A heteroterpene compound, a preparation method thereof and application thereof

Abstract

The application discloses a kind of heteroterpene compounds and preparation method and its application, the molecular formula of the heteroterpene compound is: C 26 H 26 N2O5.The preparation method of the heteroterpene compound comprises the following steps: S1, white ginseng fungus fruiting body is extracted with methanol, and the extract is concentrated to obtain extract, and the extract is obtained with ethyl acetate to obtain ethyl acetate crude extract;S2, after being separated by silica gel normal phase chromatography, gradient elution is carried out, and 8 components (Fr.1-Fr.8) with polarity from small to large are obtained;S3, component Fr.2 is separated by column chromatography of glucose gel, and dichloromethane:methanol is used as eluent with 1:1, after elution, continue to elute with dichloromethane:methanol and ethyl acetate:methanol, and the pure heteroterpene compound is separated by silica gel chromatographic column separation.The compound has significant inhibitory activity on LPS-induced NO production, with IC 50 25.2 μM, and has the potential for clinical application of anti-inflammatory treatment, and can be used for preparing anti-inflammatory drugs.

Description

TECHNICAL FIELD

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

[0002] Inflammation is a defensive response of the body to various inflammatory factors, and plays an important role in immune response. The occurrence of inflammation in local tissues will produce a series of complex changes, and the main clinical manifestations are redness, swelling, heat, pain and dysfunction. At the same time, inflammation can cause systemic reactions, including fever, lethargy, accelerated muscle protein degradation, etc. Inflammation is also an important factor in the aging process of the human body, and is closely related to many chronic diseases such as arthritis, cardiovascular disease, cancer, osteoporosis, asthma, Alzheimer's disease, dementia, obesity and type II diabetes, etc. At present, the mainstream inflammation pathogenesis believes that macrophages and the inflammatory mediators released by them are the key to triggering inflammation. Macrophages are an important immune cell in the body, are the main cells that initiate the production of inflammatory mediators in the body, and have the functions of anti-infection, anti-tumor and immune regulation. Macrophages can be activated by various inflammatory factors, such as cytokines, bacterial lipopolysaccharide LPS, extracellular matrix proteins and other chemical mediators, etc. LPS is a very important inflammatory factor, which can stimulate the synthesis and release of various endogenous active factors by macrophages in the body, and then induce inflammation. Using LPS to treat macrophages is a commonly used means of modeling in vitro inflammation model.

[0003] At present, the commonly used anti-inflammatory drugs in clinical practice mainly include non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs. Although both types of anti-inflammatory drugs have certain clinical anti-inflammatory effects, long-term and large-dose use will cause a series of adverse reactions and resistance, such as gastric mucosa damage, liver damage, kidney damage, etc. Therefore, finding and developing safer and more efficient anti-inflammatory drugs has become a hot spot in the field of anti-inflammatory drug research.

[0004] 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, and tonifying and invigorating the body, and has obvious curative effects on symptoms such as infantile night sweat, neurasthenia, dizziness and tinnitus, gynecological diseases, etc. Research reports that polysaccharide compounds isolated from white shiitake mushroom water extract have anti-tumor, anti-inflammatory and anti-bacterial activities, etc. These polysaccharide compounds can significantly inhibit the production of NO by macrophages 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

[0005] The present application aims to provide a highly oxidized heteroterpene compound from white shiitake mushroom.

[0006] Another object of the present application is to provide a preparation method of the highly oxidized heteroterpene compound from the white mushroom.

[0007] Another object of the present application is to provide an application of the highly oxidized heteroterpene compound from the white mushroom in preparing anti-inflammatory drugs.

[0008] The technical solution of the present application is a heteroterpene compound, the structural general formula of which is:

[0009] The molecular formula is: C 26 H 26 N2O5.

[0010] The heteroterpene compound is isolated from fresh fruiting bodies of the white mushroom, which is provided by Bijie City Agricultural Investment Fungus Co., Ltd.

[0011] A preparation method of a heteroterpene compound, the method comprising the following steps:

[0012] S1. Extracting the fruiting bodies of the white mushroom with methanol, concentrating the extract to obtain an extract, and obtaining an ethyl acetate crude extract by extracting the extract with ethyl acetate;

[0013] S2. Separating the ethyl acetate crude extract by silica gel normal phase chromatography and performing gradient elution, to obtain eight components (Fr.1-Fr.8) with increasing polarity from small to large;

[0014] S3. Separating component Fr.2 by column chromatography of glucose gel (Sephadex TM LH-20) and using dichloromethane:

[0015] methanol (v / v; 60:1) and ethyl acetate:methanol (v / v; 90:1) as elution solvents, and continuing to elute after the elution is completed.

[0016]

[0017] In the step S1, the number of methanol extraction is 2-5 times, and the extraction time of each time is 10 days.

[0018] In the 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.

[0019] The application of a heteroterpene compound, which can be applied to prepare anti-inflammatory drugs.

[0020] ​Compared with the prior art, the present application has the following beneficial effects:

[0021] The present application provides a heteroterpene compound from white mushroom, which has significant inhibitory activity on LPS-induced NO production, with an IC 50 25.2 μM, and can be used for preparing anti-inflammatory drugs. Therefore, the heteroterpene compound provided by the present application has clinical application potential for anti-inflammatory treatment. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below in combination with specific examples, which are only used to explain the present application and are not used to limit the scope of the present application. In the following examples, the test methods are conventional methods unless otherwise specified, and the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0023] Example 1

[0024] A preparation method of a heteroterpene compound from white mushroom, which is isolated from fresh fruiting bodies of white mushroom; the fresh fruiting bodies of white mushroom are provided by Bijie City Nongtou Fungus Industry Science and Technology Co., Ltd.

[0025] A specific preparation method of the heteroterpene compound is as follows:

[0026] 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 by extraction with ethyl acetate to obtain an ethyl acetate crude extract.

[0027] S2. The ethyl acetate crude extract is separated by silica gel normal phase chromatography; gradient elution is performed with petroleum ether / ethyl acetate to obtain 8 components (Fr. 1-Fr. 8) with increasing polarity.

[0028] S3. The component Fr. 2 is separated by column chromatography with Sephadex TM LH-20 and eluted with dichloromethane:methanol (1:1) as an eluent. After elution, further separation is performed by silica gel chromatography with dichloromethane:methanol (v / v; 60:1) and ethyl acetate:methanol (v / v; 90:1) as eluents to obtain the purified compound I.

[0029] Example 2

[0030] The new compound I is subjected to structural test analysis, and the following experimental data is obtained:

[0031] New compound I: C 26 H 26 N2O5, HRESI-MS: 469.1736 [M+H] +(Calculated 469.1734) Table 1. NMR data of compound I (CDCl3, 500 MHz / 125 MHz, ppm)

[0032]

[0033]

[0034] According to the data results of Table 1, the structural formula of the new compound I is as follows:

[0035]

[0036] Example 3

[0037] Anti-inflammatory cell screening model of compound I

[0038] 1. Cell culture and treatment

[0039] RAW 264.7 cells (Shanghai Institute of Biochemistry and Cell Biology Cell Bank) were cultured in vitro using DMEM high-sugar medium containing 10% FBS, and were routinely maintained and subcultured at 37°C and a 5% carbon dioxide concentration.

[0040] 2. Compound intervention

[0041] (1) Experimental method

[0042] The density of RAW 264.7 cells was adjusted to 1×10 5 cells / well (volume 1 mL) and was in the logarithmic growth phase, and LPS (final concentration 1 μg / mL) was added to induce macrophages to be in an inflammatory state.

[0043] Compound I indomethacin was prepared into different drug concentrations using DMSO, 3 parallel duplicate wells were set for each concentration, and positive control wells (only LPS was added), negative control wells (cells and medium), and blank control wells (medium) were set.

[0044] After 24 hours of culture, 50 μL of cell supernatant was taken and added to a new 96-well plate, 50 μL of reagent I of the NO detection kit was added, and the content of NO was determined by the Griess method.

[0045] Inhibition rate = ([NO2 - ] LPS -[NO2 - ] LPS+sample ) / ([NO2 - ] LPS -[NO2 - ] untreated )×100

[0046] (2) Results are shown in Table 2:

[0047] Table 2 Survival rate data table

[0048]

[0049] The results show that compound I exhibits better NO production inhibition activity than the positive drug indomethacin, and its IC 50 is 25.2 μM.

[0050] 3. Effect of compound on cell viability

[0051] MTT can be reduced to water-insoluble blue crystals by succinate dehydrogenase in the mitochondria of living cells, and deposited in living cells, while dead cells do not have this function.

[0052] (1) Experimental method

[0053] Take RAW 264.7 cells in the logarithmic growth phase, inoculate 1 x 10 5 cells / well in a 96-well plate, 100 μL per well. Add LPS (final concentration 1 μg / mL) to induce macrophages to be in an inflammatory state.

[0054] After 24 hours of culture, add 50 μL of 1 mg / mL MTT solution to each well, and after 4 hours of culture, aspirate the culture medium, and add 150 μL of DMSO to each well. Shake well. Measure the absorbance value (A) at 490 nm wavelength using a microplate reader. The inhibition of cell growth by the drug is expressed as survival rate, and the higher the survival rate, the lower the drug toxicity.

[0055] Survival rate (%) = [(A1-A0) / (A2-A0)] x 100%, where A0 is the absorbance value of the blank control, A1 is the absorbance value of the sample, and A2 is the absorbance value of the positive control well.

[0056] Determine the sample at 5 concentrations, draw the dose-inhibition rate curve, and obtain the IC 50 value. Each sample is measured in triplicate.

[0057] (2) Results are shown in Table 2

[0058] The results show that compound I does not show cytotoxic activity at a concentration of 25 μM, indicating that it has good potential for further preclinical research.

[0059] The details of the present application are not described, which are known to those skilled in the art. Finally, it is pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A terpenoid compound, characterized in that: The general structural formula of the heteroterpenoid compound is: The molecular formula is: C 26 H 26 N2O5.

2. The method for preparing a heteroterpenoid compound according to claim 1, wherein: The method comprises the following steps: S1. Extracting the fruiting bodies of white ginseng fungus with methanol, concentrating the extract to obtain an extract, and extracting the extract 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 and then subjected to gradient elution to obtain eight fractions Fr.1 to Fr.8 in increasing polarity; S3. Component Fr.2 was separated by column chromatography using glucose gel and an elution solvent of 1:1 dichloromethane:methanol. After the elution was completed, it was further separated by a silica gel column with an elution polarity of 60:1 dichloromethane:methanol and 90:1 ethyl acetate:methanol to obtain pure terpenoid compounds.

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

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

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