CN114544566A - Method for rapidly screening natural products with transmembrane transport activity in high throughput manner - Google Patents

Method for rapidly screening natural products with transmembrane transport activity in high throughput manner Download PDF

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CN114544566A
CN114544566A CN202210005455.7A CN202210005455A CN114544566A CN 114544566 A CN114544566 A CN 114544566A CN 202210005455 A CN202210005455 A CN 202210005455A CN 114544566 A CN114544566 A CN 114544566A
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natural products
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任长亮
贾春燕
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Xiamen University
Shenzhen Research Institute of Xiamen University
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Abstract

The invention relates to the field of ion channel screening, and discloses a method for rapidly screening a natural product with transmembrane transmission activity in a high throughput manner, which comprises the following steps: (1) taking a phospholipid bilayer chloroform solution, drying in vacuum, adding a buffer solution containing a fluorescent probe, cooling and returning the temperature for multiple times, then extruding through a membrane to obtain a phospholipid monolayer vesicle suspension, and passing through a sephadex column to obtain a vesicle stock solution; (2) inoculating vesicle stock solution coated with the fluorescent probe into a black-wall opaque microplate, adding a buffer solution containing corresponding cations or anions into micropores, respectively adding a compound to be tested into the black-wall microplate, then testing the relative fluorescence intensity change of the fluorescent probe, and when the relative fluorescence intensity is gradually quenched or increased, the compound to be tested has transmembrane transmission activity.

Description

Method for rapidly screening natural products with transmembrane transport activity in high throughput manner
Technical Field
The invention relates to the field of screening of ion channel compounds, in particular to a method for rapidly screening natural products with transmembrane transport activity in a high throughput manner.
Background
Ion channels are a class of proteins that control the exchange of material inside and outside cells, and are essential for intercellular signaling and play a key role in the cell cycle phase and other aspects of cell physiology. Defects in channel function are closely associated with the development and progression of many diseases, such as diabetes, neuropathic pain, cardiovascular disease, cerebral and peripheral vascular disease, asthma, neurodegenerative diseases, and the like. The functional regulation of ion transmembrane channels is the basis for the treatment of a variety of diseases and is also an important target for the development of new highly effective drug therapies. However, the structure of the natural channel protein is extremely complex, and the modification and regulation of the structure are extremely difficult. Therefore, ion transporters having transmembrane transport activity were developed such as: the replacement of the original functional deficient native channel protein by a channel or vector is of crucial importance for the treatment of diseases caused by channel defects. In addition, transmembrane ion transporters can rapidly destroy the ion balance inside and outside the membrane, and therefore, the transmembrane ion transporters are widely applied to the fields of antibiosis, anticancer, anti-inflammation and the like.
The natural products have wide sources and abundant structural diversity, provide a large number of sources for the discovery of medicaments and lead compounds, and small molecular compounds with transmembrane transport activity, such as Prodigiosin (Prodigiosin), amphotericin B (amphotericin B), gramicin A (Gramicin A), Valinomycin (Valinomycin), antifungal polyene amphoterinol 3 (Amphiinol 3) extracted from marine dinoflagellate and the like, which are separated from the natural products have proven to have various biological activity effects of resisting cancers, bacteria, malaria, mildew and immunosuppression. However, because of the huge amount of natural product library compounds, a high-throughput screening technology is urgently needed to rapidly realize the development and application of natural products in the field of ion transmembrane transport, and more natural products with transmembrane transport activity are found.
Currently, the usual patch clamp technique for screening compounds with transmembrane transport activity is to record the ionic current through an ion channel to reflect single or multiple ion channel molecular events in the cell membrane. However, the current electrophysiological techniques cannot realize large-scale screening of compounds in a short time, and in addition, the electrophysiological techniques have the disadvantages of expensive machines, complex and difficult operation, high technical requirements for operators and the like, and are not suitable for screening of large-scale compounds in the initial stage and the middle stage of drug development. Patent CN102884427A discloses a multi-well plate with a specific contour design, which can perform multi-stage high-flux parallel measurement on ion channels or ion transporters, but the related multi-well patch clamp device is complicated and fine, and the steps are tedious and time-consuming, so that the wide application thereof is limited. Patent CN112176020A discloses a screening method of calcium-activated chloride channel activators, which relates to the construction and transfection of more complex eukaryotic expression vectors, and screens the calcium-activated chloride channel activators through 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.
Disclosure of Invention
Therefore, a method for rapidly screening natural products with transmembrane transport activity at high throughput needs to be provided, and the problems of complicated steps, long time consumption and fine and complex equipment requirements in the existing screening technology are solved.
In order to achieve the above objects, the present invention provides a method for rapid high-throughput screening of natural products having transmembrane transport activity, comprising the steps of:
(1) taking a phospholipid bilayer chloroform solution, drying in vacuum, adding a buffer solution containing a fluorescent probe to obtain a reaction suspension, cooling and returning the reaction suspension for multiple times, then extruding the reaction suspension through a filter membrane to obtain a phospholipid monolayer vesicle suspension, and removing the uncoated fluorescent probe from the phospholipid monolayer vesicle suspension through a sephadex column to obtain a vesicle stock solution;
(2) inoculating the vesicle stock solution coated with the fluorescent probe into a black-wall opaque microplate, adding a buffer solution containing corresponding cations or anions into micropores to enable pH or ion concentration gradient to exist inside and outside the phospholipid monolayer vesicle membrane, respectively adding a compound to be tested into the black-wall microplate, then testing the relative fluorescence intensity change of the fluorescent probe, and when the relative fluorescence intensity is gradually quenched or increased, the compound to be tested has transmembrane transmission activity.
The microplate may be a 96-well or 384-well cell culture plate.
Further, in the step (1), the concentration of the phospholipid bilayer chloroform solution is 15-30mg/mL, the concentration of the nutrient probe in the buffer solution containing the fluorescent probe is 05-3.5mmol/L, and the aperture of the filter membrane is 100-300 nm.
Further, in the step (1), the Sephadex column is a Sephadex G-50 column.
Further, in the step (1), the phospholipid bilayer is one of egg yolk L-alpha-phosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, soybean phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl phosphatidylglycerol and dioleoyl lecithin.
Further, in the step (1), the fluorescent probe includes, but is not limited to, one of 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt, 5-carboxyfluorescein, 6-methoxy-N- (3-sulfopropyl) quinolinium, lucigenin, calcein, a potassium ion fluorescent probe, and a sodium ion fluorescent probe.
Further, in step (1), phosphate buffer includes, but is not limited to, 4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid buffer, NaH2PO4/Na2HPO4NaCl/HEPES, PBS buffer solution, 4-morpholine ethanesulfonic acid buffer solution and citric acid-disodium hydrogen phosphate buffer solution.
Further, in step (2), the compound to be tested is added to a final concentration of 15nM to 10. mu.M.
Further, in step (2), a buffer solution containing a corresponding cation or anion, the cation including but not limited to Li+、Na+、K+、Rb+、Cs+、Ca2+、Mg2+、H+And NMDG+Anions including but not limited to F–、Cl–、Br–、I–、NO3 –、AcO–、SCN–、HCO3 -、CO3 2-And ClO4 –One or more of (a).
Further, in the step (2), after detecting the fluorescence intensity for 300s, adding a membrane breaking agent Triton x 100 into the test solution, scanning for 30s to detect the maximum fluorescence intensity after membrane breaking, and calculating the transport speed of the compound to be tested to the ions, wherein the calculation formula is as follows:
Figure BDA0003455320540000031
wherein R istAs real-time I of the compound to be determined at the point of detection460/I403Fluorescence ratio, R0Is the first detection point I460/I403Fluorescence ratio, RfIs the maximum I after addition of Triton x 100 solution460/I403Fluorescence ratio.
The technical scheme has the following beneficial effects:
in the invention, the phospholipid bilayer is coated with the fluorescent probe to form the artificial vesicle which simulates a cell membrane, and the fluorescent probe in the vesicle can detect the transmembrane transport activity of a tested compound in real time, so that a natural product with transmembrane transport activity can be rapidly screened on a porous plate in a high-throughput manner.
The method is suitable for screening natural molecules with transmembrane transmission activity from natural products at high flux, so that the molecules are further applied to antibiosis, tumor resistance, anti-inflammation and treatment of diseases caused by channel defects.
Drawings
FIG. 1 shows the transport rate of the ansamitocin for ions in example 1.
FIG. 2 shows the ion transport rates of beauvericin in example 1.
FIG. 3 shows the ion transport rates of various concentrations of melittin in example 2.
FIG. 4 shows the ion transport process in example 3.
FIG. 5 shows the ion transport process in example 4.
FIG. 6 shows the ion transport process in example 5.
FIG. 7 shows the ion transport process in example 6.
Detailed Description
In order to explain technical contents, structural features, objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in combination with the embodiments.
The compounds used and their english abbreviation:
phospholipid bilayer: egg yolk L-alpha-phosphatidylcholine (EYPC), Palmitoyl Oleoyl Phosphatidylcholine (POPC), soybean lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), dioleoyl lecithin (DOPC).
A fluorescent probe: 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).
Phosphate buffer solution: 4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid (HEPES) buffer, NaH2PO4/Na2HPO4NaCl/HEPES, PBS buffer, 4-morpholine ethanesulfonic acid (MES) buffer, citric acid-disodium hydrogen phosphate buffer.
Example 1
A method for rapidly screening natural products with transmembrane transport activity in high throughput comprises the following steps:
step one, 1mL of an EYPC solution (20mg/mL) dissolved in dry chloroform was added to a round-bottomed flask, the mixed solvent was removed under reduced pressure at 40 ℃, and after 24 hours of vacuum drying, 1mL of a 1mmol/L HPTS-containing buffer solution (NaCl100 mM, HEPES10mM, pH 7.0) was added, and the vesicle solution was rapidly cooled and warmed up 10 times, i.e., frozen in liquid nitrogen for 1min and heated in a 37 ℃ water bath for 1.5 min. Extruding through a filter membrane with a pore size of 200nm to prepare a phospholipid unilamellar vesicle suspension having a diameter of about 200nm, passing the phospholipid unilamellar vesicle suspension through
Figure BDA0003455320540000051
Sephadex G-50 column to remove non-embedded fluorescent dye to prepare 5mL vesicle stock, final concentration 6.5 mM.
Step two, 150 μ L of each vesicle stock solution containing the HPTS fluorescent probe was dissolved in 14.85ml naci (NaCl100 mM, HEPES10mM, pH 8.0) and KCl (KCl 100mM, HEPES10mM, pH 8.0) buffer solutions, 200 μ L of the above solutions were inoculated into black-wall opaque 96-well plates, and 2 μ L of each test compound was added. Meanwhile, in order to make a comparison experiment better, the solution without the sample to be detected is used for comparison.
In this embodiment, a buffer solution of NaCl100 mM, HEPES10mM, pH 8.0, KCl 100mM, HEPES10mM, pH 8.0 is preferably used.
The dynamic change of the relative fluorescence intensity of the fluorescent probe can be detected by a multifunctional microplate reader and a fluorometer, and in the embodiment, a SYNERGY H1 microplate reader manufactured by beton instruments ltd and an F7100 fluorometer manufactured by hitachi, japan are selected and specifically set as follows: excitation light wavelength 403/460nm and emission light wavelength 510 nm; the relative fluorescence intensity change of the fluorescent probe within 330s was detected. The first 30s is an automatic oscillation 96-well plate, and aims to uniformly disperse the vesicle stock solution in the buffer solution and enable the compound to be detected to fully act with the vesicle stock solution. After the detection, 2. mu.L of 20% Triton x 100 solution was added to each 96-well plate, and the maximum fluorescence change within 30s after the LUV rupture was continuously detected under the above conditions.
The pH gradient dissipates due to the transport protein-promoted reverse transport of hydrogen or hydroxyl ions. The transmembrane transport ionic activity of the test compound can then be assessed from the pH dissipation across the lipid bilayer. The activity of the natural product with transmembrane transport activity can be reflected by a curve of the relative fluorescence intensity along with time, and the transport speed of the compound to be tested to ions can be calculated by using the following equation:
Figure BDA0003455320540000061
wherein R istAs real-time I of the compound to be determined at the point of detection460/I403Fluorescence ratio, R0Is the first detection point I460/I403Fluorescence ratio, RfIs the maximum I after addition of Triton x 100 solution460/I403Fluorescence ratio.
The compounds to be tested were 10. mu.M of enniatin B1 and 5. mu.M of beauvericin, respectively, the rate of ion transport is shown in FIGS. 1 and 2, and it can be seen that the two natural products both promote the cation M+/H+The relative fluorescence of HPTS changes due to pH gradient abrogation, indicating that both compounds have transmembrane transport activity. The literature reports that enniatin B1 is believed to be related to its ionophore properties. They can form stable lipophilic complexes with cations and transport them into lipophilic substrates such as cell membranes, leading to a disturbed cell osmotic balance, thus exerting antibacterial and insecticidal effects. Beauverine is a cyclic hexapeptide which can transport alkaline earth metal and alkali metal ions across cell membranes and destroy the ion balance inside and outside the cell membranes, thereby causing cell decay.
Example 2
Step one, 1mL of EYPC solution (20mg/mL) dissolved in dry chloroform was added to a round-bottom flask, and the mixed solvent was removed under reduced pressure at 40 ℃. After vacuum drying for 24h, 1mL,1mmol/L buffer solution (NaCl100 mM, HEPES10mM, pH 7.5) containing Carboxyfluorescein (CF) fluorescent probe was added, and the vesicle fluid was snap-cooled and warmed up 10 times, i.e., frozen in liquid nitrogen for 1min and heated in a 37 ℃ water bath for 1.5 min. Extruding through a filter membrane to prepare unilamellar vesicles having a diameter of about 200nm, passing the vesicle suspension through
Figure BDA0003455320540000062
Sephadex G-50 column to remove non-embedded CF fluorescent dye to prepare 5mL vesicle stock solution at a final concentration of 6.5 mM.
Step two, 150 μ L of each vesicle stock solution containing the CF fluorescent probe was dissolved in 14.85mL of NaCl (NaCl100 mM, HEPES10mM, pH 7.5) buffer solution. 200. mu.L of the above vesicle-containing stock buffer solution was inoculated into a black-wall opaque 96-well plate and 2. mu.L of each test compound (melittin in this example) was added. Meanwhile, in order to make a comparison experiment better, the solution without the sample to be detected is used for comparison.
The activity of the natural product with transmembrane transport activity can be reflected by a curve of the relative fluorescence intensity along with time, and the transport speed of the compound to be tested to ions can be calculated by using the following equation:
Figure BDA0003455320540000071
wherein R istAs real time I of the compound to be tested at the point of detection460/I403Fluorescence ratio, R0As the first detection point I460/I403Fluorescence ratio, RfIs the maximum I after addition of Triton x 100 solution460/I403Fluorescence ratio.
The concentrations of melittin are respectively 15nM, 60nM and 150nM, the transport speed to ions is shown in FIG. 3, under the effect of the sample concentration in the example, the change of the transport speed to ions along with time can be seen, the pore structure formed on the natural product melittin vesicle enables the CF fluorescent probe in the vesicle to diffuse out of the vesicle, the relative fluorescence value of the CF dye changes, and the melittin is shown to have transmembrane transport activity. The research shows that melittin is the main component with pharmacological action and biological activity in bee venom, can penetrate phospholipid bilayer to form pores, and can be used for researching the interaction between biological membrane and protein. In addition, melittin has various pharmacological activities such as anti-inflammatory, blood pressure lowering, pain relieving, platelet aggregation inhibiting, radioprotective, antibacterial, anti-HIV, anti-rheumatic arthritis, and anti-tumor.
The application of examples 1-2 above demonstrates the feasibility and accuracy of the present invention.
Example 3
As shown in fig. 4, a method for rapid high-throughput screening of natural products having transmembrane transport activity, example 1, compared:
in step (2), the outside of the vesicle is a buffered solution containing 100mM of MCl containing cations and having a pH of 8.0.
Such methods are useful for screening compounds having cation transport activity across membranes, where the cation M is Li+、Na+、K+、Rb+、Cs+、Ca2+、Mg2+、H+And NMDG+One or more of them.
Example 4
As shown in fig. 5, a method for rapid high-throughput screening of natural products having transmembrane transport activity, example 1, compared:
in the step (1), the vesicle stock solution contains a buffer solution containing a HPTS fluorescent probe NaX 100mM and HEPES10mM, and the pH value is 7.0;
in step (2), the outside of the vesicle is a buffer solution containing 100mM of NaX containing an anion at pH 8.0. Such methods are useful for screening compounds having anion transport across membranes, where anion X is F–、Cl–、Br–、I–、NO3 –、AcO–、SCN–、HCO3 -、CO3 2-And ClO4 –One or more of (a).
Example 5
As shown in fig. 6, a method for rapid high-throughput screening of natural products having transmembrane transport activity, example 1, compared:
the vesicle stock solution contained a CF fluorescent probe NaCl100 mM, HEPES10mM, and pH 7.5 buffer. Such methods are useful for screening compounds having a nanoscale pore size that permits transport of small molecules across a membrane.
Example 6
As shown in fig. 7, a method for rapid high-throughput screening of natural products having transmembrane transport activity, example 1, compared:
in step 1, NaNO containing chloride ion sensitive fluorescent probe SPQ is in the vesicle stock solution3200mM, HEPES10mM, pH 7.0;
in step 2, buffer solution of NaCl 200mM, HEPES10mM, pH 7.0 was added to the outside of the vesicles. Such methods are useful for screening compounds having chloride ion transport activity across membranes.
Example 7
A method for rapidly screening natural products with transmembrane transport activity in high throughput, which comprises the following steps in example 1:
in the step (1), 15mg/mL of Palmitoyl Oleoyl Phosphatidylcholine (POPC) chloroform solution is used, and after vacuum drying, NaH containing 0.5mmol/L of lucigenin fluorescent probe is contained in the prepared vesicle stock solution2PO4/Na2HPO4Buffer, using a filter membrane with a pore size of 100nm to prepare a phospholipid unilamellar vesicle suspension.
Example 8
A method for rapidly screening natural products with transmembrane transport activity in high throughput, which comprises the following steps in example 1:
in the step (1), a soybean phospholipid chloroform solution is used, vacuum drying is carried out, a PBS buffer solution containing 1.5mmol/L calcein fluorescent probe is filled in the prepared vesicle stock solution, and a filtering membrane with the aperture of 100nm is used for preparing the phospholipid unilamellar vesicle suspension.
Example 9
A method for rapidly screening natural products with transmembrane transport activity at high throughput, which comprises the following steps of 1:
in the step (1), 25mg/mL POPE chloroform solution is used, after vacuum drying, 4-morpholine ethanesulfonic acid (MES) buffer solution containing 2.5mmol/L potassium ion fluorescent Probe (PBFI) is prepared in the prepared vesicle stock solution, and a filtering membrane with the aperture of 250nm is used for preparing phospholipid unilamellar vesicle suspension.
Example 10
A method for rapidly screening natural products with transmembrane transport activity in high throughput, which comprises the following steps in example 1:
in the step (1), 30mg/mL DOPC chloroform solution is used, vacuum drying is carried out, citric acid-disodium hydrogen phosphate buffer solution containing 3.5mmol/L sodium ion fluorescent probe (SBFI) is contained in the prepared vesicle stock solution, and a filtering membrane with the aperture of 300nm is used for preparing phospholipid unilamellar vesicle suspension.
The creative contribution of the invention lies in that the artificial vesicle coated with the fluorescent probe is creatively utilized to carry out the rapid high-flux screening of the natural product with transmembrane transmission activity, hundreds or even thousands of experimental data can be obtained in one day, the cost is greatly reduced, and the artificial vesicle can be mastered by operators after simple training. Therefore, compared with the prior art such as patch clamp and the like, the method has the advantages of high speed, simple operation, low cost, strong repeatability and the like, and is very suitable for rapidly screening the compound with transmembrane transport activity from the natural product with huge compound library in high flux for further biological activity research.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.

Claims (9)

1. A method for rapidly screening natural products with transmembrane transport activity at high flux is characterized by comprising the following steps:
(1) taking a phospholipid bilayer chloroform solution, drying in vacuum, adding a buffer solution containing a fluorescent probe to obtain a reaction suspension, cooling and returning the reaction suspension for multiple times, then extruding the reaction suspension through a filter membrane to obtain a phospholipid monolayer vesicle suspension, and removing the uncoated fluorescent probe from the phospholipid monolayer vesicle suspension through a sephadex column to obtain a vesicle stock solution;
(2) inoculating the vesicle stock solution coated with the fluorescent probe into a black-wall opaque microplate, adding a buffer solution containing corresponding cations or anions into micropores to enable pH or ion concentration gradient to exist inside and outside the phospholipid monolayer vesicle membrane, respectively adding a compound to be tested into the black-wall microplate, then testing the relative fluorescence intensity change of the fluorescent probe, and when the relative fluorescence intensity is gradually quenched or increased, the compound to be tested has transmembrane transmission activity.
2. The method for rapid high-throughput screening of natural products with transmembrane transport activity according to claim 1, wherein in the step (1), the concentration of the phospholipid bilayer chloroform solution is 15-30mg/mL, the concentration of the fluorescent probe in the buffer solution containing the fluorescent probe is 0.5-3.5mmol/L, and the pore diameter of the filter membrane is 100-300 nm.
3. The method for rapid high-throughput screening of natural products having transmembrane transport activity according to claim 1, wherein in the step (1), the Sephadex column is a Sephadex G-50 column.
4. The method for rapid high-throughput screening of natural products with transmembrane transport activity according to claim 1, wherein in the step (1), the phospholipid bilayer is one of egg yolk L-alpha-phosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, soybean phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl phosphatidylglycerol and dioleoyl lecithin.
5. The method for rapid high-throughput screening of natural products with transmembrane transport activity according to claim 1, wherein in step (1), the fluorescent probe comprises but is not limited to one of 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt, 5-carboxyfluorescein, 6-methoxy-N- (3-sulfopropyl) quinolinium, lucigenin, calcein, a potassium-ion fluorescent probe, and a sodium-ion fluorescent probe.
6. The method for rapid high-throughput screening of natural products with transmembrane transport activity according to claim 1, wherein in step (1), the phosphate buffer includes but is not limited to 4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid buffer, NaH2PO4/Na2HPO4NaCl/HEPES, PBS buffer solution, 4-morpholine ethanesulfonic acid buffer solution and citric acid-disodium hydrogen phosphate buffer solution.
7. The method for rapid high-throughput screening of natural products having transmembrane transport activity according to claim 1, wherein in the step (2), the compound to be tested is added to a final concentration of 15nM to 10. mu.M.
8. The method for rapid high-throughput screening of natural products having transmembrane transport activity according to claim 1, wherein in step (2), the buffer solution contains corresponding cations or anions, wherein the cations include but are not limited to Li+、Na+、K+、Rb+、Cs+、Ca2+、Mg2+、H+And NMDG+Anions including but not limited to F–、Cl–、Br–、I–、NO3 –、AcO–、SCN–、HCO3 -、CO3 2-And ClO4 –One or more of (a).
9. The method for rapid high-throughput screening of natural products with transmembrane transport activity according to claim 1, wherein in the step (2), after the fluorescence intensity is detected for 300s, a membrane-breaking agent Triton x 100 is added into the test solution, the maximum fluorescence intensity after membrane breaking is detected for 30s, and the ion transport speed of the compound to be tested is calculated according to the following formula:
Figure FDA0003455320530000021
wherein R istAs real-time I of the compound to be determined at the point of detection460/I403Fluorescence ratio, R0Is the first detection point I460/I403Fluorescence ratio, RfIs the maximum I after addition of Triton x 100 solution460/I403Fluorescence ratio.
CN202210005455.7A 2022-01-04 2022-01-04 Method for rapidly screening natural products with transmembrane transport activity in high throughput manner Pending CN114544566A (en)

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