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

Abstract

The preparation method of the flavone enantiomer compound comprises the following steps: crushing epimedium sagittatum to prepare paste, suspending with water, extracting with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol respectively to obtain extractum of each part, carrying out gradient elution separation by using silica gel column chromatography, and concentrating to obtain the flavone enantiomer compound extracted from epimedium sagittatum. Combining the similar parts according to a thin layer chromatography (TLC) color development result to obtain a plurality of polar segments; carrying out chromatography and gradient elution on the target polar section to obtain a plurality of components, and then combining the similar components through thin-layer chromatography (TLC) dot plate absorption to obtain a plurality of sub-components; a target component is eluted and purified, fractions with different retention times are collected and dried, and 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 a flavonoid enantiomer compound extracted from Epimedium sagittatum and a preparation method and application thereof. Background Art

[0002] Traditional Chinese medicine has a history of thousands of years of civilization in treating diseases. However, with the development of science and technology, it has been found that the ingredients of traditional Chinese medicine are complex. The research on the ingredients of traditional Chinese medicine has attracted more and more attention from the medical community. Epimedium is the dried leaves of the Berberidaceae plants Epimedium, Epimedium sagittatum or Epimedium pubescens, 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 regulation, anti-atherosclerosis and antidepressant activity. According to literature reports, flavonoids are the main active ingredient type in Epimedium sagittatum, and isopentenyl 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 therapeutic drugs, their effects are limited. Since 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 has become a new trend in the development of traditional Chinese medicine. So, can we extract flavonoid enantiomer compounds that have a significant inhibitory effect on breast cancer cells MCF-7 from the active parts of Epimedium to prepare anti-breast cancer drugs? However, there has been no public report so far. Summary of the invention

[0004] In view of the above situation, in order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a flavonoid enantiomer compound extracted from Epimedium sagittatum and a preparation method and application thereof, which can effectively solve the problem of using the flavonoid enantiomer compound extracted from Epimedium sagittatum in the preparation of anti-breast cancer drugs.

[0005] The technical solution provided by the present invention is that the flavonoid enantiomeric compounds extracted from Epimedium sagittatum are enantiomeric compounds of (2"R, 3"R, 2"'R)-epimesatine M (1a) and (2"S, 3"S, 2"'S)-epimesatine M (1b), and the molecular structural formulas are respectively:

[0006]

[0007] The enantiomeric compounds of compounds (2"S, 3"S)-epimesatine N (2a) and (2"R, 3"R)-epimesatine N (2b) have molecular structural formulas:

[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.F was subjected to silica gel column chromatography, and gradient elution was performed with petroleum ether / ethyl acetate. 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 15 first group components Fr.F1...Fr.F15;

[0012] The first group component Fr.F15 was subjected to reverse phase silica gel 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 second group components Fr.F15.1...Fr.F15.9...Fr.F15.12;

[0013] Then, the fraction Fr.F15.9 was purified by passing it through a chiral chromatographic column, eluting with methanol:water=80:20 at a flow rate of 2 mL / min, and the samples with retention time t R =39.3min fraction, to obtain compound (2"S,3"S)-epimesatine N (1.3 mg), collected at retention time t R =33.3min fraction, to obtain compound (2"R,3"R)-epimesatine N (1.2 mg);

[0014] The polar segment Fr.G was chromatographed on a silica gel column, gradient eluted with petroleum ether / ethyl acetate, and recovered under reduced pressure to obtain multiple components. The multiple components were then spotted on a thin layer chromatography (TLC) plate, and components with similar absorption on the thin layer plate were combined to obtain 18 fourth group components Fr.F1...Fr.F17, Fr.F18;

[0015] The fourth group component Fr.G17 was subjected to normal phase 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 fifth group components, namely Fr.G17.1...Fr.G17.15...Fr.G17.19;

[0016] The fifth group component Fr.G17.15 was subjected to ODS column chromatography, gradient eluted with methanol / water, 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 12 sixth group components Fr.G17.15.1...Fr.G17.15.10...Fr.G17.15.12;

[0017] The sixth group component Fr.G17.15.10 was subjected to ODS column chromatography, gradient eluted with methanol / water, 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 13 components Fr.G17.15.10.1...Fr.G17.15.10.6...Fr.G17.15.10.13;

[0018] Then, component Fr.G17.15.10.6 was purified by semi-preparative HPLC using a liquid chromatography column with a mobile phase of acetonitrile / water in a volume ratio at a flow rate of 2 mL / min, and 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…Fr.G17.15.10.6.7 were separated;

[0019] The component Fr.G17.15.10.6.2 was purified again by HPLC using a liquid chromatography column with a mobile phase of acetonitrile / water in a volume ratio at a flow rate of 2 mL / min. The samples were collected at retention time t R The fraction at 63.1 min was dried to obtain compound (2"R, 3"R, 2"'R)-epimesatine M. The fraction with retention time t R The fraction at 59.1 min was dried to obtain compound (2"S,3"S,2"'S)-epimesatine M.

[0020] The isopentenyl flavonoid enantiomer compounds (2"R, 3"R, 2"'R)-epimesatine M and (2"S, 3"S, 2"'S)-epimesatine M, (2"S, 3"S)-epimesatine N and (2"R, 3"R)-epimesatine N are extracted from Epimedium sagittatum and have a significant inhibitory effect on the cell viability of human breast cancer cells MCF-7 and anti-breast cancer activity, thereby realizing application of the compounds in the preparation of drugs for treating breast cancer.

[0021] The invention has abundant raw materials and an easy-to-operate preparation method, and can effectively extract isopentenyl flavonoid enantiomer compounds (2"R, 3"R, 2"'R)-epimesatine M and (2"S, 3"S, 2"'S)-epimesatine M, (2"S, 3"S)-epimesatine N and (2"R, 3"R)-epimesatine N from the herb. The compounds have anti-breast cancer activity, have a significant inhibitory effect on the cell viability of human breast cancer cells MCF-7, and are non-toxic to human normal breast cells MCF-10A. The compounds can be effectively used for preparing drugs for treating breast cancer, and expand the medicinal value and economic value of the herb. The invention is an innovation in drugs for treating breast cancer and has huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The molecular structural formulas of the flavonoid enantiomer compounds (2"R, 3"R, 2"'R)-epimesatine M (1a) and (2"S, 3"S, 2"'S)-epimesatine M (1b), (2"S, 3"S)-epimesatine N (2a) and (2"R, 3"R)-epimesatine N (2b) of the present invention;

[0023] Figure 2 Epimesatine M of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6);

[0024] Figure 3 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6);

[0025] Figure 4 Epimesatine N of the present invention 1 H NMR spectrum (500 MHz, deuterated reagent: Acetone-d6);

[0026] Figure 5 DEPT and 13 C NMR spectrum (125 MHz, deuterated reagent: Acetone-d6). DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are described in detail below in combination with specific situations.

[0028] The technical solution provided by the present invention is that the flavonoid enantiomeric compounds extracted from Epimedium sagittatum are enantiomers, and the compounds (2"R, 3"R, 2"'R)-epimesatine M (1a) and (2"S, 3"S, 2"'S)-epimesatine M (1b) are enantiomers, and their molecular structural formulas are respectively:

[0029]

[0030] The molecular structures of the enantiomers of compounds (2"S, 3"S)-epimesatine N (2a) and (2"R, 3"R)-epimesatine N (2b) are:

[0031]

[0032] The preparation method thereof is:

[0033] (1) Preparation of polar segments

[0034] 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, and 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 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. According to the TLC color development results, similar parts were combined to obtain 8 polar segments Fr.A...Fr.F...Fr.G, Fr.H;

[0035] (2) Preparation of the first group of components

[0036] The polar segment Fr.F (13.7 g) was chromatographed on a 200-300 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 = 35:1, 1 L, 20:1, 3 L, 10:1, 3 L, 5:1, 3 L, 1:1, 2 L, 0:1, 1 L, the eluates were combined every 500 mL, 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 15 first group components Fr.F1...Fr.F15;

[0037] (3) Preparation of the second group of components

[0038] The first group component Fr.F15 (1.82 g) was further eluted by reverse phase silica gel column chromatography (ODS) with a volume gradient ratio of methanol:water = 40:60, 1 L, 50:50, 1 L, 60:40, 2 L, 70:30, 2 L, 80:20, 2 L, 90:10, 1 L, 100:0, 1 L. The eluates were combined every 200 mL and 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 12 second group components Fr.F15.1 ... Fr.F15.9 ... Fr.F15.12;

[0039] (3) Preparation of compounds (2"S, 3"S)-epimesatine N and (2"R, 3"R)-epimesatine N

[0040] The second component Fr.F15.9 was then purified by semi-preparative high performance liquid chromatography (HPLC) through a chiral chromatographic column, eluted with methanol: water = 80:20, at a flow rate of 2 mL / min, and the retention time t R =39.3min fraction, to obtain compound (2"S,3"S)-epimesatine N (1.3 mg), collected at retention time t R =33.3min fraction, to obtain compound (2"R,3"R)-epimesatine N (1.2 mg);

[0041] (4) Preparation of the fourth group of components

[0042] 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, 2 L, 0:1, 1 L, the eluate was combined every 1 L, and a plurality of components were recovered under reduced pressure, and then the plurality of components were spotted by thin layer chromatography (TLC), and the components with similar absorption on the thin layer plate were combined to obtain 18 fourth group components Fr.F1...Fr.F17, Fr.F18;

[0043] (5) Preparation of the fifth group of components

[0044] The fourth group component Fr.G17 (65.0 g) was again subjected to 200-300 mesh normal phase silica gel chromatography, 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 = 35:1, amount 1L, 20:1, amount 4L, 10:1, amount 4L, 5:1, amount 4L, 1:1, amount 2L, 0:1, amount 1L, the eluates 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 fifth group components, namely Fr.G17.1...Fr.G17.15...Fr.G17.19;

[0045] (6) Preparation of the sixth group of components

[0046] The fifth group component Fr.G17.15 (38.9 g) was eluted by ODS column chromatography, and the volume gradient ratio of the eluent was methanol: water = 40:60, used in an amount of 1L, 50:50, used in an amount of 2L, 60:40, used in an amount of 3L, 70:30, used in an amount of 3L, 80:20, used in an amount of 3L, 90:10, used in an amount of 2L, 100:0, used in an amount of 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 12 sixth group components Fr.G17.15.1...Fr.G17.15.10...Fr.G17.15.12;

[0047] (7) Preparation of compounds (2"R, 3"R, 2"'R)-epimesatine M and (2"S, 3"S, 2"'S)-epimesatine M

[0048] The sixth group component Fr.G17.15.10 was eluted by ODS column chromatography, and the volume gradient ratio of the eluent was 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 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 13 components Fr.G17.15.10.1...Fr.G17.15.10.6...Fr.G17.15.10.13;

[0049] Then, component Fr.G17.15.10.6 was purified by semi-preparative HPLC using a 10 ID × 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, to separate and obtain 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…Fr.G17.15.10.6.7;

[0050] The component Fr.G17.15.10.6.2 was purified again by HPLC using a 10 ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water (CH3CN: H2O) = 35:65 by volume at a flow rate of 2 mL / min. The retention time t R The fraction at 63.1 min was dried to obtain compound (2"R, 3"R, 2"'R)-epimesatine M (3.6 mg). The fraction with retention time t R The fraction at 59.1 min was dried to obtain compound (2"S,3"S,2"'S)-epimesatine M (2.1 mg).

[0051] The above four flavonoid enantiomeric compounds (2"R,3"R,2"'R)-epimesatine M and (2"S,3"S,2"'S)-epimesatine M, (2"S,3"S)-epimesatine N and (2"R,3"R)-epimesatine N 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 anti-breast cancer drugs.

[0052] 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 flavonoid enantiomer compound.

[0053] The present invention is a flavonoid enantiomer compound extracted from Epimedium sagittatum, which has anti-breast cancer activity and is applied in the preparation of a drug for treating breast cancer. The test results show that the compound has a very good beneficial technical effect. The relevant test data are as follows:

[0054] 1. Instruments and Materials

[0055] 1. Instruments and Materials

[0056] 1.1 Instrument: High-resolution mass spectrometer Bruker maxis HD (Bruker, Germany)

[0057] UV spectrometer Evolution 300 instrument (Thermo, MA, USA)

[0058] Infrared spectrometer Nicolet IS10 (Thermo Scientific, USA)

[0059] Nuclear magnetic resonance spectrometer Bruker AVANCEⅢ500 (Bruker, Germany)

[0060] 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)

[0061] N-1100 Rotary Evaporator and N-1111 Chilled Water Circulation Device (Shanghai Ailang Instrument Co., Ltd.)

[0062] BT25S 1 / 100,000 precision analytical balance (Sartorius)

[0063] Real-time label-free cell culture system (Agilent Biotechnology)

[0064] Carbon dioxide 3111 incubator (Thermo)

[0065] Centrifuge-5804R high-speed centrifuge (Eppendorf)

[0066] Multiskan MK3 microplate reader (Thermo Fisher)

[0067] Clean bench (Sujing Group)

[0068] HVA-85 Autoclave (Hirayama)

[0069] 1.2 Material: Reverse phase silica gel (ODS, 50 μm, YMC Group, Kyoto, Japan)

[0070] Normal phase silica gel (100–200 and 200–300 mesh, TOSOH Corp., Tokyo, Japan)

[0071] Chromatographic grade methanol and acetonitrile (Tianjin Siyou Fine Chemicals Co., Ltd.)

[0072] Analytical grade methanol, dichloromethane, ethyl acetate, petroleum ether (Beijing Chemical Plant and Tianjin Third Chemical Reagent Plant)

[0073] 16-well plate (Agilent Bio)

[0074] Culture dishes, 96-well culture plates, cell cryopreservation tubes (Corning)

[0075] Fetal bovine serum (Hangzhou Sijiqing Company), DMEM culture medium (Gibco Invitrogen)

[0076] Thiazolyl blue MTT (Beijing Solebow Technology Co., Ltd.)

[0077] Ampicillin, Streptomycin (Sigma)

[0078] Dimethyl sulfoxide (DMSO) (Shanghai MacLean Biotechnology Co., Ltd.)

[0079] Ham's F12K medium (Pnosai)

[0080] Docetaxel (Shanghai Yuanye Biotechnology Co., Ltd.)

[0081] Human normal breast cells MCF-10A, human breast cancer cells MCF-7 (Shanghai Cell Bank, Academy of Sciences)

[0082] 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.

[0083] Test drugs: flavonoid enantiomeric compounds of the present invention: (2"R, 3"R, 2"'R)-epimesatine M and (2"S, 3"S, 2"'S)-epimesatine M, (2"S, 3"S)-epimesatine N and (2"R, 3"R)-epimesatine N.

[0084] 2. Structural Identification

[0085] By comparing the H NMR spectra, optical rotation values ​​and CD effects, compounds (2”R, 3”R, 2”'R)-epimesatine M (1a) and (2”S, 3”S, 2”'S)-epimesatine M (1b) are enantiomers. UV (MeOH)λ max (logε):202(4.34),269(3.96),336(4.05)nm; IR(ν max ):3388,2932,2860,1654,1608,1365,1167,1048,843cm -1 The structural formula is:

[0086]

[0087] (2”R,3”R,2”'R)-epimesatine M(1a), yellow amorphous powder; [α] 20 D -3(c 0.3,MeOH); HRESIMS m / z491.1672[M+Na] + (calcd.for C 26 H 28 O8Na,491.1676).

[0088] (2”S,3”S,2”'S)-epimesatine M(1b), yellow amorphous powder; [α] 20 D +4(c 0.2,MeOH); HRESIMS m / z491.1675[M+Na] + (calcd.for C 26 H 28 O8Na,491.1676). 1 H and 13 C NMR data are shown in Table 1.

[0089] Table 1 Compound 1a / 1b 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0090]

[0091] By comparing the H NMR spectra, optical rotation values ​​and CD effects, compounds (2”S,3”S)-epimesatine N(2a) and (2”R,3”R)-epimesatine N(2b) are enantiomers. UV(MeOH)λmax (logε):203(4.45),268(4.19),335(4.18)nm; IR(ν max ):3415,1653,1608,1475,1437,1364,1168,1033,981cm -1 The structural formula is:

[0092]

[0093] (2”S,3”S)-epimesatine N(2a), yellow amorphous powder; [α] 20 D -7(c 0.1,MeOH); HRESIMS m / z475.1728[M+Na] + (calcd.for C 26 H 28 O7Na,475.1727).

[0094] (2”R,3”R)-epimesatine N(2b), yellow amorphous powder; [α] 20 D +10(c 0.1,MeOH); HRESIMS m / z475.1730[M+Na] + (calcd.for C 26 H 28 O7Na,475.1727). 1 H and 13 C NMR data are shown in Table 2.

[0095] Table 2 Compound 2a / 2b 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0096]

[0097] 3. Activity Test

[0098] 3.1 Cell culture

[0099] 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.

[0100] 3.2 MTT assay to detect the effects of monomeric compounds on the viability of two cell lines

[0101] 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.

[0102] 3.3 Real-time label-free dynamic cell analysis (RTCA) to detect IC50 values ​​of compounds

[0103] 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.

[0104] 3.4 Immunofluorescence detection of Sphk1 levels in MCF-7 cells

[0105] MCF-7 cells were cultured at 2×10 4The 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.

[0106] 3.5 Statistical analysis

[0107] 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.

[0108] 3.6 Activity evaluation

[0109] The experiment first used the MTT method to screen the cytotoxicity of the compounds. The experimental results are shown in Table 3. After the two pairs of enantiomeric flavonoids were treated with 10 μM, the cell viability of MCF-7 human breast cancer cells was reduced compared with the blank group. The results were statistically significant (P<0.01), that is, the two pairs of enantiomeric flavonoids at a concentration of 10 μM 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 the four compounds had no significant effect on the cell viability of MCF-10A (Table 3). Therefore, these four compounds may be the pharmacological substances in Epimedium sagittatum that exert anti-breast cancer activity.

[0110] Table 3 Effects of compounds at 10 μM concentration on the viability of MCF-7 and MCF-10A cells

[0111]

[0112] ** indicates P < 0.01; # Docetaxel is a positive drug

[0113] Then, real-time label-free dynamic cell analysis (RTCA) was used to detect the IC values ​​of the two pairs of enantiomeric flavonoids on MCF-7 cells. 50 The results are shown in Table 4. Among them, the IC values ​​of compounds 1a and 2b for MCF-7 cells 50 The values ​​were 7.45 and 8.97 μM, respectively, showing good inhibitory effects on MCF-7 cells.

[0114] Table 4 IC of compounds against MCF-7 cells 50 value

[0115]

[0116] # Docetaxel is a positive drug

[0117] Subsequently, the level of Sphk1 in MCF-7 cells treated with two pairs of enantiomeric compounds was detected by cell immunofluorescence, and the results are shown in Table 5. According to the experimental results, the fluorescence intensity of Sphk1 in MCF-7 cells treated with compound monomers was significantly different from that in the blank group (P<0.01), that is, the four compounds can significantly inhibit the expression level of Sphk1 in MCF-7 cells. This indicates that the four compounds may exert anti-breast cancer activity by inhibiting the expression level of Sphk1 in MCF-7 cells.

[0118] Table 5 Effects of compounds on the level of Sphk1 in MCF-7 cells ( n=3)

[0119]

[0120] ** P < 0.01

[0121] In summary, this study discovered two pairs of new flavonoid enantiomers from the dichloromethane extract of Epimedium sagittatum. Activity evaluation found that the four compounds could significantly reduce the cell viability of human breast cancer cells MCF-7 and were non-toxic to human normal breast cells MCF-10A, suggesting that these four compounds may be the pharmacological substances of Epimedium sagittatum to exert anti-breast cancer activity. Among them, (2”R,3”R)-epimesatine N(1a) and (2”R,3”R)-epimesatine N(2b) had an IC of 1.1747 W / m2 / cm2 on MCF-7 cells. 50 The values ​​were 7.45 and 8.97 μM, respectively. In addition, all four compounds had significant inhibitory effects on Sphk1 in MCF-7 cells, indicating that the anti-breast cancer effects of these four compounds may be related to the inhibition of Sphk1 expression levels. These four compounds are expected to become lead compounds for anti-breast cancer, providing a technical basis for the development of anti-breast cancer drugs, expanding the medicinal and economic value of Epimedium sagittatum, and are innovations in the treatment of breast cancer drugs with huge economic and social benefits.

Claims

1. Flavonoid enantiomers extracted from Epimedium sagittatum, (2'' R ,3'' R ,2''' R )-epimesatine M(1a) and (2'' S ,3'' S ,2''' S )-epimesatine M (1b) is an enantiomeric compound, and the molecular structures are: ； (2'' S ,3'' S )-epimesatine N (2a) and (2'' R ,3'' R )-epimesatine N (2b) is an enantiomeric compound, and the molecular structures are: 。 2. The flavonoid enantiomer compound extracted from Epimedium sagittatum, wherein the preparation method thereof is: (1) Preparation of polar segments The dried aerial part of Epimedium sagittatum (80 kg) was crushed and extracted three times with 120 L of 70% ethanol at 45 °C for 30 min each time. The solvent was recovered under reduced pressure to obtain an extract (6.5 kg). The extract was suspended with 3 times the volume of distilled water to obtain a suspension. The suspension was extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature for 4 times, 15 L each time, and extracted for 2 h to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract, respectively. The dichloromethane extract ( 2.1 kg), using 100-200 mesh normal phase silica gel column chromatography for gradient elution separation, the gradient volume ratio is petroleum ether-ethyl acetate = 50:1, the amount used is 12 L; 40:1, dosage 20 L; 35:1, dosage 20 L; 20:1, dosage 40 L; 10:1, dosage 40 L; 5:1, dosage 40 L; 1:1, dosage 20 L; 0:1, dosage 4 L, similar parts were combined according to the TLC color development results to obtain 8 polar segments Fr. A…Fr. F…Fr.G, Fr.H; (2) Preparation of the first group of components The polar segment Fr. F (13.7 g) was chromatographed on a 200-300 mesh normal phase silica gel column with petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was a volume ratio of petroleum ether / ethyl acetate = 35:1, 1 L, 20:1, 3 L, 10:1, 3 L, 5:1, 3 L, 1:1, 2 L, 0:1, 1 L. The eluates were combined in 500 mL, and the solvent was recovered under reduced pressure to obtain multiple components. All the components were then spotted on a thin layer chromatography (TLC) plate, and the components with similar absorption on the thin layer plate were combined to obtain 15 first group components Fr. F1...Fr. F15; (3) Preparation of the second group of components The first group component Fr. F15 (1.82 g) was further eluted by reverse phase silica gel column chromatography (ODS) gradient elution, the volume gradient ratio of the eluent was methanol: water = 40:60, 1 L, 50:50, 1 L, 60:40, 2 L, 70:30, 2 L, 80:20, 2 L, 90:10, 1 L, 100:0, 1 L, the eluents were combined together every 200 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 12 second group components Fr. F15.1…Fr. F15.9…Fr. F15.12; (3) Preparation of compound (2'' S ,3'' S )-epimesatine N and (2'' R ,3'' R )-epimesatine N The second component Fr. F15.9 was then purified by semi-preparative high performance liquid chromatography (HPLC) through a chiral column, eluted with methanol: water = 80:20, at a flow rate of 2 mL / min, and the retention time was collected. t R = 39.3 min fraction, compound (2'' S ,3'' S )-epimesatine N (1.3 mg), collected at retention time t R = 33.3 min fraction, compound (2'' R ,3'' R )-epimesatine N (1.2 mg); (4) Preparation of the fourth group of ingredients The polar segment Fr. G (100.0 g) was chromatographed on a 100-200 mesh normal phase silica gel column, and petroleum ether / ethyl acetate was used as the eluent for gradient elution. The elution gradient was a volume ratio of petroleum ether / ethyl acetate = 50:1, 1 L, 35:1, 5 L, 20:1, 5 L, 10:1, 5 L, 5:1, 5 L, 1:1, 2 L, 0:1, 1 L. The eluates were combined in 1 L, and 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 18 fourth group components Fr. F1…Fr. F17, Fr. F18; (5) Preparation of the fifth group of ingredients The fourth group fraction Fr. G17 (65.0 g) was subjected to 200-300 mesh normal phase silica gel chromatography again, and petroleum ether / ethyl acetate was used as the eluent for gradient elution. The elution gradient was petroleum ether / ethyl acetate in a volume ratio of 35:1, 1 L, 20:1, 4 L, 10:1, 4 L, 5:1, 4 L, 1:1, 2 L, 0:1, 1 L. The eluates were combined in 500 mL each, and 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 19 fifth group components, namely Fr. G17.1…Fr. G17.15…Fr. G17.19; (6) Preparation of the sixth group of ingredients The fifth group component Fr. G17.15 (38.9 g) was eluted by ODS column chromatography, and the volume gradient ratio of the eluent was 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 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 12 sixth group components Fr. G17.15.1…Fr. G17.15.10…Fr. G17.15.12; (7) Preparation of compound (2'' R ,3'' R ,2''' R )-epimesatine M and (2'' S ,3'' S ,2''' S )-epimesatine M The sixth group component Fr. G17.15.10 was eluted by ODS column chromatography, and the volume gradient ratio of the eluent was methanol: water = 40:60, 2 L, 50:50, 2 L, 60:40, 5 L, 70:30, 5 L, 80:20, 4 L, 90:10, 2 L, 100:0, 1 L. The eluents were combined together every 500 mL, and recovered under reduced pressure to obtain multiple components. 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. G17.15.10.6…Fr. G17.15.10.13; Then, 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 (CH3CN:H2O) in a volume ratio of 60:40 at a flow rate of 2 mL / min, and seven components Fr. G17.15.10.6.1, Fr. G17.15.10.6.2…Fr. G17.15.10.6.7 were separated; The component Fr. G17.15.10.6.2 was purified again by HPLC using a 10ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile: water (CH3CN:H2O) = 35:65 by volume at a flow rate of 2 mL / min. The retention time was collected. t R The fraction at 63.1 min was dried to obtain compound (2'' R ,3'' R ,2''' R )-epimesatine M (3.6 mg), collected at retention time t R The fraction at 59.1 min was dried to obtain compound (2'' S ,3'' S ,2''' S )-epimesatine M (2.1 mg).

3. The flavonoid enantiomer compound (2'' extracted from Epimedium sagittatum according to claim 1 R ,3'' R ,2''' R )-epimesatine M and (2'' S ,3'' S ,2''' S )-epimesatine M, (2'' S ,3'' S )-epimesatine N and (2'' R ,3'' R Application of )-epimesatine N in the preparation of anti-breast cancer drugs.

4. The flavonoid enantiomeric compound (2'' extracted from Epimedium sagittatum according to claim 3 R ,3'' R ,2''' R )-epimesatine M and (2'' S ,3'' S ,2''' S )-epimesatine M, (2'' S ,3'' S )-epimesatine N and (2'' R ,3'' R The use of )-epimesatine N in the preparation of anti-breast cancer drugs is characterized in that: The breast cancer cell line is human breast cancer cell MCF-7.

Images