Triterpenoid compounds, methods of making, and uses thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a triterpenoid compound extracted from a food and medicinal fungus, Ganoderma shandongense, a preparation method of the triterpenoid compound and application of the triterpenoid compound in resisting Alzheimer's disease. The triterpenoid compound is shown in a structural formula I. The triterpenoid compound provided by the application has significant acetylcholinesterase inhibitory activity, is an ideal candidate compound of acetylcholinesterase inhibitor with novel structure and high activity, and has a good application prospect in the treatment of Alzheimer's disease.

Description

TECHNICAL FIELD

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

[0002] Ganoderma is the general term of Ganoderma spp., belonging to Basidiomycetes, Polyporales, Ganodermataceae, Ganoderma, and has important economic value. It is widely used in China and some other countries in Asia and is considered to have the effects of health care and longevity. Ganoderma has a research history of over a thousand years in China and is a traditional Chinese medicinal material with a very wide range of medicinal research. Among them, G. 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. Ganoderma triterpenes are one of the active ingredients isolated from Ganoderma, have complex chemical structures, bitter taste, high lipid solubility, and have wide medicinal value. In recent years, there are more and more studies on the neuroprotective activity of Ganoderma, and the exploration of the anti-Alzheimer's disease activity of triterpenoid compounds as the main active ingredient is one of the popular directions of the pharmacological activity research of Ganoderma.

[0003] Alzheimer's disease is an irreversible chronic neurodegenerative disease that affects 4%-8% of the elderly worldwide, and its main features are progressive memory impairment, impaired daily activity, 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 cognitive function of patients. AChE is essential for neural transmission and mainly degrades acetylcholine, thereby terminating the transmission of nerve impulses. So far, the Food and Drug Administration (FDA) has approved five drugs (four of which are AChE inhibitors) for the treatment of AD, but most of these drugs have the disadvantages of short half-life or serious damage to the peripheral cholinergic system, and long-term use can seriously affect human health. Therefore, screening of natural AChE inhibitors with high activity, strong specificity, safety and no toxicity is the focus of the development of anti-AD drugs. Therefore, 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 triterpenoid compound extracted from the edible and medicinal fungus Ganoderma shandongense and a preparation method thereof and application thereof in resisting Alzheimer's disease.

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

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

[0007]

[0008] In the formula,

[0009] R1 is a structure shown as A, B, C, D or E;

[0010]

[0011] R2 is H or hydroxyl.

[0012] Preferably, the triterpenoid compound is compound 1-5, the structure of which is shown as follows:

[0013]

[0014] A preparation method of the triterpenoid compound is as follows:

[0015] (1) Preparation of seed medium: inoculate Ganoderma shandongense mycelium into malt medium, and incubate at 28°C for 5-7 days;

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

[0017] (3) Extraction: extract the fermentation culture with ethyl acetate for 3-5 times, combine the extract, and concentrate under reduced pressure to obtain a crude extract;

[0018] (4) Chromatographic separation: separate the crude extract by reduced-pressure silica gel column to obtain the compound shown as formula I.

[0019] In step (4), the crude extract is separated by reduced-pressure silica gel column 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) by gradient elution with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1) systems in order of volume ratio.

[0020] The obtained component Fr.B was subjected to silica gel column chromatography with gradient elution with petroleum ether-ethyl acetate (50:1~1:1) and dichloromethane / methanol (50:1~1:1) system by volume ratio, respectively, and the components of petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain component Fr.B3. The component Fr.B3 was subjected to ODS column chromatography with gradient elution with ethanol aqueous solution with concentration of 35%~100%, and the components of 35%, 45%, 55%, 65%, 75%, 95% were collected as Fr.B3-1, Fr.B3-2, Fr.B3-3, Fr.B3-4, Fr.B3-5, Fr.B3-6, respectively. The Fr.B3-2 was subjected to silica gel column chromatography with elution with dichloromethane-methanol with volume ratio of 1:1, and the component Fr.B3-2-1 was collected. The obtained component was subjected to ODS column chromatography with gradient elution with ethanol aqueous solution with concentration of 55%~100%, and the components of 55%~75% and 75%~100% were collected as Fr.B3-2-1-1 and Fr.B3-2-1-2, respectively. The collected component Fr.B3-2-1-1 was subjected to gel column chromatography, and then subjected to preparative HPLC with elution with methanol-water (75:25), and the chromatographic peaks a and b collected at 25.2~27.5 min and 28.2~31.5 min, respectively. The obtained sample a was subjected to semi-preparative HPLC chromatography with elution with acetonitrile-water (83:17), and the chromatographic peak collected at 24 min to obtain compound 1. The obtained sample b was subjected to semi-preparative HPLC chromatography (250x10mm, 5μm) with elution with acetonitrile-water (83:17), and the chromatographic peak collected at 28 min to obtain compound 2.

[0021] The component Fr.B3-5 was subjected to gel column chromatography to obtain component Fr.B3-5-1, and then subjected to preparative HPLC (250x20mm, 5μm) with elution with methanol / water with volume ratio of 71:29 at a flow rate of 7mL / min, and the chromatographic peaks c and d collected at 28.2~31.5 min and 77.5~81.5 min, respectively. The obtained sample c was subjected to semi-preparative HPLC chromatography (250x10mm, 5μm) with elution with acetonitrile / water (50:50) at a flow rate of 2.5mL / min, and the chromatographic peak collected at 51 min to obtain compound 3. The obtained sample d was subjected to semi-preparative HPLC chromatography (250x10mm, 5μm) with elution with acetonitrile / water (50:50) at a flow rate of 2.5mL / min, and the chromatographic peak collected at 61 min to obtain compound 4.

[0022] The component Fr.C is eluted by gradient elution with 10%-100% ethanol aqueous solution on HP20 macroporous resin, and the 10%-30% component Fr.C-1 is collected; the obtained component is separated by ODS column chromatography, gradient eluted with 45%-100% ethanol aqueous solution, and the 75%-100% component Fr.C-1-3 is collected; then the component Fr.C-1-3-5 is collected by gradient elution with petroleum ether-ethyl acetate (100:1-1:1) on silica gel column chromatography, and the compound 5 is obtained by collecting the chromatographic peak at 25.6-27.5 min eluted with methanol-water (v / v=67:33) by preparative HPLC, and collecting the chromatographic peak at 32 min eluted with acetonitrile-water (58:42) by semi-preparative HPLC.

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

[0024] A pharmaceutical composition, wherein the composition contains the triterpenoid compound shown in the formula I.

[0025] The application of the triterpenoid compound or the composition, and the application of the triterpenoid compound shown in the formula I or the pharmaceutical composition in preparing an anti-Alzheimer's disease drug.

[0026] The compounds 1-5 provided by the application are all discovered for the first time, and there is no related report about the biological activity of the four compounds;

[0027] The obtained compounds 1-5 are subjected to acetylcholinesterase inhibition activity evaluation, and the results show that the compounds 2, 3, 4 and 5 all have good acetylcholinesterase inhibition activity (IC 50 The compound 1 does not show obvious acetylcholinesterase inhibition activity. Therefore, the triterpenoid compounds 2-5 have the potential to be developed into anti-Alzheimer's disease drugs, and provide a new material basis for developing an innovative drug for treating Alzheimer's disease, developing an innovative drug of acetylcholinesterase inhibitors, and providing a scientific basis for developing and utilizing natural active substances from large fungi. In addition, the above compounds are separated from a famous edible and medicinal large fungus in China, and the safety is preliminarily ensured. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The HRESIMS spectrum of the compound 1 provided by the embodiment of the application is shown in the following table:

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

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

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

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

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

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

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

[0036] Figure 9 HRESIMS spectrum of compound 2 provided in the embodiments of the present invention;

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

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

[0039] Figure 12 The HSQC spectrum (600MHz, CDCl3) of compound 2 provided in the embodiments of the present invention;

[0040] Figure 13 The HMBC spectrum (600MHz, CDCl3) of compound 2 provided in the embodiments of the present invention;

[0041] Figure 14The H NMR spectrum (600 MHz, CDC13) of compound 2 provided for the embodiments of the present application is shown in Figure 2A. 1 H- 1 The H COSY spectrum (600 MHz, CDC13) of compound 2 provided for the embodiments of the present application is shown in Figure 2B.

[0042] Figure 15 The NOESY spectrum (600 MHz, CDC13) of compound 2 provided for the embodiments of the present application is shown in Figure 2C. l3 );

[0043] Figure 16 The R 2 and DP4 + analysis of compound 2 provided for the embodiments of the present application is shown in Figure 2D.

[0044] Figure 17 The HRESIMS spectrum of compound 3 provided for the embodiments of the present application is shown in Figure 3A.

[0045] Figure 18 The H NMR spectrum (600 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3B. 1

[0046] Figure 19 The C NMR spectrum (150 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3C. 13

[0047] Figure 20 The HSQC spectrum (600 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3D.

[0048] Figure 21 The HMBC spectrum (600 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3E.

[0049] Figure 22 The H NMR spectrum (600 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3B. 1 1 The H COSY spectrum (600 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3F.

[0050] Figure 23 The NOESY spectrum (600 MHz, CDC13) of compound 3 provided for the embodiments of the present application is shown in Figure 3G. l3 );

[0051] Figure 24 The R 2 and DP4 + analysis of compound 3 provided for the embodiments of the present application is shown in Figure 3H.

[0052] Figure 25 The HRESIMS spectrum of compound 4 provided for the embodiments of the present application is shown in Figure 4A.

[0053] ​​​Figure 26 H NMR spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2A. 1 H NMR spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2A.

[0054] Figure 27 H NMR spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2A. 13 H NMR spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2A.

[0055] Figure 28 HSQC spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2B.

[0056] Figure 29 HMBC spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2C.

[0057] Figure 30 H NMR spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2A. 1 H- 1 H COSY spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2D.

[0058] Figure 31 NOESY spectrum (600 MHz, CDC13) of compound 4 provided for embodiments of the present application is shown in Figure 2E. l3 );

[0059] Figure 32 R 2 and DP4 + analysis;

[0060] Figure 33 HRESIMS spectrum of compound 5 provided for embodiments of the present application is shown in Figure 3A.

[0061] Figure 34 H NMR spectrum (600 MHz, CDC13) of compound 5 provided for embodiments of the present application is shown in Figure 3B. 1 H NMR spectrum (600 MHz, CDC13) of compound 5 provided for embodiments of the present application is shown in Figure 3B.

[0062] Figure 35 H NMR spectrum (600 MHz, CDC13) of compound 5 provided for embodiments of the present application is shown in Figure 3B. 13 C NMR spectrum (150 MHz, CDC13) of compound 5 provided for embodiments of the present application is shown in Figure 3C.

[0063] Figure 36 HSQC spectrum (600 MHz, CDC13) of compound 5 provided for embodiments of the present application is shown in Figure 3D.

[0064] Figure 37 HMBC spectrum (600 MHz, CDC13) of compound 5 provided for embodiments of the present application is shown in Figure 3E.

[0065] Figure 38The compound 5 provided in the embodiments of the present application has the structure of formula (I) 1 H- 1 H COSY spectrum (600 MHz, CDC13);

[0066] Figure 39 The NOESY spectrum (600 MHz, CDC13) of the compound 5 provided in the embodiments of the present application is shown in Table 2. l3 );

[0067] Figure 40 The R 2 and DP4 + analysis of the compound 5 provided in the embodiments of the present application is shown in Table 3.

[0068] Figure 41 The HMBC, 1 H- 1 H COSY and NOESY correlation of the compounds 1-5 provided in the embodiments of the present application. DETAILED DESCRIPTION

[0069] 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.

[0070] 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.

[0071] Example 1: Preparation of triterpenoid compounds 1-5 in G. shandongense

[0072] 1. Fermentation conditions

[0073] Strain activation: inoculate the mycelium of G. shandongense into 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;

[0074] Strain expansion culture: take malt extract powder 10 g, distilled water 500 mL, agar 7.5 g, and autoclave at 121°C for 30 min for standby. Inoculate the cut G. shandongense into the above medium, and cultivate at 28°C for 5-6 days.

[0075] Fermentation: 100 g of rice was put into 500 mL conical flask, 100 mL water was added, 121 °C high pressure sterilization for 30 min, and standby. Inoculate 1 / 4 dish of G. shandongense mycelium G. shandongense per bottle, and incubate at 28 °C for 45 days.

[0076] 2. Extraction and separation

[0077] The culture obtained by fermentation was soaked and 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 obtained extract (350 g) was mixed with 100-200 mesh silica gel, separated by reduced pressure column silica gel (25x15 cm), and eluted with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane-methanol (50:1-1:1) systems in turn according to the volume ratio, and each fraction was identified by thin layer chromatography and high performance liquid chromatography, and combined to obtain four 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).

[0078] The component Fr. B (30.25 g) was separated by silica gel column chromatography (5x50 cm) with petroleum ether-ethyl acetate (50:1-1:1) and dichloromethane / methanol (50:1-1:1) systems in turn according to the volume ratio gradient elution, and the components of petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 were collected to obtain component Fr. B3 (9.2 g), and the Fr. B3 component was gradient eluted by ODS column chromatography with 35%-100% ethanol aqueous solution to collect 35%, 45%, 55%, 65%, 75%, and 95% components as Fr. B3-1, Fr. B3-2, Fr. B3-3, Fr. B3-4, Fr. B3-5, and Fr. B3-6, respectively.

[0079] The Fr. B3-2 (1.5 g) was gradient eluted by silica gel column chromatography with dichloromethane-methanol according to the volume ratio 1:1 to collect 20:1-10:1 component Fr. B3-2-1 (914.2 mg), which was further gradient eluted by ODS column chromatography with 55%-100% ethanol aqueous solution to collect 55%-75% component Fr. B3-2-1-1 (473.4 mg) and 75%-100% component Fr. B3-2-1-2 (365.3 mg).

[0080] The Fr.B3-2-1-1 obtained above was separated by gel column chromatography, and then eluted by preparative HPLC with methanol-water (75:25, by volume) at a flow rate of 7 mL / min, and the chromatographic peaks a and b collected at 25.2-27.5 min and 28.2-31.5 min, respectively. The sample a was further separated by semi-preparative HPLC chromatography (250x10 mm, 5 μm) with acetonitrile-water (83:17, by volume) at a flow rate of 2.5 mL / min, and the chromatographic peak collected at 24 min to obtain compound 1 (6.9 mg). The sample b was further separated by semi-preparative HPLC chromatography (250x10 mm, 5 μm) with acetonitrile-water (83:17, by volume) at a flow rate of 2.5 mL / min, and the chromatographic peak collected at 28 min to obtain compound 2 (1.1 mg).

[0081] The Fr.B3-5 component was separated by gel column chromatography to obtain Fr.B3-5-1 component, which was further separated by preparative HPLC (250x20 mm, 5 μm) with methanol / water (71:29, by volume) at a flow rate of 7 mL / min, and the chromatographic peaks c and d collected at 28.2-31.5 min and 77.5-81.5 min, respectively. The sample c was further separated by semi-preparative HPLC chromatography (250x10 mm, 5 μm) with acetonitrile / water (50:50) at a flow rate of 2.5 mL / min, and the chromatographic peak collected at 51 min to obtain compound 3 (8.6 mg). The sample d was further separated by semi-preparative HPLC chromatography (250x10 mm, 5 μm) with acetonitrile / water (50:50) at a flow rate of 2.5 mL / min, and the chromatographic peak collected at 61 min to obtain compound 4 (1.1 mg).

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

[0083] The hydrogen spectrum of compound 1 includes 7 angular methyl signals and one doublet methyl signal in the high field region, and in combination with the carbon spectrum, 31 carbon signals are given, so it is inferred that compound 1 is a triterpenoid compound. The molecular ion peak of compound 1 is at m / z 576. The molecular ion peak of compound 2 is at m / z 576. 1Two oxygen-bonded proton signals were observed in the H-NMR spectrum at δ H 3.97 (m, 1H) and δ H 3.46 (s, 3H), δ H 3.46 (s, 3H) suggested a methoxyl signal; δ H 5.65 (d, J = 5.5 Hz, 1H) was an olefinic proton signal, which, combined with the carbon spectrum, indicated that δ C 162.9, 139.6, 123.9, 132.4, the compound had two double bonds; in the13C-NMR spectrum, combined with HSQC spectrum, δ C 214.8, 198.2 were the ketone carbonyl signals at C-3 and C-7 positions of the triterpenoid; δ C 99.6, δ H 4.63 (s, 1H) was speculated to be an acetal or hemiacetal carbon signal; after comparing the nuclear magnetic data with the known compound astraeusin I, it was found that the nuclear magnetic data of the two were similar, but astraeusin I lacked a ketone carbonyl carbon, and at the same time, the compound had a six-membered oxygen ring, so it was speculated that compound 1 also had a six-membered oxygen ring; 1 H- 1 Four spin coupling systems were shown in H COSY, which were 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, δ 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 were related, δ H 3.46 (s, 3H), 1.69 (s, 3H) and δ C 99.6 were related, δ H 1.69 (brs, 3H) and δ C 132.4 (C-25) were related, and at the same time, combined with the unsaturation of the compound, these proved that compound 1 contained a six-membered oxygen ring, and thus the planar structure of compound 1 was determined, as shown in the following figure.

[0084] The stereochemical configuration of compound 1 was determined by NOESY spectrum Figure 7) were determined, the NOESY correlation between H-24 and Me-27 determined the configuration of the double bond on the six-membered oxygen ring as Z; the correlation between Me-18 / Me-19 / Me-28, Me-18 / H-20 indicated that Me-18, Me-19, Me-28, H-20 were all β type; the correlation between Me-30 / H-17 / Me-21, H-17 / H-22, Me-29 / H-5 suggested that Me-30, H-17, Me-21, H-22, Me-29, H-5 were all α type; H-22 / H-17 had NOESY correlation, indicating that compound 1 had two configurations, NMR calculation was carried out based on the configuration of C-26, and the correlation coefficient R 2 and probability analysis results DP4 + ( Figure 8 ) were analyzed, it was found that the calculation results of compound 1 and B were the highest, R 2 = 0.9983, DP4 + (all date) was 100%, so the configuration of compound C-26 was determined, through Scifinder retrieval, 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 subjected to systematic structural identification, and the results were as follows, and the corresponding spectrum was shown in the attached Figures 9-16 :

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

[0087] The hydrogen spectrum of compound 2 Figure 10 ) showed typical characteristics of triterpenoid compounds: there were 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), one doublet 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), two methine protons signals; δ H 5.67 (d, J = 5.6 Hz, 1H) is one olefinic proton signal; 13 C NMR shows 31 carbon signals, combined with HSQC spectrum, it is known that there are two double bonds in compound 2, δ C 162.9, 139.6 and δ C 132.4, 123.8; δ C 98.2, it is supposed to be one acetal or hemiacetal carbon signal, the corresponding hydrogen spectrum signal is δ H 4.74 (s, 1H); δ C 214.9, 198.3 are the ketone carbonyl carbon signals of C-3 and C-7 of lanostane type triterpenes; δ C 68.2, δ H 4.02 (dd, J = 11.5, 3.4 Hz, 1H) is one methine; δ C 63.6, δ H 3.87 (m, 1H), δ H 3.54 (m, 1H) is supposed to be one methylene; by comparing the nuclear magnetic data of compound 2 with the known compound (5α, 23E)-27-nor-3β-hydroxylanosta-8, 23-dien-7, 25-dione, it is known that compound 2 has two more carbon signals in the oxygen-containing carbon signal region than the known compound, and also lacks one ester carbonyl signal. 1 H- 1 There are four spin coupling systems in H COSY, which are 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, respectively. Figure 14 The plane structure of compound 2 is finally determined by HMBC spectrum. In the HMBC spectrum, Figure 13 63.6, δ C 4.74 (s, 1H), δ H 63.6, δ C 4.74 (s, 1H), δ C 132.4, 123.8, which indicates that the double bond exists in Δ24, 25; Me-28, H-23, δ C 68.2, which indicates that there is a five-membered oxygen ring on the branched chain of compound 2. Therefore, the plane structure of compound 2 is determined as shown in the following figure.

[0088] The stereo configuration of the compound was determined by NOESY spectrum Figure 15 ); Me-29 / Me-19 / Me-18 / H-20 had NOESY correlation, which proved that the methyl group at position 10, the methyl group at position 13 and the side chain at position 17 and Me-29 were all in β type; Me-30 / H-5 had correlation, and Me-31 / H-17 / Me-21 proved that the configuration of Me-30, H-17 and Me-21 was α; since H-23 and H-26 on the side chain were relatively far away from the mother nucleus in space, there was a lack of necessary correlation signals, and no correlation signals of the two were observed in the NOESY spectrum, indicating that compound 2 had 4 possible configurations, and NMR calculation was carried out based on the configuration of C-23 and C-26, and the correlation coefficient R 2 and probability analysis result DP4 + ( Figure 16 ) were analyzed, it was found that compound 2 had the highest calculation coincidence degree with A, R 2 value was 0.9957, DP4 + (all date) was 92.69%, so the configuration of C-23 and C-26 of the compound was determined, and through Scifinder retrieval, compound 2 was a new compound, named (23S, 26R)-8, 24-diene-23, 26-epoxytirucall-3, 7-dione.

[0089] The obtained compound 3 was subjected to systematic structure identification, and the results were as follows, and the corresponding spectrum was shown in the attached Figures 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), combined with hydrogen spectrum and carbon spectrum data, the molecular formula of the compound was determined as C 30 H 46 O4, and the unsaturation degree was calculated to be 8.

[0091] Combined with 1 H-NMR and 13 C NMR spectrum Figures 18-19 ) and HSQC spectrum, it was known that compound 3 contained 3 oxygen-containing carbon signals, δ C 76.8, 78.3, 82.0, corresponding to hydrogen spectrum data δ H (m, 4.02, 1H), 3.93 (dd, J = 6.7, 4.5, 1H), wherein δ C 82.0 was a quaternary carbon signal, δC The 76.8 peak is close to the solvent peak and can be determined using HSQC spectroscopy; 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 data of the two are similar, suggesting that the side chain of compound 3 also contains a five-membered oxygen ring. The only difference is that the C-3 position of the known compound inonotsuoxide B is a hydroxyl group, while the C-3 position of compound 3 is oxidized to a carbonyl group. 1 H- 1 H COSY shows four spin-coupled systems. Figure 22 The structures are 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, respectively. The planar structure of compound 3 was determined by HMBC spectroscopy. In the HMBC spectrum, Me-19 and δ C 163.0 is relevant; it relates to Me-18 and δ. C 139.8 is relevant, thus identifying a double bond located at C-8, C-9 ( Figure 21 Me-21 and δ C The correlation between 40.1 and 76.8 indicates the presence of a hydroxyl substitution at C-22; Me-26 and δ C The values ​​78.3, 82.0, and 76.8 are related, thus confirming that compound 3 contains a five-membered ring and has a hydroxyl substitution at position C-24. Figure 21 Thus, the planar structure of the compound was determined, as shown in the figure below.

[0092] The stereoconfiguration of compound 3 was determined by NOESY spectroscopy. Figure 23 The correlation between Me-28 / Me-19 / Me-18 / H-20 indicates that the methyl groups at C-10 and C-13 are β-type, and H-20 and Me-28 are also β-type. The correlation between H-5 / Me-29 and Me-30 / H-17 / Me-21 indicates that H-5, Me-29, Me-30, H-17, and Me-21 are α-type. Due to the relatively large spatial distance between the five-membered ring and the parent nucleus, a lack of correlation signals prevented confirmation via NOESY spectroscopy. However, a correlation was observed between H-22 and H-24 in the NOESY spectrum, indicating that compound 3 has two possible configurations. NMR calculations were performed based on the configurations of C-22 and C-24, and the correlation coefficient R0 was obtained by fitting the carbon spectral data. 2 And probability analysis results DP4 + ( Figure 24 Analysis revealed that compound 3 showed the highest agreement with the calculated results of A, R2 The value is 0.9981, DP4 + The (all date) was 99.98%, thus confirming that the configuration at positions C-22 and C-24 of the compound is S. This compound is a new compound and is named (22S,24S)-24-hydroxy-22,25-epoxytirucall-3,7-dioxo-lanosta-8-en.

[0093] The results of systematic structural identification of compound 4 are as follows, and the corresponding spectra are attached. Figures 25-32 :

[0094] Compound 4: White powder (methanol), HRESIMS gives a quasi-molecular ion peak at m / z 505.2938 [M+Na] + (calcd for C 30 H 42 O5Na, 505.2930), combined with proton and carbon spectral data, its molecular formula was determined to be C. 30 H 42 O5, the calculated degree of unsaturation is 10.

[0095] The proton spectrum of compound 4 ( Figure 26 The signal shows 7 methyl protons, presumably from a triterpenoid, and one oxygen proton, δ H 4.88 (dd, J = 8.9, 3.0 Hz, 1H); combined with the carbon spectrum of the compound ( Figure 27 As can be seen from the HSQC spectrum, compound 4 contains three ketone carbonyl carbon signals, δ C 211.0, 214.7, 198.0; a signal for an ester carbonyl carbon, δ C 179.0; a carbon-oxygen junction signal, δ C 79.0; a double bond signal, δ C 162.7, 139.4; Comparison with the NMR data of the known compound (+)-(5α,23R,24Z)-lanosta-8,24-dien-3,7-dioxo-23,26-γ-lactone shows 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 one double bond signal. 1 H- 1 H COSY shows four spin-coupled systems. Figure 30 The structures are H-1 / H-2, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-21, and H-23 / H-24 / H-25 / H-27, respectively. The planar structure of compound 4 was determined by HMBC spectroscopy, and Me-21 and δC The presence of a carbonyl substitution at position C-22 indicates that there is a carbonyl substitution present. Figure 29 Me-27 and δ C 32.7 and 179.0 are related; H-23 is related to δ. C The correlation at 179.0 indicates that the branched chain of compound 4 forms an oxygen-containing five-membered ring, and that C-26 is an ester carbonyl carbon signal. Figure 29 ).

[0096] The configuration of compound 4 is determined by the NOESY spectrum ( Figure 31 The correlation between Me-29 and H-5, and between H-17 and Me-30 and Me-21, indicates that the methyl group at C-14, H-5, H-17, Me-21, and Me-30 are of the α-type. A NOESY correlation exists between Me-18 and Me-28 and Me-19, and between Me-18 and H-20, suggesting that Me-18, Me-19, Me-28, and H-20 have a β-configuration. A correlation exists between H-23 and H-25, indicating that they have the same configuration. The chiral carbons on the five-membered rings of the side chains were determined by calculated NMR. Figure 32 ), by comparing R 2 and DP4 + The values ​​were finally determined, and the configurations of C-23 and C-25 were both S. The structure of compound 4 was thus determined, and it was identified as a new compound, named (23S,25S)-3,7,22-trioxo-lanosta-23,26-olide.

[0097] The results of the systematic structural identification of compound 5 are as follows, and the corresponding spectra are attached. Figures 33-40 :

[0098] Compound 5: White powder (methanol), HRESIMS gives a quasi-molecular ion peak at m / z 521.3247 [M+Na] + (calcd for C 31 H 46 O5Na, 521.3243), combined with proton and carbon spectral data, its molecular formula was determined to be C. 31 H 46 O5, the calculated degree of unsaturation is 9.

[0099] The proton NMR spectrum of compound 5 showed eight 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), presumably a triterpenoid compound; combining 1H and 1C spectral data, two ketone carbonyl carbon signals are present in the low-field region, δC 214.6, 199.5; two pairs of double-bonded carbon signals, δ C 158.9, 142.2 and δ C 132.4, 123.8; In the proton spectrum, there are four oxygen-bonded 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) represents a methoxy group signal; δ H 5.65 (1H, d, J = 5.6 Hz) is the signal of an olefin proton; combining the carbon spectrum and HSQC spectrum, we can see that δ C 99.6 and δ H 4.62 (1H, s), presumably a carbon signal from an acetal or hemiacetal; in addition, δ C 68.1 and δ H Connected at 3.95 (1H, dd, J = 11.6, 3.3Hz); 1 H- 1 ¹H COSY revealed two spin-coupled systems, H⁻¹ / H⁻² and H⁻¹¹ / H⁻¹². Combined with HMQC and HMBC spectra, the planar structure of compound 5 was determined. In the HMBC spectrum, Me⁻¹⁹ and δ¹²... C There is a correlation between 158.9 and 40.3, Me-30 and δ C The correlation between 142.2 and 48.4 indicates that one of the double bonds is located between C-8 and C-9; H-24 is related to δ. C 123.8 connected, Me-23 and δ C There is a correlation between 132.4 and 123.8, and between Me-27 and δ. C 99.6% correlation, H-22 and δ C 99.6% correlation proves that another double bond is located between C-24 and C-25 on the hexaoxy ring, and the methoxy group is located at C-26; Me-12 and δ C The correlation between 65.9, 158.9, and 16.8 proves that a hydroxyl group is formed at C-12; the planar structure of compound 5 is thus determined.

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

[0101] The C-H 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] 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, for later use. 20 μL of each compound, 60 μL of PBS, and 20 μL of acetylcholinesterase (0.22 U / mL) were added to 96-well plates. 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 measured at 412 nm (A3). An equal volume of PBS was used to replace both the compound and acetylcholinesterase, and the absorbance 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%.

[0110] Table 3 shows the inhibitory effects of compounds 2-5 on acetylcholinesterase.

[0111]

[0112] Experiments have confirmed that the positive control donepezil hydrochloride inhibits acetylcholinesterase IC50. 50 The value was 35.74 μM. Experiments confirmed that compounds 2-5 all have acetylcholinesterase inhibitory activity. Their extraction and separation methods are simple, which facilitates further pharmacological research and development of their application in the preparation of anti-Alzheimer's drugs.

Claims

1. A triterpenoid compound, characterized in that: The triterpenoids are selected from compounds 2-5, and their structures are shown below:

2. A method for preparing the triterpenoid compound according to claim 1, characterized in that: (1) Preparation of seed culture medium: Shandong Ganoderma mycelium Ganoderma shandongense Inoculate with malt culture medium and incubate at 28℃ for 5–7 days; (2) Fermentation: The mycelium in the above seed culture medium was inoculated into the rice solid fermentation culture medium and fermented for 40 days using solid fermentation. (3) Extraction: The culture obtained from fermentation is extracted with ethyl acetate 3-5 times, and the extracts are combined and concentrated under reduced pressure to obtain crude extract; (4) Chromatographic separation: The crude extract was separated by vacuum silica gel column chromatography using a gradient elution system of petroleum ether-ethyl acetate 50:1–1:1 and dichloromethane-methanol 50:1–1:1 at volume ratios, yielding 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). Fraction Fr. B was further separated by silica gel column chromatography using a gradient elution system of petroleum ether-ethyl acetate 50:1–1:1 and dichloromethane / methanol 50:1–1:1 at volume ratios, collecting fractions from petroleum ether-ethyl acetate 5:1 to dichloromethane / methanol 30:1 to obtain fraction Fr. B3. Fraction B3 was subjected to gradient elution with 35%–100% ethanol-water solution via ODS column chromatography, and the fractions of 35%, 45%, 55%, 65%, 75%, and 95% 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 then eluted with dichloromethane-methanol at a volume ratio of 1:1 via silica gel column chromatography, and fraction Fr. B3-2-1 was collected. The obtained fractions were then subjected to gradient elution with 55%–100% ethanol-water solution via ODS column chromatography, and the fractions of 55%–75% Fr. B3-2-1-1 and 75%–100% Fr. B3-2-1-2 were collected. After separation by gel column chromatography, the B3-2-1-1 fraction was eluted by preparative HPLC with methanol-water 75:25, 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 b was eluted by semi-preparative HPLC at 250×20 mm and 5 μm with acetonitrile-water 83:17, and the chromatographic peak at 28 min was collected to obtain compound 2. Fr.B3-5 fraction was subjected to gel column chromatography to obtain Fr.B3-5-1 fraction. Then, it was subjected to preparative HPLC at 250×20 mm, 5 μm, eluted with methanol / water at a volume ratio of 71:29, and the chromatographic peaks c and d at 7 mL / min were collected at 28.2–31.5 min and 77.5–81.5 min, respectively. The obtained sample c was subjected to semi-preparative HPLC at 250×10 mm, 5 μm, eluted with acetonitrile / water at a volume ratio of 50:50, and the chromatographic peak at 51 min was collected at a flow rate of 2.5 mL / min to obtain compound 3. The obtained sample d was subjected to semi-preparative HPLC at 250×10 mm, 5 μm, eluted with acetonitrile / water at a volume ratio of 50:50, and the chromatographic peak at 61 min was collected at a flow rate of 2.5 mL / min to obtain compound 4. Fraction Fr. C was subjected to gradient elution with 10%–100% ethanol-water solution using HP20 macroporous resin, and 10%–30% of fraction Fr. C-1 was collected. The obtained fraction was separated by ODS column chromatography, and gradient elution with 45%–100% ethanol-water solution was performed, and 75%–100% of fraction Fr. C-1-3 was collected. Then, fraction Fr. C-1-3-5 was collected by silica gel column chromatography with gradient elution with petroleum ether-ethyl acetate volume ratio of 100:1 to 1:1, and 20:1–1:1 of fraction Fr. C-1-3-5 was collected. Preparative HPLC was performed with methanol-water v / v = 67:33, and the chromatographic peak at 25.6–27.5 min was collected. The obtained sample was subjected to semi-preparative HPLC with acetonitrile-water 58:42, and the chromatographic peak at 32 min was collected to obtain compound 5.

3. The method for preparing triterpenoid compounds according to claim 2, characterized in that: The malt culture medium consists of: 20 g / L malt extract powder, 12 g / L agar, 1 L distilled water, and natural pH; the rice culture medium consists of: 35 g / bottle of rice, 45 mL / bottle of distilled water, and natural pH.

4. A pharmaceutical composition, characterized in that, The composition contains the triterpenoid compound as described in claim 1.

5. The use of a triterpenoid compound according to claim 1 or a composition according to claim 4, characterized in that: The use of the triterpenoid compound of claim 1 or the pharmaceutical composition of claim 4 in the preparation of an anti-Alzheimer's disease drug.

Images