Method for extracting flavonoid compound from epimedium sagittatum and application of flavonoid compound

Abstract

A method for extracting flavonoid compounds from epimedium sagittatum comprises the following steps: crushing epimedium sagittatum, carrying out reflux extraction, carrying out reduced pressure recovery to obtain an extract, suspending with distilled water, and extracting with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol at room temperature to respectively obtain each part; the dichloromethane part is separated, eluted and combined, and components Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G and Fr.H are obtained; eluting the component Fr.G, and combining to obtain components Fr.G1,..., Fr.G17 and Fr.G18; eluting the component Fr.G17, and combining the eluted component Fr.G17 to obtain components Fr.G 17.1,..., Fr.G 17.15,..., Fr.G 17.19; eluting a component Fr.G 17.15, recovering under reduced pressure, and merging to obtain components Fr.G 17.15. 1,..., Fr.G 17.15. 9, Fr.G 17.15. 10,..., Fr.G 17.15. 12; the preparation method comprises the following steps: carrying out semi-preparative HPLC (High Performance Liquid Chromatography) purification on the component Fr.G 17.15. 10 to obtain a compound Epimesatine P; the preparation method comprises the following steps: carrying out semi-preparative HPLC (High Performance Liquid Chromatography) purification on a component Fr.G 17.15. 9, and collecting components with different retention times, so as to respectively obtain the compounds Epimesatine Q, Epimesatine R and Epimesatine S. The preparation method comprises the following steps of: preparing the compound Fr.G 17.15. 9; the flavonoid compound can be effectively extracted from epimedium sagittatum, can be effectively used for treating breast cancer, can be applied to preparation of anti-breast cancer drugs, and has remarkable economic and social benefits.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a method for extracting flavonoid compounds from Epimedium sagittatum and application thereof. Background Art

[0002] Epimedium sagittatum is a plant of the genus Epimedium in the Berberidaceae family, also known as three-branched nine-leaf grass. It has the functions of replenishing essence and strengthening body, removing rheumatism, treating impotence, joint rheumatic pain, and leucorrhea. The Chinese Pharmacopoeia lists the dried leaves of Epimedium, Epimedium sagittatum, Epimedium pubescens, or Epimedium koreanum as the source of Epimedium. According to literature reports, the chemical components in Epimedium sagittatum include flavonoids and their glycosides, alkaloids, phenolic acids, tocopherols, etc. Among them, flavonoid compounds are the main active ingredient type in Epimedium sagittatum.

[0003] Breast cancer is a common and frequently occurring disease that seriously threatens people's life safety and physical health, especially breast cancer caused by human breast cancer cell MCF-7. Although there are therapeutic drugs available, the effects are limited. So far, there has been no public report on whether effective active ingredients (compounds) can be extracted from Epimedium sagittatum and used to prepare drugs against breast tumors (cancer). 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 a method for extracting flavonoids from Epimedium sagittatum and its application, which can effectively solve the problem of preparing flavonoids from Epimedium sagittatum and using them to prepare anti-breast tumor drugs.

[0005] The technical solution provided by the present invention is: a preparation method for extracting flavonoid compounds from Epimedium sagittatum, comprising the following steps:

[0006] (1) Extraction of polar components from Epimedium sagittatum:

[0007] The dried above-ground part of Epimedium sagittatum was crushed, ethanol was added, and refluxed for extraction for 3 times, the extracts were combined, and the solvent was recovered under reduced pressure to obtain an extract, which was suspended with distilled water. The suspension was then extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature in sequence to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction and an n-butanol fraction, respectively;

[0008] The dichloromethane fraction was separated by normal phase silica gel column chromatography, and gradient elution was performed using a volume ratio of petroleum ether: ethyl acetate. Similar fractions were combined according to the TLC color development results to obtain 8 polar fractions Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H.

[0009] (2) Extraction of target flavonoid compounds:

[0010] Component Fr.G was subjected to normal phase silica gel column chromatography, and gradient elution was performed using a volume ratio of petroleum ether: ethyl acetate; the eluate was decompressed and the solvent was recovered to obtain multiple components, and then all the components were spotted on a thin layer chromatography (TLC) plate, and components with similar absorption on the thin layer plate were combined to obtain a total of 18 components, namely, components Fr.G1...Fr.G17, Fr.G18;

[0011] Then, component Fr.G17 was eluted by gradient elution on normal phase silica gel, and the eluate was decompressed to recover the solvent to obtain multiple components. 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 a total of 19 components, namely, components Fr.G17.1, Fr.G17.2, Fr.G17.3...Fr.G17.15...Fr.G17.18, Fr.G1719;

[0012] Component Fr.G17.15 was subjected to gradient elution by ODS column chromatography, and the eluate was decompressed to recover the solvent 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 a total of 12 components, namely, components Fr.G17.15.1...Fr.G17.15.9, Fr.G17.15.10, Fr.G17.15.11, G17.1512;

[0013] The component Fr.G17.15.10 was purified by semi-preparative HPLC to obtain the compound Epimesatine P;

[0014] Component Fr.G17.15.9 was purified by semi-preparative HPLC, and components with different retention times were collected to obtain compounds Epimesatine Q, Epimesatine R, and Epimesatine S, respectively.

[0015] The flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S prepared by the method are extracted from Epimedium sagittatum and used in preparing drugs for treating breast cancer.

[0016] The invention has abundant raw materials and an easy-to-operate preparation method, and can effectively extract flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S from the Epimedium sagittatum. The obtained product has good quality and good use effect, can be effectively used for treating breast cancer, and can be applied in the preparation of anti-breast cancer drugs, thereby expanding the medicinal value and commercial value of the Epimedium sagittatum, and having significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 FIG. 4 is a structural diagram of the flavonoid compound Epimesatine P of the present invention.

[0018] Figure 2 This is the structural formula of the flavonoid compound Epimesatine Q of the present invention. (Abstract Figure)

[0019] Figure 3 FIG. 4 is a structural diagram of the flavonoid compound Epimesatine R of the present invention.

[0020] Figure 4 FIG. 4 is a structural diagram of the flavonoid compound Epimesatine S of the present invention.

[0021] Figure 5 The figures are comparative diagrams of the effects of the flavonoid compounds of the present invention on cells, wherein A is a diagram showing the effect of the compound at a concentration of 10 μM on the cell viability of human breast cancer cells MCF-7; B is a diagram showing the effect of the compound at a concentration of 10 μM on the cell viability of human normal breast cells MCF-10A; and C is a diagram showing the effect of the monomer compound on Sphk1 in MCF-7 cells.

[0022] Figure 6 Epimesatine P of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6).

[0023] Figure 7 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6).

[0024] Figure 8 Epimesatine Q of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6).

[0025] Fig. 9 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6).

[0026] Fig.10 Epimesatine R of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6).

[0027] Fig.11DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6).

[0028] Fig.12 Epimesatine S of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6).

[0029] Fig.13 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6). DETAILED DESCRIPTION

[0030] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0031] Embodiment 1:

[0032] The present invention discloses a method for preparing flavonoid compounds extracted from Epimedium sagittatum L., which comprises:

[0033] (1) Extraction of polar components from Epimedium sagittatum:

[0034] The dried aerial part of Epimedium sagittatum is crushed, and 1.5 times the weight volume of ethanol with a volume concentration of 70% is added, where the weight volume refers to the solid in kg and the liquid in L, and refluxed and extracted at 45°C for 3 times, each time for 30 minutes, and the solvent is recovered under reduced pressure to obtain an extract, and the extract is suspended with 3 times the volume of distilled water to obtain a suspension, and then extracted with 0.5-0.6 times the volume of petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature for 4 times, each time for 2 hours, to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction and an n-butanol fraction, respectively;

[0035] The dichloromethane fraction was separated by 100-200 mesh normal phase silica gel column chromatography, and gradient elution was performed by using petroleum ether: ethyl acetate in a volume ratio of 50:1, with a dosage of 12 L; 40:1, with a dosage of 20 L; 35:1, with a dosage of 20 L; 20:1, with a dosage of 40 L; 10:1, with a dosage of 40 L; 5:1, with a dosage of 40 L; 1:1, with a dosage of 20 L; 0:1, with a dosage of 4 L; similar fractions were combined according to the TLC color development results to obtain 8 polar segment components Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H;

[0036] (2) Extraction of target flavonoid compounds:

[0037] 100.0 g of component Fr.G was chromatographed on a 100-200 mesh normal phase silica gel column, and a volume ratio of petroleum ether: ethyl acetate was used for gradient elution; the gradient ratio was petroleum ether: ethyl acetate = 50:1, the amount was 1 L; 35:1, the amount was 5 L; 20:1, the amount was 5 L; 10:1, the amount was 5 L; 5:1, the amount was 5 L; 1:1, the amount was 2 L; 0:1, the amount was 1 L; the eluate was decompressed to recover the solvent 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 components in total, namely, components Fr.G1...Fr.G17, Fr.G18;

[0038] Then, 65.0 g of component Fr.G17 was taken and gradient eluted with 200-300 mesh normal phase silica gel, and the gradient ratio was petroleum ether:ethyl acetate = 35:1, the amount was 1 L; 20:1, the amount was 4 L; 10:1, the amount was 4 L; 5:1, the amount was 4 L; 1:1, the amount was 2 L; 0:1, the amount was 1 L; the eluate was decompressed to recover the solvent 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 19 components in total, namely, components Fr.G17.1, Fr.G17.2, Fr.G17.3...Fr.G17.15...Fr.G17.18, Fr.G17.19;

[0039] Component Fr.G17.15 38.9 g was subjected to gradient elution by ODS column chromatography, the gradient ratio being methanol:water=40:60, the amount being 1 L; 50:50, the amount being 2 L; 60:40, the amount being 3 L; 70:30, the amount being 3 L; 80:20, the amount being 3 L; 90:10, the amount being 2 L; 100:0, the amount being 1 L; the eluate was decompressed to recover the solvent to obtain a plurality of 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 a total of 12 components, namely, components Fr.G17.15.1 ... Fr.G17.15.9, Fr.G17.15.10, Fr.G17.15.11, G17.15.12;

[0040] The component Fr.G17.15.10 was purified by semi-preparative HPLC, the mobile phase was acetonitrile: water (CH3CN:H2O) = 65:35 by volume, the elution rate was 2.5 mL / min, and the retention time t was collected. R The fraction at 99.0 min gave 3.3 mg of compound Epimesatine P;

[0041] The component Fr.G17.15.9 was purified by semi-preparative HPLC. The mobile phase was acetonitrile: water in a volume ratio of 70:30. The elution rate was 2 mL / min. The retention time t R The fraction at 39.2 min gave 5.2 mg of Epimesatine Q; the mobile phase was acetonitrile: water in a volume ratio of 45:55, the elution rate was 2 mL / min, and the retention time t R The fraction at 55.8 min gave 8.2 mg of Epimesatine R; the mobile phase was acetonitrile: water in a volume ratio of 80:20, the elution rate was 2 mL / min, and the retention time t R The fraction at 39.2 min gave 4.0 mg of compound Epimesatine S.

[0042] Application of the flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S prepared by the above method in the preparation of drugs for treating breast cancer.

[0043] The above-mentioned embodiments are only used to illustrate the specific implementation methods of the present invention, and are not used to limit the protection scope of the present invention. Any technical solution that is essentially the same as the technology of the present application by means of equivalent replacement or change belongs to the protection scope of the present invention. According to the above-mentioned preparation method, any amount of new flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R, and Epimesatine S can be extracted and prepared as needed.

[0044] The invention has abundant raw materials and an easy-to-operate preparation method. The flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S can be effectively extracted from Epimedium sagittatum, and the cell viability of human breast cancer cells MCF-7 can be significantly reduced. The invention has no toxicity to human normal breast cells MCF-10A, and is effectively used for treating breast cancer, and is applied in the preparation of anti-breast cancer drugs. The invention has obtained very good beneficial technical effects through experiments. The relevant experimental materials are as follows:

[0045] 1. Instruments and Materials

[0046] 1.1 Instrument: High-resolution mass spectrometer Bruker maxis HD (Bruker, Germany);

[0047] UV spectrometer Evolution 300 instrument (Thermo, MA, USA);

[0048] Infrared spectrometer Nicolet IS10 (Thermo Scientific, USA);

[0049] Nuclear magnetic resonance spectrometer Bruker AVANCEⅢ500 (Bruker, Germany);

[0050] High performance liquid chromatograph Shimadzu LC-40 equipped with DAD detector and RPC18 column (10 ID × 250 mm, Cosmosil 5C18-MS-IIPacked column, nacalai tesque, China);

[0051] N-1100 rotary evaporator and N-1111 chilled water circulation device (Shanghai Ailang Instrument Co., Ltd.);

[0052] BT25S 1 / 100,000 precision analytical balance (Sartorius);

[0053] Cell real-time label-free system (Agilent Biotechnology);

[0054] CO2 3111 incubator (Thermo);

[0055] Centrifuge-5804R high-speed centrifuge (Eppendorf);

[0056] Multiskan MK3 microplate reader (Thermo Fisher);

[0057] Clean bench (Sujing Group);

[0058] HVA-85 high pressure sterilizer (Hirayama).

[0059] 1.2 Material: reverse phase silica gel (ODS, 50 μm, YMC Group, Kyoto, Japan);

[0060] Normal phase silica gel (100-200 and 200-300 mesh, TOSOH Corp., Tokyo, Japan);

[0061] Chromatographic methanol and chromatographic acetonitrile (Tianjin Siyou Fine Chemicals Co., Ltd.);

[0062] Analytical grade methanol, dichloromethane, ethyl acetate, petroleum ether (Beijing Chemical Plant and Tianjin Third Chemical Reagent Plant);

[0063] 16-well plate (Agilent Biotechnology);

[0064] Culture dishes, 96-well culture plates, and cell cryopreservation tubes (Corning);

[0065] Fetal bovine serum (Hangzhou Sijiqing Company), DMEM medium (Gibco Invitrogen);

[0066] Thiazolyl blue MTT (Beijing Solaibao Technology Co., Ltd.);

[0067] Ampicillin, streptomycin (Sigma);

[0068] Dimethyl sulfoxide (DMSO) (Shanghai MacLean Biotechnology Co., Ltd.);

[0069] Ham's F12K medium (Pnosai Corporation);

[0070] Docetaxel (Shanghai Yuanye Biotechnology Co., Ltd.);

[0071] Human normal breast cells MCF-10A, human breast cancer cells MCF-7 (Shanghai Cell Bank, Academy of Sciences);

[0072] Plant materials: The Epimedium sagittatum used in this study was collected in Zhumadian, Henan Province in September 2020 and identified by Professor Chen Suiqing of Henan University of Traditional Chinese Medicine. The specimen is preserved in Henan University of Traditional Chinese Medicine with the preservation number 20200960.

[0073] 2. Structural Identification

[0074] 2.1. Compound Epimesatine P (1)

[0075] Yellow amorphous powder, [α] 20 D -2.3(c 0.13,MeOH); UV(MeOH)λ max (logε):205(4.54),268(4.15),343(4.27)nm; IR(ν max ):3392,2980,1653,1607,1474,1439,1365,1166,1043,843cm -1 ; HRESIMS m / z 477.1509[M+Na] + (Calculated value C 25 H 26 O8Na,477.1520). 1 H NMR spectrum (500MHz) Figure 6 , 13 C (125 MHz) NMR data are shown in Table 1.

[0076] Table 1 Compound Epimesatine P 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0077]

[0078]

[0079] The structural formula of the compound Epimesatine P is:

[0080]

[0081] 2.2 Compound Epimesatine Q(2)

[0082] Yellow amorphous powder. [α] 20 D +5(c 0.1,MeOH);UV(MeOH)λ max (logε):202(4.54),268(4.13),342(4.30)nm; IR(ν max ):3414,2978,1652,1606,1474,1438,1362,1165,1046,841cm -1 ; HRESIMS m / z 549.2083[M+Na] + (Calculated value C 29 H 34 O9Na,549.2095). 1 H NMR spectrum (500MHz) Figure 7 , 13 C (125 MHz) NMR data are shown in Table 2.

[0083] Table 2 Compound Epimesatine Q 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0084]

[0085] a Signals partially overlapped.

[0086] The structural formula of the compound Epimesatine Q is:

[0087]

[0088] 2.3. Compound Epimesatine R(3)

[0089] Yellow amorphous powder. [α] 20 D -5.6(c 0.2,MeOH);UV(MeOH)λ max (logε):208(4.53),268(4.13),341(4.29)nm; IR(ν max ):3432,2979,1653,1612,1474,1439,1362,1165,1063,841cm -1 ; m / z 565.2036 [M+Na] + (Calculated value C 29 H 34 O 10 Na,565.2044). That 1 H NMR spectrum (500MHz) Figure 8 , 13 C (125 MHz) NMR data are shown in Table 3.

[0090] Table 3 Compound Epimesatine R 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0091]

[0092] a Signals partially overlapped.

[0093] The structural formula of the compound Epimesatine R is:

[0094]

[0095] 2.4. Compound Epimesatine S(4)

[0096] Yellow amorphous powder. UV(MeOH)λ max (logε):201(4.45),272(4.15),327(4.08)nm; IR(ν max ):3360,2968,2930,1658,1615,1431,1368,1259,1033,846cm-1 ; HRESIMS m / z421.2006[M+H] + (Calculated value C 26 H 29 O5,421.2010). 1 H NMR spectrum (500MHz) Fig. 9 , 13 C (125 MHz) NMR data are shown in Table 4.

[0097] Table 4 Compound Epimesatine S 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0098]

[0099] The structural formula of the compound Epimesatine S is:

[0100]

[0101] 3. Activity Test

[0102] 3.1 Cell culture

[0103] 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.

[0104] 3.2 MTT assay to detect the effects of monomeric compounds on the viability of two cell lines

[0105] 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.

[0106] 3.3 Real-time label-free dynamic cell analysis (RTCA) to detect IC50 values ​​of compounds

[0107] Baseline measurement was performed using culture medium. MCF-7 cells in the logarithmic growth phase were cultured at a rate of 2×10 4 The cells were inoculated at a density of 100 μM / mL in an E-plate. After 24 hours, the drug was administered in a gradient of 1, 5, 10, 20, 50, and 100 μM and the growth curve was continued. After the end, the Sigmoidal dose response (Vanable slope) algorithm was used to select the target time for the IC value of the analyzed drug. 50 The value is measured.

[0108] 3.4 Immunofluorescence detection of Sphk1 levels in MCF-7 cells

[0109] 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.

[0110] 3.5 Statistical analysis

[0111] 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.

[0112] Depend on Figure 5 Given, Figure 5 A and 5B show that compounds Epimesatines P, Epimesatines Q, Epimesatines R, and Epimesatines S (hereinafter referred to as compounds 1, 2, 3, and 4) can significantly inhibit the cell viability of human breast cancer cells MCF-7 at a concentration of 10 μM, and have no effect on human normal breast cells MCF-10A. IC values ​​of compounds Epimesatines P, Q, R, and S on MCF-7 cells 50 The values ​​are shown in Table 5, where the IC values ​​of Epimesatine Q for MCF-7 are50 The value is lower than that of positive drug docetaxel, showing good anti-breast cancer prospects. In addition, Epimesatines P, Q, R, and S can inhibit the expression level of Sphk1 in MCF-7 cells. Figure 5 C.

[0113] The results of cytotoxic activity evaluation showed (Table 5) that after the action of compound 1-4 (10 μM), the cell viability of MCF-7 was 0.497, 0.780, 0.743 and 0.600 compared with the blank group, and the results were statistically significant (P<0.01), that is, compound 1-4 at a concentration of 10 μM can significantly inhibit the cell viability of MCF-7 human breast cancer cells, and the results showed that after the action of compound 1-4 on MCF-10A human normal breast cells, there was basically no effect on the cell viability of MCF-10A, indicating that compound 1-4 had no obvious toxic effect on MCF-10A human normal breast cells.

[0114] Table 5 Effects of compounds at 10 μM concentration on the viability of MCF-7 and MCF-10A cells

[0115]

[0116] ** indicates P < 0.01; # Docetaxel is a positive drug

[0117] IC values ​​of compounds 1-4 on MCF-7 cells 50 The values ​​are shown in Table 6, where the IC values ​​of compound 2 for MCF-7 are 50 The value (1.27 μM) was lower than that of the positive drug docetaxel (2.13 μM), showing good anti-breast cancer prospects.

[0118] Table 6 IC of compounds 1-4 on MCF-7 cells 50 value

[0119]

[0120] # Docetaxel is a positive drug

[0121] In addition, the level of Sphk1 in MCF-7 cells treated with compounds 1-4 was detected by cell immunofluorescence, and the results are shown in Table 7. The fluorescence intensity of Sphk1 in MCF-7 cells treated with compounds 1, 3 and 4 was 0.545, 0.621 and 0.418 respectively compared with the blank group, and the results were significantly different (P<0.01), that is, compounds 1, 3 and 4 can significantly inhibit the expression level of Sphk1 in MCF-7 cells. The fluorescence intensity of Sphk1 in MCF-7 cells treated with compound 2 was 0.850 compared with the blank group, which did not show a significant difference. It is speculated that compound 2 may be involved in the partial regulation of Sphk1 in MCF-7 cells, or regulate MCF-7 cells through other pathways.

[0122] Table 7 Effects of Compounds 1-4 on Sphk1 Levels in MCF-7 Cells ( n=3)

[0123]

[0124] ** P < 0.01

[0125] This study discovered four new flavonoid components from the dichloromethane extract of Epimedium sagittatum. The activity evaluation found that all four compounds had a significant inhibitory effect on the cell viability of MCF-7 cells, and had no effect on human normal breast cells MCF-10A. In particular, Epimesatine Q had an IC 50 The value is lower than that of the positive drug docetaxel, showing good anti-breast cancer prospects. In addition, compounds 1, 3 and 4 can all significantly inhibit the expression level of Sphk1 in MCF-7 cells, suggesting that these compounds may exert anti-tumor effects by inhibiting Sphk1 in MCF-7 cells. Compound 2 did not show significant differences in Sphk1 in MCF-7 cells, and may be involved in the partial regulation of Sphk1 in MCF-7 cells, or regulate MCF-7 cells through other pathways. The compounds of the present invention are expected to become leading compounds for anti-breast cancer, providing technical support for the development of anti-tumor drugs, and the raw materials are abundant, the preparation method is easy to operate, and the flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R, and Epimesatine S can be effectively extracted from Epimedium sagittatum, which can be effectively used to treat breast cancer, realize the application in the preparation of anti-breast cancer drugs, and expand the medicinal value and commercial value of Epimedium sagittatum, with significant economic and social benefits.

Claims

1. A method for extracting flavonoids from Epimedium sagittatum, wherein the molecular structure is: The structural formula of Epimesatine P is: ； The structural formula of Epimesatine Q is: ； The structural formula of Epimesatine R is: ； The structural formula of Epimesatine S is: 。 2. The method for preparing flavonoid compounds extracted from Epimedium sagittatum according to claim 1 is: (1) Extraction of polar components from Epimedium sagittatum: The dried aerial part of Epimedium sagittatum was crushed, and 1.5 times the weight volume of ethanol with a volume concentration of 70% was added, where the weight volume refers to the solid in kg and the liquid in L, and refluxed and extracted 3 times at 45°C, each time for 30 min, and the solvent was recovered under reduced pressure to obtain an extract, and the extract was suspended with 3 times the volume of distilled water to obtain a suspension, and then extracted with 0.5-0.6 times the volume of petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature for 4 times, each time for 2 h, to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction and an n-butanol fraction, respectively; The dichloromethane fraction was separated by 100-200 mesh normal phase silica gel column chromatography, and gradient elution was performed by petroleum ether: ethyl acetate in a volume ratio of 50:1, with a dosage of 12 L; 40:1, with a dosage of 20 L; 35:1, with a dosage of 20 L; 20:1, with a dosage of 40 L; 10:1, with a dosage of 40 L; 5:1, with a dosage of 40 L; 1:1, with a dosage of 20 L; 0:1, with a dosage of 4 L; similar parts were combined according to the results of thin layer chromatography (TLC) to obtain 8 polar segment components Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F, Fr. G, and Fr. H; (2) Extraction of target flavonoid compounds: Take 100.0 g of component Fr. G and use it for normal phase silica gel column chromatography with 100-200 mesh, and use petroleum ether: ethyl acetate for gradient elution; the gradient ratio is petroleum ether: ethyl acetate = 50:1, the amount is 1 L; 35:1, the amount is 5 L; 20:1, the amount is 5 L; 10:1, the amount is 5 L; 5:1, the amount is 5 L; 1:1, the amount is 2 L; 0:1, the amount is 1 L; the eluent is decompressed to recover the solvent to obtain multiple components, and then all the components are spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate are combined to obtain 18 components in total, namely, components Fr. G1...Fr. G17, Fr. G18; Then, 65.0 g of component Fr. G17 was taken and gradient eluted with 200-300 mesh normal phase silica gel, and the gradient ratio was petroleum ether: ethyl acetate = 35:1, the amount was 1 L; 20:1, the amount was 4 L; 10:1, the amount was 4 L; 5:1, the amount was 4 L; 1:1, the amount was 2 L; 0:1, the amount was 1 L; the eluate was decompressed to recover the solvent 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 a total of 19 components, namely, components Fr. G17.1, Fr. G17.2, Fr. G17.3...Fr. G17.15...Fr. G17.18, Fr. G17.19; Component Fr. G17.15 38.9 g was subjected to gradient elution by ODS column chromatography, the gradient ratio was 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; the eluate was decompressed to recover the solvent 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 12 components in total, namely, components Fr. G17.15.1...Fr. G17.15.9, Fr. G17.15.10, Fr. G17.15.11, G17.15.12; The component Fr. G17.15.10 was purified by semi-preparative HPLC with a mobile phase of acetonitrile: water (CH3CN:H2O) in a volume ratio of 65:35 and an elution rate of 2.5 mL / min. The retention time was collected. t R The fraction at 99.0 min gave the compound Epimesatine P3.3 mg; The component Fr. G17.15.9 was purified by semi-preparative HPLC with a mobile phase of acetonitrile: water in a volume ratio of 70:30 and an elution rate of 2 mL / min. The retention time was collected. t R The fraction at 39.2 min gave 5.2 mg of Epimesatine Q; the mobile phase was acetonitrile: water in a volume ratio of 45:55, the elution rate was 2 mL / min, and the retention time was collected. t R The fraction at 55.8 min gave 8.2 mg of Epimesatine R; the mobile phase was acetonitrile: water in a volume ratio of 80:20, the elution rate was 2 mL / min, and the retention time was collected. t R The fraction at 39.2 min gave 4.0 mg of compound Epimesatine S.

3. Use of the flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S extracted from Epimedium sagittatum as claimed in claim 1 in the preparation of drugs for treating breast cancer.

4. The use of flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S extracted from Epimedium sagittatum in the preparation of a drug for treating breast cancer according to claim 3, characterized in that: The breast cancer cell line is human breast cancer cell MCF-7.

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