Preparation method and application of isoprenyl flavonoid compound extracted from epimedium sagittatum

Abstract

Isoprenyl flavonoid compounds extracted from Epimedium sagittatum and preparation method and application thereof, the preparation method is: crushing Epimedium sagittatum to paste, suspending with water, extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively to obtain various parts of extract, using silica gel column chromatography to separate by gradient elution, according to the TLC coloration result, similar parts are combined to obtain multiple polarity sections; chromatograph the target polarity section, gradient elution, obtain multiple components, then combine similar components by TLC point plate absorption to obtain multiple component groups; elute and purify the target component group, collect the flow fraction with different retention time, dry, and obtain two compounds respectively, which have significant inhibitory effect on the cell viability of human breast cancer cell MCF-7, have anti-breast cancer activity, and can be applied to prepare breast cancer treatment drugs, with huge social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, in particular to an isoprenyl flavone compound extracted from Epimedium sagittatum, and a preparation method and application thereof. BACKGROUND

[0002] Epimedium is the dry leaves of Berberidaceae Epimedium sagittatum, 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 active ingredient 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. 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. Extracting active ingredients from crude drugs for treating specific diseases is a new trend in the development of traditional Chinese medicine. Therefore, can new active ingredients (compounds) be found in Epimedium sagittatum to prepare a drug for treating breast cancer caused by human breast cancer cells MCF-7 and achieve the application in preparing a drug for treating breast tumors (cancer)? 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 an isoprenyl flavone compound extracted from Epimedium sagittatum and a preparation method and application thereof, which can effectively solve the problem of the application of the isoprenyl flavone compound extracted from Epimedium sagittatum in preparing a drug for treating breast tumors (cancer).

[0005] The technical solution solved by the present application is that the isoprenyl flavone compounds (2"S, 2"'S)-epimesatine O (1a) and (2"R, 2"'R)-epimesatine O (1b) extracted from Epimedium sagittatum have the following molecular structural formula:

[0006]

[0007] The preparation method is as follows:

[0008] The dried Epimedium sagittatum above-ground parts are pulverized, extracted with ethanol by refluxing, and the extract is suspended in water to form a suspension, which is 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 using silica gel column chromatography, and similar fractions are combined according to the thin layer chromatography (TLC) results to obtain polar fractions Fr.A…Fr.G, Fr.H;

[0009] The polar fraction Fr.G is separated by gradient elution using silica gel column chromatography with petroleum ether / ethyl acetate, and multiple components are recovered 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.

[0010] The first sub-component Fr.G17 is separated by gradient elution using silica gel column chromatography with petroleum ether / ethyl acetate, and multiple components are recovered 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 19 second sub-components Fr.G17.1, Fr.G17.2, Fr.G17.3…Fr.G17.15…Fr.G17.19.

[0011] The second sub-component Fr.G17.15 is separated by gradient elution using ODS column chromatography with methanol / water, and multiple components are recovered 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 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.

[0012] The third sub-component Fr.G17.15.10 is separated by gradient elution using ODS column chromatography with methanol / water, and multiple components are recovered 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 13 fourth sub-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.

[0013] (6) Preparation of isoprenyl flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine O

[0014] The fourth group component Fr.G17.15.10.6 was purified and separated by semi-preparative HPLC using a 10 ID x 250 mm RP C18 liquid chromatography column, with a mobile phase of acetonitrile: water = 60:40 at a flow rate of 2 mL / min, to obtain seven components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…G17.15.10.6.7;

[0015] The component Fr.G17.15.10.6.2 was purified and separated by HPLC again, with a mobile phase of acetonitrile: water = 60:40 at a flow rate of 2 mL / min, to obtain six components Fr.G17.15.10.6.2.1…Fr.G17.15.10.6.2.4…G17.15.10.6.2.6;

[0016] The component Fr.G17.15.10.6.2.4 was purified and separated by HPLC again, with a mobile phase of acetonitrile: water = 60:40 at a flow rate of 2 mL / min, to obtain five components Fr.G17.15.10.6.2.4.1…Fr.G17.15.10.6.2.4.4, Fr.G17.15.10.6.2.4.5,

[0017] wherein the retention time t R 63.6 min was dried to obtain compound (2"S, 2" 'S)-epimesatine O (11.2 mg);

[0018] The component Fr.G17.15.10.6.2.4.4 was purified by HPLC again, with a mobile phase of acetonitrile: water = 60:40 at a flow rate of 2 mL / min, and the fraction with a retention time t R 19.3 min was dried to obtain (2"R, 2" 'R)-epimesatine O (9.0 mg).

[0019] The above isoprenyl flavone compounds (2"S, 2" 'S)-epimesatine O and (2"R, 2" 'R)-epimesatine O have a significant inhibitory effect on the cell viability of human breast cancer cells MCF-7, have anti-breast cancer activity, and have an application in the preparation of a drug for treating breast cancer.

[0020] The application has rich raw materials, the preparation method is easy to operate, can effectively extract isoprenyl flavonoid compounds (2"S, 2"'S)-epimesatine O and (2"R, 2"'R)-epimesatine from Epimedium sagittatum, the above-mentioned compounds have anti-breast cancer activity, have significant inhibitory effect on cell viability of human breast cancer cells MCF-7, and have no toxicity to human normal breast cells MCF-10A, can be effectively used for preparing breast cancer treatment drugs, and develop the medicinal value and economic value of Epimedium sagittatum. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a molecular structure formula diagram of the isoprenyl flavonoid compound of the application;

[0022] Figure 2 It is a DEPT and C NMR spectrum diagram of Epimesatine O of the application; 1 It is an H NMR spectrum (500MHz, deuterated reagent: Acetone-d6) diagram;

[0023] Figure 3 It is a DEPT and C NMR spectrum diagram of Epimesatine O of the application; 13 It is an H NMR spectrum (500MHz, deuterated reagent: Acetone-d6) diagram; DETAILED DESCRIPTION

[0024] The specific embodiment of the application is described in detail in combination with specific cases.

[0025] The technical solution solved by the application is that the isoprenyl flavonoid compounds (2"S, 2"'S)-epimesatine O (1a) and (2"R, 2"'R)-epimesatine O (1b) extracted from Epimedium sagittatum have the following molecular structure formula:

[0026]

[0027] The preparation method is as follows:

[0028] (1) Preparation of the polar section

[0029] 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.

[0030] (2) Preparation of first group fractions

[0031] 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.

[0032] (3) Preparation of second group fractions

[0033] 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.

[0034] (4) Preparation of third group fractions

[0035] The second group component Fr.G17.15.10 was eluted by ODS column chromatography with a 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 a plurality of components, which were then spotted on a thin layer chromatography (TLC) plate, and the similar components on the thin layer plate were combined to obtain 13 fourth group 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;

[0036] (5) Preparation of the fourth group component

[0037] The third group component Fr.G17.15.10 was eluted by ODS column chromatography with a 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 a plurality of components, which were then spotted on a thin layer chromatography (TLC) plate, and the similar components on the thin layer plate were combined to obtain 13 fourth group 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;

[0038] (6) Preparation of isoprenyl flavonoid compounds (2”S, 2”’S)-epimesatine O and (2”R, 2”’R)-epimesatine O

[0039] The fourth group component Fr.G17.15.10.6 was purified and separated by semi-preparative HPLC using a 10 ID x 250 mm RP C18 liquid chromatography column, with a mobile phase of acetonitrile:water = 60:40, and a flow rate of 2 mL / min, to obtain 7 components Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…G17.15.10.6.7;

[0040] The component Fr.G17.15.10.6.2 is separated again by HPLC purification, the mobile phase is acetonitrile: water = 35:65, the flow rate is 2 mL / min, to obtain 6 components Fr.G17.15.10.6.2.1…Fr.G17.15.10.6.2.4…G17.15.10.6.2.6;

[0041] The component Fr.G17.15.10.6.2.4 is separated again by HPLC purification, the mobile phase is acetonitrile: water = 55:45, the flow rate is 2 mL / min, to obtain 5 components Fr.G17.15.10.6.2.4.1…Fr.G17.15.10.6.2.4.4, Fr.G17.15.10.6.2.4.5,

[0042] The retention time t R The component Fr.G17.15.10.6.2.4.5 with the retention time t63.6 min is dried to obtain compound (2"S,2"'S)-epimesatine O (11.2 mg) directly;

[0043] The component Fr.G17.15.10.6.2.4.4 is separated again by HPLC purification, the mobile phase is acetonitrile: water = 60:40, the flow rate is 2 mL / min, to collect the fraction with the retention time t R 19.3 min, and dry to obtain (2"R,2"'R)-epimesatine O (9.0 mg).

[0044] The application of the above isoprenyl flavone compounds (2"S,2"'S)-epimesatine O and (2"R,2"'R)-epimesatine O in preparing drugs for treating breast cancer.

[0045] According to the method given in the above examples, any amount of target compound can be prepared as required, the examples given are only used to illustrate the specific embodiments of the present application, and are not used to limit the protection scope of the present application, and the technical core protected by the present application is the isoprenyl flavone compound.

[0046] The present application extracts isoprenyl flavone compounds from Epimedium sagittatum, has anti-breast cancer activity, realizes the application in preparing drugs for treating breast cancer, and has very good beneficial technical effects through tests, and the relevant test data are as follows:

[0047] I. Instruments and materials

[0048] 1.1 Instruments:

[0049] High-resolution mass spectrometer Bruker maxis HD (Bruker, Germany)

[0050] UV spectrometer Evolution 300 instrument (Thermo, MA, USA)

[0051] Infrared spectrometer Nicolet IS10 (Thermo Scientific, USA)

[0052] NMR spectrometer Bruker AVANCE III 500 (Bruker, Germany)

[0053] High performance liquid chromatograph Shimadzu LC-40 equipped with DAD detector, RPC18 column (10 ID x 250 mm, Cosmosil 5C18-MS-IIPacked column, nacalai tesque, China)

[0054] Rotary evaporator N-1100 and freeze water circulation device N-1111 (Shanghai Ailang Instrument Co., Ltd.)

[0055] BT25S precision analytical balance (Sartorius)

[0056] Cell real-time label-free system (Agilent Bio)

[0057] CO2 3111 incubator (Thermo)

[0058] Centrifuge-5804R high-speed centrifuge (Eppendorf)

[0059] Multiskan MK3 microplate reader (Thermo Fisher)

[0060] Super-clean workbench (Sujing Group)

[0061] HVA-85 high-pressure sterilization pot (Hirayama)

[0062] 1.2 Materials:

[0063] Reversed-phase silica gel (ODS, 50 pm, YMC Group, Kyoto, Japan)

[0064] Normal-phase silica gel (100-200 and 200-300 mesh, TOSOH Corp., Tokyo, Japan)

[0065] Chromatographically pure methanol, acetonitrile (Tianjin Svyu Fine Chemicals Co., Ltd.)

[0066] Analytical pure methanol, dichloromethane, ethyl acetate, petroleum ether (Beijing Chemical Plant and Tianjin Third Chemical Reagent Factory)

[0067] 16-well plate (Agilent Bio)

[0068] Culture dishes, 96-well culture plates, cell freezing tubes (Corning)

[0069] Fetal bovine serum (Hangzhou Sijiqing Bioengineering Limited Company), DMEM medium (Gibco Invitrogen)

[0070] Thiazole blue MTT (Beijing Solabio Science and Technology Co., Ltd.)

[0071] Ampicillin, streptomycin (Sigma)

[0072] Dimethyl sulfoxide DMSO (Shanghai Maikelin Biological Technology Co., Ltd.)

[0073] Ham's F12K medium (Pronova Biomedic International AB)

[0074] Docetaxel (Shanghai Yuanye Bio-Technology Co., Ltd.)

[0075] Human normal breast cells MCF-10A, human breast cancer cells MCF-7 (Shanghai Cell Bank of Chinese Academy of Sciences)

[0076] Plant material: The 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, and the preservation number is 20200960

[0077] Test drug: The isoprenyl flavonoid compounds (2”S, 2”'S)-epimesatine O and (2”R, 2”'R)-epimesatine O of the present application.

[0078] II. Structure identification

[0079] By comparing nuclear magnetic resonance hydrogen spectrum, optical rotation value and CD effect, the compounds (2”S, 2”'S)-epimesatine O (1a) and (2”R, 2”'R)-epimesatine O (1b) are enantiomers. UV (MeOH) λ max (logε): 202 (4.50), 244 (4.15), 268 (4.08), 341 (4.26) nm; IR (ν max ): 3368, 2975, 1655, 1614, 1475, 1439, 1362, 1166, 1044, 841 cm -1 The structural formula is:

[0080]

[0081] (2"S,2"'S)-epimesatine O (1a), yellow amorphous powder; [a] 20 D -11 (c 0.4, MeOH); HRESIMS m / z 461.1570 [M+Na] + (calcd. for C 25 H 26 O7Na, 461.1571).

[0082] (2"R,2"'R)-epimesatine O (1b), yellow amorphous powder; [a] 20 D +16 (c 0.5, MeOH); HRESIMS m / z 461.1568 [M+Na] + (calcd. for C 25 H 26 O7Na, 461.1571). 1 H and 13 C NMR data are shown in Table 6.

[0083] Table 1 Compound 1a / 1b 1 H (500 MHz) and 13 C (125 MHz) NMR data (deuterated reagent: Acetone-d6; δ units: ppm; J units: Hz)

[0084]

[0085] III. Activity test

[0086] 3.1 Cell culture

[0087] The frozen MCF-10A cells and MCF-7 cells were thawed in a 37°C water bath until ice and water coexisted, and then immediately centrifuged (1000 rpm, 5 min). After discarding the supernatant, the cells were transferred to a culture dish containing DMEM medium with 10% FBS (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 subcultured. Fresh medium was replaced every 24 h.

[0088] 3.2 Detection of the effect of monomeric compounds on the activity of two kinds of cells based on MTT method

[0089] The cells were cultured in a 37°C, 5% CO2 incubator to the logarithmic growth phase, and the cell density was 2x10 4Cells 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.

[0090] 3.3 Immunofluorescence detection of Sphk1 levels in MCF-7 cells

[0091] MCF-7 cells were cultured at a rate of 2 × 10 4 Cells were seeded at a density of 10 μM per mL in the wells. After 24 hours, cells were divided into normal (CON) and various treatment groups (10 μM) and cultured for another 24 hours. The cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.25% Triton X-100 for 10 minutes. Cells were then blocked with 1% BSA for 30 minutes, followed by the addition of the primary antibody Sphk1 and incubation at 4°C overnight. Cells were washed three times with PBST, counterstained with DAPI for 4 minutes, and washed once with PBS. Afterwards, cells were imaged using the OperettaCLS High-Content Imaging System.

[0092] 3.4 Statistical analysis

[0093] 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.

[0094] 3.5 Activity evaluation

[0095] This experiment used the MTT assay to evaluate the effects of the compounds on MCF-7 human breast cancer cells and MCF-10A human normal breast cells. The results, shown in Table 2, show that treatment with 10 μM of compound 1a / 1b significantly reduced MCF-7 cell viability compared to the blank control group (P < 0.01). Compound 1a / 1b had no significant effect on MCF-10A cell viability. Therefore, compounds 1a / 1b significantly inhibited MCF-7 human breast cancer cell viability at a concentration of 10 μM and had no effect on MCF-10A normal breast cells, suggesting that these two compounds may be the pharmacological agents in Epimedium sagittatum that exert anti-breast cancer activity.

[0096] Table 2 Effects of compounds at 10 μM concentration on the viability of MCF-7 and MCF-10A cells

[0097]

[0098] ** P < 0.01 # Docetaxel is a positive drug

[0099] In addition, the level of Sphk1 in MCF-7 cells after treatment with compound 1a / 1b was detected by cell immunofluorescence, and the results are shown in Table 3. According to the experimental results, it can be found that the fluorescence intensity of Sphk1 in MCF-7 cells after treatment with compound monomers has significant difference compared with the blank group (P < 0.01), that is, compounds 1a / 1b can significantly inhibit the expression level of Sphk1 in MCF-7 cells. It is indicated that these compounds may play an anti-breast cancer activity by inhibiting the expression level of Sphk1 in MCF-7 cells.

[0100] Table 3 Effect of compounds on the level of Sphk1 in MCF-7 cells n = 3

[0101]

[0102] ** P < 0.01

[0103] In summary, one new isoprenylated flavonoid was found from the dichloromethane extract of Epimedium sagittatum. Activity evaluation found that two compounds can significantly reduce the cell viability of human breast cancer cells MCF-7, and have no toxicity to human normal breast cells MCF-10A, suggesting that these compounds may be the pharmacodynamic substances of Epimedium sagittatum to play the anti-breast cancer activity. In addition, two compounds have significant inhibitory effect on Sphk1 in MCF-7 cells, suggesting that the role of these two compounds in anti-breast cancer may be related to the inhibition of Sphk1 expression level in cells. These compounds are expected to become lead compounds for anti-breast cancer, providing technical basis for the development of anti-breast cancer drugs, opening up the medicinal value and economic value of Epimedium sagittatum, and making innovation in the treatment of breast cancer drugs, which has great economic and social benefits.

Claims

1. Prenylated flavonoids extracted from Epimedium sagittatum S ,2''' S )-epimesatine O (1a) and (2'' R ,2''' R )-epimesatine O (1b), the molecular structure is: ； Its 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 100.0 g of the 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 1 L at a time, and the solvent was recovered under reduced pressure to obtain multiple fractions. All fractions were then subjected to thin layer chromatography (TLC) on a spot plate, 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 65.0 g of the first group fraction 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). 500 mL of the eluates were combined 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 fractions Fr. G17.1, Fr. G17.2, Fr. G17.3, ..., Fr. G17.15, ..., Fr. G17.

19. (4) Preparation of the third group of components 38.9 g of the second group component Fr. G17.15 was subjected to gradient elution 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 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 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 the fourth group of components The third 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). 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 fourth group components, namely Fr. G17.15.10.1, Fr. G17.15.10.2, Fr. G17.15.10.3, ..., Fr. G17.15.10.6, ..., G17.15.10.

13. (6) Preparation of isopentenyl flavonoid compounds (2'' S ,2''' S )-epimesatine O and (2'' R ,2''' R )-epimesatine O The fourth group, Fr.G17.15.10.6, was purified and separated by semi-preparative HPLC using a 10 ID × 250 mm RP C18 liquid chromatography column with a mobile phase of acetonitrile:water = 60:40 at a flow rate of 2 mL / min to obtain seven components: Fr.G17.15.10.6.1, Fr.G17.15.10.6.2…G17.15.10.6.7; Component Fr.G17.15.10.6.2 was purified and separated again by HPLC with a mobile phase of acetonitrile:water = 35:65 at a flow rate of 2 mL / min to obtain 6 components Fr.G17.15.10.6.2.1…Fr. G17.15.10.6.2.4…G17.15.10.6.2.6; Component Fr.G17.15.10.6.2.4 was purified and separated again by HPLC with a mobile phase of acetonitrile:water = 55:45 at a flow rate of 2 mL / min to obtain five components: Fr. G17.15.10.6.2.4.1, Fr.G17.15.10.6.2.4.4, and Fr.G17.15.10.6.2.4.

5. Among them, retention time t R The component Fr.G17.15.10.6.2.4.5 of 63.6 min was dried to obtain compound (2'' S ,2''' S )-epimesatine O 11.2 mg; The component Fr. G17.15.10.6.2.4.4 was further purified by HPLC with a mobile phase of acetonitrile:water = 60:40 at a flow rate of 2 mL / min. The retention time was collected. t R The fraction of 19.3 min was dried to give (2'' R ,2''' R )-epimesatine O 9.0 mg.

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