Sesterterpenoids, preparation method thereof and application of sesterterpenoids in preparation of medicine for treating psoriasis

Abstract

The invention discloses a sesterterpenoid compound, a preparation method thereof and application of the sesterterpenoid compound in preparation of a medicine for treating psoriasis, and belongs to the technical field of biological medicine. Comprising a compound 1-4, and the structural formula of the compound 1-4 is as follows: # imgabs0. A new molecular template is provided for developing a new psoriasis treatment medicine, and a new thought is provided for further developing a novel treatment strategy with an immunomodulatory effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a diterpenoid compound and a preparation method thereof, and application of the compound in preparing a drug for treating psoriasis. Background Art

[0002] Psoriasis is a common chronic, systemic, inflammatory, immune-mediated disease with high incidence, long duration, unpredictable course, and great harm, which seriously damages the patient's physical health and quality of life. According to the World Health Organization, about 0.09%-11.43% of the world's population suffers from psoriasis, which is one of the most common chronic skin diseases in the world. According to the Chinese epidemiological survey, the number of psoriasis patients in my country has reached 7 million, and it is showing a trend of continuous increase. The typical histological characteristics of psoriasis plaques include excessive proliferation of epidermal keratinocytes and infiltration of dendritic cells, T lymphocytes, neutrophils and macrophages in the dermis. Traditional topical medications for the treatment of psoriasis, such as corticosteroids, calcineurin inhibitors, vitamin D analogs, anthralin and tar, are often limited in application due to their poor efficacy, large side effects and poor tolerance; therefore, the development of new psoriasis inhibitors with strong immune tolerance and small adverse reactions has become a difficult problem and research hotspot that urgently needs to be solved in clinical practice.

[0003] The exact pathogenesis of psoriasis is not fully understood, but studies have shown that genetic, immune, environmental and lifestyle factors play an important role in its onset. Current studies suggest that the interaction of immune cell keratinocytes through multiple cytokines is the most important pathogenesis of psoriasis. In the development of psoriasis, abnormally activated dendritic cells secrete a large number of cytokines, such as IL-23 and TNF-α. IL-23 can activate Th17 cells to form the IL-23 / IL-17 axis. Th17 cells play a key role in this process, and the IL-23 / IL-17 axis is considered to be another major immune pathway involved in the pathological process of psoriasis.

[0004] Two proinflammatory factors, S100A8 (myeloid-related protein 8, MRP8, calgranulin A) and S100A9 (MRP14, calgranulin A), are highly expressed in keratinocytes and leukocytes of psoriatic skin, but are not found in the skin of healthy people. Traditionally, heterodimeric S100A8 / S100A9 (calprotectin) is considered to be released upon cell stress or tissue damage and is only regarded as damage-associated pattern molecules (DAMPs) or warning molecules. However, recent studies have shown that S100A8 / A9 has a wide range of biological functions, including calcium homeostasis, cytoskeleton dynamics, inflammation regulation, energy metabolism, and antimicrobial properties. It has been reported that S100A8 / A9 is involved in the development of autoimmune-related diseases such as psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. Inhibiting the production or release of S100A8 / A9 has shown significant therapeutic effects, which highlights the potential application of targeting S100A8 / A9 in the treatment of autoimmune diseases.

[0005] Aspergillus fungi are a class of filamentous fungi that exist in different environments in nature and are widely distributed. Aspergillus has rich enzymes and can produce various types of metabolites with physiological activity. Over the past thousands of years, it has gradually become an important species in many fields such as food fermentation, medicine, and agricultural production. The chemical structure of the metabolites of Aspergillus fungi is complex, diverse, and novel, mainly including terpenes, alkaloids, polyketides, peptides, etc. These metabolites have multiple biological activities such as antibacterial, antitumor, antiviral, and antioxidant. Therefore, it is of great significance to find new active lead compounds in Aspergillus fungi. Summary of the invention

[0006] The purpose of the present invention is to provide a diterpenoid compound and a preparation method thereof and application thereof in the preparation of a medicament for treating psoriasis, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a diterpenoid compound, including compounds 1 to 4, whose structural formula is as follows:

[0009]

[0010] The second technical solution of the present invention is a method for preparing the diterpenoid compound, comprising the following steps: fermenting rice with Aspergillus undulatus to obtain a rice fermentation product; and extracting and separating the rice fermentation product to obtain the diterpenoid compound.

[0011] The third technical solution of the present invention is the use of the dipterpene compound in the preparation of a drug for treating psoriasis.

[0012] A fourth technical solution of the present invention is a drug for treating psoriasis, comprising the above-mentioned dipterpene compound.

[0013] A fifth technical solution of the present invention is the use of the diperpene compound in the preparation of immunosuppressants.

[0014] The sixth technical solution of the present invention is the use of the diterpenoid compound in the preparation of S100A8 / A9 targeted inhibitors.

[0015] The seventh technical solution of the present invention is a S100A8 / A9 targeted inhibitor, comprising the diterpenoid compound.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] (1) Compounds 1-4 have a tightly fused "6 / 5 / 5 / 6 / 5" ring system, a rare nested cyclopentadienyl[cd]indene fragment, and the presence of a bridgehead double bond makes one of the five-membered ring atoms almost coplanar.

[0018] (2) Further biological activity evaluation results showed that compound 2 could bind to S100A8 / A9 and inhibit its secretion, thereby having a significant therapeutic effect on psoriatic dermatitis.

[0019] (3) This class of compounds is the first natural product that can bind to S100A8 / A9 dimers and inhibit their secretion, which is of great significance for the development of S100A8 / A9 targeted inhibitors.

[0020] The inventors obtained four new skeleton compounds by separating and purifying the secondary metabolites of the Aspergillus undulates fungus. By using a variety of spectral analysis methods and other means, it was determined that it was a 6 / 5 / 5 / 6 / 5 pentacyclic ring system diterpenoid compound, and the specific structure is shown in formula (1). Through the evaluation of the psoriasis therapeutic activity of compounds 1-4, it was found that compounds 1-4 all had psoriasis therapeutic effects, especially compound 2. Omics results showed that compound 2 could inhibit the TNF and IL-17 signaling pathways in HaCaT cells. PharmMapper prediction showed that its highest-ranked inflammation-related target was S100A8 / A9 protein, and the combination of the two was further confirmed by in vitro CETSA experiments and dynamic simulations. In the imizomod-induced mouse psoriasis model, compounds 1 and 2 both significantly alleviated the psoriatic dermatitis of mice, and immunohistochemistry showed that the expression of IL-17, Ki67 and S100A9 proteins was significantly reduced after administration. The present invention provides a new molecular template for developing new psoriasis treatment drugs and provides a new idea for further developing new treatment strategies with immunomodulatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The crystal structure of compound 1.

[0023] Figure 2 The crystal structure of compound 2.

[0024] Figure 3 The crystal structure of compound 3.

[0025] Figure 4 The activity screening of compounds 1-4 is as follows: (A): Compounds 1-4 have an inhibitory effect on overproliferating HaCaT cells. (B): Compounds 1-4 show low cytotoxicity to normal HaCaT cells. (C): Compound 2 has a regulatory effect on the immune system.

[0026] Figure 5 Compound 2 inhibits genes related to TNF and IL-17 signaling pathways in HaCaT cells. Among them, (A): Principal component analysis (PCA) of RNA sequencing data, showing the clustering of the control group, model group and compound 2 treatment group. (BC): Volcano plots showing upregulated (red) and downregulated (green) genes in the comparison between the control group and the model group, and between the compound 2 (1.25 μM) treatment group and the model group. Genes were considered downregulated when log2 (fold change) <-1.0 and -log10 (adjusted P value) >1.0; and were considered upregulated when log2 (fold change) >1.0 and -log10 (adjusted P value) >1.0. (DE): Comparison of differentially expressed genes (DEGs) between the control group, model group and compound 2 treatment group. (F): Top 10 signaling pathways of upregulated DEGs between the control group and the model group identified by KEGG pathway enrichment analysis. (G): The number of DEGs related to IL-17 and TNF signaling pathways in the control group, model group, and compound 2 (1.25 μM)-treated group. (H): Heat map showing the expression levels of inflammation-related genes in the control group, model group, and compound 2-treated group.

[0027] Figure 6Compound 1 inhibits the expression of inflammation-related proteins in HaCaT cells and blocks the activation of NF-κB. Wherein, (A): Compound 1 inhibits the expression of inflammation-related proteins in HaCaT cells. (B): Standardization and quantification of protein band density and GAPDH band density. Data are presented as mean ± standard deviation. # p<0.05, compared with the blank control group; ***P<0.001, **P<0.01, *p<0.05, compared with the TNF-α model group. (C): Immunofluorescence results of compound 1 (scale bar=50 μm).

[0028] Figure 7 Compound 2 inhibits the expression of inflammation-related proteins in HaCaT cells and blocks the activation of NF-κB. Wherein, (A): Compound 2 inhibits the expression of inflammation-related proteins in HaCaT cells. (B): Standardization and quantification of protein band density and GAPDH band density. Data are presented as mean ± standard deviation. # p<0.05, compared with blank control group; ***P<0.001, **P<0.01, *p<0.05, compared with TNF-α model group. (C): Immunofluorescence results of compound 2 (scale bar=50 μm).

[0029] Figure 8 The effects of compounds 1 and 2 on S100A9 protein. Among them, (A): Western Blotting detection revealed that compound 2 inhibited the secretion of S100A9 protein. The data are presented as mean ± standard deviation. # p<0.05, compared with blank control group; ***P<0.001, **P<0.01, *p<0.05, compared with TNF-α model group. (B): TSA analysis of intracellular binding of compounds 1 and 2 to S100A9.

[0030] Fig. 9 The molecular docking results of compound 1-4 and calprotectin. Among them, (A): the overall structure of calprotectin and the location of the two hydrophobic pockets. (B): Compounds 1-4 are docked into the two hydrophobic pockets in sequence, and the docking scores are pocket 1 / pocket 2 respectively.

[0031] Fig.10Compound 2 binds to two hydrophobic pockets and interferes with the binding of endogenous ligands. Among them, (A): RMSD diagram of calprotectin in apo, com and com2 states and the trajectory of ligand binding to protein. (B): Rg diagram of calprotectin in apo, com and com2 states. (C): RMSF diagram of four chains of calprotectin in apo, com and com2 states. (D): Trajectory of ions (including six blue calcium ions and four gray zinc ions) of the protein in apo, com and com2 states. (E): Size and volume of two pockets in docking, apo, com and com2 states. (F): Conformational changes of two ligands (i) and residues (ii-vii) caused by them.

[0032] Fig.11 Compounds 1-4 bind to calprotectin and degrade it. (A): Affinity between compounds 1-4 and calprotectin. (B): Compounds 2 and 4 reduce the thermal stability of calprotectin. (C): Effect of compound 2 on S100A9 protein expression, with tapinarof as a positive control.

[0033] Fig.12 The therapeutic effects of compounds 1 and 2 on IMQ-induced psoriasis-like dermatitis. Wherein, (A): Skin lesions of mice. (B): Quantification of CSS of mice in each group. (C): Epidermal thickness of mice in each group. (D): Manifestations and histological characteristics of skin lesions in each group. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0040] The embodiment of the present invention provides a diterpene compound, including compounds 1 to 4, whose structural formula is as follows:

[0041]

[0042] The embodiment of the present invention also provides a method for preparing the diterpenoid compound, comprising the following steps: fermenting rice with Aspergillus undulatus to obtain a rice fermentation product; and extracting and separating the rice fermentation product to obtain the diterpenoid compound.

[0043] In some specific embodiments, the Aspergillus undulatus is from China General Microbiological Culture Collection Center, with the collection number being CGMCC No.3.6295.

[0044] In some specific embodiments, the method of fermenting rice using Aspergillus undulatus comprises: mixing rice and water in a mass ratio of 1:1, inoculating the Aspergillus undulatus, and culturing at 28° C. for 45 days to obtain the rice fermentation product.

[0045] In some specific embodiments, the extraction and separation method comprises:

[0046] (1) subjecting the rice fermentation product to alcohol extraction, and extracting with ethyl acetate to obtain a crude extract;

[0047] (2) gradient eluting the crude extract with a mixed solvent of petroleum ether: ethyl acetate: methanol = 15:1:0-5:5:1 to obtain 6 components Fr.1-Fr.6;

[0048] (3) Component Fr.4 was subjected to inversion C 18 Column chromatography gave 7 components Fr.4.1-Fr.4.7; Fr.4.4 was purified by silica gel column chromatography with a gradient elution of petroleum ether: dichloromethane: methanol = 2:1:0-0:1:0-0:50:1 to give 8 components Fr.4.4.1-Fr.4.4.8; Fr.4.4.4 was purified by reverse phase high performance liquid chromatography to give compound 1;

[0049] (4) Component Fr.3 was subjected to inversion C 18 Column chromatography yielded 21 components Fr.3.1-Fr.3.21;

[0050] (5) Fr.3.12 was subjected to gel column chromatography and then silica gel column chromatography to obtain 10 components Fr.3.12.1-Fr.3.12.10; Fr.3.12.8 was divided into 4 components Fr.3.12.8.1-Fr.3.12.8.4 by preparative liquid phase;

[0051] (6) Fr.3.12.8.3 was separated twice by HPLC to obtain three components Fr.3.12.8.3.3-Fr.3.12.8.3.3, and Fr.3.12.8.3.2 was purified again by HPLC to obtain compound 2;

[0052] (7) Fr.3.12.8.4 was separated and purified twice by HPLC to obtain compound 3;

[0053] (8) Fr.3.14 was subjected to gel column chromatography and then silica gel column chromatography to obtain four components Fr.3.14.1-Fr.3.14.4. Fr.3.14.3 was separated by high performance liquid chromatography to obtain compound 4.

[0054] In some specific embodiments, the Aspergillus undulatus is subjected to expansion culture before inoculation; the expansion culture conditions include: inoculating the Aspergillus undulatus on PDA and culturing for 10-14 days.

[0055] In some specific embodiments, the C 18 The conditions of column chromatography were methanol-water, 20%-100%.

[0056] The embodiment of the present invention also provides the use of the diterpenoid compound in preparing a drug for treating psoriasis.

[0057] The embodiment of the present invention also provides a medicine for treating psoriasis, comprising the diterpenoid compound.

[0058] The embodiment of the present invention also provides the use of the diterpenoid compound in the preparation of immunosuppressants.

[0059] The embodiment of the present invention also provides the use of the diterpenoid compound in the preparation of S100A8 / A9 targeted inhibitors.

[0060] The embodiment of the present invention further provides a S100A8 / A9 targeted inhibitor, comprising the diterpenoid compound.

[0061] The present invention uses a variety of spectral analysis methods and other means to determine that the secondary metabolites of Aspergillus undulates, the new diquat terpenoid skeleton compounds undulanoids A-D (1-4), are new diquat terpenoid skeleton compounds with a 6 / 5 / 5 / 6 / 5 ring system, and provides the structure, preparation method, structure identification and biological activity evaluation of the compounds. In addition, the compounds have a strong therapeutic effect on psoriasis, can inhibit the secretion of S100A8 / A9 protein, and promote its degradation. This is the first natural small molecule inhibitor that antagonizes the S100A8 / A9 protein target.

[0062] Example 1 Preparation and structural identification of compounds 1-4

[0063] (i) Preparation of compounds 1-4.

[0064] 1. Strain information

[0065] The Aspergillus undulates strain was purchased from the China General Microbiological Culture Collection Center, isolated from the soil of Shennongjia, Hubei, and the collection date was December 4, 2001. The collection number is CGMCC No. 3.6295. After identification, the gene sequence of the strain was as similar as 99.5% to that of Aspergillus undulates, so it was determined to be Aspergillus undulates.

[0066] 2. Extraction and separation

[0067] The strain was inoculated on PDA and cultured for 10-14 days. The PDA was then divided into small pieces and inoculated on autoclaved rice (a 1L conical flask contains 250g of rice and 250mL of water, a total of 70kg of rice, and the amount of live bacteria inoculated is 2cm 2Agar), cultured at 28°C for 45 days to obtain rice fermentation. The rice fermentation was soaked in anhydrous ethanol for 8 times, the ethanol extract was concentrated under reduced pressure to obtain an ethanol extract, the ethanol extract was suspended in water, extracted with ethyl acetate for 7 times, and the ethyl acetate extract was concentrated under reduced pressure to obtain a crude extract. The crude extract was gradient eluted with a mixed solvent of petroleum ether: ethyl acetate: methanol (15:1:0-5:5:1) to obtain 6 components (Fr.1-Fr.6).

[0068] Component Fr.4 was subjected to inversion C 18 Column chromatography (methanol-water, 20%-100%) gave 7 components (Fr.4.1–Fr.4.7). Fr.4.4 was purified by silica gel column chromatography with gradient elution of petroleum ether: dichloromethane: methanol (2:1:0-0:1:0-0:50:1) to give 8 components (Fr.4.4.1-Fr.4.4.8). Fr.4.4.4 was purified by reverse phase high performance liquid chromatography (HPLC) to give compound 1.

[0069] Component Fr.3 was reversed C 18 Column chromatography (methanol-water, 20%-100%) gave 21 components (Fr.3.1-Fr.3.21). Fr.3.12 was subjected to gel column chromatography and then silica gel column chromatography to give 10 components (Fr.3.12.1-Fr.3.12.10), of which Fr.3.12.8 was divided into 4 components (Fr.3.12.8.1-Fr.3.12.8.4) by preparative liquid chromatography, and Fr.3.12.8.3 was separated by two HPLC to give 3 components (Fr.3.12.8.3.3-Fr.3.12.8.3.3), and Fr.3.12.8.3.2 was purified again by HPLC to give compound 2. Fr.3.12.8.4 was separated and purified by two HPLC to give compound 3. Fr.3.14 was subjected to gel column chromatography and then silica gel column chromatography to obtain four components (Fr.3.14.1-Fr.3.14.4). Fr.3.14.3 was separated by high performance liquid chromatography to obtain compound 4.

[0070] (II) Structural identification of compounds 1-4

[0071] The structures of compounds 1-4 were determined by comprehensively analyzing the high-resolution mass spectra, nuclear magnetic resonance signals, circular dichroism spectra, ultraviolet spectra, infrared spectra, specific rotations, and X-ray single crystal diffraction data of compounds 1-4.

[0072] Compound 1: colorless crystals; melting point 185.5-185.8°C; (c 1.0,MeOH); UV(MeOH)λ max (logε)202(4.04)nm; ECD(MeOH)λmax (Δε)=211(+17.2)nm;IRν max 3363,2954,1633,1458,and 1385cm -1 ; 1 H and 13 C NMR data,Table 1; HRESIMS m / z 395.2925[M+Na] + (calcd.forC 25 H 40 The nuclear magnetic resonance (NMR) data of compound 1 are shown in Table 1.

[0073] Compound 2: colorless crystals; melting point 185.3-180.2°C; (c 1.0, MeOH); UV(ACN)λ max (logε)196(4.03)nm; ECD(MeOH)λ max (Δε)=200(+6.8)nm;IRν max 3363,2926,1668,1461,and 1380cm -1 ; 1 H and 13 C NMR data,Table 1; HRESIMS m / z 395.2913[M+Na] + (calcd.forC 25 H 40 The nuclear magnetic resonance (NMR) data of compound 2 are shown in Table 1.

[0074] Compound 3: colorless crystals; melting point 176.1-177.6.2°C; (c 1.0,MeOH); UV(MeOH)λ max (logε)203(4.08)nm; ECD(MeOH)λ max (Δε)=206(+6.1)nm;IRν max 3436,2954,1634,1460,and 1384cm -1 ; 1 H and 13 C NMR data,Table 1; HRESIMS m / z 395.2922[M+Na] + (calcd.forC 25 H 40The nuclear magnetic resonance (NMR) data of compound 3 are shown in Table 1.

[0075] Compound 4: colorless oil; (c 1.0,MeOH); UV(MeOH)λ max (logε)202(3.76)nm; ECD(MeOH)λ max (Δε)=209(+18.0)nm;IRν max 3415,2929,1668,1462,and 1380cm -1 ; 1 H and 13 C NMR data,Table 1; HRESIMS m / z 395.2912[M+Na] + (calcd.for C 25 H 40 The nuclear magnetic resonance (NMR) data of compound 4 are shown in Table 1.

[0076] Table 1 Hydrogen spectrum and carbon spectrum data of compounds 1-4

[0077]

[0078]

[0079] Example 2 The therapeutic effects of compounds 1-4 of the present invention on psoriasis at the cellular and animal levels

[0080] (I) In vitro cell activity

[0081] 1. Recovery, passaging and cryopreservation of HaCaT cells

[0082] (1) Recovery: The human immortalized keratinocyte (HaCaT) cell line was obtained from Procell (Wuhan, China). After taking the cells out of the -80°C freezer, they were quickly thawed, transferred to a 15 ml centrifuge tube, and resuspended with an appropriate amount of culture medium. The cells were added to a cell culture bottle and placed in a carbon dioxide incubator for static culture at 37°C and a carbon dioxide concentration of 5%. The next day, after the cells adhered to the wall, the old culture medium was discarded, and the culture medium preheated in the incubator was added, and the cell growth was observed under a microscope.

[0083] (2) Passaging: When the cells grow to 80% full, discard the old culture medium, add 4 mL of PBS to rinse 1-2 times and then pour it out, add 4 mL of preheated trypsin and place in an incubator for digestion. Take out after about 3 minutes, observe under a microscope that the cells have shrunk into a single cell, and then gently blow them off with a pipette. Collect the cells by centrifugation, add complete culture medium to resuspend them, and divide them equally into two culture dishes and culture them in an incubator.

[0084] (3) Cryopreservation: After the cells have grown to full size in the culture dish, discard the old culture medium, collect the cells, resuspend the cells in 1 mL of freezing solution, place them in a cryopreservation tube, label the cell name and freezing time, and store them in a -80°C refrigerator.

[0085] 2. Initial screening of compounds for their inhibitory activity on cell proliferation

[0086] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in a humidified environment (5% CO2, 37°C). To simulate psoriatic inflammation, HaCaT cells were treated with TNF-α (10 ng / mL) for 24 hours. 0.625, 1.25, 2.5, 5, 10, and 20 μM of compound 1 or 2 were administered to cells without stimulation and cells stimulated with TNF-α, respectively. After 24 hours, the absorbance was measured at a wavelength of 450 nm using a CCK-8 kit (KGA317; KeyGen, Nanjing, China).

[0087] 3. Flow cytometry to detect CD4, CD8, and NK cell ratios

[0088] (1) Preparation of mouse spleen lymphocyte suspension: After the mouse is killed by cervical dislocation, it is immediately soaked in 75% alcohol for 10 minutes, fixed on the dissection table, and the abdominal cavity is opened using sterile surgical instruments. The spleen is removed and repeatedly rinsed with PBS until there is no other attachment on the surface. A nylon membrane is placed at the mouth of a 50mL centrifuge tube, and the rinsed spleen is placed on the nylon membrane. The spleen is fully ground with the top of a 1mL syringe until it turns white. After grinding, the nylon membrane is rinsed with PBS two to three times, and centrifuged at 400g for 5 minutes to collect, add 2-3mL of red blood cell lysis buffer, lyse at room temperature for five minutes, centrifuge at 300g for 5 minutes, discard the supernatant, and wash with PBS 2-3 times. RPMI 1640 complete medium is added to make a spleen cell suspension, placed in an incubator, and cultured in a dish for 4 hours, and then the lymphocytes in the supernatant are aspirated.

[0089] (2) The cell suspension was evenly spread into a six-well plate, and concanavalin A (Con A, 5 μg / mL) and the corresponding concentration of compounds were added, and the plate was cultured in an incubator for 48 hours. The six-well plate was gently blown with culture medium to fully collect the cells, and centrifuged at 300g for 10 minutes. After washing the cells with culture medium containing serum, they were resuspended with buffer, and antigen CD3 / CD4 / CD8 / CD49b antibodies were added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were resuspended in 500 μL PBS, vortexed to mix, and tested on the machine.

[0090] 4. Western Blotting to detect changes in related protein levels in HaCaT cells

[0091] (1) HaCaT cell protein extraction: According to the experimental grouping, HaCaT cells were evenly plated in a six-well plate for culture, and cells from different groups were collected into 1.5 mL centrifuge tubes. The cells were gently washed 2-3 times with pre-cooled PBS, and the supernatant was discarded after centrifugation. Lysis buffer was prepared on ice at a ratio of phenylmethylsulfonyl fluoride: RIPA lysis buffer = 1:100. After preparation, 30-50 μL of lysis buffer was added to each centrifuge tube, and the cells were placed on ice for lysis for 30 minutes, with gentle shaking every 10 minutes. After lysis, the cells were centrifuged in an ultra-low temperature high-speed centrifuge for 10-15 minutes (speed: 12000 rpm; temperature: 4°C), the precipitate was discarded, the supernatant was retained, and then transferred to a new 1.5 mL EP tube and placed at -80°C for use.

[0092] (2) Determination of protein concentration by BCA method: Add 1 μL of the sample to be tested or a protein standard with a concentration gradient to a 96-well plate, then add 19 μL of PBS, prepare 200 μL of BCA working solution (A:B = 50:1) according to the BCA operating instructions, mix well, and incubate in a 37°C incubator for about 30 minutes. Use an ELISA reader to detect the absorbance of each well at 562 nm, and calculate the protein concentration based on the standard curve.

[0093] (3) The experiment used 10% and 15% SDS-PAGE gels, and the upper and lower gels were prepared using the Yazyme PAGE gel rapid preparation kit according to the instructions. Then, electrophoresis, transfer, blocking, incubation with primary antibody, incubation with secondary antibody in the dark, development and imaging were performed.

[0094] 5. Immunofluorescence detection of changes in NF-κB-P65 and S100A9 in HaCaT cells

[0095] Place sterile cell slides of appropriate size in a six-well cell culture plate (1 slide / well), collect and dilute HaCaT cells with good growth status to a density of 1.25×10 6 / mL cell suspension, then inoculated into a six-well plate, placed in an incubator and cultured for 24 hours, the cells were given compound 1 (2.5, 5.0μM) and compound 2 (1.25, 2.5μM), respectively. After 2 hours, TNF-α (10ng / mL) was added to the model and compound groups, and the supernatant was removed after 24 hours. Pour off the culture medium and wash 3 times with PBS. Fix with 4% paraformaldehyde for 20 minutes and wash 3 times with PBS. Draw circles with a tissue pen to prevent the incubation solution from flowing away in the following process, and wash with PBS. Dilute the antibody with 5% BSA (Biofroxx, 4240GR250) at a certain ratio, add an appropriate amount of primary antibody (NF-κB-P65 or NLRP3, 1:1000, Cell Signaling Technology, USA) working solution, and incubate at 4°C overnight. Rewarm and wash 3 times with PBS, 5 minutes each time. Add the working solution of the corresponding secondary antibody (SA00013-2 / AS-1111 / SA00013-1 / SA00009-3 / SA00003-3 / SA00007-7, Mitaka) in the circle, incubate in a 37°C water bath in the dark for 40 minutes, wash with PBS 3 times, 5 minutes each time. Add DAPI (Sigma, D8417-1MG) to stain the nucleus, incubate in the dark at room temperature for 20 to 30 minutes, and wash with PBS. Use anti-fluorescence quenching mounting medium (Sigma, V900155-25G) to seal the slide, and store the slide in a dark box at 4°C after sealing. Observe and take pictures under a microscope (OLYMPUS, IX51).

[0096] 6. Cellular thermal shift (CETSA) experiment

[0097] (1) Collecting and treating cells: When HaCaT cells grow to about 80% in a 15 cm culture dish, they are treated with compounds or DMSO for 1 hour and the cells are collected.

[0098] (2) Extract proteins and detect binding capacity: Wash once with PBS, then suspend in 1 mL of PBS supplemented with protease and phosphatase inhibitors, and keep the same dose of compound or DMSO as the initial treatment.

[0099] The cell suspension was distributed into 7 to 10 0.2 mL PCR tubes with a volume of 100 μL (about 1 million cells), and each tube was assigned a temperature point. The sample was heated in a metal bath at the specified temperature for 2 minutes. After heating, the test tube was immediately removed, incubated at room temperature for 3 minutes, and then the test tube was immediately quickly frozen in liquid nitrogen and stored at -80°C. To lyse the cells, three freeze-thaw cycles were performed in liquid nitrogen. After each thawing, the tube was briefly rotated. The cell lysate was collected and centrifuged at 15000 rpm for 15 minutes at 4°C to remove cell debris and precipitated and aggregated proteins. After the cell lysate sample was added with the loading buffer, it was boiled at 95°C for 5 minutes, taken out and stored in a -80°C refrigerator. Western Blotting detected the binding affinity of HaCaT cells with the target protein at different temperatures, and the protein fluorescence density and relative expression level were detected using Image J software.

[0100] (II) Dynamic simulation

[0101] 1. Molecular docking

[0102] Molecular docking is The protein model 4GGF was downloaded from the PDB crystal database and pre-processed using the protein processing tool Protein Preparation Workflow. The structures of compounds 1-4 were pre-processed by the LigPrep tool. The receptor grid was centered on the entire protein and covered the entire protein. Ligand docking was performed using default settings. Interactions were provided by Pymol software.

[0103] 2. Molecular dynamics simulation

[0104] Molecular dynamics simulations were performed in the CUDA-accelerated GROMACS-2022.2 program with the protein AMBER99SB-ILDN force field and the ligand GAFF force field (whose topology files were generated by Acpype). In the MD simulations, the complex structures of the protein and ligand were obtained from the molecular docking results and used as the original coordinate files. The complex was dissolved in a cubic periodic box using the TIP3P water molecule model, and the system charge was neutralized by sodium and chloride ions with a final ionic strength of 0.15 M. Energy minimization was performed using the gradient descent method for 5000 steps to eliminate bad contacts in the system. Then, the temperature and pressure of the system were kept constant at 310 K and 1 bar, respectively, using the microcanonical (NVT) and isothermal isobaric (NPT) ensembles. Finally, the equilibrated system was subjected to 100 ns MD simulations. Subsequent MD analysis was performed using the GROMACS built-in module, and the data were processed using Origin 2021, pymol, and VMD. Binding free energy calculations and decomposition analysis were performed using the gmx_MMPBSA program. Starting from the system equilibrium, 1 frame was spaced 20 ps apart. Other parameters were kept as default. The volume of the pocket was calculated using the SiteMap module (Evaluation of Single Binding Site Regions) of the Schrodinger software.

[0105] 3. Protein production and purification

[0106] (1) According to the literature, the sequences of S100A8 (UniProt: P05109) and S100A9 (UniProt: P06702) were synthesized by GENEWIZ and inserted into the vector pET21a (NdeI / XhoI) to construct a plasmid.

[0107] (2) For protein expression, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells and cultured at 37°C in LB medium containing antibiotics until the OD 600The induction agent IPTG (final concentration: 0.4 mM) was then added, and the culture was further incubated at 18°C ​​for 16-20 h. Equal amounts of cell pellets containing S100A8 and S100A9 were collected by centrifugation and resuspended in buffer A (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol). After lysis by ultrasonic treatment and further ultracentrifugation at 10000g, 4°C for 1 h, the inclusion bodies were resuspended in buffer A containing 0.5% (v / v) Triton X-100 and sonicated three times, and then washed three times in buffer A. The washed inclusion bodies were dissolved in 6 M guanidine hydrochloride and dialyzed three times against buffer B (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl2, 2 mM ZnSO4). The reconstituted supernatant containing calprotectin was filtered and purified by size exclusion chromatography (HiLoad TM The protein was further purified by 16 / 60 Superdex75, Cytiva).

[0108] 4. TSA experiment

[0109] Purified calprotectin was diluted to 0.5 mg / mL with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM CaCl2, and 2 mM ZnSO4 and incubated with compound or DMSO for 30 minutes at room temperature. Reactions were performed in 384-well plates with 10 μL of system per well, containing 9 μL of mixed solution and 1 μL of SYPRO TM Orange protein gel stain (Invitrogen). After centrifugation, QuantStudio TM Melting curves were monitored in a 6Pro real-time PCR system (Thermos Fisher Scientific), with a first step of incubation at 25°C for 10 minutes, followed by a gradient heating from 25°C to 95°C at a rate of 0.075°C / s. Tm values ​​were fitted and calculated by QuantstudioTM design and analysis software v1.5.2. The experiment was repeated three times.

[0110] 5. Surface plasmon resonance (SPR) determination of compound affinity

[0111] The affinity of calprotectin and the compound was determined by Biacore 1K (Cytiva). The recombinant protein was immobilized on an S-series sensor chip CM5. Binding or dissociation was performed under a predefined program (multi-cycle kinetics / affinity) with 5 gradient dilutions of the compound in 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% v / v surfactant P20, 5% v / v DMSO. The results were analyzed using a predefined program (multi-cycle kinetics) to calculate Ka, Kd and KD values.

[0112] (III) Animal experiments

[0113] 1. Animal modeling and processing

[0114] (1) Male 8-week-old Balb / C mice weighing 20-25 g were randomly divided into 5 groups after one week of adaptive feeding in an SPF barrier environment: blank control group (Con), imiquimod model group (IMQ), positive drug administration group (tapinarof), compound 1 group (10 mg / kg), and compound 2 group (10 mg / kg). The hair on the middle back of the mice was shaved with a mouse shaver one day before administration.

[0115] The method for constructing a psoriasis-like mouse model is as follows: 62.5 mg of imiquimod cream was applied daily to mice in the imiquimod model group, the positive drug administration group, the compound 1 group, and the compound 2 group. The mice developed obvious dermatitis symptoms, such as redness, swelling, scaling, epidermal thickening, desquamation, etc., indicating that the psoriasis-like mouse model was successfully constructed.

[0116] The blank control group was given corresponding amount of vaseline for seven consecutive days.

[0117] Dosage:

[0118] Compound 1 and compound 2 groups were intraperitoneally injected with 0.2 mL of saline solution (10 mg / kg) daily;

[0119] The positive drug was dissolved in a mixed solution of 60% anhydrous ethanol and 40% glycerol to a mass volume concentration of 1% and applied to the back of the mouse;

[0120] The imiquimod model group was administered with 0.2 mL of normal saline injected intraperitoneally daily.

[0121] The blank control group was given intraperitoneal injection of 0.2 mL of normal saline daily.

[0122] The dynamic changes of the skin lesions on the back of mice were observed and recorded before administration every day. On the seventh day, the eyeballs were removed to obtain blood, and the mice were killed by cervical dislocation to obtain skin and spleen.

[0123] 2. PASI score

[0124] The mouse skin was observed and photographed, and PASI scores were performed every day. These factors included erythema, scaling, and thickness, ranging from 0 to 4, including: 0: none; 1: mild; 2: moderate; 3: severe; 4: very severe. Scores were performed every day for 7 consecutive days.

[0125] 3. HE staining and epidermal thickness measurement

[0126] Most of the mouse skin lesions were stored in liquid nitrogen or -80℃, and a small part of the skin lesions were fixed with 4% paraformaldehyde. The sections were embedded in paraffin, and the specific operation was as follows: first, the sections were dehydrated from low concentration to high concentration, that is, the sections were soaked in xylene for 20 minutes, and then the xylene was discarded after repeating, and the sections were soaked in anhydrous ethanol for 5 minutes, and then 75% alcohol gradient dewaxing was added for 5 minutes, and then washed with water. Then hematoxylin staining was performed: the hydrated sections were stained with hematoxylin dye for nuclei, and dyed for a few minutes to more than ten minutes, and then acid-washed, such as 1% hydrochloric acid aqueous solution to remove excess dye and perform differentiation operations, and finally alkaline solution was used to return blue treatment to make the nuclei appear blue. Then eosin staining was performed: the sections after hematoxylin staining were stained with eosin dye for the cytoplasm, and the paraffin sections were dehydrated with gradient alcohol (85% ethanol → 95% ethanol), and placed in eosin dye solution for about a few minutes. Finally, dehydration and sealing are carried out for observation: the paraffin sections are soaked in anhydrous ethanol for several minutes and then discarded. Then they are soaked in a transparent agent (usually xylene) for a few minutes and repeated once. The sections are allowed to dry naturally and the tissues on the sections are photographed with a fluorescence microscope. In the picture, the cell nucleus is stained blue and the cytoplasm is red.

[0127] 4. Immunohistochemistry to detect changes in psoriasis-related indicators in mouse skin tissue

[0128] Sample pretreatment was the same as HE staining. The immunohistochemistry process of paraffin sections was as follows: Place the tissue sections in a repair box, fill it with EDTA (pH = 9.0) antigen repair solution, and heat it in a microwave oven. First, boil the liquid on medium heat for 8 minutes, then switch to medium-low heat for 7 minutes after stopping the fire for 8 minutes. After cooling to room temperature, wash the slides with PBS buffer (pH = 7.4) on a decolorizing shaker. Soak the slides in 3% hydrogen peroxide solution, incubate at room temperature for 25 minutes in the dark, and wash with PBS buffer. Add 3% BSA or rabbit serum liquid to the tissue circle to evenly cover the tissue, and block at room temperature for 30 minutes. Gently shake off the blocking solution, directly add the primary antibody (diluted with PBS) on the section, and incubate overnight at 4°C. The next day, reheat in a 37°C water bath for 30 minutes. After washing with PBS buffer, shake off the liquid, add the secondary antibody (HRP labeled) of the corresponding species to the primary antibody to the circle to cover the tissue, and incubate at room temperature for 50 minutes. After washing with PBS, add freshly prepared DAB colorimetric solution, observe the color development of the slide under a microscope, and rinse the slices with tap water to stop the color development. Then soak the slices in hematoxylin stain for 3 minutes, wash off the excess stain with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, soak with hematoxylin blueing solution to make the slices blue, and then rinse with running water. After dehydration and sealing, randomly take 3-5 fields of view and take pictures. The cell nucleus is blue, and the positive indicator is brown-yellow.

[0129] (IV) Results Analysis

[0130] 1. Initial screening of activity: Figure 4 As shown, TNF-α was used to stimulate the over-proliferation of HaCaT cells, and the anti-over-proliferation effects of compounds 1-4 were evaluated. Compounds 1-4 all showed significant inhibitory activity, and their IC 50 The values ​​were 2.47, 1.25, 6.72 and 8.54 μM, respectively, and the cytotoxicity to normal HaCaT cells was low. Then, the most effective compound 2 was selected to further evaluate its effectiveness and explore its mechanism of action. Flow cytometry was used to detect the proportions of CD4, CD8 and NK cells in Con A-induced mouse primary splenocytes. After Con A stimulation, the CD4 / CD8 value increased significantly (>2.5), while the level of NK cells decreased slightly. This disorder is common in autoimmune diseases. The proportion of immune cells can be restored after administration, indicating that compounds 1 and 2 have immunomodulatory effects. Compounds 3 and 4 have immunomodulatory effects comparable to compound 1.

[0131] 2. Transcriptomics analysis: Figure 5As shown in Figure 2, transcriptome RNA sequencing analysis was performed to systematically evaluate differentially expressed genes in HaCaT cells treated with TNF-α and compound 2. Principal component analysis (PCA) showed that the data were concentrated between the same groups. Compared with the control group, 280 genes were upregulated and 317 genes were downregulated in the model group, while after compound 2 treatment, 1709 genes were upregulated and 2704 genes were downregulated compared with the model group, as shown in the volcano plot, with upregulated genes represented in red and downregulated genes represented in blue. To further explore the effect of compound 2, the enrichment of upregulated and downregulated genes was analyzed, and 194 upregulated genes and 241 downregulated genes were found to return to baseline levels. In the pathway analysis, TNF-α and IL-17 signaling pathways, which are key pathways in the pathogenesis of psoriasis, were enriched. In addition, 29 genes related to IL-17 signaling and 18 differential genes related to TNF signaling were found, of which 5 and 7 overlapped, respectively. Additionally, the heatmap highlights genes involved in inflammatory pathways (e.g., CXCL1, IL1B, MMP9).

[0132] 3. Analysis of related inflammatory pathway protein expression results: Figure 6 and Figure 7 As shown in the figure, based on the omics results, the expression of proteins related to the TNF-α and IL-17 signaling pathways was studied intensively. WB detected that the levels of related proteins p-IκB, NLRP3, and COX-2 decreased after administration. At the same time, immunofluorescence showed that the administration of compounds 1-4 could reduce the phosphorylation of NF-κB p65 in the cytoplasm, thereby blocking the nuclear translocation of p65. Compound 2 had the best effect.

[0133] 4. Target speculation: Figure 8 As shown, the structures of compounds 1-4 were imported into PharmMapper (http: / / www.lilab-ecust.cn / pharmmapper / ) for target screening, and S00A8 / A9 was predicted to be the top target associated with inflammatory response. The level of S100A9 in HaCaT cells was detected by immunoblotting, and it was found that the administration of compound 2 could reduce the expression of S100A9 in a dose-dependent manner. In addition, in the cell thermal shift (CETSA) experiment, when the temperature increased, S100A9 treated with compounds 1-4 showed relative stability, especially compound 2, all of which indicated that the S100A9 protein was the direct target of this type of compound.

[0134] 5. Molecular docking: Since there are two hydrophobic binding sites for endogenous ligands to bind, the docking was performed at a ratio of protein:ligand = 1:2. 4GGF was used as a protein model. Fig. 9 As shown, compounds 1-4 can all be docked into the hydrophobic pocket of each αβ heterodimer.

[0135] 6. Molecular dynamics simulation: Combined with pharmacological experiments, the binding trajectory of compound 2 with two hydrophobic pockets was further simulated. The results showed that compound 2 could occupy the molecular pocket of calprotectin and limit the interaction between endogenous ligand and calprotectin. The specific results are shown in Fig.10 .

[0136] 7. SPR result analysis: Fig.11 As shown in Figures A and B, compounds 1-4 can bind to calprotectin, and the binding constants (KD values) are 9.59, 1.49, 5.07, and 8.10 μM, respectively. Further study of their effects on the stability of calprotectin revealed that 2 and 4 reduced its thermal stability (reduced Tm values), especially compound 2. Combined with the results of molecular dynamics simulation, 1-4 will bind to two hydrophobic pockets, destroying the stable structure of calprotectin and interfering with the binding of endogenous ligands.

[0137] 8. Analysis of immunofluorescence results: Fig.11 As shown in Figure C, the effect of compound 2 on the stability of S100A9 (chain B and chain D of calprotectin) was evaluated in cells. After incubation with TNF-α, the expression of S100A9 was upregulated, and the process was reversed after administration, even better than the positive control benvimod. This shows that compound 2 can bind to the hydrophobic pocket of calprotectin, induce its dissociation and make it unstable, thereby effectively inhibiting the inflammation caused by TNF-α and helping to reduce the expression level of S100A9 or calprotectin.

[0138] 9. Analysis of animal experiment results: Fig.12 As shown in the figure, after topical application of IMQ for 7 consecutive days, the mouse skin showed obvious symptoms of psoriasis-like dermatitis, such as skin erythema, thickening and desquamation. After treatment with 1 or 2, the above symptoms improved, and the clinical severity score (CSS) and epidermal thickness were also significantly reduced. At the same time, immunohistochemistry results showed that the expression of S100A9, IL-17 and Ki67 was significantly reduced. This shows that compounds 1 and 2 can alleviate psoriasis-like dermatitis in mice by inhibiting the secretion of S100A9 and the proliferation of keratinocytes.

[0139] In summary, the present invention has isolated 4 new diterpenoid skeleton compounds with 6 / 5 / 5 / 6 / 5 ring system from Aspergillus undulatus, which have eight chiral centers (5 quaternary carbons, 2 of which are located at the bridgehead) and a cyclopentadienyl [cd] indene fragment. In the exploration of pharmacological activity, compounds 1-4 can block the activation of inflammatory signaling pathways, thereby inhibiting immune responses and contributing to the regulation of the immune system. Reverse target prediction, CETSA analysis and molecular dynamics simulation confirmed that S100A8 / A9 is the direct target of this class of compounds. In addition, compounds 1 and 2 can inhibit S100A9 expression and excessive proliferation of stratum corneum cells to alleviate psoriatic dermatitis in mice. This class of compounds is the first natural product that inhibits the secretion of S100A8 / A9 protein dimers, providing a new lead compound for drug discovery for psoriasis and even autoimmune diseases, and opening up a new path. However, further research and clinical experiments are needed to verify its safety and efficacy, and provide a more solid foundation for its clinical application.

[0140] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A diterpenoid compound, characterized in that: Including compounds 1 to 4, the structural formulas are as follows:

2. The method for preparing a diterpenoid compound according to claim 1, characterized in that: The following steps are involved: The rice is fermented by Aspergillus undulatus to obtain a rice fermentation product; the diterpenoid compound according to claim 1 can be obtained by extracting and separating the rice fermentation product.

3. The preparation method according to claim 2, characterized in that: The Aspergillus undulatus is from the China General Microbiological Culture Collection Center, and the collection number is CGMCC No.3.6295.

4. The preparation method according to claim 2, characterized in that: The method for fermenting rice using Aspergillus undulatus comprises: mixing rice and water at a mass ratio of 1:1, inoculating the Aspergillus undulatus, and culturing at 28° C. for 45 days to obtain the rice fermentation product.

5. The preparation method according to claim 2, characterized in that: The extraction and separation method comprises: (1) subjecting the rice fermentation product to alcohol extraction, and extracting with ethyl acetate to obtain a crude extract; (2) gradient eluting the crude extract with a mixed solvent of petroleum ether: ethyl acetate: methanol = 15:1:0-5:5:1 to obtain 6 components Fr.1-Fr.6; (3) Component Fr.4 was subjected to inversion C 18 Column chromatography gave 7 components Fr.4.1-Fr.4.7; Fr.4.4 was purified by silica gel column chromatography with a gradient elution of petroleum ether: dichloromethane: methanol = 2:1:0-0:1:0-0:50:1 to give 8 components Fr.4.4.1-Fr.4.4.8; Fr.4.4.4 was purified by reverse phase high performance liquid chromatography to give compound 1; (4) Component Fr.3 was subjected to inversion C 18 Column chromatography yielded 21 components Fr.3.1-Fr.3.21; (5) Fr.3.12 was subjected to gel column chromatography and then silica gel column chromatography to obtain 10 components Fr.3.12.1-Fr.3.12.10; Fr.3.12.8 was divided into 4 components Fr.3.12.8.1-Fr.3.12.8.4 by preparative liquid phase; (6) Fr.3.12.8.3 was separated twice by HPLC to obtain three components Fr.3.12.8.3.3-Fr.3.12.8.3.3, and Fr.3.12.8.3.2 was purified again by HPLC to obtain compound 2; (7) Fr.3.12.8.4 was separated and purified twice by HPLC to obtain compound 3; (8) Fr.3.14 was subjected to gel column chromatography and then silica gel column chromatography to obtain four components Fr.3.14.1-Fr.3.14.

4. Fr.3.14.3 was separated by high performance liquid chromatography to obtain compound 4.

6. Use of the dipterpenoid compound as claimed in claim 1 in the preparation of a medicament for treating psoriasis.

7. A drug for treating psoriasis, characterized in that: The invention comprises the diterpenoid compound as claimed in claim 1.

8. Use of the diperpenoid compound as claimed in claim 1 in the preparation of immunosuppressants.

9. Use of the diperpene compound as claimed in claim 1 in the preparation of a S100A8 / A9 targeted inhibitor.

10. A S100A8 / A9 targeted inhibitor, characterized in that: The invention comprises the diterpenoid compound as claimed in claim 1.

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