CN101912425B - 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
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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 preparation method antitumor and active gingko episperm extract of immunological enhancement and extract efficient part, belong to technical field of traditional Chinese medicine pharmacy.
Background technology
Ginkgo is " living fossil " plant of world's preciousness, and China is the township of ginkgo.The fruit of ginkgo has another name called gingko, and is not only nutritious, and its existing thousands of years history of being used as medicine, and is integration of drinking and medicinal herbs treasure world-famous.Contain compounds such as flavones and terpene lactones in the Ginkgo Leaf, the Schwabe company of Germany takes the lead in having set up Folium Ginkgo extract (EGb) source mill.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 gingko, is commonly called as the gingko clothing, accounts for 3/4 of its kind reality, and in the season of every ginkgo results, just there is a large amount of exospers in the producing region and is taken as refuse and abandon, contaminate environment not only, and cause the significant wastage of Biological resources.How stinking exosper 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 exosper, 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 it is water-soluble not ideal, can only make solid dosage, limited the sick clinical application of planting of certain cancers, do not separate the go forward side by side stepping line correlation research of its efficient part 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 ofly to be had antitumor and the active gingko episperm preparation method of extract of immunological enhancement, and second purpose is to provide a kind of preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part that has.
First purpose of the present invention is achieved through the following technical solutions, and 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: 6-12W/V, filter decoction liquor respectively and merge filtered liquid;
(2) with behind the filtered liquid concentrating under reduced pressure, what add concentration and be 25%W/V is the salts solution that combines at 8: 1: 1 by sodium-chlor, yellow soda ash and sal epsom by weight, make the pH value of concentrated filtrate be 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 dialysis membrane 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, add ethanol in spissated dialyzed solution, making the ethanol final concentration is 60-85%V/V, staticly settles;
(4) with washing with alcohol 2-4 final vacuum drying or the lyophilize of throw out, obtain the gingko episperm extract with 90-98%.
It is 1.02-1.08 that filtered liquid 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 visual inspection under 1%W/V concentration of gingko episperm extract in the described step (4) is dissolved in water fully, and its polysaccharide content and protein content sum are greater than 60%.
Second purpose of the present invention is achieved through the following technical solutions, and has preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part, 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: 6-12W/V, decoction liquor is filtered respectively and merge, filtered liquid is evaporated to after density is 1.02-1.08, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts solution that combines at 8: 1: 1 by weight, make the pH value of concentrated filtrate be 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 dialysis membrane 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, throw out with 2-4 final vacuum drying of 90-98% washing with alcohol or lyophilize, is got the gingko episperm extract;
(2) the gingko episperm extract is water-soluble, the concentration that makes the aqueous solution is 1%W/V;
(3) with 1%W/V gingko episperm solution of extract suction filtration, subsequent filtrate is crossed macroporous resin column;
(4) use the distilled water wash-out earlier, when treating that the test of elutriant sugar is negative, use the ethanol elution of the 65-95% of 10BV, collect ethanol eluate, concentrating under reduced pressure, lyophilize obtains gingko episperm extract efficient part.
The height and the diameter ratio of big pore adsorption resin chromatography 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 visual inspection under 2%W/V concentration of resulting gingko episperm extract efficient part is dissolved in water fully in the described step (4), and its polysaccharide 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 sedimentation helps removing the water-insoluble in the gingko episperm extract and carries out the efficient part separation, the gingko episperm solution of extract is crossed the innoxious solvent wash-out that the low-pole macroporous adsorbent resin is also used opposed polarity, make separate efficient part content height, security is good.Polysaccharide content and protein content sum are greater than 60% in the gingko episperm extract that this method makes, meet national correlated quality standard-required, polysaccharide content and protein content sum are greater than 90% in the gingko episperm extract efficient part 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.What this method made has antitumor and active gingko episperm extract of immunological enhancement and efficient part good water solubility thereof, both can make solid dosage, also can make liquid dosage form, and preparation technology is simple and feasible, is fit to industrialized production.
1, peptide polysaccharide (polysaccharide content+protein content) Determination on content
Adopt the phenol sulfuric acid process to survey polysaccharide content: it is an amount of that precision takes by weighing the anhydrous grape saccharide, is made into the glucose reference liquid of 1mg/ml with distilled water, and further dilution is different concns again.It is that the glucose standardized 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, after mixing, add the 5ml vitriol oil, mix, boiling water bath 15min, cooling bath 30min.Measure absorbancy in the 490nm place with ultraviolet spectrophotometer, glucose solution concentration and absorbancy are carried out linear regression, set up regression equation.Get gingko episperm extract (GBE) and efficient part (GBEE) need testing solution 0.5ml thereof, press aforesaid operations and survey absorbance, the substitution regression equation calculates polysaccharide content.
Adopt the Xylene Brilliant Cyanine G 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 different concns 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 Xylene Brilliant Cyanine G G-250 solution again, after shaking up, place 5～10min, after treating that it fully reacts, measure absorbancy in the 595nm place with ultraviolet spectrophotometer, bovine serum albumin solution concentration and absorbancy 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 %=(polysaccharide 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 logarithmic phase, after tryptic digestion, the washing of Hank ' s liquid, adjust cell to 1 * 10 with containing 10% calf serum RPMI1640 nutrient solution
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 different concns that contains the preparation of 10% calf serum RPMI1640 nutrient solution, each concentration is established 6 multiple holes, cultivates 48h.Cultivate and finish preceding 4h adding MTT (final concentration 5mg/ml), discard nutrient solution after continuing to cultivate 4h, add the acidifying Virahol, the 10min that vibrates uses microplate reader to measure the OD value in each hole in the 570nm place.By formula " inhibiting rate=(1-medicine hole OD value/control wells OD value) * 100% " calculates external inhibiting rate to SGC-7901 cell proliferation.The results are shown in Table 2.
Table 2GBE and GBEE are to the effect of people's cancer of the stomach SGC-7901 cell inhibiting
Table 2 result shows that GBE and the GBEE restraining effect to stomach cancer cell line under 12.5-200 μ g/ml dosage has dose-effect relationship, and GBEE reaches the inhibiting rate 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 mouse, 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 nutrient solution 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 different concns of the RPMI1640 nutrient solution 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 acidifying Virahol 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 splenic lymphocyte.The results are shown in Table 3.
Table 3GBE and GBEE are to the promoter action of mouse immune cell proliferation
Table 3 result shows, GBE and GBEE have remarkable promoter action to Con A inductive mouse spleen T 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-soluble mensuration
GBE and GBEE respectively under 1%W/V and 2%W/V concentration visual inspection be dissolved in water fully.
Embodiment:
Have antitumor and the active gingko episperm preparation method of extract of immunological enhancement
Embodiment 1
Fresh exosper 100kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 8W/V.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 filtered liquid, 70 ℃ of filtered liquids are evaporated to after density is 1.02, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts 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 dialysis membrane distill water dialysis 24h with 50 times of volumes that the filtered liquid 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, throw out with 2 final vacuum dryings of 90% washing with alcohol, is got the gingko episperm extract, and the visual inspection under 1%W/V concentration of this extract is dissolved in water fully.Adopt phenol sulfuric acid process and Xylene Brilliant Cyanine G method to detect polysaccharide and protein content respectively, the result is respectively 37.6% and 24.8%, and peptide polysaccharide total content (polysaccharide content+protein content) is 62.4%.Adopt the influence of mtt assay vitro culture 48h detection to SGC-7901 cell proliferation.The result shows that the gingko episperm extract can directly suppress growth of tumour cell, and its restraining effect 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 inhibiting rate (%) is respectively 50 under the 12.5 μ g/ml, 38,21,11,5.Adopt mtt assay 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 splenic lymphocyte.The result shows that the gingko episperm extract has remarkable promoter action to Con A inductive mouse spleen T lymphopoiesis, in giving dosage range, strengthens gradually along with dosage increases its promoter action.The cell control 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 exosper 200kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 6W/V.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 filtered liquid, 70 ℃ of filtered liquids are evaporated to after density is 1.04, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts 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 dialysis membrane distill water dialysis 12h with 80 times of volumes that the filtered liquid 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, throw out with 3 final vacuum dryings of 95% washing with alcohol, is got the gingko episperm extract, and the visual inspection under 1%W/V concentration of this extract is dissolved in water fully.Adopt phenol sulfuric acid process and Xylene Brilliant Cyanine G method to detect polysaccharide and protein content respectively, the result is respectively 40.1% and 21.1%, and peptide polysaccharide total content (polysaccharide content+protein content) is 61.2%.Adopt the influence of mtt assay vitro culture 48h detection to SGC-7901 cell proliferation.The result shows that the gingko episperm extract can directly suppress growth of tumour cell, and its restraining effect has dose-effect relationship, is 200 μ g/ml at final concentration, l00 μ g/ml, 50 μ g/ml, 25 μ g/ml, its inhibiting rate (%) is respectively 54 under the 12.5 μ g/ml, 40,22,14,5.Adopt mtt assay 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 splenic lymphocyte.The result shows that the gingko episperm extract has remarkable promoter action to Con A inductive mouse spleen T lymphopoiesis, in giving dosage range, strengthens gradually along with dosage increases its promoter action.The cell control 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 exosper 500kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 12W/V.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 filtered liquid, 70 ℃ of filtered liquids are evaporated to after density is 1.08, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts 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 dialysis membrane distill water dialysis 18h with 100 times of volumes that the filtered liquid 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, throw out with 4 postlyophilizations of 98% washing with alcohol, is got the gingko episperm extract, and the visual inspection under 1%W/V concentration of this extract is dissolved in water fully.Adopt phenol sulfuric acid process and Xylene Brilliant Cyanine G method to detect polysaccharide and protein content respectively, the result is respectively 43.9% and 20.4%, and peptide polysaccharide total content (polysaccharide content+protein content) is 64.3%.Adopt the influence of mtt assay vitro culture 48h detection to SGC-7901 cell proliferation.The result shows that the gingko episperm extract can directly suppress growth of tumour cell, and its restraining effect 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 inhibiting rate (%) is respectively 52 under the 12.5 μ g/ml, 37,24,16,7.Adopt mtt assay 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 splenic lymphocyte.The result shows that the gingko episperm extract has remarkable promoter action to Con A inductive mouse spleen T lymphopoiesis, in giving dosage range, strengthens gradually along with dosage increases its promoter action.The cell control 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.
Has preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part
Embodiment 4
Fresh exosper 500kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 12W/V.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 filtered liquid, 70 ℃ of filtered liquids are evaporated to after density is 1.08, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts 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 dialysis membrane distill water dialysis 18h with 100 times of volumes that the filtered liquid 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, throw out 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 the aqueous solution is 1%, suction 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 wash-out, treat to use instead when the test of elutriant sugar is negative the ethanol elution of 10BV65%, flow velocity is 2BV/h during ethanol elution.Collect ethanol eluate, concentrating under reduced pressure, lyophilize gets gingko episperm extract efficient part, and the visual inspection under 2% (W/V) concentration of this efficient part is dissolved in water fully.Adopt phenol sulfuric acid process and Xylene Brilliant Cyanine G 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 (polysaccharide content+protein content) is 61.6%, gingko episperm extract efficient part polysaccharide and protein content are respectively 61.8% and 33.3%, and peptide polysaccharide total content (polysaccharide content+protein content) is 95.1%.Adopt the influence of mtt assay vitro culture 48h detection to SGC-7901 cell proliferation.The result shows, gingko episperm extract and efficient part thereof all can directly suppress growth of tumour cell, in giving dosage range, its restraining effect has dose-effect relationship, and required dosage was about the latter's 50% when gingko episperm extract efficient part reached the retarding effect similar to the gingko episperm extract, showed that the function of tumor inhibition potency of gingko episperm extract efficient part 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 inhibiting rate (%) under the 12.5 μ g/ml, the gingko episperm extract is respectively 52,39,23,13,8, gingko episperm extract efficient part is respectively 69,57,35,23,14.Adopt mtt assay 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 splenic lymphocyte.The result shows, gingko episperm extract and efficient part thereof have remarkable promoter action to Con A inductive mouse spleen T lymphopoiesis, in giving dosage range, along with increasing its promoter action, dosage strengthens gradually, and required dosage was about the latter's 50% when gingko episperm extract efficient part reached the accelerating effect similar to the gingko episperm extract, showed that the immunologic enhancement potency of gingko episperm extract efficient part is higher than the gingko episperm extract.The cell control 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 efficient part 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 exosper 200kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 6W/V.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 filtered liquid, 70 ℃ of filtered liquids are evaporated to after density is 1.04, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts 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 dialysis membrane distill water dialysis 12h with 80 times of volumes that the filtered liquid 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, throw out 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 the aqueous solution is 1%, suction 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 wash-out, treat to use instead when the test of elutriant sugar is negative the ethanol elution of 10BV95%, flow velocity is 1BV/h during ethanol elution.Collect ethanol eluate, concentrating under reduced pressure, lyophilize gets gingko episperm extract efficient part, and the visual inspection under 2%W/V concentration of this efficient part is dissolved in water fully.Adopt phenol sulfuric acid process and Xylene Brilliant Cyanine G 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 (polysaccharide content+protein content) is 65.3%, gingko episperm extract efficient part polysaccharide and protein content are respectively 62.0% and 34.2%, and peptide polysaccharide total content (polysaccharide content+protein content) is 96.2%.Adopt the influence of mtt assay vitro culture 48h detection to SGC-7901 cell proliferation.The result shows, gingko episperm extract and efficient part thereof all can directly suppress growth of tumour cell, in giving dosage range, its restraining effect has dose-effect relationship, and required dosage was about the latter's 50% when gingko episperm extract efficient part reached the retarding effect similar to the gingko episperm extract, showed that the function of tumor inhibition potency of gingko episperm extract efficient part 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 inhibiting rate (%) under the 12.5 μ g/ml, the gingko episperm extract is respectively 53,36,24,12,5, gingko episperm extract efficient part is respectively 71,59,41,21,17.Adopt mtt assay 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 splenic lymphocyte.The result shows, gingko episperm extract and efficient part thereof have remarkable promoter action to Con A inductive mouse spleen T lymphopoiesis, in giving dosage range, along with increasing its promoter action, dosage strengthens gradually, and required dosage was about the latter's 50% when gingko episperm extract efficient part reached the accelerating effect similar to the gingko episperm extract, showed that the immunologic enhancement potency of gingko episperm extract efficient part is higher than the gingko episperm extract.The cell control 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 efficient part 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 exosper 100kg placed to boil carry in the pot, add water and make solid-liquid ratio be about 1: 8W/V.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 filtered liquid, 70 ℃ of filtered liquids are evaporated to after density is 1.02, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts 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 dialysis membrane distill water dialysis 24h with 50 times of volumes that the filtered liquid 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, throw out with 2 final vacuum dryings of 90% washing with alcohol, is got the gingko episperm extract.This extract is multiple water-soluble, and the concentration that makes the aqueous solution is 1%, suction filtration, it is that the 10cm height is the macroporous adsorption resin chromatography post of 200cm that subsequent filtrate is crossed diameter, earlier use the distilled water wash-out, treat to use instead when the test of elutriant sugar is negative the ethanol elution of 10BV85%, flow velocity is 1.5BV/h during ethanol elution.Collect ethanol eluate, concentrating under reduced pressure, lyophilize gets gingko episperm extract efficient part, and the visual inspection under 2%W/V concentration of this efficient part is dissolved in water fully.Adopt phenol sulfuric acid process and Xylene Brilliant Cyanine G 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 (polysaccharide content+protein content) is 62.5%, gingko episperm extract efficient part polysaccharide and protein content are respectively 63.2% and 33.8%, and peptide polysaccharide total content (polysaccharide content+protein content) is 97.0%.Adopt the influence of mtt assay vitro culture 48h detection to SGC-7901 cell proliferation.The result shows, gingko episperm extract and efficient part thereof all can directly suppress growth of tumour cell, in giving dosage range, its restraining effect has dose-effect relationship, and required dosage was about the latter's 50% when gingko episperm extract efficient part reached the retarding effect similar to the gingko episperm extract, showed that the function of tumor inhibition potency of gingko episperm extract efficient part 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 inhibiting rate (%) under the 12.5 μ g/ml, the gingko episperm extract is respectively 55,40,20,15,6, gingko episperm extract efficient part is respectively 68,60,39,24,14.Adopt mtt assay 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 splenic lymphocyte.The result shows, gingko episperm extract and efficient part thereof have remarkable promoter action to Con A inductive mouse spleen T lymphopoiesis, in giving dosage range, along with increasing its promoter action, dosage strengthens gradually, and required dosage was about the latter's 50% when gingko episperm extract efficient part reached the accelerating effect similar to the gingko episperm extract, showed that the immunologic enhancement potency of gingko episperm extract efficient part is higher than the gingko episperm extract.The cell control 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 efficient part 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: 6-12W/V, filter decoction liquor respectively and merge filtered liquid;
(2) with behind the filtered liquid concentrating under reduced pressure, what add concentration and be 25%W/V is the salts solution that combines at 8: 1: 1 by sodium-chlor, yellow soda ash and sal epsom by weight, make the pH value of concentrated filtrate be 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 dialysis membrane 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, add ethanol in spissated dialyzed solution, making the ethanol final concentration is 60-85%V/V, staticly settles;
(4) with washing with alcohol 2-4 final vacuum drying or the lyophilize of throw out, obtain the gingko episperm extract with 90-98%.
2. according to claim 1 have antitumor and the active gingko episperm preparation method of extract of immunological enhancement, it is characterized in that it is 1.02-1.08 that filtered liquid in the described step (2) is evaporated to density.
3. according to claim 1 have antitumor and the 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 the active gingko episperm preparation method of extract of immunological enhancement, it is characterized in that the gingko episperm extract visual inspection under 1%W/V concentration in the described step (4) is dissolved in water fully, its polysaccharide content and protein content sum are greater than 60%.
5. one kind has preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part, 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: 6-12W/V, decoction liquor is filtered the back respectively to be merged, 70 ℃ of filtered liquids are evaporated to after density is 1.02-1.08, add concentration and be 25%W/V by sodium-chlor, yellow soda ash and sal epsom are the salts solution that combines at 8: 1: 1 by weight, make the pH value of concentrated filtrate be 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 dialysis membrane 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, throw out with 2-4 final vacuum drying of 90-98% washing with alcohol or lyophilize, is got the gingko episperm extract;
(2) the gingko episperm extract is water-soluble, the concentration that makes the aqueous solution is 1%W/V;
(3) with 1%W/V gingko episperm solution of extract suction filtration, subsequent filtrate is crossed the macroporous adsorbent resin chromatography column;
(4) use the distilled water wash-out earlier, when treating that the test of elutriant sugar is negative, use the ethanol elution of the 65-95% of 10BV, collect ethanol eluate, concentrating under reduced pressure, lyophilize obtains gingko episperm extract efficient part.
6. according to claim 5 have a preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part, it is characterized in that the height and the diameter ratio of big pore adsorption resin chromatography column in the described step (3) is 20: 1.
7. according to claim 5 have a preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part, and flow velocity is 1-2BV/h when it is characterized in that ethanol elution used in the described step (4).
8. according to claim 5 have a preparation method antitumor and the active gingko episperm extract of immunological enhancement efficient part, it is characterized in that the gingko episperm extract efficient part visual inspection under 2%W/V concentration described in the described step (4) is dissolved in water fully, its polysaccharide content and protein content sum are greater than 90%.
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