Flavonoid C-glycoside compound as well as extraction method and application thereof

Abstract

The invention belongs to the technical field of natural product extraction, and provides a flavone C-glycoside compound as well as an extraction method and application thereof. The structural formula of the novel flavone C-glycoside compound is # imgabs0 #. The compound has good inhibitory activity on EV71 virus, has no toxic effect on cells, has the potential of developing anti-EV71 virus drugs, and can be used as a raw material and a parent structure for research and development of new drugs.

Description

Technical Field

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

[0002] Disclosing the information of this background art aims 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 Galls", etc., is the dried flower of Trollius chinensis Bunge, a perennial herb of the genus Trollius in the family Ranunculaceae. It is mainly distributed in the temperate and cold temperate mountainous regions 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 and gingival bleeding, earache, eye pain, with the effects of improving eyesight and relieving mountain miasma. Clinically, it is widely used for treating respiratory tract infections, tonsillitis, otitis media, acute conjunctivitis, lymphangitis, sore throat, carbuncle and sore toxin, 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. 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 pharmaceutical 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 objects, the present invention adopts the following technical solutions.

[0009] A flavone C-glycoside compound, named trollchivone A, with the molecular formula C 28 H 30 O 12, the chemical structural formula is as follows: .

[0010] The preparation method of the above compound includes the following steps: (1) The extract obtained by heating and refluxing the Trollius chinensis Bunge medicinal material in ethanol is removed the solvent to obtain the crude extract; (2) After the crude extract is suspended in water, it is successively extracted with petroleum ether and ethyl acetate. The ethyl acetate extract is removed the solvent to obtain the ethyl acetate extract; (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 F 1 -F 6 , collect and combine the fractions eluted with the 90:10 eluent to obtain the elution fraction F 6 ; (4) The elution fraction F 6 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 F 6 S 1 -F 6 S 7 are obtained. Collect the elution fraction F 6 S 4 corresponding to the fourth chromatographic peak; (5) The elution fraction F 6 S 4 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; Collect the eluate of the component with an elution time of 35 min, remove the solvent to obtain the target compound trollchivone A.

[0011] 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).

[0012] In step (2), the volume-mass ratio of petroleum ether or ethyl acetate to the crude extract is 1.25:1 (L / kg).

[0013] In step (3), the fineness of the silica gel filler in the silica gel column is 200 - 300 mesh.

[0014] 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.

[0015] The present invention has the following advantages: (1) The present invention provides a flavonoid 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; (2) The flavonoid C - glycoside compound, trollchivone A, provided by the present invention can inhibit EV71 virus and has no toxic effect on cells, has the potential for developing drugs against EV71 virus, and can be used as the raw material and parent structure for new drug research and development; (3) The extraction and separation method of the flavonoid C - glycoside compound, trollchivone A, provided by the present invention is simple, rapid, low - cost, and the extracted compound has high purity. Brief Description of the Drawings

[0016] Figure 1 is the medium - pressure liquid chromatography diagram of step (4) in Example 1; Figure 2 is the high - resolution mass spectrum (HRESIMS) of trollchivone A; Figure 3 is the nuclear magnetic resonance hydrogen spectrum ( 1 ¹H - NMR) of trollchivone A; Figure 4 is the nuclear magnetic resonance carbon spectrum ( 13 ¹³C - NMR) of trollchivone A; Figure 5 is the heteronuclear single - quantum correlation spectrum (HSQC) of trollchivone A; Figure 6 is the heteronuclear multiple - bond correlation spectrum (HMBC) of trollchivone A; Figure 7 is the result diagram of the cytotoxicity experiment of trollchivone A on RD cells; Figure 8 is the result diagram of the inhibitory effect of trollchivone A on EV71 virus. Detailed Embodiments

[0017] The present invention will be further described below in conjunction with the embodiments and the drawings, but the present invention is not limited by the following embodiments.

[0018] Example 1 Isolation and identification of compound trollchivone A 1. Isolation of target compound (1) Weigh 20 kg of Trollius chinensis medicinal materials, add 80% ethanol, and extract three times by heating and reflux, wherein the added volume (L) of 80% ethanol is 15 times the mass (kg) of Trollius chinensis medicinal materials, and the reflux extraction time for each time is 2 h; collect the extracts obtained by three heating and reflux extractions, filter and combine the filtrates; decompress and concentrate the filtrate until there is no alcohol taste, cool it to room temperature, and obtain a crude extract for later use; (2) After the crude extract was suspended in water, petroleum ether and ethyl acetate were used for extraction, respectively, 4 times and 2 times at room temperature, and the ratio of the volume of petroleum ether and ethyl acetate to the mass of the crude extract was 1.25:1 (L / kg) each time; the extracts were combined and recovered under reduced pressure to obtain a petroleum ether extract and an ethyl acetate extract, respectively; (3) The ethyl acetate extract was separated by silica gel column chromatography using 200-300 mesh silica gel and gradient elution 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 F. 1 -F 6 , collect fraction F eluted with 90:10 eluent 6 ; (4) F 6 The medium pressure liquid chromatograph was used to analyze the 18 The column (80 mm × 40 mm, 30 μm) was used for chromatographic separation, and the gradient elution was carried out with methanol-water (30:70-90:10, v / v, 40 mL / min) for 1 h. Figure 1 ) can obtain 7 elution fractions F 6 S 1 -F 6 S 7 , where peak 4 corresponds to fraction F 6 S 4 ; (5) Fraction F 6 S 4 Semi-preparative HPLC, using ODS C 18 The product was separated by chromatography on a column (250 mm × 10 mm, 5 μm) and isocratically eluted with methanol-water (47:53, v / v, 2.5 mL / min). The corresponding fractions with an elution time of 35 min were collected and recovered to dryness under reduced pressure at 40°C to obtain the target compound as a yellow powder.

[0019] 2. Identification of the target compound 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 .

[0020] 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 , with an unsaturation degree of 14, as shown in Figure 2 .

[0021] Its 1 1H-NMR and 13 13C-NMR nuclear magnetic data were as follows: 1 1H-NMR (800 MHz, DMSO- d 6 ) 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, H 3 -4′′′), 0.73 (3H, d, J J = 7.2 Hz, H 3 -5′′′), 1.91 (3H, s, H 3 -2′′′′), 13.09 (1H, br s, 5-OH).

[0022] 13 C-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′′′′).

[0023] 1 1H-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 the sugar δ H 4.96 (1H, d, J J = 10.4 Hz).

[0024] 13 13C-NMR is as Figure 4 , combined with the HSQC spectrum ( Figure 5 ) showing 28 carbon signals, including 15 carbon signals of the flavonoid aglycone, 6 carbon signals of glucose ( δ C 71.3, 71.4, 75.6, 70.7, 78.3, 63.9), 3 methyl groups, one sp 3Hybrid methylene, 1 methine, 2 ester carbonyls ( δ C 174.7, 170.5). The above data indicate that compound trollchivone A is a flavonoid glycoside.

[0025] 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 spectroscopic 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 shown that the sugar moiety is attached to the C-8 position of the 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 C-glycoside. According to CH 3 -4′′′ having correlations with C-2′′′ ( δ C 40.4), C-3′′′ ( δ C 25.9), and H-2′′′, H 2 -3′′′, CH 3 -5′′′ and H-2′′ having correlations with C-1′′′ ( δ C 174.7), it is shown that one 2-methylbutyryl group forms an ester bond with the hydroxyl group at the C-2′′ position of the sugar. According to CH 3 -2′′′′, H 2 -6′′ having correlations with C-1′′′′ ( δ C 170.5), it is shown that one acetyl group forms an ester bond with the hydroxyl group at the C-6′′ position of the sugar.

[0026] In summary, the molecular formula of the finally determined target compound isolated is C 28 H 30 O12 , with the chemical structural formula: , named trollchivone A.

[0027] Example 2 Antiviral activity of trollchivone A against EV71 virus (1) Toxicity experiment of trollchivone A on RD cells RD cells were placed in DMEM medium containing 10% fetal bovine serum and cultured at 37 °C under 5% CO 2 conditions. 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 replicates were set for each concentration, and a normal cell control group was also established. Incubate at 37 °C and 5% CO 2 in an incubator. Observe the cytopathic effect daily for 72 h continuously. Determine the cell viability by MTT method, measure the OD value at 490 nm wavelength with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the median toxic concentration TC 50 .

[0028] The results of the toxicity 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.

[0029] (2) Inhibitory effect of trollchivone A on EV71 virus After the RD cells grew into a monolayer on a 96-well plate, the supernatant culture medium was discarded. After washing 3 times with PBS, 100 μL of 100-fold TCID 50 of EV71 virus solution was inoculated into each well. After adsorbing at 37 °C for 1 h, the virus solution was discarded. 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 respectively. Three replicates were set for each concentration, and a normal control group and a virus control group were also established. Incubate at 37 °C and 5% CO 2 in an incubator for continued culture. Observe the cell morphology for 72 h, determine the cell viability by MTT method, measure the absorbance at 490 nm, and calculate the virus inhibition rate.

[0030] The results of the inhibitory effect of trollchivone A on EV71 virus are as Figure 8As shown, when the concentration of trollchivone A is 100 µg / mL, the virus inhibition rate is 50.2%, indicating good inhibitory activity against EV71 virus.

[0031] The above are only examples 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 flavonoid carbon glycoside compound, the chemical structural formula is: 。 2. A method for preparing flavonoid glycoside compounds as claimed in claim 1, characterized in that: The following steps are involved: (1) heating the extract of the golden lotus medicinal material in ethanol and refluxing it, and removing the solvent to obtain a crude extract; (2) After suspending the crude extract in water, extracting with petroleum ether and ethyl acetate in sequence, 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, using dichloromethane-methanol as the eluent for gradient elution, the volume ratio of dichloromethane-methanol was 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, and the elution volumes were 7 times, 6 times, 7 times, 7 times, 7 times, and 7 times the column volume, respectively, to obtain 6 elution fractions F1-F6, and the fractions eluted with the 90:10 eluent were collected and combined to obtain elution fraction F6; (4) The eluted fraction F6 was separated by medium pressure liquid chromatography using an ODS C column. 18 The column was used for gradient elution for 1 h with methanol-water as the mobile phase, the ratio of methanol-water gradient elution was 30:70-90:10, v / v, and the flow rate of the mobile phase was 40 mL / min; 7 elution fractions F6S1-F6S7 were obtained, and the elution fraction F6S4 corresponding to the fourth chromatographic peak was collected; (5) The eluted fraction F6S4 was separated by semi-preparative HPLC using an ODS C column. 18 The column was isocratically eluted with methanol-water as the mobile phase, the methanol-water ratio was 47:53, v / v, and the flow rate of the mobile phase was 2.5 mL / min; the eluate with the peak time of 35 min was collected, the solvent was removed, and the target compound was obtained.

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 nasturtium 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. A use of the flavonoid glycoside compound as claimed in claim 1 in the preparation of anti-EV71 virus drugs.

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