Chemical composition analysis method of heigu tengui chui feng huoluo capsule and research method of mechanism of action of heigu tengui chui feng huoluo capsule in treating rheumatoid arthritis

Abstract

This invention relates to the field of traditional Chinese medicine research technology, specifically to the chemical composition analysis method of Heiguteng Zhuifeng Huoluo Capsules and its mechanism of action in treating rheumatoid arthritis. This application utilizes ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), network pharmacology, molecular docking, and in vitro cell experiments to explore the material basis and mechanism of action of Heiguteng Zhuifeng Huoluo Capsules in treating rheumatoid arthritis (RA). Compounds were identified by combining retention time, precise molecular weight, and secondary fragment information. A total of 56 potential active compounds were obtained through the SwissADME database and supplementary data. Target information was mined using databases such as SwissTargetPrediction and GeneCards to collect compound targets and RA targets, identifying common targets. Core components and key targets were screened by constructing a medicinal material-active ingredient-intersection target diagram and a PPI network diagram.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine research technology, specifically to the chemical composition analysis method of Heiguteng Zhuifeng Huoluo Capsules and the research method on its mechanism of action in treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by symmetrical, chronic, progressive arthritis. In Traditional Chinese Medicine (TCM), it falls under the categories of "Bi syndrome," "Li Jie," and "Bai Hu Bing." The pathogenesis of RA is complex, and it is speculated to be primarily related to immune cells (macrophages, synovial cells, synovial T cells, etc., causing local inflammatory responses during infection), inflammatory factors (cyclooxygenase-2, 5-lipoxygenase, arachidonic acid, etc.), matrix metalloproteinases (MMPs), abnormal oxidative stress, genetic factors, and environmental factors. Currently, clinical treatment for RA mainly focuses on controlling the inflammatory response and regulating immune function; the main treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GCs), disease-modifying antirheumatic drugs (DMARDs), biologics, and traditional Chinese medicine. However, biologics are expensive; NSAIDs cause significant gastrointestinal and renal damage; and DMARDs and GCs have significant adverse reactions and toxic side effects. Traditional Chinese medicine (TCM) treatment for rheumatoid arthritis (RA) is characterized by syndrome differentiation and individualized treatment. Traditional Chinese medicine has advantages such as synergistic effects of multiple components and multiple targets and low toxicity and side effects, and has become one of the hot topics in the research and development of new anti-RA drugs. However, the complexity of the components of Chinese medicine compound prescriptions and the unclear mechanism of action have limited the promotion of its clinical application.

[0003] Black Bone Vine Wind-Chasing and Blood-Activating Capsules (HZHC) are composed of three medicinal herbs: Green Wind Vine, Black Bone Vine, and Wind-Chasing Umbrella. Derived from the "Compendium of Miao Medicine of Western Hunan," this is a widely circulated and effective traditional Chinese medicine formula. It has been developed into a proprietary new drug by Sinopharm Tongjitang (Guizhou) Pharmaceutical Co., Ltd. In this formula, Green Wind Vine is bitter and neutral in nature, entering the liver and spleen meridians; it has the effects of dispelling wind and dampness, clearing the meridians, relieving pain, and promoting urination. Black Bone Vine is a commonly used medicinal herb in Miao medicine, bitter and pungent in taste, warm in nature, and slightly toxic; it is mainly used to treat rheumatic joint pain, traumatic injuries, and menstrual disorders. Wind-Chasing Umbrella is an effective traditional Chinese medicine used for treating arthralgia, with the effects of dispelling wind, clearing the meridians, activating blood circulation, and relieving pain. The combination of these three herbs, in Miao medicine, is mainly used to treat "excessive wind and dampness, clearing the meridians and relieving pain," which corresponds to the traditional Chinese medicine concept of "dispelling wind and dampness, clearing the meridians and relieving pain." It can be used for wind-cold-dampness arthralgia, shoulder, arm, waist, and leg pain, and has shown good clinical efficacy for patients with rheumatoid arthritis (RA).

[0004] Currently, there are few reports on the material basis of Heiguteng Zhuifeng Huoluo Capsules, and since all three herbs are mainly used to treat rheumatic joint pain, the mechanism of action of treating RA through multiple components, multiple targets, and multiple pathways is unknown. Therefore, to fully explore and rationally develop Heiguteng Zhuifeng Huoluo Capsules, to investigate its scientific connotation in treating RA, and to provide a theoretical basis for the rational use and clinical promotion of this drug, is the current focus of research. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this invention provides a method for analyzing the chemical components of Heiguteng Zhuifeng Huoluo Capsules and a method for studying their mechanism of action in treating rheumatoid arthritis, including the following:

[0006] A method for determining multiple components in Heiguteng Zhuifeng Huoluo Capsules, used to simultaneously determine the content of multiple components (see Table 1 for details) in Heiguteng Zhuifeng Huoluo Capsules, characterized in that the determination is performed using UPLC-Q-TOF-MS analysis method;

[0007] The specific chromatographic conditions are as follows: Column: Waters Acquity UPLC HSS T3 (2.1) Column temperature: 100 mm, 1.8 μm; Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B); Elution gradient: 0~1 min, 0% B; 1~20 min, 0%~28% B; 20~40 min, 28%~80% B; 40~42 min, 80%~100% B; 42~45 min, 100% B; 45-45.1 min, 100%~0% B; 45.1-48 min, 0% B. Injection volume: 1 μL; Column temperature: 35℃; Flow rate: 0.36 mL / min;

[0008] The specific mass spectrometry conditions were as follows: Electrospray ionization (ESI) was used in positive / negative ion scanning mode, with a mass scan range of 50–1500 Da; N2 was used as the desolventizing gas at a flow rate of 500 L / h and a temperature of 400 °C; N2 was used as the conical gas at a flow rate of 100 L / h and a voltage of 40 eV; the ionization source temperature was 120 °C; and the capillary voltage was 3500 eV. Argon was used as the collision gas, with a low collision energy of 6 eV and a high collision energy of 30–60 eV. Sodium formate and leucine-enkephalin solution were used as real-time calibration standard solutions at a flow rate of 5 μl / min, and the positive source reference ion was set to m / z 556.2771 [M+H]. + The negative source reference ion has a m / z of 554.2615 [M–H]. - .

[0009] Furthermore, the test solution of Heiguteng Zhuifeng Huoluo Capsules was analyzed, and baseline chromatograms (BPI chromatograms, see [link]) were obtained under positive and negative ion modes. Figure 1 Mass spectrometry data were acquired using Mass Lynx 4.1 software, and information on quasi-molecular ion peaks and fragment peaks of each chromatographic peak was extracted. Combined with reference standards and literature data, the compounds were analyzed and their structures were identified.

[0010] Furthermore, the test solution was prepared as follows: the contents of the Heiguteng Zhuifeng Huoluo capsules were mixed, accurately weighed, and placed in a 50 mL stoppered conical flask. 25 mL of methanol was added and extracted by ultrasonication (350 W power, 50 Hz frequency) for 30 min. The mixture was cooled to room temperature, shaken well, and filtered through a 0.22 μm microporous membrane to obtain the test solution.

[0011] Black Bone Vine Wind-Chasing and Blood-Activating Capsules are composed of three medicinal herbs: Green Wind Vine, Black Bone Vine, and Wind-Chasing Umbrella.

[0012] Furthermore, for the reference solutions used, appropriate amounts of each standard were accurately weighed and dissolved in methanol to prepare 0.5–1 mg / mL reference solutions. Then, appropriate amounts of each of these solutions were used to prepare mixed standard solutions for later use, thus obtaining the reference solutions. Standards: Magnolia alkaloid (98.0%, DST180226-004) was purchased from Chengdu Dester Biotechnology Co., Ltd.; chlorogenic acid (99.3%, 1N7K-9CB1) was purchased from the China National Institute for Food and Drug Control; neochlorogenic acid (98.0%, 3209), cryptochlorogenic acid (98.0%, 3208), and uridine (98.0%, 3555) were purchased from Shanghai Shidander Technology Co., Ltd.

[0013] A research method for studying the mechanism of action of Heiguteng Zhuifeng Huoluo Capsules in treating rheumatoid arthritis includes the following steps:

[0014] (1) Screening of active ingredients: The identified compound SMILES numbers were imported into the SwissADME database. The potential active ingredients were obtained by using the criteria of high oral bioavailability and two or more "Yes" in drug-like properties.

[0015] (2) Acquisition of active ingredient compound targets; the potential active compounds obtained by screening are further searched through the SwissTargetPrediction database, with the species attribute set to "Homo sapiens", and targets with P>0 are screened;

[0016] (3) Enrichment analysis of KEGG and Reactome pathways for active ingredient targets: Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis were performed on the screened targets (FDR < 10). -4 ), thus obtaining the signal path;

[0017] (4) RA target acquisition: GeneCards and DisGeNET databases were searched using “rheumatoid arthritis” as the keyword, and disease targets with a score greater than or equal to the median were screened; the intersection of active ingredient targets and RA targets was plotted using the Draw Venn Diagram platform to obtain the potential target of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA.

[0018] (5) Constructing a network of medicinal materials, active ingredients, and intersection targets: Using Cytoscape 3.9.1 software, a network model of medicinal materials, active ingredients, and intersection targets was constructed and the network topology properties were analyzed to screen out the top ten active ingredients with the highest degree values;

[0019] (6) Constructing a PPI network: Protein-protein interaction (PPI) analysis was performed on the intersection targets using the STRING platform. The species attribute was set to "Homo sapiens", the threshold was 0.4, and unconnected targets were removed. Finally, the PPI network was visualized and its topological properties were analyzed using Cytoscape 3.9.1 software to determine the key targets of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA.

[0020] (7) KEGG pathway enrichment analysis: KEGG pathway enrichment analysis was performed on the intersection targets (FDR<10). -4 The top 20 KEGG pathways were selected based on FDR value and a bubble chart was drawn to determine the main signal pathways.

[0021] Furthermore, in step (1), the identified compound is a compound obtained by the method for determining multiple components in the Black Bone Vine Wind-Chasing and Blood-Activating Capsules as described in claim 1 (see Table 1 for details).

[0022] Furthermore, in step (1), chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and salicylic acid are added to the list of active ingredients obtained by screening using the SwissADME database.

[0023] Furthermore, in step (3), the KEGG and Reactome pathways belonging to the immune system are integrated, and PPI analysis is performed on the targets in the pathways. Figure 4Based on the degree value, the key signaling pathway target is determined.

[0024] Furthermore, in step (3), the lipid metabolism-related targets of the Heiguteng Zhuifeng Huoluo capsules are integrated, and its intervention ability on lipid metabolism and cardiovascular complications in the course of RA is analyzed through PPI network.

[0025] Compared with the prior art, the technical effects of this invention are reflected in:

[0026] (1) The present invention uses UPLC-Q-TOF-MS method to comprehensively characterize the chemical composition of Heiguteng Zhuifeng Huoluo capsules. It can simultaneously identify 82 active ingredient compounds in Heiguteng Zhuifeng Huoluo capsules, including alkaloids, phenylpropanoids, steroids and other types, and basically clarify the material basis of Heiguteng Zhuifeng Huoluo capsules.

[0027] (2) This application screened the identified compounds and obtained targets through multiple databases. The network pharmacology method was used to construct the medicinal material-active ingredient-intersection target network diagram and the target PPI network diagram. Finally, it was determined that 6-O-demethylmenisporphine, Sinoracutine, Menisporphine, linolenic acid, 8-oxobamatin, Corydinemethyl ether, Periforgenin A, berberine, dehydrocorydalmine, and cyperiforgenin are the key components of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA, and AKT1, TNF, and IL6 are its key targets.

[0028] (3) The network pharmacology study results of this application indicate that Heiguteng Zhuifeng Huoluo capsules may exert a therapeutic effect on RA through related targets and pathways in anti-inflammatory and immunomodulatory aspects.

[0029] (4) This application uses UPLC-Q-TOF-MS technology to establish an analytical method for the chemical components in Heiguteng Zhuifeng Huoluo capsules, elucidates its material basis, explores its potential mechanism of action in treating RA using network pharmacology, and conducts preliminary verification using molecular docking and in vitro cell experiments, in order to provide a theoretical basis for the rational use and clinical promotion of the drug. Attached Figure Description

[0030] Figure 1 This is the base peak chromatogram of Heiguteng Zhuifeng Huoluo Capsules (A. Positive ion mode, B. Negative ion mode).

[0031] Figure 2 It is the alkaloid cleavage pathway (A. Peak 29; B. Peak 60; C. Peak 36; D. Peak 40).

[0032] Figure 3 This is the cleavage pathway of peak 31.

[0033] Figure 4 This is a network diagram of PPIs (Protein Targets) of the immune system. The size and color of the nodes are proportional to the node's degree value.

[0034] Figure 5 This is a network diagram of lipid metabolism targets (PPIs). The size and color of the nodes are directly proportional to the node's degree value.

[0035] Figure 6 This is a Venn diagram of the active ingredients and RA target points of the Black Bone Vine Wind-Chasing and Blood-Activating Capsules.

[0036] Figure 7 This is a network diagram of medicinal materials, active ingredients, and intersection targets. QFT, HGT, and ZFS are abbreviations for *Sinomenium acutum*, *Sinomenium styracifolium*, and *Symplocos buergerianum*, respectively; squares represent intersection targets; triangles represent medicinal materials; and circles represent active ingredients (the size of the circle is proportional to the degree value of the ingredient's node).

[0037] Figure 8 (A) Network diagram of PPI, common target of active ingredient-RA in Heiguteng Zhuifeng Huoluo Capsules; (B) Bubble diagram of TOP 20 KEGG pathway.

[0038] Figure 9 This is a diagram of compound-target molecular docking. A: 6- O -demethylmenisporphine interacts with TNF-α; B: Periforgenin A interacts with IL-6; C: 6- O -demethylmenisporphine interacts with AKT1.

[0039] Figure 10 The study investigated the effects of (A) Heiguteng Zhuifeng Huoluo Capsules on TNF-α release and (B) Heiguteng Zhuifeng Huoluo Capsules on IL-6 release. DXMS was used for dexamethasone. All data are presented as mean ± SD (n=3). Significant differences between the control group and the model group were expressed as: ## p < 0.01, ### p < 0.001; significant differences between the treated group and the model group were expressed as: p<0.05, p<0.01. Detailed Implementation

[0040] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description.

[0041] Example:

[0042] 1. Instruments and reagents

[0043] 1.1 Instruments

[0044] Quadrupole time-of-flight mass spectrometer (Waters Xevo G2, Waters Corporation, USA); purified water system (Milli-Q International, Millipore, USA); microplate reader (EPOCH, Berten Instruments, USA); microscope (Olympus Corporation, CX23); biosafety cabinet (HFsafe-1200LC, Likang Biomedical Technology Holding Co., Ltd.); pipettes (Eppendorf, Germany); CO2 incubator (Thermofisher Scientific, USA, 3111); electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd., MS204S); medical CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., KQ-250DE).

[0045] 1.2 Materials

[0046] Black Bone Vine Wind-Chasing and Blood-Activating Capsules (Sinopharm Group Tongjitang (Guizhou) Pharmaceutical Co., Ltd., 180816); Magnolia flower alkaloid (98.0%, DST180226-004) was purchased from Chengdu Dester Biotechnology Co., Ltd.; Chlorogenic acid (99.3%, 1N7K-9CB1) was purchased from China National Institute for Food and Drug Control; Neochlorogenic acid (98.0%, 3209), cryptochlorogenic acid (98.0%, 3208), and uridine (98.0%, 3555) were purchased from Shanghai Shidander Technology Co., Ltd.; Acetonitrile (LC-MS grade) was purchased from Shanghai Titan Technology Co., Ltd.; Formic acid (LC-MS grade) was purchased from Thermo Fisher Scientific Co., Ltd.; All other reagents were of analytical grade; RAW264.7 cells were purchased from ATCC, USA; DMEM high glucose, RPMI 1640, PBS (pH 7.4), and fetal bovine serum were purchased from Gibco; Antibiotic-Antimycotic, Trypsin-EDTA (0.05%), and bovine serum albumin were purchased from Lifetechnologies; ELISA kits were purchased from Shanghai Asia-Pacific Biotechnology Service Center; cell culture dishes, 96-well cell culture plates, 75cm² cell culture flasks, cell culture plates, and 96-well microplates were purchased from Coning Biotechnology, USA; mouse TNF-α ELISA kits and mouse IL-6 ELISA kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0047] 2 Methods

[0048] 2.1 Solution Preparation

[0049] 2.1.1 Preparation of the test solution

[0050] Take several capsules of Heiguteng Zhuifeng Huoluo Capsules, mix them well, weigh them accurately, place them in a 50 mL stoppered conical flask, add 25 mL of methanol, extract by ultrasonication (350 W power, 50 Hz frequency) for 30 min, cool to room temperature, shake well, filter through a 0.22 μm microporous membrane to obtain the test solution.

[0051] 2.1.2 Preparation of the reference solution

[0052] Take appropriate amounts of each standard, accurately weigh them, and add methanol to prepare a 0.5~1 mg / mL reference solution. Then, take appropriate amounts of the above solutions to prepare a mixed standard solution for later use, which is the reference solution.

[0053] 2.2 UPLC-Q-TOF-MS Analysis Method

[0054] 2.2.1 Chromatographic conditions

[0055] Chromatographic column: Waters Acquity UPLC HSS T3 (2.1) Column temperature: 100 mm, 1.8 μm; Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B); Elution gradient: 0~1 min, 0% B; 1~20 min, 0%~28% B; 20~40 min, 28%~80% B; 40~42 min, 80%~100% B; 42~45 min, 100% B; 45-45.1 min, 100%~0% B; 45.1-48 min, 0% B. Injection volume: 1 μL; Column temperature: 35℃; Flow rate: 0.36 mL / min.

[0056] 2.2.2 Mass Spectrometry Conditions

[0057] Electrospray ionization (ESI) was used in positive / negative ion scanning mode with a mass scan range of 50–1500 Da. N2 was used as the desolvation gas at a flow rate of 500 L / h and a temperature of 400 °C. N2 was used as the conical gas at a flow rate of 100 L / h and a voltage of 40 eV. The ionization source temperature was 120 °C, and the capillary voltage was 3500 eV. Argon was used as the collision gas, with a low collision energy of 6 eV and a high collision energy of 30–60 eV. Sodium formate and leucine-enkephalin solution were used as real-time calibration standard solutions at a flow rate of 5 μl / min. The positive source reference ion was set to m / z 556.2771 [M+H]. + The negative source reference ion has a m / z of 554.2615 [M–H]. - .

[0058] 2.3 Chemical Composition Analysis

[0059] Under the conditions described in section 2.2, the test solution of Heiguteng Zhuifeng Huoluo Capsules was analyzed to obtain baseline chromatograms (BPI chromatograms, see section 2.2) in both positive and negative ion modes. Figure 1 Mass spectrometry data were acquired using Mass Lynx 4.1 software, and the quasi-molecular ion peaks and fragment peaks of each chromatographic peak were extracted. Combined with reference standards and literature data, the compounds were analyzed and their structures were identified.

[0060] 2.4 Network Pharmacological Analysis

[0061] 2.4.1 Screening of active ingredients

[0062] The identified compound SMILES numbers were imported into the SwissADME database. Potential active ingredients were identified by screening for high oral bioavailability and two or more "Yes" results in drug-likeness, supplemented by relevant literature. The identified potential active compounds were further searched in the SwissTargetPrediction database with the species attribute set to "Homo sapiens," and targets with p>0 were selected.

[0063] 2.4.2 Regulatory effects of active ingredients on biological systems and processes

[0064] Since the intervention of traditional Chinese medicine in disease progression is often related to its regulation of multiple biological systems and processes, we further investigated the regulatory effects of the active ingredients of Heiguteng Zhuifeng Huoluo Capsules on biological systems and processes. We conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis (FDR < 10). -4 ).

[0065] 2.4.3 Acquisition of RA Targets

[0066] Using "rheumatoid arthritis" as the keyword, the GeneCards and DisGeNET databases were searched to screen for disease targets with a score greater than or equal to the median. The intersection of the active ingredient targets and RA targets was plotted using the Draw Venn Diagram platform to obtain the potential therapeutic targets of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA.

[0067] 2.4.4 Construction of the Network Diagram of Medicinal Materials-Active Ingredients-Intersecting Targets

[0068] Using Cytoscape 3.9.1 software, a network model of medicinal materials, active ingredients, and intersection targets was constructed, and the network topology properties were analyzed.

[0069] 2.4.5 Construction of PPI network and enrichment of KEGG pathway

[0070] Protein-protein interaction (PPI) analysis was performed on the intersection targets using the STRING platform. The species attribute was set to "Homo sapiens," the threshold was 0.4, and unconnected targets were removed. Finally, the data was imported into Cytoscape 3.9.1 software for PPI network visualization and topological property analysis. KEGG pathway enrichment analysis was performed on the intersection targets (FDR < 10). -4 The top 20 KEGG pathways were selected based on their FDR values ​​and a bubble chart was generated.

[0071] 2.5 Network pharmacology analysis validation

[0072] 2.5.1 Molecular docking

[0073] Molecular docking was used to verify the interactions between key components and the target. The 3D protein structure of the target was downloaded from the PDB database and processed using PyMOL software. The 3D structure of the compound was downloaded from the PubChem database. Finally, molecular docking was performed using Auldock Vina software, and the docking results were visualized using PyMOL software.

[0074] 2.5.2 In vitro cell validation

[0075] In vitro experiments were conducted using an LPS-induced RAW 264.7 cell model, with dexamethasone as a positive control, to verify the effects of Heifengteng Zhuifeng Huoluo capsules on TNF-α and IL-6, two inflammatory factors. Cells were cultured in cell culture medium containing 10% fetal bovine serum at 37℃, 5% CO2, and 90% relative humidity. Cells were seeded into 96-well plates and cultured overnight, then pre-cultured with the contents of Heifengteng Zhuifeng Huoluo capsules for 48 hours, followed by stimulation with 10 μL LPS for 8 hours. The concentrations of TNF-α and IL-6 in the cell supernatant were determined using an ELISA kit according to the manufacturer's instructions.

[0076] 3 Results

[0077] 3.1 Chemical composition identification

[0078] Molecular formulas were determined using quasi-molecular ion peaks, and the fragmentation patterns of compounds were analyzed using secondary fragmentation information. Combined with reference standards and relevant literature data, a total of 82 chemical components were identified. Among the identified compounds, 46 were alkaloids, 9 were phenylpropanoids, 7 were steroids, and 20 were other types. By comparing with the chromatographic information of single medicinal materials, the medicinal sources of the 82 compounds were attributed. Among these, 43 were specific to *Sinomenium acutum*, 11 to *Gynostemma pentaphyllum*, 8 to *Ulmus pumila*, and 20 were shared components (see Table 1).

[0079] Table 1. UPLC-Q-TOF-MS identification results of chemical components in Heiguteng Zhuifeng Huoluo Capsules

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Note:" "Indicates confirmation after comparison with the reference standard: S. Qingfengteng, P. Heiguteng, L. Zhuifengsan."

[0089] 3.1.1 Alkaloids

[0090] These compounds are mostly derived from *Sinomenium acutum*, primarily consisting of five isoquinoline alkaloids: benzylisoquinoline type (peaks 17, 18, 23, 25, 27, 30, 36, 38, 39, 44), apophene type (peaks 40, 42, 47, 50, 51, 77, 78, 80, 82), morphine type (peaks 14, 15, 19, 20, 24, 28, 29, 32, 41, 59), protoberberine type, and berberine type (peaks 46, 49, 52, 53, 58, 60, 65, 74, 76), as well as other alkaloids (peaks 1, 6, 8, 35, 37, 63, 64, 66). These compounds exhibit a high response in positive ion mode.

[0091] Morphine-type alkaloids are most common in *Sinomenium acutum*. These compounds readily lose the nitrogen-containing substituent and nitrogen-bridged ring. Taking peak 29 as an example, in positive ion mode, the quasi-molecular ion peak is... m / z 330.1685, the possible molecular formula is C 19 H 23 NO4, MS E Mass spectrum generation m / z 273.1120[M+H–C3H7N] + ,241.0844[M+H–C3H7N–CH4O] + ,239.0708[M+H–C3H7N–CH4O–2H] + ,213.0885[M+H–C3H7N–CH4O–CO] + ,211.0737[M+H–C3H7N–CH4O–2H–CO] + ,209.0597[M+H–C3H7N–2CH4O] + ,207.0435[M+H–C3H7N–2CH4O–2H] + ,181.0634[M+H–C3H7N–2CH4O–CO] + ,153.0695[M+H–C3H7N–2CH4O–2CO] + Plasma ions (see Figure 2 A) was identified as sinomenine.

[0092] The main structural difference between the original berberine type and the berberine type lies in the degree of hydrogenation of the C ring. Their mass spectrometric fragmentation mechanisms are the same: they readily lose hydroxyl and methoxy substituents on the ring first, followed by the loss of CO and other groups from the parent nucleus, resulting in a p-π conjugated system in the parent nucleus. Therefore, the N atom in the parent nucleus is more difficult to lose. The quasi-molecular ion peak of compound 60 is 336.1228, suggesting a molecular formula of C. 20 H 18 NO4 + MS E Generate in the map m / z 321.0948[M+H–CH3] + 320.0908[M+H–CH3–H] + 306.0708[M+H–2CH3] + ,292.0981[M+H–CH3–H–CO] + 278.0800[M+H–2CH3–CO] + Plasma ions (see Figure 2 B), consistent with the major cleavage of Pseudoberberine.

[0093] The parent nucleus of benzylisoquinoline alkaloids often cleaves first, resulting in the loss of [NH(CH3)2].+ [NH2CH3] + [NH3] + These groups form [M–45]. + [M–31] + [M–17] + On the one hand, it produces fragment peaks, and on the other hand, it is prone to α-fragmentation, forming... m / z Relatively small fragments. The quasi-molecular ion peak of peak 36 is 286.1440, suggesting a molecular formula of C. 17 H 19 NO3, MS E The spectrum shows that, on the one hand, the loss of a molecule of NH3 due to the breakage of the parent nucleus produces... m / z Fragment ions of 269.1163 then break down and lose CH3OH, forming an epoxy structure, producing... m / z Fragment ions of 237.0940; on the other hand, the parent nucleus undergoes α-fractionation, producing m / z Fragment ions of 178.0797 and 107.0492 (see...) Figure 2 C), consistent with the main cleavage of linderine.

[0094] Apophene-type alkaloids readily break down the benzene ring by substituents such as the methoxy and hydroxyl groups, and the methyl group on the nitrogen atom. The quasi-molecular ion peak at peak 40 is 342.1681, suggesting a molecular formula of C4. 20 H 24 NO4 + MS E Generate in the map m / z 297.1142[M+H–(CH3)2NH] + ,282.0907[M+H–(CH3)2NH–CH3] + ,265.0860[M+H–(CH3)2NH–CH3OH] + , 237.0909[M+H–(CH3)2NH–CH3OH–CO] + Plasma ions (see Figure 2 D), after confirmation, was identified as magnoflorine.

[0095] 3.1.2 Phenylpropanoids

[0096] These compounds (peaks 21, 26, 31, 33, 43, 45, 54, 56, and 61) are distributed in all three medicinal materials, and are mostly chlorogenic acid isomers. The excimer values ​​of peaks 21, 31, and 33 in negative ion mode are all... m / z 353.0883, indicating the molecular formula is C 16 H 18 O9. MS EIn the graph, many are generated m / z Fragment ions at 191, 179, 161, and 135 are fragment ions generated during the removal of caffeic acid or quinic acid, resulting from the breaking of ester or ether bonds. Therefore, it is inferred that all three compounds are isomers of chlorogenic acid. Confirmation with reference standards indicates that peak 21 is neochlorogenic acid and peak 31 is chlorogenic acid (see [link to fragmentation pathway]). Figure 3 Peak 33 is cryptochlorogenic acid.

[0097] 3.1.3 Steroidal compounds

[0098] The steroidal compounds were mainly derived from *Gynostemma pentaphyllum* (peaks 67, 68, 69, 70, 72, 73, 75), primarily consisting of cardiac glycosides (Type A), followed by C. 21 Steroid compounds. Cardiac alpha-glycosides and C 21 Most steroid compounds contain glycosyl substitution structures, so their mass spectrometry fragmentation mainly involves the shedding of glycosyl fragments.

[0099] 3.1.4 Other types of compounds

[0100] Based on relevant literature and secondary fragments, this formulation also identified other types of compounds, including amino acids, sugars, and organic acids.

[0101] 3.2 Network pharmacology analysis

[0102] 3.2.1 Screening of active ingredients and acquisition of targets

[0103] The 82 identified chemical components were screened using the SwissADME database, resulting in 49 potential active ingredients. Based on relevant literature, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and salicylic acid were further included, ultimately leading to 56 potential active ingredients. A search of the SwissTargetPrediction database yielded 834 compound targets (Appendix 1-S1).

[0104] 3.2.2 Regulatory effects of active ingredients on biological systems and processes

[0105] We performed KEGG and Reactome pathway enrichment analysis on 834 targets and obtained 279 signaling pathways (Appendix 1-S2). The biological systems and processes with the most pathways were the immune system and metabolism, respectively.

[0106] Rabies (RA) is a heterogeneous, systemic autoimmune disease caused by immune abnormalities. Currently, TNF-α and IL-6 inhibitors are mainly used to treat RA by exerting anti-inflammatory and immunomodulatory effects, and this approach has been recommended in domestic and international guidelines. We integrated the KEGG and Reactome pathways, which belong to the immune system, and performed PPI analysis on targets within these pathways. Figure 4 Based on the degree value, it was found that TNF-α and IL-6 occupy key positions in the entire network, suggesting that these two targets may play an important role in the treatment of RA with Heiguteng Zhuifeng Huoluo Capsules.

[0107] Cardiovascular disease is a common complication of rheumatoid arthritis (RA), and coronary heart disease and other cardiovascular diseases are the leading causes of death in RA patients. The possible pathological mechanism is inflammation-related lipid metabolism disorders. Therefore, we integrated lipid metabolism-related targets from Heiguteng Zhuifeng Huoluo capsules and analyzed their intervention ability on lipid metabolism and cardiovascular complications in the progression of RA using a PPI network. Figure 5 As shown in the figure, Heiguteng Zhuifeng Huoluo Capsules mainly regulate lipid metabolism in RA patients by acting on cytochrome P450 enzymes (such as CYP1A1, CYP1A2, CYP3A4, etc.), suggesting that Heiguteng Zhuifeng Huoluo Capsules may have a certain therapeutic effect on cardiovascular diseases complicated by RA.

[0108] 3.2.3 RA Target Acquisition

[0109] 3546 RA targets were identified through screening using the GeneCards and DisGeNET databases (Appendix 1-S3). Venn diagrams revealed 382 overlapping targets between the active ingredient targets of the Heiguteng Zhuifeng Huoluo capsules and the RA targets. Figure 6 )

[0110] 3.2.4 Network diagram of medicinal materials-active ingredients-intersection targets

[0111] See the network diagram of medicinal materials, active ingredients, and intersection targets. Figure 7 The network contains 443 nodes and 2044 relationships. The top ten active ingredients by degree value are 6- O -demethylmenisporphine, sinoracutine, menisporphine, linolenic acid, 8-oxobarbitine, corydine methyl ether, periforgenin A, berberine, dehydrocorydalmine, salicylic acid, of which 6- OSeven alkaloids, including -demethylmenisporphine, sinoracutine, menisporphine, and 8-oxobamatin, are derived from *Sinomenium acutum*; two cardiac glycosides, Periforgenin A and cyperiforgenin, are derived from *Heiguteng*; linolenic acid is a common component of *Sinomenium acutum*, *Sinomenium acutum*, and *Heiguteng*, and these may be key components of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA (Table 2).

[0112] Table 2 Key Active Ingredients (Top Ten)

[0113]

[0114] 3.2.5 Construction of PPI network and enrichment of KEGG pathway

[0115] The PPI network results are shown below. Figure 8 A shows that it involves 381 nodes and 7109 edges. The top three targets in terms of degree value are AKT1, TNF-α, and IL-6, which may be key targets for the treatment of RA with Heiguteng Zhuifeng Huoluo Capsules. Figure 8 The 20 pathways in B mainly involve multiple inflammation-related pathways such as the PI3K-Akt signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, and TNF signaling pathway, as well as immune regulation-related pathways such as the chemokine signaling pathway, C-type lectin receptor signaling pathway, and T-cell receptor signaling pathway. This indicates that Heiguteng Zhuifeng Huoluo Capsules treat RA through multiple targets and pathways.

[0116] 3.3 Validation of Network Pharmacology Results

[0117] 3.3.1 Molecular docking

[0118] Using molecular docking techniques, the key active ingredient 6- O Demethylmenisporphine, sinoracutine, menisporphine, linolenic acid, 8-oxobarbital, corydine methyl ether, periforgenin A, berberine, dehydrocorydalmine, and salicylic acid were docked with key targets AKT1, TNF-α, and IL-6, respectively, to preliminarily verify the interaction between active ingredients and targets. The results (Table 3) show that the binding energy of all 30 groups of compounds to their targets was <0 kcal / mol, indicating that they can spontaneously bind. The docking combinations with low binding energy to each target and capable of forming hydrogen bonds were visualized. Figure 9 As shown, 6- OThe carbonyl oxygen atom, hydroxyl oxygen atom, and methoxy oxygen atom in the structure of -demethylmenisporphine form hydrogen bond interactions with the amino acid residues lysine (Lys) 174 and isoleucine (Ile) 173 of TNF-α; the hydroxyl oxygen atom in the structure of Periforgenin A forms hydrogen bond interactions with the amino acid residues arginine (Arg) 179 and (Arg) 182 of IL-6; 6- O When -demethylmenisporphine binds to AKT1, it forms hydrogen bond interactions with its amino acid residues asparagine (Asn) 204 and serine (Ser) 205.

[0119] Table 3 Molecular docking results (Kcal / mol)

[0120]

[0121] 3.3.2 In vitro cell activity verification

[0122] IL-6 and TNF-α are both involved in the pathological process of RA, and their abnormal expression and dysregulation are two important factors promoting the onset of RA. Network pharmacology research shows that IL-6 and TNF-α are two key targets for Qingfengteng Zhuifeng Huoluo Capsule to exert its anti-RA effect. Therefore, an in vitro inflammation model of LPS-induced RAW 264.7 cells was selected to verify the effects of Heiguteng Zhuifeng Huoluo Capsule on the two inflammatory factors TNF-α and IL-6.

[0123] Heiguteng Zhuifeng Huoluo Capsule at three concentrations (50, 100, 200 μg / ml) had no obvious effect on the viability of RAW 264.7 cells (viability > 85%). The results of in vitro anti-inflammatory activity showed ( Figure 10 ) that Heiguteng Zhuifeng Huoluo Capsule at a high concentration (200 μg / ml) had a significant inhibitory effect on the release of TNF-α; each concentration had an inhibitory trend on the release of IL-6, but there was no significant difference.

[0124] 4 Discussion

[0125] In this experiment, UPLC-Q-TOF-MS was used to comprehensively characterize the chemical constituents of Heiguteng Zhuifeng Huoluo Capsule. A total of 82 compounds were identified, including alkaloids, phenylpropanoids, steroids, and other types, and the material basis of Heiguteng Zhuifeng Huoluo Capsule was basically clarified. The mass spectrometry fragmentation rules of major components were summarized, providing a theoretical basis for the rapid identification of corresponding components.

[0126] Compounds were screened and targets were identified using multiple databases. Network pharmacology methods were employed to construct a network diagram of medicinal materials, active ingredients, and intersection targets, as well as a target PPI network diagram. Results showed that 6- O Demethylmenisporphine, sinoracutine, menisporphine, linolenic acid, 8-oxobarbital, corydinemethyl ether, periforgenin A, berberine, dehydrocorydalmine, and cytosine aglycone are key components of Heiguteng Zhuifeng Huoluo Capsules for the treatment of rheumatoid arthritis (RA). AKT1, TNF, and IL6 are its key targets. Linolenic acid is a polyunsaturated fatty acid, and studies have found that it can exert anti-inflammatory effects by reducing the content of nuclear factor I-κBα, thereby decreasing the expression and content of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. Cytosine aglycone has a significant inhibitory effect on histamine release from mast cells in vitro and in vivo, and this effect is dose-dependent. Berberine has been used to treat various inflammatory diseases. 8-oxobarbital is an oxidation product of berberine. Current research has found that berberine and 8-oxobarbital can alleviate dextran sulfate sodium (DSS)-induced colitis by activating the Nrf2 pathway and inhibiting the NLRP3 inflammasome. The main pathological features of rheumatoid arthritis (RA) include inflammatory cell and factor infiltration in the synovium and excessive proliferation of synovial cells. The AKT1 gene plays a role in regulating cell proliferation and growth, primarily by participating in cellular processes, including regulating apoptosis and controlling glucose metabolism, thereby influencing the disease progression of RA. TNF-α plays a dominant role in inducing synovial cell proliferation in rheumatoid arthritis and can induce an increase in local IL-6 concentration in synovial cells of RA patients. Therefore, TNF-α and IL-6 are expressed in joint pathological tissues. High expression of joint inflammatory factors promotes osteoclast maturation and activation, reduces chondrocyte proliferation, stimulates synovial tissue proliferation, and ultimately leads to joint damage.

[0127] Among the 20 KEGG signaling pathways, the PI3K / AKT pathway and the TNF signaling pathway are closely related to rheumatoid arthritis. The PI3K / AKT pathway is involved in apoptosis, proliferation, and differentiation. Growth factors, hormones, cytokines, and chemokines can all activate PI3K protein. After activation, PI3K protein continues to activate downstream kinases, of which AKT is an important kinase. After AKT activation, it can mediate the activation of NF-κB by regulating the degradation of IkBα, thereby triggering a series of inflammatory responses. Studies have shown that Heiguteng Zhuifeng Huoluo capsules can significantly reduce the expression of p-PI3K and p-AKT proteins in the synovial tissue of collagen-induced rheumatoid arthritis (CIA) rats and improve the degree of paw swelling in CIA rats. Anti-TNF therapy is a major treatment for rheumatoid arthritis (RA). Serum and synovial fluid TNF-α levels in RA patients are positively correlated with disease activity. TNF-α has been reported to promote the proliferation and autophagy of high-intensity lipoprotein atherosclerosis (HFLS), thereby increasing its tolerance to apoptotic stimuli. Furthermore, in diseased joints, TNF-α can stimulate the synthesis of PGE2 and collagenase in synovial and chondrocytes, disrupting bone and cartilage resorption and thus promoting HFLS proliferation. It has been reported that Heiguteng Zhuifeng Huoluo capsules can significantly reduce serum and synovial fluid TNF-α levels in CIA rats, decrease paw edema, and improve thymus and spleen indices, thus alleviating symptoms. Network pharmacology studies suggest that Heiguteng Zhuifeng Huoluo capsules may exert their therapeutic effect on RA through related targets and pathways in anti-inflammation and immunomodulation.

[0128] The network pharmacology results were validated using molecular docking and in vitro activity assays. The results showed that the binding energies of the 10 key active ingredients to the 3 key targets were all less than 0, indicating that they could all bind spontaneously. In vitro activity experiments demonstrated that Heiguteng Zhuifeng Huoluo Capsules could inhibit the release of TNF-α and IL-6 from inflammatory cells, and exhibited a significant inhibitory effect on TNF-α release at high concentrations.

[0129] In summary, this study integrates UPLC-Q-TOF-MS technology, network pharmacology, molecular docking technology, and in vitro activity experiments to preliminarily elucidate the material basis and mechanism of action of Heiguteng Zhuifeng Huoluo Capsules in treating RA. This lays the foundation for explaining the scientific connotation of Heiguteng Zhuifeng Huoluo Capsules in treating RA and provides a basis for its rational clinical use and promotion.

[0130] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for determining multiple components in Heiguteng Zhuifeng Huoluo Capsules, used to simultaneously determine the content of multiple components in Heiguteng Zhuifeng Huoluo Capsules, characterized in that, The determination was performed using UPLC-Q-TOF-MS analysis. The specific chromatographic conditions are as follows: Chromatographic column: Waters Acquity UPLC HSS T3, 2.1 100 mm, 1.8 μm; Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B); Elution gradient: 0~1 min, 0% B; 1~20 min, 0%~28% B; 20~40 min, 28%~80% B; 40~42 min, 80%~100% B; 42~45 min, 100% B; 45-45.1 min, 100%~0% B; 45.1-48 min, 0% B; Injection volume: 1 μL; Column temperature: 35℃; Flow rate: 0.36 mL / min; The specific mass spectrometry conditions are as follows: an electrospray ionization source was used, with positive / negative ion scanning mode and a mass scan range of 50–1500 Da; N2 was used as the desolvation gas at a flow rate of 500 L / h and a temperature of 400 °C; N2 was used as the orifice gas at a flow rate of 100 L / h and a voltage of 40 eV; the ionization source temperature was 120 °C; the capillary voltage was 3500 eV; argon was used as the collision gas with a low collision energy of 6 eV and a high collision energy of 30–60 eV; sodium formate and leucine-enkephalin solution were used as real-time calibration standard solutions at a flow rate of 5 μl / min, and the positive source reference ion was set to m / z 556.2771 [M+H]. + The negative source reference ion has a m / z of 554.2615 [M–H]. - ; The test solution of Heiguteng Zhuifeng Huoluo Capsules was analyzed to obtain base peak chromatograms in positive and negative ion modes. Mass spectrometry data were acquired using MassLynx 4.1 software, and the quasi-molecular ion peaks and fragment peaks of each chromatographic peak were extracted. Combined with reference standards and literature data, the compounds were analyzed and their structures were identified. The compounds were confirmed to include magnoflorine, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, and uridine by comparison with reference standards. The test solution was prepared as follows: Take the contents of the Black Bone Vine Wind-Chasing and Blood-Activating Capsules, mix them well, weigh them accurately, place them in a 50 mL stoppered conical flask, add 25 mL of methanol, sonicate at 350 W power and 50 Hz frequency for 30 min, cool to room temperature, shake well, and filter through a 0.22 μm microporous membrane to obtain the test solution.

2. The method for determining multiple components in the Black Bone Vine Wind-Chasing and Blood-Activating Capsules according to claim 1, characterized in that, The reference solution used is prepared by accurately weighing each standard, adding methanol to prepare a 0.5~1 mg / mL reference solution, and then separately preparing a mixed standard solution for later use.

3. A method for studying the mechanism of action of Heiguteng Zhuifeng Huoluo Capsules in treating rheumatoid arthritis, characterized in that... Includes the following steps: (1) Screening of active ingredients: The identified compound SMILES numbers were imported into the SwissADME database. The potential active ingredients were obtained by using the screening criteria of high oral bioavailability and two or more "Yes" in drug-like properties. (2) Acquisition of active ingredient compound targets; the potential active compounds obtained by screening are further searched through the SwissTargetPrediction database, with the species attribute set to "Homo sapiens", and targets with P>0 are screened; (3) Enrichment analysis of KEGG and Reactome pathways for active ingredient targets: Kyoto Encyclopedia of Genes and Genomes and Reactome pathway enrichment analysis were performed on the screened targets. FDR < 10 -4 Thus, a signal path is obtained; (4) RA target acquisition: GeneCards and DisGeNET databases were searched using "rheumatoid arthritis" as the keyword, and disease targets with a score greater than or equal to the median were screened; the intersection of active ingredient targets and RA targets was plotted using the Draw Venn Diagram platform to obtain the potential target of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA. (5) Constructing a network of medicinal materials, active ingredients, and intersection targets: Using Cytoscape 3.9.1 software, a network model of medicinal materials, active ingredients, and intersection targets was constructed and the network topology properties were analyzed to screen out the top ten active ingredients with the highest degree values; (6) Constructing a PPI network: Protein-protein interaction analysis was performed on the intersection targets using the STRING platform. The species attribute was set to "Homo sapiens", the threshold was 0.4, and unconnected targets were removed. Finally, the PPI network was visualized and its topological properties were analyzed using Cytoscape 3.9.1 software to determine the key targets of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA. (7) KEGG pathway enrichment analysis: KEGG pathway enrichment analysis was performed on the intersection targets, with FDR < 10. -4 The top 20 KEGG pathways were screened based on FDR value and bubble diagrams were drawn to identify the main signaling pathways. Ultimately, 6-O-demethylmenisporphine, sinoracutine, menisporphine, linolenic acid, 8-oxobamatin, corydinemethyl ether, periforgenin A, berberine, dehydrocorydalmine, and salicylic acid were identified as key components of Heiguteng Zhuifeng Huoluo Capsules for the treatment of RA, and AKT1, TNF, and IL6 were identified as key targets. In step (1), the identified compound is a compound determined by the method for determining multiple components in the Black Bone Vine Wind-Chasing and Blood-Activating Capsule as described in claim 1.

4. The method for studying the mechanism of action of the Black Bone Vine Wind-Chasing and Blood-Activating Capsule in treating rheumatoid arthritis according to claim 3, characterized in that, In step (1), chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and salicylic acid are added to the list of active ingredients obtained by screening using the SwissADME database.

5. The method for studying the mechanism of action of the Black Bone Vine Wind-Chasing and Blood-Activating Capsule in treating rheumatoid arthritis according to claim 3, characterized in that, In step (3), the KEGG and Reactome pathways belonging to the immune system are integrated, and PPI analysis is performed on the targets in the pathways. Based on the degree value, the key signaling pathway targets are determined.

6. The method for studying the mechanism of action of the Black Bone Vine Wind-Chasing and Blood-Activating Capsule in treating rheumatoid arthritis according to claim 3, is characterized in that, In step (3), the lipid metabolism-related targets of the Heiguteng Zhuifeng Huoluo capsules are integrated, and its intervention ability on lipid metabolism and cardiovascular complications in the course of RA is analyzed through PPI network.

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