Ganoderma triterpenic acid compound, preparation method thereof and application of ganoderma triterpenic acid compound in prevention and treatment of influenza

Abstract

The invention belongs to the field of medicines, and discloses a ganoderma triterpenic acid compound, a preparation method thereof and application of the ganoderma triterpenic acid compound in prevention and treatment of influenza viruses. The ganoderma triterpenic acid compound (24E)-3alpha-hydroxy-7-oxo-lanosta-8, 24-dien-26-oic acid is as shown in a formula I which is described in the specification. Experiments prove that the compound disclosed by the invention has relatively good influenza virus neuraminidase inhibitory activity, and the IC50 of the compound is 18.3 mu M. Researches show that the compound has an application prospect in further development of novel anti-influenza drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a novel Ganoderma lucidum triterpenoid compound (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid isolated from Ganoderma lucidum, its preparation method, and its use in anti-influenza virus therapy. Background Technology

[0002] Reishi mushroom, or Ganoderma lucidum, is a general term for fungi belonging to the genus Ganoderma in the family Polyporaceae. There are 120 species worldwide, with 106 found in my country. It is a fungus used both as food and medicine. Reishi was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), listed as a superior herb, sweet, neutral, and non-toxic, beneficial to essence and qi, good for tendons and bones, and improves complexion. The *Chinese Pharmacopoeia* stipulates that the dried fruiting bodies of *Ganoderma lucidum* (Leyss. ex Fr.) Karst. or *Ganoderma sinense* Zhao, Xuet Zhang are legally recognized medicinal materials. The main chemical components of reishi include triterpenes, heteroterpenes, polysaccharides, steroids, alkaloids, and sesquiterpenes. Reishi has the effects of tonifying qi and calming the mind, relieving cough and asthma, and is mainly used for restlessness, insomnia, palpitations, cough and asthma due to lung deficiency, shortness of breath due to weakness, and loss of appetite. Currently, artificially cultivated Ganoderma lucidum is mainly Ganoderma lucidum (Leyss. ex Fr.) Karst.

[0003] Influenza is an acute respiratory infection caused by the influenza virus. Seasonal influenza is common worldwide. Symptoms of influenza include acute fever, cough, sore throat, body aches, and fatigue. There are four types of influenza viruses: A, B, C, and D. Types A and B influenza viruses cause seasonal epidemics, while only type A influenza viruses can cause pandemics. Currently, the H1N1 and H3N2 influenza virus subtypes are prevalent in humans. Neuraminidase (NA) is one of the important targets of antiviral drugs for influenza. When the NA of the influenza virus is inhibited, newly synthesized progeny viruses accumulate on the surface of host cells and cannot be released, thus inhibiting further spread and infection of new cells. Currently, clinically used NA inhibitors are designed based on the sialic acid isomer 2,3-disehydro-2-deoxy-N-acetylneuraminic acid (DANA). The U.S. Food and Drug Administration (FDA) classifies drugs into five categories (A, B, C, D, and X) based on animal studies and clinical experience regarding their teratogenic effects on fetuses. Category C drugs are those for which animal studies have shown harm to the fetus (teratogenicity or embryonic death, etc.), or for which there are no controlled studies in pregnant women, or for which no studies have been conducted on pregnant women and animals. These drugs can only be used after weighing the benefits to the pregnant woman against the risks to the fetus. Because no controlled trials have been conducted on pregnant women, NA inhibitors are all classified as Category C according to the FDA. Ganoderma lucidum is considered a superior herb, non-toxic, suitable for long-term use, and has almost no toxic side effects.

[0004] The novel Ganoderma lucidum triterpene acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid involved in this study is a new compound, and its inhibitory activity against influenza virus neuraminidase is being discovered for the first time. To date, no patents or literature reports have been found regarding these research findings.

[0005] The purpose of this invention is to fully develop my country's traditional Chinese medicine resources and to find compounds with unique chemical structures, high activity, and low toxicity and side effects that resist influenza viruses, in order to provide new anti-influenza virus drugs for clinical research. Summary of the Invention

[0006] The technical problem solved by this invention is to provide Ganoderma lucidum triterpenic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid, its preparation method, and its use in preventing and treating influenza viruses.

[0007] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0008] The first aspect of the present invention is to provide ganoderic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid. Its characteristic is that the compound has the following structural formula:

[0009]

[0010] The second aspect of the present invention provides a method for preparing the ganoderic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid described in the first aspect:

[0011] 1) The dried fruiting bodies of Ganoderma lucidum were extracted three times by heating and refluxing with 90% ethanol-water. The extracts were combined and concentrated under reduced pressure to obtain an extract, which was then mixed with macroporous resin.

[0012] 2) Macroporous resin chromatography was performed, with gradient elution using 30% ethanol-water, 60% ethanol-water, 80% ethanol-water, and 95% ethanol-water to obtain four fractions, namely DK-1-DK-4. DK-3 was further eluted by silica gel column chromatography with a gradient elution using a mixture of petroleum ether and ethyl acetate as the eluent, sequentially eluted in volume ratios, to obtain 25 fractions, namely Fr.1-Fr.25. Fr.17 was eluted by medium-pressure silica gel column chromatography to obtain 14 subfractions, namely Fr.17-1-Fr.17-14. Fr.17-7 was further eluted by C... 18 Reverse-phase column chromatography yielded 25 subfractions, namely Fr.17-7-1-Fr.17-7-25. Fr.17-7-21 was separated by semi-preparative reversed-phase high-performance liquid chromatography with isocratic elution using 55% acetonitrile-water as eluent to obtain ganoderic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid (number LZ-GO-44).

[0013] LZ-GO-44 is a white amorphous powder. UV(MeOH)λ max (logε)211(4.04),252(3.88)nm. IR spectrum indicates 3363cm. -1 The absorption peaks are for hydroxyl groups, at 1690 and 1650 cm⁻¹. -1 The presence of two carbonyl groups is indicated, 1583 cm⁻¹ -1 This is a double bond absorption peak. HR-ESI-MS shows a quasi-molecular ion peak at m / z 471.3478 [M+H]. + (calcd.for C 30 H 47 O4,471.3469), its molecule is determined to be C30 H 46 O4 has an unsaturation degree of 8.

[0014] 1 The 1H-NMR spectrum (Table 1) shows 44 hydrogen signals. One double-bonded hydrogen signal is observed in the low-field region [δ]. H 6.89 (1H,t,J=7.4Hz,H-24)], 1 hydroxymethyl hydrogen signal [δ H 3.49 (br s, H-3)]; 7 methyl hydrogen signals were observed in the high-field region [δ H [1.84(3H,s,H-27), 1.17(3H,s,H-19), 0.96(3H,s,H-28), 0.95(3H,d,J=6.1Hz,H-21), 0.94(3H,s,H-29), 0.92(3H,s,H-30), 0.66(3H,s,H-18)], the remainder being the methylene or methine hydrogen signals of the compound.

[0015] 13 The C-NMR spectrum (Table 1) shows 30 carbon signals, including 2 carbonyl carbon signals [δ]. C 199.2(C-7), 172.3(C-26)]; 4 carbon signals from 2 double bonds [δ C 165.7 (C-9), 145.6 (C-24), 138.8 (C-8), 126.6 (C-25)], 1 oxygen-carbon signal [δ C 75.0(C-3)] and 7 methyl carbon signals [δ C 27.3 (C-28), 25.0 (C-30), 21.8 (C-29), 18.6 (C-21), 18.3 (C-19), 15.8 (C-18), 12.0 (C-27)], plus 16 other aliphatic carbon signals [δ C 48.9(C-17),47.8(C-14),45.0(C-13),43.9(C-5),39.8(C-10),37.6(C-4),36.5(C-6),36.3(C-20), 3 4.8(C-22),32.1(C-15),30.1(C-12),29.1(C-1),28.8(C-16),25.9(C-23),25.3(C-2),23.5(C-11)].

[0016] By HR-ESI-MS, 1 H-NMR and 13 C-NMR spectra suggest that LZ-GO-44 is a lanostane-type triterpenoid.

[0017] Combined with HMBC spectrum, H3-28(δ) H 0.96), H3-29(δ) C 0.94) and C-3(δ C 75.0) and C-5 (δ C 43.9) is related, indicating that the C-3 position is a hydroxyl group; H-5 (δ H 2.15) and C-7(δ) C The correlation of 199.2 indicates that the C-7 position is a carbonyl group; H3-19 (δ H 1.17) and C-9(δ) C 165.7) related, H3-30(δ) H 0.92) and C-8(δ) C 138.8) is related, indicating that C-8 and C-9 are double bonds; H-24 (δ H 6.89) and C-22 (δ C 34.8), C-26 (δ) C 172.3), C-27 (δ) C The correlation with 12.0 indicates that the C-24 and C-25 positions of the side chain are double bonds, and the C-26 position is a carboxyl group.

[0018] In the NOESY spectrum, H2-23(δ) H 2.25, 2.11) and H3-27 (δ H 1.84) related, indicating that the two large groups, methylene (C-23) and carboxyl (C-26), are on opposite sides, and the double bond is in E configuration.

[0019] Thus, the planar structure of the compound was established.

[0020] The chemical shift and peak shape of the H-3-hydroxymethyl hydrogen signal are broad singlets [δ] H From [3.49(br s,H-3)], we know that H-3 is in an equatorial bond, with a β-configuration, and 3-OH is in an α-configuration. The configurations of the remaining chiral carbons are determined based on the lanostane skeleton. Finally, the absolute configuration of LZ-GO-44 is determined to be 3R,5R,10S,13R,14R,17R,20R.

[0021] The structure of LZ-GO-44 was determined and named (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid. A Sci-finder search confirmed it as a novel compound.

[0022] Table 1. (24E)-3α-Hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid1 H NMR and 13 CNMR data

[0023]

[0024] 1 H NMR 400MHz and 13 C NMR 125MHz, in CDCl3.

[0025] A third aspect of the present invention is to provide a pharmaceutical composition, characterized in that: the pharmaceutical composition contains the ganoderic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid described in the first aspect, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0026] The fourth aspect of the present invention is the use of the ganoderic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid described in the first aspect of the present invention in the preparation of medicaments for the prevention and / or treatment of influenza.

[0027] Beneficial technical effects

[0028] The inventors of this invention discovered natural Ganoderma lucidum triterpenic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid in natural products. Based on this, the inhibitory activity of the obtained compound against influenza virus neuraminidase was evaluated, and a novel structural compound (IC) exhibiting significant inhibitory activity against influenza virus neuraminidase was obtained. 50 The concentration was 18.3 μM. The study revealed that (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid is a novel active compound with potential for further development as a new drug for treating influenza-related diseases. To date, no structural and activity studies of this compound have been reported in the literature. Existing literature and techniques do not report on (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid or its medically acceptable salts, nor on the use of this compound for treating influenza. Therefore, the present invention is significantly innovative. Attached Figure Description

[0029] LZ-GO-44

[0030] Figure 1The structure of Ganoderma triterpenic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid

[0031] Figure 2 High-resolution mass spectrometry of (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid

[0032] Figure 3 UV spectrum of (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid Figure 4 Infrared spectrum of (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid Figure 5 (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid 1 H NMR spectrum

[0033] Figure 6 (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid 13 C NMR spectrum

[0034] Figure 7 HSQC spectrum of (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid Figure 8 HMBC spectrum of (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid Figure 9 NOESY spectrum of (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid Detailed Implementation

[0035] Example 1: Isolation of Ganoderma lucidum triterpenic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oicacid

[0036] Molecular formula: C 30 H 46 O4

[0037] Nomenclature: (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid

[0038] Separation and preparation route:

[0039] The dried fruiting bodies of Ganoderma lucidum were extracted three times by heating and reflux with 90% ethanol-water. The extracts were combined and concentrated under reduced pressure to obtain a paste, which was then mixed with macroporous resin. Macroporous resin chromatography was performed, with gradient elution using 30% ethanol-water, 60% ethanol-water, 80% ethanol-water, and 95% ethanol-water to obtain four fractions, namely DK-1 to DK-4. DK-3 was further eluted by silica gel column chromatography with a gradient elution of a mixed solvent of petroleum ether and ethyl acetate at volume ratios of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50. The column was then washed sequentially with ethyl acetate and methanol to obtain 25 fractions, namely Fr.1 to Fr.25. Fr.17 was eluted by gradient chromatography on a medium-pressure silica gel column with petroleum ether:ethyl acetate (80:20-50:50, v / v). The column was then washed with ethyl acetate to obtain 14 subfractions, namely Fr.17-1-Fr.17-14. Fr.17-7 was subjected to C... 18 Reverse-phase column chromatography with gradient elution using methanol:water (50:50-75:25, v / v) as the eluent, followed by column washing with 100% methanol, yielded 25 subfractions, namely Fr.17-7-1-Fr.17-7-25. Fr.17-7-21 was separated using a semi-preparative reversed-phase column YMC-Pack ODS-A (250×20 mm) with isocratic elution of 55% acetonitrile-water as the mobile phase at a flow rate of 12 mL / min. R =57 min yielded Ganoderma triterpenic acid (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid (number LZ-GO-44).

[0040] Pharmacological experiments

[0041] Experimental Example 1:

[0042] 1.1. Inhibitory effect of (24E)-3α-Hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid on influenza virus neuraminidase

[0043] The influenza virus neuraminidase is located on the surface of the influenza virus and can cause the virus to be released from the host cell. The compound 4-methylumbelliferyl-N-acetyl-α-D-neuraminidase sodium hydrate (MUNANA) is a specific substrate of neuraminidase (NA). The metabolites produced under the action of neuraminidase can produce 450 nm fluorescence when excited by 360 nm irradiation. The change in fluorescence intensity can sensitively reflect the activity of neuraminidase.

[0044] Experimental methods:

[0045] In the enzyme reaction system, a certain concentration of the analyte compound and influenza virus neuraminidase were suspended in a reaction buffer (pH 6.5). The fluorescent substrate MUNANA was added to start the reaction, and the mixture was incubated at 37°C for 60 minutes. The reaction was then terminated by adding a stop solution. The fluorescence intensity was measured under the parameters of an excitation wavelength of 360 nm and an emission wavelength of 450 nm. The inhibition rate of the compound on neuraminidase activity was calculated based on the decrease in fluorescence intensity.

[0046] Experimental results:

[0047] The IC50 of compound (24E)-3α-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid inhibits influenza neuraminidase activity. 50 The IC50 value of the positive control drug zanamivir was 18.3 μM. 50 It is 7.8×10 -4 μM.

Claims

1. A Ganoderma lucidum triterpenoid compound or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound is 2. The method for preparing the compound according to claim 1, characterized in that, 1) The dried fruiting bodies of Ganoderma lucidum were extracted three times by heating and refluxing with 90% ethanol-water. The extracts were combined and concentrated under reduced pressure to obtain an extract, which was then mixed with macroporous resin. 2) Macroporous resin chromatography was performed with gradient elution using 30% ethanol-water, 60% ethanol-water, 80% ethanol-water, and 95% ethanol-water to obtain four fractions, namely DK-1-DK-4. DK-3 was further eluted by silica gel column chromatography with a gradient elution of a mixture of petroleum ether and ethyl acetate at volume ratios of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50, followed by washing the column with ethyl acetate and methanol to obtain 25 fractions, namely Fr.1-Fr.

25. Fr.17 was eluted by medium-pressure silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate (80:20-50:50, v / v), followed by washing the column with ethyl acetate to obtain 14 subfractions, namely Fr.17-1-Fr.17-14. Fr.17-7 was further eluted by C... 18 Reverse-phase column chromatography with gradient elution using methanol:water (50:50-75:25, v / v) as the eluent, followed by washing the column with 100% methanol, yielded 25 subfractions, namely Fr.17-7-1-Fr.17-7-25. Fr.17-7-21 was separated by semi-preparative reversed-phase high-performance liquid chromatography with isocratic elution using 55% acetonitrile-water as the eluent to obtain the compound of claim 1.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

4. The use of the compound of claim 1 and its pharmaceutically acceptable salt in the preparation of medicaments for the prevention and / or treatment of influenza.

5. The application according to claim 4, characterized in that, The influenza mentioned includes influenza A and influenza B, among others.

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