Medicine for treating cerebral diseases

Abstract

The invention provides a medicine for treating cerebral diseases. Specifically, the invention provides a pharmaceutical composition, and the active components of the pharmaceutical composition comprise (a) and (b): (a) a first active component which is a pharmaceutically acceptable salt of salvianolic acid; and (b) a second active ingredient, wherein the second active ingredient is ginsenoside or a pharmaceutically acceptable salt thereof; wherein the pharmaceutically acceptable salt of salvianolic acid is selected from the following group: salvianolic acid B magnesium salt, salvianolic acid B calcium salt, salvianolic acid A magnesium salt, rosmarinic acid sodium salt or a combination thereof; the ginsenoside is selected from the following group: ginsenoside Rg1, ginsenoside Rh2, ginsenoside Rb1 or a combination thereof; moreover, the weight ratio of the first active component to the second active component is 1: (0.1-10), and the weight ratio is based on the pharmaceutically acceptable salt of salvianolic acid and ginsenoside. The invention also relates to the use of the pharmaceutical composition in the prevention or treatment of cerebral diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a medicine for treating brain diseases and application thereof. Background Art

[0002] Brain diseases refer to disorders that result from various causes and lead to abnormal brain function. The most common brain diseases are cerebrovascular diseases, including cerebral arteriosclerosis, cerebral ischemia, ischemic stroke, and cerebral hemorrhage. Other degenerative diseases of the nervous system include Alzheimer's disease, Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy.

[0003] The brain, as a vital central nervous system, controls various physiological and psychological activities. Patients with these brain diseases experience varying degrees of impairment in functional abilities, such as speech and walking, and often suffer from disabling sequelae, such as hemiplegia, slurred speech, intellectual impairment, joint stiffness and contractures, and even dementia. These conditions severely impact patients' quality of life and place a heavy burden on their families.

[0004] Therefore, the present invention needs to provide more excellent candidate drugs to prevent or treat brain diseases. Summary of the Invention

[0005] The object of the present invention is to provide a drug capable of preventing or treating brain diseases.

[0006] In a first aspect, the present invention provides a pharmaceutical composition, wherein the active ingredients of the pharmaceutical composition include (a) and (b):

[0007] (a) a first active ingredient, wherein the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid; and (b) a second active ingredient, wherein the second active ingredient is ginsenoside, or a pharmaceutically acceptable salt thereof;

[0008] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of salvianolic acid and ginsenoside.

[0009] In another preferred embodiment, the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid B; and the second active ingredient is ginsenoside Rg1, or a pharmaceutically acceptable salt thereof;

[0010] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of salvianolic acid B and ginsenoside Rg1.

[0011] In another preferred embodiment, the pharmaceutically acceptable salt of salvianolic acid is selected from the group consisting of calcium salt, magnesium salt, sodium salt, potassium salt, lithium salt, ammonium salt (such as diethylamine, trimethylamine, diethanolamine), piperazine salt, zinc salt, silver salt, amino acid salt, or a combination thereof, preferably magnesium salt.

[0012] In another preferred embodiment, the active ingredients of the pharmaceutical composition include (a) and (b):

[0013] (a) a first active ingredient, wherein the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid; and (b) a second active ingredient, wherein the second active ingredient is ginsenoside, or a pharmaceutically acceptable salt thereof;

[0014] Wherein, the pharmaceutically acceptable salt of salvianolic acid is selected from the following group: salvianolic acid B magnesium salt, salvianolic acid B calcium salt, salvianolic acid A magnesium salt, rosmarinic acid sodium salt, or a combination thereof;

[0015] The ginsenoside is selected from the group consisting of ginsenoside Rg1, ginsenoside Rh2, ginsenoside Rb1, or a combination thereof;

[0016] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of salvianolic acid and ginsenoside.

[0017] In another preferred embodiment, the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid B; and (b) a second active ingredient, the second active ingredient is ginsenoside, or a pharmaceutically acceptable salt thereof;

[0018] Wherein, the pharmaceutically acceptable salt of salvianolic acid B is selected from the following group: salvianolic acid B magnesium salt, salvianolic acid B calcium salt, or a combination thereof;

[0019] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is based on the pharmaceutically acceptable salt of salvianolic acid B and ginsenosides. Preferably, the pharmaceutical composition further comprises a third active ingredient: other salvianolic acids, other ginsenosides, or a combination thereof, such as magnesium salt of salvianolic acid A, sodium salt of rosmarinic acid, ginsenoside Rb1, ginsenoside Rh1, or a combination thereof.

[0020] In another preferred embodiment, the first active ingredient is magnesium salt of salvianolic acid B.

[0021] In another preferred embodiment, the purity of the pharmaceutically acceptable salt of salvianolic acid B is ≥80%, preferably ≥85%, more preferably ≥90%, ≥95%, 98%, ≥99% or 99.5%, by weight.

[0022] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient: ginsenoside Rg1, or a pharmaceutically acceptable salt thereof.

[0023] In another preferred embodiment, the first active ingredient is magnesium salt of salvianolic acid B, and (b) the second active ingredient is ginsenoside Rg1, or a pharmaceutically acceptable salt thereof;

[0024] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the magnesium salt of salvianolic acid B and ginsenoside Rg1.

[0025] In another preferred embodiment, the first active ingredient is magnesium salt of salvianolic acid B, and (b) the second active ingredient is ginsenoside Rg1;

[0026] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the magnesium salt of salvianolic acid B and ginsenoside Rg1.

[0027] In another preferred embodiment, the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid A; and the second active ingredient is ginsenoside Rb1, or a pharmaceutically acceptable salt thereof;

[0028] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of salvianolic acid A and ginsenoside Rb1, preferably salvianolic acid A magnesium salt and ginsenoside Rb1 or ginsenoside Rh1.

[0029] In another preferred embodiment, the first active ingredient is a pharmaceutically acceptable salt of rosmarinic acid; and the second active ingredient is ginsenoside Rb1, or a pharmaceutically acceptable salt thereof;

[0030] Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of rosmarinic acid and ginsenoside Rb1, preferably rosmarinic acid sodium salt and ginsenoside Rb1.

[0031] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.2-5), preferably 1:(0.4-0.6) or 1:(2-3).

[0032] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.3-0.5), preferably 1:(0.35-0.45), more preferably 1:(0.38-0.42), more preferably 1:(0.39-0.41), or 1:0.4.

[0033] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.38, 1:0.39, 1:0.4, 1:0.41, 1:0.42, 1:0.45, 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0. :0.9, 1:0.95, 1:1, 1:1.2, 1:1.4, 1:1.5, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10. In another preferred embodiment, the pharmaceutical composition further comprises (c) a pharmaceutically acceptable carrier.

[0034] In another preferred embodiment, the purity of the pharmaceutically acceptable salt of salvianolic acid B in the first active ingredient is ≥80%, preferably ≥90%, more preferably ≥95%, 98%, ≥99% or 99.5%.

[0035] In another preferred embodiment, the purity of ginsenoside Rg1 in the second active ingredient is ≥80%, preferably ≥90%, more preferably ≥95%, 98%, ≥99% or 99.5%.

[0036] 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.

[0037] 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 (such as lozenge, drops), respiratory tract administration preparation, skin administration preparation, mucosal administration preparation, more preferably, the dosage form is an injection.

[0038] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for oral administration.

[0039] In another preferred embodiment, the dosage form of the oral pharmaceutical composition is an orally disintegrating tablet, a mucosal adhesive tablet, an orally disintegrating film, an orally disintegrating granule, a powder, a sublingual tablet, a buccal tablet, a chewable tablet, an effervescent tablet, or a solution.

[0040] In a second aspect, the present invention provides a pharmaceutically acceptable salt of salvianolic acid B, wherein the salt is salvianolic acid B magnesium salt or salvianolic acid B calcium salt.

[0041] In another preferred embodiment, the pharmaceutically acceptable salt of salvianolic acid B is salvianolic acid B (mono) magnesium salt.

[0042] In another preferred embodiment, the purity of the pharmaceutically acceptable salt of salvianolic acid B is ≥80%, ≥85%, preferably ≥90%, more preferably ≥95%, 98%, ≥99% or 99.5%, by weight.

[0043] In a third aspect, the present invention provides a method for preparing magnesium salt of salvianolic acid B, comprising the steps of reacting free salvianolic acid B with an inorganic salt or an inorganic base containing magnesium ions in water or an aqueous solvent to produce magnesium salt of salvianolic acid B.

[0044] In another preferred embodiment, the inorganic salt or inorganic base containing magnesium ions is selected from the following group: magnesium carbonate, magnesium sulfate, magnesium nitrate, magnesium hydroxide, magnesium chloride, magnesium bicarbonate or a combination thereof.

[0045] In another preferred embodiment, the method comprises the steps of: adding an aqueous solution of free salvianolic acid B to an aqueous solution of an inorganic salt or an inorganic base containing magnesium ions, thereby reacting to form salvianolic acid B magnesium salt.

[0046] In another preferred embodiment, the molar ratio of free salvianolic acid to the inorganic salt or inorganic base containing magnesium ions is 1:0.98-1.02, preferably, 1:0.99-1.01, and more preferably, 1:1.

[0047] In another preferred embodiment, the method further comprises the steps of: loading the reaction solution onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, 15% methanol-water solution, and 20% methanol-water solution in a gradient manner, collecting fractions, combining fractions with qualified purity, removing methanol, and re-loading the sample after methanol removal onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, and 20% methanol-water solution in a gradient manner, combining fractions with qualified purity, and drying to obtain magnesium salt of salvianolic acid B.

[0048] In a fourth aspect, the present invention provides a method for preparing calcium salt of salvianolic acid B, comprising the steps of reacting free salvianolic acid B with an inorganic salt or inorganic base containing calcium ions in water or an aqueous solvent to generate calcium salt of salvianolic acid B.

[0049] In another preferred embodiment, the inorganic salt or inorganic base containing calcium ions is selected from the following group: calcium carbonate, calcium sulfate, calcium nitrate, calcium hydroxide, calcium bicarbonate, or a combination thereof.

[0050] In another preferred embodiment, the method comprises the steps of: adding an aqueous solution of free salvianolic acid B to an aqueous solution of an inorganic salt or an inorganic base containing calcium ions, thereby reacting to generate salvianolic acid B calcium salt.

[0051] In another preferred embodiment, the molar ratio of free salvianolic acid to the inorganic salt or inorganic base containing calcium ions is 1:0.98-1.02, preferably, 1:0.99-1.01, and more preferably, 1:1.

[0052] In another preferred embodiment, the method further comprises the steps of: loading the reaction solution onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, 15% methanol-water solution, and 20% methanol-water solution in a gradient manner, collecting fractions, combining fractions with qualified purity, removing methanol, and re-loading the sample after methanol removal onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, and 20% methanol-water solution in a gradient manner, combining fractions with qualified purity, and drying to obtain salvianolic acid B calcium salt.

[0053] In another preferred embodiment, the magnesium salt of salvianolic acid B or the calcium salt of salvianolic acid B of the present invention is a high-purity salt prepared by the above method, rather than being isolated from natural products.

[0054] In a fifth aspect, the present invention provides use of the pharmaceutical composition according to the first aspect of the present invention or the pharmaceutically acceptable salt of salvianolic acid B according to the second aspect of the present invention in the preparation of a medicament for preventing and / or treating brain diseases.

[0055] In another preferred embodiment, the brain disease is cerebrovascular disease or cerebral ischemic disease. Preferably, the cerebrovascular disease or cerebral ischemic disease is selected from the following group: ischemic stroke, cerebral arteriosclerosis, cerebral ischemia, cerebral hemorrhage, cerebral edema, or a combination thereof.

[0056] In another preferred embodiment, the brain disease is Alzheimer's disease, vascular dementia, cerebral atrophy, dementia caused by unexplained cerebral atrophy, and loss of nerve cell number, behavioral disorders, sensory disorders, or a combination thereof caused by chronic brain damage.

[0057] The brain disease is a neurodegenerative disease. Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), hippocampal sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, multiple system atrophy, progressive supranuclear palsy, or a combination thereof.

[0058] In another preferred embodiment, the prevention and / or treatment of brain diseases includes improving biomarkers of neurodegenerative diseases, and the biomarkers are selected from the following group: Iba1, neurofilament light chain, GFAP, tau, α-synuclein, or a combination thereof.

[0059] In another preferred embodiment, the prevention and / or treatment of brain diseases is the inhibition of white matter atrophy, inhibition of hippocampal atrophy, inhibition of lateral ventricle enlargement, inhibition of striatal atrophy, or a combination thereof.

[0060] In another preferred embodiment, the prevention and / or treatment of brain diseases is the inhibition of white matter neuronal cell loss, the inhibition of hippocampal neuronal cell loss, the inhibition of lateral ventricle enlargement, the inhibition of striatal neuronal cell loss, or a combination thereof.

[0061] In another preferred embodiment, the prevention and / or treatment of brain diseases is the inhibition of chronic necrosis of brain tissue, especially liquefaction necrosis.

[0062] In another preferred embodiment, the patient is in the acute, subacute or chronic phase after an ischemic stroke, such as 0-4.5h, 4.5-6h or 6-24h, such as 8h, 12, 16, 18h, 20h or even 24h after an ischemic stroke.

[0063] In another preferred embodiment, the pharmaceutical composition is used before, during or after reperfusion therapy (such as intravenous thrombolysis or mechanical thrombectomy) for patients with cerebral ischemic disease, preferably before perfusion therapy so that reperfusion therapy can be performed while it continues to exert its protective effect.

[0064] In another preferred embodiment, the patient is a patient who is about to receive, is receiving or has received reperfusion therapy.

[0065] In another preferred embodiment, the patient is a stroke patient who cannot receive reperfusion therapy.

[0066] In another preferred embodiment, the pharmaceutical composition of the present invention can be used to prevent or treat other organ damage caused by ischemic stroke, such as heart damage, lung damage, thymus damage, kidney damage, or a combination thereof; systemic hypoxia caused by ischemic stroke, or secondary damage caused by hypoxia; or reperfusion injury after ischemic stroke, such as tissue and organ damage caused by reperfusion, such as heart damage, lung damage, thymus damage, kidney damage, or a combination thereof.

[0067] The sixth aspect of the present invention provides a method for preventing or treating brain diseases, wherein the pharmaceutical composition described in the first aspect of the present invention or the pharmaceutically acceptable salt of salvianolic acid B described in the second aspect of the present invention is administered to a subject in need, thereby preventing or treating the disease.

[0068] In another preferred embodiment, the subject is a human or non-human mammal, such as a rat or a mouse.

[0069] 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

[0070] Figure 1The infarct volume-reducing effects of various salt forms of SalB / Rg1 are shown; ***P<0.001 compared with the Sham group; ###P<0.001 compared with the MCAO group; $$P<0.01 compared with the free SalB / Rg1 group.

[0071] Figure 2 The results showed that the MLB / Rg1 combination had a more significant effect on improving the structure of brain tissue than the free SalB / Rg1 combination.

[0072] Figure 3 It was shown that the MLB / Rg1 combination better maintained the number of neurons than the free SalB / Rg1 combination.

[0073] Figure 4 The results showed that the MLB / Rg1 combination has a synergistic effect in the treatment of stroke; among them, the representative TTC staining image of rat brain (A) and the quantitative results of infarct volume (B), as well as the evaluation based on the Bliss independent statistical model, proved that MLB and Rg1 have a very significant synergistic effect in reducing infarct volume (C); compared with Sham, ***P<0.001; compared with MCAO, #P<0.05, ###P<0.001; compared with the MLB group alone, &&P<0.01; compared with the Rg1 group alone, $$$P<0.001.

[0074] Figure 5 Representative images of the whole brain and cortex and striatum stained with HE in coronal sections (scale bar: 50 μm) show that the MLB / Rg1 combination improves brain tissue structure after stroke.

[0075] Figure 6 The results of Garcia JH scores on the 1st day (A), 3rd day (B), 5th day (C), and 7th day (D) after stroke are shown; compared with Sham, ***P<0.001; compared with the MCAO group, #P<0.05, ##P<0.01, ###P<0.001; compared with the MLB group alone, &&P<0.01; compared with the Rg1 group alone, $P<0.05, $$P<0.01,

[0076] $$$P<0.001.

[0077] Figure 7The results of Longa scores on the 1st day (A), 3rd day (B), 5th day (C), and 7th day (D) after stroke are shown; compared with Sham, ***P < 0.001; compared with the MCAO group, #P < 0.05, ##P < 0.01, ###P < 0.001; compared with the MLB group alone, &P < 0.05, &&P < 0.01; compared with the Rg1 group alone, $$P < 0.01.

[0078] Figure 8 Representative images of Nissl staining of coronal sections of the whole brain, cerebral cortex, and striatum (A) and quantification of neuronal numbers in the cortex and striatum (B) are shown; ***P < 0.001 compared with sham; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the MCAO group; &P < 0.05 compared with the MLB group alone; $P < 0.05 compared with the Rg1 group alone (Scale bar: 50 μm).

[0079] Figure 9 Research plan for the treatment of brain atrophy with MLB / Rg1 (A), as well as gross images of brain atrophy and representative images of TTC staining (B).

[0080] Figure 10 Quantitative results of brain atrophy; compared with the Sham group, *p<0.05, **p<0.01, ***p<0.001; compared with MCAO, #p<0.05, ##p<0.01, ###p<0.001; compared with Rg1, &p<0.05, &&p<0.01, &&&p<0.001; compared with MLB, $p<0.05, $$p<0.01, $$$p<0.001; ^ represents p<0.05, ^^p<0.01, ^^^p<0.001 compared with 5 (1:10) mg / kg MLB / Rg1.

[0081] Figure 11 The statistical results of the balance beam test are shown; *p<0.05, **p<0.01, compared with the Sham group

[0082] ***p<0.001; #p<0.05, ##p<0.01, ###p<0.001 compared with MCAO.

[0083] Figure 12 Representative images of HE staining.

[0084] Figure 13 Representative images of Nissl staining.

[0085] Figure 14Nissl staining quantitative images; *p<0.05, **p<0.01, ***p<0.001 compared with the Sham group; #p<0.05 compared with MCAO. ## p<0.01, ### p<0.001; compared with Rg1&p<0.05, && p<0.01, &&& p<0.001; compared with MLB $p<0.05, $$ p<0.01, $$$ p<0.001; compared with 5 (1:10) mg / kg MLB / Rg1^p<0.05,

[0086] ^^p<0.01,^^^p<0.001.

[0087] Figure 15 The synergistic effects of MLB and Rg1 in reducing infarct volume (A) and inhibiting brain atrophy (B) were evaluated by the Bliss method.

[0088] Figure 16 This is the whole-body TTC staining image of the brain.

[0089] Figure 17 Quantitative graph of brain atrophy. Compared with the Sham group *p<0.05, **p<0.01, ***p<0.001; compared with MCAO #p<0.05, ## p<0.01, ### p<0.001;

[0090] Figure 18 Representative images of HE staining.

[0091] Figure 19 Representative images of Nissl staining.

[0092] Figure 20 Quantitative graph of Nissl staining; *p<0.05, **p<0.01, ***p<0.001 compared with the Sham group; represents #p<0.05, ##p<0.01, ###p<0.001 compared with MCAO.

[0093] Figure 21 To evaluate the downregulatory effect of different ratios of MLB / Rg1 on infarct volume by TTC staining.

[0094] Figure 22 TTC staining was used to evaluate the downregulation effect of SalA magnesium salt / Rb1 on infarct volume (A) and the improvement of neurobehavior (B, C).

[0095] Figure 23TTC staining was used to evaluate the downregulation effect of SalA magnesium salt / Rh2 group on infarct volume (A) and the improvement of neurobehavior (B, C).

[0096] Figure 24 The downregulatory effect of different ratios of RosA-Na / Rb1 on infarct volume was evaluated by TTC staining (A). The neurobehavioral evaluation of the different ratios of RosA-Na / Rb1 groups was performed using BWT Score (B) and Garcia JH score (C).

[0097] Figure 25 The downregulatory effect of sublingual administration of MLB / Rg1 on infarct volume was evaluated by TTC staining (AB), BWT evaluation (C), and Garcia JH score (D).

[0098] Figure 26 Survival curves of different groups, compared with the Sham group, **P<0.01; compared with the tMCAO group, #P<0.05.

[0099] Figure 27 showed that MLB / Rg1 treatment reduced brain edema in tMCAO mice.

[0100] Figure 28 It shows that MLB / Rg1 improves the neurobehavior of mice by protecting the brain's nerve cells.

[0101] Figure 29 Magnetic resonance images of mouse cerebral blood flow (CBF).

[0102] Figure 30 MLB / Rg1 significantly improved cerebral blood flow in the infarct edge zone.

[0103] Figure 31 It shows that MLB / Rg1 can still reduce infarct volume when administered 24 hours after infarction, representative image of TTC staining (A), quantitative results of infarct volume (B).

[0104] Figure 32 Bliss-independent model statistics showed that MLB / Rg1 reduced infarct volume 24 hours after infarction and had a synergistic effect.

[0105] Figure 33 MLB / Rg1 administration 24 hours after infarction showed prolonged coagulation time (A) and improved neurobehavioral performance (B) as assessed by tissue sampling seven days later.

[0106] Figure 34 MLB / Rg1 administration 24 hours after infarction improved blood oxygen levels (A) and enhanced cardiac contractile function (B) as assessed by imaging seven days later.

[0107] Figure 35 MLB / Rg1 is shown to reduce post-stroke thymic (A) and lung (B) damage when administered 24 hours after infarction and assessed seven days later. DETAILED DESCRIPTION

[0108] After extensive and intensive research, screening, and testing, the present inventors have developed a drug with significantly improved efficacy in preventing or treating brain diseases. The present inventors unexpectedly discovered for the first time that, compared to the combination of free salvianolic acid B (SalB) and ginsenoside Rg1, the combination of a specific salt of salvianolic acid B and ginsenoside Rg1 has significantly improved therapeutic effects in treating acute, subacute, and chronic brain injuries or neurodegenerative diseases. This invention was completed based on this discovery.

[0109] the term

[0110] 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.

[0111] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0112] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0113] Active ingredient

[0114] In the present invention, the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid, preferably a pharmaceutically acceptable salt of salvianolic acid B. As used herein, "pharmaceutically acceptable salt of salvianolic acid" refers to a salt formed by salvianolic acid and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed between the compound of the active ingredient of the present invention and an acid. Acids suitable for forming salts 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, phenylmethanesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. A preferred class of salts is a salt formed between the compound of the active ingredient 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, sodium carbonate, sodium bicarbonate, sodium phosphate, calcium carbonate, and magnesium carbonate; and organic bases such as aqueous ammonia, trimethylamine, triethylamine, diethylamine, diethanolamine, and piperazine. Another preferred salt type is salts formed between the active ingredient of the present invention and metal ions, including, but not limited to, (mono)magnesium salts, (mono)calcium salts, (di)sodium salts, (di)potassium salts, (di)lithium salts, zinc salts, and (di)silver salts, as well as salts formed with (natural) amino acids such as arginine and lysine.

[0115] Preferably, the salt is a magnesium salt of salvianolic acid B.

[0116]

[0117] In another preferred embodiment, the purity of the magnesium salt of salvianolic acid B in the first active ingredient is ≥98%, preferably ≥98.5%, more preferably ≥99% or 99.5%, by weight.

[0118] In the present invention, the second active ingredient is ginsenoside, or a pharmaceutically acceptable salt thereof, preferably ginsenoside Rg1, or a pharmaceutically acceptable salt thereof.

[0119]

[0120] In the present invention, "pharmaceutically acceptable salts" of ginsenosides include salts of ginsenosides formed with acids or bases 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, sodium carbonate, sodium bicarbonate, and sodium phosphate; and organic bases such as ammonia water, trimethylamine, triethylamine, diethylamine, diethanolamine, and piperazine. 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 salts, sodium salts, calcium salts, potassium salts, lithium salts, zinc salts and silver salts, etc., as well as salts formed with (natural) amino acids such as arginine, lysine, etc.

[0121] Preferably, the active ingredient of the present invention includes only the first active ingredient and the second active ingredient described above, excluding inevitable impurities.

[0122] Preferably, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on magnesium salt of salvianolic acid B and ginsenoside Rg1 (original compound).

[0123] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.2-5), preferably 1:(0.4-0.6) or 1:(2-3).

[0124] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0.9, 1:0 .95, 1:1, 1:1.2, 1:1.4, 1:1.5, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, 1:3.2, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, or 1:10.

[0125] Preferably, the present invention provides a pharmaceutically acceptable salt of salvianolic acid B, wherein the salt is salvianolic acid B magnesium salt or salvianolic acid B calcium salt.

[0126] In another preferred embodiment, the purity of the magnesium salt of salvianolic acid B is ≥80%, preferably ≥90%, more preferably ≥95%, 98%, ≥99% or 99.5%.

[0127] In another preferred embodiment, the purity of the calcium salt of salvianolic acid B is ≥80%, preferably ≥90%, more preferably ≥95%, 98%, ≥99% or 99.5%.

[0128] In the present application, when the magnesium salt or calcium salt of salvianolic acid B is used as an active ingredient in a pharmaceutical composition, its purity refers to its purity in a separable single drug substance.

[0129] In another preferred embodiment, the pharmaceutical composition further comprises (c) a pharmaceutically acceptable carrier.

[0130] blood-brain barrier

[0131] The blood-brain barrier refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can prevent certain substances from entering the brain tissue from the blood.

[0132] The existence of the blood-brain barrier is of great significance in preventing harmful substances from entering the brain from the blood, but it also blocks the transfer of most small molecule and large molecule drugs (such as peptides, proteins and nucleic acids), seriously limiting the treatment of central nervous system diseases (such as neurodegenerative diseases, brain tumors, brain infections and strokes).

[0133] In most cases, blood-brain barrier penetration depends on the compound's lipophilicity. Lipophilicity, defined as the equilibrium partition coefficient of a non-ionized compound between two immiscible phases (e.g., n-octanol and water / buffer), is one of the most important physicochemical properties of a compound. Lipophilicity influences a compound's absorption, distribution, metabolism, excretion, toxicity, and pharmacological activity, and is a parameter closely related to blood-brain barrier penetration. Improving lipophilicity can enhance drug penetration across the blood-brain barrier.

[0134] However, the present application unexpectedly discovered that the combination of a salt of salvianolic acid B (especially a magnesium salt) and Rg1 (the hydrophilicity is enhanced and the fat solubility is reduced after salt formation) significantly improves the protection of the brain and the therapeutic effect on brain diseases compared to the combination of the free acid of salvianolic acid B and Rg1. This is not only reflected in cerebrovascular diseases such as stroke, but it is also unexpected that the combination of salvianolic acid B salt / Rg1 has a significantly improved and unexpected therapeutic effect on degenerative diseases represented by Alzheimer's disease and vascular dementia, which is extremely surprising to those skilled in the art.

[0135] In addition, the present application also found that the combination of magnesium salt of salvianolic acid B and Rg1 has significantly higher bioavailability and efficacy when administered sublingually / orally / mucosally, and is very suitable for preparing emergency drugs for sublingual administration of brain diseases.

[0136] Pharmaceutical compositions and uses

[0137] The present invention provides a pharmaceutical composition, wherein the active ingredients of the pharmaceutical composition may include (a) and (b):

[0138] (a) a first active ingredient, which is a pharmaceutically acceptable salt of salvianolic acid; (b) a second active ingredient, which is a ginsenoside, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier.

[0139] Typically, the salvianolic acids that can be used in the present invention include but are not limited to: salvianolic acid B, salvianolic acid A, salvianolic acid C, lithospermic acid, rosmarinic acid, danshensu, protocatechuic aldehyde, yunnanic acid A, ferulic acid, salvianolic acid D, salvianolic acid E, salvianolic acid F, salvianolic acid G, salvianolic acid L, caffeic acid, isoferulic acid, rosmarinus officinalis, salviaflaside, protolithospermic acid, salvinal, dimethyl lithospermate, etc., preferably salvianolic acid B or salvianolic acid A.

[0140] Typically, ginsenosides that can be used in the present invention include but are not limited to: ginsenosides Ra3, Rb1, Rb2, Rb3, Rc, Rd, Rg3, F, CK, Mc, Rg3, Rh2, Re, Rg1, Rg2, Rh1, F1, R1; notoginsenosides R1, R2, R3, R6, M, N, Rt, T3, FP1, N, Rw1, etc., preferably ginsenoside Rg1 or ginsenoside Rb1.

[0141] Preferably, the pharmaceutical composition of the present invention comprises: (a) a first active ingredient, which is a pharmaceutically acceptable salt of salvianolic acid B; (b) a second active ingredient, which is ginsenoside Rg1, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier. The pharmaceutically acceptable salt of salvianolic acid B is selected from the group consisting of salvianolic acid B magnesium salt, salvianolic acid B calcium salt, or a combination thereof. More preferably, the first active ingredient is salvianolic acid B magnesium salt, and the second active ingredient is ginsenoside Rg1.

[0142] Preferably, the pharmaceutical composition of the present invention comprises: (a) a first active ingredient, which is a pharmaceutically acceptable salt of salvianolic acid A; and (b) a second active ingredient, which is ginsenoside Rh2, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier.

[0143] Preferably, the pharmaceutical composition of the present invention comprises: (a) a first active ingredient, which is a pharmaceutically acceptable salt of salvianolic acid A; and (b) a second active ingredient, which is ginsenoside Rb1, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier.

[0144] Preferably, the pharmaceutical composition of the present invention comprises: (a) a first active ingredient, which is a pharmaceutically acceptable salt of rosmarinic acid; and (b) a second active ingredient, which is ginsenoside Rb1, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier.

[0145] The present application unexpectedly discovered that the salt of salvianolic acid B (especially the magnesium salt) significantly improves the brain protection and therapeutic effect on brain diseases compared to the combination of salvianolic acid B free acid and Rg1. This is not only reflected in cerebrovascular diseases such as stroke, but it is also unexpectedly discovered that the combination of salvianolic acid B salt / Rg1 has a significantly improved and unexpected therapeutic effect on degenerative diseases represented by Alzheimer's disease and vascular dementia.

[0146] The "prevention" and "treatment" described in the present invention include delaying and stopping the progression of the disease, or eliminating the disease, and do not require 100% inhibition, elimination and reversal. In some embodiments, the composition or pharmaceutical composition of the present invention prevents, alleviates, inhibits and / or reverses ischemia-reperfusion injury by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the level observed in the absence of the composition or pharmaceutical composition of the present invention.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0153] 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.

[0154] A preferred dosage form is a formulation for oral administration, such as a sublingual dosage form or a buccal mucosal dosage form, such as an orally disintegrating tablet, a mucoadhesive tablet, a sublingual tablet, a buccal tablet, a chewable tablet, an effervescent tablet, a solution, and the like. Furthermore, a person of ordinary skill in the art can make various modifications to the tablet as needed. It can be an orally dispersible formulation, such as an orally disintegrating film, an orally disintegrating tablet, a suspension, a suspension tablet, a rapidly disintegrating tablet, an orally disintegrating granule, an orally disintegrating lozenge, a sublingual tablet, a powder, and / or a chewable tablet. Taking into account the circumstances under which the pharmaceutical formulation is administered, portability, and various purposes, the pharmaceutical formulation according to the present disclosure can be prepared as a suspension formulation, an orally disintegrating film formulation, a rapidly disintegrating tablet, or an orally disintegrating granule. In particular, 80% or more of the formulation can be dissolved, dispersed, or disintegrated within 10 minutes or 5 minutes after oral administration.

[0155] The term orally dissolving film refers to a preparation that adheres to the oral cavity, such as on the tongue, on the oral mucosa, under the tongue, etc. When the orally administered pharmaceutical preparation according to the present disclosure is prepared as an orally dissolving film, it may contain a polymer to form a film, such as pullulan, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), polyvinyl pyrrolidone (PVP), starch, polyethylene glycol-polyvinyl alcohol copolymer, copovidone, hydroxyethyl cellulose, hydroxypropyl starch, polyethylene oxide, poloxamer or a mixture thereof. Based on the weight of the dry film, the content of the polymer may be 20% to 50% by weight, but is not limited thereto.

[0156] Preferably, the oral administration preparation taste masking agent is such as β-cyclodextrin, α-cyclodextrin and γ-cyclodextrin, or their derivatives containing hydroxypropyl or methyl groups. Cyclodextrin can not only mask the bitter taste, but also help to evenly disperse the active ingredient.

[0157] 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.

[0158] 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.

[0159] 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 the salt 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 being 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.

[0160] Preparation method

[0161] The present invention provides a method for preparing a pharmaceutically acceptable salt of salvianolic acid, comprising the steps of: reacting free salvianolic acid with a substance containing a desired cation in water or an aqueous solvent to generate a pharmaceutically acceptable salt of salvianolic acid.

[0162] Preferably, the present invention provides a method for preparing a pharmaceutically acceptable salt of salvianolic acid B, comprising the steps of: reacting free salvianolic acid B with a substance containing a desired cation in water or an aqueous solvent to generate a pharmaceutically acceptable salt of salvianolic acid B.

[0163] More preferably, the present invention provides a method for preparing magnesium salt of salvianolic acid B, comprising the steps of: reacting free salvianolic acid B with an inorganic salt or inorganic base containing magnesium ions in water or an aqueous solvent to produce magnesium salt of salvianolic acid B.

[0164] In another preferred embodiment, the inorganic salt or inorganic base containing magnesium ions is selected from the following group: magnesium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium hydroxide, magnesium bicarbonate, or a combination thereof.

[0165] In another preferred embodiment, the method comprises the steps of: adding an aqueous solution of free salvianolic acid B to an aqueous solution of an inorganic salt or an inorganic base containing magnesium ions, thereby reacting to form salvianolic acid B magnesium salt.

[0166] In another preferred embodiment, the molar ratio of free salvianolic acid to the inorganic salt or inorganic base containing magnesium ions is 1:0.98-1.02, preferably, 1:0.99-1.01, and more preferably, 1:1.

[0167] In another preferred embodiment, the method further comprises the steps of: loading the reaction solution onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, 15% methanol-water solution, and 20% methanol-water solution in a gradient manner, collecting fractions, combining fractions with qualified purity, removing methanol, and re-loading the sample after methanol removal onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, and 20% methanol-water solution in a gradient manner, combining fractions with qualified purity, and drying to obtain magnesium salt of salvianolic acid B.

[0168] The present invention also provides a method for preparing the calcium salt of salvianolic acid B, comprising the steps of: reacting free salvianolic acid B with an inorganic salt or inorganic base containing calcium ions in water or an aqueous solvent to generate the calcium salt of salvianolic acid B.

[0169] In another preferred embodiment, the inorganic salt or inorganic base containing calcium ions is selected from the following group: calcium carbonate, calcium sulfate, calcium nitrate, calcium hydroxide, or a combination thereof.

[0170] In another preferred embodiment, the method comprises the steps of: adding an aqueous solution of free salvianolic acid B to an aqueous solution of an inorganic salt or an inorganic base containing calcium ions, thereby reacting to generate salvianolic acid B calcium salt.

[0171] In another preferred embodiment, the molar ratio of free salvianolic acid to the inorganic salt or inorganic base containing calcium ions is 1:0.98-1.02, preferably, 1:0.99-1.01, and more preferably, 1:1.

[0172] In another preferred embodiment, the method further comprises the steps of: loading the reaction solution onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, 15% methanol-water solution, and 20% methanol-water solution in a gradient manner, collecting fractions, combining fractions with qualified purity, removing methanol, and re-loading the sample after methanol removal onto a C18 reverse phase chromatography column, sequentially eluting with pure water, 10% methanol-water, and 20% methanol-water solution in a gradient manner, combining fractions with qualified purity, and drying to obtain salvianolic acid B calcium salt.

[0173] Generally, the purity in the “qualified purity” refers to a purity standard defined according to needs, which can be selected according to actual needs, such as purity ≥80%, preferably ≥90%, more preferably ≥95%, 98%, ≥99% or 99.5%.

[0174] Furthermore, the present invention also provides a method for preparing the pharmaceutical composition of the present invention, comprising the steps of:

[0175] The first active ingredient, the second active ingredient and the pharmaceutically acceptable carrier are uniformly mixed in any order to obtain the pharmaceutical composition.

[0176] The main advantages of the present invention include:

[0177] The present invention unexpectedly discovered for the first time that, compared with the combination of free salvianolic acid B and ginsenoside Rg1, the combination of a specific salt of salvianolic acid B and ginsenoside Rg1 has significantly improved synergistic effects and therapeutic effects in preventing or treating brain diseases, thereby providing a drug with better efficacy.

[0178] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0179] Table 1 Abbreviations

[0180] abbreviation meaning SalB Free salvianolic acid B SalB-Ca Salvianolic acid B calcium salt MLB Salvianolic acid B magnesium salt SalB / Rg1 Combination of free salvianolic acid B and ginsenoside Rg1 SalB-Ca / Rg1 Combination of salvianolic acid calcium salt and ginsenoside Rg1 MLB / Rg1 Combination of magnesium salt of salvianolic acid B and ginsenoside Rg1

[0181] General Methods

[0182] 1. Experimental Materials and Instruments

[0183] 1. Materials: Danshen tablets, methanol (analytical grade), acetonitrile (analytical grade), phosphoric acid (analytical grade); column chromatography silica gel; magnesium carbonate; chromatographic grade acetonitrile; hair clips; surgical scissors, small curved forceps, ophthalmic scissors, and arteriolar clamps; sutures and needles; medical absorbent cotton and cotton swabs (Sinopharm Chemical Reagent Co., Ltd.); syringes; suture plugs (Shanghai Yuyan Scientific Instrument Co., Ltd., model: M8510); aluminum foil; 6-well cell culture plates (NUNC); microporous filter membranes (Millex-GP Filter Unit); Shutai 50 (Vickers Co., Ltd., France); iodophor (Shanghai Likang Disinfection High-Tech Co., Ltd.); 2,3,5-triphenyltetrazolium chloride (Sigma); sodium chloride injection (Heilongjiang Qitai Animal Health Products Co., Ltd.); 75% ethanol (Sinopharm Chemical Reagent Co., Ltd.). The storage conditions of salvianolic acid B, salvianolic acid B magnesium salt (MLB), salvianolic acid B calcium salt (SalB-Ca) and ginsenoside Rg1 were all -20°C and kept away from light.

[0184] 2. Instruments: medium-pressure C18 reverse-phase chromatographic column, high-pressure C18 reverse-phase chromatographic column, HPLC analytical column, medium-pressure preparative instrument, analytical high-performance liquid chromatography instrument, preparative high-performance liquid chromatography instrument, ultrasonic cleaner, rotary evaporator, vacuum drying oven, freeze dryer, low-temperature refrigerator, vacuum pump, magnetic stirrer, cold light source, animal constant temperature system, electronic balance, electronic balance, vertical pressure steam sterilizer, centrifuge, vortex mixer, ultra-low temperature refrigerator, stereo microscope, electric blast drying oven.

[0185] 3. Experimental Animals: SD rats and C57BL / 6 mice were housed in the SPF animal facility of the Shanghai Institute of Materia Medica with free access to food and water, a 12-hour light-and-dark cycle, and a temperature controlled at 22 ± 2°C. Experiments were conducted after three days of acclimatization. All animal care and experimental procedures were approved by the Experimental Animal Committee of the Shanghai Institute of Materia Medica and strictly adhered to the Guide for the Care and Use of Laboratory Animals.

[0186] 2. General model preparation and testing methods

[0187] 1. Establishment of Permanent Cerebral Ischemia Animal Model

[0188] 1.1 Preoperative preparation

[0189] Before surgery, sterilize surgical instruments, cotton swabs, and other surgical supplies in a pressure cooker at 120°C for 30 minutes and set aside. Take out the test solution needed for the day from a -20°C refrigerator and allow it to return to room temperature. Weigh and conduct behavioral tests on all experimental animals before surgery. Clean and disinfect the surgical area and prepare surgical supplies.

[0190] 1.2 Surgical anesthesia

[0191] Rats or mice were anesthetized with Zotai 50 via intraperitoneal injection. Anesthesia was maintained until the animal was unable to turn over freely. After preparing the neck with a razor and depilatory cream, the animal was secured to a sterile surgical board and the neck was disinfected again with iodine.

[0192] 1.3 Establishing a permanent cerebral ischemia animal model by occluding the middle cerebral artery

[0193] Using ophthalmic scissors, the skin of the animal's neck was incised along the midline, separating the subcutaneous tissue and fat to expose the deep cervical fascia and anterior tracheal muscles. Care was taken to avoid nerve damage during vascular dissection. After isolating the carotid artery, a 5-0 silk suture of appropriate length was passed proximally to occlude blood flow to the left common carotid artery. A small incision was made proximally at the common carotid artery using microscissors, and a silicone suture was gently inserted. The insertion depth and resistance of the suture were used to determine whether the suture had reached the bifurcation of the anterior and middle cerebral arteries. The suture was secured with a slipknot at the distal end of the common carotid artery. According to the experimental design, samples were obtained 24 hours after occluding the middle cerebral artery to evaluate the protective effect during the acute phase of infarction; 7 days to evaluate the effect during the subacute phase of infarction; and 28 days to evaluate the chronic phase of infarction and the therapeutic effects on diseases such as cerebral atrophy, vascular dementia, and Alzheimer's disease.

[0194] 2. Establishment of Reperfusion Cerebral Ischemia Animal Model

[0195] An intraperitoneal injection of Zolpidem 50 was administered. After anesthesia, the animal was shaved from the neck to the mandible with a razor. The animal was tied to a mouse cage with a leather tether and disinfected with iodine-containing cotton balls. The skin on the right side of the neck was cut open. The subcutaneous connective tissue between the two tympanic glands was dissected mid-sentence, and the anterior cervical muscles between the right muscle mass were separated. The vessels and nerves adjacent to the common carotid artery (CCA) were carefully dissected, and the CCA was isolated. The bifurcation of the CCA was temporarily occluded with a micro-arterial clamp. The CCA was secured proximally with sutures and the distal end was set with a hanging line. A small oblique incision was made at the upper end of the CCA between the two ligatures using ophthalmic scissors. A suture plug was grasped with ophthalmic forceps and inserted through the small incision in the CCA. After the suture plug was inserted, it was gently tied with a thin thread wrapped around the distal end of the CCA. The suture plug was gently pushed with ophthalmic forceps, and the distance from the bifurcation was measured. The insertion depth and resistance of the silicone suture plug were used to determine whether it had reached the middle cerebral artery (MCA). After trimming off any excess sutures not inserted into the blood vessels, 1 ml of a 4X double-antibody solution was dripped into the wound to prevent infection. The wound was then sutured and placed on a constant temperature pad. After the ischemia reached the specified time (3 hours, 6 hours, 9 hours, 12 hours, or 24 hours), the sutures were removed from the wound and the sutures were pulled out to achieve reperfusion. 1 ml of a 4X double-antibody solution was dripped into the wound to prevent infection. The wound was then sutured and placed on a constant temperature heating pad until the animal regained consciousness and was placed in a breeding room. According to the experimental design, samples were taken 24 hours or 72 hours after reperfusion to evaluate the protective effect in the acute phase of infarction; samples were taken 7 days after reperfusion to evaluate the effect in the subacute phase of infarction; and samples were taken 28 days after reperfusion to evaluate the chronic phase of infarction, as well as the therapeutic effects on cerebral atrophy, vascular dementia, and Alzheimer's disease.

[0196] 3. Behavioral evaluation

[0197] Behavioral assessment is one of the key indicators used by clinicians to assess the severity of ischemic stroke. It determines the extent of neurological impairment based on the patient's behavioral abilities. According to the experimental design, researchers performed behavioral assessments on animals using Longa and Garcia JH scores on days 1, 3, 5, 7, 14, and 28 after establishing either a permanent cerebral ischemia model or a reperfused cerebral ischemia model.

[0198] 3.1 Longa Rating

[0199] The Longa score is a classic method used in animal behavior assessment both domestically and internationally, providing a comprehensive assessment of an animal's motor behavior. Taking left cerebral ischemia as an example, the scoring criteria are as follows: 0 represents normal limb function, indicating no neurobehavioral deficits; 1 represents inability to fully extend the right limb, indicating mild neurobehavioral deficits; 2 represents circling to the right while walking, indicating moderate neurobehavioral deficits; 3 represents falling to the right while walking, indicating severe neurobehavioral deficits; and 4 represents inability to walk spontaneously, indicating loss of consciousness. In other words, higher Longa scores indicate more severe neurobehavioral deficits in rats.

[0200] 3.2 Garcia JH score

[0201] The Garcia JH score is also a commonly used method for animal behavioral assessment. Patients with ischemic stroke may experience significant impairments in motor, sensory, visual, and language functions after onset. The role of post-stroke neurological recovery is not fully understood, and neurological recovery in stroke patients may be related not only to the size of the infarct but also to the location and extent of the defect. Therefore, the Garcia JH score is used to assess motor and sensory function in animals. The scoring criteria for the Garcia JH score are shown in Table 2. The first four items assess motor function, and the last two items assess sensory function. The minimum score is 3, and the maximum is 18. Higher scores indicate better neurological function. Scores of 3-7 indicate severe neurological impairment, 8-12 indicate moderate neurological impairment, and 13-16 indicate mild neurological impairment. The sensory score in this article is a comprehensive assessment of bilateral body tactile reflexes and bilateral whisker tactile reflexes (see the table below).

[0202] Table 2 Garcia JH scoring criteria

[0203]

[0204]

[0205] 3.3 Beam walking test (BWT)

[0206] The horizontal beam test can assess the integration and coordination of an animal's motor function. A horizontal wooden board is placed, and the animals are placed one by one onto it. The animals are then asked to stand upright and cross a narrow elevated beam to a safe platform. The BWT score ranges from 1 to 7: 7 points: able to climb over the balance beam smoothly, with full function of the paralyzed limbs and no obvious signs of neurological damage; 6 points: able to climb over the balance beam with >50% function of the paralyzed limbs; 5 points: able to climb over the balance beam with <50% function of the paralyzed limbs; 4 points: unable to climb over the balance beam smoothly, with a fall rate of <50%; 3 points: unable to climb over the balance beam smoothly, with a fall rate of >50%; 2 points: unable to crawl on the balance beam, but able to sit on it; 1 point: completely unable to crawl, unable to place the hind legs in a horizontal position, and would fall if placed on the balance beam.

[0207] 3.4 Forelimb placement test (FPT) scoring: The evaluation range is 0 to 10, with higher scores indicating better neurobehavioral status of the animal. The scoring rules are as follows:

[0208] The whisker-induced forelimb placement test (also known as the paw-whisker test) can be used to assess asymmetry in the sensorimotor cortex and striatum. The examiner holds the rat's back skin so that the limbs are suspended in the air, and brushes the whiskers against the edge of the table corner to test the activity of the ipsilateral forelimb. Uninjured individuals can quickly place the forelimb on the table. This action is impaired to varying degrees in brain injuries. The rat is tested 10 times on each side, and the percentage of the number of times the forelimb touches the edge of the table corner is the score for that side. The rat is gripped gently, with the forelimbs hanging freely. Before the test, the rat is gently moved up and down to try to relax it. If the rat struggles, has muscle tension, or places its limbs on the experimenter's hand, it will not be counted.

[0209] 4. TTC Staining and Infarct Volume Quantification

[0210] 2,3,5-Triphenyltetrazolium chloride (TTC) is a lipid-soluble, light-sensitive compound. TTC staining is a routine method for detecting ischemic blockage in mammalian tissues such as the brain or heart in preclinical studies.

[0211] After euthanasia, the whole brain was cut into five consecutive 3-mm-thick slices, divided along the midline between the ipsilateral (Ips) and contralateral (Con). After TTC staining, the non-infarcted area appears red, and the infarcted area appears white. Because white infarcted areas can be edematous, correction was performed using the ipsilateral non-infarcted area. After imaging with a stereomicroscope, the infarcted or non-infarcted area of each slice was quantified using Image-ProPlus analysis software, and the volume was calculated based on the area.

[0212] Formula for calculating ipsilateral non-infarct volume

[0213] V (Ips(non)) =[(A1+A2) / 2]*D(mm3)

[0214] V (Ips(non)) is the ipsilateral non-infarct volume of each brain slice (mm3). [A1 and A2 are the ipsilateral non-infarct area of the rostral and caudal sides of the brain slice (mm3) 2 ); D is the slice thickness (mm)]; similarly, the contralateral hemisphere infarction volume V can be calculated Con The total brain infarct volume was the sum of the infarct volumes of the five brain slices.

[0215] Considering the irregularity of the left and right hemisphere volumes caused by edema, the infarct volume ratio was corrected by the brain volume of the contralateral hemisphere to the infarction. Infarct volume (%) = (volume of the contralateral hemisphere to the infarction - ipsilateral non-infarct volume) / volume of the contralateral hemisphere to the infarction × 100%

[0216] That is: V infarc t(%)=[(V Con -V (Ips(non) ) / V Con ] 100%

[0217] V infarct (%) is the percentage of infarct volume, V (Ips(non)) is the ipsilateral non-infarct volume, V con is the contralateral volume.

[0218] 5. Quantification of Brain Atrophy Volume

[0219] After euthanasia, the whole brain was cut into five consecutive 3-mm-thick slices, with the infarcted and contralateral sides divided along the midline. Images were taken using a stereomicroscope, and the hemispheric area of each slice was quantified using Image-Pro Plus analysis software. Hemispheric volume was calculated based on the hemispheric area.

[0220] The brain atrophy volume ratio was corrected by the brain volume of the contralateral hemisphere to the infarction. Atrophy volume ratio (%) = (volume of the contralateral hemisphere to the infarction - volume of the ipsilateral hemisphere) / volume of the contralateral hemisphere to the infarction × 100%

[0221] That is: V atrophy (%)=(V Con -V Ips ) / V Con 100%

[0222] Where V atrophy is the shrinkage volume. Here, V Ips V is the volume of the hemisphere ipsilateral to the infarction; Con is the volume of the hemisphere contralateral to the infarction.

[0223] 6. Brain Tissue Paraffin Sections

[0224] Brain sections fixed with 4% paraformaldehyde for at least 72 hours were placed in an embedding frame. After rinsing with running water for 4 hours, sections were placed in a dehydrator and immersed in 75% ethanol for 2 hours, 95% ethanol for 2 hours, 100% ethanol for 2 hours, xylene for 2 hours, and paraffin for 2 hours. After removal, tissue was embedded using a paraffin embedding machine. Slides were soaked in acetone containing 2% APES for 30 seconds. After removal, they were rinsed twice in distilled water and air-dried in a fume hood. The paraffin tissue was sliced into 4 μm sections using a microtome and mounted flatly on slides for subsequent histological staining and observation.

[0225] 7. Hematoxylin-eosin staining

[0226] HE staining is a commonly used tissue staining method for observing pathological tissue structure. Hematoxylin stain is alkaline and primarily stains chromatin within the cell nucleus and nucleic acids within the cytoplasm purple-blue. Eosin, an acidic dye, primarily stains components of the cytoplasm and extracellular matrix red.

[0227] Paraffin sections of brain tissue were heated in a slide oven set at 65°C for 60 minutes. The sections were then sequentially treated with xylene for 15 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 75% ethanol for 5 minutes, followed by a 5-minute rinse in distilled water. The sections were then stained in hematoxylin solution for 15 minutes, rinsed in running water for 5 minutes, and then differentiated in 1% hydrochloric acid-ethanol solution for 3-5 seconds (the appropriate degree of differentiation was observed under a microscope). The sections were then rinsed in running water for 5 minutes. The sections were then stained in eosin solution for 5 minutes, removed, and rapidly rinsed in running water for 1 second. The sections were then sequentially treated with 75% ethanol for 30 seconds, 95% ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and xylene for 15 minutes. The sections were then removed and mounted with neutral resin. Stained images of the brain, cortex, and striatum were obtained using a pathology section fluorescence scanner.

[0228] 8. Nissl staining

[0229] Nissl staining is a staining method used to assess neuronal count by staining Nissl bodies in the neuronal cytoplasm. Neurons appear bluish-purple after staining and are often used to visualize the basic neural structures of the brain or spinal cord. Large and numerous Nissl bodies in neurons indicate robust protein synthesis. Conversely, when neurons are damaged, the number of Nissl bodies decreases or even disappears.

[0230] Paraffin sections of brain tissue were heated in a slide oven set at 65°C for 60 minutes. The sections were then sequentially treated with xylene for 15 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 75% ethanol for 5 minutes, followed by a 5-minute rinse in distilled water. The sections were then stained with Nissl stain for 3-10 minutes, washed twice with distilled water, and immersed in anhydrous ethanol for 5 seconds. After clearing in xylene for a few seconds, the sections were removed and mounted with neutral resin. Stained images of the whole brain, cortex, and striatum were obtained using a pathology section fluorescence scanner. Finally, the number of cells in each region was counted using Image J software.

[0231] 9. Bliss Independent Statistical Model

[0232] The definition of drug synergy is crucial in the fields of pharmacology, toxicology, and medicine. Drug synergy refers to the ability of two or more drugs to enhance each other's effects when used in combination.

[0233] In order to explore the synergistic effect of MLB and Rg1 in treating ischemic stroke, the Bliss independent statistical model was used to illustrate the statistical method using the infarct volume index as an example. First, the following calculations were performed: V MCAO 、V MLB 、V Rg1 and V MLB / Rg1 represent the infarct volumes of MCAO, MLB, Rg1 and MLB / Rg1 groups, respectively. MLV , I Rg1 and I MLB / Rg1 represent the ratios of the infarct volumes of the MLV, Rg1, and MLV / Rg1 groups to those of the MCAO group, respectively. The I values were calculated as shown in formulas 1, 2, and 3.

[0234] I MLB =(V MLB / V MCAO )×100%(Formula 1)

[0235] I Rg1 =(V Rg1 / V MCAO )×100%(Formula 2)

[0236] I MLB / Rg1 =(V MLB / Rg1 / V MCAO )×100%(Formula 3)

[0237] The synergistic effect of MLB and Rg1 in the treatment of ischemic stroke was evaluated using the Bliss independence statistical model. Independence was assumed to be the theoretical value of synergy calculated for MLB and Rg1. Based on the Bliss independence statistical model, independence was calculated as shown in Equation 4.

[0238] Independence=I MLB ×I Rg1 (Formula 4)

[0239] In order to simplify the statistical test of the synergistic effect, after the Kolmogorov-Smirnov test to see if the data conform to the normal distribution, the data were transformed on the logarithmic scale to obtain the additive equation shown in Formula 5.

[0240] InV SalB + InV Rg1 -InV MCAO -InV SalB / Rg1 > 0 (Formula 5)

[0241] 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. MLB / Rg1 If there is a statistical difference between MLB and Independence, it is considered that MLB and Rg1 have a synergistic effect.

[0242] 10. Small Animal Magnetic Resonance Imaging (MRI)

[0243] 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.

[0244] T2-weighted imaging (T2WI): Whole-brain T2WI was acquired with a rapid acquisition with relaxation enhancement (RARE) sequence, with a flip angle of 90°, a repetition time (TR) of 2500 ms, an echo time (TE) of 6.5 ms, and a spatial resolution of 0.063 × 0.063 × 0.5 mm3. For T2AMP, a multislice multiecho (MSME) sequence was used with a flip angle of 90°, a TR of 2200 ms, and an effective TE of 30, ranging from 7.5 ms to 225 ms.

[0245] 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, spatial resolution = 0.08 × 0.08 mm2, and slice thickness = 0.8 mm.

[0246] Arterial spin labeling (ASL): ASL images were acquired using a drift-corrected steady pulsed imaging and labeling (dSPIL) sequence with a time interval of 3.0 ms, a time interval of 5000 ms, and 32 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 T2-weighted images. Signal intensity data were fitted to a monoexponential decay curve using a least-squares algorithm, and whole-brain T2-relaxation times were calculated based on T2-MAP. Cerebral blood flow (CBF) images were processed using a custom program written in MATLAB. ROIs for different MRI modalities were manually created using ITK-SNAP software. Method for evaluating cerebral blood flow: The brain tissue is divided into the infarct area, the infarct edge area and the contralateral area, and the CBF value in the area is measured.

[0247] 11. Activated Partial Thromboplastin Time (APTT)

[0248] A four-channel semi-automatic coagulation analyzer (MC-4000, Metron GmbH, Germany) was used to detect the activated partial thromboplastin time (APTT). The activated partial thromboplastin time (APTT) detection kit was purchased from Beijing Wokai Biotechnology Co., Ltd.; the reaction cup was purchased from Taizhou Kangjin Medical Equipment Co., Ltd., KB005), and the operation was carried out according to the instrument and reagent instructions.

[0249] 12. Cardiac function testing

[0250] The MPA Cardiac Function Analysis System (ALC-MPA, Shanghai Alcott Biotechnology Co., Ltd.) uses sensors to monitor, record, store, and analyze changes in cardiac function and hemodynamics in real time. Before the experiment begins, the PE catheter and pressure sensor must be filled with normal saline, ensuring that no bubbles remain. If bubbles are present, they must be ejected by gently pushing the syringe while flicking, allowing the water flow to dislodge them. After anesthesia, the animal is shaved from the neck to the mandible with a razor. The animal is strapped to a mouse board and disinfected with iodine-containing cotton balls. The skin on the right side of the neck is incised, and the subcutaneous connective tissue between the two tympanic glands is dissected mid-sentence. The anterior cervical muscles between the right muscle mass are separated. The common carotid artery (CCA) is carefully dissected from the surrounding vessels and nerves, and the CCA is isolated. The distal end is ligated with a 4-0 suture, and the proximal end is clamped with an artery clamp. A suture is placed on the proximal end. The proximal tee is opened and the distal end is closed. The PE catheter is filled with 10% sodium heparin and placed flat on a table. Cut a small incision on the CCA, insert the PE catheter, tie the line at the proximal end to the artery where the PE catheter is inserted, open the arterial clamp and continue to insert the PE catheter to collect cardiac function indicators.

[0251] 13. Blood oxygen saturation detection

[0252] A PowerLab system (PowerLab8 / 30, AD Instruments) was used to connect the oximeter and the blood oxygen probe, and the infrared probe of the blood oxygen probe was clamped on the sole of the animal's left hind foot. Data were collected according to the instrument instructions.

[0253] 14. Animal culling conditions

[0254] Animals that died of surgical reasons before drug administration;

[0255] Animals that died of subarachnoid hemorrhage after drug administration. Animals with model failure: Animals in which the suture plug was not inserted long enough, resulting in an infarct volume less than 10%, or the suture plug did not enter the internal carotid artery during modeling.

[0256] 15. Animal Addition Conditions

[0257] For animals that meet the elimination criteria, a corresponding number of animals need to be added to supplement the group to ensure that there are 8 animals in each group.

[0258] 16. Statistics

[0259] Data are presented as x ± SEM. After confirming homogeneity of variance, differences between groups were compared using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. Statistical differences between two groups were compared using the t-test. Ordinal data, such as the Longa and Garcia scores, were compared using the rank-sum test. Synergistic effects were analyzed using the Bliss independent model. P < 0.05 was considered statistically significant.

[0260] Preparation Example

[0261] Preparation method of magnesium salt, calcium salt and free salvianolic acid B

[0262] 1. Extraction of Free Salvianolic Acid B (SalB): 200g of Danshen slices were sonicated twice with 10-fold deionized water. The combined aqueous solution was filtered through a Büchner funnel (30 cm diameter, 10 L suction flask) lined with normal-phase silica gel. After filtration, the silica gel was rinsed with a small amount of deionized water. The combined aqueous solution, approximately 5 L in total, was applied to a medium-pressure C18 reverse-phase column (60 x 450 mm) and eluted with a gradient of 10%, 20%, and 30% methanol, with each gradient volume exceeding 1000 mL. Fractions were collected every 100 mL.

[0263] Each fraction was analyzed by HPLC. HPLC conditions: acetonitrile-0.3% phosphoric acid (30:70) and detection wavelength: 286 nm. The retention time of salvianolic acid B was approximately 8 minutes. Fractions containing salvianolic acid B were combined. The eluent was concentrated and subjected to high-pressure preparative chromatography using acetonitrile:0.3% phosphoric acid (30:70) as the mobile phase and detection wavelength: 286 nm to separate and prepare salvianolic acid B. The eluent containing salvianolic acid B of acceptable purity was collected and concentrated on a rotary evaporator at 40°C to remove the acetonitrile. Approximately 2 L of free salvianolic acid B solution was prepared. This solution was applied to a C18 reverse-phase column (15 x 100 mm) and eluted with pure methanol to obtain approximately 50 mL of concentrated solution. This was concentrated on a rotary evaporator at 40°C to remove the methanol and then dissolved in a small amount of water to 50 mL. After freeze-drying, pure free salvianolic acid B (pale yellow powder) was obtained.

[0264] 2. Preparation of magnesium salt of salvianolic acid B (MLB): Dissolve 60 mg of magnesium carbonate in 1000 mL of deionized water. Heat and stir to accelerate dissolution at approximately 80°C. Allow to cool and store in a refrigerator at approximately 4°C. Stir the solution containing free salvianolic acid B into the magnesium carbonate solution.

[0265] The salvianolic acid B magnesium salt solution that has reached the reaction endpoint is subjected to a medium-pressure C18 reverse-phase chromatography column (60 x 450 mm) and gradient eluted with 1000 mL of pure water, 1000 mL of 10% methanol solution, 2000 mL of 15% methanol solution, and 1000 mL of 20% methanol solution, collecting 100 mL fractions. The fractions are analyzed by HPLC, and the salvianolic acid B magnesium salt of acceptable purity is combined and the methanol is removed by rotary evaporation at 40°C. The salvianolic acid B magnesium salt solution after methanol removal is again subjected to a medium-pressure C18 reverse-phase chromatography column (40 x 400 mm) and gradient eluted with 500 mL of pure water, 500 mL of 10% methanol solution, and 500 mL of 20% methanol solution. The fractions are analyzed by HPLC, and the salvianolic acid B magnesium salt solution of acceptable purity, approximately 500 mL, is combined and concentrated by rotary evaporation at 40°C. The concentrate is frozen in a -30°C refrigerator and lyophilized the next day for 2-3 days. After freeze-drying, the product was dried in a vacuum oven at 40°C for 48 hours. The HPLC purity was 99.1%.

[0266] 3. Preparation of Salvianolic Acid B Calcium Salt (SalB-Ca): Dissolve 100 mg of calcium bicarbonate in 1000 mL of deionized water. Heat and stir to accelerate dissolution at approximately 80°C. Allow to cool and store in a refrigerator at approximately 4°C. Stir the solution containing free salvianolic acid B into the calcium bicarbonate solution.

[0267] The salvianolic acid B calcium salt solution that has reached the reaction endpoint is subjected to a medium-pressure C18 reverse-phase chromatography column (60 x 450 mm) and gradient eluted with 1000 mL of pure water, 1000 mL of 10% methanol solution, 2000 mL of 15% methanol solution, and 1000 mL of 20% methanol solution, collecting 100 mL fractions. The fractions are assayed by HPLC, and the salvianolic acid B calcium salt of acceptable purity is combined and the methanol is removed by rotary evaporation at 40°C. The salvianolic acid B calcium salt solution after methanol removal is again subjected to a medium-pressure C18 reverse-phase chromatography column (40 x 400 mm) and gradient eluted with 500 mL of pure water, 500 mL of 10% methanol solution, and 500 mL of 20% methanol solution. The fractions are assayed by HPLC, and the salvianolic acid B calcium salt solution of acceptable purity is combined to approximately 500 mL, concentrated by rotary evaporation at 40°C. The concentrate is frozen in a -30°C refrigerator and lyophilized the next day for 2-3 days. After freeze-drying, the product was dried in a vacuum oven at 40°C for 48 hours. The HPLC purity was 99.5%.

[0268] (4)-OES refers to an inductively coupled plasma optical emission spectrometer, which can be used for qualitative and quantitative analysis of more than 70 metal elements and some non-metal elements in samples from geology, environmental protection, chemical industry, biology, medicine, food, metallurgy, agriculture, etc. The ICP-OES test results are as follows:

[0269]

[0270] " / " detection value is less than 0.01%

[0271] Example 1. Comparative study on the effects of free salvianolic acid B (SalB), magnesium salt of salvianolic acid B (MLB), and calcium salt of salvianolic acid B (SalB-Ca)

[0272] Using an animal model of permanent cerebral ischemia, different combinations were investigated for their ability to protect rats during the acute phase of 24-hour ischemia. Simulating a clinical scenario, 3 hours after middle cerebral artery occlusion, the test substance was injected into the tail vein at a volume of 10 ml / kg according to the experimental animal grouping and dosing schedule. Samples were collected 24 hours after surgery. This experiment was conducted in a double-blind manner. The required injection reagents were prepared before the experiment began and the blind was opened after all statistically analyzed indicators were complete. The weight ratio of SalB, MLB, and SalB-Ca to Rg1 was 5:2.

[0273] Group (n=8) Injection reagents Sham Group 0.9% sodium chloride injection MCAO group 0.9% sodium chloride injection SalB / Rg1 group Free SalB / Rg1 combination (10 mg / kg) MLB / Rg1 Group SalB magnesium salt / Rg1 combination (10 mg / kg) SalB-Ca / Rg1 group SalB calcium salt / Rg1 combination (10 mg / kg)

[0274] 1. Survival rate

[0275] The survival rates of the sham, MCAO, free SalB / Rg1, SalB-Ca / Rg1 and MLB / Rg1 groups were 100%, 71.4%, 87.5%, 75% and 100%, respectively.

[0276] 2. Infarct volume

[0277] Compared with the MCAO model group (57.7±2.8%), infarct volumes were significantly reduced in the free SalB / Rg1 group (40.1±6.4%, P<0.001), the SalB-Ca / Rg1 group (35.8±7.0%, P<0.001), and the MLB / Rg1 group (30.4±5.9%, P<0.001). Surprisingly, the infarct volume in the SalB-Ca / Rg1 group was further reduced compared with the free SalB / Rg1 group, although the difference was not significant. However, the infarct volume in the MLB / Rg1 group was further significantly reduced compared with the free SalB / Rg1 group (40.1±6.4% versus 30.4±5.9%, P<0.01), indicating that the calcium and magnesium salts of salvianolic acid B have a better ability to cross the blood-brain barrier and exert their therapeutic effects than free salvianolic acid B. Among them, MLB / Rg1 has a better effect in reducing infarct volume and treating stroke ( Figure 1 ).

[0278] These results demonstrate that, compared to free SalB / Rg1, the use of salvianolic acid salts provides superior brain protection, particularly in the MLB / Rg1 group, where the survival rate reached 100%. This is of significant significance for reducing the mortality rate of cerebral infarction. Those skilled in the art understand that, to treat brain diseases, drugs must penetrate the blood-brain barrier to exert their effects. The drugs of the present invention also need to penetrate the blood-brain barrier after intravenous injection to reach the infarcted area. Surprisingly, while the SalB salts of the present invention have reduced lipid solubility compared to free SalB (typically making it more difficult to penetrate the blood-brain barrier), experiments have demonstrated that the SalB salt / Rg1 combination provides significantly greater brain protection and therapeutic effects, a finding that is highly unexpected to those skilled in the art.

[0279] 3. HE staining

[0280] Middle cerebral artery occlusion causes necrosis or apoptosis of neurons in the corresponding areas of the rat brain's cortex and striatum. We analyzed the morphological and structural changes of neurons in the cerebral cortex and striatum by HE staining. Figure 2 Representative images of HE staining of coronal sections of the brain are shown. The neurons in the cortex and striatum of rats in the sham group were densely arranged, structurally intact, and morphologically normal. In the MCAO group, due to cerebral ischemia, disorganized neurons, neuronal necrosis, and nuclear condensation were observed in the cerebral cortex and striatum. Treatment with free SalB / Rg1 showed a trend toward improving pathological tissue abnormalities after ischemic stroke. Compared with free SalB / Rg1 or SalB-Ca / Rg1, the morphology and structure of neurons in the cerebral cortex and striatum of the MLB / Rg1 group were closer to those of the sham group, indicating that MLB / / Rg1 has a better protective effect on neurons after cerebral ischemia.

[0281] 4. Nissl staining

[0282] We examined the number of surviving neurons in the cerebral cortex, striatum, and hippocampus using Nissl staining. Figure 3 Representative images of Nissl staining of coronal brain sections are shown, showing a significant decrease in the number of surviving neurons in the cerebral cortex, striatum, and hippocampus in the MCAO group compared to the sham group. Compared to the MCAO group, the number of neurons in the cortex, striatum, and hippocampus in the MLB / Rg1 group increased significantly. Furthermore, compared to the number of neurons in the cortex, striatum, and hippocampus in the free SalB / Rg1 and SalB-Ca / Rg1 groups, the MLB / Rg1 group showed a stronger trend in protecting neurons, suggesting that MLB / Rg1 may improve behavioral function in animals after stroke.

[0283] Example 2. Study on the synergistic effect of magnesium salt of salvianolic acid B (MLB) combined with ginsenoside Rg1 in the treatment of ischemic stroke

[0284] To determine whether SalB magnesium salt (MLB) and Rg1 have a synergistic effect, the study strictly followed the requirements of the Bliss independent statistical model to evaluate synergy. Briefly, a permanent cerebral ischemia animal model was used. MLB and Rg1 were combined in a 5:2 ratio. Assessment was performed during the 7-day subacute phase of cerebral ischemia. Rats were randomly divided into a sham operation group (Sham), a model control group (MCAO), an MLB group, an Rg1 group, and an MLB / Rg1 combination group, with an n value of 10 for each group. Both the Sham and MCAO groups were given 0.9% sodium chloride injection. The MLB group received 7.14 mg / kg SalB magnesium salt, the Rg1 group received 2.86 mg / kg Rg1, and the MLB / Rg1 group received 10 mg / kg MLB / Rg1.

[0285] To simulate a clinical scenario, animals were occluded for 3 hours and injected into the tail vein according to the experimental animal grouping and dosing schedule. The injection volume was 10 ml / kg once daily for 7 consecutive days. This study was conducted in a double-blind format. The required injection reagents were prepared before the experiment began and the blind was opened after all indicators were statistically analyzed.

[0286] Group (n=10) Injection reagents Sham Group 0.9% sodium chloride injection MCAO group 0.9% sodium chloride injection MLB / Rg1 Group Salvianolic acid B magnesium salt / Rg1 (10mg / kg) MLB Group Salvianolic acid B magnesium salt (7.14 mg / kg) Rg1 group Rg1 (2.86 mg / kg)

[0287] 1. MLB and Rg1 have a synergistic effect in reducing infarct volume

[0288] Although the efficacy of the SalB magnesium salt / Rg1 combination (MLB / Rg1) in a MCAO rat model has been previously evaluated, it remains unclear whether MLB / Rg1, as a combination, has a synergistic effect with Rg1 in the treatment of ischemic stroke. This is a question that must be answered before MLB / Rg1 can be translated into clinical practice. The Bliss independent statistical model is an important method for evaluating multicomponent drug interactions. By establishing a rat model of permanent middle cerebral artery ischemia and using the Bliss independent statistical model to assess cerebral infarction volume, the synergistic effect of MLB and Rg1 was determined.

[0289] like Figure 4 As shown in (A), after TTC staining, the non-infarcted area of the brain appears red and the infarcted area appears white. Figure 4As shown in (B), compared with the sham group, the MCAO group significantly increased infarct volume (57.1±6.6% versus 0±0%, P<0.001), indicating that a permanent cerebral infarction model in rats has been successfully established. Compared with the MCAO group (57.1±6.6%), the MLB group (48.4±5.6%) and the Rg1 group (53.0±3.8%) showed a downward trend, but without statistical significance. The MLB / Rg1 group (34.4±9.3%, P<0.001) significantly reduced infarct volume, and MLB / Rg1 still significantly enhanced the downward regulation of infarct volume compared with the MLB (P<0.01) and Rg1 groups (P<0.001).

[0290] like Figure 4 (C) The percentage of infarct volume in the different treatment groups relative to the MCAO group is plotted according to the Bliss independence statistical model. Data were normally distributed according to the Kolmogorov-Smirnov test and homogeneity of variance was confirmed by the F test. Relative to the MCAO group, the percentage of infarct volume in the MLB / Rg1 group was 61.2±16.9%, the percentage of infarct volume in the MLB group was 85.9±13.4%, and the percentage of infarct volume in the Rg1 group was 95.2±13.4%. The theoretical effect of combined treatment with MLB and Rg1 was calculated using the Bliss independence statistical model, resulting in a percentage of infarct volume of 82.7±21.7% in the Independence group. Compared with the Independence group, the MLB / Rg1 group showed a significant reduction in infarct volume (P<0.01). Therefore, Bliss independence statistical analysis demonstrated a significant synergistic effect of MLB and Rg1 in reducing infarct volume.

[0291] 2 Compared with MLB or Rg1 alone, MLB / Rg1 enhances the protective effect of neurons

[0292] Middle cerebral artery occlusion causes necrosis or apoptosis of neurons in the corresponding areas of the rat brain's cortex and striatum. We analyzed the changes in neurons and structures in the cerebral cortex and striatum using HE staining. Figure 5Representative HE staining images of coronal sections of the brain are shown, in which the red box area is the cortex and the blue box area is the striatum. The neurons in the cortex and striatum of the rats in the Sham group are tightly arranged, with intact structure and normal morphology. Due to cerebral ischemia in the rats in the MCAO group, disordered arrangement of neurons, neuronal necrosis and nuclear condensation can be observed in the cerebral cortex and striatum. Treatment with MLB or Rg1 showed a trend of improving pathological tissue abnormalities after ischemic stroke. Compared with MLB or Rg1 alone, the morphology and structure of neurons in the cerebral cortex and striatum of the MLB / Rg1 group were closer to those of the Sham group, indicating that MLB / Rg1 has a better protective effect on neurons after cerebral ischemia.

[0293] 3. MLB / Rg1 improves neural function compared to MLB or Rg1 alone

[0294] Ischemic stroke is a highly disabling disease, in which neuronal necrosis or apoptosis leads to the development of vascular dementia. Vascular dementia is primarily manifested by cognitive impairment, including decreased attention, mobility, situational analysis, and decision-making abilities. The cerebral cortex is the highest-level center for controlling somatic movement, and the striatum primarily regulates muscle tone and coordinates various delicate and complex movements. Assessing the severity of a patient's neurological dysfunction through behavioral testing is an important diagnostic tool for clinicians. Therefore, we evaluated rat neurological function and neurobehavior using the Garcia JH and Longa scores on days 1, 3, 5, and 7 after MCAO surgery.

[0295] like Figure 6As shown in the data, compared with the Sham group, the Garcia JH scores of the MCAO group were significantly reduced on the 1st day (9.1±0.6 versus 18±0, P<0.001), 3rd day (8.7±0.8 versus 18±0, P<0.001), 5th day (8.0±1.2 versus 18±0, P<0.001), and 7th day (7.6±0.9 versus 18±0, P<0.001) after surgery, indicating that the motor and sensory functions of the rats were severely impaired, and MLB / Rg1 treatment significantly improved the neurological dysfunction. Compared with MLB alone, MLB / Rg1 significantly upregulated the Garcia JH scores of rats on day 1 (11.6±0.7 versus 10.6±0.7, P<0.01), day 3 (11.0±0.8 versus 9.6±0.7, P<0.01), day 5 (10.4±0.5 versus 8.4±1.3, P<0.01), and day 7 (10.6±0.5 versus 8.5±1.0, P<0.01). Compared with Rg1 alone, MLB / Rg1 significantly upregulated the Garcia JH scores of rats on day 1 (11.6±0.7 versus 10.1±1.4, P<0.001), day 3 (11.0±0.8 versus 9.3±0.5, P<0.01), day 5 (10.4±0.5 versus The GarciaJH scores at 7 days (9.2±0.8, P<0.05) and 7 days (10.6±0.5 versus 9.2±0.4, P<0.001) were significantly improved, indicating that MLB / Rg1 could improve the neurological function of rats.

[0296] 4. MLB / Rg1 improves behavioral performance compared to either MLB or Rg1 alone

[0297] We used the Longa score to comprehensively evaluate the behavioral ability of rats. Figure 7As shown in the data, compared with the Sham group, the Longa scores of the MCAO group were significantly increased on the 1st day (2.1±0.3 versus 0±0, P<0.001), 3rd day (2.1±0.4 versus 0±0, P<0.001), 5th day (2.6±0.5 versus 0±0, P<0.001), and 7th day (2.8±0.4 versus 0±0, P<0.001) after surgery, indicating that the rats had severe behavioral deficits, which were significantly improved after MLB / Rg1 treatment. Compared with MLB alone, MLB / Rg1 significantly downregulated the Longa scores of rats on day 3 (1.1±0.4 versus 1.9±0.4, P<0.01) and day 5 (1.7±0.5 versus 2.3±0.5, P<0.05). Compared with Rg1 alone, MLB / Rg1 significantly downregulated the Longa scores of rats on day 3 (1.1±0.4 versus 1.8±0.4, P<0.01) and day 5 (1.7±0.5 versus 2.2±0.4, P<0.05). The Longa score results indicate that MLB / Rg1 can improve the behavioral ability of rats.

[0298] 5 Compared with MLB or Rg1 alone, MLB / Rg1 enhances the inhibitory effect on neuronal damage

[0299] We detected the number of surviving neurons in the cerebral cortex and striatum by Nissl staining. Figure 8 Representative images of Nissl staining of coronal brain sections (A) and quantification of the number of surviving neurons (B) are shown. Figure 8 (B) Quantitative results showed that the number of neurons in the cortex (223.6±30.2 versus 363.2±22.2, P<0.001) and striatum (138.2±20.7 versus 249.2±9.0, P<0.001) of the MCAO group was significantly decreased compared with that of the Sham group. Compared with MLB alone, the number of neurons in the MLB / Rg1 group was significantly increased in the cortex (332.4±33.9 versus 287.6±8.9, P<0.05) and striatum (201.6±14.7 versus 172.8±19.6, P<0.05) regions; compared with Rg1 alone, the number of neurons in the MLB / Rg1 group was significantly increased in the cortex (332.4±33.9 versus 267.8±42.1, P<0.05) and striatum (201.6±14.7 versus 174.2±20.9, P<0.05), showing a trend of stronger neuronal protection, suggesting that MLB / Rg1 is related to the improvement of behavioral function of animals after stroke.

[0300] Example 3. Study on the synergistic inhibitory effect of magnesium salt of salvianolic acid B (MLB) combined with ginsenoside Rg1 on vascular dementia

[0301] An animal model of reperfused cerebral ischemia was used, and the pathological changes of vascular dementia were caused by long-term feeding after injury. SD rats with qualified neurological behavior were randomly divided according to body weight, with 10 rats in each group. The weight of the animals after grouping was tested to ensure that there was no difference in animal weight between the groups. An animal model of reperfused cerebral ischemia was used, and the drug was administered immediately after reperfusion 6 hours after middle cerebral artery infarction, and then once a day for 7 consecutive days, and the tail vein was injected according to the experimental animal grouping and dosing schedule; the animals were continuously fed after 7 days of administration until they were collected on the 28th day. In order to meet the energy needs of the animals that were unable to eat due to modeling, 4 ml of enteral nutrition suspension was gavaged daily after modeling to maintain the required energy. Behavioral evaluations were performed 1d, 3d, 5d, 7d, 14d, 21d, and 28d after surgery. The experimental plan is as follows Figure 9 A:

[0302]

[0303]

[0304] like Figure 9 As shown in Figure B, whether it is the overall image of the brain or the slices after TTC staining, the animals in the model group showed significant brain atrophy. After receiving different types of treatment, the degree of atrophy decreased.

[0305] To investigate the effects of MLB / Rg1 on long-term vascular dementia in an ischemia-reperfusion model, we quantified brain atrophy volume. After euthanasia, the rats were cut into five consecutive brain slices 3 mm thick, with the infarcted side and the contralateral side divided along the midline. After photographing with a stereomicroscope, the hemispheric area of each brain slice was quantified using Image-Pro Plus analysis software, and the hemispheric volume was calculated based on the hemispheric area. The results are shown in Figure 2. Figure 10As shown in the results, compared with the normal control group, the model group had a significantly increased atrophy volume (37.81±6.64%), indicating that cerebral vascular occlusion caused significant brain atrophy. Compared with the model control group, 5 mg / kg of MLB / Rg1 (5:2, 10.31±2.95, P<0.01) and 5 mg / kg of MLB / Rg1 (1:10; 21.93±5.51%) significantly reduced atrophy volume. SalB magnesium salt alone (20.83±5.61%) and Rg1 alone (21.93±5.51%) also reduced atrophy volume. However, 5 mg / kg MLB / Rg1 (5:2) further significantly reduced the atrophy volume compared with the MLB group (P<0.01) and the Rg1 group (P<0.01), indicating that MLB and Rg1 have a significant synergistic effect in inhibiting brain atrophy and thus treating vascular dementia. However, from the results of SalB magnesium salt / Rg1 (1:10), it can be seen that SalB magnesium salt and Rg1 only have a synergistic effect under a specific preferred ratio.

[0306] Within 28 days after modeling, we conducted a general evaluation of the rats' behavior. The results of the balance beam evaluation on the 28th day were as follows: Figure 11 As shown in Figure 2, the behavioral function of the MCAO model group was the most severely damaged. Compared with the model group, all drug-treated groups were able to improve the behavioral function, but only the 5 mg / kg MLB / Rg1 (5:2) group had a significant difference ( Figure 11 ).

[0307] In order to evaluate the effect of drugs on pathological morphology, the sections were stained with HE ( Figure 12 ), the results showed that consistent with the quantitative results of each brain slice area, 5mg / kg MLB / Rg1 (5:2) showed a decrease in atrophy volume and necrotic cells in the injury site.

[0308] Neuronal morphology and number were assessed using Nissl staining, based on the Nissl staining results of the third brain section ( Figure 13 ), the model group showed obvious nuclear condensation and disappearance of Nissl bodies, and the quantitative results ( Figure 14 ) showed that the model group showed a significant decrease in neurons in both the cortex and striatum compared to the sham group. Compared with MLB, Rg1, and MLB / Rg1 (1:10), 5mg / kg MLB / Rg1 (5:2) significantly reduced neuronal death.

[0309] The Bliss method was used to evaluate the synergistic effect of MLB and Rg1 in reducing infarct volume and inhibiting brain atrophy. The results showed that p < 0.05, with significant differences, indicating that MLB / Rg1 (5:2) has a significant therapeutic effect in improving the progression of neurodegenerative diseases compared with MLB and Rg1, and MLB and Rg1 have a synergistic effect ( Figure 15 ).

[0310] In addition, further experimental results show that the effect of the SalB magnesium salt / Rg1 (5:2) of the present invention in reducing vascular dementia induced by cerebral ischemia reperfusion is significantly better than that of free SalB / Rg1 (5:2) and SalB calcium salt / Rg1 (5:2).

[0311] Example 4. Study on the inhibitory effect of magnesium salt of salvianolic acid B (MLB) combined with ginsenoside Rg1 on vascular dementia

[0312] SD rats were randomly divided into groups based on body weight, and the weight differences of the animals after grouping were tested to ensure that there were no differences in animal weight between groups. An animal model of permanent cerebral ischemia of the middle cerebral artery was used. Drugs were administered 3 hours after infarction and then once daily for 7 consecutive days. Tail vein injections were performed according to the experimental animal grouping and dosing schedule, and samples were collected 28 days after surgery. This experiment was double-blind. Drugs were administered by tail vein injection 3 hours, 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days after surgery according to the experimental animal grouping and dosing schedule, for a total of seven doses. Behavioral evaluations were performed 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, and 28 days after surgery.

[0313] Group (n=10) Injection reagents Sham (sham operation control group) 0.9% sodium chloride injection MCAO (model control group) 0.9% sodium chloride injection 5mg / kg MLB / Rg1 group 5mg / kg MLB / Rg1 (SalB magnesium salt: Rg1 = 5:2) 10mg / kg MLB / Rg1 group 10 mg / kg MLB / Rg1 (SalB magnesium salt: Rg1 = 5:2)

[0314] like Figure 16 As shown in the gross image of the brain and the slices after TTC staining, the model group animals showed significant brain atrophy. After receiving different types of treatment, the degree of atrophy was significantly reduced ( Figure 17 ).

[0315] In order to evaluate the pathological improvement effect of MLB / Rg1 (5:2) on vascular dementia, HE staining was performed on brain sections. The results showed that consistent with the quantitative results of each brain section area, 5 mg / kg MLB / Rg1 showed a decrease in atrophy volume and necrotic cells in the injury site ( Figure 18 ).

[0316] Because stroke can lead to long-term cognitive impairment, we chose to use Nissl staining to evaluate the morphology and number of neurons in the hippocampus, a brain region primarily responsible for learning and memory. Based on the Nissl staining results of the fourth brain section ( Figure 19 ), the model group showed obvious nuclear condensation and disappearance of Nissl bodies, and the model group showed a significant decrease in neurons compared with the sham operation group. Compared with the model group, the MLB / Rg1 group showed a significant decrease in neuronal death in the CA3 and dentate gyrus regions of the hippocampus ( Figure 20 ).

[0317] In addition, further experimental results show that the MLB / Rg1 (5:2) of the present invention is significantly better than free SalB / Rg1 (5:2) and SalB-Ca / Rg1 (5:2) in reducing brain atrophy induced by permanent middle cerebral artery infarction.

[0318] Example 5. Protective effects of different ratios of MLB and Rg1 on acute stroke

[0319] SD rats meeting behavioral assessment criteria were randomly assigned based on body weight, with 10 rats per group. Animal weights were checked after grouping to ensure no differences between groups. This study employed a double-blind approach using a reperfused cerebral ischemia model. Drugs were administered via a single tail vein injection 6 hours after reperfusion. Brain infarct volume was quantified 24 hours after reperfusion.

[0320] Group Injection reagents Sham (sham operation control group) 0.9% sodium chloride injection MCAO (model control group) 0.9% sodium chloride injection (1:10)MLB / Rg1 Group 5mg / kg magnesium salt of salvianolic acid B / ginsenoside Rg1 (1:10) (5:5)MLB / Rg1 Group 5mg / kg magnesium salt of salvianolic acid B / ginsenoside Rg1 (5:5) (10:1)MLB / Rg1 group 5mg / kg magnesium salt of salvianolic acid B / ginsenoside Rg1 (10:1)

[0321] Results: The effect of different ratios of MLB / Rg1 on reducing infarct volume was evaluated by TTC staining. The results showed that the degree of reducing infarct volume by MLB / Rg1 (1:10), MLB / Rg1 (5:5), and MLB / Rg1 (10:1) was basically maintained between 15% and 20%. Figure 21 ).

[0322] Example 6: Protective effect of the combination of SalA magnesium salt and Rb1 on acute stroke

[0323] Normal Sprague-Dawley rats were randomly divided according to body weight into groups of 10. Animal weights were checked after grouping to ensure no differences between groups. This experiment was double-blind. A reperfused cerebral ischemia model was used. A single dose of the drug was administered via tail vein injection 6 hours after reperfusion. Behavioral assessments were performed 24 hours after reperfusion. Following behavioral assessments, brain infarct volume was quantified.

[0324] Group Injection reagents Sham (sham operation control group) 0.9% sodium chloride injection MCAO (model control group) 0.9% sodium chloride injection SalA magnesium salt / Rb1 group 5mg / kg magnesium salt of salvianolic acid A / ginsenoside Rb1 (5:5)

[0325] 3. Experimental results: The effect of SalA magnesium salt / Rb1 on infarct volume reduction was evaluated by TTC staining. The results showed that SalA magnesium salt / Rb1 was about 15%. From the behavioral evaluation, SalA-Mg / Rb1 significantly improved the balance beam (BWT) score (1.6±0.2, P<0.05) and the perception ability of Garcia JH (P<0.01) ( Figure 22 ).

[0326] Example 7: Protective effect of the combination of SalA magnesium salt and Rh2 on acute stroke

[0327] Normal Sprague-Dawley rats were randomly divided according to body weight, with 10 rats per group. Animal weights were checked after grouping to ensure no differences between groups. This experiment was double-blind. A reperfused cerebral ischemia model was used. A single dose of the drug was administered via tail vein injection immediately 6 hours after reperfusion. Behavioral assessments were performed 24 hours after reperfusion. After behavioral assessments, brain infarct volume was quantified.

[0328] Group Injection reagents Sham (sham operation control group) 0.9% sodium chloride injection MCAO (model control group) 0.9% sodium chloride injection SalA magnesium salt / Rh2 group 5mg / kg magnesium salt of salvianolic acid A / ginsenoside Rh2 (10:1)

[0329] 3. Experimental results: SalA magnesium salt / Rh2 group reduced infarct volume by approximately 15% as assessed by TTC staining. From the behavioral evaluation, SalA-Mg / Rb1 significantly improved balance beam (BWT) score (1.7±0.19, P<0.05) and Garcia JH perception ability (4.0±0.23, P<0.01) ( Figure 23 ).

[0330] Example 8: Protective effects of different ratios of rosmarinic acid sodium salt / Rb1 combinations on acute stroke

[0331] Normal Sprague-Dawley rats were randomly divided according to body weight, with 10 rats per group. Animal weights were checked after grouping to ensure no differences between groups. This experiment was double-blind. A reperfused cerebral ischemia model was used. A single dose of the drug was administered via tail vein injection immediately 6 hours after reperfusion. Behavioral assessments were performed 24 hours after reperfusion. After behavioral assessments, brain infarct volume was quantified.

[0332] Group Injection reagents Sham (sham operation control group) 0.9% sodium chloride injection MCAO (model control group) 0.9% sodium chloride injection RosA-Na / Rb1 group 5mg / kg rosmarinic acid sodium salt / ginsenoside Rb1 (1:10) RosA-Na / Rb1 group 5mg / kg rosmarinic acid sodium salt / ginsenoside Rb1 (10:1)

[0333] 4. Experimental results: TTC staining was used to evaluate the effect of different ratios of RosA-Na / Rb1 on the reduction of infarct volume. The results showed that RosA-Na / Rb1 had a tendency to reduce infarct volume. Different ratios of RosA-Na / Rb1 had a tendency to improve neurobehavior. The (1:10) RosA-Na / Rb1 group significantly improved the perception ability of Garcia JH (4.0±0, P<0.05) ( Figure 24 ).

[0334] Example 9: Therapeutic Effect of Sublingual Administration of MLB / Rg1 on Stroke

[0335] Normal Sprague-Dawley rats were randomly divided according to body weight, with 10 rats per group. Animal weights were checked after grouping to ensure no differences between groups. This experiment was double-blind. A reperfused cerebral ischemia model was used. Sublingual administration of the drug was performed immediately after reperfusion 6 hours after infarction. Behavioral assessment was performed 24 hours after reperfusion using the Long score, Garcia JH score, forelimb placement test, and balance beam test. Following behavioral assessment, cerebral infarction volume was quantified.

[0336] Group Injection reagents Sham (sham operation control group) 0.9% sodium chloride injection MCAO (model control group) 0.9% sodium chloride injection MLB / Rg1 sublingual administration group 5mg / kg magnesium salt of salvianolic acid B / ginsenoside Rg1 (5:2)

[0337] Experimental results: TTC staining was used to evaluate infarct volume. Compared with the MCAO model group (47.4±4.1), sublingual administration of MLB / Rg1 significantly reduced infarct volume (26.4±3.6, p<0.01); significantly improved neurobehavioral function, and showed a very significant improvement in balance beam BWT score (P<0.05) and Garcia JH perception ability (P<0.01). Figure 25 ).

[0338] Example 10: MLB / Rg1 synergistically inhibits brain edema and improves cerebral blood flow

[0339] The purity of both salvianolic acid B magnesium salt and ginsenoside Rg1 was above 99% as determined by high-performance liquid chromatography. MLB / Rg1 was prepared by mixing salvianolic acid B magnesium salt and ginsenoside Rg1 in a ratio of 5:2. The mixture was prepared with normal saline to the desired concentration and administered via tail vein injection according to the study protocol.

[0340] All mice were evaluated behaviorally before the experiment to ensure that all mice had normal neurobehavioral functions. A reperfusion cerebral ischemia mouse model was used. The experiment set up a sham operation group, a model group, and a magnesium salt of salvianolic acid B / Rg1 group (5 mg / kg MLB / Rg1). The preparation of magnesium salt of salvianolic acid B / Rg1 was a ratio of magnesium salt of salvianolic acid B to Rg1 of 5:2. To ensure the objectivity of the research results, the experiment was conducted in a double-blind manner until the experiment was completed and the conclusion was reached. All mice with normal neurological function were randomly divided into three groups (n=10), namely, a sham operation group (Sham), a cerebral ischemia reperfusion model group (tMCAO), and an MLB / Rg1 group (5 mg / kg MLB / Rg1). Three hours after middle cerebral artery occlusion, reperfusion was achieved by removing the suture plug and administering alteplase. Simultaneously with reperfusion, MLB / Rg1 was administered via the tail vein. MLB / Rg1 was administered a second time 24 hours after reperfusion, a third time 48 hours after reperfusion, and a fourth time 70 hours after reperfusion. Samples were collected at 72 hours after reperfusion, the endpoint of the experiment. The dose of MLB / Rg1 was 5 mg / kg. The model group received injections of normal saline at the same time points, and alteplase was administered at the time of reperfusion. In the sham group, only the common carotid artery was isolated, ligated proximally with sutures, and the wound was immediately sutured. Dosing was administered at the same time as in the other groups, with normal saline administered.

[0341] Edema volume = infarcted hemisphere volume / contralateral hemisphere volume

[0342] To ensure objectivity, the experiment was conducted using a double-blind method. The experimenters prepared the test substances and aliquoted them the day before the experiment began. Researchers not affiliated with the project assigned numbers to each group, replacing the names of the test substances. The blind was not revealed until the experiment was completed and conclusions reached.

[0343] 1. MLB / Rg1 improves the survival rate of reperfused mice

[0344] This study used a mouse cerebral ischemia-reperfusion model prepared by the suture embolization method. The process of suture removal simulates the reperfusion process of mechanical thrombectomy. Over time, reperfused mice continued to die. The 72-hour mortality rate of tMCAO mice exceeded 50%, reaching 45.6%, while MLB / Rg1 significantly improved the 72-hour survival rate, reaching 90% (90% vs. 45.6%, P<0.05) ( Figure 26 ).

[0345] 2MLB / Rg1 improves edema in reperfused mice

[0346] As expected, as infarction occurred, brain slices of reperfused mice swelled to varying degrees, as evidenced by an increase in the volume ratio of the infarcted side to the normal side, while MLB / Rg1 significantly reduced the volume of brain edema. The brain tissue of the mice was weighed and dried, and the weight after drying was weighed again to determine the water content of the mouse brain tissue. It was found that the brain water content of the model group mice was significantly higher than that of the sham group, indicating the occurrence of edema. Compared with the model group, MLB / Rg1 treatment reduced brain edema in tMCAO mice ( Figure 27 ).

[0347] 3MLB / Rg1 increases the number of nerves

[0348] The brain is the highest control center for regulating body movement. Brain damage can lead to decreased balance and stimulate muscle movement. Damage to brain neurons can severely impact neurobehavioral function in mice. Based on the finding that MLB / Rg1 improves neurobehavioral function in mice with stroke, Nissl staining was performed on brain tissue sections to determine whether MLB / Rg1 protects neurons by counting Nissl bodies.

[0349] In the brain tissue images after Nissl staining, the same location in the cerebral cortex of each image was selected for quantification. The number of Nissl-positive cells in the cortex after modeling was significantly decreased compared with the Sham group (P<0.001). The number of Nissl-positive cells in the MLB / Rg1 group was significantly increased compared with the tMCAO group (P<0.05) ( Figure 28 This result corresponds to the above behavioral results, proving that MLB / Rg1 improves the neurobehavior of mice by protecting the nerve cells in the brain.

[0350] 4. MRI imaging revealed that MLB / Rg1 increased cerebral blood flow

[0351] Magnetic resonance imaging (MRI) is a sufficiently sensitive clinical diagnostic tool that can detect abnormalities in brain tissue and its surroundings. 11.7T small animal MRI can achieve non-invasive detection of multiple parameters. This project uses an 11.7T small animal MRI system to evaluate cerebral blood flow. According to the distance from the infarct area, the brain tissue is divided into the infarct core area (Infarct Zone), the border area (Border Zone) and the contralateral area (Contralateral side), and the cerebral blood flow (CBF) of the three areas is evaluated separately ( Figure 29 ).

[0352] By measuring cerebral blood flow, MLB / Rg1 significantly improved CBF in the infarct edge area. Figure 30The CBF value of the MLB / Rg1 group was 63.92±10.73ml / 100g / min, while that of the Model group was only 43.36±11.24ml / 100g / min. There was a significant difference between the two groups (P<0.05).

[0353] Example 11: Synergistic protective effect of MLB / Rg1 on subacute stroke

[0354] Behavioral evaluation: Fifty normal male SD rats were randomly divided into five groups according to their body weight. The weight of the animals after grouping was tested for differences to ensure that there was no difference in the weight of the animals among the groups.

[0355] Group (n=10) Injection reagents Normal control group 0.9% sodium chloride injection Model control group 0.9% sodium chloride injection 5mg / kg MLB / Rg1 group Salvianolic acid B magnesium salt / ginsenoside Rg1 (5:2) MLB Group 3.57mg / kg MLB Rg1 group 1.43mg / kg Rg1

[0356] The normal control group, model control group, 5mg / kg MLB / Rg1 group (5:2), MLB group, and Rg1 group were administered at a volume of 5ml / kg, with the vehicle being 0.9% sodium chloride injection. This study employed a double-blind approach. This study employed a rat model of reperfused cerebral ischemia, with reperfusion performed 24 hours after middle cerebral artery occlusion. This extended the reperfusion window to 24 hours, a highly demanding research system. Reperfusion was performed immediately 24 hours after modeling. Drugs were then administered via tail vein injection seven times, one, two, three, four, five, and six days after reperfusion, according to the experimental animal grouping and dosing schedule. To meet the energy requirements of the animals due to the modeling process, 4ml of enteral nutrition suspension was administered orally twice daily after modeling to maintain energy requirements. Seven days after reperfusion, samples were collected to evaluate the protective effect of extending the reperfusion window to 24 hours on the subacute phase of stroke.

[0357] 1. MLB / Rg1 significantly prolonged the reperfusion time window and had a significant synergistic effect in reducing infarct volume

[0358] TTC staining was used to evaluate infarct volume ( Figure 31 A), quantitative results of infarct volume showed that compared with the MCAO model group (46.07±0.28%), the MLB group alone (39.51±0.65%) and Rg1 alone (37.67±0.88%) showed a trend of decreasing infarct volume; the MLB / Rg1 group significantly decreased infarct volume compared with the model group (20.78±0.69 vs 46.07±0.28%, p<0.001), and also significantly decreased infarct volume compared with MLB alone (p<0.01) and Rg1 alone (p<0.01) ( Figure 31 B).

[0359] This study was designed according to the requirements of the Bliss independent statistical model, and the percentage of infarct volume of different treatment groups relative to the MCAO group was plotted. The data of each group were in accordance with the normal distribution after the Kolmogorov-Smirnov test and the variance homogeneity after the F test. Compared with the MCAO group, the percentage of infarct volume in the MLB / Rg1 group was (40.66±0.88%), the percentage of infarct volume in the MLB group was (85.77±0.87%), and the percentage of infarct volume in the Rg1 group was (68.75±1.13%). According to the Bliss independent statistical model calculation formula, the theoretical effect of the combination of the MLB group and the Rg1 group was calculated, that is, the percentage of infarct volume in the Independence group was 59.04±1.53%. Compared with the Independence group, the MLB / Rg1 group significantly reduced the infarct volume (P<0.01). Therefore, the Bliss independent statistical analysis showed that MLB and Rg1 have a significant synergistic effect in reducing infarct volume ( Figure 32 ).

[0360] 2. MLB / Rg1 significantly prolongs the reperfusion time window, delays blood clotting time and improves neurobehavioral performance

[0361] Blood was collected 7 days after reperfusion for testing. Compared with the Sham group, the APTT values of the MCAO group and the MLB group showed a significant decrease, indicating a significant shortening of the coagulation time. Compared with the MCAO group, the APTT values of the Rg1 group and the MLB / Rg1 group showed a significant increase, indicating that the coagulation time was prolonged. The above results show that Rg1 and MLB / Rg1 have a certain anticoagulant effect on blood coagulation in vivo. * represents *p<0.05, **p<0.01, ***p<0.001 compared with the Sham group; $ represents $p<0.05, $$p<0.01, $$$p<0.001 compared with the MCAO group ( Figure 33 A).

[0362] Animal behavior was evaluated by FPT. There were significant differences between the MLB / Rg1 and MCAO groups, indicating that MLB / Rg1 significantly improved the behavioral abilities of animals. * indicates p < 0.05, ** p < 0.01, *** p < 0.001 compared with the sham group; $ indicates $ p < 0.05, $$ p < 0.01, $$ p < 0.001 compared with the MCAO group. Figure 33 B).

[0363] 3. MLB / Rg1 significantly prolongs the reperfusion time window, delays blood clotting time and improves neurobehavioral performance

[0364] According to the blood oxygen measurement results, compared with the sham group, the MLB and MCAO groups showed a significant decrease; compared with the MCAO group, the blood oxygen content of the MLB / Rg1 group animals was significantly increased. * represents p < 0.05, **p < 0.01, ***p < 0.001 compared with the sham group; $ represents $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with the MCAO group ( Figure 34 A).

[0365] dp / dt represents cardiac contractile function. Compared with the sham group, the cardiac contractile function of the MLB, MCAO, and Rg1 groups was significantly decreased, while that of the MLB / Rg1 group was significantly improved compared with the MCAO group. * indicates p<0.05, **p<0.01, ***p<0.001 compared with the sham group; $ indicates $p<0.05, $$p<0.01, $$$p<0.001 compared with the tMCAO group. Figure 34 B).

[0366] 4. MLB / Rg1 significantly prolongs the reperfusion time window and reduces multi-organ damage after stroke

[0367] After a stroke, multiple organs can be damaged, resulting in a poor prognosis for the patient. Thymus evaluation by HE staining showed that the thymic medulla of the Sham group was larger, and the boundary between the medulla and the cortex was clear and distinct. In the MCAO group, the thymus tissue of each group atrophied, and the medulla also shrank. At the same time, the medulla and cortex showed obvious disorder, with unclear boundaries and more obvious tissue fragmentation ( Figure 35 A).

[0368] HE staining was used to evaluate lung tissue. In the Sham group, the lung tissue cells were neatly arranged, the alveolar walls were intact, and no red blood cell infiltration or lymphocyte infiltration was found in the alveoli and trachea. In the MCAO group, red blood cells were found in the alveoli, indicating that bleeding occurred in the lung tissue. In addition, red blood cell deposition was found in the blood vessels of the MCAO group, and the proportion of pulmonary embolism was the highest ( Figure 35 B), while treatment with MLB / Rg1 resulted in varying degrees of relief.

[0369] Reperfusion therapy for ischemic brain diseases, such as stroke, aims to restore cerebral blood flow as quickly as possible to reduce brain damage caused by ischemia. The wider the window for reperfusion therapy, the more time patients have to receive treatment. Typically, the reperfusion therapy window for acute ischemic stroke is within 4.5 hours of onset. Treatment becomes increasingly complex and risky over time. Early treatment (0-4.5 hours): During this timeframe, intravenous thrombolysis is the most effective treatment. Mid-term treatment (4.5-6 hours or longer): In addition to intravenous thrombolysis, interventional therapies such as mechanical thrombectomy may also be considered. However, the risk of hemorrhagic transformation increases with delayed treatment. Late treatment (over 6 hours): Although studies have shown that reperfusion therapy can be performed beyond the typical window under specific conditions, such as through the use of advanced imaging techniques to identify patients likely to benefit, overall treatment efficacy is reduced and the risk of complications increases. Regardless of the approach, the earlier treatment is initiated, the better the outcome. Treatment complexity and risk increase with delayed treatment.

[0370] The above results show that the combination of MLB / Rg1 still has a significant synergistic protective effect on animals that underwent blood reperfusion 24 hours after middle cerebral artery occlusion, which means that it can prolong the reperfusion time window of patients with cerebral ischemia, and is expected to improve the therapeutic effect of late-stage treatment of patients with cerebral ischemia, reduce complications, and improve mobility, which is crucial to improving patients' self-care ability and quality of life.

[0371] 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. A pharmaceutical composition, wherein The active ingredients of the pharmaceutical composition include (a) and (b): (a) a first active ingredient, wherein the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid; and (b) a second active ingredient, wherein the second active ingredient is ginsenoside, or a pharmaceutically acceptable salt thereof; Wherein, the pharmaceutically acceptable salt of salvianolic acid is selected from the following group: salvianolic acid B magnesium salt, salvianolic acid B calcium salt, salvianolic acid A magnesium salt, rosmarinic acid sodium salt, or a combination thereof; The ginsenoside is selected from the group consisting of ginsenoside Rg1, ginsenoside Rh2, ginsenoside Rb1, or a combination thereof; Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of salvianolic acid and ginsenoside.

2. The pharmaceutical composition according to claim 1, wherein The first active ingredient is selected from the group consisting of magnesium salt of salvianolic acid B, calcium salt of salvianolic acid B, or a combination thereof; And the second active ingredient is ginsenoside Rg1, or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1, wherein The first active ingredient is magnesium salt of salvianolic acid B; and the second active ingredient is ginsenoside Rg1; Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the magnesium salt of salvianolic acid B and ginsenoside Rg1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The weight ratio of the first active ingredient to the second active ingredient is 1:(0.2-5).

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein The weight ratio of the first active ingredient to the second active ingredient is 1:(0.38-0.42).

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein The weight ratio of the first active ingredient to the second active ingredient is 1:0.

4.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein The dosage form of the pharmaceutical composition is selected from the following group: liquid preparation, solid preparation, gaseous dosage form, and semisolid dosage form.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein The dosage form is selected from the group consisting of injections, oral preparations, sublingual preparations, respiratory tract administration preparations, skin administration preparations, and mucosal administration preparations.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein The dosage form is selected from the group consisting of injection, powder injection, capsule, tablet, pill, powder, granule, syrup, oral solution, tincture, lozenge, and drops.

10. A pharmaceutical composition for oral administration, characterized in that: The active ingredients of the pharmaceutical composition include (a) and (b): (a) a first active ingredient, wherein the first active ingredient is a pharmaceutically acceptable salt of salvianolic acid B; and (b) a second active ingredient, which is a ginsenoside, or a pharmaceutically acceptable salt thereof; Wherein, the pharmaceutically acceptable salt of salvianolic acid B is selected from the following group: salvianolic acid B magnesium salt, salvianolic acid B calcium salt, or a combination thereof; Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the pharmaceutically acceptable salt of salvianolic acid B and ginsenosides.

11. The pharmaceutical composition according to claim 10, wherein The first active ingredient is magnesium salt of salvianolic acid B; and the second active ingredient is ginsenoside Rg1; Furthermore, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10), wherein the weight ratio is calculated based on the magnesium salt of salvianolic acid B and ginsenoside Rg1.

12. The pharmaceutical composition according to claim 10 or 11, wherein The dosage form of the pharmaceutical composition is orally disintegrating tablet, mucosal adhesive tablet, orally disintegrating film, orally disintegrating granule, powder, sublingual tablet, buccal tablet, chewable tablet, effervescent tablet, and solution.

13. Use of the pharmaceutical composition according to any one of claims 1 to 12 in the preparation of a medicament for preventing and / or treating brain diseases.

14. The use according to claim 13, characterized in that The brain disease is cerebrovascular disease or cerebral ischemic disease. Preferably, the cerebrovascular disease or cerebral ischemic disease is selected from the group consisting of ischemic stroke, cerebral arteriosclerosis, cerebral ischemia, cerebral hemorrhage, cerebral edema, or a combination thereof.

15. The use according to claim 13, characterized in that The brain disease is Alzheimer's disease, vascular dementia, cerebral atrophy, dementia caused by unexplained cerebral atrophy, and loss of nerve cell number, behavioral disorders, sensory disorders, or a combination thereof caused by chronic brain damage.

16. The use according to claim 13, characterized in that The brain disease is a neurodegenerative disease. Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), hippocampal sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, multiple system atrophy, progressive supranuclear palsy, or a combination thereof.

17. The use according to claim 13, wherein The prevention and / or treatment of brain diseases is selected from the following group: inhibiting white matter atrophy, inhibiting hippocampal atrophy, inhibiting lateral ventricle enlargement, inhibiting striatal atrophy, or a combination thereof.

18. The use according to claim 13, wherein The prevention and / or treatment of brain diseases is selected from the following group: inhibiting white matter nerve cell loss, inhibiting hippocampal nerve cell loss, inhibiting lateral ventricle enlargement, inhibiting striatal nerve cell loss, or a combination thereof.

19. The use according to claim 13, wherein The patient is in the acute, subacute or chronic stage after an ischemic stroke.

20. The use according to claim 13, wherein The pharmaceutical composition is used for preventing or treating heart damage, lung damage, thymus damage, kidney damage, or a combination thereof caused by ischemic stroke; Systemic hypoxia caused by ischemic stroke, or secondary damage caused by hypoxia; and / or Reperfusion injury after ischemic stroke, such as reperfusion-induced heart injury, lung injury, thymus injury, kidney injury, or a combination thereof.

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