Preparation method for extracting isopentenyl flavone compound from epimedium sagittatum and application of isopentenyl flavone compound

Abstract

The preparation method comprises the following steps: crushing epimedium sagittatum to prepare paste, suspending with water, extracting with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol respectively to obtain extractum of each part, carrying out gradient elution separation by using silica gel column chromatography, and concentrating to obtain the isopentenyl flavone compound extracted from epimedium sagittatum, thereby obtaining the isopentenyl flavone compound extracted from epimedium sagittatum. Combining the similar parts according to a thin layer chromatography (TLC) color development result to obtain a plurality of polar segments; carrying out chromatography and gradient elution on the target polar section to obtain a plurality of components, and then combining the similar components through thin-layer chromatography (TLC) dot plate absorption to obtain a plurality of sub-components; a target component is eluted and purified, fractions with different retention times are collected and dried, and two compounds are respectively obtained, have a remarkable inhibition effect on the cell viability of human breast cancer cells MCF-7, have anti-breast cancer activity, can be applied to preparation of drugs for treating breast cancer, and have huge social and economic benefits.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to an isopentenyl flavonoid compound extracted from Epimedium sagittatum and a preparation method and application thereof. Background Art

[0002] Epimedium is the dried leaves of the plants Epimedium, Epimedium sagittatum, Epimedium pubescens or Epimedium koreana of the Berberidaceae family, among which Epimedium sagittatum is the most widely distributed and used. Modern pharmacological studies have shown that Epimedium sagittatum has a wide range of pharmacological effects, such as anti-tumor, antioxidant, anti-osteoporosis, immune function regulation, anti-atherosclerosis and antidepressant activity. According to literature reports, flavonoids are the main active ingredient type in Epimedium sagittatum, and prenylated flavonoids are a type of structure with isopentenyl substitution on the flavonoid skeleton, which usually has better lipophilicity and pharmacological activity.

[0003] Breast cancer is a common and frequently occurring disease that seriously threatens people's life safety and physical health. Although there are therapeutic drugs available, their effects are limited. The active ingredients of traditional Chinese medicine are complex, have multiple targeting properties, and have unique therapeutic effects in treating diseases. Extracting active ingredients from the original medicine for the treatment of specific diseases is a new trend in the development of traditional Chinese medicine. So, can new active ingredients (compounds) be discovered from Epimedium sagittatum to prepare drugs for treating breast cancer caused by human breast cancer cells MCF-7 and realize their application in the preparation of drugs for treating breast tumors (cancer)? However, there has been no public report so far. Summary of the invention

[0004] In view of the above situation, in order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide an isopentenyl flavonoid compound extracted from Epimedium sagittatum and its preparation method and application, which can effectively solve the problem of using the isopentenyl flavonoid compound extracted from Epimedium sagittatum in the preparation of drugs for treating breast tumors (cancer).

[0005] The technical solution provided by the present invention is that the isopentenyl flavonoid compounds (2"S, 2"'S)-epimesatine O(1a) and (2"R, 2"'R)-epimesatine O(1b) are extracted from Epimedium sagittatum, and the molecular structure formula is:

[0006]

[0007] The preparation method thereof is:

[0008] The dried above-ground part of Epimedium sagittatum is crushed, extracted with ethanol by reflux to obtain an extract, suspended with water to form a suspension, and then extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract respectively; the dichloromethane extract is separated by gradient elution by silica gel column chromatography, and similar parts are combined according to the color development results of thin layer chromatography (TLC) to obtain polar segments Fr.A...Fr.G, Fr.H;

[0009] The polar segment Fr.G was chromatographed on a silica gel column, eluted with petroleum ether / ethyl acetate in a gradient manner, and the solvent was recovered under reduced pressure to obtain multiple components. Then all the components were spotted on a thin layer chromatography (TLC) plate, and the components with similar absorption on the thin layer plate were combined to obtain 18 first group components Fr.F1...Fr.F17, Fr.F18;

[0010] The first group component Fr.G17 was subjected to silica gel chromatography, gradient elution was performed with petroleum ether / ethyl acetate, and multiple components were recovered under reduced pressure. Then, the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 19 second group components Fr.G17.1, Fr.G17.2, Fr.G17.3...Fr.G17.15...Fr.G17.19;

[0011] The second group component Fr.G17.15 was subjected to ODS column chromatography, gradient elution was performed with methanol / water, and the solvent was recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 12 third group components Fr.G17.15.1, Fr.G17.15.2, Fr.G17.15.3...Fr.G17.15.9, Fr.G17.15.9...Fr.G17.15.12;

[0012] The third group component Fr.G17.15.10 was subjected to ODS column chromatography, gradient elution was performed with methanol / water, and the solvent was recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 13 fourth components Fr.G17.15.10.1, Fr.G17.15.10.2, Fr.G17.15.10.3...Fr.G17.15.10.6...G17.15.10.13;

[0013] (6) Preparation of isopentenyl flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine O

[0014] The fourth group component Fr.G17.15.10.6 was purified and separated by semi-preparative HPLC using a 10ID×250mm RP C18 liquid chromatography column, with a mobile phase of acetonitrile:water=60:40 and a flow rate of 2mL / min, to obtain 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…G17.15.10.6.7;

[0015] The component Fr.G17.15.10.6.2 was purified and separated again by HPLC, with the mobile phase being acetonitrile:water=60:40 and the flow rate being 2 mL / min, to obtain 6 components Fr.G17.15.10.6.2.1…Fr.G17.15.10.6.2.4…G17.15.10.6.2.6;

[0016] The component Fr.G17.15.10.6.2.4 was purified and separated again by HPLC, with the mobile phase being acetonitrile:water=60:40 and the flow rate being 2 mL / min, to obtain 5 components Fr.G17.15.10.6.2.4.1…Fr.G17.15.10.6.2.4.4, Fr.G17.15.10.6.2.4.5,

[0017] Among them, the retention time t R The component Fr.G17.15.10.6.2.4.5 at 63.6 min was dried to obtain compound (2"S,2"'S)-epimesatine O (11.2 mg);

[0018] The component Fr.G17.15.10.6.2.4.4 was purified by HPLC again, the mobile phase was acetonitrile: water = 60:40, the flow rate was 2mL / min, and the retention time t was collected. R The fraction at 19.3 min was dried to give (2"R,2"'R)-epimesatine O (9.0 mg).

[0019] The isopentenyl flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine O have a significant inhibitory effect on the cell viability of human breast cancer cells MCF-7, have anti-breast cancer activity, and can be used in the preparation of drugs for treating breast cancer.

[0020] The invention has abundant raw materials and an easy-to-operate preparation method, and can effectively extract isopentenyl flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine from the epimedium sagittatum. The compounds have anti-breast cancer activity, have a significant inhibitory effect on the cell viability of human breast cancer cells MCF-7, and are non-toxic to human normal breast cells MCF-10A. The compounds can be effectively used to prepare drugs for treating breast cancer, and expand the medicinal value and economic value of the epimedium sagittatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The molecular structural formula of the isopentenyl flavonoid compound of the present invention is shown in FIG.

[0022] Figure 2 Epimesatine O of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6);

[0023] Figure 3 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6). DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described in detail below in combination with specific situations.

[0026] The technical solution provided by the present invention is that the isopentenyl flavonoid compounds (2"S, 2"'S)-epimesatine O(1a) and (2"R, 2"'R)-epimesatine O(1b) are extracted from Epimedium sagittatum, and the molecular structure formula is:

[0027]

[0028] The preparation method thereof is:

[0029] (1) Preparation of polar segments

[0030] The dried aerial part of Epimedium sagittatum (80 kg) was crushed, and refluxed and extracted three times at 45°C with 120 L of 70% ethanol each time, each time for 30 min, and the solvent was recovered under reduced pressure to obtain an extract (6.5 kg). The extract was suspended with 3 times the volume of distilled water to obtain a suspension. The suspension was extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol at room temperature for 4 times, each time with 15 L, and the extraction was carried out for 2 h to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract, respectively. The alkane extract (2.1 kg) was separated by gradient elution using a 100-200 mesh normal phase silica gel column chromatography method, with a gradient volume ratio of petroleum ether-ethyl acetate = 50:1, 12 L; 40:1, 20 L; 35:1, 20 L; 20:1, 40 L; 10:1, 40 L; 5:1, 40 L; 1:1, 20 L; 0:1, 4 L. Similar parts were combined according to the TLC color development results to obtain 8 polar segments Fr.A...Fr.G, Fr.H;

[0031] (2) Preparation of the first group of components

[0032] The polar segment Fr.G (100.0 g) was chromatographed on a 100-200 mesh normal phase silica gel column, with petroleum ether / ethyl acetate as the eluent, and gradient elution was performed, the elution gradient being a volume ratio of petroleum ether / ethyl acetate = 50:1, 1 L, 35:1, 5 L, 20:1, 5 L, 10:1, 5 L, 5:1, 5 L, 1:1, 5 L, 0:1, 5 L, the eluates were combined every 1 L, the solvent was recovered under reduced pressure to obtain multiple components, and then all the components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 18 first group components Fr.F1...Fr.F17, Fr.F18;

[0033] (3) Preparation of the second group of components

[0034] The first group component Fr.G17 (65.0 g) was subjected to gradient elution through 200-300 mesh normal phase silica gel, and the volume gradient ratio of the eluent was petroleum ether / ethyl acetate = 35:1, 1L, 20:1, 4L, 10:1, 4L, 5:1, 4L, 1:1, 2L, 0:1, 1L, and the eluents were combined together every 500mL, and recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 19 second group components Fr.G17.1, Fr.G17.2, Fr.G17.3 ... Fr.G17.15 ... Fr.G17.19;

[0035] (4) Preparation of the third group of components

[0036] The second group of components Fr.G17.15 (38.9 g) was subjected to ODS column chromatography gradient elution, the volume gradient ratio of the eluent was MeOH:H2O (methanol: water) = 40:60, the amount was 1L, 50:50, the amount was 2L, 60:40, the amount was 3L, 70:30, the amount was 3L, 80:20, the amount was 3L, 90:10, the amount was 2L, 100:0, the amount was 1L, and the eluent was 50 0mL were combined together, the solvent was recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 12 third group components Fr.G17.15.1, Fr.G17.15.2, Fr.G17.15.3...Fr.G17.15.9, Fr.G17.15.9...Fr.G17.15.12;

[0037] (5) Preparation of the fourth group of components

[0038] The third group component Fr.G17.15.10 was eluted by ODS column chromatography, and the volume gradient ratio of the eluent was MeOH:H2O (methanol:water) = 40:60, used in 2L, 50:50, used in 2L, 60:40, used in 5L, 70:30, used in 5L, 80:20, used in 4L, 90:10, used in 2L, 100:0, used in 1L, and the eluents were combined in every 500mL, and the solvent was recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 13 fourth group components Fr.G17.15.10.1, Fr.G17.15.10.2, Fr.G17.15.10.3 ... Fr.G17.15.10.6 ... G17.15.10.13;

[0039] (6) Preparation of isopentenyl flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine O

[0040] The fourth group component Fr.G17.15.10.6 was purified and separated by semi-preparative HPLC using a 10ID×250mm RP C18 liquid chromatography column, with a mobile phase of acetonitrile:water=60:40 and a flow rate of 2mL / min, to obtain 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…G17.15.10.6.7;

[0041] The component Fr.G17.15.10.6.2 was purified and separated again by HPLC, with the mobile phase being acetonitrile:water=35:65 and the flow rate being 2 mL / min, to obtain 6 components Fr.G17.15.10.6.2.1…Fr.G17.15.10.6.2.4…G17.15.10.6.2.6;

[0042] The component Fr.G17.15.10.6.2.4 was purified and separated again by HPLC, with the mobile phase being acetonitrile:water=55:45 and the flow rate being 2 mL / min, to obtain 5 components Fr.G17.15.10.6.2.4.1…Fr.G17.15.10.6.2.4.4, Fr.G17.15.10.6.2.4.5,

[0043] Among them, the retention time t R The component Fr.G17.15.10.6.2.4.5 at 63.6 min was dried to obtain compound (2"S,2"'S)-epimesatine O (11.2 mg);

[0044] The component Fr.G17.15.10.6.2.4.4 was purified by HPLC again, the mobile phase was acetonitrile: water = 60:40, the flow rate was 2mL / min, and the retention time t was collected. R The fraction at 19.3 min was dried to give (2"R,2"'R)-epimesatine O (9.0 mg).

[0045] Application of the prenylated flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine O in the preparation of drugs for treating breast cancer.

[0046] According to the method given in the above embodiment, any amount of the target compound can be prepared as needed. The given embodiment is only used to illustrate the specific implementation mode of the present invention, rather than to limit the protection scope of the present invention. The technical core protected by the present invention is the isopentenyl flavonoid compound.

[0047] The present invention is to extract isopentenyl flavonoid compounds from Epimedium sagittatum, which have anti-breast cancer activity and are used in the preparation of drugs for treating breast cancer. The test results show that very good beneficial technical effects have been achieved. The relevant test data are as follows:

[0048] 1. Instruments and Materials

[0049] 1.1 Instrument:

[0050] High-resolution mass spectrometer Bruker maxis HD (Bruker, Germany)

[0051] UV spectrometer Evolution 300 instrument (Thermo, MA, USA)

[0052] Infrared spectrometer Nicolet IS10 (Thermo Scientific, USA)

[0053] Nuclear magnetic resonance spectrometer Bruker AVANCEⅢ500 (Bruker, Germany)

[0054] High performance liquid chromatograph Shimadzu LC-40, equipped with DAD detector, RPC18 chromatographic column (10ID×250mm, Cosmosil 5C18-MS-IIPacked column, Nacalai Tesque, China)

[0055] N-1100 Rotary Evaporator and N-1111 Chilled Water Circulation Device (Shanghai Ailang Instrument Co., Ltd.)

[0056] BT25S 1 / 100,000 precision analytical balance (Sartorius)

[0057] Real-time label-free cell culture system (Agilent Biotechnology)

[0058] Carbon dioxide 3111 incubator (Thermo)

[0059] Centrifuge-5804R high-speed centrifuge (Eppendorf)

[0060] Multiskan MK3 microplate reader (Thermo Fisher)

[0061] Clean bench (Sujing Group)

[0062] HVA-85 Autoclave (Hirayama)

[0063] 1.2 Materials:

[0064] Reverse phase silica gel (ODS, 50 μm, YMC Group, Kyoto, Japan)

[0065] Normal phase silica gel (100–200 and 200–300 mesh, TOSOH Corp., Tokyo, Japan)

[0066] Chromatographic grade methanol and acetonitrile (Tianjin Siyou Fine Chemicals Co., Ltd.)

[0067] Analytical grade methanol, dichloromethane, ethyl acetate, petroleum ether (Beijing Chemical Plant and Tianjin Third Chemical Reagent Plant)

[0068] 16-well plate (Agilent Bio)

[0069] Culture dishes, 96-well culture plates, cell cryopreservation tubes (Corning)

[0070] Fetal bovine serum (Hangzhou Sijiqing Company), DMEM culture medium (Gibco Invitrogen)

[0071] Thiazolyl blue MTT (Beijing Solebow Technology Co., Ltd.)

[0072] Ampicillin, Streptomycin (Sigma)

[0073] Dimethyl sulfoxide (DMSO) (Shanghai MacLean Biotechnology Co., Ltd.)

[0074] Ham's F12K medium (Pnosai)

[0075] Docetaxel (Shanghai Yuanye Biotechnology Co., Ltd.)

[0076] Human normal breast cell MCF-10A, human breast cancer cell MCF-7 (Shanghai Cell Bank, Academy of Sciences)

[0077] Plant materials: The Epimedium sagittatum used in this study was collected from Zhumadian, Henan Province in September 2020. The specimen is deposited in Henan University of Traditional Chinese Medicine with the deposit number 20200960

[0078] Test drugs: the prenylated flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine O of the present invention.

[0079] 2. Structural Identification

[0080] By comparing the H NMR spectra, optical rotation values ​​and CD effects, compounds (2”S, 2”'S)-epimesatine O(1a) and (2”R, 2”'R)-epimesatine O(1b) are enantiomers. UV(MeOH)λ max (logε):202(4.50),244(4.15),268(4.08),341(4.26)nm; IR(ν max ):3368,2975,1655,1614,1475,1439,1362,1166,1044,841cm -1 The structural formula is:

[0081]

[0082] (2”S,2”'S)-epimesatine O(1a), yellow amorphous powder; [α] 20 D -11(c 0.4,MeOH); HRESIMS m / z461.1570[M+Na] + (calcd.for C 25 H 26 O7Na,461.1571).

[0083] (2”R,2”'R)-epimesatine O(1b), yellow amorphous powder; [α] 20 D +16(c 0.5,MeOH); HRESIMS m / z461.1568[M+Na] + (calcd.for C 25 H 26 O7Na,461.1571). 1 H and 13 C NMR data are shown in Table 6.

[0084] Table 1 Compound 1a / 1b 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0085]

[0086] 3. Activity Test

[0087] 3.1 Cell culture

[0088] Take the frozen MCF-10A cells and MCF-7 cells and thaw them in a 37℃ water bath until ice and water coexist. Immediately centrifuge (1000rpm, 5min). After discarding the supernatant, transfer the cells to a culture dish containing DMEM medium with 10% FBS (penicillin and streptomycin are both 100kU / L), and culture them in a 37℃ constant temperature incubator containing 5% CO2. When the cells grow to 80% to 90% of the dish, subculture them. Replace the fresh culture medium every 24h.

[0089] 3.2 MTT assay to detect the effects of monomeric compounds on the viability of two cell lines

[0090] The cells were cultured in a 37°C, 5% CO2 incubator until the logarithmic growth phase and the cell density was 2×10 4 / mL, 200μL per well was inoculated in a 96-well plate, and divided into a normal control group (CON) and each drug group (10μM) after 24h. After culturing for 24h, 20μL of MTT solution (5mg / mL) was added to each well and cultured for another 4h. Carefully aspirate the culture medium, add 150μL of DMSO to each well, and shake for 10min to completely dissolve the blue-purple crystals in the well. The absorbance value (OD) of each well was measured by an enzyme-labeled instrument at 490nm to calculate the cell viability. The experiment was repeated three times in parallel.

[0091] 3.3 Immunofluorescence detection of Sphk1 levels in MCF-7 cells

[0092] MCF-7 cells were cultured at 2×10 4 The cells were inoculated at a density of 10 μM / mL in the wells. After 24 hours, they were divided into normal (CON) and each drug group (10 μM) and cultured for another 24 hours. They were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.25% Triton X-100 for 10 minutes. Then 1% BSA was added for blocking for 30 minutes, and then the primary antibody Sphk1 was added for overnight incubation at 4°C. The cells were washed with PBST three times, counterstained with DAPI for 4 minutes, and washed with PBS once, and then the cells were photographed using the OperettaCLS high-content imaging analysis system.

[0093] 3.4 Statistical analysis

[0094] The experimental data are expressed as mean ± standard deviation. SPSS 26.0 was used for statistical analysis. One-way ANOVA was used for comparison among groups. * P<0.05 indicated a significant difference. ** P<0.01 indicated a very significant difference.

[0095] 3.5 Activity evaluation

[0096] The experiment used the MTT method to evaluate the effects of the compounds on MCF-7 human breast cancer cells and MCF-10A human normal breast cells. The experimental results are shown in Table 2. After the action of 10μM compound 1a / 1b, the cell viability of MCF-7 was significantly reduced compared with the blank group, and the results were statistically significant (P<0.01), and compound 1a / 1b had no significant effect on the cell viability of MCF-10A. Therefore, compound 1a / 1b can significantly inhibit the cell viability of MCF-7 human breast cancer cells at a concentration of 10μM, and has no effect on MCF-10A normal breast cells, indicating that these two compounds may be the pharmacological substances in Epimedium sagittatum that exert anti-breast cancer activity.

[0097] Table 2 Effects of compounds at 10 μM concentration on the viability of MCF-7 and MCF-10A cells

[0098]

[0099] ** indicates P < 0.01; # Docetaxel is a positive drug

[0100] In addition, the level of Sphk1 in MCF-7 cells treated with compound 1a / 1b was detected by cell immunofluorescence, and the results are shown in Table 3. According to the experimental results, the fluorescence intensity of Sphk1 in MCF-7 cells treated with compound monomers was significantly different from that in the blank group (P<0.01), that is, compound 1a / 1b can significantly inhibit the expression level of Sphk1 in MCF-7 cells. This indicates that these compounds may exert anti-breast cancer activity by inhibiting the expression level of Sphk1 in MCF-7 cells.

[0101] Table 3 Effects of compounds on Sphk1 levels in MCF-7 cells ( n=3)

[0102]

[0103] ** P < 0.01

[0104] In summary, this study discovered a pair of new isopentenyl flavonoid components from the dichloromethane extraction part of Epimedium sagittatum. Activity evaluation found that both compounds could significantly reduce the cell viability of human breast cancer cells MCF-7, and were non-toxic to human normal breast cells MCF-10A, suggesting that these compounds may be the pharmacological substances of Epimedium sagittatum to exert anti-breast cancer activity. In addition, both compounds had a significant inhibitory effect on Sphk1 in MCF-7 cells, suggesting that the anti-breast cancer effect of these two compounds may be related to the inhibition of Sphk1 expression levels in cells. These compounds are expected to become lead compounds for anti-breast cancer, provide a technical basis for the development of anti-breast cancer drugs, expand the medicinal value and economic value of Epimedium sagittatum, and are innovations in the treatment of breast cancer drugs with huge economic and social benefits.

Claims

1. Prenylated flavonoids extracted from Epimedium sagittatum S ,2''' S )-epimesatine O (1a) and (2'' R ,2''' R )-epimesatine O (1b), the molecular structure is: 。 2. Prenylated flavonoids extracted from Epimedium sagittatum S ,2''' S )-epimesatine O and (2'' R ,2''' R )-epimesatine O, the preparation method thereof is: (1) Preparation of polar segments The dried aerial part of Epimedium sagittatum (80 kg) was crushed and extracted three times with 120 L of 70% ethanol at 45 °C for 30 min each time. The solvent was recovered under reduced pressure to obtain an extract (6.5 kg). The extract was suspended with 3 times the volume of distilled water to obtain a suspension. The suspension was extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature for 4 times, 15 L each time, and extracted for 2 h to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract, respectively. The dichloromethane extract ( 2.1 kg), using 100-200 mesh normal phase silica gel column chromatography for gradient elution separation, the gradient volume ratio is petroleum ether-ethyl acetate = 50:1, the amount used is 12 L; 40:1, dosage 20 L; 35:1, dosage 20 L; 20:1, dosage 40 L; 10:1, dosage 40 L; 5:1, dosage 40 L; 1:1, dosage 20 L; 0:1, dosage 4 L, similar parts were combined according to the TLC color development results to obtain 8 polar segments Fr. A...Fr.G, Fr.H; (2) Preparation of the first group of components The polar segment Fr. G (100.0 g) was chromatographed on a 100-200 mesh normal phase silica gel column with petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was a volume ratio of petroleum ether / ethyl acetate = 50:1, 1 L, 35:1, 5 L, 20:1, 5 L, 10:1, 5 L, 5:1, 5 L, 1:1, 5 L, 0:1, 5 L. The eluates were combined in 1 L, and the solvent was recovered under reduced pressure to obtain multiple components. All the components were then spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 18 first group components Fr. F1...Fr. F17, Fr. F18; (3) Preparation of the second group of components The first group component Fr. G17 (65.0 g) was gradient eluted through 200-300 mesh normal phase silica gel, and the volume gradient ratio of the eluent was petroleum ether / ethyl acetate = 35:1, 1 L, 20:1, 4 L, 10:1, 4 L, 5:1, 4 L, 1:1, 2 L, 0:1, 1 L, and the eluents were combined together every 500 mL, and recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 19 second group components Fr. G17.1, Fr. G17.2, Fr. G17.3...Fr. G17.15...Fr. G17.19; (4) Preparation of the third group of ingredients The second group component Fr. G17.15 (38.9 g) was subjected to gradient elution by ODS column chromatography, and the volume gradient ratio of the eluent was MeOH:H2O (methanol:water) = 40:60, used for 1 L, 50:50, used for 2 L, 60:40, used for 3 L, 70:30, used for 3 L, 80:20, used for 3 L, 90:10, used for 2 L, 100:0, used for 1 L, and the eluents were combined in 500 mL, and the solvent was recovered under reduced pressure to obtain multiple components, and then the multiple components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 12 third group components Fr. G17.15.1, Fr. G17.15.2, Fr. G17.15.3...Fr. G17.15.9, Fr. G17.15.9…Fr.G17.15.12; (5) Preparation of the fourth group of ingredients The third group component Fr. G17.15.10 was eluted by ODS column chromatography, and the volume gradient ratio of the eluent was MeOH:H2O (methanol:water) = 40:60, 2 L, 50:50, 2 L, 60:40, 5 L, 70:30, 5 L, 80:20, 4 L, 90:10, 2 L, 100:0, 1 L. The eluents were combined in 500 mL, and the solvent was recovered under reduced pressure to obtain multiple components. The multiple components were then spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 13 fourth group components Fr. G17.15.10.1, Fr. G17.15.10.2, Fr. G17.15.10.3...Fr.G17.15.10.6...G17.15.10.13; (6) Preparation of isopentenyl flavonoid compounds (2'' S ,2''' S )-epimesatine O and (2'' R ,2''' R )-epimesatine O The fourth component Fr.G17.15.10.6 was purified and separated by semi-preparative HPLC using a 10ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water = 60:40 and a flow rate of 2 mL / min to obtain 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…G17.15.10.6.7; The component Fr.G17.15.10.6.2 was purified and separated again by HPLC, with the mobile phase being acetonitrile:water=35:65 and the flow rate being 2 mL / min, to obtain 6 components Fr.G17.15.10.6.2.1…Fr. G17.15.10.6.2.4…G17.15.10.6.2.6; The component Fr.G17.15.10.6.2.4 was purified and separated again by HPLC, with the mobile phase of acetonitrile:water = 55:45 and the flow rate of 2 mL / min, to obtain 5 components Fr. G17.15.10.6.2.4.1…Fr.G17.15.10.6.2.4.4, Fr.G17.15.10.6.2.4.5, Among them, retention time t R The component Fr.G17.15.10.6.2.4.5 of 63.6 min was dried to obtain compound (2'' S ,2''' S )-epimesatine O (11.2 mg); The component Fr. G17.15.10.6.2.4.4 was purified by HPLC again, the mobile phase was acetonitrile: water = 60:40, the flow rate was 2 mL / min, and the retention time was collected. t R The fraction at 19.3 min was dried to give (2'' R ,2''' R )-epimesatine O (9.0 mg).

3. Use of the isopentenyl flavonoid compound according to claim 1 in the preparation of a drug for treating breast cancer.

4. The use of the isopentenyl flavonoid compound according to claim 3 in the preparation of a drug for treating breast cancer, characterized in that: The breast cancer cell line is human breast cancer cell MCF-7.

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