CN111249271A

CN111249271A - Medicament for treating diabetes and preparation method and application thereof

Info

Publication number: CN111249271A
Application number: CN202010195390.8A
Authority: CN
Inventors: 杨新洲
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-06-09

Abstract

The invention relates to the technical field of medicines, in particular to a medicament for treating diabetes and a preparation method and application thereof, the medicament comprises at least one of active components of butin, butein-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone and butin, and the medicament can obtain the active components through extraction.

Description

Medicament for treating diabetes and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a medicament for treating diabetes, and a preparation method and application thereof.

Background

Sophora alopecuroides L, also called Sophora alopecuroides root, Sophora alopecuroides L, belongs to perennial herbs or basal lignified plants in Sophora of Leguminosae, grows in alkaline sandy soil of desert, semi-fixed sand dune and fixed sand dune, and is mainly distributed in Xinjiang, Gansu, Qinghai, Ningxia, inner Mongolia, Shaanxi provinces and the like in China. The sophora alopecuroides is saline-alkali resistant, barren resistant and drought resistant, and is an important vegetation for preventing wind, fixing sand and resisting saline-alkali erosion in northwest regions. The whole plant of sophora alopecuroide is bitter in taste and cold in nature, has the effects of clearing heat, drying dampness, relieving pain and the like, and is mainly used for treating diseases such as dysentery, stomachache, eczema, sore and furuncle, stubborn dermatitis and the like. Sophora alopecuroides L is listed as a road medicinal material which is mainly protected in part of provinces in the northwest of China and is included in the action plan of the modern science and technology industry of traditional Chinese medicine.

Currently, many reports about chemical components and pharmacological actions of sophora alopecuroides are reported, most of researches on sophora alopecuroides are concentrated on alkaloid components unique to sophora medicinal plants, YaWen and the like, 7 alkaloid components are separated from sophora alopecuroides seeds, namely matrine, oxymatrine, sophocarpine, 9 α -hydroxysophocarpine, oxymatrine, 9 α -hydroxysophoramine, (-) -13, 14-dehydrosophoridine, a non-alkaloid compound 3-indolylamine, homoerythrin and the like are obtained for the first time, 9 α -hydroxymatrine is obtained from the seeds of Xinjiang sophora alopecuroides, matrine, sophoridine and oxymatrine are obtained through the separation, flavonoids are also contained in sophora alopecuroides, hot cumin keiya and the like, 3 flavonoid chemical components are separated from new sophora alopecuroides for the first time, namely 3',4' -dihydroxyisoflavone-7-O- β -D-glucopyranoside, 3-dihydropicrin, ketopyranoside, keton-3-7-dihydropicrin, and the like are obtained through a-2-7-dihydropicrola-7-dihydropicrin, and the like are obtained through the gas chromatography, and the separation of sophora alopecuroide, and the steps of a alophane and the steps of a lactone, the steps of a alopecuroide, the steps of obtaining 3-2-7-2-dihydropicrin, the steps of a alophanthomsonianin, the steps of a, the steps of extracting the steps of obtaining the steps of the first-7-.

In the aspect of pharmacological activity evaluation, Zhang Shuang and other researches find that matrine and sophoridine have obvious inhibition effect on pain response of mice. The strong of the residual construction and the like find that the oxysophoridine and the oxymatrine have central inhibition effect on mice and show sedative-hypnotic effect. The research of Hou Yan Hui and the like finds that matrine, sophocarpine and the like can regulate the thermoregulation center, lower the normal body temperature and inhibit the rectal warming. This suggests that Sophora alopecuroides has an effect on the central nervous system. Studies such as Liu Jing, etc. find that the alkaloid of sophora alopecuroides has an anti-tumor effect, and the mechanism of the alkaloid is mainly an important target spot in the process of regulating and controlling cell apoptosis such as Bcl-2, Caspase protein family, ASK1-p38 signal pathway, etc. Modern pharmacological experimental research also shows that the sophora alopecuroides total alkaloids have anti-tumor effects in vivo and in vitro. And the alkaloid derivative of the compound, namely the schofirauda also has obvious anti-tumor effect. Korean swallow and the like find that alkaloid components of sophora alopecuroides can obviously reduce inflammatory reaction in mice. Tian A and the like find that the sophora alopecuroides total alkaloids have obvious inhibition effect on inflammatory reaction caused by helicobacter pylori. In addition, the aloperine can obviously reduce the liver fibrosis degree of rats. Shenlinghu et al found that the polysaccharide of sophora alopecuroides has the ability of eliminating hydroxyl free radicals after carboxymethylation modification. In general, the pharmacological activities of sophora alopecuroides mainly have the effects of resisting cancer, diminishing inflammation, inhibiting bacteria, influencing the central nervous system, resisting oxidation and the like.

The traditional Chinese medicine is a valuable cultural heritage in China, and makes a great contribution to the human health career. China has abundant Chinese herbal medicine resources and clinical experience of preventing and treating diseases by using natural medicines for thousands of years, and besides the effects of resisting inflammation, influencing the central nervous system, resisting bacteria, resisting tumors and the like, the method still has development space for researching the medicinal value of the sophora alopecuroides in other fields.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a medicament for treating diabetes and a preparation method and application thereof, so as to obtain the medicinal value of sophora alopecuroides in a new field.

The invention is realized by the following steps:

in a first aspect, embodiments of the present invention provide a medicament for treating diabetes comprising an active ingredient comprising at least one of butin, butein-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone, and butin.

In a second aspect, embodiments of the present invention provide a method for preparing a medicament according to the previous embodiments, comprising: extracting to obtain the active component;

optionally, separating and purifying from herba Sophorae Alopecuroidis to obtain active component;

optionally, the separating and purifying comprises: extracting Sophora alopecuroides with solvent to obtain Sophora alopecuroides extract, removing organic solvent from Sophora alopecuroides extract to obtain residue, diluting with water to obtain suspension, and extracting with ethyl acetate to obtain Sophora alopecuroides suspension; optionally, the extractive solution is alcohol or alcohol-water mixture, and optionally, the extraction method comprises refluxing, soaking or percolating herba Sophorae Alopecuroidis powder with the extractive solution.

Optionally, after removing the solvent from the sophora alopecuroide extracting solution, adding water for mixing to obtain a suspension, and then extracting the suspension by using the ethyl acetate;

optionally, the using amount ratio of the solvent to the sophora alopecuroide is 13-17: 1, preferably 15: 1; the dilution ratio of the residue to water is 1: 5-15, preferably 1: 10; the dosage ratio of the ethyl acetate to the sophora alopecuroide suspension is 0.8-1.2: 1, preferably 1: 1.

optionally, the separating and purifying further comprises: passing the extract obtained after ethyl acetate extraction through a macroporous resin column, and performing gradient elution by using an alcohol-water mixed solution; further optionally, gradient elution is carried out by using alcohol solutions with the mass concentrations of 10% -30%, 30% -50%, 50% -70%, 70% -90% and 90% -100% respectively; more optionally, gradient elution is carried out with alcoholic solutions with mass concentrations of 10%, 30%, 50%, 70% and 90%, respectively; optionally, collecting an eluent obtained by eluting with an alcohol solution with the mass concentration of 70% -90% as the sophora alopecuroide ethyl acetate extract; optionally, the alcohol solution is an ethanol solution.

In a third aspect, the embodiments of the present invention further provide a use of the pharmaceutical preparation according to any one of the previous embodiments or the pharmaceutical preparation prepared by the preparation method of the previous embodiments in preparing an agonist for promoting glucose uptake.

In a fourth aspect, the embodiments of the present invention further provide a use of the pharmaceutical agent according to any one of the previous embodiments or the pharmaceutical agent prepared by the preparation method of the previous embodiments in preparing a glucose transporter 4 agonist;

optionally, a glucose transporter 4 agonist is used to facilitate transport of the glucose transporter 4 vesicle to the cell membrane.

In a fifth aspect, the embodiments of the present invention further provide a use of the pharmaceutical agent according to any one of the previous embodiments or the pharmaceutical agent prepared by the preparation method of the previous embodiments in preparing a protein kinase C agonist;

alternatively, protein kinase C agonists are used to stimulate an increase in PKC phosphorylation, resulting in activation of the PKC signaling pathway.

In a sixth aspect, the embodiment of the present invention further provides an application of the pharmaceutical preparation according to any one of the foregoing embodiments or the pharmaceutical preparation prepared by the preparation method of the foregoing embodiments in preparing a drug or a health food for treating or preventing diseases caused by metabolic abnormalities;

optionally, the disease caused by metabolic abnormality is a disease caused by a decrease in total cholesterol and/or triglyceride levels in the body;

alternatively, the disease caused by metabolic abnormality is obesity or hyperlipidemia.

In a seventh aspect, the present invention also provides a use of the medicament according to any one of the previous embodiments or the medicament prepared by the preparation method of the previous embodiments in preparing a medicament for treating type ii diabetes.

The invention has the following beneficial effects:

the active components with therapeutic activity on diabetes mellitus, namely the butin, the butein-4 ' -O- β -D-glucoside, the daidzein, the 7,3',4' -trihydroxyflavone, the 7,3' -dihydroxy-5 ' -methoxyisoflavone and the butin, are separated and purified from the traditional Chinese medicine sophora alopecuroides, and the six active components have good hypoglycemic activity and can be used for preparing clinical diabetes mellitus medicaments no matter being used singly or used in combination.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing the effect of the flavonoid site of Sophora alopecuroides on glucose uptake by L6 cells in example 2;

FIG. 2 is a graph showing the effect of the flavonoid site of Sophora alopecuroides in example 2 on GLUT4 translocation activity of L6 cells;

FIG. 3 shows that the flavonoid site of Sophora alopecuroides L in example 2 promotes GLUT4 translocation in L6 cell via PKC signaling pathway;

FIG. 4 shows that the flavonoid site of Sophora alopecuroides in example 2 promotes the expression of GLUT4 in L6 cells through PKC signaling pathway;

FIG. 5 is a graph showing the effect of the flavonoid site of Sophora alopecuroides in example 2 on fasting plasma glucose in type II diabetic mice;

FIG. 6 is a graph showing the effect of the flavonoid site of Sophora alopecuroides in example 2 on oral glucose tolerance in type II diabetic mice;

FIG. 7 shows the effect of the flavonoid sites of Sophora alopecuroides L in example 2 on serum insulin and insulin resistance index of type II diabetic mice;

FIG. 8 is the effect of the flavonoid sites of Sophora alopecuroides in example 2 on serum lipid levels in type II diabetic mice;

FIG. 9 is a graph showing the effect of the flavonoid site of Sophora alopecuroides in example 2 on the liver morphology of type II diabetic mice;

FIG. 10 is a graph showing the effect of the flavonoid site of Sophora alopecuroides in example 2 on the improvement of pancreatic injury in type II diabetic mice;

FIG. 11 is a graph showing the effect of the flavonoid site of Sophora alopecuroides in example 2 on pPKC, GLUT4 protein in insulin target tissues of type II diabetic mice.

In FIGS. 1 to 4, control represents the normal group, Insulin represents the Insulin positive group, PMA represents the PKC pathway agonist positive control group, and SA-FRE represents the flavonoid site of Sophora alopecuroides L.

In FIGS. 5 to 11, NC represents the normal group, DC represents the model group, SFL represents the group of low dose of flavonoid site of sophora alopecuroides, SFH represents the group of high dose of flavonoid site of sophora alopecuroides, and MET represents the group of positive metformin.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following is a detailed description of the pharmaceutical agent for treating diabetes according to the embodiments of the present invention, and a method for preparing and synthesizing the same.

Some embodiments of the present invention provide a medicament for treating diabetes, comprising an active ingredient comprising at least one of butin, butein-4 '-O- β -D-glucoside, daidzein, 7,3',4 '-trihydroxyflavone, 7,3' -dihydroxy-5 '-methoxyisoflavone and butin, i.e., the active ingredient can be any one of butin, butin-4' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3 '-dihydroxy-5' -methoxyisoflavone and butin alone or in combination of two or more thereof.

In some preferred embodiments, the active ingredients include butin, butein-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone, and butin.

Preferably, the active components comprise 16-20 parts of butin, 3-5 parts of butin-4 '-O- β -D-glucoside, 22-26 parts of daidzein, 5-8 parts of 7,3',4 '-trihydroxyflavone, 38-42 parts of 7,3' -dihydroxy-5 '-methoxyisoflavone and 6-10 parts of butin by weight, for example, the active components comprise 18 parts of butin, 4 parts of butin-4' -O- β -D-glucoside, 24 parts of daidzein, 6 parts of 7,3',4' -trihydroxyflavone, 40 parts of 7,3 '-dihydroxy-5' -methoxyisoflavone and 8 parts of butin.

Wherein the structural formula of butin is shown as

The structural formula of butein-4' -O- β -D-glucoside is shown in the specification

The structural formula of the daidzein is shown as

The structural formula of the 7,3',4' -trihydroxyflavone is shown in the specification

The structural formula of the 7,3 '-dihydroxy-5' -methoxy isoflavone is shown in the specification

The structural formula of the butein is shown as

In an alternative embodiment, the medicament comprises an extract of Sophora alopecuroides which comprises butin, butin-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone and butin.

In some embodiments, the extract of sophora alopecuroides is an ethyl acetate extract of sophora alopecuroides. The preparation process of the sophora alopecuroide ethyl acetate extract comprises the following steps: extracting Sophora alopecuroides with alcohol-water mixed solution, diluting the obtained Sophora alopecuroides residue with water, and extracting with ethyl acetate to obtain Sophora alopecuroides ethyl acetate extract.

In alternative embodiments, the medicament further comprises a pharmaceutically acceptable carrier and/or adjuvant.

It is noted that the term "pharmaceutically acceptable" means that the compound is physiologically acceptable when administered to a human and does not cause allergic reactions such as gastrointestinal disorders, dizziness or systemic allergic reactions similar to these allergic reactions.

"pharmaceutically acceptable carriers or adjuvants" include, but are not limited to: binders (such as microcrystalline cellulose, alginates, gelatin, and polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose, and anhydrous lactic acid), disintegrants (such as crosslinked PVP, sodium crosslinked carboxymethyl starch, sodium crosslinked carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose), lubricants (magnesium stearate, aluminum stearate, talc, polyethylene glycol, sodium benzoate), wetting agents (such as glycerin), surfactants (such as cetyl alcohol), and absorption enhancers, flavors, sweeteners, diluents, coating agents, and the like. The carriers or adjuvants are employed in the amounts customary for a person skilled in the art in the light of the prior art.

In some embodiments, the above medicament may be a composition comprising a plurality of components of the above active ingredient, or may be a preparation containing the above active ingredient composition, and the preparation may be a tablet, a soft capsule, a hard capsule, an oral liquid, a pill, a suppository, a powder, a granule, an emulsion, a syrup, an aerosol, a sterile injection, a sterile powder, or the like. In order to allow the drug to release the active ingredient rapidly, continuously and over a long period of time, the pharmaceutical composition or formulation of the present invention may be manufactured according to conventional methods disclosed in those technical fields. The route of administration of the agents of the embodiments of the invention may be oral, nasal inhalation or parenteral.

Some embodiments of the present invention also provide a method of preparing a medicament as in the previous embodiments, including but not limited to: extracting to obtain the above active components. That is, the above active ingredients, including but not limited to sophora alopecuroides, can be obtained by extraction in plants. Of course, the above active ingredients can be obtained directly from the market by purchase or the like.

In some preferred embodiments, the pharmaceutical agent can be prepared by: extracting active components from herba Sophorae Alopecuroidis. Specifically, the sophora alopecuroides can be extracted by adopting a solvent to obtain sophora alopecuroides extract, the sophora alopecuroides extract is subjected to organic solvent removal to obtain residues, water is added for dilution to obtain suspension, and then the sophora alopecuroides suspension is extracted by ethyl acetate; optionally, the extractive solution is alcohol or alcohol-water mixture, and optionally, the extraction method comprises refluxing, soaking or percolating herba Sophorae Alopecuroidis powder with the extractive solution.

Optionally, removing solvent from herba Sophorae Alopecuroidis extractive solution, mixing with water to obtain suspension, and extracting with ethyl acetate; optionally, the ratio of the solvent to the sophora alopecuroide is 15: 1; diluting the residue B and water at a ratio of 1: 10; the dosage ratio of the ethyl acetate to the sophora alopecuroide suspension is 1: 1.

in some embodiments, the sophora alopecuroide powder is refluxed, soaked or percolated by alcohol or alcohol-water mixed solution, and an extracting solution is obtained after filtration; evaporating the solvent in the extractive solution to obtain extract; adding water into the extract, and stirring to obtain a suspension; finally, the suspension is extracted with ethyl acetate to obtain an ethyl acetate extract and a water-soluble fraction.

Further, the extract obtained after ethyl acetate extraction is passed through a macroporous resin column, and gradient elution is carried out by using an alcohol-water mixed solution; preferably, the gradient elution is carried out by using alcohol solutions with the mass concentration of 10-30%, 30-50%, 50-70%, 70-90% and 90-100% respectively. More preferably, the gradient elution is performed with alcoholic solutions having mass concentrations of 10%, 30%, 50%, 70%, 80% and 90%, respectively. Preferably, collecting the eluent obtained by eluting with 70-90% alcohol solution as the sophora alopecuroide ethyl acetate extract. The components obtained by eluting different gradient alcohol solutions are respectively combined and respectively subjected to activity screening, and the activity screening result shows that the component eluted by the alcohol solution with the mass concentration of 70-90% has the best activity, and can be used as the sophora alopecuroides ethyl acetate extract. In some embodiments, the alcohol solution is an ethanol solution.

Some embodiments of the invention also provide a use of an agent of any of the preceding embodiments in the preparation of an agonist for promoting glucose uptake.

The inventor discovers, through research, that the alopecuroide flavone extract and the alopecuroide ethyl acetate extract containing the active components, and the butin, the butin-4 '-O- β -D-glucoside, the daidzein, the 7,3',4 '-trihydroxyflavone, the 7,3' -dihydroxy-5 '-methoxyisoflavone and the butin can improve the glucose uptake level, wherein the alopecuroide flavone extract, the butin-4' -O- β -D-glucoside, the daidzein, the 7,3',4' -trihydroxyflavone, the 7,3 '-dihydroxy-5' -methoxyisoflavone and the butin have remarkable promoting effects on the glucose uptake at a basic level and under the stimulation of insulin.

Some embodiments of the invention also provide a use of an agent of any of the preceding embodiments for the preparation of a glucose transporter 4 agonist.

Glucose transporter 4(GLUT4), a transmembrane protein, is expressed primarily in adipose and muscle cells and is the major protein responsible for transport of the grape lining in the animal body. In adipocytes and skeletal muscle cells, GLUT4 is present in a specific membrane structure, called GLUT4 vesicles. Insulin stimulation is a direct cause of the massive transport of intracellular GLUT4 vesicles to the cell membrane. Insulin stimulates intracellular GLUT4 vesicle transport to the membrane for release, resulting in an increase in GLUT4 content on the membrane. GLUT4 with activity on membrane transports glucose into muscle, fat and other tissues for metabolism and storage, so as to maintain the sugar metabolism balance of body. In the unstimulated state, about 95% of GLUT4 was located in the vesicles within the reservoir cells. When movement or signal stimulation stimulates insulin to bind to the receptor, a series of signal responses are triggered, resulting in movement of GLUT 4-rich vesicles to the plasma membrane, GLUT4 translocates to the plasma membrane and increases activity, binding to glucose and conformational changes occur, at which time glucose is transported into the cell.

In the early experiments, a cell model with GLUT4 as a screening target is established, and effective extracts or monomeric compounds with GLUT4 as the target are searched by detecting the fluorescence intensity on cell membranes. Human has high sequence homology with mouse GLUT4, and mouse L6 myotube cells can be used to replace human muscle cells in vitro experiments. In the unstimulated state, about 95% of GLUT4 was located in the vesicles within the reservoir cells. When movement or signal stimulation stimulates insulin to bind to the receptor, a series of signal responses are triggered, resulting in movement of GLUT 4-rich vesicles to the plasma membrane, GLUT4 translocates to the plasma membrane and increases activity, binding to glucose and conformational changes occur, at which time glucose is transported into the cell. Based on the above, the inventor creates a drug screening system based on GLUT4 fluorescent marker of L6 cells; the degree of influence of the sample on the GLUT4 translocation activity was determined by directly tracking the movement of fluorescently labeled GLUT4 in L6 cells using a confocal laser microscope.

The inventors found, through the above studies, that the aloperine extract and the aloperine extract containing the above active ingredients, and butin, butin-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone and butin, all of which are capable of promoting the transport of GLUT4 vesicles to the cell membrane, binding of glucose and transport of glucose into the cell, thereby increasing the uptake of glucose by the cell, and thus found that the above glucose transporter 4 agonist is useful for promoting the transport of glucose transporter 4 vesicles to the cell membrane.

Some embodiments of the invention also provide a use of an agent according to any of the preceding embodiments for the preparation of a protein kinase C agonist.

In eukaryotic cells, there are mainly two signaling pathways that regulate the transport of GLUT 4. One is the insulin-induced GLUT4 translocation pathway; the second is the non-insulin signal-dependent pathway for GLUT4 transport. Wherein, the signal path induced by insulin comprises PI3K/AKT/PKB, PKCs, CAP/CBL/TC10, MAPKs and the like. Protein kinase c (pkc), a calcium-or/and phospholipid-dependent protein phosphorylase, is an effector in G protein-coupled receptor systems, and is also a cellular esterase, widely present in various tissue cells of the human body, mainly distributed in the cytoplasm of unstimulated cells and on the cell membrane of stimulated cells. It can be activated by extracellular bioactive factors (growth factors, neurotransmitters, cytokines, etc.), complete phosphorylation of target cell protein, complete response of cell exogenous signals through change of bioactivity after protein phosphorylation, and constitute an important intracellular information network system. Activation of PKC and a series of unknown pathways downstream of PKC in the insulin signaling pathway will facilitate transport of GLUT 4. Therefore, it can activate enzymes in cytoplasm and participate in the regulation of biochemical reaction. PKC is also an important target of organism metabolism, and has important effects in controlling liver glycogen metabolism, regulating cell differentiation, regulating lipid metabolism, regulating gene expression, inhibiting and regulating tumor growth for a long time. Numerous studies have shown that PKC regulates a number of different metabolic pathways, which are safe and effective targets for the treatment of type ii diabetes and lipid metabolism disorders. GLUT4 is the rate-limiting step in glucose uptake by skeletal muscle and adipose tissue, while PKC is one of the major regulatory proteins of GLUT 4. During glycolipid metabolism, external stimulation increases PKC phosphorylation, promotes PKC signal pathway activation, thereby promoting GLUT4 to be transported to a cell membrane from cytoplasm, and finally enhancing glucose uptake of cells.

To further investigate the specific signaling pathway that the above active ingredients contribute to the GLUT4, the inventors used AMPK signaling pathway inhibitors (Compound C), PI3K/Akt signaling pathway inhibitors (Wortmannin), and PKC signaling pathway inhibitors

Studying whether the active component can inhibit the expression and translocation of the GLUT4 so as to preliminarily determine the signal path of the action of the active component. The inventor discovers through research that the sophora alopecuroides flavone extract and the sophora alopecuroides ethyl acetate extract containing the active components can increase the phosphorylation of PKC and activate a PKC signal path, thereby promoting the transmembrane and expression of GLUT4 protein and finally playing a role in improving the symptoms of type II diabetes. Therefore, protein kinase C agonists are used to stimulate the increase of PKC phosphorylation, which leads to the activation of the PKC signaling pathway.

Some embodiments of the present invention also provide a use of the agent according to any one of the preceding embodiments in the preparation of a medicament or health food for treating or preventing a disease caused by metabolic abnormality. Optionally, the disease caused by metabolic abnormality is a disease caused by a decrease in total cholesterol and/or triglyceride levels in the body; alternatively, the disease caused by metabolic abnormality is obesity or hyperlipidemia.

Some embodiments of the invention also provide a use of an agent according to any of the preceding embodiments in the manufacture of a medicament for the treatment of type ii diabetes.

The active components can activate PKC which is an important target for regulating metabolic diseases, and researches show that the sophora alopecuroides flavone extract and the active components of butein, butein-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone and butein can reduce the content of total cholesterol and triglyceride in diseases caused by metabolic abnormality, so that the active components can be used for relieving or treating or preventing the diseases caused by metabolic abnormality, such as diabetes, obesity and hyperlipidemia.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The whole plant of herba Sophorae Alopecuroidis is purchased from West Ning medicinal material market in Qinghai province, and is dried seed of herba Sophorae Alopecuroidis (Sophora alopecuroides L). Soaking 500g of herba Sophorae Alopecuroidis seed dried powder in 95% ethanol water solution at a material-to-liquid ratio of 1:15(1.0kg of medicinal material corresponds to 15.0L of solvent) for 3 days, filtering to obtain filtrate, extracting the residue with 95% ethanol water solution for 4 times according to the above method, filtering, mixing the filtrates, and concentrating under reduced pressure to obtain 88g of brown residue; dissolving in water at a volume ratio of 1:10 for dilution, extracting the water solution with ethyl acetate with the same volume as the water part for 4 times, and recovering and concentrating under reduced pressure to obtain 16g of herba Sophorae Alopecuroidis ethyl acetate extract.

Example 2

Dissolving 12g of sophora alopecuroides ethyl acetate extract with 50mL of chloroform, adding 25g of macroporous resin (D101 type), uniformly stirring, drying under reduced pressure, uniformly dispersing, fully adding the mixture to a macroporous resin column (D101, 300g), carrying out gradient elution by using aqueous ethanol with the mass concentration of 10%, 30%, 50%, 70%, 80% and 90%, respectively concentrating and drying alcohol-water eluates with different concentrations under reduced pressure, screening the eluates with different concentrations by using a GLUT4 membrane conversion activity screening method, and displaying good GLUT4 membrane conversion promoting activity on 70-90% of parts, namely sophora alopecuroides flavone parts (SA-FREE, 4.2 g). 100mg of SA-FRE was dissolved in 2.0mL of methanol and filtered. 200 μ L of SD-PRE was separated from Waters semi-preparative liquid phase using Waters Sunfire C18HPLC column (5 μm, 19X 250mm i.d.), eluted with a gradient of pure water and acetonitrile as mobile phase (two phases containing 0.1% formic acid, respectively), 0-40 min, 20-35% acetonitrile; 40-45 min, 35-95% acetonitrile; 45-50min, 95% acetonitrile. Manually collecting 6 main chromatographic peaks, repeating the sample injection for 20 times, combining the fractions of the same time period, collecting 6 peaks using a Sephadex column (350X 10mm, 65% MeOH: 35% CH)₂Cl₂Containing 0.1% formic acid, respectively) to give 6 pure compounds, butin (16.6mg), butin-4 ' -O- β -D-glucoside (2.9mg), daidzein (21.3mg), 7,3',4' -trihydroxyflavone (5.6mg), 7,3' -dihydroxy-5 ' -methoxyisoflavone (35.5mg) and butin (6.7mg), respectively.

Example 3

Effect of the flavonoid site of sophora alopecuroide (extract containing six active ingredients) on glucose uptake by L6 muscle cells:

thawing frozen L6 cells, culturing with α -MEM medium containing 10% FBS and 1% double antibody, when they grow to logarithmic phase, differentiating with α -MEM medium containing 2% FBS and 1% double antibody, after they completely differentiate into myotubes, according to 1 × 10⁴–5×10⁴The cells/well were seeded in a 96-well plate at a density of 100. mu.L per well, and then L6 cells in the 96-well plate were differentiated for 6 days with α -MEM medium containing 2% FBS, after differentiation, the cells in the 96-well plate were starved for 2h with α -MEM medium without serum, the Sophora alopecuroides flavone fraction in example 2 was dissolved in DMSO solution at a concentration of 1 mg/50. mu.L to prepare a Sophora alopecuroides flavone fraction mother solution at 20. mu.g/. mu.L, and then 0.25. mu.L of the Sophora alopecuroides flavone fraction mother solution was added to each 100. mu.L of α -MEM medium to prepare α -MEM medium having a Sophora flavone concentration of 50. mu.g/mL, to prepare L6 cells as a drug-containing medium, the mother solution of 2-NBDG was prepared as described above and diluted in α -MEM medium to make the drug-containing α -NBDG at a concentration of 150. mu.g/mL.

Cells were placed at 37 ℃ and 5% CO₂The cells were incubated in an incubator for 30min, each group had more than three replicates and a blank control, an insulin positive control (100 nM). After 30min the 96-well plates were centrifuged at 400g for 5min at room temperature. The supernatant was discarded, 200. mu.L of the kit buffer was added to each well and mixed well, and centrifugation was continued at 400g for 5min at room temperature. Abandoning the supernatant again, and finally every timeAdd 100. mu.L of kit buffer to the wells and mix well. The fluorescence absorption of 2-NBDG from each well was measured using a microplate reader at an excitation wavelength of 485nm and an emission wavelength of 535 nm.

The results are shown in fig. 1, and the flavonoid site of sophora alopecuroides was able to significantly enhance glucose uptake by L6 cells at a dose of 50 μ g/mL compared to the normal group (blank control group) and exhibited significant differences (. about.p < 0.01).

Example 4

The flavonoid part of the sophora alopecuroide (extract containing six active components) is applied to GLUT4 membrane transposition:

l6 cells stably expressing GLUT4-mOrange were seeded in a 6cm dish, 10% fetal bovine serum-containing MEM- α medium was added thereto, and 5% CO was added thereto at 37 ℃₂And (5) culturing in a cell culture box. First, GLUT4-mOrange-L6 cells were trypsinized and plated on sterile coverslips at 37 ℃ and 5% CO₂The cells are cultured in the incubator overnight to be completely attached. The medium was then discarded, starved for 2 hours with serum-free medium added, and gently washed with PBS solution. 50 μ g/mL of the flavonoid fraction from Sophora alopecuroides of example 2 was added, and the change in intracellular fluorescence intensity was observed by monitoring the dynamic change in GLUT4-mOrange transport after drug addition using a laser scanning confocal microscope to reflect the membrane condition on GLUT 4.

As can be seen from FIG. 2, the flavonoid site of Sophora alopecuroides shows a strong effect of promoting GLUT4 to translocate and membrane. Specifically, the membrane GLUT4 fluorescence intensity was gradually increased after the addition of 50 μ g/mL of the aloperine site in a in fig. 2, and as can be seen from B in fig. 2, the membrane GLUT4-mOrange fluorescence intensity was gradually increased within 30min after the addition of 50 μ g/mL of SA-FRE, and reached 1.85-fold at 50min (. about.p <0.01,. about.p <0.001, compared with the normal group).

Example 5

Based on the experimental procedure of example 2, L6IRAP-mOrange cells were selected and Compound C (10. mu.M), Wortmannin (100nM) and,

(10. mu.M) incubation of cells for 30min before addition of assayAnd testing the drug, and observing the change of fluorescence on the membrane to indirectly reflect the membrane condition on the GLUT 4.

As can be seen from fig. 3, after the inhibitor Compound C of AMPK (fig. 3B) was added, the fluorescence intensity did not change significantly, and did not inhibit the translocation of GLUT4 due to the flavonoid site of sophora alopecuroides, nor did the inhibitor Wortmannin of PI3K (fig. 3A) inhibit the translocation of GLUT4 due to the flavonoid site of sophora alopecuroides, and therefore, the inhibitors Wortmannin of PI3K and the inhibitor Compound C of (B) AMPK could not inhibit the translocation of GLUT4 of L6 cells due to SA-FRE. Inhibitors of PKC

(FIG. 3C) significantly inhibited GLUT4 translocation induced by Sophora alopecuroides flavone site ((FIG. 3C))^***P<0.001，^**P<0.01, compared to the normal group). Thus, test compounds promote translocation of GLUT4 to the upper membrane via the PKC signaling pathway, thereby increasing glucose uptake.

Further detection was then carried out by Western blot, and as shown in fig. 2, it was found that after incubation at the site where 50 μ g/mL of sophora alopecuroides flavone was added, the expression level of GLUT4 was significantly enhanced in L6 cells, and the phosphorylation level of PKC was significantly increased (fig. 4A and 4B). Specifically, in fig. 4A, 50 μ g/mL SA-FRE significantly elevated PKC phosphorylation levels in L6 cells; in FIG. 4B, 50 μ g/mL SA-FRE significantly promoted the expression level of GLUT4 in L6 cells (II) ((III))^**P<0.01，^*P<0.05, compared to the normal group). Combining the above data, it is shown that the flavonoid site of Sophora alopecuroides of example 1 promotes GLUT4 translocation and expression in L6 cells via PKC signaling pathway.

Animal experiments: hypoglycemic effect of sophora alopecuroide flavone part on diabetic mice

1. Materials and instruments

1.1 Experimental animals

8 week old male C57/BL6J mice, SPF grade, purchased from Beijing Huafukang Biotechnology GmbH, Inc., license number SCXK (Kyoto) 2009-. The rat material was sterilized at 60 ℃ and supplied by Beijing Huafukang Biotech GmbH. High fat feeds were purchased from Jiangsu Meditson biomedical Co. A breeding environment: the temperature of the room temperature is 23 +/-2 ℃, the humidity is 50-60%, water is freely fed, and the illumination period is 12 h.

1.2 drugs and reagents

The flavonoid fraction of Sophora alopecuroides obtained in example 2, insulin radioimmunoassay kit (Tianjin Jiuding medical bioengineering Co., Ltd.), glucose (national group chemical reagent Co., Ltd., lot No. 20130328), metformin hydrochloride tablet (Shanghai Shibaopharmacy Co., Ltd., Yang Zhong), RIPA Cell lysate (Biyuntian Biotech Co., Ltd.), PMSF (Biyuntian Biotechnology Co., Ltd.), BCA protein concentration kit (Biyuntian Biotechnology Co., Ltd.), GLUT4, β -actin, pPKC, secondary antibody, etc. (Cell Signaling Technology).

1.3 Experimental instruments

Glucometer (trinoagulant glucometer), glucose test strips (trinoagulant glucose test strips), analytical balance CP214 (aohaus instruments shanghai ltd), ALLEG RAX-22R high speed refrigerated centrifuge (beckmann coulter, usa), fully automatic biochemical analyzer (hiti 7180+ ISE).

2. Experimental methods

2.1 preparation of samples to be tested

A sample to be detected is obtained by referring to a preparation method of the sophora alopecuroides flavone part and is used for the following experiments.

2.2 establishment of Experimental model

65C 57BL/6J mice are fed with common feed for one week to adapt to the environment, 10C 57BL/6J mice are used as a normal control group, the other 50 mice are fed with high-fat feed for three weeks to induce the mice to generate obesity and insulin resistance, then 40mg/kg of STZ solution (PH 4.5) dissolved in ammonium citrate solution is injected into the abdominal cavity of the mice, three days later, tail blood is taken to measure the fasting blood glucose concentration of the mice, the blood glucose concentration is more than 11mmol/L, the mice are qualified model mice, and the mice can be used for the next experiment. Randomly dividing into 4 groups of model control group, positive control group (metformin hydrochloride tablet 200mg/kg), and herba Sophorae Alopecuroidis flavone part administration group (100mg/kg, 200mg/kg) according to fasting blood glucose value, wherein each group contains 8 patients. The groups are administrated by equal-volume and unequal-concentration gavage (normal control group and model control group are administrated with equal-volume normal saline), the gavage is administrated once every day at regular time, the gavage volume is 0.1mL/10g, and the gavage is continuously carried out for the periphery.

2.3 Experimental data Collection

Blood glucose values of mice were measured by taking blood from tail veins before administration, after administration, and at 7d, 15d, 21d, and 28d, respectively. Body weights of mice were measured before, after, and every two days between administrations, respectively. After the last administration, the mice were fasted for 12 hours, the blood glucose concentration of the mice was measured as the blood glucose value for 0 hour, the blood glucose values of the mice were measured at 2.0g/kg for intragastric administration for 30min, 60min, 90min and 120min, respectively, the change in the blood glucose values of the mice in each group was observed, and the differences between the groups were compared.

After the blood sugar index measurement is finished, blood is taken by taking eyeballs, and serum is separated by centrifuging at the rotating speed of 3000rpm for 15 min. The serum cholesterol, triglyceride, high density lipoprotein cholesterol and low density lipoprotein cholesterol contents of total cholesterol and triglyceride in the mouse serum are respectively measured by using a full-automatic biochemical analyzer, and the free fatty acid content is detected by using a kit. The immunoassay method is used for measuring the content of serum insulin.

After blood collection was completed, the mice were sacrificed and then skeletal muscle, liver, and adipose tissues of the mice were collected. A portion of the liver and pancreas was fixed in 4% formaldehyde solution. These tissues were then embedded with paraffin and finally mounted with neutral resin and dried. These tissues were cut into 5 μm thick sections and stained with eosin and hematoxylin. The pancreas was immunohistochemically stained. The tissue sections were observed under a microscope and photographed for morphological analysis.

The detection of mouse tissue related protein includes extracting total protein with RIPA lysate, determining protein concentration with BCA protein kit, taking equal amount of protein from each group of samples, SDS-PAGE gel separating protein, transferring the protein to PVDF membrane electrically, sealing with TBST containing 0.5% defatted milk powder for 1 hr, adding diluted anti-GLUT 4, pPKC and β -actin antibody at 4 deg.c overnight, washing with horse radish peroxidase labeled goat anti-rabbit or goat anti-mouse IgG, shaking at room temperature for 1 hr, washing completely, adding ECL developing liquid, and imaging analysis with gel imaging system (APLEGEN, INC, USA).

3. Results of the experiment

Data are presented as mean ± standard error (mean ± SEM), and tests for significant differences between groups were performed using t-test and correlation analysis.

3.1 Effect on fasting plasma glucose in type II diabetic mice

After being raised for four weeks with high fat, the fasting blood sugar value of the diabetic mice in the model group is obviously higher than that of the diabetes blood sugar value of the C57 mice in the normal group, and the differences are significant (⁺⁺⁺P < 0.001), while the blood glucose levels of the administered group and the model group were not significantly different. Four weeks after administration, blood glucose levels were significantly reduced in each of the administered groups, with significant differences compared to the model group (. about.p < 0.001). Therefore, the flavonoid part of the sophora alopecuroides has the effect of reducing the blood sugar of the mice with type II diabetes. The results of the experiment are shown in FIG. 5, in which the ordinate represents the blood glucose level and the abscissa represents the time in unit of week in FIG. 5.

3.2 Effect on oral glucose tolerance in type II diabetic mice

The blood glucose level reached a maximum 30min after oral glucose administration in normal group mice, and blood glucose had returned to essentially the pre-dose level 120 min. The blood glucose values of the model group reached a maximum after 30min of oral glucose, and remained at a higher level thereafter, although somewhat decreased. The blood sugar value of the low-dose and high-dose treatment group with low or high flavonoid part of sophora alopecuroides reaches the highest level 30min after glucose is orally taken, is remarkably reduced after 30min, and basically reaches the level before glucose is orally taken 120min later. The area under the curve of blood sugar (AUC) is counted, and the results show that the low-position and high-dose groups of the aloperine have significant difference compared with the model group (P < 0.001). Therefore, the flavonoid part of the sophora alopecuroides can improve the oral glucose tolerance of the type II diabetic mice. The experimental results are shown in FIG. 6, wherein A in FIG. 6 is the blood glucose level of the mice within 2h after intragastric administration of 2.0g/kg glucose; b in fig. 6 is the area under the blood glucose curve (AUC).

3.4 Effect on serum insulin and insulin resistance index in type II diabetic mice

The insulin of the mice in the normal group is in the normal level, the insulin level of the mice in the model group is obviously higher than that of the mice in the normal group, and the insulin level of the mice in the model group has significant difference (A)⁺⁺⁺P < 0.001), indicating that the diabetic mouse has insulin resistance. Sophora alopecuroides flavone part treatment group insulin waterThe average was significantly lower than the model group, and there was a significant difference in the high dose group (. about.p < 0.01). A steady state model method proposed by MATTHEWS et al in 1985 was used to evaluate the insulin resistance state, with a resistance index (HOMA-IR) of fasting plasma glucose (mmol/L) x (mIU/L)/22.5. Compared with the mice in the model group, the insulin resistance index of the diabetic mice can be obviously reduced in each administration group. These results show that the insulin resistance of diabetic mice is obviously reduced after the treatment of the flavonoid part of the sophora alopecuroides, the hypoglycemic effect of the flavonoid part of the sophora alopecuroides is probably related to the improvement of the insulin resistance, and the experimental result is shown in figure 7.

3.5 Effect of Sophora alopecuroides flavone part on serum lipid index of type II diabetic mice

Serum cholesterol (TC), Triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and Free Fatty Acid (FFA) levels of the mice in the normal group are at normal levels, and each index of the mice in the model group is obviously higher than that of the mice in the normal group. After four weeks of treatment by the sophora alopecuroides flavone part, the contents of TC, TG, FFA and LDL-C in the serum of mice in each treatment group are obviously lower than those of a model group, and the HDL-C level is obviously higher than that of the model group, which shows that the sophora alopecuroides flavone part has the function of improving the dyslipidemia of the diabetic mice, and the experimental result is shown in figure 8, wherein A, B, C, D, E in figure 8 respectively and sequentially corresponds to the influence of the sophora alopecuroides flavone part on triglyceride, total cholesterol, free fatty acid, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol of the diabetic mice.

3.6 Effect of Sophora alopecuroides flavone part on liver morphology of type II diabetic mice

Liver section HE staining results As shown in FIG. 9 (scale, 50 μm), liver sections from normal group C57BL/6J mice showed normal morphology, model group mice showed marked symptoms of fatty liver, and liver sections contained many vacuoles of fat. Although some liver steatosis was seen in the flavonoid sites of sophora alopecuroides and in liver sections of diabetic mice after metformin treatment, the symptoms were significantly reduced compared to the model group, and the effect was best in the high dose group with metformin and sophora alopecuroides flavonoid sites. The results show that the flavonoid part of the sophora alopecuroides can be orally taken to effectively prevent the occurrence and the development of the hepatic steatosis of the diabetic mice.

3.7 improvement of flavonoid part of Sophora alopecuroides on pancreatic function damage of mice with type II diabetes

The immunohistochemical staining result of the pancreatic section is shown in FIG. 10 (ruler, 50 μm), the islet and peripheral exocrine region of the normal group C57BL/6J mice are clearly defined, the shape is normal, the islet cells are closely arranged, the insulin antibody staining area is large, and the shape is regular; the pancreatic tissues of the diabetic mice in the model group have a plurality of pathological morphological changes such as islet volume reduction, severe damage of islet cells, unclear peripheral edges and the like. After the intragastric administration, compared with the model group, the islet form of each treatment group is close to the rule, the volume is larger, and the immune signal is gradually enhanced.

3.8 Effect of Sophora alopecuroides flavone site on related proteins in insulin target tissues (skeletal muscle, adipose tissue, liver) of type II diabetic mice

The pPKC and GLUT4 protein levels in mouse skeletal muscle, adipose tissue, and liver were determined by the corresponding antibodies. As can be seen from FIG. 11, the low and high dose groups of flavonoid sites in Sophora alopecuroides L all increased the level of GLUT4 and pPKC in skeletal muscle and adipose tissue of diabetic mice and increased the level of pPKC in liver of diabetic mice compared to the model group. Combining the research result of an in vitro mechanism, the flavonoid part of the sophora alopecuroides can promote the transposition and the expression of GLUT4 mainly through a PKC signal path, improve the insulin resistance of diabetic mice, regulate the glycolipid metabolism and improve the symptoms of type II diabetes.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A medicament for the treatment of diabetes comprising an active ingredient comprising at least one of butin, butein-4 ' -O- β -D-glucoside, daidzein, 7,3',4' -trihydroxyflavone, 7,3' -dihydroxy-5 ' -methoxyisoflavone and butein.

2. The medicament of claim 1, wherein said active ingredients comprise said butin, said butein-4 ' -O- β -D-glucoside, said daidzein, said 7,3',4' -trihydroxyflavone, said 7,3' -dihydroxy-5 ' -methoxyisoflavone, and said butin;

preferably, the active components comprise, by weight, 16-20 parts of butin, 3-5 parts of butin-4 '-O- β -D-glucoside, 22-26 parts of daidzein, 5-8 parts of 7,3',4 '-trihydroxyflavone, 38-42 parts of 7,3' -dihydroxy-5 '-methoxyisoflavone and 6-10 parts of butin, more effectively, 18 parts of butin, 4 parts of butin-4' -O- β -D-glucoside, 24 parts of daidzein, 6 parts of 7,3',4' -trihydroxyflavone, 40 parts of 7,3 '-dihydroxy-5' -methoxyisoflavone and 8 parts of butin;

preferably, the structural formula of the butin is shown in the specification

The structural formula of the butein-4' -O- β -D-glucoside is shown in the specification

The structural formula of the daidzein is shown as

The structural formula of the butein is shown as

3. The agent according to claim 1 or 2, wherein the agent comprises an extract of sophora alopecuroides containing the butin, the butin-4 ' -O- β -D-glucoside, the daidzein, the 7,3',4' -trihydroxyflavone, the 7,3' -dihydroxy-5 ' -methoxyisoflavone and the butin;

preferably, the sophora alopecuroides extract is sophora alopecuroides ethyl acetate extract.

4. The medicament of claim 1 or 2, further comprising a pharmaceutically acceptable carrier or adjuvant, preferably the carrier or adjuvant comprises at least one of a binder, a filler, a disintegrant, a lubricant, a wetting agent, a surfactant, an absorption enhancer, a flavoring agent, a sweetener, a diluent, and a coating agent;

preferably, the medicament is a composition of multiple components, or the medicament is a preparation containing the composition, and the preparation is tablets, soft capsules, hard capsules, oral liquid, pills, suppositories, powder, granules, emulsions, syrups, aerosols or sterile injections.

5. A process for the preparation of a medicament according to any one of claims 1 to 4, which comprises: extracting to obtain the active component;

preferably, the active component is obtained by separating and purifying the sophora alopecuroides;

preferably, the separation and purification comprises: extracting Sophora alopecuroides with solvent to obtain Sophora alopecuroides extract, removing organic solvent from Sophora alopecuroides extract to obtain residue, diluting with water to obtain suspension, and extracting with ethyl acetate to obtain Sophora alopecuroides suspension; optionally, the extract is alcohol or alcohol-water mixture, optionally, the extraction method comprises refluxing, soaking or percolating herba Sophorae Alopecuroidis powder with the extract;

preferably, after the solvent of the sophora alopecuroide extracting solution is removed, water is added for mixing to obtain a suspension, and then the suspension is extracted by the ethyl acetate;

preferably, the using amount ratio of the solvent to the sophora alopecuroide is 13-17: 1, preferably 15: 1; the dilution ratio of the residue to water is 1: 5-15, preferably 1: 10; the dosage ratio of the ethyl acetate to the sophora alopecuroide suspension is 0.8-1.2: 1, preferably 1: 1;

preferably, the separation and purification further comprises: passing the extract obtained after the ethyl acetate extraction through a macroporous resin column, and performing gradient elution by using an alcohol-water mixed solution; more preferably, the gradient elution is carried out by using alcohol solutions with the mass concentrations of 10-30%, 30-50%, 50-70%, 70-90% and 90-100% respectively; more preferably, gradient elution is performed with alcohol solutions having mass concentrations of 10%, 30%, 50%, 70%, 80% and 90%, respectively; preferably, collecting an eluent obtained by eluting with an alcohol solution with the mass concentration of 70-90% as the sophora alopecuroide ethyl acetate extract; preferably, the alcohol solution is an ethanol solution.

6. Use of the agent of any one of claims 1 to 4 or the agent prepared by the preparation method of claim 5 in the preparation of an agonist for promoting glucose uptake.

7. Use of the agent of any one of claims 1 to 4 or the agent prepared by the method of claim 5 for the preparation of a glucose transporter 4 agonist;

preferably, the glucose transporter 4 agonist is used to facilitate transport of the glucose transporter 4 vesicle to a cell membrane.

8. Use of the agent of any one of claims 1 to 4 or the agent prepared by the preparation method of claim 5 for preparing a protein kinase C agonist;

preferably, the protein kinase C agonist is used to stimulate an increase in PKC phosphorylation, resulting in activation of the PKC signaling pathway.

9. The use of the agent according to any one of claims 1 to 4 or the agent prepared by the preparation method according to claim 5 for preparing a medicament or health food for treating or preventing diseases caused by metabolic disorders;

preferably, the disease caused by metabolic abnormality is a disease caused by a decrease in total cholesterol and/or triglyceride levels in the body;

preferably, the disease caused by metabolic abnormality is obesity or hyperlipidemia.

10. Use of the agent of any one of claims 1 to 4 or the agent prepared by the method of claim 5 in the preparation of a medicament for the treatment of type ii diabetes.