CN106389484A - Methods for preparing water-soluble ginkgo exopleura extract and preparation thereof - Google Patents
Methods for preparing water-soluble ginkgo exopleura extract and preparation thereof Download PDFInfo
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- CN106389484A CN106389484A CN201610916393.XA CN201610916393A CN106389484A CN 106389484 A CN106389484 A CN 106389484A CN 201610916393 A CN201610916393 A CN 201610916393A CN 106389484 A CN106389484 A CN 106389484A
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- water
- extract
- preparation
- ethanol
- exopleura
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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)
-
- 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
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- 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
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention relates to methods for preparing a water-soluble ginkgo exopleura extract and a preparation thereof. The method for preparing the water-soluble ginkgo exopleura extract comprises the following steps of putting ethanol-soaked ginkgo exopleura into a handled cooking pot, conventionally adding water into the handled cooking pot, decocting an obtained first mixture, filtering the decocted first mixture, carrying out vacuum concentration on the filtered first mixture, slowly adding ethanol into a concentrated solution, quickly agitating an obtained second mixture by adopting a magnetic agitator, making the agitated second mixture stand and precipitate, and carrying out freeze drying or vacuum drying on a precipitate, so that the water-soluble ginkgo exopleura extract is obtained. The main component of the extract is proteoglycan, and has anti-tumor activity. The extract is added into an excipient or a flavoring agent and a preservative to be made into anti-tumor capsule preparations, tablets or oral solution preparations. The ginkgo exopleura extract obtained by the method is small in particle size and good in water solubility; the content of the proteoglycan and the anti-tumor potency are both improved; the water-soluble ginkgo exopleura extract can be prepared into anti-tumor solid and liquid preparations.
Description
Technical field
The present invention relates to the preparation method of a kind of water-solubility ginkgo episperm extract and its preparation, belong to herbal pharmaceutical neck
Domain.
Background technology
Semen Ginkgo is the rare tree of China, is also the important plant resourceses that Jiangsu Province's agricultural is earned foreign exchange.In order to preferably develop
Using gingko resource, from the episperm of Semen Ginkgo, extraction has antitumor action and the polysaccharide of purposes carries earliest for our seminars
Take thing, have been achieved for a series of achievements in research.However, due to water miscible problem, making the dosage form of extraction fromginkgoseed coat
Selection receives a definite limitation.We are once in traditional handicraft（ZL00134363.7）On the basis of improved, after improvement
Technique（ZL201010251068.9）Although water solublity improves, technique is loaded down with trivial details, and preparation time is longer, increased cost.With
Based on improvement water solublity, syllabus target process optimization is always the research emphasis of this seminar.
Content of the invention
The purpose of the present invention is for the deficiencies in the prior art, provides a kind of water-solubility ginkgo episperm extract and its system
The preparation method of agent.
First purpose of the present invention is achieved through the following technical solutions, a kind of water-solubility ginkgo episperm extract
Preparation method, comprise the following steps：
The gingko episperm being soaked by ethanol is placed in boil and puies forward routine in pot and add water to cook 2-3 times and filter respectively, will
Filtrate merging is evaporated to proportion in 1.21-1.30；By solution with container with 0.05-0.15（V/V）Ratio by concentrated solution
It is transferred in container, container is placed on the pedestal of magnetic stirring apparatuss, ethanol is slowly added dropwise in concentrated solution, make ethanol dense eventually
Degree reaches 60-80% in 1.5-2.5h（V/V）, start magnetic stirring apparatuss while adding ethanol, with 1400- at 20-30 DEG C
1800rpm/min stirs, and precipitate is washed with high concentration ethanol after standing lyophilization or vacuum drying again after for several times, obtains
Water-solubility ginkgo episperm extract.
Second object of the present invention is achieved through the following technical solutions, a kind of water-solubility ginkgo episperm extract
The preparation method of preparation, water-solubility ginkgo episperm extract prepared by said method adds excipient or correctivess and anti-corrosion
Antitumor capsule preparations, tablet or oral liquid formulations are made in agent.
Compared with prior art, the invention has the advantages that：
Ethanol fully mixed by the way of continuous magnetic agitation by the present invention with concentrated solution, it is to avoid hand operated mixing mixing is uneven
Phenomenon, thus obtain that particle diameter is little, good water solubility, effective site content and all higher gingko episperm of antitumor potency
Extract.The method design science is feasible, and operation is simply controlled, suitable industrialized great production.Gained water-solubility ginkgo episperm
Extract can be made into capsule, tablet and oral liquid formulations.
1st, water miscible mensure
Gingko episperm crude extract is 1.5%（W/V）Under concentration, perusal is dissolved completely in water, and water-solubility ginkgo episperm
Extract is 3.0%（W/V）Under concentration, perusal is dissolved completely in water, points out the extraction fromginkgoseed coat that the method is obtained
Water solublity improves.
2nd, the mensure of peptide polysaccharide content
Polyoses content is surveyed using Phenol sulfuric acid procedure：Precision weighs anhydrous grape saccharide in right amount, is made into 1mg/ml with distilled water
Glucose standard, be further diluted to variable concentrations.Precision measure concentration be 30 μ g/ml, 60 μ g/ml, 90 μ g/ml,
120 μ g/ml, 150 μ g/ml, glucose standards solution 0.5ml of 180 μ g/ml are filled in test tube in 6 10ml tools, add 50mg/
The re-distilled phenol solution 1ml of ml, after mix homogeneously, adds 5ml concentrated sulphuric acid, mix homogeneously, boiling water bath 15min, psychrolusia 30min.
With ultraviolet spectrophotometer at 490nm mensuration absorbance, linear regression is carried out to glucose concentration and absorbance, set up
Regression equation.Take need testing solution 0.5ml, survey absorbance by aforesaid operations, substitute into regression equation, calculate polyoses content.
Protein content is surveyed using Coomassie Brilliant Blue：Precision weighs bovine serum albumin standard substance in right amount, uses distilled water
It is made into the protein standard solution of 100 μ g/ml, be further diluted to variable concentrations.It is 20 μ g/ml, 40 μ g/ that precision measures concentration
Ml, 60 μ g/ml, 80 μ g/ml, 100 μ g/ml protein standard solution 0.5ml, respectively at 5 10ml tool plug test tubes, add 2.5ml
Coomassie brilliant G-250 solution, after shaking up, place 5～10min, after it fully reacts, with ultraviolet spectrophotometer in
Mensuration absorbance at 595nm, carries out linear regression to bovine serum albumin solution concentration and absorbance, sets up regression equation.Take
Need testing solution 0.5ml, surveys absorbance by aforesaid operations, substitutes into regression equation, calculates protein content.
Qualitative analyses are carried out using infrared spectrometric analyzer.Result shows extraction fromginkgoseed coat amide bond, points out
For peptide polysaccharide.Peptide polysaccharide content %=(polyoses content+protein content) %.
Its polyoses content of gingko episperm crude extract prepared using the method is 36%, protein content is 28%, so
Peptide polysaccharide content is 36%+28%=64%.And water-solubility ginkgo its polyoses content of episperm extract be 45%, protein content be
31%, so peptide polysaccharide content is 45%+31%=76%, point out water-solubility ginkgo episperm extract anti-tumor effective component content
Increase.
3rd, particle size determination
Precision weighs gingko episperm crude extract and each 2.5mg of water-solubility ginkgo episperm extract, is dissolved in distilled water respectively, fixed
Hold to 50 ml, obtain the GBEP aqueous solution that concentration is 50 μ g/mL.This aqueous solution is taken to be placed in tool plug test tube, ultrasonic 20min
Afterwards, using Malvern laser particle analyzer, measure particle diameter.Result shows, the particle diameter of gingko episperm crude extract is 1.5-2.0 μm,
And adopt the water-solubility ginkgo episperm extract particle diameter that the method is obtained to be 150-200nm.
4th, zeta potential measures
Precision weighs gingko episperm crude extract and each 2.5mg of water-solubility ginkgo episperm extract, is dissolved in distilled water respectively, fixed
Hold to 50 ml, obtain the GBEP aqueous solution that concentration is 50 μ g/mL.This aqueous solution is taken to be placed in tool plug test tube, ultrasonic 20min
Afterwards, using Malvern laser particle analyzer, measure zeta potential.Result shows, gingko episperm crude extract zeta potential is (- 14)-(- 11)
MV, and adopt the water-solubility ginkgo episperm extract zeta potential that the method is obtained to be (- 27)-(- 25) mV, point out water-soluble silver
Fructus Pruni episperm solution of extract is more stable.
5th, drug efficacy study
Take the H22 cell being in exponential phase, after being washed with trypsinization, Hank ' s liquid, with containing 10% calf serum
RPMI1640 culture fluid adjustment cell is to 1 × 105/ml.It is separately added into above-mentioned cell suspension 100 μ in each hole of 96 well culture plates
L, in 5%CO2, 37 DEG C of saturated humidities incubator in after culture 24h, add with joining containing 10% calf serum RPMI1640 culture fluid
The each 100 μ l of sample of the variable concentrations of system, each concentration sets 6 multiple holes, cultivates 48h.Culture terminates front 4h and adds MTT（Dense eventually
Degree 5mg/ml）, discard culture fluid after continuing culture 4h, add acidifying isopropanol, vibrate 10min, surveyed at 570nm with microplate reader
The OD value in fixed each hole.By formula " suppression ratio=(1- medicine hole OD value/control wells OD value) × 100% ", calculate in vitro to H22 cell
The suppression ratio of propagation.The results are shown in Table 1.
The inhibitory action to H22 hepatoma carcinoma cell for table 1 extraction fromginkgoseed coat
Table 1 result shows, extraction fromginkgoseed coat has direct suppression under 10-320 μ g/ml dosage and makees to hepatoma carcinoma cell
With increasing with dosage, its inhibitory action also gradually strengthens.And, magnetic agitation gained water-solubility ginkgo episperm extract
Half-inhibition concentration IC50（μg/ml）It is substantially less than the extraction fromginkgoseed coat of hand operated mixing gained, illustrate potency more
High.
Specific embodiment
Embodiment 1
Take 50 grams of the dry gingko episperm being soaked by ethanol, add water 800 grams, be placed in boil carry in pot in 98 DEG C decoct, 1 is little every time
When, decoct 2 times altogether.Decoction liquor is merged, being evaporated to proportion in 70 DEG C is 1.21, in 0.05V/V ratio by concentrated solution
It is transferred in container, container is placed on the pedestal of magnetic stirring apparatuss, ethanol is slowly added dropwise in concentrated solution, make ethanol dense eventually
Degree reaches 80% in 1.5h（V/V）, start magnetic stirring apparatuss while adding ethanol, 1800rpm/min stirring at 20 DEG C,
By precipitate centrifuge dripping after standing 12 hours, then use high concentration ethanol（Volumetric concentration more than 98%）Washing freezes for 2 times dry afterwards
Dry, obtain water-solubility ginkgo episperm extract.Prepared water-solubility ginkgo episperm extract is 3.0%（W/V）Meat under concentration
Eye is observed and is dissolved completely in water, detects its polysaccharide and protein content respectively using Phenol sulfuric acid procedure and Coomassie Brilliant Blue, knot
Fruit is respectively 45% and 31%, and peptide polysaccharide total content is 76%, and particle diameter is 121.4nm, and zeta potential is -25.9mV.External using mtt assay
The impact to H22 cell proliferation for the detection, adds the water-solubility ginkgo episperm extract of variable concentrations, continues after inoculating cell 24h
Continuous culture 48h detection.Result shows, water-solubility ginkgo episperm extract final concentration is in 320 μ g/ml, 160 μ g/ml, 80 μ g/
Ml, 40 μ g/ml, 20 μ g/ml, can directly suppress H22 liver cancer cell growth under 10 μ g/ml, and increase with dosage, and its suppression is made
With gradually strengthening, suppression ratio（%）It is respectively 78,73,61,48,36 and 23, half-inhibition concentration IC50（μg/ml）For 84.5.Take
Water-solubility ginkgo episperm extract dry powder 100g, adds excipient 50g, mixes thoroughly, loads Capsuleses, system by every 150mg
Become water-solubility ginkgo episperm extract capsule preparation.
Embodiment 2
Take 100 grams of the dry gingko episperm being soaked by ethanol, add water 2000 grams, be placed in boil to carry in pot and decoct in 95 DEG C, every time 1
Hour, decoct 3 times altogether.Decoction liquor is merged, being evaporated to proportion in 70 DEG C is 1.26, will concentrate in the ratio of 0.15V/V
Liquid is transferred in container, and container is placed on the pedestal of magnetic stirring apparatuss, and ethanol is slowly added dropwise in concentrated solution, makes ethanol eventually
Concentration reaches 60% in 2.5h（V/V）, start magnetic stirring apparatuss while adding ethanol, stirred with 1400rpm/min at 30 DEG C
Mix, by precipitate centrifuge dripping after standing 24 hours, then wash lyophilization after 3 times with high concentration ethanol, obtain water-soluble silver
Fructus Pruni episperm extract.Prepared water-solubility ginkgo episperm extract is 3.0%（W/V）Under concentration, perusal is completely dissolved
Yu Shui, detects its polysaccharide and protein content respectively using Phenol sulfuric acid procedure and Coomassie Brilliant Blue, result be respectively 46% and
23%, peptide polysaccharide total content is 69%, and particle diameter is 143.6nm, and zeta potential is -25.0mV.Using mtt assay vitro detection to H22 cell
The impact of propagation, adds the water-solubility ginkgo episperm extract of variable concentrations after inoculating cell 24h, continue culture 48h detection.
Result shows, water-solubility ginkgo episperm extract final concentration 320 μ g/ml, 160 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ g/
Ml, can directly suppress H22 liver cancer cell growth under 10 μ g/ml, and increases with dosage, and its inhibitory action gradually strengthens, suppression
Rate（%）It is respectively 76,70,59,46,33 and 21, half-inhibition concentration IC50（μg/ml）For 99.6.Water-soluble gingko episperm
Extract dry powder 100g is dissolved in pure water, adds appropriate correctivess and preservative by drug quality requirement, shakes up, by 10ml
Specification is bottled, and makes water-solubility ginkgo episperm extract oral liquid formulation.
Embodiment 3
Take 80 grams of the dry gingko episperm being soaked by ethanol, add water 1500 grams, be placed in boil to carry in pot and decoct in 97 DEG C, every time 1
Hour, decoct 2 times altogether.Decoction liquor is merged, being evaporated to proportion in 70 DEG C is 1.30, will concentrate in the ratio of 0.11V/V
Liquid is transferred in container, and container is placed on the pedestal of magnetic stirring apparatuss, and ethanol is slowly added dropwise in concentrated solution, makes ethanol eventually
Concentration reaches 70% in 2.0h（V/V）, start magnetic stirring apparatuss while adding ethanol, stirred with 1600rpm/min at 25 DEG C
Mix, standing by precipitate centrifuge dripping, then was washed with high concentration ethanol after 2 times and is vacuum dried after 16 hours, obtained water-soluble silver
Fructus Pruni episperm extract.Prepared water-solubility ginkgo episperm extract is 3.0%（W/V）Under concentration, perusal is completely dissolved
Yu Shui, detects its polysaccharide and protein content respectively using Phenol sulfuric acid procedure and Coomassie Brilliant Blue, result be respectively 46% and
25%, peptide polysaccharide total content is 71%, and particle diameter is 133.4nm, and zeta potential is -25.2mV.Using mtt assay vitro detection to H22 cell
The impact of propagation, adds the water-solubility ginkgo episperm extract of variable concentrations after inoculating cell 24h, continue culture 48h detection.
Result shows, water-solubility ginkgo episperm extract final concentration is in 320 μ g/ml, 160 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ
G/ml, can directly suppress H22 liver cancer cell growth under 10 μ g/ml, and increases with dosage, and its inhibitory action gradually strengthens, suppression
Rate processed（%）It is respectively 77,71,60,48,34 and 22, half-inhibition concentration IC50（μg/ml）For 92.0.Plant outside water-soluble Semen Ginkgo
Peel extract dry powder 150g, adds 50 grams of excipient, mixes thoroughly, make tablet by every 100mg, and this is to plant outside water-solubility ginkgo
Peel extract tablet.
Claims (7)
1. a kind of preparation method of water-solubility ginkgo episperm extract, is characterized in that, described preparation method includes：Will be by ethanol
The gingko episperm soaking is placed in boil and puies forward routine in pot and add water to cook 2-3 time and filter respectively, will filtrate to merge decompression dense
Contracting, obtains concentrated solution；Concentrated solution is transferred in container, container is placed on the pedestal of magnetic stirring apparatuss, ethanol is slowly dripped
Add in concentrated solution, make ethanol final concentration reach 60-80%（V/V）, start magnetic stirrer while adding ethanol,
After standing, precipitate is washed lyophilization or vacuum drying after for several times with high concentration ethanol again, obtain water-solubility ginkgo episperm
Extract.
2. the preparation method of water-solubility ginkgo episperm extract according to claim 1, is characterized in that, described concentrated solution
Proportion be 1.21-1.30.
3. the preparation method of water-solubility ginkgo episperm extract according to claim 1, is characterized in that, described concentrated solution
With described volume of a container than for 0.05-0.15V/V.
4. the preparation method of water-solubility ginkgo episperm extract according to claim 1, is characterized in that, described ethanol delays
Slowly the time for adding being added dropwise to concentrated solution is 1.5h-2.5h.
5. the preparation method of water-solubility ginkgo episperm extract according to claim 1, is characterized in that, described magnetic force stirs
The whipping temp mixing device is 20-30 DEG C.
6. the preparation method of water-solubility ginkgo episperm extract according to claim 1, is characterized in that, described magnetic force stirs
Mixing device rotating speed is 1400-1800rpm/min.
7. a kind of preparation method of water-solubility ginkgo episperm extract formulation, is characterized in that, by the water of claim 1-6 preparation
Dissolubility extraction fromginkgoseed coat adds excipient or correctivess and preservative to make antitumor capsule preparations, tablet or antitumor
Oral liquid formulations.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107362783A (en) * | 2017-08-30 | 2017-11-21 | 齐鲁工业大学 | A kind of method that gingko episperm sorbing material is prepared using ball milling means |
| CN110558337A (en) * | 2019-07-19 | 2019-12-13 | 湖南科技学院 | biocontrol preparation for preventing and treating rice blast and preparation method thereof |
| CN110558336A (en) * | 2019-07-19 | 2019-12-13 | 湖南科技学院 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
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| CN1299834A (en) * | 2000-12-07 | 2001-06-20 | 扬州大学 | Ginkgo Testa Polysaccharide and the preparation of pharmaceutical thereof |
| CN101912426A (en) * | 2010-08-06 | 2010-12-15 | 扬州大学 | Method for preparing ginkgo exocarp anti-tumor effective part and preparation thereof |
| CN101912425A (en) * | 2010-08-06 | 2010-12-15 | 扬州大学 | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof |
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2016
- 2016-10-21 CN CN201610916393.XA patent/CN106389484A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1299834A (en) * | 2000-12-07 | 2001-06-20 | 扬州大学 | Ginkgo Testa Polysaccharide and the preparation of pharmaceutical thereof |
| CN101912426A (en) * | 2010-08-06 | 2010-12-15 | 扬州大学 | Method for preparing ginkgo exocarp anti-tumor effective part and preparation thereof |
| CN101912425A (en) * | 2010-08-06 | 2010-12-15 | 扬州大学 | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107362783A (en) * | 2017-08-30 | 2017-11-21 | 齐鲁工业大学 | A kind of method that gingko episperm sorbing material is prepared using ball milling means |
| CN107362783B (en) * | 2017-08-30 | 2020-07-31 | 齐鲁工业大学 | Method for preparing ginkgo biloba exocarp adsorbing material by adopting ball milling means |
| CN110558337A (en) * | 2019-07-19 | 2019-12-13 | 湖南科技学院 | biocontrol preparation for preventing and treating rice blast and preparation method thereof |
| CN110558336A (en) * | 2019-07-19 | 2019-12-13 | 湖南科技学院 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
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