A diterpenoid compound, its preparation method and anti-inflammatory use thereof

Abstract

The application discloses a novel diterpenoid compound with anti-inflammatory activity separated from Origanum vulgare Linn. 20 H 18 O4, and a structural formula is as shown in formula I. A preparation method of the compound comprises the following steps: crushing dried medicinal materials of Origanum vulgare Linn., extracting by using ethanol, concentrating under reduced pressure to obtain total extract; after extraction by using petroleum ether, the target compound is obtained through silica gel column chromatography, Sephadex LH-20 gel column chromatography and preparation type high performance liquid phase chromatography separation and purification. In vitro anti-inflammatory experiments show that the compound can significantly inhibit NO generation in a LPS-induced RAW264.7 macrophage model, and the inhibition rate reaches 62.97%, and the compound shows good anti-inflammatory activity. The compound can be used for preparing medicines for preventing or treating inflammatory diseases such as enteritis, hepatitis and meningitis.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to a diterpenoid compound isolated from oregano, its preparation method, and its anti-inflammatory uses. Background Technology

[0002] Oregano (Origanum vulgare) is a plant belonging to the genus Origanum Linn. of the family Lamiaceae. Oregano is a commonly used folk herbal medicine and also a popular edible tea. It is recorded in books such as "Folk Medicines of Guizhou", "Materia Medica of Yunnan", and "Pharmacopoeia of Fujian" as being used for symptoms such as heatstroke, colds, and abdominal pain. It has the effects of clearing heat and relieving exterior symptoms, regulating qi and relieving summer heat, and promoting diuresis and reducing swelling.

[0003] Modern research shows that its extract contains a variety of anti-inflammatory active ingredients such as carvacrol, thymol, oleanolic acid, and rosmarinic acid, which can balance the levels of pro-inflammatory and anti-inflammatory factors and alleviate inflammatory responses. Some progress has been made in the study of its mechanism of action.

[0004] In vitro studies have shown that the alcoholic extract of oregano can inhibit the production of inflammatory factors in macrophages and alleviate lipopolysaccharide-induced macrophage inflammatory damage. Currently, there is limited research on the material basis and application development of oregano in the prevention and treatment of inflammatory diseases. Therefore, as a common plant used for both food and medicine, oregano is abundant and readily available, and has great potential for development in the food and pharmaceutical fields. Summary of the Invention

[0005] In view of the above background, the main objective of this invention is to provide a diterpenoid compound isolated from oregano, its preparation method, and its anti-inflammatory uses.

[0006] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0007] This invention first provides a diterpenoid compound with the molecular formula: C 20 H 18 O4, named 4-hydroxy-2,3,3-trimethyl-3,8-dihydro-2H-dibenzo[1,2-f:1',2'-b]furano[2,3-d]oxecyclooctam-8-one, has the following structural formula:

[0008] .

[0009] Furthermore, this invention provides a method for preparing the above-mentioned diterpenoid compounds, wherein the diterpenoid compounds are extracted, separated, and purified from oregano, comprising the following steps:

[0010] 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;

[0011] S2. The total extract is concentrated under vacuum to obtain the total extract.

[0012] S3. Dissolve the total extract in water and then extract with petroleum ether to obtain the petroleum ether extract fraction.

[0013] 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.

[0014] S5. Separate fraction N5 by Sephdex LH-20 dextran gel column chromatography to obtain subfractions N5-1 to N5-10.

[0015] S6. N5-5 was purified by preparative high performance liquid chromatography to prepare diterpenoid compounds.

[0016] As some embodiments of the preparation method of the present invention, in step S1, the volume fraction of the solvent used for extraction is 40% to 100%.

[0017] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S1, the amount of extraction solvent used is 6 to 20 times the mass of the medicinal material.

[0018] As some embodiments of the preparation method of the present invention, in step S1, the extraction method is heating and reflux, the extraction time is 1h to 6h each time, and the number of extractions is 2 to 6 times.

[0019] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S2, the total extract is concentrated under reduced pressure at 30-60°C.

[0020] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S2, the vacuum degree of the reduced pressure concentration is 0.1MPa to 0.5MPa.

[0021] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S3, the volume ratio of total extract to water is 1:3 to 1:5.

[0022] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S3, the amount of petroleum ether used is 1.5 to 2 times the total volume of the extract.

[0023] As some embodiments of the preparation method of the present invention, in step S4, 100-200 mesh normal phase silica gel is used for silica gel column chromatography separation.

[0024] As some embodiments of the preparation method of the present invention, in step S4, the organic solvent for gradient elution is chloroform-methanol, petroleum ether-acetone, petroleum ether-ethyl acetate, or cyclohexane-ethyl acetate.

[0025] As some embodiments of the preparation method of the present invention, in step S4, when using petroleum ether-ethyl acetate as a gradient elution organic solvent, the volume ratio of petroleum ether-ethyl acetate is 200:1 to 1:2.

[0026] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S4, gradient elution is performed sequentially using petroleum ether-ethyl acetate volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1 as eluents.

[0027] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S4, when the silica gel thin film is identified, a 12% concentrated sulfuric acid ethanol solution is sprayed onto the silica gel thin film, and color comparison is performed under heating conditions.

[0028] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S5, when performing Sephdex LH-20 dextran gel column chromatography on fraction N5, the eluent is petroleum ether-dichloromethane-methanol (volume ratio 5:5:1) or dichloromethane-methanol (volume ratio 2:1).

[0029] As a preferred embodiment of some embodiments of the preparation method of the present invention, 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.

[0030] As a preferred embodiment of some embodiments of the preparation method of the present invention, in step S6, during preparative high performance liquid chromatography purification, the mobile phase is 86% acetonitrile-water, the flow rate is 18 mL / min, the detection wavelength is 290 nm, and the preparative column is Waters SunFire C18 19×250 mm, 5 μm. One chromatographic peak appears in 15 min. This chromatographic peak is repeatedly prepared and enriched to obtain new diterpenoid compounds.

[0031] The present invention also provides the use of the above-mentioned diterpenoid compounds in the preparation of drugs for inflammatory diseases.

[0032] The aforementioned inflammatory disease medications are for treating enteritis, hepatitis, meningitis, and other similar conditions.

[0033] The present invention also provides a pharmaceutical formulation comprising the above-mentioned diterpenoid compound, and one or more pharmaceutically acceptable carriers or excipients.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. Novel Compound Structure and Significant Activity: This invention is the first to isolate a novel diterpenoid compound from oregano, which has been confirmed as a novel compound not previously reported in the literature. In vitro anti-inflammatory experiments showed that this compound exhibited significant inhibitory activity against LPS-induced nitric oxide (NO) production by RAW264.7 macrophages at a concentration of 50 μM, with an inhibition rate as high as 62.97%, demonstrating excellent anti-inflammatory potential and providing a valuable lead compound for the development of novel anti-inflammatory drugs.

[0036] 2. The preparation method is efficient and reproducible: This invention provides a complete method for preparing this compound. By organically combining multiple 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 well-defined. It can efficiently and accurately separate and purify the target compound from oregano raw materials. Moreover, the method is stable and reproducible, laying a technological foundation for the further research and development of this compound.

[0037] 3. Abundant raw material sources and great development potential: This invention uses oregano, a plant used for both medicinal and edible purposes, as raw material. The resources are abundant and readily available, and the cost is low, which is conducive to the sustainable acquisition and future industrialization of this compound. The obtained compound can be used as an active ingredient to prepare drugs or health products for treating inflammatory diseases such as enteritis, hepatitis, and meningitis, expanding the medicinal and economic value of oregano and showing broad application prospects in the pharmaceutical and health fields. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The structure of the compound prepared in Example 1 of this invention is shown as structural formula I.

[0040] Figure 2 The image shows the infrared (IR) spectrum of the compound prepared in Example 1 of this invention.

[0041] Figure 3 The compound prepared in Example 1 of this invention 1 H NMR spectrum.

[0042] Figure 4 The compound prepared in Example 1 of this invention 13 C10 NMR spectrum.

[0043] Figure 5The image shows the HSQC spectrum of the compound prepared in Example 1 of this invention.

[0044] Figure 6 The image shows the HMBC spectrum of the compound prepared in Example 1 of this invention.

[0045] Figure 7 This is the COSY spectrum of the compound prepared in Example 1 of the present invention.

[0046] Figure 8 The cell survival rate under different LPS concentrations in Example 2 of this invention (**P<0.01, *P<0.05 compared with the control group).

[0047] Figure 9 The NO content in cells under different LPS concentrations in Example 2 of this invention (**P<0.01, *P<0.05 compared with the control group). Detailed Implementation

[0048] To make the technical means, creative features, objectives and effects of this invention easier to understand, the technical solutions in the specific embodiments of this invention are described clearly and completely below to further illustrate this invention. Obviously, the specific embodiments described are only a part of the embodiments of this invention, and not all of them.

[0049] Example 1: This example describes a method for preparing the diterpenoid compound of the present invention using oregano, comprising the following steps:

[0050] S1. Dry the whole oregano plant. In this example, a total of 30 kg of dried oregano was used. After appropriate crushing, it was passed through a 50-mesh sieve and extracted three times by heating and reflux with solvents of 90%, 80% and 70% ethanol and water, respectively. Each extraction time was 2 hours. The extracts obtained from each extraction were combined to obtain the total extract.

[0051] S2. The total extract obtained in step S1 is evaporated and concentrated under heating at 55°C and vacuum of 0.1 MPa, and dried to obtain 2.0 kg of total oregano extract.

[0052] S3. The total extract obtained in step S2 is sonicated in water to form a uniform suspension. The volume ratio of the total extract to water is 1:3 (V / V). The extract is then extracted with petroleum ether in a 5L separatory funnel to obtain the corresponding petroleum ether fraction (300g).

[0053] S4. Take the petroleum ether fraction obtained in step S3, mix it with normal-phase silica gel (100-200 mesh), and separate it in an open glass column. Perform gradient elution with petroleum ether-ethyl acetate volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1. Identify the eluted fractions using thin-layer chromatography and perform colorimetric comparison with 12% concentrated sulfuric acid ethanol under heating conditions. The Rf value of the target point is 0.1-0.6. Merge the same eluted fractions with similar points to obtain 20 fractions N1-N20.

[0054] S5. The fraction N5 obtained in step S4 is purified by Sephadex LH-20 gel column chromatography. The eluent is petroleum ether-dichloromethane-methanol (volume ratio of 5:5:1) for isocratic elution. Based on the thin-layer chromatography spotting, the same eluted fractions are combined to obtain 10 sub-fractions N5-1 to N5-10.

[0055] S6. Separation and preparation were carried out on a preparative high-performance liquid chromatography (HPLC) column (N5-5) under the following conditions: mobile phase: 86% acetonitrile-water, flow rate: 18 mL / min, detection wavelength: 290 nm, and preparative column: Waters SunFire C18 19×250 mm, 5 μm. There was one main peak in the HPLC, with an elution time of 15 min. After repeated preparation and enrichment, a new diterpenoid compound I was obtained, with a weight of 10 mg.

[0056] In this embodiment, the structure of the newly isolated diterpenoid compound I was identified:

[0057] The compound is a brown powder. HR-ESI-MS shows an m / z of 323.1283 [M+H]. + Peak (calculated value: C) 20 H 19 O4, 323.1283), its molecular formula was determined to be C 20 H 18 O4 has an unsaturation degree of 12. 1 There is one active hydrogen in the low field of the H-NMR spectrum. δ H 12.06, 6 olefin proton signals δ H 7.72, 7.67, 7.58, 7.31, 7.24, 7.21, 7.09 (each 1H, s). Three methyl proton signals are observed in the high field, including one bimodal proton signal δ. H 1.47 (3H, d, J =6.2Hz), 2 single-peak proton signals δ H 1.41, 1.33;13 The 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.

[0058] 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.

[0059] Table 1. New diterpenoid compound I 13 C-NMR data (measurement solvent: CD3OD); δ :ppm; J :Hz)

[0060]

[0061] Example 2: This example demonstrates the in vitro anti-inflammatory activity of the diterpenoid compounds prepared in Example 1.

[0062] 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%.

[0063] NO content determination: Add 8×10⁻⁶ to a 96-well plate. 3RAW264.7 cells in the logarithmic growth phase were incubated overnight at 37°C with 5% CO2. Different concentrations of diterpenoids were added to the experimental groups, while the blank and model groups were added with equal volumes of culture medium. Pretreatment was performed for 2 hours, with three replicates per group. LPS solution was then added, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, the NO content in the supernatant was measured according to the NO reagent kit instructions. 50 μL of supernatant was collected and added to a 96-well plate, followed by 50 μL of Griess Reagent I and then 50 μL of Griess Reagent II. The plate was shaken at room temperature in the dark for 5 minutes, and the OD value was measured at 540 nm using a microplate reader. A standard curve of absorbance versus NaNO2 was established, and the NO content was calculated based on the standard curve. NO inhibition rate (%) = (NO content in the model group - NO content in the experimental group) / NO content in the model group × 100%.

[0064] Data processing: Data analysis was performed using IBM SPSS 21 statistical software, and plotting was done using Origin 9.1 software. The t-test was used to compare data between two groups. In the significance analysis, *P < 0.05 indicated a significant difference, and **P < 0.01 indicated a highly significant difference, which was statistically significant.

[0065] RAW264.7 cell viability assay results showed that LPS concentrations of 0.1–5 μg / ml had no significant effect on cell viability, while a concentration of 50 μg / ml had a highly significant effect. Figure 8 As shown in the figure. The NO content test results indicate that the highest NO content was produced when LPS was 5 ug / ml. Figure 9 As shown in the figure. Therefore, considering all factors, 5 μg / ml was selected as the optimal stimulation concentration of LPS for RAW264.7 cells.

[0066] The novel diterpenoid compound I isolated from oregano in Example 1 was screened for its inhibitory effect on NO in LPS-induced RAW264.7 macrophages. The results showed that most of the screened novel diterpenoid compounds I had no significant effect on cell viability at a concentration of 50 μM, but inhibited NO production, exhibiting certain anti-inflammatory activity. The NO inhibition rate of the novel diterpenoid compound I was 62.97%, as shown in Table 2.

[0067] Table 2. Effects of compounds on inhibiting LPS-induced NO production in RAW264.7 cells.

[0068]

[0069] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments to this invention, without departing from the essence and scope of this invention, still fall within the scope of protection of this invention.

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. The diterpenoid compound was prepared by purifying N5-5 by preparative high performance liquid chromatography. The gradient elution uses petroleum ether-ethyl acetate volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1 as the eluent. When inspecting silica gel thin-layer plates, a 12% concentrated sulfuric acid ethanol solution is sprayed onto the silica gel thin-layer plates, and color development and comparison are performed under heating conditions. When performing Sephdex LH-20 dextran gel column chromatography on fraction N5, the eluent was petroleum ether-dichloromethane-methanol in a volume ratio of 5:5:

1. The chromatographic column used in the preparative high performance liquid chromatography purification system is C18; the mobile phase is 86% acetonitrile-water.

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 is performed using 100-200 mesh normal-phase silica gel.

6. The use of the diterpenoid compound of claim 1 in the preparation of a drug for inflammatory diseases.

7. The application according to claim 6, characterized in that, The inflammatory disease medications mentioned are those used to treat enteritis, hepatitis, and / or meningitis.

8. 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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