Sterol compounds, methods of making, and medicaments for treating pulmonary fibrosis

Abstract

The present application relates to a sterol compound, a preparation method and a drug for treating pulmonary fibrosis, and belongs to the technical field of medicines. A desmethyl cryptosterol separated from a fungus of the genus Podospora has good anti-pulmonary fibrosis activity, and can be used as a lead compound for developing a drug for treating idiopathic pulmonary fibrosis. The present application provides an alternative compound for developing a new drug for treating idiopathic pulmonary fibrosis, and has very important significance for the development and utilization of natural product resources.

Description

Sterol compounds, preparation methods, and drugs for treating pulmonary fibrosis Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to sterol compounds, the isolation and preparation of drugs for the treatment of idiopathic pulmonary fibrosis, and especially to the process of isolation and purification of desmethylcryptostol, structural confirmation and anti-pulmonary fibrosis activity. Background Technology

[0002] Epidemiological meta-analyses show that the global incidence of idiopathic pulmonary fibrosis (IPF) is 0.09–1.30 cases per 10,000 person-years, with a sharp increase in incidence with age. Current drug treatments, including glucocorticoids, broad-spectrum immunosuppressants, and the antifibrotic drug combination of pirfenidone / nintedanib, can only moderately slow the decline in forced vital capacity and are limited by dose-limiting toxicities. Therefore, the median survival after diagnosis remains only 3–5 years, making IPF one of the most deadly chronic respiratory diseases and placing an increasingly heavy burden on global public health systems.

[0003] Histopathologically, IPF is characterized by persistent fibroblast proliferation and the continuous accumulation of pathological extracellular matrix (ECM), ultimately leading to thickening and obstruction of the alveolar septa and irreversible distortion of lung structure. Transforming growth factor-β1 (TGF-β1) plays a central role in this process, triggering a series of self-amplifying cascade reactions that promote fibroblast transdifferentiation into myofibroblasts (FMT), clonal proliferation, and excessive synthesis and deposition of ECM. The resulting positive feedback loop continuously increases tissue stiffness, leading to progressive respiratory failure. The significant treatment gap underscores the urgent need to discover and develop next-generation antifibrotic drugs from the fields of natural products and synthetic chemistry.

[0004] Fungi are an important source of sterols. These compounds possess a unique tetracyclic steroid core structure and are widely found in fungal metabolites. Thanks to advances in natural product chemistry, numerous fungal sterols have been discovered and elucidated in recent years, highlighting their increasing medicinal value. Studies have shown that fungal sterols possess diverse pharmacological activities, exhibiting significant therapeutic potential in anti-inflammatory, anticancer, immunomodulatory, and cardiovascular protective effects. Based on their unique mechanism of action and excellent biological activity, fungal sterols are considered potential drug candidates for the treatment of idiopathic pulmonary fibrosis. Summary of the Invention

[0005] The objective of this invention is to provide a method for isolating and purifying compounds with anti-pulmonary fibrosis properties from the fungus *Paraciothyrium* sp., and their applications. The compounds of this invention are potential dual inhibitors of fibroblast activation and extracellular matrix deposition, exhibiting excellent anti-pulmonary fibrosis activity and can serve as lead compounds for the development of anti-pulmonary fibrosis drugs. According to a first aspect of this invention, a highly degraded sterol compound is provided; furthermore, this compound has been found to possess excellent anti-pulmonary fibrosis activity, showing promising development potential.

[0006] A sterol compound, the structural formula of which is shown in formula (1):

[0007] Equation (1).

[0008] The method for preparing the sterol compound includes the following steps:

[0009] (1) After fermenting the fungi of the genus Pseudomonas with rice, the alcohol was extracted, the alcohol was recovered by vacuum concentration, and then extracted with ethyl acetate to obtain the ethyl acetate extract.

[0010] (2) The ethyl acetate fraction obtained in step (1) was subjected to column chromatography and then eluted with petroleum ether-ethyl acetate gradient to obtain 7 components with increasing polarity. The 6th component was then separated by gel chromatography, silica gel column chromatography and high performance liquid chromatography to obtain the sterol compound.

[0011] The paraconiothyrium sp. strain was purchased from the China Marine Microbial Culture Collection Center, and its strain number is MCCC 3A00275.

[0012] The fungus *Pseudomonas scutellatus* was inoculated into a solid rice fermentation medium and cultured at 25-28 ℃ for 28 days.

[0013] The alcohol mentioned in step (1) is ethanol, and the mass concentration of the alcohol is 95% or higher.

[0014] In step (2), petroleum ether-ethyl acetate-methanol gradient elution is performed under the following conditions: 50-1:1:0-1.

[0015] Separation was achieved through gel chromatography, normal-phase and reverse-phase silica gel column chromatography, and high-performance liquid chromatography. Gel chromatography was performed using a 1:1 (v / v) dichloromethane-methanol elution solution.

[0016] The separation conditions for silica gel column chromatography were elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20-1:1:0-1.

[0017] The high-performance liquid chromatography (HPLC) separation conditions are methanol-water elution with a volume ratio of 50-75:25-50.

[0018] A drug for treating pulmonary fibrosis, comprising the aforementioned sterol compounds.

[0019] The medications for treating pulmonary fibrosis also include pharmaceutically acceptable excipients.

[0020] The use of the sterol compounds in the preparation of drugs with anti-pulmonary fibrosis activity and / or for the treatment of pulmonary fibrosis.

[0021] Overall, the technical solutions conceived by this invention have the following main advantages compared with the prior art: 1. Sterol compounds were discovered from the fungus paraconiothyrium sp.; 2. Further bioactivity evaluation results show that the new compound has a strong inhibitory effect on fibroblast activation and can effectively combat idiopathic pulmonary fibrosis. Attached Figure Description

[0022] Figure 1 is the single-crystal diffraction pattern of compound demethylcryptostol A2.

[0023] Figure 2 is the proton NMR spectrum of compound demethylcryptostol A2.

[0024] Figure 3 is the carbon spectrum of compound demethylcryptostol A2.

[0025] Figure 4 shows the pharmacological effects of compound desmethylcryptosidol A2 on antifibrosis, verified by the CCK-8 assay. In this figure: A represents the cytotoxicity of compound desmethylcryptosidol A2 on NIH / 3T3 cells; B represents the cell proliferation of TGF-β1-activated NIH / 3T3 cells after treatment with different concentrations of compound desmethylcryptosidol A2 or solvent for 48 hours, as detected by the CCK-8 assay.

[0026] Figure 5 shows the cell proliferation of activated and quiescent cells after 48 hours of treatment with different doses of the compound desmethylcryptostrol A2 or the solvent, as assessed by the EdU assay.

[0027] Figure 6 shows the cell migration ability assessed by the wound healing experiment under the same treatment conditions;

[0028] Figure 7 shows the levels of α-SMA and COL1A1 proteins detected by Western blot analysis under the same experimental protocol.

[0029] Figure 8 illustrates the effect of compound desmethylcryptostrol A2 on improving the pathological changes of bleomycin (BLM)-induced pulmonary fibrosis in vivo, using methods such as body weight monitoring, lung coefficient calculation, and hydroxyproline content determination. In the figure: A represents the change in mouse body weight throughout the experiment; B represents the lung coefficient of the mice calculated at the experimental endpoint; and C represents the quantitative detection of hydroxyproline content in lung tissue to assess collagen deposition.

[0030] Figure 9 shows the effect of compound desmethylcryptostol A2 on improving the pathological changes of bleomycin (BLM)-induced pulmonary fibrosis in vivo, verified by tissue staining and pathological scoring methods. Among them, A is a representative micrograph of lung tissue sections stained with hematoxylin-eosin (H&E) and Masson trichrome; BC is a semi-quantitative analysis of lung tissue pathological changes using fibrosis scoring and Ashcroft scoring. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0032] The compound isolated and purified from the fungus *Pseudomonas* in this invention belongs to the sterol class and has the chemical name (3aS,5aR,6R,8aR)-6-((2R,5S,E)-5,6-dimethylhept-3-en-2-yl)-3a-hydroxy-5a-methyl-3a,4,5,5a,6,7,8,8a-octahydro-2H-indeno[5,4-b]furan-2-one. It is named demethylcryptostrol A2, and its structural formula is shown in Formula 1.

[0033]

[0034] Formula 1

[0035] The method for preparing sterol compounds in this invention includes the following steps: extracting rice fermentation product of the fungus *Pseudomonas* genus with industrial alcohol (95% ethanol), concentrating under reduced pressure to recover the industrial alcohol, and then extracting with ethyl acetate to obtain an ethyl acetate fraction extract; subjecting the ethyl acetate fraction extract to column chromatography: specifically, mixing with silica gel and dry packing the column; then eluting with a gradient of petroleum ether-ethyl acetate, combining the same components to obtain a total of 7 components with polarity from smallest to largest; wherein component 6 is further separated by repeated gel chromatography, reversed-phase silica gel column chromatography, and high-performance liquid chromatography to obtain the compound demethylcryptostrol A2.

[0036] The application of sterol compounds in the anti-pulmonary fibrosis of this invention is that the compounds can inhibit fibrosis-related inflammatory responses by targeting and inhibiting TGF-β1-driven fibroblast activation and extracellular matrix production, and can also inhibit fibroblast proliferation and migration and reduce collagen deposition, thereby alleviating the symptoms of lung tissue structural damage and functional impairment caused by pulmonary fibrosis.

[0037] In summary, this invention obtained a sterol compound, a secondary metabolite of *paraconiothyrium* sp., through extraction and column chromatography of the fermentation broth. Its structure was determined using various spectroscopic analyses and other methods, as shown in Formula 1. Evaluation of the anti-pulmonary fibrosis activity of the compound of Formula 1 revealed that it significantly inhibited TGF-β1-driven fibroblast activation and extracellular matrix production, exhibiting therapeutic activity against pulmonary fibrosis. Therefore, it can serve as a lead compound for the development of drugs for the treatment of pulmonary fibrosis.

[0038] The following are specific embodiments.

[0039] Example 1

[0040] 1. Isolation and preparation of compound demethylcryptostrol A2 as shown in Formula 1

[0041] The fungi of the genus *Pseudomonas* were inoculated into 100 kg of rice solid fermentation medium and cultured at 28 ℃ for 28 days. The rice fermentation product was extracted 8 times with industrial alcohol. The industrial alcohol was concentrated and recovered under reduced pressure at below 50 ℃ and then extracted with ethyl acetate to obtain 1.12 kg of ethyl acetate extract.

[0042] The ethyl acetate fraction extract was subjected to column chromatography: specifically, the sample was mixed with 200-300 mesh silica gel and dry-packed into a column, eluted with a gradient of petroleum ether-ethyl acetate-methanol (50:1:1), detected by TLC, and the same components were combined to obtain 7 fractions with increasing polarity. Fraction 6 was then separated by repeated gel chromatography, normal and reverse phase silica gel column chromatography, and high performance liquid chromatography (gel chromatography separation conditions were elution with dichloromethane-methanol at a volume ratio of 1:1; normal phase silica gel column chromatography separation conditions were elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:1; high performance liquid chromatography separation conditions were elution with methanol-water at a volume ratio of 75:25) to obtain compound desmethylcryptostrol A2 (113.2 mg), which is the compound of formula 1 of this application.

[0043] 2. Structural identification of compound demethylcryptostrol A2 as shown in Formula 1

[0044] Nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses of compound demethylcryptostol A2 revealed that its proton and carbon spectra were similar to those of compounds reported in the literature. 1 The high-field methyl region of its ¹³C NMR spectrum, except for the H3-19 methyl singlet, shows the characteristic methyl signal of steroids. However, its ¹³C NMR data shows only 21 carbon atoms, indicating that it is chemically a degradation sterol. 13 The presence of C NMR signals at δ 173.6, 172.9, 112.6, and 104.1 indicates the presence of a γ-hydroxy-α,β-unsaturated γ-lactone system in the molecule. 1 The triplet doublet at δ5.36 and the doublet doublet at δ5.24 in the 1H NMR spectrum were identified as the olefin protons H-15 and H-16 in the side chain. Further X-ray single-crystal diffraction data analysis of demethylcryptostol A2 confirmed the molecular formula of the compound as C2. 21 H 32 Based on O3, combined with the Flake parameters and chiral center data, the absolute stereoconfiguration of this molecule can be confirmed as 4S,7R,8R,11R,13R,15E,17S, thus confirming that the structure of this compound is consistent with the literature reports. The crystal structure is shown in Figure 2, and the crystal structure data are as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 13 C NMR and 1 The 1H NMR data are shown in Table 1, the 1H NMR spectrum is shown in Figure 2, and the 1C NMR spectrum is shown in Figure 3.

[0045] Compound demethylcryptostol A2: Molecular formula: C 21 H 32O3, molecular weight: 332.46 g / mol, unit cell parameters: a = 7.98040(10) Å, b = 6.67820(10) Å, c = 18.6960(2) Å, V = 993.15(2) Å 3 ,α= 90°,β= 94.6260(10)°,γ= 90°, cell volume (V): 993.15(2) ų, temperature (T): 99.99(10) K, space group: P21, number of molecules in the cell (Z): 2, absorption coefficient (μ, Cu Kα): 0.568 mm -1 Number of diffraction points measured: 22315; Number of independent diffraction points: 3860; Diffraction point repeatability (R0) int ): 0.0486, Structural refinement result: R1 (I>2σ(I)): 0.0337, wR(F 2 (I>2σ(I)): 0.0881, R1 (full data): 0.0339, wR(F) 2 (Full data): 0.0883, Goodness of fit (GOFonF²): 1.049, Flack parameter: 0.10(10).

[0046] Table 1. Compounds of Formula (1) 1 H-NMR (400MHz, CD3OD) and 13 C-NMR (100MHz, CD3OD) data

[0047]

[0048] Example 2

[0049] 1. In vitro pharmacodynamics and mechanism studies

[0050] (1) Evaluation of the inhibitory activity against TGF-β1-induced fibroblast proliferation

[0051] The basic safety of compound desmethylcryptostol A2 in fibroblasts and its specific antagonistic activity against the proliferative effect of TGF-β1 were verified by CCK-8 assay and EdU detection.

[0052] CCK-8 Method: NIH / 3T3 cells were seeded at a density of 8 × 10³ cells / well in 96-well plates. After treatment with a series of compound concentrations for 48 hours, the cells were analyzed according to the kit instructions. In constructing a TGF-β1-induced pulmonary fibrosis cell model, NIH / 3T3 cells (at a density of 5 × 10³ cells / well) were used. 4Cells (100 μL / mL) were seeded in each well of a 96-well plate. After cell adhesion, the cells were divided into a blank control group, a model group (containing 5 ng / mL TGF-β1), and compound treatment groups (containing different concentrations of the compound in the presence of TGF-β1). After treatment, each well was incubated with CCK-8 reagent for 1 hour, and the absorbance was measured at 450 nm. Cell viability was calculated using the formula: Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%, where As, Ac, and Ab are the absorbance values ​​of the experimental group, normal control group, and blank well (containing only culture medium), respectively. The inhibition rate of the compound on TGF-β1-induced proliferation was calculated based on the absorbance values, and the dose-response relationship was analyzed using GraphPad Prism software. As shown in Figure 4A, the toxicity test results showed that at concentrations below 2.5 μM, the cell viability of the demethylcryptostol A2 treatment group was not statistically different from that of the normal control group, indicating that the compound had no significant cytotoxicity within this concentration range. As shown in Figure 4B, in the inhibition activity experiment, the cell viability of the TGF-β1-stimulated group was significantly higher than that of the normal control group, confirming that it successfully induced cell proliferation. Furthermore, desmethylcryptostol A2 significantly reversed this pro-proliferative effect of TGF-β1 in a concentration-dependent manner. At a concentration of 1.25 μM, its inhibition rate against TGF-β1-induced proliferation reached a significant level, and the inhibitory effect further increased with increasing concentration, showing superior efficacy compared to the positive control SB431542.

[0053] 5-Ethynyl-2'-deoxyuridine (EdU) labeling detection method: NIH / 3T3 cells were labeled with 1.5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 10 ng / mTGF-β1 in 6-well plates. After adherence and 80% confluence, cells were divided into control and experimental groups. Both groups received 10 ng / mTGF-β1, while the experimental group received a gradient concentration of the compound for 48 hours. Cells were then incubated with EdU for 2 hours, fixed at room temperature, stained with an EdU cell proliferation assay kit, and quantitatively analyzed using ImageJ software. As shown in Figure 5, fluorescence microscopy images revealed a significant increase in the number of EdU-positive (red fluorescent) cells in the TGF-β1-stimulated group compared to the normal control group, directly confirming the proliferative effect of TGF-β1. In the group co-treated with normethazine A2 and TGF-β1, the proportion of EdU-positive cells decreased in a dose-dependent manner. Quantitative analysis further confirmed that at a concentration of 1.25 μM, normethazine A2 significantly reduced the TGF-β1-induced EdU incorporation rate in a concentration-dependent manner.

[0054] (2) Evaluation of the inhibitory activity against TGF-β1-induced fibroblast migration ability

[0055] The scratch assay was used to simulate key steps in the migration of fibroblasts to the injury site during the fibrosis process, and to examine the intervention effect of compounds on cell motility.

[0056] Scratch healing assay: NIH / 3T3 cells in logarithmic growth phase were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in pre-marked 12-well plates and cultured overnight at 37°C and 5% CO2 until a dense monolayer formed. Uniform scratches were made using a 200 μL sterile pipette tip. After washing with PBS to remove exfoliated cells, the medium was replaced with low-serum medium containing 1% FBS (to reduce the impact of basal proliferation), and different treatments were added: control group, TGF-β1 (5 ng / mL) stimulation group, and TGF-β1 co-treatment group with different concentrations of compounds. Images were taken at the same location at 0 and 24 hours after scratching, and the scratch area was quantified using ImageJ software. The scratch healing rate (%) was calculated using the formula: [(A0- A] 24 [) / A0] × 100%, where A0 is the initial scratch area at hour 0, A 24 The remaining scratch area after 24 hours is shown in Figure 6. After 24 hours of culture, the control group exhibited slow cell migration and a lower scratch closure rate due to the low serum environment. In the TGF-β1-stimulated group, cells at the scratch edge significantly extended into the blank area, resulting in a significantly higher scratch closure rate than the control group, confirming that TGF-β1 strongly promoted cell migration. In contrast, the extension of cells towards the scratch center was significantly inhibited in groups treated with different concentrations of desmethylcryptosidol A2. Even at a concentration of 1.25 μM, the compound significantly inhibited scratch healing, exhibiting a dose-dependent trend.

[0057] (3) Regulatory effects on key signaling pathways and effector protein expression in fibrosis

[0058] Based on the confirmed effects of the compound on cell function (proliferation and migration), the regulatory role of demethylcryptostrol A2 on the expression of key effector molecules in the downstream core fibrosis signaling pathway of TGF-β1 was directly detected and quantitatively analyzed at the protein molecular level, thereby elucidating the specific molecular mechanism by which it exerts its anti-fibrotic activity.

[0059] Total protein was extracted from NIH / 3T3 cells after different treatments (control group, TGF-β1 stimulation group, and TGF-β1 co-treatment group with different concentrations of compounds). After electrophoresis, transfer to a membrane, and blocking, the samples were sequentially incubated with primary and secondary antibodies targeting α-smooth muscle actin, type I collagen α1 chains, and the internal reference protein GAPDH. Finally, the images were developed using chemiluminescence. As shown in Figure 7, compared with the control group, TGF-β1 stimulation significantly upregulated the protein expression levels of α-SMA and COL1A1. Co-treatment with normethazine A2 and TGF-β1 reversed this upregulation effect in a dose-dependent manner, significantly reducing the expression levels of the aforementioned fibrosis marker proteins. Quantitative analysis showed that at a concentration of 1.25 μM, the expression level of COL1A1 protein recovered to near or even below the basal state. These results confirm that demethylcryptosidol A2 can directly block key processes that lead to cell activation into myofibroblasts (decreased α-SMA expression) and excessive extracellular matrix deposition (decreased COL1A1 expression) by inhibiting TGF-β1 signaling, thus elucidating the molecular basis of its antifibrotic effect.

[0060] 1. In vivo pharmacodynamic validation

[0061] Based on the established in vitro mechanism, the anti-pulmonary fibrosis activity of desmethylcryptosidol A2 was further evaluated at the whole animal level. A pulmonary fibrosis model of C57BL / 6 mice induced by bleomycin intratracheal infusion was established. A model group, a desmethylcryptosidol A2 treatment group, and a normal control group were set up. Body weight was monitored regularly. After euthanasia, the body weight and wet weight of the separated and washed lung tissue were measured. The lung coefficient was calculated as (lung wet weight / body weight) × 100%. Lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. HE staining and Masson staining were performed according to the kit instructions. After mounting, the sections were used for later use. The degree of inflammatory infiltration, collagen deposition, and structural damage in the stained sections was scored according to the Ashcroft criteria.

[0062] As shown in Figure 8, desmethylcryptosidol A2 has a comprehensive therapeutic effect on the bleomycin-induced pulmonary fibrosis model. The progressive weight loss trend in the treated mice was significantly alleviated, effectively improving the disease-related systemic wasting state; the abnormal increase in the lung coefficient was also significantly suppressed, directly indicating a reduction in the pathological burden on lung tissue. Crucially, the fibrosis pathological score of the lung tissue in the treated group was significantly reduced, quantitatively confirming its effective ability to reverse structural remodeling and abnormal repair of lung tissue.

[0063] As shown in Figure 9, HE and Masson staining results of lung tissue provide direct histopathological evidence. HE staining in the model group revealed severe alveolar structural damage, extensive inflammatory cell infiltration, and interstitial thickening; Masson staining showed widespread and dense collagen fiber deposition (large and deep blue-stained areas). In contrast, the desmethylcryptostol A2 treatment group showed better alveolar structural integrity, reduced inflammatory infiltration, significantly smaller and lighter blue-stained areas in Masson staining, and significantly reduced collagen deposition. These results, corroborated by macroscopic indicators, confirm that this compound intervenes in the core pathological processes of pulmonary fibrosis through multiple targets: protecting alveolar structure, inhibiting inflammatory responses, and reducing abnormal collagen deposition.

[0064] 2. Results and Analysis:

[0065] In summary, the compound desmethylcryptostol A2 provided by this invention can intervene in the pathological process of pulmonary fibrosis through multiple targets and multiple links: in vitro, it can effectively inhibit the proliferation and migration of fibroblasts and the transdifferentiation of myofibroblasts; in vivo, it can significantly improve inflammatory infiltration and fibrotic pathological damage in lung tissue. Therefore, desmethylcryptostol A2 has significant pharmaceutical development value and can be used to prepare drugs for the treatment of pulmonary fibrosis and related fibrotic diseases.

[0066] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing sterol compounds, characterized in that, The process includes the following steps: (1) After fermenting *Paraconiothyrium* with rice, it is extracted with alcohol, concentrated under reduced pressure to recover the alcohol, and then extracted with ethyl acetate to obtain an ethyl acetate extract. The *Paraconiothyrium* sp. was purchased from the China Marine Microbial Culture Collection Center with the accession number MCCC 3A00275. *Paraconiothyrium* was inoculated into rice solid fermentation medium and cultured at 25-28℃ for 28 days; (2) The ethyl acetate extract obtained in step (1) is subjected to column chromatography and then eluted with a gradient of petroleum ether-ethyl acetate-methanol to obtain 7 components with increasing polarity. The 6th component is then separated by gel chromatography, silica gel column chromatography, and high performance liquid chromatography to obtain the sterol compound. The structural formula of the sterol compound is shown in formula (1). Formula (1); the separation conditions for gel chromatography are elution with dichloromethane-methanol at a volume ratio of 1:1; the separation conditions for silica gel column chromatography are elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20-1:1:0-1; the separation conditions for high performance liquid chromatography are elution with methanol-water at a volume ratio of 50-75:25-50.

2. The preparation method according to claim 1, characterized in that, The alcohol mentioned in step (1) is ethanol, and the mass concentration of the alcohol is above 95%. In step (2), petroleum ether-ethyl acetate-methanol gradient elution is performed under the gradient elution conditions of 50-1:1:0-1.

3. The use of the sterol compound prepared by the method of claim 1 or 2 in the preparation of a drug with anti-pulmonary fibrosis activity.

4. The application as described in claim 3, characterized in that, The application of the sterol compounds in the preparation of drugs for treating pulmonary fibrosis.

5. The application as described in claim 3, characterized in that, Drugs with anti-pulmonary fibrosis activity also include pharmaceutically acceptable excipients.

Images