Preparation method of scutellarin A and its application in preparing antitumor drugs

Abstract

The present invention relates to a method for preparing withangulatin A and its use in preparing anti-tumor drugs, which belongs to the field of medical technology. Specifically, it relates to a method for preparing withangulatin A and its use in preparing anti-tumor drugs. The present invention provides a rapid preparation method for withangulatin A, which has a simple preparation process and a high extraction rate, thereby ensuring the large-scale enrichment of withangulatin A. The preparation process of the present invention does not use toxic and harmful solvents, and the process is environmentally friendly. The product yield is high, the content is high, the process is simple and safe, the equipment requirements are low, and the production cost is low. In addition, the present invention also discovered that withangulatin A has anti-tumor effects against various types of cancer, especially that withangulatin A enhances the sensitivity of PI3K inhibitors to triple-negative breast cancer cells, and the two have a synergistic anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a preparation method of scutellarin A and application of the same in the preparation of anti-tumor drugs. Background Art

[0002] In recent years, cancer has become increasingly widespread and is affecting younger people. It has become the second leading cause of death worldwide, with incidence rates increasing rapidly each year. The cancer epidemic in my country is influenced by multiple factors, including an aging population, increasing environmental pollution, and infections, which have severely impacted both the health of residents and social development. Numerous researchers are dedicated to the development of various anticancer drugs. Natural products have diverse structures and, consequently, diverse biological activities. Identifying active lead compounds from these products is a key avenue for drug discovery. According to the latest literature, a total of 1,394 small molecule drugs were approved for treatment between 1981 and 2019, of which approximately 64.5% are related to natural products.

[0003] Withania lactide compounds are a class of steroidal compounds with excellent activity. Due to their structural diversity, these compounds have excellent pharmacological activities in many aspects, such as anti-tumor, anti-inflammatory, antibacterial, hypoglycemic, and immunomodulatory. Some genera of Solanaceae plants mainly contain these compounds, among which the main distribution is in plants such as Physalis, Withania, Datura, and Solanum (Natural Product Reports, 2021, 10.1039 / d1np00055a.). Physalis plants are widely distributed in China and have multiple species, including Physalis, Lantern, Physalis spp., Bitterroot, Bitterroot, Physalis spp., and Lantern fruit.

[0004] Physalis minima L. is an annual herbaceous plant of the Solanaceae family and the genus Physalis. It is primarily distributed in Guangdong, Guangxi, Yunnan, and Sichuan provinces of my country. The whole plant is used medicinally, with a bitter taste and cooling properties. The whole plant has the effects of clearing heat and dampness, removing phlegm and relieving cough, softening and dispersing lumps, reducing inflammation, killing insects, and detoxifying. It is primarily used to treat damp-heat jaundice, dysuria, chronic cough and asthma, malnutrition, scrofula, and can be applied externally to treat pemphigus, eczema, and furuncles. Previous literature research revealed that this plant primarily contains withanolides and flavonoids, making it of high research and development value. With the deepening of research on the plant's chemical components, research on its pharmacological effects is also increasing. Physalis minima L. and its isolated compounds have demonstrated a variety of pharmacological activities, including anti-tumor, anti-inflammatory, and antibacterial. Ma, L. et al. used the MTT assay to test the cytotoxic effects of withanolide compounds isolated from Physalis alkekengi on colorectal cancer cells HCT-116 and human non-small cell lung cancer cells NCI-H460. The IC values ​​of most of these compounds were 50The values ​​are between 10-60μM (Helvetica Chimica Acta 2007,90:1406-1419.). Wu Jiang et al. tested the inhibitory activity of multiple withanolide compounds isolated from Physalis alkekengi against lung adenocarcinoma cell A549, liver cancer cell SMMC-7721 and breast cancer cell MCF-7 by MTT assay. The IC 50 The values ​​ranged from 40.01 to 82.17 μM, indicating moderate cytotoxic activity (Phytochemistry 2018, 155:164-170). Preliminary studies have shown that extracts from Physalis alkekengi exhibit antitumor cell proliferation activity. To further clarify the active chemical components of Physalis alkekengi, the extracts were isolated to obtain the main active components and their antitumor activity was studied. However, there are currently no reports of a high-purity preparation of scutellarin A from Physalis alkekengi. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a method for preparing withangulatin A.

[0006] Another object of the present invention is to provide the use of scutellarin A in the preparation of anti-tumor drugs.

[0007] In the first aspect, the scutellarin A used in the present invention is extracted and isolated from Physalis minima L. and is a white amorphous powder (MeOH). Nuclear magnetic resonance data is consistent with the literature (Heterocycles, 1990, 31(7), 1371-5.), confirming its structure as scutellarin A with a purity of >96%. The scutellarin A described in the present invention has the following structural formula:

[0008]

[0009] The present invention provides a method for preparing scutellarin A, which is achieved through the following technical scheme.

[0010] The withanolide compound is obtained from the aerial part of the medicinal plant Physalis spp. of the Solanaceae family as a raw material through organic solvent extraction, extraction, silica gel column separation, and high performance liquid chromatography separation steps, specifically comprising the following steps:

[0011] (1) Organic solvent extraction process: using the aerial part of Physalis alkekengi as raw material, adding 8 to 12 times the mass of the raw material and having a volume concentration of 60% to 80% ethanol aqueous solution, reflux extraction 2 to 4 times, each extraction time 2 to 4 hours, combining to obtain the extract, decompressing and recovering the solvent, and concentrating to obtain the total extract; wherein, the 8 to 12 times the mass of the raw material specifically refers to the volume mass ratio of the added ethanol aqueous solution to the raw material being (8 to 12):1 in mL / g;

[0012] (2) Organic solvent extraction: The total extract is dispersed in 5-10 times the mass of water, extracted with petroleum ether and ethyl acetate in sequence, and the extract is concentrated to recover the solvent to obtain a petroleum ether extract concentrate, an ethyl acetate extract concentrate and an aqueous phase respectively;

[0013] (3) Silica gel column chromatography: The ethyl acetate extract concentrate was separated by silica gel column chromatography, and gradient elution was performed with dichloromethane-methanol in a volume ratio of 100:1, 50:1, 20:1, 15:1, 10:1, 5:1, 1:1 and 0:1, respectively, to obtain fraction E3 (the ratio of dichloromethane to methanol was 50:1); the filler used for the column chromatography was 100-300 mesh column chromatography silica gel, and the weight ratio of the extract to the filler after the extract was concentrated was 1:10-1:20; fraction E3 was concentrated and separated by silica gel column chromatography, and gradient elution was performed with dichloromethane-acetone in a volume ratio of 70:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 and 0:1, respectively, to obtain fraction E34 (the ratio of dichloromethane to acetone was 10:1);

[0014] (4) HPLC separation and purification: Fraction E34 was dissolved in methanol, and 60%-70% acetonitrile or methanol was used as the mobile phase at a flow rate of 8 mL / min. A C18 reverse phase column was used as the stationary phase, the UV detector was 210 nm, the injection volume was 50-500 μL, and the chromatographic peak was collected for 30-60 min. After multiple accumulation, the compound was evaporated to dryness to obtain the compound zearalenone A.

[0015] The present invention provides another rapid preparation method of scutellarin A, which comprises the following steps:

[0016] 1) refluxing the whole herb of Physalis alkekengi with an extraction solvent to obtain an extract; the whole herb of Physalis alkekengi includes the stems, leaves, flowers, and fruits of the plant; the extraction solvent is alcohol or an aqueous solution of 40-100% alcohol by volume; the alcohol is methanol or ethanol; the amount of the extraction solvent added each time is 5-10 times the weight of the whole herb of Physalis alkekengi, and refluxing extraction is performed at 40-100° C. for 1-5 times, each time for 1-5 hours;

[0017] 2) concentrating the extract obtained in step 1), dispersing it with water, and then extracting it once or multiple times with a low-polarity organic solvent with a polarity between 0 and 0.2 to remove low-polarity impurities; the low-polarity organic solvent is at least one of petroleum ether, n-hexane, and cyclohexane;

[0018] 3) extracting the remaining aqueous layer after the extraction in step (2) once or multiple times with a medium-polarity organic solvent with a polarity between 3 and 5.6 to obtain an extract, concentrating the extract, and separating the extract by column chromatography. Eluting the extract with an elution system consisting of dichloromethane and methanol to obtain a first mixed component containing scutellarin A, wherein the filler for the column chromatography is 100-300 mesh column chromatography silica gel; the medium-polarity organic solvent is at least one of chloroform, dichloromethane and ethyl acetate; and the elution system consisting of dichloromethane and methanol is eluted, specifically, the volume ratio of dichloromethane to methanol is 100:1, 70:1, 50:1, 20:1, 10:1, and 1:1 in a gradient elution manner.

[0019] or

[0020] The remaining aqueous layer in step 2) is adsorbed by a macroporous adsorption resin and gradient eluted with an elution system consisting of ethanol and water to obtain a second mixed component containing zearalenone A; the macroporous adsorption resin model is selected from any one of AB-8, D101, DM130, ADS-17, HPD-100, HPD-300, and HPD-600; the elution system consisting of ethanol and water is a gradient elution, and the elution gradient is specifically as follows: first, 3-10 volumes of 40-50% ethanol-water solution are used to elute the impurities, and then 3-10 volumes of 60-90% ethanol-water solution are used to elute to obtain a second mixed component containing zearalenone A; the obtained second mixed component containing zearalenone A is separated by column chromatography, and gradient eluted with an elution system consisting of methanol and water, and a single component containing zearalenone A is quickly locked in based on the behavior of thin layer chromatography; the filler used in the column chromatography is octadecylsilane bonded silica gel RP-C 18 The weight ratio of the single component containing scutellarin A to the filler is 1:20 to 1:50; the elution system composed of methanol and water is a gradient elution, and the elution gradient is: first eluting with 30-40% methanol-water solution, then eluting with 50-60% methanol-water solution; and finally eluting with 90-100% methanol-water solution;

[0021] 4) The first mixed component containing zearalenone A or the single component containing zearalenone A obtained in step 3) is subjected to preparative high performance liquid chromatography, eluted and concentrated with an elution system consisting of methanol and water to obtain high-purity zearalenone A; the preparative liquid chromatography column packing is RP-C 18The elution system in this step is a methanol-water solution containing 50-80% methanol by volume. Finally, high-purity, single-stranded scutellarin A is obtained.

[0022] In a second aspect, the present invention provides a use of scutellarin A in the preparation of anti-tumor drugs.

[0023] Preferably, the scutellarin A is prepared using the preparation method of the present invention.

[0024] As an optional mode, in the above application, the tumor is renal cancer, breast cancer, lung adenocarcinoma, colon cancer, fibrosarcoma, pancreatic cancer or ovarian cancer,

[0025] Preferably, the breast cancer is triple-negative breast cancer (eg, MDA-MB-231 cells).

[0026] As an optional method, in the above application, the present invention also provides an anti-tumor mechanism of scutellarin A, wherein scutellarin A regulates HO-1 protein and GPX4 protein, thereby causing Fe 2+ The accumulation of lipid ROS promotes ferroptosis of tumor cells and exerts anti-tumor effects.

[0027] As an optional mode, in the above application, the drug is prepared from scutellarin A and a PI3K inhibitor, and the scutellarin A enhances the sensitivity of the PI3K inhibitor to triple-negative breast cancer cells, and the two have a synergistic anti-tumor effect.

[0028] Preferably, the PI3K inhibitor is 3-MA.

[0029] As an optional manner, in the above application, the ratio of the scutellarin A to the 3-MA is 1:1-1:10 by weight.

[0030] As an option, in the above application, the drug further comprises a pharmaceutically acceptable excipient.

[0031] Preferably, the dosage form of the drug is powder, tablet, capsule, pill, suppository, drop pill, enteric solvent, injection, syrup, emulsion, suspension or tincture.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] Existing methods for the preparation and purification of zearalenone A (such as Chinese Herbal Medicine, 2018, 1: 62-68) mostly use solvent extraction, silica gel column separation, ODS column separation, and gel column separation in the steps. However, the method has many steps and the extraction rate is low, and it is impossible to effectively enrich. The present invention provides a rapid preparation method of zearalenone A. The preparation process is simple to operate and the extraction rate is high, which provides a guarantee for the large-scale enrichment of zearalenone A. The advantages of the present invention are that no toxic and harmful solvents are used in the preparation process, the process has no environmental pressure, the product yield is high, the content is high, the process is simple and safe, the equipment requirements are low, and the production cost is low. In addition, the present invention also found that zearalenone A has anti-tumor effects against various types of cancer, especially zearalenone A enhances the sensitivity of PI3K inhibitors to triple-negative breast cancer cells, and the two have a synergistic anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the liquid phase analysis chromatogram of scutellarin A prepared by the existing method.

[0035] Figure 2 The figure is a liquid phase analysis chromatogram of scutellarin A obtained by the rapid preparation method of the present invention.

[0036] Figure 3 Figure 1 shows the inhibitory effect of zeararin A on MDA-MB-231 cell proliferation in vitro. (A) Cloning assay evaluating the effect of different concentrations of zeararin A on MDA-MB-231 cell proliferation. (B) 3D spheroid assay evaluating the effect of different concentrations of zeararin A on MDA-MB-231 cell proliferation.

[0037] Figure 4 Figure 1 shows the inhibitory effect of scutellarin A on MDA-MB-231 cell proliferation in vivo. (A) Changes in tumor volume. (B) Changes in nude mouse body weight. (C) Tumor mass. (D) Images of tumor tissues under different dosing conditions.

[0038] Figure 5 For functional and pathway enrichment analysis of proteomics data.

[0039] Figure 6 To investigate the anti-tumor mechanism of zearalin A. (A) Western Blot evaluation of HO-1 and GPX4 expression in MDA-MB-231 cells after zearalin A treatment. (B) Western Blot evaluation of HO-1 and GPX4 expression in MDA-MB-231 cell xenografts after zearalin A treatment. (C) Detection of Fe 2+ Levels, (D) Detection of lipid ROS levels.

[0040] Figure 7 To investigate the combined effect of scutellarin A and PI3K inhibitor 3-MA. (A) Western Blot evaluation of AKT and mTOR phosphorylation levels, (B) CCK8 assay evaluation of the effect of combined administration on MDA-MB-231 cell proliferation, (C) cloning assay evaluation of the effect of combined administration, (D) detection of Fe 2+ (E) Detection of lipid ROS levels, (F) Western Blot evaluation of HO-1 and GPX4 expressions. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only 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 making creative efforts are within the scope of protection of the present invention.

[0042] Example 1: Separation and preparation of scutellarin A standard

[0043] 1. Experimental Methods

[0044] 1 kg of the aerial part of Physalis alkekengi was placed in an extraction tank, and 10 L of a 75% ethanol-water solution was added. The mixture was heated under reflux and extracted three times for 2 hours each time. The combined extracts were concentrated under reduced pressure, diluted to 1 L with water, and extracted three times with an equal volume of petroleum ether to remove low-polarity impurities. The extract was then extracted three times with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated to yield 22.2 g of an extract. The extract was separated by silica gel column chromatography using a gradient elution of dichloromethane and methanol (volume ratios: 100:1, 70:1, 50:1, 20:1, 10:1, and 1:1). Using thin-layer chromatography (TLC) as a guide, a mixed component (E3) containing styracin A was quickly identified. E3 was then subjected to preparative high-performance liquid chromatography (HPLC) using a 70% methanol-water solvent to yield styracin A (11 mg) of high purity.

[0045] 2. Experimental Results

[0046] The HPLC purity test of kusarabinin A is shown in the following figure: Figure 1 As shown, the peak time is 7.06 minutes and the purity is 97.4% (YMC-Triart C 18 5 μm, 250×4.6 mm; 70% methanol-water; 0.8 mL / min).

[0047] The obtained scutellarin A was a white powder (MeOH), and the NMR data were as follows: 1H-NMR (pyridine-d5, 600MHz): δ6.53(1H,dd,J=10.0,2.0Hz,H-2), 7.32(1H,ddd,J=10.0,6.0,2.0Hz,H-3), 2.03(3H, s,OAc-15), 1.33(3H,s,H-18),1.51(3H,s,H-19),1.22(3H,d,J=7.0Hz,H-21),1.87(3H,s,H-27),1.84(3H,s,H-28). 13 C-NMR (pyridine-d5, 150MHz): δ203.1(C-1), 132.2(C-2), 146.2(C-3), 70.6(C-4), 61.3(C-5), 64.3( C-6), 27.3(C-7), 35.6(C-8), 40.9(C-9), 48.1(C-10), 21.8(C-11), δ37.9(C-12), 53.2(C-13), 81.8( C-14), 84.9(C-15), 122.7(C-16), 162.6(C-17), 18.3(C-18), 16.7(C-19), 36.6(C-20), 17.3(C-21), 79.2(C-22), 32.8(C-23), 149.8(C-24), 122.1(C-25), 166.6(C-26), 12.9(C-27), 20.0(C-28), 21.6( Me CO-15), 170.3(Me C The NMR data were consistent with the literature (Heterocycles, 1990, 31(7), 1371-5.), and its structure was confirmed to be scutellarin A.

[0048] Example 2: Rapid separation and preparation method of scutellarin A

[0049] 1. Experimental Methods

[0050] 1 kg of the whole plant of Physalis alkekengi was placed in an extraction tank, and 10 L of 75% ethanol-water solution was added. The mixture was heated and refluxed for 3 times, each time for 2 hours. The extracts were combined, concentrated under reduced pressure, dispersed into 1 L of water, and extracted 3 times with an equal volume of petroleum ether to remove small polar impurities. The remaining water layer was adsorbed by D101 macroporous adsorption resin (resin volume 200 mL), first eluted with 800 mL of 40% ethanol-water solution to remove impurities, and then eluted with 800 mL of 80% ethanol-water solution. The eluate was collected to obtain a second mixed component containing zearalenone A. The obtained second mixed component of zearalenone A was purified by RP-C 18ODS column chromatography was used for separation, with a methanol-water gradient elution (methanol / water, volume ratio of 30:70, 50:50, 90:10). Guided by thin layer chromatography behavior, a single component (D2) containing zearalenone A was quickly identified. D2 was subjected to preparative high performance liquid chromatography, eluted with a 60% volume fraction methanol-water solvent, to obtain zearalenone A (140 mg) of high purity.

[0051] 2. Experimental Results

[0052] The obtained scutellarin A and the reference substance scutellarin A were analyzed by high performance liquid chromatography, and it was confirmed that it was scutellarin A with a purity of 96.8% (YMC-Triart C 18 5 μm, 250 × 4.6 mm; 70% methanol-water; 0.8 mL / min), such as Figure 2 shown.

[0053] Example 3: Study on the anti-tumor effect and mechanism of scutellarin A

[0054] 1. CCK8 assay to detect the effect of scutellarin A on tumor cell survival rate

[0055] A variety of tumor cells in the logarithmic growth phase, including (MCF-7, MDA-MB-231, Caki-2, HepG2, A549, HCT116, SW1990, U2OS, ES-2, HCC1806 and HT1080 cells) were seeded at 5000 cells / well in a 96-well plate and cultured for 12 hours. The cells were treated with different concentrations of zearalenone A (100, 50, 25, 12.5, 6.25 and 3.125 μmol / L). The cell wells to which the corresponding volume of DMSO was added were used as blank controls. After 24 hours, the culture medium was discarded, and 100 μL of culture medium containing 10% CCK8 was added to each well. After culturing for 20 minutes, the OD value of each well at 450 nm was detected by a microplate reader to calculate the IC 50 According to the results of CCK8 experiments (Table 1), scutellarin A was more sensitive to breast cancer cells MCF-7 and MDB-MA-231. Finally, scutellarin A was selected to study the anti-tumor mechanism in breast cancer cells MDB-MA-231.

[0056] Table 1 Cytotoxic activity of scutellarin A (IC 50 :μM)

[0057]

[0058]

[0059] 2. Weichakurin A inhibits the proliferation of MDB-MA-231 cells in vitro and in vivo

[0060] The activity of withangulatin A (WA) in inhibiting tumor cell proliferation was detected using plate cloning and 3D cell spheroid assays. Figure 3 As shown, the compound significantly inhibited the proliferation of tumor cells at a concentration of 1 μM.

[0061] After injection of MDB-MA-231 cells, nude mice were randomly divided into three groups: a control group and two scutellarin A treatment groups (20 and 40 mg / kg). After successful model establishment, the control group was gavaged with normal saline, while the treatment groups were gavaged with scutellarin A. After 24 days, a significant reduction in tumor volume was observed in the experimental groups. The average tumor weight in the experimental groups was significantly reduced. Furthermore, no significant differences in body weight were observed between the groups throughout the study ( Figure 4 ), indicating that the dose of scutellarin A used in this experiment had no obvious side effects on the growth of animals.

[0062] 3. Weichakurin A induces ferroptosis in triple-negative breast cancer cells

[0063] To explore the mechanism of action of zearalenone A in inhibiting triple-negative breast cancer cell proliferation, the tumor tissues obtained from the above experiment were subjected to proteomic analysis. The up-regulated and down-regulated proteins in the zearalenone A group were analyzed, and their functional enrichment and pathway enrichment were performed ( Figure 5 The results showed that the regulation of ferric ferric acid A on tumor cells involved ribosomes, RNA transport, RNA degradation, ferroptosis and other related signals.

[0064] The mechanism of action of zearalenone A was then verified in cells and tumor tissues. MDA-MB-231 cells were seeded in 6-well plates and cultured for 12 hours. After the experimental groups were treated with different concentrations of zearalenone A (0.5, 1, and 2 μmol / L) for 24 hours, the culture medium was discarded, the cells were washed, and the expression of proteins HO-1 and GPX4 was measured. The exposed bands were analyzed for grayscale using a Gel-Pro analyzer. In addition, tissues of mice bearing xenograft tumors treated with zearalenone A were taken, tissue proteins were extracted, and the expression of proteins HO-1 and GPX4 was measured. The exposed bands were analyzed for grayscale using a Gel-Pro analyzer. Figure 6 A and Figure 6 Results B showed that ferroptosis-induced ferroptosis in the cells was detected by Fe 2+ and lipid ROS, the results showed that scutellarin A increased Fe 2+ and lipid ROS levels, and the addition of the ferroptosis inhibitor Fer-1 inhibited the effect of ferroptosis inhibitor A ( Figure 6 C and Figure 6 D) In ​​conclusion, ferroptosis of MDA-MB-231 cells was induced by ferroptosis of ferroptosis-induced MDA-MB-231 cells.

[0065] 4. Weichakurin A and PI3K inhibitors have synergistic anti-tumor effects.

[0066] From the pathway signal enrichment of proteomics, it was determined that scutellarin A can regulate the PI3K pathway. First, the effect of scutellarin A on the PI3K signaling pathway in MDA-MB-231 cells was evaluated. The results showed that the compound activated the PI3K-AKT-mTOR signaling pathway ( Figure 7 A). Subsequently, the combined use of zearalenone A (1 μmol / L) and PI3K inhibitor 3-MA (10 μmol / L) was investigated, and the CCK8 experiment was used to evaluate its effect on the proliferation of MDA-MB-231 cells. The results showed that the inhibition rate of the zearalenone A group was 47.54±2.20%, the inhibition rate of the 3-MA group was 10.79±1.15%, and the inhibition rate of the combined drug group was 71.13±0.90%. According to the calculation formula of Kim Jong-kyun known in the art (see, Kim Jong-kyun, "Addition in combined medication", Chinese Pharmacological Acta, December 1980, 1 (2) 70-76), the synergistic index q value was determined, q>1, proving that the combination of the two drugs has a synergistic effect ( Figure 7 B). Figure 7 The results of cell cloning in C also verified this conclusion. To further determine whether 3-MA can synergistically induce ferroptosis or whether 3-MA can increase the sensitivity of ferroptosis to ferroptosis, we analyzed the Fe 2+ The results showed that ferroptosis of MDA-MB-231 cells was induced by 3-MA and ferroptosis of MDA-MB-231 cells. Figure 7 D. Figure 7 E and Figure 7 F).

[0067] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing scutellarin A, characterized in that: The following steps are involved: (1) Using the whole plant of Physalis alkekengi as raw material, reflux extraction is performed with a methanol or ethanol aqueous solution with a volume fraction of 40% to 100% and a liquid-to-material weight ratio of 5:1 to 10:1 to obtain an extract; (2) concentrating the extract obtained in step (1), dispersing it with water, and then extracting it once or multiple times with a low-polarity organic solvent; the low-polarity organic solvent is at least one of petroleum ether, n-hexane, and cyclohexane; (3) extracting the remaining aqueous layer after the extraction in step (2) once or multiple times with a medium-polarity organic solvent to obtain an extract, concentrating the extract, and separating the extract by column chromatography using an elution system consisting of dichloromethane and methanol in a volume ratio of 100:1, 70:1, 50:1, 20:1, 10:1, and 1:1 to obtain a first mixed component containing scutellarin A; the filler for the column chromatography is 100-300 mesh column chromatography silica gel; the medium-polarity organic solvent is at least one of chloroform, dichloromethane, and ethyl acetate; or The remaining aqueous layer after extraction in step (2) is adsorbed by a macroporous adsorption resin, and eluted with an elution system consisting of ethanol and water to obtain a second mixed component containing zearalenone A, specifically: first eluted with a 40-50% by volume ethanol-water solution, and then eluted with a 60-90% by volume ethanol-water solution; the second mixed component containing zearalenone A is then separated by column chromatography, the filler of the column chromatography is octadecylsilane bonded silica gel, and eluted with an elution system consisting of methanol and water to obtain a single component containing zearalenone A, specifically: first eluted with a 30-40% by volume methanol-water solution, then eluted with a 50-60% by volume methanol-water solution, and finally eluted with a 90-100% by volume methanol-water solution; (4) The first mixed component containing zearalenone A or the single component containing zearalenone A obtained in step (3) is subjected to preparative high performance liquid chromatography, eluted with a 50-80% by volume methanol-water solution and concentrated to obtain zearalenone A; the filler used in the preparative high performance liquid chromatography is octadecylsilane bonded silica gel.

2. The method for preparing scutellarin A according to claim 1, wherein The macroporous adsorption resin is any one of AB-8, D101, DM130, ADS-17, HPD-100, HPD-300 and HPD-600.

3. The use of scutellarin A in the preparation of anti-triple-negative breast cancer drugs, characterized in that: The anti-triple-negative breast cancer drug is prepared from scutellarin A and a PI3K inhibitor. The scutellarin A enhances the sensitivity of the PI3K inhibitor to triple-negative breast cancer cells, and the two have a synergistic anti-tumor effect.

4. The use according to claim 3, characterized in that In terms of weight ratio, the ratio of the scutellarin A to the PI3K inhibitor is 1:1-1:

10.

5. The use according to claim 3, characterized in that The anti-triple-negative breast cancer drug further comprises a pharmaceutically acceptable excipient, and the dosage form of the drug is powder, tablet, capsule, pill, suppository, drop pill, enteric solvent, injection, syrup, emulsion, suspension or tincture.

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