Indole alkaloid compound, extraction method and application thereof

Abstract

The invention discloses an indole alkaloid compound and an extraction method and application thereof, belonging to the field of biomedical technology. The structural formula of the indole alkaloid compound is shown in Formula 1:. The invention takes a milk vine plant and refluxes it in an organic solvent I for extraction or leaching, recovers the organic solvent until it is tasteless, suspends it in water, extracts it with an organic solvent II under acidic conditions; extracts the water layer after the organic solvent II with an organic solvent III under alkaline conditions, and separates the organic layer by chromatography to obtain an indole alkaloid compound. The indole alkaloid compound is extracted from the milk vine plant, and the indole alkaloid compound can inhibit the proliferation of HepG2 human liver cancer cells, and has the potential to be developed into a drug for preventing or treating liver cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an indole alkaloid compound and an extraction method and application thereof. Background Art

[0002] Bousigonia mekongensis is a plant of the genus Bousigonia in the Apocynaceae family, mainly distributed in southwest my country. Bousigonia mekongensis plants are rich in monoterpene indole alkaloid compounds with novel structures and significant activities, some of which have biological activities such as anti-tumor and anti-diabetic nephropathy.

[0003] For most patients diagnosed with advanced liver cancer, it is difficult to undergo surgical treatment. Current clinical treatment drugs are not ideal in terms of efficacy, safety, and survival benefits. There is an urgent need to develop safer and more effective treatment drugs. Lead compounds from natural sources have always been the source of anti-tumor drug research and development. Therefore, finding and developing safe, effective, and low-adverse-reaction anti-liver cancer drug lead compounds from natural medicinal plants is an important way to develop anti-liver cancer drugs. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides an indole alkaloid compound and an extraction method and application thereof. The indole alkaloid compound has good activity in inhibiting the proliferation of HepG2 cells and has the prospect of being further developed into a new anti-liver cancer drug.

[0005] The present invention is achieved through the following technical solutions:

[0006] An indole alkaloid compound, the structural formula of the indole alkaloid compound is shown in Formula 1:

[0007]

[0008] A method for extracting the above-mentioned indole alkaloid compound, wherein the indole alkaloid compound is extracted from the plant of Glechoma longituba, specifically comprising the following steps:

[0009] (1) extracting or leaching the milk vine plant in solvent I by reflux, recovering the organic solvent until it is tasteless, suspending it in water, and extracting it with organic solvent II under acidic conditions;

[0010] (2) After the extraction with organic solvent II in step (1), the aqueous layer is extracted with organic solvent III under alkaline conditions, and the organic layer is separated by chromatography to obtain an indole alkaloid compound.

[0011] Furthermore, the solvent I is C 1-6 Alcohol solution; the organic solvent II and organic solvent III are C 1-6 Halogenated hydrocarbons.

[0012] Furthermore, the solvent I is a 95% ethanol solution; the organic solvent II and the organic solvent III are dichloromethane or chloroform.

[0013] Furthermore, the pH under acidic conditions in step (1) is 2-3; and the pH under alkaline conditions in step (2) is 9-10.

[0014] Furthermore, the organic layer after extraction with organic solvent III in step (2) is subjected to ODS medium pressure chromatography, reversed phase ODS column chromatography, and Sephadex LH-20 gel chromatography to prepare a liquid phase to obtain an indole alkaloid compound.

[0015] Furthermore, ODS medium pressure chromatography was eluted with 15%, 30%, 60%, and 100% methanol-water solutions, and after HPLC analysis and merging, 7 parts Fr.1~7 were obtained; Fr.6 was eluted by reverse phase ODS column chromatography with 60%, 70%, 80%, 90%, and 100% methanol-water solutions and methanol solution containing 0.1wt% TFA, respectively, and after HPLC analysis and merging, 3 parts Fr.6-1~Fr.6-3 were obtained; Fr.6-2 was isocratically eluted by gel column chromatography with a methanol-dichloromethane eluent with a volume ratio of 20:1, and after HPLC analysis and merging, 11 parts Fr.6-2-1~Fr.6-2-11 were obtained, and Fr.6-2-5 was eluted by preparative liquid chromatography with 85% methanol-water solution at 8mL / min to obtain an indole alkaloid compound.

[0016] In the present invention, the indole alkaloid compound is used in the preparation of an anti-liver cancer preparation.

[0017] Beneficial Effects

[0018] The invention discloses a novel indole alkaloid compound bousimekofruine A extracted from milk vine, and the compound can be further prepared into a pharmaceutically acceptable salt thereof; the indole alkaloid compound bousimekofruine A can inhibit the proliferation of HepG2 human liver cancer cells and has the potential to be developed into a drug for preventing or treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 For bousimekofruine A 1 H NMR spectra (CDCl3);

[0020] Figure 2 For bousimekofruine A 13 C NMR spectrum (CDCl3);

[0021] Figure 3 For bousimekofruine A1 H- 1 H COSY pattern (CDCl3);

[0022] Figure 4 HSQC diagram of bousimekofruine A (CDCl3);

[0023] Figure 5 is the HMBC diagram of bousimekofruine A (CDCl3);

[0024] Figure 6 NOESY diagram of bousimekofruine A (CDCl3);

[0025] Figure 7 This is the UV image of bousimekofruine A (Methanol);

[0026] Figure 8 This is the IR map of bousimekofruine A;

[0027] Fig. 9 The NMR calculated images of two possible configurations (β or α) of the 2-hydroxyl group of bousimekofruine A, a is the β configuration, b is the α configuration;

[0028] Fig.10 The calculated ECD and measured ECD diagram of bousimekofruine A;

[0029] Fig.11 This is the structural formula of bousimekofruine A. DETAILED DESCRIPTION

[0030] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0031] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0032] Example 1

[0033] (1) Take 15 kg of air-dried milk vine fruit, extract it with 95% ethanol solution by percolation for 5 times, recover the solvent until there is no alcohol taste, add water to suspend it, adjust the pH to 2-3 with 2% HCl, and extract it with dichloromethane for 4 times;

[0034] (2) The aqueous layer after extraction in step (1) was adjusted to pH 9.5 with 10% NaOH, and eluted with 15%, 30%, 60%, and 100% methanol aqueous solution by ODS medium pressure chromatography. After HPLC analysis, seven fractions (Fr.1-7) were obtained after merging. Fr.6 was eluted with 60%, 70%, 80%, 90%, and 100% methanol aqueous solution and methanol solution containing 0.1wt% TFA, respectively, by reverse phase ODS column chromatography. After HPLC analysis, three fractions (Fr.6-1-Fr.6-3) were obtained after merging. Fr.6-2 was eluted isocratically by gel column chromatography with methanol-dichloromethane eluent in a volume ratio of 20:1. After HPLC analysis, 11 fractions (Fr.6-2-1-Fr.6-2-11) were obtained after merging. Fr.6-2-5 was eluted with 85% methanol aqueous solution at 8 mL / min by preparative liquid chromatography to obtain the indole alkaloid compound bousimekofruine A.

[0035] The structural formula of bousimekofruine A was determined by infrared spectroscopy, mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy. Infrared spectral data: IR (max, KBr): 3381, 3309, 2960, 2924, 2878, 1679, 1604, 1581, 1466, 1249, 1026, 918, 895, 748 cm -1 ; Mass spectral data: HR-ESI-MS (m / z): 593.3856 [M+H] + (Calcd.593.3856 [M+H] + ); 1 H NMR and 13 C NMR data are shown in Table 1. 1 H NMR spectrum (CDCl3) Figure 1 As shown, bousimekofruine A 13 C NMR diagram (CDCl3) Figure 2 As shown, bousimekofruineA 1 H- 1 H COSY diagram (CDCl3) Figure 3 As shown, the HSQC diagram of bousimekofruine A (CDCl3) is as follows Figure 4 As shown, the HMBC diagram (CDCl3) of bousimekofruine A is as follows Figure 5 As shown in the NOESY diagram of bousimekofruine A (CDCl3), Figure 6 As shown, the UV image of bousimekofruine A (Methanol) is as follows Figure 7 As shown; the IR diagram of bousimekofruine A is shown Figure 8 The NMR calculation diagrams of the two possible configurations (β or α) of the 2-hydroxyl group of bousimekofruine A are shown in Fig. 9 As shown in the figure (where a is β configuration and b is α configuration), the calculated ECD and measured ECD of bousimekofruine A are shown in the figure Fig.10 As shown, the structural formula of bousimekofruine A is shown in Fig.11 shown.

[0036] Bousimekofruine A is a white powder. High-resolution mass spectrometry shows that its m / z is 593.3856 (theoretical value is 593.3856). Its molecular formula is inferred to be C 38 H 48 N4O2. Infrared spectrum IR at 3381, 3309 cm -1 There is absorption at the position, indicating that OH or NH may exist in the structure. 1 H NMR showed six aromatic hydrogen signals δ H 7.17 (1H, d, J = 7.2 Hz), 7.11 (1H, d, J = 7.2 Hz), 7.06 (1H, d, J = 8.0 Hz), 7.01 (1H, td, J = 1.6, 7.2 Hz), 6.82 (1H, td, J = 0.8, 7.2 Hz), 6.45 (1H, d, J = 8.0 Hz), one NH active hydrogen signal δ H 4.67 (1H, s), two methyl signals δ H 0.96 (3H, t, J = 7.2 Hz) and 0.41 (3H, t, J = 7.2 Hz). 13 C NMR and HSQC spectra showed that the compound had 38 carbon signals, including 2 methyl groups, 14 methylene groups, 11 methine groups, and 11 quaternary carbon groups. Further analysis of its 1D NMR and 2D NMR suggested that the compound might be an akuammicine-Aspidosperma type double alkaloid.

[0037] In unit A, 1 H- 1The H COSY spectrum showed six connected fragments a (C-15-C-16-C-17), b (C-15-C-14-C-3), c (C-15-C-20-C-21), d (C-15-C-20-C-19-C-18), e (C-5-C-6) and f (C-9-C-10-C-11-C-12), suggesting that the unit may be an akuammicine alkaloid. In the HMBC spectrum, H-9 correlated with C-7 / C-11 / C-12, NH-1 / H-5 correlated with C-7, H-3 / H-6 / H-16 / H-17 correlated with C-2, H-16 correlated with C-15 / C-20, H-18 correlated with C-19 / C-20, and H-19 correlated with C-15 / C-21, which further confirmed the above inference.

[0038] In unit B, 1 H- 1 The H COSY spectrum showed five connecting fragments, namely a (C-5′-C-6′), b (C-11′-C-12′), c (C-3′-C-14′-C-15′), d (C-16′-C-17′) and e (C-18′-C-19′). In the HMBC spectrum, H-18′ was related to C-19′ / C-20′, H-19′ was related to C-15′ / C-17′, H-21′ was related to C-2′ / C-3′ / C-6′ / C-7′ / C-8′ / C-15′ / C-17′, H-17′ was related to C-2′, H-11′ was related to C-13′, and H-12′ was related to C-8′ / C-10′. The above information suggests that unit B may be an Aspidosperma type alkaloid. In addition, H-11′ and C-9′ / C-17, and H-21′ and C-9′ respectively have HMBC correlations, suggesting that units A and B are connected via C-17-C-10′ and that C-9′ is substituted with a hydroxyl group.

[0039] The relative configuration of bousimekofruine A was determined by NOESY spectrum. H 2.16) is related to H-3 / H-9, H-16 is related to H-6β (δ H 2.89) / H-19, H-6β is related to H-19, H-15 is related to H-17, suggesting that H-3 / H-15 / H-20 are α-configuration and H-16 is β-configuration. H-21′ is related to H-18′, H-19′ is related to H-17′α (δ H 1.48), H-17′α and H-16′α (δ H 2.62) related, H-16′β (δH 3.03) and H-6′β (δ H 2.01), suggesting that H-21′ and CH2-19′ are in α-configuration. To further determine the configuration of the hydroxyl group connected to C-2, we performed NMR calculations (1-a: 2-OHβ, 1-b: 2-OHα), and the results showed that 2-OH was in β-configuration. The absolute configuration of bousimekofruine A was determined by comparing the calculated ECD with the measured ECD. Through calculation, its absolute configuration was finally determined to be (2R, 3S, 7R, 15S, 16S, 20S, 7′R, 20′S, 21′S), and the structural formula is shown in Fig.11 shown.

[0040] Table 1 Bousimekofruine A 1 H NMR and 13 C NMR data (CDCl3, *shown are overlapping signals)

[0041]

[0042] Example 2

[0043] Experimental study on the inhibitory activity of bousimekofruine A prepared in Example 1 on HepG2 liver cancer cells:

[0044] (1) Experimental materials:

[0045] Cell line: HepG2 cell line, positive control is doxorubicin; other reagent materials are shown in Table 2 below:

[0046] Table 2 Experimental reagents and materials

[0047]

[0048] (2) Experimental methods

[0049] Hep G2 cells in logarithmic growth phase (cell concentration 1×10 6Cells were inoculated into 96-well culture plates with 100 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 incubator for 12 h. The cells were divided into a normal control group and a sample group (bousimekofruine A and doxorubicin, respectively), each with 6 replicate wells. In the normal control group, 100 μL of 1640 complete medium containing 10% FBS was added to each well. Sample group: The samples (bousimekofruine A and doxorubicin) were dissolved and diluted in 1640 complete medium containing 10% FBS to prepare six concentrations of 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM and 0.78125 μM, respectively. Six replicate wells were set for each concentration, and 100 μL was added to each well. Each group was cultured in a 37°C, 5% CO2 incubator for 48 h, and 10 μL of CCK8 solution (5 mg / ml) was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 2 h. The absorbance of each well was measured at 450 nm by a microplate reader, and the cell proliferation inhibition rate was calculated. The half inhibitory concentration (IC) was calculated using GraphPad Prism8 software. 50 ), bousimekofruine A and IC of doxorubicin 50 The values ​​are shown in Table 3 below. As can be seen from Table 3, bousimekofruine A extracted by the present invention has the activity of inhibiting the proliferation of liver cancer cells and is expected to be developed into an anti-liver cancer drug.

[0050] Table 3 Data table of cell proliferation activity of indole alkaloid compounds

[0051]

[0052] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Although the present application is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An indole alkaloid compound, characterized in that The structural formula of the indole alkaloid compound is shown in Formula 1:

2. A method for extracting indole alkaloid compounds according to claim 1, characterized in that: The indole alkaloid compound is extracted from the plant of Glehnia littoralis, and specifically comprises the following steps: (1) Take 15 kg of air-dried milk vine fruit, extract it with 95% ethanol solution by percolation for 5 times, recover the solvent until there is no alcohol taste, add water to suspend it, adjust the pH to 2-3 with 2% HCl, and extract it with dichloromethane for 4 times; (2) The aqueous layer after extraction in step (1) was adjusted to pH 9.5 with 10% NaOH, and eluted with 15%, 30%, 60%, and 100% methanol aqueous solution by ODS medium pressure chromatography. After HPLC analysis and merging, 7 fractions Fr.1 to 7 were obtained; Fr.6 was eluted with 60%, 70%, 80%, 90%, and 100% methanol aqueous solution and methanol solution containing 0.1wt% TFA, respectively, by reverse phase ODS column chromatography, and 3 fractions Fr.6-1 to Fr.6-3 were obtained after HPLC analysis and merging; Fr.6-2 was eluted isocratically by gel column chromatography with methanol-dichloromethane eluent in a volume ratio of 20:1, and 11 fractions Fr.6-2-1 to Fr.6-2-11 were obtained after HPLC analysis and merging. Fr.6-2-5 was eluted with 85% methanol aqueous solution at 8 mL / min by preparative liquid chromatography to obtain an indole alkaloid compound.

3. Use of the indole alkaloid compound according to claim 1 in the preparation of an anti-liver cancer preparation.

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