CN106279317B - A method of preparing Quercetin -3-o- glucoside from ginkgo biloba p.e - Google Patents
A method of preparing Quercetin -3-o- glucoside from ginkgo biloba p.e Download PDFInfo
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- CN106279317B CN106279317B CN201510247609.3A CN201510247609A CN106279317B CN 106279317 B CN106279317 B CN 106279317B CN 201510247609 A CN201510247609 A CN 201510247609A CN 106279317 B CN106279317 B CN 106279317B
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Abstract
The method that the invention discloses a kind of to prepare Quercitrin-3-O-glucoside 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 Quercitrin-3-O-glucoside.A kind of specific medicinal compound-Quercitrin-3-O-glucoside is prepared 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
The method that Quercetin -3-o- glucoside is prepared in extract, 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, thus can not accurately again it is exact reflection product material base.It is well known that ginkgo biloba p.e
Extracting method it is different, active compound and its content are different in obtained extract, then its drug effect is not also identical, by its system
The applicable symptom and function of standby obtained drug must be not exactly the same；Strictly, even if extracting method is identical, due to ginkgo leaf
Raw material batch is different, product is different, and the content of chemical component is also not quite similar in finally obtained extract, and above-mentioned factor
Different degrees of influence can be generated to the drug effect of drug eventually, therefore, only with existing quality standard to ginkgo biloba p.e and its system
Agent carries out the effect of quality control not very strictly with specification, and largely the presence of unknown substances affects the accurate survey to its content
It is fixed, more constrain the 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-Quercitrin-3-O-glucoside, to in ginkgo biloba p.e and its preparation it is medicinal at
The assay divided provides foundation.
For this purpose, the technical solution that the application takes is,
A method of preparing Quercitrin-3-O-glucoside from ginkgo biloba p.e, including,
(1) ginkgo biloba p.e is dissolved in the ethanol-water solution that volumetric concentration is 40-80%, ginkgo leaf is prepared
The concentration of extract is the extract solution of 10-1000mg/mL；(2) one is carried out to the extract solution that the step (1) obtains
Liquid chromatogram separation is tieed up, one-dimensional crude product is obtained；(3) the one-dimensional crude product that step (2) obtains is dissolved in volumetric concentration is 40-
In 80% methanol-water solution or ethanol-water solution, the concentration for obtaining the one-dimensional crude product is the thick production of 20-200mg/mL
Product solution；(4) two-dimensional liquid chromatography preparation is carried out to the crude product solution that the step (3) obtains, obtains the Portugal Quercetin -3-O-
Polyglycoside.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, it is one-dimensional in the step (2)
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 or methanol, water phase is water；Elution requirement is down to the progress of 90% gradient or isocratic progress with 0-15min organic phase 95%；
The component that retention time is 15~20min is collected, the one-dimensional crude product is dried to obtain.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, it is one-dimensional in the step (2)
Liquid chromatogram separation, organic phase also contain formic acid, and formic acid volumetric concentration is 0.1%；Also contain formic acid in water phase, formic acid volume is dense
Degree is 0.1%.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, it is two-dimentional in the step (4)
Liquid phase chromatogram condition, which is that chromatographic column is used, is bonded C18 reverse phase filler by matrix of silica gel；Organic phase in mobile phase be ethyl alcohol or
Methanol, water phase are water；Elution requirement increases to the progress of 80% gradient or isocratic progress with 0-60min organic phase 15%；It collects and retains
Time is the component of 30-40min, is dried to obtain Quercitrin-3-O-glucoside.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, step (4) Two-dimensional Liquid
Prepared by phase chromatography, formic acid is also contained in mobile phase, and formic acid volumetric concentration is 0.1%；Also contain formic acid in water phase, formic acid volume is dense
Degree is 0.1%.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, it is two-dimentional in the step (4)
Prepared by liquid chromatogram, the organic phase in mobile phase is methanol, and water phase is water.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, in the step (1), will
Ginkgo biloba p.e is dissolved in the ethanol-water solution that volumetric concentration is 50-60%, and the concentration that ginkgo biloba p.e is prepared is
The extract solution of 250-550mg/mL.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, in the step (3), will
The one-dimensional crude product that step (2) obtains is dissolved in the methanol-water solution or ethanol-water solution that volumetric concentration is 50-60%, described
Crude product solution concentration is 60-100mg/mL.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, one-dimensional liquid chromatogram, flowing
Phase flow velocity is 60-120mL/min, and column temperature is room temperature or 25-40 DEG C, and sample volume is 200-3000 μ L/ needle, and detector is DAD purple
External 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.
It is above-mentioned from the method for preparing Quercitrin-3-O-glucoside in ginkgo biloba p.e, two-dimensional liquid chromatography, flowing
Phase flow velocity is 80-100mL/min, and column temperature is room temperature or 25-40 DEG C, and sample volume is 2000-2500 μ L/ needle, UV detector inspection
Survey wavelength is 360nm.
Compared with prior art, the invention has the advantages that,
(1) a kind of specific medicinal compound-Quercetin -3-O- grape is prepared in the application from ginkgo biloba p.e
Glucosides, preparation method is simple, and lock out operation is convenient, isolates a specific compound using two-dimensional liquid chromatography method, thus
Index components are increased in the research of ginkgo biloba p.e quality control, enrich the test object of active material, are conducive to silver
The raising of apricot leaf extract and its drug standard is improved particularly the quality of existing widely used Shu Xuening 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 interference of the Multiple components to Quercitrin-3-O-glucoside in ginkgo biloba p.e is avoided,
Quercitrin-3-O-glucoside of the purity 90% or more is prepared.
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-
Aqueous solution " indicate methanol aqueous solution wherein methanol percentage by volume be 40%；" ethyl alcohol (containing 0.1% formic acid) " indicates ethyl alcohol
Percentage by volume with the mixed solution of formic acid wherein formic acid is 0.1%；" water (contain 0.1% formic acid) " indicates the mixed of water and formic acid
Close solution wherein formic acid percentage by volume be 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, prepares Quercitrin-3-O-glucoside crude product to be one-dimensional.Quercitrin is dissolved with 50% ethanol-water solution
Element -3-O- glucoside crude product, concentration 80mg/mL carry out two-dimensional liquid chromatography preparation, chromatographic column through filtering with microporous membrane
To be selected by hydrophilic chromatograph packing material (50 × 150mm, 5 μm, the Hua Puxinchuan Science and Technology Ltd.) mobile phase of matrix of silica gel
Methanol containing 0.1% volumetric concentration formic acid is organic phase, and the water containing 0.1% volumetric concentration formic acid is water phase, using 0-60min's
15-80% organic phase gradient elution.Absorbing wavelength is selected using DAD UV detector 360nm, preparation temperature is room temperature, sample introduction
Measuring is 1000 μ L/ needles, flow rate of mobile phase 90mL/min, fraction of the collection retention time in 30-40min, rotary evaporated to dryness,
Quercitrin-3-O-glucoside compound is obtained, through liquid-phase chromatographic analysis, purity 95.5% is one-dimensional to prepare Quercetin -3-O-
The content of Quercitrin-3-O-glucoside is 30% in glucoside crude product.
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,
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, molecular weight 464.0959,
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 prepares Quercitrin-3-O-glucoside crude product to be one-dimensional.It is dissolved with 40% ethanol-water solution
Quercitrin-3-O-glucoside crude product, concentration 20mg/mL carry out two-dimensional liquid chromatography preparation, color through filtering with microporous membrane
Composing column is to be bonded C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) by matrix of silica gel, mobile phase
Selecting the ethyl alcohol containing 0.1% volumetric concentration formic acid is organic phase, and the water containing 0.1% volumetric concentration formic acid is water phase, and use is isocratic
Type of elution, organic phase concentration are 20% totally 60 minutes.Absorbing wavelength, preparation temperature are selected using DAD UV detector 360nm
For room temperature, sample volume is 1000 μ L/ needles, and flow rate of mobile phase 100mL/min collects the fraction of retention time 30-40min, rotation
Turn to be evaporated to dryness, obtain Quercitrin-3-O-glucoside compound, through liquid-phase chromatographic analysis, the Quercetin -3-O- of two dimension preparation
The content of Quercitrin-3-O-glucoside is 94.0% in glucoside crude product, and one-dimensional to prepare Quercitrin-3-O-glucoside thick
The content of Quercitrin-3-O-glucoside is 32% in product.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 prepares Quercetin -3-O- grape to be one-dimensional
Glucosides crude product.Quercitrin-3-O-glucoside crude product, concentration 80mg/mL, through micropore are dissolved with 60% ethanol-water solution
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 μ, Hua Puxinchuan Science and Technology Ltd.), it is organic phase that mobile phase, which selects ethyl alcohol (containing 0.1% formic acid), and water (contains 0.1% formic acid)
For water phase, 80% gradient elution is risen to from 20% using organic phase.Absorbing wavelength, system are selected using DAD UV detector 360nm
Standby temperature is 25 DEG C, and sample volume is 3000 μ L/ needles, and flow rate of mobile phase 120mL/min collects evaporating for retention time 35-40min
Point, rotary evaporated to dryness obtains Quercitrin-3-O-glucoside compound, through liquid-phase chromatographic analysis, purity 96.5%, and one
The content that dimension prepares Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product is 29%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 Quercitrin-3-O-glucoside crude product.Quercitrin-3-O-glucoside crude product is dissolved with 80% ethanol-water solution, it is dense
Degree is 50mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, chromatographic column is that bonding C18 is anti-by matrix of silica gel
Phase filling (50 × 150mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.), it is organic that mobile phase, which selects ethyl alcohol (containing 0.1% formic acid),
Phase, water (containing 0.1% formic acid) is water phase, is eluted using 15% organic phase concentration.It selects to inhale using DAD UV detector 360nm
Wavelength is received, preparation temperature is 40 DEG C, and sample volume is 200 μ L/ needles, and flow rate of mobile phase 60mL/min collects retention time in 30-
35min fraction, rotary evaporated to dryness obtain Quercitrin-3-O-glucoside compound, and through liquid-phase chromatographic analysis, purity is
94.2%, the one-dimensional content for preparing Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product is 35%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 Quercitrin-3-O-glucoside crude product.Quercitrin-3-O-glucoside crude product is dissolved with 55% methanol-water solution, it is dense
Degree is 60mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, chromatographic column is that bonding C18 is anti-by matrix of silica gel
Phase filling (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), it is organic that mobile phase, which selects methanol (containing 0.1% formic acid),
Phase, water (containing 0.1% formic acid) is water phase, and using gradient elution mode, 0-60 minutes organic phase concentrations increase from 20% to 80%.It adopts
Absorbing wavelength is selected with DAD UV detector 360nm, preparation temperature is room temperature, and sample volume is 2000 μ L/ needles, flow rate of mobile phase
For 90mL/min, the fraction of retention time 30-40min is collected, rotary evaporated to dryness obtains Quercitrin-3-O-glucoside
Object is closed, Quercitrin-3-O-glucoside in the Quercitrin-3-O-glucoside crude product prepared through liquid-phase chromatographic analysis, two dimension
Content is 98.2.0%, and the one-dimensional content for preparing Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product is
36%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 Quercitrin-3-O-glucoside crude product.Quercitrin-3-O-glucoside crude product is dissolved with 55% ethanol-water solution, it is dense
Degree is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, chromatographic column is that C18 is bonded by matrix of silica gel
Reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), mobile phase select ethyl alcohol (containing 0.1% formic acid) to have
Machine phase, water (containing 0.1% formic acid) is water phase, and 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, and sample volume is 2500 μ L/ needles, mobile phase stream
Speed is 90mL/min, collects the fraction of retention time 30-40min, and rotary evaporated to dryness obtains Quercitrin-3-O-glucoside
Compound, through liquid-phase chromatographic analysis, Quercitrin-3-O-glucoside in the Quercitrin-3-O-glucoside crude product of two-dimentional preparation
Content be 97.8%, the one-dimensional content for preparing Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product is
36%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 prepares Quercitrin-3-O-glucoside crude product to be one-dimensional.Quercetin-is dissolved with 50% methanol-water solution
3-O- glucoside crude product, concentration 70mg/mL carry out two-dimensional liquid chromatography preparation through filtering with microporous membrane, chromatographic column be with
Silica gel is that matrix is bonded C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), and mobile phase selects methanol
(containing 0.1% formic acid) is organic phase, and water (containing 0.1% formic acid) is water phase, using isocratic elution mode, organic phase concentration 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,
Flow rate of mobile phase is 80mL/min, collects the fraction of retention time 35-40min, and rotary evaporated to dryness obtains Quercetin -3-O-
Glucoside compounds, through liquid-phase chromatographic analysis, Quercetin -3-O- in the Quercitrin-3-O-glucoside crude product of two-dimentional preparation
The content of glucoside is 94.6%, one-dimensional to prepare Quercitrin-3-O-glucoside in Quercitrin-3-O-glucoside crude product
Content is 34%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 prepares Quercitrin-3-O-glucoside crude product to be one-dimensional.Quercetin-is dissolved with 60% ethanol-water solution
3-O- glucoside crude product, concentration 200mg/mL carry out two-dimensional liquid chromatography preparation through filtering with microporous membrane, and chromatographic column is
It is bonded C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.) by matrix of silica gel, mobile phase selects second
Alcohol (containing 0.1% formic acid) is organic phase, and water (containing 0.1% formic acid) is water phase, and using isocratic elution mode, organic phase concentration is
15% totally 60 minutes.Absorbing wavelength is selected using DAD UV detector 360nm, preparation temperature is room temperature, and sample volume is 2500 μ
L/ needle, flow rate of mobile phase 100mL/min collect the fraction of retention time 30-35min, and rotary evaporated to dryness obtains quercitrin
Element -3-O- glucoside compounds, through liquid-phase chromatographic analysis, quercitrin in the Quercitrin-3-O-glucoside crude product of two-dimentional preparation
The content of element -3-O- glucoside is 95.7%, one-dimensional to prepare the Portugal Quercetin -3-O- in Quercitrin-3-O-glucoside crude product
The content of polyglycoside is 33%.
With embodiment 1 be respectively adopted mass spectrum,1H-NMR and13C-NMR (MeOD) analyzes obtained final product, finally
Confirmation obtains being Quercitrin-3-O-glucoside.
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 (8)
1. a kind of method that Quercitrin-3-O-glucoside is prepared from ginkgo biloba p.e, 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 or methanol, water phase is water；Elution requirement is down to the progress of 90% gradient or isocratic progress with 0-15min organic phase 95%；
The component that retention time is 15~20min is collected, the one-dimensional crude product is dried to obtain；
(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 Quercetin -3-O- grape
Glucosides；Wherein two-dimensional liquid chromatography condition is that chromatographic column is used using silica gel as matrix bonding C18 reverse phase filler；In mobile phase
Organic phase is ethyl alcohol or methanol, and water phase is water；Elution requirement with 0-60min organic phase 15% increase to 80% gradient carry out or it is isocratic
It carries out；The component that retention time is 30-40min is collected, Quercitrin-3-O-glucoside is dried to obtain.
2. the method according to claim 1 for preparing Quercitrin-3-O-glucoside from ginkgo biloba p.e, feature
It is,
One-dimensional liquid chromatogram separation in the step (2), organic phase also contain formic acid, and formic acid volumetric concentration is 0.1%；In water phase
Also contain formic acid, formic acid volumetric concentration is 0.1%.
3. the method according to claim 2 for preparing Quercitrin-3-O-glucoside from ginkgo biloba p.e, feature
It is,
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%.
4. the method according to claim 3 for preparing Quercitrin-3-O-glucoside from ginkgo biloba p.e, feature
It is,
In the step (4) prepared by two-dimensional liquid chromatography, and the organic phase in mobile phase is methanol, and water phase is water.
5. the method according to claim 1 to 4 that Quercitrin-3-O-glucoside is prepared from ginkgo biloba p.e,
It 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.
6. the method for preparing Quercitrin-3-O-glucoside in -5 any slave ginkgo biloba p.es according to claim 1,
It 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.
7. the method for preparing Quercitrin-3-O-glucoside in -6 any slave ginkgo biloba p.es according to claim 1,
It 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.
8. the method according to claim 7 for preparing Quercitrin-3-O-glucoside from ginkgo biloba p.e, feature
It is,
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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