Isodhillane type meroterpenoid compound as well as preparation method and application of isodhillane type meroterpenoid compound

Abstract

The invention discloses an Isodhillane type meroterpenoid compound as well as a preparation method and application thereof, the general formula of the Isodhillane type meroterpenoid compound is as shown in a formula I to a formula III, in the formula (I) and the formula (II), the cyclic skeleton is 6 / 7 / 6 / 5 / 6 / 5; in the formula (III), the ring skeleton is 6 / 7 / 6 / 5 / 6; r1 is selected from hydrogen or hydroxyl, and R2 is selected from methyl or hydroxymethyl. According to the present invention, the Isodhillane type meroterpenoid compound is firstly used in the anti-hepatic fibrosis treatment aspect, and the anti-hepatic fibrosis experiment results show that the Isodhillane type meroterpenoid compound has low cytotoxicity and good anti-hepatic fibrosis activity, and is expected to be developed into the novel anti-hepatic fibrosis drug.

Description

Technical Field

[0001] This invention relates to the field of heteroterpenoid compound technology, and particularly to a class of Isodhilarane-type heteroterpenoid compounds, their preparation methods, and applications. Background Technology

[0002] Liver fibrosis is a dynamic, reversible pathological process characterized by abnormal liver tissue damage and repair, and excessive extracellular matrix (ECM) deposition due to the activation or transformation of hepatic stellate cells (HSCs) and hepatocytes. The gradual accumulation of fibrotic tissue accelerates liver function loss and may eventually develop into cirrhosis or liver cancer, seriously threatening human health. Persistent liver damage in various chronic liver diseases (such as viral hepatitis, alcoholic and non-alcoholic fatty liver disease, cholestasis, and autoimmune hepatitis) can lead to liver fibrosis. Without timely intervention and treatment, 75-80% of patients may develop cirrhosis or liver cancer. Furthermore, with the continued high incidence of obesity and metabolic disorders, the number of patients with liver fibrosis will continue to increase. Due to the multiple pathogenic factors and complex pathological mechanisms of liver fibrosis, the development of anti-fibrotic drugs is extremely challenging. Although significant progress has been made in etiological treatment (such as antiviral drugs for chronic hepatitis B and C, and immunosuppressants for autoimmune hepatitis, which can effectively slow the fibrotic process of certain liver diseases), no targeted anti-fibrotic drugs have yet been approved. Therefore, the development of anti-fibrotic drugs with novel structures and mechanisms is of great significance. Summary of the Invention

[0003] The present invention aims to provide a class of Isodhilarane-type heteroterpenoid compounds, their preparation methods and applications, which can be used in the development of anti-liver fibrosis drugs.

[0004] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0005] This invention provides a class of Isodhilarane-type heteroterpenoid compounds, with general formulas as shown in Formulas I-III:

[0006] In formulas (I) and (II), the ring system skeleton is 6 / 7 / 6 / 5 / 6 / 5; in formula (III), the ring system skeleton is 6 / 7 / 6 / 5 / 6.

[0007] R1 is selected from hydrogen or hydroxyl, and R2 is selected from methyl or hydroxymethyl.

[0008] Preferably, the Isodhilarane-type heteroterpenoid compound specifically comprises: .

[0009] The present invention also provides the use of the above-mentioned Isodhilarane-type heteroterpenoid compounds or pharmaceutically acceptable derivatives thereof in the preparation of anti-hepatic fibrosis drugs.

[0010] Preferably, the Isodhilarane-type heteroterpenoid compound is selected from at least one of compounds 2, 3, 11, 12, and 13.

[0011] More preferably, compound 11 can downregulate the expression of fibrosis markers, including fibronectin, collagen I, and α-smooth muscle actin.

[0012] More preferably, compound 11 can reduce plasma alanine aminotransferase and aspartate aminotransferase levels and improve liver damage.

[0013] This invention also provides a method for preparing a class of Isodhilarane-type heteroterpenoid compounds, comprising the following steps:

[0014] 1) The fungi are activated and cultured on a large scale to obtain the fermentation product;

[0015] 2) The fungal ferment was soaked in ethyl acetate, filtered, concentrated under reduced pressure, and the solvent was recovered to obtain an ethyl acetate extract of the fungus;

[0016] 3) Extract the extract with ethyl acetate, then perform normal-phase silica gel column chromatography with gradient elution and collect the eluent;

[0017] 4) The eluent was concentrated and separated by reversed-phase silica gel column chromatography and high-performance liquid chromatography to obtain various Isodhilarane-type terpenoid compounds.

[0018] Preferably, in step 1), the fungus is selected from the genus Penicillium (…). sp.) or genus Cyanobacteria ( sp.).

[0019] Preferably, in step 3), the eluent for the normal-phase silica gel column chromatography is chloroform-methanol or dichloromethane-methanol.

[0020] Preferably, in step 4), the eluent for the reversed-phase silica gel column chromatography is methanol-water or acetone-water.

[0021] The beneficial effects of this invention are:

[0022] The inventors prepared a new class of Isodhilarane-type heteroterpenoids and applied them to the treatment of liver fibrosis for the first time. The results of the anti-liver fibrosis experiment showed that the Isodhilarane-type heteroterpenoids all had low cytotoxicity and good anti-liver fibrosis activity, and are expected to be developed into new anti-liver fibrosis drugs or health products. In particular, compound 11 has great development potential. Attached Figure Description

[0023] Figures 1-2 The proton and carbon NMR spectra of compound 2 of the present invention are shown below.

[0024] Figures 3-4 The proton and carbon NMR spectra of compound 3 of the present invention are shown below.

[0025] Figures 5-6 The proton and carbon NMR spectra of compound 11 of the present invention are shown below.

[0026] Figures 7-8 The proton and carbon NMR spectra of compound 12 of the present invention are shown below.

[0027] Figures 9-10 The proton and carbon NMR spectra of compound 13 of the present invention are shown below.

[0028] Figure 11 The results of screening for the inhibitory effects of the compounds of this invention on fibronectin levels in TGF-β1 model cells;

[0029] Figure 12 The effect of compound 11 of the present invention on the expression level of fibrosis marker proteins in TGF-β1 model cells;

[0030] Figure 13 The therapeutic effect of compound 11 of the present invention on a mouse model of CCl4 liver fibrosis. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to embodiments. Unless otherwise specified, the specific materials and methods described below can be implemented using conventional technical means in the art.

[0032] Example 1: Preparation of Isodhilarane-type heteroterpenoids

[0033] In this embodiment of the invention, Penicillium fungi are used. Taking sp.F0836 as an example, Penicillium was studied. sp.F0836 was deposited on December 24, 2025, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO: M 20253002.

[0034] I. Preparation Method

[0035] Includes the following steps:

[0036] S1. Penicillium fungi Sp. F0836 was removed from the -80℃ freezer and pre-cultured in a 28℃ incubator for 24 hours. It was then transferred to sterile PDA plates for activation and incubated for 2-4 days. Endophytic fungi the size of a broad bean were collected after activation. sp.F0836 cells were inoculated into sterile PDB liquid medium and cultured in a shaker at 28°C for 2-3 days to obtain seed culture. The cultured seed culture was then transferred to sterile rice solid medium in a sterile laminar flow hood using a pipette and cultured in a static fermentation culture at 28°C for 28-31 days to obtain fermentation product.

[0037] S2. The fermentation product obtained in step S1 was extracted with ethyl acetate three times, each time for 48 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain a crude extract. The crude extract was then extracted with ethyl acetate three times, each time for 24 hours, to obtain an ethyl acetate extract.

[0038] S3. The ethyl acetate extract obtained in step S2 was subjected to normal-phase silica gel column chromatography with chloroform-methanol as the eluent. Gradient elution was performed with chloroform:methanol (v / v) = 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, and 0:1 to obtain 8 fractions (Frs. AH), namely fraction A (100:0), fraction B (100:1), C (50:1), D (20:1), E (10:1), F (5:1), G (2:1 and 1:1), and H (1:2 and 0:1).

[0039] S4. Perform ODS column chromatography on fraction D obtained in step S3, using acetone-water as the eluent, and sequentially using acetone:water (v / v) = 30:70, 40:60, 55:45, 70:30, 85:15, and 100:0 as solvents. Each 500 mL fraction was evaporated to dryness and collected as one fraction, resulting in a total of 42 fractions.

[0040] S5. The fraction 15 obtained in step S4 was separated and purified by high performance liquid chromatography (mobile phase: 45% acetonitrile / water, flow rate: 3 mL / min, detection wavelength: 210 nm) to obtain compounds 2, 12 and 13.

[0041] S6. The fraction 16 obtained in step S4 was separated and purified by high performance liquid chromatography (mobile phase: 48% acetonitrile / water, flow rate: 3 mL / min, detection wavelength: 210 nm) to obtain compound 11.

[0042] S7. Perform ODS column chromatography on fraction E obtained in step S3, using methanol-water as the eluent, and sequentially use methanol:water (v / v) = 30:70, 40:60, 55:45, 70:30, 85:15, 100:0 as solvents for gradient elution. Each 500 mL fraction is evaporated to dryness and collected as one fraction, for a total of 55 fractions.

[0043] S8. The fraction 18 obtained in step S7 is separated and purified by high performance liquid chromatography (mobile phase is 40% acetonitrile / water, flow rate is 3 mL / min, detection wavelength is 210 nm) to obtain compound 3.

[0044] The structural formulas of these five compounds are shown below:

[0045] .

[0046] II. Structural Identification

[0047] The chemical structures of compounds 2, 3, 11, 12, and 13 were determined using structural identification techniques such as spectrometry and mass spectrometry.

[0048] The structural assessment data is as follows:

[0049] Compound 2: 1H NMR data are as follows (ppm, 400 MHz, CD3OD): 6.36 (s, 1H, H-2), 1.92 (dd, J=12.4, 2.8 Hz, 1H, H-5), 2.08 (dd, J=14.4, 12.4 Hz, 1H, H-6a), 1.84 (dd, J=14.4, 2.8 Hz, 1H, H-6b), 4.34 (q, J=6.8 Hz, 1H, H-9), 4.99 (d, J=2.8 Hz, 1H, H-13), 6.27 (br d, J=2.8, 1H, H-14), 1.59 (s, 3H, H3-17), 1.66(s, 3H, H3-18), 1.42 (s, 3H, H3-19), 1.37 (d, J=6.8 Hz, 3H, H3-21), 5.68 (s, 1H, H-23), 1.29 (s, 3H, H3-24), 3.90 (d, J=12.0 Hz, 1H, H-25a), 3.43 (d, J=12.0 Hz, 1H, H-25b); The carbon NMR data are as follows: (ppm, 100 MHz, CD3OD): 165.7 (C, C-1), 121.0 (CH, C-2), 156.6 (C, C-3), 77.6 (C, C-4), 44.6 (CH, C-5), 29.1 (CH2, C-6), 50.8 (C, C-7), 178.3 (C, C-8), 64.7 (CH, C-9), 154.6 (C, C-10), 110.0 (C, C-11), 48.9 (C, C-12), 92.3 (CH, C-13), 130.1 (CH, C-14), 133.5 (C, C-15), 85.3 (C, C-16), 25.8 (CH3, C-17), 26.7 (CH3, C-18), 21.7 (CH3, C-19), 17.9 (CH3, C-21), 73.7 (C, C-22), 104.3 (CH, C-23), 20.5 (CH3, C-24), 50.8 (CH2, C-25). like Figure 1 and 2 As shown.

[0050] Compound 3: 1H NMR data are as follows (ppm, 400 MHz, acetone-d6): 6.36 (s, 1H,H-2), 1.93 (m, 1H, H-5), 2.11 (m, 1H, H-6a), 1.90 (m, 1H, H-6b), 4.34 (q, J=6.8 Hz, 1H, H-9), 5.00 (d, J=2.8 Hz, 1H, H-13), 6.23 (br d, J=2.8, 1H, H-14), 1.62 (s, 3H, H3-17), 1.65 (s, 3H, H3-18), 1.34 (s, 3H, H3-19), 1.30 (d, J=6.8,3H, H3-21), 3.32 (d, J=4.8, 1H, H-22), 6.20 (d, J=4.8, 1H, H-23), 3.74 (m, 2H, H2-24), 3.86 (d, J=12.0, 1H, H-25a), 3.58 (d, J=12.0, 1H, H-25b); The carbon NMR data are as follows: (ppm,100MHz, CD3OD): 163.1 (C, C-1), 121.3 (CH, C-2), 154.6 (C,C-3), 77.4 (C, C-4), 43.7 (CH, C-5), 25.7 (CH2, C-6), 54.2 (C, C-7), 176.9(C, C-8), 63.6 (CH, C-9), 151.0 (C, C-10), 107.6 (C, C-11), 48.6 (C, C-12), 91.4 (CH, C-13), 129.2 (CH, C-14), 133.4 (C, C-15), 84.0 (C, C-16), 25.8 (CH3, C-17), 26.7 (CH3, C-18), 19.5 (CH3, C-19), 18.1 (CH3, C-21), 39.1 (CH, C-22), 99.2 (CH, C-23), 68.9 (CH2, C-24), 51.1 (CH2, C-25). (e.g.) Figure 3 and 4 As shown.

[0051] Compound 11: 1H NMR data are as follows (ppm, 400 MHz, CD3OD): 6.01 (s, 1H, H-2), 2.55 (dd, J=12.0, 4.5 Hz, 1H, H-5), 169 (m, 1H, H-6a), 1.63 (dd, J=14.5,12.0 Hz, 1H, H-6b), 4.43 (q, J=6.5 Hz, 1H, H-9), 4.96 (d, J=2.5 Hz, 1H, H-13), 6.13 (d, J=2.5 Hz, 1H, H-14), 1.46 (s, 3H, H3-17), 1.65 (s, 3H, H3-18),1.30 (s, 3H, H3-19), 1.35 (d, J = 6.5 Hz, 3H, H3-21), 5.70 (s, 1H, H-23), 1.25 (s, 3H, H3-24), 3.25 (d, J = 5.5 Hz, 1H, H-25a), 2.57 (d, J = 5.5 Hz, 1H, H-25b); CMR data are as follows: (ppm, 100 MHz, CD3OD): 165.5 (C, C-1), 115.9 (CH, C-2), 157.8 (C, C-3), 58.9 (C, C-4), 39.1 (CH, C-5), 26.6 (CH2, C-6), 50.4 (C, C-7), 178.2 (C, C-8), 64.8 (CH, C-9), 154.7 (C, C-10), 109.1 (C, C-11), 48.8 (C, C-12), 92.2 (CH, C-13), 129.7 (CH, C-14), 134.3 (C, C-15), 84.5 (C, C-16), 26.1(CH3, C-17), 25.9 (CH3, C-18), 21.7 (CH3, C-19), 17.8 (CH3, C-21), 73.4 (C, C-22), 104.2 (CH, C-23), 20.4 (CH3, C-24), 56.6 (CH2, C-25). like Figure 5 and 6 As shown.

[0052] Compound 12: 1H NMR data are as follows (ppm, 400 MHz, CD3OD): 5.94 (s, 1H, H-2), 2.48 (dd, J=12.0, 3.5 Hz, 1H, H-5), 1.72 (dd, J=14.0, 2.0 Hz, 1H, H-6a), 1.62 (dd, J=14.0, 4.0 Hz, 1H, H-6b), 4.36 (q, J=6.5, 1H, H-9), 5.07 (d, J=2.5Hz, 1H, H-13), 6.29 (m, 1H, H-14), 1.50 (s, 3H, H3-17), 1.66 (s, 3H, H3-18), 1.33 (s, 3H, H3-19), 1.32 (d, J=6.5 Hz, 3H, H3-21), 3.02 (d, J=2.5 Hz, 1H, H-22), 6.12 (d, J=2.5 Hz, 1H, H-23), 1.29 (s, 3H, H3-24), 3.20 (d, J=5.5 Hz, 1H, H-25a), 2.54 (d, J=5.5 Hz, 1H, H-25b); The carbon NMR data are as follows: (ppm, 100 MHz, CD3OD): 165.6 (C, C-1), 115.9 (CH, C-2), 157.9 (C, C-3), 58.9 (C, C-4), 38.8 (CH, C-5), 28.4 (CH2, C-6), 46.6 (C, C-7), 179.7 (C, C-8), 64.5 (CH, C-9), 151.4 (C, C-10), 106.8 (C, C-11), 47.9 (C, C-12), 91.7 (CH, C-13), 130.0 (CH, C-14), 134.1 (C, C-15), 84.5 (C, C-16), 26.1 (CH3, C-17), 25.9 (CH3, C-18), 19.8 (CH3, C-19), 18.0 (CH3, C-21), 41.7 (CH, C-22), 99.4 (CH, C-23), 24.1 (CH3, C-24), 56.5 (CH2, C-25). like Figure 7 and 8 As shown.

[0053] Compound 13: 1H NMR data are as follows (ppm, 400 MHz, CD3OD): 5.97 (s, 1H, H-2), 1.81 (dd, J=13.8, 3.2 Hz, 1H, H-5), 2.36 (dd, J=13.8, 3.2 Hz, 1H, H-6a), 1.54 (m, 1H, H-6b), 4.25 (q, J=7.0, 1H, H-9), 3.68 (d, J=18.2 Hz, 1H, H-13a),2.51 (dd, J=18.2, 8.5 Hz, 1H, H-13b), 6.23 (d, J=8.5 Hz, 1H, H-14), 1.44 (s,3H, H3-17), 1.62 (s, 3H, H3-18), 0.85 (s, 3H, H3-19), 1.37 (d, J=6.5, 3H, H3-21), 4.12 (d, J=6.5 Hz, 1H, H-22), 6.30 (d, J=6.5 Hz, 1H, H-23), 1.46 (s, 3H, H3-24), 2.70 (d, J=5.5 Hz, 1H, H-25a), 2.68 (d, J=5.5 Hz, 1H, H-25b), 3.88 (s, 3H, H3-26); The carbon NMR data are as follows: (ppm, 100 MHz, CD3OD): 165.9 (C, C-1), 115.6 (CH, C-2), 153.7 (C, C-3), 60.7 (C, C-4), 47.1 (CH, C-5), 29.5 (CH2, C-6), 45.5 (C, C-7), 178.6 (C, C-8), 74.4 (CH, C-9), 210.2 (C, C-10), 64.3 (C, C-11), 41.9 (C, C-12), 35.8 (CH, C-13), 133.4 (CH, C-14), 138.2 (C, C-15), 85.1 (C, C-16), 28.8 (CH3, C-17), 26.3 (CH3, C-18), 18.0 (CH3, C-19), 168.9(C, C-20), 18.8 (CH3, C-21), 51.5 (CH, C-22), 99.5 (CH, C-23), 27.4 (CH3, C-24), 57.8 (CH2, C-25), 53.7 (CH3, C-26). like Figure 9and 10 As shown.

[0054] Ultimately, from marine Penicillium The structures of the Isodhilarane-type heteroterpenoids isolated from sp. F0836 are as follows:

[0055] .

[0056] Example 2: High-throughput screening for antifibrotic effects of Isodhilarane-type heteroterpenoids derived from *Penicillium marineum*.

[0057] In this embodiment, *Penicillium marineum* is used. The isodhilarane-type heteroterpenoids isolated from sp. F0836 were used as test objects. High content screening was used to evaluate the effect of the compounds on fibronectin expression in TGF-β1-treated human fibroblast LX-2 cells.

[0058] I. Experimental Methods

[0059] LX-2 fibroblasts were seeded at a density of 5000 cells / 100 μL in 96-well plates and cultured overnight. LX-2 cells were then induced with TGF-β1 (10 ng / mL) for 48 hours and fixed. After permeabilization and blocking, cells were stained with fibronectin antibody at a 1:100 ratio at 4°C for 8 hours, followed by co-incubation with Alexa 647-labeled secondary antibody and 2 μg / mL DAPI (for nuclear staining). Fluorescence signals of fibronectin and DAPI were acquired sequentially using excitation light at 640 nm and 405 nm. Intracellular fluorescence signals were measured using an Image Xpress Micro confocal imaging system (Molecular Devices, Inc., USA). Approximately 5000 cells were analyzed per sample, and the average fluorescence intensity of fibronectin in each cell was obtained.

[0060] II. Experimental Results

[0061] See results Figure 11 To evaluate the antifibrotic activity of the screened compounds, this study established a TGF-β1-induced human LX-2 cell fibrosis model, which effectively simulates the pathological activation process of fibrosis in vivo, following previously published methods. The isolated and identified compounds and the antifibrotic drug bifenidone (PFD) were incubated at concentrations of 10 μM and 500 μM, respectively, for 48 hours, and high-throughput screening was performed using a high-content image analysis system. Figure 11 A). Notably, compared to the TGF-β1 model control group, 11 compounds exhibited significant anti-fibrotic effects, reducing the signal of the fibrosis marker fibronectin by more than 50%. Figure 11 B). Compounds 2, 3, 11, 12, and 13, after incubation at 10 μM for 48 hours, reduced fibronectin signal by more than 70% without showing significant cytotoxicity. Figure 11 C).

[0062] Example 3: Determination of the antifibrotic effect of Isodhilarane-type heteroterpenoid compound 11 derived from Penicillium marineis in TGF-β1-treated LX-2 fibroblasts (immunoblotting)

[0063] In this embodiment, Isodhilarane-type heteroterpenoids derived from marine Penicillium were used as test subjects, and Western blotting was employed to study the anti-liver fibrosis effects of TGF-β1-treated human LX-2 cells.

[0064] I. Experimental Methods

[0065] Total protein was extracted and quantified after cell or tissue lysis. Proteins were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked for 30 minutes at room temperature with 5% bovine serum albumin-TBST blocking buffer, followed by overnight incubation with primary antibody at 4°C, and then incubation with secondary antibody. Protein bands were visualized using an ECL chemiluminescence assay kit. Density analysis was performed using Quantity One software (BioRad Laboratories, USA). The protein levels of control cells or mice were set as 1, and the relative fold expression levels of each group were calculated.

[0066] II. Experimental Results

[0067] See results Figure 12 We systematically evaluated the antiproliferative and antifibrotic effects of compound 11 on TGF-β1-treated LX-2 cells. After 48 hours of treatment, compound 11 treatment downregulated the expression of fibrosis markers at the protein level, including fibronectin, collagen I, and α-smooth muscle actin.

[0068] Example 4: Effects of Isodhilarane-type heteroterpenoid compound 11 derived from marine Penicillium on liver fibrosis

[0069] In this embodiment, the isodhilarane-type heteroterpenoid compound 11, derived from Penicillium marineis, was used as a test subject for in vivo validation experiments in a mouse model of liver fibrosis induced by corn oil in 20% carbon tetrachloride (CCl4).

[0070] I. Experimental Methods

[0071] (1) Constructing a mouse model of liver fibrosis:

[0072] Eight-week-old male C57BL / 6J mice (weighing 18-20 grams) were used as subjects. They were given physiological saline or corn oil containing 20% ​​carbon tetrachloride (CCl4) by intraperitoneal injection (ip) for 7 weeks.

[0073] (2) Preparation of compound 11 solution:

[0074] Prepare solutions of compound 11 with concentrations of 1 mg / mL and 0.5 mg / mL using physiological saline solutions containing 5% Tween 80 and 1% DMSO, and mix thoroughly to dissolve.

[0075] (3) Intervention treatment with solvent or compound J183:

[0076] Mice injected with corn oil containing 20% ​​carbon tetrachloride (CCl4) for four weeks were randomly divided into four groups (CCl4 model control group, high-dose J183 group, low-dose J183 group, and bifenidone group), with eight mice in each group. Normal mice and the CCl4 model control group received intraperitoneal injections of saline, while the high-dose and low-dose J183 groups received intraperitoneal injections of compound 11 solution. Compound 11 was administered intraperitoneally every other day (at doses of 10 mg / kg and 30 mg / kg), with oral administration of the antifibrotic drug pirfenidone (PFD, 200 mg / kg) as a positive control. Food intake and body weight were observed and recorded. Seven weeks after administration, all mice were anesthetized, blood was collected, and the mice were euthanized by cervical dislocation and dissected. Body weight and liver weight were recorded for each group.

[0077] (4) Analysis of mouse blood samples:

[0078] The mouse blood samples collected in step (3) were centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The levels of alanine aminotransferase and aspartate aminotransferase in the serum were detected.

[0079] (5) Mouse liver histopathology:

[0080] Liver tissue was fixed in 4% formaldehyde solution and then embedded in paraffin according to standard procedures. The tissue was cut into 4-micrometer thick sections and stained with hematoxylin and eosin (H&E) (for assessing liver injury) and Masson's trichrome (for assessing fibrosis), respectively. The sections were observed under a microscope, and histological images were acquired at 200x magnification.

[0081] II. Experimental Results

[0082] See results Figure 13 Carbon tetrachloride treatment led to a significant decrease in body weight and an increase in liver weight in mice. Figure 13 AB), which is related to histopathological changes in liver tissue and liver damage, specifically manifested as a rough liver surface and abnormal liver H&E staining ( Figure 13C) and elevated plasma AST and ALT levels ( Figure 13 (DE). Compared with the model group, treatment with compound 11 and bifenidone had little effect on mouse body weight, but significantly reversed the increase in liver weight in carbon tetrachloride-treated mice. In addition, treatment with compound 11 and bifenidone both reduced plasma alanine aminotransferase and aspartate aminotransferase levels, thus improving liver injury.

[0083] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A class of isodhilarane-type heteroterpenoids, with general formulas as shown in Formulas I-III: ； In formulas (I) and (II), the ring system skeleton is 6 / 7 / 6 / 5 / 6 / 5; in formula (III), the ring system skeleton is 6 / 7 / 6 / 5 / 6. R1 is selected from hydrogen or hydroxyl, and R2 is selected from methyl or hydroxymethyl.

2. The Isodhilarane-type heteroterpene compound according to claim 1, characterized in that, The Isodhilarane-type heteroterpenoids are specifically: 。 3. The use of the Isodhilarane-type heteroterpenoid compound or its pharmaceutically acceptable derivative as described in claim 1 or 2 in the preparation of an anti-hepatic fibrosis drug.

4. The application according to claim 3, characterized in that, The Isodhilarane-type heteroterpenoid compound is selected from at least one of compounds 2, 3, 11, 12, and 13.

5. The application according to claim 4, characterized in that, Compound 11 can downregulate the expression of fibrosis markers, including fibronectin, collagen I, and α-smooth muscle actin.

6. The application according to claim 4, characterized in that, Compound 11 can reduce plasma alanine aminotransferase and aspartate aminotransferase levels and improve liver damage.

7. A method for preparing a class of Isodhilarane-type heteroterpenoids, comprising the following steps: 1) The fungi are activated and cultured on a large scale to obtain the fermentation product; 2) The fungal ferment was soaked in ethyl acetate, filtered, concentrated under reduced pressure, and the solvent was recovered to obtain an ethyl acetate extract of the fungus; 3) Extract the extract with ethyl acetate, then perform normal-phase silica gel column chromatography with gradient elution and collect the eluent; 4) The eluent was concentrated and separated by reversed-phase silica gel column chromatography and high-performance liquid chromatography to obtain various Isodhilarane-type terpenoid compounds.

8. The preparation method according to claim 7, characterized in that, In step 1), the fungus is selected from the genus Penicillium ( sp.) or genus Cyanobacteria ( sp.).

9. The preparation method according to claim 7, characterized in that, In step 3), the eluent for the normal phase silica gel column chromatography is chloroform-methanol or dichloromethane-methanol.

10. The preparation method according to claim 7, characterized in that, In step 4), the eluent for the reversed-phase silica gel column chromatography is methanol-water or acetone-water.

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