Triterpenoids in ganoderma lucidum from shandong province and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a triterpenoid compound in medicinal fungus Shandong ganoderma, a preparation method of the triterpenoid compound, and application of the triterpenoid compound in resisting Alzheimer's disease. The compound is shown in general formula I, and the application also provides application of the compound 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] This invention belongs to the field of pharmaceutical technology, specifically relating to triterpenoid compounds from the medicinal fungus Ganoderma lucidum (Shandong Ganoderma), their preparation methods, and the application of these compounds in the treatment of Alzheimer's disease. Background Technology

[0002] Reishi is a general term for fungi of the genus *Ganoderma*, belonging to the subphylum Basidiomycetes, order Polyporales, family Ganodermataceae, and genus *Ganoderma*. Reishi has a research history of over a thousand years in my country and is a traditional Chinese medicine with a very wide range of medicinal applications. Since the publication of the "Monthly Treatise on Medicinal Fungi in my country" in 1974, *Ganoderma lucidum* has been considered the scientific name of the well-known medicinal fungus *Ganoderma*. China has a rich variety of medicinal *Ganoderma* species, including *Amauroderma*, *G. atrum*, *G. sinense*, and *G. gibbosum*, mainly distributed in Guizhou, Yunnan, Fujian, and Guangxi provinces. *Ganoderma* contains polysaccharides, triterpenes, alkaloids, steroids, and trace elements such as calcium, zinc, and magnesium. Currently, 103 species of Ganoderma have been reported in my country, but current research focuses primarily on species such as Ganoderma lucidum, Ganoderma sinense, and Ganoderma applanatum, with research on other species being relatively lacking. These species are important resources for drug development. Among them, Ganoderma shandongense was a new species discovered and identified by Zhao Jiding et al. in 1982. Later, Sun et al., through phylogenetic analysis, found that Ganoderma shandongense formed an independent branch, establishing a new genus (Sinoganoderma gen.nov.) and classifying G. shandongense under S. shandongense. Currently, there are no reports on its chemical composition and biological activities. With the development of science and technology, research on the medicinal uses of Ganoderma is becoming increasingly in-depth. Ganoderma has antioxidant, anti-tumor, and hepatoprotective effects, and is clinically used to treat chronic bronchitis and some respiratory diseases, diabetes, hypertension, Alzheimer's disease, and as an adjunct therapy for tumors, becoming a research hotspot in recent years.

[0003] Alzheimer's disease (AD) is an irreversible, chronic neurodegenerative disease characterized by progressive memory impairment, impaired daily activities, and decreased acquired learning abilities. It is reported that two-thirds of the 50 million dementia cases worldwide are AD patients, and the number is projected to reach 152 million globally by 2050, with China's AD patient population potentially reaching 2.35 times that of 2015. Beta-amyloid plaques and neurofibrillary tangles caused by excessive phosphorylation of tau protein are the main pathogenic mechanisms of AD. Neuronal death, oxidative stress, and neurotransmitter imbalances are also associated with the occurrence and development of AD. The COVID-19 pandemic in 2020 may have exacerbated AD-related deaths. Currently, N-methyl-D-aspartate (NMDA) receptor antagonists and cholinesterase inhibitors are mainly used clinically, but side effects such as dizziness, gastrointestinal discomfort, worsening of stroke, and even death can occur. Therefore, it is urgent to design and develop new and highly effective drugs for the treatment of Alzheimer's disease. Summary of the Invention

[0004] The primary objective of this invention is to provide a triterpenoid compound prepared from Ganoderma shandongense.

[0005] Another object of the present invention is to provide a method for preparing the triterpenoid compound and the application of the triterpenoid compound in the preparation of anti-Alzheimer's disease drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A triterpenoid compound, as shown in general formula 1.

[0008]

[0009] In the formula,

[0010] R1 is H;

[0011] R2 is a hydroxyl group or an oxo group;

[0012] R3 is H or oxo;

[0013] R4 is H, hydroxyl, or oxo; or forms a C5-C8 heterocycle containing at least one heteroatom with R1 and adjacent atoms;

[0014] R5 is H or a hydroxyl group;

[0015] R6 represents the structure shown in A, B, C, or D;

[0016]

[0017] Preferably, the compound has the following structures 1-13:

[0018]

[0019] A method for preparing the aforementioned triterpenoid compound:

[0020] (1) Activation of strain: G. shandongense mycelium of Ganoderma lucidum was inoculated onto malt extract medium and cultured at 28℃ in the dark for 5-7 days until ready for use;

[0021] (2) Enrichment culture of strains: The activated strains were inoculated into enrichment medium and cultured at 28°C for 5-6 days.

[0022] (3) Fermentation: The enriched strain obtained in (2) was inoculated into rice solid fermentation medium and fermented using solid fermentation.

[0023] (4) The culture obtained by fermentation in (3) is purified to obtain the compound shown in Formula 1.

[0024] The purification step (4) involves extracting the obtained fermentation culture with ethyl acetate 3-5 times, combining the extracts and concentrating under reduced pressure to obtain an extract, which is then separated by silica gel column chromatography under reduced pressure. The extract is eluted with petroleum ether-ethyl acetate (50:1 to 1:1) and dichloromethane-methanol (50:1 to 1:1). The fractions are identified and combined by thin-layer chromatography and high-performance liquid chromatography to obtain four components: 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).

[0025] Fraction Fr.B was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate (50:1 to 1:1) and dichloromethane / methanol (50:1 to 1:1) systems in volume ratios. Fractions from petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain fraction Fr.B3, and fractions from dichloromethane / methanol 20:1 to 5:1 were collected to obtain fraction Fr.B4.

[0026] Fr.B3 was separated by ODS column chromatography with ethanol / water gradient elution. The fractions of 70%–85% were collected as Fr.B3-3, 85%–90% as Fr.B3-4, and 90%–95% as Fr.B3-5.

[0027] Fr.B3-3 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peak at 36-37 min was collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peak at 36 min was collected to obtain compound 1, and the chromatographic peak at 38 min was collected to obtain compound 6.

[0028] Fr.B3-4 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peaks at 20-35 min were collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peaks at 22 min were collected to obtain compound 5, compound 11 at 24 min, compound 3 at 26 min, and compound 8 at 32 min.

[0029] Fr.B3-5 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peaks at 25-55 min were collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peaks at 27 min were collected to obtain compound 7, and the chromatographic peaks at 52 min were collected to obtain compound 9.

[0030] Fr.B4 was separated by ODS column chromatography with ethanol / water gradient elution. The 40%–50% fraction was collected as Fr.B4-1 and the 50%–60% fraction was collected as Fr.B4-2.

[0031] Fr.B4-1 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peak at 15-30 min was collected. The resulting sample was then subjected to semi-preparative HPLC, eluted with acetonitrile / water. The chromatographic peak at 26 min yielded compound 4, the chromatographic peak at 31 min yielded compound 10, and the chromatographic peak at 40 min yielded compound 12.

[0032] Fr.B4-2 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peak at 35-40 min was collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peak at 31 min yielded compound 2, and the chromatographic peak at 35 min yielded compound 13.

[0033] 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%.

[0034] The flow rate for the preparative HPLC separation was 7 ml / min;

[0035] The flow rate for the semi-preparative HPLC separation was 2.5 ml / min;

[0036] The methanol / water concentration was 75%–80% during the methanol / water elution of compounds 1 and 6 by the preparative HPLC separation.

[0037] The methanol concentration for methanol / water elution of compounds 3, 5, 8 and 11 in the preparative HPLC separation was 75%–80%.

[0038] The methanol / water concentration was 70%–75% during the methanol / water elution of compounds 7 and 9 by the preparative HPLC separation.

[0039] The methanol / water concentration was 85%–90% during the methanol / water elution of compounds 4, 10, and 12 by the preparative HPLC separation.

[0040] The methanol / water concentration was 65%–70% during the methanol / water elution of compounds 2 and 13 by the preparative HPLC separation.

[0041] The semi-preparative HPLC separation of compounds 1 and 6 uses methanol / water elution and acetonitrile / water elution with an acetonitrile concentration of 60%–70%.

[0042] The acetonitrile concentration for eluting compounds 3, 5, 8 and 11 by the semi-preparative HPLC method was 55%–60% during acetonitrile / water elution.

[0043] The acetonitrile concentration was 45%–50% during acetonitrile / water elution of compounds 7 and 9 in the semi-preparative HPLC separation.

[0044] The acetonitrile concentration was 60%–65% during acetonitrile / water elution of compounds 4, 10, and 12 in the semi-preparative HPLC separation.

[0045] The acetonitrile concentration was 40%–45% during acetonitrile / water elution of compounds 2 and 13 in the semi-preparative HPLC separation.

[0046] The malt extract culture medium consists of: 10g malt extract, 500mL distilled water, and 7.5g agar; the culture conditions are: 25-28℃ for 5-6 days.

[0047] 7. The method for preparing triterpenoid compounds according to claim 3, characterized in that: 1 / 4 dish of Ganoderma lucidum mycelium (G. shandongense) is added to every 80-120g of rice solid fermentation medium, and then the mixture is allowed to stand for culture until the mycelium fully colonizes the substrate; wherein, the rice solid fermentation medium is prepared by adding 80-120g of rice to every 100-250mL of water.

[0048] The systematic structural identification results of compounds 1-11 are as follows, and the corresponding spectra are shown below. Figure 2 :

[0049] Ganoshandongone A(1): Yellow crystals; (c 0.10,CH3OH),HRESIMS m / z469.3368[M+H] + (calcd for C 30 H 45 O4,469.3318), the molecular formula is determined to be C 30 H 44 O4, through analysis of compound 1 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 1 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and Mosher assay, identifying it as a new compound.

[0050] Ganoshandongone B(2): white powder; (c 0.10,CH3OH),HRESIMS m / z521.2861[M+Na] + (calcd for C 30 H 42 O6Na, 521.2879), the molecular formula is determined to be C 30 H 42 O6, through analysis of compound 2 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 2 was determined by ¹H COSY, NOESY, and NMR calculations, identifying it as a new compound.

[0051] Ganoshandongone C(3): white powder; (c 0.50,CH3OH),HRESIMS m / z549.3190[M+Na] + (calcd for C 32 H 48 O5Na, 549.3192), the molecular formula is determined to be C 32 H 46 O6, through analysis of compound 3 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 3 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and Mosher assay, identifying it as a new compound.

[0052] Ganoshandongone D(4): white powder; (c 0.50,CH3OH),HRESIMS m / z471.3470[M+H] + (calcd for C 30 H 47 O4,471.3474), the molecular formula is determined to be C 30 H 46 O4, through analysis of compound 4 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 4 was determined by ¹H COSY, NOESY, and NMR calculations, identifying it as a new compound.

[0053] Ganoshandongone E(5): white powder, (c 0.50,CH3OH),HRESIMS m / z471.3470[M+H] + (calcd for C 30 H 47 O4,471.3474), the molecular formula is determined to be C 30 H 46 O4, through analysis of compound 5 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 5 was determined by ¹H COSY, NOESY, and NMR calculations, identifying it as a new compound.

[0054] Ganoshandongone F(6): white powder; (c 0.20,CH3OH),HRESIMS m / z453.3368[MH] - (calcd for C 30 H 45 O3, 453.3374), the molecular formula is determined to be C 30 H 46 O3, through analysis of compound 6 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum, NOESY spectrum 1 H- 1 The structure of compound 6 was determined by HCl COSY spectroscopy and Mosher method, which identified it as a new compound.

[0055] Ganoshandongone G(7): white powder; (c 0.50,CH3OH),HRESIMS m / z493.3291[M+Na] + (calcd for C 30 H 46 O4Na, 493.3294), the molecular formula is determined to be C 30 H 46 O4, through analysis of compound 7 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 7 was determined by HCl COSY, NOESY, and CD spectra, identifying it as a new compound.

[0056] Ganoshandongone H(8): white powder; (c 0.50,CH3OH),HRESIMS m / z535.3395[M+Na] + (calcd for C 32 H 48 O5Na, 535.3394), the molecular formula is determined to be C 32 H 48 O5, through analysis of compound 8 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 8 was determined by HCl COSY spectroscopy, NOESY spectroscopy, and Mosher assay, identifying it as a new compound.

[0057] Ganoshandongone I(9): white powder; (c 0.50,CH3OH),HRESIMS m / z493.3251[M+Na] + (calcd for C 30 H 46 O4Na, 493.3294), the molecular formula is determined to be C 30 H 46 O4, through analysis of compound 9 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum 1 H- 1 The structure of compound 9 was determined by ¹H COSY, NOESY, and NMR calculations, identifying it as a new compound.

[0058] Ganoshandongone J(10): white powder; (c 0.50,CH3OH),HRESIMS m / z471.3477[M+H] + (calcd for C 30 H 47 O4,471.3474), the molecular formula is determined to be C 30 H 46 O4, through analysis of compound 10 1 H NMR, 13 C NMR, HMQC spectrum, HMBC spectrum, NOESY spectrum 1 H- 1 The structure of compound 10 was determined by 1H COSY spectroscopy and NMR calculation, identifying it as a new compound.

[0059] Ganoshandongone K(11): white powder; (c 0.50,CH3OH),HRESIMS m / z467.3159[M+H] + (calcd for C 30 H 43 O4,467.3156), the molecular formula is determined to be C 30 H 42 O4, through analysis of compound 11 1 H NMR, 13 C NMR, HMQC spectrum 1 H- 1 The structure of compound 11 was determined by ¹H COSY, ¹HMBC, and ¹NOESY spectra, as well as by calculated NMR, and it is a new compound.

[0060] A pharmaceutical composition comprising the aforementioned triterpenoid compound, a pharmaceutically acceptable excipient and / or carrier.

[0061] An application of the compound, the triterpenoid compound, or the pharmaceutical composition described herein, in the preparation of an anti-Alzheimer's disease drug.

[0062] The obtained compounds 1-13 were evaluated for their acetylcholinesterase inhibitory activity, and the results showed that the compounds exhibited good inhibitory activity against acetylcholinesterase. Therefore, the triterpenoid compounds described in this invention have the potential to be developed into anti-Alzheimer's disease drugs. Attached Figure Description

[0063] Figure 1 Δδ of (S)- and (R)-MTPA esters of compounds 1, 6, and 8 provided in embodiments of the present inventionS-R value;

[0064] Figure 2 Calculated carbon spectrum data of compounds 2-5 and 9-11 provided in the embodiments of the present invention; Detailed Implementation

[0065] The embodiments listed below are intended to help those skilled in the art better understand the present invention, but do not limit the invention in any way.

[0066] Example 1: Preparation of triterpenoid compounds 1-13 from Ganoderma lucidum in Shandong

[0067] 1. Fermentation conditions

[0068] Strain activation: Ganoderma lucidum mycelium (G. shandongense) 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 0.5cm pieces. 2 Small pieces are to be used later; the components of the malt extract culture medium are: 10g malt extract, 500mL distilled water, 7.5g agar, pH natural;

[0069] Enrichment culture of the strain: Take 10g of malt extract powder, 500mL of distilled water, and 7.5g of agar, and autoclave at 121℃ for 30min. Inoculate the cut pieces of G. shandongense into the above culture medium and incubate at 28℃ for 5–6 days.

[0070] Fermentation: Place 100g of rice into a 500mL Erlenmeyer flask, add 100mL of water, and autoclave at 121℃ for 30min. Inoculate each flask with 1 / 4 dish of Ganoderma lucidum mycelium (G. shandongense) and incubate at 25-28℃ for 40-50 days.

[0071] 2. Extraction and separation

[0072] The fermented culture was extracted with ethyl acetate 3-5 times at room temperature. The extracts were combined and concentrated under reduced pressure at 40°C to obtain an extract. The obtained extract (350.0 g) was mixed with 100-200 mesh silica gel and subjected to reduced pressure silica gel column chromatography (25×15 cm) with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1). 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 30:1-1:1), Fr.C (dichloromethane-methanol 50:1-10:1), and Fr.D (dichloromethane-methanol 8:1-1:1).

[0073] The obtained fraction Fr.B was subjected to silica gel column chromatography (5×50cm) with gradient elution using petroleum ether-ethyl acetate (50:1 to 1:1) and dichloromethane / methanol (50:1 to 1:1) systems at volume ratios. Fractions from petroleum ether-ethyl acetate at 5:1 to dichloromethane / methanol at 30:1 were collected to obtain fraction Fr.B3 (9.32g). Fractions from dichloromethane / methanol at 20:1 to 5:1 were collected to obtain fraction Fr.B4 (5.26g).

[0074] The obtained Fr.B3 was further separated by ODS column chromatography with ethanol / water gradient elution, and 70%-85% of the fraction was collected as Fr.B3-3, 85%-90% as Fr.B3-4, and 90%-95% as Fr.B3-5.

[0075] Fr.B3-3 was subjected to preparative HPLC, eluted with 75%–80% methanol / water, and the chromatographic peak at 36–37 min was collected at a flow rate of 7 mL / min. The obtained sample was then subjected to semi-preparative HPLC, eluted with 60%–70% acetonitrile / water, and the chromatographic peak at 36 min was collected to give compound 1 (28.9 mg, t). R =36 min), the chromatographic peak at 38 min gave compound 6 (188.5 mg, t R =38min);

[0076] Fr.B3-4 was then subjected to preparative HPLC, eluted with 75%–80% methanol / water, and the chromatographic peaks at 20–35 min were collected. The resulting sample was subjected to semi-preparative HPLC, eluted with 55%–60% acetonitrile / water, and the chromatographic peaks at 22 min were collected to obtain compound 5 (3.7 mg), compound 11 (15 mg) was collected at 24 min, compound 3 (1.4 mg) was collected at 26 min, and compound 8 (3 mg) was collected at 32 min.

[0077] Fr.B3-5 was subjected to preparative HPLC, eluted with 70%–75% methanol / water, and the chromatographic peaks at 25–55 min were collected. The resulting sample was subjected to semi-preparative HPLC, eluted with 45%–50% acetonitrile / water, and the chromatographic peaks at 27 min were collected to obtain compound 9 (1.1 mg), and the chromatographic peaks at 52 min were collected to obtain compound 7 (2.2 mg).

[0078] Fr.B4 was separated by ODS column chromatography, eluted with an ethanol / water gradient (35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), and the 40%–50% fraction was collected as Fr.B4-1, and the 50%–60% fraction was collected as Fr.B4-2.

[0079] Fr.B4-1 was prepared by HPLC, eluted with 85%–90% methanol / water, and the chromatographic peaks at 15–30 min were collected. The resulting sample was then subjected to semi-preparative HPLC, eluted with 60%–65% acetonitrile / water. The chromatographic peak at 26 min yielded compound 4 (7 mg), the chromatographic peak at 31 min yielded compound 10 (4.1 mg), and the chromatographic peak at 40 min yielded compound 12 (8.2 mg).

[0080] Fr.B4-2 was prepared by HPLC, eluted with 65%–70% methanol / water, and the chromatographic peak at 35–40 min was collected. The obtained sample was then subjected to semi-preparative HPLC, eluted with 40%–45% acetonitrile / water, and the chromatographic peak at 31 min yielded compound 2 (5.3 mg), and the chromatographic peak at 35 min yielded compound 13 (10.3 mg).

[0081] Table 1. Compounds 1-6 1 1H NMR (600MHz, CDCl3) data (δin ppm, multi, J in Hz)

[0082]

[0083] Compound 7-11 in Table 2 1 1H NMR (600MHz, CDCl3) data (δin ppm, multi, J in Hz)

[0084]

[0085]

[0086] Table 3 Compounds 1-11 13 C NMR (150MHz, CDCl3) data (δin ppm)

[0087]

[0088]

[0089] Example 2: Acetylcholinesterase inhibitory activity of compounds 1-13

[0090] The compounds obtained above were diluted with PBS to concentrations of 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, and 5 μmol / L, respectively, for later use. 20 μL of each compound, 60 μL of PBS, and 20 μL of acetylcholinesterase (0.22 U / mL) were added to each well of a 96-well plate. After reacting at 4°C for 10 min, 20 μL of substrate AchI (15 mM) and DNTB were added. 100 μL was added, and the reaction was repeated three times in parallel. The absorbance (A1) was measured at 412 nm after 20 min of further reaction. An equal volume of PBS was used to replace acetylcholinesterase, and the reaction was repeated three times in parallel, with the absorbance measured at 412 nm (A2). An equal volume of PBS was used to replace the compound, and the absorbance was measured at 412 nm (A3). An equal volume of PBS was used to replace both the compound and acetylcholinesterase, and the absorbance was measured at 412 nm (A4). Donepezil hydrochloride diluted to concentrations of 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, and 5 μmol / L served as a positive control. Triple replicates were performed, and the average value was used to calculate the acetylcholinesterase inhibition rate and IC50. 50 The value is calculated using the formula: inhibition rate = [(A3-A4)-(A1-A2)] / (A3-A4)×100%.

[0091] Table 4 shows the inhibitory effects of compounds 1-12 on acetylcholinesterase.

[0092]

[0093] Experiments have confirmed that compounds 1-13 all possess acetylcholinesterase inhibitory activity, with compounds 1, 5-7, and 12 showing the highest IC50 values. 50 Its value is superior to that of donepezil hydrochloride, a positive control drug. Its extraction and separation method is simple, which facilitates further pharmacological and clinical research and development of its application in the preparation of anti-Alzheimer's drugs.

Claims

1. A triterpenoid compound, characterized in that: The compound is shown in general formula 1. In the formula, R1 is H; R2 is a hydroxyl group or an oxo group; R3 is H or oxidized; R4 is H, hydroxyl, or oxo; or forms a C5-C8 heterocycle containing at least one heteroatom with R1 and adjacent atoms; R5 is H or a hydroxyl group; R6 represents the structure shown in A, B, C, or D; 2. The triterpenoid compound according to claim 1, characterized in that: The compound has the following structures 1-13:

3. A method for preparing the triterpenoid compound according to claim 1, characterized in that: (1) Activation of strain: G. shandongense mycelium of Ganoderma lucidum was inoculated onto malt extract medium and cultured at 28℃ in the dark for 5-7 days until ready for use; (2) Enrichment culture of strains: The activated strains were inoculated into enrichment medium and cultured at 28°C for 5-6 days. (3) Fermentation: The enriched strain obtained in (2) was inoculated into rice solid fermentation medium and fermented using solid fermentation. (4) The culture obtained by fermentation in (3) is purified to obtain the compound shown in Formula 1.

4. The method for preparing triterpenoid compounds according to claim 3, characterized in that: The purification step (4) involves extracting the obtained fermentation culture with ethyl acetate 3-5 times, combining the extracts and concentrating under reduced pressure to obtain an extract, which is then separated by silica gel column chromatography under reduced pressure. The extract is eluted with petroleum ether-ethyl acetate (50:1 to 1:1) and dichloromethane-methanol (50:1 to 1:1). The fractions are identified and combined by thin-layer chromatography and high-performance liquid chromatography to obtain four components: 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 and gradient elution with petroleum ether-ethyl acetate (50:1 to 1:1) and dichloromethane / methanol (50:1 to 1:1) systems in volume ratios. Fractions from petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain fraction Fr.B3, and fractions from dichloromethane / methanol 20:1 to 5:1 were collected to obtain fraction Fr.B4. Fr.B3 was separated by ODS column chromatography with ethanol / water gradient elution. The fractions of 70%–85% were collected as Fr.B3-3, 85%–90% as Fr.B3-4, and 90%–95% as Fr.B3-5. Fr.B3-3 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peak at 36-37 min was collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peak at 36 min was collected to obtain compound 1, and the chromatographic peak at 38 min was collected to obtain compound 6. Fr.B3-4 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peaks at 20-35 min were collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peaks at 22 min were collected to obtain compound 5, compound 11 at 24 min, compound 3 at 26 min, and compound 8 at 32 min. Fr.B3-5 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peaks at 25-55 min were collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peaks at 27 min were collected to obtain compound 7, and the chromatographic peaks at 52 min were collected to obtain compound 9. Fr.B4 was separated by ODS column chromatography with ethanol / water gradient elution. The 40%–50% fraction was collected as Fr.B4-1 and the 50%–60% fraction was collected as Fr.B4-2. Fr.B4-1 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peak at 15-30 min was collected. The resulting sample was then subjected to semi-preparative HPLC, eluted with acetonitrile / water. The chromatographic peak at 26 min yielded compound 4, the chromatographic peak at 31 min yielded compound 10, and the chromatographic peak at 40 min yielded compound 12. Fr.B4-2 was subjected to preparative HPLC, eluted with methanol / water, and the chromatographic peak at 35-40 min was collected. The resulting sample was subjected to semi-preparative HPLC, eluted with acetonitrile / water, and the chromatographic peak at 31 min yielded compound 2, and the chromatographic peak at 35 min yielded compound 13.

5. The method for preparing the triterpenoid compound according to claim 4, 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 / water concentration was 75%–80% during the methanol / water elution of compounds 1 and 6 by the preparative HPLC separation. The methanol concentration for methanol / water elution of compounds 3, 5, 8 and 11 in the preparative HPLC separation was 75%–80%. The methanol / water concentration was 70%–75% during the methanol / water elution of compounds 7 and 9 by the preparative HPLC separation. The methanol / water concentration was 85%–90% during the methanol / water elution of compounds 4, 10, and 12 by the preparative HPLC separation. The methanol / water concentration was 65%–70% during the methanol / water elution of compounds 2 and 13 by the preparative HPLC separation. The semi-preparative HPLC separation of compounds 1 and 6 uses methanol / water elution and acetonitrile / water elution with an acetonitrile concentration of 60%–70%. The acetonitrile concentration for eluting compounds 3, 5, 8 and 11 by the semi-preparative HPLC method was 55%–60% during acetonitrile / water elution. The acetonitrile concentration was 45%–50% during acetonitrile / water elution of compounds 7 and 9 in the semi-preparative HPLC separation. The acetonitrile concentration was 60%–65% during acetonitrile / water elution of compounds 4, 10, and 12 in the semi-preparative HPLC separation. The acetonitrile concentration was 40%–45% during acetonitrile / water elution of compounds 2 and 13 in the semi-preparative HPLC separation.

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

7. The method for preparing the triterpenoid compound according to claim 3, characterized in that: Inoculate with 1 / 4 dish of Ganoderma lucidum mycelium (G. shandongense) per 80-120g of rice solid fermentation medium, and then let it stand in the culture until the mycelium has covered the substrate; wherein, the rice solid fermentation medium is 80-120g of rice per 100-250mL of water.

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

9. An application of the compound according to claim 1, characterized in that: The use of the triterpenoid compound of claim 1 or the pharmaceutical composition of claim 8 in the preparation of an anti-Alzheimer's disease drug.

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