Application of sesquiterpene dimer compound of Hubei chloranthus spicatus in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention discloses application of a Hubei chloranthus spicatus sesquiterpene dimer compound in preparation of a medicine for treating a non-alcoholic fatty liver disease. The structural formula of the sesquiterpene dimer compound is shown as a formula (I). The invention finds that the compound has the effects of reducing liver cell lipid accumulation and improving the non-alcoholic fatty liver disease for the first time. And compared with pioglitazone recommended by international guidelines, the compound has the advantages of better non-alcoholic fatty liver disease resistance drug effect and lower toxicity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the fields of natural medicinal chemistry and pharmacology, and in particular to application of a Hubei Jinsulan sesquiterpene dimer compound in preparing a drug for treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease unrelated to alcohol. Its core characteristic is the excessive accumulation of fat within liver cells, which may be accompanied by inflammation, fibrosis, and even cirrhosis. Currently, there is only one approved new drug for NAFLD: Resmetirom, which was approved by the US FDA in March 2024, but it has not yet entered the Chinese market. Currently, antioxidants, insulin sensitizers, and lipid-lowering drugs are used to alleviate NAFLD symptoms in China, and some Western medications are still in clinical trials. Active ingredients derived from traditional Chinese medicine are a research hotspot for new anti-NAFLD drugs.

[0003] Chloranthus henryi (Chloranthus henryi Hemsl. var. hupehensis (Pamp.) KFWu) is a perennial herbaceous plant of the Chloranthus family. It is widely distributed in Hubei and Sichuan provinces of China. Its roots, rhizomes, or the entire plant are primarily used as medicine. It is pungent and bitter, warm in nature, and toxic. It has the effects of dispelling wind and cold, relaxing muscles and strengthening bones, promoting blood circulation and dispersing blood stasis, and relieving swelling and pain. With in-depth research on this genus, its chemical composition and pharmacological effects have attracted widespread attention. Sesquiterpenes and their dimers in Chloranthus henryi plants exhibit a variety of biological activities, including anti-inflammatory, anti-tumor, anti-malarial, antioxidant, and neuroprotective effects, and have significant potential for drug development. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide a method for preparing a Hubei Jinsulan sesquiterpene dimer compound.

[0005] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned compound in the preparation of anti-NAFLD drugs.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] The use of a Hubei Jinsulan sesquiterpene dimer compound in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the Hubei Jinsulan sesquiterpene dimer compound has a structural formula as shown in formula (I) and is hereinafter named Cycloshizukaol A:

[0008]

[0009] The preparation method of the above-mentioned Hubei Jinsulan sesquiterpene dimer compound comprises the following steps:

[0010] a. Extract the roots of Hubei Jinsulan using ethanol and aqueous solution, and concentrate to obtain an extract;

[0011] b. The extract was dissolved in methanol, adsorbed by diatomaceous earth, and then the solvent was evaporated;

[0012] c. The substance obtained in step b was separated by column chromatography, eluted with ethyl acetate, and concentrated to obtain the ethyl acetate elution portion;

[0013] d. The ethyl acetate elution fraction was dissolved with methanol, applied to a silica gel column, and subjected to a first gradient elution using a mixed solution of petroleum ether and ethyl acetate. The first gradient elution was performed sequentially using a volume ratio of petroleum ether: ethyl acetate = 100:1, 100:2, 100:3, 100:5, 100:10, 100:15, 100:30, 2:1, and 1:1 gradient elution; similar terms were combined using a thin layer silica gel plate to obtain 9 components Fr.1 to Fr.9 according to the compound polarity from small to large;

[0014] e. The component Fr.7 obtained in step d was dissolved in methanol and applied to a silica gel column. The mixture was eluted with a mixed solution of dichloromethane and methanol for a second gradient elution. The second gradient elution was performed sequentially using a volume ratio of dichloromethane: methanol = 100:1, 100:2, 100:3, 100:4, 100:5, 100:6, 100:7, 100:8, 100:9, 100:10 gradient elution; similar items were combined using a thin layer silica gel plate to obtain 12 components Fr.7-1 to Fr.7-12 according to the compound polarity from small to large;

[0015] f. The component Fr.7-3 obtained in step e was separated by medium pressure column chromatography, and then separated by preparative liquid phase to obtain the Hubei Jinsulan sesquiterpene dimer compound.

[0016] In step a, the roots of the Hubei Chrysanthemi orchid are pre-crushed before extraction; the concentration of the ethanol aqueous solution is 50-99% v / v (preferably 75% v / v); the extraction is performed by heating and reflux extraction at a temperature of 75-85° C., 2-3 times (preferably 3 times), and each extraction takes 2-3 hours (preferably 3 hours).

[0017] In step b, the mass ratio of the extract to the diatomaceous earth is 1:0.8-1.2 (preferably 1:1).

[0018] In step a or step c, the concentration is performed by reducing pressure until there is no solvent smell.

[0019] In step d, the ethyl acetate elution portion can be dissolved with methanol; the volume ratio of the sample loading amount to the silica gel column is 1:5 to 1:10 (preferably 1:8).

[0020] In step e, the component Fr.7 obtained in step d can be dissolved in methanol; the volume ratio of the sample loading amount to the silica gel column is 1:8 to 1:12 (preferably 1:10).

[0021] In step f, the medium-pressure column chromatography method is a uniform linear elution of 20-60% v / v methanol aqueous solution for 8 hours (within 8 hours, the concentration of methanol aqueous solution is uniformly increased from 20% to 60%), a flow rate of 20 mL / min, starting from 0 o'clock, and collecting 1 fraction every 30 minutes, collecting the 9th fraction; the preparative liquid phase separation is carried out under the condition of using 35% v / v acetonitrile aqueous solution.

[0022] The Hubei Jinsulan sesquiterpene dimer compound treats non-alcoholic fatty liver disease by downregulating the contents of lipid droplets, triglycerides (TG) and cholesterol (TC) in liver cells.

[0023] A pharmaceutical preparation comprising the above-mentioned compound (I) and pharmaceutically acceptable excipients; the preferred dosage form of the preparation is granules, tablets, capsules, oral liquids, pills, emulsions, suspensions, injections, infusions or sprays.

[0024] Preferably, the dosage form is a tablet, and pharmaceutically acceptable excipients include diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets or multi-layer tablets.

[0025] Preferably, the dosage form is a capsule, and pharmaceutically acceptable excipients include diluents, glidants, binders, and disintegrants. The active ingredient and excipients can be mixed and the mixture can be directly placed in a hard capsule or soft capsule, or the mixture can be made into granules or pellets and then placed in a hard capsule or soft capsule. Among them, the diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate; the wetting agent can be water, ethanol, isopropyl alcohol; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate; the lubricant and glidant can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol.

[0026] Preferably, the dosage form is an injection, and water, ethanol, isopropyl alcohol, propylene glycol, or a mixture thereof can be used as a solvent, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, or hydroxypropyl-β-cyclodextrin; the pH adjuster can be phosphate, acetate, hydrochloric acid, or sodium hydroxide; and the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, or acetate.

[0027] Preferably, colorants, preservatives, spices, flavoring agents or other additives are added to the pharmaceutical preparation as excipients.

[0028] Compared with the prior art, the present invention has the following significant advantages:

[0029] 1. This study first discovered that Cycloshizukaol A, a sesquiterpenoid dimer compound derived from Hubei Jinsulan, can reduce lipid accumulation in hepatocytes, and confirmed its anti-NAFLD activity through in vivo and in vitro experiments.

[0030] 2. Compared with the commonly used clinical drug pioglitazone, this compound has better anti-NAFLD activity and lower toxicity, and has the potential to be further developed into a new anti-NAFLD drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0032] Figure 1This is a separation process of the sesquiterpene dimer compound Cycloshizukaol A;

[0033] Figure 2 This is the hydrogen spectrum of the sesquiterpene dimer compound Cycloshizukaol A;

[0034] Figure 3 This is the carbon spectrum of the sesquiterpene dimer compound Cycloshizukaol A;

[0035] Figure 4 This is the high-resolution mass spectrum of the sesquiterpene dimer compound Cycloshizukaol A;

[0036] Figure 5 This is a HepG2 cell toxicity test of the sesquiterpene dimer compound Cycloshizukaol A;

[0037] Figure 6 The anti-lipid accumulation effect of sesquiterpenoid dimer compound Cycloshizukaol A on HepG2 cells;

[0038] Figure 7 The effect of sesquiterpene dimer compound Cycloshizukaol A on reducing TG and TC in HepG2 cells;

[0039] Figure 8 The sesquiterpene dimer compound Cycloshizukaol A improves liver pathology in the MAFLD zebrafish model;

[0040] Figure 9 The inhibitory effect of sesquiterpenoid dimer compound Cycloshizukaol A on lipid accumulation in the MAFLD zebrafish model;

[0041] Figure 10 The inhibitory effect of the sesquiterpene dimer compound Cycloshizukaol A on TG and TC in the MAFLD zebrafish model. DETAILED DESCRIPTION

[0042] Example 1: Isolation and physicochemical characterization of the sesquiterpene dimer compound Cycloshizukaol A

[0043] like Figure 1 As shown, in this embodiment, the preparation steps of the sesquiterpene dimer compound Cycloshizukaol A are as follows:

[0044] a. The dried root materials of Hubei Jin Sulan (50.0kg) were crushed and soaked in a 75% v / v aqueous ethanol solution (80L) for 24h, then extracted three times under reflux at 85°C for 3h each. The extract was concentrated under reduced pressure until it had no solvent taste to obtain an extract (5.0kg);

[0045] b. Dissolve the extract in methanol, add an equal amount of diatomaceous earth and mix thoroughly for adsorption (the mass ratio of extract to diatomaceous earth is 1:1), let it dry, grind into powder, and load into the chromatography column;

[0046] c. Sequentially eluted with petroleum ether, ethyl acetate and methanol, and concentrated under reduced pressure to obtain three elution fractions;

[0047] d. The ethyl acetate elution fraction (1.25 kg) was dissolved in methanol and loaded onto a silica gel column at a volume ratio of 1:8. Gradient elution was performed using petroleum ether:ethyl acetate = 100:1, 100:2, 100:3, 100:5, 100:10, 100:15, 100:30, 2:1, and 1:1, in that order. After TLC analysis, similar compounds were combined in ascending order of polarity to obtain nine fractions (Fr.1 to Fr.9).

[0048] e. The component Fr.7 obtained in step d was dissolved in methanol and loaded onto a silica gel column. The volume ratio of the sample to the silica gel column was 1:10. Gradient elution was performed using dichloromethane: methanol = 100:1, 100:2, 100:3, 100:4, 100:5, 100:6, 100:7, 100:8, 100:9, 100:10, and TLC analysis was performed. The compounds were separated by combining similar items from small to large polarity to obtain 12 components (Fr.7-1 to Fr.7-12).

[0049] f. The component Fr.7-3 obtained in step e was separated by medium pressure column chromatography, and 20-60% v / v methanol aqueous solution was linearly eluted at a uniform rate for 8 hours (within 8 hours, the concentration of methanol aqueous solution was uniformly increased from 20% to 60%), at a flow rate of 20 mL / min, starting from 0 o'clock, and 1 fraction was collected every 30 minutes. The 9th fraction was collected and then prepared by liquid phase (conditions of 35% v / v acetonitrile aqueous solution) to obtain a sesquiterpene dimer compound CycloshizukaolA (240 mg) from Fr.7-3-9.

[0050] The physicochemical properties and spectral data of the sesquiterpene dimer compound Cycloshizukaol A are as follows:

[0051] Physical and chemical properties: The compound is a light yellow solid.

[0052] Spectral data ( Figures 2-4 ): UV(CH3OH)λmax :223, 335nm. HRESI-MSm / z 549.2470[M+H] + , determine the molecular formula C 32 H 36 O8, NMR data see Table 1.

[0053] Table 1 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of Cycloshizukaol A (in CDCl3)

[0054]

[0055] Example 2: In vitro investigation of the effect of Cycloshizukaol A on NAFLD model hepatocytes

[0056] ① Cycloshizukaol A toxicity to HepG2 cells

[0057] HepG2 cells in good condition were counted at 2×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured in 200 μL of serum-free DMEM medium for 24 hours. The total volume per well was 200 μL, with DMEM as the primary solvent. Other components included 1% bovine serum albumin (BSA) without free fatty acids (FFA), oleic acid, palmitic acid, and varying concentrations of Cycloshizukaol A and pioglitazone. 1 mM FFA was prepared by dissolving oleic acid and palmitic acid in a 2:1 volume ratio of 1% BSA without FFA. Experiments were divided into a control group (1% BSA without FFA), a model group (containing 1.0 mM FFA), and a treatment group. For the half-inhibitory assay of HepG2 cells, Cycloshizukaol A was administered at concentrations of 0, 0.1, 1.0, 10, 100, and 200 μM. In HepG2 cytotoxicity experiments, different concentrations of Cycloshizukaol A (5μM), Cycloshizukaol A (10μM), Pioglitazone (10μM), and their combination with free fatty acids (FFA, 1.0mM) were administered to the HepG2 cells and incubated for 24 hours. The supernatant was then discarded, the cells were washed twice with PBS, and DMEM supplemented with 10% CCK-8 was added for further incubation. After a one-hour incubation, the absorbance at 450nm was measured. Cell viability was calculated based on the absorbance.

[0058] ②HepG2 cell grouping and drug administration

[0059] HepG2 cells with good growth status were digested and passaged, and 5×10 5 Cells were seeded at a density of 10 cells / mL in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. The total volume per well was 2 mL, with DMEM as the primary solvent. Other components included FFA-free 1% BSA, oleic acid, palmitic acid, and varying concentrations of Cycloshizukaol A and pioglitazone. Experimental groups included: normal control group (1% BSA without FFA); model group (1 mM FFA); low-dose Cycloshizukaol A group (1 mM FFA + 5 μM); high-dose Cycloshizukaol A group (1 mM FFA + 10 μM); and pioglitazone group (1 mM FFA + 10 μM). FFA, Cycloshizukaol A, and pioglitazone were added simultaneously to serum-free DMEM and cultured for 24 hours, after which cells were harvested.

[0060] ③ Oil red O staining of hepatocytes

[0061] Aspirate the medium from the 6-well plate and wash three times with 37°C PBS. Fix with 4% paraformaldehyde at 4°C for 15 minutes, discard the paraformaldehyde, and wash three times with tap water, each for 5 minutes. Add 1.0 mL of Oil Red O working solution to each well and stain for 30 minutes. Discard the Oil Red O staining solution, wash three times with triple-distilled water, each for 5 minutes, and photograph under an inverted microscope. Then, add 100 μL of isopropanol to each well and incubate at room temperature for 15 minutes. Gently shake to mix, transfer to a 96-well plate, and measure absorbance using a microplate reader at a wavelength of 520 nm.

[0062] ④Determination of triglyceride and cholesterol content in hepatocytes

[0063] Cells were harvested from 6-well plates and assayed for TG and TC levels according to the TG and TC assay kit instructions. Total cellular protein was measured using the BCA protein concentration assay.

[0064] like Figure 5 As shown in Figure 2, 1 mM FFA induced HepG2 cells for 24 hours to establish a hepatic steatosis model. Cycloshizukaol A cytotoxicity to HepG2 cells was detected by CCK8 and showed that: Cycloshizukaol A IC 50 The value was 38.92 μM. Compared with the positive drug pioglitazone, the cell viability after treatment with Cycloshizukaol A (5 μM) was equivalent to that after treatment with pioglitazone (10 μM). The cell viability after treatment with Cycloshizukaol A (10 μM) was higher than that after treatment with pioglitazone (10 μM). The experimental results showed that Cycloshizukaol A had lower toxicity.

[0065] like Figure 6 and Figure 7 As shown, the lipid droplet content, TG, and TC content in the model group were significantly higher than those in the control group, indicating successful modeling. Compared with the model group, Cycloshizukaol A (5 and 10 μM) significantly reduced the lipid droplet, TG, and TC content in HepG2 cells in a dose-dependent manner. Pioglitazone (10 μM) also significantly reduced the lipid droplet, TG, and TC content in HepG2 cells. However, at the same dose (10 μM), Cycloshizukaol A was more effective than pioglitazone.

[0066] Example 3: In vivo investigation of the lipid-lowering effect of Cycloshizukaol A in a MAFLD zebrafish model

[0067] ① Zebrafish grouping and drug administration

[0068] Cholesterol (purity 92.5%) was purchased from Sigma-Aldrich, and zebrafish basal larval feed (AP100) was purchased from Zeigler. The feed contains 12% crude fat and 50% crude protein.

[0069] High cholesterol diet (HCD) was prepared as follows: cholesterol was first mixed with basal diet in ether (2 ml / g diet). The ether was then evaporated from the HCD in a 60°C water bath. The final cholesterol concentration in HCD was 5% (w / w). Normal diet (ND) was prepared using the same method as HCD, except that only ether was used.

[0070] Wild-type AB zebrafish were maintained in a 14 / 10-hour day / night cycle with a water temperature of (28±1)°C, a pH of 7.4, and a salt concentration of approximately 0.004%. Healthy AB juveniles 6 days post fertilization (dpf) were transferred to 100 mm glass culture plates (1 L / plate). A MAFLD zebrafish model was established by inducing 5% high cholesterol for 2 weeks: either the high cholesterol diet (HCD) group or the drug group was fed HCD (20 mg / plate daily) starting on day 8 post fertilization (dpf); the control group was fed ND starting on day 8. The zebrafish juveniles were divided into four groups, each containing 100 fish. The experimental groups were designed as follows: blank control group fed with normal diet (ND), model group fed with high cholesterol diet (HCD), low-dose Cycloshizukaol A group (HCD+2.5μM Cycloshizukaol A), and high-dose Cycloshizukaol A group (HCD+10μM Cycloshizukaol A).

[0071] ② Zebrafish HE staining

[0072] After administration, zebrafish from each group (n=10) were collected, fixed, embedded in paraffin, and sliced, stained with HE, and mounted with neutral gum. Images were collected under an optical microscope to analyze the pathological changes in the liver tissue of zebrafish larvae in each group.

[0073] ② Zebrafish Oil Red O staining

[0074] Zebrafish (n=10) were collected from each group and fasted for 24 hours after modeling. They were then fixed with 4% paraformaldehyde and allowed to stand at room temperature for 48 hours. After removal from the fixative, residual paraformaldehyde was washed away with distilled water. The juveniles were then permeabilized with 40%, 60%, 80%, and 100% 1,2-propylene glycol solutions, each for 20 minutes. Following permeabilization, the fish were stained with 0.3% Oil Red O solution, and the floating color was washed away with 60°C isopropanol. The samples were then preserved in a 50% glycerol-water solution. The fish were observed and photographed under a stereomicroscope for staining. Image J software was used to calculate the Oil Red O staining area on the abdomen of the juveniles, and the average optical density (A value) of the liver was measured.

[0075] ③Determination of triglyceride and cholesterol content in zebrafish

[0076] Zebrafish from each group (n=20) were collected and the TG and TC levels in cells were determined according to the instructions of the TG and TC kits. The total protein content in cells was determined using the BCA protein concentration assay.

[0077] like Figure 8 Figure 2 shows a magnified image of the zebrafish abdominal liver (×200). Compared with the control group, the hepatocytes of zebrafish larvae in the model group were less tightly packed, had sparse cytoplasm, and showed more hepatocyte vacuolation. Compared with the model group, treatment with 2.5μM and 10μM CycloshizukaolA resulted in more compact hepatocytes, tighter cytoplasm, and significantly less hepatocyte vacuolation.

[0078] Oil red O staining was used to detect the lipid accumulation in the zebrafish liver area, and Image J software was used to calculate the average optical density of the stained liver area of ​​the larvae. Figure 9 As shown in the figure, compared with the control group, the average A value of the zebrafish liver region in the model group was significantly increased (P < 0.01), indicating that a large amount of lipid droplets were accumulated in the liver tissue of the larval fish. Compared with the model group, the lipid accumulation in the liver of the larval fish in the low-dose and high-dose CycloshizukaolA groups (2.5 and 10 μM) was significantly reduced (P < 0.05 and 0.01), indicating that CycloshizukaolA can reduce lipid accumulation in liver tissue in a dose-dependent manner.

[0079] The TG and TC contents of zebrafish were detected by TG and TC kits (such as Figure 10 The TG and TC contents in the model group were significantly higher than those in the control group. Compared with the model group, the TG and TC contents in the juvenile fish of the low-dose and high-dose Cycloshizukaol A groups (2.5 and 10 μM) decreased in a dose-dependent manner.

[0080] The present invention provides a method for preparing and applying a Hubei Jinsulan sesquiterpene dimer compound. There are many methods and approaches for implementing this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Any components not specified in this embodiment can be implemented using existing technologies.

Claims

1. Application of Hubei Jinsulan sesquiterpene dimer compound in the preparation of drugs for treating non-alcoholic fatty liver disease, wherein: The structural formula of the Hubei Jinsulan sesquiterpene dimer compound is shown in formula (I):

2. The use according to claim 1, characterized in that The Hubei Jinsulan sesquiterpene dimer compound is prepared according to the following steps: a. Extract the roots of Hubei Jinsulan using ethanol and aqueous solution, and concentrate to obtain an extract; b. The extract was dissolved in methanol, adsorbed by diatomaceous earth, and then the solvent was evaporated; c. The substance obtained in step b was separated by column chromatography, eluted with ethyl acetate, and concentrated to obtain the ethyl acetate elution portion; d. The ethyl acetate elution fraction was dissolved with methanol, applied to a silica gel column, and subjected to a first gradient elution using a mixed solution of petroleum ether and ethyl acetate. The first gradient elution was performed sequentially using a volume ratio of petroleum ether: ethyl acetate = 100:1, 100:2, 100:3, 100:5, 100:10, 100:15, 100:30, 2:1, and 1:1 gradient elution; similar terms were combined using a thin layer silica gel plate to obtain 9 components Fr.1 to Fr.9 according to the compound polarity from small to large; e. The component Fr.7 obtained in step d was dissolved in methanol and applied to a silica gel column. The mixture was eluted with a mixed solution of dichloromethane and methanol for a second gradient elution. The second gradient elution was performed sequentially using a volume ratio of dichloromethane: methanol = 100:1, 100:2, 100:3, 100:4, 100:5, 100:6, 100:7, 100:8, 100:9, 100:10 gradient elution; similar items were combined using a thin layer silica gel plate to obtain 12 components Fr.7-1 to Fr.7-12 according to the compound polarity from small to large; f. The component Fr.7-3 obtained in step e was separated by medium pressure column chromatography, and then separated by preparative liquid phase to obtain the Hubei Jinsulan sesquiterpene dimer compound.

3. The use according to claim 2, characterized in that In step a, the roots of the Hubei Chrysanthemi orchid are crushed before extraction; the concentration of the ethanol aqueous solution is 50-99% v / v; the extraction is performed by heating and reflux extraction at a temperature of 75-85° C., 2-3 times, and each extraction lasts 2-3 hours.

4. The use according to claim 2, characterized in that In step b, the mass ratio of the extract to the diatomaceous earth is 1:0.8-1.

2.

5. The use according to claim 2, characterized in that In step a or step c, the concentration is performed by reducing pressure until there is no solvent smell.

6. The use according to claim 2, characterized in that In step d, the volume ratio of the sample loading amount to the silica gel column is 1:5 to 1:

10.

7. The use according to claim 2, characterized in that In step e, the volume ratio of the sample loading amount to the silica gel column is 1:8 to 1:

12.

8. The use according to claim 2, characterized in that In step f, the medium pressure column chromatography is performed by uniform linear elution with 20-60% v / v methanol aqueous solution for 8 hours at a flow rate of 20 mL / min. Starting from 0 o'clock, 1 fraction is collected every 30 minutes, and the 9th fraction is collected.

9. The use according to claim 1, characterized in that The Hubei Jinsulan sesquiterpene dimer compound can treat non-alcoholic fatty liver disease by downregulating the contents of fat droplets, triglycerides and cholesterol in liver cells.

10. The use according to claim 1, characterized in that The drug is in the form of granules, tablets, capsules, oral liquids, pills, emulsions, suspensions, injections, infusions or sprays.

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