A steroidal compound, and a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, in particular to a kind of steroid compound and its preparation method and application.The structural formula of the steroid compound is as shown in formula (1).The steroid compound has inhibitory activity on LPS-induced inflammatory response, and can be used as a lead compound for developing drugs for treating neuroinflammation-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a steroidal compound, its preparation method, and its application. Background Technology

[0002] Neuroinflammation is an immune response activated by microglia and astrocytes in the central nervous system. Its pathological causes and processes are complex and diverse, arising from various factors such as central nervous system injury, infection, ischemia, and toxin accumulation. It involves the activation of glial cells, the release of inflammatory mediators (such as cytokines and chemokines), and the production of reactive oxygen species and nitrogenous substances. The interaction between risk factors and the development of neuroinflammation may lead to vascular damage, oxidative stress, and ultimately, brain injury. In recent years, increasing research has shown that neuroinflammation is a major pathological process in neurodegenerative diseases and some central nervous system disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, exploring effective drugs for treating neuroinflammation has long been an important research focus in this field.

[0003] Natural products are the most common source of drugs. Statistics show that since the 1980s, 30-40% of marketed drugs each year are directly or indirectly derived from natural products, with the proportion reaching as high as 50% in 2010. Natural products are also an important source of drugs for treating neuroinflammatory diseases in clinical practice. For example, huperzine A from *Huperzine spp.* and galantamine from *Eleocharis dulcis* are important drugs for treating Alzheimer's disease. Due to the scarcity of plant resources, researchers have focused on discovering natural products with anti-neuroinflammatory activity from microbial sources in recent years. An increasing number of compounds with novel and complex chemical structures and significant activity have been discovered, laying a foundation for new drug development. Therefore, isolating new natural products with anti-neuroinflammatory effects from microbial secondary metabolites is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned problems existing in the prior art and to provide a steroidal compound, its preparation method and application.

[0005] To achieve the above objectives, the first aspect of the present invention provides a steroidal compound, the structural formula of which is shown in formula (1);

[0006]

[0007] A second aspect of the present invention provides a method for preparing the steroidal compounds described above, comprising the following steps:

[0008] (1) Inoculate Penicillium fungi onto PDA medium and then incubate at a constant temperature to obtain seed culture medium;

[0009] (2) Cut the seed culture medium containing mycelium into pieces and inoculate it into sterilized rice culture medium for fermentation culture;

[0010] (3) The fermentation product obtained in step (2) is extracted with ethanol and then concentrated under reduced pressure to obtain the total extract;

[0011] (4) The total extract was mixed with water, then extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract;

[0012] (5) The ethyl acetate extract was separated by chromatography to obtain the steroidal compound shown in formula (1).

[0013] Preferably, the Penicillium fungus is Penicillium citrinum, the strain of which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20231078, deposit address: Wuhan University, Wuhan, China, deposit date: June 21, 2023, and classified as Penicillium citrinum ZYT 1.

[0014] Preferably, in step (1), the conditions for constant temperature culture include: a temperature of 25-30℃ and a time of 3-10 days;

[0015] Preferably, in step (2), the fermentation conditions include a temperature of 25-28°C and a time of 30-40 days.

[0016] Preferably, the specific process of step (5) includes:

[0017] S1: The ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate-methanol solution with a volume ratio of 20:1:0-0:0:1. TLC detection was performed, and similar fractions were combined to obtain 5 components.

[0018] S2; Component 2 was separated by reversed-phase silica column chromatography, gel chromatography, normal-phase silica column chromatography and high performance liquid chromatography to obtain the steroidal compound shown in formula (1).

[0019] Preferably, the reversed-phase silica column chromatography separation is performed by elution with methanol-water at a volume ratio of 30:70-100:0.

[0020] Preferably, the gel chromatography separation is performed by elution with dichloromethane-methanol at a volume ratio of 1:1.

[0021] Preferably, the normal-phase silica gel column chromatography separation is performed by elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0-1:1:1.

[0022] Preferably, the high performance liquid chromatography separation is performed by elution with methanol-water at a volume ratio of 75:25.

[0023] A third aspect of the present invention provides the use of the steroidal compounds described above in the preparation of medicaments for treating neuroinflammatory diseases.

[0024] This invention provides a steroidal compound represented by formula (1) and a method for obtaining the steroidal compound by isolating and purifying it using the Penicillium fungus *Penicillium citrinum*. Evaluation of its anti-neuroinflammatory activity revealed that this steroidal compound exhibits inhibitory activity against LPS-induced inflammatory responses, and it can serve as a lead compound for the development of drugs for the treatment of neuroinflammatory-related diseases (such as Alzheimer's disease and Parkinson's disease). Attached Figure Description

[0025] Figure 1 This is a graph showing the test results of test example 1, which uses CCK8 and other methods to verify the protective effect of compound citrinum dimethoate B on LPS-induced BV-2 cells.

[0026] Figure 2 This is a graph showing the test results of the inhibitory effect of compound citrinum dimethoate B on LPS-induced inflammation in BV-2 cells in Test Example 2;

[0027] Figure 3 This is a graph showing the test results of test example 3, which uses immunocytochemical analysis to test the effects of compound citrinum dimethoate B on TNF-α, NF-κB and iNOS in BV cells;

[0028] Figure 4 This is a graph showing the results of Western blot analysis of the effects of compound citrinum dimethoate B on the expression of COX-2, iNOS, and NF-κB in LPS-induced BV-2 cells in test example 4. Detailed Implementation

[0029] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] The first aspect of the present invention provides a steroidal compound, the structural formula of which is shown in formula (1), and is named Citrulline B;

[0032]

[0033] The steroidal compound (a steroidal compound with an ortho-diol structure on the C-17 side chain) shown in Formula (1) of this invention has a good anti-neuroinflammatory effect, and its main mechanism may be through inhibiting the NF-κB signaling pathway.

[0034] A second aspect of the present invention provides a method for preparing the steroidal compounds described above, comprising the following steps:

[0035] (1) Inoculate Penicillium fungi onto PDA medium and then incubate at a constant temperature to obtain seed culture medium;

[0036] (2) Cut the seed culture medium containing mycelium into pieces and inoculate it into sterilized rice culture medium for fermentation culture;

[0037] (3) The fermentation product obtained in step (2) is extracted with ethanol and then concentrated under reduced pressure to obtain the total extract;

[0038] (4) The total extract was mixed with water, then extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract;

[0039] (5) The ethyl acetate extract was separated by chromatography to obtain the steroidal compound shown in formula (1).

[0040] In a preferred embodiment, the Penicillium fungus is *Penicillium citrinum*. This strain was derived from the leaves of *Hypericum hypericum* from Sichuan and Hubei provinces, and was named *Penicillium citrinum*. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20231078, at Wuhan University, Wuhan, China, on June 21, 2023, and is classified as *Penicillium citrinum* ZYT 1.

[0041] Preferably, in step (1), the conditions for constant temperature culture include: a temperature of 25-30℃ and a time of 3-10 days; specifically, the temperature for constant temperature culture can be 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃, and the time for constant temperature culture can be 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

[0042] In this invention, the PDA medium refers to potato dextrose agar medium.

[0043] Preferably, in step (2), the fermentation conditions include: a temperature of 25-28℃ and a time of 30-40 days; specifically, the fermentation temperature can be 25℃, 26℃, 27℃ or 28℃, and the fermentation time can be 30 days, 32 days, 35 days, 36 days, 38 days or 40 days.

[0044] In this invention, in step (3), the fermentation product obtained in step (2) refers to the material containing the culture medium obtained after fermentation and mycelium.

[0045] In a preferred embodiment, the ethanol extraction is performed 10 times in step (3).

[0046] In a preferred embodiment, in step (4), the ethyl acetate extraction is performed 10 times.

[0047] In a preferred embodiment, step (5) includes the following specific process:

[0048] S1: The ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate-methanol solution with a volume ratio of 20:1:0-0:0:1. TLC detection was performed, and similar fractions were combined to obtain 5 components.

[0049] S2; Component 2 was separated by reversed-phase silica column chromatography, gel chromatography, normal-phase silica column chromatography and high performance liquid chromatography to obtain the steroidal compound shown in formula (1).

[0050] Preferably, the reversed-phase silica column chromatography separation is performed by elution with methanol-water at a volume ratio of 30:70-100:0.

[0051] Preferably, the gel chromatography separation is performed by elution with dichloromethane-methanol at a volume ratio of 1:1.

[0052] Preferably, the normal-phase silica gel column chromatography separation is performed by elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0-1:1:1.

[0053] Preferably, the high performance liquid chromatography is performed using a methanol-water elution solution with a volume ratio of 75:25.

[0054] According to a specific embodiment of the present invention, the specific process of step (5) is as follows:

[0055] S1: Mix the ethyl acetate extract with 200-300 mesh silica gel and dry pack it into a column. Elute with a gradient of petroleum ether-ethyl acetate-methanol (20:1:0-0:0:1). Detect by TLC. Combine the same components to obtain a total of 5 components (component 1-component 5) with polarity from smallest to largest.

[0056] S2: Separate component 2 obtained in step S1 by reversed-phase silica gel column chromatography, eluting with methanol-water at a volume ratio of 30:70-100:0 to obtain 6 components (components 2.1-2.6);

[0057] Component 2.3 was separated by gel chromatography using dichloromethane-methanol at a volume ratio of 1:1 to obtain four components (2.3.1-2.3.4).

[0058] Component 2.3.2 was separated by normal-phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0-1:1:1, to obtain 5 components (2.3.2.1-2.3.2.5);

[0059] Component 2.3.2.2 was separated by high performance liquid chromatography and eluted with methanol-water at a volume ratio of 75:25 to obtain compound citridin B.

[0060] A third aspect of the present invention provides the use of the steroidal compounds described above in the preparation of medicaments for treating neuroinflammatory diseases.

[0061] The steroidal compound represented by formula (1) of this invention has good anti-neuroinflammatory activity. This steroidal compound can reduce the release of lipopolysaccharide-induced inflammatory cytokines (including COX-2, IL-1β, iNOS, NF-κB, IL-18, AKT-1, Caspase1, and MCP1) in microglia by regulating the TNF-α signaling pathway.

[0062] Preferably, the microglia are BV-2 cells.

[0063] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0064] The Penicillium fungus used in the following examples is Penicillium citrinum. This strain was derived from the leaves of Hypericum chuanxiong and is currently deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20231078, deposited on June 21, 2023, and classified as Penicillium citrinum ZYT 1.

[0065] Example 1

[0066] 1. Preparation of compound citrinum dimethoate B as shown in formula (1):

[0067] (1) Inoculate Penicillium fungi onto PDA medium (potato dextrose agar medium) and incubate at 28°C for 4 days to obtain seed culture medium;

[0068] (2) Cut the seed culture medium containing mycelium into pieces and inoculate it into 100 kg of sterilized rice culture medium for fermentation culture. The fermentation culture temperature is 28℃ and the time is 35 days.

[0069] (3) The rice solid fermentation culture was extracted 10 times with industrial alcohol, and the industrial alcohol was recovered by vacuum concentration at below 50°C to obtain the total extract.

[0070] (4) Mix the total extract with water, then extract with ethyl acetate 10 times, and then concentrate under reduced pressure to obtain 2 kg of ethyl acetate extract;

[0071] (5) The ethyl acetate extract was subjected to column chromatography. Specifically, the sample was mixed with 200-300 mesh silica gel and packed into a column by dry method. The column was eluted with a gradient of petroleum ether-ethyl acetate-methanol with a volume ratio of 20:1:0-0:0:1. The TLC was used for detection. The same components were combined to obtain a total of 5 components with polarity from smallest to largest, which were denoted as components 1-5.

[0072] Component 2 was separated by reversed-phase silica gel column chromatography and eluted with methanol-water at a volume ratio of 30:70-100:0 to obtain 6 components, denoted as components 2.1-2.6.

[0073] Component 2.3 was separated into four components using gel chromatography with methanol-dichloromethane (volume ratio 1:1), denoted as 2.3.1-2.3.4.

[0074] Component 2.3.2 was further separated by normal-phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0-1:1:1, to obtain 5 components, denoted as 2.3.2.1-2.3.2.5;

[0075] Finally, component 2.3.2.2 was separated by high performance liquid chromatography, eluted with methanol-water at a volume ratio of 75:25, to obtain compound citridin B (3.0 mg).

[0076] 2. Structural identification of the prepared compounds

[0077] The prepared compounds were subjected to nuclear magnetic resonance, mass spectrometry, optical rotation, infrared spectroscopy, ultraviolet spectroscopy, circular dichroism spectroscopy, and molybdenum salt-induced circular dichroism spectroscopy data, which were then compared with literature data to determine the structure of the compounds.

[0078] Test result: Yellow oily substance; UV(MeOH)λ max (logε)203(4.26)nm; ECD(MeOH)λ max (Δε)206(-3.36),364(+13.56)nm; IR(KBr)v max 3413,2925,1649,1585,and 1221cm -1 The data for the NMR H- and C- spectra are shown in Table 1.

[0079] Table 1. HCl and C-ray spectral data (δin ppm and δin Hz) of compound citrinum dimethoate B

[0080]

[0081]

[0082] Note: a The test solvent is deuterated methanol, and the test instrument is a 400M nuclear magnetic resonance spectrometer.

[0083] Test Example 1

[0084] The protective effect of compound citrinum dimethoate B against LPS-induced BV-2 cells was verified using CCK8 assays and other methods. The test results are as follows: Figure 1 As shown. Wherein: Figure 1 A is the result observed under a microscope after BV-2 cells were stimulated with LPS and then given the compound citrinum dimethoate B; Figure 1 B represents the inhibitory effect of compound citrinum dimethoate B on LPS-induced NO release; Figure 1 C represents the results of evaluating the cytotoxic effects of compound citrinin B.

[0085] The NO detection process is as follows:

[0086] Following the instructions for the NO assay kit, the induced NO concentration in cells was determined using the nitrate reduction method and Griess reagent, followed by absorbance measurement at 540 nm using a microplate reader. The NO concentration was calculated using a sodium nitrite standard solution.

[0087] The CCK8 assay was used to test the cytotoxic activity of BV-2 microglia as follows:

[0088] BV-2 microglia were seeded into 96-well plates and treated with different concentrations of citrinum dimethoate B (2.5 μM, 5 μM, 10 μM). Cells treated with DMSO (0.1%, v / v) served as a control. After 24 hours of incubation, the 96-well plates were centrifuged, the supernatant was removed, and 100 μL of 10% CCK8 solution (diluted with BV-2 cell-specific medium) was added to each well. The plates were then incubated at 37°C for 0.5 hours. The absorbance was measured at 450 nm using a microplate reader.

[0089] Depend on Figure 1 It is known that citrinum dimethoate B has a protective effect on BV-2 cells. Citrinum dimethoate B not only significantly reduces the release of LPS-induced NO and pro-inflammatory cytokines from microglia, but also inhibits the production of cellular ROS.

[0090] Test Example 2

[0091] The inhibitory effect of compound citrinum dimethoate B on LPS-induced inflammation in BV-2 cells was tested, and the results are as follows: Figure 2 As shown. Among them, Figure 2 AI-treated BV-2 cells with LPS or PBS for 24 h, followed by treatment with DMSO or citrinin B for 60 min. RT-PCR was used to analyze the mRNA expression of inflammatory cytokines or chemokines.

[0092] The method for quantitative RT-PCR analysis is as follows:

[0093] Total RNA was extracted from BV-2 cells, and RNA samples were reverse transcribed using the PrimeScript™ RT kit. RNA integrity was confirmed by electrophoresis prior to the reverse transcriptase reaction. RNA quantification was performed spectrophotometrically at 260 nm. The Fast qPCRMix Kit and real-time quantitative PCR detection system were used to determine relative mRNA levels via real-time PCR. Data are expressed as Ct values ​​normalized to GAPDH, and the fold change between the control and treatment groups was determined using the 2-ΔΔCt method.

[0094] Depend on Figure 2It is known that the steroidal compound (Citrus dimethoprim B) described in this invention can reduce the release of lipopolysaccharide-induced inflammatory cytokines (including COX-2, IL-1β, iNOS, NF-κB, IL-18, AKT-1, Caspase1, and MCP1) in microglia by regulating the TNF-α signaling pathway.

[0095] Test Example 3

[0096] The effects of compound citrinum dimethoate B on TNF-α, NF-κB, and iNOS in BV cells were tested using immunocytochemical analysis. The results are as follows: Figure 3 As shown. Wherein: Figure 3 A represents the expression of TNF-α and iNOS analyzed by immunohistochemistry; Figure 3 B represents the expression levels of TNF-α and NF-κB analyzed using immunohistochemistry.

[0097] The process of immunocytochemical analysis is as follows:

[0098] Cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.3% (v / v) Triton X-100 for 15 minutes. After incubation for 1 hour with blocking buffer PBS and 10% (v / v) donkey serum and 1% (w / v) BSA, cells were incubated overnight at 4°C with either rabbit anti-NF-κB p65 primary antibody or rabbit anti-Nrf2 primary antibody. A negative control was incubated without primary antibody. Cells were then washed and washed in the dark with either anti-rabbit IgG H&L secondary antibody Alexa Fluor 647 or anti-rabbit IgG H&L secondary antibody Alexa Fluor 488, respectively, and then injected with DAPI (1 μg·mL⁻¹). -1 The cell nuclei were counterstained, and cell localization was examined under a laser scanning confocal microscope. The exposure settings were the same for each control group.

[0099] Depend on Figure 3 It is known that the steroidal compound (citrinin B) described in this invention can reduce the release of lipopolysaccharide-induced inflammatory cytokines (iNOS, NF-κB) in microglia by regulating the TNF-α signaling pathway.

[0100] Test Example 4

[0101] The effects of compound citrinum dimethoate B on the expression of COX-2, iNOS, and NF-κB in LPS-induced BV-2 cells were tested by Western blot. The results are as follows: Figure 4 As shown. Wherein: Figure 4 A represents the expression level of COX-2. Figure 4 D is Figure 4 Visualization results of COX-2 expression levels in A; Figure 4B represents the expression level of iNOS. Figure 4 E is Figure 4 Visualization results of iNOS expression levels in B; Figure 4 C represents the expression level of NF-κB. Figure 4 F is Figure 4 Visualization results of NF-κB expression levels in C.

[0102] The Western blot analysis process is as follows:

[0103] Total protein was obtained by adding a mixture of protease and phosphatase inhibitors to BV-2 cells in ice-cold RIPA lysis buffer. Protein concentration was quantified using a BCA protein assay kit. Protein samples were loaded onto SDS-PAGE gels and separated, transferred to Biodyne membranes, blocked in protease-free bovine serum albumin, and then incubated overnight at 5°C with indicated antibodies, followed by incubation at room temperature for 1 hour with horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive bands of proteins were detected using ECL-Plus chemiluminescence reagent. Spectra™ multicolor wide-range protein molecular weight standards were included in all gels. The following antibodies were used: rabbit anti-inducible nitric oxide synthase (iNOS) IgG, rabbit anti-NF-κBp65 IgG, rabbit anti-GAPDH IgG, rabbit anti-COX-2 IgG, goat anti-rabbit IgG, and HRP horse anti-mouse IgG. ImageJ software v1.60 was used to scan the density of each immunoblot band, and the ratios of phosphorylated protein / total protein, target protein / β-actin or histone H3 in the corresponding samples were calculated.

[0104] Depend on Figure 4 It is evident that, at the protein expression level, the compound citrinum dimethoate B does indeed reduce the expression of related inflammatory proteins, such as iNOS, COX-2, and NF-κB.

[0105] Based on the above test results, it is speculated that citrinum dimethoate B mainly exerts its anti-inflammatory effect by inhibiting the NF-κB inflammatory cytokine pathway. Combined with existing literature, the overall mechanism by which citrinum dimethoate B exerts its anti-inflammatory and neuroprotective effects is likely through binding to TLR4, thereby reducing TLR4 receptor activity and decreasing LPS binding to TLR4 to reduce inflammatory damage. Citrinum dimethoate B can significantly reduce the activity of TNF-α inflammatory cytokine. According to immunofluorescence results, citrinum dimethoate B also significantly reduces the process of NF-κB entering the cell core. These results indicate that citrinum dimethoate B simultaneously reduces the activity of both TNF-α and TLR4 receptors, decreases the TLR4 damage pathway, and reduces TNF-α inflammatory cytokine. Finally, combined with iNOS and immunofluorescence results, it is shown that citrinum dimethoate B reduces the inflammatory cytokine iNOS, thereby reducing NO production caused by LPS-induced damage, ultimately exerting an anti-inflammatory effect.

[0106] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A steroidal compound, characterized in that, The structural formula of the steroidal compound is shown in formula (1); Equation (1).

2. A method for preparing the steroidal compound as described in claim 1, characterized in that, Includes the following steps: (1) Inoculate Penicillium fungi onto PDA medium and then incubate at a constant temperature to obtain seed culture medium; (2) Cut the seed culture medium containing mycelium into pieces and inoculate it into sterilized rice culture medium for fermentation culture; (3) The fermentation product obtained in step (2) is extracted with ethanol and then concentrated under reduced pressure to obtain the total extract; (4) The total extract was mixed with water, then extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract; (5) The ethyl acetate extract was separated by chromatography to obtain the steroidal compound shown in formula (1); The Penicillium fungus in question is *Penicillium citrinum*, and the strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20231078, on June 21, 2023. It is classified and named as follows: Penicillium citrinum ZYT 1; In step (1), the conditions for constant temperature culture include: temperature of 25-30℃ and time of 3-10 days; In step (2), the fermentation conditions include: a temperature of 25-28℃ and a time of 30-40 days; The specific process of step (5) includes: S1: The ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate-methanol solution with a volume ratio of 20:1:0–0:0:

1. TLC detection was performed, and similar fractions were combined to obtain 5 components. S2; Component 2 was separated by reversed-phase silica column chromatography, gel chromatography, normal-phase silica column chromatography and high performance liquid chromatography to obtain the steroidal compound shown in formula (1); The reversed-phase silica column chromatography separation conditions were elution with methanol-water at a volume ratio of 30:70–100:

0. The gel chromatography separation conditions were elution with dichloromethane-methanol at a volume ratio of 1:

1. The normal-phase silica gel column chromatography separation conditions were elution with petroleum ether-ethyl acetate-methanol at a volume ratio of 20:1:0–1:1:

1. The high-performance liquid chromatography separation conditions are methanol-water elution with a volume ratio of 75:

25.

3. Use of the steroidal compound of claim 1 in the preparation of a medicament for treating neuroinflammatory diseases.

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