CN106589155B - A kind of preparation method and applications of the water-soluble polysaccharide from horsedung sea urchin gonad - Google Patents
A kind of preparation method and applications of the water-soluble polysaccharide from horsedung sea urchin gonad Download PDFInfo
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Abstract
The present invention relates to field of medicaments, and in particular to a kind of preparation method and applications of the water-soluble polysaccharide from horsedung sea urchin gonad.Specific method of the present invention is that high-temperature heating inactivates the enzyme in sea urchin gonad, then polysaccharide is extracted with lower than 60 DEG C water, extracting solution centrifugation, supernatant is concentrated under reduced pressure, successively use papain enzymolysis and Sevage method removing protein, aqueous solution is through dialysis, alcohol precipitation, dry that sea urchin total starches, total starches are isolated and purified to obtain two polysaccharide with gel column chromatography.Extracting temperature used in the present invention is low, does not influence polysaccharide structures and activity, and impurity is few in extracting solution, and post-processing approach is simple, significantly reduces production cost.Gained total starches character and solubility are good, easily prepared a variety of pharmaceutical preparations.Total starches and its two kinds of polysaccharide contained have preferable antitumor action, can be developed into the anti-tumor drug having no toxic side effect.
Description
Technical field
The present invention relates to the preparation methods and its antitumor application thereof of a kind of horsedung sea urchin gonad polysaccharide, belong to medical science
Field.
Background technique
Polysaccharide is a kind of high-molecular compound being polymerized by monosaccharide by glycosidic bond, is distributed widely in animals and plants and ocean
In organism, treat cardiovascular disease, antitumor, anti-hepatitis and in terms of there is unique bioactivity, therefore
It is very active always to the research of carbohydrate.
Due to particular surroundings such as with high salt, high pressure, low temperature, anoxics, unique structure and activity are often metabolized out in marine organisms body
Significant substance obtains various active polysaccharose substance from marine animal in recent years.Sea urchin is Echinodermata Echinoidea biology,
China coast sea area is widely distributed, and the economical sea urchin that can be eaten is mainly Anthocidaris crassispina (Anthocidaris
Crassispina), Strongylocentrotus nudus (Strongylocentrotus nudus) and horsedung sea urchin (Hemicentrotus
Several kinds such as pulcherrimus).Sea urchin shell spine is long as Chinese medicine usage history, have " resolving hard lump, resolving sputum detumescence,
Disappear scrofula subcutaneous nodule, and product phlegm is not changed, sternal rib pain " the effect of.The sea urchin gonad of yellow is reached maturity in sea urchin breeding period, is contained
The nutritional ingredients such as unsaturated fatty acid, amino acid and microelement abundant are a kind of advanced MNarine food webs.In recent years it sends out
Existing, the polysaccharide component that sea urchin gonad contains has reducing blood lipid, inhibits a variety of physiology such as gastric ulcer, enhancing human body immune function
Activity has very high medical value.
Polysaccharide is polymerized by monosaccharide, and as extent of polymerization increases, molecular weight increases, and water solubility decreases, and increases water
Solubility can be improved in temperature.The molecular weight of the polysaccharide found in sea urchin at present is mostly million grades, and molecule aggregation degree is high, therefore is extracted
Temperature setting is at 80 DEG C -100 DEG C, to increase polysaccharide solubility.Increasing temperature can be improved the extraction efficiency of polysaccharide, but in extracting solution
Impurity increase, will affect the purification effect of polysaccharide, also increase later-period purification process, cause the loss of polysaccharide.
In addition, polysaccharide is high-molecular compound, equally there is higher structure with protein, and closely related with its activity,
Long-time heating may change its higher structure, influence its biological activity.
The polysaccharide structures that metabolism generates in the sea urchin body of different germlines are different, and physicochemical property is different, of the present invention more
Sugar is that extraction and purifying obtain from horsedung sea urchin gonad.Optimize extracting method the experimental results showed that, under lower temperature
(40-50 DEG C) and 10 times of amount water reach optimum extraction efficiency (Fig. 1), this illustrates that polysaccharide contained by horsedung sea urchin is soluble easily in water, is lower than
Ordinary temperature can realize high efficiency extraction.
Parallel laboratory test compares 50 DEG C of parameters that polysaccharide is mentioned with 90 DEG C of water, and low temperature extraction has the advantage that
(1) extracting solution transparency is higher, and impurities are less, not only simplifies later-period purification process, but also low temperature extraction subtracts
Few energy consumption, can be greatly reduced production cost.
(2) the polysaccharide extraction liquid stability that low temperature obtains is more preferable, is long placed in no Precipitation.Much through same purification process institute
Sugar is white powder, soluble easily in water under room temperature, conducive to being further purified and biological activity screening experiment.
(3) good water solubility of polyose, stabilized aqueous solution not only have better bioavilability, after being also beneficial to
The design and development of phase drug different dosage forms.
(4) total starches of low temperature acquisition and its two polysaccharide obtained through column chromatography separating purification are to tumour cell BEL-
7402, Panc-28, U20S, HepG2, SGC7901 and THP-1 have different degrees of inhibiting effect (Fig. 3-Fig. 8), and 90 DEG C
It extracts obtained total starches and inhibitory activity is not shown to above-mentioned cell.
On the basis of soluble easily in water under finding polysaccharide room temperature contained by horsedung sea urchin, present system is screened and has been determined
A kind of efficient, inexpensive extraction and purification process of sea urchin polysaccharide, compared with high-temperature water mentions polysaccharide, present invention gained polysaccharide tool
There are better water-soluble, stability and anti-tumor activity.Rarely have both at home and abroad at present and inquires into what Extracting temperature influenced active polysaccharide
Research.
Summary of the invention
The present invention is intended to provide a kind of water-soluble polysaccharide extracted from horsedung sea urchin gonad, and also disclose described
The extraction preparation method of water-soluble polysaccharide, it is soluble easily in water under water-soluble polysaccharide room temperature of the invention, have good water-soluble, steady
Qualitative and anti-tumor activity；The present invention goes back while providing application of the water-soluble polysaccharide in anti-tumor drug.
For this purpose, the technical scheme adopted by the invention is that:
A kind of preparation method of the water-soluble polysaccharide from horsedung sea urchin gonad, is characterized in that, will be fresh
After the yellow gonad of horsedung sea urchin crushes, successively mentioned by acetone degreasing, short time high temperature water inactivator, water at low temperature, Aqueous extracts
Be concentrated under reduced pressure after merging, and after digested, de- albumen, then through dialysis, alcohol precipitation, it is dry after obtain total starches (HPP)；Total starches water
After molten, isolate and purify to obtain two polysaccharide (HPP-1 and HPP-2) through Sephacryl S-300 gel column chromatography.
The above extraction process is carried out by following specific steps:
A, the sea urchin gonad for taking fresh horsedung sea urchin takes off ester with acetone after crushing, obtains sea urchin Huang defatted seed flour；
B, defatted seed flour measures 90-100 DEG C of hot water treatment 10-30min of volume with 2 times；8 times of amount volumes are then added
It is extracted 2-6 hours in 20-60 DEG C of water, then 20-60 DEG C of water for measuring volume with 10-20 times extracts 2-3 times, combined extract is concentrated into
The half of total volume；
C, press defatted seed flour: protease=100:(4-8) mass ratio, to step b obtain concentrate in pawpaw is added
Protease digests 20-30h, uses Sevage method removing protein 10-15 times after heat inactivation, be concentrated under reduced pressure again with Rotary Evaporators；
D, for the polysaccharide solution that step c is obtained through flowing distilled water dialysis, 4 times of anhydrous second of amount are added in the aqueous after dialysis
Alcohol is centrifuged to obtain polysaccharide precipitation, is washed with dehydrated alcohol, dry horsedung sea urchin total starches HPP；
E, excrement sea urchin total starches obtained by step d are configured to the aqueous solution of 50mg/ml, with Sephacryl S-300 gel
Pillar layer separation, distilled water are mobile phase elution, and every 10ml collects a pipe, and phend-sulphuric acid measures every pipe polyoses content, according to
Elution curve merges eluent, and freeze-drying obtains two horsedung sea urchin polysaccharide, is respectively designated as HPP-1 and HPP-2.
More specifically, purification step of the invention are as follows:
A, the horsedung sea urchin of 6-9 month fresh acquisition is chosen, shell is peeled off, takes the sea urchin gonad of its yellow, use tissue
Refiner sufficiently crushes, and the 1-2 times of anhydrous propanone for measuring volume is added, and stirring at normal temperature takes off ester, repeats degreasing 7-10 times, and centrifugation is filled
After dividing drying, sea urchin Huang defatted seed flour is obtained；
B, the water of 2 times of amount volumes is added to defatted seed flour, is heated to 90-100 DEG C of maintenance 10-30min；Then it is added 8 times
The water for measuring volume maintains 20-60 DEG C of extraction 2-6h, then the water for measuring volume with 10-20 times maintains 20-60 DEG C to extract 2-3 times, merges
Extracting solution is concentrated into the half of total volume；
C, press defatted seed flour: protease=100:(4-8) mass ratio, to step b obtain concentrate in pawpaw is added
Protease is heated to 85-95 DEG C of inactivation 10-15min in 50-60 DEG C of enzymatic hydrolysis 20-26h, then by system, supernatant is collected by centrifugation
Liquid；The Sevage reagent (chloroform: n-butanol=4:1) of its volume fraction values 1/5 of supernatant extracts 10-15 times, and it is molten to abandon lower layer
Agent and albuminate, upper layer aqueous solution are concentrated under reduced pressure with Rotary Evaporators；
D, the polysaccharide solution that step c is obtained is packed into the bag filter that aperture is 3500Da, for 24 hours through the dialysis of flowing distilled water,
The dehydrated alcohol of 4 times of amount volumes is added into the polysaccharide solution after dialysis, stands 12h, precipitates from collecting, is washed with dehydrated alcohol
It washs 3-5 times, obtains horsedung sea urchin total starches HPP after dry；
E, excrement sea urchin total starches obtained by step d are configured to the aqueous solution of 50mg/ml, with Sephacryl S-300 gel
Pillar layer separation, distilled water are mobile phase elution, and every 10ml collects a pipe, and phend-sulphuric acid measures every pipe polyoses content, according to
Elution curve merges eluent, and freeze-drying obtains two horsedung sea urchin polysaccharide, is respectively designated as HPP-1 and HPP-2.
The sea urchin total starches purity of above method preparation is up to 90%, good water solubility under room temperature, and aqueous solution light transmittance is high.Through
Two polysaccharide and total starches that Sephacryl S-300 gel column chromatography isolates and purifies can inhibit the increasing of tumour cell
It grows, there is preferable antitumor action, can be developed into the anti-tumor drug having no toxic side effect.Extracting temperature used in the present invention
It is low, polysaccharide structures and activity are not influenced, impurity is few in extracting solution, and post-processing approach is simple, significantly reduces production cost.
The present invention sets 10 DEG C -100 DEG C of different Extracting temperatures in preferred extracting method, the results showed that, with temperature
Degree is promoted, and total starches recovery rate increases, but water temperature too high extraction is in a slight decrease instead, and optimum extraction effect is reached at 50 DEG C
(Fig. 1 a).It is preferred that material/water is also indicated that than the experimental result of, extraction time, the good water solubility (Fig. 1) of the polysaccharide is final according to just
Experiment is handed over to determine the water temperature in extracting method, extraction time, material/water than the optimal parameter (Tables 1 and 2) with extraction time.
1. Polyose extraction factor level table of table
The intuitive analytical table of 2. orthogonal experiment of table
Since Extracting temperature is low, to avoid the intracorporal enzyme hydrolysis of glycocalix sea urchin, first short time high temperature heating makes enzyme-deactivating,
Temperature is reduced again to extract.
Compared with 90 DEG C of extracting solution, the extracting solution color obtained under 50 DEG C of water temperatures is shallower, and gained polysaccharide is water-soluble after centrifugation
Liquid system is stablized, and no Precipitation is long placed in.Because of polysaccharide good water solubility, extract small with the total volume of water, it is only necessary to be concentrated into original volume
Half, successively using papain enzymolysis and sevage method removing binding protein and floating preteins, then through sufficiently thoroughly
Analysis removes small molecular weight impurity, and the higher total starches of purity (HPP) can be obtained.
Total starches are isolated and purified with Sephacryl S-300 gel column chromatography, can be by all polysaccharide by mobile phase of water
Column is eluted, if the elution speed of polysaccharide can be accelerated with the saline solution of gradient concentration.The result shows that horsedung sea urchin total starches contain
There are two types of polysaccharide, merge eluent respectively, are freeze-dried to obtain two purified polysaccharides HPP-1 and HPP-2.Under room temperature, HPP, HPP-1
It is soluble easily in water with HPP-2, tumor cell line BEL-7402, Panc-28, U20S, HepG2, SGC7901 and THP-1 are shown
Inhibiting effect out.
Detailed description of the invention
Fig. 1: horsedung sea urchin polysaccharide water extracts single factor test optimization experiment (a: the recovery rate under different water temperatures；B: difference is extracted
The recovery rate of time；C: different material/water ratio recovery rate；D: the recovery rate of different extraction times)；
Fig. 2: Sephacryl S-300 gel column chromatography elution curve；
Fig. 3: sea urchin polysaccharide is to the inhibiting effect of BEL-7402 cell strain, compared with the control group, P < 0.01 * P < 0.05, * *；
Fig. 4: sea urchin polysaccharide is to the inhibiting effect of panc-28 cell strain, compared with the control group, P < 0.01 * P < 0.05, * *；
Fig. 5: sea urchin polysaccharide is to the inhibiting effect of U20S cell strain, compared with the control group, P < 0.01 * P < 0.05, * *；
Fig. 6: sea urchin polysaccharide is to the inhibiting effect of HepG2 cell strain, compared with the control group, P < 0.01 * P < 0.05, * *；
Fig. 7: sea urchin polysaccharide is to the inhibiting effect of SGC7901 cell strain, compared with the control group, P < 0.01 * P < 0.05, * *；
Fig. 8: sea urchin polysaccharide is to the inhibiting effect of THP-1 cell strain, compared with the control group, P < 0.01 * P < 0.05, * *.
Specific embodiment
Several specific embodiments of the invention are given below, to elaborate to technical solution of the present invention.
Embodiment 1
The horsedung sea urchin of 6-9 month fresh acquisition is selected, shell is peeled off, takes the sea urchin gonad of its yellow, it is even with tissue
Pulp grinder sufficiently crushes, and the anhydrous propanone of 2 times of amount volumes is added, and stirring at normal temperature degreasing repeats degreasing 7 times, centrifugation, sufficiently after drying
Obtain defatted seed flour.
Defatted seed flour adds the water of 2 times of amount volumes, is heated to 90 DEG C -95 DEG C and maintains 10min, adds 8 times of amount water and maintains
20 DEG C of -30 DEG C of extraction 2h are extracted 2 times with 10 times of amount water in 20 DEG C -30 DEG C again, combined extract be concentrated into total volume two/
One.
By defatted seed flour: papain is added into concentrate for protease=100:4 (mass ratio), digests in 55 DEG C
For 24 hours, then by system 90 DEG C of inactivation 15min are warming up to, be centrifuged, collect supernatant.
The sevage reagent (chloroform: n-butanol=4:1) of its volume fraction 1/5 of supernatant extracts 10 times, and it is molten to abandon lower layer
Agent and albuminate, upper layer aqueous solution are concentrated under reduced pressure, and concentrate is packed into the bag filter that aperture is 3500Da, saturating with flowing distilled water
Analysis is for 24 hours.
4 times of amount dehydrated alcohols are added into the polysaccharide solution after dialysis, stands 12h, precipitating is collected by centrifugation, with anhydrous second
Alcohol washs 3 times, dry sea urchin total starches.
Sea urchin total starches are configured to the aqueous solution of 50mg/ml, with the good Sephacryl S-300 gel column of pre-balance
Chromatographic isolation makees mobile phase with distilled water.Every mono- pipe of 10ml receives eluent, measures every pipe eluent with phend-sulphuric acid
Polyoses content draws elution curve with pipe number and absorbance.Merge eluent according to curve, freeze-drying obtains two purifying seas
Gallbladder polysaccharide.
Embodiment 2
The horsedung sea urchin of 6-9 month fresh acquisition is selected, shell is peeled off, takes the sea urchin gonad of its yellow, it is even with tissue
Pulp grinder sufficiently crushes, and the anhydrous propanone of 1 times of amount volume is added, and stirring at normal temperature degreasing repeats degreasing 10 times, and centrifugation is sufficiently dry
Defatted seed flour is obtained afterwards.
Defatted seed flour adds the water of 2 times of amount volumes, is heated to 95 DEG C -100 DEG C and maintains 30min, adds 8 times of amount water and maintains
50 DEG C of -60 DEG C of extraction 6h are extracted 3 times with 20 times of amount water in 50 DEG C -60 DEG C again, combined extract be concentrated into total volume two/
One.
By defatted seed flour: papain is added into concentrate for protease=100:8 (mass ratio), digests in 55 DEG C
For 24 hours, then by system 90 DEG C of inactivation 15min are warming up to, be centrifuged, collect supernatant.
The sevage reagent (chloroform: n-butanol=4:1) of its volume fraction 1/5 of supernatant extracts 15 times, and it is molten to abandon lower layer
Agent and albuminate, upper layer aqueous solution are concentrated under reduced pressure, and concentrate is packed into the bag filter that aperture is 3500Da, saturating with flowing distilled water
Analysis is for 24 hours.
4 times of amount dehydrated alcohols are added into the polysaccharide solution after dialysis, stands 12h, precipitating is collected by centrifugation, with anhydrous second
Alcohol washs 5 times, dry sea urchin total starches.
Sea urchin total starches are configured to the aqueous solution of 50mg/ml, with the good Sephacryl S-300 gel column of pre-balance
Chromatographic isolation makees mobile phase with distilled water.Every mono- pipe of 10ml receives eluent, measures every pipe eluent with phend-sulphuric acid
Polyoses content draws elution curve with pipe number and absorbance.Merge eluent according to curve, freeze-drying obtains two purifying seas
Gallbladder polysaccharide.
Embodiment 3
The horsedung sea urchin 20Kg for taking acquire in Weihai in Shandong province July, strips sea urchin shell, obtains yellow sea urchin gonad 3.5Kg.
1.15kg is taken, is crushed with tissue pulverizer, interval is primary within every two minutes, and repetitive operation to sea urchin Huang is homogenate.To sea urchin
Acetone (2400mL) stirring at normal temperature 8h of two volumes is added in Huang homogenate, precipitating is collected by centrifugation.Precipitating repeats degreasing 8 times extremely
Acetone layer is colourless, the dry defatted seed flour 220g that weighs to obtain.450ml water is added, is heated to 90 DEG C of maintenance 10min, adds
1.8L water maintains 50 DEG C of extraction 4h.Centrifugation, precipitating are extracted 2 times with 2.2L water again.After extracting solution merges, it is concentrated under reduced pressure into 3.3L,
1.9g papain is added to digest for 24 hours in 55 DEG C, is then heated to 90 DEG C of inactivation 15min, supernatant is collected by centrifugation.To supernatant
Sevage reagent (Lv Fang ︰ n-butanol=4 ︰ 1 of its volume fraction 1/5 are added in liquid) extraction, discard lower liquid and denaturation egg
It is white, repeat the operation 10 times.Upper layer polysaccharide solution is concentrated under reduced pressure into 1.5L, is packed into the bag filter that aperture is 3500Da,
For 24 hours with the dialysis of flowing distilled water.6L dehydrated alcohol is added into the polysaccharide solution after dialysis, stands 12h, centrifugation, precipitating is used
Dehydrated alcohol washs 3 times, sufficiently dry white sea urchin total starches powder 22g.Phend-sulphuric acid surveys its polyoses content
89%.
Embodiment 4
The horsedung sea urchin 50Kg for taking August part to acquire in Weihai in Shandong province, strips sea urchin shell, obtains yellow sea urchin gonad
9.53Kg is carried out crushing to obtain homogenate with tissue pulverizer.With anhydrous propanone degreasing 8 times of two volumes, defatted seed flour is obtained
1.7Kg.Extracting degreasing powder 230g is added 500ml water in 90 DEG C of heating 30min, adds 3.0L water and maintain 50 DEG C of extraction 6h, from
The heart.Precipitating is extracted 2 times with 3.5L water again.Combined extract is concentrated under reduced pressure into 4.5L.Its volume fraction 1/ is added into supernatant
5 sevage reagent (Lv Fang ︰ n-butanol=4 ︰ 1) extraction, lower liquid and albuminate are discarded, is repeated the operation 11 times.On
Layer polysaccharide solution is concentrated under reduced pressure into 1.5L, is packed into the bag filter that aperture is 3500Da, for 24 hours with the dialysis of flowing distilled water.To
6.0L dehydrated alcohol is added in polysaccharide solution after dialysis, stands 12h, centrifugation, precipitating washs 5 times with dehydrated alcohol, sufficiently
Dry white sea urchin total starches powder 32.5g.It is 91% that phend-sulphuric acid, which surveys its polyoses content,.
It takes 1g total starches 20ml water to dissolve, the good Sephacryl S-300 gel column of pre-balance is added, with distilled water
For mobile phase elution, every 10ml receives 1 pipe, and phend-sulphuric acid measures polyoses content in every pipe, merges the 7th pipe respectively to the 15th
Pipe eluent, the 16th pipe to the 24th pipe eluent are freeze-dried to obtain HPP-1 (630mg) and HPP-2 (50mg) (Fig. 2).
Two, cell growth inhibition assay
1. the recovery of tumor cell line is cultivated and is frozen
(1) cell recovery: by cryovial after taking out in liquid nitrogen container or in -80 DEG C of refrigerators, being put into rapidly in 37 DEG C of water-baths,
When the state of mixture of ice and water is presented in cell, cryopreservation tube is taken out from water-bath, after 75% alcohol sterilization, is moved into sterile
In super-clean bench.It is added in the centrifuge tube added with culture solution in advance, mixes after cell in cryopreservation tube is sucked out, 1000rpm centrifugation
5min.Liquid is discarded supernatant, lower confluent monolayer cells is collected, is washed 2 times with culture solution, be added in the culture bottle equipped with appropriate culture solution, be put into
37 DEG C, cultivate in the constant incubator of 5%CO2.The growth conditions of daily observation cell, it is laggard to 80%-90% to cell length
Row secondary culture.(preparation of culture solution: the culture medium of type is suitble to add 10% fetal calf serum, 1% penicillin streptomycin mixing
Liquid)
(2) passage of cell: culture bottle is taken out from CO2 incubator and is put into super-clean bench, attached cell directly discards
Culture solution is digested after PBS buffer solution flushing twice is added with pancreatin, is terminated and is digested with culture solution, and sub-bottle culture is carried out.37℃,
It is cultivated in the constant incubator of 5%CO2.
2.MTT method detects influence of the polysaccharide to tumor cell proliferation
Mtt assay detects polysaccharide to the inhibiting effect of variety classes growth of tumour cell:
Cell passage after, with 5 × 103/hole be added 96 orifice plates in, every hole 200l, add sterile measuring samples (0,
32,64,128,256,512,1024-g/ml) in 37 DEG C, the culture of 5%CO2 incubator for 24 hours, then suck supernatant.Finally use
Mtt assay is detected: the MTT dyeing liquor (5mg/ml) of 20l is added in every hole, abandons supernatant after reacting 4h, DMSO 150l is added, keeps away
With the light absorption value at microplate reader detection 570nm after light concussion dissolution 10min, cell survival rate is calculated.
3. statistical procedures
As a result the data in show that application software SPSS 13.0 carries out data processing in the form of average value ± SD, guarantee
3 experiments repeat.Multi-group data is compared using one-way analysis of variance method, the difference between control group and each experimental group passes through t
Inspection compares and analyzes, and as P < 0.01, control group and experimental group difference are extremely significant, control group and experiment as P < 0.05
Group has significant difference.
Claims (2)
1. a kind of preparation method of the water-soluble polysaccharide from horsedung sea urchin gonad, is characterized in that
It is carried out by following specific steps:
A, the sea urchin gonad for taking fresh horsedung sea urchin uses acetone degreasing after crushing, obtain sea urchin Huang defatted seed flour；
B, defatted seed flour measures 90-100 DEG C of hot water treatment 10-30min of volume with 2 times；The 20-60 of 8 times of amount volumes is then added
It is extracted 2-6 hours in DEG C water, then 20-60 DEG C of water for measuring volume with 10-20 times extracts 2-3 times, combined extract is concentrated into totality
Long-pending half；
C, papain is added in the concentrate obtained to step b, after enzymatic hydrolysis, use Sevage method removing protein after heat inactivation again
It 10-15 times, is concentrated under reduced pressure with Rotary Evaporators；
D, for the polysaccharide solution that step c is obtained through flowing distilled water dialysis, 4 times of amount dehydrated alcohols are added in the aqueous after dialysis, from
Gains in depth of comprehension polysaccharide precipitation is washed, dry horsedung sea urchin total starches HPP with dehydrated alcohol；
E, horsedung sea urchin total starches obtained by step d are configured to the aqueous solution of 50mg/ml, with SephacrylS-300 gel column color
Spectrum separation, distilled water are mobile phase elution, and every 10ml collects a pipe, and phend-sulphuric acid measures every pipe polyoses content, according to elution
Curve merges eluent, and freeze-drying obtains two horsedung sea urchin polysaccharide, is respectively designated as HPP-1 and HPP-2；
In step a, after sea urchin gonad is sufficiently crushed using tissue refiner, the 1-2 times of anhydrous propanone for measuring volume, room temperature is added
Degreasing is stirred, is repeated degreasing 7-10 times；
In step b, by defatted seed flour: protease=100:(4-8) mass ratio papain is added into concentrate, digest
20-30h。
2. application of the water-soluble polysaccharide that preparation method as described in claim 1 obtains in anti-tumor drug.
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| CN101974097B (en) * | 2010-10-29 | 2012-05-23 | 中国药科大学 | Scale separation and purification method of pentaxanthin amylose |
| CN104231105B (en) * | 2014-09-28 | 2016-06-22 | 浙江工业大学 | The preparation method of a kind of sea anemone polysaccharide and antitumor application thereof |
| CN104987434B (en) * | 2015-07-15 | 2017-10-03 | 中国科学院烟台海岸带研究所 | A kind of method that water at low temperature extracts inulin in use |
| CN105777924B (en) * | 2016-03-04 | 2018-05-29 | 浙江大学 | A kind of Phellinus fructification active anticancer polysaccharide PBPP and preparation method thereof |
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