Flavonoid compound in lomatogonium radiatum as well as preparation method and application of flavonoid compound

Abstract

The invention belongs to the technical field of natural pharmaceutical chemistry, and discloses a flavonoid compound in lomatogonium radiatum as well as a preparation method and application thereof.The flavonoid compound is separated from lomatogonium radiatum of a gentiaceae lomatogonium plant, the medicinal material of lomatogonium radiatum is extracted and concentrated, and the flavonoid compound is obtained. And sequentially carrying out macroporous adsorption resin separation, gel column chromatography separation, medium-low pressure ODS column chromatography separation and semi-preparative high performance liquid chromatography separation. Pharmacological studies show that the compound provided by the invention can effectively inhibit the levels of COX2, IL-6 and TNF-alpha in mouse macrophages RAW246.7 caused by lipopolysaccharide (LPS), which indicates that the compound has obvious anti-inflammatory activity and can be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, and in particular relates to a flavonoid compound from a radially ribbed flower, its preparation method and application. Background Technology

[0002] Radial ribbed flowers ( Lomatogonium rotatum *Gentiana* is a plant belonging to the genus *Gentiana* in the family Gentianaceae. It has a bitter taste and cold properties, and is believed to have calming, heat-clearing, stomach-strengthening, and wound-healing effects. It is one of the commonly used varieties of medicinal materials in the *Gentiana* class. Clinically, it is mainly used to treat jaundice, liver and gallbladder heat, etc. Modern pharmacological studies have shown that it has hepatoprotective, anti-inflammatory, antioxidant, and anti-tumor pharmacological effects, and mainly contains ketones, iridoids, and other types of compounds.

[0003] Currently, there are few literature reports on *Radiate Ribaceous Flower*, and even fewer studies on its medicinal substances. To further utilize natural medicinal resources, it is necessary to conduct in-depth research on the medicinal substances of *Radiate Ribaceous Flower*. Summary of the Invention

[0004] In view of the shortcomings and deficiencies of the prior art, this invention provides a flavonoid compound from *Pterocarya radiata*, its preparation method, and its application. This compound exhibits good anti-inflammatory activity, providing another direction for anti-inflammatory drugs. This invention also relates to a method for preparing this compound.

[0005] The technical solution of the present invention is as follows: A flavonoid compound from a radially ribbed flower has the following chemical structural formula: .

[0006] The present invention also provides a method for preparing the flavonoid compounds, comprising the following steps: (1) Take the radially ribbed flowers, extract them with ethanol solution, collect the extract, filter and concentrate it until the volume fraction of ethanol in the extract is 20% to obtain the concentrate; (2) The concentrated solution obtained in step (1) was loaded onto a macroporous adsorption resin for column chromatography separation. Gradient elution was performed with ethanol aqueous solutions with volume concentrations of 30%, 50%, 70%, and 95% to obtain four fractions: 1, 2, 3, and 4. (3) Take fraction 2 obtained in step (2), dissolve it in methanol, filter it, and load it onto a gel column for column chromatography separation. Elute it with methanol to obtain 26 fractions A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z; (4) Take fraction C obtained in step (3), dissolve it in methanol, filter it, and load it onto a medium-low pressure ODS column for column chromatography separation. Use methanol aqueous solution with volume concentrations of 40%, 50%, 60%, and 100% for gradient elution to obtain four fractions C1, C2, C3, and C4. (5) Take the fraction C1 obtained in step (4) and dissolve it in methanol. After filtration, perform semi-preparative high performance liquid chromatography separation, elute with acetonitrile-water at a volume ratio of 2:8, and retain for 35 min to obtain the flavonoids.

[0007] Preferably, in step (1), the extraction method is any one of cold soaking, percolation, microwave extraction, ultrasonic extraction, and reflux extraction. The number of extractions is 1-5 times, the extraction time is 1-6 hours, and the extraction temperature is 20-120 ℃. More preferably, the extraction method is reflux extraction 2-3 times, each extraction time is 2-3 hours, and the extraction temperature is 85-95 ℃.

[0008] 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 70-80%. The amount of ethanol solution added during extraction is 6-30 times the weight of the medicinal material, more preferably 10-20 times.

[0009] Preferably, in step (2), the macroporous adsorption resin is HP-20 type.

[0010] Preferably, in step (3), the gel column is of type LH-20.

[0011] Preferably, in step (4), the ODS packing used in the medium and low pressure ODS column has the following specifications: C18, particle size 25 μm.

[0012] Preferably, in step (5), the column specifications of the semi-preparative high performance liquid chromatography are: 20 mm × 250 mm, C18 column, and 10 μm particle size.

[0013] The present invention also provides the application of the flavonoids in the preparation of anti-inflammatory drugs.

[0014] The present invention also provides a pharmaceutical composition comprising the aforementioned flavonoid compounds.

[0015] The present invention also provides a pharmaceutical formulation comprising a therapeutically effective amount of the flavonoid compound and a pharmaceutically acceptable carrier or excipient.

[0016] 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.

[0017] Preferably, the oral preparation is a tablet, capsule, granule, fat emulsion, microcapsule, or droplet.

[0018] Preferably, the injectable preparation is an injection solution or a powder for injection.

[0019] The beneficial effects of this invention are as follows: The preparation method of the compounds of this invention is simple, and the flavonoid compounds can be isolated from *Pterocarya radiata*. Pharmacological studies have shown that the compounds of this invention can effectively inhibit the levels of COX2, IL-6, and TNF-α in lipopolysaccharide (LPS)-induced mouse macrophage RAW246.7 cells, indicating that the compounds have significant anti-inflammatory activity and can be used in the preparation of anti-inflammatory drugs. Attached Figure Description

[0020] Figure 1 This is the chemical structural formula of the flavonoids obtained in this invention; Figure 2 This is the mass spectrum of the flavonoids obtained in this invention; Figure 3 The flavonoids obtained in this invention are 1 H-NMR spectrum; Figure 4 The flavonoids obtained in this invention are 13 C-NMR spectrum; Figure 5 This is the DEPT spectrum of the flavonoids obtained in this invention; Figure 6 This is the HSQC spectrum of the flavonoids obtained in this invention; Figure 7 This is the HMBC spectrum of the flavonoids obtained in this invention; Figure 8 The flavonoids obtained in this invention are 1 H- 1 H-COSY spectrum; Figure 9 This invention describes the effects of flavonoids prepared in this invention on the expression levels of LPS-induced inflammatory factors TNF-α, IL-6, and COX2 in RAW264.7 cells. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0023] Example 1: Preparation of the compound This embodiment provides a method for preparing flavonoids from radially ribbed flowers, the steps of which are as follows: (1) Take radially ribbed flowers, add 10 times the volume of 70% ethanol aqueous solution, reflux extract twice at 85℃, extract for 2 hours each time, filter after extraction, combine the extracts, concentrate until the volume fraction of ethanol in the extract is 20%, and obtain concentrated solution.

[0024] (2) The concentrated solution obtained in step (1) was loaded onto HP-20 macroporous adsorption resin for column chromatography separation. Gradient elution was performed with ethanol aqueous solutions with volume concentrations of 30%, 50%, 70%, and 95% to obtain four fractions: 1, 2, 3, and 4.

[0025] (3) Take fraction 2 obtained in step (2), dissolve it in methanol, filter it, and then load it onto an LH-20 gel column for column chromatography. Elute it with methanol to obtain 26 fractions A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z.

[0026] (4) Take the fraction C obtained in step (3) and dissolve it in methanol. After filtration, load it onto a medium-low pressure ODS column (ODS packing: C18, particle size 25 μm) for column chromatography separation. Use methanol aqueous solution with volume concentrations of 40%, 50%, 60% and 100% for gradient elution to obtain 4 fractions C1, C2, C3 and C4.

[0027] (5) Take the fraction C1 obtained in step (4) and dissolve it in methanol. After filtration, separate it using a semi-preparative high performance liquid chromatograph (20 mm × 250 mm column, C18 column, 10 μm particle size). Elute with acetonitrile-water gradient at a volume ratio of 2:8 and retain for 35 min to obtain the target compound.

[0028] I. Structural analysis and identification of compounds Structural analysis of compounds: primarily utilizing spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). 1H-NMR, 13 Its structure was identified by C-NMR and 2D-NMR, and the specific spectra are shown below. Figures 2-8 As shown, its spectral data and analysis process are as follows: (1) The compound is a yellow powder. High-resolution mass spectrometry (HR-ESI-MS): 599.1611 [M+H] + (Theoretical value C) 26 H 30 O 16 (599.1607). The molecular formula of the compound is C. 26 H 30 O 16 The degree of unsaturation is 12. Comprehensive analysis 1 H-NMR, 13 C-NMR and HSQC spectra show that the compound contains one carbonyl carbon. δ C :181.8], 2 methoxy groups[ δ C The hydrogen proton signals corresponding to [61.6, 57.1] are respectively. δ H [3.83, 3.82], 2 glycan-terminal proton signals [ δ H The carbon signals corresponding to [4.90, 4.21] are respectively δ C [102.3, 104.6], three active hydrogen proton signals [ δ H [7.44, 6.98, 6.80]. Then, combining two-dimensional nuclear magnetic resonance spectroscopy data such as HMBC, DEPT, and HSQC spectra, the structure of the compound was determined as follows: Figure 1 As shown.

[0029] (2) The spectral data of the compound are as follows: 1 H-NMR (600 MHz, DMSO-d6) δ H 7.45 (d, 1H, J =9.1 Hz, H-6), 6.98 (d, 1H, J =9.1 Hz, H-7), 6.80(s, 1H, H-4), 4.91 (d, 1H, J =7.6 Hz, H-1'), 4.21 (d, 1H, J =7.6 Hz, H-1''), 4.00(dd, 1H, J=5.8,11.5 Hz, H-6'), 3.83 (s, 3H), 3.82 (s, 3H),3.67(m, 1H, H-5'),3.54(m, 1H, H-3'), 3.41(m, 1H, H-2'), 3.32 (m, 1H, H-3''),3.31 (m, 1H, H-4''), 3.29(m, 1H, H-4'), 3.03 (m, 1H, H-2''), 3.00 (t, 1H, J =5.3, 11.3 Hz, H-5'').

[0030] 13 C-NMR (150 MHz, DMSO-d6) δ C :181.8 (C=O),157.9 (C-3), 155.0(C-1),151.9(C-8), 150.6(C-4a), 148.6(C-4b), 143.0(C-5), 130.1(C-2), 120.8(C-6),108.7(C-8a), 105.7(C-7), 104.6(C-1''), 104.4(C-8b), 102.3(C-1'), 100.9(C-4),76.8(C-2''), 76.5(C-2'), 76.5(C-3'), 73.9(C-4'), 73.8(C-3''), 70.1(C-5'), 69.7(C-4''), 68.4(C-6'), 66.1(C-5''), 61.3(5-OCH3), 57.0(2-OCH3).

[0031] II. Drug Efficacy Experimental Study 1. Cytotoxicity detection of the compounds of this invention The toxicity of the flavonoids prepared in Example 1 to RAW264.7 cells was detected using the CCK8 assay. RAW264.7 cells in logarithmic growth phase were used, and the cell density was adjusted to 1×10⁻⁶ cells using culture medium containing 10% fetal bovine serum (FBS). 6Cells were seeded at a density of 100 μg / ml in 96-well plates. Cell culture media containing different concentrations of the compound (100 µM, 50 µM, 25 µM, 12.5 µM) were added to the 96-well plates. A blank group (containing culture media without cells) and a normal cell control group (containing cell culture media without the drug) were also set up. The cells were incubated at 37 °C and 5% CO2 for 24 h. The supernatant was discarded and CCK8 solution was added. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the cell viability was calculated. The results are shown in Table 1 below. The compound had no significant effect on cell viability at concentrations of 12.5 µM to 100 µM.

[0032] Cell viability = (A 给药组 -A 空白组 ) / (A 正常细胞对照组 -A 空白组 ).

[0033] Table 1. Effects of different concentrations of compounds on cell viability of RAW264.7 cells.

[0034] 2. Anti-inflammatory activity test of the compounds of this invention (1) Cell culture Mouse macrophages RAW264.7 were cultured in DMEM medium containing 1% penicillin-streptomycin mixture and 10% fetal bovine serum in an incubator at 37 ℃ and 5% CO2.

[0035] (2) Cell passage and counting RAW264.7 cells: When the cell density in the culture dish reaches 80-90% under a microscope, discard the old culture medium and add 2 mL of preheated phosphate-buffered saline (PBS) at 37 °C to prepare a cell suspension. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 3 min. Discard the supernatant, add 1 mL of complete culture medium, and resuspend the cells. Take 10 μL of the mixed cell suspension and horizontally transfer it into a cell counting chamber for microscopic counting.

[0036] (3) Cell modeling Inflammation model: RAW264.7 cells were induced with 1 μg / mL lipopolysaccharide (LPS) for 24 h to construct an in vitro inflammation model.

[0037] (4) Experimental treatment and qPCR detection of inflammatory factor levels RAW264.7 cells were seeded in 6-well plates with approximately 1 × 10⁶ cells per well. 6Cells were cultured in an incubator for 24 hours, and the supernatant was discarded. In the experimental setup, both the blank group and the model group were treated with 2 mL of complete culture medium, and the drug treatment group was treated with 100 µM of flavonoids (prepared in Example 1). After 2 hours of pre-drug administration, 2 µL of LPS was added to the drug treatment group and the model group to make the final concentration 1 µg / mL, and the modeling was carried out for 24 hours.

[0038] After each experimental treatment group was completed, total RNA was extracted according to the total RNA kit instructions, and the total RNA from each group was quantified using a microplate reader. A reverse transcription reaction system was prepared, and gDNA removal was performed according to the kit instructions. Subsequently, qPCR analysis was conducted, and the relative mRNA expression levels of inflammatory factors TNF-α, IL-6, and COX2 were measured (see Table 2). Figure 9 .

[0039] Table 2. Effects of compounds on the expression levels of LPS-induced inflammatory factors TNF-α, IL-6, and COX2 in RAW264.7 cells.

[0040] The results showed that after LPS induction, the expression of inflammatory factors TNF-α, IL-6 and COX2 in cells increased significantly. The intervention of the flavonoids in this invention could effectively restore the level of inflammatory factors, proving that the flavonoids have a good protective effect against LPS-induced inflammatory damage to RAW264.7 cells.

[0041] 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. A flavonoid compound from radially ribbed flowers, characterized in that: The chemical structural formulas of the flavonoids are as follows: 。 2. A method for preparing flavonoids according to claim 1, characterized in that: Includes the following steps: (1) Take the radially ribbed flowers, extract them with ethanol solution, collect the extract, filter and concentrate to obtain the concentrated solution; (2) The concentrated solution obtained in step (1) was loaded onto a macroporous adsorption resin for column chromatography separation. Gradient elution was performed with ethanol aqueous solutions with volume concentrations of 30%, 50%, 70%, and 95% to obtain four fractions: 1, 2, 3, and 4. (3) Take fraction 2 obtained in step (2), load it onto a gel column for column chromatography separation, and elute with methanol to obtain 26 fractions A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z; (4) Take fraction C obtained in step (3), load it onto a medium-low pressure ODS column for column chromatography separation, and perform gradient elution with methanol aqueous solution with volume concentrations of 40%, 50%, 60% and 100% to obtain 4 fractions C1, C2, C3 and C4. (5) Take the fraction C1 obtained in step (4) and perform semi-preparative high performance liquid chromatography separation. Elute with acetonitrile-water at a volume ratio of 2:8 and retain for 35 min to obtain the flavonoids.

3. The preparation method according to claim 2, characterized in that: In step (2), the macroporous adsorption resin is HP-20 type.

4. The preparation method according to claim 2, characterized in that: In step (3), the gel column is of type LH-20.

5. The preparation method according to claim 2, characterized in that: In step (4), the specifications of the ODS packing used in the medium and low pressure ODS column are: C18, particle size 25 μm.

6. The preparation method according to claim 2, characterized in that: In step (5), the column specifications for the semi-preparative high performance liquid chromatography are: 20 mm × 250 mm, C18 column, and 10 μm particle size.

7. The use of a flavonoid compound according to claim 1 in the preparation of an anti-inflammatory drug.

8. A pharmaceutical composition, characterized in that: Includes the flavonoids described in claim 1.

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

10. The pharmaceutical preparation according to claim 9, characterized in that: The pharmaceutical preparation is an oral or injectable preparation.

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