Method for screening active ingredients of traditional Chinese medicine compound with effect of treating central nervous system diseases

Abstract

The invention discloses a method for screening active ingredients of a traditional Chinese medicine compound with a function of treating central nervous system diseases, which comprises the following steps of: constructing a novel magnetic bead-assisted cell membrane chromatography system by taking coptis chinensis detoxification decoction as a model prescription based on a neurovascular unit protection function, and screening active ingredients of a traditional Chinese medicine compound with a function of treating central nervous system diseases by combining a brain microdialysis in-vivo sampling technology; cerebrospinal fluid of a traditional Chinese medicine compound with the effect of treating the central nervous system diseases acts on a mixed cell membrane bionic magnetic nanoparticle composite system related to the central nervous system diseases for the first time, and an in-vitro nerve and blood vessel unit model screening system is constructed to screen active ingredients of the traditional Chinese medicine compound for treating the central nervous system. Therefore, the quality marker is determined, and the migration characteristics of components in the traditional Chinese medicine compound for treating the central nervous system diseases, such as coptis chinensis detoxification decoction, in blood and brain are also clarified.

Description

Technical field:

[0001] The invention relates to a method for screening active ingredients of a traditional Chinese medicine compound having the function of treating central nervous system diseases. Background technology:

[0002] The elucidation of active ingredients of traditional Chinese medicine is not only an important part of drug discovery, but also the basis and core of quality control of traditional Chinese medicine and traditional Chinese medicine compound. Cell membrane chromatography is a powerful tool for screening active ingredients. In recent years, it has been widely used in the study of the material basis of the efficacy of traditional Chinese medicine. A large number of research results have confirmed that cell membrane chromatography has the advantages of specificity and high throughput in elucidating the active ingredients of traditional Chinese medicine compound, but the research team also found that it still has some shortcomings during the research process. First, the screening method based on the construction of a single cell membrane is inefficient and difficult to reflect the multi-target effects of traditional Chinese medicine compound on complex diseases; secondly, traditional cell membrane chromatography often uses traditional Chinese medicine compound extracts to directly incubate with effector cell membranes to achieve specific binding of active ingredients to effector cell membrane receptors, which is easy to cause interference from non-effect components and omission of active metabolites in the body; finally, traditional cell membrane chromatography requires multiple centrifugation operations to achieve the washing of non-specific binding components and the dissociation of specific binding components, which is not only time-consuming, but also easy to cause cell membrane loss, thus affecting the accuracy of the results.

[0003] Brain diseases, especially stroke and Alzheimer's disease, are extremely complex processes, and single treatment strategies such as neuroprotection often fail to achieve the desired results. More and more studies have shown that the components of the neurovascular unit are highly involved in the occurrence and development of brain diseases. Therefore, based on the protective effect of the neurovascular unit, Chinese herbal compound with the effect of treating central nervous system diseases is screened. At present, cell membrane chromatography technology uses a single cell membrane to construct an active ingredient screening system, which not only limits the research efficiency of the active ingredients of Chinese herbal compound, but also can only obtain the active ingredients of the Chinese herbal compound that exert a certain pharmacological effect. It cannot reflect the overall picture of the pharmacological material basis of the entire compound, nor can it reflect the multi-component and multi-target characteristics of the Chinese herbal compound. Summary of the invention:

[0004] The purpose of the present invention is to provide a method for screening active ingredients of a traditional Chinese medicine compound having the effect of treating central nervous system diseases.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for screening active ingredients of a Chinese medicinal compound having the effect of treating central nervous system diseases, the method comprising the following steps:

[0007] S1. Characterization and identification of chemical components of traditional Chinese medicine compound: The main chemical components of traditional Chinese medicine compound for the treatment of central nervous system diseases were systematically identified by thin layer chromatography or ultra-high pressure liquid chromatography-mass spectrometry, and fingerprints were established;

[0008] S2. Characterize and identify the components and metabolites of the Chinese herbal compound that enter the blood and cerebrospinal fluid: insert the blood probe toward the rat's heart, and the semipermeable membrane of the probe should be completely immersed in the blood vessel. Suture the mesh on the probe to the muscle tissue on both sides to fix the probe, suture the skin, start perfusion at 1.5μL / min, and perform microdialysis sampling after 1h of equilibrium. Give the Chinese herbal compound solution for the treatment of central nervous system diseases by gavage, and continuously collect the microdialysis fluid. Give the drug once every 2h for 8h. After the obtained microdialysis samples are treated with solid phase extraction, high-resolution mass spectrometry is used to identify the composition of the dialysate; compare the total ion flow diagram of the Chinese herbal compound, blank blood microdialysis fluid, and rat blood microdialysis fluid after administration of the Chinese herbal compound for the treatment of central nervous system diseases, and screen and analyze the prototype drug components and metabolites in the blood;

[0009] A brain microdialysis probe was implanted into a specific brain region of a rat. One hour after implantation, the brain microdialysis tissue fluid before administration was collected as a blank control. Then, a suspension of a Chinese herbal compound for treating central nervous system diseases was gavaged into the rats. After 5 minutes, the suspension was continuously collected for 8 hours. The obtained cerebrospinal fluid samples were subjected to solid phase extraction and then analyzed by LC-MS / MS. The chromatograms of the Chinese herbal compound, the cerebrospinal fluid samples of the rats and the blank control samples were compared to screen and analyze the prototype drug components and metabolites in the cerebrospinal fluid.

[0010] S3. The Chinese herbal compound obtained in step S2 enters the cerebrospinal fluid and acts on the mixed cell membrane bionic magnetic nanoparticle composite system related to central nervous system diseases to construct an in vitro neurovascular unit model, and further determine the active ingredients with neurovascular unit protective effects in the Chinese herbal compound for treating central nervous system diseases, thereby determining its quality markers.

[0011] The hybrid cell membrane bionic magnetic nanoparticle composite system related to central nervous system diseases has magnetic nanoparticles as the core and a hybrid cell membrane composed of human umbilical vein endothelial cells (HUVECs), neuronal cells (HT22) and microglial cells (BV2) as the shell. The preparation method is as follows:

[0012] 1) Human umbilical vein endothelial cells (HUVECs), microglia (BV2) and neuronal cells (HT22 cells) in logarithmic growth phase and in good growth condition were collected and washed with phosphate buffered saline (PBS), suspended in 0.25× phosphate buffered saline (PBS) at 4°C for 30 minutes, and then centrifuged to remove the cell contents. The obtained precipitate was washed several times, and finally the collected cell membranes were suspended and stored in distilled water, and ultrasonically treated in an ice water bath for 2 minutes, and then mixed at a protein concentration of 1:1:1 and extruded through 1μm, 400nm and 200nm polycarbonate porous membranes for multiple times to promote membrane fusion. Finally, the mixed cell membranes (HM) were collected by centrifugation, resuspended in phosphate buffered saline (PBS) and set aside.

[0013] 2) dispersing the nanomagnetic beads in phosphate buffered saline (PBS), mixing with the hybrid cell membrane (HM) prepared in step 1), repeatedly extruding the mixed solution through a 200 nm polycarbonate porous membrane on a small extruder, and removing excess hybrid cell membrane (HM) by centrifugation, and keeping the obtained hybrid cell membrane bionic magnetic nanoparticle composite system (HM-MB) at 4° C. for use.

[0014] Preferably, in step 2), the mass ratio of the amount of nanomagnetic beads to the total amount of mixed cell membrane protein is 1:1.

[0015] The Chinese herbal compound prescriptions for treating central nervous system diseases include Huanglian Jiedu Decoction, Buyang Huanwu Decoction, Gualou Guizhi Decoction, Bushen Yizhi Decoction, Liuwei Dihuang Pills, Ditan Decoction and the like.

[0016] When the Chinese herbal compound for treating central nervous system diseases is Huanglian Jiedu Decoction, the preparation of the test sample in step S1 is as follows: prepare the Huanglian Jiedu Decoction water extract by reflux extraction according to the prescription recorded in "Wai Tai Mi Yao", concentrate under reduced pressure and then freeze-dry, shake the freeze-dried powder with 50wt% methanol-water to prepare a solution with a freeze-dried powder concentration of 10-20mg / mL, centrifuge, and take the supernatant as the test sample solution.

[0017] When the Chinese herbal compound for treating central nervous system diseases is Huanglian Jiedu Decoction, the developing solvent in step S1 using thin layer chromatography is a mixture of ethyl acetate, ammonia solution and methanol in a volume ratio of 5:1:1; the chromatographic conditions in the ultra-high pressure liquid chromatography-mass spectrometry technology in step S1 are: chromatographic column: Acquity UPLC CSH C18 column; flow rate: 0.3 mL / min; mobile phase: acetonitrile (A)-0.1% formic acid water, elution gradient: 0-20 min, 8-23% A; 20-30 min, 23-70% A; detection wavelength: 250 nm.

[0018] When the traditional Chinese medicine compound for treating central nervous system diseases is Huanglian Jiedu Decoction, the five ingredients of magnolia alkaloids, epiberberine, bamipine, coptisine and baicalin were finally confirmed to be the potential active ingredients in Huanglian Jiedu Decoction that play a protective role on the neurovascular unit.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1) The present invention uses Huanglian Jiedu Decoction as a model prescription, constructs a novel magnetic bead-assisted cell membrane chromatography system based on the protective effect of the neurovascular unit, and combines it with the in vivo brain microdialysis sampling technology. For the first time, the cerebrospinal fluid of a Chinese herbal compound with the effect of treating central nervous system diseases is used to act on a mixed cell membrane bionic magnetic nanoparticle composite system related to central nervous system diseases, and an in vitro neurovascular unit model screening system is constructed to screen the active ingredients of the Chinese herbal compound for treating the central nervous system, thereby determining its quality markers; for the first time, microdialysis sampling technology and ultra-high performance liquid chromatography combined with high-resolution mass spectrometry (UPLC-Q / TOF-MS) are applied to the analysis and research of the components, blood-entering and brain-entering components of a Chinese herbal compound with the effect of treating central nervous system diseases, such as Huanglian Jiedu Decoction, to clarify the migration characteristics of the components in the Chinese herbal compound with the effect of treating central nervous system diseases, such as Huanglian Jiedu Decoction, in the blood and brain.

[0021] 2) This screening method is accurate and comprehensive, which solves the problem that the existing technology cannot reflect the overall picture of the pharmacological material basis of the entire compound, nor can it reflect the multi-component and multi-target action characteristics of the traditional Chinese medicine compound. Description of the drawings:

[0022] Figure 1 These are the thin layer chromatograms of 15 batches (AO) of Huanglian Jiedu Decoction.

[0023] Figure 2 This is the fingerprint of Huanglian Jiedu Decoction, where A, 15 batches of Huanglian Jiedu Decoction samples; B, control map.

[0024] Figure 3 This is the total ion current diagram of blank brain microdialysis fluid and brain microdialysis fluid after administration of Huanglian Jiedu Decoction: among them, a, positive ion mode of brain microdialysis fluid after administration of Huanglian Jiedu Decoction; b, positive ion mode of blank brain microdialysis fluid; c, negative ion mode of brain microdialysis fluid after administration of Huanglian Jiedu Decoction; d, negative ion mode of blank brain microdialysis fluid.

[0025] Figure 4 It is the total ion current diagram of blank blood microdialysis fluid and blood microdialysis fluid after administration of Huanglian Jiedu Decoction, among which, a, positive ion mode of blood microdialysis fluid after administration of Huanglian Jiedu Decoction; b, positive ion mode of blank blood microdialysis fluid; c, negative ion mode of blood microdialysis fluid after administration of Huanglian Jiedu Decoction; d, negative ion mode of blank blood microdialysis fluid.

[0026] Figure 5 These are the SEM (small picture) and TEM images of blank magnetic nanoparticles and mixed cell membrane composite magnetic nanoparticles, where A, blank magnetic nanoparticles; B, mixed cell membrane composite magnetic nanoparticles.

[0027] Figure 6 These are the mixed cell membrane laser confocal microscopy observation images (×20), among which, A, HT22 cell membrane labeled with fluorescein isothiocyanate (DSPE-PEG-FITC-HT22); B, HUVECs cell membrane labeled with polyethylene glycol-rhodamine B (DSPE-PEG-RohB-HUVECs); C, BV2 cell membrane labeled with polyethylene glycol-cy5 (DSPE-PEG-cy5-BV2); D, Merge.

[0028] Figure 7 It is the total ion chromatogram of the dissociated solution under positive and negative ionization modes; among them, A, positive ion mode of the control group; B, positive ion mode of the HLJDD treatment group; C, negative ion mode of the control group; D, negative ion mode of the HLJDD treatment group. Specific implementation method:

[0029] The following is a further description of the present invention, rather than a limitation of the present invention.

[0030] Embodiment 1:

[0031] 1. Preparation and quality control of Huanglian Jiedu Decoction

[0032] The UPLC, TLC and fingerprint methods of Huanglian Jiedu Decoction were established to carry out qualitative analysis of Huanglian Jiedu Decoction.

[0033] (1) Sample preparation

[0034] According to the prescription recorded in "Wai Tai Mi Yao", the water extract of Huang Lian Jie Du Tang was prepared by reflux extraction, and then lyophilized after vacuum concentration. 15 mg of HLJDD lyophilized powder was mixed with 1 mL of 50% methanol-water, centrifuged, and the supernatant was taken as the test solution.

[0035] (2) Thin layer chromatography identification

[0036] Sample volume: 10 μL

[0037] Developing solvent: ethyl acetate, ammonia solution, methanol (5:1:1)

[0038] Unfolding distance: 8cm

[0039] Inspection: 365nm

[0040] The results of TLC of 15 batches (AO) of Huanglian Jiedu Decoction can be found in Figure 1 .

[0041] (3) Fingerprint

[0042] Chromatographic conditions: chromatographic column: Acquity UPLC CSH C18 column (2.1×100mm, 1.7μm); flow rate: 0.3mL / min; column temperature: 40℃; mobile phase: acetonitrile (A)-0.1% formic acid water, elution gradient: 0–20min, 8-23%A; 20–30min, 23–70%A; detection wavelength: 250nm; injection volume: 5μL.

[0043] See the sample and reference spectra of 15 batches of Huanglian Jiedu Decoction for details. Figure 2 .

[0044] 2. Microdialysis and Preparation of Cerebrospinal Fluid

[0045] (1) Brain probe and cerebrospinal fluid collection

[0046] SD rats were anesthetized with chloral hydrate and fixed on a brain stereotaxic apparatus to mark the location of the hypothalamus or striatum. After drilling a hole with an electric drill, a guide tube with a stylet was inserted and fixed with dental powder. The rats were fed for 24 hours, the stylet was removed, and the brain probe was inserted to complete the implantation of the brain microdialysis probe into the specific brain region of the rat. After the brain microdialysis probe was implanted and equilibrated for 1 hour, the brain microdialysis tissue fluid before administration was collected as a blank control. Rats were given a suspension of lyophilized powder of Huanglian Jiedu Decoction by oral gavage at a dose of 0.615 g / kg, and continuous collection began 5 minutes later for 8 hours. The cerebrospinal fluid samples were subjected to solid phase extraction and LC-MS / MS analysis. The results are shown in Tables 1 and Figure 3 .

[0047] Table 1 Composition of components in brain microdialysis fluid

[0048]

[0049] (2) Blood probe implantation and dialysate collection

[0050] SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the neck was shaved. The rats were fixed on the operating table in a supine position, and the neck was cut about 1.5 cm from the right side of the midline of the neck. The jugular vein was bluntly separated, and the distal end of the jugular vein was ligated. A small cut was made at the distal end, and the blood probe was inserted toward the heart. The semipermeable membrane of the probe should be completely immersed in the blood vessel. The mesh on the probe was sutured with the muscle tissue on both sides to fix the probe. The skin was sutured and perfusion was started at 1.5 μL / min. Microdialysis sampling was performed after 1 hour of equilibrium. HLJDD solution (0.0615 g / 100 g) was administered by gavage, and microdialysis fluid was continuously collected. The drug was administered once every 2 hours for 8 hours. The microdialysis samples were treated with solid phase extraction and high-resolution mass spectrometry was used to identify the components in the dialysate. The results are shown in Tables 2 and Figure 4 .

[0051] Table 2 Composition of blood microdialysis fluid

[0052]

[0053] 3. Cell Culture

[0054] Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell culture medium (ECM) containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Cells in logarithmic growth phase and in good growth state were selected for the experiment.

[0055] Microglial cells (BV2) were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase and in good growth condition were selected for the experiment.

[0056] Neuronal cells (HT22) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and high glucose in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase and in good growth condition were selected for the experiment.

[0057] 4. Preparation of Hybrid Membranes (HM)

[0058] The collected HUVECs, BV2 and HT22 cells in logarithmic growth phase and good growth status were washed with phosphate buffered saline (PBS), suspended in 0.25× phosphate buffer (phosphate buffer with a concentration of 0.0025 mol / L, a hypotonic solution diluted with distilled water) at 4°C for 30 minutes, and then centrifuged at 15000×g for 7 minutes to remove the cell contents. The obtained precipitate was washed several times with 1× phosphate buffer (phosphate buffer with normal osmotic pressure, concentration of 0.01 mol / L), and finally the collected cell membranes were suspended and stored in distilled water. The protein concentration of each cell membrane was determined using a protein quantification kit. The HUVECs, BV2 and HT22 cell membranes were ultrasonically treated in an ice water bath for 2 minutes, and then mixed at a protein concentration of 1:1:1 and extruded through 1μm, 400nm and 200nm polycarbonate porous membranes for multiple times to promote membrane fusion. Finally, the mixed cell membranes (HM) were collected by centrifugation (21000×g, 30 min, 4°C) and resuspended in PBS for later use.

[0059] 5. Preparation and characterization of hybrid cell membrane-magnetic bead composites (HM-MB)

[0060] 50 μg of nanomagnetic beads (MB, 200 nm) were dispersed in 1 mL of PBS and mixed with the hybrid cell membrane HM prepared in the above steps. The mixed solution was repeatedly extruded through a 200 nm polycarbonate porous membrane on a small extruder and centrifuged at 1000 g to remove excess HM. Finally, the obtained HM-MB was kept at 4 °C for use. The morphology of MB and HM-MB was characterized by using SEM and TEM. The results are shown in Figure 5 .

[0061] HUVECs, BV2 and HT22 cell membranes were labeled with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-fluorescein isothiocyanate (DSPE-PEG-FITC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-rhodamine B, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-rhodamine red B (DSPE-PEG-RohB), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-Cy5 (DSPE-PEG-Cy5), respectively, to prepare HM-MBs. MBs and HM-MBs were analyzed using laser confocal microscopy to observe cell membrane fusion. The results are shown in Figure 6 .

[0062] 6. Construction of HM-MB Screening System

[0063] The HM-MB suspension was centrifuged at 1000r / min for 10min, the supernatant was discarded, and the cell pellet was obtained and divided into a blank control group and a drug group. The blank group was added with PBS, and the drug group was added with cerebrospinal fluid containing Huanglian Jiedu Decoction, and they were placed in a constant temperature shock box for incubation at 37°C for 2h. An external magnetic field was applied to separate the nonspecific binding components from HM-MB. After magnetic separation, PBS was used to wash away the nonspecifically adsorbed inactive components. After repeated washing 8 times, 20wt% acetic acid solution was added, and the cells were placed in a constant temperature shock box for further incubation at 37°C. The eluate was collected after 2h. After solid phase extraction, the samples were subjected to mass spectrometry analysis using a high-resolution mass spectrometer. The accurate mass data of the primary mass spectrometer was used to qualitatively study the unknown substances in the dissociation solution. The binding components in the dissociation solution were analyzed in combination with the accurate mass data of the secondary mass spectrometer. The molecular structure of each active component was determined in combination with the existing literature and the spectral information of the standard. 35 specific binding components were successfully separated and identified, see Table 3.

[0064] Table 3 Components in Huanglian Jiedu Decoction that specifically bind to neurovascular units

[0065]

[0066] Continued from the table above

[0067]

[0068] Confirmation of active ingredients:

[0069] Combining the 11 components that enter the blood and brain and the potential active ingredients obtained by the screening system based on the neurovascular unit, 5 ingredients (magnolia alkaloids, epiberberine, palmatine, coptisine, and baicalin) were finally confirmed as potential active ingredients in Huanglian Jiedu Decoction that play a protective role in the neurovascular unit. The structures are as follows:

[0070]

Claims

1. A method for screening active ingredients of a Chinese medicinal compound having the effect of treating central nervous system diseases, characterized in that: The method comprises the following steps: S1. Characterization and identification of chemical components of traditional Chinese medicine compound: The main chemical components of traditional Chinese medicine compound for the treatment of central nervous system diseases were systematically identified by thin layer chromatography or ultra-high pressure liquid chromatography-mass spectrometry, and fingerprints were established; S2. Characterize and identify the components and metabolites of the Chinese herbal compound that enter the blood and cerebrospinal fluid: insert the blood probe toward the rat's heart, and the semipermeable membrane of the probe should be completely immersed in the blood vessel. Suture the mesh on the probe to the muscle tissue on both sides to fix the probe, suture the skin, start perfusion at 1.5μL / min, and perform microdialysis sampling after 1h of equilibrium. Give the Chinese herbal compound solution for the treatment of central nervous system diseases by gavage, and continuously collect the microdialysis fluid. Give the drug once every 2h for 8h. After the obtained microdialysis samples are treated with solid phase extraction, high-resolution mass spectrometry is used to identify the composition of the dialysate; compare the total ion flow diagram of the Chinese herbal compound, blank blood microdialysis fluid, and rat blood microdialysis fluid after administration of the Chinese herbal compound for the treatment of central nervous system diseases, and screen and analyze the prototype drug components and metabolites in the blood; A brain microdialysis probe was implanted into a specific brain region of a rat. One hour after implantation, the brain microdialysis tissue fluid before administration was collected as a blank control. Then, a suspension of a Chinese herbal compound for treating central nervous system diseases was gavaged into the rats. After 5 minutes, the suspension was continuously collected for 8 hours. The obtained cerebrospinal fluid samples were subjected to solid phase extraction and then analyzed by LC-MS / MS. The chromatograms of the Chinese herbal compound, the cerebrospinal fluid samples of the rats and the blank control samples were compared to screen and analyze the prototype drug components and metabolites in the cerebrospinal fluid. S3. The Chinese herbal compound obtained in step S2 enters the cerebrospinal fluid and acts on the mixed cell membrane bionic magnetic nanoparticle composite system related to central nervous system diseases to construct an in vitro neurovascular unit model, and further determine the active ingredients with neurovascular unit protective effects in the Chinese herbal compound for treating central nervous system diseases, thereby determining its quality markers.

2. The method according to claim 1, characterized in that The mixed cell membrane bionic magnetic nanoparticle composite system related to central nervous system diseases has magnetic nanoparticles as the core and a mixed cell membrane composed of human umbilical vein endothelial cells, neuronal cells and microglial cells as the shell.

3. The method according to claim 2, characterized in that The preparation method of the hybrid cell membrane bionic magnetic nanoparticle composite system is as follows: 1) Collect human umbilical vein endothelial cells, microglia and neuronal cells in logarithmic growth phase and in good growth state, wash them with phosphate buffer respectively, suspend them in phosphate buffer at 4°C for 30 minutes, then centrifuge to remove cell contents, wash the obtained precipitate multiple times, finally suspend and store the collected cell membranes in distilled water, and ultrasonically treat them in an ice water bath for 2 minutes, then mix them at a protein concentration of 1:1:1 and extrude them through 1 μm, 400 nm and 200 nm polycarbonate porous membranes multiple times to promote membrane fusion, finally collect the mixed cell membranes by centrifugation, resuspend them in phosphate buffer and set aside; 2) dispersing the nanomagnetic beads in phosphate buffer and mixing with the mixed cell membrane prepared in step 1), repeatedly extruding the mixed solution through a 200 nm polycarbonate porous membrane on a small extruder, and removing excess mixed cell membrane by centrifugation, and keeping the obtained mixed cell membrane bionic magnetic nanoparticle composite system at 4° C. for use.

4. The method according to claim 3, characterized in that Step 2) The mass ratio of the amount of nanomagnetic beads to the total amount of mixed cell membrane protein is 1:

1.

5. The method according to claim 1, characterized in that The Chinese medicinal compound for treating central nervous system diseases includes Huanglian Jiedu Decoction, Buyang Huanwu Decoction, Gualou Guizhi Decoction, Bushen Yizhi Decoction, Liuwei Dihuang Pills and Ditan Decoction.

6. The method according to claim 5, characterized in that When the Chinese herbal compound for treating central nervous system diseases is Huanglian Jiedu Decoction, the preparation of the test sample in step S1 is as follows: prepare the Huanglian Jiedu Decoction water extract by reflux extraction according to the prescription recorded in "Wai Tai Mi Yao", concentrate under reduced pressure and then freeze-dry, shake the freeze-dried powder with 50wt% methanol-water to prepare a solution with a freeze-dried powder concentration of 10-20mg / mL, centrifuge, and take the supernatant as the test sample solution.

7. The method according to claim 5, characterized in that When the Chinese herbal compound for treating central nervous system diseases is Huanglian Jiedu Decoction, the developing solvent in step S1 using thin layer chromatography is a mixture of ethyl acetate, ammonia solution and methanol in a volume ratio of 5:1:1; the chromatographic conditions in step S1 ultra-high pressure liquid chromatography-mass spectrometry technology are: chromatographic column: Acquity UPLC CSHC18column; flow rate: 0.3 mL / min; mobile phase: acetonitrile-0.1% formic acid water, elution gradient: 0-20 min, 8-23% A; 20-30 min, 23-70% A; detection wavelength: 250 nm.

8. The method according to claim 5, characterized in that When the traditional Chinese medicine compound for treating central nervous system diseases is Huanglian Jiedu Decoction, the five ingredients of magnolia alkaloids, epiberberine, bamipine, coptisine and baicalin were finally confirmed to be the potential active ingredients in Huanglian Jiedu Decoction that play a protective role on the neurovascular unit.

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