CN112724113A - Flavonoid compound with anti-tumor activity and preparation method and application thereof - Google Patents
Flavonoid compound with anti-tumor activity and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a flavonoid compound with anti-tumor activity, which is named as 5-hydroxy-6, 7,3 ', 4 ', 5 ' -pentamethoxyl flavonoid and has a chemical structural formula as follows:
the flavonoid compound separated from murraya paniculata has good inhibition effect on tumor cells, and can significantly inhibit the proliferation of mouse melanoma cells B16 and human breast cancer cells MDA-MB-231. The compound provided by the invention can provide a material basis for the development of novel antitumor drugs, and is beneficial to the further development of the medicinal value of murraya paniculata.
Description
Technical Field
The invention belongs to the technical field of natural medicinal chemistry, and particularly relates to a flavonoid compound with anti-tumor activity, and a preparation method and application thereof.
Background
Cancer is one of the most common diseases threatening human health, and the morbidity and mortality rate thereof is second to cardiovascular diseases and has a tendency to exceed the mortality rate of heart diseases, thereby bringing a heavy economic and mental burden to the society and families. Traditional chemotherapy and radiotherapy become the main methods for treating cancer, and although the traditional chemotherapy and radiotherapy have certain curative effect clinically, the traditional chemotherapy and radiotherapy have great side effect and are easy to generate drug resistance. Therefore, the discovery of the anti-tumor compound with high efficiency, low toxicity and strong specificity has important significance, and the anti-tumor compound can provide a lead compound for the development of novel anti-tumor drugs and provide a new way for the treatment of tumors.
Chinese herbal medicines contain secondary metabolites with various structures, and have become one of important sources for searching antitumor lead compounds and candidate medicines. Murraya paniculata (Murraya tetramera) is a plant of Murraya genus of Rutaceae family, and is distributed in Western Guangxi (Bai Cao, Debao, etc.) and southeast Yunnan (inkstone, Funing, Wenshan, Western Domain, etc.), and can be used for treating rheumatism, common cold, cough, etc. The drug mainly contains various components such as coumarin, flavone, carbazole alkaloid and the like, and has certain cytotoxicity on breast cancer cells MCF7, liver cancer cells SMMC7721, lung cancer cells A549 and the like. Therefore, the discovery of the compound with anti-tumor activity from the murraya paniculata has important significance for the research and development of novel anti-tumor drugs and the development of the medicinal value of the murraya paniculata.
Through detection, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a flavonoid compound with anti-tumor activity and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the flavonoid compound with the anti-tumor activity is named as 5-hydroxy-6, 7,3 ', 4 ', 5 ' -pentamethoxyl flavonoid, and has the chemical structural formula:
moreover, the steps are as follows:
preparing a methanol extract: taking dried branches and leaves of murraya paniculata, crushing, ultrasonically extracting with methanol for 2-3 times, wherein the solvent amount is 6-8 times, ultrasonically extracting for 30-60min each time, combining filtrates, recovering the solvent to obtain extract, and weighing to obtain methanol extract, wherein the extract yield is 4-5%;
and the separation of the compounds: taking methanol extract of murraya paniculata, and extracting according to the following methanol extract: mixing the methanol extract and the silica gel at a mass ratio of 1:1, stirring the mixture by a dry method, performing silica gel column chromatography, and sequentially performing petroleum ether-ethyl acetate 60: 1. 30: 1. 10: 1. 5: 1. 1:1, chloroform-methanol 20: 1. 10: 1. 1: gradient elution is carried out for 1, thin-layer chromatography detection is carried out, and the same components are combined to obtain 90 fractions;
passing fraction 66-67 through resin column, eluting with ethanol-water gradient, separating and purifying by HPLC preparative liquid phase chromatography, and purifying with C18Performing gradient elution with methanol-water as mobile phase to obtain the flavone compounds with antitumor activity.
Moreover, the silica gel in the step II is 160-200 meshes.
Moreover, the step of passing the segments 66-67 through MCI GEL CHP20P fast resin columns in the second phase.
The flavonoid compound with antitumor activity is applied to the preparation of antitumor drugs.
Moreover, the tumor cells are mouse melanoma cells B16 and human breast cancer cells MDA-MB-231.
The invention has the following advantages and positive effects:
1. the flavonoid compound separated from murraya paniculata has good inhibition effect on tumor cells, and can significantly inhibit the proliferation of mouse melanoma cells B16 and human breast cancer cells MDA-MB-231. The compound provided by the invention can provide a material basis for the development of novel antitumor drugs, and is beneficial to the further development of the medicinal value of murraya paniculata.
2. The preparation method has simple process, and the flavonoid compound with obvious effect of inhibiting tumor cells can be obtained by the method.
Drawings
FIG. 1 shows flavonoid compounds of the present invention1H-NMR spectrum;
FIG. 2 shows the flavonoid compounds of the present invention13C-NMR spectrum.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
The flavonoid compound with the anti-tumor activity is named as 5-hydroxy-6, 7,3 ', 4 ', 5 ' -pentamethoxyl flavonoid, and has the chemical structural formula:
preferably, the steps are as follows:
preparing a methanol extract: taking dried branches and leaves of murraya paniculata, crushing, ultrasonically extracting with methanol for 2-3 times, wherein the solvent amount is 6-8 times, ultrasonically extracting for 30-60min each time, combining filtrates, recovering the solvent to obtain extract, and weighing to obtain methanol extract, wherein the extract yield is 4-5%;
and the separation of the compounds: taking methanol extract of murraya paniculata, and extracting according to the following methanol extract: mixing the methanol extract and the silica gel at a mass ratio of 1:1, stirring the mixture by a dry method, performing silica gel column chromatography, and sequentially performing petroleum ether-ethyl acetate 60: 1. 30: 1. 10: 1. 5: 1. 1:1, chloroform-methanol 20: 1. 10: 1. 1: gradient elution is carried out for 1, thin-layer chromatography detection is carried out, and the same components are combined to obtain 90 fractions;
passing fraction 66-67 through resin column, eluting with ethanol-water gradient, separating and purifying by HPLC preparative liquid phase chromatography, and purifying with C18Performing gradient elution with methanol-water as mobile phase to obtain the flavone compounds with antitumor activity.
Preferably, the silica gel in the step II is 160-200 meshes.
Preferably, the second step is to pass the segments 66-67 through MCI GEL CHP20P fast resin columns.
The flavonoid compound with antitumor activity is applied to the preparation of antitumor drugs.
Preferably, the tumor cells are mouse melanoma cells B16 and human breast cancer cells MDA-MB-231.
Specifically, the preparation and detection examples are as follows:
EXAMPLE 1 preparation of flavonoids according to the invention
1) Preparing a methanol extract: taking dried branches and leaves of murraya paniculata, crushing, performing ultrasonic extraction for 3 times by adopting methanol, wherein the solvent amount is respectively 8 times, 6 times and 6 times, performing ultrasonic extraction for 30min each time, combining filtrates, recovering the solvent to obtain an extract, and weighing to obtain the methanol extract, wherein the extract yield is 4.4%.
2) Compound separation: taking 110g of murraya paniculata methanol extract, mixing samples according to a dry method of 1:1 (extract amount: silica gel amount), performing silica gel (160-mesh and 200-mesh) column chromatography, sequentially performing petroleum ether-ethyl acetate 60: 1. 30: 1. 10: 1. 5: 1. 1:1Chloroform-methanol 20: 1. 10: 1. 1: gradient elution is carried out by 1, thin layer chromatography detection is carried out, and the same components are combined to obtain 90 fractions. Passing fractions 66-67 through MCI GEL CHP20P flash resin column, eluting with ethanol-water gradient, and separating and purifying by HPLC preparative liquid phase chromatography using C18Performing gradient elution with methanol-water as mobile phase to obtain the final product (50 mg).
Example 2 structural identification of Flavonoids of the invention
Yellow needle crystal, easily soluble in chloroform, and exhibits yellow-green fluorescence under ultraviolet lamp (365 nm).
13C-NMR(125MHz，CDCl3) In the spectrum, a 21 carbon signal is given, with 182.6ppm being the carbonyl carbon signal of C-4; 163.8ppm is the C-2 signal, the carbon on the-O ethylene bond signal; 105.5ppm is the C-3 signal, the carbon on olefin bond signal; these are typical flavone backbone carbon signals. 158.9, 153.6, 153.3, 153.1, 141.5, 132.8ppm are signals of carbon on the benzene ring of the bi-O; 126.5, 106.2, 103.8 and 90.7ppm are carbon signals on a benzene ring; the above is the carbon signal on the flavone parent nucleus. 61.1, 60.9, 56.4, 56.4ppm are-OCH3Carbon-up signal.
1H-NMR(500MHz，CDCl3) In the spectrum, at 12.72ppm, the signal of 1 s peak of H is given, which is a typical flavone 5-OH signal; at 7.11ppm, the signal giving the s peak of 2H, is typical of the flavone H-2 ', H-6' signal; signals of s-peaks at 6.64, 6.59ppm, each giving 1H, are H-3, H-8 signals; at 4.01ppm, an s-peak signal of 3H is given as-OCH3A hydrogen uptake signal; at 4.00ppm, an s-peak signal of 6H is given, which is 2-OCH3A hydrogen uptake signal; at 3.96ppm, an s-peak signal of 6H is given, which is 2-OCH3A hydrogen uptake signal; thus, the compound is determined to be a5, 6,7,3 ', 4 ', 5 ' substituted flavonoid component.
Based on the above analysis, the molecular formula is presumed to be C20H20O8Reference to[1](Rwangabo P.C.,Claeys M.,Pieters L.,et al.Umuhengerin,a new antimicrobially active flavonoid fromLantana trifolia[J].Journal of NaturalProducts,1988,51(5):966-968.), and the flavonoid compound of the invention is identified as 5-hydroxy-6, 7,3 ', 4 ', 5 ' -pentamethoxyl flavone, and the hydrogen spectrum and the carbon spectrum data are shown in figures 1 and 2.
EXAMPLE 3 antitumor Activity of Flavonoids of the invention
Experiment medication: flavonoids according to example 1 of the present invention;
positive control drug: doxorubicin hydrochloride;
CCK-8 is adopted to test the in-vitro inhibitory activity of the flavonoid compound on tumor cells, and the screened tumor cell strains are B16 and MDA-MB-231. The specific process is as follows: the flavonoid compound is prepared into mother solution with the concentration of 20mg/mL by DMSO, and then diluted into sample solutions with the concentration of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 mu g/mL by culture medium. Culturing B16 cells in RPMI 1640(Gibco) medium, and culturing MDA-MB-231 in DMEM (Gibco) medium containing 10% fetal bovine serum and 1% streptomycin at 37 deg.C and 5% CO2In a cell culture box. Cells in logarithmic growth phase were seeded in 96-well plates in a volume of 100. mu.L per well containing approximately 6X 10 cells per well3Cells, incubated at 37 ℃ in 5% CO2Culturing for 12-24h in a cell culture box to allow the cells to adhere to the wall. Discard the culture medium in the wells, then add 100. mu.L of sample solutions of different concentrations to each well, incubate at 37 ℃ for 48h, make 7 concentrations for each sample, make 5 wells for each concentration, and repeat the experiment 3 times. Removing culture medium in the wells, adding 100 μ L culture medium and 10 μ L CCK-8 reagent (Shanghai Biyun day) into each well, developing at 37 deg.C for 1-2 hr, measuring absorbance at 450nm with Bio-Rad enzyme labeling instrument, and analyzing and calculating IC with Probit (IBM SPSS V20.0)50The results are shown in Table 1.
TABLE 1 test results of tumor cytotoxic activity of flavonoids of the present invention
The experimental results show that the flavonoid compound has obvious inhibition effect on two tumor cells.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Claims (6)
1. A flavonoid compound with anti-tumor activity is characterized in that: the name of the flavonoid compound is 5-hydroxy-6, 7,3 ', 4 ', 5 ' -pentamethoxyl flavone, and the chemical structural formula is as follows:
2. the method for preparing flavonoids compounds with antitumor activity according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
preparing a methanol extract: taking dried branches and leaves of murraya paniculata, crushing, ultrasonically extracting with methanol for 2-3 times, wherein the solvent amount is 6-8 times, ultrasonically extracting for 30-60min each time, combining filtrates, recovering the solvent to obtain extract, and weighing to obtain methanol extract, wherein the extract yield is 4-5%;
and the separation of the compounds: taking methanol extract of murraya paniculata, and extracting according to the following methanol extract: mixing the methanol extract and the silica gel at a mass ratio of 1:1, stirring the mixture by a dry method, performing silica gel column chromatography, and sequentially performing petroleum ether-ethyl acetate 60: 1. 30: 1. 10: 1. 5: 1. 1:1, chloroform-methanol 20: 1. 10: 1. 1: gradient elution is carried out for 1, thin-layer chromatography detection is carried out, and the same components are combined to obtain 90 fractions;
passing fraction 66-67 through resin column, eluting with ethanol-water gradient, separating and purifying by HPLC preparative liquid phase chromatography, and purifying with C18Performing gradient elution with methanol-water as mobile phase to obtain the flavone compounds with antitumor activity.
3. The method for preparing flavonoids compounds with antitumor activity according to claim 2, characterized in that: the silica gel in the step II is 160-mesh and 200-mesh.
4. The method for preparing flavonoids compounds with antitumor activity according to claim 2 or 3, characterized in that: the step is to pass the segments 66-67 through MCI GEL CHP20P fast resin columns in the second phase.
5. The use of flavonoids according to claim 1 with antitumor activity in the preparation of antitumor drugs.
6. Use according to claim 5, characterized in that: the tumor cells are mouse melanoma cells B16 and human breast cancer cells MDA-MB-231.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292263A (en) * | 2021-12-22 | 2022-04-08 | 上海工程技术大学 | Novel symmetrical biflavonoid compound and preparation and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864740A (en) * | 2012-12-18 | 2014-06-18 | 中国科学院兰州化学物理研究所 | Preparation method of gardenia flower flavones with anticancer activity |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864740A (en) * | 2012-12-18 | 2014-06-18 | 中国科学院兰州化学物理研究所 | Preparation method of gardenia flower flavones with anticancer activity |
Non-Patent Citations (2)
| Title |
|---|
| CHUN-XUE YOU ET AL.: "Cytotoxic Flavonoids from the Leaves and Twigs of Murraya tetramera", 《MOLECULES》 * |
| JIA-YU ZHANG ET AL.: "Characterization of seventy polymethoxylated flavonoids (PMFs) in the leaves of Murraya paniculata by on-line high-performance liquid chromatography coupled to photodiode array detection and electrospray tandem mass spectrometry", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292263A (en) * | 2021-12-22 | 2022-04-08 | 上海工程技术大学 | Novel symmetrical biflavonoid compound and preparation and application thereof |
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