Compounds for treating hemorrhagic diseases and compositions thereof

Abstract

The invention relates to a pharmaceutical composition for treating hemorrhagic diseases. The pharmaceutical composition comprises salvianolic acid as a first active ingredient; and ginsenoside as a second active ingredient. The pharmaceutical composition can be used for preventing and / or treating hemorrhagic diseases of tissues and organs, especially hemorrhagic cerebral apoplexy, and can improve neurological symptoms related to the hemorrhagic cerebral apoplexy.

Description

Compounds and compositions for treating bleeding disorders Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a group of compounds and compositions thereof and their use in preventing and / or treating hemorrhagic diseases. Background Art

[0002] Hemorrhagic diseases pose a huge threat to human life and safety. Based on different pathological conditions, bleeding can occur in organs such as the brain and spleen. Bleeding caused by trauma, vascular rupture, etc. is a common intracranial hemorrhagic cerebrovascular disease. Hemorrhagic stroke accounts for about 30% of all strokes and is caused by bleeding due to lesions, necrosis, and rupture of the cerebral blood vessel walls. Compared with ischemic stroke, hemorrhagic stroke has a higher mortality rate and long-term disability risk. At present, clinical treatment strategies for hemorrhagic stroke include timely diagnosis, active blood pressure control, correction of underlying coagulation defects, and possible surgical intervention. In terms of therapeutic drugs for hemorrhagic stroke, patients have very limited options, and more therapeutic drugs need to be developed to meet patients' treatment needs of reducing neurological damage and optimizing neurological function. The existing technology has systematically studied the application of monomeric compounds and their compositions to treat hemorrhagic stroke. In the past, the effects of the above-mentioned compounds and compositions in the field of hemorrhagic stroke were unknown.

[0003] Therefore, there is a need in the art to develop pharmaceutical compositions that can prevent and / or treat hemorrhagic diseases of tissues and organs, especially hemorrhagic stroke.

[0004] Summary of the Invention

[0005] The present invention aims to provide a pharmaceutical composition for preventing and / or treating hemorrhagic diseases of tissues and organs, particularly cerebral hemorrhage and hemorrhagic stroke. Specifically, the present invention provides the use of a salvianolic acid B / ginsenoside Rg1 pharmaceutical composition for preparing a medicament for treating, alleviating, and / or preventing hemorrhagic diseases.

[0006] In a first aspect of the present invention, there is provided a use of a combination of active ingredients for preparing a medicament for treating, alleviating and / or preventing a hemorrhagic disease, wherein the combination of active ingredients comprises:

[0007] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid, or a combination thereof; and

[0008] (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof.

[0009] In another preferred embodiment, the combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.

[0010] In another preferred embodiment, the salvianolic acid is selected from the group consisting of salvianolic acid B (Sal B), salvianolic acid A (Sal A), salvianolic acid C (Sal C), isosalvianolic acid C, salvianolic acid D (Sal D), salvianolic acid E (Sal E), salvianolic acid F (Sal F), salvianolic acid G (Sal G), salvianolic acid H (Sal H), salvianolic acid I (Sal I), salvianolic acid J (Sal J), salvianolic acid K (SalK), salvianolic acid L (Sal L), salvianolic acid M (Sal M), salvianolic acid N (Sal N, Sal N), Salvianolic acid T (Salvianolic acid T, Sal T), Salvianolic acid U (Salvianolic acid U, Sal U), Salvianolic acid Y (Salvianolic acid Y,Sal Y), Caffeic Acid, Danshensu, Protocatechuic acid, Protocatechuic aldehyde, Ferulic acid, Isoferulic Acid, Salvinal, Ailanthoidol, Rosmarinic acid, Lithospermic acid, Salviaflaside, Catechin, dimethyl lithospermate B, tanshinone I, dihydrotanshinone I, tanshindiol-A, tanshinone IIA, cryptotanshinone, miltirone, dehydro miltirone, isotanshinone I, Danshenxinquinone A A), or a combination thereof.

[0011] In another preferred embodiment, the ginsenosides are selected from the group consisting of ginsenoside Rg1, ginsenoside Re, ginsenoside Rg2, ginsenoside Rh1, ginsenoside F1, notoginsenoside R1, notoginsenoside R2, notoginsenoside R3, notoginsenoside R3, notoginsenoside R6, notoginsenoside M, notoginsenoside N, notoginsenoside Rt, notoginsenoside T3, notoginsenoside FP1, notoginsenoside RW1, notoginsenoside RW1), Notoginsenoside Fh7, Yesanchinoside D, Yesanchinoside U, 20-O-glucoginsenoside Rf (20-glucoginsenoside R), Malonyl-ginsenoside Rg1, Chikusetsu saponin L5, Koryoginsenoside R1, Pseudoginsenoside RT3, Saponin 10, Saponin 1, Saponin 2, Saponin 3, Ginsenoside Ra3, Ginsenoside Rb1, Ginsenoside Rb2 Ginsenoside Rb2), Ginsenoside Rb3, Ginsenoside Rc, Ginsenoside Rd, Ginsenoside Rg3, Malonyl-ginsenoside Rb1, Malonyl-ginsenoside Rd, 6'-O-acetylginsenoside Rg3Rg3), ginsenoside F2, ginsenoside CK, ginsenoside Mc, 20(S)-ginsenoside Rg3 (20(S)-Ginsenoside Rg3), 20(R)-ginsenoside Rg3 (20(R)-Ginsenoside Rg3), 20(R)-ginsenoside Rh2 (20(R)-Ginsenoside Rh2), 20(S)-ginsenoside Rh2 (20(S)-Ginsenoside Rh2), notoginsenoside R4, notoginsenoside K, gypenoside XVII, gypenoside IX, gypenoside XV, gypenoside VIII (gypenoside Notoginsenoside VIII), notoginsenoside Fa, notoginsenoside Fc, notoginsenoside Fc, notoginsenoside Fc, notoginsenoside D, notoginsenoside L, notoginsenoside L*, notoginsenoside O, notoginsenoside P, notoginsenoside Q, notoginsenoside S, notoginsenoside T, notoginsenoside FP2, notoginsenoside Ftl, notoginsenoside ST-4, notoginsenoside FZ, notoginsenoside Fh1, or a combination thereof.

[0012] In another preferred embodiment, the salvianolic acid is salvianolic acid B, and the ginsenoside is ginsenoside Rg1.

[0013] In another preferred embodiment, the first active ingredient includes a purified product of salvianolic acid or a pharmaceutically acceptable salt thereof.

[0014] In another preferred embodiment, the purity of the purified product, calculated as salvianolic acid, is ≥90%, preferably ≥95%, more preferably ≥98% or 99%, based on the total weight of the purified product.

[0015] In another preferred embodiment, the first active ingredient comprises a salvianolic acid extract having a salvianolic acid content C1 ≥ 30 wt %, wherein the content C1 is calculated based on the weight of salvianolic acid.

[0016] In another preferred embodiment, the content C1 of salvianolic acid in the extract is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0017] In another preferred embodiment, the second active ingredient comprises a total saponin extract having a ginsenoside content C2 ≥ 30 wt%, wherein the content C2 is calculated based on the weight of the total saponins.

[0018] In another preferred embodiment, the content of ginsenoside C2 in the extract is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0019] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1-100:100-1, 1-50:50-1, 1-20:20-1, 1-10:10-1, 1-9:9-1, 1-8:8-1, 1-7:7-1, 1-6:6-1, 1-5:5-1, 1-4:4-1, 1-3:3-1, or 1-2:2-1.

[0020] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:2-1:10, for example, 5:2, 5:5, 2:5, 1:5, or 1:10.

[0021] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.0), or 5:(1.2-3.8), or 5:(1.5-3.5).

[0022] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1.8-3.2), or 5:(1.9-3.1), or 5:(2-3).

[0023] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 2:(2-20.0), or 2:(5-20), or 2:(10-20).

[0024] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 2:(5-20), or 2:(19.9-20.1), or 1:10.

[0025] In another preferred embodiment, the first active ingredient is salvianolic acid B, and the second active ingredient is ginsenoside Rg1.

[0026] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as freeze-dried preparations), gaseous dosage forms, and semisolid dosage forms.

[0027] In another preferred embodiment, the hemorrhagic disease is selected from: hemorrhagic diseases of various tissues and organs.

[0028] In another preferred embodiment, the tissue organ is selected from the group consisting of brain, heart, liver, spleen, lung, kidney, stomach, intestine, artery, vein, muscle, nerve, or a combination thereof.

[0029] In another preferred embodiment, the hemorrhagic disease is a cerebral hemorrhagic disease.

[0030] In another preferred embodiment, the cerebral hemorrhagic disease is selected from the following group: hemorrhagic stroke, brain trauma, intracranial aneurysm rupture, cerebral vascular malformation bleeding, intracranial malignant tumor-induced bleeding, post-infarction bleeding, bleeding after anticoagulation or thrombolytic therapy, cerebral vascular rupture bleeding, traumatic cerebral hemorrhage, hyperglycemia-induced cerebral hemorrhage, or a combination thereof.

[0031] In another preferred embodiment, the hemorrhagic stroke is caused by hemorrhagic transformation.

[0032] In another preferred embodiment, the cerebral hemorrhagic disease includes drug-induced cerebral hemorrhage, preferably cerebral hemorrhage caused by thrombolytic drugs, and more preferably cerebral hemorrhage caused by alteplase.

[0033] In another preferred embodiment, the drug is used for a purpose selected from the group consisting of:

[0034] a) Reduce cerebral hematoma;

[0035] b) Improve neurobehavioral function in animals with cerebral hemorrhage;

[0036] c) Protecting the cerebral cortex and striatum neurons in animals with cerebral hemorrhage;

[0037] d) Improve the survival rate of animals with cerebral hemorrhage.

[0038] In another preferred embodiment, the dosage form is selected from the following groups: injection (such as injection solution or powder injection), oral preparation (such as capsule, tablet, pill, powder, granule, syrup, oral solution or tincture), sublingual preparation, respiratory tract administration preparation, skin administration preparation, mucosal administration preparation, more preferably, the dosage form is an injection.

[0039] In a second aspect of the present invention, there is provided a use of a pharmaceutical composition for preparing a medicament for treating, alleviating and / or preventing a hemorrhagic disease, the pharmaceutical composition comprising:

[0040] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid, or a combination thereof;

[0041] (b) a second active ingredient selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof; and

[0042] (c) a pharmaceutically acceptable carrier.

[0043] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1-100:100-1, 1-50:50-1, 1-20:20-1, 1-10:10-1, 1-9:9-1, 1-8:8-1, 1-7:7-1, 1-6:6-1, 1-5:5-1, 1-4:4-1, 1-3:3-1, or 1-2:2-1.

[0044] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:2-1:10, for example, 5:2, 5:5, 2:5, 1:5, or 1:10.

[0045] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-4.0), or 5:(1.2-3.8), or 5:(1.5-3.5).

[0046] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 5:(1.8-3.2), or 5:(1.9-3.1), or 5:(2-3).

[0047] In another preferred embodiment, the salvianolic acid is salvianolic acid B, and the ginsenoside is ginsenoside Rg1.

[0048] In another preferred embodiment, the first active ingredient is salvianolic acid B, and the second active ingredient is ginsenoside Rg1.

[0049] In a third aspect of the present invention, there is provided a use of an active ingredient for preparing a medicament for treating, alleviating and / or preventing a hemorrhagic disease, wherein the active ingredient is selected from the group consisting of:

[0050] (1) salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, or its derivatives, and extracts containing salvianolic acid; and

[0051] (2) Ginsenosides, their stereoisomers, their crystal forms, their pharmaceutically acceptable salts or esters, or their derivatives, and extracts containing ginsenosides.

[0052] In a fourth aspect of the present invention, a method for treating, alleviating and / or preventing a bleeding disorder in a subject in need thereof is provided, the method comprising administering to a subject in need thereof a drug comprising the following active ingredients:

[0053] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid, or a combination thereof; and

[0054] (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof.

[0055] In another preferred embodiment, the hemorrhagic disease is selected from: hemorrhagic diseases of various tissues and organs.

[0056] In another preferred embodiment, the tissue organ is selected from the group consisting of brain, heart, liver, spleen, lung, kidney, stomach, intestine, artery, vein, muscle, nerve, or a combination thereof.

[0057] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows representative images of cerebral hemorrhage (A) and the quantitative results of SalB / Rg1 reducing hemorrhage compared with the hemorrhage model group (B).

[0059] Figure 2 shows the synergistic effect of SalB / Rg1 in preventing and treating collagenase-induced intracerebral hemorrhage. (A) Representative images of hemorrhage; (B) Quantitative results of the reduction of hemorrhage by SalB / Rg1 compared with the hemorrhage model group; (C) Percentage of hemorrhage inhibition in each group compared with the model group; (D) Results of synergistic effect evaluated by the Bliss assay. Compared with the model group, #p < 0.05; compared with the Rg1 group, &p < 0.05, &&p < 0.01.

[0060] Figure 3 shows the experimental scheme for SalB / Rg1 to inhibit alteplase (tPA)-induced cerebral hemorrhage and prolong the tPA thrombolytic window. (A) Alteplase (tPA)-induced hemorrhage model 4.5 hours after infarction and reperfusion. (B) Alteplase (tPA)-induced hemorrhage model 6 hours after infarction and reperfusion. (C) Alteplase (tPA)-induced hemorrhage model 9 hours after infarction and reperfusion.

[0061] Figure 4 shows that SalB / Rg1 reduces the bleeding score in tPA-induced intracerebral hemorrhage. (A) Representative images of hemorrhage. (B) Hemorrhage score. ***P < 0.001 compared with the sham group; #P < 0.05, ##P < 0.01 compared with the hemorrhage model group; n = 10.

[0062] Figure 5 shows behavioral assessments using the Longa score, Bederson score, mNSS score, and hanging test. Compared with the sham operation group, ***P < 0.001; compared with the hemorrhage model group, #P < 0.05, ##P < 0.01.

[0063] Figure 6 shows: (A) Representative images of Nissl staining of SalB / Rg1-protected neurons after tPA administration; (B) Quantification of Nissl bodies in the cortex; (C) Quantification of Nissl bodies in the striatum. ***P < 0.001 compared with the sham-operated group; #P < 0.05, ##P < 0.01 compared with the hemorrhage model group. Scale bar = 250 μm, n = 6.

[0064] Figure 7 shows that SalB / Rg1 improves the survival rate of animals with tPA-induced cerebral hemorrhage. Compared with the sham operation group, ***P<0.01, **P<0.001; compared with the hemorrhage model group, ###P<0.001.

[0065] Figure 8 shows representative SWI images of brain tissues and quantitative results of hemorrhage scores in mice in which SalB / Rg1 reduced hemorrhage scores. n=6-7.

[0066] Figure 9 shows that SalB and Rg1 have a synergistic effect in reducing tPA-induced bleeding. (A) Representative images of bleeding. (B) Bleeding score. (C) Bliss model assessment of synergistic effect. ***P < 0.001 compared with the sham group; #P < 0.05, ##P < 0.01 compared with the bleeding model group; &&P < 0.01 compared with the Rg1 group; n = 10.

[0067] Figure 10 shows representative images (top) and quantitative data (bottom) of the hemorrhage score for evaluating the therapeutic effect of SalB / Rg1 in an animal model of hemorrhagic stroke. ***P<0.001 compared with the sham group; &P<0.05 compared with the hemorrhage model group.

[0068] Figure 11 shows that SalB / Rg1 improves the neurological behavior of animals with hyperglycemia-induced bleeding. Compared with the sham operation group, ***P<0.001; compared with the bleeding model group, &P<0.05. DETAILED DESCRIPTION

[0069] After extensive and in-depth research, screening, and testing, the present inventors have developed a pharmaceutical composition containing salvianolic acid B and ginsenoside Rg1 as active ingredients for the treatment of hemorrhagic diseases. Animal experiments have demonstrated that the composition can effectively reduce the amount of intracerebral hemorrhage in mice with hemorrhagic stroke and improve neurological behavior in animals with cerebral hemorrhage. The pharmaceutical composition exhibits superior therapeutic effects on hemorrhagic diseases, particularly hemorrhagic stroke, and can be used to treat hemorrhagic diseases in a variety of tissues and organs. This is the basis for the completion of the present invention.

[0070] the term

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0072] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0073] As used herein, the term "stereoisomer" is intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformers)): for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the active ingredients of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformers) are all within the scope of the present invention.

[0074] The active ingredient of the present invention may be in an amorphous form, a crystalline form or a mixture thereof.

[0075] As used herein, "pharmaceutically acceptable salt" refers to a salt formed between the active ingredient compound of the present invention and an acid or base that is suitable for pharmaceutical use. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred salt type is a salt formed between the active ingredient compound of the present invention and an acid. Suitable acids for salt formation include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. A preferred salt type is a salt formed between the active ingredient compound of the present invention and a base. Suitable bases for salt formation include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, magnesium carbonate, calcium carbonate, sodium carbonate, sodium bicarbonate, and sodium phosphate; and organic bases such as ammonia, triethylamine, and diethylamine. Another preferred salt is a salt formed between the active ingredient of the present invention and a metal ion, including but not limited to magnesium salt, sodium salt, calcium salt, potassium salt and the like.

[0076] As used herein, "pharmaceutically acceptable ester" refers to an ester suitable for use as a drug formed by a compound of the active ingredient of the present invention and an acid or alcohol. A preferred type of ester is an ester formed by one or more hydroxyl groups of the active ingredient of the present invention and an acid. Acids suitable for forming esters include but are not limited to: phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, etc.; another preferred type of ester is an ester formed by the carboxyl group of the active ingredient of the present invention and an alcohol. Alcohols suitable for forming esters include but are not limited to: C1-C6 alkyl-OH, such as methanol, ethanol, n-propanol, isopropanol, etc.

[0077] The "prevention" and "treatment" described herein include delaying and stopping the progression of the disease, or eliminating the disease, and do not require 100% inhibition, elimination, and reversal. In some embodiments, the composition or pharmaceutical composition of the present invention prevents, alleviates, inhibits, and / or reverses cerebral hemorrhage damage by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the level observed in the absence of the composition or pharmaceutical composition of the present invention.

[0078] In the present invention, the term "salvianolic acid" used refers to a compound selected from the following group: salvianolic acid B, salvianolic acid A, salvianolic acid C, isosalC, salvianolic acid D, salvianolic acid E, salvianolic acid F, salvianolic acid G, salvianolic acid H, salvianolic acid I, salvianolic acid J, salvianolic acid K, salvianolic acid L, salvianolic acid M, salvianolic acid N, salvianolic acid T, salvianolic acid U, salvianolic acid Y, caffeic acid, danshensu, protocatechuic acid, protocatechuic aldehyde, ferulic acid, isoferulic acid, Salvinal, Ailanthoidol, rosmarinic acid, lithospermic acid, danshen glycoside, catechol, dimethyllithospermic acid B, tanshinone I, dihydrotanshinone I, tanshinone diol-A, tanshinone IIA, cryptotanshinone, tanshinone, dehydrotanshinone, isotanshinone I, tanshinone A, or a combination thereof.

[0079] In the present invention, the term "ginsenoside" used refers to a compound selected from the group consisting of ginsenoside Rg1, ginsenoside Re, ginsenoside Rg2, ginsenoside Rh1, ginsenoside F1, notoginsenoside R1, notoginsenoside R2, notoginsenoside R3, notoginsenoside R6, notoginsenoside M, notoginsenoside N, notoginsenoside Rt, notoginsenoside T3, notoginsenoside FP1, notoginsenoside RW1, notoginsenoside Saponin Fh7, Camelliaside D, Camelliaside U, 20-glucose ginsenoside Rf, malonyl ginsenoside Rg1, bamboo saponin L5, safflower ginsenoside R1, pseudo-ginsenoside RT3, saponin 10, saponin 1, saponin 2, saponin 3, ginsenoside Ra3, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Rg3, Malonoside ginsenoside Rb1, Malonyl ginsenoside Rd, 6'-O-acetyl ginsenoside Rg3, Ginsenoside F2, Ginsenoside CK, Ginsenoside Mc, 20(S)-ginsenoside Rg3, 20(R)-ginsenoside Rg3, 20(R)-ginsenoside Rh2, 20(S)-ginsenoside Rh2, Notoginsenoside R4, Notoginsenoside K, Gynostemma pentaphyllum saponin XVII ... Gynostemma saponin IX, Gynostemma saponin XV, Gynostemma saponin VIII, Notoginsenoside Fa, Notoginsenoside Fc, Notoginsenoside Fc, Notoginsenoside D, Notoginsenoside L, Notoginsenoside L*, Notoginsenoside O, Notoginsenoside P, Notoginsenoside Q, Notoginsenoside S, Notoginsenoside T, Notoginsenoside FP2, Notoginsenoside Ftl, Notoginsenoside ST-4, Notoginsenoside FZ, Notoginsenoside Fh1, or a combination thereof.

[0080] As used in the present invention, the term "SalB" and "salvianolic acid B" can be used interchangeably; the term "Rg1" and "ginsenoside Rg1" can be used interchangeably.

[0081] First active ingredient

[0082] The present invention provides a pharmaceutical composition that can be used to prepare a pharmaceutical composition for treating, alleviating and / or preventing hemorrhagic diseases, comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is selected from the following group: salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid, or a combination thereof.

[0083] In a preferred embodiment of the present invention, the first active ingredient is selected from the group consisting of salvianolic acid B, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, extracts containing salvianolic acid B, or combinations thereof. The salvianolic acid B is represented by the following formula (I):

[0084] In another preferred embodiment, the first active ingredient includes a purified product of salvianolic acid B or a pharmaceutically acceptable salt thereof.

[0085] In another preferred embodiment, the purity of the purified product, calculated as salvianolic acid B, is ≥90%, preferably ≥95%, more preferably ≥98% or 99%, based on the total weight of the purified product.

[0086] In another preferred embodiment, the first active ingredient comprises a salvianolic acid extract having a salvianolic acid B content C1 ≥ 30 wt %, wherein the content C1 is calculated based on the weight of salvianolic acid.

[0087] In another preferred embodiment, the content C1 of salvianolic acid B in the extract is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0088] Second active ingredient

[0089] In the present invention, the second active ingredient is selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof.

[0090] In a preferred embodiment of the present invention, the second active ingredient is selected from the group consisting of ginsenoside Rg1, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, extracts containing ginsenoside Rg1, or combinations thereof. The ginsenoside Rg1 is represented by the following formula (II):

[0091] In another preferred embodiment, the second active ingredient comprises a total saponin extract having a ginsenoside Rg1 content C2 ≥ 30 wt%, wherein the content C2 is calculated based on the weight of the total saponins.

[0092] In another preferred embodiment, the content C2 of ginsenoside Rg1 in the extract is ≥70%, preferably ≥80%, more preferably ≥90% or ≥95%, based on the dry weight of the extract.

[0093] Pharmaceutical compositions, active ingredient combinations, and kits

[0094] The present invention provides a use of a pharmaceutical composition, comprising:

[0095] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts, an extract containing salvianolic acid, or a combination thereof;

[0096] (b) a second active ingredient selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts thereof, extracts containing ginsenosides, or combinations thereof; and (c) a pharmaceutically acceptable carrier.

[0097] The present invention also provides a use of an active ingredient combination, wherein the active ingredient combination comprises:

[0098] (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts, an extract containing salvianolic acid, or a combination thereof;

[0099] (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts thereof, extracts containing ginsenosides, or combinations thereof.

[0100] In another preferred embodiment, the combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.

[0101] In a preferred embodiment of the present invention, in the pharmaceutical composition and active ingredient combination described above, the weight ratio of the first active ingredient to the second active ingredient is 1-100:100-1, 1-50:50-1, 1-20:20-1, 1-10:10-1, 1-9:9-1, 1-8:8-1, 1-7:7-1, 1-6:6-1, 1-5:5-1, 1-4:4-1, 1-3:3-1, or 1-2:2-1.

[0102] Preferably, the weight ratio of the first active ingredient to the second active ingredient is 5:(1-5.0), or 5:(1.2-3.8), or 5:(1.5-3.5).

[0103] More preferably, the weight ratio of the first active ingredient to the second active ingredient is 5:(1.8-3.2), or 5:(1.9-3.1), or 5:(2-3).

[0104] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 2:(2-20.0), or 2:(5-20), or 2:(10-20).

[0105] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 2:(5-20), or 2:(19.9-20.1), or 1:10.

[0106] In a preferred embodiment of the present invention, the salvianolic acid is salvianolic acid B, and the ginsenoside is ginsenoside Rg1.

[0107] In a further preferred embodiment of the present invention, the first active ingredient is salvianolic acid B, and the second active ingredient is ginsenoside Rg1.

[0108] The dosage form of the pharmaceutical composition is selected from the following groups: liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as freeze-dried preparations).

[0109] In another preferred embodiment, the dosage form is selected from the following group: injection (such as injection solution or powder injection), oral preparation (such as capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), more preferably, preferably, the dosage form is injection.

[0110] In the pharmaceutical composition of the present invention, the first active ingredient and the second active ingredient can be prepared into preparations separately or mixed together into a preparation.

[0111] The pharmaceutical composition of the present invention comprises a first active ingredient and / or a second active ingredient within a safe and effective amount. "Safe and effective amount" means an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the active ingredient of the present invention per dose, more preferably 10-500 mg per dose. Preferably, "one dose" refers to a capsule, tablet, injection, etc.

[0112] In the present invention, "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the first active ingredient and / or the second active ingredient without significantly reducing the efficacy of the first active ingredient and / or the second active ingredient. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0113] In the active ingredient combination, the first active ingredient and the second active ingredient may be independent of each other, or they may be combined together to form an active ingredient composition.

[0114] The pharmaceutical composition and active ingredient combination of the present invention can be prepared using conventional methods and equipment.

[0115] Use and application method

[0116] The present invention provides a use of the pharmaceutical composition, active ingredient, combination of active ingredients or kit described herein for preparing a medicament or kit for preventing and / or treating a hemorrhagic disease, preferably preventing and / or treating hemorrhagic stroke.

[0117] In the present invention, the hemorrhagic disease is selected from hemorrhagic diseases of various tissues and organs, including but not limited to brain, heart, liver, spleen, lung, kidney, stomach, intestine, artery, vein, muscle, nerve, or a combination thereof.

[0118] In the present invention, hemorrhagic stroke refers to intracerebral hemorrhage caused by lesions, necrosis, or rupture of the cerebral blood vessels. Clinical manifestations include headache, vomiting, impaired consciousness, hemiplegia, sensory impairment, or a combination thereof. The hemorrhagic stroke may be caused by hemorrhagic transformation.

[0119] In the present invention, the prevention and / or treatment of hemorrhagic diseases, the prevention and / or treatment of hemorrhagic stroke, etc., include preventive applications and also include post-treatment ameliorative applications. For example, for hemorrhagic stroke, the pharmaceutical composition or active ingredient combination of the present invention may be administered before, during, and / or after the hemorrhagic stroke to protect, repair, or improve or enhance the function of tissues and organs after the hemorrhagic stroke.

[0120] Preferably, the pharmaceutical composition or combination of active ingredients used for the purpose of the present invention only contains the first active ingredient salvianolic acid B and the second active ingredient ginsenoside Rg1.

[0121] In the pharmaceutical composition or combination of active ingredients 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, antioxidants, tissue dehydrating drugs, neuroprotectants, etc.

[0122] In the pharmaceutical composition, combination of active ingredients or kit of the present invention, the first active ingredient and the second active ingredient may be administered simultaneously, separately or sequentially.

[0123] In the pharmaceutical composition, combination of active ingredients or kit of the present invention, the administration mode of the first active ingredient and the second active ingredient is not particularly limited. Representative administration modes include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0124] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0125] Solid dosage forms such as tablets, drupes, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain portion of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If desired, the active ingredient can also be microencapsulated with one or more of the above-mentioned excipients.

[0126] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0127] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0128] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0129] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0130] 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.

[0131] In the pharmaceutical compositions, combinations of active ingredients, or kits of the present invention, the therapeutically effective dose of the active ingredient, calculated based on the total amount of salvianolic acid B and ginsenoside Rg1, generally ranges from about 1 to 2000 mg / day, about 10 to 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 administered in one or more doses. However, it should be understood that the specific dose of the active ingredient of the present invention for any particular patient will depend on a variety of factors, such as the patient's age, sex, weight, general health, diet, individual response, timing of administration, severity of the disease to be treated, dosage form, mode of application, and concomitant medications. The therapeutically effective amount for a given situation can be determined by routine experimentation and is within the ability and judgment of the clinician or physician. In any case, the active ingredient will be administered in multiple doses based on the patient's individual circumstances and in a manner that allows for the delivery of a therapeutically effective amount.

[0132] The main advantages of the present invention include:

[0133] 1. Compared with the prior art, the present invention discovered the use of the salvianolic acid B / ginsenoside Rg1 pharmaceutical composition in the treatment of hemorrhagic diseases.

[0134] 2. The present invention discovered that the salvianolic acid B / ginsenoside Rg1 pharmaceutical composition has a synergistic effect in the treatment of hemorrhagic stroke, and can significantly reduce the amount of intracerebral hemorrhage.

[0135] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0136] Example 1 Effect of different ratios of combinations of salvianolic acid B and ginsenoside Rg1 (SalB / Rg1) on inhibiting cerebral hemorrhage

[0137] 1. Experimental Materials

[0138] 1.1 Experimental Animals

[0139] Male SD rats weighing 260–300 g were purchased from Beijing Huafukang Biotechnology Co., Ltd. Rats were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature of 22 ± 2°C, a 12-h light-dark cycle, and free access to food and water.

[0140] 1.2 Main reagents and drugs

[0141] Table 1 Materials and reagents

[0142] 1.3 Main instruments

[0143] Table 2 Experimental instruments

[0144] 2. Experimental Methods

[0145] 2.1 Experimental Grouping

[0146] Forty-eight male SD rats were randomly divided into eight groups according to their body weight, with six rats in each group. The collagenase concentration used for modeling was 0.5 U / μl.

[0147] Salvianolic acid B is abbreviated as SalB, and ginsenoside Rg1 is abbreviated as Rg1. Mice in the bleeding model control, (10:1) SalB / Rg1, (5:1) SalB / Rg1, (5:2) SalB / Rg1, (5:5) SalB / Rg1, (2:5) SalB / Rg1, (1:5) SalB / Rg1, and (1:10) SalB / Rg1 groups were administered 5 ml / kg of the drug in 0.9% sodium chloride injection. This study was conducted in a double-blind manner. All EP tubes and pipette tips used in the preparation of the drug formulations were sterilized in a vertical pressure steam autoclave.

[0148] 2.2 Construction of Collagenase VII-S-Induced Intracerebral Hemorrhage Animals

[0149] Surgical supplies were sterilized in an autoclave at 120°C for 30 minutes before surgery. Rats anesthetized with Zolpidem were placed in a prone position on the operating table, and the scalp was disinfected with alcohol and iodine. A longitudinal incision approximately 2 cm long was made at the midpoint of the line connecting the two ears. Forceps were used to dissect the subcutaneous tissue to expose the skull. 3% H₂O₂ was used to disrupt the periosteum, exposing the anterior and posterior bregma. The stereotaxic apparatus was adjusted to align the anterior and posterior bregma in the same plane. Using a 0.5 mm diameter dental drill, a small hole approximately 1.0 mm in diameter was drilled 0.5 mm anterior to the anterior bregma and 3.5 mm to the right of the midline. Collagenase VII (0.5 U / μl) and 1 μl were slowly injected into the striatum via a microsyringe. The hole was sealed with bone wax, the skin was sutured, and the rats were given ample food and water. Immediately after collagenase injection, 5 mg / kg of SalB / Rg1 was injected into the tail vein.

[0150] 2.3 Hematoma volume quantification

[0151] After euthanasia, the rats were sliced ​​into seven consecutive 2 mm thick slices. Image Pro Plus (version 6.0) software was used to quantify the hematoma area in the red area of ​​each slice, and the hematoma volume was calculated based on the hematoma area. The hematoma volume calculation formula is V = (∑(A1+A2) / 2)*D (mm 3 ). V is the hematoma volume of each brain slice (mm 3 ); A1 and A2 are the red hemorrhage areas on the rostral and caudal sides of the brain slice (mm 2 ); D is slice thickness (mm); the total brain hematoma volume is the sum of the hematoma volumes of the seven brain slices.

[0152] Considering the irregularity of the left and right hemisphere volumes caused by hematoma, the hematoma volume ratio was corrected using the brain volume of the hemisphere contralateral to the hemorrhage. Hematoma volume (%) = (hematoma volume / brain volume of the hemisphere contralateral to the hemorrhage) × 100%.

[0153] 2.4 Data Statistics

[0154] Data Data were expressed as % and processed using Graph Pad Prism 9.0. For data that conformed to a normal distribution, one-way ANOVA was used for statistical analysis, and Tukey's test was used for inter-group differences. For data that did not conform to a normal distribution, the Kruskal-Wallis test was used. A significant difference between the groups was considered when p < 0.05.

[0155] 3. Results

[0156] The collagenase VII-induced hemorrhage animal model is a classic model for studying cerebral hemorrhage. Because collagenase is injected into the striatum, Figure 1A shows the presence of distinct hemorrhage foci in the striatum. By quantifying the number of hemorrhage foci and comparing the inhibitory effects of different ratios of salvianolic acid B / Rg1 combinations on cerebral hemorrhage, the hematoma volume inhibition rate was calculated using the following formula: (hematoma volume in the hemorrhage model group - hematoma volume in the salvianolic acid B / Rg1 group) / hematoma volume in the hemorrhage model group. The hematoma inhibition rate of (10:1) SalB / Rg1 was 0.30±0.20%; the hematoma inhibition rate of (5:1) SalB / Rg1 was -10.05±0.34%; the hematoma inhibition rate of (5:2) SalB / Rg1 was 13.31±0.13%; the hematoma inhibition rate of (5:5) SalB / Rg1 was 11.92±0.09%; the hematoma inhibition rate of (2:5) SalB / Rg1 was 21.33±0.27%; the hematoma inhibition rate of (1:5) SalB / Rg1 was 13.14±0.28%; the hematoma inhibition rate of (1:10) SalB / Rg1 was 23.95±0.24%.

[0157] Example 2 Salvianolic acid B and Rg1 have a synergistic effect in reducing collagenase-induced cerebral hemorrhage

[0158] 1. Experimental Materials

[0159] 1.1 Experimental Animals

[0160] Male Sprague-Dawley rats weighing 260–300 g were purchased from Beijing Huafukang Biotechnology Co., Ltd. Rats were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, at a constant temperature of 22 ± 2°C, with a 12-h light-dark cycle, and free access to food and water.

[0161] 1.2 Main reagents, drugs and instruments are the same as above

[0162] 2. Experimental Methods

[0163] 2.1 Preparation of collagenase VII-S-induced cerebral hemorrhage animal model

[0164] 2.2 Experimental Grouping

[0165] As mentioned above, different ratios of salvianolic acid B and Rg1 have different inhibitory effects on cerebral hematoma, and the inhibitory effect of salvianolic acid B and Rg1 at a ratio of 1:10 is more prominent. This example investigates the synergistic effect of the salvianolic acid B / Rg1 combination and uses the Bliss independent statistical model to evaluate the synergistic effect.

[0166] Male Sprague-Dawley rats were randomly divided into four groups: a bleeding model group, a SalB group, an Rg1 group, and a SalB / Rg1 combination group, with 10 animals in each group. The bleeding model group received normal saline; the SalB group received 0.45 mg / kg of salvianolic acid B; the Rg1 group received 4.55 mg / kg of ginsenoside Rg1; and the SalB / Rg1 group received 5 mg / kg of salvianolic acid B / Rg1 (with salvianolic acid B and Rg1 prepared at a mass ratio of 1:10). Following collagenase treatment, the rats were injected into the tail vein with normal saline, salvianolic acid B, Rg1, or the SalB / Rg1 combination.

[0167] 2.3 Hematoma volume quantification is the same as above

[0168] 2.4 Bliss Independent Statistical Model

[0169] In order to explore whether SalB and Rg1 have a synergistic effect in treating cerebral hemorrhage, the Bliss independent statistical model was used to verify the inhibition of hematoma volume. First, the following calculation was performed: 模型 、S SalB 、S Rg1 and S SalB / Rg1 Represent the hematoma volumes of the bleeding model, SalB, Rg1 and SalB / Rg1 groups, respectively. SalB , I Rg1 and I SalB / Rg1 Respectively represent the ratio of hematoma volume of SalB, Rg1 and SalB / Rg1 groups to the hemorrhage model group. The I value is calculated as shown in formulas 1, 2 and 3. SalB =(S SalB / S 模型 )×100%(Formula 1) I Rg1 =(S Rg1 / S 模型 )×100%(Formula 2) I SalB / Rg1 =(S SalB / Rg1 / S 模型 )×100%(Formula 3)

[0170] The synergistic effect of SalB and Rg1 in treating cerebral hemorrhage was evaluated using the Bliss independence statistical model. Assume that Independence is the theoretical value of synergy calculated from SalB and Rg1. According to the Bliss independence statistical model, Independence is calculated as shown in Formula 4. Independence = I SalB ×I Rg1 (Formula 4)

[0171] In order to simplify the statistical test of synergistic effect, after the Kolmogorov-Smirnov test, the data were transformed on the logarithmic scale to obtain the equation shown in Formula 5. SalB +InS Rg1 -InS 模型 -InS SalB / Rg1 >0(Formula 5)

[0172] According to the statistical method, one-way analysis of variance and Tukey's multiple comparison test were used to compare the differences between the two. If P < 0.05, SalB and Rg1 were considered to have a synergistic effect.

[0173] 2.5 Statistics

[0174] Statistical analysis of quantitative data in research Data were expressed as % and processed using Graph Pad Prism 9.0. For data that conformed to a normal distribution, one-way ANOVA was used for statistical analysis, and Tukey's test was used for inter-group differences. For data that did not conform to a normal distribution, the Kruskal-Wallis test was used. A significant difference between the groups was considered when p < 0.05.

[0175] 3. Experimental Results

[0176] Figure 2A shows representative images of each group undergoing collagenase-induced cerebral hemorrhage, with the hematoma site appearing dark red. Figure 2B shows the quantitative results of hematoma volume. In the hemorrhage model group, the hematoma volume ratio was 10.97±3.97%. Neither SalB (8.54±3.63%) nor Rg1 (11.03±2.38%) alone significantly reduced hematoma volume. However, 5mg / kg of SalB / Rg1 significantly reduced hematoma volume (6.92±2.36%, p<0.05), achieving statistically significant efficacy compared to Rg1 (p<0.05). Figure 2C shows the quantitative results of hematoma volume inhibition rate. 5mg / kg of SalB / Rg1 significantly increased the inhibition rate (36.95±0.22%) compared to Rg1, demonstrating superior efficacy (p<0.01).

[0177] The synergistic effect was evaluated using the Bliss independent statistical model. Based on the formula given above, the results showed that the Sal / Rg1 group had a statistically significant difference from the independent group (p < 0.05), indicating that the combination of SalB and Rg1 has a very significant synergistic effect in reducing cerebral hematoma.

[0178] Example 3: Salvianolic acid B combined with ginsenoside Rg1 inhibits cerebral hemorrhage induced by alteplase (tPA)

[0179] 1 Experimental Materials

[0180] Male C57BL / 6 mice (26 ± 3 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature (22 ± 2°C) and a 12-h light and dark cycle, and had free access to food and water.

[0181] 2 The drugs, reagents and instruments used in the experiment are mentioned above.

[0182] The special reagents used in this example are as follows:

[0183] Table 3 Experimental drugs and reagents information

[0184] 3. Experimental Methods

[0185] 3.1 Alteplase (tPA)-induced intracerebral hemorrhage model in mice

[0186] All mice underwent behavioral assessments before the experiment to ensure normal neurobehavioral function. Mice were anesthetized with an intraperitoneal injection of a combination of Zolpidem and Xylazine. The neck skin was prepared and disinfected with iodine. The skin was incised along the midline of the neck to expose the deep cervical fascia and anterior tracheal muscles. The common carotid artery was isolated and threaded with a slipknot to occlude blood flow. The common carotid artery was dissected upwards, and a knot was tied at the proximal end of the common carotid artery. A suture was prepared at the distal end but not yet tightened. A small incision was made at the proximal end of the common carotid artery with microscissors. A silicone suture was gently inserted through the incision, occluding the origin of the main trunk of the middle cerebral artery. The slipknot prepared at the distal end of the common carotid artery was tightened to secure the suture, and the skin was sutured. After the middle cerebral artery was temporarily occluded for the prescribed period, the suture was opened, the suture was removed, and the knot at the distal end of the common carotid artery was tightened for reperfusion. Three experimental studies were conducted based on the timing of tPA administration. The mice underwent middle cerebral artery occlusion for 4.5, 6, and 9 hours, followed by suture removal to achieve reperfusion and immediate administration of tPA to induce a bleeding model. Each experiment included a sham operation group, a model group, and a salvianolic acid B / Rg1 group (5 mg / kg salvianolic acid B / Rg1). The salvianolic acid B / Rg1 formulation was formulated with a 5:2 ratio of salvianolic acid B to Rg1.

[0187] In this study, both the bleeding model group and the Salvianolic Acid B / Rg1 group were administered 10 mg / kg tPA, while the Sham group received normal saline at the corresponding time points. SalB / Rg1 was formulated with a mass ratio of Salvianolic Acid B to Rg1 of 5:2. The SalB / Rg1 treatment timelines are shown in Figure 3, with a total of three administrations. The experimental endpoint was 48 hours after reperfusion, and the experimental design for each time window is shown in Figure 3. To ensure objectivity, the study was conducted using a double-blind approach, with the blind removed until the experiment was completed and concluded.

[0188] 3.2 Behavioral evaluation

[0189] 3.2.1. Longa score

[0190] The Longa score is assessed on a scale of 0 to 4:

[0191] 0 points, normal, no neurological deficit;

[0192] 1 point: the right limb (paralysis test) cannot be fully extended, with mild neurological deficit;

[0193] 2 points: turning toward the paralyzed side (right side) while walking, moderate neurological deficit;

[0194] 3 points: falling toward the paralysis side (right side) when walking, severe neurological deficit;

[0195] 4 points: unable to walk spontaneously and has lost consciousness.

[0196] 3.2.2 Bederson score

[0197] The Bederson score is scored on a 5-point scale:

[0198] 0 points, no defects;

[0199] 1 point, loss of forelimb flexion;

[0200] 2 points, loss of forelimb flexion and low resistance to lateral pushing;

[0201] 3 points, one-way circling;

[0202] 4 points, longitudinal rotation or epileptic activity;

[0203] 5 points, no exercise.

[0204] 3.2.3 Suspension test

[0205] Place the mouse on a conventional cage lid and gently shake the lid to encourage the mouse to grip the lid. Then, quickly flip the lid over and record the longest latency for the hind limbs to release the lid. 90 seconds is the cutoff value; if the latency exceeds 90 seconds, it is counted as 90 seconds; if the latency is less than 90 seconds, the actual time is recorded. Repeat each experiment three times, and the best result is recorded.

[0206] 3.2.4 mNSS (Modified Neurological Severity Score) score

[0207] Table 4 mNSS score table

[0208] 3.3 Bleeding score

[0209] A 2% Evans blue dye solution was prepared with normal saline for injection and injected into the tail vein at a dose of 4 mg / kg. Two hours later, the heart was perfused with 10 ml of 0.9% normal saline. Brain tissue was removed and photographed using an OLYMPUS-BX51 microscope. The degree of hemorrhage was scored using the HT scale: 0 = no blood; 1 = H1, small petechiae at the edge of the infarct; 2 = H1 II, numerous confluent petechiae within the infarct area but no mass effect; 3 = PH I, blood clot not exceeding 30% of the infarct area with mild mass effect; 4 = PH II, dense blood clot exceeding 30% of the infarct volume with significant mass effect. The scores for each section were summed, and the total score was used as the evaluation index.

[0210] 3.4 Nissl staining

[0211] After fixation in 4% neutral formaldehyde for 48 hours, the brain tissue was removed and placed in an embedding frame. After rinsing with running water for 3-5 hours, the tissue was placed in an automatic dehydrator and dehydrated according to the programmed program. After dehydration, the tissue was embedded in paraffin. The embedded tissue was cut into 4μm thick paraffin sections. The sections were heated at 68°C for 60 minutes in a microtome oven, then removed and deparaffinized in xylene for 15 minutes, followed by anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 75% ethanol for 5 minutes. The sections were then rinsed with running water for 5 minutes. The sections were then stained with Nissl stain for 10 minutes and washed twice with distilled water.

[0212] 3.5 Data Statistical Methods

[0213] Data For normally distributed variables, analysis of variance (ANOVA) with Bonferroni post hoc test was used to assess differences between groups. For comparisons between only two groups, an unpaired, two-tailed Student's t test was used. Differences between ratios between two groups were examined using the chi-squared test. P < 0.05 was considered statistically significant.

[0214] 4 Results

[0215] 4.1 SalB / Rg1 reduces alteplase (tPA)-induced bleeding

[0216] Representative images of brain tissue hemorrhage are shown in Figure 4A. As the time of tPA administration was delayed, the bleeding score in the hemorrhage model group also increased, indicating that the bleeding became more severe with delayed tPA administration. Figure 4B shows the bleeding score. It can be seen that SalB / Rg1 intervention significantly reduced the bleeding score, regardless of whether tPA was administered 4.5, 6, or 9 hours after ischemia (P < 0.01). When tPA was administered 4.5 hours after ischemia, the bleeding score in the hemorrhage model group was 3.75 ± 0.71. After SalB / Rg1 administration, the bleeding score decreased to 2.20 ± 1.93, which was significantly different from the bleeding model group (P < 0.01). After tPA was administered 6 hours after ischemia, the bleeding score in the hemorrhage model group increased to 4.75 ± 1.98. After SalB / Rg1 administration, the bleeding score decreased to 1.70 ± 2.34, which was significantly different from the bleeding model group (P < 0.01). Even in the experiment of administering tPA 9 hours after cerebral ischemia, there was still a very significant difference between the SalB / Rg1 group and the hemorrhage model group (5.4±1.58 versus 3.6±1.43, P<0.01).

[0217] 4.2 SalB / Rg1 improves neurobehavior

[0218] As shown in Figure 5, the neurobehavioral scores of the mice after modeling showed extremely significant differences compared to the sham-operated group. In the experiment in which tPA was administered 4.5 hours after cerebral ischemia, the SalB / Rg1 group showed significant reductions in the three scores except the hanging test. In the experiment in which tPA was administered 4.5 hours after cerebral ischemia, the SalB / Rg1 group showed a significant reduction in the mNSS score and prolonged the hanging time (P<0.05). Although the Longa score and Bederson score did not decrease significantly, there was a trend towards a decrease in the scores. Neurobehavioral results indicate that SalB / Rg1 can improve the neurobehavioral function of animals with cerebral hemorrhage.

[0219] 4.3 SalB / Rg1 protects neurons in the cortex and striatum

[0220] Damage to neurons in the cortex and striatum can have a serious impact on the behavior of mice. Therefore, brain tissue sections were Nissl-stained, and the number of Nissl bodies was counted to determine whether SalB / Rg1 could protect neurons. The Nissl staining results are shown in Figure 6. In the Nissl-stained brain tissue images, the same locations in the cerebral cortex and striatum were selected for quantification in each image. In the 4.5-hour and 6-hour experiments, the number of Nissl-positive cells in the cortex and striatum after modeling was significantly decreased compared with the sham-operated group (P<0.001). However, the number of Nissl-positive cells in the SalB / Rg1 group was significantly increased compared with the hemorrhage model group (P<0.05). This result is consistent with the above-mentioned behavioral results, suggesting that SalB / Rg1 may improve the neurological behavior of mice by protecting neurons in the cerebral cortex and striatum.

[0221] Although SalB / Rg1 intervention did not improve the neurobehavioral scores in the tPA experiment 9 hours after cerebral ischemia, the number of positive cells in the cortex (2513.67±429.51 versus 1694.50±272.71, P<0.001) and striatum (2963.33±508.18 versus 2026.17± 481.92, P<0.001) of the SalB / Rg1 group was still significantly different from that of the hemorrhage model group in Nissl staining.

[0222] SalB / Rg1 improves survival after delayed alteplase administration

[0223] The survival curves for each group of mice are shown in Figure 7. Whether tPA was administered 4.5 hours, 6 hours, or 9 hours after cerebral ischemia, the survival rate of mice was significantly reduced after model establishment (P<0.01). However, administration of SalB / Rg1 improved the survival rate of mice. In the experiment where tPA was administered 4.5 hours after cerebral ischemia, the survival rate after SalB / Rg1 intervention was significantly improved compared to the hemorrhage model group (80% versus 20%, P<0.001). In the experiment where tPA was administered 6 hours after cerebral ischemia, the survival rate of the SalB / Rg1 group was 71.43% after 12 hours of reperfusion, while the survival rate of the hemorrhage model group was 44%. At 48 hours after reperfusion, the survival rate of the SalB / Rg1 group was 50%, while the survival rate of the hemorrhage model group was 36%. Similar results were obtained in the 9-hour experiment. Twelve hours after reperfusion, the survival rate of the SalB / Rg1 group was 90%, while that of the hemorrhage model group was 60%. At 48 hours after reperfusion, the survival rate of the SalB / Rg1 group was 70%, while that of the hemorrhage model group was 40%. These results indicate that SalB / Rg1 can prolong the survival of mice.

[0224] Example 4. MRI imaging evaluation of the effect of SalB / Rg1 in inhibiting cerebral hemorrhage

[0225] 1 Experimental Materials

[0226] 1.1 The purchase and breeding conditions of experimental animals are the same as above

[0227] 1.2 Experimental drugs and reagents are the same as before

[0228] 1.3 Experimental instruments and materials

[0229] Table 5 Experimental instruments and materials information

[0230] 2 Experimental methods

[0231] 2.1 Model preparation and treatment scheme are the same as Example 3, Scheme B in Figure 3.

[0232] 2.2 Small Animal MRI

[0233] MRI data acquisition was performed on an 11.7T BioSpec 117 / 16USR MRI system. A 2×2 phased array coil and a 72mm volume resonator were used for transmission and image acquisition, respectively. All animals were anesthetized with 1% to 1.5% isoflurane and maintained at 37°C to ensure proper immobilization during MRI scanning. Respiration was monitored and maintained at a rate of 95–110 breaths / minute.

[0234] T2-weighted imaging (T2WI): Whole-brain T2WI was acquired using a rapid acquisition and relaxation enhancement (RARE) sequence with a 90° flip angle, 2500 ms repetition time (TR), 6.5 ms echo time (TE), and a 0.063 × 0.063 × 0.5 mm 3 For T2AMP, a multislice multi-echo (MSME) sequence was used with a flip angle of 90°, a 2200 ms TR, and a 30 ms effective TE, ranging from 7.5 ms to 225 ms.

[0235] Susceptibility Weighted Imaging (SWI): For SWI, a flow-compensated Fast Low-Angle Shot (FLASH) sequence was used to obtain vascular abnormalities, with TR = 350 ms, TE = 12 ms, matrix size = 200 × 200, and spatial resolution = 0.08 × 0.08 mm. 2 , slice thickness = 0.8 mm.

[0236] Arterial spin labeling (ASL): ASL images were acquired using a drift-corrected steady pulsed imaging and labeling (dSPIL) sequence with a TE of 3.0 ms, a TR of 5000 ms, and 32 temporal repetitions. Image analysis and data processing: Motion correction and alignment were performed using SPMMouse, which is based on SPM8 (http: / / www.fil.ion.ucl.ac.uk / spm / software / spm8 / ). Data analysis was performed using MATLAB 2021b (MathWorks, Natick, USA). Other MRI modalities were rigidly coregistered with T2WI.

[0237] Signal intensity data were fitted to a single exponential decay curve using the least-squares algorithm, and T2-relaxation time was calculated for the entire brain based on the T2-MAP. Cerebral blood flow (CBF) images were processed using a custom program written in MATLAB. ROIs were manually created using ITK-SNAP software under different MRI modes. Cerebral blood flow evaluation methods: The brain tissue was divided into the infarct area, the infarct margin area, and the contralateral local area, and CBF values ​​were measured within each area.

[0238] 2.3 Data Statistical Methods

[0239] Data For normally distributed variables, one-way analysis of variance (ANOVA) with Bonferroni post hoc test was used to assess differences between groups. If only two groups were compared, an unpaired two-tailed Student's t test was used, and P < 0.05 was considered statistically significant.

[0240] 3 Results

[0241] 3.1 Magnetic resonance imaging shows that SalB / Rg1 reduces bleeding scores

[0242] Hemorrhage within the brain parenchyma is detected by SWI in MRI. SWI is a high-resolution 3D gradient-echo MRI sequence that highlights the difference between paramagnetic substances (such as deoxyhemoglobin, hemosiderin, and ferritin) and the brain parenchyma, making this sequence sensitive for detecting microbleeds. Paramagnetic substances (such as hemosiderin, deoxyhemoglobin, and ferritin) show low signal intensity on SWI images.

[0243] SWI scanned multiple layers from the olfactory bulb to the cerebellum, and the degree of bleeding was scored for each layer. The sum of the scores for each layer was used as the bleeding score for that mouse and statistically analyzed. The results are shown in Figure 8. Consistent with the above, no bleeding occurred in the sham group. Among the six mice in the hemorrhage model group, four had severe parenchymal hemorrhage. In the SalB / Rg1 group, only two of the six mice had hemorrhage detected on SWI (7.00±7.83 versus 0.50±0.76).

[0244] Example 5 Salvianolic acid B and ginsenoside Rg1 have a synergistic effect in inhibiting alteplase-induced cerebral hemorrhage

[0245] 1. Experimental Materials

[0246] Male C57BL / 6 mice (26 ± 3 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were housed in an SPF-grade animal room at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, with a constant temperature (22 ± 2°C) and a 12-h light and dark cycle, and had free access to food and water.

[0247] 2. The experimental drugs, reagents and instruments are mentioned above.

[0248] 3. Experimental Methods

[0249] 3.1 Alteplase (tPA)-induced mouse cerebral hemorrhage model is shown in Example 3, the same as the scheme B in Figure 3 above.

[0250] 3.2 Bleeding Scoring The same bleeding scoring method as in Example 3

[0251] 3.3 Experimental Animal Grouping and Dosing

[0252] Male C57BL / 6 mice were randomly divided into four groups: a bleeding model group, a SalB group, an Rg1 group, and a SalB / Rg1 combination group, with 10 animals in each group. The bleeding model group received normal saline; the SalB group received 3.57 mg / kg of salvianolic acid B; the Rg1 group received 1.43 mg / kg of ginsenoside Rg1; and the SalB / Rg1 group received 5 mg / kg of salvianolic acid B / Rg1 (with salvianolic acid B and Rg1 prepared at a mass ratio of 5:2). Depending on the group, animals were injected via the tail vein with normal saline, salvianolic acid B, ginsenoside Rg1, or the SalB / Rg1 combination.

[0253] 3.4 Bliss independent statistical model as described in Example 2

[0254] 3.5 Data Statistical Methods

[0255] Data For normally distributed variables, analysis of variance (ANOVA) with Bonferroni post hoc test was used to assess differences between groups. For comparisons between only two groups, an unpaired, two-tailed Student's t test was used. Differences between ratios between two groups were examined using the chi-squared test. P < 0.05 was considered statistically significant.

[0256] 4. Results

[0257] SalB / Rg1 have a synergistic effect in reducing alteplase (tPA)-induced bleeding

[0258] Figure 9A shows representative images of each group of tPA-induced intracerebral hemorrhage, with the bleeding site displayed in red. Figure 9B shows the quantitative results of the bleeding severity. The bleeding score in the hemorrhage model group was 4.75±1.98. Neither SalB (3.88±1.76) nor Rgl (4.5±1.86) alone significantly reduced the bleeding score. SalB / Rgl at 5mg / kg significantly reduced the bleeding score (1.7±2.34 vs. 4.75±1.98, p<0.01), and the efficacy was statistically significant compared to Rg1 (1.7±2.34 vs. 4.5±1.86, p<0.01).

[0259] The synergistic effect was evaluated using the Bliss independent statistical model (Figure 9C). Based on the formula given above, the results showed a significant statistical difference between the Sal / Rg1 group and the independent group (p < 0.01), indicating that the combination of SalB and Rg1 has a very significant synergistic effect in reducing thrombolytic drug-induced intracerebral hemorrhage.

[0260] Example 6. SalB / Rg1 inhibits cerebral hemorrhage exacerbated by hyperglycemia

[0261] 1. Experimental Materials

[0262] Healthy male C57BL / 6 mice, weighing 25–28 g, were purchased from Shanghai Slack Laboratory Animal Company. The animals were housed in the SPF animal facility at the Shanghai Institute of Materia Medica with free access to food and water, a 12-hour light / dark cycle, and a temperature controlled at 22 ± 2°C. Experiments were conducted after three days of acclimatization. Before the experiments began, the mice underwent behavioral training, including neurological damage severity scoring and hanging time training.

[0263] 2. Experimental Methods

[0264] 2.1 Preparation of hemorrhagic mouse model

[0265] A mouse intracerebral hemorrhage model was established using high glucose and reperfusion stimulation. 7 mg / kg of glucose was injected intraperitoneally 15 minutes before ischemia, and the suture plug was removed 3 hours after ischemia to restore blood flow for 21 hours. A sham operation was performed using the same procedure, without the suture plug.

[0266] 2.2 Experimental design and grouping

[0267] To evaluate the effects of SalB / Rg1 on hyperglycemia-induced intracerebral hemorrhage, mice were randomly divided into three groups (n=8-10). Sham-operated mice were given saline as a normal control. The other two groups were induced to bleed with high glucose followed by reperfusion. Mice in the hemorrhage model group were given saline as a model control. Mice in the SalB / Rg1 treatment group were given 6.25 mg / kg of SalB / Rg1 to assess the therapeutic effect. Neurobehavioral testing was performed 24 hours later.

[0268] 2.3 Medication and Dosage Regimen

[0269] To ensure the objectivity of the research results, the experiment was conducted double-blind and the blind was not revealed until the end of the study. Preparation of SaB / Rg1: SalB and Rg1 powders were weighed and thoroughly mixed in a 5:2 ratio. After complete dissolution on a vortex shaker, the mixture was filtered through a microporous filter membrane into sterile EP tubes and stored frozen at -20°C until use.

[0270] 2.4 Behavioral assessment, see the method introduction in Example 3

[0271] 2.5 Bleeding Assessment

[0272] Twenty-two hours after ischemia, mice were intravenously injected with 2% Evans blue to assess hemorrhagic transformation. After 2 hours of circulation, mice were transcardially perfused with saline, and the brains were rapidly removed. Hemorrhagic transformation was then assessed using the Heidelberg hemorrhagic classification (Yaghi, S. & Willey, J.Z. 2017). Briefly, hemorrhagic transformation events were scored as follows: 0 for the absence of blood; 1 for type I hemorrhagic infarction (HI), with small petechiae at the infarct margin; 2 for type II HI, with more confluent petechiae within the infarct area but no mass effect; 3 for type I parenchymal hemorrhage (PH), with a clot not exceeding 30% of the infarct area and a mild mass effect; and 4 for type II PH, with a dense clot exceeding 30% of the infarct area and a significant mass effect.

[0273] 2.6 Statistical methods

[0274] GraphPad Prism 6.0 was used for data analysis, and all quantitative data were The homogeneity of variance was tested by one-way ANOVA, and the differences among the groups were compared by t-test, with p < 0.05 indicating statistical significance.

[0275] 3 Experimental results

[0276] 3.1 SalB / Rg1 alleviates hyperglycemia-induced cerebral hemorrhage

[0277] Intravenous injection of Evans blue facilitated the observation of bleeding spots, which were consistently located at the edge of Evans blue staining (Figure 10A). The bleeding score was determined based on the number and extent of bleeding spots. Bleeding spots were quite prominent in the bleeding model group. Compared with the bleeding model group, 6.25 mg / kg SalB / Rg1 significantly reduced the bleeding score (1.5±0.20 vs 2.22±0.22, p<0.05). Neurological function outcomes were assessed using modified Bederson and Longa scores, hanging time, and mNSS scores. Neurobehavioral outcomes showed an improvement trend with SalB / Rg1 treatment (Figure 11).

[0278] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of a combination of active ingredients for preparing a medicament for treating, alleviating and / or preventing a hemorrhagic disease, characterized in that: The combination of active ingredients includes: (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid, or a combination thereof; and (b) a second active ingredient, wherein the second active ingredient is selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof.

2. The use according to claim 1, characterized in that The salvianolic acid is selected from the following group: salvianolic acid B, salvianolic acid A, salvianolic acid C, isosalC, salvianolic acid D, salvianolic acid E, salvianolic acid F, salvianolic acid G, salvianolic acid H, salvianolic acid I, salvianolic acid J, salvianolic acid K, salvianolic acid L, salvianolic acid M, salvianolic acid N, salvianolic acid T, salvianolic acid U, salvianolic acid Y, caffeic acid, danshensu, protocatechuic acid, protocatechuic aldehyde, ferulic acid, isoferulic acid, Salvinal, Ailanthoidol, rosmarinic acid, lithospermic acid, danshen glycoside, catechol, dimethyl lithospermic acid B, tanshinone I, dihydrotanshinone I, tanshinone diol-A, tanshinone IIA, cryptotanshinone, tanshinone, dehydrotanshinone, isotanshinone I, tanshinone A, or a combination thereof.

3. The use according to claim 1, characterized in that The ginsenosides are selected from the group consisting of ginsenoside Rg1, ginsenoside Re, ginsenoside Rg2, ginsenoside Rh1, ginsenoside F1, notoginsenoside R1, notoginsenoside R2, notoginsenoside R3, notoginsenoside R6, notoginsenoside M, notoginsenoside N, notoginsenoside Rt, notoginsenoside T3, notoginsenoside FP1, notoginsenoside RW1, notoginsenoside Fh7, camelliaside D, and notoginsenoside N. Camelliaside U, 20-glucose ginsenoside Rf, malonate ginsenoside Rg1, bamboo saponin L5, safflower ginsenoside R1, pseudo-ginsenoside RT3, saponin 10, saponin 1, saponin 2, saponin 3, ginsenoside Ra3, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Rg3, malonate ginsenoside Rb 1. Malonylginsenoside Rd, 6'-O-acetylginsenoside Rg3, Ginsenoside F2, Ginsenoside CK, Ginsenoside Mc, 20(S)-ginsenoside Rg3, 20(R)-ginsenoside Rg3, 20(R)-ginsenoside Rh2, 20(S)-ginsenoside Rh2, Notoginsenoside R4, Notoginsenoside K, Gynostemma saponin XVII, Gynostemma saponin IX , Gynostemma saponin XV, Gynostemma saponin VIII, Notoginsenoside Fa, Notoginsenoside Fc, Notoginsenoside Fc, Notoginsenoside D, Notoginsenoside L, Notoginsenoside L*, Notoginsenoside O, Notoginsenoside P, Notoginsenoside Q, Notoginsenoside S, Notoginsenoside T, Notoginsenoside FP2, Notoginsenoside Ftl, Notoginsenoside ST-4, Notoginsenoside FZ, Notoginsenoside Fh1, or a combination thereof.

4. The use according to claim 1, wherein The salvianolic acid is salvianolic acid B.

5. The use according to claim 1, characterized in that The ginsenoside is ginsenoside Rg1.

6. The use according to claim 1, wherein The combination of active ingredients consists of (a) a first active ingredient and (b) a second active ingredient.

7. The use according to claim 1, characterized in that The weight ratio of the first active ingredient to the second active ingredient is 5:2-1:

10.

8. The use according to claim 1, characterized in that The hemorrhagic disease is a cerebral hemorrhagic disease.

9. The use according to claim 8, characterized in that The cerebral hemorrhagic disease includes drug-induced cerebral hemorrhage, preferably cerebral hemorrhage caused by thrombolytic drugs, and more preferably cerebral hemorrhage caused by alteplase.

10. The use according to claim 1, characterized in that The drug is used for a purpose selected from the group consisting of: a) Reduce cerebral hematoma; b) Improve neurobehavioral function in animals with cerebral hemorrhage; c) Protecting the cerebral cortex and striatum neurons in animals with cerebral hemorrhage; d) Improve the survival rate of animals with cerebral hemorrhage.

11. Use of a pharmaceutical composition for preparing a medicament for treating, alleviating and / or preventing a hemorrhagic disease, characterized in that: The pharmaceutical composition comprises: (a) a first active ingredient, wherein the first active ingredient is selected from the group consisting of salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, an extract containing salvianolic acid, or a combination thereof; (b) a second active ingredient selected from the group consisting of ginsenosides, stereoisomers thereof, crystal forms thereof, pharmaceutically acceptable salts or esters thereof, extracts containing ginsenosides, or combinations thereof; and (c) a pharmaceutically acceptable carrier.

12. The use according to claim 11, characterized in that The salvianolic acid is salvianolic acid B.

13. The use according to claim 11, characterized in that The ginsenoside is ginsenoside Rg1.

14. The use according to claim 11, characterized in that The weight ratio of the first active ingredient to the second active ingredient is 5:2-1:

10.

15. Use of an active ingredient for preparing a medicament for treating, alleviating and / or preventing a bleeding disorder, characterized in that: The active ingredient is selected from the following group: (1) Salvianolic acid, its stereoisomers, its crystal forms, its pharmaceutically acceptable salts or esters, or its derivatives, and extracts containing salvianolic acid; as well as (2) Ginsenosides, their stereoisomers, their crystal forms, their pharmaceutically acceptable salts or esters, or their derivatives, and extracts containing ginsenosides.

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