Benzofuran compounds in jinying and preparation method and application thereof

Abstract

The application belongs to the technical field of natural medicinal chemistry, and discloses a benzofuran compound in Jinyunming and a preparation method and application thereof. The benzofuran compound is separated from Jinyunming of the plant Dalbergia hupeana Tutch in the family of Leguminosae. The heartwood of Jinyunming is extracted and concentrated to obtain extract, the extract is sequentially separated by multiple silica gel column chromatography, medium-pressure ODS column chromatography and gel column chromatography, and then purified by semi-preparative high-performance liquid chromatography, so that the benzofuran compound can be obtained. The application establishes an inflammation model by inducing RAW 264.7 cells with lipopolysaccharide (LPS), and shows that the compound has good anti-inflammatory activity, the IC50 is 14.33 μM, and the expression of inflammatory factors interleukin-6 (IL-6) and interleukin-1β (IL-1β) can be inhibited.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, and particularly relates to benzofuran compounds in Dalbergia odorifera, their preparation methods and applications. Background Technology

[0002] Fragrance ( Dalbergia odorifera *Dalbergia odorifera* (T. Chen) is the dried heartwood of a plant in the genus *Dalbergia* of the legume family. It is pungent and warm in nature, and has the effects of promoting blood circulation, stopping bleeding, regulating qi, and relieving pain. It is often used as a traditional Chinese medicine to treat cardiovascular diseases such as coronary heart disease and angina pectoris. The active components of *Dalbergia odorifera* are mainly flavonoids and volatile oils, possessing pharmacological activities such as anti-myocardial ischemia, antioxidant, anti-inflammatory, and anti-tumor effects. Current research on the pharmacodynamic material basis of *Dalbergia odorifera* mainly focuses on volatile oils and flavonoids, with limited research on the chemical composition of other types of compounds.

[0003] Benzofurans and their derivatives are widely found in natural products. By introducing different substituents at various positions of benzofurans, a series of derivatives with practical value have been formed. Modern pharmacological studies have shown that these compounds have a variety of biological activities, such as anti-inflammatory, anti-tumor, antioxidant, antibacterial, and antiviral effects, which are of great significance for the treatment of diseases and human development. Summary of the Invention

[0004] In view of the shortcomings and deficiencies of the prior art, the present invention provides a benzofuran compound isolated from Dalbergia odorifera, which has been shown to have anti-inflammatory activity, providing another direction for anti-inflammatory drugs. The present invention also relates to a method for preparing the compound.

[0005] The technical solution of the present invention is as follows:

[0006] The benzofuran compounds in Dalbergia odorifera have the following chemical structural formulas:

[0007] .

[0008] This application names the compound 7-hydroxy-6-methoxy-2-(2´-hydroxy-3´,4´-dimethoxyphenyl)benzofuran (Odoriferol G).

[0009] The present invention also provides a method for preparing the benzofuran compounds, comprising the following steps:

[0010] (1) Take dried heartwood of Dalbergia odorifera, extract it with ethanol solution, collect the extract, filter and concentrate it to obtain crude extract;

[0011] (2) The extract obtained in step (1) was subjected to silica gel column chromatography, and eluted with petroleum ether, dichloromethane, ethyl acetate and methanol, respectively, for 5 column volumes, to obtain petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction and methanol fraction, respectively;

[0012] (3) The dichloromethane fraction obtained in step (2) was subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate as mobile phase (volume ratio was 100:1, 95:5, 90:10, 0:1, respectively) for isocratic elution, to obtain 19 fractions A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, C1, C2, C3, D1, D2, D3, E;

[0013] (4) The fraction B4 obtained in step (3) was subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate as mobile phase (volume ratio was 80:1, 50:1, 30:1, 15:1, 10:1, 5:1, 1:1, 0:1, respectively) for gradient elution, to obtain 9 fractions B4A1, B4A2, B4A3, B4A4, B4A5, B4B1, B4B2, B4B3, B4B4;

[0014] (5) The fraction B4B1 obtained in step (4) was subjected to medium pressure ODS column chromatography, and eluted with methanol-water as mobile phase (volume ratio was 1:9, 7:3, 5:5, 7:3, 1:0, respectively) for gradient elution, to obtain 6 fractions B4B1O1, B4B1O2, B4B1O3, B4B1O4, B4B1O5, B4B1O6;

[0015] (6) The fraction B4B1O1 obtained in step (5) was subjected to silica gel column chromatography, and eluted with petroleum ether-dichloromethane as mobile phase (volume ratio was 19:1, 9:1, 5:1, 1:1, 0:1, respectively) for gradient elution, to obtain 9 fractions B4B1O1G1, B4B1O1G2, B4B1O1G3, B4B1O1G4, B4B1O1G5, B4B1O1G6, B4B1O1G7, B4B1O1G8, B4B1O1G9;

[0016] (7) The fraction B4B1O1G2 obtained in step (6) was subjected to gel column chromatography, and eluted with dichloromethane-methanol (volume ratio was 1:1) as mobile phase, to obtain 7 fractions B4B1O1G2N1, B4B1O1G2N2, B4B1O1G2N3, B4B1O1G2N4, B4B1O1G2N5, B4B1O1G2N6, B4B1O1G2N7;

[0017] (8) The fraction B4B1O1G2N2 obtained in step (7) is purified by semi-preparative high performance liquid chromatography (HPLC) with isocratic elution of acetonitrile-water (volume ratio 57:43), the chromatographic peak with a retention time of 28 min is collected, the solvent is recovered, concentrated, and dried to obtain the compound.

[0018] Preferably, the extraction method in step (1) is one of cold soaking method, percolation method, microwave extraction method, ultrasonic extraction method, and reflux extraction method. The extraction is performed 1-5 times, the extraction time is 1-6 hours, and the extraction temperature is 20-120 DEG C. More preferably, the extraction is performed 3 times by reflux extraction, the extraction time is 2 hours each time, and the extraction temperature is 105 DEG C.

[0019] Preferably, the ethanol solution used in step (1) is an ethanol aqueous solution with a volume concentration of 5-95%, and more preferably, it is an ethanol aqueous solution with a volume concentration of 75%. The amount of the ethanol solution added during the extraction is 6-30 times the mass of the medicinal material, and more preferably, it is 10 times.

[0020] Preferably, in steps (2), (3), (4), and (6), the mesh number of the silica gel used in the silica gel column is 20-40, 100-200, 200-300, and 300-400, respectively.

[0021] Preferably, in step (5), the ODS filler used in the medium-pressure ODS column has a pore size of 120 angstrom and a particle size of 50 microns.

[0022] Preferably, in step (7), the chromatographic conditions of the gel column chromatography are as follows: Sephadex LH-20 chromatographic column, chromatographic size 2.5 cm x 2.5 cm x 2.0 m, particle size 18-111 microns, and flow rate 3.0 ml / min.

[0023] Preferably, in step (8), the chromatographic column used in the semi-preparative high performance liquid chromatography has a size of 10 mm x 250 mm and a particle size of 5 microns.

[0024] The application further provides a use of the benzofuran compound in the preparation of an anti-inflammatory drug.

[0025] Preferably, the anti-inflammatory drug is an NO inhibitor, IC 50 is 14.33 micromoles.

[0026] Preferably, the benzofuran compound has good anti-inflammatory activity in an inflammation model of RAW 264.7 cells induced by lipopolysaccharide (LPS) and can inhibit the expression of inflammatory factors IL-6 and IL-1 beta.

[0027] The present invention provides a pharmaceutical composition comprising the benzofuran compound.

[0028] The present invention provides a pharmaceutical formulation comprising a therapeutically effective amount of the benzofuran compound and a pharmaceutically acceptable carrier or excipient.

[0029] Those skilled in the art can directly or indirectly add the benzofuran compounds to various pharmaceutically acceptable excipients required for the preparation of different dosage forms, such as fillers, disintegrants, lubricants, and binders, to produce commonly used oral or injectable formulations using conventional pharmaceutical formulation methods.

[0030] Preferably, the oral preparation is a tablet, capsule, granule, fat emulsion, microcapsule, or droplet.

[0031] Preferably, the injectable preparation is an injection solution or a powder for injection.

[0032] The beneficial effects of this invention are as follows:

[0033] The compound of this invention is a benzofuran compound isolated from Dalbergia odorifera. Its chemical structure was determined using modern spectroscopic techniques (one-dimensional and two-dimensional NMR, high-resolution mass spectrometry, and infrared spectroscopy, etc.), and a method for its preparation is also provided. Cellular level experiments have demonstrated that this compound possesses good anti-inflammatory activity and can be used to prepare anti-inflammatory drugs, providing another direction for anti-inflammatory drug development. Attached Figure Description

[0034] Figure 1 This is the HR-ESI-MS spectrum of the benzofuran compounds obtained in this invention;

[0035] Figure 2 This is the UV spectrum of the benzofuran compounds obtained in this invention;

[0036] Figure 3 This is the IR spectrum of the benzofuran compounds obtained in this invention;

[0037] Figure 4 The benzofuran compounds obtained by this invention 1 H-NMR spectrum;

[0038] Figure 5 The benzofuran compounds obtained by this invention 13 C-NMR spectrum;

[0039] Figure 6 This is the HSQC spectrum of the benzofuran compounds obtained in this invention;

[0040] Figure 7 This is the HMBC spectrum of the benzofuran compounds obtained in this invention;

[0041] Figure 8 is a benzofuran compound prepared by the present application 1 H- 1 H-COSY spectrum. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0043] Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels.

[0044] Example 1 Preparation of compounds

[0045] The present embodiment provides a preparation method of a benzofuran compound, and the steps are as follows:

[0046] (1) Take the dry heartwood coarse powder of Dalbergia odorifera (25.0 Kg), add 10 times the volume of 75% ethanol aqueous solution, heat (105 ℃) and reflux extract 3 times, each for 2 hours, combine the extract, filter and concentrate under reduced pressure, recover ethanol, and obtain the crude extract extract (7.2 Kg).

[0047] (2) Take the crude extract extract (6.5 Kg) obtained in step (1) to perform silica gel column chromatography, use 20-40 mesh silica gel wet column as separation layer, dissolve the extract in methanol and mix with 20-40 mesh silica gel to dry, then dry column sample as sample layer, and then elute with petroleum ether, dichloromethane, ethyl acetate and methanol, each for 5 column volumes, to obtain petroleum ether part (52.3 g), dichloromethane part (877.2 g), ethyl acetate part (3.7 kg) and methanol part (679.0 g), respectively.

[0048] (3) Take the dichloromethane part (827 g) obtained in step (2) to perform silica gel column chromatography, use 100-200 mesh silica gel wet column as separation layer, dissolve the dichloromethane part extract in methanol and mix with 100-200 mesh silica gel to dry, then dry column sample as sample layer, and then use petroleum ether-ethyl acetate (100:1, 95:5, 90:10, 0:1, v / v) as mobile phase for isocratic elution, to obtain 19 fractions A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, C1, C2, C3, D1, D2, D3, E after elution.

[0049] (4) The fraction B4 (35.0 g) was subjected to silica gel column chromatography, 200-300 mesh silica gel was wet-packed to form a separation layer, the extract of the fraction B4 was dissolved in methanol and mixed with 200-300 mesh silica gel, and then the mixture was dried and packed as a sample layer, and then petroleum ether-methanol (80:1, 50:1, 30:1, 15:1, 10:1, 5:1, 1:1, 0:1, v / v) was used as a mobile phase for gradient elution, to obtain 9 fractions B4A1, B4A2, B4A3, B4A4, B4A5, B4B1, B4B2, B4B3, B4B4.

[0050] (5) The fraction B4B1 (5.3 g) was subjected to medium-pressure ODS column chromatography, ODS filler (pore size 120 Å, particle size 50 μm) was wet-packed to form a separation layer, the extract of the fraction B4B1 was dissolved in methanol, and then wet-packed as a sample layer, and then methanol-water (1:9, 7:3, 5:5, 7:3, 1:0, v / v) was used as a mobile phase for gradient elution, to obtain 6 fractions B4B1O1, B4B1O2, B4B1O3, B4B1O4, B4B1O5, B4B1O6.

[0051] (6) The fraction B4B1O1 (1.3 g) was subjected to silica gel column chromatography, 300-400 mesh silica gel was dry-packed to form a separation layer, the extract of the fraction B4B1O1 was dissolved in methanol and mixed with 200-300 mesh silica gel, and then dry-packed as a sample layer, and then petroleum ether-dichloromethane (19:1, 9:1, 5:1, 1:1, 0:1, v / v) was used as a mobile phase for gradient elution, to obtain 9 fractions B4B1O1G1, B4B1O1G2, B4B1O1G3, B4B1O1G4, B4B1O1G5, B4B1O1G6, B4B1O1G7, B4B1O1G8, B4B1O1G9.

[0052] (7) The fraction B4B1O1G2 (0.51 g) was subjected to Sephadex LH-20 column chromatography, eluted with dichloromethane-methanol (1:1, v / v), to obtain 7 fractions B4B1O1G2N1, B4B1O1G2N2, B4B1O1G2N3, B4B1O1G2N4, B4B1O1G2N5, B4B1O1G2N6, B4B1O1G2N7.

[0053] The chromatographic conditions of the Sephadex LH-20 column chromatography were as follows: column size 2.5 cm x 2.5 cm x 2.0 m, particle size 18-111 μm, and flow rate 3.0 ml / min.

[0054] (8) The fraction B4B1O1G2N2 (12.1 mg) was purified by semi-preparative HPLC. It was eluted with acetonitrile-water (57:43, v / v), and the chromatographic peak with a retention time of 28 min was collected. The solvent was recovered, concentrated, and dried to obtain the compound (4.6 mg).

[0055] The specifications of the semi-preparative HPLC column are: column size 10 mm × 250 mm, particle size 5 μm.

[0056] I. Structural Identification of Compounds

[0057] The structural spectrum of the compound of this invention is shown in the figure. Figures 1-8 Therefore, we can conclude that:

[0058] (1) The compound is a purple oil and is soluble in methanol. Figure 1 The HR-ESI-MS spectrum of this compound is shown, with the quasi-molecular ion peak at m / z: 317.1016 [M+H]. + The calculated value is 317.1019, thus determining the molecular formula of the compound to be C. 17 H 16 O6, with an unsaturation degree of 10; Figure 2 The UV spectrum of the compound is shown, with maximum absorption wavelengths of 218.7 nm and 316.0 nm. Figure 3 The IR spectrum of this compound is shown, with characteristic peaks at 3423 cm⁻¹. -1 2926 cm -1 1620 cm -1 1152cm -1 .

[0059] (2) 1 H-NMR spectrum (see) Figure 4 The signal shows a single peak of hydrogen with a double bond. δ H 7.20 (1H, s), two sets of ortho-coupled aromatic hydrogen signals δ H 7.61 (1H, d, 8.9Hz) and δ H 6.60 (1H, d, 8.9Hz) δ H 7.11 (1H, d, 8.3Hz) and δ H 6.87 (1H, d, 8.3Hz), 3 methoxyl signals δ H 4.30 (3H, s) δ H 3.97 (3H, s) δH 3.92 (3H, s); 13 C-NMR spectrum (see Figure 5 ) and HSQC spectrum (see Figure 6 ) showed 17 carbons in common, including 1 carbon of double bond with oxygen (C-2, δ C 151.5), 1 carbon of double bond (C-3, δ C 104.2), 5 carbons of double bond with oxygen (C-4, δ C 131.6, δ C 135.8, δ C 144.7, δ C 146.7, δ C 152.1), 1 carbon of quaternary carbon with oxygen (C-8, δ C 144.4) and 2 carbons of quaternary carbon (C-9, δ C 111.5, δ C 125.3), 4 carbons of methine aromatic (C-4, δ C 104.7, δ C 111.3, δ C 114.6, δ C 121.0), 3 carbons of methoxyl (C-5, δ C 56.1, δ C 60.8, δ C 61.2), it was initially inferred that the compound was a compound with 2 hydroxyl groups, 3 methoxyl groups, and a benzofuran nucleus.

[0060] (3) HMBC spectrum (see δ ) showed that, δ H 7.20 (1H, s, H-3) and δ C 151.5 (C-2), δ C 144.4 (C-8), δ C 125.3 (C-9) were carbon-hydrogen long-range correlations, δ H 7.11 (1H, d, 8.3 Hz, H-4) andδ C 131.6 (C-6), 144.7 (C-7), Figure 8 C 144.4 (C-8), δ C 125.3 (C-9) long-range carbon-hydrogen correlation present, δ H 6.87 (1H, d, 8.3 Hz, H-5) and δ C 131.6 (C-6), 144.7 (C-7), δ C 125.3 (C-9) long-range carbon-hydrogen correlation present, in addition δ H 4.30 (3H, s, -OCH3) and ​ C 131.6 (C-6) long-range carbon-hydrogen correlation present, indicating the methoxy group at C-6; ​ H 6.60 (1H, d, 8.9 Hz, H-5') and ​ C 111.5 (C-1'), 135.8 (C-3'), ​ C 152.1 (C-4') long-range carbon-hydrogen correlation present, ​ H 7.61 (1H, d, 8.9 Hz, H-6') and ​ C 151.5 (C-2), ​ C 146.7 (C-2'), ​ C 152.1 (C-4') long-range carbon-hydrogen correlation present, in addition ​ H 3.97 (3H, s, -OCH3) and ​ C 135.8 (C-3') long-range carbon-hydrogen correlation present, indicating the methoxy group at C-3', ​ H 3.92 (3H, s, -OCH3) and ​ C 152.1 (C-4') long-range carbon-hydrogen correlation present, indicating the methoxy group at C-4'; 1 H- 1 The H-COSY spectrum (see ​ ) shows, ​ H 7.11 (1H, d, 8.3 Hz, H-4) and ​H 6.87 (1H, d, 8.3 Hz, H-5) with remote hydrogen-hydrogen correlation signals, ​ H 6.60 (1H, d, 8.9 Hz, H-5') with ​ H 7.61 (1H, d, 8.9 Hz, H-6') with remote hydrogen-hydrogen correlation signals. In combination with the molecular formula, it can be determined that there are two hydroxyl groups respectively connected to C-7 and C-2' positions.

[0061] The hydrogen spectrum and carbon spectrum of the compound are as follows in Table 1:

[0062] Table 1 Hydrogen spectrum and carbon spectrum of the compound 1 H-NMR (600 MHz) and 13 C-NMR (150MHz) data (CDCl3- d 6, ​ (ppm)

[0063]

[0064] In combination with the above analysis, the structure of the compound collected in Example 1 is determined as 7-hydroxy-6-methoxy-2-(2'-hydroxy-3',4'-dimethoxyphenyl)benzofuran. The chemical structural formula is as follows:

[0065] .

[0066] II. Pharmacodynamic experimental research

[0067] 1. Toxicity detection of the compound of the present application on mouse monocyte macrophage leukemia cells (RAW264.7)

[0068] The CCK-8 method is used to detect the toxicity of the compound prepared in Example 1 on mouse monocyte macrophage leukemia cells (RAW264.7). RAW264.7 cells are divided into three groups, a blank group (without cells, only DMEM culture medium is added), a normal group (cells are seeded without drug, i.e. cell culture solution is added), and a drug group (cells are seeded with drug, i.e. cell culture solution containing the compound drug is added). RAW264.7 cells are cultured in high-sugar medium (DMEM) containing 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml), and at the same time, the cells are cultured in a 37 ℃, 5% carbon dioxide humidified incubator. When the cells grow to more than 80%, the cells are scraped off with a cell scraper, and then the cells are counted by using trypan blue and a cell counting plate, and then 3.0×10 4The density of 100 μL / well was inoculated into a 96-well plate, and incubated for 24 h. The drug administration group was added with the compound at a final concentration of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM, respectively, and 6 replicate wells were set for each concentration, and incubated for 24 h; then 10 μL of CCK-8 solution was added to each well, and after incubation for 2 h, the OD value of each well was measured at 450 nm using a SPECTRAMAX190 enzyme labeler, and the values of 3 replicate wells were taken. According to the calculation formula: cell inhibition rate % = [1- (drug administration group OD value-blank group OD value) / (normal group OD value-blank group OD value)] x 100%, the relative inhibition rate of the compound at different concentrations on RAW264.7 cells was calculated, and the results are shown in Table 2 below.

[0069] Table 2 Toxicity of compounds at different concentrations on RAW264.7 cells

[0070]

[0071] The results in Table 2 show that the compound does not exhibit obvious toxicity at 50 μM and below.

[0072] 2, Protection activity detection of the compound of the present application on LPS (lipopolysaccharide) induced RAW264.7 cell inflammatory injury

[0073] The nitric oxide (NO) kit was used to detect the protective activity of the compound prepared in Example 1 on LPS induced RAW264.7 cell inflammatory injury. RAW264.7 cells were cultured in high-sugar medium (DMEM) containing 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml), and the cells were cultured in a humidified incubator containing 5% carbon dioxide at 37 ℃. When the cells grew to more than 80%, the cells were scraped off with a cell scraper, and then counted using trypan blue and a cell counting plate, and then 1.0 x 10 5 The density of 100 μL / well was inoculated into a 96-well plate, and incubated for 24 h.

[0074] The incubated RAW264.7 cells are randomly divided into 3 groups: (1) normal group: the cells are cultured in DMEM medium; (2) model group: the cells are cultured in DMEM medium containing 1 μg / mL LPS; (3) drug administration group: the cells are cultured in DMEM medium containing 1 μg / mL LPS, and different concentrations of compound (3.125 μM, 6.25 µM, 12.5 µM, 25 µM, 6 replicates for each concentration) are added, and the cells are cultured in a 37 ℃, 5% carbon dioxide humidified incubator for 24 hours. After the culture is completed, the OD value of each well is measured at 540 nm by using a SPECTRA MAX190 enzyme labeler, and the NO inhibition rate % is calculated as follows: [1- (drug administration group OD value-normal group OD value) / (model group OD value-normal group OD value)]x100%. The protective activity of different concentrations of compound on LPS-induced inflammatory injury of RAW264.7 cells is analyzed, and then the dose-effect curve is fitted, the IC 50 value of the compound is calculated, and the conclusion is obtained through statistical analysis, and the results are shown in Table 3.

[0075] Table 3 Protective effect of different concentrations of compound on LPS-induced inflammatory injury of RAW264.7 cells

[0076]

[0077] The results show that the IC 50 value of the compound is 14.33 μM, and the data has a significant statistical difference. The compound shows obvious protective effect on LPS-induced inflammatory injury of RAW264.7 cells at a concentration of 25 µM.

[0078] 3, Effect of the compound of the present application on LPS-induced inflammatory factors of RAW264.7 cells

[0079] The effect of the compound prepared in Example 1 on LPS-induced inflammatory factors interleukin-6 (IL-6) and interleukin-1β (IL-1β) of RAW264.7 cells is detected by using real-time fluorescence quantitative PCR. RAW264.7 cells are cultured in high-sugar medium (DMEM) containing 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml), and the cells are cultured in a 37 ℃, 5% carbon dioxide humidified incubator. When the cells grow to more than 80%, the cells are scraped off with a cell scraper, and then the cells are counted by using trypan blue and a cell counting plate, and then the cells are inoculated on a 6-well plate at a density of 5.0×10 6 / well (100 µL / well), and the cells are incubated for 24 h and then treated.

[0080] The incubated RAW264.7 cells were randomly divided into 3 groups: (1) normal group: the cells were cultured in DMEM medium; (2) model group: the cells were cultured in DMEM medium containing 1 μg / mL LPS; (3) drug administration group: the cells were cultured in DMEM medium containing 1 μg / mL LPS, and 25 μM concentration of compound was added (3 replicate wells were set), and the culture was carried out in a 37 ℃, 5% carbon dioxide humidified incubator for 24 hours. After the culture was completed, 1 mL of total RNA extraction reagent (Trizol) was added to each well of each group to fully lyse the sample, and the sample was allowed to stand at room temperature for 5 minutes; 200 μL of chloroform was added, and the sample was mixed and allowed to stand at room temperature for 5 minutes, and then centrifuged at 12000 g / min for 15 minutes; about 100 μL of supernatant was taken and 400 μL of isopropanol was added, and the sample was mixed up and down until the liquid was clear, and then allowed to stand at room temperature for 10 minutes, and then centrifuged at 12000 g / min for 15 minutes; the supernatant was discarded, and the sample was washed with 75% ethanol, and then centrifuged at 12000 g / min for 15 minutes; the supernatant was discarded, and after the precipitate was dried, 20 μL of diethyl pyrocarbonate (DEPC) treated water was added for dissolution, and then the RNA concentration and purity were determined using a nucleic acid protein detector, and then the sample was stored in a -80 ℃ refrigerator. Reverse transcription, reverse transcription polymerase chain reaction and other experiments were performed according to the kit instructions. β-actin was used as an internal reference, and the 2-ΔΔCT method was used for calculation. The real-time fluorescence quantitative PCR primers were designed by Nanchang Yang'er Biotechnology Co., Ltd., and the mRNA relative expression contents of inflammatory factors IL-6 and IL-1β were measured, as shown in Table 4.

[0081] Table 4 Effect of the compound on the expression level of inflammatory factors IL-6 and IL-1β of LPS-induced RAW264.7 cells

[0082]

[0083] The results show that the compound can significantly inhibit the expression of inflammatory factors IL-6 and IL-1β at 25 μM, and the data has a significant statistical difference. Therefore, the compound can be used for preparing anti-inflammatory drugs, and provides another direction for developing anti-inflammatory drugs.

[0084] Finally, it should be noted that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing benzofuran compounds from sandalwood, characterized in that: The chemical structural formula of the compound is as follows: ； The method for preparing the compound includes the following steps: (1) Take dried heartwood of Dalbergia odorifera, extract it with ethanol solution, collect the extract, filter and concentrate it to obtain crude extract; (2) The extract obtained in step (1) was purified by silica gel column chromatography. It was eluted with petroleum ether, dichloromethane, ethyl acetate and methanol for 5 column volumes each to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction and methanol fraction respectively. (3) The dichloromethane fraction obtained in step (2) was purified by silica gel column chromatography. The mobile phase was eluted isocratically with petroleum ether-ethyl acetate in volume ratios of 100:1, 95:5, 90:10 and 0:1 to obtain 19 fractions: A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, C1, C2, C3, D1, D2, D3 and E. (4) The fraction B4 obtained in step (3) was purified by silica gel column chromatography. The mobile phase was petroleum ether-ethyl acetate with volume ratios of 80:1, 50:1, 30:1, 15:1, 10:1, 5:1, 1:1 and 0:1 for gradient elution to obtain 9 fractions B4A1, B4A2, B4A3, B4A4, B4A5, B4B1, B4B2, B4B3 and B4B4. (5) Take the fraction B4B1 obtained in step (4) and purify it by medium-pressure ODS column chromatography. Use methanol-water with volume ratios of 1:9, 7:3, 5:5, 7:3, and 1:0 as the mobile phase for gradient elution to obtain 6 fractions B4B1O1, B4B1O2, B4B1O3, B4B1O4, B4B1O5, and B4B1O6. (6) The fraction B4B1O1 obtained in step (5) was purified by silica gel column chromatography. The mobile phase was petroleum ether-dichloromethane with volume ratios of 19:1, 9:1, 5:1, 1:1 and 0:1 for gradient elution to obtain 9 fractions B4B1O1G1, B4B1O1G2, B4B1O1G3, B4B1O1G4, B4B1O1G5, B4B1O1G6, B4B1O1G7, B4B1O1G8 and B4B1O1G9. (7) Take the fraction B4B1O1G2 obtained in step (6) and purify it by gel column chromatography. Use dichloromethane-methanol with a volume ratio of 1:1 as the mobile phase to elute and obtain 7 fractions B4B1O1G2N1, B4B1O1G2N2, B4B1O1G2N3, B4B1O1G2N4, B4B1O1G2N5, B4B1O1G2N6, and B4B1O1G2N7. (8) The fraction B4B1O1G2N2 obtained in step (7) was purified by semi-preparative high performance liquid chromatography, eluted isocratically with acetonitrile-water at a volume ratio of 57:43, and retained for 28 min to obtain the compound.

2. The method for preparing benzofuran compounds in Dalbergia odorifera according to claim 1, characterized in that: In steps (2), (3), (4), and (6), the silicone used in the silicone column is, in order, 20-40 mesh, 100-200 mesh, 200-300 mesh, and 300-400 mesh.

3. The method for preparing benzofuran compounds in Dalbergia odorifera according to claim 1, characterized in that: In step (5), the specifications of the ODS packing used in the medium-pressure ODS column are: pore size 120 Å, particle size 50 μm.

4. The method for preparing benzofuran compounds in Dalbergia odorifera according to claim 1, characterized in that: In step (7), the chromatographic conditions for gel column chromatography are: Sephadex LH-20 column, column size 2.5 cm × 2.5 cm × 2.0 m, particle size 18-111 μm, and flow rate 3.0 ml / min.

5. The method for preparing benzofuran compounds in Dalbergia odorifera according to claim 1, characterized in that: In step (8), the column specifications for the semi-preparative high performance liquid chromatography are: 10 mm × 250 mm in size and 5 μm in particle size.

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