Method for analyzing active components of Xuanhushan against liver cancer based on multi-target affinity ultrafiltration

Abstract

The application discloses a method for analyzing active ingredients of Xuanhushan against liver cancer based on multi-target affinity ultrafiltration, and the method comprises the following steps: preparing water extract of Xuanhushan; respectively incubating with four target proteins of COX-2, HKDC1, STAT3 and NGF; identifying the combined ingredients after ultrafiltration separation through LC-MS / MS and calculating the binding degree; verifying the HepG2 cell proliferation inhibition activity through a CCK-8 method; simulating the binding mode and free energy through molecular docking, and constructing an active ingredient-multi-target interaction network; the application integrates experimental and calculation technologies, systematically clarifies the active ingredients and molecular mechanisms of Xuanhushan against liver cancer, and provides a new strategy for multi-target analysis of traditional Chinese medicine compounds.

Description

Technical Field

[0001] The present invention belongs to the technical fields of bioinformatics and natural pharmaceutical analysis, and specifically relates to a method for systematically analyzing anti-hepatocellular carcinoma active ingredients in the traditional Chinese medicine compound Xuanhusuo San and their interactions with multiple targets based on multi-target affinity ultrafiltration technology, liquid chromatography-mass spectrometry, cell activity verification and molecular docking. Background Art

[0002] Current clinical treatment methods, such as surgical resection, radiotherapy, chemotherapy and targeted drug therapy, have certain curative effects, but still have problems such as high postoperative recurrence rate, strong chemotherapeutic drug resistance, limited selectivity of targeted drugs and obvious side effects. Therefore, searching for high-efficiency, low-toxicity novel anti-hepatocellular carcinoma lead compounds or drug combinations from natural products has become an important direction of current drug research and development.

[0003] With the overall regulation characteristic of "multi-component, multi-target, multi-pathway" synergistic effect, traditional Chinese medicine compounds show unique advantages in the prevention and treatment of complex diseases such as tumors. Xuanhusuo San, a classic herb pair composed of Corydalis Rhizoma and Curcumae Rhizoma, has the effects of promoting blood circulation to remove blood stasis and moving qi to alleviate pain, and is clinically commonly used for the treatment of qi stagnation and blood stasis syndrome. Modern pharmacological studies have preliminarily shown that Corydalis Rhizoma is rich in alkaloids, while Curcumae Rhizoma is rich in volatile oil and sesquiterpenoids, both of which have been reported to have anti-inflammatory and anti-tumor activities. However, as a whole, the specific pharmacodynamic material basis for its anti-hepatocellular carcinoma effect, the key active ingredient group and their synergistic mechanism are still unclear, which seriously restricts its modern development and clinical application.

[0004] For the research on active ingredients in the complex system of traditional Chinese medicine, traditional methods such as solvent extraction and separation, activity tracking and the like have cumbersome processes, low efficiency, and are easy to miss active ingredients with trace and medium affinity. In recent years, bio-affinity-based screening technologies, such as affinity ultrafiltration-liquid chromatography-mass spectrometry technology, have become a powerful tool for efficiently discovering active ingredients in natural products because they can directly screen ligands that bind to specific biological targets from complex mixtures and maintain the natural conformation of proteins. In the prior art, there have been reports of using a single target for affinity ultrafiltration screening, but for hepatocellular carcinoma, a multi-factor and multi-pathway disease, the ingredients obtained by single-target screening are often difficult to comprehensively explain the overall curative effect of traditional Chinese medicine compounds. Therefore, constructing a simultaneous analysis model of multiple targets closely related to the occurrence and development of hepatocellular carcinoma (such as COX-2, STAT3, HKDC1, NGF related to inflammation, proliferation, metabolism, and neural invasion) is crucial for systematically elucidating the material basis and action mechanism of Xuanhusuo San against hepatocellular carcinoma.

[0005] In summary, current technologies lack an integrated method that can efficiently and accurately analyze the active ingredient groups and their molecular mechanisms acting on the multi-target network of liver cancer from traditional Chinese medicine compound formulas such as Xuanhu Suo San, and directly verify the analytical results with cellular-level anti-liver cancer activity. Developing such a method would not only help reveal the scientific implications of Xuanhu Suo San's anti-liver cancer effects, but also provide new strategies and technical support for the research on the pharmacodynamic material basis of other traditional Chinese medicine compound formulas and the development of innovative drugs.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] Purpose of the invention

[0008] This invention provides a method for analyzing the anti-hepatocellular carcinoma active ingredients in Corydalis Rhizoma Powder and their interactions with targets based on multi-target affinity ultrafiltration technology. This method integrates affinity ultrafiltration experiments, LC-MS / MS identification, cell activity verification, and molecular docking, and can systematically elucidate the active ingredient group, target sites, and signaling network of Corydalis Rhizoma Powder in anti-hepatocellular carcinoma.

[0009] Technical solution

[0010] To address the shortcomings of existing technologies, this invention provides a method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration includes the following steps:

[0013] (1) Mix Corydalis rhizome and Curcuma rhizome in a 1:1 mass ratio, add 10 times the amount of ultrapure water, soak for 30 minutes, boil, keep at a gentle boil for 1 hour, filter, repeat the extraction twice, combine the supernatant, concentrate and dry to obtain Corydalis rhizome water extract.

[0014] (2) Prepare a sample solution from the water extract obtained in step (1), and incubate the sample solution separately with each of the target proteins COX-2, HKDC1, STAT3 and NGF. Each target protein is incubated independently to form its corresponding ligand-protein complex.

[0015] (3) The incubated solution was subjected to ultrafiltration and centrifugation to separate the ligand-protein complex;

[0016] (4) Elute and collect the active ligands that bind to the target protein to obtain an active ingredient enrichment solution;

[0017] (5) The active ingredient enrichment solution was analyzed by liquid chromatography-mass spectrometry, and the binding degree was calculated by using the corresponding inactivated protein solution as a control to identify the potential active ingredients that specifically bind to the target protein.

[0018] (6) Using the CCK-8 method, with human hepatocellular carcinoma HepG2 cells as a model, the cell proliferation inhibition rate of the potential active ingredients identified in step (5) was measured to verify their anti-hepatocellular carcinoma activity.

[0019] (7) Using molecular docking technology, simulate the binding mode and binding free energy of the potential active ingredients identified in step (5) with the active pockets of the target proteins, verify their interaction, construct an active ingredient-multi-target interaction network, and elucidate its anti-liver cancer molecular mechanism.

[0020] Preferably, the concentration of the sample solution in step (2) is 0.5~2 mg / mL; the concentration of COX-2 in the target protein is 0.1~1.0 U / mL, and the concentrations of HKDC1, STAT3 and NGF are each 0.01~1 mg / mL independently; the incubation conditions are incubation at 35~39℃ for 30~60 minutes.

[0021] Preferably, in step (3), an ultrafiltration tube with a molecular weight cutoff of 30 kDa is used to separate the complexes bound to COX-2, HKDC1, and NGF, and an ultrafiltration tube with a molecular weight cutoff of 50 kDa is used to separate the complexes bound to STAT3; the ultrafiltration centrifugation conditions are centrifugation at 8000~12000 rpm for 8~15 minutes.

[0022] Preferably, in step (4), the unbound components are first washed with buffer solution 2 to 4 times, and then the bound ligands are eluted with methanol solution of 85% to 95% by volume 2 to 4 times, and the eluents are combined.

[0023] Preferably, the formula for calculating the binding degree in step (5) is: BD=(A_active-A_inactive) / A_active×100%, where A_active is the chromatographic peak area of ​​the target component in the active protein experimental group, and A_inactive is the chromatographic peak area of ​​the same component in the inactivated protein control group; BD≥30% indicates that the component has specific binding potential with the target protein.

[0024] Preferably, the determination of the cell proliferation inhibition rate in step (6) includes: seeding HepG2 cells in a 96-well plate and culturing for 24 hours; adding culture medium containing different concentrations of the test sample and culturing for another 24-72 hours; adding CCK-8 reagent and incubating for 1-4 hours, measuring absorbance at 450 nm; and calculating the inhibition rate and half-maximal inhibitory concentration (IC50). 50 .

[0025] Preferably, the molecular docking in step (7) includes: obtaining the crystal structure or homology model of the target protein from the protein database and performing preprocessing; using molecular docking software to simulate the binding conformation, calculate the binding free energy, and analyze key interactions such as hydrogen bonds and hydrophobic interactions.

[0026] Preferably, the potential active ingredient identified in step (5) includes at least one of grandiol, protopine, berberine, coptisine, baicalein, corydaline, berberine, dehydrocorydaline, atractylodes lactone II, curcuminol, eugenol, and germaconone-4,5-epoxide.

[0027] The present invention also provides a pharmaceutical composition for treating liver cancer, comprising a therapeutically effective amount of the active ingredient obtained by the method screening, and a pharmaceutically acceptable carrier or excipient.

[0028] Preferably, the dosage form is tablet, capsule, granule, oral liquid or injection.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) This invention, targeting the specific traditional Chinese medicine compound Xuanhu Suo San, for the first time combines four liver cancer-related targets—COX-2, HKDC1, STAT3, and NGF—into a single affinity ultrafiltration analysis system, constructing a systematic analysis method covering multiple dimensions of inflammation, signal transduction, metabolic reprogramming, and neural regulation. Compared with existing single-target or purely computational methods, the multi-target combination constructed in this invention covers multiple dimensions of inflammation (COX-2), signal transduction (STAT3), metabolic reprogramming (HKDC1), and neural regulation (NGF), reflecting a systematic consideration of the heterogeneity of liver cancer. This method maintains the native conformation of the target proteins during affinity binding, more realistically reflecting interactions under physiological conditions, and overcoming the shortcomings of purely computational predictions lacking experimental verification.

[0031] (2) This invention closely integrates efficient multi-target affinity ultrafiltration experimental technology with cell-level pharmacodynamic verification (HepG2 cell inhibition experiment) and theoretical calculation verification (molecular docking), forming a four-in-one research strategy of "experimental integration - mass spectrometry identification - cell verification - simulation analysis". This not only accurately identified the potential active ingredients in Corydalis powder that specifically bind to each target, but also directly confirmed its anti-hepatocellular carcinoma activity through the CCK-8 method. Furthermore, through molecular docking (with binding free energy < -5.0 kcal / mol as the criterion), the molecular basis of the interaction and key binding sites (such as hydrogen bonds, π-π stacking, etc.) were theoretically elucidated, systematically revealing part of the pharmacodynamic material basis and mechanism of action of Corydalis powder against hepatocellular carcinoma.

[0032] (3) The analytical method provided by this invention has high throughput, good selectivity, and simple operation, and is suitable for the systematic analysis of active ingredients in complex systems such as traditional Chinese medicine compound prescriptions. This method is highly reproducible and can be used not only for Xuanhu Suo San, but also extended to the study of anti-tumor mechanisms of other traditional Chinese medicine compound prescriptions or natural products. The group of active ingredients identified by this method (including 12 kinds such as grandiol, protopine, palmatine, berberine, fentanyl, corydaline, berberine, dehydrocorydaline, atractylodes lactone II, curcuminol, eugenol, and gemmaconone-4,5-epoxide) provides an important material basis and clear candidate molecules for the development of novel anti-hepatocellular carcinoma drugs or lead compounds characterized by multi-target synergistic effects. Among them, the anti-hepatocellular carcinoma effects of fentanyl and corydaline are reported for the first time in this invention. At the same time, this method also provides new evidence and research strategies for clarifying the modern scientific connotation of Xuanhu Suo San's "blood-activating and stasis-removing" anti-hepatocellular carcinoma effect. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the technical route and implementation scheme of the present invention;

[0034] Figure 2 Comparative LC-MS / MS chromatograms of affinity ultrafiltration screening of Corydalis powder active ingredients and target protein HKDC1;

[0035] Figure 3 Comparative LC-MS / MS chromatograms of affinity ultrafiltration screening of active ingredients of Corydalis powder with target protein STAT3;

[0036] Figure 4 Comparative LC-MS / MS chromatograms of affinity ultrafiltration screening of active ingredients of Corydalis powder with target protein COX-2;

[0037] Figure 5 Comparative LC-MS / MS chromatograms of affinity ultrafiltration screening of active ingredients of Corydalis powder with target protein NGF;

[0038] Figure 6 The effect of some active ingredients in Corydalis powder on the proliferation rate of HepG2 cells (camptothecin was a positive control).

[0039] Figure 7 This is a schematic diagram of the molecular docking between some active ingredients in Corydalis Rhizome Powder and the target protein HKDC1.

[0040] Figure 8 This is a schematic diagram of the molecular docking pattern between some active ingredients in Corydalis Rhizome Powder and the target protein STAT3.

[0041] Figure 9 This is a schematic diagram of the molecular docking pattern between some active ingredients in Corydalis Rhizome Powder and the target protein COX-2.

[0042] Figure 10 This is a schematic diagram of the molecular docking pattern between some active ingredients in Corydalis Rhizome Powder and the target protein NGF. Detailed Implementation

[0043] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0044] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0045] Example 1: Preparation of Corydalis Rhizome Water Extract

[0046] Take 100g each of dried Corydalis Rhizoma and Curcumae Rhizoma, and mix them (1:1, w / w). Add 10 times the volume (w / v, 2L) of ultrapure water and soak for 30 minutes. Then heat to boiling and reflux for 1 hour at a gentle boil. After cooling, centrifuge at 8000rpm for 15 minutes and collect the supernatant. Add 8 times the volume (1.6L) of ultrapure water to the residue again and reflux for 1 hour using the same method. Combine the supernatants from the two extractions, concentrate under reduced pressure at 60℃ using a rotary evaporator, and then freeze-dry to obtain a freeze-dried powder of Corydalis Rhizoma aqueous extract, which is stored in a desiccator for later use.

[0047] Example 2: Screening of active ingredients based on multi-target affinity ultrafiltration technology

[0048] (1) Preparation of buffer solution and sample solution:

[0049] Prepare buffer solutions suitable for different target proteins: COX-2, HKDC1, and NGF were prepared using 0.1 mol / L phosphate-buffered saline (PBS) at pH 7.4; STAT3 was prepared using Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl. Accurately weigh the lyophilized Corydalis powder prepared in Example 1, dissolve and dilute to volume with the appropriate buffer to prepare a sample stock solution with a concentration of 1 mg / mL. Filter the stock solution through a 0.22 μm microporous membrane for later use.

[0050] (2) Preparation of target protein solution:

[0051] Human recombinant COX-2, HKDC1, STAT3, and NGF proteins were dissolved in their respective buffer solutions to prepare the following working concentrations: COX-2: 0.5 U / mL; HKDC1: 0.2 mg / mL; STAT3: 0.2 mg / mL; NGF: 0.2 mg / mL. An equal volume of each protein solution was then inactivated by heating in a 100°C water bath for 10 minutes, serving as a negative control group.

[0052] (3) Affinity binding and ultrafiltration separation:

[0053] Mix 100 μL of Corydalis yanhusuo powder sample solution (1 mg / mL) with an equal volume of single-target protein solution in a centrifuge tube. Incubate at 37°C with shaking for 40 minutes to allow the small molecule ligand to fully bind to the protein. After incubation, transfer the mixture to an ultrafiltration centrifuge tube with the appropriate molecular weight cutoff (30 kDa membrane for COX-2, HKDC1, and NGF; 50 kDa membrane for STAT3). Centrifuge at 4°C and 10,000 rpm for 10 minutes and discard the filtrate. Add 200 μL of the corresponding buffer solution to the ultrafiltration tube, let stand for 10 minutes, and then centrifuge and wash under the same conditions. Repeat this step 3 times to fully elute unbound components. Finally, 200 μL of 90% (v / v) methanol aqueous solution was added to the ultrafiltration tube and allowed to stand at room temperature for 10 minutes to dissociate the ligands bound to the protein. The tube was then centrifuged under the same conditions and the filtrate was collected. This elution step was repeated 3 times. All filtrates containing ligands were combined, concentrated by nitrogen blowing, reconstituted with methanol, and analyzed by LC-MS.

[0054] Example 3: Screening and Identification of Active Ingredients by LC-MS / MS

[0055] The collected filtrate was dried with mild nitrogen gas, and the residue was redissolved in 100 μL of methanol. After filtration through a 0.22 μm filter membrane, it was analyzed by LC-MS / MS.

[0056] Chromatographic conditions: Agilent 1290 Infinity II liquid chromatography system, ZORBAX Eclipse Plus C18 column (2.1×100mm, 1.8μm); mobile phase: 0.1% formic acid aqueous solution (A) and acetonitrile (B); gradient elution program: 0-30 min, 5%B→95%B; 30-31 min, 95%B→5%B; 31-36 min, 5%B hold; flow rate: 0.3 mL / min; column temperature: 30℃; injection volume: 2 μL.

[0057] Mass spectrometry conditions: Agilent 6545 Q-TOF mass spectrometer, electrospray ionization (ESI) source, positive ion mode; scan range m / z 100-1500; drying gas temperature 325℃; drying gas flow rate 8L / min; nebulizer pressure 35psi; capillary voltage 3500V; fragmentation voltage 135V.

[0058] Combination degree calculation: BD ≥ 30% was considered a specific binding. A total of 12 active ingredients were identified, some examples include: grandipine (49.96% BD with NGF), berberine (62.55% BD with COX-2), berberine (43.00% BD with COX-2 and 45.23% BD with NGF), and atractylodes lactone II (48.67% BD with STAT3), etc.

[0059] By comparing the LC-MS / MS chromatograms of the active protein experimental group and the inactivated protein control group, the binding ability of the compound to the target protein was evaluated by comparing the changes in peak area or response value of specific chromatographic peaks. The binding degree (BD value) was used for quantification, and the calculation formula is as follows: Where A_active and A_inactive are the chromatographic peak areas of the same compound in the active group and the inactive group, respectively.

[0060] Screening thresholds were set as follows: binding affinity (BD value) to COX-2 target ≥ 30%; BD value to NGF target ≥ 30%; BD value to HKDC1 target ≥ 30%; BD value to STAT3 target ≥ 30%. Compounds meeting any one of the above conditions were identified as potential active ingredients, and compounds meeting any two of the above conditions were identified as potential multi-target active ingredients.

[0061] Finally, several potential active ingredients were screened and identified from the aqueous extract of Corydalis rhizome, including grandialdehyde, protopine, berberine, corydaline, berberine, dehydrocorydaline, atractylodes lactone II, curcuminol, eugenol, and geranione-4,5-epoxide (BD values ​​are shown in the attached data table).

[0062]

[0063] Example 4: Validation of CCK-8 cell viability

[0064] (1) Cell culture and inoculation:

[0065] Human hepatocellular carcinoma HepG2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-antibody solution at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were harvested, digested, centrifuged, resuspended in complete medium, counted, and their density adjusted to 5 × 10⁶ cells / year.4 cells / mL. The cell suspension was seeded at 100 μL per well in a 96-well plate (i.e., 5 × 10⁶ cells / mL). 3 (1 cell), placed in an incubator for pre-culture for 24 hours to allow the cells to fully adhere to the incubator.

[0066] (2) Sample processing and incubation:

[0067] Accurately weigh the lyophilized powder of total extract of Corydalis Rhizoma Sinensis prepared in Example 1 or the standard of the single potential active ingredient identified in Example 2, dissolve it in DMSO to prepare a high-concentration stock solution, and then dilute it stepwise with serum-free medium to ensure that the concentration of DMSO in the final treatment medium does not exceed 0.1%. Set up 5-7 concentration gradients, as well as a blank control group (medium containing only 0.1% DMSO) and a zeroing group (medium containing only medium, without cells).

[0068] Discard the old culture medium from the 96-well plate, and add 100 μL of treatment medium containing different concentrations of the sample to each well, with 6 replicates for each concentration. Return the cells to the incubator and continue culturing for 24 hours.

[0069] (3) Cell viability assay:

[0070] After incubation, carefully add 10 μL of CCK-8 solution to each well, gently shake to mix, and continue incubation for 1.5 hours. Measure the absorbance (OD) value of each well at 450 nm using a microplate reader.

[0071] (4) Data analysis:

[0072] Calculate cell viability and proliferation inhibition rate: Cell proliferation inhibition rate (%) = 100% - cell survival rate.

[0073] Using GraphPad Prism 8.0 software, a nonlinear regression was performed with the logarithm of sample concentration on the x-axis and the cell proliferation inhibition rate on the y-axis to calculate the half-maximal inhibitory concentration (IC50). 50 ).

[0074] The results showed that the total extract of Corydalis Rhizome Powder had an IC50 effect on HepG2 cells after 24 hours of treatment. 50 The concentration was 42.76 μg / mL. Several screened active ingredients, such as berberine and coptisine, all exhibited significant dose-dependent inhibitory activity, with an IC50 of 42.76 μg / mL. 50 Values ​​in the range of 10-600 μM confirmed its potential anti-liver cancer efficacy.

[0075] Example 5: Molecular docking verification

[0076] To theoretically verify the interaction between the screened active ingredients and the target proteins, molecular docking technology was used for simulation.

[0077] (1) Protein and ligand preparation:

[0078] Crystal structures of the target proteins were downloaded from the RCSB protein database (https: / / www.rcsb.org): COX-2 (PDB ID: 5IKR), STAT3 (PDB ID: 6NJS), and NGF (PDB ID: 1WWW). Since HKDC1 lacks a crystal structure, its 3D model was obtained using homology modeling. The proteins were preprocessed using AutoDock Tools software: water molecules and protoligands were removed, polar hydrogen was added, and Kollman charges were calculated.

[0079] Download the 3D structure files (sdf format) of the active ingredients (such as berberine, curcumin, etc.) identified in Example 2 from the PubChem database, convert them to pdbqt format using Open Babel software, and perform energy minimization.

[0080] (2) Molecular docking:

[0081] Molecular docking was performed using AutoDock Vina 1.1.2 software. Based on literature and active site prediction results, a docking grid box was defined to cover the known active pockets of the protein (such as the cyclooxygenase active site of COX-2, the SH2 domain of STAT3, etc., with camptothecin as a positive control). The docking search space was set to 60×60×60 Å, with a grid spacing of 0.375 Å. The docking results generated multiple binding conformations, and the conformation with the lowest binding free energy (ΔG) was selected as the optimal binding mode.

[0082] (3) Results analysis:

[0083] Interactions in the optimal binding conformation were analyzed, including hydrogen bonds, π-π stacking, and hydrophobic interactions. For example, docking results showed that berberine can bind to key residues such as LYS-468 and TYR-122 in the COX-2 active center through multiple hydrogen bonds.

[0084] The binding free energy between the active ingredients and their corresponding targets in all tests was below -5.0 kcal / mol, as shown in the table below:

[0085]

[0086] Based on the molecular docking experiments, a binding energy (BE) ≤ -5.0 kcal / mol was used as the threshold for judging whether a compound has moderate to high binding activity with the target protein, and a Ki value of less than approximately 200 μmol / L (nanomolar to micromolar level) was considered effective inhibition. At the HKDC1 target site, purslane (-5.61 kcal / mol), berberine (-5.25 kcal / mol), curcuminol (-5.58 kcal / mol), and corydaline (-5.13 kcal / mol) all reached the threshold, with curcuminol having the lowest binding energy. Purslane formed a hydrogen bond with ILE-163, berberine formed a hydrogen bond with LEU-611, and corydaline formed hydrogen bonds with MET-29 and ASP-23. Among the STAT3 targets, atractylodes lactone II (-5.09 kcal / mol) reached the threshold, while the binding energies of the remaining compounds were all above -5.0. Cyperine (-4.09 kcal / mol) was below the threshold and was judged as a weak binding. Among the COX-2 targets, only berberine (-5.21 kcal / mol) reached the threshold, forming hydrogen bonds with TYR-122 and LYS-468. Among the NGF targets, grandiol (-9.18 kcal / mol, Ki 185.87 nM) and berberine (-8.88 kcal / mol) had binding energies far below -7.0, even lower than the positive control, indicating strong binding. Protopinine (-6.26 kcal / mol), berberine (-5.87 kcal / mol), and dehydrocorydaline (-5.83 kcal / mol) also had binding energies below -5.0, showing moderate binding activity. The above quantitative judgment criteria provide an objective basis for screening potential active ingredients.

[0087] Example 6: Preparation of the pharmaceutical composition

[0088] (1) Tablets: Take 50 mg of the active ingredient composition selected in Example 2 (such as berberine and curcumin mixed in a weight ratio of 1:2), mix it evenly with 150 mg of pregelatinized starch, 60 mg of microcrystalline cellulose and 4 mg of magnesium stearate, and compress it directly to make tablets containing 50 mg of active ingredients per tablet.

[0089] (2) Lyophilized powder for injection: Take 100 mg of total extract of Corydalis Rhizome powder, add 200 mg of mannitol, dissolve in water for injection and bring the volume to 10 mL, filter sterile through a 0.22 μm filter membrane, dispense into vials, 2 mL per vial, freeze dry, and cap. Reconstitute with physiological saline before use.

[0090] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration, characterized in that, Includes the following steps: (1) Mix Corydalis rhizome and Curcuma rhizome in a 1:1 mass ratio, add 10 times the amount of ultrapure water, soak for 30 minutes, boil, keep at a gentle boil for 1 hour, filter, repeat the extraction twice, combine the supernatant, concentrate and dry to obtain Corydalis rhizome water extract. (2) Prepare a sample solution from the water extract obtained in step (1), and incubate the sample solution separately with each of the target proteins COX-2, HKDC1, STAT3 and NGF. Each target protein is incubated independently to form its corresponding ligand-protein complex. (3) The incubated solution was subjected to ultrafiltration and centrifugation to separate the ligand-protein complex; (4) Elute and collect the active ligands that bind to the target protein to obtain an active ingredient enrichment solution; (5) The active ingredient enrichment solution was analyzed by liquid chromatography-mass spectrometry, and the binding degree was calculated by using the corresponding inactivated protein solution as a control to identify the potential active ingredients that specifically bind to the target protein. (6) Using the CCK-8 method, with human hepatocellular carcinoma HepG2 cells as a model, the cell proliferation inhibition rate of the potential active ingredients identified in step (5) was measured to verify their anti-hepatocellular carcinoma activity. (7) Using molecular docking technology, simulate the binding mode and binding free energy of the potential active ingredients identified in step (5) with the active pocket of the target protein, verify their interaction, construct an active ingredient-multi-target interaction network, and elucidate its anti-liver cancer molecular mechanism. In step (4), the unbound components are washed 2-4 times with buffer solution, and the bound ligands are eluted 2-4 times with 85%-95% methanol solution. The eluents are then combined. The potential active ingredients identified in step (5) are grandiol, protopine, berberine, coptisine, baicalein, corydaline, berberine, dehydrocorydaline, atractylodes lactone II, curcuminol, eugenol, and germaconone-4,5-epoxide.

2. The method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration according to claim 1, characterized in that, The concentration of the sample solution in step (2) is 0.5~2 mg / mL; the concentration of COX-2 in the target protein is 0.1~1.0 U / mL, and the concentrations of HKDC1, STAT3 and NGF are each 0.01~1 mg / mL independently; the incubation conditions are incubation at 35~39℃ for 30~60 minutes.

3. The method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration according to claim 1, characterized in that, In step (3), an ultrafiltration tube with a molecular weight cutoff of 30 kDa was used to separate the complexes bound to COX-2, HKDC1, and NGF, and an ultrafiltration tube with a molecular weight cutoff of 50 kDa was used to separate the complexes bound to STAT3; the ultrafiltration centrifugation conditions were 8000~12000 rpm for 8~15 minutes.

4. The method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration according to claim 1, characterized in that, The formula for calculating the degree of binding in step (5) is: Where A_active is the chromatographic peak area of ​​the target component in the active protein experimental group, and A_inactive is the chromatographic peak area of ​​the same component in the inactivated protein control group; BD≥30% indicates that the component has the potential to specifically bind to the target protein.

5. The method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration according to claim 1, characterized in that, The determination of cell proliferation inhibition rate in step (6) includes: seeding HepG2 cells in 96-well plates and culturing for 24 hours; adding culture medium containing different concentrations of the test sample and culturing for another 24-72 hours; adding CCK-8 reagent and incubating for 1-4 hours, and measuring absorbance at 450 nm; calculating the inhibition rate and half-maximal inhibitory concentration (IC50). 50 .

6. The method for analyzing the anti-hepatocellular carcinoma active ingredients of Corydalis Rhizome Powder based on multi-target affinity ultrafiltration according to claim 1, characterized in that, The molecular docking described in step (7) includes: obtaining the crystal structure or homology model of the target protein from the protein database and performing preprocessing; using molecular docking software to simulate the binding conformation, calculate the binding free energy, and analyze hydrogen bonds and hydrophobic interactions.

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