CN102786530B - Plant flavanoid compound, preparation method and application thereof - Google Patents
Plant flavanoid compound, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a plant flavanoid compound, a preparation method and application of the flavanoid compound and application. The flavanoid compound is separated from tobacco, and is named as fistulaflavonoidC, has a molecular formula as C18H1606; and the flavanoid compound has the structure in the description; the preparation method of the flavanoid compound comprises the steps of extracting, silica column chromatography and high-pressure liquid-phase chromatographic separation, and specifically comprises the steps of crushing a plant sample, ultrasonically extracting via ethanol, combining the extractive liquids, filtering, and releasing pressure to concentrate to obtain an extract; packing the extract through 200 to 300 meshes of silica column, carrying out silica column chromatography, and then carrying out gradient elute through a chloroform-methanol solution based on volume ratio of chloroform to methanol being 20: 1 to 1: 1, combining the same parts, collecting the eluent of each part, and condensing; and carrying out high-pressure liquid phase chromatographic separation and purifying on the part of the eluent where the volume ratio of chloroform to methanol is 9: 1 so as to obtain the flavanoid compound. Tests show that the compound has high resistance to activity of tobacco mosaic virus. The compound provided by the invention has a simple structure, is easy to artificially synthesize, has high activity, and can be served as a piloting compound for resisting the activity of tobacco mosaic virus.
Description
Technical field
The invention belongs to vegetable chemistry technical field, be specifically related to a kind of plant that derives from, especially derive from the flavonoid compound and preparation method thereof and application of tobacco.
Background technology
A Bole (formal name used at school:
cassia fistula), have another name called golden anxious rain, laburnum, gold rain, Persian Chinese honey locust, Fructus cassiae fistulae (Cassia fistula L.), long fruit tree, Cassia fistula L., ox horn tree etc., Hong Kong claims chitling beans more, is a kind of plant of Caesalpinoidea.A Bole is extensively in the torrid zone and subtropical zone plantation, except can doing landscape tree or shade tree, its function cure mainly into profit just, strong muscle, opens retardance, diarrhea.For halitosis, have sore throat, the hot dysentery causing of enteron aisle, sacroiliitis, stimulate the menstrual flow.
A Bole pericarp is containing flavones (flavonoids), anthraquinone (anthraquinones), chromone (chromones), alkaloid (alkaloids), sterol (sterols), triterpene (triterpenes), half-dried seed is containing a large amount of free fatty acidies (free fatty acid), wax (waxes) and hydrocarbon polymer.Pulp is containing unsaturated wax, aloin (barbaloin), the glycoside of hydroxyl methoxyl group anthraquinone, and 11 seed amino acid, as arginine (arginine), leucine (leuine), methionine(Met) (methionine), phenylalanine (phenylalanine), tryptophane (tryptophan), aspartic acid (aspartic acid), L-glutamic acid (glutamic acid).
Flavones (chromone) is the biologically active substance that a class occurring in nature extensively exists, and because flavone component structure type in plant is many, stereochemistry is complicated, has multiple biological activity, very active to the research in this field both at home and abroad; No matter be naturally occurring, or the flavonoid compound that obtains of synthetic, chemist's extensive concern all caused.
Summary of the invention
The first object of the present invention is to provide a kind of vegetable flavonoid; The second object is to provide the preparation method of described vegetable flavonoid; The 3rd object is to provide described vegetable flavonoid preparing the application in resisting tobacco mosaic virus medicine.
The first object of the present invention is achieved in that the separation from tobacco of described vegetable flavonoid obtains, called after fistulaflavonoid C, and its molecular formula is C
18h
16o
6, there is following structure:
The present invention's the second object is achieved in that and comprises extraction, silica gel column chromatography, high pressure liquid chromatography separating step, specifically comprises:
A, medicinal extract extract: plant sample is crushed to 20 ~ 40 orders, and with 60 ~ 80% ethanol ultrasonic extraction 2 ~ 4 times, each 4 ~ 8h, extracting solution merges, and filters, and concentrating under reduced pressure becomes medicinal extract;
B, silica gel column chromatography: medicinal extract carries out silica gel column chromatography with 200 ~ 300 order silica gel dress posts of 2 ~ 5 times of amounts of weight ratio; The chloroform-methanol solution that the volume proportion of take is 20:1 ~ 1:1 carries out gradient elution, merges identical part, collects each several part elutriant concentrated;
C, high pressure liquid chromatography separation: the 9:1 part of B step elutriant further obtains described flavonoid compound with high pressure liquid chromatography separation and purification.
The structure of the flavonoid compound of preparing with aforesaid method is to measure out by the following method:
The compounds of this invention is yellow; UV spectrum (solvent is methyl alcohol),
λ max(log
ε) 348 (2.28), 297 (4.21), 210 (4.69) nm; Infrared spectra (pressing potassium bromide troche)
ν max3384,2962,2876,1625,1530,1483,1422,1269,1038,872 cm
-1; High resolution mass spectrum (HRESIMS, Fig. 3) provides quasi-molecular ion peak
m/z[351.0852 M+Na]
+(calculated value 301.1076).In conjunction with
1h and
13c NMR spectrum provides a molecular formula C
18h
16o
6, degree of unsaturation is 11.From
1in H NMR spectrogram, can find out a structure of pterocarpan
δ h4.28 (dd,
j=4.6,10.5 Hz, H-2
α),
δ h3.51 (t,
j=10.5 Hz, H-2
β),
δ h3.44 (m, H-3), and
δ h5.42 (d,
j=6.6 Hz, H-4
β), simultaneously from hydrogen spectrum
δ h6.94 (d,
j=8.5 Hz, H-5),
δ h6.60 (d,
j=8.5 Hz, H-6),
δ h6.51 (d,
j=8.1 Hz, H-5 ￠), and
δ h6.73 (d,
j=8.1 Hz, H-6 ￠), also can see 7,8,3 ￠, the aromatic ring structure fragment that 4 ￠ replace.In carbon spectrum, can see that this compound has 17 carbon atoms, comprise 1 methoxyl group, 2 methylene radical, 6 methynes and 8 unhydrided carbon.HMBC
δ hin spectrogram 3.80 (OMe) and
δ c150.8 (C-4 ') are relevant, (
δ h3.80) and C-3 ' (
δ c132.1) relevant, show that 2 methoxyl groups are connected to C-4 ' and C-3 '.
δ h5.86,5.91 (OCH
2o-) with
δ c146.7 (C-7), and
δ cthe relevant proof of 134.0 (C-8) methylenedioxy group is connected to C-7 and C-8, and therefore, remaining 1 hydroxyl must be connected to C-3 '.
In addition, from document and biological regularity, the H-3 on B/C ring and H-4 are generally cis, for example
α,
αor
β,
β.General by the analysis of CD (circular dichroism) and ORD (optical rotatory dispersion), optically-active is (-), and H-3 and H-4 are generally
α,
α(3
r, 4
r), optically-active is (+), H-3 and H-4 are generally
β,
β(3
s, 4
s).Therefore, this compound optically-active is (+), and the absolute configuration that shows H-3 and H-4 is (3
s, 4
s).So far the structure of compound is confirmed, and compound is named as: fistulaflavonoid C.
The compound of table-1.
1h NMR and
13(solvent is CD to C NMR data
3oD)
The present invention's the 3rd object be achieved in that vegetable flavonoid preparation to the application in resisting tobacco mosaic virus medicine.
Vegetable flavonoid of the present invention is separated first from Caesalpinoidea plant A Bole, has determined for flavonoid compound, and characterized its concrete structure by nucleus magnetic resonance and measuring method of mass spectrum.The relative inhibition of this compound of evidence is 31.2%, and the relative inhibition 24.5% far above contrast Ningnanmycin, illustrates that compound has good activity of resisting tobacco mosaic virus.The compounds of this invention is simple in structure, and synthetic is easily realized, and the activity of compound is good; Can be used as the guiding compound of activity of resisting tobacco mosaic virus.
Accompanying drawing explanation
Fig. 1 be the compounds of this invention proton nmr spectra (
1h NMR) figure.
Fig. 2 be the compounds of this invention carbon-13 nmr spectra (
13c NMR) figure.
Fig. 3 is the main HMBC correlogram of the compounds of this invention.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further illustrated, but never in any form the present invention is limited, and any conversion or the improvement based on training centre of the present invention, done, all fall into protection scope of the present invention.
Vegetable flavonoid C of the present invention
18h
16o
6preparation method, comprise extraction, silica gel column chromatography, high pressure liquid chromatography separating step, specifically comprise:
It is that plant sample is crushed to 20 ~ 40 orders that described medicinal extract extracts, with 60 ~ 80% ethanol ultrasonic extraction 2 ~ 4 times, and each 4 ~ 8h, extracting solution merges, and filters, and concentrating under reduced pressure becomes medicinal extract;
Described silica gel column chromatography is that medicinal extract is carried out to silica gel column chromatography with 200 ~ 300 order silica gel dress posts of 2 ~ 5 times of amounts of weight ratio; The chloroform-methanol solution that the volume proportion of take is 20:1 ~ 1:1 carries out gradient elution, merges identical part, collects each several part elutriant concentrated;
Described high pressure liquid chromatography separation is that the 9:1 part of elutriant is further obtained to target flavonoid compound with high pressure liquid chromatography separation and purification.
Described extraction alcohol concn used is 65 ~ 75%.
The described supersound extraction time is 5 ~ 7h.
Described medicinal extract, before silica gel column chromatography rough segmentation, weighs 80 ~ 100 order silica gel silica gel mixed samples of 2 ~ 3 times with medicinal extract after 80 ~ 100% dissolve with methanol by 1.5 ~ 3 times of amounts of weight ratio, further removes impurity.
Described chloroform-methanol volume proportion is 20:1,9:1,8:2,7:3,6:4,5:5.
Described high pressure liquid chromatography separation and purification is that the methanol-water that employing volume proportion is 7:3 is moving phase, 20 * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 10 ~ 14mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 180 ~ 220 mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness.
Material after described high pressure lipuid chromatography (HPLC) separation and purification is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with gel filtration chromatography, with further separation and purification.
What described gel filtration chromatography was used is Sephadex LH-20 gel column, and the gel column of other types can be realized object of the present invention equally.
Flavonoid compound application of the present invention is to the application in resisting tobacco mosaic virus medicine in preparation.
The present invention is raw materials used not limited by area and kind, is not limited only to tobacco yet, all can realize object of the present invention, take below to derive from Yunnan Dehong tobacco leaf the present invention is described further as example as raw materials, but the present invention is not done to any restriction:
test example 1---compound extracts with separated
Plant sample is adopted in Dehong county, Yunnan, A Bole complete stool is sampled to 4.5 kg and be crushed to 20～40 orders, and with ethanol supersound extraction 3 times of 70%V/V, each 6 hours, extracting solution merged, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 364 g.After total appropriate methyl alcohol medicinal extract for (medicinal extract weight 1.5～3 times) dissolves, with silica gel (80～100 order), mix sample, silica gel (200～300 order) fills post and carries out silica gel column chromatography.Chloroform-methanol (20:1,9:1,8:2,7:3,6:4,5:5) gradient elution, TLC monitoring merges identical part, obtains 6 parts, wherein chloroform-acetone (9:1) wash-out is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 45% the methyl alcohol of take is moving phase, the C of (20 mm * 250 mm, 5mm)
18preparative column is stationary phase, and flow velocity is 12 mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 200mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
the evaluation of test example 2---compound:
The compounds of this invention is yellow; UV spectrum (solvent is methyl alcohol), λ
max(log ε) 348 (2.28), 297 (4.21), 210 (4.69) nm; Infrared spectra (pressing potassium bromide troche)
ν max3384,2962,2876,1625,1530,1483,1422,1269,1038,872 cm
-1; High resolution mass spectrum (HRESIMS, Fig. 3) provides quasi-molecular ion peak
m/z[351.0852 M+Na]
+(calculated value 301.1076).In conjunction with
1h and
13c NMR spectrum provides a molecular formula C
18h
16o
6, degree of unsaturation is 11.From
1in H NMR spectrogram, can find out a structure of pterocarpan
δ h4.28 (dd,
j=4.6,10.5 Hz, H-2
α),
δ h3.51 (t,
j=10.5 Hz, H-2
β),
δ h3.44 (m, H-3), and
δ h5.42 (d,
j=6.6 Hz, H-4
β), simultaneously from hydrogen spectrum
δ h6.94 (d,
j=8.5 Hz, H-5),
δ h6.60 (d,
j=8.5 Hz, H-6),
δ h6.51 (d,
j=8.1 Hz, H-5 ￠), and
δ h6.73 (d,
j=8.1 Hz, H-6 ￠), also can see 7,8,3 ￠, the aromatic ring structure fragment that 4 ￠ replace.In carbon spectrum, can see that this compound has 17 carbon atoms, comprise 1 methoxyl group, 2 methylene radical, 6 methynes and 8 unhydrided carbon.HMBC
δ hin spectrogram 3.80 (OMe) and
δ c150.8 (C-4 ') are relevant, (
δ h3.80) and C-3 ' (
δ c132.1) relevant, show that 2 methoxyl groups are connected to C-4 ' and C-3 '.
δ h5.86,5.91 (OCH
2o-) with
δ c146.7 (C-7), and
δ cthe relevant proof of 134.0 (C-8) methylenedioxy group is connected to C-7 and C-8, and therefore, remaining 1 hydroxyl must be connected to C-3 '.
In addition, from document and biological regularity, the H-3 on B/C ring and H-4 are generally cis, for example
α,
αor
β,
β.General by the analysis of CD (circular dichroism) and ORD (optical rotatory dispersion), optically-active is (-), and H-3 and H-4 are generally
α,
α(3
r, 4
r), optically-active is (+), H-3 and H-4 are generally
β,
β(3
s, 4
s).Therefore, this compound optically-active is (+), and the absolute configuration that shows H-3 and H-4 is (3
s, 4
s).
The compound of table-1.
1h NMR and
13(solvent is CD to C NMR data
3oD)
test example 3---activity of resisting tobacco mosaic virus detects.
Adopt half leaf method, when the mass concentration of medicament is 50mg/L, the compounds of this invention is carried out to activity of resisting tobacco mosaic virus mensuration.5～6 age flue-cured tobacco plant on, choose be applicable to test blade (leaf is capable normal, anosis without worm), first blade is evenly sprinkled to fine emery powder, with writing brush, standby tobacco mosaic virus (TMV) source (3.0 * 10-3) is evenly put on the blade sprinkled with silicon carbide, after the blade of all middle choosings connects poison and finishes, be placed on immediately and in the culture dish that fills liquid, process 20 min, take out, spill the globule and about liquid on defoliation sheet, two and half leaves are restored and to be emitted on the enamel son of an influential official that is covered with toilet paper moisturizing and to add cover glass, temperature control (23 ± 2) ℃, be placed on greenhouse natural light irradiation, 2～3 d are visible withered spot. each is processed and establishes second half leaf for contrast, be provided with in addition 1 group be commodity Ningnanmycin processing as a comparison, press formula and calculate relative inhibition.
XI％=(CK-T)／CK×100％
X: relative inhibition (%), CK: be soaked in the withered spot number (individual) that half sheet in clear water connects malicious leaf, T is soaked in the withered spot number (individual) that half sheet in liquid connects malicious leaf.
The relative inhibition of bright compound of result is 31.2%, and the relative inhibition 24.5% far above contrast Ningnanmycin, illustrates that compound has good activity of resisting tobacco mosaic virus.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
embodiment 1
Plant sample is adopted in Dehong county, Yunnan, and Turkish tobaccos complete stool is sampled to 4.5 kg and be crushed to 20 orders, supersound extraction 3 times of the ethanol with 70%, each 6h, extracting solution merges, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 364 g.After 90% dissolve with methanol that medicinal extract use weight ratio is 3 times, with the 80 order silica gel mixed samples of 3 times, the 300 order silica gel dress posts of 2 times carry out silica gel column chromatography.With volume proportion, be 20:1,9:1,8:2,7:3,6:4, the chloroform-methanol solution gradient wash-out of 5:5, TLC monitoring merges identical part, obtains 6 parts, chloroform-acetone wash-out that wherein volume proportion is 9:1 is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 45% the methyl alcohol of take is moving phase, 20 mm * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 12mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 200mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
Embodiment 2
Plant sample is adopted in Dehong county, Yunnan, and Turkish tobaccos complete stool sampling 3kg is crushed to 40 orders, supersound extraction 4 times of the ethanol with 60%, and each 4h, extracting solution merges, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 278g.After 95% dissolve with methanol that medicinal extract use weight ratio is 1.5 times, with the 100 order silica gel mixed samples of 2 times, the 200 order silica gel dress posts of 3 times carry out silica gel column chromatography.With volume proportion, be 20:1,9:1,8:2,7:3,6:4, the chloroform-methanol solution gradient wash-out of 5:5, TLC monitoring merges identical part, obtains 6 parts, chloroform-acetone wash-out that wherein volume proportion is 9:1 is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 40% the methyl alcohol of take is moving phase, 20 mm * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 10mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 180mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
Embodiment 3
Plant sample is adopted in Dehong county, Yunnan, and Turkish tobaccos complete stool sampling 3.5kg is crushed to 40 orders, supersound extraction 2 times of the ethanol with 65%, and each 5h, extracting solution merges, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 321g.Medicinal extract with after 100% dissolve with methanol of 2 times of weight ratios with the 90 order silica gel mixed samples of 2.5 times,
the 250 order silica gel dress posts of 2.5 times carry out silica gel column chromatography.With volume proportion, be 20:1,9:1,8:2,7:3,6:4, the chloroform-methanol solution gradient wash-out of 5:5, TLC monitoring merges identical part, obtains 6 parts, chloroform-acetone wash-out that wherein volume proportion is 9:1 is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 50% the methyl alcohol of take is moving phase, 20 mm * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 14mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 220mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
Embodiment 4
Plant sample is adopted in Dehong county, Yunnan, and Turkish tobaccos complete stool sampling 5kg is crushed to 30 orders, supersound extraction 3 times of the ethanol with 75%, and each 7h, extracting solution merges, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 479g.After 85% dissolve with methanol that medicinal extract use weight ratio is 3 times, with the 100 order silica gel mixed samples of 2 times, the 200 order silica gel dress posts of 2.5 times carry out silica gel column chromatography.With volume proportion, be 20:1,9:1,8:2,7:3,6:4, the chloroform-methanol solution gradient wash-out of 5:5, TLC monitoring merges identical part, obtains 6 parts, chloroform-acetone wash-out that wherein volume proportion is 9:1 is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 50% the methyl alcohol of take is moving phase, 20 mm * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 11mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 190mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
Plant sample is adopted in Dehong county, Yunnan, and Turkish tobaccos complete stool sampling 4.5kg is crushed to 40 orders, supersound extraction 3 times of the ethanol with 80%, and each 8h, extracting solution merges, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 396g.The 100 order silica gel mixed samples of 1.5 times after 80% dissolve with methanol that medicinal extract use weight ratio is 2.5 times, the 300 order silica gel dress posts of 2 times carry out silica gel column chromatography.With volume proportion, be 20:1,9:1,8:2,7:3,6:4, the chloroform-methanol solution gradient wash-out of 5:5, TLC monitoring merges identical part, obtains 6 parts, chloroform-acetone wash-out that wherein volume proportion is 9:1 is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 45% the methyl alcohol of take is moving phase, 20 mm * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 12 mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 200mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
Embodiment 6
Plant sample is adopted in Dehong county, Yunnan, and Turkish tobaccos complete stool sampling 5.2kg is crushed to 30 orders, supersound extraction 3 times of the ethanol with 80%, and each 6h, extracting solution merges, filters, and concentrating under reduced pressure becomes medicinal extract, obtains total medicinal extract 492g.After 85% dissolve with methanol that medicinal extract use weight ratio is 3 times, with the 100 order silica gel mixed samples of 2 times, the 200 order silica gel dress posts of 5 times carry out silica gel column chromatography.With volume proportion, be 20:1,9:1,8:2,7:3,6:4, the chloroform-methanol solution gradient wash-out of 5:5, TLC monitoring merges identical part, obtains 6 parts, chloroform-acetone wash-out that wherein volume proportion is 9:1 is partly separated by the prompt logical sequence 1,100 half preparation high pressure liquid chromatography of peace, 50% the methyl alcohol of take is moving phase, 20 mm * 250 mm, the C of 5mm
18preparative column is stationary phase, and flow velocity is 11mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 190mL, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness; Gained material is used pure dissolve with methanol again, then take pure methyl alcohol as moving phase, separated with Sephadex LH-20 gel filtration chromatography, gets final product to obtain target compound.
Embodiment 7
The compound of getting embodiment 1 preparation is yellow.
Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique, identify structure.
1) UV spectrum (solvent is methyl alcohol),
λ max(log
ε) 348 (2.28), 297 (4.21), 210 (4.69) nm;
2) infrared spectra (pressing potassium bromide troche)
ν max3384,2962,2876,1625,1530,1483,1422,1269,1038,872 cm
-1;
3) high resolution mass spectrum (HRESIMS, Fig. 3) provides quasi-molecular ion peak
m/z[351.0852 M+Na]
+(calculated value 301.1076).In conjunction with
1h and
13c NMR spectrum provides a molecular formula C
18h
16o
6, degree of unsaturation is 11.
From
1in H NMR spectrogram, can find out a structure of pterocarpan
δ h4.28 (dd,
j=4.6,10.5 Hz, H-2
α),
δ h3.51 (t,
j=10.5 Hz, H-2
β),
δ h3.44 (m, H-3), and
δ h5.42 (d,
j=6.6 Hz, H-4
β), simultaneously from hydrogen spectrum
δ h6.94 (d,
j=8.5 Hz, H-5),
δ h6.60 (d,
j=8.5 Hz, H-6),
δ h6.51 (d,
j=8.1 Hz, H-5 ￠), and
δ h6.73 (d,
j=8.1 Hz, H-6 ￠), also can see 7,8,3 ￠, the aromatic ring structure fragment that 4 ￠ replace.In carbon spectrum, can see that this compound has 17 carbon atoms, comprise 1 methoxyl group, 2 methylene radical, 6 methynes and 8 unhydrided carbon.HMBC
δ hin spectrogram 3.80 (OMe) and
δ c150.8 (C-4 ') are relevant, (
δ h3.80) and C-3 ' (
δ c132.1) relevant, show that 2 methoxyl groups are connected to C-4 ' and C-3 '.
δ h5.86,5.91 (OCH
2o-) with
δ c146.7 (C-7), and
δ cthe relevant proof of 134.0 (C-8) methylenedioxy group is connected to C-7 and C-8, and therefore, remaining 1 hydroxyl must be connected to C-3 '.
In addition, from document and biological regularity, the H-3 on B/C ring and H-4 are generally cis, for example
α,
αor
β,
β.General by the analysis of CD (circular dichroism) and ORD (optical rotatory dispersion), optically-active is (-), and H-3 and H-4 are generally
α,
α(3
r, 4
r), optically-active is (+), H-3 and H-4 are generally
β,
β(3
s, 4
s).Therefore, this compound optically-active is (+), and the absolute configuration that shows H-3 and H-4 is (3
s, 4
s).So far the structure of compound is confirmed, and compound is named as: fistulaflavonoid C.
Embodiment 8
The compound of getting embodiment 2 preparations is yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 2 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 9
The compound of getting embodiment 3 preparations is yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 3 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 10
The compound of getting embodiment 4 preparations is yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 4 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 11
The compound of getting embodiment 5 preparations is yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 5 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 12
The compound of getting embodiment 6 preparations is yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 6 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 13
The activity of resisting tobacco mosaic virus of arbitrary chromocor compound that embodiment 1 ~ 6 is prepared detects:
Adopt half leaf method, when the mass concentration of medicament is 50mg/L, the compounds of this invention is carried out to activity of resisting tobacco mosaic virus mensuration.5～6 age flue-cured tobacco plant on, choose be applicable to test blade (leaf is capable normal, anosis without worm), first blade is evenly sprinkled to fine emery powder, with writing brush, standby tobacco mosaic virus (TMV) source (3.0 * 10-3) is evenly put on the blade sprinkled with silicon carbide, after the blade of all middle choosings connects poison and finishes, be placed on immediately and in the culture dish that fills liquid, process 20 min, take out, spill the globule and about liquid on defoliation sheet, two and half leaves are restored and to be emitted on the enamel son of an influential official that is covered with toilet paper moisturizing and to add cover glass, temperature control (23 ± 2) ℃, be placed on greenhouse natural light irradiation, 2～3 d are visible withered spot. each is processed and establishes second half leaf for contrast, be provided with in addition 1 group be commodity Ningnanmycin processing as a comparison, press formula and calculate relative inhibition.
XI％=(CK-T)／CK×100％
X: relative inhibition (%), CK: be soaked in the withered spot number (individual) that half sheet in clear water connects malicious leaf, T is soaked in the withered spot number (individual) that half sheet in liquid connects malicious leaf.
Result shows that the relative inhibition of this compound is 31.2%, and the relative inhibition 24.5% far above contrast Ningnanmycin, illustrates that compound has good activity of resisting tobacco mosaic virus.
Claims (10)
1. a vegetable flavonoid, is characterized in that separation obtains from tobacco, called after fistulaflavonoid C, and its molecular formula is C
18h
16o
6, there is following structure:
。
2. a preparation method for vegetable flavonoid described in claim 1, is characterized in that comprising extraction, silica gel column chromatography, high pressure liquid chromatography separating step, specifically comprises:
A, medicinal extract extract: plant sample is crushed to 20 ~ 40 orders, and with 60 ~ 80% ethanol ultrasonic extraction 2 ~ 4 times, each 4 ~ 8h, extracting solution merges, and filters, and concentrating under reduced pressure becomes medicinal extract; Described plant sample is the Turkish tobaccos complete stool of adopting in Dehong county, Yunnan;
B, silica gel column chromatography: medicinal extract carries out silica gel column chromatography with 200 ~ 300 order silica gel dress posts of 2 ~ 5 times of amounts of weight ratio; The chloroform-methanol solution that the volume proportion of take is 20:1 ~ 1:1 carries out gradient elution, merges identical part, collects each several part elutriant concentrated;
C, high pressure liquid chromatography separation: the 9:1 part of B step elutriant further obtains described flavonoid compound with high pressure liquid chromatography separation and purification.
3. the preparation method of vegetable flavonoid according to claim 2, is characterized in that in described A step, alcohol concn is 65 ~ 75%.
4. the preparation method of vegetable flavonoid according to claim 2, is characterized in that in described A step that the supersound extraction time is 5 ~ 7h.
5. the preparation method of vegetable flavonoid according to claim 2, it is characterized in that in described B step that medicinal extract is before silica gel column chromatography rough segmentation, with weigh 80 ~ 100 order silica gel silica gel mixed samples of 2 ~ 3 times after 80 ~ 100% dissolve with methanol of 1.5 ~ 3 times of amounts of weight ratio with medicinal extract, further remove impurity.
6. the preparation method of vegetable flavonoid according to claim 2, is characterized in that in described B step, chloroform-methanol volume proportion is 20:1,9:1,8:2,7:3,6:4,5:5.
7. the preparation method of vegetable flavonoid according to claim 2, is characterized in that the separation and purification of described C step mesohigh liquid chromatography is that to adopt the methanol-water that volume proportion is 40 ~ 50:50 ~ 60 be moving phase, 20 * 250 mm, the C of 5 μ m
18preparative column is stationary phase, and flow velocity is 10 ~ 14mL/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 180 ~ 220 μ L, the chromatographic peak of collection 26.4 min, repeatedly cumulative rear evaporate to dryness.
8. the preparation method of vegetable flavonoid according to claim 2, it is characterized in that the material after the separation and purification of described C step mesohigh liquid phase chromatography uses pure dissolve with methanol again, take pure methyl alcohol as moving phase again, separated with gel filtration chromatography, with further separation and purification.
9. the preparation method of vegetable flavonoid according to claim 8, what it is characterized in that described gel filtration chromatography uses is Sephadex LH-20 gel column.
10. described in a claim 1, vegetable flavonoid is being prepared the application in resisting tobacco mosaic virus medicine.
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| CN103664862B (en) * | 2013-08-27 | 2015-10-14 | 云南烟草科学研究院 | Polyphenolic compound and its preparation method and application in a kind of Turkish tobaccos |
| CN104370874B (en) * | 2014-08-06 | 2016-05-25 | 云南中烟工业有限责任公司 | A kind of parallel heptatomic ring biphenyl compound and its preparation method and application |
| CN104292202B (en) * | 2014-09-15 | 2015-12-09 | 云南民族大学 | A kind of flavonoid compound and its preparation method and application |
| CN104497000B (en) * | 2014-12-15 | 2016-06-01 | 云南中烟工业有限责任公司 | A kind of resisting tobacco mosaic virus vegetable flavonoid and its preparation method and application |
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| CN102584845A (en) * | 2011-12-30 | 2012-07-18 | 云南烟草科学研究院 | Furan flavonoid compound in nicotiana tobacum and application thereof |
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| CN102584845A (en) * | 2011-12-30 | 2012-07-18 | 云南烟草科学研究院 | Furan flavonoid compound in nicotiana tobacum and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Flavonoids from Tephrosia aequilata;Paul K. Tarus, et al.;《Phytochemistry》;20021231;第60卷;375-379 * |
| JOHNL.INGHAM et al..TEPHROCARPIN |
| Paul K. Tarus, et al..Flavonoids from Tephrosia aequilata.《Phytochemistry》.2002,第60卷375-379. |
| TEPHROCARPIN, A PTEROCARPAN PHYTOALEXIN FROM TEPHROSIA BIDWILL AND A STRUCTURE PROPOSAL FOR ACANTHOCARPAN;JOHN L. INGHAM, et al.;《Phytochemistry》;19821231;第21卷(第12期);2969-2972 * |
| 耿召良,等.植物源抗烟草花叶病毒天然产物研究进展.《中国烟草科学》.2011,第32卷(第1期),84-91. * |
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