Application of active ingredient composition of astragalus-curcuma zedoary drug pair in preparation of anti-tumor metastasis drug

Abstract

The invention discloses an application of an active ingredient composition of an astragalus-curcuma zedoary drug pair in preparation of an anti-tumor metastasis drug. The composition is prepared from calycosin glycoside and curcumin. The composition can effectively inhibit trans-endothelial invasion and vascular mimicry generation of tumor cells under the non-toxic dosage, and has the advantages of high efficiency and low toxicity in treatment of tumor metastasis. The important point of the invention is that the preferable composition is found to be capable of remarkably reducing the loading level of exosome LAP-TGF-beta 1 protein, so that the tumor metastasis process caused by an exosome mediated TGF-beta signal channel is greatly inhibited. The discovery provides a new perspective for the development of targeting exosome LAP-TGF-beta1 protein loaded anti-tumor metastasis drugs, and overcomes the defects of unclear treatment effect caused by unclear compatibility of effective components and uncontrollable ratio of traditional Chinese medicines. Therefore, the traditional Chinese medicine active ingredient composition has an application prospect in preparation of anti-tumor metastasis drugs.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and relates to an effective component composition of an astragalus-curcuma zedoaria drug pair and application thereof in preparing an anti-tumor metastasis drug. Background Art

[0002] Malignant tumors are one of the diseases that seriously threaten human health. Metastasis is a characteristic biological behavior of malignant tumors and is also the main cause of treatment failure and patient death. Therefore, it is urgent to develop new target 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 the distal site through fluid circulation. Among them, abnormal activation of the TGF-β (Transforming growth factor-β, TGF-β) signaling pathway is closely related to tumor metastasis. TGF-β1 (Transforming growth factor-β1, TGF-β1) is one of the main members of the TGF-β family and an important pathway for cell signal transduction. It can promote tumor cell invasion, epithelial-mesenchymal transition, vascular mimicry and other metastasis-related biological reactions. Epithelial-mesenchymal transition and vascular mimicry are two key events in tumor metastasis and invasion. They interact through complex molecular mechanisms and signaling pathways to jointly promote the spread and deterioration of tumors. Tumor-derived exosomal TGF-β1 has been widely reported for its role in promoting tumor epithelial-mesenchymal transition and vascular mimicry. The inactive form of this molecule, LAP-TGF-β1, is a protein dimer composed of latency-associated peptide (LAP) and TGF-β1 active subunits. Exosomal LAP-TGF-β1 is tumor-specific and promotes the TGF-β signaling cascade. Therefore, targeting exosomal LAP-TGF-β1 protein provides a new target for combating tumor metastasis.

[0004] In recent years, although a variety of strategies to block TGF-β signaling have been developed and advanced into clinical practice, their severe side effects limit their clinical application. Most importantly, the presence of TGF-β in exosomes has hampered the progress of TGF-β blockers. Therefore, it is of great significance to develop highly effective, low-toxic anti-tumor metastasis drugs that target exosomal LAP-TGF-β1.

[0005] my country's traditional Chinese medicine resources are a treasure trove, providing a rich material basis for new anti-tumor drugs. Discovering effective anti-tumor drugs from traditional Chinese medicine has unique advantages and broad prospects. However, due to the many shortcomings and limitations of traditional Chinese medicine and compound prescriptions, such as unknown ingredients, unclear mechanisms, difficult quality control, and inability to evaluate clinical efficacy, the role of traditional Chinese medicine in tumor treatment is restricted. The combination of effective ingredients of traditional Chinese medicine is an innovative strategy for the combination of traditional Chinese medicine, which has the advantages of high safety, clear clinical indications and good pertinence, relatively clear ingredients and mechanisms of action, and stable and controllable quality. In our previous studies, we found that the combination of Astragalus and Curcuma has the advantages of safe and effective inhibition of tumor growth, especially the significant inhibition of tumor cell metastasis, which is of great significance to make up for the current lack of anti-tumor drugs. Therefore, it is of great clinical significance to study the pharmacodynamic combination of Astragalus and Curcuma against tumor metastasis and to develop drugs targeting exosome LAP-TGF-β1 protein loading. This research direction is expected to provide a new theoretical basis and practical guidance for the development of therapeutic drugs for tumor metastasis. Summary of the invention

[0006] The present invention aims to provide an application of an Astragalus-Erdoblast drug pair effective ingredient composition in the preparation of an anti-tumor metastasis drug, and the drug development thereof in terms of targeted exosome LAP-TGF-β1 protein loading.

[0007] Another object of the present invention is to provide a method for preparing tumor-derived exosomes, which is simple, easy to operate and low in cost.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] The invention discloses an effective component composition of astragalus and zedoaria medicinal pair, which consists of calycosin isoflavone glycosides and curcumin.

[0010] The molecular structures of the calycosin isoflavone glycosides and curcumin are as follows (I) and (II):

[0011]

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

[0013] The invention discloses an application of the effective component composition of the Astragalus membranaceus-Zhuozhu medicinal pair in the preparation of a drug for inhibiting tumors and / or resisting tumor metastasis.

[0014] As a preferred embodiment of the present invention, the effective components of the Astragalus-Ezekield 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-Ezekield drug pair inhibit the formation of vascular mimicry in tumor cells, thereby reducing the dependence of tumor cells on blood supply and effectively curbing the growth and spread of tumors.

[0016] As a preferred embodiment of the present invention, the effective components of the Astragalus-Ezekield drug pair reduce the exosome LAP-TGF-β1 protein loading level, thereby greatly preventing the tumor metastasis process induced by the TGF-β signaling pathway.

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

[0018] Beneficial effects of the present invention:

[0019] The present invention found that the combination of the effective ingredients of the Astragalus-Curcuma drug pair, calycosin and curcumin, can synergistically inhibit the loading of LAP-TGF-β1 protein in tumor cell exosomes, and significantly inhibit transendothelial invasion and vascular mimicry during tumor metastasis. This discovery is helpful to develop a composition with clear ingredients, good efficacy, safety and no toxic side effects to replace the Astragalus-Curcuma drug pair for auxiliary treatment of anti-tumor metastasis. Its anti-tumor metastasis target is clear, and it inhibits the amplification effect of tumor metastasis-related TGF-β signaling pathway conduction by downregulating the loading of exosome LAP-TGF-β1 protein. It is of great significance for the prevention of malignant tumors and has broad prospects in the preparation of drugs for the treatment of tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 Effects of different doses of the optimal ratio of calycosin 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 calycosin and curcumin (1:2) on transendothelial invasion of osteosarcoma HOS cells;

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

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

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

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

[0027] Figure 8 Calycosin and curcumin synergistically inhibit breast tumor growth;

[0028] Fig. 9 Calycosin and curcumin synergistically inhibit breast cancer lung metastasis;

[0029] Fig.10 Calycosin and curcumin synergistically inhibit the growth of osteosarcoma;

[0030] Fig.11 Calycosin and curcumin synergistically inhibit lung metastasis of osteosarcoma;

[0031] Fig.12 Calycosin and curcumin synergistically inhibit melanoma growth;

[0032] Fig.13 Calycosin and curcumin synergistically inhibit melanoma lung metastasis. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below in conjunction with examples, but embodiments of the present invention are not limited thereto. The present invention will be further described in detail below with reference to examples, but it should be appreciated by those skilled in the art that the present invention is not limited to these examples and the preparation method used. Moreover, those skilled in the art can carry out equivalent replacement, combination or improvement to the present invention according to 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 active components of the Astragalus-Erdoblastus drug pair, calycosin and curcumin, on the LAP-TGF-β1 protein loading of exosomes of breast cancer cells

[0035] 1. Isolation of Tumor Cell Exosomes

[0036] Different concentrations of calycosin (CG) and curcumin (CU) were treated alone or in combination with MDA-MB-231 breast cancer cells (cultured in 1640 medium containing 10% fetal bovine serum, with a confluence of 80-90%) for 24 hours, then washed twice with PBS and replaced with medium without exosome serum. After 48 hours of culture, the supernatant of the cells was collected and exosomes were separated by differential centrifugation:

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

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

[0039] c. Centrifuge for the third time (110,000 g, 70 min), collect the precipitate, and resuspend it in PBS.

[0040] d. Centrifuge for the fourth time (110,000 g, 70 min), collect and resuspend the exosomes in an appropriate amount of PBS.

[0041] 2. Purification of Exosomes

[0042] First, 50% iodixanol solution was diluted with 0.85% NaCl and 10mM Tris-HCl (pH 7.4) to prepare a certain concentration gradient of iodixanol solution, namely 50% (w / v), 40% (w / v) and 10% (w / v). The exosomes were resuspended in 3.8mL of 50% (w / v) iodixanol solution and then transferred to a Beckman centrifuge tube with a total volume of 10.8mL, and then 3mL of 40% (w / v) solution and 2.5mL of 10% (w / v) solution were added to the centrifuge tube in sequence. Ultrahigh-speed centrifugation (4°C, 200000g, 2h). The solution in the centrifuge tube was divided into 10 parts from top to bottom, and the density (g / mL) was determined by weighing the mass of each part. The 10 parts were resuspended with PBS and ultrahigh-speed centrifugation (4°C, 110000g, 70min). The pellet was resuspended in RIPA buffer for the next experiment or stored at -80°C.

[0043] 3. Quantification of Exosomes

[0044] The number and size distribution of exosomes were determined using the NTA nanoparticle size analyzer. The purified exosomes were filtered through a 0.22 μm filter and diluted 1000 to 2000 times with PBS for detection. Each determination was performed 3 times. The 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 10 Particles / mL, and the expression of LAP-TGF-β1 in exosomes was detected using an ELISA kit (#DB100B, R&D, USA). The operation steps are as follows:

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

[0048] b. Add 20 μL 1.2N NaOH / 0.5M HEPES to neutralize the acidified sample, mix well, and incubate at room temperature for 10 minutes. c. Take out the microplate that has been equilibrated to room temperature and add 50 μL of RD1-21 or RD1-73 dilution to each well. d. Add 50 μL of different concentrations of standards and samples (1500 pg / mL standard as high standard, calibration dilution

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

[0050] 2h.

[0051] e. Discard the liquid in the plate, add 400 μL of washing solution to each well, repeat 4 times, and finally absorb the residual 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 color substrate (mix 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 enzyme 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 calycosin and curcumin (CG-CU) significantly reduced the exosome LAP-TGF-β1 protein loading level of breast cancer cells, thereby preventing the tumor metastasis process induced by the TGF-β signaling pathway to a greater extent.

[0057] Example 2 Effect of synergistic effects of calycosin and curcumin on LAP-TGF-β1 protein loading in exosomes of breast cancer cells

[0058] Take the MDA-MB-231 cells in the logarithmic growth phase and inoculate them in a 96-well plate. After 24 hours, when the cells are completely attached to the wall, add different ratios of calycosin (CG) and curcumin (CU) respectively, and continue to culture for 24 hours. The concentration settings of different ratios of CG and CU are as follows:

[0059] Table 2-1 Concentration setting of CG and CU with a molar ratio of 1:1

[0060]

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

[0062]

[0063] Table 2-3 Concentration settings for CG and CU molar ratio of 1:3

[0064]

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

[0066] When CI<1, it means that the combination of the two drugs has a synergistic effect; CI=1, it means that the combination of the two drugs has an additive effect; when CI>1, it means that the combination of the two drugs has an antagonistic effect.

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

[0068]

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

[0070]

[0071] The results are shown in Tables 2-5. When calycosin isoflavone glycosides and curcumin are combined in a ratio of 1:1, 1:2, and 1:3, they have a synergistic effect, among which CG-CU (1:2) has the smallest synergistic index. Therefore, when calycosin isoflavone glycosides and curcumin are combined in a molar ratio of 1:2, the synergistic ability is the strongest, which significantly reduces the LAP-TGF-β1 protein loading level of breast cancer cell exosomes, and can be used as the optimal ratio.

[0072] Example 3 Effect of synergistic effects of calycosin and curcumin on LAP-TGF-β1 protein loading in exosomes of osteosarcoma cells

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

[0074] Osteosarcoma HOS cells in the logarithmic growth phase were inoculated in 96-well plates. After 24 hours, when the cells were completely attached to the wall, different ratios of calycosin (CG) and curcumin (CU) were added and cultured for another 24 hours. After the drug action time 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.

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

[0076]

[0077] The results are shown in Table 3-1. When calycosin isoflavone glycosides and curcumin are combined in a ratio of 1:1, 1:2, and 1:3, they have a synergistic effect, among which CG-CU (1:2) has the smallest synergistic index. Therefore, when calycosin isoflavone glycosides and curcumin are combined in a molar ratio of 1:2, the synergistic ability is the strongest, which significantly reduces the exosome LAP-TGF-β1 protein loading level of osteosarcoma cells, and can be used as the optimal ratio.

[0078] Example 4 Effect of synergistic effects of calycosin and curcumin on LAP-TGF-β1 protein loading in melanoma cell exosomes

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

[0080] A-375 cells in the logarithmic growth phase were inoculated in a 96-well plate. After 24 hours, when the cells were completely attached to the wall, different ratios of calycosin (CG) and curcumin (CU) were added and cultured for another 24 hours. After the drug action time 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.

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

[0082]

[0083] The results are shown in Table 4-1. When calycosin isoflavone glycosides and curcumin are combined in a ratio of 1:1, 1:2, and 1:3, they have a synergistic effect, among which CG:CU=1:2 has the smallest synergistic index. Therefore, when calycosin isoflavone glycosides and curcumin are combined in a molar ratio of 1:2, the synergistic ability is the strongest, which significantly reduces the LAP-TGF-β1 protein loading level in melanoma cell exosomes, and can be used as the optimal ratio.

[0084] Example 5 Effects of different doses of the optimal ratio (1:2) of the active components of the Astragalus-Erdoblast drug pair, calycosin and curcumin, on transendothelial invasion of breast cancer cells

[0085] CG-CU (1:2) was divided into three dose groups for the experiment: low dose group: CG and CU were 62.5nM and 125nM respectively; medium dose group: CG and CU were 125nM and 250nM respectively; high dose group: CG and CU were 250nM and 500nM respectively. The doses in the following examples are the same as those in this example.

[0086] HUVEC cells in the logarithmic growth phase were digested with EDTA trypsin and cultured at 2×104 The density of cells / well was inoculated in the Transwell chamber and placed in the incubator for overnight culture. MDA-MB-231 cells were pretreated with CG-CU (1:2) at different concentrations for 24 h and then inoculated in a 6-well plate. Single cell suspension was prepared after trypsin digestion and counted. 5×10 4 The density of cells / well was inoculated on the upper layer of HUVEC cells. 750 μL of complete culture medium was added to the lower chamber. After 12-16 hours, the Transwell chamber was removed, the culture medium was aspirated, PBS was rinsed twice, 4% paraformaldehyde was used to fix for 20-30 minutes, PBS was rinsed three times, and the cells that did not penetrate the chamber were gently wiped with a cotton swab. After drying, it was observed under an inverted microscope, and 5 areas were randomly selected for photography to calculate the number of invasive cells.

[0087] The results are as follows Figure 2 As shown, the optimal combination of calycosin and curcumin (1:2) reduced the number of transendothelial cells of MDA-MB-231 cells in a dose-dependent manner, indicating that the optimal combination of calycosin 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 active components of the Astragalus-Edward zedoaria drug pair, calycosin and curcumin, on transendothelial invasion of osteosarcoma cells

[0089] HUVEC cells in the logarithmic growth phase were digested with EDTA trypsin and cultured at 2×10 4 The density of cells / well was inoculated in the Transwell chamber and placed in the incubator for overnight culture. HOS cells were pretreated with CG-CU (1:2) at different concentrations for 24 h and then inoculated in a 6-well plate. After trypsin digestion, single cell suspension was prepared and counted. 4×10 4 The density of cells / well was inoculated on the upper layer of HUVEC cells. 750 μL of complete culture medium was added to the lower chamber. After 12-16 hours, the Transwell chamber was removed, the culture medium was aspirated, PBS was rinsed twice, 4% paraformaldehyde was used to fix for 20-30 minutes, PBS was rinsed three times, and the cells that did not penetrate the chamber were gently wiped with a cotton swab. After drying, it was observed under an inverted microscope, and 5 areas were randomly selected for photography to calculate the number of invasive cells.

[0090] The results are as follows Figure 3 As shown, the optimal combination of calycosin and curcumin (1:2) reduced the number of transendothelial cells of HOS cells in a dose-dependent manner, indicating that the optimal combination of calycosin 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 active components of the Astragalus-Edward zedoaria drug pair, calycosin and curcumin, on transendothelial invasion of melanoma cells

[0092] HUVEC cells in the logarithmic growth phase were digested with EDTA trypsin and cultured at 2×10 4 The density of cells / well was inoculated in the Transwell chamber and placed in the incubator for overnight culture. A-375 cells were pretreated with CG-CU (1:2) at different concentrations for 24 h and then inoculated in a 6-well plate. After trypsin digestion, single cell suspension was prepared and counted. 5×10 4 The density of cells / well was inoculated on the upper layer of HUVEC cells. 750 μL of complete culture medium was added to the lower chamber. After 12-16 hours, the Transwell chamber was removed, the culture medium was aspirated, PBS was rinsed twice, 4% paraformaldehyde was used to fix for 20-30 minutes, PBS was rinsed three times, and the cells that did not penetrate the chamber were gently wiped with a cotton swab. After drying, it was observed under an inverted microscope, and 5 areas were randomly selected for photography to calculate the number of invasive cells.

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

[0094] Example 8 Effects of different doses of the optimal ratio (1:2) of the active components of the Astragalus-Erdoblast drug pair, calycosin and curcumin, on tubule formation in breast cancer cells

[0095] Place the matrix gel in an ice box and allow it to melt naturally into a liquid state, then evenly spread on the angiogenesis slide. Add 10 μL of matrix gel (preferably without bubbles and evenly) to each well and place it in a cell culture incubator at 37°C, containing 5% CO2 and 95% air for 30 minutes to solidify. Pretreat MDA-MB-231 cells with different concentrations of CG-CU (1:2) composition for 24 hours, digest with trypsin, resuspend in DMEM medium containing 1% fetal bovine serum, and take 50 μL of cell suspension and spread on the solidified matrix gel surface (2.0×10 4 The cells were incubated in a cell culture incubator for 2 to 8 hours, and 5 areas were randomly selected for observation and photographing under an inverted microscope. Image J software was used for quantitative analysis to evaluate the degree of in vitro vascular mimicry formation, including the total number of nodes, total number of connection points, total number of meshes, and length of tubes.

[0096] The results are as follows Figure 5As shown, the optimal combination of calycosin and curcumin (1:2) can dose-dependently reduce the total number of nodes, total number of connection points, total number of meshes and length of tubules formed in MDA-MB-231 cells, indicating that the optimal combination of calycosin and curcumin can significantly inhibit the vascular mimicry formation of breast cancer cells.

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

[0098] Place the matrix gel in an ice box and allow it to melt naturally into a liquid state, then evenly spread it on the angiogenesis slide. Add 10 μL of matrix gel (preferably without bubbles and evenly) to each well and place it in a cell culture incubator at 37°C, containing 5% CO2 and 95% air for 30 minutes to solidify. Pretreat HOS cells with different concentrations of CG-CU (1:2) composition for 24 hours, digest with trypsin, resuspend in DMEM medium containing 1% fetal bovine serum, and take 50 μL of cell suspension and spread it on the solidified matrix gel surface (2.0×10 4 The cells were incubated in a cell culture incubator for 2 to 8 hours, and 5 areas were randomly selected for observation and photographing under an inverted microscope. Image J software was used for quantitative analysis to evaluate the degree of in vitro vascular mimicry formation, including the total number of nodes, total number of connection points, total number of meshes, and length of tubes.

[0099] The results are as follows Figure 6 As shown, the optimal combination of calycosin and curcumin (1:2) can reduce the total number of nodes, total number of connection points, total number of meshes and length of tubules formed in HOS cells in a dose-dependent manner, indicating that the optimal combination of calycosin and curcumin can significantly inhibit the vascular mimicry formation of osteosarcoma cells.

[0100] Example 10 Effects of different doses of the optimal ratio (1:2) of the active components of the Astragalus-Erdoblast drug pair, calycosin and curcumin, on tubule formation in melanoma cells

[0101] Place the matrix gel in an ice box and allow it to melt naturally into a liquid state, then evenly spread it on the angiogenesis slide. Add 10 μL of matrix gel (preferably without bubbles and evenly) to each well and place it in a cell culture incubator at 37°C, containing 5% CO2 and 95% air for 30 minutes to solidify. Pretreat A-375 cells with different concentrations of CG-CU (1:2) composition for 24 hours, digest with trypsin, resuspend in DMEM medium containing 1% fetal bovine serum, and take 50 μL of cell suspension and spread it on the solidified matrix gel surface (1.5×10 4The cells were incubated in a cell culture incubator for 2 to 8 hours, and 5 areas were randomly selected for observation and photographing under an inverted microscope. Image J software was used for quantitative analysis to evaluate the degree of in vitro vascular mimicry formation, including the total number of nodes, total number of connection points, total number of meshes, and length of tubes.

[0102] The results are as follows Figure 7 As shown, the optimal combination of calycosin and curcumin (1:2) can reduce the total number of nodes, total number of connection points, total number of meshes and length of tubules formed in A-375 cells in a dose-dependent manner, indicating that the optimal combination of calycosin and curcumin can significantly inhibit the vascular mimicry formation of melanoma cells.

[0103] Example 11 The synergistic effect of the active components of the Astragalus-Ezema drug pair, calycosin and curcumin, on breast cancer metastasis

[0104] To investigate the synergistic effects of the active components of the Astragalus-Curcuma drug pair, calycosin and curcumin, on breast cancer metastasis. MDA-MB-231 breast cancer cells were inoculated into the mammary glands of 7-8 week-old NCG-HLA-A2.1 female mice. The mice were randomly divided into 6 groups: control group, calycosin group (CG), curcumin (CU), calycosin and curcumin (1:1) group (CG-CU (1:1)), calycosin and curcumin (1:2) group (CG-CU (1:2)), and calycosin and curcumin (1:3) group (CG-CU (1:3)). According to the clinical use of Astragalus and Curcuma, the contents of calycosin and curcumin in the water extract of the Astragalus-Curcuma drug pair were 30g and 15g, respectively. According to our previous analysis, the contents of calycosin and curcumin in the water extract of the Astragalus-Curcuma drug pair were 0.79% and 0.81%, respectively. The human-mouse dose conversion method provided in the Chinese Medicine Pharmacology Research Methodology was used for calculation. The final dosages of 0.0395 g / kg and 0.0243 g / kg were used as the dosages of the single-drug group of calycosin and the single-drug group of curcumin, respectively; 0.079, 0.1185, and 0.158 g / kg were used as the dosages of the combination of calycosin and curcumin (1:1, 1:2, and 1:3), respectively. From the second day after tumor implantation until the end of the experiment, each group was gavaged once a day at a fixed time for 27 consecutive days; the control group was gavaged with an equal amount of normal saline once a day for 27 consecutive days. The results of tumor weight analysis of each group showed that as the ratio of calycosin and curcumin increased, the tumor burden of mice decreased significantly. However, when the ratio of calycosin and curcumin exceeded 1:2, the inhibitory effect did not increase. Therefore, the tumor inhibition effect was optimal when the ratio of calycosin and curcumin was 1:2 ( Figure 8 In addition, H&E staining results of lung tissue showed that the ratio of calycosin to curcumin was 1:2, which significantly reduced the occurrence of lung metastasis of breast cancer ( Fig. 9 ).

[0105] Example 12 The synergistic effect of the effective components of the Astragalus-Edwardius drug pair, calycosin and curcumin, on the metastasis of osteosarcoma

[0106] The dosages of calycosin and curcumin in this example are consistent with those in Example 11.

[0107] To investigate the synergistic effects of the active components of the Astragalus-Curcuma drug pair, calycosin and curcumin, on osteosarcoma metastasis. The passaged osteosarcoma HOS cells were orthotopically injected into the tibia of 6-8 week-old NCG-HLA-A2.1 male mice. The mice were randomly divided into 6 groups: control group, calycosin group (CG), curcumin (CU), calycosin and curcumin (1:1) group (CG-CU (1:1)), calycosin and curcumin (1:2) group (CG-CU (1:2)), and calycosin and curcumin (1:3) group (CG-CU (1:3)). From the second day after tumor implantation to the end of the experiment, each group was intragastrically administered with medication once a day at a fixed time for 27 consecutive days; the control group was also intragastrically administered with an equal amount of normal saline once a day for 27 consecutive days. The results of tumor weight analysis of each group showed that the tumor burden of mice treated with the combination of calycosin and curcumin was significantly reduced, and the ratio of calycosin to curcumin was 1:2, which significantly reduced the tumor weight ( Fig.10 ). The results of H&E staining of lung tissue showed that the ratio of calycosin to curcumin was 1:2, which significantly reduced the occurrence of lung metastasis of osteosarcoma ( Fig.11 ).

[0108] Example 13 The synergistic effect of the active components of the Astragalus-Ezema drug pair, calycosin and curcumin, on melanoma metastasis

[0109] The dosages of calycosin and curcumin in this example are consistent with those in Example 11.

[0110] In order to investigate the synergistic effect of the active components of the Astragalus-Curcuma drug pair, calycosin and curcumin, on melanoma metastasis, the animals were divided into 6 groups, namely the control group, calycosin group (CG), curcumin (CU), calycosin and curcumin (1:1) group (CG-CU (1:1)), calycosin and curcumin (1:2) group (CG-CU (1:2)), and calycosin and curcumin (1:3) group (CG-CU (1:3)). The passaged human malignant melanoma A-375 cells were injected subcutaneously into 6-8 week old NCG-HLA-A2.1 male mice. From the second day after tumor implantation to the end of the experiment, each group was intragastrically administered once a day at a fixed time for 27 consecutive days; the control group was also intragastrically administered with an equal amount of normal saline once a day for 27 consecutive days. The results of tumor weight analysis of each group showed that the tumor burden of mice treated with the combination of calycosin and curcumin was significantly reduced, and the ratio of calycosin to curcumin was 1:2, which significantly reduced the tumor weight ( Fig.12 ). The results of H&E staining of lung tissue showed that the ratio of calycosin to curcumin was 1:2, which significantly reduced the occurrence of melanoma lung metastasis ( Fig.13 ).

Claims

1. A composition of active ingredients of Radix Astragali and Rhizoma Curcumae, characterized in that: The composition consists of calycosin isoflavone glycosides and curcumin.

2. The composition according to claim 1, characterized in that The molar ratio of calycosin to curcumin is 1:(1-3).

3. The composition according to claim 2, characterized in that The molar ratio of calycosin to curcumin is 1:

2.

4. Use of the effective ingredient composition of the Astragalus membranaceus-Zhuozhu medicinal pair according to any one of claims 1 to 3 in the preparation of a drug for inhibiting tumors and / or resisting tumor metastasis.

5. The use according to claim 4, characterized in that: The composition can significantly reduce the exosomal LAP-TGF-β1 protein loading level and significantly inhibit the transendothelial invasion and vascular mimicry of tumor cells, thereby greatly inhibiting the metastasis process of tumor cells.

6. The use according to claim 4, characterized in that The tumor is selected from breast cancer, osteosarcoma or melanoma.

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