CN106279317A - A kind of method preparing Quercetin-3-o-glucoside from Folium Ginkgo extract - Google Patents
A kind of method preparing Quercetin-3-o-glucoside from Folium Ginkgo extract Download PDFInfo
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- CN106279317A CN106279317A CN201510247609.3A CN201510247609A CN106279317A CN 106279317 A CN106279317 A CN 106279317A CN 201510247609 A CN201510247609 A CN 201510247609A CN 106279317 A CN106279317 A CN 106279317A
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Abstract
The invention discloses a kind of method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, including, Folium Ginkgo extract is dissolved in ethanol-water solution, prepares Folium Ginkgo extract solution；Described extract solution is carried out one-dimensional liquid chromatograph separation, obtains one-dimensional thick product；One-dimensional thick product is dissolved in methanol-water solution or ethanol-water solution, obtains described thick product solution；Described thick product solution is carried out two-dimensional liquid chromatography prepare, obtain Quercitrin-3-O-glucoside.The application prepares a kind of clear and definite medicinal compound-Quercitrin-3-O-glucoside from Folium Ginkgo extract, its preparation method is simple, lock out operation is convenient, two-dimensional liquid chromatography method is used to isolate a specific compound, thus add index components in the research of Folium Ginkgo extract quality control, enrich the detection object of active substance, beneficially Folium Ginkgo extract and the raising of drug standard thereof.
Description
Technical field
The present invention relates to the extraction preparation method of an effective ingredient in Folium Ginkgo extract, be specifically related to one
Plant the method preparing Quercetin-3-o-glucoside from Folium Ginkgo extract, belong to compound and prepare skill
Art field.
Background technology
Folium Ginkgo is the dried leaves of Ginkgoaceae plant Ginkgo biloba, and Folium Ginkgo extract is through modern extraction process
The enriched products of active substance extracted from Folium Ginkgo, can be used for Alzheimer, depression,
Diabetes, sacred disease, sexual impotence, dysmnesia, peripheral blood vessel, intermittent claudication, vertigo
The treatment of the diseases such as tinnitus.Its main active is flavonoid and terpenoid.Flavones ingredient includes list
Flavone, flavonol glycosides, acetyl-flavones alcohol glycosides, bisflavone, flavan-3-alcohol class and proanthocyanidin etc..
Terpenoid bilobalide has Ginkgolide A. B. C, J, M and bilobalide.
State food pharmaceuticals administration general bureau (CFDA) have approved tens of kinds of Folium Ginkgo extract at present
Dosage form, including Folium Ginkgo, capsule, granule, soft capsule, dispersible tablet, ball, tincture, drop, mouth
Taking liquid, gingko leaf extract injection etc., the quality control of above-mentioned preparation and Folium Ginkgo extract is adopted more
Carrying out by the method for finger printing, the quality index of the Folium Ginkgo extract that It is generally accepted in the world is for containing
Flavone more than 24%, lactone more than 6%, what domestic committee of pharmacopeia promulgated is former with Folium Ginkgo extract
Material SHUXUENING ZHUSHEYE quality standard specify total flavonoids amount be no less than 24%, ginkalide A,
The total content of ginkalide B, ginkalide C and 4 kinds of compositions of bilobalide is no less than 6%.But it is silver-colored
Folium Pruni extract is an extremely complex compound enriched products, containing from inorganic matter to Organic substance,
From polarity to nonpolar, from the various compositions of little molecule to biomacromolecule, Semen Ginkgo according to incompletely statistics
Containing more than 240 chemical composition in leaf, and the finger printing of common Folium Ginkgo extract only has
Ten several peaks, thus the material base of reflection product that cannot be the most definite.It is well known that
The extracting method of Folium Ginkgo extract is different, and in its extract obtained, active compound and content thereof are not
With, then its drug effect also differs, it applicable symptom of the medicine prepared and function must be incomplete
Identical；Strictly, even if extracting method is identical, due to Folium Ginkgo raw material batch difference, product not
With, in its extract finally given, the content of chemical composition is also not quite similar, and the meeting eventually of above-mentioned factor
The drug effect of medicine is produced impact in various degree, therefore, only with existing quality standard, Folium Ginkgo is carried
Take thing and preparation carries out that the effect of quality control is the strictest and specification, the existence of a large amount of not clear materials
Have impact on the Accurate Determining to its content, more constrain the raising of drug standard.
Summary of the invention
It is an object of the invention to the chemical composition in Folium Ginkgo extract be separated and prepares, enter
One step confirms and high-purity extracts a kind of chemical composition-Quercitrin-3-O-glucoside, to Folium Ginkgo
In extract and preparation thereof, the assay of medicinal ingredient provides foundation.
To this end, the technical scheme that the application takes is,
A kind of method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, including,
(1) Folium Ginkgo extract is dissolved in the ethanol-water solution that volumetric concentration is 40-80%, preparation
Obtain the extract solution that concentration is 10-1000mg/mL of Folium Ginkgo extract；(2) to described step
(1) extract solution obtained carries out one-dimensional liquid chromatograph separation, obtains one-dimensional thick product；(3) will
The one-dimensional thick product that step (2) obtains is dissolved in the methanol-water solution or second that volumetric concentration is 40-80%
In alcohol-water solution, obtain the thick product solution that concentration is 20-200mg/mL of described one-dimensional thick product；
(4) the thick product solution obtaining described step (3) carries out two-dimensional liquid chromatography and prepares, and obtains Mongolian oak
Pi Su-3-O-glucoside.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(2) condition that in, one-dimensional liquid chromatograph separates is, chromatographic column uses the hydrophilic color with silica gel as substrate
Spectrum filler；Organic facies in flowing mutually is ethanol or methanol, and aqueous phase is water；Elution requirement is with 0-15min
Organic facies 95% is down to 90% gradient and is carried out or isocratic carry out；Collecting retention time is 15～20min
Component, is dried to obtain described one-dimensional thick product.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(2) in, one-dimensional liquid chromatograph separates, and organic facies is possibly together with formic acid, and formic acid volumetric concentration is 0.1%；Water
Possibly together with formic acid in mutually, formic acid volumetric concentration is 0.1%.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(4) in, two-dimensional liquid chromatography condition is, chromatographic column uses with silica gel for substrate-bound C18 reverse phase filler；
Organic facies in flowing mutually is ethanol or methanol, and aqueous phase is water；Elution requirement is with 0-60min organic facies
15% increases to 80% gradient carries out or isocratic carries out；Collecting retention time is the component of 30-40min, dry
Dry obtain Quercitrin-3-O-glucoside.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(4) prepared by two-dimensional liquid chromatography, and possibly together with formic acid in flowing mutually, formic acid volumetric concentration is 0.1%；Water
Possibly together with formic acid in mutually, formic acid volumetric concentration is 0.1%.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(4) in prepared by two-dimensional liquid chromatography, and the organic facies in flowing mutually is methanol, and aqueous phase is water.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(1) in, Folium Ginkgo extract is dissolved in the ethanol-water solution that volumetric concentration is 50-60%, preparation
Obtain the extract solution that concentration is 250-550mg/mL of Folium Ginkgo extract.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, described step
(3), in, the one-dimensional thick product that step (2) obtains is dissolved in the methanol that volumetric concentration is 50-60%
-aqueous solution or ethanol-water solution, described thick product solution concentration is 60-100mg/mL.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, one-dimensional liquid phase
Chromatograph, flow rate of mobile phase is 60-120mL/min, and column temperature is room temperature or 25-40 DEG C, and sample size is
200-3000 μ L/ pin, detector is DAD UV-detector；
Two-dimensional liquid chromatography, flow rate of mobile phase is 60-120mL/min, and column temperature is room temperature or 25-40 DEG C,
Sample size is 1000-3000 μ L/ pin, and UV-detector detection wavelength is 360nm.
In the above-mentioned method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, two dimension liquid phase
Chromatograph, flow rate of mobile phase is 80-100mL/min, and column temperature is room temperature or 25-40 DEG C, and sample size is
2000-2500 μ L/ pin, UV-detector detection wavelength is 360nm.
Compared with prior art, the invention have the advantages that,
(1) the application prepares a kind of clear and definite medicinal compound-Cortex querci dentatae from Folium Ginkgo extract
Element-3-O-glucoside, its preparation method is simple, and lock out operation is convenient, uses two-dimensional liquid chromatography method
Isolate a specific compound, thus add finger in the research of Folium Ginkgo extract quality control
Mark composition, enriches the detection object of active substance, beneficially Folium Ginkgo extract and drug quality mark thereof
Accurate raising, is improved particularly the quality of existing widely used SHUXUENING ZHUSHEYE.
(2) applicant is by the connected applications to one-dimensional liquid chromatograph Yu two-dimensional liquid chromatography, respectively
Flowing phase, chromatographic column and elution requirement selected, it is to avoid Multiple components in Folium Ginkgo extract
Interference to Quercitrin-3-O-glucoside, prepares purity Quercetin-3-O-Portugal more than 90%
Polyglycoside.
Accompanying drawing explanation
In order to make present disclosure be more likely to be clearly understood, below according to the concrete reality of the present invention
Executing example and combine accompanying drawing, the present invention is further detailed explanation, wherein
The mass spectrum of the compound prepared in Fig. 1 embodiment of the present invention 1；
The H spectrum of the compound prepared in Fig. 2 embodiment of the present invention 1；
The C spectrum of the compound prepared in Fig. 3 embodiment of the present invention 1.
Detailed description of the invention
The percent % occurred in the embodiment of the present invention, represent if no special instructions is percentage by volume, such as,
" ethanol-water solution of 40% " represents that the percentage by volume of the aqueous solution wherein ethanol of ethanol is 40%；
" methanol-water solution of 40% " represents that the percentage by volume of the aqueous solution wherein methanol of methanol is 40%；
" ethanol (containing 0.1% formic acid) " represents the volume basis of ethanol and the mixed solution wherein formic acid of formic acid
Number is 0.1%；
" water (containing 0.1% formic acid) " represents that water with the percentage by volume of the mixed solution wherein formic acid of formic acid is
0.1%；
Embodiment 1
Weigh Folium Ginkgo extract 10g, be dissolved in the ethanol-water solution of 50mL 40%, prepare Semen Ginkgo
Leaf extract solution, concentration is 200mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid phase
Chromatographic isolation.One-dimensional liquid chromatograph uses the hydrophilic chromatograph packing material 50 × 250mm with silica gel as substrate,
10 μm (Hua Puxinchuan Science and Technology Ltd.), flowing uses the methanol containing 0.1% volumetric concentration formic acid mutually
For organic facies, the water containing 0.1% volumetric concentration formic acid is aqueous phase, gradient elution mode: 0-15min has
Machine phase concentration is down to 90% stepwise gradient from 95% and is carried out.Use the choosing of DAD UV-detector 360nm
Selecting absorbing wavelength, preparation temperature is room temperature, and sample size is 500 μ L/ pins, and flow rate of mobile phase is 90
ML/min, collects the fraction of 15～20 minutes, carries out rotary evaporation and be concentrated to dryness, prepare Mongolian oak for one-dimensional
Pi Su-3-O-glucoside crude product.Ethanol-water solution with 50% dissolves Quercitrin-3-O-glucoside
Crude product, concentration is 80mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, chromatograph
Post is that (China's spectrum newly creates science and technology to the hydrophilic chromatograph packing material with silica gel as substrate for 50 × 150mm, 5 μm
Company limited) flowing selects containing the methanol of 0.1% volumetric concentration formic acid to be organic facies, containing 0.1% volume mutually
The water of concentration formic acid is aqueous phase, uses the 15-80% organic facies gradient elution of 0-60min.Use DAD
UV-detector 360nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 1000 μ L/
Pin, flow rate of mobile phase is 90mL/min, collects the retention time fraction at 30-40min, rotates and steam
Sending to do, obtain Quercitrin-3-O-glucoside compound, through liquid-phase chromatographic analysis, purity is 95.5%,
It is one-dimensional that to prepare the content of Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product be 30%.
Be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end-product to obtaining is carried out point
Analysis, wherein mass spectrum,1H-NMR spectrum as illustrated in fig. 1 and 2,
13C-NMR (MeOD) resolves as follows,
Quercetin female ring: 157.64 (C2), 134.24 (C3), 178.10 (C4), 161.64 (C5),
98.49 (C6), 164.60 (C7), 93.32 (C8), 157.07 (C9), 104.31 (C10), 121.69
(C1 '), 114.61 (C2 '), 144.51 (C3 '), 148.45 (C4 '), 116.18 (C5 '), 121.81
(C6’)
3-O-glucose: 102.95 (C1), 74.34 (C2 "), 76.98 (C3 "), 69.83 (C4 "),
76.72 (C5 "), 61.17 (C6 ").
Comprehensive identification is Quercitrin-3-O-glucoside, and the molecular formula of compound is C21H20O12, molecule
Amount 464.0959, structural formula is as follows
Embodiment 2
Weigh Folium Ginkgo extract 1g, be dissolved in the ethanol-water solution of 100mL volumetric concentration 80%,
Preparing Folium Ginkgo extract solution, concentration is 10mg/mL, crosses 0.45 μm microporous filter membrane, carries out
Prepared by one-dimensional liquid chromatograph.One-dimensional liquid chromatograph uses the hydrophilic chromatograph packing material with silica gel as substrate
(50 × 250mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), flowing uses ethanol to be organic mutually
Phase, water is aqueous phase, and organic phase concentration is 90% isocratic, uses the choosing of DAD UV-detector 360nm
Selecting absorbing wavelength, preparation temperature is 40 DEG C, and sample size is 200 μ L/ pins, and flow rate of mobile phase is 60
ML/min, collects the fraction of 15～20 minutes, carries out rotary evaporation and be concentrated to dryness, prepare Mongolian oak for one-dimensional
Pi Su-3-O-glucoside crude product.Ethanol-water solution with 40% dissolves Quercitrin-3-O-glucoside
Crude product, concentration is 20mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, chromatograph
Post is that for substrate-bound C18 reverse phase filler, (China's spectrum newly creates science and technology for 50 × 150mm, 10 μm with silica gel
Company limited), flowing selects the ethanol containing 0.1% volumetric concentration formic acid to be organic facies mutually, containing 0.1% body
The water of volume concentrations formic acid is aqueous phase, uses isocratic elution mode, and organic phase concentration is 20% totally 60 points
Clock.Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature, sample introduction
Amount is 1000 μ L/ pins, and flow rate of mobile phase is 100mL/min, collects evaporating of retention time 30-40min
Point, rotary evaporated to dryness, obtain Quercitrin-3-O-glucoside compound, through liquid-phase chromatographic analysis,
In the Quercitrin-3-O-glucoside crude product of two dimension preparation, the content of Quercitrin-3-O-glucoside is
94.0%, one-dimensional prepare the content of Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product
It is 32%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Embodiment 3
Weigh Folium Ginkgo extract 100g, be dissolved in the ethanol-water solution of 200mL 50%, prepare silver
Folium Pruni extract solution, concentration is 500mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid
Prepared by phase chromatograph.One-dimensional liquid chromatograph uses the hydrophilic chromatograph packing material (50 × 250 with silica gel as substrate
Mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.), flowing uses the ethanol (containing 0.1% formic acid) to be mutually
Organic facies, water (containing 0.1% formic acid) is aqueous phase, uses 95% organic facies isocratic mode eluting.Use
DAD UV-detector 360nm selects absorbing wavelength, and preparation temperature is 30 DEG C, and sample size is 3000
μ L/ pin, flow rate of mobile phase is 120mL/min, collects the fraction of 15～20 minutes, carries out rotating and steams
Send out and be concentrated to dryness, prepare Quercitrin-3-O-glucoside crude product for one-dimensional.Alcohol-water with 60% is molten
Liquid dissolves Quercitrin-3-O-glucoside crude product, and concentration is 80mg/mL, through filtering with microporous membrane, enters
Prepared by row two-dimensional liquid chromatography, chromatographic column is with silica gel for substrate-bound C18 reverse phase filler (50 × 150
Mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.), flowing selects the ethanol (containing 0.1% formic acid) to be mutually
Organic facies, water (containing 0.1% formic acid) is aqueous phase, uses organic facies to rise to 80% gradient elution from 20%.
Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is 25 DEG C, and sample size is
3000 μ L/ pins, flow rate of mobile phase is 120mL/min, collects the fraction of retention time 35-40min,
Rotary evaporated to dryness, obtains Quercitrin-3-O-glucoside compound, through liquid-phase chromatographic analysis, purity
It is 96.5%, one-dimensional prepares containing of Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product
Amount is 29%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Embodiment 4
Weigh Folium Ginkgo extract 100g, be dissolved in the ethanol-water solution of 100mL40%, prepare Semen Ginkgo
Leaf extract solution, concentration is 1000mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid
Prepared by phase chromatograph.One-dimensional liquid chromatograph uses the hydrophilic chromatograph packing material (50 × 250 with silica gel as substrate
Mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.), flowing uses the ethanol (containing 0.1% formic acid) to be mutually
Organic facies, water (containing 0.1% formic acid) is aqueous phase, uses gradient elution: organic phase concentration is by 95% warp
Within 15 minutes, it is reduced to 90%, uses DAD UV-detector 360nm to select absorbing wavelength, preparation temperature
Degree is 25 DEG C, and sample size is 2000 μ L/ pins, and flow rate of mobile phase is 80mL/min, collects 15～20
Minute fraction, carry out rotary evaporation and be concentrated to dryness, to prepare Quercitrin-3-O-glucoside thick for one-dimensional
Product.Ethanol-water solution with 80% dissolves Quercitrin-3-O-glucoside crude product, and concentration is 50
Mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is with silica gel as base
Matter bonding C18 reverse phase filler (50 × 150mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.), flowing
Selecting ethanol (containing 0.1% formic acid) mutually is organic facies, and water (containing 0.1% formic acid) is aqueous phase, uses
15% organic phase concentration eluting.DAD UV-detector 360nm is used to select absorbing wavelength, preparation
Temperature is 40 DEG C, and sample size is 200 μ L/ pins, and flow rate of mobile phase is 60mL/min, when collecting reservation
Between in 30-35min fraction, rotary evaporated to dryness, obtain Quercitrin-3-O-glucoside compound, warp
Liquid-phase chromatographic analysis, purity is 94.2%, one-dimensional prepares Cortex querci dentatae in Quercitrin-3-O-glucoside crude product
The content of element-3-O-glucoside is 35%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Embodiment 5
Folium Ginkgo extract is dissolved in the ethanol-water solution of volumetric concentration 55%, prepares Folium Ginkgo and extract
Thing solution, concentration is 550mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph system
Standby.One-dimensional liquid chromatograph use hydrophilic chromatograph packing material with silica gel as substrate (50 × 250mm, 10
μm, Hua Puxinchuan Science and Technology Ltd.), it is organic facies that flowing uses ethanol (containing 0.1% formic acid) mutually,
Water (containing 0.1% formic acid) is aqueous phase, and type of elution 0-15min organic phase concentration is down to 90% from 95%
Gradient is carried out, and uses DAD UV-detector 360nm to select absorbing wavelength, and preparation temperature is 30 DEG C,
Sample size is 2000 μ L/ pins, and flow rate of mobile phase is 100mL/min, collects the fraction of 15～20 minutes,
Carry out rotary evaporation to be concentrated to dryness, prepare Quercitrin-3-O-glucoside crude product for one-dimensional.With 55%
Methanol-water solution dissolves Quercitrin-3-O-glucoside crude product, and concentration is 60mg/mL, filters through micropore
Membrane filtration, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is for fill out with silica gel for substrate-bound C18 is anti-phase
Material (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), flowing selects methanol (to contain mutually
0.1% formic acid) it is organic facies, water (containing 0.1% formic acid) is aqueous phase, uses gradient elution mode, 0-60
Minute organic phase concentration increases to 80% from 20%.DAD UV-detector 360nm is used to select to absorb
Wavelength, preparation temperature is room temperature, and sample size is 2000 μ L/ pins, and flow rate of mobile phase is 90mL/min,
Collect the fraction of retention time 30-40min, rotary evaporated to dryness, obtain Quercitrin-3-O-glucoside
Compound, Quercetin in liquid-phase chromatographic analysis, the Quercitrin-3-O-glucoside crude product of two dimension preparation
The content of-3-O-glucoside is 98.2.0%, one-dimensional prepares Mongolian oak in Quercitrin-3-O-glucoside crude product
The content of Pi Su-3-O-glucoside is 36%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Embodiment 6
Folium Ginkgo extract is dissolved in the ethanol-water solution of volumetric concentration 60%, prepares Folium Ginkgo and extract
Thing solution, concentration is 250mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph system
Standby.One-dimensional liquid chromatograph use hydrophilic chromatograph packing material with silica gel as substrate (50 × 250mm, 10
μm, Hua Puxinchuan Science and Technology Ltd.), it is organic facies that flowing uses ethanol (containing 0.1% formic acid) mutually,
Water (containing 0.1% formic acid) is aqueous phase, and type of elution 0-15min organic phase concentration is down to 90% from 95%
Gradient is carried out, and uses DAD UV-detector 360nm to select absorbing wavelength, and preparation temperature is 30 DEG C,
Sample size is 2000 μ L/ pins, and flow rate of mobile phase is 100mL/min, collects the fraction of 15～20 minutes,
Carry out rotary evaporation to be concentrated to dryness, prepare Quercitrin-3-O-glucoside crude product for one-dimensional.With 55%
Ethanol-water solution dissolves Quercitrin-3-O-glucoside crude product, and concentration is 100mg/mL, through micropore
Membrane filtration, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is anti-phase with silica gel for substrate-bound C18
Filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), flowing selects ethanol (to contain mutually
0.1% formic acid) it is organic facies, water (containing 0.1% formic acid) is aqueous phase, uses gradient elution mode, 0-60
Minute organic phase concentration increases to 80% from 20%.DAD UV-detector 360nm is used to select to absorb
Wavelength, preparation temperature is room temperature, and sample size is 2500 μ L/ pins, and flow rate of mobile phase is 90mL/min,
Collect the fraction of retention time 30-40min, rotary evaporated to dryness, obtain Quercitrin-3-O-glucoside
Compound, Quercetin in liquid-phase chromatographic analysis, the Quercitrin-3-O-glucoside crude product of two dimension preparation
The content of-3-O-glucoside is 97.8%, one-dimensional prepares Cortex querci dentatae in Quercitrin-3-O-glucoside crude product
The content of element-3-O-glucoside is 36%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Embodiment 7
Folium Ginkgo extract is dissolved in the ethanol-water solution of volumetric concentration 45%, prepares Folium Ginkgo and extract
Thing solution, concentration is 400mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph system
Standby.One-dimensional liquid chromatograph use hydrophilic chromatograph packing material with silica gel as substrate (50 × 250mm, 10
μm, Hua Puxinchuan Science and Technology Ltd.), flowing uses ethanol to be organic facies mutually, and water is aqueous phase, has
Machine phase concentration is 90% isocratic, uses DAD UV-detector 360nm to select absorbing wavelength, preparation
Temperature is 40 DEG C, and sample size is 2500 μ L/ pins, and flow rate of mobile phase is 70mL/min, collects 15-20
Minute fraction, carry out rotary evaporation and be concentrated to dryness, to prepare Quercitrin-3-O-glucoside thick for one-dimensional
Product.Methanol-water solution with 50% dissolves Quercitrin-3-O-glucoside crude product, and concentration is 70
Mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is with silica gel as base
Matter bonding C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), stream
Moving and selecting methanol (containing 0.1% formic acid) mutually is organic facies, and water (containing 0.1% formic acid) is aqueous phase, uses
Isocratic elution mode, organic phase concentration is 20% totally 60 minutes.Use DAD UV-detector 360
Nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 2000 μ L/ pins, and flow rate of mobile phase is
80mL/min, collects the fraction of retention time 35-40min, rotary evaporated to dryness, obtains Quercetin-3-O-
Glucoside compounds, through liquid-phase chromatographic analysis, the Quercitrin-3-O-glucoside crude product of two dimension preparation
The content of middle Quercitrin-3-O-glucoside is 94.6%, and one-dimensional to prepare Quercitrin-3-O-glucoside thick
In product, the content of Quercitrin-3-O-glucoside is 34%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Embodiment 8
Folium Ginkgo extract is dissolved in the ethanol-water solution of volumetric concentration 70%, prepares Folium Ginkgo and extract
Thing solution, concentration is 150mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph system
Standby.One-dimensional liquid chromatograph use hydrophilic chromatograph packing material with silica gel as substrate (50 × 250mm, 10
μm, Hua Puxinchuan Science and Technology Ltd.), flowing uses ethanol to be organic facies mutually, and water is aqueous phase, has
Machine phase concentration is 95% isocratic, uses DAD UV-detector 360nm to select absorbing wavelength, preparation
Temperature is room temperature, and sample size is 800 μ L/ pins, and flow rate of mobile phase is 110mL/min, collects 15-20
Minute fraction, carry out rotary evaporation and be concentrated to dryness, to prepare Quercitrin-3-O-glucoside thick for one-dimensional
Product.Ethanol-water solution with 60% dissolves Quercitrin-3-O-glucoside crude product, and concentration is 200
Mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is with silica gel as base
Matter bonding C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), stream
Moving and selecting ethanol (containing 0.1% formic acid) mutually is organic facies, and water (containing 0.1% formic acid) is aqueous phase, uses
Isocratic elution mode, organic phase concentration is 15% totally 60 minutes.Use DAD UV-detector 360
Nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 2500 μ L/ pins, and flow rate of mobile phase is
100mL/min, collects the fraction of retention time 30-35min, rotary evaporated to dryness, obtains Quercetin
-3-O-glucoside compounds, through liquid-phase chromatographic analysis, the Quercitrin-3-O-glucoside of two dimension preparation
In crude product, the content of Quercitrin-3-O-glucoside is 95.7%, one-dimensional prepares Quercetin-3-O-glucose
In glycosides crude product, the content of Quercitrin-3-O-glucoside is 33%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13The C-NMR (MeOD) end to obtaining
Product is analyzed, final confirm to obtain for Quercitrin-3-O-glucoside.
Obviously, above-described embodiment is only for clearly demonstrating example, and not to embodiment party
The restriction of formula.For those of ordinary skill in the field, the most also may be used
To make other changes in different forms.Here without also all of embodiment being given
With exhaustive.And the obvious change thus extended out or variation are still in the guarantor of the invention
Protect among scope.
Claims (10)
1. the method preparing Quercitrin-3-O-glucoside from Folium Ginkgo extract, including,
(1) Folium Ginkgo extract is dissolved in the ethanol-water solution that volumetric concentration is 40-80%, preparation
Obtain the extract solution that concentration is 10-1000mg/mL of Folium Ginkgo extract；
(2) extract solution obtaining described step (1) carries out one-dimensional liquid chromatograph separation,
To one-dimensional thick product；
(3) the one-dimensional thick product that step (2) obtains is dissolved in the methanol that volumetric concentration is 40-80%
In-aqueous solution or ethanol-water solution, the concentration obtaining described one-dimensional thick product is 20-200mg/mL's
Thick product solution；
(4) the thick product solution obtaining described step (3) carries out two-dimensional liquid chromatography and prepares,
To Quercitrin-3-O-glucoside.
The most according to claim 1 from Folium Ginkgo extract, prepare Quercitrin-3-O-glucoside
Method, it is characterised in that
The condition that in described step (2), one-dimensional liquid chromatograph separates is, chromatographic column uses with silica gel as base
The hydrophilic chromatograph packing material of matter；Organic facies in flowing mutually is ethanol or methanol, and aqueous phase is water；Eluting
Condition is down to 90% gradient with 0-15min organic facies 95% and is carried out or isocratic carry out；Collect retention time
It is the component of 15～20min, is dried to obtain described one-dimensional thick product.
The most according to claim 2 from Folium Ginkgo extract, prepare Quercitrin-3-O-glucoside
Method, it is characterised in that
In described step (2), one-dimensional liquid chromatograph separates, and organic facies is possibly together with formic acid, and formic acid volume is dense
Degree is 0.1%；Possibly together with formic acid in aqueous phase, formic acid volumetric concentration is 0.1%.
4. described from Folium Ginkgo extract, prepare Quercetin-3-O-Portugal according to claim 1-3 is arbitrary
The method of polyglycoside, it is characterised in that
In described step (4), two-dimensional liquid chromatography condition is, chromatographic column uses with silica gel as substrate-bound
C18 reverse phase filler；Organic facies in flowing mutually is ethanol or methanol, and aqueous phase is water；Elution requirement with
0-60min organic facies 15% increases to 80% gradient and carries out or isocratic carry out；Collection retention time is
The component of 30-40min, is dried to obtain Quercitrin-3-O-glucoside.
The most according to claim 4 from Folium Ginkgo extract, prepare Quercitrin-3-O-glucoside
Method, it is characterised in that
Prepared by described step (4) two-dimensional liquid chromatography, possibly together with formic acid in flowing mutually, formic acid volume is dense
Degree is 0.1%；Possibly together with formic acid in aqueous phase, formic acid volumetric concentration is 0.1%.
The most according to claim 5 from Folium Ginkgo extract, prepare Quercitrin-3-O-glucoside
Method, it is characterised in that
In described step (4) prepared by two-dimensional liquid chromatography, and the organic facies in flowing mutually is methanol, aqueous phase
For water.
7. described from Folium Ginkgo extract, prepare Quercetin-3-O-Portugal according to claim 1-6 is arbitrary
The method of polyglycoside, it is characterised in that
In described step (1), Folium Ginkgo extract is dissolved in the alcohol-water that volumetric concentration is 50-60%
In solution, prepare the extract solution that concentration is 250-550mg/mL of Folium Ginkgo extract.
8. described from Folium Ginkgo extract, prepare Quercetin-3-O-Portugal according to claim 1-7 is arbitrary
The method of polyglycoside, it is characterised in that
In described step (3), the one-dimensional thick product that step (2) obtains is dissolved in volumetric concentration is
The methanol-water solution of 50-60% or ethanol-water solution, described thick product solution concentration is
60-100mg/mL。
9. described from Folium Ginkgo extract, prepare Quercetin-3-O-Portugal according to claim 1-8 is arbitrary
The method of polyglycoside, it is characterised in that
One-dimensional liquid chromatograph, flow rate of mobile phase is 60-120mL/min, and column temperature is room temperature or 25-40 DEG C,
Sample size is 200-3000 μ L/ pin, and detector is DAD UV-detector；
Two-dimensional liquid chromatography, flow rate of mobile phase is 60-120mL/min, and column temperature is room temperature or 25-40 DEG C,
Sample size is 1000-3000 μ L/ pin, and UV-detector detection wavelength is 360nm.
The most according to claim 9 from Folium Ginkgo extract, prepare Quercetin-3-O-glucose
The method of glycosides, it is characterised in that
Two-dimensional liquid chromatography, flow rate of mobile phase is 80-100mL/min, and column temperature is room temperature or 25-40 DEG C,
Sample size is 2000-2500 μ L/ pin, and UV-detector detection wavelength is 360nm.
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