CN101912425A - Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof - Google Patents
Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof Download PDFInfo
- Publication number
- CN101912425A CN101912425A CN 201010251050 CN201010251050A CN101912425A CN 101912425 A CN101912425 A CN 101912425A CN 201010251050 CN201010251050 CN 201010251050 CN 201010251050 A CN201010251050 A CN 201010251050A CN 101912425 A CN101912425 A CN 101912425A
- Authority
- CN
- China
- Prior art keywords
- extract
- gingko episperm
- water
- antitumor
- filtrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a ginkgo episperm extract with antitumor and immunostimulating activity and a preparation method of an effective part thereof and belongs to the technical field of traditional Chinese pharmaceutics. Ginkgo episperm is decocted for 2-3 times in hot water, decocted fluid is filtered respectively, a combined salt solution is added to filtrate after vacuum concentration, and after water insoluble matters are filtered or removed by centrifugation, dialysis is carried out by distilled water; after dialysis internal fluid is concentrated, precipitation is carried out by ethanol, and the precipitate is dried, thus obtaining the ginkgo episperm extract; the ginkgo episperm extract is dissolved in water and is then pumped and filtered, continued filtrate flows through a macroporous resin column, is eluted by distilled water and is then eluted by the ethanol when the sugar test result of the eluent is negative, and then ethanol eluent is collected, vacuum-concentrated and dried, thus obtaining the effective part of the ginkgo episperm extract. The content sums of polysaccharide and protein in the extract and the effective part are greater than 60 percent and 90 percent respectively, the water solubility is good, the antitumor and immunostimulating activity is high, solid formulation and fluid formulation can be prepared, and the preparation process is simple, convenient and feasible and is suitable for industrial mass production.
Description
Technical field
The present invention relates to have the preparation method of antitumor and active gingko episperm extract of immunological enhancement and extract effective site, belong to technical field of traditional Chinese medicine pharmacy.
Background technology
Semen Ginkgo is " living fossil " plant of world's preciousness, and China is the township of Semen Ginkgo.The fruit of Semen Ginkgo has another name called Semen Ginkgo, and is not only nutritious, and its existing thousands of years history of being used as medicine, and is integration of edible and medicinal herbs treasure world-famous.Contain chemical compounds such as flavone and terpene lactones in the Folium Ginkgo, the Schwabe company of Germany takes the lead in having set up Folium Ginkgo extract (EGb) processing factory.Through nearly 40 years lasting research both at home and abroad, gingko leaf preparation has been widely used in the treatment cardio-cerebrovascular diseases at present, and this lays a good foundation for the comprehensive utilization of gingko resource.Gingko episperm is the outermost meat skin of Semen Ginkgo, is commonly called as the Semen Ginkgo clothing, accounts for 3/4 of its kind reality, and in the season of every Semen Ginkgo results, just there is a large amount of episperm in the producing region and is taken as refuse and abandon, contaminated environment not only, and cause the significant wastage of living resources.How stinking episperm is turned waste into wealth, comprehensive development and utilization gingko resource better, our seminar has carried out the research of more than ten years around this problem, once from episperm, extracted polysaccharide compound and carried out series of basic and clinical principium on probation, obtained a series of achievements in research.Though but adopt the polysaccharide compound of prepared in the past also to have certain biological activity, but because its water solublity is not ideal, can only make solid dosage forms, limited the sick clinical practice of planting of certain cancers, do not separate the go forward side by side stepping line correlation research of its effective site again, thus the process that has influenced new drug development and declared.
Summary of the invention
The present invention is exactly the deficiency that exists at above-mentioned prior art, first purpose provides a kind of have antitumor and the active gingko episperm preparation method of extract of immunological enhancement, and second purpose is to provide a kind of preparation method with antitumor and the active gingko episperm extract of immunological enhancement effective site.
First purpose of the present invention is achieved through the following technical solutions, and has the active gingko episperm preparation method of extract of antitumor and immunological enhancement, it is characterized in that may further comprise the steps:
(1) gingko episperm is adopted 90-99 ℃ of hot water decoct, each 1-2h decocts 2-3 time altogether, and solid-liquid ratio is 1: 6-12 (V/W), filter decoction liquor respectively and merge filtrate;
(2) with behind the filtrate concentrating under reduced pressure, what add concentration and be 25% (W/V) is the saline solution that combines at 8: 1: 1 by sodium chloride, sodium carbonate and magnesium sulfate by weight, the pH value that makes concentrated filtrate is 7.0-7.4, leave standstill 2-4h after fully stirring, filter or press 800-1200 rev/min of centrifugal 3-5 minute removal water-insoluble once more;
(3) to adopt cutoff value be the distill water dialysis 12-24h of the dialyzer of 7000-10000 with 50-100 times of volume to the concentrated filtrate that will remove water-insoluble, behind the dialyzed solution concentrating under reduced pressure, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 60-85% (V/V), staticly settles;
(4) with washing with alcohol 2-4 final vacuum drying or the lyophilization of precipitate, obtain the gingko episperm extract with 90-98%.
It is 1.02-1.08 that filtrate in the described step (2) is evaporated to density;
It is 1.09-1.15 that dialyzed solution in the described step (3) is evaporated to density;
The perusal under 1% (W/V) concentration of gingko episperm extract in the described step (4) is dissolved in water fully, and its polyoses content and protein content sum are greater than 60%.
Second purpose of the present invention is achieved through the following technical solutions, and has the preparation method of the active gingko episperm extract of antitumor and immunological enhancement effective site, it is characterized in that may further comprise the steps:
(1) gingko episperm is adopted 90-99 ℃ of hot water decoct, each 1-2h, decoct 2-3 time altogether, solid-liquid ratio is 1: 6-12 (V/W), decoction liquor is filtered respectively and merge, filtrate is evaporated to after density is 1.02-1.08, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.0-7.4, leave standstill 2-4h after fully stirring, filter or press 800-1200 rev/min of centrifugal 3-5 minute removal water-insoluble once more, it is the distill water dialysis 12-24h of the dialyzer of 7000-10000 with 50-100 times of volume that the concentrated filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.09-1.15, add ethanol in spissated dialyzed solution, making the ethanol final concentration is 60-85% (V/V), staticly settles, precipitate with 2-4 final vacuum drying of 90-98% washing with alcohol or lyophilization, is got the gingko episperm extract;
(2) the gingko episperm extract is water-soluble, the concentration that makes aqueous solution is 1% (W/V);
(3) with 1% (W/V) gingko episperm solution of extract sucking filtration, subsequent filtrate is crossed macroporous resin column;
(4) use the distilled water eluting earlier, when treating that the test of eluent sugar is negative, use the ethanol elution of the 65-95% of 10BV, collect ethanol elution, concentrating under reduced pressure, lyophilization obtains gingko episperm extract effective site.
The height and the diameter ratio of big pore adsorption resin chromatographic column are 20: 1 in the described step (3);
In the described step (4) during used ethanol elution flow velocity be 1-2BV/h;
The perusal under 2% (W/V) concentration of resulting gingko episperm extract effective site is dissolved in water fully in the described step (4), and its polyoses content and protein content sum are greater than 90%.
Science of the present invention, reasonable, simple, adding the combined salt solution that contains multiple acid group before ethanol precipitation helps removing the water-insoluble in the gingko episperm extract and carries out the effective site separation, the gingko episperm solution of extract is crossed the innoxious solvent eluting that the low pole macroporous adsorbent resin is also used opposed polarity, make separate effective site content height, safety is good.Polyoses content and protein content sum are greater than 60% in the gingko episperm extract that this method makes, meet national correlated quality standard-required, polyoses content and protein content sum are greater than 90% in the gingko episperm extract effective site that makes, make antitumor and promote the immunization potency to increase, also help it is further carried out chemical analysis and Anticancer Effect and Mechanism research.This method makes has antitumor and active gingko episperm extract of immunological enhancement and effective site good water solubility thereof, both can make solid dosage forms, also can make liquid dosage form, and preparation technology is simple and feasible, is fit to industrialized great production.
1, peptide polysaccharide (polyoses content+protein content) Determination on 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 gingko episperm extract (GBE) and effective site (GBEE) need testing solution 0.5ml thereof, 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 GBE and GBEE 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 all amide containing keys of GBE and GBEE, and prompting is all peptide polysaccharide.
Peptide polysaccharide content %=(polyoses content+protein content) %
GBE and GBEE peptide polysaccharide content results see Table 1.
Table 1GBE and GBEE peptide polysaccharide content
Table 1 result shows that the peptide polysaccharide content of GBE is greater than 60%, and GBEE peptide polysaccharide content is greater than 90%.
2, antitumor cytolytic activity
Get the SGC-7901 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 GBE and each 100 μ l of GBEE 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 SGC-7901 cell proliferation.The results are shown in Table 2.
Table 2GBE and GBEE are to the effect of people's gastric cancer SGC-7901 cell inhibiting
Table 2 result shows that GBE and the GBEE inhibitory action to stomach cancer cell line under 12.5-200 μ g/ml dosage has dose-effect relationship, and GBEE reaches the suppression ratio required dosage similar to GBE and is about the latter's 50%, and the antitumor action potency increases.
3, immunological enhancement determination of activity
Get 6 of ICR mices, 20 ± 2g, male and female half and half, taking off cervical vertebra puts to death, aseptic condition takes out spleen down behind body surface 75% alcohol disinfecting, prepare splenocyte suspension according to a conventional method, use the washing of Hank ' s liquid behind the lysed erythrocyte for several times, adjust cell to 1 * 10 with the RPMI1640 culture fluid that contains 10% calf serum
7/ ml.Get above-mentioned cell suspension 100 μ l and add in each hole of 96 well culture plates, add Con A (final concentration is 5 μ g/ml) again and with GBE and each 100 μ l of GBEE of the variable concentrations of the RPMI RPMI-1640 preparation that contains 10% calf serum.Culture plate is placed 5%CO
2, 37 ℃ of saturated humidities incubator in cultivate 48h.4h sucking-off 100 μ l supernatants from each hole discarded before cultivation finished, and added MTT (final concentration 5mg/ml), added the acidify isopropyl alcohol behind the continuation cultivation 4h, and vibration 10min is with the OD value of microplate reader in each hole of mensuration, 570nm place.The positive correlation that how much becomes of the height of OD value and splenocyte.The results are shown in Table 3.
Table 3GBE and GBEE are to the facilitation of mouse immune cell proliferation
Table 3 result shows, GBE and GBEE have remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis under 12.5-200 μ g/ml dosage, required dosage was about 50% of GBE when GBEE reached the accelerating effect similar to GBE, promoted the immunization potency to increase.
4, water solublity is measured
GBE and GBEE respectively under 1% (V/W) and 2% (V/W) concentration perusal be dissolved in water fully.
The specific embodiment:
Has the active gingko episperm preparation method of extract of antitumor and immunological enhancement
Embodiment 1
Fresh episperm 100kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 8 (V/W).To boil to carry and pour out decoction liquor after kettle temperature remains on 90-99 ℃ of 1.5h, repeat 1 time, decoct altogether 2 times.Decoction liquor is filtered the back respectively merge filtrate, 70 ℃ of filtrate are evaporated to after density is 1.02, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.0, leave standstill 2h after fully stirring, filter or press 800 rev/mins of centrifugal 5 minutes removal water-insolubles once more, it is 10000 the dialyzer distill water dialysis 24h with 50 times of volumes that the filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.09, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 60% (V/V), staticly settle, precipitate with 2 final vacuum dryings of 90% washing with alcohol, is got the gingko episperm extract, and the perusal under 1% (W/V) concentration of this extract is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect polysaccharide and protein content respectively, the result is respectively 37.6% and 24.8%, and peptide polysaccharide total content (polyoses content+protein content) is 62.4%.Adopt the influence of mtt assay In vitro culture 48h detection to the SGC-7901 cell proliferation.The result shows that the gingko episperm extract can directly suppress growth of tumour cell, and its inhibitory action has dose-effect relationship, is 200 μ g/ml at final concentration, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, its suppression ratio (%) is respectively 50 under the 12.5 μ g/ml, 38,21,11,5.Adopt mtt assay In vitro culture 48h to detect influence, adopt Con A (final concentration is 5 μ g/ml) to induce mouse spleen lymphocyte propagation, with the OD value of microplate reader in each hole of mensuration, 570nm place, the positive correlation that how much becomes of the height of OD value and splenocyte.The result shows that the gingko episperm extract has remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis, in giving dosage range, strengthens gradually along with dosage increases its facilitation.The cell matched group OD value that this embodiment does not add processing is 0.173 ± 0.018, gingko episperm extract final concentration is at 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the cell experiment group OD value that 12.5 μ g/ml handle down is respectively 0.303 ± 0.015,0.290 ± 0.012,0.231 ± 0.017,0.223 ± 0.009,0.201 ± 0.016.
Embodiment 2
Fresh episperm 200kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 6 (V/W).To boil to carry and pour out decoction liquor after kettle temperature remains on 90-99 ℃ of 1h, repeat 2 times, decoct altogether 3 times.Decoction liquor is filtered the back respectively merge filtrate, 70 ℃ of filtrate are evaporated to after density is 1.04, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.2, 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 12h with 80 times of volumes that the filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.15, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 70% (V/V), staticly settle, precipitate with 3 final vacuum dryings of 95% washing with alcohol, is got the gingko episperm extract, and the perusal under 1% (W/V) concentration of this extract is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect polysaccharide and protein content respectively, the result is respectively 40.1% and 21.1%, and peptide polysaccharide total content (polyoses content+protein content) is 61.2%.Adopt the influence of mtt assay In vitro culture 48h detection to the SGC-7901 cell proliferation.The result shows that the gingko episperm extract can directly suppress growth of tumour cell, and its inhibitory action has dose-effect relationship, is 200 μ g/ml at final concentration, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, its suppression ratio (%) is respectively 54 under the 12.5 μ g/ml, 40,22,14,5.Adopt mtt assay In vitro culture 48h to detect influence, adopt Con A (final concentration is 5 μ g/ml) to induce mouse spleen lymphocyte propagation, with the OD value of microplate reader in each hole of mensuration, 570nm place, the positive correlation that how much becomes of the height of OD value and splenocyte.The result shows that the gingko episperm extract has remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis, in giving dosage range, strengthens gradually along with dosage increases its facilitation.The cell matched group OD value that this embodiment does not add processing is 0.169 ± 0.008, gingko episperm extract final concentration is at 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the cell experiment group OD value that 12.5 μ g/ml handle down is respectively 0.289 ± 0.011,0.278 ± 0.014,0.259 ± 0.013,0.199 ± 0.010,0.186 ± 0.012.
Embodiment 3
Fresh episperm 500kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 12 (V/W).To boil to carry and pour out decoction liquor after kettle temperature remains on 90-99 ℃ of 2h, repeat 2 times, decoct altogether 3 times.Decoction liquor is filtered the back respectively merge filtrate, 70 ℃ of filtrate are evaporated to after density is 1.08, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.4, leave standstill 4h after fully stirring, filter or press 1200 rev/mins of centrifugal 3 minutes removal water-insolubles once more, it is 7000 the dialyzer distill water dialysis 18h with 100 times of volumes that the filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.12, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 85% (V/V), staticly settle, precipitate with 4 postlyophilizations of 98% washing with alcohol, is got the gingko episperm extract, and the perusal under 1% (W/V) concentration of this extract is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect polysaccharide and protein content respectively, the result is respectively 43.9% and 20.4%, and peptide polysaccharide total content (polyoses content+protein content) is 64.3%.Adopt the influence of mtt assay In vitro culture 48h detection to the SGC-7901 cell proliferation.The result shows that the gingko episperm extract can directly suppress growth of tumour cell, and its inhibitory action has dose-effect relationship, is 200 μ g/ml at final concentration, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, its suppression ratio (%) is respectively 52 under the 12.5 μ g/ml, 37,24,16,7.Adopt mtt assay In vitro culture 48h to detect influence, adopt Con A (final concentration is 5 μ g/ml) to induce mouse spleen lymphocyte propagation, with the OD value of microplate reader in each hole of mensuration, 570nm place, the positive correlation that how much becomes of the height of OD value and splenocyte.The result shows that the gingko episperm extract has remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis, in giving dosage range, strengthens gradually along with dosage increases its facilitation.The cell matched group OD value that this embodiment does not add processing is 0.192 ± 0.019, gingko episperm extract final concentration is at 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the cell experiment group OD value that 12.5 μ g/ml handle down is respectively 0.301 ± 0.019,0.290 ± 0.016,0.266 ± 0.011,0.248 ± 0.018,0.208 ± 0.014.
Preparation method with the active gingko episperm extract of antitumor and immunological enhancement effective site
Embodiment 4
Fresh episperm 500kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 12 (V/W).To boil to carry and pour out decoction liquor after kettle temperature remains on 90-99 ℃ of 2h, repeat 2 times, decoct altogether 3 times.Decoction liquor is filtered the back respectively merge filtrate, 70 ℃ of filtrate are evaporated to after density is 1.08, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.4, leave standstill 4h after fully stirring, filter or press 1200 rev/mins of centrifugal 3 minutes removal water-insolubles once more, it is 7000 the dialyzer distill water dialysis 18h with 100 times of volumes that the filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.12, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 85% (V/V), staticly settle, precipitate with 4 postlyophilizations of 98% washing with alcohol, is got the gingko episperm extract.This extract is multiple water-soluble, and the concentration that makes aqueous solution is 1%, sucking filtration, it is that the 20cm height is the macroporous adsorption resin chromatography post of 400cm that subsequent filtrate is crossed diameter, earlier use the distilled water eluting, treat to use instead when the test of eluent sugar is negative the ethanol elution of 10BV 65%, flow velocity is 2BV/h during ethanol elution.Collect ethanol elution, concentrating under reduced pressure, lyophilization gets gingko episperm extract effective site, and the perusal under 2% (W/V) concentration of this effective site is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect polysaccharide and protein content respectively, the result shows, gingko episperm extract polysaccharide and protein content are respectively 38.1% and 23.5%, peptide polysaccharide total content (polyoses content+protein content) is 61.6%, gingko episperm extract effective site polysaccharide and protein content are respectively 61.8% and 33.3%, and peptide polysaccharide total content (polyoses content+protein content) is 95.1%.Adopt the influence of mtt assay In vitro culture 48h detection to the SGC-7901 cell proliferation.The result shows, gingko episperm extract and effective site thereof all can directly suppress growth of tumour cell, in giving dosage range, its inhibitory action has dose-effect relationship, and required dosage was about the latter's 50% when gingko episperm extract effective site reached the depression effect similar to the gingko episperm extract, showed that the function of tumor inhibition potency of gingko episperm extract effective site is higher than the gingko episperm extract, was 200 μ g/ml at final concentration, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the suppression ratio (%) under the 12.5 μ g/ml, the gingko episperm extract is respectively 52,39,23,13,8, gingko episperm extract effective site is respectively 69,57,35,23,14.Adopt mtt assay In vitro culture 48h to detect influence, adopt Con A (final concentration is 5 μ g/ml) to induce mouse spleen lymphocyte propagation, with the OD value of microplate reader in each hole of mensuration, 570nm place, the positive correlation that how much becomes of the height of OD value and splenocyte.The result shows, gingko episperm extract and effective site thereof have remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis, in giving dosage range, along with increasing its facilitation, dosage strengthens gradually, and required dosage was about the latter's 50% when gingko episperm extract effective site reached the accelerating effect similar to the gingko episperm extract, showed that the immunologic enhancement potency of gingko episperm extract effective site is higher than the gingko episperm extract.The cell matched group OD value that this embodiment does not add processing is 0.178 ± 0.011, final concentration is at 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 the cell experiment group OD value that μ g/ml handles down, the gingko episperm extract is respectively 0.291 ± 0.016,0.274 ± 0.010,0.222 ± 0.016,0.203 ± 0.011,0.188 ± 0.015, gingko episperm extract effective site is respectively 0.311 ± 0.017,0.299 ± 0.008,0.276 ± 0.012,0.253 ± 0.012,0.200 ± 0.009.
Embodiment 5
Fresh episperm 200kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 6 (V/W).To boil to carry and pour out decoction liquor after kettle temperature remains on 90-99 ℃ of 1h, repeat 2 times, decoct altogether 3 times.Decoction liquor is filtered the back respectively merge filtrate, 70 ℃ of filtrate are evaporated to after density is 1.04, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.2, 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 12h with 80 times of volumes that the filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.15, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 70% (V/V), staticly settle, precipitate with 3 final vacuum dryings of 95% washing with alcohol, is got the gingko episperm extract.This extract is multiple water-soluble, and the concentration that makes aqueous solution is 1%, sucking filtration, it is that the 15cm height is the macroporous adsorption resin chromatography post of 300cm that subsequent filtrate is crossed diameter, earlier use the distilled water eluting, treat to use instead when the test of eluent sugar is negative the ethanol elution of 10BV 95%, flow velocity is 1BV/h during ethanol elution.Collect ethanol elution, concentrating under reduced pressure, lyophilization gets gingko episperm extract effective site, and the perusal under 2% (W/V) concentration of this effective site is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect polysaccharide and protein content respectively, the result shows, gingko episperm extract polysaccharide and protein content are respectively 38.3% and 27.0%, peptide polysaccharide total content (polyoses content+protein content) is 65.3%, gingko episperm extract effective site polysaccharide and protein content are respectively 62.0% and 34.2%, and peptide polysaccharide total content (polyoses content+protein content) is 96.2%.Adopt the influence of mtt assay In vitro culture 48h detection to the SGC-7901 cell proliferation.The result shows, gingko episperm extract and effective site thereof all can directly suppress growth of tumour cell, in giving dosage range, its inhibitory action has dose-effect relationship, and required dosage was about the latter's 50% when gingko episperm extract effective site reached the depression effect similar to the gingko episperm extract, showed that the function of tumor inhibition potency of gingko episperm extract effective site is higher than the gingko episperm extract, was 200 μ g/ml at final concentration, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the suppression ratio (%) under the 12.5 μ g/ml, the gingko episperm extract is respectively 53,36,24,12,5, gingko episperm extract effective site is respectively 71,59,41,21,17.Adopt mtt assay In vitro culture 48h to detect influence, adopt Con A (final concentration is 5 μ g/ml) to induce mouse spleen lymphocyte propagation, with the OD value of microplate reader in each hole of mensuration, 570nm place, the positive correlation that how much becomes of the height of OD value and splenocyte.The result shows, gingko episperm extract and effective site thereof have remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis, in giving dosage range, along with increasing its facilitation, dosage strengthens gradually, and required dosage was about the latter's 50% when gingko episperm extract effective site reached the accelerating effect similar to the gingko episperm extract, showed that the immunologic enhancement potency of gingko episperm extract effective site is higher than the gingko episperm extract.The cell matched group OD value that this embodiment does not add processing is 0.208 ± 0.018, final concentration is at 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 the cell experiment group OD value that μ g/ml handles down, the gingko episperm extract is respectively 0.290 ± 0.018,0.277 ± 0.017,0.249 ± 0.015,0.231 ± 0.010,0.219 ± 0.013, gingko episperm extract effective site is respectively 0.344 ± 0.015,0.303 ± 0.012,0.281 ± 0.007,0.260 ± 0.010,0.233 ± 0.011.
Embodiment 6
Fresh episperm 100kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 8 (V/W).To boil to carry and pour out decoction liquor after kettle temperature remains on 90-99 ℃ of 1.5h, repeat 1 time, decoct altogether 2 times.Decoction liquor is filtered the back respectively merge filtrate, 70 ℃ of filtrate are evaporated to after density is 1.02, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.0, leave standstill 2h after fully stirring, filter or press 800 rev/mins of centrifugal 5 minutes removal water-insolubles once more, it is 10000 the dialyzer distill water dialysis 24h with 50 times of volumes that the filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.09, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 60% (V/V), staticly settle, precipitate with 2 final vacuum dryings of 90% washing with alcohol, is got the gingko episperm extract.This extract is multiple water-soluble, the concentration that makes aqueous solution is 1%, sucking filtration, it is that the 10cm height is the macroporous adsorption resin chromatography post of 200cm that subsequent filtrate is crossed diameter, use earlier the distilled water eluting, treat to use instead when the test of eluent sugar is negative the ethanol elution of 10BV 85%, flow velocity is 1.5BV/h during ethanol elution.Collect ethanol elution, concentrating under reduced pressure, lyophilization gets gingko episperm extract effective site, and the perusal under 2% (W/V) concentration of this effective site is dissolved in water fully.Adopt phenol sulfuric acid process and Coomassie brilliant blue method to detect polysaccharide and protein content respectively, the result shows, gingko episperm extract polysaccharide and protein content are respectively 39.6% and 22.9%, peptide polysaccharide total content (polyoses content+protein content) is 62.5%, gingko episperm extract effective site polysaccharide and protein content are respectively 63.2% and 33.8%, and peptide polysaccharide total content (polyoses content+protein content) is 97.0%.Adopt the influence of mtt assay In vitro culture 48h detection to the SGC-7901 cell proliferation.The result shows, gingko episperm extract and effective site thereof all can directly suppress growth of tumour cell, in giving dosage range, its inhibitory action has dose-effect relationship, and required dosage was about the latter's 50% when gingko episperm extract effective site reached the depression effect similar to the gingko episperm extract, showed that the function of tumor inhibition potency of gingko episperm extract effective site is higher than the gingko episperm extract, was 200 μ g/ml at final concentration, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, the suppression ratio (%) under the 12.5 μ g/ml, the gingko episperm extract is respectively 55,40,20,15,6, gingko episperm extract effective site is respectively 68,60,39,24,14.Adopt mtt assay In vitro culture 48h to detect influence, adopt Con A (final concentration is 5 μ g/ml) to induce mouse spleen lymphocyte propagation, with the OD value of microplate reader in each hole of mensuration, 570nm place, the positive correlation that how much becomes of the height of OD value and splenocyte.The result shows, gingko episperm extract and effective site thereof have remarkable facilitation to the inductive mouse spleen T of Con A lymphopoiesis, in giving dosage range, along with increasing its facilitation, dosage strengthens gradually, and required dosage was about the latter's 50% when gingko episperm extract effective site reached the accelerating effect similar to the gingko episperm extract, showed that the immunologic enhancement potency of gingko episperm extract effective site is higher than the gingko episperm extract.The cell matched group OD value that this embodiment does not add processing is 0.189 ± 0.015, final concentration is at 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 the cell experiment group OD value that μ g/ml handles down, the gingko episperm extract is respectively 0.311 ± 0.020,0.289 ± 0.009,0.251 ± 0.015,0.230 ± 0.017,0.211 ± 0.016, gingko episperm extract effective site is respectively 0.341 ± 0.014,0.309 ± 0.003,0.294 ± 0.010,0.264 ± 0.019,0.236 ± 0.008.
Claims (8)
1. one kind has antitumor and the active gingko episperm preparation method of extract of immunological enhancement, it is characterized in that may further comprise the steps:
(1) gingko episperm is adopted 90-99 ℃ of hot water decoct, each 1-2h decocts 2-3 time altogether, and solid-liquid ratio is 1: 6-12 (V/W), filter decoction liquor respectively and merge filtrate;
(2) with behind the filtrate concentrating under reduced pressure, what add concentration and be 25% (W/V) is the saline solution that combines at 8: 1: 1 by sodium chloride, sodium carbonate and magnesium sulfate by weight, the pH value that makes concentrated filtrate is 7.0-7.4, leave standstill 2-4h after fully stirring, filter once more or 800-1200 rev/min of centrifugal 3-5 minute removal water-insoluble;
(3) to adopt cutoff value be the distill water dialysis 12-24h of the dialyzer of 7000-10000 with 50-100 times of volume to the concentrated filtrate that will remove water-insoluble, behind the dialyzed solution concentrating under reduced pressure, in spissated dialyzed solution, add ethanol, making the ethanol final concentration is 60-85% (V/V), staticly settles;
(4) with washing with alcohol 2-4 final vacuum drying or the lyophilization of precipitate, obtain the gingko episperm extract with 90-98%.
2. according to claim 1 have antitumor and an active gingko episperm preparation method of extract of immunological enhancement, it is characterized in that it is 1.02-1.08 that filtrate in the described step (2) is evaporated to density.
3. according to claim 1 have antitumor and an active gingko episperm preparation method of extract of immunological enhancement, it is characterized in that it is 1.09-1.15 that dialyzed solution in the described step (3) is evaporated to density.
4. according to claim 1 have antitumor and an active gingko episperm preparation method of extract of immunological enhancement, it is characterized in that the gingko episperm extract perusal under 1% (W/V) concentration in the described step (4) is dissolved in water fully, its polyoses content and protein content sum are greater than 60%.
5. preparation method with antitumor and the active gingko episperm extract of immunological enhancement effective site is characterized in that may further comprise the steps:
(1) gingko episperm is adopted 90-99 ℃ of hot water decoct, each 1-2h, decoct 2-3 time altogether, solid-liquid ratio is 1: 6-12 (V/W), decoction liquor is filtered the back respectively to be merged, 70 ℃ of filtrate are evaporated to after density is 1.02-1.08, add concentration and be 25% (W/V) by sodium chloride, sodium carbonate and magnesium sulfate are the saline solution that combines at 8: 1: 1 by weight, the pH value that makes concentrated filtrate is 7.0-7.4, leave standstill 2-4h after fully stirring, filter or press 800-1200 rev/min of centrifugal 3-5 minute removal water-insoluble once more, it is the distill water dialysis 12-24h of the dialyzer of 7000-10000 with 50-100 times of volume that the concentrated filtrate of having removed water-insoluble is adopted cutoff value, dialyzed solution is evaporated to after density is 1.09-1.15, add ethanol in spissated dialyzed solution, making the ethanol final concentration is 60-85% (V/V), staticly settles, precipitate with 2-4 final vacuum drying of 90-98% washing with alcohol or lyophilization, is got the gingko episperm extract;
(2) the gingko episperm extract is water-soluble, the concentration that makes aqueous solution is 1% (W/V);
(3) with 1% (W/V) gingko episperm solution of extract sucking filtration, subsequent filtrate is crossed the macroporous adsorbent resin chromatographic column;
(4) use the distilled water eluting earlier, when treating that the test of eluent sugar is negative, use the ethanol elution of the 65-95% of 10BV, collect ethanol elution, concentrating under reduced pressure, lyophilization obtains gingko episperm extract effective site.
6. the preparation method with antitumor and the active gingko episperm extract of immunological enhancement effective site according to claim 5 is characterized in that the height and the diameter ratio of big pore adsorption resin chromatographic column in the described step (3) is 20: 1.
7. the preparation method with antitumor and the active gingko episperm extract of immunological enhancement effective site according to claim 5, flow velocity is 1-2BV/h when it is characterized in that ethanol elution used in the described step (4).
8. the preparation method with antitumor and the active gingko episperm extract of immunological enhancement effective site according to claim 5, it is characterized in that the perusal under 2% (W/V) concentration of the gingko episperm extract effective site described in the described step (4) is dissolved in water fully, its polyoses content and protein content sum are greater than 90%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102510509A CN101912425B (en) | 2010-08-06 | 2010-08-06 | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102510509A CN101912425B (en) | 2010-08-06 | 2010-08-06 | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101912425A true CN101912425A (en) | 2010-12-15 |
| CN101912425B CN101912425B (en) | 2011-12-14 |
Family
ID=43320172
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102510509A Expired - Fee Related CN101912425B (en) | 2010-08-06 | 2010-08-06 | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101912425B (en) |
Cited By (5)
| 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 |
| CN106344645A (en) * | 2016-10-09 | 2017-01-25 | 锦州医科大学 | Traditional Chinese medicine immunity enhancer suitable for poultry and preparation method 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 |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281050A (en) * | 1999-07-15 | 2001-01-24 | 沈阳药科大学 | Process for preparing ginkgolic polyose by culturing ginkgolic cells |
| CN1299834A (en) * | 2000-12-07 | 2001-06-20 | 扬州大学 | Ginkgo Testa Polysaccharide and the preparation of pharmaceutical thereof |
-
2010
- 2010-08-06 CN CN2010102510509A patent/CN101912425B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1281050A (en) * | 1999-07-15 | 2001-01-24 | 沈阳药科大学 | Process for preparing ginkgolic polyose by culturing ginkgolic cells |
| CN1299834A (en) * | 2000-12-07 | 2001-06-20 | 扬州大学 | Ginkgo Testa Polysaccharide and the preparation of pharmaceutical thereof |
Non-Patent Citations (6)
| Title |
|---|
| 《中国新药杂志》 20010830 贾玲昌等 2种银杏外种皮多糖的成分及药效学比较研究 592-594 1-8 , 第08期 2 * |
| 《中药材》 19970930 宋根萍等 银杏外种皮多糖的成分分析 461-463 1-8 , 第09期 2 * |
| 《中药材》 20061025 仰榴青等 大孔吸附树脂纯化银杏外种皮多糖的研究 1098-1100 1-8 , 第10期 2 * |
| 《安徽农业科学》 20081001 王桃云等 超声辅助提取银杏外种皮多糖工艺优化研究 12072-12074 1-8 , 第28期 2 * |
| 《山东科学》 20030715 曹仲伟等 银杏外种皮多糖的初步研究 57-60 1-8 , 第02期 2 * |
| 《山地农业生物学报》 20040428 王椰等 银杏外种皮多糖浸提的影响因素分析 139-141 1-8 , 第02期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106344645A (en) * | 2016-10-09 | 2017-01-25 | 锦州医科大学 | Traditional Chinese medicine immunity enhancer suitable for poultry and preparation method thereof |
| CN106344645B (en) * | 2016-10-09 | 2019-10-22 | 锦州医科大学 | Traditional Chinese medicine for livestock immunopotentiator and preparation method |
| 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 |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101912425B (en) | 2011-12-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102600219B (en) | Total flavone extract of abelmoschus manihot and preparing method of total flavone extract | |
| CN108503719A (en) | A method of extraction Dendrobium officinale polysaccharide | |
| CN103768534B (en) | A kind of Chinese medicine composition with antitumor activity | |
| CN101912425B (en) | Ginkgo episperm extract with antitumor and immunostimulating activity and preparation method of effective part thereof | |
| CN103432193A (en) | Microwave-assisted aqueous two-phase extraction and separation method of kudzu root total flavones | |
| CN103040917A (en) | Method for preparing total saponins and polysaccharides from Apios americana flowers simultaneously, and application of total saponins and polysaccharides | |
| CN102146142B (en) | Method for preparing astragalus polysaccharides | |
| CN104127463B (en) | A kind of Echinacea Purpurea Herb P.E and its preparation method and application | |
| CN101974045A (en) | Method for preparing salidroside | |
| CN101507740B (en) | Lysimachia capillipes Hemsl. total saponin extract preparation method using macroporous resin and use thereof | |
| CN107638431A (en) | A kind of preparation technology of P-Cymene and its application | |
| CN102895294B (en) | Astragalus mongholicus injection and preparation method thereof | |
| CN101884788B (en) | Traditional Chinese medicine astragalus polysaccharide immunopotentiator | |
| CN105311075A (en) | Preparation method of manyprickle acanthopanax root extract | |
| CN104857435B (en) | A kind of Traditional Chinese medicine compound composition with antitumor activity and its preparation method and application | |
| CN101468104B (en) | Chinese medicinal compound preparation for treating osteoporosis and method for preparing the same | |
| CN105147716B (en) | A kind of newcastle disease vaccine Yupingfeng polysaccharide immunopotentiator | |
| CN102048770B (en) | Artificially-cultured cordceps militaris sporocarp extract and preparation process thereof | |
| CN101167755B (en) | Method for preparing centipede polysaccharide protein composition with anti-tumor activity and use | |
| CN102617893A (en) | Preparation and application of composition of panaxoside Rg3, ginseng polysaccharide and ganoderan | |
| WO2014000119A1 (en) | Anti-tachyarrhythmia formulation and preparation method thereof | |
| CN101912426B (en) | Method for preparing ginkgo exocarp anti-tumor effective part and preparation thereof | |
| CN106496346A (en) | A kind of isolation and purification method of white polysaccharide | |
| CN1590383A (en) | Rod sage root extract and making method of its preparation | |
| CN103040857B (en) | Application of phyllanthus urinaria polysaccharide component in preparing drug for resisting hepatitis B virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111214 Termination date: 20150806 |
|
| EXPY | Termination of patent right or utility model |