Triterpenoids as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a triterpenoid extracted from edible and medicinal fungus ganoderma lucidum, a preparation method of the triterpenoid and application of the triterpenoid in the aspect of resisting Alzheimer's disease. The triterpenoid compound is shown as a structural formula I. The triterpenoid compound provided by the invention has remarkable acetylcholin esterase inhibitory activity, is an ideal candidate compound of an acetylcholin esterase inhibitor which is novel in structure and efficient in activity, and has a good application prospect in the aspect of treatment of Alzheimer's disease. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a triterpenoid compound extracted from the edible and medicinal fungus Shandong Ganoderma lucidum, a preparation method thereof, and an application thereof in combating Alzheimer's disease. Background Art

[0002] Lingzhi is a general term for fungi of the genus Ganoderma (Ganoderma spp.), belonging to the subphylum Basidiomycetes, the order Polyporales, the family Ganodermataceae, and the genus Ganoderma. It has important economic value and is widely used in my country and some other Asian countries. It is believed to have the effects of health care and longevity. Lingzhi has a history of thousands of years of research in my country. It is a traditional Chinese medicinal material in my country and has a very wide range of medicinal research. Among them, Shandong Lingzhi (G. shandongense) is a new species discovered and identified by Zhao Jiding et al. in 1982. There are currently no reports on its chemical composition and biological activity. Lingzhi triterpenes are one of the active ingredients isolated from Lingzhi. They have a complex chemical structure, bitter taste, high fat solubility, and a wide range of medicinal value. In recent years, more and more studies have been conducted on the neuroprotective activity of Lingzhi. As the main active ingredient, triterpenoid compounds are one of the hot topics in the current research on the pharmacological activity of Lingzhi.

[0003] Alzheimer's disease is an irreversible chronic neurodegenerative disease that affects 4%-8% of the elderly worldwide. Its main characteristics are progressive memory deficits, impaired daily activities, and decreased acquired learning ability. At present, the most common treatment for AD is to inhibit the activity of acetylcholinesterase (AChE) in the brain to improve the patient's cognitive function. AChE is essential for nerve conduction and mainly degrades acetylcholine, thereby terminating the transmission of nerve impulses. So far, the U.S. Food and Drug Administration (FDA) has approved a total of 5 drugs (4 of which are AChE inhibitors) for the treatment of AD, but most of these drugs have shortcomings such as short half-life or severe peripheral cholinergic system damage, and long-term use will seriously affect human health. Therefore, screening highly active, specific, safe and non-toxic natural source AChE inhibitors is currently the focus of anti-AD drug development. Therefore, it is of great significance to find and develop new acetylcholinesterase inhibitors from natural products. Summary of the invention

[0004] The primary purpose of the present invention is to provide a triterpenoid compound extracted from the edible and medicinal fungus Shandong Ganoderma lucidum and a preparation method thereof, as well as the application of such compound in anti-Alzheimer's disease.

[0005] To achieve the above purpose, the present invention adopts the technical solution as follows:

[0006] A triterpenoid compound, characterized in that: the triterpenoid compound is as shown in structural formula 1,

[0007]

[0008] In the formula,

[0009] R1 is the structure represented by A, B, C, D or E;

[0010]

[0011] R2 is H or hydroxyl.

[0012] Preferably, the triterpenoid compound is compound 1-5 whose structure is shown below:

[0013]

[0014] A method for preparing the triterpenoid compounds is as follows:

[0015] (1) Preparation of seed culture medium: Ganoderma shandongense mycelium was inoculated into malt culture medium and cultured at a constant temperature of 28° C. for 5 to 7 days;

[0016] (2) fermentation: inoculating the mycelium in the seed culture medium into a rice solid fermentation medium, and performing a fermentation culture for 40 days by solid-state fermentation;

[0017] (3) Extraction: The culture obtained by fermentation is extracted with ethyl acetate for 3-5 times, and the combined extracts are concentrated under reduced pressure to obtain a crude extract;

[0018] (4) Chromatographic separation: The crude extract is separated by a reduced pressure silica gel column to obtain the compound represented by formula 1.

[0019] The crude extract of step (4) is separated by a reduced pressure silica gel column and gradient eluted with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1) systems in order by volume ratio to obtain four crude fractions: Fr.A (petroleum ether-ethyl acetate 50:1-40:1), 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);

[0020] The obtained component Fr.B was subjected to silica gel column chromatography with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane / methanol (50:1-1:1) systems in a volume ratio for gradient elution, and the components with petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain component Fr.B3. The Fr.B3 component was subjected to ODS column chromatography with an ethanol aqueous solution with a concentration of 35%-100% for gradient elution, and the 35%, 45%, 55%, 65%, 75%, and 95% components were collected as Fr.B3-1, Fr.B3-2, Fr.B3-3, Fr.B3-4, Fr.B3-5, and Fr.B3-6, respectively. Fr.B3-2 was subjected to silica gel column chromatography with dichloromethane-methanol in a volume ratio of 1:1 to collect component Fr.B3-2-1. The obtained components were further subjected to OD The S column chromatography was gradient eluted with 55% to 100% ethanol-water solution, and 55% to 75% of the components Fr.B3-2-1-1 and 75% to 100% of the components Fr.B3-2-1-2 were collected respectively; the collected Fr.B3-2-1-1 components were separated by gel column chromatography and then subjected to preparative HPLC, eluted with methanol-water (75:25), and chromatographic peaks a and b at 25.2 to 27.5 min and 28.2 to 31.5 min were collected respectively; the obtained sample a was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water (83:17), and the chromatographic peak at 24 min was collected to obtain compound 1; the obtained sample b was subjected to semi-preparative HPLC chromatography (250×10 mm, 5 μm), eluted with acetonitrile-water (83:17), and the chromatographic peak at 28 min was collected to obtain compound 2.

[0021] The Fr.B3-5 component was subjected to gel column chromatography to obtain the Fr.B3-5-1 component, which was subjected to preparative HPLC (250×20mm, 5μm) and eluted with methanol / water in a volume ratio of 71:29 at a flow rate of 7mL / min, and the chromatographic peaks c and d at 28.2-31.5min and 77.5-81.5min were collected respectively. The obtained sample c was subjected to semi-preparative HPLC chromatography (250×10mm, 5μm) and eluted with acetonitrile / water (50:50) at a flow rate of 2.5mL / min, and the chromatographic peak at 51min was collected to obtain compound 3. The obtained sample d was subjected to semi-preparative HPLC chromatography (250×10mm, 5μm) and eluted with acetonitrile / water (50:50) at a flow rate of 2.5mL / min, and the chromatographic peak at 61min was collected to obtain compound 4.

[0022] Component Fr.C was subjected to HP20 macroporous resin and gradient eluted with 10% to 100% ethanol aqueous solution, and 10% to 30% component Fr.C-1 was collected; the obtained components were separated by ODS column chromatography and gradient eluted with 45% to 100% ethanol aqueous solution, and 75% to 100% component Fr.C-1-3 were collected; then silica gel column chromatography was performed with a petroleum ether-ethyl acetate volume ratio of 100:1 to 1:1 for gradient elution, and 20:1 to 1:1 components Fr.C-1-3-5 were collected; preparative HPLC was performed with methanol-water (v / v=67:33) for elution, and the chromatographic peaks of 25.6 to 27.5 min were collected; the obtained sample was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water (58:42), and the chromatographic peak of 32 min was collected to obtain compound 5.

[0023] The components of the malt extract powder culture medium are: 20 g / L malt extract powder, 12 g / L agar, 1 L distilled water, and natural pH; the components of the rice culture medium are: 35 g / bottle rice, 45 mL / bottle distilled water, and natural pH.

[0024] A pharmaceutical composition comprising the triterpenoid compound represented by formula 1.

[0025] An application of the triterpenoid compound or the composition, and an application of the triterpenoid compound represented by formula 1 or the pharmaceutical composition in the preparation of an anti-Alzheimer's disease drug.

[0026] Compounds 1-5 provided by the present invention are all discovered for the first time, and there are no relevant reports on the biological activities of these four compounds;

[0027] The obtained compounds 1-5 were evaluated for their acetylcholinesterase inhibitory activity. The results showed that compounds 2, 3, 4, and 5 all had good acetylcholinesterase inhibitory activity (IC 50 The values ​​were 36.66, 37.02, 43.76, and 53.32 μM, respectively). Compound 1 did not show obvious acetylcholinesterase inhibitory activity. Therefore, the triterpenoid compounds 2-5 described in the present invention have the potential to be developed as anti-Alzheimer's disease drugs, providing a new material basis for the development of innovative drugs for the treatment of Alzheimer's disease and the development of innovative drugs for acetylcholinesterase inhibitors, and providing a scientific basis for the development and utilization of natural active substances from large fungi. In addition, the above compounds are separated from my country's famous large fungi for both food and medicine, and their safety is preliminarily guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The HRESIMS spectrum of compound 1 provided in the embodiment of the present invention;

[0029] Figure 2Compound 1 provided in the embodiment of the present invention 1 H NMR spectrum (600 MHz, CDCl3);

[0030] Figure 3 Compound 1 provided in the embodiment of the present invention 13 C NMR spectrum (150 MHz, CDCl3);

[0031] Figure 4 HSQC spectrum (600 MHz, CDCl3) of compound 1 provided in the embodiment of the present invention;

[0032] Figure 5 HMBC spectrum (600 MHz, CDCl3) of compound 1 provided in the embodiment of the present invention;

[0033] Figure 6 Compound 1 provided in the embodiment of the present invention 1 H- 1 H COSY spectrum (600 MHz, CDCl3);

[0034] Figure 7 NOESY spectrum (600 MHz, CDC) of compound 1 provided in the embodiment of the present invention l3 );

[0035] Figure 8 R of compound 1 provided in the embodiment of the present invention 2 and DP4 + Computational analysis;

[0036] Fig. 9 The HRESIMS spectrum of compound 2 provided in the embodiment of the present invention;

[0037] Fig.10 Compound 2 provided in the embodiment of the present invention 1 H NMR spectrum (600 MHz, CDCl3);

[0038] Fig.11 Compound 2 provided in the embodiment of the present invention 13 C NMR spectrum (150 MHz, CDCl3);

[0039] Fig.12 HSQC spectrum (600 MHz, CDCl3) of compound 2 provided in the embodiment of the present invention;

[0040] Fig.13 HMBC spectrum (600 MHz, CDCl3) of compound 2 provided in the embodiment of the present invention;

[0041] Fig.14Compound 2 provided in the embodiment of the present invention 1 H- 1 H COSY spectrum (600 MHz, CDCl3);

[0042] Fig.15 NOESY spectrum (600 MHz, CDC) of compound 2 provided in the embodiment of the present invention l3 );

[0043] Fig.16 R of compound 2 provided in the embodiment of the present invention 2 and DP4 + Computational analysis;

[0044] Fig.17 The HRESIMS spectrum of compound 3 provided in the embodiment of the present invention;

[0045] Fig.18 Compound 3 provided in the embodiment of the present invention 1 H NMR spectrum (600 MHz, CDCl3);

[0046] Fig.19 Compound 3 provided in the embodiment of the present invention 13 C NMR spectrum (150 MHz, CDCl3);

[0047] Fig. 20 HSQC spectrum (600 MHz, CDCl3) of compound 3 provided in the embodiment of the present invention;

[0048] Fig.21 HMBC spectrum (600 MHz, CDCl3) of compound 3 provided in the embodiment of the present invention;

[0049] Fig. 22 Compound 3 provided in the embodiment of the present invention 1 H- 1 H COSY spectrum (600 MHz, CDCl3);

[0050] Fig.23 NOESY spectrum (600 MHz, CDC) of compound 3 provided in the embodiment of the present invention l3 );

[0051] Fig.24 R of compound 3 provided in the embodiment of the present invention 2 and DP4 + Computational analysis;

[0052] Fig.25 The HRESIMS spectrum of compound 4 provided in the embodiment of the present invention;

[0053] Fig.26 Compound 4 provided in the embodiment of the present invention 1 H NMR spectrum (600 MHz, CDCl3);

[0054] Fig. 27 Compound 4 provided in the embodiment of the present invention 13 C NMR spectrum (150 MHz, CDCl3);

[0055] Fig.28 HSQC spectrum (600 MHz, CDCl3) of compound 4 provided in the embodiment of the present invention;

[0056] Fig.29 HMBC spectrum (600 MHz, CDCl3) of compound 4 provided in the embodiment of the present invention;

[0057] Fig.30 Compound 4 provided in the embodiment of the present invention 1 H- 1 H COSY spectrum (600 MHz, CDCl3);

[0058] Fig.31 NOESY spectrum (600 MHz, CDC) of compound 4 provided in the embodiment of the present invention l3 );

[0059] Fig.32 R of compound 4 provided in the embodiment of the present invention 2 and DP4 + Computational analysis;

[0060] Fig.33 The HRESIMS spectrum of compound 5 provided in the embodiment of the present invention;

[0061] Fig.34 Compound 5 provided in the embodiment of the present invention 1 H NMR spectrum (600 MHz, CDCl3);

[0062] Fig.35 Compound 5 provided in the embodiment of the present invention 13 C NMR spectrum (150 MHz, CDCl3);

[0063] Fig.36 HSQC spectrum (600 MHz, CDCl3) of compound 5 provided in the embodiment of the present invention;

[0064] Fig.37 HMBC spectrum (600 MHz, CDCl3) of compound 5 provided in the examples of the present invention;

[0065] Fig.38Compound 5 provided in the embodiment of the present invention 1 H- 1 H COSY spectrum (600 MHz, CDCl3);

[0066] Fig.39 NOESY spectrum (600 MHz, CDC) of compound 5 provided in the embodiment of the present invention l3 );

[0067] Fig.40 R of compound 5 provided in the embodiment of the present invention 2 and DP4 + Computational analysis;

[0068] Fig.41 HMBC of compound 1-5 provided in the embodiments of the present invention, 1 H- 1 H COSY and NOESY are related. DETAILED DESCRIPTION

[0069] The following examples are intended to help those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

[0070] In the following examples, G. shandongense is a well-known strain recorded in Acta Mycologica Sinica 1986(02):86-92. The strain used in the present invention was donated by Professor Dai Yucheng of Beijing Forestry University.

[0071] Example 1: Preparation of triterpenoid compounds 1-5 from Shandong Ganoderma lucidum

[0072] 1. Fermentation conditions

[0073] Strain activation: Inoculate the mycelium of Shandong Ganoderma lucidum G.shandongense on the malt extract powder medium and culture it at 28℃ in dark conditions for 5-7 days. Then cut the mycelium on the plate into 0.5cm 2 The components of the malt extract medium are: 10g malt extract, 500mL distilled water, 7.5g agar, natural pH;

[0074] Expansion culture of the strain: Take 10g of malt extract powder, 500mL of distilled water, and 7.5g of agar, sterilize at 121℃ for 30min, and set aside. Inoculate the cut G.shandongense into the above culture medium and culture at 28℃ for 5-6 days.

[0075] Fermentation: Put 100g rice into a 500mL conical flask, add 100mL water, sterilize at 121℃ for 30min, and set aside. Inoculate 1 / 4 dish of Shandong Ganoderma mycelium G.shandongense into each flask, and culture at 28℃ for 45 days.

[0076] 2. Extraction and separation

[0077] The culture obtained by fermentation is extracted by soaking with ethyl acetate at room temperature for 3-5 times, the extracts are combined, and the extracts are concentrated under reduced pressure at 40°C to obtain an extract. The obtained extract (350g) is mixed with 100-200 mesh silica gel, separated by vacuum column silica gel (25×15cm), and gradient eluted in turn by petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1) systems according to volume ratio. Each fraction is identified and combined by thin layer chromatography and high performance liquid chromatography to obtain four components Fr.A (petroleum ether-ethyl acetate 50:1-40:1), 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).

[0078] Component Fr.B (30.25 g) was eluted by silica gel column chromatography (5×50 cm) with petroleum ether-ethyl acetate (50:1~1:1) and dichloromethane / methanol (50:1~1:1) systems in sequence by volume ratio, and the components from petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain component Fr.B3 (9.2 g). The Fr.B3 component was eluted by ODS column chromatography with a gradient elution solution of 35% to 100% ethanol aqueous solution, and the 35%, 45%, 55%, 65%, 75%, and 95% components were collected as Fr.B3-1, Fr.B3-2, Fr.B3-3, Fr.B3-4, Fr.B3-5, and Fr.B3-6, respectively.

[0079] Fr.B3-2 (1.5 g) was subjected to silica gel column chromatography with a gradient elution of dichloromethane-methanol in a volume ratio of 1:1, and the 20:1-10:1 component Fr.B3-2-1 (914.2 mg) was collected. It was then subjected to ODS column chromatography with a gradient elution of 55%-100% ethanol aqueous solution, and the 55%-75% component Fr.B3-2-1-1 (473.4 mg) and the 75%-100% component Fr.B3-2-1-2 (365.3 mg) were collected respectively.

[0080] The Fr.B3-2-1-1 obtained above was separated by gel column chromatography and then subjected to preparative HPLC, eluted with methanol-water in a volume ratio of 75:25 at a flow rate of 7 mL / min, and the chromatographic peaks a and b at 25.2-27.5 min and 28.2-31.5 min were collected respectively. The obtained sample a was subjected to semi-preparative HPLC chromatography (250×10 mm, 5 μm), eluted with acetonitrile-water in a volume ratio of 83:17 at a flow rate of 2.5 mL / min, and the chromatographic peak at 24 min was collected to obtain compound 1 (6.9 mg). The obtained sample b was subjected to semi-preparative HPLC chromatography (250×10 mm, 5 μm), eluted with acetonitrile-water in a volume ratio of 83:17 at a flow rate of 2.5 mL / min, and the chromatographic peak at 28 min was collected to obtain compound 2 (1.1 mg).

[0081] The Fr.B3-5 component was subjected to gel column chromatography to obtain the Fr.B3-5-1 component, which was subjected to preparative HPLC (250×20mm, 5μm) and eluted with methanol / water (volume ratio of 71:29) at a flow rate of 7mL / min, and the chromatographic peaks c and d at 28.2-31.5min and 77.5-81.5min were collected respectively. The obtained sample c was subjected to semi-preparative HPLC chromatography (250×10mm, 5μm) and eluted with acetonitrile / water (50:50) at a flow rate of 2.5mL / min, and the chromatographic peak at 51min was collected to obtain compound 3 (8.6mg). The obtained sample d was subjected to semi-preparative HPLC chromatography (250×10mm, 5μm) and eluted with acetonitrile / water (50:50) at a flow rate of 2.5mL / min, and the chromatographic peak at 61min was collected to obtain compound 4 (1.1mg).

[0082] Component Fr.C (40 g) was subjected to HP20 macroporous resin and gradient eluted with 10% to 100% ethanol aqueous solution, and 10% to 30% component Fr.C-1 (8.1 g) was collected; the obtained components were separated by ODS column chromatography and gradient eluted with 45% to 100% ethanol aqueous solution, and 75% to 100% component Fr.C-1-3 (1.61 g) was collected; then silica gel column chromatography was performed with a petroleum ether-ethyl acetate volume ratio of 100:1 to 1:1 for gradient elution, and 20:1 to 1:1 component Fr.C-1-3-5 (329.6 mg) was collected, and preparative HPLC was performed with methanol-water (67:33) to collect the chromatographic peaks of 25.6 to 27.5 min. The obtained sample was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water (58:42), and the chromatographic peak of 32 min was collected to obtain compound 5.

[0083] The hydrogen spectrum of compound 1 includes 7 angular methyl signals and a double-peak methyl signal in the high-field region. Combined with the carbon spectrum, 31 carbon signals are given, so it is speculated that compound 1 is a triterpenoid compound. 1In the H-NMR spectrum, there are two oxygen proton signals δ H 3.97 (m, 1H) and δ H 3.46(s,3H),δ H 3.46 (s, 3H) indicates a methoxy signal; δ H 5.65 (d, J = 5.5 Hz, 1H), is an olefin proton signal. Combined with the carbon spectrum, δ C 162.9, 139.6, 123.9, 132.4, the compound has two double bonds; in the 13C-NMR spectrum, combined with the HSQC spectrum, it can be seen that δ C 214.8, 198.2 are the keto carbonyl signals at C-3 and C-7 of triterpenoids; δ C 99.6, δ H 4.63 (s, 1H), which is speculated to be an acetal or hemiacetal carbon signal. After comparing with the NMR data of the known compound astraeusin I, it was found that the NMR data of the two were similar, but astraeusin I lacked a keto carbonyl carbon. At the same time, there was a six-membered oxygen ring on the side chain of the compound, so it was speculated that compound 1 also had a six-membered oxygen ring. 1 H- 1 H COSY showed four spin coupling systems, namely H-1 / H-2, H-5 / H-6, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-22(H-21) / H-23 / H-24. The final structure of compound 1 was determined by HMBC spectrum. H 0.98 (d, J = 6.8 Hz, 1H), δ H 4.63 (s, 1H) and δ H 5.65 (d, J = 5.5 Hz, 1H) and δ C 68.2 is related, δ H 3.46 (s, 3H), 1.69 (s, 3H) and δ C 99.6 is related, δ H 1.69 (brs, 3H) and δ C 132.4(C-25), and combined with the unsaturation of the compound, these all prove that compound 1 contains a six-membered oxygen ring, and thus the planar structure of compound 1 is determined, as shown in the figure below.

[0084] The stereo configuration of compound 1 was determined by NOESY spectrum ( Figure 7) was determined, the NOESY correlation of H-24 and Me-27 determined that the configuration of the double bond on the six-membered oxygen ring was Z; the correlations of Me-18 / Me-19 / Me-28 and Me-18 / H-20 showed that Me-18, Me-19, Me-28, and H-20 were all β-type; the correlations of Me-30 / H-17 / Me-21, H-17 / H-22, and Me-29 / H-5 suggested that the configurations of Me-30, H-17, Me-21, H-22, Me-29, and H-5 were α-type; H-22 / H-17 had a NOESY correlation, indicating that compound 1 had two configurations. NMR calculations were performed based on the configuration of C-26, and the correlation coefficient R of the carbon spectrum data was fitted. 2 And probability analysis results DP4 + ( Figure 8 ) were analyzed and it was found that the calculated results of compound 1 and B were the most consistent, R 2 The value is 0.9983, DP4 + (all date) was 100%, thus determining the configuration of the compound at position C-26. According to Scifinder search, compound 1 was a new compound named (22S,26R,24Z)-8,24-diene-22,26-epoxytirucall-3,7-dione.

[0085] The obtained compound 2 was systematically identified and the results are as follows. The corresponding spectrum is attached. Figure 9-16 :

[0086] Compound 2: white powder (methanol), HRESIMS gave a quasi-molecular ion peak m / z 505.3263 [M+Na] + (calcd for C 31 H 46 O4Na, 505.3294), and the molecular formula was determined to be C by combining the hydrogen and carbon spectrum data. 31 H 46 O4, the calculated degree of unsaturation is 9.

[0087] The hydrogen spectrum of compound 2 ( Fig.10 ) shows the typical characteristics of triterpenoids: there are 6 single-peak methyl proton signals in the high field region, δ H 1.54 (s, 3H), 1.34 (s, 3H), 1.12 (s, 3H), 1.10 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.70 (s, 3H), a double peak methyl proton signal, 1.27 (d, J = 7.1 Hz, 3H); δ H4.74 (s, 1H), 4.02 (dd, J = 11.5, 3.4 Hz, 1H), presumably two oxygen-linked methine proton signals; δ H 5.67 (d, J = 5.6 Hz, 1H) is an olefin proton signal; 13 C NMR showed 31 carbon signals. Combined with the HSQC spectrum, it can be seen that compound 2 has two double bonds, δ C 162.9, 139.6 and δ C 132.4, 123.8; δ C 98.2, which is speculated to be an acetal or hemiacetal carbon signal, and the corresponding hydrogen spectrum signal is δ H 4.74(s,1H);δ C 214.9, 198.3 are the keto carbonyl carbon signals at C-3 and C-7 of lanostane triterpenes; δ C 68.2, δ H 4.02 (dd, J = 11.5, 3.4 Hz, 1H) is an oxygen-linked methine; δ C 63.6, δ H 3.87 (m, 1H), δ H 3.54 (m, 1H) is speculated to be an oxygen-linked methylene group. Comparing the NMR data of compound 2 with those of the known compound (5α, 23E)-27-nor-3β-hydroxylanosta-8,23-dien-7,25-dione, it can be seen that compound 2 has two more carbon signals in the oxygen-linked carbon signal area than the known compound, and also lacks an ester carbonyl signal. 1 H- 1 H COSY shows that there are four spin-coupled systems ( Fig.14 ), respectively H-1 / H-2, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-22(H-21), H-25 / H-26 / H-28; the planar structure of compound 2 was finally determined by HMBC spectrum. Fig.13 ) in which Me-21 and δ C 63.6 is related, δ H 4.74(s,1H) and δ C 63.6, thus proving that C-23 is an acetal or hemiacetal carbon; H-27 is related to δ C 132.4, 123.8 are correlated, indicating that the double bond exists in Δ24, 25; Me-28, H-23 and δ C 68.2 is correlated, indicating that there is a five-membered oxygen ring on the side chain of compound 2. Therefore, the planar structure of compound 2 is determined, as shown in the figure below.

[0088] The stereo configuration of the compound was determined by NOESY spectrum ( Fig.15 ) confirmed, Me-29 / Me-19 / Me-18 / H-20 have NOESY correlation, proving that the methyl group at position 10, the methyl group at position 13, the side chain at position 17 and Me-29 are all β-type; Me-30 / H-5 have correlation, Me-31 / H-17 / Me-21 proves that the configuration of Me-30, H-17 and Me-21 is α; Since the spatial distance between H-23 and H-26 on the side chain and the parent nucleus is relatively far, the necessary correlation signals are missing, and no correlation signals between the two are observed in the NOESY spectrum, indicating that compound 2 has 4 possible configurations. NMR calculations were performed based on the configurations of C-23 and C-26. The correlation coefficient R of the carbon spectrum data fitting was 2 And probability analysis results DP4 + ( Fig.16 ) were analyzed and it was found that compound 2 had the highest agreement with the calculated results of A. 2 The value is 0.9957, DP4 + (all date) was 92.69%, thus determining the configuration of the compound at C-23 and C-26. After searching on Scifinder, compound 2 was identified as a new compound named (23S,26R)-8,24-diene-23,26-epoxytirucall-3,7-dione.

[0089] The obtained compound 3 was systematically identified and the results are as follows. The corresponding spectrum is attached. Figure 17-24 :

[0090] Compound 3: white powder (methanol), HRESIMS gave a quasi-molecular ion peak m / z 493.3286 [M+Na] + (calcd for C 30 H 46 O4Na, 493.3294), and the molecular formula was determined to be C by combining the hydrogen and carbon spectrum data. 30 H 46 O4, the calculated unsaturation is 8.

[0091] Combination 1 H-NMR and 13 C NMR spectrum ( Figure 18-19 ) and HSQC spectra show that compound 3 contains three oxygen-linked carbon signals, δ C 76.8, 78.3, 82.0, and the corresponding hydrogen spectrum data are δ H (m, 4.02, 1H), 3.93 (dd, J = 6.7, 4.5, 1H), where δ C 82.0 is a quaternary carbon signal, δC 76.8 is close to the solvent peak and can be confirmed by HSQC spectrum; δ C 163.0 and 139.8 are two olefin proton signals. After comparing with the NMR data of the known compound inonotsuoxide B, it was found that the two data were similar. It was speculated that the side chain of compound 3 also contained a five-membered oxygen ring. The only difference was that the C-3 position of the known compound inonotsuoxide B was a hydroxyl group, while the C-3 position in compound 3 was oxidized to a carbonyl group. 1 H- 1 H COSY shows that there are four spin-coupled systems ( Fig. 22 ), respectively H-1 / H-2, H-5 / H-6, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-22(H-21) / H-23 / H-24; the planar structure of compound 3 was determined by HMBC spectrum. In the HMBC spectrum, Me-19 and δ C 163.0 is related to Me-18 and δ C 139.8, thus confirming that a double bond is located at C-8, C-9 ( Fig.21 ); Me-21 and δ C 40.1, 76.8 are correlated, indicating that there is a hydroxyl substitution at C-22; Me-26 and δ C 78.3, 82.0, 76.8 were correlated, which confirmed that compound 3 contained a five-membered ring and a hydroxyl group substituted at the C-24 position ( Fig.21 ). Thus, the planar structure of the compound was determined, as shown in the figure below.

[0092] The stereo configuration of compound 3 was confirmed by NOESY spectroscopy ( Fig.23 ) was determined by the NOESY spectrum. Me-28 / Me-19 / Me-18 / H-20 were correlated, indicating that the methyl groups on C-10 and C-13 were β-type, and H-20 and Me-28 were also β-type; H-5 / Me-29, Me-30 / H-17 / Me-21 were correlated, indicating that H-5, Me-29, Me-30, H-17, and Me-21 were α-type; since the five-membered ring was relatively far away from the parent nucleus and there was a lack of relevant signals, it could not be determined by the NOESY spectrum. However, in the NOESY spectrum, it was observed that H-22 and H-24 were correlated, thus determining that compound 3 had two possible configurations. NMR calculations were performed based on the configurations of C-22 and C-24, and the correlation coefficient R of the carbon spectrum data was fitted. 2 And probability analysis results DP4 + ( Fig.24 ) were analyzed and it was found that compound 3 had the highest agreement with the calculation results of A.2 The value is 0.9981, DP4 + (all date) was 99.98%, thus confirming that the configurations of C-22 and C-24 of the compound were both S. The compound was a new compound named (22S,24S)-24-hydroxy-22,25-epoxytirucall-3,7-dioxo-lanosta-8-en.

[0093] The obtained compound 4 was systematically identified and the results are as follows. The corresponding spectrum is attached. Figure 25-32 :

[0094] Compound 4: white powder (methanol), HRESIMS gave a quasi-molecular ion peak m / z 505.2938 [M+Na] + (calcd for C 30 H 42 O5Na, 505.2930), and the molecular formula was determined to be C by combining the hydrogen and carbon spectrum data. 30 H 42 O5, calculated unsaturation is 10.

[0095] The hydrogen spectrum of compound 4 ( Fig.26 ) showed 7 methyl proton signals, which were presumed to be triterpenoid compounds, and one oxygen proton signal, δ H 4.88 (dd, J = 8.9, 3.0 Hz, 1H); carbon spectrum of the combined compound ( Fig. 27 ) and HSQC spectra show that compound 4 contains three keto carbonyl carbon signals, δ C 211.0, 214.7, 198.0; one ester carbonyl carbon signal, δ C 179.0; one oxygen-linked carbon signal, δ C 79.0; one double bond signal, δ C 162.7, 139.4; compared with the NMR data of the known compound (+)-(5α,23R,24Z)-lanosta-8,24-dien-3,7-dioxo-23,26-γ-lactone, it can be seen that the two have the same skeleton. The difference is that compound 4 has one more ester carbonyl carbon signal than the known compound, but lacks a double bond signal. 1 H- 1 H COSY shows that there are four spin-coupled systems ( Fig.30 ), respectively H-1 / H-2, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-21, H-23 / H-24 / H-25 / H-27; the planar structure of compound 4 was determined by HMBC spectrum, Me-21 and δC 211.0 is related, indicating that there is a carbonyl substitution at C-22 ( Fig.29 ); Me-27 and δ C 32.7, 179.0 are related, H-23 and δ C 179.0 is correlated, indicating that the branched chain of compound 4 forms an oxygen-containing five-membered ring, and C-26 is an ester carbonyl carbon signal ( Fig.29 ).

[0096] The configuration of compound 4 was confirmed by NOESY spectrum ( Fig.31 ) was determined, Me-29 was correlated with H-5, H-17 with Me-30, and Me-21, thus confirming that the methyl group at C-14, H-5, H-17, Me-21, and Me-30 were α-type; Me-18, Me-28 and Me-19, Me-18 and H-20 had NOESY correlations, suggesting that the configurations of Me-18, Me-19, Me-28 and H-20 were β; H-23 and H-25 were correlated, indicating that the two had the same configuration, and the chiral carbon on the five-membered ring on the side chain was determined by calculation NMR ( Fig.32 ), by comparing R 2 and DP4 + The configurations of C-23 and C-25 were finally determined to be S. The structure of compound 4 was determined to be a new compound named (23S,25S)-3,7,22-trioxo-lanosta-23,26-olide.

[0097] The obtained compound 5 was systematically identified and the results are as follows. The corresponding spectrum is attached. Figure 33-40 :

[0098] Compound 5: white powder (methanol), HRESIMS gave a quasi-molecular ion peak m / z 521.3247 [M+Na] + (calcd for C 31 H 46 O5Na, 521.3243), and the molecular formula was determined to be C by combining the hydrogen and carbon spectrum data. 31 H 46 O5, calculated unsaturation is 9.

[0099] The hydrogen spectrum of compound 5 showed 8 methyl signals δ H 3.46 (3H, s), 1.70 (3H, s), 1.41 (3H, s), 1.18 (3H, s), 1.12 (3H, s), 1.11 (3H, s), 0.97 (3H, m), 0.70 (3H, s), which is speculated to be a triterpenoid compound; combined with the hydrogen spectrum and carbon spectrum data, there are two keto carbonyl carbon signals in the low field region, δC 214.6,199.5; two pairs of double bond carbon signals, δ C 158.9, 142.2 and δ C 132.4,123.8; In the hydrogen spectrum, there are four oxygen proton signals δ H 4.62(1H,s),4.52(1H,s),3.95(1H,dd,J=11.6,3.3Hz),3.46(3H,s); δ H 3.46 (3H, s) is a methoxy signal; δ H 5.65 (1H, d, J = 5.6 Hz), which is an olefin proton signal; combined with the carbon spectrum and HSQC spectrum, it can be seen that δ C 99.6 and δ H 4.62 (1H, s), which is speculated to be an acetal or hemiacetal carbon signal; in addition, δ C 68.1 and δ H 3.95 (1H, dd, J = 11.6, 3.3 Hz); 1 H- 1 H COSY showed two spin coupling systems, H-1 / H-2 and H-11 / H-12. Combining HMQC and HMBC spectra, the planar structure of compound 5 was determined. In the HMBC spectrum, Me-19 and δ C 158.9, 40.3 are related, Me-30 and δ C 142.2,48.4 are correlated, which confirms that one of the double bonds is located between C-8 and C-9; H-24 and δ C 123.8 connected, Me-23 and δ C 132.4,123.8 are related, Me-27 and δ C 99.6 correlation, H-22 and δ C 99.6 correlation, indicating that another double bond is located between C-24 and C-25 on the six-membered ring, and the methoxy group is located on C-26; Me-12 and δ C 65.9,158.9,16.8 were correlated, proving that a hydroxyl group was substituted at C-12; the planar structure of compound 5 was determined.

[0100] The relative configuration of compound 5 was determined by NOESY spectrum. H-5 was correlated with Me-28, and H-17 was correlated with Me-21 and Me-30, which proved that Me-21, Me-28, Me-30, H-5, and H-17 were α-configuration; Me-18 / Me-19 / Me-29 / H-20 were correlated, indicating that Me-29, Me-18, Me-19, and H-20 were β-type; In summary, the structure of compound 5 was determined to be a new compound named ganoshandong A.

[0101] The carbon and hydrogen signals of compounds 1-5 are assigned as follows:

[0102] Table 1 1 H NMR(600MHz)data for compounds 1-5(CDCl3)

[0103]

[0104]

[0105] Table 2 13 CNMR(150MHz)data for compounds 1-5(CDCl3)

[0106]

[0107]

[0108] Example 2: Acetylcholinesterase inhibitory activity of compounds 1-5

[0109] The compounds 1-5 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, and then 20 μL of the compound, 60 μL of PBS, and 20 μL of acetylcholinesterase (0.22 U / mL) were added to a 96-well plate. After reacting at 4°C for 10 min, 20 μL of substrate AChI (15 mM) and DNTB were added. 100μL, parallel 3 times, continue the reaction for 20min, and measure the absorbance A1 at 412nm; take an equal volume of PBS instead of acetylcholinesterase, parallel 3 times, and measure the absorbance A2 at 412nm; take an equal volume of PBS instead of the compound, and measure the absorbance A3 at 412nm; take an equal volume of PBS instead of the compound and acetylcholinesterase, and measure the absorbance A4 at 412nm; use donepezil hydrochloride diluted to 200μmol / L, 100μmol / L, 50μmol / L, 25μmol / L, and 5μmol / L as the positive control, parallel 3 times, take the average value, and calculate the acetylcholinesterase inhibition rate and IC50 The formula is inhibition rate = [(A3-A4)-(A1-A2)] / (A3-A4)х100%.

[0110] Table 3 Inhibitory effect of compounds 2-5 on acetylcholinesterase

[0111]

[0112] The positive control donepezil hydrochloride has been confirmed to have an effect on acetylcholinesterase IC 50 The value is 35.74 μM. Experiments have confirmed that compounds 2-5 all have acetylcholinesterase inhibitory activity. The extraction and separation methods are simple, which is convenient for further pharmacological research and development of their application in the preparation of anti-Alzheimer's disease drugs.

Claims

1. A triterpenoid compound, characterized in that: The triterpenoid compounds are shown in the structural formula 1. In the formula, R1 is the structure represented by A, B, C, D or E; R2 is H or hydroxyl.

2. The triterpenoid compound according to claim 1, characterized in that: The triterpenoid compounds are compounds 1-5 whose structures are shown below:

3. A method for preparing the triterpenoid compound according to claim 1, characterized in that: (1) Preparation of seed culture medium: Ganoderma shandongense mycelium was inoculated into malt culture medium and cultured at a constant temperature of 28° C. for 5 to 7 days; (2) fermentation: inoculating the mycelium in the seed culture medium into a rice solid fermentation medium, and performing a fermentation culture for 40 days by solid-state fermentation; (3) Extraction: The culture obtained by fermentation is extracted with ethyl acetate for 3-5 times, and the combined extracts are concentrated under reduced pressure to obtain a crude extract; (4) Chromatographic separation: The crude extract is separated by a reduced pressure silica gel column to obtain the compound represented by formula 1.

4. The method for preparing triterpenoids according to claim 3, characterized in that: The crude extract of step (4) is separated by vacuum silica gel column and gradient eluted with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1) systems in order of volume ratio to obtain four crude fractions: Fr.A (petroleum ether-ethyl acetate 50:1-40:1), 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); the obtained component Fr.B is subjected to silica gel column chromatography. Gradient elution was performed with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane / methanol (50:1-1:1) systems in a volume ratio, and the components ranging from petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain component Fr.B3. The Fr.B3 component was subjected to ODS column chromatography and gradient eluted with 35%-100% ethanol aqueous solution, and 35%, 45%, 55%, 65%, 75%, and 95% components were collected as Fr.B3-1, Fr.B3-2, Fr.B3-3, Fr.B3-4, Fr.B3-5, Fr.B3-6, Fr.B3-2 was eluted by silica gel column chromatography with dichloromethane-methanol in a volume ratio of 1:1, and the component Fr.B3-2-1 was collected; the obtained components were further subjected to ODS column chromatography with a gradient elution of 55% to 100% ethanol aqueous solution to collect 55% to 75% of the components Fr.B3-2-1-1 and 75% to 100% of the components Fr.B3-2-1-2; the collected Fr.B3-2-1-1 components were separated by gel column chromatography and then Preparative HPLC, eluted with methanol-water (75:25), collected chromatographic peaks a and b at 25.2-27.5min and 28.2-31.5min, respectively, and the obtained sample a was subjected to semi-preparative HPLC (250×20mm, 5μm), eluted with acetonitrile-water (83:17), and the chromatographic peak at 24min was collected to obtain compound 1; the obtained sample b was subjected to semi-preparative HPLC (250×20mm, 5μm), eluted with acetonitrile-water (83:17), and the chromatographic peak at 28min was collected to obtain compound 2; The Fr.B3-5 component was subjected to gel column chromatography to obtain the Fr.B3-5-1 component, which was subjected to preparative HPLC (250×20mm, 5μm) and eluted with methanol / water in a volume ratio of 71:29 at a flow rate of 7mL / min to collect chromatographic peaks c and d at 28.2-31.5min and 77.5-81.5min, respectively. The obtained sample c was subjected to semi-preparative HPLC chromatography (250×10mm, 5μm) and eluted with acetonitrile / water (50:50) at a flow rate of 2.5mL / min to collect the chromatographic peak of 51min to obtain compound 3. The obtained sample d was subjected to semi-preparative HPLC chromatography (250×10mm, 5μm) and eluted with acetonitrile / water (50:50) at a flow rate of 2.5mL / min to collect the chromatographic peak of 61min to obtain compound 4; Component Fr.C was subjected to HP20 macroporous resin and gradient eluted with 10% to 100% ethanol aqueous solution, and 10% to 30% component Fr.C-1 was collected; the obtained components were separated by ODS column chromatography and gradient eluted with 45% to 100% ethanol aqueous solution, and 75% to 100% component Fr.C-1-3 were collected; then silica gel column chromatography was performed with a petroleum ether-ethyl acetate volume ratio of 100:1 to 1:1 for gradient elution, and 20:1 to 1:1 components Fr.C-1-3-5 were collected; preparative HPLC was performed with methanol-water (v / v=67:33) for elution, and the chromatographic peaks of 25.6 to 27.5 min were collected; the obtained sample was subjected to semi-preparative HPLC chromatography, eluted with acetonitrile-water (58:42), and the chromatographic peak of 32 min was collected to obtain compound 5.

5. The method for preparing triterpenoids according to claim 3, characterized in that: The components of the malt extract powder culture medium are: 20 g / L malt extract powder, 12 g / L agar, 1 L distilled water, and natural pH; the components of the rice culture medium are: 35 g / bottle rice, 45 mL / bottle distilled water, and natural pH.

6. A pharmaceutical composition, characterized in that The composition contains the triterpenoid compound represented by formula 1 as claimed in claim 1.

7. Use of the triterpenoid compound according to claim 1 or the composition according to claim 6, characterized in that: The use of the triterpenoid compound shown in formula 1 of claim 1 or the pharmaceutical composition described in claim 6 in the preparation of anti-Alzheimer's disease drugs.

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