Triterpenoid in ganoderma sinensis fermented mycelium and anti-inflammatory application of triterpenoid

Abstract

The invention provides a triterpenoid compound in ganoderma sinensis fermented mycelium. The structural formulas of the triterpenoid compound are structural formulas 1, 3, 4 and 7. The invention further provides application of the triterpenoid in the ganoderma sinensis fermented mycelium as a medicine for preventing or treating inflammation. The triterpenoid in the ganoderma sinensis mycelium is one or more of compounds with structural formulas 1-8. The triterpenoids separated and purified from ganoderma sinensis mycelia are subjected to anti-inflammatory activity test for the first time, and the results show that the compounds with the structural formulas 1-8 can inhibit release of TNF-alpha and IL-6 and have remarkable anti-inflammatory activity.

Description

Technical Field

[0001] This invention relates to the field of edible fungi applications, specifically to a triterpenoid compound in fermented Ganoderma lucidum mycelium and its anti-inflammatory application. Background Technology

[0002] "Lingzhi" is a general term for fungi of the genus Ganoderma P. Karst., belonging to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, and family Ganodermataceae. Ganoderma sinense (purple Ganoderma) JDZhao, LWHsu & X.Q.Zhang is a type of Lingzhi listed in the Chinese Pharmacopoeia as suitable for medicinal use (Pharmacopoeia of the People's Republic of China, 2020 edition). Currently, due to limitations such as the limited number of commercially available varieties and low strain resistance, the yield of artificially cultivated Ganoderma sinense in China is far lower than that of Ganoderma rubrum. Compared to the cultivation of fruiting bodies, the fermentation of Ganoderma sinense has the advantages of a shorter production cycle, controllable conditions, and unique fermentation products. Furthermore, the fermented mycelium of Ganoderma sinense is rich in triterpenoid active compounds.

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

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

[0005] This invention first provides a triterpenoid compound from fermented Ganoderma lucidum mycelium, the structural formula of which is as follows: compounds of structural formulas 1, 3, 4, and 7, or their medicinal salts, crystals, or hydrates:

[0006]

[0007] The present invention also provides an anti-inflammatory preparation comprising one or more compounds of structural formulas 1-8 or pharmaceutical salts thereof and a medically acceptable carrier.

[0008] The compounds with structural formulas 1-8 mentioned above are:

[0009]

[0010]

[0011] This invention also provides the application of triterpenoid compounds in fermented Ganoderma lucidum mycelium as drugs for the prevention or treatment of inflammation; wherein the triterpenoid compounds in fermented Ganoderma lucidum mycelium are one or more of the following compounds with structural formulas 1-8:

[0012] The compounds with structural formulas 1-8 mentioned above are:

[0013]

[0014]

[0015] The chemical names of the compounds with structural formulas 1-8 above are as follows:

[0016] Compound 1: (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oicacid

[0017] Compound 2: (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid Compound 3: (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid Compound 4: (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid

[0018] Compound 5: (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid Compound 6: (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid

[0019] Compound 7: (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid Compound 8: lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid

[0020] The compounds of structural formulas 1-8 provided by this invention can also be used as preparations or foods for the prevention and / or treatment of inflammation or for adjuvant purposes during or after the treatment of inflammation.

[0021] The eight triterpenoid compounds isolated from Ganoderma lucidum mycelium provided by this invention all have significant anti-inflammatory effects, especially compounds 2, 7, and 8, which have outstanding anti-inflammatory effects and have broad market application prospects. Attached Figure Description

[0022] Figure 1 Fr3 semi-preparative liquid chromatogram

[0023] Figure 2 Fr5 semi-preparative liquid chromatogram

[0024] Figure 3 Fr8 semi-preparative liquid chromatogram Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Ganoderma sinense (strain number: Dai20076) is currently preserved at the Institute of Microbiology, College of Ecology and Nature Conservation, Beijing Forestry University.

[0028] Mouse monocytes RAW264.7 were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, product catalog number SCSP-5036.

[0029] DMEM culture medium and fetal bovine serum (FBS): purchased from Gibco, USA;

[0030] Penicillin and streptomycin: purchased from Amresco, USA;

[0031] Mouse-derived TNF-α and IL-6 reagent kits: purchased from Beijing Sizhengbai Biotechnology Co., Ltd.;

[0032] Glucose potato agar (PDA): purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0033] Glucose, sucrose, agar, MgSO4·H2O, KH2PO4, CaSO4, as well as analytical grade methanol, chloroform, petroleum ether, acetone, ethanol, acetonitrile, and glacial acetic acid: all were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] Chromatographic grade methanol and acetonitrile: purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0035] Silymarin, deuterated methanol, and deuterated chloroform: purchased from Sigma-Aldrich;

[0036] YMC*GEL C18 silica gel (50μm, 12nm): Beijing Yuanbaoshan Chromatography Technology Co., Ltd.;

[0037] Zorbax Eclipse Plus C18 column (5μm, 4.6mm × 250mm): Agilent Technologies, USA;

[0038] Zorbax Eclipse Plus-C18 semi-preparative chromatography column (5μm, 21.2mm × 250mm): Agilent Technologies, USA;

[0039] GF254 silicone rubber sheet (0.2-0.25mm): Qingdao Ocean Chemical Plant;

[0040] LC3000 High Performance Liquid Chromatograph: Beijing Innovation Tongheng Technology Co., Ltd.

[0041] Agilent LC1290 Infinity II Ultra-High Performance Liquid Chromatograph: Agilent Technologies, Inc.

[0042] Agilent 6495 triple quadrupole mass spectrometer: Agilent Technologies, Inc., USA;

[0043] Bruker AV II-600 nuclear magnetic resonance spectrometer: Bruker GmbH, Germany;

[0044] Synergy HT Multifunctional Microplate Reader: BIO-TEK, USA;

[0045] Thermo Scientific Q Exactive HF Orbitrap-FTMS High-Resolution Mass Spectrometer: Thermo Scientific, Inc.

[0046] Example 1: Fermentation of Ganoderma lucidum mycelium

[0047] Culture medium formulation: 3% soluble starch, 1.2% glucose, 1% yeast extract, 0.15% MgSO4·7H2O, 0.3% KH2PO4 (all weight percentages), pH: natural.

[0048] The bacterial strain stored in a 4°C refrigerator was activated and cultured in a shake flask under dark conditions for 7 days (150 r / min, 25°C). Then it was transferred to a secondary shake flask for static culture for 21 days to obtain mycelium.

[0049] Example 2: Extraction and extraction of Ganoderma lucidum mycelium

[0050] The dried mycelium was soaked in a 95% ethanol aqueous solution at room temperature for 24 hours each time. The extracts were combined and concentrated to obtain the ethanol extract of Ganoderma lucidum mycelium.

[0051] The ethanol extract of Ganoderma lucidum mycelium was further extracted three times with an equal volume of petroleum ether. The petroleum ether phase was discarded, and the remaining phase was extracted three times with an equal volume of ethyl acetate. The ethyl acetate extracts were combined, concentrated under reduced pressure, and dried to obtain the ethyl acetate phase.

[0052] Example 3 Preparation of Compounds

[0053] The ethyl acetate phase was separated by medium-pressure chromatography using YMC C18 (50 μm, 12 nm) as the packing material and eluting with a gradient of acetonitrile (A)-0.01% glacial acetic acid-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%) at a flow rate of 200 mL / min. One fraction was collected every 500 mL, for a total of 60 fractions. The fractions were numbered in the order of collection and subjected to thin-layer chromatography. Fractions with the same Rf value on the thin-layer plates were combined to obtain Fr1-Fr13.

[0054] After thin-layer chromatography analysis, the 60 fractions 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), and Fr13 (47-60, 3.17 g).

[0055] Fr1-Fr13 were analyzed by HPLC. According to the liquid phase detection results, Fr3, Fr5, and Fr8 are rich in characteristic absorption peaks of triterpenoids. Therefore, Fr3, Fr5, and Fr8 were further separated and purified.

[0056] Isolation and purification of Fr3

[0057] A Zorbax Eclipse Plus-C18 semi-preparative chromatographic column was selected, and gradient elution was performed using acetonitrile (A)-0.01% glacial acetic acid aqueous solution (0.0-18.0 min, A: 50%-50%; 18.0-18.5 min, A: 50%-100%; 18.5-23.0 min, A: 100%-100%), with detection at a wavelength of 240 nm. The preparation was repeated multiple times, and the chromatographic peak at 15.5 min was collected to obtain Fr3-8.

[0058] Fr3-8 used a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column, eluted with a gradient of acetonitrile (A) and 0.01% glacial acetic acid in 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 detected at a wavelength of 240 nm. The preparation was repeated multiple times, and the chromatographic peak at 8.2 min was collected to obtain compound 1 (100.00 mg); the chromatographic peak at 18.0 min was collected to obtain compound 2 (140.00 mg).

[0059] Isolation and purification of Fr5

[0060] Fr5 was analyzed using a Zorbax Eclipse Plus-C18 semi-preparative column with gradient elution of acetonitrile (A) and 0.01% glacial acetic acid in 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%), detected at 240 nm. The peak at 15.0 min was collected to obtain Fr5-4; the peak at 20.8 min was collected to obtain compound 3 (101.00 mg); and the peak at 23.0 min was collected to obtain Fr5-6.

[0061] Fr5-4 used a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column and performed gradient elution with acetonitrile (A) and 0.01% glacial 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 performed at 240 nm. The preparation was repeated multiple times, and the chromatographic peak at 12.2 min was collected to obtain compound 4 (100.00 mg). The chromatographic peak at 25.0 min was collected to obtain compound 5 (110.00 mg).

[0062] Fr5-6 used a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column, employing a gradient elution of acetonitrile (A) and 0.01% glacial 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%), with detection at 240 nm. The preparation was repeated multiple times, and the chromatographic peak at 15.0 min was collected to obtain compound 6 (280.00 mg).

[0063] Fr8 separation and purification

[0064] Fr8 used a Zorbax Eclipse Plus-C18 semi-preparative chromatographic column, employing a gradient elution of acetonitrile (A) and 0.01% glacial 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%), with detection at 240 nm. The preparation was repeated multiple times, collecting the peak at 35.0 min to obtain compound 7 (612.00 mg), and collecting the peak at 40.0 min to obtain compound 8 (109.00 mg).

[0065] Example 3: Confirmation of compounds 1-8.

[0066] Using mass spectrometry and nuclear magnetic resonance (NMR) detection, the structures of eight compounds obtained from Ganoderma lucidum mycelium were analyzed, and their precise chemical structures were determined. The specific confirmation information of the eight compounds is as follows:

[0067] Compound 1 [(22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oicacid]

[0068] The molecular formula is C 30 H 44 O6, a pale yellow amorphous powder, readily soluble in methanol and chloroform. HRTOFMSm / z: 501.3216 [M+H] + (C 30 H 45 O6, calculated value 501.3211). 1 H-NMR(CD3OD,500MHz)δ:6.83(1H,m,H-24),4.24(1H,dd,J=5.0,10.0Hz,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=5.0Hz,H-21),0.78(3H,s,H-18); 13C-NMR(CD3OD,125MHz)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).

[0069] Compound 2 [(22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid]

[0070] Molecular formula C 30 H 44 O5, a pale yellow amorphous powder, readily soluble in methanol. ESIMSm / z (negative ion): 483.0 [MH] - . 11H-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, 那么 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 13C-NMR(CD3OD, 125 MHz) δ: 219.4 (C-3), 17: 18 (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).

[0071] Compound 3 [(22S, 24E)-3β-acetoxy-15α, 22β-dihydroxylanosta-7, 9(11), 24-trien-26-oic acid]

[0072] Molecular formula is C 32 H 48O6, a pale yellow amorphous powder, readily soluble in methanol. HRTOFMSm / z: 551.3350 [M+Na] + (C 32 H 48 O6Na, calculated value 551.3343). 1 H-NMR(CD3OD,500MHz)δ:6.83(1H,m,H-24),5.91(1H,d,J=10.0Hz,H-7),5.37(1H,d,J=5.0Hz,H-11),4.45(1H,m,H-3),4.23(1H,dd,J=10.0,10 .0Hz,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=5.0Hz,H-21),0.64(3H,s,H-18). 13 C-NMR (CD3OD, 125MHz) 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-1 3),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).

[0073] Compound 4 [(22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oicacid]

[0074] The molecular formula is C 34 H 44 O8, yellow crystals, readily soluble in methanol. HRTOFMSm / z: 557.3114 [M+H] + (C 32 H 45 O8, calculated value 557.3109). 1 H-NMR(CD3OD,500MHz)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=5.0Hz,H-21),0.91(3H,s,H-18). 13C-NMR(CD3OD,125MHz)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-1 3),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).

[0075] Compound 5 [(22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oicacid]

[0076] The molecular formula is C 32 H 46 O6, a pale yellow amorphous powder, readily soluble in methanol. ESIMSm / z (negative ion): 525.1 [MH] - . 11H-NMR (CD3OD, 500 MHz) δ: 6.69 (1H, m, H-24), 5.95 (1H, d, J = 10.0 Hz, H-7), 5.45 (1H, d, J = 5.0 Hz, H-11), 5.07 (1H, m, H-22), 4.24 (1H, d, J = 5.0, 10.0 Hz, 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 = 5.0 Hz, H-21), 0.91 (3H, s, H-30), 0.67 (3H, s, H-18). 13 13C-NMR (CD3OD, 125 MHz) 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-19), 17.9 (C-30), 16.4 (C-18), 13.2 (C-21), 12.7 (C-27).

[0077] Compound 6 [(22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]

[0078] The molecular formula is C34 H 48 O7, a white solid, readily soluble in methanol. ESIMSm / z: 567.1 [MH] - . 1 H-NMR (CD3OD, 500MHz) δ: 6.67 (1H, m, H-24), 5.58 (1H, d, J = 5.0Hz, H-7), 5.49 (1H ,d,J=5.0Hz,H-11),5.07(1H,dd,J=5.0,10.0Hz,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=5.0,10.0Hz,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=5.0Hz,H-21),1.01(3H,s,H-30),0.72(3H,s,H-18). 13C-NMR (CD3OD, 125MHz) 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-1 7),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).

[0079] Compound 7 [(22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid]

[0080] The molecular formula is C 34 H 50 O7, a white amorphous powder, readily soluble in methanol. HRTOFMSm / z: 569.3485 [MH] - (C 34 H 49 O7, calculated value 569.3484). 1H-NMR(CD3OD,500MHz)δ:6.71(1H,m,H-24),5.94(1H,d,J=5.0Hz,H-7),5.38(1H,d,J=5.0Hz,H-11),5.08(1H,t,J=5.0Hz,H-22),4.47(1H,m,H-3),4.24(1H,dd,J=5.0,10.0Hz,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=5.0Hz,H-21),0.94(3H,s,H-30),0.92(3H,s,H-28),0.67(3H,s,H-18). 13 C-NMR(CD3OD,125MHz)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),21.2or20.9(OCOCH3-3),20.9or 21.2(OCOCH3-22),18.0(C-30),17.4(C-29),16.4(C-18),13.2(C-21),12.7(C-27)。

[0081] Compound 8 [lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid]

[0082] The molecular formula is C 36 H 52 O8, white crystals, readily soluble in methanol. ESIMSm / z (negative ion): 611.2 [MH] - . 1 H-NMR (CD3OD, 500MHz) δ: 6.67 (1H, m, H-24), 5.53 (1H, d, J = 5.0Hz, H-7), 5.40 (1 H,d,J=5.0Hz,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),2.1 2(1H,m,H-12),2.09(1H,m,H-6),2.07(3H,s,OCOCH3-15),2.06(1H,m,H-6),2.0 5(1H,m,H-1),2.04(3H,s,OCOCH3-3),2.04(3H,s,OCOCH3-22),1.83(1H,m,H-1 6),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=5.0Hz,H-2 1),1.00(3H,s,H-30),0.98(3H,s,H-29),0.90(3H,s,H-28),0.69(3H,s,H-18). 13C-NMR (CD3OD, 125MHz) 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),2 4.1(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).

[0083] After structural analysis, four new compounds and four known compounds were identified as lanosterane-type triterpenoids. Their names and structural formulas are shown below:

[0084] Compound 1, (22S,24E)-3,7-dioxo-15α,22β-dihydroxylanosta-8,24-dien-26-oicacid, is a new compound.

[0085]

[0086] Compound 2, (22S,24E)-3-oxo-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid.

[0087]

[0088] Compound 3, (22S,24E)-3β-acetoxy-15α,22β-dihydroxylanosta-7,9(11),24-trien-26-oic acid, is a new compound.

[0089]

[0090] Compound 4, (22S,24E)-3,7,11-trioxo-15α-hydroxy-22β-acetoxylanosta-8,24-dien-26-oic acid, is a new compound.

[0091]

[0092] Compound 5, (22S,24E)-3-oxo-15α-hydroxy-22β-acetoxylanosta-7,9(11),24-trien-26-oic acid.

[0093]

[0094] Compound 6, (22S,24E)-3-oxo-15α,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid.

[0095]

[0096] Compound 7, (22S,24E)-15α-hydroxy-3β,22β-diacetoxylanosta-7,9(11),24-trien-26-oic acid, is a new compound.

[0097]

[0098] Compound 8, lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid.

[0099]

[0100] Implementation Example 4: Detection of the anti-inflammatory activity of compounds

[0101] In this embodiment, an LPS-induced RAW264.7 cell model was used. The extracellular NO content was detected by the Griess reagent method, and the inhibitory effect of the compounds with structural formulas 1-8 on NO release was determined.

[0102] Culture and treatment of mouse macrophage RAW264.7 cells

[0103] RAW264.7 cells were cultured in DMEM high-sugar phenol red medium containing a mixture of 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin in a constant temperature incubator at 37°C and 5% CO2.

[0104] (1) Effects of compounds 1-8 on NO release from LPS-stimulated RAW264.7 cells

[0105] RAW264.7 cells in the logarithmic growth phase were diluted to 1×10⁻⁶ cells using colorless RPMI 1640 medium. 5 Single-cell suspensions of 194 μL / mL were seeded into 96-well plates, with three parallel wells per group. After culturing in a CO2 incubator for 24 h, 1 μL of compound 1-6 (2 μmol / mL) and the positive control silymarin (both with a final concentration of 10 nmol / mL) were added. After incubation for another 1 h, 5 μL of LPS (100 μg / mL) was added. An LPS group (1 μL PBS + 5 μL LPS) and a blank control group (1 μL PBS + 5 μL PBS) were also established. After 48 h of culture, 100 μL of culture supernatant was transferred to an ELISA plate, and 50 μL of Leriess reagent was added to each well. After incubation at room temperature in the dark for 10 min, the OD value was measured at 543 nm using an ELISA reader. The concentration of NO in the cell culture supernatant and the inhibition rate of NO release in each group were calculated based on the NaNO2 standard curve. The cell inhibition rate of the samples was calculated according to Formula 1.

[0106]

[0107] The experimental results are shown in Table 1 below:

[0108] Table 1 Comparison of the inhibition rates of compounds on NO release

[0109]

[0110]

[0111] Experimental results show that compounds 1-8 all have strong activity in inhibiting NO production.

[0112] (2) Effects of the compound on LPS-induced expression of inflammatory factors in RAW264.7 cells

[0113] RAW264.7 macrophages in the logarithmic growth phase were seeded into 96-well culture plates, with the cell number controlled to be approximately 1 × 10⁻⁶. 5Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours of CO2 incubation, 1 μL of compound 1-6 (2 μmol / mL) and the positive control silymarin (both with a final concentration of 10 nmol / mL) were added, and incubation continued for another hour. Then, 5 μL of LPS (100 μg / mL) was added. An LPS group (1 μL PBS + 5 μL LPS) and a blank control group (1 μL PBS + 5 μL PBS) were established. After 48 hours of culture, the cell culture supernatant was collected, and the levels of TNF-α and IL-6 were detected by ELISA.

[0114] The experimental results are shown in Tables 2 and 3, respectively. It should be noted that in Tables 2 and 3, * P<0.05, ** The difference between P<0.01 and the model group # P<0.05, ## P<0.01 difference from the control group.

[0115] Table 2. Effects of compounds on LPS-induced TNF-α release in RAW264.7 cells.

[0116]

[0117] Table 3. Effects of compounds on LPS-induced IL-6 release in RAW264.7 cells.

[0118]

[0119]

[0120] The experimental results (Tables 2 and 3) showed that TNF-α and IL-6 were significantly elevated in the model group. Compounds with structures 1-8 significantly inhibited TNF-α release activity, with compounds 1, 2, 7, and 8 showing significantly better inhibition than the positive control silymarin. Except for compound 6, compounds 1-5, 7, and 8 significantly inhibited IL-6 release activity, with compounds 2, 7, and 8 showing significantly better inhibitory effects than the positive control silymarin. These results indicate that compounds 1-8 can effectively inhibit the expression of inflammatory factors.

Claims

1. A triterpenoid compound in fermented Ganoderma lucidum mycelium, characterized in that... Its structural formula is as follows: compounds with structural formulas 3 and 7, or their medicinal salts or crystals:

2. An anti-inflammatory preparation, characterized in that... It contains one or two compounds represented by structural formula 3 or 7, or their pharmaceutical salts, and a medically acceptable carrier; The compounds with structural formulas 3 and 7 mentioned above are:

3. The application of triterpenoid compounds in fermented Ganoderma lucidum mycelium for the preparation of drugs for the prevention or treatment of inflammation; wherein the triterpenoid compounds in fermented Ganoderma lucidum mycelium are one or two of the following compounds with structural formula 3 or 7: The compounds with structural formulas 3 and 7 mentioned above are:

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