Flavonoid derivatives in jiangxiang and preparation method and application thereof

Abstract

The application belongs to the technical field of natural medicine chemistry, and discloses flavone derivatives in Jinyunhuang, and a preparation method and application thereof.The flavone derivatives are separated from Jinyunhuang of the plant Dalbergia hupeana, and the heartwood of Jinyunhuang is extracted and concentrated to obtain extractum, the extractum is separated by silica gel column chromatography, gel column chromatography, and semi-preparative high performance liquid chromatography, and the flavone derivatives can be obtained.The application establishes an inflammation model by inducing RAW 264.7 cells with lipopolysaccharide (LPS), and confirms that the compound has good anti-inflammatory activity and can be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural medicinal chemistry technology, and particularly relates to flavonoid derivatives in Dalbergia odorifera and 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 flavonoid derivatives.

[0003] Flavonoids are a general term for a series of compounds consisting of two benzene rings connected by three carbon atoms. By introducing different substituents at various positions, a series of derivatives with practical value are formed. Modern pharmacological studies have shown that these compounds have good anti-inflammatory, antioxidant, estrogen-like, and cardiovascular protective effects.

[0004] Existing anti-inflammatory drugs generally suffer from insufficient targeting and significant side effects, making it difficult to meet the long-term treatment needs of patients with chronic inflammatory diseases. Flavonoids, compared to commonly used nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and biologics, avoid many of the adverse reactions of traditional anti-inflammatory drugs, and are of great significance for disease treatment and human development. Summary of the Invention

[0005] In view of the shortcomings and deficiencies of the prior art, the present invention provides a flavonoid derivative 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 flavonoid derivative.

[0006] The technical solution of the present invention is as follows:

[0007] Two flavonoid derivatives from Dalbergia odorifera, namely compound I and compound II, have the following chemical structural formulas:

[0008] .

[0009] Compound I comprises two isomers, compound 1R and compound 1S, and compound II comprises two isomers, compound 2R and compound 2S, with the following chemical structural formulas:

[0010] .

[0011] The compound I is named as (±)-7-hydroxy-8-methoxy-2-methyl-5-phenyl-2,5-methanobenzo[d][1,3]dioxepin-4(5H)-one ((±)-Odoriferol B), and the compound II is named as (±)-3-(2',4'-dimethoxyphenyl)-3-hydroxy-7-methoxycoumaran-4-one ((±)-Odoriferol D).

[0012] The application further provides a preparation method of the flavone derivative or an enantiomer thereof, comprising the following steps:

[0013] (1) taking dry heartwood of Daphne odora, extracting with an ethanol solution, collecting the extract, filtering and concentrating to obtain a crude extract;

[0014] (2) taking the extract obtained in step (1) to perform silica gel column chromatography, sequentially eluting with 5 column volumes of petroleum ether, dichloromethane, ethyl acetate and methanol to obtain petroleum ether parts, dichloromethane parts, ethyl acetate parts and methanol parts respectively;

[0015] (3) taking the dichloromethane parts obtained in step (2) to perform silica gel column chromatography, sequentially eluting with petroleum ether-ethyl acetate with a volume ratio of 100:1, 95:5, 90:10 and 0:1 as a mobile phase to obtain 19 fractions A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, C1, C2, C3, D1, D2, D3 and E;

[0016] (4) taking the fraction B6 obtained in step (3) to perform silica gel column chromatography after dissolving with a solvent, gradient eluting with petroleum ether-ethyl acetate with a volume ratio of 80:1, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1 and 0:1 as a mobile phase to obtain 15 fractions B6A1, B6A2, B6A3, B6A4, B6A5, B6B1, B6B2, B6B3, B6B4, B6B5, B6C1, B6C2, B6C3, B6C4 and B6C5;

[0017] (5) taking the fraction B6B3 obtained in step (4) to perform silica gel column chromatography, gradient eluting with petroleum ether-dichloromethane with a volume ratio of 19:1, 15:1, 12:1, 10:1, 5:1, 1:1 and 0:1 as a mobile phase to obtain 11 fractions B6B3G1, B6B3G2, B6B3G3, B6B3G4, B6B3G5, B6B3G6, B6B3G7, B6B3G8, B6B3G9 and B6B3G11;

[0018] (6) The fraction B6B3G2 obtained in step (5) is purified by semi-preparative high performance liquid chromatography (HPLC) with isocratic elution of acetonitrile-water (volume ratio 25:75), the chromatographic peak with retention time of 29 min is collected, the solvent is recovered, concentrated, and dried to obtain the purified compound I;

[0019] (7) The compound I obtained in step (6) is subjected to chiral resolution by chiral semi-preparative HPLC with isocratic elution of acetonitrile-water (volume ratio 50:50), the chromatographic peaks with retention times of 18 min and 19.5 min are collected, the solvent is recovered, concentrated, and dried to obtain the chiral resolution of compound 1R and compound 1S;

[0020] (8) The fraction B5 obtained in step (3) is subjected to silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate (volume ratio 80:1, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1, and 0:1, respectively) to obtain 13 fractions B5A1, B5A2, B5A3, B5A4, B5A5, B5B1, B5B2, B5B3, B5B4, B5B5, B5C1, B5C2, and B5C3;

[0021] (9) The fraction B5B1 obtained in step (8) is subjected to gel column chromatography with elution of dichloromethane-methanol (volume ratio 1:1) to obtain 6 fractions B5B1N1, B5B1N2, B5B1N3, B5B1N4, B5B1N5, and B5B1N6;

[0022] (10) The fraction B5B1N4 obtained in step (9) is purified by semi-preparative high performance liquid chromatography (HPLC) with isocratic elution of acetonitrile-water (volume ratio 50:50), the chromatographic peak with retention time of 34 min is collected, the solvent is recovered, concentrated, and dried to obtain the purified compound II;

[0023] (11) The compound II obtained in step (10) is subjected to chiral resolution by chiral semi-preparative HPLC with isocratic elution of acetonitrile-water (volume ratio 60:40), the chromatographic peaks with retention times of 11.5 min and 20.5 min are collected, the solvent is recovered, concentrated, and dried to obtain the chiral resolution of compound 2R and compound 2S.

[0024] Preferably, the extraction method in step (1) is one of cold soaking, percolation, microwave extraction, ultrasonic extraction, and reflux extraction. The extraction is performed 1-5 times, the extraction time is 1-6 h, and the extraction temperature is 20-120℃. More preferably, the extraction is performed 3 times by reflux extraction, the extraction time is 2 h each time, and the extraction temperature is 105℃.

[0025] Preferably, the ethanol solution used in step (1) is an aqueous ethanol solution with a volume concentration of 5-95%, more preferably an aqueous ethanol solution with a volume concentration of 75%.

[0026] Preferably, in steps (2), (3), (4), (5), and (8), the mesh size of the silica gel used in the silica gel column is 20-40 mesh, 100-200 mesh, 200-300 mesh, 300-400 mesh, and 200-300 mesh, respectively.

[0027] Preferably, in step (9), 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 μm, and flow rate 3.0 ml / min.

[0028] Preferably, in steps (6), (7), (10), and (11), the conditions of semi-preparative high performance liquid chromatography (HPLC) and chiral semi-preparative high performance liquid chromatography (HPLC) are as follows: size 10 mm x 250 mm, particle size 5 μm.

[0029] The application also provides the use of the flavonoid derivative or its enantiomer in the preparation of an anti-inflammatory drug.

[0030] Preferably, the anti-inflammatory drug is an NO inhibitor, IC 50 of 10-20 μM.

[0031] Preferably, the flavonoid derivative has good anti-inflammatory activity in an inflammation model established by lipopolysaccharide (LPS) induction of RAW 264.7 cells, IC 50 of compound 1R is 12.18 μM, IC 50 of compound 1S is 10.25 μM, IC 50 of compound 2R is 15.86 μM, and IC 50 of compound 2S is 19.90 μM.

[0032] The application provides a pharmaceutical composition comprising the flavonoid derivative or its enantiomer.

[0033] The application provides a pharmaceutical preparation comprising a therapeutically effective amount of the flavonoid derivative or its enantiomer, and a pharmaceutically acceptable carrier or excipient.

[0034] The flavone derivatives or the enantiomers thereof can be directly or indirectly added by those skilled in the art into various common pharmaceutically acceptable adjuvants required in preparation of different dosage forms, such as fillers, disintegrants, lubricants, binders, etc., to prepare common oral preparations or injection preparations by conventional pharmaceutical preparation methods.

[0035] Preferably, the oral preparations are tablets, capsules, granules, fat emulsions, microcapsules or drop pills.

[0036] Preferably, the injection preparations are injection solutions or powder injections.

[0037] The present application has the following beneficial effects:

[0038] The compound of the present application is two flavone derivatives and enantiomers thereof isolated from Jinyiang, the chemical plane structure of which is determined by modern spectroscopic techniques (one-dimensional and two-dimensional nuclear magnetic resonance, high-resolution mass spectrometry, infrared spectroscopy, etc.), and the absolute configuration of the compound is determined by circular dichroism, optical instrument and quantum chemical calculation, and the preparation method of the compound is provided. The two compounds are confirmed by cell level experiments to have good anti-inflammatory activity, and can be used for preparing anti-inflammatory drugs, and provide another direction for developing anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the HR-ESI-MS spectrum of the flavone derivative (compound I) prepared in the present application;

[0040] Figure 2 is the UV spectrum of the flavone derivative (compound I) prepared in the present application;

[0041] Figure 3 is the IR spectrum of the flavone derivative (compound I) prepared in the present application;

[0042] Figure 4 is the 1 H-NMR spectrum of the flavone derivative (compound I) prepared in the present application;

[0043] Figure 5 is the 13 C-NMR spectrum of the flavone derivative (compound I) prepared in the present application;

[0044] Figure 6 is the HSQC spectrum of the flavone derivative (compound I) prepared in the present application;

[0045] Figure 7 is the HMBC spectrum of the flavone derivative (compound I) prepared in the present application;

[0046] Figure 8is a chiral resolution spectrum of the flavonoid derivative (compound I) prepared in the present application;

[0047] Figure 9 is a spectrum after fitting the measured CD and the calculated CD of the flavonoid derivative (compound I and its enantiomer) prepared in the present application;

[0048] Figure 10 is an HR-ESI-MS spectrum of the flavonoid derivative (compound II) prepared in the present application;

[0049] Figure 11 is a UV spectrum of the flavonoid derivative (compound II) prepared in the present application;

[0050] Figure 12 is an IR spectrum of the flavonoid derivative (compound II) prepared in the present application;

[0051] Figure 13 is a H-NMR spectrum of the flavonoid derivative (compound II) prepared in the present application; 1

[0052] Figure 14 is a C-NMR spectrum of the flavonoid derivative (compound II) prepared in the present application; 13

[0053] Figure 15 is a HSQC spectrum of the flavonoid derivative (compound II) prepared in the present application;

[0054] Figure 16 is a HMBC spectrum of the flavonoid derivative (compound II) prepared in the present application;

[0055] Figure 17 is a chiral resolution spectrum of the flavonoid derivative (compound II) prepared in the present application;

[0056] Figure 18 is a spectrum after fitting the measured CD and the calculated CD of the flavonoid derivative (compound II and its enantiomer) prepared in the present application. DETAILED DESCRIPTION

[0057] 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 the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0058] 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.

[0059] ​​Preparation of compound I, compound II and its enantiomer of example 1

[0060] The present example provides a preparation method of flavonoid derivatives, comprising the following steps:

[0061] (1) Take the dry heartwood coarse powder of Dalbergia odorifera T. Chen (25.0 Kg), add 10 times the volume of 75% ethanol aqueous solution, heat (105°C) 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 infusion (7.2 Kg).

[0062] (2) Take the crude extract infusion (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 infusion in methanol, mix with 20-40 mesh silica gel, dry, and then dry column as sample layer, and then sequentially elute with 5 column volumes of petroleum ether, dichloromethane, ethyl acetate and methanol, respectively, 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).

[0063] (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 infusion in methanol, mix with 100-200 mesh silica gel, dry, and then dry column as sample layer, and then perform isocratic elution with petroleum ether-ethyl acetate (100:1, 95:5, 90:10, 0:1, v / v) as mobile phase, to obtain 19 fractions A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, C1, C2, C3, D1, D2, D3, E.

[0064] (4) Take fraction B6 (36.0 g) to perform silica gel column chromatography, use 200-300 mesh silica gel wet column as separation layer, dissolve fraction B6 infusion in methanol, mix with 200-300 mesh silica gel, dry, and then dry column as sample layer, and then perform gradient elution with petroleum ether-ethyl acetate (80:1, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1, 0:1, v / v) as mobile phase, to obtain 15 fractions B6A1, B6A2, B6A3, B6A4, B6A5, B6B1, B6B2, B6B3, B6B4, B6B5, B6C1, B6C2, B6C3, B6C4, B6C5.

[0065] (5) Take fraction B6B3 (2.4g) for silica gel column chromatography. Use 300-400 mesh silica gel dry loading as the separation layer. Dissolve fraction B6B3 extract in methanol and mix with 300-400 mesh silica gel. Dry the sample and then dry load it as the sample layer. Then use petroleum ether: dichloromethane (19:1, 15:1, 12:1, 10:1, 5:1, 1:1, 0:1, v / v) as the mobile phase for gradient elution to obtain 11 fractions B6B3G1, B6B3G2, B6B3G3, B6B3G4, B6B3G5, B6B3G6, B6B3G7, B6B3G8, B6B3G9, B6B3G11.

[0066] (6) The fraction B6B3G2 (132.0 mg) was purified by semi-preparative HPLC. It was eluted with acetonitrile-water (25:75, v / v), and the chromatographic peak with a retention time of 29 min was collected. The solvent was recovered, concentrated, and dried to obtain compound I (10 mg).

[0067] (7) Take compound I (10 mg) and perform chiral resolution by chiral semi-preparative HPLC. Elute with acetonitrile-water (50:50, v / v) to obtain the enantiomers of compound I, namely compound 1R (4.6 mg) and compound 1S (4.4 mg).

[0068] (8) Take fraction B5 (93.3g) for silica gel column chromatography. Use 200-300 mesh silica gel wet loading as the separation layer. Dissolve fraction B5 extract in methanol and mix with 200-300 mesh silica gel. Dry the sample and then dry load it as the sample layer. Then use petroleum ether-ethyl acetate (80:1, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1, 0:1, v / v) as the mobile phase for gradient elution to obtain 13 fractions B5A1, B5A2, B5A3, B5A4, B5A5, B5B1, B5B2, B5B3, B5B4, B5B5, B5C1, B5C2, B5C3.

[0069] (9) Take fraction B5B1 (2.64 g) for Sephadex LH-20 column chromatography. The extract of fraction B5B1 was dissolved in methanol and wet-loaded to form the sample layer. Then, isocratic elution was performed using dichloromethane-methanol with a volume ratio of 1:1 as the mobile phase to obtain 6 fractions B5B1N1, B5B1N2, B5B1N3, B5B1N4, B5B1N5, and B5B1N6.

[0070] The chromatographic conditions for the Sephadex LH-20 column were: column size 2.5cm × 2.5cm × 2.0m, particle size 18-111μm, and flow rate 3.0ml / min.

[0071] (10) The fraction B5B1N4 (33.9 mg) was purified by semi-preparative HPLC. It was eluted with acetonitrile-water (50:50, v / v), and the chromatographic peak with a retention time of 34 min was collected. The solvent was recovered, concentrated, and dried to obtain compound II (5.1 mg).

[0072] (11) Compound II (5.1 mg) was chirally separated by chiral semi-preparative HPLC and eluted with acetonitrile-water (50:50, v / v) to obtain the enantiomers of compound II, namely compound 2R (2.4 mg) and compound 2S (2.4 mg).

[0073] In the above steps, the specifications of the semi-preparative HPLC column and the chiral semi-preparative HPLC column are: 5 μm particle size and column size 10 mm × 250 mm.

[0074] I. Structural Identification of Compounds

[0075] 1. Structural identification of compound I and its enantiomers

[0076] The structural spectrum of compound I of this invention is shown in the figure. Figures 1-9 Therefore, we can conclude that:

[0077] (1) Compound I is a yellow amorphous powder that is soluble in chloroform. Figure 1 The HR-ESI-MS spectrum of this compound is shown, with the quasi-molecular ion peak at m / z: 335.0892 [M+Na]. + The calculated value is 335.0895, thus determining the molecular formula of the compound to be C. 18 H 16 O5 has an unsaturation degree of 11; Figure 2 The UV spectrum of the compound is shown, with a maximum absorption wavelength of 298.1 nm; Figure 3 The IR spectrum of this compound is shown, with characteristic peaks at 3429 cm⁻¹. -1 2925cm -1 1784cm -1 1023 cm -1 .

[0078] (2) 1 H-NMR spectrum (see) Figure 4 This displays a set of proton hydrogen signals for monosubstituted benzene rings. δ H 7.74 (2H, brs), δ H 7.45 (2H, t, J = 8.1 Hz), δ H7.42 ~ 7.36 (1H, m)], a set of aromatic proton hydrogen signals of 1,4-para-substituted aromatics [ δ H 6.50 (1H, s), δ H 6.34 (1H, s)], a methoxy hydrogen signal [ δ H 3.84 (3H, s)], a hydroxyl hydrogen signal [ δ H 5.22 (1H, s)], a set of methylene hydrogen signals [ δ H 2.66 (1H, d, J = 11.5 Hz), δ H 2.61 (1H, d, J = 11.5 Hz)], a set of methyl signals [ δ H 1.89 (3H, s)]; 13 C-NMR spectrum (see) Figure 5 ) and HSQC spectrum (see Figure 6 The data shows a total of 18 carbons, including one carbonyl carbon signal. δ C 172.6), 4 oxygen-carbon signals ( δ C 103.7, δ C 140.7, δ C 147.8, δ C 145.0), 3 seasonal carbon signals ( δ C 50.3, δ C 116.7, δ C 134.8), 7 methylene carbon signals ( δ C 128.8, δ C 128.8, δ C 128.2, δ C 128.2, δ C 128.2, δ C 112.2, δ C 100.3), 1 methylene carbon signal ( δC 42.0), 1 methyl carbon signal ( δ C 23.4), 1 methoxy carbon signal ( δ C 56.3), it is preliminarily inferred that compound I is a compound with one hydroxyl group and one methoxy group substituted in the C-6 and C-7 rings of the flavonoid A ring, and the parent nucleus is a flavonoid.

[0079] (3) HMBC spectrum (see) Figure 7 The key signal indicates that... δ H 1.89 (1H, s, 2-CH3) and δ C 103.7 (C-2) δ C 42.0 (C-2a) δ C 145.0 (C-10) exhibits hydrocarbon long-range correlation. δ H 2.61 (1H, d, H-2a) and δ C 103.7 (C-2) δ C 172.6 (C-4) δ C 50.3 (C-5) δ C 116.7 (C-11) δ C The presence of a long-range hydrogen-carbon correlation signal at 134.8 (C-1′) suggests that the compound is a flavonoid, in which the lactone ring on the flavonoid is replaced by a dioxane seven-membered ring, and C-2 and C-5 on the seven-membered ring are connected by a carbon bridge (C-2a), with C-5 connected to a benzene ring. δ H 6.34 (1H, s, H-6) and δ C 50.3 (C-5) δ C 140.7 (C-7) δ C 145.0 (C-10) δ C 116.7 (C-11) exhibits a hydrocarbon long-range correlation. δ H 5.22 (1H, s, 7-OH) and δ C 140.7 (C-7) δ C 147.8 (C-8) exhibits a hydrocarbon long-range correlation.δ H 6.50 (1H, s, H-9) and δ C 147.8 (C-8) δ C The presence of a long-range hydrogen-carbon correlation signal at 145.0 (C-10) indicates that the methoxy group is located at the C-8 position of the benzene ring and the hydroxyl group is located at the C-7 position of the benzene ring.

[0080] Based on the above analysis, the planar structure of compound I collected in Example 1 is determined as follows, and its chemical name is: 7-hydroxy-8-methoxy-2-methyl-5-phenyl-2,5-methylbridged benzo[d][1,3]dioxane-4(5H)-one.

[0081] .

[0082] The specific data of the proton and carbon spectra of the compounds are shown in Table 1 below:

[0083] Table 1. Compounds 1 H-NMR (600 MHz) and 13 C-NMR (150MHz) data (CDCl3, δ (ppm)

[0084]

[0085] (4) Compound I has a specific rotation value less than 0, suggesting the possible existence of a pair of enantiomers. It was separated using a chiral semi-preparative HPLC column, and the separation chromatogram is shown below. Figure 8 As shown, compounds 1R[α]25 D:+8, ECD(MeOH): λ (Δε)= 201(+5.69), 249 (-3.25), 300 (-0.95) and 1S[α]25 D:-3, ECD(MeOH): λ (Δε)= 201(-7.37), 249 (+4.90), 300 (+1.4) were obtained.

[0086] (5) The electronic circular dichroism (ECD) chromatogram was calculated using Gaussian09 software. Finally, the calculated ECD values ​​in the Boltzmann fitted chromatogram were compared with the measured ECD values ​​in the experimental chromatogram. The results are shown in […]. Figure 9 The absolute configuration of compound 1R was determined to be R, and the absolute configuration of compound 1S was determined to be S. HR-ESI-MS, UV, IR, and other spectra of compounds 1R and 1S were analyzed. 1 H-NMR, 13 C-NMR, HSQC and HMBC spectra and compound I ( Figures 1-7 The basic consistency is as follows.

[0087] Based on the above analysis, the structures of the enantiomers (compound 1R and compound 1S) of compound I collected in Example 1 were determined to be (±)-7-hydroxy-8-methoxy-2-methyl-5-phenyl-2,5-methylbridged benzo[d][1,3]dioxane-4(5H)-one. Their chemical structural formulas are as follows:

[0088] .

[0089] 2. Structural identification of compound II and its enantiomers

[0090] The structural spectrum of compound II of the present invention is shown in the figure. Figures 10-18 Therefore, we can conclude that:

[0091] (1) Compound II is a yellow amorphous powder that is soluble in acetone. Figure 10 The HR-ESI-MS spectrum of this compound is shown, with the quasi-molecular ion peak at m / z: 313.1072 [M-H₂O+H]. + The calculated value is 313.1076, thus determining the molecular formula of the compound to be C. 18 H 18 O6, with an unsaturation degree of 10; Figure 11 The UV spectrum of the compound is shown, with maximum absorption wavelengths of 275.5 nm and 311.2 nm. Figure 12 The IR spectrum of this compound is shown, with characteristic peaks at 3307 cm⁻¹. -1 1599 cm -1 1235 cm -1 1027 cm -1 .

[0092] (2) 1 H-NMR spectrum (see) Figure 13 The display shows the hydrogen proton signals of the two sets of benzene ring ABX coupling systems. δ H 7.83 (1H, d, J = 8.8 Hz), δ H 6.67 (1H, dd, J = 8.8, 2.4 Hz), δ H 6.50 (1H, d, J = 2.4 Hz); δ H 7.55 (1H,d, J = 8.4 Hz), δ H 6.59 (1H, dd, J = 8.5, 2.4 Hz),δ H 6.50 (1H, d, J = 2.4 Hz)], three methoxy hydrogen signals [ δ H 3.88 (3H, s) δ H 3.80 (3H, s) δ H 3.65 (3H, s)], a hydroxyl hydrogen signal [ δ H 5.28 (3H, s)], a set of hydrogen signals of the hydroxymethylene group [ δ H 4.79 (1H, d, J = 11.8 Hz), δ H 4.18 (1H, d, J = 11.9 Hz)]; 13 C-NMR spectrum (see) Figure 14 ) and HSQC spectrum (see Figure 15 The data shows a total of 18 carbons, including one carbonyl signal. δ C 189.6), 4 oxygen-carbon signals ( δ C 166.5, δ C 163.8, δ C 162.0, δ C 158.3), 2 seasonal carbon signals ( δ C 121.4, δ C 114.7), 6 methylene carbon signals ( δ C 130.1, δ C 129.3, δ C 101.4, δ C 110.7, δ C 105.7, δ C 100.1), 1 methylene carbon signal ( δ C 75.2), 1 oxygen-tertiary carbon signal ( δ C 75.0), 3 methoxy carbon signals ( δ C 56.1,δ C 56.0, δ C 55.6).

[0093] (3) HMBC spectrum (see) Figure 16 The key signal indicates that... δ H 4.18 (1H, d, H-2) and δ C 121.4 (C-1′) δ C 75.0 (C-3) δ C 189.6 (C-4) δ C A long-range hydrogen-carbon correlation signal exists at 163.8 (C-9). δ H 7.83 (1H, d, H-5) and δ C 189.6 (C-4) δ C 166.5 (C-7) δ C A long-range hydrogen-carbon correlation signal exists at 163.8 (C-9). δ H 7.55 (1H, d, H-6′) and δ C 75.0 (C-3) δ C 158.3 (C-2′) δ C A long-range hydrogen-carbon correlation signal exists at 162.1 (C-4′). δ H 5.28 (1H, s, 3-OH) shows a long-range hydrogen-carbon correlation signal with C-3, indicating that the compound is a dihydroisoflavone compound with a hydroxyl group at C-3, a methoxy group on ring A at C-7 via a methoxy group, and two methoxy groups on ring B at C-2′ and C-4′.

[0094] Based on the above analysis, the planar structure of compound II collected in Example 1 was determined as follows, and its chemical name is: 3-(2′,4′-dimethoxyphenyl)-3-hydroxy-7-methoxychroman-4-one.

[0095] .

[0096] The specific data for the proton and carbon spectra of compound II are shown in Table 2 below:

[0097] Table 2 Compound II 1H-NMR (600 MHz) and 13 C-NMR (150MHz) data (CD3COCD3, δ (ppm)

[0098]

[0099] (4) Compound II has a specific rotation value of less than 0, suggesting the possible existence of a pair of enantiomers. It was separated using a chiral semi-preparative HPLC column, and the separation chromatogram is shown below. Figure 17 As shown, compounds 2R [α]25 D:+25, ECD(MeOH):λ (Δε)=212 (+9.5), 237 (-17.55), 310 (-5.34), 336 (+5.00) and 2S [α]25 D:-6, ECD(MeOH):λ (Δε)= 212 (-13.89), 237 (+24.62), 310 (+7.90), 336 (-6.37) were obtained.

[0100] (5) The electronic circular dichroism (ECD) chromatogram was calculated using Gaussian09 software. Finally, the calculated ECD values ​​in the Boltzmann fitted chromatogram were compared with the measured ECD values ​​in the experimental chromatogram. The results are shown in […]. Figure 18 The absolute configuration of compound 2R was determined to be R, and the absolute configuration of compound 2S was determined to be S. HR-ESI-MS, UV, and IR spectra of compounds 2R and 2S were analyzed. 1 H-NMR, 13 C-NMR, HSQC and HMBC spectra and compound II ( Figures 11-17 The basic consistency is as follows.

[0101] Based on the above analysis, the structures of the enantiomers (compound 2R and compound 2S) of compound II collected in Example 1 were determined to be (±)-3-(2′,4′-dimethoxyphenyl)-3-hydroxy-7-methoxychroman-4-one. Their chemical structural formulas are as follows:

[0102] .

[0103] II. Experimental Study on the Efficacy of Compounds

[0104] 1. Toxicity test of the compound of the present invention against mouse mononuclear macrophage leukemia cells (RAW264.7)

[0105] The toxicity of the compound prepared in Example 1 to mouse mononuclear macrophage leukemia cells (RAW264.7) was detected using the CCK-8 assay. RAW264.7 cells were divided into three groups: a blank group (no cell seeding, only DMEM medium), a normal group (cell seeding without drug administration, i.e., cell culture medium), and a drug administration group (cell seeding with drug administration, i.e., cell culture medium containing the compound). RAW264.7 cells were cultured in DMEM complete medium (high glucose medium containing 10% fetal bovine serum, penicillin (100 U / ml), and streptomycin (100 μg / ml)) in a humidified incubator at 37°C with 5% carbon dioxide. When the cells reached over 80% confluence, they were gently scraped off with a cell scraper, and counted using trypan blue and a cell counting chamber. The cells were then counted at 3.0 × 10⁻⁶ cells / mL. 4 The cells were seeded at a density of 100 µL / well in 96-well plates and incubated for 24 h. The drug-treated groups were treated with compounds at final concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, with six replicates for each concentration, and incubated for 24 h. Then, 10 μL of CCK-8 solution was added to each well, and after incubation for another 2 hours, the OD value of each well was measured at 450 nm using a SPECTRAMAX 190 microplate reader. The values ​​from three replicates were used, and the cell viability % was calculated as: (OD value of drug-treated group - OD value of control group) / (OD value of normal group - OD value of control group) × 100%. The results are shown in Table 3 below.

[0106] Table 3. Toxicity of different concentrations of compounds on RAW264.7 cells

[0107]

[0108] The results in Table 3 show that compounds 1R, 1S, and 2R did not exhibit significant toxicity at or below 50 μM, and compound 2S did not exhibit significant toxicity at or below 25 μM.

[0109] 2. Detection of the protective activity of the compounds of this invention against LPS (lipopolysaccharide)-induced inflammatory damage in RAW264.7 cells.

[0110] The protective activity of the compounds prepared in Example 1 against LPS-induced inflammatory damage in RAW264.7 cells was detected using a nitric oxide (NO) assay kit. RAW264.7 cells were cultured in high-glucose medium (DMEM) containing 10% fetal bovine serum, penicillin (100 U / ml), and streptomycin (100 μg / ml) in a humidified incubator at 37°C with 5% carbon dioxide. When the cells reached confluence of more than 80%, they were gently scraped off with a cell scraper, and counted using trypan blue and a cell counting chamber. The cells were then analyzed at a concentration of 1.0 × 10⁻⁶ cells / mL.5 The cells were seeded at a density of 100 µL / well in 96-well plates and incubated for 24 h before further treatment.

[0111] RAW264.7 cells were randomly divided into three groups after incubation: (1) Normal group: cells were cultured normally in DMEM medium; (2) Model group: cells were cultured in DMEM medium containing 1 μg / mL LPS; (3) Drug treatment group: cells were cultured in DMEM medium containing 1 μg / mL LPS, and different concentrations of compounds (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, with 6 replicates for each concentration) were added and cultured for 24 hours in a humidified incubator containing 5% carbon dioxide at 37°C. After culture, each well of each group was tested with a NO kit, and another plate was used to detect different concentrations of NaNO2 and a standard curve was plotted. The NO concentration (μM) of each well was measured at 540 nm using a SPECTRA MAX190 microplate reader, and the relative NO content (μM) was calculated as: NO concentration in the treatment group or model group (μM) - NO concentration in the normal group (μM). The protective activity of different concentrations of the compound against LPS-induced inflammatory damage in RAW264.7 cells was analyzed, and dose-response curves were fitted to calculate the IC50 of the compound. 50 The values ​​were then analyzed statistically to draw conclusions, and the results are shown in Table 4.

[0112] Table 4. Protective effects of different concentrations of compounds on LPS-induced inflammatory damage in RAW264.7 cells.

[0113]

[0114] The results showed that the IC50 of compound 1R was... 50 The value was 12.18 μM, and the IC50 of compound 1S was... 50 The value was 10.25 μM, and the IC50 of compound 2R was [value missing]. 50 The value was 15.86 μM, and the IC50 of compound 2S was... 50 The value was 19.90 μM, indicating a statistically significant difference. All four compounds at a concentration of 25 µM showed significant protective effects against LPS-induced inflammatory damage in RAW264.7 cells, with compound 1S exhibiting the best effect.

[0115] Finally, it should be noted that the above description is merely a preferred 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Flavonoid derivatives from Dalbergia odorifera, characterized in that: The chemical structural formula of the flavonoid derivative is as follows: 。 2. A method for preparing flavonoid derivatives according to claim 1, characterized in that: 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 B6 obtained in step (3) was purified by silica gel column chromatography. The mobile phase was eluted with petroleum ether:ethyl acetate in volume ratios of 80:1, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1, and 0:1 to obtain 15 fractions: B6A1, B6A2, B6A3, B6A4, B6A5, B6B1, B6B2, B6B3, B6B4, B6B5, B6C1, B6C2, B6C3, B6C4, and B6C5. (5) The fraction B6B3 obtained in step (4) was purified by silica gel column chromatography. The mobile phase was petroleum ether-dichloromethane with volume ratios of 19:1, 15:1, 12:1, 10:1, 5:1, 1:1, and 0:1 for gradient elution to obtain fractions B6B3G1, B6B3G2, B6B3G3, B6B3G4, B6B3G5, B6B3G6, B6B3G7, B6B3G8, B6B3G9, and B6B3G11. (6) The fraction B6B3G2 obtained in step (5) was purified by semi-preparative high performance liquid chromatography, and isocratic eluted with acetonitrile-water at a volume ratio of 25:75 for a retention time of 29 min to obtain compound I; (7) Take the compound I obtained in step (6) and perform chiral separation by chiral semi-preparative high performance liquid chromatography. Elute with acetonitrile-water at a volume ratio of 50:

50. The retention times are 18 min and 19.5 min, respectively, to obtain the chiral separated compounds 1R and 1S. (8) The fraction B5 obtained in step (3) was purified by silica gel column chromatography. The mobile phase was eluted with petroleum ether-ethyl acetate in volume ratios of 80:1, 50:1, 25:1, 15:1, 10:1, 5:1, 1:1, and 0:1 to obtain 13 fractions: B5A1, B5A2, B5A3, B5A4, B5A5, B5B1, B5B2, B5B3, B5B4, B5B5, B5C1, B5C2, and B5C3. (9) Take the fraction B5B1 obtained in step (8) and purify it by gel column chromatography. Elute it with dichloromethane-methanol at a volume ratio of 1:1 to obtain 6 fractions B5B1N1, B5B1N2, B5B1N3, B5B1N4, B5B1N5, and B5B1N6. (10) The fraction B5B1N4 obtained in step (9) was purified by semi-preparative high performance liquid chromatography and eluted isocratically with acetonitrile-water at a volume ratio of 50:50 for a retention time of 34 min to obtain compound II; (11) Compound II obtained in step (10) was chirally separated by chiral semi-preparative high performance liquid chromatography. It was eluted isocratically with acetonitrile-water at a volume ratio of 60:40, with retention times of 11.5 min and 20.5 min, respectively, to obtain chiral separated compounds 2R and 2S. The chemical structural formulas of compounds 2R and 2S are as follows: 。 3. The method for preparing flavonoid derivatives according to claim 2, characterized in that: In steps (2), (3), (4), (5), and (8), the silicone used in the silicone column is, in order, 20-40 mesh, 100-200 mesh, 200-300 mesh, 300-400 mesh, and 200-300 mesh.

4. The method for preparing flavonoid derivatives according to claim 2, characterized in that: In step (9), the chromatographic conditions for gel column chromatography are: Sephadex LH-20 column, column size 2.5cm×2.5cm×2.0m, particle size 18-111μm, flow rate 3.0ml / min.

5. The method for preparing flavonoid derivatives according to claim 2, characterized in that: In steps (6), (7), (10), and (11), the column specifications for semi-preparative high-performance liquid chromatography and chiral semi-preparative high-performance liquid chromatography are: 10 mm × 250 mm in size and 5 μm in particle size.

6. The use of a flavonoid derivative from Dalbergia odorifera according to claim 1 in the preparation of an anti-inflammatory drug.

7. The application according to claim 6, characterized in that: The anti-inflammatory drug is a NO inhibitor, IC50. 50 The value is 10-20 μM.

8. A pharmaceutical composition, characterized in that: Includes the flavonoid derivatives as described in claim 1.

9. A pharmaceutical preparation, characterized in that: It includes a therapeutically effective amount of the flavonoid derivative of claim 1, and a pharmaceutically acceptable carrier or excipient.

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