Method for optimizing traditional Chinese medicine compound based on network pharmacology and material basis analysis

Abstract

The invention belongs to the field of traditional Chinese medicine modernization and pharmaceutical preparations, and particularly relates to a method for optimizing a traditional Chinese medicine compound based on network pharmacology and material basis analysis. The method comprises the following steps: S1, acquiring medicinal material compositions, screening active ingredients and action targets, intersecting with disease targets to obtain a potential target set, and constructing a PPI network to screen core targets; s2, analyzing the compound extract / preparation by adopting a separation technology, and identifying main components and source medicinal materials; s3, tracing active ingredients and source medicinal materials based on core targets, evaluating the contribution degree of the medicinal materials in combination with high-abundance ingredients, and screening key medicinal materials to form an optimized simple formula.

Description

Technical Field

[0001] This invention relates to the field of modernization of traditional Chinese medicine and pharmaceutical preparations, and in particular to a method for optimizing traditional Chinese medicine compound prescriptions based on network pharmacology and material basis analysis. Background Technology

[0002] Lipid metabolism disorders refer to abnormal changes in the lipid profile, including various diseases associated with elevated total cholesterol (TC) and / or triglycerides (TG), elevated low-density lipoprotein (LDL), and / or decreased high-density lipoprotein (HDL). Studies have shown that hyperlipidemia (especially elevated LDL) is closely related to lipid metabolism disorders, such as atherosclerotic vascular disease, abnormal vasomotor function, and non-alcoholic fatty liver disease (NAFLD). Therefore, regulating abnormal lipid levels to the normal range is an effective strategy to reduce the incidence of these diseases.

[0003] In clinical practice, statins, omega-3 polyunsaturated fatty acids (PUFAs), bile acid conjugating resins, fibrates, niacin, and proprotein convertase / matrix metalloproteinase-like protein 9 (PCSK9) inhibitors are widely used for the prevention and treatment of hyperlipidemia. However, in some cases, patients may not achieve the desired therapeutic effect due to side effects or high treatment costs. Combining two or more treatment regimens with different mechanisms of action can effectively improve symptoms of specific diseases with fewer side effects and is considered a potential strategy for treating a variety of intractable diseases. Traditional Chinese medicine (TCM) is often used in combination with conventional therapies due to its lower cost and fewer side effects. Given the different cholesterol-lowering mechanisms of TCM and Western medicine, combined lipid-lowering therapies integrating TCM and Western medicine have been widely reported.

[0004] Heze Lipid-Lowering Oral Solution is an in-house preparation developed by Shanghai First People's Hospital in the 1980s based on the teachings of the renowned traditional Chinese medicine master Zhang Jingren and on traditional theories such as "spleen deficiency generates phlegm and dampness" and "liver disease indicates liver-to-spleen transmission." The formula uses stir-fried Atractylodes macrocephala as the chief ingredient to tonify the Taiyin meridian; Alisma plantago-aquatica promotes diuresis and eliminates dampness; Pinellia ternata and Citrus reticulata peel resolve phlegm and regulate Qi; Xuanming San (a traditional Chinese medicine formula) raises Yang, improves vision, and stimulates thinking; Salvia miltiorrhiza invigorates blood and regulates nutrition; Crataegus pinnatifida resolves phlegm and dissipates nodules; Pinellia ternata clears heat, nourishes Yin, resolves phlegm, and lowers lipids; and Nelumbo nucifera leaf (a traditional Chinese medicine formula) promotes the upward movement of Yang, brightens the eyes, and stimulates thinking; Salvia miltiorrhiza invigorates blood and regulates nutrition; Crataegus pinnatifida resolves phlegm and dissipates nodules; Pinellia ternata clears heat, nourishes Yin, resolves phlegm, and lowers lipids; and Nelumbo nucifera leaf (a traditional Chinese medicine formula) promotes the upward movement of clear Qi, lowers turbidity, and eliminates phlegm and turbidity. This formula (originally named "Ningzhi Fang") is used to treat hyperlipidemia, obesity, and other diseases with significant efficacy. This study aims to explore the lipid-lowering pharmacological mechanism of this formula and optimize its formulation. While reducing the number of Chinese herbs required, the lipid-lowering effect of the formula will not be weakened and may even be enhanced, and adverse reactions will not increase and may even be reduced. By optimizing the formula composition, the effects of increased efficacy, reduced toxicity, Summary of the Invention

[0005] In view of this, the present invention provides a method for optimizing traditional Chinese medicine (TCM) compound prescriptions based on network pharmacology and material basis analysis. The present invention aims to systematically analyze the pharmacodynamic material basis and key molecular targets of TCM compound prescriptions that exert their lipid-lowering effects, thereby elucidating the overall mechanism of action of the compound prescription in synergistically regulating lipid metabolism through multiple active ingredients, multiple targets, and multiple signaling pathways. Based on this, through a prescription optimization strategy based on mechanism and network analysis, the lipid-lowering efficacy is maintained or even enhanced while reducing the number of TCM herbs contained in the prescription, and adverse reactions are not increased or are even further reduced. This optimization approach aims to achieve a comprehensive improvement in the efficacy, safety, and resource utilization efficiency of the compound prescription, achieving the modern formulation design goals of "increased efficacy, reduced toxicity, and material conservation."

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for optimizing traditional Chinese medicine compound prescriptions based on network pharmacology and material basis analysis, comprising the following steps: S1. Network pharmacology analysis: Obtain the medicinal material composition of the target compound; screen the potential active ingredients of the medicinal materials through the database and predict the target of the potential active ingredients; compare with disease targets related to the target disease; take the intersection of the target of the active ingredients and the disease targets to obtain the potential target set; construct a protein-protein interaction (PPI) network and screen the core targets based on topological parameters. S2. Material basis identification: Prepare the total extract of the target Chinese herbal medicine compound and the single extracts of each herb that make up the target Chinese herbal medicine compound; use separation and identification technology to analyze the chemical composition of the total extract and each single extract to identify the main chemical components whose relative abundance in the total ion chromatogram of the total extract is greater than a preset threshold, and determine the source herbs of the main chemical components based on the analysis results of each single extract. S3. Formula Optimization: Calculate the overall contribution of each medicinal material and select the key medicinal materials with the highest overall contribution in a predetermined quantity to form an optimized simplified formula.

[0008] In step S1, the target compound is a compound used to treat or prevent hyperlipidemia.

[0009] The separation and identification techniques include ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis.

[0010] The UPLC-MS analysis was performed using electrospray ionization in positive and negative ion mode. The chromatographic column was a BEH C18 column (1.7 μm, 2.1 × 100 mm). The mobile phase consisted of an acetonitrile solution containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid, with gradient elution.

[0011] Furthermore, the UPLC-MS analysis and detection conditions are as follows: Chromatographic conditions: A BEH C18 1.7 μm packed column (2.1 × 100 mm) was used. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution. Gradient elution program: 0–3 min, mobile phase B concentration 5%–20%; 3–10 min, mobile phase B concentration 20%–100%; 10–12 min, mobile phase B concentration 100%; 12–15 min, 100%–5% mobile phase; 15– Flow rate 0.4 mL / min. Column temperature: 45 ℃. Injection volume: 3 μL.

[0012] Mass spectrometry conditions: VION quadrupole time-of-flight mass spectrometer (Shanghai Waters Technology Co., Ltd.). Ion mode: electrospray ionization (ESI) with positive and negative ions; capillary voltage: 2 kV; cone voltage: 40 V; ion source temperature: 115 °C; ion source temperature: 40 V. Ion source temperature: 0.5 μL / min.

[0013] Signal acquisition range: 50 to 1000 m / z, ion source temperature: 115℃, scanning range: 50 to 1000 m / z.

[0014] In step S1, the selection of core target points is based on network topology parameters, including node degree, betweenness centrality, and proximity centrality.

[0015] In step S3, the calculation of the comprehensive contribution includes: normalizing and weighting the total number of target points hit by compounds from each medicinal material in the core target point after merging and deduplicating, and the relative abundance of compounds from each medicinal material in the mass spectrometry analysis results.

[0016] Secondly, the present invention provides a simplified traditional Chinese medicine formula obtained by the method described above, comprising three medicinal materials: Atractylodes macrocephala, Pinellia ternata, and lotus leaf.

[0017] The formulation exerts its lipid-regulating effect by acting on PTGS2, ADRB2 and / or ACHE targets, and / or regulating the PI3K-Akt signaling pathway and / or lipid and atherosclerosis pathway through its flavonoid, terpene and / or phenolic acid active ingredients.

[0018] In one specific embodiment, the raw materials are composed of the following parts by weight: 8-15 parts of Atractylodes macrocephala, 6-12 parts of Citrus reticulata peel, and 4-9 parts of Pinellia ternata.

[0019] The drug is used to lower serum and / or liver levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and / or to raise high-density lipoprotein cholesterol (HDL-C) levels.

[0020] Thirdly, a pharmaceutical composition comprising an extract of a simplified traditional Chinese medicine formula as described above, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is used to treat or prevent hyperlipidemia.

[0021] The composition exerts its lipid-regulating effect by acting on PTGS2, ADRB2 and / or ACHE targets, and / or regulating the PI3K-Akt signaling pathway and / or lipid and atherosclerosis pathway through its flavonoid, terpene and / or phenolic acid active ingredients.

[0022] The whole extract of Heze Lipid-Lowering Oral Solution (HZ), the various optimized simplified extracts and single herbal extracts used in this invention are all prepared by standardized processes to ensure the stability and experimental homology of the pharmacodynamic material basis.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention addresses the current situation where Heze Lipid-Lowering Oral Liquid only has clinical efficacy verification but lacks mechanistic and material basis research. For the first time, it systematically elucidates the synergistic mechanism of its multi-component and multi-target effects by integrating network pharmacology and liquid chromatography-mass spectrometry, filling the gap in the pharmacological research of this compound. On this basis, it innovatively establishes an integrated strategy of "mechanism analysis - substance identification - formulation optimization". Without changing the original ratio of medicinal materials, it achieves the overall optimization goal of "enhancing efficacy, reducing toxicity, and saving materials" of the compound simply by scientifically screening the types of medicinal materials. This provides a repeatable and verifiable technical path for the precise design and modern development of traditional Chinese medicine compound formulas.

[0024] 2. Mechanism studies of this invention show that the compound, through multiple active ingredients such as flavonoids, terpenes and phenolic acids (e.g., neohesperidin, 6-gingerol, atractylodes lactone, etc.), synergistically acts on key targets such as PTGS2, ADRB2, and ACHE, and regulates biological networks closely related to lipid metabolism and inflammation, such as the PI3K-Akt signaling pathway and the lipid and atherosclerosis pathway, thereby exerting a systemic regulatory effect of "multi-component-multi-target-multi-pathway".

[0025] 3. Based on the above mechanism analysis, this invention further proposes a prescription optimization strategy centered on "network efficacy." Through network pharmacology and multidimensional network analysis of "herbs-components-targets," Atractylodes macrocephala, Citrus reticulata peel, and Pinellia ternata were precisely identified as key herbs for maintaining the core efficacy network from the original nine herbs in Heze Lipid-Lowering Oral Liquid (HZ). Based on this, an optimized compound composed of the above three herbs was successfully obtained. This optimization theoretically preserves the core mechanism of action of the original formula and focuses on key pharmacodynamic substances.

[0026] 4. Further, animal experiments have shown that this optimized compound can significantly improve the lipid profile of hyperlipidemic model mice (increase HDL-C, decrease TC and TG), reduce pathological damage to the liver and adipose tissue, and its overall efficacy is superior to or equivalent to the positive control drug simvastatin, with good safety.

[0027] 5. This invention not only systematically elucidates the synergistic mechanism of compound prescriptions, but also provides a data-driven, mechanism-oriented method for simplifying prescriptions, offering a research paradigm that can be referenced for the modern research and development of traditional Chinese medicine compound prescriptions, and has significant scientific research value and transformation potential. Attached Figure Description

[0028] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is the network pharmacology result (drug-compound-disease target network) of Heze lipid-lowering oral liquid in the treatment of hyperlipidemia; Figure 2 This study analyzed the bioaccumulation of the core target of Heze Lipid-Lowering Oral Solution in the treatment of hyperlipidemia (A-KEGG analysis; B-GO analysis). Figure 3 This is a traceability map of the core components of medicinal materials obtained by UPLC-MS identification of small molecule compounds in extract samples; Figure 4 These are indicators used to evaluate the results of hyperlipidemia modeling, where A is body weight; B is low-density lipoprotein; C is total cholesterol; and D is triglycerides. Figure 5 The study compared the effects of ginseng and peony lipid-lowering oral liquid (using a complete formula and a simplified formula) on lipid regulation with the positive control drug simvastatin. A represents high-density lipoprotein (HDL), B represents low-density lipoprotein (LDL), C represents total cholesterol, and D represents total triglycerides. E represents HDL (liver), F represents LDL (liver), G represents total cholesterol (liver), and H represents total triglycerides (liver). Figure 6 This shows the staining of liver tissue pathological sections; the scale bar in the image is 100μm. Figure 7 This shows the staining of adipose tissue pathological sections; the scale bar in the image is 100 μm. Figure 8 These are the results of safety biochemical indicator tests; among them, A is alanine aminotransferase; B is aspartate aminotransferase; C is alkaline phosphatase; D is cholinesterase; ECK is creatine kinase; F is creatine kinase isoenzyme; G is creatinine; H is blood urea nitrogen; and I is uric acid. Detailed Implementation

[0029] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0030] The whole extract of Heze Lipid-Lowering Oral Liquid (HZ) used in this invention, as well as the extracts of each single herb (Atractylodes macrocephala, Alisma plantago-aquatica, Pinellia ternata, Citrus reticulata peel, Salvia miltiorrhiza, Crataegus pinnatifida, and Nelumbo nucifera leaf) that make up the compound, were all prepared and provided by the Traditional Chinese Medicine Preparation Center of Shanghai First People's Hospital according to a unified standardized process, ensuring the stability of the pharmacodynamic material basis and the homology of the experimental materials. The optimized simplified extract was prepared by mixing the single herb extracts in equal amounts according to their concentration and ratio in the compound.

[0031] Example 1: Research on the Material Basis of Network Pharmacology 1. Network pharmacology analysis To elucidate the potential mechanism of action of Heze Lipid-Lowering Oral Solution (HZ), the following systematic analysis workflow was adopted: 1.1 Screening of active ingredients and prediction of targets First, based on the TCMSP and TCMID databases and literature reports, the chemical components of the nine medicinal herbs in HZ formula were collected. Preliminary screening was conducted using the SwissADME platform according to Lipinski's Rule of Five. With oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 as criteria, 166 potential active compounds were initially identified; those with a degree greater than twice the network median were designated as core targets. Subsequently, the SwissTargetPrediction and STITCH platforms were used to perform reverse virtual screening and prediction of potential targets for the screened compounds.

[0032] 1.2 Disease Target Collection and Intersecting Target Acquisition Using the GeneCards, DisGeNET, OMIM, and DrugBank databases, and employing the keywords "hyperlipidemia" and "dyslipidemia," disease targets related to hyperlipidemia were retrieved and integrated. After deduplication, a disease target set was established. A Venn analysis was performed on the predicted drug component target set and the disease target set, and the intersection was used to obtain the potential direct target set for Heze Lipid-Lowering Oral Solution (HZ) in the intervention of hyperlipidemia.

[0033] 1.3 Network Construction and Analysis: The aforementioned intersection targets were imported into the STRING database (confidence > 0.7) to construct a protein-protein interaction (PPI) network. The PPI network was visualized and its topology analyzed using Cytoscape software, and core targets were selected based on parameters such as node degree and betweenness centrality. Furthermore, information on medicinal materials, active ingredients, core targets, and diseases was integrated to construct a multi-layered interaction network of "medicinal materials-ingredients-targets-diseases," visually representing the multi-component, multi-target action characteristics of the compound formula.

[0034] 1.4. Enrichment analysis of biological functions and pathways Using online analysis platforms such as DAVID or Metascape, gene ontology (GO) functional enrichment analysis (including biological processes, cellular components, and molecular functions) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the potential direct-acting target set. A corrected p-value (adj. p-value) <0.05 was set as the significant enrichment criterion.

[0035] Finally, to improve the reliability of predictions and clarify key mechanisms, this invention further employs molecular docking technology to simulate the binding mode of core active ingredients and core target proteins (such as HMGCR, PPARα, etc.), and evaluates the feasibility of their interaction. This provides a focused direction and theoretical basis for subsequent verification through cell or animal experiments of its regulation of specific processes such as cholesterol synthesis and fatty acid oxidation.

[0036] 2. Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) Instrument: VION quadrupole time-of-flight mass spectrometer (Shanghai Waters Technology Co., Ltd.). Liquid chromatography analysis was performed using a BEH C18 1.7μm packed column, 2.1×100mm (pre-column used). Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution. Gradient elution program: 0-3 min, mobile phase B concentration 5%-20%; 3-10 min, mobile phase B concentration 20%-100%; 10-12 min, mobile phase B concentration 100%; 12-15 min, 100-5% mobile phase; flow rate 0.4 mL / min. Column temperature: 45 ℃. Injection volume: 3 μL.

[0037] The mass spectrometry detection conditions were as follows: ion mode was electrospray ionization (ESI) with positive and negative ions; capillary voltage was 2 kV; cone voltage was 40 V; ion source temperature was 115℃; and nebulizer temperature was 450℃. The ion source temperature was 0.5 μL / min.

[0038] Signal acquisition range: 50 to 1000 m / z; ion source temperature: 115℃; scan range: 50 to 1000 m / z. In this time-of-flight secondary ion mass spectrometry, ion mobility techniques can eliminate mass spectrometry interference and generate collision cross section (CCS) characteristic values ​​for each ion, thereby obtaining more sample information. This method can be used for qualitative analysis of the material basis of Heze lipid-lowering oral liquid extract. Millipore ultrapure water was used as the water source, and formic acid, acetonitrile, and other organic reagents were all Fischer Scientific Optima LC / MS grade products.

[0039] 3. Network pharmacology results To systematically elucidate the multi-component, multi-target mechanism of action of Heze Lipid-Lowering Oral Solution (HZ) in intervening in lipid metabolism disorders, this invention conducted the following analysis based on network pharmacology: First, active ingredient screening and target prediction were conducted. Using the TCMSP database and the SwissADME platform, drug-likeness screening (Lipinski's five rules, etc.) was performed, initially identifying 166 potential active compounds with good oral bioavailability (OB) and drug-likeness (DL) from the Heze lipid-lowering oral liquid compound. Subsequently, reverse target prediction was performed on these compounds using the SwissTargetPrediction database, and corresponding potential targets were collected.

[0040] Secondly, a disease target set was constructed and common targets were screened. Lipid metabolism disorder-related disease targets were systematically retrieved from the OMIM, TTD, and GeneCards databases using keywords such as "hyperlipidemia," "dyslipidemia," and related terms. After deduplication and standardization, the intersection of these targets with the predicted drug targets was calculated, ultimately yielding 130 potential key targets for Heze Lipid-Lowering Oral Liquid to intervene in lipid metabolism disorders. A multidimensional network of "drug-component-target" was constructed. The compound composition of Heze Lipid-Lowering Oral Liquid, predicted active ingredients, common targets, and their interactions were imported into Cytoscape to create a visual network diagram. Figure 1 This visually demonstrates the complex interactions of multiple components and targets in compound preparations.

[0041] Next, protein-protein interaction (PPI) network analysis and core target discovery were performed. 130 common targets were imported into the STRING database, with a minimum interaction confidence level > 0.7, to obtain PPI network data. Figure 1The Cytoscape software was used for visualization and topological attribute analysis, with "degree," "betweenness centrality," and "closeness centrality" as key parameters for evaluating node importance. Higher node topological parameters indicate a more central and controlling position within the network. Based on this, a subset of core target nodes was selected.

[0042] Furthermore, a systematic screening and reverse tracing analysis of potential targets were conducted based on network topology parameters. The specific steps are as follows: First, based on the protein-protein interaction network, core targets with high node degree, high betweenness centrality, and low affinity centrality were calculated and screened. These topology parameters collectively reflect the pivotal role and regulatory importance of the targets in the network (Table 1). Subsequently, through the "drug-component-target" interaction network, the aforementioned core targets were back-mapped to the active compounds that directly interact with them, and further traced back to their plant-derived medicinal materials, thus constructing a three-level association system of "key target-active component-source medicinal material." This analytical method aims to systematically reveal the main material basis and medicinal material contribution of Heze Lipid-Lowering Oral Liquid in exerting its core pharmacological effects. Finally, functional and pathway enrichment analyses were conducted to elucidate the biological significance. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on 130 common targets. GO analysis covered three levels: biological processes (BP), cellular components (CC), and molecular functions (MF). Based on the corrected P-values ​​(adj. P-value) in ascending order, the top 20 significantly enriched items in each level were selected for visualization. Figure 2 (B) to reveal the main biological functions involved in potential targets. KEGG pathway analysis was also sorted by significance, with the top significantly enriched pathways including the PI3K-Akt signaling pathway, lipid and atherosclerosis pathway, Rap1 signaling pathway, phospholipase D signaling pathway, and hepatitis C-related pathways (B). Figure 2 (A). These pathways collectively suggest that Heze lipid-lowering oral liquid may exert a comprehensive lipid-regulating effect by regulating networked processes such as lipid metabolism, inflammatory response, cell proliferation and survival.

[0043] Table 1. Topological parameters of core targets and the main medicinal materials to which their active components belong.

[0044] To clarify the material basis of the efficacy of Heze Lipid-Lowering Oral Solution (HZ), this invention first systematically identified the chemical components of its whole-formula extract. The extract was diluted with ultrapure water to a clinically equivalent concentration, filtered through a 0.22 μm microporous membrane, and then qualitatively analyzed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). By comparing the mass spectrometry fragment information, retention time, and isotope distribution with reference standards, literature reports, and public mass spectrometry databases (such as PubChem, MassBank, and mzCloud), 82 major chemical components were identified in the whole-formula sample, matching 166 potential active ingredients predicted by network pharmacology. This indicates a high consistency between the predicted results and the experimentally identified material basis, verifying the reliability of the network prediction. Arranged in descending order of mass spectrometry response signal intensity, representative components with high abundance and potential pharmacological activity include: Neohesperidin, leucodelphinidin, 6-shogaol, Naphthol[2,3-b]furan-2(4H)-one, 9a-ethoxy-4a,5,6,7,8,8a,9,9a-octahydro-3,8a-dimethyl-5-methylene-,[4aS-(4aα,8aβ,9aβ)]- (9CI)rel-(1R,3aS,4S,7S,8R,8aS)-7-isopropyl-1,4-dimethyldecahydro-4,7-epoxyazulene-1,8-diol-12-senecioyl-2E,8E,10E-atractylentriol, Neocryptotanshinone, alisol C, Madecassic acidAtractylenolide III. Atractylenolide-1, etc. Detailed information on all identified compounds (including compound name, molecular formula, retention time, major ion fragments, and data sources) is summarized in Table 2.

[0045] Table 2. Detailed data on small molecule compounds in medicinal extracts analyzed by UOLC

[0046] To clarify the contribution of each medicinal material in the compound to the overall chemical composition, the above 82 compounds were further attributed to their corresponding single medicinal materials based on their plant origin, and a "medicinal material-component" traceability distribution map was drawn. Figure 3). The central prototype icon represents the identified small molecule compounds, and the depth of its color is positively correlated with the relative content in the compound prescription. The visualization results show that Atractylodes macrocephala, Pinellia ternata, and Lotus leaf contribute more high-abundance components, indicating that they play a dominant role in the compound chemical system and may be the key medicinal materials contributing to the efficacy. This traceability analysis not only intuitively reveals the weights of each medicinal material in the overall chemical profile of the compound prescription but also provides a key basis for subsequent interpretation of the "sovereign, ministerial, adjuvant, and guiding" compatibility rules and formula optimization based on the material basis. Accordingly, the optimized simple formula extract is prepared by equally blending the extracts of single medicinal materials according to their concentrations and ratios in the compound prescription.

[0047] Example 2 verifies the lipid-regulating effect of Heze Jiangzhi Oral Liquid and the results of formula optimization 1. Animal grouping and model establishment The present invention selects 42 SPF-grade C57BL / 6J male mice (body weight 20±2 g, 8 weeks old), purchased from Shanghai Lingchang Experimental Animal Co., Ltd., and the experimental animal production license number is SCXK(Shanghai)2023-0002. After all mice are adaptively raised in a standard SPF-grade animal experiment facility (temperature 22±2°C, humidity 50±10%, 12 h light-dark cycle) for 1 week, the formal experiment begins. The experimental protocol has been reviewed and approved by the Experimental Animal Ethics Committee of this unit (approval number: A2024089). <000014​​​​​​​​​​​​​​​​​​(6) Simple formula low dose group (HZS-L): On the basis of feeding high-fat feed, the optimized simple formula extract of Heze lipid-lowering oral liquid (Atractylodes macrocephala, Pinellia ternata, lotus leaf) was administered by gavage daily at a dose of 350 mg / kg (the amount of raw herbs is calculated based on the amount of the principal herb Atractylodes macrocephala; the proportion of each herb is the same as the whole formula group).

[0054] (7) High-dose group of simplified prescription (HZS-H): On the basis of feeding high-fat diet, the optimized simplified prescription extract (Atractylodes macrocephala, Pinellia ternata, lotus leaf) was administered by gavage daily at a dose of 1050 mg / kg (the amount of raw herbs is calculated based on the amount of Atractylodes macrocephala, the principal herb; the proportion of each herb is the same as the whole prescription group).

[0055] Except for the normal control group, all other groups were fed a high-fat diet continuously from the start of the experiment. All oral administration was performed at a fixed time every morning, with a uniform volume of 10 mL / kg. The model control group and the normal control group were simultaneously administered the same volume of solvent (distilled water) via oral gavage.

[0056] At the end of the intervention, the modeling effect was evaluated. Compared with the normal control group, the serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels in the model control group mice were all significantly increased (P<0.05), accompanied by a decrease in high-density lipoprotein cholesterol (HDL-C) levels, indicating that the hyperlipidemia animal model was successfully induced. Figure 4 (where A is body weight; B is low-density lipoprotein; C is total cholesterol; and D is triglycerides) can be used for subsequent pharmacodynamic evaluation.

[0057] 2. Pharmacodynamic evaluation indicators In terms of pharmacodynamic evaluation, this invention comprehensively evaluated the regulatory effects of each intervention group on the hyperlipidemia (HLP) model from two levels: systemic (serum) and local (liver), using a multi-dimensional approach that combines biochemical indicators with histopathology.

[0058] Serum lipid profile assay: Serum samples collected at the experimental endpoint were directly analyzed using a fully automated biochemical analyzer (CMax Plus). The instrument was equipped with original manufacturer calibrators and quality control samples, and the concentrations of serum total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were determined strictly in accordance with standard operating procedures.

[0059] Lipid content determination in liver tissue: To investigate the direct effects of drugs on the liver, a core organ for lipid metabolism, we determined the lipid content in liver tissue homogenates. Liver tissue samples from the same location were weighed, homogenized with pre-cooled physiological saline, and the concentrations of TC, TG, and LDL-C in the liver tissue homogenates were determined using a commercially available biochemical kit provided by Elabscience (Wuhan, China). All procedures were strictly performed according to the kit instructions. These results reveal the direct intervention effect of drugs on hepatic lipid metabolism.

[0060] The results show that: The complete formula (HZF) showed significant effects in increasing high-density lipoprotein cholesterol (HDL-C) levels, with its efficacy in raising both liver and serum HDL-C levels superior to the positive control drug simvastatin. P <0.05. However, in terms of reducing hepatic low-density lipoprotein cholesterol (LDL-C), serum total cholesterol (TC), and serum triglycerides (TG), the whole formula showed no statistically significant difference compared with the simvastatin group. P >0.05); however, in terms of reducing liver TC and liver TG, the overall effect was slightly lower than that of the simvastatin group ( Figure 5 Where A is high-density lipoprotein, B is low-density lipoprotein, C is total cholesterol, D is total triglycerides; E is high-density lipoprotein (liver), F is low-density lipoprotein (liver), G is total cholesterol (liver), and H is total triglycerides (liver).

[0061] The simplified formulation (HZS), optimized through network pharmacology and herbal traceability analysis, exhibited more comprehensive lipid-regulating advantages. This formulation was significantly superior to the simvastatin group in increasing liver and serum HDL-C, and reducing serum TC and TG. P <0.01). Furthermore, in reducing key indicators such as hepatic LDL-C, hepatic TG, and serum LDL-C, the simplified formula showed comparable efficacy to the positive control group. P >0.05). The only indicator where the simplified formula showed a slightly weaker regulatory effect was liver TC, with a slightly lower reduction than the simvastatin group. It is worth emphasizing that this formula optimization did not adjust the dosage of any single herb in the original formula, nor did it introduce any new excipients; it was entirely based on a systematic analysis of the original formula's composition and pharmacological mechanisms. Through simplification, the original nine herbs were reduced to a core simplified formula containing only three herbs. This simplified formula not only showed outstanding effects in increasing HDL-C but also demonstrated significant improvement in reducing key lipid parameters contributing to atherosclerosis, such as LDL-C, TC, and TG.

[0062] 3. Liver histopathological analysis To directly observe lipid deposition and histological changes in the liver, we performed a systematic pathological examination on liver samples: Oil Red O (ORO) staining: Frozen sections of liver tissue were prepared and stained with Oil Red O. Oil Red O is a lipid-soluble dye that specifically binds to neutral fats (mainly triglycerides and cholesterol esters) in the liver, turning them bright red. The number, size, and area of ​​lipid droplets in hepatocytes were observed using an optical microscope and semi-quantitatively assessed using image analysis software, directly revealing the lipid accumulation in the liver. The results of Oil Red O staining of the liver showed that a large number of orange-red lipid droplets accumulated in the hepatocytes of mice in the high-fat diet model group, while lipid droplet deposition was significantly reduced in all treatment groups, showing a dose-dependent decreasing trend. Figure 6 H&E staining further revealed that the model group showed disordered hepatocyte arrangement, obvious fatty degeneration, and punctate necrosis; while the positive control group and each HZ-treated group showed basically normal hepatocyte morphology, more orderly arrangement, and significantly reduced inflammatory infiltration.

[0063] Hematoxylin-eosin (H&E) staining: Adjacent liver lobe tissues were embedded in paraffin, sectioned, and then subjected to routine H&E staining. This staining clearly shows hepatocyte morphology, hepatic cord arrangement, central vein, and portal structure, and is used to assess whether a high-fat diet and drug intervention lead to pathological changes such as hepatocyte ballooning degeneration, fatty degeneration, and inflammatory cell infiltration. Pathological changes in white adipose tissue: H&E staining analysis of perirenal white adipose tissue (WAT) in each group of mice showed ( Figure 7 In the normal control group, adipocytes were small and tightly packed; in the model group, adipocytes showed significant hypertrophy, irregular shape, and intracellular lipid accumulation. All drug treatment groups alleviated the above pathological changes to varying degrees, manifested as reduced adipocyte size, more regular shape, and decreased lipid accumulation.

[0064] By integrating serum biochemical indicators (systemic metabolic level), liver lipid content (local metabolic load), and histopathological images (morphological evidence), this invention provides a comprehensive and objective evaluation of the lipid-lowering efficacy of Heze Lipid-Lowering Oral Solution and its optimized formula, from functional to morphological perspectives and from macroscopic to microscopic levels, ensuring the reliability of the efficacy conclusions.

[0065] 4. Safety assessment This invention, while evaluating the lipid-lowering efficacy of Heze Lipid-Lowering Oral Solution (HZ) and its optimized simplified formula, simultaneously conducted a systematic safety assessment. The safety evaluation was based on the following background: Common adverse reactions of widely used lipid-lowering drugs in clinical practice—statins (such as simvastatin, the positive control drug used in this invention)—mainly focus on muscle and liver / kidney function effects, including myalgia, rhabdomyolysis, elevated liver enzymes, and potential kidney damage risk. Therefore, to comprehensively examine the safety characteristics of the compound preparation and provide a reference for its clinical translation, this invention selected simvastatin as a positive control and systematically monitored key serum biochemical indicators reflecting liver and kidney function and muscle damage.

[0066] Specific assessment indicators include: ① Liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and cholinesterase (CHE), used to evaluate the effects of drugs on hepatocyte integrity, cholestasis, and synthetic function; ② Kidney function indicators: blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA), used to reflect glomerular filtration function and the accumulation of nitrogen metabolites; ③ Muscle-related indicators: creatine kinase (CK) and its cardiac isoenzyme (CK-MB), used to monitor potential muscle damage, especially the risk of myotoxicity associated with statins.

[0067] All serum samples were collected uniformly at the experimental endpoint and analyzed using a fully automated biochemical analyzer (CMax Plus, with original reagents) strictly according to standard operating procedures. Internal quality control measures were implemented to ensure the accuracy and repeatability of the test results. By comparing the differences in the above indicators between each treatment group (including high and low dose groups of the full formula and different simplified formulas) and the model control group and positive control group, it is possible to comprehensively determine whether the Heze Lipid-Lowering Oral Liquid compound has better safety characteristics for liver, kidney and muscle tissue while exerting lipid-lowering effects, thus providing key experimental evidence for its optimization goal of "enhancing efficacy and reducing toxicity".

[0068] At the experimental endpoint, serum was collected from each group of mice, and related biomarkers such as liver function, myocardial enzyme profile, and kidney function were systematically detected. Figure 8 These are the results of safety biochemical indicator tests; among them, A is alanine aminotransferase; B is aspartate aminotransferase; C is alkaline phosphatase; D is cholinesterase; ECK is creatine kinase; F is creatine kinase isoenzyme; G is creatinine; H is blood urea nitrogen; and I is uric acid.

[0069] Analysis revealed that serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly higher in the high-fat diet model group and all drug treatment groups than in the normal diet control group, suggesting that the pathological state of hyperlipidemia itself has already placed a certain burden and caused damage to the liver. When comparing the safety indicators of the full formula, simplified formula, and the positive control drug simvastatin of Heze Lipid-Lowering Oral Solution, no statistically significant differences were found in parameters reflecting liver and kidney function damage and hemolysis risk among the groups, indicating that Heze Lipid-Lowering Oral Solution at this dosage has a safety and tolerability comparable to commonly used Western medicines.

[0070] Furthermore, both Heze lipid-lowering oral liquid and simvastatin significantly reduced serum urea nitrogen levels. It is worth noting that since there was no significant difference in baseline urea nitrogen levels between the normal control group and the high-fat model group, the exact physiological significance of this reduction still needs careful interpretation: it may suggest that the drug has a certain renal protective effect, or it may stem from its indirect influence on liver metabolism or other physiological processes; this point requires further clarification in subsequent studies.

[0071] A noteworthy finding is that the optimized simplified formula of Heze lipid-lowering oral liquid exhibited a significant uric acid-lowering effect. The serum uric acid level in the model group was 6.7 mg / dL, while the low- and high-dose simplified formula groups decreased to 4.1 mg / dL and 3.9 mg / dL, respectively. As an end product of purine metabolism and a potential cardiovascular risk factor, the reduction in uric acid levels may be an additional metabolic benefit of this simplified formula beyond lipid regulation; however, its specific mechanisms and clinical significance warrant further investigation.

[0072] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for optimizing traditional Chinese medicine compound prescriptions based on network pharmacology and material basis analysis, characterized in that, Includes the following steps: S1. Network pharmacology analysis: Obtain the medicinal material composition of the target compound; screen the potential active ingredients of the medicinal materials through the database and predict the target of the potential active ingredients; compare with disease targets related to the target disease; take the intersection of the target of the active ingredients and the disease targets to obtain the potential target set; construct a protein-protein interaction (PPI) network and screen the core targets based on topological parameters. S2. Material basis identification: Prepare the total extract of the target Chinese herbal medicine compound and the single extracts of each herb that makes up the target Chinese herbal medicine compound. Separation and identification techniques were used to analyze the chemical composition of the total extract and each single extract to identify the main chemical components whose relative abundance in the total ion chromatogram of the total extract was greater than a preset threshold, and the source medicinal materials of the main chemical components were determined based on the analysis results of each single extract. S3. Formula Optimization: Calculate the overall contribution of each medicinal material and select the key medicinal materials with the highest overall contribution in a predetermined quantity to form an optimized simplified formula.

2. The method according to claim 1, characterized in that, The separation and identification techniques include ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis.

3. The method according to claim 2, characterized in that, The UPLC-MS analysis was performed using electrospray ionization in positive and negative ion mode. The chromatographic column was a BEH C18 column, and the mobile phase consisted of an acetonitrile solution containing 0.1% formic acid and an aqueous solution containing 0.1% formic acid, with gradient elution.

4. The method according to claim 1, characterized in that, In step S1, the selection of core target points is based on network topology parameters, including node degree, betweenness centrality, and proximity centrality.

5. The method according to claim 1, characterized in that, In step S3, the calculation of the comprehensive contribution includes: normalizing and weighting the total number of target points hit by compounds from each medicinal material in the core target point after merging and deduplicating, and the relative abundance of compounds from each medicinal material in the mass spectrometry analysis results.

6. The method according to claim 1, characterized in that, In step S1, the target compound is a compound used to treat or prevent hyperlipidemia.

7. A simplified traditional Chinese medicine formula obtained by the method according to any one of claims 1-6, characterized in that, Including Atractylodes macrocephala, Pinellia ternata, and lotus leaf.

8. The simplified traditional Chinese medicine formula according to claim 7, characterized in that, The formulation exerts its lipid-regulating effect by acting on PTGS2, ADRB2 and / or ACHE targets, and / or regulating the PI3K-Akt signaling pathway and / or lipid and atherosclerosis pathway through its flavonoid, terpene and / or phenolic acid active ingredients.

9. The simplified traditional Chinese medicine formula according to claim 8, characterized in that, The drug is used to lower serum and / or liver levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and / or to raise high-density lipoprotein cholesterol (HDL-C) levels.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises extracts prepared from three medicinal herbs: Atractylodes macrocephala, Pinellia ternata, and Nelumbo nucifera leaf; and pharmaceutically acceptable carriers or excipients, and is used to treat or prevent hyperlipidemia.

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