CN106317247B - A kind of preparation method of water-solubility ginkgo exosper polysaccharide and its preparation - Google Patents
A kind of preparation method of water-solubility ginkgo exosper polysaccharide and its preparation Download PDFInfo
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Abstract
The present invention relates to the preparation methods of a kind of water-solubility ginkgo exosper polysaccharide and its preparation.The gingko episperm impregnated by ethyl alcohol is placed in boil to mention in pot and is routinely added water to cook, filtering, it is concentrated under reduced pressure, ethyl alcohol is added into concentrate, staticly settles, the sediment is placed in ball grinder, revolving speed, positive and negative alternate operation are adjusted, circulation crushes for several times, vacuum drying or freeze-drying, can be obtained water-solubility ginkgo exosper polysaccharide.Excipients or corrigent is added in the polysaccharide and antitumor capsule preparations or antineoplastic oral liquid preparation is made in preservative.The gingko exocarp polysaccharide partial size that this method obtains reduces, and water-soluble and antitumor potency all significantly improves, and can be prepared into antitumor solid and liquid preparation.
Description
Technical field
The present invention relates to the preparation methods of a kind of water-solubility ginkgo exosper polysaccharide and its preparation, belong to herbal pharmaceutical neck
Domain.
Background technique
We extract polysaccharide compound from gingko episperm and have carried out series of basic and clinical principium examination
With, discovery gingko exocarp polysaccharide (Ginkgo bilobaExocarp polysaccharide, GBEP) there is direct killing
The effect of tumour cell and enhancing immune function.Its capsule preparations, in tumor patient many years, achieves good in clinical application
Economic and social benefit.However, with traditional handicraft (ZL00134363.7) prepare GBEP water solubility be not it is too ideal, can only make
At solid pharmaceutical preparation；And improved technique (ZL201010251068.9) prepares solid and liquid although water solubility can be improved
Preparation, but cumbersome preparation time is longer, thus cost is also higher.Therefore, it is water-soluble same in improvement to carry out process modification
When, improving preparation efficiency is the problem of this seminar pays close attention to for many years.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of water-solubility ginkgo exosper polysaccharide and its preparations
Preparation method.
The first purpose of this invention is achieved through the following technical solutions, the preparation of water-solubility ginkgo exosper polysaccharide
Method, comprising the following steps:
(1) gingko episperm impregnated by ethyl alcohol is placed in boil to mention and routinely adds water to cook 2-3 times and distinguishes in pot
Filtrate is merged and is concentrated under reduced pressure into specific gravity in 1.21-1.30, ethyl alcohol is slowly added in concentrate, ethyl alcohol final concentration is made by filtering
In 60-80%(V/V), side edged quickly stirs, and supernatant is abandoned in centrifugation after standing, then for several times with high concentration ethanol centrifuge washing, is obtained
Ethanol pellet；
(2) planetary ball mill is opened, it is respectively 20mm, 10mm that diameter is packed into ball grinder for 40-60% by filling rate
With the large, medium and small agate abrading-ball of 6mm, the quantity ratio of large, medium and small agate abrading-ball is 1:(28-32): (152-155),
Ethanol pellet as described in step (1) is added by ratio of grinding media to material 15-20:1(W/W)；The operation of positive and negative alternate interval, turns
Speed is 300-600rpm/min, is recycled 3-5 times, is dried in vacuo or is freeze-dried after sieving, can be obtained outer kind of water-solubility ginkgo
Skin polysaccharide.
Shutdown 0.5-1.0h is reversely transported again after positive and negative alternate interval described in step (2) operates to positive operation 0.8-1.2h
Row 0.8-1.2h shuts down 0.5-1.0h.
Second object of the present invention is achieved through the following technical solutions, water-solubility ginkgo exosper polysaccharide formulation
A small amount of excipients or corrigent and preservative system is added in the water-solubility ginkgo exosper polysaccharide of above method preparation by preparation method
At antitumor capsule preparations or antineoplastic oral liquid preparation.
Compared with prior art, the invention has the following advantages:
The present invention makes hard sphere carry out strong shock to raw material, grinds and stir using the rotation or vibration of planetary ball mill
It mixes, raw material is crushed as water insoluble particulate.This method operation and preparation flow are simply controllable, gained gingko exocarp polysaccharide grain
Diameter reduces, and even particle size distribution, water-soluble and antitumor potency all significantly improves, and can be prepared into antitumor solid and liquid
Body preparation.This method is easy to operate, at low cost, is suitble to industrialized production.
1, water-soluble measurement
GBEP before crushing is in 1.5%(W/V) it visually observes under concentration and is dissolved completely in water, and smashed GBEP exists
It 2.5%(W/V) is visually observed under concentration and is dissolved completely in water, prompt water-soluble improve.
2, particle size determination
Precision, which weighs, crushes preceding and each 2.5mg of smashed GBEP, is dissolved in distilled water respectively, is settled to 50 ml, obtains dense
Degree is the GBEP aqueous solution of 50 μ g/mL.The aqueous solution is taken to be placed in tool plug test tube, after ultrasonic 20min, using Malvern laser
Particle size analyzer measures partial size.The results show that the partial size of the gingko exocarp polysaccharide coarse powder before crushing is 1.5-2.0 μm, and using should
Water-solubility ginkgo exosper polysaccharide partial size made from method is 200-250nm.
3, zeta potential measures
Precision, which weighs, crushes preceding and each 2.5mg of smashed GBEP, is dissolved in distilled water respectively, is settled to 50 ml, obtains dense
Degree is the GBEP aqueous solution of 50 μ g/mL.The aqueous solution is taken to be placed in tool plug test tube, after ultrasonic 20min, using Malvern laser
Particle size analyzer measures zeta potential.The results show that the gingko exocarp polysaccharide zeta potential before crushing is (- 14)-(- 11) mV, and use
Gingko exocarp polysaccharide zeta potential made from this method is (- 26)-(- 24) mV, prompts smashed gingko exocarp polysaccharide water
Solution is more stable.
4, the measurement of peptide polysaccharide content
Survey polyoses content using Phenol sulfuric acid procedure: it is appropriate that precision weighs anhydrous grape saccharide, is made into distilled water
The glucose standard of 1mg/ml, is further diluted to various concentration.It is 30 μ g/ml, 60 μ g/ml, 90 μ that precision, which measures concentration,
G/ml, 120 μ g/ml, 150 μ g/ml, 180 μ g/ml glucose standards solution 0.5ml in 6 10ml tool plug test tubes, be added
The 5ml concentrated sulfuric acid is added after mixing in the re-distilled phenol solution 1ml of 50mg/ml, is uniformly mixed, boiling water bath 15min, cold bath
30min.Absorbance is measured at 490nm with ultraviolet specrophotometer, and glucose concentration and absorbance are linearly returned
Return, establishes regression equation.Test solution 0.5ml is taken, absorbance value is surveyed by aforesaid operations, substitutes into regression equation, calculate more
Sugared content.
Survey protein content using Coomassie Brilliant Blue: it is appropriate that precision weighs bovine serum albumin(BSA) standard items, uses distilled water
It is made into the protein standard solution of 100 μ g/ml, is further diluted to various concentration.It is 20 μ g/ml, 40 μ g/ that precision, which measures concentration,
Ml, 60 μ g/ml, 80 μ g/ml, 100 μ g/ml protein standard solution 0.5ml add 2.5ml respectively at 5 10ml tool plug test tubes
Coomassie brilliant G-250 solution, after shaking up, place 5~10min, after it is sufficiently reacted, with ultraviolet specrophotometer in
Absorbance is measured at 595nm, and linear regression is carried out to bovine serum albumin solution concentration and absorbance, establishes regression equation.It takes
Test solution 0.5ml surveys absorbance value by aforesaid operations, substitutes into regression equation, calculate protein content.
Qualitative analysis is carried out using infrared spectrometric analyzer.Gingko exocarp polysaccharide amide bond as the result is shown prompts for
Peptide polysaccharide.Peptide polysaccharide content %=(polyoses content+protein content) %.
Gingko exocarp polysaccharide its polyoses content before crushing is 36%, protein content 28%, so peptide polysaccharide content
For 36%+28%=64%.Use its polyoses content of water-solubility ginkgo exosper polysaccharide made from this method for 40%, protein content
It is 26%, so peptide polysaccharide content is 40%+26%=66%, gingko exocarp polysaccharide anti-tumor effective component before and after crushing is prompted to contain
Amount is held essentially constant.
5, drug efficacy study
The HepG-2 cell in logarithmic growth phase is taken, it is small with containing 10% after being washed with trypsin digestion, Hank ' s liquid
Cow's serum RPMI1640 culture solution adjusts cell to 1 × 105/ml.It is outstanding that above-mentioned cell is separately added into each hole of 96 well culture plates
100 μ l of liquid, in 5%CO2, 37 DEG C of saturated humidities incubator in cultivate for 24 hours after, be added and trained with containing 10% calf serum RPMI1640
Each 100 μ l of sample for the various concentration that nutrient solution is prepared, each concentration set 6 multiple holes, cultivate 48h.MTT is added in 4h before culture terminates
(final concentration 5mg/ml) continues to discard culture solution after cultivating 4h, acidification isopropanol is added, vibrates 10min, with microplate reader in
The OD value in each hole is measured at 570nm.By formula " inhibiting rate=(1- drug hole OD value/control wells OD value) × 100% ", calculate external
To the inhibiting rate of HepG-2 cell Proliferation.It the results are shown in Table 1.
Table 1 is the results show that gingko exocarp polysaccharide has directly inhibition to human liver cancer cell under 10-320 μ g/ml dosage
Effect, as dosage increases, inhibiting effect is also gradually increased.Moreover, the half of smashed gingko exocarp polysaccharide inhibits
Concentration IC50Before (μ g/ml) is substantially less than crushing, illustrate that potency is higher.
Specific embodiment
Embodiment 1
120 grams of dry gingko episperm impregnated by ethyl alcohol are taken, is placed in boil and mentions in pot plus 1500 grams of water, are decocted when 95 DEG C
1 hour, continues after filtering plus water repeats above-mentioned decocting method 1 time and filters, 2 filtrate is merged, is concentrated under reduced pressure in 70 DEG C
It is 1.21 to specific gravity, 95% ethyl alcohol is slowly added in concentrate, makes ethyl alcohol final concentration 80%, side edged quickly stirs,
It is washed 2 times after standing 6 hours by sediment centrifuge dripping, then with high concentration ethanol (98% or more volumetric concentration), obtains ethanol precipitation
Object；Planetary ball mill is opened, is packed into quantity in ball grinder by filling rate 50% than the large, medium and small agate mill for 1:28:155
Ball is 20:1(W/W by ratio of grinding media to material) above-mentioned ethanol pellet is added；1.0h inverted running 1.0h again is shut down after forward direction operation 1.0h
After shut down 1.0h；Revolving speed is 600rpm/min, is recycled 3 times, obtained water-solubility ginkgo exosper polysaccharide is in 2.5%(W/V) concentration
Under visually observe and be dissolved completely in water, partial size 219.3nm, zeta potential is -25.4mV.It is bright using Phenol sulfuric acid procedure and coomassie
Blue laws detects its polysaccharide and protein content respectively, and as a result respectively 40% and 26%, peptide polysaccharide total content is 66%.Using mtt assay
Influence of the vitro detection to HepG-2 cell Proliferation, inoculating cell are added water-solubility ginkgo exosper polysaccharide afterwards for 24 hours, continue to cultivate
48h detection.The results show that water-solubility ginkgo exosper polysaccharide final concentration is in 320 μ g/ml, 160 μ g/ml, 80 μ g/ml, 40 μ g/
Hepatoma cell growth can directly be inhibited under ml, 20 μ g/ml, 10 μ g/ml, and as dosage increases, inhibiting effect gradually increases
By force, inhibiting rate (%) is respectively 70,65,56,48,31 and 15, half-inhibitory concentration IC50(μ g/ml) is 122.5.Take water-soluble silver
Apricot exosper polysaccharide dry powder 200g is dissolved in pure water, and appropriate corrigent and preservative is added by drug quality requirement, shakes up,
It bottles by 10ml specification, water-solubility ginkgo exosper polyoses oral liquid formulation is made.
Embodiment 2
160 grams of dry gingko episperm impregnated by ethyl alcohol are taken, is placed in boil and mentions in pot plus 2000 grams of water, are decocted when 98 DEG C
1 hour, continues after filtering plus water repeats above-mentioned decocting method 1 time and filters, 2 filtrate is merged, is concentrated under reduced pressure in 70 DEG C
It is 1.26 to specific gravity, 95% ethyl alcohol is slowly added in concentrate, makes ethyl alcohol final concentration 60%, side edged quickly stirs,
It is washed 2 times after standing 10 hours by sediment centrifuge dripping, then with high concentration ethanol, obtains ethanol pellet；Open planetary ball
Grinding machine, by filling rate be 60% in ball grinder be packed into quantity than the large, medium and small agate abrading-ball for 1:32:152, be by ratio of grinding media to material
Above-mentioned ethanol pellet 15:1(W/W) is added；0.5h, which is shut down, after forward direction operation 0.8h shuts down 0.5h after inverted running 0.8h again；Turn
Speed is 300 rpm/min, is recycled 5 times, obtained water-solubility ginkgo exosper polysaccharide is in 2.5%(W/V) it has visually observed under concentration
Fully dissolved is -25.1mV in water, partial size 246.2nm, zeta potential.It is detected respectively using Phenol sulfuric acid procedure and Coomassie Brilliant Blue
Its polysaccharide and protein content, as a result respectively 36% and 25%, peptide polysaccharide total content is 61%.Using mtt assay vitro detection pair
Water-solubility ginkgo exosper polysaccharide is added in the influence of HepG-2 cell Proliferation, inoculating cell afterwards for 24 hours, continues to cultivate 48h detection.Knot
Fruit shows, water-solubility ginkgo exosper polysaccharide final concentration in 320 μ g/ml, 160 μ g/ml, 80 μ g/ml, 40 μ g/ml, 20 μ g/ml,
Hepatoma cell growth can directly be inhibited under 10 μ g/ml, and as dosage increases, inhibiting effect is gradually increased, inhibiting rate (%)
Respectively 67,63,55,47,28 and 13, half-inhibitory concentration IC50(μ g/ml) is 137.2.Water intaking dissolubility gingko exocarp polysaccharide
Dry powder 100g is added excipients 50g, mixes thoroughly, is packed into Capsules by every 150mg, water-solubility ginkgo exosper polysaccharide is made
Capsule preparations.
Embodiment 3
100 grams of dry gingko episperm impregnated by ethyl alcohol are taken, is placed in boil and mentions in pot plus 1500 grams of water, are decocted when 96 DEG C
1 hour, continues after filtering plus water repeats above-mentioned decocting method 1 time and filters, 2 filtrate is merged, is concentrated under reduced pressure in 70 DEG C
It is 1.30 to specific gravity, 95% ethyl alcohol is slowly added in concentrate, makes ethyl alcohol final concentration 70%, side edged quickly stirs,
It is washed 3 times after standing 12 hours by sediment centrifuge dripping, then with high concentration ethanol, obtains ethanol pellet；Open planetary ball
Grinding machine, by filling rate be 40% in ball grinder be packed into quantity than the large, medium and small agate abrading-ball for 1:31:154, be by ratio of grinding media to material
Above-mentioned ethanol pellet 18:1(W/W) is added；It is shut down after shutting down 0.8h, then reverse direction operation 1.2h after forward direction operation 1.2h
0.8h, revolving speed are 500 rpm/min, are recycled 4 times, obtained water-solubility ginkgo exosper polysaccharide is in 2.5%(W/V) meat under concentration
Eye observation is dissolved completely in water, partial size 238.5nm, and zeta potential is -25.2mV.Using Phenol sulfuric acid procedure and Coomassie Brilliant Blue
Its polysaccharide and protein content are detected respectively, as a result respectively 38% and 26%, peptide polysaccharide total content is 64%.It is external using mtt assay
The influence to HepG-2 cell Proliferation is detected, water-solubility ginkgo exosper polysaccharide is added in inoculating cell afterwards for 24 hours, continues to cultivate 48h
Detection.The results show that water-solubility ginkgo exosper polysaccharide final concentration is in 320 μ g/ml, 160 μ g/ml, 80 μ g/ml, 40 μ g/ml,
Hepatoma cell growth can directly be inhibited under 20 μ g/ml, 10 μ g/ml, and as dosage increases, inhibiting effect is gradually increased,
Inhibiting rate (%) is respectively 69,64,55,48,30 and 14, half-inhibitory concentration IC50(μ g/ml) is 128.5.Take water-solubility ginkgo
Exosper polysaccharide dry powder 200g is dissolved in pure water, and appropriate corrigent and preservative is added by drug quality requirement, shakes up, presses
The bottling of 20ml specification, is made water-solubility ginkgo exosper polyoses oral liquid formulation.
Claims (5)
1. a kind of preparation method of water-solubility ginkgo exosper polysaccharide, characterized in that the preparation method comprises the following steps:
(1) gingko episperm impregnated by ethyl alcohol is placed in boil to mention and routinely adds water to cook 2-3 times in pot and filters respectively,
Filtrate is merged and is concentrated under reduced pressure into specific gravity as 1.21-1.30, ethyl alcohol is slowly added in concentrate, ethyl alcohol volume final concentration is made
In 60-80%(V/V), side edged quickly stirs, and supernatant is abandoned in centrifugation after standing, then is washed with the centrifugation of 98% or more ethyl alcohol of volumetric concentration
It washs 2 times or 3 times, obtains ethanol pellet；
(2) open planetary ball mill, be packed into large, medium and small abrading-ball in ball grinder, large, medium and small abrading-ball be respectively diameter 20mm,
The agate ball of 10mm and 6mm, large, medium and small abrading-ball quantity ratio are 1:(28-32): second as described in step (1) is added in (152-155)
Alcohol sediment；0.5-1.0h is shut down after operating to positive operation 0.8-1.2h in the operation of positive and negative alternate interval, positive and negative alternate interval, then
0.5-1.0h is shut down after inverted running 0.8-1.2h；Circulation 3-5 times is dried in vacuo or is freeze-dried after sieving, water can be obtained
Dissolubility gingko exocarp polysaccharide.
2. the preparation method of water-solubility ginkgo exosper polysaccharide according to claim 1, characterized in that institute in step (2)
The filling rate of large, medium and small abrading-ball is 40-60% in the ball grinder stated.
3. the preparation method of water-solubility ginkgo exosper polysaccharide according to claim 1, characterized in that institute in step (2)
The weight ratio for stating large, medium and small abrading-ball and ethanol pellet is (15-20): 1(W/W).
4. the preparation method of water-solubility ginkgo exosper polysaccharide according to claim 1, characterized in that institute in step (2)
The revolving speed of the positive operation or inverted running stated is 300-600rpm.
5. a kind of preparation method of water-solubility ginkgo exosper polysaccharide formulation, characterized in that obtain any one of claim 1-4
To water-solubility ginkgo exosper polysaccharide a small amount of excipients are added antitumor capsule preparations are made, or will be in claim 1-4
A small amount of corrigent is added in the water-solubility ginkgo exosper polysaccharide that any one obtains and antineoplastic oral liquid preparation is made in preservative.
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| CN103524638A (en) * | 2013-10-16 | 2014-01-22 | 江南大学 | Method for preparing soluble yeast glucan |
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