Triterpenoid alihong terpene acid A and its preparation method and application

Abstract

The present invention relates to the technical field of separation and purification of iris radiata, and relates to a triterpenoid compound, iris radiata terpene acid A, and its preparation method and application. The dried fruiting bodies of iris radiata are taken, dried in the shade, crushed, and extracted with an ethanol solution under heating and reflux. The reflux extracts are combined, recovered under reduced pressure, and concentrated to obtain a total extract of iris radiata. The extract is then dispersed into a suspension with water, extracted with dichloromethane, and the extract is concentrated. The dichloromethane extract is separated by gradient elution on a silica gel column to obtain five fractions. The third of the five fractions is purified and separated by gradient elution on a high-performance liquid chromatography column, and the eluate is collected to obtain the triterpenoid compound, iris radiata terpene acid A, at 33.5 minutes. This invention discloses the triterpenoid compound, iris radiata terpene acid A, for the first time, and determines and characterizes its structure and physicochemical properties. Experiments have shown that it has certain anti-inflammatory activity, thereby enabling its application in the preparation of preventive and anti-inflammatory drugs.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of iridium truncatum, and is a triterpenoid compound, iridium truncatum terpene acid A, and a preparation method and application thereof. Background Art

[0002] Fomes officinalis is derived from the fruiting body of the medicinal fungus Fomitopsis officinalis. The triterpenoid compounds with a 6 / 6 / 6 / 5 tetracyclic ring system in Fomes officinalis have significant anti-inflammatory activity, and Fomes officinalis has a wide range of applications, which has attracted the interest of many researchers.

[0003] The medicinal effects of Alihong mainly come from terpenoid compounds. Therefore, developing and utilizing the triterpenoid compound Alihong terpene acid A with a 6 / 6 / 6 / 5 tetracyclic ring system in Alihong, further exploring its potential medicinal value, and determining and characterizing its structure and physicochemical properties are of great significance to the development and utilization of Alihong. Summary of the Invention

[0004] The present invention provides a triterpenoid compound, alihong terpene acid A, and a preparation method and application thereof, which overcome the shortcomings of the above-mentioned prior art. It discloses the triterpenoid compound, alihong terpene acid A for the first time, and enables it to be used in the preparation of preventive anti-inflammatory drugs and the preparation of anti-inflammatory drugs.

[0005] One of the technical solutions of the present invention is achieved by the following measures: a triterpenoid compound, alihong terpene acid A, whose chemical structure is:

[0006] .

[0007] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions:

[0008] The above-mentioned triterpenoid compound Alihong terpene acid A is obtained according to the following method: the first step is to obtain the dried fruiting body of Alihong, dry it in the shade, crush it, and extract it with ethanol solution after heating and refluxing, then combine the reflux extract and recover it under reduced pressure and concentrate it to obtain the total extract of Alihong; the second step is to disperse the total extract of Alihong into a suspension with water, extract it with dichloromethane, and concentrate the extract to obtain the dichloromethane extract; the third step is to take the dichloromethane extract and separate it by gradient elution on silica gel column chromatography to obtain 5 fractions; the fourth step is to purify and separate the third fraction of the 5 fractions obtained by high performance liquid chromatography gradient elution, and collect the eluate to obtain the triterpenoid compound Alihong terpene acid A at 33.5 minutes.

[0009] In the first step, the volume ratio of the ethanol solution is 95%, and the extraction is performed by heating under reflux for 3 times, each time for 3 hours.

[0010] In the first step above, 8 mL to 12 mL of ethanol solution was added to every 1 g of the fruiting body of the Alihong plant.

[0011] In the third step, the gradient eluent of the silica gel column chromatography comprises petroleum ether and ethyl acetate, and the volume ratios of petroleum ether and ethyl acetate are 50:1, 20:1, 10:1, 5:1 and 2:1, respectively.

[0012] In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:35.

[0013] The second technical solution of the present invention is achieved through the following measures: a preparation method of the triterpenoid compound Alihong terpene acid A, which is carried out according to the following steps: the first step is to take the dried fruiting body of Alihong, dry it in the shade, crush it, and extract it with ethanol solution after heating and refluxing, combine the reflux extract and reduce the pressure to recover and concentrate it to obtain the total extract of Alihong; the second step is to disperse the total extract of Alihong into a suspension with water, extract it with dichloromethane, and concentrate the extract to obtain the dichloromethane extract; the third step is to take the dichloromethane extract and separate it by gradient elution on a silica gel column chromatography to obtain 5 fractions; the fourth step is to purify and separate the third fraction of the 5 fractions obtained by high performance liquid chromatography gradient elution, and collect the eluate to obtain the triterpenoid compound Alihong terpene acid A at 33.5 minutes.

[0014] The following is a further optimization and / or improvement of the second technical solution of the above invention:

[0015] In the above-mentioned first step, the volume ratio of the ethanol solution is 95%, wherein the heating reflux extraction is performed 3 times, each time for 3 hours, and 8mL to 12mL of ethanol solution is added to every 1g of Alihong fruiting body; or / and, in the third step, the silica gel column chromatography gradient eluent includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether and ethyl acetate are 50:1, 20:1, 10:1, 5:1, and 2:1 respectively; or / and, in the fourth step, the high performance liquid chromatography eluent is a mixture of methanol and water, wherein the volume ratio of methanol and water is 65:35.

[0016] The third technical solution of the present invention is achieved through the following measures: a triterpenoid compound, alihong terpene acid A, is used in the preparation of preventive anti-inflammatory drugs.

[0017] The fourth technical solution of the present invention is achieved through the following measures: a triterpenoid compound, alihong terpene acid A, is used in the preparation of anti-inflammatory drugs.

[0018] The present invention discloses for the first time the triterpenoid compound alihong terpene acid A, determines and characterizes its structure and physicochemical properties, and proves through experiments that it has certain anti-inflammatory activity, thereby enabling it to be used in the preparation of preventive anti-inflammatory drugs and anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attachment Figure 1 The triterpenoid compound Alihong terpene acid A obtained in Example 10 of the present invention 1 H-NMR spectrum.

[0020] Attachment Figure 2 The triterpenoid compound Alihong terpene acid A obtained in Example 10 of the present invention 13 C-APT diagram. DETAILED DESCRIPTION

[0021] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals known and used in the prior art.

[0022] Example 1: The triterpenoid compound, terpene acid A, has the chemical structural formula:

[0023] .

[0024] Example 2: As an optimization of the above example, the following method is used to obtain: the first step is to obtain dried fruiting bodies of Alihong, dry them in the shade, crush them, and extract them with ethanol solution by heating and refluxing. The reflux extracts are combined and recovered under reduced pressure and concentrated to obtain the total extract of Alihong; the second step is to disperse the total extract of Alihong into a suspension with water, extract it with dichloromethane, and concentrate the extract to obtain the dichloromethane extract; the third step is to obtain the dichloromethane extract by silica gel column chromatography gradient elution and separation to obtain 5 fractions; the fourth step is to purify and separate the third fraction of the 5 obtained fractions by high performance liquid chromatography gradient elution, and collect the eluate to obtain the triterpenoid compound Alihong terpene acid A at 33.5 minutes.

[0025] Example 3: As an optimization of the above example, in the first step, the volume ratio of the ethanol solution is 95%, wherein the heating reflux extraction is performed 3 times, each time for 3 hours.

[0026] Example 4: As an optimization of the above example, in the first step, 8 mL to 12 mL of ethanol solution is added to every 1 g of Alihong fruiting body.

[0027] Example 5: As an optimization of the above example, in the third step, the gradient eluent of the silica gel column chromatography includes petroleum ether and ethyl acetate, and the volume ratios of petroleum ether and ethyl acetate are 50:1, 20:1, 10:1, 5:1, and 2:1, respectively.

[0028] Example 6: As an optimization of the above example, in the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:35.

[0029] Example 7: The preparation method of the triterpenoid compound Alihong terpene acid A is carried out according to the following steps: the first step is to obtain the dried fruiting body of Alihong, dry it in the shade, crush it, and extract it with ethanol solution after heating and refluxing, then combine the reflux extract and recover it under reduced pressure and concentrate it to obtain the total extract of Alihong; the second step is to disperse the total extract of Alihong into a suspension with water, extract it with dichloromethane, and concentrate the extract to obtain the dichloromethane extract; the third step is to take the dichloromethane extract and separate it by gradient elution on silica gel column chromatography to obtain 5 fractions; the fourth step is to purify and separate the third fraction of the 5 fractions obtained by high performance liquid chromatography gradient elution, and collect the eluate to obtain the triterpenoid compound Alihong terpene acid A at 33.5 minutes.

[0030] Example 8: Use of the triterpenoid compound alihong terpene acid A in the preparation of preventive anti-inflammatory drugs.

[0031] Example 9: Use of the triterpenoid compound alihong terpene acid A in the preparation of anti-inflammatory drugs.

[0032] Example 10: The preparation method of the triterpenoid compound Alihong terpene acid A is carried out according to the following steps: the first step is to take the dried fruiting body of Alihong, dry it in the shade, crush it, and heat it with a 95% by volume ethanol solution for reflux extraction 3 times, each time for 3 hours, combine the reflux extracts and reduce the pressure to recover and concentrate to obtain the total extract of Alihong, wherein 10mL of ethanol solution is added to every 1g of Alihong fruiting body; the second step is to disperse the total extract of Alihong into a suspension with water, extract it with dichloromethane, and concentrate the extract to obtain the dichloromethane extract. ; The third step is to take the extract of the dichloromethane part and use petroleum ether and ethyl acetate with a volume ratio of 50:1, 20:1, 10:1, 5:1, and 2:1 as eluents for silica gel column chromatography gradient elution and separation to obtain 5 fractions; the fourth step is to purify and separate the third fraction of the 5 fractions obtained by high performance liquid chromatography gradient elution, and collect the eluate to obtain the triterpenoid compound alihong terpene acid A at 33.5 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water with a volume ratio of 65:35.

[0033] The triterpenoid compound Alihong terpene acid A obtained in Example 10 was subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) and carbon nuclear magnetic resonance spectroscopy ( 13 C-APT) analysis, 1 H-NMR spectrum Figure 1 As shown, 1 H-NMR spectrum Figure 2 As shown, Figure 1 and Figure 2 Perform spectrum analysis and compare spectrum 1 and Figure 2 Each peak is assigned, Figure 1 and Figure 2 The peak attribution is shown in Table 1. From the data in Table 1, it can be seen that the chemical structure of the triterpenoid compound Alihong terpene acid A of the present invention is

[0034] .

[0035] The triterpenoid compound Alihong terpene acid A obtained in Example 10 of the present invention was subjected to cell anti-inflammatory activity screening, and the Griess method was used to perform a cell anti-inflammatory activity screening experiment on the triterpenoid compound Alihong terpene acid A.

[0036] Mouse mononuclear macrophage RAW264.7 cells were cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 incubator. The cells were then diluted to 1×10 5 / mL, take 200μL / well and inoculate in 96-well culture plates, place in a 37℃, 5% CO2 cell culture incubator for 24h, add LPS with a final concentration of 1μg / mL and triterpenoid compound alihong terpene acid A, gently shake the 96-well plate, and continue to culture for 24h, centrifuge, take 100μL of culture supernatant and add Griess reagent, place at room temperature for 20 minutes, perform colorimetry by spectrophotometry, measure the absorbance at 546nm with an enzyme reader, and make a standard curve. Calculate the content of No in the supernatant according to the standard curve, and then calculate the IC of triterpenoid compound alihong terpene acid A by Excel software 50 The value is 18.72 μM. Therefore, it can be seen that the triterpenoid compound alihong terpene acid A of the present invention has certain anti-inflammatory activity.

[0037] In summary, the present invention discloses the triterpenoid compound alihong terpene acid A for the first time, determines and characterizes its structure and physicochemical properties, and proves through experiments that it has certain anti-inflammatory activity, thereby enabling it to be used in the preparation of preventive anti-inflammatory drugs and anti-inflammatory drugs.

[0038] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the requirements of different situations.

[0039]

Claims

1. A method for preparing triterpenoid compound Alihong terpene acid A, characterized in that The process is as follows: first, taking dried fruiting bodies of Alihong, drying in the shade, crushing, and extracting with ethanol solution by heating and refluxing, combining the reflux extracts and recovering under reduced pressure and concentrating to obtain the total extract of Alihong; second, dispersing the total extract of Alihong into a suspension with water, extracting with dichloromethane, and concentrating the extract to obtain the dichloromethane extract; third, taking the dichloromethane extract and separating it by silica gel column chromatography gradient elution to obtain 5 fractions, wherein the silica gel column chromatography gradient eluent is petroleum ether and ethyl acetate. The volume ratios of ester, petroleum ether and ethyl acetate are 50:1, 20:1, 10:1, 5:1 and 2:1 respectively; in the fourth step, the third fraction of the obtained five fractions is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the triterpenoid compound Alihong terpene acid A at 33.5 minutes. The high performance liquid chromatography eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 65:

35. The chemical structure of the triterpenoid compound Alihong terpene acid A is: 。 2. The method for preparing the triterpenoid compound A11 according to claim 1, characterized in that In the first step, the volume ratio of the ethanol solution is 95%, wherein the heating reflux extraction is performed 3 times, each time for 3 hours, and 8 mL to 12 mL of ethanol solution is added to every 1 g of Alihong fruiting body.

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