Preparation method and application of astragalus flavonoid extract with effect of treating systemic lupus erythematosus

Abstract

The invention discloses a preparation method and application of an astragalus flavonoid extract with an effect of treating systemic lupus erythematosus, and belongs to the technical field of pharmaceutical preparations. The preparation method disclosed by the invention comprises the following steps: screening components for treating systemic lupus erythematosus target protein in astragalus membranaceus by adopting reverse network pharmacology; the preparation method comprises the following steps: preparing an astragalus flavonoid substance by adopting an ultrasonic-assisted deep eutectic solvent method, and extracting the astragalus flavonoid substance by using ethyl acetate to obtain an astragalus flavonoid ethyl acetate extract; preparing magnetic nanoparticles of which the surfaces are adsorbed with the target protein; and carrying out micromolecule fishing on the astragalus flavone ethyl acetate extract by adopting the magnetic nanoparticles, so as to obtain the astragalus flavone extract with the effect of treating systemic lupus erythematosus. The radix astragali flavonoid extract prepared by the invention has a higher curative effect on systemic lupus erythematosus, and has a better effect than the existing medicine under the same dosage.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a preparation method and application of an astragalus flavonoid extract capable of treating systemic lupus erythematosus. Background Art

[0002] Astragalus flavonoids, a general term for a series of compounds derived from 2-phenylchromone as a basic skeleton, have demonstrated positive effects on many aspects of human physiology and health. In recent years, research on their pharmacological activity has increased, and flavonoids also hold great potential as therapeutic agents for systemic lupus erythematosus (SLE). However, practical applications of Astragalus have been plagued by low efficacy. Over the years, researchers have developed a variety of extraction and purification methods for Astragalus flavonoids, including hot water extraction, alkaline extraction, alkaline-alcohol extraction, and enzymatic hydrolysis. However, these methods suffer from low extraction yields, long extraction times, low purity, high energy consumption, complex procedures, and potential damage to the compound structure, all of which limit the further application of Astragalus flavonoids. Furthermore, the extracts obtained by these methods are complex in composition and contain a high concentration of impurities without therapeutic effects, which hinders the estimation of effective drug concentration and indirectly contributes to the loss of Astragalus. Furthermore, the main active components, therapeutic mechanisms, and targets of astragalus flavonoids in the treatment of SLE remain unclear, hindering in-depth research into the pathogenesis and therapeutic mechanisms of systemic lupus erythematosus. Therefore, analyzing the components of astragalus flavonoids that are effective in treating SLE and preparing astragalus flavonoids that can enhance the therapeutic efficacy of SLE are of great significance for in-depth research on the therapeutic mechanisms of SLE and the further development and application of astragalus medicinal materials. Summary of the Invention

[0003] The present invention aims to provide a method for preparing an astragalus flavonoid extract with therapeutic effects on systemic lupus erythematosus and its use, in order to address the problems of the prior art described above. The present invention utilizes reverse network pharmacology, ultrasound-assisted deep eutectic solvent, and small molecule fishing technology to prepare an astragalus flavonoid extract. The resulting extract has clear ingredients with few impurities, exhibits enhanced efficacy against systemic lupus erythematosus, and, at equivalent dosages, demonstrates superior efficacy compared to existing drugs.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a method for preparing an astragalus flavonoid extract having the effect of treating systemic lupus erythematosus, comprising the following steps:

[0006] (1) Reverse network pharmacology was used to screen target proteins in Astragalus for the treatment of systemic lupus erythematosus;

[0007] (2) preparing astragalus flavonoids by an ultrasonic-assisted deep eutectic solvent method, and extracting with ethyl acetate to obtain an astragalus flavonoid ethyl acetate extract;

[0008] (3) preparing magnetic nanoparticles with the target protein adsorbed on the surface; using the magnetic nanoparticles to carry out small molecule fishing on the astragalus flavonoid ethyl acetate extract to obtain the astragalus flavonoid extract having the effect of treating systemic lupus erythematosus.

[0009] Furthermore, the steps of the ultrasound-assisted deep eutectic solvent method are as follows:

[0010] Choline chloride, glycerol and water are mixed to obtain a deep eutectic solvent; astragalus medicinal material powder is mixed with the deep eutectic solvent, and astragalus flavonoids are obtained through ultrasonic treatment and centrifugation.

[0011] Furthermore, the molar ratio of the choline chloride to the glycerol is 1:(2-4); and / or,

[0012] The water content of the deep eutectic solvent is 40% to 60%; and / or,

[0013] The ratio of the astragalus powder to the deep eutectic solvent is 1 g: (20-35) mL; and / or,

[0014] The power during the ultrasonic treatment is 1160W-1700W, the temperature is 60-80°C, and the time is 30-60 minutes.

[0015] Furthermore, the fishing method is: mixing the magnetic nanoparticles with the target protein adsorbed on the surface with the astragalus flavonoid ethyl acetate extract, and then performing magnetic separation; washing the magnetic nanoparticles after magnetic separation with methanol, collecting the eluate, and obtaining the astragalus flavonoid extract with the effect of treating systemic lupus erythematosus.

[0016] Furthermore, the temperature during the mixing is 35-38° C., and the mixing time is 12-20 min.

[0017] Optionally, the magnetic nanoparticles having the target protein adsorbed on the surface include Fe3O4@SiO2 magnetic nanoparticles having the target protein adsorbed on the surface.

[0018] Optionally, the target protein includes TLR7.

[0019] The present invention also provides an astragalus flavonoid extract prepared by the above preparation method, wherein the astragalus flavonoid extract contains quercetin, rutin, liquiritigenin, luteolin, daidzein and genistin.

[0020] The present invention also provides the use of the astragalus flavonoid extract in preparing medicine for treating systemic lupus erythematosus.

[0021] The present invention also provides the use of daidzein in the astragalus flavonoid extract in the preparation of a medicine for treating systemic lupus erythematosus.

[0022] The present invention also provides the use of genistein in the astragalus flavonoid extract in the preparation of a medicine for treating systemic lupus erythematosus.

[0023] The present invention discloses the following technical effects:

[0024] The present invention first adopts the reverse network pharmacology method in combination with molecular docking to screen the target of systemic lupus erythematosus, uses ultrasound to assist low eutectic solvent to efficiently extract astragalus flavonoid extract, and uses molecular fishing technology to "fish" the effective ingredients from the astragalus flavonoid extract according to the screened targets. It has been verified that the effective ingredients have a good therapeutic effect on systemic lupus erythematosus. Under the same dosage, the astragalus flavonoid extract prepared by the present invention is more effective than the existing drugs. The astragalus flavonoid preparation method of the present invention using reverse network pharmacology-ultrasound-assisted low eutectic solvent-small molecule fishing technology can improve the defect of traditional astragalus flavonoid extract in treating systemic lupus erythematosus, improve the therapeutic effect, provide technical guidance for studying the mechanism of action of astragalus flavonoid on systemic lupus erythematosus and the reason for its effective treatment, provide a theoretical basis for the development of new flavonoid compounds or drugs with the effect of treating systemic lupus erythematosus, and open up new paths for the application of traditional Chinese medicine in the treatment of systemic lupus erythematosus in modern medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The effect of different extraction solvents on the extraction amount of TFAR;

[0027] Figure 2 The results show that the UDES extraction conditions are optimized. A to F represent the effects of solid-liquid ratio, ultrasonic time, ultrasonic temperature, ultrasonic power, molar ratio of HBA to HBD in DES, and water content in DES on the extraction yield of TFAR.

[0028] Figure 33D response surface curve diagram of the two-factor interaction; a to f represent the effects of the interaction of solid-liquid ratio-water content, solid-liquid ratio-ultrasonic power, solid-liquid ratio-ultrasonic temperature, water content-ultrasonic power, water content-ultrasonic temperature, and ultrasonic power-ultrasonic temperature on the TFAR extraction amount;

[0029] Figure 4 Fourier transform-infrared spectra of Fe3O4 nanoparticles, Fe3O4@SiO2 magnetic nanoparticles and APTES-modified Fe3O4@SiO2 magnetic nanoparticles;

[0030] Figure 5 is the appearance of Fe3O4@SiO2-TLR7 magnetic nanoparticles; A is the state of Fe3O4@SiO2-TLR7 without magnetic field, and B is the state of Fe3O4@SiO2-TLR7 with magnetic field;

[0031] Figure 6 The following are the structural diagrams of 36 flavonoid standards;

[0032] Figure 7 The cluster analysis results of Astragalus flavonoids are shown in Figure 1; A-1, A-2, and A-3 are TR-TFARs, and B-1, B-2, and B-3 are UDES-TFARs;

[0033] Figure 8 This is the volcano plot of Astragalus flavonoids;

[0034] Figure 9 The results of TFAR cytotoxicity to RAW264.7 cells;

[0035] Figure 10 The cytotoxicity results of the six different components on RAW264.7 cells;

[0036] Figure 11 The effects of different groups on LPS-induced cellular inflammatory factors and oxidative stress indicators; a to f represent the expression levels of TNF-α, IL-6, TGF-β, SOD, MDA and GSH in cell culture medium, respectively;

[0037] Figure 12 The effect of different groups on LPS-induced cellular NO expression levels;

[0038] Figure 13 The effect of six different components on LPS-induced cellular NO expression levels;

[0039] Figure 14 The changes of mouse autoantibodies anti-dsDNA and anti-smith before and after UDES-TFAR treatment;

[0040] Figure 15 The effect of UDES-TFAR on inflammatory factors and oxidative stress indicators in mouse serum; a to f represent the levels of IL-6, TGF-β, TNF-α, MDA, SOD and GSH in serum, respectively;

[0041] Figure 16 Histopathological sections of the heart, liver, spleen, and kidney of mice in each group (HE staining, 200×);

[0042] Figure 17 Histopathological sections of the heart and liver of mice in each group (HE staining, 400×);

[0043] Figure 18 The histopathological sections of the kidneys and spleens of mice in each group (HE staining, 400×). DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] Reverse network pharmacology is a disease-to-drug research approach. Its fundamental principle is to start from known disease-associated targets and work backwards to identify drug components that act on these targets, thereby identifying corresponding drugs. This approach combines computer simulation with experimental validation to discover new therapeutic strategies. Using reverse network pharmacology, we systematically analyze the complex relationships between the syndrome differentiation and treatment of systemic lupus erythematosus (SLE) using astragalus flavonoid extracts, revealing the traditional Chinese medicine mechanism of action of astragalus flavonoid extracts in the treatment of SLE.

[0050] Deep eutectic solvents (DES) offer broad application prospects in a variety of fields due to their low cost, ease of synthesis, environmental friendliness, ease of storage, and high efficiency. Ultrasonic-assisted deep eutectic solvents (UDES) combine DES with ultrasound technology, leveraging the cavitation effect of ultrasound to increase the frequency of molecular motion. This helps improve drug extraction efficiency compared to traditional extraction methods.

[0051] Small molecule fishing, a cutting-edge experimental technique, plays a key role in identifying specific molecules that interact with known biomolecules within complex sample systems. Using known receptors or target proteins as "bait," small molecule compounds that bind to them, such as ligands or drug candidates, are "fished" from complex mixtures. This technique provides a highly efficient screening method for disease targets and drug development, enabling the rapid identification of target molecules with specific binding.

[0052] Example 1 Ultrasonic-assisted deep eutectic solvent (UDES) method for extracting astragalus flavonoid extract (TFAR)

[0053] 1. Preparation of deep eutectic solvent (DES)

[0054] Choline chloride was used as the hydrogen bond acceptor (HBA), and ethylene glycol, 1,2-propylene glycol, glycerol, 1,4-butanediol, glucose, and lactic acid were used as hydrogen bond donors (HBD). HBA, HBD, and water were added to a sample vial in the proportions listed in Table 1. The mixture was heated in an 80°C oil bath and magnetically stirred for 3 hours until a clear liquid formed. The mixture was then allowed to cool to room temperature.

[0055] Table 1 Composition formula of DES

[0056] NO. HBA HBD molar ratio Moisture content w / w% DES-1 Choline chloride Ethylene glycol 1:3 30 DES-2 Choline chloride 1,2-Propanediol 1:3 30 DES-3 Choline chloride Glycerol 1:3 30 DES-4 Choline chloride 1,4-Butanediol 1:3 30 DES-5 Choline chloride glucose 1:3 30 DES-6 Choline chloride lactic acid 1:3 30

[0057] 2. Screening of DES types

[0058] Astragalus flavonoid extract (TFAR) was prepared using both the traditional method (TR) and the ultrasound-assisted deep eutectic solvent method (UDES). The traditional method used a 70% ethanol solution as the extraction solvent, while the UDES used the DES listed in Table 1. The preparation process involved adding the DES listed in Table 1 or a 70% ethanol solution to Astragalus powder. Ultrasonic treatment was performed at a material-liquid ratio of 1 g to 20 mL at a temperature of 60°C, a duration of 40 min, and a power of 1400 W. The extract was cooled to room temperature and centrifuged. The filtrate was the TFAR extract.

[0059] The effects of 6 DES and 70% ethanol solution on TFAR extraction were compared under the same conditions. Figure 1 As shown in the results, compared with 70% ethanol solution, there was no significant difference in the effects of DES-1, 2, and 4 on the TFAR extraction amount (p>0.05), the TFAR extraction amount of DES-3 was significantly increased, and the TFAR extraction amount of DES-5 and 6 was significantly decreased (p<0.01), so DES-3 was selected as the extraction solvent for subsequent experiments.

[0060] 3. Process optimization of UDES extraction of TFAR

[0061] A single-factor experiment investigated the effects of different ultrasonic time, material-liquid ratio, ultrasonic temperature, ultrasonic power, HBA / HBD molar ratio, and water content (mass ratio) in DES on the TFAR extraction yield. The TFAR extraction yield was calculated using the following formula:

[0062] W=(C×V0×V1) / (m×V2)

[0063] Where: W is the amount of TFAR extracted, mg / g; C is the TFAR concentration of the extract calculated according to the rutin standard curve, mg / mL; m is the mass of Astragalus powder, g; V0 is the volume of the extract after constant volume, mL; V1 is the volume of the extract, mL; V2 is the volume of the sample used for measurement, mL.

[0064] 3.1 Effect of solid-liquid ratio on TFAR extraction yield

[0065] DES with a water content of 30% and a molar ratio of choline chloride to glycerol of 1:3 was selected as the extraction solvent. The total ultrasonic power was 2330W. Under the conditions of ultrasonic power reaching 60%, ultrasonic time 40min, and ultrasonic temperature 60℃, a single factor experiment was conducted on the material-liquid ratio to detect the TFAR extraction amount. The material-liquid ratios were selected as 1:10, 1:15, 1:20, 1:25, and 1:30, respectively. The test results are as follows: Figure 2As shown in Figure a, increasing the solvent dosage decreases the concentration of components surrounding the sample, facilitating their diffusion into the solvent. However, excessively increasing the solvent dosage can lead to the dissolution of other components, thus affecting extraction efficiency. When the solid-liquid ratio reaches 1:25 g / mL, further decreasing the ratio decreases the TFAR extraction yield. Therefore, a 1:25 g / mL ratio was selected, achieving an average TFAR extraction yield of 2.18 mg / g.

[0066] 3.2 Effect of ultrasonic time on TFAR extraction yield

[0067] Under the conditions of material-liquid ratio of 1:20, ultrasonic temperature of 60℃ and ultrasonic power of 60%, a single factor experiment on ultrasonic time was conducted. The ultrasonic time was set at 20, 30, 40, 50, 60 and 70 minutes respectively. The test results are as follows: Figure 2 As shown in Figure (b), the mechanical action and cavitation during ultrasonic extraction increase the solvent's penetration, facilitating the dissolution of the active ingredient. When the ultrasonication time ranged from 20 to 40 minutes, the TFAR extraction yield gradually increased to 2.34 mg / g before declining significantly. Therefore, an extraction time of 40 minutes was selected.

[0068] 3.3 Effect of ultrasonic temperature on TFAR extraction yield

[0069] Under the conditions of material-liquid ratio of 1:20, ultrasonic time of 40min and ultrasonic power of 60%, a single factor experiment on ultrasonic temperature was conducted. The ultrasonic temperatures were selected as 40, 50, 60, 70 and 80℃ respectively. The test results are as follows: Figure 2 As shown in Figure c, increasing the temperature decreases the viscosity of DES, increases the diffusion coefficient, and accelerates the diffusion of the active ingredients. However, excessively high temperatures can destroy the active ingredients. When the ultrasonic temperature is raised to 60°C, the TFAR extraction yield averages 2.10 mg / g. Above 60°C, the extraction yield decreases, so 60°C was selected as the extraction temperature.

[0070] 3.4 Effect of ultrasonic power on TFAR extraction yield

[0071] Under the conditions of material-liquid ratio 1:20, ultrasonic time 40min, and ultrasonic temperature 60℃, a single factor experiment on ultrasonic power was conducted. The ultrasonic power was set to 40%, 50%, 60%, 70%, 80% and 90% respectively. The test results are as follows: Figure 2 As shown in Figure d, the powerful penetration of ultrasound facilitates the dissolution and diffusion of ingredients. When the ultrasonic power reaches 60% of the total power, the TFAR extraction yield reaches its maximum. Further increasing the power significantly decreases the extraction yield. This may be due to the excessive penetration of ultrasound, which causes the decomposition of certain flavonoid components. Alternatively, it may be due to unstable temperature control at high power, which destroys the structure of flavonoid compounds. Therefore, the ultrasonic power of 60% was selected.

[0072] Effect of the molar ratio of HBA to HBD in 3.5DES on the extraction yield of TFAR

[0073] Under the conditions of material-liquid ratio of 1:20, ultrasonic time of 40 min, ultrasonic temperature of 60 ° C, and ultrasonic power of 60%, a single factor experiment on the molar ratio of choline chloride to glycerol was conducted. The molar ratios of HBA and HBD were set at 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6 respectively. The test results are as follows Figure 2 As shown in Figure 5, increasing the HBD ratio effectively modulates the viscosity and surface tension of the DES, promoting the diffusion of flavonoids. However, excessive HBD content weakens the effect of HBA and affects the interaction between flavonoids and the solvent. When the molar ratio is 1:3, the extraction yield reaches its highest value, averaging 2.02 mg / g.

[0074] 3.6 Effect of DES moisture content on TFAR extraction yield

[0075] Under the conditions of material-liquid ratio of 1:20, ultrasonic time of 40min, ultrasonic temperature of 60℃ and ultrasonic power of 60%, a single factor experiment on the moisture content of DES was conducted. The moisture content was set to 20%, 30%, 40%, 50%, 60% and 70% respectively. The test results are shown in Figure 2. Figure 2 As shown in Figure 5, adding an appropriate amount of water to the DES can reduce solvent viscosity and adjust solvent polarity. When the water content in the DES increases from 10% to 50%, the average TFAR extraction yield increases from 1.38 mg / mL to 2.35 mg / mL. As the water content further increases, the viscosity of the extractant decreases, leading to a decrease in extraction efficiency. Therefore, a solvent system with a water content of 50% was selected as the extraction solvent.

[0076] 3.7 Optimization of extraction conditions by response surface methodology

[0077] Through single-factor experiments, the effects of different ultrasonic time, solid-liquid ratio, ultrasonic temperature, ultrasonic power, HBA and HBD molar ratio and water content (mass ratio) in DES on the extraction yield of astragalus flavonoids extract were investigated. Based on the influence of each factor on the TFAR extraction yield, the BBD-RSM method was selected to further optimize the extraction conditions.

[0078] The multiple regression equation for the response variable and the independent variable can be obtained by modifying the following third-order polynomial model. Taking the TFAR extraction amount as the response value, the quadratic polynomial regression model equation was fitted using Design Expert 13.0 software, and the regression equation was obtained: Astragalus flavonoid extraction amount = 2.41200-0.099167A-0.051667B-0.049167C-0.078333D+0.092500AB+0.002500AC+0.172500AD+0.075000BC-0.077500BD+0.015000CD-0.142250A 2 -0.128500B 2 -0.239750C 2 -0.138500D 2 .

[0079] According to the fitting equation and model and the response surface 3D curve diagram of BBD-RSM experiment (see Figure 3 ) The optimal extraction conditions were a solid-liquid ratio of 1:25 g / mL, a water content of 50%, an ultrasonic power of 60%, an ultrasonic temperature of 60°C, and a ultrasonic time of 40 minutes, resulting in a predicted extraction yield of 2.55 mg / g. To verify the predicted extraction yield, three parallel experiments were conducted under the optimal extraction conditions, yielding 2.50 ± 0.12 mg / g of astragalus flavonoids, close to the predicted value and demonstrating the reliability of the model. The traditional method yielded only 2.12 ± 0.10 mg / g, with a p < 0.05 indicating statistical significance.

[0080] Example 2 Reverse Network Pharmacology-Small Molecule Fishing Technology Combined with UDES Method to Prepare Astragalus Flavonoid Extract for the Treatment of SLE

[0081] 1. Reverse network pharmacology

[0082] Reverse network pharmacology was used to analyze and screen disease targets for SLE. The process was as follows:

[0083] (1) The databases GeneCards (https: / / www.genecards.org / ), DisGeNET (https: / / www.disgenet.org / ), OMIM (https: / / omim.org / ) and DrugBank (https: / / go.drugbank.com / ) were used in combination with the latest treatment guidelines and literature search. The search terms were “systemic lupus erythematosus,” “lupus erythematosus,” “lupus nephritis,” “discoid lupus erythematosus,” “subacute cutaneous lupus erythematosus,” “deep lupus erythematosus,” and “neonatal lupus erythematosus.” GeneCards retrieved 2,686 disease targets, with Relevance Index (RI) as the search term. Duplicates were removed by screening with a score ≥ 10, resulting in 689 disease-related targets. DisGnet searched for targets with a score ≥ 0.1, merging and removing duplicates yielded 219 targets. The OMIM database was screened and removed for duplicates, yielding 285 targets. 1010 disease-related targets were merged and removed from the databases, resulting in 632 relevant targets after removing non-human targets and duplicates. Bioinformatics analysis software Venn diagrams were used to identify intersecting targets. A target protein PPI network was constructed using the STRING database. Network topology analysis identified core target proteins, allowing for the development of a small molecule compound library relevant to the treatment of SLE.

[0084] (2) By querying the TCM-ID, TCMS, and HIT databases and combining literature retrieval, we collected the flavonoid compound library 2 from Astragalus. The compound libraries 1 and 2 intersected, and the Venn diagram showed the intersection component library 3.

[0085] (3) Construction of “compound-target” network: The Astragalus flavonoid extract library 3 and potential targets were imported into Cytoscape software according to their properties to construct a compound-regulated target network and analyze the core components of Astragalus flavonoids in the treatment of SLE.

[0086] (4) Molecular docking: The core target and flavonoid compounds were molecularly docked and verified using SYBYL-X2.0 software. The 2D structure of the compound was obtained from the PubChem database and saved in mol.2 format after optimal energy quantification using Chem3D, and used as the small molecule ligand in the docking. The small molecule ligand TLR7 was finally screened and used as the target for subsequent small molecule fishing. The crystal structure of the core target protein was downloaded from the RSCD-PDB database and saved in pdb format as a macromolecular protein receptor.

[0087] 2. Small molecule fishing technology

[0088] Magnetic nanoparticles were prepared using the core target proteins screened by reverse network pharmacology to target Astragalus flavonoid compounds that can treat SLE. The process is as follows:

[0089] (1) Synthesis of Fe3O4@SiO2-TLR7 magnetic nanoparticles

[0090] a. Preparation of Fe₃O₄@SiO₂ Nanoparticles: Weigh 60 mg of Fe₃O₄ nanoparticles into a 250 mL three-necked flask. Pour 238 mL of ethanol, 70 mL of water, and 7.5 mL of aqueous ammonia. Add 3 mL of tetraethyl silicate and stir at room temperature for 8 h. After magnetic separation, rinse thoroughly with water and dry at 50°C to obtain Fe₃O₄@SiO₂ magnetic nanoparticles.

[0091] b. Weigh 25 mg of Fe3O4@SiO2 magnetic nanoparticles and place them in 50 mL of ethanol and ultrasonicate for 10 min to uniformly disperse the nanoparticles. Add 0.5 mL of APTES and stir at room temperature for 6 h. Magnetic separation was performed to obtain APTES-modified Fe3O4@SiO2 nanoparticles.

[0092] c. Fourier transform infrared spectroscopy (FT-IR) was performed on Fe3O4 nanoparticles, Fe3O4@SiO2 magnetic nanoparticles and APTES modified Fe3O4@SiO2 magnetic nanoparticles prepared in the above process. The results are as follows Figure 4 As shown, APTES was successfully modified onto Fe3O4@SiO2 magnetic nanoparticles.

[0093] d. TLR7 was immobilized on APTES-modified Fe3O4@SiO2 magnetic nanoparticles by classic glutaraldehyde activation method: 2 mg of APTES-modified Fe3O4@SiO2 nanoparticles were dispersed in 2 mL of 5% glutaraldehyde solution and allowed to stand for 1 hour to obtain glutaraldehyde-activated nanoparticles. Then, 1 mL of TLR solution (1 mg / mL) was added and shaken for 2 hours. After magnetic separation and washing three times with buffer solution, Fe3O4@SiO2-TLR7 magnetic nanoparticles were obtained, whose appearance and morphology are shown in FIG. Figure 5As shown, A is the state of Fe3O4@SiO2-TLR7 without magnetic field, and B is the state of Fe3O4@SiO2-TLR7 with magnetic field.

[0094] (2) Preparation of Astragalus flavonoid ethyl acetate extract

[0095] The Astragalus flavonoid extract was prepared according to the optimized UDES method of Example 1, and the process was as follows:

[0096] a. Choline chloride and glycerol were mixed in a molar ratio of 1:3, and water was added to make the water content of the solvent 50% to prepare DES;

[0097] b. The Astragalus medicinal material powder was mixed with DES in a material-liquid ratio of 1 g: 25 mL, and the ultrasonic power was set to 60% (total power was 2330 W), the ultrasonic temperature was 60°C, and the ultrasonic time was 40 min. After the ultrasonication was completed, the mixture was cooled to room temperature and centrifuged to obtain the supernatant, which was freeze-dried to obtain Astragalus flavonoid freeze-dried powder.

[0098] Weigh 2.4 g of lyophilized astragalus flavonoids powder and dissolve it in 20 mL of water. Liquid-liquid extraction was performed with equal volumes of petroleum ether and ethyl acetate in increasing polarity to obtain the ethyl acetate extract of astragalus flavonoids. The extract was concentrated by rotary evaporation at 60°C and lyophilized to a powder, which was stored in a sealed, dry place until use.

[0099] (3) Fe3O4@SiO2-TLR7 ligand magnetic nanoparticles fishing

[0100] 20 mg of Fe3O4@SiO2-TLR7 magnetic nanoparticles were mixed with a 5 mg / mL solution of Astragalus flavonoid ethyl acetate extract and incubated at 37°C for 15 minutes. After magnetic separation, the particles were washed three times with 2 mL of buffer to remove unbound components from the surface of the magnetic particles. The extracted components were eluted from the surface of the magnetic particles with 3 mL of methanol, and the eluate was collected (the eluted magnetic particles Fe3O4@SiO2-TLR7 can be reused for small molecule extraction after repeated rinsing with buffer). After filtration through a 0.45 μm filter membrane, the Astragalus flavonoid extract was obtained. HPLC analysis was performed.

[0101] (4) HPLC determination of flavonoids extract of Astragalus membranaceus

[0102] Using calycosin as a standard, HPLC was used to determine the presence of calycosin in the extract. Chromatographic conditions were: a Diamomsil-C18 column (5 μm, 250 mm × 4.6 mm), a mobile phase consisting of methanol (A) and pure water (B), a flow rate of 1.0 mL / min, a detection wavelength of 267 nm, an injection volume of 10 μL, and a gradient elution according to Table 2. The results showed that the Astragalus flavonoid extract contained calycosin, indicating that the component obtained using small molecule elution technology is an Astragalus flavonoid.

[0103] Table 2 HPLC gradient elution program

[0104]

[0105]

[0106] Example 3 Determination of Astragalus Flavonoids for the Treatment of SLE

[0107] 1. Detection of Astragalus flavonoids by UPLC-MS combined with SRM / MRM technology

[0108] The astragalus flavonoid extract prepared by the traditional method (the preparation process is shown in Example 1, referred to as TR-TFAR) and the astragalus flavonoid extract prepared by the reverse network pharmacology-small molecule fishing technology combined with the UDES method (the preparation process is shown in Example 2, referred to as UDES-TFAR) were used as samples. Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) technology was combined with selective reaction monitoring / multi-reaction monitoring (SRM / MRM) technology to analyze the 36 common flavonoid standards available in the laboratory ( Figure 6 ) was used as a reference to test the samples.

[0109] UPLC chromatographic conditions: Waters ACQUITY UPLC7I-Class system, chromatographic column: HSS T3 2.5 μm, 2.1 mm x 150 mm, Waters; mobile phase: phase A water (0.1% formic acid): phase B acetonitrile (0.1% formic acid); flow rate: 400 μL / min; column temperature: 4°C; injection volume: 2 μL; gradient elution program is shown in Table 3.

[0110] Mass spectrometry conditions: MRM analysis was performed on an AB SCIEX 5500QTRAP mass spectrometer in positive and negative ion switching modes. Positive ion mode source parameters: Source temperature, 550°C; Gas 1, 5; Gas 2, 50; CRU, 30; ISVF, 5500 V. Negative ion mode source parameters: Source temperature, 550°C; Gas 1, 55; Gas 2, 50; CRU, 30; ISVF, -4500 V.

[0111] Table 3 UPLC gradient elution program

[0112] Time (min) Phase A - Water (0.1% formic acid) Phase B - acetonitrile (0.1% formic acid) 0 95 5 3 80 20 4.3 80 20 9 55 45 11 2 98 13 2 98 13.1 95 5 15 95 5

[0113] The UPLC-MS identification results of astragalus flavonoids in UDES-TFAR are shown in Table 4.

[0114] Table 4 Qualitative results of flavonoids

[0115]

[0116]

[0117] 2. Screening of differential components

[0118] 2.1 Orthogonal partial least squares discriminant analysis (OPLS-DA) is a supervised pattern recognition method that manually classifies samples before analysis to remove data irrelevant to the classification. This allows the results to better reflect intergroup differences, making the model easier to interpret and identify differential components. R2x and R2y represent the explanatory power of the model, while Q2 indicates the model's predictive power. The closer the R2 and Q2 values ​​are to 1, the better the model. In OPLS-DA model analysis, VIP reflects the contribution of a variable to the model's explanation. The larger the VIP value of a variable, the more important it is to the model. A VIP value > 1 was used as the screening criterion to identify potential differential components (Table 5).

[0119] Table 5 Potential difference components

[0120] name VIP value name VIP value Genistin 1.03502 Licorice 1.03222 Quercetin 1.03493 Eriochoride 1.03219 Isoliquiritigenin 1.03443 Formononetin 1.03217 Puerarin 1.03442 Kaempferol 1.03217 Naringenin 1.03436 Chrysin 1.03028 Daidzein 1.03427 Luteolin 1.02765 Naringin 1.03407 Rutin 1.02416 Glycitin 1.03395 Sakura extract 1.02283 Biochanin A 1.0337 Daidzein 1.02168 Butebase 1.03353 Myricetin 1.01615 Genistein 1.03341 Soybean 1.01493 Apigenin 1.03339 / /

[0121] 2.2 Cluster analysis (CA) of Astragalus flavonoids was performed. CA is the process of classifying data into different categories. When the analysis is reasonable and accurate, similar samples can appear in the same cluster through clustering. Samples in the same cluster have similarities, and samples in different clusters have certain differences. The cluster analysis results are as follows Figure 7 shown.

[0122] 2.3 All data were analyzed using t-test and fold change analysis and a volcano plot was drawn. The horizontal axis is log2 (Fold Change), the vertical axis is -lg (p-value), and each point represents a compound. The two lines parallel to the Y-axis are X=1 and X=-1. The points on the left side of X=-1 are components that are down-regulated by more than 2 times, and the points on the right side of X=1 are components that are up-regulated by more than 2 times. There is a dotted line Y=1.30 parallel to the x-axis, that is, lg (0.05). The points above the dotted line represent components with p less than 0.05. The components above the dotted line Y=1.30, to the right of X=1 and to the left of X=-1 are recorded as components with significant differences. Figure 8 As shown in the figure, compared with TR-TFAR, there were 6 components that were significantly upregulated and 8 components that were downregulated in UDES-TFAR, and there was no significant difference in the remaining 13 components.

[0123] 2.4 Results

[0124] According to the OPLS-DAVIP value, combined with cluster analysis, t-test and fold difference analysis, six differential components were screened out: Quercitrin, Rutin, Glycitin, Luteolin, Daidzin and Genistin. These six compounds are the differential components between the two extraction methods.

[0125] There was no significant difference between the two methods in the total content of 36 specific flavonoids (|Log2FC|<1), but the UDES method significantly increased the content of the above six different components (|Log2FC| was close to 1), and has the potential to be developed into a dedicated method for extracting this type of flavonoids.

[0126] Example 4 Functional Verification of Astragalus Flavonoids

[0127] 1. Sample

[0128] TR-TFAR, UDES-TFAR, differential component (Quercitrin, Rutin, Glycitin, Luteolin, Daidzin, Genistin) standards, berberine, LPS.

[0129] 2. Experimental process

[0130] 2.1 Cell experiments

[0131] The cell experiment used a preventive drug administration method, with cells divided into a control group, a model group (400 ng / mL LPS treatment for 24 hours), a positive group (15 μmol / L berberine and 400 ng / mL LPS treatment for 24 hours), a TFAR dosage group (50-1000 μg / mL UDES-TFAR and TR-TFAR treatment for 24 hours), and a Flavonoids group (5-120 μmol / L differential components treatment for 400 ng / mL LPS treatment for 24 hours). The experimental process is as follows:

[0132] RAW 264.7 cells were seeded into 96-well plates. After 24 hours, culture medium containing TFAR and differential component standards was added and cultured for 24 hours. The culture medium was discarded, and 100 μL of serum-free culture medium and 10 μL of MTT (5 mg / mL) were added to each well. The cells were cultured in a constant temperature incubator for another 4 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well. The cells were mixed and incubated in the dark for 10 minutes. The absorbance (OD) was measured at 490 nm, and the cell viability was calculated. Cell viability (%) = (OD of drug group / OD of blank group) × 100%.

[0133] The cell survival rate results of TFAR dosage groups are as follows Figure 9 As shown in the figure, compared with the control group, when the TFAR concentration was 600 μg / mL, there was no statistically significant difference in cell viability (p>0.05). When the TFAR concentration was 800 μg / mL, the cell survival rate was less than 90%, which showed obvious cytotoxicity (p<0.05). There was no significant difference between UDES-TFAR and TR-TFAR. The cell survival rate results of the flavonoids group are shown in the figure. Figure 10 As shown in the figure, among the six different component standards, the cell activity was higher than 90% when the concentration of quercetin was below 100 μmol / L, rutin was below 70 μmol / L, glycyrrhizin was below 60 μmol / L, luteolin and genistin were below 50 μmol / L, and daidzein was below 40 μmol / L, indicating that the drugs had no cytotoxicity within the above concentration range. In actual application, the dosage can be designed within this range.

[0134] Effects of the control group, model group, positive group and TFAR dose group on the expression levels of LPS-induced cellular inflammatory factors (TNF-α, IL-6 and TGF-β) and oxidative stress indicators (SOD, MDA and GSH) Figure 11 The effects of LPS on NO expression in cells were shown in Figure 12As shown in the figure, the expression levels of TNF-α, IL-6, and MDA in the model group cells were significantly higher than those in the control group, while TGF-β, SOD, and GSH were lower than those in the control group, indicating that the model was successfully established. The positive drug berberine downregulated the expression levels of TNF-α, IL-6, and MDA in the cells and upregulated the expression levels of TGF-β, SOD, and GSH in the cells. TFAR has the same function as the positive drug and is concentration-dependent. Among them, UDES-TFAR is more effective than TR-TFAR. The NO expression level in the model group cells was higher than that in the control group, while the positive drug berberine significantly inhibited NO expression. Similarly, TFAR also inhibited NO expression in a concentration-dependent manner. Among them, UDES-TFAR had a better inhibitory effect on NO expression than TR-TFAR.

[0135] To further explore the reason why UDES-TFAR has a higher inhibitory effect on NO expression than TR-TFAR, the effects of six different components on intracellular NO content were detected. Figure 13 As shown. The NO content in the model group was significantly higher than that in the control group, indicating that the model was successfully established. The six differential components can all reduce the NO content in cells, and are concentration-dependent; among them, daidzin and daidzin can significantly reduce the NO content in cells at a concentration of 5μmol / L, and the remaining components can also significantly reduce NO production in cells at medium doses (10μmol / L) and high doses (15μmol / L) (p<0.01). Moreover, at a concentration of 15μmol / L, the inhibitory effect of the six differential components on NO expression is comparable to that of berberine. In summary, the significant increase in the content of the six differential components in UDES-TFAR is the reason why UDES-TFAR has higher biological activity.

[0136] 2.2 Animal experiments

[0137] Forty-two SPF female BALB / c mice were acclimated to the SPF environment for 7 days and then randomly divided into six groups of seven mice each: a normal control group (Control), a model group (Model), a HCQ group (hydroxychloroquine, 100 mg / kg), and a UDES-TFAR group (doses of 25 mg / kg, 50 mg / kg, and 100 mg / kg). A systemic lupus erythematosus model was established in the model, HCQ, and UDES-TFAR groups by a single intraperitoneal injection of 0.5 mL of pristane. Ten weeks after injection, mice in the HCQ and UDES-TFAR groups received oral administration of the drug.

[0138] Urinary protein was measured using urine protein test strips before and monthly thereafter using pristane injection. Results showed an increase in antibodies and a significant increase in urine protein between 8 and 10 weeks of modeling, indicating successful modeling. Following oral administration of the positive drugs hydroxychloroquine and UDES-TFAR, urine protein decreased by 14 weeks.

[0139] At 4, 8, 10, 14, 16, 18, 22, and 26 weeks after intraperitoneal injection of pristane, peripheral blood was collected from the orbital venous plexus of the mice, and the levels of anti-dsDNA and anti-smith in the serum were determined according to the instructions of the ELISA kit. The results are as follows: Figure 14 As shown in the data, the anti-dsDNA and anti-smith levels showed an upward trend from 4 to 10 weeks. At the 14th week, the anti-dsDNA and anti-smith levels continued to increase. Starting from the 16th week, the anti-dsDNA and anti-smith levels gradually decreased and eventually reached the same level as the control group.

[0140] At 16 weeks after intraperitoneal injection of pristane, the levels of inflammatory markers TNF-α, IL-6, and TGF-β in the peripheral blood of the orbital venous plexus of mice, as well as the levels of oxidative markers MDA, SOD, and GSH were detected by ELISA to regularly monitor the inflammatory status of the mice. Figure 15 As shown in the figure, the IL-6, TNF-α and MDA contents of the model group mice were higher than those of the control group, and the TGF-β, SOD and GSH contents were lower than those of the control group, indicating that the model was successfully established. After oral administration of hydroxychloroquine and UDES-TFAR, the IL-6, TNF-α and MDA contents were lower than those of the model group, and the TGF-β, SOD and GSH contents were higher than those of the model group, and returned to normal or near normal levels.

[0141] At 28 weeks after intraperitoneal injection of pristane, the mice were killed, and their hearts, livers, spleens, and kidneys were removed. HE-stained pathological sections were prepared, and the morphology of each organ was observed under a microscope. Figure 16-18 As shown in the data, after oral administration of hydroxychloroquine and UDES-TFAR, the edema, degeneration and necrosis of liver tissue cells in the UDES-TFAR group were alleviated compared with those in the model group, the lymphocyte infiltration and white pulp lesions in the spleen were significantly alleviated, the glomerular and tubular lesions changed slightly, and the lymphocyte infiltration was significantly reduced.

[0142] The above results show that UDES-TFAR has a therapeutic effect on pristane-induced systemic lupus erythematosus, and at the same dosage, it has better therapeutic effect than the existing drug hydroxychloroquine.

[0143] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing an astragalus flavonoid extract having a therapeutic effect on systemic lupus erythematosus, characterized in that: The steps include: (1) Use reverse network pharmacology to screen target proteins in Astragalus for the treatment of systemic lupus erythematosus; (2) preparing astragalus flavonoids by an ultrasonic-assisted deep eutectic solvent method, and extracting with ethyl acetate to obtain an astragalus flavonoid ethyl acetate extract; (3) preparing magnetic nanoparticles with the target protein adsorbed on the surface; using the magnetic nanoparticles to perform small molecule fishing on the astragalus flavonoid ethyl acetate extract to obtain the astragalus flavonoid extract with the effect of treating systemic lupus erythematosus.

2. The preparation method according to claim 1, characterized in that: The steps of the ultrasonic-assisted deep eutectic solvent method are as follows: Choline chloride, glycerol and water are mixed to obtain a low eutectic solvent; Astragalus medicinal material powder is mixed with the low eutectic solvent, and astragalus flavonoids are obtained through ultrasonic treatment and centrifugation.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the choline chloride to the glycerol is 1:(2-4); and / or, The water content of the deep eutectic solvent is 40% to 60%; and / or, The ratio of the astragalus powder to the deep eutectic solvent is 1 g: (20-35) mL; and / or, The power of the ultrasonic treatment is 1160W-1700W, the temperature is 60-80°C, and the time is 30-60min.

4. The preparation method according to claim 1, characterized in that: The fishing method is: after mixing the magnetic nanoparticles with the target protein adsorbed on the surface with the astragalus flavonoid ethyl acetate extract, perform magnetic separation; use methanol to wash the magnetic nanoparticles after magnetic separation, collect the eluate, and obtain the astragalus flavonoid extract with the effect of treating systemic lupus erythematosus.

5. The preparation method according to claim 1, characterized in that: The magnetic nanoparticles with the target protein adsorbed on the surface include Fe3O4@SiO2 magnetic nanoparticles with the target protein adsorbed on the surface.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The target protein includes TLR7.

7. An Astragalus flavonoid extract prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The astragalus flavonoid extract contains quercetin, rutin, liquiritigenin, luteolin, daidzein and genistein.

8. Use of the astragalus flavonoid extract according to claim 7 in the preparation of a medicament for treating systemic lupus erythematosus.

9. Use of daidzein in the astragalus flavonoid extract according to claim 7 in the preparation of a medicament for treating systemic lupus erythematosus.

10. Use of genistein in the astragalus flavonoid extract according to claim 7 in the preparation of a medicament for treating systemic lupus erythematosus.

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