Application of Rupikang capsules in preparation of anti-breast cancer drugs
By applying Rupikang capsules to anti-breast cancer drugs, the adverse reaction problem of existing treatment methods has been solved, achieving a highly effective and low-toxicity treatment effect for breast cancer, with significant inhibitory and apoptosis-promoting effects.
Patent Information
- Application Number
- CN202511947889.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-01-23
AI Technical Summary
Existing breast cancer treatments have many adverse reactions and lack highly effective and low-toxicity natural drug resources to improve cure rates and quality of life.
Rupikang capsules are used in the preparation of anti-breast cancer drugs. They reduce tumor volume and weight by inhibiting the proliferation and migration of breast cancer cells and promoting apoptosis. They are used in the form of capsules, tablets, granules and other forms.
It significantly inhibits the proliferation and migration of breast cancer cells and promotes apoptosis. Both in vivo and in vitro experiments showed anti-breast cancer effects, and the safety profile was high, with no significant impact on mouse body weight.
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Figure CN121371108A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Rupikang capsules in preparation of anti-breast cancer drugs. BACKGROUND
[0002] Breast cancer is a high-incidence female malignant tumor worldwide, which has the characteristics of complex pathogenesis and rapid disease development. The number of female breast cancer patients and the number of deaths in China rank first in the world. Breast cancer has become the main cause of female cancer death, which seriously endangers the life and health safety of women. Breast cancer is a highly heterogeneous disease, and different subtypes have great differences in the choice of treatment methods, sensitivity and efficacy. Current treatment methods for different subtypes include endocrine therapy, targeted therapy, chemotherapy, immunotherapy and surgery. Although these treatment methods have certain effects on breast cancer patients, they also have many adverse reactions. Therefore, it is necessary to further optimize the treatment strategy and find new treatment drugs to improve the cure rate and quality of life of patients.
[0003] Traditional Chinese medicine prescriptions have the characteristics of multiple components and multiple targets, can produce biological regulation on target genes and signal pathways, effectively inhibit breast cancer cell proliferation and induce apoptosis or reduce the drug resistance of cancer cells to chemotherapy and endocrine therapy, and are an important source of breast cancer treatment drugs. Rupikang capsules are traditional Chinese medicine, which has the functions of soothing liver and regulating qi, promoting blood circulation and removing blood stasis, and are often used in clinical practice for breast hyperplasia and breast swelling caused by liver stagnation and blood stasis. The drug is a capsule, the content is brown to brownish particles, and the drug composition includes spirea prunifolia, orange leaf, salvia miltiorrhiza, safflower, curcuma zedoary, kudzu vine, and earthworm. Currently, there is no literature report on the research and application of Rupikang capsules in the treatment of breast cancer. SUMMARY
[0004] The purpose of the present application is to find new natural drug resources with high efficiency and low toxicity for breast cancer treatment drugs, and to improve the cure rate and improve the prognosis and quality of life of patients with breast cancer.
[0005] In order to achieve the above purpose, the application provides the application of Rupikang capsules in the preparation of anti-breast cancer drugs, and the breast cancer cells include human breast cancer cells MDA-MB-231, MDA-MB-468, BT-549 and mouse breast cancer cells 4T1-Luc.
[0006] The application of Rupikang capsules in the preparation of anti-breast cancer drugs has at least one of the following effects:
[0007] (1) Inhibiting the proliferation and migration of breast cancer cells and promoting the apoptosis of breast cancer cells.
[0008] (2) reducing the volume and weight of breast tumors.
[0009] The anti-breast cancer drug of the present application is prepared by directly preparing the Rupikang capsule into an anti-breast cancer drug, or combining with other anti-breast cancer drugs to prepare a compound drug, or combining with a pharmaceutically acceptable carrier to prepare an anti-breast cancer drug. The drug is any one of injection, capsule, tablet, granule allowed in pharmacy.
[0010] The beneficial effects of the present application are as follows:
[0011] The present application first discovers the new efficacy of Rupikang capsule in resisting breast cancer. In vitro studies show that Rupikang capsule can significantly inhibit the viability of human breast cancer cells MDA-MB-231, MDA-MB-468 and BT-549, inhibit the proliferation and migration of breast cancer cells, promote the apoptosis of breast cancer cells, and change the morphology of breast cancer cells, thereby achieving the effect of treating breast cancer; in vivo studies show that after Rupikang capsule treatment, the bioluminescence signal intensity of mouse breast tumors can be significantly reduced, the tumor volume and tumor weight of tumor-bearing mice can be reduced, the growth of mouse breast tumors can be inhibited, and the effect of resisting breast cancer can be played, and Rupikang capsule treatment has no significant effect on the body weight of mice, and has high safety. Therefore, Rupikang capsule has potential value in the preparation of drugs for preventing and treating breast cancer or breast cancer cell proliferation inhibition. And Rupikang capsule, as a traditional Chinese medicine that has been used in clinical practice, can be directly applied to the human body without the need for further clinical safety evaluation, has good application prospect, and can quickly improve the market share of this product. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 is a cell morphology real photo of human breast cancer cells MDA-MB-231, MDA-MB-468 and BT-549 treated with different concentrations of Rupikang capsules.
[0013] Figure 2 is the effect of different concentrations of Rupikang capsules on the proliferation of MDA-MB-231, MDA-MB-468 and BT-549 cells.
[0014] Figure 3 is the detection of the number of colonies formed after 24 h of MDA-MB-231, MDA-MB-468 and BT-549 cells treated with different concentrations of Rupikang capsules.
[0015] Figure 4 is the effect of different concentrations of Rupikang capsules on the horizontal migration of MDA-MB-231, MDA-MB-468 and BT-549 cells.
[0016] Figure 5PI staining detection results of MDA-MB-231, MDA-MB-468 and BT-549 cells after 24 h of action of different concentrations of Rupikang capsules.
[0017] Figure 6 Flow cytometry detection results of apoptosis of MDA-MB-231, MDA-MB-468 and BT-549 cells after 24 h of action of different concentrations of Rupikang capsules.
[0018] Figure 7 A is a representative image of in vivo imaging of tumor-bearing mice in different groups, B is the change of tumor size in different groups, C is the change of tumor weight in different groups, and D is the tumor growth curve in different groups.
[0019] Figure 8 Effects of Rupikang capsules on the histopathological changes of tumors, hearts, livers, kidneys, spleens and lungs of tumor-bearing mice.
[0020] Figure 9 Effects of Rupikang capsules on the body weight of tumor-bearing mice. DETAILED DESCRIPTION
[0021] The application will be further described in detail below in conjunction with the examples and the accompanying drawings, but the scope of protection of the application is not limited to these examples.
[0022] Example 1
[0023] Preparation of sample solution: The contents of Rupikang capsules were prepared into a sample stock solution with a concentration of 100 mg / mL using PBS buffer. Different concentrations of Rupikang capsules were prepared by diluting the stock solution with PBS buffer.
[0024] Preparation of 5 mg / mL MTT solution: 0.2 g of MTT powder was weighed, 40 mL of sterilized double-distilled water was added, and it was dissolved at 60°C. The solution was filtered through a 0.2 µm filter membrane to remove bacteria and stored at 4°C.
[0025] Cell lines: Human breast cancer cells MDA-MB-231, MDA-MB-468, BT-549 and mouse breast cancer cells 4T1-Luc were all preserved by Guizhou Province Center for Natural Products Research. BT-549 cells were cultured in RPMI Medium 1640 medium containing 10% FBS, and other cells were cultured in DMEM medium containing 10% FBS in a 37°C, 5% CO2 incubator.
[0026] 1. Cell morphology observation
[0027] MDA-MB-231, MDA-MB-468 and BT-549 cells were respectively inoculated at 1.5×10 5The cells were inoculated in 6-well plates at 1,000 cells / well and incubated in an incubator at 37°C overnight. After the cells adhered, MDA-MB-231 and BT-549 cells were treated with Rupikang capsules at different concentrations (2.5, 3.5 and 4.5 mg / mL) and MDA-MB-468 cells were treated with Rupikang capsules at different concentrations (3, 4 and 5 mg / mL) for 24 h, with PBS buffer treatment as a control. The morphological changes of the cells in each group were observed under an inverted microscope at 200-fold magnification and a scale of 100 μm, as shown in FIG. 1. Figure 1 The results showed that, as the concentration of Rupikang capsules increased, the cell density in the drug-treated groups decreased significantly in a concentration-dependent manner, and the cell shrinkage and damage increased with the increase of the concentration, as compared with the control group.
[0028] 2. Detection of cell proliferation by MTT method
[0029] The cells in good condition were collected and inoculated in 96-well plates (MDA-MB-231, MDA-MB-468 and BT-549 cells: 6,000 cells / well). 100 μL sterilized water was added around the 96-well plates. After the cells adhered and grew, they were treated with Rupikang capsules at different concentrations (5, 4, 3, 2 and 1 mg / mL) and with PBS buffer treatment as a control. After the drug treatment for 0 h, 24 h, 48 h, 72 h and 96 h, 10 μL of 5 mg / mL MTT solution was added to each well, and the plates were placed in an incubator for 4 h. After centrifugation, the blue-purple crystals were retained at the bottom of the plates, 160 μL of DMSO was added to each well, and the OD value of each well was determined by an enzyme-labeled instrument at a wavelength of 490 nm after complete dissolution. The inhibition rate (I) of Rupikang capsules on the cells was calculated according to Formula (1).
[0030] I=[(A C -A D -A B ) / A C ]×100% (1)
[0031] In the formula, A C is the OD value of the blank control group; A D is the OD value of the drug-treated group; and A B is the OD value of the drug solution at the same concentration.
[0032] Figure 2 The results of the detection showed that, as compared with the control group, Rupikang capsules could significantly inhibit the proliferation of breast cancer cells MDA-MB-231, MDA-MB-468 and BT-549 in a concentration-dependent manner.
[0033] 3. Detection of cell viability by plate colony formation method
[0034] MDA-MB-231, MDA-MB-468, and BT-549 cells were cultured at 2.0 × 10⁻⁶. 5 Cells were seeded at 2000 cells / well in 6-well plates. After cell adhesion, MDA-MB-231 and BT-549 cells were treated with different concentrations (final concentrations of 2.5, 3.5, and 4.5 mg / mL) of Rupikang capsules; MDA-MB-468 cells were treated with different concentrations (final concentrations of 3, 4, and 5 mg / mL) of Rupikang capsules. PBS buffer treatment served as a control. All treatments lasted 24 h. Cells were collected and counted. Cells were re-seeded at 2000 cells / well in new 6-well plates, with the culture medium changed every 3 days for 14 consecutive days. The supernatant was removed, and cells were fixed with 1 mL of methanol for 30 min. Then, 500 μL of crystal violet was added to each well for staining in the dark for 15 min. Cells were washed with PBS buffer, air-dried, and images were taken.
[0035] Figure 3 The results showed that, compared with the control group, Rupikang capsules significantly reduced the number of colonies formed by MDA-MB-231, MDA-MB-468 and BT-549 cells.
[0036] 4. Scratch-call assay for detecting cell-level migration
[0037] Draw a straight line every 1 cm on the back of a 6-well plate using a marker. Seed MDA-MB-231, MDA-MB-468, and BT-549 cells in the labeled 6-well plates and culture until the cells adhere and congregate on the bottom of the plate. Use a 10 μL pipette tip perpendicular to the wells and the lines to scratch the cells. Discard the old culture medium, gently wash the cells three times with sterile PBS buffer to remove the scratched cells, and then treat MDA-MB-231 and BT-549 cells with culture medium containing 2.5, 3.5, and 4.5 mg / mL of Rupikang capsules, respectively; treat MDA-MB-468 cells with culture medium containing 3, 4, and 5 mg / mL of Rupikang capsules, respectively. PBS buffer treatment serves as a control. Incubate at 37°C and photograph at 0 and 24 h. Measure the distance of the scratches at each time point using Image Pro Plus 6.0 software and calculate the cell migration rate using the following formula.
[0038] Migration rate (%) = (0 h scratch distance - 24 h scratch distance) / 0 h scratch distance × 100%
[0039] Figure 4 Scratch-call results showed that, compared with the control group, Rupikang capsules inhibited the horizontal migration ability of MDA-MB-231, MDA-MB-468 and BT-549 cells.
[0040] 5. PI staining to detect the degree of cell damage
[0041] MDA-MB-231, MDA-MB-468 and BT-549 cells were seeded at 1.5 x 10 5 cells / well in 6-well plates and incubated in a 37°C incubator overnight. After the cells adhered, MDA-MB-231 and BT-549 cells were treated with different concentrations (final concentrations of 2.5, 3.5 and 4.5 mg / mL) of Rupikang capsules, and MDA-MB-468 cells were treated with different concentrations (final concentrations of 3, 4 and 5 mg / mL) of Rupikang capsules, while PBS buffer treatment was used as a control, all for 24 h. The cells were washed with culture medium for 3 times, 500 μL of PI solution prepared with culture medium (10 μL of PI staining solution was added to 500 μL of culture medium to make the final concentration of PI staining solution 20 μg / mL) was added, and the cells were incubated in a 37°C incubator for 20 min. After washing with culture medium for 3 times, the cells were photographed under an inverted fluorescence microscope, magnification: 200x, scale: 100 μm.
[0042] Figure 5 The PI staining results showed that there were more PI positive cells in the drug-treated groups than in the control group, indicating that Rupikang capsules increased the degree of cell membrane damage in a concentration-dependent manner.
[0043] 6. Flow cytometry to detect cell apoptosis
[0044] MDA-MB-231, MDA-MB-468 and BT-549 cells were seeded at 1.5 x 10 5 cells / well in 6-well plates and incubated in a 37°C incubator overnight. After the cells adhered, MDA-MB-231 and BT-549 cells were treated with different concentrations (final concentrations of 2.5, 3.5 and 4.5 mg / mL) of Rupikang capsules, and MDA-MB-468 cells were treated with different concentrations (final concentrations of 3, 4 and 5 mg / mL) of Rupikang capsules, while PBS buffer treatment was used as a control, all for 24 h. Centrifugation (1000 rpm, 5 min), collect cells, resuspend with 1 mL PBS buffer, centrifugation (1200 rpm, 5 min), remove supernatant, resuspend cells with 50 μL of 1x binding buffer, and add 2.5 μL of FITC-Annexin V and PI dye respectively, incubate in the dark for 15 min, centrifugation (1000 rpm, 5 min), remove supernatant. Resuspend with 400 μL of 1x binding buffer, detect cell apoptosis by flow cytometry, and analyze data with NovoExpress 1.3.4 software.
[0045] Figure 6The flow cytometry results showed that, compared with the control group, different concentrations of Rupikang capsules could promote the apoptosis of MDA-MB-231, MDA-MB-468 and BT-549 cells, and the apoptosis rate increased with the increase of concentration.
[0046] 7. In vivo experiment of mice
[0047] (1) Establishment of mouse breast cancer model
[0048] 4-6 week old female BALB / c mice were raised in sterile cages with free access to food and water. 4T1-Luc cells were cultured in DMEM medium containing 10% fetal bovine serum. When the cells were subcultured to a stable state and the density was close to 80%, the cells were collected at a density of 5.0 x 10 5 4T1-Luc cells were inoculated into the mammary fat pad of mice at a density of 5.0 x 10
[0049] After the modeling was completed, the body weight and tumor size were measured every other day, and the tumor volume was calculated using the formula Volume (mm 3 )=1 / 2 x a x b 2 , where a and b are the long and short diameters of the tumor, respectively. After the model was successfully established, the mice were divided into groups (control group, low concentration group, high concentration group, 10 mice in each group), the low concentration group was given Rupikang capsules at a concentration of 1.32 g / kg for gavage, the high concentration group was given Rupikang capsules at a concentration of 2.64 g / kg for gavage, and the control group was given the same volume of normal saline for gavage every day. The tumor size of the mice was observed, and when the tumor volume of the mice reached the preset observation standard, D-Luciferin sodium salt was injected, and the bioluminescence intensity of the tumor in the mice was detected using a mouse in vivo imaging system. After the mice were anesthetized and sacrificed, the tumor weight was recorded.
[0050] (2) HE staining
[0051] The tissues separated from different groups were fixed with 4% paraformaldehyde for 24 h, then the excess fixing solution was removed and dehydrated with different concentrations of ethanol (70%, 80% and anhydrous ethanol in turn). After dehydration, residual ethanol was removed with xylene, then paraffin embedding was performed. After embedding, slices were cut at a thickness of 5 μm, placed in heated water to flatten, then attached to glass slides and placed in an incubator for drying. After drying, deparaffinization was performed with xylene, then different concentrations of ethanol (70%, 80% and anhydrous ethanol in turn) were used for treatment, then the slices were placed in distilled water for sufficient hydration. Then hematoxylin staining was performed for 7 min, the dye was washed away with running water, 1% hydrochloric acid alcohol differentiation solution was used for differentiation for 30 s, and the slices were washed with running water for 10 min. After eosin staining for 30 s, the dye was washed away with running water. After drying in an incubator, the slices were cooled and observed under a microscope for photography.
[0052] AsFigure 7 As shown in Figures A, B, and C, compared to the control group, the bioluminescence intensity of the tumor site in the Rupikang capsule treatment group was significantly reduced, and the tumor volume and weight in the 2.64 g / kg Rupikang capsule treatment group were significantly lower than those in the control group. Figure 7 As shown in Figure D, the tumor volume measurement results in mice showed that, compared with the control group, the tumor growth rate in the 2.64 g / kg Rupikang capsule treatment group was significantly lower after 12 days. Histopathological observation after HE staining showed that the tumor tissue in the control group exhibited frequent mitotic figures, inconsistent nuclei size, and an imbalanced nucleus-cytoplasm ratio. In the 2.64 g / kg Rupikang capsule treatment group, the tumor tissue showed reduced mitotic figures, relatively uniform nuclei, and a relatively balanced nucleus-cytoplasm ratio. To clarify the effects of Rupikang capsules on the heart, liver, spleen, lungs, and kidneys of mice, the pathological characteristics of these organs were observed after HE staining. Figure 8 The results showed that, compared with the control group, no significant pathological changes were observed in the heart, liver, spleen, lungs, and kidneys in any group. Figure 9 The results of mouse weight measurement showed that, compared with the control group, the Rupikang capsule treatment group had no significant effect on mouse weight.
Claims
1. Use of Rupikang Capsule in the preparation of a drug for resisting breast cancer.
2. Use of the Rupikang Capsules according to claim 1 in the preparation of an anti-breast cancer drug, characterized in that: The drug is a drug for inhibiting the proliferation and migration of breast cancer cells.
3. Use of the Rupikang Capsules according to claim 1 in the preparation of an anti-breast cancer medicament, characterized in that: The drug is a drug for promoting the apoptosis of breast cancer cells.
4. The use of the Rupiqiang capsule according to any one of claims 1 to 3 in the preparation of an anti-breast cancer drug, characterized in that: The breast cancer cells are any one or more of human breast cancer cells MDA-MB-231, MDA-MB-468, BT-549 and mouse breast cancer cells 4T1-Luc.
5. The use of the Rupiqiang capsule according to any one of claims 1 to 3 in the preparation of an anti-breast cancer drug, characterized in that: The drug is a drug for resisting breast cancer prepared directly from Rupikang Capsule, a compound drug prepared in combination with other drugs for resisting breast cancer, or a drug for resisting breast cancer prepared in combination with a pharmaceutically acceptable carrier.
6. The use of the rupi kang capsule according to claim 5 in the preparation of an anti-breast cancer drug, characterized in that: The drug is any one of injection, capsule, tablet and granule allowed in pharmacy.