AU2022352631B2

AU2022352631B2 - Azulene compound, and preparation method therefor and use thereof

Info

Publication number: AU2022352631B2
Application number: AU2022352631A
Authority: AU
Inventors: Yulin FENG; Mingzhen HE; Junmao LI; Zhifeng Li; Zhiqiang Li; Hui Ouyang; Shilin Yang
Original assignee: Jiangxi Bencao Tiangong Technology Co Ltd; Jiangxi University of Traditional Chinese Medicine
Current assignee: Jiangxi Bencao Tiangong Technology Co Ltd; Jiangxi University of Traditional Chinese Medicine
Priority date: 2021-09-23
Filing date: 2022-08-12
Publication date: 2024-05-16
Anticipated expiration: 2042-08-12
Also published as: CN113563172B; JP2024505106A; AU2022352631A1; JP7501847B2; CN113563172A; WO2023045612A1

Abstract

The present application belongs to the technical field of medicine, and discloses an azulene compound, and a preparation method therefor and the use thereof. The compound comprises two azulene compounds that are mutually isomers, and the chemical name thereof is 8-(1,2-dihydroxypropane-2-yl)-2-hydroxy-1,10-dimethyl-9-dihydroazulene-3,4-dione. The preparation method of the compound of the present invention is simple in terms of operation, and can obtain a pure compound by means of separating. Experiments show that the two compounds of the present invention have no significant effect on the growth of RAW 264.7 macrophages within 24 h, can significantly inhibit LPS-induced NO release of RAW264.7 cells and inhibit excessive secretion of IL-1β, IL-6 and TNF-α in cells, and have an obvious anti-inflammatory effect.

Description

AZULENE COMPOUND, AND PREPARATION METHOD THEREFOR AND USE THEREOF

Technical Field

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

Background Art

Inflammation is a complex biological response of tissues to harmful stimuli such as pathogens, damaged cells, or irritants. Inflammation is usually a protective attempt by an organism to remove harmful stimuli and initiate the process of tissue healing. However, inadequately controlled inflammation might lead to incidence of a variety of diseases. When the body's immune response is abnormal, it might cause an inappropriate or excessive immune response, and further lead to inflammation due to damage of tissues and cells. Tissue damage caused by the immune response is most common in various types of hypersensitivity reactions: type I allergies such as allergic rhinitis and urticaria, type II allergies such as anti-basement membrane glomerulonephritis, type III allergies such as glomerulonephritis caused by immune complex deposition, type IV allergies such as tuberculosis, typhoid fever, etc.; and many autoimmune diseases such as lymphocytic thyroiditis, ulcerative colitis, and so on.

Lilac is Syringa oblata Lindl. Its roots, stems, leaves, and heartwood can all be used as medicine. It has the curative effects of clearing away heat, drying dampness, and being antibacterial and anti-hepatitis. In Mongolian medicine, lilac roots and heartwood are used to treat heart heat, heart tingling, dizziness, insomnia, palpitation, asthma, and "Heyi" disease. China is rich in lilac an ornamental flower, but only its leaves are mainly applied to the medicinal field currently, and there are few researches and reports on use of its stems. Therefore, its medicinal effects and medicinal substances need to be further studied and developed.

Summary of the Invention

The present invention attempts to conduct an in-depth study on the chemical components of lilac stems, and provides a preparation method and use of azulene compounds in the lilac stems.

An azulene compound is provided, and the azulene compound includes a compound 1 and a compound 2 that are isomers respective to each other. The chemical name of the azulene compound is 8-(1,2-dihydroxypropane-2-yl)-2-Hydroxy-1,10-dimethyl-9-dihydroazulene-3,4 dione, and the structural formula therefor is as follows:

OH OH OH OH

K 0 0 oH OOO 00

Compound 1 Compound 2

Further, the preparation method for the azulene compound includes the following steps:

(1) taking out a lilac stem, adding an ethanol solution to extract, collecting extracted liquid after filtering, and concentrating the extracted liquid into an extract;

(2) taking out the extract, dispersing it with water, extracting with dichloromethane and ethyl acetate sequentially, collecting two solvent extracts and an aqueous solution, concentrating the solvent under reduced pressure, and drying to obtain a dichloromethane extract, an ethyl acetate extract, and a water extract; and

(3) taking out the ethyl acetate extract obtained in step (2) and performing chromatographic separation and purification to obtain the azulene compound.

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

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

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

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

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

Further, the number of extractions in step (1) is 1-3, and the mass ratio of the amount of the ethanol solution added to the lilac stem is (8-30):1; and the number of dichloromethane and ethyl acetate extractions in step (2) is 4-6, and the amount of dichloromethane and ethyl acetate each time is 1/2-1/4 of the volume of the extracted liquid.

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

Further, in step B, the volume concentrations of methanol-water for gradient elution are 5%, 15%, 30%, 50%, 70%, and 100%, respectively, and the chromatographic conditions of the ODS medium pressure preparative chromatography are as follows: ODS-C18 chromatographic columns, a chromatographic column size of 800*25 mm, a particle size of 20-45 [m, a flow rate of 30 ml/min, and a column temperature of 25°C.

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

The present invention further provides a use of the azulene compound in the preparation of anti-inflammatory drugs.

In the present invention, the azulene compound can be directly or indirectly added to various pharmaceutically acceptable common excipients, such as fillers, disintegrants, lubricants, binders, etc., to prepare oral preparations or injectable preparations by means of conventional pharmaceutical preparation methods.

The oral preparations exist in the forms of tablets, capsules, granules, lipid emulsions, microcapsules or dripping pills, and the injectable preparations include liquid injections or powder injections.

Beneficial effects: the preparation method for the azulene compound of the present invention is simple to operate, through which a pure compound can obtained via separation. Experiments have shown that the azulene compounds of the present invention has no significant effect on the growth of RAW 264.7 macrophages within 24 hours, but can significantly inhibit LPS-induced NO release in RAW264.7 cells, and excessive secretion of IL- , IL-6 and TNF-a in the cells. Therefore, the compound of the present invention can be applied to the preparation of anti-inflammatory drugs.

Description of the Drawings

FIG. 1 shows a chemical plane structure of an azulene compound of the present invention;

FIG. 2 is a spectrum on the measured value and calculated value of electronic circular dichroism (ECD) of the azulene compound of the present invention;

FIG. 3 is a1 H nuclear magnetic resonance (H-NMR) spectrum of a compound 1 of the present invention;

FIG. 4 is a carbon-13 nuclear magnetic resonance 1( 3 C-NMR) spectrum of the compound 1 of the present invention;

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

FIG. 6 is a Distortionless Enhancement by Polarization Transfer (DEPT) spectrum of the compound 1 of the present invention;

FIG. 7 is a heteronuclear multiple bond correlation (HMBC) spectrum of the compound 1 of the present invention;

FIG. 8 is a heteronuclear single quantum coherence (HSQC) spectrum of the compound 1 of the present invention;

FIG. 9 is a nuclear overhauser effect spectroscopy (NOESY) spectrum of the compound 1 of the present invention;

FIG. 10 is an H-H correlation spectroscopy (H-H-COSY) spectrum of the compound 1 of the present invention;

FIG. 11 is a H-NMR spectrum of a compound 2 of the present invention;

FIG. 12 is a1 3 C-NMR spectrum of the compound 2 of the present invention;

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

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

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

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

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

FIG. 18 is an H-H-COSY spectrum of the compound 2 of the present invention;

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

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

FIG. 21 is a schematic diagram of the effect of the compound 1 of the present invention on LPS-induced lipid peroxidation in RAW264.7 cells; and

FIG. 22 is a schematic diagram of the effect of the compound 2 of the present invention on LPS-induced lipid peroxidation in RAW264.7 cells.

Detailed Description of Embodiments

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are merely some rather than all of the embodiments of the present invention. On the basis of the embodiments in the present invention, all other embodiments acquired by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products, and all can be purchased through commercial channels.

Example 1

This example provides a lilac azulene compound, and the azulene compound includes a compound 1 and a compound 2 that are isomers respective to each other. The chemical name of the azulene compound is 8-(1,2 (dihydroxypropane-2-yl)-2-Hydroxy-1,10-dimethyl-9

dihydroazulene-3,4-dione, and the structural formula therefor is as follows:

OH OH. OH OH

o= OH OH o 0 Compound 1 Compound 2

The preparation method for the lilac azulene compound is as follows:

(1) a dried lilac stem was taken out, 8 times the amount of 70% (volume concentration) ethanol were added, heating reflux extraction was performed for 2 hours, 2 hours, and 1 hour, respectively, and extracted liquid was filtered, merged, and concentrated under reduced pressure to be non-alcoholic, to obtain an extract;

(2) the extract was dispersed with water, and extracted with dichloromethane and ethyl acetate 5 times sequentially (the amount of dichloromethane and ethyl acetate used each time is 1/3 of the volume of the extracted liquid), two solvent extracts and an aqueous solution were collected, the solvent was concentrated under reduced pressure and dried to obtain a dichloromethane extract, an ethyl acetate extract, and a water extract; and

(3) the ethyl acetate extract obtained in step (2) was fully dissolved with methanol, 100-200 mesh silica gel was added to mix a sample through the dry method, the sample was loaded into a pre-filled 200-300 mesh silica gel column for column chromatography separation, petroleum ether-acetone (volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1) was used for gradient elution, silica gel GF254 and G TLC (thin layer chromatography) plates were used to identify, and fractions containing similar spots were merged to obtain 11 fractions numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively; fraction 4 was dissolved in methanol and filtered, the obtained filtrate was separated by ODS medium pressure preparative chromatography, methanol-water (with the concentration of 5%, 15%, 30%, 50%, 70%, or 100%) was used for gradient elution, the silica gel GF254 TCL plates were checked, and the fractions with similar spots were merged, concentrated, and dried to obtain 5 fractions numbered A, B, C, D, and E; fraction B was dissolved in 50% acetonitrile, filtered, separated by preparative liquid chromatography, and eluted with 13% acetonitrile-water, and the fractions were collected according to the appearance time of each chromatographic peak, and concentrated to obtain the pure crystals of the compound 1 (200mg) and the compound 2 (450mg) respectively. The purity of the two compounds was detected by means of the area normalization method for high performance liquid chromatography, and the purity was above 98%.

The chromatographic conditions of the ODS medium pressure preparative chromatography are as follows: ODS-C18 chromatographic columns, a chromatographic column size of 800*25 mm, a particle size of 20-45 m, a flow rate of 30 ml/min, and a column temperature of 25°C.

The chromatographic conditions of the preparative liquid chromatography are as follows: YMC-Triart C18 chromatographic columns, a chromatographic column size of 250*20 mm, a particle size of 5 m, and a flow rate of 10 ml/min.

Example 2

This example provides a lilac azulene compound, and its preparation method is as follows:

(1) a dried lilac stem was taken out, 15 times the amount of 80% ethanol were added, cold soaking extraction was performed twice (24 hours each time), and extracted liquid was filtered, merged, and concentrated under reduced pressure to be non-alcoholic, to obtain an extract;

(2) the extract was dispersed with water, and extracted with dichloromethane and ethyl acetate 4 times sequentially (the amount of dichloromethane and ethyl acetate used each time is 1/2 of the volume of the extracted liquid), two solvent extracts and an aqueous solution were collected, the solvent was concentrated under reduced pressure and dried to obtain a dichloromethane extract, an ethyl acetate extract, and a water extract; and

(3) the ethyl acetate extract obtained in step (2) was fully dissolved with methanol, 100-200 mesh silica gel was added to mix a sample through the dry method, the sample was loaded into a pre-filled 200-300 mesh silica gel column for column chromatography separation, petroleum ether-acetone (volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1) was used for gradient elution, silica gel GF254 and G TLC (thin layer chromatography) plates were used to identify, and fractions containing similar spots were merged to obtain 11 fractions numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively; fraction 4 was dissolved in methanol and filtered, the obtained filtrate was separated by ODS medium pressure preparative chromatography, methanol-water (with the concentration of 5%, 15%, 30%, 50%, 7 0 % , or 100%) was used for gradient elution, the silica gel GF254 TCL plates were checked, and the fractions with similar spots were merged, concentrated, and dried to obtain 5 fractions numbered A, B, C, D, and E; fraction B was dissolved in 50% acetonitrile, filtered, separated by preparative liquid chromatography, and eluted with 13% acetonitrile-water, and the fractions were collected according to the appearance time of each chromatographic peak, and concentrated to obtain the pure crystals of the compound 1 and the compound 2 respectively. The purity of the two compounds was detected by means of the area normalization method for high performance liquid chromatography, and the purity was above 98%.

The rest are the same as in Example 1.

Example 3

(1) a dried lilac stem was taken out, 30 times the amount of 95% ethanol were added, percolation extraction was performed for 1 week, and extracted liquid was filtered, merged, and concentrated under reduced pressure to be non-alcoholic, to obtain an extract;

(2) the extract was dispersed with water, and extracted with dichloromethane and ethyl acetate 6 times sequentially (the amount of dichloromethane and ethyl acetate used each time is 1/4 of the volume of the extracted liquid), two solvent extracts and an aqueous solution were collected, the solvent was concentrated under reduced pressure and dried to obtain a dichloromethane extract, an ethyl acetate extract, and a water extract; and

(3) the ethyl acetate extract obtained in step (2) was fully dissolved with methanol, 100-200 mesh silica gel was added to mix a sample through the dry method, the sample was loaded into a pre-filled 200-300 mesh silica gel column for column chromatography separation, petroleum ether-acetone (volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1) was used for gradient elution, silica gel GF254 and G TLC (thin layer chromatography) plates were used to identify, and fractions containing similar spots were merged to obtain 11 fractions numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively; fraction 4 was dissolved in methanol and filtered, the obtained filtrate was separated by ODS medium pressure preparative chromatography, methanol-water (with the concentration of 5%, 15%, 30%, 50%, 70%, or 100%) was used for gradient elution, the silica gel GF254 TCL plates were checked, and the fractions with similar spots were merged, concentrated, and dried to obtain 5 fractions numbered A, B, C, D, and E; fraction B was dissolved in 50% acetonitrile, filtered, separated by preparative liquid chromatography, and eluted with 13% acetonitrile-water, and the fractions were collected according to the appearance time of each chromatographic peak, and concentrated to obtain the pure crystals of the compound 1 and the compound 2 respectively. The purity of the two compounds was detected by means of the area normalization method for high performance liquid chromatography, and the purity was above 98%.

The rest are the same as in Example 1.

Example 4

This example provides a lilac azulene compound, and step (1) of its preparation method is as follows:

a dried lilac stem was taken out, 20 times the amount of 5% (volume concentration) ethanol were added, heating reflux extraction was performed for 3 hours, 2 hours, and 1 hour, respectively, and extracted liquid was filtered, merged, and concentrated under reduced pressure to be non-alcoholic, to obtain an extract.

The rest are the same as in Example 1.

I. Structure analysis of the compound of the present invention

Spectroscopy techniques, including ultraviolet, infrared, mass spectrometry, nuclear magnetic resonance (1H-NMR, "C-NMR, 2D-NMR), etc., are mainly used to identify the structure of the compound of the present invention. The specific wave spectrogram is shown in FIGs. 2-18, and its spectral data and analysis process are as follows:

(1) The compounds 1 and 2 obtained in Example 1 were taken for structural analysis. Compounds 1 and 2 are pale yellow amorphous powders. The high-resolution mass spectrum is ESI-TOF-MS m/z: 301.1046[M+Na]*.

The molecular formulaofC 15 Hi 8 05applies to both the compound 1 and the compound 2, the exact molecular weight is 278.1154, and the degree of unsaturation is 7. And the 'H NMR, "C NMR, HMBC, DEPT, and HSQC spectra of the two compounds are basically the same.

Comprehensive analysis of 1H NMR and 13 C NMR shows that there are 3 methyl groups in the structure of the compound [6C 24.2, 23.5, and 11.0 (corresponding to C-11, C-15, and C-12 respectively), 6H 2.06 (s, Me-12 ), 6H 1.38 (s, Me-15), and 6H1.37 (s, Me-l1)], 2 methylene groups [6C 69.1 and 38.6 (C-14 and C-9, respectively), 6H 3.65 (d , J=11.3 Hz, H-14a) and 6H 3.53 (d, J= 11.3 Hz, H-14b), 6H 3.02 (d, J= 17.7 Hz, H-9a) and 2.00 (dd, J= 2.0, 17.6 Hz, H 9b)], 2 methine groups [6C 126.2 and 125.5 (corresponding to C-5 and C-7 respectively), 6H 6.78 (s, H-5), 6H 6.49 (d, J= 2.4 Hz, H-7)], and 8 quaternary carbons [6C189.3, 181.8, 171.2, 160.0, 147.0, 135.9, 76.6, and 45.7 (respectively C-3, C-4, C-8, C-6, C -2, C-1, C-13, and C 10)].

In combination with two-dimensional NMR spectroscopy data such as HMBC, DEPT and HSQC spectra, a planar structure of the compound of the present invention is determined. Its chemical planar structure is shown in FIG. 1 and its chemical name is 8-(1,2-dihydroxypropane 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 there are four configurations for this planar structure: (10R, 13S), (lOS, 13R), (10R, 13R), and (OS, 13S). By comparing the measured electronic circular dichroism (ECD) spectrum with the theoretically calculated ECD spectrum (calculated by Gaussian 09W software), it is determined that the absolute configuration of the compound is (10R, 13R) and (10R, 13S). The specific spectrum is shown in FIG. 2. According to the NOESY spectrum data, it is found that the C-11, C-12, and C-15 methyl hydrogen of the compound 1 have related signals, while the compound 2 only produces the related signals of C-i land C-12 methyl hydrogen, so the absolute configurations of the compound 1 and the compound 2 can be judged to be (10R, 13R) and (10 R, 13S), respectively, and the structural formula is as shown in Example 1.

(2) The spectral data of the compounds 1 and 2 are summarized as follows:

Compound 1: 1H NMR (600 MHz, MeOD) 6 6.79 (s, 1H, H-5), 6.49 (d, J= 2.4Hz, 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).

"C NMR (150 MHz, MeOD) 6 187.9 (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) 6 6.79 (s, 1H, H-5), 6.51 (d, J= 2.4Hz, 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).

"C NMR (150 MHz, MeOD) 6 187.9 (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).

II. Research on the anti-inflammatory effect of the compound of the present invention

(1) Experimental materials and reagents

1. Drugs and reagents

The two compounds 1 and 2 prepared in Example 1 (self-extracted, with a purity of more than 98%); mouse peritoneal macrophages RAW264.7 (Shanghai Xuanke Biotechnology Co., Ltd.); fetal bovine serum (FBS) (Thermo Fisher/ Gibco). CCK-8 kit, IL- Ikit, IL-6 kit, TNF-a kit, DMEM medium, penicillin streptomycin mixture (100x) (Beijing Solarbio Science &

Technology Co.,Ltd.); DMSO, LPS (American sigma company); Nitric oxide detection kit (Shanghai Beyotime Biotechnology Co., Ltd.).

2. Laboratory apparatus

BT 125D electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); SPECTRO star Nano full-wavelength microplate reader (BMGLABTECH); Form 311 carbon dioxide incubator (Thermo Scientific Company).

3.Test drugs and treatment methods

27.82 mg of the compound 1 and 27.79 mg of the compound 2 were weighed respectively, placed in a 5 ml volumetric flask, first dissolved with a small amount of DMSO, and then diluted with a DMEM complete medium to a metered volume to obtain a drug-containing DMEM medium stock solution with compound concentrations of 20 mmol/L and 19.98 mmol/L, respectively. In the experiment, the DMEM medium was used for diluting to the required concentration. The volume ratio of DMSO is controlled within three thousandths.

(2) Experimental methods

1. Cell culture: RAW 264.7 cells were added to the DMEM medium containing a mixture of 10% FBS and 1% penicillin streptomycin, subcultured in an incubator containing 5% C02 at 37 °C, and subcultured the next day.

2. Evaluation of the effects of two compounds on the viability of RAW264.7 cells

RAW264.7 cells in the logarithmic growth phase were inoculated in a 96-well plate after the cell density was adjusted to 1x104 ml' with a DMEM medium containing 10% FBS culture solution. The cell culture solution containing the compounds with different drug concentrations was added to the 96-well plate at a ratio of 100 pL/well. The administration groups with drug concentrations of 12.5, 25, 50, 100, 200, and 400 [mol/L were set up. In contrast, normal cell control groups were set up, and a drug-free cell culture solution was added, with 6 replicate wells per group. After incubating at 37 °C in a solution containing 5% C02 for 24 hours, the supernatant was discarded, and 10% CCK-8 solution was added, with 100 pL per well. The absorbance (OD value) at a wavelength of 450 nm was measured with a microplate reader to calculate the cell viability.

3. Evaluation of the effects of two compounds on LPS-induced lipid peroxidation in RAW264.7 cells

(1) Standard curve plotting: a standard was diluted into a solution with a concentration of 0, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 mM, the same amount of Griess reagent was added, and the absorbance was measured at a wavelength of 550 nm with a microplate reader to draw a standard curve.

(2) The Griess method was used to determine the inhibitory effect of the compounds on LPS-induced NO production in RAW264.7 cells. RAW264.7 cells in the logarithmic growth phase were inoculated in a 96-well plate (5x104 cells/well) and cultured at 37°C in a solution containing 5% CO2 for 24 hours, the old medium was discarded, and 100 pL/well of a drug- containing DMEM medium (low dose of 50 pmol/L, medium dose of 100 pmol/L, high dose of 200 [mol/L) was added for each administration group. For the model group and the blank group, 100 [L/well of a blank medium was added. After culturing for 1 hour, 100 pL of an LPS (2 ptg/ml) solution was added for each group rather than the blank group. After continued incubation for 24 hours, 50 pL of supernatant was sucked from each well into a new 96-well plate. 50 L of a Griess I solution and 50 pL of a GriessII solution were added successively, and the OD value at 550 nm was measured with a microplate reader to calculate the NO inhibition rate.

4. Using ELISA method to detect the contents of inflammatory factors IL-I, IL-6, and TNF-a

The experiment grouping, modeling and administration procedures are the same as those in step 3(2). The cell supernatant was collected, and the secretion volumes of IL-I, IL-6, and TNF-a in the RAW264.7 cell supernatant at 3, 6, 12, and 24 hours, were determined according to the cytokine detection kit operating instructions for each test group.

5. Data processing and analysis

The data was analyzed by t-test using excel software, and the results were expressed as "x s". Graph Pad Prism8.0.2 software was used to draw a histogram.

(3) Experimental results

1. Evaluation results of the effects of two compounds on the viability of RAW264.7 cells

The results of detection by the CCK-8 method are shown in FIGs. 19 and 20. At 400 ptmol/L, the two compounds had no significant effect on the growth of RAW264.7 macrophages within 24 hours.

2. Evaluation of the effects of two compounds on LPS-induced lipid peroxidation in RAW264.7 cells

The results on the effects of the two compounds on the LPS-induced NO release in RAW264.7 cells are shown in Table 1 and FIGs. 21 and 22.

Analysis results showed that the model group of the compounds 1 and 2 had extremely significant differences compared with the control group (P<0.01), indicating that LPS stimulation significantly increased the production of NO in RAW264.7 cells, and the modeling was successfully completed. Compared with the model group, the high-dose and medium-dose groups (administration groups) of the compounds 1 and 2 had extremely significant differences

(P < 0.01), and the low-dose group had significant differences (P < 0.05), indicating that compounds 1 and 2 had a significant inhibitory effect on production of NO in RAW264.7 cells after LPS stimulation.

Table 1 Effects of two compounds on LPS-induced lipid peroxidation in RAW264.7 cells

NO dilution amount

Group Compound 1 Compound 2

Blank group 5.55+0.42 5.55+0.42

Model group 19.7±1.17## 21.17±0.96##

Low-dose group 11.32+1.15* 12.8+1.12*

Medium-dose group 9.67+0.84** 9.93±0.89**

High-dose group 6.5+0.6** 6.83+0.56**

3. Using the ELISA method to detect the contents of inflammatory factors IL-I, IL-6, and TNF-a

The experimental results are shown in Tables 2 and 3. Compared with those of the blank group, the contents of IL- I, IL-6, and TNF-a in the cell supernatant of the model group were significantly increased after LPS stimulation, indicating that the modeling was successfully completed. Compared with the model group, the high-dose group of the compounds 1 and compound 2 and the medium-dose group of the compound 2 showed an extremely significant decline in the IL- Icontent at 4 time points, while the medium-dose group of the compound 1 also showed a certain downward trend in the IL- Pcontent, and both compounds were dose dependent at the same time point; the high-dose group of the compound 1 showed an extremely significant decline in the IL-6 content at 4 time points, and the medium-dose group of the compound 1 and the high-dose and medium-dose groups of the compound 2 also showed a downward trend. The high-dose group of the compounds 1 and 2 showed an extremely significant decline in the content of TNF-a, while the medium-dose and low-dose groups of the compounds 1 and 2 also had a certain inhibitory effect on the content of TNF-a. It indicates that the compounds 1 and 2 could significantly inhibit the excessive secretion of IL-I, IL-6, and TNF-a in RAW264.7 cells under LPS-induced conditions to a certain extent, and the compounds 1 and 2 had significant anti-inflammatory effects.

Table 2 The effect of the compound 1 on the secretion of LPS-induced inflammatory factors IL- I, IL-6, and TNF-a in RAW264.7 cells

Inflam T Blank group Model group Compound 1 matory I factors m High-dose Medium-dose Low-dose e group group group

h )

3 2043A$023 39.z1032 339A $10.g5 433364 3f1 4267f 2

12 204431k73 $4 i L t1 412 11927 413M*110.9 4 79,1L7

24 86 9 9 4`3199,9 279 L 32047±9 97 39439t1578

S126 33+2,40 =0 375b38 l 9690e1L56 409 8 a31

n4

6 41669 4 5097*15 9 3)5 2,2 49 3

12 16490&l9 4233449 31 12 32334Zl 3.14tNi3

24 143934::15 24,15108 357jVI.0 2 2 S 737 1 SS99 24.4Jt 2977 30 3 7.2313a,,1

Table 3 The effect of the compound 2 on the secretion of LPS-induced inflammatory factors IL- I, IL-6, and TNF-a in RAW264.7 cells

h )

ILl `3 2044 3i:0 21 4*

9N 10 44 412 6 4 ~¶~~2O0 U $rnt 0-t 413$±24 4S Al28±32 p)490472"9 6~ ~~V0 19A010 2 20133=9.3 . e 519b5±892 54306*(K04 4n2341$42 45994dt 24 18I56i8 98 3949z}5S

6 12L$StS.99 *. 4119O0i136 44937Z*II.74 M3S&3112' 35S09461 TF9 . 498~~ 0411M 449 72231 I9 P<0.5,ad*1*m1e 32557P.003 4 UsA3'ztU46" Fhimt12 oh e

-44 42L~h2IiX~t4., 434116, 23 24 X941t

Remarks: In the above tables 1-3 and FIGs. 21 and 22, #refers tothe comparison between the model group and the blank group, #indicates P <0.05, ## indicates P <0.01; *refers to the comparison of the compound (high, medium, low) dose groups with the model group, *means P<0.05, and ** means P<0.01.

Finally, it should be noted that the above is only apreferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiment, for those skilled in the art, it is still apparent that the technical solutions described in the above embodiment may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention are intended to fall within the scope of protection of the present invention.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

It will be understood that the terms "comprise" and "include" and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

In some cases, a single embodiment may, for succinctness and/or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to "at least one of' a list of items refers to any combination of those items, including single members. As an example, "at least one of: a, b, or c" is intended to cover: a,b, c, a-b, a-c,b-c, and a-b-c.

It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An azulene compound, wherein the azulene compound is a compound 1 or a compound 2, and its structural formula is as follows:

OH OH OH

0 0 H OH 0 .0 Compound I Compound 2

2. A preparation method for the azulene compound according to claim 1, comprising the following steps:

(3) taking out the ethyl acetate extract obtained in step (2) and performing chromatographic separation and purification to obtain the azulene compound,

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

C. separating the fraction B by means of preparative liquid chromatography, eluting with acetonitrile-water with a volume concentration of 13%, collecting fractions according to the appearance time of each chromatographic peak, and concentrating each fraction to obtain pure crystals of the compounds 1 and 2 respectively, in step A, the silica gel used for sample mixing has 100-200 meshes, the mass ratio of the ethyl acetate extract to the silica gel used for sample mixing is 1:10, the silica gel column has 200-300 meshes, and the volume ratios of petroleum ether-acetone for gradient elution are 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively; in step B, the volume concentrations of methanol-water for gradient elution are 5%, 15%, %, 50%, 70%, and 100%, respectively, and the chromatographic conditions of the ODS medium pressure preparative chromatography are as follows: ODS-C18 chromatographic columns, a chromatographic column size of 800*25 mm, a particle size of 20-45 [m, a flow rate of 30 ml/min, and a column temperature of 25°C; and in step C, the chromatographic conditions of the preparative liquid chromatography are as follows: YMC-Triart C18 chromatographic columns, a chromatographic column size of 250*20 mm, a particle size of 5 m, and a flow rate of 10 ml/min.

3. The preparation method for the azulene compound according to claim 2, wherein the ethanol solution in step (1) is an ethanol aqueous solution with a volume concentration of 5-95%, and the extraction method refers to cold soaking, percolation, microwave extraction, ultrasonic extraction, reflux extraction or continuous reflux extraction.

4. The preparation method for the azulene compound according to either claim 2 or claim 3, wherein the number of extractions in step (1) is 1-3, and the mass ratio of the amount of the ethanol solution added to the lilac stem is (8-30):1; and the number of dichloromethane and ethyl acetate extractions in step (2) is 4-6, and the amount of dichloromethane and ethyl acetate each time is %-1/4 of the volume of the extracted liquid.

5. Use of the azulene compound according to claim 1 or obtained according to any one of claims 2 to 4 in the preparation of anti-inflammatory drugs.

6. The use of the azulene compound in the preparation of anti-inflammatory drugs according to claim 5, wherein the azulene compound is directly or indirectly added to various pharmaceutically acceptable carriers or excipients to prepare an oral preparation or an injectable preparation.

7. The use of the azulene compound in the preparation of anti-inflammatory drugs according to claim 6, wherein the oral preparation is in the form of tablets, capsules, granules, lipid emulsions, microcapsules or dripping pills, and the injectable preparation is in the form of a liquid injection or a powder injection.

8. A method of treating inflammation, which comprises administering an effective amount of the azulene compound according to claim 1 or obtained according to any one of claims 2 to 4 to a subject in need.