CN113185484A - Chromone compound and preparation method and application thereof - Google Patents

Chromone compound and preparation method and application thereof Download PDF

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CN113185484A
CN113185484A CN202110385027.7A CN202110385027A CN113185484A CN 113185484 A CN113185484 A CN 113185484A CN 202110385027 A CN202110385027 A CN 202110385027A CN 113185484 A CN113185484 A CN 113185484A
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organic solvent
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chromone compound
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CN113185484B (en
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高雪梅
李斌
张再
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Jiangxi University of Traditional Chinese Medicine
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Abstract

The chromone compound is prepared by using dried branches and leaves of plants in the genus of cassia of leguminosae as raw materials and performing the steps of extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation. Through the experiment of resisting tobacco mosaic virus, the compound has the inhibition rate of 28.7 +/-4.1%, has good activity of resisting the tobacco mosaic virus, and is similar to the inhibition rate (32.2 +/-3.2%) of a positive control nandinmycin. The test proves that the compound has good activity of resisting tobacco mosaic virus, and the compound has simple structure and good activity; can be used as a guiding compound of a tobacco mosaic virus resistant medicament.

Description

Chromone compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of extraction of effective components of plants, in particular to a chromone compound and a preparation method and application thereof.
Background
The chromone compound is widely existed in nature, generally has the functions of bacteriostasis, anti-inflammation and the like, and is an important component in bacteriostasis and anti-inflammation medicines. The chromone compounds are currently generally extracted from plants, such as plants of the genus Caragana of the family Leguminosae.
About 600 species of plants of the Cassia genus, distributed in tropical and subtropical regions of the world, with a few distributed in temperate regions; more than 10 original species of China, including more than 20 species for introduction and cultivation, are widely distributed in the regions of south and north. The cassia plant is one of the main sources of natural flavonoids. In addition to flavonoids, anthraquinones and polysaccharides are characteristic components of plants of this genus and have various biological activities.
Few researches on plants in the genus of cassia have been conducted in the prior art, and few reports have been reported on extraction of chromone compounds from plants in the genus of cassia. In order to more effectively utilize the resources of the cassia plants in China, active ingredients with development prospects are searched from the cassia plants, and the method is an important work for the current research of the cassia plants.
Disclosure of Invention
In view of the above, there is a need to provide a chromone compound, a preparation method and applications thereof based on the extraction and research of active ingredients of plants in the genus of cassia.
In one aspect, the invention provides a chromone compound having the following chemical structural formula:
Figure BDA0003014424540000011
the chromone compound is obtained by using dried branches, leaves or fruits of plants in the genus of Cassia as raw materials and performing extract extraction, organic solvent extraction, silica gel column chromatography and high pressure liquid chromatography separation, and the molecular formula of the compound is C13H12O5The compound is named as 7-hydroxy-5- (3-hydroxy-2-oxopyryl) -2-methyl-4H-chromen-4-one.
The invention also provides a preparation method of the chromone compound, which comprises the following steps:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of a cassia plant to 20-40 meshes, performing ultrasonic extraction for 2-4 times by using an organic solvent for 30-60 min each time, combining extracting solutions, filtering the extracting solutions, concentrating the extracting solutions under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain a first extract;
B. organic solvent extraction: adding water into the obtained extract a, wherein the weight of the added water is 1-2 times of the weight of the extract, extracting for 3-5 times by using an organic solvent with the same volume as that of the water, combining organic solvent extraction phases, and concentrating under reduced pressure to obtain an extract b;
C. silica gel column chromatography: dissolving the extract b by using an organic solvent with the weight of 1.5-3 times that of the extract b, mixing the sample by using 200-300 meshes of silica gel with the weight of 0.8-1.2 times that of the extract b, performing silica gel column chromatography, wherein the silica gel filled in the column is 200-300 meshes, and the using amount of the silica gel filled in the column is 6-8 times that of the extract b; gradient eluting with two mixed organic solvents in a volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. reversed-phase column chromatography: c, subjecting the eluent combined with the same part in the step C to reverse phase column chromatography, wherein the reverse phase column is filled with a reverse phase material; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluents of all parts and concentrating by adopting three solutions with concentration ratios with sequentially increased methanol concentration in the gradient elution process, monitoring by TLC, and combining the same parts to obtain 3 components;
E. performing gradient elution on the 1 st component of the eluent obtained in the step D by using a normal-phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the petroleum ether-ethyl acetate solution with 8 concentration ratios and with sequentially increased ethyl acetate concentration is adopted in the gradient elution process to obtain 8 components;
F. high performance liquid chromatography separation: and E, separating and purifying the concentrated solution of the component 5 of the eluent in the step E by high performance liquid chromatography to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
In the invention, a novel chromone compound is obtained by researching plants in the genus of cassia and separating and preparing active ingredients from the plants in the genus of cassia. The chromone compound is determined by nuclear magnetic resonance and other spectrum technology measuring methods, and the specific structure of the chromone compound is characterized as follows:
Figure BDA0003014424540000031
further, the preparation method of the chromone compound, wherein the step of separating and purifying the concentrated solution of the 5 th component of the eluent in the step E by high performance liquid chromatography comprises the following steps:
and E, loading the concentrated solution of the 5 th component of the eluent in the step E into a high performance liquid chromatography column, taking 45-65% methanol as a mobile phase, taking a reversed-phase preparation column with the flow rate of 8-14 ml/min and the flow rate of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 45-60 mu L of sample each time, collecting chromatographic peaks for 15-33 min, accumulating for multiple times and evaporating to dryness to obtain the chromone compound.
Further, in the preparation method of the chromone compound, the organic solvent adopted for extracting the extract in the step A is 80-100% of acetone, ethanol or methanol.
Further, in the preparation method of the chromone compound, in the step B, the organic solvent adopted for extracting the extract a is ethyl acetate, chloroform, diethyl ether, petroleum ether or benzene.
Further, the preparation method of the chromone compound is characterized in that the mixed organic solvent in the step C is n-hexane-acetone, chloroform-methanol, petroleum ether-acetone and petroleum ether-ethyl acetate.
Further, the volume ratio of the mixed organic solvent in the step C is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, and 0: 1.
The invention also provides application of the chromone compound in preparing a tobacco mosaic virus resistant medicament. The anti-tobacco mosaic virus medicine takes the chromone compound as the only active component.
Through the experiment of resisting tobacco mosaic virus, the compound has the inhibition rate of 28.7 +/-4.1%, has good activity of resisting the tobacco mosaic virus, and is similar to the inhibition rate (32.2 +/-3.2%) of a positive control nandinmycin. The test proves that the compound has good activity of resisting tobacco mosaic virus, and the compound has simple structure and good activity; can be used as a guiding compound of a tobacco mosaic virus resistant medicament.
Drawings
FIG. 1 is a compound
7-hydroxy-5-(3-hydroxNuclear magnetic resonance carbon spectrum of y-2-oxopropyl) -2-methyl-4H-chromen-4-one (C:)13C NMR)；
FIG. 2 is a compound
NMR spectrum of 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one (f: (F))1H NMR)；
FIG. 3 is a compound
The major HMBC (→) association of 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Detailed Description
In order to make the objects, features and advantages of the present invention comprehensible, specific embodiments accompanied with examples are described in detail below. Several embodiments of the invention are given in the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1
The preparation method of the chromone compound in the embodiment comprises the following steps:
a1, collecting dried branch, leaf and/or fruit of Cassia plant 6.5kg, pulverizing into 20 mesh powder, ultrasonic extracting with 80% acetone for 30min for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/4; standing, filtering out precipitates, and concentrating to 380g of extract a;
b1, adding 480g of water into the extract a, extracting for 5 times by using chloroform with the same volume as the water, combining extract phases, and concentrating under reduced pressure to 154g of extract B;
c1, packing 1300g of 200-mesh silica gel into a column, adding 450g of acetone into the extract b for dissolving, then adding 180g of 100-mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; gradient eluting with dichloromethane-ethyl acetate mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 8 components, wherein the eluate obtained by eluting 5 th component, namely dichloromethane-ethyl acetate mixed organic solvent at volume ratio of 7:3 is 60 g;
d1, loading the eluate of the 5 th component obtained by elution in the step C1 into a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three solutions with concentration ratios, the methanol concentrations of which are sequentially increased, in the gradient elution process, collecting the eluate of each part, concentrating, monitoring by TLC, and combining the same parts to obtain 3 components; in the step, the three concentration ratios are 20%, 40%, 70% and 100% in sequence, the obtained 3 components have different polarities, and the chromone compound only exists in the 1 st component;
e1, performing gradient elution on the eluent of the 1 st component in the step D1 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F1, eluting an organic solvent with the volume ratio of 7:3 in the step E1 to obtain an eluent, taking a methanol aqueous solution with the volume content of 45-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 8ml/min and the volume content of 21.2mm multiplied by 25cm and the volume content of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 50 mu L of sample each time, collecting a chromatographic peak for 21min, and evaporating to dryness after multiple accumulation to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxyphenyl) -2-methyl-4H-chromen-4-one.
Example 2
The preparation method of the chromone compound in the embodiment comprises the following steps:
a2, collecting dried branch, leaf and/or fruit of Cassia plant 3.3kg, pulverizing into 20 mesh powder, ultrasonic extracting with 100% ethanol for 2 times (30 min each time), and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/3; standing, filtering out precipitates, and concentrating to 280g of extract a;
b2, adding 280g of water into the extract a, extracting for 3 times by using chloroform with the same volume as the water, combining the extract phases, and concentrating under reduced pressure to obtain 110g of extract B;
c2, packing the extract b into a column by using 680g of 160-mesh silica gel, adding 150g of acetone for dissolving, then adding 90g of 80-mesh silica gel for sample mixing, and loading the mixture onto the column after sample mixing; gradient eluting with petroleum ether-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain eluate of 8 components, wherein the eluate of 5 th component, i.e. petroleum ether-acetone mixed organic solvent at volume ratio of 7:3, is 48 g;
d2, loading the eluent of the 5 th component in the step C2 on a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three solutions with concentration ratios, the methanol concentration of which is increased in sequence, in the gradient elution process, collecting the eluent of each part, concentrating, monitoring by TLC, and combining the same parts to obtain 3 components;
e2, performing gradient elution on the eluent of the 1 st component in the step D1 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F2, eluting an organic solvent with the volume ratio of 7:3 in the step E2 to obtain an eluent, taking a methanol water solution with the volume content of 40-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 8ml/min and the volume content of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 50 mu L of sample each time, collecting a chromatographic peak for 22min, and evaporating to dryness after multiple accumulation to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxypyr) -2-methyl-4H-chromen-4-one.
Example 3
The preparation method of the chromone compound in the embodiment comprises the following steps:
a3, collecting dried branch, leaf and/or fruit of Cassia plant 6.5kg, pulverizing into 30 mesh powder, ultrasonic extracting with 80% methanol for 30min each time for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitates, and concentrating to obtain 416g of extract a;
b3, adding 460g of water into the extract a, extracting for 4 times by using ether with the same volume as the water, combining extraction phases, and concentrating under reduced pressure to obtain 243g of extract B;
c3, packing 1400g of 180-mesh silica gel into a column, adding 340g of acetone into the extract b for dissolution, then adding 250g of 90-mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; gradient eluting with dichloromethane-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 8 components, wherein the eluate of the 5 th component, namely dichloromethane-acetone mixed organic solvent at volume ratio of 7:3 is 50 g;
d3, loading the eluent of the 3 rd component in the step C3 into a reversed-phase column by using a reversed-phase material ODS (oxide dispersion strengthened) column, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three concentration ratios of solutions with sequentially increased methanol concentration in the gradient elution process, collecting and concentrating the eluent of each part, monitoring by TLC (thin layer chromatography), and combining the same parts to obtain 3 components;
e3, performing gradient elution on the eluent of the 1 st component in the step D3 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F3, eluting an organic solvent with the volume ratio of 7:3 in the step E3 to obtain an eluent, taking a methanol water solution with the volume content of 40-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 10ml/min and the volume content of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 50 mu L of sample each time, collecting chromatographic peaks for 24min, and evaporating to dryness after multiple accumulation to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxypyr) -2-methyl-4H-chromen-4-one.
Example 4
The preparation method of the chromone compound in the embodiment comprises the following steps:
a4, collecting dried branch, leaf and/or fruit of Cassia plant 5.5kg, coarse pulverizing to 40 mesh, extracting with 90% ethanol for 3 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/4; standing, filtering out precipitates, and concentrating to obtain 420g of extract a;
b4, adding 490g of water into the extract a, extracting for 4 times by using petroleum ether with the same volume as the water, combining the extract phases, and concentrating under reduced pressure to 260g of extract B;
c4, packing 1400g of 160-mesh silica gel into a column, adding 300g of acetone into the extract b for dissolving, then adding 260g of 80-mesh silica gel for sample mixing, and loading the mixture onto the column after sample mixing; gradient eluting with chloroform-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 8 components, wherein the eluate obtained by eluting 5 th component, namely chloroform-acetone mixed organic solvent at volume ratio of 7:3, is 55 g;
d4, loading the 5 th component in the step C3 into a reversed phase column by using a reversed phase material C-8, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three solutions with concentration ratios with sequentially increased methanol concentration in the gradient elution process, collecting and concentrating eluent of each part, monitoring by TLC, and combining the same parts to obtain 3 components;
e4, performing gradient elution on the eluent of the 1 st component in the step D4 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F4, eluting an organic solvent with the volume ratio of 7:3 in the step E3 to obtain an eluent, taking a methanol water solution with the volume content of 40-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 10ml/min and the volume content of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, collecting a chromatographic peak for 20min, and evaporating to dryness after multiple accumulation to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxypyropyl) -2-methyl-4H-chromen-4-one.
Example 5
The preparation method of the chromone compound in the embodiment comprises the following steps:
a5, collecting dried branch, leaf and/or fruit of Cassia plant 5.7kg, pulverizing into 20 mesh powder, ultrasonic extracting with 70% methanol for 35min for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitates, and concentrating to obtain 310g of extract a;
b3, adding 500g of water into the extract a, extracting for 5 times by using benzene with the same volume as the water, combining the extract phases, and concentrating under reduced pressure to obtain 120g of extract B;
c5, loading 850g of 200-mesh silica gel into a column, adding 130g of acetone into the extract b for dissolution, then adding 120g of 100-mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; gradient eluting with petroleum ether-chloroform mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 8 components, wherein the eluate obtained from 5 th component (petroleum ether-chloroform mixed organic solvent system at volume ratio of 7: 3) is 58 g;
d5, loading the eluent of the 5 th component obtained by elution in the step C1 into a reversed-phase column by using a reversed-phase material ODS (oxide dispersion strengthened) column, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three concentration ratios of solutions with sequentially increased methanol concentration in the gradient elution process, collecting and concentrating the eluent of each part, monitoring by TLC (thin layer chromatography), and combining the same parts to obtain 3 components;
e5, performing gradient elution on the eluent of the 1 st component in the step D1 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F5, eluting an organic solvent with the volume ratio of 7:3 in the step E5 to obtain an eluent, taking a methanol water solution with the volume content of 40-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 12ml/min and the thickness of 21.2mm multiplied by 25cm and the thickness of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, collecting a chromatographic peak for 21min, accumulating for multiple times and evaporating to dryness to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxypyropyl) -2-methyl-4H-chromen-4-one.
Example 6
The preparation method of the chromone compound in the embodiment comprises the following steps:
a6, pulverizing dried branch, leaf and/or fruit of Cassia plant 11kg into 20 mesh coarse powder, ultrasonic extracting with 100% ethanol for 2 times (30 min each time), and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/3; standing, filtering out precipitate, and concentrating to 1300g extract a;
b6, adding 1300g of water into the extract a, extracting for 3 times by using chloroform with the same volume as the water, combining the extract phases, and concentrating under reduced pressure to 550g of extract B;
c6, loading 3500g of 160-mesh silica gel into a column, adding 540g of acetone into the extract b for dissolution, then adding 550g of 80-mesh silica gel for sample mixing, and loading the mixture onto the column after sample mixing; gradient eluting with n-hexane-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 8 components, wherein the 5 th component is 120g of n-hexane-acetone mixed organic solvent at volume ratio of 7: 3;
d6, loading the eluate of the 5 th component obtained by elution in the step C6 into a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three solutions with concentration ratios, the methanol concentrations of which are sequentially increased, in the gradient elution process, collecting the eluate of each part, concentrating, monitoring by TLC, and combining the same parts to obtain 3 components;
e6, performing gradient elution on the eluent of the 1 st component in the step D6 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F6, eluting an organic solvent with the volume ratio of 7:3 in the step E6 to obtain an eluent, taking a methanol water solution with the volume content of 40-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 13ml/min and the volume content of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 50 mu L of sample each time, collecting chromatographic peaks for 16min, and evaporating to dryness after multiple accumulation to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxypyr) -2-methyl-4H-chromen-4-one.
Example 7
The preparation method of the chromone compound in the embodiment comprises the following steps:
a7, collecting dried branch, leaf and/or fruit of Cassia plant 1.2kg, pulverizing into 30 mesh powder, ultrasonic extracting with 80% methanol for 30min each time for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitates, and concentrating to obtain 130g of extract a;
b7, adding 130g of water into the extract a, extracting for 4 times by using ether with the same volume as the water, combining the extract phases, and concentrating under reduced pressure to obtain 45g of extract B;
c7, loading 450g of 180-mesh silica gel into a column, adding 110g of acetone into the extract b for dissolution, then adding 45g of 90-mesh silica gel for sample mixing, and loading the mixture onto the column after sample mixing; gradient eluting with dichloromethane-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, 0:1, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 8 components, wherein the 5 th component is 7g of dichloromethane-acetone mixed organic solvent at volume ratio of 7: 3;
d7, loading the eluent of the 5 th component in the step C7 on a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol water solution with the volume content of 20-100%, adopting three solutions with concentration ratios, the methanol concentration of which is increased in sequence, in the gradient elution process, collecting the eluent of each part, concentrating, monitoring by TLC, and combining the same parts to obtain 3 components;
e7, performing gradient elution on the eluent of the 1 st component in the step D1 by using a normal phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the volume ratio of the petroleum ether-ethyl acetate solution in the gradient elution process is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2 and 0:1, so as to obtain 8 components;
and F7, eluting an organic solvent with the volume ratio of 7:3 in the step E7 to obtain an eluent, eluting the eluent by using a methanol aqueous solution with the volume content of 40-60% as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 12ml/min and the size of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by using an ultraviolet detector, injecting 50 mu L of the sample each time, collecting chromatographic peaks for 20min, and evaporating the chromatographic peaks after multiple accumulation to dryness to obtain the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Example 8
The compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one prepared in example 1 was taken as a light brown powder;
the determination method comprises the following steps: nuclear magnetic resonance, in combination with other spectroscopic techniques, was used to identify structures.
(1) Ultraviolet spectrum (methanol as solvent), lambdamax(logε)：296(3.29),252(3.98)nm；
(2) Infrared spectrum (Potassium bromide tablet) vmax 3419，2971，2928，1648，1622，1577，1396，1273，1160，944，840，601cm–1；
(3) HRESIMS shows that the ion peak of the quasi-molecule of the compound of the invention is M/z 249.0765[ M + H]+(calculated 249.0757), combined13C and1h NMR spectrum (FIG. 1 and FIG. 2, the data of carbon spectrum and hydrogen spectrum are shown in Table 1) shows that the molecular formula is C13H12O5。1H NMR(400MHz，CD3OD) and13CNMR(100MHz,CD3OD) data, see table 1.
HRESIMS shows that the peak of the excimer ion is the peak M/z 249.0765[ M + H ]]+(calculated 249.0757), combined13C NMR spectrum to confirm the molecular formula as C13H12O5The unsaturation degree is 8. The infrared spectrum showed hydroxyl groups (3419 cm)-1) Conjugated carbonyl group (1648 cm)-1) The absorption peak of (1). In that13In C-NMR, C-4 (. delta.)C179.3) also confirmed the presence of conjugated carbonyl groups. Of the compound1H-NMR data showed that it contained 1 methyl group (. delta.)H2.02), 2 methylene groups (. delta.))H4.54,4.94),1 alkene hydrocarbon (. delta.)H6.03) and 1 meta-substituted phenyl [ delta ]H 7.09(1H,d,J＝2.3Hz),7.01(1H,d,J＝2.3Hz)]. In addition to the compound13C-NMR and DEPT spectra (Table 1) show 13 carbon signals, including 2 carbonyl carbons (. delta.)C209.15,179.28), 6 aromatic carbons (. delta.)C 162.57,160.67,139.27,120.63,114.87,103.42) 2 olefin carbons (. delta.)C164.72,110.80), 2 methylene groups (. delta.))C45.47,69.54, 1 of which is oxymethylene) and 1 methyl group (. delta. )C19.47). The above spectral data indicate that the compound is a chromone compound, and is similar to the spectral data of the known compound 5-acetyl-7-hydroxy-2-methyl chromone. Except that 5-acetyl-7-hydroxy-2-methyl chromone has one acetonyl group connected to C-5 position, but one more hydroxyl group connected to the acetonyl group. In the HMBC spectrum of the compound, H-3 (. delta.)H6.03) and C-2 (. delta.))C164.72)，C-10(δC 114.87)，C-14(δC19.47) has a clear correlation signal, H-6 (delta)H7.01) and C-8 (. delta.)C 103.42)，C-10(δC 114.87)，C-11(δC45.47) correlation, H-8 (. delta.))H7.09) and C-6 (. delta.))C 120.63)，C-9(δC 160.67)，C-10(δC114.87), H-14 (delta)H2.02) and C-2 (. delta.))C 164.72)，C-3(δC110.80) has obvious correlation signal to determine the connection position of methyl as C-2, H-11 (delta)H4.54) and C-5 (. delta.))C139.27)，C-12(δC209.15) has significant correlation signals that confirm the 3-hydroxy-acetonyl linkage position as C-5, H-13 (. delta.))H4.94) and C-12 (. delta.))C209.15) had a clear correlation signal confirming that the methylene group is attached to C-12. The structure of this compound is now defined and named 7-hydroxy-5- (3-hydroxy-2-oxopyryl) -2-methyl-4H-chromen-4-one.
Example 9
The compound prepared in example 2 was taken as a light brown powder; the structure determination was carried out as in example 8, with the results: the structure is the same as that of example 8, and the molecular formula is C13H12O5. It was confirmed that the compound produced in example 2 was the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Example 10
The compound prepared in example 3 was taken as a light brown powder; the structure determination was carried out as in example 8, with the results: the structure is the same as that of example 8, and the molecular formula is C13H12O5. It was confirmed that the compound produced in example 3 was the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Of the compounds of Table 11H and13c NMR data (400/100MHz, C5D5N)
Figure BDA0003014424540000121
Example 11
The compound prepared in example 4 was taken as a light brown powder; the structure determination was carried out as in example 8, with the results: the structure is the same as that of example 8, and the molecular formula is C13H12O5. It was confirmed that the compound produced in example 4 was the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Example 12
The compound prepared in example 5 was taken as a light brown powder; the structure determination was carried out as in example 8, with the results: the structure is the same as that of example 8, and the molecular formula is C13H12O5. It was confirmed that the compound produced in example 4 was the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Example 13
The compound prepared in example 6 was taken as a light brown powder; the structure determination was carried out as in example 8, with the results: the structure is the same as that of example 8, and the molecular formula is C13H12O5. It was confirmed that the compound produced in example 4 was the chromone compound 7-hydroxy-5- (3-hydroxy-2-oxopropyl) -2-methyl-4H-chromen-4-one.
Example 14
The compound prepared in example 7 was taken as a light brown powder; the structure determination was carried out as in example 8, with the results: the structure is the same as that of example 8, and the molecular formula is C13H12O5. It was confirmed that the compound prepared in example 4 was the chromone compound 7-hydroxy-5- (3-hy)droxy-2-oxopropyl)-2-methyl-4H-chromen-4-one。
Example 15
Any chromone compound prepared in the embodiments 1-7 is used for an activity detection test of tobacco mosaic virus resistance, and the test conditions are as follows:
the test host is TMV dry spot host tobacco Nicotiana luteinosa L, TMV system infects host tobacco Nicotiana tabacum L.K326, and the test host is cultivated in an insect-proof greenhouse.
Testing a toxic source: tobacco mosaic virus (TMV, U1 strain) was stored on common cigarette K326 by Nicotiana tabacum chemical Key laboratory, Yunnan province.
Virus purification: with reference to the method of Gooding et al, a few modifications are made. The typical symptomatic leaves are centrifuged with differential speed, PEG precipitated and centrifuged with 10% -40% sucrose discontinuous density gradient to purify the virus. The purified virus is determined to have a mass concentration of 20mg/mL by ultraviolet scanning
Figure BDA0003014424540000131
Figure BDA0003014424540000132
The purified virus was stored at-20 ℃ and diluted to 32. mu.g/mL with 0.01M PBS before use.
And (3) determination of infection inhibition: local scorch method is adopted. Test compounds were dissolved in DMSO and diluted to the desired concentration with distilled water, ningnanmycin was used as a positive control. Healthy and exuberant-growing heart-leaf tobacco in 5-6 leaf stages is selected, the left half leaf is inoculated with a mixed solution of a compound and viruses in equal volume, and the right half leaf is inoculated with a mixed solution of distilled water (containing a small amount of DMSO) and viruses in equal volume to serve as a negative control. After inoculation, the cells were rinsed with water. 4-5 leaves are inoculated in each treatment, the treatment is repeated for 3 times, the number of the scorched spots is counted after 3-4 days, and the inhibition rate is calculated. Specifically, the inhibition ratio was (control number of dead spots-number of treated dead spots)/control number of dead spots X100%
Test results
The chromone compound is separated for the first time, is determined to be the chromone compound by a nuclear magnetic resonance and mass spectrometry method, and represents the specific structure of the chromone compound. Through the experiment of resisting tobacco mosaic virus, the inhibition rate reaches 28.7 +/-4.1%, and the tobacco mosaic virus resisting activity is very good and is similar to the inhibition rate (32.2 +/-3.2%) of a positive control nanningmycin. The results show that the compound has good application prospect in the preparation of the anti-tobacco mosaic virus medicine. The compound has simple structure and good activity, and can be used as a guiding compound of a tobacco mosaic virus resistant medicament.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. A chromone compound having the chemical structure:
Figure FDA0003014424530000011
2. a method for producing a chromone compound of claim 1, comprising the steps of:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of a cassia plant to 20-40 meshes, performing ultrasonic extraction for 2-4 times by using an organic solvent for 30-60 min each time, combining extracting solutions, filtering the extracting solutions, concentrating the extracting solutions under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain a first extract;
B. organic solvent extraction: adding water into the obtained extract a, wherein the weight of the added water is 1-2 times of the weight of the extract, extracting for 3-5 times by using an organic solvent with the same volume as that of the water, combining organic solvent extraction phases, and concentrating under reduced pressure to obtain an extract b;
C. silica gel column chromatography: dissolving the extract b by using an organic solvent with the weight of 1.5-3 times that of the extract b, mixing the sample by using 200-300 meshes of silica gel with the weight of 0.8-1.2 times that of the extract b, performing silica gel column chromatography, wherein the silica gel filled in the column is 200-300 meshes, and the using amount of the silica gel filled in the column is 6-8 times that of the extract b; gradient eluting with two mixed organic solvents in a volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. reversed-phase column chromatography: c, performing reverse phase column chromatography on the eluent after the same parts are combined in the step C, wherein the reverse phase column is filled with a reverse phase material; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluents of all parts and concentrating by adopting three solutions with concentration ratios with sequentially increased methanol concentration in the gradient elution process, monitoring by TLC, and combining the same parts to obtain 3 components;
E. performing gradient elution on the 1 st component of the eluent obtained in the step D by using a normal-phase silica gel column, wherein the eluent is petroleum ether-ethyl acetate solution, and the petroleum ether-ethyl acetate solution with 8 concentration ratios and with sequentially increased ethyl acetate concentration is adopted in the gradient elution process to obtain 8 components;
F. high performance liquid chromatography separation: and E, separating and purifying the concentrated solution of the 5 th component of the eluent in the step E by using high performance liquid chromatography to obtain the chromone compound.
3. The process for producing a chromone compound according to claim 2, wherein the step of separating and purifying the concentrate of the 5 th component of the eluate from step E by high performance liquid chromatography comprises:
and E, loading the concentrated solution of the 5 th component of the eluent in the step E into a high performance liquid chromatography column, taking 45-65% methanol as a mobile phase, taking a reversed-phase preparation column with the flow rate of 8-14 ml/min and the flow rate of 21.2mm multiplied by 25cm and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 45-60 mu L of sample each time, collecting chromatographic peaks for 15-33 min, and evaporating to dryness after multiple accumulation.
4. The method for producing a chromone compound according to claim 2, wherein the organic solvent used for the extraction of the extract in step a is 80 to 100% acetone, ethanol, or methanol.
5. The method for producing a chromone compound according to claim 2, wherein the organic solvent used for extraction of the extract a in step B is ethyl acetate, chloroform, diethyl ether, petroleum ether, or benzene.
6. The method of producing a chromone compound in claim 2, wherein the mixed organic solvent in step C is n-hexane-acetone, chloroform-methanol, petroleum ether-acetone, or petroleum ether-ethyl acetate.
7. The method of producing a chromone compound according to claim 2, wherein the volume ratio of the mixed organic solvent in step C is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:2, or 0: 1.
8. Use of the chromone compound of claim 1 in the preparation of a medicament against tobacco mosaic virus.
9. The use of a chromone compound in the preparation of a medicament against tobacco mosaic virus according to claim 8, wherein the medicament against tobacco mosaic virus has the chromone compound as the only active ingredient.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HU, QIU-FEN,ET AL: "Antiviral Chromones from the Stem of Cassia siamea", 《JOURNAL OF NATURAL PRODUCTS 》 *
HU, QIU-FEN,ET AL: "Chromones from the twigs of Cassia fistula and their anti-tobacco mosaic virus activities", 《HETEROCYCLES》 *
LI, YINKE,ET AL: "Chromones from the stems of Cassia fistula and their anti-tobacco mosaic virus activities", 《PHYTOCHEMISTRY LETTERS 》 *

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