CN106279316B - A method of Kaempferol -3-O- is prepared from ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained - Google Patents
A method of Kaempferol -3-O- is prepared from ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained Download PDFInfo
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Abstract
The invention discloses a kind of to prepare Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained method from ginkgo biloba p.e, including, ginkgo biloba p.e is dissolved in ethanol-water solution, ginkgo biloba p.e solution is prepared；One-dimensional liquid chromatogram separation is carried out to the extract solution, obtains one-dimensional crude product；One-dimensional crude product is dissolved in methanol-water solution or ethanol-water solution, the crude product solution is obtained；Two-dimensional liquid chromatography preparation is carried out to the crude product solution, obtains Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.A kind of specific medicinal compound-Kaempferol -3-O- is prepared to coumaric acyl glucose rhamnoside contained in the application from ginkgo biloba p.e; preparation method is simple; lock out operation is convenient; one specific compound is isolated using two-dimensional liquid chromatography method; to increase index components in the research of ginkgo biloba p.e quality control; the test object of active material is enriched, conducive to the raising of ginkgo biloba p.e and its drug standard.
Description
Technical field
The present invention relates to the extraction preparation methods of an effective component in ginkgo biloba p.e, and in particular to a kind of from ginkgo leaf
Kaempferol -3-O- is prepared in extract to coumaric acyl glucose rhamnoside contained method, belongs to technical field of compound preparation.
Background technique
Ginkgo leaf be Ginkgoaceae plant Ginkgo biloba dried leaf, ginkgo biloba p.e be through modern extraction process from ginkgo leaf
The enriched products of the active material of extraction can be used for Alzheimer disease, depression, diabetes, neurological disease, impotence, memory
The treatment of the diseases such as obstacle, peripheral artery disease, intermittent claudication, vertigo and tinnitus.Its main active is flavonoids and terpene
Class.Flavones ingredient includes single flavones, flavonol glycosides, acetyl-flavones alcohol glycosides, biflavone, flavan-3-alcohol class and former pattern
Element etc..Terpene ginkgolides has Ginkgolide A. B. C, J, M and Bilobalide.
State food pharmaceuticals administration general bureau (CFDA) has approved tens of kinds of ginkgo biloba p.e dosage forms, including silver at present
Apricot blade, capsule, particle, soft capsule, dispersible tablet, ball, tincture, drops, oral solution, gingko leaf extract injection etc., above-mentioned system
The method that the control of the quality of agent and ginkgo biloba p.e mostly uses finger-print carries out, and generally accepted ginkgo leaf extracts in the world
The quality index of object is containing 24% or more flavones, 6% or more lactone, and what domestic pharmacopoeia commission promulgated is with ginkgo biloba p.e
The Shu Xuening injection quality standard of raw material provides that the amount of total flavonoids is no less than 24%, ginkalide A, ginkolide B, silver
The total content of 4 kinds of ingredients of apricot lactone C and Bilobalide is no less than 6%.But ginkgo biloba p.e is an extremely complex change
Close object enriched products, containing from inorganic matter to organic matter, from polarity to nonpolarity, from small molecule to large biological molecule it is various at
Point, contain more than 240 a chemical components in ginkgo leaf according to incompletely statistics, and in the finger-print of common ginkgo biloba p.e
Only more than ten of peak, to can not accurately reflect the material base of product.It is well known that the extraction of ginkgo biloba p.e
Method is different, and active compound and its content are different in obtained extract, then its drug effect is not also identical, is prepared by it
Drug applicable symptom and function must be not exactly the same；Strictly, even if extracting method is identical, due to ginkgo leaf raw material batch
Secondary difference, product are different, and the content of chemical component is also not quite similar in finally obtained extract, and above-mentioned factor eventually can be right
The drug effect of drug generates different degrees of influence, therefore, is only carried out with existing quality standard to ginkgo biloba p.e and its preparation
For the effect of quality control not very strictly with specification, largely the presence of unknown substances affects the Accurate Determining to its content, more makes
The about raising of drug standard.
Summary of the invention
It is an object of the invention to which the chemical component in ginkgo biloba p.e is separated and is prepared, further confirm that and
High-purity extracts a kind of chemical component-Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained, to ginkgo biloba p.e
And its assay of medicinal ingredient provides foundation in preparation.
For this purpose, the technical solution that the application takes is,
A method of Kaempferol -3-O- is prepared from ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained, packet
It includes, ginkgo biloba p.e is dissolved in the ethanol-water solution that volumetric concentration is 40-80% by (1), and ginkgo biloba p.e is prepared
Concentration be 10-1000mg/mL extract solution；(2) one-dimensional liquid phase is carried out to the extract solution that the step (1) obtains
Chromatographic isolation obtains one-dimensional crude product；(3) the one-dimensional crude product that step (2) obtains is dissolved in volumetric concentration is 40-80%'s
In methanol-water solution or ethanol-water solution, the concentration for obtaining the one-dimensional crude product is the crude product solution of 20-200mg/mL；
(4) two-dimensional liquid chromatography preparation is carried out to the crude product solution that the step (3) obtains, obtains Kaempferol -3-O- to coumaric acyl
Base glucose rhamnoside.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, the condition that one-dimensional liquid chromatogram separates in the step (2) is that chromatographic column is used fills out by the hydrophilic chromatography of matrix of silica gel
Material；Organic phase in mobile phase is ethyl alcohol or methanol, and water phase is water；Elution requirement is down to 90% with 0-15min organic phase 95%
Gradient carries out or isocratic progress；The component that retention time is 15~20min is collected, the one-dimensional crude product is dried to obtain.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, one-dimensional liquid chromatogram separation in the step (2), organic phase also contains formic acid, and formic acid volumetric concentration is 0.1%；In water phase
Also contain formic acid, formic acid volumetric concentration is 0.1%.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, two-dimensional liquid chromatography condition is in the step (4), and chromatographic column, which is used, is bonded C18 reverse phase filler by matrix of silica gel；Flowing
Organic phase in phase is ethyl alcohol or methanol, and water phase is water；Elution requirement increases to 80% gradient with 0-60min organic phase 15% and carries out
Or isocratic progress；The component that retention time is 50-55min is collected, is dried to obtain Kaempferol -3-O- to coumaric acyl glucose mouse
Lee's glucosides.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, prepared by step (4) two-dimensional liquid chromatography, formic acid is also contained in mobile phase, and formic acid volumetric concentration is 0.1%；In water phase
Also contain formic acid, formic acid volumetric concentration is 0.1%.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, in the step (4) prepared by two-dimensional liquid chromatography, and the organic phase in mobile phase is methanol, and water phase is water.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, in the step (1), ginkgo biloba p.e is dissolved in the ethanol-water solution that volumetric concentration is 50-60%, is prepared
The concentration of ginkgo biloba p.e is the extract solution of 250-550mg/mL.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, in the step (3), it is molten that the one-dimensional crude product that step (2) obtains is dissolved in the methanol-water that volumetric concentration is 50-60%
Liquid or ethanol-water solution, the crude product solution concentration are 60-100mg/mL.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, one-dimensional liquid chromatogram, flow rate of mobile phase 60-120mL/min, column temperature is room temperature or 25-40 DEG C, sample volume 200-3000
μ L/ needle, detector are DAD UV detector；
Two-dimensional liquid chromatography, flow rate of mobile phase 60-120mL/min, column temperature are room temperature or 25-40 DEG C, and sample volume is
1000-3000 μ L/ needle, UV detector Detection wavelength are 360nm.
Kaempferol -3-O- is prepared in above-mentioned slave ginkgo biloba p.e to coumaric acyl glucose rhamnoside contained method
In, two-dimensional liquid chromatography, flow rate of mobile phase 80-100mL/min, column temperature is room temperature or 25-40 DEG C, sample volume 2000-
2500 μ L/ needles, UV detector Detection wavelength are 360nm.
Compared with prior art, the invention has the advantages that,
(1) a kind of specific medicinal compound-Kaempferol -3-O- is prepared to perfume (or spice) in the application from ginkgo biloba p.e
Beans acyl glucose rhamnoside, preparation method is simple, and lock out operation is convenient, isolates a spy using two-dimensional liquid chromatography method
Fixed compound enriches active material to increase index components in the research of ginkgo biloba p.e quality control
Test object is improved particularly existing widely used Floium Ginkgo conducive to the raising of ginkgo biloba p.e and its drug standard
The quality of injection.
(2) applicant is by the connected applications to one-dimensional liquid chromatogram and two-dimensional liquid chromatography, respectively to mobile phase, chromatography
Column and elution requirement are selected, and avoid in ginkgo biloba p.e Multiple components to Kaempferol -3-O- to coumaric acyl grape
Purity is prepared in 90% or more Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained in the interference of sugared rhamnoside.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
The mass spectrum for the compound being prepared in Fig. 1 embodiment of the present invention 1；
The H for the compound being prepared in Fig. 2 embodiment of the present invention 1 is composed；
The C for the compound being prepared in Fig. 3 embodiment of the present invention 1 is composed.
Specific embodiment
The percentage % occurred in the embodiment of the present invention, what is indicated unless otherwise specified is percentage by volume, for example,
" 40% ethanol-water solution " indicate ethyl alcohol aqueous solution wherein ethyl alcohol percentage by volume be 40%；
" 40% methanol-water solution " indicate methanol aqueous solution wherein methanol percentage by volume be 40%；
" ethyl alcohol (contain 0.1% formic acid) " indicates that the percentage by volume of the mixed solution of ethyl alcohol and formic acid wherein formic acid is
0.1%；
" water (contain 0.1% formic acid) " indicates that the percentage by volume of the mixed solution of water and formic acid wherein formic acid is 0.1%；
Embodiment 1
Ginkgo biloba p.e 10g is weighed, the ethanol-water solution of 50mL 40% is dissolved in, ginkgo biloba p.e solution is made,
Concentration is 200mg/mL, crosses 0.45 μm of miillpore filter, carries out one-dimensional liquid chromatogram separation.One-dimensional liquid chromatogram is used is with silica gel
50 × 250mm of hydrophilic chromatograph packing material of matrix, 10 μm (Hua Puxinchuan Science and Technology Ltd.), mobile phase, which uses, contains 0.1% body
The methanol of product concentration formic acid is organic phase, and the water containing 0.1% volumetric concentration formic acid is water phase, and gradient elution mode: 0-15min has
Machine phase concentration is down to 90% stepwise gradient from 95% and is carried out.Absorbing wavelength, preparation temperature are selected using DAD UV detector 360nm
Degree is room temperature, and sample volume is 500 μ L/ needles, and flow rate of mobile phase 90mL/min collects 15~20 minutes fractions, rotated
It is concentrated by evaporation to doing, is the one-dimensional Kaempferol -3-O- for preparing to coumaric acyl glucose rhamnoside contained crude product.With 50% ethyl alcohol-
Aqueous dissolution Kaempferol -3-O- is to coumaric acyl glucose rhamnoside contained crude product, concentration 80mg/mL, through miillpore filter mistake
Filter, carry out two-dimensional liquid chromatography preparation, chromatographic column be using silica gel as matrix hydrophilic chromatograph packing material (50 × 150mm, 5 μm, China
Pu Xinchuan Science and Technology Ltd.) to select the methanol containing 0.1% volumetric concentration formic acid be organic phase to mobile phase, it is dense to contain 0.1% volume
The water for spending formic acid is water phase, using the 15-80% organic phase gradient elution of 0-60min.It is selected using DAD UV detector 360nm
Absorbing wavelength is selected, preparation temperature is room temperature, and sample volume is 1000 μ L/ needles, and flow rate of mobile phase 90mL/min collects retention time
In the fraction of 50-55min, rotary evaporated to dryness obtains Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained compound,
Through liquid-phase chromatographic analysis, purity 95.5%, the one-dimensional Kaempferol -3-O- for preparing is in coumaric acyl glucose rhamnoside contained crude product
Kaempferol -3-O- is 30% to coumaric acyl glucose rhamnoside contained content.
Be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, wherein mass spectrum,1H-
NMR spectra is as illustrated in fig. 1 and 2,
13C-NMR (MeOD) parsing is as follows,
Kaempferol female ring: 156.98 (C2), 135.42 (C3), 178.14 (C4), 161.75 (C5), 98.40 (C6),
164.18 (C7), 93.46 (C8), 156.88 (C9), 104.68 (C10), 121.25 (C1 '), 130.56 (C2 ', C6 '),
115.00 (C3 ', C5 '), 159.67 (C4 ').
3-O- rhamnose: 101.12 (C1 "), 82.26 (C2 "), 70.38 (C3 "), 72.10 (C4 "), 70.55 (C5 "),
16.33(C6”)。
2 "-glucose: 105.66 (C1 " '), 73.99 (C2 " '), 76.29 (C3 " '), 70.78 (C4 " '), 73.73
(C5 " '), 63.12 (C6 " ').
6 "-to coumaric acyl: 167.40 (C1 " "), 113.29 (C2 " "), 145.16 (C3 " "), 125.58 (C4 " "),
129.62 (C5 " ", C9 " "), 115.23 (C6 " ", C8 " "), 160.02 (C7 " ").
Comprehensive identification is Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained, and the molecular formula of compound is
C36H36O17, molecular weight 740.1982, structural formula is as follows
Embodiment 2
Ginkgo biloba p.e 1g is weighed, the ethanol-water solution of 100mL volumetric concentration 80% is dissolved in, ginkgo leaf is made and extracts
Object solution, concentration 10mg/mL cross 0.45 μm of miillpore filter, carry out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram use with
Silica gel is the hydrophilic chromatograph packing material (50 × 250mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) of matrix, and mobile phase uses second
Alcohol is organic phase, and water is water phase, and organic phase concentration is 90% isocratic, selects absorbing wavelength using DAD UV detector 360nm,
Preparation temperature is 40 DEG C, and sample volume is 200 μ L/ needles, and flow rate of mobile phase 60mL/min collects 15~20 minutes fractions, into
Row rotary evaporation is concentrated to dryness, and is the one-dimensional Kaempferol -3-O- for preparing to coumaric acyl glucose rhamnoside contained crude product.With 40%
Ethanol-water solution dissolves Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained crude product, concentration 20mg/mL, through micropore
Membrane filtration, carry out two-dimensional liquid chromatography preparation, chromatographic column be by matrix of silica gel be bonded C18 reverse phase filler (50 × 150mm,
10 μm, Hua Puxinchuan Science and Technology Ltd.), it is organic phase that mobile phase, which selects the ethyl alcohol containing 0.1% volumetric concentration formic acid, is contained
The water of 0.1% volumetric concentration formic acid is water phase, and using isocratic elution mode, organic phase concentration is 20% totally 60 minutes.Using DAD
UV detector 360nm selects absorbing wavelength, and preparation temperature is room temperature, and sample volume is 1000 μ L/ needles, and flow rate of mobile phase is
100mL/min, collects the fraction of retention time 50-55min, and rotary evaporated to dryness obtains Kaempferol -3-O- to coumaric acyl Portugal
Grape sugar rhamnoside compound, through liquid-phase chromatographic analysis, the Kaempferol -3-O- of two dimension preparation is to coumaric acyl glucose rhamnose
Kaempferol -3-O- is 94.0% to coumaric acyl glucose rhamnoside contained content in glycosides crude product,
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Embodiment 3
Ginkgo biloba p.e 100g is weighed, the ethanol-water solution of 200mL 50% is dissolved in, it is molten that ginkgo biloba p.e is made
Liquid, concentration 500mg/mL cross 0.45 μm of miillpore filter, carry out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram is used with silicon
Glue is the hydrophilic chromatograph packing material (50 × 250mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.) of matrix, and mobile phase uses ethyl alcohol
(containing 0.1% formic acid) is organic phase, and water (containing 0.1% formic acid) is water phase, is eluted using 95% organic equality mode.Using
DAD UV detector 360nm selects absorbing wavelength, and preparation temperature is 30 DEG C, and sample volume is 3000 μ L/ needles, and flow rate of mobile phase is
120mL/min collects 15~20 minutes fractions, carries out rotary evaporation and is concentrated to dryness, and is the one-dimensional Kaempferol -3-O- for preparing to perfume (or spice)
Beans acyl glucose rhamnoside crude product.With 60% ethanol-water solution dissolution Kaempferol -3-O- to coumaric acyl glucose
Rhamnoside crude product, concentration 80mg/mL carry out two-dimensional liquid chromatography preparation through filtering with microporous membrane, and chromatographic column is with silica gel
It is bonded C18 reverse phase filler (50 × 150mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.) for matrix, mobile phase selection ethyl alcohol (contains
0.1% formic acid) it is organic phase, water (containing 0.1% formic acid) is water phase, rises to 80% gradient elution from 20% using organic phase.It adopts
Absorbing wavelength is selected with DAD UV detector 360nm, preparation temperature is 25 DEG C, and sample volume is 3000 μ L/ needles, flow rate of mobile phase
For 120mL/min, the fraction of retention time 50-55min is collected, rotary evaporated to dryness obtains Kaempferol -3-O- to coumaric acyl
Glucose rhamnoside compound, through liquid-phase chromatographic analysis, purity 96.5%, the one-dimensional Kaempferol -3-O- for preparing is to coumaric acyl
Kaempferol -3-O- is 29% to coumaric acyl glucose rhamnoside contained content in base glucose rhamnoside crude product.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Embodiment 4
Ginkgo biloba p.e 100g is weighed, the ethanol-water solution of 100mL40% is dissolved in, it is molten that ginkgo biloba p.e is made
Liquid, concentration 1000mg/mL cross 0.45 μm of miillpore filter, carry out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram is used with silicon
Glue is the hydrophilic chromatograph packing material (50 × 250mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.) of matrix, and mobile phase uses ethyl alcohol
(containing 0.1% formic acid) is organic phase, and water (containing 0.1% formic acid) is water phase, and using gradient elution: organic phase concentration is by 95% through 15
Minute is reduced to 90%, selects absorbing wavelength using DAD UV detector 360nm, preparation temperature is 25 DEG C, sample volume 2000
μ L/ needle, flow rate of mobile phase 80mL/min collect 15~20 minutes fractions, carry out rotary evaporation and are concentrated to dryness, and are one-dimensional system
Standby Kaempferol -3-O- is to coumaric acyl glucose rhamnoside contained crude product.Kaempferol -3-O- is dissolved with 80% ethanol-water solution
To coumaric acyl glucose rhamnoside contained crude product, concentration 50mg/mL carries out two-dimensional liquid chromatography system through filtering with microporous membrane
Standby, chromatographic column is to be bonded C18 reverse phase filler (50 × 150mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.) by matrix of silica gel, stream
It is organic phase that dynamic phase, which selects ethyl alcohol (containing 0.1% formic acid), and water (containing 0.1% formic acid) is water phase, is washed using 15% organic phase concentration
It is de-.Absorbing wavelength is selected using DAD UV detector 360nm, preparation temperature is 40 DEG C, and sample volume is 200 μ L/ needles, mobile phase
Flow velocity is 60mL/min, collects retention time in 50-55min fraction, rotary evaporated to dryness obtains Kaempferol -3-O- to tonka-bean
Acyl glucose rhamnoside compound, through liquid-phase chromatographic analysis, purity 94.2%, the one-dimensional Kaempferol -3-O- for preparing is to perfume (or spice)
Kaempferol -3-O- is 35% to coumaric acyl glucose rhamnoside contained content in beans acyl glucose rhamnoside crude product.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Embodiment 5
Ginkgo biloba p.e is dissolved in the ethanol-water solution of volumetric concentration 55%, ginkgo biloba p.e solution is made, it is dense
Degree is 550mg/mL, crosses 0.45 μm of miillpore filter, carries out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram is used using silica gel as base
The hydrophilic chromatograph packing material (50 × 250mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) of matter, mobile phase (are contained using ethyl alcohol
0.1% formic acid) it is organic phase, water (containing 0.1% formic acid) is water phase, and type of elution 0-15min organic phase concentration is down to from 95%
90% gradient carries out, and selects absorbing wavelength using DAD UV detector 360nm, preparation temperature is 30 DEG C, and sample volume is 2000 μ
L/ needle, flow rate of mobile phase 100mL/min collect 15~20 minutes fractions, carry out rotary evaporation and are concentrated to dryness, and are one-dimensional system
Standby Kaempferol -3-O- is to coumaric acyl glucose rhamnoside contained crude product.Kaempferol -3-O- is dissolved with 55% methanol-water solution
To coumaric acyl glucose rhamnoside contained crude product, concentration 60mg/mL carries out two-dimensional liquid chromatography system through filtering with microporous membrane
Standby, chromatographic column is to be bonded C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) by matrix of silica gel,
It is organic phase that mobile phase, which selects methanol (containing 0.1% formic acid), and water (containing 0.1% formic acid) is water phase, using gradient elution mode, 0-
60 minutes organic phase concentrations increase from 20% to 80%.Absorbing wavelength is selected using DAD UV detector 360nm, preparation temperature is
Room temperature, sample volume are 2000 μ L/ needles, and flow rate of mobile phase 90mL/min collects the fraction of retention time 50-55min, and rotation is steamed
It is sent to dry, obtains Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained compound, through liquid-phase chromatographic analysis, two dimension preparation
Kaempferol -3-O- to Kaempferol -3-O- in coumaric acyl glucose rhamnoside contained crude product to coumaric acyl glucose rhamnose
The content of glycosides is 98.2.0%, and the one-dimensional Kaempferol -3-O- for preparing is to Kaempferol -3- in coumaric acyl glucose rhamnoside contained crude product
O- is 36% to coumaric acyl glucose rhamnoside contained content.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Embodiment 6
Ginkgo biloba p.e is dissolved in the ethanol-water solution of volumetric concentration 60%, ginkgo biloba p.e solution is made, it is dense
Degree is 250mg/mL, crosses 0.45 μm of miillpore filter, carries out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram is used using silica gel as base
The hydrophilic chromatograph packing material (50 × 250mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) of matter, mobile phase (are contained using ethyl alcohol
0.1% formic acid) it is organic phase, water (containing 0.1% formic acid) is water phase, and type of elution 0-15min organic phase concentration is down to from 95%
90% gradient carries out, and selects absorbing wavelength using DAD UV detector 360nm, preparation temperature is 30 DEG C, and sample volume is 2000 μ
L/ needle, flow rate of mobile phase 100mL/min collect 15~20 minutes fractions, carry out rotary evaporation and are concentrated to dryness, and are one-dimensional system
Standby Kaempferol -3-O- is to coumaric acyl glucose rhamnoside contained crude product.Kaempferol -3-O- is dissolved with 55% ethanol-water solution
To coumaric acyl glucose rhamnoside contained crude product, concentration 100mg/mL carries out two-dimensional liquid chromatography system through filtering with microporous membrane
Standby, chromatographic column is to be bonded C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) by matrix of silica gel,
It is organic phase that mobile phase, which selects ethyl alcohol (containing 0.1% formic acid), and water (containing 0.1% formic acid) is water phase, using gradient elution mode, 0-
60 minutes organic phase concentrations increase from 20% to 80%.Absorbing wavelength is selected using DAD UV detector 360nm, preparation temperature is
Room temperature, sample volume are 2500 μ L/ needles, and flow rate of mobile phase 90mL/min collects the fraction of retention time 50-55min, and rotation is steamed
It is sent to dry, obtains Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained compound, through liquid-phase chromatographic analysis, two dimension preparation
Kaempferol -3-O- to Kaempferol -3-O- in coumaric acyl glucose rhamnoside contained crude product to coumaric acyl glucose rhamnose
The content of glycosides is 97.8%, and the one-dimensional Kaempferol -3-O- for preparing is to Kaempferol -3-O- in coumaric acyl glucose rhamnoside contained crude product
It is 36% to coumaric acyl glucose rhamnoside contained content.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Embodiment 7
Ginkgo biloba p.e is dissolved in the ethanol-water solution of volumetric concentration 45%, ginkgo biloba p.e solution is made, it is dense
Degree is 400mg/mL, crosses 0.45 μm of miillpore filter, carries out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram is used using silica gel as base
The hydrophilic chromatograph packing material (50 × 250mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) of matter, mobile phase use ethyl alcohol to be organic
Phase, water are water phase, and organic phase concentration is 90% isocratic, select absorbing wavelength, preparation temperature using DAD UV detector 360nm
It is 40 DEG C, sample volume is 2500 μ L/ needles, and flow rate of mobile phase 70mL/min collects 15-20 minutes fractions, carries out rotation steaming
Hair is concentrated to dryness, and is the one-dimensional Kaempferol -3-O- for preparing to coumaric acyl glucose rhamnoside contained crude product.With 50% methanol-water
Solution dissolves Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained crude product, concentration 70mg/mL, through filtering with microporous membrane,
Carry out two-dimensional liquid chromatography preparation, chromatographic column be by matrix of silica gel be bonded C18 reverse phase filler (50 × 150mm, 10 μm, Hua Pu
Xin Chuan Science and Technology Ltd.), it is organic phase that mobile phase, which selects methanol (containing 0.1% formic acid), and water (containing 0.1% formic acid) is water phase,
Using isocratic elution mode, organic phase concentration is 20% totally 60 minutes.Absorbing wavelength is selected using DAD UV detector 360nm,
Preparation temperature is room temperature, and sample volume is 2000 μ L/ needles, and flow rate of mobile phase 80mL/min collects retention time 50-55min's
Fraction, rotary evaporated to dryness obtain Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained compound, through liquid chromatogram point
Analysis, the Kaempferol -3-O- of two dimension preparation is to Kaempferol -3-O- in coumaric acyl glucose rhamnoside contained crude product to coumaric acyl Portugal
The content of grape sugar rhamnoside is 94.6%, and the one-dimensional Kaempferol -3-O- for preparing is in coumaric acyl glucose rhamnoside contained crude product
Kaempferol -3-O- is 34% to coumaric acyl glucose rhamnoside contained content.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Embodiment 8
Ginkgo biloba p.e is dissolved in the ethanol-water solution of volumetric concentration 70%, ginkgo biloba p.e solution is made, it is dense
Degree is 150mg/mL, crosses 0.45 μm of miillpore filter, carries out one-dimensional liquid chromatogram preparation.One-dimensional liquid chromatogram is used using silica gel as base
The hydrophilic chromatograph packing material (50 × 250mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) of matter, mobile phase use ethyl alcohol to be organic
Phase, water are water phase, and organic phase concentration is 95% isocratic, select absorbing wavelength, preparation temperature using DAD UV detector 360nm
For room temperature, sample volume is 800 μ L/ needles, and flow rate of mobile phase 110mL/min collects 15-20 minutes fractions, carries out rotation steaming
Hair is concentrated to dryness, and is the one-dimensional Kaempferol -3-O- for preparing to coumaric acyl glucose rhamnoside contained crude product.With 60% alcohol-water
Solution dissolves Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained crude product, concentration 200mg/mL, through miillpore filter mistake
Filter, carry out two-dimensional liquid chromatography preparation, chromatographic column be by matrix of silica gel be bonded C18 reverse phase filler (50 × 150mm, 10 μm, China
Pu Xinchuan Science and Technology Ltd.), it is organic phase that mobile phase, which selects ethyl alcohol (containing 0.1% formic acid), and water (containing 0.1% formic acid) is water
Phase, using isocratic elution mode, organic phase concentration is 15% totally 60 minutes.It selects to absorb wave using DAD UV detector 360nm
Long, preparation temperature is room temperature, and sample volume is 2500 μ L/ needles, and flow rate of mobile phase 100mL/min collects retention time 50-
The fraction of 55min, rotary evaporated to dryness obtain Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained compound, through liquid phase
Chromatography, the Kaempferol -3-O- of two dimension preparation is to Kaempferol -3-O- in coumaric acyl glucose rhamnoside contained crude product to tonka-bean
The content of acyl glucose rhamnoside is 95.7%, and the one-dimensional Kaempferol -3-O- for preparing is to coumaric acyl glucose rhamnoside contained
Kaempferol -3-O- is 33% to coumaric acyl glucose rhamnoside contained content in crude product.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Kaempferol -3-O- to coumaric acyl glucose rhamnoside contained.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (7)
1. a kind of Kaempferol -3-O- for preparing from ginkgo biloba p.e is to coumaric acyl glucose rhamnoside contained method, including,
(1) ginkgo biloba p.e is dissolved in the ethanol-water solution that volumetric concentration is 40-80%, ginkgo leaf extraction is prepared
The concentration of object is the extract solution of 10-1000mg/mL；
(2) one-dimensional liquid chromatogram separation is carried out to the extract solution that the step (1) obtains, obtains one-dimensional crude product, it is one-dimensional
The condition of liquid chromatogram separation is that chromatographic column uses the hydrophilic chromatograph packing material using silica gel as matrix；Organic phase in mobile phase
For ethyl alcohol, water phase is water；Elution requirement is down to the progress of 90% gradient or isocratic progress with 0-15min organic phase 95%；It collects and protects
Staying the time is the component of 15~20min, is dried to obtain the one-dimensional crude product, wherein organic phase also contains formic acid, formic acid volume
Concentration is 0.1%；Also contain formic acid in water phase, formic acid volumetric concentration is 0.1%；
(3) the one-dimensional crude product that step (2) obtains is dissolved in the methanol-water solution or alcohol-water that volumetric concentration is 40-80%
In solution, the concentration for obtaining the one-dimensional crude product is the crude product solution of 20-200mg/mL；
(4) two-dimensional liquid chromatography preparation is carried out to the crude product solution that the step (3) obtains, obtains Kaempferol -3-O- to perfume (or spice)
Beans acyl glucose rhamnoside；
Two-dimensional liquid chromatography condition is in the step (4), and chromatographic column, which is used, is bonded C18 reverse phase filler by matrix of silica gel；Stream
Organic phase in dynamic phase is ethyl alcohol or methanol, and water phase is water；Elution requirement with 0-60min organic phase 15% increase to 80% gradient into
Capable or isocratic progress；The component that retention time is 50-55min is collected, is dried to obtain Kaempferol -3-O- to coumaric acyl glucose
Rhamnoside.
2. Kaempferol-the 3-O- according to claim 1 for preparing from ginkgo biloba p.e is to coumaric acyl glucose sandlwood
The method of glucosides, which is characterized in that
Prepared by step (4) two-dimensional liquid chromatography, formic acid is also contained in mobile phase, and formic acid volumetric concentration is 0.1%；In water phase
Also contain formic acid, formic acid volumetric concentration is 0.1%.
3. Kaempferol-the 3-O- according to claim 2 for preparing from ginkgo biloba p.e is to coumaric acyl glucose sandlwood
The method of glucosides, which is characterized in that
In the step (4) prepared by two-dimensional liquid chromatography, and the organic phase in mobile phase is methanol, and water phase is water.
4. Kaempferol-the 3-O- according to claim 1 to 3 for preparing from ginkgo biloba p.e is to coumaric acyl grape
The method of sugared rhamnoside, which is characterized in that
In the step (1), ginkgo biloba p.e is dissolved in the ethanol-water solution that volumetric concentration is 50-60%, is prepared
The concentration of ginkgo biloba p.e is the extract solution of 250-550mg/mL.
5. Kaempferol-the 3-O- according to claim 4 for preparing from ginkgo biloba p.e is to coumaric acyl glucose sandlwood
The method of glucosides, which is characterized in that
In the step (3), it is molten that the one-dimensional crude product that step (2) obtains is dissolved in the methanol-water that volumetric concentration is 50-60%
Liquid or ethanol-water solution, the crude product solution concentration are 60-100mg/mL.
6. Kaempferol-the 3-O- according to claim 5 for preparing from ginkgo biloba p.e is to coumaric acyl glucose sandlwood
The method of glucosides, which is characterized in that
One-dimensional liquid chromatogram, flow rate of mobile phase 60-120mL/min, column temperature are room temperature or 25-40 DEG C, sample volume 200-
3000 μ L/ needles, detector are DAD UV detector；
Two-dimensional liquid chromatography, flow rate of mobile phase 60-120mL/min, column temperature are room temperature or 25-40 DEG C, sample volume 1000-
3000 μ L/ needles, UV detector Detection wavelength are 360nm.
7. Kaempferol-the 3-O- according to claim 6 for preparing from ginkgo biloba p.e is to coumaric acyl glucose sandlwood
The method of glucosides, which is characterized in that
Two-dimensional liquid chromatography, flow rate of mobile phase 80-100mL/min, column temperature are room temperature or 25-40 DEG C, sample volume 2000-
2500 μ L/ needles, UV detector Detection wavelength are 360nm.
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