CN106279315A - One prepares Quercetin-3-O-2 ' ', the method for 6 ' '-two rhamanopyranosyl glucosides from Folium Ginkgo extract - Google Patents
One prepares Quercetin-3-O-2 ' ', the method for 6 ' '-two rhamanopyranosyl glucosides from Folium Ginkgo extract Download PDFInfo
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- CN106279315A CN106279315A CN201510246875.4A CN201510246875A CN106279315A CN 106279315 A CN106279315 A CN 106279315A CN 201510246875 A CN201510246875 A CN 201510246875A CN 106279315 A CN106279315 A CN 106279315A
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- quercetin
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Abstract
The invention discloses and a kind of from Folium Ginkgo extract, prepare Quercetin-3-O-2 ", 6 " method of-two rhamanopyranosyl glucosides, 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, obtains Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides.The application prepares a kind of clear and definite medicinal compound-Quercetin-3-O-2 from Folium Ginkgo extract "; 6 "-two rhamanopyranosyl glucosides, 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
Kind from Folium Ginkgo extract, prepare Quercetin-3-O-2 ", 6 " method of-two rhamanopyranosyl glucosides,
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-Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl Portugals
Polyglycoside, 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,
One prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucosides
Method, including, Folium Ginkgo extract is dissolved in the ethanol-water solution that volumetric concentration is 40-80% by (1)
In, prepare the extract solution that concentration is 10-1000mg/mL of Folium Ginkgo extract；(2) right
The extract solution that described 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 the methanol-water that volumetric concentration is 40-80% molten
In liquid or ethanol-water solution, obtain the thick product that concentration is 20-200mg/mL of described one-dimensional thick product
Solution；(4) the thick product solution obtaining described step (3) carries out two-dimensional liquid chromatography and prepares,
To Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, in described step (2) one-dimensional liquid chromatograph separate condition be, chromatographic column use with
Silica gel is the hydrophilic chromatograph packing material of substrate；Organic facies in flowing mutually is ethanol or acetonitrile, 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
Retention time is the component of 28-32min, is dried to obtain described one-dimensional thick product.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, in described step (2), one-dimensional liquid chromatograph separates, and organic facies is possibly together with formic acid, first
Acid volumetric concentration is 0.1%；Possibly together with formic acid in aqueous phase, formic acid volumetric concentration is 0.1%.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, in described step (4), two-dimensional liquid chromatography condition is, chromatographic column uses and with silica gel is
Substrate-bound C18 reverse phase filler；Organic facies in flowing mutually is ethanol or acetonitrile, and aqueous phase is water；
Isocratic elution in 0-60min, during eluting, the volumetric concentration of organic facies is 15-25%；Collect retention time
For the component of 40-45min, it is dried to obtain Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, prepared by described step (4) two-dimensional liquid chromatography, possibly together with formic acid, first in flowing mutually
Acid volumetric concentration is 0.1%；Possibly together with formic acid in aqueous phase, formic acid volumetric concentration is 0.1%.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, in described step (4) prepared by two-dimensional liquid chromatography, and the organic facies in flowing mutually is second
Nitrile, aqueous phase is water.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, in described step (1), it is 50-60% that Folium Ginkgo extract is dissolved in volumetric concentration
Ethanol-water solution in, prepare the extraction that concentration is 250-550mg/mL of Folium Ginkgo extract
Thing solution.
Above-mentioned prepares Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, in described step (3), the one-dimensional thick product that step (2) obtains is dissolved in body
Volume concentrations 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 Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl glucoses
In the method for glycosides, 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 Quercetin-3-O-2 from Folium Ginkgo extract ", 6 "-two rhamanopyranosyl Fructus Vitis viniferaes
In the method for glucosides, 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
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-2 ", 6 "-two rhamanopyranosyl glucosides, its preparation method is simple, and lock out operation is convenient,
Two-dimensional liquid chromatography method is used to isolate a specific compound, thus in Folium Ginkgo extract quality control
Adding index components in the research of system, enrich the detection object of active substance, beneficially Folium Ginkgo carries
Take the raising of thing and drug standard thereof, be improved particularly existing widely used SHUXUENING ZHUSHEYE
Quality.
(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
To Quercetin-3-O-2 ", 6 " interference of-two rhamanopyranosyl glucosides, prepare purity 90%
Above Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides.
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%；
" acetonitrile-aqueous solution of 40% " represents that the percentage by volume of the aqueous solution wherein acetonitrile of acetonitrile 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.), it is organic that flowing uses acetonitrile (containing 0.1% formic acid) mutually
Phase, water (containing 0.1% formic acid) is aqueous phase, gradient elution mode: 0-15min organic phase concentration is from 95%
It is down to 90% stepwise gradient carry out.DAD UV-detector 360nm is used to select absorbing wavelength, system
Standby temperature is room temperature, and sample size is 500 μ L/ pins, and flow rate of mobile phase is 90mL/min, collects 28-
The fraction of 30 minutes, carries out rotary evaporation and is concentrated to dryness, and prepares Quercetin-3-O-2 for one-dimensional ", 6 "
-two rhamanopyranosyl glucoside crude products.Ethanol-water solution with 50% dissolves Quercetin-3-O-2 ", 6 "
-two rhamanopyranosyl glucoside crude products, concentration is 80mg/mL, through filtering with microporous membrane, carries out two dimension
Prepared by liquid chromatograph, chromatographic column be hydrophilic chromatograph packing material with silica gel as substrate (50 × 150mm, 5
μm, Hua Puxinchuan Science and Technology Ltd.) to use acetonitrile (containing 0.1% formic acid) mutually be organic facies in flowing,
Water (containing 0.1% formic acid) is aqueous phase, uses 0-60min20% organic facies isocratic elution.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 40-45min, rotates and steam
Send to do, obtain Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoside compounds, through liquid phase color
Analysis of spectrum, purity is 95.5%, one-dimensional prepares Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Quercetin-3-O-2 in glycosides crude product ", 6 " content of-two rhamanopyranosyl 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,
Quercetin female ring: 157.04 (C2), 133.07 (C3), 177.88 (C4), 161.73 (C5),
98.38 (C6), 164.24 (C7), 93.32 (C8), 157.53 (C9), 104.51 (C10), 122.16
(C1 '), 116.04 (C2 '), 144.51 (C3 '), 148.14 (C4 '), 114.68 (C5 '), 122.08
(C6’)
Glucose: 101.24 (C1 "), 72.48 (C2 "), 75.66 (C3 "), 70.47 (C4 "),
72.67 (C5 "), 66.89 (C6 ").
2 "-rhamnose: 100.85 (C1 " '), 78.64 (C2 " '), 70.74 (C3 " '), 71.01 (C4 " '),
68.32 (C5 " '), 16.13 (C6 " ').
6 "-rhamnose: 99.10 (C1 " "), 77.51 (C2 " "), 70.87 (C3 " "), 70.91 (C4 " "),
68.56 (C5 " "), 16.43 (C6 " ").
Comprehensive identification is Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides, the molecule of compound
Formula is C33H40O20, molecular weight 756.2103, 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 28～32 minutes, carries out rotary evaporation and be concentrated to dryness, prepare Mongolian oak for one-dimensional
Pi Su-3-O-2 ", 6 "-two rhamanopyranosyl glucoside crude products.Ethanol-water solution with 40% dissolves
Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoside crude products, concentration is 20mg/mL, through micro-
Hole membrane filtration, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is anti-with silica gel for substrate-bound C18
Phase filling (50 × 150mm, 10 μm, Hua Puxinchuan Science and Technology Ltd.), flowing selects ethanol mutually
(containing 0.1% formic acid) 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.DAD UV-detector 360nm is used to select to absorb ripple
Long, preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 100mL/min,
Collect the fraction of retention time 40-45min, rotary evaporated to dryness, obtain Quercetin-3-O-2 ", 6 "
-two rhamanopyranosyl glucoside compounds, through liquid-phase chromatographic analysis, the Quercetin of two dimension preparation
-3-O-2 ", 6 " Quercetin-3-O-2 in-two rhamanopyranosyl glucoside crude products ", 6 " and-two rhamnose
The content of base glucoside 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 28～32 minutes, carries out rotating and steams
Send out and be concentrated to dryness, prepare Quercetin-3-O-2 for one-dimensional ", 6 "-two rhamanopyranosyl glucoside crude products.
Ethanol-water solution with 60% dissolves Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoside crude products,
Concentration is 80mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, chromatographic column be with
Silica gel is that (50 × 150mm, 10 μ, China's spectrum is new creates scientific and technological limited public affairs to substrate-bound C18 reverse phase filler
Department), it is organic facies that flowing selects ethanol (containing 0.1% formic acid) mutually, and water (containing 0.1% formic acid) is water
Phase, uses organic facies 25% isocratic elution.DAD UV-detector 360nm is used to select to absorb ripple
Long, 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 40-45min, rotary evaporated to dryness, obtain Quercetin-3-O-2 ", 6 "
-two rhamanopyranosyl glucoside compounds, through liquid-phase chromatographic analysis, purity is 96.5%, one-dimensional preparation
Quercetin-3-O-2 ", 6 " Quercetin-3-O-2 in-two rhamanopyranosyl glucoside crude products ", 6 "-
The content of two rhamanopyranosyl glucosides 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 28-32
Minute fraction, carry out rotary evaporation and be concentrated to dryness, prepare Quercetin-3-O-2 for one-dimensional ", 6 "-
Two rhamanopyranosyl glucoside crude products.Ethanol-water solution with 80% dissolves Quercetin-3-O-2 ", 6 "
-two rhamanopyranosyl glucoside crude products, concentration is 50mg/mL, through filtering with microporous membrane, carries out two dimension
Prepared by liquid 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,
Water (containing 0.1% formic acid) is aqueous phase, uses 15% organic phase concentration eluting.Use the inspection of DAD ultraviolet
Surveying device 360nm and select absorbing wavelength, preparation temperature is 40 DEG C, and sample size is 200 μ L/ pins, flowing
Phase flow velocity is 60mL/min, and collection retention time, in 30-35min fraction, rotary evaporated to dryness, obtains
To Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoside compounds are through liquid-phase chromatographic analysis, pure
Degree is 94.2%, one-dimensional prepares Quercetin-3-O-2 ", 6 " Mongolian oak in-two rhamanopyranosyl glucoside crude products
Pi Su-3-O-2 ", 6 " content of-two rhamanopyranosyl glucosides 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 28-32 minute,
Carry out rotary evaporation to be concentrated to dryness, prepare Quercetin-3-O-2 for one-dimensional ", 6 "-two rhamanopyranosyl Fructus Vitis viniferaes
Glucosides crude product.Methanol-water solution with 55% dissolves Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl Portugals
Polyglycoside crude product, concentration is 60mg/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 acetonitrile (containing 0.1% formic acid) mutually, and water is (containing 0.1%
Formic acid) it is aqueous phase, 0-60 minute organic facies 15% isocratic elution.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
90mL/min, collects the fraction of retention time 40-45min, rotary evaporated to dryness, obtains Quercetin
-3-O-2 ", 6 "-two rhamanopyranosyl glucoside compounds, through liquid-phase chromatographic analysis, two dimension preparation
Quercetin-3-O-2 ", 6 " Quercetin-3-O-2 in-two rhamanopyranosyl glucoside crude products ", 6 "-
The content of two rhamanopyranosyl glucosides is 98.2.0%, one-dimensional prepares Quercetin-3-O-2 ", 6 "-two
Quercetin-3-O-2 in rhamanopyranosyl glucoside crude product ", 6 " content of-two rhamanopyranosyl glucosides
It 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 28-30 minute,
Carry out rotary evaporation to be concentrated to dryness, prepare Quercetin-3-O-2 for one-dimensional ", 6 "-two rhamanopyranosyl Fructus Vitis viniferaes
Glucosides crude product.Ethanol-water solution with 55% dissolves Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl Portugals
Polyglycoside crude product, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography system
Standby, chromatographic column is with silica gel for substrate-bound C18 reverse phase filler (50 × 150mm, 10 μm, Hua Pu
Xin Chuan Science and Technology Ltd.), it is organic facies that flowing selects acetonitrile (containing 0.1% formic acid) mutually, and water (contains
0.1% formic acid) it is aqueous phase, 0-60 minute organic facies 20% isocratic elution.Use DAD ultraviolet detection
Device 360nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 2500 μ L/ pins, and flow phase
Flow velocity is 90mL/min, collects the fraction of retention time 40-45min, rotary evaporated to dryness, obtains
Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoside compounds, through liquid-phase chromatographic analysis, two dimension
Quercetin-the 3-O-2 of preparation ", 6 " Quercetin-3-O-2 in-two rhamanopyranosyl glucoside crude products ", 6 "
The content of-two rhamanopyranosyl glucosides is 97.8%, one-dimensional prepares Quercetin-3-O-2 ", 6 "-two
Quercetin-3-O-2 in rhamanopyranosyl glucoside crude product ", 6 " content of-two rhamanopyranosyl glucosides
It 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 30-32
Minute fraction, carry out rotary evaporation and be concentrated to dryness, prepare Quercetin-3-O-2 for one-dimensional ", 6 "-
Two rhamanopyranosyl glucoside crude products.Methanol-water solution with 50% dissolves Quercetin-3-O-2 ", 6 "
-two rhamanopyranosyl glucoside crude products, concentration is 70mg/mL, through filtering with microporous membrane, carries out two dimension
Prepared by liquid 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 that flowing selects acetonitrile (containing 0.1% formic acid) mutually
Phase, water (containing 0.1% formic acid) is aqueous phase, uses isocratic elution mode, and organic phase concentration is 25% altogether
60 minutes.Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature,
Sample size is 2000 μ L/ pins, and flow rate of mobile phase is 80mL/min, collects retention time 40-45min
Fraction, rotary evaporated to dryness, obtain Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucuronidation
Compound, through liquid-phase chromatographic analysis, the Quercetin-3-O-2 of two dimension preparation ", 6 "-two rhamanopyranosyl Fructus Vitis viniferaes
Quercetin-3-O-2 in glucosides crude product ", 6 " content of-two rhamanopyranosyl glucosides is 94.6%, one
Dimension prepares Quercetin-3-O-2 ", 6 " Quercetin-3-O-2 in-two rhamanopyranosyl glucoside crude products ", 6 "
The content of-two rhamanopyranosyl 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 30-32
Minute fraction, carry out rotary evaporation and be concentrated to dryness, prepare Quercetin-3-O-2 for one-dimensional ", 6 "-
Two rhamanopyranosyl glucoside crude products.Ethanol-water solution with 60% dissolves Quercetin-3-O-2 ", 6 "
-two rhamanopyranosyl glucoside crude products, concentration is 200mg/mL, through filtering with microporous membrane, carries out two
Dimension liquid chromatograph prepare, 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 that flowing selects acetonitrile (containing 0.1% formic acid) mutually
Phase, water (containing 0.1% formic acid) is aqueous phase, uses isocratic elution mode, and organic phase concentration is 20% altogether
60 minutes.Using DAD UV-detector 360nm to select absorbing wavelength, preparation temperature is room temperature,
Sample size is 2500 μ L/ pins, and flow rate of mobile phase is 100mL/min, collects retention time 40-45min
Fraction, rotary evaporated to dryness, obtain Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucuronidation
Compound, through liquid-phase chromatographic analysis, the Quercetin-3-O-2 of two dimension preparation ", 6 "-two rhamanopyranosyl Fructus Vitis viniferaes
Quercetin-3-O-2 in glucosides crude product ", 6 " content of-two rhamanopyranosyl glucosides is 95.7%, one
Dimension prepares Quercetin-3-O-2 ", 6 " Quercetin-3-O-2 in-two rhamanopyranosyl glucoside crude products ", 6 "
The content of-two rhamanopyranosyl glucosides 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucoses
Glycosides.
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 Quercetin-3-O-2 for one kind ", 6 "-two rhamanopyranosyl Fructus Vitis viniferaes
The method of glucosides, 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 Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides.
The most according to claim 1 from Folium Ginkgo extract, prepare Quercetin-3-O-2 ", 6 "
The method of-two rhamanopyranosyl glucosides, 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 acetonitrile, 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
For the component of 28-32min, it is dried to obtain described one-dimensional thick product.
The most according to claim 2 from Folium Ginkgo extract, prepare Quercetin-3-O-2 ", 6 "
The method of-two rhamanopyranosyl glucosides, 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 according to claim 1-3 is arbitrary
-3-O-2 ", 6 " method of-two rhamanopyranosyl glucosides, 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 acetonitrile, and aqueous phase is water；In 0-60min etc.
Degree eluting, during eluting, the volumetric concentration of organic facies is 15-25%；Collection retention time is 40-45min
Component, be dried to obtain Quercetin-3-O-2 ", 6 "-two rhamanopyranosyl glucosides.
The most according to claim 4 from Folium Ginkgo extract, prepare Quercetin-3-O-2 ", 6 "
The method of-two rhamanopyranosyl glucosides, 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 Quercetin-3-O-2 ", 6 "
The method of-two rhamanopyranosyl glucosides, it is characterised in that
In described step (4) prepared by two-dimensional liquid chromatography, and the organic facies in flowing mutually is acetonitrile, aqueous phase
For water.
7. described from Folium Ginkgo extract, prepare Quercetin according to claim 1-6 is arbitrary
-3-O-2 ", 6 " method of-two rhamanopyranosyl glucosides, 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 according to claim 1-7 is arbitrary
-3-O-2 ", 6 " method of-two rhamanopyranosyl glucosides, 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 according to claim 1-8 is arbitrary
-3-O-2 ", 6 " method of-two rhamanopyranosyl glucosides, 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-2 ", 6 "
The method of-two rhamanopyranosyl glucosides, 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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