Use of astragalus-momordica pair active ingredient composition in preparation of anti-tumor metastasis drugs

Abstract

The application discloses application of a Chinese medicine pair effective component composition of Astragalus membranaceus and Curcuma zedoary in preparation of anti-tumor metastasis drugs. The composition is composed of calycosin-7-glucoside and curcumin. The composition can effectively inhibit transendothelial invasion and vasculogenic mimicry generation of tumor cells at a non-toxic dose, and has the advantages of high efficiency and low toxicity in treating tumor metastasis. The important point of the application is that the preferred composition can significantly reduce the exosome LAP-TGF-beta1 protein loading level, thereby greatly inhibiting the tumor metastasis process caused by the exosome-mediated TGF-beta signal pathway. The finding provides a new perspective for the development of drugs for targeting exosome LAP-TGF-beta1 protein loading to resist tumor metastasis, and overcomes the defects of unclear effective components, uncontrollable proportioning and unclear treatment effect of traditional Chinese medicine compatibility. Therefore, the Chinese medicine effective component composition has application prospects in preparation of anti-tumor metastasis drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and it relates to the composition of the effective components of the Astragalus-Curcuma zedoaria drug pair and its application in the preparation of anti-tumor metastasis drugs. Background Technology

[0002] Malignant tumors are among the most serious threats to human health. Metastasis is a characteristic biological behavior of malignant tumors and a major cause of treatment failure and patient death. Therefore, there is an urgent need to develop new targeted drugs that can effectively inhibit tumor metastasis.

[0003] Tumor metastasis is a complex and multi-stage process in which tumor cells or messengers containing tumor genetic information escape from the primary site to distant sites via fluid circulation. Aberrant activation of the TGF-β (Transforming Growth Factor-β) signaling pathway is closely related to tumor metastasis. TGF-β1 (Transforming Growth Factor-β1) is a major member of the TGF-β family and an important pathway for cell signaling, promoting various metastasis-related biological responses such as tumor cell invasion, epithelial-mesenchymal transition (EMT), and vascular mimicry. EMT and vascular mimicry, as two key events in tumor metastasis and invasion, interact through complex molecular mechanisms and signaling pathways, jointly promoting tumor spread and progression. Tumor-derived exosomal TGF-β1 has been widely reported for its role in promoting tumor EMT and vascular mimicry. The inactive form of this molecule, LAP-TGF-β1, is a protein dimer composed of a latency-related peptide (LAP) and an active TGF-β1 subunit. Exosomal LAP-TGF-β1 exhibits tumor specificity and promotes the amplification of the TGF-β signaling cascade. Therefore, targeting exosomal LAP-TGF-β1 protein provides a novel target for combating tumor metastasis.

[0004] In recent years, although various strategies for blocking TGF-β signaling have been developed and advanced clinically, their severe side effects have limited their clinical application. Most importantly, the presence of TGF-β in exosomes hinders the development of TGF-β inhibitors. Therefore, the development of highly effective, low-toxicity anti-tumor metastasis drugs that target exosomal LAP-TGF-β1 is of great significance.

[0005] my country's traditional Chinese medicine (TCM) resources are a treasure trove, providing a rich material basis for novel anti-tumor drugs. Discovering effective anti-tumor drugs from TCM has unique advantages and broad prospects. However, due to the lack of clarity regarding the components and mechanisms of traditional Chinese medicine and its compound preparations, difficulty in quality control, and the inability to evaluate clinical efficacy, the role of TCM in tumor treatment is constrained. The combination of effective components of TCM is an innovative strategy in traditional Chinese medicine formulation, offering advantages such as high safety, clear and targeted clinical indications, relatively clear components and mechanisms of action, and stable and controllable quality. Our previous research found that the combination of Astragalus membranaceus and Curcuma zedoaria safely and effectively inhibits tumor growth, especially significantly inhibiting tumor cell metastasis, which is of great significance in addressing the current lack of anti-tumor drugs. Therefore, research on the pharmacodynamic combination of Astragalus membranaceus and Curcuma zedoaria against tumor metastasis, and the development of drugs targeting exosomal LAP-TGF-β1 protein loading, has extremely important clinical significance. This research direction is expected to provide a new theoretical basis and practical guidance for the development of drugs for the treatment of tumor metastasis. Summary of the Invention

[0006] The purpose of this invention is to provide an application of the Astragalus-Curcuma zedoaria drug pair active ingredient composition in the preparation of anti-tumor metastasis drugs, and its drug development in targeting exosomal LAP-TGF-β1 protein loading.

[0007] Another objective of this invention is to provide a method for preparing tumor-derived exosomes that is simple, easy to operate, and low in cost.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A composition of active ingredients of the Astragalus-Curcuma zedoaria herbal pair, consisting of verbascoside and curcumin.

[0010] The molecular formulas of the verbascoflavonoid glycosides and curcumin are as follows: (I) and (II):

[0011]

[0012] As a preferred embodiment of the present invention, the molar ratio of verbascoflavonoid glycoside and curcumin is 1:(1-3), and the optimal ratio is 1:2.

[0013] The application of the Astragalus-Curcuma zedoaria drug pair active ingredient composition described in this invention in the preparation of drugs for inhibiting tumors and / or preventing tumor metastasis.

[0014] As a preferred embodiment of the present invention, the effective components of the Astragalus-Curcuma zedoaria drug pair inhibit the transendothelial invasion of tumor cells, thereby preventing the invasiveness of tumor cells to the surrounding environment and effectively curbing the growth and spread of tumors.

[0015] As a preferred embodiment of the present invention, the effective components of the Astragalus-Curcuma zedoaria drug pair reduce the dependence of tumor cells on blood supply by inhibiting the formation of angiogenesis in tumor cells, thereby effectively curbing the growth and spread of tumors.

[0016] As a preferred embodiment of the present invention, the effective components of the Astragalus-Curcuma zedoaria drug pair greatly inhibit the tumor metastasis process induced by the TGF-β signaling pathway by reducing the loading level of exosomal LAP-TGF-β1 protein.

[0017] As a preferred embodiment of the present invention, the tumor includes breast cancer, osteosarcoma, and melanoma; preferably breast cancer.

[0018] The beneficial effects of this invention are:

[0019] This invention discovers that the combination of the active ingredients, verrucoside and curcumin, of the Astragalus-Curcuma zedoaria pair can synergistically inhibit the loading of LAP-TGF-β1 protein on tumor cell exosomal tissues and significantly inhibit transendothelial invasion and angiogenesis during tumor metastasis. This discovery contributes to the development of compositions with well-defined components, good efficacy, and a safe, non-toxic approach to replace the Astragalus-Curcuma zedoaria pair as adjuvant therapy for anti-tumor metastasis. Its anti-tumor metastasis target is clearly defined; it inhibits the amplification effect of the tumor metastasis-related TGF-β signaling pathway by downregulating the loading of LAP-TGF-β1 protein on exosomal tissues. This has significant implications for the prevention of malignant tumors and shows broad promise in the preparation of drugs for treating tumor metastasis. Attached Figure Description

[0020] Figure 1 Effects of verbascoside and curcumin on LAP-TGF-β1 protein loading in breast cancer cell exosomes;

[0021] Figure 2 Effects of different doses of the optimal ratio of verbascoflavonoids and curcumin (1:2) on transendothelial invasion of breast cancer MDA-MB-231 cells;

[0022] Figure 3 Effects of different doses of the optimal ratio of verbascoflavonoids and curcumin (1:2) on transendothelial invasion of osteosarcoma HOS cells;

[0023] Figure 4 Effects of different doses of the optimal ratio of verbascoflavonoids and curcumin (1:2) on transendothelial invasion of melanoma A-375 cells;

[0024] Figure 5 Effects of different doses of the optimal ratio of verbascoflavonoids and curcumin (1:2) on tubule formation in MDA-MB-231 breast cancer cells;

[0025] Figure 6 Effects of different doses of the optimal ratio of verbascoflavonoids and curcumin (1:2) on tubule formation in osteosarcoma HOS cells;

[0026] Figure 7 Effects of different doses of the optimal ratio of verbascoflavonoids and curcumin (1:2) on tubule formation in melanoma A-375 cells;

[0027] Figure 8 Verbena isoflavone glycosides and curcumin synergistically inhibit the growth of breast tumors;

[0028] Figure 9 Verbena isoflavone glycosides and curcumin synergistically inhibit lung metastasis of breast cancer;

[0029] Figure 10 Verbena isoflavone glycosides and curcumin synergistically inhibit osteosarcoma growth;

[0030] Figure 11 Verbena isoflavone glycosides and curcumin synergistically inhibit osteosarcoma lung metastasis;

[0031] Figure 12 Verbena isoflavone glycosides and curcumin synergistically inhibit melanoma growth;

[0032] Figure 13 Verbena isoflavone glycosides and curcumin synergistically inhibit lung metastasis of melanoma. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. The present invention will be further described in detail below with reference to embodiments, but those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Moreover, those skilled in the art can make equivalent substitutions, combinations or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0034] Example 1: Effects of the effective components of Astragalus-Curcuma zedoaria on the loading of LAP-TGF-β1 protein in breast cancer cell exosomes.

[0035] 1. Isolation of tumor cell exosomes

[0036] MDA-MB-231 breast cancer cells were treated with different concentrations of verrucoside (CG) and curcumin (CU) alone or in combination for 24 h. The cells were cultured in 1640 medium containing 10% fetal bovine serum, with a confluence of 80-90%. After washing twice with PBS, the culture medium was replaced with serum-free exosome medium. The cells were cultured for 48 h, and the supernatant was collected. Exosomes were then separated using differential centrifugation.

[0037] a. Centrifuge for the first time (2000g, 10min) and collect the supernatant.

[0038] b. Centrifuge a second time (10000g, 30min) and collect the supernatant again.

[0039] c. Centrifuge a third time (110000g, 70min), collect the precipitate, and resuspend it in PBS.

[0040] d. Centrifuge for the fourth time (110000g, 70min), collect and resuspend the exosomes in an appropriate amount of PBS.

[0041] 2. Purification of exosomes

[0042] First, dilute a 50% iodixanol solution with 0.85% NaCl and 10 mM Tris-HCl (pH 7.4) to prepare iodixanol solutions of varying concentrations: 50% (w / v), 40% (w / v), and 10% (w / v). Resuspend the exocrine secretions in 3.8 mL of 50% (w / v) iodixanol solution and transfer to a 10.8 mL Beckman centrifuge tube. Then, add 3 mL of 40% (w / v) solution and 2.5 mL of 10% (w / v) solution to the centrifuge tube. Centrifuge at ultrahigh speed (4°C, 200,000 g, 2 h). Divide the solution in the centrifuge tube into 10 fractions from top to bottom. Determine the density (g / mL) by weighing each fraction. Resuspend all 10 fractions in PBS and centrifuge at ultrahigh speed (4°C, 110,000 g, 70 min). The precipitate is resuspended in RIPA buffer for further experiments or stored at -80°C.

[0043] 3. Quantitative analysis of exosomes

[0044] The number and size distribution of exosomes were determined using an NTA nanoparticle size analyzer. Purified exosomes were filtered through a 0.22 μm filter and diluted 1000 to 2000 times with PBS before analysis; each assay was performed three times. Data were viewed using the software included with the NTA instrument.

[0045] 4. ELISA detection of LAP-TGF-β1 content in exosomes

[0046] The extracted tumor cell exosomes were diluted with PBS to a concentration of 1x10⁻⁶. 10 The expression level of LAP-TGF-β1 in exosomes was detected using an ELISA kit (#DB100B, R&D, USA) at a particle density of 1 mL. The procedure is as follows:

[0047] a. Mix the sample, take 100 μL into a 1.5 mL EP tube, add 20 μL of 1N HCl, mix well, and incubate at room temperature for 10 min.

[0048] b. Add 20 μL of 1.2N NaOH / 0.5M HEPES to neutralize the acidified sample, mix well, and incubate at room temperature for 10 min. c. Remove the microplate after equilibration to room temperature and add 50 μL of RD1-21 or RD1-73 dilution buffer to each well. d. Add 50 μL of standards and samples of different concentrations (1500 pg / mL standard as high standard, calibrated by dilution).

[0049] Liquid RD6-31 (diluted 1:2) was used as the zero standard (0 pg / mL). The sample was sealed with sealing gel and incubated at room temperature.

[0050] 2h.

[0051] e. Discard the liquid in the plate, add 400 μL of washing buffer to each well, repeat 4 times, and finally aspirate the remaining liquid. f. Add 100 μL of LAP-TGF-β1 binding antibody to each well, seal with sealing glue, and incubate at room temperature for 2 hours.

[0052] g. Add 100 μL of chromogenic substrate (equal volumes of reagents A and B) to each well, seal the plate, and incubate at room temperature in the dark.

[0053] Incubate for 30 minutes.

[0054] h. Add 100 μL of stop solution to each well.

[0055] i. Measure the absorbance at 450 nm and 540 nm using an ELISA reader within 30 min.

[0056] The results are as follows Figure 1 As shown, compared with the control group and the single-drug group, the combination of verrucoside and curcumin (CG-CU) significantly reduced the loading level of LAP-TGF-β1 protein in breast cancer cell exosomes, thereby inhibiting the tumor metastasis process induced by the TGF-β signaling pathway to a greater extent.

[0057] Example 2: Effect of the synergistic effect of verbascoside and curcumin on LAP-TGF-β1 protein loading in breast cancer cell exosomes

[0058] Logarithmic growth phase MDA-MB-231 cells were seeded into 96-well plates. After 24 hours, once the cells had fully adhered, different ratios of vermouthroside (CG) and curcumin (CU) were added, and the cells were cultured for another 24 hours. The concentrations of CG and CU at different ratios are shown below:

[0059] Table 2-1 Concentration settings for a CG to CU molar ratio of 1:1

[0060]

[0061] Table 2-2 Concentration settings for a CG to CU molar ratio of 1:2

[0062]

[0063] Table 2-3 shows the concentration settings for a CG to CU molar ratio of 1:3.

[0064]

[0065] After the drug action period ended, the exosomal LAP-TGF-β1 content was detected using an ELISA kit, and then the combination index (CI) of CG and CU was calculated using CompuSyn software.

[0066] When CI < 1, it indicates that the two drugs have a synergistic effect when used together; when CI = 1, it indicates that the two drugs have an additive effect when used together; when CI > 1, it indicates that the two drugs have an antagonistic effect when used together.

[0067] Table 2-4 Analyzing the synergistic or antagonistic effects of drug combinations using CI equations

[0068]

[0069] Table 2-5 Effects of different CG and CU ratios on CI values ​​of MDA-MB-231 cells

[0070]

[0071] The results are shown in Tables 2-5. Verbena isoflavone glycoside and curcumin exhibit synergistic effects when combined in ratios of 1:1, 1:2, and 1:3. Among these, CG-CU (1:2) has the lowest synergistic index. Therefore, the synergistic effect is strongest when verbena isoflavone glycoside and curcumin are combined in a molar ratio of 1:2, which significantly reduces the loading level of LAP-TGF-β1 protein in breast cancer cell exosomes and can be considered the optimal ratio.

[0072] Example 3: Effect of the synergistic effect of verrucoside and curcumin on LAP-TGF-β1 protein loading in osteosarcoma cell exosomes

[0073] The concentration settings for different ratios of CG and CU are consistent with those in Example 2.

[0074] Osteosarcoma HOS cells in logarithmic growth phase were seeded into 96-well plates. After 24 hours, once the cells had fully adhered, different ratios of verrucoside (CG) and curcumin (CU) were added, and the cells were cultured for another 24 hours. After the drug treatment period, the exosomal LAP-TGF-β1 content was detected using an ELISA kit, and the combined index (CI) of CG and CU was calculated using CompuSyn software.

[0075] Table 3-1 Effects of different CG and CU ratios on CI values ​​of HOS cells

[0076]

[0077] The results are shown in Table 3-1. Verbena isoflavone glycoside and curcumin exhibit synergistic effects when combined in ratios of 1:1, 1:2, and 1:3. Among these, CG-CU (1:2) has the lowest synergistic index. Therefore, the synergistic effect is strongest when verbena isoflavone glycoside and curcumin are combined in a molar ratio of 1:2, which significantly reduces the loading level of LAP-TGF-β1 protein in osteosarcoma cell exosomes and can be considered the optimal ratio.

[0078] Example 4: Effect of the synergistic effect of verrucoside and curcumin on the loading of LAP-TGF-β1 protein in melanoma cell exosomes

[0079] The concentration settings for different ratios of CG and CU are consistent with those in Example 2.

[0080] A-375 cells in logarithmic growth phase were seeded into 96-well plates. After 24 hours, once the cells had fully adhered, different ratios of verrucoside (CG) and curcumin (CU) were added, and the cells were cultured for another 24 hours. After the drug treatment period, the exosomal LAP-TGF-β1 content was detected using an ELISA kit, and the combined index (CI) of CG and CU was calculated using CompuSyn software.

[0081] Table 4-1 Effects of different CG and CU ratios on CI values ​​of B16-F10 cells

[0082]

[0083] The results are shown in Table 4-1. Verbena isoflavone glycoside and curcumin exhibit synergistic effects when combined in ratios of 1:1, 1:2, and 1:3. Among these, the synergistic index is the smallest when CG:CU = 1:2. Therefore, the synergistic effect is strongest when verbena isoflavone glycoside and curcumin are combined in a molar ratio of 1:2, which significantly reduces the loading level of LAP-TGF-β1 protein in melanoma cell exosomes and can be considered the optimal ratio.

[0084] Example 5: Effects of different doses of the optimal ratio (1:2) of the effective components of the Astragalus-Curcuma zedoaria pair, verbascoside and curcumin, on transendothelial invasion of breast cancer cells.

[0085] The CG-CU (1:2) was tested in three dosage groups: low-dose group: CG and CU were 62.5 nM and 125 nM, respectively; medium-dose group: CG and CU were 125 nM and 250 nM, respectively; high-dose group: CG and CU were 250 nM and 500 nM, respectively. The dosages in the following examples are the same as in this example.

[0086] HUVEC cells in the logarithmic growth phase were digested with EDTA trypsin and then subjected to 2×10⁻⁶ ppm.4 Cells were seeded at a density equal to the number of wells in Transwell chambers and incubated overnight. MDA-MB-231 cells were pretreated with different concentrations of CG-CU (1:2) at the optimal ratio for 24 hours, then seeded into 6-well plates. After trypsin digestion, single-cell suspensions were prepared, counted, and cultured at 5 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 100 / well on the upper layer of HUVEC cells. 750 μL of complete culture medium was added to the lower chamber. After 12–16 h, the Transwell chamber was removed, the culture medium was aspirated, and the cells were rinsed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 20–30 min, rinsed three times with PBS, and any cells that had not penetrated the chamber were gently wiped with a cotton swab. After air-drying, the cells were observed under an inverted microscope, and five randomly selected areas were photographed to count the number of invading cells.

[0087] The results are as follows Figure 2 As shown, the optimal combination of verbascoside and curcumin (1:2) reduced the number of transendothelial cells in MDA-MB-231 cells in a dose-dependent manner, indicating that the optimal combination of verbascoside and curcumin can inhibit the transendothelial invasion ability of breast cancer cells in a dose-dependent manner.

[0088] Example 6: Effects of different doses of the optimal ratio (1:2) of the effective components of the Astragalus-Curcuma herb pair, verbascoside and curcumin, on transendothelial invasion of osteosarcoma cells.

[0089] HUVEC cells in the logarithmic growth phase were digested with EDTA trypsin and then subjected to 2×10⁻⁶ ppm. 4 Cells were seeded at a density of 4 × 10⁻⁶ cells / well in Transwell chambers and incubated overnight. HOS cells were pretreated with different concentrations of CG-CU (1:2) at the optimal ratio for 24 hours and then seeded into 6-well plates. After trypsin digestion, single-cell suspensions were prepared, counted, and cultured at 4 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 100 / well on the upper layer of HUVEC cells. 750 μL of complete culture medium was added to the lower chamber. After 12–16 h, the Transwell chamber was removed, the culture medium was aspirated, and the cells were rinsed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 20–30 min, rinsed three times with PBS, and any cells that had not penetrated the chamber were gently wiped with a cotton swab. After air-drying, the cells were observed under an inverted microscope, and five randomly selected areas were photographed to count the number of invading cells.

[0090] The results are as follows Figure 3 As shown, the optimal combination of verbascoside and curcumin (1:2) reduced the number of transendothelial cells in HOS cells in a dose-dependent manner, indicating that the optimal combination of verbascoside and curcumin can inhibit the transendothelial invasion ability of osteosarcoma cells in a dose-dependent manner.

[0091] Example 7: Effects of different doses of the optimal ratio (1:2) of the effective components of the Astragalus-Curcuma herb pair, verbascoside and curcumin, on the transendothelial invasion of melanoma cells.

[0092] HUVEC cells in the logarithmic growth phase were digested with EDTA trypsin and then subjected to 2×10⁻⁶ ppm. 4 Cells were seeded at a density equal to the number of wells in Transwell chambers and incubated overnight. A-375 cells were pretreated with different concentrations of CG-CU (1:2) at the optimal ratio for 24 hours, then seeded into 6-well plates. After trypsin digestion, single-cell suspensions were prepared, counted, and cultured at 5 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 100 / well on the upper layer of HUVEC cells. 750 μL of complete culture medium was added to the lower chamber. After 12–16 h, the Transwell chamber was removed, the culture medium was aspirated, and the cells were rinsed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 20–30 min, rinsed three times with PBS, and any cells that had not penetrated the chamber were gently wiped with a cotton swab. After air-drying, the cells were observed under an inverted microscope, and five randomly selected areas were photographed to count the number of invading cells.

[0093] The results are as follows Figure 4 As shown, the optimal combination of verbascoside and curcumin (1:2) reduced the number of transendothelial cells in A-375 cells in a dose-dependent manner, indicating that the optimal combination of verbascoside and curcumin can inhibit the transendothelial invasive ability of melanoma cells in a dose-dependent manner.

[0094] Example 8: Effects of different doses of the optimal ratio (1:2) of the effective components of the Astragalus-Curcuma zedoaria pair, verbascoside and curcumin, on tubule formation in breast cancer cells.

[0095] Place the matrix gel in an ice box to allow it to melt naturally into a liquid state, then spread it evenly onto angiogenesis slides. Add 10 μL of matrix gel (preferably bubble-free and uniform) to each well, and incubate at 37°C in a cell culture incubator containing 5% CO2 and 95% air for 30 min to allow it to solidify. Pretreat MDA-MB-231 cells for 24 h with different concentrations of CG-CU (1:2) mixture, digest with trypsin, resuspend in DMEM medium containing 1% fetal bovine serum, and take 50 μL of the cell suspension and spread it onto the solidified matrix gel surface (2.0 × 10⁻⁶). 4 Cells / well). Incubate in a cell culture incubator for 2–8 h, observe under an inverted microscope, and photograph 5 randomly selected areas. Quantitative analysis was performed using ImageJ software to assess the degree of in vitro vascular mimicry formation, including the total number of nodes, total number of connection points, total number of grids, and tube length.

[0096] The results are as follows Figure 5As shown, the optimal combination of verbenafiloside and curcumin (1:2) can reduce the total number of nodes, total number of connection points, total number of grids and tube length of tubule formation in MDA-MB-231 cells in a dose-dependent manner, indicating that the optimal combination of verbenafiloside and curcumin can significantly inhibit angiogenesis in breast cancer cells.

[0097] Example 9: Effects of different doses of the optimal ratio (1:2) of the effective components of the Astragalus-Curcuma drug pair, verbascoside and curcumin, on tubule formation in osteosarcoma cells.

[0098] Place the matrix gel in an ice box to allow it to melt naturally into a liquid state, then spread it evenly onto angiogenesis slides. Add 10 μL of matrix gel (preferably bubble-free and uniform) to each well, and incubate at 37°C in a cell culture incubator containing 5% CO2 and 95% air for 30 min to allow it to solidify. Pretreat HOS cells for 24 h with different concentrations of CG-CU (1:2) mixture, digest with trypsin, resuspend in DMEM medium containing 1% fetal bovine serum, and take 50 μL of the cell suspension and spread it onto the solidified matrix gel surface (2.0 × 10⁻⁶). 4 Cells / well). Incubate in a cell culture incubator for 2–8 h, observe under an inverted microscope, and photograph 5 randomly selected areas. Quantitative analysis was performed using ImageJ software to assess the degree of in vitro vascular mimicry formation, including the total number of nodes, total number of connection points, total number of grids, and tube length.

[0099] The results are as follows Figure 6 As shown, the optimal combination of verbascoside and curcumin (1:2) can reduce the total number of nodes, total number of connections, total number of grids and tube length of tubule formation in HOS cells in a dose-dependent manner, indicating that the optimal combination of verbascoside and curcumin can significantly inhibit angiogenesis in osteosarcoma cells.

[0100] Example 10: Effects of different doses of the optimal ratio (1:2) of the effective components of the Astragalus-Curcuma herb pair, verbascoside and curcumin, on melanoma cell tubule formation.

[0101] Place the matrix gel in an ice box to allow it to melt naturally into a liquid state, then spread it evenly onto angiogenesis slides. Add 10 μL of matrix gel (preferably bubble-free and uniform) to each well, and incubate at 37°C in a cell culture incubator containing 5% CO2 and 95% air for 30 min to allow it to solidify. Pretreat A-375 cells for 24 h with different concentrations of CG-CU (1:2) mixture, digest with trypsin, resuspend in DMEM medium containing 1% fetal bovine serum, and take 50 μL of the cell suspension and spread it onto the solidified matrix gel surface (1.5 × 10⁻⁶). 4Cells / well). Incubate in a cell culture incubator for 2–8 h, observe under an inverted microscope, and photograph 5 randomly selected areas. Quantitative analysis was performed using ImageJ software to assess the degree of in vitro vascular mimicry formation, including the total number of nodes, total number of connection points, total number of grids, and tube length.

[0102] The results are as follows Figure 7 As shown, the optimal combination of verbenafiloside and curcumin (1:2) can reduce the total number of nodes, total number of connections, total number of grids and tube length of tubule formation in A-375 cells in a dose-dependent manner, indicating that the optimal combination of verbenafiloside and curcumin can significantly inhibit angiogenesis in melanoma cells.

[0103] Example 11: The synergistic effect of the effective components of the Astragalus-Curcuma zedoaria pair, verbascoside and curcumin, on breast cancer metastasis.

[0104] To investigate the synergistic effect of the effective components of the Astragalus-Curcuma zedoaria pair, verrucoside and curcumin, on breast cancer metastasis, passaged breast cancer MDA-MB-231 cells were seeded into the mammary glands of 7-8 week old female NCG-HLA-A2.1 mice. Mice were randomly divided into 6 groups: control group, verrucoside group (CG), curcumin (CU), verrucoside and curcumin (1:1) group (CG-CU(1:1)), verrucoside and curcumin (1:2) group (CG-CU(1:2)), and verrucoside and curcumin (1:3) group (CG-CU(1:3)). Based on the clinical usage of 30g and 15g of Astragalus and Curcuma zedoaria, respectively, and considering our previous analysis of the verrucoside and curcumin contents in the aqueous extract of the Astragalus-Curcuma zedoaria pair (0.79% and 0.81%, respectively), the results were analyzed. Referring to the human-to-mouse dose conversion method provided in the methodology for pharmacological research of traditional Chinese medicine, the final dosages were 0.0395 g / kg and 0.0243 g / kg for the isoflavone glycoside monotherapy group and the curcumin monotherapy group, respectively; and 0.079, 0.1185, and 0.158 g / kg for the isoflavone glycoside and curcumin (1:1, 1:2, and 1:3) combinations, respectively. From day 2 after tumor inoculation to the end of the experiment, mice in each group were administered the drugs by gavage once daily at a fixed time for 27 consecutive days; the control group was simultaneously administered an equal volume of physiological saline by gavage once daily for 27 consecutive days. Tumor weight analysis showed that as the ratio of isoflavone glycoside to curcumin increased, the tumor burden in mice significantly decreased. However, when the ratio of isoflavone glycoside to curcumin exceeded 1:2, the inhibitory effect was not improved. Therefore, the tumor-inhibiting effect was optimal when the ratio of isoflavone glycoside to curcumin was 1:2. Figure 8 Furthermore, H&E staining of lung tissue showed that a ratio of verbascoside to curcumin of 1:2 significantly reduced the incidence of lung metastases from breast cancer. Figure 9 ).

[0105] Example 12: The synergistic effect of the effective components of the Astragalus-Curcuma herb pair, verbascoside and curcumin, on osteosarcoma metastasis.

[0106] In this embodiment, the dosages of verbascoside and curcumin are the same as in Example 11.

[0107] To investigate the synergistic effect of the Astragalus-Curcuma synergistic combination of its active components, verrucoside and curcumin, on osteosarcoma metastasis, passaged osteosarcoma HOS cells were injected orally into the tibia of 6-8 week old male NCG-HLA-A2.1 mice. Mice were randomly divided into six groups: control group, verrucoside group (CG), curcumin (CU), verrucoside and curcumin (1:1) group (CG-CU(1:1)), verrucoside and curcumin (1:2) group (CG-CU(1:2)), and verrucoside and curcumin (1:3) group (CG-CU(1:3)). From day 2 after tumor inoculation until the end of the experiment, all groups received the drugs via gavage once daily for 27 consecutive days; the control group received an equal volume of physiological saline via gavage once daily for 27 consecutive days. Tumor weight analysis results showed that the tumor burden of mice treated with the combination of verbenafil and curcumin was significantly reduced, with a 1:2 ratio of verbenafil to curcumin significantly reducing tumor weight. Figure 10 H&E staining of lung tissue showed that a ratio of versicolor isoflavone glycoside to curcumin of 1:2 significantly reduced the incidence of osteosarcoma lung metastasis. Figure 11 ).

[0108] Example 13: The synergistic effect of the effective components of the Astragalus-Curcuma herbal pair, verbascoside and curcumin, on melanoma metastasis.

[0109] In this embodiment, the dosages of verbascoside and curcumin are the same as in Example 11.

[0110] To investigate the synergistic effect of the effective components of Astragalus membranaceus-Curcuma zedoaria pair, verrucoside and curcumin, on melanoma metastasis, animals were divided into 6 groups: control group, verrucoside group (CG), curcumin (CU), verrucoside and curcumin (1:1) group (CG-CU (1:1)), verrucoside and curcumin (1:2) group (CG-CU (1:2)), and verrucoside and curcumin (1:3) group (CG-CU (1:3)). Passaged human malignant melanoma A-375 cells were injected subcutaneously into 6-8 week old male NCG-HLA-A2.1 mice. From day 2 after tumor inoculation until the end of the experiment, each group received the drug via gavage once daily for 27 consecutive days; the control group received an equal volume of physiological saline via gavage once daily for 27 consecutive days. Tumor weight analysis results showed that the tumor burden of mice treated with the combination of verbenafil and curcumin was significantly reduced, with a 1:2 ratio of verbenafil to curcumin significantly reducing tumor weight. Figure 12 H&E staining of lung tissue showed that a 1:2 ratio of versicolor isoflavone glycosides to curcumin significantly reduced the incidence of lung metastases from melanoma. Figure 13 ).

Claims

1. The application of the Astragalus-Curcuma zedoaria herbal pair active ingredient composition in the preparation of drugs for inhibiting tumors and / or preventing tumor metastasis, characterized in that, The tumor is selected from breast cancer, osteosarcoma, or melanoma, and the composition consists of verrucoside and curcumin in a molar ratio of 1:2.

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