Flavonoid enantiomer compound extracted from Epimedium sagittatum and its preparation method and application

Abstract

The invention discloses a flavonoid enantiomer compound extracted from Epimedium sagittatum and its preparation method and application. The preparation method comprises the following steps: crushing Epimedium sagittatum to prepare a paste, suspending the paste with water, extracting the paste with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively to obtain extracts of each part, performing gradient elution separation by silica gel column chromatography, combining similar parts according to the color development results of thin layer chromatography (TLC) to obtain multiple polar segments; chromatographing the target polar segment and performing gradient elution to obtain multiple components, and then combining similar components by thin layer chromatography (TLC) spot plate absorption to obtain multiple sub-components; eluting and purifying the target sub-components, collecting fractions with different retention times, and drying to obtain four compounds respectively. The four compounds have a significant inhibitory effect on the cell viability of human breast cancer MCF-7 cells and have anti-breast cancer activity. The four compounds can be used in the preparation of drugs for treating breast cancer, with huge social and economic benefits.

Description

Technical Field

[0001] The present 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 (TCM) has been used to treat diseases for thousands of years. However, with the advancement of science and technology, the complex composition of TCM has been discovered, and research on its components has attracted increasing attention in the medical community. Epimedium is the dried leaves of the Berberidaceae plants Epimedium, Epimedium sagittatum, or Epimedium pubescens, with Epimedium sagittatum being the most widely distributed and used. Modern pharmacological studies have shown that E. sagittatum exhibits a wide range of pharmacological effects, including anti-tumor, antioxidant, anti-osteoporosis, immune-regulating, anti-atherosclerotic, and antidepressant activities. According to literature reports, flavonoids are the primary active ingredient in E. sagittatum. Prenylated flavonoids, a type of structure with an isopentenyl group substituted on the flavonoid backbone, generally exhibit enhanced lipophilicity and pharmacological activity.

[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 treatment drugs, their effectiveness is limited. Due to the complexity of the active ingredients in traditional Chinese medicine, their multi-targeting properties, and their unique therapeutic efficacy 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. Therefore, is it possible to extract flavonoid enantiomers from the active part of Epimedium brevicornum that have a significant inhibitory effect on breast cancer MCF-7 cells to prepare anti-breast cancer drugs? However, there have been no published reports to date. 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 flavonoid enantiomer compound extracted from Epimedium sagittatum and its preparation method and application, 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 their molecular structural formulas are:

[0006]

[0007] The enantiomeric compounds (2"S, 3"S)-epimesatine N (2a) and (2"R, 3"R)-epimesatine N (2b) have the following molecular structures:

[0008]

[0009] Its preparation method is:

[0010] The dried aerial part of Epimedium sagittatum is crushed and extracted with ethanol under reflux to obtain an extract, which is then suspended in water to form a suspension. The extract is then extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain extracts of the petroleum ether portion, the dichloromethane portion, the ethyl acetate portion, and the n-butanol portion, respectively. The dichloromethane portion extract is separated by gradient elution on a silica gel column. Similar portions are combined based on the results of thin layer chromatography (TLC) to obtain polar segments Fr.A...Fr.G, and Fr.H.

[0011] The polar fragment Fr.F was subjected to silica gel column chromatography with a petroleum ether / ethyl acetate gradient elution, and the solvent was recovered under reduced pressure to obtain multiple fractions. All fractions were then spotted on a thin layer chromatography (TLC) plate, and fractions with similar absorption on the TLC plate were combined to obtain 15 first-group fractions Fr.F1 to Fr.F15.

[0012] The first group fraction Fr.F15 was subjected to reverse phase silica gel column chromatography with a methanol / water gradient elution, and the solvent was recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted on a thin layer chromatography (TLC) plate, and fractions with similar absorption on the TLC plate were combined to obtain 12 second group fractions Fr.F15.1...Fr.F15.9...Fr.F15.12;

[0013] Then, the fraction Fr.F15.9 was purified by chiral chromatography using methanol:water = 80:20 as eluent at a flow rate of 2 mL / min. The residues with retention time t R =39.3min fraction, to obtain compound (2"S,3"S)-epimesatine N (1.3 mg), which was collected at retention time t R =33.3min fraction, yielding compound (2"R,3"R)-epimesatine N (1.2 mg);

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

[0015] The fourth group fraction Fr.G17 was subjected to normal phase silica gel chromatography with a petroleum ether / ethyl acetate gradient elution and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted on a thin layer chromatography (TLC) plate, and fractions with similar absorption on the TLC plate were combined to obtain 19 fifth group fractions, namely Fr.G17.1...Fr.G17.15...Fr.G17.19.

[0016] The fifth group fraction Fr.G17.15 was subjected to ODS column chromatography, gradient elution was performed with methanol / water, and multiple fractions were recovered under reduced pressure. The multiple fractions were then spotted on a thin layer chromatography (TLC) plate, and fractions with similar absorption on the TLC plate were combined to obtain 12 sixth group fractions Fr.G17.15.1…Fr.G17.15.10…Fr.G17.15.12;

[0017] The sixth group, Fr.G17.15.10, was subjected to ODS column chromatography, gradient eluted with methanol / water, 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 TLC 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 (volume ratio) at a flow rate of 2 mL / min to separate and obtain seven components: Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…Fr.G17.15.10.6.7;

[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 with 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 invention discloses prenyl 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 extracted from Epimedium sagittatum. The compounds have a significant inhibitory effect on the cell viability of human breast cancer MCF-7 cells and have anti-breast cancer activity, thereby realizing application in the preparation of drugs for treating breast cancer.

[0021] The invention has abundant raw materials and an easy-to-operate preparation method. Prenylated 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 can be effectively extracted from the herb. The compounds have anti-breast cancer activity, significantly inhibit the cell viability of human breast cancer MCF-7 cells, and are non-toxic to human normal breast cells MCF-10A. The compounds can be effectively used in the preparation of drugs for treating breast cancer, thus expanding the medicinal 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 enantiomeric 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 are shown;

[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 of the present invention is described in detail below in combination with specific situations.

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

[0029]

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

[0031]

[0032] Its preparation method is:

[0033] (1) Preparation of polar segments

[0034] The dried aerial part of Epimedium sagittatum (80 kg) was crushed and refluxed for 3 times at 45°C with 120 L of 70% ethanol each time 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 was The extract (2.1 kg) was separated by gradient elution using a 100-200 mesh normal phase silica gel column chromatography method. The gradient volume ratio was petroleum ether-ethyl acetate = 50:1, used in 12 L; 40:1, used in 20 L; 35:1, used in 20 L; 20:1, used in 40 L; 10:1, used in 40 L; 5:1, used in 40 L; 1:1, used in 20 L; 0:1, used in 4 L. Similar parts were combined according to the results of thin layer chromatography (TLC) to obtain 8 polar segments Fr.A...Fr.F...Fr.G, Fr.H;

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

[0036] Polar fragment Fr.F (13.7 g) was chromatographed on a 200-300 mesh normal phase silica gel column using petroleum ether / ethyl acetate as the eluent, with a gradient elution. The elution gradient was: petroleum ether / ethyl acetate = 35:1 (volume ratio) in 1 L, 20:1 (volume ratio) in 3 L, 10:1 (volume ratio) in 3 L, 5:1 (volume ratio) in 3 L, 1:1 (volume ratio) in 2 L, and 0:1 (volume ratio) in 1 L. The eluates were combined in 500 mL increments, and the solvent was recovered under reduced pressure to obtain multiple fractions. All fractions were then spotted by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 15 first-group fractions Fr.F1 to Fr.F15.

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

[0038] The first fraction Fr.F15 (1.82 g) was further eluted using reverse phase silica gel column chromatography (ODS) with a gradient elution 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), and 100:0 (1 L). 200 mL of the eluates were combined and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 12 second fractions 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 residues with retention time t R =39.3min fraction, to obtain compound (2"S,3"S)-epimesatine N (1.3 mg), which was collected at retention time t R =33.3min fraction, yielding compound (2"R,3"R)-epimesatine N (1.2 mg);

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

[0042] Polar fragment Fr.G (100.0 g) was chromatographed on a 100-200 mesh normal phase silica gel column using petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was: petroleum ether / ethyl acetate = 50:1 by volume (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 increments and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted by thin layer chromatography (TLC). Fractions with similar absorption on the TLC plate were combined to obtain 18 fourth group fractions, Fr.F1 to Fr.F17, and Fr.F18.

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

[0044] The fourth fraction, Fr.G17 (65.0 g), was again chromatographed on 200-300 mesh normal phase silica gel using petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was: petroleum ether / ethyl acetate = 35:1 (volume ratio) in 1 L, 20:1 (volume ratio) in 4 L, 10:1 (volume ratio) in 4 L, 5:1 (volume ratio) in 4 L, 1:1 (volume ratio) in 2 L, and 0:1 (volume ratio) in 1 L. The eluates were combined in 500 mL increments and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 19 fifth fractions, namely, Fr.G17.1 ... Fr.G17.15 ... Fr.G17.19.

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

[0046] The fifth subgroup, Fr.G17.15 (38.9 g), was eluted by ODS column chromatography with a gradient volume ratio of methanol:water of 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). 500 mL of the eluates were combined and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 12 sixth subgroups, 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 with a volume gradient ratio of 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), and 100:0 (1 L). The eluates were combined in 500 mL increments and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spotted by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 13 fractions, Fr.G17.15.10.1 ... Fr.G17.15.10.6 ... Fr.G17.15.10.13.

[0049] Component Fr.G17.15.10.6 was then 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 seven components: Fr.G17.15.10.6.1, Fr.G17.15.10.6.2, …, and 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) in a volume ratio of 35:65 at a flow rate of 2 mL / min. The retention time t R The fraction at 63.1 min was dried to give compound (2"R, 3"R, 2"'R)-epimesatine M (3.6 mg). The fraction was 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).

[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 MCF-7 cells, 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 target compound can be prepared as needed. The given embodiment is only used to illustrate the specific implementation of the present invention, and is not used to limit the scope of protection of the present invention. The technical core protected by the present invention is the flavonoid enantiomer compound.

[0053] The present invention relates to a flavonoid enantiomer compound extracted from Epimedium sagittatum, which has anti-breast cancer activity and is used in the preparation of a drug for treating breast cancer. Tests have shown excellent beneficial technical effects, and 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 AVANCE III 500 (Bruker, Germany)

[0060] High-performance liquid chromatography Shimadzu LC-40 equipped with DAD detector and RPC18 column (10 ID × 250 mm, Cosmosil 5C18-MS-II Packed column, Nacalai Tesque, China)

[0061] N-1100 Rotary Evaporator and N-1111 Chilled Water Circulation Device (Shanghai Airan 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 Biotechnology)

[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 Solaibao Technology Co., Ltd.)

[0077] Ampicillin, streptomycin (Sigma)

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

[0079] Ham's F12K medium (Pnosel Corporation)

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

[0081] Human normal breast cells MCF-10A, human breast cancer cells MCF-7 (Shanghai Cell Bank, Chinese 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 at Henan University of Traditional Chinese Medicine with the accession 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 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 (125 MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0090]

[0091] By comparing 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 (125 MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0096]

[0097] 3. Activity Test

[0098] 3.1 Cell culture

[0099] 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.

[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 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.

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

[0103] 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.

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

[0105] MCF-7 cells were cultured at a rate of 2 × 10 4Cells 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.

[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 indicates a significant difference. ** P<0.01 indicates a very significant difference.

[0108] 3.6 Activity Evaluation

[0109] The experiment first screened the compounds for cytotoxicity using the MTT assay. The results, shown in Table 3, indicate that treatment with 10 μM of each of the two enantiomeric flavonoids significantly reduced the viability of MCF-7 human breast cancer cells compared to the blank control group (P < 0.01). This indicates that both enantiomeric flavonoids significantly inhibited the viability of MCF-7 human breast cancer cells at a concentration of 10 μM. The MTT assay then examined the compounds' effects on MCF-10A normal human breast cells, revealing that none of the four compounds significantly affected MCF-10A cell viability (Table 3). Therefore, these four compounds may be the pharmacological agents 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 two pairs of flavonoid enantiomers 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, cell immunofluorescence was used to detect Sphk1 levels in MCF-7 cells treated with the two pairs of enantiomeric compounds. The results are shown in Table 5. The experimental results showed that the fluorescence intensity of Sphk1 in MCF-7 cells treated with the compound monomers was significantly different from that in the blank group (P < 0.01), indicating that all four compounds significantly inhibited Sphk1 expression in MCF-7 cells. This suggests that these four compounds may exert their anti-breast cancer activity by inhibiting Sphk1 expression in MCF-7 cells.

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

[0119]

[0120] ** Indicates P < 0.01

[0121] In summary, this study discovered two new pairs of flavonoid enantiomers from the dichloromethane extract of Epimedium sagittatum. Activity evaluation found that all four compounds could significantly reduce the cell viability of human breast cancer cells MCF-7 and had no toxicity 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 1.177 for MCF-7 cells. 50 The values ​​were 7.45 and 8.97 μM, respectively. Furthermore, all four compounds showed significant inhibitory effects on Sphk1 in MCF-7 cells, suggesting that their anti-breast cancer effects may be related to the inhibition of Sphk1 expression. These four compounds are expected to become lead compounds for anti-breast cancer treatment, providing a technical basis for the development of anti-breast cancer drugs. This expands the medicinal and economic value of Epimedium sagittatum, representing an innovation in breast cancer treatment with significant economic and social benefits.

Claims

1. The flavonoid enantiomer compound extracted from Epimedium sagittatum is (2'' R ,3'' R ,2''' R )-epimesatine M1a and (2'' S ,3'' S ,2''' S )-epimesatine M 1b is an enantiomeric compound, and the molecular structures are: 。 2. The method for preparing the flavonoid enantiomer compound extracted from Epimedium sagittatum according to claim 1 is: (1) Preparation of polar segments 80 kg of the dried aerial parts of Epimedium sagittatum were 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 6.5 kg of extract. The extract was suspended in 3 times the volume of distilled water to obtain a suspension. The suspension was extracted four times with petroleum ether, dichloromethane, ethyl acetate, and n-butanol at room temperature, 15 L each time, and 2 h each time, to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract, respectively. 2.1 kg of the dichloromethane extract was separated by gradient elution on a 100-200 mesh normal phase silica gel column with a gradient volume ratio of petroleum ether to ethyl acetate = 50:1, using 12 L of the column. 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, using 4 L, similar parts were combined according to the TLC results to obtain 8 polar segments Fr. A…Fr. F…Fr. G, Fr. H; (2) Preparation of the first group of components 3.7 g of the polar fragment Fr. F1 was chromatographed on a 200–300 mesh normal phase silica gel column using petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was: petroleum ether / ethyl acetate = 35:1 (volume ratio) for 1 L, 20:1 (volume ratio) for 3 L, 10:1 (volume ratio) for 3 L, 5:1 (volume ratio) for 3 L, 1:1 (volume ratio) for 2 L, and 0:1 (volume ratio) for 1 L. 500 mL of the eluate was combined, and the solvent was recovered under reduced pressure to obtain multiple fractions. All fractions were then spotted on a thin layer chromatography (TLC) plate, and fractions with similar absorption on the TLC plate were combined to obtain 15 first-group fractions Fr. F1 to Fr. F15. (3) Preparation of the second group of components 1.82 g of the first fraction, Fr. F15, was further subjected to reverse-phase silica gel column chromatography with an 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), and 100:0 (1 L). 200 mL of the eluents were combined and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spot-plated by thin-layer chromatography (TLC). Fractions with similar absorption on the TLC plate were combined to obtain 12 second fractions, Fr. F15.1…Fr. F15.9…Fr. F15.

12. (3) Preparation of compound (2'' S ,3'' S )-epimesatine N 2a and (2'' R ,3'' R )-epimesatine N 2b The second component Fr. F15.9 was then purified by semi-preparative HPLC and passed through a chiral column with methanol:water = 80:20 as eluent at a flow rate of 2 mL / min. 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, (2'' S ,3'' S )-epimesatine N 2a and (2'' R ,3'' R The molecular structure of )-epimesatine N 2b is: ； (4) Preparation of the fourth group of components Polar fragment Fr. G (100.0 g) was chromatographed on a 100–200 mesh normal phase silica gel column using petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was: petroleum ether / ethyl acetate = 50:1 (volume ratio) (1 L), 35:1 (volume ratio) (5 L), 20:1 (volume ratio) (5 L), 10:1 (volume ratio) (5 L), 5:1 (volume ratio) (5 L), 1:1 (volume ratio) (2 L), 0:1 (volume ratio) (1 L). The eluates were combined in 1 L increments and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spot-plated by thin layer chromatography (TLC). Fractions with similar absorption on the TLC plate were combined to obtain 18 fourth group fractions, Fr. F1…Fr. F17, and Fr. F18. (5) Preparation of the fifth group of components The fourth fraction, Fr. G1765.0 g, was again chromatographed on 200–300 mesh normal phase silica gel with petroleum ether / ethyl acetate as the eluent for gradient elution. The elution gradient was: petroleum ether / ethyl acetate = 35:1 (volume ratio) for 1 L, 20:1 (volume ratio) for 4 L, 10:1 (volume ratio) for 4 L, 5:1 (volume ratio) for 4 L, 1:1 (volume ratio) for 2 L, and 0:1 (volume ratio) for 1 L. 500 mL of the eluates were combined and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spot-chromated on a TLC plate, and fractions with similar absorption on the plate were combined to obtain 19 fifth fractions, namely, Fr. G17.1…Fr. G17.15…Fr. G17.

19. (6) Preparation of the sixth group of components The fifth group fraction, Fr. G17.1538.9 g, was eluted by ODS column chromatography with a volume 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). 500 mL of the eluate was combined and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spot-plated by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 12 sixth group fractions, 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, Fr. G17.15.10, was eluted by ODS column chromatography with a volume gradient ratio of 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), and 100:0 (1 L). 500 mL of the eluates were combined and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then spot-plated by thin layer chromatography (TLC), and fractions with similar absorption on the TLC plate were combined to obtain 13 fractions, Fr. G17.15.10.1…Fr. G17.15.10.6…Fr. G17.15.10.

13. Component Fr. G17.15.10.6 was then purified by semi-preparative HPLC using a 10 ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile:water (60:40 by volume) at a flow rate of 2 mL / min to separate seven components: Fr. G17.15.10.6.1, Fr. G17.15.10.6.2, …, and Fr. G17.15.10.6.

7. 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 in a volume ratio of 35:65 at a flow rate of 2 mL / min. The retention time was collected. t R The fraction at 63.1 min was dried to give 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 give 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 Application of )-epimesatine M in the preparation of anti-breast cancer drugs.

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