Compound extracted from cyatheaspinulosa and extraction method thereof and application of compound in preparation of medicine for preventing and / or treating non-alcoholic fatty liver disease

Abstract

This invention relates to a compound extracted from *Dryopteris macrocarpa*, its extraction method, and its application in the preparation of drugs for the prevention and / or treatment of non-alcoholic fatty liver disease. This invention provides a novel natural compound obtained from *Dryopteris macrocarpa* for the first time, along with a method for its preparation. Activity studies were conducted, and experimental data show that the compound exhibits low cytotoxicity to normal cells. It can inhibit fatty acid uptake in cells by suppressing the expression of fatty acid uptake-related genes FATP2 and CD36, thereby inhibiting lipid droplet accumulation and reducing cellular lipid levels. This compound can be applied in the preparation of drugs for the prevention and / or treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to an extract compound from *Dryopteris macrocarpa*, its extraction method, and its application in the preparation of drugs for the prevention and / or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. It refers to a clinicopathological syndrome characterized by diffuse hepatocellular steatosis, excluding alcohol and other known liver-damaging factors (such as viral hepatitis and drug-induced liver injury). Its spectrum includes simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), progressive liver fibrosis, and cirrhosis. In recent years, with the global prevalence of obesity, type 2 diabetes, and hyperlipidemia, the prevalence of NAFLD has experienced explosive growth.

[0003] Currently, the treatment of NAFLD focuses on slowing disease progression and preventing complications, emphasizing individualized comprehensive management. Lifestyle modifications remain the mainstream treatment approach for NAFLD. In addition, drug therapy can be used as an adjunct. Traditional adjunctive drugs include lipid-lowering agents such as statins and fibrates; insulin sensitizers such as metformin and pioglitazone; and some antioxidant and hepatoprotective drugs such as vitamin E and N-acetylcysteine. Newer drugs such as smegglutide and remetrotinol are also available. The former can improve insulin resistance and reduce hepatic fat deposition and inflammation; while the latter can improve liver fibrosis by selectively activating thyroid hormone receptor-β. However, the current range of drugs is still insufficient to address the diverse clinical manifestations of non-alcoholic fatty liver disease caused by different etiologies and with varying degrees of severity.

[0004] Therefore, there is an urgent need to develop more safe and effective new therapeutic drugs to meet clinical needs and expand treatment strategies for non-alcoholic fatty liver disease. Summary of the Invention

[0005] This invention addresses the deficiency of the limited variety of existing drugs for treating non-alcoholic fatty liver disease by providing a compound that can inhibit lipid synthesis.

[0006] A second objective of this invention is to provide a method for preparing the compound.

[0007] A third object of the present invention is to provide an extract containing the compound.

[0008] A fourth object of the present invention is to provide a pharmaceutical composition comprising the compound or the extract.

[0009] A fifth object of the present invention is to provide the use of the compound, the extract, or the pharmaceutical composition in the preparation of a medicament for the treatment and / or prevention of non-alcoholic fatty liver disease.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention protects a compound having the following structure:

[0012] .

[0013] The present invention also protects compounds that include their pharmaceutically acceptable salts.

[0014] This invention also protects a method for preparing the compound, comprising the following steps:

[0015] S1: Prepare a suspension by adding water to the alcohol extract of the underground part of Dryopteris macrocarpa, extract with petroleum ether, remove the organic layer to remove the solvent, and obtain the extract paste.

[0016] S2: The extract obtained in step S1 is separated by silica gel column chromatography, and the extract is eluted sequentially with a petroleum ether-ethyl acetate mixed solvent at volume ratios of 100:0, 100:1, 20:1 and 5:1. The fractions eluted by the petroleum ether-ethyl acetate mixed solvent at volume ratios of 20:1 and 5:1 are collected and combined to obtain the fraction Fr.D.

[0017] S3: The Fr.D fraction obtained in step S2 is separated by silica gel column chromatography, and eluted sequentially with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 10:1 and 5:1. The fraction eluted with the petroleum ether-ethyl acetate mixed solvent with a volume ratio of 5:1 is collected to obtain the fraction Fr.D5.

[0018] S4: The Fr.D5 fraction obtained in step S3 is separated by gel column chromatography and eluted with a 1:1 volume ratio of dichloromethane-methanol mixed solvent to obtain the fraction Fr.D5-b.

[0019] S5: The Fr.D5-b fraction obtained in step S4 is separated by ODS column chromatography, and is eluted sequentially with methanol-water solutions of 0 vol%, 70 vol%, 80 vol%, 85 vol%, 90 vol%, 95 vol%, and 100 vol%. The fractions eluted by 90 vol% and 95 vol% methanol-water solutions are collected and combined to obtain the fraction Fr.D5-b-20.

[0020] S6: The Fr.D5-b-20 fraction obtained in step S5 is purified by high performance liquid chromatography to obtain the compound.

[0021] In step S6, the conditions of the purified mobile phase are as follows: mobile phase A is an aqueous solution containing 0.1 vol% formic acid, mobile phase B is methanol, and the volume ratio of mobile phase A to mobile phase B is 5:95.

[0022] Preferably, in step S1, the ethanol extract of the underground part of the giant feather fern is obtained by maceration.

[0023] Furthermore, the impregnation method is a repeated impregnation method (i.e., multiple impregnation methods).

[0024] Preferably, the impregnation method uses a 95 vol% ethanol solution as the impregnation agent.

[0025] Specifically, the impregnation method includes the following steps: after pretreatment of the dried underground part of Dryopteris macrocarpa, it is impregnated with 95 vol% ethanol solution as impregnation agent, and the resulting impregnation solution is desolventized to obtain the ethanol extract of the underground part of Dryopteris macrocarpa.

[0026] Preferably, the thorough impregnation is carried out at room temperature.

[0027] Preferably, the number of times the substance is fully impregnated is 1 to 5.

[0028] Furthermore, the number of times the substance is fully impregnated is four.

[0029] Preferably, the soaking time is 2 to 23 days, more preferably 15 days.

[0030] Furthermore, when using multiple immersions, the soaking time should be gradually extended each time.

[0031] Furthermore, the soaking time for each soaking is 2 to 8 days, preferably 2 to 6 days.

[0032] Specifically, the process of thorough impregnation is as follows: extracting and collecting the effluent by impregnation at room temperature, and repeating the impregnation process on the resulting residue; finally, combining all the effluent to obtain the impregnation solution.

[0033] Preferably, the mass-to-volume ratio of the dried underground part of the Dryopteris macrocarpa to the ethanol solution is (1~4) kg:10L.

[0034] Furthermore, the mass-to-volume ratio of the dried underground part of the Dryopteris macrocarpa to the ethanol solution is 3 kg: 10 L.

[0035] Furthermore, the pretreatment includes pulverization.

[0036] Preferably, the solvent removal is performed by vacuum distillation.

[0037] Preferably, in step S1, the mass-to-volume ratio of the ethanol extract of the underground part of the giant feather fern to water is (5~7) kg: 7 L.

[0038] Further, in step S1, the mass-to-volume ratio of the ethanol extract of the underground part of the giant feather fern to water is (5.5~6.5) kg:7 L.

[0039] Preferably, in step S1, the volume ratio of the suspension to petroleum ether is 1:(2~4).

[0040] Further, in step S1, the volume ratio of the suspension to petroleum ether is 1:(2.5~3.5).

[0041] More preferably, in step S1, the volume ratio of the suspension to petroleum ether is 1:3.

[0042] Preferably, in step S1, the solvent removal is performed by vacuum distillation.

[0043] Preferably, in steps S2 and S3, the silicone column is a normal phase silicone column.

[0044] Preferably, in steps S2 and S3, the silica gel particles in the normal phase silica gel column have a mesh size of 80 to 400 mesh.

[0045] Furthermore, the silica gel particles in the silica gel column have a particle size of 200-300 mesh.

[0046] Preferably, the mass of the silicone column is 2-5 kg.

[0047] Furthermore, the mass of the silicone column is 3.5 kg.

[0048] Preferably, in step S4, the gel column is a dextran gel column.

[0049] Furthermore, in step S4, the dextran gel column is a Sephadex LH-20 dextran gel column.

[0050] Specifically, in step S4, elution is performed using a 1:1 volume ratio of dichloromethane-methanol mixed solvent, combined with thin-layer chromatography (using a 1:1 volume ratio of dichloromethane-methanol mixed solvent as the developing solvent), and the fractions with fluorescent spots at 254 nm are collected and combined to obtain the Fr.D5-b fraction.

[0051] Preferably, in step S6, the high-performance liquid chromatography purification is performed using a preparative high-performance liquid chromatography column for separation and purification.

[0052] Specifically, the preparative high-performance liquid chromatography column is preferably an octadecylsilane-bonded silica column, model Cosmosil C18 column (20 × 250 mm, 5 μm).

[0053] Furthermore, in step S6, the compound can be distinguished from other impurities by the elution time of the chromatographic peak. In the method of this application, the retention time of the compound is approximately 26 min. Within the determined retention time range, the components corresponding to each chromatographic peak can be collected separately, and then the structures of the obtained components can be confirmed by nuclear magnetic resonance (NMR) technology.

[0054] The present invention also protects an extract containing the compound.

[0055] Furthermore, the extract is a plant extract, and even more specifically, a *Dryopteris macrocarpa* extract.

[0056] The present invention also protects a pharmaceutical composition comprising the compound or the extract of the fern *Dryopteris macrocarpa*.

[0057] The present invention also protects the use of the compound, the extract of the fern *Tetracentron sinense*, or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0058] Preferably, the cellular administration concentration of the compound is 1-100 μM.

[0059] Furthermore, the cellular administration concentration of the compound is 6.25–50 μM.

[0060] More preferably, the cellular administration concentration of the compound is 12.5 to 50 μM.

[0061] Most preferably, the cellular administration concentration of the compound is 25 μM.

[0062] Furthermore, the prevention and / or treatment of non-alcoholic fatty liver disease involves inhibiting the accumulation of lipid droplets in cells and reducing lipid levels in cells.

[0063] Furthermore, the prevention and / or treatment of non-alcoholic fatty liver disease involves inhibiting the expression of fatty acid uptake genes.

[0064] Furthermore, the fatty acid uptake genes include FATP2 and / or CD36.

[0065] The present invention has the following beneficial effects:

[0066] This invention provides a novel natural compound obtained from *Dryopteris macrocarpa* for the first time, along with a method for its preparation. Activity studies were conducted, and experimental data show that the compound exhibits low toxicity to normal cells. It can inhibit fatty acid uptake in cells by suppressing the expression of fatty acid uptake-related genes FATP2 and CD36, thereby inhibiting lipid droplet accumulation and reducing lipid levels in cells. This compound can be applied to the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease. Attached Figure Description

[0067] Figure 1 The hydrogen nuclear magnetic resonance spectrum of DWR-12, an extract of Dryopteris macrocarpa.

[0068] Figure 2 The carbon nuclear magnetic resonance spectrum of DWR-12, an extract of Dryopteris macrocarpa.

[0069] Figure 3 The graph shows the cytotoxicity of different concentrations of DWR-12 extract from Dryopteris macrocarpa on HepG2 cells. ns indicates no significant difference in results.

[0070] Figure 4 The figure shows the effect of DWR-12, an extract of Dryopteris macrocarpa, on oleic acid-induced lipid droplet accumulation in HepG2 cells.

[0071] Figure 5 The figure shows the effect of DWR-12 extract from Dryopteris macrocarpa on triglyceride levels in oleic acid-induced HepG2 cells. *** indicates P<0.001.

[0072] Figure 6 The figure shows the effect of treatment with DWR-12 extract from Dryopteris macrocarpa on the expression levels of fatty acid uptake-related genes FATP2 and CD36. ** indicates P<0.01. Detailed Implementation

[0073] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0074] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0075] Example 1: Isolation and extraction of DWR-12 from Dryopteris macrocarpa extract

[0076] 30 kg of dried underground parts of *Dryopteris macrocarpa* were pulverized and extracted four times at room temperature using 100 L of 95 vol% ethanol. The extraction was performed for two days, and the eluent was collected. The residue was then soaked for three days, and the eluent was collected again. The residue was then soaked for four days, and the eluent was collected again. The residue was then soaked for six days, and the eluent was collected again. The four extracts were combined and concentrated under reduced pressure to recover the solvent, yielding a crude extract (6.1 kg). The extract was suspended in 7.0 L of water, and the suspension was extracted with three times the volume of petroleum ether. The solvent was recovered under reduced pressure to obtain the petroleum ether fraction extract (714.4 g). The petroleum ether extract was subjected to silica gel column chromatography using a normal-phase silica gel column (silica gel particle size 200-300 mesh, weight 3.5 kg). Gradient elution was performed using petroleum ether-ethyl acetate mixed solvents at volume ratios of 100:0, 100:1, 20:1, and 5:1. The fractions eluted by the 20:1 and 5:1 volume ratios of the petroleum ether-ethyl acetate mixed solvents were collected and combined to obtain fraction Fr.D. Fraction Fr.D (208.2 g) was then subjected to silica gel column chromatography using a normal-phase silica gel column (silica gel particle size 200-300 mesh, weight 3.5 kg). Further purification was achieved by elution using petroleum ether-ethyl acetate mixed solvents at volume ratios of 10:1 and 5:1. The fraction eluted by the 5:1 volume ratio of the petroleum ether-ethyl acetate mixed solvent was collected to obtain fraction Fr.D5. The fraction Fr.D5 (49.1 g) was subjected to gel column chromatography using a Sephadex LH-20 dextran column, eluted with a 1:1 (v / v) dichloromethane-methanol mixture. Thin-layer chromatography (TLC) was then performed, with the fractions exhibiting fluorescent spots at 254 nm collected and combined to obtain fraction Fr.D5-b. The Fr.D5-b fraction was then subjected to ODS column chromatography, eluted sequentially with 0 vol%, 70 vol%, 80 vol%, 85 vol%, 90 vol%, and 95 vol% methanol-water solutions. The fractions eluted with 90 vol% and 95 vol% methanol-water solutions were collected and combined to obtain fraction Fr.D5-b-20. Finally, the fraction Fr.D5-b-20 was purified by HPLC using a Cosmosil C18 column (20 × 250 mm, 5 μm). The mobile phase conditions were: mobile phase A was an aqueous solution containing 0.1 vol% formic acid, and mobile phase B was methanol, with a volume ratio of 5:95. Compound DWR-12 was obtained, with a retention time of approximately 26 min. Its structure is shown below:

[0077] .

[0078] The structural identification data of compound DWR-12 are shown in Table 1, and its hydrogen atom nuclear magnetic resonance spectrum is shown in Table 1. Figure 1 Carbon atom nuclear magnetic resonance spectrum see Figure 2 .

[0079] Table 1. Nuclear magnetic resonance (NMR) spectra of compound DWR-12 (H NMR and carbon NMR spectra, CDCl3, ...). δ , J (inHz) a

[0080]

[0081] Example 2: Application of DWR-12 extract from Dryopteris macrocarpa

[0082] 1. Cell Culture

[0083] The HepG2 cell line used in this experiment was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. It was cultured in a 37°C cell culture incubator containing 5% CO2 in high-glucose DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0084] 2. Cytotoxicity test

[0085] Take HepG2 cells in good growth condition and administer them at a rate of 1.5 × 10⁻⁶. 4 Each well was inoculated with one sample of *Dryopteris macrocarpa* extract DWR-12 into a 96-well plate. A 50 mM stock solution was prepared by dissolving *Dryopteris macrocarpa* extract DWR-12 in DMSO, and seven concentration gradients were established (0, 3.125, 6.25, 12.5, 25, 50, 100 μM). The medium in the 96-well plates was replaced with medium containing different concentration gradients of *Dryopteris macrocarpa* extract DWR-12, and cultured for 24 hours. The medium was then discarded, and 100 μL of CCK-8 working solution was added to each well. The plates were then incubated in the dark for 2 hours, followed by measurement of absorbance at 450 nm.

[0086] To determine the cytotoxicity of DWR-12, an extract from *Dryopteris macrocephala*, against HepG2 cells, cell viability was measured using a CCK-8 assay kit. Results are shown below. Figure 3 No significant cytotoxicity was observed after treatment with 3.125, 6.25, 12.5, 25, 50 and 100 μM, and 25 μM was selected as the drug concentration for subsequent experiments.

[0087] 3. Cell staining and fluorescence experiments

[0088] Cells were seeded at an appropriate density onto cell slides and, after adhesion, treated with OA and DWR-12 extract from *Dryopteris macrocarpa*. After treatment, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes. After washing three times with PBS, 2 mg / mL Bodipy dye was diluted 1:5000 with PBS, and 1 mL of dye was added to each well. The cells were incubated at room temperature in the dark for 20 minutes. After staining, the dye was removed, and the cell nuclei were stained with DAPI. Lipid droplet accumulation in the cells was observed using a Leica SP8 confocal microscope.

[0089] See results Figure 4 In the negative control CT group, intracellular lipid droplet accumulation was low, while in the positive control OA treatment group, intracellular lipid droplets significantly increased. However, after treatment with OA and DWR-12 extract from *Dryopteris macrocarpa*, intracellular lipid droplet accumulation was significantly reduced compared to the positive control group, indicating that DWR-12 extract from *Dryopteris macrocarpa* can effectively inhibit lipid accumulation in HepG2 cells induced by OA.

[0090] 4. Triglyceride content detection

[0091] Cells were seeded at an appropriate density into well plates and, after adhesion, treated with OA and DWR-12 extract from *Dryopteris macrocarpa*. Cells were digested with trypsin, collected by centrifugation, and then lysis buffer was added. The cells were allowed to stand at room temperature for 10 minutes. A suitable amount of supernatant was transferred to a 1.5 mL centrifuge tube. The tube was heated at 70°C for 15 minutes. The remaining lysis buffer was centrifuged at 8000×g for 10 minutes at 4°C, and the supernatant was used for BCA quantification of protein concentration. The sample heated at 70°C was centrifuged at 12000×g for 15 minutes at room temperature; the supernatant was then ready for enzymatic assays. The working solution for the enzymatic assay was prepared by mixing 4 mL of reagent R1 and 1 mL of reagent R2 at a 4:1 ratio. 10 μL of the supernatant was added to a 96-well plate, followed by 100 μL of the working solution. The plate was incubated at 37°C for 15 minutes.

[0092] See results Figure 5 The determination of triglyceride content further confirmed that DWR-12 can effectively inhibit lipid accumulation. After OA treatment, the intracellular triglyceride level of HepG2 cells increased significantly. After OA + DWR-12 extract of Dryopteris macrocephala, the intracellular triglyceride level was significantly reduced compared with the positive control group.

[0093] 5. RNA extraction

[0094] When cell confluence reaches 90%, remove the culture medium, wash once with PBS, and add 500 μL of RNAiso Plus to each well. After shaking for 2 minutes, transfer the RNAiso from the wells to RNase-free EP tubes. Add 1 / 5 volume of chloroform to the EP tube and shake vigorously until the liquid becomes emulsion. Let it stand at room temperature for 5 minutes. Centrifuge the EP tubes at 12000×g, 4°C for 15 minutes. After centrifugation, carefully remove the EP tubes, avoiding shaking. At this point, the bottom layer of the EP tube is the organic phase, the middle layer contains protein (gDNA), and the upper aqueous phase contains RNA. Carefully transfer the upper aqueous phase to a new RNase-free EP tube, avoiding contact with the middle and bottom layers. Let it stand for at least 10 minutes to precipitate the RNA. Centrifuge at 12000×g, 4°C for 10 minutes. A white precipitate will be visible at the bottom of the EP tube. Carefully aspirate the supernatant, being careful not to touch the precipitate. Add an equal volume of 75% ethanol to the tube (equal to the volume of RNAiso Plus), and invert the tube to allow the precipitate to float. Centrifuge at 7500×g, 4℃ for 10 minutes. Carefully aspirate the supernatant, and place the EP tube in a clean bench to air dry until the precipitate becomes transparent. Dissolve the precipitate with an appropriate amount of DEPC H2O, depending on the volume.

[0095] 6. Reverse transcription

[0096] Mix the RNA sample thoroughly, take 1 μL and measure the concentration using Nano Drop One to calculate the required volume of 1 μg RNA sample (x μL). Perform the first step of reverse transcription according to the system in Table 2. Mix the sample thoroughly, incubate at 65°C for 5 minutes, then remove the sample and place it on ice for later use.

[0097] Table 2 Reverse Transcription System

[0098]

[0099] Calculate the required amount of enzyme mix by adding 0.5 μL of AB Script II enzyme to each sample vial, and mix well. Add 5.5 μL of enzyme mix to each vial and mix well. Incubate at room temperature for 5 minutes, then perform reverse transcription in a 42°C water bath for 1 hour. Terminate the reaction by heating in an 80°C metal bath for 5 minutes. Dilute the sample to the working concentration by adding 90 μL of ddH2O to each vial.

[0100] 7. Real-time quantitative PCR (RT-qPCR)

[0101] Primer sequences are shown in Table 3. The primers were synthesized by Guangzhou Aiji Biotechnology.

[0102] Table 3 RT-qPCR primer sequences

[0103]

[0104] Configure the qPCR reaction system according to the system in Table 4.

[0105] Table 4 RT-qPCR System

[0106]

[0107] The qPCR program was set up as follows: 95℃ for 30 s, 95℃ for 10 s, 65℃ for 10 s, for a total of 30 cycles. β-actin was used as an internal control, and 2... -△△CT The method calculates the relative expression level of the target gene.

[0108] The experimental results are shown in Figure 6 Real-time quantitative PCR experiments demonstrated that DWR-12, an extract from Dryopteris macrocephala, affects lipid accumulation in cells by inhibiting fatty acid uptake. Compared with the control DMSO group, the expression of lipid synthesis-related genes FATP2 and CD36 was significantly downregulated after DWR-12 treatment.

[0109] The experimental data above indicate that the compound has low toxicity to normal cells. It can inhibit the uptake of fatty acids in cells by inhibiting the expression of fatty acid uptake-related genes FATP2 and CD36, thereby inhibiting the accumulation of lipid droplets in cells and reducing lipid levels in cells. It can be applied to the field of preparing drugs for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound, characterized in that, The compound has the following structure: 。 2. The compound according to claim 1, characterized in that, The compounds also include their pharmaceutically acceptable salts.

3. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: S1: Prepare a suspension by adding water to the alcohol extract of the underground part of Dryopteris macrocarpa, extract with petroleum ether, remove the organic layer to remove the solvent, and obtain the extract paste. S2: The extract obtained in step S1 is separated by silica gel column chromatography, and the extract is eluted sequentially with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 100:0, 100:1, 20:1 and 5:

1. The fractions eluted with the petroleum ether-ethyl acetate mixed solvent with a volume ratio of 20:1 and 5:1 are collected and combined to obtain the fraction Fr.D. S3: The Fr.D fraction obtained in step S2 is separated by silica gel column chromatography, and eluted sequentially with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 10:1 and 5:

1. The fraction eluted with the petroleum ether-ethyl acetate mixed solvent with a volume ratio of 5:1 is collected to obtain the fraction Fr.D5. S4: The Fr.D5 fraction obtained in step S3 is separated by gel column chromatography and eluted with a 1:1 volume ratio of dichloromethane-methanol mixed solvent to obtain the fraction Fr.D5-b. S5: The Fr.D5-b fraction obtained in step S4 is separated by ODS column chromatography, and the fraction is eluted sequentially with 0 vol%, 70 vol%, 80 vol%, 85 vol%, 90 vol%, and 95 vol% methanol-water solution. The fractions eluted with 90 vol% and 95 vol% methanol-water solution are collected and combined to obtain the fraction Fr.D5-b20. S6: The Fr.D5-b-20 fraction obtained in step S5 is purified by high performance liquid chromatography to obtain the compound; In step S6, the conditions of the purified mobile phase are as follows: mobile phase A is an aqueous solution containing 0.1 vol% formic acid, mobile phase B is methanol, and the volume ratio of mobile phase A to mobile phase B is 5:

95.

4. The preparation method according to claim 3, characterized in that, In step S1, the ethanol extract of the underground part of the giant feather fern is obtained by maceration.

5. The preparation method according to claim 3, characterized in that, In steps S2 and S3, the silicone column is a normal phase silicone column.

6. The preparation method according to claim 3, characterized in that, In step S4, the gel column is a dextran gel column.

7. The preparation method according to claim 6, characterized in that, The dextran gel column is a Sephadex LH-20 dextran gel column.

8. An extract, characterized in that, The extract contains the compound of claim 1.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1 or 2 or the extract of claim 8.

10. The use of the compound of claim 1 or 2, the extract of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of non-alcoholic fatty liver disease.

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