Traditional Chinese medicine components used to inhibit or reduce breast cancer cells and their applications in the preparation of anticancer drugs.

Abstract

A traditional Chinese medicine composition for inhibiting or reducing breast cancer cells comprises Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare. The weight ratio of Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare in the traditional Chinese medicine composition is 1-2:4-6:2-3:3-5:2-4:2-3. This traditional Chinese medicine composition is intended for the preparation of anticancer drugs. The composition is administered to breast cancer cells at an effective dose between 0.1 and 10 mg / ml for culturing. Cell experiments have confirmed that the traditional Chinese medicine composition exhibits inhibitory activity against breast cancer cells, thereby achieving the purpose of treating breast cancer symptoms or inhibiting breast cancer cell activity.

Description

Technical Field

[0001] This invention relates to traditional Chinese medicines that inhibit breast cancer cells; in particular, it relates to a traditional Chinese medicine composition for inhibiting or reducing breast cancer cells and its use in preparing anticancer drugs. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women. The main treatments for breast cancer include thoracotomy, radiation therapy, and targeted drug therapy. Targeted drug therapy involves administering targeted drugs to patients to inhibit the growth of breast cancer cells, slow the spread of breast cancer cells, and prevent cancer recurrence.

[0003] However, the drugs currently used in targeted therapy can have significant side effects on the human body. The existing targeted drug for breast cancer is tamoxifen, which is a hormone inhibitor. It works by binding to estrogen receptors and preventing the transmission of cell signals, thus achieving the effect of tumor treatment. However, long-term use of tamoxifen may lead to endometrial cancer and venous thrombosis, and may even cause breast cancer cells to develop drug resistance, ultimately leading to treatment failure.

[0004] In order to improve the side effects of breast cancer drugs, some related companies have developed natural Chinese herbal medicines as an adjunct therapy. However, current research has not yet been able to actually develop Chinese herbal medicine compound formulas that can provide efficacy in inhibiting drug-resistant breast cancer cells. Therefore, the technical issue of how to select Chinese herbal medicine compound formulas to effectively treat or inhibit breast cancer symptoms is the problem that this invention aims to solve. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a traditional Chinese medicine composition for inhibiting or reducing breast cancer cells and its use in preparing anticancer drugs. The traditional Chinese medicine composition basically includes Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides and Hordeum vulgare. Experiments have confirmed that the traditional Chinese medicine composition has the effect of inhibiting the activity of conventional breast cancer cells and drug-resistant breast cancer cells.

[0006] To achieve the above objectives, the present invention provides a traditional Chinese medicine composition for inhibiting or reducing breast cancer cells, comprising Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare. The weight ratio of Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare in the traditional Chinese medicine composition is 1-2:4-6:2-3:3-5:2-4:2-3.

[0007] The present invention provides another embodiment of the use of the aforementioned traditional Chinese medicine composition for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells. The traditional Chinese medicine composition includes Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare. The traditional Chinese medicine composition is administered to the breast cancer cells at an effective dose between 0.1 and 10 mg / ml for culture. The traditional Chinese medicine composition exhibits the ability to inhibit the activity of the breast cancer cells. The breast cancer cells are selected from one of the populations consisting of conventional breast cancer cells, tamoxifen-resistant breast cancer cells, and tamoxifen-sensitive breast cancer cells.

[0008] The effect of this invention is that the weight ratio of the selected herbs—Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare—in the herbal composition, as confirmed by cell experiments, can effectively inhibit or reduce the activity of breast cancer cells. Furthermore, the effective dose of the herbal composition, between 0.1 and 10.0 mg / ml, can effectively inhibit the activity of conventional breast cancer cells, tamoxifen-resistant breast cancer cells, and tamoxifen-sensitive breast cancer cells for at least 24 hours, and correspondingly reduce the number of these cells. When the breast cancer cells are conventional breast cancer cells, the herbal composition… An effective dose of 5.0–10.0 mg / ml exhibits superior inhibitory activity against conventional breast cancer cells. When the breast cancer cells are anti-tamoxifen breast cancer cells, an effective dose of the traditional Chinese medicine composition between 1.25–10.0 mg / ml exhibits superior inhibitory activity against tamoxifen-sensitive breast cancer cells. When the breast cancer cells are tamoxifen-sensitive breast cancer cells, an effective dose of the traditional Chinese medicine composition between 2.5–10.0 mg / ml exhibits superior inhibitory activity against tamoxifen-sensitive breast cancer cells. Therefore, the formulation of the traditional Chinese medicine composition, combined with a specific effective dose, is applied to achieve the purpose of treating breast cancer symptoms or inhibiting the activity of breast cancer cells. Attached Figure Description

[0009] Figure 1AThe following are cell images of conventional breast cancer cells (MCF-7) in experimental groups 1-6 after being treated with effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to a preferred embodiment of the present invention, and after 24 hours, 48 ​​hours and 72 hours of culture.

[0010] Figure 1B The graph shows the cell survival rate data after 24 hours of culture, obtained by administering the herbal composition of the present invention to conventional breast cancer cells (MCF-7) in experimental groups 1-6 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, according to a preferred embodiment of the present invention.

[0011] Figure 1C The graph shows the cell survival rate data after 48 hours of culture, obtained by administering the herbal composition of the present invention to conventional breast cancer cells (MCF-7) in experimental groups 1-6 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, according to the dosage of the herbal composition of the present invention.

[0012] Figure 1D The graph shows the cell survival rate data after 72 hours of culture, obtained by administering the herbal composition of the present invention to conventional breast cancer cells (MCF-7) in experimental groups 1-6 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, according to the dosage of the herbal composition of the present invention.

[0013] Figure 2A The following are cell images of a preferred embodiment of the present invention, showing the effective doses of the herbal composition administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, after 24 hours, 48 ​​hours and 72 hours of culture.

[0014] Figure 2B The graph shows the cell survival rate data after 24 hours of culture, obtained by administering the herbal composition of the present invention to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, according to the dosage of the herbal composition of the present invention.

[0015] Figure 2CThe graph shows the cell survival rate data after 48 hours of culture, obtained by administering the herbal composition of the present invention to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, according to the effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, as a preferred embodiment of the present invention.

[0016] Figure 2D The graph shows the cell survival rate data after 72 hours of culture, obtained by administering the herbal composition of the present invention to experimental groups 7-12 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1).

[0017] Figure 3A In a preferred embodiment of the present invention, the herbal composition was administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, and the images of breast cancer cells with cell migration were observed.

[0018] Figure 3B The image shows the effective doses of the herbal composition of a preferred embodiment of the present invention administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml, and 10 mg / ml, respectively, and the corresponding data on the migration of breast cancer cells after 24 hours of culture.

[0019] Figure 4A In a preferred embodiment of the present invention, the herbal composition was administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, and the imaging effects on the invasion of breast cancer cells were observed.

[0020] Figure 4BThe images show the protein expression of ATG5, ATG7 and LC3-II of breast cancer cells corresponding to effective doses of the herbal composition administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to a preferred embodiment of the present invention.

[0021] Figure 5A Electrophoresis images of the proteins ATG5, ATG7, and LC3-II of breast cancer cells, corresponding to effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml, and 10 mg / ml administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12, as described in a preferred embodiment of the present invention.

[0022] Figure 5B The following is a graph showing the protein data of ATG5, ATG7 and LC3-II corresponding to the effective doses of the herbal composition administered to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7-12 at doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to a preferred embodiment of the present invention.

[0023] Figure 6A The following are cell images of a preferred embodiment of the present invention, showing the effective doses of the herbal composition administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, after 24 hours, 48 ​​hours and 72 hours of culture.

[0024] Figure 6B The graph shows the cell survival rate data after 24 hours of culture, obtained by administering the herbal composition of the present invention to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml respectively, according to a preferred embodiment of the present invention.

[0025] Figure 6CThe graph shows the cell survival rate data after 48 hours of culture, obtained by administering the herbal composition of the traditional Chinese medicine to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to the experimental group.

[0026] Figure 6D The graph shows the cell survival rate data after 72 hours of culture, obtained by administering the herbal composition of the traditional Chinese medicine to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to the experimental group.

[0027] Figure 7A In a preferred embodiment of the present invention, the herbal composition was administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 13-18 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, and the images of breast cancer cells with cell migration were observed.

[0028] Figure 7B The image shows the effective doses of the herbal composition of a preferred embodiment of the present invention administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 13-18 at 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml, and 10 mg / ml, respectively, and the corresponding data on the migration of breast cancer cells after 24 hours of culture.

[0029] Figure 8A In a preferred embodiment of the present invention, the herbal composition was administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, and the imaging effects on breast cancer cell invasion were observed.

[0030] Figure 8BThe images show the expression of ATG5, ATG7 and LC3-II proteins in breast cancer cells corresponding to effective doses of the herbal composition administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to a preferred embodiment of the present invention.

[0031] Figure 9A Electrophoresis images of the proteins ATG5, ATG7, and LC3-II of breast cancer cells, corresponding to effective doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml, and 10 mg / ml administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18, as described in a preferred embodiment of the present invention.

[0032] Figure 9B The following is a graph showing the protein data of ATG5, ATG7 and LC3-II corresponding to the effective doses of the herbal composition administered to tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13-18 at doses of 0.1 mg / ml, 0.5 mg / ml, 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml and 10 mg / ml, respectively, according to a preferred embodiment of the present invention. Detailed Implementation

[0033] A preferred embodiment of the present invention provides a traditional Chinese medicine composition for inhibiting or reducing breast cancer cells. The basic components include Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare. The Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare are all extracted from natural plants.

[0034] The pharmacological properties of each component in the traditional Chinese medicine composition are described below:

[0035] Euphorbia kansui: The active ingredients contain diterpenoids and triterpenoids. The diterpenoids mainly contain euphorbia diterpenol and pseudo-elemenone. The euphorbia diterpenol has significant anti-cancer and antiviral activity. The triterpenoids mainly contain euphol and tirucallol, and the triterpenoids also have anti-cancer activity.

[0036] White peony: The active ingredients include paeoniflorin and its derivatives, flavonoids, organic acids, etc. Paeoniflorin is the main effective component of white peony and has a variety of pharmacological effects such as anti-inflammatory, analgesic, antioxidant and immune-regulating functions.

[0037] Licorice: The active ingredients contain flavonoids, triterpenoid saponins and glycyrrhiza polysaccharide (GPS). The active ingredients of licorice have shown good anti-inflammatory, anti-tumor, liver-protective and immune-enhancing effects in clinical trials.

[0038] Dandelion: The active ingredients include flavonoids, terpenes, sterols, phenols, choline and organic acids, etc. The active ingredients of dandelion have been shown in clinical trials to have effects such as protecting the liver, antithrombosis, lowering blood sugar, anti-inflammation, anti-oxidation, antibacterial and anti-tumor effects.

[0039] Gardenia: The active ingredients contain iridoids, monoterpenoids, diterpenoids, triterpenoids, volatile oils, organic acid esters, flavonoids, polysaccharides and trace elements. The active ingredients of gardenia have been shown in clinical trials to have anti-inflammatory, antithrombotic, antiviral and antitumor effects.

[0040] Malt: The active ingredients include α- and β-amylase, catalytic enzyme, maltose and malt alkaloid, adenine, choline, protein, amino acids, vitamins B, D, E, cytochrome C, etc. The active ingredients of malt have digestive aid, hypoglycemic effect and antibacterial effect.

[0041] The weight ratio of the following herbs in the herbal composition is 1-2:4-6:2-3:3-5:2-4:2-3. The weight ratio of these herbs can be adjusted according to the spread of breast cancer cells.

[0042] In a preferred embodiment, in actual clinical research of traditional Chinese medicine, if the patient's breast cancer cells severely compress other organs and cause obvious tumor pain, the weight ratio of the following traditional Chinese medicine components—Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare—is configured as 1:6:3:3:2:2, and the traditional Chinese medicine components are administered to the patient regularly. The traditional Chinese medicine components can indeed effectively improve the patient's breast cancer symptoms.

[0043] In another preferred embodiment, in actual clinical research of traditional Chinese medicine, if the patient develops severe lymphedema after surgery, the weight ratio of the following ingredients in the traditional Chinese medicine composition is configured as 2:4:2:3:2:3, and the traditional Chinese medicine composition is regularly administered to the postoperative patient. This not only inhibits and reduces the number of active breast cancer cells, but also improves the symptoms of lymphedema.

[0044] In another preferred embodiment, in actual clinical research of traditional Chinese medicine, if the patient experiences severe skin toxicity during chemotherapy, the weight ratio of the following traditional Chinese medicine components—Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare—is configured as 1:4:2:5:4:2. Regular administration of this traditional Chinese medicine component to the patient undergoing chemotherapy can not only inhibit and reduce the number and activity of breast cancer cells but also effectively improve the symptoms of lymphedema.

[0045] Furthermore, the traditional Chinese medicine composition is used to prepare anticancer drugs that inhibit or reduce the activity of breast cancer cells. The traditional Chinese medicine composition is administered to the breast cancer cells at an effective dose between 0.1 and 10.0 mg / ml for culture. In this embodiment, the culture time of the traditional Chinese medicine composition on the breast cancer cells is between 24 and 72 hours. The effective dose of the traditional Chinese medicine composition includes 0.1 mg / ml and 10.0 mg / ml, and the culture time of the breast cancer cells includes 24 hours and 72 hours. The breast cancer cells are selected from one of the populations consisting of conventional breast cancer cells, anti-tamoxifen breast cancer cells, and tamoxifen-sensitive breast cancer cells. The traditional Chinese medicine composition has the effect of inhibiting the activity of the breast cancer cells, thereby achieving the purpose of treating breast cancer symptoms or inhibiting the activity of breast cancer cells.

[0046] To fully understand the purpose, features, and efficacy of the present invention, the content of the traditional Chinese medicine components described in the first embodiment is provided, and different effective doses are applied to conventional breast cancer cells (MCF-7), tamoxifen-resistant breast cancer cells (MCF-7 / TAMR1), and tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) for cell experiments. The conventional breast cancer cells (MCF-7), tamoxifen-resistant breast cancer cells (MCF-7 / TAMR1), and tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) after application are then compared with those of conventional breast cancer cells (MCF-7), tamoxifen-resistant breast cancer cells (MCF-7 / TAMR1), and tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5). 5) Cell viability was analyzed at different culture times. The conventional breast cancer cells (MCF-7), tamoxifen-resistant breast cancer cells (MCF-7 / TAMR1), and tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were purchased from Merck Millipore Corporation. The weight ratio of Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare in the herbal composition of this experiment was 1:6:3:3:2:2, indicating that the herbal composition of this experiment did indeed provide the effect of inhibiting or reducing the activity of breast cancer cells.

[0047] The following specific embodiments further demonstrate the practical applicability of the present invention, and any person skilled in the art can make appropriate changes to its parameters or settings after referring to the present invention, and such changes should still fall within the scope of the present invention.

[0048] I. To investigate the cell survival rate of the conventional breast cancer cells (MCF-7) corresponding to the application of the aforementioned traditional Chinese medicine composition:

[0049] 1. This experiment includes control group 1 and experimental groups 1 to 6:

[0050] Control group 1: The conventional breast cancer cells (MCF-7) were not treated with the herbal medicine composition, indicating that the effective dose of the herbal medicine composition was 0 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours and 72 hours.

[0051] Experimental Group 1: The herbal composition was administered to the conventional breast cancer cells (MCF-7) at an effective dose of 0.1 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours, and 72 hours after administration.

[0052] Experimental Group 2: The herbal composition was administered to the conventional breast cancer cells (MCF-7) at an effective dose of 0.5 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours, and 72 hours after administration.

[0053] Experimental Group 3: The herbal composition was administered to the conventional breast cancer cells (MCF-7) at an effective dose of 1.25 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0054] Experimental group 4: The herbal composition was administered to the conventional breast cancer cells (MCF-7) at an effective dose of 2.5 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0055] Experimental group 5: The herbal composition was administered to the conventional breast cancer cells (MCF-7) at an effective dose of 5.0 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0056] Experimental Group 6: The herbal composition was administered to the conventional breast cancer cells (MCF-7) at an effective dose of 10.0 mg / ml, and the conventional breast cancer cells (MCF-7) were cultured for 24 hours, 48 ​​hours, and 72 hours after administration.

[0057] 2. Analyze the cell viability of the conventional breast cancer cells (MCF-7) corresponding to experimental groups 1-6:

[0058] In this experiment, conventional breast cancer cells (MCF-7) were implanted at a density of 5 × 10⁶ cells per well. 3Cells were cultured in 96-well dishes for 24 hours. After the conventional breast cancer cells (MCF-7) adhered to the wells, the cell culture medium was removed. Then, according to the effective doses of the herbal medicine components prepared for control group 1 and experimental groups 1-6, the cells were injected into the corresponding wells of the conventional breast cancer cells (MCF-7) for 24, 48, and 72 hours of cell culture. During the 24, 48, and 72-hour culture periods of control group 1 and experimental groups 1-6, the cell numbers were observed using an electron microscope to record the cell count. Subsequently, the culture medium was removed from the culture dishes, and the conventional breast cancer cells (MCF-7) were washed twice with sterile phosphate-buffered saline (PBS) in the wells of control group 1 and experimental groups 1-6. 0.2 mL of culture medium was added to the wells of experimental groups 1-6 corresponding to the conventional breast cancer cells (MCF-7), and the culture dishes were placed in a constant temperature incubator for 2 hours. Subsequently, the culture medium in the culture dishes was removed, and the wells of control group 1 and experimental groups 1-6 corresponding to the conventional breast cancer cells (MCF-7) were washed once with unsterilized phosphate-buffered saline (PBS). Finally, 0.2 mL of DMSO was added to the wells of control group 1 and experimental groups 1-6 corresponding to the conventional breast cancer cells (MCF-7) to dissolve the crystals. The optical density (OD) value was measured at a wavelength of 570 nm using an Enzyme-linked Immunosorbent Assay (ELISA reader) analyzer, and the relative cell viability rate was calculated.

[0059] The cell viability data measured in each of the experimental groups 1 to 6 were calculated by taking the absorbance of the control group 1 as 100%, dividing the absorbance of each of the experimental groups 1 to 6 by the control group 1, and calculating the cell viability of each of the experimental groups 1 to 6 relative to the conventional breast cancer cells (MCF-7).

[0060] Observation results as follows Figures 1A to 1D As shown, where Figure 1A The images are of conventional breast cancer cells (MCF-7) corresponding to experimental groups 1-6, and after 24 hours, 48 ​​hours, and 72 hours of culture, respectively. Figures 1B to 1D The graphs show the cell viability data of conventional breast cancer cells (MCF-7) in experimental groups 1-6 after 24 hours, 48 ​​hours, and 72 hours of culture. Figures 1B to 1DThe standard error (SE) data results of experimental groups 1 to 6 are shown. These are the detection values ​​of three independent experiments (n=3) for each of the experimental groups 1 to 6. "*" indicates the comparison of the values ​​of experimental groups 1 to 6 with respect to control group 1, and p < 0.05.

[0061] like Figure 1A , Figure 1B As shown, based on the data from experimental groups 1-6 after 24 hours of culture, the cell viability of conventional breast cancer cells (MCF-7) in experimental groups 1 and 2 did not show a significant decrease compared to control group 1. However, the cell viability of conventional breast cancer cells (MCF-7) in experimental groups 3 and 4 showed a gradual decreasing trend compared to control group 1, indicating that the effective doses of the herbal medicine components administered to experimental groups 3 and 4 (1.25 mg / ml and 2.5 mg / ml) could appropriately inhibit or reduce the cell growth of conventional breast cancer cells (MCF-7). The cell viability of conventional breast cancer cells (MCF-7) in experimental groups 5 and 6 was significantly reduced to below 10% compared to control group 1, with experimental group 6 showing a more significant decrease in cell viability compared to experimental group 5. Figure 1A As shown, the cell density observed in experimental groups 5 and 6 was significantly reduced compared to experimental groups 1-4. This indicates that the effective doses of the herbal medicine composition administered to experimental groups 5 and 6, at 5.0 mg / ml and 10.0 mg / ml respectively, after 24 hours of culture, could significantly inhibit or reduce the growth of conventional breast cancer cells (MCF-7). Furthermore, the effective dose of the herbal medicine composition administered to experimental group 6, at 10.0 mg / ml, showed a more effective inhibitory effect on the growth of conventional breast cancer cells (MCF-7).

[0062] like Figure 1A , Figure 1C As shown, based on the data from experimental groups 1-6 after 48 hours of culture, the cell viability of conventional breast cancer cells (MCF-7) in experimental groups 1 and 2 was not significantly reduced compared to control group 1. However, the cell viability of conventional breast cancer cells (MCF-7) in experimental groups 3 and 4 showed a gradual decreasing trend compared to control group 1. Figure 1B and Figure 1CIt can be seen that the 48-hour culture of experimental groups 3 and 4 did not significantly reduce the cell viability of the conventional breast cancer cells (MCF-7), while the cell viability of the conventional breast cancer cells (MCF-7) in experimental groups 5 and 6 was also reduced to below 10% compared with the control group 1. Among them, the 48-hour culture of experimental group 6 reduced the cell viability of the conventional breast cancer cells (MCF-7) more than the 24-hour culture.

[0063] like Figure 1A , Figure 1D As shown, based on the data from experimental groups 1-6 after 72 hours of culture, the cell viability of conventional breast cancer cells (MCF-7) in experimental groups 1-4 showed a decreasing trend compared to control group 1. Furthermore, the cell viability of conventional breast cancer cells (MCF-7) in experimental groups 5 and 6 also decreased to below 10% compared to control group 1. Figure 1C and Figure 1D It can be seen that the experimental group 6, after 72 hours of culture, further reduced the cell viability of the conventional breast cancer cells (MCF-7) compared to 48 hours of culture.

[0064] Therefore, the effective dose of the herbal composition between 0.1 and 10.0 mg / ml can indeed effectively inhibit or reduce the activity of conventional breast cancer cells (MCF-7). The effective dose of the herbal composition between 1.25 and 10.0 mg / ml can effectively reduce the cell activity rate of conventional breast cancer cells. In a preferred embodiment, the effective dose of the herbal composition between 5.0 and 10.0 mg / ml more significantly inhibits the activity of conventional breast cancer cells (MCF-7).

[0065] II. To investigate the cell survival rate of the aforementioned anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) corresponding to the traditional Chinese medicine composition administered:

[0066] 1. This experiment included control group 2 and experimental groups 7–12:

[0067] Control group 2: The anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were not administered the herbal medicine composition, indicating that the effective dose of the herbal medicine composition was 0 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours.

[0068] Experimental group 7: The herbal composition was administered to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) at an effective dose of 0.1 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0069] Experimental Group 8: The herbal composition was administered to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) at an effective dose of 0.5 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0070] Experimental Group 9: The herbal composition was administered to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) at an effective dose of 1.25 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0071] Experimental group 10: The herbal composition was administered to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) at an effective dose of 2.5 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0072] Experimental group 11: The herbal composition was administered to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) at an effective dose of 5.0 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0073] Experimental group 12: The herbal composition was administered to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) at an effective dose of 10.0 mg / ml, and the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0074] 2. Analyze the cell viability of experimental groups 7–12 corresponding to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1):

[0075] The cell viability test steps for control group 2 and experimental groups 7-12 corresponding to the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) are the same as those for control group 1 and experimental groups 1-6, and will not be repeated here.

[0076] Observation results as follows Figures 2A to 2D As shown, where Figure 2A Cell images of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7–12 after 24 hours, 48 ​​hours, and 72 hours of culture, respectively. Figures 2B to 2D The graph shows the cell viability data of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7–12 after 24 hours, 48 ​​hours, and 72 hours of culture. Figures 2B to 2D The standard error (SE) data results of experimental groups 7-12 are shown. These are the detection values ​​of three independent experiments (n=3) for each of the experimental groups 7-12. "*" indicates the comparison of the values ​​of experimental groups 7-12 with respect to control group 2, and p<0.05.

[0077] like Figure 2A , Figure 2B As shown, based on the data from experimental groups 7-12 after 24 hours of culture, the cell viability of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7 and 8 showed a gradually decreasing trend compared to control group 2. This indicates that the effective doses of the herbal medicine components administered to experimental groups 7 and 8, at 0.1 mg / ml and 0.5 mg / ml respectively, could appropriately inhibit or reduce the cell growth of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). Furthermore, the cell viability of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 9-12 was significantly reduced to below 10% compared to control group 2, and the combination with... Figure 2AAs shown, the cell density observed in experimental groups 9-12 was significantly reduced compared to experimental groups 7 and 8, indicating that the effective dose of the herbal medicine composition administered to experimental groups 9-12, ranging from 1.25 mg / ml to 10.0 mg / ml, under 24-hour culture conditions, could significantly inhibit or reduce the growth of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1).

[0078] like Figure 2A , Figure 2C As shown, in the data results corresponding to 48 hours of culture in experimental groups 7-12, the cell viability rate of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7 and 8 also showed a significant decreasing trend compared to control group 2. Figure 2B and Figure 2C It can be seen that the cell viability rate of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental group 8 was significantly reduced after 48 hours of culture compared to 24 hours of culture. Furthermore, the cell viability rate of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental group 8 after 48 hours of culture was reduced to below 50%. Similarly, the cell viability rate of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 9-12 was also reduced to below 10% compared to the control group 2. The cell viability rate of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 9-12 was slightly reduced after 48 hours of culture compared to 24 hours of culture.

[0079] like Figure 2A , Figure 2D As shown, based on the data from experimental groups 7-12 after 72 hours of culture, the cell viability of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental group 7 was not significantly reduced compared to control group 2. However, the cell viability of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental group 8 decreased to below 50%, and the cell viability of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 9-12 also decreased to below 10% compared to control group 2. Furthermore, the cell viability of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 9-12 was further reduced after 72 hours of culture compared to 48 hours of culture.

[0080] Therefore, the effective dose of the herbal medicine composition between 0.1 and 10.0 mg / ml can indeed effectively inhibit or reduce the activity of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). The effective dose of the herbal medicine composition between 0.5 and 10.0 mg / ml can effectively reduce the cell activity rate of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). In a preferred embodiment, the effective dose of the herbal medicine composition between 1.25 and 10.0 mg / ml more significantly inhibits the activity of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1).

[0081] III. To investigate the inhibitory effect of experimental groups 7-12 on the migration of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1):

[0082] Cell migration assay: A 24-well suspended cell culture inserter (EMDMillipore Corporation, USA) was prepared. The upper chamber of the inserter contained the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1), and different concentrations of the herbal composition from experimental groups 7-12 were added accordingly. Serum-free medium was used in the upper chamber to limit cell growth, while the lower chamber contained standard culture medium to attract cells. The upper and lower chambers of the inserter were combined and placed in a cell culture incubator at 37°C for 24 hours. After culture... Unmigrated anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) were removed from the upper chamber of the cell culture inserter, while the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) that migrated to the lower chamber of the cell culture inserter were fixed with 4% paraformaldehyde and then stained with 0.05% Giemsa. The number of migrating anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) was observed and analyzed.

[0083] Observation results as follows Figure 3A , Figure 3B As shown, where Figure 3A Cell images of experimental groups 7–12 after 24 hours of culture for anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in migration assays. Figure 3BThe image shows cell migration data of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 7–12. Figure 3B The standard error (SE) data results of experimental groups 7-12 are shown. These are the detection values ​​of three independent experiments (n=3) for each of the experimental groups 7-12. "*" indicates the comparison of the values ​​of experimental groups 7-12 with respect to control group 2, and p<0.05.

[0084] like Figure 3A , Figure 3B As shown, in the data results corresponding to 24 hours of culture in experimental groups 7-12, the cell migration of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental group 7 was not significantly different from that in control group 2. This indicates that the effective dose of the herbal medicine composition administered to experimental group 7, 0.1 mg / ml, could not effectively inhibit the cell migration of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). Meanwhile, the cell migration of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 8 and 9 was also significantly different. The cell migration of MCF-7 / TAMR1 cells was significantly lower than that of the control group 2. In experimental groups 10-12, the cell migration of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) was significantly reduced to less than 10% compared with the control group 2. This indicates that the effective dose of the herbal medicine composition administered to experimental groups 10-12, ranging from 2.5 mg / ml to 10.0 mg / ml, under 24-hour culture conditions, can significantly inhibit the cell migration of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1).

[0085] IV. To investigate the inhibitory effect of experimental groups 7-12 on the invasion of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1):

[0086] The cell invasion assay, similar to the aforementioned cell migration assay, involves preparing a suspended cell culture inserter. A 0.4 mg / mL Matrigel is coated onto the filter membrane of the upper chamber of the inserter. Anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) are seeded onto the Matrigel, and different concentrations of the herbal medicine components from experimental groups 7-12 are added to simulate the barriers in organ tissues. This requires cells to not only migrate but also invade the Matrigel. The lower chamber of the inserter contains a standard culture medium to attract cells. After assembling the upper and lower chambers, the cells were placed in a cell culture incubator at 37°C and cultured for 24 hours. After the culture was completed, the unmigrated anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in the upper chamber of the cell culture inserter were removed, while the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) that had migrated to the lower chamber of the cell culture inserter were fixed with 4% paraformaldehyde and then stained with 0.05% Giemsa. The number of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) that migrated after invasion was observed and analyzed.

[0087] Observation results as follows Figure 4A , Figure 4B As shown, the migration rate of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental group 7 after invasion was not significantly different from that in control group 2, indicating that the effective dose of the herbal medicine composition administered to experimental group 7 (0.1 mg / ml) could not effectively inhibit the cell invasion of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). Meanwhile, the migration rate of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 8 and 9 after invasion was similar. Compared to the control group 2, the migration rate of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 10-12 was significantly reduced to below 50% after invasion, while the migration rate of these cells was significantly reduced to below 10% compared to the control group 2. This indicates that the effective doses of the herbal medicine components administered to experimental groups 10-12, ranging from 2.5 mg / ml to 10.0 mg / ml, under 24-hour culture conditions, significantly inhibited the cell invasion of anti-tamoxifen breast cancer cells (MCF-7 / TAMR1).

[0088] V. To investigate the autophagy effect of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) on experimental groups 7-12:

[0089] Autophagy Assay - Western Blot:

[0090] Each experimental group (7-12) corresponding to anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) was cultured for 24 hours to detect the autophagy activity corresponding to antibodies ATG5, ATG7, and LC3-II. First, the polyvinylidene fluoride (PVDF) membrane was cultured with the antibodies for 3 hours, then washed with phosphate-buffered saline (PBS), followed by the addition of horse-radish peroxidase (HRP) antibody and culturing for another hour. Finally, the antigen-antibody complex was detected using Immobilion Western spectroscopy, and protein expression was detected using a densitometer (GE ImageQuant TL8.1; GE Healthcare Life Sciences).

[0091] Specifically, autophagy is an intracellular physiological process that primarily involves the breakdown of old proteins within the cell, the metabolism of inactive organelles, and the removal of foreign microorganisms. Many proteins are involved in the regulation of autophagy. Microtubule-associated protein 1A / 1B-light chain 3 (LC3), located in the cytoplasm, is often used as a fluorescent marker for autophagy activation. When autophagy is activated, LC3 binds to the lipid phosphatidyl-ethanolamine to form LC3-II, which then embeds itself on the membrane of phagocytic vesicles. Therefore, the amount of LC3-II produced within vesicles can serve as an indicator of autophagy activation.

[0092] Observation results are as follows Figure 5A , Figure 5BCompared to control group 2, experimental groups 8-10 showed significantly increased LC3-II protein expression, indicating that the effective dose of the herbal composition administered to experimental groups 8-10, ranging from 0.5 mg / ml to 2.5 mg / ml, significantly induced LC3-II protein expression in anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). Experimental group 10 showed the most significant LC3-II protein expression. This demonstrates that the herbal composition, at a specific effective dose, can effectively induce autophagy in anti-tamoxifen breast cancer cells (MCF-7 / TAMR1), thereby effectively inhibiting and reducing the activity of these cells. In experimental groups 11 and 12, LC3-II expression in anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) was significantly increased. The degradation of protein I indicates that the herbal ingredients in experimental groups 11 and 12, at effective doses of 5 mg / ml and 10 mg / ml, can significantly activate LC3-II protein and induce autophagy in the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1). Figure 2B The cell activity of the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) in experimental groups 11 and 12 was significantly reduced. After the anti-tamoxifen breast cancer cells (MCF-7 / TAMR1) completed autophagy, the effective dose of the herbal medicine composition increased the expression of ATG5 and ATG7 and the degradation of LC3 I protein.

[0093] VI. To investigate the cell survival rate of the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) corresponding to the application of the traditional Chinese medicine composition:

[0094] 1. This experiment included control group 3 and experimental groups 13–18:

[0095] Control group 3: The tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were not administered the herbal composition, indicating that the effective dose of the herbal composition was 0 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours.

[0096] Experimental group 13: The herbal composition was administered to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at an effective dose of 0.1 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0097] Experimental group 14: The herbal composition was administered to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at an effective dose of 0.5 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0098] Experimental group 15: The herbal composition was administered to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at an effective dose of 1.25 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0099] Experimental group 16: The herbal composition was administered to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at an effective dose of 2.5 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0100] Experimental group 17: The herbal composition was administered to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at an effective dose of 5.0 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0101] Experimental group 18: The herbal composition was administered to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at an effective dose of 10.0 mg / ml, and the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) were cultured for 24 hours, 48 ​​hours and 72 hours after administration.

[0102] 2. Analyze the cell viability of the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) corresponding to experimental groups 13-18:

[0103] The cell viability assay procedures for the control group 3 and the experimental groups 13-18 corresponding to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) are the same as those for the control group 1 and the experimental groups 1-6, and will not be repeated here.

[0104] Observation results as follows Figures 6A to 6D As shown, where Figure 6A Images of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) from experimental groups 13–18, after 24, 48, and 72 hours of culture, respectively. Figures 6B to 6D The graphs show the cell viability data of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) from experimental groups 13-18 after 24 hours, 48 ​​hours, and 72 hours of culture. Figures 6B to 6D The standard error (SE) data results of experimental groups 13-18 are shown. These are the detection values ​​of three independent experiments (n=3) of cell experiments of each experimental group 13-18. "*" indicates the comparison of the values ​​of experimental groups 13-18 with respect to control group 3, and p<0.05.

[0105] like Figure 6A , Figure 6BAs shown, based on the data from experimental groups 13-18 after 24 hours of culture, the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 13-15 was not significantly reduced compared to control group 3. However, the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 16-18 was significantly reduced to below 50%, with the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 17 and 18 further reduced to below 10%. The cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 18 was significantly reduced compared to experimental group 17, and... Figure 6A As shown, the cell density observed in experimental groups 16-18 was significantly reduced compared to experimental groups 13-15. This indicates that the effective doses of the herbal medicine composition administered to experimental groups 16-18, ranging from 2.5 mg / ml to 10.0 mg / ml, under 24-hour culture conditions, significantly inhibited or reduced the growth of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5). Furthermore, the effective dose of the herbal medicine composition administered to experimental group 18, at 10.0 mg / ml, showed a more effective inhibitory effect on the growth of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5).

[0106] like Figure 6A , Figure 6CAs shown, in the data results of 48 hours of culture for each of the experimental groups 13-18, the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13 did not show a significant decrease. However, the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 14 and 15 showed a gradually decreasing trend compared to the control group 3. This indicates that the effective doses of the herbal medicine components administered to experimental groups 14 and 15 (0.5 mg / ml and 1.25 mg / ml, respectively) after 48 hours of culture could appropriately inhibit or reduce the cell growth of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5). Furthermore, the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 16-18 was significantly reduced to below 50%. Figure 6B and Figure 6C It can be seen that the experimental group 18, after 48 hours of culture, reduced the cell viability of the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) more than after 24 hours of culture.

[0107] like Figure 6A , Figure 6D As shown, in the data results corresponding to 72 hours of culture for each of the experimental groups 13-18, the cell viability rate of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13 did not show a significant decrease. However, the cell viability rate of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 14 and 15 showed a further decreasing trend compared to the control group 3. Figure 6C and Figure 6D It can be seen that the 72-hour culture time in experimental group 15 significantly reduced the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) compared to 48 hours of culture. Similarly, the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 16-18 was also significantly reduced to below 50%.

[0108] Therefore, the effective dose of the herbal medicine composition between 0.5 and 10.0 mg / ml can indeed effectively inhibit or reduce the activity of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5). The effective dose of the herbal medicine composition between 2.5 and 10.0 mg / ml can effectively reduce the cell activity rate of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5). In a preferred embodiment, the effective dose of the herbal medicine composition between 5.0 and 10.0 mg / ml more significantly inhibits the activity of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5).

[0109] VII. To investigate the inhibitory effect of experimental groups 13-18 on the migration of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5):

[0110] The migration assay and invasion assay procedures for the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in control group 3 and experimental groups 13-18 are the same as those for control group 2 and experimental groups 7-13, and will not be repeated here.

[0111] Cell migration assay results as follows Figure 7A , Figure 7B As shown, where Figure 7A Cell images of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) cultured for 24 hours in each of the experimental groups 13-18 during the migration assay; Figure 7B The image shows cell migration data of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) corresponding to experimental groups 13-18. Figure 7B The standard error (SE) data results of experimental groups 13-18 are shown. These are the detection values ​​of three independent experiments (n=3) of cell experiments of each experimental group 13-18. "*" indicates the comparison of the values ​​of experimental groups 13-18 with respect to control group 3, and p<0.05.

[0112] like Figure 7A , Figure 7BAs shown, in the data results corresponding to 24 hours of culture for each of the experimental groups 13-18, the cell migration of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 13 was slightly lower than that in control group 3. However, the cell migration of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 14 and 15 began to show a significant decrease compared to control group 3. In experimental groups 16-18... The migration rate of the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) was significantly reduced to less than 10% compared to the control group 3, indicating that the effective doses of the herbal composition administered to experimental groups 16-18, ranging from 2.5 mg / ml to 10.0 mg / ml, under 24-hour culture conditions, could significantly inhibit the cell migration of the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5).

[0113] 8. To investigate the inhibitory effect of experimental groups 13-18 on the invasion of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5):

[0114] Cell invasion results as follows Figure 8A , Figure 8B As shown, the migration rate of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 13-18 after invasion was significantly lower than that in control group 3. Specifically, the migration rate of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 14-18 decreased to below 50%, and the migration rate of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental group 18 was significantly reduced to below 10%. This indicates that the effective dose of the herbal medicine composition administered to experimental groups 13-18, ranging from 0.1 mg / ml to 10.0 mg / ml, under 24-hour culture conditions, could effectively inhibit the cell invasion of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5).

[0115] IX. To investigate the autophagy effect on tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) corresponding to experimental groups 13-18:

[0116] The Western ink dot procedure and detection of ATG5, ATG7 and LC3-II protein expression in control group 3 and experimental groups 13-18 corresponding to the tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) are the same as those in control group 2 and experimental groups 7-13, and will not be repeated here.

[0117] See protein expression results Figure 9A , Figure 9B Compared to the control group 3, experimental groups 16-18 showed a significant increase in LC3-II protein expression, indicating that the effective dose of the herbal composition administered to experimental groups 16-18, ranging from 2.5 mg / ml to 10.0 mg / ml, could significantly induce LC3-II protein expression in tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5), equivalent to... Figure 6B The results showed that the cell viability of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) in experimental groups 16-18 was significantly reduced to below 50%. Figure 9B The experimental group 17 showed the most significant expression of LC3-II protein, indicating that the herbal composition can effectively induce autophagy in tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) at a specific effective dose, thereby effectively inhibiting and reducing the activity of tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5).

[0118] In summary, the weight ratio of the selected herbs (Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare) in the herbal composition, as confirmed by cell experiments, can effectively inhibit or reduce the activity of breast cancer cells. Furthermore, the effective dose of the herbal composition, between 0.1 and 10.0 mg / ml, can effectively inhibit the activity of conventional breast cancer cells (MCF-7), anti-tamoxifen breast cancer cells (MCF-7 / TAMR1), and tamoxifen-sensitive breast cancer cells (MCF-7 / SO.5) within a culture time of at least 24 hours, and correspondingly reduce the number of these cells. The breast cancer cells in question are the conventional breast cancer cells (MCF-7). When the breast cancer cells are tamoxifen-resistant breast cancer cells (MCF-7), the effective dose of the traditional Chinese medicine composition is between 5.0 and 10.0 mg / ml, exhibiting superior inhibitory activity against conventional breast cancer cells (MCF-7). When the breast cancer cells are tamoxifen-sensitive breast cancer cells (MCF-7 / 0.5), the effective dose of the traditional Chinese medicine composition is between 2.5 and 10.0 mg / ml, exhibiting superior inhibitory activity against tamoxifen-sensitive breast cancer cells (MCF-7 / 0.5). Therefore, the appropriate proportions of the traditional Chinese medicine composition, combined with specific effective doses, are used to treat breast cancer symptoms or inhibit breast cancer cell activity.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art, upon referring to the above teachings, can make appropriate modifications to the content of the above embodiments or achieve the technical effects claimed by the present invention according to different experimental methods.

Claims

1. A traditional Chinese medicine composition for inhibiting or reducing breast cancer cells, comprising Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare, wherein the weight ratio of Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare in the traditional Chinese medicine composition is 1-2:4-6:2-3:3-5:2-4:2-3.

2. The traditional Chinese medicine composition for inhibiting or reducing breast cancer cells as described in claim 1, wherein the breast cancer cells are selected from one of the populations consisting of conventional breast cancer cells, anti-tamoxifen breast cancer cells, and tamoxifen-sensitive breast cancer cells.

3. The use of a traditional Chinese medicine composition for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the traditional Chinese medicine composition comprises Euphorbia kansui, Paeonia lactiflora, Glycyrrhiza uralensis, Taraxacum mongolicum, Gardenia jasminoides, and Hordeum vulgare malt, wherein the traditional Chinese medicine composition is administered to the breast cancer cells at an effective dose between 0.1 mg / ml and 10.0 mg / ml for culture, and wherein the traditional Chinese medicine composition exhibits inhibitory activity against the breast cancer cells, wherein the breast cancer cells are selected from one of the populations consisting of conventional breast cancer cells, anti-tamoxifen breast cancer cells, and tamoxifen-sensitive breast cancer cells.

4. The use of the traditional Chinese medicine composition as described in claim 3 for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the weight ratio of the following herbs in the traditional Chinese medicine composition is 1-2:4-6:2-3:3-5:2-4:2-3.

5. The use of the traditional Chinese medicine composition as described in claim 4 for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the breast cancer cells are conventional breast cancer cells, the effective dose of the traditional Chinese medicine composition is between 5.0 mg / ml and 10.0 mg / ml, and the culture time of the traditional Chinese medicine composition applied to the breast cancer cells is between 24 hours and 72 hours.

6. The use of the traditional Chinese medicine composition as described in claim 4 for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the breast cancer cells are anti-tamoxifen breast cancer cells, the effective dose of the traditional Chinese medicine composition is between 0.5 mg / ml and 10.0 mg / ml, and the culture time of the traditional Chinese medicine composition applied to the breast cancer cells is between 24 hours and 72 hours.

7. The use of the traditional Chinese medicine composition as described in claim 6 for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the traditional Chinese medicine composition is administered to the anti-tamoxifen breast cancer cells at an effective dose between 0.5 mg / ml and 2.5 mg / ml to induce the expression of LC3-II protein and trigger autophagy in the anti-tamoxifen breast cancer cells.

8. The use of the traditional Chinese medicine composition as described in claim 4 for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the breast cancer cells are tamoxifen-sensitive breast cancer cells, the effective dose of the traditional Chinese medicine composition is between 2.5 mg / ml and 10.0 mg / ml, and the culture time of the traditional Chinese medicine composition applied to the breast cancer cells is between 24 hours and 72 hours.

9. The use of the traditional Chinese medicine composition as described in claim 8 for preparing an anticancer drug that inhibits or reduces the activity of breast cancer cells, wherein the traditional Chinese medicine composition is administered to the tamoxifen-sensitive breast cancer cells at an effective dose between 2.5 mg / ml and 10.0 mg / ml to induce the expression of LC3-II protein and trigger autophagy in the tamoxifen-sensitive breast cancer cells.

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