CN109265503B - Flavonoid compound and preparation method and application thereof - Google Patents
Flavonoid compound and preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
Abstract
The invention discloses a flavonoid compound and a preparation method and application thereof, wherein the molecular formula of the flavonoid compound is C24H24O10The compound is named kaempferol-3-O- [ (4 ', 5' -O-isopropylidene) -a-L-rhamnopyranoside]Having the following structural formula:
the preparation method of the flavonoid compound is characterized in that dried branches, leaves or fruits of an arbor plant of leguminosae cassia are used as raw materials, and the flavonoid compound is obtained by the steps of extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation. The application is the application of the flavonoid compound in the neuraminidase inhibitory activity. Through the experiments of neuraminidase inhibitory activity, oseltamivir carboxylate is used as a control, kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]IC for neuraminidase50Value of 187.40μmol/L, it has better neuraminidase inhibitory activity. The compound has simple structure and good activity, can be used as a leading compound of an anti-influenza virus medicament, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of extraction of effective components of plants, and particularly relates to a flavonoid compound and a preparation method and application thereof.
Background
Cassia genus of Caesalpinia subfamily of Leguminosae (A. Megaeae)Cassia LOver 600 plants, widely distributed in tropical and subtropical regions of the earth, especially in tropical america and tropical africa, are common throughout the world. There are 25 species in our country, which are widely distributed in each province of south, and all species are distributed in Yunnan. The main chemical components of Cassia plant are triterpene, anthraquinone, steroid, alkaloid, flavone, and stilbene; the compounds have anticancer, anti-HIV, anti-hepatitis, insecticidal, anti-anxiety and anti-tranquilization activities. In recent years, the active new compounds found by domestic researchers from the plants of the genus cassia Dai are as follows: an alkaloid having antimalarial activity; chromone compounds; cycloartane triterpenes and saponins, anthraquinones and lignans having anti-HIV-1 activity; xanthones and polyketones; and chromone, aurone and flavonoid compounds with activity against tobacco mosaic virus and HIV-1. In order to more effectively utilize the Caesalpinia subfamily plant resources in China and search for active ingredients with development prospects, systematic active ingredient research work is selected to be carried out on the Caesalpinia subfamily plants.
Influenza (flu) is an upper respiratory disease caused by influenza virus, affecting the life health of millions of people worldwide each year, and thus posing serious economic and social problems to some extent. However, until now, the current medical level of human beings has always lacked a safe and effective control method for influenza viruses. Neuraminidase is a key enzyme in the life cycle of influenza A and B viruses, and is an ideal target in the development of anti-influenza virus medicaments. Neuraminidase inhibitors are anti-influenza virus drugs, are not easy to generate drug resistance, have good tolerance, have inhibition effect on influenza A and B viruses, have great significance for clinical treatment by researching high-efficiency and low-toxicity neuraminidase inhibitors, and have wide market prospect.
The invention separates a new flavone compound from leguminous plants, and the compound has better neuraminidase inhibitory activity.
Disclosure of Invention
The first purpose of the invention is to provide a flavonoid compound; the second purpose is to provide a preparation method of the flavonoid compound; the third purpose is to provide the application of the flavonoid compound in preparing anti-influenza virus medicines.
The first purpose of the invention is realized by that the flavonoid compound is obtained by using dried branches, leaves or fruits of an arbor plant of leguminosae cassia as a raw material 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 C24H24O10The compound is named kaempferol-3-O- [ (4 ', 5' -O-isopropylidene) -a-L-rhamnopyranoside]Having the following structural formula:
the second purpose of the invention is realized by that the preparation method of the flavonoid compound is obtained by using dried branches, leaves or fruits of an arbor plant of leguminosae cassia as a raw material and performing extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of an arbor plant of the genus Cassia of the family Leguminosae to 20-40 meshes, ultrasonically extracting for 30-60 min each time for 2-4 times by using an organic solvent, and mixing extracting solutions; filtering the extracting solution, concentrating the extracting solution under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain an extract a;
B. organic solvent extraction: adding 1-2 times of water by weight into the extract a, extracting for 3-5 times by using an organic solvent with the same volume as 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 ratio of 1.5-3 times, mixing the sample by using 100-200 meshes of silica gel with the weight of 0.8-1.2 times of the extract, and 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 is 6-8 times of the weight of the extract b; gradient eluting with a mixed organic solvent with 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: subjecting the eluate obtained by eluting with organic solvent at ratio of 4:1 to reverse phase column chromatography, wherein the reverse phase column is filled with reverse phase material C-18, C-8 or ODS; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluent of each part, concentrating, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: separating and purifying an eluent obtained by eluting with 30-50% methanol aqueous solution by volume through high performance liquid chromatography to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ];
F. and E, performing high performance liquid chromatography separation and purification by taking 30-50% methanol as a mobile phase, taking a reverse phase preparation column with the flow rate of 3ml/min and the flow rate of 21.2 x 250 mm as a stationary phase and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by using an ultraviolet detector, feeding 20-50 mu L of sample each time, collecting chromatographic peaks for 10-30 min, and evaporating to dryness after accumulating for multiple times. So as to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ].
The flavonoid compound is separated for the first time, the structure of the flavonoid compound prepared by the method is determined by the following method, and the specific structure is characterized by:
the compound is light yellow amorphous powder; ultraviolet spectrum (the solvent is methanol),λ max(log ε): 345 (3.76), 265 (3.91) nm; infrared spectrum (Potassium bromide tablet) vmax 3425, 1656, 1609, 1507, 1450, 1361, 1176, 1086, 839 cm–1(ii) a HRESIMS shows the peak of the excimer of the compound of the inventionm/z 472.4369 [M]+(calculated 472.4404), combined13C（CD3OD, 100 MHz) and1H NMR (CD3OD, 400 MHz) spectrum (FIG. 1)FIG. 2, attribution of the hydrogen spectrum data of carbon spectrum in Table 1) shows that the molecular formula is C24H24O10。
HRESIMS shows that the peak of the excimer ion ism/z472.4369 [M]+(calculated 472.4404), combined13C NMR spectrum to confirm the molecular formula as C24H24O10The unsaturation degree was 13. The infrared spectrum showed that the compound contained hydroxyl groups (3425 cm)-1) Carbonyl group (1656 cm)-1) And phenyl (1609 cm)-1And 1507 cm-1) And the like. The ultraviolet spectrum has maximum absorption at 265 nm and 345 nm, and the compound is supposed to be flavonoid compound1H-and13C-NMR data are assigned, see Table 1. The presence of two coupled signals at the A ring of the flavoneδ H6.33 (1H, s, H-8) and 6.15 (1H, s, H-6)]And deducing the B ring [ 2 ], [ H-2 '/H-6' and [ H-3 '/H-5' symmetryδ H 7.75 (2H, d, J = 8.0 Hz, H-2 ', 6') and 6.88 (2H, d,J = 8.0 Hz, H-3′, 5′)]is a benzene ring A2X2The spin system, furthermore, was characterized by chemical shifts of 5.66 (1H, s, H-1 '') and 0.73 (3H, d,Jproton signal of = 4.0 Hz, H-9 '') and HMBC spectra (FIG. 1),δ5.66 (H-1 '') sugar terminal hydrogen signals withδ135.5 (C-3) carbon signals, suggesting that one is linked to the 3-position of flavoneα-a rhamnose. Of the compound13The C NMR spectrum (table 1) shows the presence of 24 carbon signals (two of which are symmetric carbons,δ C 132.1 andδ C c-2'/C-6' and C-3'/C-5' at 116.7. the 24 carbon signals include 15 carbons on the flavone backbone, 6 carbons on the rhamnopyranose and 3 carbons on the dioxyisopropyl group [ alpha ], [ betaδ C 110.5 (s, C-6 ''), 26.7 (q, C-7 ''), 28.5 (q, C-8 ''). By consulting the literature and comparing the relevant data, the compound is found to be kaempferol-3-O-aL-rhamnopyranoside is most similar, differing by chemical shift changes of the rhamnose and additional dioxo-isopropyl signal. In that1Conversion to H-5 '' in H NMR spectrumThe chemical shifts increased from 3.99 to 4.47 (. DELTA. + 0.48 ppm) and the chemical shifts of H-4 "increased from 3.52 to 3.90 (. DELTA. + 0.38 ppm).1In the C NMR spectrum, the chemical shift of C-1 "decreased from 102.3 to 99.8 (. DELTA. -2.5 ppm) and the chemical shift of C-3" increased from 71.6 to 74.8 (. DELTA. + 3.2 ppm), the chemical shift of C-4 "increased from 71.1 to 79.6 (. DELTA. + 8.5 ppm) and the chemical shift of C-5" increased from 70.8 to 77.3 (. DELTA. + 6.5 ppm); in the HMBC spectrum (figure 1),δ3.90（H-4′′），δ4.47（H-5′′），δ1.33（H-7′′），δ1.40 (H-8 '') hydrogen signal withδThe 110.5 (C-6 '') carbon signal is related, suggesting that the 4 '' and 5 '' positions of the rhamnopyranose are linked to a dioxyisopropylidene group. Based on the above, the structure of the compound can be deduced, and the compound is determined to be kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside] 。
The third purpose of the invention is realized by the application of the flavonoid compound in preparing anti-influenza virus medicines. Through the experiments of neuraminidase inhibitory activity, oseltamivir carboxylate is used as a control, kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]IC for neuraminidase50Value of 187.40μmol/L, it has better neuraminidase inhibitory activity. The compound has simple structure and good activity, can be used as a leading compound of an anti-influenza virus medicament, and has good application prospect.
Drawings
FIG. 1 shows the compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranosi de]Nuclear magnetic resonance carbon spectrum of (13C NMR）；
FIG. 2 shows the compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranosi de]Nuclear magnetic resonance hydrogen spectrum of (1H NMR）；
FIG. 3 is a graph of the major HMBC (→) association of the compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranosi de ].
Detailed Description
The invention is further described with reference to the accompanying drawings, but the invention is not limited in any way, and any alterations or modifications based on the teaching of the invention are within the scope of the invention.
The flavonoid compound is obtained by taking dried branches, leaves or fruits of an arbor plant of leguminosae cassia as a raw material 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 C24H24O10Named kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]Having the following structural formula:
the second purpose of the invention is realized by that the preparation method of the flavonoid compound is obtained by using dried branches, leaves or fruits of an arbor plant of leguminosae cassia as a raw material and performing extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of an arbor plant of the genus Cassia of the family Leguminosae to 20-40 meshes, ultrasonically extracting for 30-60 min each time for 2-4 times by using an organic solvent, and mixing extracting solutions; filtering the extracting solution, concentrating the extracting solution under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain an extract a;
B. organic solvent extraction: adding 1-2 times of water by weight into the extract a, extracting for 3-5 times by using an organic solvent with the same volume as 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 ratio of 1.5-3 times, mixing the sample by using 100-200 meshes of silica gel with the weight of 0.8-1.2 times of the extract, and 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 is 6-8 times of the weight of the extract b; gradient eluting with a mixed organic solvent with 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: subjecting the eluate obtained by eluting with organic solvent at ratio of 4:1 to reverse phase column chromatography, wherein the reverse phase column is filled with reverse phase material C-18, C-8 or ODS; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluent of each part, concentrating, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: separating and purifying an eluent obtained by eluting with 30-50% methanol aqueous solution by volume through high performance liquid chromatography to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ];
F. and E, performing high performance liquid chromatography separation and purification by taking 30-50% methanol as a mobile phase, taking a reverse phase preparation column with the flow rate of 3ml/min and the flow rate of 21.2 x 250 mm as a stationary phase and the size of 5 mu m as a stationary phase, detecting the wavelength of 254nm by using an ultraviolet detector, feeding 20-50 mu L of sample each time, collecting chromatographic peaks for 10-30 min, and evaporating to dryness after accumulating for multiple times. So as to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ].
The flavonoid compound disclosed by the invention is applied to preparation of anti-influenza virus medicines.
The leguminous plants disclosed by the invention are not limited by regions and varieties and can be realized.
Example 1
Pulverizing dried branch, leaf and/or fruit of arbor of Leguminosae 12kg into 20 mesh coarse powder, ultrasonic extracting with 90% ethanol water 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 obtain 2767g of extract a; adding 2760g 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 1430g of extract b; loading 14000g of 200-mesh silica gel into a column, adding 1000g of methanol into the extract b for dissolving, then adding 1400g of 100-mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; gradient eluting with dichloromethane-methanol mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, 0:1 respectively, collecting gradient eluent, concentrating, monitoring by TLC, mixing the same parts to obtain 9 parts, wherein the volume ratio of dichloromethane-methanol mixed organic solvent eluent c is 25 g; loading the eluate C on a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC, and combining the same parts; and (2) taking an eluent obtained by eluting with 30-50% of methanol aqueous solution by volume, taking 40% of methanol as a mobile phase, taking an Agilent Zorbax SB-C18 reversed phase preparation column with the flow rate of 3ml/min, the thickness of 22 multiplied by 250 mm and the thickness of 5 mu m as a stationary phase, taking a sample of 50 mu L each time with the detection wavelength of an ultraviolet detector of 254nm, collecting a chromatographic peak for 19 min, accumulating for multiple times and evaporating to dryness to obtain the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isoproylene) -a-L-rhamnopyranoside ].
Example 2
Collecting dried branch, leaf and/or fruit of arbor of Leguminosae 4.8kg, coarse pulverizing to 20 mesh, ultrasonic extracting with 100% ethanol for 30min for 3 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/3; standing, filtering out precipitates, and concentrating to 560g of extract a; adding 560g of water into the extract a, extracting for 3 times by using chloroform with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 320g of extract b; 4000g of 200-mesh 300-mesh silica gel is used for filling a column, 340g of ethyl acetate is added into the extract b for dissolving, 320g of 100-mesh silica gel is added for sample mixing, and the mixture is loaded on the column after the sample mixing; gradient eluting with dichloromethane-ethyl acetate mixed organic solvent at volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; 16g of dichloromethane-ethyl acetate mixed organic solvent eluent c in the volume ratio of 6: 4; loading the eluate C on a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC, and combining the same parts; and (2) taking an eluent obtained by eluting with a methanol aqueous solution with the volume content of 30-60%, taking 35% methanol as a mobile phase, taking an Agilent Zorbax SB-C18 reversed-phase preparation column with the flow rate of 3ml/min, the size of 22 multiplied by 250 mm and the size of 5 mu m as a stationary phase, taking 30 mu L of ultraviolet detector sample introduction each time, collecting a chromatographic peak for 26min, accumulating for multiple times, and evaporating to dryness to obtain the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isoproylene) -a-L-rhamnopyranoside ].
Example 3
Taking 6.5kg of dried branches, leaves and/or fruits of arbor of Leguminosae, coarsely pulverizing to 30 mesh, ultrasonically extracting with 80% methanol for 30min for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitate, and concentrating to obtain 675g extract a; adding 700g of water into the extract a, extracting for 4 times by using ether with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 342g of extract b; 2800g of 180-mesh silica gel is filled into a column, 930g of acetone is added into the extract b for dissolution, 360g of 90-mesh silica gel is added for sample mixing, and the mixture is loaded on 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, 3:2, 1:1, 1:2, and 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; the volume ratio of the chloroform-acetone mixed organic solvent eluent c is 45 g; loading a reversed-phase material ODS into a column, loading the eluent c into the reversed-phase column, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluent of each part, monitoring by TLC, and combining the same parts; taking an eluent obtained by eluting with 40-60% of methanol aqueous solution by volume content, taking 45% of methanol as a mobile phase, taking an Agilent Zorbax SB-C18 reversed phase preparation column with the flow rate of 3ml/min, the thickness of 22 multiplied by 250 mm and the thickness of 5 mu m as a stationary phase, taking a sample of 50 mu L each time by an ultraviolet detector, collecting a chromatographic peak for 13min, accumulating for multiple times and evaporating to dryness to obtain the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isoproylene) -a-L-rhamnopyranoside ].
Example 4
Collecting dried branch, leaf and/or fruit of arbor of Leguminosae 5.9kg, 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 810g of extract a; 880g of water is added into the extract a, petroleum ether with the same volume as the water is used for extraction for 4 times, extraction phases are combined, and pressure reduction and concentration are carried out to obtain 265g of extract b; 1450g of 160-mesh silica gel is filled into a column, 290g of methanol is added into the extract b for dissolution, 265g of 80-mesh silica gel is added for sample mixing, and the mixture is loaded into the column after the sample mixing; gradient eluting with petroleum ether-ethyl acetate mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; 52g of eluent c of petroleum ether-ethyl acetate mixed organic solvent with the volume ratio of 1: 2; loading the eluate C on a reversed-phase column by using a reversed-phase material C-8, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC, and combining the same parts; and (2) taking an eluent obtained by eluting with 30-50% of methanol aqueous solution by volume content, taking 43% methanol as a mobile phase, taking an Agilent Zorbax SB-C18 reversed phase preparation column with the flow rate of 3ml/min, the size of 22 multiplied by 250 mm and the size of 5 mu m as a stationary phase, collecting a chromatographic peak of 16min, accumulating for multiple times and evaporating to dryness to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ].
Example 5
Collecting dried branch, leaf and/or fruit of arbor of Leguminosae 5.6 kg, coarse pulverizing to 20 mesh, 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 700g of extract a; adding 1400g of water into the extract a, extracting for 5 times by using benzene with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 310g of extract b; loading 200 mesh silica gel 1860g into a column, adding 420g of methanol into the extract b for dissolving, then adding 310g of 100 mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; performing gradient elution with chloroform-methanol mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; the volume ratio of chloroform-methanol mixed organic solvent eluent c is 56 g; loading a reversed-phase material ODS into a column, loading the eluent c into the reversed-phase column, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluent of each part, monitoring by TLC, and combining the same parts; taking an eluent obtained by eluting with 30-50% methanol aqueous solution by volume content, taking 370% methanol as a mobile phase, taking an Agilent Zorbax SB-C18 reversed phase preparation column with the flow rate of 3ml/min, the size of 22 multiplied by 250 mm and the size of 5 mu m as a stationary phase, collecting a chromatographic peak of 22min, accumulating for a plurality of times and evaporating to dryness to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ].
Example 6
The compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ] prepared in example 1 was taken as a pale yellow amorphous 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 (the solvent is methanol),λ max（logε）：345 (3.76), 265 (3.91)nm；
(2) infrared spectrum (Potassium bromide tablet) vmax 3425, 1656, 1609, 1507, 1450, 1361, 1176, 1086, 839 cm–1；
(3) HRESIMS shows that the peak of the excimer of the compound ism/z472.4369 [M]+(calculated 472.4404), combined13C and1the H NMR spectrum (FIGS. 1 and 2, attribution of hydrogen spectrum data in carbon spectrum in Table 1) gave a molecular formula C24H24O10。1H NMR(400 MHz,CD3OD) and13C NMR(100 MHz,CD3OD) data, see table 1.
HRESIMS shows that the peak of the excimer ion ism/z472.4369 [M]+(calculated 472.4404), combined13C NMR spectrum to confirm the molecular formula as C24H24O10The unsaturation degree was 13. The infrared spectrum showed that the compound contained hydroxyl groups (3425 cm)-1) Carbonyl group (1656 cm)-1) And phenyl (1609 cm)-1And 1507 cm-1) And the like. The ultraviolet spectrum of the compound has maximum absorption at 265 nm and 345 nm, and the compound is supposed to be a flavonoid compoundCompound of (i) a1H-and13C-NMR data are assigned, see Table 1. The presence of two coupled signals at the A ring of the flavoneδ H6.33 (1H, s, H-8) and 6.15 (1H, s, H-6)]And deducing the B ring [ 2 ], [ H-2 '/H-6' and [ H-3 '/H-5' symmetryδ H 7.75 (2H, d, J = 8.0 Hz, H-2 ', 6') and 6.88 (2H, d,J = 8.0 Hz, H-3′, 5′)]is a benzene ring A2X2The spin system, furthermore, was characterized by chemical shifts of 5.66 (1H, s, H-1 '') and 0.73 (3H, d,Jproton signal of = 4.0 Hz, H-9 '') and HMBC spectra (FIG. 1),δ5.66 (H-1 '') sugar terminal hydrogen signals withδ135.5 (C-3) carbon signals, suggesting that one is linked to the 3-position of flavoneα-a rhamnose. Process for preparing compounds13The C NMR spectrum (table 1) shows the presence of 24 carbon signals (two of which are symmetric carbons,δ C 132.1 andδ C c-2'/C-6' and C-3'/C-5' at 116.7. the 24 carbon signals include 15 carbons on the flavone backbone, 6 carbons on the rhamnopyranose and 3 carbons on the dioxyisopropyl group [ alpha ], [ betaδ C 110.5 (s, C-6 ''), 26.7 (q, C-7 ''), 28.5 (q, C-8 ''). By consulting the literature and comparing the relevant data, the compound is found to be kaempferol-3-O-aL-rhamnopyranoside is most similar, differing by chemical shift changes of the rhamnose and additional dioxo-isopropyl signal. In that1In the H NMR spectrum, the chemical shift of H-5 "increased from 3.99 to 4.47 (. DELTA. + 0.48 ppm) and the chemical shift of H-4" increased from 3.52 to 3.90 (. DELTA. + 0.38 ppm).1In the C NMR spectrum, the chemical shift of C-1 "decreased from 102.3 to 99.8 (. DELTA. -2.5 ppm) and the chemical shift of C-3" increased from 71.6 to 74.8 (. DELTA. + 3.2 ppm), the chemical shift of C-4 "increased from 71.1 to 79.6 (. DELTA. + 8.5 ppm) and the chemical shift of C-5" increased from 70.8 to 77.3 (. DELTA. + 6.5 ppm); in the HMBC spectrum (figure 1),δ3.90（H-4′′），δ4.47（H-5′′），δ1.33（H-7′′），δ1.40 (H-8 '') hydrogen signal withδ110.5 (C-6 '') carbon signals are correlatedThis suggests that the 4 "and 5" positions of rhamnose are linked to a dioxoisopropylidene group. Based on the above, the structure of the compound can be deduced, and the compound is determined to be kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]。
Example 7
The compound prepared in example 2 was taken as a pale yellow amorphous powder; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C24H24O10. The compound prepared in example 2 was confirmed to be the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]。
Example 8
The compound prepared in example 3 was taken as a pale yellow amorphous powder; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C24H24O10. The compound prepared in example 3 was confirmed to be the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]。
Of the compounds of Table 11H and13c NMR data (400/100 MHz, CD)3OD)
Example 9
The compound prepared in example 4 was taken as a pale yellow amorphous powder; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C24H24O10. The compound prepared in example 4 was confirmed to be the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]。
Example 10
The compound prepared in example 5 was taken as a pale yellow amorphous powder; the structure determination was carried out as in example 6, with the results: it is composed ofThe structure is the same as example 6, the molecular formula is C24H24O10. The compound prepared in example 5 was confirmed to be the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]。
Example 11
Any flavonoid compound prepared in examples 1-5 is subjected to a neuraminidase inhibitory activity detection test, and the test conditions are as follows:
(a) activity measurement method
1. Preparation of positive control oseltamivir carboxylate solution
a. 5 granules of the oseltamivir capsule are taken, the capsule powder is evenly ground in a glass mortar, and the quality is precisely weighed.
b. Taking 25 mg of oseltamivir powder into a 25 mL volumetric flask, adding 15 mL of distilled water, and ultrasonically dissolving for 15 min.
c. Adding distilled water to constant volume of 25 mL, shaking, and centrifuging at 10000 r/min for 10 min.
d. 8 mL of the supernatant was transferred to a 20 mL beaker and 2 mL of 0.1 g/mL sodium hydroxide solution was added.
e. After stirring at room temperature for 5 hours, the pH was adjusted to about 7.5 with glacial acetic acid.
2. Preparation of test Compound solution
Accurately weighing 1 mg of the compound to be detected, preparing 10% DMSO aqueous solution with the concentration of 1 mg/mL by using distilled water and DMSO, and dissolving by ultrasonic. Then diluting according to multiple ratio, respectively preparing into 200μg/mL，40μg/mL，8 μg/mL of aqueous solution for use.
3. Preparation of sample detection:
a. in a 96-well plate, 70 samples per well were applied using a microsyringeμl neuraminidase detection buffer.
b. 10 more per wellμl neuraminidase.
c. Adding 0-10 per holeμSamples of neuraminidase inhibitors to be screened.
d. Adding 0-10 per holeμThe total volume of the I Mili-Q water per well was 90μl。
4. And (3) detection:
a. vibrating and mixing for about l min.
b．37 oIncubation for 2min allowed full interaction of the inhibitor and neuraminidase.
c. Adding 10 per holeμl neuraminidase fluorogenic substrate.
d. Then vibrating and mixing for about 1 min.
e．37 oAnd C, performing fluorescence measurement after incubation for 20 min. The excitation wavelength was 360 nm and the emission wavelength was 440 nm.
The oseltamivir carboxylic acid is used as a control for each plate, and the sample adding amount is 0-10μl。
Results of the experiment
The inhibition rate was calculated according to the following formula:
inhibition rate ═ (enzyme activity fluorescence intensity-sample fluorescence intensity) ÷ (enzyme activity fluorescence intensity-blank)
The experimental results show that: through the experiments of neuraminidase inhibitory activity, oseltamivir carboxylate is used as a control, kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]IC for neuraminidase50Value of 187.40μmol/L, it has better neuraminidase inhibitory activity.
Neuraminidase inhibitory Activity of the Compounds of Table 2
| No. | IC50 (µM) |
| kaempferol-3-O-[(4′′, 5′′-O-isopropylidene)-a-L-rhamnopyranoside] | 187.40±1.02 |
| Oseltamivir carboxylate | 94.63±1.03 |
aAll data are expressed as mean ± SD (standard deviation), n = 3.
Claims (7)
1. A flavonoid compound is characterized in that the flavonoid compound is obtained by taking dried branches, leaves or fruits of an arbor plant of Leguminosae cassia as a raw material 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 C24H24O10Named kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside]Having the following structural formula:
2. a method for preparing flavonoids according to claim 1, which is characterized in that dried branches, leaves or fruits of an arbor plant belonging to the genus Cassia of the family Leguminosae are used as raw materials, and the flavonoids are obtained by extraction of extractum, extraction with an organic solvent, silica gel column chromatography and high pressure liquid chromatography separation, and specifically comprise the following steps:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of an arbor plant of the genus Cassia of the family Leguminosae to 20-40 meshes, ultrasonically extracting for 30-60 min each time for 2-4 times by using an organic solvent, and mixing extracting solutions; filtering the extracting solution, concentrating the extracting solution under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain an extract a;
B. organic solvent extraction: adding 1-2 times of water by weight into the extract a, extracting for 3-5 times by using an organic solvent with the same volume as 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 ratio of 1.5-3 times, mixing the sample by using 100-200 meshes of silica gel with the weight of 0.8-1.2 times of the extract, and 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 is 6-8 times of the weight of the extract b; gradient eluting with a mixed organic solvent with 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: subjecting the eluate obtained by eluting with organic solvent at ratio of 4:1 to reverse phase column chromatography, wherein the reverse phase column is filled with reverse phase material C-18, C-8 or ODS; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluent of each part, concentrating, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: separating and purifying an eluent obtained by eluting with 30-50% methanol aqueous solution by volume through high performance liquid chromatography to obtain the flavonoid compound kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ];
F. and E, performing high performance liquid chromatography separation and purification by taking 30-50% methanol as a mobile phase, taking a reversed phase preparation column with the flow rate of 3ml/min, the flow rate of 21.2 x 250 mm and the thickness of 5 mu m as a stationary phase, detecting the wavelength of 254nm by an ultraviolet detector, feeding 20-50 mu L of sample each time, collecting a chromatographic peak of 10-30 min, accumulating for multiple times, and evaporating to dryness to obtain the flavonoid kaempferol-3-O- [ (4 '', 5 '' -O-isopropylidene) -a-L-rhamnopyranoside ].
3. The method for producing a flavonoid compound according to claim 2, wherein: the organic solvent in the step A is 80-100% of acetone, ethanol or methanol.
4. The method for producing a flavonoid compound according to claim 2, wherein: and the organic solvent in the step B is ethyl acetate, chloroform, diethyl ether, petroleum ether or benzene.
5. The method for preparing flavonoids according to claim 2, wherein the mixed organic solvent in step C is dichloromethane-methanol, dichloromethane-ethyl acetate, petroleum ether-acetone or petroleum ether-ethyl acetate.
6. The method for preparing flavonoids compounds 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, 3:2, 1:1, 1:2, 0: 1.
7. Use of a flavonoid compound as claimed in claim 1 in the preparation of a medicament for the treatment of influenza.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4211772A (en) * | 1977-06-02 | 1980-07-08 | Laboratoires Sarget | Benzopyran glycoside acetals and ketals |
| CN105566306A (en) * | 2014-10-14 | 2016-05-11 | 四川农业大学 | Preparation of flavone derivative and application to neuraminidase inhibition |
| CN105769894A (en) * | 2014-12-15 | 2016-07-20 | 房学迅 | Applications of rutin in preparing medicines for preventing and treating influenza A |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4211772A (en) * | 1977-06-02 | 1980-07-08 | Laboratoires Sarget | Benzopyran glycoside acetals and ketals |
| CN105566306A (en) * | 2014-10-14 | 2016-05-11 | 四川农业大学 | Preparation of flavone derivative and application to neuraminidase inhibition |
| CN105769894A (en) * | 2014-12-15 | 2016-07-20 | 房学迅 | Applications of rutin in preparing medicines for preventing and treating influenza A |
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| Title |
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| Inhibitors of Nitric Oxide Production from the Bark of Myrica rubra: Structures of New Biphenyl Type Diarylheptanoid Glycosides and Taraxerane Type Triterpene;Jing Tao，等;《Bioorganic & Medicinal Chemistry》;20021231;第4005-4012页 * |
| The Flavonoid Fraction from Rhoeo discolor Leaves Acting as Antiviral Against Influenza A Virus Antiviral Against Influenza A Virus;Yazmin Sánchez-Roque，等;《Rec. Nat. Prod.》;20170707;第11卷(第6期);第532-546页 * |
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