CN106279316A - A kind of method preparing kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract - Google Patents
A kind of method preparing kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract Download PDFInfo
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Abstract
The invention discloses a kind of method preparing kaempferol-3-O-p-coumaroyl glucose rhamnoside 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.The application prepares a kind of clear and definite medicinal compound-kaempferol-3-O-p-coumaroyl glucose rhamnoside 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract,
Belong to technical field of compound preparation.
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 product cannot be reflected accurately.It is well known that Folium Ginkgo
The extracting method of extract is different, and in its extract obtained, active compound and content thereof are different, then
Its drug effect also differs, it applicable symptom of the medicine prepared and function must be incomplete same；
Strictly, even if extracting method is identical, owing to Folium Ginkgo raw material batch is different, product is different, its
In the extract finally given, the content of chemical composition is also not quite similar, and above-mentioned factor eventually can be to medicine
Drug effect produce impact in various degree, therefore, only with existing quality standard to Folium Ginkgo extract and
Its preparation carries out that the effect of quality control is the strictest and specification, and the existence of a large amount of not clear materials have impact on
Accurate Determining to its content, more constrains 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-kaempferol-3-O-p-coumaroyl glucose Fructus rhamni (Rhamnus davurica Pall.)
Glucosides, to providing foundation to the assay of medicinal ingredient in Folium Ginkgo extract and preparation thereof.
To this end, the technical scheme that the application takes is,
A kind of kaempferol-3-O-p-coumaroyl glucose rhamnoside prepared from Folium Ginkgo extract
Method, including, Folium Ginkgo extract is dissolved in the ethanol-water solution that volumetric concentration is 40-80% by (1),
Prepare the extract solution that concentration is 10-1000mg/mL of Folium Ginkgo extract；(2) to described
The extract solution that step (1) obtains carries out one-dimensional liquid chromatograph separation, obtains one-dimensional thick product；(3)
The one-dimensional thick product that step (2) obtains is dissolved in methanol-water solution that volumetric concentration is 40-80% or
In ethanol-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 mountain
How phenol-3-O-p-coumaroyl glucose rhamnoside.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, in described step (2) one-dimensional liquid chromatograph separate condition be, chromatographic column use with silicon
Glue is the hydrophilic chromatograph packing material of substrate；Organic facies in flowing mutually is ethanol or methanol, and aqueous phase is water；
Elution requirement is down to 90% gradient with 0-15min organic facies 95% and is carried out or isocratic carry out；Collect and retain
Time is the component of 15～20min, is dried to obtain described one-dimensional thick product.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, in described step (2) one-dimensional liquid chromatograph separate, organic facies is possibly together with formic acid, formic acid
Volumetric concentration is 0.1%；Possibly together with formic acid in aqueous phase, formic acid volumetric concentration is 0.1%.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, in described step (4), two-dimensional liquid chromatography condition is, chromatographic column use with silica gel as base
Matter bonding C18 reverse phase filler；Organic facies in flowing mutually is ethanol or methanol, and aqueous phase is water；Eluting
Condition increases to 80% gradient with 0-60min organic facies 15% and carries out or isocratic carry out；Collect retention time
For the component of 50-55min, it is dried to obtain kaempferol-3-O-p-coumaroyl glucose rhamnoside.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, prepared by described step (4) two-dimensional liquid chromatography, flowing mutually in possibly together with formic acid, formic acid
Volumetric concentration is 0.1%；Possibly together with formic acid in aqueous phase, formic acid volumetric concentration is 0.1%.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, in described step (4) prepared by two-dimensional liquid chromatography, flowing mutually in organic facies be methanol,
Aqueous phase is water.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, in described step (1), it is 50-60%'s that Folium Ginkgo extract is dissolved in volumetric concentration
In ethanol-water solution, prepare the extract that concentration is 250-550mg/mL of Folium Ginkgo extract
Solution.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, in described step (3), the one-dimensional thick product that step (2) obtains is dissolved in volume
Concentration is methanol-water solution or the ethanol-water solution of 50-60%, and described thick product solution concentration is
60-100mg/mL。
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, one-dimensional liquid chromatograph, flow rate of mobile phase is 60-120mL/min, column temperature be 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.
Above-mentioned prepares kaempferol-3-O-p-coumaroyl glucose rhamnoside from Folium Ginkgo extract
Method in, two-dimensional liquid chromatography, flow rate of mobile phase is 80-100mL/min, column temperature be room temperature or
25-40 DEG C, sample size is 2000-2500 μ L/ pin, and 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-Rhizoma Kaempferiae from Folium Ginkgo extract
Phenol-3-O-p-coumaroyl glucose rhamnoside, its preparation method is simple, and lock out operation is convenient, adopts
A specific compound is isolated by two-dimensional liquid chromatography method, thus at Folium Ginkgo extract quality control
Research in add index components, enrich the detection object of active substance, beneficially Folium Ginkgo extracts
Thing and the raising of drug standard thereof, be improved particularly the matter of existing widely used SHUXUENING ZHUSHEYE
Amount.
(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 kaempferol-3-O-p-coumaroyl glucose rhamnoside, prepare purity 90% with
On kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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, for one-dimensional preparation mountain
How phenol-3-O-p-coumaroyl glucose rhamnoside crude product.Ethanol-water solution with 50% dissolves mountain
How phenol-3-O-p-coumaroyl glucose rhamnoside crude product, concentration is 80mg/mL, through microporous filter membrane
Filtering, carry out two-dimensional liquid chromatography and prepare, chromatographic column is the hydrophilic chromatograph packing material with silica gel as substrate
(50 × 150mm, 5 μm, Hua Puxinchuan Science and Technology Ltd.) flowing selects containing 0.1% volume dense mutually
The methanol of degree formic acid is organic facies, and the water containing 0.1% volumetric concentration formic acid is aqueous phase, uses 0-60min
15-80% organic facies gradient elution.DAD UV-detector 360nm is used to select absorbing wavelength,
Preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 90mL/min, collects and protects
Stay the time in the fraction of 50-55min, rotary evaporated to dryness, obtain kaempferol-3-O-p-coumaroyl Portugal
Grape sugar rhamnoside compound, through liquid-phase chromatographic analysis, purity is 95.5%, one-dimensional prepares kaempferol
Kaempferol-3-O-p-coumaroyl glucose Mus in-3-O-p-coumaroyl glucose rhamnoside crude product
The content of Lee's glucosides is 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,
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 "-p-coumaroyl: 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-p-coumaroyl glucose rhamnoside, the molecular formula of compound
For C36H36O17, molecular weight 740.1982, 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, for one-dimensional preparation mountain
How phenol-3-O-p-coumaroyl glucose rhamnoside crude product.Ethanol-water solution with 40% dissolves mountain
How phenol-3-O-p-coumaroyl glucose rhamnoside crude product, concentration is 20mg/mL, through microporous filter membrane
Filtering, carry out two-dimensional liquid chromatography and prepare, chromatographic column is with silica gel for substrate-bound C18 reverse phase filler
(50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), flowing selects mutually containing 0.1% volume
The ethanol of concentration formic acid is organic facies, and the water containing 0.1% volumetric concentration formic acid is aqueous phase, uses isocratic washing
Off-square formula, organic phase concentration is 20% totally 60 minutes.Use the choosing of DAD UV-detector 360nm
Selecting absorbing wavelength, preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 100
ML/min, collects the fraction of retention time 50-55min, rotary evaporated to dryness, obtains kaempferol-3-O-
P-coumaroyl glucose rhamnoside compound, through liquid-phase chromatographic analysis, the kaempferol of two dimension preparation
Kaempferol-3-O-p-coumaroyl glucose Mus in-3-O-p-coumaroyl glucose rhamnoside crude product
The content of Lee's glucosides is 94.0%,
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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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 kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product for one-dimensional.With
The ethanol-water solution of 60% dissolves kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product, dense
Degree is 80mg/mL, and through filtering with microporous membrane, carrying out two-dimensional liquid chromatography prepares, and chromatographic column is with silicon
Glue is substrate-bound C18 reverse phase filler (50 × 150mm, 10 μ, Hua Puxinchuan Science and Technology Ltd.),
It is organic facies that flowing selects ethanol (containing 0.1% formic acid) mutually, and water (containing 0.1% formic acid) is aqueous phase, adopts
80% gradient elution is risen to from 20% by organic facies.DAD UV-detector 360nm is used to select to inhale
Receiving wavelength, preparation temperature is 25 DEG C, and sample size is 3000 μ L/ pins, and flow rate of mobile phase is 120mL/min,
Collect the fraction of retention time 50-55min, rotary evaporated to dryness, obtain kaempferol-3-O-to coumaric acyl
Base glucose rhamnoside compound, through liquid-phase chromatographic analysis, purity is 96.5%, one-dimensional prepares Rhizoma Kaempferiae
Kaempferol-3-O-p-coumaroyl glucose in phenol-3-O-p-coumaroyl glucose rhamnoside crude product
The content of rhamnoside 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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, prepare kaempferol-3-O-p-coumaroyl for one-dimensional
Glucose rhamnoside crude product.Ethanol-water solution with 80% dissolves kaempferol-3-O-p-coumaroyl
Glucose rhamnoside crude product, concentration is 50mg/mL, through filtering with microporous membrane, carries out two dimension liquid phase
Prepared by chromatograph, chromatographic column be with silica gel for substrate-bound C18 reverse phase filler (50 × 150mm, 10 μ,
Hua Puxinchuan Science and Technology Ltd.), it is organic facies that flowing selects ethanol (containing 0.1% formic acid) mutually, and water (contains
0.1% formic acid) it is aqueous phase, use 15% organic phase concentration eluting.Use DAD UV-detector 360
Nm selects absorbing wavelength, and preparation temperature is 40 DEG C, and sample size is 200 μ L/ pins, and flow rate of mobile phase is
60mL/min, collection retention time, in 50-55min fraction, rotary evaporated to dryness, obtains kaempferol-3-O-
P-coumaroyl glucose rhamnoside compound, through liquid-phase chromatographic analysis, purity is 94.2%, one-dimensional
Prepare in kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product kaempferol-3-O-to coumaric acyl
The content of base glucose rhamnoside 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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 kaempferol-3-O-p-coumaroyl glucose rhamnose for one-dimensional
Glycosides crude product.Methanol-water solution with 55% dissolves kaempferol-3-O-p-coumaroyl glucose rhamnose
Glycosides crude product, concentration is 60mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, color
Spectrum post is with silica gel for substrate-bound C18 reverse phase filler (50 × 150mm, 10 μm, Hua Puxinchuan section
Skill company limited), it is organic facies that flowing selects methanol (containing 0.1% formic acid) mutually, and water is (containing 0.1% first
Acid) it is aqueous phase, use gradient elution mode, within 0-60 minute, organic phase concentration increases to 80% from 20%.
Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature, and sample size is
2000 μ L/ pins, flow rate of mobile phase is 90mL/min, collects the fraction of retention time 50-55min,
Rotary evaporated to dryness, obtains kaempferol-3-O-p-coumaroyl glucose rhamnoside compound, through liquid
Analysis of hplc, mountain in the kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product of two dimension preparation
How the content of phenol-3-O-p-coumaroyl glucose rhamnoside is 98.2.0%, one-dimensional prepares kaempferol
Kaempferol-3-O-p-coumaroyl glucose Mus in-3-O-p-coumaroyl glucose rhamnoside crude product
The content of Lee's glucosides 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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 kaempferol-3-O-p-coumaroyl glucose rhamnose for one-dimensional
Glycosides crude product.Ethanol-water solution with 55% dissolves kaempferol-3-O-p-coumaroyl glucose rhamnose
Glycosides crude product, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares,
Chromatographic column is with silica gel for substrate-bound C18 reverse phase filler (50 × 150mm, 10 μm, the new wound of China's spectrum
Science and Technology Ltd.), it is organic facies that flowing selects ethanol (containing 0.1% formic acid) mutually, and water is (containing 0.1%
Formic acid) it is aqueous phase, use gradient elution mode, within 0-60 minute, organic phase concentration increases to 80% from 20%.
Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature, and sample size is
2500 μ L/ pins, flow rate of mobile phase is 90mL/min, collects the fraction of retention time 50-55min,
Rotary evaporated to dryness, obtains kaempferol-3-O-p-coumaroyl glucose rhamnoside compound, through liquid
Analysis of hplc, mountain in the kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product of two dimension preparation
How the content of phenol-3-O-p-coumaroyl glucose rhamnoside is 97.8%, one-dimensional prepares kaempferol
Kaempferol-3-O-p-coumaroyl glucose Mus in-3-O-p-coumaroyl glucose rhamnoside crude product
The content of Lee's glucosides 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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, prepare kaempferol-3-O-p-coumaroyl for one-dimensional
Glucose rhamnoside crude product.Methanol-water solution with 50% dissolves kaempferol-3-O-p-coumaroyl
Glucose rhamnoside crude product, concentration is 70mg/mL, through filtering with microporous membrane, carries out two dimension liquid phase
Prepared by chromatograph, chromatographic column be with silica gel for substrate-bound C18 reverse phase filler (50 × 150mm, 10 μm,
Hua Puxinchuan Science and Technology Ltd.), it is organic facies that flowing selects methanol (containing 0.1% formic acid) mutually, and water (contains
0.1% formic acid) it is aqueous phase, use isocratic elution mode, organic phase concentration is 20% totally 60 minutes.
Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature, and sample size is
2000 μ L/ pins, flow rate of mobile phase is 80mL/min, collects the fraction of retention time 50-55min,
Rotary evaporated to dryness, obtains kaempferol-3-O-p-coumaroyl glucose rhamnoside compound, through liquid
Analysis of hplc, mountain in the kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product of two dimension preparation
How the content of phenol-3-O-p-coumaroyl glucose rhamnoside is 94.6%, one-dimensional prepares kaempferol
Kaempferol-3-O-p-coumaroyl glucose Mus in-3-O-p-coumaroyl glucose rhamnoside crude product
The content of Lee's glucosides 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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, prepare kaempferol-3-O-p-coumaroyl for one-dimensional
Glucose rhamnoside crude product.Ethanol-water solution with 60% dissolves kaempferol-3-O-p-coumaroyl
Glucose rhamnoside crude product, concentration is 200mg/mL, through filtering with microporous membrane, carries out Two-dimensional Liquid
Prepared by phase chromatograph, chromatographic column be with silica gel for substrate-bound C18 reverse phase filler (50 × 150mm, 10
μm, Hua Puxinchuan Science and Technology Ltd.), it is organic facies that flowing selects ethanol (containing 0.1% formic acid) mutually,
Water (containing 0.1% formic acid) is aqueous phase, uses isocratic elution mode, and organic phase concentration is 15% totally 60
Minute.Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature, enters
Sample amount is 2500 μ L/ pins, and flow rate of mobile phase is 100mL/min, collects retention time 50-55min
Fraction, rotary evaporated to dryness, obtain kaempferol-3-O-p-coumaroyl glucose rhamnoside compound,
Through liquid-phase chromatographic analysis, the kaempferol-3-O-p-coumaroyl glucose rhamnoside crude product of two dimension preparation
The content of middle kaempferol-3-O-p-coumaroyl glucose rhamnoside is 95.7%, one-dimensional prepares Rhizoma Kaempferiae
Kaempferol-3-O-p-coumaroyl glucose in phenol-3-O-p-coumaroyl glucose rhamnoside crude product
The content of rhamnoside 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
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. from Folium Ginkgo extract, prepare kaempferol-3-O-p-coumaroyl glucose rhamnoside for one kind
Method, 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 kaempferol-3-O-p-coumaroyl glucose rhamnoside.
Kaempferol-the 3-O-for preparing from Folium Ginkgo extract the most according to claim 1 is to coumaric acyl
The method of base glucose rhamnoside, 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.
Kaempferol-the 3-O-for preparing from Folium Ginkgo extract the most according to claim 2 is to coumaric acyl
The method of base glucose rhamnoside, 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 kaempferol-3-O-pair according to claim 1-3 is arbitrary
Coumaric acyl glucose rhamnoside contained method, 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 50-55min, is dried to obtain kaempferol-3-O-p-coumaroyl glucose rhamnoside.
Kaempferol-the 3-O-for preparing from Folium Ginkgo extract the most according to claim 4 is to coumaric acyl
The method of base glucose rhamnoside, 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%.
Kaempferol-the 3-O-for preparing from Folium Ginkgo extract the most according to claim 5 is to coumaric acyl
The method of base glucose rhamnoside, 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 kaempferol-3-O-pair according to claim 1-6 is arbitrary
Coumaric acyl glucose rhamnoside contained method, 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 kaempferol-3-O-pair according to claim 1-7 is arbitrary
Coumaric acyl glucose rhamnoside contained method, 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 kaempferol-3-O-pair according to claim 1-8 is arbitrary
Coumaric acyl glucose rhamnoside contained method, 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.
Kaempferol-the 3-O-for preparing from Folium Ginkgo extract the most according to claim 9 is to tonkabean
The method of acyl glucose rhamnoside, 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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