CN102786530A - 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 the 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., the chitling beans are claimed in Hong Kong more, are a kind of plants 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 is opened retardance, diarrhea.Be used for halitosis, have sore throat, the hot dysentery that causes of enteron aisle, sacroiliitis, stimulate the menstrual flow.
The A Bole pericarp contains flavones (flavonoids); Anthraquinone (anthraquinones), chromone (chromones), vegeto-alkali (alkaloids); Sterol (sterols); Triterpene (triterpenes), half-dried seed contain a large amount of free fatty acids (free fatty acid), wax (waxes) and hydrocarbon polymer.Pulp contains unsaturated wax, aloin (barbaloin), the glycoside of hydroxyl methoxyl group anthraquinone, and 11 seed amino acids; Like l-arginine (arginine), leucine (leuine), methionine(Met) (methionine); Phenylalanine(Phe) (phenylalanine); Tryptophane (tryptophan), aspartic acid (aspartic acid), L-glutamic acid (glutamic acid).
Flavones (chromone) is the biologically active substance that one type of occurring in nature extensively exists, because the flavone component structure type is many in the plant, stereochemistry is complicated, has multiple biological activity, and is 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
First purpose of the present invention is to provide a kind of vegetable flavonoid; Second purpose is to provide the preparation method of said vegetable flavonoid; The 3rd purpose is to provide said vegetable flavonoid preparing the application in the resisting tobacco mosaic virus medicine.
First purpose of the present invention is achieved in that said vegetable flavonoid separates and obtains from tobacco, called after fistulaflavonoid C, and its molecular formula is C
18H
16O
6, have following structure:
The present invention's second purpose is achieved in that and comprises extraction, silica gel column chromatography, HPLC 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 post of 2 ~ 5 times of amounts of weight ratio; The chloroform-methanol solution that with the volume proportion is 20:1 ~ 1:1 carries out gradient elution, merges identical part, collects the each several part elutriant and concentrates;
C, HPLC are separated: the 9:1 part of B step elutriant further promptly gets described flavonoid compound with the HPLC separation and purification.
Structure with the flavonoid compound of method for preparing is measured out through following method:
The compounds of this invention is yellow; UV spectrum (solvent is a methyl alcohol),
λ Max(log
ε) 348 (2.28), 297 (4.21), 210 (4.69) nm; Ir spectra (pressing potassium bromide troche)
ν Max3384,2962,2876,1625,1530,1483,1422,1269,1038,872 cm
-1(HRESIMS Fig. 3) provides quasi-molecular ion peak to high resolution mass spectrum
M/z[351.0852 M+Na]
+(calculated value 301.1076).In conjunction with
1H with
13C NMR spectrum provides a molecular formula C
18H
16O
6, degree of unsaturation is 11.From
1Can find out a structure of pterocarpan in the H NMR spectrogram
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 the 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 substituted aromatic ring structure fragment of 4 ￠.Can see that this compound has 17 carbon atoms in the carbon spectrum, comprise 1 methoxyl group, 2 methylene radical, 6 methynes and 8 unhydrided carbon.HMBC
δ HIn the spectrogram 3.80 (OMe) with
δ C(150.8 C-4 ') is 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 C134.0 (C-8) relevant proof methylenedioxy group is connected C-7 and C-8, therefore, remaining 1 hydroxyl must be connected C-3 '.
In addition, from document and biology rule, H-3 on the B/C ring and H-4 generally are cis, for example
α,
αOr
β,
βGeneral analysis through CD (circular dichroism) and ORD (optical rotatory dispersion), optically-active is (-), 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 obtains confirming that compound is named as: fistulaflavonoid C.
The compound of table-1.
1H NMR with
13(solvent is CD to C NMR data
3OD)
The present invention's the 3rd purpose is achieved in that vegetable flavonoid is preparing the application in the resisting tobacco mosaic virus medicine.
Vegetable flavonoid of the present invention is separated from Caesalpinoidea plant A Bole first, has confirmed to be flavonoid compound through nucleus magnetic resonance and measuring method of mass spectrum, and has characterized its concrete structure.The relative inhibition of this compound of evidence is 31.2%, far above the relative inhibition 24.5% of contrast Ningnanmycin, explains that compound has good activity of resisting tobacco mosaic virus.The compounds of this invention is simple in structure, and synthetic realizes easily, compound active good; Can be used as the guiding compound of activity of resisting tobacco mosaic virus.
Description of drawings
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 described, but never in any form the present invention is limited, any conversion or improvement based on training centre of the present invention is done all fall into protection scope of the present invention.
Vegetable flavonoid C according to the invention
18H
16O
6The preparation method, comprise extraction, silica gel column chromatography, HPLC 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 medicinal extract to be adorned post with 200 ~ 300 order silica gel of 2 ~ 5 times of amounts of weight ratio carry out silica gel column chromatography; The chloroform-methanol solution that with the volume proportion is 20:1 ~ 1:1 carries out gradient elution, merges identical part, collects the each several part elutriant and concentrates;
It is that 9:1 part with elutriant further promptly gets the target flavonoid compound with the HPLC separation and purification that described HPLC is separated.
The used alcohol concn of said extraction is 65 ~ 75%.
The said supersound extraction time is 5 ~ 7h.
Said medicinal extract is before the silica gel column chromatography rough segmentation, with 80 ~ 100 order silica gel silica gel mixed samples that weigh 2 ~ 3 times behind 80 ~ 100% dissolve with methanol of 1.5 ~ 3 times of amounts of weight ratio with medicinal extract, further removes impurity.
Said chloroform-methanol volume proportion is 20:1,9:1,8:2,7:3,6:4,5:5.
Said HPLC separation and purification is that to adopt volume proportion be that the methanol-water of 7:3 is a moving phase, 20 * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 10 ~ 14mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 180 ~ 220 mL collect the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up.
Material after the said HPLC separation and purification is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with gel filtration chromatography, with further separation and purification.
What said gel filtration chromatography used is Sephadex LH-20 gel column, and the gel column of other types can be realized the object of the invention equally.
It is to the application in the resisting tobacco mosaic virus medicine in preparation that flavonoid compound of the present invention is used.
Raw materials used area and the kind of not receiving of the present invention limits, and also is not limited only to tobacco, all can realize the object of the invention, is that example is done further explanation to the present invention to derive from Yunnan Dehong tobacco leaf as the preparation raw material below, but the present invention do not done any restriction:
Test Example 1---compound extracts and separates
Plant sample is adopted the Dehong county in Yunnan, and A Bole complete stool 4.5 kg that take a sample are crushed to 20～40 orders, and with supersound extraction 3 times, each 6 hours, extracting solution merged, filters with the ethanol of 70%V/V, and concentrating under reduced pressure becomes medicinal extract, total medicinal extract 364 g.Total medicinal extract is mixed appearance with an amount of methyl alcohol (medicinal extract weight 1.5～3 times) dissolving back with silica gel (80～100 order), and silica gel (200～300 order) is adorned post and carried out silica gel column chromatography.Chloroform-methanol (20:1,9:1,8:2; 7:3,6:4,5:5) gradient elution; The TLC monitoring merges identical part, obtains 6 parts, and wherein chloroform-acetone (9:1) wash-out partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 45% is moving phase, (20 mm * 250 mm, C 5mm)
18Preparative column is a stationary phase, and flow velocity is 12 mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 200mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
The evaluation of Test Example 2---compound:
The compounds of this invention is yellow; UV spectrum (solvent is a methyl alcohol), λ
Max(log ε) 348 (2.28), 297 (4.21), 210 (4.69) nm; Ir spectra (pressing potassium bromide troche)
ν Max3384,2962,2876,1625,1530,1483,1422,1269,1038,872 cm
-1(HRESIMS Fig. 3) provides quasi-molecular ion peak to high resolution mass spectrum
M/z[351.0852 M+Na]
+(calculated value 301.1076).In conjunction with
1H with
13C NMR spectrum provides a molecular formula C
18H
16O
6, degree of unsaturation is 11.From
1Can find out a structure of pterocarpan in the H NMR spectrogram
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 the 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 substituted aromatic ring structure fragment of 4 ￠.Can see that this compound has 17 carbon atoms in the carbon spectrum, comprise 1 methoxyl group, 2 methylene radical, 6 methynes and 8 unhydrided carbon.HMBC
δ HIn the spectrogram 3.80 (OMe) with
δ C(150.8 C-4 ') is 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 C134.0 (C-8) relevant proof methylenedioxy group is connected C-7 and C-8, therefore, remaining 1 hydroxyl must be connected C-3 '.
In addition, from document and biology rule, H-3 on the B/C ring and H-4 generally are cis, for example
α,
αOr
β,
βGeneral analysis through CD (circular dichroism) and ORD (optical rotatory dispersion), optically-active is (-), 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 with
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 activity of resisting tobacco mosaic virus and measure.5～6 age flue-cured tobacco plant on, choose the blade (leaf is capable normal, anosis no worm) that is applicable to test; Earlier blade is evenly sprinkled fine emery powder, with writing brush will subsequent use tobacco mosaic virus(TMV) source (3.0 * 10-3) evenly are put on the blade that spreads silicon carbide, treat that the blade that selects in all connects the poison end after; Be placed on immediately and handle 20 min in the petridish that fills soup, take out, spill the globule and about liquid on the defoliation sheet; Two and half leaves are restored to be emitted on be covered with the enamel son of an influential official that toilet paper preserves moisture and add cover glass; Temperature control (23 ± 2) ℃ is placed on the greenhouse natural light irradiation, the promptly visible withered spot of 2～3 d. and each is handled and all establishes second half leaf and be contrast; Be provided with in addition 1 group be the 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 the clear water connects malicious leaf, T is soaked in the withered spot number (individual) that half sheet in the soup connects malicious leaf.
The relative inhibition of bright compound of result is 31.2%, far above the relative inhibition 24.5% of contrast Ningnanmycin, explains that compound has good activity of resisting tobacco mosaic virus.
The above is merely preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of within spirit of the present invention and principle, being done, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Plant sample is adopted the Dehong county in Yunnan, and Turkish tobaccos complete stool 4.5 kg that take a sample are crushed to 20 orders, and the ethanol with 70% is with supersound extraction 3 times, and each 6h, extracting solution merge, filter, and concentrating under reduced pressure becomes medicinal extract, total medicinal extract 364 g.Medicinal extract with 90% dissolve with methanol of 3 times of weight ratios after with 3 times 80 order silica gel mixed samples, 2 times 300 order silica gel dress post carries out silica gel column chromatography.Use volume proportion to 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, and wherein volume proportion is that chloroform-acetone wash-out of 9:1 partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 45% is moving phase, 20 mm * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 12mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 200mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
Embodiment 2
Plant sample is adopted the Dehong county in Yunnan, and Turkish tobaccos complete stool sampling 3kg is crushed to 40 orders, and the ethanol with 60% is with supersound extraction 4 times, and each 4h, extracting solution merge, filter, and concentrating under reduced pressure becomes medicinal extract, gets total medicinal extract 278g.Medicinal extract with 95% dissolve with methanol of 1.5 times of weight ratios after with 2 times 100 order silica gel mixed samples, 3 times 200 order silica gel dress post carries out silica gel column chromatography.Use volume proportion to 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, and wherein volume proportion is that chloroform-acetone wash-out of 9:1 partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 40% is moving phase, 20 mm * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 10mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 180mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
Embodiment 3
Plant sample is adopted the Dehong county in Yunnan, and Turkish tobaccos complete stool sampling 3.5kg is crushed to 40 orders, and the ethanol with 65% is with supersound extraction 2 times, and each 5h, extracting solution merge, filter, and concentrating under reduced pressure becomes medicinal extract, gets total medicinal extract 321g.Medicinal extract with 100% dissolve with methanol of 2 times of weight ratios after with 2.5 times 90 order silica gel mixed samples,
2.5 the dress of 250 order silica gel doubly post carries out silica gel column chromatography.Use volume proportion to 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, and wherein volume proportion is that chloroform-acetone wash-out of 9:1 partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 50% is moving phase, 20 mm * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 14mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 220mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
Embodiment 4
Plant sample is adopted the Dehong county in Yunnan, and Turkish tobaccos complete stool sampling 5kg is crushed to 30 orders, and the ethanol with 75% is with supersound extraction 3 times, and each 7h, extracting solution merge, filter, and concentrating under reduced pressure becomes medicinal extract, gets total medicinal extract 479g.Medicinal extract with 85% dissolve with methanol of 3 times of weight ratios after with 2 times 100 order silica gel mixed samples, 2.5 times 200 order silica gel dress post carries out silica gel column chromatography.Use volume proportion to 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, and wherein volume proportion is that chloroform-acetone wash-out of 9:1 partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 50% is moving phase, 20 mm * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 11mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 190mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
Embodiment 5
Plant sample is adopted the Dehong county in Yunnan, and Turkish tobaccos complete stool sampling 4.5kg is crushed to 40 orders, and the ethanol with 80% is with supersound extraction 3 times, and each 8h, extracting solution merge, filter, and concentrating under reduced pressure becomes medicinal extract, gets total medicinal extract 396g.Medicinal extract with 80% dissolve with methanol of 2.5 times of weight ratios after 1.5 times 100 order silica gel mixed samples, 2 times 300 order silica gel dress post carries out silica gel column chromatography.Use volume proportion to 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, and wherein volume proportion is that chloroform-acetone wash-out of 9:1 partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 45% is moving phase, 20 mm * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 12 mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 200mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
Embodiment 6
Plant sample is adopted the Dehong county in Yunnan, and Turkish tobaccos complete stool sampling 5.2kg is crushed to 30 orders, and the ethanol with 80% is with supersound extraction 3 times, and each 6h, extracting solution merge, filter, and concentrating under reduced pressure becomes medicinal extract, gets total medicinal extract 492g.Medicinal extract with 85% dissolve with methanol of 3 times of weight ratios after with 2 times 100 order silica gel mixed samples, 5 times 200 order silica gel dress post carries out silica gel column chromatography.Use volume proportion to 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, and wherein volume proportion is that chloroform-acetone wash-out of 9:1 partly separates with the prompt logical sequence 1,100 half preparation HPLies of peace; Methyl alcohol with 50% is moving phase, 20 mm * 250 mm, the C of 5mm
18Preparative column is a stationary phase, and flow velocity is 11mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 190mL collects the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up; The gained material is used pure dissolve with methanol once more, is moving phase with pure methyl alcohol again, separates with Sephadex LH-20 gel filtration chromatography, get final product target compound.
Embodiment 7
Get the compound of embodiment 1 preparation, be yellow.
Measuring method is: use nucleus magnetic resonance, identify structure in conjunction with other spectroscopic technique.
1) UV spectrum (solvent is a methyl alcohol),
λ Max(log
ε) 348 (2.28), 297 (4.21), 210 (4.69) nm;
2) ir spectra (pressing potassium bromide troche)
ν Max3384,2962,2876,1625,1530,1483,1422,1269,1038,872 cm
-1
3) (HRESIMS Fig. 3) provides quasi-molecular ion peak to high resolution mass spectrum
M/z[351.0852 M+Na]
+(calculated value 301.1076).In conjunction with
1H with
13C NMR spectrum provides a molecular formula C
18H
16O
6, degree of unsaturation is 11.
From
1Can find out a structure of pterocarpan in the H NMR spectrogram
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 the 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 substituted aromatic ring structure fragment of 4 ￠.Can see that this compound has 17 carbon atoms in the carbon spectrum, comprise 1 methoxyl group, 2 methylene radical, 6 methynes and 8 unhydrided carbon.HMBC
δ HIn the spectrogram 3.80 (OMe) with
δ C(150.8 C-4 ') is 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 C134.0 (C-8) relevant proof methylenedioxy group is connected C-7 and C-8, therefore, remaining 1 hydroxyl must be connected C-3 '.
In addition, from document and biology rule, H-3 on the B/C ring and H-4 generally are cis, for example
α,
αOr
β,
βGeneral analysis through CD (circular dichroism) and ORD (optical rotatory dispersion), optically-active is (-), 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 obtains confirming that compound is named as: fistulaflavonoid C.
Get the compound of embodiment 2 preparations, be yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 2 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 9
Get the compound of embodiment 3 preparations, be yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 3 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 10
Get the compound of embodiment 4 preparations, be yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 4 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 11
Get the compound of embodiment 5 preparations, be yellow.Measuring method is identical with embodiment 7, confirms that the compound of embodiment 5 preparations is described flavonoid compound---fistulaflavonoid C.
Embodiment 12
Get the compound of embodiment 6 preparations, be 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 activity of resisting tobacco mosaic virus and measure.5～6 age flue-cured tobacco plant on, choose the blade (leaf is capable normal, anosis no worm) that is applicable to test; Earlier blade is evenly sprinkled fine emery powder, with writing brush will subsequent use tobacco mosaic virus(TMV) source (3.0 * 10-3) evenly are put on the blade that spreads silicon carbide, treat that the blade that selects in all connects the poison end after; Be placed on immediately and handle 20 min in the petridish that fills soup, take out, spill the globule and about liquid on the defoliation sheet; Two and half leaves are restored to be emitted on be covered with the enamel son of an influential official that toilet paper preserves moisture and add cover glass; Temperature control (23 ± 2) ℃ is placed on the greenhouse natural light irradiation, the promptly visible withered spot of 2～3 d. and each is handled and all establishes second half leaf and be contrast; Be provided with in addition 1 group be the 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 the clear water connects malicious leaf, T is soaked in the withered spot number (individual) that half sheet in the soup connects malicious leaf.
The result shows that the relative inhibition of this compound is 31.2%, far above the relative inhibition 24.5% of contrast Ningnanmycin, explains that compound has good activity of resisting tobacco mosaic virus.
Claims (10)
1. a vegetable flavonoid is characterized in that from tobacco, separating obtaining, called after fistulaflavonoid C, and its molecular formula is C
18H
16O
6, have following structure:
。
2. the preparation method of the said vegetable flavonoid of claim 1 is characterized in that comprising extraction, silica gel column chromatography, HPLC 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 post of 2 ~ 5 times of amounts of weight ratio; The chloroform-methanol solution that with the volume proportion is 20:1 ~ 1:1 carries out gradient elution, merges identical part, collects the each several part elutriant and concentrates;
C, HPLC are separated: the 9:1 part of B step elutriant further promptly gets described flavonoid compound with the HPLC separation and purification.
3. according to the preparation method of the said vegetable flavonoid of claim 2, it is characterized in that alcohol concn is 65 ~ 75% in the said A step.
4. according to the preparation method of the said vegetable flavonoid of claim 2, it is characterized in that the supersound extraction time is 5 ~ 7h in the said A step.
5. according to the preparation method of the said vegetable flavonoid of claim 2; It is characterized in that medicinal extract is before the silica gel column chromatography rough segmentation in the said B step; With 80 ~ 100 order silica gel silica gel mixed samples that weigh 2 ~ 3 times behind 80 ~ 100% dissolve with methanol of 1.5 ~ 3 times of amounts of weight ratio with medicinal extract, further remove impurity.
6. according to the preparation method of the said vegetable flavonoid of claim 2, it is characterized in that the chloroform-methanol volume proportion is 20:1 in the said B step, 9:1,8:2,7:3,6:4,5:5.
7. according to the preparation method of the said vegetable flavonoid of claim 2, it is characterized in that the separation and purification of said C step mesohigh liquid chromatography is that to adopt volume proportion be that the methanol-water of 40 ~ 50:50 ~ 60 is a moving phase, 20 * 250 mm, the C of 5 μ m
18Preparative column is a stationary phase, and flow velocity is 10 ~ 14mL/min, and it is 254 nm that UV-detector detects wavelength, and each sample introduction 180 ~ 220 μ L collect the chromatographic peak of 26.4 min, and the back evaporate to dryness repeatedly adds up.
8. according to the preparation method of the said vegetable flavonoid of claim 2; It is characterized in that the material after the separation and purification of said C step mesohigh liquid phase chromatography uses pure dissolve with methanol once more; Be moving phase with pure methyl alcohol again, separate with gel filtration chromatography, with further separation and purification.
9. the preparation method of said vegetable flavonoid according to Claim 8, what it is characterized in that said gel filtration chromatography uses is Sephadex LH-20 gel column.
10. the said vegetable flavonoid of claim 1 is preparing the application in the resisting tobacco mosaic virus medicine.
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| CN103664862A (en) * | 2013-08-27 | 2014-03-26 | 云南烟草科学研究院 | Polyphenol compounds in aromatic tobaccos as well as preparation method and application of polyphenol compounds |
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| CN104292202A (en) * | 2014-09-15 | 2015-01-21 | 云南民族大学 | Flavonoid compound as well as preparation method and application of flavonoid compound |
| 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 |
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