CN113563172B

CN113563172B - Azulene compound and preparation method and application thereof

Info

Publication number: CN113563172B
Application number: CN202111114354.5A
Authority: CN
Inventors: 何明珍; 冯育林; 李志强; 李志峰; 欧阳辉; 李军茂; 杨世林
Original assignee: Jiangxi University of Traditional Chinese Medicine; Jiangxi Bencao Tiangong Technology Co Ltd
Current assignee: Jiangxi University of Traditional Chinese Medicine; Jiangxi Bencao Tiangong Technology Co Ltd
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2021-12-17
Anticipated expiration: 2041-09-23
Also published as: AU2022352631B2; AU2022352631A1; JP2024505106A; WO2023045612A1; CN113563172A

Abstract

The application belongs to the technical field of medicines and discloses an azulene compound and a preparation method and application thereof, wherein the compound comprises two azulene compounds which are isomers of each other, and the chemical name of the compound is 8- (1, 2-dihydroxypropane-2-yl) -2-hydroxy-1, 10-dimethyl-9-dihydroazulene-3, 4-diketone. The preparation method of the compound is simple to operate, pure compounds can be obtained through separation, and experiments show that the two compounds have NO obvious influence on the growth of RAW264.7 macrophages within 24h, can obviously inhibit NO release of RAW264.7 cells induced by LPS, inhibit over secretion of IL-1 beta, IL-6 and TNF-alpha in the cells, and have obvious anti-inflammatory effect.

Description

Azulene compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an azulene compound, a preparation method and an application thereof, in particular to an application in preparation of anti-inflammatory drugs.

Background

Inflammation is a complex biological response of a tissue to a noxious stimulus, such as a pathogen, damaged cells, or an irritant. It is often a protective attempt by the organism to clear the noxious stimuli and initiate the tissue healing process. However, poorly controlled inflammation can lead to the development of a variety of diseases. When the immune response state of the body is abnormal, inappropriate or excessive immune response can be caused, so that tissues and cells are damaged, and inflammation is caused. Tissue damage caused by immune reactions is most commonly seen in various types of hypersensitivity reactions: type I allergy such as allergic rhinitis and urticaria, type II allergy such as anti-basement membrane glomerulonephritis, type III allergy such as glomerulonephritis caused by immune complex deposition, type IV allergy such as tuberculosis and typhoid fever; in addition, there are many autoimmune diseases such as lymphocytic thyroiditis, ulcerative colitis, and the like.

The lilac is herba Violae Murilowii of genus lilac of family Oleaceae (lilac)Syringa oblataLindl.). The root, stem, leaf and heartwood of lilac root and heartwood can be used as medicine, has the functions of clearing heat and drying dampness, resisting bacteria and hepatitis, etc., and can be used for treating heart heat, cardiodynia, dizziness, insomnia, palpitation, asthma and hei disease in mongolian medicine. Lilac has a large amount of cultivation and rich resources in China as ornamental flowers, but the medicinal development of lilac mainly focuses on the development of leaves, the research and report on stems are less, and the medicinal effect and medicinal effect substances of lilac are deeply researched and developed.

Disclosure of Invention

The invention aims to deeply research the chemical components of lilac stems and provides a preparation method and application of azulene compounds in lilac stems.

The compound comprises a compound 1 and a compound 2 which are isomers, and has a chemical name of 8- (1, 2-dihydroxypropane-2-yl) -2-hydroxy-1, 10-dimethyl-9-dihydroazulene-3, 4-diketone, and a structural formula as follows:

。

further, the preparation method of the azulene compounds comprises the following steps:

(1) adding an ethanol solution into the lilac stem medicinal material for extraction, filtering, collecting an extracting solution, and concentrating the extracting solution into an extract;

(2) dispersing the extract with water, sequentially extracting with dichloromethane and ethyl acetate, collecting two solvent extractive solutions and water solution, concentrating the solvent under reduced pressure, and drying to obtain dichloromethane extraction part, ethyl acetate extraction part, and water part;

(3) and (3) taking the ethyl acetate extraction part obtained in the step (2), and carrying out chromatographic separation and purification to obtain the azulene compounds.

Further, the chromatographic separation and purification in the step (3) specifically comprises the following steps:

A. dissolving the ethyl acetate extraction part obtained in the step (2) with methanol, adding silica gel to mix with a sample, loading the sample to a silica gel column, performing column chromatography separation, and performing gradient elution with petroleum ether-acetone with different volume ratios to obtain 11

fractions

1,2, 3,4, 5, 6, 7, 8, 9, 10 and 11;

B. separating fraction 4 by ODS medium pressure preparative chromatography, and performing gradient elution with methanol-water with different volume concentrations to obtain 5 fractions A, B, C, D, E;

C. separating fraction B by preparative liquid chromatography, eluting with 13% volume acetonitrile-water, collecting fractions according to peak emergence time of each chromatographic peak, and concentrating each fraction to obtain pure compound 1 and 2 crystals respectively.

Further, the ethanol solution in the step (1) is ethanol water solution with volume concentration of 5-95%, and the extraction method is cold soaking method, percolation method, microwave extraction method, ultrasonic extraction method, reflux extraction method or continuous reflux extraction method.

Further, the extraction times in the step (1) are 1-3, and the mass ratio of the addition amount of the ethanol solution to the syringyl stem medicinal material is (8-30): 1; the times of extraction of the dichloromethane and the ethyl acetate in the step (2) are respectively 4-6, and the dosage of the dichloromethane and the ethyl acetate is 1/2-1/4 of the volume of the extracted liquid.

Further, in the step A, the silica gel used for sample mixing is 100-200 meshes, and the mass ratio of the ethyl acetate extraction part to the silica gel used for sample mixing is 1: 10, the silica gel column is 200-mesh 300-mesh silica gel column, and the volume ratio of petroleum ether-acetone subjected to gradient elution is respectively 5: 1. 4: 1. 3: 1. 2: 1. 1: 1. 0: 1.

further, in step B, the volume concentrations of methanol-water subjected to gradient elution are respectively 5%, 15%, 30%, 50%, 70% and 100%, and the chromatographic conditions of ODS medium pressure preparative chromatography are as follows: ODS-C18 chromatographic column, size of chromatographic column 800 × 25mm, particle size 20-45 μm, flow rate 30ml/min, column temperature 25 deg.C.

Further, in step C, the chromatographic conditions of the preparative liquid chromatography are: and a YMC-Triart C18 chromatographic column, wherein the size of the chromatographic column is 250 x 20mm, the particle size is 5 mu m, and the flow rate is 10 ml/min.

The invention also provides application of the azulene compounds in preparation of anti-inflammatory drugs.

The azulene compounds can also be directly or indirectly added with various pharmaceutically acceptable common auxiliary materials, such as a filler, a disintegrating agent, a lubricant, an adhesive and the like, and prepared into oral preparations or injection preparations by a conventional pharmaceutical preparation method.

The oral preparation is tablet, capsule, granule, fat emulsion, microcapsule or dripping pill, and the injection preparation is injection or powder for injection.

Has the advantages that: the preparation method of the azulene compounds is simple to operate, and pure compounds can be obtained by separation. Experiments show that the azulene compounds have NO obvious influence on the growth of RAW264.7 macrophages within 24h, and can obviously inhibit NO release of RAW264.7 cells induced by LPS and over-secretion of IL-1 beta, IL-6 and TNF-alpha in the cells. Therefore, the compound of the invention can be applied to preparing anti-inflammatory drugs.

Drawings

FIG. 1 is a chemical plane structural diagram of the azulene compounds according to the invention;

FIG. 2 is an ECD measured value and a calculated value spectrogram of the azulene compounds of the invention;

FIG. 3 is a drawing showing a scheme of Compound 1 of the present invention¹An H-NMR spectrum;

FIG. 4 shows Compound 1 of the present invention¹³A C-NMR spectrum;

FIG. 5 is a mass spectrum of Compound 1 of the present invention;

FIG. 6 is a DEPT carbon spectrum of Compound 1 of the present invention;

FIG. 7 is an HMBC carbon spectrum of compound 1 of the present invention;

FIG. 8 is an HSQC carbon spectrum of Compound 1 of the present invention;

FIG. 9 is a NOESY carbon spectrum of Compound 1 of the present invention;

FIG. 10 is a carbon spectrum of H-H-COSY of Compound 1 of the present invention;

FIG. 11 is a drawing showing Compound 2 of the present invention¹An H-NMR spectrum;

FIG. 12 shows Compound 2 of the present invention¹³A C-NMR spectrum;

FIG. 13 is a mass spectrum of Compound 2 of the present invention;

FIG. 14 is a DEPT carbon spectrum of Compound 2 of the present invention;

FIG. 15 is an HMBC carbon spectrum of compound 2 of the present invention;

FIG. 16 is an HSQC carbon spectrum of Compound 2 of the present invention;

FIG. 17 is a NOESY carbon spectrum of Compound 2 of the present invention;

FIG. 18 is a carbon spectrum of H-H-COSY of Compound 2 of the present invention;

FIG. 19 is a schematic representation of the effect of Compound 1 of the present invention on the viability of RAW264.7 cells;

FIG. 20 is a schematic representation of the effect of Compound 2 of the present invention on the viability of RAW264.7 cells;

FIG. 21 is a graph showing the effect of Compound 1 of the present invention on LPS-induced lipid peroxidation in RAW264.7 cells;

FIG. 22 is a graph showing the effect of Compound 2 of the present invention on LPS-induced lipid peroxidation in RAW264.7 cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1

This example provides an azulene compounds in lilac, which includes two compounds 1 and 2 that are isomers of each other, and its chemical name is 8- (1, 2-dihydroxypropane-2-yl) -2-hydroxy-1, 10-dimethyl-9-dihydroazulene-3, 4-dione, and the structural formula is as follows:

。

the preparation method of the azulene compounds in the lilac comprises the following steps:

(1) taking dry syringa vulgaris stem medicinal material, adding 70% (volume concentration) ethanol of 8 times, heating and refluxing for 3 times, wherein the time is 2 hours, 2 hours and 1 hour respectively, filtering, combining extracting solutions, and concentrating under reduced pressure until no alcohol smell exists to obtain extract;

(2) dispersing the extract with water, sequentially extracting with dichloromethane for 5 times and ethyl acetate for 5 times, wherein the amount of dichloromethane and ethyl acetate is 1/3 of the volume of the extract, collecting two solvent extracts and water solution, concentrating the solvent under reduced pressure, and drying to obtain dichloromethane extract part, ethyl acetate extract part and water part;

(3) completely dissolving the ethyl acetate extraction part obtained in the step (2) by using methanol, adding 100-mesh 200-mesh silica gel into the mixture to mix the sample by a dry method, loading the sample into a 200-mesh 300-mesh silica gel column filled in advance, carrying out column chromatography separation, carrying out gradient elution by using petroleum ether-acetone (the volume ratio is 5: 1, 4: 1, 3: 1, 2: 1, 1: 1 and 0: 1), identifying by using silica gel GF254 and G thin layer plates, and combining the fractions containing approximate spots to obtain 11 fractions 1,2, 3,4, 5, 6, 7, 8, 9, 10 and 11; dissolving fraction 4 with methanol, filtering, subjecting the filtrate to ODS medium pressure preparative chromatography, gradient eluting with 5%, 15%, 30%, 50%, 70%, and 100% methanol-water, inspecting on silica gel GF254 thin layer plate, mixing fractions containing similar spots, concentrating, and drying to obtain 5 fractions A, B, C, D, E; dissolving fraction B with 50% acetonitrile, filtering, separating by preparative liquid chromatography, eluting with 13% acetonitrile-water, collecting fractions according to peak emergence time of each chromatographic peak, concentrating each fraction to obtain pure compound 1 (200 mg) and 2 (450 mg) crystals respectively, and detecting the purity of the two compounds by high performance liquid chromatography area normalization method to reach above 98%.

Wherein, the chromatographic conditions of the ODS medium-pressure preparative chromatography are as follows: ODS-C18 chromatographic column, size of chromatographic column 800 × 25mm, particle size 20-45 μm, flow rate 30ml/min, column temperature 25 deg.C.

The chromatographic conditions for preparative liquid chromatography were: and a YMC-Triart C18 chromatographic column, wherein the size of the chromatographic column is 250 x 20mm, the particle size is 5 mu m, and the flow rate is 10 ml/min.

Example 2

The embodiment provides an azulene compound in lilac, and the preparation method comprises the following steps:

(1) taking dry syringa vulgaris stem medicinal material, adding 15 times of 80% ethanol, extracting by cold soaking method for 2 times, each time for 24 hours, filtering, combining the extracting solutions, and concentrating under reduced pressure until no alcohol smell exists to obtain extract;

(2) dispersing the extract with water, sequentially extracting with dichloromethane for 4 times and ethyl acetate for 4 times, wherein the amount of dichloromethane and ethyl acetate is 1/2 of the volume of the extract, collecting two solvent extracts and water solution, concentrating the solvent under reduced pressure, and drying to obtain dichloromethane extract part, ethyl acetate extract part and water part;

(3) completely dissolving the ethyl acetate extraction part obtained in the step (2) by using methanol, adding 100-mesh 200-mesh silica gel into the mixture to mix the sample by a dry method, loading the sample into a 200-mesh 300-mesh silica gel column filled in advance, carrying out column chromatography separation, carrying out gradient elution by using petroleum ether-acetone (the volume ratio is 5: 1, 4: 1, 3: 1, 2: 1, 1: 1 and 0: 1), identifying by using silica gel GF254 and G thin layer plates, and combining the fractions containing approximate spots to obtain 11 fractions 1,2, 3,4, 5, 6, 7, 8, 9, 10 and 11; dissolving fraction 4 with methanol, filtering, subjecting the filtrate to ODS medium pressure preparative chromatography, gradient eluting with 5%, 15%, 30%, 50%, 70%, and 100% methanol-water, inspecting on silica gel GF254 thin layer plate, mixing fractions containing similar spots, concentrating, and drying to obtain 5 fractions A, B, C, D, E; dissolving fraction B with 50% acetonitrile, filtering, separating by preparative liquid chromatography, eluting with 13% acetonitrile-water, collecting fractions according to peak emergence time of each chromatographic peak, concentrating each fraction to obtain pure compounds 1 and 2 crystals, respectively, and detecting the purity of the two compounds by high performance liquid chromatography area normalization method to obtain the purity of more than 98%.

The rest is the same as in example 1.

Example 3

(1) taking dry syringa vulgaris stem medicinal material, adding 30 times of 95% ethanol, extracting by percolation method, percolating for 1 week, filtering, mixing extractive solutions, and concentrating under reduced pressure until no alcohol smell exists to obtain extract;

(2) dispersing the extract with water, sequentially extracting with dichloromethane for 6 times and ethyl acetate for 6 times, wherein the amount of dichloromethane and ethyl acetate is 1/4 of the volume of the extract, collecting two solvent extracts and water solution, concentrating the solvent under reduced pressure, and drying to obtain dichloromethane extract part, ethyl acetate extract part and water part;

The rest is the same as in example 1.

Example 4

The embodiment provides an azulene compound in lilac, and the preparation method comprises the following steps (1):

taking dried syringa vulgaris stem medicinal material, adding 5% (volume concentration) ethanol of 20 times, heating and refluxing for 3 times, wherein the time is 3 hours, 2 hours and 1 hour respectively, filtering, combining extracting solutions, and concentrating under reduced pressure until no alcohol smell exists to obtain extract.

The rest is the same as in example 1.

First, structural analysis of the Compound of the present invention

Mainly uses the wave spectrum technology, including ultraviolet, infrared, mass spectrum, nuclear magnetic resonance (¹H-NMR、¹³C-NMR, 2D-NMR) and the like, wherein specific spectrograms are shown in figures 2-18, and the spectral data and the analysis process are as follows:

(1) taking the compounds 1 and 2 obtained in the example 1 for structural analysis, wherein the compounds 1 and 2 are light yellow amorphous powder, and carrying out high-resolution mass spectrometry ESI-TOF-MSm/z：301.1046[M+Na]⁺。

The molecular formulas of the compound 1 and the compound 2 are both C₁₅H₁₈O₅The exact molecular weight is 278.1154 and the unsaturation is 7. And of two compounds¹H NMR、¹³The C NMR, HMBC, DEPT and HSQC spectra were essentially identical.

Comprehensive analysis¹H NMR、¹³C NMR showed that 3 methyl groups [ delta C24.2, 23.5 and 11.0 (corresponding to C-11, C-15 and C-12, respectively), delta H2.06 (s, Me-12), delta H1.38 (s, Me-15) and delta H1.37 (s, Me-11) were present in the compound structure]2 methylene groups [ Delta C69.1 and 38.6 (C-14 and C-9, respectively), Delta H3.65 (d,Jh-14a) and δ H3.53 (d,J = 11.3 Hz, H-14b), δH 3.02 (d, Jh-9a) and 2.00 (dd,J = 2.0, 17.6 Hz, H-9b)]2 methines [ δ C126.2 and 125.5 (corresponding to C-5 and C-7, respectively), δ H6.78 (s, H-5), δ H6.49 (d,J = 2.4 Hz, H-7)]and also 8 quaternary carbons [ Delta C189.3, 181.8, 171.2, 160.0, 147.0, 135.9, 76.6 and 45.7 (C-3, C-4, C-8, C-6, C-2, C-1, C-13 and C-10, respectively)]。

The planar structure of the compound is determined by combining two-dimensional nuclear magnetic resonance spectrum data such as HMBC, DEPT, HSQC spectrum and the like, the chemical planar structural formula of the compound is shown in the attached drawing 1, and the compound is chemically named as: 8- (1, 2-dihydroxypropan-2-yl) -2-hydroxy-1, 10-dimethyl-9-dihydroazulene-3, 4-dione. The compound has two chiral centers, namely C-10 and C-13, so that four configurations are respectively shown in the plane structure: (10R, 13S), (10S, 13R), (10R, 13R), (10S, 13S). The absolute configuration of the compound is determined to be (10R, 13R) and (10R, 13S) by comparing an actually measured electronic circular dichroism map (ECD) with an ECD spectrogram calculated by theoretical calculation (calculated by Gaussian 09W software), and the specific spectrogram is shown in figure 2. The NOESY spectrum data shows that the C-11, C-12 and C-15 methyl hydrogen of the compound 1 has related signals, while the compound 2 only generates related signals of the C-11 and C-12 methyl hydrogen, so that the absolute configurations of the compound 1 and the compound 2 can be judged to be (10R, 13R) and (10R, 13S) respectively, and the structural formula is shown in the example 1.

(2) The spectral data for compounds 1,2 are summarized below:

compound 1:¹H NMR (600 MHz, MeOD) δ 6.79 (s, 1H, H-5), 6.49 (d, J = 2.4 Hz, 1H, H-7), 3.70 (d, J = 11.3 Hz, 1H, H-14a), 3.62 (d, J = 11.3 Hz, 1H, H-14b), 3.14 (d, J = 17.7 Hz, 1H, H-9a), 2.43 (dd, J = 17.6, 2.0 Hz, 1H, H-9b), 2.07 (s, 3H, H-12), 1.38 (s, 3H, H-15), 1.36 (s, 3H, H-11)。

¹³C NMR (150 MHz, MeOD) δ 187.9 (C-3), 180.5 (C-4), 169.7 (C-8), 158.7 (C-6), 145.6 (C-2), 134.6 (C-1), 124.7 (C-5), 124.1 (C-7), 74.9 (C-13), 68.1 (C-14), 44.2 (C-10), 37.0 (C-9), 22.9 (C-11), 22.1 (C-15), 9.6 (C-12)。

compound 2:¹H NMR (600 MHz, MeOD) δ 6.79 (s, 1H, H-5), 6.51 (d, J = 2.4 Hz, 1H, H-7), 3.68 (d, J = 11.3 Hz, 1H, H-14a), 3.56 (d, J = 11.3 Hz, 1H, H-14b), 3.05 (d, J = 17.7 Hz, 1H, H-9a), 2.47 (dd, J = 17.6, 2.0 Hz, 1H, H-9b), 2.08 (s, 3H, H-12), 1.40 (s, 3H, H-15), 1.38 (s, 3H, H-11)。

¹³C NMR (150 MHz, MeOD) δ 187.9 (C-3), 180.4 (C-4), 169.8 (C-8), 158.6 (C-6), 145.6 (C-2), 134.5 (C-1), 124.8 (C-5), 123.6 (C-7), 75.2 (C-13), 67.7 (C-14), 44.2 (C-10), 37.2 (C-9), 22.8 (C-11), 22.1 (C-15), 9.6 (C-12)。

secondly, research on anti-inflammatory effect of the compound

(I) test materials and reagents

1. Drugs and agents

Two compounds 1,2 prepared in example 1 (self-extracted, purity 98% or more); mouse peritoneal macrophage RAW264.7 (Shanghai Xuan Biotech Co., Ltd.); fetal Bovine Serum (FBS) (Thermo Fisher/Gibco Co.). CCK-8 kit, IL-1 beta kit, IL-6 kit, TNF-alpha kit, DMEM culture medium, streptomycin mixed solution (100 x) (Beijing Soilebao science and technology Co., Ltd.); DMSO, LPS (sigma corporation, usa); nitric oxide detection kit (Shanghai Biyuntian biotechnology limited).

2. Laboratory apparatus

BT 125D type electronic balance (beijing sydolis scientific instruments ltd); SPECTRA star Nano full-wavelength microplate reader (BMGLABTECH); form 311 type carbon dioxide incubator (Thermo Scientific Co.).

3. Test drug and treatment method

127.82 mg and 227.79 mg of compounds are precisely weighed respectively and placed in a 5ml volumetric flask, dissolved by a small amount of DMSO, diluted by DMEM complete culture medium and fixed to the scale, drug-containing DMEM culture medium mother liquor with the compound concentration of 20mmol/L and 19.98 mmol/L is respectively obtained, and the DMEM culture medium is diluted to the required concentration during the experiment. The volume ratio of DMSO is controlled within three thousandths.

(II) Experimental method

1. Cell culture: RAW264.7 cells were added to DMEM medium containing 10% FBS and 1% streptomycin in 5% CO₂Subculturing in an incubator at 37 ℃ and subculturing every other day.

2. Evaluation of the Effect of two Compounds on RAW264.7 cell viability

Taking RAW264.7 cells in logarithmic growth phase, and adjusting cell density to 1 × 10 with DMEM medium containing 10% FBS culture solution⁴ ml^-1And inoculating the cells into a 96-well plate, adding cell culture solution containing compounds with different drug concentrations into the 96-well plate at a rate of 100 mu L/well, designing administration groups with drug concentrations of 12.5, 25, 50, 100, 200 and 400 mu mol/L respectively, additionally arranging a normal cell control group, and adding cell culture solution without drugs into the normal group, wherein each group has 6 duplicate wells. At 37 ℃ with 5% CO₂After 24h of culture, the supernatant was discarded and 10% CCK-8 solution was added to each well at 100. mu.L, and the cell viability was calculated by measuring the absorbance (OD value) at 450 nm in a microplate reader.

3. Evaluation of the Effect of two Compounds on LPS-induced lipid peroxidation of RAW264.7 cells

(1) Drawing a standard curve: the standard was diluted to a solution of 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100mM, and an equal amount of Griess reagent was added, and absorbance was measured at a wavelength of 550nm with a microplate reader to prepare a standard curve.

(2) The inhibition of LPS-induced NO production by RAW264.7 cells by the compound was determined by the Griess method. RAW264.7 cells in logarithmic growth phase were seeded in 96-well plates (5X 10)⁴Individual cells/well), 37 ℃ 5% CO₂Culturing for 24h under the condition, discarding the old culture medium, adding 100 mu L of DMEM culture medium containing medicines (low dose 50 mu mol/L, medium dose 100 mu mol/L and high dose 200 mu mol/L) into each well of the administration group, adding 100 mu L of blank culture medium into each well of the model group and the blank group, culturing for 1h, adding 100 mu L of LPS (2 mu g/ml) solution into each group except the blank group, continuously culturing for 24h, sucking 50 mu L of supernatant into each well, adding 50 mu L of Griess I solution and 50 mu L of Griess II solution into a new 96-well plate, measuring the OD value at 550nm by using an enzyme-labeling instrument, and calculating the NO inhibition rate.

4. ELISA method for detecting content of inflammatory factors IL-1 beta, IL-6 and TNF-alpha

The test grouping, modeling and administration processes are the same as the step 3(2), cell supernatant is collected, and the secretion amounts of IL-1 beta, IL-6 and TNF-alpha in the supernatant of RAW264.7 cells of each test group at 3, 6, 12 and 24 hours are determined according to the operation instructions of a cytokine detection kit.

5. Data processing and analysis

Data were analyzed by t-test using excel software, and results are expressed as "x ± s". Histograms were plotted using Graph Pad Prism 8.0.2 software.

(III) results of the experiment

1. Evaluation results of the Effect of two Compounds on RAW264.7 cell viability

The CCK-8 method detection results are shown in FIGS. 19 and 20, and the two compounds have no obvious influence on the growth of RAW264.7 macrophages within 24 hours when the concentration of the two compounds is 400 mu mol/L.

2. Evaluation of the Effect of two Compounds on LPS-induced lipid peroxidation of RAW264.7 cells

The results of the effect of the two compounds on LPS-induced release of NO from RAW264.7 cells are shown in table 1 below and fig. 21 and 22.

Analysis shows that the model groups of the compounds 1 and 2 have very significant difference (P < 0.01) compared with the control group, which indicates that LPS stimulation significantly increases NO production in RAW264.7 cells and the modeling is successful. Compared with the model group, the high-dose group and the medium-dose group of the compounds 1 and 2 have very significant difference (P is less than 0.01) and the low-dose group has significant difference (P is less than 0.05), which shows that the compounds 1 and 2 have obvious inhibition effect on NO generation in RAW264.7 cells after LPS stimulation.

TABLE 1 Effect of two compounds on LPS-induced lipid peroxidation of RAW264.7 cells

3. ELISA method for detecting content of inflammatory factors IL-1 beta, IL-6 and TNF-alpha

The experimental results are shown in tables 2 and 3, and compared with the blank group, after LPS stimulation, the contents of IL-1 beta, IL-6 and TNF-alpha in the cell supernatant of the model group are all remarkably increased, which indicates that the modeling is successful. Compared with the model group, the IL-1 beta content in the high-dose group of the compound 1 and the compound 2 and the medium-dose group of the compound 2 show extremely remarkable descending trend at 4 time points in the administration group, while the IL-1 beta content in the medium-dose group of the compound 1 also shows a certain descending trend, and both the compounds show dose dependence at the same time point; the IL-6 content in the compound 1 high dose group showed a very significant decrease at all 4 time points, as did the compound 1 medium dose group and the compound 2 high and medium dose groups. The content of TNF-alpha in the high-dose groups of the compounds 1 and 2 is extremely obviously reduced, and the medium-low dose groups of the compounds 1 and 2 also have certain inhibition effect on the content of the TNF-alpha; the compounds 1 and 2 can obviously inhibit the excessive secretion of IL-1 beta, IL-6 and TNF-alpha of RAW264.7 cells under the condition of LPS induction to a certain extent, and the compounds 1 and 2 have obvious anti-inflammatory action.

TABLE 2 Effect of Compound 1 on secretion of RAW264.7 cytokines IL-1 beta, IL-6 and TNF-alpha induced by LPS

TABLE 3 Effect of Compound 2 on LPS-induced secretion of the inflammatory factors IL-1 beta, IL-6 and TNF-alpha from RAW264.7 cells

Remarking: in tables 1,2 and 3 above and figures 21 and 22,^#it is meant that the model set is compared to the blank set,^#the expression P is less than 0.05,^##represents P < 0.01;^*means that the compound (high, medium and low) dose groups are compared with the model group,^*the expression P is less than 0.05,^**represents P < 0.01.

Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and not intended to limit the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or some technical features thereof can be replaced. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An azulene compound, which is characterized in that: the azulene compounds are compound 1 or compound 2, and the structural formula is as follows:

。

2. a process for preparing azulene compounds according to claim 1, characterized in that: the method comprises the following steps:

(3) carrying out chromatographic separation and purification on the ethyl acetate extraction part obtained in the step (2) to obtain the azulene compounds;

the chromatographic separation and purification in the step (3) specifically comprises the following steps:

A. dissolving the ethyl acetate extraction part obtained in the step (2) with methanol, adding silica gel to mix with a sample, loading the sample to a silica gel column, performing column chromatography separation, and performing gradient elution with petroleum ether-acetone with different volume ratios to obtain 11 fractions 1,2, 3,4, 5, 6, 7, 8, 9, 10 and 11;

C. separating fraction B by preparative liquid chromatography, eluting with 13% volume acetonitrile-water, collecting fractions according to peak emergence time of each chromatographic peak, and concentrating each fraction to obtain pure compound 1 and 2 crystals respectively;

in the step A, the silica gel used for sample mixing is 100-200 meshes, and the mass ratio of the ethyl acetate extraction part to the silica gel used for sample mixing is 1: 10, the silica gel column is 200-mesh 300-mesh silica gel column, and the volume ratio of petroleum ether-acetone subjected to gradient elution is respectively 5: 1. 4: 1. 3: 1. 2: 1. 1: 1. 0: 1;

in the step B, the volume concentration of methanol-water for gradient elution is respectively 5%, 15%, 30%, 50%, 70% and 100%, and the chromatographic conditions of ODS medium pressure preparative chromatography are as follows: ODS-C18 chromatographic column with size of 800 × 25mm, particle size of 20-45 μm, flow rate of 30ml/min, and column temperature of 25 deg.C;

in the step C, the chromatographic conditions of the preparative liquid chromatography are as follows: and a YMC-Triart C18 chromatographic column, wherein the size of the chromatographic column is 250 x 20mm, the particle size is 5 mu m, and the flow rate is 10 ml/min.

3. The process for producing the azulene compounds according to claim 2, characterized in that: the ethanol solution in the step (1) is ethanol water solution with volume concentration of 5-95%, and the extraction method is cold soaking method, percolation method, microwave extraction method, ultrasonic extraction method, reflux extraction method or continuous reflux extraction method.

4. The process for producing the azulene compounds according to claim 2, characterized in that: the extraction times in the step (1) are 1-3 times, and the mass ratio of the addition amount of the ethanol solution to the syringyl stem medicinal material is (8-30): 1; the times of extraction of the dichloromethane and the ethyl acetate in the step (2) are respectively 4-6, and the dosage of the dichloromethane and the ethyl acetate is 1/2-1/4 of the volume of the extracted liquid.

5. Use of the azulene compounds according to claim 1 in the preparation of anti-inflammatory drugs.

6. The use of the azulene compounds according to claim 5 in the preparation of anti-inflammatory drugs, characterized in that: the azulene compounds are directly or indirectly added with pharmaceutically acceptable carriers or excipients to prepare oral preparations or injection preparations, wherein the oral preparations are tablets, capsules, granules, fat emulsion, microcapsules or dropping pills, and the injection preparations are injection or powder injection.