CN106166164B - Preparation method of ginkgo leaf extract and product thereof - Google Patents
Preparation method of ginkgo leaf extract and product thereof Download PDFInfo
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- 239000009429 Ginkgo biloba extract Substances 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 70
- 241000218628 Ginkgo Species 0.000 claims abstract description 64
- 235000011201 Ginkgo Nutrition 0.000 claims abstract description 64
- 235000008100 Ginkgo biloba Nutrition 0.000 claims abstract description 64
- 239000000284 extract Substances 0.000 claims abstract description 51
- 238000010992 reflux Methods 0.000 claims abstract description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002244 precipitate Substances 0.000 claims abstract description 27
- 238000000605 extraction Methods 0.000 claims abstract description 25
- 239000011347 resin Substances 0.000 claims abstract description 22
- 229920005989 resin Polymers 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 19
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- 230000001376 precipitating effect Effects 0.000 claims abstract description 9
- 238000007873 sieving Methods 0.000 claims abstract description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- 239000006260 foam Substances 0.000 claims description 16
- 238000001291 vacuum drying Methods 0.000 claims description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 14
- YXHVCZZLWZYHSA-UHFFFAOYSA-N (Z)-6-[8-pentadecenyl]salicylic acid Natural products CCCCCCC=CCCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-UHFFFAOYSA-N 0.000 claims description 12
- YXHVCZZLWZYHSA-FPLPWBNLSA-N Ginkgoic acid Chemical compound CCCCCC\C=C/CCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-FPLPWBNLSA-N 0.000 claims description 12
- SVINQHQHARVZFF-UHFFFAOYSA-N Ginkgotoxin Chemical compound COCC1=C(CO)C=NC(C)=C1O SVINQHQHARVZFF-UHFFFAOYSA-N 0.000 claims description 12
- SQGLUEWZRKIEGS-UHFFFAOYSA-N Ginkgetin Natural products C1=CC(OC)=CC=C1C1=CC(=O)C2=C(O)C=C(OC)C(C=3C(=CC=C(C=3)C=3OC4=CC(O)=CC(O)=C4C(=O)C=3)O)=C2O1 SQGLUEWZRKIEGS-UHFFFAOYSA-N 0.000 claims description 11
- 229930045534 Me ester-Cyclohexaneundecanoic acid Natural products 0.000 claims description 11
- 229930003944 flavone Natural products 0.000 claims description 11
- 235000011949 flavones Nutrition 0.000 claims description 11
- AIFCFBUSLAEIBR-UHFFFAOYSA-N ginkgetin Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C(C=1)=CC=C(OC)C=1C1=C(O)C=C(O)C(C(C=2)=O)=C1OC=2C1=CC=C(O)C=C1 AIFCFBUSLAEIBR-UHFFFAOYSA-N 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
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- 150000002212 flavone derivatives Chemical class 0.000 claims description 9
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims description 9
- UFJORDZSBNSRQT-UHFFFAOYSA-N 3-(4-oxo-2-phenylchromen-3-yl)-2-phenylchromen-4-one Chemical compound O1C2=CC=CC=C2C(=O)C(C=2C(C3=CC=CC=C3OC=2C=2C=CC=CC=2)=O)=C1C1=CC=CC=C1 UFJORDZSBNSRQT-UHFFFAOYSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 8
- 229920000053 polysorbate 80 Polymers 0.000 claims description 8
- 229940068052 ginkgo biloba extract Drugs 0.000 claims description 7
- 235000020686 ginkgo biloba extract Nutrition 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 229930184727 ginkgolide Natural products 0.000 claims description 5
- -1 diterpene lactone Chemical class 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000000703 high-speed centrifugation Methods 0.000 claims description 2
- 238000009775 high-speed stirring Methods 0.000 claims description 2
- 229930004069 diterpene Natural products 0.000 claims 3
- 239000002994 raw material Substances 0.000 abstract description 6
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- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- MOLPUWBMSBJXER-YDGSQGCISA-N bilobalide Chemical compound O([C@H]1OC2=O)C(=O)[C@H](O)[C@@]11[C@@](C(C)(C)C)(O)C[C@H]3[C@@]21CC(=O)O3 MOLPUWBMSBJXER-YDGSQGCISA-N 0.000 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- SQFSKOYWJBQGKQ-UHFFFAOYSA-N kaempferide Chemical compound C1=CC(OC)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 SQFSKOYWJBQGKQ-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
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- 150000003505 terpenes Chemical class 0.000 description 4
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- 238000012216 screening Methods 0.000 description 3
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- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 2
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 150000002213 flavones Chemical class 0.000 description 2
- IZQSVPBOUDKVDZ-UHFFFAOYSA-N isorhamnetin Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 IZQSVPBOUDKVDZ-UHFFFAOYSA-N 0.000 description 2
- 235000008800 isorhamnetin Nutrition 0.000 description 2
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 2
- 150000002596 lactones Chemical class 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000005875 quercetin Nutrition 0.000 description 2
- 229960001285 quercetin Drugs 0.000 description 2
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- 238000001694 spray drying Methods 0.000 description 2
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- 239000004480 active ingredient Substances 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
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- 239000010432 diamond Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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Classifications
-
- 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)
-
- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- 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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- 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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- 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 discloses a preparation method of a high-purity ginkgo leaf standard extract, which comprises the following steps: weighing raw materials, crushing, sieving, and putting into an extraction reflux tank; reflux extraction; recovering ethanol, and vacuum concentrating to obtain extract with specific gravity of 1.0-1.5; fully stirring the extract with pure water, and centrifuging at high speed to remove precipitate to obtain supernatant; passing the supernatant through macroporous resin, gradient eluting, collecting 40-60% ethanol eluate, and acidifying to obtain acidified solution; precipitating the acidified solution at 0-5 deg.C overnight, and collecting precipitate; washing the precipitate with pure water, and drying under vacuum condition to obtain standard folium Ginkgo extract. The method has the advantages of simple adopted equipment, simple and feasible process and convenience for industrial production; the used solvent is nontoxic and has no residue, and no environmental pollution is caused; the ginkgo leaf extract has high removal rate of harmful substances, and the produced standard extract meets the requirements of the American FDA.
Description
Technical Field
The invention relates to the technical field of medicines, and in particular relates to a preparation method of a high-purity ginkgo leaf standard extract and a product thereof.
Background
The ginkgo standard extract is widely applied to cardiovascular and cerebrovascular diseases, is one of the most extensive traditional Chinese medicine varieties, and with the deep research on the ginkgo extract, the side effects of active ingredients and other ingredients in the ginkgo extract on human bodies are more and more discovered, the ginkgolic acid causes allergic dermatitis to the skin of the human bodies, and 4-O-methyl pyridoxine causes convulsion and unconsciousness. The ginkgo biloba extract mainly contains more than 30 bioactive flavones, wherein the structure of more than 20 kinds of the bioactive flavones is analyzed and clarified, and various researches show that the ginkgo biloba extract has wide biological activities of inhibiting platelet aggregation, regulating blood fat, expanding blood vessels, improving brain circulation, improving brain function, resisting atherosclerosis, resisting tumors and the like; the ginkgolide is a diterpenoid compound, mainly comprises ginkgolides A, B, C, J, M and bilobalide, is a recognized platelet activating factor receptor antagonist, has great effects on cerebral nerve transmission, repair and protection of cerebral tissues, and has good development prospects for heart and cerebral vessels. Chinese ginkgo is the world major production place of ginkgo and accounts for 70 percent of the world output, but most of ginkgo extracts in China only have primary extracts, the content is low, the lactone content does not meet the requirements, toxic and harmful components in the ginkgo extracts cannot be separated, and consequently, primary products can be provided only for foreign countries, and the value is very low. General study of extraction process of ginkgo biloba extract: many manufacturers of ginkgo extracts, most of which are obtained by water extraction or low-concentration alcohol extraction, then passing through macroporous resin D101, and then spray-drying. Others are still in the laboratory research stage and have not gone into production applications such as molecular membrane filtration, reverse liquid chromatography, supercritical CO2 extraction, etc., and such reports are numerous. Now, a new method for improving the content of ginkgo flavone is urgently needed to be developed.
Disclosure of Invention
Technical problem to be solved
The invention aims to solve the technical problem of how to improve the content of ginkgo flavone and effectively remove harmful components, and provides a preparation method of a high-purity ginkgo leaf standard extract and a product thereof.
(II) technical scheme
In order to solve the technical problems, the invention provides a preparation method of a high-purity ginkgo leaf standard extract, which comprises the following steps (raw materials are all purchased in the market):
the method comprises the following steps: preparing: weighing raw materials, crushing, sieving with a 18-22 mesh sieve, drying in the sun, and then putting the dried raw materials into an extraction reflux tank;
step two: reflux extraction: adding 60-75% ethanol 8-12 times of folium Ginkgo, refluxing at 65-70 deg.C for 2.5-3.5 hr;
step three: secondary reflux extraction: adding 60-75% ethanol 4-6 times the weight of folium Ginkgo, and refluxing at 65-70 deg.C for 1.5-2.5 hr;
step four: concentration: mixing the two reflux liquids, recovering alcohol, and vacuum concentrating to obtain extract with specific gravity of 1.0-1.5;
step five: removing bubbles: stirring the extract with 8-12 times of pure water, alkalifying with KOH to pH 8.0-10, adding 0.2% Tween 80, stirring at high speed to automatically remove foam until no foam is generated, and centrifuging at high speed to remove precipitate to obtain supernatant;
step six: filtering with macroporous resin: passing the supernatant through macroporous resin, gradient eluting, collecting 40-60% ethanol eluate, collecting 5 BV eluates, recovering ethanol, stirring with 8-12 times of pure water, and acidifying with hydrochloric acid to pH3.5-4.5 to obtain acidified solution;
step seven: precipitating the acidified liquid obtained in the sixth step at 0-5 ℃ overnight, and collecting the precipitate;
step eight: and (3) vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH of 6.5-7.0, and vacuum drying to obtain standard folium Ginkgo extract with ginkgetin content of 31%, bilobalide 11%, ginkgolic acid content of less than 1PPM, 4-O-methyl pyridoxine content of less than 1PPM, and biflavone content of less than 1 PPM; the yield thereof was found to be 2.3%.
Preferably, in the first step, the sieve is 20 meshes.
Preferably, in step two and step three, the reflux temperature is 65 ℃.
Preferably, in step four, the recovered alcohol is recovered to an alcohol content of less than 15%.
Preferably, in the fourth step, the vacuum degree is-0.08-0.09 MPa; the specific gravity is 1.15-1.2.
Preferably, in step six, the macroporous resin used is a D201 macroporous resin.
Preferably, the preparation method comprises the following steps:
the method comprises the following steps: preparing: weighing raw materials, crushing, sieving with a 20-mesh sieve, drying in the sun, and then putting the dried raw materials into an extraction reflux tank;
step two: reflux extraction: adding 65% alcohol 10 times of folium Ginkgo weight, refluxing at 65 deg.C for 3 hr;
step three: secondary reflux extraction: adding 65% alcohol 5 times of folium Ginkgo weight, controlling temperature at 65 deg.C, refluxing for 2 hr;
step four: concentration: mixing the two reflux liquids, recovering alcohol until the alcohol content is below 15%, and vacuum concentrating to obtain extract with specific gravity of 1.15-1.2;
step five: removing bubbles: fully stirring the extract with 10 times of pure water by weight, alkalifying the extract with KOH to pH9.0, adding 0.2% Tween 80, stirring at high speed to automatically remove foam until no foam is generated, and centrifuging at high speed to remove precipitate to obtain supernatant;
step six: filtering with macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40-60% ethanol eluate, collecting 5 BV eluates, recovering ethanol, stirring with 10 times of pure water, and acidifying with hydrochloric acid to pH4.0 to obtain acidified solution;
step seven: precipitating the acidified liquid obtained in the sixth step at 0-5 ℃ overnight, and collecting the precipitate;
step eight: and (3) vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH 7.0, and vacuum drying to obtain standard folium Ginkgo extract with ginkgetin content of 31%, bilobalide 11%, ginkgolic acid content of less than 1PPM, 4-O-methyl pyridoxine content of less than 1PPM, and biflavone content of less than 1 PPM; the yield thereof was found to be 2.3%.
The invention also provides the standard ginkgo biloba extract prepared by the preparation method of the standard high-purity ginkgo biloba extract.
(III) advantageous effects
The method has the advantages of simple adopted equipment, simple and feasible process and convenience for industrial production; the used solvent is nontoxic and has no residue, and no environmental pollution is caused; the method has high removal rate of harmful substances in ginkgo, and the produced standard extract meets the requirements of the American FDA.
Detailed Description
The following examples further describe embodiments of the present invention in detail. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The solvents, KOH, HCL used below were all food grade or analytical grade, and commercial grade should not be used.
Example 1
1, taking 500KG of ginkgo leaves (dried), crushing and screening the ginkgo leaves (dried) with a 20-mesh sieve according to HPLC detection, wherein the ginkgo leaves contain 0.72 percent of ginkgetin and 0.29 percent of terpene dilactone, and putting the ginkgo leaves into a reflux extraction tank;
2, first reflux extraction: 5000 kilograms of 65 percent alcohol, the temperature is controlled at 65 ℃ and the reflux time is 3 hours;
and 3, second refluxing: 2500 kg of 65% alcohol, the temperature is controlled at 65 ℃, and the reflux is carried out for 2 hours;
4, concentrating: and combining the two reflux liquids, recovering alcohol, and concentrating in vacuum to 173 kg of extract with the specific gravity of 1.2.
5, defoaming: 1730 kg of pure water is added into 173 kg of the extract, KOH is alkalized to pH9.0, 0.2 percent of Tween 80 is added, the mixture is stirred at high speed to automatically remove foam until no foam is generated, and then the mixture is centrifuged at high speed to remove precipitates, so 1860 kg of supernatant is obtained.
6, filtering by macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40% -60% ethanol eluate, 5 BV eluates, recovering ethanol, stirring with 10 times of water, and acidifying with hydrochloric acid to pH 4.0;
7, precipitating the acidizing fluid at 0-5 ℃ overnight, and collecting 39 kg of precipitate;
and 8, washing and vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH of 6.5-7.0, vacuum drying to obtain standard folium Ginkgo extract, and analyzing by HPLC to obtain ginkgetin content of 30.69%, bilobalide 11.72%, ginkgolic acid, 4-O-methyl pyridoxine, and biflavone content lower than 1PPM, and collecting standard folium Ginkgo extract 11.36 kg.
Example 2
1, taking 500KG of ginkgo leaves (dried), crushing and screening the ginkgo leaves (dried) with a 20-mesh sieve according to HPLC detection, wherein the ginkgo leaves contain 0.76 percent of ginkgetin and 0.31 percent of terpene dilactone, and putting the ginkgo leaves into a reflux extraction tank;
2, first reflux extraction: 5000 kilograms of 65 percent alcohol, the temperature is controlled at 65 ℃ and the reflux time is 3 hours;
and 3, second refluxing: 2500 kg of 65% alcohol, the temperature is controlled at 65 ℃, and the reflux is carried out for 2 hours;
4, concentrating: and combining the two reflux liquids, recovering alcohol, and concentrating in vacuum to 179 kg of extract with the specific gravity of 1.2.
5, defoaming: adding 1790 kg of pure water into 179 kg of extract, alkalifying KOH to pH9.0, adding 0.2% Tween 80, stirring at high speed to automatically remove foam until no foam is generated, and centrifuging at high speed to remove precipitate to obtain 1910 kg of supernatant.
6, filtering by macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40% -60% ethanol eluate, 5 BV eluates, recovering ethanol, stirring with 10 times of water, and acidifying with hydrochloric acid to pH 4.0;
7, precipitating the acidizing fluid at 0-5 ℃ overnight, and collecting 46 kg of precipitate;
and 8, washing and vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH of 6.5-7.0, vacuum drying to obtain standard folium Ginkgo extract, and analyzing by HPLC to obtain ginkgetin content of 30.27%, bilobalide 10.97%, ginkgolic acid, 4-O-methyl pyridoxine, and biflavone content lower than 1PPM, and collecting standard folium Ginkgo extract 12.77 kg.
Example 3
1, taking 500KG of ginkgo leaves (dried), crushing and screening the ginkgo leaves (containing 0.69% of ginkgetin and 0.25% of terpene dilactone by HPLC detection) through a 20-mesh sieve, and putting the ginkgo leaves and the terpene dilactone into a reflux extraction tank;
2, first reflux extraction: 5000 kilograms of 65 percent alcohol, the temperature is controlled at 65 ℃ and the reflux time is 3 hours;
and 3, second refluxing: 2500 kg of 65% alcohol, the temperature is controlled at 65 ℃, and the reflux is carried out for 2 hours;
4, concentrating: and combining the two reflux liquids, recovering alcohol, and concentrating in vacuum to obtain 165 kg of extract with the specific gravity of 1.2.
5, defoaming: 1650 kg of pure water is added into 165 kg of extract, KOH is alkalized to PH9.0, 0.2% Tween 80 is added, foam is automatically removed by high-speed stirring until no foam is generated, and then high-speed centrifugation is carried out to remove precipitate, so as to obtain 1790 kg of supernatant.
6, filtering by macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40% -60% ethanol eluate, 5 BV eluates, recovering ethanol, stirring with 10 times of water, and acidifying with hydrochloric acid to pH 4.0;
7, precipitating the acidizing fluid at 0-5 ℃ overnight, and collecting 33 kg of precipitate;
and 8, washing and vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH of 6.5-7.0, vacuum drying to obtain standard folium Ginkgo extract, and analyzing by HPLC to obtain ginkgetin content of 30.19%, bilobalide 11.05%, ginkgolic acid, 4-O-methyl pyridoxine, and biflavone content lower than 1PPM, and collecting standard folium Ginkgo extract 10.15 kg.
Comparative example 1
The ginkgo extraction and purification process of Shandong agriculture university and the ethanol extraction-resin adsorption method are adopted, and the process comprises the following steps:
leaching folium Ginkgo with 70% alcohol 12 times of the weight of folium Ginkgo, leaching at 55-60 deg.C for 3 hr for 2 times, mixing filtrates, concentrating to specific gravity of 1.2, diluting with 10 times of water, stirring, centrifuging at high speed, removing supernatant, passing through D101 macroporous resin column, washing with water until colorless, eluting with 70% alcohol to obtain eluate, concentrating the eluate, and spray drying to obtain folium Ginkgo extract.
More specifically, the ginkgo leaf extract is extracted by the process of more than 500kg of ginkgo leaves (the content of analyzed flavone is 0.81%, and the content of lactone is 0.32%) to obtain 16.7 kg of ginkgo leaf extract.
Experimental example 1
HPLC determination of ginkgo total flavone (food science and technology 2006, Beijing Nutrition element food research institute such as 03 WangJing Jing)
1, a detection method:
1) materials and instruments
Phosphoric acid, hydrochloric acid, methanol, standard substances (kaempferide, quercetin, isorhamnetin), and purified water
WATER 2487 high pressure liquid chromatography, ultrasonic oscillator, DHT magnetic stirrer
2) Method of producing a composite material
Chromatographic conditions are as follows: chromatographic column diamond C18 (250X 4.65 micron) column temperature 40 deg.C, detection wavelength 370nm
Mobile phase: methanol: 0.4% phosphoric acid 60: 40 (volume ratio), flow rate 1.0ML/MIN, sample size 20UL
3) The quantitative method comprises the following steps: external standard method
4) And (3) calculating:
the total flavone amount is 2.51 quercetin amount +2.63 kaempferide amount +2.39 isorhamnetin amount
Experimental example 2
Bilobalide assay
RP-HPLC method for measuring bilobalide content (Judaming, Changchun Chinese medicine university, Jilin agriculture university school newspaper 2013, 35(4))
Experimental example 3
4-O-methyl pyridoxine assay
GC-MS detection of 4-O-methyl pyridoxine
Experimental example 4
Determination of ginkgolic acid
RP-HPLC method for measuring content of ginkgolic acid (Jiangfeng, Jiangsu Huaiyin institute of Industrial and chemical institute, proceedings 200810)
Table: the result of the detection
| Sample (I) | Ginkgo flavone | Bilobalide | 4-O-methyl pyridoxine | Ginkgolic acid |
| Example 1 | 30.69％ | 11.72％ | Less than 1PPM | Less than 1PPM |
| Example 2 | 30.27％ | 10.97％ | Less than 1PPM | Less than 1PPM |
| Example 3 | 30.19％ | 11.05％ | Less than 1PPM | Less than 1PPM |
| Comparative example | 24.9％ | 6.82％ | 15 mg/kg | 27 mg/kg |
The results in the table show that: compared with the traditional extraction process, the method has obvious improvement effect on removing harmful substances in the ginkgo biloba extract, and has good popularization and application significance.
The above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is covered by the claims of the present invention.
Claims (4)
1. A preparation method of a ginkgo leaf extract is characterized by comprising the following steps:
1) taking 500Kg of dried ginkgo leaves, crushing, sieving by a 20-mesh sieve, and putting into a reflux extraction tank; the dry folium Ginkgo contains ginkgetin 0.72% and diterpene lactone 0.29% by HPLC detection;
2) first reflux extraction: 5000 kilograms of 65 percent alcohol, the temperature is controlled at 65 ℃ and the reflux time is 3 hours;
3) and (3) second reflux: 2500 kg of 65% alcohol, the temperature is controlled at 65 ℃, and the reflux is carried out for 2 hours;
4) concentration: mixing the two reflux liquids, recovering alcohol, and vacuum concentrating to obtain 173 kg of extract with the specific gravity of 1.2;
5) removing bubbles: 1730 kg of pure water is added into 173 kg of the extract, KOH is alkalized to pH9.0, 0.2 percent of Tween 80 is added, the mixture is stirred at high speed to automatically remove foam until no foam is generated, and then the mixture is centrifuged at high speed to remove precipitates, thus obtaining 1860 kg of supernatant;
6) filtering with macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40% -60% ethanol eluate, 5 BV eluates, recovering ethanol, stirring with 10 times of water, and acidifying with hydrochloric acid to pH4.0;
7) precipitating the acidified solution at 0-5 ℃ overnight, and collecting 39 kg of precipitate;
8) washing and vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH 6.5-7.0, vacuum drying to obtain folium Ginkgo extract, and analyzing by HPLC to obtain folium Ginkgo extract with ginkgo flavone content of 30.69%, ginkgolide of 11.72%, ginkgolic acid, 4-O-methyl pyridoxine, and biflavone content of less than 1ppm, and collecting folium Ginkgo extract 11.36 kg.
2. A preparation method of a ginkgo leaf extract is characterized by comprising the following steps:
1) taking 500Kg of dried ginkgo leaves, crushing, sieving by a 20-mesh sieve, and putting into a reflux extraction tank; the dry folium Ginkgo contains ginkgetin 0.76% and diterpene lactone 0.31% by HPLC detection;
2) first reflux extraction: 5000 kilograms of 65 percent alcohol, the temperature is controlled at 65 ℃ and the reflux time is 3 hours;
3) and (3) second reflux: 2500 kg of 65% alcohol, the temperature is controlled at 65 ℃, and the reflux is carried out for 2 hours;
4) concentration: mixing the two reflux liquids, recovering alcohol, and vacuum concentrating to obtain 179 kg of extract with the specific gravity of 1.2;
5) removing bubbles: adding 1790 kg of pure water into 179 kg of extract, alkalifying KOH to pH9.0, adding 0.2% Tween 80, stirring at high speed to automatically remove foam until no foam is generated, and centrifuging at high speed to remove precipitate to obtain 1910 kg of supernatant;
6) filtering with macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40% -60% ethanol eluate, 5 BV eluates, recovering ethanol, stirring with 10 times of water, and acidifying with hydrochloric acid to pH4.0;
7) precipitating the acidified solution at 0-5 ℃ overnight, and collecting 46 kg of precipitate;
8) washing and vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH 6.5-7.0, vacuum drying to obtain folium Ginkgo extract, and analyzing by HPLC to obtain folium Ginkgo extract with ginkgo flavone content of 30.27%, ginkgolide 10.97%, ginkgolic acid, 4-O-methyl pyridoxine, and biflavone content of less than 1ppm, and collecting folium Ginkgo extract 12.77 kg.
3. A preparation method of a ginkgo leaf extract is characterized by comprising the following steps:
1) taking 500Kg of dried ginkgo leaves, crushing, sieving by a 20-mesh sieve, and putting into a reflux extraction tank; the dry folium Ginkgo contains ginkgetin 0.69% and diterpene lactone 0.25% by HPLC detection;
2) first reflux extraction: 5000 kilograms of 65 percent alcohol, the temperature is controlled at 65 ℃ and the reflux time is 3 hours;
3) and (3) second reflux: 2500 kg of 65% alcohol, the temperature is controlled at 65 ℃, and the reflux is carried out for 2 hours;
4) concentration: mixing the two reflux liquids, recovering alcohol, and vacuum concentrating to obtain 165 kg of extract with the specific gravity of 1.2;
5) removing bubbles: 1650 kg of pure water is added into 165 kg of extract, KOH is alkalized to PH9.0, 0.2% Tween 80 is added, foam is automatically removed by high-speed stirring until no foam is generated, and then high-speed centrifugation is carried out to remove precipitate, so as to obtain 1790 kg of supernatant;
6) filtering with macroporous resin: passing the supernatant through D201 macroporous resin, gradient eluting, collecting 40% -60% ethanol eluate, 5 BV eluates, recovering ethanol, stirring with 10 times of water, and acidifying with hydrochloric acid to pH4.0;
7) precipitating the acidified solution at 0-5 ℃ overnight, and collecting 33 kg of precipitate;
8) washing and vacuum drying: washing the precipitate with pure water at 0-5 deg.C to pH 6.5-7.0, vacuum drying to obtain folium Ginkgo extract, and analyzing by HPLC to obtain folium Ginkgo extract with ginkgo flavone content of 30.19%, ginkgolide of 11.05%, ginkgolic acid, 4-O-methyl pyridoxine, and biflavone content of less than 1ppm, and collecting folium Ginkgo extract 10.15 kg.
4. The ginkgo biloba extract produced by the method of any one of claims 1 to 3.
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