A method for quality evaluation of cinobufagin capsules based on efficacy / toxicity synchronous analysis

Abstract

The present application belongs to the technical field of traditional Chinese medicine quality evaluation, and relates to a quality evaluation method for cinobufagin capsules based on efficacy / toxicity synchronous analysis, which comprises the following steps: S1, screening a plurality of key efficacy / toxicity components in the cinobufagin capsules; S2, obtaining the contents of the plurality of key efficacy / toxicity components; S3, obtaining the IC 50 values of the plurality of key efficacy / toxicity components; S4, determining the weight coefficients of the plurality of key efficacy / toxicity components by using an AHP (analytic hierarchy process) method according to the IC 50 value sizes; S5, obtaining the weighted total contents of the plurality of key efficacy / toxicity components according to the contents and the weight coefficients of the plurality of key efficacy / toxicity components; and S6, comparing the weighted total contents with the clinical safety content limit to complete the quality evaluation. The method can balance the conversion relationship between the efficacy and toxicity of various components in the cinobufagin capsules, and can ensure the safety and effectiveness of the cinobufagin capsules for clinical medication by using the synchronous analysis of efficacy and toxicity.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality evaluation technology, and relates to a quality evaluation method for Huachansu capsules based on simultaneous efficacy / toxicity analysis. Background Technology

[0002] Huachansu Capsules (HCSc) are a broad-spectrum anti-tumor drug independently developed in my country. They are refined from dried toad skin, a single traditional Chinese medicine, and are widely used clinically for various malignant tumors such as liver cancer, colon cancer, and breast cancer. However, the raw material for HCSc, dried toad skin, has certain toxicity, which leads to various potential toxic side effects in Huachansu preparations, mainly cardiotoxicity, hepatotoxicity, and nephrotoxicity. This greatly limits its clinical application. Therefore, evaluating the active and toxic components of HCSc is of great significance for its clinical use.

[0003] Currently, the specific quality evaluation and control methods for Huachansu capsules include: The national drug standard (YBZ30992005-2011Z-2021) uses thin-layer chromatography (TLC), alcohol-soluble extracts, content determination, and general inspection items for control. TLC compares the components in the drug with those in the original medicinal material to control the main active substances. Ultimately, a dual-index quantitative quality control system is established for the content of the active ingredient indole alkaloids (5-hydroxytryptamine) and bufotoxin / bufotoxin ester. The 5-hydroxytryptamine content is limited to no less than 0.1 mg per capsule, and the sum of the contents of bufotoxin and bufotoxin ester is limited to 35-90 μg / capsule. This standard ensures its therapeutic effect and, to a certain extent, avoids some adverse reactions in the clinical application of HCSc. However, studies have found that HCSc and related preparations made from dried toad skin contain various types of components. Among them, alkaloids have little activity in organisms, and their toxicity is far weaker than that of bufotalene components, with their activity mainly manifested in anti-inflammatory effects. Furthermore, the Chinese Pharmacopoeia (2025 edition) has not yet established specific quality control standards for dried toad skin; existing standards are mostly based on qualitative identification and lack a multi-indicator quantitative control system. Quality evaluation studies on dried toad skin and related preparations made from it have also largely focused on the content determination of a single component or a few components such as bufotalin, bufotoxin, and bufotalin ester, without specifying regulations for other bufotalene components. This poses serious potential safety issues for the clinical application of such preparations.

[0004] Current literature reports that bufotalene, the main active ingredient in Huachansu preparations, is both its primary pharmacological component and its toxic component. It exhibits a narrow therapeutic window and a low safety margin; animal studies show that doses exceeding twice the clinical dose can cause significant toxic side effects. Furthermore, existing research on the efficacy-toxicity conversion of Huachansu preparations is limited. Quality standards only limit the levels of bufotalene esters and bufotalin, while neglecting other bufotalene components. Improper dosage control can easily lead to poisoning. Moreover, due to the inherent characteristics of traditional Chinese medicine (TCM) such as multi-component synergistic effects, multi-dimensional biological effects, and multi-target network regulation, the controllability of toxic TCM is extremely complex. Coupled with the dynamic changes in clinical compatibility and the cumulative metabolic effects of long-term use, traditional quality control strategies based on single or multiple indicators have significant limitations in terms of quality specificity, efficacy correlation, and safety assessment.

[0005] Therefore, how to balance the conversion relationship between the efficacy and toxicity of HCSc, establish controllable quality standards, and ensure its safe and effective clinical use has become a key issue that urgently needs to be addressed. Summary of the Invention

[0006] In view of the limited research on the efficacy-toxicity conversion of Huachansu capsules and the significant limitations of quality control strategies based on single or multiple indicators in terms of quality specificity, efficacy correlation, and safety assessment, this invention provides a quality evaluation method for Huachansu capsules based on simultaneous efficacy / toxicity analysis.

[0007] This invention calculates the weighted total content of multiple key efficacy / toxicity components in Huachansu capsules using network pharmacology, high-performance liquid chromatography, and AHP hierarchical analysis. The weighted total content is then compared with the clinical safety limits of Huachansu capsules to evaluate their quality. This method balances the conversion relationship between the efficacy and toxicity of various components in Huachansu capsules and utilizes simultaneous efficacy-toxicity correlation analysis to ensure the safety and effectiveness of Huachansu capsules in clinical use. It also establishes a controllable quality evaluation standard for Huachansu capsules.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A quality evaluation method for Huachansu capsules based on simultaneous efficacy / toxicity analysis includes the following steps: Step S1: Several key active / toxic components in Huachansu capsules were screened using network pharmacology and molecular docking technology; Step S2: Obtain the content of multiple key active / toxic components in Huachansu capsules based on high performance liquid chromatography; Step S3: Obtain the ICs of multiple key active / toxic components. 50 value; Step S4: Based on the IC50 of multiple key active / toxic components 50 The weight coefficients of multiple key efficacy / toxicity components were obtained using the AHP (Analytical Hierarchy Process) method to determine the value. Step S5: Based on the content of multiple key active / toxic components in step S2 and the weighting coefficients of multiple key active / toxic components in step S4, obtain the weighted total content of multiple key active / toxic components in Huachansu capsules. Step S6: Compare the weighted total content of multiple key active / toxic components in Huachansu capsules with the clinical safety content limit of Huachansu capsules to complete the quality evaluation of Huachansu capsules.

[0009] Further specifying, the specific steps in step S1 for screening multiple key active / toxic components in Huachansu capsules using network pharmacology and molecular docking technology are as follows: S1.1 Identification of main components: The main components of Huachansu capsules were identified based on UPLC-Q-Exactive Orbitrap MS; S1.2 Screening of active ingredients: Using oral bioavailability ≥30% and drug similarity ≥0.18 as screening criteria, the main components of Huachansu capsules in step S1.1 were screened through the database and analysis platform of traditional Chinese medicine systems pharmacology to determine the active ingredients of Huachansu capsules; S1.3 Obtaining efficacy-toxicity targets: Using "Cardiotoxicity" and "Colon cancer" as keywords, the targets of Huachansu capsules on cardiotoxicity and colon cancer were obtained from the CTD, OMIM and Gene Cards databases, and then efficacy-toxicity targets were obtained through integrated analysis. S1.4 Constructing a multidimensional pharmacodynamic-toxicity network: Utilizing active ingredients and efficacy-toxicity targets, a multidimensional pharmacodynamic-toxicity network of "drug-ingredient-efficacy / toxicity targets" is constructed using network pharmacology; S1.5 Identify key efficacy / toxicity components and core targets: Visualize and analyze the multidimensional efficacy-toxicity network of "drug-component-efficacy / toxicity target", and use the degree value as the screening criterion to obtain the top-ranked active ingredients and targets as key efficacy / toxicity components and core targets. S1.6 Screening: Molecular docking technology was used to calculate the binding energy between the top-ranked active ingredients and the target sites, and the identified key efficacy / toxicity components were verified to complete the screening of multiple key efficacy / toxicity components in Huachansu capsules.

[0010] Further specifying, in step S1.1, the detection conditions for UPLC-Q-Exactive Orbitrap MS include: UPLC chromatographic conditions: Waters BEH C18 column (2.1 × 100 mm, 1.7 μm), mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile, flow rate 0.4 mL / min. -1 The injection volume was 10 μL, and the column temperature was 40 ℃; the elution gradient is shown in the table below: Mass spectrometry conditions: ESI source, positive and negative ion detection mode, sheath gas flow rate 30 Arb, auxiliary gas flow rate 10 Arb, spray voltage 3.5 kV, capillary temperature 275 ℃, auxiliary gas temperature 350 ℃; scanning mode: Full MS / dd-MS2, Full MS resolution 60000, dd-MS2 resolution 15000, scanning range 100 ~ 1500 m / z.

[0011] Further specifying, in steps S1.5 and S1.6, the key active / toxic components are bufotoxin, bufotoxin base, far-reaching bufotoxin, ester bufotoxin, sand bufotoxin, bufotoxin, bufotoxin, and bufotoxin.

[0012] Further specifying, the process of obtaining the content of multiple key active / toxic components in Huachansu capsules based on high performance liquid chromatography is as follows: S2.1 Solution Preparation Preparation of reference solutions: Using bufotalin, bufotalin, far-reaching bufotalin, bufotalin ester, bufotalin, bufotalin, bufotalin and bufotalin as reference standards, reference solutions were prepared using methanol solution; Preparation of the test solution: Accurately weigh 0.25 g of the contents of Huachansu capsules, add methanol to make up to 10 mL, sonicate for 30 min, and filter the supernatant through a 0.22 μm organic filter membrane to obtain the test solution; S2.2 Obtain the reference chromatogram and the test sample chromatogram. Take the reference solution and the test solution from step S2.1, and obtain the reference chromatogram and the test chromatogram respectively by high performance liquid chromatography; The conditions for high-performance liquid chromatography (HPLC) were as follows: the chromatographic column was Betasil. TM C18; mobile phase: acetonitrile and 0.5% potassium dihydrogen phosphate aqueous solution; detection wavelength: 296 nm; flow rate: 0.9 mL / min -1 The injection volume was 10 μL; the column temperature was 35 ℃; and the gradient elution conditions are shown in the table below. S2.3 Calculate the content of key active / toxic components. The reference chromatogram and the test sample chromatogram from step S2.2 are processed to generate the corresponding reference fingerprint chromatogram and test fingerprint chromatogram. Peak matching was performed on the test fingerprint spectrum and the control fingerprint spectrum, and the characteristic peaks were marked sequentially according to the common peak numbers. Based on the peak positions of multiple key effective / toxic components in the control spectrum, the corresponding peak numbers of multiple key effective / toxic components in the test fingerprint spectrum were determined. Calculate the content of several key active / toxic components on the fingerprint spectrum of the test sample.

[0013] Further specifying, the peak numbers corresponding to multiple key active / toxic components in the tested fingerprint spectrum are: Peak 1: Bufotalin; Peak 4: Bufotalin; Peak 7: Bufotalin; Peak 9: Bufotalin; Peak 10: Bufotalin; Peak 11: Bufotalin; Peak 12: Bufotalin; Peak 13: Bufotalin ester.

[0014] Further specifying, in step S3, the IC50 of multiple key potentiogenic / toxic components is determined by assessing their proliferative activity against AC116 cells. 50 value.

[0015] Further specifying, in step S4, the specific process of obtaining the weight coefficients of multiple key efficacy / toxic components using the AHP (Analytic Hierarchy Process) is as follows: S4.1. The Satty 1-9 scale was used to determine the IC50 values ​​of several key active / toxic components. 50 The values ​​are compared pairwise to determine the importance scale of each key efficacy / toxic component relative to other key efficacy / toxic components, and a comprehensive judgment matrix is ​​constructed: in: Indicates the first i The key efficacy / toxic component relative to the first j The importance scale of key efficacy / toxicity components, and meets the requirements. m ij >0, m ij =1 / m ji , m ii =1; S4.2 Calculate the weight vector Calculate the first in the comprehensive judgment matrix i geometric mean of row elements ; =1, 2, ..., 8 in: Indicates the first iThe key efficacy / toxic component relative to the first j The importance scale of key efficacy / toxicity components For the first i The 8th root of the product of all elements in the row, i.e., the [number]th [row]. i The geometric mean of the row elements; The vector is processed according to the following formula. = After normalization, we obtain the first... Normalized weighting coefficients of key efficacy / toxic components; =1, 2, ..., 8 in: For the first Normalized weighting coefficients of key efficacy / toxicity components, For the first i The geometric mean of the row elements. For the first k Normalized weighting coefficients of key efficacy / toxic components; According to the The normalized weight coefficients of each key efficacy / toxic component are used to obtain the weight vector. ; in: For the weight vector, This is the normalized weighting coefficient for the first key efficacy / toxic component. The normalized weighting coefficient for the second key efficacy / toxic component; For the first Normalized weighting coefficients of key efficacy / toxicity components, =1, 2, ..., 8; S4.3. Perform a consistency check on the comprehensive judgment matrix and calculate the consistency ratio. The weight vector obtained in step S4.2 The results are reliable; S4.4, The weight vector after passing the consistency test various Converting this to percentages yields the weighting coefficients for each key efficacy / toxic component.

[0016] Further specifying, in step S5, the weighted total content of multiple key active / toxic components in the Huachansu capsules is: in: For the first i The content of key active / toxic components, For the first iThe weighting coefficients of key efficacy / toxicity components, i The number of key active / toxic components is 1, 2, ..., 8.

[0017] Further specifying, in step S6, the clinically safe content limit of Huachansu capsules is 35μg / capsule to 80μg / capsule.

[0018] Compared with existing technologies, this invention calculates the weighted total content of multiple key efficacy / toxicity components in Huachansu capsules (HCSc) using network pharmacology, high-performance liquid chromatography, and AHP hierarchical analysis. This weighted total content is then compared with the clinical safety limits of Huachansu capsules to evaluate their quality. This method balances the conversion relationship between the efficacy and toxicity of multiple components in Huachansu capsules and utilizes simultaneous efficacy-toxicity correlation analysis to ensure the safety and effectiveness of Huachansu capsules in clinical use. It also establishes a controllable quality evaluation standard for Huachansu capsules. The beneficial effects of this invention are specifically manifested in the following aspects: 1. This invention identifies chemical components using UPLC-Q-Exactive Orbitrap MS technology, and screens and assigns the identified chemical components based on a traditional Chinese medicine systems pharmacology database to obtain candidate active ingredients. Through integrated analysis of the CTD, OMIM, and GeneCards databases, the intersection gene set of colorectal cancer treatment targets and cardiotoxicity-related targets is obtained. A multidimensional pharmacodynamic-toxicity network of "drug-component-efficacy / toxicity target" is constructed using Cytoscape, and key efficacy / toxicity components are screened based on network topology parameters (Degree values). This invention not only intuitively displays the key efficacy / toxicity components in Huachansu capsules, a traditional Chinese medicine preparation, but also effectively balances the conversion relationship between the efficacy and toxicity of various components in Huachansu capsules, improving the accuracy of the evaluation results.

[0019] 2. This invention establishes a fingerprint spectrum using high-performance liquid chromatography (HPLC), and then determines the content of key active / toxic components based on the fingerprint spectrum. Through methodological research, this method ensures the authenticity, accuracy, and reliability of the determination results, improving the accuracy of quality evaluation, and is applicable to the qualitative and quantitative detection of Huachansu capsules. Furthermore, this invention improves the quality evaluation standard of HCSc by determining the content of multiple components in different batches of HCSc and comprehensively evaluating the toxicity and efficacy of different batches of HCSc from the perspective of simultaneous efficacy / toxicity analysis.

[0020] 3. This invention utilizes the Analytic Hierarchy Process (AHP) to determine the weighting coefficients of key efficacy / toxicity components. Specifically, it constructs a hierarchical model with target and criterion layers, transforming complex multidimensional problems into an operable hierarchical structure. This retains a holistic approach while assigning calculable weights to the contributions of each key efficacy / toxicity component, achieving quantifiable quality standards. Simultaneously, it examines the effects of key efficacy / toxicity components in Huachansu capsules on cellular IC50. 50 By constructing a comprehensive judgment matrix based on the strength of the values, the weight coefficients of each key efficacy / toxicity component can be determined, which can balance the mutual influence between multiple efficacy / toxicity components and improve the accuracy of quality evaluation.

[0021] 4. Animal experiments of this invention have yielded the clinically safe content limit of HCSc, ensuring that the active ingredient achieves the anti-tumor effect while avoiding the harm of toxic ingredients to the human body, thus realizing the simultaneous "efficacy-toxicity" analysis of HCSc quality evaluation. Attached Figure Description

[0022] Figure 1 This is a full scan mass spectrum of HCSc; Note: (A) positive ion mode; (B) negative ion mode; Figure 2 It is a potential target for HCSc to mediate cardiotoxicity and anti-colon cancer activity; Figure 3 A network diagram of drug-component-efficacy / toxicity targets; Figure 4 A heatmap showing the binding energy between key active / toxic components of HCSc and their core targets; Figure 5 Molecular docking diagram of key efficacy / toxic components of HCSc with core targets; Figure 6 Fingerprints (A) and reference fingerprints (B) of 15 batches of HCSc HPLC are shown (S1-S15); Note: 1. Bufotalin, 4. Bufotalin, 7. Bufotalin, 9. Bufotalin, 10. Bufotalin, 11. Bufotalin, 12. Bufotalin base, 13. Ester bufotalin base; Figure 7 The effects of eight key potentiogenic / toxic components on HCT116 cell viability; Figure 8 The effects of eight potent / toxic components on AC16 cell viability; Figure 9 The effects of different cycles and doses of HCSc intervention on animal electrocardiograms (x) (±SD, n=3); (A) Changes in animal electrocardiograms over 7 days; (B) Changes in animal electrocardiograms over 14 days; (C) Changes in animal electrocardiograms over 21 days; (D) Changes in animal electrocardiograms over 28 days; Figure 10The effects of different cycles and doses of HCSc intervention on ejection fraction; Figure 11 The effects of HCSc intervention with different cycles and doses on short axis ratio; Figure 12 To evaluate the echocardiography of animals at different cycles and doses, including 7 days (a), 14 days (b), 21 days (c), and 28 days (d); *p<0.05, **p<0.01, which were significantly different from the control group; Figure 13 The effects of different cycles and doses of HCSc intervention on animal biochemical indicators (x) ±SD, n=6); (AE) five myocardial enzyme profiles; (F) BNP; (GH) myocardial troponin cTnI and cTnT; *p<0.05, **p<0.01, significantly different from the control group; Figure 14 HE staining results of cardiac tissues from different cycles and different dose groups (200×). Figure 15 Tumor morphology was the experimental endpoint for evaluating the efficacy of different treatment cycles and drug administration groups in mice with colon cancer, including 7 days (a), 14 days (b), 21 days (c), and 28 days (d). Figure 16 Tumor weight was the experimental endpoint for evaluating the efficacy of different treatment cycles and different drug administration groups in mice with colon cancer; *p<0.05, **p<0.01, which was significantly different from the control group. Figure 17 HE staining results of tumor tissues from different cycles and different dose groups (200×). Figure 18 TUNEL staining results (20×) of tumor tissues from different cycles and different dose groups; Figure 19 IHC results for tumor tissues from different cycles and different dose groups (x) (±SD, n=3); (A) Ki-67 immunohistochemical analysis; (B) Cleaved caspase-3 immunohistochemical analysis; Figure 20 The positive rate of Ki-67 protein expression in different cycles and different dose groups; * p<0.05, ** p<0.01, which were significantly different from the control group; Figure 21 The positive rate of Cleaved caspase-3 protein expression in different cycle and dose groups; * p < 0.05, ** p < 0.01, which were significantly different from the control group; Figure 22The HPLC chromatograms are of the reference standard (top) and the test sample (bottom) solutions for the specificity test results; Note: 1. Bufotalin, 2. Bufotalin, 3. Bufotalin, 4. Bufotalin, 5. Bufotalin, 6. Bufotalin, 7. Bufotalin base, 8. Ester bufotalin. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0024] The materials and instruments used in this invention are shown in Tables 1 and 2 below.

[0025] Table 1 Main materials used Table 2 lists the main instruments used. The quality evaluation method for Huachansu capsules based on simultaneous efficacy / toxicity analysis provided by this invention includes the following steps: S1. Several key efficacy / toxic components in Huachansu capsules were screened using network pharmacology and molecular docking technology; Step S1 of the present invention specifically includes the following steps: S1.1 Identification of main components Specifically, the main components of Huachansu capsules (HCSc) were identified using UPLC-Q-Exactive Orbitrap MS.

[0026] (1) Preparation of the test solution Huachansu capsules (HCSc) were purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd. An appropriate amount of HCSc contents was dissolved in methanol and extracted by ultrasonication (frequency 40 kHz, power 250 W) for 20 min. After cooling to room temperature, the extract was filtered. The filtrate was centrifuged at 4000 r / min for 5 min to obtain a clear supernatant. The supernatant was then filtered through a 0.22 μm microporous membrane and analyzed by UPLC-Q-Exactive Orbitrap MS.

[0027] (2) Chromatographic conditions Chromatographic column: Waters BEH C18 (2.1 × 100 mm, 1.7 μm); mobile phase: 0.1% formic acid aqueous solution (A) and acetonitrile (B); flow rate: 0.4 mL·min -1Injection volume: 10 μL, column temperature: 40 ℃. Elution gradient is shown in Table 3.

[0028] Table 3 Mobile phase elution gradient table (3) Mass spectrometry conditions The ion source was an ESI source, with positive and negative ion detection modes. The sheath gas flow rate was 30 Arb, the auxiliary gas flow rate was 10 Arb, the spray voltage was 3.5 kV, the capillary temperature was 275 ℃, and the auxiliary gas temperature was 350 ℃. The scanning modes were Full MS / dd-MS2, with a Full MS resolution of 60000 and a dd-MS2 resolution of 15000. The scanning range was 100 ~ 1500 m / z.

[0029] A total of 75 compounds were analyzed and identified from HCSc under both positive and negative ion modes. Literature review and comparative identification of some results revealed the following main compounds: 9 bufotalenes, 22 alkaloids, 6 lipids, 11 amino acids, 11 organic acids, and 16 steroids and others. (See [link to relevant documentation]). Figure 1 .

[0030] S1.2, Screening of active ingredients The main components of HCSc obtained by UPLC-Q-Exactive Orbitrap mass spectrometry analysis were screened and assigned using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The active ingredients of Huachansu capsules were determined using oral bioavailability (OB) ≥30% and drug similarity (DL) ≥0.18 as screening criteria. Specifically, 75 compounds identified in the analysis were screened, and 50 HCSc active ingredients were determined, as shown in Table 4.

[0031] Table 4. Identification information of 50 major components of HCSc S1.3, Obtaining the target of efficacy-toxicity Using "Cardiotoxicity" and "Colon cancer" as keywords, the targets of Huachansu capsules on cardiotoxicity and colon cancer were obtained from the CTD, OMIM, and Gene Cards databases. The results from the above databases were then integrated and analyzed, and the targets of the active ingredient HCSc were further merged with the disease targets. The overlapping targets are the potential targets of HCSc on cardiotoxicity and colon cancer, i.e., efficacy-toxicity targets.

[0032] Specifically, the CTD, OMIM, and Gene Cards databases were used to search for targets related to "Cardiotoxicity" and "Coloncancer." A Venn diagram was constructed by comparing the potential targets of 50 HCSc active ingredients with disease targets related to cardiotoxicity and colon cancer, identifying 54 intersection targets. These intersection targets were identified as potential efficacy / toxicity targets for HCSc-mediated cardiotoxicity and anti-colon cancer activity, such as... Figure 2 As shown.

[0033] S1.4 Constructing a multidimensional pharmacodynamic-toxicity network: Using network pharmacology, a multidimensional pharmacodynamic-toxicity network of "drug-component-efficacy / toxicity target" is constructed; S1.5 Identify key efficacy / toxicity components and core targets: Perform visual analysis on the multidimensional efficacy-toxicity network of "drug-component-efficacy / toxicity target", and use the degree value as the screening criterion to obtain the top-ranked active ingredients and targets as key efficacy / toxicity components and core targets.

[0034] Specifically, a "drug-component-active / toxic target" network was constructed using Cytoscape 3.7 software and visualized for analysis. The network topology parameters (Degree values) of the drug components and targets were obtained. Based on these parameters, the priority order of active / toxic components and targets was determined, with the top-ranked active components being the key active / toxic components and core targets of HCSc.

[0035] like Figure 3 As shown, the drug-component-target network contains 105 nodes and 658 edges. Triangles represent drugs, hexagons represent components, and rhombuses represent targets. The degree value of a node reflects the number and strength of links connecting it to the node, and is a measure of how well the node is embedded in the network. The higher the degree value, the more tightly connected the node is, and the more important the component or target it represents.

[0036] Degree values ​​directly reflect the importance of the corresponding drug components and targets. Therefore, using degree values ​​as the screening criterion, the top 8 key active / toxic components of HCSc were obtained using Cytoscape 3.7.1 software, namely bufotoxin, bufotalin, bufotalin ester, bufotalin, bufotalin, bufotalin spp., as shown in Table 5. Furthermore, as shown in Table 6, the top 5 targets with the highest degree values ​​are AMPK, IL1β, BAX, cAMP, and mTOR. Based on the above criteria, these targets were identified as the core targets with active / toxic effects in HCSc.

[0037] Table 5. Top 8 key active / toxic components by Degree value. Table 6. Top 5 Core Targets by Degree Value S1.6, Molecular docking The binding affinity of the top eight components by degree ranking to the core target was examined to verify whether they are key efficacy / toxic components of HCSc. The three-dimensional structures of the components were obtained using the PubChem database, and the three-dimensional structure of the core protein receptor was obtained using the UniProt database. Subsequently, PyMOL software was used to dehydrogenate the protein and remove small molecule ligands. Finally, AutoDockTools 1.5.6 software was used to perform molecular docking between the processed protein receptor and ligands, and the binding energy was recorded.

[0038] Molecular docking was used to evaluate the binding of the eight key active / toxic components to the five core targets. A binding free energy less than 0 indicates spontaneous binding, and a lower binding fraction results in a more stable complex. As shown in Table 7, the binding energies between the eight key active / toxic components and the five proteins (core targets) are all <-5 kcal / mol, indicating strong binding energies between these compounds and the five proteins (core targets). Furthermore, heatmap analysis more clearly reveals the differences in binding energies between the eight key active / toxic components and the five core targets. Figure 4 By observing the chemical bonds and amino acid residues of each compound, it was found that the corresponding compounds can interact with the five core target sites through conventional hydrogen bonds, π-alkyl interactions, and π-π stacking interactions. (See...) Figure 5 .

[0039] Table 7. Binding energies (kcal / mol) between key active / toxic components of HCSc and core target molecules A multidimensional pharmacodynamic-toxicological network of HCSc (Cellular Drug-Component-Efficacy / Toxicity Target) was constructed using network pharmacology, and key efficacy / toxicity components and core targets of HCSc were screened based on topological parameters. Molecular docking technology was then used to examine and validate these key efficacy / toxicity components and core targets. Molecular docking results showed that the eight key efficacy / toxicity components could bind to the core target through conventional hydrogen bonds, π-alkyl interactions, and π-π stacking interactions, with binding energies all less than -5 kcal / mol, indicating good binding ability. Ultimately, the eight key efficacy / toxicity components in Huachansu capsules were identified as bufotoxin, bufotalin, bufotalin ether, bufotalin ether, bufotalin ether, and bufotalin ether.

[0040] S2. The contents of several key active / toxic components in Huachansu capsules were obtained by high performance liquid chromatography. The specific process for this step is as follows: S2.1 Solution Preparation Prepare reference solution and test solution separately. (1) Preparation of reference solutions: Accurately weigh appropriate amounts of eight reference standards, add methanol to dissolve them and dilute to volume to obtain the reference standard stock solution. Take another 5 mL volumetric flask, add appropriate amounts of the above stock solution according to the proportion, and dilute to volume with methanol to obtain the mixed reference standard. The mass concentrations of bufotoxin, bufotalin, far-infrared bufotalin, bufotalin ester, bufotalin, bufotalin, bufotalin, and far-infrared bufotalin are 112 μg·mL, respectively. -1 173 μg·mL -1 74 μg·mL -1 126 μg·mL -1 154 μg·mL -1 124 μg·mL -1 82 μg·mL -1 149 μg·mL -1 .

[0041] (2) Preparation of test solution: Accurately weigh 0.25 g of HCSc contents, add methanol to make up to 10 mL, sonicate for 30 min, and filter the supernatant through a 0.22 μm organic filter membrane to obtain the test solution.

[0042] Preferably, the present invention uses 15 batches of HCSc to prepare test solutions. All 15 batches of HCSc were purchased from Shaanxi Dongtai Pharmaceutical Co., Ltd., and each batch of HCSc was prepared according to the above method to obtain 15 test solutions.

[0043] S2.2 Obtain the reference chromatogram and the test sample chromatogram. Take the reference solution and the test solution from step S2.1, and obtain the reference chromatogram and the test chromatogram respectively by high performance liquid chromatography (HPLC); The chromatographic conditions for high performance liquid chromatography are as follows: Column: Betasil TM C18 (4.6 mm × 250 mm; 5 μm); mobile phase: acetonitrile (A) - 0.5% potassium dihydrogen phosphate aqueous solution (B); detection wavelength: 296 nm; flow rate: 0.9 mL·min -1 Injection volume: 10 μL; Column temperature: 35 ℃. Gradient elution conditions are shown in Table 8.

[0044] Table 8 Mobile Phase Elution Gradient Table The present invention obtains one reference chromatogram and 15 HCSc test sample chromatograms (15 test sample chromatograms) through the above high performance liquid chromatography.

[0045] S2.3 Calculate the content of key active / toxic components. The control chromatogram and the test sample chromatogram from step S2.2 are processed to generate corresponding control fingerprint chromatogram and test fingerprint chromatogram.

[0046] Specifically, the control fingerprint (1) and test sample fingerprints (15) were processed using a multi-point calibration method and the averaging method, resulting in 15 fingerprint profiles and 1 control fingerprint profile. (See attached image.) Figure 6 .

[0047] Then, peak matching was performed on 15 fingerprint spectra and 1 control fingerprint spectra, and characteristic peaks were marked sequentially according to the common peak numbers. Based on the peak positions of the 8 key active / toxic components in the control spectrum, the corresponding peak numbers of the 8 key active / toxic components in the 15 fingerprint spectra were further determined. Preferably, a total of 13 common peaks were marked, and the peak numbers of the 8 key active / toxic components in the 15 fingerprint spectra were determined as follows: Peak 1 (bufotalin), Peak 4 (bufotoxin), Peak 7 (bufotoxin), Peak 9 (bufotalin), Peak 10 (bufotalin), Peak 11 (bufotalin), Peak 12 (bufotalin), and Peak 13 (bufotalin ester). Because bufotalin has good separation and symmetry, it was selected as the reference peak.

[0048] The contents of eight key active / toxic components were determined. The results are shown in Table 9.

[0049] Table 98 Contents of Key Active / Toxic Components Furthermore, data analysis was performed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using S1 as the control fingerprint chromatogram, the similarity of chromatograms of 15 batches of samples was calculated.

[0050] The similarity of fingerprint spectra of 15 batches of HCSc samples was calculated. According to the calculation results, the similarity of samples S1-S15 is shown in Table 10.

[0051] Table 10. Similarity analysis results of 15 batches of HCSc (S1-S15) The results showed that the similarity of these 15 batches of HCSc was greater than 0.9, indicating that the quality properties of HCSc were stable.

[0052] Step S3: Obtain the IC50 of multiple key active / toxic components in Huachansu capsules. 50value; In this step, the MTT assay was used to examine the toxic effects of eight key active / toxic components in Huachansu capsules (S1 batch) on HCT116 and AC16 cells, and the IC50 of several key active / toxic components was determined. 50 value.

[0053] In this step, HCT116 human colon cancer epithelial cell line (Wuhan Pronosai Life Science Technology Co., Ltd.) and AC16 human cardiomyocytes (Suzhou Haixing Biotechnology Co., Ltd.) were used.

[0054] The specific method is as follows: HCT116 and AC16 cells in logarithmic growth phase were seeded into 96-well culture plates with a cell suspension concentration adjusted to 5 × 10³ cells / well (PBS was added to the outer wells). The culture plates were pre-cultured at 37°C with 5% CO2 for 12 hours to allow for full cell attachment, followed by removal of the original culture medium. The test solution (experimental group) was prepared using the Huachansu capsules from batch S1, following step S2. The culture medium in the experimental group was replaced with a gradient concentration of the drug solution, while the control group was supplemented with an equal volume of complete culture medium and cultured for another 24 hours. 20 μL of MTT working solution was added to each well, and the reaction was terminated after 4 hours of incubation in the dark. The liquid in the wells was carefully aspirated. 150 μL of DMSO was added and shaken to dissolve the crystals. The absorbance of each well was measured at 490 nm using a multi-mode microplate reader. The relative viability of the two cell lines was calculated by comparing the absorbance ratios of the experimental and control groups.

[0055] The effects of eight key components on HCT116 cell viability were investigated using the MTT assay. Results are shown below. Figure 7 IC50 of eight key components: Bufotalin, Bufotalin, Bufotalin ester, Bufotalin, Bufotalin, Bufotalin, Bufotalin, Bufotalin, Bufotalin 50 The values ​​were 3.271 ng·mL -1 21.79 ng·mL -1 49.11 ng·mL -1 23.20 ng·mL -1 23.45 ng·mL -1 35.75 ng·mL -1 69.50 ng·mL -1 2.005 ng·mL -1 The antitumor abilities of the eight key active / toxic components, from strongest to weakest, are as follows: Bufotalin > Bufotalin > Bufotalin base > Bufotalin ester > Bufotalin > Bufotalin > Bufotalin > Bufotalin > Bufotalin > Bufotalin.

[0056] The effects of eight key components on AC116 cell viability were investigated using the MTT assay. Results are shown below. Figure 8IC50 of eight key components: Bufotalin, Bufotalin, Bufotalin ester, Bufotalin, Bufotalin, Bufotalin, Bufotalin, Bufotalin, Bufotalin 50 The values ​​were 3.835 ng·mL -1 5.524 ng·mL -1 35.59 ng·mL -1 19.24 ng·mL -1 27.81 ng·mL -1 29.06 ng·mL -1 43.68 ng·mL -1 25.85 ng·mL -1 The toxicity of the eight key active / toxic components, ranked from strongest to weakest, is as follows: Bufotalin > Bufotalin-based > Bufotalin-based > Bufotalin > Bufotalin > Bufotalin > Bufotalin > Bufotalin > Bufotalin > Bufotalin > Bufotalin.

[0057] Based on the relative strengths of the antitumor and toxic effects of the eight key active / toxic components, except for bufalin, the ranking of their antitumor and toxic effects is consistent. However, considering the safety of bufalin capsules, the IC50 of the eight key active / toxic components was determined based on their effects on AC116 cell viability. 50 value.

[0058] S4. The weight coefficients of the eight key efficacy / toxicity components were obtained using the Analytic Hierarchy Process (AHP). Based on the IC50 of multiple key active / toxic components 50 The values ​​were determined, and the weight coefficients of multiple key efficacy / toxicity components were constructed using the AHP (Analytical Hierarchy Process) method. Based on the eight key active / toxic components in Huachansu capsules (bufotoxin, bufotoxin base, bufotoxin ether, bufotoxin ester, bufotoxin extract, bufotoxin ether, far-reaching bufotoxin extract, and bufotoxin ether), IC 50 The values ​​were used to compare the eight key efficacy / toxicity components pairwise to determine their relative importance weights and establish an AHP evaluation structure.

[0059] (1) Target layer (A): Determination of the weights of 8 key efficacy / toxicity components; (2) Protocol layer (P): 8 key efficacy / toxicity components to be evaluated (P1, P2, ..., P8).

[0060] S4.1 Constructing a comprehensive judgment matrix The Satty 1-9 scale was used to determine the IC50 values ​​of several key active / toxic components. 50 The values ​​are compared pairwise to determine the importance scale of each key efficacy / toxic component relative to other key efficacy / toxic components, and a comprehensive judgment matrix is ​​constructed: Specifically, the Satty 1-9 scale (as shown in Table 11) was used to determine the IC50 values ​​of the eight key active / toxic components. 50 The values ​​are compared pairwise to construct an 8th-order positive-reciprocal judgment matrix and then construct a comprehensive judgment matrix: in: Indicates the first i The key efficacy / toxic component relative to the first j The importance scale of key efficacy / toxicity components, and meets the requirements. m ij >0, m ij =1 / m ji , m ii =1.

[0061] The comprehensive judgment matrix constructed by the Satty 1-9 scaling method in this invention is shown in Table 12.

[0062] Table 11. Matrix Scale and Its Meaning Table 128 Comprehensive Judgment Matrix of Key Active / Toxic Components S4.2 Calculate the weight vector (1) Calculate the first value in the comprehensive judgment matrix. i geometric mean of row elements ; =1, 2, ..., 8 in: Indicates the first i The key efficacy / toxic component relative to the first j The importance scale of the key efficacy / toxicity components, i.e., the first one in the comprehensive judgment matrix. i Line number j Column elements; For the first i The 8th root of the product of all elements in the row, i.e., the [number]th [row]. i The geometric mean of the row elements; (2) Apply the following formula to the vector = After normalization, we obtain the first... Normalized weighting coefficients of key efficacy / toxic components; =1, 2, ..., 8 in: For the first Normalized weighting coefficients of key efficacy / toxicity components, For the first i The geometric mean of the row elements. For the first k Normalized weighting coefficients of key efficacy / toxic components; (3) According to the first The normalized weight coefficients of each key efficacy / toxic component are used to obtain the weight vector. ; in: For the weight vector, This is the normalized weighting coefficient for the first key efficacy / toxic component. The normalized weighting coefficient for the second key efficacy / toxic component; For the first Normalized weighting coefficients of key efficacy / toxicity components, =1, 2, ..., 8; S4.3 Perform a consistency check Perform a consistency check on the comprehensive judgment matrix and determine the weight vector. The reliability of the results is as follows: (1) Calculate the largest eigenvalue of the comprehensive judgment matrix. in: M It is an n×n comprehensive judgment matrix (n=8, corresponding to 8 key efficacy / toxic components); W =(w1,w2,…,w n ) T : Normalized weight coefficients (column vector) obtained by the square root method; :matrix M with vector W After multiplication, the first element in the resulting new vector is... One element; Weight vector W The Middle Normalized weighting coefficients of key efficacy / toxic components; n: The order of the matrix to be determined, n=8; (2) Calculate the consistency index (3) Refer to the RI value for n=8 in the random consistency index table; for n=8, RI=1.404 (4) Calculate the consistency ratio Judgment criteria: When When the value is less than 0.10, the consistency of the comprehensive judgment matrix is ​​considered to be within an acceptable range, and the weight allocation is reasonable.

[0063] This step involves calculations to obtain... =0, which meets the requirement. =0 < 0.10, passing the consistency test, indicating that the constructed comprehensive judgment matrix has good logical consistency, and the weight vector in step 4.2... The calculation results are reliable.

[0064] S4.4 Calculate the weighting coefficients The weight vector obtained after passing the consistency test Each component in Convert to percentage form, For the first The normalized weight coefficients of each key active / toxic component are obtained, which are the weight coefficients of each key active / toxic component relative to the overall target. Based on the weight coefficients, the relative importance order of each key active / toxic component in Huachansu capsules can be determined, as shown in Table 13.

[0065] Table 138 Weighting coefficients for key active / toxic components Note: The eigenvectors in the table refer to the vectors before normalization. That is, the first i The geometric mean of the row elements; the weighting coefficients are normalized percentage values, i.e., the weight vector. Each component in The result is converted to a percentage.

[0066] Based on the above efficacy-toxicity synchronization analysis, the weight coefficients of 8 key efficacy / toxic components were constructed, and the priority order of the 8 key efficacy / toxic components was determined as follows: Bufotalin > Bufotalin > Bufotalin > Bufotalin ester > Bufotalin > Bufotalin > Bufotalin > Bufotalin = Bufotalin.

[0067] S5. Obtain the weighted content of the active / toxic components. Based on the content of the eight key active / toxic components in step S2 and the weighting coefficients of the eight key active / toxic components in step S4, the weighted total content of the eight key active / toxic components in each batch of Huachansu capsules is obtained.

[0068] The weighted total content of multiple key active / toxic components in Huachansu capsules = in: For the firsti The content of the i weight coefficient of the i key effective / toxic components, where the number of key effective / toxic components takes values of 1, 2, ……, 8.

[0069] Substitute the results of Table 9 and Table 13 to calculate the weighted total content of 8 key effective / toxic components in the Huachansu capsules of batch S1 as shown in Table 14. Since the similarity of the chromatograms of 15 batches of Huachansu capsules is greater than 0.9, it indicates that the quality of the selected 15 batches of Huachansu capsules is consistent and stable. Therefore, the weight coefficients of the 8 key effective / toxic components in the remaining batches S2 - 15 of Huachansu capsules are all in accordance with the weight coefficients of batch S1 in Table 13, and the weighted total content of the 8 key effective / toxic components in the batches S2 - 15 of Huachansu capsules is shown in Table 14.

[0070] Table 14 Weighted Total Content of Key Effective / Toxic Components S6. Quality Evaluation Compare the weighted total content of multiple key effective / toxic components in the Huachansu capsules in step S5 with the clinical safety content limit of the Huachansu capsules to complete the quality evaluation of the Huachansu capsules.

[0071] In this step, the clinical safety content limit of the Huachansu capsules is determined through animal experiments. Specifically, a subcutaneous transplantation tumor model of HCT116 - Luc cells in BALB / c - nu mice is established to investigate the effective / toxic effects of HCSc (batch S1) on colon cancer mice at different administration cycles and different administration doses, and then the clinical safety content limit of the Huachansu capsules is determined.

[0072] 1. Experimental cells: HCT116 - Luc human colon cancer epithelial cell line (Wuhan Punosai Life Science and Technology Co., Ltd.).

[0073] 2. Experimental animals: 240 female SPF - level BALB / c - nu (20 ± 2 g) mice, purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Certificate number: SCXK (Chuan) 2020 - 0030). All animal experiments are carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" of the National Research Council of the United States and are approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine (Permit number: SUCMDL20241022001).

[0074] 3. Experimental methods 3.1. Establishment of subcutaneous tumor model Using HCT116 - Luc cells in the logarithmic growth phase, prepare approximately 5×10 7Cell suspensions of 100 cells / mL were subcutaneously inoculated into BALB / c-nu mice via the flank. During the experiment, all mice were randomly assigned to groups and placed in clean cages at a controlled temperature of (23±1)℃. The mice's mental state and the time of tumor appearance were observed. Mice that failed to establish the model were excluded, and tumors with a size of approximately 150 mm were observed. 3 Start administering medication at that time.

[0075] 3.2 Grouping and Administration One hundred and ninety-two BALB / c-nu mice were divided into four different cycles (7 days, 14 days, 21 days, and 28 days). Within each cycle, the animals were randomly divided into eight groups (n=6): a control group and HCSc treatment groups 1, 2, 3, 4, 5, 6, and 7. The control group received saline, while the treatment groups received HCSc 0.26 g·kg⁻¹. -1 0.35 g·kg -1 0.44 g·kg -1 0.52 g·kg -1 0.69 g·kg -1 0.86 g·kg -1 1.04 g·kg -1 The dosage design of HCSc is based on the results of subacute toxicity studies, and is increased sequentially from the clinical dose (where 0.26 g·kg). -1 This is based on clinical human dosage converted to mouse dosage), and ensures that all doses administered to mice are within the dosage range of the subacute toxicity test (the high-dose group in the subacute toxicity study was converted to a mouse dosage of 1.04 g·kg). -1 The dosage volume is 1 mL / 100g. -1 Administer once daily. Observe animals for clinical signs of toxicity after administration, such as changes in fur, eyes, mucous membranes, diarrhea, salivation, sleep, and lethargy within 30 minutes of administration, and check regularly within 24 hours. In addition, examine overall behavioral changes in body posture, movement, standing, gait, and tremors during this period until the end of the experiment.

[0076] 3.3 Examination of Animal Tumor Quality After the experiment, the mice in each group were euthanized by cervical dislocation. The tumor tissue of each group of mice was washed in physiological saline and dried. Finally, the removed tumors were photographed and recorded.

[0077] 3.4 Electrocardiogram (ECG) testing Electrocardiograms (ECGs) were recorded in the animals to assess the effects of HCSc on cardiac function. Animals in each group were immediately anesthetized with 5% isoflurane after HCSc administration, and maintained anesthesia with 1% isoflurane for 120 minutes. Heart rate and ECGs were analyzed according to cardiac histology atlas guidelines.

[0078] 3.5 Echocardiography Four cycles of animal echocardiography were performed after the HCSc intervention. A VINNO 6 portable Doppler ultrasound diagnostic instrument with a 10–23 MHz probe was used for echocardiography. Mice were anesthetized with 3.5% isoflurane gas and maintained with 1.5% isoflurane gas. The mice were fixed in a supine position, and the probe was placed on the right side of the sternum, perpendicular to the sternum, with the landmark pointing towards the right shoulder at the 12 o'clock position and tilted 15° to the left. Fraction shortening (FS) and ejection fraction (EF) values ​​were recorded and calculated. The values ​​of each parameter were measured over three consecutive cardiac cycles and averaged.

[0079] 3.6. Examination of complete blood cell count and biochemical markers After the experiment, blood samples were first collected from the animals using EDTA tubes, and complete blood cell counts were performed using a fully automated blood and body fluid analyzer. Separate blood samples were placed in EDTA-free tubes for biochemical analysis. Serum levels of AST, CK, LDH, HBDH, CKMB, BNP, cTnI, and cTnT were measured to assess the cardiotoxicity of HCSc at different time points and doses in a mouse model of colon cancer.

[0080] 3.7. TUNEL assay for tumor cell apoptosis Tumor tissues were fixed in 4% paraformaldehyde for 30–60 min, washed with PBS, dehydrated with ethanol, and then immersed in 20 μg / mL ethanol solution. -1 Incubate in Proteinase K working solution and Equilibration Buffer at 37°C for 30 min. Then, use a solution containing 0.1% Tritium X-100 and 5 g·L⁻¹. -1 The tissue was washed again with BSA and PBS. Finally, the slide was immersed in DAPI solution (2 mg / L) under light-protected conditions. -1 After 5 minutes, the slides were mounted with neutral resin. Tumor tissue apoptosis was observed under a fluorescence microscope, with three image fields randomly selected. Cells exhibiting brownish-yellow granules were recorded as positive expression.

[0081] 3.8 Histopathological Analysis Mouse heart and tumor tissue samples were fixed in 4% paraformaldehyde solution and then immersed in decalcification solution for 3 days. The samples were then prepared into paraffin blocks and sectioned. Hematoxylin-eosin (HE) staining was performed on the sections following standardized procedures, and histological images of major organs were obtained using a panoramic slide scanner.

[0082] 3.9 Immunohistochemical analysis Immunohistochemical staining analysis of cleaved caspase-3 and Ki-67 proteins in collected tumor tissues was performed. The slides were sequentially immersed in xylene three times for 15 minutes each time, followed by gradient elution with ethanol, washing with distilled water, antigen retrieval, blocking of endogenous peroxidase, serum blocking, incubation with primary antibody, incubation with secondary antibody, DAB staining, counterstaining of cell nuclei, and finally dehydration, mounting, microscopic examination, and image acquisition and analysis.

[0083] 3.10 Data Statistics Data are expressed as mean ± standard deviation. Differences between two groups were analyzed using t-tests, and differences among multiple groups were analyzed using one-way ANOVA. Statistical analysis and visualization were performed in SPSS 27 and GraphPad Prism 10. p A value <0.05 is considered statistically significant.

[0084] 4. Results 4.1 Cardiotoxicity of HCSc in a mouse model of colon cancer 4.1.1 Effects of HCSc intervention at different cycles and doses on animal electrocardiograms To evaluate the effects of four cycles and seven different doses on animal electrocardiograms, animals were anesthetized after HCSc intervention, and electrocardiogram changes were continuously monitored using an electrocardiogram monitor. The results are as follows: Figure 9 .

[0085] The electrocardiogram results showed that, compared with the control group, there were no significant changes in the electrocardiograms of animals after 7 days of HCSc intervention. Figure 9 A). At 14 days after intervention, the animals had HCSc at 0.69 g / kg. -1 ST segment elevation began to appear in the group. Figure 9 B); and during the 21-day and 28-day dosing cycles, the animal electrocardiogram changed from HCSc-0.52g·kg -1 The group already showed obvious ST segment elevation ( Figure 9 CD).

[0086] 4.1.2 Effects of HCSc intervention with different cycles and doses on animal echocardiography Echocardiographic results of animals in different cycles and different dosage groups are as follows: Figure 10 , Figure 11 and Figure 12 .

[0087] The results showed that, compared with the control group, there were no significant changes in echocardiography, LVEF, and LVFS in animals after 7 days of HCSc intervention, and the heartbeats of the animals were basically the same. After 14 and 21 days of intervention, HCSc-0.69 g·kg⁻¹-1 The echocardiograms of the group of animals showed varying degrees of change. p <0.05)( Figure 12 bc). During the 28-day dosing cycle, the interval between the two peaks in the animal echocardiogram was 0.52 g·kg⁻¹. -1 The group already showed significant widening, and the LVEF and LVFS values ​​decreased significantly. p <0.01)( Figure 12 d).

[0088] 4.1.3 Effects of different cycles and doses of HCSc intervention on whole blood cell counts in animals At the end of the experiment, on day 28, blood samples were collected from each experimental group for comprehensive hematological analysis, as shown in Table 15. The results indicated that 7 days of HCSc intervention did not cause significant cardiotoxicity in the animals. As shown in Table 16, compared to a 7-day dosing cycle, after 14 days of HCSc intervention, the animals' complete blood cell count was 0.69 g / kg. -1 Significant changes were observed at certain doses. p <0.05). During the 21-day and 28-day dosing cycles, multiple blood cell counts and biochemical indicators in the animals were within 0.52 g·kg⁻¹. -1 Significant changes were observed even at the dosage level. p <0.01 (Tables 17 and 18).

[0089] Table 15 Effects of different doses of intervention on complete blood cell counts during a 7-day dosing cycle ( ±SD, n=6) Table 16 Effects of different doses of intervention on complete blood cell counts during a 14-day dosing cycle ( ±SD, n=6) Table 17 Effects of different doses of intervention on complete blood cell counts during a 21-day dosing cycle ( ±SD, n=6) Table 18 Effects of different doses of intervention on complete blood cell counts during a 28-day dosing cycle ( ±SD, n=6) In Tables 15-18 above, * shows a significant difference from the control group (p<0.05); ** shows a significant difference from the control group (p<0.01).

[0090] 4.1.4 Effects of different cycles and doses of HCSc intervention on animal biochemical indicators The effects of different dosing cycles and different dosing groups on the animal heart were evaluated by detecting five myocardial enzyme parameters, heart failure, and myocardial troponin. Figure 13 As shown, compared with the control group, there were no significant effects on the eight cardiac-related indicators in each dose group during the 7-day dosing cycle, indicating that HCSc intervention for 7 days did not produce significant cardiotoxicity in the animals. Compared with the 7-day dosing cycle, the eight cardiac-related indicators showed varying degrees of significant changes at 14-day, 21-day, and 28-day dosing cycles. At 14-day and 21-day dosing cycles, the levels of the five myocardial enzymes, heart failure indicators, and troponin were significantly different at 0.69 g·kg⁻¹. -1 Significant changes occurred at different doses, and during a 28-day dosing cycle, eight cardiac-related parameters remained constant at 0.52 g / kg. -1 Significant changes occurred at different doses. Changes in cardiac-related parameters further illustrate the cardiotoxicity of HCSc. This also suggests that the cardiac damage caused by HCSc in animals may be time- and dose-dependent.

[0091] 4.1.5 Effects of HCSc intervention at different cycles and doses on animal heart tissue Cardiac histopathological results showed that, Figure 14 As shown, in the control group, the cardiomyocytes exhibited clear striations, centered nuclei, and no degeneration or necrosis, with no inflammatory response in any location. Compared to the control group, there were no significant changes in any dose group during the 7-day dosing cycle. However, the groups treated with HCSc for 14 and 21 days showed varying degrees of lesions, with the inflammatory response in cardiomyocytes becoming more pronounced with increasing HCSc dosage. Furthermore, compared to other groups, during the 28-day dosing cycle, HCSc-1.04 g / kg... -1 The group exhibited the most significant cardiac damage, with marked inflammatory cell infiltration in the myocardial interstitium.

[0092] 4.2 Therapeutic effect of HCSc on a mouse model of colon cancer 4.2.1 Effects of different cycles and doses of HCSc on tumor growth The in vivo antitumor effects of different drug administration groups were studied using a BALB / c-nu mouse subcutaneous tumor transplantation model, such as... Figure 15 and Figure 16 As shown.

[0093] like Figure 15As shown, under dosing cycles of 7, 14, and 21 days, the tumor volume of the seven different dose groups showed varying degrees of reduction compared to the control group. However, compared to the 7, 14, and 21-day dosing cycles, the tumor volume of the animals in each treatment group showed a more significant reduction compared to the control group under 28-day HCSc intervention. Furthermore, at the end of the experiment, the tumor weight of the animals treated with different dosing cycles and doses also showed results consistent with the above findings. Figure 16 Therefore, HCSc has a significant inhibitory effect on tumor growth in animals, and the inhibitory effect is more obvious with the increase of the dosing cycle and dosage.

[0094] 4.2.2. HE staining to observe the effects of different cycles and dosages on tumor tissue. HE staining of tumor tissues from four different drug administration cycles is shown in the figure. Figure 17 In the control group, tumor cells in the animal tumor tissues showed a high-density, orderly distribution, clear nucleocytoplasmic boundaries, and significantly enhanced morphology and structure, maintaining typical proliferative characteristics. In contrast, tumor cells in each of the treatment groups exhibited varying degrees of necrosis and a decrease in cell number. Specifically, within the same treatment cycle, during the 7-day cycle, compared to the control group, tumor cells in all seven dosage groups showed mild necrosis in a dose-dependent manner. During the 14-day and 21-day cycles, compared to the control group, tumor cells in all seven dosage groups showed significant moderate to severe necrosis. During the 28-day cycle, compared to the control group, the seven dosage groups, from low to high concentration, successively showed severe tumor cell necrosis, a decrease in cell number, and the formation of microparticles. HE staining results were consistent with these indicators.

[0095] 4.2.3. TUNEL staining to observe the effects of different cycles and dosages on tumor cell apoptosis. The effects of different dosing cycles and doses on tumor tissue cell apoptosis are shown in the figure. Figure 18 Compared with the control group, the number of apoptotic cells stained with strong green fluorescence was significantly increased in different dosage groups during the four dosing cycles, indicating that HCSc can promote tumor tissue cell apoptosis. The HCSc dosage in the 28-day dosing cycle was 1.04 g / kg. -1 The group had the most apoptotic cells.

[0096] 4.2.4 Immunohistochemical analysis of the effects of different cycles and doses on the expression of Cleaved caspase-3 and Ki-67 proteins in tumor tissues. Immunohistochemical results of the effects of different dosing cycles and doses on the expression of Cleaved Caspase-3 and Ki-67 proteins in animal tumor tissues are shown in [reference needed]. Figure 19 , Figure 20 and Figure 21The results showed that all different dosage groups increased the expression of cleaved caspase-3 and decreased the expression of Ki-67 in tumor tissue. Among them, the HCSc dosage of 1.04 g·kg⁻¹ was highest during the 28-day dosing cycle. -1 The group had the most significant effect ( p <0.01).

[0097] In summary, the longer the intervention time of HCSc and the higher the dosage, the more severe the cardiotoxicity. During 14-day and 21-day dosing cycles, HCSc at 0.69 g / kg... -1 Cardiotoxicity began to appear at doses of 0.52 g / kg, while during a 28-day dosing cycle, HCSc showed improvement at 0.52 g / kg. -1 Toxic side effects have already begun to occur at certain doses.

[0098] Based on its standard clinical dosing period (28 days), the minimum effective dose of Huachansu capsules in BALB / c-nu mice was determined to be 0.26 g·kg⁻¹. -1 The minimum toxic dose is 0.52 g·kg. -1 This provides key threshold parameters for optimizing the clinical safety dose window.

[0099] Converting the above animal experimental results into human effective doses, and taking into account the actual content of 15 batches of Huachansu capsules, the clinical safe content limit of Huachansu capsules was determined to be 35μg / capsule to 80μg / capsule.

[0100] In this invention, when the weighted total content of the effective / toxic components of Huachansu capsules is between 35 μg / capsule and 80 μg / capsule, it indicates that Huachansu capsules have the best efficacy and low toxicity, and that the quality of Huachansu capsules is qualified.

[0101] The technical effects of the present invention are verified through the following research.

[0102] Verification 1 The following is a comprehensive methodological validation of the qualitative and quantitative analysis of the content of several key active / toxic components in Huachansu capsules using high-performance liquid chromatography (HPLC).

[0103] (1) Specificity examination like Figure 22 As shown, the peak shapes were good and there were no impurities in the reference and test solutions, indicating that the method has good specificity.

[0104] (2) Examination of linear relationships The reference solution was diluted with chromatographically pure methanol to different concentration levels, injected for analysis, and the chromatograms and peak areas were recorded. Linear regression was performed with mass concentration as the x-axis and peak area as the y-axis. The results are shown in Table 19, indicating that the key active / toxic components have good linear relationships within a certain concentration range.

[0105] Table 19 Results of linear relationship examination of key efficacy / toxicity components (3) Precision test Take the reference solution and perform repeated injections and determinations 6 times under the chromatographic conditions, recording the chromatograms. Calculate the RSD value of the peak area to evaluate the precision of the instrument.

[0106] Referring to Table 20, the RSDs of the peak areas of bufotalin, bufotoxin, bufotalin, bufotalin, bufotalin, bufotalin, and bufotalin ester were 0.86%, 0.91%, 0.85%, 0.79%, 0.87%, 0.91%, 0.83%, and 0.69%, respectively. This indicates that the instrument has good precision.

[0107] Table 20 Results of Precision Study of Key Phenolic / Toxic Components (4) Stability test Take the test solution and inject it at 0, 2, 4, 8, 12, and 24 h after preparation according to the chromatographic conditions, and record the chromatograms. Calculate the RSD value of the peak area to evaluate the stability of the test solution within 24 h.

[0108] Referring to Table 21, the RSDs of the peak areas of bufotalin, bufotoxin, bufotalin, bufotalin, bufotalin, bufotalin, and bufotalin ester were 1.10%, 1.13%, 1.07%, 0.63%, 1.81%, 1.08%, 0.65%, and 0.64%, respectively. This indicates that the test solution exhibited good stability within 24 hours.

[0109] Table 21 Results of stability studies on key active / toxic components (5) Repeatability test Take HCSc from batch S1, prepare 6 test solutions, inject them according to chromatographic conditions, and record the chromatograms. Calculate the RSD values ​​of each key active / toxic component and evaluate the repeatability of this method.

[0110] Referring to Table 22, the RSDs of the contents of bufotalin, bufotoxin, bufotalin, bufotalin, bufotalin, bufotalin, and bufotalin ester were 1.56%, 1.18%, 1.02%, 1.46%, 1.66%, 1.04%, 1.23%, and 1.20%, respectively. This indicates that the method has good reproducibility.

[0111] Table 22 Results of repeatability studies for key efficacy / toxicity components (6) Recovery test Accurately weigh nine portions of HCSc(S1) with known content, and add them to the reference standard at ratios of 80%, 100%, and 120%, respectively. Inject and determine the samples according to chromatographic conditions, and calculate the average recovery rate and RSD of each key active / toxic component.

[0112] As shown in Table 23, the results show that the average recovery rate and RSD value of the eight components meet the requirements, indicating that the quantitative analysis method has high accuracy in detecting the content of the eight key active / toxic components in HCSc.

[0113] Table 23 Results of Recovery Rate Study of Key Active / Toxic Components The specificity, linearity, precision, stability, repeatability, and recovery rate of the established high-performance liquid chromatography method were verified through the above tests. The results show that the test method of the present invention is suitable for the qualitative and quantitative detection of Huachansu capsules, ensuring that the test results are true, accurate, and reliable, and improving the accuracy of quality evaluation.

[0114] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present invention.

Claims

1. A method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis, characterized in that, Includes the following steps: Step S1: Several key active / toxic components in Huachansu capsules were screened using network pharmacology and molecular docking technology; Step S2: Obtain the content of multiple key active / toxic components in Huachansu capsules based on high performance liquid chromatography; Step S3: Obtain the ICs of multiple key active / toxic components. 50 value; Step S4: Based on the IC50 of multiple key active / toxic components 50 The weight coefficients of multiple key efficacy / toxicity components were obtained using the AHP (Analytical Hierarchy Process) method to determine the value. Step S5: Based on the content of multiple key active / toxic components in step S2 and the weighting coefficients of multiple key active / toxic components in step S4, obtain the weighted total content of multiple key active / toxic components in Huachansu capsules. Step S6: Compare the weighted total content of multiple key active / toxic components in Huachansu capsules from step S5 with the clinical safety content limit of Huachansu capsules to complete the quality evaluation of Huachansu capsules.

2. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 1, characterized in that, The specific steps in step S1, which employ network pharmacology and molecular docking technology to screen for multiple key efficacy / toxic components in Huachansu capsules, are as follows: S1.1 Identification of main components: The main components of Huachansu capsules were identified based on UPLC-Q-Exactive Orbitrap MS; S1.2 Screening of active ingredients: Using oral bioavailability ≥30% and drug similarity ≥0.18 as screening criteria, the main components of Huachansu capsules in step S1.1 were screened through the database and analysis platform of traditional Chinese medicine systems pharmacology to determine the active ingredients of Huachansu capsules; S1.3 Obtaining efficacy-toxicity targets: Using "Cardiotoxicity" and "Colon cancer" as keywords, the targets of Huachansu capsules on cardiotoxicity and colon cancer were obtained from the CTD, OMIM and Gene Cards databases, and then efficacy-toxicity targets were obtained through integrated analysis. S1.4 Constructing a multidimensional pharmacodynamic-toxicity network: Utilizing active ingredients and efficacy-toxicity targets, a multidimensional pharmacodynamic-toxicity network of "drug-ingredient-efficacy / toxicity targets" is constructed using network pharmacology; S1.5 Identify key efficacy / toxicity components and core targets: Visualize and analyze the multidimensional efficacy-toxicity network of "drug-component-efficacy / toxicity target", and use the degree value as the screening criterion to obtain the top-ranked active ingredients and targets as key efficacy / toxicity components and core targets. S1.6 Screening: Molecular docking technology was used to calculate the binding energy between the top-ranked active ingredients and the target sites, and the identified key efficacy / toxicity components were verified to complete the screening of multiple key efficacy / toxicity components in Huachansu capsules.

3. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 2, characterized in that, In step S1.1, the detection conditions for UPLC-Q-Exactive Orbitrap MS include: UPLC chromatographic conditions: Waters BEH C18 column, 2.1 × 100 mm, 1.7 μm; mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile; flow rate 0.4 mL / min. -1 The injection volume was 10 μL, and the column temperature was 40 ℃; the elution gradient is shown in the table below: Mass spectrometry conditions: ESI source, positive and negative ion detection mode, sheath gas flow rate 30 Arb, auxiliary gas flow rate 10 Arb, spray voltage 3.5 kV, capillary temperature 275 ℃, auxiliary gas temperature 350 ℃; scanning mode: Full MS / dd-MS2, Full MS resolution 60000, dd-MS2 resolution 15000, scanning range 100 ~ 1500 m / z.

4. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 2, characterized in that, In steps S1.5 and S1.6, the key active / toxic components are bufotoxin, bufotoxin base, far-reaching bufotoxin, bufotoxin ester, sand bufotoxin, bufotoxin, bufotoxin and bufotoxin.

5. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 4, characterized in that, The process of obtaining the content of multiple key active / toxic components in Huachansu capsules based on high performance liquid chromatography is as follows: S2.1 Solution Preparation Preparation of reference solutions: Using bufotalin, bufotalin, far-reaching bufotalin, bufotalin ester, bufotalin, bufotalin, bufotalin and bufotalin as reference standards, reference solutions were prepared using methanol solution; Preparation of the test solution: Accurately weigh 0.25 g of the contents of Huachansu capsules, add methanol to make up to 10 mL, sonicate for 30 min, and filter the supernatant through a 0.22 μm organic filter membrane to obtain the test solution; S2.2 Obtain the reference chromatogram and the test sample chromatogram. Take the reference solution and the test solution from step S2.1, and obtain the reference chromatogram and the test chromatogram respectively by high performance liquid chromatography; The conditions for high-performance liquid chromatography (HPLC) were as follows: the chromatographic column was Betasil. TM C18; mobile phase: acetonitrile and 0.5% potassium dihydrogen phosphate aqueous solution; detection wavelength: 296 nm; flow rate: 0.9 mL / min -1 The injection volume was 10 μL; the column temperature was 35 ℃; and the gradient elution conditions are shown in the table below. S2.3 Calculate the content of key active / toxic components. The reference chromatogram and the test sample chromatogram from step S2.2 are processed to generate the corresponding reference fingerprint chromatogram and test fingerprint chromatogram. Peak matching was performed on the test fingerprint spectrum and the control fingerprint spectrum, and the characteristic peaks were marked sequentially according to the common peak numbers. Based on the peak positions of multiple key effective / toxic components in the control spectrum, the corresponding peak numbers of multiple key effective / toxic components in the test fingerprint spectrum were determined. Calculate the content of several key active / toxic components on the fingerprint spectrum of the test sample.

6. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 5, characterized in that, The peak numbers corresponding to multiple key active / toxic components in the tested fingerprint spectrum are: Peak 1: Bufotalin; Peak 4: Bufotalin; Peak 7: Bufotalin; Peak 9: Bufotalin; Peak 10: Bufotalin; Peak 11: Bufotalin; Peak 12: Bufotalin; Peak 13: Bufotalin ester.

7. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 1, characterized in that, In step S3, the IC50 of several key effector / toxicant components on the proliferation activity of AC116 cells is determined. 50 value.

8. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 4, characterized in that, In step S4, the specific process of obtaining the weight coefficients of multiple key efficacy / toxicity components using the AHP (Analytical Hierarchy Process) is as follows: S4.

1. The Satty 1-9 scale was used to determine the IC50 values ​​of several key active / toxic components. 50 The values ​​are compared pairwise to determine the importance scale of each key efficacy / toxic component relative to other key efficacy / toxic components, and a comprehensive judgment matrix is ​​constructed: in: Indicates the first i The key efficacy / toxic component relative to the first j The importance scale of key efficacy / toxicity components, and meets the requirements. m ij >0, m ij =1 / m ji , m ii =1; S4.2 Calculate the weight vector Calculate the first in the comprehensive judgment matrix i geometric mean of row elements ; =1，2，…，8 in: Indicates the first i The key efficacy / toxic component relative to the first j The importance scale of key efficacy / toxicity components For the first i The 8th root of the product of all elements in the row, i.e., the [number]th [row]. i The geometric mean of the row elements; The vector is processed according to the following formula. = After normalization, we obtain the first... Normalized weighting coefficients of key efficacy / toxic components; =1，2，…，8 in: For the first Normalized weighting coefficients of key efficacy / toxicity components, For the first i The geometric mean of the row elements. For the first k Normalized weighting coefficients of key efficacy / toxic components; According to the The normalized weight coefficients of each key efficacy / toxic component are used to obtain the weight vector. ; in: For the weight vector, This is the normalized weighting coefficient for the first key efficacy / toxic component. The normalized weighting coefficient for the second key efficacy / toxic component; For the first Normalized weighting coefficients of key efficacy / toxicity components, =1, 2, ..., 8; S4.

3. Perform a consistency check on the comprehensive judgment matrix and calculate the consistency ratio. The weight vector obtained in step S4.2 The results are reliable; S4.4, The weight vector after passing the consistency test various Converting this to percentages yields the weighting coefficients for each key efficacy / toxic component.

9. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to claim 4, characterized in that, In step S5, the weighted total content of multiple key active / toxic components in the Huachansu capsules is: in: For the first i The content of key active / toxic components, For the first i The weighting coefficients of key efficacy / toxicity components, i The number of key active / toxic components is 1, 2, ..., 8.

10. The method for quality evaluation of Huachansu capsules based on simultaneous efficacy / toxicity analysis according to any one of claims 1-9, characterized in that, In step S6, the clinically safe content limit of Huachansu capsules is 35μg / capsule to 80μg / capsule.

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