Steroidal compounds having anti-parkinsonian activity, processes for their preparation and use

Abstract

The application discloses a kind of steroidal compounds with anti-parkinson disease activity and its preparation method and application.The steroidal compound has the structure as shown in formula (I) or formula (II), or stereoisomer, tautomer, pharmaceutically acceptable salt or prodrug of the structure shown in formula (I) or formula (II).The application is fermented by fungal co-cultivation to alternaria brassicae and penicillium granatum, and the expression of silent gene of strain is promoted by adopting co-cultivation and optimizing medium condition strategy, novel secondary metabolite is obtained, and new steroidal compound with treatment parkinson disease value and application prospect is extracted and separated from co-cultivation ferment, which provides great prospect for the research and development of parkinson disease treatment drugs.

Description

Technical Field

[0001] This application relates to the field of anti-Parkinson's disease drug technology, specifically to a steroidal compound with anti-Parkinson's disease activity, its preparation method, and its application. Background Technology

[0002] Parkinson's disease (PD) is a chronic neurodegenerative disease caused by neurotransmitter dysfunction due to a deficiency of dopamine and other neurotransmitters. The main clinical manifestations of PD are resting tremor, bradykinesia, rigidity, and postural and gait abnormalities, accompanied by neurological symptoms, which significantly impact patients' daily activities. Currently, there are approximately 4 million people with PD in my country. Given the significant population growth in China over the past few decades, the number of people with PD in my country is expected to increase annually in the future.

[0003] Currently, Parkinson's disease (PD) is treated with either surgery or medication. Based on surgical cases and prognoses, surgery carries certain risks, and patient tolerance is a concern. Furthermore, it requires a long period of rehabilitation and adjustment after surgery. In medication, drugs primarily composed of levodopa (L-DOPA) have shown good efficacy in treating PD. However, long-term use of levodopa can lead to side effects such as the "on-off phenomenon"—sudden onset of limb rigidity and inability to move. Moreover, these drugs have minimal effect on patients in the middle and late stages of the disease. Therefore, there is a need to actively develop cost-effective anti-PD drugs.

[0004] Microorganisms are now a rich resource for drug discovery, due to the novelty and stability of their products, as well as the mature fermentation processes that allow for large-scale production of stable natural products. This makes them an important research subject for drug discovery both domestically and internationally. However, in recent years, with the increasing scale of research, the repetitive occurrence of secondary metabolites in microorganisms has slowed the development of lead compounds. In seeking solutions to this problem, the emergence of microbial co-culture has provided a new research approach to increase metabolite diversity. Furthermore, with the application of this method in microbial culture, compounds with novel skeletons and activities have been discovered. Therefore, using co-culture methods and optimizing culture conditions to mine the metabolites and bioactivities of fermentation products from co-cultured fungi is of paramount importance for developing novel drugs for the treatment of Parkinson's disease. Summary of the Invention

[0005] This application provides a steroidal compound with anti-Parkinson's disease activity, its preparation method, and its application. By co-culturing and fermenting Alternaria brassicae and Penicillium granulosum, and by adopting co-culturing and optimizing culture medium conditions, the expression of silent genes in the strains is promoted, novel secondary metabolites are obtained, and new steroidal compounds with therapeutic value and application prospects for Parkinson's disease are extracted and isolated from the co-culture fermentation product.

[0006] Therefore, the embodiments disclose at least the following technical solutions:

[0007] In a first aspect, embodiments of this application disclose a steroidal compound with anti-Parkinson's disease activity, wherein the steroidal compound has a structure as shown in formula (I) or formula (II), or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug of the structure shown in formula (I) or formula (II):

[0008]

[0009] Secondly, embodiments of this application disclose an anti-Parkinson's disease formulation, the formulation containing a therapeutically effective amount of the steroidal compound described in the first aspect, as well as pharmaceutically acceptable excipients.

[0010] Thirdly, embodiments of this application disclose a method for preparing a steroidal compound as described in the first aspect, comprising the following steps:

[0011] Activated colonies containing Alternaria brassicae and Penicillium granulosum were obtained, respectively.

[0012] The activated colonies were inoculated into liquid culture medium and cultured to form seed culture.

[0013] The seed culture was inoculated into a solid fermentation medium for large-scale fermentation to obtain fermentation products;

[0014] The fermentation product is extracted and separated to obtain the steroidal compound;

[0015] Among them, Alternaria brassicicola, with the Latin name Alternaria brassicicola, is from the China Agricultural Microbial Culture Collection Center, with accession number ACCC 37296;

[0016] Penicillium granulatum, from the China Marine Microbial Culture Collection Center, accession number: MCCC 3A00475.

[0017] Fourthly, embodiments of this application disclose the use of a steroidal compound as described in the first aspect or prepared by the method described in the third aspect in the preparation of anti-Parkinson's drugs.

[0018] Compared with the prior art, this application has at least the following beneficial effects:

[0019] 1. This application uses Alternaria brassicae and Penicillium granulosum for co-culture. By changing the culture medium and culture conditions, and the mixing ratio, the silent biosynthetic genes of Alternaria brassicae are epigenetically regulated, and novel secondary metabolite compounds 1-3 are obtained.

[0020] 2. The novel compound 1 provided in this application exhibited good biological activity against both cell and animal models of Parkinson's disease. Compound 1 exerted dose-dependent effects on MPP at concentrations ranging from 0 to 50 μM. + The protective effect of compound 1 on SH-SY5Y cell PD model; compound 1 significantly improved the motor ability of MPTP-induced zebrafish PD model at a concentration of 80.0 μg / mL. This demonstrates the great potential of these compounds to be developed into drugs for the treatment of Parkinson's disease.

[0021] 3. In terms of process flow, this application only requires reviving and subculturing the frozen strains according to the experimental plan, and culturing them under optimized culture conditions to achieve scale-up fermentation. Furthermore, the yield of compounds can be purposefully controlled by adjusting the total amount of fermentation products, ultimately obtaining the target product with anti-Parkinson's disease properties. Attached Figure Description

[0022] Figure 1 The single-crystal structure diagram of the compound shown in Formula I-1 provided in the embodiments of this application is shown.

[0023] Figure 2 A comparison chart of experimental and calculated ECD values ​​for the compounds shown in Formula I-2 provided in the embodiments of this application.

[0024] Figure 3 The single-crystal structure diagram of the compound shown in Formula II-1 provided in the embodiments of this application is shown.

[0025] Figure 4 A diagram illustrating the cytotoxic effects of the compounds provided in this application and the positive control drug L-DOPA on SH-SY5Y cells.

[0026] Figure 5 The compounds provided in the embodiments of this application and the positive control drug L-DOPA against MPP + The protective effect of induced SH-SY5Y cell PD model.

[0027] Figure 6The compound shown in I-1 provided in the embodiments of this application and the positive control drug L-DOPA against MPP + The protective effect of induced SH-SY5Y cell PD model.

[0028] Figure 7 The diagram shows the effect of the compound shown in I-1 provided in the embodiments of this application on the movement distance of the MPTP-induced zebrafish PD model.

[0029] Figure 8 The graph shows the effect of the compound shown in I-1 provided in the embodiments of this application on improving the movement of the MPTP-induced zebrafish PD model. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0031] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence, nor do they substantially limit the technical features thereafter. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0032] For the purpose of better understanding this application and not limiting its scope, all figures indicating quantities, percentages, and other numerical values ​​used herein should, in all cases, be understood to be modified by the word "approximately." Therefore, unless otherwise stated, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0033] This application discloses a steroidal compound with anti-Parkinson's disease activity, wherein the steroidal compound has a structure as shown in formula (I) or formula (II), or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug of the structure shown in formula (I) or formula (II):

[0034]

[0035] In some embodiments, the steroid compound has a structure as shown in formula (I-1), formula (I-2), or formula (II-1), or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug of the structure shown in formula (I-1), formula (I-2), or formula (II-1):

[0036]

[0037] In some embodiments, the steroidal compound is as shown in formula (I-1).

[0038] In some embodiments, the steroidal compound is as shown in formula (I-2).

[0039] In some embodiments, the steroidal compound is as shown in formula (II-1).

[0040] In some embodiments, the steroid compound is a stereoisomer of the structure shown in formula (I-1).

[0041] In some embodiments, the steroid compound is a stereoisomer of the structure shown in formula (I-2).

[0042] In some embodiments, the steroid compound is a stereoisomer of the structure shown in formula (II-1).

[0043] In some embodiments, the steroid compound is a tautomer of the structure shown in formula (I-1).

[0044] In some embodiments, the steroid compound is a tautomer of the structure shown in formula (I-2).

[0045] In some embodiments, the steroid compound is a tautomer of the structure shown in formula (II-1).

[0046] In some embodiments, the steroid compound is a pharmaceutically acceptable salt of the structure shown in formula (I-1).

[0047] In some embodiments, the steroidal compound is a pharmaceutically acceptable salt of the structure shown in formula (I-2).

[0048] In some embodiments, the steroid compound is a pharmaceutically acceptable salt of the structure shown in formula (II-1).

[0049] In some embodiments, the steroid compound is a prodrug with the structure shown in formula (I-1).

[0050] In some embodiments, the steroid compound is a prodrug with the structure shown in formula (I-2).

[0051] In some embodiments, the steroid compound is a prodrug with the structure shown in formula (II-1).

[0052] This application also discloses an anti-Parkinson's disease formulation containing a therapeutically effective amount of the steroidal compound and pharmaceutically acceptable excipients.

[0053] In some embodiments, the pharmaceutically acceptable excipients include at least one of a diluent, a carrier, and an excipient.

[0054] This application also discloses a method for preparing the steroidal compound as described above, comprising the following steps:

[0055] Activated colonies containing Alternaria brassicae and Penicillium granulosum were obtained, respectively.

[0056] The activated colonies were inoculated into liquid culture medium and cultured to form seed culture.

[0057] The seed culture was inoculated into a solid fermentation medium for large-scale fermentation to obtain fermentation products;

[0058] The fermentation product is extracted and separated to obtain the steroidal compound;

[0059] Among them, Alternaria brassicicola, with the Latin name Alternaria brassicicola, is from the China Agricultural Microbial Culture Collection Center, with accession number ACCC 37296;

[0060] Penicillium granulatum, from the China Marine Microbial Culture Collection Center, accession number: MCCC 3A00475.

[0061] In some embodiments, the activated colonies are obtained by inoculating a strain of *Alternaria brassicae* or *Penicillium granulosum* into a PDA medium and culturing it.

[0062] In some embodiments, the seed culture is obtained by inoculating activated strains from PDA medium into PDB medium and culturing them.

[0063] In some embodiments, the culture temperature of the PDA or PDB medium is 25-32°C.

[0064] In some embodiments, the culture temperature of the PDA or PDB medium is 28°C.

[0065] In some embodiments, the solid fermentation medium comprises 40-60 wt% rice.

[0066] In some embodiments, the solid fermentation medium comprises 50 wt% rice.

[0067] In some embodiments, the step of extracting and separating the fermentation products includes:

[0068] A crude extract is obtained, wherein the crude extract is obtained by extracting the fermentation broth with anhydrous methanol;

[0069] A refined extract is obtained by sequentially extracting the crude extract with petroleum ether, dichloromethane and ethyl acetate.

[0070] To obtain a steroidal compound with the structure shown in formula (I) or formula (II), wherein the steroidal compound of formula (I) or formula (II) is obtained by purifying the ethyl acetate fraction of the refined extract by normal-phase silica gel chromatography, reversed-phase silica gel chromatography, and high-performance preparative chromatography; and

[0071] To obtain a steroidal compound with the structure shown in formula (I-1), formula (I-2), or formula (II-1), wherein the steroidal compound described in formula (I-1), formula (I-2), or formula (II-1) is obtained by preparing and purifying the steroidal compound described in formula (I) or formula (II) using a chiral chromatographic column.

[0072] In some embodiments, the steps for obtaining the steroidal compound as described in formula (I) or (II) specifically include:

[0073] The first component is obtained by loading the refined extract onto a normal-phase silica gel column and collecting the eluent.

[0074] A second component is obtained by loading the first component onto a reversed-phase silica column and collecting the eluent.

[0075] A third component is obtained by loading the second component onto a high-performance liquid chromatography column and collecting the eluent, the third component comprising a steroidal compound as shown in formula (I) or formula (II).

[0076] This application also discloses the use of a steroidal compound as described above, or a steroidal compound prepared by any of the methods described above, in the preparation of anti-Parkinson's drugs.

[0077] The technical solution of this application will be clearly and completely described below with reference to the embodiments of this application.

[0078] 1. Preparation of compounds as shown in Formula I or Formula II

[0079] (1)Strain

[0080] The compounds disclosed in the embodiments of this application, as shown in Formula I-1, Formula I-2 and Formula II, are obtained by extracting and separating their secondary metabolites through fungal fermentation culture.

[0081] The bacterial strains involved in the embodiments of this application come from the following sources:

[0082] Alternaria brassicicola, from the China Agricultural Microbial Culture Collection Center, accession number: ACCC 37296;

[0083] Penicillium granulatum, from the China Marine Microbial Culture Collection Center, accession number: MCCC 3A00475.

[0084] (2) Activation of strains

[0085] Prepare the culture medium according to the following formula ratio, and follow the steps below:

[0086] 1) Pour the weighed PDA into the blue-capped bottle, add sterile water and mix well. Adjust the pH to 7.0 with NaOH, and put it into an autoclave set at 121℃ for 30 minutes.

[0087] 2) Sterilize by UV exposure for 30 minutes, and allow to cool to approximately 50°C before pouring into plates.

[0088] 3) Irradiate with ultraviolet light for 15 minutes. After the culture medium has completely solidified, inoculate with Alternaria brassicicola ACCC 37296 and Penicillium granulatum MCCC 3A00475 respectively. After incubation at 28°C for 1 week, plate colonies can be obtained.

[0089] 4) Add PDB culture medium to a 250mL Erlenmeyer flask, add sterile water and mix well. At the same time, add antibiotics and histone deacetylase inhibitor (nicotinamide) and put it into an autoclave set at 121℃ for 30min.

[0090] 5) Sterilize by UV exposure for 30 minutes, and after cooling to approximately room temperature, inoculate Alternaria brassicae and Penicillium granulosum from PDA medium into PDB medium at a ratio of approximately 4:1 (size of agar block). Incubate in a shaker incubator at 28°C and 180 rpm for 5 days to form a seed culture.

[0091] (3) Fermentation

[0092] The seed culture obtained above was inoculated into a sterile solid fermentation medium and fermented for 28 days under normal pressure and oxygen content to obtain the fermentation broth. The fermentation medium was a 50 wt% (mass percentage) rice aqueous solution, and the rice could be soaked and crushed in advance;

[0093] (4) Extraction and separation

[0094] 1) Obtain crude extract and refined extract

[0095] The fermentation broth was extracted with anhydrous methanol, and the solvent was recovered under reduced pressure to obtain 374.8 g of crude extract. Then, it was suspended in 5 L of 50 °C hot water and extracted sequentially with equal volumes of petroleum ether, dichloromethane and ethyl acetate. The solvent was recovered from the ethyl acetate extract to obtain 183.5 g of refined extract.

[0096] 2) Obtain the first component

[0097] 183.5g of the refined extract was loaded onto a normal phase silica gel chromatographic column (60-80 mesh) and eluted with a petroleum ether-acetone gradient (100% petroleum ether → 10% petroleum ether) to obtain 10 fractions, which were named Fr.1-Fr.10 in sequence.

[0098] Fraction Fr.4 was loaded again onto a normal-phase silica column and eluted with a gradient of petroleum ether-ethyl acetate (100% petroleum ether → 25% petroleum ether) to obtain nine fractions, which were named Fr.6.1-Fr.6.9 respectively; among them, Fr.6.4 is the first component.

[0099] 3) Obtain the second component

[0100] The first fraction, Fr.6.4, was loaded onto a reversed-phase silica column and eluted with a methanol-water gradient (30% methanol → 100% methanol) to obtain six fractions, which were named Fr.6.4.1-Fr.6.4.6 respectively; among them, Fr.6.4.5 is the second fraction.

[0101] 4) Obtain the third component

[0102] The second fraction, Fr.6.4.5, was loaded onto a reversed-phase high-performance chromatography preparative column (Acclaim™ Trinity Q1LC, catalog number: 083244; mobile phase: methanol:water = 85:15, 2 ml / min); compounds of formula I and formula II were obtained respectively.

[0103] If the compounds shown in Formula I are loaded onto chiral chromatographic columns (Chilalpak IC column, mobile phase: acetonitrile:water = 70:30, 2 ml / min) respectively, the compounds shown in Formula I-1 and Formula I-2 can be obtained respectively.

[0104] If the compound shown in Formula II is loaded onto a chiral chromatographic column (Chilalpak IC column, mobile phase: acetonitrile:water = 70:30, 2 ml / min) to prepare the compound shown in Formula II-1, the compound shown in Formula II-1 can be obtained.

[0105] 2. Structural identification of compounds as shown in Formula I or Formula II

[0106] The absolute configurations of compounds I-1, I-2 and II-1 were obtained using NMR (nuclear magnetic resonance), computational ECD (electron circular diffraction), and X-ray (X-single crystal diffraction).

[0107] (1) Nuclear magnetic resonance

[0108] The compound shown in Formula I-1: colorless bulk crystals; HRESIMS: m / z 495.3447 [M+Na] + (calcd forC 30 H 48 O4Na + ,495.3445);[α]2D0+36.2(c 0.06,CHCl3);UV(CH3OH)λ max (logε)203(5.23)nm; IR(KBr)ν max 2956,2866,1708,1379,1267,1095,1026cm –1 ECDλ max (Δε)216(+0.53)nm; 1 H and 13 CNMR data are shown in Table 1.

[0109] The compound shown in Formula I-2 is a colorless, oily liquid; HRESIMS: m / z 495.3457 [M+Na] + (calcd forC 30 H 48 O4Na +,495.3445);[α]2D0 -78.2(c 0.05,CHCl3);UV(CH3OH)λ max (logε)203(5.13)nm; IR(KBr)ν max 2956,2872,1718,1452,1375,1251,1024cm –1 ECDλ max (Δε)206(-12.86)nm; 1 H and 13 C NMR data are shown in Table 1.

[0110] The compound shown in Formula II-1: colorless bulk crystals; HRESIMS: m / z 453.3313 [M+Na] + (calcd forC 28 H 46 O3Na + ,453.3345);[α]2D0+51.3(c 0.03,CHCl3);UV(CH3OH)λ max (logε)204(4.51)nm; IR(KBr)ν max 3726,3315,2956,2872,2299,1456,1373cm –1 ; 1 H and 13 C NMR data are shown in Table 1.

[0111] Table 1. 1 H and 13 C NMR data of Compounds 1-3.(CDCl3,δin ppm,J in Hz)

[0112]

[0113]

[0114] (2) Electronic circular dichroism (ECD)

[0115] The absolute configuration of the compound shown in Formula I-2 (named compound 2) was verified by electronic circular dichroism (ECD). Results: The Cotton effect measured by the experimental ECD was in good agreement with the calculated value, see [see figure]. Figure 2 .

[0116] (3) X-ray single-crystal diffraction analysis

[0117] The absolute configuration of compound I-1 (named compound 1) was determined by copper target X-ray analysis, crystal number: CCDC 2163265, see [link to crystal analysis]. Figure 1 .

[0118] The absolute configuration of compound II-1 (named compound 3) was determined by copper-target X-ray analysis of the compound shown in formula II-1. Crystal number: CCDC 2169685. See [link to crystal analysis]. Figure 3 .

[0119] The final compound obtained was identified as...

[0120]

[0121] The compound shown.

[0122] 3. Cytotoxicity studies

[0123] The steps for assessing cell viability using the CCK-8 assay include:

[0124] The SH-SY5Y cell line was selected and cultured in DMEM high glucose + 10% FBS + 1% P / S. The culture was performed at a rate of 1×10⁶ cells / year. 4 Cells were seeded at a density of 100 cells / mL into 96-well plates and cultured in an incubator. Cell status was then observed. The following experimental groups were set up:

[0125] Control group: No cells were added, only culture medium and CCK-8 were added;

[0126] Normal group: group with added cells, no drug administered;

[0127] Experimental group 1: Cells were added and treated with 20 μM levodopa (L-DOPA);

[0128] Experimental group 2: Cells were added and treated with 50 μM compound 1;

[0129] Experimental group 3: Cells were added and treated with 50 μM compound 2;

[0130] Experimental group 4: Cells were added and treated with 50 μM compound 3;

[0131] After incubation with 10 μL of CCK-8 for 4 h, the absorbance at 450 nm was measured, and the cell viability (n=3) was calculated using the formula:

[0132]

[0133] Among them, compounds 1, 2, and 3 are the compounds shown in formula I-1, formula I-2, and formula II-1, respectively.

[0134] Result: As Figure 4 As shown, analysis of CCK-8 assay data revealed that compounds 1-3 showed no significant cytotoxic effect on SH-SY5Y cells at a concentration of 50 μM; the positive control drug levodopa (L-DOPA) showed no significant cytotoxic effect on SH-SY5Y cells at a concentration of 20 μM.

[0135] 4. The compounds shown in Formula I and Formula II are effective against MPP. + Activity assay of induced SH-SY5Y cell PD model

[0136] SH-SY5Y cells were cultured in DMEM complete medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. Cells in the logarithmic growth phase were seeded into 96-well cell culture plates (2000-4000 cells / well) and cultured for 1 day. The CCK-8 assay was used for experiments. The experiment included a blank control group, a normal control group, a model group, and an experimental group.

[0137] Control group: No cells were added, only culture medium and CCK-8 were added;

[0138] Normal group: group with added cells, no drug administered;

[0139] Model group: Add cells and use 100 μM MPP + Induced injury for 24 hours;

[0140] Experimental group a: Cells were added, pretreated with a compound or positive control for 12 hours, and then 100 μM MPP was added. + Induced injury for 24 hours. Compound concentration: 50 μM; Positive agent concentration: 20 μM.

[0141] Experimental group b: Cells were added, pretreated with a compound or positive control for 12 hours, and then 100 μM MPP was added. + Induced injury for 24 hours. Compound 1 concentrations: 6.25, 12.5, 25, 50 μM; Positive agent concentration: 20 μM.

[0142] Set up 3 replicates, n=3. Calculate the SH-SY5Y cell viability using the formula:

[0143]

[0144] Among them, compounds 1, 2, and 3 are the compounds shown in formula I-1, formula I-2, and formula II-1, respectively.

[0145] Results of experimental group a: as follows Figure 5 As shown, analysis of CCK-8 data revealed that at 100 μM MPP +In the PD model induced by SH-SY5Y cells, cell survival rate was significantly reduced. After pretreatment of cells with 50 μM compound 1-3 or 20 μM positive control levodopa, the compounds significantly improved cell survival rate compared with the model group, showing certain anti-PD activity.

[0146] Compound 1 showed the best results, so it was further screened.

[0147] Results of experimental group b: as follows Figure 6 As shown, analysis of CCK-8 assay data revealed that, compared to the model group, compound 1 improved cell viability in a dose-dependent manner within the 50 μM concentration range.

[0148] 5. Animal behavioral experiments of the compound shown in Formula I-1 (compound 1) on MPTP-induced PD model in zebrafish.

[0149] Zebrafish were cultured in aquarium water at 28°C (water quality: 200 mg of readily soluble sea salt added per 1 L of reverse osmosis water; conductivity 450–550 μS / cm; pH 6.5–8.5; hardness 50–100 mg / L CaCO3). Wild-type AB strain zebrafish were bred through natural pair mating. Zebrafish aged 4 days post-fertilization (4 dpf) were used for the determination of the maximum detectable concentration (MTC) of compound 1 for improving motility and behavior, and for efficacy evaluation.

[0150] Wild-type AB strain zebrafish at 4 dpf were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Compound 1 (concentrations of 40.0, 60.0, and 80.0 μg / mL) was administered in water, and a normal control group and a model control group were also set up, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were given MPTP in water to establish a zebrafish Parkinson's disease model. Treatment was carried out at 28℃ for 48 h, with daily medium changes. At 6 dpf, 10 zebrafish from each experimental group were randomly selected and transferred to 96-well plates, 100 μL / zebrafish, 1 zebrafish / well. Data were collected using a behavioral analyzer, and the total movement distance of the zebrafish within 10 min was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of compound 1 in improving movement behavior.

[0151] result:

[0152] As shown in Table 2, the MTC of compound 1 in model zebrafish was 80.0 μg / mL.

[0153] like Figure 7 and Figure 8 As shown, at this concentration, compound 1 has a behavioral-improving effect on the zebrafish PD model, demonstrating its anti-Parkinson's disease activity.

[0154] Table 2. Results of the concentration exploratory experiment for improving motor behavior of compound 1 (n=30)

[0155]

[0156]

[0157] In summary, this application uses Alternaria brassicae and Penicillium granulosum for co-culture, and through research methods such as changing the culture medium and culture conditions, and the mixing ratio, epigenetic regulation of their silenced biosynthetic genes was carried out, resulting in novel secondary metabolite compounds 1-3.

[0158] The novel compound 1 exhibited good biological activity against both cell and animal models of Parkinson's disease. Compound 1 exerted dose-dependent effects on MPP at concentrations ranging from 0 to 50 μM. + The protective effect of compound 1 on SH-SY5Y cell PD model; compound 1 significantly improved the motor ability of MPTP-induced zebrafish PD model at a concentration of 80.0 μg / mL. This demonstrates the great potential of these compounds to be developed into drugs for the treatment of Parkinson's disease.

[0159] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for preparing a steroidal compound, comprising the following steps: Activated colonies containing Alternaria brassicae and Penicillium granulosum were obtained, respectively. The activated colonies were inoculated into liquid culture medium and cultured to form seed culture. The seed culture was inoculated into a solid fermentation medium for large-scale fermentation to obtain fermentation products; The fermentation product is extracted and separated to obtain the steroidal compound; Among them, Alternaria brassicicola, with the Latin name Alternaria brassicicola, is from the China Agricultural Microbial Culture Collection Center, with accession number ACCC 37296; Penicillium granulatum, from the China Marine Microbial Culture Collection Center, accession number: MCCC 3A00475; The steroid compound has a structure as shown in formula (I-1), (I-2), or (II-1): 、 、 。 2. The preparation method according to claim 1, wherein the activated colonies are obtained by inoculating a strain of *Alternaria brassicae* or *Penicillium granulosum* into PDA medium and culturing them; The seed culture is obtained by inoculating activated strains from PDA medium into PDB medium and culturing them. The culture temperature of the PDA or PDB medium is 25-32℃; The solid fermentation medium comprises 40-60 wt% rice.

3. The preparation method according to claim 1, wherein the step of extracting and separating the fermentation product includes: A crude extract is obtained, wherein the crude extract is obtained by extracting the fermentation broth with anhydrous methanol; A refined extract is obtained by sequentially extracting the crude extract with petroleum ether, dichloromethane, and ethyl acetate.

4. The preparation method according to claim 3, wherein the step of obtaining the steroidal compound according to claim 1 specifically includes: The first component is obtained by loading the refined extract onto a normal-phase silica gel column and collecting the eluent. A second component is obtained by loading the first component onto a reversed-phase silica column and collecting the eluent. A third component is obtained by loading the second component onto a high-performance liquid chromatography column and collecting the eluent, the third component containing the steroidal compound.

5. The use of steroidal compounds having structures as shown in formula (I-1), (I-2), or (II-1) prepared by the preparation method according to any one of claims 1 to 4 in the preparation of anti-Parkinson's drugs.

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