Ganoderma lucidum triterpenoid compound, preparation method thereof, and use thereof in preventing and treating liver damage

Abstract

The present invention belongs to the field of pharmaceutical technology and discloses Ganoderma triterpenoid compounds, their preparation method, and their use in preventing and treating liver damage. The Ganoderma triterpenoid compounds described herein are obtained by isolating compound 1, compound 2, or compound 3 from the dried fruiting bodies of Ganoderma lucidum. The dried fruiting bodies of Ganoderma lucidum are subjected to alcohol extraction, and the extract is concentrated to obtain an extract. The extract is then separated by macroporous resin chromatography, silica gel column chromatography, medium-pressure silica gel column chromatography, and semi-preparative reversed-phase high-performance liquid chromatography to obtain compound 1. Compound 1 is then separated by reversed-phase column chromatography, and different fractions are separated and purified by semi-preparative reversed-phase high-performance liquid chromatography to obtain compound 2 and compound 3, respectively. The preparation method of the present invention is simple to operate and can isolate and obtain the Ganoderma triterpenoid compounds. Experimental results show that the compounds, at a concentration of 10 μM, significantly protect human HepG2 liver cancer cells damaged by APAP, with cell viability rates of 84.06%, 53.43%, and 53.54%, respectively. These compounds can be used in the preparation of drugs for preventing or treating liver damage.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a Ganoderma lucidum triterpenoid compound, a preparation method thereof, and an application thereof in preventing and treating liver damage. Background Art

[0002] Ganoderma, collectively known as Lingzhi, is a traditional Chinese medicine. Its medicinal value has long been documented in ancient texts such as the Shennong Bencao Jing. The Chinese Pharmacopoeia designates the dried fruiting bodies of Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense (Zhao, Xu et Zhang) as legal medicinal materials, primarily used to treat restlessness, insomnia, palpitations, lung deficiency, cough and asthma, shortness of breath, and loss of appetite. The main chemical components of Ganoderma include triterpenes, miscellaneous terpenes, polysaccharides, steroids, alkaloids, and sesquiterpenes. Among these, triterpenes, the main active ingredients, have become a research hotspot due to their significant biological activities in immunomodulation, anti-tumor activities, and liver protection.

[0003] As a vital organ in the human body, the liver performs numerous important physiological functions, including metabolism, detoxification, synthesis, and storage. Liver injury refers to damage to liver tissue due to various causes, resulting in structural and functional abnormalities. Liver injury can be caused by a variety of factors, including viral infection, excessive alcohol consumption, drug or toxin effects, fatty liver disease, and autoimmune reactions. These factors can lead to hepatocyte degeneration and necrosis, causing liver fibrosis and cirrhosis, impairing liver function, and threatening the patient's health and life.

[0004] The treatment of liver damage has long been a hot topic in medical research, with the search for effective liver-protective ingredients in natural medicines being a key research direction. Ganoderma triterpenes, the primary active ingredient in Ganoderma lucidum, have recently demonstrated potential efficacy in preventing and treating liver damage, offering new insights into its prevention and treatment. Summary of the Invention

[0005] The purpose of this invention is to fully exploit my country's traditional Chinese medicine resources and identify compounds with unique chemical structures, strong activity, and minimal toxic side effects that have hepatoprotective activity, thereby providing novel drugs for the prevention and treatment of liver damage in clinical research. This invention provides Ganoderma lucidum triterpenoid compounds, their preparation methods, and their use in the prevention and treatment of liver damage. Research has revealed that three Ganoderma lucidum triterpenoid compounds of this invention exhibit significant hepatoprotective activity. To date, these research findings have not been reported in patents or literature.

[0006] The technical solutions of the present invention are as follows:

[0007] The first aspect of the present invention provides a Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof. The Ganoderma triterpenoid compound is Compound 1, Compound 2 or Compound 3, and its chemical structure is as follows:

[0008]

[0009] The second aspect of the present invention is to provide a method for preparing the Ganoderma lucidum triterpenoid compound, comprising the following steps:

[0010] (1) extracting the dried fruiting bodies of Ganoderma lucidum with an ethanol solution, combining the extracts, and concentrating to obtain an extract;

[0011] (2) The extract obtained in step (1) was mixed with a macroporous resin, and subjected to macroporous resin chromatography, using an ethanol-water gradient elution with a volume ratio of 30:70, 60:40, 80:20, and 95:5 to obtain four fractions DK-1, DK-2, DK-3, and DK-4;

[0012] (3) Fraction DK-3 was subjected to silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50, and then the column was rinsed with ethyl acetate and methanol in sequence to obtain 25 fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11, Fr.12, Fr.13, Fr.14, Fr.15, Fr.16, Fr.17, Fr.18, Fr.19, Fr.20, Fr.21, Fr.22, Fr.23, Fr.24, and Fr.25;

[0013] (4) Fraction Fr.17 was chromatographed on a medium-pressure silica gel column using a gradient elution of petroleum ether and ethyl acetate in a volume ratio of 80:20, 70:30, 60:40, and 50:50, followed by rinsing the column with ethyl acetate to obtain 14 subfractions: Fr.17-1, Fr.17-2, Fr.17-3, Fr.17-4, Fr.17-5, Fr.17-6, Fr.17-7, Fr.17-8, Fr.17-9, Fr.17-10, Fr.17-11, Fr.17-12, Fr.17-13, and Fr.17-14;

[0014] (5) Subfraction Fr.17-5 was separated by semi-preparative reverse-phase HPLC and isocratically eluted with methanol-water at a volume ratio of 85:15 to obtain compound 1;

[0015] (6) Subfraction Fr.17-7 was subjected to reverse phase column chromatography and eluted with a gradient of methanol-water in a volume ratio of 50:50, 60:40, and 75:25, and then the column was flushed with methanol to obtain 25 subfractions Fr.17-7-1, Fr.17-7-2, Fr.17-7-3, Fr.17-7-4, Fr.17-7-5, Fr.17-7-6, Fr.17-7-7, Fr.17-7-8, Fr.17-7-9, Fr.17-7-10, Fr.17-7-11, Fr.17-7-12, Fr.17-7-13, Fr.17-7-14, Fr.17-7-15, Fr.17-7-16, Fr.17-7-17, Fr.17-7-18, Fr.17-7-19, Fr.17-7-20, Fr.17-7-21, Fr.17-7-22, Fr.17-7-23, Fr.17-7-24, Fr.17-7-25, Fr.17-7-26, Fr.17-7-27, Fr.17-7-28, Fr.17-7-29, Fr.17-7-30, Fr.17-7-31, Fr.17-7-32, Fr.17-7-33, Fr.17-7-34, Fr.17-7-35, Fr.17-7-36, Fr.17-7-37, Fr.17-7-38, Fr.17-7-39, Fr.17-7-40, Fr.1 10. Fr.17-7-11, Fr.17-7-12, Fr.17-7-13, Fr.17-7-14, Fr.17-7-15, Fr.17-7-16, Fr.17-7-17, Fr.1 7-7-18, Fr.17-7-19, Fr.17-7-20, Fr.17-7-21, Fr.17-7-22, Fr.17-7-23, Fr.17-7-24, Fr.17-7-25;

[0016] (7) Subfraction Fr.17-7-12 was separated by semi-preparative reverse-phase HPLC and isocratically eluted with acetonitrile-water at a volume ratio of 45:55 to obtain compound 2;

[0017] (8) Subfraction Fr.17-7-14 was separated by semi-preparative reverse-phase high performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 45:55 to obtain compound 3.

[0018] Preferably, the extraction method in step (1) is cold soaking, percolation, microwave extraction, ultrasonic extraction or reflux extraction. More preferably, reflux extraction is performed 2 to 4 times, each extraction time is 1 to 3 hours, and the extraction temperature is 80 to 90°C.

[0019] Preferably, the ethanol solution used in step (1) is an ethanol solution with a volume concentration of 5% to 95%, more preferably a 90% ethanol solution. The amount of ethanol solution added during extraction is 3 to 50 times the volume of the medicinal material, more preferably 10 times.

[0020] Preferably, in steps (5), (7) and (8), the semi-preparative reverse-phase high performance liquid chromatography is of model YMC-PackODS-A with a size of 250×20 mm.

[0021] Preferably, in step (5), the elution flow rate is 16 mL / min, and the retention time t R =25min.

[0022] Preferably, in step (6), the reverse phase column chromatography is specifically C18 reverse phase column chromatography.

[0023] Preferably, in step (7), the elution flow rate is 12 mL / min, and the retention time t R =62min.

[0024] Preferably, in step (8), the elution flow rate is 12 mL / min, and the retention time t R =56min.

[0025] The third aspect of the present invention provides a pharmaceutical composition comprising the Ganoderma lucidum triterpenoid compound or a pharmaceutically acceptable salt thereof.

[0026] The fourth aspect of the present invention provides a pharmaceutical preparation comprising a therapeutically effective amount of a Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0027] Those skilled in the art can directly or indirectly add the compound to various commonly used pharmaceutically acceptable excipients required for preparing different dosage forms, such as fillers, disintegrants, lubricants, adhesives, etc., and use conventional pharmaceutical preparation methods to prepare commonly used oral preparations or injection preparations.

[0028] Preferably, the oral preparation is a tablet, capsule, granule, fat emulsion, microcapsule, or pill.

[0029] Preferably, the injection preparation is an injection solution or a powder injection.

[0030] The fifth aspect of the present invention provides the use of the Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating liver damage.

[0031] Preferably, the liver injury is acute liver injury or chronic liver injury.

[0032] The beneficial effects of the present invention are:

[0033] The invention discovers three Ganoderma lucidum triterpenoid compounds from the dried fruiting body of the natural product Ganoderma lucidum, and the preparation method is simple.

[0034] Based on this, the hepatocellular protective activity of the three compounds was evaluated. The results showed that the three Ganoderma triterpenoids, at 10 μM, significantly protected human HepG2 liver cancer cells damaged by APAP, with cell viability rates of 84.06%, 53.43%, and 53.54%, respectively, compared to 40.81% in the model group. Furthermore, the hepatocellular protective activity of the three Ganoderma triterpenoids was comparable to that of the positive control drug glutathione (55.30% at 40 μM), suggesting potential for further development as new drugs for the treatment of liver injury-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The chemical structural formula of the Ganoderma triterpenoid compound prepared by the present invention;

[0036] Figure 2 This is a high-resolution mass spectrum of compound 1 prepared in the present invention;

[0037] Figure 3 This is the UV spectrum of compound 1 prepared in the present invention;

[0038] Figure 4 IR spectrum of compound 1 prepared in the present invention;

[0039] Figure 5 Compound 1 prepared by the present invention 1 H NMR spectrum;

[0040] Figure 6 Compound 1 prepared by the present invention 13 C NMR spectrum;

[0041] Figure 7 HSQC spectrum of compound 1 prepared in the present invention;

[0042] Figure 8 This is the HMBC spectrum of compound 1 prepared in the present invention;

[0043] Figure 9 NOESY spectrum of compound 1 prepared in the present invention;

[0044] Figure 10 This is a high-resolution mass spectrum of compound 2 prepared in the present invention;

[0045] Figure 11 This is the UV spectrum of compound 2 prepared in the present invention;

[0046] Figure 12 IR spectrum of compound 2 prepared in the present invention;

[0047] Figure 13 Compound 2 prepared by the present invention 1 H NMR spectrum;

[0048] Figure 14 Compound 2 prepared by the present invention 13 C NMR spectrum;

[0049] Figure 15 HSQC spectrum of compound 2 prepared in the present invention;

[0050] Figure 16 This is the HMBC spectrum of compound 2 prepared in the present invention;

[0051] Figure 17NOESY spectrum of compound 2 prepared in the present invention;

[0052] Figure 18 This is a high-resolution mass spectrum of compound 3 prepared in the present invention;

[0053] Figure 19 This is the UV spectrum of compound 3 prepared in the present invention;

[0054] Figure 20 IR spectrum of compound 3 prepared in the present invention;

[0055] Figure 21 Compound 3 prepared by the present invention 1 H NMR spectrum;

[0056] Figure 22 Compound 3 prepared by the present invention 13 C NMR spectrum;

[0057] Figure 23 HSQC spectrum of compound 3 prepared in the present invention;

[0058] Figure 24 This is the HMBC spectrum of compound 3 prepared in the present invention;

[0059] Figure 25 This is the NOESY spectrum of compound 3 prepared in the present invention. DETAILED DESCRIPTION

[0060] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0061] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0062] Example 1 Preparation of Compound

[0063] This embodiment provides a method for preparing a guaiacyl-type sesquiterpene dimer compound, comprising the following steps:

[0064] (1) Take the dried fruiting body of Ganoderma lucidum, add 10 times the volume of ethanol aqueous solution (volume concentration 90%), and reflux extract at 85°C for 3 times, each extraction for 2 hours. After extraction, filter, combine the extracts, and concentrate under reduced pressure until there is no alcohol taste to obtain an extract;

[0065] (2) The extract obtained in step (1) was mixed with a macroporous resin, and subjected to macroporous resin chromatography, using an ethanol-water gradient elution with a volume ratio of 30:70, 60:40, 80:20, and 95:5 to obtain four fractions DK-1, DK-2, DK-3, and DK-4;

[0066] (3) Fraction DK-3 was subjected to silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50, and then the column was rinsed with ethyl acetate and methanol in sequence to obtain 25 fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11, Fr.12, Fr.13, Fr.14, Fr.15, Fr.16, Fr.17, Fr.18, Fr.19, Fr.20, Fr.21, Fr.22, Fr.23, Fr.24, and Fr.25;

[0067] (4) Fraction Fr.17 was chromatographed on a medium-pressure silica gel column using a gradient elution of petroleum ether and ethyl acetate in a volume ratio of 80:20, 70:30, 60:40, and 50:50, followed by rinsing the column with ethyl acetate to obtain 14 subfractions: Fr.17-1, Fr.17-2, Fr.17-3, Fr.17-4, Fr.17-5, Fr.17-6, Fr.17-7, Fr.17-8, Fr.17-9, Fr.17-10, Fr.17-11, Fr.17-12, Fr.17-13, and Fr.17-14;

[0068] (5) The subfraction Fr.17-5 was separated by semi-preparative reversed-phase HPLC YMC-Pack ODS-A (250×20 mm) and isocratically eluted with methanol-water (volume ratio 85:15) at a flow rate of 16 mL / min. R =25min to obtain compound 1;

[0069] (6) Subfraction Fr.17-7 was chromatographed on a C18 reverse phase column using a gradient elution of methanol-water with a volume ratio of 50:50, 60:40, and 75:25, and then the column was flushed with methanol to obtain 25 subfractions Fr.17-7-1, Fr.17-7-2, Fr.17-7-3, Fr.17-7-4, Fr.17-7-5, Fr.17-7-6, Fr.17-7-7, Fr.17-7-8, Fr.17-7-9, and Fr.17- 7-10, Fr.17-7-11, Fr.17-7-12, Fr.17-7-13, Fr.17-7-14, Fr.17-7-15, Fr.17-7-16, Fr.17-7-17, Fr. 17-7-18, Fr.17-7-19, Fr.17-7-20, Fr.17-7-21, Fr.17-7-22, Fr.17-7-23, Fr.17-7-24, Fr.17-7-25;

[0070] (7) The subfraction Fr.17-7-12 was separated by semi-preparative reversed-phase HPLC YMC-Pack ODS-A (250×20 mm) and isocratically eluted with acetonitrile-water (volume ratio 45:55) at a flow rate of 12 mL / min. R =62min to obtain compound 2;

[0071] (8) The subfraction Fr.17-7-14 was separated by semi-preparative reversed-phase HPLC YMC-Pack ODS-A (250×20 mm) and isocratically eluted with acetonitrile-water (volume ratio 45:55) at a flow rate of 12 mL / min. R =56min to obtain compound 3.

[0072] 1. Structural analysis and identification of compounds

[0073] The structures of compounds 1, 2 and 3 were analyzed by spectroscopy, including mass spectrometry (HR-ESI-MS), infrared spectroscopy, ultraviolet spectroscopy, nuclear magnetic resonance (NMR) and so on. 1 H-NMR, 13 C-NMR, 2D-NMR) and other methods to identify its structure and obtain its structural formula as shown in the attached figure. Figure 1 , the specific spectrum is as attached Figures 2 to 25 As shown, the spectrum data and analysis process are as follows:

[0074] 1. Compound 1 is a white amorphous powder. UV(MeOH)λ max (logε)234(4.33)nm,240(4.27)nm. IR shows 3487cm -1is the hydroxyl absorption peak, 1697 cm -1 and 1682cm -1 The absorption peak at 1642 cm -1 It is a double bond absorption peak. The mass spectrum HR-ESI-MS shows a quasi-molecular ion peak m / z 475.3181[M+Na] + (calcd.for C 30 H 44 O3Na, 475.3183), and its molecular formula is determined to be C 30 H 44 O3, unsaturation is 9.

[0075] 1 The H-NMR spectrum (Table 1) showed 43 hydrogen signals. The low field region showed one aldehyde hydrogen signal [δ H 9.43 (s, H-26)], 3 double bond hydrogen signals [δ H 6.62 (t, J = 7.6 Hz, H-24), 5.51 (br d, J = 6.7 Hz, H-7), 5.38 (br d, J = 6.2 Hz, H-11)] and one oxymethylene hydrogen signal [δ H 4.37 (2H, s, H-27)]; six methyl signals were shown in the high field region [δ H 1.20(3H,s,H-19),1.13(3H,s,H-29),1.09(3H,s,H-28),0.95(3H,d,J=6.3Hz,H-21),0.88(3H,s,H-30),0.60(3H,s,H-18)].

[0076] 13 The C-NMR spectrum (Table 1) showed 30 carbon signals, including one carbonyl carbon signal [δ C 216.8 (C-3)]; 1 aldehyde carbon signal [196.0 (C-26)]; 6 carbon signals of 3 double bonds [δ C 157.5 (C-24), 144.6 (C-9), 142.7 (C-8), 141.2 (C-25), 120.1 (C-7), 117.1 (C-11)]; 1 oxygen-linked carbon signal [δ C 56.0 (C-27)] and 6 methyl carbon signals [δ C 25.4 (C-28, C-30), 22.5 (C-19), 22.1 (C-29), 18.3 (C-21), 15.8 (C-18)], and 15 other aliphatic carbon signals [δ C50.8(C-17),50.7(C-5),50.3(C-14),47.5(C-4),43.9(C-13),37.8(C-12),37.2(C-10),36.6( C-1),36.2(C-20),35.1(C-22),34.9(C-2),31.5(C-16),28.0(C-15),26.0(C-23),23.7(C-6)].

[0077] By HR-ESI-MS, 1 H-NMR and 13 The C-NMR spectrum suggests that compound 1 is a lanostane triterpene. The NMR data of compound 1 are similar to those of the known lanostane triterpene ganoderiol F (Tsuyoshi Nishitoba, et al. Agric. Biol. Chem., 1988, 52, 367–372.), with the only difference being that the chemical shifts of H-26 in the hydrogen spectrum and C-26 in the carbon spectrum are significantly different from those of ganoderiol F. Combined with the HMBC spectrum, the H-24 (δ H 6.62) and C-26(δ C 196.0), indicating that C-26 of compound 1 is an aldehyde group, while C-26 of ganoderiol F is a hydroxymethyl group. Thus, the planar structure of compound 1 was established.

[0078] NOESY spectrum of H-24 (δ H 6.62) and H-26(δ H 9.43) and H2-23(δ H 2.36) and H2-27(δ H 4.37), demonstrating that the olefinic hydrogen at H-24 and the aldehyde hydrogen at H-26 are on the same side of the double bond, while the methylene hydrogen at H2-23 and the oxymethylene hydrogen at H2-27 are on opposite sides of the double bond. Because the two large groups, the methylene (C-23) and the aldehyde (C-26), are on opposite sides of the double bond, the C-24–C-25 double bond has an E configuration. The absolute configuration of the chiral carbon atoms in compound 1, determined based on the lanostane skeleton, is 5R, 10S, 13R, 14R, 17R, 20R.

[0079] The structure of compound 1 was determined and its structural formula is shown in Figure 1 , named (24E)-3-oxo-27-hydroxy-lanosta-7,9(11),24-trien-26-al. It was identified as a new compound by Sci-finder search.

[0080] 2. Compound 2 is a white amorphous powder. UV(MeOH)λ max (logε)203(3.76)nm,249(3.78)nm. IR shows 3392cm -1 is the hydroxyl absorption peak, 1712 cm -1 and 1655cm -1 The absorption peak indicates the presence of two carbonyl groups, 1587 cm -1 It is a double bond absorption peak. The mass spectrum HR-ESI-MS shows a quasi-molecular ion peak of m / z 471.3468 [M+H] + (calcd.for C 30 H 47 O4,471.3469), and its molecular formula was determined to be C 30 H 46 O4, unsaturation is 8.

[0081] 1 The H-NMR spectrum (Table 1) showed 44 hydrogen signals. The low field region showed one olefinic hydrogen signal [δ H 5.39 (t, J = 7.0 Hz, H-24)], one oxymethylene signal [δ H 4.00 (2H, s, H-26)] and one oxymethylene hydrogen signal [δ H 4.52 (dd, J = 9.2, 5.1 Hz, H-11)]; 7 methyl signals were shown in the high field region [δ H 1.67(3H,s,H-27),1.40(3H,s,H-19),1.15(3H,s,H-30),1.12(3H,s,H- 29), 1.11 (3H, s, H-28), 0.94 (3H, d, J = 6.5 Hz, H-21), 0.69 (3H, s, H-18)].

[0082] 13 The C-NMR spectrum (Table 1) showed 30 carbon signals, including two carbonyl carbon signals [δ C 214.5 (C-3), 199.4 (C-7)]; 4 carbon signals of 2 double bonds [δ C 158.7 (C-9), 142.2 (C-8), 134.6 (C-25), 126.8 (C-24)]; 2 oxygen-linked carbon signals [δ C 69.1 (C-26), 65.9 (C-11)] and 7 methyl carbon signals [δ C 25.4 (C-30), 25.1 (C-28), 21.6 (C-29), 19.3 (C-19), 18.5 (C-21), 17.0 (C-18), 13.7 (C-27)], and 15 other aliphatic carbon signals [δC 50.9(C-5),49.8(C-17),48.2(C-4),47.6(C-14),47.5(C-13),44.7(C-12),40.1(C-10),37.6( C-6),36.1(C-20),35.8(C-22),34.9(C-1),34.7(C-2),32.7(C-15),28.0(C-16),24.5(C-23)].

[0083] By HR-ESI-MS, 1 H-NMR and 13 The C-NMR spectrum suggests that compound 2 is a lanostane triterpene. Compound 2 is structurally similar to the known lanostane triterpene leucocontextin R (Zhen-Zhu Zhao, et al. Fitoterapia, 2016, 109: 91-98.), with the only difference being that the C-27 position of leucocontextin R is a hydroxymethyl group, while the C-27 position of compound 2 is a methyl group. This can be seen from the H-24 (δ H 5.39) and C-27(δ C 13.7) was confirmed by HMBC correlation. Thus, the planar structure of compound 2 was established.

[0084] NOESY spectrum of H-24 (δ H 5.39) and H2-26(δ H 4.00) and H2-23 (δ H 2.10) and H3-27(δ H 1.67) correlation, indicating that the H-24 olefinic hydrogen and H2-26 oxymethylene hydrogen are on the same side of the double bond, and the H2-23 methylene hydrogen and H3-27 methyl hydrogen are on the other side of the double bond. Since the two large groups, the oxymethylene (C-26) and the methylene (C-23) are on opposite sides of the double bond, the C-24–C-25 double bond is E-configuration. In addition, the NOESY correlation of H-11 (δ H 4.52) / H3-19(δ H 1.40), H3-18(δ H 0.69) confirms that the hydrogen atom at position 11 is in the β configuration, and that the 11-OH group is in the α configuration. The absolute configurations of the remaining chiral carbon atoms were determined based on the lanostane skeleton. Ultimately, the absolute configurations of compound 2 were determined to be 5R, 10S, 11R, 13R, 14R, 17R, and 20R.

[0085] The structure of compound 2 was determined and named (24E)-3,7-dioxo-lanosta-8,24-dien-11α,26-diol. Figure 1 . It was confirmed to be a new compound through Sci-finder search.

[0086] 3. Compound 3 is a white amorphous powder. UV(MeOH)λ max (logε)217(4.14)nm,255(3.81)nm. IR shows 3478cm -1 is the hydroxyl absorption peak, 1728 cm -1 、1712cm -1 and 1651cm -1 The absorption peaks indicate the presence of three carbonyl groups, 1583 cm -1 It is a double bond absorption peak. The mass spectrum HR-ESI-MS shows a quasi-molecular ion peak of m / z 529.3526 [M+H] + (calcd.for C 32 H 49 O6,529.3524), and its molecular formula was determined to be C 32 H 48 O6, unsaturation is 9.

[0087] 1 The H-NMR spectrum (Table 1) shows 46 hydrogen signals. Among them, the low field region shows one olefinic hydrogen signal [δ H 6.83 (td, J = 7.4, 1.3 Hz, H-24)] and two oxymethine signals [δ H 5.17 (dd, J = 9.6, 5.6 Hz, H-15), 3.23 (dd, J = 11.5, 4.8 Hz, H-3)]; 8 methyl signals were shown in the high field region [δ H 2.09(3H,s,H-32),1.83(3H,d,J=1.3Hz,H-27),1.24(3H,s,H-30),1.11(3H,s,H-19), 1.03(3H,s,H-28),0.92(3H,s,H-18),0.90(3H,d,J=6.5Hz,H-21),0.83(3H,s,H-29)].

[0088] 13 The C-NMR spectrum (Table 1) showed 32 carbon signals, including 3 carbonyl carbon signals [δ C 198.3 (C-11), 171.6 (C-26), 170.8 (C-31)], four carbon signals of two double bonds [δ C161.9 (C-8), 144.7 (C-24), 140.1 (C-9), 126.8 (C-25)], two oxygen-linked carbon signals [δ C 78.7 (C-3), 75.3 (C-15)] and 8 methyl carbon signals [δ C 28.3 (C-28), 21.3 (C-32), 20.1 (C-30), 19.0 (C-19), 17.9 (C-21), 17.0 (C-18), 15.7 (C-29), 12.1 (C-27)]; there are 15 other aliphatic carbon signals [δ C 52.7(C-14),51.7(C-12,C-5),48.9(C-17),46.9(C-13),39.0(C-4),37.8(C-10),35.9( C-20),35.7(C-16),34.4(C-1,C-22),29.8(C-7),28.0(C-2),25.8(C-23),17.3(C-6)].

[0089] By HR-ESI-MS, 1 H-NMR and 13 C-NMR spectrum suggests that compound 3 is a lanostane triterpene. Comparison of NMR data shows that compound 3 is structurally similar to the known lanostane triterpene resinacein C (Xin-Qiang Chen, et al. Phytochemistry, 2018, 149: 103–115.), with the difference that compound 3 has an additional acetyl group compared to resinacein C, and the chemical shift of H-15 is shifted to the downfield (δ H 4.36→5.17). Combined with HMBC spectrum, H-15 (δ H 5.17) and C-31(δ C The HMBC correlation of 170.8) indicated that the acetoxy group of compound 3 was attached to the C-15 position. Thus, the planar structure of compound 3 was established.

[0090] NOESY spectrum of H2-23(δ H 2.25) and H3-27(δ H 1.83), indicating that the methylene hydrogen of H2-23 and the methyl hydrogen of H3-27 are on the same side of the double bond. Since the two large groups, carboxyl (C-26) and methylene (C-23), are on opposite sides of the double bond, the C-24–C-25 double bond is in E configuration. H 5.17) / H3-18(δ HA correlation of 0.92 indicates that H-15 is in the β position, indicating that 15-OAc is in the α configuration. Furthermore, since the peak pattern of H-3 is dd (J = 11.5, 4.8 Hz), H-3 is in the α configuration, identical to resinacein C. The absolute configurations of the remaining chiral carbon atoms were determined based on the lanostane backbone. Ultimately, the absolute configuration of compound 3 was determined to be 3S, 5R, 10S, 13R, 14R, 15S, 17R, and 20R.

[0091] The structure of compound 3 was determined, and its structural formula is shown in Figure 1 The product was named (24E)-3β-hydroxy-11-oxo-15α-acetoxy-lanosta-8,24-dien-26-oic acid. It was identified as a new compound by Sci-finder search.

[0092] 4. The three Ganoderma triterpenoid compounds obtained by the present invention 1 H NMR and 13 C NMR data are shown in Table 1 below.

[0093] Table 1 Ganoderma lucidum triterpenoids 1 H NMR and 13 C NMR data (CDCl3, δ in ppm, J in Hz)

[0094]

[0095]

[0096] a1 H NMR 400 MHz and 13 C NMR 100MHz.

[0097] b1 H NMR 500 MHz and 13 C NMR 125MHz.

[0098] 2. Drug efficacy test research

[0099] Human hepatoma cell lines HepG2 retain the characteristics of normal human hepatocytes. By measuring the survival rate of APAP-induced damage in human hepatoma cell lines HepG2 treated with Ganoderma triterpenoids, the hepatoprotective activity of the compounds can be better evaluated.

[0100] 1. Experimental methods:

[0101] (1) Cytotoxicity detection of Ganoderma triterpenoids

[0102] Human liver cancer cells HepG2 were inoculated into 96-well cell culture plates. After culturing for 24 hours, 10 μM of the tested Ganoderma lucidum triterpenoid compounds (Compound 1, Compound 2, Compound 3 obtained in Example 1) were added. A solvent blank control group (DMSO) was set up at the same time, and 3 parallel wells were set up in each group. After the drug acted on the cells for 24 hours, the culture medium was discarded, and 100 μL of MTT (thiazolyl blue, 0.5 mg / mL) solution was added to each well. The cells were cultured for 4 hours, the MTT solution was discarded, and 150 μL of DMSO (dimethyl sulfoxide) was added to each well. The mixture was shaken on a shaker, and the absorbance was measured at a wavelength of 570 nm on a microplate reader, and the cell survival rate was calculated. This experiment was used to confirm whether the compound was cytotoxic to human liver cancer cells HepG2. The non-toxic compound was used in subsequent efficacy evaluation experiments at a concentration of 10 μM.

[0103] (2) Protective effect of Ganoderma lucidum triterpenoids on APAP-induced hepatocellular damage

[0104] Human hepatocellular carcinoma cells HepG2 were seeded in a 96-well cell culture plate. After 24 hours of culture, 10 μM of the tested Ganoderma lucidum triterpenoid compounds (Compounds 1, 2, and 3 prepared in Example 1) and APAP (final concentration 8 mM) were added to the wells. This was set as the drug-treated group. A positive drug control group (glutathione (GSH), a solvent blank control group (DMSO), and a model group were also set up. The cells were exposed to the medium for another 24 hours. The culture medium was discarded, and 100 μL of MTT solution (thiazolyl blue, 0.5 mg / mL) was added to each well. The cells were cultured for another 4 hours, the MTT solution was discarded, and 150 μL of DMSO was added to each well. The cells were mixed and shaken on a shaker. The absorbance was measured at a wavelength of 570 nm on a microplate reader, and the cell survival rate was calculated.

[0105] Cell survival rate (%) = (mean OD value of drug administration group, model group or positive drug control group / mean OD value of solvent blank control group) × 100%.

[0106] 2. Experimental results

[0107] After 24 hours of treatment with Compounds 1, 2, and 3, cell viability was greater than 90%. This indicates that these three Ganoderma triterpenoids have no significant toxicity to HepG2 cells. The protective effects of Ganoderma triterpenoids against APAP-induced hepatocellular damage are shown in Table 2.

[0108] Table 2 Protective effects of three Ganoderma triterpenoids on human hepatocellular carcinoma cells HepG2 induced by APAP

[0109]

[0110] Note: ### p<0.001, compared with the blank control group;* p<0.05, ** p<0.01, compared with the APAP model group.

[0111] As shown in Table 2, APAP (8 mM) treated human HepG2 liver cancer cells for 24 hours significantly damaged the cells, with the cell survival rate (40.81%) in the model group significantly lower than that in the blank control group. Compounds 1, 2, and 3 significantly protected human HepG2 liver cancer cells damaged by APAP at a concentration of 10 μM, with cell survival rates of 84.06%, 53.43%, and 53.54%, respectively. The positive drug GSH had a cell survival rate of 55.30% at 40 μM. These experimental results demonstrate that compounds 1, 2, and 3 have significant protective effects against human HepG2 liver cancer cells damaged by APAP, without cytotoxicity, and have promising medicinal prospects.

[0112] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof, characterized in that: The Ganoderma triterpenoid compound is Compound 1, Compound 2 or Compound 3, and its chemical structure is as follows: 。 2. A method for preparing a Ganoderma lucidum triterpenoid compound according to claim 1, characterized in that: The following steps are involved: (1) extracting the dried fruiting bodies of Ganoderma lucidum with an ethanol solution, combining the extracts, and concentrating to obtain an extract; (2) The extract obtained in step (1) was mixed with a macroporous resin, and subjected to macroporous resin chromatography, using an ethanol-water gradient elution with a volume ratio of 30:70, 60:40, 80:20, and 95:5 to obtain four fractions DK-1, DK-2, DK-3, and DK-4; (3) Fraction DK-3 was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate in a volume ratio of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50 to obtain 25 fractions: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11, Fr.12, Fr.13, Fr.14, Fr.15, Fr.16, Fr.17, Fr.18, Fr.19, Fr.20, Fr.21, Fr.22, Fr.23, Fr.24, and Fr.25; (4) Fraction Fr.17 was chromatographed on a medium-pressure silica gel column using a gradient elution of petroleum ether and ethyl acetate in a volume ratio of 80:20, 70:30, 60:40, and 50:50 to obtain 14 subfractions: Fr.17-1, Fr.17-2, Fr.17-3, Fr.17-4, Fr.17-5, Fr.17-6, Fr.17-7, Fr.17-8, Fr.17-9, Fr.17-10, Fr.17-11, Fr.17-12, Fr.17-13, and Fr.17-14; (5) Subfraction Fr.17-5 was separated by semi-preparative reverse-phase HPLC and isocratically eluted with methanol-water at a volume ratio of 85:15 to obtain compound 1; (6) Subfraction Fr.17-7 was subjected to reverse phase column chromatography and gradient elution with methanol-water in a volume ratio of 50:50, 60:40, and 75:25 to obtain 25 subfractions Fr.17-7-1, Fr.17-7-2, Fr.17-7-3, Fr.17-7-4, Fr.17-7-5, Fr.17-7-6, Fr.17-7-7, Fr.17-7-8, Fr.17-7-9, Fr.17-7-10, and Fr. .17-7-11, Fr.17-7-12, Fr.17-7-13, Fr.17-7-14, Fr.17-7-15, Fr.17-7-16, Fr.17-7-17, Fr.17- 7-18, Fr.17-7-19, Fr.17-7-20, Fr.17-7-21, Fr.17-7-22, Fr.17-7-23, Fr.17-7-24, Fr.17-7-25; (7) Subfraction Fr.17-7-12 was separated by semi-preparative reverse-phase HPLC and isocratically eluted with acetonitrile-water at a volume ratio of 45:55 to obtain compound 2; (8) Subfraction Fr.17-7-14 was separated by semi-preparative reverse-phase high performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 45:55 to obtain compound 3.

3. The method for preparing the Ganoderma lucidum triterpenoid compound according to claim 2, wherein: In steps (5), (7) and (8), the semi-preparative reverse-phase high performance liquid chromatography was of model YMC-Pack ODS-A, with a size of 250×20 mm.

4. The method for preparing Ganoderma lucidum triterpenoids according to claim 2, wherein: In step (5), the elution flow rate is 16 mL / min, and the retention time t R =25min.

5. The method for preparing Ganoderma lucidum triterpenoids according to claim 2, wherein: In step (7), the elution flow rate is 12 mL / min, and the retention time t R =62min.

6. The method for preparing Ganoderma lucidum triterpenoids according to claim 2, wherein: In step (8), the elution flow rate is 12 mL / min, and the retention time t R =56min.

7. A pharmaceutical composition, characterized in that: The invention comprises the Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1.

8. Use of the Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a drug for preventing or treating liver damage.

9. Use according to claim 8, characterized in that The liver damage is acute liver damage or chronic liver damage.

10. A pharmaceutical preparation, characterized in that: The invention comprises a therapeutically effective amount of the Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier or excipient.

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