A plant endophytic fungus producing eupatilin and its application

Abstract

The present invention discloses an endophytic fungus Septoriella phragmitis and its application. This strain can produce eupatilin under liquid fermentation conditions. This strain was deposited at the China Center for Type Culture Collection (Wuhan University) on September 13, 2022, with the deposit number: CCTCC M 20221413. The present invention also discloses a method for preparing eupatilin using the deposited strain, including culturing and fermenting the bacteria, extracting secondary metabolites, and purifying by column chromatography. The present invention provides a new strain for producing eupatilin by microbial fermentation. The fermentation cycle of eupatilin by this strain is short, and the yield of eupatilin in shake flask fermentation can reach 159.6 μg / L. The eupatilin produced by this strain was α tested for α-glucosidase and PTP1B inhibitory activities. At a concentration of 5 μg / ml, it has varying degrees of inhibitory activities, and the inhibition rates are 22.34% and 51.32% respectively. Eupatilin also has good α α-glucosidase and PTP1B inhibitory effects and can be used as a lead compound for anti-diabetic dual targets.

Description

Technical Field

[0001] The present invention relates to an endophytic fungus producing euparin, a method for preparing euparin by using the strain, and the application of euparin in the treatment of diabetes, belonging to the fields of microbial biotechnology and biopharmaceutical technology. Background Art

[0002] Euparin (also known as euparin) is one of the active ingredients in the roots of Eupatorium chinense. Its chemical structure is 5-acetyl-6-hydroxy-2-isopropenylbenzofuran, belonging to the benzofuran class of compounds. According to literature reports, euparin has various biological activities such as antifeedant, antiviral, antibacterial, antioxidant, antidepressant, antitumor, participating in immune regulation and treating cardiovascular diseases, etc., but its biological activities related to diabetes have not been reported. Natural euparin is mainly isolated from the roots of Eupatorium plants. Among them, the content in the roots of Eupatorium chinense is the highest, but it only accounts for one ten-thousandth of the Eupatorium chinense sample. It should be emphasized that so far, euparin from microbial sources has not been reported.

[0003] At present, it has been confirmed that many natural compounds from plants are closely related to endophytic fungi of plants and are even secondary metabolites of endophytic fungi. Obtaining natural products with medicinal value from plant endophytic fungi can not only break through the limitations of lack of plant resources and long regeneration cycles, but also realize the large-scale, low-cost and pollution-free production of natural active compounds by industrial fermentation [Research on active ingredients of plant endophytic fungi. Chinese Traditional and Herbal Drugs, 2005, 36(5): 772-776.]. In the present invention, an endophytic fungus Septoriella phragmitis is isolated from the roots of Eupatorium chinense. This strain can produce euparin under liquid fermentation conditions, which is the first discovery that microorganisms can produce euparin.

[0004] Diabetes has become the third major disease affecting human health after cardiovascular and cerebrovascular diseases and tumors, with as many as 463 million patients worldwide. Among them, 90% of diabetes is type 2 diabetes (T2DM). T2DM is a series of metabolic disorder syndromes mainly characterized by insulin resistance and / or insulin cell function failure, often accompanied by various acute and chronic complications. 80% of T2DM patients are also accompanied by obesity. However, at present, drugs cannot effectively prevent the necrosis of pancreatic islet β cells and have many side effects. For example, long-term use of hypoglycemic drugs such as metformin and α-glucosidase inhibitors can easily cause serious gastrointestinal side effects such as abdominal distension, nausea, and vomiting in diabetic patients, and even cause serious damage to the kidneys and livers of patients [Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020 Edition). Chinese Journal of Diabetes, 2021, 13(4): 315-409.].

[0005] Alpha-glucosidase in the small intestine can hydrolyze polysaccharides in the body into monosaccharides, thereby increasing the blood glucose level in the body. Its inhibitor can reduce postprandial blood glucose. Protein tyrosine phosphatase 1B (PTP1B) plays a key negative regulatory role in insulin and leptin signaling, and its inhibitor has the effects of enhancing insulin sensitivity, improving blood glucose level, and reducing body weight. Inhibiting both the alpha-glucosidase and PTP1B targets can, on the one hand, reduce postprandial blood glucose, and on the other hand, enhance insulin sensitivity, reduce the load on pancreatic islet beta cells, and can reduce the body weight of obese T2DM patients by increasing the leptin level in the body. The dual-target inhibitor of alpha-glucosidase and PTP1B can interact with two T2DM-related targets, alpha-glucosidase and PTP1B, simultaneously to jointly maintain the stability of blood glucose level in the body. In this way, it can not only be used for the treatment of T2DM patients, but also be a very valuable treatment method for individuals with impaired glucose tolerance, making its dual-target inhibitory effect reasonably integrated [Wang Meiyan. Research on the Design, Synthesis, Activity and Mechanism of Action of Dual-Target Inhibitors of Alpha-Glucosidase and PTP1B. Doctoral Thesis of Tianjin Medical University, 2017.]. Therefore, the use of multi-target drugs for treatment can improve the treatment effect of T2DM, avoid some adverse interactions caused by combination drug use, and has great research value.

[0006] The eupatilin produced by this strain was tested for its inhibitory activities against alpha-glucosidase and PTP1B. At a concentration of 5 μg / ml, it showed varying degrees of inhibitory activities. The preparation method of the compound of the present invention is simple, and eupatilin has good inhibitory effects on both alpha-glucosidase and PTP1B, and can be used as a lead compound for anti-diabetic dual targets, having good application prospects. Summary of the Invention

[0007] The present invention relates to a plant endophytic fungus Septoriella phragmitis that produces eupatilin, a method for preparing eupatilin using this strain, and the application of eupatilin in the treatment of diabetes.

[0008] The present invention obtained a series of endophytic fungal strains from the roots of Eupatorium chinense. After isolation and screening, a strain Septoriella phragmitis that can produce eupatilin was obtained. Through morphological identification and analysis of the ITS sequence of the strain, it was identified as Septoriella phragmitis. It was deposited in the China Center for Type Culture Collection on September 13, 2022, with the deposit number CCTCC NO: M 20221413, classified and named Septoriella phragmitis LH-1, and the deposit address is Wuhan University, Wuhan, Hubei.

[0009] The colony characteristics of the strain Septoriella phragmitis of the present invention are as follows: on PDA medium, when cultured in an inverted position at 28°C, the growth rate is relatively fast. After 3 days, the colony diameter is about 2.5 cm, and after 6 days, the colony diameter reaches 6.0 cm. The middle part of the colony is white (mixed with a little yellow mycelium), the edge is white, with aerial mycelium, the whole is radial, and the edge is serrated. The mycelium is dark-colored, septate, and no conidia are seen.

[0010] The application of the endophytic fungus Septoriella phragmitis strain of the present invention in the preparation of eupatilin is specifically to prepare eupatilin from the metabolites of Septoriella phragmitis. The preparation method is as follows:

[0011] (1) Cultivation and fermentation of Septoriella phragmitis:

[0012] Cultivation of the strain: Inoculate the preserved strain of the endophytic fungus Septoriella phragmitis onto a PDA medium plate for constant temperature cultivation;

[0013] Fermentation of the strain: Inoculate the cultivated strain into a PDA liquid medium, and perform constant temperature shaking cultivation to obtain a seed solution. Then inoculate the seed solution into a PDA liquid medium and perform constant temperature shaking cultivation to obtain a fermentation broth;

[0014] (2) Extraction of secondary metabolites: After the fermentation is completed, collect the fermentation broth and mycelium respectively. Extract the fermentation broth and mycelium with ethyl acetate respectively. After the obtained extract is concentrated under reduced pressure to an extract, then mix to obtain secondary metabolites;

[0015] (3) Isolation and purification of eupatilin: The secondary metabolites are separated by normal-phase silica gel column chromatography, and then subjected to horizontal gradient elution with a mixture of petroleum ether - ethyl acetate. Collect the 5th fraction and refine and purify it by semi-preparative high performance liquid chromatography to obtain eupatilin produced by Septoriella phragmitis.

[0016] In the steps (1) above, both the cultivation of the strain and the fermentation process of the strain are carried out under a constant temperature condition of 25 - 28°C; the constant temperature shaking cultivation is carried out on a shaker at 120 rpm - 180 rpm.

[0017] In the step (2) above, the volume ratio of the fermentation broth to ethyl acetate is 1:1 - 3;

[0018] Before extraction, the mycelium is first dried, crushed, and then soaked in ethyl acetate with a volume 8 - 12 times or more for overnight, and then ultrasonic extraction is carried out to obtain an ethyl acetate extract.

[0019] In step (3), the volume ratio of petroleum ether to ethyl acetate is 30:1 - 3.

[0020] In step (3), the elution conditions of semi-preparative high performance liquid chromatography are as follows: the mobile phase is methanol and water with a volume ratio of 95:5, the flow rate is 1 - 3.0 mL / min, the detection wavelength is 260 nm, the running time is 10 - 30 min, and the retention time of eupatilin in HPLC is 10 - 15 min.

[0021] The present invention relates to the application of the endophytic fungus Septoriella phragmitis or eupatilin in the preparation of drugs for inhibiting α-glucosidase or PTP1B activity.

[0022] Determination of eupatilin content in the shake flask of the strain: The HPLC method is adopted (conditions: the mobile phase is acetonitrile and water with a volume ratio of 85:15, the flow rate is 1.0 mL / min, the detection wavelength is 260 nm, and the running time is 30 min).

[0023] The eupatilin produced by the Septoriella phragmitis strain of the present invention has been confirmed, and its structure is completely consistent with that of the eupatilin standard. The present invention provides the possibility and basis for future strain improvement, optimization of fermentation culture conditions, and selection of strains suitable for industrial large-scale production of eupatilin.

[0024] The present invention also tested the inhibitory activities of eupatilin produced by this strain against α-glucosidase and PTP1B. At a concentration of 5 μg / ml, it has good inhibitory effects on both α-glucosidase and PTP1B, and can be used as a lead compound for anti-diabetic dual targets, with good application prospects. Description of the Drawings

[0025] Figure 1 It is the colony morphology of Septoriella phragmitis of the present invention, where 1 - 1 is the front schematic diagram and 1 - 2 is the back schematic diagram.

[0026] Figure 2 It is the hydrogen spectrum ( 1 1H-NMR) diagram of eupatilin produced by the strain of the present invention.

[0027] Figure 3 It is the carbon spectrum ( 13 13C-NMR) diagram of eupatilin produced by the strain of the present invention.

[0028] Figure 4 It is the HPLC standard curve of eupatilin.

[0029] Figure 5It is the HPLC analysis chart of eupatilin and the secondary metabolites of this strain. Among them, 5-1 is the HPLC chart of eupatilin, and 5-2 is the HPLC chart of the secondary metabolites of this strain. Specific implementation mode

[0030] The following is further to illustrate the present invention in conjunction with embodiments, but the scope of protection required by the present invention is not limited to the scope expressed in the embodiments.

[0031] Example 1

[0032] Isolation and preservation of Septoriella phragmitis strain:

[0033] In October 2016, in Huoshaoping Township, Changyang County, Hubei Province (longitude: 110.744578, latitude: 30.507959), Eupatorium chinense was relatively concentrated in distribution and rich in resources. The average altitude of this township is 1,800 meters, the average annual temperature is 7.6 °C, and the annual frost-free period is 200 days. It is known as "the Tibet of Changyang". Remove the surface soil from the collected roots of Eupatorium chinense, wash them clean with tap water, and then wash them 3 times with sterile water. After disinfecting the roots of Eupatorium chinense with 75% alcohol for 5 minutes, wash them 3 times with sterile water. After fully grinding and standing the disinfected roots of Eupatorium chinense to obtain a bacterial suspension, after gradient dilution, 100 μL was respectively taken and spread on the PDA (potato dextrose agar solid medium) culture medium plate, and placed in a constant temperature culture at 28 °C. Observe regularly. After the mycelia grow, the mycelium tip purification method is used. Pick the mycelium tip and purify it on a new PDA culture medium plate. When colonies with different colors or morphologies grow out, continue the purification operation with the mycelium tip purification method until each colony is a single pure culture. The pure strain isolated therefrom is inoculated into a PDA slant, cultured in an incubator at 28 °C for 48 hours, sealed with sterile liquid paraffin, and stored in a 4 °C refrigerator to complete the isolation of the endophytic fungus Septoriella phragmitis, as shown in Figure 1 .

[0034] Example 2

[0035] Identification of Septoriella phragmitis strain:

[0036] Sequence determination of Septoriella phragmitis strain: After the strain was cultured in PDA liquid and the mycelia were collected by filtration, an appropriate amount of mycelia was quickly ground into powder with liquid nitrogen, and then the DNA of the strain was extracted using an Ezup column genomic DNA extraction kit (for fungi). The extracted DNA was eluted with sterile double-distilled water for PCR amplification. A pair of universal primers ITS5 / ITS4 was used for PCR amplification of ITS rDNA.

[0037] Forward primer ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3', reverse primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3'.

[0038] Amplified using a ready-to-use PCR amplification kit. The PCR reaction system was as follows: 1 μL of template DNA, 2 μL of dNTP (2.5 mmol / L each), 1 μL of each primer, 2 μL of 10×PCR buffer, 0.2 μL of Taq enzyme, 12.8 μL of ddH 2 O, with a total volume of 20 μL. The reaction program was: denaturation at 94°C for 1 min, annealing at 56°C for 30 s, extension at 72°C for 1 min, for a total of 33 cycles; the final extension was carried out at 72°C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis, with a 5 μL sample loading, at 80 V for 30 min. The amplified target bands were detected by a gel imaging system, and the target fragments were recovered using a DNA purification and recovery kit. Sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0039] The strain was identified as Septoriella phragmitis by alignment with the sequences deposited in GenBank through Blast. The measured sequence was submitted to GenBank and obtained the accession number MZ664268.

[0040] Example 3

[0041] A method for preparing eupatilin from the metabolites of Septoriella phragmitis strain, comprising the following steps: Step 1: Cultivation and fermentation of Septoriella phragmitis strain: The preserved strain was inoculated onto a PDA medium plate and cultured in a constant temperature incubator at 28°C for 48 hours. Fermentation of the strain: A piece of about 1 cm × 1 cm of the cultured strain was picked with an inoculation needle and inoculated into a container containing PDA liquid medium. Under the conditions of a temperature of 28°C and a rotation speed of 180 rpm, it was cultured with a constant temperature shaking incubator for 5 days to obtain a seed liquid. The seed liquid was inoculated into a container containing 200 mL of PDA liquid medium at an inoculation amount of 5 mL and continued to be cultured with a constant temperature shaking incubator at a temperature of 28°C and a rotation speed of 120 rpm for 30 days;

[0042] Step 2: Extraction of secondary metabolites: After fermentation, the fermentation broth and mycelia were collected separately. The fermentation broth was extracted with ethyl acetate three times, with the volume ratio of fermentation broth to ethyl acetate being 1:1 each time. All the ethyl acetate extracts were combined and concentrated under reduced pressure to an extract state to obtain the ethyl acetate extract of the fermentation broth. The mycelia were dried at 45 °C and then ground into powder. Ethyl acetate was added according to the weight of mycelia: volume of ethyl acetate = 1:10 and soaked overnight, followed by ultrasonic extraction for 30 min. The extraction was repeated three times. All the ethyl acetate extracts were combined and concentrated under reduced pressure to an extract state to obtain the ethyl acetate extract of the mycelia. The combined ethyl acetate extract of the fermentation broth and the ethyl acetate extract of the mycelia was the total extract of the secondary metabolites;

[0043] Step 3: Isolation and purification of eupatilin: The total extract of the secondary metabolites was separated by normal-phase silica gel column chromatography, and gradient elution was carried out with petroleum ether - ethyl acetate = 30:1 (volume ratio). A total of 10 fractions were obtained. The 5th fraction was refined and purified by semi-preparative high-performance liquid chromatography. The mobile phase was methanol and water with a volume ratio of 95:5, the flow rate was 3.0 mL / min, the detection wavelength was 260 nm, the running time was 30 min, and the retention time of eupatilin in HPLC was 15 min. The peak of eupatilin was collected to obtain eupatilin produced by Septoriella phragmitis.

[0044] Example 4

[0045] Structural identification of eupatilin produced by the Septoriella phragmitis strain obtained in Example 3: Physical constants and spectral data of eupatilin produced by the Septoriella phragmitis strain: Pale yellow needle-shaped crystals, m.p. 152 - 153 °C; UVλ max = 260, 322 nm; EI-MS m / z: 216.08 [M] + , C 13 H 12 O 3 . 1 H-NMR (400 MHz, CDCl 3 ) δ H : 12.49 (1H, s, 6-OH), 7.80 (H, s, H-4), 6.90 (1H, s, H-3), 6.46 (1H, s, H-7), 5.71 (1H, s, H-13a), 5.15 (1H, s, H-13b), 2.61 (3H, s, H-14), 2.06 (3H, s, H-11), see Figure 2 ; 13 C-NMR (100 MHz, CDCl 3 ) δ c: 203.8 (C-10), 161.4 (C-8), 159.4 (C-6), 157.7 (C-2), 131.9 (C-12), 123.4 (C-4), 121.7 (C-5), 116.6 (C-9), 113.5 (C-13), 102.3 (C-7), 99.2 (C-3), 26.6 (C-11), 19.0 (C-14), see Figure 3 . After consulting the literature, the nuclear magnetic resonance spectroscopy data of this compound are completely consistent with those of the eupatilin standard [Study on Chemical Constituents from Roots of Eupatorium chinense L. Chinese Traditional and Herbal Drugs, 2018, 49(20): 4798-4802.]. Therefore, this compound was identified as eupatilin, and its structure is as follows:

[0046] Example 5

[0047] Determination of eupatilin content in Septoriella phragmitis strain in shake flasks:

[0048] Using the HPLC method (conditions: the mobile phase is acetonitrile and water in a volume ratio of 85:15, the flow rate is 1.0 mL / min, the detection wavelength is 260 nm, and the running time is 30 min), a standard curve of eupatilin was established, and the eupatilin content in the strain shake flask was calculated to be 159.6 μg / L. It includes the following steps:

[0049] Step 1: Preparation of the reference substance (eupatilin) solution: Take an appropriate amount of eupatilin, weigh it accurately, and dissolve it in acetone to make a 1 mg / mL solution.

[0050] Step 2: Preparation of the standard curve: Accurately measure 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of the reference substance solution respectively, place them in 10 mL volumetric flasks, make up the volume, and use a high-performance liquid chromatograph to measure the peak area at 260 nm. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa, as Figure 4 shown.

[0051] Step 3: Preparation of the test solution: Take about 0.5 g of the strain fermentation product, add 25 mL of acetone solution, filter, accurately take 0.2 mL of the filtrate, place it in a 10 mL volumetric flask, and make up the volume to obtain the test solution.

[0052] Step 4: Content determination: Take an appropriate amount of the test solution and use a high-performance liquid chromatograph to measure the peak area (see Figure 5 ), HPLC conditions: the mobile phase is acetonitrile and water in a volume ratio of 85:15, the flow rate is 1.0 mL / min, the detection wavelength is 260 nm, and the running time is 30 min. The total eupatilin content in the test solution was calculated to be 159.6 μg / L through the standard curve.

[0053] Example 6

[0054] Inhibitory activities of eupatilin against α-glucosidase and PTP1B:

[0055] The present invention also tested the inhibitory activities of eupatilin produced by this strain against α-glucosidase and PTP1B. When the concentration was 5 μg / ml, it had good inhibitory effects on both α-glucosidase and PTP1B, and could be used as a lead compound for anti-diabetic dual targets, showing good application prospects. It includes the following steps:

[0056] Step 1: Test for α-glucosidase inhibitory activity: The determination method of α-glucosidase refers to the literature [Rapidscreening and identification of α-glucosidase inhibitors from mulberry leavesusing enzyme-immobilized magnetic beads coupled with HPLC / MS andNMR.Biomedical Chromatography,2013,27(2):148-155.]. Add 40 μL of eupatilin (concentration 5 μg / mL, solvent DMSO) to 40 μL of reaction buffer solution (PBS), add 20 μL of α-glucosidase solution (2 U / mL) and mix in each well. Preheat the reaction mixture for 15 min using a block heater at 37 °C, add 20 μL of reaction substrate PNPG (5 mM), react at 37 °C for 15 min, and add Na 2 CO 3 solution (80 μL, 0.2 M) to stop the reaction. Record the absorbance at 405 nm. Set up an enzyme activity group (enzyme + reaction buffer + substrate), an enzyme blank group (reaction buffer + reaction substrate), a sample group (sample + reaction buffer + enzyme + reaction substrate), and a sample blank group (sample + reaction buffer + reaction substrate), and use acarbose solution as a positive control.

[0057] Step 2: PTP1B inhibitory activity test: The determination method of PTP1B refers to the literature [New triterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B. Journal of Functional Foods, 2018, 41: 232-239; Chemical constituents of Cydonia oblongata Mill. seeds and their PTP1B inhibitory activity. Acta Pharmaceutica Sinica, 2019, 54(3): 510-513.]. Add 10 μL of eupatilin (concentration 5 μg / mL, solvent DMSO) to 170 μL of reaction buffer solution, which consists of 50 mM citric acid (pH 7.4), 50 mM NaCl, 2 mM dithiothreitol (DTT), 1.1 mM EDTA. Add 20 μL of recombinant PTP1B solution (1 mg / mL, 1 μL) and mix in each well. Preheat the reaction mixture for 15 min using a block heater at 37 °C. Add 10 μL of the reaction substrate p-nitrophenyl phosphate pNPP (33 mM), and react at 37 °C for 15 min. Add NaOH solution (10 μL, 0.1 M) to stop the reaction. Record the absorbance at 405 nm. Set up an enzyme activity group (enzyme + reaction buffer + substrate), an enzyme blank group (reaction buffer + reaction substrate), a sample group (sample + reaction buffer + enzyme + reaction substrate), a sample blank group (sample + reaction buffer + reaction substrate), and use an aqueous solution of sodium orthovanadate as a positive control.

[0058] The results of the inhibitory activities of eupatilin against α-glucosidase and PTP1B are shown in Table 1.

[0059] Table 1. Results of the inhibitory activities of eupatilin against α-glucosidase and PTP1B

[0060]

Claims

1. Endophytic fungus Septoriella phragmitis LH-1 was deposited at the China Center for Type Culture Collection on September 13, 2022, with the deposit number CCTCC NO: M 20221413, and the taxonomic name is Septoriella phragmitis LH-1. The deposit address is Wuhan University, Wuhan, Hubei.

2. Endophytic fungi Septoriella phragmitis Use in the preparation of euparin, wherein the endophytic fungi Septoriella phragmitis are the endophytic fungi Septoriella phragmitis LH-1 as claimed in claim 1.

3. The application according to claim 2, characterized in that, Application for preparing eupatilin from the metabolites of Septoriella phragmitis .

4. The application according to claim 3, characterized in that, The specific preparation method is as follows: (1) Septoriella phragmitis Cultivation and fermentation: Cultivation of strains: The endophytic fungi Septoriella phragmitis The preserved strains were inoculated onto PDA medium plates and cultured at a constant temperature; Fermentation of the strain: The cultured strain is inoculated into a PDA liquid medium, and cultured with constant temperature oscillation to obtain a seed liquid. The seed liquid is then inoculated into the PDA liquid medium and cultured with constant temperature oscillation to obtain a fermentation broth; (2) Extraction of secondary metabolites: After the fermentation is completed, the fermentation broth and mycelium are collected separately. The fermentation broth and mycelium are extracted with ethyl acetate respectively. After the obtained extract is concentrated under reduced pressure to an extract, the secondary metabolites are obtained by mixing; (3)Separation and purification of eupatilin: The secondary metabolites were separated by normal-phase silica gel column chromatography, and then subjected to horizontal gradient elution with a mixture of petroleum ether and ethyl acetate. The 5th fraction was collected and purified by semi-preparative high performance liquid chromatography to obtain Septoriella phragmitis the produced eupatilin.

5. The application according to claim 4, characterized in that, In step (1), the cultivation of the strain and the fermentation process of the strain are both carried out under the constant temperature condition of 25-28 °C; The constant temperature oscillation culture is carried out on a shaker at 120 rpm - 180 rpm.

6. The application according to claim 4, characterized in that, In step (2), the volume ratio of the fermentation broth to ethyl acetate is 1:1 - 3; The mycelium is dried, crushed before extraction, and then soaked in ethyl acetate with a volume 8 - 12 times or more for overnight, and then the ethyl acetate extract is obtained by ultrasonic extraction.

7. The application according to claim 4, characterized in that, In step (3), the volume ratio of petroleum ether - ethyl acetate is 30:1 - 3.

8. The application according to claim 4, characterized in that, In step (3), the elution conditions of semi-preparative high performance liquid chromatography: The mobile phase is methanol and water with a volume ratio of 95:5, the flow rate is 1 - 3.0 mL / min, the detection wavelength is 260 nm, the running time is 10 - 30 min, and the retention time of eupatilin in HPLC is 10 - 15 min.

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