In-vitro bionic screening method and device for effect components of traditional Chinese medicine

Abstract

The invention discloses an in-vitro bionic screening method and device for traditional Chinese medicine effect components, and relates to the technical field of screening and analysis of traditional Chinese medicine effect components. The method comprises the following steps: preparing a traditional Chinese medicine sample extracting solution, putting hollow fibers fixed with normal cells and pathological cells into the traditional Chinese medicine sample extracting solution diluted by a culture medium, and screening the traditional Chinese medicine effect components in an in-vitro bionic environment. After screening is finished, desorption and LC-MS (liquid chromatography-mass spectrometry) or GC-MS (gas chromatography-mass spectrometry) determination are respectively performed on effect components combined on the two cells in the fiber cavity. The cell capture factor is used for evaluating the difference between the traditional Chinese medicine effect component and the two cell binding capacities, so that the potential effect component of the traditional Chinese medicine is preliminarily confirmed. The invention provides a simple, convenient, efficient and accurate screening method for the effect components of the traditional Chinese medicine for specific diseases, and the screening method has important significance on development of research on the action mechanism and quality control of the traditional Chinese medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of screening and analyzing effective components of traditional Chinese medicine, and particularly relates to a method and device for in-vitro biomimetic screening of effective components of traditional Chinese medicine by using living cells. BACKGROUND

[0002] Traditional Chinese medicines are various in types and complex in components, and are usually used in the form of compound prescriptions. The characteristics of multiple components, multiple targets and multiple pathways in the curative effect of traditional Chinese medicines are both the features of traditional Chinese medicines and the factors restricting the modernization of traditional Chinese medicines. Efficient and accurate analysis of effective components of traditional Chinese medicines is the basis for elucidating the mechanism of pharmacodynamic action, and also affects the selection of quality control indicators of traditional Chinese medicines.

[0003] The research of active ingredients of traditional Chinese medicine is mainly based on the characteristics that target points on cell membranes can specifically bind to active ingredients. Cell membranes are loaded onto different carriers to screen active compounds, such as cell membrane chromatography (CMC) that fixes cell membranes on chromatographic packing, cell membrane-coated magnetic nanomaterials, and cell membrane-coupled capillary electrophoresis. CMC can identify the structure of known or unknown active compounds in traditional Chinese medicinal materials or complex prescriptions by combining with HPLC or LC-MS. For example, Pan et al. proposed a stop-flow comprehensive two-dimensional cell membrane chromatography comparative system to screen active ingredients in Pyrrosia calvata (Bak.) Ching against crystal-induced kidney injury (HK-2 / CIKI) using HK-2 cells and HK-2 / CIKI model cells (A stop-flow comprehensive two-dimensional HK-2 and HK-2 / CIKI cell membrane chromatography comparative analysis system for screening the active ingredients from Pyrrosia calvata (Bak.) Ching against crystal-induced kidney injury, Journal of Pharmaceutical and Biomedical Analysis, 2021, 195: 113825).Cell membrane-coated magnetic materials combine magnetic separation technology with cell membrane coating. Hu et al. developed a bifunctionalized carbon nanotube that combined magnetic nanoparticles and HEK 293 cell membranes with high expression of α1A-adrenergic receptors as a drug screening platform (Magnetic carbon nanotubes camouflaged with cell membrane as a drug discovery platform for selective extraction of bioactive compounds from natural products, Chemical Engineering Journal, 2019, 364: 269-279). In order to improve the aggregation of magnetic materials, magnetic graphene oxide was modified with polyethylene glycol, and active compounds in Angelica sinensis were screened (Accurate construction of cell membrane biomimetic graphene nanodecoys via purposeful surface engineering to improve screening efficiency of active components of traditional Chinese medicine, Acta Pharmaceutica Sinica B, 2022, 12(1): 394-405). CMC can effectively and specifically screen active ingredients, but cell membrane chromatography columns require specialized preparation techniques; cell membrane coating technology does not require a cumbersome operation process and has high scalability, but the coated membrane is easily lost during the extraction process of cell membrane-coated magnetic materials, the combination between cell membranes and carriers is weak and unstable, and satisfactory screening efficiency cannot be obtained in practical applications.

[0004] It is known that drug targets exist not only in cell membrane receptors but also in intracellular receptors (such as messenger substances, enzymes and gene transcription molecules, etc.), and only using cell membranes for activity screening research will result in the loss of subcellular membrane targets and the missing of part of active ingredients. Therefore, the whole cell as a screening model can be more comprehensive for the research of effective components of traditional Chinese medicine. Normal cell morphology is helpful for the normal functioning of cells; when the cells become pathological, the cell receptors and morphology, intracellular pathway regulation or intracellular factors can change, leading to changes in cell function. A large number of diseases occur and progress due to abnormal physiological changes of cells, compared with normal cells, the effective components of traditional Chinese medicine have higher affinity and specific binding with pathological cells, therefore, using pathological cells and normal cells as controls, investigating the differences in the binding of effective components with the two can better reflect the real disease condition, pathological research and screening of effective components are more targeted, and the effective components of traditional Chinese medicine that specifically bind with diseases can be more accurately and efficiently screened and the effect of the effective components can be judged. SUMMARY

[0005] The purpose of the present application is to provide an in vitro biomimetic screening method and device for effective components of traditional Chinese medicine, which can biomimetically screen the effective components of traditional Chinese medicine under the condition of maintaining the inherent biological characteristics of cells and avoid the activity screening errors caused by non-specific binding.

[0006] To achieve the above-mentioned purpose, according to one aspect of the present application, an in vitro biomimetic screening method for effective components of traditional Chinese medicine is provided, comprising: Step one, normal cell suspension and model pathological cell suspension treated by an inducing agent are respectively injected into the inner cavity of hollow fibers, and are cultured in complete culture medium to make the cells adhere to the inner wall of the hollow fibers and be fixed, so as to obtain the hollow fibers fixed with living cells; Step two, the hollow fibers fixed with living cells are immersed in the extract of traditional Chinese medicine samples for screening of effective components; Step three, after the screening is completed, the effective components combined with the living cells in the inner cavity of the hollow fibers are desorbed and determined; Step four, based on the determination results of step three, the cell trapping factor CTF is calculated, ; Wherein, C model cell and A model cell respectively represent the concentration and chromatographic peak area of the effective components screened from the hollow fibers fixed with model pathological cell living cells; C normal cell and A normal cellrespectively represent the concentration of the effective component screened from the hollow fiber fixed with normal cells and the chromatographic peak area; CTF 0, indicating that the effective component in the traditional Chinese medicine is specifically recognized and combined by the model pathological cells, and is used for preliminary confirmation of the potential effective component of the traditional Chinese medicine.

[0007] Further, in step one, when fixing normal cells, the normal cells obtained by digestion are resuspended with complete culture medium to obtain a cell suspension, the cell suspension is taken with a sterile syringe without a needle, and then the needle is installed and inserted into one end of the hollow fiber, which is placed in a culture bottle. The cell suspension is injected into the lumen of the hollow fiber and filled; the hollow fiber is added with complete culture medium, and the culture bottle is placed in a CO2 incubator for culture, so as to obtain a hollow fiber fixed with normal cell living cells.

[0008] Further, in step one, when fixing model pathological cells, the normal cells after digestion are resuspended with complete culture medium containing an inducer to obtain a cell suspension, the cell suspension is taken with a sterile syringe without a needle, and then the needle is installed and inserted into one end of the hollow fiber, which is placed in a culture bottle. The cell suspension is injected into the lumen of the hollow fiber and filled; the hollow fiber is added with complete culture medium containing an inducer, and the culture bottle is placed in a CO2 incubator for culture, so as to obtain a hollow fiber fixed with model pathological cell living cells.

[0009] Further, the culture is ended when the cell growth density reaches 70%-80%.

[0010] Further, in step one, the material of the hollow fiber is polypropylene, the membrane wall pore size is 0.2 μm, the lumen inner diameter is 0.6 mm, the length of the small section is 5-7 cm, and the small section is sterilized for standby.

[0011] Further, in step two, water or an organic solvent or a combination of water and an organic solvent is used as an extraction solvent for extraction of the traditional Chinese medicine, the extract is concentrated and treated, the residue after evaporation of the extraction solvent is dissolved with dimethyl sulfoxide, and then the basic culture medium is added for dilution to a working concentration, so as to obtain the traditional Chinese medicine sample extract.

[0012] Further, the organic solvent is selected from any one or a mixture of several of methanol, ethanol, butanol, diethyl ether, ethyl acetate, and chloroform.

[0013] Further, in step two, the two ends of the hollow fiber fixed with living cells are tied with sterilized cotton thread, folded into a U shape, and placed in a sample bottle with a magnetic rotor, completely immersed in the traditional Chinese medicine sample extract, and subjected to screening under the conditions of 37℃ water bath and 500-700 rpm magnetic stirring.

[0014] Further, in step three, the inner wall of the hollow fiber is rinsed with a methanol solution, the eluate is collected, the protein is removed by centrifugation, the supernatant is dried, redissolved and centrifuged, and LC-MS or GC-MS is used for analysis.

[0015] According to another aspect of the present application, a device for implementing the in-vitro biomimetic screening method of the effective components of traditional Chinese medicine is provided, comprising: a constant-temperature magnetic stirrer, a water bath, a hollow fiber and a sample bottle. The water bath is placed on the upper part of the constant-temperature magnetic stirrer, and the water bath is filled with water; the sample bottle is placed in the water bath, the sample bottle contains the traditional Chinese medicine sample extract and is provided with a magnetic rotor; the hollow fiber is folded into a U shape after being sealed at both ends and is immersed in the traditional Chinese medicine sample extract.

[0016] The present application takes traditional Chinese medicine as the research object, screens the effective components in traditional Chinese medicine through the hollow fiber fixed with living cells, and evaluates the difference in affinity of the effective components to normal cells / diseased cells through the capture factor, which can be applied to screen the components (group) effective for specific diseases in traditional Chinese medicine.

[0017] Compared with the prior art, the present application has the following beneficial effects: (1) without special materials and complex technology, simple and economical materials are used to guarantee the cell viability and function, an in-vitro high-throughput traditional Chinese medicine activity screening platform at the cell level is constructed, and the operation is convenient; (2) the present application is an activity screening research on complete cells, which avoids the omission of effective components when only the cell membrane is used for research; (3) the normal cells are fixed in the hollow fiber cavity with good biocompatibility, and a disease cell model is established, the screening results of the two are compared, the “cell capture factor” is proposed, and the components capable of combining with normal cells but with weak binding force are excluded, which is conducive to more accurately screening the effective components specifically combined with diseased cells; (4) the amount of cells loaded in the hollow fiber does not need to be too much to achieve good screening effect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The schematic diagram of the in-vitro biomimetic screening device of the effective components of traditional Chinese medicine provided by the present application; Figure 1 In the figure, 1 is a constant-temperature magnetic stirrer, 2 is a water bath, 3 is water, 4 is a magnetic rotor, 5 is a hollow fiber, 6 is a traditional Chinese medicine sample extract, and 7 is a sample bottle.

[0019] Figures 2-4 The LC-MS graph for screening the anti-kidney fibrosis effective components in Piper longum medicinal materials by the present application; Among them, Figure 2 The LC-MS graph of the mixed control; Figure 3 The LC-MS graph of screening Piper longum extract by the hollow fiber fixed with HK-2 cells; Figure 4LC-MS image of Piper longum extract for screening diseased cells in a hollow fiber fixation model.

[0020] Figures 5-7 This is an LC-MS chromatogram showing the effect of Banxia Xiexin Decoction on the anti-chronic atrophic gastritis. in, Figure 5 The image shows the LC-MS chromatogram of the mixed reference standard. In the figure, 1: palmatine, 2: glycyrrhizin, 3: naringenin, 4: sennain A-7-O-β-D-glucuronide, 5: isorhamnetin, 6: 6-gingerol, 7: baicalin, and 8: acaciain. Figure 6 LC-MS image of Banxia Xiexin Decoction for screening GES-1 cells fixed in hollow fibers; Figure 7 LC-MS image of Banxia Xiexin Decoction for screening diseased cells in hollow fiber fixation model.

[0021] Figure 8 The present invention aims to screen for capture factors of the effective components in Banxia Xiexin Decoction. Detailed Implementation

[0022] The basic concept of this invention is based on the theory of interaction between the effective components of traditional Chinese medicine and cells. Normal cells and diseased cells are respectively immobilized in the lumen of hollow fibers, and suitable conditions are set to simulate the physiological environment so that the cells maintain normal vitality and function. After screening, based on the difference in the binding force of normal cells and diseased cells to the effective components of traditional Chinese medicine, active components (groups) that are effective against specific diseases in traditional Chinese medicine materials or compound prescriptions are preliminarily screened.

[0023] Based on the above inventive concept, a typical embodiment of the present invention provides an in vitro biomimetic screening method for the active ingredients of traditional Chinese medicine, comprising the following steps one to three.

[0024] Step 1: Cell Culture and Fixation Normal cell suspension and model diseased cell suspension treated with an inducer were injected into the lumen of hollow fibers and cultured in a complete culture medium to allow the cells to adhere and grow to the inner wall of the hollow fibers, thus obtaining hollow fibers with immobilized live cells.

[0025] In this step, normal cells in the logarithmic growth phase, such as human renal tubular epithelial (HK-2) cells and human gastric mucosal epithelial (GES-1) cells, are selected, washed with PBS, and then digested with trypsin.

[0026] Model disease cells are normal cells resuspended in complete culture medium containing specific disease inducers. The inducers used to induce model disease cells are selected based on the chosen cell line and the type of disease to be simulated. For example, TGF-β is selected as the fibrosis inducer for HK-2 cells, and MNNG is selected as the lesion inducer for GES-1 cells.

[0027] The culture medium preferably adopts DMEM / F-12 or DMEM medium.

[0028] The hollow fiber is a material with a tubular cavity in the axial direction and a membrane wall densely covered with micropores, which has the advantages of low cost, cell adhesion, flexibility and easy handling, biocompatibility and the like. Using the fiber membrane as a cell carrier, the cells adhere and grow on the inner wall of the tubular structure of the fiber membrane, forming a three-dimensional structure similar to the in-vivo tubular space in vitro, which can study the effective components of traditional Chinese medicine under conditions close to physiological conditions. In addition, the micrometer-scale wall pores of the hollow fiber have selective permeability, allowing solvents and small molecule active ingredients to pass through, similar to the action of small molecule active ingredients and cells, while larger molecule solutes are blocked by the membrane wall, realizing simultaneous screening and sample purification.

[0029] The material of the hollow fiber described in the embodiment is polypropylene, the membrane wall pore size is 0.2 μm, and the lumen inner diameter is 0.6 mm. The small sections with a length of 5-7 cm, preferably 6 cm, are cut and sterilized by a high-pressure steam sterilization pot for standby use.

[0030] The hollow fibers with fixed living cells include hollow fibers with fixed normal cell living cells and hollow fibers with fixed model pathogenic cell living cells, and the preparation methods are as follows.

[0031] Method for fixing normal cell living cells in hollow fibers: resuspend the digested normal cells with complete culture medium to obtain a cell suspension, use a sterile syringe without a needle to suck the cell suspension, then insert the end of the hollow fiber after installing the needle, and place it in a culture bottle. The cell suspension is injected into the lumen of the hollow fiber and filled; add complete culture medium to cover the hollow fiber, and place the culture bottle in a CO2 incubator for culture.

[0032] Method for fixing model pathogenic cell living cells in hollow fibers: resuspend the digested normal cells with complete culture medium containing an inducing agent to obtain a cell suspension, use a sterile syringe without a needle to suck the cell suspension, then insert the end of the hollow fiber after installing the needle, and place it in a culture bottle. The cell suspension is injected into the lumen of the hollow fiber and filled; add complete culture medium containing an inducing agent to cover the hollow fiber, and place the culture bottle in a CO2 incubator for culture to obtain the hollow fiber with fixed model pathogenic cell living cells.

[0033] The complete culture medium is injected into the lumen of the hollow fiber, and after culture, it is used as a blank control group.

[0034] The cell suspension is sucked by a sterile syringe without a needle, and then the end of the hollow fiber is inserted after installing the needle, which is to avoid damaging the cells.

[0035] Preferably, each culture bottle contains at most 12 fibers, which are placed dispersedly and avoid overlapping.

[0036] During the culture, the culture solution needs to be changed every other day, and the culture is ended when the cell density in the cavity reaches 70-80%, and then the hollow fiber fixed with the living cells is taken out.

[0037] Step two, effect component screening The hollow fiber fixed with the living cells is immersed into the traditional Chinese medicine sample extract for effect component screening.

[0038] The traditional Chinese medicine to be tested in the present application can be single medicine material or compound.

[0039] The traditional Chinese medicine is extracted by using water or organic solvent or a combination of water and organic solvent as the extraction solvent, the extract is concentrated and treated, the residue after the extraction solvent is evaporated is dissolved by dimethyl sulfoxide, and then diluted to working concentration by adding base culture medium, so as to obtain the traditional Chinese medicine sample extract.

[0040] The organic solvent is selected from any one or a mixture of several of methanol, ethanol, butanol, diethyl ether, ethyl acetate and chloroform.

[0041] Specifically, after the culture is ended, the hollow fiber fixed with the living cells is taken out. The two ends of the hollow fiber fixed with the living cells are tied by sterilized cotton thread, folded into U shape and put into a sample bottle with a magnetic rotor, completely immersed into the traditional Chinese medicine sample extract, simulated physiological environment, screened under the condition of 37℃ water bath and 500-700 rpm magnetic stirring, and the rotation speed of the magnetic stirring is preferably 600 rpm.

[0042] Step three, effect component desorption After the screening is ended, the effect component combined with the living cells in the cavity of the hollow fiber is desorbed and determined.

[0043] Specifically, after the screening is ended, the hollow fiber is taken out, the outer wall is wiped dry, the two end seals are cut open, and the liquid in the cavity of the hollow fiber is blown out by a sterile syringe and placed in an Ep tube. The inner wall of the hollow fiber is eluted by 2-5 times (80-200 μL) of methanol, the eluate is combined and collected, vortexed, and high-speed centrifuged to remove protein, and the supernatant is vacuum dried at 50℃, and the residue is redissolved by an appropriate amount of methanol and centrifuged, and the supernatant is subjected to LC-MS or GC-MS analysis.

[0044] Step four, preliminary confirmation of effect component Based on the determination result of step three, the cell trapping factor (cell trapping factor, CTF , see formula 1), (formula 1); Wherein, C model cell And A model cellThese represent the concentration and chromatographic peak area of ​​the effector components screened from hollow fibers immobilized with live model disease cells, respectively. C normal cell and A normal cell These represent the concentration and peak area of ​​the effect component selected from hollow fibers immobilized with normal cells, respectively.

[0045] Cell capture factors CTF Represented as C model cell and C normal cell Difference and C normal cell The ratio. Within a defined concentration range, the concentration of the captured effective components of traditional Chinese medicine is directly proportional to its peak area; therefore, peak area can also be used instead of concentration for calculations.

[0046] CTF A value > 0 indicates that the effective components of traditional Chinese medicine are specifically recognized and bound by the model diseased cells, which is used to preliminarily identify potential effective components of traditional Chinese medicine.

[0047] The technical solutions claimed in this invention will be further described below through some embodiments. However, the embodiments are for explaining the implementation of the present invention and do not exceed the scope of the subject matter of the present invention. The scope of protection of the present invention is not limited by the embodiments. Unless otherwise specified, the materials and reagents used in this invention can be obtained from commercially available products in the art.

[0048] like Figure 1 As shown in the following embodiments, the apparatus for the in vitro biomimetic screening method of the active ingredients of traditional Chinese medicine includes a constant temperature magnetic stirrer 1, a water bath 2, a hollow fiber 5, and a sample bottle 7.

[0049] The water bath 2 is placed on top of the thermostatic magnetic stirrer 1. The water bath 2 is filled with water 3. The water level must be consistent with the height of the Chinese medicine sample extract 6 in the sample bottle 7. The sample bottle 7 is placed in the water bath 2. The sample bottle 7 contains the Chinese medicine sample extract 6 and is equipped with a magnetic rotor 4. The hollow fiber 5 is sealed at both ends and folded into a U-shape and immersed in the Chinese medicine sample extract 6. Example 1

[0050] Human renal tubular epithelial (HK-2) cells were fixed onto hollow fibers: Hollow fibers were cut into 6 cm segments and autoclaved for later use. HK-2 cells in logarithmic growth phase were washed with PBS, digested with trypsin for 3 min, and digestion was terminated with DMEM / F-12 complete medium. The cells were centrifuged at 1000 rpm, the supernatant was discarded, and the cells were resuspended in DMEM / F-12 complete medium for cell counting and dilution to a density of 6 × 10⁻⁶. 5Cell suspension at 1 mL / cell density. Aspirate the cell suspension using a 1 mL syringe (without needle), attach the needle, insert it into one end of the hollow fiber, and place it in a culture flask. Slowly and evenly fill the hollow fiber lumen with the cell suspension. Add DMEM / F-12 complete culture medium to cover the hollow fiber, and then incubate the flask in a CO2 incubator for 36 h.

[0051] Immobilizing renal fibrotic cells in hollow fibrosis: Digested HK-2 cells were resuspended in DMEM / F-12 complete medium containing 10 ng / mL TGF-β (HK-2 cell fibrosis inducer) to obtain 6 × 10⁶ cells / mL. 5 Cell suspension was prepared at a density of [number] cells / mL. The cell suspension was drawn up using a 1 mL syringe without a needle, and after attaching the needle, it was inserted into one end of the hollow fiber and placed in a culture flask. The cell suspension was slowly and evenly injected into the hollow fiber lumen. DMEM / F-12 complete culture medium containing the same concentration of TGF-β inducer as inside the lumen was added to the culture flask, ensuring the hollow fiber was completely submerged. The culture flask was then incubated in a CO2 incubator for 36 h. Separately, DMEM / F-12 complete culture medium was injected into the hollow fiber lumen and cultured thereafter as a blank control group.

[0052] Preparation of the Piper longum solution for screening: Weigh 8 g of Piper longum powder into an Erlenmeyer flask, add 160 mL of 60% ethanol, soak for 1 h, and extract by ultrasonication for 30 min. After cooling the extract to room temperature, centrifuge to collect the supernatant, evaporate to dryness in a 60℃ water bath, add 0.5% DMEM / F-12 basal medium to reconstitute the residue, transfer to a 20 mL volumetric flask, dilute to the mark with DMEM / F-12 basal medium, and shake well to obtain a solution with a concentration of 0.4 mg / mL for later use.

[0053] The following method was used to screen for the anti-renal fibrosis components of Piper longum: The 0.4 mg / mL Piper longum extract was diluted with complete culture medium to a 0.1 mg / mL sample solution. 6 mL of this solution was placed in a sample vial equipped with a magnetic stirrer. The ends of the hollow fiber were sealed, folded into a U-shape, and immersed in the sample solution. The vial was placed on a magnetic stirrer and screened at 37°C and 600 rpm for 1.5 h. After screening, the fiber was removed, the outer wall was dried, the seal was cut open, and the liquid inside the hollow fiber was blown out into an Eppendorf tube using a syringe. The inner wall of the fiber was eluted with 40 μL of methanol, repeated twice. The eluents were combined, vortexed, and centrifuged at 11000 rpm for 10 min at 4°C. The supernatant was collected in a new Eppendorf tube and vacuum dried at 50°C. The residue was reconstituted with 30 μL of methanol, sonicated for 1 min, vortexed for 1 min, and centrifuged at 11000 rpm for 10 min. The supernatant was then analyzed by LC-MS. By calculating cell trapping factors, the anti-renal fibrosis effect components in Piper longum were preliminarily screened.

[0054] Chromatographic conditions: Accucore C18 column (2.6 μm, 100 mm × 2.1 mm id, ThermoFisher); column temperature: 40℃; flow rate: 0.2 mL / min; injection volume: 2 μL; autosampler temperature: 4℃; mobile phase: acetonitrile (A) and 0.1% formic acid (B); gradient elution program: 0–11.7 min, 35% A; 11.7–12.0 min, 35% A–55% A; 12.0–39.0 min, 55% A–80% A.

[0055] Mass spectrometry conditions: HESI ion source in positive and negative ion modes; spray voltages of 3.5 kV and 3.0 kV, respectively; ion transfer tube and evaporator temperatures of 320℃ and 400℃, respectively; pressures of sheath gas, auxiliary gas, and purge gas of 55, 15, and 5 Arb, respectively; mass spectrometry scan range of 150-2000 m / z; resolution of 60000.

[0056] The results are as follows Figures 2-4 As shown, in high-resolution mass spectrometry analysis, the response values ​​of each component are lower in negative ion mode, and the number of component peaks is less than in positive ion mode. Therefore, total ion chromatograms in positive ion mode are used for analysis. Figure 2 shows the total ion chromatograms and selected ion monitoring chromatograms of the reference standards of long peppermint, long peppermintine, piperine, and sesamin in positive ion mode. Figure 3 and Figure 4 Calculation of measurement results CTF Piperamide and piperic acid CTF The highest concentration among the 24 screened compounds indicates that it has the strongest affinity for model diseased cells, and its effect in delaying renal fibrosis may be superior to other active ingredients; N-(2,5-dimethoxyphenyl)-4-p-methoxybenzamide, piperine, dihydropiperine, piperine, 1-[1-carbonyl-9(3,4-methylenedioxyphenyl)-2E,8E-nonadienyl]-pyrrole, pipecallosine, pseudopiperine B, styraxine, ZINC03996196, (E,E,E)-11-(1,3-Benzodioxol-5-yl)-N-(2-methylpropyl)-2,4,10-undecatrienenamide, piperine, piperine, pipechabamide D. Pipernonaline, dehydropipernonaline, methyl piperate, guinea piperamide, pipernonaline, and pipenonaline CTF All values ​​were greater than 0, indicating that it has a strong affinity for the model diseased cells and can be used as a potential active ingredient for the treatment of renal fibrosis. Example 2

[0057] Human gastric mucosal epithelial (GES-1) cells were fixed in hollow fiber: GES-1 cells in logarithmic growth phase were washed with PBS and then digested with trypsin for 3 min. Once the cells transitioned from adherence to suspension, DMEM complete medium was immediately added to terminate the digestion. After centrifugation, the cells were resuspended in DMEM complete medium to a density of 5 × 10⁶ cells / mL. 6 Cell suspension at 1 mL / cell density. Draw up the cell suspension using a 1 mL syringe (without the needle), then attach the needle and insert it into one end of the hollow fiber. Slowly and evenly fill the hollow fiber with the cell suspension in the cell culture flask until it flows out from the other end. Add DMEM complete culture medium to cover the hollow fiber in the culture flask and incubate in a CO2 cell culture incubator for 24 h.

[0058] Immobilize model diseased cells in hollow fibers: Digested GES-1 cells were resuspended in DMEM complete medium containing 20 μM MNNG (GES-1 cell pathogenesis inducer) to obtain 5 × 10⁶ cells. 6 Cell suspension at 1 mL / cell density. Aspirate the cell suspension using a 1 mL syringe (without the needle), then insert the syringe back into the hollow fiber and slowly and evenly fill the hollow fiber with the cell suspension until it flows out from the other end. Add DMEM complete medium containing the same concentration of MNNG inducer as inside the cavity to completely submerge the hollow fiber, and incubate in a CO2 cell culture incubator for 24 h. Separately, inject DMEM complete medium into the hollow fiber cavity and incubate as a blank control group.

[0059] The present invention was used to screen the active components of Banxia Xiexin Decoction for its anti-chronic atrophic gastritis effect: The lyophilized powder of Banxia Xiexin Decoction was prepared into a sample solution with a concentration of 20 mg / mL using DMEM basal medium. 6 mL of this solution was added to a 10 mL sample bottle containing a magnetic stir bar. Hollow fibers were sealed at both ends with cotton thread, folded into a U-shape, and immersed in the sample solution. Screening was performed at 37℃ and 600 rpm for 3 h. After screening, the hollow fibers were removed, and the surface sample solution was gently wiped away with a paper towel. The sealed ends were cut along the base of the cotton thread. One end was placed in a 0.5 mL Eppendorf tube, and the other end was inserted into a 10 mL syringe to expel the liquid. 40 μL of methanol was used to elute the fiber lumen, repeated twice, and the liquids were combined in an Eppendorf tube. The tube was vortexed for 1 min, centrifuged at 4℃ and 11000 rpm for 10 min, and the supernatant was evaporated in a vacuum drying oven to remove the solvent. The residue was redissolved with 30 μL of methanol, sonicated for 1 min until completely dissolved, and then vortexed and centrifuged again. The supernatant was then analyzed by LC-MS. The analysis was performed by calculation. CTF Preliminary screening was conducted to identify the active components of Banxia Xiexin Decoction in treating chronic atrophic gastritis.

[0060] Chromatographic conditions: Waters T3 column (1.8 μm, 100 mm × 2.1 mm id); mobile phase: methanol (A) – 0.1% formic acid solution (B), gradient elution program (0–1 min, 5% A; 1–2 min, 5%–20% A; 2–3 min, 20%–40% A; 3–10 min, 40%–52% A; 10–14 min, 52%–55% A; 14–19 min, 55%–60% A; 19–22 min, 60%–70% A; 22–24 min, 70%–75% A; 24–24.5 min, 75%–5% A; 24.5–27 min, 5% A); column temperature: 40 °C; flow rate: 0.25 mL / min; injection volume: 5 μL.

[0061] Mass spectrometry conditions: Thermo Scientific Q Exactive high-resolution liquid chromatography-mass spectrometry system, HESI ion source in positive and negative ion modes; spray voltage 3500V, capillary temperature 320℃; scan mode: full scan / data-dependent two-stage scan (Full MS / DD-MS2); analyzer: electrostatic track trap, scan range 80-1200 m / z, resolution 70000.

[0062] The results are as follows Figures 5-7 As shown, in high-resolution mass spectrometry analysis, the response values ​​of each component are lower in negative ion mode, and the number of component peaks is less than in positive ion mode; therefore, all analyses are performed in positive ion mode. Figure 8 As shown, the 8 effect components CTF All values ​​are greater than 0, which preliminarily identifies palmatine, glycyrrhizin, naringenin, and phloroglucinol A-7-. O Eight components, including β-D-glucuronide, isorhamnetin, 6-gingerol, baicalin, and acaciain, can be considered potential active ingredients in Banxia Xiexin Decoction for treating chronic atrophic gastritis. Among them, palmatine... CTF The highest concentration indicates that it has the strongest affinity for the model diseased cells, and its efficacy in treating chronic atrophic gastritis may be superior to other active ingredients.

[0063] The scope of protection claimed by this invention is not limited to the specific embodiments described above. For those skilled in the art, this invention can have various modifications and alterations. Any modifications, improvements, and equivalent substitutions made within the concept and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for in vitro biomimetic screening of active ingredients of traditional Chinese medicine, characterized in that, It comprises: Step one, normal cell suspension and induced model pathogenic cell suspension are injected into the inner cavity of hollow fiber respectively, and cultured in complete medium to make cells adhere to the inner wall of hollow fiber and be fixed, thus obtaining hollow fiber with living cells fixed; Step two, the hollow fiber with living cells fixed is immersed in traditional Chinese medicine sample extract to screen effective components; Step three, after screening, the effective components combined with living cells in the inner cavity of hollow fiber are desorbed and determined; Step four, based on the determination result of step three, cell trapping factor CTF is calculated, ； wherein, C model cell and A model cell respectively represent the concentration of the effector component selected from the hollow fiber in which the model lesion cells are immobilized and the chromatographic peak area; C normal cell and A normal cell respectively represent the concentration of the effector component selected from the hollow fiber in which the normal cells are immobilized and the chromatographic peak area; CTF > 0, indicating that the effective components in traditional Chinese medicine are specifically recognized and combined by the model diseased cells, which is used for preliminary confirmation of potential traditional Chinese medicine effective components.

2. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 1, characterized in that: In step one, when fixing normal cells, the normal cells obtained by digestion are resuspended in complete medium to obtain cell suspension, and the cell suspension is taken by a sterile syringe without needle, then the needle is installed and inserted into one end of the hollow fiber, and the cell suspension is injected into the inner cavity of the hollow fiber and filled; complete medium is added to cover the hollow fiber, and the culture bottle is placed in a CO2 incubator for culture, thus obtaining hollow fiber with normal cell living cells fixed.

3. The in vitro biomimetic screening method of effective components of traditional Chinese medicine according to claim 1 or 2, characterized in that: In step one, when fixing model pathological cells, the normal cells after digestion are resuspended in complete medium containing an inducer to obtain cell suspension, and the cell suspension is taken by a sterile syringe without needle, then the needle is installed and inserted into one end of the hollow fiber, and the cell suspension is injected into the inner cavity of the hollow fiber and filled; complete medium containing an inducer is added to cover the hollow fiber, and the culture bottle is placed in a CO2 incubator for culture, thus obtaining hollow fiber with model pathogenic cell living cells fixed.

4. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 3, characterized in that: The culture is ended when the cell growth density reaches 70%-80%.

5. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 4, characterized in that: In step one, the material of the hollow fiber is polypropylene, the membrane wall pore size is 0.2 μm, the tube cavity inner diameter is 0.6 mm, and the hollow fiber is cut into small pieces with a length of 5-7 cm and sterilized for standby.

6. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 1 or 5, characterized in that: In step two, water or organic solvent or a combination of water and organic solvent is used as extraction solvent for extraction of traditional Chinese medicine, and the extract is concentrated and treated, and the residue after evaporation of the extraction solvent is dissolved in dimethyl sulfoxide and then diluted to working concentration with basic medium, thus obtaining the traditional Chinese medicine sample extract.

7. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 6, characterized in that: The organic solvent is selected from any one or a mixture of several of methanol, ethanol, butanol, diethyl ether, ethyl acetate, and chloroform.

8. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 1 or 7, characterized in that: In step two, the two ends of the hollow fiber with living cells fixed are tied with sterilized cotton thread, folded into a U shape, and placed in a sample bottle with a magnetic rotor, completely immersed in the traditional Chinese medicine sample extract, and screened under the conditions of 37℃ water bath and 500-700 rpm magnetic stirring.

9. The method for screening the active ingredients of traditional Chinese medicines in vitro according to claim 8, characterized in that: In step three, the inner wall of the hollow fiber is washed with methanol solution, the eluate is collected, the protein is removed by centrifugation, the supernatant is dried, redissolved and centrifuged, and LC-MS or GC-MS is used for analysis.

10. A device for carrying out the in vitro biomimetic screening method of the active ingredients of traditional Chinese medicine according to any one of claims 1-9, characterized in that, It comprises: Constant temperature magnetic stirrer (1), water bath (2), hollow fiber (5) and sample bottle (7); The water bath (2) is placed on the upper part of the constant temperature magnetic stirrer (1), and the water bath (2) is provided with water (3); the sample bottle (7) is placed in the water bath (2), the sample bottle (7) is provided with the traditional Chinese medicine sample extract (6) and a magnetic rotor (4); and the hollow fiber (5) is folded into a U shape after being sealed at two ends and is immersed in the traditional Chinese medicine sample extract (6).

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