Method for separating active components in tobacco based on liquid-phase subsection separation, SPR (Surface Plasmon Resonance) and affinity chromatography enrichment, product and application

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating active components in tobacco based on liquid-phase segmented separation, SPR, affinity chromatography enrichment and mass spectrometry detection, a product and application. According to the method, liquid chromatography separation, SPR detection screening, affinity chromatography enrichment and mass spectrum identification technologies are fused, and active small molecules are screened and enriched from a complex system by virtue of high sensitivity of SPR and accuracy of mass spectrum. The method comprises the following steps: firstly, carrying out liquid chromatography segmented separation on extract components to improve SPR detection precision and affinity enrichment efficiency; by means of specific binding of Ni Sepharose FF (NTA) resin and a His tag, more target proteins are fixed, and the micromolecule enrichment effect is further enhanced; and finally, identifying the molecular structure by using mass spectrum, and locking the bioactive small molecule combined with the target protein. According to the strategy, efficient enrichment of small molecule ligands is achieved, and a new way is provided for early-stage development of small molecules in drug discovery. 6.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a method, product and application for separating active ingredients in tobacco based on liquid phase segmentation separation, SPR and affinity chromatography enrichment. Background Art

[0002] In the fields of drug discovery and active ingredient screening, the separation and identification of active small molecules that bind to specific proteins from complex plant extracts has always been a research focus. Currently, a variety of technologies have been applied in this field.

[0003] Liquid chromatography is a commonly used technique for separating substances. Due to the varying properties of molecules in a liquid system (such as polarity, molecular weight, and charge), their distribution between the stationary and mobile phases varies, leading to varying migration rates within the separation column. Gradient elution allows for the segmented separation of liquid substances.

[0004] Surface plasmon resonance (SPR) technology is a classic method for monitoring molecular interactions, suitable for monitoring interactions between proteins and small molecules, proteins and proteins, proteins and nucleic acids, etc. This technology is based on the plasma resonance induced by light at the metal-dielectric interface and the resulting evanescent wave phenomenon. By monitoring the changes in the refractive index of light caused by the interaction of biomolecules on the surface of the SPR biosensor, molecular interactions can be analyzed. For example, when analyzing the interaction between proteins and small molecules, the target protein is fixed to the surface of the SPR sensor, and the analyte to be measured flows through the sensor surface in the form of a solution. When they interact, the refractive index changes, which is then detected and used to calculate the binding and dissociation of the target protein and the analyte. SPR can provide information including k on 、k off , affinity and molecule-protein interaction time. It also has the advantages of high sensitivity and accuracy, label-free and real-time detection. Therefore, SPR is now regarded as the "gold standard" for studying biomolecular interactions and is widely used in small molecule ligand screening. For example, Chen et al. used SPR and mass spectrometry to identify 5 small molecule ligands of STAT3 protein from extracts of 32 Chinese herbal medicines. Cao et al. also identified small molecule ligands of TNF-R1 protein from rhubarb extracts based on SPR technology. Affinity chromatography technology, which utilizes the affinity specificity between molecules and specific matrices, can achieve the separation and enrichment of target substances through a binding-elution procedure. It is also a commonly used method for researchers to separate and enrich target products from complex systems.

[0005] Over the past few decades, mass spectrometry has become one of the fastest-growing and most widely used analytical techniques for molecular structure identification, primarily due to its exceptionally high accuracy and low detection limits. The technique is based on molecular ionization, followed by analysis using mass-to-charge ratio detection. Initially used to identify the structure of a single substance, mass spectrometry has evolved into a high-throughput technology capable of simultaneously examining the chemical structures of multiple substances in complex systems, as exemplified by its applications in proteomics and metabolomics.

[0006] While the aforementioned technologies have demonstrated promising applications in active ingredient screening, several challenges remain in efficiently enriching active small molecules that bind to specific proteins from complex systems such as plant extracts. First, plant extracts are complex, containing numerous structurally diverse compounds. Direct screening for active small molecules can interfere with detection, reducing accuracy and efficiency. Traditional techniques often struggle to efficiently isolate and enrich target active small molecules from such complex systems. Second, in SPR assays, the complex extract composition can lead to false-positive or false-negative results, compromising accurate assessment of small molecule-protein binding activity. Furthermore, the low concentrations of target active small molecules in extracts make traditional methods ineffective in enrichment, hindering subsequent structural identification. Furthermore, affinity chromatography, when applied to complex extracts, presents challenges in efficiently immobilizing the target protein and improving the efficiency of enriching low-abundance active small molecules. Summary of the Invention

[0007] To address the above-mentioned shortcomings, the present invention is based on liquid chromatography separation, surface plasmon resonance (SPR) detection and screening, affinity chromatography enrichment and mass spectrometry identification technology, making full use of the high sensitivity and high accuracy of SPR detection and mass spectrometry identification technology, as well as the advantages of simple operation of liquid chromatography and affinity chromatography, to provide a method for screening and enriching active small molecule compounds targeting specific proteins from complex systems, so as to achieve more efficient and accurate separation and identification of active small molecules binding to specific proteins from plant extracts, and promote the early development of small molecules in the drug discovery process.

[0008] The technical solution of the present invention is: In one aspect, the present invention provides a method for separating and identifying active ingredients in plants based on liquid phase segmented separation, SPR, affinity chromatography enrichment and mass spectrometry detection, characterized in that it comprises the following steps: S1. Using liquid chromatography to separate the plant extract into sections to obtain multiple components; S2. Immobilize the target protein on an SPR chip, use SPR technology to detect the binding activity of each component obtained in step S1 with the target protein, and screen out components with high responsiveness; S3, immobilizing the target protein on an affinity chromatography matrix, and enriching the high-responsiveness components screened out in step S2 for small molecule compounds that bind to the target protein through affinity chromatography; S4. Use mass spectrometry technology to perform structural identification on the small molecule compounds enriched in step S3.

[0009] Specifically, the liquid chromatography in step S1 is semi-preparative liquid chromatography.

[0010] Preferably, the semi-preparative liquid chromatography column is a C18 bonded silica gel chromatography column.

[0011] Further preferably, the semi-preparative liquid chromatography column is Agilent Zorbax SB-C18 (5 μm, 9.4 × 250 mm).

[0012] Specifically, the mobile phase of the semi-preparative liquid chromatography is phase A: an aqueous solution containing 0.1% formic acid, phase B: methanol; The gradient elution program of the semi-preparative liquid chromatography was as follows: 0-3.75 minutes, 98% A; 3.75-7.5 minutes, 98% A→15% A; 7.5-25 minutes, 15% A→0% A; 25.25-40 minutes, 98% A; The sample loading volume of the semi-preparative liquid chromatography was 900 μL, and the flow rate was 1 mL / min.

[0013] Specifically, the components in step S1 are eluted and collected as one component every 1 minute from 10 minutes to 40 minutes after the injection.

[0014] Specifically, the target protein in step S2 is a Kras protein with a His tag, which is fixed to an SPR chip in a sodium acetate buffer with a pH of 4.5-5.5.

[0015] Preferably, the pH of the sodium acetate buffer is 5.0.

[0016] Specifically, the buffer solution for the SPR detection in step S2 is a PBS solution supplemented with 0.05% Tween 20 and 5 μM GDP.

[0017] Specifically, the affinity chromatography matrix in step S3 is Ni Sepharose FF (NTA) resin.

[0018] Specifically, the enrichment process in step S3 includes: incubating the target protein with the matrix, loading the high-responsiveness component, and eluting the bound small molecule compounds with a 0.5% formic acid solution after washing.

[0019] 11. The method according to claim 1, characterized in that the mass spectrometry technology in step S4 is LC-MS / MS, and the small molecule structure is identified by comparison with a metabolite structure database.

[0020] Specifically, the small molecule compound in step S4 is (R,S)-Anatabine, 7-[4-(tert-butyl)phenyl]-2-methyl-5-(methylthio)pyrazolo[1,5-a]pyrimidine-6-carbonitrile, Carbamazepine, NP-007866, 9-Oxo-4,5,6,7,8,9-hexahydro-pyrazolo[5,1-b]quinazoline-3-carbonitrile, 4,4-dimethyl-1-(3-nitrophenyl)-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-one, Decarbamoyl-neosaxitoxin, NP-022229, Di(2-ethylhexyl) phthalate, Thiacloprid, Didecyldimethylammonium chloride, PEG Monolaurate 5, and n-butylene glycol. n6, 4-[5-(5-anilino-1,2,4-thiadiazol-3-yl)-4-methylpyrimidin-2-yl]benzamide and 6-(4-methoxyphenyl)pyrimidine-2,4-diamine.

[0021] Specifically, the plant in step S1 is tobacco, wolfberry, liquorice, astragalus, chrysanthemum, snow lotus, salvia miltiorrhiza, mint, ginseng, yew, tea tree, lavender, olive, cordyceps or dendrobium.

[0022] Preferably, the plant is tobacco leaves, and the extraction solvent is 80% methanol solution.

[0023] In another aspect, the present invention provides a kit for enriching active small molecules that bind to specific proteins, wherein the kit comprises the reagents or standards of the aforementioned method.

[0024] Specifically, the kit also includes a positive sample.

[0025] In another aspect, the present invention provides use of the aforementioned method or kit for enriching active small molecules that bind to specific proteins from plant extracts.

[0026] Specifically, the plant is tobacco, wolfberry, liquorice, astragalus, chrysanthemum, snow lotus, salvia miltiorrhiza, mint, ginseng, yew, tea tree, lavender, olive, cordyceps or dendrobium.

[0027] Preferably, the plant is tobacco leaves, and the extraction solvent is 80% methanol solution.

[0028] The beneficial effects of the present invention are: First, liquid chromatography was used to fractionate the metabolite extract. This process reduced the complexity of the initial metabolite extract composition, improved the accuracy of subsequent SPR detection, and enhanced the efficiency of subsequent affinity enrichment. Furthermore, through the specific binding of Ni Sepharose FF (NTA) resin and the His tag, more target proteins could be loaded into the affinity chromatography system. This, in turn, made it possible to enrich more target small molecule compounds, thereby improving the efficiency of enriching low-abundance target small molecules in complex metabolite systems. Finally, mass spectrometry analysis was used to identify the molecular structures of the enriched compounds, and some bioactive small molecules that may bind to the target proteins were discovered. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the liquid chromatography separation components of tobacco leaf extract. The ultraviolet absorption detection wavelength is 210 nm. The numbers 1-29 represent the different separation components of tobacco leaf extract.

[0030] Figure 2 The adsorption effect of Kras (G12C) protein on the chip SPR High Capacity AmineSensor under different pH buffer conditions.

[0031] Figure 3 After the chip was activated with EDC and NHS (a), Kras (G12C) protein and a control group (protein-free buffer) were immobilized to two detection points on the chip by amino coupling at pH 5.0 (b). The Kras (G12C) and control groups were then blocked with ethanolamine (c).

[0032] Figure 4 Figure 2 shows the SPR detection curves of BI 2865 small molecule and Kras (G12C) protein at concentrations of 1.56 nM, 3.12 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. The Kd value (3.03 nM) for the binding of BI 2865 to Kras (G12C) protein was calculated by fitting.

[0033] Figure 5Binding and dissociation curves of components 2-12 (a), 13-22 (b), 23-29 and 1 (c) of tobacco leaf extract with SPR chip-Kras (G12C) protein.

[0034] Figure 6 This is a protein gel analysis of the effluent from each wash and elution step during affinity chromatography enrichment. All samples were separated by 12% polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue, and destained with 30% methanol + 10% glacial acetic acid before gel imaging. Lane 1: Kras(G12C) protein control; lane 2: Kras(G12C) protein flow-through after column loading; lane 3: PBS (0.05% Tween 20, 5 μM GDP) wash flow-through; lane 4: metabolite fraction flow-through; lane 5: ddH2O wash flow-through; lane 6: 0.5% formic acid eluate. M: protein maker.

[0035] Figure 7 is the chemical structural formula of (R,S)-Anatabine.

[0036] Figure 8 The chemical structural formula is 7-[4-(tert-butyl)phenyl]-2-methyl-5-(methylthio)pyrazolo[1,5-a]pyrimidine-6-carbonitrile.

[0037] Figure 9 is the chemical structural formula of Carbamazepine.

[0038] Figure 10 is the chemical structural formula of NP-007866.

[0039] Figure 11 It is the chemical structural formula of 9-Oxo-4,5,6,7,8,9-hexahydro-pyrazolo[5,1-b]quinazoline-3-carbonitrile.

[0040] Figure 12 The chemical structural formula is 4,4-dimethyl-1-(3-nitrophenyl)-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-one.

[0041] Figure 13 is the chemical structural formula of Decarbamoyl-neosaxitoxin.

[0042] Figure 14 is the chemical structural formula of NP-022229.

[0043] Figure 15 It is the chemical structural formula of Di(2-ethylhexyl) phthalate.

[0044] Figure 16 is the chemical structural formula of Thiacloprid.

[0045] Figure 17 It is the chemical structural formula of Didecyldimethylammonium.

[0046] Figure 18 is the chemical structural formula of PEG Monolaurate n5.

[0047] Figure 19 is the chemical structural formula of PEG Monolaurate n6.

[0048] Figure 20 The chemical structural formula is 4-[5-(5-anilino-1,2,4-thiadiazol-3-yl)-4-methylpyrimidin-2-yl]benzamide.

[0049] Figure 21 This is the chemical structural formula of 6-(4-methoxyphenyl)pyrimidine-2,4-diamine. DETAILED DESCRIPTION

[0050] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0051] Example 1 1.1 Materials and Reagents Tobacco leaves were obtained from the laboratory plant culture room. The protein Kras (G12C) was purchased from ACROBiosystems (Cat. No. KRS-H51H3-100 μg), guanosine-5-diphosphate disodium salt (GDP) was purchased from Beijing Solebao Technology Co., Ltd., and the small molecule inhibitor BI 2865 was purchased from Taoshu Biotechnology Co., Ltd. (Cat. No. T72062-1 mg BI-2865). HPLC-grade methanol, formic acid, and acetonitrile were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Ni Sepharose FF (NTA) matrix was purchased from Nanjing Novozymes Biotechnology Co., Ltd., and an SPR chip (SPR High Capacity AmineSensor) was purchased from Bruker (Beijing) Technology Co., Ltd.

[0052] 1.2 Extraction of tobacco leaf metabolites Grind 1 g of tobacco leaves in liquid nitrogen, add 5 mL of 80% (v / v) methanol, sonicate for 15 minutes, and place in an ice bath for 5 minutes. Centrifuge at 15,000 × g for 20 minutes, remove the supernatant, and filter through a 0.45 μm filter for later use.

[0053] 1.3 Preparation of leaf metabolite components The supernatant prepared in 1.2 was subjected to semipreparative chromatography to obtain the different fractions of the extract. The instrument model was an Agilent 1260 Infinity II, and the column was an Agilent Zorbax SB-C18 (5 μm, 9.4 × 250 mm). The mobile phase was phase A (H2O, 0.1% formic acid (v / v)) and phase B (methanol). The elution gradient was: 98% A from 0 to 3.75 minutes; 98% A to 15% A from 3.75 to 7.5 minutes; 15% A to 0% A from 7.5 to 25 minutes; and 98% A from 25.25 to 40 minutes. The sample volume was 900 μL, and the flow rate was 1 mL / min. A UV absorbance peak appeared at 10 minutes, and fractions were collected every 1 minute from 10 minutes to 40 minutes. Each component was freeze-dried and reconstituted with 250 μL PBS (supplemented with 0.05% Tween 20 (v / v), 5 μM GDP) for later use.

[0054] 1.4 Preliminary experimental selection of the appropriate pH for protein immobilization on the chip Kras (G12C) protein was diluted and dissolved in 10 mM sodium acetate buffer at pH 4, 4.5, 5, and 5.5 to 20 μg / mL. Using the Bruker Sierra SPR-32 instrument control program, the Kras (G12C) protein diluted and dissolved at different pH values ​​was loaded onto different detection points of the SPR High Capacity Amine Sensor chip. The loading time was 60 s and the flow rate was 5 μL / min. The response [RU] was recorded. Each detection point was then washed with 0.1 M HCl to remove the protein loaded at each detection point and prepare it for reuse in the next detection point.

[0055] 1.5 Immobilization of target protein on SPR chip Immobilization of the target protein followed the standard protocol for the Bruker Sierra SPR-32 instrument. Briefly, the detection point of the HighCapacity Amine Sensor chip was activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, 20 μg / mL of Kras(G12C) protein was loaded onto the chip detection point at a rate of 10 μL / min at pH 5.0 for 360 s. The detection point was then blocked with 1 M ethanolamine. A separate detection point was set up as a control detection point. No protein was added during loading, and only the protein-dissolving buffer was passed through. All other steps remained unchanged.

[0056] 1.6 Activity verification after target protein immobilization After immobilizing Kras(G12C) protein to the chip, the activity of the immobilized Kras(G12C) protein was verified using BI2865, a previously reported small molecule inhibitor of Kras(G12C). BI 2865 was diluted in PBS (supplemented with 0.05% Tween 20 (v / v) and 5 μM GDP) to concentrations of 1.56 nM, 3.12 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM, respectively. The sample was then loaded onto the Kras(G12C) protein-immobilized assay spot and a control assay spot. The assay was run at a flow rate of 25 μL / min with an association time of 120 s and a dissociation time of 180 s. RU values ​​were recorded, and Kd values ​​were calculated from the curve.

[0057] 1.7 SPR detection of each component After immobilizing the target protein Kras(G12C) to the SPR chip, leaf fractions were sampled and each fraction was loaded for analysis. Each fraction was split into two aliquots, one flowing through a Kras(G12C)-loaded detection spot and the other through a control unloaded detection spot. The system used PBS (supplemented with 0.05% v / v Tween 20 and 5 μM GDP) as the buffer. The association time was 120 s, the dissociation time was 180 s, and the fraction flow rate was 25 μL / min. Response (RU) values ​​were recorded.

[0058] Enrichment of small molecule ligands for Kras(G12C) protein in fraction 1.8 For fractions with high SPR detection reactivity, affinity chromatography was used to enrich small molecule ligands for the Kras(G12C) protein. The procedure was as follows: 500 μL of Ni Sepharose FF (NTA) matrix (Vazyme) was added to a 5 mL gravity chromatography column. The matrix was washed with 10 mL of PBS (supplemented with 0.05% Tween 20 (v / v) and 5 μM GDP), and the waste solution was drained. 12 μg (500 μL) of protein was loaded onto the matrix and incubated at room temperature for 20 minutes with gentle shaking. The waste solution was drained. Subsequently, the matrix was washed with 1 mL of PBS (supplemented with 0.05% Tween 20 and 5 μM GDP), and the waste solution was drained. 60 μL of the upper fraction was added to the matrix system and incubated at room temperature for 20 minutes with gentle shaking. The waste solution was drained. The system was rinsed with 1 mL of ddH2O, and the waste solution was drained. Then, 500 μL of 0.5% formic acid solution was added to the system, incubated at room temperature for 5 min, and the effluent was collected.

[0059] 1.9 Mass spectrometry detection and structural identification The enriched fraction from affinity chromatography was filtered through a 3K filter membrane. The filtrate was freeze-dried and resuspended in 20 μL of 50% methanol. The filtrate was centrifuged at 10,000 x g for 20 minutes at 4°C. The supernatant was analyzed by mass spectrometry. The mass spectrometer was an Orbitrap Eclipse (Thermo Fisher Scientific) using an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm, Waters). The mobile phases were phase A (H2O, 0.1% formic acid (v / v)) and phase B (acetonitrile, 0.1% formic acid (v / v)). The injection volume was 5 μL. The elution gradient was: 0-1 min, 1% A; 1-10 min, 1% B → 60% B; 10-20 min, 60% B → 99% B; 20-22 min, 99% B; 22-25 min, 99% B to 1% B; 25-30 min, 1% B. The flow rate was 0.25 mL / min. Mass spectrometry detection was performed in positive ion mode. The mass spectrometer scan range was m / z 70-800. Ion source H-ESI conditions included: spray voltage: 3.5 kV; sheath gas flow rate: 50 Arb; auxiliary gas flow rate: 10 Arb; ion transfer tube temperature: 325°C; RF lenth: 30%; purge gas flow rate: 1 Arb; and nebulizer temperature: 350°C. The resulting raw files were imported into Compound Discovery for comparison. The selected analysis workflow was the Max ID workflow: Detect and identify all compounds in a single sample (with ddMS2), even compounds with very low abundance. The relevant molecular ion peaks and fragment ion information were compared with the mzCloud, mzVault, ChemSpider, and MassList databases to predict molecular structures. The analysis results were further filtered using the criteria of peak area ≥ 100,000 and mzCloud Best Match ≥ 85. The resulting molecules were considered identified.

[0060] 2.0 Results and Discussion 1. Liquid chromatography segmented separation of tobacco leaf extract One-month-old K326 tobacco leaves were selected and ground with liquid nitrogen and ultrasonically extracted with 80% methanol to obtain leaf extracts. Subsequently, different components were obtained by semi-preparative liquid chromatography using gradient elution. The UV absorption of the eluate was monitored at a wavelength of 210 nm. The results showed that the extract had a UV absorption peak at a retention time of approximately 10 minutes and disappeared around 40 minutes ( Figure 1Therefore, from the 10th minute to the 40th minute, one fraction was collected every minute to obtain 29 fractions of tobacco leaf extract ( Figure 1 ).

[0061] 2. Optimization of immobilization conditions, immobilization effect, and activity verification of Kras(G12C) protein on SPR chip When the SPR High Capacity Amine Sensor chip fixes the protein, the protein first adheres to the chip surface by electrostatic adsorption to complete the subsequent amino coupling fixation. Different pH conditions will affect the adsorption efficiency of the protein. pH 4.0, 4.5, 5.0, and 5.5 conditions were selected respectively to conduct pH Scouting experiments to select the most suitable fixation pH conditions. The results showed that under the four pH conditions, Kras (G12C) protein can be well adsorbed on the chip surface, and the RU values ​​are 7000 (pH 4.0), 4400 (pH 4.5), 3800 (pH 5.0), and 3400 (pH 5.5). Figure 2 The responsiveness under these four pH conditions can meet the requirements of subsequent experiments. Considering that excessive pH may affect the activity of the protein, pH 5.0 was selected for protein fixation.

[0062] Under pH 5.0 buffer conditions, the protein was fixed to the SPR High Capacity Amine Sensor chip. The fixation results are shown in Figure 2. Figure 3 As shown in the figure, after the chip was activated by EDC and NHS, Kras(G12C) protein was fixed to the chip by amino coupling, and the RU value reached 11000, indicating that the protein was well fixed. However, the control point (no protein flow) had no response value (RU=0) during the fixation process ( Figure 3 Since the protein was fixed to the chip by amino coupling, whether it still had activity after fixation needed further verification. Therefore, the reported small molecule inhibitor of Kras (G12C) protein BI 2865 was used as the test substance to verify whether the protein still had activity after being fixed to the chip. Figure 4 As shown in Figure 2, 1.56 nM, 3.12 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM concentrations of BI 2865 were passed through the chip immobilized with Kras(G12C) protein, and the binding and dissociation curves of each concentration were obtained ( Figure 4Calculations revealed a Kd value of 3.03 nM for the binding of BI 2865 to the Kras(G12C) protein, close to the 4.5 nM reported in the literature (Nature. 2023 Jul;619(7968):160-166. doi:10.1038 / s41586-023-06123-3). This suggests that the Kras(G12C) protein remains active after immobilization on the chip and can be used for screening active small molecules.

[0063] 3. Analysis of the binding ability of each extract component with Kras (G12C) protein Each component was divided into three batches and subjected to SPR detection of Kras (G12C) protein chip. The detection response results of each component were as follows: Figure 5 As shown in the figure, the response values ​​range from approximately 20 RU to approximately 900 RU, indicating that the content of active small molecules that can bind to the Kras(G12C) protein varies significantly among different fractions. This also indirectly demonstrates the effectiveness of the liquid-phase fractionation process, which can isolate fractions with different binding activities for the Kras(G12C) protein. Selecting fractions with high responsiveness for subsequent enrichment can improve enrichment efficiency.

[0064] Based on the above considerations, and to ensure comprehensive enrichment of active small molecules, three fractions with high responsiveness were selected from each batch for the next step of affinity chromatography enrichment of active small molecules. These fractions were fractions 3, 4, and 12 from batch 1, fractions 13, 14, and 15 from batch 2, and fractions 1, 26, and 27 from batch 3, for a total of nine fractions.

[0065] 4. Enrichment of active components that specifically bind to Kras (G12C) protein in the extract fractions Utilizing the affinity between the Ni-NTA matrix and the protein histidine tag, the His-tagged Kras (G12C) protein was loaded onto the matrix. Fractions with high SPR response values ​​were then passed through the system. After washing with ddH2O, the eluate was eluted with 0.5% formic acid (v / v). The eluate was enriched with small molecules that bind to the Kras (G12C) protein. Compared to the aforementioned SPR chip-based enrichment method, the enrichment method of the present invention can immobilize larger amounts of protein (up to mg levels), thus enriching more small molecular weight compounds, facilitating subsequent structural identification. Based on this method, tobacco extract fractions 1, 3, 4, 12, 13, 14, 15, 26, and 27 were enriched for active small molecules by affinity chromatography. During the enrichment process, it was necessary to ensure that the proteins remained active at all times. Therefore, the effluent of each step was subjected to polyacrylamide gel electrophoresis to detect whether the effluent of each washing and elution step contained Kras (G12C) protein, so as to determine whether each step of the enrichment operation would lead to protein inactivation and dissociation from the matrix. Figure 6 It can be seen that the effluent of each step does not contain Kras(G12C) protein ( Figure 6 ), indicating that the protein remains active throughout the entire affinity chromatography process and the elution steps do not cause the protein to lose activity, verifying the feasibility and effectiveness of the present invention based on affinity chromatography enrichment.

[0066] 5. Chemical structure identification of enriched components After the enrichment procedure, the structures of these enriched components were elucidated using liquid chromatography-mass spectrometry (LC-MS / MS)-based metabolomics. Raw primary and secondary mass-to-charge ratio data were collected on the LC-MS / MS system and then compared to an online metabolite structure database using Compound Discovery software. Similar metabolomics analyses were performed on a sample pool obtained through the same enrichment procedure using PBS buffer as the response component, as well as a sample pool obtained using whole plant extract as the response component but enriched in a matrix without Kras(G12C) protein (all other steps were identical). These samples served as negative controls. The analysis identified 19, 14, 13, 12, 15, 16, 12, and 10 small molecules in enriched fractions 3, 4, 5, 12, 13, 14, and 26, and fractions 1 and 27, respectively. It is worth noting that some small molecules were identified simultaneously in different fractionation groups, but many were only identified in samples enriched in specific fractionation groups, indicating that the initial liquid chromatography separation was effective. After further confirmation combined with mass spectrometry peak shape, 15 reliable active small molecules were finally screened. These molecules are believed to be able to bind to the KRAS (G12C) protein and therefore may serve as small molecule inhibitors of the protein (Table 1). The chemical formula is as follows Figure 7-Figure 21 shown.

[0067] Table 1 Small molecule information identified by mass spectrometry

[0068] Note: The content increase multiple refers to the increase multiple of the content after enrichment treatment compared with that before enrichment treatment.

[0069] Table 1 Small molecule information identified by mass spectrometry

[0070] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A method for separating active ingredients in plants based on liquid phase segmentation separation, SPR, affinity chromatography enrichment and mass spectrometry detection, characterized in that: The following steps are involved: S1. Using liquid chromatography to separate the plant extract into sections to obtain multiple components; S2. Immobilize the target protein on an SPR chip, use SPR technology to detect the binding activity of each component obtained in step S1 with the target protein, and screen out components with high responsiveness; S3, immobilizing the target protein on an affinity chromatography matrix, and enriching the high-responsiveness components screened out in step S2 for small molecule compounds that bind to the target protein through affinity chromatography; S4. Use mass spectrometry technology to perform structural identification on the small molecule compounds enriched in step S3.

2. The method according to claim 1, characterized in that The liquid chromatography in step S1 is semi-preparative liquid chromatography.

3. The method according to claim 2, characterized in that The column for the semi-preparative liquid chromatography is a C18 bonded silica gel chromatography column.

4. The method according to claim 3, characterized in that The column for the semi-preparative liquid chromatography was Agilent Zorbax SB-C18, 5 μm, 9.4 × 250 mm.

5. The method according to claim 4, characterized in that The mobile phases of the semi-preparative liquid chromatography were phase A: an aqueous solution containing 0.1% formic acid, and phase B: methanol; The gradient elution program of the semi-preparative liquid chromatography was as follows: 0-3.75 minutes, 98% A; 3.75-7.5 minutes, 98% A→15% A; 7.5-25 minutes, 15% A→0% A; 25.25-40 minutes, 98% A; The sample loading volume of the semi-preparative liquid chromatography was 900 μL, and the flow rate was 1 mL / min.

6. The method according to claim 5, characterized in that The components in step S1 are eluted and collected as one component every 1 minute from 10 minutes to 40 minutes after the injection.

7. The method according to claim 1, characterized in that In step S2, the target protein is a Kras protein with a His tag, which is fixed to an SPR chip in a sodium acetate buffer with a pH of 4.5-5.

5.

8. The method according to claim 7, characterized in that The pH value of the sodium acetate buffer solution is 5.

0.

9. The method according to claim 1, characterized in that The buffer for the SPR assay in step S2 is PBS solution supplemented with 0.05% Tween 20 and 5 μM GDP.

10. The method according to claim 1, characterized in that The affinity chromatography matrix in step S3 is NiSepharose FF NTA resin.

11. The method according to claim 1, wherein The enrichment process in step S3 includes: incubating the target protein with the matrix, loading the high-responsiveness component, and eluting the bound small molecule compounds with a 0.5% formic acid solution after washing.

12. The method according to claim 1, characterized in that The mass spectrometry technique described in step S4 is LC-MS / MS, which identifies the small molecule structure by comparing it with a metabolite structure database.

13. The method according to claim 1, characterized in that The small molecule compounds described in step S4 are (R,S)-Anatabine, 7-[4-(tert-butyl)phenyl]-2-methyl-5-(methylthio)pyrazolo[1,5-a]pyrimidine-6-carbonitrile, Carbamazepine, NP-007866, 9-Oxo-4,5,6,7,8,9-hexahyd ro-pyrazolo[5,1-b]quinazoline-3-carbonitrile, 4,4-dimethyl-1-(3-nitrophenyl)-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-one, Decarbamoyl-neosaxitoxin, NP-022229, Di(2-ethylhexyl) Phthalate, Thiacloprid, Didecyldimethylammonium, PEG Monolaurate n5, PEG Monolaurate n6, 4-[5-(5-anilino-1,2,4-thiadiazol-3-yl)-4-methylpyrimidin-2-yl]benzamide and 6-(4-methoxyphenyl)pyrimidine-2,4-diamine.

14. The method according to claim 1, characterized in that The plant in step S1 is tobacco, wolfberry, liquorice, astragalus, chrysanthemum, snow lotus, salvia miltiorrhiza, mint, ginseng, yew, tea tree, lavender, olive, cordyceps or dendrobium.

15. The method according to claim 14, characterized in that The plant is tobacco leaves, and the extraction solvent is 80% methanol solution.

16. A kit for enriching active small molecules that bind to specific proteins, characterized in that: The kit comprises the reagents or standards of any one of claims 1 to 15.

17. The kit according to claim 16, characterized in that The kit also includes a positive sample.

18. Use of the method according to any one of claims 1 to 15 or the kit according to claim 16 or 17 for enriching active small molecules that bind to specific proteins from plant extracts.

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