Isoflavone compound, preparation method thereof and application in pharmaceutical manufacturing

Abstract

The present invention relates to the field of biomedicine technology, and more particularly to an isoflavone compound, a preparation method thereof, and its application in pharmaceutical manufacturing. The isoflavone compound having a structure shown in Formula I provided by the present invention has a novel structure and a significant inhibitory effect on the production of nitric oxide (NO). As shown in the test results of the examples, the isoflavone compound provided by the present invention has a higher inhibition rate on NO production than the positive control drug N-monomethyl-L-arginine monoacetate at the same concentration. The IC value of the isoflavone compound for inhibiting NO production is 2.34. 50 The value is 40.52±1.06μM. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an isoflavone compound, a preparation method thereof, and application thereof in pharmaceutical manufacturing, a pharmaceutically acceptable salt of the isoflavone compound, and application thereof in pharmaceutical manufacturing, and a pharmaceutical composition. Background Art

[0002] Inflammation is a common and frequently occurring disease that threatens human health. The primary function of anti-inflammatory drugs is to inhibit inflammatory responses and alleviate inflammatory symptoms. Based on their mechanisms of action, anti-inflammatory drugs can be divided into non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), and immunosuppressants. A large number of plant-derived secondary metabolites, such as aescin, glycyrrhizin, puerarin, moringin, danzhi acid A, and corosolic acid, have been found to possess significant anti-inflammatory effects and demonstrate promising therapeutic efficacy against various acute and chronic inflammatory conditions.

[0003] Agriophyllum squarrosum, an annual herbaceous plant in the Chenopodiaceae family, is a Mongolian medicinal herb with heat-clearing, detoxifying, and diuretic properties. It is used to treat plague, headaches, conjunctivitis, jaundice, kidney heat, urethral burning, stomach ulcers, oral and tongue sores, and tooth ulcers. However, there are currently no reports on Agriophyllum squarrosum isoflavones with anti-inflammatory properties. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an isoflavone compound, a preparation method thereof, and use thereof in pharmaceutical preparation, a pharmaceutically acceptable salt of an isoflavone compound, and use thereof in pharmaceutical preparation, and a pharmaceutical composition. The isoflavone compound provided by the present invention has excellent anti-inflammatory effects.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an isoflavone compound having a structure shown in Formula I:

[0007]

[0008] The present invention provides a method for preparing the isoflavone compounds described in the above technical solution, comprising the following steps:

[0009] Mixing the safflower and a lower alcohol aqueous solution, performing extraction to obtain an alcohol extract; wherein the volume fraction of the lower alcohol in the lower alcohol aqueous solution is 50 to 100%;

[0010] The alcohol extract was subjected to a first silica gel column chromatography to obtain 25 fractions, and the 11th fraction was collected and recorded as Fr.11; the eluent used in the first silica gel column chromatography included a petroleum ether-ethyl acetate solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solvent was 100:0 to 0:100;

[0011] Fr.11 was subjected to a second silica gel column chromatography to obtain 8 fractions, and the sixth fraction was collected and recorded as Fr.11.6; the eluent used in the second silica gel column chromatography included a first methanol-dichloromethane solvent, wherein the volume ratio of dichloromethane to methanol in the first methanol-dichloromethane solvent was 100:0 to 98:2;

[0012] Fr.11.6 was subjected to gel column chromatography to obtain four fractions, and the first fraction was collected and recorded as Fr.11.6.1; the eluent used in the gel column chromatography included a dichloromethane-methanol mixed solvent, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent was 1:4 to 4:1;

[0013] The Fr.11.6.1 is crystallized to obtain a racemic mixture;

[0014] The racemic mixture is subjected to chiral preparative chromatographic column separation to obtain the isoflavone compound; the eluent used in the chiral preparative chromatographic column separation includes a n-hexane-ethanol mixed solvent, and the volume ratio of n-hexane to ethanol in the n-hexane-ethanol mixed solvent is 50:50 to 95:5.

[0015] Preferably, the ratio of the dry weight of the Sempervivum sempervivum to the volume of the lower alcohol aqueous solution for single extraction is 1 kg: 2 to 20 L;

[0016] The lower alcohol includes methanol and / or ethanol;

[0017] The extraction temperature is 15-40° C., the number of extractions is 2-4 times, and the time for a single extraction is 24-120 hours.

[0018] Preferably, the elution mode of the first silica gel column chromatography is gradient elution, and the volume ratios of petroleum ether and ethyl acetate during the gradient elution process are 100:0, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40, 50:50 and 0:100, respectively.

[0019] Preferably, the elution mode of the second silica gel column chromatography is gradient elution, and the volume ratios of dichloromethane and methanol during the gradient elution process are 100:0, 99:1 and 98:2 respectively.

[0020] Preferably, the crystallization solvent used in the crystallization comprises a second methanol-dichloromethane solvent, and the volume ratio of dichloromethane to methanol in the second methanol-dichloromethane solvent is 100:0 to 0:100.

[0021] The present invention provides a pharmaceutically acceptable salt of an isoflavone compound, wherein the isoflavone compound is the isoflavone compound described in the above technical solution or the isoflavone compound prepared by the preparation method described in the above technical solution.

[0022] The present invention provides the use of the isoflavone compounds described in the above technical solution, the isoflavone compounds prepared by the preparation method described in the above technical solution, or the pharmaceutically acceptable salts of the isoflavone compounds described in the above technical solution in the preparation of drugs, wherein the drugs include anti-inflammatory drugs.

[0023] The present invention provides a pharmaceutical composition comprising an active component and a pharmaceutically acceptable excipient, wherein the active component comprises an isoflavone compound and / or a pharmaceutically acceptable salt thereof; the isoflavone compound is the isoflavone compound described in the above technical solution or the isoflavone compound prepared by the preparation method described in the above technical solution.

[0024] Preferably, the content of the active ingredient in the pharmaceutical composition is 0.1 to 99 wt%.

[0025] The isoflavone compounds provided by the present invention have a novel structure and have a significant inhibitory effect on the production of nitric oxide (NO). As shown in the test results of the examples, the isoflavone compounds provided by the present invention have a higher inhibition rate on NO production than the positive control drug N-monomethyl-L-arginine monoacetate at the same concentration. The IC value of the isoflavone compounds for inhibiting NO production is 2.34. 50 The value was 40.52±1.06μM.

[0026] The preparation method of isoflavone compounds provided by the present invention has simple process, simple operation, low production cost, is green and environmentally friendly, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Graph showing the experimental and calculated ECD values ​​of isoflavone compounds. DETAILED DESCRIPTION

[0028] The present invention provides an isoflavone compound having a structure shown in Formula I:

[0029]

[0030] The present invention provides a method for preparing the isoflavone compounds described in the above technical solution, comprising the following steps:

[0031] Mixing the safflower and a lower alcohol aqueous solution, performing extraction to obtain an alcohol extract; wherein the volume fraction of the lower alcohol in the lower alcohol aqueous solution is 50 to 100%;

[0032] The alcohol extract was subjected to a first silica gel column chromatography to obtain 25 fractions, and the 11th fraction was collected and recorded as Fr.11; the eluent used in the first silica gel column chromatography included a petroleum ether-ethyl acetate solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solvent was 100:0 to 0:100;

[0033] Fr.11 was subjected to a second silica gel column chromatography to obtain 8 fractions, and the sixth fraction was collected and recorded as Fr.11.6; the eluent used in the second silica gel column chromatography included a first methanol-dichloromethane solvent, wherein the volume ratio of dichloromethane to methanol in the first methanol-dichloromethane solvent was 100:0 to 98:2;

[0034] Fr.11.6 was subjected to gel column chromatography to obtain four fractions, and the first fraction was collected and recorded as Fr.11.6.1; the eluent used in the gel column chromatography included a dichloromethane-methanol mixed solvent, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent was 1:4 to 4:1;

[0035] The Fr.11.6.1 is crystallized to obtain a racemic mixture;

[0036] The racemic mixture is subjected to chiral preparative chromatographic column separation to obtain the isoflavone compound; the eluent used in the chiral preparative chromatographic column separation includes a n-hexane-ethanol mixed solvent, and the volume ratio of n-hexane to ethanol in the n-hexane-ethanol mixed solvent is 50:50 to 95:5.

[0037] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0038] The present invention mixes S. sabdariffa with a lower alcohol aqueous solution and extracts the mixture to obtain an alcohol extract. In the present invention, the volume fraction of the lower alcohol in the lower alcohol aqueous solution is preferably 50-100%, more preferably 60-90%, and even more preferably 70-80%; the lower alcohol comprises methanol and / or ethanol. In the present invention, the ratio of the dry weight of S. sabdariffa to the volume of the lower alcohol aqueous solution used for a single extraction is preferably 1 kg:2-20 L, more preferably 1 kg:5-15 L, and even more preferably 1 kg:5-10 L. In the present invention, the extraction temperature is preferably 15-40°C, more preferably 20-30°C; the number of extractions is preferably 2-4, more preferably 3; and the duration of a single extraction is preferably 24-120 hours, more preferably 30-100 hours, and even more preferably 40-50 hours.

[0039] After the extraction is completed, the present invention preferably further comprises: combining the extracts and concentrating to obtain an ethanol extract. The present invention is not particularly limited to the concentration, and a concentration method well known to those skilled in the art can be used until the volume no longer changes, specifically, concentration under reduced pressure.

[0040] After obtaining the alcohol extract, the present invention performs a first silica gel column chromatography on the alcohol extract to obtain 25 components, and collects the 11th component, which is recorded as Fr.11; the eluent used in the first silica gel column chromatography includes a petroleum ether-ethyl acetate solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solvent is 100:0 to 0:100; the elution mode of the first silica gel column chromatography is preferably gradient elution, and the volume ratio of petroleum ether to ethyl acetate during the gradient elution process is preferably 100:0, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40, 50:50 and 0:100 in sequence; the particle size of the silica gel column chromatography filler used in the first silica gel column chromatography is preferably 200 to 300 meshes.

[0041] After obtaining Fr.11, the present invention subjects Fr.11 to a second silica gel column chromatography to obtain 8 components, and collects the 6th component, which is recorded as Fr.11.6; the eluent used in the second silica gel column chromatography includes a first methanol-dichloromethane solvent, and the volume ratio of dichloromethane and methanol in the first methanol-dichloromethane solvent is 100:0 to 98:2; the elution mode of the second silica gel column chromatography is preferably gradient elution, and the volume ratio of dichloromethane and methanol during the gradient elution process is preferably 100:0, 99:1 and 98:2, respectively; the particle size of the silica gel column chromatography filler used in the second silica gel column chromatography is preferably 200 to 300 mesh.

[0042] After obtaining Fr.11.6, the present invention performs gel column chromatography on the Fr.11.6 to obtain 4 components, and collects the first component, which is recorded as Fr.11.6.1; the eluent used in the gel column chromatography includes a dichloromethane-methanol mixed solvent, and the volume ratio of dichloromethane and methanol in the dichloromethane-methanol mixed solvent is 1:4 to 4:1, preferably 1:2 to 2:1, and more preferably 1:1; the elution mode of the gel column chromatography is preferably isocratic elution.

[0043] After obtaining Fr.11.6.1, the present invention crystallizes the Fr.11.6.1 to obtain a racemic mixture. In the present invention, the crystallization solvent used in the crystallization preferably includes a second methanol-dichloromethane solvent, and the volume ratio of dichloromethane to methanol in the second methanol-dichloromethane solvent is preferably 100:0 to 0:100, more preferably 30:70 to 70:30, further preferably 40:60 to 60:40, and most preferably 50:50 (i.e., 1:1). In the present invention, the crystallization is preferably natural crystallization (i.e., volatile crystallization).

[0044] After the crystallization is completed, the present invention preferably further comprises washing the resulting crystalline product with water and then drying it to obtain a racemic mixture. In the present invention, the number of water washings is preferably 3 to 4 times. The present invention does not particularly limit the drying conditions; drying to a constant weight is sufficient.

[0045] After obtaining the racemic mixture, the present invention performs chiral preparative chromatography on the racemic mixture to obtain the isoflavone compounds; the eluent used in the chiral preparative chromatography includes a n-hexane-ethanol mixed solvent, and the volume ratio of n-hexane to ethanol in the n-hexane-ethanol mixed solvent is 50:50 to 95:5, preferably 70:30 to 90:10, more preferably 80:20 to 85:15, and most preferably 84:16. The elution mode of the chiral preparative chromatography is preferably isocratic elution.

[0046] The present invention provides a pharmaceutically acceptable salt of an isoflavone compound, wherein the isoflavone compound is the isoflavone compound described in the above technical solution or the isoflavone compound produced by the preparation method described in the above technical solution. In the present invention, the pharmaceutically acceptable salt preferably comprises a sodium salt or a potassium salt, preferably obtained by reacting the isoflavone compound with sodium hydroxide or potassium hydroxide, with the salt-forming site being a phenolic hydroxyl group. The present invention does not particularly limit the preparation conditions of the pharmaceutically acceptable salt of the isoflavone compound, and preparation conditions familiar to those skilled in the art can be employed.

[0047] The present invention provides the use of the isoflavone compounds described in the above technical solution, the isoflavone compounds prepared by the preparation method described in the above technical solution, or the pharmaceutically acceptable salts of the isoflavone compounds described in the above technical solution in the preparation of drugs, wherein the drugs include anti-inflammatory drugs.

[0048] The present invention provides a pharmaceutical composition comprising an active component and a pharmaceutically acceptable excipient, wherein the active component comprises an isoflavone compound and / or a pharmaceutically acceptable salt thereof; the isoflavone compound is the isoflavone compound described in the above technical solution or the isoflavone compound prepared by the preparation method described in the above technical solution.

[0049] In the present invention, the content of the active ingredient in the pharmaceutical composition is preferably 0.1 to 99 wt%, more preferably 0.5 to 90 wt%, and further preferably 5 to 50 wt%.

[0050] In the present invention, the pharmaceutical composition preferably includes an oral agent or an injection. In the present invention, the oral agent preferably includes a powder, tablet, capsule, pill, or solution, wherein the tablet includes a sugar-coated tablet; the solution includes a syrup; and the injection preferably includes a powder injection or a solution injection.

[0051] In the present invention, the pharmaceutically acceptable excipient preferably includes a pharmaceutical carrier or excipient, and the pharmaceutically acceptable excipient is non-toxic to humans and animals and is an inert excipient; the pharmaceutically acceptable excipient includes one or more of liquid excipients, solid excipients and semi-solid excipients, specifically one or more of lactose, starch, magnesium stearate and sodium chloride solution.

[0052] In the present invention, the pharmaceutical composition is preferably used in the form of a dosage per unit body weight, and the administration method is preferably oral administration and / or injection, and the injection preferably includes intravenous injection or intramuscular injection.

[0053] To further illustrate the present invention, the following detailed description of isoflavone compounds, their preparation methods and applications in pharmaceutical manufacturing, pharmaceutically acceptable salts of isoflavone compounds and their applications, and pharmaceutical compositions is provided in conjunction with the following examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] S1: 31 kg of the dried aerial parts of Salicornia herba were soaked and extracted with methanol (single dosage: 60 L) at room temperature for 72 h. The extracts were combined and concentrated under reduced pressure at 40°C until the volume no longer changed, to obtain 1.72 kg of alcohol-extracted extract.

[0056] S2: The alcohol extract was subjected to silica gel column chromatography (silica gel 200-300 mesh) with petroleum ether-ethyl acetate solvents of volume ratios of 100:0, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40, 50:50, and 0:100, respectively, to obtain 25 fractions. The 11th fraction was collected and recorded as Fr.11.

[0057] S3: gradient elution of Fr.11 by silica gel column chromatography using a dichloromethane-methanol mixed solvent with a volume ratio of 100:1, 99:1, and 98:2, to obtain 8 fractions. The sixth fraction was collected and recorded as Fr.11.6.

[0058] S4: Fr.11.6 was subjected to gel column chromatography using a 1:1 volume ratio of dichloromethane / methanol mixed solvent to obtain four fractions, and the first fraction was collected and recorded as Fr.11.6.1;

[0059] S5: The Fr.11.6.1 is volatilized and crystallized with a dichloromethane-methanol mixed solvent in a volume ratio of 1:1 to obtain a solid product, which is washed with water 3 to 4 times to obtain a racemic mixture.

[0060] S6: The racemic mixture collected in S5 can be separated by chiral preparative chromatography using a mixed solvent of n-hexane and ethanol in a volume ratio of 84:16 to obtain an isoflavone compound represented by Formula I (denoted as (+)-AS12).

[0061] Among them, the number of separations by silica gel column chromatography, gel column chromatography, crystallization and chiral preparative chromatography column was once.

[0062] Structural Analysis of Isoflavone Compound (+)-AS12

[0063] The (+)-AS12 white powder product prepared in Example 1 was subjected to mass spectrometry, ultraviolet, infrared analysis, and ECD calculation analysis. The results are as follows:

[0064] Molecular formula C 18 H 18 O6; ESIMS (pos.): m / z 353[M+Na] + ;HRESIMS: m / z 353.0993[M+Na] + (353.0996calcd for C 18 H 18 O6Na); (c0.10,MeOH); UV(MeOH)λ max :219,270(sh)nm;IR(KBr)ν max :2924,2868,1757,1739,1693,1642,1509,1307,1234,1160,1094,1006,949cm -1 ; NMR spectroscopy data are shown in Table 1.

[0065] Table 1 Nuclear magnetic resonance spectrum data (CDCl3)

[0066]

[0067] The electronic circular dichroism (ECD) data of (+)-AS12 were calculated using quantum chemical calculation methods, and the calculated curves were compared with the experimental curves ( Figure 1) to determine the absolute configuration of AS12. The results showed that the ECD calculated spectrum of (S)-AS12 was consistent with the cotton effect of the experimental spectrum, confirming that the absolute configuration of compound (+)-AS12 was S.

[0068] Combining mass spectrometry, ultraviolet and infrared analysis, and ECD calculation analysis, it can be known that the (+)-AS12 prepared by the present invention has the following structure:

[0069]

[0070] Test Example 1

[0071] The obtained compound (+)-AS12 has an inhibitory effect on NO

[0072] Mouse mononuclear macrophage RAW264.7 cells were treated with lipopolysaccharide (LPS) to induce the production of nitric oxide synthase, and the test compound was added at the same time. The culture medium was aspirated and the absorbance at 570 nm was measured by the Griess method to detect nitrite (NO2 - ).

[0073] Experimental Methods (Primary Screening): RAW264.7 cells were seeded into 96-well plates and stimulated with 1 μg / mL LPS. The test compound (+)-AS12 (final concentration 50 μM) was then added. A drug-free group and a positive control group treated with N-monomethyl-L-arginine monoacetate (L-NMMA) were set up as controls. After overnight incubation, the culture medium was collected to measure NO production by measuring absorbance at 570 nm. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added to the remaining culture medium to assess cell viability and rule out any cytotoxic effects of the compound. This experiment evaluated the inhibitory activity of (+)-AS12 against nitric oxide (NO) production. The inhibitory percentage at a 50 μM concentration is shown in Table 2.

[0074] NO production inhibition rate (%) = (OD 570nm - Sample group OD 570nm ) / OD of non-drug treated group 570nm ×100%

[0075] Table 2 Inhibition rate of NO generation

[0076] sample Atlas No. Concentration (μM) NO generation inhibition rate (%) L-NMMA Positive control 50 52.01±1.96 (+)-AS12 (+)-AS12 50 58.58±0.85

[0077] Experimental method (rescreening): RAW264.7 cells were seeded into 96-well plates and stimulated with 1 μg / mL LPS. The test compound (2-fold dilution starting from a final concentration of 50 μM) was added simultaneously. A drug-free group and an L-NMMA-positive drug group were set up as controls. After overnight cell culture, the culture medium was collected to detect NO production and the absorbance was measured at 570 nm. MTS was added to the remaining culture medium to determine cell viability and exclude the toxic effects of the compound on the cells. IC 50 (50% Concentration of Inhibition) was calculated according to the Reed & Muench method. The experimental results are shown in Table 3.

[0078] Table 3 IC of samples for inhibition of NO generation 50 value

[0079] Compound <![CDATA[IC 50 (μM)]]> (+)-AS12 40.52±1.06

[0080] As shown in Table 3, (+)-AS12 inhibits NO production with IC 50 The value was 40.52±1.06μM.

[0081] Example 2

[0082] The prepared compound (+)-AS12 is made into a usable medicine. According to different medication habits, the compound (+)-AS12 can be made into powder, tablets, sugar-coated tablets, capsules, solutions, syrups or pills.

[0083] (1) Tablets

[0084] Formula: Compound (+)-AS12 10 mg, lactose 180 mg, starch 55 mg, magnesium stearate 5 mg;

[0085] Preparation method: Compound (+)-AS12, lactose and starch are mixed and evenly moistened with water. The moistened mixture is sieved and dried. After sieving, magnesium stearate is added, and the mixture is compressed into tablets. Each tablet weighs 250 mg and contains 10 mg of compound (+)-AS12.

[0086] (2) Capsules

[0087] Formulation: Compound (+)-AS12 100 mg, starch 100 mg, magnesium stearate capsule shell;

[0088] Preparation: The compound is mixed with starch, sieved and the resulting mixture is filled into hard gelatin capsules.

[0089] (3) Ampoules

[0090] Formula: Compound (+)-AS12 2 mg, sodium chloride 10 mg, water for injection 100 mL;

[0091] Preparation method: Dissolve compound (+)-AS12 and sodium chloride in an appropriate amount of water for injection, filter, and fill the resulting mixed solution into an ampoule under sterile conditions and seal it.

[0092] Although the above embodiments provide a detailed description of the present invention, they are only part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing isoflavone compounds, characterized in that: The isoflavone compound has a structure shown in Formula I: The preparation method comprises the following steps: Mixing the safflower and a lower alcohol aqueous solution, performing extraction to obtain an alcohol extract; wherein the volume fraction of the lower alcohol in the lower alcohol aqueous solution is 50 to 100%; The alcohol extract was subjected to a first silica gel column chromatography to obtain 25 fractions, and the 11th fraction was collected and recorded as Fr.11; the eluent used in the first silica gel column chromatography was a petroleum ether-ethyl acetate solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solvent was 100:0 to 0:100; Fr.11 was subjected to a second silica gel column chromatography to obtain 8 fractions, and the sixth fraction was collected and recorded as Fr.11.6; the eluent used in the second silica gel column chromatography was a first methanol-dichloromethane solvent, and the volume ratio of dichloromethane to methanol in the first methanol-dichloromethane solvent was 100:0 to 98:2; Fr.11.6 was subjected to gel column chromatography to obtain four fractions, and the first fraction was collected and recorded as Fr.11.6.1; the eluent used in the gel column chromatography was a dichloromethane-methanol mixed solvent, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solvent was 1:4 to 4:1; The Fr.11.6.1 is crystallized to obtain a racemic mixture; The racemic mixture is subjected to chiral preparative chromatographic column separation to obtain the isoflavone compounds; the eluent used in the chiral preparative chromatographic column separation is a n-hexane-ethanol mixed solvent, and the volume ratio of n-hexane to ethanol in the n-hexane-ethanol mixed solvent is 50:50 to 95:

5.

2. The preparation method according to claim 1, characterized in that The ratio of the dry weight of the said S. sempervirens to the volume of the lower alcohol aqueous solution used for a single extraction is 1 kg: 2 to 20 L; The lower alcohol is methanol and / or ethanol; The extraction temperature is 15-40° C., the number of extractions is 2-4 times, and the time for a single extraction is 24-120 hours.

3. The preparation method according to claim 1, characterized in that The elution mode of the first silica gel column chromatography is gradient elution, and the volume ratios of petroleum ether and ethyl acetate during the gradient elution process are 100:0, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40, 50:50 and 0:100, respectively.

4. The preparation method according to claim 1, characterized in that The elution mode of the second silica gel column chromatography is gradient elution, and the volume ratios of dichloromethane and methanol during the gradient elution process are 100:0, 99:1 and 98:2 respectively.

5. The preparation method according to claim 1, characterized in that The crystallization solvent used in the crystallization is a second methanol-dichloromethane solvent, and the volume ratio of dichloromethane to methanol in the second methanol-dichloromethane solvent is 100:0 to 0:100.

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