WO2021037244A1 - 一种药物组合物及其应用 - Google Patents

一种药物组合物及其应用 Download PDF

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Publication number
WO2021037244A1
WO2021037244A1 PCT/CN2020/112258 CN2020112258W WO2021037244A1 WO 2021037244 A1 WO2021037244 A1 WO 2021037244A1 CN 2020112258 W CN2020112258 W CN 2020112258W WO 2021037244 A1 WO2021037244 A1 WO 2021037244A1
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Prior art keywords
active ingredient
group
pharmaceutical composition
salvianolic acid
ginsenoside
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PCT/CN2020/112258
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English (en)
French (fr)
Inventor
姜宝红
王琳琳
王小玉
付瑶
边慧淼
邢蓉蓉
Original Assignee
中国科学院上海药物研究所
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Application filed by 中国科学院上海药物研究所 filed Critical 中国科学院上海药物研究所
Priority to JP2022513868A priority Critical patent/JP2022545968A/ja
Priority to EP20858285.8A priority patent/EP4023227A4/en
Priority to US17/639,271 priority patent/US20220331281A1/en
Publication of WO2021037244A1 publication Critical patent/WO2021037244A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4808—Preparations in capsules, e.g. of gelatin, of chocolate characterised by the form of the capsule or the structure of the filling; Capsules containing small tablets; Capsules with outer layer for immediate drug release
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure

Definitions

  • the present invention relates to the field of medicine, in particular to a pharmaceutical composition and its application in preventing and/or treating ischemic diseases and/or ischemia-reperfusion injury and other diseases.
  • Blood vessels are responsible for supplying blood to the tissues and organs of the whole body. Once the blood vessels are blocked, the tissues and organs will suffer from diseases due to insufficient blood supply, especially the heart, brain and other tissues and organs that require high blood oxygen supply; and with the dredging of the microcirculation during shock, the coronary arteries
  • the establishment and application of methods such as spasm relief, arterial bypass, thrombolytic therapy, percutaneous transluminal coronary angioplasty, cardiopulmonary bypass, cardiopulmonary cerebral resuscitation, amputated limb replantation, and organ transplantation have made many tissues and organs
  • the blood supply can be restored after ischemia (ie reperfusion).
  • the Chinese application with application number CN2011102229806 discloses a pharmaceutical composition containing the compound salvianolic acid B and ginsenoside Rg1, which has a curative effect on cardiac ischemia-reperfusion injury.
  • the research on the compatibility of the two is not yet sufficient, and it is necessary to carry out further in-depth research on the compatibility of the prescription to provide a pharmaceutical composition with curative effect on ischemia-reperfusion injury of different tissues and organs.
  • the purpose of the present invention is to provide a pharmaceutical composition for preventing and/or treating tissue and organ ischemia, as well as ischemia-reperfusion injury and other diseases.
  • a pharmaceutical composition including:
  • the first active ingredient, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and salvianolic acid B-containing Extract, or their combination;
  • the second active ingredient which is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and an extract containing ginsenoside Rg1 , Or a combination of them; and
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.5), wherein the weight ratio is based on salvianolic acid B and ginsenoside Rg1.
  • the first active ingredient includes a purified product of salvianolic acid B or a pharmaceutically acceptable salt thereof.
  • the purity of the purified product is ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98% or 99%, based on the total amount of the purified product Weight meter.
  • the first active ingredient includes a salvianolic acid extract with a salvianolic acid B content C1 ⁇ 30wt%, wherein the content C1 is based on the weight of the salvianolic acid.
  • the content of salvianolic acid B in the extract is C1 ⁇ 70%, preferably ⁇ 80%, more preferably ⁇ 90% or ⁇ 95%, based on the dry weight of the extract.
  • the second active ingredient includes a total saponin extract with a content of ginsenoside Rg1 C2 ⁇ 30wt%, wherein the content C2 is based on the weight of the total saponin.
  • the content C2 of ginsenoside Rg1 in the extract is greater than or equal to 70%, preferably greater than or equal to 80%, more preferably greater than or equal to 90% or greater than or equal to 95%, based on the dry weight of the extract.
  • the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.0), preferably 5:(1.2-3.8), more preferably 5:(1.5-3.5) .
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1.8-3.2), preferably 5:(1.9-3.1), more preferably 5:(2- 3), best 5:2.
  • the first active ingredient is salvianolic acid B
  • the second active ingredient is ginsenoside Rg1.
  • 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), gas dosage forms, and semi-solid dosage forms. .
  • the dosage form is selected from the following group: injection (such as injection or powder injection), oral preparation (such as capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), Sublingual oral preparations, respiratory tract administration preparations, skin administration preparations, mucosal administration preparations, and more preferably, the dosage form is an injection.
  • a combination of active ingredients includes:
  • the first active ingredient, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and salvianolic acid B-containing Extract, or their combination;
  • the second active ingredient which is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and an extract containing ginsenoside Rg1 , Or their combination;
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.5), wherein the weight ratio is based on salvianolic acid B and ginsenoside Rg1.
  • the combination of the active ingredients consists of (a) the first active ingredient and (b) the second active ingredient.
  • a medicine kit in a third aspect of the present invention, includes:
  • a first pharmaceutical composition comprising: (a) a first active ingredient, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal form, and its A pharmaceutically acceptable salt or ester, an extract containing salvianolic acid B, or a combination thereof; and a pharmaceutically acceptable carrier;
  • a second pharmaceutical composition comprising: (b) a second active ingredient, the second active ingredient is selected from the following group: ginsenoside Rg1, its stereoisomers, its crystal form, and its pharmacy The above-acceptable salt or ester, the extract containing ginsenoside Rg1, or a combination thereof; and a pharmaceutically acceptable carrier;
  • first pharmaceutical composition and the second pharmaceutical composition are administered in combination, wherein the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.5), and the weight ratio is Salvianolic acid B and ginsenoside Rg1.
  • first pharmaceutical composition and the second pharmaceutical composition are different (or independent) pharmaceutical compounds, or the same pharmaceutical composition.
  • the fourth aspect of the present invention provides the pharmaceutical composition according to the first aspect of the present invention, the combination of the active ingredients in the second aspect of the present invention, or the use of the kit according to the third aspect of the present invention, for preparing a medicine Or a kit for (i) prevention and/or treatment of ischemic diseases; (ii) prevention and/or treatment of ischemia-reperfusion injury; (iii) inhibition of lactate dehydrogenase.
  • the medicine or kit is used for (i) prevention and/or treatment of ischemic heart disease; (ii) prevention and/or treatment of ischemia-reperfusion injury; (iii) inhibition of lactate dehydrogenase And/or (iv) prevention and/or treatment of ischemic diseases.
  • the ischemic disease is selected from the group consisting of tissue and organ damage caused by acute ischemia and/or tissue and organ damage caused by chronic ischemia.
  • the ischemic disease is selected from the following group: tissue and organ ischemic damage caused by primary tissue and vascular disease, and/or ischemic disease caused by secondary causes, such as trauma The resulting vascular severance, vascular occlusion caused by inflammation, and vascular compression caused by tumors.
  • the ischemic disease is selected from the group consisting of ischemic heart disease, ischemic stroke (such as acute cerebral infarction), ischemic liver injury, pulmonary embolism, ischemic kidney injury , Ischemic nerve injury, or a combination thereof.
  • the ischemic heart disease includes: coronary heart disease, myocardial infarction, angina pectoris, myocardial fibrosis, heart failure, or a combination thereof.
  • the ischemia-reperfusion injury is tissue and organ injury caused by reperfusion.
  • the tissue organ is selected from the group consisting of heart, brain, liver, spleen, lung, kidney, muscle, nerve, or a combination thereof.
  • the tissues and organs are selected from the following group: liver, spleen, lung, kidney, brain, nerve, or a combination thereof.
  • the tissue organ is selected from the following group: heart, brain, or a combination thereof.
  • the medicine or kit is also used to improve myocardial hypertrophy induced by pulmonary embolism.
  • the medicine or the kit is also used to improve the diastolic function of the reperfusion-injured heart.
  • the diastolic function of the heart includes the diastolic rate of the heart.
  • the medicine or the kit is also used to improve the contraction function of the heart (such as the contraction rate of the heart).
  • the medicine or kit is used to improve the reperfusion injury of the kidney, preferably including improving the structure of the kidney.
  • the tissue and organ injury is post-operative reperfusion injury.
  • the operation is selected from the following group: arterial bypass, thrombectomy or thrombolytic therapy, percutaneous transluminal coronary angioplasty Reperfusion injury caused by surgery, cardiac surgery under cardiopulmonary bypass, heart, lung and/or brain resuscitation after cardiac arrest, replantation of amputated limbs or organ transplantation, or other major operations.
  • Figure 1 shows the effect of salvianolic acid B/ginsenoside Rg1 on reducing the area of myocardial infarction in an animal model of myocardial infarction;
  • A TTC staining representative image of heart slices;
  • B quantitative infarct area (infarct area accounted for Percentage of the area of the entire heart).
  • Figure 2 shows the effect of salvianolic acid B/ginsenoside Rg1 on the blood lactate dehydrogenase content in an animal model of myocardial infarction.
  • Figure 3 shows the protective effect of salvianolic acid B/ginsenoside Rg1 on the structure of heart tissue in an animal model of myocardial infarction.
  • Figure 4 shows that in the animal model of myocardial ischemia-reperfusion injury, the salvianolic acid B/ginsenoside Rg1 (5:2) group significantly reduced the heart rate than the salvianolic acid B/ginsenoside Rg1 (2:5) group Infarct area and improve heart structure;
  • A TTC staining representative image of heart slice;
  • B Infarct area quantification (infarct area occupies the area percentage of the whole heart);
  • C HE staining representative image of heart tissue.
  • Figure 5 shows the blood flow of rats in the salvianolic acid B/ginsenoside Rg1 (2:5) group and the salvianolic acid B/ginsenoside Rg1 (5:2) group on the animal model of myocardial ischemia-reperfusion injury Kinetics (maximum relaxation rate and maximum relaxation rate) test results.
  • "*" means P ⁇ 0.05 compared with the salvianolic acid B/ginsenoside Rg1 (2:5) group.
  • Figure 6 shows the blood flow of rats in the salvianolic acid B/ginsenoside Rg1 (2:5) group and the salvianolic acid B/ginsenoside Rg1 (5:2) group on the animal model of myocardial ischemia-reperfusion injury Kinetics (end diastolic blood pressure and mean arterial pressure) test results.
  • Figure 7 shows the sham operation group, the renal ischemia reperfusion model group, the salvianolic acid B/ginsenoside Rg1 (2:5) group and the salvianolic acid B/ginsenoside Rg1 on the renal ischemia-reperfusion injury model. (5:2) HE staining results of rat kidneys in group.
  • Figure 8 shows the sham operation group, the renal ischemia reperfusion model group, the salvianolic acid B/ginsenoside Rg1 (2:5) group and the salvianolic acid B/ginsenoside Rg1 on the renal ischemia-reperfusion injury model. (5:2) The results of periodic acid Schiff staining (PAS) of rats in the group.
  • Figure 9 shows the therapeutic effect of salvianolic acid B/Rg1 (5:2) on pulmonary embolism on a pulmonary embolism model;
  • A left lung index;
  • B right lung index;
  • C representative of lung HE staining Figure,
  • D quantitative results of lung interstitial area
  • E representative image of HE staining of heart,
  • F average optical density of lung neutrophils.
  • Figure 10 shows 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 heart HE staining, (B) cross-sectional area of myocardial cells Quantitative graphs.
  • Figure 11 shows that in the acute cerebral infarction model, salvianolic acid B/Rg1 (5:2) significantly reduced the infarct size, (A) representative image of brain TTC staining, (B) quantitative results of infarct size.
  • Figure 12 shows the results of salvianolic acid B/Rg1 (5:2) improving the behavioral scores of rats after cerebral infarction in the acute cerebral infarction model.
  • Figure 13 shows the protective effect of salvianolic acid B/Rg1 (5:2) on cerebral cortex neurons in an acute cerebral infarction model
  • A is a representative image of HE staining of the cerebral cortex
  • B is HE staining Quantitative results of the number of nerve cells
  • 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.
  • Figure 14 shows representative images of HE staining of hippocampus CA1, CA2, and CA3 on an acute cerebral infarction model.
  • Figure 15 shows representative images of Nissan staining of hippocampus CA1, CA2, and CA3 on an acute cerebral infarction model and their quantitative results.
  • Figure 16 shows that on a cerebral ischemia-reperfusion injury model, salvianolic acid B/Rg1 (5:2) reduces the cerebral infarct area of reperfusion injury, (A) representative image of TTC staining of brain tissue, (B) infarct area Quantitative results.
  • Figure 17 shows that on a cerebral ischemia-reperfusion injury model, salvianolic acid B/Rg1 (5:2) improves the behavioral score of rats after cerebral ischemia-reperfusion.
  • Figure 18 shows the protective effect of salvianolic acid B/Rg1 on cerebral cortex neurons in a cerebral ischemia-reperfusion injury model.
  • A is the HE staining of the cerebral cortex;
  • B is the Nissan staining of the cerebral cortex.
  • Figure 19 shows representative images of HE staining of hippocampus CA1, CA2, and CA3 on a rat brain ischemia-reperfusion injury model.
  • Figure 20 shows a representative image of Nissan staining of hippocampus CA1, CA2, and CA3 on a rat brain ischemia-reperfusion injury model.
  • Figure 21 shows a representative image of HE staining of liver tissue on a rat liver ischemia-reperfusion injury model.
  • the inventor provided a pharmaceutical composition containing salvianolic acid B and ginsenoside Rg1 as the active ingredient ratio through a large number of screenings and tests.
  • the pharmaceutical composition of the present invention The compound shows a more excellent therapeutic effect on ischemic diseases and ischemia-reperfusion injury of tissues and organs; and surprisingly, the composition of the present invention is not only on the heart, brain, liver, but also on the kidneys and other organs. Ischemia-reperfusion injury has curative effect and can be used for ischemia-reperfusion injury of various tissues and organs. The present invention has been completed on this basis.
  • the terms “including”, “including”, and “containing” are used interchangeably, and include not only closed definitions, but also semi-closed and open definitions. In other words, the term includes “consisting of” and “consisting essentially of”.
  • stereoisomers is intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, containing The R and S configurations of the center of symmetry, the (Z) and (E) isomers of the double bond, etc. Therefore, a single stereochemical isomer of the active ingredient of the present invention or a mixture of its enantiomers, diastereomers or geometric isomers (or conformational isomers) all belong to the scope of the present invention.
  • the active ingredient of the present invention may be amorphous, crystalline, or a mixture thereof.
  • pharmaceutically acceptable salt refers to a salt formed by a compound of the active ingredient of the present invention and an acid or base suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • a preferred type of salt is the salt formed with an acid of the compound of the active ingredient of the present invention.
  • Acids suitable for salt formation include but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • a preferred type of salt is the salt of the compound of the active ingredient of the present invention with a base.
  • Suitable bases for salt formation include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium phosphate, and organic bases such as ammonia, triethylamine, and diethylamine.
  • inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium phosphate
  • organic bases such as ammonia, triethylamine, and diethylamine.
  • Another type of preferred salt is the salt formed by the active ingredient of the present invention and metal ion, including but not limited to magnesium salt, sodium salt, calcium salt, potassium salt and the like.
  • esters refers to the ester formed by the compound of the active ingredient of the present invention with an acid or alcohol and is suitable for use as a medicine.
  • a preferred class of esters is one or more hydroxyl groups of the active ingredient of the present invention and the acid. Formed esters.
  • Acids suitable for ester formation 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, benzene methanesulfonic acid, benzenesulfonic acid, etc.; another preferred ester is the ester formed by the carboxyl group of the active ingredient of the present invention and an alcohol.
  • the alcohol suitable for forming an ester includes but is not limited to: C1 -C6 alkyl-OH, such as methanol, ethanol, n-propanol, isopropanol, etc.
  • the weight ratio is calculated in the form of the original compound of salvianolic acid B and ginsenoside Rg1.
  • the "prevention" and “treatment” in the present invention include delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • the composition or pharmaceutical composition of the present invention prevents, reduces, inhibits, and/or inhibits ischemia-reperfusion injury compared to the level observed in the absence of the composition or pharmaceutical composition of the present invention. Or reversal, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.
  • the first active ingredient is selected from the following group: salvianolic acid B, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, an extract containing salvianolic acid B, or their combination.
  • the first active ingredient includes a purified product of salvianolic acid B or a pharmaceutically acceptable salt thereof.
  • the purity of the purified product is ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98% or 99%, based on the total amount of the purified product Weight meter.
  • the first active ingredient includes a salvianolic acid extract with a salvianolic acid B content C1 ⁇ 30wt%, wherein the content C1 is based on the weight of the salvianolic acid.
  • the content of salvianolic acid B in the extract is C1 ⁇ 70%, preferably ⁇ 80%, more preferably ⁇ 90% or ⁇ 95%, based on the dry weight of the extract.
  • the second active ingredient is selected from the following group: ginsenoside Rg1, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, an extract containing ginsenoside Rg1, or a combination thereof.
  • the second active ingredient includes a total saponin extract with a content of ginsenoside Rg1 C2 ⁇ 30wt%, wherein the content C2 is based on the weight of the total saponin.
  • the content C2 of ginsenoside Rg1 in the extract is greater than or equal to 70%, preferably greater than or equal to 80%, more preferably greater than or equal to 90% or greater than or equal to 95%, based on the dry weight of the extract.
  • composition active ingredient combination, medicine box
  • the present invention provides a pharmaceutical composition, including:
  • the first active ingredient, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and salvianolic acid B-containing Extract, or their combination;
  • the second active ingredient which is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and an extract containing ginsenoside Rg1 , Or a combination of them; and
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.5), wherein the weight ratio is based on salvianolic acid B and ginsenoside Rg1.
  • the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.0), preferably 5:(1.2-3.8), more preferably 5:(1.5-3.5) .
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1.8-3.2), preferably 5:(1.9-3.1), more preferably 5:(2- 3), best 5:2.
  • the first active ingredient is salvianolic acid B
  • the second active ingredient is ginsenoside Rg1.
  • 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).
  • the dosage form is selected from the following group: injection (such as injection or powder injection), oral preparation (such as capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), More preferably, preferably, the dosage form is an injection.
  • the first active ingredient and the second active ingredient can be prepared separately or mixed together to prepare a preparation.
  • the pharmaceutical composition of the present invention contains the first active ingredient and/or the second active ingredient in a safe and effective amount.
  • the "safe and effective amount” refers to: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of the active ingredient/dose of the present invention, more preferably, 10-500 mg of the active ingredient/dose of the present invention.
  • the "one dose” is a capsule, tablet, injection or the like.
  • 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 .
  • Cosmetic here means that the components in the composition can be blended with the first active ingredient and/or the second active ingredient without significantly reducing the first active ingredient and/or the second active ingredient. effect.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as Tween
  • wetting agents such as sodium lauryl sulfate
  • coloring agents such as sodium lauryl sulfate
  • flavoring agents such as pepperminophen, sorbitol, etc.
  • antioxidants
  • the present invention also provides a combination of active ingredients, said combination of active ingredients comprising:
  • the first active ingredient, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and salvianolic acid B-containing Extract, or their combination;
  • the second active ingredient which is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal form, its pharmaceutically acceptable salt or ester, and an extract containing ginsenoside Rg1 , Or their combination;
  • the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.5), wherein the weight ratio is based on salvianolic acid B and ginsenoside Rg1.
  • the combination of the active ingredients consists of (a) the first active ingredient and (b) the second active ingredient.
  • 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.
  • the present invention also provides a medicine box, which comprises:
  • a first pharmaceutical composition comprising: (a) a first active ingredient, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal form, and its A pharmaceutically acceptable salt or ester, an extract containing salvianolic acid B, or a combination thereof; and a pharmaceutically acceptable carrier;
  • a second pharmaceutical composition comprising: (b) a second active ingredient, the second active ingredient is selected from the following group: ginsenoside Rg1, its stereoisomers, its crystal form, and its pharmacy The above-acceptable salt or ester, the extract containing ginsenoside Rg1, or a combination thereof; and a pharmaceutically acceptable carrier;
  • first pharmaceutical composition and the second pharmaceutical composition are administered in combination, wherein the weight ratio of the first active ingredient and the second active ingredient is 5:(1-4.5), and the weight ratio is Salvianolic acid B and ginsenoside Rg1.
  • kit further includes instructions.
  • first pharmaceutical composition and the second pharmaceutical composition are different (or independent) pharmaceutical compounds, or the same pharmaceutical composition.
  • the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously, separately or sequentially during administration.
  • the pharmaceutical composition, active ingredient combination, and medicine box of the present invention can all be prepared by using conventional methods and equipment.
  • the present invention provides the use of a pharmaceutical composition, a combination of active ingredients or a kit as described herein for preparing a medicine or a kit for (i) prevention and/or treatment of ischemia Disease; (ii) prevention and/or treatment of ischemia-reperfusion injury; and/or (iii) inhibition of lactate dehydrogenase.
  • the ischemic disease refers to tissue or organ damage or disease caused by ischemia.
  • the "ischemia” means that the blood supply of a tissue or organ is lower than the normal value, especially that the blood supplied in the tissue or organ cannot meet the metabolic needs of the tissue or organ.
  • 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.
  • the ischemic heart disease is a heart disease caused by myocardial ischemia and hypoxia caused by changes in coronary circulation.
  • Common ischemic heart diseases include (but are not limited to) the following groups: coronary heart disease, myocardial infarction, myocardial fibrosis, angina pectoris, or a combination thereof.
  • the ischemia-reperfusion injury includes tissue and organ injury caused by reperfusion.
  • the tissues and organs include (but are not limited to) the following group: heart, liver, spleen, lung, kidney, brain, muscle, nerve, or a combination thereof.
  • the tissue and organ damage also includes postoperative reperfusion injury, and the surgery includes the following groups (but not limited to): arterial bypass, thrombolytic therapy, percutaneous transluminal coronary angioplasty, cardiac surgery under extracorporeal circulation, Heart, lung and/or brain resuscitation, replantation of severed limbs or organ transplantation after cardiac arrest.
  • the reperfusion injury also includes: reperfusion injury after microcirculation dredging after shock, and reperfusion injury after coronary artery spasm is relieved.
  • the application of prevention and/or treatment of ischemic diseases, prevention and/or treatment of ischemia-reperfusion injury, etc. includes preventive applications as well as applications for improvement afterwards.
  • it includes administering the pharmaceutical composition, active ingredient composition or kit of the present invention before, during, and/or after reperfusion to protect and repair tissues and organs after reperfusion injury. Or functional improvement or enhancement.
  • the combination of active ingredients or the kit of the present invention, the first active ingredient and the second active ingredient can also be administered in combination with other pharmaceutically acceptable compounds, including (but not limited to): antihypertensive drugs , Lipid-lowering drugs, hypoglycemic drugs, anti-platelet aggregation drugs, etc.
  • the pharmaceutical composition, active ingredient combination 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. It exists in various organs and tissues throughout the body, such as liver, heart, pancreas, kidney, skeletal muscle, and lymph. Tissue and blood cells. Lactate dehydrogenase is involved in the final step of glycolysis in which pyruvate is converted to lactic acid. Although normal tissues usually use glycolysis only when oxygen is insufficient, cancer tissues rely heavily on aerobic glycolysis, which is incompatible with oxygen. Regardless of the level of supply, LDH inhibitors are used in pathologies that involve metabolic conversion from oxidative phosphorylation to glycolysis.
  • lactate dehydrogenase is the enzyme responsible for the conversion of glyoxylate to oxalate in the mitochondrial/peroxisome glycine metabolism pathway of the liver and pancreas.
  • Inhibiting LDH can be used to treat chronic kidney disease, such as hyperoxaluria.
  • the pharmaceutical composition of the present invention can reduce the concentration of LDH in the blood and/or inhibit the activity of LDH, and can be used as an LDH inhibitor.
  • the combination of active ingredients or the kit of the present invention the first active ingredient and the second active ingredient may be administered simultaneously, separately or sequentially during administration.
  • the combination of active ingredients or the kit of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, rectal, gastrointestinal External (intravenous, intramuscular or subcutaneous), and topical administration.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and gly
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of active ingredients in such compositions may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient can also be formed into a microcapsule form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents 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-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous 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.
  • 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.
  • the therapeutically effective dose of the active ingredient will generally range from about 1-2000 mg/day, about 10 mg/day. -About 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day, about 10 to about 100 mg/day, or about 10 to about 80 mg/day.
  • the therapeutically effective dose will be given in one or more doses.
  • the specific dosage of the active ingredient of the present invention for any specific patient will depend on a variety of factors, such as the age, gender, weight, general health, diet, individual response, administration time, and treatment of the patient to be treated.
  • the severity, dosage form, application mode and concomitant drugs of the disease to be treated can be determined by routine experiments and is within the ability and judgment of the clinician or physician.
  • the active ingredient will be administered in multiple doses based on the individual condition of the patient and in a manner that allows delivery of a therapeutically effective amount.
  • the pharmaceutical composition of the present invention has a better active ingredient distribution ratio, and has a better curative effect on the treatment of ischemic diseases and ischemia-reperfusion injury (such as excellent reduction in infarct area and improvement The effect of organ function).
  • the pharmaceutical composition of the present invention has a therapeutic effect not only on the heart, but also on the reperfusion injury of different organs such as the brain, liver, lung and kidney, and can be widely used in the ischemia-reperfusion injury of various tissues and organs.
  • the pharmaceutical composition of the present invention also has the effect of inhibiting lactate dehydrogenase, can quickly reduce the content and/or activity of lactate dehydrogenase in the blood, and can be used as a lactate dehydrogenase inhibitor to be related to lactate dehydrogenase. disease.
  • Wistar and SD rats were provided by the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences, and were bred in the SPF animal room of the Laboratory Animal Center of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, at a constant temperature of 22 ⁇ 2°C, 12h light, standard diet, and free drinking water.
  • Mode of administration Unless otherwise specified, the mode of administration in the examples is tail vein injection.
  • the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg) and fixed on the rat board in the supine position. Cut the abdomen along the midline with scissors, take out the contents of the abdominal cavity, dry the liquid in the abdominal cavity with a dry cotton ball, take blood from the abdominal aorta with a 5mL syringe to a 2ml EP tube; place the blood on ice for 0.5 hours Afterwards, centrifuge the collected blood for 10 minutes at 4°C and 8000r/min, take the supernatant, and dispense the serum into 0.5ml EP tubes, and store them in a refrigerator at -80°C for later use.
  • sodium pentobarbital 40 mg/kg
  • TTC powder Dissolve 0.5g TTC powder in 100ml PBS, store it in the dark, and prepare it for current use. Cut the fresh tissue into multiple pieces, place it in a small box with a lid, add TTC solution, place in a wet box to protect from light, incubate in a constant temperature oven at 37°C for 20 minutes, turn several times during the period to make the tissue evenly contact the dye solution. Take out a picture after 20 minutes.
  • the dehydrated tissue is embedded in paraffin and poured into the embedding box.
  • Use heated tweezers to put the waxed tissue block into the embedding frame, gently move it to the cold table, and take it out after the paraffin has solidified. Place the paraffin block to prepare for sectioning.
  • the slicer Before slicing, put the wax block in the refrigerator to pre-cool, after cooling, the slicer continuously slices paraffin slices with a thickness of 5 ⁇ m. The slices were spread out on a spreader with warm water at 38°C. The slides were taken out with polylysine-coated glass slides, and they were air-dried naturally for subsequent histopathological staining.
  • LDH detection is based on the lactate dehydrogenase assay kit (lactic acid substrate method, Sysmex Biotechnology (Wuxi) Co., Ltd., batch number: R8004), using an automatic biochemical analyzer (JCA-BM6010/C, Sysmex Medical Electronics (Shanghai) Co., Ltd. tested rat serum.
  • the Powerlab 8/30 physiological recorder (ML870, ADINSTRUMENTS) was used to record the carotid artery pressure, the maximum contraction rate of the left ventricle, Hemodynamic indicators such as the maximum diastolic rate of the left ventricle and the diastolic pressure at the end of the left ventricle.
  • the rats were weighed, they were anesthetized by intraperitoneal injection of 30 mg/kg Shutai, 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 lung, right lung and body weight.
  • NSS score Longa score, NSS score and EBST were tested on all experimental animals two days before surgery and one day after surgery.
  • NSS scoring method 0 points: normal nerve function; 1 point: mild neurological deficit (flexion of the left forelimb when the tail is lifted); 2 points: moderate neurological deficit (turning to the left when walking); 3 points: Moderate neurological deficit (inclined to the left); 4 points: unable to walk, decreased consciousness; 5 points: death related to ischemia.
  • Elevated Body Swing Test When measuring, first lift the rat's tail base by hand. The rat's head hangs about 5cm from the plane. At this time, the rat's head will rotate to the left or right, the angle of unilateral rotation When it is greater than 100, it is the counting standard. Record the direction and angle of rotation. Let the rat rest for one minute after one test, and repeat 20 times for the next test. Record the total direction and number of times.
  • the auricle tip, pulmonary artery cone, and atrial appendage junction were threaded and ligated 1mm. After the coronary artery was ligated, the myocardial tissue immediately turned from rosy to pale. In sham-operated animals, except that the left anterior descending coronary artery was not ligated, the rest of the operations were exactly the same.
  • mice 56 rats were randomly divided into the following 7 groups: sham operation group, ischemia model group, salvianolic acid B and ginsenoside Rg1 combined administration group (according to 5:4, 5:3, 5:2, 5:1 respectively) And 2:5 ratio), 8 in each group.
  • the drug is administered in a double-blind manner, that is, the operator does not participate in the drug, and the data statistician does not know the grouping information.
  • the salvianolic acid B and ginsenoside Rg1 were mixed according to 5:4, 5:3, 5:2, 5:1, 2:5 respectively, numbered randomly after mixing, and after dissolution, filtered with a microporous membrane for use.
  • the sham operation group and myocardial infarction model group were given equal volumes of normal saline according to body weight. Immediately after the operation, 15 mg/kg was given by tail vein injection once, and then again 24 hours later, and then blood was taken from the abdominal aorta and the heart was collected for cardiac histological examination.
  • the 5:3 group and 5:2 group have a more significant effect in reducing the infarct size compared to the 2:5 group.
  • the infarct size is reduced by 1.50 times (5:3) and 1.42 (5:2). ) Times.
  • the heart structure of the infarct area (upper), the infarct marginal area (middle), and the distal infarct area (lower) of the heart were analyzed respectively (as shown in Figure 3).
  • the structure of the infarct area of the heart was severely damaged, with a large number of inflammatory cells infiltrated, and the myocardial cells showed edema, necrosis, and loss of nuclei. Wavy.
  • SalB/Rg1 showed different degrees of improvement on the above-mentioned damages, whether in the infarct zone or the infarct marginal zone.
  • the 5:2 group had the most obvious improvement in the myocardial tissue structure: in the infarct zone, inflammatory cells Infiltration is significantly reduced, cardiomyocyte edema and necrosis are weakened, the number of lost nuclei is reduced, and muscle fibers are arranged more regularly; in the marginal area of infarction, inflammatory cell infiltration is reduced, and myocardial fibers are arranged regularly.
  • the cells of each group of animals in the distal area of the infarction are arranged in an orderly and tightly regular manner, and there is no significant difference.
  • the combined administration of SalB/Rg1 has an effect on reducing the infarct size, and surprisingly, it is the best ratio of SalB/Rg1 disclosed in the prior art (2:5)
  • the infarct area of the rat heart is smaller, and it exhibits a more excellent curative effect in reducing LDH content in the blood and improving the structure of the heart. .
  • Pentobarbital sodium (40mg/kg) was injected intraperitoneally, and the rat was fixed on a surgical board, and the chest was covered with hair. Insert the pen-type venous indwelling needle sleeve into the trachea to connect to the animal ventilator, disinfect with iodine, cut the skin bluntly separated muscles between the 3-4 ribs on the left side of the chest, open the third and fourth ribs and fix and expose them On the upper part of the heart, tear open the heart bag, the needle holder holds a 5-0 suture needle with a thread, marking the left coronary vein, and threading the thread 1mm below the junction of the left atrial appendage, pulmonary artery cone and atrial appendage, and inserting the two silk threads into the knotted place A 2-0 line was ligated and the myocardial tissue immediately turned from rosy to pale after the coronary artery was ligated.
  • Rats of about 220 g were randomly divided into the following two groups: salvianolic acid B and ginsenoside Rg1 combined administration group (which was prepared according to the ratio of 5:2 and 2:5), with 8 rats in each group. Rats were reperfused after 40 minutes of myocardial ischemia. The rats were given 15 mg/kg tail vein injection immediately after reperfusion. After 1 hour of reperfusion, the hemodynamic test was performed, and then the heart was collected for cardiac histology test.
  • Figure A is a representative image of infarct area TTC staining
  • Figure B is a quantitative result of infarct area
  • Figure C is a representative image of HE staining.
  • SalB/Rg1 Compared with SalB/Rg1 (2:5), SalB/Rg1 (5:2) in the infarct zone and the infarct marginal zone more significantly inhibits the infiltration of inflammatory cells, necrosis of myocardial cells, loss of nuclei and other damages.
  • the cells of each group of animals in the distal area of the infarction are arranged in an orderly and tightly regular manner, and there is no significant difference.
  • Figures 5 and 6 show the results of hemodynamics. Compared with SalB/Rg1 (2:5), SalB/Rg1 (5:2) increased the maximum relaxation rate by 18.2% (P ⁇ 0.05) and increased the maximum contraction rate by 11.6% , which shows that SalB/Rg1(5:2) is more effective than SalB/Rg1(2:5) in improving cardiac function; Figure 6 shows that there is no significant difference between terminal diastolic blood pressure and mean arterial pressure, which shows that SalB/Rg1(5: 2) Compared with SalB/Rg1 (2:5), it has no adverse effect on blood pressure regulation.
  • SalB/Rg1(5:2) and SalB/Rg1(2:5) were further compared in the renal ischemia-reperfusion injury model.
  • the right and left kidneys are operated in the same way. After 40 minutes, the arterial clamps of the left and right kidneys were removed. After 24 hours, renal ischemia-reperfusion injury was caused, and the muscles and skin were sutured. In the sham-operated animals, except that the kidney pedicles on both sides were not clamped, the rest of the operation was exactly the same.
  • sham operation group Thirty-two rats were randomly divided into the following 4 groups: sham operation group, ischemia-reperfusion model group, salvianolic acid B and ginsenoside Rg1 combined administration group (among them according to the ratio of 5:2 and 2:5), each group 8 pieces.
  • the sham operation group and the renal ischemia reperfusion model group were given equal volumes of normal saline according to body weight. Rats were reperfused after 40 minutes of renal ischemia. At the same time as the rats were reperfused, they were given a 15 mg/kg tail vein injection once, and then again after 24 hours. The kidneys were collected for renal histological examination.
  • Wistar rats Thirty male Wistar rats were randomly divided into the following 3 groups: normal control group, model control group, and salvianolic acid B/Rg1 group (20 mg/kg), each with 10 rats.
  • the normal control group was given normal saline at a dose of 5ml/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 is 200,000 particles/ml, and the diameter is 45 ⁇ m.
  • Wistar rats were injected with polystyrene microspheres in the tail vein at a dose of 1 million grains/kg (5ml/kg) on days 0, 7, 14, and 21, respectively.
  • Two groups of animals were given polystyrene microspheres. One group was injected with saline every day on the 7th day as a model control group for 28 consecutive days; the other group was injected with salvianol at a dose of 20 mg/kg per day on the 7th day. Acid B/Rg1 (ratio 5:2) for 28 consecutive days. All animals were collected on the 35th day for heart and lung histological examination.
  • salvianolic acid B/Rg1 can improve microsphere-induced pulmonary embolism.
  • salvianolic acid B/Rg1 significantly reduced left lung index (A) and right lung index (B); from the tissue staining chart, it can be seen that salvianolic acid B/Rg1 significantly improved Lung structure (C), the area of the lung interstitial is significantly down-regulated (D), which proves the effect of salvianolic acid B/Rg1 on improving lung function; in addition, it can be seen from (E) that salvianolic acid B/Rg1 also It can inhibit the infiltration of neutrophils in lung tissue, and its inhibitory effect is statistically significant (F), suggesting that salvianolic acid B/Rg1 significantly inhibits the occurrence of pulmonary embolism.
  • Salvianolic acid B/Rg1 combined use to improve myocardial hypertrophy caused by pulmonary embolism
  • the main complication of pulmonary embolism is myocardial hypertrophy.
  • hypertrophic cardiomyocytes can be evaluated by the size of cell cross-sectional area.
  • A is a representative image of HE staining of the heart
  • B is a quantitative image of the cross-sectional area of cardiomyocytes.
  • Salvianolic acid B/Rg1 significantly reduces the occurrence of cardiac hypertrophy induced by pulmonary embolism.
  • the rats are anesthetized, fixed, and sterilized by chest coat and iodine, separate the muscle, subcutaneous connective tissue, anterior cervical muscles, and expose the common carotid artery (CCA); cut a small mouth 4mm from the bifurcation of CCA and embed it
  • CCA common carotid artery
  • the thread is inserted into the internal carotid artery (ICA) from a small opening, and the thread plug is pushed about 20mm from the branch of the blood vessel to block the middle cerebral artery (MCA) to induce cerebral infarction.
  • ICA internal carotid artery
  • MCA middle cerebral artery
  • 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 treated with saline (model group); animals in the sham operation group were treated with normal saline (model group); The operation was exactly the same, and normal saline was given at the same time point (sham operation group).
  • FIG. 11 The area of cerebral infarction was evaluated by TTC staining, as shown in Figure 11, where Figure A is a representative image of brain TTC staining. The infarcted area is stained white and the non-infarcted area is stained red. Figure B shows the quantitative results of infarct area (white area area/(red area area + white area area) percentage). Salvianolic acid B/Rg1 significantly reduced the infarct area. Compared with the model group, the salvianolic acid B/Rg1 group had cerebral infarction The area is reduced by 39.42%. Note: ***p ⁇ 0.001VS sham operation group, &&p ⁇ 0.01VS model group.
  • NSS score and EBST were used to evaluate the neuromotor function of rats before and after operation. The results are shown in Figure 12. According to the three scoring methods, there were no obvious behavioral differences in the three groups of animals before surgery. After the cerebral infarction model was established, according to the EBST score (p ⁇ 0.001), Longa score (p ⁇ 0.05) and NSS score (p ⁇ 0.05), salvianolic acid B/Rg1 significantly improved neuromotor function.
  • the cerebral cortex is the high-level center that regulates and controls body movements.
  • A is a representative image of HE staining of the cerebral cortex
  • B is a quantitative result of the number of nerve cells under a single image field of HE staining
  • C is a representative image of Neys staining of the cerebral cortex
  • D is a representative image of Nissl of the cerebral cortex under a single image field Quantitative result of body number.
  • the results showed that whether it was HE staining or Nissl staining, salvianolic acid B/Rg1 showed a significant protective effect on nerve cells.
  • the hippocampus is often seen as a medial bulge of the temporal horn of the lateral ventricle, which consists of four areas CA1, CA2, CA3, and CA4.
  • the cell body of the nerve cell and its neural network area are arranged in layers.
  • Figure 14 is a HE staining diagram of CA1, CA2, and CA3.
  • the animals in the model group showed obvious vacuolization, shrinkage, loss and number of neuron cell bodies; compared with the model group, the survival of nerve cells in the salvianolic acid B/Rg1 group increased, and the nerve cells were arranged The sequence and the number of nerve cells have increased significantly.
  • Fig. 15 is a representative graph of the Nissan staining of CA1, CA2, and CA3 and their quantitative results.
  • the representative graphs (A, B, C) of Nissl staining in CA1, CA2, and CA3 showed the number of cells 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 gray difference between the left and right brain/image field reflects the degree of damage to the nerve cells of the model animal.
  • the quantitative results of Figure B, Figure D and Figure F show that salvianolic acid B/Rg1 protects the integrity of nerve cells. From Figures 15-16, it can be seen that regardless of HE staining or Nissl staining, salvianolic acid B/Rg1 showed protective effects on hippocampal CA1, CA2, and CA3.
  • Salvianolic acid B/Rg1 combined (5:2) treatment of cerebral ischemia-reperfusion injury in rats
  • the rats are anesthetized, fixed, and sterilized by chest coat and iodine, separate the muscle, subcutaneous connective tissue, anterior cervical muscles, and expose the common carotid artery (CCA); cut a small mouth 4mm from the bifurcation of CCA and embed it
  • CCA common carotid artery
  • the thread is inserted into the internal carotid artery (ICA) from a small opening, and the thread plug is pushed about 20mm from the bifurcation of the blood vessel, and the middle cerebral artery (MCA) is blocked for 2 hours to induce cerebral infarction. After 2 hours, the thread plug is pulled out to make cerebral ischemia and 2h reperfusion. 24h model.
  • the cerebral infarct area was evaluated by TTC staining.
  • the representative image of the staining results of each group is shown in Figure A, and the quantitative results of the infarct area are shown in Figure B.
  • the salvianolic acid B/Rg1 and edaravone Compared with butylphthalide, the effect of reducing infarct size is more significant. Note: ***p ⁇ 0.001, *p ⁇ 0.1VS sham operation group, &&p ⁇ 0.01VS model group.
  • Salvianolic acid B/Rg1 improves the behavioral score of rats after cerebral ischemia and reperfusion
  • the Longa score was used to evaluate the neuromotor function of rats before and after surgery, and the results are shown in Figure 17. According to the Longa scoring method, there were no obvious behavioral differences in the five groups of animals before surgery. After modeling, according to Longa's evaluation, salvianolic acid B/Rg1 showed a better effect on improving neuromotor function than edaravone and butylphthalide.
  • A is the HE staining of the cerebral cortex
  • B is the Nissan staining of the cerebral cortex.
  • the model group showed obvious vacuolization, shrinkage, loss, and decrease of neuron cell bodies.
  • the survival of nerve cells in the salvianolic acid B/Rg1 group was significantly higher than that in the model group, edaravone group and butylphthalide group.
  • salvianolic acid B/Rg1 showed a protective effect on nerve cells, and showed a more excellent curative effect than edaravone and butylphthalide.
  • FIG. 19 is a representative image of HE staining of CA1, CA2, and CA3
  • FIG. 20 is a representative image of Nepalese staining of CA1, CA2, and CA3.
  • salvianolic acid B/Rg1 all showed protective effects on hippocampal CA1, CA2, and CA3, and showed a better curative effect than edaravone and butylphthalide.
  • the preparation method of hepatic ischemia-reperfusion injury model is as follows; weigh the rat, anesthetize the abdomen, and fix it on the operating table; sterilize with iodophor, make a longitudinal incision of 5cm in the middle of the abdomen to the xiphoid process; cut the skin, muscle and peritoneum in turn , Fully expose the liver and gastrointestinal tract; separate the perihepatic ligaments; clamp the middle lobe of the liver with microscopic hemostatic clips; expose the left portal vein, hepatic artery and bile duct; the color of the liver lobe gradually changes from red to pale, indicating that hepatic blood flow is blocked success.
  • liver lobe gradually became rosy, indicating successful liver reperfusion.
  • the model control group was given normal saline at the same time of reperfusion; the salvianolic acid B/Rg1 combination group was given 10mg/kg of salvianolic acid B/ginsenoside Rg1 (5:2) at the same time of reperfusion, 6 hours after reperfusion Take materials to evaluate the effect of liver protection.
  • HE staining was performed on the above liver tissue. The results are shown in Fig. 21 (the upper picture is 200X, the lower picture is 400X). After 6 hours of ischemia-reperfusion, the liver lobule structure in the model group is incomplete, the liver cells are arranged disorderly, hyperemia, swelling, and cell necrosis is obvious. Acid B/Rg1 can improve the structure of liver cells, but the effect is not as obvious as the heart and brain.
  • the pharmaceutical composition of the present invention (salvianolic acid B weight> ginsenoside Rg1) is effective in treating ischemic diseases such as myocardial infarction, cerebral infarction, and pulmonary embolism. And heart, cerebral ischemia-reperfusion injury (such as reducing the area of infarction, improving the structure and function of tissues and organs) show more excellent effects.
  • 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.
  • the pharmaceutical composition of the present invention not only has an excellent effect on improving organ function against ischemia-reperfusion injury of the heart, but also the brain, kidney, liver and other organs, and can be widely used in various tissues and organs. Of ischemia-reperfusion injury.
  • the SalB/Rg1 combination therapy has a better therapeutic effect on heart and brain ischemic diseases and ischemia-reperfusion injury than other tissues and organs, especially on ischemic stroke and ischemic stroke.
  • the protective effect of post-reperfusion injury is particularly surprising, not only significantly reducing the infarct size, but also significantly improving animal behavior, suggesting that the pharmaceutical composition of the present invention has excellent therapeutic prospects for heart disease and brain disease.

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Abstract

一种药物组合物及其应用，具体地，所述药物组合物包括：丹酚酸B作为第一活性成分；和人参皂苷Rg1作为第二活性成分，并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)。该药物组合物具有比现有技术更佳的活性成分配比，在预防和/或治疗缺血性疾病、缺血再灌注组织脏器损伤方面具有更优异的疗效，并且可用于治疗不同组织器官的缺血和/或缺血再灌注损伤。

Description

一种药物组合物及其应用 技术领域
本发明涉及医药领域，具体涉及一种药物组合物及其在预防和/或治疗缺血性疾病和/或缺血再灌注损伤等疾病中的应用。
背景技术
血管负责对全身组织器官供血，一旦血管受阻，组织器官因供血不足而导致疾病，尤其是心脏、脑等对血液供氧需求很高的组织器官；而随着休克时微循环的疏通、冠状动脉痉挛的缓解、动脉搭桥术、溶栓疗法、经皮腔内冠脉血管成形术、心脏外科体外循环、心肺脑复苏、断肢再植和器官移植等方法的建立和推广应用，使许多组织器官缺血后可以重新恢复供血(即再灌注)。但这种缺血后再灌注，有时不仅不能使组织、器官功能恢复，反而加重组织、器官的功能障碍和结构损伤。这种在缺血基础上恢复血流后组织损伤反而加重，甚至发生不可逆性损伤的现象称为缺血再灌注损伤。
申请号为CN2011102229806的中国申请公开了一种包含化合物丹酚酸B和人参皂苷Rg1的药物组合物，对心脏缺血再灌注损伤具有疗效。但二者配伍使用的研究尚不充分，有必要开展进一步的深入组方配伍研究，以提供对不同组织器官的缺血再灌注损伤具有疗效的药物组合物。
发明内容
本发明的目的是提供一种用于预防和/或治疗组织、器官缺血，以及缺血再灌注损伤等疾病的药物组合物。
本发明第一方面，提供了一种药物组合物，包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；和
(c)药学上可接受的载体；
并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
在另一优选例中，所述的第一活性成分包括丹酚酸B或其药学上可接受的盐的纯化产物。
在另一优选例中，所述的纯化产物中，按丹酚酸B计，纯度≥90％，较佳地≥95％，更佳地≥98％或99％，按所述纯化产物的总重量计。
在另一优选例中，所述的第一活性成分包括丹酚酸B含量C1≥30wt％的丹酚酸提取物，其中所述含量C1按丹酚酸的重量计。
在另一优选例中，所述提取物中，丹酚酸B的含量C1≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
在另一优选例中，所述的第二活性成分包括人参皂苷Rg1的含量C2≥30wt％的总皂苷提取物，其中所述含量C2按总皂苷的重量计。
在另一优选例中，所述提取物中，人参皂苷Rg1的含量C2≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
在另一优选例中，所述第一活性成分和第二活性成分的重量比5:(1-4.0)，较佳地5:(1.2-3.8)，更佳地5:(1.5-3.5)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:(1.8-3.2)，较佳地，5:(1.9-3.1)，更佳地5:(2-3)，最佳地5:2。
在另一优选例中，第一活性成分为丹酚酸B，而第二活性成分为人参皂苷Rg1。
在另一优选例中，所述药物组合物的剂型选自下组：液体制剂(如溶液、乳液、悬浮液)、固体制剂(如冻干制剂)、气体剂型、半固体剂型。。
在另一优选例中，所述剂型选自下组：注射剂(如注射液或粉针剂)、口服制剂(如胶囊剂、片剂、丸剂、散剂、颗粒剂、糖浆、口服液或酊剂)、舌下含服制剂、呼吸道给药制剂、皮肤给药制剂、粘膜给药制剂，更佳地，所述剂型为注射剂。
本发明第二方面，提供了一种活性成分的组合，所述的活性成分的组合包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；
并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
在另一优选例中，所述的活性成分的组合由(a)第一活性成分和(b)第二活性成分构成。
本发明第三方面，提供了一种药盒，所述的药盒包括：
第一药物组合物，所述第一药物组合物包括：(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；和药学上可接受的载体；
第二药物组合物，所述第二药物组合物包括：(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；和药学上可接受的载体；
并且，所述第一药物组合物和第二药物组合物是联合施用的，其中所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
在另一优选例中，所述第一药物组合物和第二药物组合物是不同的(或独立的) 药物化合物、或是同一药物组合物。
本发明第四方面，提供了如本发明第一方面所述的药物组合物、本发明第二方面活性成分的组合、或本发明第三方面所述的药盒的用途，用于制备一药物或药盒，所述药物或药盒用于(i)预防和/或治疗缺血性疾病；(ii)预防和/或治疗缺血再灌注损伤；(iii)抑制乳酸脱氢酶。
另一优选例中，所述药物或药盒用于(i)预防和/或治疗缺血性心脏病；(ii)预防和/或治疗缺血再灌注损伤；(iii)抑制乳酸脱氢酶和/或(iv)预防和/或治疗缺血性疾病。
在另一优选例中，所述缺血性疾病选自下组：急性缺血导致的组织器官损伤和/或慢性缺血导致组织器官损伤。
在另一优选例中，所述缺血性疾病选自下组：由于组织和血管原发性病变导致的组织器官缺血损伤，和/或继发性原因导致的缺血性病变，如外伤导致的血管离断、炎症引起的血管闭塞、肿瘤导致的血管压迫。
在另一优选例中，所述缺血性疾病选自下组：缺血性心脏病、缺血性脑卒中(如急性脑梗死)、缺血性肝损伤、肺栓塞、缺血性肾损伤、缺血性神经损伤，或其组合。
在另一优选例中，所述缺血性心脏病包括：冠心病、心肌梗死、心绞痛、心肌纤维化、心衰，或其组合。
在另一优选例中，所述的缺血再灌注损伤为再灌注所导致的组织器官损伤。
在另一优选例中，所述组织器官选自下组：心、脑、肝、脾、肺、肾、肌肉、神经，或其组合。在另一优选例中，所述的组织器官选自下组：肝、脾、肺、肾、脑、神经，或其组合。
在另一优选例中，所示组织器官选自下组：心、脑，或其组合。
在另一优选例中，所述的药物或药盒还用于改善肺栓塞诱导的心肌肥大。在另一优选例中，所述的药物或药盒还用于改善再灌注损伤的心脏的舒张功能，较佳地，所述的心脏的舒张功能包括心脏的舒张速率。
在另一优选例中，所述的药物或药盒还用于改善心脏的收缩功能(如心脏的收缩速率)。
在另一优选例中，所述药物或药盒用于改善肾脏的再灌注损伤，较佳地，包括改善肾脏结构。
在另一优选例中，所述组织器官损伤为手术后再灌注损伤，较佳地，所述手术选自下组：动脉搭桥术、取栓或溶栓疗法、经皮腔内冠脉血管成形术、体外循环下心脏手术、心脏骤停后心、肺和/或脑复苏、断肢再植或器官移植、或其他重大手术带来的再灌注损伤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1示出了在心肌梗死的动物模型上，丹酚酸B/人参皂苷Rg1对减小心脏梗死面积的效果；(A)心脏切片TTC染色代表图；(B)梗死面积定量(梗死区占整个心脏的面积百分比)。
图2示出了在心肌梗死的动物模型上，丹酚酸B/人参皂苷Rg1对血液中乳酸脱氢酶含量的影响。
图3示出了在心肌梗死的动物模型上，丹酚酸B/人参皂苷Rg1对心脏组织结构的保护作用。
图4示出了在心肌缺血再灌注损伤的动物模型上，丹酚酸B/人参皂苷Rg1(5:2)组比丹酚酸B/人参皂苷Rg1(2:5)组明显减小心脏梗死面积并且改善心脏结构；(A)心脏切片TTC染色代表图；(B)梗死面积定量(梗死区占整个心脏的面积百分比)；(C)心脏组织HE染色代表图。
图5示出了在心肌缺血再灌注损伤的动物模型上，丹酚酸B/人参皂苷Rg1(2:5)组和丹酚酸B/人参皂苷Rg1(5:2)组大鼠血流动力学(最大舒张速率和最大舒张速率)检测结果。“*”表示与丹酚酸B/人参皂苷Rg1(2:5)组比较P﹤0.05。
图6示出了在心肌缺血再灌注损伤的动物模型上，丹酚酸B/人参皂苷Rg1(2:5)组和丹酚酸B/人参皂苷Rg1(5:2)组大鼠血流动力学(末端舒张压和平均动脉压)检测结果。
图7示出了在肾脏缺血再灌注损伤模型上，假手术组、肾脏缺血再灌模型组、丹酚酸B/人参皂苷Rg1(2:5)组以及丹酚酸B/人参皂苷Rg1(5:2)组大鼠肾脏HE染色结果。
图8示出了在肾脏缺血再灌注损伤模型上，假手术组、肾脏缺血再灌模型组、丹酚酸B/人参皂苷Rg1(2:5)组以及丹酚酸B/人参皂苷Rg1(5:2)组大鼠过碘酸雪夫染色(PAS)结果。
图9示出了在肺栓塞模型上，丹酚酸B/Rg1(5：2)对肺栓塞的治疗效果；(A)左肺指数；(B)右肺指数；(C)肺脏HE染色代表图，(D)肺间质面积定量结果(E)心脏HE染色代表图，(F)肺脏中性粒细胞平均光密度。
图10示出了在肺栓塞模型上，丹酚酸B/Rg1(5：2)降低肺栓塞诱导的心肌肥大的发生，(A)心脏HE染色的代表图，(B)心肌细胞横截面积的定量图。
图11示出了在急性脑梗死模型上，丹酚酸B/Rg1(5：2)显著降低梗死面积，(A)大脑TTC染色代表图，(B)梗死面积定量结果。
图12示出了在急性脑梗死模型上，丹酚酸B/Rg1(5：2)改善脑梗死后大鼠行为学评分结果。
图13示出了在急性脑梗死模型上，丹酚酸B/Rg1(5：2)对大脑皮层神经细胞的保护作用，(A)为大脑皮层的HE染色代表图；(B)为HE染色的神经细胞数定量结果；(C)为大脑皮层的尼式染色代表图；(D)为大脑皮层尼氏小体数的定量结果。
图14示出了在急性脑梗死模型上，海马体CA1、CA2、CA3的HE染色代表图。
图15示出了在急性脑梗死模型上，海马体CA1、CA2、CA3的尼式染色代表图及其定量结果。
图16示出了在脑缺血再灌注损伤模型上，丹酚酸B/Rg1(5：2)降低再灌注损伤的脑梗死面积，(A)脑组织TTC染色代表图，(B)梗死面积定量结果。
图17示出了在脑缺血再灌注损伤模型上，丹酚酸B/Rg1(5：2)改善脑缺血再灌注后大鼠行为学评分。
图18示出了在脑缺血再灌注损伤模型上，丹酚酸B/Rg1对大脑皮层神经细胞的保护作用，A为大脑皮层的HE染色；B为大脑皮层的尼式染色。
图19示出了大鼠脑缺血再灌注损伤模型上，海马体CA1、CA2、CA3的HE染色代表图。
图20示出了大鼠脑缺血再灌注损伤模型上，海马体CA1、CA2、CA3的尼式染色代表图。
图21示出了大鼠肝缺血再灌注损伤模型上，肝组织的HE染色代表图。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一种包含丹酚酸B与人参皂苷Rg1作为活性成分比的药物组合物，与现有技术相比，本发明的药物组合物对缺血性疾病、组织脏器的缺血再灌注损伤表现出更优异的改善治疗效果；且令人惊讶地，本发明的组合物不仅对心脏，脑、肝脏而且还对肾脏等器官的缺血再灌注损伤具有疗效，可用于多种组织器官的缺血再灌注损伤。在此基础上完成了本发明。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“立体异构体”意在包括所有的同分异构形式(如对映异构，非对映异构和几何异构体(或构象异构体))：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体等。因此，本发明活性成分的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
本发明活性成分可以是无定形的、晶型或其混合物。
如本文所用，“药学上可接受的盐”指本发明活性成分的化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明活性成分的化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。一类优选的盐是本发明活性 成分的化合物与碱形成的盐。适合形成盐的碱包括但并不限于：氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸钠等无机碱，氨水、三乙胺、二乙胺等有机碱。另一类优选的盐是本发明活性成分与金属离子形成的盐，包括但不限于镁盐、钠盐、钙盐、钾盐等等。
如本文所用，“药学上可接受的酯”指本发明活性成分的化合物与酸或醇形成的适合用作药物的酯一类优选的酯为本发明的活性成分的一个或多个羟基与酸形成的酯，适合形成酯的酸包括但并不限于：磷酸、甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等；另一类优选的酯为本发明的活性成分的羧基与醇形成的酯，适合形成酯的醇包括但并不限于：C1-C6烷基-OH，如甲醇、乙醇、正丙醇、异丙醇等。
如无特别说明，药物组合物中，所述重量比以丹酚酸B和人参皂苷Rg1的原化合物形式计算。
本发明所述的“预防”和“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述组合物或药物组合物时观察到的水平相比，本发明所述组合物或药物组合物将缺血再灌注损伤预防、减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％。
如本发明所用，术语“SalB”与“丹酚酸B”可以互换使用；术语“Rg1”与“人参皂苷Rg1”可以互换使用。
第一活性成分
本发明中，第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合。
Figure PCTCN2020112258-appb-000001
在另一优选例中，所述的第一活性成分包括丹酚酸B或其药学上可接受的盐的纯化产物。
在另一优选例中，所述的纯化产物中，按丹酚酸B计，纯度≥90％，较佳地≥95％，更佳地≥98％或99％，按所述纯化产物的总重量计。
在另一优选例中，所述的第一活性成分包括丹酚酸B含量C1≥30wt％的丹酚酸提取物，其中所述含量C1按丹酚酸的重量计。
在另一优选例中，所述提取物中，丹酚酸B的含量C1≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
第二活性成分
本发明中，第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合。
Figure PCTCN2020112258-appb-000002
在另一优选例中，所述的第二活性成分包括人参皂苷Rg1的含量C2≥30wt％的总皂苷提取物，其中所述含量C2按总皂苷的重量计。
在另一优选例中，所述提取物中，人参皂苷Rg1的含量C2≥70％，较佳地≥80％，更佳地≥90％或≥95％，按提取物的干重计。
药物组合物、活性成分组合、药盒
本发明提供了一种药物组合物，包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；和
(c)药学上可接受的载体；
并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
在另一优选例中，所述第一活性成分和第二活性成分的重量比5:(1-4.0)，较佳地5:(1.2-3.8)，更佳地5:(1.5-3.5)。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为5:(1.8-3.2)，较佳地，5:(1.9-3.1)，更佳地5:(2-3)，最佳地5:2。
在另一优选例中，第一活性成分为丹酚酸B，而第二活性成分为人参皂苷Rg1。
所述药物组合物的剂型选自下组：液体制剂(如溶液、乳液、悬浮液)、固体制剂(如冻干制剂)。
在另一优选例中，所述剂型选自下组：注射剂(如注射液或粉针剂)、口服制剂(如胶囊剂、片剂、丸剂、散剂、颗粒剂、糖浆、口服液或酊剂)，更佳地，优选地，所 述剂型为注射剂。
本发明的药物组合物中，第一活性成分和第二活性成分可以分别制成制剂或混合在一起制成制剂。
本发明的药物组合物包含安全有效量范围内第一活性成分和/或第二活性成分。其中“安全有效量”指的是：活性成分的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明活性成分/剂，更佳地，含有10-500mg本发明活性成分/剂。较佳地，所述的“一剂”为一个胶囊、药片、针剂等。
本发明中，“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和第一活性成分和/或第二活性成分相互掺和，而不明显降低第一活性成分和/或第二活性成分的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2020112258-appb-000003
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明还提供了一种活性成分的组合，所述的活性成分的组合包括：
(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；
(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；
并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
在另一优选例中，所述的活性成分的组合由(a)第一活性成分和(b)第二活性成分构成。
在所述的活性成分组合中，第一活性成分和第二活性成分可以是相互独立的，也可以使组合在一起，以活性成分组合物的形成存在。
本发明还提供了一种药盒，所述的药盒包括：
第一药物组合物，所述第一药物组合物包括：(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；和药学上可接受的载体；
第二药物组合物，所述第二药物组合物包括：(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；和药学上可接受的载体；
并且，所述第一药物组合物和第二药物组合物是联合施用的，其中所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂 苷Rg1计。
在另一优选例中，所述的药盒还包括说明书。
在另一优选例中，所述第一药物组合物和第二药物组合物是不同的(或独立的)药物化合物、或是同一药物组合物。
在另一优选例中，所述的第一药物组合物和第二药物组合物在施用时同时给药、分别给药或顺序给药。
本发明的药物组合物、活性成分组合、药盒均可采用常规方法和设备进行制备。
用途和施用方法
本发明提供了一种本文所述药物组合物、活性成分的组合或药盒的用途，用于制备一药物或药盒，所述药物或药盒用于(i)预防和/或治疗缺血性疾病；(ii)预防和/或治疗缺血再灌注损伤；和/或(iii)抑制乳酸脱氢酶。
在本发明中，所述缺血性疾病是指组织或器官由于缺血而造成的损伤或病变。所述“缺血”指组织或器官的供血较正常值降低，尤其是指组织或器官中所供应的血液不能满足所述组织或器官新陈代谢的需要。
本发明的活性成分对缺血性疾病具有明显的治疗效果。常见的缺血性疾病包括但并不限于：缺血性心脏病、缺血性脑卒中、缺血性肝损伤、缺血性肺损伤、缺血性肾损伤，或其组合。
本发明中，所述的缺血性心脏病是由冠状动脉循环改变而导致心肌缺血、缺氧而引起的心脏病。常见的缺血性心脏病包括下组(但并不限于)：冠心病、心肌梗死、心肌纤维化、心绞痛，或其组合。
本发明中，所述的缺血再灌注损伤包括再灌注所导致的组织器官损伤。所述组织器官包括下组(但并不限于)：心、肝、脾、肺、肾、脑、肌肉、神经，或其组合。所述组织器官损伤还包括手术后再灌注损伤，所述手术包括下组(但并不限于)：动脉搭桥术、溶栓疗法、经皮腔内冠脉血管成形术、体外循环下心脏手术、心脏骤停后心、肺和/或脑复苏、断肢再植或器官移植。所述再灌注损伤还包括：休克后微循环疏通后的再灌注损伤、冠状动脉痉挛缓解后的再灌注损伤。
本发明中，所述预防和/或治疗缺血性疾病、预防和/或治疗缺血再灌注损伤等应用，包括在预防性的应用，也包括事后改善性的应用。例如，对于再灌注损伤，包括在再灌注之前、之中、和/或之后施用本发明的药物组合物、活性成分的组合物或药盒，以对再灌注损伤后的组织器官的保护、修复或功能的改善或增强。
本发明的药物组合物、活性成分的组合或药盒中，第一活性成分与第二活性成分还可以与其他药学上可接受的化合物联合给药，包括(但并不限于)：降血压药、降血脂药、降糖药、抗血小板聚集药等。
本发明的药物组合物、活性成分的组合或药盒还可用于抑制乳酸脱氢酶。乳酸脱氢酶(lactate dehydrogenase，LDH)是将糖转化为细胞能量过程中所需的一种酶， 存在于全身多种器官和组织中，例如：肝脏、心脏、胰腺、肾脏、骨骼肌、淋巴组织和血细胞。乳酸脱氢酶在丙酮酸被转化成乳酸的糖酵解的最后步骤中涉及，尽管正常组织通常仅当氧气供应不足时使用糖酵解，但癌症组织严重依赖有氧糖酵解，而与氧气供应水平无关，LDH抑制剂被用于涉及从氧化磷酸化到糖酵解的代谢转化的病理，例如，可用于(但并不限于)：治疗患有其中出现从氧化磷酸化到糖酵解的代谢转化的癌症、纤维化或其它病症的患者。同时乳酸脱氢酶是负责肝脏和胰脏的线粒体/过氧化物酶体甘氨酸代谢通路中将乙醛酸转化为草酸的酶，抑制LDH可用于治疗慢性肾病，如高草酸尿症。本发明的药物组合物可使血液中LDH浓度降低和/或抑制LDH的活性，可作为LDH抑制剂。
本发明的药物组合物、活性成分的组合或药盒中，第一活性成分和第二活性成分可以在施用时同时给药、分别给药或顺序给药。
本发明的药物组合物、活性成分的组合或药盒中，第一活性成分和第二活性成分的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明活性成分的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明的药物组合物、活性成分的组合或药盒中，按丹酚酸B和人参皂苷Rg1总量计，活性成分的治疗有效剂量的一般范围将是：约1-2000mg/天、约10-约1000mg/天、约10-约500mg/天、约10-约250mg/天、约10-约100mg/天，或约10-约80mg/天。治疗有效剂量将以一个或多个剂量给予。然而，应理解，对于任何特定患者的本发明活性成分的特定剂量将取决于多种因素，例如，待治疗的患者的年龄、性别、体重、一般健康状况、饮食、个体响应，给予时间、待治疗的疾病的严重性、剂型、应用模式和伴用药物。给定情况的治疗有效量能用常规实验测定，并在临床医生或医师能力和判断范围内。在任何情况中，所述活性成分将基于患者的个体情况以多个剂量给予并以允许递送治疗有效量的方式给予。
本发明的主要优点包括：
1.与现有技术相比，本发明的药物组合物具更优地活性成分配比，对治疗缺血性疾病、缺血再灌注损伤具有更佳疗效(如优异的减小梗死面积、改善组织脏器功能的效果)。
2.本发明的药物组合物不仅对心脏，而且还对脑、肝、肺和肾脏等不同器官的再灌注损伤具有治疗效果，可广泛应用于多种组织器官的缺血再灌注损伤。
3.本发明的药物组合物还具有抑制乳酸脱氢酶的效果，能快速降低血液中乳酸脱氢酶含量和/或活性，可作为乳酸脱氢酶抑制剂，用于与乳酸脱氢酶相关疾病。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
一、试剂和材料
1.1动物
清洁级雄性Wistar大鼠134只，体重220±10g；其中56只用于不同配比组合药物对心肌梗死大鼠心脏的保护作用研究；16只用于组合药物对缺血再灌注心脏的保护作用研究；32只用于组合药物对缺血再灌注肾脏的保护作用研究；30只用于评价组合药物对于肺栓塞及其并发症的防治效果。
清洁级雄性SD大鼠96只，体重220±10g；其中30只用于组合药物对缺血性脑梗死的保护作用研究；50只用于组合药物对脑缺血再灌注损伤的保护作用研究；16 只用于组合药物对缺血再灌注肝脏的保护作用研究。
Wistar和SD大鼠由中国科学院上海实验动物中心提供，饲养于中国科学院上海药物研究所实验动物中心SPF级动物室，恒温22±2℃，12h光照，标准饮食，自由饮水。
给药方式：如未特别说明，实施例中的给药方式为尾静脉注射。
1.2主要试剂与耗材
Figure PCTCN2020112258-appb-000004
1.3主要仪器
Figure PCTCN2020112258-appb-000005
1.4通用实验方法
血液的分离与处理
腹腔注射戊巴比妥钠(40mg/kg)麻醉剂麻醉大鼠，仰卧固定在鼠板上。用剪刀沿正中线剪开腹部，将腹腔内容物取出，用干棉球将腹腔内的液体擦干，在腹主动脉用5mL注射器取血至2ml EP管中；血液于冰上静置0.5小时后，在4℃，8000r/min条件下将采集到的血液离心10min，取上清液，并将血清分装于0.5ml EP管内，置于-80℃冰箱保存，待用。
TTC染色
0.5g TTC粉末溶解于100ml PBS，避光保存，现用现配。将新鲜的组织横切为多片，放置在带盖小盒中加入TTC溶液，置于湿盒中避光，在37℃恒温烘箱内温育 20min，期间翻动几次使组织均匀接触染液，20min后拿出拍照。
样品固定、脱水、石蜡包埋、切片
甲醛100ml，磷酸二氢钠4g，磷酸氢二钠6.5g，溶于900mL蒸馏水中，配制成体积比为10％的多聚甲醛固定液。将组织于多聚甲醛固定液中固定72h后，自来水冲洗2h，放置于脱水机中设定程序自动脱水，依次经75％乙醇1.5h，95％乙醇1.5h，100％乙醇1.5h，二甲苯1.5h、石蜡1.5h。提前2h打开石蜡包埋机熔化石蜡，温度控制在60℃。待石蜡融化后，脱水后的组织进行石蜡包埋，倒入包埋盒，用加热的镊子将浸蜡后的组织块放入包埋框内，轻轻移至冷台，待石蜡凝固后取下石蜡块准备切片。切片前，将蜡块放入冰箱内预冷，冷却后切片机连续切5μm厚度的石蜡切片。切片在摊片机38℃温水上展开，用涂有多聚赖氨酸的载玻片捞片，自然风干用于后续的病理组织学染色。
HE染色
将组织切片放置在65℃烤片机上烤60min，迅速放入二甲苯脱蜡15min，依次在100％、95％、75％乙醇中浸泡5min，流水冲洗5min后，苏木素染液中置15min，水洗5min，再放入1％盐酸乙醇分化液(配制：把3mL浓盐酸加入到300mL75％乙醇中，搅拌均匀即可)3s，流水返蓝5min，放入1％伊红溶液10min，快速冲洗掉多余的伊红染液后转移至75％乙醇浸泡4min，95％乙醇中4min，100％乙醇中5min，二甲苯透明15min，最后用二甲苯中性树胶封片，待树胶风干后，采用体视显微镜拍摄大体图，BX51显微镜拍摄局部放大图。
PAS染色
将组织切片放置在65℃烤片机上烤60min，迅速放入二甲苯脱蜡15min，依次在95％、70％、30％乙醇中浸泡2min，蒸馏水浸泡2分钟，将配制好的试剂一应用染液滴加在玻片的样本上，使其全部覆盖样本，将此玻片轻轻平放于染色架上，室温避光孵育8-15分钟。取出染色玻片，自来水流水缓慢冲洗玻片3-5分钟。玻片未完全干透前，滴加试剂二应用液于玻片样本上，用洗耳球将染液吹匀并全部覆盖样本，然后置于室温孵育8-15分钟。孵育结束后，取出染色玻片，自来水流水缓慢冲洗玻片30-60秒，自然晾干。滴加试剂三复染液染色20-30秒，然后流水冲净，待干后用封片剂封片，待封片剂风干后，采用体视显微镜拍摄大体图，BX51显微镜拍摄局部放大图。
生化指标检测
LDH检测按照乳酸脱氢酶测定试剂盒(乳酸底物法，希森美康生物科技(无锡)有限公司，批号：R8004)，使用全自动生化分析仪(JCA-BM6010/C，希森美康医用电子(上海)有限公司)对大鼠血清进行检测。
血流动力学检测
大鼠腹腔注射40mg/kg戊巴比妥钠麻醉剂麻醉后，分离右颈总动脉并插入米勒导管，用Powerlab8/30生理记录仪(ML870，ADINSTRUMENTS)记录颈动脉压、左心室最大收缩速率、左心室最大舒张速率、左心室末端舒张压等血流动力学指标。
左、右肺指数的计算
大鼠称重后，腹腔注射30mg/kg舒泰麻醉，分离左肺、右肺并称重。依据左肺、 右肺与体重的比值分别计算左、右肺指数。
肺间质面积的定量
针对苏木素-伊红染色的肺组织进行定量分析。每个样本除去气管、支气管和肺泡等肺实质，其余紫色部分代表肺间质，每个组织样本在相同倍率下拍照，对肺间质面积进行定量。
免疫组化染色考察中性粒细胞的浸润
将石蜡包埋的组织在65℃，烤片45-50分钟；然后二甲苯15分钟、无水乙醇5分钟、95％乙醇5分钟、75％乙醇3分钟、流水冲洗1分钟、脱蜡至水相；微波条件下，通过柠檬酸修复液修复抗原；于37℃，10％山羊血清孵浴30分钟；4℃，CD44抗体孵浴过夜；37℃，二抗孵浴1小时；滴入DAB溶液40s；苏木素复染15分钟；1％盐酸乙醇分化5秒；二甲苯透化；中性树胶封片。采用Image Pro Plus软件定量每个样本的阳性细胞面积和光密度，平均光密度值通过光密度与阳性细胞面积的比值计算。
动物的行为学检测
手术前两天和术后一天对所有实验动物进行Longa评分、NSS评分和EBST检测。
a.Longa评分法：0分：正常，无神经功能损伤；1分：左侧前爪不能完全伸展，轻度神经功能损伤；2分：行走时，大鼠向左侧(偏瘫侧)转圈，中度神经功能损伤；3分：行走时，大鼠身体向左侧(偏瘫侧)倾倒，重度神经功能损伤；4分：不能自发行走，有意识丧失。
b.NSS评分法：0分：神经功能正常；1分：轻度神经功能缺损(提尾时左前肢屈曲)；2分：中度神经功能缺损(行走时向左侧转圈)；3分：中度神经功能缺损(向左侧倾斜)；4分：无法行走，意识减退；5分：与缺血有关的死亡。
c.Elevated Body Swing Test：测量时首先用手提起大鼠的尾根部，大鼠头部悬垂距平面5cm左右，这时大鼠的头部会向左或向右旋转，向单侧旋转的角度大于100时为计数标准，记录旋转的方向和角度，一次试验后让大鼠休息一分钟，在进行下一次试验，重复20次，记录总的方向和次数。
数据统计方法
使用GraphPad Prism 6.0(GraphPad软件，LA Jolla，CA，美国)进行数据分析，所有计量数据均用平均值±标准差表示，均数间采用单因素方差分析(One-way ANOVA)确认方差是否齐性。若n值一致，采用Tukey法进行比较；若n值不一致，采用Bonferroni法进行比较，P﹤0.05具有统计学意义。
二、 动物实验
实施例1
1.1心肌梗死模型的制备
腹腔注射戊巴比妥钠(40mg/kg)，将大鼠固定于手术板上胸部被毛将笔型静脉留置针套插入气管连接动物呼吸机碘酒消毒在胸部左侧3-4肋间剪开皮肤钝性分离肌肉，撑开第三根和第四根肋骨间隙并固定暴露出心脏上部，撕开心包，持针器夹持5-0带线缝合针以左冠状静脉为标志，从左心耳尖、肺动脉圆锥和心耳交界下1mm处 穿线并结扎，结扎冠状动脉后心肌组织立即由红润转为苍白。假手术动物除不结扎冠脉动脉左前降支以外，其余手术操作完全相同。
1.2动物分组及给药方式
56只大鼠随机分为以下7组：假手术组、缺血模型组、丹酚酸B与人参皂苷Rg1联合给药组(分别按照5:4、5:3、5:2、5:1和2:5比例配制)，每组8只。采用双盲的方式给药，即手术者不参与给药，数据统计人员不知道分组信息。将丹酚酸B与人参皂苷Rg1分别按5:4、5:3、5:2、5:1、2:5混合，混匀后随机编号，溶解后，用微孔滤膜过滤待用。假手术组和心肌梗死模型组按照体重给等体积生理盐水。手术后立即按15mg/kg尾静脉注射给药一次，24小时后再给药一次，随后腹主动脉取血以及收集心脏进行心脏组织学检测。
1.3实验结果与分析
1.3.1 SalB/Rg1减小心脏梗死面积
如图1所示，假手术组无梗死区(0％)。与缺血模型组相比，2:5组梗死面积下降了23.4％；5:4组梗死面积下降了15.3％；5:3组梗死面积下降了35.2％(P＜0.01)；5:2组梗死面积下降了33.2％(P＜0.01)；5:1组梗死面积下降了25.1％；*表示与假手术组相比，***P＜0.001； #表示与缺血模型组相比， #P＜0.05。并且，5:3组和5:2组相较于2:5组降低梗死面积效果更为明显，梗死面积相较于2:5组分别降低1.50倍(5:3)和1.42(5:2)倍。
1.3.2 SalB/Rg1减少血液中LDH含量
血液中LDH含量结果如图2所示，与缺血模型组相比，2:5组LDH含量下降了19.7％；5:4组LDH含量下降了7.5％；5:3组LDH含量下降了23.0％；5:2组LDH含量下降了37.9％；5:1组LDH含量下降了13.7％；。*表示与假手术组相比，*P＜0.05。与2:5组相比，5:2组LDH含量下降了1.9倍。
1.3.3 SalB/Rg1改善心脏结构
为进一步评价SalB/Rg1对心脏组织结构的保护，分别分析心脏的梗死区(上)，梗死边缘区(中)，梗死远端区(下)的心脏结构(如图3所示)。缺血模型组的心脏梗死区结构破坏严重，有大量炎症细胞浸润，心肌细胞表现为水肿、坏死、细胞核丢失，肌纤维呈条索状；在梗死边缘区，炎症细胞明显浸润，心肌纤维变长呈波浪状。与缺血模型组比较，无论在梗死区还是梗死边缘区，SalB/Rg1对上述损伤都表现出不同程度的改善，其中5:2组大鼠心肌组织结构改善最为明显：在梗死区，炎症细胞浸润明显减少，心肌细胞水肿、坏死减弱，细胞核丢失数量减少，肌纤维排列较规则；在梗死边缘区，炎症细胞浸润减少，心肌纤维排列规则。各组动物在梗死远端区的细胞排列有序而紧密规则，并无明显差异。
上述结果说明，相对缺血模型组，SalB/Rg1联合给药对减小梗死面积均有效果，且令人惊讶的，与现有技术中公开的SalB/Rg1最佳配比(2:5)相比，本发明中SalB/Rg1为5:3和5:2的组给药后，大鼠心脏的梗死面积更小，且在降低血液中LDH含量和心脏结构改善方面展现出更优异的疗效。
实施例2
使用SalB/Rg1为5:2的组合和现有技术中公开的SalB/Rg1为2：5的最佳配比组合在心肌缺血再灌注损伤模型中进行进一步对比。
2.1心肌缺血再灌注损伤模型的制备
腹腔注射戊巴比妥钠(40mg/kg)，将大鼠固定于手术板上，胸部被毛。将笔型静脉留置针套插入气管连接动物呼吸机，碘酒消毒，在胸部左侧3-4肋间剪开皮肤钝性分离肌肉，撑开第三根和第四根肋骨间隙并固定暴露出心脏上部，撕开心包，持针器夹持5-0带线缝合针以左冠状静脉为标志，从左心耳尖、肺动脉圆锥和心耳交界下1mm处穿线，两根丝线打结的地方放入一根2-0号线并结扎，结扎冠状动脉后心肌组织立即由红润转为苍白。心肌缺血40分钟后，将丝线剪开，拿掉2-0号线进行心肌缺血再灌注，1h后进行各项检测评价。假手术动物除不结扎冠脉动脉左前降支以外，其余手术操作完全相同。
2.2动物分组及给药方式
16只220g左右大鼠随机分为以下2组：丹酚酸B与人参皂苷Rg1联合给药组(其中按照5:2和2:5比例配制)，每组8只。大鼠心肌缺血40分钟后再灌，再灌的同时立即按15mg/kg尾静脉注射给药，再灌注1小时后进行血流动力学检测，随后收集心脏进行心脏组织学检测。
2.3实验结果
在心脏缺血再灌注损伤模型上，实验结果如图4，图A为梗死面积TTC染色代表图，图B为梗死面积定量结果，图C为HE染色代表图。与SalB/Rg1(2:5)相比，SalB/Rg1(5:2)大鼠心脏梗死面积下降了15.03％。为进一步评价SalB/Rg1对缺血再灌注损伤心脏组织结构的保护，分别分析心脏的梗死区(上)，梗死边缘区(中)，梗死远端区(下)的心脏结构(图C)。与SalB/Rg1(2:5)相比，SalB/Rg1(5:2)在梗死区和梗死边缘区，更加显著地抑制炎症细胞浸润，心肌细胞坏死、细胞核丢失等损害。各组动物在梗死远端区的细胞排列有序而紧密规则，并无明显差异。
图5和图6显示血流动力学结果，与SalB/Rg1(2:5)相比，SalB/Rg1(5:2)提高最大舒张速率18.2％(P＜0.05)、提高最大收缩速率11.6％，说明SalB/Rg1(5:2)相较于SalB/Rg1(2:5)改善心脏功能的效果更加优异；图6显示末端舒张压和平均动脉压无明显差别，说明SalB/Rg1(5:2)相较于SalB/Rg1(2:5)在血压调控方面无不利效果。
实施例3
SalB/Rg1(5:2)与SalB/Rg1(2:5)在肾脏缺血再灌注损伤模型中进行进一步对比。
3.1肾脏缺血再灌注损伤模型的制备
腹腔注射戊巴比妥钠(40mg/kg)，将大鼠固定于手术板上，腹部被毛碘酒消毒腹部正中切口2.5cm，分离肠道暴露出左侧肾脏，分离肾脏周围脂肪，动脉夹夹闭左侧肾蒂(包括肾动脉、肾静脉、肾盂)40分钟造成肾脏缺血，右侧肾脏和左侧肾脏同样操作。40分钟后，取下左右两侧肾脏的动脉夹，24h后造成肾脏缺血再灌注损伤， 缝合肌肉和皮肤。假手术动物除不夹闭两侧肾蒂以外，其余手术操作完全相同。
3.2动物分组及给药方式
32只大鼠随机分为以下4组：假手术组、缺血再灌注模型组、丹酚酸B与人参皂苷Rg1联合给药组(其中按照5:2和2:5比例配制)，每组8只。假手术组和肾脏缺血再灌注模型组按照体重给等体积生理盐水。大鼠肾脏缺血40分钟后再灌，再灌的同时立即按15mg/kg尾静脉注射给药一次，24小时后再给药一次，随后收集肾脏进行肾脏组织学检测。
3.3实验结果
为评价SalB/Rg1对缺血再灌注损伤肾脏组织结构的保护，分析肾脏皮质的结构(如图7)。缺血再灌模型组的肾脏皮质有大量炎症细胞浸润，红细胞大量渗出，细胞间隙增大。与缺血再灌模型组比较，SalB/Rg1对上述损伤都表现出不同程度的改善，而5:2组大鼠肾脏组织结构改善最为明显：肾脏皮质炎症细胞浸润和红细胞渗出均明显减少，细胞间隙减小。
为评价SalB/Rg1对缺血再灌注损伤肾脏组织糖原堆积的作用，分析肾脏皮质糖原堆积的情况(如图8)。缺血再灌模型组的肾脏系膜区糖原聚集，肾小球系膜细胞增生，基底膜增厚，细胞核脱落。与缺血再灌模型组比较，SalB/Rg1对上述损伤都表现出不同程度的改善，与2:5组相比，5:2组大鼠肾脏糖原堆积明显减少，肾小球系膜细胞增生改善，细胞核脱落数量减少。
实施例4
评价丹酚酸B/人参皂苷Rg1联用(5:2)对大鼠肺栓塞及其并发症的防治效果
4.1实验动物及模型制备
30只雄性Wistar大鼠随机分为以下3组：正常对照组、模型对照组、丹酚酸B/Rg1组(20mg/kg)，每组10只。正常对照组分别于第0、7、14、21天，按5ml/kg的剂量给予生理盐水，另外两组在对应时间给予聚苯乙烯微球。聚苯乙烯微球的浓度为20万粒/ml，直径为45μm。分别于第0、7、14、21天，按100万粒/kg(5ml/kg)的剂量，对Wistar大鼠尾静脉注射聚苯乙烯微球。
给予聚苯乙烯微球的两组动物，一组于第7天开始每天注射生理盐水作为模型对照组，连续施用28天；另一组第7天开始每天以20mg/kg的剂量腹腔注射丹酚酸B/Rg1(比例5：2)，连续28天。所有动物在第35天取材，进行心、肺组织学检测。
4.2实验结果
4.2.1丹酚酸B/Rg1(5:2)联用可改善微球诱导的肺栓塞
丹酚酸B/Rg1联用可改善微球诱导的肺栓塞。如图9所示，与模型组相比，丹酚酸B/Rg1显著降低左肺指数(A)和右肺指数(B)；从组织染色图可以看出，丹酚酸B/Rg1显著改善肺脏结构(C)，使肺间质的面积明显下调(D)，这证明丹酚酸B/Rg1对肺功能的改善效果；另外，从(E)可以看出，丹酚酸B/Rg1还可以抑制中性粒细胞浸润于肺脏组织，其抑制效果具有显著的统计学意义(F),提示丹酚酸B/Rg1显著抑制肺栓塞的发生。
4.2.2丹酚酸B/Rg1联用改善肺栓塞引起的心肌肥大
肺栓塞的主要并发症是心肌肥大，在HE染色的心肌组织上，肥大的心肌细胞可以通过细胞横截面积的大小评价。如图10所示，A是心脏HE染色的代表图，B是心肌细胞横截面积的定量图，丹酚酸B/Rg1显著减少了肺栓塞诱导的心肌肥大的发生。
实施例5
丹酚酸B/Rg1联用(5:2)治疗大鼠急性脑梗死
5.1实验动物及模型制备
30只雄性SD大鼠，随机分为以下3组：假手术组、急性脑梗死模型组、丹酚酸B/人参皂苷Rg1联用组(联用组配比为5:2，给药及剂量为10mg/kg)，每组10只。对大鼠实施麻醉、固定、胸部被毛和碘酒消毒后，依次分离肌肉、皮下结缔组织、颈前肌群、暴露颈总动脉(CCA)；距CCA分叉部4mm处剪一小口、栓线从小口插入到颈内动脉(ICA)，从血管分叉处推动线栓约20mm,阻塞大脑中动脉(MCA)诱导脑梗的发生。脑梗死的动物给予10mg/kg丹酚酸B/人参皂苷Rg1治疗(丹酚酸B/Rg1组)；脑梗死的动物给予生理盐水(模型组)；假手术组动物除不插入线栓，其余手术操作完全相同，并在相同的时间点给予生理盐水(假手术组)。
5.2实验结果
5.2.1丹酚酸B/Rg1降低脑梗死面积
脑梗死面积通过TTC染色进行评价，如图11所示，其中图A为大脑TTC染色代表图，梗死区域被染为白色，非梗死区域被染为红色。图B为梗死面积定量结果(白色区域面积/(红色区域面积+白色区域面积)百分比)，丹酚酸B/Rg1显著降低梗死面积，与模型组相比，丹酚酸B/Rg1组脑梗死面积降低了39.42％。注：***p<0.001VS假手术组，&&p<0.01VS模型组。
5.2.2丹酚酸B/Rg1改善脑梗死后大鼠行为学评分
采用Longa评分、NSS评分和EBST评价术前和术后大鼠神经运动功能，结果如图12所示。依据三种评分方法，三组动物在手术前都未见明显行为学差异。在脑梗死造模后，依据EBST评分(p<0.001)、Longa评分(p<0.05)和NSS评分(p<0.05)，丹酚酸B/Rg1显著改善神经运动功能。
5.2.3丹酚酸B/Rg1保护大脑皮层神经细胞
大脑皮层是调节、控制躯体运动的高级中枢。如图13所示，A为大脑皮层的HE染色代表图；B为HE染色单个图像视野下神经细胞数定量结果；C为大脑皮层尼式染色代表图；D是单个图像视野下大脑皮层尼氏小体数的定量结果。结果显示，无论是HE染色，还是尼氏染色，丹酚酸B/Rg1都显现出显著的神经细胞的保护效果。注：***p<0.001VS假手术组，&&p<0.01，&&&p<0.001VS模型组。
5.2.4丹酚酸B/Rg1保护海马神经细胞
从解剖学的角度来看，海马常被看做侧脑室颞角的一个内侧凸起，它由CA1、CA2、CA3和CA4四个区域组成。神经细胞的细胞体与其神经网区域呈层状排列。图14是CA1、CA2、CA3的HE染色图。与假手术组相比，模型组动物呈现明显空泡化、神经元胞体皱 缩、丢失和数量减少；与模型组相比，丹酚酸B/Rg1组存活的神经细胞增多，神经细胞排列有序、神经细胞数量明显提升。
图15是CA1、CA2、CA3的尼式染色代表图及其定量结果。CA1、CA2和CA3区尼氏染色的代表图(A、B、C)呈现的细胞数量，与HE染色的趋势相一致。本动物模型的左脑是损伤部位，右脑呈现正常组织学结构。左右脑灰度差值/图像视野，反映了模型动物神经细胞的损害程度，图B、图D和图F的定量结果显示丹酚酸B/Rg1保护神经细胞的完整性。从图15-16可以看出无论HE染色，还是尼氏染色，丹酚酸B/Rg1都显现出都对海马CA1、CA2、CA3的保护效果。
实施例6
丹酚酸B/Rg1联用(5:2)治疗大鼠脑缺血再灌注损伤
6.1实验动物及模型制备
30只雄性SD大鼠随机分为以下3组：假手术组、脑缺血再灌注损伤模型组、依达拉奉(购自国药集团国瑞药业有限公司，批号：1909116)组、丁苯酞(购自石药集团恩必普药业有限公司，批号：6182002117)组、丹酚酸B/Rg1联用组(5:2，给药及剂量为5mg/kg)，每组10只。
对大鼠实施麻醉、固定、胸部被毛和碘酒消毒后，依次分离肌肉、皮下结缔组织、颈前肌群、暴露颈总动脉(CCA)；距CCA分叉部4mm处剪一小口、栓线从小口插入到颈内动脉(ICA)，从血管分叉处推动线栓约20mm,阻塞2h大脑中动脉(MCA)诱导脑梗的发生,2h后拔出线栓制作脑缺血2h再灌注24h的模型。拔出线栓前10分钟进行给药(尾静脉注射)：给予5mg/kg丹酚酸B/人参皂苷Rg1(丹酚酸B/Rg1组)；给予5mg/kg依达拉奉(依达拉奉组，按有效成分计)；给予5mg/kg丁苯酞(丁苯酞组，按有效成分计)；给予5mg/kg生理盐水(模型组)；假手术组动物，除不插入线栓，所有手术操作完全相同，给予5mg/kg生理盐水。
6.2实验结果
6.2.1丹酚酸B/Rg1降低再灌注损伤的脑梗死面积
如图16所示，通过TTC染色对脑梗死面积进行评价，各组染色结果的代表图见图A，梗死面积定量结果见图B，可以看出，丹酚酸B/Rg1与依达拉奉、丁苯酞相比，降低梗死面积的效果更加显著。注：***p<0.001,*p<0.1VS假手术组，&&p<0.01VS模型组。
6.2.2丹酚酸B/Rg1改善脑缺血再灌注后大鼠行为学评分
采用Longa评分评价术前和术后大鼠神经运动功能，结果如图17所示。依据Longa评分方法，五组动物在手术前都未见明显行为学差异。在造模后，依据Longa评价，丹酚酸B/Rg1相较于依达拉奉、丁苯酞呈现更佳的改善神经运动功能的效果。
6.2.3丹酚酸B/Rg1保护大脑皮层神经细胞
如图18所示，A是大脑皮层的HE染色；B是大脑皮层的尼式染色。与假手术组相比，模型组可见明显空泡化、神经元胞体皱缩、丢失、数量减少。与模型组相比，丹酚酸B/Rg1组存活的神经细胞显著高于模型组、依达拉奉组和丁苯酞组。无论HE染色， 还是尼氏染色，丹酚酸B/Rg1都显现出都神经细胞的保护效果，且呈现出相较于依达拉奉、丁苯酞更加优异的疗效。
6.2.4丹酚酸B/Rg1保护海马神经细胞
图19是CA1、CA2、CA3的HE染色代表图，图20是CA1、CA2、CA3的尼式染色代表图。无论HE染色，还是尼氏染色，丹酚酸B/Rg1都显现出都对海马CA1、CA2、CA3的保护效果，且呈现出相较于依达拉奉、丁苯酞更加优异的疗效。
实施例7
丹酚酸B/人参皂苷Rg1联用(5:2)对大鼠肝缺血再灌注损伤的保护作用
7.1实验动物及模型制备
16只雄性SD大鼠，随机分成两组，一组是肝缺血再灌注损伤模型组，一组是丹酚酸B/Rg1(5：2)组合治疗组。
肝缺血再灌注损伤的模型制备方法如下；对大鼠称重麻醉、腹部被毛、固定于手术台；碘伏消毒，腹部正中纵行切口5cm至剑突；依次剪开皮肤、肌肉和腹膜，充分显露肝脏及胃肠道；分离肝周围韧带；使用显微止血夹夹闭肝脏中叶；暴露左叶门静脉、肝动脉及胆管；肝叶颜色由红色逐渐变为苍白，说明肝血流阻断成功。血流阻断1h后，去除止血夹；肝叶颜色逐渐红润，提示肝脏再灌注成功。模型对照组在再灌注的同时给予生理盐水；丹酚酸B/Rg1联用组在再灌注的同时给予10mg/kg的丹酚酸B/人参皂苷Rg1(5：2)，复灌6小时后取材评价肝脏保护效果。
7.2实验结果
对上述肝脏组织进行HE染色。结果如图21(上图为200X，下图为400X)所示，缺血再灌注6h后，模型组肝小叶结构不完整，肝细胞排列紊乱，充血，肿胀，并明显可见细胞坏死，丹酚酸B/Rg1能够改善肝细胞结构,但效果不如心、脑明显。
讨论
在现有技术中，实验结果显示不同药物对减小梗死面积的效果：丹酚酸B<人参皂苷Rg1<<丹酚酸B:人参皂苷Rg1＝2:5给药组，(参见CN2011102229806，图3)，所以容易认为人参皂苷Rg1对减小心脏梗死面积的效果优于丹酚酸B，联合用药时人参皂苷Rg1占比更大效果更好，但令人惊奇地，与现有技术中公开的最佳配比组合(SalB/Rg1为2:5)的相比，本发明的药物组合物(丹酚酸B重量>人参皂苷Rg1)在治疗心肌梗死、脑梗死、肺栓塞等缺血性疾病和心脏、脑缺血再灌注损伤(如减小梗死面积、改善组织脏器结构、功能)方面展现出更优异的效果。意外地，本发明的药物组合物还可使血液中LDH浓度降低和/或抑制LDH的活性，可作为LDH抑制剂。
不同组织器官(如：心、肝、脾、肺、肾、脑以及肌肉、神经等)，由于其组织结构、功能、血管分布，以及对血液的需求等各不相同，药物对不同器官的缺血灌注损伤是否都具有治疗作用，结果难以预测。令人惊讶地，本发明的药物组合物不仅对心脏，而且还对脑、肾脏、肝脏等器官的缺血再灌注损伤也展现出优异的改善器官功能的效果，可广泛用于多种组织器官的缺血再灌注损伤。
更意外地，SalB/Rg1联合用药对心、脑的缺血性疾病、缺血再灌注损伤表现出优于其他组织器官的治疗效果，尤其是对缺血性脑卒中、和缺血性脑卒中后的再灌注损伤的保护效果尤为令人惊讶，不但显著降低梗死面积，还能显著改善动物的行为，提示该本发明的药物组合物对心脏疾病和脑疾病具有优异的治疗前景。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种药物组合物，其特征在于，包括：
    (a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；
    (b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；和
    (c)药学上可接受的载体；
    并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
  2. 如权利要求1所述的药物组合物，其特征在于，所述第一活性成分和第二活性成分的重量比5:(1-4.0)，较佳地5:(1.2-3.8)，更佳地5:(1.5-3.5)。
  3. 如权利要求1所述的药物组合物，其特征在于，所述第一活性成分和第二活性成分的重量比为5:(1.8-3.2)，较佳地，5:(1.9-3.1)，更佳地5:(2-3)，最佳地5:2。
  4. 如权利要求1-3中任一所述的药物组合物，其特征在于，第一活性成分为丹酚酸B，而第二活性成分为人参皂苷Rg1。
  5. 如权利要求1所述的药物组合物，其特征在于，所述药物组合物的剂型选自下组：液体制剂(如溶液、乳液、悬浮液)、固体制剂(如冻干制剂)、气体剂型、半固体剂型。
  6. 如权利要求5所述的药物组合物，其特征在于，所述剂型选自下组：注射剂(如注射液或粉针剂)、口服制剂(如胶囊剂、片剂、丸剂、散剂、颗粒剂、糖浆、口服液或酊剂)、舌下含服制剂、呼吸道给药制剂、皮肤给药制剂、粘膜给药制剂，更佳地，所述剂型为注射剂。
  7. 一种活性成分的组合，其特征在于，所述的活性成分的组合包括：
    (a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；
    (b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；
    并且，所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
  8. 一种药盒，其特征在于，所述的药盒包括：
    第一药物组合物，所述第一药物组合物包括：(a)第一活性成分，所述第一活性成分选自下组：丹酚酸B、其立体异构体、其晶型、其药学上可接受的盐或酯、含丹酚酸B的提取物、或它们的组合；和药学上可接受的载体；
    第二药物组合物，所述第二药物组合物包括：(b)第二活性成分，所述第二活性成分选自下组：人参皂苷Rg1、其立体异构体、其晶型、其药学上可接受的盐或酯、含人参皂苷Rg1的提取物、或它们的组合；和药学上可接受的载体；
    并且，所述第一药物组合物和第二药物组合物是联合施用的，其中所述第一活性成分和第二活性成分的重量比为5:(1-4.5)，其中所述重量比以丹酚酸B和人参皂苷Rg1计。
  9. 如权利要求7的活性成分的组合、包含其的药物组合物或药盒的用途，其特征在于，用于制备一药物或药盒，所述药物或药盒用于(i)预防和/或治疗缺血性疾病；(ii)预防和/或治疗缺血再灌注损伤；和/或(iii)抑制乳酸脱氢酶。
  10. 如权利要求9所述的用途，其特征在于，所述缺血性疾病选自下组：由于组织和血管原发性病变导致的组织器官缺血损伤，和/或继发性原因导致的缺血性病变，如外伤导致的血管离断、炎症引起的血管闭塞、肿瘤导致的血管压迫。
  11. 如权利要求10所述的用途，其特征在于，所述缺血性疾病选自下组：缺血性心脏病、缺血性脑卒中、肺栓塞、缺血性肝损伤、缺血性肾病、缺血性神经损伤，或其组合。
  12. 如权利要求11所述的用途，其特征在于，所述缺血性心脏病包括：冠心病、心肌梗死、心绞痛、心肌纤维化、心衰，或其组合。
  13. 如权利要求9所述的用途，其特征在于，所述的缺血再灌注损伤为再灌注所导致的组织器官损伤。
  14. 如权利要求13所述的用途，其特征在于，所述组织器官选自下组：心、脑、肝、脾、肺、肾、肌肉、神经，或其组合。
  15. 如权利要求14所述的用途，其特征在于，所述组织器官损伤为手术后再灌注损伤和/或溶栓治疗后的再灌注损伤，较佳地，所述手术选自下组：动脉搭桥术、取栓疗法、经皮腔内冠脉血管成形术、体外循环下心脏手术、心脏骤停后心、肺和/或脑复苏、断肢再植或器官移植。
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