Extraction of isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum and preparation method and application thereof

Abstract

Isopentenyl flavonoid enantiomer compounds are extracted from Epimedium sagittatum and their preparation method and application. The preparation method comprises the following steps: crushing the 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 various parts, 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, then combining similar components through 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 application relates to the field of traditional Chinese medicine, in particular to extraction of isoprenyl flavone enantiomer compounds from Epimedium sagittatum, and preparation method and application thereof. BACKGROUND

[0002] Epimedium is the dry leaves of Epimedium, Epimedium sagittatum, Epimedium pubescens or Epimedium koreanum, 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, anti-oxidation, anti-osteoporosis, immune function regulation, anti-atherosclerosis and anti-depression activity, etc. According to literature reports, flavones are the main effective component type in Epimedium sagittatum, and isoprenyl flavones are a structural type with isoprenyl substitution on the flavone skeleton, which usually has better lipophilicity and pharmacological activity.

[0003] Breast cancer is a common 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 treatment drugs, the effect is limited. Traditional Chinese medicine active ingredients are complex, have multiple targeting, and have unique therapeutic effects for treating diseases. Therefore, one of the technical problems to be solved by the present application is to find new active ingredients (compounds) from Epimedium sagittatum, the second is how to extract isoprenyl flavone enantiomer compounds from Epimedium sagittatum, and the third is to realize the application in the preparation of drugs for treating breast tumors (cancer). However, there is no public report so far. SUMMARY

[0004] In view of the above situation, in order to overcome the shortcomings of the prior art, the purpose of the present application is to provide isoprenyl flavone enantiomer compounds extracted from Epimedium sagittatum and a preparation method and application thereof, which can effectively solve the problem of extracting isoprenyl flavone enantiomer compounds from Epimedium sagittatum and realize the application in the preparation of drugs for treating breast tumors (cancer).

[0005] The technical solution solved by the present application is to extract isoprenyl flavone enantiomer compounds from Epimedium sagittatum. The compounds (R)-epimesatine J (1a) and (S)-epimesatine J (1b) are enantiomers, and their molecular structural formulas are respectively:

[0006]

[0007] The compounds (2”R,2”'S)-epimesatine K (2a) and (2”S,2”'R)-epimesatine K (2b) are enantiomers, and their molecular structural formulas are respectively:

[0008]

[0009] The preparation method is:

[0010] The dried Epimedium sagittatum above-ground parts are pulverized, extracted with ethanol by reflux, and a decoction is obtained. The decoction is suspended in water to form a suspension, and then extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol, respectively, to obtain petroleum ether part extract, dichloromethane part extract, ethyl acetate part extract, and n-butanol part extract. The dichloromethane part extract is separated by gradient elution using silica gel column chromatography, and similar parts are combined according to the thin layer chromatography (TLC) coloration results to obtain polar segments Fr.A…Fr.G, Fr.H.

[0011] The polar segment Fr.G is separated by gradient elution using silica gel column chromatography with petroleum ether / ethyl acetate, and multiple components are obtained by recovering the solvents under reduced pressure. Then, all the components are spotted on a thin layer chromatography (TLC) plate, and similar components on the thin layer plate are combined to obtain 18 first sub-components Fr.F1…Fr.F17, Fr.F18.

[0012] The first sub-component Fr.G17 is separated by gradient elution using silica gel chromatography with petroleum ether / ethyl acetate, and multiple components are obtained by recovering the solvents under reduced pressure. Then, the multiple components are spotted on a thin layer chromatography (TLC) plate, and similar components on the thin layer plate are combined to obtain 19 second sub-components Fr.G17.1, Fr.G17.2, Fr.G17.3…Fr.G17.15…Fr.G17.19.

[0013] The second sub-component Fr.G17.15 is separated by gradient elution using ODS column chromatography with methanol / water, and multiple components are obtained by recovering the solvents under reduced pressure. Then, the multiple components are spotted on a thin layer chromatography (TLC) plate, and similar components on the thin layer plate are combined to obtain 12 third sub-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 sub-component Fr.G17.15.9 is purified by semi-preparative HPLC using a liquid chromatography column with a mobile phase of acetonitrile:water at a flow rate of 1 mL / min. Fractions with retention times of t R 14.0 min are collected and dried to obtain compound (R)-epimesatine J. Fractions with retention times of t R 13.7 min are collected and dried to obtain compound (S)-epimesatine J.

[0015] The third group 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, which were then spotted on a thin layer chromatography (TLC) plate, and the thin layer plate was combined with similar components 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 subjected to semi-preparative HPLC purification, using a liquid chromatography column, with a mobile phase of acetonitrile: water, at a flow rate of 2 mL / min, to separate 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2, ……Fr.G17.15.10.6.7, and the component Fr.G17.15.10.6.1 was subjected to HPLC purification again, using a liquid chromatography column, with a mobile phase of acetonitrile: water, at a flow rate of 2 mL / min, to collect the fractions with retention times t R 42.2 min, dried to obtain compound (2”R, 2”'S)-epimesatine K. R 48.9 min, dried to obtain compound (2”S, 2”'R)-epimesatine K.

[0017] The four isoprenyl flavone 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 to prepare drugs for treating breast cancer.

[0018] The present application has abundant raw materials, the preparation method is easy to operate, and the isoprenyl flavone enantiomer compounds (R)-epimesatine J, (S)-epimesatine J, (2”R, 2”'S)-epimesatine K and (2”S, 2”'R)-epimesatine K can be effectively extracted from Epimedium sagittatum, the above-mentioned 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, can be effectively used to prepare drugs for treating breast cancer, and develop the medicinal value and economic value of Epimedium sagittatum, which is an innovation in the treatment of breast cancer drugs, and has great economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Molecular structure formula of the isoprenyl flavone enantiomer compound of the present application;

[0020] Figure 2 DEPT and C NMR spectra of the compound Epimesatine J of the present application 1 H NMR spectrum (500MHz, deuterated reagent: Acetone-d6) chart;

[0021] Figure 3 DEPT and C NMR spectra of the compound Epimesatine J of the present application 13 C NMR spectrum (125MHz, deuterated reagent: Acetone-d6) chart;

[0022] Figure 4 DEPT and C NMR spectra of the compound Epimesatine K of the present application 1 H NMR spectrum (500MHz, deuterated reagent: Acetone-d6) chart;

[0023] Figure 5 DEPT and C NMR spectra of the compound Epimesatine K of the present application 13 C NMR spectrum (125MHz, deuterated reagent: Acetone-d6) chart. DETAILED DESCRIPTION

[0024] The specific embodiments of the present application are described in detail below in combination with specific cases.

[0025] The technical solution solved by the present application is to extract isoprenyl flavone enantiomer compounds from Epimedium sagittatum, and the compounds (R)-epimesatine J (1a) and (S)-epimesatine J (1b) are enantiomers, and their molecular structure formulas are respectively:

[0026]

[0027] The compounds (2”R,2”'S)-epimesatine K (2a) and (2”S,2”'R)-epimesatine K (2b) are enantiomers, and their molecular structure formulas are respectively:

[0028]

[0029] The preparation method is:

[0030] (1) Preparation of the polar segment

[0031] Dry Epimedium sagittatum Maxim. aerial parts (80 kg) were pulverized, extracted with 120 L of 70% ethanol by volume at 45°C for 30 min three times, and the solvent was recovered under reduced pressure to obtain an extract (6.5 kg). The extract was suspended in 3 times the volume of distilled water to obtain a suspension, which was sequentially extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butanol at room temperature for 15 L each time for 2 h to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract. The dichloromethane extract (2.1 kg) was separated by gradient elution using a 100-200 mesh normal silica gel column with 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 thin layer chromatography (TLC) results. Similar fractions were combined to obtain 8 polar fractions Fr.A…Fr.G, Fr.H.

[0032] (2) Preparation of first group fractions

[0033] The polar fraction Fr.G (100.0 g) was separated by gradient elution using a 100-200 mesh normal silica gel column with petroleum ether / ethyl acetate as the eluent. The elution gradient was petroleum ether / ethyl acetate (50:1, 1 L; 35:1, 5 L; 20:1, 5 L; 10:1, 5 L; 5:1, 5 L; 1:1, 5 L; 0:1, 5 L). The eluent was combined every 1 L, and the solvent was recovered under reduced pressure to obtain multiple fractions. Then, the fractions were spotted on thin layer chromatography (TLC) plates, and similar fractions on the thin layer plates were combined to obtain 18 first group fractions Fr.F1…Fr.F17, Fr.F18.

[0034] (3) Preparation of second group fractions

[0035] The first group fraction Fr.G17 (65.0 g) was separated by gradient elution using a 200-300 mesh normal silica gel column with a volume gradient of petroleum ether / ethyl acetate (35:1, 1 L; 20:1, 4 L; 10:1, 4 L; 5:1, 4 L; 1:1, 2 L; 0:1, 1 L). The eluent was combined every 500 mL, and the solvent was recovered under reduced pressure to obtain multiple fractions. Then, the fractions were spotted on thin layer chromatography (TLC) plates, and similar fractions on the thin layer plates were combined to obtain 19 second group fractions Fr.G17.1, Fr.G17.2, Fr.G17.3…Fr.G17.15…Fr.G17.19.

[0036] (4) Preparation of third group fractions

[0037] The second group component Fr.G17.15.9 was purified by semi-preparative HPLC using a 4.6 ID x 250 mm RP C18 column with a mobile phase of acetonitrile: water (CH3CN:H2O) = 60:40 at a flow rate of 1 mL / min. Fractions with retention times of t

[0038] (5) Preparation of isoprenyl flavone enantiomer compounds (R)-epimesatine J and (S)-epimesatine J

[0039] The second group component Fr.G17.15.9 was purified by semi-preparative HPLC using a 4.6 ID x 250 mm RP C18 column with a mobile phase of acetonitrile: water (CH3CN:H2O) = 60:40 at a flow rate of 1 mL / min. Fractions with retention times of t R 14.0 min were collected, dried and yielded compound (R)-epimesatine J (5.6 mg). Fractions with retention times of t R 13.7 min were collected, dried and yielded compound (S)-epimesatine J (9.9 mg).

[0040] (6) Preparation of isoprenyl flavone enantiomer compounds (2”R,2”’S)-epimesatine K and (2”S,2”’R)-epimesatine K

[0041] The third fraction Fr.G17.15.10 was eluted by ODS column chromatography with a volume ratio gradient of MeOH:H2O (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, and the eluate was combined every 500 mL, and the solvent was recovered under reduced pressure to obtain several fractions, which were then spotted on a thin layer chromatography (TLC) plate, and the thin layer plate was combined with similar fractions to obtain 13 fractions 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 fraction Fr.G17.15.10.6 was purified by semi-preparative HPLC using a 10 ID x 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile:water (CH3CN:H2O) = 60:40 (volume ratio) at a flow rate of 2 mL / min to separate 7 fractions Fr.G17.15.10.6.1, Fr.G17.15.10.6.2, …, Fr.G17.15.10.6.7, and the fraction Fr.G17.15.10.6.1 was further purified by HPLC using a 10 ID x 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile:water (CH3CN:H2O) = 60:40 (volume ratio) at a flow rate of 2 mL / min to collect the fractions with retention times t R 42.2 min, dried to obtain compound (2”R, 2”’S)-epimesatine K (2.2 mg), and the fractions with retention times t R 48.9 min, dried to obtain compound (2”S, 2”’R)-epimesatine K (1.9 mg).

[0043] The four isoprenyl flavone enantiomer compounds (R)-epimesatine J and (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.

[0044] The target compounds can be prepared in any amount as needed according to the method given in the above examples, and the examples given are only used to illustrate the specific embodiments of the present application, but not to limit the protection scope of the present application, and the technical core protected by the present application is the isoprenyl flavone enantiomer compound.

[0045] The application is to extract isoprenyl flavone enantiomer compounds from Epimedium sagittatum, which has anti-breast cancer activity, realizes the application in preparing breast cancer treatment drugs, and has very good beneficial technical effects through tests. The relevant test data are as follows:

[0046] I. Instruments and materials

[0047] 1.1 Instruments:

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

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

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

[0051] Nuclear magnetic resonance spectrometer Bruker AVANCE III 500 (Bruker, Germany)

[0052] High-performance liquid chromatograph Shimadzu LC-40 equipped with DAD detector and RPC18 chromatographic column (10 ID x 250 mm, Cosmosil 5C18-MS-IIPacked column, nacalai tesque, China)

[0053] N-1100 rotary evaporator and N-1111 refrigerated water circulating device (Shanghai Ailang Instrument Co., Ltd.)

[0054] BT25S ten-millionth precision analytical balance (Sartorius)

[0055] Cell real-time label-free system (Agilent)

[0056] Carbon dioxide 3111 type incubator (Thermo)

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

[0058] Multiskan MK3 enzyme marker (Thermo Fisher)

[0059] Super-clean workbench (Sujing Group)

[0060] HVA-85 high-pressure sterilization pot (Hirayama)

[0061] 1.2 Materials:

[0062] Reversed-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] Chromatographically pure methanol, acetonitrile (Tianjin Suiyou Fine Chemical Co., Ltd.)

[0065] Analytically pure methanol, dichloromethane, ethyl acetate, petroleum ether (Beijing Chemical Plant and Tianjin Third Chemical Reagent Factory)

[0066] 16-well plates (Agilent Bio)

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

[0068] Fetal bovine serum (Hangzhou Sijiqing Bioengineering Ltd.), DMEM medium (Gibco Invitrogen)

[0069] Thiazolyl blue MTT (Beijing Solabio Science and Technology Co., Ltd.)

[0070] Ampicillin, streptomycin (Sigma)

[0071] Dimethyl sulfoxide DMSO (Shanghai McLean Biotech Co., Ltd.)

[0072] Ham’s F12K medium (Ponyase Co., Ltd.)

[0073] Docetaxel (Shanghai Yuanye Bio-Technology Co., Ltd.)

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

[0075] Plant material: Epimedium sagittatum used in this subject was collected from Zhumadian, Henan Province in September 2020. The specimen is preserved in Henan University of Chinese Medicine, with the preservation number 20200960.

[0076] Test drugs: The isoprenyl flavone 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 application.

[0077] II. Structure identification

[0078] By comparing 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 (125 MHz) NMR data (deuterated reagent: Acetone-d6; δ unit: ppm; J unit: Hz)

[0082]

[0083] By comparing 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(log ε): 208 (4.32), 240 (3.98), 268 (3.93), 341 (4.01) nm; IR (ν max ): 3444, 2975, 1699, 1653, 1436, 1363, 1260, 1170, 1052 cm -1 . The structural formula is:

[0084]

[0085] (2"R,2"'S)-epimesatine K (2a), yellow amorphous powder; [a] 20 D -3 (c 0.2, MeOH); HRESIMS m / z 479.1665 [M + Na] + (calcd. for C 25 H 28 O8Na, 479.1676). (2"S,2" 'R)-epimesatine K (2b), yellow amorphous powder; [a] 20 D +3 (c 0.2, MeOH); HRESIMS m / z 479.1669 [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 2.1H (500 MHz) and 1 H (500 MHz) and 13 C (125 MHz) NMR data (deuterated reagent: Acetone-d6; δ units: ppm; J units: Hz)

[0087]

[0088]

[0089] III. Activity test

[0090] 3.1 Cell culture

[0091] The frozen MCF-10A cells and MCF-7 cells were thawed to ice-water coexistence state at 37°C water bath, and then immediately centrifuged (1000 rpm, 5 min). After discarding the supernatant, the cells were transferred to a culture dish containing 10% FBS DMEM medium (both penicillin and streptomycin were 100 kU / L), and cultured in a 37°C constant temperature incubator containing 5% CO2. When the cells grew to 80%-90% of the dish, they were passaged. Fresh medium was replaced every 24 h.

[0092] 3.2 Detection of the effect of monomer compounds on the viability of two cells based on MTT method

[0093] The cells were cultured at 37°C in a 5% CO2 incubator to the logarithmic growth phase, and then inoculated in a 96-well plate at a cell density of 2×10 4 After 24 h, they were divided into a normal control group (CON) and each drug group (10 μM). After 24 h of culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the culture was continued for 4 h. The culture solution was carefully aspirated, 150 μL of DMSO was added to each well, and the blue-purple crystals in the well were completely dissolved by shaking for 10 min. The absorbance value (OD) of each well was measured by a microplate reader at 490 nm, and the cell viability was calculated. The experiment was repeated three times in parallel.

[0094] 3.3 Detection of Sphk1 level in MCF-7 cells by cell immunofluorescence

[0095] MCF-7 cells were inoculated in the wells at a density of 2×10 4 After 24 h, they were divided into a normal (CON) and each drug group (10 μM) for continuous culture for 24 h. They were fixed in 4% paraformaldehyde for 15 min, and then permeated with 0.25% Triton X-100 for 10 min. Then, 1% BSA was added for blocking for 30 min, and then the primary antibody Sphk1 was added for incubation at 4°C overnight. After washing with PBST for 3 times, DAPI was added for staining for 4 min, and then PBS was added for washing once. The cells were photographed by using Operetta CLS high-content imaging analysis system.

[0096] 3.4 Statistical analysis

[0097] The experimental data were expressed as mean ± standard deviation , and the statistical analysis was performed by using SPSS 26.0. One-way ANOVA was used for comparison between groups. * P<0.05 indicates a significant difference, ** P<0.01 indicates a very significant difference.

[0098] 3.5 Activity evaluation

[0099] This study first screened the cytotoxicity of the compounds using the MTT assay. The results, shown in Table 3, indicate that treatment with 10 μM of compounds 1a / 1b and 2a / 2b significantly reduced the viability of MCF-7 human breast cancer cells compared to the blank control group (P < 0.01). These results indicate that these compounds significantly inhibited the viability of MCF-7 human breast cancer cells at a concentration of 10 μM. Subsequently, the MTT assay was used to examine the effects of the compounds on MCF-10A normal human breast cells. None of the compounds significantly affected MCF-10A cell viability (Table 3). This suggests that these compounds may be the pharmacological agents 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 effects of compounds 1a / 1b and 2a / 2b on Sphk1 expression in MCF-7 cells were examined using cell-based immunofluorescence assays. The results are shown in Table 4. The fluorescence intensity of Sphk1 in MCF-7 cells treated with compounds 1a / 1b and 2a / 2b alone was significantly different from that in the blank control group (P < 0.01). This indicates that all four compounds, 1a / 1b and 2a / 2b, significantly reduced Sphk1 expression in MCF-7 cells. This suggests that these compounds may exert their anti-breast cancer activity by inhibiting Sphk1 expression in MCF-7 cells.

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

[0105]

[0106] ** Indicates P < 0.01

[0107] In conclusion, two pairs of new isoprenylated flavones enantiomers were discovered from dichloromethane extract of Epimedium sagittatum. Activity evaluation showed that four compounds could significantly reduce the cell viability of human breast cancer cell MCF-7, and had no toxicity to human normal breast cell MCF-10A, suggesting that these compounds might be the pharmacodynamic substances of Epimedium sagittatum to exert the anti-breast cancer activity. In addition, four compounds could significantly inhibit Sphk1 in MCF-7 cells, suggesting that these compounds might exert the anti-breast cancer effect by inhibiting the expression level of Sphk1. These compounds are expected to become the lead compounds for anti-breast cancer, provide technical basis for the development of anti-breast cancer drugs, open up the medicinal value and economic value of Epimedium sagittatum, and are the innovation of breast cancer drugs, which has great economic and social benefits.

Claims

1. A method for extracting isopentenyl flavonoid enantiomer compounds from Epimedium sagittatum, characterized in that: The isopentenyl flavonoid enantiomeric compound is an enantiomeric compound ( R )-epimesatine J (1a) and ( S )-epimesatineJ (1b), the molecular structure is: ； Enantiomeric compound (2'' R ,2''' S )-epimesatine K (2a) and (2'' S ,2''' R )-epimesatine K(2b) is an enantiomer with the molecular structure: ； The preparation method 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, dosage 4 L, based on the TLC results, similar parts were combined to obtain 8 polar segments Fr. A...Fr.G, Fr.H; (2) Preparation of the first group of components Polar fragment Fr. 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) for 1 L, 35:1 (volume ratio) for 5 L, 20:1 (volume ratio) for 5 L, 10:1 (volume ratio) for 5 L, 5:1 (volume ratio) for 5 L, 1:1 (volume ratio) for 5 L, and 0:1 (volume ratio) for 5 L. The eluates were combined in 1 L increments and the solvent was recovered under reduced pressure to obtain multiple fractions. All fractions were then subjected to thin layer chromatography (TLC) spotting, and fractions with similar absorption on the TLC plate were combined to obtain 18 first-group fractions, Fr. F1…Fr. F17, and 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 with a volume gradient ratio of petroleum ether / ethyl acetate = 35:1 (1 L), 20:1 (4 L), 10:1 (4 L), 5:1 (4 L), 1:1 (2 L), and 0:1 (1 L). The eluates were combined in 500 mL increments and recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then subjected to thin layer chromatography (TLC) and fractions with similar absorption on the TLC 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 components The second group component Fr. G17.15 was subjected to gradient elution by ODS column chromatography, with the volume gradient ratio of eluent being 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 eluents were combined, and the solvent was recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then subjected to thin layer chromatography (TLC) spotting, and fractions with similar absorption on the TLC 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, and 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, Fr. G17.15.10, was eluted by ODS column chromatography with a volume gradient ratio of 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), and 100:0 (1 L). 500 mL of the eluates were combined and the solvent was recovered under reduced pressure to obtain multiple fractions. The multiple fractions were then subjected to 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. G17.15.10.2, 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. The sub-component Fr. G17.15.10.6.1 was purified again by 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 the retention time was collected. t R The fraction at 42.2 min was dried to give compound (2'' R ,2''' S )-epimesatine K, collection retention time t R The fraction at 48.9 min was dried to give compound (2'' S ,2''' R )-epimesatine K.

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