Method for high-throughput screening of active components of natural product based on equilibrium dialysis-mass spectrometry

Abstract

The invention discloses a method for high-throughput screening of active components of a natural product based on equilibrium dialysis-mass spectrometry, which comprises the following steps: adding a protein solution on one side of an activated and moist 96-hole microdialysis plate, and adding a to-be-detected solution of a traditional Chinese medicine crude extract on the other side of the activated and moist 96-hole microdialysis plate; respectively recovering samples from the upper solution and the lower solution of the incubated 96-hole microdialysis plate, adding methanol, mixing, standing to precipitate denatured protein, centrifuging to remove precipitate, and taking supernate for LC-MS (liquid chromatography-mass spectrometry) analysis; and carrying out characteristic peak annotation through LC-MS data of the supernate, comparing with a standard product database, and carrying out differential analysis and screening to obtain the required active ingredients of the natural product. According to the method for screening the active ingredients of the natural product, the equilibrium dialysis technology, a high-throughput screening device and mass spectrometry are combined, and the efficient, accurate and anti-interference screening effect is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical analysis technology, and in particular to a method for high-throughput screening of active ingredients of natural products based on balanced dialysis-mass spectrometry. BACKGROUND

[0002] Natural products, as an important source of small molecule compounds, have always been a key resource for drug development. However, due to the complexity of "multi-component, multi-target, multi-pathway", it is difficult to directly screen active ingredients from complex mixtures. Especially when dealing with multi-component and high complexity samples, traditional pharmacological screening methods face the bottleneck of low screening efficiency and poor recognition accuracy. In recent years, based on affinity selection-mass spectrometry (AS-MS) and affinity ultrafiltration-mass spectrometry (AU-MS) and other technologies, it has become the mainstream method for screening potential active ingredients in natural products.

[0003] However, the traditional screening method often uses single-well or small-scale batch processing, which leads to low screening efficiency and cannot meet the high-throughput screening needs of multi-component and high-complexity samples in natural product extracts. Moreover, natural product crude extracts usually contain a large amount of macromolecular impurities such as proteins and polysaccharides. These interferents may non-specifically bind to small molecule ligands or affect the analysis signal, resulting in a decrease in signal-to-noise ratio and further masking the true signal of target binding.

[0004] The following key technical problems exist in the current screening process of active ingredients of natural products:

[0005] (1) The existing screening method represented by ultrafiltration-mass spectrometry relies on the centrifugation process to separate free small molecules from target proteins, but under the action of centrifugal shear force, the non-covalent interaction between weakly bound small molecules and target proteins is easily destroyed, which leads to the inability of these molecules with potential biological activity to be effectively retained and recognized, thereby affecting the comprehensiveness of the screening results.

[0006] (2) Traditional screening techniques are mostly single-well or small-scale batch processing, which has low operation efficiency and cannot meet the parallel screening needs of multi-component and high-complexity samples in natural product extracts, limiting the application potential of the method in high-throughput screening.

[0007] (3) Natural product crude extracts often contain macromolecular impurities such as proteins and polysaccharides. These interferents may non-specifically bind or affect the analysis signal during screening, resulting in a decrease in signal-to-noise ratio and masking the true target binding event.

[0008] CN115524422A discloses a method for screening active ingredients of traditional Chinese medicine. The method collects verified chemical components and related data of traditional Chinese medicine through big data mining technology, combines high performance liquid chromatography and mass spectrometry technology for screening, and finally screens out active ingredients combined with serum albumin. The method can improve the efficiency and accuracy of traditional Chinese medicine component screening, but it may be affected by sample interference when dealing with complex natural product extracts, especially in the process of high-throughput screening, there are certain operation difficulties and sample singleization problems.

[0009] Equilibrium dialysis (ED) is a separation technique based on the principle of semi-permeable membrane, mainly used for determining the binding between small molecules and macromolecules (such as proteins, etc.) in solution. The method achieves equilibrium state by free diffusion of small molecules between the two sides of dialysis membrane, so as to determine the proportion of free and bound small molecules. The typical device is composed of a plate dialysis unit, a constant temperature oscillation incubator and an automatic sampling LC-MS; the key steps include membrane activation and pre-rinse, double-side buffer matching, constant temperature oscillation to equilibrium, two-cavity sampling, quantification and non-specific adsorption correction, etc. The equilibrium dialysis technology has low experimental cost and high accuracy, and can effectively retain the weak non-covalent binding between small molecules and target proteins. It is widely used in drug discovery, toxicology research and protein-ligand interaction analysis. Natural product-dialysis and mass spectrometry (NP-DiaMS) screening system allows direct use of natural product crude extract for screening, and retains the interaction information of small molecule active ingredients and target proteins by selectively retaining macromolecular interferents through dialysis membrane.

[0010] At present, equilibrium dialysis has been used for screening of natural products, but there are still some problems in practical application. For example, the traditional equilibrium dialysis method is usually more complicated in operation, and is only suitable for processing of specific samples. SUMMARY

[0011] The present application aims to solve the technical problems of low throughput, impurity interference, complicated steps and many interference factors in the current screening method of active ingredients of natural products, and provides a new screening method of active ingredients of natural products, which combines equilibrium dialysis technology, high-throughput screening device and mass spectrometry analysis, and realizes the effect of efficient, accurate and anti-interference screening.

[0012] To achieve the above purpose, the technical scheme adopted by the present application is:

[0013] A method for screening active ingredients of natural products based on equilibrium dialysis-mass spectrometry high-throughput screening, characterized in that it comprises the following steps:

[0014] Step 1, prepare a traditional Chinese medicine crude extract as a test solution by decoction or concentration;

[0015] Step 2, add a protein solution to one side of an activated and moistened 96-well microdialysis plate, and add the test solution to the other side;

[0016] Step 3, incubate the 96-well microdialysis plate loaded with the solution in step 2 in a constant temperature oscillator in a dark environment at 0-5°C for 18-26 hours;

[0017] Step 4, recover samples from the upper and lower layers of the incubated 96-well microdialysis plate, mix after adding methanol, stand to precipitate denatured proteins, remove the precipitate by centrifugation, and take the supernatant for LC-MS analysis;

[0018] Step 5, annotate the LC-MS data of the supernatant in step 4, and screen the desired natural product active ingredients by differential analysis and comparison with a standard database.

[0019] The present application uses equilibrium dialysis technology to screen natural product active ingredients, has the advantages of high throughput, strong weak binding recognition ability, and anti-interference performance, etc., to improve the screening efficiency and recognition accuracy, and overcome the limitations of the prior art in component retention, throughput processing and signal recognition.

[0020] The traditional Chinese medicine crude extract includes a crude extract of aconite; the traditional Chinese medicine crude extract has not been subjected to polysaccharide removal treatment.

[0021] The crude extract of aconite is obtained by crushing aconite and other traditional Chinese medicines, heating reflux or decoction extraction with water or an organic solvent (such as ethanol or methanol), filtration, combining the filtrate, and concentrating to remove part of the solvent, without other treatments such as polysaccharide removal, and contains target active ingredients, similar derivatives, and polysaccharides, proteins, pigments, and other substances, and the components are relatively complex, so there are great interference and difficulty in using ordinary screening methods. In the present application, a 96-well microdialysis plate is used, which has the advantages of high throughput and strong anti-interference ability, so that efficient screening can be achieved in traditional Chinese medicine crude extracts containing various impurities.

[0022] The 96-well microdialysis plate has a molecular weight cut-off of 3kD-30kD. Preferably, the molecular weight cut-off is 8-15kD, so that large molecular impurities can be filtered out.

[0023] The protein solution includes any one or more of PKM2, STAT3, APP, GLUL, etc.

[0024] The mass concentration of the protein in the protein solution is 0.05-0.5mg / ml;

[0025] The solvent used for the test solution and the protein solution includes an ammonium acetate buffer solution;

[0026] The pH of the to-be-tested solution and the protein solution is 7.0-8.0;

[0027] The mass concentration of the to-be-tested substance in the to-be-tested solution is 0.1-0.5 g / ml;

[0028] In step 4, standing at-20℃ to-15℃ for 0.5-1.5 h.

[0029] In step 5, the differential analysis is carried out by performing multivariate statistical analysis on the mass spectrum data of the experimental group and the control group samples, using a local database to annotate the characteristic ion peaks in the LC-MS data; and based on the annotation results, volcano plot analysis is used for differential screening, and the screening standard is based on the fold change value (Fold Change) of the compound between groups and the statistical significance (p-value) of the hypothesis test, so that potential active ingredients with biological significance can be efficiently and intuitively identified.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The present application proposes a high-throughput screening technology based on balanced dialysis-mass spectrometry, which introduces balanced dialysis to replace the traditional centrifugal separation process, effectively avoiding the problem of weakly bound molecules being lost, and can more accurately retain low-affinity molecules in natural products. At the same time, combined with the high-throughput design of the 96-well plate and the characteristics of the dialysis membrane selectively retaining macromolecular impurities, the screening efficiency is greatly improved, meeting the needs of large-scale natural product screening. By breaking through the limitations of traditional screening technology, a new type of screening technology with high efficiency, precision and anti-interference is provided, which is suitable for various complex natural product systems and has strong universality, providing important technical support for high-throughput screening of active ingredients of natural products. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a schematic diagram of the traditional ultrafiltration and balanced dialysis process, wherein (A) is a schematic diagram of the ultrafiltration method; (B) is a schematic diagram of the balanced dialysis method.

[0033] Figure 2A It is the balanced dialysis experiment result of PKM2 and standard product mixture.

[0034] Figure 2B It is the balanced dialysis experiment result of STAT3 and standard product mixture.

[0035] Figure 2C It is the balanced dialysis experiment result of APP protein and standard mixture.

[0036] Figure 2D It is the balanced dialysis experiment result of GLUL protein and standard mixture.

[0037] Figure 3A The ultrafiltration of the crude extract of Fuzi.

[0038] Figure 3B The total ion chromatogram of the crude extract of Fuzi.

[0039] Figure 3C The results of the equilibrium dialysis experiment of the polysaccharide-free extract of Fuzi.

[0040] Figure 3D The results of the equilibrium dialysis experiment of the crude extract of Fuzi.

[0041] Figure 4A The results of the equilibrium dialysis experiment of the mixture of PKM2 and standard.

[0042] Figure 4A The results of verifying the binding of deoxyaconitine and PKM2 by SPR.

[0043] Figure 4B The results of the molecular docking of PKM2 and noracoinitine.

[0044] Figure 5A The results of verifying the binding of noracoinitine and PKM2 by CETSA.

[0045] Figure 5B The results of verifying the binding of noracoinitine and PKM2 by SPR.

[0046] Figure 5C The cell viability of A549-PKM2 overexpressed cells and normal A549 cells after administration of noracoinitine.

[0047] Figure 5D The extracellular acidification rate and glycolytic capacity of A549 cell line cells after administration of noracoinitine. DETAILED DESCRIPTION

[0048] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application. Any modification or equivalent replacement made by those skilled in the art based on the technical solutions of the present application without departing from the spirit and scope of the present application should be covered within the protection scope of the present application.

[0049] The raw materials used in the following specific embodiments are all purchased from the market, and the 96-well microdialysis plate adopts SWISSCII Diaplate TM with a molecular weight cut-off of 10 kDa.

[0050] Example 1

[0051] Step 1, use 150 mM ammonium acetate to activate 96-well microdialysis plate (SWISSCII dialysis plate TM , 10 kD), add 10 ul to each well in the upper layer and 300 ul to each well in the lower layer to activate the semi-permeable membrane. After standing for 30 minutes, quickly discard the upper and lower liquids to ensure that the semi-permeable membrane is always wet. Step 2, prepare a mixed solution containing 14 standard aconite alkaloids, with a total alkaloid concentration of 100 mM. The specific names are shown in Table 1. The concentration of each component in the standard mixture solution is accurately calculated to ensure the accuracy of the experimental results. Add the prepared solution to be screened to the standard mixture solution, and adjust the pH to 7.4.

[0052] Arrange 100 pg / ml of PKM2 protein ammonium acetate buffer solution in 8 pL per well on a 10 kDa MWCO 96-well microdialysis plate, set up three repeats, and seal with an aluminum foil seal to create a protein chamber. On the back, add 300 pL of the above standard mixture solution to each protein chamber, and seal with the same aluminum foil seal to form a traditional Chinese medicine standard chamber.

[0053] Step 3, place the loaded microdialysis plate on a rotary shaker (120 rpm) in the dark at 4°C and incubate for 24 hours. Recover 5 ul of the upper and lower solutions, add 4 volumes of methanol to the recovered solutions, vortex mix for 5 s, and then place in a -20°C refrigerator for 1 hour to completely denature and precipitate the proteins. Then centrifuge to remove the proteins, and take the supernatant for analysis on LC-MS.

[0054] Step 3, use the local library to annotate the characteristic peaks in the data obtained from LC-MS analysis, and finally perform differential analysis on the annotated results to screen out compounds with significant differences. The overall experimental steps are shown in Figure 5E (B).

[0055] Similarly, using the 100 μM standard mixture solution prepared in step 2 above as a control, 100 μL of the standard mixture solution and 100 μL of PKM2 buffer (100 μg / ml) were mixed to obtain a sample solution with a total volume of 200 μL. The mixture was mixed for 30 seconds, incubated at 37°C for 30 minutes, and then transferred to a YM-10 ultrafiltration tube (Millipore, Bedford, MA) with a molecular weight of 10 kD, and centrifuged at 13000 rpm / min at 25°C for 15 minutes. The unbound components were washed away by centrifuging three times with 100 μL of PBS buffer solution at 25°C. Then 200 μL of methanol was injected into the filter membrane, and centrifuged at 13000 rpm / min for 10 minutes to release the bound protein ligand, and repeated three times. In the control group, the same volume of inactivated CDC123 solution was used instead. The ultrafiltrate was collected and subjected to LC-MS analysis, and the step diagram is shown in Fig. 1 (A). Figure 1

[0056] The screening results of the standard mixture by equilibrium dialysis and ultrafiltration are shown in Fig. 2, and the binding rate data obtained by ultrafiltration (Table 2) are compared with the compounds with significant differences obtained by equilibrium dialysis screening Figure 1 ), and the results show that the small molecules with the best binding effect to PKM2 obtained by the two methods are consistent, which are pennogenine-N-oxide, as Figure 2A shown, proving the reliability of this method. At the same time, three different proteins (STAT3, APP, GLUL) were used for verification, the experimental process remained unchanged, only the protein was replaced, and the results are shown in Figs. Figure 2A 、 Figure 2B 、 Figure 2C The different screening results prove that the above results have specificity.

[0057] Table 1: 14 kinds of aconite alkaloids in English

[0058]

[0059] Table 2: PKM2 ultrafiltration binding rate results of standard mixture

[0060]

[0061]

[0062] Example 2: Screening of natural crude extract for target binding components by equilibrium dialysis method

[0063] To verify the application of the equilibrium dialysis method in the screening of natural crude extract, this embodiment screens the crude extract of aconite and compares the screening results before and after removing polysaccharides.

[0064] ​First, weigh 10g of processed aconite root, add 1L of water, bring to a boil over high heat, then simmer over low heat for 1 hour. Next, add another 1L of water, bring to a boil, and simmer over low heat for 40 minutes. Filter the liquid through a 200-mesh screen. Transfer the filtrate to a rotary evaporator and concentrate under reduced pressure to 50mL to obtain a 0.2g / ml crude aconite extract solution. Part of this solution was subjected to overnight precipitation with 4 times its volume of 75% ethanol to remove polysaccharides, while the other part remained in its original state as a control.

[0065] Subsequently, the polysaccharide-free and polysaccharide-free Aconitum carmichaelii extract solutions were used for equilibrium dialysis and ultrafiltration experiments, respectively. All experimental conditions were the same as in Example 1, and the same local database and alkaloid compounds were selected for difference analysis as in Example 1.

[0066] Ultrafiltration experiments showed that crude Aconitum carmichaelii extract without polysaccharide removal adhered to the ultrafiltration tube, severely affecting the ultrafiltration effect; for example... Figure 2D As shown, except for polysaccharides, the Aconitum carmichaelii extract can be used normally in ultrafiltration experiments, and the total ion chromatogram is as follows. Figure 3A As shown in Table 3, the binding rate results are as follows.

[0067] Table 3. Results of ultrafiltration binding rate of PKM2 with Aconitum carmichaelii extract (excluding polysaccharides).

[0068]

[0069]

[0070] The results of the equilibrium dialysis experiment showed that, except for polysaccharides, the small alkaloid molecules in the Aconitum carmichaelii extract had a relatively weak binding to PKM2. Figure 3B ), while the alkaloid molecules in the crude extract of Aconitum carmichaelii without polysaccharide removal showed stronger binding ( ), Figure 3C The results were consistent with those of ultrafiltration, indicating that balanced dialysis has high sensitivity for screening natural products and can effectively identify small molecules that bind to target proteins, thus verifying the application value of balanced dialysis in natural product screening.

[0071] Example 3: Validation of the method for screening weakly binding molecules using equilibrium dialysis

[0072] Regarding PKM2 and 14 standards in Example 1, in the comparative experiment of Example 1, we found that although the equilibrium dialysis method and ultrafiltration method showed high consistency in the screening results of most Aconitum carmichaelii alkaloids, the equilibrium dialysis method could additionally identify small molecule components that were not detected by ultrafiltration. To explore the source of this difference, we compared and analyzed the screening results of the two methods (ultrafiltration binding rate results are shown in Table 2, equilibrium dialysis method experimental results are shown in Table 3). Figure 3D The specific experimental steps were the same as in Example 1. The results showed that one of the different compounds was deoxyaconitine, as shown in Table 2.Figure 4A The phenomenon is presumably related to the weak binding complex dissociation caused by centrifugal shear force in ultrafiltration.

[0073] To verify this presumption, we further used surface plasmon resonance technology to detect the binding of deoxyaconitine and PKM2 protein. SPR experiments showed that the dissociation constant of the two was 175 μM, which belonged to the level of weak binding. Figure 4A This result is consistent with the screening data of equilibrium dialysis method, while in the ultrafiltration experiment, the weak binding complex is easily dissociated during centrifugation, so it cannot be identified.

[0074] In summary, the equilibrium dialysis method has natural advantages in maintaining binding balance and avoiding mechanical shear interference, and can effectively screen weakly bound small molecules that are difficult to detect by ultrafiltration. This example not only verifies the sensitivity of the equilibrium dialysis method in weakly bound ligand recognition, but also provides a reliable technical path for the discovery of weakly interacting molecules in complex natural product systems.

[0075] Example 4: Small molecule and target binding verification

[0076] Human non-small cell lung cancer cell line: A549 cells

[0077] 1. Molecular docking: AutoDock 4.2.6 was used to dock PKM2 protein and deoxyaconitine. The receptor protein was pretreated by removing water, adding hydrogen and assigning charges, etc. before docking, and the small molecule was imported into the calculation after energy minimization and rotatable bond setting. The results showed that the binding free energy (ΔG) of the two was -7.6 kJ / mol, indicating a strong binding tendency. Figure 4B

[0078] 2. CETSA experiment: Cell thermal shift assay (CETSA) was used to evaluate the binding ability of deoxyaconitine and PKM2. 50 μM deoxyaconitine was mixed with cell lysate in proportion, and the control group used PBS solution containing 0.1% DMSO, incubated at room temperature for 3 min, then heated in PCR instrument for 3 min, and transferred to ice to terminate the reaction. After centrifugation to remove the precipitate, the supernatant was subjected to Western blot analysis. The results showed that in the deoxyaconitine treatment group, the stability of PKM2 protein under elevated temperature was significantly enhanced, indicating that the compound can directly bind to PKM2 and improve its thermal stability. Figure 5A

[0079] ​​3. SPR experiment: Surface plasmon resonance (SPR) analysis was performed using Biacore 8K platform. PKM2 was immobilized on the CM7 chip surface (about 25,000 RU), and the binding and dissociation curves were measured under the condition of different concentrations of nor-laudanosine in the flow phase, and the kinetic parameters were obtained by multi-cycle analysis. The results showed that nor-laudanosine had moderate affinity binding with PKM2, and the equilibrium dissociation constant (Kd) was 14.4 μM, further verifying the direct interaction between the two. Figure 5B

[0080] 4. Overexpression cell efficacy verification: PKM2 overexpression A549 cell model and control group A549 cell model were constructed respectively, and different concentrations of nor-laudanosine were used for cell administration, and MTT method was used to evaluate the proliferation and survival of cells. By measuring cell viability, it was found that the PKM2 overexpression group cells showed significantly higher survival rate under the treatment of all concentrations of nor-laudanosine, and with the increase of drug concentration, the cell viability decreased less than the normal cell group, as shown in Figure 5C . It showed that PKM2 overexpression A549 cells exhibited stronger drug resistance under the treatment of nor-laudanosine, further supporting the role of PKM2 as a potential target.

[0081] 5. Seahorse experiment: Seahorse XF96 analysis platform was used to detect glycolysis (ECAR) parameters. Cells were seeded in Seahorse cell culture plates, and when the density reached 70%-80%, drug treatment was performed. After 24 h of administration, the medium was replaced with XFRPMI without sugar and starved for 1 h, and then the substrate and inhibitor were loaded according to the experimental type: ECAR experiment was injected with glucose, oligomycin and 2-deoxyglucose (2-DG) in turn; OCR experiment was injected with oligomycin, FCCP and rotenone / antimycin A in turn. Each stage during the detection process included 3 mixing and 3 measurements. The results are shown in Figure 5D Figure 5E . Nor-laudanosine significantly reduced the basal glycolytic rate and maximum glycolytic capacity in a dose-dependent manner, suggesting that the compound may affect the glycolysis pathway by regulating PKM2.​

Claims

1. A method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry, characterized in that, Including the following steps: Step 1: Prepare crude extracts of traditional Chinese medicine by decoction or concentration as the test solution; Step 2: Add protein solution to one side of the activated and moistened 96-well microdialysis plate, and add the test solution to the other side; Step 3: After loading the solution in Step 2, the 96-well microdialysis plate was incubated with constant temperature shaking at 0-5℃ in the dark for 18-26 hours. Step 4: Collect the samples from the upper and lower layers of the incubated 96-well microdialysis plate, add methanol and mix, let stand to precipitate denatured proteins, centrifuge to remove the precipitate and take the supernatant for LC-MS analysis. Step 5: Annotate the characteristic peaks of the LC-MS data of the supernatant in Step 4, and screen the desired natural product active ingredients by comparing them with the standard database.

2. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The crude extracts of the traditional Chinese medicine include crude extracts of Aconitum carmichaelii.

3. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The crude extract of the Chinese herbal medicine was not subjected to polysaccharide removal treatment.

4. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The molecular weight cutoff of the 96-well microdialysis plate is 3-30 kDa.

5. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The protein solution includes any one or more of PKM2, STAT3, APP, and GLUL.

6. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The protein concentration in the protein solution is 0.05-0.5 mg / ml.

7. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The solvent used for the test solution and the protein solution is ammonium acetate buffer solution.

8. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The pH of the test solution and the protein solution is 7.0-8.

0.

9. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, The concentration of crude extract of traditional Chinese medicine in the test solution is 0.1-0.5 g / ml.

10. The method for high-throughput screening of active ingredients in natural products based on equilibrium dialysis-mass spectrometry according to claim 1, characterized in that, In step 4, let it stand at -20℃ to -15℃ for 0.5-1.5 hours.

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