Ganoderma triterpenoids, preparation method thereof and application of ganoderma triterpenoids in preventing and treating liver injury

Abstract

The invention belongs to the technical field of medicine, and discloses a ganoderma triterpenoid compound and a preparation method and application thereof in prevention and treatment of liver injury, the ganoderma triterpenoid compound is a compound 1, a compound 2 or a compound 3 separated from dry sporocarp of ganoderma lucidum, the dry sporocarp of ganoderma lucidum is taken to be subjected to alcohol extraction, an extracting solution is concentrated to obtain an extract, and the extract is subjected to freeze drying to obtain the ganoderma triterpenoid compound. The extract is sequentially separated through macroporous resin chromatography, silica gel column chromatography, medium-pressure silica gel column chromatography and semi-preparative reversed-phase high performance liquid chromatography to obtain a compound 1, then the compound 1 is separated through reversed-phase column chromatography, different fractions are taken and separated and purified through the semi-preparative reversed-phase high performance liquid chromatography, and a compound 2 and a compound 3 are obtained. The preparation method is simple to operate, and the ganoderma triterpenoids can be obtained through separation. Experiments show that the compound can significantly protect human hepatoma carcinoma cells HepG2 damaged by APAP at the concentration of 10 [mu] M, the cell survival rates are 84.06%, 53.43% and 53.54% respectively, and the compound can be used for preparing drugs for preventing or treating liver injury.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to ganoderic triterpenoids, a preparation method thereof, and a use for preventing and treating liver injury. Background Art

[0002] Fungi of the genus Ganoderma in the family Ganodermataceae are collectively called Ganoderma lucidum, which is a traditional Chinese medicine, and its medicinal value has been recorded in ancient books such as "Shennong Ben Cao Jing". "Chinese Pharmacopoeia" stipulates that the dried fruiting bodies of Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang are the legal medicinal materials, which are mainly used for treating restlessness, insomnia and palpitation, cough and asthma due to lung deficiency, general debility and shortness of breath, loss of appetite, etc. The main chemical components of Ganoderma lucidum include triterpenoids, diterpenoids, polysaccharides, steroids, alkaloids and sesquiterpenoids, etc. Among them, the main active ingredient - ganoderic triterpenoids has become a research hotspot because of its significant biological activities in immunomodulation, anti-tumor, liver protection, etc.

[0003] As an important organ of the human body, the liver undertakes various important physiological functions such as metabolism, detoxification, synthesis and storage. Liver injury refers to the damage of liver tissue due to various reasons, resulting in abnormal structure and function. Liver injury can be caused by a variety of factors, including viral infection, excessive alcohol, drug or poison action, fatty liver, autoimmune reaction, etc. These factors can lead to degeneration and necrosis of hepatocytes, cause liver fibrosis and cirrhosis, affect liver function, and threaten the life and health of patients.

[0004] The treatment of liver injury has always been a research hotspot in medicine, and searching for effective hepatoprotective active ingredients in natural medicines is one of the key research directions. As the main active ingredient of Ganoderma lucidum, ganoderic triterpenoids have shown potential efficacy in the prevention and treatment of liver injury in recent years, providing a new idea for the prevention and treatment of liver injury. Summary of the Invention

[0005] The purpose of the present invention is to fully develop the traditional Chinese medicine resources in our country, search for compounds with unique chemical structures, strong activities and low toxic and side effects for hepatocyte protection, so as to provide new drugs for the clinical research of preventing and treating liver injury. The present invention provides ganoderic triterpenoids, a preparation method thereof, and a use for preventing and treating liver injury. Through research, it is found that three ganoderic triterpenoids of the present invention have significant hepatocyte protection activities. So far, the above research results have not been reported in patents or literatures.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, ganoderic triterpenoids or pharmaceutically acceptable salts thereof are provided. The ganoderic triterpenoids are Compound 1, Compound 2 or Compound 3, and their chemical structural formulas are as follows:

[0008]

[0009] In the second aspect of the present invention, a preparation method of the ganoderic triterpenoids is provided, including the following steps:

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

[0011] (2) Mix the extract obtained in step (1) with macroporous resin, perform macroporous resin chromatography, and elute with ethanol-water gradients with volume ratios of 30:70, 60:40, 80:20, and 95:5 to obtain 4 fractions DK-1, DK-2, DK-3, and DK-4;

[0012] (3) Fraction DK-3 is subjected to silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradients with volume ratios of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50, and then the chromatographic column is 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 is subjected to medium-pressure silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradients with volume ratios of 80:20, 70:30, 60:40, and 50:50, and then the chromatographic column is rinsed with ethyl acetate to obtain 14 sub-fractions 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) Sub-fraction Fr.17-5 is separated by semi-preparative reverse-phase high-performance liquid chromatography and isocratically eluted with methanol-water with a volume ratio of 85:15 to obtain Compound 1;

[0015] (6) The sub-fraction Fr.17-7 was subjected to reverse-phase column chromatography and eluted with a methanol-water gradient of 50:50, 60:40, and 75:25 by volume. Then, the chromatographic column was rinsed with methanol to obtain 25 sub-fractions: 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;

[0016] (7) The sub-fraction Fr.17-7-12 was separated by semi-preparative reverse-phase high performance liquid chromatography and isocratically eluted with acetonitrile-water at a volume ratio of 45:55 to obtain Compound 2;

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

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

[0019] Preferably, the ethanol solution used in step (1) is an ethanol aqueous solution with a volume concentration of 5% to 95%, more preferably 90% ethanol aqueous solution. The addition amount of the ethanol aqueous solution 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 model of the semi-preparative reverse-phase high performance liquid chromatography is YMC-Pack ODS-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 = 25 min.

[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 = 62 min.

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

[0025] The third aspect of the present invention is to provide a pharmaceutical composition comprising the ganoderic triterpenoid compound or a pharmaceutically acceptable salt thereof.

[0026] The fourth aspect of the present invention is to provide a pharmaceutical preparation comprising a therapeutically effective amount of the ganoderic 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 various commonly used pharmaceutically acceptable excipients required for preparing different dosage forms, such as fillers, disintegrants, lubricants, binders, etc., and prepare common oral preparations or injection preparations by conventional pharmaceutical preparation methods.

[0028] Preferably, the oral preparation is tablets, capsules, granules, fat emulsions, microcapsules, or dripping pills.

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

[0030] The fifth aspect of the present invention is to provide the use of the ganoderic triterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating liver injury.

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

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

[0033] Three ganoderic triterpenoid compounds were discovered from the dried fruiting bodies of Ganoderma lucidum, a natural product, and the preparation method is simple.

[0034] On this basis, the hepatoprotective activities of the three obtained compounds were evaluated, indicating that these three ganoderic triterpenoid compounds can significantly protect human hepatocellular carcinoma cells HepG2 damaged by APAP at 10 μM, and the cell survival rates are 84.06%, 53.43% and 53.54% respectively, while the model group is 40.81%. In addition, the hepatoprotective activities of these three ganoderic triterpenoid compounds are comparable to those of the positive control drug glutathione (40 μM, 55.30%), and have potential application value for further development into new drugs for treating liver injury-related diseases. Description of the Drawings

[0035] Figure 1Chemical structural formula of ganoderic triterpenoids prepared according to the present invention;

[0036] Figure 2 High-resolution mass spectrum of compound 1 prepared according to the present invention;

[0037] Figure 3 UV spectrum of compound 1 prepared according to the present invention;

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

[0039] Figure 5 of compound 1 prepared according to the present invention 1 H NMR spectrum;

[0040] Figure 6 of compound 1 prepared according to the present invention 13 C NMR spectrum;

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

[0042] Figure 8 HMBC spectrum of compound 1 prepared according to the present invention;

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

[0044] Figure 10 High-resolution mass spectrum of compound 2 prepared according to the present invention;

[0045] Figure 11 UV spectrum of compound 2 prepared according to the present invention;

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

[0047] Figure 13 of compound 2 prepared according to the present invention 1 H NMR spectrum;

[0048] Figure 14 of compound 2 prepared according to the present invention 13 C NMR spectrum;

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

[0050] Figure 16 HMBC spectrum of compound 2 prepared according to the present invention;

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

[0052] Figure 18 High-resolution mass spectrum of compound 3 prepared according to the present invention;

[0053] Figure 19 UV spectrum of compound 3 prepared according to the present invention;

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

[0055] Figure 21 For compound 3 prepared according to the present invention 1 1H NMR spectrum;

[0056] Figure 22 For compound 3 prepared according to the present invention 13 13C NMR spectrum;

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

[0058] Figure 24 HMBC spectrum of compound 3 prepared according to the present invention;

[0059] Figure 25 NOESY spectrum of compound 3 prepared according to the present invention. Detailed implementation manners

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

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

[0062] Example 1 Preparation of compounds

[0063] This example provides a preparation method of a guaiane-type sesquiterpene dimer compound, and the steps are as follows:

[0064] (1) Take the dried fruiting bodies of Ganoderma lucidum, add an ethanol aqueous solution (volume concentration 90%) with a volume 10 times that of the raw material, reflux and extract 3 times at 85°C, each extraction for 2 h, filter after extraction, combine the extraction solutions, and concentrate under reduced pressure until the alcohol smell disappears to obtain an extract;

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

[0066] (3) Fraction DK-3 was subjected to silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradients of volume ratios 85:15, 80:20, 75:25, 70:30, 60:40, 50:50, and the chromatographic column was rinsed successively with ethyl acetate and methanol 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, Fr.25;

[0067] (4) Fraction Fr.17 was subjected to medium-pressure silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradients of volume ratios 80:20, 70:30, 60:40, 50:50, and the chromatographic column was rinsed with ethyl acetate to obtain 14 sub-fractions 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, Fr.17-14;

[0068] (5) Sub-fraction Fr.17-5 was separated by semi-preparative reversed-phase high performance liquid chromatography YMC-Pack ODS-A (250×20 mm), isocratically eluted with methanol-water of volume ratio 85:15, the elution flow rate was 16 mL / min, and compound 1 was obtained at retention time t R = 25 min;

[0069] (6) The sub-fraction Fr.17-7 was subjected to C18 reverse-phase column chromatography and eluted with a methanol-water gradient of 50:50, 60:40, and 75:25 by volume. The chromatographic column was then rinsed with methanol to obtain 25 sub-fractions: 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;

[0070] (7) The sub-fraction Fr.17-7-12 was separated by semi-preparative reverse-phase high performance liquid chromatography on a YMC-Pack ODS-A column (250×20 mm), isocratically eluted with acetonitrile-water (45:55 by volume) at a flow rate of 12 mL / min, and compound 2 was obtained at a retention time of t R = 62 min;

[0071] (8) The sub-fraction Fr.17-7-14 was separated by semi-preparative reverse-phase high performance liquid chromatography on a YMC-Pack ODS-A column (250×20 mm), isocratically eluted with acetonitrile-water (45:55 by volume) at a flow rate of 12 mL / min, and compound 3 was obtained at a retention time of t R = 56 min.

[0072] I. Structure Analysis and Identification of Compounds

[0073] Structure analysis of compound 1, compound 2, and compound 3: The structures were mainly identified using spectroscopic techniques, including mass spectrometry (HR-ESI-MS), infrared spectroscopy, ultraviolet spectroscopy, nuclear magnetic resonance ( 1 1H-NMR, 13 13C-NMR, 2D-NMR), etc. The structural formulas are as shown in the appendix Figure 1 , and the specific spectra are as shown in the appendix Figures 2 to 25 as follows. The spectral 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 3487 cm -1is the hydroxyl absorption peak, 1697 cm -1 and the absorption peaks at 1682 cm -1 suggest the presence of two carbonyl groups. The absorption peak at 1642 cm -1 is the double bond absorption peak. The HR-ESI-MS mass spectrum shows a quasi-molecular ion peak at m / z 475.3181 [M+Na] + (calcd. for C 30 H 44 O3Na, 475.3183), determining its molecular formula to be C 30 H 44 O3, with an unsaturation degree of 9.

[0075] 1 The 1H-NMR spectrum (Table 1) shows 43 hydrogen signals. In the low field region, there is 1 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 1 oxygenated methylene hydrogen signal [δ H 4.37 (2H, s, H-27)]; in the high field region, there are 6 methyl signals [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 = 6.3 Hz, H-21), 0.88 (3H, s, H-30), 0.60 (3H, s, H-18)].

[0076] 13 The 13C-NMR spectrum (Table 1) shows 30 carbon signals, including 1 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 oxygenated 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 there are 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] From HR-ESI-MS, 1 1H-NMR and 13 13C-NMR spectra, it was speculated that compound 1 was a lanostane-type triterpenoid. The NMR data of compound 1 were similar to those of the known lanostane-type triterpenoid ganoderiol F (Tsuyoshi Nishitoba, et al. Agric. Biol. Chem., 1988, 52, 367–372.), except that the chemical shifts of H-26 in the 1H spectrum and C-26 in the 13C spectrum differed significantly from those of ganoderiol F. Combining with the HMBC spectrum, H-24 (δ H 6.62) of compound 1 was correlated with C-26 (δ C 196.0), indicating that C-26 of compound 1 was an aldehyde group, while C-26 of ganoderiol F was a hydroxymethyl group. Thus, the planar structure of compound 1 was established.

[0078] In the NOESY spectrum, H-24 (δ H 6.62) was correlated with H-26 (δ H 9.43), and H2-23 (δ H 2.36) was correlated with H2-27 (δ H 4.37), proving that the vinylic hydrogen of H-24 and the aldehyde hydrogen of H-26 were on the same side of the double bond, and the methylene hydrogen of H2-23 and the oxygenated methylene hydrogen of H2-27 were on the other side of the double bond. Due to the two large groups, the methylene (C-23) and the aldehyde group (C-26) were on the opposite sides of the double bond, so the C-24–C-25 double bond was of the E configuration. The absolute configuration of the chiral carbons of compound 1 was determined according to the lanostane-type skeleton, which was 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. After searching in Sci-finder, it was determined to be a new compound.

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

[0081] 1 The 1H-NMR spectrum (Table 1) shows 44 hydrogen signals. The low field region shows 1 vinylic hydrogen signal [δ H 5.39 (t, J = 7.0 Hz, H-24)], 1 oxygenated methylene signal [δ H 4.00 (2H, s, H-26)] and 1 oxygenated methine hydrogen signal [δ H 4.52 (dd, J = 9.2, 5.1 Hz, H-11)]; the high field region shows 7 methyl signals [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 13C-NMR spectrum (Table 1) shows 30 carbon signals, including 2 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 oxygenated 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 another 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] From HR-ESI-MS, 1 H-NMR and 13 C-NMR spectra, it can be speculated that compound 2 is a lanostane-type triterpenoid. Compound 2 is structurally similar to the known lanostane-type triterpenoid leucocontextin R (Zhen-Zhu Zhao, et al. Fitoterapia, 2016, 109: 91-98.), except that the C-27 position of leucocontextin R is hydroxymethyl, while the C-27 of compound 2 is methyl. This can be proven by the HMBC correlation of H-24 (δ H 5.39) and C-27 (δ C 13.7) of compound 2. Thus, the planar structure of compound 2 is established.

[0084] In the NOESY spectrum, H-24 (δ H 5.39) is correlated with H2-26 (δ H 4.00), and H2-23 (δ H 2.10) is correlated with H3-27 (δ H 1.67), proving that the vinylic hydrogen of H-24 and the oxygenated methylene hydrogen of H2-26 are on the same side of the double bond, and the methylene hydrogen of H2-23 and the methyl hydrogen of H3-27 are on the other side of the double bond. Due to the two large groups, the oxygenated methylene (C-26) and the methylene (C-23) are on the opposite sides of the double bond, so the C-24–C-25 double bond is of the E configuration. In addition, from the NOESY correlation H-11 (δ H 4.52) / H3-19 (δ H 1.40), H3-18 (δ H 0.69), it can be determined that the hydrogen atom at the 11 position is of the β configuration, so 11-OH is of the α configuration. The absolute configurations of the remaining chiral carbons are determined according to the lanostane-type skeleton. Finally, the absolute configuration of compound 2 is determined to be 5R, 10S, 11R, 13R, 14R, 17R, 20R.

[0085] The structure of Compound 2 was determined and named as (24E)-3,7-dioxo-lanosta-8,24-dien-11α,26-diol, and its structural formula is shown in Figure 1 . Through Sci-finder search, it was determined to be a new compound.

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

[0087] 1 The 1H-NMR spectrum (Table 1) shows 46 hydrogen signals. Among them, in the low-field region, there is 1 vinylic hydrogen signal [δ H 6.83 (td, J = 7.4, 1.3 Hz, H-24)] and 2 oxygenated methine 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)]; in the high-field region, there are 8 methyl signals [δ H 2.09 (3H, s, H-32), 1.83 (3H, d, J = 1.3 Hz, 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 = 6.5 Hz, H-21), 0.83 (3H, s, H-29)].

[0088] 13 The 13C-NMR spectrum (Table 1) shows 32 carbon signals, including 3 carbonyl carbon signals [δ C 198.3 (C-11), 171.6 (C-26), 170.8 (C-31)], 4 carbon signals of 2 double bonds [δ C[161.9 (C-8), 144.7 (C-24), 140.1 (C-9), 126.8 (C-25)], two oxygenated carbon signals [δ C 78.7 (C-3), 75.3 (C-15)] and eight 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 also 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] From HR-ESI-MS, 1 H-NMR and 13 C-NMR spectra, it can be deduced that compound 3 is a lanostane-type triterpenoid. Comparing the NMR data, compound 3 is similar in structure to the known lanostane-type triterpenoid resinacein C (Xin-Qiang Chen, etal. Phytochemistry, 2018, 149: 103–115.), except that compound 3 has an additional acetoxy group compared to resinacein C, and the chemical shift of H-15 moves downfield (δ H 4.36 → 5.17). Combining with the HMBC spectrum, the HMBC correlation between H-15 (δ H 5.17) and C-31 (δ C 170.8) indicates that the acetoxy group of compound 3 is attached to C-15. Thus, the planar structure of compound 3 is established.

[0090] In the NOESY spectrum, the correlation between H2-23 (δ H 2.25) and H3-27 (δ H 1.83) proves that the methylene hydrogen of H2-23 and the methyl hydrogen of H3-27 are on the same side of the double bond. Due to the two large groups, the carboxyl group (C-26) and the methylene group (C-23) are on the opposite side of the double bond. Therefore, the C-24–C-25 double bond is of the E configuration. From the H-15 (δ H 5.17) / H3-18 (δ H0.92), it can be determined that H-15 is in the β position, so 15-OAc is in the α configuration. In addition, since the peak pattern of H-3 is dd (J = 11.5, 4.8 Hz), H-3 is in the α configuration, which is the same as that of resinacein C. The absolute configurations of the remaining chiral carbons are determined according to the lanostane skeleton. Finally, the absolute configuration of compound 3 is determined to be 3S, 5R, 10S, 13R, 14R, 15S, 17R, 20R.

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

[0092] 4. The 1 H NMR and 13 C NMR data of the three ganoderic triterpenoids obtained in the present invention are shown in Table 1 below.

[0093] Table 1 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 100 MHz.

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

[0098] II. Pharmacodynamic experiment research

[0099] Human hepatoma cell line HepG2 well retains the characteristics of normal human hepatocytes. By detecting the survival rate of APAP-induced damaged human hepatoma cell line HepG2 treated with ganoderic triterpenoids, the hepatoprotective activity of the compounds can be better evaluated.

[0100] 1. Experimental method:

[0101] (1) Detection of cytotoxicity of ganoderic triterpenoids

[0102] Human hepatoma cells HepG2 were inoculated into 96-well cell culture plates. After culturing for 24 h, 10 μM of the ganoderic triterpenoid compounds to be tested (Compound 1, Compound 2, and Compound 3 prepared in Example 1) were added. At the same time, a solvent blank control group (DMSO) was set, and each group had 3 parallel wells. After the drugs acted on the cells for 24 h, the culture medium was discarded. 100 μL of MTT (thiazole blue, 0.5 mg / mL) solution was added to each well, and the cells were cultured for another 4 h. Then the MTT solution was discarded, and 150 μL of DMSO (dimethyl sulfoxide) was added to each well. The mixture was shaken on a mixing oscillator, and the absorbance value was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. This experiment was used to confirm whether the compounds had cytotoxicity to human hepatoma cells HepG2. Non-toxic compounds were used in the subsequent pharmacodynamic evaluation experiment at a concentration of 10 μM.

[0103] (2) Protective effect of ganoderic triterpenoid compounds on hepatocyte injury induced by acetaminophen (APAP)

[0104] Human hepatoma cells HepG2 were inoculated into 96-well cell culture plates. After culturing for 24 h, 10 μM of the ganoderic triterpenoid compounds to be tested (Compound 1, Compound 2, and Compound 3 prepared in Example 1) and APAP (final concentration 8 mM) were added, which was set as the drug administration group. At the same time, a positive drug control group of glutathione (GSH), a solvent blank control group (DMSO), and a model group were set. The cells were continuously treated for 24 h. The culture medium was discarded. 100 μL of MTT solution (thiazole blue, 0.5 mg / mL) was added to each well, and the cells were cultured for another 4 h. Then the MTT solution was discarded, and 150 μL of DMSO was added to each well. The mixture was shaken on a mixing oscillator. The absorbance value was measured at a wavelength of 570 nm using an ELISA reader, and the cell survival rate was calculated.

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

[0106] 2. Experimental results

[0107] After Compound 1, Compound 2, and Compound 3 acted on human hepatoma cells HepG2 for 24 h, the cell survival rate was greater than 90%. This indicated that these three ganoderic triterpenoid compounds had no obvious toxicity to human hepatoma cells HepG2. The results of the protective effect of ganoderic triterpenoid compounds on hepatocyte injury induced by acetaminophen (APAP) are shown in Table 2.

[0108] Table 2 Protective effect of three ganoderic triterpenoid compounds on human hepatoma cells HepG2 damaged 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 can be seen from Table 2, when APAP (8 mM) acts on human hepatocarcinoma cells HepG2 for 24 h, it causes significant damage to the cells, and the cell survival rate of the model group (40.81%) is significantly lower than that of the blank control group. Compounds 1, 2, and 3 can significantly protect human hepatocarcinoma cells HepG2 damaged by APAP at a concentration of 10 μM, and the cell survival rates are 84.06%, 53.43%, and 53.54% respectively. The cell survival rate of the positive drug GSH is 55.30% at 40 μM. The experimental results show that Compounds 1, 2, and 3 have significant protective effects on human hepatocarcinoma cells HepG2 damaged by APAP, have no cytotoxicity, and have good medicinal prospects.

[0112] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof, characterized in that: The Ganoderma lucidum triterpenoid compound is compound 1, compound 2 or compound 3, and its chemical structure is as follows: 。 2. A method for preparing triterpenoid compounds of Ganoderma lucidum according to claim 1, characterized in that: The following steps are involved: (1) extracting 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, and eluted with an ethanol-water gradient 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 eluted 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 subjected to medium pressure silica gel column chromatography and gradient eluted with petroleum ether-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) The subfraction Fr.17-5 was separated by semi-preparative reverse-phase high performance liquid chromatography and isocratically eluted with methanol-water in a volume ratio of 85:15 to obtain compound 1; (6) Subfraction Fr.17-7 was subjected to reverse phase column chromatography and gradient eluted 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. .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) The subfraction Fr.17-7-12 was separated by semi-preparative reverse-phase HPLC and isocratically eluted with acetonitrile-water in a volume ratio of 45:55 to obtain compound 2; (8) Subfraction Fr.17-7-14 was separated by semi-preparative reverse-phase HPLC and isocratically eluted with acetonitrile-water in a volume ratio of 45:55 to obtain compound 3.

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

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

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

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

7. A pharmaceutical composition, characterized in that: The invention comprises the Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof as described in 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. The 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: It comprises a therapeutically effective amount of the Ganoderma triterpenoid compound or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable carrier or excipient.

Images