Steroids, processes for their preparation and use

Abstract

The application belongs to the technical field of medicine, and particularly relates to a steroidal compound in medicinal fungus Shandong ganoderma, a preparation method of the steroidal compound and application of the steroidal compound in resisting Alzheimer's disease. The structures of the steroidal compounds 1-4 are shown in the following; the application further provides application of the compounds in preparing medicines for resisting Alzheimer's disease. The preparation method is simple, reproducible, the obtained compound has high purity, and has good inhibitory activity on acetylcholinesterase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a steroidal compound in medicinal fungus Ganoderma shandongense, a preparation method of the steroidal compound, and application of the steroidal compound in resisting Alzheimer's disease. BACKGROUND

[0002] Ganoderma spp. is a collective term for various fungi of the genus Ganoderma, belonging to Basidiomycetes, Polyporales, Polyporaceae, Ganoderma, and has important economic value. Ganoderma lucidum is widely used in China and some other Asian countries, with sweet and flat nature, and heart, lung, liver and kidney meridians, and has the effects of tonifying qi, soothing the nerves, relieving cough and asthma, and is used for dizziness, insomnia, palpitation, shortness of breath, and is considered to have the effects of health care and longevity, and is mainly distributed in Guizhou, Yunnan, Fujian, Guangxi and other places. As a traditional Chinese medicine, Ganoderma has a relatively definite medicinal value. Modern research on Ganoderma shows that the main chemical components contained in Ganoderma include sugars, proteins, amino acids, alkaloids, triterpenoid compounds and trace elements such as calcium, zinc and magnesium. Pharmacological studies have shown that Ganoderma has various pharmacological activities such as anti-tumor, immune regulation and anti-diabetic, and has very important research value. At present, 103 species of Ganoderma have been reported, but the research focus is mainly on Ganoderma lucidum, Ganoderma sinense and Ganoderma applanatum, and other species are relatively lacking in research, which is an important resource for drug development. Among them, Ganoderma shandongense was discovered and identified as a new species by Zhao Jiding et al. in 1982, and there is no report on its chemical composition and biological activity.

[0003] Alzheimer's disease (AD), commonly known as senile dementia, is a complex degenerative disease of the central nervous system, and its clinical manifestations include progressive cognitive impairment, memory impairment, aphasia, visual spatial impairment, executive dysfunction, personality and behavior changes, etc. Therefore, AD-related symptoms can be alleviated by maintaining or restoring the level of acetylcholine in the brain. Acetylcholinesterase can promote the degradation of acetylcholine in synapses and neuromuscular junctions, thereby causing the termination of nerve impulses. However, existing acetylcholinesterase inhibitors generally have different degrees of adverse reactions such as diarrhea, vomiting, insomnia, etc., which reduces the medication compliance of AD patients. Therefore, it is urgent to design and develop new and efficient AD treatment drugs, and it is of great significance to find and develop new acetylcholinesterase inhibitors from natural products. SUMMARY

[0004] The primary object of the present application is to provide a steroidal compound in medicinal fungus G.shandongense and its preparation method and the application of the compound in resisting Alzheimer's disease.

[0005] To achieve the above object, the technical scheme adopted by the present application is

[0006] A steroidal compound, 1-4 is shown as follows:

[0007]

[0008] A preparation method of the steroidal compound in claim 1:

[0009] (1) Strain activation: inoculate G.shandongense mycelium on malt powder medium, and place it in 28℃, dark condition for 5-7 days. Then cut the mycelium on the plate into 0.5cm 2 small pieces for use; the components of the malt powder medium are: malt powder 10g, distilled water 500mL, agar 7.5g, pH natural;

[0010] (2) Enlarged culture of the strain: take malt powder 10g, distilled water 500mL, agar 7.5g, and sterilize them at 121℃ for 30min for standby. Inoculate the above cut G.shandongense into the above medium, and incubate it at 28℃ for 5-6d;

[0011] (3) Fermentation: inoculate the mycelium obtained in (2) into rice solid fermentation medium, and carry out fermentation culture by solid state fermentation;

[0012] (4) the culture obtained from the fermentation in (3) is extracted with ethyl acetate for 3-5 times, the combined extract is concentrated under reduced pressure to obtain a extract, which is then separated by silica gel column under reduced pressure, eluted with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1), and each fraction is identified by thin layer chromatography and high performance liquid chromatography, and then combined to obtain four fractions Fr.A (petroleum ether-ethyl acetate 50:1), Fr.B (petroleum ether-ethyl acetate 30:1-1:1), Fr.C (dichloromethane-methanol 50:1-10:1), and Fr.D (dichloromethane-methanol 8:1-1:1), and the fraction Fr.B is further separated by silica gel column chromatography, eluted with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane / methanol (50:1-1:1) in gradient, and the fractions eluted with petroleum ether-ethyl acetate 5:1 and dichloromethane / methanol 50:1-30:1 are collected to obtain the fraction Fr.B3, and the obtained fraction is separated by ODS column chromatography, eluted with ethanol / water in gradient, and the fractions Fr.B3-1 with ethanol concentration of 90%-95% and Fr.B3-2 with ethanol concentration of 70%-85% are collected;

[0013] The collected fraction Fr.B3-1 is separated by preparative HPLC, eluted with methanol / water, and the chromatographic peaks at 45-50 min are collected, and the obtained sample is separated by semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peak at 18 min is collected to obtain compound 1, the chromatographic peak at 32 min is collected to obtain compound 2, and the chromatographic peak at 54 min is collected to obtain compound 4;

[0014] The collected fraction Fr.B3-2 is further separated by preparative HPLC, eluted with methanol / water, and the chromatographic peaks at 40-70 min are collected, and the obtained sample is separated by semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peak at 68 min is collected to obtain compound 3.

[0015] The ethanol concentration gradient in the elution with ethanol / water in gradient is: 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%;

[0016] The methanol concentration in the elution with methanol / water in the preparative HPLC separation of compounds 1, 2 and 4 is 90%-95%;

[0017] The methanol concentration in the elution with methanol / water in the preparative HPLC separation of compound 3 is 80%-90%;

[0018] The acetonitrile concentration in the elution with acetonitrile / water in the semi-preparative HPLC separation of compounds 1, 2 and 4 is 85%-90%.

[0019] The acetonitrile concentration in acetonitrile / water elution for separating compound 3 by the semi-preparative HPLC is 85%-90%.

[0020] The G.shandongense is a known strain described in Mycosystema 1986(02):86-92, and the strain used in the present application is obtained from Professor Dai Yucheng of Beijing Forestry University.

[0021] The mycelium in preparing the compound is inoculated into a malt extract medium plate, and cultured at 28℃ in dark for 5-7 days. Then the mycelium on the plate is uniformly cut into 0.5cm 2 small pieces for use; the components of the malt extract medium are: malt extract 10g, distilled water 500mL, agar 7.5g, pH natural;

[0022] Take malt extract 10g, distilled water 500mL, agar 7.5g, and autoclave at 121℃ for 30min for standby. The cut G.shandongense is inoculated into the above medium, and incubated at 28℃ for 5-6d;

[0023] Inoculate 1 / 4 dish of G.shandongense mycelium into 80-120g rice solid fermentation medium per 80-120g rice, and then incubate until the mycelium covers the substrate; wherein the rice solid fermentation medium is 80-120g rice added into 100-250mL water.

[0024] A pharmaceutical composition comprising the steroidal compound, a pharmaceutically acceptable excipient or / and carrier.

[0025] The steroidal compound or the pharmaceutical composition for preparing an anti-Alzheimer's disease drug.

[0026] The obtained compound 1-4 is evaluated for acetylcholinesterase inhibitory activity, and the results show that the obtained compound has good acetylcholinesterase inhibitory activity (IC 50 values are 77.93, 29.35, 33.21, and 29.35μmol / L, respectively). Therefore, the steroidal compound has the potential to be developed as an anti-Alzheimer's disease drug. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The HRESIMS spectrum of compound 1 provided for the embodiment of the present application;

[0028] Figure 2 The HRESIMS spectrum of compound 1 provided for the embodiment of the present application; 1H NMR spectrum (600 MHz, CDC13) ;

[0029] Figure 3 HSQC spectrum (600 MHz, CDC13) of Compound 1 provided for the embodiments of the present application; 13 C NMR spectrum (150 MHz, CDC13) ;

[0030] Figure 4 HMBC spectrum (600 MHz, CDC13) of Compound 1 provided for the embodiments of the present application;

[0031] Figure 5 HMBC spectrum (600 MHz, CDC13) of Compound 1 provided for the embodiments of the present application;

[0032] Figure 6 HSQC spectrum (600 MHz, CDC13) of Compound 1 provided for the embodiments of the present application; 1 H- 1 H COSY spectrum (600 MHz, CDC13) ;

[0033] Figure 7 NOESY spectrum (600 MHz, CDC13) of Compound 1 provided for the embodiments of the present application;

[0034] Figure 8 Experimental and calculated data of Compound 1 provided for the embodiments of the present application 13 Regression analysis of CNMR chemical shifts; DETAILED DESCRIPTION

[0035] The following examples are helpful for those skilled in the art to better understand the present application, but do not limit the present application in any way.

[0036] In the following examples, G. shandongense is a known strain described in Mycosystema 1986 (02): 86-92, and the strain used in the present application is obtained from Professor Dai Yucheng of Beijing Forestry University.

[0037] Example 1: Preparation of steroidal compounds 1-4 in G. shandongense

[0038] 1. Fermentation conditions

[0039] Strain activation: inoculate the mycelium of G. shandongense on malt extract powder medium, and cultivate at 28°C in the dark for 5-7 days. Then evenly cut the mycelium on the plate into 0.5 cm 2 small pieces for use; the components of the malt extract powder medium are: malt extract powder 10 g, distilled water 500 mL, agar 7.5 g, pH natural;

[0040] G.shandongense was inoculated into the above medium and incubated at 28°C for 5-6 days.

[0041] Fermentation: 100 g of rice was put into a 500 mL conical flask, 100 mL of water was added, and the mixture was autoclaved at 121°C for 30 min. G.shandongense mycelium was inoculated at a ratio of 1 / 4 dish per bottle, and the mixture was incubated at 28°C for 45 days.

[0042] 2. Extraction and separation

[0043] The fermented culture was extracted with ethyl acetate at room temperature for 3-5 times, and the extract was combined and concentrated under reduced pressure at 40°C to obtain a extract. The extract (350.0 g) was mixed with 100-200 mesh silica gel, and gradient elution was performed with petroleum ether-ethyl acetate (50:1, 30:1, 10:1, 5:1, 3:1, 1:1) and dichloromethane-methanol (50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1) systems. The same fractions were combined by TLC detection, and petroleum ether-ethyl acetate 50:1 was component Fr.A (20.5 g), petroleum ether-ethyl acetate 30:1-1:1 was component Fr.B (30.3 g), dichloromethane-methanol 50:1-10:1 was component Fr.C (40.0 g), and dichloromethane-methanol 8:1-1:1 was Fr.D (36.2 g). Fr.B (30.3 g) was separated by silica gel column chromatography (diameter 80 mm, length 610 mm) with gradient elution with petroleum ether-ethyl acetate (50:1, 30:1, 10:1, 5:1, 3:1, 1:1) and dichloromethane-methanol (50:1, 30:1, 10:1, 5:1, 3:1, 1:1) systems in order of volume ratio, and the same fractions were combined by TLC and HPLC detection to obtain four fractions (petroleum ether-ethyl acetate 50:1 was Fr.B1, petroleum ether-ethyl acetate 30:1-1:1 was Fr.B2, dichloromethane-methanol 50:1-10:1 was Fr.B3, and dichloromethane-methanol 5:1-1:1 was Fr.B4). Fr.B3 (9.3 g) was separated by ODS column chromatography (diameter 50 mm, length 600 mm) with gradient elution with ethanol-water (35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), and the components Fr.B3-1 at 90%-95% and Fr.B3-2 at 70%-85% were collected.

[0044] Fr.B3-1 (945.0 mg) was subjected to column chromatography on silica gel (diameter 20 mm, length 400 mm) eluted with petroleum ether: ethyl acetate system in gradient of 20:1 to 1:1 by volume. Fraction Fr.B3-1-1 (165.5 mg) was collected at 20:1 to 10:1. Fraction Fr.B3-1-1 was subjected to preparative HPLC (250 x 20 mm, 5 μm) eluted with methanol / water at a flow rate of 7 mL / min. The sample was collected at 45-50 min. The sample was subjected to semi-preparative HPLC (250 x 10 mm, 5 μm) eluted with acetonitrile / water at a flow rate of 2.5 mL / min. Compound 1 (2.3 mg, t R = 18 min) was collected at 32 min and Compound 2 (43.9 mg, t R = 32 min) was collected at 54 min and Compound 4 (16.2 mg, t R = 54 min) was collected.

[0045] Fr.B3-2 (1.5 g) was subjected to column chromatography on silica gel (diameter 30 mm, length 450 mm) eluted with dichloromethane:methanol system in gradient of 100:1 to 1:1 by volume. Fraction Fr.B3-2-1 (457.0 mg) was collected at 100:1 to 30:1. Fraction Fr.B3-2-1 was subjected to preparative HPLC (250 x 20 mm, 5 μm) eluted with methanol / water at a flow rate of 7 mL / min. The sample was collected at 40-70 min. The sample was subjected to semi-preparative HPLC (250 x 10 mm, 5 μm) eluted with acetonitrile / water at a flow rate of 2.5 mL / min. Compound 3 (2.4 mg, t R = 68 min) was collected at 68 min.

[0046] The obtained compound 1 was subjected to systematic structural identification. The results are as follows, and the corresponding spectra are shown in the attached Figures 1-8 :

[0047] Compound 1: white powder, HRESIMS gave the pseudo-molecular ion peak m / z 411.3262 [M-H] - (calcd for C 28 H 43 O2, 411.3269), combined with the hydrogen spectrum and carbon spectrum data, the molecular formula of C 28 H 44 O2, the calculated unsaturation is 7.

[0048] In the hydrogen spectrum of compound 1, the high field region showed 6 methyl signals at δ H1.33 (3H, s), 1.00 (3H, s), 0.90 (3H, d, J = 6.5 Hz), 0.85 (3H, d, J = 6.9 Hz), 0.78 (3H, d, J = 2.3 Hz), 0.77 (3H, d, J = 2.3 Hz), presumed to be a steroidal compound; low field region shows presence of 2 olefinic proton signals, δ H 6.17 (1H, d, J = 9.8 Hz), 6.09 (1H, d, J = 9.8 Hz); carbon spectral data shows presence of one ketone carbonyl carbon signal in low field region, δ C 199.7, presumed to be at C-3 position; 4 olefinic carbon signals, δ C 163.3, 139.1, 127.8, 125.2, in conjunction with hydrogen spectral data, presence of 2 double bond signals in compound 1; one oxygen-bearing carbon signal, δ C 71.9; assignment of relevant hydrogen signals was also made by HSQC spectral data. The planar structure of compound 1 was determined by HMBC spectral data, Me-19 with δ C 36.2, 53.1, 163.3 have correlation, δ H 6.17, 6.09 with δ C 163.3 have correlation, δ H 6.09 with δ C 125.2 have correlation, indicating that the 2 double bonds in compound 1 are located at Δ 4,5 , Δ 6,7 . δ H 6.17 with δ C 71.9 have correlation, indicating that there is a hydroxyl group at C-8 position; thus, the planar structure of compound 1 was determined.

[0049] The configuration of compound 1 was determined by NOESY spectral data, in NOESY spectral data, Me-18 / Me-19 / H-20 have correlation, from which it can be determined that Me-19, Me-18, H-20 are all β type, Me-30 / H-17 / Me-21 correlation signals indicate that Me-30, H-17, Me-21 are all α type. Based on the relative configuration of C-28, NMR calculation was carried out, based on carbon spectral data fitting, the correlation coefficient R 2 and DP4+ analysis, by comparing the R 2 and DP4+ between the two possible configurations of compound 1 A-B, it is found that the calculation results of compound 1 and A are the highest, R 2 value is 0.999, carbon and hydrogen spectral DP4+ probability is 100%, through Scifinder retrieval, it is a new compound, named 8β-hydroxy-4,6-diene-3-en. The assignment of all carbon and hydrogen signals is shown in Table 1.

[0050] Table 1.1H NMR(600MHz,CDCl3)and 1 H NMR(600MHz,CDCl3)and 13 C NMR(150MHz,CDCl3)data

[0051]

[0052] HMBC of compound 1, 1 H- 1 H COSY and NOESY correlations are as follows:

[0053]

[0054] The obtained compound 2 was systematically identified as follows:

[0055] Compound 2: colorless solid, molecular formula C 28 H 42 O2, HRESIMS gave the pseudo-molecular ion peak m / z 411.32[M+H] + , confirming its molecular weight as 410. In 1 H-NMR(600MHz,CDCl3), five olefinic proton signals were observed in the low field region at δ H 6.15(d,J=9.7Hz,1H),6.07(d,J=9.8Hz,1H),5.71(s,1H),5.21(dd,J=15.2,7.8Hz,1H)and 5.12(dd,J=15.3,8.4Hz,1H), which were assigned to H-7, H-6, H-4, H-23 and H-22 signals, respectively. Six methyl signals were observed in the high field region at δ H 1.32(s,3H),1.01(s,3H),0.98(d,J=6.6Hz,3H),0.90(d,J=6.8Hz,3H),0.82(d,J=6.8Hz,3H)and 0.81(d,J=6.8Hz,3H), which suggested that the compound was a steroidal compound due to the presence of two singlet methyl signals. 13 C NMR(150MHz,CDCl3) spectrum, six olefinic carbon signals were observed in the low field region at δ C 139.4,135.4,132.3,128.1and 125.3; one carbonyl signal at δ C 199.8; one oxygen-bearing carbon signal at δ C 71.9; six methyl signals were observed in the high field region at δ C 20.1,19.8,19.1,18.0,17.86and 14.6; δ C35.1, 33.8, 18.0, 40.9, 19.0, 28.2 are methylene signals; δ C 53.3, 57.2, 56.6, 39.8, 42.9, 33.2 are methine signals, compound 2 is identified as isocyathisterol.

[0056] The obtained compound 3 was systematically identified as follows:

[0057] Compound 3: white powder, molecular formula is C 28 H 40 O3, HRESIMS gives quasi-molecular ion peak m / z 425.30 [M+H] + , the molecular weight is 424, in 1 H-NMR (600MHz, CDCl3) in the low field region gives four olefinic protons δ H 5.74 (s, 1H), 5.72 (s, 1H), 5.25 (dd, J = 15.2, 7.8 Hz, 1H), 5.14 (dd, J = 15.2, 8.5 Hz, 1H). In the high field region, six methyl signals are given respectively δ H 1.23 (s, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.63 (s, 3H), because there are two single methyl signals, it is suggested that the compound is a steroid. In 13 C NMR (150MHz, CDCl3) spectrum, 28 carbon signals are given, six olefinic carbon signals in the low field region are δ C 114.8, 174.0, 113.5, 159.6, 134.8, 133.0, respectively, C-4, C-5, C-7, C-8, C-22 and C-23 signals; two carbonyl signals δ C 198.6 and 162.6 are C-3 and C-6 signals, respectively; six methyl signals in the high field region are δ C 12.6, 20.1, 21.2, 20.1, 12.6, 17.7, respectively, C-18, C-19, C-21, C-26, C-27 and C-29 signals; δ C 34.1, 33.3, 25.5, 22.7, 27.8 are methylene signals; δ C 47.4, 58.3, 56.5, 40.5, 43.0, 33.2 are methine signals, compound 3 is identified as herbarulide.

[0058] The obtained compound 4 was systematically identified as follows:

[0059] Compound 4: colorless crystal, the molecular formula of which was determined as C 29 H 48 O2by combining high molecular mass spectrometry (ESI-MS). HRESIMS gave a quasi-molecular ion peak m / z 429.37 [M+H] + , determining the molecular weight of 428. In combination with hydrogen spectrum and carbon spectrum data, six methyl signals were given in the high field region, which were δ H 1.38 (s, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.84 (d, J = 6.1 Hz, 3H), 0.81 (d, J = 6.1 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H) and 0.74 (s, 3H), corresponding to carbon spectrum signals δ C 20.0, 19.7, 19.2, 18.9, 12.2, 12.1, wherein the hydrogen spectrum signal δ H 0.85 (t, J = 7.3 Hz, 3H) indicated that it was connected to a methylene group; in the low field region, one olefinic proton δ H 5.81 (s, 1H) and one oxymethylene signal δ H 4.35 (t, J = 3.1 Hz, 1H) were given, corresponding to carbon spectrum signals δ C 73.4, indicating the presence of a hydroxyl group; in the 13 C NMR (150 MHz, CDCl3) spectrum, 28 carbon signals were given, one carbonyl carbon signal δ C 200.6; δ C 37.3 (C-1), 34.4 (C-2), 38.7 (C-7), 21.1 (C-11), 39.7 (C-12), 24.3 (C-15), 28.3 (C-16), 34.0 (C-22), 26.2 (C-23), 23.2 (C-28) were methylene signals; δ C 29.9 (C-8), 53.8 (C-9), 56.0 (C-14), 56.2 (C-17), 36.3 (C-20), 46.0 (C-24), 29.3 (C-25) were methine signals. Compound 4 was determined to be 6β-hydroxystigmast-4-en-3-one.

[0060] Example 2: Acetylcholinesterase inhibitory activity of compounds 1-4

[0061] The above obtained compounds 1-4 were respectively diluted with PBS to 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, 5 μmol / L for use, 20 μL of the compound, 60 μL of PBS, 20 μL of acetylcholinesterase (0.22 U / mL) were added in a 96-well plate, 20 μL of substrate AchI (15 mM) and 100 μL of DNTB were added after reaction at 4°C for 10 min, parallel 3 times, and the reaction was continued for 20 min, the absorbance A1 was measured at 412 nm; equal volume of PBS was taken instead of acetylcholinesterase, parallel 3 times, the absorbance A2 was measured at 412 nm; equal volume of PBS was taken instead of the compound, the absorbance A3 was measured at 412 nm; equal volume of PBS was taken instead of the compound and acetylcholinesterase, the absorbance A4 was measured at 412 nm; donepezil hydrochloride diluted to 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, 5 μmol / L was used as a positive control, parallel 3 times, and the average value was taken to calculate the acetylcholinesterase inhibition rate and IC 50 value, the formula was inhibition rate = [(A3-A4)-(A1-A2)] / (A3-A4) x 100%.

[0062] Experiments proved that the positive control donepezil hydrochloride had an acetylcholinesterase IC 50 value of 35.74 μmol / L, and the acetylcholinesterase IC 50 values of the compounds 1-4 were 77.93, 29.35, 33.21, and 29.35 μmol / L respectively, indicating that they all had acetylcholinesterase inhibition effect, the extraction and separation method was simple, and it was convenient for further pharmacological and clinical research, and the application in the preparation of anti-Alzheimer's disease drugs.

Claims

1. A steroidal compound, characterized in that: The structures of steroidal compounds are shown below: 。 2. A method for preparing the steroidal compound according to claim 1, characterized in that: (1) Activation of strain: Shandong Ganoderma lucidum G. shandongense Mycelium was inoculated onto malt extract medium and cultured at 28°C in the dark for 5-7 days; then the mycelium on the plate was evenly cut into 0.5 cm pieces. 2 Small pieces are ready for use; (2) Large-scale culture of the strain: The above-mentioned cut pieces G. shandongense Inoculate into malt extract medium and incubate at 28°C for 5-6 days; (3) Fermentation: The mycelium obtained from (2) culture was inoculated into rice solid fermentation medium and fermented using solid fermentation method; (4) The culture obtained from fermentation in (3) was extracted with ethyl acetate 3-5 times. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was separated by silica gel column chromatography under reduced pressure, eluted with petroleum ether-ethyl acetate at a ratio of 50:1 to 1:1 and dichloromethane-methanol at a ratio of 50:1 to 1:

1. The fractions were identified and combined by thin-layer chromatography and high-performance liquid chromatography to obtain four fractions: Fr. A: petroleum ether-ethyl acetate 50:1, Fr. B: petroleum ether-ethyl acetate 30:1 to 1:1, Fr. C: dichloromethane-methanol 50:1 to 10:1, and Fr. D: dichloromethane-methanol 8:1 to 1:

1. Fraction Fr. B was subjected to silica gel column chromatography, eluted sequentially with petroleum ether-ethyl acetate at a ratio of 50:1 to 1:1 and dichloromethane / methanol at a ratio of 50:1 to 1:1, and the fractions of petroleum ether-ethyl acetate 5:1 and dichloromethane / methanol 50:1 to 30:1 were collected to obtain fraction Fr. B3 was separated by ODS column chromatography with ethanol / water gradient elution. The fraction Fr. B3-1 with ethanol concentration of 90%~95% and the fraction Fr. B3-2 with ethanol concentration of 70%~85% were collected. Fr. B3-1 was eluted by preparative HPLC with methanol / water and the chromatographic peak was collected at 45-50 min. The resulting sample was chromatographically separated by semi-preparative HPLC with acetonitrile / water and the chromatographic peak was collected at 18 min to obtain compound 1.

3. The method for preparing the steroidal compound according to claim 2, characterized in that: The malt extract culture medium consists of: 10 g malt extract, 500 mL distilled water, and 7.5 g agar; the culture conditions are: 25-28℃ for 5-6 days.

4. The method for preparing the steroidal compound according to claim 2, characterized in that: Add 1 / 4 dish of Shandong Ganoderma lucidum to every 80-120g of rice solid fermentation culture medium. G. shandongense The mycelium is inoculated in a certain proportion, and then statically cultured until the mycelium fully grows into the substrate; the rice solid fermentation medium is made by adding 80-120 g of rice to every 100-250 mL of water.

5. The method for preparing the steroidal compound according to claim 2, characterized in that: The ethanol concentration gradient for elution with an ethanol / water gradient is: 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. The methanol concentration was 90%~95% during methanol / water elution of compound 1 by the preparative HPLC method. The acetonitrile concentration was 85%~90% during the acetonitrile / water elution of compound 1 by the semi-preparative HPLC.

6. A pharmaceutical composition, characterized in that, Includes the steroidal compound of claim 1, a pharmaceutically acceptable excipient and / or carrier.

7. The application of the steroidal compound according to claim 1, characterized in that: The use of the steroidal compound of claim 1 in the preparation of anti-Alzheimer's disease drugs.

8. The use of the pharmaceutical composition according to claim 6, characterized in that: The use of the pharmaceutical composition of claim 6 in the preparation of an anti-Alzheimer's disease drug.

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