CN116019791A - Application of diphenyl ether compound in preparation of beta-glucuronidase inhibitor - Google Patents

Abstract

The invention discloses application of diphenyl ether compounds in preparation of beta-glucuronidase inhibitors. The diphenyl ether compound is obtained from the fungi Aspergillus versicolor ZJUTE2 through a series of processes such as organic solvent leaching, chromatographic column separation and the like by simple extraction and separation, and the preparation method has the advantages of simplicity, rapidness, high purity of the obtained compound and the like; the diphenyl ether compound extracted by the invention has better beta-glucuronidaseInhibitory Activity, IC ₅₀ The value is 56.59 +/-1.79 mu M, is equivalent to the positive medicine D-glucaric acid 1,4-lactone, and can be used for treating drug-induced diarrhea caused by irinotecan Kang Huofei steroidal anti-inflammatory drugs. In addition, the compound is used as a lead, and the structure optimization is developed, so that the compound has important significance for the research and development of novel medicine for treating the drug-induced diarrhea.

Description

Application of diphenyl ether compound in preparation of beta-glucuronidase inhibitor

Field of the art

The invention belongs to the technical field of biological medicine, and in particular relates to application of 7-hydroxy-dimeric orcinol of diphenyl ether compound separated from fungi Aspergillus versicolor ZJUTE in inhibiting beta-glucuronidase activity of escherichia coli source.

(II) background art

Glucuronidation is one of the most important detoxification processes of human bodies, most endogenous substances and medicines can be transported to the duodenum through a single tube after glucuronidation, beta-glucuronidase secreted by intestinal flora is hydrolyzed to form aglycone, and free aglycone is reabsorbed into the blood circulation system through liver and intestine circulation. The beta-glucuronidase of the intestinal bacteria plays an important role in the glucuronidation process of endogenous substances and medicines, and is highly related to the gastrointestinal toxicity of the medicines.

Irinotecan is a clinical therapeutic drug for metastatic colorectal cancer, gastric cancer, non-small cell lung cancer and other diseases. After injection, irinotecan is first hydrolyzed by liver carboxylesterase to release active metabolite SN-38; SN-38 is then converted to glucuronidation product SN38G by UDP-glucuronyl transferase, which is inactivated and enters the gastrointestinal tract via bile. Under the action of intestinal bacteria beta-glucuronidase, SN-38G is hydrolyzed to generate SN-38 again, and strong cytotoxicity of the SN-38 causes intestinal epithelial cell injury, thereby causing diarrhea and limiting clinical application of irinotecan. Thus, the intestinal bacteria beta-glucuronidase is considered to be one of the important targets for the treatment of irinotecan-induced drug-induced diarrhea. In addition, intestinal bacteria beta-glucuronidase has direct connection with gastrointestinal toxicity caused by non-steroidal anti-inflammatory drugs such as ketoprofen, diclofenac and the like.

7-hydroxy-dimeric orcinol (7-hydroxy-diorcinol) as diphenyl ether compound, colorless solid, molecular formula C ₁₄ H ₁₄ O ₄ The molecular weight is 246, the compound is easily dissolved in methanol and can be separated from fungi Aspergillus versicolor ZJUTE2, and the chemical structure is shown as a formula (I):

at present, the research on whether the compounds have beta-glucuronidase inhibition activity has not been reported in related documents; that is, whether the compounds can be developed as β -glucuronidase inhibitors or not is a blank area of investigation in the art.

(III) summary of the invention

The invention aims to solve the defects of the prior art, provides an application of diphenyl ether compound separated from fungi Aspergillus versicolor ZJUTE in preparing a beta-glucuronidase inhibitor, and can provide a lead structure for research and development of a novel beta-glucuronidase inhibitor.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of a diphenyl ether compound in preparing a beta-glucuronidase inhibitor, wherein the diphenyl ether compound is 7-hydroxy-dimeric orcinol shown in a formula (I), and the molecular formula is C ₁₄ H ₁₄ O ₄ ；

Preferably, the beta-glucuronidase is of E.coli origin.

The invention also provides application of the diphenyl ether compound in preparing medicines for treating drug-induced diarrhea caused by irinotecan Kang Huofei steroid anti-inflammatory drugs.

The 7-hydroxy-dimeric orcinol is prepared according to the following method:

(1) Inoculating Aspergillus versicolor (Aspergillus versicolor) ZJUTE2 to rice solid culture medium, and dark culturing at 20-30deg.C for 15-30 days to obtain rice fermentation product; the aspergillus versicolor ZJUTE2 is preserved in China center for type culture Collection, the preservation date is 2022, 1 month and 13 days, the preservation number is CCTCCNO: M2022064, the address is China, wuhan, university of Wuhan, and the method is disclosed in patent application CN 114806888A; the rice solid culture medium is a mixture of rice and distilled water, wherein the distilled water dosage is 1-5mL/g (preferably 1.35 mL/g) based on the mass of the rice;

(2) Adding organic solvent into rice fermentation product (preferably mashed), leaching at room temperature (25-30deg.C), concentrating the extractive solution until no liquid flows out to obtain crude extract;

(3) Suspending the crude extract in the step (2) by water, extracting with an organic solvent, collecting an organic phase, and concentrating under reduced pressure to dryness to obtain an extract;

(4) Dissolving the extract of the step (3) by methanol, performing MCI CHP20P column chromatography, and sequentially performing gradient elution by taking a methanol/water mixed solvent with the volume ratio of 30-100:70-0 as an eluent, wherein each gradient elution is 2-5 (preferably 2) column volumes, and the flow rate is 10-20mL/min (preferably 15 mL/min); collecting methanol-water eluting part with volume ratio of 45:55-55:45, concentrating under reduced pressure to dry to obtain concentrate; dissolving the concentrate with methanol, recrystallizing at room temperature, and filtering to obtain 7-hydroxy-dimerized orcinol shown in formula (I).

Preferably, before the aspergillus versicolor ZJUTE2 is inoculated to the rice solid culture medium in the step (1), performing activation culture, and then inoculating the activated bacterial suspension to the rice solid culture medium in an inoculum size of 0.1-1% (preferably 1%) by volume, wherein the activation refers to that the aspergillus versicolor ZJUTE2 is inoculated to Potato Dextrose Agar (PDA) culture medium, the bacterial strain is activated by culturing for 7 days at 28 ℃, and the activated bacterial strain is resuspended by sterile water containing tween 80 with the volume concentration of 2%, so as to obtain bacterial suspension; the concentration of thallus in the bacterial suspension is 1 multiplied by 10 ⁵ -1×10 ⁸ Per mL, preferably 1X 10 ⁶ individual/mL; the PDA culture medium consists of 200g/L potato, 20g/L glucose and 18g/L agar, and the solvent is distilled water with natural pH.

Preferably, the fermentation culture conditions in step (1) are: dark culture at 28℃for 20 days.

Preferably, the organic solvent in step (2) is 95% ethanol, methanol or acetone; the volume and the dosage of the organic solvent are 2-8 mL/g (preferably 5-6 mL/g) based on the dry weight of the rice in the rice solid culture medium; the leaching is carried out for at least 3 times, the time of each extraction is 3-5 days, and the volume dosage of the organic solvent for each leaching is 4mL/g based on the dry weight of rice in the rice solid culture medium.

Preferably, the water volume in step (3) is 2-5mL/g (preferably 3.1 mL/g) based on the weight of the crude extract; the organic solvent is ethyl acetate, and the volume ratio of the organic solvent to water is 1:0.5-3; the extraction is carried out 3-5 times, and the volume ratio of the organic solvent to water for each extraction is preferably 1:1.

Preferably, the method of step (4) is as follows: 1) Dissolving the extract with methanol, performing MCI CHP20P column chromatography, and sequentially performing gradient elution with methanol/water mixed solvents with volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 as eluent, wherein each gradient elution comprises 2-5 (preferably 2) columns with flow rates of 10-20mL/min (preferably 15 mL/min); collecting methanol-water eluting part with volume ratio of 45:55-55:45, concentrating under reduced pressure to dry to obtain concentrate; 2) Dissolving the concentrate in the step 1) by methanol, recrystallizing and filtering to obtain the compound shown in the formula (I).

The diphenyl ether compound is obtained from the fungi Aspergillus versicolor ZJUTE2 through simple extraction and separation, has better beta-glucuronidase inhibition activity and is equivalent to the positive medicament D-glucaric acid 1,4-lactone (DSL).

Compared with the prior art, the invention has the beneficial effects that:

(1) The diphenyl ether compound is obtained from the fungi Aspergillus versicolor ZJUTE through a series of processes such as organic solvent leaching, chromatographic column separation and the like by simple extraction and separation, and the preparation method has the advantages of simplicity, rapidness, high purity of the obtained compound and the like, the culture time is 20 days, the yield is 260mg/Kg rice, and the purity can reach more than 95%;

(2) The diphenyl ether compound extracted by the invention has better beta-glucuronidase inhibition activity and IC ₅₀ The value is 56.59 +/-1.79 mu M, is equivalent to the positive medicine D-glucaric acid 1,4-lactone, and is expected to be used for treating the drug-induced diarrhea caused by the irinotecan Kang Huofei steroid anti-inflammatory drug. In addition, the compounds are used as a lead to develop structural optimizationHas important significance for developing novel medicine for treating drug-induced diarrhea.

(IV) description of the drawings

FIG. 1 is a diagram of Compound 1 ¹ H-NMR spectrum.

FIG. 2 is a diagram of Compound 1 ¹³ C-NMR spectrum.

FIG. 3 is a high performance liquid chromatogram of Compound 1.

FIG. 4 is a concentration-dependent curve of Compound 1 for beta-glucuronidase.

FIG. 5 is a concentration-dependent curve of D-glucaric acid 1,4-lactone versus beta-glucuronidase.

FIG. 6 is a graph of the double reciprocal of Lineweaver Burk for determining the type of inhibition of Compound 1.

(fifth) detailed description of the invention

The foregoing will be further described in detail with reference to the drawings and examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, and all techniques based on the foregoing are within the scope of the present invention.

The room temperature of the invention is 25-30 ℃. The PDA culture medium comprises the following components: 200g/L of potato, 20g/L of glucose and 18g/L of agar, wherein the solvent is distilled water, and the pH is natural.

EXAMPLE 1 fermentation culture of Cordyceps sinensis colonizing fungi Aspergillus versicolor (Aspergillus versicolor) ZJUTE2

1. Strain activation

Taking out Aspergillus versicolor (Aspergillus versicolor) ZJUTE2 strain freezing tube stored in-80deg.C refrigerator, thawing at 4deg.C, dipping appropriate amount of spore suspension in super clean bench with sterile cotton stick, inoculating to PDA culture medium, and standing in 28 deg.C incubator for 7 days for activating strain.

2. Strain fermentation

After 7 days of activation, spores of the strain were washed with 10mL of sterile water containing Tween 80 at a volume concentration of 2%, and placed in 100mL of sterile water containing Tween 80 at a volume concentration of 2% to give a spore suspension (spore concentration: 1X 10) ⁶ personal/mL); 1mL of spore suspension was removed separatelyInoculating into 50 triangular flask containing rice culture medium (100 g of rice, 135mL of distilled water, sterilized at 121deg.C for 20min, and cooled), and dark culturing at 28deg.C for 20 days to obtain 50 bottles of rice fermentation product.

Example 2 preparation of Compound 1

(1) 50 bottles of the rice fermentation product prepared in example 1 were mixed and mashed, added with 95% ethanol for 20 liters, leached at room temperature for 4 days, filtered, and the filter cake was repeatedly leached 3 times (each time with 3 liters of 95% ethanol for 4 days), and the extracts were combined and concentrated to dryness under reduced pressure to obtain 475g of crude extract.

(2) The crude extract (475 g) from step (1) was suspended in 1.5L of distilled water, extracted with ethyl acetate 1.5L each for 3 times, the ethyl acetate phases were combined and concentrated to dryness under reduced pressure to give 45g of ethyl acetate extract.

(3) Dissolving 45g of the ethyl acetate extract obtained in the step (2) in 100mL of methanol, performing MCI CHP20P column chromatography (diameter d=4 cm, height h=60 cm), and sequentially performing gradient elution by using methanol/water mixed solvents with volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 as eluent, wherein each gradient elution is 1.5L (2 column volumes), and the flow rate is 15mL/min; collecting the eluting parts of methanol/water=45:55-55:45 (v/v), mixing, and concentrating under reduced pressure to dry to obtain concentrate 2.1g.

(4) To 2.1g of the concentrate obtained in the step (3) was added 10mL of methanol for dissolution, and recrystallization was performed at room temperature, followed by filtration to obtain compound 1 (1.3 g).

Example 3 structural identification and purity detection of Compound 1

1. Structural identification of Compound 1

Compound 1: colorless solid. Molecular formula C ₁₄ H ₁₄ O ₄ The method comprises the steps of carrying out a first treatment on the surface of the Which is a kind of ¹ H-NMR (FIG. 1) and ¹³ the C-NMR (FIG. 2) data are as follows: ¹ H NMR(600MHz,CD ₃ OD):δ _H 6.55(1H,m,H-6),6.47(1H,m,H-2),6.38(1H,s,H-6’),6.31(1H,t,J＝2.2Hz,H-4),6.30(1H,s,H-4’),6.23(1H,t,J＝2.1Hz,H-2’),4.50(2H,s,H-7),2.24(3H,s,H-7’)。 ¹³ C NMR(150MHz,CD ₃ OD):δ _C 159.9(C-5),159.8(C-3),159.6(C-1’),159.5 (C-3 '), 145.6 (C-1), 141.7 (C-5'), 112.1 (C-6 '), 118.9 (C-4'), 109.5 (C-6), 109.2 (C-2), 105.7 (C-4), 104.4 (C-2 '), 64.9 (C-7), 21.5 (C-7'). The above spectral data are consistent with 7-hydroxy-dimeric orcinol (7-hydroxy-diorcinol) reported in the literature (HeP, tian S, xu Y, et al, three new phenyl ether derivatives from Aspergillus carneus HQ889708.Helv. Chim. Acta,2015,98,819-822.).

2. Purity detection of Compound 1

(1) Compound 1 prepared in example 2 was dissolved in methanol and quantitatively diluted to prepare a sample solution having a concentration of 200 μg/mL.

(2) The sample solution was taken for high performance liquid chromatography under the following chromatographic conditions. Instrument: agilent 1200 services; chromatographic column: cosmosil 5C18-PAQ (4.6 mm i.d.×250 mm); mobile phase: methanol-water (35:65-80:20, v/v); flow rate: 1mL/min; detection wavelength: 254nm; sample injection amount: 50. Mu.L; column temperature: room temperature. The record chromatogram is detected.

(3) The result of the HPLC detection of the sample to be detected is shown in FIG. 3. The retention time of the compound 1 is 16.523min, and the peak area normalization analysis shows that the purity of the compound is 96.5%.

Example 4 evaluation of in vitro beta-glucuronidase inhibitory Activity of Compound 1

1. Screening of EcGUS inhibitor (final inhibitor concentration 100. Mu.M)

(1) Reagent(s)

Inhibitors: compound 1 was made up with Dimethylsulfoxide (DMSO) to a solution of 10mM for use.

A substrate: 4-Nitrophenyl-beta-D-glucopyranoside (PNPG, available from Roen's reagent) was prepared in 2.5mM solution in PBS buffer for use.

Reaction enzyme solution: beta-glucuronidase (EcGUS) lyophilized powder (from sigma, enzyme activity 4724.48 kU/g) was dissolved in PBS buffer and diluted to 2.5. Mu.g/mL as a reaction enzyme solution.

Positive control (DSL): d-glucaric acid-1, 4-lactone (D-sacchoric acid 1,4-lactone, DSL, available from Sigma-Aldrich) was dissolved in DMSO to prepare a 10mM solution as a positive control.

(2) The reaction: the cells were divided into a blank group, an experimental group and a positive control group in 96-well plates.

Blank group: 10 mu L of the reaction enzyme solution+79 mu L of PBS+1 mu L of 10% volume concentration DMSO aqueous solution+10 mu L of substrate;

experimental group: 10. Mu.L of the reaction enzyme solution+79. Mu.L of PBS+1. Mu.L of inhibitor+10. Mu.L of substrate;

positive control group: 10. Mu.L of the reaction enzyme solution+79. Mu.L of PBS+10 mM positive control 1. Mu.L+10. Mu.L of substrate.

3 replicates were prepared for each group, samples were added in the order of the reaction enzyme solution, PBS, inhibitor/positive control, and substrate, OD values (incubation at 37℃during period) were measured for 0min and 30min, respectively, using an enzyme-labeled instrument, and calculated to give an inhibition ratio of 71.63% for EcGUS for Compound 1 at a final concentration of 100. Mu.M, which was higher than that for the positive control DSL (inhibition ratio 68.85%).

The specific calculation process is as follows:

ΔOD＝OD _30min -OD _0min ；

ΔC _PNP =Δod/0.003262 (0.003262 is the correlation coefficient of absorbance and PNP solubility of the present invention);

relative activity (%) = experimental group Δc _PNP Blank ΔC _PNP ；

Inhibition (%) =1-relative activity (%).

2、IC ₅₀ Measurement of values: determination of IC of Compound 1 ₅₀ Values, a series of inhibitor concentration points (e.g., 0.1, 1, 10, 20, 30, 50, 70, 100. Mu.M) were set up within 0.1-100. Mu.M final concentration, and reactions were performed in 96-well plates, with blank, experimental, and positive control groups set up.

Blank group: 10 mu L of reaction enzyme solution+79 mu L of PBS+1 mu L of volume fraction 10% DMSO+10 mu L of substrate;

experimental group: 10 mu L of reaction enzyme solution+79 mu L of PBS+1 mu L of inhibitor with different concentrations+10 mu L of substrate;

positive control group: 10. Mu.L of the reaction enzyme solution+79. Mu.L of PBS+1. Mu.L of positive control+10. Mu.L of substrate.

Each group is provided with 3 parallel groups, adding the sample in the sequence of the reaction enzyme solution, PBS, inhibitor/positive control and substrate, and then performing enzyme labelingOD values (incubation at 37 ℃ during period) of 0min and 30min are respectively measured at 405nm wavelength, relative activity values of the inhibitor on the EcGUS under different concentration conditions are obtained through calculation, and finally, the derivative taking the concentration (mu M) of the inhibitor as the base of 10 is used for obtaining an lg value, the lg value is taken as an abscissa, relative activity is taken as an ordinate, and the IC is drawn by using Graphad prism8.0 software ₅₀ Graphs (FIGS. 4, 5) and analysis by this software gave the IC of inhibitor/positive control versus EcGUS ₅₀ Value, IC for Compound 1 against EcGUS ₅₀ The value was 56.59.+ -. 1.79. Mu.M, which was compared with the positive control DSL (IC ₅₀ = 53.70 ±0.85 μm).

3. Inhibition type study of EcGUS by compound 1: compound 1 was formulated with PBS as 0.3, 0.5, 0.7mM solutions (i.e., 30, 50, 70 μm final concentrations) and the substrate was formulated with PBS as 2, 3, 5, 10mM solutions (i.e., 200, 300, 500, 1000 μm final concentrations). The alignment of the different concentrations of substrate and compound 1 is shown in table 1.

TABLE 1 alignment and combination Table of pNPG and Compound at different concentrations

Annotation: c (C) _PNPG Represents the final concentration of the substrate, C _IN Represents the final concentration of compound 1, o represents one well of a 96-well plate, and three parallel groups are made for each concentration combination.

The reaction was performed in 96-well plates, the reaction system was as follows: 10 mu L of reaction enzyme solution, 79 mu L of PBS and 1 mu L of inhibitor with different concentrations and 10 mu L of substrate with different concentrations are respectively arranged, 3 parallel combinations are arranged, the sample is added according to the sequence of the reaction enzyme solution, the PBS, the inhibitor and the substrate, an enzyme label instrument is used for respectively measuring the absorbance of 0min and 30min (incubation at 37 ℃ in the period) at 405nm, PNP concentration difference values corresponding to the combinations with different concentrations are calculated according to the step 1, and finally 1/V (mu mol/min/mg) and 1/PNPG value, V (mu mol/min/mg) are calculated, namely the catalytic speed of the enzyme, and the molar quantity of the catalytic produced product per milligram of enzyme per minute is expressed under the conditions of certain temperature, pH value and substrate concentration;

the calculation process is as follows:

1/V(μmol/min/mg)＝1/(ΔC _PNP ﹡100/10/30/1)；1/PNPG＝1/ΔC _PNP ；

wherein DeltaC _PNP The concentration difference of PNP in the system of 0min and 30min is represented by 100. Mu.L of the reaction system, 10 by 30 by the reaction time, and 1 by the enzyme preparation solubility of 1. Mu.g/mL.

Finally, a graph of the double reciprocal inhibition is drawn by using Graphad Prism8.0 software (figure 6), the type of the inhibitor is judged according to the intersection point of the curves, and the image can be found to have the intersection point in the third quadrant from figure 5, which shows that the inhibition of the compound 1 on the beta-glucuronidase belongs to mixed inhibition and can be combined with the active site and the allosteric site of the enzyme.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the invention in any way, but other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims (10)

1. An application of diphenyl ether compound shown in formula (I) in preparing beta-glucuronidase inhibitor,

2. the use of claim 1, wherein the inhibitor is a medicament for the treatment of drug-induced diarrhea caused by irinotecan Kang Huofei steroid anti-inflammatory drugs.

3. Use according to claim 1, characterized in that the beta-glucuronidase is of escherichia coli origin.

4. The use according to claim 1, wherein the diphenyl ether compound is prepared by the steps of:

(1) Inoculating Aspergillus versicolor (Aspergillus versicolor) CCTCC NO: M2022064 to rice solid culture medium, and dark culturing at 20-30deg.C for 15-30 days to obtain rice fermentation product; the rice solid culture medium is a mixture of rice and distilled water;

(2) Adding an organic solvent into the rice fermentation product, leaching at room temperature, and concentrating the extracting solution until no liquid flows out to obtain a crude extract;

(3) Suspending the crude extract in the step (2) by water, extracting with an organic solvent, collecting an organic phase, and concentrating under reduced pressure to dryness to obtain an extract;

(4) Dissolving the extract of the step (3) by methanol, performing MCI CHP20P column chromatography, and sequentially performing gradient elution by taking a methanol/water mixed solvent with the volume ratio of 30-100:70-0 as an eluent, wherein each gradient elution is 2-5 column volumes, and the flow rate is 10-20mL/min; collecting methanol-water eluting part with volume ratio of 45:55-55:45, concentrating under reduced pressure to dry to obtain concentrate; dissolving the concentrate with methanol, recrystallizing at room temperature, and filtering to obtain diphenyl ether compound shown in formula (I).

5. The use according to claim 4, wherein the aspergillus versicolor CCTCC NO: M2022064 in the step (1) is subjected to activation culture before being inoculated into a rice solid culture medium, the activated bacterial suspension is inoculated into the rice solid culture medium with the inoculation amount of 0.1-1% of volume concentration, the activation means that the aspergillus versicolor CCTCC NO: M2022064 is inoculated into a PDA culture medium, the bacterial strain is activated by culturing for 7 days at 28 ℃, and the activated bacterial strain is resuspended with sterile water containing tween 80 with the volume concentration of 2% to obtain the bacterial suspension; the concentration of thallus in the bacterial suspension is 1 multiplied by 10 ⁵ -1×10 ⁸ And each mL.

6. The use according to claim 4, wherein distilled water is used in step (1) in an amount of 1 to 5mL/g based on the mass of rice.

7. The use according to claim 4, wherein the organic solvent in step (2) is 95% ethanol, methanol or acetone; the volume and the dosage of the organic solvent are 2-8 mL/g based on the dry weight of rice in the rice solid culture medium.

8. The use according to claim 4, wherein the leaching in step (2) is carried out at least 3 times, each for 3 to 5 days, the volume of organic solvent used for each leaching being 4mL/g based on the dry weight of rice in the solid medium of rice.

9. The use according to claim 4, wherein the water volume in step (3) is 2-5mL/g based on the weight of the crude extract; the organic solvent is ethyl acetate, and the volume ratio of the organic solvent to water is 1:0.5-3.

10. The use of claim 4, wherein the method of step (4) is: 1) Dissolving the extract with methanol, performing MCI CHP20P column chromatography, and sequentially performing gradient elution with methanol/water mixed solvents with volume ratios of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 as eluent, wherein each gradient elution is 2-5 column volumes, and the flow rate is 10-20mL/min; collecting methanol-water eluting part with volume ratio of 45:55-55:45, concentrating under reduced pressure to dry to obtain concentrate; 2) Dissolving the concentrate in the step 1) by methanol, recrystallizing at room temperature, and filtering to obtain the compound shown in the formula (I).

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