Application of 4-phenylcoumarin compound MF1 and composition thereof in preparation of medicine for treating triple negative breast cancer

Abstract

The invention relates to a 4-phenylcoumarin compound MF1 and a composition containing the same. The structural formula of the 4-phenylcoumarin compound MF1 is as shown in the specification: # imgabs0 #. According to the invention, the 4-phenylcoumarin compound MF1 and the MF1-containing dry flower bud extract of the ferrugina are subjected to anti-tumor activity screening by an MTT method, and the in-vivo anti-TNBC activity mechanism of the 4-phenylcoumarin compound MF1 and the MF1-containing dry flower bud extract is researched. Results show that the 4-phenylcoumarin compound MF1 and the MF1-containing dried ferrugina flower bud extract have a good effect of treating the triple-negative breast cancer, can treat the triple-negative breast cancer by inhibiting proliferation, migration, apoptosis and the like of breast cancer cells, can be used for preparing the medicine for treating the triple-negative breast cancer, and can be used for preparing the medicine for treating the triple-negative breast cancer. The compound is especially used for preparing drugs for resisting triple-negative breast cancer metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and specifically relates to a 4-phenylcoumarin compound MF1 (chemical structure shown in Figure 1 ) and its combination in the preparation of drugs for treating triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) refers to a type of breast cancer in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) are all negative. Compared with other breast cancer subtypes, TNBC has the characteristics of high histological grade and early distant metastasis. Research data show that the 5-year survival rate of patients with early breast cancer after surgery can reach 91%, but for TNBC patients, due to high metastasis, the overall survival period is only 18 months.

[0003] Tumor-associated macrophages (TAMs) can be polarized into two different states: one is anti-tumor M1 macrophages, which express inflammatory factors such as TNFα, IL-6, IL-8, and chemokines such as CXCL10; the other is pro-tumor M2 macrophages, which synthesize and release anti-inflammatory cytokines, immunosuppressive factors, and various cytokines, such as IL-10, TGFβ, and VEGFA. These factors promote tumor angiogenesis and degradation and remodeling of the extracellular matrix, thereby promoting tumor cell migration, invasion, and distant metastasis.

[0004] The clinical treatment of TNBC is mainly surgical resection and chemotherapy, and there is no particularly good treatment strategy. In the past few years, the development and research of small molecule targeted drugs such as AKT inhibitors and PARP inhibitors, gene therapy and immunotherapy have made rapid progress, but their application in triple-negative breast cancer is in its infancy and the prospects are unclear.

[0005] Ironwood, (scientific name Iron rod L.), an evergreen tree of the genus Tieli in the family Garcinia. Tieli is native to southern, western and southwestern Yunnan, Guangdong and Guangxi in China. Its main components are coumarin compounds. Modern pharmacological studies have shown that its main pharmacological effects include anti-tumor, antibacterial, anti-inflammatory and other effects.

[0006] The present invention studied the dried flower bud extract of Ironwood and found that it contained compound MF1, and unexpectedly found that the compound and its composition can effectively treat triple-negative breast cancer. This discovery has not been reported at home and abroad, and its results have great practical significance for the development of drugs for the treatment of triple-negative breast cancer. Based on this, this application was developed. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art and provide a 4-phenylcoumarin compound MF1 and its composition for use in the preparation of drugs for treating triple-negative breast cancer, especially in the preparation of drugs against metastasis of triple-negative breast cancer, that is, it has the efficacy of treating triple-negative breast cancer.

[0008] To achieve the above object, the present invention adopts the following technical solution: A 4-phenylcoumarin compound MF1 (named: Mesuaferol A), the structural formula of which is shown below: .

[0009] The present invention provides a composition, which comprises the 4-phenylcoumarin compound MF1.

[0010] Specifically, the composition includes but is not limited to a Chinese medicine extract. Further, the Chinese medicine extract may be an extract of dried flower buds of the ironwood tree. The dried flower buds of the ironwood tree are ironwood of the genus Ironwood of the family Luteaceae ( Mesua iron L.) of dried flower buds.

[0011] Specifically, the dried flower bud extract of Ironwood can be prepared by the following steps: Take the dried bud powder of the ironwood, extract it 2-4 times with 8-12 times by weight of organic solvent, evaporate and concentrate the extract to obtain an extract, then disperse the extract with 8-12 times by weight of pure water (ultrasound-assisted dissolution and suspension can be used), add petroleum ether at a volume ratio of 1:1-2 and extract it 2-4 times to obtain a petroleum ether layer extract, elute the petroleum ether layer extract with a silica gel column, and the eluent is a petroleum ether-acetone mixture with a volume ratio of 100:0 to 0:100. According to the polarity, a total of 11 components MF-P-1 to MF-P-11 are obtained from small to large, and the 11 components (MF-P-1 to MF-P-11) are screened for anti-tumor activity by the MTT method. The results show that MF-P-5 has the best activity, so MF-P-5 is the dried bud extract of the ironwood. At the same time, the present invention selects the active part MF-P-5 to study the in vivo anti-TNBC activity mechanism.

[0012] Furthermore, the organic solvent includes but is not limited to methanol or ethanol with a volume concentration of 75% to 95%, wherein methanol and 95% ethanol are the most preferred. The extraction methods include: ultrasonic extraction, 25-35 minutes each time; flash extraction, 3-6 minutes each time; reflux extraction, 1-3 hours each time; immersion extraction, 36-48 hours each time. Among them, ultrasonic extraction, 30 minutes each time and flash extraction, 4.5 minutes each time are the most preferred.

[0013] The present invention also provides a method for extracting the above-mentioned 4-phenylcoumarin compound MF1, which specifically comprises the following steps: 1) Take dried bud powder of Tetrapanax ferox, extract it with an organic solvent, evaporate and concentrate the extract to obtain an extract, then disperse the extract with purified water, extract it with petroleum ether to obtain a petroleum ether layer extract, elute the petroleum ether layer extract with a silica gel column, and the eluent is a petroleum ether-acetone mixture with a volume ratio of 100:0 to 0:100, and obtain 11 components MF-P-1 to MF-P-11 in descending order of polarity; 2) Component MF-P-5 was again loaded onto a silica gel column and eluted with a petroleum ether-acetone mixture in a volume ratio of 100:0-0:100, and 11 fractions A1-A11 were obtained in order of polarity. Fraction A7 was subjected to open ODS column chromatography and gradient eluted with a methanol-water mixture in a volume ratio of 30:70-100:0, and 14 subfractions A7B1-A7B14 were obtained in order of polarity. Subfraction A7B11 was separated by reverse phase high performance liquid chromatography using a methanol-water mixture in a volume ratio of 90:10 as the mobile phase to obtain 4-phenylcoumarin compound MF1.

[0014] The present invention also provides the use of the above-mentioned 4-phenylcoumarin compound MF1 and / or the composition in the preparation of a drug for treating triple-negative breast cancer, especially in the preparation of a drug for resisting metastasis of triple-negative breast cancer, that is, it has the efficacy of treating triple-negative breast cancer. Triple-negative breast cancer (TNBC) refers to a type of breast cancer in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) are all negative, which has a high degree of malignancy and is easy to metastasize and has poor prognosis.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered and screened a new 4-phenylcoumarin compound MF1 and a Chinese medicine extract containing MF1, especially a dried flower bud extract of the ironwood; and further explored its extraction process. In addition, the present invention also screened the anti-tumor activity of the 4-phenylcoumarin compound MF1 and the dried flower bud extract of the ironwood containing MF1 by the MTT method, and studied its in vivo anti-TNBC activity mechanism. The results showed that the 4-phenylcoumarin compound MF1 and the dried flower bud extract of the ironwood containing MF1 have good efficacy in treating triple-negative breast cancer, which can treat triple-negative breast cancer by inhibiting breast cancer cell proliferation, migration, apoptosis, etc., and can be used to prepare drugs for treating triple-negative breast cancer, especially for preparing drugs against metastasis of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the chemical structure of MF1; Figure 2 For MF1 1 H NMR spectrum; Figure 3 For MF1 13 C NMR spectrum; Figure 4 is the HSQC spectrum of MF1; Figure 5 For MF1 1 H- 1 H COSY spectrum; Figure 6 is the HMBC spectrum of MF1; Figure 7 is the HR-ESI-MS spectrum of MF1; Figure 8 is the IR spectrum of MF1; Figure 9 is the UV spectrum of MF1; Figure 10 For MF1 1 H-NMR and 13 C-NMR data attribution; Figure 11 MF-P-4~MF-P-10 inhibits the proliferation of breast cancer cells. (A) (C) MDA-MB-231, (B) (D) 4T1 cells were treated with different concentrations of MF-P-4~MF-P-10 for 24 h and 48 h, and the cell viability was measured by MTT assay. Figure 12To show that MF1 inhibits the proliferation of breast cancer cells, (A) MDA-MB-231 and (B) 4T1 cells were treated with different concentrations of MF1 for 24 h and 48 h, respectively, and then the cell viability was determined by MTT assay; (C) MDA-MB-231 and (D) 4T1 cells were treated with different concentrations of MF1 for cell clone formation experiment; (E) Quantitative results of MDA-MB-231 and (F) 4T1 cells, all data are expressed as mean ± standard error of the mean (SEM), compared with the Control group, ***p<0.001, ****p<0.0001; Figure 13 To show that MF1 inhibits the migration of breast cancer cells, (A): MDA-MB-231 cells and (B) 4T1 cells were treated with different concentrations of MF1 for 24 h; (C): The expression level of E-cadherin was detected by Western blot after MDA-MB-231 and 4T1 cells were treated with MF1 for 48 h; (D): The relative wound healing area bar graph of MDA-MB-231 cells and (E) 4T1 cells; (F): The relative gene expression of MDA-MB-231 cells and (G) 4T1 cells, β -actin was used as an internal reference, and Image J was used for statistical analysis; Note: All data are expressed as mean ± SEM, and all data were obtained from at least 3 independent experiments. Compared with the Control group, ns, no significant difference, ***p<0.001, ****p<0.0001; Figure 14 Figure MF1 induces apoptosis of breast cancer cells. (A): After MDA-MB-231 and (B) 4T1 cells were treated with different concentrations of MF1 for 48 hours, flow cytometry was used to detect the apoptosis of MDA-MB-231 and 4T1 cells stained with Annexin V-PI. (C): Cell apoptosis rate after MDA-MB-231 and (D) 4T1 cells were treated with different concentrations of MF1. (E): After MDA-MB-231 and 4T1 cells were treated with MF1 for 48 hours, the expression levels of Bax and PARP were detected by Western blot. (F): The expression results of Bax and PARP were detected by Western blot. β -actin was used as an internal reference, and Image J was used for statistical analysis; Note: All data are expressed as mean ± SEM, all data were subjected to at least 3 independent experiments, compared with the Control group, compared with the Control group, ns, no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; &&&&p<0.0001, ####p<0.0001; Figure 15 The anti-tumor effect of MF-P-5 in vivo was evaluated in the nude mouse xenograft MDA-MB-231 model. (A): Representative tumor images of each group were collected on the last day of the experiment; (B): The nude mouse model transplanted with MDA-MB-231 was used to evaluate the inhibitory effect of MF-P-5 on tumor growth rate in this model; (C): Statistics of the body weight of each group of nude mice during the treatment period; (D) (E): Statistics of the changes in tumor volume and tumor weight during the treatment period of each group, n=6; (F): The expression level of the proliferation marker Ki-67 in tumor tissue was evaluated by immunohistochemistry; (H): The expression of the proliferation marker Ki-67 in tumor tissue was quantitatively analyzed using Image J; all data are expressed as mean ± SEM. (G): The expression levels of MMP-2, E-cadherin and Bcl-2 in tumor tissues after MF-P-5 treatment were detected by Western blot, and the expression levels of MMP-2, E-cadherin and Bcl-2 proteins in tumor tissues were evaluated by Western blot; (I): The expression of MMP-2, E-cadherin and Bcl-2 in tumor tissues was evaluated by Western blot. β -actin was used as an internal reference, and Image J was used for statistical analysis; Note: All data are expressed as mean ± SEM. All data were obtained from at least 3 independent experiments. Compared with the Control group, **p<0.01, ****p<0.0001. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0018] Example 1 Preparation of 4-phenylcoumarin compound MF1 and its composition and separation and structural confirmation of MF1 This embodiment provides a method for extracting 4-phenylcoumarin compound MF1, which specifically comprises the following steps: (1) Extraction: Take 4 kg of dried flower bud powder of the ironwood tree, extract it with 10 times the weight of methanol by ultrasonic at room temperature for 3 times, each time for 30 minutes. The extract is evaporated and concentrated to obtain about 600 g of extract concentrate.

[0019] (2) Extraction: The extract was dissolved in 10 times the weight of pure water, and then extracted with petroleum ether for 3 times (the extraction solvent was added in a volume ratio of 1:1) to obtain 250 g of petroleum ether layer extract.

[0020] (3) Separation: 200 g of the petroleum ether extract was subjected to silica gel column chromatography using a petroleum ether-acetone mixture with a volume ratio of (100:0, 100:1, 100:2, 100:4, 100:8, 100:12, 100:18, 100:25, 50:50, 0:100) gradient elution, with 20 column volumes for each gradient (a single column volume is approximately 2500 mL). A total of 11 components (MF-P-1 to MF-P-11) were obtained in order of polarity from small to large. The component MF-P-5 was loaded onto the silica gel column again and gradient elution was performed using a petroleum ether-acetone mixture with a volume ratio of (100:0, 100:1, 100:2, 100:4, 100:8, 100:12, 100:18, 100:25, 50:50, 0:100) as the eluent, with 20 column volumes for each gradient (a single column volume was approximately 750 mL). A total of 11 fractions (A1~A11) were obtained in order of polarity from small to large. Fraction A7 (2.33 g) was subjected to open ODS column chromatography and gradient eluted with methanol-water (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, volume ratio), each gradient was about 30 column volumes (single column volume was about 30 mL), and 14 subfractions (A7B1~A7B14) were obtained in descending order of polarity. Subfraction A7B11 (246.2 mg) was separated by reverse phase high performance liquid chromatography (RP-HPLC) with methanol-water (90:10, volume ratio) as the mobile phase to prepare 4-phenylcoumarin compound MF1 (9.4 mg).

[0021] The structure of compound MF1 was confirmed: Yellow oil (methanol), easily soluble in organic solvents such as dichloromethane, chloroform, methanol, acetonitrile, etc., dark spots are observed under ultraviolet 254 nm, blue fluorescence is observed at 365 nm, and 10% sulfuric acid-ethanol color developer is used, and it turns yellow-brown after heating. Figure 7 The HR-ESI-MS spectrum of MF1 is given. m / z 489.2282 [MH] - (calcd for 489.2277, C 30 H 33 O6 - ), suggesting the molecular formula of the compound: C 30 H 34 O6, unsaturation is 14. In the IR spectrum (see Figure 8 ), 3425 cm -1 、1604 cm -1 The absorption bands show that it contains hydroxyl and acyl groups. Figure 9), 345 nm (logℇ 3.92), 282nm (lo ℇ 4.33) and 226nm (log ℇ A strong absorption peak appears at 4.09.

[0022] Figure 2 Given the MF1 1 H NMR spectrum. 1 H NMR (600 MHz, CDCl3) Mid- and low-field regions d H 14.60 (1H, s) is the active hydrogen signal, and the chemical shift moving to the downfield indicates that the active hydrogen forms an intramolecular hydrogen bond with the adjacent carbonyl group; d H 7.38~7.39 (3H, m) and 7.29~7.30 (2H, m) are the hydrogen signals of monosubstituted benzene ring; d H 5.93 (1H, s) is the hydrogen proton signal at position 3 of coumarin, and the single peak indicates that the carbon at position 4 is substituted; d H 5.15 (1H, t, J =7.07 Hz)、4.95 (1H, t, J =9.57Hz) is the unsaturated olefinic hydrogen or oxygen-linked methine proton signal; 5 methyl proton signals are given in the high field region, including 3 singlet methyls: d H 1.71 (3H, s), 1.65 (3H, s), 1.38 (3H, s), 1 doublet of methyl: d H 1.16 (3H, d, J =6.70 Hz), 1 triplet of methyl: d H 0.90 (3H, d, J =7.45 Hz).

[0023] Figure 3 Given the MF1 13 C NMR spectrum. 13 C NMR (100 MHz, CDCl3) gave 30 carbon signals, including 1 carbonyl carbon signal ( d C 209.5), 1 lactone carbon signal ( d C 159.9), 16 SP 2 The hybridized carbon signals are ( d C165.0, 156.7, 155.7, 139.3, 132.9, 128.3, 127.7×2,127.3×2, 123.7, 112.2,105.2, 103.9, 102.9, 102.6), 2 oxygen-carbon signals ( d C 92.1, 73.7).

[0024] Figure 4 The HSQC spectrum of MF1 is given. The carbon and hydrogen atoms of the compound are assigned by the HSQC spectrum, and the planar structure of the compound is determined by 1 H- 1 H COSY and HMBC spectra were confirmed. Figure 10 Given the MF1 1 H-NMR and 13 C-NMR data assignment. Figure 5 Given the MF1 1 H- 1 H COSY spectrum. 1 H- 1 In the H COSY spectrum, H-2' ( d H 7.30)、H-6' ( d H 7.30) and H-3' ( d H 7.39)、H-5' ( d H 7.39) related, H-4' ( d H 7.39) and H-3' ( d H 7.39)、H-5' ( d H 7.39), and is speculated to contain a monosubstituted phenyl fragment.

[0025] Figure 6 The HMBC spectrum of MF1 is given. In the HMBC spectrum, H-3 ( d H 5.93) and C-4 ( d C 156.7), C-4a ( d C 102.6)、C-1'( d C 139.3), it is inferred that the monosubstituted phenyl group is located at the 4-carbon. 1 H- 1 In the H COSY spectrum, H-2" ( dH 3.59) and CH3-3" ( d H 1.16)、CH2-4"( d H 1.41, 1.80) related, H2-4" ( d H 1.41, 1.80) and CH3-5"( d H 0.90) and 5-OH ( d H 14.60) and C-5 ( d C 165.0)、C-6 ( d C 102.9), C-4a ( d C 102.6), it is speculated that the compound contains a 2-methyl-1-butyryl fragment, and the fragment is located at the 6-carbon position. 1 H- 1 In the H COSY spectrum, H-2"' ( d H 4.95) and H2-1"' ( d H 3.28, 3.35) related; in the HMBC spectrum, CH3-4"' ( d H 1.38) respectively with C-2"' ( d C 92.1)、C-3"' ( d C 73.7)、C-5"' ( d C 37.7) related, H2-1"' ( d H 3.28, 3.35) and C-7 ( d C 163.9)、C-8 ( d C 105.2), C-8a ( d C 155.7), it is speculated that the compound contains a cyclized isopentyl fragment located at the 7- and 8-carbon positions. 1 H- 1 In the H COSY spectrum, H2-5"' ( d H 1.59)、H-7"' ( d H 5.15) and H2-6"' ( d H 2.13, 2.19) related; in the HMBC spectrum, CH3-9"' ( d H 1.65)、CH3-10"' ( d H 1.71) respectively with C-7"' ( d C 123.7)、C-8"' ( d C 132.9), it is speculated that the compound contains another isopentene fragment, and the fragment is located at the 5" carbon. The above results suggest that the compound has 2"'-(1-hydroxy-1,5-dimethyl-Δ 5 -hexenyl)-7,8-dihydrofuran ring system.

[0026] In summary, the planar structure of the compound was determined, and the structural formula is shown below (see Figure 1 ).

[0027] .

[0028] Example 2 Preparation of active site MF-P-5 In the above Example 1, the petroleum ether layer extract was eluted with a silica gel column, and 11 fractions (MF-P-1 to MF-P-11) were obtained in order of polarity from small to large. The 11 fractions (MF-P-1 to MF-P-11) were screened for anti-tumor activity by the MTT method, and the results showed that MF-P-5 had the best activity.

[0029] Example 3 MTT anti-tumor activity screening Experimental purpose: To detect whether MF-P-1~MF-P-11 has an inhibitory effect on the proliferation of two breast cancer cells (MDA-MB-231 and 4T1). First, different concentrations of MF-P-1~MF-P-11 (200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 μg / mL) were incubated in the above two breast cancer cells for 24 h and 48 h, respectively. MTT was used to evaluate the inhibitory ability of MF-P-1~MF-P-11 on the short-term proliferation of the two breast cancer cells.

[0030] Experimental method: MTT experiment: Take cells in the logarithmic growth phase, inoculate the cells at a density of 5000 cells / well in a 96-well plate, add 100 μL of cell suspension to each well, and continue to incubate in a cell culture incubator at 37°C with 5% CO2 for 24 hours with DMEM culture medium (containing 10% fetal bovine serum). Then add 100 μL of drug for 24 hours or 48 hours. Then add 20 μL of 5 mg / mL MTT solution to each well and incubate in an incubator for 3 hours. After the reaction is sufficient, discard the supernatant, add 150 μL of DMSO to each well, oscillate at a low speed on a 96-well plate oscillator for about 5 minutes to fully dissolve the crystal violet, and measure the absorbance value with an enzyme marker at a wavelength of 490nm. Analysis and calculation of cell activity: Cell survival rate = (experimental well - blank well) / (control well - blank well) * 100%. (Note: Due to the poor solubility of MF-P-1~MF-P-3 and MF-P-11, the activity was not measured. The wells with drug-containing culture medium are experimental wells, the wells with drug-free culture medium are control wells, and the wells without any treatment in the 96-well plate are blank wells).

[0031] Experimental test results The results are as follows Figure 11 The results showed that MF-P-5 had the best anti-tumor activity among the seven fractions (MF-P-4~MF-P-10) screened by MTT method. In MDA-MB-231 cells, the IC 50 The IC value of 48 h was 48.01 ± 1.64 μg / mL. 50 The IC value of MF-P-5 on 4T1 cells at 24 h was 13.35 ± 1.15 μg / mL. 50 The IC value of 45.39 ± 1.70 μg / mL at 48 h 50 is 13.13 ± 1.14 μg / mL. Therefore, MF-P-5 has an inhibitory effect on the proliferation of both breast cancer cells.

[0032] Example 4: Discovery of the inhibitory effect of 4-phenylcoumarin MF1 on the proliferation of triple-negative breast cancer cells Experimental purpose: To detect whether MF1 has an inhibitory effect on the proliferation of two breast cancer cells (MDA-MB-231 and 4T1). First, different concentrations of MF1 (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 μM) were incubated with the above two breast cancer cells for 24 h and 48 h, respectively. The inhibitory ability of MF1 on the short-term proliferation of the two breast cancer cells was evaluated by MTT. Secondly, the inhibitory ability of MF1 on the long-term proliferation of the two breast cancer cells was evaluated by cell cloning experiments.

[0033] Experimental method: MTT experiment: Take cells in the logarithmic growth phase, inoculate the cells at a density of 5000 cells / well in a 96-well plate, add 100 μL of cell suspension to each well, and continue to incubate in a cell culture incubator at 37°C with 5% CO2 for 24 hours with DMEM culture medium (containing 10% fetal bovine serum). Then add 100 μL of drug for 24 hours or 48 hours, and then add 20 μL of 5mg / mL MTT solution to each well and incubate in the incubator for 3 hours. After the reaction is sufficient, discard the supernatant, add 150 μL of DMSO to each well, oscillate at a low speed on a 96-well plate oscillator for about 5 minutes to fully dissolve the crystal violet, and measure the absorbance value with an enzyme marker at a wavelength of 490nm. Analysis and calculation of cell activity: Cell survival rate = (experimental wells - blank wells) / (control wells - blank wells) * 100%. (Wells with drug-containing culture medium are experimental wells, wells with drug-free culture medium are control wells, and wells without any treatment in the 96-well plate are blank wells).

[0034] Cell cloning experiment: Take cells in the logarithmic growth phase and inoculate them in a 6-well plate at a density of 800 cells / well. Each well has 2 mL of cell suspension. After culturing in a cell culture incubator at 37°C and 5% CO2 for 24 h, treat the cells with different concentrations of MF1 and continue to culture for about 2 weeks. Fix cells: After the culture is completed, discard the culture medium, wash with PBS, add 2 mL of 4% paraformaldehyde to each well, and fix for 30 min; Cell staining: After complete fixation, discard 4% paraformaldehyde, wash with PBS, add 2 mL of crystal violet staining solution (C0121, Beyotime) to each well, and stain for 20 min at room temperature; After staining, remove the crystal violet staining solution and wash with PBS 5 times. The culture plate was inverted on a white background, dried and photographed. Image J was used to count the cell colonies in each well.

[0035] Evaluation results: 1) MTT assay results like Figure 12 As shown in A and B, the results showed that in MDA-MB-231 cells, the IC 50 The IC value of 16.71±1.52 μM for 48 h 50 The IC value of MF1 on 4T1 cells for 24 h was 9.53±1.28 μM. 50 The IC value of 7.92±1.20 μM for 48 h 50is 6.98±1.14 μM. Therefore, MF1 has an inhibitory effect on the proliferation of both breast cancer cells.

[0036] Cell clone test results Cell clone formation assay was used to evaluate the effect of MF1 on the long-term proliferation of MDA-MB-231 and 4T1 cells. Figure 12 As shown in C and D, clones formed by treating MDA-MB-231 and 4T1 cells with different concentrations (0, 5, 10, 20 μM) of MF1 were obtained. The experimental results showed that MF1 exhibited good activity in inhibiting long-term proliferation of both MDA-MB-231 and 4T1 cells.

[0037] Example 5 Discovery of the inhibitory effect of 4-phenylcoumarin MF1 on the metastasis of triple-negative breast cancer cells Experimental purpose: To detect whether MF1 has an inhibitory effect on the metastasis of two breast cancer cells (MDA-MB-231 and 4T1). First, MDA-MB-231 and 4T1 cells were treated with MF1 at different concentrations (0, 6.25, 12.5, 25 μM) for 24 h, and then cell scratches were detected to verify whether MF1 would inhibit the migration of tumor cells. Secondly, the expression level of tumor metastasis marker E-cadherin was detected by Western Blot.

[0038] Experimental method: Cell scratch test: Take cells in the logarithmic growth phase and press 100×10 4 The cells were inoculated at a density of 100 / well in a 6-well plate, placed in an incubator and cultured in a cell culture incubator at 37°C and 5% CO2 with DMEM medium (containing 10% fetal bovine serum). When the cells were fully grown, a horizontal line was vertically drawn on the bottom of the dish with a 1ml sterile pipette tip, and then gently washed with PBS and added with serum-free medium for culture. Photos were taken at 0 h and 24 h to record the migration of tumor cells.

[0039] Western Blot: Cells were treated with different doses of MF1 for 48 h, and adherent and floating cells were collected. The cell pellet was resuspended in cell lysis buffer (P0013B, Beyotime) and lysed at 4°C for 1 h. Then, after centrifugation at 12,000 rpm for 20 min, the protein content in the supernatant was determined using the BCA protein concentration assay kit (P0011, Beyotime). Equal amounts of total protein were then separated by 10-15% SDS-PAGE and transferred to a PVDF membrane, which was soaked in blocking buffer (5% skim milk or BSA). Next, the proteins were detected with primary antibodies, followed by HRP-conjugated secondary antibodies, and ECL was used as an HRP substrate for color development. Immunoblots were quantified using Image J.

[0040] Evaluation results: 1) Cell scratch test results like Figure 13 As shown in A and B, the results of the cell scratch assay showed that MF1 inhibited the migration of MDA-MB-231 and 4T1 cells in a dose-dependent manner.

[0041] Experimental test results The expression level of tumor metastasis marker E-cadherin was further detected by Western Blot. Figure 13 As shown in C, Western blot results also showed that MF1 upregulated E-cadherin, proving that MF1 inhibited the migration of MDA-MB-231 and 4T1 cells.

[0042] Example 6 Discovery of the effect of 4-phenylcoumarin MF1 in inducing apoptosis of triple-negative breast cancer cells Experimental purpose: To detect whether MF1 can induce apoptosis in two breast cancer cells (MDA-MB-231 and 4T1). First, after treating MDA-MB-231 and 4T1 cells with different concentrations of MF1 (0, 5, 10, 20, 40 μM) for 24 h, Annexin V-PI cell apoptosis assay was used to verify whether MF1 can induce apoptosis in two breast cancer cells (MDA-MB-231 and 4T1). Secondly, Western Blot was used to detect the expression levels of tumor apoptosis markers to evaluate whether MF1 can induce apoptosis in two breast cancer cells.

[0043] Experimental method: Annexin V-PI cell apoptosis detection: cells in the logarithmic growth phase were taken and the cells were plated at 30×10 4 Cells were inoculated in a 6-well plate at a density of 100 / well, with 2 mL of cell suspension per well. After incubation in a cell culture incubator with DMEM medium (containing 10% fetal bovine serum) at 37°C and 5% CO2 for 24 h, the cells were treated with different concentrations of MF1 for 48 h. After MF1 treatment for 48 h, the cells were collected, washed once with PBS, and the PBS was discarded. The collected cell pellet was resuspended in 100 μL Binding Buffer (FXP018-100, Tetraseiboshi), 5 μL Annexin V / FITC (FXP018-100, Tetraseiboshi) was added and mixed, and then incubated at 37°C in the dark for 30 min. After the incubation, 10 μL of 20 μg / mL propidium iodide solution (PI) was added, and 400 μL of 1× binding buffer was added and mixed, and flow cytometry was performed immediately.

[0044] Western Blot: Cells were treated with different doses of MF1 for 48 h, and adherent and floating cells were collected. The cell pellet was resuspended in cell lysis buffer (P0013B, Beyotime) and lysed at 4°C for 1 h. After centrifugation at 12,000 rpm for 20 min, the protein content in the supernatant was determined using the BCA protein concentration assay kit (P0011, Beyotime). Equal amounts of total protein were then separated by 10-15% SDS-PAGE and transferred to a PVDF membrane, which was soaked in blocking buffer (5% skim milk or BSA). Next, the proteins were detected with primary antibodies, followed by HRP-conjugated secondary antibodies, and ECL was used as an HRP substrate for color development. Immunoblots were quantified using Image J.

[0045] Evaluation results: 1) Annexin V-PI staining test results After 48 h of treatment with different concentrations of MF1 (0, 5, 10, 20, 40 μM), MDA-MB-231 and 4T1 cells were stained with Annexin V-PI by flow cytometry. The results of Annexin V-PI staining showed that MF1 induced apoptosis of MDA-MB-231 and 4T1 cells in a dose-dependent manner. Figure 14 A and B in Chinese.

[0046] Experimental test results The expression levels of classic apoptosis markers were detected by Western blot. The results of Western blot experiments showed that MF1 significantly stimulated the mitochondrial apoptosis pathway by upregulating Bax. At the same time, MF1 also promoted the cleavage of PARP. Figure 14 Middle C.

[0047] Example 7 Study on the anti-breast cancer activity and mechanism of action of MF-P-5 in vivo Experimental purpose: To evaluate the anti-tumor activity of MF-P-5 in nude mice with MDA-MB-231 cell xenografts in terms of tumor growth rate, tumor volume and tumor weight. The proliferation markers of tumor tissues were detected by immunohistochemistry, and the proliferation and metastasis markers of tumor tissues were also detected by Western Blot.

[0048] Experimental methods: Establishment of nude mouse xenograft tumor model: Twelve female BALB / c nude mice were selected and fed normally for one week to adapt to the environment. MDA-MB-231 cells (5×10 6 When the tumor volume reaches 0.1cm3 Around 24 hours after treatment, the mice were randomly divided into 2 groups (n = 6): blank control group (normal saline 50 mg / kg), MF-P-5 (50 mg / kg). During the treatment period, the drug was administered by gavage, 200 μL per mouse per day, the animals were weighed every day, and the tumor size was measured with an electronic vernier caliper. After the treatment, all mice were killed, and the tumor tissues were taken, weighed, and photographed. Then, the tumor tissues were immediately frozen in liquid nitrogen or fixed in formalin for further study.

[0049] Western Blot: Cells were treated with different doses of MF-P-5 for 48 h, and adherent and floating cells were collected. The cell pellet was resuspended in cell lysis buffer (P0013B, Beyotime) and lysed at 4°C for 1 h. Then, after centrifugation at 12,000 rpm for 20 min, the protein content in the supernatant was determined using the BCA protein concentration assay kit (P0011, Beyotime). Equal amounts of total protein were then separated by 10-15% SDS-PAGE and transferred to a PVDF membrane, which was soaked in blocking buffer (5% skim milk or BSA). Next, the proteins were detected with primary antibodies, followed by HRP-conjugated secondary antibodies, and ECL was used as an HRP substrate for color development. Immunoblots were quantified using Image J.

[0050] Evaluation results: 1) MDA-MB-231 nude mouse xenograft tumor experimental test results like Figure 15 As shown in A, B, C, D, and E, Figure A shows that the tumor volume has decreased, Figure B shows the weight of nude mice weighed daily, and Figure C shows the results of statistical analysis of tumor volume using Image J and compared with the Control group. Figure E shows the results of statistical analysis of tumor weight using Image J and compared with the Control group. It can be seen from the figure that the tumor proliferation rate has slowed down, the tumor volume has decreased, and the tumor weight has decreased. The results of tumor proliferation rate, tumor volume, and tumor weight show that MF-P-5 can effectively slow down the proliferation of MDA-MB-231 tumor cells.

[0051] Results of immunohistochemical experiments on nude mouse xenograft tumors Immunohistochemistry was used to evaluate the effect of MF-P-5 on the proliferation marker Ki-67 in tumor tissues. The experimental results showed that the expression of the proliferation marker Ki-67 was reduced after MF-P-5 treatment, and there was a statistically significant difference (e.g. Figure 15 (as shown in F).

[0052] Western blot test results of nude mouse xenograft tumors The expression levels of MMP-2, E-cadherin and Bcl-2 proteins in tumor tissues were detected by Western blot. The experimental results showed that the expression of E-cadherin in tumor tissues increased significantly after MF-P-5 treatment, and the expression level of MMP-2 was reduced. This indicates that MF-P-5 can also inhibit the migration of breast cancer cells in vivo. At the same time, the expression of Bcl-2 in tumor tissues was also detected by Western blot, and the experimental results showed that the expression level of Bcl-2 was significantly reduced. This indicates that MF-P-5 significantly stimulated the mitochondrial apoptosis pathway (such as Figure 15 (shown in G).

Claims

1. A 4-phenylcoumarin compound MF1, characterized in that: The structural formula is as follows: 。 2. A composition, characterized in that Contains the 4-phenylcoumarin compound MF1 described in claim 1.

3. The composition according to claim 2, characterized in that The composition is a traditional Chinese medicine extract.

4. The composition according to claim 3, characterized in that The Chinese medicine extract is an extract of dried flower buds of Tetrapanax ferox.

5. The composition according to claim 4, characterized in that The ironwood dried flower bud extract is prepared by the following steps: taking ironwood dried flower bud powder, extracting with an organic solvent, evaporating and concentrating the extract to obtain an extract, then dispersing the extract with pure water, extracting with petroleum ether to obtain a petroleum ether layer extract, eluting the petroleum ether layer extract with a silica gel column, the eluent being a petroleum ether-acetone mixture with a volume ratio of 100:0 to 0:100, and obtaining a total of 11 components MF-P-1 to MF-P-11 in ascending polarity, and MF-P-5 is the ironwood dried flower bud extract.

6. The composition according to claim 5, characterized in that The organic solvent is methanol or ethanol with a volume concentration of 75% to 95%. The extraction methods include: ultrasonic extraction, each time for 25-35 minutes; flash extraction, each time for 3-6 minutes; reflux extraction, each time for 1-3 hours; immersion extraction, each time for 36-48 hours.

7. The method for extracting 4-phenylcoumarin compound MF1 according to claim 1, characterized in that: The steps include: 1) Take dried bud powder of Tetrapanax ferox, extract it with an organic solvent, evaporate and concentrate the extract to obtain an extract, then disperse the extract with purified water, extract it with petroleum ether to obtain a petroleum ether layer extract, elute the petroleum ether layer extract with a silica gel column, and the eluent is a petroleum ether-acetone mixture with a volume ratio of 100:0 to 0:100, and obtain 11 components MF-P-1 to MF-P-11 in descending order of polarity; 2) Component MF-P-5 was again loaded onto a silica gel column and eluted with a petroleum ether-acetone mixture in a volume ratio of 100:0-0:100, and 11 fractions A1-A11 were obtained in order of polarity. Fraction A7 was subjected to open ODS column chromatography and gradient eluted with a methanol-water mixture in a volume ratio of 30:70-100:0, and 14 subfractions A7B1-A7B14 were obtained in order of polarity. Subfraction A7B11 was separated by reverse phase high performance liquid chromatography using a methanol-water mixture in a volume ratio of 90:10 as the mobile phase to obtain 4-phenylcoumarin compound MF1.

8. Use of the 4-phenylcoumarin compound MF1 according to claim 1 and / or the composition according to any one of claims 2 to 6 in the preparation of a drug for treating triple-negative breast cancer.

9. The use according to claim 8, characterized in that The triple-negative breast cancer refers to a type of breast cancer in which estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 are all negative.

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