Biofilms for screening and evaluating V2R inhibitors, their preparation and applications

Abstract

This invention discloses a biomembrane for screening and evaluating V2R inhibitors, its preparation method, and its application. The preparation method includes: amylating and carboxylating a glass fiber membrane to obtain a carboxylated glass fiber membrane; and reacting the carboxylated glass fiber membrane with O... 6 -Benzylguanine reacts to produce O 6 -Benzylguanine-modified glass fiber membrane; and, making the O 6 A novel biomembrane for screening and evaluating V2R inhibitors was prepared by incubating a benzylguanine-modified glass fiber membrane with a cell membrane. This invention provides a novel biomembrane for screening and evaluating V2R inhibitors, and this biomembrane can be used for targeted screening and evaluation of potentially V2R-antagonistic bioactive compounds in natural products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomembrane for screening and evaluating V2R inhibitors, its preparation method, and its application. Background Technology

[0002] Arginine vasopressin V2 receptor (V2R) antagonists, with their pharmacological effects in maintaining the body's water and electrolyte balance, are now used to treat hyponatremia, cirrhosis, and arginine vasopressin secretion disorder. However, tolvaptan and conivatan require monitoring during use and are therefore limited to hospital use. Furthermore, V2R antagonists may be associated with serious adverse reactions such as liver failure and hemorrhagic shock.

[0003] Traditional V2R inhibitor screening methods are mainly at the cellular level, while direct protein screening methods are limited. Existing screening methods are time-consuming and cannot achieve rapid and accurate screening. Finding suitable membrane protein stabilization methods and establishing rapid and accurate screening methods while maintaining the correct conformation and good activity of V2Rs is a research challenge.

[0004] Natural products play a crucial role in drug discovery, serving as the source and foundation for most early-stage drugs. However, due to the chemical complexity of traditional Chinese medicine and other natural products, efficient screening of active ingredients is currently key to the research and development of highly effective lead drugs. Rapid screening and identification of bioactive components from natural products remains a technical challenge. Traditionally, screening methods for bioactive compounds are time-consuming and inefficient. Affinity-based screening methods offer a convenient and efficient way to separate potential ligands from complex mixtures. By selectively enriching drug active ingredients with different affinities to the target, highly selective and high-throughput screening analysis of drug ligands can be achieved. Summary of the Invention

[0005] The main objective of this invention is to provide a biofilm for screening and evaluating V2R inhibitors, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0007] This invention provides a method for preparing a biofilm for screening and evaluating V2R inhibitors, comprising:

[0008] Amination and carboxylation treatments were performed on the glass fiber membrane to obtain a carboxylated glass fiber membrane;

[0009] The carboxylated glass fiber membrane is then mixed with O 6 -Benzylguanine reacts to produce O 6 -Benzylguanine-modified glass fiber membrane;

[0010] And, make the O 6 - A benzylguanine-modified glass fiber membrane was mixed with a cell membrane and incubated to prepare a biomembrane for screening and evaluating V2R inhibitors.

[0011] In some more specific implementations, the cell membrane is derived from HEK293 cells that highly express SNAP-tag-V2R.

[0012] The present invention also provides a biomembrane prepared by the aforementioned method for screening and evaluating V2R inhibitors.

[0013] This invention also provides the use of the aforementioned biomembrane for screening and evaluating V2R inhibitors in the targeted screening and / or evaluation of V2R antagonistic bioactive compounds in natural products.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses GMF as a carrier and modifies and connects O 6 SNAP-V2R@GMF-BG biomembrane was prepared by fusing a SNAP-tag as a fixed label to a cell membrane that highly expresses V2R. Cys145, located at the active site of the SNAP-tag, reacts with the benzyl group of BG. This biomembrane can be used for targeted screening and evaluation of potential V2R antagonistic bioactive compounds in natural products. Specifically, the SNAP-V2R@GMF-BG biomembrane can screen for rosmarinic acid and baicalin in the natural product *Tea sibiricum*, and the inhibition constant Ki and pharmacological activity of the compounds were verified. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the preparation of the biofilm in Example 1 of the present invention;

[0017] Figures 2a-2b These are SEM images of GMF and biofilm in Embodiment 1 of the present invention;

[0018] Figure 3 These are the infrared spectra of GMF and GMF-BG in Embodiment 1 of the present invention;

[0019] Figures 4a-4bThis is a selective adsorption diagram of the biofilm in Example 2 of the present invention;

[0020] Figure 5 This is a graph showing the adsorption rate of compounds with different affinities for the biofilm in Example 3 of this invention. Detailed Implementation

[0021] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention. The technical solution of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0022] Specifically, as one aspect of the technical solution of this invention, a method for preparing a biofilm for screening and evaluating V2R inhibitors includes:

[0023] Amination and carboxylation treatments were performed on a glass fiber membrane (denoted as GMF) to obtain a carboxylated glass fiber membrane;

[0024] The carboxylated glass fiber membrane is then mixed with O 6 -Benzylguanine (denoted as BG) reacts to produce O 6 -Benzylguanine-modified glass fiber membrane;

[0025] And, make the O 6 - A benzylguanine-modified glass fiber membrane was mixed with a cell membrane and incubated to prepare a biofilm sheet for screening and evaluating V2R inhibitors (denoted as: SNAP-V2R@GMF-BG).

[0026] Specifically, in this invention, GMF is used as a carrier and modified to connect O. 6 -Benzylguanine (BG) molecules were fused to cell membranes that highly expressed V2R using a SNAP-tag as a fixed label. Cys145, located at the active site of the SNAP-tag, reacted with the benzyl group of BG to prepare a biofilm SNAP-V2R@GMF-BG.

[0027] The glass fiber membrane in this invention can be replaced with a glass fiber membrane analogue.

[0028] In some preferred embodiments, the cell membrane is derived from HEK293 cells that highly express SNAP-tag-V2R.

[0029] In some preferred embodiments, the preparation method specifically includes: shaking a first mixed reaction system containing glass fiber membrane, dopamine, Tris-HCl and polyethyleneimine at room temperature for 10-12 hours to obtain an aminated glass fiber membrane.

[0030] In some preferred embodiments, the preparation method specifically includes: shaking a second mixed reaction system containing the aminated glass fiber membrane, succinic anhydride, triethylamine and dichloromethane at room temperature for 48-50 hours to obtain a carboxylated glass fiber membrane.

[0031] In some preferred embodiments, the preparation method specifically includes: preparing a glass fiber membrane containing the carboxylated glass fiber membrane, 1-hydroxybenzotriazole, triethylamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and O 6 The third mixed reaction system of benzylguanine and dichloromethane was reacted at room temperature for 20-24 h to prepare O. 6 -Benzylguanine-modified glass fiber membrane.

[0032] In some preferred embodiments, the preparation method specifically includes: making the O 6 - Benzylguanine-modified glass fiber membranes were mixed with cell membranes and incubated at 4°C for 15-30 min to obtain the biomembrane used for screening and evaluating V2R inhibitors.

[0033] In some more specific embodiments, the method for preparing the biofilm for screening and evaluating V2R inhibitors includes:

[0034] 1. Washing of GMF (Glass Fiber Membrane)

[0035] The GMF membrane was cut into 6mm*6mm squares, washed with 1mol / L NaOH solution for 2h, then washed with 2mol / L HCl solution for 2h, and finally rinsed in 95% ethanol for 10min. It was then transferred to an oven at 45℃ for 2h.

[0036] 2. Amination of glass fiber membrane (GMF-NH2)

[0037] Take 10 cleaned GMF membranes and place them in 5 mL of 2 mg / mL dopamine Tris-HCl (10 mM pH = 8.5) solution. Add 10 μL of polyethyleneimine and shake at 100 r / min for 10-12 h at room temperature.

[0038] 3. Carboxylation of glass fiber membranes (GMF-COOH)

[0039] Add 10 tablets of GMF-NH2 to 20 mL of dichloromethane, add 1.0 g of succinic anhydride and 1 mL of triethylamine, shake at 100 r / min for 48-50 h at room temperature, and elute repeatedly in dichloromethane to obtain GMF-COOH.

[0040] 4. Glass fiber membrane BG (GMF-BG)

[0041] Ten GMF-COOH tablets were added to 20 mL of dichloromethane, and 75 mg of 1-hydroxybenzotriazole (HOBT) was added to 4 mL of triethylamine. The mixture was stirred in an ice bath for 20-30 min. 284 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) was added, and the reaction was allowed to proceed for 10-12 h. Then, 10 mg of BG was added, and the reaction was allowed to proceed at room temperature for 20-24 h. After repeated washing with dichloromethane, GMF-BG was obtained.

[0042] 5. Extraction of cell membranes containing target sites

[0043] Cells were scraped off and washed with PBS (pH=7.4), centrifuged at 1000×g and 4℃ for 10 min, and the washing process was repeated three times. Cells were resuspended in Tris-HCl (50 mM, pH=7.4), homogenized 50 times using a homogenizer at speed 6, and sonicated for 12 cycles. After disruption, cells were centrifuged at 1000×g and 4℃ for 10 min. The supernatant was collected, centrifuged at 140000×g and 4℃ for 30 min, the supernatant was discarded, and the cell membranes were resuspended in 1 mL PBS.

[0044] 6. Construction of SNAP-V2R@GMF-BG biofilm

[0045] 1 mL of cell membrane solution was incubated with the GMF-BG membrane in an EP tube for 30 min at 4 °C to obtain SNAP-V2R@GMF-BG. The membrane was then washed three times with PBS before use.

[0046] Another aspect of the present invention provides a biomembrane prepared by the aforementioned method for screening and evaluating V2R inhibitors.

[0047] Another aspect of the present invention provides the use of the aforementioned biomembrane for screening and evaluating V2R inhibitors in targeted screening and / or evaluation of V2R inhibitors in natural products.

[0048] Furthermore, the SNAP-V2R@GMF-BG can be used to evaluate the inhibitory activity of V2R ligands.

[0049] In some preferred embodiments, the biofilm is used for targeted screening and / or evaluation of rosmarinic acid and baicalin in kidney tea.

[0050] Furthermore, the inhibition constant K of the baicalein i The value is 150–200 μM; and / or, the inhibition constant K of the rosmarinic acid is... i The value is 50–100 μM.

[0051] This invention utilizes the screening of the natural product *Tea sibiricum* to identify two V2R inhibitors, rosmarinic acid and baicalein, and verifies their inhibition constant Ki and pharmacological activity. Specifically, SNAP-V2R@GMF-BG biofilm was used to screen *Tea sibiricum* extract to obtain baicalein and rosmarinic acid, and the inhibition constant Ki was calculated. i The values ​​were 150–200 μM for baicalein and 50–100 μM for rosmarinic acid, demonstrating that baicalein and rosmarinic acid have the potential to become V2R antagonists.

[0052] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0053] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0054] Example 1

[0055] This embodiment constructs a SNAP-tagged V2RHEK293 cell membrane and O based on the GMF vector. 6 The specific method for preparing SNAP-V2R@GMF-BG, a carrier specifically covalently bound to a benzylguanine derivative BG, is as follows (see schematic diagram). Figure 1 As shown):

[0056] 1. Washing of GMF (Glass Fiber Membrane)

[0057] The GMF membrane was cut into 6mm*6mm squares, washed with 1mol / L NaOH solution for 2h, then washed with 2mol / L HCl solution for 2h, and finally rinsed in 95% ethanol for 10min. It was then transferred to an oven at 45℃ for 2h.

[0058] 2. Amination of glass fiber membrane (GMF-NH2)

[0059] Ten cleaned GMF membranes were placed in 5 mL of 2 mg / mL dopamine Tris-HCl (10 mM pH = 8.5) solution, and 10 μL of polyethyleneimine was added. The mixture was then shaken at 100 r / min for 11 h at room temperature.

[0060] 3. Carboxylation of glass fiber membranes (GMF-COOH)

[0061] Ten GMF-NH2 tablets were added to 20 mL of dichloromethane, along with 1.0 g of succinic anhydride and 1 mL of triethylamine. The mixture was shaken at 100 r / min for 49 h at room temperature. After repeated elution in dichloromethane, GMF-COOH was obtained.

[0062] 4. Glass fiber membrane BG (GMF-BG)

[0063] Ten GMF-COOH tablets were added to 20 mL of dichloromethane, and 75 mg of 1-hydroxybenzotriazole (HOBT) was added to 4 mL of triethylamine. The mixture was stirred in an ice bath for 20-30 min. 284 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) was added, and the reaction was allowed to proceed for 10-12 h. Then, 10 mg of BG was added, and the reaction was allowed to proceed at room temperature for 23 h. After repeated washing with dichloromethane, GMF-BG was obtained.

[0064] 5. Extraction of cell membranes containing target sites

[0065] Cells were scraped off and washed with PBS (pH=7.4), centrifuged at 1000×g and 4℃ for 10 min, and the washing process was repeated three times. Cells were resuspended in Tris-HCl (50 mM, pH=7.4), homogenized 50 times using a homogenizer at speed 6, and sonicated for 12 cycles. After disruption, cells were centrifuged at 1000×g and 4℃ for 10 min. The supernatant was collected, centrifuged at 140000×g and 4℃ for 30 min, the supernatant was discarded, and the cell membranes were resuspended in 1 mL PBS.

[0066] 6. Construction of SNAP-V2R@GMF-BG biofilm

[0067] 1 mL of cell membrane solution and a GMF-BG membrane were incubated together in an EP tube for 30 min at 4 °C to obtain SNAP-V2R@GMF-BG. The membrane was then washed three times with PBS before use.

[0068] Material characterization methods:

[0069] The morphology and structure of the material were characterized using SEM; the chemical composition was identified using XPS; and the structure was further characterized using zeta potential analysis at 400-4000 cm⁻¹. -1 Within the range, record the infrared absorption peaks of the material.

[0070] SEM Visualization Analysis

[0071] As shown in the SEM image, the surface of the GMF membrane is composed of fibers with a diameter of approximately 1 μm, and its surface is smooth. Figure 2a As for the SEM image of SNAP-V2R@GMF-BG ( Figure 2bV2R membrane proteins were observed in the spaces between interlaced fibers due to the functional sites of BG on the fiber surface.

[0072] FT-IR spectral characterization analysis

[0073] To further confirm the successful modification of BG on the GMF film surface, Fourier transform infrared (FT-IR) spectroscopy was used for comparison and analysis. Figure 3 In the FT-IR plot of GMF, 1065.99 cm -1 The strong adsorption band at 3448 cm⁻¹ is a Si-O-Si bond, a characteristic peak of the GMF film; -1 The peak at 1566 cm⁻¹ represents -OH, originating from water and hydroxyl groups in the GMF membrane. The FT-IR spectrum of GMF-BG shows a peak at 1566 cm⁻¹. -1 An absorption peak for the benzene ring C=C was found at 1486 cm⁻¹. -1 A high-intensity NH stretching absorption peak and a carbon and nitrogen absorption peak are present at 1651 cm⁻¹. -1 There is a new amide C=O tensile vibration band nearby, at 3352 cm. -1 The presence of a new NH2 stretching vibration nearby indicates successful BG bonding.

[0074] Example 2

[0075] The adsorption performance of the material (SNAP-V2R@GMF-BG prepared in Example 1) was investigated using tolvaptan, a representative compound, as the detection index. In addition, the adsorption specificity of the material was evaluated by selecting V2R antagonists conivatan and lishivaptan as representative positive drugs and pioglitazone and A1R antagonist T62 as negative drugs.

[0076] The fixed material size was 6mm*6mm. The sample volume was 0.6mL of methanol-PBS (v / v, 5 / 95) standard solution containing 4.5μg / mL tolvaptan and three other representative compounds. The mixture was incubated at 4℃ for 30min. After incubation, SNAP-V2R@GMF-BG was dissociated using 0.6mL methanol-PBS (v / v, 1 / 1) as the eluent. The eluent was filtered through a 0.22μm microporous membrane and then analyzed by HPLC. The peak areas of the five representative compounds were recorded, and their concentrations and adsorption rates were calculated using the standard curve formula. Three sets of experiments were performed in parallel. The adsorption effect is as follows: Figure 4a and Figure 4b As shown in the figure, the SNAP-V2R@GMF-BG material exhibits the best adsorption effect on V2R antagonists, which is significantly better than the adsorption effect on other receptor antagonists. This indicates that the SNAP-V2R@GMF-BG material has adsorption specificity, and this result is consistent with the results of this study.

[0077] Example 3

[0078] Different K i Adsorption rate ranking of compounds

[0079] A series of compounds were obtained by structural modification of V2R antagonists, and their affinity was ranked by V2R ligand binding experiments to obtain the corresponding K. i Values. (Table 1)

[0080] Table 1: K-type modified V2R antagonists i value

[0081]

[0082] To investigate the effects of SNAP-V2R@GMF-BG on different K... i To assess the adsorption capacity of V2R antagonists, five V2R antagonists were prepared into standard solutions using the same method as tolvaptan. These solutions were then dissociated using the same method. The eluent was filtered through a 0.22 μm microporous membrane and analyzed by HPLC. The peak areas of six representative compounds were recorded, and their concentrations and adsorption rates were calculated using the standard curve formula. The adsorption effect graph shows that the SNAP-V2R@GMF-BG material exhibits a certain adsorption effect on all V2R antagonists. With the increase of K... i With increasing concentration, the adsorption capacity of the SNAP-V2R@GMF-BG material decreases linearly. This indicates that the SNAP-V2R@GMF-BG material has different adsorption capacities for V2R antagonists with different affinities, and this result also proves that the synthesized material is consistent with the expected results. Figure 5 Therefore, this is defined as a quantitative standard for V2R affinity, used as a preliminary quantitative standard for screening the binding affinity of unknown compounds to V2R.

[0083] Example 4: Application in the screening of V2R antagonists in natural drugs

[0084] The SNAP-V2R@GMF-BG biofilm was 6mm x 6mm. 0.6mL of a 500-fold diluted methanol-PBS (v / v, 5 / 95) standard solution of kidney tea extract was added. After processing using the same method, the sample was analyzed by LC-MS. The SNAP-V2R@GMF-BG biofilm yielded two components, baicalin and rosmarinic acid, from the kidney tea extract; the formulas are shown below:

[0085]

[0086] To investigate whether baicalein and rosmarinic acid have agonist or antagonist effects on V2R, a cAMP experiment was conducted. The rise or fall of cAMP levels was used to determine whether the ligand was an agonist or antagonist. The results showed that both baicalein and rosmarinic acid had antagonist effects on V2R.

[0087] Substituting it into the V2R affinity quantitative standard curve yields K i The values ​​were 177.83 μM for baicalein and 62.82 μM for rosmarinic acid. The results obtained were consistent with the K values ​​obtained from the V2R ligand affinity experiment. i The values ​​are within an order of magnitude, demonstrating that baicalein and rosmarinic acid have the potential to become V2R antagonists.

[0088] Conclusion: This invention is the first to utilize a novel cell membrane-coated GMF biofilm and apply it to the targeted screening and evaluation of potential V2R antagonistic bioactive compounds in natural products. Using this membrane to screen the natural product *Tea styrax*, two inhibitory components were identified, and the inhibition constant K of the compounds was verified. i The study also examined the pharmacological activity of the biofilm, indicating that the SNAP-V2R@GMF-BG biofilm has broad market application prospects.

[0089] Example 5

[0090] This embodiment constructs a SNAP-tagged V2R HEK293 cell membrane and O based on the GMF vector. 6 The specific method for preparing SNAP-V2R@GMF-BG, a carrier specifically covalently bound to a benzylguanine derivative BG, is as follows (see schematic diagram). Figure 1 As shown):

[0091] 1. Washing of GMF (Glass Fiber Membrane)

[0092] The GMF membrane was cut into 6mm*6mm squares, washed with 1mol / L NaOH solution for 2h, then washed with 2mol / L HCl solution for 2h, and finally rinsed in 95% ethanol for 10min. It was then transferred to an oven at 45℃ for 2h.

[0093] 2. Amination of glass fiber membrane (GMF-NH2)

[0094] Take 10 cleaned GMF membranes and place them in 5 mL of 2 mg / mL dopamine Tris-HCl (10 mM pH = 8.5) solution, add 10 μL of polyethyleneimine, and shake at 100 r / min for 10 h at room temperature;

[0095] 3. Carboxylation of glass fiber membranes (GMF-COOH)

[0096] Ten GMF-NH2 tablets were added to 20 mL of dichloromethane, along with 1.0 g of succinic anhydride and 1 mL of triethylamine. The mixture was shaken at 100 r / min for 48 h at room temperature. After repeated elution in dichloromethane, GMF-COOH was obtained.

[0097] 4. Glass fiber membrane BG (GMF-BG)

[0098] Ten GMF-COOH tablets were added to 20 mL of dichloromethane, and 75 mg of 1-hydroxybenzotriazole (HOBT) was added to 4 mL of triethylamine. The mixture was stirred in an ice bath for 20-30 min. 284 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) was added, and the reaction was allowed to proceed for 10-12 h. Then, 10 mg of BG was added, and the reaction was allowed to proceed at room temperature for 20 h. After repeated washing with dichloromethane, GMF-BG was obtained.

[0099] 5. Extraction of cell membranes containing target sites

[0100] Cells were scraped off and washed with PBS (pH=7.4), centrifuged at 1000×g and 4℃ for 10 min, and the washing process was repeated three times. Cells were resuspended in Tris-HCl (50 mM, pH=7.4), homogenized 50 times using a homogenizer at speed 6, and sonicated for 12 cycles. After disruption, cells were centrifuged at 1000×g and 4℃ for 10 min. The supernatant was collected, centrifuged at 140000×g and 4℃ for 30 min, the supernatant was discarded, and the cell membranes were resuspended in 1 mL PBS.

[0101] 6. Construction of SNAP-V2R@GMF-BG biofilm

[0102] 1 mL of cell membrane solution was incubated with the GMF-BG membrane in an EP tube for 15 min at 4 °C to obtain SNAP-V2R@GMF-BG. The membrane was then washed three times with PBS before use.

[0103] Example 6

[0104] This embodiment uses a GMF vector to construct a SNAP-tagged V2RHEK293 cell membrane and O 6 The specific method for preparing SNAP-V2R@GMF-BG, a carrier specifically covalently bound to a benzylguanine derivative BG, is as follows (see schematic diagram). Figure 1 As shown):

[0105] 1. Washing of GMF (Glass Fiber Membrane)

[0106] The GMF membrane was cut into 6mm*6mm squares, washed with 1mol / L NaOH solution for 2h, then washed with 2mol / L HCl solution for 2h, and finally rinsed in 95% ethanol for 10min. It was then transferred to an oven at 45℃ for 2h.

[0107] 2. Amination of glass fiber membrane (GMF-NH2)

[0108] Take 10 cleaned GMF membranes and place them in 5 mL of 2 mg / mL dopamine Tris-HCl (10 mM pH = 8.5) solution. Add 10 μL of polyethyleneimine and shake at 100 r / min for 12 h at room temperature.

[0109] 3. Carboxylation of glass fiber membranes (GMF-COOH)

[0110] Ten GMF-NH2 tablets were added to 20 mL of dichloromethane, along with 1.0 g of succinic anhydride and 1 mL of triethylamine. The mixture was shaken at 100 r / min for 50 h at room temperature. After repeated elution in dichloromethane, GMF-COOH was obtained.

[0111] 4. Glass fiber membrane BG (GMF-BG)

[0112] Ten GMF-COOH tablets were added to 20 mL of dichloromethane, and 75 mg of 1-hydroxybenzotriazole (HOBT) was added to 4 mL of triethylamine. The mixture was stirred in an ice bath for 20-30 min. 284 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) was added, and the reaction was allowed to proceed for 12 h. Then, 10 mg of BG was added, and the reaction was allowed to proceed at room temperature for 20-24 h. After repeated washing with dichloromethane, GMF-BG was obtained.

[0113] 5. Extraction of cell membranes containing target sites

[0114] Cells were scraped off and washed with PBS (pH=7.4), centrifuged at 1000×g and 4℃ for 10 min, and the washing process was repeated three times. Cells were resuspended in Tris-HCl (50 mM, pH=7.4), homogenized 50 times using a homogenizer at speed 6, and sonicated for 12 cycles. After disruption, cells were centrifuged at 1000×g and 4℃ for 10 min. The supernatant was collected, centrifuged at 140000×g and 4℃ for 30 min, the supernatant was discarded, and the cell membranes were resuspended in 1 mL PBS.

[0115] 6. Construction of SNAP-V2R@GMF-BG biofilm

[0116] 1 mL of cell membrane solution was incubated with the GMF-BG membrane in an EP tube for 20 min at 4 °C to obtain SNAP-V2R@GMF-BG. The membrane was then washed three times with PBS before use.

[0117] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0118] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A method for preparing a biomembrane for targeted screening and / or evaluation of rosmarinic acid and baicalin in kidney tea, characterized in that, include: The first mixed reaction system containing glass fiber membrane, dopamine, Tris-HCl and polyethyleneimine was shaken and reacted at room temperature for 10-12 h to obtain an aminated glass fiber membrane. The second mixed reaction system, comprising the aminated glass fiber membrane, succinic anhydride, triethylamine, and dichloromethane, was subjected to a shaking reaction at room temperature for 48-50 hours to obtain a carboxylated glass fiber membrane. The carboxylated glass fiber membrane is then mixed with O 6 -Benzylguanine reacts to produce O 6 -Benzylguanine-modified glass fiber membrane; And, make the O 6 - A benzylguanine-modified glass fiber membrane was mixed with a cell membrane and incubated to prepare a biomembrane for targeted screening and / or evaluation of rosmarinic acid and baicalin in kidney tea; wherein the cell membrane was derived from HEK293 cells that highly expressed SNAP-tag-V2R.

2. The preparation method according to claim 1, characterized in that, Specifically, it includes: The mixture comprises the carboxylated glass fiber membrane, 1-hydroxybenzotriazole, triethylamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and O 6 The third mixed reaction system of benzylguanine and dichloromethane was reacted at room temperature for 20-24 h to prepare O. 6 -Benzylguanine-modified glass fiber membrane.

3. The preparation method according to claim 1, characterized in that, Specifically, it includes: Make the O 6 - A benzylguanine-modified glass fiber membrane was mixed with a cell membrane and incubated at 4°C for 15-30 min to obtain the biomembrane used for targeted screening and / or evaluation of rosmarinic acid and baicalin in kidney tea.

4. The use of the biofilm prepared by the method of any one of claims 1-3 for targeted screening and / or evaluation of rosmarinic acid and baicalin in kidney tea in targeted screening and / or evaluation of rosmarinic acid and baicalin in kidney tea.

5. The use according to claim 4, characterized in that: The inhibition constant K of baicalin i The value is 150~200 μM; and / or, the inhibition constant K of the rosmarinic acid is... i The value is 50~100μM.

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