Preparation method of Kash berberis extract and protection effect of Kash berberis extract on myocardial cells

Abstract

The invention discloses a preparation method of a Kash berberis thunbergii extract and a protective effect of the Kash berberis thunbergii extract on myocardial cells, and belongs to the field of medical preparations. The invention aims to solve the technical problem of how to apply the Kush berberis to the field of myocardial cell protection or / and how to obtain the Kush berberis extract with a myocardial cell protection effect. In order to solve the technical problem, the invention provides the application of the Kash berberis thunbergii extract in preparation of the medicine for reducing myocardial cell injury. Experimental results show that the fat-soluble extract FSA of the Berberis thunbergii or the water-soluble extract WSA of the Berberis thunbergii has a protective effect on the activity of a myocardial cell injury model. Compared with a control group, the cell viability of a treatment group of the water-soluble extract WSA of the Berberis thunbergii or the fat-soluble extract FSA of the Berberis thunbergii is improved, the cell migration rate is improved, and the cell SOD activity is improved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of a Berberis kashgarensis extract and its protective effect on myocardial cells. Background Art

[0002] Myocardial cell injury usually refers to the destruction of the integrity of normal myocardial cell membranes. Myocardial cells are terminally differentiated cells. Once damaged, they cannot regenerate and can only rely on scar tissue to repair them. Myocardial injury has different causes and different degrees of injury. Depending on the cause and the extent of the injury, the patient's clinical manifestations vary. The most severe may cause acute myocardial infarction. There are many causes of myocardial injury, including hypoxia, ischemia, immune response, various infections caused by pathogens such as viruses, Mycoplasma pneumoniae, and bacteria, as well as drug-induced cardiotoxicity. Doxorubicin (DOX) is an effective topoisomerase II inhibitor that prevents cancer cell DNA replication and is a first-line drug for cancer treatment. However, it is accompanied by severe side effects such as cardiotoxicity, so the clinical application of doxorubicin is limited.

[0003] Berberis kaschgarica Rapr. is a deciduous shrub belonging to the genus Berberis in the Berberidaceae family. It is a native plant of Xinjiang, growing in the highlands of Kashgar, Xinjiang, at altitudes of 2,000-3,000 meters. Preliminary analysis of the chemical composition of Berberis kaschgarica fruit using a classical in vitro preparatory method (color reaction) has been reported. The results indicate that Berberis kaschgarica fruit contains a variety of bioactive substances, including amino acids, proteins, sugars and their glycosides, saponins, phenols, organic acids, flavonoids, coumarins, and volatile oils. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: how to apply Berberis schrenkiana to the field of myocardial cell protection and / or how to obtain a Berberis schrenkiana extract with myocardial cell protective effect. To solve this technical problem, the present invention provides the following technical solutions:

[0005] The present invention provides the use of a Berberis dahurica extract in any of the following:

[0006] A1) Application in reducing myocardial cell damage;

[0007] A2) Use in the preparation of a product for reducing myocardial cell damage;

[0008] A3) Application in improving cell survival rate;

[0009] A4) Use in the preparation of products for improving cell viability;

[0010] A5) Application in improving cell viability;

[0011] A6) Use in the preparation of products for improving cell viability;

[0012] A7) Application in promoting cell migration;

[0013] A8) Use in the preparation of products that promote cell migration;

[0014] A9) Application in increasing superoxide dismutase activity in cells;

[0015] A10) in the preparation of a product for increasing the activity of superoxide dismutase in cells.

[0016] In the present invention, the Berberis kashiensis extract is a substance extracted from Berberis kashiensis.

[0017] In the present invention, the Berberis kashiensis extract is a substance extracted from Berberis kashiensis fruits.

[0018] In the present invention, the Berberis dahurica extract may be Berberis dahurica extract FSA or / and Berberis dahurica extract WSA, and the Berberis dahurica extract FSA is prepared according to a method comprising the following steps:

[0019] S1) mixing the powder of Berberis kashgarensis fruit with an ethanol aqueous solution, performing reflux extraction, and collecting the extract;

[0020] S2) removing ethanol from the extract and collecting the aqueous solution;

[0021] S3) mixing the aqueous solution with the hydrochloric acid solution, adjusting the pH to 3 to obtain an acidic mixed solution, and then letting it stand, and filtering to collect the filtrate;

[0022] S4) mixing the filtrate with petroleum ether, extracting, removing the petroleum ether phase, and collecting the non-petroleum ether phase;

[0023] S5) mixing the non-petroleum ether phase with diethyl ether, extracting, removing the diethyl ether phase, and collecting the non-diethyl ether phase;

[0024] S6) mixing the non-ether phase with concentrated aqueous ammonia, adjusting the pH to 10 to obtain an alkaline mixed solution, extracting with chloroform, collecting the chloroform phase, and removing chloroform from the chloroform phase to obtain Berberis chinensis extract FSA;

[0025] The Barberry extract WAS is prepared according to the following steps:

[0026] S1′) mixing the powder of Berberis kashiensis fruit with an ethanol aqueous solution, performing reflux extraction, and collecting the extract;

[0027] S2') removing ethanol from the extract and collecting the aqueous solution;

[0028] S3') mixing the aqueous solution with concentrated ammonia water, adjusting the pH to 10 to obtain an alkaline mixed solution, letting it stand and then filtering to obtain the filtrate;

[0029] S4') mixing the filtrate with n-butanol for extraction, collecting the n-butanol phase, and removing the n-butanol from the n-butanol phase to obtain Berberis kashgarensis extract WSA.

[0030] In the present invention, the particle size of the powder is not higher than 380 μm.

[0031] In the present invention, the cells are animal cells.

[0032] In the present invention, the animal cells are cardiomyocytes.

[0033] In some embodiments of the present invention, the cardiomyocytes are rat cardiomyocytes, and specifically, the cardiomyocytes are rat cardiomyocyte H9C2 cell line.

[0034] The above method for preparing Berberis rubra extract FSA is also protected by the present invention.

[0035] The present invention also provides Berberis schneiderii extract FSA and / or Berberis schneiderii extract WSA prepared by the above method.

[0036] The present invention also provides a pharmaceutical composition having a protective effect on cells, wherein the pharmaceutical composition comprises the above-mentioned Berberis dahurica extract FSA and / or Berberis dahurica extract WSA.

[0037] The present invention also provides a method for improving the cell survival rate of a myocardial cell injury model, which comprises contacting the myocardial cell injury model with a Berberis dahurica extract to improve the cell survival rate of the myocardial cell injury model.

[0038] The present invention also provides a method for studying the effect of Berberis schrenkiana extract on cardiomyocytes, which comprises contacting a cardiomyocyte injury model with the Berberis schrenkiana extract and detecting the cell survival rate of the cardiomyocyte injury model to study the effect of the Berberis schrenkiana extract on cardiomyocytes.

[0039] The myocardial cell injury model is a cell obtained by treating isolated myocardial cells with adriamycin.

[0040] The myocardial cell injury model is an in vitro cell model.

[0041] The present invention also provides the use of the Berberis kashiensis extract in preparing a medicine having a myocardial cell protective effect.

[0042] The use of substance X or a composition comprising substance X as a medicine also falls within the protection scope of the present invention. The substance X may be the above-mentioned Berberis schneiderii extract FSA and / or Berberis schneiderii extract WSA.

[0043] In certain embodiments of the present invention, the drug may be a drug for preventing and / or treating diseases of myocardial cell damage.

[0044] The present invention also provides use of the above-mentioned Berberis schneiderii extract FSA and / or Berberis schneiderii extract WSA, medicine or pharmaceutical composition for preventing and / or treating myocardial cell damage.

[0045] The present invention also provides a method for preventing and / or treating myocardial cell damage, comprising the step of administering an effective dose of the above-mentioned single-domain antibody, polypeptide, drug or pharmaceutical composition to a subject to prevent and / or treat myocardial cell damage.

[0046] In the present invention, the subject exhibits myocardial cell damage and / or myocardial damage.

[0047] In the present invention, the medicine or pharmaceutical composition may further comprise a pharmaceutically acceptable carrier in addition to the Berberis kashiiense extract.

[0048] In the present invention, the myocardial cell protective effect includes but is not limited to at least one of the following:

[0049] B1) Improve cell viability;

[0050] B2) Improve the survival rate of damaged myocardial cells;

[0051] B3) Increase the migration rate of myocardial damage;

[0052] B4) Increase SOD activity in myocardial injury.

[0053] In the present invention, the kashgar barberry extract FSA contains the following at least 18 compounds: Caffeoylcholine (CAS: 87189-10-4), Apoglaziovine (CAS: 2128-77-0), DemethylenBerberine (Chinese name: Demethylenberberine, CAS: 25459-91-0), Norrisocorydine (CAS: 475-70-7), Palmat-Rubine (Chinese name: Palmatine Red Base, CAS: 16176-68-4), Columbamine (African tetrandrine, CAS: 3621-36-1), Corydine (Chinese name: Purple Benzyl Alcohol, CAS: 476-69-7), bernumidine, Magnoflorine (Chinese name: Magnolia alkaloid, CAS: 2141-09-5), Cocamidopropylβine (Chinese name: lauramide propyl betaine, CAS: 4292-10-8), Oxyberberine (Chinese name: 8-oxidized berberine or berberine alkaloids, CAS: 549-21-3), Lennoxamine (CAS: 95530-38-4), Argemonine (CAS: 6901-16-2), 13-deoxychilenine, khyberine (CAS: 77795-10-9), Espinine (CAS: 26137-40-6), Berbamine (Chinese name: berbamine, CAS: 478-61-5), Isotetrandrine (Chinese name: isohanfangjisu, CAS: 477-57-6), and other 18 compounds.

[0054] In the present invention, the water extract WSA of Barberry contains at least the following seven compounds: Betaine (Chinese name: betaine, CAS: 107-43-7), Aminopurine (Chinese name: 2-aminopurine, CAS: 452-06-2), Trigonelline (Chinese name: trigonelline, CAS: 535-83-1), Dopamine (Chinese name: dopamine hydrochloride, CAS: 62-31-7), Magnocurarine (Chinese name: magnolia curarine, CAS: 6801-40-7), 10-Formyl-THF (CAS: 2800-34-2), Procyanidin C2 (Chinese name: proanthocyanidin C2, CAS: 37064-31-6), etc.

[0055] The beneficial technical effects achieved by the present invention are as follows:

[0056] The present invention demonstrates through examples that both FSA and WSA, a fat-soluble extract of Berberis schrenkiana, have protective effects on the activity of cardiomyocytes in a cardiomyocyte injury model. Compared to the control group, the WSA or FSA-treated groups showed increased cell viability, cell migration rate, and cellular SOD activity.

[0057] Compared with the water-soluble extract of Berberis kashiensis (WSA), the fat-soluble extract of Berberis kashiensis (FSA) treated cardiomyocytes showed higher viability in the injured cardiomyocyte model; cardiomyocytes treated with the fat-soluble extract of Berberis kashiensis (FSA) showed higher cell migration rate and SOD enzyme activity. Compared with the water-soluble extract of Berberis kashiensis (WSA), the fat-soluble extract of Berberis kashiensis (FSA) showed an unexpected protective effect on cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the total ion current of the mixed sample quality control QC sample in negative ion mode.

[0059] Figure 2 This is the total ion current of the mixed quality control QC sample in positive ion mode.

[0060] Figure 3 This is a multi-peak diagram for MRM metabolites detected in negative ion mode.

[0061] Figure 4 This is a multi-peak diagram for MRM metabolites detected in positive ion mode.

[0062] Figure 5 These are the results of doxorubicin concentration screening for modeling.

[0063] Figure 6 These are the results of the effects of doxorubicin and Berberis dahurica extract on cardiomyocyte viability.

[0064] Figure 7 The results show that the extract of Berberis dahurica has a protective effect on adriamycin-induced cardiomyocyte injury.

[0065] Figure 8 Effects of Berberis dahurica liposoluble extract on cardiomyocyte migration.

[0066] Figure 9 Effects of water-soluble extract of Berberis dahurica on cardiomyocyte migration.

[0067] Figure 10 Effects of Berberis dahurica extract on cardiomyocyte migration.

[0068] Figure 11 This study was to detect the SOD activity of Berberis dahurica extract on adriamycin-induced cardiomyocyte injury. DETAILED DESCRIPTION

[0069] 1. Terms used in this invention

[0070] When used in a list of two or more items, the term "or / and" or "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A or / and B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B, or / and C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0071] In the present invention, the term "extract" specifically refers to plant extracts, which are substances extracted or processed from plants (all or part of a plant, in this case, the fruit of Berberis dahurica) using appropriate solvents or methods. They can be used in the pharmaceutical, food, daily chemical, and other industries. There is a conceptual overlap between plant extracts and Chinese herbal medicine extracts. The raw materials for plant extracts in my country are mainly derived from Chinese herbal medicines, so domestic plant extracts can also be referred to as Chinese herbal medicine extracts to some extent.

[0072] The term "treat" or "treatment" refers to therapeutic treatment, wherein the purpose is to slow down or alleviate undesirable physiological changes or diseases, or to provide beneficial or desired clinical results during treatment. Beneficial or desired clinical results include alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., stopping deterioration), delay or slowing of disease progression, improvement or alleviation of disease state, and / or relief (whether partial relief or complete relief, and whether detectable or undetectable). "Treatment" can also mean prolonged survival compared to the expected survival of the subject without treatment. Those subjects in need of treatment include those subjects who have already suffered from undesirable physiological changes or diseases and those subjects who tend to suffer from physiological changes or diseases. Treatment can involve therapeutic agents, also referred to herein as "medicaments or medications," which can be intended to help achieve the beneficial or desired clinical results of interest through their effects. Therapeutic agents or drugs can be administered to subjects by many approaches, including at least intravenous and oral routes. The term "intravenous" relevant to the administration of therapeutic agents or drugs refers to administering the therapeutic agent or drug in one or more intravenous regions. The term "oral," in reference to the administration of a therapeutic agent or drug, refers to the administration of the therapeutic agent or drug via the oral passage, such as the mouth.

[0073] In the present invention, "subject" includes a person who is being treated for a disease or prevented as a patient. The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, rodents (order Rodentia) mammals, such as mice and hamsters, and lagomorphs (order Logomorpha) mammals, such as rabbits. The mammal can be of the order Carnivora, including felines (cats) and canines (dogs). The mammal can be of the order Artiodactyla, including bovines (cows) and suids (pigs), or of the order Perssodactyla, including equines (horses). The mammal can be of the order Primate, Ceboid, or Simoid (monkeys) or a humanoid. In the present invention, the subject can also be an isolated cell. For example, an isolated myocardial injury cell model.

[0074] The term "effective," as applied to dosage or amount, refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, an effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0075] The phrase "pharmaceutically acceptable" used in conjunction with the compositions described herein refers to the molecular entities and other ingredients of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" means listed by a recognized pharmacopoeia for use in mammals, and more particularly in humans.

[0076] For example, the term "pharmaceutically acceptable carrier" includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, perfumes, flavorings, sweeteners or other materials may be added to the pharmaceutical preparations.

[0077] 2. Example

[0078] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0079] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0080] The fresh fruits of Berberis kaschgarica Rupr. used in the following examples were harvested in Akto County, Kashgar Prefecture, Xinjiang. Rat cardiomyocytes (H9C2) were obtained from Zishan Biotechnology Co., Ltd., catalog number STCC30008G. Doxorubicin (DOX) was obtained from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S17092. Methanol, acetonitrile, and ethanol used in the following examples were all chromatographically pure (#Merck), and the standard samples used in the following examples were chromatographically pure (BioPha / Sigma-Aldrich). The Enhanced Cell Counting Kit 8 (WST-8 / CCK8) used was obtained from Elabscience, catalog number E-CK-A362.

[0081] BioPha / Sigma-Aldrich chromatographic grade

[0082] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged. Data processing and plotting were performed using SPSS 26.0 and Graph Pad Prism 10.0. Differences between groups were compared using one-way analysis of variance. Pairwise comparisons were performed using the LSD-t test. Nonparametric tests were used when variances were unequal. P < 0.05 was considered statistically significant.

[0083] Example 1 Qualitative analysis of components of Berberis kashgarensis fruit

[0084] 1.1 Sample extraction process

[0085] Fresh Berberis kashgarensis fruit was first freeze-dried in a vacuum oven and ground into a powder using a grinder. A certain amount of the powder was weighed and dissolved in a methanol-water solution (methanol:water = 8:2 by volume). The solution was vortexed 6-10 times to increase the extraction yield. The dissolved sample was placed in a 4°C biosafety cabinet overnight. After centrifugation, the supernatant was carefully aspirated and filtered through a microporous filter membrane (0.22 μm proesize). The filtrate was collected and stored in an injection vial for subsequent UPLC-MS / MS analysis.

[0086] 1.2. Chromatographic mass spectrometry acquisition conditions

[0087] The data acquisition instrument system mainly consists of two parts: ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS).

[0088] Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 1.8 μm, 2.1 mm*100 mm;

[0089] Mobile phase: Phase A is ultrapure water pre-mixed with 0.03% volume acetic acid, and phase B is acetonitrile pre-mixed with 0.05% volume acetic acid; elution gradient: the proportion of phase B is 5% at 0.00 min, the proportion of phase B increases linearly to 95% within 10.00 min and is maintained at 95% for 1 min, and the proportion of phase B is reduced to 5% from 11.00 to 11.10 min; and equilibrated at 5% for 14 min; flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 μL.

[0090] The electrospray ionization (ESI) source temperature was 550°C, the mass spectrometer voltage was 5500 V, the curtain gas (CUR) was 30 psi, and the collision-activated dissociation (CAD) parameter was set to high. In a triple quadrupole (QQQ), each ion transition was scanned based on the optimized declustering potential (DP) and collision energy (CE).

[0091] In the present invention, the UPLC-MS / MS detection method is the internal standard method. The detection principle and confirmation process of the compounds in the Berberis dahurica extract are as follows:

[0092] The instrument used is an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer (UPLC-MS / MS), commonly referred to as QQQ in mass spectrometry. Triple quadrupole tandem mass spectrometry is commonly used for the quantitative detection of low-concentration compounds due to its exceptional sensitivity. QQQ refers to the three quadrupoles within the mass spectrometer. Typically, the outer quadrupoles Q1 and Q3 perform scanning functions, while the center quadrupole Q2 does not perform analytical functions but serves as a collision cell. In quantitative analysis, ion pairs are typically selected based on the compound's precursor ion and characteristic fragments. After liquid separation, the compound is sprayed, charged by an electric field, and vaporized and desolvated at high temperatures to form gas-phase ions. These ions are then sorted in Q1. Only ions with a pre-selected mass-to-charge ratio are allowed to pass through Q1 and enter the Q2 collision cell. In Q2, high-purity inert gas collides with the resulting fragments, which are then fed to the third analytical quadrupole, Q3. Similar to Q1, only fragments with the specified mass-to-charge ratio pass through Q3, ultimately being detected by the detector. That is to say, only compounds that can pass the double screening of Q1 and Q3 can detect the corresponding signal. If the mass-to-charge ratio is not what we set, the ion will be screened out when passing through Q1 or Q3.

[0093] At the same time, we use liquid chromatography-mass spectrometry detection, with ultra-high performance liquid chromatography at the front end to separate compounds based on polarity. The smaller the polarity, the easier it is to be eluted from the reversed-phase chromatography column by the organic solvent in the liquid phase. Conversely, the greater the polarity, the more difficult it is to elute, which corresponds to an increase in the retention time of the compound.

[0094] Therefore, in the UPLC-MS / MS detection and analysis process, the peak compound is confirmed based on two dimensions of information: the compound's retention time and characteristic ion pairs (MS fragment analysis).

[0095] 1.3 Qualitative analysis of chemical components (secondary metabolites) of Berberis kashgarensis

[0096] The mass spectrometry data were processed using Analys 1.6.3 software. Total ion current (TIC) chromatograms (i.e., the sum of the intensities of all ions in the mass spectrum at each time point and the resulting continuous plot) and multi-peak graphs (XIC chromatograms of multi-substance extraction) of the mixed sample QC samples were obtained. Based on the chemical composition information provided by the database and its analytical accuracy, 105 confirmed secondary metabolites with an analytical accuracy of more than 95% were screened out ( Figures 1 to 4 , Table 1).

[0097] Figure 1 and Figure 2It is the total ion current (TIC) of the mixed quality control QC sample, that is, the spectrum obtained by continuously plotting the intensities of all ions in the mass spectrum at each time point. Figure 1 In negative ion mode, Figure 2 The horizontal axis represents the retention time (Rt) of metabolite detection, and the vertical axis represents the ion current intensity (in cps, counts per second) of ion detection.

[0098] Figure 3 and Figure 4 Multi-peak diagram for MRM metabolite detection (multi-substance extracted ion current spectrum, XIC). Figure 3 In negative ion mode, Figure 4 The MRM metabolite detection multi-peak diagram shows the substances that can be detected in the sample, and each mass spectrum peak of a different color represents a detected metabolite.

[0099] In Table 1, retention time refers to the time it takes for a metabolite to be detected by the detector after separation on the chromatographic column, molecular weight is the sum of the relative atomic masses of the atoms in the chemical formula, MS fragments are the characteristic product ions produced by collision-induced dissociation (CID) of the parent ion when analyzing the target metabolite by mass spectrometry, and ID is the compound identified based on retention time, molecular weight, and MS characteristic fragment analysis. CAS is the CAS number corresponding to the compound. The ID and CAS numbers for the compounds in Table 1 are publicly available from the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The results in Table 1 indicate that Berberis dahurica contains 105 secondary metabolites with an accuracy of over 95%.

[0100] Table 1 Identification of chemical components of Berberis kashgarensis by UPLC-MS / MS

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Example 2, Preparation process of Berberis kashgarensis extract

[0110] 2.1 Extraction and separation of fat-soluble alkaloids (FSA) from Berberis chinensis (alcohol extraction)

[0111] Place the fruits of Berberis dahurica in a cool and ventilated place to air dry naturally. Then use a grinder to crush the dried fruits and sieve the dry powder with a 40-mesh sieve. Select powder with a particle size of less than 380 μm for subsequent extraction. Accurately weigh 500 g of dry powder of Berberis dahurica fruit and pour it into a round-bottom flask containing two liters of 95% ethanol aqueous solution. Mix the powder with the 95% ethanol aqueous solution, reflux extract at 80°C for 4 hours, and collect the ethanol aqueous solution (extract). Continue to add one liter of 95% ethanol aqueous solution to the medicinal residue, continue to reflux extract the medicinal residue and 95% ethanol for 2 hours, and collect the ethanol aqueous solution (extract). Combine the ethanol aqueous solutions obtained twice. Use for subsequent processing.

[0112] Filter the combined ethanol-water solution through 25 μm filter paper and collect the filtrate. Transfer the filtrate to a 2-liter round-bottom flask and recover the solvent using a rotary evaporator at 50°C under reduced pressure. Pour out the remaining concentrated aqueous solution in the flask and allow it to cool naturally to room temperature (20°C).

[0113] The concentrated aqueous solution was then mixed with an appropriate amount of 5% aqueous hydrochloric acid to obtain an acidic mixture. 5% aqueous hydrochloric acid was added until the pH of the acidic mixture reached 3. The mixture was stirred to dissolve the acid and allowed to stand overnight. The next day, the precipitated acid-insoluble resin and oil components were filtered out using filter paper. The filtrate was collected and used for the next reaction.

[0114] The filtrate was then extracted 5-7 times with an equal volume of petroleum ether to remove fat-soluble impurities (the petroleum ether phase was removed and the non-petroleum ether phase was retained). The extraction conditions were Soxhlet extraction at room temperature for 1 hour.

[0115] The non-petroleum ether phase was mixed with an equal volume of diethyl ether and extracted to remove acidic and neutral impurities. The diethyl ether phase was removed and the non-diethyl ether phase was collected. This non-diethyl ether phase was designated as the acidic aqueous solution. Soxhlet extraction was performed at room temperature for 1 hour.

[0116] The acidic aqueous solution was mixed with concentrated ammonia water to obtain an alkaline mixed solution, wherein the amount of concentrated ammonia water added was such that the pH of the alkaline mixed solution was 10. The alkaline mixed solution was mixed with twice the volume of chloroform, and the extract was collected after extraction (i.e., the chloroform phase was collected). The extraction conditions were Soxhlet extraction at room temperature for 1 hour. The medicinal residues were mixed with chloroform and the extraction was continued for 6 times. The extracts collected multiple times were mixed, and the chloroform solvent was recovered under reduced pressure to obtain the fat-soluble extract of Berberis chinensis (FSA).

[0117] 2.2 Extraction and separation of water soluble alkaloids (WSA) from Berberis chinensis (water extraction)

[0118] Place the fruits of Berberis dahurica in a cool and ventilated place to air dry. Then, crush the dried fruits with a grinder and sieve the dry powder with a 40-mesh sieve. Select powder with a particle size of less than 380 μm for subsequent product extraction. Accurately weigh 500 g of the dried powder of Berberis dahurica fruits and pour it into a round-bottom flask containing two liters of 95% ethanol aqueous solution. Mix the powder with the 95% ethanol aqueous solution and reflux extract at 80°C for 4 hours. Collect the ethanol extract. Add 1 L of ethanol aqueous solution to the medicinal residue and continue reflux extraction at 80°C for 2 hours, and collect the ethanol extract again.

[0119] The two extracts were combined and filtered through 25 μm filter paper. The filtrate was collected and transferred to a 2-liter round-bottom flask. The ethanol solvent was recovered under reduced pressure using a rotary evaporator at 50°C. The remaining concentrated aqueous solution in the flask was decanted and allowed to cool naturally to room temperature (20°C). The cooled concentrated aqueous solution was mixed with concentrated ammonia to obtain an alkaline mixture. The pH of the mixture was adjusted to 12. The solution was stirred, dissolved, and allowed to stand overnight. The next day, the precipitated alkaline-insoluble components were filtered out. The filtrate was then extracted with an equal volume of n-butanol using the Soxhlet method at 50°C for 3 hours. The n-butanol phase and the non-n-butanol phase were collected. The non-n-butanol phase was further mixed with an equal volume of n-butanol and extracted a second time at 50°C. The n-butanol phase was again collected. A total of eight n-butanol extractions were performed. The n-butanol phases collected from these extractions were combined and the n-butanol solvent was recovered under reduced pressure. The remaining mixture was the water-soluble extract (WSA) of Berberis kashiensis.

[0120] The obtained Berberis schrenkiana fat-soluble extract (FSA) and Berberis schrenkiana water-soluble extract (WSA) were filtered and set aside. UPLC-MS / MS analysis was performed according to "1.2. Chromatographic Mass Spectrometry Acquisition Conditions" in Example 1. The results are shown in Tables 2 and 3.

[0121] UPLC-MS / MS analysis showed that the fat-soluble extract of Berberis chinensis contained Caffeoylcholine (CAS: 87189-10-4), Apoglaziovine (CAS: 2128-77-0), Demethylen Berberine (Chinese name: Demethylen Berberine, CAS: 25459-91-0), Norrisocorydine (CAS: 475-70-7), Palmat-Rubine (Chinese name: Palmatine Red Alkaloid, CAS: 16176-68-4), Columbamine (African Stephania Tetrandrakine, CAS: 3621-36-1), Corydine (Chinese name: Purple Benzyl Alcohol, CAS: 476-69-7), bernumidine-Magnoflorine (Chinese name: Magnolia Flower Alkaloid, CAS: 2141-09-5), Cocamidopropylβine (Chinese name: 18 compounds including lauramidopropyl betaine, CAS: 4292-10-8), Oxyberberine (Chinese name: 8-oxidized berberine or berberine alkaloids, CAS: 549-21-3), Lennoxamine (CAS: 95530-38-4), Argemonine (CAS: 6901-16-2), 13-deoxychilenine, Espinine (CAS: 26137-40-6), Berbamine (Chinese name: berbamine, CAS: 478-61-5), and Isotetrandrine (Chinese name: isotetrandrine, CAS: 477-57-6).

[0122] The water-soluble extract of Berberis kashgarensis contains seven compounds: Betaine (Chinese name: Betaine, CAS: 107-43-7), Aminopurine (Chinese name: 2-aminopurine, CAS: 452-06-2), Trigonelline (Chinese name: Trigonelline, CAS: 535-83-1), Dopamine (Chinese name: Dopamine hydrochloride, CAS: 62-31-7), Magnocurarine (Chinese name: Magnolia curarine, CAS: 6801-40-7), 10-Formyl-THF (CAS: 2800-34-2), and Procyanidin C2 (Chinese name: Proanthocyanidin C2, CAS: 37064-31-6).

[0123] Table 2 18 fat-soluble alkaloids (FSA) from Berberis chinensis

[0124]

[0125]

[0126] Table 3 Seven water-soluble alkaloids (WSA) of Berberis chinensis

[0127]

[0128] Example 3, drug efficacy study

[0129] 3.1 Cell Culture

[0130] H9C2 cardiomyocytes were cultured in DMEM containing 10% fetal bovine serum. When the cells filled 70%-80% of the culture flask, the culture medium was discarded and the cells were rinsed twice with 1 mL of PBS buffer. 1 mL of 0.25% trypsin was added and digested for 1 min. When the cells began to detach from the flask wall, 2 mL of complete culture medium was added to terminate the digestion. The cells were divided into new culture flasks in appropriate proportions and cultured according to the cell culture method. Subsequent experiments were performed after the second to third passage.

[0131] 3.2. CCK-8 assay for drug toxicity

[0132] Resuspend the H9C2 cardiomyocytes cultured in 3.1 in complete culture medium to prepare a cell suspension of 5000 cells / well. Plate the cells in a 96-well plate at a density of 100 μL / well. The experiment was divided into three groups, depending on the drug added: FSA, WSA, and doxorubicin (DOX). An equal volume of complete culture medium was added as a control (Ctrol).

[0133] FSA group: 100 μL of fat-soluble extract (FSA) was added to the cell culture system. The final concentrations of FSA were 1.5, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mmol / L, respectively;

[0134] WSA group: 100 μL of Berberis kashii water-soluble extract (WSA) was added to the cell culture system. The final concentrations of WSA were: 1.5, 3.125, 6.25, 12.5, 25, 50, 100, 200 mmol / L;

[0135] Doxorubicin group: 100 μL of modeling drug doxorubicin (DOX) was added to the plate wells. The final concentrations of FSA in the cell culture system were: 0.125, 0.25, 0.5, 1, 2, 4, 8 mmol / L.

[0136] Control group (Ctrol): add 100 μL of complete culture medium to the plate wells.

[0137] Blank group: 100 μL of PBS solution was added to the well plate.

[0138] Three parallel wells were set up in each group. After 24 hours, the cell suspension (i.e., supernatant) was discarded and washed once with 100 μL PBS buffer. According to the instructions of the Enhanced Cell Counting Kit 8 (WST-8 / CCK8), 100 μL of CCK-8 solution was added to each well to measure cell viability. The cells were cultured in an incubator for 2 hours. After obvious color development, the OD value of each well at a wavelength of 450 nm was measured using a multifunctional microplate reader.

[0139] Wherein, cell survival rate (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0140] The results are as follows Figure 5 and Figure 6 As shown, Figure 5 The results of the doxorubicin concentration screening for modeling showed that compared with the control group, cardiomyocyte viability gradually decreased with increasing doxorubicin doses within the range of 0.125 to 8 mmol / L. Cell viability reached its highest level at 2 mmol / L, approaching 50%. This suggests that 2 mmol / L is the optimal concentration for doxorubicin. Therefore, in subsequent experiments, 2 mmol / L of doxorubicin was used for modeling and inducing a cardiomyocyte injury model.

[0141] Figure 6 The results show the effects of doxorubicin and Berberis schrenkiana extract on cardiomyocyte viability. The results show that compared with the control group, cell viability gradually decreased with increasing doses of Berberis schrenkiana fat-soluble extract above 100 mmol / L, suggesting that the optimal concentration range for subsequent experiments is 0-100 mmol / L. Compared with the control group, cell viability gradually decreased with increasing doses of Berberis schrenkiana water-soluble extract above 50 mmol / L, suggesting that the optimal concentration range for subsequent experiments is 0-50 mmol / L.

[0142] 3.3 Protective Effect of Berberis dahurica Extract on Adriamycin-Induced Cardiomyocyte Injury

[0143] A cardiomyocyte injury model was established using the doxorubicin concentration (2 mmol / L) determined in Experiment 3.2. The specific method was as follows: H9C2 cardiomyocytes cultured in 3.1 were resuspended in complete culture medium to prepare a cell suspension of 5000 cells / well. The suspension was seeded into a 96-well plate at a density of 100 μL / well. Depending on the type of drug administered, the following drugs were added (mixed in complete culture medium; administration volume: 100 μL):

[0144] Fat-soluble extract group (labeled as FSA): Adriamycin and Berberis schrenkiana fat-soluble extract were administered to the cell culture system, with the final concentration of adriamycin being 2 mmol / L and the concentrations of Berberis schrenkiana fat-soluble extract being 0.75, 1.5, 3.125, 6.25, 12.5, 25, 50, and 100 mmol / L, respectively;

[0145] Berberis schreberi water-soluble extract group (labeled as WSA): Adriamycin and Berberis schreberi water-soluble extract were administered to the cell culture system at a final concentration of 2 mmol / L for adriamycin and 0.75, 1.5, 3.125, 6.25, 12.5, 25, and 50 mmol / L for Berberis schreberi water-soluble extract, respectively.

[0146] Model control (labeled as DOX): Doxorubicin was administered to the cell culture system at a final concentration of 2 mmol / L.

[0147] Control group: Complete culture medium was added to the culture system.

[0148] The cells were cultured for 24 hours, with three replicate wells per group. After 24 hours, the cell suspension was discarded and washed once with 100 μL of PBS buffer. Cell viability was measured by adding 100 μL of CCK-8 solution to each well according to the instructions of the Enhanced Cell Counting Kit 8 (WST-8 / CCK8). The cells were incubated in an incubator for 2 hours. After significant color development, the OD value of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader, and the cell viability was calculated.

[0149] Figure 7 These are the statistical results of the protective effect of Berberis dahurica extract on adriamycin-induced cardiomyocyte injury. Figure 7 In the table, "##" indicates that the difference between the modeling group and the blank group is statistically significant, and "**" indicates that the difference between the drug-treated group and DOX is statistically significant. "##" indicates P < 0.01, "#" indicates P < 0.05, "**" indicates P < 0.01, and "*" indicates P < 0.05.

[0150] The results showed that compared with the model control group (DOX), the fat-soluble extract of Berberis schrenkiana and the water-soluble extract of Berberis schrenkiana both had a certain protective effect on doxorubicin-induced cardiomyocyte injury, that is, the cell survival rate of the doxorubicin-induced cardiomyocyte injury model was significantly improved after adding the fat-soluble extract of Berberis schrenkiana and the water-soluble extract of Berberis schrenkiana.

[0151] And at the same added concentration, the fat-soluble extract of Barberry has a greater protective effect on the myocardial cell injury model, that is, at the same added concentration, the cell survival rate of the Barberry fat-soluble extract group is higher than that of the Barberry water-soluble extract group.

[0152] The effective concentration of the fat-soluble extract of Berberis kashiensis is 0.75mmol / L to 100mmol / L, and the optimal concentration is 12.5mmol / L. The effective concentration of the water-soluble extract of Berberis kashiensis is 0.75mmol / L to 100mmol / L, and the optimal concentration is 6.25mmol / L.

[0153] The results of significance analysis of the differences between the FSA and WSA groups showed that compared with DOX, the blank group was added with complete culture medium, the modeling group was added with 100 μL of 2 mmol / L doxorubicin, the FSA administration group was added with corresponding concentrations of FSA and 2 mmol / L doxorubicin at the same time, and the WSA group was added with corresponding concentrations of WSA and 2 mmol / L doxorubicin at the same time.

[0154] 3.4 Cell migration assay

[0155] According to the different added substances, they are divided into three groups:

[0156] FSA group: added with Berberis kashii extract FSA;

[0157] WSA group: added with Berberis kashii extract WSA;

[0158] Control group: only an equal volume of complete culture medium was added.

[0159] Taking the FSA group as an example, the experimental steps are as follows: H9C2 cells in the logarithmic growth phase are seeded in a 6-well plate at 500,000 / well, and then 2 mL of complete culture medium containing FSA of Berberis dahurica extract at concentrations of 3.125 mmol / L, 6.25 mmol / L and 12.5 mmol / L are added respectively, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The cell density is observed. When the cell density reaches 80%, three straight lines are drawn perpendicular to the horizontal line on the back of the bottom using a 10 μL pipette tip. Immediately after the lines are drawn, the 6-well plate is inverted under a microscope, the observation site is selected, the situation of the lines is observed and photographed, and the scratch area is recorded as A0. Three replicate wells are set up, and the results are averaged. Then continue to culture for 24 hours, invert the 6-well plate under a microscope, continue to select the observation site, observe the healing of the line site and take photos, and the scratch area is recorded as A 24 Three replicate wells were set up and the results were averaged.

[0160] Cell migration rate (%) = A0-A 24 / A0

[0161] A0: scratch area at 0h, A 24: Scratch area after 24 hours.

[0162] The operations of the WSA group and the control group referred to those of the FSA group, with the only difference being that in the WSA group, the Berberis schrenkiana extract FSA in the complete culture medium was replaced with Berberis schrenkiana extract WSA containing concentrations of 1.5 mmol / L, 3.125 mmol / L, and 6.25 mmol / L, respectively; and in the control group, the supplemented culture medium was replaced with complete culture medium.

[0163] The results are as follows Figure 8 、 Figure 9 and Figure 10 The results showed that compared with the blank control group, the extract of Berberis dahurica had a promoting effect on the migration of cardiomyocytes at 24 hours. Among them, FSA significantly promoted the migration of cardiomyocytes in a dose-dependent manner.

[0164] 3.5 SOD detection

[0165] The H9C2 cardiomyocytes cultured in 3.1 were resuspended in complete culture medium to prepare 1×10 5 Cell suspension of 10 cells / well was inoculated into 6-well plates, 1 mL / well. The groups were divided into the following groups according to the added substances:

[0166] Control group: only 2 mL of complete culture medium was added;

[0167] DOX group: 2 mL of complete culture medium containing 2 mmol / L doxorubicin was added;

[0168] FSA group: Complete medium containing corresponding concentrations of Berberis dahurica extract FSA and 2 mmol / L DOX were added to H9C2 cells cultured for 24 hours, and the cells were cultured for another 24 hours.

[0169] WSA group: Complete medium containing corresponding concentrations of Berberis kashii extract WSA and 2 mmol / L DOX were added to H9C2 cells cultured for 24 hours, with a total volume of 2 mL, and cultured for another 24 hours;

[0170] Taking the FSA group as an example, the experimental steps are as follows: H9C2 cardiomyocytes cultured in 3.1 were resuspended in complete culture medium to prepare 1×10 5 Cell suspensions of 1 mL / well were seeded into 6-well plates. The plates were incubated in a humidified incubator for 24 hours. Complete medium containing 2 mmol / L doxorubicin and FSA from Berberis dahurica extract (3.125 mmol / L, 6.25 mmol / L, and 12.5 mmol / L, respectively) was then added to a total volume of 2 mL. The cells were incubated for 24 hours. The cells were then placed in a 37°C, 5% CO2 incubator for 24 hours, and then SOD activity was measured.

[0171] The detection of SOD in cells is a common procedure in the art. An exemplary detection method is provided as follows: 24 hours after cell treatment, the cell culture supernatant in each well is collected, and the SOD activity in the cell supernatant is detected at a wavelength of 550 nm using a colorimetric method according to the instructions of the SOD detection kit (Elabscience; E-BC-K019-M). H9C2 cells are plated at 1×10 5 After the above grouping treatment, the SOD detection kit (Elabscience; BL521A) was used for detection, with 3 replicates and the results were averaged.

[0172] Figure 11 The results of the test on the SOD activity of adriamycin-induced cardiomyocyte injury by the extract of Berberis dahurica showed that adriamycin significantly reduced SOD activity, while the extract of Berberis dahurica increased SOD activity compared with the adriamycin group, with FSA significantly increasing SOD activity in a dose-dependent manner.

[0173] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, the present invention is intended to include any variation, use or improvement of the present invention, including changes that depart from the disclosed scope of the present invention and are made using conventional techniques known in the art.

Claims

1. Application, characterized in that, The application is the application of the Berberis dahurica extract in any of the following: A1) Application in reducing myocardial cell damage; A2) Use in the preparation of a product for reducing myocardial cell damage; A3) Application in improving cell survival rate; A4) Use in the preparation of products for improving cell viability; A5) Application in improving cell viability; A6) Use in the preparation of products for improving cell viability; A7) Application in promoting cell migration; A8) Use in the preparation of products that promote cell migration; A9) Application in increasing superoxide dismutase activity in cells; A10) Use in the preparation of a product for increasing the activity of superoxide dismutase in cells; The Berberis kashiensis extract is a substance extracted from Berberis kashiensis.

2. The use according to claim 1, characterized in that: The Berberis rubrum extract is Berberis rubrum extract FSA or / and Berberis rubrum extract WSA, and the Berberis rubrum extract FSA is prepared according to a method comprising the following steps: S1) mixing the powder of Berberis kashgarensis fruit with an ethanol aqueous solution, performing reflux extraction, and collecting the extract; S2) removing ethanol from the extract and collecting the aqueous solution; S3) mixing the aqueous solution with the hydrochloric acid solution, adjusting the pH to 3 to obtain an acidic mixed solution, and then letting it stand, and filtering to collect the filtrate; S4) mixing the filtrate with petroleum ether, extracting, removing the petroleum ether phase, and collecting the non-petroleum ether phase; S5) mixing the non-petroleum ether phase with diethyl ether, extracting, removing the diethyl ether phase, and collecting the non-diethyl ether phase; S6) mixing the non-ether phase with concentrated aqueous ammonia, adjusting the pH to 10 to obtain an alkaline mixed solution, extracting with chloroform, collecting the chloroform phase, and removing chloroform from the chloroform phase to obtain Berberis chinensis extract FSA; The Barberry extract WAS is prepared according to the following steps: S1′) mixing the powder of Berberis kashiensis fruit with an ethanol aqueous solution, performing reflux extraction, and collecting the extract; S2') removing ethanol from the extract and collecting the aqueous solution; S3') mixing the aqueous solution with concentrated ammonia water, adjusting the pH to 10 to obtain an alkaline mixed solution, letting it stand and then filtering to obtain the filtrate; S4') mixing the filtrate with n-butanol for extraction, collecting the n-butanol phase, and removing the n-butanol from the n-butanol phase to obtain Berberis kashgarensis extract WSA.

3. The use according to claim 2, characterized in that: The powder particle size is not higher than 380 μm.

4. The use according to any one of claims 1 to 3, characterized in that: The cells are animal cells.

5. The use according to claim 4, characterized in that: The animal cells are cardiomyocytes.

6. Berberis kashiensis extract FSA and / or Berberis kashiensis extract WSA obtained according to the method of claim 2 or 3.

7. Use of the Berberis kashiensis extract according to claim 6 in the preparation of a medicament for protecting myocardial cells.

8. A pharmaceutical composition having a cell-protective effect, characterized in that: The pharmaceutical composition comprises the Berberis schneiderii extract FSA and / or the Berberis schneiderii extract WSA according to claim 6.

9. The pharmaceutical composition according to claim 8, characterized in that: The cells are animal cells.

10. A method for improving the cell survival rate of a cardiomyocyte injury model, comprising contacting the Berberis kashiiense extract of claim 6 with the cardiomyocyte injury model to improve the cell survival rate of the cardiomyocyte injury model.

Images