H9C2 cell membrane bionic magnetic nano material and application thereof

Abstract

The invention discloses an H9C2 cell membrane bionic magnetic nano material. The material is prepared from graphene oxide, citric acid, N-(2-hydroxyethyl) maleimide and an H9C2 cell membrane. The constructed cell membrane bionic magnetic nano material system is applied to active ingredient fishing of the fructus choerospondiatis extracting solution for treating the coronary heart disease, the specificity of the target protein contained on the surface of the H9C2 cell membrane is utilized, and the target protein can be specifically combined with potential active ingredients in the traditional Chinese medicine extracting solution, so that the screening purpose is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of functional materials, and in particular relates to an H9C2 cell membrane bionic magnetic nanomaterial and application thereof. Background Art

[0002] Graphene oxide (GO) is relatively stable in aqueous solution and can be easily modified by hydrophilic or hydrophobic compounds in a covalent or non-covalent form. Its two-dimensional planar structure, chemical / mechanical stability, excellent photosensitivity, excellent conductivity, high specific surface area and good biocompatibility for cancer treatment are favored by researchers. Screening of potential active ingredients in traditional Chinese medicine requires efficient and simple methods. Magnetic nanomaterials can quickly separate materials from samples in the form of an external magnetic field. Therefore, integrating GO and Fe3O4 into a composite material can greatly reduce the workload of screening. According to the characteristics of cell membranes that can bind to specific groups, the surface of graphene oxide can be modified by covalent bonding to synthesize target groups that can bind to cell membranes, and fixed materials can be prepared. In addition, the enrichment ability of GO can assist trace amounts of potential active ingredients in traditional Chinese medicine to bind to targets on the cell membrane.

[0003] Traditional Chinese medicine is a treasure of the Chinese nation with a long history and the characteristics of multiple components and multiple targets. However, the complexity and diversity of its components and the unclear material basis and mechanism of action of its efficacy have become obstacles in the process of modernization. The screening and identification of active ingredients of traditional Chinese medicine are the prerequisites for studying the material basis and mechanism of action of traditional Chinese medicine. The traditional screening method is generally to separate the categories by chromatography and then conduct pharmacological experiments on single components one by one. The traditional screening method is not only time-consuming but also has a low success rate. Nowadays, targeted fishing technology is widely used in the screening of active ingredients of natural products due to its high specificity, easy separation, short time consumption and small sample volume. Magnetic nanomaterials have the advantages of easy modification and rapid separation, and have great application potential in separation; on the other hand, cell membranes have a complete biological structure and are rich in a large number of receptors, making them good multi-target targeted screening materials. Therefore, the magnetic bionic nanomaterials designed and developed by combining cell membranes with magnetic materials have the advantages of rapid separation and high specificity.

[0004] Choerospondias australis comes from the dried mature fruit of the plant Choerospondias australis of the Anacardiaceae family. It is one of the common Mongolian medicinal materials for the treatment of coronary heart disease because of its anti-myocardial ischemia, anti-myocardial fibrosis, antioxidant, and anti-inflammatory effects. Its role in the treatment of coronary heart disease was first recorded in "Yuewang Yaodian", in which the antioxidant and anti-inflammatory effects are mainly related to its flavonoid chemical components. Modern pharmacological studies have shown that a large number of active ingredients in Choerospondias australis have multiple physiological activities and are used in the cardiovascular system through multiple mechanisms to fight against cardiovascular diseases such as arrhythmias and myocardial ischemia. Therefore, it is critical to find the active ingredients that play a therapeutic role, and it is an important task to study the mechanism of action of Choerospondias australis in the treatment of coronary heart disease. Summary of the invention

[0005] The purpose of the present invention is to design a H9C2 cell membrane bionic magnetic nanomaterial and its application, starting from graphene oxide, attaching Fe3O4 to the surface of graphene oxide through synthesis, and then obtaining a magnetic material containing maleimide groups (SMGO@M) after a series of surface functional modifications.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The first object of the present invention is to provide a H9C2 cell membrane bionic magnetic nanomaterial, characterized in that the material is prepared from graphene oxide, citric acid, N-(2-hydroxyethyl) maleimide, and H9C2 cell membrane, and the specific method is as follows: S1: 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HOBt were activated by citric acid in alkaline conditions regulated by triethylamine, reacted in N,N-dimethylformamide, collected by magnet and washed and dried with distilled water and ethanol respectively to obtain silica magnetic graphene oxide with carboxyl groups; S2: Activate the carboxyl-containing silica magnetic graphene oxide material using 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole in N,N-dimethylformamide, and add N-(2-hydroxyethyl) maleimide to the reaction solution for overnight reaction; collect the material, wash it with distilled water and ethanol, and dry it to obtain a magnetic material with maleimide groups; S3: adding a phosphate buffer solution composed of phosphatase inhibitor, protease inhibitor and Tris-HCl to the collected H9C2 cells and then sonicating; the broken cells are centrifuged twice to obtain a precipitate, which is the H9C2 cell membrane; S4: The magnetic material with maleimide groups is bound to the cell membrane suspension, and the unbound cell membrane is washed away with PBS.

[0007] The second purpose of the present invention is to provide an application of H9C2 cell membrane bionic magnetic nanomaterials in the extraction of active ingredients from Choerospondias axillaris extract for treating coronary heart disease.

[0008] The application method is as follows: (1) After the medicinal material of Fructus Australis is crushed and sieved, the Fructus Australis powder is extracted with ethanol and repeated twice, the combined extract is filtered and freeze-dried, dissolved in ddH2O and fixed to scale to obtain Fructus Australis extract; the Fructus Australis extract is added to a magnetic material with a maleimide group for fishing, and the active components are separated from the non-specific components by magnetic separation; and methanol is used for elution to obtain the fishing solution; (2) The active ingredients screened were analyzed using UHPLC-Q-Exactive Orbitrap-MS / MS.

[0009] The following beneficial effects can be obtained through the above technical solution: The present invention applies the constructed cell membrane bionic magnetic nanomaterial system to active ingredient fishing in Choerospondias axillaris extract for treating coronary heart disease, and utilizes the specificity of the target protein contained on the surface of H9C2 cell membrane. The target protein can specifically bind to the potential active ingredients in the traditional Chinese medicine extract, thereby achieving the purpose of screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Fourier infrared spectra of SMGO@M and SMGO@M-HCMs obtained in Example 1 of the present invention; Figure 2 The hysteresis regression lines of SMGO@M and SMGO@M-HCMs obtained in Example 1 of the present invention; Figure 3 Fluorescence confocal images of SMGO@M and SMGO@M-HCMs obtained in Example 1 of the present invention; Figure 4 Transmission electron microscope characterization images of SMGO@M and SMGO@M-HCMs obtained in Example 1; Figure 5 The adsorption performance results of SMGO@M and SMGO@M-HCMs, where a is the static adsorption result and b is the Freundlich isotherm fitting result.

[0011] Figure 6 The elution results of positive drugs in SMGO@M and SMGO@M-CMs using five different solutions. DETAILED DESCRIPTION

[0012] The present invention will be further described below in conjunction with the accompanying drawings: like Figure 1-6 As shown, a cell membrane-coated magnetic material is used, starting from graphene oxide. Fe3O4 is attached to the surface of graphene oxide through synthesis, and then a series of surface functional modifications are performed to obtain a magnetic material containing maleimide groups (SMGO@M).

[0013] The method comprises the following steps: Step 1: Synthesis of SMGO@M magnetic materials Step 101: Weigh graphene oxide (GO) in a 250 mL three-necked flask, add distilled water to dissolve, stir mechanically and heat. l2·4H2O is dissolved and added to the flask to form a suspension under vigorous stirring. The pH value is adjusted to alkaline with NH3·H2O. Stir continuously at a certain temperature. After the reaction is completed, the material is separated from the mixture with a magnet, and MGO is obtained after washing and drying.

[0014] Step 102: Weigh MGO and add it to ethanol, stir tetraethyl silicate to form a suspension. Add the suspension to the ethanol solution containing MGO and stir mechanically, then absorb NH3·H2O and add it to the solution and continue stirring. Collect the product with a magnet, wash and dry it to obtain SMGO.

[0015] Step 103: EDC, HOBT, citric acid, and triethylamine were sequentially placed in a round-bottom flask containing DMF. After activation at room temperature for 1 h, an appropriate amount of SMGO material with amino groups was added to the reaction, and mechanical stirring was continued for 2.5 h. The material was separated from the mixture by an external magnetic field, and washed with ethanol and ddH2O for 3 times. SMGO with carboxyl groups was obtained by drying at 40°C for 6 h. 50 mL of DMF was placed in a three-necked flask, and the SMGO material with carboxyl groups, EDC, and HOBT were sequentially placed in the solvent for activation at room temperature for 1 h. N-(2-hydroxyethyl)maleimide was added to the reaction solution for overnight reaction. After the materials were collected, they were washed with distilled water and ethanol for 3 times, respectively, and dried at 60°C for 4 h to obtain a material with maleimide groups (SMGO@M).

[0016] Step 2: Preparation of SMGO@M-HCMs magnetic composites Step 201: H9C2 cells were cultured in a complete medium consisting of 10% FBS, DMEM medium, and 1% double antibody, in a 5% carbon dioxide, 37°C environment. When the cell coverage area reached 80-90% of the volume, the cells were washed 3 times with PBS to remove cell debris and other impurities, 3 mL each time, and then 1 mL of trypsin was added to the 37°C incubator for digestion for 1-2 min. The inner wall of the culture flask was blown off with a pipette to remove the cells, and the cell suspension was collected in a centrifuge tube and centrifuged at 1000 r·min -1 After centrifugation for 5 min, the supernatant was discarded to obtain H9C2 cells. Phosphate buffered saline (PBS) consisting of phosphatase inhibitor (1%), protease inhibitor (1%) and Tris-HCl (pH=7.5, 1%) was added to the H9C2 cells and ultrasonicated in a 480 W ultrasonicator for 30 min; the disrupted cells were rotated at 1000 r·min -1 Centrifuge for 10 min, discard other organelles, collect the supernatant, and spin at 12000 r·min -1Centrifuge for 20 min, and the precipitate is H9C2 cell membranes (HCMs); resuspend with 1 mL PBS to obtain cell membrane suspension, and store at 4°C for later use.

[0017] Step 202: Incubate the SMGO@M material and the HCMs suspension in a specific shaker, and wash away the unbound cell membrane with PBS to obtain SMGO@M-HCMs.

[0018] Step 3: Fishing for active ingredients in traditional Chinese medicine Step 301: Prepare Chinese medicine extract Step 302: Add the Chinese medicine extract prepared in step 301 to the SMGO@M-HCMs prepared in step 2, put them in a shaker for fishing, and then wash them with PBS for 3 times, each time for 30 seconds to wash away the non-specifically bound extract. Shake the SMGO@M-HCMs with methanol to elute the specifically bound active ingredients. After magnetic solid-liquid separation, a fishing solution containing the active ingredients of Chinese medicine is obtained.

[0019] In the above application, ultra-high performance liquid chromatography-mass spectrometry is used to analyze the active chemical components in the fishing liquid in step 2 to obtain an ion chromatogram of the chemical components of the fishing liquid, and the active components are determined by comparing with the reference substance information and the compound database.

[0020] The present invention takes graphene oxide as a starting point, and attaches Fe3O4 to its surface under alkaline and N2 protection conditions. It is coated with tetraethyl silicate Fe3O4, and the surface is functionalized and modified by a silanizing agent with amino groups to obtain a magnetic material containing amino groups, and further functionalized and modified by citric acid and N-(2-hydroxyethyl) maleimide to obtain a material with maleimide groups, which can react with the sulfhydryl groups on the cell membrane to form a stable thioether bond. The cell membrane obtained by ultrasound and centrifugation steps is combined with SMGO@M, and the unbound cell membrane is washed off with PBS, and SMGO@M-HCMs is obtained by magnetic separation.

[0021] The present invention utilizes the maleimide groups on the magnetic material to combine with the thiol groups exposed on the cell membrane to form a stable thioether bond, thereby obtaining a magnetic material wrapped based on the cell membrane. Example

[0022] This embodiment includes the following steps: Step 1: Synthesis of SMGO@M Step 101: Weigh 0.5 g of graphene oxide (GO) into a 250 mL three-necked flask, add 100 mL of distilled water to dissolve, stir mechanically and heat to 70°C. l2·4H2O was dissolved in 40 mL of distilled water to form a suspension under vigorous stirring, and the pH value was adjusted to 10 with NH3·H2O. Stirring was continued at 70°C for 60 min. After the reaction was completed, the material was separated from the mixture with a magnet, washed with distilled water and ethanol three times respectively, and dried at 60°C for 6 h to obtain MGO.

[0023] Step 102: Weigh 0.5 g of MGO and add it to 50 mL of ethanol, stir 6.3 mL of water and 2.1 mL of tetraethyl silicate for 3 min, and ultrasonicate for 1 min to form a suspension. Add the suspension to the ethanol solution containing MGO and stir at 0°C for 10 min, then absorb 2 mL of NH3·H2O and add it to the solution and continue stirring at 0°C for 10 h. Collect the product with a magnet, wash it with 2% HNO3, water, and ethanol three times respectively, and dry it at 40°C for 6 h to obtain SMGO.

[0024] Step 103: Add 0.2 g SMGO and 100 mL of 50% ethanol solution into a 250 mL three-necked flask, adjust the pH to 3-4 with 0.1 mol / L HCl, and add 2 mL APES dropwise into the solution. Stir at 50 °C, react for 5 h, and then wash with ethanol three times to obtain an amino SMGO material.

[0025] Step 104: EDC, HOBT, citric acid, and triethylamine were sequentially placed in a round-bottom flask containing DMF. After activation at room temperature for 1 h, an appropriate amount of SMGO material with amino groups was added to the reaction, and mechanical stirring was continued for 2.5 h. The material was separated from the mixture by an external magnetic field, and washed with ethanol and ddH2O for 3 times. SMGO with carboxyl groups was obtained by drying at 40°C for 6 h. 50 mL of DMF was placed in a three-necked flask, and the SMGO material with carboxyl groups, EDC, and HOBT were sequentially placed in the solvent for activation at room temperature for 1 h. N-(2-hydroxyethyl)maleimide was added to the reaction solution for overnight reaction. After the materials were collected, they were washed with distilled water and ethanol for 3 times, respectively, and dried at 60°C for 4 h to obtain a material with maleimide groups (SMGO@M).

[0026] Step 2: Preparation of SMGO@M-HCMs magnetic composite materials Step 201: H9C2 cells were cultured in a complete medium consisting of 10% FBS, DMEM medium, and 1% double antibody, in a 5% carbon dioxide, 37°C environment. When the cell coverage area reached 80-90% of the volume, the cells were washed 3 times with PBS to remove cell debris and other impurities, 3 mL each time, and then 1 mL of trypsin was added to the 37°C incubator for digestion for 1-2 min. The inner wall of the culture flask was blown off with a pipette to remove the cells, and the cell suspension was collected in a centrifuge tube and centrifuged at 1000 r·min -1 After centrifugation for 5 min, the supernatant was discarded to obtain H9C2 cells. Phosphate buffered saline (PBS) consisting of phosphatase inhibitor (1%), protease inhibitor (1%) and Tris-HCl (pH=7.5, 1%) was added to the H9C2 cells and ultrasonicated in a 480 W ultrasonicator for 30 min; the disrupted cells were rotated at 1000 r·min -1 Centrifuge for 10 min, discard other organelles, collect the supernatant, and spin at 12000 r·min -1 Centrifuge for 20 min, and the precipitate is H9C2 cell membranes (HCMs); resuspend with 1 mL PBS to obtain cell membrane suspension, and store at 4°C for later use.

[0027] Step 202: 10 mg of SMGO@M material and the cell membrane suspension are incubated in a shaker at 37° C. and 200 rpm, and the unbound cell membrane is washed away with PBS to obtain the SMGO@M-HCMs composite material.

[0028] Step 3: Investigation of adsorption performance of SMGO@HCMs In order to investigate the adsorption performance of SMGO@M-HCMs, the adsorption experiments were carried out as follows. All the following experiments were repeated three times.

[0029] Step 301: Investigate the maximum adsorption capacity of SMGO@M-HCMs and SMGO@M, and set the concentration to 0 mg·L -1 , 0.1 mg·L -1 , 0.2 mg·L -1 , 0.8 mg·L -1 , 1.0 mg·L -1 , 1.2 mg·L -110 mg of valsartan solution was added to 10 mg of SMGO@M-HCMs and SMGO@M, and adsorbed at 37°C and 200 rpm for 1 h. The samples were separated by magnet, and the unadsorbed positive drugs were discarded. The samples were vortex-washed with PBS for 3 times, each time for about 30 s, and finally eluted with 1 mL of methanol solution for 1 h. The valsartan concentration in the eluate was detected by HPLC, and the adsorption amount of valsartan by the material was calculated according to the following formula. The Langmuir and Freundlich models were used to fit the data.

[0030] Adsorption amount ( Q ) is calculated as (1): (1) in, Q (µg·g -1 ), C e (mg / L) is the elution concentration, V (mL) is the sample volume, m (g) is the mass of adsorbent. Langmuir equation: Q e =Q max K L C e / （1+K L C e ） Among them, Q max (µg·g -1 ) is the maximum adsorption amount of valsartan per unit mass of adsorbent; Q e (µg·g -1 ) is the adsorption amount at equilibrium; C e (mg·L -1 ) is the concentration of valsartan in the solution at adsorption equilibrium, K L (L mg -1 ) is the Langmuir constant. Freundlich equation: QUR e = lgK F + mlgC e in, Q e (µg·g-1 ) is the adsorption amount at equilibrium; C e (mg·L -1 ) is the concentration of valsartan in the solution at adsorption equilibrium, K F (L mg -1 ) is the Freundlich constant.

[0031] The cell membrane-wrapped magnetic materials were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM).

[0032] Infrared spectra of SMGO@M-HCMs and SMGO@M are shown in Figure 1 As shown. 60 cm -1 is the stretching vibration of Fe-O, indicating that Fe3O4 is successfully attached to GO. 1100 cm -1 The characteristic peak at 2980 cm-1 belongs to the oxygen-silicon bond, which is a proof of the successful use of silane coupling agent to wrap the material. After various compounds modified its surface, the FI-IR spectrum showed 2980 cm-1. -1 , which is the characteristic peak of CH2 and CH3. In the synthesis, citric acid and N-(2-hydroxyethyl)maleimide both have methyl and methylene groups. This result proves that N-(2-hydroxyethyl)maleimide was successfully synthesized.

[0033] VSM studies the magnetic properties of the two materials. Figure 2 It can be seen that MGO is synthesized and attached to GO. Since there are no other modified groups on the surface, the magnetic strength reaches 26.38 emu g -1 , which has strong magnetism, which is related to the synthesis of ferroferric oxide. After various surface modifications, the magnetization intensity decreased significantly, especially the SMGO@M-HCMs with cell membranes on the surface, whose magnetic intensity dropped to 11.51 emu g -1 , but the magnetic strength is sufficient to separate the material from the solution when an external magnetic field is applied.

[0034] Fluorescein-5-maleimide dye was used to stain the cell membrane, and the fluorescence confocal results showed that ( Figure 3 ), SMGO@M did not produce fluorescence under green excitation light, while SMGO@M-HCMs produced a very obvious fluorescence response under the same conditions, proving that the cell membrane was fixed on the material.

[0035] The morphological changes of SMGO@M and SMGO@M-CMs were observed and photographed using a transmission electron microscope. Figure 4As shown, the bare material surface is rough and granular attached to the GO sheet, while the SMGO@M-HCMs material has a cell membrane on its surface, which is covered by a thin film. By comparing the two materials, it can be proved that the cell membrane is successfully grafted onto the SMGO@M material.

[0036] To investigate the adsorption performance of the material, valsartan solutions of different concentrations were prepared and incubated with SMGO@M-CMs and SMGO@M for 1 h, respectively, washed with PBS three times, 30 s each time, and finally eluted with 1 mL of methanol for 1 h. The binding amount was monitored using HPLC. Figure 5 As shown, the results are Figure 5 As shown in Figure 2, the adsorption amount of SMGO@M-HCMs increased with the increase of valsartan concentration in the early stage, and the adsorption amount slowed down significantly in the later stage. The initial concentration was 1000 ug·mL -1 When the maximum adsorption capacity of the material is reached, it is in equilibrium, and the adsorption capacity of MGO@M-HCMs no longer changes with concentration. However, at the same initial concentration, the adsorption capacity of SMGO@M is significantly lower than that of SMGO@M-HCMs. -1 Equilibrium began to appear when the adsorption amount at this concentration was 1512.60 µg·g -1 , while the adsorption capacity of SMGO@M-HCMs at this concentration was 3503.19 µg·g -1 . The adsorption amount of SMGO@M is lower than that of SMGO@M-HCMs because it lacks cell membrane on its surface and has no specific receptors to bind to drugs. It only relies on physical adsorption to interact with drugs. After processing and analyzing the data, it was found that the adsorption of the two materials is closer to the Freundlich model, and SMGO@M failed to fit the Langmuir model. The r values ​​of SMGO@M and SMGO@M-HCMs in the Freundlich isotherm fitting are 0.9499 and 0.9168, respectively. This result shows that both materials belong to multilayer adsorption and there is no interaction between molecules.

[0037] In order to determine the best elution solvent, the concentrations of methanol, acetonitrile, PBS, 0.1% acetic acid, and 1% acetic acid were tested by HPLC. Figure 6 As shown in the figure, the relative recovery rate of methanol in the eluent of SMGO@M-HCMs is 100%. The elution ability of methanol in SMGO@M (100%) is superior to that of ddH2O, 0.1% acetic acid, and 1% acetic acid. In general, methanol has the best ability to elute valsartan from SMGO@M and SMGO@M-HCMs.

[0038] Step 4: Practical Application Step 401: Add the coarse powder of Fructus Australis to a 250 mL round-bottom flask, add 8 times the amount of 70% ethanol (m:m), soak for 30 min, extract for 2 h, filter with gauze to obtain the filtrate, extract the residue again for 2 h, filter, and combine the filtrate. Evaporate the ethanol to dryness using a rotary evaporator, freeze-dry the concentrate in a freeze dryer after placing it at -20 °C, and obtain freeze-dried powder for use. Take 10 mg of freeze-dried powder of Fructus Australis, weigh it accurately, place it in a volumetric flask, add ddH2O to dissolve it and dilute it to the mark to obtain a concentration of 2 mg·mL -1 of Choerospondias axillaris extract.

[0039] Step 402: Add 1 mL of the extract to 10 mg of SMGO@M-HCMs and fish for 1 h. Discard the supernatant by magnetic separation, wash with PBS vortex three times, 30 s each time, then elute with 1 ml of methanol for 50 min, and filter through a 0.22 µm filter to obtain the eluate.

[0040] Step 403: The fishing liquid is subjected to UHPLC-Q-Exactive Orbitrap-MS / MS technology to identify the active ingredients obtained by fishing.

[0041] The liquid chromatography system was Dionex Ultimate 3000 RSLC, the chromatographic column used was C18 (4.6×250 mm, 1.9μm), the liquid chromatography conditions were 0.4% formic acid-water mixed solution (aqueous phase), acetonitrile mixed solution (organic phase), gradient elution program: 0-2min, 95-85%, 2-3min, 85-75%, 2-7min, 75-55%, 7-9 min, 55-40%, 9-10min, 40-25%, 10-10.01 min, 25-95%, 10-13 min, 95%, flow rate: 0.3 mL·min -1 The mass spectrometry system used ThermoScientific Q Exactive Series, HESI-II as the ion source, positive and negative ion mode detection, and scanning parameters were Full MS-ddms 2, 100 to 1500m / z, Profile, Full MS:70,000, MS / MS:17,500. The eluent was subjected to secondary mass spectrometry analysis and identification of each potential active ingredient by UPLC-Q-Exactive Orbitrap-MS / MS. According to the comparison of retention time, molecular weight, ion fragments and reported literature, it can be seen from the table that a total of 7 compounds were screened out.

[0042] Table 2 Compound information NO Rt(min) Compounds Molecular formula m / z Fragments △m / z(ppm) 1 3.36 4-Hydroxybenzoic acid <![CDATA[C7H6O3]]> 137.02 93.03,65.04 -0.15 2 4.23 p-Hydroxybenzaldehyde <![CDATA[C7H6O2]]> 121.03 93.03,108.02 -0.35 3 7.21 7-Hydroxycoumarin <![CDATA[C9H6O3]]> 163.04 119.05,107.05 -0.27 4 8.39 Diacerein <![CDATA[C 19 H 12 O8]]> 367.05 281.05,239.04 4.71 5 10.2 Atractylodin <![CDATA[C 13 H 10 O]]> 183.08 105.03,95.03 -0.14 6 10.87 Embelin <![CDATA[C 17 H 26 O4]]> 293.18 195.18,247.17 0.46 7 11.34 8-Desoxygartanin <![CDATA[C 23 H 24 O5]]> 379.15 281.05,321.08 -0.51 The present invention aims to provide a strategy for quickly establishing cell membrane bionic magnetic nanomaterials, and to use this strategy for screening active ingredients in traditional Chinese medicine. The reaction can occur at 37°C and pH 6.5-7.5, and the cell membrane is wrapped on the magnetic material, which reduces the workload of screening active ingredients in traditional Chinese medicine, saves time, and the material has good stability.

[0043] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, modifications to various equivalent forms of the present invention all belong to the protection scope of the claims attached to this application. The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, modifications to various equivalent forms of the present invention all belong to the protection scope of the claims attached to this application.

Claims

1. A H9C2 cell membrane bionic magnetic nanomaterial, characterized in that: The material is prepared from graphene oxide, citric acid, N-(2-hydroxyethyl)maleimide, and H9C2 cell membrane. The specific method is as follows: S1: 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HOBt were activated by citric acid in alkaline conditions regulated by triethylamine, reacted in N,N-dimethylformamide, collected by magnet and washed and dried with distilled water and ethanol respectively to obtain silica magnetic graphene oxide with carboxyl groups; S2: Activate the carboxyl-containing silica magnetic graphene oxide material using 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole in N,N-dimethylformamide, and add N-(2-hydroxyethyl) maleimide to the reaction solution for overnight reaction; collect the material, wash it with distilled water and ethanol, and dry it to obtain a magnetic material with maleimide groups; S3: adding a phosphate buffer solution composed of phosphatase inhibitor, protease inhibitor and Tris-HCl to the collected H9C2 cells and then sonicating; the broken cells are centrifuged twice to obtain a precipitate, which is the H9C2 cell membrane; S4: The magnetic material with maleimide groups is bound to the cell membrane suspension, and the unbound cell membrane is washed away with PBS.

2. An application of H9C2 cell membrane bionic magnetic nanomaterials, characterized in that: Application of active ingredients in the extraction of Choerospondias axillaris for the treatment of coronary heart disease.

3. The use according to claim 2, characterized in that: The application method is as follows: (1) After the medicinal material of Fructus Australis is crushed and sieved, the Fructus Australis powder is extracted with ethanol and repeated twice, the combined extract is filtered and freeze-dried, dissolved in ddH2O and fixed to scale to obtain Fructus Australis extract; the Fructus Australis extract is added to a magnetic material with a maleimide group for fishing, and the active components are separated from the non-specific components by magnetic separation; Use methanol to elute to obtain fishing liquid; (2) The active ingredients screened were analyzed using UHPLC-Q-Exactive Orbitrap-MS / MS.

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