Composition, application of composition in preparation of medicine for treating liver cancer or / and breast cancer and screening method

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a composition, application of the composition in preparation of medicine for treating liver cancer or / and breast cancer and a screening method. According to the invention, the composition is obtained through screening by combining the hemogenic component of the marsdenia tenacissima tablets with targeted network pharmacological research, and the composition comprises neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, marsdenia tenacissima glycoside I and marsdenia tenacissima glycoside H. Each active component in the composition corresponds to a plurality of targets, each target is connected with a plurality of active components, and the plurality of targets correspond to a plurality of pathways, so that the plurality of active components in the composition act on the liver cancer or / and the breast cancer from the plurality of targets and the plurality of pathways in a synergistic manner, and a new research direction is provided for developing medicines for treating the liver cancer or / and the breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a composition, its application in the preparation of a drug for treating liver cancer or / and breast cancer, and a screening method. Background Art

[0002] With the development of social economy, the acceleration of the aging process of the population, and the prevalence of carcinogenic factors such as bad living habits and environmental pollution, the mortality rate of malignant tumors continues to rise, and the disease burden is increasing day by day. According to the latest research report of the Global Cancer Research Center (IARC), the incidence and mortality rates of tumors worldwide show an increasing trend year by year. Therefore, the research on tumors has received extensive attention. Common malignant tumors include breast cancer, liver cancer, gastric cancer, lung cancer, and colorectal cancer, etc.

[0003] Marsdenia tenacissima, also known as Cynanchum auriculatum Royle ex Wight, Marsdenia tenacissima, Lactagogue Sida Rhombifolia Root, etc., is the dried stem of the plant of the genus Marsdenia in the family Asclepiadaceae. Recent research has pointed out that Marsdenia tenacissima has been proven to have anti-tumor activity both in vivo and in vitro, such as liver cancer, lung cancer, esophageal cancer, gastric cancer, and breast cancer, etc. C21 steroidal saponins, as the primary characteristic of Marsdenia tenacissima, are considered to be its main anti-tumor active ingredients. It can inhibit the growth of cancer cells, regulate the angiogenesis of tumor cells, and disrupt the blood supply of tumor tissues, thereby inhibiting the growth and spread of tumors. It can also promote the differentiation of cancer cells and make them transform into normal cells.

[0004] The journal document "Discussion on the Mechanism of Action of Marsdenia tenacissima in the Treatment of Breast Cancer Based on Network Pharmacology and Experimental Verification" provides 37 active ingredients in Marsdenia tenacissima that can be used for breast cancer, and the journal document "Discussion on the Mechanism of Action of Marsdenia tenacissima Against Hepatocellular Carcinoma Based on Network Pharmacology" provides 50 active ingredients in Marsdenia tenacissima that can be used for hepatocellular carcinoma. However, these active ingredients are all screened based on the active ingredient database of Marsdenia tenacissima. The active ingredients in the database have not been verified through the in vivo metabolic process. Some active ingredients may undergo structural changes after in vivo metabolism, resulting in the possibility that the screened active ingredients may be ineffective precursors that are not actually absorbed; it is also possible to miss the true active ingredients produced by the metabolism of the original components of Marsdenia tenacissima.

[0005] Currently, there are also some patent documents that screen effective active ingredients based on blood components. For example, a modified analysis method for the pharmacodynamic material basis of Naoxinqing tablets disclosed in the patent document with the publication number CN118010862A applies UPLC-Q / TOF-MS technology to the identification and analysis of the chemical components and blood components of Naoxinqing tablets, combines with the network pharmacology platform, discovers the pharmacodynamic material components and action mechanisms of Naoxinqing tablets, and reveals the complex pharmacodynamic network mechanism of Naoxinqing tablets.

[0006] However, there is currently no research on the key active ingredients and action mechanisms of Marsdenia tenacissima in the treatment of liver cancer or / and breast cancer. Summary of the Invention

[0007] The present invention aims to solve the above problems and provides a composition, its application in the preparation of drugs for treating liver cancer and / or breast cancer, and a screening method.

[0008] The technical solution for solving the problems of the present invention is as follows: First, a composition is provided, which includes neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H.

[0009] In the present invention, the inventors screened a composition through the research on the blood-forming components and targeted network pharmacology of Tongguanteng tablets. Multiple active ingredients in the composition coexist and synergistically act in the blood components.

[0010] Secondly, the present invention also provides the application of the above composition in the preparation of drugs for treating liver cancer and / or breast cancer.

[0011] As a preference of the present invention, the composition is particularly suitable for the application in the preparation of drugs for treating liver cancer and breast cancer.

[0012] As a preference of the present invention, the composition is applied to the preparation of drugs for treating liver cancer and / or breast cancer by at least affecting the PI3K-Akt signaling pathway, GnRH signaling pathway, and IL-17 signaling pathway, and is particularly suitable for the application in the preparation of drugs for treating liver cancer and breast cancer.

[0013] As a preference of the present invention, the composition acts on at least the STAT3, PIK3CA, KDR, HSR90AA1, MET, PIK3CD, and PIK3CB targets to at least affect the PI3K-Akt signaling pathway, GnRH signaling pathway, and IL-17 signaling pathway, and is applied to the preparation of drugs for treating liver cancer and / or breast cancer, and is particularly suitable for the application in the preparation of drugs for treating liver cancer and breast cancer.

[0014] In the present invention, multiple active ingredients in the composition coexist and synergistically act in the blood components. Each active ingredient corresponds to multiple targets, each target connects multiple active ingredients, and multiple targets correspond to multiple pathways, so that multiple active ingredients in the composition synergistically act on liver cancer and / or breast cancer from multiple targets and multiple pathways.

[0015] When applied alone, the dosage of chlorogenic acid is not less than 10 μg / mL. For example, it can be 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, all of which can inhibit the viability of MCF-7 cells and HepG2 cells, and are effectively applied to breast cancer and liver cancer.

[0016] Preferably, the dosage of chlorogenic acid is not less than 100 g / mL, which can further improve the inhibitory effect on the viability of MCF-7 cells and HepG2 cells. Meanwhile, it can reduce the MDA content in MCF-7 and HepG2 cells, increase the SOD enzyme activity in MCF-7 and HepG2 cells, and increase the GSH content in MCF-7 and HepG2 cells.

[0017] More preferably, the dosage of chlorogenic acid is not less than 400 g / mL, which can further improve the inhibitory effect on the viability of MCF-7 cells and HepG2 cells. Meanwhile, it can further reduce the MDA content in MCF-7 and HepG2 cells, further increase the SOD enzyme activity in MCF-7 and HepG2 cells, and further increase the GSH content in MCF-7 and HepG2 cells.

[0018] When applied alone, the dosage of neochlorogenic acid is not less than 50 μg / mL. For example, it can be 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, all of which can inhibit the viability of MCF-7 cells and HepG2 cells and can be effectively applied to breast cancer and liver cancer.

[0019] Preferably, the dosage of neochlorogenic acid is not less than 100 g / mL, which can further improve the inhibitory effect on the viability of MCF-7 cells and HepG2 cells. Meanwhile, it can reduce the MDA content in MCF-7 and HepG2 cells, increase the SOD enzyme activity in MCF-7 and HepG2 cells, and increase the GSH content in MCF-7 and HepG2 cells.

[0020] More preferably, the dosage of neochlorogenic acid is not less than 400 g / mL, which can further improve the inhibitory effect on the viability of MCF-7 cells and HepG2 cells. Meanwhile, it can further reduce the MDA content in MCF-7 and HepG2 cells, further increase the SOD enzyme activity in MCF-7 and HepG2 cells, and further increase the GSH content in MCF-7 and HepG2 cells.

[0021] When applied alone, the dosage of cryptochlorogenic acid is not less than 25 μg / mL. For example, it can be 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, all of which can inhibit the viability of MCF-7 cells and HepG2 cells and can be effectively applied to breast cancer and liver cancer.

[0022] Preferably, the dosage of cryptochlorogenic acid is not less than 100 g / mL, which can further improve the inhibition of MCF-7 cell viability and HepG2 cell viability. At the same time, it can reduce the MDA content in MCF-7 and HepG2 cells, increase the SOD enzyme activity in MCF-7 and HepG2 cells, and increase the GSH content in MCF-7 and HepG2 cells.

[0023] More preferably, the dosage of cryptochlorogenic acid is not less than 400 g / mL, which can further improve the inhibition of MCF-7 cell viability and HepG2 cell viability. At the same time, it can further reduce the MDA content in MCF-7 and HepG2 cells, further increase the SOD enzyme activity in MCF-7 and HepG2 cells, and further increase the GSH content in MCF-7 and HepG2 cells.

[0024] When used alone, the dosage of tenacissoside I is not less than 50 μg / mL. For example, it can be 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, all of which can inhibit the viability of MCF-7 cells and HepG2 cells.

[0025] Preferably, the dosage of tenacissoside I is not less than 100 μg / mL, which can further improve the inhibition of MCF-7 cell viability and HepG2 cell viability. At the same time, it can reduce the MDA content in MCF-7 and HepG2 cells, increase the SOD enzyme activity in MCF-7 and HepG2 cells, and increase the GSH content in MCF-7 and HepG2 cells.

[0026] More preferably, the dosage of tenacissoside I is not less than 600 μg / mL, which can further improve the inhibition of MCF-7 cell viability and HepG2 cell viability. At the same time, it can further reduce the MDA content in MCF-7 and HepG2 cells, further increase the SOD enzyme activity in MCF-7 and HepG2 cells, and further increase the GSH content in MCF-7 and HepG2 cells.

[0027] When used alone, the dosage of tenacissoside H is not less than 50 μg / mL. For example, it can be 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, all of which can inhibit the viability of MCF-7 cells and HepG2 cells.

[0028] Preferably, the dosage of tenacissoside H is not less than 100 μg / mL, which can further improve the inhibitory effect on the viability of MCF-7 cells and HepG2 cells. Meanwhile, it can reduce the MDA content in MCF-7 and HepG2 cells, increase the SOD enzyme activity in MCF-7 and HepG2 cells, and increase the content of GSH in MCF-7 and HepG2 cells.

[0029] More preferably, the dosage of tenacissoside H is not less than 600 μg / mL, which can further improve the inhibitory effect on the viability of MCF-7 cells and HepG2 cells. Meanwhile, it can further reduce the MDA content in MCF-7 and HepG2 cells, further increase the SOD enzyme activity in MCF-7 and HepG2 cells, and further increase the content of GSH in MCF-7 and HepG2 cells.

[0030] When used in combination, the dosage of the composition is not less than 100 μg / mL. For example, it can be 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL.

[0031] The dosage ratio of phenolic acid compounds to glycoside compounds in the composition is 1:(0.5 - 5). For example, it can be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5. The phenolic acid compounds refer to neochlorogenic acid or chlorogenic acid or cryptochlorogenic acid, and the glycoside compounds refer to tenacissoside I or tenacissoside H.

[0032] Preferably, the dosage ratio of phenolic acid compounds to glycoside compounds is 1:(1 - 2).

[0033] Finally, the present invention also provides a method for screening the above composition from Marsdenia tenacissima tablets, including the following steps: S1. After administration, use UPLC-Q / TOF-MS technology to analyze the blood components in the administered plasma; S2. Use relevant databases of network pharmacology to collect the action targets corresponding to the blood components, construct a target library of Marsdenia tenacissima tablets; construct an intersection target library of liver cancer and breast cancer; take the intersection of the Marsdenia tenacissima tablet target library and the liver cancer and breast cancer intersection target library to obtain potential action targets; S3. Use the blood components and the potential action targets to construct a visualization network diagram of "blood components - targets - diseases" of Marsdenia tenacissima tablets, and obtain the main active components in the blood components according to network analysis. The main active components are the composition.

[0034] In step S1, preferably in the present invention, after administering the drug to the experimental rats, their heads are chopped off to collect blood. After centrifuging the blood sample, the supernatant is taken to obtain a plasma sample, which is stored at -80 °C for later use. After preprocessing the plasma sample, the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q / TOF-MS) technique is used to screen out the blood components.

[0035] More preferably, the preprocessing of the plasma sample includes the following steps: Take out the plasma sample and thaw it at 0 - 4 °C. Add 3 - 5 mL of cold methanol to 1 mL of plasma, vortex for 3 - 8 min, then shake for 15 - 25 min, centrifuge at 0 - 4 °C and 6000 - 10000 r / min for 5 - 15 min, take the supernatant and dry it with nitrogen, redissolve it with 80 - 120 µL of methanol, and centrifuge again at 0 - 4 °C and 12000 - 15000 r / min for 5 - 15 min. Take the supernatant for UPLC-Q-TOF-MS analysis.

[0036] More preferably, the HPLC chromatographic conditions are as follows: Column: Agilent Poroshell-C18 (4.6 mm × 150 mm, 2.7 μm), column temperature: 30 °C, flow rate: 0.5 mL / min, injection volume: 5 μL; Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid in water, and the gradient elution program is: 0 - 4 min, 5 - 20% A; 4 - 15 min, 20 - 25% A; 15 - 25 min, 25 - 35% A; 25 - 45 min, 35 - 50% A; 45 - 50 min, 50 - 75% A; 50 - 55 min, 75 - 95% A; 55 - 57 min, 95 - 100% A; 57 - 62 min, 100% A; 62 - 67 min, 100 - 5% A; 67 - 72 min, 5% A.

[0037] Mass spectrometry conditions: Use an ESI electrospray ion source, MSE full scan mode, collect data in both positive and negative ion modes, scan time 0.3 s, scan range 50 - 1200 Da. Use argon as the collision gas, low collision energy is 6 V, and high collision energies are 20 - 30 V, 30 - 50 V, 50 - 60 V. Use sodium formate and leucine enkephalin to calibrate and perform real-time calibration of the mass spectrometer. The real-time reference ions generated by leucine enkephalin are [M + H]+ (m / z 556.2771) in the positive ion mode and [M - H]- (m / z 554.2615) in the negative ion mode. In step S2, preferably in the present invention, the identified blood components are imported into the Swiss TargetPrediction database and the Uniprot database to search for the target proteins corresponding to each blood component, thereby obtaining the target library of Marsdenia tenacissima tablets.

[0038] Disease-related targets of liver cancer and breast cancer are obtained in the GeneCards database to obtain the intersection target library of liver cancer and breast cancer. Among them, further preferably, an algorithm for calculating the median is used to remove genes with relatively low correlation values.

[0039] Using the Venny 2.2.0 online analysis tool, upload the target library of Marsdenia tenacissima tablets and the intersection target library of liver cancer and breast cancer, so as to obtain the potential action targets of Marsdenia tenacissima tablets in the treatment of liver cancer and breast cancer.

[0040] In step S3, preferably in the present invention, the blood components in Marsdenia tenacissima tablets obtained in step S1 and the potential action targets obtained in step S2 are respectively imported into the Cytoscape software to construct a "blood component-core target-disease" network of Marsdenia tenacissima tablets, and key blood components are obtained according to network analysis.

[0041] Preferably in the present invention, the following steps are further included: S4. Establish a PPI network for the potential action targets to obtain key action targets.

[0042] In step S4, further preferably, the potential action targets are imported into the String database for PPI network construction, and after removing free proteins, a network analysis diagram is obtained, and the key action targets of Marsdenia tenacissima tablets in the treatment of liver cancer and breast cancer are obtained through visualization display by the Cytoscape software.

[0043] Preferably in the present invention, the following steps are further included: S5. Perform GO enrichment analysis and KEGG pathway enrichment on the potential action targets to obtain signal pathways with high enrichment significance.

[0044] In step S5, further preferably, visit the official website of the Metascape database, import the potential action targets, obtain the results of GO enrichment analysis and KEGG pathway enrichment analysis, and draw a triple-bar chart of BP, CC and MF and a KEGG enrichment analysis bubble chart, and analyze the pathways with high enrichment significance respectively.

[0045] Preferably in the present invention, the following steps are further included: S6. Verify the effect of the composition on the proliferation of HepG2 and MCF-7 cells through the CCK-8 experiment; perform cell experiments to detect the effects of the pharmacodynamic components on the MDA content, SOD enzyme activity and GSH content of HepG2 and MCF-7 cells using an oxidative stress factor kit.

[0046] Advantages of the present invention: Through the study of the original forming components entering the blood of Tongguanteng tablets combined with targeted network pharmacology, a composition was screened. Multiple active ingredients (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, tenacissoside H) in the composition coexist in the components entering the blood. Each active ingredient corresponds to multiple targets, each target is connected to multiple active ingredients, and multiple targets correspond to multiple pathways. Thus, multiple active ingredients in the composition act on liver cancer and / or breast cancer synergistically from multiple targets and multiple pathways, providing a new research direction for the development of drugs for treating liver cancer and / or breast cancer. Brief Description of the Drawings

[0047] Figure 1 is the total ion current chromatogram of rat plasma samples (A. blank plasma ES+; B. plasma sample ES+; C. blank plasma ES-; D. plasma sample ES-); Figure 2 is the Venn diagram of potential action targets of Tongguanteng tablets in treating liver cancer and breast cancer; Figure 3 is the visualization network diagram of "components entering the blood - targets - diseases" of Tongguanteng tablets; Figure 4 is the detection result of the survival rate of MCF-7 cells measured by CCK-8; Figure 5 is the detection result of the survival rate of HepG2 cells measured by CCK-8; Figure 6 is the result of the effect of administration of active ingredients at different concentrations on the MDA content of MCF-7 and HepG2 cells; Figure 7 is the result of the effect of administration of active ingredients at different concentrations on the SOD enzyme activity of MCF-7 and HepG2 cells; Figure 8 is the result of the effect of administration of active ingredients at different concentrations on the GSH content of MCF-7 and HepG2 cells. Detailed Embodiments

[0048] The following are the detailed embodiments of the present invention. In combination with the drawings, the technical solutions of the present invention are further described, but the present invention is not limited to these embodiments.

[0049] Example 1 A method for screening a composition from Tongguanteng tablets for preparing a drug for treating liver cancer and / or breast cancer, comprising the following steps: S1. After administration, use UPLC-Q / TOF-MS technology to analyze the components entering the blood in the administered plasma, specifically including the following steps: Experimental animals: Male SD rats, weighing 200±20 g.

[0050] Drug administration regimen: Weigh an appropriate amount of Tongguanteng tablets, grind them finely, add an appropriate amount of ultrapure water, mix them evenly with a vortex mixer, and then intragastrically administer the drug to the experimental rats (2.08 g / kg).

[0051] Sample collection: Continuously intragastrically administer the suspension (2.08 g / kg) to the rats in the drug administration group for 7 days, and the blank group is given an equal amount of ultrapure water. All experimental rats are fasted for 12 h before intragastric administration on the seventh day. After the fasting period ends, decapitate the rats to collect blood at 0.25, 0.5, 1, 2, and 4 h after intragastric administration, place the blood in heparin tubes, centrifuge the collected samples under the conditions of centrifugation (3500 r / min, 4 °C) for 10 min, aspirate the supernatant to obtain plasma samples, and store them in a -80 °C refrigerator for later use.

[0052] Sample pretreatment and detection: When analyzing the samples, take out the plasma samples and thaw them at 4 °C. Add 4 mL of cold methanol to 1 mL of plasma, vortex for 5 min, shake for 20 min, centrifuge at 4 °C and 8000 r / min for 10 min, take the supernatant and dry it with nitrogen, re-dissolve it with 100 μL of methanol, and centrifuge again at 4 °C and 13000 r / min for 10 min. Take the supernatant for UPLC-Q-TOF-MS analysis.

[0053] Among them, the HPLC chromatographic conditions are as follows: chromatographic column: Agilent Poroshell-C18 (4.6 mm×150 mm, 2.7 μm), column temperature: 30 °C, flow rate: 0.5 mL / min, injection volume: 5 μL; mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid in water, and the gradient elution program is: 0 - 4 min, 5 - 20% A; 4 - 15 min, 20 - 25% A; 15 - 25 min, 25 - 35% A; 25 - 45 min, 35 - 50% A; 45 - 50 min, 50 - 75% A; 50 - 55 min, 75 - 95% A; 55 - 57 min, 95 - 100% A; 57 - 62 min, 100% A; 62 - 67 min, 100 - 5% A; 67 - 72 min, 5% A.

[0054] Mass spectrometry conditions: ESI electrospray ionization source was used, in MSE full scan mode, data was collected in positive and negative ion modes, the scan time was 0.3 s, and the scan range was 50 - 1200 Da. Argon was used as the collision gas, the low collision energy was 6 V, and the high collision energies were 20 - 30 V, 30 - 50 V, and 50 - 60 V. Sodium formate and leucine enkephalin were used to calibrate the mass spectrometer and for real-time calibration. Leucine enkephalin generated real-time reference ions, which were [M + H]+ (m / z 556.2771) in positive ion mode and [M - H]- (m / z 554.2615) in negative ion mode.

[0055] As Figure 1 shown, the total ion chromatogram of rat plasma samples treated with methanol was obtained by analysis, and a total of 22 blood components were identified.

[0056] Using relevant databases of network pharmacology, the action targets corresponding to the blood components were collected to construct the target library of Marsdenia tenacissima tablets; the intersection target library of liver cancer and breast cancer was constructed; the intersection of the target library of Marsdenia tenacissima tablets and the intersection target library of liver cancer and breast cancer was taken to obtain potential action targets, which specifically included the following steps: For the 22 identified blood prototype components, the corresponding target proteins of each component were searched through SwissTargetPrediction and Uniprot databases. After removing duplicate values, a total of 316 compound-related targets were obtained, which was the target library of Marsdenia tenacissima tablets.

[0057] In the GeneCards database, "liver cancer" or "breast cancer" was entered as the keyword, and the disease-related targets of liver cancer and breast cancer were retrieved. Among them, the algorithm of calculating the median was used to remove genes with relatively low relevance values, obtaining 2467 disease targets for liver cancer and 7783 for breast cancer. These target results were combined, and duplicate gene information was removed. An intersection target library of liver cancer and breast cancer containing 2842 targets was obtained.

[0058] Through the online tool interface of Venny, 316 target points related to the blood components of Marsdenia tenacissima tablets and 2842 disease-related target points were imported into venny 2.1.0, and the intersection of the two was taken to obtain the Venn diagram as Figure 2 shown. 146 potential action targets of Marsdenia tenacissima tablets for treating liver cancer and breast cancer were obtained, and the potential action targets are shown in Table 1 below.

[0059] Table 1. S3. Using the blood components and potential action targets, construct a visualization network diagram of "blood components - targets - diseases" for Tongguanteng tablets, and obtain the main active components in the blood components according to network analysis. The main active components are a composition, which specifically includes the following steps: Import the blood components and potential action targets in Tongguanteng tablets into the Cytoscape software respectively to construct a "blood components - targets - diseases" network for Tongguanteng tablets, as Figure 3 shown. According to the principle of network analysis, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tengonin I and tengonin H in the blood components are connected to more targets and are the main active components.

[0060] Establish a PPI network for the potential action targets to obtain the key action targets, which specifically includes the following steps: Import 146 potential action targets into the String database for PPI network construction. After hiding the free proteins, a network analysis diagram is obtained, and through visualization display using the Cytoscape software, the key action targets for Tongguanteng tablets to treat liver cancer and breast cancer are obtained: HSP90AA1, PTPN11, PIK3CB, PIK3CD, KDR, MAPK3, ESR1, ERB2, JAK2, RXRA, MET, IL6, CDK1, STAT3, EGFR, PIK3CA, MAPK1.

[0061] At the same time, based on the main active components obtained in step S3, further confirm that the key action targets connected to them include STAT3, PIK3CA, KDR, HSR90AA1, MET, PIK3CD and PIK3CB.

[0062] Conduct GO enrichment analysis and KEGG pathway enrichment on the potential action targets to obtain signal pathways with high enrichment significance, which specifically includes the following steps: Import the potential action targets into the Metascape database website to obtain the results of GO enrichment analysis and KEGG pathway enrichment analysis, and draw a triple-bar graph of BP, CC and MF and a KEGG enrichment analysis bubble graph, and analyze the pathways with high enrichment significance respectively. It is found that the five components of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tengonin I and tengonin H in Tongguanteng tablets can play a therapeutic role through key pathways such as the PI3K-Akt signaling pathway, GnRH signaling pathway, IL-17 signaling pathway, and tumor signaling pathway.

[0063] Based on the above steps, a composition is obtained, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I and tenacissoside H. This composition exerts its therapeutic effects on liver cancer and breast cancer by acting on at least key targets STAT3, PIK3CA, KDR, HSR90AA1, MET, PIK3CD and PIK3CB, and at least affecting key pathways such as PI3K-Akt signaling pathway, GnRH signaling pathway, IL-17 signaling pathway, tumor signaling pathway, etc.

[0064] The CCK-8 assay was used to verify the effect of the composition on the proliferation of HepG2 and MCF-7 cells, which specifically included the following steps: HepG2 cells and MCF-7 cells were incubated in an incubator at 37 °C and 5% CO2. Logarithmic growth phase HepG2 and MCF-7 cells were respectively seeded into 96-well plates, with a density of 1×10 4 cells / mL, and a total of 10 plates were seeded. After 24 hours of culture, the supernatant was discarded, and drugs (doxorubicin, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, tenacissoside H) were administered to HepG2 and MCF-7 cells respectively. Each drug was set at 8 different concentration gradients and cultured in the incubator for 24 h.

[0065] The concentrations of the drug solutions that need to be prepared separately before drug administration are as follows: doxorubicin: 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2, 4 μg / mL; neochlorogenic acid: 2.5, 5, 10, 25, 50, 100, 200, 400 μg / mL; chlorogenic acid: 2.5, 5, 10, 25, 50, 100, 200, 400 μg / mL; cryptochlorogenic acid: 2.5, 5, 10, 25, 50, 100, 200, 400 μg / mL; tenacissoside I: 10, 20, 50, 100, 200, 400, 600, 800 μg / mL; tenacissoside H: 10, 20, 50, 100, 200, 400, 600, 800 μg / mL.

[0066] After 24 hours of culture, a mixture of CCK-8 reagent and complete culture medium in a ratio of 1:10 was added to each well, and incubated in the dark. After incubation, the optical density (OD) value of the well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0067] As Figure 4As shown, it is the detection result of the survival rate of MCF-7 cells measured by CCK-8 (Note: compared with the blank group, *P<0.05, **P<0.01). It can be seen that when the concentrations of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid are ≥100 μg / mL, they can inhibit the viability of MCF-7 cells (P<0.05). When the concentration of tenacissoside I is ≥50 μg / mL, it can inhibit the viability of MCF-7 cells (P<0.05). When tenacissoside H ≥50 μg / mL, it can inhibit the viability of MCF-7 cells (P<0.05).

[0068] As Figure 5 shown, it is the detection result of the survival rate of HepG2 cells measured by CCK-8 (Note: compared with the blank group, *P<0.05, **P<0.01). It can be seen that when the concentration of chlorogenic acid is ≥10 μg / mL, it can inhibit the viability of HepG2 cells (P<0.05). Further, when the concentration of chlorogenic acid is ≥100 μg / mL, the inhibitory effect is more significant. And at the same concentration, the inhibitory effect of chlorogenic acid on the viability of HepG2 cells is more significant than that on the viability of MCF-7 cells. When neochlorogenic acid ≥50 μg / mL, it can inhibit the viability of HepG2 cells (P<0.05). When cryptochlorogenic acid ≥25 μg / mL, it can inhibit the viability of HepG2 cells (P<0.05). When the concentration of tenacissoside I is ≥100 μg / mL, it can inhibit the viability of HepG2 cells (P<0.05). When tenacissoside H ≥50 μg / mL, it can inhibit the viability of HepG2 cells (P<0.05).

[0069] The effects of the pharmacodynamic components on the MDA content, SOD enzyme activity, and GSH content of HepG2 and MCF-7 cells were detected by the oxidative stress factor kit through cell experiments, and the specific steps are as follows: Among them, since neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid are isomers, it is considered that the effects of the three on cells are basically the same. Therefore, the detection by the oxidative stress factor kit of chlorogenic acid can represent neochlorogenic acid and cryptochlorogenic acid.

[0070] The cells were randomly divided into a blank control group, a positive drug group, high- and low-concentration drug administration groups. The drug administration groups were the tenacissoside I group (600 μg / mL, 100 μg / mL), the tenacissoside H group (600 μg / mL, 100 μg / mL), the chlorogenic acid group (400 μg / mL, 100 μg / mL), and the positive drug doxorubicin group 0.25 μg / mL. After culturing for 24 h, the differences in MDA content, SOD enzyme activity, and GSH content among different groups were compared. SPSS 20.0 software was used for statistical analysis, and GraphPad Prism8.0 software was used for drawing. P<0.05 represents that the difference is statistically significant.

[0071] AsFigure 6 As shown, it is the influence of administering active ingredients at different concentrations on the MDA content of MCF-7 and HepG2 cells (Note: compared with the blank group, *P<0.05, **P<0.01). It can be seen that for chlorogenic acid, both chlorogenic acid at concentrations of 100 μg / mL and 400 μg / mL can reduce the MDA content of MCF-7 and HepG2 cells, and there are significant differences; among them, the reduction effect of chlorogenic acid at the higher concentration of 400 μg / mL is more significant. For tenacissoside I, tenacissoside I at the higher concentration of 600 μg / mL can reduce the MDA content of MCF-7 and HepG2 cells, and there are significant differences. For tenacissoside H, both tenacissoside H at concentrations of 100 μg / mL and 600 μg / mL can reduce the MDA content of MCF-7 and HepG2 cells, and there are significant differences; among them, the reduction effect of tenacissoside H at the higher concentration of 600 μg / mL is more significant.

[0072] As Figure 7 As shown, it is the influence of administering active ingredients at different concentrations on the SOD enzyme activity of HepG2 and MCF-7 cells (Note: compared with the blank group, *P<0.05, **P<0.01). It can be seen that for chlorogenic acid, both chlorogenic acid at concentrations of 100 μg / mL and 400 μg / mL can increase the SOD enzyme activity of HepG2 and MCF-7 cells, and there are significant differences; among them, the increase effect of chlorogenic acid at the concentration of 400 μg / mL is more significant. For tenacissoside I, tenacissoside I at the higher concentration of 600 μg / mL can increase the SOD enzyme activity of MCF-7 and HepG2 cells, and there are significant differences. For tenacissoside H, tenacissoside H at the higher concentration of 600 μg / mL can increase the SOD enzyme activity of MCF-7 and HepG2 cells, and there are significant differences.

[0073] As Figure 8As shown in the figure, it is the effect of administering active ingredients at different concentrations on the GSH content of MCF-7 and HepG2 cells (Note: Compared with the blank group, *P<0.05, **P<0.01). It can be seen that for chlorogenic acid, chlorogenic acid at concentrations of 100 μg / mL and 400 μg / mL can increase the GSH content of MCF-7 and HepG2 cells, and there are significant differences; among them, the improvement effect of chlorogenic acid at the higher concentration of 400 μg / mL is more significant. For tenacissoside I, tenacissoside I at a higher concentration of 600 μg / mL can increase the GSH content of MCF-7 and HepG2 cells, and there are significant differences. For tenacissoside H, tenacissoside H at concentrations of 100 μg / mL and 600 μg / mL can increase the GSH content of MCF-7 and HepG2 cells, and there are significant differences; among them, the improvement effect of tenacissoside H at the higher concentration of 600 μg / mL is more significant.

[0074] Example 2 A composition includes neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I and tenacissoside H. Administration concentration: 20 μg / mL neochlorogenic acid, 20 μg / mL chlorogenic acid, 20 μg / mL cryptochlorogenic acid, 20 μg / mL tenacissoside I and 20 μg / mL tenacissoside H, a total of 100 μg / mL.

[0075] Using the same method as in step S6 of Example 1, the effect of the composition of Example 2 on the proliferation of HepG2 and MCF-7 cells was verified by CCK-8 experiment.

[0076] The detection results and the data in Example 1 were integrated as shown in Table 2 below.

[0077] Table 2. As can be seen from Table 2, when using the same administration concentration of 100 μg / mL, in Example 2, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I and tenacissoside H were used simultaneously. Compared with using chlorogenic acid alone, neochlorogenic acid alone, cryptochlorogenic acid alone, tenacissoside I alone, and tenacissoside H alone in Example 1, there was a significant improvement in inhibiting the viability of MCF-7 and HepG2 cells. This shows that the active ingredients neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I and tenacissoside H in the composition coexist and act synergistically to act on liver cancer or / and breast cancer.

[0078] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art to which the present invention pertains may make various modifications or supplements to the described specific embodiments or use similar means for substitution, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A composition, characterized in that: It includes neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, marsdenoside I, and marsdenoside H.

2. Use of a composition as described in claim 1 in the preparation of a drug for treating liver cancer and / or breast cancer.

3. Use of a composition according to claim 2 in the preparation of a medicament for treating liver cancer and / or breast cancer, characterized in that: The composition is used in the preparation of a drug for treating liver cancer and / or breast cancer by at least affecting the PI3K-Akt signaling pathway, GnRH signaling pathway, and IL-17 signaling pathway.

4. Use of a composition according to claim 3 in the preparation of a medicament for treating liver cancer and / or breast cancer, characterized in that: The composition is used in the preparation of a drug for treating liver cancer and / or breast cancer by acting on at least the targets STAT3, PIK3CA, KDR, HSR90AA1, MET, PIK3CD, and PIK3CB.

5. Use of a composition according to claim 2 in the preparation of a medicament for treating liver cancer and / or breast cancer, characterized in that: The dosage of the composition is not less than 100 μg / mL.

6. Use of a composition according to claim 5 in the preparation of a medicament for treating liver cancer and / or breast cancer, characterized in that: The dosage ratio of phenolic acid compounds to glycoside compounds is 1:(0.5 - 5); the phenolic acid compounds are neochlorogenic acid or chlorogenic acid or cryptochlorogenic acid, and the glycoside compounds are marsdenoside I or marsdenoside H.

7. Use of a composition according to claim 6 in the preparation of a medicament for treating liver cancer and / or breast cancer, characterized in that: The dosage ratio of phenolic acid compounds to glycoside compounds is 1:(1 - 2).

8. A method for screening the composition as described in claim 1 from Marsdenia tenacissima Roxb. leaves tablets, characterized in that: It includes the following steps: S1. After administration, use UPLC-Q / TOF-MS technology to analyze the blood components in the administered plasma. S2. Use relevant databases of network pharmacology to collect the action targets corresponding to the blood components, construct the target library of Marsdenia tenacissima tablets; construct the intersection target library of liver cancer and breast cancer; take the intersection of the target library of Marsdenia tenacissima tablets and the intersection target library of liver cancer and breast cancer to obtain potential action targets. S3. Use the blood components and the potential action targets to construct a visualization network diagram of "blood components - targets - diseases" of Marsdenia tenacissima tablets, and obtain the main active components in the blood components according to network analysis. The main active components are the composition.

9. A method for screening a composition from Marsdenia tenacissima tablets according to claim 8, characterized in that: It also includes the following steps: S4. Establish a PPI network for the potential action targets to obtain key action targets.

10. A method for screening a composition from Marsdenia tenacissima tablets according to claim 8, characterized in that: It also includes the following steps: S5. Conduct GO enrichment analysis and KEGG pathway enrichment on the potential action targets to obtain signal pathways with high enrichment significance.

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