CN105175567B - The extracting method of crocodile blood polysaccharide and its application in lung cancer therapy - Google Patents
The extracting method of crocodile blood polysaccharide and its application in lung cancer therapy Download PDFInfo
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Abstract
Extracting method of the present invention for crocodile blood polysaccharide and its application in lung cancer therapy, the crocodile blood polysaccharide of the present invention is the polysaccharide component purified first from crocodile blood blood meal, the crocodile blood polysaccharide and the regulation of immunologic function simultaneously, the identification of cell and cell, the transport of intercellular substance, the Clinics and Practices etc. of cancer suffer from close relationship, have antiviral, antitumor and immunoregulation effect；Meanwhile the crocodile blood polysaccharide component pharmaceutically can be with acid, alkali, salt, hydrate or ester；And other auxiliary materials combine and prepare piece agent, capsule, oral liquid, emulsion, parenteral solution or combinations thereof, are used as treating cancer or tumour medicine, are advantageous to the research of antitumor activity.
Description
Technical field
The invention belongs to medicinal chemistry art, and in particular in crocodile blood freeze-dried powder the extracting method of crocodile blood polysaccharide and its
Application in lung cancer therapy.
Background technology
Crocodile is most ancient reptile, has been survived on earth more than 200,000,000 3 thousand ten thousand years, thus has " living fossil "
Title.
Traditional Chinese medical science ancient book is recorded, and crocodile blood has clearing heat and detoxicating, anti-inflammatory, treating tumor and other effects.In recent years, foreign scholar is to crocodile
The effect of fish blood, is studied, and existing crocodile blood has the report that antibacterial, antiviral etc. act on, and helps to be lifted
The primary immune response ability of experimental animal under pathological conditions.Modern study shows that crocodile blood has two big significant functions special
Point：Crocodile whole blood, hemoglobin, serum, blood plasma and leucocyte have antibacterium, fungi, virus and active anticancer.Another is notable
Functional characteristics is that the oxygen carrying content of crocodile hemoglobin exceedes more than 100 times of other animals, and this special hemoglobin enters
The blood oxygen carrying content of human body can be improved after human body and human body hemoglobin synthesis, enough oxygen is sent to each of brain and body
Position, strengthen vigor and the anti-mutation ability of body fluid and cell.
Why crocodile blood has the effect of so many, it is believed that there may be very big relation with the polysaccharide in blood, because
Show for numerous studies, the regulation of polysaccharide and immunologic function, the identification of cell and cell, the transport of intercellular substance, cancer
Clinics and Practices etc. suffer from close relationship, have antiviral, antitumor and immunoregulation effect.Therefore we are by crocodile blood
In Polyose extraction come out, carried out the research of antitumor activity.
The content of the invention
In view of this, the present invention is intended to provide going out a kind of extracting method of crocodile blood polysaccharide and its answering in lung cancer therapy
With to solve problems of the prior art.
To reach above-mentioned purpose, the technical proposal of the invention is realized in this way：
Crocodile blood polysaccharide, the crocodile blood polysaccharide are at least one more to isolate and purify containing for rear gained from crocodile blood blood meal
The mixture of saccharic composition.
Further, the crocodile blood polysaccharide is the application in preparing treatment or suppressing cancer, tumour medicine.
Further, the crocodile blood polysaccharide is answering in the medicine for suppressing lung cancer growth and transfer is prepared
With.
Further, the medicine can be prepared in piece agent, capsule, oral liquid, emulsion and parenteral solution at least
It is a kind of.
Further, the crocodile blood polysaccharide includes 1~3 kind of polysaccharide component.
Relative to prior art, crocodile blood polysaccharide of the present invention has the advantage that：
The crocodile blood polysaccharide is the polysaccharide component that is purified first from crocodile blood blood meal, at the same the crocodile blood polysaccharide with
The regulation of immunologic function, the identification of cell and cell, the transport of intercellular substance, the Clinics and Practices etc. of cancer suffer from closely
Relation, there is antiviral, antitumor and immunoregulation effect；Meanwhile the crocodile blood polysaccharide component pharmaceutically can with acid,
Alkali, salt, hydrate or ester；And other auxiliary materials combine prepare piece agent, capsule, oral liquid, emulsion, parenteral solution or it
Combination, used as treating cancer or tumour medicine, be advantageous to the research of antitumor activity.
The present invention also provides a kind of preparation method of crocodile blood polysaccharide crude extract, and the preparation method is to freeze crocodile blood
Dry powder presses solid-liquid ratio 1：(20~40) add in distilled water, the break process in ultrasonic cell-break machine；It is then centrifuged for 3~4
Secondary merging supernatant, adds the absolute ethyl alcohol left undisturbed overnight of 3~4 times of volumes, then centrifuges, and abandons supernatant, and precipitation is used appropriate anhydrous
Ethanol cleans 2~3 times, after supernatant is abandoned in centrifugation, with the piping and druming of appropriate ether into powdered, obtains crocodile blood polysaccharide crude extract.
Further, the distilled water is distilled water.
Further, the centrifugation 10 that the centrifugal process is 4000~4600rpm/min of holding under 4 DEG C of environment~
20min。
Relative to prior art, the preparation method of crocodile blood polysaccharide crude extract of the present invention has the advantage that：
The crocodile blood polysaccharide crude extract that the present invention is obtained using the heavy alcohol extracting method processing crocodile blood freeze-dried powder of water, can be accurate
Show that the crocodile blood polysaccharide in crocodile blood has and significantly improve anti-tumor function, and efficiently control the growth of tumour cell.
In addition, the present invention also provides a kind of one pack system method for purifying and separating of crocodile blood polysaccharide, wherein, the purifying
Step obtains polysaccharide solution for crocodile blood polysaccharide crude extract is dissolved in distilled water, by 1:4 volume ratio is in polysaccharide solution
Chloroform-butanol solution is added, 20~30min is acutely vibrated after mixing, the denatured protein in intermediate layer is removed after centrifugation, is repeated
For several times untill intermediate layer separates out without protein, lower floor's polysaccharide solution is then separated out, is placed in bag filter, dialyse 48~72h, no
It is disconnected to change water, to remove the small molecular weight impurities such as oligosaccharide, then concentrated, be finally freeze-dried the crocodile blood polysaccharide purified
Powder；
The separating step is further separated for the crocodile blood polysaccharide powder of purifying is crossed into sephadex column, is collected
Go out peak position and accordingly elute polysaccharide solution in pipe, and merge the polysaccharide under unified peak type, obtain 1~3 kind of polysaccharide component.
Further, the distilled water is distilled water, and the chloroform-butanol solution is that chloroform presses 4 with n-butanol:1 body
Product ratio mixes.
Relative to prior art, the one pack system method for purifying and separating of crocodile blood polysaccharide of the present invention has following excellent
Gesture：
The crocodile blood polysaccharide that the inventive method obtains is used as the crocodile blood polysaccharide after purifies and separates, contains polysaccharide in 1~3
Component, can be widely used as medicine, the raw material of functional food, and therapeutic effect is more notable.Meanwhile preparation method work provided by the invention
Skill is simple, is easier to realize industrialization production.
Brief description of the drawings
The accompanying drawing for forming the part of the present invention is used for providing a further understanding of the present invention, schematic reality of the invention
Apply example and its illustrate to be used to explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the elution song that the polysaccharide crude extract extracted in crocodile blood dry powder is crossed after Sephadex G-200 posts
Line；
Fig. 2 shows suppression of three kinds of different components to NCI-H446 proliferation of lung cancer cells in MTT detection crocodile blood polysaccharide
Effect；
Fig. 3 shows suppression of three kinds of different components to NCI-H460 proliferation of lung cancer cells in MTT detection crocodile blood polysaccharide
Effect；
Fig. 4 shows that three kinds of different components are to the inhibitory action of A549 proliferation of lung cancer cells in MTT detection crocodile blood polysaccharide；
Fig. 5 shows that component ABPSI is to the inhibitory action of NCI-H446 cell migrations in crocodile blood polysaccharide；
Fig. 6 shows that component ABPSII is to the inhibitory action of NCI-H446 cell migrations in crocodile blood polysaccharide；
Fig. 7 shows that component ABPSIII is to the inhibitory action of NCI-H446 cell migrations in crocodile blood polysaccharide；
Fig. 8 shows that component ABPSI is to the inhibitory action of NCI-H460 cell migrations in crocodile blood polysaccharide；
Fig. 9 shows that component ABPSII is to the inhibitory action of NCI-H460 cell migrations in crocodile blood polysaccharide；
Figure 10 shows that component ABPSIII is to the inhibitory action of NCI-H460 cell migrations in crocodile blood polysaccharide；
Figure 11 shows that component ABPSI is to the inhibitory action of A549 cell migrations in crocodile blood polysaccharide；
Figure 12 shows that component ABPSII is to the inhibitory action of A549 cell migrations in crocodile blood polysaccharide；
Figure 13 shows that component ABPSIII is to the inhibitory action of A549 cell migrations in crocodile blood polysaccharide.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is necessary to illustrate, described embodiment is only part of the embodiment of the present invention, rather than whole implementation
Example.Based on the embodiment in the present invention, every other embodiment that those of ordinary skill in the art are obtained belongs to the present invention
The scope of protection.
Wherein, in embodiment, test material used and its source include：
1. cell line
NCI-H446 (human small cell lung carcinoma cell)：Purchased from Nanjing KaiJi Biology Science Development Co., Ltd；
NCI-H460 (human large cell lung cancer cell):Purchased from Nanjing KaiJi Biology Science Development Co., Ltd；
A549 (Lu-csf-1):Purchased from Nanjing KaiJi Biology Science Development Co., Ltd；
Experimental agents and reagent
Crocodile blood freeze-dried powder：It is powdered, provided by Beijing Bai Shengkang bio tech ltd.
Tetramethyl azo azoles salt (MTT)：Shanghai Sheng Gong bioengineering Co., Ltd, article No.：TB0799-1g, lot number：
XP1008B3012J, rank：Analyze pure, specification 1g.
Dimethyl sulfoxide (DMSO) (DMSO)：Purchased from Shanghai Sheng Gong bioengineering Co., Ltd, article No.：DN3039A, lot number：
SJ0731S4013J, rank：Pure, specification 500mL is analyzed, dissolves purple crystal for mtt assay.
Dimethyl sulfoxide (DMSO) (DMSO) cell culture level：Purchased from Beijing Suo Laibao Science and Technology Ltd, article No.：D8370-500,
Lot number：2P005970, rank：Cell culture level, specification 500mL, prepared for cell cryopreservation, and medicine.
The culture mediums of RPMI 1640：Purchased from Thermo Fisher Scientific Inc., lot number：NYG0920, specification：500mL.
Trypsase：Purchased from Shanghai Sheng Gong bioengineering Co., Ltd, article No.：T0458-10, lot number：0301C314, level
Not：USP, specification：10g.
3. key instrument
AKTA systems：GE companies, model：UPC-900
Biohazard Safety Equipment：Thailand of Spain thing reaches (Telstar), model：Bio II A.
CO2Incubator：Shi Doukai instrument and equipments (Shanghai) Co., Ltd., model：STIK IL-161HI.
Inverted phase contrast microscope：Producer：Olympus, model：CKX41.
Electronic balance：Shanghai Ao Haosi Instrument Ltd., model：AR2202CN；
Tabletop refrigerated centrifuge：Thermo Fischer Scient Inc., model:ST16R；
Ultrasonic cell-break machine：Sai Mo flies generation that (Thermo Fisher), model：SCIENTZ-IID；
Rotary Evaporators：Shanghai Yarong Biochemical Instrument Plant, model：RE-5205；
4 DEG C of refrigerators：Qingdao HaiEr Co., Ltd, model：BCD-192KJ；
Vortex vortex mixer:Its woods Bel's instrument manufacturing Co., Ltd of Haimen City, model：QZ-861.
4. the compound method of agents useful for same includes：
Tetramethyl azo azoles salt (MTT) liquid storage：MTT powder 1.0g are taken, are dissolved in 200ml phosphate buffers (PBS), it is dense eventually
Spend for 5mg/ml, with 0.22 μm of membrane filtration to remove the bacterium in solution, dispense, -20 DEG C are kept in dark place.Wherein, solution
Preparation and use all should operate in sterile biological safety cabinet.
Describe the present invention in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
The preparation method of the crocodile blood polysaccharide of embodiment 1
1st, the preparation of crocodile blood polysaccharide crude extract
Crocodile blood freeze-dried powder 4.0g is weighed, by solid-liquid ratio 1：20 add corresponding distilled water (ddH2O), in supersonic cell
In disintegrating machine, 5min is crushed；4600rpm/min in 4 DEG C of centrifuges, 10min is centrifuged, supernatant is taken, is repeated 3 times, merge supernatant
Liquid, the absolute ethyl alcohol left undisturbed overnight of 3 times of volumes is added, 4600rpm/min, after centrifuging 10min, abandoning supernatant, precipitation is used suitable
Washes of absolute alcohol 3 times is measured, after supernatant is abandoned in centrifugation, with the piping and druming of appropriate ether into powdered, obtains crocodile blood polysaccharide coarse extraction
Thing.
2nd, crocodile blood polysaccharide different component isolates and purifies
The characteristics of being denatured according to protein in chloroform equal solvent, chloroform and n-butanol are pressed 4:After 1 volume mixture, it is placed in
Preserved in brown bottle.Crocodile blood polysaccharide crude extract is dissolved in ddH2O, by 1:4 add chloroform-n-butanol in polysaccharide solution
Solution, after mixing, 20min is acutely vibrated, the denatured protein in intermediate layer is removed after centrifugation, is repeated several times to intermediate layer without albumen
Untill matter separates out, lower floor's polysaccharide solution is then separated out, is placed in bag filter, dialyse 72h, water is constantly changed, to remove oligosaccharide etc.
Small molecular weight impurity, then concentrated, final freeze-drying is polysaccharide powder.
Polysaccharide uses ddH after deproteinized, removal of impurities2Crocodile blood polysaccharide is configured to polysaccharide solution by O, using isolating and purifying
System (AKTA systems) is crossed Sephadex G-200 sephadex columns and further separated, and ddH is used before loading2O is flat first
Weigh pillar, and system uses ddH automatically after loading pin loading2O carries out the elution of polysaccharide, and system exists according to molecular size range difference
Appearance at different elution volumes, collect out peak position and accordingly elute polysaccharide solution in pipe, and merge more under unified peak type
Sugar.
As shown in figure 1, the crude extract of crocodile blood polysaccharide is distinguished after Sephadex G-200 molecular sieves isolate and purify
There is top at 5.6,11.4,17.3 elution volumes, so that the polysaccharide component of isolated three kinds of different molecular weight sizes,
It is designated as respectively：ABPSI、ABPSII、ABPSIII.
The influence that the different component of the crocodile blood polysaccharide of embodiment 2 is bred to human lung carcinoma cell
Experimental method is MTT colorimetric methods：Cleaning Principle can make exogenous for the succinate dehydrogenase in living cells mitochondria
MTT is reduced to the bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) of water-insoluble and is deposited in cell, and dead cell is without this function.Two
Methyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, and its absorbance value is determined at 490nm wavelength with enzyme-linked immunosorbent assay instrument,
Living cells quantity can be reflected indirectly.In the range of certain cell number, it is directly proportional to cell number that first a ceremonial jade-ladle, used in libation crystallizes the amount to be formed.This method
Be widely used in the Activity determinations of some bioactie agents, large-scale screening anti-tumor medicine, cell toxicity test and
Tumor radiosensitivity measure etc..
Method and step is as follows：
(1) cell recovery and culture
The human lung carcinoma cell frozen is taken out from liquid nitrogen, put into 37 DEG C of water-baths melts cell immediately.Biology peace
Cell suspension is drawn onto in the centrifuge tube equipped with appropriate culture medium in full cabinet, 800rpm/min is centrifuged 5 minutes；Supernatant is abandoned, is used
1mL culture medium suspension cells, it is drawn onto in the Tissue Culture Dish equipped with appropriate culture medium, cell is placed in 37 DEG C, 5%CO2, saturation
Cultivated under conditions of humidity.Reach when cell when 80%-90% contacts converge and passed on, 0.2% pancreatin is digested to unicellular
Suspension, 800rpm/min are centrifuged 5 minutes；Supernatant is abandoned, adds 1-2mL culture medium suspension cells, cell is passed to 2-3 equipped with appropriate
In the culture dish of culture medium, continue to cultivate.
(2) inhibitory action of mtt assay measure medicine cell growth
By the attached cell in exponential phase after Trypsin Induced, individual cells are dispersed into, and make its suspension
In corresponding cell culture medium.Cell is inoculated on 96 well culture plates, per the μ L cell suspensions of hole 100,3000cells/ holes.Will
Above-mentioned 96 well culture plate is placed in 37 DEG C, is incubated overnight 24 hours in carbon dioxide (5%) incubator, makes cell attachment.
After 24 hours cell completely it is adherent after, discard nutrient solution, test group is separately added into containing various concentrations
ABPSI, ABPSII, ABPSIII cell culture fluid, and set and be not added with the control wells (crocodile that medicine only adds relative medicine solvent
Blood polysaccharide solvent is ddH2O), while setting only adds the not celliferous zeroing hole of culture medium.Every group sets 6 parallel holes, then will
96 orifice plates are placed in 37 DEG C, are cultivated 48 hours in carbon dioxide (5%) incubator.
After 48 hours, 25 μ L MTT (concentration 5mg/mL) are added per hole, continue to be incubated 4h.Then nutrient solution is gently inhaled
Go out, 150 μ L DMSO are added per hole and make solvent dissolving, with the absorbance at ELIASA measure 570nm after dissolving.
(3) data analysis
The survival rate and inhibiting rate of cell are calculated, and calculates half-inhibition concentration IC50 of the medicine to cell.Survival rate %
=(experimental group OD values-zeroing hole OD values)/(negative model group OD values-zeroing hole OD values) × 100%；(1- is deposited inhibiting rate %=
Motility rate) × 100%.The calculating of IC50 values is carried out with the softwares of Graphpad Prism 5；Measurement result is shown in Table 1.
Inhibitory action (IC50 value) of the crocodile blood polysaccharide different component of table 1 to lung carcinoma cell
| Group | NCI-H446 | NCI-H460 | A549 |
| ABPSI | 1.559 | 2.944 | 2.013 |
| ABPSII | 2.092 | 2.397 | 1.883 |
| ABPSIII | 2.534 | 1.147 | 1.935 |
From table 1 it follows that crocodile blood polysaccharide has obvious Inhibit proliferaton to human lung carcinoma cell in low concentration
Effect.
As in Figure 2-4, be the crocodile blood polysaccharide medicine of various concentrations different component that is detected in embodiment 2 to it is a variety of not
The inhibitory action curve map of the growth of same human lung carcinoma cell, can draw concentration with being grown in human lung carcinoma cell from figure
Obvious inhibitory action is presented during low concentration, with the increase of crocodile blood polysaccharide concentration, to the inhibitory action of human lung carcinoma cell
Gradually enhancing, and obvious concentration dependent is presented.
The influence that the different component of the crocodile blood polysaccharide of embodiment 3 migrates to human lung carcinoma cell
Method and step is as follows：
(1) cell recovery and culture
The human lung carcinoma cell frozen is taken out from liquid nitrogen, put into 37 DEG C of water-baths melts cell immediately.Biology peace
Cell suspension is drawn onto in the centrifuge tube equipped with appropriate culture medium in full cabinet, 800rpm/min is centrifuged 5 minutes；Supernatant is abandoned, is used
1mL culture medium suspension cells, it is drawn onto in the Tissue Culture Dish equipped with appropriate culture medium, cell is placed in 37 DEG C, 5%CO2, saturation
Cultivated under conditions of humidity.Reach when cell when 80%-90% contacts converge and passed on, 0.2% pancreatin is digested to unicellular
Suspension, 800rpm/min are centrifuged 5 minutes；Supernatant is abandoned, adds 1-2mL culture medium suspension cells, cell is passed to 2-3 equipped with appropriate
In the culture dish of culture medium, continue to cultivate.
(2) plating cells
It is uniform to draw horizontal line first with marker pens and ruler at the plate bottom of 24 orifice plates, via is crossed, per two, hole horizontal line,
Spacing 0.2-0.5cm.By the attached cell in exponential phase after Trypsin Induced, individual cells are dispersed into, and make
It is suspended in corresponding cell culture medium.Cell is inoculated on 24 well culture plates, per the μ L cell suspensions of hole 500,
500000cells/ holes.Above-mentioned 24 well culture plate is placed in 37 DEG C, is incubated overnight 24 hours in carbon dioxide (5%) incubator,
Make cell attachment.
After 24 hour cells completely it is adherent cover with after, the horizontal line cut with 20 μ L pipette tips perpendicular to plate bottom, pipette tips are vertical,
It can not tilt.After PBS 3 times, test group is separately added into the cell of ABPSI, ABPSII, ABPSIII containing various concentrations
Serum free medium, blank control group add the serum free medium of equal volume.It is glimmering in being inverted in 0h, 24h, 48h respectively
Viewed under light microscopy simultaneously photographs to record.
(3) data analysis
The calculating of inhibition of metastasis rate is carried out with the softwares of Graphpad Prism 5.
(image in Fig. 5-13 is Y mobility-X time graphs, represents 0,1,2 μm of ol tri- respectively as shown in figures 5-13
Influence of the dosage and administration time of individual various concentrations to mobility), it is different to three kinds to illustrate three kinds of crocodile blood polysaccharide
The inhibitory action of human lung carcinoma cell travel motion, it is illustrated that there is different degrees of three kinds of crocodile blood polysaccharide after middle display effect 48h
Suppress the effect of lung carcinoma cell migration.
Wherein, as illustrated in figs. 5-7, the migration acted on for three kinds of crocodile blood polysaccharide components NCI-H446 cellular migration inhibitions
Inhibiting rate, can as seen from the figure, and polysaccharide component concentration is higher, and the suppression migration to cancer cell is more obvious.
As seen in figs. 8-10, the inhibition of metastasis that three kinds of crocodile blood polysaccharide components are acted on NCI-H460 cellular migration inhibitions
Rate, can as seen from the figure, and polysaccharide component concentration is higher, and the suppression migration to cancer cell is more obvious.
As figs 11-13, the inhibition of metastasis rate that three kinds of crocodile blood polysaccharide components are acted on A549 cellular migration inhibitions, can
So that as seen from the figure, polysaccharide component concentration is higher, the suppression migration to cancer cell is more obvious.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (9)
1. crocodile blood polysaccharide, it is characterised in that：The crocodile blood polysaccharide is that containing for rear gained is isolated and purified from crocodile blood blood meal
The mixture of at least one polysaccharide component, and the crocodile blood polysaccharide has treatment or suppresses cancer, the activity of tumour.
2. crocodile blood polysaccharide according to claim 1, it is characterised in that：The crocodile blood polysaccharide, which has, suppresses lung cancer growth
And the activity of transfer.
3. crocodile blood polysaccharide according to claim 1 or 2, it is characterised in that：The crocodile blood polysaccharide is prepared in application
At least one of piece agent, capsule, oral liquid, emulsion and parenteral solution.
4. crocodile blood polysaccharide according to claim 1, it is characterised in that：The crocodile blood polysaccharide includes 1~3 kind of polysaccharide group
Point.
5. the preparation method of crocodile blood polysaccharide crude extract, it is characterised in that：The preparation method is to press crocodile blood freeze-dried powder
Solid-liquid ratio 1：(20~40) add in distilled water, the break process in ultrasonic cell-break machine；It is then centrifuged for 3~4 merging
Supernatant, adds the absolute ethyl alcohol left undisturbed overnight of 3~4 times of volumes, then centrifuges, and abandons supernatant, and precipitation is clear with appropriate absolute ethyl alcohol
Wash 2~3 times, after supernatant is abandoned in centrifugation, with the piping and druming of appropriate ether into powdered, obtain crocodile blood polysaccharide crude extract.
6. the preparation method of crocodile blood polysaccharide crude extract according to claim 5, it is characterised in that：The distilled water is
Distilled water.
7. the preparation method of crocodile blood polysaccharide crude extract according to claim 5, it is characterised in that：The centrifugal process
To keep 4000~4600r/min to centrifuge 10~20min under 4 DEG C of environment.
8. the one pack system method for purifying and separating of crocodile blood polysaccharide, it is characterised in that：The purification step is that crocodile blood polysaccharide is thick
Extract, which is dissolved in distilled water, obtains polysaccharide solution, by 1:4 volume ratio adds chloroform-butanol solution in polysaccharide solution,
20~30min is acutely vibrated after mixing, the denatured protein in intermediate layer is removed after centrifugation, is repeated several times to intermediate layer without protein
Untill precipitation, lower floor's polysaccharide solution is then separated out, is placed in bag filter, dialyse 48-72h, constantly changes water, small to remove oligosaccharide
Molecular impurity, then concentrated, be finally freeze-dried the crocodile blood polysaccharide powder purified；
The separating step is further separated for the crocodile blood polysaccharide powder of purifying is crossed into sephadex column, collects appearance
Position accordingly elutes the polysaccharide solution in pipe, and merges the polysaccharide under unified peak type, obtains 1~3 kind of polysaccharide component.
9. according to the method for claim 8, it is characterised in that：The distilled water is distilled water, and the chloroform-n-butanol is molten
Liquid is that chloroform presses 4 with n-butanol:1 volume ratio mixes.
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