A flavonoid C-glycoside compound, its extraction method and application

Abstract

The present invention belongs to the technical field of natural product extraction, and provides a flavone C-glycoside compound, an extraction method thereof and an application. The structural formula of the novel flavone C-glycoside compound is:. This compound has good inhibitory activity against EV71 virus, has no toxic effect on cells, has the potential to develop anti-EV71 virus drugs, and can be used as a raw material and a parent structure for new drug research and development.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting flavone C-glycosides from Trollius chinensis Bunge and its medical use. Background Art

[0002] Disclosing the information of this background art is intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Trollius chinensis Bunge, also known as "Tropaeolum majus", "Tropaeolum minus", "Golden Hibiscus", "Golden Meicao", "Golden Knot", etc., is the dried flower of the perennial herb Trollius chinensis Bunge of the genus Trollius in the Ranunculaceae family. It is mainly distributed in the temperate and cold temperate mountainous areas of the Northern Hemisphere, and in China, it is mainly distributed in Shanxi, Hebei, Inner Mongolia and other regions. "Compendium of Materia Medica Supplements" records that it is bitter in taste, cold in nature, non-toxic, and is mainly used for treating oral ulcers, throat swelling, floating heat in the teeth, earache, eye pain, and has the effects of improving eyesight and relieving mountain miasma. Clinically, it is widely used to treat respiratory infections, tonsillitis, otitis media, acute conjunctivitis, lymphangitis, sore throat, carbuncle and sore, oral ulcers and other diseases. Trollius chinensis Pharmacological studies have shown that Trollius chinensis Bunge has various biological activities such as antibacterial, antiviral, antitumor, antioxidant, anti-inflammatory, and analgesic effects. Trollius chinensis Bunge contains various types of chemical components such as flavonoids, flavonoid glycosides, fatty acids, alkaloids, sterols, and phenyl ethylenes. The rich chemical components provide a material basis for its diverse biological activities. Therefore, in-depth research on the chemical components in Trollius chinensis Bunge can not only obtain more natural products with novel structures and drug lead compounds with good biological activities, but also provide theoretical support for the rational development and utilization of Trollius chinensis Bunge resources.

[0004] Summary of the Invention

[0005] In order to further develop the medical use of Trollius chinensis Bunge, the present invention provides a flavone C-glycoside compound derived from Trollius chinensis Bunge.

[0006] Another object of the present invention is to provide a method for extracting the above-mentioned flavone C-glycoside compound from Trollius chinensis Bunge.

[0007] Another object of the present invention is to provide the use of the above-mentioned flavone C-glycoside compound in the preparation of anti-EV71 virus drugs.

[0008] To achieve the above object, the present invention adopts the following technical solutions.

[0009] A flavone C-glycoside compound, named trollchivone A, with the molecular formula CH 30 O 12 ​, the chemical structural formula is as follows:

[0010] .

[0011] The preparation method of the above compound includes the following steps:

[0012] (1) The extract obtained by heating and refluxing the Trollius chinensis Bunge medicinal material in ethanol, and the solvent is removed to obtain a crude extract;

[0013] (2) After the crude extract is suspended in water, it is successively extracted with petroleum ether and ethyl acetate. The solvent of the ethyl acetate extract is removed to obtain an ethyl acetate extract;

[0014] (3) The ethyl acetate extract is separated by silica gel column chromatography, and gradient elution is carried out with dichloromethane-methanol as the eluent. The volume ratios of dichloromethane-methanol are 100:0, 98:2, 96:4, 94:6, 92:8, 90:10 respectively, and the elution volumes are 7 times, 6 times, 7 times, 7 times, 7 times, 7 times the column volume respectively, corresponding to obtaining 6 elution fractions F1-F6. The fractions eluted with the 90:10 eluent are collected and combined to obtain the elution fraction F6;

[0015] (4) The elution fraction F6 is separated by a medium-pressure liquid chromatograph, and the chromatographic column is an ODS C 18 column. Gradient elution is carried out with methanol-water as the mobile phase for 1 h. The ratio of methanol-water gradient elution is 30:70-90:10, v / v, and the flow rate of the mobile phase is 40 mL / min; 7 elution fractions F6S1-F6S7 are obtained, and the elution fraction F6S4 corresponding to the fourth chromatographic peak is collected;

[0016] (5) The elution fraction F6S4 is separated by a semi-preparative high-performance liquid chromatograph, and the chromatographic column is an ODS C 18 column. Isocratic elution is carried out with methanol-water as the mobile phase, and the ratio of methanol-water is 47:53, v / v. The flow rate of the mobile phase is 2.5 mL / min; The eluent of the component with an elution time of 35 min is collected, and the solvent is removed to obtain the target compound trollchivone A.

[0017] In step (1), the concentration of ethanol is 80% (v / v); the volume-mass ratio of ethanol to Trollius chinensis Bunge is 15:1 (L / kg).

[0018] In step (2), the volume-mass ratio of petroleum ether or ethyl acetate to the crude extract is 1.25:1 (L / kg).

[0019] In step (3), the fineness of the silica gel filler in the silica gel column is 200-300 mesh.

[0020] The above-mentioned compound has the activity of inhibiting EV71 virus and has no toxic effect on cells, and can be used for preparing drugs against EV71 virus.

[0021] The present invention has the following advantages:

[0022] (1) The present invention provides a flavone C-glycoside compound, trollchivone A, which is extracted and isolated from Trollius chinensis Bunge for the first time, and the separation method and pharmacological activity research have not been reported.

[0023] (2) The flavone C-glycoside compound, trollchivone A, provided by the present invention can inhibit EV71 virus and has no toxic effect on cells, and has the potential to develop drugs against EV71 virus, and can be used as the raw material and parent structure for new drug research and development.

[0024] (3) The extraction and separation method of the flavone C-glycoside compound, trollchivone A, provided by the present invention is simple, rapid, low-cost, and the extracted compound has high purity. Description of the Drawings

[0025] Figure 1 is the medium-pressure liquid chromatography diagram of step (4) of Example 1;

[0026] Figure 2 is the high-resolution mass spectrum (HRESIMS) of trollchivone A;

[0027] Figure 3 is the nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) of trollchivone A;

[0028] Figure 4 is the nuclear magnetic resonance carbon spectrum ( 13 13C-NMR) of trollchivone A;

[0029] Figure 5 is the heteronuclear single quantum correlation spectrum (HSQC) of trollchivone A;

[0030] Figure 6 is the heteronuclear multiple bond correlation spectrum (HMBC) of trollchivone A;

[0031] Figure 7 is the result diagram of the cytotoxicity experiment of trollchivone A on RD cells;

[0032] Figure 8 is the result diagram of the inhibitory effect of trollchivone A on EV71 virus. Detailed Embodiments

[0033] The present invention will be further described below in conjunction with embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments.

[0034] Example 1 Separation and Identification of Compound Trollchivone A

[0035] 1. Separation of the Target Compound

[0036] (1) Weigh 20 kg of Trollius chinensis Bunge medicinal materials, add ethanol with a concentration of 80%, and extract three times by heating under reflux. Among them, the added volume (L) of 80% ethanol is 15 times the mass (kg) of the Trollius chinensis Bunge medicinal materials, and the reflux extraction time each time is 2 h; collect the extraction solutions obtained from the three heating reflux extractions, filter and combine the filtrates; concentrate the filtrates under reduced pressure until the alcohol smell disappears, cool to room temperature to obtain a crude extract for standby;

[0037] (2) After suspending the crude extract in water, extract it successively with petroleum ether and ethyl acetate, extract 4 times and 2 times respectively at room temperature, and the volume ratio of petroleum ether and ethyl acetate to the mass of the crude extract is 1.25:1 (L / kg) each time; combine the extraction solutions and recover them under reduced pressure to obtain petroleum ether extract and ethyl acetate extract respectively;

[0038] (3) Separate the ethyl acetate extract by silica gel column chromatography. The silica gel used is 200 - 300 mesh, and gradient elution is carried out successively with dichloromethane - methanol (100:0 v / v, 7 column volumes, 98:2 v / v, 6 column volumes, 96:4 v / v, 7 column volumes, 94:6 v / v, 7 column volumes, 92:8 v / v, 7 column volumes, 90:10 v / v, 7 column volumes) to obtain 6 fractions F1 - F6, and collect the fraction F6 eluted with the 90:10 eluent;

[0039] (4) Load the fraction F6 onto a medium - pressure liquid chromatograph and perform chromatographic separation using an ODS C 18 column (80 mm × 40 mm, 30 μm), and carry out gradient elution with methanol - water (30:70 - 90:10, v / v, 40 mL / min) for 1 h. According to the chromatogram ( Figure 1 ) 7 elution fractions F6S1 - F6S7 can be obtained, and collect the fraction F6S4 corresponding to peak 4;

[0040] (5) Load the fraction F6S4 onto a semi - preparative high - performance liquid chromatograph and perform chromatographic separation using an ODS C 18 column (250 mm × 10 mm, 5μm), and carry out isocratic elution with methanol - water (47:53, v / v, 2.5 mL / min). Collect the corresponding fraction with an elution time of 35 min, and recover it to dryness under reduced pressure at 40℃ to obtain the target compound, which is a yellow powder.

[0041] 2. Identification of the target compound

[0042] The target compounds separated in the above process were respectively subjected to high-resolution mass spectrometry, 1 1H-NMR, 13 13C-NMR, HSQC, and HMBC tests, and the following results were obtained respectively Figures 2 - 6 .

[0043] The HRESIMS m / z of the above target compound was 559.17963 [M + H] + (calculated value 559.18100), indicating that its molecular formula was C 28 H 30 O 12 , and the degree of unsaturation was 14, as shown in Figure 2 .

[0044] Its 1 1H-NMR and 13 13C-NMR nuclear magnetic data were as follows: 1 1H-NMR (800 MHz, DMSO- d d6) δ : 6.69 (1H, s, H-3), 6.04 (1H, s, H-6), 8.00 (2H, d, J J = 8.8 Hz, H-2′, 6′), 6.94 (2H, d, J J = 8.0 Hz, H-3′, 5′), 4.96 (1H, d, J J = 10.4 Hz, H-1′′), 5.43 (1H, t, J J = 9.6 Hz, H-2′′), 3.53 (1H, overlapped, H-3′′), 3.52 (1H, overlapped, H-4′′), 3.56 (1H, m, H-5′′), 4.38 (1H, d, J J = 12.8 Hz, H-6′′a), 4.09 (1H, dd, J J = 12.8, 6.4 Hz, H-6′′b), 2.06 (1H, m, H-2′′′), 1.28 (1H, m, H-3′′′a), 1.15 (1H, m, H-3′′′b), 0.62 (3H, t, J J = 7.2 Hz, H3-4′′′), 0.73 (3H, d, J J = 7.2 Hz, H3-5′′′), 1.91 (3H, s, H3-2′′′′), 13.09 (1H, br s, 5-OH).

[0045] 13C-NMR (200 MHz, DMSO- d 6) δ : 163.0 (C-2), 102.2 (C-3), 181.2 (C-4), 160.8 (C-5), 98.8 (C-6), 160.9 (C-7), 102.3 (C-8), 156.8 (C-9), 103.2 (C-10), 121.9 (C-1′), 128.4 (C-2′), 115.9 (C-3′), 161.1 (C-4′), 115.9 (C-5′), 128.4 (C-6′), 71.3 (C-1′′), 71.4 (C-2′′), 75.6 (C-3′′), 70.7 (C-4′′), 78.3 (C-5′′), 63.9 (C-6′′), 174.7 (C-1′′′), 40.4 (C-2′′′), 25.9 (C-3′′′), 11.3 (C-4′′′), 16.6 (C-5′′′), 170.5 (C-1′′′′), 20.7 (C-2′′′′).

[0046] 1 H-NMR is as Figure 3 , showing one AA′XX′ system δ H 8.00 (2H, d, J J = 8.8 Hz), 6.94 (2H, d, J J = 8.0 Hz)], one vinylic hydrogen signal δ H 6.69 (1H, s), one aromatic proton signal δ H 6.04 (1H, s), one hydroxyl proton signal δ H 13.09 (1H, br s) and one anomeric hydrogen signal of sugar δ H 4.96 (1H, d, J J = 10.4 Hz).

[0047] 13 C-NMR is as Figure 4 , combined with the HSQC spectrum ( Figure 5 ) showing 28 carbon signals, including 15 flavonoid nucleus carbon signals, 6 carbon signals of glucose ( δ C 71.3, 71.4, 75.6, 70.7, 78.3, 63.9), 3 methyl groups, one sp 3 -hybridized methylene, one methine, 2 ester carbonyl groups ( δ C174.7, 170.5). The above data indicate that compound trollchivone A is a flavonoid glycoside compound.

[0048] The HMBC spectrum ( Figure 6 ) shows that 5-OH has correlations with C-5 ( δ C 160.8) and C-6 ( δ C 98.8), suggesting that there is one hydroxyl group at the C-5 position of the flavone nucleus. According to the chemical shifts of C-6 and C-7 ( δ C 160.9), it is suggested that there is one hydroxyl group at the C-7 position. According to the correlations of H-2′, H-3′, H-5′, H-6′ with C-4′ ( δ C 161.1), it is proved that there is one hydroxyl group at the C-4′ position. The above spectral data indicate that the aglycone of compound trollchivone A is 5,7,4′-trihydroxyflavone. According to the anomeric hydrogen H-1′′ of the sugar having correlations with C-8 ( δ C 102.3), C-9 ( δ C 156.8), it is indicated that the sugar moiety is attached to the C-8 position of the mother nucleus. According to the coupling constant of the anomeric hydrogen of the sugar and the chemical shifts of the anomeric hydrogen and anomeric carbon, it is inferred that the glycosidic bond is a β configuration carbon glycoside. According to the correlations of CH3-4′′′ with C-2′′′ ( δ C 40.4), C-3′′′ ( δ C 25.9), and the correlations of H-2′′′, H2-3′′′, CH3-5′′′ and H-2′′ with C-1′′′ ( δ C 174.7), it is indicated that one 2-methylbutyryl group forms an ester bond with the hydroxyl group at the C-2′′ position of the sugar. According to the correlations of CH3-2′′′′, H2-6′′ with C-1′′′′ ( δ C 170.5), it is indicated that one acetyl group forms an ester bond with the hydroxyl group at the C-6′′ position of the sugar.

[0049] In summary, the molecular formula of the finally isolated target compound is determined to be C 28 H 30 O 12 , and the chemical structural formula is: , named trollchivone A.

[0050] Example 2 Anti-EV71 virus activity of trollchivone A

[0051] (1)Cytotoxicity experiment of trollchivone A on RD cells

[0052] RD cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C under 5% CO2. Trollchivone A was prepared into 5 concentration gradients (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) by 2-fold dilution with cell maintenance medium, and then inoculated into a 96-well plate with a monolayer of RD cells, 100 μL per well. Three replicate wells were set for each concentration. At the same time, a normal cell control group was established and cultured in an incubator at 37 °C and 5% CO2. The cytopathic effect was observed daily for 72 h. The cell viability was determined by the MTT method. The OD value was measured at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the half-toxic concentration TC was calculated. 50 。

[0053] The results of the cytotoxicity experiment of trollchivone A on RD cells are as Figure 7 shown. The TC 50 value of trollchivone A is greater than 100 μg / mL, so 100 μg / mL was used as the maximum concentration for subsequent experiments.

[0054] (2)Inhibitory effect of trollchivone A on EV71 virus

[0055] After the RD cells grew into a monolayer on a 96-well plate, the supernatant culture medium was discarded, and after washing 3 times with PBS, 100 μL of EV71 virus solution with 100-fold TCID 50 was inoculated into each well. After adsorption at 37 °C for 1 h, the virus solution was discarded, and after washing 2 times with PBS, different concentration gradients of trollchivone A were inoculated into the virus-infected RD cells at 100 μL per well. Three replicate wells were set for each concentration, and a normal control group and a virus control group were also set. The cells were continued to be cultured in an incubator at 37 °C and 5% CO2. The cell morphology was observed for 72 h. The cell viability was determined by the MTT method, the absorbance was measured at 490 nm, and the virus inhibition rate was calculated.

[0056] The results of the inhibitory effect of trollchivone A on EV71 virus are as Figure 8 shown. When the concentration of trollchivone A was 100 μg / mL, the virus inhibition rate was 50.2%, indicating that it had good inhibitory activity against EV71 virus.

[0057] The above are only embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A flavone C-glycoside compound, the chemical structural formula of which is: 。 2. A method for preparing a flavonoid C-glycoside compound as described in claim 1, characterized in that, Comprising the following steps: (1) Heating and refluxing the Trollius chinensis Bunge medicinal material in ethanol, and removing the solvent to obtain a crude extract; (2) Suspending the crude extract in water, and successively extracting with petroleum ether and ethyl acetate. Removing the solvent from the ethyl acetate extract to obtain an ethyl acetate extract; (3) Separating the ethyl acetate extract by silica gel column chromatography, and performing gradient elution with dichloromethane-methanol as the eluent. The volume ratios of dichloromethane-methanol are 100:0, 98:2, 96:4, 94:6, 92:8, 90:10 respectively, and the elution volumes are 7 times, 6 times, 7 times, 7 times, 7 times, 7 times the column volume respectively, corresponding to obtaining 6 elution fractions F1-F6. Collecting and combining the fractions eluted with the 90:10 eluent to obtain the elution fraction F6; (4) Separate the elution fraction F6 using a medium-pressure liquid chromatograph. The chromatographic column is an ODS C 18 column. Perform gradient elution for 1 h with methanol-water as the mobile phase. The ratio of methanol-water for gradient elution is 30:70 - 90:10, v / v, and the flow rate of the mobile phase is 40 mL / min. Obtain 7 elution fractions F6S1 - F6S7, and collect the elution fraction F6S4 corresponding to the fourth chromatographic peak; (5) The elution fraction F6S4 was separated by semi-preparative high performance liquid chromatography. The chromatographic column was an ODS C 18 column. Isocratic elution was carried out with methanol-water as the mobile phase. The ratio of methanol to water was 47:53, v / v, and the flow rate of the mobile phase was 2.5 mL / min. The eluent with an elution peak time of 35 min was collected, and the solvent was removed to obtain the target compound.

3. The preparation method according to claim 2, characterized in that, In step (1), the concentration of ethanol is 80% (v / v); the volume-mass ratio of ethanol to Trollius chinensis Bunge is 15:1 (L / kg); In step (2), the volume-mass ratio of petroleum ether or ethyl acetate to the crude extract is 1.25:1 (L / kg); In step (3), the fineness of the silica gel filler in the silica gel column is 200-300 mesh.

4. Use of a flavone C-glycoside compound as described in claim 1 in the preparation of an anti-EV71 virus drug.

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