A method for extracting flavonoids from Epimedium sagittatum and its application

Abstract

A method for extracting flavonoids from Epimedium sagittatum is as follows: crushing the Epimedium sagittatum, performing reflux extraction, and recovering under reduced pressure to obtain an extract; suspending the extract in distilled water; and then extracting the extract with petroleum ether, dichloromethane, ethyl acetate, and n-butanol at room temperature to obtain various fractions; separating the dichloromethane fraction, eluting, and combining to obtain fractions Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H; eluting and combining fraction Fr.G to obtain fractions Fr.G1, Fr.G17, and Fr.G17. , Fr.G18; Fraction Fr.G17 was eluted and combined to obtain fractions Fr.G17.1, Fr.G17.15, and Fr.G17.19; Fraction Fr.G17.15 was eluted, recovered under reduced pressure, and combined to obtain fractions Fr.G17.15.1, Fr.G17.15.9, Fr.G17.15.10, and Fr.G17.15.12; Fraction Fr.G17.15.10 was purified by semi-preparative HPLC to obtain compound Epimesatine P; Fraction Fr.G17.15.9 was purified by semi-preparative HPLC, and fractions with different retention times were collected to obtain compounds Epimesatine Q, Epimesatine R, and Epimesatine S, respectively. This method can effectively extract flavonoids from Epimedium sagittatum, can be effectively used to treat breast cancer, and can be used in the preparation of anti-breast cancer drugs, with significant economic and social benefits.

Description

Technical Field

[0001] The present 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, also known as three-branched nine-leaf grass, is a plant of the genus Epimedium in the Berberidaceae family. It is known for its properties of tonifying essence and strengthening body, dispelling rheumatism, and treating impotence, rheumatic joint pain, and leucorrhea. The Chinese Pharmacopoeia identifies the dried leaves of Epimedium, Epimedium sagittatum, Epimedium pubescens, or Epimedium koreanatum as the source of Epimedium. Literature reports indicate that the chemical components of Epimedium sagittatum include flavonoids and their glycosides, alkaloids, phenolic acids, and tocopherols. Flavonoids are the primary active ingredients in Epimedium sagittatum.

[0003] Breast cancer is a common and frequently occurring disease that seriously threatens people's life and health, especially breast cancer caused by human breast cancer cells MCF-7. Although there are currently available therapeutic drugs, their effects are limited. Therefore, whether it is possible to extract effective active ingredients (compounds) from Epimedium sagittatum and use them to prepare drugs against breast tumors (cancer) has not been publicly reported so far. Summary of the Invention

[0004] In view of the above situation, in order to overcome the shortcomings of the existing technology, 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 aerial part of Epimedium sagittatum was crushed, ethanol was added, and reflux extraction was performed three times. The extracts were combined and the solvent was recovered under reduced pressure to obtain an extract. The extract was suspended in distilled water. The suspension was then extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence at room temperature 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 using a gradient elution of petroleum ether:ethyl acetate by volume. Similar fractions were combined based on the results of thin layer chromatography (TLC) to obtain eight polar fractions: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, and Fr.H.

[0009] (2) Extraction of target flavonoids:

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

[0011] Then, component Fr.G17 was eluted with a normal phase silica gel gradient, and the eluate was decompressed and the solvent was recovered 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 a total of 19 components, namely components Fr.G17.1, Fr.G17.2, Fr.G17.3...Fr.G17.15...Fr.G17.18, and 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 fractions. All the fractions were then spotted on a thin layer chromatography (TLC) plate, and fractions with similar absorption on the TLC plate were combined to obtain a total of 12 fractions, namely, components Fr.G17.15.1...Fr.G17.15.9, Fr.G17.15.10, Fr.G17.15.11, and G17.1512;

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

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

[0015] The invention relates to an application of flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S extracted from Epimedium sagittatum by the method in preparing a drug 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 sagittal epimedium. The obtained product has good quality and good use effect, can be effectively used for treating breast cancer, and can be used in the preparation of anti-breast cancer drugs, thereby expanding the medicinal value and commercial value of the sagittal epimedium and having significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 FIG1 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 FIG1 is a structural diagram of the flavonoid compound Epimesatine R of the present invention.

[0020] Figure 4 FIG1 is a structural diagram of the flavonoid compound Epimesatine S of the present invention.

[0021] Figure 5 Figures 1 and 2 are comparative graphs showing the effects of the flavonoid compounds of the present invention on cells, wherein A is a graph showing the effect of the compound at a concentration of 10 μM on the viability of human breast cancer cells MCF-7; B is a graph showing the effect of the compound at a concentration of 10 μM on the viability of human normal breast cells MCF-10A; and C is a graph showing the effect of the monomeric 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] Figure 9 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6).

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

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

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

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

[0030] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations.

[0031] Example 1:

[0032] The present invention provides a preparation method for extracting flavonoid compounds from Epimedium sagittatum L. comprising:

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

[0034] 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. The mixture was refluxed and extracted at 45°C for 3 times, each time for 30 minutes. The solvent was recovered under reduced pressure to obtain an extract. The extract was suspended with 3 times the volume of distilled water to obtain a suspension. The suspension was 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 using a volume ratio of petroleum ether: ethyl acetate, with the gradient ratio being petroleum ether: ethyl acetate = 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 thin layer chromatography (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 flavonoids:

[0037] 100.0 g of component Fr.G was chromatographed on a 100-200 mesh normal phase silica gel column using a gradient elution of petroleum ether:ethyl acetate by volume; the gradient ratio was 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 (2 L); and 0:1 (1 L). The eluate was decompressed and the solvent was recovered to obtain multiple fractions. All fractions were then spotted by thin layer chromatography (TLC). Fractions with similar absorption on the TLC plate were combined to obtain a total of 18 fractions, namely, fractions Fr.G1...Fr.G17, and Fr.G18.

[0038] Then, 65.0 g of component Fr.G17 was eluted with a 200-300 mesh normal phase silica gel gradient elution at a gradient ratio of petroleum ether:ethyl acetate = 35:1 (1 L); 20:1 (4 L); 10:1 (4 L); 5:1 (4 L); 1:1 (2 L); and 0:1 (1 L). The eluate was decompressed and the solvent was recovered to obtain multiple components. All components were then spotted by thin layer chromatography (TLC), and components with similar absorption on the TLC 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, and Fr.G1719.

[0039] Component Fr.G17.15 (38.9 g) was subjected to gradient elution with an ODS column chromatography at a ratio of methanol:water = 40:60 (1 L); 50:50 (2 L); 60:40 (3 L); 70:30 (3 L); 80:20 (3 L); 90:10 (2 L); and 100:0 (1 L). The eluate was decompressed and the solvent was recovered to obtain multiple components. All components were then spotted by thin layer chromatography (TLC). Components with similar absorption on the TLC 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, and G17.1512.

[0040] 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 at an elution rate of 2.5 mL / min. The retention time t R The fraction at 99.0 min gave 3.3 mg of compound Epimesatine P;

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

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

[0043] The above examples are intended only to illustrate specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any technical solution essentially equivalent to the technology of the present application, produced by equivalent substitution or modification, shall fall within the scope of protection of the present invention. The above preparation method can be used to extract and produce any amount of the new flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R, and Epimesatine S as needed.

[0044] The present 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. The flavonoid compounds can significantly reduce the cell viability of human breast cancer MCF-7 cells and are non-toxic to human normal breast cells MCF-10A. The flavonoid compounds can be effectively used to treat breast cancer and can be used in the preparation of anti-breast cancer drugs. Experiments have shown very good beneficial technical effects. 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 AVANCE III 500 (Bruker, Germany);

[0050] High performance liquid chromatography 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 incubator model 3111 (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 Materials: Reversed-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, and 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 freezing tubes (Corning);

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

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

[0067] ampicillin and streptomycin (Sigma);

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

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

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

[0071] Human normal breast cells MCF-10A and human breast cancer cells MCF-7 (Shanghai Cell Bank, Chinese 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 deposited at Henan University of Traditional Chinese Medicine under the accession 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 (500 MHz) 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 (125 MHz) 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 (500 MHz) 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 (125 MHz) 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 (500 MHz) 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 (125 MHz) 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 (500 MHz) Figure 9 , 13 C (125 MHz) NMR data are shown in Table 4.

[0097] Table 4 Compound Epimesatine S 1 H(500MHz) and 13 C (125 MHz) 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] Thaw frozen MCF-10A and MCF-7 cells in a 37°C waterbath until ice-water coexists. Immediately centrifuge (1000 rpm, 5 min). Discard the supernatant and transfer the cells to culture dishes containing DMEM (100 kU / L penicillin and streptomycin) supplemented with 10% FBS. Culture in a 37°C incubator with 5% CO2. Subculture cells when they reach 80%-90% of the dish. Replace with fresh medium every 24 hours.

[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 Cells were plated in a 96-well plate at a concentration of 200 μL per well. After 24 hours, cells were divided into a normal control group (CON) and various treatment groups (10 μM). After 24 hours of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. The culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The cells were shaken for 10 minutes to completely dissolve the blue-purple crystals. The absorbance (OD) of each well was measured at 490 nm using a microplate reader, and cell viability was calculated. The experiment was repeated three times.

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

[0107] Baseline determination 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 drug being analyzed. 50 The value is measured.

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

[0109] MCF-7 cells were cultured at a rate of 2 × 10 4 Cells were seeded at a density of 10 μM per mL in the wells. After 24 hours, cells were divided into normal (CON) and various treatment groups (10 μM) and cultured for another 24 hours. The cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.25% Triton X-100 for 10 minutes. Cells were then blocked with 1% BSA for 30 minutes, followed by the addition of the primary antibody Sphk1 and incubation at 4°C overnight. Cells were washed three times with PBST, counterstained with DAPI for 4 minutes, and washed once with PBS. Afterwards, cells were imaged using the OperettaCLS High-Content Imaging 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 indicates a significant difference. ** P<0.01 indicates 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 treatment with 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, respectively. 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. The results also showed that after the treatment with compound 1-4 and 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 against 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 against 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. 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. 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] ** Indicates P < 0.01

[0125] This study discovered four new flavonoids from the dichloromethane extract of Epimedium sagittatum. Activity evaluation showed 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 values ​​were lower than those of the positive drug docetaxel, demonstrating promising anti-breast cancer potential. Furthermore, compounds 1, 3, and 4 all significantly inhibited Sphk1 expression in MCF-7 cells, suggesting that these compounds may exert their anti-tumor effects by inhibiting Sphk1 in MCF-7 cells. Compound 2 showed no significant difference in Sphk1 expression in MCF-7 cells, suggesting that it may be involved in the partial regulation of Sphk1 in MCF-7 cells or modulate MCF-7 cells through other pathways. The compounds of this invention are expected to become lead compounds for breast cancer treatment, providing technical support for the development of anti-tumor drugs. Furthermore, the raw materials are abundant, the preparation method is easy to use, and the flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R, and Epimesatine S can be effectively extracted from Epimedium sagittatum. These compounds can be effectively used to treat breast cancer and can be used in the preparation of anti-breast cancer drugs. This expands the medicinal and commercial value of Epimedium sagittatum and has significant economic and social benefits.

Claims

1. A flavonoid compound extracted from Epimedium sagittatum, the molecular structure of which is: The structural formula of Epimesatine P is: ； The structural formula of Epimesatine Q is: ； The structural formula of Epimesatine R is: 。 2. The method for extracting flavonoids from Epimedium sagittatum according to claim 1: (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. The mixture was refluxed and extracted at 45°C for 3 times, each time for 30 minutes. The solvent was recovered under reduced pressure to obtain an extract. The extract was suspended with 3 times the volume of distilled water to obtain a suspension. The suspension was 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. The dichloromethane fraction was separated by 100-200 mesh normal phase silica gel column chromatography, using a gradient elution of petroleum ether:ethyl acetate in a volume ratio of 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); and 0:1 (4 L). Similar fractions were combined based on the TLC results to obtain eight polar fractions: Fr. A, Fr. B, Fr. C, Fr. D, Fr. E, Fr. F, Fr. G, and Fr. H. (2) Extraction of target flavonoids: 100.0 g of component Fr. G was chromatographed on a 100–200 mesh normal phase silica gel column using a gradient elution of petroleum ether:ethyl acetate in a volume ratio of 50:1 (1 L); 35:1 (5 L); 20:1 (5 L); 10:1 (5 L); 5:1 (5 L); 1:1 (2 L); and 0:1 (1 L). The eluate was decompressed and the solvent was recovered to obtain multiple fractions. All fractions were then subjected to thin layer chromatography (TLC) spotting. Fractions with similar absorption on the TLC plate were combined to obtain a total of 18 fractions, namely, fractions Fr. G1…Fr. G17, and Fr. G18. Then, 65.0 g of component Fr. G17 was eluted with a 200–300 mesh normal phase silica gel gradient elution at a gradient ratio of petroleum ether:ethyl acetate = 35:1 (1 L); 20:1 (4 L); 10:1 (4 L); 5:1 (4 L); 1:1 (2 L); and 0:1 (1 L). The eluate was decompressed and the solvent was recovered to obtain multiple components. All components were then subjected to thin layer chromatography (TLC) spotting, and 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, and Fr. G17.

19. Component Fr. G17.15 (38.9 g) was subjected to gradient elution on an ODS column chromatography with a gradient ratio of methanol:water = 40:60 (1 L); 50:50 (2 L); 60:40 (3 L); 70:30 (3 L); 80:20 (3 L); 90:10 (2 L); and 100:0 (1 L). The eluate was decompressed and the solvent was recovered to obtain multiple components. All components were then subjected to thin layer chromatography (TLC) spotting, and components with similar absorption on the TLC 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, and G17.1512. The component Fr. G17.15.10 was purified by semi-preparative HPLC with a mobile phase of acetonitrile: water in a volume ratio of 65:35 at an elution rate of 2.5 mL / min. The retention time was collected. t R The fraction at 99.0 min gave compound Epimesatine P 3.3 mg; The fraction Fr. G17.15.9 was purified by semi-preparative HPLC with a mobile phase of acetonitrile and water in a volume ratio of 70:30 at 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 compound 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 the compound Epimesatine S. The structural formula of Epimesatine S is: 。 3. Use of the flavonoid compounds Epimesatine P, Epimesatine Q, Epimesatine R and Epimesatine S extracted from Epimedium sagittatum by the method of claim 2 in the preparation of a drug for treating breast cancer.

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