A cell membrane capillary monolithic column, its preparation method and application

Abstract

The application discloses a cell membrane capillary whole column and a preparation method and application thereof, and the preparation method is as follows: (1) preparing a cell membrane suspension; (2) pretreating a capillary; (3) preparing a modified capillary whole column; (4) injecting an HCl aqueous solution into the modified capillary whole column, replacing methanol and reacting, injecting an NaIO4 aqueous solution, oxidizing, washing with water, and balancing with a borate buffer; and injecting the cell membrane suspension obtained in the step (1), replacing the borate buffer, so that a cell membrane capillary whole column is obtained. The cell membrane capillary whole column is used for separating active ingredients of traditional Chinese medicines, does not need to balance a chromatographic column, and is simple and easy to operate. In the process of discovering active chemical ingredients, unnecessary separation steps can be avoided, and the separation period is shortened in the research of effective substances of traditional Chinese medicines. Compared with a traditional separation and identification method, the method is more suitable for the research of effective substances of traditional Chinese medicines containing complex components.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of screening of Chinese medicine effective substances by affinity chromatography, and particularly relates to a cell membrane capillary whole column and a preparation method and application thereof. BACKGROUND

[0002] The Chinese medicine effective substance basis generally refers to the effective substance produced after water decoction of the medicine. The effective substance produced after water decoction has complex components, some of which contain dozens of components or even hundreds of components. The determination of the Chinese medicine effective substance often needs to be carried out through separation and purification of chemical components and combined with pharmacological experiments. Limited to the existing chromatographic separation technology, most of the components obtained through separation and active screening from the Chinese medicine are constant components, and the research on components with low content is less. Therefore, revealing the Chinese medicine effective substance basis is still a difficult problem attracting attention in the academic field.

[0003] In recent years, the development of receptor pharmacology makes people realize that various membrane receptors or channels are not only the targets for studying the action characteristics of drugs, but also the targets for screening and discovering active components of Chinese medicine. Cell membrane chromatography is a new drug screening method for determining the effective substance by combining high performance liquid chromatography, cell biology and receptor pharmacology, utilizing the specific affinity between drugs and membrane receptors, and dynamically simulating the action process of drugs in the body in the chromatographic column. Since the cell membrane chromatography is to fix the active tissue cell membranes of human or animals on the surface of the carrier to form a carrier cell membrane, which maximally maintains the integrity of the cell membrane, the three-dimensional structure of the membrane receptor and the surrounding environment, not only has the characteristics of ordinary cell membrane preparations, but also has a certain rigidity, so it can be used as a biologically active filler for liquid chromatography to form a chromatographic system that can simulate the interaction between drugs and targets under dynamic conditions. The system can avoid the complicated separation and purification steps of Chinese medicine, and directly determine the effective substance of Chinese medicine, especially compound Chinese medicine.

[0004] Professor He Langchong's research group has carried out research on cell membrane chromatography for many years, established a white blood cell membrane chromatography, and screened TLR4 receptor antagonists contained in Chinese medicine atractylodes; the research on the use of HEK293 cell membrane chromatography to distinguish adrenaline and other alpha-adrenergic receptor agonists has also received good results. In addition, cell membrane chromatography established by using beta adrenergic cells, vascular smooth muscle cells, macrophages and the like has also been reported and applied to the study of Chinese medicine.

[0005] However, at present, the above-mentioned cell membrane chromatography mainly uses the cell membrane fixed on the surface of the silica gel carrier to pack the chromatographic column, and relies on the interaction between the cell membrane receptor and the different active chemical components in the Chinese medicine to play a separation role. In this process, the chromatographic system needs to be balanced, and the chromatograph needs to be connected for operation.

[0006] Capillary monolithic column is a continuous rod-like monolithic material which is in-situ polymerized in an empty column by organic or inorganic polymerization method. Compared with conventional packed extraction column, monolithic column is simple to prepare and can avoid complicated packing process of chromatographic column; monolithic column contains special pore structure, has good permeability and small mass transfer resistance, and can improve extraction efficiency; and the sample can be enriched, separated and purified through selective adsorption and selective elution. However, the technology can only enrich chemical components by chemical method and cannot enrich active components of traditional Chinese medicine in a targeted manner. SUMMARY

[0007] The present application aims at overcoming the deficiencies of the prior art and providing a cell membrane capillary monolithic column.

[0008] The second object of the present application is to provide a preparation method of the cell membrane capillary monolithic column.

[0009] The third object of the present application is to provide an application of the cell membrane capillary monolithic column in separation of active components of traditional Chinese medicine.

[0010] The technical scheme of the present application is summarized as follows:

[0011] A preparation method of a cell membrane capillary monolithic column comprises the following steps:

[0012] (1) 0.1-1 mM benzylsulfonyl fluoride is added to cell suspension and mixed; the cell is broken by a cell ultrasonic disrupter at 4-20℃ for 5-10 min, centrifuged at 200-1000xg at 4-20℃ for 5-10 min, and the supernatant is taken; then the supernatant is discarded, the precipitate is washed with water or 5-50 mM phosphate buffer with pH of 7.2-7.4, the buffer is added to the precipitate to adjust the concentration to 1-3.5 mg / mL, and cell membrane suspension is obtained;

[0013] (2) 50% mass concentration methanol solution of γ-(methacryloyloxy) propyl trimethoxysilane is injected into a capillary with an inner diameter of 200-530 μm, both ends are sealed, and the capillary is reacted at 30℃ for 12-18 h to obtain a pretreated capillary;

[0014] 1 g of fumed SiO2 nanoparticles, 25 mL of ethanol and 5.2 mL of 0.05 mM-0.15 mM hydrochloric acid aqueous solution are mixed uniformly by ultrasonic mixing, stirred and heated to 80℃, and 3.6 mL of γ-(methacryloyloxy) propyl trimethoxysilane is added dropwise during the heating process, and the reaction is carried out for 6 h to obtain methacryloylated fumed SiO2 nanoparticles;

[0015] (3) proportionally, 0.04 g of the methacrylated fumed SiO2 nanoparticles are mixed with 0.75 mL of glycidyl methacrylate, 0.25 mL of ethylene glycol dimethacrylate, 2.7 mL of cyclohexanol, 0.3 mL of dodecanol and 0.04 g of azobisisobutyronitrile, vortexed and ultrasonicated to uniformly disperse the methacrylated fumed SiO2 nanoparticles; after purging with N2 for 3-10 min, a mixed solution I is obtained; the mixed solution I is injected into the pretreated capillary with a syringe, both ends are sealed, and the reaction is carried out at 55-65℃ for 12-24 h; a capillary monolithic column is obtained; a toluene solution of dimethyloctadecylchlorosilane with a mass concentration of 4% is injected into the capillary monolithic column, and the reaction is carried out at 80℃ for 6-12 h, and then the capillary monolithic column is washed with methanol to obtain a modified capillary monolithic column;

[0016] (4) 20 μL of 3.16 M HCl aqueous solution is injected into the modified capillary monolithic column to replace the methanol and react for 60-96 h, 20 μL of 1.5% NaIO4 aqueous solution is injected to oxidize for 20-40 min, the column is washed with water, and then equilibrated with a borate buffer with pH = 6.8-8.2 for 10 min; 20 μL of the cell membrane suspension obtained in step (1) is injected to replace the borate buffer, and a cell membrane capillary monolithic column is obtained.

[0017] The cell membrane capillary monolithic column prepared by the preparation method.

[0018] The application of the cell membrane capillary monolithic column in separating active ingredients of traditional Chinese medicine.

[0019] Advantages of the present application:

[0020] The cell membrane capillary monolithic column of the present application is used for separating active ingredients of traditional Chinese medicine, and does not need to be equilibrated with a chromatographic column, so the method is simple and easy to operate. In the process of discovering active chemical ingredients, unnecessary separation steps can be avoided, and the separation period is shortened in the research of active substances of traditional Chinese medicine. Compared with the traditional separation and identification method, the present application is more suitable for the research of active substances of traditional Chinese medicine containing complex components. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 To characterize the polymer material of the fixed cell membrane by using a scanning electron microscope;

[0022] Figure 2 To characterize the cell membrane capillary monolithic column by using a scanning electron microscope;

[0023] Figure 3 It is a total ion chromatogram of the water decoction of Atractylodes lancea;

[0024] Figure 4 It is a total ion chromatogram of the water decoction of Atractylodes lancea treated by using a cell membrane capillary monolithic column. DETAILED DESCRIPTION

[0025] The cell suspension is obtained by adding physiological saline, DMEM medium or 50 mM phosphate buffer solution with pH = 7.2-7.4 to the blood cells of rats, the blood cells of rabbits or cultured cells.

[0026] The following examples are used to illustrate the present application, but the scope of protection of the present application is not limited to the following examples. The ordinary skilled in the art can realize the present application according to the above disclosure and the range of each parameter.

[0027] Example 1

[0028] 1. Preparation of cell membrane suspension

[0029] Leukocytes are obtained from rabbit blood and a cell suspension is prepared using 50 mM phosphate buffer solution with pH = 7.4;

[0030] 0.1 mM benzylsulfonyl fluoride is added to the cell suspension to obtain a final concentration, and the mixture is mixed. The cell suspension is crushed by a cell sonicator at 4°C for 5 min, and then centrifuged at 1000 x g at 4°C for 5 min. The supernatant is collected, and then centrifuged at 20000 x g for 30 min. The supernatant is discarded, and the precipitate is washed with 5 mM phosphate buffer solution with pH = 7.4 for 3 times. The precipitate is added with the buffer solution and adjusted to obtain a cell membrane suspension;

[0031] The total protein content of the cell membrane suspension is 3.5 mg / mL, which is determined by using a total protein quantitative test kit from Nanjing Jiancheng Biological Engineering Institute;

[0032] 2. Pretreatment of capillary and synthesis of methylacryloylized gas phase SiO2 nanoparticles

[0033] A capillary with an inner diameter of 530 μm is washed with 0.1 M NaOH aqueous solution for 1 h, and then washed with water until the outlet solution has pH = 7.0, and washed with methanol for 10 min;

[0034] A methanol solution of γ-(methacryloyloxy)propyl trimethoxysilane with a mass concentration of 50% is injected into the above capillary, both ends are sealed, and the reaction is carried out at 30°C for 12 hours. The excess γ-(methacryloyloxy)propyl trimethoxysilane methanol solution is washed away with methanol; and a pretreated capillary is obtained.

[0035] Take 1 g of fumed SiO2 nanoparticles in a 50 mL round bottom flask, add 25 mL of ethanol and 5.2 mL of 0.1 mM hydrochloric acid aqueous solution, ultrasonic mixing, magnetic stirring and heating to 80°C, and in the process of heating, dropwise add 3.6 mL of γ-(methacryloyloxy) propyl trimethoxysilane, reaction for 6 h; after the reaction, wash with water and anhydrous ethanol in turn to neutral, vacuum drying box 45°C drying for standby, the product obtained is the methacryloyl fumed SiO2 nanoparticles;

[0036] 3. Preparation of modified capillary monolithic column

[0037] Take 0.04 g of methacryloyl fumed SiO2 nanoparticles, mix with 0.75 mL of glycidyl methacrylate, 0.25 mL of ethylene glycol dimethacrylate, 2.7 mL of cyclohexanol, 0.3 mL of dodecanol and 0.04 g of azobisisobutyronitrile, vortex and ultrasonic to disperse the methacryloyl fumed SiO2 nanoparticles uniformly; after 7 min of N2, the mixed solution is obtained; inject the mixed solution into the pretreated capillary with a syringe, seal both ends, and react at 60°C for 12 h; obtain the capillary monolithic column; inject the toluene solution of dimethyloctadecylchlorosilane with a mass concentration of 4% into the capillary monolithic column, react at 80°C for 8 h, rinse with methanol for 10 min, and obtain the modified capillary monolithic column;

[0038] 4. Preparation of capillary cell membrane monolithic column

[0039] Inject 20 μL of 3.16 M HCl aqueous solution into the modified capillary monolithic column, replace methanol and react for 60 h, inject 20 μL of 1.5% NaIO4 aqueous solution, oxidize for 30 min, rinse with water, and equilibrate with pH = 8.2 borate buffer for 10 min; inject 20 μL of the cell membrane suspension obtained in step (1), replace the borate buffer, and obtain a cell membrane capillary monolithic column. The polymer material for immobilizing the cell membrane is shown in Figure 1 , and the scanning electron microscope image of the cell membrane capillary monolithic column is shown in Figure 2 .

[0040] Example 2

[0041] 1. Preparation of cell membrane suspension

[0042] Obtain white blood cells from rabbit blood and prepare a cell suspension with pH = 7.4 50 mM phosphate buffer;

[0043] The benzylsulfonyl fluoride was added to the cell suspension to a final concentration of 0.5 mM, mixed well; the cells were broken by a cell sonicator at 6°C for 8 min, centrifuged at 500 x g for 8 min at 4°C, and the supernatant was taken; the supernatant was further centrifuged at 15000 x g for 15 min, and the supernatant was discarded; the precipitate was washed with 5 mM phosphate buffer at pH 7.4 for 3 times, and the precipitate was suspended in the buffer and adjusted to a certain concentration to obtain a cell membrane suspension; the content of cell membrane protein in the cell membrane suspension was determined to be 1 mg / mL by using a total protein quantitative test kit from Nanjing Jiancheng Bioengineering Institute;

[0044] 2. Pretreatment of capillary and synthesis of methylacryloylated fumed SiO2 nanoparticles

[0045] The capillary with an inner diameter of 300 μm was washed with 0.1 M aqueous NaOH solution for 1 h, and then washed with water until the pH of the outlet solution was 7.0, and washed with methanol for 10 min;

[0046] A methanol solution of γ-(methacryloyloxy)propyltrimethoxysilane with a mass concentration of 50% was injected into the capillary, both ends were sealed, and the reaction was carried out at 30°C for 15 h; the excess γ-(methacryloyloxy)propyltrimethoxysilane methanol solution was washed away with methanol; and a pretreated capillary was obtained.

[0047] 1 g of fumed SiO2 nanoparticles was weighed into a 50 mL round-bottom flask, 25 mL of ethanol and 5.2 mL of 0.05 mM hydrochloric acid aqueous solution were added thereto; after ultrasonic mixing, magnetic stirring was performed and the temperature was raised to 80°C, and 3.6 mL of γ-(methacryloyloxy)propyltrimethoxysilane was added dropwise during the temperature raising process, and the reaction was continued for 6 h; after the reaction was completed, washing with water and anhydrous ethanol was sequentially performed until neutral, and drying was performed in a vacuum drying oven at 45°C for standby; and the obtained product was methylacryloylated fumed SiO2 nanoparticles;

[0048] 3. Preparation of modified capillary monolithic column

[0049] 0.04 g of methylacryloylated fumed SiO2 nanoparticles was weighed, mixed with 0.75 mL of glycidyl methacrylate, 0.25 mL of ethylene glycol dimethacrylate, 2.7 mL of cyclohexanol, 0.3 mL of dodecanol, and 0.04 g of azobisisobutyronitrile, vortexed and ultrasonically mixed to uniformly disperse the methylacryloylated fumed SiO2 nanoparticles; after N2 was passed for 3 min, a mixed solution one was obtained; the mixed solution one was injected into the pretreated capillary by using a syringe, both ends were sealed, and the reaction was carried out at 55°C for 24 h; a capillary monolithic column was obtained; a toluene solution of dimethyloctadecylchlorosilane with a mass concentration of 4% was injected into the capillary monolithic column, and the reaction was carried out at 80°C for 12 h; and washing with methanol was carried out for 10 min to obtain a modified capillary monolithic column.

[0050] 4. Preparation of the capillary cell membrane monolith column

[0051] Into the modified capillary monolith column, 20 μL of 3.16 M aqueous HCl solution was injected to displace the methanol and react for 72 h, 20 μL of 1.5% (mass concentration) aqueous NaIO4 solution was injected to oxidize for 20 min, the capillary was washed with water and equilibrated with pH 7.8 borate buffer for 10 min; 20 μL of the cell membrane suspension obtained in step (1) was injected to displace the borate buffer, thus obtaining a cell membrane capillary monolith column.

[0052] Example 3

[0053] 1. Preparation of the cell membrane suspension

[0054] White blood cells were obtained from rabbit blood and a cell suspension was prepared with 50 mM phosphate buffer at pH 7.4;

[0055] White blood cells of rats or cultured cells can be used to replace the white blood cells obtained from rabbit blood in this example, and other operations are the same as in this example, thus obtaining a corresponding cell membrane capillary monolith column; physiological saline, DMEM medium or 50 mM phosphate buffer at pH 7.2 can be used to replace the 50 mM phosphate buffer at pH 7.4 in this example, and other operations are the same as in this example, thus obtaining a corresponding cell membrane capillary monolith column.

[0056] PMSF was added to the cell suspension to a final concentration of 1 mM and mixed well; the cell suspension was broken by a cell sonicator at 20°C for 10 min, centrifuged at 200 x g at 20°C for 10 min, and the supernatant was taken; the supernatant was further centrifuged at 10 000 x g for 60 min, and the supernatant was discarded and the precipitate was washed with 50 mM phosphate buffer at pH 7.2 for 3 times; the precipitate was added to the buffer and adjusted to a certain concentration to obtain a cell membrane suspension; the total protein content of the cell membrane suspension was determined to be 2.4 mg / mL by using a total protein quantification test kit from Nanjing Jiancheng Biological Engineering Institute;

[0057] 2. Pretreatment of the capillary and synthesis of the gas-phase SiO2nanoparticles modified with methacryl groups

[0058] The capillary with an inner diameter of 200 μm was washed with 0.1 M aqueous NaOH solution for 1 h, then washed with water until the pH of the outlet solution was 7.0, and washed with methanol for 10 min;

[0059] A methanol solution of 50% γ-(methacryloyloxy)propyltrimethoxysilane was injected into the capillary tube, both ends were sealed, and the reaction was carried out at 30°C for 18 hours. Excess γ-(methacryloyloxy)propyltrimethoxysilane methanol solution was washed away with methanol to obtain the pretreated capillary tube.

[0060] Weigh 1g of fumed SiO2 nanoparticles into a 50mL round-bottom flask, add 25mL of ethanol and 5.2mL of 0.15mM hydrochloric acid aqueous solution; after ultrasonic mixing, stir magnetically and heat to 80℃, and add 3.6mL of γ-(methacryloyloxy)propyltrimethoxysilane dropwise during the heating process. The reaction continues for 6h. After the reaction is completed, wash with water and anhydrous ethanol until neutral, and dry in a vacuum drying oven at 45℃ for later use. The product obtained is methacryloylated fumed SiO2 nanoparticles.

[0061] 3. Preparation of modified monolithic capillary columns

[0062] Weigh 0.04 g of methacrylamide-modified fumed SiO2 nanoparticles and mix them with 0.75 mL of glycidyl methacrylate, 0.25 mL of ethylene glycol dimethacrylate, 2.7 mL of cyclohexanol, 0.3 mL of dodecanol, and 0.04 g of azobisisobutyronitrile. Vortex and sonicate to uniformly disperse the methacrylamide-modified fumed SiO2 nanoparticles. After passing N2 through the mixture for 10 min, obtain mixture one. Inject mixture one into a pretreated capillary tube using a syringe, seal both ends, and react at 65 °C for 12 h to obtain a monolithic capillary column. Inject a 4% (w / w) solution of dimethyloctadecylchlorosilane in toluene into the monolithic capillary column and react at 80 °C for 6 h. Rinse with methanol for 10 min to obtain the modified monolithic capillary column.

[0063] 4. Preparation of monolithic capillary cell membrane columns

[0064] 20 μL of 3.16M HCl aqueous solution was injected into the modified capillary monolithic column to replace methanol and react for 96 h. Then, 20 μL of 1.5% NaIO4 aqueous solution was injected and oxidized for 40 min. The column was rinsed with water and equilibrated with pH 6.8 borate buffer for 10 min. Then, 20 μL of the cell membrane suspension obtained in step (1) was injected to replace the borate buffer, thus obtaining a cell membrane capillary monolithic column.

[0065] Example 4

[0066] Screening of medicinal substances in traditional Chinese medicine using cell membrane capillary monolithic column

[0067] 10g of Atractylodes lancea powder was extracted with 80mL of hot water under reflux for 1 hour to obtain Atractylodes lancea decoction.

[0068] The cell membrane capillary monolithic column prepared in Example 1 was injected with the aqueous decoction of Atractylodes lancea at 37℃, and incubated for 30 min to allow the pharmacodynamic substances to specifically bind to the target of the cell membrane capillary monolithic column. Then, the column was washed with 50 mM phosphate buffer at pH 7.4, and the dissociation solution (20% acetic acid aqueous solution) was added to dissociate the pharmacodynamic substances from the target on the surface of the cell membrane monolithic column. The effluent was collected and analyzed by GC / MS. Figure 3 , Figure 4 )。

[0069] As can be seen from Figure 3 , Figure 4 , the chromatographic peaks in the total ion chromatogram of the aqueous decoction of Atractylodes lancea treated by the cell membrane capillary monolithic column Figure 4 ) are significantly reduced compared with the total ion chromatogram of the aqueous decoction of Atractylodes lancea Figure 3 ). The chemical components in the effluent were identified by comparison with the NIST database, and it was determined that the chemical component contained β-eudesmol. The cell membrane on the surface of the prepared cell membrane capillary monolithic column contains MD-2 protein and other anti-inflammatory related proteins, which can interact with small molecule substances to exert anti-inflammatory effect. The identified β-eudesmol has strong anti-inflammatory effect, indicating that the prepared cell membrane capillary monolithic column can be used for rapid determination of pharmacodynamic substances of anti-inflammatory traditional Chinese medicines. Compared with conventional methods, this method does not require tedious separation and purification process.

[0070] Experiments proved that the cell membrane capillary monolithic column prepared in Examples 2 and 3 has similar effect in screening the pharmacodynamic substance β-eudesmol of traditional Chinese medicines as the cell membrane capillary monolithic column prepared in Example 1.

Claims

1. A method for preparing a cell membrane capillary monolith column, characterized by It comprises the following steps: (1) adding benzylsulfonyl fluoride to a cell suspension with a final concentration of 0.1-1 mM, mixing; using a cell ultrasonic disrupter to break the cells at 4-20℃ for 5-10 min, centrifuging at 200-1000 x g at 4-20℃ for 5-10 min, and taking the supernatant; further centrifuging at 10000-20000 x g for 15-60 min, discarding the supernatant, and washing the precipitate with water or a 5-50 mM phosphate buffer with pH=7.2-7.4, adding the buffer to the precipitate to adjust the concentration to 1-3.5 mg / mL, and obtaining a cell membrane suspension; (2) injecting a 50% mass concentration methanol solution of γ-(methacryloyloxy)propyltrimethoxysilane into a capillary with an inner diameter of 200-530 μm, sealing both ends, and reacting at 30℃ for 12-18 h to obtain a pretreated capillary; Proportionally, 1 g of fumed SiO2 nanoparticles, 25 mL of ethanol, and 5.2 mL of a 0.05 mM-0.15 mM volume concentration hydrochloric acid aqueous solution are uniformly mixed by ultrasonic mixing, stirred, and heated to 80℃, and 3.6 mL of γ-(methacryloyloxy)propyltrimethoxysilane is added dropwise during the heating process, and the reaction is carried out for 6 h to obtain methacryloylated fumed SiO2 nanoparticles; (3) proportionally, 0.04 g of methacryloylated fumed SiO2 nanoparticles are mixed with 0.75 mL of glycidyl methacrylate, 0.25 mL of ethylene glycol dimethacrylate, 2.7 mL of cyclohexanol, 0.3 mL of dodecanol, and 0.04 g of azobisisobutyronitrile, and the mixture is vortexed and ultrasonically mixed to uniformly disperse the methacryloylated fumed SiO2 nanoparticles; after N2 is passed for 3-10 min, a mixed solution one is obtained; the mixed solution one is injected into the pretreated capillary with a syringe, both ends are sealed, and the reaction is carried out at 55-65℃ for 12-24 h; a capillary monolithic column is obtained; a 4% mass concentration dimethyloctadecylchlorosilane toluene solution is injected into the capillary monolithic column, and the reaction is carried out at 80℃ for 6-12 h, and the column is washed with methanol to obtain a modified capillary monolithic column; (4) 20 μL of a 3.16 M HCl aqueous solution is injected into the modified capillary monolithic column, methanol is replaced, and the reaction is carried out for 60-96 h; 20 μL of a 1.5% mass concentration NaIO4 aqueous solution is injected, and oxidation is carried out for 20-40 min; the column is washed with water, and equilibrated with a pH=6.8-8.2 borate buffer for 10 min; 20 μL of the cell membrane suspension obtained in step (1) is injected, and the borate buffer is replaced, thereby obtaining a cell membrane capillary monolithic column.

2. A cell membrane capillary monolithic column prepared by the preparation method of claim 1.

3. Application of the cell membrane capillary monolithic column of claim 2 to separation of active components of traditional Chinese medicines.

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