CN104523771A - Ginkgo leaf total flavones and preparing method thereof - Google Patents
Ginkgo leaf total flavones and preparing method thereof Download PDFInfo
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- CN104523771A CN104523771A CN201510019560.6A CN201510019560A CN104523771A CN 104523771 A CN104523771 A CN 104523771A CN 201510019560 A CN201510019560 A CN 201510019560A CN 104523771 A CN104523771 A CN 104523771A
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- folium ginkgo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
- A61K9/2004—Excipients; Inactive ingredients
- A61K9/2022—Organic macromolecular compounds
- A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
- A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention provides ginkgo leaf total flavones and a preparing method of the ginkgo leaf total flavones and particularly relates to high-content total flavones made from ginkgo leaf extracts and a separation and purification method. The flavone content is increased by removing impurities except flavone. Compared with other existing high-content flavone preparing methods, the method has the advantages that the flavone content of the total flavones prepared with the method is equal to or higher than 42% and is higher than the existing standard flavone content of total flavones made from standard ginkgo leaf extracts by 24% or more, the yield is equal to or higher than 73%, and the varieties of flavones are rich.
Description
Technical field
The present invention relates to a kind of Folium Ginkgo total flavones and preparation method thereof, be specifically related to a kind of method preparing total flavones in Folium Ginkgo extract, relate to the production technology of separation and purification Ginkgo total flavones from Folium Ginkgo extract.
Background technology
Folium Ginkgo extract (Ginko Biloba Extract, GBE) be for raw material with the leaf of Semen Ginkgo Ginkgo biloba L., adopt suitable solvent, one series products of the effective ingredient enrichment of extracting, for light yellowish brown flowable powder, have the fragrance that this product is intrinsic, bitter in the mouth, have invigorate blood circulation, blood stasis dispelling, dredging collateral effect.Folium Ginkgo extract quality standard international at present, i.e. standard silver Fructus Pruni extract (Ginkgo total flavones >=24%, terpene lactones >=6%, ginkgoic acid≤10ppm), taken the lead in formulating by the seventies Germany in last century.
Folium Ginkgo extract is except having significant antagonism paf receptor; drug effect can also be played in antiinflammatory, antiallergic, blood vessel dilating, protection cardiovascular and cerebrovascular vessel, peripheral blood circulation of improving, reduction serum cholesterol and anticancer adjuvant etc., be widely used in control and the health care of the systemic disease such as cardiovascular and cerebrovascular vessel, nerve.Flavones ingredient in Folium Ginkgo, tool resists myocardial ischemia preferably, anticoagulant, the pharmacological action such as anti-hypoxia and enhancing short term memory, scavenging free radicals, slow down aging.
Research shows, highly purified ginkgetin has better curative effect than common standard silver Fructus Pruni extract in treatment cardiovascular and cerebrovascular disease.But, in the extraction separation method report of current high-purity flavone, mostly be that extracting directly is separated from Folium Ginkgo, its flavones content is less, only collects the flow point of a certain Concentraton gradient, and its productive rate is lower, few containing flavone kind, and mostly be active poor rutin etc., compared with standard Folium Ginkgo extract, differ greatly.Relate to a kind of separation method of flavonoid active material in CN 102138945 A, its complex operation step, process are complicated, and when ensureing yield 25%, its flavones content only reaches 22%.A kind of preparation method of high-purity silver flavone is mentioned in CN 102078341 A, when it crosses macroporous resin column separation, in order to obtain high-purity flavone, only collect the eluent at 50%-70% ethanol high flavones content position, the flavones ingredient obtained has two aspects not enough: the first, and pursuit purity simply, causes its yield greatly to decline, less than 20%, cause the great wasting of resources; The second, this technique only obtains low pole and middle polarity ginkgetin, the flavone component contained by it after being separated, and compared with standard Folium Ginkgo extract, Species differences is comparatively large, is difficult to compare with Folium Ginkgo extract.
The preparation technology of current Folium Ginkgo extract still becomes perfect, but the extraction key technology of high-purity silver flavone needs to be further developed especially, possesses important medical treatment and significance of scientific research.
Technical scheme
1. the present invention relates to a kind of Folium Ginkgo total flavones, flavones content >=42%.
2. the source of a kind of Folium Ginkgo total flavones of the present invention comprises and is not restricted to Folium Ginkgo extract; The present invention relates to a kind of method preparing total flavones in Folium Ginkgo extract.
3. the present invention is by Folium Ginkgo extract dissolve with ethanol, and D101 type macroporous resin mixes sample loading, crosses macroporous resin column and is separated, the ethanol water eluting of different gradient, collect the flow point between 10%-70%, concentrated, dry.Dissolution with solvents, leaves standstill, sucking filtration, and waste, gets filtrate, vacuum drying, porphyrize, and to get final product.
4. the present invention is by silver containing dissolve with ethanol under leaf extract 40 DEG C of water bath condition, and D101 type macroporous resin mixes sample loading, crosses macroporous resin column and is separated, the ethanol water eluting of different gradient, collect the flow point between 10%-70%, concentrated, dry.Dissolution with solvents, leaves standstill, sucking filtration, and waste, gets filtrate, vacuum drying, porphyrize, and to get final product.
5. the gradient of ethanol water respectively: water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol.
6. the present invention relates to a kind of preparation method of Folium Ginkgo total flavones.It is characterized in that: take Folium Ginkgo extract as raw material, its processing step comprises:
(1) to Folium Ginkgo extract sample pretreatment: after dissolve with ethanol, add macroporous resin and mix sample;
(2) macroporous resin column eluting: that step (1) is handled well mixes sample macropore loading, and the ethanol of different gradient carries out eluting to macroporous resin column respectively; The gradient of ethanol water is respectively: water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol;
(3) eluent is collected: the eluent collecting the 10%-70% ethanol position of flowing out in macroporous resin column in step (2);
(4) concentrating under reduced pressure: the eluent in step (3) is carried out concentrating under reduced pressure by gradient, merges concentrated solution, evaporate to dryness; Temperature control 45-60 DEG C; Vacuum degree control 0.1MPa;
(5) dissolve: the precipitation that step (4) evaporate to dryness obtains, adds organic solvent dissolution;
(6) leave standstill;
(7) filter, waste, gets filtrate;
(8) vacuum drying, porphyrize powder, to obtain final product.Or;
(9) assay: measure according to total flavonoids content assaying method in " Chinese Pharmacopoeia " Folium Ginkgo
Total flavonoids content=(quercetin content+kaempferide content+isorhamnetin content) * 2.51
The present invention relates to a kind of method preparing total flavones in Folium Ginkgo extract, it is characterized in that:
(1)-(6) are the same
(7) filter, waste, gets filtrate; Use organic solvent washing residue, get washing liquid; Merging filtrate and washing liquid; Organic solvent is preferably ethanol;
(8) vacuum drying, porphyrize powder, to obtain final product;
In the active substance of gained of the present invention, flavones content >=42%; Productive rate >=73%;
In step (5), solvent selected by resolution of precipitate is the combination in any in ethanol, methanol, ethyl acetate, acetone; Organic solvent is preferably ethanol;
The invention provides a kind of Folium Ginkgo total flavones, contained Main Flavonoids constituents: Quercetin-3-O-2 ", 6 "-two rhamnose glucosides, kaempferol-3-O-2 ", 6 "-two rhamnose glucosides, isorhamnetin-3-O-2 ", 6 "-two rhamnose glucosides, ampelopsin-3-O-rutinoside, ampelopsin-3-O-glucoside, Quercetin-3-O-rutinoside, Quercitrin-3-O-glucoside, Quercetin-2 "-glucose rhamnoside, Isorhamnetin-3-O-neohespeidoside, isorhamnetin-3-O-2 "-glucose rhamnoside, FNS, isorhamnetin-3-O-rutinoside, kaempferol-3-O-glucose rhamnoside, Quercetin-3-O-2 "-(6 "-p-coumaroyl)-glucose rhamnoside, kaempferol-3-O-2 "-(6 "-p-coumaroyl)-glucose rhamnoside, kaempferol-3-O-2 "-(6 "-p-coumaroyl)-Glucose Glucose glucosides etc.
Assay:
Measure according to total flavonoids content assaying method in " Chinese Pharmacopoeia " Folium Ginkgo, efficient liquid phase detection method is: take octadecylsilane chemically bonded silica as filler; Methanol-0.4% phosphoric acid (50: 50) is mobile phase; Determined wavelength is 360nm; Chromatographic column temperature: 25 DEG C; Sample size: 10 μ L.Theoretical cam curve calculates should be not less than 2500 with Quercetin peak.
The preparation of reference substance solution: get Quercetin reference substance, kaempferide reference substance, isorhamnetin reference substance respectively in right amount, accurately weighed, add methanol and make every 1ml respectively containing the mixed solution of 30 μ g, 30 μ g, 20 μ g, product solution in contrast.
The preparation of need testing solution: the total flavones sample of Example 1 gained Folium Ginkgo extract is about 1g, accurately weighed, puts in apparatus,Soxhlet's, add chloroform and reflux 2 hours, discard chloroform liquid, medicinal residues volatilize; Add methanol eddy and extract 4 hours, extracting solution volatilizes, and residue adds methanol-25% hydrochloric acid solution (4: 1) mixed solution 25 milliliters, and reflux 30 minutes, lets cool, and is transferred in 50ml measuring bottle, and adds methanol to scale, shakes up, to obtain final product.
Total flavonoids content=(quercetin content+kaempferide content+isorhamnetin content) * 2.51
Cubage:
Reference substance concentration:
C
kaempferide=30.4 μ g/ml
C
isorhamnetin=18.8 μ g/ml
C
quercetin=33.0 μ g/ml
(1) standard silver Folium Pruni extract concentrations:
Recorded by HPLC,
Reference substance peak area:
A
kaempferide=1267220
A
isorhamnetin=1085827
A
quercetin=1345106
Test sample peak area:
A
kaempferide'=41044508
A
isorhamnetin'=11578117
A
quercetin'=35765399
By A
1/ A
1'=C
1/ C
1' known,
C
kaempferide'=(A
kaempferide' * C
kaempferide)/A
kaempferide=(36646061*30.4) μ g/ml/1267220=879.12 μ g/ml in like manner,
C
isorhamnetin'=141.68 μ g/ml
C
quercetin'=922.49 μ g/ml
W=(C
kaempferide+ C
isorhamnetin+ C
quercetin) * 50ml/1g*2.51*100%
W＝(879.12+141.68+922.49)μg/ml*50ml/1g*2.51*100％＝24.39％
(2) gained extractum content of the present invention
Recorded by HPLC,
Reference substance peak area:
A
kaempferide=1267220
A
isorhamnetin=1085827
A
quercetin=1345106
Test sample peak area:
A
kaempferide'=72122856
A
isorhamnetin'=18343000
A
quercetin'=65896590
By A
1/ A
1'=C
1/ C
1' known,
C
kaempferide'=(A
kaempferide' * C
kaempferide)/A
kaempferide=(72122856*30.4) μ g/ml/1267220=1552.02 μ g/ml in like manner,
C
isorhamnetin'=309.66 μ g/ml
C
quercetin'=1514.65 μ g/ml
W=(C
kaempferide+ C
isorhamnetin+ C
quercetin) * 50ml/1g*2.51*100%
W＝(1552.02+309.66+1514.65)μg/ml*50ml/1g*2.51*100％＝42.37％
7. the analytical method of a kind of Folium Ginkgo total flavones of gained of the present invention, HPLC optimum condition
Instrument: Shimadzu LC-20A chromatograph of liquid
The testing conditions of HPLC: detect under ultraviolet 360nm condition
HPLC mobile phase:
A:(0.1%-5%) formic acid-water
B: acetonitrile
C: methanol (washing post)
D: water (washing post)
Flow velocity: 1ml/min
Temporal sequence:
The present invention relates to a kind of tablet of Folium Ginkgo total flavones,
Folium Ginkgo total flavones 25-150g, starch 25-150g, dextrin 25-150g, calcium sulfate 150-500g, crosslinked ketopyrrolidine 2-40g; Flavones content >=42% in Folium Ginkgo total flavones.
The present invention relates to a kind of preferred tablet of total flavones of Folium Ginkgo extract,
Folium Ginkgo total flavones 80g, starch 75g, dextrin 75g, calcium sulfate 255g, crosslinked ketopyrrolidine 10g; Flavones content >=42% in Folium Ginkgo total flavones;
Method for making: get Folium Ginkgo total flavones, add starch, dextrin, calcium sulfate and crosslinked ketopyrrolidine, make granule, tabletting, sugar coating or film-coat, to obtain final product.
Make 1000 (sheets) or 2000 (small pieces).
The every sheet of this product must not be less than (small pieces) 16.8mg, (sheet) 32.6mg containing total flavonoids.
Accompanying drawing illustrates:
Fig. 1 embodiment of the present invention 1 gained Ginkgo total flavones (powder) HPLC chromatogram (1mg/ml)
Fig. 2 standard silver Folium Pruni extractive HPLC chromatogram (1mg/ml)
1 removal of impurity of Fig. 3 embodiment of the present invention
The HPLC (1mg/ml) of 10% alcohol elution of Fig. 4 embodiment of the present invention 1
The HPLC (1mg/ml) at the 50%-70% eluting position of Fig. 5 embodiment of the present invention 1
The HPLC (1mg/ml) at the 70%-100% eluting position of Fig. 6 embodiment of the present invention 1
Reference numeral:
1.quercetin 3-O-2″，6″-dirhamnosylglucoside
Quercetin-3-O-2 ", 6 "-two rhamnose glucosides
2.myricetin 3-O-rutinoside
Ampelopsin-3-O-rutinoside
3.quercetin 3-O-6″-rhamnosyl-2″-(6″-p-coumaroylglucosyl)glucoside
Quercetin-3-O-6 "-rhamanopyranosyl-2 "-(6 "-p-coumaroyl) glucoside
4.kaempferol 3-O-2″，6″-dirhamnosylglucoside
Kaempferol-3-O-2 ", 6 "-two rhamnose glucosides
5.isorhamnetin 3-O-2″，6″-dirhamnosylglucoside
Isorhamnetin-3-O-2 ", 6 "-two rhamnose glucosides
6.kaempferol 3-O-6″-rhamnosyl-2″-(6″-p-coumaroylglucosyl)glucoside
Kaempferol-3-O-6 "-rhamanopyranosyl-2 "-(6 "-p-coumaroyl) glucoside
7.quercetin 3-O-rutinoside
Quercetin-3-O-rutinoside
8.quercetin 3-O-glucoside
Quercitrin-3-O-glucoside
9.quercetin 2″-glucosylrhamnoside
Quercetin-2 "-glucose rhamnoside
10.kaempferol 3-O-rutinoside
FNS
11.isorhamnetin3-O-rutinoside
Isorhamnetin-3-O-rutinoside
12.quercetin 3-O-2″-(6″-p-coumaroyl)glucosylrhamnoside
Quercetin-3-O-2 "-(6 "-p-coumaroyl) glucose rhamnoside
13.kaempferol 3-O-2″-(6″-p-coumaroyl)glucosylrhamnoside
Kaempferol-3-O-2 "-(6 "-p-coumaroyl) glucose rhamnoside
Gained total flavones HPLC of the present invention sample preparation:
The embodiment of the present invention 1 gained Ginkgo total flavones (powder), chromatograph dissolve with methanol, concentration is 1mg/ml
Standard Folium Ginkgo extract, chromatograph dissolve with methanol, concentration is 1mg/ml
The embodiment of the present invention 1 removal of impurity, chromatograph dissolve with methanol, concentration is 1mg/ml
10% alcohol elution of the embodiment of the present invention 1, evaporate to dryness, chromatograph dissolve with methanol, concentration is 1mg/ml
The 50%-70% alcohol elution of the embodiment of the present invention 1, evaporate to dryness, chromatograph dissolve with methanol, concentration is 1mg/ml
The 70%-100% eluting position of the embodiment of the present invention 1, evaporate to dryness, chromatograph dissolve with methanol, concentration is 1mg/ml
The explanation of the relation of gained total flavones HPLC of the present invention and preparation technology:
Fig. 1 is the embodiment of the present invention 1 gained Ginkgo total flavones (powder) HPLC chromatogram (1mg/ml), compared with Fig. 2 standard silver Folium Pruni extractive HPLC chromatogram (1mg/ml), by obviously increasing the removal flavones content of non-flavones ingredient, but its Main Flavonoids composition does not change.
Fig. 3 is the embodiment of the present invention 1 removal of impurity, the institute of the present invention removal of impurity (HPLC is as Fig. 3), has a large amount of grey matter to remain, through physical and chemical identification, mostly be inorganic salts composition after lighting.Institute's filtrate of getting (HPLC is as Fig. 1) is rufous supernatant liquid after concentrated, and without muddy, main component is different types of flavonoid glycoside.Compared with existing 2010 editions " Chinese Pharmacopoeias " and all kinds of invention, while guarantee medicine stability, greatly improve drug effect, safety is controlled.Can be used as raw material for formulation development.
Fig. 4 is the HPLC of 10% alcohol elution of the embodiment of the present invention 1, and in the present invention, by the removal to non-flavones ingredients such as 10% alcohol elution pigment, tannins, improve general flavone content, the utilization of resources is reasonable, and waste is few.
Fig. 5 is the HPLC at the 50-70% eluting position of the embodiment of the present invention 1, and 50-70% flow point gained flavone purity is high, but its flavone kind is less, and yield is less than 20% (as Fig. 5).
Fig. 6 is the HPLC at the 70%-100% eluting position of the embodiment of the present invention 1.
The invention provides a kind of method of separation and purification high-load total flavones from Folium Ginkgo extract, its preparation technology simply, reliable, cost is low, do not use toxic reagent in process, safe, controlled.Its gained ginkgetin, content is high, waste is few, and definite ingredients is stablized, and ensure that drug effect greatly.
This product is chocolate brown powder, and compared with standard silver Fructus Pruni extract, the good mobility of tool, adheres to less.Can be used as preparation raw material exploitation.Preparation technology of the present invention is simple, reliable, and cost is low, the etoh solvent used in process, water, economy, environmental protection, nontoxic, will have wide market value and important social meaning.
The invention provides a kind of preparation method of total Content of Flavone Glycosides from Ginkgo biloba Extract, main purpose increases flavones content by removing non-flavonoid impurity, compared with existing 2010 editions " Chinese Pharmacopoeias " and all kinds of invention, gained flavones content many (>=42%), yield high (>=73%), and flavone abundant species, compared with other high-load flavone preparation methoies current, impurities is few, is better than the existing standard of standard silver Fructus Pruni extract.The institute of the present invention removal of impurity, analyzes through HPLC and does not almost have flavones ingredient, has a large amount of grey matter to remain, through physical and chemical identification, mostly be inorganic salts composition after lighting.Be rufous supernatant liquid after institute's concentrating filter liquor of getting, without muddy, main component is different types of flavonoid glycoside.While guarantee medicine stability, greatly improve drug effect, safety is controlled.
Function of the present invention cures mainly: blood circulation promoting and blood stasis dispelling, thin network of promoting blood circulation, and for the thoracic obstruction, apoplexy that blood stasis causes, disease sees uncomfortable in chest, cardiopalmus stiff tongue and retardation in speech, hemiplegia.
Detailed description of the invention
Embodiment 1
(1) precision takes Folium Ginkgo extract (flavones content 24%) 20g and puts in 100mL beaker, adds 50ml ethanol, 40 DEG C of heating make it entirely molten after, pour in evaporating dish, add 20g macroporous resin and mix sample;
(2) get macroporous resin D101 appropriate, after pretreatment, wet method dress post, after 95% alcohol flushing, is washed to without alcohol taste, loading;
(3) with variable concentrations graded ethanol, eluting is carried out to macroporous resin column respectively, collect the eluent at 10%-70% ethanol position, 50 DEG C of reclaim under reduced pressure evaporates to dryness.The gradient of ethanol water is respectively: water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol;
(4) by the precipitation that step 3 evaporate to dryness obtains, dissolve with ethanol, standing, sucking filtration, waste gets filtrate, and vacuum drying, porphyrize powder, obtain the total flavones of Folium Ginkgo extract;
(5) flavones content is 43%, productive rate 74%.
Embodiment 2
(1) precision takes Folium Ginkgo extract (flavones content about 24%) 10g and puts in 50mL beaker, adds 25mL methanol, 40 DEG C of heating make it entirely molten after, pour in evaporating dish, add 10g macroporous resin and mix sample;
(2) get macroporous resin D101 appropriate, after pretreatment, wet method dress post, after 95% alcohol flushing, is washed to without alcohol taste, loading; With the ethanol of variable concentrations gradient, eluting is carried out to macroporous resin column respectively, collect the eluent at 10%-70% ethanol position, 50 DEG C of reclaim under reduced pressure evaporates to dryness.The gradient of ethanol water is respectively: water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol
(3) by the precipitation that step 3 evaporate to dryness obtains, dissolve with ethanol, standing, sucking filtration, waste gets filtrate, and vacuum drying, porphyrize powder, obtain the total flavones of Folium Ginkgo extract.
(4) flavones content is 42%, productive rate 76%.
Embodiment 3
(1) take Folium Ginkgo extract (flavones content about 24%) 500g to put in 3L beaker, add 2L methanol, 40 DEG C of heating make it entirely molten after pour pallet into, add about 500g macroporous resin and mix sample;
(2) get macroporous resin D101 appropriate, after pretreatment, wet method dress post, after 95% alcohol flushing, is washed to without alcohol taste, loading;
(3) with different graded ethanol, eluting is carried out to macroporous resin column respectively, collect the eluent at 10%-70% ethanol position, 50 DEG C of reclaim under reduced pressure evaporates to dryness.The gradient of ethanol water is respectively: water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol
(4) by the precipitation that step (3) institute evaporate to dryness obtains, dissolve with ethanol, standing, sucking filtration, waste gets filtrate, and vacuum drying, porphyrize powder, obtain the total flavones of Folium Ginkgo extract.
(5) flavones content is 47%, productive rate 73%.
Embodiment 4
(1) take Folium Ginkgo extract (flavones content about 24%) 500g to put in 3L beaker, add 2L methanol, 40 DEG C of heating make it entirely molten after pour pallet into, add about 500g macroporous resin and mix sample;
(2) get macroporous resin D101 appropriate, after pretreatment, wet method dress post, after 95% alcohol flushing, is washed to without alcohol taste, loading;
(3) with different graded ethanol, eluting is carried out to macroporous resin column respectively, collect the eluent at 10%-70% ethanol position, 50 DEG C of reclaim under reduced pressure evaporates to dryness.The gradient of ethanol water is respectively: water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol;
(4) by the precipitation that step 3 evaporate to dryness obtains, dissolve with ethanol, leave standstill;
(5) sucking filtration, waste, gets filtrate; Use organic solvent washing residue, get washing liquid; Merging filtrate and washing liquid;
(6) vacuum drying, porphyrize powder, obtain the total flavones of Folium Ginkgo extract.
Embodiment 5
Extraction solvent is investigated
1. experimental procedure:
(1) take 5 grams of standard Folium Ginkgo extract 6 parts, put in 100ml beaker;
(2) in beaker, add water, methanol, ethanol, acetone, ethyl acetate, each 20ml of dichloromethane respectively;
(3) heated and stirred 30min makes dissolving;
(4) observe extract and dissolve situation, and sucking filtration.
2. experimental result:
Water: solution bronzing, volume precipitation is not dissolved, and is precipitated quality 2.4g after sucking filtration;
Ethanol: solution dark reddish brown, precipitation is all dissolved, and sucking filtration is not precipitated;
Methanol: solution dark reddish brown, precipitation is all dissolved, and sucking filtration is not precipitated;
Acetone: solution bronzing, volume precipitation is not dissolved, and is precipitated quality 2.1g after sucking filtration;
Ethyl acetate: solution light reddish brown color, volume precipitation is not dissolved, and is precipitated quality 3.3g after sucking filtration;
Dichloromethane: solution colour is lighter, shows slightly faint yellow, and precipitation is not almost dissolved, and is precipitated quality 4.7g after sucking filtration.
3. interpretation of result:
The present embodiment research display, methanol, ethanol are better to Folium Ginkgo extract dissolubility.Water, acetone, ethyl acetate extraction efficiency are poor, and dichloromethane extraction almost can not obtain flavones ingredient.Methanol has stronger toxicity, has the greatest impact to the nervous system of human body and blood system, and it is taken in through digestive tract, respiratory tract or skin all can produce toxic reaction, the respiratory mucosa that methanol vapor energy loss victimizes and vision.Comparatively speaking, alcohol toxicity is less, and safety is higher, so preferred alcohol is Extraction solvent.
Embodiment 6
Solution temperature is investigated
1. experimental procedure:
(1) take 5 grams of standard Folium Ginkgo extract 6 parts, put in 100ml beaker;
(2) in beaker, add each 20ml of ethanol;
(3) respectively 0 DEG C, 10 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C be stirred to dissolve;
(4) dissolving situation is observed, and sucking filtration.
2. experimental result:
0 DEG C: most precipitation is not dissolved, and is precipitated quality 3.4g after sucking filtration;
10 DEG C: most precipitation is not dissolved, and is precipitated quality 3.1g after sucking filtration;
20 DEG C: most precipitation is not dissolved, and is precipitated quality 2.8g after sucking filtration;
30 DEG C: half precipitation is not dissolved, and is precipitated quality 2.4g after sucking filtration;
40 DEG C: all dissolve, without precipitation;
50 DEG C: all dissolve, without precipitation;
60 DEG C: all dissolve, without precipitation.
3. interpretation of result: during 0-30 DEG C, extract dissolubility is poor, and extraction efficiency is low.40-60 DEG C of heated and stirred, extract all dissolves, and extraction efficiency is higher.In order to avoid, temperature is too high has impact to chemical composition, and preferably 40 DEG C are heated to be solution temperature.
Embodiment 7
1. assay:
Measure according to total flavonoids content assaying method in " Chinese Pharmacopoeia " Folium Ginkgo, efficient liquid phase detection method is: take octadecylsilane chemically bonded silica as filler; Methanol-0.4% phosphoric acid (50: 50) is mobile phase; Determined wavelength is 360nm; Chromatographic column temperature: 25 DEG C; Sample size: 10 μ L.Theoretical cam curve calculates should be not less than 2500 with Quercetin peak.
(1) preparation of reference substance solution: get Quercetin reference substance, kaempferide reference substance, isorhamnetin reference substance respectively in right amount, accurately weighed, add methanol and make every 1ml respectively containing the mixed solution of 30 μ g, 30 μ g, 20 μ g, product solution in contrast.
(2) preparation of need testing solution: the total flavones sample of Example 1 gained Folium Ginkgo extract is about 1g, accurately weighed, puts in apparatus,Soxhlet's, add chloroform and reflux 2 hours, discard chloroform liquid, medicinal residues volatilize; Add methanol eddy and extract 4 hours, extracting solution volatilizes, and residue adds methanol-25% hydrochloric acid solution (4: 1) mixed solution 25 milliliters, and reflux 30 minutes, lets cool, and is transferred in 50ml measuring bottle, and adds methanol to scale, shakes up, to obtain final product.
(3) total flavonoids content=(quercetin content+kaempferide content+isorhamnetin content) * 2.51
2. cubage
Reference substance concentration:
C
kaempferide=30.4 μ g/ml
C
isorhamnetin=18.8 μ g/ml
C
quercetin=33.0 μ g/ml
(1) standard silver Folium Pruni extract concentrations:
Recorded by HPLC,
Reference substance peak area:
A
kaempferide=1267220
A
isorhamnetin=1085827
A
quercetin=1345106
Test sample peak area:
A
kaempferide'=41044508
A
isorhamnetin'=11578117
A
quercetin'=35765399
By A
1/ A
1'=C
1/ C
1' known,
C
kaempferide'=(A
kaempferide' * C
kaempferide)/A
kaempferide=(36646061*30.4) μ g/ml/1267220=879.12 μ g/ml in like manner,
C
isorhamnetin'=141.68 μ g/ml
C
quercetin'=922.49 μ g/ml
W=(C
kaempferide+ C
isorhamnetin+ C
quercetin) * 50ml/1g*2.51*100%
W＝(879.12+141.68+922.49)μg/ml*50ml/1g*2.51*100％＝24.39％
(2) gained extractum content of the present invention
Recorded by HPLC,
Reference substance peak area:
A
kaempferide=1267220
A
isorhamnetin=1085827
A
quercetin=1345106
Test sample peak area:
A
kaempferide'=72122856
A
isorhamnetin'=18343000
A
quercetin'=65896590
By A
1/ A
1'=C
1/ C
1' known,
C
kaempferide'=(A
kaempferide' * C
kaempferide)/A
kaempferide=(72122856*30.4) μ g/ml/1267220=1552.02 μ g/ml in like manner,
C
isorhamnetin'=309.66 μ g/ml
C
quercetin'=1514.65 μ g/ml
W=(C
kaempferide+ C
isorhamnetin+ C
quercetin) * 50ml/1g*2.51*100%
W＝(1552.02+309.66+1514.65)μg/ml*50ml/1g*2.51*100％＝42.37％
Embodiment 8
Gained total flavones HPLC optimum condition of the present invention
The embodiment of the present invention 1 gained Ginkgo total flavones (powder), chromatograph dissolve with methanol, concentration is 1mg/ml
Sample size: 10 μ L.
Instrument: Shimadzu LC-20A chromatograph of liquid
The testing conditions of HPLC: detect under ultraviolet 360nm condition
HPLC mobile phase:
A:0.1% formic acid-water
B: acetonitrile
C: methanol (washing post)
D: water (washing post)
Flow velocity: 1ml/min
Temporal sequence:
Embodiment 9
The total flavones of the Folium Ginkgo extract in Example 1 and each 5.0g of standard Folium Ginkgo extract, measure angle of repose by fixing cone method.After measured, total flavones angle of repose of the Folium Ginkgo extract in embodiment 1 is 31 °, and standard Folium Ginkgo extract angle of repose 47 °, this product is chocolate brown powder, and compared with standard silver Fructus Pruni extract, the good mobility of tool, adheres to less.
Embodiment 10
The total flavones 80g that Folium Ginkgo extract in Example 1 prepares, starch 75g, dextrin 75g, calcium sulfate 255g, crosslinked ketopyrrolidine 10g, makes 1000 (sheets) or 2000 (small pieces);
Method for making: the total flavones that the Folium Ginkgo extract in Example 1 prepares, add starch, dextrin, calcium sulfate and crosslinked ketopyrrolidine, make granule, tabletting, sugar coating or film-coat, to obtain final product.
The every sheet of this product must not be less than (small pieces) 16.8mg, (sheet) 32.6mg containing total flavonoids.
Claims (10)
1. a preparation method for Folium Ginkgo total flavones, is characterized in that: by Folium Ginkgo extract dissolution with solvents, and macroporous resin mixes sample loading, cross macroporous resin column to be separated, the ethanol water eluting of different gradient, collects the flow point between 10%-70%, concentrated, dry; Dissolve with ethanol, leaves standstill, sucking filtration, and waste, gets filtrate, vacuum drying, porphyrize, and to get final product.
2. a Folium Ginkgo total flavones, is characterized in that: flavones content >=42%.
3. the preparation method of a kind of Folium Ginkgo total flavones according to claim 2, is characterized in that: take Folium Ginkgo extract as raw material, its processing step comprises:
(1) to Folium Ginkgo extract sample pretreatment: after dissolve with ethanol, add macroporous resin and mix sample;
(2) macroporous resin column eluting: that step (1) is handled well mixes sample macropore loading, and the ethanol of different gradient carries out eluting to macroporous resin column respectively;
(3) eluent is collected: the eluent collecting the 10%-70% ethanol position of flowing out in macroporous resin column in step (2);
(4) concentrating under reduced pressure: the eluent in step (3) is carried out concentrating under reduced pressure by gradient, merges concentrated solution, evaporate to dryness; Temperature control 45-60 DEG C; Vacuum degree control 0.1MPa;
(5) dissolve: the precipitation that step (4) evaporate to dryness obtains, adds organic solvent dissolution;
(6) leave standstill;
(7) filter, waste, gets filtrate;
(8) dry, porphyrize powder, to obtain final product.
4. the preparation method of a kind of Folium Ginkgo total flavones according to claim 3, is characterized in that:
(7) filter, waste, gets filtrate; Use organic solvent washing residue, get washing liquid; Merging filtrate and washing liquid;
(8) dry, porphyrize powder, to obtain final product.
5. the preparation method of a kind of Folium Ginkgo total flavones according to claim 3, it is characterized in that macroporous resin used in (1) is take styrene as the macroporous resin D101 of main skeleton, and mixing solvent selected by sample is ethanol.
6. the preparation method of a kind of Folium Ginkgo total flavones according to claim 3, it is characterized in that (2)-(3) middle eluent collected is 10%-70% ethanol position.
7. the preparation method of a kind of Folium Ginkgo total flavones according to claim 3, is characterized in that
In step (5), solvent selected by resolution of precipitate is the combination in any in ethanol, methanol, ethyl acetate, acetone;
In step (6), time of repose is 6 hours;
In step (7), filter type is vacuum filtration.
8. the preparation method of a kind of Folium Ginkgo total flavones according to claim 3, is characterized in that: in (7) after sucking filtration, preferred site is filtrate; In step (5), ethanol contend is the precipitation quality of 10 times amount.
9. the analytical method of a kind of Folium Ginkgo total flavones according to claim 2, is characterized in that:
The condition of HPLC:
Detect under ultraviolet 360nm condition
HPLC mobile phase:
A:0.1%-5% formic acid-water
B: acetonitrile
C: methanol, washes post
D: water, washes post
Flow velocity: 1ml/min;
Temporal sequence:
The composition mobile phase total volume percent of time min unit mobile phase
。
10. the tablet of a kind of Folium Ginkgo total flavones according to claim 2, is characterized in that:
Folium Ginkgo total flavones 25-150g, starch 25-150g, dextrin 25-150g, calcium sulfate 150-500g, crosslinked ketopyrrolidine 2-40g.
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| CN110196291A (en) * | 2019-04-30 | 2019-09-03 | 贵州中医药大学 | The detection method of 7 kinds of flavones ingredients in a kind of Sabia parviflora Wall.ex Roxb medicinal material |
| CN112439000A (en) * | 2019-08-31 | 2021-03-05 | 北京化工大学 | Method for extracting flavonoid compounds from ginkgo leaves by combining eutectic solvent with macroporous resin purification |
| CN111537656A (en) * | 2020-06-19 | 2020-08-14 | 劲牌有限公司 | Identification method of tartary buckwheat extract |
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