Use of natural dihydroflavonoids diplacone in preparation of medicine for treating breast cancer and medicine composition

Abstract

The application discloses application of a natural dihydroflavone compound Diplacone in preparation of a drug for treating breast cancer and a medicine composition, and belongs to the field of natural medicines and antitumor drugs. The Diplacone is extracted from a Paulownia fortune fruit peel, and the application first systematically expounds the breast cancer resistance mechanism of the Diplacone. The compound can significantly inhibit the proliferation and clonogenic ability of Luminal type breast cancer MCF-7 cells and triple-negative breast cancer 4T1 cells; by activating a TP53 signal path, up-regulating CDKN1A expression, down-regulating the level of cycle-related genes and proliferation markers, the compound can induce G1 / S phase cycle arrest of tumor cells; meanwhile, the compound can up-regulate pro-apoptotic protein Bax, down-regulate anti-apoptotic protein Bcl-2, activate a mitochondrial apoptosis path, and cooperatively exert the anticancer effect in multiple ways. The application is fully verified through experiments such as transcriptome analysis, molecular biology and flow cytometry, can provide experimental support for development of a safe and effective novel natural medicine for resisting breast cancer, and has a good research and clinical application prospect.

Description

Technical Field

[0001] This invention relates to the fields of natural medicines and antitumor drugs, and in particular to the application of the natural dihydroflavonoid compound Diplacone in the preparation of drugs for treating breast cancer and pharmaceutical compositions thereof. Background Technology

[0002] Breast cancer is one of the leading causes of cancer-related deaths among women worldwide. Luminal and triple-negative breast cancer (TNBC) are the two most common subtypes, accounting for over 90% of all breast cancer cases. Clinical data shows that approximately 70-80% of patients with early-stage non-metastatic breast cancer can achieve clinical cure with existing treatments. However, advanced breast cancer with distant metastases remains incurable and is a leading cause of death. Breast cancer exhibits high heterogeneity, with key characteristics including abnormal activation of human epidermal growth factor receptor 2 (HER2), hormone receptor (estrogens receptor ER and progesterone receptor PR) expression, and BRCA gene mutations. These characteristics lead to different subtypes, and treatment strategies become highly individualized. With the development of precision medicine, breast cancer treatment has evolved into a multidisciplinary system encompassing both local and systemic therapies. Treatment of metastatic breast cancer is even more complex, requiring the selection of targeted or immunotherapies based on molecular profiles. Current treatments still face many challenges, such as the tendency for endocrine and targeted drugs to develop acquired resistance, the limited response rate of immunosuppressants and the potential for adverse reactions, and the high cost of innovative drugs, which increases the burden on patients and the healthcare system. Therefore, there is an urgent clinical need to develop new natural anti-breast cancer drugs with novel mechanisms of action, definite efficacy and better safety.

[0003] Natural products, due to their structural diversity, multi-target action, and relatively low toxicity, have become an important source for the development of anti-tumor drugs. Diplacone is a geranyl dihydroflavonoid natural compound isolated from the fruit of Paulownia tomentosa. It has anti-inflammatory, antioxidant, antibacterial, and broad-spectrum anti-tumor activities. However, existing publicly available data do not report the in vitro inhibitory activity of this compound against breast cancer cells, nor are there any studies on related molecular regulatory pathways. Summary of the Invention

[0004] To overcome the above shortcomings, the present invention aims to provide the application of the natural dihydroflavonoid compound Diplacone in the preparation of drugs for treating breast cancer and a pharmaceutical composition thereof. The present invention reveals for the first time the molecular mechanism of action of this compound against breast cancer, providing experimental evidence for the drug development and clinical application of this natural active ingredient.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides the application of the natural dihydroflavonoid compound Diplacone in the preparation of a drug for treating breast cancer, wherein the structural formula of the natural dihydroflavonoid compound Diplacone is as follows: .

[0006] Furthermore, the breast cancer is Luminal breast cancer and / or triple-negative breast cancer.

[0007] Furthermore, the Diplacone exerts its anti-breast cancer effect by activating the TP53 signaling pathway.

[0008] Furthermore, the Diplacone upregulates the expression of TP53 and CDKN1A genes and proteins, and downregulates the expression of CCNE2, CDK1, and PCNA genes and proteins.

[0009] Furthermore, the Diplacone induces G1 / S phase cell cycle arrest in breast cancer cells.

[0010] Furthermore, the Diplacone activates the mitochondrial apoptosis pathway in breast cancer cells.

[0011] Furthermore, the Diplacone upregulates the expression of the pro-apoptotic protein Bax and downregulates the expression of the anti-apoptotic protein Bcl-2, thereby increasing the Bax / Bcl-2 protein ratio.

[0012] Furthermore, the Diplacone is extracted from the pericarp of Paulownia tomentosa.

[0013] The present invention also provides a pharmaceutical composition for treating breast cancer, comprising the active ingredient Diplacone described above and pharmaceutically acceptable excipients; Furthermore, the dosage form of the pharmaceutical composition is a tablet, capsule, injection, granule, suspension, or topical preparation.

[0014] The beneficial effects of this application, based on the above technical solutions, are as follows: This invention provides a novel use of Diplacone in the preparation of drugs for treating breast cancer. This compound induces G1 / S phase cell cycle arrest in breast cancer cells by activating the TP53 signaling pathway, upregulating the expression of the downstream target gene CDKN1A (P21), and activating the mitochondrial apoptosis pathway, thereby inhibiting abnormal tumor cell proliferation. This invention was validated using transcriptome sequencing GSEA enrichment analysis, qRT-PCR, Western blotting, and flow cytometry. Results showed that Diplacone can dose-dependently upregulate the transcriptional levels and protein expression of TP53 and CDKN1A, while significantly downregulating the expression of cell cycle driver genes CCNE2 and CDK1, as well as the proliferation marker PCNA. This compound can induce significant G1 phase cell cycle arrest in human Luminal breast cancer MCF-7 cells, while upregulating the expression of the pro-apoptotic protein Bax, increasing the Bax / Bcl-2 protein ratio, and activating the mitochondrial apoptosis pathway. Through the synergistic effect of cell cycle arrest and apoptosis induction, it exerts an anti-breast cancer proliferation effect. Diplacone possesses good anti-breast cancer pharmacological activity and can be considered as a candidate drug for anti-breast cancer treatment, with broad prospects for research and application. Attached Figure Description

[0015] Figure 1 This is the proton NMR spectrum of Diplacone, a compound from an embodiment of the present invention. Figure 2 The carbon spectrum of compound Diplacone from an embodiment of the present invention is shown below. Figure 3 The results of the experiment on CCK8 breast cancer cells using the compound Diplacone; Figure 4 The results of the clonogenic assay of the compound Diplacone on breast cancer 4T1 cells; Figure 5 The results of the clonogenic assay of the compound Diplacone on breast cancer MCF-7 cells; Figure 6 To analyze the enrichment of the compound Diplacone in the apoptosis signaling pathway of breast cancer MCF-7 cells using transcriptome sequencing combined with GSEA enrichment analysis; Figure 7 To analyze the enrichment of the compound Diplacone in the TP53 signaling pathway of breast cancer MCF-7 cells using transcriptome sequencing combined with GSEA enrichment analysis; Figure 8 To detect the effect of Diplacone on TP53 gene expression in breast cancer MCF-7 cells using qRT-PCR; Figure 9 To detect the effect of Diplacone on CDKN1A gene expression in breast cancer MCF-7 cells using qRT-PCR; Figure 10 To detect the effect of Diplacone on PCNA gene expression in breast cancer MCF-7 cells using qRT-PCR; Figure 11 To detect the effect of Diplacone on CCNE2 gene expression in breast cancer MCF-7 cells using qRT-PCR; Figure 12 To detect the effect of Diplacone on CDK1 gene expression in breast cancer MCF-7 cells using qRT-PCR; Figure 13 To detect the effects of Diplacone on the protein expression of TP53, P21, pro-apoptotic protein Bax, anti-apoptotic protein Bcl-2, and cell cycle-related protein Cyclin E2 in breast cancer cells by immunoblotting; Figure 14 The results of flow cytometry analysis were used to detect the effect of 0 μM Diplacone on the cell cycle distribution of breast cancer MCF-7 cells. Figure 15 The results of flow cytometry analysis were used to detect the effect of 10 μM Diplacone on the cell cycle distribution of breast cancer MCF-7 cells. Figure 16 The results of flow cytometry analysis were used to detect the effect of 20 μM Diplacone on the cell cycle distribution of breast cancer MCF-7 cells. Figure 17 The results of flow cytometry analysis were used to detect the effect of 40 μM Diplacone on the cell cycle distribution of breast cancer MCF-7 cells. Figure 18 Statistical results of flow cytometry analysis of the cell cycle distribution of 0 μM, 10 μM, 20 μM and 40 μM Diplacone in breast cancer MCF-7 cells; Figure 19 The results of flow cytometry analysis were used to detect the effect of 0 μM Diplacone on apoptosis in breast cancer MCF-7 cells. Figure 20 The results of flow cytometry analysis were used to detect the effect of 10 μM Diplacone on apoptosis in breast cancer MCF-7 cells. Figure 21 The results of flow cytometry analysis were used to detect the effect of 20 μM Diplacone on apoptosis in breast cancer MCF-7 cells. Figure 22 The results of flow cytometry analysis were used to detect the effect of 40 μM Diplacone on apoptosis in breast cancer MCF-7 cells. Figure 23Statistical results for the effect of 0 μM, 10 μM, 20 μM and 40 μM Diplacone on apoptosis in breast cancer MCF-7 cells by flow cytometry. Detailed Implementation

[0016] To fully illustrate the preparation concept of this invention, the following examples and comparative examples further illustrate the invention. It should be understood that the examples and comparative examples of this invention are merely illustrative and not intended to limit the invention. Therefore, any simple modifications to this invention based on the method described herein are within the scope of protection claimed by this invention.

[0017] The MCF-7 cell line (SCSP-531) and the 4T1 cell line (SCSP-5056) were provided by the Stem Cell Bank of the Chinese Academy of Sciences (Shanghai). The compound Diplacone was extracted and isolated by the applicant's laboratory and its structure was confirmed.

[0018] Example 1: Extraction, separation and structural confirmation of compound Diplacone (1) Extraction and enrichment process: Take 1.0 kg of white-flowered paulownia fruit bark, crush it, add 80% ethanol, and reflux extract for 0.5 h at a material-to-liquid ratio of 1:10. After extraction once, combine the filtrates, filter, and concentrate to obtain an extract. Dissolve the extract in 95% ethanol, pack it into a column using a pretreated HPD 100 macroporous adsorption resin using a wet method, and load the sample using a dry method. After loading the sample, elute with 40% ethanol solution. When the eluent is nearly colorless, replace it with 80% ethanol solution and continue eluting. Collect the eluent in the 80% ethanol stage, concentrate under reduced pressure, and obtain a yellow powder.

[0019] (2) Separation: Take 10.0 g of the above yellow powder, add 15 g of 200–300 mesh silica gel, add the sample in small amounts several times and mix well. After drying, grind into a fine powder for later use. Silica gel column chromatography (wet packing, dry loading) was used with petroleum ether-ethyl acetate system as eluent. Gradient elution was performed sequentially with 100% petroleum ether, petroleum ether-ethyl acetate (9:1, 8:2, 7:3, 6:4, 1:1) and 100% ethyl acetate. The elution progress was judged based on the color change and concentration of the eluent. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used for analysis, and fractions with the same composition were combined. Further separation and purification were performed by silica gel column chromatography, Sephadex LH-20 gel column chromatography, ODS reversed-phase column chromatography, and preparative high-performance liquid chromatography to obtain the target compound Diplacone monomer.

[0020] (3) Structural confirmation: Through high-resolution electrospray mass spectrometry (HRESIMS), ¹H-NMR, and ¹³C-NMR spectral data analysis, combined with literature comparison, the structure of the obtained compound was identified as Diplacone. Its hydrogen and carbon spectra are shown in Figure 1 and Figure 2. Figure 2 As shown, the specific structural confirmation data is as follows: The compound is a light yellow powder, ESI-MS m / z: 425 [M+H] + Its molecular weight is 424.

[0021] ¹H-NMR (800 MHz, CD3OD) δ 12.28 (1H, s, 5-OH), 6.92 (1H, br.s, H-2′), 6.79 (2H, br.s, H-5′,6′), 5.94 (1H, s, H-8), 5.24 (1H,dd, J = 12.9, 3.0 Hz,H-2), 3.04 (1H, dd, J = 17.0, 12.9 Hz, H-3a), 2.68 (1H,dd, J = 17.0, 3.1 Hz,H-3b), 3.22 (2H, d, J = 7.2, H-1′′), 5.24 (H, d, J = 7.3, H-2′′), 1.75 (3H,s, H-4′′), 2.04 (2H, q, J = 7.5 Hz, H-5′′), 1.94 (2H, t, J = 7.6 Hz, H-6′′), 5.05 (1H, t, J = 7.1 Hz, H-7′′), 1.63 (3H, s, H-9′′), 1.57 (3H, s, H-10′′); ¹³C-NMR (201 MHz, CD3OD): δ 80.45 (C-2), 44.24 (C-3), 197.80 (C-4), 162.49 (C-5), 109.67 (C-6), 165.97 (C-7), 95.40 (C-8), 162.44 (C-9), 103.20(C-10), 131.92 (C-1′), 114.66 (C-2′), 146.82 (C-3′), 146.46 (C-4′), 116.22(C-5′), 119.22 (C-6′), 21.79 (C-1′′), 123.95 (C-2′′), 135.22 (C-3′′), 16.20(C-4′′), 40.90 (C-5′′), 27.72 (C-6′′), 125.47 (C-7′′), 132.00 (C-8′′), 17.70(C-9′′), 25.85 (C-10′′).

[0022] Example 2: Diplacone inhibits breast cancer cell proliferation in CCK8 and colony formation assays. Experimental methods: (1) CCK8 experiment: MCF-7 and 4T1 cells in logarithmic growth phase were selected, washed once with PBS, digested with trypsin, centrifuged, resuspended, and counted. The cells were then subjected to a 3×10⁻⁶ ppm incubation period. 3 Cells were seeded into 96-well culture plates at the specified concentration. PBS was added to the edge of the plate to eliminate edge effects. After cell attachment, cells were treated with 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM Diplacone for 48 h. CCK8 was then added at a volume ratio of 10 μL of CCK8 per 100 μL of culture medium. The plates were incubated for another 2 h, and the OD value at 450 nm was measured for each well. The inhibitory rate of the drug on cells was calculated using the following formula: Cell viability (%) = [(OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells)] × 100%; Cell inhibition rate (%) = 100% - cell survival rate (%).

[0023] (2) Clonogenic assay: Logarithmic growth phase breast cancer cells were routinely digested and counted, with 4 × 10⁶ cells per well. 3The cells were seeded into 6-well plates, and the plates were gently shaken to ensure even distribution. After 24 hours, the cells were stimulated with different inhibitors or drugs as needed. 24 hours after drug administration, the drug-containing medium was aspirated and replaced with fresh, complete medium. The plates were then incubated in a cell culture incubator. The cell culture medium was changed every four days. After two weeks of culture, clones were visible to the naked eye in the culture plates, at which point the culture was terminated. The medium was discarded, and the cells were rinsed once with fresh PBS. 1 mL of tissue fixative was added to each well for 15 min. The fixative was discarded, and the cells were washed three times with PBS. 1 mL of crystal violet staining solution was added to each well, and the plates were stained on a shaker at room temperature for 10 min. The crystal violet staining solution was then discarded. The cells were washed three times with PBS on a shaker for 5 min each time. The staining was then photographed and recorded.

[0024] Experimental results: Experimental results are as follows Figure 3 , Figure 4 and Figure 5 As shown, Diplacone has a significant inhibitory effect on the proliferation of breast cancer cells. Specifically, it exhibits a high half-maximal inhibitory concentration (IC50) against mouse triple-negative breast cancer 4T1 cells. 50 The concentration was 4.434 μM, and the IC50 value for human Luminal breast cancer MCF-7 cells was 4.434 μM. 50 The concentration was 18.35 μM. Simultaneously, Diplacone reduced the number of colonies formed in both types of breast cancer cells in a dose-dependent manner, indicating that Diplacone can significantly inhibit the proliferation and colony formation of breast cancer cells, demonstrating clear anti-breast cancer activity.

[0025] Example 3: Transcriptome sequencing and GSEA enrichment analysis of the regulatory effects of Diplacone on breast cancer cell signaling pathways Experimental methods: Log-phase MCF-7 cells were collected and divided into a control group and a Diplacone treatment group (20 μM, 48 h). After treatment, cells were collected, and total RNA was extracted using TRIzol reagent. After quality control, cDNA libraries were constructed, and transcriptome sequencing was performed using a high-throughput sequencing platform. After quality control and alignment, gene set enrichment analysis (GSEA) was used with the MSigDB database as a reference to perform pathway enrichment analysis on differentially expressed genes and screen signaling pathways significantly regulated by Diplacone.

[0026] Experimental results: Experimental results are as follows Figure 6 and Figure 7As shown in the GSEA enrichment analysis, Diplacone treatment significantly enriched both the apoptosis pathway and the TP53 signaling pathway in breast cancer cells, indicating that Diplacone can exert its anti-breast cancer effect by activating these two key pathways. This result provides direct transcriptomic evidence for the mechanism by which Diplacone induces cell cycle arrest and apoptosis in breast cancer cells.

[0027] Example 4: qRT-PCR detection of the effect of Diplacone on the transcriptional levels of breast cancer cell-related genes Experimental Methods: MCF-7 cells in logarithmic growth phase were resuspended, counted, and plated. A control group and groups treated with different concentrations of Diplacone were set up. After 48 h, the culture medium was discarded, and the cells were washed with pre-cooled PBS. mRNA was extracted using a rapid RNA extraction kit. cDNA was synthesized via reverse transcription. Real-time quantitative PCR (qRT-PCR) was used, with GAPDH as an internal reference gene, to detect the mRNA expression levels of TP53, CDKN1A (P21), CCNE2 (Cyclin E2), CDK1, and the proliferation marker PCNA. 2⁻ ΔΔCt The relative expression levels of each gene can be calculated.

[0028] Experimental results: Experimental results are as follows Figures 8-12 As shown, compared with the control group, the mRNA expression levels of TP53 and CDKN1A in breast cancer cells treated with Diplacone were upregulated in a dose-dependent manner; simultaneously, the mRNA expression levels of downstream cell cycle driver genes CCNE2 and CDK1, as well as the proliferation marker PCNA, were downregulated in a dose-dependent manner. These results indicate that Diplacone can activate the TP53 / CDKN1A pathway at the transcriptional level and inhibit the expression of key cell cycle genes and proliferation markers, providing molecular-level evidence for its ability to induce G1 / S phase cell cycle arrest and inhibit tumor cell proliferation.

[0029] Example 5: Detection of the effect of Diplacone on the expression of breast cancer cell-related proteins by Western blot. Experimental methods: (1) Western blot experiment: Prepare the required concentration of the lower gel solution mixture according to the YARN gel kit, i.e., take equal volumes of lower gel solution and lower gel buffer, add modified coagulant to a coagulant volume ratio of 1%, mix well, and pour into pre-aligned and clamped glass plates, leaving space for the stacking gel and comb teeth. Add anhydrous ethanol on top of the lower gel. After the lower gel solidifies, discard the ethanol, prepare the upper gel mixture, pour it between the glass plates, and slowly insert the comb teeth to avoid the generation of air bubbles. After the upper gel solidifies, place the glass plates on the electrode holder, transfer them to the gel running tank, and add an appropriate amount of running buffer. Vertically pull out the comb teeth for electrophoresis. Load 4 μL of pre-stained protein marker or 30 μg of total protein per well. After loading, electrophoresis at a constant voltage of 80 V for 120 min until bromophenol blue reaches the bottom of the separating gel, and then end the electrophoresis. Place the membrane in a transfer clamp in a sandwich configuration: sponge, filter paper, PVDF membrane, protein-containing gel strips, filter paper, and sponge. Place the membrane in an electrophoresis tank, add an appropriate amount of transfer buffer, and maintain a constant current of 300 mA. Adjust the transfer time according to the size of the target protein. After transfer, block the PVDF membrane by incubating it in 5% skim milk prepared with TBST at room temperature for 2 h. Wash three times with TBST solution for 10 min each. Add the appropriate primary antibody and incubate overnight at 4°C. Wash three times with TBST solution for 10 min each time. Add secondary antibody prepared with 5% skim milk and incubate at room temperature for 2 h. After secondary antibody incubation, wash three times with TBST solution for 10 min each time, and develop using ECL developing solution on a gel imaging system.

[0030] Experimental results: Experimental results are as follows Figure 13 As shown, compared with the control group, the expression levels of TP53, P21, and the pro-apoptotic protein Bax in breast cancer cells treated with Diplacone were upregulated in a dose-dependent manner, while the expression level of the anti-apoptotic protein Bcl-2 was downregulated in a dose-dependent manner. Simultaneously, the expression level of the cell cycle-related protein Cyclin E2 was decreased in a dose-dependent manner. These results indicate that Diplacone can activate the TP53 / P21 pathway at the protein level, regulate the Bax / Bcl-2 balance, and inhibit Cyclin E2 expression, thereby inducing G1 / S phase cell cycle arrest in breast cancer cells and activating the mitochondrial apoptosis pathway, providing direct protein-level evidence for its anti-breast cancer effect.

[0031] Example 6: Flow cytometry detection of the effect of Diplacone on the cell cycle distribution of breast cancer MCF-7 cells Experimental methods: Logarithmic growth phase MCF-7 cells were seeded at an appropriate density in culture plates. After cell attachment, a control group and groups treated with different concentrations of Diplacone were set up and cultured for 24 h. After treatment, the drug-containing medium was discarded, and the cells were washed twice with pre-chilled PBS. The cells were collected, and pre-chilled 70% ethanol was added to fix the cells overnight at 4°C. The next day, the cells were centrifuged at 3000 rpm for 5 min to remove the fixative, washed twice with pre-chilled PBS, and incubated with propidium iodide (PI) staining solution at room temperature in the dark for 30 min. Cell cycle distribution was detected by flow cytometry, and the proportion of cells in each phase (G0 / G1, S, and G2 / M phases) was analyzed using ModFit software.

[0032] Experimental results: Experimental results are as follows Figures 14-18 As shown, compared with the control group, the proportion of MCF-7 cells in the Diplacone-treated group was significantly increased in the G0 / G1 phase, and the proportion of cells in the S phase was correspondingly decreased, while the proportion of cells in the G2 / M phase remained unchanged. These results indicate that Diplacone can induce significant G1 / S phase cell cycle arrest in breast cancer MCF-7 cells, exerting an anti-proliferative effect by inhibiting cell cycle progression. This is consistent with the aforementioned findings regarding TP53 / CDKN1A pathway activation and changes in the expression of cell cycle-related proteins.

[0033] Example 7: Flow cytometry detection of the effect of Diplacone on apoptosis in breast cancer MCF-7 cells Experimental methods: Logarithmic growth phase MCF-7 cells were seeded at an appropriate density in culture plates. After cell attachment, a control group and groups treated with different concentrations of Diplacone were set up and cultured for 24 h. After treatment, the drug-containing medium was discarded, and the cells were washed twice with pre-chilled PBS. The cells were collected, centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cell pellet was collected. The cells were resuspended in pre-chilled PBS solution to adjust the cell concentration to 1×10⁻⁶ cells / mL. 6 Cells / mL: Take 100 μL of cell suspension into a flow cytometry tube, add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, mix gently, and incubate at room temperature in the dark for 15 min; then add 400 μL of Annexin V binding buffer to each tube, mix well, and use flow cytometry to detect apoptosis within 1 h.

[0034] Experimental results: Experimental results are as follows Figures 19-23As shown, Diplacone can significantly induce apoptosis in MCF-7 cells, and the apoptosis-inducing effect is significantly concentration-dependent. With the increase of Diplacone concentration, the proportion of viable MCF-7 cells gradually decreases, while the proportion of early and late apoptotic cells increases significantly. This confirms that Diplacone can exert its anti-breast cancer effect by inducing apoptosis, providing direct experimental evidence for its development as an anti-breast cancer drug.

Claims

1. The application of a natural dihydroflavonoid compound, Diplacone, in the preparation of a drug for treating breast cancer, characterized in that, The structural formula of the natural dihydroflavonoid compound Diplacone is: 。 2. The application according to claim 1, characterized in that, The breast cancer referred to is Luminal breast cancer and / or triple-negative breast cancer.

3. The application according to claim 1, characterized in that, Diplacone exerts its anti-breast cancer effect by activating the TP53 signaling pathway.

4. The application according to claim 3, characterized in that, The Diplacone upregulated the expression of TP53 and CDKN1A genes and proteins, and downregulated the expression of CCNE2, CDK1, and PCNA genes and proteins.

5. The application according to claim 1, characterized in that, Diplacone induces G1 / S phase cell cycle arrest in breast cancer cells.

6. The application according to claim 1, characterized in that, Diplacone activates the mitochondrial apoptosis pathway in breast cancer cells.

7. The application according to claim 6, characterized in that, The Diplacone upregulates the expression of the pro-apoptotic protein Bax and downregulates the expression of the anti-apoptotic protein Bcl-2, thereby increasing the Bax / Bcl-2 protein ratio.

8. The application according to claim 1, characterized in that, Diplacone is extracted from the pericarp of Paulownia tomentosa.

9. A pharmaceutical composition for treating breast cancer, characterized in that, It comprises the Diplacone as described in claim 1 and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, injections, granules, suspensions, or topical preparations.

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