Application of Tibetan capillary extract in preparation of medicine for treating breast cancer
The inhibition of the proliferation and tumor-promoting factor expression of cancer-related fibroblasts through the extract of cephalosing alcohol has solved the problem of insufficient early screening and diagnosis in breast cancer treatment, and achieved effective inhibition of breast cancer.
Patent Information
- Application Number
- CN202510204608.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-24
- Publication Date
- 2025-05-16
AI Technical Summary
In the prior art, no application of cynomolectomycetes in the treatment of breast cancer, and the treatment of breast cancer still has problems of early screening and insufficient diagnosis, resulting in poor treatment effect.
By extracting alcohol extracts from Cangyinchen, the apoptosis of cancer-related fibroblasts is promoted, and its proliferation is inhibited, and the expression of tumor-promoting factors such as NF-κB and VEGF are downregulated, thereby inhibiting angiogenesis and metastasis of breast cancer.
It effectively inhibits the invasion and metastasis of breast cancer mediated by cancer-related fibroblasts, provides a new strategy for the treatment of breast cancer, and provides a scientific basis for the development and utilization of Tibetan Yinchen.
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Figure CN120000697A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of Tibetan Artemisia Capillaris extract and new application thereof, and in particular to application of the Tibetan Artemisia Capillaris extract in preparing a medicine for treating breast cancer. Background Art
[0002] Breast cancer, the most common malignant tumor in women worldwide, accounts for 24.5% of all new cancers in women, according to the World Health Organization's 2020 Global Cancer Report. Especially in areas with scarce medical resources, due to the lack of early screening and diagnosis, many patients are already in the late stage when diagnosed, and the treatment effect is greatly reduced, and the mortality rate also rises. Therefore, in-depth research on the occurrence and treatment mechanism of breast cancer, especially the expression of key molecules and their regulatory mechanisms in tumor invasion and metastasis, is particularly urgent, which is directly related to the improvement of patient survival rate.
[0003] Tumor microenvironment (TME) plays a core role in the occurrence, development and metastasis of tumors. Among them, cancer-associated fibroblasts (CAFs), as a key component of TME, significantly promote the proliferation, invasion and migration of tumor cells by secreting growth factors, cytokines and remodeling the extracellular matrix. Research on CAFs is expected to provide new strategies for the treatment of breast cancer.
[0004] Epithelial membrane protein 1 (EMP1), a transmembrane protein belonging to the PMP22 / EMP family, has attracted widespread attention in recent years for its abnormal expression in a variety of tumors. It is involved in the proliferation, invasion and metastasis of tumors, but its role is complex and dual: it promotes cancer in some tumors, while it suppresses cancer in others. Even at different stages of development of the same cancer, the biological behavior characteristics of EMP1 may be completely different. There is still controversy in the academic community about the specific role of EMP1 in the progression of breast cancer and colorectal cancer.
[0005] Tibetan Artemisia capillaris, also known as Swertiamussotii Franch., is one of the eight treasures of Tibetan medicine that enjoys a high reputation in the history of Tibetan medicine. It is famous for its multiple functions such as clearing away heat and detoxification, soothing the liver and regulating qi, promoting bile and relieving jaundice, and protecting the liver. It was first recorded in the Tibetan medicine classic "Four Medical Classics". It not only performs well in the treatment of common diseases such as colds, fever, sore throats, and oral ulcers, but also has significant therapeutic effects in improving liver function, treating icteric hepatitis, and cholecystitis. Modern pharmacological studies have also confirmed that Tibetan Artemisia capillaris has a significant liver-protecting effect, can effectively reduce the increase of alanine aminotransferase in mice caused by carbon tetrachloride, alleviate liver cell lesions, and antagonize liver damage caused by acetaminophen. Tibetan Artemisia capillaris, which is known as the "Three Wonders of Tibetan Medicine" together with saffron and cordyceps sinensis, has a profound cultural heritage and a wide application base among the Tibetan people.
[0006] However, despite the rich application history and remarkable therapeutic effects of Tibetan Artemisia Capillaris in traditional Chinese medicine, there are no reports on its application in the treatment of breast cancer in the prior art. Summary of the invention
[0007] In order to solve the above problems, the present invention provides the use of Tibetan Artemisia Capillaris extract in preparing a medicine for treating breast cancer.
[0008] In a first aspect, the present invention provides a use of a Tibetan Artemisia capillaris extract in preparing a medicament for treating breast cancer.
[0009] Furthermore, the breast cancer includes triple-negative breast cancer.
[0010] Furthermore, the Tibetan Artemisia capillaris extract promotes apoptosis of cancer-associated fibroblasts and inhibits their proliferation, thereby inhibiting angiogenesis and metastasis of breast cancer mediated by cancer-associated fibroblasts.
[0011] Furthermore, the Tibetan Artemisia capillaris extract inhibits the growth and metastasis of breast cancer mediated by cancer-associated fibroblasts by down-regulating the expression levels of tumor-promoting factors NF-κB and VEGF secreted by cancer-associated fibroblasts.
[0012] Furthermore, the Tibetan Artemisia capillaris extract combines with EMP-1 and its related NF-κB pathway proteins to inhibit breast cancer tumor growth and metastasis.
[0013] Furthermore, the EMP1 is downregulated in Lumina breast cancer; the EMP1 is upregulated in TNBC breast cancer; and the EMP1 is positively correlated with the expression of cancer-associated fibroblast marker αSMA.
[0014] Furthermore, the Tibetan Artemisia capillaris extract inhibits the invasive ability of breast cancer mediated by cancer-associated fibroblasts by downregulating EMP1 and its downstream inflammatory pathway.
[0015] Furthermore, the Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris;
[0016] The preparation method of the alcohol extract of Tibetan Artemisia capillaris comprises the following steps:
[0017] The crushed Tibetan Artemisia capillaris is added into a 90% by volume ethanol solution for soaking at room temperature, and then reflux extraction and filtration are performed to obtain an ethanol extract;
[0018] The ethanol extract is concentrated and dried to obtain the alcohol extract of Artemisia capillaris.
[0019] In a second aspect, the present invention provides an EMP1 inhibitor, wherein the EMP1 inhibitor is an extract of Capillaris ternata.
[0020] Furthermore, the Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris;
[0021] The preparation method of the alcohol extract of Tibetan Artemisia capillaris comprises the following steps:
[0022] The crushed Tibetan Artemisia capillaris is added into a 90% by volume ethanol solution for soaking at room temperature, and then reflux extraction and filtration are performed to obtain an ethanol extract;
[0023] The ethanol extract is concentrated and dried to obtain the alcohol extract of Artemisia capillaris.
[0024] In a third aspect, the present invention provides a preparation for inhibiting CAF infiltration in triple-negative breast cancer, wherein the effective ingredient of the preparation is a Tibetan Artemisia capillaris extract.
[0025] Furthermore, the Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris;
[0026] The preparation method of the alcohol extract of Tibetan Artemisia capillaris comprises the following steps:
[0027] The crushed Tibetan Artemisia capillaris is added into a 90% by volume ethanol solution for soaking at room temperature, and then reflux extraction and filtration are performed to obtain an ethanol extract;
[0028] The ethanol extract is concentrated and dried to obtain the alcohol extract of Artemisia capillaris.
[0029] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:
[0030] The embodiments of the present invention provide the use of Tibetan Artemisia Capillaris extract in the preparation of a drug for treating breast cancer. The present invention discovers for the first time that the Tibetan Artemisia Capillaris extract inhibits the invasive ability of breast cancer mediated by cancer-associated fibroblasts by downregulating EMP1 and its downstream inflammatory pathway, thereby providing a scientific basis for the use of Tibetan Artemisia Capillaris traditional Chinese medicine in the prevention and treatment of breast cancer, promoting the development and utilization of Tibetan Artemisia Capillaris traditional Chinese medicine, and having broad practical application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.
[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.
[0033] Figure 1 The in vitro and in vivo experimental test results in Example 2 of the present invention are Figure 1 .
[0034] Figure 2 The in vitro and in vivo experimental test results in Example 2 of the present invention are Figure 2 .
[0035] Figure 3 The in vitro and in vivo experimental test results in Example 2 of the present invention are Figure 3 .
[0036] Figure 4 The in vitro and in vivo experimental test results in Example 2 of the present invention are Figure 4 .
[0037] Figure 5 The in vitro and in vivo experimental test results in Example 2 of the present invention are Figure 5 .
[0038] Figure 6 The molecular docking technology and transcriptome analysis results in Example 3 of the present invention Figure 1 .
[0039] Figure 7 The molecular docking technology and transcriptome analysis results in Example 3 of the present invention Figure 2 .
[0040] Figure 8 The molecular docking technology and transcriptome analysis results in Example 3 of the present invention Figure 3 . DETAILED DESCRIPTION
[0041] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0042] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. At the same time, unless otherwise specified or specified, the steps involved in the present invention can be carried out according to the steps and parameters disclosed in the prior art or using existing equipment, and the present invention document will not repeat them one by one.
[0043] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.
[0044] Example 1
[0045] This case investigates the inhibitory effect of Tibetan Artemisia capillaris extract on breast cancer proliferation and invasion, including the following processes:
[0046] 1) Preparation of Tibetan Artemisia Capillaris extract
[0047] The Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris, and its preparation method comprises the following process:
[0048] The dried whole herb of Artemisia capillaris was air-dried in a cool place away from direct sunlight, and then crushed into coarse powder; 1000 g of the coarse powder was added with 90% ethanol (solid-liquid ratio 1:10, w / v), and soaked at room temperature for 12 hours; then, all the filtrates were combined by reflux extraction; the combined ethanol extracts were concentrated to dryness under reduced pressure using a rotary evaporator in a 40°C water bath to obtain a dark green alcohol extract, which was the Artemisia capillaris extract.
[0049] 2) In vitro cell experiments
[0050] Cell culture: TNBC cell lines MDA-MB-231 and MDA-MB-468, and immortalized normal breast epithelial cells MCF-10A were used as control cells. All cells were cultured in DMEM medium containing 10% FBS and 1% PS (100 U / mL) at 37°C and 5% CO 2 Cultured in a constant temperature incubator.
[0051] Drug treatment: Tibetan Artemisia capillaris alcohol extract was prepared in serum-free culture medium at different concentrations (0, 5, 10, 20, 40 μg / mL). The cells of the treatment group and the control group were inoculated in 96-well plates, 6-well plates or other suitable culture plates, and the drug treatment time was set at 24 hours, 48 hours and 72 hours to evaluate the time-dependent effect. At least three replicates were set for each concentration to ensure the reliability of the data.
[0052] Cell proliferation detection: CCK-8, Edu and other methods were used for cell proliferation detection. After the cells were treated, CCK-8 reagent was added and incubated for 1-4 hours, and the cell activity was measured according to the absorbance (450nm). At the same time, a clone formation experiment was performed. The treated cells were inoculated in a 6-well plate at a certain density, stained with crystal violet after 14 days of culture, and the number of clones was counted. The cell proliferation inhibition curve was drawn and the IC50 value was calculated using nonlinear regression analysis.
[0053] Cell invasion and migration assay: Transwell chambers were used for cell invasion experiments. Matrigel was pre-coated on the membrane to simulate the extracellular matrix. The treated cells migrated to the lower chamber containing 10% FBS in serum-free medium. After 24 hours, the number of invasive cells was stained and counted. In the scratch healing experiment, a straight scratch was made on the cell monolayer with a sterile pipette tip. After washing with PBS, the drug was treated, and the healing status at different time points was photographed to calculate the healing rate.
[0054] Cell apoptosis detection: Annexin V-FITC / PI double staining flow cytometry was used. After the cells were treated, they were stained with Annexin V-FITC and PI, and the apoptosis rate was analyzed by flow cytometry. Early apoptosis, late apoptosis and necrotic cells were distinguished, and the induction effect of Tibetan Artemisia capillaris on cell apoptosis was evaluated.
[0055] 3) In vivo animal experiments
[0056] Animal model establishment: 4-6 week old female BALB / c nude mice weighing between 18-22 g were selected. MDA-MB-231 breast cancer cells (1×10 6 The cells / 100 μL PBS) were inoculated subcutaneously under the right shoulder blade of nude mice, and the tumor growth was monitored regularly. When the tumor volume reached 50-100 mm 3 Start drug intervention.
[0057] Drug administration and grouping: Tumor-bearing nude mice were randomly divided into a control group, a low-dose group of Tibetan Artemisia Capillaris (100 mg / kg), a high-dose group (200 mg / kg), and a positive control group (5 mg / kg of rudorubicin). The drug was administered by intraperitoneal injection once a day for 3-4 weeks. Ensure that there are at least 6 nude mice in each group to improve statistical power. Tumor growth monitoring: The length and width of the tumor were measured with a caliper every 3 days, and the tumor volume was calculated using the formula volume = (length × width) / 2. The tumor growth curve was recorded, and the tumor was weighed at the end of the experiment to calculate the inhibition rate.
[0058] Histological analysis: After the tumor tissue was removed, it was fixed, dehydrated, embedded and sliced. HE staining was used to observe the cell morphological changes of tumor tissue, such as nuclear size, chromatin distribution, and cell apoptosis. Immunohistochemical staining was used to detect the expression levels of proliferation-related protein Ki-67 and invasion-related proteins MMP-2 and MMP-9. Western blot was used to analyze the expression changes of the same proteins to quantitatively evaluate the molecular effects of Tibetan Artemisia Capillaris on tumor tissue.
[0059] The experimental results showed that the alcohol extract of Tibetan Artemisia capillaris was observed to inhibit the proliferation and invasion of breast cancer cells in vitro. The alcohol extract and active ingredients of Tibetan Artemisia capillaris could significantly inhibit the proliferation and invasion of triple-negative breast cancer (TNBC) cell line MDA-MB-231. The CCK8 proliferation of breast cancer cells treated with the alcohol extract of Tibetan Artemisia capillaris was inhibited, the number of clones formed was reduced, and the invasion and migration abilities were significantly reduced.
[0060] Example 2
[0061] This case investigates the inhibitory effect of Tibetan Artemisia capillaris extract on CAF-mediated breast cancer proliferation and invasion, including the following processes:
[0062] 1) Preparation of Tibetan Artemisia Capillaris extract
[0063] The Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris, and its preparation method is as shown in Example 1.
[0064] 2) Through in vitro and in vivo experiments, it was found that Swertiamarin, the active ingredient of Tibetan Artemisia Capillaris, can promote apoptosis of cancer-associated fibroblasts (CAFs) and inhibit their proliferation, thereby inhibiting CAF-mediated breast cancer angiogenesis and metastasis. In the co-culture model of breast cancer cells and CAFs, the proliferation and invasion ability of breast cancer cells were significantly reduced after treatment with Tibetan Artemisia Capillaris Swer. At the same time, Tibetan Artemisia Capillaris can downregulate the expression levels of tumor-promoting factors NF-κB and VEGF secreted by CAFs, such as Figure 1 , Figure 2 and Figure 3 shown.
[0065] The test method includes the following procedures:
[0066] 1) Use HUVEC (human umbilical vein endothelial cells) to conduct the experiment. The following are the steps of a common Tubeformation test method. The appropriate cell density (1×105 cells / mL) is suspended in the culture medium. Add 100μL of cell suspension to each well and distribute the cells evenly. Tube formation: Place the well plate at 37 degrees Celsius. Tube formation is usually observed within 6 to 24 hours. Observation and analysis: Use an inverted microscope to observe the formation of cell tubes. Consider the possibility of adherent cells. 2) The inhibitory effect on angiogenesis in tumor tissues was performed by immunofluorescence experiments. Fluorescently labeled primary antibodies against EMP1 and fluorescently labeled secondary antibodies against the vascular marker VEGFa were added separately and observed under a laser confocal microscope to evaluate the inhibition of EMP1 and angiogenesis in tumor tissues by Swer drugs.
[0067] The experimental results are as follows Figure 4-5 shown.
[0068] Example 3
[0069] This case investigates the molecular mechanism by which Tibetan Artemisia capillaris extract promotes the development of breast cancer through the EMP-1 signaling pathway, including the following processes:
[0070] 1) Preparation of Tibetan Artemisia Capillaris extract
[0071] The Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris, and its preparation method is as shown in Example 1.
[0072] 2) Through TCGA and breast cancer databases and clinical pathological analysis, the inventors found that EMP-1 is highly expressed in TNBC breast cancer and is positively correlated with the expression of CAFs marker αSMA. In in vitro and in vivo experiments, after knocking down EMP-1 with lentivirus, the proliferation, invasion and migration abilities of TNBC cells were significantly reduced:
[0073] 3) In addition, the inventors used molecular docking technology and transcriptome analysis, such as Figure 6 , Figure 7 and Figure 8 As shown, it was found that Swertiamarin from Tibetan Artemisia Capillaris could bind to EMP-1 and its related NF-κB pathway proteins, and inhibit their mRNA and protein expression levels.
[0074] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.
[0075] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.
Claims
1. Use of Tibetan Artemisia Capillaris extract in preparing medicine for treating breast cancer.
2. The use according to claim 1, characterized in that The breast cancer includes triple-negative breast cancer.
3. The use according to claim 1, characterized in that The Tibetan Artemisia Capillaris extract promotes the apoptosis of cancer-associated fibroblasts and inhibits their proliferation, so as to inhibit the angiogenesis and metastasis of breast cancer mediated by cancer-associated fibroblasts.
4. The use according to claim 1, characterized in that The Tibetan Artemisia capillaris extract down-regulates the expression levels of tumor-promoting factors NF-κB and VEGF secreted by cancer-associated fibroblasts, thereby inhibiting the growth and metastasis of breast cancer tumors mediated by cancer-associated fibroblasts.
5. The use according to claim 1, characterized in that: The Tibetan Artemisia capillaris extract combines with EMP-1 and its related NF-κB pathway proteins to inhibit breast cancer tumor growth and metastasis.
6. The use according to claim 5, characterized in that The EMP1 is downregulated in Lumina breast cancer; the EMP1 is upregulated in TNBC breast cancer; and the EMP1 is positively correlated with the expression of αSMA, a cancer-associated fibroblast marker.
7. The use according to claim 1, characterized in that The Tibetan Artemisia capillaris extract inhibits the invasive ability of breast cancer mediated by cancer-associated fibroblasts by downregulating EMP1 and its downstream inflammatory pathway.
8. The use according to any one of claims 1 to 7, characterized in that: The Tibetan Artemisia Capillaris extract is an alcohol extract of Tibetan Artemisia Capillaris; The preparation method of the alcohol extract of Tibetan Artemisia capillaris comprises the following steps: The crushed Tibetan Artemisia capillaris is added into a 90% by volume ethanol solution for soaking at room temperature, and then reflux extraction and filtration are performed to obtain an ethanol extract; The ethanol extract is concentrated and dried to obtain the alcohol extract of Artemisia capillaris.
9. An EMP1 inhibitor, characterized in that The effective component of the EMP1 inhibitor is a Tibetan Artemisia Capillaris extract.
10. A preparation for inhibiting CAF infiltration in triple-negative breast cancer, characterized in that: The effective component of the preparation is the extract of Capillaris ternata.