CN102399207B - Extraction method of ginkgetin or isoginkgetin - Google Patents
Extraction method of ginkgetin or isoginkgetin Download PDFInfo
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Abstract
The invention discloses an extraction method of ginkgetin or isoginkgetin. According to the invention, ginkgetin or isoginkgetin is first extracted from Capparis spinosa L., so a new approach is provided for the obtaining of ginkgetin or isoginkgetin.
Description
Technical field
The present invention relates to the extracting method of a kind of ginkgetin or isoginkgetin.
Background technology
Hyoscyamus niger L Capparis spinosa L. is that Capparidaceae Capparidaceae Chinese lime belongs to Capparis plant, has another name called Euphorbia lathyris, water melon button, wild watermelon, Semen Capparis, perennial liana shape half sprawling shrub.Happiness is born on low hillside that arid has sandstone, sand ground.Originate in Mediterranean.Now be common in South of France, Italy and the area such as Algerian, the areas such as Xinjiang of China, Tibet also produce.Hyoscyamus niger L taste is arduous, warm in nature, and its root skin, leaf and fruit have a wide range of applications the Uygur nationality is among the people, are used for the treatment of rheumatosis, have dispel the wind, the effect of loose cold, dehumidifying.The main component of Hyoscyamus niger L is phytomelin, capric acid, forulic acid, sinapinic acid, unsaturated fatty acids containing 18 carbon.Up to now, not yet there is document openly to cross about the report that extracts ginkgetin or isoginkgetin from Hyoscyamus niger L.
In prior art, ginkgetin and isoginkgetin be separation and Extraction from gingko episperm, Ginkgo Leaf or seed of Schisandra propinqua conventionally.
Pan Jingxian, Zhang Huyi, the plant resourceses such as Yang Xianbin and environment 1995,4 (2): 17-21 has reported the method for extracting ginkgetin and isoginkgetin from gingko episperm.After the method is pulverized gingko episperm 930g, soak with except the little composition of depolarization with sherwood oil, then soak 2 times by ethyl acetate, each 3 days, filter the concentrated ethyl acetate extract 40g that to obtain.Through silica gel sherwood oil, methylene dichloride, ethyl acetate and methanol-eluted fractions for reduced pressure chromatography, obtain 4 parts.Wherein ethyl acetate part 6.8g is through polyamide column chromatography, and methylene chloride-methanol gradient elution, obtains 01～12 sample.07 has 231.5mg, and wherein 50mg is through Preparative TLC chromatography (PTLC), developping agent: methylene dichloride: methyl alcohol (96: 4), launches 4 times to obtain ginkgetin 3.3mg, isoginkgetin 27.3mg.
Tang Yuping, Lou Fengchang, Wang Jinghua, Li Yanfang Chinese Pharmaceutical Journal 2001,36 (4): 231-233 has reported the method for extracting ginkgetin and isoginkgetin from Ginkgo Leaf.The method will be dried Ginkgo Leaf 10.0kg and pulverize, with 95% alcohol reflux 3 times.Extracting solution merges, and concentrating under reduced pressure, mixes after sample with silicon dried soil, puts in cable type extractor according, extracts successively with sherwood oil, ethyl acetate, methanol eddy.Ethyl acetate extract is through silicagel column (silica gel granularity: 200～300 orders) chromatography repeatedly, chloroform-methanol (100: 1～100: 100) gradient elution, obtain the crude product of Compound I～VII, use again SephadexLH-20 column chromatography (using pure methanol-eluted fractions) to carry out purifying, obtain respectively Compound I (50mg), II (16mg), III (10mg), IV (10mg), V (800mg), ginkgetin VI (100mg), the sterling of isoginkgetin VII (40mg).
Li Jun, Li Fu, the GUIHAIA such as Lu Yuanyuan 2006,26 (5): 690-691 has reported the method for extracting ginkgetin from seed of Schisandra propinqua.The method is pulverized seed of Schisandra propinqua 2kg, by 75% ethanol 10L refluxing extraction 3 times, and 2h at every turn, united extraction liquid, concentrating under reduced pressure obtains crude extract 268g.To after this extract water suspendible, extract 3 times with sherwood oil, chloroform, ethyl acetate and propyl carbinol are each successively, after recovery solvent, obtain acetic acid ethyl ester extract 48g.Acetic acid ethyl ester extract, through silica gel column chromatography, is carried out to gradient elution with chloroform-methanol (volume ratio 100: 0～50: 50).95: 5 wash-out parts are merged, through silica gel column chromatography, SephadexLH-20 column chromatography and silica gel Preparative TLC chromatographic separation obtain ginkgetin 32mg repeatedly.
Summary of the invention
Technical problem to be solved by this invention has been to provide extracting method a kind of and the diverse ginkgetin of prior art or isoginkgetin.The raw material of extracting method of the present invention is completely different from prior art.
The extracting method that the present invention relates to a kind of ginkgetin, it comprises the following step:
(1) the water extract-alcohol precipitation liquid of Hyoscyamus niger L fruit is filtered, concentrated, obtain medicinal extract;
(2) medicinal extract step (1) being obtained is water-soluble, uses successively sherwood oil and chloroform extraction, obtains chloroform extract;
(3) chloroform extract of step (2) gained is carried out to gradient elution with polyamide column, eluent is followed successively by the aqueous ethanolic solution that water and volume percent are 30%～95%, collect and detect with silicon thin-layer chromatography, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.5～0.8;
(4) step (3) gained material is carried out to wash-out with gel column, collect with silicon thin-layer chromatography and detect, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.6～0.8;
(5) step (4) gained material is carried out to silica gel column chromatography separation, or silica gel Preparative TLC chromatographic separation, collect with silicon thin-layer chromatography and detect, developping agent is volume ratio while being the chloroform of 15: 1 and methyl alcohol, the cut that Rf value is 0.45;
(6) step (5) gained material is prepared to liquid phase separation, collect the component detecting under following testing conditions: column temperature is 25～30 ℃, Waters510 pump, Waters484 detector, Global-C18 pillar, detect wavelength 270nm, moving phase is the mixed solution of methyl alcohol and water, and the two volume ratio is 90: 10, and flow velocity is 3.40ml/min, retention time is 24.7min, obtains ginkgetin.
In step (1), the fruit that described Hyoscyamus niger L fruit is preferably dried; Described Hyoscyamus niger L dry fruit preferably uses for after pulverizing, and the particle diameter after pulverizing is preferably 3～7 orders; The concentrating degree of described medicinal extract is preferably to detect without alcohol taste, and its density is preferably 1.0～2.0g/ml.
Described water extract-alcohol precipitation liquid can be made by following method: by Hyoscyamus niger L fruit water extraction, filters, and filtrate is concentrated, add aqueous ethanolic solution, leave standstill.Wherein, the volume of described water is preferably 8～10 times of Hyoscyamus niger L dry fruit volume, preferably 8 times.With the number of times of water extraction be preferably 2～3 times, preferably 2 times; With the time of water extraction be preferably each 1～2 hour, preferably 2 hours.The density of described concentrated rear filtrate is preferably 1.0～2.0g/ml, preferably 1.2g/ml.The described concentration that adds the ethanol in solution after aqueous ethanolic solution is preferably volume percent 65～85%, preferably 70%.
In step (2), 10～15 times of the medicinal extract volume that the consumption of described water preferably obtains for step (1); 10～15 times of the medicinal extract volume that the consumption of described sherwood oil preferably obtains for step (1); 15～20 times of the medicinal extract volume that the consumption of described chloroform preferably obtains for step (1).With the number of times of petroleum ether extraction be preferably 2～3 times, best 3 times, with the number of times of chloroform extraction be preferably 3～4 times, the best 4 times.
In step (3), the specification of described polyamide column is preferably 14-30 order, 30-60 order, 60-100 order, 100-120 order or 200-400 order, and better is 100-200 order; Polymeric amide filler in polyamide column and the weight ratio of upper pillaring substance are preferably 50: 1～80: 1; The eluent of described gradient elution is preferably: water → 30% aqueous ethanolic solution → 50% aqueous ethanolic solution → 70% aqueous ethanolic solution → 90% aqueous ethanolic solution (being above volume percent); Wherein, the consumption of water is preferably 3～5 times of polyamide column volume, and the consumption of 30% aqueous ethanolic solution is preferably 4～6 times of polyamide column volume; The consumption of 50% aqueous ethanolic solution is preferably 4～6 times of polyamide column volume; The consumption of 70% aqueous ethanolic solution is preferably 4～6 times of polyamide column volume; The consumption of 90% aqueous ethanolic solution is preferably 4～6 times of polyamide column volume.
In step (4), described gel column is preferably Sephadex LH
20.Described with the eluent that gel column carries out wash-out be preferably methyl alcohol.The consumption of described eluent is preferably 1～2 times of gel column volume.
In step (5), during described silica gel column chromatography separates, eluent is preferably the mixed solvent of chloroform and methyl alcohol.In described silica gel Preparative TLC chromatographic separation, developping agent is preferably the mixed solvent of chloroform and methyl alcohol, and both volume ratios are preferably 15: 1.
Step (5) preferably comprises the following step: step (4) gained material is carried out silica gel column chromatography separation by (1 '), collect and detect with silicon thin-layer chromatography, the volume ratio of developping agent chloroform and methyl alcohol is 15: 1 o'clock, the cut that Rf value is 0.3～0.5; Step (1 ') gained material is carried out silica gel column chromatography separation by (2 '), or silica gel Preparative TLC chromatographic separation, collect with silicon thin-layer chromatography and detect, developping agent is volume ratio while being the chloroform of 15: 1 and methyl alcohol, the cut that Rf value is 0.45.
In step (1 '), during described silica gel column chromatography separates, the granularity of silica gel is preferably 100～200 orders; The weight ratio of silica gel and upper pillaring substance is preferably 50: 1～80: 1.
Eluent during described silica gel column chromatography separates is preferably the mixed solvent of sherwood oil and ethyl acetate or the mixed solvent of normal hexane and ethyl acetate, better is the mixed solvent of sherwood oil and ethyl acetate, and described sherwood oil and the volume ratio of ethyl acetate are preferably 6: 1～1: 1; Elution process during described silica gel column chromatography separates is preferably gradient elution, in the time that eluent is the mixed solvent of sherwood oil and ethyl acetate, gradient is preferably sherwood oil: ethyl acetate=6: 1 → 4: 1 → 2: 1 → 1: 1, and the eluent consumption of each gradient is preferably 4～6 times of silicagel column volume.
In step (2 '), during described silica gel column chromatography separates, eluent is preferably the mixed solvent of chloroform and methyl alcohol.In described silica gel Preparative TLC chromatographic separation, developping agent is preferably the mixed solvent of chloroform and methyl alcohol, and both volume ratios are preferably 15: 1.
In step (6), the condition of described preparation liquid phase separation can be the normal condition that liquid phase is prepared in this area, as long as obtain the material of the retention time under above-mentioned testing conditions by preparing the method for liquid phase separation.The condition of preferred preparation liquid phase separation is as follows: column temperature is preferably 20～30 ℃; Chromatographic column is preferably reverse C18 post, and better is Global-C18 pillar; Pump is preferably waters510 pump; Detector is preferably waters484 detector; Detecting wavelength is preferably 270nm; Moving phase is preferably the mixed solution of first alcohol and water, and its volume ratio is preferably 90: 10; Flow velocity is preferably 3～5ml/min.
The extracting method that the invention still further relates to a kind of isoginkgetin, it comprises the following step:
(1) the water extract-alcohol precipitation liquid of Hyoscyamus niger L fruit is filtered, concentrated, obtain medicinal extract;
(2) medicinal extract step (1) being obtained is water-soluble, uses successively sherwood oil and chloroform extraction, obtains chloroform extract;
(3) chloroform extract of step (2) gained is carried out to gradient elution with polyamide column, eluent is followed successively by the aqueous ethanolic solution that water and volume percent are 30%～95%, collect and detect with silicon thin-layer chromatography, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.5～0.8;
(4) step (3) gained material is carried out to wash-out with gel column, collect with silicon thin-layer chromatography and detect, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.6～0.8;
(5) step (4) gained material is carried out to silica gel column chromatography separation, or silica gel Preparative TLC chromatographic separation, collect with silicon thin-layer chromatography and detect, developping agent is volume ratio while being the chloroform of 15: 1 and methyl alcohol, the component that Rf value is 0.45;
(6) step (5) gained material is prepared to liquid phase separation, collect the component detecting under following testing conditions: column temperature is 25～30 ℃, waters510 pump, waters484 detector, Global-C18 pillar, detect wavelength 270nm, moving phase is the mixed solution of methyl alcohol and water, and the two volume ratio is 90: 10, and flow velocity is 3.40ml/min, retention time is 21.8min, obtains isoginkgetin.
Wherein, the preferred method of step (1)～(6) is all identical with the extracting method of aforesaid ginkgetin with condition.
In the present invention, the each optimum condition in above-mentioned preparation method can carry out arbitrary combination without prejudice to the field on the basis of common sense, obtains each preferred embodiment of the present invention.
Except specified otherwise, the raw material the present invention relates to and reagent is commercially available obtaining all.
Positive progressive effect of the present invention is:
The present invention has extracted ginkgetin or isoginkgetin for the first time from Hyoscyamus niger L, for obtaining of ginkgetin and isoginkgetin provides a new approach.
Embodiment
Further illustrate the present invention with embodiment below, but the present invention is not limited.
Embodiment 1 extracts ginkgetin
Hyoscyamus niger L dry fruit 50kg, pulverizes (particle diameter after pulverizing is 6 orders), with the water extraction of 8 times of volumes, extracts twice, and each 2 hours, filter, merge twice filtrate, concentrating under reduced pressure, to density 1.2g/ml.Add ethanol, to determining alcohol be 70%, alcohol precipitation hold over night.Filtration obtains filtrate, is evaporated to without alcohol taste and obtains medicinal extract.
Medicinal extract is water-soluble, use successively sherwood oil, chloroform extraction, obtain petroleum ether part 40g, chloroform extract 168g.
Chloroform extract is separated with polyamide column, and water, 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol are washed post successively.Collect with silicon thin-layer chromatography and detect, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.5～0.8.
After above-mentioned gained cut is concentrated, use Sephadex LH
20post separates, and methyl alcohol is washed post, collects with silicon thin-layer chromatography and detects, and the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.6～0.8.
After above-mentioned gained cut is concentrated, carry out silica gel column chromatography separation, carry out gradient elution (gradient was as 6: 1 → 4: 1 → 2: 1 → 1: 1) take petroleum ether-ethyl acetate (volume ratio was as 6: 1～1: 1), collect and detect with silicon thin-layer chromatography, the volume ratio of developping agent chloroform and methyl alcohol is 15: 1 o'clock, the cut that Rf value is 0.3～0.5.
After above-mentioned gained cut is concentrated, carry out silica gel Preparative TLC chromatography (silica-gel plate model HSGF-254, by Yantai, Jiang You silica gel development corporation, Ltd. provides), developping agent is chloroform-methanol (volume ratio is 15: 1), collect the cut that Rf value is 0.45, obtain 29.3mg mixture.
Above-mentioned gained 29.3mg mixture is prepared to liquid phase separation, condition is: column temperature is 25～30 ℃, waters510 pump, waters484 detector, global-C18 pillar (global chromatogram limited liability company provides by Jiangsu), detect wavelength 270nm, moving phase: methyl alcohol: water=90: 10 (volume ratios), flow velocity 3.40ml/min.The component that collection retention time is 24.7min, obtains ginkgetin 17.6mg.It is 98% that HPLC area normalization method records its purity.Its Structural Identification data are as follows:
Ginkgetin Ginkgetin, yellow crystals, mp 347-349 ℃, ESIMS:m/z=565.18[M-H]
-,
1h-NMR (DMSO-d
6) ppm: δ 3.77 (3H, s, 4 '-OCH
3), 3.81 (3H, s, 7-OCH
3), 6.21 (1H, s, H-6), 6.34 (1H, s, H-6 "), 6.68 (1H, s, H-3 "), 6.70 (2H, d, J=8.5Hz, H-3 " ', 5 " '), 6.77 (1H, s, H-8), 6.96 (1H, s, H-3), 7.32 (1H, d, J=9.0Hz, H-5 '), 7.46 (2H, d, J=8.5Hz, H-2 " ', 6 " '), 8.04 (1H, d, J=2.0Hz, H-2 '), 8.15 (1H, dd, J=9.0, 2.0Hz, H-6 '), 12.92 (1H, s, 5-OH), 13.04 (1H, s, 5 " OH),
13c-NMR (DMSO-d
6) ppm:55.9 (4 '-OCH
3), 56.1 (7-OCH
3), 92.7 (C-8), 98.2 (C-6 "), 99.5 (C-6), 102.2 (C-3 "), 103.7 (C-3, C-10 "), 103.9 (C-8 "), 104.8 (C-10), 111.6 (C-5 '), 115.9 (C-3 " ', C-5 " '), 121.3 (C-1 " '), 122.2 (C-1 '), 123.1 (C-3 '), 127.8 (C-6 ', C-2 " ', C-6 " '), 131.2 (C-2 '), 154.5 (C-9 "), 157.4 (C-9), 160.6 (C-4 ', C-5 "), 161.0 (C-4 " '), 161.2 (C-5), 161.7 (C-7 "), 163.9 (C-2, C-2 "), 165.2 (C-7), 181.4 (C-4), 182.0 (C-4 ").
It is consistent with bibliographical information that (document is: Konda, Y., Sasaki, T., Kagawa, H., Takayanagi, H., Harigaya, Y., Sun, X.-L., et a1. (1995) .Conformational analysis of c3 '-c8connected biflavones.J Heterocyclic Chem, 32 (5), 1531-1535).
Embodiment 2 extracts isoginkgetin
Hyoscyamus niger L dry fruit 50kg, pulverizes (particle diameter after pulverizing is 6 orders), with the water extraction of 8 times of volumes, extracts twice, and each 2 hours, filter, merge twice filtrate, concentrating under reduced pressure, to density 1.2g/ml.Add ethanol, to determining alcohol be 70%, alcohol precipitation hold over night.Filtration obtains filtrate, is evaporated to without alcohol taste and obtains medicinal extract.
Medicinal extract is water-soluble, use successively sherwood oil, chloroform extraction, obtain petroleum ether part 40g, chloroform extract 168g.
Chloroform extract is separated with polyamide column, and water, 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol are washed post successively.Collect with silicon thin-layer chromatography and detect, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.5～0.8.
After above-mentioned gained cut is concentrated, use Sephadex LH
20post separates, and methyl alcohol is washed post, collects with silicon thin-layer chromatography and detects, and the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.6～0.8.
After above-mentioned gained cut is concentrated, carry out silica gel column chromatography separation, carry out gradient elution (gradient was as 6: 1 → 4: 1 → 2: 1 → 1: 1) take petroleum ether-ethyl acetate (volume ratio was as 6: 1～1: 1), collect and detect with silicon thin-layer chromatography, the volume ratio of developping agent chloroform and methyl alcohol is 15: 1 o'clock, the cut that Rf value is 0.3～0.5.
After above-mentioned gained cut is concentrated, carry out silica gel Preparative TLC chromatography (silica-gel plate model HSGF-254, by Yantai, Jiang You silica gel development corporation, Ltd. provides), developping agent is chloroform-methanol (volume ratio is 15: 1), collect the cut that Rf value is 0.45, obtain 29.3mg mixture.
Above-mentioned gained 29.3mg mixture is prepared to liquid phase separation, condition is: column temperature is 25～30 ℃, waters510 pump, waters484 detector, global-C18 pillar (global chromatogram limited liability company provides by Jiangsu), detect wavelength 270nm, moving phase: methyl alcohol: water=90: 10 (volume ratios), flow velocity 3.40ml/min.The component that collection retention time is 21.8min, obtains isoginkgetin 9.1mg.It is 98% that HPLC area normalization method records its purity.Its Structural Identification data are as follows:
Isoginkgetin Isoginkgetin, yellow crystals mp 349-351 ℃, ESIMS:m/z=565.16[M-H]
-,
1h-NMR (DMSO-d
6) ppm: δ 3.75 (3H, s, 4 " '-OCH
3), 3.78 (3H, s, 4 '-OCH
3), 6.20 (1H, s, H-6), 6.42 (1H, s, H-6 "), 6.49 (1H, s, H-8), 6.90 (1H, s, H-3 "), 6.92 (1H, s, H-3), 6.94 (2H, d, J=8.5Hz, H-3 " ', 5 " '), 7.36 (1H, d, J=10.0Hz, H-5 '), 7.61 (2H, d, J=8.5Hz, H-2 " ', 6 " '), 8.05 (1H, s, H-2 '), 8.18 (1H, d, J=10.0Hz, H-6 '), 12.92 (1H, s, 5-OH), 13.06 (1H, s, 5 " OH),
13c-NMR (DMSO-d
6) ppm: δ 55.5 (4 '-OCH
3), 56.0 (4 " '-OCH
3), 94.2 (C-8), 98.7 (C-6 "), 98.9 (C-6), 103.3 (C-3 "), 103.7 (C-3), 103.8 (C-10, 8 ", 10 "), 111.8 (C-5 '), 114.6 (C-3 " ', 5 " '), 121.6 (C-3 '), 122.6 (C-1 '), 122.9 (C-1 " '), 127.8 (C-2 " ', C-6 " '), 128.3 (C-6 '), 130.9 (C-2 '), 154.5 (C-9 "), 157.5 (C-9), 160.5 (C-5 "), 160.7 (C-4 '), 161.5 (C-7 "), 161.8 (C-5), 162.3 (C-4 " '), 163.1 (C-7), 163.4 (C-2), 164.3 (C-2 "), 181.8 (C-4), 182.01 (C-4 ").
It is consistent with bibliographical information that (document is: Markham, K.R., Sheppard, C., & Geiger, H. (1987) .13C NMR studies of some naturally occurring amentoflavone and hinokiflavone biflavonoids.Phytochemistry, 26 (12), 3335-3337).
Comparative example 1
By the chloroform extract medicinal extract 100mg in embodiment 1, through Sephadex LH
20post separates repeatedly, and methyl alcohol is washed post, collects with silicon thin-layer chromatography and detects, and the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.5～0.8.After above-mentioned gained cut is concentrated, be greater than 85% through HPLC detection ginkgetin and isoginkgetin content sum, separate with anti-phase C18 post column chromatography, use respectively 30%, 40%, 50% methanol-eluted fractions, HPLC (condition: acetonitrile-0.1% formic acid water gradient, 0min → 40min acetonitrile 45% → 85%; Instrument: Agilent HP1100; Chromatographic column: Diamonsil C-
18, 5 μ, 250 × 4.6mm) and detect cut, ginkgetin and isoginkgetin are not separated.
Comparative example 2
By the chloroform extract medicinal extract 100mg in embodiment 1, use 95% alcohol reflux, 2 hours, to filter, filtrate is concentrated.After above-mentioned gained is concentrated, carrying out repeatedly silica gel column chromatography separates, carry out gradient elution (gradient was as 1: 2: 0 → 1: 8: 0 → 1: 1: 0.01 → 1: 2: 0.01 → 0: 1: 0.01) take sherwood oil-chloroform-methanol (volume ratio was as 1: 2: 0～0: 100: 1), collect and detect with silicon thin-layer chromatography, the volume ratio of developping agent chloroform and methyl alcohol is 8: 1 o'clock, the cut that Rf value is 0.5～0.8.After above-mentioned gained cut is concentrated, be greater than 80% through HPLC detection ginkgetin and isoginkgetin content sum.After above-mentioned gained is concentrated, carrying out repeatedly silica gel column chromatography separates, carry out gradient elution (gradient was as 1: 2: 0 → 1: 4: 0 → 1: 8: 0 → 1: 1: 0.01 → 1: 2: 0.01 → 1: 4: 0.01 → 0: 1: 0.01) take sherwood oil-chloroform-methanol (volume ratio was as 1: 2: 0～0: 100: 1), HPLC (condition: acetonitrile-0.1% formic acid water gradient, 0min → 40min acetonitrile 45% → 85%; Instrument: Agilent HP1100; Chromatographic column: Diamonsil C-
18, 5 μ, 250 × 4.6mm) and detect cut, ginkgetin and isoginkgetin are not separated.
Claims (18)
1. an extracting method for ginkgetin, it comprises the following step:
(1) the water extract-alcohol precipitation liquid of Hyoscyamus niger L fruit is filtered, concentrated, obtain medicinal extract;
(2) medicinal extract step (1) being obtained is water-soluble, uses successively sherwood oil and chloroform extraction, obtains chloroform extract;
(3) chloroform extract of step (2) gained is carried out to gradient elution with polyamide column, eluent is followed successively by the aqueous ethanolic solution that water and volume percent are 30%～95%, collect and detect with silicon thin-layer chromatography, when the volume ratio of developping agent chloroform and methyl alcohol is 8:1, the cut that Rf value is 0.5～0.8;
(4) step (3) gained material is carried out to wash-out with gel column, collect with silicon thin-layer chromatography and detect, when the volume ratio of developping agent chloroform and methyl alcohol is 8:1, the cut that Rf value is 0.6～0.8;
(5) step (4) gained material is carried out to silica gel column chromatography separation, or silica gel Preparative TLC chromatographic separation, collect with silicon thin-layer chromatography and detect, developping agent is volume ratio while being the chloroform of 15:1 and methyl alcohol, the cut that Rf value is 0.45;
(6) step (5) gained material is prepared to liquid phase separation, collect the component detecting under following testing conditions: column temperature is 25～30 ℃, Waters510 pump, Waters484 detector, Global-C18 pillar, detect wavelength 270nm, moving phase is the mixed solution of methyl alcohol and water, and the two volume ratio is 90:10, and flow velocity is 3.40ml/min, retention time is 24.7min, obtains ginkgetin.
2. preparation method as claimed in claim 1, is characterized in that: in step (1), described Hyoscyamus niger L fruit is dry fruit; Described Hyoscyamus niger L fruit uses after pulverizing, and the particle diameter after pulverizing is 3～7 orders.
3. preparation method as claimed in claim 1, is characterized in that: in step (1), described water extract-alcohol precipitation liquid is made by following method: by Hyoscyamus niger L fruit water extraction, filter, and filtrate is concentrated, add aqueous ethanolic solution, leave standstill; The volume of described water is 8～10 times of Hyoscyamus niger L dry fruit volume, with the number of times of water extraction be 2～3 times, with the time of water extraction be each 1～2 hour; The density of described concentrated rear filtrate is 1.0～2.0g/ml; The described concentration that adds the ethanol in solution after aqueous ethanolic solution is volume percent 65～85%.
4. preparation method as claimed in claim 1, is characterized in that: in step (2), the consumption of described water is 10～15 times of the medicinal extract volume that obtains of step (1); The consumption of described sherwood oil is 10～15 times of the medicinal extract volume that obtains of step (1); The consumption of described chloroform is 15～20 times of the medicinal extract volume that obtains of step (1); The described number of times with petroleum ether extraction is 2～3 times; The described number of times with chloroform extraction is 3～4 times.
5. preparation method as claimed in claim 1, it is characterized in that: in step (3), the specification of described polyamide column is 14-30 order, 30-60 order, 60-100 order, 100-120 order or 200-400 order, and the polymeric amide filler in described polyamide column and the weight ratio of upper pillaring substance are 50:1～80:1; The eluent of described gradient elution is: water → 30% aqueous ethanolic solution → 50% aqueous ethanolic solution → 70% aqueous ethanolic solution → 90% aqueous ethanolic solution, more than be volume percent, in described gradient elution, the consumption of water is 3～5 times of polyamide column volume, the consumption of 30% aqueous ethanolic solution is 4～6 times of polyamide column volume, the consumption of 50% aqueous ethanolic solution is 4～6 times of polyamide column volume, the consumption of 70% aqueous ethanolic solution is 4～6 times of polyamide column volume, and the consumption of 90% aqueous ethanolic solution is 4～6 times of polyamide column volume.
6. preparation method as claimed in claim 1, is characterized in that: in step (4), described gel column is Sephadex LH
20; Described with the eluent that gel column carries out wash-out be methyl alcohol, the consumption of eluent is 1～2 times of gel column volume.
7. preparation method as claimed in claim 1, it is characterized in that: step (5) comprises the following step: step (4) gained material is carried out silica gel column chromatography separation by (1 '), collect and detect with silicon thin-layer chromatography, when the volume ratio of developping agent chloroform and methyl alcohol is 15:1, the cut that Rf value is 0.3～0.5; Step (1 ') gained material is carried out silica gel column chromatography separation by (2 '), or silica gel Preparative TLC chromatographic separation, collects with silicon thin-layer chromatography and detect, and developping agent is volume ratio while being the chloroform of 15:1 and methyl alcohol, the cut that Rf value is 0.45.
8. preparation method as claimed in claim 7, is characterized in that: in step (1 '), during described silica gel column chromatography separates, the granularity of silica gel is 100～200 orders; The weight ratio of silica gel and upper pillaring substance is 50:1～80:1; Eluent is the mixed solvent of sherwood oil and ethyl acetate or the mixed solvent of normal hexane and ethyl acetate; In the time that eluent is the mixed solvent of sherwood oil and ethyl acetate, the volume ratio of sherwood oil and ethyl acetate is 6:1～1:1; Elution process is gradient elution, and in the time that eluent is the mixed solvent of sherwood oil and ethyl acetate, gradient is sherwood oil: ethyl acetate=6:1 → 4:1 → 2:1 → 1:1, and the eluent consumption of each gradient is 4～6 times of silicagel column volume.
9. preparation method as claimed in claim 7, is characterized in that: in step (2 '), during described silica gel column chromatography separates, eluent is the mixed solvent of chloroform and methyl alcohol; In described silica gel Preparative TLC chromatographic separation, developping agent is the mixed solvent of chloroform and methyl alcohol.
10. an extracting method for isoginkgetin, it comprises the following step:
(1) the water extract-alcohol precipitation liquid of Hyoscyamus niger L fruit is filtered, concentrated, obtain medicinal extract;
(2) medicinal extract step (1) being obtained is water-soluble, uses successively sherwood oil and chloroform extraction, obtains chloroform extract;
(3) chloroform extract of step (2) gained is carried out to gradient elution with polyamide column, eluent is followed successively by the aqueous ethanolic solution that water and volume percent are 30%～95%, collect and detect with silicon thin-layer chromatography, when the volume ratio of developping agent chloroform and methyl alcohol is 8:1, the cut that Rf value is 0.5～0.8;
(4) step (3) gained material is carried out to wash-out with gel column, collect with silicon thin-layer chromatography and detect, when the volume ratio of developping agent chloroform and methyl alcohol is 8:1, the cut that Rf value is 0.6～0.8;
(5) step (4) gained material is carried out to silica gel column chromatography separation, or silica gel Preparative TLC chromatographic separation, collect with silicon thin-layer chromatography and detect, developping agent is volume ratio while being the chloroform of 15:1 and methyl alcohol, the cut that Rf value is 0.45;
(6) step (5) gained material is prepared to liquid phase separation, collect the component detecting under following testing conditions: column temperature is 25～30 ℃, waters510 pump, waters484 detector, Global-C18 pillar, detect wavelength 270nm, moving phase is the mixed solution of methyl alcohol and water, and the two volume ratio is 90:10, and flow velocity is 3.40ml/min, retention time is 21.8min, obtains isoginkgetin.
11. preparation methods as claimed in claim 10, is characterized in that: in step (1), described Hyoscyamus niger L fruit is dry fruit; Described Hyoscyamus niger L fruit uses after pulverizing, and the particle diameter after pulverizing is 3～7 orders.
12. preparation methods as claimed in claim 10, is characterized in that: in step (1), described water extract-alcohol precipitation liquid is made by following method: by Hyoscyamus niger L fruit water extraction, filter, and filtrate is concentrated, add aqueous ethanolic solution, leave standstill; The volume of described water is 8～10 times of Hyoscyamus niger L dry fruit volume, with the number of times of water extraction be 2～3 times, with the time of water extraction be each 1～2 hour; The density of described concentrated rear filtrate is 1.0～2.0g/ml; The described concentration that adds the ethanol in solution after aqueous ethanolic solution is volume percent 65～85%.
13. preparation methods as claimed in claim 10, is characterized in that: in step (2), the consumption of described water is 10～15 times of the medicinal extract volume that obtains of step (1); The consumption of described sherwood oil is 10～15 times of the medicinal extract volume that obtains of step (1); The consumption of described chloroform is 15～20 times of the medicinal extract volume that obtains of step (1); The described number of times with petroleum ether extraction is 2～3 times; The described number of times with chloroform extraction is 3～4 times.
14. preparation methods as claimed in claim 10, it is characterized in that: in step (3), the specification of described polyamide column is 14-30 order, 30-60 order, 60-100 order, 100-120 order or 200-400 order, and the polymeric amide filler in described polyamide column and the weight ratio of upper pillaring substance are 50:1～80:1; The eluent of described gradient elution is: water → 30% aqueous ethanolic solution → 50% aqueous ethanolic solution → 70% aqueous ethanolic solution → 90% aqueous ethanolic solution, more than be volume percent, in described gradient elution, the consumption of water is 3～5 times of polyamide column volume, the consumption of 30% aqueous ethanolic solution is 4～6 times of polyamide column volume, the consumption of 50% aqueous ethanolic solution is 4～6 times of polyamide column volume, the consumption of 70% aqueous ethanolic solution is 4～6 times of polyamide column volume, and the consumption of 90% aqueous ethanolic solution is 4～6 times of polyamide column volume.
15. preparation methods as claimed in claim 10, is characterized in that: in step (4), described gel column is Sephadex LH
20; Described with the eluent that gel column carries out wash-out be methyl alcohol, the consumption of eluent is 1～2 times of gel column volume.
16. preparation methods as claimed in claim 10, it is characterized in that: step (5) comprises the following step: step (4) gained material is carried out silica gel column chromatography separation by (1 '), collect and detect with silicon thin-layer chromatography, when the volume ratio of developping agent chloroform and methyl alcohol is 15:1, the cut that Rf value is 0.3～0.5; Step (1 ') gained material is carried out silica gel column chromatography separation by (2 '), or silica gel Preparative TLC chromatographic separation, collects with silicon thin-layer chromatography and detect, and developping agent is volume ratio while being the chloroform of 15:1 and methyl alcohol, the cut that Rf value is 0.45.
17. preparation methods as claimed in claim 16, is characterized in that: in step (1 '), during described silica gel column chromatography separates, the granularity of silica gel is 100～200 orders; The weight ratio of silica gel and upper pillaring substance is 50:1～80:1; Eluent is the mixed solvent of sherwood oil and ethyl acetate or the mixed solvent of normal hexane and ethyl acetate; In the time that eluent is the mixed solvent of sherwood oil and ethyl acetate, the volume ratio of sherwood oil and ethyl acetate is 6:1～1:1; Elution process is gradient elution, and in the time that eluent is the mixed solvent of sherwood oil and ethyl acetate, gradient is sherwood oil: ethyl acetate=6:1 → 4:1 → 2:1 → 1:1, and the eluent consumption of each gradient is 4～6 times of silicagel column volume.
18. preparation methods as claimed in claim 16, is characterized in that: in step (2 '), during described silica gel column chromatography separates, eluent is the mixed solvent of chloroform and methyl alcohol; In described silica gel Preparative TLC chromatographic separation, developping agent is the mixed solvent of chloroform and methyl alcohol.
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