CN101912426A - Method for preparing ginkgo exocarp anti-tumor effective part and preparation thereof - Google Patents
Method for preparing ginkgo exocarp anti-tumor effective part and preparation thereof Download PDFInfo
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Abstract
The invention relates to a method for preparing ginkgo exocarp anti-tumor effective part and a preparation thereof, and belongs to the field of Chinese pharmaceutical technology. The method comprises the following steps of: soaking dry ginkgo exocarp by using ethanol, filtering out the ethanol, performing air drying, and decocting for 2 to 3 times with hot water; respectively filtering, merging the filtered fluid, adding combined salt solution into filtered fluid concentrated under reduced pressure, filtering or centrifuging to remove water-insoluble substances, and dialyzing by using distilled water; concentrating dialyzed solution under reduced pressure, adding the ethanol, standing and precipitating; washing and drying the precipitate by using the ethanol to prepare the protein and polysaccharide component-containing ginkgo exocarp anti-tumor effective part; and adding an excipient or a flavoring agent and a preservative into the ginkgo exocarp anti-tumor effective part to prepare an anti-tumor capsule preparation or an anti-tumor oral liquid preparation. The ginkgo exocarp anti-tumor effective part has the advantages of sum of the content of the polysaccharide and protein of over 60 percent, high water solubility, good anti-tumor effect, capacities of being prepared into a solid formulation and a liquid formulation, simple and practicable preparation technology and suitability for industrial production.
Description
Technical field
The present invention relates to the preparation method of a kind of ginkgo exocarp anti-tumor effective part and preparation thereof, belong to technical field of traditional Chinese medicine pharmacy.
Background technology
Semen Ginkgo has another name called Semen Ginkgo, and the outermost layer meat skin of Semen Ginkgo is a gingko episperm, and is annual 9, and Semen Ginkgo in October sophisticated season, the episperm that the producing region is stinking just is seen everywhere, and has become the main source of contaminated environment.Semen Ginkgo once was the precious Chinese medicine and the nutriment of 1 jin of dozens of yuan, but in the last few years for various reasons, and the Semen Ginkgo price of only 2-5 per jin unit has seriously influenced orchard worker's enthusiasm.China has the gingko resource in the whole world 3/4, in order to fully utilize gingko resource well, our seminar has carried out the research of more than ten years, mainly is to extract the polysaccharide compound with antitumor action and purposes from episperm, has obtained a series of achievements in research.Because poorly water-soluble is the common problem of present many crude polysaccharides preparations, its water solublity of gingko exocarp polysaccharide of our employings prepared in the past neither be too desirable, thereby can only make solid dosage forms, and clinical practice is restricted.Antitumor drug not only needs solid dosage forms, more needs liquid dosage form, how to improve the water solublity of prepared gingko exocarp polysaccharide, is that those skilled in the art want the technical barrier that solves always.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, the preparation method of a kind of ginkgo exocarp anti-tumor effective part and preparation thereof is provided.
The objective of the invention is to be achieved through the following technical solutions, the preparation method of ginkgo exocarp anti-tumor effective part is characterized in that may further comprise the steps:
(1) with dried gingko episperm with elimination ethanol behind the soak with ethanol 24-96h of 90-98%, the air-dry back of episperm is decocted 2-3 time in 90-99 ℃ of hot water, decoct 1-2h at every turn, filter back merging filtrate respectively;
(2) filtrate is evaporated to the liquid material than for 600-800% (V/W) back adds 25% (W/V) combined salt solution, the pH value that makes concentrated filtrate is 7.0-7.4, after fully stirring and leaving standstill 4-6h, filters or removed in centrifugal 3-5 minute water-insoluble once more;
(3) will remove the concentrated filtrate distill water dialysis 12-24h of water-insoluble, dialyzed solution is evaporated to the liquid material than after being 300-400% (V/W), add ethanol in spissated dialyzed solution, making the ethanol final concentration is 60-80% (V/V), staticly settles;
(4) with washing with alcohol 2-4 final vacuum drying or the lyophilization of precipitate, obtain ginkgo exocarp anti-tumor effective part with 90-98%.
The solid-liquid ratio that hot water described in the step (1) decocts is 1: 10-1: 20 (V/W);
Combined salt solution described in the step (2) is made up of sodium chloride, sodium carbonate and magnesium sulfate, and the ratio of three's weight is 8: 1: 1;
It is the dialyzer of 7000-10000 that cutoff value is adopted in dialysis described in the step (3), and the volume of used distilled water is 50-100 a times of concentrated filtrate volume;
The perusal under 1.5% (W/V) concentration of ginkgo exocarp anti-tumor effective part described in the step (4) is dissolved in water fully, and its polyoses content and protein content sum are greater than 60%.
The preparation method of ginkgo exocarp anti-tumor effective part preparation is characterized in that the ginkgo exocarp anti-tumor effective part of method for preparing is added excipient or correctives and antiseptic makes antitumor capsule preparations or antineoplastic oral liquid preparation.
Science of the present invention, rationally, simply, before decocting, adopt hot water soak with ethanol to help defat and decolouring earlier gingko episperm, help removing medicine such as ginkgoic acid in the gingko episperm material of limiting the quantity of, help the hot water decoction and make oozing out of ginkgo exocarp anti-tumor effective part, adding is by sodium chloride, the saline solution that contains multiple acid group of sodium carbonate and magnesium sulfate reasonable combination helps removing water-insoluble, it is that the dialyzer of 7000-10000 can be removed various small-molecule substances through the distill water dialysis of 50-100 times of volume that the filtrate of removing water-insoluble is adopted cutoff value, dialyzed solution is evaporated to the liquid material than being 300-400% (V/W) back adding ethanol, water solublity and the polysaccharide and the protein content of the ginkgo exocarp anti-tumor effective part that further raising finally makes.Gingko episperm effective site polyoses content that this method makes and protein content sum are greater than 60%, and good water solubility has anti-tumor activity, both can make solid dosage forms, also can make liquid dosage form, preparation technology is simple and feasible, is fit to industrialized great production.
1, water miscible mensuration
Ginkgo exocarp anti-tumor effective part perusal under 1.5% (W/V) concentration is dissolved in water fully.
2, the mensuration of peptide polysaccharide content (polyoses content+protein content)
Adopt the phenol sulfuric acid process to survey polyoses content: it is an amount of that precision takes by weighing the anhydrous grape saccharide, is made into the glucose titer of 1mg/ml with distilled water, and further dilution is variable concentrations again.It is that the glucose standard solution 0.5ml of 30 μ g/ml, 60 μ g/ml, 90 μ g/ml, 120 μ g/ml, 150 μ g/ml, 180 μ g/ml is in 6 10ml tool plug test tubes that precision is measured concentration, the re-distilled phenol solution 1ml that adds 50mg/ml, behind the mix homogeneously, add the 5ml concentrated sulphuric acid, mix homogeneously, boiling water bath 15min, psychrolusia 30min.Measure absorbance in the 490nm place with ultraviolet spectrophotometer, glucose solution concentration and absorbance are carried out linear regression, set up regression equation.Get ginkgo exocarp anti-tumor effective part need testing solution 0.5ml, press aforesaid operations and survey absorbance, the substitution regression equation calculates polyoses content.
Adopt the Coomassie brilliant blue method to survey protein content: it is an amount of that precision takes by weighing the bovine serum albumin standard substance, is made into the protein standard solution of 100 μ g/ml with distilled water, and further dilution is variable concentrations again.It is that 20 μ g/ml, 40 μ g/ml, 60 μ g/ml, 80 μ g/ml, 100 μ g/ml protein standard solution 0.5ml are respectively at 5 10ml tool plug test tubes that precision is measured concentration, add 2.5ml Coomassie brilliant blue G-250 solution again, after shaking up, place 5～10min, after treating that it fully reacts, measure absorbance in the 595nm place with ultraviolet spectrophotometer, bovine serum albumin solution concentration and absorbance are carried out linear regression, set up regression equation.Get ginkgo exocarp anti-tumor effective part need testing solution 0.5ml, press aforesaid operations and survey absorbance, the substitution regression equation calculates protein content.
Adopt infrared spectrometric analyzer to carry out qualitative analysis.The result shows ginkgo exocarp anti-tumor effective part amide containing key, prompts for peptide polysaccharide.Peptide polysaccharide content %=(polyoses content+protein content) %.
Its polyoses content of ginkgo exocarp anti-tumor effective part that adopts the preparation of this method is 36%, protein content is 28%, so peptide polysaccharide content is 36%+28%=64%.
3, drug efficacy study
Get the SMMC-7721 cell that is in exponential phase, after trypsinization, the washing of Hank ' s liquid, adjust cell to 1 * 10 with containing 10% calf serum RPMI1640 culture fluid
5/ ml.In each hole of 96 well culture plates, add above-mentioned cell suspension 100 μ l respectively, at 5%CO
2, 37 ℃ of saturated humidities incubator in cultivate 24h after, add each 100 μ l of ginkgo exocarp anti-tumor effective part with the variable concentrations that contains the preparation of 10% calf serum RPMI1640 culture fluid, each concentration is established 6 multiple holes, cultivates 48h.Cultivate and finish preceding 4h adding MTT (final concentration 5mg/ml), discard culture fluid after continuing to cultivate 4h, add the acidify isopropyl alcohol, the 10min that vibrates uses microplate reader to measure the OD value in each hole in the 570nm place.By formula " suppression ratio=(1-medicine hole OD value/control wells OD value) * 100% " calculates external suppression ratio to the SMMC-7721 cell proliferation.The results are shown in Table 1.
Table 1 ginkgo exocarp anti-tumor effective part is to the inhibitory action of SMMC-7721 human liver cancer cell
Table 1 result shows that ginkgo exocarp anti-tumor effective part has direct repression to human liver cancer cell under 10-160 μ g/ml dosage, and along with dosage increases, its inhibitory action strengthens gradually.
The specific embodiment:
Embodiment 1
Get dried gingko episperm 200kg, elimination ethanol behind the soak with ethanol 24h of adding 95% decocts the air-dry back of gingko episperm in 95 ℃ of hot water, and each 1h decocts 2 times altogether, and solid-liquid ratio is 1: 10 (V/W).Decoction liquor is filtered merging respectively, with filtrate be evaporated to liquid material ratio be 600% (V/W) back add concentration be 25% (W/V) be the saline solution that combines at 8: 1: 1 by weight by sodium chloride, sodium carbonate and magnesium sulfate, the pH value that makes concentrated filtrate is 7.2, leave standstill 2h after fully stirring, filter or press 800 rev/mins of centrifugal 5 minutes removal water-insolubles once more; It is 7000 the dialyzer distill water dialysis 12h through 50 times of volumes that the concentrated filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after liquid material ratio is 300% (V/W) once more, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 60% (V/V), staticly settles; Precipitate with 2 final vacuum dryings of 90% washing with alcohol or lyophilization, is made ginkgo exocarp anti-tumor effective part dry powder, and the perusal under 1.5% (W/V) concentration of this dry powder is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect its polysaccharide and protein content respectively, the result is respectively 45% and 23%, and the peptide polysaccharide total content is 68%.Adopt of the influence of mtt assay vitro detection, add ginkgo exocarp anti-tumor effective part behind the inoculating cell 24h, continue to cultivate 48h and detect the SMMC-7721 cell proliferation.The result shows, the ginkgo exocarp anti-tumor effective part final concentration is at 160 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ g/ml, can directly suppress the SMMC-7721 hepatoma cell growth under the 10 μ g/ml, and along with dosage increases, its inhibitory action strengthens gradually, suppression ratio (%) is respectively 60,52,39,26 and 8.Get ginkgo exocarp anti-tumor effective part dry powder 100g, add excipient 100g, mix thoroughly,, make the ginkgo exocarp anti-tumor effective part capsule preparations by every 200mg Capsules of packing into.
Embodiment 2
Get dried gingko episperm 400kg, elimination ethanol behind the soak with ethanol 72h of adding 90% decocts the air-dry back of gingko episperm in 90 ℃ of hot water, and each 1.5h decocts 3 times altogether, and solid-liquid ratio is 1: 15 (V/W).Decoction liquor is filtered merging respectively, with filtrate be evaporated to liquid material ratio be 700% (V/W) back add concentration be 25% (W/V) be the saline solution that combines at 8: 1: 1 by weight by sodium chloride, sodium carbonate and magnesium sulfate, the pH value that makes concentrated filtrate is 7.4, leave standstill 3h after fully stirring, filter or press 1000 rev/mins of centrifugal 4 minutes removal water-insolubles once more; It is 8000 the dialyzer distill water dialysis 18h through 80 times of volumes that the concentrated filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after liquid material ratio is 350% (V/W) once more, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 70% (V/V), staticly settles; Precipitate with 3 final vacuum dryings of 98% washing with alcohol or lyophilization, is made ginkgo exocarp anti-tumor effective part dry powder, and the perusal under 1.5% (W/V) concentration of this dry powder is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect its polysaccharide and protein content respectively, the result is respectively 44% and 22%, and the peptide polysaccharide total content is 66%.Adopt of the influence of mtt assay vitro detection, add ginkgo exocarp anti-tumor effective part behind the inoculating cell 24h, continue to cultivate 48h and detect the SMMC-7721 cell proliferation.The result shows, the ginkgo exocarp anti-tumor effective part final concentration is at 160 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ g/ml, can directly suppress the SMMC-7721 hepatoma cell growth under the 10 μ g/ml, and along with dosage increases, its inhibitory action strengthens gradually, suppression ratio (%) is respectively 58,49,37,21 and 10.Get ginkgo exocarp anti-tumor effective part dry powder 200g and be dissolved in the purified water, add an amount of correctives and antiseptic, shake up, press the bottling of 10ml specification, make the ginkgo exocarp anti-tumor effective part oral liquid formulations by the drug quality requirement.
Embodiment 3
Get dried gingko episperm 200kg, elimination ethanol behind the soak with ethanol 96h of adding 98% decocts the air-dry back of gingko episperm in 99 ℃ of hot water, and each 2h decocts 2 times altogether, and solid-liquid ratio is 1: 20 (V/W).Decoction liquor is filtered merging respectively, with filtrate be evaporated to liquid material ratio be 800% (V/W) back add concentration be 25% (W/V) be the saline solution that combines at 8: 1: 1 by weight by sodium chloride, sodium carbonate and magnesium sulfate, the pH value that makes concentrated filtrate is 7.0, leave standstill 4h after fully stirring, filter or press 1200 rev/mins of centrifugal 3 minutes removal water-insolubles once more; It is 10000 the dialyzer distill water dialysis 24h through 100 times of volumes that the concentrated filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after liquid material ratio is 400% (V/W) once more, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 80% (V/V), staticly settles; Precipitate with 4 final vacuum dryings of 95% washing with alcohol or lyophilization, is made ginkgo exocarp anti-tumor effective part dry powder, and the perusal under 1.5% (W/V) concentration of this dry powder is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect its polysaccharide and protein content respectively, the result is respectively 41% and 21%, and the peptide polysaccharide total content is 62%.Adopt of the influence of mtt assay vitro detection, add ginkgo exocarp anti-tumor effective part behind the inoculating cell 24h, continue to cultivate 48h and detect the SMMC-7721 cell proliferation.The result shows, the ginkgo exocarp anti-tumor effective part final concentration is at 160 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ g/ml, can directly suppress the SMMC-7721 hepatoma cell growth under the 10 μ g/ml, and along with dosage increases, its inhibitory action strengthens gradually, suppression ratio (%) is respectively 64,54,40,23 and 12.Get ginkgo exocarp anti-tumor effective part dry powder 100g, add excipient 100g, mix thoroughly,, make the ginkgo exocarp anti-tumor effective part capsule preparations by every 200mg Capsules of packing into.
Claims (6)
1. the preparation method of a ginkgo exocarp anti-tumor effective part is characterized in that may further comprise the steps:
(1) with dried gingko episperm with elimination ethanol behind the soak with ethanol 24-96h of 90-98%, the air-dry back of gingko episperm is decocted 2-3 time in 90-99 ℃ of hot water, decoct 1-2h at every turn, filter back merging filtrate respectively;
(2) filtrate is evaporated to the liquid material than being 600-800% (V/W) back adding 25% (W/V) combined salt solution, the pH value that makes concentrated filtrate is 7.0-7.4, leaves standstill 2-4h after fully stirring, and filters or removed in centrifugal 3-5 minute water-insoluble once more;
(3) will remove the concentrated filtrate distill water dialysis 12-24h of water-insoluble, dialyzed solution is evaporated to the liquid material than after being 300-400% (V/W), add ethanol in spissated dialyzed solution, making the ethanol final concentration is 60-80% (V/V), staticly settles;
(4) with washing with alcohol 2-4 final vacuum drying or the lyophilization of precipitate, obtain ginkgo exocarp anti-tumor effective part with 90-98%.
2. the preparation method of ginkgo exocarp anti-tumor effective part according to claim 1 is characterized in that the solid-liquid ratio that the hot water described in the step (1) decocts is 1: 10-1: 20 (V/W).
3. the preparation method of ginkgo exocarp anti-tumor effective part according to claim 1, it is characterized in that the combined salt solution described in the step (2) is made up of sodium chloride, sodium carbonate and magnesium sulfate, the ratio of three's weight is 8: 1: 1, and centrifugal speed is 800-1200 rev/min.
4. the preparation method of ginkgo exocarp anti-tumor effective part according to claim 1, it is characterized in that the concentrated filtrate described in the step (3) is 7000-10000 with the dialyzer cutoff value of distill water dialysis, the volume of distilled water is 50-100 a times of concentrated filtrate volume.
5. the preparation method of ginkgo exocarp anti-tumor effective part according to claim 1, it is characterized in that the perusal under 1.5% (W/V) concentration of the ginkgo exocarp anti-tumor effective part described in the step (4) is dissolved in water fully, its polyoses content and protein content sum are greater than 60%.
6. the preparation method of a ginkgo exocarp anti-tumor effective part preparation is characterized in that ginkgo exocarp anti-tumor effective part with method for preparing adds excipient or correctives and antiseptic and makes antitumor capsule preparations or antineoplastic oral liquid preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106265779A (en) * | 2016-10-21 | 2017-01-04 | 扬州大学 | The extraction fromginkgoseed coat with anti-tumor activity of a kind of decolouring and the preparation method of preparation thereof |
| CN106389484A (en) * | 2016-10-21 | 2017-02-15 | 扬州大学 | Methods for preparing water-soluble ginkgo exopleura extract and preparation thereof |
| CN106632711A (en) * | 2016-10-21 | 2017-05-10 | 扬州大学 | Preparation method for submicron-scale ginkgo biloba exocarp polysaccharides and preparation thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100063C (en) * | 2000-12-07 | 2003-01-29 | 扬州大学 | Ginkgo Testa Polysaccharide and the preparation of pharmaceutical thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100063C (en) * | 2000-12-07 | 2003-01-29 | 扬州大学 | Ginkgo Testa Polysaccharide and the preparation of pharmaceutical thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《山地农业生物学报》 20041231 王椰，何照范 银杏外种皮多糖浸提的影响因素分析 第140页 1-6 第23卷, 第2期 2 * |
| 《食品科学》 20061231 吴向阳等 银杏外种皮粗多糖提取工艺研究 第373页 1-6 第27卷, 第10期 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106265779A (en) * | 2016-10-21 | 2017-01-04 | 扬州大学 | The extraction fromginkgoseed coat with anti-tumor activity of a kind of decolouring and the preparation method of preparation thereof |
| CN106389484A (en) * | 2016-10-21 | 2017-02-15 | 扬州大学 | Methods for preparing water-soluble ginkgo exopleura extract and preparation thereof |
| CN106632711A (en) * | 2016-10-21 | 2017-05-10 | 扬州大学 | Preparation method for submicron-scale ginkgo biloba exocarp polysaccharides and preparation thereof |
| CN106632711B (en) * | 2016-10-21 | 2019-03-05 | 扬州大学 | A kind of preparation method of submicron order gingko exocarp polysaccharide and its preparation |
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| CN101912426B (en) | 2012-07-04 |
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