Isopentenyl flavone enantiomer compound extracted from epimedium sagittatum as well as preparation method and application of isopentenyl flavone enantiomer compound

Abstract

The preparation method of the isopentenyl flavone enantiomer compound comprises the following steps: crushing epimedium sagittatum to prepare paste, suspending with water, respectively extracting with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol to obtain extractum of each part, carrying out gradient elution separation by using silica gel column chromatography, and concentrating to obtain the isopentenyl flavone enantiomer compound, namely the isopentenyl flavone enantiomer compound. 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 four compounds are obtained respectively, 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 enantiomer 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, especially breast cancer caused by human breast cancer cells MCF-7. Although there are currently available therapeutic drugs, 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. Therefore, one of the technical problems to be solved in this application is to discover new active ingredients (compounds) from Epimedium sagittatum, the second is how to extract isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum, and the third is to realize the application in the preparation of drugs for the treatment of breast tumors (cancer), but 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 a method for extracting isopentenyl flavonoid enantiomers from Epimedium sagittatum and a preparation method and application thereof, which can effectively solve the problem of extracting isopentenyl flavonoid enantiomers from Epimedium sagittatum and realize the application of the compounds in the preparation of drugs for treating breast tumors (cancer).

[0005] The technical solution provided by the present invention is to extract isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum, wherein the compounds (R)-epimesatine J(1a) and (S)-epimesatine J(1b) are enantiomers, and their molecular structural formulas are respectively:

[0006]

[0007] Compounds (2"R, 2"'S)-epimesatine K (2a) and (2"S, 2"'R)-epimesatine K (2b) are enantiomers, and their molecular structures are:

[0008]

[0009] The preparation method thereof is:

[0010] 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;

[0011] 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;

[0012] 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;

[0013] 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;

[0014] The third group fraction Fr.G17.15.9 was purified by semi-preparative HPLC using a liquid chromatography column with acetonitrile:water as the mobile phase at a flow rate of 1 mL / min. The samples with retention time t R The fraction at 14.0 min was dried to obtain compound (R)-epimesatine J. The fraction with retention time t R The fraction at 13.7 min was dried to obtain compound (S)-epimesatine J.

[0015] 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 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;

[0016] The component Fr.G17.15.10.6 was purified by semi-preparative HPLC using a liquid chromatography column with acetonitrile: water as the mobile phase at a flow rate of 2 mL / min, and 7 sub-components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…Fr.G17.15.10.6.7 were separated. The sub-component Fr.G17.15.10.6.1 was purified again by HPLC using a liquid chromatography column with acetonitrile: water as the mobile phase at a flow rate of 2 mL / min, and the samples with retention time t R The fraction at 42.2 min was dried to obtain the compound (2"R, 2"'S)-epimesatine K, and the retention time was collected. R The fraction at 48.9 min was dried to obtain compound (2"S,2"'R)-epimesatine K.

[0017] The above four isopentenyl flavonoid enantiomer compounds (R)-epimesatine J, (S)-epimesatine J, (2"R,2"'S)-epimesatine K and (2"S,2"'R)-epimesatine K 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.

[0018] The invention has abundant raw materials and an easy-to-operate preparation method, and can effectively extract isopentenyl flavonoid enantiomer compounds (R)-epimesatine J, (S)-epimesatine J, (2"R,2"'S)-epimesatine K and (2"S,2"'R)-epimesatine K 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 invention can be effectively used for preparing drugs for treating breast cancer, expands the medicinal value and economic value of the epimedium sagittatum, is an innovation in drugs for treating breast cancer, and has huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a molecular structural formula diagram of the isopentenyl flavonoid enantiomer compound of the present invention;

[0020] Figure 2 The compound Epimesatine J of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6);

[0021] Figure 3 is the DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6);

[0022] Figure 4 The compound Epimesatine K of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6);

[0023] Figure 5 is the DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6). DETAILED DESCRIPTION

[0024] The specific implementation modes of the present invention are described in detail below in combination with specific situations.

[0025] The technical solution provided by the present invention is to extract isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum, wherein the compounds (R)-epimesatine J(1a) and (S)-epimesatine J(1b) are enantiomers, and their molecular structural formulas are respectively:

[0026]

[0027] Compounds (2"R, 2"'S)-epimesatine K (2a) and (2"S, 2"'R)-epimesatine K (2b) are enantiomers, and their molecular structures are:

[0028]

[0029] The preparation method thereof is:

[0030] (1) Preparation of polar segments

[0031] 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;

[0032] (2) Preparation of the first group of components

[0033] 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;

[0034] (3) Preparation of the second group of components

[0035] 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;

[0036] (4) Preparation of the third group of components

[0037] 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;

[0038] (5) Preparation of isopentenyl flavonoid enantiomers (R)-epimesatine J and (S)-epimesatine J

[0039] The third group fraction Fr.G17.15.9 was purified by semi-preparative HPLC using a 4.6 ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water (CH3CN:H2O) = 60:40 by volume at a flow rate of 1 mL / min. The samples with retention time t R The fraction at 14.0 min was dried to obtain compound (R)-epimesatine J (5.6 mg). R The fraction at 13.7 min was dried to obtain compound (S)-epimesatine J (9.9 mg).

[0040] (6) Preparation of isopentenyl flavonoid enantiomer compounds (2"R,2"'S)-epimesatine K and (2"S,2"'R)-epimesatine K

[0041] 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 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;

[0042] The component Fr.G17.15.10.6 was purified by semi-preparative HPLC using a 10ID×250mm RP C18 liquid chromatography column, the mobile phase was acetonitrile: water (CH3CN: H2O) in a volume ratio of 60:40, and the flow rate was 2mL / min, and 7 sub-components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2...Fr.G17.15.10.6.7 were separated, and the sub-component Fr.G17.15.10.6.1 was purified again by HPLC using a 10ID×250mm RP C18 liquid chromatography column, the mobile phase was acetonitrile: water (CH3CN: H2O) in a volume ratio of 60:40, and the flow rate was 2mL / min, and the retention time t R The fraction at 42.2 min was dried to obtain compound (2"R, 2"'S)-epimesatine K (2.2 mg). R The fraction at 48.9 min was dried to obtain compound (2"S,2"'R)-epimesatine K (1.9 mg).

[0043] The above four isopentenyl flavonoid enantiomer compounds (R)-epimesatine J and (S)-epimesatine J, (2"R,2"'S)-epimesatine K and (2"S,2"'R)-epimesatine K have significant inhibitory effects 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.

[0044] 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 enantiomer compound.

[0045] The present invention is to extract isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum, which has anti-breast cancer activity and is applied in the preparation of drugs for treating breast cancer. The test results show that the compounds have very good beneficial technical effects. The relevant test data are as follows:

[0046] 1. Instruments and Materials

[0047] 1.1 Instrument:

[0048] High-resolution mass spectrometer Bruker maxis HD (Bruker, Germany)

[0049] UV spectrometer Evolution 300 instrument (Thermo, MA, USA)

[0050] Infrared spectrometer Nicolet IS10 (Thermo Scientific, USA)

[0051] Nuclear magnetic resonance spectrometer Bruker AVANCEⅢ500 (Bruker, Germany)

[0052] High performance liquid chromatograph Shimadzu LC-40, equipped with DAD detector, RPC18 column (10 ID × 250 mm, Cosmosil 5C18-MS-IIPacked column, Nacalai Tesque, China)

[0053] N-1100 Rotary Evaporator and N-1111 Chilled Water Circulation Device (Shanghai Ailang Instrument Co., Ltd.)

[0054] BT25S 1 / 100,000 precision analytical balance (Sartorius)

[0055] Real-time label-free cell culture system (Agilent Biotechnology)

[0056] Carbon dioxide 3111 incubator (Thermo)

[0057] Centrifuge-5804R high-speed centrifuge (Eppendorf)

[0058] Multiskan MK3 microplate reader (Thermo Fisher)

[0059] Clean bench (Sujing Group)

[0060] HVA-85 Autoclave (Hirayama)

[0061] 1.2 Materials:

[0062] Reverse phase silica gel (ODS, 50 μm, YMC Group, Kyoto, Japan)

[0063] Normal phase silica gel (100–200 and 200–300 mesh, TOSOH Corp., Tokyo, Japan)

[0064] Chromatographic grade methanol and acetonitrile (Tianjin Siyou Fine Chemicals Co., Ltd.)

[0065] Analytical grade methanol, dichloromethane, ethyl acetate, petroleum ether (Beijing Chemical Plant and Tianjin Third Chemical Reagent Plant)

[0066] 16-well plate (Agilent Bio)

[0067] Culture dishes, 96-well culture plates, cell cryopreservation tubes (Corning)

[0068] Fetal bovine serum (Hangzhou Sijiqing Company), DMEM culture medium (Gibco Invitrogen)

[0069] Thiazolyl blue MTT (Beijing Solebow Technology Co., Ltd.)

[0070] Ampicillin, Streptomycin (Sigma)

[0071] Dimethyl sulfoxide (DMSO) (Shanghai MacLean Biotechnology Co., Ltd.)

[0072] Ham's F12K medium (Pnosai)

[0073] Docetaxel (Shanghai Yuanye Biotechnology Co., Ltd.)

[0074] Human normal breast cell MCF-10A, human breast cancer cell MCF-7 (Shanghai Cell Bank, Academy of Sciences)

[0075] 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 preservation number 20200960.

[0076] Test drugs: the isopentenyl flavonoid enantiomer compounds (R)-epimesatine J and (S)-epimesatine J, (2"R, 2"'S)-epimesatine K and (2"S, 2"'R)-epimesatine K of the present invention.

[0077] 2. Structural Identification

[0078] By comparing the H NMR spectra, optical rotation values ​​and CD effects, compounds (R)-epimesatine J(1a) and (S)-epimesatine J(1b) are enantiomers. UV(MeOH)λ max (logε):207(4.24),242(3.89),268(3.82),342(3.96)nm; IR(ν max ):3400,2976,1653,1616,1477,1439,1359,1166,1031,842cm -1 The structural formula is:

[0079]

[0080] (R)-epimesatine J(1a), yellow amorphous powder; [α] 20 D -5(c 0.5,MeOH); HRESIMS m / z463.1725[M+Na] + (calcd.for C 25 H 28 O7Na, 463.1727). (S)-epimesatine J(1b), yellow amorphous powder; [α] 20 D +6(c 0.5,MeOH); HRESIMS m / z463.1713[M+Na] + (calcd.for C 25 H 28 O7Na,463.1727). 1 H and 13 The CNMR data are shown in Table 1.

[0081] Table 1 Compound 1a / 1b 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0082]

[0083] By comparing the H NMR spectra, optical rotation values ​​and CD effects, compounds (2”R, 2”'S)-epimesatine K(2a) and (2”S, 2”'R)-epimesatine K(2b) are enantiomers. UV(MeOH)λ max(logε):208(4.32),240(3.98),268(3.93),341(4.01)nm; IR(ν max ):3444,2975,1699,1653,1436,1363,1260,1170,1052cm -1 The structural formula is:

[0084]

[0085] (2”R,2”'S)-epimesatine K(2a), yellow amorphous powder; [α] 20 D -3(c 0.2,MeOH); HRESIMS m / z479.1665[M+Na] + (calcd.for C 25 H 28 O8Na, 479.1676). (2”S,2”'R)-epimesatine K(2b), yellow amorphous powder; [α] 20 D +3(c 0.2,MeOH); HRESIMS m / z479.1669[M+Na] + (calcd.for C 25 H 28 O8Na,479.1676). 1 H and 13 C NMR data are shown in Table 2.

[0086] Table 2 Compound 2a / 2b 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0087]

[0088]

[0089] 3. Activity Test

[0090] 3.1 Cell culture

[0091] 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.

[0092] 3.2 MTT assay to detect the effects of monomeric compounds on the viability of two cell lines

[0093] 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.

[0094] 3.3 Immunofluorescence detection of Sphk1 levels in MCF-7 cells

[0095] 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.

[0096] 3.4 Statistical analysis

[0097] 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.

[0098] 3.5 Activity evaluation

[0099] In this experiment, the MTT method was first used to screen the cytotoxicity of the compounds. The experimental results are shown in Table 3. After the action of 10μM compounds 1a / 1b and 2a / 2b, the cell viability of MCF-7 human breast cancer cells was reduced compared with the blank group, and the results were statistically significant (P<0.01), that is, these compounds at a concentration of 10μM can significantly inhibit the cell viability of MCF-7 human breast cancer cells. Then, the MTT method was used to detect the effect of the compounds on MCF-10A human normal breast cells, and it was found that these compounds had no significant effect on the cell viability of MCF-10A (Table 3). This shows that these compounds may be the pharmacological substances in Epimedium sagittatum that exert anti-breast cancer activity.

[0100] Table 3 Effects of compounds at 10 μM concentration on the viability of MCF-7 and MCF-10A cells

[0101]

[0102] ** indicates P < 0.01; # Docetaxel is a positive drug

[0103] Subsequently, the effect of compounds 1a / 1b and 2a / 2b on the expression level of Sphk1 in MCF-7 cells was detected by cell immunofluorescence method, and the results are shown in Table 4. According to the experimental results, the fluorescence intensity of Sphk1 in MCF-7 cells treated with compound 1a / 1b and 2a / 2b monomers was significantly different from that in the blank group (P<0.01), that is, the four compounds 1a / 1b and 2a / 2b can significantly reduce 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.

[0104] Table 4 Effects of compounds on Sphk1 levels in MCF-7 cells ( , n=3)

[0105]

[0106] ** P < 0.01

[0107] In summary, the present invention discovered two pairs of new isopentenyl flavonoid enantiomers from the dichloromethane extraction part of Epimedium sagittatum. Activity evaluation found that the four compounds can significantly reduce the cell viability of human breast cancer cells MCF-7, and are 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, all four compounds have a significant inhibitory effect on Sphk1 in MCF-7 cells, suggesting that these compounds may exert anti-breast cancer effects by inhibiting the expression level of Sphk1. These compounds are expected to become leading compounds against breast cancer, provide a technical basis for the development of anti-breast cancer drugs, expand the medicinal value and economic value of Epimedium sagittatum, are innovations in drugs for the treatment of breast cancer, and have huge economic and social benefits.

Claims

1. Extraction of isopentenyl flavonoid enantiomers from Epimedium sagittatum R )-epimesatine J (1a) and ( S )-epimesatine J (1b) is an enantiomer, and its molecular structure is:

2. Compound (2'' R ,2''' S )-epimesatine K (2a) and (2'' S ,2''' R )-epimesatine K (2b) is an enantiomer with the molecular structure: 。 3. The preparation method of extracting isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum is: (1) Preparation of polar segments 80 kg of the dried aerial part of Epimedium sagittatum was crushed, and 120 L of 70% ethanol was used each time for reflux extraction at 45°C for three times, each time for 30 min, and the solvent was recovered under reduced pressure to obtain 6.5 kg of extract. 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 for 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. 2.1 kg of dichloromethane extract was separated by gradient elution using 100-200 mesh normal phase silica gel column chromatography, with a gradient volume ratio of petroleum ether-ethyl acetate = 50:1, and the amount was 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 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, and 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, and the eluates were combined in 1 L, and 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; (3) Preparation of the second group of components The first group component Fr. G17 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 for 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 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 isopentenyl flavonoid enantiomers (R)-epimesatine J and (S)-epimesatine J The third group fraction Fr. G17.15.9 was purified by semi-preparative HPLC using a 4.6 ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water = 60:40 by volume at a flow rate of 1 mL / min. The retention time was collected. t R The fraction at 14.0 min was dried to obtain compound (R)-epimesatine J. The retention time was collected. t R The fraction at 13.7 min was dried to obtain compound (S)-epimesatine J; (6) Preparation of isopentenyl flavonoid enantiomers (2'' R ,2''' S )-epimesatine K and (2'' S ,2''' R )-epimesatine K 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 =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 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; The component Fr. G17.15.10.6 was purified by semi-preparative HPLC using a 10ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water in a volume ratio of 60:40 at a flow rate of 2 mL / min, and 7 sub-components Fr. G17.15.10.6.1, Fr. G17.15.10.6.2…Fr. G17.15.10.6.7 were separated and the sub-component Fr. G17.15.10.6.1 was purified by HPLC again using a 10ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water (CH3CN:H2O) in a volume ratio of 60:40 at a flow rate of 2 mL / min. The retention time t R The fraction at 42.2 min was dried to obtain compound (2'' R ,2''' S )-epimesatine K, collection retention time t R The fraction at 48.9 min was dried to obtain compound (2'' S ,2''' R )-epimesatine K.

4. Use of the prenylated flavonoid enantiomer compound extracted from Epimedium sagittatum as claimed in claim 1 in the preparation of anti-breast cancer drugs.

5. The use of the isopentenyl flavonoid enantiomer compound extracted from Epimedium sagittatum in the preparation of anti-breast cancer drugs according to claim 3, characterized in that: The breast cancer is breast cancer cell MCF-7.

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