A sesquiterpenoid compound, a preparation method therefor and an application thereof

Abstract

The application discloses a sesquiterpenoid compound, a preparation method thereof and application of the sesquiterpenoid compound. 15 H 18 O7。 The preparation method of the sesquiterpenoid compound comprises the following steps: S1. extracting white ginseng mushroom bodies with methanol, concentrating the extract to obtain an extract, and obtaining an ethyl acetate crude extract by extracting the extract with ethyl acetate; S2. separating the ethyl acetate crude extract by silica gel normal phase chromatography, eluting, and then separating by silica gel, gel and C-18 reverse phase column chromatography, so that the sesquiterpenoid compound is obtained. The compound has a significant inhibitory effect on the expression of interleukin-6 (IL-6) at a concentration of 25 muM, and the inhibition rate reaches 71.6%, and the compound can be used for preparing anti-inflammatory drugs and has a clinical application potential in anti-inflammatory treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical compounds, in particular, to a highly oxidized sesquiterpenoid compound from the fungus of white shiitake mushroom and a preparation method thereof and an application thereof in preparing anti-inflammatory drugs. BACKGROUND

[0002] Inflammation is a basic defense mechanism of immunity, which can protect the body from harmful stimuli such as pathogens and poisons. The inflammation process itself can cause tissue damage, leading to many diseases and even cancer. Several cell types such as macrophages and T cells and extracellular mediators of the immune system are key to regulating the inflammation process, one of the most important cytokines is interleukin-6 (IL-6). IL-6 is a pro-inflammatory cytokine produced by a variety of cells, which plays an important role in the regulation of inflammation, immune response and acute phase response. They act at local and systemic levels, affecting the function of several cell types, including not only immune system cells, but also hepatocytes, skeletal muscle cells and adipocytes. IL-6 is also a widely functional and multi-effective cytokine, which can regulate the growth and differentiation of various cells, has the function of regulating immune response, acute phase response and hematopoietic function. In addition, IL-6 is considered to be a key mediator of the pathogenesis of chronic diseases in various pathological inflammatory conditions, including end-stage renal disease and rheumatoid arthritis. Tocilizumab is the first humanized monoclonal antibody drug targeting IL-6 receptor (IL-6R) in the world, which can block IL-6 signaling by targeting IL-6R to play an anti-inflammatory role. Therefore, non-toxic compounds with IL-6 inhibitory activity can be developed as new anti-inflammatory drugs.

[0003] White shiitake mushroom is a rare edible and medicinal mushroom, which is usually consumed as a health product in Asia. In traditional Chinese medicine, it has the effects of clearing liver and eyesight, tonifying and strengthening body, and is particularly effective for treating infantile night sweat, neurasthenia, dizziness and tinnitus, gynecological diseases, etc. Research reports that polysaccharide compounds isolated from the water extract of white shiitake mushroom have anti-tumor, anti-inflammatory and anti-bacterial activities, etc. These polysaccharide compounds can significantly inhibit the production of macrophage NO and down-regulate the expression of inflammatory factor nuclear factor-kappa (NF-κB). Therefore, it has important medicinal and economic value to develop new anti-inflammatory drugs by mining organic molecules in the organic extract of white shiitake mushroom. SUMMARY

[0004] The purpose of the present application is to provide a highly oxidized sesquiterpenoid compound from the fungus of white shiitake mushroom.

[0005] Another purpose of the present application is to provide a preparation method of the highly oxidized sesquiterpenoid compound from the fungus of white shiitake mushroom.

[0006] Another object of the present application is to provide the use of the highly oxidized sesquiterpenes from the white mushroom in the preparation of anti-inflammatory drugs.

[0007] The technical solution of the present application is a sesquiterpene compound, the structural general formula of which is:

[0008] The molecular formula is: C 15 H 18 O7.

[0009] The sesquiterpene compound is isolated from fresh fruiting bodies of white mushrooms, which are provided by Bijie City Agricultural Investment Fungus Co., Ltd.

[0010] A preparation method of a sesquiterpene compound, the method comprising the following steps:

[0011] S1. Extracting white mushroom fruiting bodies with methanol, concentrating the extract to obtain an extract, and extracting the extract with ethyl acetate to obtain an ethyl acetate crude extract;

[0012] S2. Separating the ethyl acetate crude extract by silica gel normal phase chromatography, gradient elution, and then separating by silica gel, gel, and C-18 reverse phase column chromatography to obtain the sesquiterpene compound.

[0013] In step S1, the number of methanol extraction is 2-5 times, and the extraction time is 10 days.

[0014] In step S2, the solvent used for gradient elution is petroleum ether / ethyl acetate, and the 10%-80% ethyl acetate / petroleum ether part is collected after elution.

[0015] The sesquiterpene compound can be used in the preparation of anti-inflammatory drugs.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application provides a highly oxidized sesquiterpene compound from white mushrooms, which has a significant inhibitory effect on interleukin-6 at a concentration of 25 muM, with an inhibition rate of 71.6%, and can be used in the preparation of anti-inflammatory drugs. Therefore, the highly oxidized sesquiterpene compound provided by the present application has clinical application potential for anti-inflammatory treatment. DETAILED DESCRIPTION

[0018] The application will be further described in conjunction with specific examples, which are used to explain the application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0019] Example 1

[0020] A preparation method of a highly oxidized sesquiterpenoid compound from a white mushroom, the highly oxidized sesquiterpenoid compound being isolated from fresh fruiting bodies of the white mushroom; the fresh fruiting bodies of the white mushroom being provided by Bijie City Agricultural Investment Fungus Co., Ltd.

[0021] The specific preparation method of the highly oxidized sesquiterpenoid compound is as follows:

[0022] S1. The white mushroom fruiting bodies are repeatedly extracted with methanol, and the extract is concentrated to obtain an extract residue, and the extract residue is obtained with ethyl acetate to obtain an ethyl acetate crude extract.

[0023] S2. The ethyl acetate crude extract is separated by silica gel normal phase chromatography; eluted with petroleum ether / ethyl acetate, and then separated by silica gel, gel, and C-18 reverse phase column chromatography to obtain the highly oxidized sesquiterpenoid compound of the general formula.

[0024] Example 2

[0025] The new compound is subjected to structural test analysis, and the following experimental data are obtained:

[0026] New compound I: C 15 H 18 O7, HRESI-MS: 333.0948 [M+Na] + (calculated value 333.0945).

[0027] Table 1. NMR data of compound I (CD3OD, 600MHz / 150MHz, ppm)

[0028]

[0029]

[0030] According to the above data results, the structural formula of compound I is as follows:

[0031]

[0032] Example 3

[0033] IL-6 expression inhibition screening model of compound I

[0034] 1) Total RNA extraction

[0035] The cells were randomly divided into normal control group, stimulation group and drug group, and 3 replicates were set up in each group. The normal control group was cultured with complete culture medium, the stimulation group was stimulated by adding LPS (final concentration 1 μg / mL), and the drug group was added with LPS (final concentration 1 μg / mL) and drugs, respectively. After incubation in an incubator for 12 h, the culture medium was discarded, and 1 mL of TRIzol lysis was added to extract total RNA. The total method is as follows: take the above cell lysate, transfer to a centrifuge tube, and stand for 5 min. Add 0.2 mL of chloroform, shake well, stand for 5 min on ice, centrifuge at 4°C, 12000 rpm for 15 min. Transfer the supernatant to a new centrifuge tube, add 0.8 mL of isopropanol, mix well, stand for 30 min on ice, centrifuge at 4°C, 10000 rpm for 10 min. Carefully remove the supernatant, add 0.5 mL of 75% ethanol / water to wash the precipitate, centrifuge at 4°C, 7500 rpm for 5 min. Carefully remove the supernatant, dry on ice for 5-10 min, add 20 μL of DEPC-H2O to dissolve, and store in a -70°C refrigerator. RNA quality detection: 1% agarose gel electrophoresis to determine the concentration of RNA, and ultraviolet spectrophotometer to detect the ratio of A 260 and A 280 .

[0036] 2) Reverse transcription reaction

[0037] First step: remove genomic DNA, room temperature for 5 min, then store at 4°C for standby. Reaction solution preparation method: 5×gDNAEraser Buffer, 2.0 μL; gDNAErase, 1.0 μL; Total RNA, *1; RNase FreedH2O, Up to 10 μL.

[0038] Second step: reverse transcription reaction, 37°C for 15 min; 85 degrees for 5 seconds; 4°C for standby. Prepare the reaction solution and operate on ice. Reverse transcription reaction enzyme and inhibitor ratio: reaction solution of step 1, 10.0 μL; Prime Script RT Enzyme Mix 1, 1.0 μL; RT Primer Mix *4 , 1.0 μL, 5×Prime Script Buffer 2 (for Real Time), 4.0 μL; RNase Free dH2O, 4.0 μL; Toal 20 μL *5 .

[0039] 3) Real-time quantitative PCR determination

[0040] The products were quantitatively analyzed by using 10 μL reaction system, denaturation temperature 95 °C, 10 min; 95 °C, 15 s, annealing temperature 60 °C, 30 s, extension 72 °C, 30 s, 40 cycles in total. The difference between the CT value of the target gene and the internal reference gene GADPH in the same sample, namely the so-called ΔCT value, was used for the relative quantitative detection of the gene in the sample according to the difference of the ΔCT value of the same gene in different samples. The screening results are shown in Table 2. -△△CT

[0041] Table 2: Screening data table

[0042] Compound No. IL-6 (25 μM) I 71.6％ Dexamethasone IL-6 (25 μM) Dexamethasone IL-6 (25 μM 85.9％

[0043] The results show that the compound I has strong inhibitory activity on the expression of IL-6, and the inhibition rate reaches 71.6% at a concentration of 25 μM.

[0044] The part not described in the present application is the known technology of the person skilled in the art. Finally, it is stated that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.​

Claims

1. A sesquiterpenoid compound, characterized in that: The structural formula of the sesquiterpenoid compound is: 。 2. The method for preparing a sesquiterpenoid compound according to claim 1, wherein: The method comprises the following steps: S1. The fruiting bodies of white ginseng fungus were extracted with methanol, and the extract was concentrated to obtain an extract, which was extracted with ethyl acetate to obtain a crude ethyl acetate extract; S2. The crude ethyl acetate extract was separated by normal phase chromatography on silica gel followed by gradient elution, and then separated by silica gel, gel, and C-18 reverse phase column chromatography to obtain the sesquiterpenoid compound.

3. The method for preparing a sesquiterpenoid compound according to claim 2, wherein: In step S1, the number of methanol extractions is 2-5 times, and the extraction time for one time is 10 days.

4. The method for preparing a sesquiterpenoid compound according to claim 2, wherein: In step S2, the solvent used for gradient elution is petroleum ether / ethyl acetate, and after elution, 10% to 80% ethyl acetate / petroleum ether fractions are collected.

5. The use of the sesquiterpenoid compound according to claim 1, characterized in that: The sesquiterpenoid compound is used for preparing anti-inflammatory drugs.

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