A phenolic acid compound Retusiusines H, its preparation method and application

Abstract

The present invention provides a novel phenolic acid compound, Retusiusines H. The present invention also provides the use of the phenolic acid compound Retusiusines H in the preparation of a drug for treating and / or preventing melanoma. The phenolic acid compound can inhibit human cutaneous melanoma cells with significant activity at a low concentration, and the IC 50 value is 9.59 μmol / L. Thus, the phenolic acid compound has great application potential in the development of anti-melanoma drugs. The present invention also provides a preparation method of the phenolic acid compound Retusiusines H, which can rapidly, accurately and efficiently prepare the compound from Dendrobium huoshanense

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial activity research, and particularly relates to a phenolic acid compound Retusiusines H, a preparation method thereof, and an application thereof. Background Art

[0002] Melanoma is a skin cancer transformed from malignant transformation of melanocytes, which is the most metastatic and dangerous. It accounts for 90% of all skin cancer deaths. Approximately 90% of melanoma deaths are attributed to metastasis, and the median progression-free survival of stage IV melanoma patients after metastasis is only 1.7 months. Currently, the World Health Organization estimates that the mortality rate of newly diagnosed melanoma patients can reach 30% annually, seriously threatening human health.

[0003] Currently, the main chemotherapy drugs for inhibiting melanoma metastasis clinically are temozolomide, dacarbazine, and sorafenib. However, the existing chemotherapy drugs have an objective response rate of only 10% - 15% for advanced melanoma, and there are problems such as severe drug resistance and toxic side effects. Therefore, finding new low-toxic and highly effective therapeutic drugs has become a major need to achieve the strategic goal of a healthy China.

[0004] Natural products have shown unique curative effects in the treatment of various diseases, and have the characteristics of reducing toxicity and increasing efficacy, low cost, and low circulation. A large number of new structures with significant anti-cancer effects have emerged continuously in the research and development of anti-cancer drugs from natural products, such as the taxol amide derivative AVE8062. From January 1981 to September 2019, a total of 185 small molecule anti-tumor new drugs were approved for marketing worldwide, and a total of 156 drugs related to natural products (accounting for 84%). Therefore, finding new anti-tumor drugs from natural products is a research hotspot in this field. Summary of the Invention

[0005] The main purpose of the present invention is to provide a phenolic acid compound Retusiusines H, a preparation method thereof, and an application thereof in anti-melanoma drugs. The phenolic acid compound Retusiusines H can effectively inhibit the growth of human skin melanoma A2058 at a low concentration and has great application potential.

[0006] In the first aspect, the present invention provides a phenolic acid compound Retusiusines H represented by formula (I).

[0007]

[0008] In the second aspect, the present invention provides an application of the phenolic acid compound Retusiusines H represented by the above formula (I) in the preparation of drugs for treating and / or preventing melanoma.

[0009] In some embodiments, the melanoma is A2058 human cutaneous melanoma.

[0010] In a third aspect, the present invention provides a method for preparing the phenolic acid compound Retusiusines H shown in the above formula (I), and the preparation method comprises the following steps:

[0011] Step (1): Take Dendrobium huoshanense, extract to obtain an extract, and concentrate it under reduced pressure to obtain a Dendrobium huoshanense extract.

[0012] Step (2): Dissolve the extract obtained in step (1) in water, extract with ethyl acetate, take the ethyl acetate phase and concentrate it under reduced pressure to obtain a Dendrobium huoshanense extract paste.

[0013] Step (3): Separate the extract paste obtained in step (2) by normal-phase silica gel column chromatography, elute with a gradient, collect the gradient eluates of each gradient and concentrate them. Monitor by TLC, combine the same parts to obtain 7 components Fr.1 - Fr.7.

[0014] Step (4): Separate the component Fr.2 obtained in step (3) by normal-phase silica gel column chromatography, elute with a gradient, collect the gradient eluates of each gradient and concentrate them. Monitor by TLC, combine the same parts to obtain 6 sub-components 2A1 - 2A6.

[0015] Step (5): Separate the sub-component 2A2 obtained in step (4) by semi-preparative high performance liquid chromatography to obtain the phenolic acid compound Retusiusines H shown in formula (I).

[0016] In some embodiments, in step (1), the extraction method of Dendrobium huoshanense is maceration extraction, ultrasonic extraction, reflux extraction, etc., preferably maceration extraction;

[0017] And / or, the extraction solvent is ethanol;

[0018] And / or, the extraction time is 5 - 9 days, such as 7 days;

[0019] And / or, the extraction times are 1 - 5 times, such as 3 times.

[0020] In some embodiments, step (1) is specifically: Take fresh Dendrobium huoshanense samples, remove the leaves and keep the stems, crush them with a crusher, macerate and extract 3 times with 20 L of anhydrous ethanol at room temperature for 7 days each time, then filter to obtain the filtrate and concentrate it under reduced pressure to obtain a Dendrobium huoshanense extract.

[0021] In some embodiments, the preparation method comprises the following steps:

[0022] Step (1): Take fresh samples of Dendrobium huoshanense, remove the leaves and keep the stems, crush them with a pulverizer, and extract them with 20 L of absolute ethanol at room temperature for 3 times, 7 days each time. Then filter to obtain the filtrate, and concentrate it under reduced pressure to obtain the Dendrobium huoshanense extract.

[0023] Step (2): Dissolve the extract obtained in step (1) in water, extract it with ethyl acetate, take the ethyl acetate phase and concentrate it under reduced pressure to obtain an extract paste.

[0024] Step (3): Load the extract paste obtained in step (2) onto a column by dry loading method, separate it by normal-phase silica gel column chromatography, then perform gradient elution, collect the gradient eluates of each gradient and concentrate them. Monitor by TLC, combine the same parts to obtain 7 fractions Fr.1 - Fr.7.

[0025] Step (4): Load the fraction Fr.2 obtained in step (3) onto a column by dry loading method, separate it by normal-phase silica gel column chromatography, then perform gradient elution, collect the gradient eluates of each gradient and concentrate them. Monitor by TLC, combine the same parts to obtain 6 sub-fractions 2A1 - 2A6.

[0026] Step (5): Purify the sub-fraction 2A2 obtained in step (4) by semi-preparative high performance liquid chromatography to obtain the phenolic acid compound shown in formula (I).

[0027] In some embodiments, in step (2),

[0028] The volume ratio of ethyl acetate to water is 0.5 - 3:1, for example 1:1;

[0029] and / or, perform the extraction operation by ultrasound, and let it stand overnight after extraction;

[0030] and / or, the number of extraction times is 3 - 5 times.

[0031] In some embodiments, specifically in step (2):

[0032] Dissolve the extract obtained in step (1) in water, add an equal volume of ethyl acetate, ultrasonicate at room temperature for 0.5 - 1.5 h, then let it stand at room temperature for 8 - 12 h, take the supernatant, and concentrate it under reduced pressure; the number of extraction times is 3 - 5 times.

[0033] In some embodiments, during the normal-phase silica gel column chromatography separation in step (3), dry loading method is used.

[0034] In some embodiments, in step (3), the specific method of dry loading the extract paste is: dissolve the extract paste with 10 times the volume of ethyl acetate, adsorb and mix it with silica gel of 80 - 100 mesh with the same mass as the extract paste, and then load it onto the column.

[0035] In some embodiments, in step (3), the silica gel packed in the normal-phase silica gel column is 200-300 mesh normal-phase silica gel.

[0036] In some embodiments, in step (3), the mobile phase used for gradient elution is a mixed solvent of dichloromethane and methanol;

[0037] Preferably, in the mixed solvent of dichloromethane and methanol, the volume ratios of dichloromethane to methanol are 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1, 0:1 in sequence;

[0038] Preferably, after each gradient elution until there are no spots on the TLC plate, the next gradient elution is replaced to obtain a total of 7 components, namely Fr.1-Fr.7.

[0039] In some embodiments, during the normal-phase silica gel column chromatography separation in step (4), dry loading is adopted.

[0040] In some embodiments, in step (4), the specific method of dry loading is as follows: Component Fr.2 is dissolved in methanol with a volume 10 times that of Fr.2, adsorbed and mixed with silica gel of 80-100 mesh with the same mass as component Fr.2, and then loaded onto the column.

[0041] In some embodiments, in step (4), the silica gel packed in the normal-phase silica gel column is 200-300 mesh normal-phase silica gel.

[0042] In some embodiments, in step (4), the mobile phase used for gradient elution is a mixed solvent of petroleum ether and ethyl acetate;

[0043] Preferably, the volume ratios of petroleum ether to ethyl acetate are 100:1, 60:1, 30:1, 10:1, 1:1 in sequence;

[0044] Preferably, after each gradient elution until there are no spots on the TLC plate, the next gradient elution is replaced to obtain a total of 6 sub-components, namely 2A1-2A6.

[0045] In some embodiments, in step (5), gel chromatography purification is first carried out, and then semi-preparative high performance liquid chromatography purification is carried out.

[0046] In some embodiments, in step (5), the gel column used for gel chromatography purification is Sephadex LH-20, and a mixed solvent of dichloromethane and methanol with a volume ratio of 1:1 is used as the mobile phase.

[0047] In some embodiments, during gel chromatography purification in step (5), sub-component 2A2 is dissolved in methanol with a volume 2 times that of 2A2 and then loaded onto the column. After monitoring by TLC, the same parts are combined.

[0048] In some embodiments, in step (5), the chromatographic conditions for semi-preparative high performance liquid chromatography are as follows:

[0049] The mobile phase is a mixed solvent of methanol and water with a volume ratio of 9:1;

[0050] The flow rate is 1 mL / min;

[0051] Collect the components with the starting peak time of 5.6 min;

[0052] Use a C 18 reverse chromatographic column; the specification is preferably: 4.6 mm x 250 mm, 5 μm;

[0053] Column temperature: 30 °C;

[0054] Sample injection volume each time: 20 μl;

[0055] Use an ultraviolet detector with a detection wavelength of 210 nm.

[0056] In some embodiments, during gradient elution, TLC monitoring is used and the same parts are combined.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0058] (1) The present invention provides a novel phenolic acid compound, Retusiusines H (see the attached Figure 1 ) for the chemical structure of the compound.

[0059] (2) The present invention also provides the use of the phenolic acid compound Retusiusines H in the preparation of a drug for treating and / or preventing melanoma. The phenolic acid compound can inhibit human skin melanoma cells at a low concentration and exhibits significant activity, with an IC 50 value of 9.59 μmol / L. Thus, the phenolic acid compound has great application potential in the development of anti-melanoma drugs.

[0060] (3) The present invention also provides a preparation method of the phenolic acid compound Retusiusines H, which can quickly, accurately and efficiently prepare the compound from Dendrobium huoshanense. Brief Description of the Drawings

[0061] Figure 1 is the chemical structural formula of the phenolic acid compound Retusiusines H described in the present invention;

[0062] Figure 2 is the 1 H NMR spectrum of the phenolic acid compound Retusiusines H described in the present invention;

[0063] Figure 3 For the phenolic acid compound Retusiusines H of the present invention 13 13C NMR spectrum. Specific embodiments

[0064] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only illustrative explanations of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0065] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0066] Application Example 1 Preparation of phenolic acid compound Retusiusines H

[0067] The preparation method of the phenolic acid compound Retusiusines H includes the following steps:

[0068] 1) Take 20 kg of fresh Dendrobium huoshanense samples, remove the leaves and keep the stems, crush them with a crusher, soak and extract with 20 L of anhydrous ethanol at room temperature for 3 times, 7 days each time, then filter to obtain the filtrate, and concentrate it under reduced pressure to obtain the Dendrobium huoshanense extract;

[0069] 2) Dissolve the extract obtained in step 1) with water, extract with ethyl acetate, the volume ratio of water to ethyl acetate is 1:1, take the ethyl acetate phase and concentrate it under reduced pressure to obtain 967 g of extract;

[0070] 3) Dissolve the extract obtained in step (2) with ethyl acetate, adsorb and mix it with silica gel, and perform rough fractionation and gradient elution through a normal-phase silica gel column to obtain fractions Fr.1-Fr.7. The stationary phase of the silica gel column chromatography is 200-300 mesh normal-phase silica gel, and the mobile phases are dichloromethane / methanol (100:0 → 0:100) in sequence;

[0071] Specifically: dissolve the extract with 10 times the volume of ethyl acetate, adsorb and mix it with 80-100 mesh silica gel equal in mass to the extract, and then load the sample;

[0072] The mobile phases used for gradient elution are mixed solvents of dichloromethane and methanol in sequence. The volume ratios of dichloromethane and methanol in the mixed solvent of dichloromethane and methanol are 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1, 0:1 in sequence. After each gradient elution until there is no spot on the TLC plate, change to the next gradient elution to obtain a total of 7 fractions Fr.1-Fr.7.

[0073] 4) The fraction Fr.2 was subjected to normal-phase silica gel column chromatography, monitored by TLC, and the same fractions were combined to obtain 6 sub-fractions (2A1 - 2A6). The stationary phase of the silica gel column chromatography was normal-phase silica gel with a mesh size of 200 - 300, and the mobile phase was petroleum ether / ethyl acetate (100:1 → 1:1).

[0074] Specifically: The fraction Fr.2 was dissolved in 10 volumes of methanol, adsorbed and mixed with silica gel of the same mass as Fr.2 with a mesh size of 80 - 100, and then loaded onto the column. The mobile phase for gradient elution was a mixed solvent of petroleum ether and ethyl acetate, and the volume ratios of petroleum ether to ethyl acetate were 100:1, 60:1, 30:1, 10:1, and 1:1 in sequence. After each gradient elution until no spots appeared on the TLC plate, the next gradient elution was carried out, and a total of 6 sub-fractions 2A1 - 2A6 were obtained.

[0075] 5) 2A2 was purified by gel column chromatography Sephadex LH-20 and HPLC. The mobile phase of the gel was dichloromethane / methanol (1:1); the mobile phase of the HPLC was methanol / water (9:1), with a flow rate of 1 mL / min. The fraction with the starting peak time of 5.6 min was collected to obtain compound Retusiusines H (5 mg).

[0076] During gel chromatography purification, the sub-fraction 2A2 was dissolved in 2 volumes of methanol and then loaded onto the column; 10 ml test tubes were used for collection, 5 ml was collected in each tube, and a total of 60 tubes were collected. After monitoring by TLC and combining the same parts, tubes 10 - 20 were combined, concentrated under reduced pressure, and then purified by HPLC.

[0077] The HPLC chromatographic conditions were as follows: the mobile phase was a mixed solvent of methanol and water (volume ratio 9:1), the flow rate was 1 mL / min, the fraction with the starting peak time of 5.6 min was collected, and an Agilent C 18 reverse chromatographic column was used, with a specification of: 4.6 mm x 250 mm, 5 μm; column temperature: 30 °C, injection volume each time: 20 μl, and a UV detector was used with a detection wavelength of 210 nm.

[0078] The structure of the obtained monomeric compound Retusiusines H was determined by NMR spectra, and the properties and spectral data of the obtained compound were as follows:

[0079] The structural formula of the phenolic acid compound Retusiusines H is shown in formula (Ⅰ);

[0080]

[0081] Its English name is Retusiusines H (3-(4-hydroxy-3-methoxyphenyl)propyl 3-phenylpropanoate), which is a colorless oily liquid with the molecular formula C 19 H 22 O4 and a molecular weight of 314.1518.

[0082] 1 1H NMR (CDCl3, 500 MHz) δ H : 6.65 (1H, s, H-2), 6.83 (1H, d, J = 8.39 Hz, H-5), 6.64 ((1H, d, J = 7.00 Hz, H-6), 2.57 (2H, t, J = 7.50 Hz, H-7), 1.90 (2H, m, H-8), 4.09 (2H, t, J = 6.56 Hz, H-9), 3.87 (3H, s, H-10), 7.21 (1H, d, J = 7.72 Hz, H-2′), 7.29 (1H, t, J = 8.16 Hz, H-3′), 7.19 (1H, t, J = 7.89 Hz, H-4′), 7.29 (1H, t, J = 8.16 Hz, H-5′), 7.21 (1H, d, J = 7.72 Hz, H-6′), 2.96 (2H, t, J = 7.78 Hz, H-7′), 2.65 (2H, t, J = 8.10 Hz, H-8′), 5.51 (1H, s, 4-OH).

[0083] 13 13C NMR (CDCl3, 125 MHz) δ C : 133.2 (C-1), 111.0 (C-2), 146.5 (C-3), 144.0 (C-4), 114.4 (C-5), 121.1 (C-6), 31.1 (C-7), 30.6 (C-8), 63.9 (C-9), 56.0 (C-10), 140.6 (C-1′), 128.4 (C-2′), 128.6 (C-3′), 126.4 (C-4′), 128.6 (C-5′), 128.4 (C-6′), 31.9 (C-7′), 36.0 (C-8′), 173.1 (C-9′).

[0084] Application Example 2 Research on the Activity of Phenolic Acid Compound Retusiusines H in Inhibiting Human Melanoma A2058

[0085] 1 Materials

[0086] 1.1 Cells

[0087] Human melanoma cells (A2058) were purchased from the Cell Bank of Chinese Academy of Sciences (Wuhan).

[0088] 1.2 Materials

[0089] Fetal bovine serum was purchased from Hangzhou Sijiqing Biological Products Co., Ltd. DMEM medium, phosphate buffer solution (PBS), penicillin-streptomycin, and 0.25% trypsin were all purchased from Gibco (USA), and the CCK8 kit was purchased from Abcam (USA).

[0090] 1.3 Instruments

[0091] Carbon dioxide incubator (Hong Kong HLC Medical Technology Co., Ltd.), bench-top high-speed centrifuge (Kunshan Ultrasonic Instruments Co., Ltd.), microplate reader (Bio-TEK, USA)

[0092] 2 Methods

[0093] 2.1 Experimental grouping

[0094] To study the inhibitory effect of different concentrations of Retusiusines H on A2058 cells, cells in different groups were cultured with Retusiusines H at 40 μmol / L, 20 μmol / L, 10 μmol / L, 5 μmol / L, and 1 μmol / L respectively.

[0095] 2.2 Cell culture

[0096] A2058 cells were cultured in DMEM medium containing 10% fetal bovine serum by volume, cultured in an incubator at 5% CO2 and 37 °C, digested with trypsin, and passaged at a ratio of 1:3.

[0097] 2.3 CCK8 assay for cell proliferation

[0098] A2058 cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 4 - 5×10 4 cells / ml. 100 μl of cells were inoculated into each well of a 96-well plate and cultured in an incubator (37 °C, 5% CO2) for 24 h. The 96-well plate was taken out, and the medium was discarded. 100 μl of A2058 medium with different concentrations (the medium was DMEM medium containing 10% fetal bovine serum by volume) was added to each well of the experimental group, and 5 replicates were set for each concentration. After continuous culture for 24 h, the medium containing the drug was aspirated and discarded, and the cells were washed 3 times with PBS. After observation, 10 μl of 10% CCK8 by volume was added to each well, and the plate was incubated at 37 °C in the dark for 4 h. The absorbance (OD value) was measured at 450 nm with a microplate reader, and the inhibition rate was calculated.

[0099] 3 Experimental results

[0100] The inhibitory rates of Retusiusines H at concentrations of 40 μmol / L, 20 μmol / L, 10 μmol / L, 5 μmol / L, and 1 μmol / L on A2058 cells were 99.03%, 80.36%, 67.69%, 16.69%, and 4.99% respectively (Table 1). Therefore, Retusiusines H has significant inhibitory activity on A2058 cells, and the IC 50 value was 9.59 μmol / L.

[0101] Table 1 Inhibitory rates of Retusiusines H at different concentrations on A2058 cells

[0102] 40 μmol / L 20 μmol / L 10 μmol / L 5 μmol / L 1 μmol / L Inhibition rate % 99.03±0.06 80.36±0.05 67.69±0.07 16.69±0.08 4.49±0.11

[0103] 4 Experimental conclusions

[0104] Retusiusines H can significantly inhibit the proliferation of A2058 cells, and the IC 50 value was 9.59 μmol / L. Thus, it can be seen that the phenolic acid compound Retusiusines H has great application potential in the application of anti-melanoma drugs.

[0105] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A phenolic acid compound Retusiusines H represented by formula (I) 2. Use of the phenolic acid compound Retusiusines H represented by formula (I) according to claim 1 in the preparation of a medicament for treating and / or preventing melanoma 3. The application according to claim 2, characterized in that, The melanoma is A2058 human cutaneous melanoma 4. Preparation method of the phenolic acid compound Retusiusines H represented by formula (I) according to claim 1, the preparation method comprising the following steps Step (1): Take Dendrobium huoshanense, extract to obtain an extract, and concentrate under reduced pressure to obtain a Dendrobium huoshanense extract Step (2): Dissolve the extract obtained in step (1) in water, extract with ethyl acetate, concentrate the ethyl acetate phase under reduced pressure to obtain a Dendrobium huoshanense extract paste Step (3): Separate the extract paste obtained in step (2) by normal-phase silica gel column chromatography, elute with a gradient, collect the gradient eluates of each gradient and concentrate, monitor by TLC, and combine the same parts to obtain 7 components Fr.1 - Fr.7 Step (4): Separate the component Fr.2 obtained in step (3) by normal-phase silica gel column chromatography, elute with a gradient, collect the gradient eluates of each gradient and concentrate, monitor by TLC, and combine the same parts to obtain 6 sub-components 2A1 - 2A6 Step (5): Separate the sub-component 2A2 obtained in step (4) by semi-preparative high performance liquid chromatography to obtain the phenolic acid compound Retusiusines H represented by formula (I) 5. The preparation method according to claim 4, wherein In step (1) The extraction method of Dendrobium huoshanense is maceration extraction, ultrasonic extraction, reflux extraction and / or, the extraction solvent is ethanol and / or, the extraction time is 5 - 9 days and / or, the extraction times are 1 - 5 times 6. The preparation method according to claim 5, characterized in that, Step (1) is specifically: Take fresh Dendrobium huoshanense samples, remove leaves and keep stems, crush with a crusher, macerate with 20L of absolute ethanol at room temperature for 3 times, 7 days each time, then filter to obtain the filtrate, and concentrate under reduced pressure to obtain a Dendrobium huoshanense extract 7. The preparation method according to any one of claims 4 to 6, characterized in that, In step (2) The volume ratio of ethyl acetate to water is 0.5 - 3:1 and / or, ultrasonic extraction is used for the extraction operation, and let it stand overnight after extraction and / or, the extraction times are 3 - 5 times 8. The preparation method according to claim 7, wherein In step (2), specifically Dissolve the extract obtained in step (1) in water, add an equal volume of ethyl acetate, ultrasonically extract for 0.5 - 1.5h at room temperature, then let it stand for 8 - 12h at room temperature, take the supernatant, and concentrate under reduced pressure; the extraction times are 3 - 5 times 9. The preparation method according to any one of claims 4-6, characterized in that, In step (3) During the normal-phase silica gel column chromatography separation, dry loading is used and / or, the silica gel for packing the column in the normal-phase silica gel column is 200 - 300 mesh normal-phase silica gel and / or, the mobile phase used for gradient elution is a mixed solvent of dichloromethane and methanol 10. The preparation method according to claim 9, characterized in that, In step (3), the method of dry loading the extract paste is: Dissolve the extract paste with 10 times the volume of ethyl acetate, adsorb and mix with silica gel of 80 - 100 mesh equal to the mass of the extract paste, and then load the sample And / or, in step (3), the volume ratios of dichloromethane to methanol in the mixed solvent of dichloromethane and methanol are successively 100:0, 100:1, 80:1, 50:1, 30:1, 10:1, 5:1, 1:1, 0:

1.

11. The preparation method according to any one of claims 4-6, characterized in that, In step (4), During the normal-phase silica gel column chromatography separation, dry loading is adopted; And / or, the silica gel for packing the normal-phase silica gel column is 200 - 300 mesh normal-phase silica gel; And / or, the mobile phase used for gradient elution is a mixed solvent of petroleum ether and ethyl acetate.

12. The preparation method according to claim 11, wherein In step (4), the specific method of dry loading is: Component Fr.2 is dissolved in 10 times its volume of methanol, adsorbed and mixed with silica gel of 80 - 100 mesh equal in mass to Component Fr.2, and then loaded onto the column; And / or, in step (4), the volume ratios of petroleum ether to ethyl acetate are successively 100:1, 60:1, 30:1, 10:1, 1:

1.

13. The preparation method according to any one of claims 4-6, characterized in that, In step (5), gel chromatography purification is first carried out, and then semi-preparative high-performance liquid chromatography purification is carried out.

14. The preparation method according to claim 13, characterized in that, The gel column used for gel chromatography purification is Sephadex LH-20, and a mixed solvent of dichloromethane and methanol with a volume ratio of 1:1 is used as the mobile phase.

15. The preparation method according to claim 14, characterized in that, During gel chromatography purification, sub-component 2A2 is dissolved in 2 times its volume of methanol and then loaded onto the column. After monitoring by TLC, the same parts are combined.

16. The preparation method according to claim 13, wherein, The chromatographic conditions for semi-preparative high-performance liquid chromatography purification are: The mobile phase is a mixed solvent of methanol and water with a volume ratio of 9:1; The flow rate is 1 mL / min; Collect the component with the starting peak time of 5.6 min; Using C 18 Reverse-phase chromatographic column; specifications: 4.6 mm x 250 mm, 5 μm; Column temperature: 30 °C; Each injection volume: 20 μL; An ultraviolet detector is used, and the detection wavelength is 210 nm.

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