Triterpenoid Compounds in Ganoderma sinense Fermented Mycelium and Their Anti-Inflammatory Applications

Abstract

The present invention provides triterpenoid compounds in Ganoderma sinense fermented mycelia. The structural formulas of the triterpenoid compounds are Structural Formulas 1, 3, 4, and 7. The present invention also provides the application of the triterpenoid compounds in Ganoderma sinense fermented mycelia as a drug for preventing or treating inflammation; the triterpenoid compounds in the Ganoderma sinense mycelia are one or more of the compounds with Structural Formulas 1-8. The present invention for the first time conducts an anti-inflammatory activity test on the triterpenoid compounds isolated and purified from Ganoderma sinense mycelia, and finds that the compounds with Structural Formulas 1-8 can all inhibit the release of TNF-α and IL-6, and have significant anti-inflammatory activity.

Description

Technical Field

[0001] The present invention relates to the field of edible mushroom applications, and specifically to triterpenoid compounds in Ganoderma sinense fermented mycelium and their anti-inflammatory applications. Background Art

[0002] "Ganoderma lucidum" is a general term for fungi of the genus Ganoderma P.Karst., belonging to the Basidiomycota phylum, Agaricomycetes class, Polyporales order, and Ganodermataceae family. Ganoderma sinense J.D.Zhao, L.W.Hsu & X.Q.Zhang is a type of Ganoderma lucidum that can be used as a medicine as specified in the Chinese Pharmacopoeia (Chinese Pharmacopoeia, 2020 Edition). Currently, due to limitations such as few commercial varieties and low strain resistance, the yield of artificially cultivated Ganoderma sinense in China is far lower than that of Ganoderma lucidum. Compared with the cultivation of fruiting bodies, the fermentation of Ganoderma sinense has the characteristics of a short production cycle, controllable conditions, and unique fermentation products. In addition, the fermented mycelium of Ganoderma sinense is rich in triterpenoid active compounds.

[0003] Inflammation is a physiological defense response of living tissues to harmful stimuli such as pathogens, damaged cells, or irritants. When the body is stimulated by inflammatory factors, it will activate macrophages in the body to synthesize and release various inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), etc., increase vascular permeability, and promote the chemotaxis of inflammatory cells, thereby inducing the occurrence of inflammation.

[0004] Searching for anti-inflammatory drugs or lead compounds from the fermentation products of Ganoderma sinense will be a promising direction for the development of Ganoderma sinense resources. Summary of the Invention

[0005] The present invention first provides a triterpenoid compound in Ganoderma sinense fermented mycelium, which is a compound represented by Structural Formulas 1, 3, 4, and 7 below or its medicinal salt, crystal, or hydrate:

[0006]

[0007]

[0008] The present invention also provides a preparation for anti-inflammatory, which contains one or more compounds represented by Structural Formulas 1-8 or their medicinal salts and a pharmaceutically acceptable carrier;

[0009] Wherein the compounds of Structural Formulas 1-8 are:

[0010]

[0011]

[0012] The present invention also provides the use of triterpenoid compounds in Ganoderma sinense fermented mycelia as a drug for preventing or treating inflammation; wherein the triterpenoid compounds in Ganoderma sinense fermented mycelia are one or more of the compounds with the following structural formulas 1-8:

[0013] Wherein the compounds with structural formulas 1-8 are:

[0014]

[0015]

[0016] The chemical names of the above compounds with structural formulas 1-8 are as follows:

[0017] Compound 1: (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid

[0018] Compound 2: (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid

[0019] Compound 3: (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid

[0020] Compound 4: (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic

[0021] acid

[0022] Compound 5: (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid

[0023] Compound 6: (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid

[0024] Compound 7: (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid

[0025] Compound 8: lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid

[0026] The compounds of Structural Formulas 1-8 provided by the present invention can also be used as preparations or foods for preventing and / or treating inflammation or assisting in the treatment process of inflammation during or after the treatment.

[0027] All eight triterpenoid compounds isolated from Ganoderma sinense mycelium provided by the present invention have significant anti-inflammatory effects. In particular, Compounds 2, 7, and 8 have prominent anti-inflammatory effects and have broad market application prospects. Description of the Drawings

[0028] Figure 1 Semi-preparative liquid chromatography diagram of Fr3

[0029] Figure 2 Semi-preparative liquid chromatography diagram of Fr5

[0030] Figure 3 Semi-preparative liquid chromatography diagram of Fr8 Detailed Embodiments

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0032] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0033] Ganoderma sinense (strain number: Dai20076), and the strain is currently preserved in the Institute of Microbiology, School of Ecology and Nature Conservation, Beijing Forestry University.

[0034] Mouse mononuclear macrophage RAW264.7 was purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and the product catalog number is SCSP-5036.

[0035] DMEM medium, fetal bovine serum (FBS): purchased from Gibco, USA;

[0036] Penicillin and streptomycin: purchased from Amresco, USA;

[0037] Mouse-derived TNF-α and IL-6 kits: purchased from Beijing Sizhengbai Biotechnology Co., Ltd.;

[0038] Glucose Potato Agar (PDA): Purchased from Beijing Solarbio Science & Technology Co., Ltd.;

[0039] Glucose, sucrose, agar, MgSO4·H2O, KH2PO4, CaSO4, and analytical grade methanol, chloroform, petroleum ether, acetone, ethanol, ethanol, acetonitrile, glacial acetic acid: All purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0040] Chromatographic grade methanol, acetonitrile: Purchased from Shanghai ANPEL Laboratory Technologies Inc.;

[0041] Silymarin, deuterated methanol, deuterated chloroform: Purchased from Sigma;

[0042] YMC*GEL C18 silica gel (50μm, 12nm): Beijing Yuanbaoshan Chromatography Technology Co., Ltd.;

[0043] Zorbax Eclipse Plus C18 chromatographic column (5μm, 4.6mm×250mm): Agilent Technologies, USA;

[0044] Zorbax Eclipse Plus-C18 semi-preparative chromatographic column (5μm, 21.2mm×250mm): Agilent Technologies, USA;

[0045] GF254 silica gel plate (0.2 - 0.25mm): Qingdao Marine Chemical Factory;

[0046] LC3000 high performance liquid chromatograph: Beijing Innovation Tongheng Technology Co., Ltd.;

[0047] Agilent LC1290 infinity II ultra-high performance liquid chromatograph: Agilent Technologies, USA;

[0048] Agilent 6495 triple quadrupole mass spectrometer: Agilent Technologies, USA;

[0049] Bruker AB II-600 nuclear magnetic resonance spectrometer: Bruker, Germany;

[0050] Synergy HT multi-functional microplate reader: BIO-TEK Instruments, Inc., USA;

[0051] Thermo Scientific Q Exactive HF Orbitrap-FTMS high resolution mass spectrometer: Thermo Fisher Scientific, USA

[0052] Example 1: Fermentation of Ganoderma sinense mycelium

[0053] Culture medium formula: soluble starch 3%, glucose 1.2%, yeast powder 1%, MgSO4·7H2O 0.15%, KH2PO4 0.3% (the above are all weight percentages), natural pH.

[0054] The strains stored in a 4°C refrigerator were activated and subjected to first-stage shaking culture in a shake flask for 7 days (150 r / min, 25°C) under dark conditions, and then transferred to a second-stage shake flask for static culture for 21 days to obtain mycelia.

[0055] Example 2 Extraction and extraction of Ganoderma sinense mycelia

[0056] The dried mycelia were soaked and extracted 3 times with an aqueous solution of 95% ethanol at room temperature for 24 hours each time. The extraction solutions were combined and concentrated to obtain an ethanol extract of Ganoderma sinense mycelia.

[0057] Furthermore, the ethanol extract of Ganoderma sinense mycelia was extracted three times with an equal volume of petroleum ether. The petroleum ether phase was discarded, and the remaining phase was then extracted three times with an equal volume of ethyl acetate. The ethyl acetate extraction solutions were combined and concentrated under reduced pressure to dryness to obtain the ethyl acetate phase.

[0058] Example 3 Preparation of compounds

[0059] The ethyl acetate phase was separated by medium-pressure chromatography using YMC C18 (50 μm, 12 nm) as the packing material, and gradient elution was performed with acetonitrile (A)-0.01% acetic acid in water (0 - 15 min, A: 55% - 55%; 15 - 85 min, A: 55% - 75%; 85 - 105 min, A: 75% - 85%; 105 - 115 min, A: 85% - 100%; 115 - 135 min, A: 100% - 100%). The flow rate was 200 ml / min, and one fraction was collected every 500 mL. A total of 60 fractions were collected, numbered in the order of collection and subjected to thin-layer chromatography analysis. The fractions with the same retention factor (Rf) on the thin-layer chromatography plate were combined to obtain Fr1 - Fr13.

[0060] After thin-layer chromatography analysis of the 60 fractions, they were combined into 13 components: Fr1 (1 - 10, 5.02 g), Fr2 (11 - 14, 1.14 g), Fr3 (15 - 16, 1.65 g), Fr4 (17 - 18, 2.68 g), Fr5 (19 - 20, 3.89 g), Fr6 (21 - 22, 3.95 g), Fr7 (23 - 24, 4.3 g), Fr8 (25 - 33, 24.88 g), Fr9 (34 - 36, 2.78 g), Fr10 (36 - 38, 2.46 g), Fr11 (39 - 41, 1.33 g), Fr12 (42 - 46, 1.72 g), Fr13 (47 - 60, 3.17 g).

[0061] Perform HPLC detection on Fr1 - Fr13. According to the liquid phase detection results, it can be seen that Fr3, Fr5, and Fr8 are rich in characteristic absorption peaks of triterpenoid compounds. Therefore, Fr3, Fr5, and Fr8 are further separated and purified.

[0062] Separation and purification of Fr3

[0063] Select a Zorbax Eclipse Plus - C18 semi - preparative chromatographic column, and perform gradient elution with acetonitrile (A) - 0.01% glacial acetic acid water (0.0 - 18.0 min, A: 50% - 50%; 18.0 - 18.5 min, A: 50% - 100%; 18.5 - 23.0 min, A: 100% - 100%), and detect at a wavelength of 240 nm. Repeat the preparation multiple times, collect the chromatographic peak at 15.5 min, and obtain Fr3 - 8.

[0064] For Fr3 - 8, select a Zorbax Eclipse Plus - C18 semi - preparative chromatographic column, and perform gradient elution with acetonitrile (A) - 0.01% glacial acetic acid water (0.0 - 12.0 min, A: 52% - 52%; 12.0 - 12.5 min, A: 52% - 75%; 12.5 - 20.0 min, A: 75% - 75%; 20.0 - 21.0 min, A: 75% - 100%; 21.0 - 26.0 min, A: 100% - 100%), and detect at a wavelength of 240 nm. Repeat the preparation multiple times, collect the chromatographic peak at 8.2 min, and obtain Compound 1 (100.00 mg); collect the chromatographic peak at 18.0 min, and obtain Compound 2 (f140.00 mg).

[0065] Separation and purification of Fr5

[0066] For Fr5, select a Zorbax Eclipse Plus - C18 semi - preparative chromatographic column, and perform gradient elution with acetonitrile (A) - 0.01% glacial acetic acid water (0.0 - 19.0 min, A: 80% - 80%; 19.0 - 19.5 min, A: 80% - 83%; 19.5 - 25.0 min, A: 83% - 83%; 25.0 - 25.5 min, A: 83% - 100%; 25.5 - 30.0 min, A: 100% - 100%), and detect at a wavelength of 240 nm. Collect the chromatographic peak at 15.0 min, and obtain Fr5 - 4; collect the chromatographic peak at 20.8 min, and obtain Compound 3 (101.00 mg), collect the chromatographic peak at 23.0 min, and obtain Fr5 - 6.

[0067] For Fr5-4, a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column was selected, and gradient elution was carried out with acetonitrile (A)-0.01% acetic acid in water (0.0 - 18.0 min, A: 53% - 53%; 18.0 - 18.5 min, A: 53% - 79%; 18.5 - 27.0 min, A: 79% - 79%; 27.0 - 28.0 min, A: 79% - 100%; 28.0 - 32.0 min, A: 100% - 100%). Detection was carried out at a wavelength of 240 nm. After repeated preparation for many times, the chromatographic peak at 12.2 min was collected to obtain Compound 4 (100.00 mg), and the chromatographic peak at 25.0 min was collected to obtain Compound 5 (110.00 mg).

[0068] For Fr5-6, a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column was selected, and gradient elution was carried out with acetonitrile (A)-0.01% acetic acid in water (0.0 - 17.0 min, A: 89% - 89%; 17.0 - 17.5 min, A: 89% - 100%; 17.5 - 22.0 min, A: 100% - 100%). Detection was carried out at a wavelength of 240 nm. After repeated preparation for many times, the chromatographic peak at 15.0 min was collected to obtain Compound 6 (280.00 mg).

[0069] Separation and purification of Fr8

[0070] For Fr8, a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column was selected, and gradient elution was carried out with acetonitrile (A)-0.01% acetic acid in water (0.0 - 20.0 min, A: 80% - 80%; 20.0 - 20.5 min, A: 80% - 85%; 20.5 - 32.0 min, A: 85% - 85%; 32.00 - 40.5 min, A: 85% - 100%; 40.50 - 45.0 min, A: 100% - 100%). Detection was carried out at a wavelength of 240 nm. After repeated preparation for many times, the chromatographic peak at 35.0 min was collected to obtain Compound 7 (612.00 mg), and the chromatographic peak at 40.0 min was collected to obtain Compound 8 (109.00 mg).

[0071] Example 3 Confirmation of Compounds 1 - 8

[0072] By means of mass spectrometry and nuclear magnetic resonance detection, etc., the structures of 8 compounds obtained from Ganoderma lucidum mycelium were analyzed to determine their precise chemical structures. The confirmation information of the specific eight compounds is as follows:

[0073] Compound 1 [(22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid]

[0074] The molecular formula is C 30 H 44 O6, a pale yellow amorphous powder, soluble in methanol and chloroform. HRTOFMS m / z: 501.3216 [M+H] + (C 30 H 45 O6, calculated value 501.3211). 1 1H-NMR (CD3OD, 500 MHz) δ: 6.83 (1H, m, H-24), 4.24 (1H, dd, J = 5.0, 10.0 Hz, H-15), 3.75 (1H, m, H-22), 2.80 (1H, m, H-2), 2.77 (1H, m, H-6), 2.50 (2H, m, H-11), 2.40 (2H, m, H-2, 6), 2.28 (1H, m, H-23), 2.19 (1H, m, H-1), 2.18 (1H, m, H-5), 2.05 (1H, m, H-17), 2.04 (1H, m, H-16), 1.95 (1H, m, H-12), 1.92 (1H, m, H-16), 1.84 (1H, m, H-1), 1.83 (1H, m, H-23), 1.83 (3H, s, H-27), 1.81 (1H, m, H-12), 1.41 (1H, m, H-20), 1.40 (3H, s, H-19), 1.14 (3H, s, H-29), 1.08 (3H, s, H-28), 0.95 (3H, s, H-30), 0.92 (3H, d, J = 5.0 Hz, H-21), 0.78 (3H, s, H-18); 1313C-NMR(CD3OD, 125 MHz) δ: 217.0 (C-3), 203.6 (C-7), 171.9 (C-26), 171.3 (C-9), 141.2 (C-24), 140.1 (C-8), 130.5 (C-25), 74.0 (C-15), 73.4 (C-22), 53.0 (C-14), 51.7 (C-5), 48.5 (C-4), 47.2 (C-13), 46.4 (C-17), 42.7 (C-20), 41.5 (C-10), 38.1 (C-6), 37.6 (C-16), 36.4 (C-1), 36.0 (C-23), 35.6 (C-2), 32.3 (C-12), 25.8 (C-28), 25.5 (C-11), 22.2 (C-29), 19.0 (C-30), 16.9 (C-18), 18.1 (C-19), 13.1 (C-27), 12.5 (C-21).

[0075] Compound 2 [(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid]

[0076] Molecular formula C 30 H 44 O5, pale yellow amorphous powder, soluble in methanol. Negative ion ESI MS m / z: 483.0 [M-H] – . 1H-NMR(CD3OD, 500 MHz) δ: 6.83 (1H, m, H-24), 5.95 (1H, d, J = 10.0 Hz, H-7), 5.46 (1H, d, J = 5.0 Hz, H-11), 4.24 (1H, dd, J = 5.0, 10.0 Hz, H-15), 3.75 (1H, m, H-22), 2.85 (1H, m, H-2), 2.43 (1H, m, H-23), 2.35 (2H, m, H-1, 12), 2.28 (2H, m, H-2, 23), 2.23 (1H, m, H-6), 2.12 (1H, m, H-6), 2.09 (2H, m, H-12, 16), 2.09 (1H, m, H-17), 1.89 (1H, m, H-16), 1.83 (3H, s, H-27), 1.71 (1H, m, H-1), 1.54 (1H, dd, J = 5.0, 10.0 Hz, H-5), 1.38 (1H, m, H-20), 1.22 (3H, s, H-19), 1.13 (3H, s, H-29), 1.07 (3H, s, H-28), 0.96 (3H, s, H-30), 0.90 (3H, d, J = 5.0 Hz, H-21), 0.67 (3H, s, H-18). 13 C-NMR(CD3OD, 125 MHz) δ: 219.4 (C-3), 171.8 (C-26), 146.5 (C-9), 142.9 (C-8), 141.3 (C-24), 130.4 (C-25), 122.5 (C-7), 118.5 (C-11), 75.3 (C-15), 73.6 (C-22), 53.5 (C-14), 52.5 (C-5), 48.8 (C-4), 46.7 (C-17), 45.5 (C-13), 42.5 (C-20), 40.0 (C-11, 16), 38.8 (C-10), 38.1 (C-1), 36.0 (C-2, 23), 26.2 (C-28), 24.9 (C-6), 23.2 (C-29), 22.8 (C-19), 18.2 (C-30), 16.8 (C-18), 13.1 (C-27), 12.5 (C-21).

[0077] Compound 3 [(22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid]

[0078] The molecular formula is C 32 H 48O6, a pale yellow amorphous powder, soluble in methanol. HRTOFMS m / z: 551.3350 [M+Na] + (C 32 H 48 O6Na, calculated value 551.3343). 1 1H-NMR (CD3OD, 500 MHz) δ: 6.83 (1H, m, H-24), 5.91 (1H, d, J = 10.0 Hz, H-7), 5.37 (1H, d, J = 5.0 Hz, H-11), 4.45 (1H, m, H-3), 4.23 (1H, dd, J = 10.0, 10.0 Hz, H-15), 3.73 (1H, m, H-22), 2.41 (1H, m, H-23), 2.35 (1H, m, H-12), 2.28 (1H, m, H-23), 2.13 (1H, m, H-6), 2.08 (1H, m, H-17), 2.07 (3H, m, H-6, 12, 16), 2.04 (1H, m, H-1), 2.04 (3H, s, OCOCH3-3), 1.87 (1H, m, H-16), 1.83 (3H, s, H-27), 1.72 (1H, m, H-2), 1.47 (2H, m, H-1, 2), 1.37 (1H, m, H-20), 1.18 (1H, m, H-5), 1.03 (3H, s, H-19), 0.99 (3H, s, H-29), 0.97 (3H, s, H-30), 0.90 (3H, s, H-28), 0.90 (3H, d, J = 5.0 Hz, H-21), 0.64 (3H, s, H-18). 13 13C-NMR (CD3OD, 125 MHz) δ: 173.0 (OCOCH3-3), 171.9 (C-26), 147.6 (C-9), 142.7 (C-8), 141.3 (C-24), 130.4 (C-25), 122.5 (C-7), 117.7 (C-11), 82.6 (C-3), 75.4 (C-15), 73.6 (C-22), 53.6 (C-14), 51.0 (C-5), 46.7 (C-17), 45.6 (C-13), 42.5 (C-20), 40.1 (C-16), 40.0 (C-12), 38.9 (C-4), 38.8 (C-10), 36.9 (C-1), 36.0 (C-23), 28.9 (C-28), 25.5 (C-2), 24.1 (C-6), 23.7 (C-19), 21.4 (OCOCH3-3), 18.3 (C-30), 17.7 (C-29), 16.8 (C-18), 13.1 (C-27), 12.5 (C-21).

[0079] Compound 4 [(22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid]

[0080] The molecular formula is C 34 H 44 O8, a yellow crystal, soluble in methanol. HRTOFMS m / z: 557.3114 [M+H] + (C 32 H 45 O8, calculated value 557.3109). 1 1H-NMR (CD3OD, 500 MHz) δ: 6.68 (1H, m, H-24), 5.07 (1H, m, H-22), 4.33 (1H, m, H-15), 2.93 (1H, m, H-12), 2.87 (1H, m, H-1), 2.80 (1H, m, H-6), 2.68 (1H, m, H-2), 2.54 (2H, m, H-12, 23), 2.47 (1H, m, H-6), 2.45 (1H, m, H-2), 2.40 (1H, m, H-23), 2.39 (1H, m, H-5), 2.04 (3H, s, OCOCH3-22), 1.98 (1H, m, H-17), 1.97 (1H, m, H-16), 1.86 (1H, m, H-1), 1.84 (3H, s, H-27), 1.62 (1H, m, H-20), 1.27 (3H, s, H-19), 1.16 (3H, s, H-30), 1.12 (3H, s, H-28), 1.11 (3H, s, H-29), 0.99 (1H, m, H-16), 0.99 (3H, d, J = 5.0 Hz, H-21), 0.91 (3H, s, H-18). 1313C-NMR(CD3OD, 125 MHz) δ: 218.3 (C-3), 206.6 (C-7), 202.5 (C-11), 171.2 (C-26), 153.9 (C-9), 152.0 (C-8), 138.3 (C-24), 131.5 (C-25), 75.9 (C-22), 73.5 (C-15), 54.0 (C-14), 52.8 (C-12), 50.1 (C-5), 48.7 (C-13), 47.8 (C-4), 46.3 (C-17), 40.6 (C-20), 40.4 (C-10), 37.7 (C-6), 36.8 (C-16), 36.3 (C-1), 35.0 (C-2), 32.5 (C-23), 27.6 (C-28), 20.7 (C-29), 20.4 (C-30), 18.2 (C-19), 17.5 (C-18), 13.1 (C-21), 12.7 (C-27).

[0081] Compound 5 [(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid]

[0082] The molecular formula is C 32 H 46 O6, a pale yellow amorphous powder, soluble in methanol. Negative ion ESI MS m / z: 525.1 [M-H] – . 1H-NMR(CD3OD,500MHz)δ:6.69(1H,m,H-24),5.95(1H,d,J=10.0Hz,H-7),5.45(1H,d,J=5.0Hz,H-11),5.07(1H,m,H-22),4.24(1H,d,J=5.0,10.0Hz,H-15),2.84(1H,m,H-2),2.57(1H,m,H-23),2.40(1H,m,H-23),2.34(1H,m,H-1),2.33(1H,m,H-12),2.27(1H,m,H-2),2.23(1H,m,H-6),2.11(2H,m,H-6),2.10(1H,m,H-12),2.04(3H,s,OCOCH3-22),1.95(1H,m,H-16),1.86(1H,m,H-16),1.84(1H,s,H-27),1.80(1H,m,H-17),1.71(1H,m,H-1),1.55(1H,m,H-20),1.53(1H,m,H-5),1.22(3H,s,H-19),1.13(3H,s,H-29),1.06(3H,s,H-28),1.00(3H,d,J=5.0Hz,H-21),0.91(3H,s,H-30),0.67(3H,s,H-18). 13 C-NMR(CD3OD,125MHz)δ:219.1(C-3),171.2(C-26),146.3(C-9),142.4(C-8),138.5(C-24),131.3(C-25),122.5(C-7),118.1(C-11),76.2(C-22),74.9(C-15),53.2(C-14),52.2(C-5),48.6(C-4),46.8(C-17),45.3(C-13),40.6(C-20),40.0(C-16),39.7(C-12),38.5(C-10),37.9(C-1),35.8(C-2),32.6(C-23),25.9(C-28),24.6(C-6),22.9(C-29),22.5(C-1),17.9(C-30),16.4(C-18),13.2(C-21),12.7(C-27).

[0083] Compound 6 [(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]

[0084] The molecular formula is C34 H 48 O7, a white solid, soluble in methanol. Negative ion ESI MS m / z: 567.1 [M-H] – . 1 H-NMR (CD3OD, 500 MHz) δ: 6.67 (1H, m, H-24), 5.58 (1H, d, J = 5.0 Hz, H-7), 5.49 (1H, d, J = 5.0 Hz, H-11), 5.07 (1H, dd, J = 5.0, 10.0 Hz, H-15), 5.03 (1H, m, H-22), 2.84 (1H, m, H-2), 2.56 (1H, m, H-23), 2.39 (1H, m, H-23), 2.36 (1H, m, H-16), 2.35 (1H, m, H-12), 2.29 (1H, m, H-2), 2.23 (1H, m, H-6), 2.15 (1H, m, H-12), 2.14 (1H, m, H-16), 2.11 (1H, m, H-6), 2.07 (3H, s, OCOCH3-15), 2.04 (3H, s, OCOCH3-22), 1.84 (3H, s, H-27), 1.83 (1H, m, H-17), 1.82 (1H, m, H-1), 1.70 (1H, m, H-1), 1.61 (1H, m, H-20), 1.52 (1H, dd, J = 5.0, 10.0 Hz, H-5), 1.22 (3H, s, H-19), 1.13 (3H, s, H-29), 1.06 (3H, s, H-28), 1.01 (3H, d, J = 5.0 Hz, H-21), 1.01 (3H, s, H-30), 0.72 (3H, s, H-18). 1313C-NMR(CD3OD, 125 MHz) δ: 218.9 (C-3), 173.0 (OCOCH3-15), 172.6 (OCOCH3-22), 171.5 (C-26), 146.0 (C-9), 141.7 (C-8), 138.2 (C-24), 131.6 (C-25), 122.5 (C-7), 118.4 (C-11), 78.4 (C-15), 76.1 (C-22), 52.7 (C-14), 52.1 (C-5), 48.5 (C-4), 46.9 (C-17), 45.1 (C-13), 40.8 (C-20), 39.2 (C-12), 38.5 (C-10), 37.8 (C-16), 37.7 (C-1), 35.7 (C-2), 32.6 (C-23), 25.9 (C-28), 24.6 (C-6), 22.9 (C-29), 22.5 (C-19), 21.2 (OCOCH3-15), 20.9 (OCOCH3-22), 18.8 (C-30), 16.3 (C-18), 13.1 (C-21), 12.8 (C-27).

[0085] Compound 7 [(22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]

[0086] The molecular formula is C 34 H 50 O7, a white amorphous powder, soluble in methanol. HRTOFMS m / z: 569.3485 [M-H] - (C 34 H 49 O7, calculated value 569.3484). 11H-NMR (CD3OD, 500 MHz) δ: 6.71 (1H, m, H-24), 5.94 (1H, d, J = 5.0 Hz, H-7), 5.38 (1H, d, J = 5.0 Hz, H-11), 5.08 (1H, t, J = 5.0 Hz, H-22), 4.47 (1H, m, H-3), 4.24 (1H, dd, J = 5.0, 10.0 Hz, H-15), 2.58 (1H, m, H-23), 2.41 (1H, m, H-23), 2.34 (1H, m, H-12), 2.16 (1H, m, H-6), 2.10 (1H, m, H-6), 2.08 (1H, m, H-1), 2.09 (1H, m, H-12), 2.06 (3H, s, OCOCH3-3), 2.06 (3H, s, OCOCH3-22), 1.94 (1H, m, H-16), 1.86 (3H, s, H-27), 1.85 (1H, m, H-16), 1.82 (1H, m, H-17), 1.74 (1H, m, H-2), 1.62 (1H, m, H-20), 1.49 (2H, m, H-1, 2), 1.19 (1H, m, H-5), 1.05 (3H, s, H-19), 1.01 (3H, s, H-29), 1.01 (3H, d, J = 5.0 Hz, H-21), 0.94 (3H, s, H-30), 0.92 (3H, s, H-28), 0.67 (3H, s, H-18). 13 13C-NMR (CD3OD, 125 MHz) δ: 172.8 (OCOCH3-3), 172.5 (OCOCH3-22), 171.2 (C-26), 147.4 (C-9), 142.3 (C-8), 138.5 (C-24), 131.3 (C-25), 122.5 (C-7), 117.3 (C-11), 82.3 (C-3), 76.2 (C-22), 75.0 (C-15), 53.3 (C-14), 50.7 (C-5), 46.8 (C-13, 17), 40.6 (C-20), 40.0 (C-16), 39.6 (C-12), 38.7 (C-4), 38.6 (C-10), 36.6 (C-1), 32.6 (C-23), 28.7 (C-28), 25.2 (C-2), 23.8 (C-6), 23.4 (C-19), 21.2 or 20.9 (OCOCH3-3), 20.9 or 21.2 (OCOCH3-22), 18.0 (C-30), 17.4 (C-29), 16.4 (C-18), 13.2 (C-21), 12.7 (C-27).

[0087] Compound 8 [lanosta - 7,9(11),24 - trien - 3β,15α,22β - triacetoxy - 26 - oic acid]

[0088] The molecular formula is C 36 H 52 O8, a white crystal, soluble in methanol. Negative ion ESI MS m / z: 611.2 [M - H] – . 1 1H - NMR(CD3OD, 500 MHz) δ: 6.67(1H, m, H - 24), 5.53(1H, d, J = 5.0 Hz, H - 7), 5.40(1H, d, J = 5.0 Hz, H - 11), 5.06(1H, m, H - 15), 5.03(1H, m, H - 22), 4.45(1H, m, H - 3), 2.57(1H, m, H - 23), 2.38(1H, m, H - 23), 2.35(1H, m, H - 12), 2.13(1H, m, H - 16), 2.12(1H, m, H - 12), 2.09(1H, m, H - 6), 2.07(3H, s, OCOCH3 - 15), 2.06(1H, m, H - 6), 2.05(1H, m, H - 1), 2.04(3H, s, OCOCH3 - 3), 2.04(3H, s, OCOCH3 - 22), 1.83(1H, m, H - 16), 1.83(3H, s, H - 27), 1.81(1H, m, H - 17), 1.73(1H, m, H - 2), 1.59(1H, m, H - 20), 1.47(1H, m, H - 1), 1.16(1H, m, H - 5), 1.03(3H, s, H - 19), 1.00(3H, d, J = 5.0 Hz, H - 21), 1.00(3H, s, H - 30), 0.98(3H, s, H - 29), 0.90(3H, s, H - 28), 0.69(3H, s, H - 18). 1313C-NMR (CD3OD, 125 MHz) δ: 173.3 (OCOCH3-15), 173.1 (OCOCH3-3), 172.8 (OCOCH3-22), 171.4 (C-26), 147.3 (C-9), 141.8 (C-8), 138.7 (C-24), 131.6 (C-25), 122.8 (C-7), 117.8 (C-11), 82.5 (C-3), 78.8 (C-15), 76.3 (C-22), 52.9 (C-14), 50.9 (C-5), 47.1 (C-17), 45.4 (C-13), 41.0 (C-20), 39.4 (C-12), 38.9 (C-4), 38.8 (C-10), 38.0 (C-16), 36.9 (C-1), 32.9 (C-23), 28.9 (C-28), 25.5 (C-2), 24.1 (C-6), 23.6 (C-19), 21.4 (OCOCH3-15, 22), 21.1 (OCOCH3-3), 19.2 (C-30), 17.7 (C-29), 16.6 (C-18), 13.4 (C-21), 13.0 (C-27).

[0089] After structural analysis, 4 new compounds and 4 known compounds are all lanostane-type triterpenoids. Their names and structural formulas are as follows:

[0090] Compound 1, (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oic acid, is a new compound.

[0091]

[0092] Compound 2, (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid.

[0093]

[0094] Compound 3, (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid, is a new compound.

[0095]

[0096] Compound 4, (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid, is a new compound.

[0097]

[0098] Compound 5, (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid.

[0099]

[0100] Compound 6, (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid.

[0101]

[0102] Compound 7, (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid, is a new compound.

[0103]

[0104] Compound 8, lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid.

[0105]

[0106]

[0107] Detection of the anti-inflammatory activity of the compounds in Example 4

[0108] In this example, the LPS-stimulated RAW264.7 cell model was used to detect the extracellular NO content by the Griess reagent method, and the inhibitory effect of the compounds with the above structural formulas 1-8 on NO release was determined.

[0109] Culture and treatment of mouse macrophage RAW264.7 cells

[0110] RAW264.7 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin mixture in a constant temperature incubator at 37 °C and 5% CO2.

[0111] (1) Effects of Compounds 1-8 on NO Release from LPS-Stimulated RAW264.7 Cells

[0112] RAW264.7 cells in the logarithmic phase were diluted with colorless RPMI1640 medium to a single cell suspension of 1×10 5 cells / mL and inoculated into 96-well plates (194 μL per well), with three parallel wells in each group. After culturing in a CO2 incubator for 24 h, 1 μL of Compounds 1-6 with a concentration of 2 μmol / mL and the positive control silymarin (the final concentration of the samples was 10 nmol / mL) were added respectively, and after continuing to incubate for 1 h, 5 μL of LPS with a concentration of 100 μg / mL was added. At the same time, an LPS group (1 μL PBS + 5 μL LPS) and a blank control group (1 μL PBS + 5 μL PBS) were set. After culturing for 48 h, 100 μL of the culture supernatant was aspirated into an enzyme-linked immunosorbent assay (ELISA) plate, 50 μL of Griess reagent was added to each well, and after incubating at room temperature in the dark for 10 min, the OD value was measured at 543 nm with an ELISA reader. The concentration of NO in the cell culture supernatant of each group and the inhibition rate of NO release were calculated according to the NaNO2 standard curve, and the inhibition rate of the samples on the cells was calculated according to Equation 1.

[0113]

[0114] The experimental results are shown in Table 1 below:

[0115] Table 1 Comparison of the Inhibition Rates of Compounds on NO Release

[0116]

[0117] The experimental results showed that Compounds 1-8 all had strong activities in inhibiting NO production.

[0118] (2) Effects of Compounds on the Expression of Inflammatory Factors in LPS-Induced RAW264.7 Cells

[0119] RAW264.7 macrophages in the logarithmic phase were inoculated into 96-well culture plates, and the cell number was controlled to be about 1×10 5cells / mL. Then it was placed in a 37 °C, 5% CO2 cell culture incubator and cultured for 24 h. After culturing for 24 h in the CO2 incubator, 1 μL of compounds 1-6 with a concentration of 2 μmol / mL and the positive control silymarin (the final concentration of the samples was 10 nmol / mL) were added respectively. After continuing to incubate for 1 h, 5 μL of LPS with a concentration of 100 μg / mL was added. At the same time, an LPS group (1 μL of PBS + 5 μL of LPS) and a blank control group (1 μL of PBS + 5 μL of PBS) were set. After culturing for 48 h, the cell culture supernatant was collected, and the contents of TNF-α and IL-6 were detected by ELISA method.

[0120] The experimental results are shown in Tables 2 and 3 respectively. It should be noted that in Tables 2 and 3, * P < 0.05, ** P < 0.01 indicates the difference from the model group, # P < 0.05, ## P < 0.01 indicates the difference from the blank group.

[0121] Table 2 Effects of Compounds on the Release of TNF-α from LPS-Induced RAW264.7 Cells

[0122]

[0123] Table 3 Effects of Compounds on the Release of IL-6 from LPS-Induced RAW264.7 Cells

[0124]

[0125] The experimental results (Tables 2 and 3) show that: in the model group, TNF-α and IL-6 were significantly increased. Compounds with structural formula 1-8 could all significantly inhibit the activity of TNF-α release, among which compounds 1, 2, 7, and 8 were significantly better than the positive control silymarin. Except for compound 6, compounds 1-5, 7, and 8 could all significantly inhibit the activity of IL-6 release, among which compounds 2, 7, and 8 had significantly better inhibitory ability than the positive control silymarin. The above results indicate that compounds 1-8 can effectively inhibit the expression of inflammatory factors.

Claims

1. Triterpenoid compounds in Ganoderma sinense fermented mycelium, characterized in that A compound with the following structural formula 1 or 4, or its medicinal salt or crystal:

2. A preparation for anti - inflammation, characterized in that It contains one or more compounds with the structural formula 1 or 4, or their medicinal salts, and a medically acceptable carrier; The compounds with the structural formula 1 and 4 are as follows:

3. Application of triterpenoid compounds in Ganoderma sinense fermented mycelia for preparing drugs for preventing or treating inflammation; the triterpenoid compounds in Ganoderma sinense fermented mycelia are compounds with the following structural formula 1 or 4: The compounds with the structural formula 1 and 4 are as follows:

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