A method for rapid high-throughput screening of natural products with transmembrane transport activity

Abstract

The present invention relates to the field of ion channel screening, and discloses a method for rapid high-throughput screening of natural products with transmembrane transport activity, comprising the following steps: (1) taking a chloroform solution of a phospholipid bilayer, vacuum drying it, adding a buffer solution containing a fluorescent probe, cooling and warming it up multiple times, extruding it through a membrane sheet to obtain a phospholipid monolayer vesicle suspension, and passing it through a dextran gel column to obtain a vesicle stock solution; (2) inoculating the vesicle stock solution coated with the fluorescent probe into a black-walled opaque microplate, adding a buffer solution containing corresponding cations or anions into the microplate, adding a compound to be tested into the black-walled well plate, and then testing the change in the relative fluorescence intensity of the fluorescent probe. When the relative fluorescence intensity is gradually quenched or increased, the compound to be tested has transmembrane transport activity. The method of the present invention can rapidly and high-throughput screen natural products with transmembrane transport activity based on artificial vesicles coated with fluorescent probes.

Description

Technical Field

[0001] The invention relates to the field of ion channel compound screening, in particular to a method for rapid high-throughput screening of natural products with transmembrane transport activity. Background Art

[0002] Ion channels are a class of proteins that control the exchange of substances inside and outside the cell. These proteins are essential for intercellular signal transduction and play a key role in the cell cycle stage and other aspects of cell physiology. Defects in channel function are closely related to the occurrence and development of many diseases, such as diabetes, neuropathic pain, cardiovascular disease, brain and peripheral vascular disease, asthma, neurodegenerative diseases, etc. Functional regulation of ion transmembrane channels is the basis for the treatment of many diseases and an important target for the development of new and efficient drug treatments. However, the structure of natural channel proteins is extremely complex, and it is extremely difficult to modify and regulate their structure. Therefore, the development of ion transporters with transmembrane transport activity, such as channels or carriers, to replace the original functionally defective natural channel proteins is crucial for the treatment of diseases caused by channel defects. In addition, transmembrane ion transporters can quickly destroy the ion balance inside and outside the membrane, so they are also widely used in antibacterial, anti-cancer and anti-inflammatory fields.

[0003] Natural products have a wide range of sources and rich structural diversity, providing a large number of sources for the discovery of drugs and lead compounds. Small molecule compounds with transmembrane transport activity isolated from natural products, such as Prodigiosin, Amphotericin B, Gramicidin A, Valinomycin isolated from secondary metabolites of bacteria, and antifungal polyene amphidinol 3 extracted from marine dinoflagellates, have been proven to have multiple biological activities such as anticancer, antibacterial, antimalarial, antifungal, and immunosuppressive effects. However, due to the large number of compounds in the natural product library, high-throughput screening technology is urgently needed to quickly realize the development and application of natural products in the field of ion transmembrane transport and discover more natural products with transmembrane transport activity.

[0004] At present, the commonly used patch clamp technique for screening compounds with transmembrane transport activity is to record the ion current through the ion channel to reflect the single or multiple ion channel molecular activities of the cell membrane. However, the current electrophysiological technology cannot realize the work of screening a large number of compounds in a short time. In addition, there are disadvantages such as expensive machines, cumbersome and difficult operation, and high technical requirements for operators, which are not suitable for screening a large number of compounds in the early and mid-term drug development. Patent CN102884427A discloses a porous plate with a specific contour design that can perform multi-stage high-throughput parallel determination of ion channels or ion transporters, but the porous patch clamp device involved is complex and delicate, and the steps are cumbersome and time-consuming, which limits its wide application. Patent CN112176020A discloses a screening method for calcium-activated chloride channel activators, involving the construction and transfection of a relatively complex eukaryotic expression vector, and screening calcium-activated chloride channel activators by the relative fluorescence intensity of fluorescent protein. The method involves living cell culture and protein expression, and has high technical and equipment requirements, and large cost and time investment. Summary of the invention

[0005] Therefore, it is necessary to provide a method for rapid high-throughput screening of natural products with transmembrane transport activity to solve the problems of the existing screening technology, which is cumbersome, time-consuming, and requires sophisticated equipment.

[0006] To achieve the above object, the present invention provides a method for rapid high-throughput screening of natural products with transmembrane transport activity, comprising the following steps:

[0007] (1) taking a chloroform solution of a phospholipid bilayer, vacuum drying it, adding a buffer solution containing a fluorescent probe to obtain a reaction suspension, cooling the reaction suspension several times, and then extruding it through a filter membrane to obtain a phospholipid monolayer vesicle suspension, and passing the phospholipid monolayer vesicle suspension through a dextran gel column to remove uncoated fluorescent probes to obtain a vesicle stock solution;

[0008] (2) The vesicle stock solution coated with the fluorescent probe is inoculated into a black-walled opaque microplate, and a buffer solution containing corresponding cations or anions is added into the microwells so that there is a pH or ion concentration gradient inside and outside the phospholipid monolayer vesicle membrane. The test compounds are added into the black-walled well plate respectively, and then the relative fluorescence intensity change of the fluorescent probe is tested. When the relative fluorescence intensity is gradually quenched or increased, the test compound has transmembrane transport activity.

[0009] Microplates can be 96-well or 384-well cell culture plates.

[0010] Furthermore, in step (1), the concentration of the phospholipid bilayer chloroform solution is 15-30 mg / mL, the concentration of the nutrient probe in the buffer solution containing the fluorescent probe is 0.5-3.5 mmol / L, and the pore size of the filter membrane is 100-300 nm.

[0011] Furthermore, in step (1), the dextran gel column is a Sephadex G-50 column.

[0012] Furthermore, in step (1), the phospholipid bilayer is one of egg yolk L-α-phosphatidylcholine, palmitoyloleylphosphatidylcholine, soybean lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoylphosphatidylglycerol, and dioleoylphosphatidylcholine.

[0013] Furthermore, in step (1), the fluorescent probe includes but is not limited to 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, 5-carboxyfluorescein, 6-methoxy-N-(3-sulfopropyl)quinolinium, lucigenin, calcein, potassium ion fluorescent probe and sodium ion fluorescent probe.

[0014] Furthermore, in step (1), the phosphate buffer includes, but is not limited to, one of 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid buffer, NaH2PO4 / Na2HPO4, NaCl / HEPES, PBS buffer, 4-morpholineethanesulfonic acid buffer, and citric acid-disodium hydrogen phosphate buffer.

[0015] Furthermore, in step (2), the final concentration of the compound to be tested is added in a range of 15 nM to 10 μM.

[0016] Further, in step (2), a buffer solution containing corresponding cations or anions, wherein the cations include but are not limited to Li + 、Na + , K + , Rb + , Cs + , Ca 2+ Mg 2+ , H + and NMDG + One or more of, anions include but are not limited to F – , Cl – Br – ,I – 、NO3 – ,AcO – 、SCN – 、HCO3 - 、CO3 2- and ClO4 – One or more of .

[0017] Furthermore, in step (2), after the fluorescence intensity is detected for 300 seconds, the membrane permeabilization agent Triton x100 is added to the test solution, and the maximum fluorescence intensity after membrane permeabilization is detected by scanning for 30 seconds, and the transport rate of the test compound to the ion is calculated. The calculation formula is as follows:

[0018]

[0019] Where R t The real-time I 460 / I 403 Fluorescence ratio, R0 is the I of the first detection point 460 / I 403 Fluorescence ratio, R f is the maximum I after adding Triton x 100 solution 460 / I 403 Fluorescence ratio.

[0020] The above technical solution has the following beneficial effects:

[0021] In the present invention, a phospholipid bilayer is coated with a fluorescent probe to form an artificial vesicle to simulate a cell membrane. The fluorescent probe in the vesicle can detect the transmembrane transport activity of the tested compound in real time, thereby enabling rapid high-throughput screening of natural products with transmembrane transport activity on a multi-well plate.

[0022] The method of the present invention is suitable for high-throughput screening of natural molecules with transmembrane transport activity from natural products, so as to further apply such molecules to antibacterial, anti-tumor, anti-inflammatory and treatment of diseases caused by channel defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the transport rate of ions by enfusin in Example 1.

[0024] Figure 2 It is the transport rate of ions by beauvericin in Example 1.

[0025] Figure 3 The transport rate of ions by melittin at different concentrations in Example 2.

[0026] Figure 4 Example 3 is the ion transport process.

[0027] Figure 5 Example 4 is the ion transport process.

[0028] Figure 6 Example 5 is the ion transport process.

[0029] Figure 7Example 6 is the ion transport process. DETAILED DESCRIPTION

[0030] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0031] Compounds used and their English abbreviations:

[0032] Phospholipid bilayer: egg yolk L-α-phosphatidylcholine (EYPC), palmitoyloleoylphosphatidylcholine (POPC), soybean lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), dioleoylphosphatidylcholine (DOPC).

[0033] Fluorescent probes: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), 5-carboxyfluorescein (CF), 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), lucigenin, calcein, potassium ion fluorescent probe (PBFI) and sodium ion fluorescent probe (SBFI).

[0034] Phosphate buffer: 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) buffer, NaH2PO4 / Na2HPO4, NaCl / HEPES, PBS buffer, 4-morpholineethanesulfonic acid (MES) buffer, citrate-disodium hydrogen phosphate buffer.

[0035] Example 1

[0036] A method for rapid high-throughput screening of natural products with transmembrane transport activity, comprising the following steps:

[0037] Step 1: Add 1 mL of EYPC solution (20 mg / mL) dissolved in dry chloroform to a round-bottom flask, remove the mixed solvent under reduced pressure at 40°C, and add 1 mL, 1 mmol / L buffer solution containing HPTS (NaCl 100 mM, HEPES 10 mM, pH = 7.0) after vacuum drying for 24 hours. Rapidly cool the vesicle solution and warm it up 10 times, i.e., freeze it in liquid nitrogen for 1 minute and heat it in a 37°C water bath for 1.5 minutes. Extrude it through a filter membrane with a pore size of 200 nm to prepare a phospholipid monolayer vesicle suspension with a diameter of about 200 nm. Pass the phospholipid monolayer vesicle suspension through Sephadex G-50 column was used to remove unencapsulated fluorescent dye to prepare 5 mL of vesicle stock solution with a final concentration of 6.5 mM.

[0038] Step 2: Take 150 μL of the above vesicle stock solution containing HPTS fluorescent probe, dissolve in 14.85 mL NaCl (NaCl 100 mM, HEPES 10 mM, pH = 8.0) and KCl (KCl 100 mM, HEPES 10 mM, pH = 8.0) buffer solution, take 200 μL of the above solution and inoculate it in a black wall opaque 96-well plate and add 2 μL of the test compound. At the same time, in order to make a better comparison experiment, a solution without the test sample is used for comparison.

[0039] As a preference, in this embodiment, buffer solutions of NaCl 100 mM, HEPES 10 mM, pH=8.0 and KCl 100 mM, HEPES 10 mM, pH=8.0 are selected.

[0040] The dynamic changes of the relative fluorescence intensity of the fluorescent probe can be detected by a multifunctional microplate reader and a fluorescence meter. In this embodiment, the SYNERGY H1 microplate reader produced by Berton Instrument Co., Ltd. in the United States and the F7100 fluorescence meter produced by Hitachi, Japan are selected. The specific settings are as follows: the excitation light wavelength is 403 / 460nm, and the emission light wavelength is 510nm; the relative fluorescence intensity changes of the fluorescent probe within 330s are detected. The first 30s are for automatic shaking of the 96-well plate, the purpose is to make the vesicle stock solution evenly dispersed in the buffer solution, and to allow the test compound to fully react with the vesicle stock solution. After the detection, 2μL of 20% Triton x 100 solution was added to the 96-well plate, and the changes in the highest fluorescence value within 30s after the LUV membrane was broken were continued under the above conditions.

[0041] The pH gradient dissipates due to the reverse transport of hydrogen ions or hydroxide ions promoted by the transport protein. The transmembrane ion transport activity of the test compound can then be evaluated based on the pH dissipation across the lipid bilayer. The activity of natural products with transmembrane transport activity can be reflected by the relative fluorescence intensity versus time curve, and the transport rate of ions by the test compound can be calculated using the following equation:

[0042]

[0043] Where R t The real-time I 460 / I 403 Fluorescence ratio, R0 is the I of the first detection point 460 / I 403 Fluorescence ratio, R f is the maximum I after adding Triton x 100 solution 460 / I 403 Fluorescence ratio.

[0044] When the tested compounds were 10 μM enfusin B1 and 5 μM beauvericin, the transport rate of the ions was Figure 1 , 2 shows that the change of ion transport rate over time shows that both natural products promote the transport of cations M + / H + The reverse transport of β-catenin leads to the dissipation of pH gradient and causes the change of relative fluorescence value of HPTS, indicating that both compounds have transmembrane transport activity. It has been reported in the literature that enfusin B1 is considered to be related to its ion carrier properties. They can form stable lipophilic complexes with cations and transport them to lipophilic matrices such as cell membranes, causing cell osmotic balance disorders, thereby exerting antibacterial and insecticidal effects. Beauvericin is a cyclic hexapeptide that can transport alkaline earth metal and alkali metal ions across the cell membrane, destroying the ion balance inside and outside the cell membrane and causing cell death.

[0045] Example 2

[0046] Step 1: Add 1 mL of EYPC solution (20 mg / mL) dissolved in dry chloroform into a round-bottom flask and remove the mixed solvent under reduced pressure at 40°C. After vacuum drying for 24 hours, add 1 mL, 1 mmol / L buffer solution containing carboxyfluorescein (CF) fluorescent probe (NaCl 100 mM, HEPES 10 mM, pH = 7.5), and rapidly cool the vesicle solution and warm it up 10 times, i.e., freeze it in liquid nitrogen for 1 minute and heat it in a 37°C water bath for 1.5 minutes. Extrude through a filter membrane to prepare unilamellar vesicles with a diameter of about 200 nm, and pass the vesicle suspension through Sephadex G-50 column was used to remove the unentrapped CF fluorescent dye to prepare 5 mL of vesicle stock solution with a final concentration of 6.5 mM.

[0047] Step 2: Take 150 μL of the above vesicle stock solution containing the CF fluorescent probe and dissolve it in 14.85 mL of NaCl (NaCl 100 mM, HEPES 10 mM, pH = 7.5) buffer solution. Take 200 μL of the above buffer solution containing the vesicle stock solution and inoculate it into a black-walled opaque 96-well plate and add 2 μL of the test compound (in this example, bee venom peptide). At the same time, in order to make a better comparison experiment, a solution without the test sample is used for comparison.

[0048] The activity of natural products with transmembrane transport activity can be reflected by the curve of relative fluorescence intensity versus time. The transport rate of ions by the test compound can be calculated using the following equation:

[0049]

[0050] Where R t The real-time I460 / I 403 Fluorescence ratio, R0 is the I of the first detection point 460 / I 403 Fluorescence ratio, R f is the maximum I after adding Triton x 100 solution 460 / I 403 Fluorescence ratio.

[0051] The concentrations of bee venom peptide were 15nM, 60nM, and 150nM, respectively, and the transport rate of ions was Figure 3 As shown, under the action of the sample concentration in this example, the change in ion transport rate over time shows that a pore structure is formed on the natural product bee venom peptide vesicle, allowing the CF fluorescent probe in the vesicle to diffuse out of the vesicle, and the relative fluorescence value of the CF dye changes, indicating that bee venom peptide has transmembrane transport activity. Studies have shown that bee venom peptide is the main component with pharmacological and biological activities in bee venom, which can pass through the phospholipid bilayer to form pores and can be used to study the interaction between biological membranes and proteins. In addition, bee venom peptide also has multiple pharmacological activities such as anti-inflammatory, antihypertensive, analgesic, platelet aggregation inhibition, anti-radiation, antibacterial, anti-HIV, anti-rheumatoid arthritis and anti-tumor.

[0052] The application of the above examples 1-2 proves the feasibility and accuracy of the present invention.

[0053] Example 3

[0054] like Figure 4 As shown, a method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0055] In step (2), the outside of the vesicle is a buffer solution containing 100 mM MCl and pH = 8.0 containing cations.

[0056] This method is used to screen compounds with cation transmembrane transport activity, where the cation M is Li + 、Na + , K + , Rb + , Cs + , Ca 2+ Mg 2+ , H + and NMDG + One or more of.

[0057] Example 4

[0058] like Figure 5 As shown, a method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0059] In step (1), the vesicle stock solution contains a buffer solution containing 100 mM HPTS fluorescent probe NaX, 10 mM HEPES, and pH = 7.0;

[0060] In step (2), the outside of the vesicle is a buffer solution containing 100 mM NaX and pH 8.0 containing anions. This type of method is used to screen compounds with anion transmembrane transport activity, and the anion X is F – , Cl – Br – ,I – 、NO3 – ,AcO – 、SCN – 、HCO3 - 、CO3 2- and ClO4 – One or more of .

[0061] Example 5

[0062] like Figure 6 As shown, a method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0063] The vesicle stock solution contains a buffer solution containing CF fluorescent probe NaCl 100mM, HEPES 10mM, pH = 7.5. This type of method is used to screen compounds with nanoscale pores that can transport small molecules across the membrane.

[0064] Example 6

[0065] like Figure 7 As shown, a method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0066] In step 1, the vesicle stock solution contains a buffer solution of 200 mM NaNO3, 10 mM HEPES, and pH = 7.0, which contains the chloride ion-sensitive fluorescent probe SPQ;

[0067] In step 2, the outside of the vesicle is a buffer solution of 200 mM NaCl, 10 mM HEPES, pH = 7.0. This type of method is used to screen compounds with chloride ion transmembrane transport activity.

[0068] Example 7

[0069] A method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0070] In step (1), 15 mg / mL palmitoyloleoylphosphatidylcholine (POPC) chloroform solution is used. After vacuum drying, the prepared vesicle stock solution contains NaH2PO4 / Na2HPO4 buffer containing 0.5 mmol / L lucigenin fluorescent probe, and a phospholipid monolayer vesicle suspension is prepared using a filter membrane with a pore size of 100 nm.

[0071] Example 8

[0072] A method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0073] In step (1), a soybean lecithin chloroform solution is used, and after vacuum drying, the prepared vesicle stock solution contains a PBS buffer solution containing 1.5 mmol / L calcein fluorescent probe, and a phospholipid monolayer vesicle suspension is prepared using a filter membrane with a pore size of 100 nm.

[0074] Example 9

[0075] A method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0076] In step (1), a 25 mg / mL POPE chloroform solution is used, and after vacuum drying, the prepared vesicle stock solution contains a 4-morpholineethanesulfonic acid (MES) buffer containing 2.5 mmol / L potassium ion fluorescent probe (PBFI), and a phospholipid monolayer vesicle suspension is prepared using a filter membrane with a pore size of 250 nm.

[0077] Example 10

[0078] A method for rapid high-throughput screening of natural products with transmembrane transport activity, compared with Example 1:

[0079] In step (1), a 30 mg / mL DOPC chloroform solution is used, and after vacuum drying, the prepared vesicle stock solution contains a citric acid-disodium hydrogen phosphate buffer solution containing 3.5 mmol / L sodium ion fluorescent probe (SBFI), and a phospholipid monolayer vesicle suspension is prepared using a filter membrane with a pore size of 300 nm.

[0080] The creative contribution of the present invention is the pioneering use of artificial vesicles coated with fluorescent probes to conduct rapid high-throughput screening of natural products with transmembrane transport activity. Hundreds or even thousands of experimental data can be obtained in one day, the cost is greatly reduced, and operators can master it after simple training. Therefore, compared with existing patch clamp and other related technologies, the method of the present invention not only has a greater advantage in speed, but also has the advantages of simple operation, low cost, strong repeatability, etc., and this method is very suitable for rapid high-throughput screening of compounds with transmembrane transport activity from natural products in a large compound library for further biological activity research.

[0081] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "include..." or "comprise..." do not exclude the existence of other elements in the process, method, article or terminal device including the elements. In addition, in this article, "greater than", "less than", "exceed" and the like are understood to exclude the number itself; "above", "below", "within" and the like are understood to include the number itself.

[0082] Although the above embodiments have been described, once those skilled in the art know the basic creative concepts, they can make additional changes and modifications to these embodiments. Therefore, the above description is only an embodiment of the present invention and does not limit the patent protection scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the specification and drawings of the present invention, or directly or indirectly used in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Rapid high-throughput screening of natural products with transmembrane transport activity, It is characterized in that The steps include: (1) taking a chloroform solution of a phospholipid bilayer, vacuum drying it, adding a buffer solution containing a fluorescent probe to obtain a reaction suspension, cooling the reaction suspension several times, and then extruding it through a filter membrane to obtain a phospholipid monolayer vesicle suspension, and passing the phospholipid monolayer vesicle suspension through a dextran gel column to remove uncoated fluorescent probes to obtain a vesicle stock solution; (2) inoculating the vesicle stock solution coated with the fluorescent probe into a black-walled opaque microplate, adding a buffer solution containing corresponding cations or anions into the microplate, so that there is a pH or ion concentration gradient inside and outside the phospholipid monolayer vesicle membrane, adding the test compound into the black-walled well plate, and then testing the change in the relative fluorescence intensity of the fluorescent probe. When the relative fluorescence intensity is gradually quenched or increased, the test compound has transmembrane transport activity; After the fluorescence intensity was detected for 300 seconds, the membrane permeabilization agent Triton x 100 was added to the test solution, and the maximum fluorescence intensity after membrane permeabilization was detected by scanning for 30 seconds. The transport rate of the tested compound to the ion was calculated according to the following formula: Where R t The real-time I 460 / I 403 Fluorescence ratio, R 0 I is the first detection point 460 / I 403 Fluorescence ratio, R f is the maximum I after adding Triton x 100 solution 460 / I 403 Fluorescence ratio.

2. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (1), the concentration of the phospholipid bilayer chloroform solution is 15-30 mg / mL, the concentration of the fluorescent probe in the buffer solution containing the fluorescent probe is 0.5-3.5 mmol / L, and the pore size of the filter membrane is 100-300 nm.

3. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (1), the dextran gel column is a Sephadex G-50 column.

4. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (1), the phospholipid bilayer is one of egg yolk L-α-phosphatidylcholine, palmitoyloleoylphosphatidylcholine, soybean lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoylphosphatidylglycerol, and dioleoylphosphatidylcholine.

5. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (1), the fluorescent probe is one of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, 5-carboxyfluorescein, 6-methoxy-N-(3-sulfopropyl)quinolinium, lucigenin, calcein, potassium ion fluorescent probe and sodium ion fluorescent probe.

6. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (1), the phosphate buffer is 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid buffer, NaH 2 PO 4 / Na 2 HPO 4 , NaCl / HEPES, PBS buffer, 4-morpholineethanesulfonic acid buffer, and citrate-disodium hydrogen phosphate buffer.

7. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (2), the final concentration of the compound to be tested is added to be 15 nM-10 μM.

8. The method for rapid high-throughput screening of natural products with transmembrane transport activity as claimed in claim 1, It is characterized in that In step (2), a buffer solution containing the corresponding cation or anion, the cation is Li + 、Na + , K + , Rb + , Cs + , Ca 2+ Mg 2+ , H + and NMDG + One or more of the following, the anion is F – , Cl – Br – ,I – 、NO 3 – ,AcO – 、SCN – , HCO 3 - , CO 3 2- and ClO 4 – One or more of .

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