CN117736219A - Sesquiterpenoids derived from white ginseng fungus, preparation method thereof and application thereof in preparing anti-inflammatory drugs - Google Patents
Sesquiterpenoids derived from white ginseng fungus, preparation method thereof and application thereof in preparing anti-inflammatory drugs Download PDFInfo
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a sesquiterpenoid from white ginseng fungus, a preparation method thereof and application thereof in preparing anti-inflammatory drugs. The sesquiterpenoids disclosed by the invention comprise a compound I and a compound II, have good anti-inflammatory activity, can obviously inhibit the expression of interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) of macrophages at the concentration of 25 mu M, the inhibition rates of the compound I on the IL-6 and the IL-1 beta are 67.9% and 65.8%, and the inhibition rates of the compound II on the IL-6 and the IL-1 beta are 86.0% and 70.3%, respectively. Therefore, the sesquiterpenoids provided by the invention can be used for preparing anti-inflammatory drugs and have clinical application potential of anti-inflammatory treatment.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical compounds. More particularly relates to sesquiterpenoids derived from white ginseng fungus, a preparation method thereof and application thereof in preparing anti-inflammatory drugs.
Background
Inflammation is a defensive response of the body to various inflammatory factors and plays an important role in the immune response. It is widely accepted that inflammation occurs mainly in association with macrophages and inflammatory mediators released by macrophages. Macrophages are an important immune cell in the body that can be activated by a variety of inflammatory stimuli such as cytokines, bacterial lipopolysaccharides, extracellular matrix proteins, and other chemical mediators, among others. Wherein, bacterial Lipopolysaccharide (LPS) is also called endotoxin, has strong effect of activating mononuclear macrophages, can activate macrophages and induce the macrophages to synthesize and release various inflammatory mediators, such as pro-inflammatory cytokines IL-6, IL-1 beta and the like, so that a series of inflammatory reactions occur in organisms.
The anti-inflammatory agents currently in common clinical use mainly include non-steroidal anti-inflammatory agents and steroidal anti-inflammatory agents. Although both of these anti-inflammatory agents have some clinical anti-inflammatory effects, a number of adverse effects and tolerability such as gastric mucosal injury, liver injury, kidney injury, etc. can result from prolonged use in large amounts. Therefore, searching and developing safer and more efficient anti-inflammatory drugs is a hotspot in the field of anti-inflammatory drug research.
The white ginseng fungus (also called schizophyllum commune, schizophyllum commune Fr.) is a rare mushroom fungus used as both medicine and food, and the theory of traditional Chinese medicine considers that the white ginseng fungus has the effects of clearing liver and improving vision, nourishing and strengthening body, and has obvious curative effects on children night sweat, neurasthenia, dizziness, tinnitus, gynecological diseases and the like. Therefore, the white ginseng fungus contains various active ingredients, and the comprehensive utilization value of the white ginseng fungus can be increased by fully utilizing the active ingredients. Studies on anti-inflammatory action of crude polysaccharide fermented by Schizophyllum commune liquid, liu Weifeng, the schizophyllum commune polysaccharide is extracted from fruiting bodies, mycelia and fermentation liquor of Schizophyllum commune (Schizophyllum commune Fr.), and the study shows that the schizophyllum commune polysaccharide with high dosage has anti-inflammatory action. However, schizophyllan in this study is a crude extract, which is subjected to fermentation, and moreover, high doses are required for the development of anti-inflammatory activity.
Therefore, the development of novel anti-inflammatory drugs by mining more active ingredients of white ginseng fungus has important medicinal and economic values. Moreover, the white ginseng fungus is a common food material, does not cause adverse reaction to human bodies, and has high safety as a raw material for developing medicaments.
Disclosure of Invention
The invention aims to provide sesquiterpenoids derived from white ginseng fungus.
The invention also aims to provide a preparation method of the sesquiterpenoids derived from the white ginseng fungus.
The invention also aims to provide application of the sesquiterpenoids derived from the white ginseng fungus in preparing anti-inflammatory drugs.
The above object of the present invention is achieved by the following technical scheme:
the invention extracts a new sesquiterpene compound from fruiting bodies of white ginseng fungus, the structural formula of the sesquiterpene compound is shown as a formula (I), wherein R=OH or R=H
Namely, the compound comprises two compounds, namely a compound I and a compound II, and the structural formulas of the compound I and the compound II are shown in the formula I or the formula II respectively:
accordingly, the present application protects the above sesquiterpenoids.
The sesquiterpenoids have good anti-inflammatory activity and can be used for preparing anti-inflammatory drugs, so the invention also claims the application of the sesquiterpenoids from the white ginseng fungus in preparing anti-inflammatory drugs.
Based on this, anti-inflammatory drugs containing the above sesquiterpenoids are also claimed.
In addition, although the sesquiterpenoids are isolated from the fruiting body of the white ginseng fungus, in practical application, they may be isolated from the fruiting body of the white ginseng fungus or obtained by other production means, such as isolation from other organisms or chemical synthesis.
In particular, the invention provides a method for separating the sesquiterpenoids from the fruiting bodies of the white ginseng fungus.
As a preferred embodiment, the method for separating the sesquiterpenoids from the fruiting bodies of white ginseng comprises the following steps:
s1, leaching fruiting bodies of white ginseng fungus with methanol, concentrating an extracting solution to obtain an extract, and extracting the extract with ethyl acetate to obtain an ethyl acetate crude extract;
s2, separating the ethyl acetate crude extract obtained in the step S1 by silica gel normal phase chromatography: eluting with petroleum ether/ethyl acetate to obtain eluent;
s3, separating the eluent in the step S2 through gel, silica gel and C-18 reversed phase column chromatographic separation technology to obtain the sesquiterpene compound shown in the formula (I).
The specific method of step S3 is specifically preferably as follows:
s31, performing glucose gel column chromatography separation on the eluent obtained in the step S2: eluting and separating with dichloromethane/methanol as eluent; after the elution is finished, collecting eluent;
s32, separating the eluent obtained in the step S31 by a silica gel chromatographic column: eluting with dichloromethane/methanol and ethyl acetate/methanol as eluent, and separating to obtain a mixture of the compound I and the compound II;
s33, using methanol/water as an eluent, carrying out high performance liquid chromatography resolution on the mixture of the compound I and the compound II obtained in the step S32, and collecting eluent with retention time of 12.0-12.6 min to obtain sesquiterpenoids shown in the formula I; collecting the eluent with retention time of 14.5-15.0 min to obtain sesquiterpenoids shown in formula II.
Preferably, the number of times of leaching the white ginseng fungus fruiting body with methanol in the step S1 is 2-3, and the time of leaching is 8-10 days; the extracts were combined.
More preferably, the number of times of leaching the fruiting body of the white ginseng fungus with methanol in step S1 is 3, and the time of one leaching is 10 days.
Preferably, the volume ratio of petroleum ether/ethyl acetate in step S2 is 3:7 (v/v).
Preferably, the volume ratio of dichloromethane/methanol in step S31 is 1:1 (v/v).
Preferably, the volume ratio of dichloromethane to methanol in the step S32 is 4:1-5:1 (v/v), and the volume ratio of ethyl acetate to methanol is 9:1-10:1 (v/v).
More preferably, the volume ratio of dichloromethane/methanol in step S32 is 5:1 (v/v), and the volume ratio of ethyl acetate/methanol is 10:1 (v/v).
Preferably, the volume ratio of methanol/water in step S33 is 95:5 (v/v).
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a sesquiterpenoid compound from white ginseng fungus, which can obviously inhibit the expression of interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) at the concentration of 25 mu M, and the inhibition rates are 67.9% and 65.8% (compound I), 86.0% and 70.3% (compound II) respectively. Therefore, the sesquiterpenoids provided by the invention can be used for preparing anti-inflammatory drugs and have clinical application potential of anti-inflammatory treatment.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Unless otherwise indicated, reagents and materials used in the following examples were those available commercially.
The white ginseng fungus fruiting bodies used in the following examples are provided by the company of the industrial scientific and technological Co.Ltd.
EXAMPLE 1 preparation of sesquiterpenoids
The preparation method comprises the following steps:
1. extracting fruiting body of white ginseng fungus with methanol for three times, wherein each extraction time is 10 days; combining the three leaching solutions, concentrating to obtain a viscous extract, and extracting the extract with ethyl acetate to obtain 28.6g of ethyl acetate crude extract.
2. Separating the ethyl acetate crude extract by using a silica gel chromatographic column: and eluting by using petroleum ether/ethyl acetate with the volume ratio of 3:7 as an eluent to obtain an eluent. (in the research process, gradient elution is carried out on petroleum ether/ethyl acetate with the volume ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7 and 2:8 in sequence to obtain 8 components Fr.1-Fr.8 with the polarity from small to large, and subsequent purification and identification are carried out on the 8 components, wherein the obtained component Fr.7 contains a compound I and a compound II, so that petroleum ether/ethyl acetate with the volume ratio of 3:7 is selected as an eluent.)
3. Separating the eluent obtained in the step 2 by using a glucose gel (Sephadex (TM) LH-20) column chromatography: eluting and separating by using methylene dichloride/methanol with the volume ratio of 1:1 as an eluent; after the elution is finished, collecting eluent;
4. separating the eluent obtained in the step 3 by using a silica gel chromatographic column: eluting and separating sequentially with dichloromethane/methanol with volume ratio of 5:1 and ethyl acetate/methanol with volume ratio of 10:1 as eluent to obtain a mixture of the compound I and the compound II.
5. Subjecting the mixture of compound I and compound II obtained in step 4 to high performance liquid chromatography using a C18 reverse column (Ultimate XB-C18,5 μm, 10X1250 mm) under the following conditions: collecting eluents when the retention time is respectively 12.0-12.6 min and 14.5-15.0 min by taking methanol/water with the volume ratio of 95:5 as an eluent, namely the sesquiterpenoids shown in the formula I and the formula II are respectively obtained (the eluent when the retention time is 12.0-12.6 min is the sesquiterpenoids shown in the formula I, and the eluent when the retention time is 14.5-15.0 min is the sesquiterpenoids shown in the formula II).
EXAMPLE 2 structural resolution of sesquiterpenoids
And (3) carrying out structural test analysis on the novel compounds I and II to obtain the following experimental data:
novel compounds I: c (C) 15 H 20 O 6 ，HRESI-MS：319.1155[M+Na] + (calculated 319.1152);
novel compounds II: c (C) 15 H 20 O 5 ，HRESI-MS：303.1199[M+Na] + (calculated 303.1203).
The NMR data for novel compounds I and II are shown in Table 1.
TABLE 1 NMR data for Compounds I and II (CD 3 OD，600MHz/150MHz，ppm)
From the above data, it was confirmed that the structural formulas of the compound I and the compound II are shown in the following formula I or formula II, respectively:
EXAMPLE 3 detection of anti-inflammatory Activity of sesquiterpenoids
Extraction of Total RNA
RAW264.7 macrophages in the logarithmic growth phase were randomly divided into a normal control group, a stimulated group and an administration group, each group being provided with 3 duplicate wells.
The normal control group was cultured with DMEM complete medium, the stimulation group was stimulated with LPS (final concentration 1. Mu.g/mL), the administration group was respectively added with LPS (final concentration 1. Mu.g/mL) and 10. Mu.L of the drug (Compound I or II) (final concentration 25. Mu.M), after culturing in an incubator for 12 hours, the medium was discarded, and 1mL of TRIzol was added for cleavage to extract total RNA. The total extraction method is as follows:
(1) Transferring the cell lysate into a centrifuge tube, and standing for 5min;
(2) Adding 0.2mL of chloroform, shaking and mixing, standing on ice for 5min, and centrifuging at 12000rpm at 4 ℃ for 15min;
(3) Transferring the supernatant into a new centrifuge tube, adding 0.8mL of isopropanol, mixing, standing on ice for 30min, and centrifuging at 4 ℃ and 10000rpm for 10min;
(4) Carefully remove the supernatant, add 0.5mL 75% ethanol solution to wash the precipitate, 4 ℃, 7500rpm centrifugal 5min;
(5) Carefully removing the supernatant, drying on ice for 5-10min, adding 20 μL DEPC.H 2 O is dissolved and stored in a refrigerator at the temperature of-70 ℃;
(6) And (3) RNA quality detection: the concentration of RNA was determined by 1% agarose gel electrophoresis, and the ratio of A260 to A280 was measured by an ultraviolet spectrophotometer.
(II) reverse transcription reaction
1. Removal of genomic DNA
The preparation method of the reaction solution is shown in Table 1.
TABLE 1
The reaction solution is placed at room temperature for reaction for 5min, and then the reaction product is placed at 4 ℃ for standby.
2. Reverse transcription reaction
(1) The reaction solution was prepared on ice according to the preparation method shown in Table 2.
TABLE 2
(2) The prepared reaction solution is subjected to reverse transcription according to the following reaction procedures:
37℃15min；
85℃5s；
preserving at 4 ℃ for standby.
3. Real-time quantitative PCR assay
(1) The change in the expression level of the cytokines (IL-6 and IL-1. Beta.) was measured by q-PCR: q-PCR was performed on the reaction product of step 2, and the specific procedure is shown in Table 3 below:
TABLE 3 Table 3
(2) Quantitative analysis of the products
According to the procedure of the Promega company quantitative kit (cat# NG 1201) instruction, according to the same gene 2 in different samples -ΔΔCT The difference in the values was measured for the relative quantification of IL-6 and IL-1. Beta. Genes in different samples, and the results are shown in Table 4.
TABLE 4 Table 4
IL-6 inhibition = ([ IL-6)] LPS -[IL-6] LPS+sample )/([IL-6] LPS -[IL-6] untreated )×100％
IL-1β inhibition rate= ([ IL-1β)] LPS -[IL-1β] LPS+sample )/([IL-1β] LPS -[IL-1β] untreated )×100％
Wherein,
[IL-6] LPS and [ IL-1 beta ]] LPS 2 of the stimulated groups IL-6 and IL-1 beta, respectively -ΔΔCT A value;
[IL-6] LPS+sample and [ IL-1 beta ]] LPS+sample 2 of IL-6 and IL-1 beta, respectively, in the administration group -ΔΔCT A value;
[IL-6] untreated and [ IL-1 beta ]] untreated 2 of IL-6 and IL-1β, respectively, of the normal control group -ΔΔCT Values.
The results show that both the compound I and the compound II show strong IL-6 expression inhibition activity, and the inhibition rate reaches 67.9% and 86.0% respectively at the concentration of 25 mu M, and the compound II has anti-inflammatory activity equivalent to that of positive medicament dexamethasone. In addition, at the concentration of 25 mu M, the inhibition rate of the compound I and the compound II on IL-1 beta is more than 50%, which shows that the compound I and the compound II also show stronger IL-1 beta expression inhibition activity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A sesquiterpene compound is characterized in that the structural formula of the sesquiterpene compound is shown as the following formula (I), wherein R=OH or R=H
2. The process for producing a sesquiterpene compound according to claim 1, wherein the sesquiterpene compound is isolated from a white ginseng fungus.
3. The process for producing a sesquiterpene compound according to claim 1, wherein the sesquiterpene compound is isolated from a fruiting body of white ginseng.
4. A method of preparing a sesquiterpene compound according to claim 3, comprising the steps of:
s1, leaching fruiting bodies of white ginseng fungus with methanol, concentrating an extracting solution to obtain an extract, and extracting the extract with ethyl acetate to obtain an ethyl acetate crude extract;
s2, separating the ethyl acetate crude extract obtained in the step S1 by silica gel normal phase chromatography: eluting with petroleum ether/ethyl acetate to obtain eluent;
s3, separating the eluent in the step S2 through gel, silica gel and C-18 reversed phase column chromatographic separation technology to obtain the sesquiterpene compound shown in the formula (I).
5. The preparation method according to claim 4, wherein the specific method of step S3 is as follows:
s31, performing glucose gel column chromatography separation on the eluent obtained in the step S2: eluting and separating with dichloromethane/methanol as eluent; after the elution is finished, collecting eluent;
s32, separating the eluent obtained in the step S31 by a silica gel chromatographic column: eluting with dichloromethane/methanol and ethyl acetate/methanol as eluent, and separating to obtain a mixture of the compound I and the compound II;
s33, using methanol/water as an eluent, carrying out high performance liquid chromatography resolution on the mixture of the compound I and the compound II obtained in the step S32, and collecting eluent with retention time of 12.0-12.6 min to obtain sesquiterpenoids shown in the formula I; collecting the eluent with retention time of 14.5-15.0 min to obtain sesquiterpenoids shown in formula II.
6. The preparation method according to claim 4, wherein the fruiting body of the white ginseng fungus is leached with methanol in the step S1 for 2-3 times, and one time is 8-10 days.
7. The method according to claim 4, wherein the volume ratio of petroleum ether/ethyl acetate in step S2 is 3:7.
8. The method according to claim 5, wherein the volume ratio of dichloromethane/methanol in step S32 is 4:1-5:1, and the volume ratio of ethyl acetate/methanol is 9:1-10:1.
9. Use of the sesquiterpenoids of claim 1 for the preparation of anti-inflammatory drugs.
10. An anti-inflammatory agent comprising the sesquiterpenoid of claim 1.
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