrogenase inhibitor for diseases related to lactate dehydrogenase.

[0140] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0141] 1. Reagents and Materials

[0142] 1.1 Animals

[0143] 134 clean male Wistar rats, weighing 220±10g; 56 of them were used to study the protective effect of different ratios of combination drugs on the heart of rats with myocardial infarction; 16 were used to study the protective effect of combination drugs on ischemia-reperfusion heart; 32 were used to study the protective effect of combination drugs on ischemia-reperfusion kidneys; 30 were used to evaluate the preventive and therapeutic effects of combination drugs on pulmonary embolism and its complications.

[0144] 96 clean male SD rats, weighing 220±10g; 30 of them were used to study the protective effect of the combination drug on ischemic cerebral infarction; 50 were used to study the protective effect of the combination drug on cerebral ischemia-reperfusion injury; 16 were used to study the protective effect of the combination drug on ischemia-reperfusion liver.

[0145] Wistar and SD rats were provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences, and were housed in an SPF-grade animal room at the Experimental Animal Center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature of 22±2℃, 12h light, standard diet, and free access to drinking water.

[0146] Administration: Unless otherwise specified, the administration method in the examples is tail vein injection.

[0147] 1.2 Main reagents and consumables

[0148]

[0149] 1.3 Main instruments

[0150]

[0151] 1.4 General experimental methods

[0152] Blood separation and processing

[0153] Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg) and fixed on a mouse board in a supine position. The abdomen was cut open along the midline with scissors, the contents of the abdominal cavity were taken out, the fluid in the abdominal cavity was wiped dry with a dry cotton ball, and blood was collected from the abdominal aorta with a 5 mL syringe into a 2 ml EP tube; after the blood was left to stand on ice for 0.5 hours, the collected blood was centrifuged at 4°C, 8000 r / min for 10 minutes, the supernatant was taken, and the serum was divided into 0.5 ml EP tubes and stored in a -80°C refrigerator for later use.

[0154] TTC staining

[0155] Dissolve 0.5g TTC powder in 100ml PBS, store in dark, and prepare before use. Cut fresh tissue into multiple slices, place in a small box with a lid, add TTC solution, place in a wet box, avoid light, and incubate in a 37℃ constant temperature oven for 20 minutes. During this period, turn the tissue several times to make it evenly contact with the dye solution. Take it out and take pictures after 20 minutes.

[0156] Sample fixation, dehydration, paraffin embedding, sectioning

[0157] 100ml of formaldehyde, 4g of sodium dihydrogen phosphate, and 6.5g of disodium hydrogen phosphate were dissolved in 900mL of distilled water to prepare a 10% paraformaldehyde fixative by volume. After the tissue was fixed in the paraformaldehyde fixative for 72 hours, it was rinsed with tap water for 2 hours and placed in a dehydrator with a program set for automatic dehydration, followed by 75% ethanol for 1.5 hours, 95% ethanol for 1.5 hours, 100% ethanol for 1.5 hours, xylene for 1.5 hours, and paraffin for 1.5 hours. The paraffin embedding machine was opened 2 hours in advance to melt the paraffin, and the temperature was controlled at 60°C. After the paraffin melted, the dehydrated tissue was embedded in paraffin and poured into the embedding box. The wax-soaked tissue block was placed in the embedding frame with heated tweezers and gently moved to the cold stage. After the paraffin solidified, the paraffin block was removed for sectioning. Before sectioning, the wax block was placed in a refrigerator for precooling. After cooling, the slicer continuously cut paraffin sections with a thickness of 5μm. The sections were spread on a slide spreader at 38°C, scooped out with a glass slide coated with poly-lysine, and air-dried for subsequent pathological histological staining.

[0158] HE staining

[0159] Place the tissue sections on a 65℃ slide oven for 60 minutes, quickly put them into xylene for dewaxing for 15 minutes, soak them in 100%, 95%, and 75% ethanol for 5 minutes, rinse them with running water for 5 minutes, place them in hematoxylin solution for 15 minutes, rinse them with water for 5 minutes, and then put them in 1% hydrochloric acid ethanol differentiation solution (preparation: add 3mL concentrated hydrochloric acid to 300mL75% ethanol and stir well) for 3 seconds, turn blue with running water for 5 minutes, put them in 1% eosin solution for 10 minutes, quickly rinse off the excess eosin solution, transfer them to 75% ethanol and soak them for 4 minutes, 95% ethanol for 4 minutes, 100% ethanol for 5 minutes, make them transparent in xylene for 15 minutes, and finally seal them with xylene neutral gum. After the gum is air-dried, use a stereo microscope to take gross pictures and a BX51 microscope to take local magnified pictures.

[0160] PAS staining

[0161] Place the tissue slices on a 65℃ baking machine for 60 minutes, quickly put them into xylene for dewaxing for 15 minutes, soak them in 95%, 70%, and 30% ethanol for 2 minutes, soak them in distilled water for 2 minutes, and drop the prepared reagent 1 application dye solution on the sample on the slide to cover the sample completely. Gently place the slide flat on the staining rack and incubate at room temperature away from light for 8-15 minutes. Take out the stained slide and slowly rinse the slide with running water for 3-5 minutes. Before the slide is completely dry, drop reagent 2 application liquid on the slide sample, blow the dye solution evenly with an ear wash bulb and cover the sample completely, and then incubate at room temperature for 8-15 minutes. After the incubation, take out the stained slide, slowly rinse the slide with running water for 30-60 seconds, and dry it naturally. Add reagent triple counterstain solution and stain for 20-30 seconds, then rinse with running water, seal the slide with sealing medium after it dries, and after the sealing medium is air-dried, use a stereo microscope to take a gross picture, and a BX51 microscope to take a local magnified picture.

[0162] Biochemical index detection

[0163] LDH detection was performed on rat serum using a fully automatic biochemical analyzer (JCA-BM6010 / C, Sysmex Medical Electronics (Shanghai) Co., Ltd.) according to the lactate dehydrogenase assay kit (lactate substrate method, Sysmex Biotech (Wuxi) Co., Ltd., batch number: R8004).

[0164] Hemodynamic testing

[0165] After the rats were anesthetized by intraperitoneal injection of 40 mg / kg sodium pentobarbital, the right common carotid artery was isolated and a Miller catheter was inserted. The hemodynamic indices such as carotid artery pressure, maximum left ventricular contraction rate, maximum left ventricular relaxation rate, and left ventricular end diastolic pressure were recorded using a Powerlab8 / 30 physiological recorder (ML870, ADINSTRUMENTS).

[0166] Calculation of left and right lung index

[0167] After the rats were weighed, 30 mg / kg of Zotal was injected intraperitoneally for anesthesia, and the left and right lungs were separated and weighed. The left and right lung indexes were calculated based on the ratio of the left and right lungs to body weight.

[0168] Quantification of lung interstitial area

[0169] Quantitative analysis was performed on hematoxylin-eosin-stained lung tissue. The lung parenchyma, including the trachea, bronchi, and alveoli, was removed from each sample, and the remaining purple part represented the lung interstitium. Each tissue sample was photographed at the same magnification to quantify the area of ​​the lung interstitium.

[0170] Immunohistochemical staining to investigate neutrophil infiltration

[0171] The paraffin-embedded tissue was baked at 65°C for 45-50 minutes; then xylene for 15 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, rinsed with running water for 1 minute, and dewaxed to the aqueous phase; antigens were repaired with citric acid repair solution under microwave conditions; incubated at 37°C with 10% goat serum for 30 minutes; incubated with CD44 antibody at 4°C overnight; incubated with secondary antibody at 37°C for 1 hour; DAB solution was dropped for 40 seconds; hematoxylin counterstaining for 15 minutes; differentiated with 1% hydrochloric acid ethanol for 5 seconds; permeabilized with xylene; and sealed with neutral gum. Image Pro Plus software was used to quantify the positive cell area and optical density of each sample, and the average optical density value was calculated by the ratio of optical density to positive cell area.

[0172] Behavioral testing of animals

[0173] Longa score, NSS score and EBST test were performed on all experimental animals two days before surgery and one day after surgery.

[0174] a. Longa scoring method: 0 points: normal, no neurological damage; 1 point: the left front paw cannot be fully extended, mild neurological damage; 2 points: when walking, the rat turns to the left (hemiplegic side), moderate neurological damage; 3 points: when walking, the rat's body leans to the left (hemiplegic side), severe neurological damage; 4 points: unable to walk spontaneously, loss of consciousness.

[0175] b. NSS scoring method: 0 points: normal neurological function; 1 point: mild neurological deficit (flexion of the left forelimb when lifting the tail); 2 points: moderate neurological deficit (turning to the left when walking); 3 points: moderate neurological deficit (leaning to the left); 4 points: unable to walk, decreased consciousness; 5 points: death related to ischemia.

[0176] c. Elevated Body Swing Test: When measuring, first lift the tail root of the rat with your hand, and let the rat's head hang about 5 cm above the plane. At this time, the rat's head will rotate to the left or right. When the angle of unilateral rotation is greater than 100, it is the counting standard. Record the direction and angle of rotation. After one test, let the rat rest for one minute before the next test. Repeat 20 times and record the total direction and number of times.

[0177] Data Statistical Methods

[0178] GraphPad Prism 6.0 (GraphPad Software, LA Jolla, CA, USA) was used for data analysis. All measured data were expressed as mean ± standard deviation, and one-way ANOVA was used to confirm whether the variance was homogeneous. If the n values ​​were consistent, the Tukey method was used for comparison; if the n values ​​were inconsistent, the Bonferroni method was used for comparison, and P < 0.05 was considered statistically significant.

[0179] two, Animal experiments

[0180] Example 1

[0181] 1.1 Preparation of myocardial infarction model

[0182] Sodium pentobarbital (40 mg / kg) was injected intraperitoneally, and the rats were fixed on the surgical board. The chest hair was inserted into the trachea with a pen-type intravenous indwelling needle sleeve and connected to the animal ventilator. The skin was cut open between the 3rd and 4th ribs on the left side of the chest, and the muscles were bluntly separated. The third and fourth ribs were opened and fixed to expose the upper part of the heart. The pericardium was torn open, and the needle holder clamped a 5-0 suture needle with thread and used the left coronary vein as a mark. The thread was threaded and ligated from the left atrial appendage apex, the pulmonary artery cone and the junction of the atrial appendage 1mm below. After the coronary artery was ligated, the myocardial tissue immediately turned from ruddy to pale. The sham-operated animals were operated in exactly the same way except that the left anterior descending branch of the coronary artery was not ligated.

[0183] 1.2 Animal grouping and drug administration

[0184] Fifty-six rats were randomly divided into the following seven groups: sham operation group, ischemia model group, and combined administration group of salvianolic acid B and ginsenoside Rg1 (prepared in the ratio of 5:4, 5:3, 5:2, 5:1 and 2:5, respectively), with 8 rats in each group. The administration was done in a double-blind manner, that is, the surgeon did not participate in the administration, and the data statisticians did not know the grouping information. Salvianolic acid B and ginsenoside Rg1 were mixed in the ratio of 5:4, 5:3, 5:2, 5:1, and 2:5, respectively, and randomly numbered after mixing. After dissolution, they were filtered with a microporous filter membrane for use. The sham operation group and the myocardial infarction model group were given an equal volume of normal saline according to body weight. Immediately after the operation, the rats were injected once with a tail vein at 15 mg / kg, and then once again after 24 hours. Then, blood was collected from the abdominal aorta and the heart was collected for cardiac histology.

[0185] 1.3 Experimental Results and Analysis

[0186] 1.3.1 SalB / Rg1 reduces myocardial infarction area

[0187] like Figure 1As shown, there was no infarct area in the sham operation group (0%). Compared with the ischemic model group, the infarct area in the 2:5 group decreased by 23.4%; the infarct area in the 5:4 group decreased by 15.3%; the infarct area in the 5:3 group decreased by 35.2% (P < 0.01); the infarct area in the 5:2 group decreased by 33.2% (P < 0.01); the infarct area in the 5:1 group decreased by 25.1%; * indicates that compared with the sham operation group, ***P < 0.001; # Compared with the ischemia model group, # P<0.05. In addition, the 5:3 group and the 5:2 group had a more significant effect on reducing the infarct size than the 2:5 group, and the infarct size was reduced by 1.50 times (5:3) and 1.42 times (5:2) respectively compared with the 2:5 group.

[0188] 1.3.2 SalB / Rg1 reduces LDH levels in the blood

[0189] The results of LDH content in blood are as follows Figure 2 As shown in the figure, compared with the ischemic model group, the LDH content in the 2:5 group decreased by 19.7%; the LDH content in the 5:4 group decreased by 7.5%; the LDH content in the 5:3 group decreased by 23.0%; the LDH content in the 5:2 group decreased by 37.9%; and the LDH content in the 5:1 group decreased by 13.7%. * indicates that compared with the sham operation group, *P<0.05. Compared with the 2:5 group, the LDH content in the 5:2 group decreased by 1.9 times.

[0190] 1.3.3 SalB / Rg1 improves cardiac structure

[0191] To further evaluate the protective effect of SalB / Rg1 on cardiac tissue structure, the cardiac structures of the infarcted area (upper), infarcted edge area (middle), and infarcted distal area (lower) were analyzed. Figure 3 As shown). In the ischemic model group, the structure of the infarcted area of ​​the heart was severely damaged, with a large number of inflammatory cells infiltrating, myocardial cells showing edema, necrosis, cell nucleus loss, and myocardial fibers in the shape of cords; in the infarct edge area, inflammatory cells infiltrated significantly, and myocardial fibers became elongated and wavy. Compared with the ischemic model group, SalB / Rg1 showed different degrees of improvement on the above damage in both the infarct area and the infarct edge area, among which the myocardial tissue structure of rats in the 5:2 group was the most obvious: in the infarct area, the infiltration of inflammatory cells was significantly reduced, the edema and necrosis of myocardial cells were weakened, the number of cell nucleus loss was reduced, and the myocardial fibers were arranged more regularly; in the infarct edge area, the infiltration of inflammatory cells was reduced, and the myocardial fibers were arranged regularly. The cells in the distal area of ​​the infarction of each group of animals were arranged in an orderly, tight and regular manner, with no significant difference.

[0192] The above results indicate that, relative to the ischemic model group, the combined administration of SalB / Rg1 is effective in reducing the infarct area. Surprisingly, compared with the optimal SalB / Rg1 ratio (2:5) disclosed in the prior art, after administration to the groups with SalB / Rg1 of 5:3 and 5:2 in the present invention, the infarct area of ​​the rat heart was smaller, and better therapeutic effects were shown in reducing the LDH content in the blood and improving the heart structure.

[0193] Example 2

[0194] A further comparison was performed in a myocardial ischemia-reperfusion injury model using a combination of SalB / Rg1 of 5:2 and the optimal combination of SalB / Rg1 of 2:5 disclosed in the prior art.

[0195] 2.1 Preparation of myocardial ischemia-reperfusion injury model

[0196] Sodium pentobarbital (40 mg / kg) was injected intraperitoneally, and the rats were fixed on the operating board, with hair on the chest. The pen-type intravenous indwelling needle set was inserted into the trachea and connected to the animal ventilator, iodine disinfection, the skin was cut open between the 3rd and 4th ribs on the left side of the chest, and the muscles were bluntly separated. The third and fourth ribs were opened and fixed to expose the upper part of the heart, and the pericardium was torn open. The needle holder clamped a 5-0 suture needle with a thread and marked the left coronary vein. The thread was threaded from the left atrial appendage apex, the pulmonary artery cone and the atrial appendage 1mm below the junction. A 2-0 thread was placed in the place where the two silk threads were knotted and ligated. After the coronary artery was ligated, the myocardial tissue immediately turned from ruddy to pale. After 40 minutes of myocardial ischemia, the silk thread was cut open, and the 2-0 thread was removed for myocardial ischemia reperfusion, and various tests and evaluations were performed 1h later. The sham-operated animals were operated exactly the same except that the left anterior descending coronary artery was not ligated.

[0197] 2.2 Animal grouping and drug administration

[0198] 16 rats weighing about 220g were randomly divided into the following 2 groups: Salvianolic acid B and ginsenoside Rg1 co-administration group (prepared in a ratio of 5:2 and 2:5), 8 rats in each group. The rats were myocardially ischemic for 40 minutes and then reperfused. Immediately at the same time of reperfusion, 15 mg / kg of the drug was injected into the tail vein. Hemodynamics were tested 1 hour after reperfusion, and then the hearts were collected for cardiac histology.

[0199] 2.3 Experimental Results

[0200] In the cardiac ischemia-reperfusion injury model, the experimental results are as follows Figure 4Figure A is a representative picture of TTC staining of infarct area, Figure B is the quantitative result of infarct area, and Figure C is a representative picture of HE staining. Compared with SalB / Rg1(2:5), the infarct area of ​​SalB / Rg1(5:2) rats decreased by 15.03%. To further evaluate the protection of SalB / Rg1 on the cardiac tissue structure of ischemia-reperfusion injury, the cardiac structure of the infarct area (top), infarct edge area (middle), and infarct distal area (bottom) was analyzed respectively (Figure C). Compared with SalB / Rg1(2:5), SalB / Rg1(5:2) significantly inhibited inflammatory cell infiltration, myocardial cell necrosis, cell nuclear loss and other damages in the infarct area and infarct edge area. The cells in the distal area of ​​the infarct of each group of animals were arranged in an orderly and tight manner, with no significant difference.

[0201] Figure 5 and Figure 6 The hemodynamic results showed that compared with SalB / Rg1(2:5), SalB / Rg1(5:2) increased the maximum diastolic rate by 18.2% (P<0.05) and the maximum systolic rate by 11.6%, indicating that SalB / Rg1(5:2) was more effective than SalB / Rg1(2:5) in improving cardiac function; Figure 6 The results showed that there was no significant difference in terminal diastolic blood pressure and mean arterial pressure, indicating that SalB / Rg1(5:2) had no adverse effect on blood pressure regulation compared with SalB / Rg1(2:5).

[0202] Example 3

[0203] SalB / Rg1(5:2) and SalB / Rg1(2:5) were further compared in the renal ischemia-reperfusion injury model.

[0204] 3.1 Preparation of renal ischemia-reperfusion injury model

[0205] Intraperitoneal injection of sodium pentobarbital (40mg / kg), rats are fixed on the operating board, the abdomen is disinfected with iodine tincture, the midline incision of the abdomen is 2.5cm, the intestine is separated to expose the left kidney, the fat around the kidney is separated, the artery clamp is clamped on the left renal pedicle (including renal artery, renal vein, renal pelvis) for 40 minutes to cause renal ischemia, and the right kidney and the left kidney are operated the same. After 40 minutes, the artery clamps of the kidneys on both sides are removed, and renal ischemia-reperfusion injury is caused after 24h, and muscles and skin are sutured. Sham-operated animals are completely identical except that the renal pedicles on both sides are not clamped.

[0206] 3.2 Animal grouping and drug administration

[0207] Thirty-two rats were randomly divided into the following four groups: sham operation group, ischemia-reperfusion model group, and combined administration group of salvianolic acid B and ginsenoside Rg1 (prepared in a ratio of 5:2 and 2:5), with 8 rats in each group. The sham operation group and the renal ischemia-reperfusion model group were given an equal volume of saline according to body weight. The rats were reperfused after 40 minutes of renal ischemia, and immediately injected once into the tail vein at 15 mg / kg at the same time of reperfusion, and then administered once again 24 hours later, and then the kidneys were collected for renal histology.

[0208] 3.3 Experimental Results

[0209] To evaluate the protective effect of SalB / Rg1 on renal tissue structure in ischemia-reperfusion injury, the structure of renal cortex (e.g. Figure 7 ). In the renal cortex of the ischemia-reperfusion model group, a large number of inflammatory cells infiltrated, a large number of red blood cells infiltrated, and the intercellular space was enlarged. Compared with the ischemia-reperfusion model group, SalB / Rg1 showed different degrees of improvement on the above damage, and the improvement of the renal tissue structure of rats in the 5:2 group was the most obvious: the infiltration of inflammatory cells and the infiltration of red blood cells in the renal cortex were significantly reduced, and the intercellular space was reduced.

[0210] To evaluate the effect of SalB / Rg1 on glycogen accumulation in renal tissues after ischemia-reperfusion injury, the accumulation of glycogen in the renal cortex was analyzed (e.g. Figure 8 ). In the ischemia-reperfusion model group, glycogen accumulation in the renal mesangial area, proliferation of glomerular mesangial cells, thickening of basement membrane, and detachment of nuclei were observed. Compared with the ischemia-reperfusion model group, SalB / Rg1 showed different degrees of improvement on the above injuries. Compared with the 2:5 group, the renal glycogen accumulation of rats in the 5:2 group was significantly reduced, the proliferation of glomerular mesangial cells was improved, and the number of detached nuclei was reduced.

[0211] Example 4

[0212] To evaluate the preventive and therapeutic effects of salvianolic acid B / ginsenoside Rg1 combination (5:2) on pulmonary embolism and its complications in rats

[0213] 4.1 Experimental animals and model preparation

[0214] Thirty male Wistar rats were randomly divided into the following three groups: normal control group, model control group, and salvianolic acid B / Rg1 group (20 mg / kg), with 10 rats in each group. The normal control group was given normal saline at a dose of 5 ml / kg on days 0, 7, 14, and 21, and the other two groups were given polystyrene microspheres at the corresponding time. The concentration of polystyrene microspheres was 200,000 particles / ml and the diameter was 45 μm. Polystyrene microspheres were injected into the tail vein of Wistar rats at a dose of 1 million particles / kg (5 ml / kg) on ​​days 0, 7, 14, and 21.

[0215] Two groups of animals were given polystyrene microspheres. One group was injected with normal saline every day from the 7th day as a model control group for 28 consecutive days. The other group was injected with salvianolic acid B / Rg1 (ratio 5:2) at a dose of 20 mg / kg every day from the 7th day for 28 consecutive days. All animals were sampled on the 35th day for heart and lung histology.

[0216] 4.2 Experimental Results

[0217] 4.2.1 Salvianolic acid B / Rg1 (5:2) combination can improve microsphere-induced pulmonary embolism

[0218] The combination of salvianolic acid B / Rg1 can improve microsphere-induced pulmonary embolism. Fig. 9 As shown, compared with the model group, salvianolic acid B / Rg1 significantly reduced the left lung index (A) and the right lung index (B); from the tissue staining diagram, it can be seen that salvianolic acid B / Rg1 significantly improved the lung structure (C) and significantly reduced the area of ​​pulmonary interstitium (D), which proves the improvement effect of salvianolic acid B / Rg1 on lung function; in addition, it can be seen from (E) that salvianolic acid B / Rg1 can also inhibit the infiltration of neutrophils in the lung tissue, and its inhibitory effect has significant statistical significance (F), indicating that salvianolic acid B / Rg1 significantly inhibits the occurrence of pulmonary embolism.

[0219] 4.2.2 Salvianolic acid B / Rg1 combination improves myocardial hypertrophy caused by pulmonary embolism

[0220] The main complication of pulmonary embolism is myocardial hypertrophy. In HE-stained myocardial tissue, hypertrophic myocardial cells can be evaluated by the size of the cell cross-sectional area. Fig.10 As shown, A is a representative image of cardiac HE staining, and B is a quantitative image of the cross-sectional area of ​​cardiomyocytes. Salvianolic acid B / Rg1 significantly reduced the occurrence of cardiac hypertrophy induced by pulmonary embolism.

[0221] Example 5

[0222] Treatment of acute cerebral infarction in rats with salvianolic acid B / Rg1 combination (5:2)

[0223] 5.1 Experimental animals and model preparation

[0224] Thirty male SD rats were randomly divided into the following three groups: sham operation group, acute cerebral infarction model group, and salvianolic acid B / ginsenoside Rg1 combination group (combination group ratio of 5:2, drug administration and dosage of 10 mg / kg), with 10 rats in each group. After anesthesia, fixation, chest hair and iodine disinfection, the muscles, subcutaneous connective tissue, anterior cervical muscles were separated in sequence to expose the common carotid artery (CCA); a small incision was made 4 mm away from the bifurcation of the CCA, and the suture was inserted from the small incision into the internal carotid artery (ICA), and the suture was pushed about 20 mm from the bifurcation of the blood vessel to block the middle cerebral artery (MCA) to induce cerebral infarction. Animals with cerebral infarction were treated with 10 mg / kg salvianolic acid B / ginsenoside Rg1 (salvianolic acid B / Rg1 group); animals with cerebral infarction were given normal saline (model group); animals in the sham operation group were subjected to the same surgical procedures except that the suture was not inserted, and normal saline was given at the same time point (sham operation group).

[0225] 5.2 Experimental Results

[0226] 5.2.1 Salvianolic acid B / Rg1 reduces cerebral infarction area

[0227] The infarct area was evaluated by TTC staining. Fig.11 As shown, Figure A is a representative TTC staining image of the brain, the infarct area is stained white, and the non-infarct area is stained red. Figure B is the quantitative result of infarct area (white area / (red area+white area) percentage). Salvianolic acid B / Rg1 significantly reduced the infarct area. Compared with the model group, the cerebral infarction area in the Salvianolic acid B / Rg1 group was reduced by 39.42%. Note: ***p<0.001VS sham operation group, &&p<0.01VS model group.

[0228] 5.2.2 Salvianolic acid B / Rg1 improves behavioral scores in rats after cerebral infarction

[0229] Longa score, NSS score and EBST were used to evaluate the neuromotor function of rats before and after surgery. Fig.12 As shown. According to the three scoring methods, there was no significant behavioral difference among the three groups of animals before surgery. After cerebral infarction modeling, according to EBST score (p<0.001), Longa score (p<0.05) and NSS score (p<0.05), salvianolic acid B / Rg1 significantly improved neuromotor function.

[0230] 5.2.3 Salvianolic acid B / Rg1 protects cerebral cortical neurons

[0231] The cerebral cortex is the higher center for regulating and controlling body movements. Fig.13As shown, A is a representative picture of HE staining of the cerebral cortex; B is the quantitative result of the number of neurons in a single image field of view of HE staining; C is a representative picture of Nissl staining of the cerebral cortex; D is the quantitative result of the number of Nissl bodies in the cerebral cortex in a single image field of view. The results showed that whether it was HE staining or Nissl staining, salvianolic acid B / Rg1 showed a significant protective effect on neurons. Note: ***p<0.001VS sham operation group, &&p<0.01, &&&p<0.001VS model group.

[0232] 5.2.4 Salvianolic acid B / Rg1 protects hippocampal neurons

[0233] From an anatomical point of view, the hippocampus is often seen as a medial bulge in the temporal horn of the lateral ventricle. It consists of four regions: CA1, CA2, CA3, and CA4. The cell bodies of nerve cells and their neural network regions are arranged in layers. Fig.14 This is the HE staining of CA1, CA2, and CA3. Compared with the sham operation group, the animals in the model group showed obvious cavitation, shrinkage, loss, and reduction of neuronal cell bodies; compared with the model group, the number of surviving neurons in the salvianolic acid B / Rg1 group increased, the neurons were arranged in an orderly manner, and the number of neurons increased significantly.

[0234] Fig.15 The following are representative Nissl staining images of CA1, CA2, and CA3 and their quantitative results. The number of cells presented in the representative Nissl staining images (A, B, and C) of the CA1, CA2, and CA3 regions is consistent with the trend of HE staining. The left brain of this animal model is the site of injury, and the right brain presents a normal histological structure. The grayscale difference / image field of the left and right brains reflects the degree of damage to the nerve cells of the model animal. The quantitative results of Figures B, D, and F show that salvianolic acid B / Rg1 protects the integrity of nerve cells. Figure 15-16 It can be seen that whether HE staining or Nissl staining, salvianolic acid B / Rg1 shows a protective effect on hippocampal CA1, CA2, and CA3.

[0235] Example 6

[0236] Treatment of cerebral ischemia-reperfusion injury in rats with combination of salvianolic acid B and Rg1 (5:2)

[0237] 6.1 Experimental animals and model preparation

[0238] Thirty male SD rats were randomly divided into the following three groups: sham operation group, cerebral ischemia-reperfusion injury model group, edaravone (purchased from Sinopharm Guorui Pharmaceutical Co., Ltd., batch number: 1909116) group, butylphthalide (purchased from CSPC Enbi Pharmaceutical Co., Ltd., batch number: 6182002117) group, and salvianolic acid B / Rg1 combination group (5:2, administration and dosage of 5 mg / kg), with 10 rats in each group.

[0239] After the rats were anesthetized, fixed, and disinfected with chest hair and iodine, the muscles, subcutaneous connective tissue, and anterior cervical muscles were separated in turn to expose the common carotid artery (CCA); a small incision was made 4 mm away from the bifurcation of the CCA, and the suture was inserted from the small incision into the internal carotid artery (ICA). The suture was pushed about 20 mm from the bifurcation of the blood vessel, and the middle cerebral artery (MCA) was blocked for 2 hours to induce cerebral infarction. After 2 hours, the suture was pulled out to make a model of cerebral ischemia for 2 hours and reperfusion for 24 hours. Drugs were administered 10 minutes before the suture was pulled out (tail vein injection): 5 mg / kg of salvianolic acid B / ginsenoside Rg1 (salvianolic acid B / Rg1 group); 5 mg / kg of edaravone (edaravone group, calculated as active ingredient); 5 mg / kg of butylphthalide (butylphthalide group, calculated as active ingredient); 5 mg / kg of normal saline (model group); for the animals in the sham operation group, all surgical operations were exactly the same except that the suture was not inserted, and 5 mg / kg of normal saline was given.

[0240] 6.2 Experimental Results

[0241] 6.2.1 Salvianolic acid B / Rg1 reduces the infarct size in patients with reperfusion injury

[0242] like Fig.16 As shown, the cerebral infarction area was evaluated by TTC staining. The representative pictures of the staining results of each group are shown in Figure A, and the quantitative results of the infarction area are shown in Figure B. It can be seen that compared with edaravone and butylphthalide, salvianolic acid B / Rg1 has a more significant effect in reducing the infarction area. Note: ***p<0.001, *p<0.1VS sham operation group, &&p<0.01VS model group.

[0243] 6.2.2 Salvianolic acid B / Rg1 improves behavioral scores in rats after cerebral ischemia-reperfusion

[0244] Longa score was used to evaluate the neuromotor function of rats before and after surgery. Fig.17 As shown. According to the Longa scoring method, there was no significant behavioral difference among the five groups of animals before surgery. After modeling, according to the Longa evaluation, salvianolic acid B / Rg1 showed a better effect of improving neuromotor function than edaravone and butylphthalide.

[0245] 6.2.3 Salvianolic acid B / Rg1 protects cerebral cortical neurons

[0246] like Fig.18As shown, A is HE staining of the cerebral cortex; B is Nissl staining of the cerebral cortex. Compared with the sham operation group, the model group showed obvious cavitation, shrinkage, loss and reduction of neuronal cell bodies. Compared with the model group, the number of surviving neurons in the salvianolic acid B / Rg1 group was significantly higher than that in the model group, edaravone group and butylphthalide group. Whether HE staining or Nissl staining, salvianolic acid B / Rg1 showed a protective effect on neurons, and showed a better therapeutic effect than edaravone and butylphthalide.

[0247] 6.2.4 Salvianolic acid B / Rg1 protects hippocampal neurons

[0248] Fig.19 This is a representative picture of HE staining of CA1, CA2, and CA3. Fig. 20 This is a representative picture of Nissl staining of CA1, CA2, and CA3. Regardless of HE staining or Nissl staining, salvianolic acid B / Rg1 showed a protective effect on hippocampal CA1, CA2, and CA3, and showed a more excellent therapeutic effect than edaravone and butylphthalide.

[0249] Example 7

[0250] Protective effect of salvianolic acid B / ginsenoside Rg1 combination (5:2) on hepatic ischemia-reperfusion injury in rats

[0251] 7.1 Experimental animals and model preparation

[0252] Sixteen male SD rats were randomly divided into two groups: one was the liver ischemia-reperfusion injury model group, and the other was the salvianolic acid B / Rg1 (5:2) combination treatment group.

[0253] The model of liver ischemia-reperfusion injury was prepared as follows: rats were weighed, anesthetized, and fixed on the operating table with abdominal hair; iodine disinfection was performed, and a 5-cm longitudinal incision was made in the middle of the abdomen to the xiphoid process; the skin, muscle, and peritoneum were cut in sequence to fully expose the liver and gastrointestinal tract; the ligaments around the liver were separated; the middle lobe of the liver was clamped with a micro-hemostatic clamp; the portal vein, hepatic artery, and bile duct of the left lobe were exposed; the color of the liver lobe gradually changed from red to pale, indicating that the hepatic blood flow was successfully blocked. After 1 hour of blood flow blockage, the hemostatic clamp was removed; the color of the liver lobe gradually became ruddy, indicating that the liver was successfully reperfused. The model control group was given normal saline at the same time as reperfusion; the salvianolic acid B / Rg1 combination group was given 10 mg / kg of salvianolic acid B / ginsenoside Rg1 (5:2) at the same time as reperfusion, and the liver protection effect was evaluated 6 hours after reperfusion.

[0254] 7.2 Experimental Results

[0255] HE staining was performed on the above liver tissues. Fig.21(The upper picture is 200X, the lower picture is 400X) as shown, 6 hours after ischemia-reperfusion, the liver lobule structure in the model group was incomplete, the liver cells were disordered, congested, swollen, and cell necrosis was obvious. Salvianolic acid B / Rg1 can improve the liver cell structure, but the effect is not as obvious as that of the heart and brain.

[0256] discuss

[0257] In the prior art, experimental results show the effect of different drugs on reducing infarction area: Salvianolic acid B < ginsenoside Rg1 << Salvianolic acid B: ginsenoside Rg1 = 2:5 administration group, (see CN2011102229806, Figure 3 ), so it is easy to think that ginsenoside Rg1 is better than salvianolic acid B in reducing the area of ​​myocardial infarction, and the greater the proportion of ginsenoside Rg1, the better the effect when combined with other drugs. However, surprisingly, compared with the best ratio combination disclosed in the prior art (SalB / Rg1 is 2:5), the pharmaceutical composition of the present invention (salvianolic acid B weight> ginsenoside Rg1) shows a better effect in treating ischemic diseases such as myocardial infarction, cerebral infarction, pulmonary embolism, and heart and brain ischemia-reperfusion injury (such as reducing the area of ​​infarction, improving the structure and function of tissue organs). Unexpectedly, the pharmaceutical composition of the present invention can also reduce the concentration of LDH in the blood and / or inhibit the activity of LDH, and can be used as an LDH inhibitor.

[0258] Different tissues and organs (such as heart, liver, spleen, lung, kidney, brain, muscle, nerve, etc.) have different tissue structures, functions, vascular distribution, and blood requirements, and it is difficult to predict whether the drug has a therapeutic effect on ischemic reperfusion injury of different organs. Surprisingly, the pharmaceutical composition of the present invention not only shows excellent effects on improving organ function for ischemic reperfusion injury of the heart, but also for ischemic reperfusion injury of organs such as the brain, kidney, and liver, and can be widely used for ischemic reperfusion injury of various tissues and organs.

[0259] More surprisingly, the SalB / Rg1 combination showed superior therapeutic effects on ischemic diseases and ischemic reperfusion injuries of the heart and brain than on other tissues and organs, especially the protective effect on ischemic stroke and reperfusion injury after ischemic stroke was particularly surprising. It not only significantly reduced the infarct area, but also significantly improved the behavior of animals, indicating that the pharmaceutical composition of the present invention has excellent therapeutic prospects for heart diseases and brain diseases.

[0260] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition consists of components (a)-(c): (a) a first active ingredient, wherein the first active ingredient is salvianolic acid B; (b) a second active ingredient, wherein the second active ingredient is ginsenoside Rg1; (c) a pharmaceutically acceptable carrier; The weight ratio of the first active ingredient to the second active ingredient is 5:

2.

2. The pharmaceutical composition according to claim 1, characterized in that The dosage form of the pharmaceutical composition is selected from liquid preparations, solid preparations, gaseous preparations or semisolid preparations.

3. The pharmaceutical composition according to claim 2, characterized in that The dosage form of the pharmaceutical composition is selected from injection, oral preparation, sublingual preparation, respiratory tract administration preparation or transdermal administration preparation.

4. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition is selected from injection, powder injection, capsule, tablet, pill, powder, granule, syrup, oral solution or tincture.

5. Use of the pharmaceutical composition as claimed in claim 1 in the preparation of a medicament for preventing and / or treating myocardial infarction, ischemic stroke, pulmonary embolism, myocardial ischemia-reperfusion injury, cerebral ischemia-reperfusion injury, liver ischemia-reperfusion injury or renal ischemia-reperfusion injury.

Images