Diterpenoid compound as well as preparation method and anti-inflammatory application thereof

Abstract

The invention discloses a novel diterpenoid compound which is separated from origanum vulgare and has anti-inflammatory activity, the molecular formula of the diterpenoid compound is C20H18O4, and the structural formula of the diterpenoid compound is shown as a formula I. The preparation method of the compound comprises the following steps: crushing a dried medicinal material of origanum vulgare, extracting with ethanol, and concentrating under reduced pressure to obtain a total extract; and after extracting with petroleum ether, separating and purifying through silica gel column chromatography, Sephadex LH-20 gel column chromatography and preparative high performance liquid chromatography to obtain a target compound. In-vitro anti-inflammatory experiments show that the compound can significantly inhibit NO generation in an LPS-induced RAW264.7 macrophage model, the inhibition rate reaches 62.97%, and good anti-inflammatory activity is shown. The compound can be used for preparing medicines for preventing or treating inflammatory diseases such as enteritis, hepatitis and meningitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural medicinal chemistry, and particularly relates to a diterpenoid compound isolated from Origanum vulgare and a preparation method and anti-inflammatory use thereof. BACKGROUND

[0002] Origanum vulgare is a plant of Origanum Linn. in Lamiaceae, and is a commonly used folk herbal medicine and a popular edible tea. It is recorded in Guizhou Folk Medicine, Dian Nan Ben Cao and Fujian Medicine, etc. to be used for symptoms such as heatstroke, cold, abdominal pain, etc., and has the effects of clearing heat and relieving superficies, regulating qi and relieving summer-heat, and diuresis and detumescence.

[0003] Modern research shows that the extract contains various anti-inflammatory active ingredients such as carvacol, thymol, oleanolic acid, rosmarinic acid, etc., can balance the levels of pro-inflammatory factors and anti-inflammatory factors, and relieve inflammatory reaction, and certain progress has been made in the study of its mechanism of action.

[0004] In vitro studies have shown that the alcohol extract of Origanum vulgare can inhibit the production of inflammatory factors in macrophages, and can reduce the inflammatory damage of macrophages induced by lipopolysaccharide. At present, there are few studies on the material basis of Origanum vulgare in the prevention and treatment of inflammatory diseases and the development of application fields. Therefore, as a common medicinal and edible plant, Origanum vulgare is rich in resources and easy to obtain, and has great development prospects in the fields of food and medicine. SUMMARY

[0005] In view of the above background, the main purpose of the present application is to provide a diterpenoid compound isolated from Origanum vulgare and a preparation method and anti-inflammatory use thereof.

[0006] In order to achieve the above-mentioned purpose, the specific scheme adopted by the present application is as follows: The present application first provides a diterpenoid compound, the molecular formula of which is C 20 H 18 O4, the name of which is 4-hydroxy-2,3,3-trimethyl-3,8-dihydro-2H-dibenzo[1,2-f:1',2'-b]furo[2,3-d]oxocin-8-one, and the structural formula of which is: .

[0007] In addition, the present application provides a preparation method of the above-mentioned diterpenoid compound, which is obtained by extraction, separation and purification from Origanum vulgare, comprising the following steps: S1, grinding Origanum vulgare dried medicinal materials to 50-100 meshes, and extracting several times with ethanol water as the extraction solvent to obtain total extract by combining the extraction solutions; S2, concentrating the total extract under vacuum to obtain a total extract; S3, dissolving the total extract in water and then extracting with petroleum ether to obtain a petroleum ether extract; S4, subjecting the petroleum ether extract obtained in step S3 to silica gel column chromatography separation, then gradient eluting with organic solvents, and then combining to obtain sub-fractions N1-N20 in sequence after identification by silica gel thin layer plate; S5, subjecting the sub-fraction N5 to Sephdex LH-20 dextran gel column chromatography separation to obtain sub-fractions N5-1-N5-10; S6, purifying N5-5 by preparative high performance liquid chromatography to obtain the anti-inflammatory diterpenoid compound.

[0008] As some embodiments of the preparation method of the present application, in step S1, the volume fraction of the extraction solvent is 40%-100%.

[0009] As a preferred embodiment of the preparation method of the present application, in step S1, the amount of the extraction solvent is 6-20 times the mass of the medicinal material.

[0010] As some embodiments of the preparation method of the present application, in step S1, the extraction method is heating reflux, and the extraction time is 1h-6h each time, and the extraction times are 2-6 times.

[0011] As a preferred embodiment of the preparation method of the present application, in step S2, the total extract is concentrated under reduced pressure at 30-60℃.

[0012] As a preferred embodiment of the preparation method of the present application, in step S2, the vacuum degree of the reduced pressure concentration is 0.1MPa-0.5MPa.

[0013] As a preferred embodiment of the preparation method of the present application, in step S3, the volume ratio of the total extract to water is 1:3-1:5.

[0014] As a preferred embodiment of the preparation method of the present application, in step S3, the amount of petroleum ether is 1.5-2 times the volume of the total extract.

[0015] As some embodiments of the preparation method of the present application, in step S4, normal silica gel with a mesh size of 100-200 is used for silica gel column chromatography separation.

[0016] As some embodiments of the preparation method of the present application, in step S4, the organic solvents for gradient elution are chloroform-methanol, petroleum ether-acetone, petroleum ether-ethyl acetate, or cyclohexane-ethyl acetate.

[0017] As some embodiments of the preparation method of the present application, in step S4, when petroleum ether-ethyl acetate is used as the gradient elution organic solvent, the volume ratio of petroleum ether-ethyl acetate is 200:1 to 1:2.

[0018] As a preferred embodiment of the preparation method of the present application, in step S4, gradient elution is performed using petroleum ether-ethyl acetate with volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1 and 1:1 as eluents in sequence.

[0019] As a preferred embodiment of the preparation method of the present application, in step S4, when the silica gel thin layer plate is detected, 12% concentrated sulfuric acid ethanol solution is sprayed on the silica gel thin layer plate, and color development comparison is performed under heating conditions.

[0020] As a preferred embodiment of the preparation method of the present application, in step S5, when the Sephdex LH-20 dextran gel column chromatography is performed on the flow N5, the eluent is petroleum ether-dichloromethane-methanol (volume ratio 5:5:1) or dichloromethane-methanol (volume ratio 2:1).

[0021] As a preferred embodiment of the preparation method of the present application, in step S6, the chromatographic column used in the preparative high performance liquid chromatography purification system is C4, C8, C6 or C18; wherein the mobile phase is chromatographic methanol-water or acetonitrile-water.

[0022] As a preferred embodiment of the preparation method of the present application, in step S6, when the preparative high performance liquid chromatography purification is performed, the mobile phase is 86% acetonitrile water, the flow rate is 18 mL / min, the detection wavelength is 290 nm, the preparative column is Waters SunFire C18 19x250mm, 5μm, and one chromatographic peak appears in 15 min. The new diterpenoid compound is obtained by repeatedly preparing and enriching the chromatographic peak.

[0023] The present application also provides the use of the above-mentioned diterpenoid compound in the preparation of an inflammatory disease drug.

[0024] The above-mentioned inflammatory disease drug is a drug for treating enteritis, hepatitis, meningitis, etc.

[0025] The present application also provides a pharmaceutical preparation comprising the above-mentioned diterpenoid compound and one or more pharmaceutically acceptable carriers or excipients.

[0026] Compared with the prior art, the present application has the following beneficial effects: 1. Novel structure and significant activity: The application first separates a novel diterpenoid compound from oregano, which is confirmed to be a new compound not reported in the literature. In vitro anti-inflammatory experiments show that the compound has significant inhibitory activity on the production of nitric oxide (NO) by LPS-induced RAW264.7 macrophages at a concentration of 50 μM, with an inhibition rate of up to 62.97%, showing excellent anti-inflammatory potential and providing a valuable lead compound for the development of new anti-inflammatory drugs.

[0027] 2. Efficient and reproducible preparation method: The application provides a complete method for preparing the compound, which combines various separation and purification techniques such as ethanol extraction, petroleum ether extraction, silica gel column chromatography, gel column chromatography, and preparative high-performance liquid chromatography. The process is clear and the parameters are clear, which can efficiently and accurately separate and purify the target compound from oregano raw materials, and the method is stable and reproducible, laying a process foundation for further research and development of the compound.

[0028] 3. Abundant raw material source and great development potential: The application uses the medicinal and edible plant oregano as raw material, which is abundant and easy to obtain, and has low cost, which is beneficial to the sustainable acquisition and future industrialization of the compound. The obtained compound can be used as an active ingredient for preparing drugs or health products for treating inflammatory diseases such as enteritis, hepatitis, and meningitis, expanding the medicinal and economic value of oregano, and having a broad application prospect in the fields of medicine and health. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are only one embodiment of the application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 The structural formula I of the compound prepared in Example 1 of the application.

[0031] Figure 2 The infrared spectrum (IR) graph of the compound prepared in Example 1 of the application.

[0032] Figure 3 The infrared spectrum (IR) graph of the compound prepared in Example 1 of the application. 1 H NMR spectrum of the compound prepared in Example 1 of the application.

[0033] Figure 4 The infrared spectrum (IR) graph of the compound prepared in Example 1 of the application. 13 C NMR spectrum of the compound prepared in Example 1 of the application.

[0034] Figure 5HSQC spectrum of the compound prepared in Example 1 of the present application.

[0035] Figure 6 HMBC spectrum of the compound prepared in Example 1 of the present application.

[0036] Figure 7 COSY spectrum of the compound prepared in Example 1 of the present application.

[0037] Figure 8 Cell survival rate under different LPS concentrations in Example 2 of the present application (compared with the control group **P<0.01, *P<0.05).

[0038] Figure 9 NO content of cells under different LPS concentrations in Example 2 of the present application (compared with the control group **P<0.01, *P<0.05). DETAILED DESCRIPTION

[0039] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the technical solutions in the specific embodiments of the present application will be described clearly and completely below to further illustrate the present application. Obviously, the described specific embodiments are only a part of the embodiments of the present application, not all.

[0040] Example 1: This embodiment is a method for preparing the diterpenoid compound described in the present application by using oregano, which comprises the following steps: S1, take the whole plant of oregano for drying, 30 kg of dried oregano is used in this embodiment, after being crushed properly, pass through a 50 mesh sieve, and then sequentially refluxed with solvents of 90%, 80% and 70% ethanol water, 3 times, each time for 2 hours, and then combine the extractives obtained each time to obtain the total extractive; S2, evaporate and concentrate the total extractive obtained in step S1 under the conditions of heating at 55℃ and vacuum degree of 0.1 MPa, and then dry to obtain the total extractive of oregano 2.0 kg; S3, uniformly suspend the total extractive obtained in step S2 in water, the volume ratio of the total extractive to water is 1:3, V / V, and then extract with petroleum ether in a 5L separatory funnel to obtain the corresponding petroleum ether fraction (300g); S4, the petroleum ether part obtained in step S3 was taken, and normal phase silica gel (100-200 mesh) was used for sample mixing, and separation was performed in an open glass column, gradient elution was performed with petroleum ether-ethyl acetate with a volume ratio of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, respectively, and the eluted fractions were identified by thin layer chromatography, and 12% concentrated sulfuric acid ethanol was used for color development under heating conditions, the Rf value of the target point was 0.1-0.6, and the same elution part was combined according to the similar point, and 20 fractions N1-N20 were obtained; S5, the fraction N5 obtained in step S4 was purified by Sephadex LH-20 gel column chromatography, isocratic elution was performed with petroleum ether-dichloromethane (volume ratio of 5:5:1) as the eluent, and the same elution part was combined according to the thin layer chromatography point plate, and 10 sub-fractions N5-1-N5-10 were obtained; S6, the sub-fraction N5-5 was separated and prepared by preparative high performance liquid chromatography, and the preparation conditions were as follows: mobile phase: 86% acetonitrile water, flow rate: 18 mL / min, detection wavelength: 290 nm, preparation column: Waters SunFire C18 19x250mm, 5μm, there was one main peak in the liquid phase, and the peak time was 15 min, and after repeated preparation and enrichment, a new diterpenoid compound I was obtained, and the weight was 10 mg.

[0041] In this embodiment, the structure of the new diterpenoid compound I obtained by separation was identified: The compound was brown powder. HR-ESI-MS showed m / z 323.1283 [M+H] + Peak (calculated value: C 20 H 19 O4, 323.1283), the molecular formula of which was C 20 H 18 O4, and the unsaturation degree was 12. 1 H-NMR spectrum had one active hydrogen delta H 12.06, 6 olefinic hydrogen proton signals delta H 7.72, 7.67, 7.58, 7.31, 7.24, 7.21, 7.09 (each 1H, s). There were three methyl proton signals in the high field, of which one was a doublet proton signal δ H 1.47 (3H, d, J =6.2Hz), two singlet proton signals delta H 1.41, 1.33; 13The C-NMR spectrum showed 20 carbon signals, and combined with the HSQC spectrum, it was determined that there were 3 methyl carbons, 8 methine carbons, and 9 quaternary carbons.

[0042] Combined with the infrared spectrum of the new diterpenoid compound I ( Figure 2 )H1N and 1C NMR spectra ( Figure 3 , 4 ), and through comprehensive analysis of the compound's two-dimensional NMR spectra, including HSQC, 1 H- 1 Analyzing H COSY and HMBC spectra ( Figure 5 , 6 (7) The planar structure of the compound was determined. A search of the SciFinder database confirmed that the compound is a novel diterpenoid, with structural formula I as shown below. Figure 1 As shown, no other literature has reported this. Its carbon spectral data are shown in Table 1 below.

[0043] Table 1. New diterpenoid compound I 13 C-NMR data (measurement solvent: CD3OD); delta :ppm; J :Hz)

[0044] Example 2: This example demonstrates the in vitro anti-inflammatory activity of the diterpenoid compounds prepared in Example 1. CCK-8 assay for cell viability: Add 8 × 10⁸ cells to a 96-well plate. 3 RAW264.7 cells in the logarithmic growth phase were cultured overnight in a 5% CO2 incubator at 37°C with 100 μL / well. Different concentrations of diterpenoid compounds were added to different experimental groups, or an equal volume of culture medium was added to the blank control group for 24 h. Each group was repeated in triplicate. Then, following the instructions of the CCK-8 kit, 10 μL of CCK-8 reagent was added directly to each well, and the cells were incubated at 37°C for 2 hours. The OD value was measured at 450 nm using a microplate reader. Cell viability (%) = mean of drug group / control group × 100%.

[0045] NO content determination: Add 8×10⁻⁶ to a 96-well plate. 3The RAW264.7 cells in logarithmic phase were incubated overnight at 37℃ in a 5% CO2 incubator. Different concentrations of diterpenoid compounds were added to the experimental groups, and the same volume of medium was added to the blank and model groups. Each group was repeated in triplicate. Then, LPS solution was added, and the cells were incubated at 37℃ in a 5% CO2 incubator for 24 hours. After incubation, the NO content in the supernatant was detected according to the NO kit operation manual. 50 μL of the supernatant was added to a 96-well plate, followed by the addition of Griess Reagent I 50 μL, Griess Reagent II 50 μL, and shaking for 5 minutes at room temperature in the dark. Then, the OD value at 540 nm was measured in an enzyme-labeled instrument. Meanwhile, a standard curve of absorbance and NaNO2 was established, and the NO content was calculated according to the standard curve. NO inhibition rate (%) = (model group NO content-experimental group NO content) / model group NO content x 100%.

[0046] Data processing: IBM SPSS 21 statistical software was used for data analysis and processing, and Origin 9.1 software was used for plotting. The data between two groups were compared by t-test. In the significance analysis, *P<0.05 indicates a significant difference, **P<0.01 indicates a very significant difference, and it has statistical significance.

[0047] The RAW264.7 cell viability test results showed that LPS at a concentration of 0.1-5 ug / ml had no significant effect on cell viability, and reached 50 ug / ml, which had a very significant effect on cell viability, as shown in Figure 8 The NO content test results showed that the NO content produced by LPS at 5 ug / ml was the highest, as shown in Figure 9 Therefore, considering comprehensively, 5 ug / ml was selected as the optimal stimulating concentration of LPS on RAW264.7 cells.

[0048] The new diterpenoid compound I isolated from O. basilicum in Example 1 was screened for its NO inhibition effect on LPS-induced RAW264.7 macrophages. The results showed that most of the screened new diterpenoid compound I had no significant effect on cell viability at a concentration of 50 μM, and had an inhibitory effect on NO production, showing certain anti-inflammatory activity. The NO inhibition rate of the new diterpenoid compound I was 62.97%, as shown in Table 2.

[0049] Table 2 Effect of compounds on inhibition of LPS-induced RAW264.7 cells to produce NO

[0050] It should be noted that the above-mentioned embodiments are only illustrative, but not limiting the protection scope of the present application, and the protection scope of the present application is defined by the claims. Some non-essential improvements and adjustments of the present application made by those skilled in the art without departing from the spirit and scope of the present application shall still fall within the protection scope of the present application.

Claims

1. A diterpenoid compound, characterized in that: The structural formula of the diterpenoid compound is: 。 2. A method for preparing the diterpenoid compound as described in claim 1, characterized in that, Includes the following steps: S1. Pulverize the dried oregano into 50-100 mesh, extract several times using ethanol and water as the extraction solvent, and combine the extracts to obtain the total extract; S2. The total extract is concentrated under vacuum to obtain the total extract. S3. Dissolve the total extract in water and then extract with petroleum ether to obtain the petroleum ether extract fraction. S4. The petroleum ether extract obtained in step S3 is separated by silica gel column chromatography, then eluted with organic solvents in a gradient, and after identification by silica gel thin-layer plate, the fractions N1 to N20 are combined sequentially. S5. Separate fraction N5 by Sephdex LH-20 dextran gel column chromatography to obtain subfractions N5-1 to N5-10. S6. N5-5 was purified by preparative high performance liquid chromatography to prepare anti-inflammatory diterpenoid compounds.

3. The preparation method according to claim 2, characterized in that, In step S1, the volume fraction of the extraction solvent is 40%–100%; The amount of extraction solvent used is 6 to 20 times the mass of the medicinal material; The extraction method is heating and reflux, with each extraction lasting 1 to 6 hours; the number of extractions is 2 to 6. In step S2, the total extract is concentrated under reduced pressure at a temperature of 30℃ to 60℃ and a vacuum of 0.1MPa to 0.5MPa.

4. The preparation method according to claim 2, characterized in that, In step S3, the volume ratio of total extract to water is 1:3; The amount of petroleum ether used is 1.5 to 2 times the total volume of the extract.

5. The preparation method according to claim 2, characterized in that, In step S4, silica gel column chromatography separation is performed using 100-200 mesh normal-phase silica gel; The organic solvent used for gradient elution is chloroform-methanol, petroleum ether-acetone, petroleum ether-ethyl acetate, or cyclohexane-ethyl acetate. When inspecting silica gel thin-layer plates, a 12% concentrated sulfuric acid ethanol solution is sprayed onto the silica gel thin-layer plate, and color development and comparison are performed under heating conditions.

6. The preparation method according to claim 2, characterized in that, In step S5, the eluent for Sephdex LH-20 dextran gel column chromatography of fraction N5 is petroleum ether-dichloromethane-methanol (volume ratio 5:5:1) or dichloromethane-methanol (volume ratio 2:1).

7. The preparation method according to claim 2, characterized in that, In step S6, the chromatographic column used in the preparative high-performance liquid chromatography purification system is C4, C8, C6 or C18; wherein the mobile phase is chromatographic methanol-water or acetonitrile-water.

8. The use of the diterpenoid compound of claim 1 in the preparation of a medicament for inflammatory diseases.

9. The application according to claim 8, characterized in that, The inflammatory disease medications mentioned are those used to treat enteritis, hepatitis, and / or meningitis.

10. A pharmaceutical preparation, characterized in that, It comprises the diterpenoid compound of claim 1, and one or more pharmaceutically acceptable carriers or excipients.

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