A class of sesquiterpenoids derived from *Tremella fuciformis* Berk. and their preparation methods and applications in the preparation of anti-inflammatory drugs

Abstract

The present invention discloses a class of sesquiterpenoid compounds derived from white ginseng fungus and its preparation method and application in the preparation of anti-inflammatory drugs. The sesquiterpenoid compounds disclosed in the present invention, including compound I and compound II, all have good anti-inflammatory activity, and can significantly inhibit the expression of macrophage interleukin-6 (IL-6) and interleukin-1β (IL-1β) at a concentration of 25μM, and the inhibition rates of compound I to IL-6 and IL-1β are 67.9% and 65.8% respectively, and the inhibition rates of compound II to IL-6 and IL-1β are 86.0% and 70.3% respectively. Therefore, the sesquiterpenoid compounds provided by the present invention can be used for the preparation of anti-inflammatory drugs, with the clinical application potential of anti-inflammatory treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical compounds, and more specifically relates to a class of sesquiterpenoid compounds derived from white ginseng fungus, a preparation method thereof, and an application thereof in the preparation of anti-inflammatory drugs. Background Art

[0002] Inflammation is the body's defensive response to various inflammatory factors and plays an important role in the immune response. Current research generally believes that the occurrence of inflammation is mainly related to macrophages and the inflammatory mediators they release. Macrophages are an important immune cell in the body and can be activated by a variety of inflammatory stimuli, such as cytokines, bacterial lipopolysaccharides, extracellular matrix proteins and other chemical mediators. Among them, bacterial lipopolysaccharide (LPS), also known as endotoxin, has a strong effect in activating mononuclear macrophages, which can activate macrophages and induce them to synthesize and release a variety of inflammatory mediators, such as pro-inflammatory cytokines IL-6, IL-1β, etc., leading to a series of inflammatory reactions in the body.

[0003] Currently, commonly used anti-inflammatory drugs in clinical practice primarily include nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SADs). While both classes of anti-inflammatory drugs have some clinical anti-inflammatory effects, long-term and excessive use can lead to a range of adverse reactions and intolerance, such as gastric mucosal damage, liver damage, and kidney damage. Therefore, the search for and development of safer and more effective anti-inflammatory drugs has become a hot topic in the field of anti-inflammatory drug research.

[0004] White ginseng mushroom (also known as Schizophyllum commune Fr.) is a rare mushroom used as both a medicine and a food. According to Traditional Chinese Medicine (TCM), it has the benefits of clearing the liver and improving eyesight, as well as nourishing and strengthening the body. It is particularly effective in treating pediatric night sweats, neurasthenia, dizziness, tinnitus, and gynecological disorders. As can be seen, white ginseng mushrooms contain multiple active ingredients, and their full utilization can increase the overall utilization value of white ginseng mushrooms. The study "Study on the Anti-inflammatory Effect of Crude Polysaccharides from Liquid Fermentation of Schizophyllum Commune Fr.," by Liu Weifeng, extracted Schizophyllum polysaccharides from the fruiting bodies, mycelium, and fermentation broth of Schizophyllum commune Fr., demonstrating that high doses of Schizophyllum polysaccharides exhibit anti-inflammatory effects. However, the Schizophyllum polysaccharide used in this study was a crude extract that required fermentation, and high doses were required for its anti-inflammatory activity to be effective.

[0005] Therefore, developing new anti-inflammatory drugs by exploring more active ingredients from white ginseng mushrooms has important medicinal and economic value. Furthermore, white ginseng mushrooms are a common food ingredient that does not cause adverse reactions in the human body, making them highly safe as a raw material for drug development. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of sesquiterpenoid compounds derived from white ginseng fungus.

[0007] Another object of the present invention is to provide a method for preparing the sesquiterpenoid compounds derived from the above-mentioned white ginseng fungus.

[0008] Another object of the present invention is to provide the use of the sesquiterpenoid compounds derived from the white ginseng fungus in the preparation of anti-inflammatory drugs.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention extracts a new type of sesquiterpenoid compound from the fruiting body of white ginseng fungus, and its structural formula is shown in formula (I). In formula (I), R=OH or R=H

[0011]

[0012] That is, it includes two compounds, compound I and compound II, whose structural formulas are shown in Formula I or Formula II respectively:

[0013]

[0014] Therefore, the present invention applies to protect the above-mentioned sesquiterpenoid compounds.

[0015] The sesquiterpenoid compounds of the present invention have good anti-inflammatory activity and can be used for the preparation of anti-inflammatory drugs. Therefore, the present invention also claims the use of the sesquiterpenoid compounds derived from the white ginseng fungus in the preparation of anti-inflammatory drugs.

[0016] Based on this, an application is also made to protect anti-inflammatory drugs containing the above-mentioned sesquiterpenoid compounds.

[0017] In addition, although the above-mentioned sesquiterpenoid compounds are isolated from the fruiting bodies of the white ginseng fungus, in actual application, they can be isolated from the fruiting bodies of the white ginseng fungus or obtained through other production means, such as isolation from other organisms or chemical synthesis, which should be possible.

[0018] Specifically, the present invention provides a method for separating the above-mentioned sesquiterpenoid compounds from the fruiting body of the white ginseng fungus.

[0019] As a preferred embodiment, the method for isolating the above-mentioned sesquiterpenoid compounds from the fruiting body of the white ginseng fungus comprises the following steps:

[0020] S1. Extracting the fruiting body of the 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;

[0021] S2. The crude ethyl acetate extract of step S1 was separated by normal phase chromatography on silica gel: eluted with petroleum ether / ethyl acetate to obtain an eluent;

[0022] S3. The eluate from step S2 is separated by gel, silica gel, or C-18 reverse phase column chromatography to obtain the sesquiterpenoid compound represented by formula (I).

[0023] Specifically preferably, the specific method of step S3 is as follows:

[0024] S31 The eluate obtained in step S2 was subjected to glucose gel column chromatography: using dichloromethane / methanol as an eluent for elution and separation; after the elution is completed, the eluate was collected;

[0025] S32. The eluate obtained in step S31 was separated by silica gel chromatography: dichloromethane / methanol and ethyl acetate / methanol were used as eluents to separate the elution, thereby obtaining a mixture of compound I and compound II;

[0026] S33. Using methanol / water as eluent, the mixture of compound I and compound II obtained in step S32 is separated by high performance liquid chromatography, and the eluate with a retention time of 12.0 to 12.6 min is collected to obtain a sesquiterpenoid compound represented by formula I; the eluate with a retention time of 14.5 to 15.0 min is collected to obtain a sesquiterpenoid compound represented by formula II.

[0027] Preferably, in step S1, the number of times of extracting the fruiting body of the white ginseng fungus with methanol is 2 to 3 times, and the time of each extraction is 8 to 10 days; and the extracts are combined.

[0028] More preferably, the methanol extraction of the white ginseng fungus fruiting body in step S1 is performed three times, and the extraction time is 10 days.

[0029] Preferably, the volume ratio of petroleum ether / ethyl acetate in step S2 is 3:7 (v / v).

[0030] Preferably, the volume ratio of dichloromethane / methanol in step S31 is 1:1 (v / v).

[0031] Preferably, the volume ratio of dichloromethane / methanol in step S32 is 4:1 to 5:1 (v / v), and the volume ratio of ethyl acetate / methanol is 9:1 to 10:1 (v / v).

[0032] More preferably, in step S32, the volume ratio of dichloromethane / methanol is 5:1 (v / v), and the volume ratio of ethyl acetate / methanol is 10:1 (v / v).

[0033] Preferably, the volume ratio of methanol / water in step S33 is 95:5 (v / v).

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention provides a class of sesquiterpenoid compounds derived from the fungus Leucorum leucogenum. These compounds can significantly inhibit the expression of interleukin-6 (IL-6) and interleukin-1β (IL-1β) at a concentration of 25 μM, with inhibition rates of 67.9% and 65.8% (Compound I), and 86.0% and 70.3% (Compound II), respectively. Therefore, the sesquiterpenoid compounds provided by the present invention can be used to prepare anti-inflammatory drugs and have clinical application potential for anti-inflammatory treatment. DETAILED DESCRIPTION

[0036] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0038] The fruiting bodies of the white ginseng mushroom used in the following examples were provided by Bijie Agricultural Investment Fungi Technology Co., Ltd.

[0039] Example 1 Preparation of Sesquiterpenoids

[0040] The preparation method steps are as follows:

[0041] 1. The fruiting body of the white ginseng fungus was extracted with methanol three times, each extraction time was 10 days; the three extracts were combined and concentrated to obtain a viscous extract, which was extracted with ethyl acetate to obtain 28.6 g of ethyl acetate crude extract.

[0042] 2. The crude ethyl acetate extract was separated using a silica gel chromatography column: elution was performed using a petroleum ether / ethyl acetate ratio of 3:7 by volume to obtain an eluent. (During the study, gradient elution was performed using petroleum ether / ethyl acetate ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, and 2:8 by volume, yielding eight fractions, Fr.1 to Fr.8, with increasing polarity. These eight fractions were subsequently purified and identified, yielding fraction Fr.7, which contained Compound I and Compound II. Therefore, a petroleum ether / ethyl acetate ratio of 3:7 by volume was selected as the eluent.)

[0043] 3. Separate the eluate obtained in step 2 by Sephadex™ LH-20 column chromatography using a 1:1 volume ratio of dichloromethane to methanol as the eluent for elution and separation; after elution, collect the eluate;

[0044] 4. The eluate obtained in step 3 was separated by silica gel chromatography: using dichloromethane / methanol with a volume ratio of 5:1 and ethyl acetate / methanol with a volume ratio of 10:1 as eluents, eluting and separating to obtain a mixture of compound I and compound II.

[0045] 5. The mixture of compound I and compound II obtained in step 4 was separated by high performance liquid chromatography using a C18 reversed phase column (Ultimate XB-C18, 5 μm, 10 × 250 mm). The separation conditions were as follows: using methanol / water with a volume ratio of 95:5 as the eluent, and collecting the eluents at retention times of 12.0 to 12.6 min and 14.5 to 15.0 min, respectively, to obtain the sesquiterpenoid compounds represented by Formula I and Formula II, respectively (the eluent at a retention time of 12.0 to 12.6 min is the sesquiterpenoid compound represented by Formula I; the eluent at a retention time of 14.5 to 15.0 min is the sesquiterpenoid compound represented by Formula II).

[0046] Example 2 Structural analysis of sesquiterpenoids

[0047] Structural analysis of new compounds I and II yielded the following experimental data:

[0048] New compound I:C 15 H 20 O6, HRESI-MS: 319.1155[M+Na] + (calculated value 319.1152);

[0049] New compound II: C 15 H 20 O5, HRESI-MS: 303.1199[M+Na] + (Calculated value 303.1203).

[0050] The NMR data of new compounds I and II are shown in Table 1.

[0051] Table 1 NMR data of compounds I and II (CD3OD, 600MHz / 150MHz, ppm)

[0052]

[0053]

[0054] Based on the above data, the structural formulas of Compound I and Compound II are shown in Formula I or Formula II, respectively:

[0055]

[0056] Example 3 Detection of anti-inflammatory activity of sesquiterpenoids

[0057] (1) Extraction of total RNA

[0058] RAW264.7 macrophages in the logarithmic growth phase were randomly divided into normal control group, stimulation group and drug treatment group, with 3 replicates in each group.

[0059] The normal control group was cultured in complete DMEM medium. The stimulation group was stimulated with LPS (final concentration 1 μg / mL). The drug-treated group was treated with LPS (final concentration 1 μg / mL) and 10 μL of drug (Compound I or II) (final concentration 25 μM). After 12 hours of incubation, the culture medium was discarded, and 1 mL of TRIzol was added to lyse the cells. Total RNA was extracted. The extraction method is as follows:

[0060] (1) Take the above cell lysate, transfer it into a centrifuge tube, and let it stand for 5 minutes;

[0061] (2) Add 0.2 mL of chloroform, shake and mix, let stand on ice for 5 min, and centrifuge at 12,000 rpm at 4°C for 15 min;

[0062] (3) Pipette the supernatant into a new centrifuge tube, add 0.8 mL of isopropanol and mix well, let it stand on ice for 30 min, and centrifuge at 4°C and 10,000 rpm for 10 min;

[0063] (4) Carefully remove the supernatant, add 0.5 mL of 75% ethanol solution to wash the precipitate, and centrifuge at 7500 rpm at 4°C for 5 min;

[0064] (5) Carefully remove the supernatant, dry on ice for 5-10 min, dissolve in 20 μL DEPC·H2O, and store in a -70°C refrigerator.

[0065] (6) RNA quality detection: The RNA concentration was determined by 1% agarose gel electrophoresis, and the ratio of A260 to A280 was measured by UV spectrophotometer.

[0066] (2) Reverse transcription reaction

[0067] 1. Removal of genomic DNA

[0068] The preparation method of the reaction solution is shown in Table 1.

[0069] Table 1

[0070]

[0071] The reaction solution was placed at room temperature for 5 min, and then the reaction product was stored at 4°C for later use.

[0072] 2. Reverse Transcription Reaction

[0073] (1) Prepare the reaction solution on ice according to the preparation method in Table 2.

[0074] Table 2

[0075]

[0076] (2) The prepared reaction solution was subjected to reverse transcription reaction according to the following reaction procedure:

[0077] 37℃15min;

[0078] 85℃5s;

[0079] Store at 4℃ until use.

[0080] 3. Real-time Quantitative PCR Assay

[0081] (1) Quantitative PCR was used to determine the changes in the expression levels of inflammatory factors (IL-6 and IL-1β): The reaction products of step 2 were subjected to q-PCR reaction. The specific steps are shown in Table 3 below:

[0082] Table 3

[0083]

[0084] (2) Quantitative analysis of each product

[0085] According to the instructions of the Promega quantitative kit (Cat. No. NG1201), the expression of the same gene in different samples was detected by 2 -ΔΔCT Based on the differences in values, relative quantitative detection of IL-6 and IL-1β genes in different samples was performed, and the detection results are shown in Table 4.

[0086] Table 4

[0087]

[0088]

[0089] IL-6 inhibition rate = ([IL-6] LPS -[IL-6] LPS+sample ) / ([IL-6] LPS -[IL-6] untreated )×100%

[0090] IL-1β inhibition rate = ([IL-1β] LPS -[IL-1β] LPS+sample ) / ([IL-1β] LPS -[IL-1β] untreated )×100%

[0091] in,

[0092] [IL-6] LPS and [IL-1β] LPS 2 of IL-6 and IL-1β in the stimulation group -ΔΔCT value;

[0093] [IL-6] LPS+sample and [IL-1β] LPS+sample 2 of IL-6 and IL-1β in the treatment group -ΔΔCT value;

[0094] [IL-6] untreated and [IL-1β] untreated 2 of IL-6 and IL-1β in the normal control group -ΔΔCT value.

[0095] The above results show that both Compound I and Compound II exhibited strong inhibitory activity against IL-6 expression, with inhibition rates reaching 67.9% and 86.0%, respectively, at a concentration of 25 μM. Compound II exhibited anti-inflammatory activity comparable to that of the positive drug dexamethasone. In addition, at a concentration of 25 μM, the inhibition rates of Compound I and Compound II against IL-1β were both above 50%, indicating that Compound I and Compound II also exhibited strong inhibitory activity against IL-1β expression.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A sesquiterpenoid compound, characterized in that: The structural formula of the sesquiterpenoid compound is shown in the following formula (I), wherein R=OH or R=H 2. The method for preparing the sesquiterpenoid compound according to claim 1, characterized in that: The sesquiterpenoid compound is separated and obtained from white ginseng fungus.

3. The method for preparing the sesquiterpenoid compound according to claim 1, characterized in that: The sesquiterpenoid compound is separated and obtained from the fruiting body of the white ginseng fungus.

4. The preparation method according to claim 3, characterized in that The preparation method of the sesquiterpenoid compound comprises the following steps: S1. Extracting the fruiting body of the 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 of step S1 was separated by normal phase chromatography on silica gel: eluted with petroleum ether / ethyl acetate to obtain an eluent; S3. The eluate from step S2 is separated by gel, silica gel, or C-18 reverse phase column chromatography to obtain the sesquiterpenoid compound represented by formula (I).

5. The preparation method according to claim 4, characterized in that The specific method of step S3 is as follows: S31 The eluate obtained in step S2 was subjected to glucose gel column chromatography: using dichloromethane / methanol as an eluent for elution and separation; after the elution is completed, the eluate was collected; S32. The eluate obtained in step S31 was separated by silica gel chromatography: dichloromethane / methanol and ethyl acetate / methanol were used as eluents to separate the elution, thereby obtaining a mixture of compound I and compound II; S33. The mixture of compound I and compound II obtained in step S32 was separated by high performance liquid chromatography using methanol / water as the eluent, and the eluate with a retention time of 12.0 to 12.6 min was collected to obtain a sesquiterpenoid compound represented by formula I; Collecting the eluate at a retention time of 14.5 to 15.0 minutes to obtain a sesquiterpenoid compound represented by formula II; Wherein, the structural formulas of compound I and compound II are shown in Formula I or Formula II below, respectively:

6. The preparation method according to claim 4, characterized in that The methanol extraction of the fruiting body of the white ginseng fungus in step S1 is performed 2 to 3 times, each time for 8 to 10 days.

7. The preparation method according to claim 4, characterized in that The volume ratio of petroleum ether / ethyl acetate in step S2 is 3:

7.

8. The preparation method according to claim 5, characterized in that In step S32, the volume ratio of dichloromethane to methanol is 4:1 to 5:1, and the volume ratio of ethyl acetate to methanol is 9:1 to 10:

1.

9. Use of the sesquiterpenoid compound according to claim 1 in the preparation of anti-inflammatory drugs.

10. An anti-inflammatory drug, characterized in that Contains the sesquiterpenoid compound according to claim 1.

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