CN106986948A - A kind of Pacific oyster neutral polysaccharide and its preparation method and application - Google Patents

A kind of Pacific oyster neutral polysaccharide and its preparation method and application Download PDF

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CN106986948A
CN106986948A CN201710144246.XA CN201710144246A CN106986948A CN 106986948 A CN106986948 A CN 106986948A CN 201710144246 A CN201710144246 A CN 201710144246A CN 106986948 A CN106986948 A CN 106986948A
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oyster
polysaccharide
neutral polysaccharide
neutral
crude extract
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CN106986948B (en
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刘杨
夏李轩
张杰良
肖湘
钟名其
伦镜盛
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Shantou University
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof

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Abstract

The invention discloses a kind of preparation method of Pacific oyster neutral polysaccharide, comprise the following steps：（1）By Pacific oyster dry powder by acetone degreasing, degreasing ostreae testa pulverata is obtained after drying；（2）Degreasing ostreae testa pulverata is soluble in water, handled with ultrasonic-microwave extraction instrument, by the solution centrifugal after processing, supernatant is concentrated by evaporation, and obtains oyster polysaccharide crude extract；（3）Oyster polysaccharide crude extract is added into ethanol (NH4)2SO4Double-aqueous phase system carries out extract and separate, and the lower phase after extraction is oyster neutral polysaccharide crude extract；（4）Oyster neutral polysaccharide crude extract is dialysed, oyster polysaccharide refining liquid is obtained, oyster neutral polysaccharide is can obtain after concentrate drying.Extraction process of the present invention is simple, cost is low; suitable for intermittent and large-scale production process high-purity, in high yield, the oyster neutral polysaccharide finished product of high bioactivity; and obtained oyster neutral polysaccharide can long time stored, purity it is high; mouse spleen lymphocyte immunocompetence can be improved, suppresses the liver cancer cells of Hepg 2 activity.

Description

A kind of Pacific oyster neutral polysaccharide and its preparation method and application
Technical field
Be related to natural products field of deep the present invention relates to one kind, and in particular to a kind of Pacific oyster neutral polysaccharide and Its extracting method and application,
Background technology
Oyster is the first big cultivated shellfish in the world, is also China important marine shellfish, belongs to Ostreidae, and Bivalve software is moved Thing, is distributed in temperate zone and tropical each ocean stretch of coastal water, main species have Crassostrea rivularis, Pacific oyster, ostrea talienwhanensis Crosse and Close squama oyster.Pacific oyster has two shells in left and right, is connected with ligament and closed shell flesh etc..Left housing is recessed, thick greatly, can be used for Adhere to his thing, there is more radial rib on shell surface, it is clear denumerable；Right shell is smaller and flat, and there is the scale of multilayer concentric ring-type on surface, does not have There is significant radial rib.
Oyster is a kind of marine commercial molluscs animal with medical value, according to analysis, is removed in its meat and contains what is enriched Also there is the polysaccharide of a large amount of glycogen structures outside protein, the content of polysaccharide accounts for 20%-40% of oyster dry weight or so.Oyster is slightly more Sugar mainly has neutral and two kinds of acidic polysaccharose.As other polysaccharide molecules, the structure of oyster glycogen is divided into primary structure and height Level structure (including two grades, three-level, quaternary structure).
For the extraction of polysaccharide, for same materials, the extracting method taken is different, and resulting polysaccharide structures are not yet Together, the polysaccharide of the different parts of same materials also has different structures, therefore process step is also not quite similar.Therefore, select Suitable method is most important, on the basis of destruction glycogen original structure as few as possible, obtains higher recovery rate.
At present, the extracting method of oyster polysaccharide mainly has water extraction method, alkali extraction method and enzymatic extraction method.
Water extraction method has the advantages that equipment and simple to operate, widely applicable, but the operating time is long, and efficiency is low, And need to operate repeatedly, therefore energy consumption is higher.Water extraction method process is substantially：After fresh oyster meat is accurately weighed, after cleaning Homogenate, the triangular flask that then will be equipped with oyster slurry is put into boiling water bath and boils 1h, and cold filtration concentrates alcohol precipitation.
Alkali extracting method can more completely be extracted from tissue to polysaccharide, and this is based in animal body, sugared many and albumen Glycoprotein is combined to form, its covalent bond chance alkali is unstable, so as to reach the purpose of release polysaccharide；It is general to use the alkali solubles such as NaOH, KOH Liquid is as extractant, and recovery rate is higher, but polysaccharide molecule is possible to be degraded, and the structure and activity to polysaccharide have certain shadow Ring.Alkali extraction method process is substantially：Oyster meat is homogenized, 30% potassium hydroxide is added, 100 DEG C of heating 1h are cooled to after room temperature Add 95%
Ethanol, filtering to precipitate, i.e. oyster Thick many candies
Enzymatic extraction method extracts active polysaccharide using biology enzyme, because the selectivity of enzyme and selectivity are stronger, so method has Mild condition, does not destroy active polysaccharide, the advantages of purity of polysaccharide of extraction is higher, but can improve production cost, to extraction conditions It is required that also higher.Enzyme formulation enzyme is generally the oyster Thick many candies after pepsin and trypsase, enzymolysis and uses ion exchange layer Analysis is further purified, and can obtain different components.
Also contain pigment in Thick many candies, protein, the impurity such as oligosaccharide.Frequently with activated carbon decolorizing or H2O2Decolourize, this Neutral polysaccharide can be caused to be adsorbed, the problems such as oxidation.Column chromatography is the method for removing these impurity more universal at present.
In a word, the above extract and go deimpurity method to exist operating procedure is more, product yield is low, the operation cycle The problems such as long, introducing impurity, activity reduction.
The content of the invention
It is an object of the present invention to the defect for overcoming prior art, there is provided a kind of new Pacific oyster neutral polysaccharide Preparation method, comprise the following steps：
(1) by Pacific oyster dry powder by acetone degreasing, degreasing ostreae testa pulverata is obtained after drying；
(2) it is degreasing ostreae testa pulverata is soluble in water, handled with ultrasonic-microwave extraction instrument, by the solution centrifugal after processing, supernatant steams Hair concentration, obtains oyster polysaccharide crude extract；
(3) oyster polysaccharide crude extract is added into ethanol-(NH4)2SO4Double-aqueous phase system carries out extract and separate, and the lower phase after extraction is Oyster neutral polysaccharide crude extract；
(4) oyster neutral polysaccharide crude extract is dialysed, obtains oyster polysaccharide refining liquid, oyster is can obtain after concentrate drying Neutral polysaccharide.
As the further improvement to above-mentioned technical proposal, wherein ultrasonic-microwave extraction instrument processing time is in step (2) 20-30 minutes, treatment temperature was 80-90 DEG C.
It is used as the further improvement to above-mentioned technical proposal, wherein ethanol-(NH in step (3)4)2SO4In double-aqueous phase system Ethanol, (NH4)2SO4Weight ratio with PBS is 0.5:0.32:1, and the ratio of double-aqueous phase system and oyster polysaccharide crude extract is： 10：1, crude extract concentration is about 9mg/mL.
Another object of the present invention is to provide Pacific oyster neutral polysaccharide prepared by a kind of use above method.
The yet another object of the present invention be to provide a kind of above-mentioned Pacific oyster neutral polysaccharide prepare it is Ia Application in medicine.
The yet another object of the present invention is to provide a kind of Pacific oyster neutral polysaccharide in antineoplastic is prepared Using.Preferably, wherein the tumour is liver cancer.
The present invention has advantages below：
(1) side that the present invention is extracted using ultrasonic-microwave, the ethanol-extraction of (NH4) 2SO4 double-aqueous phase systems and dialysis is combined Formula extracts oyster neutral polysaccharide, it is to avoid the cost of traditional chromatographic technique is high, yield it is low (<5% oyster neutral polysaccharide/oyster Powder), the drawback such as time-consuming, extraction process is simple, cost is low, suitable for intermittent and large-scale production processing high-purity, in high yield Oyster neutral polysaccharide finished product.
(2) oyster polysaccharide that the method for prior art is extracted is typically the mixture of neutral polysaccharide and acidic polysaccharose, and this The oyster polysaccharide that the method for invention is extracted is pure neutral polysaccharide.The present inventor's research finds that oyster neutral polysaccharide is compared to acid Property polysaccharide can preferably improve the activity of mice spleen lymphocyte immune cell, have stronger suppression to human liver cancer cell Hepg-2 Propagation, apoptosis-induced effect.
(3) present invention prepared by Pacific oyster neutral polysaccharide can for a long time at 4 DEG C storage, activity will not reduce or Lose.
(4) the Pacific oyster neutral polysaccharide prepared by the present invention can promote the immunocompetence of mouse spleen lymphocyte, There is Inhibit proliferaton to human liver cancer cell Hepg-2, apoptosis-induced effect.
Brief description of the drawings
Fig. 1 is the Pacific oyster neutral polysaccharide extraction process flow chart of the present invention.
Fig. 2 is traditional oyster polysaccharide extraction process flow chart.
Fig. 3 is to use ethanol-(NH4)2SO4The result that double-aqueous phase system is enriched with to oyster polysaccharide crude extract.
Fig. 4 is ethanol-(NH using the present invention4)2SO4The Pacific oyster Thick many candies that double-aqueous phase system method is extracted Finished product.
Fig. 5 be comparative example in using traditional method for extracting Pacific oyster Thick many candies finished product.
Fig. 6 is the purity that the oyster neutral polysaccharide prepared by the inventive method that photodetector is determined is evaporated using HPLC- Collection of illustrative plates.
Fig. 7 (a) (b) is respectively the scanning of the ultraviolet specrophotometer of oyster polysaccharide crude extract and oyster neutral polysaccharide finished product Collection of illustrative plates.
Fig. 8 is that the oyster neutral polysaccharide monose prepared by the inventive method that HPLC-ELSD instrument is detected determines collection of illustrative plates, with Portugal Grape saccharide collection of illustrative plates coincide.
Fig. 9 is the oyster neutral polysaccharide (OGN2) prepared by traditional oyster neutral polysaccharide (OGN1), the inventive method, tradition Oyster acidic polysaccharose (OGA2) and the present invention side abandoned after acidic polysaccharose (OGA1), the inventive method aqueous two-phase extraction in liquid Polysaccharide crude extract (OG) is determined to the proliferation rate of mouse spleen lymphocyte in method.
Survey of oyster neutral polysaccharides of the Figure 10 prepared by the inventive method to the IL-2 burst sizes of mouse spleen lymphocyte It is fixed.
Figure 11 is inhibiting rate of the oyster neutral polysaccharide prepared by the inventive method to human liver cancer cell Hepg-2.
Figure 12 is double detections of the dye method to human liver cancer cell Hepg-2 Apoptosis of Annexin V-FITC, wherein (a), , (c), (b) (d) corresponding blank group, positive group (the μ g/mL of 5-Fluorouracil 50), oyster prepared by the inventive method respectively Neutral polysaccharide (200 μ g/mL), the oyster neutral polysaccharide (200 μ g/mL) obtained using traditional extraction process.
Embodiment
The present invention is further described below in conjunction with specific embodiment.Embodiments of the invention are intended to illustrate this hair Bright technical scheme, is not construed as limiting to technical scheme.
Embodiment 1：The Pacific oyster neutral polysaccharide finished product prepared using the inventive method
Prepared by the technique of Pacific oyster neutral polysaccharide finished product as shown in Figure 1, comprise the following steps that：
(1) degreasing ostreae testa pulverata is prepared：Oyster dry powder after crushed, crosses 40 mesh sieves, and 60 DEG C of dryings are to constant weight in an oven.Weigh 6g Oyster powder add in 60ml deionized water, add 6000rpm centrifugations 20min at the acetone washing of 3 times of volumes, 4 DEG C, it is low Temperature drying, obtains degreasing ostreae testa pulverata.
(2) oyster polysaccharide crude extract is prepared：Degreasing ostreae testa pulverata obtained by step (1) is put into 1000mL glass extraction In container, add 600mL deionized waters and be placed in handling in 500W ultrasonic-microwave extraction instrument, 86 DEG C of temperature, time 26min is carried Cooled down after taking.Extract solution is centrifuged (4000rpm, 10min), merges supernatant, concentrated by rotary evaporation obtains oyster polysaccharide and slightly carried Liquid, freeze-drying obtains yellow Thick many candies powder, and carries out protein and measurement of the polysaccharide content.
(3) oyster neutral polysaccharide refining liquid is prepared：Ethanol-(NH4)2SO4Double-aqueous phase system solution phase composition is： 17.7wt% (NH4)2SO4, 27.29wt% ethanol, 55.01wt%PBS.3.54g40wt% is sequentially added in 10mL centrifuge tubes (NH4)2SO4, 0.8g Thick many candies solution (concentration 9mg/mL), 1.4768g 0.01M PBS (pH=7.2), 2.1832g ethanol, mix Even 30min, stands 10min.As shown in figure 3, using ethanol-(NH4)2SO4Double-aqueous phase system extracts to oyster polysaccharide crude extract After taking, pigment impurity etc. is enriched in phase on ethanol, and the acidic polysaccharose and protein-enriched of macromolecule are in intermediate layer, (NH4)2SO4 Lower phase is mainly enriched oyster neutral polysaccharide.
Phase is discarded, removes and oyster neutral polysaccharide crude extract is mutually made.
(4) oyster neutral polysaccharide finished product is prepared：The oyster neutral polysaccharide refining liquid prepared by step (3) is taken to be placed in 2000KD Bag filter in flowing water dialyse 24h, then in deionized water dialyse 24h, dialyzate rotary evaporation concentration, freeze-drying be made White powder oyster neutral polysaccharide finished product (OGN2) (as shown in Figure 3).
Comparative example 1：Pacific oyster polysaccharide finished product is prepared using conventional method
Polysaccharide in Pacific oyster is extracted using traditional handicraft, concrete technology is as shown in Figure 2.Resulting product is neutral many The mixture of sugar and acidic polysaccharose, is the floccule (as shown in Figure 4) of yellow.
Embodiment 1 with pass through phend-sulphuric acid and polysaccharide in the different extraction steps of BCA methods measure in comparative example 1 The content of polysaccharide and protein in solution.
Phend-sulphuric acid：Precision is weighed and dried at 105 DEG C to the glucose 20mg of constant weight, is put in 500mL measuring bottles, is added water To scale, shake up, with 40 μ g/mL glucose standards solution.Precision draw glucose standards solution 0.4,0.6,0.8, 1.0th, 1.2,1.4,1.6,1.8mL, is respectively placed in 10mL tool plug scale test tubes, is respectively mended to 2.0mL, be then respectively adding with water 5% phenol solution 1mL, shakes up, and is rapidly added concentrated sulfuric acid 5mL, and shaking room temperature places 5min, is put into 15min in boiling water, taking-up is put 30min is cooled down in cold water, scale is finally added water to, shaken up, using 2mL distilled water by same color operation as blank, with purple Outer spectrophotometer determines absorbance at 490nm.Standard curve is drawn to concentration of glucose by ordinate of A values, with standard Curve blank is control, dilutes the polysaccharide solution of certain multiple, sample is found from standard curve according to the light absorption value of sample Polyoses content.Calculation formula is as follows：
Polyoses content (%)=(COyster polysaccharide·D)/WOyster polysaccharide finished product× 100% (1).
C in formulaOyster polysaccharideFor test liquid polysaccharide concentration, D is the extension rate of test liquid；WOyster polysaccharide finished productFor oyster polysaccharide finished product Weight.
BCA methods：Take 50 parts of reagent As to be well mixed with 1 part of reagent B, accurately draw 25 μ L samples solution in enzyme mark hole, plus Enter the μ L of BCA reagents 200, jog is incubated 30min in 37 DEG C, is cooled to after room temperature, using blank as control, the 562nm on ELIASA Locate colorimetric, using bovine serum albumin content as abscissa, using light absorption value as ordinate, draw standard curve.It is empty with standard curve White is control, finds the protein content of sample from standard curve according to the light absorption value of sample.Calculation formula is as follows：
Protein content (%)=(CProtein·D)/WOyster polysaccharide finished product× 100% (2).
C in formulaProteinFor test liquid protein concentration, D is the extension rate of test liquid；WOyster polysaccharide finished productFor oyster polysaccharide finished product Weight.
Statistical analysis is carried out to the yield of oyster neutral polysaccharide in different extraction steps simultaneously, and observes different extraction steps The outward appearance of middle oyster neutral polysaccharide.
Learnt by detection, the yield of the oyster neutral polysaccharide extracted using the inventive method compare conventional method from 2.86% brings up to 11.43%, increases significantly, calculation formula is as follows：
Neutral sugar yield (%)=WOyster neutral sugar finished product/WOyster dry powder× 100% (3).
Fig. 7 (a), (b) is respectively the scanning spectra of the ultraviolet specrophotometer of oyster polysaccharide crude extract and neutral polysaccharide, in Property polysaccharide at 280nm without obvious absorption peaks, illustrate to reach the effect for removing removing protein well.
Embodiment 2：The molecular weight and purity for the Pacific oyster neutral polysaccharide that HPLC detections are prepared using the inventive method
Condition：Splitter：TSKgel G4000SW(300mm×7.5mm)；Mobile phase：Ammonium acetate solution (0.05mol/L)；Sample Product concentration：Detailed data below；Sample size：20μL；Flow velocity：0.5mL/min, drift tube temperature：55 DEG C, flow rate of carrier gas：2L/ Min, ram：Open.As a result as shown in figure 5, being the narrower symmetrical peak type of peak width.
Embodiment 3：Detection is using monose composition measuring in the Pacific oyster neutral polysaccharide of the inventive method preparation
(1) the Pacific oyster neutral polysaccharide 10mg prepared using the inventive method is weighed, is placed in 10mL tool plug scale test tubes, Plus 1mol/L H2SO42mL, sealing.In 100 DEG C of constant temperature hydrolysis 4h, saturation Ba (OH) is used after hydrolyzate cooling2It is neutralized to neutrality, 3000rpm centrifugations 10min removes BaSO4Precipitation.Supernatant is fully transferred in 10mL measuring bottles, with distilled water diluting to quarter Degree, solution obtains oyster neutral polysaccharide hydrolyzate after 0.45 μm of filtering with microporous membrane.
(2) HPLC-ELSD instrument is detected：Dextrose standard sample (500 μ g/mL), oyster neutral polysaccharide hydrolyzate, chromatographic column Carbohydrate ES 5u(250mm×4.6mm)；Mobile phase：Acetonitrile-water (75%-25%), sample size：20mL, flow velocity： 1mL/min, drift tube temperature：83 DEG C, flow rate of carrier gas 2.1L/min.
Fig. 8 is monose survey in Pacific oyster neutral polysaccharide prepared by use the inventive method that HPLC-ELSD instrument is detected Determine collection of illustrative plates, coincide with dextrose standard sample collection of illustrative plates, it is glucose to illustrate monose composition.
Embodiment 4：Propagation of the Pacific oyster neutral polysaccharide prepared using the inventive method to mouse spleen lymphocyte Determine
1. the preparation of mouse spleen lymphocyte suspension
(1) mouse is moved into super-clean bench with after 75% ethanol soaking disinfection 3min, takes out mouse and be placed on sterile plastics platform, Left abdomen upward, cuts off osculum in the middle part of abdomen, exposure spleen is lifted spleen with tweezers, following connective group is separated with eye scissors Knit, take out spleen, the cell filtration for being placed in 70 μm is online, is placed below on 50mL centrifuge tubes, with appropriate (about 15mL) sterile PBS Liquid (pH7.2-7.4) is slowly rinsed, while being ground spleen with grinding rod, individual cells is flushed to by PBS in centrifuge tube, it Lapping liquid 1000rpm is centrifuged into 10min afterwards, supernatant liquor is discarded.
(2) erythrocyte cracked liquid 6mL is added in precipitating, piping and druming is mixed, static 5min, centrifugation 10min (1000rpm) is abandoned Supernatant is removed, plasma fraction, cell fragment and some red blood cells are removed.Add appropriate RPMI1640 complete mediums (RPMI- 1640 culture mediums：Hyclone：The 89 of penicillin/streptomycin:10:1 proportions) suspension splenocyte, blown and beaten with liquid-transfering gun equal It is even, it is placed in 5%CO2, in 37 DEG C of incubator after culture 2h, RPMI1640 complete medium suspensions splenocyte is changed and cultivated Bottle, to remove the heteroproteose cell of adherent growth, is placed in 5%CO again afterwards2, cultivate 24h in 37 DEG C of incubator.Use cell counting count board The number of cell is counted, it is 1 × 10 to add appropriate culture medium adjustment cell concentration6Individual/mL.
2. the propagation of mouse spleen lymphocyte is determined using mtt assay
(1) by the cell suspension inoculation prepared to 96 orifice plates, per the μ L of hole 100, add containing oyster polysaccharide, concanavalin A (ConA), the μ L of RPMI-1640 nutrient solutions 100 (Con A final concentration is 5.0 μ g/m L), makes to prepare using the inventive method Pacific oyster neutral polysaccharide final concentration of 10,50,250 μ g/mL), every group be all provided with control group (only plus RPMI-1640 training Nutrient solution), each sample sets 4 multiple holes, is placed in 5%CO2, cultivate 48h in 37 DEG C of incubator.
(2) the μ L/ holes of MTT solution 20 are added after culture terminates, continues to cultivate 4h, adds three liquid (10g dodecyl sulphates Sodium, 5mL isobutanols, 0.1mL 12mol/L HCI are dissolved in distilled water, are settled to 100mL) 80 μ L/ holes, stood overnight in 37 DEG C Afterwards, wavelength 570nm absorbance A is surveyed with ELIASA570nmIt is worth for experimental result, using proliferation rate as index, judges that lymphocyte turns Change degree：
Fig. 9 is that oyster polysaccharide is determined to the proliferation rate of mouse spleen lymphocyte, OGN1, OGN2, OGA1, OGA2, OG difference To be abandoned after the oyster neutral polysaccharide prepared by traditional oyster neutral polysaccharide, the inventive method, convention acidic polysaccharide, aqueous two-phase extraction Polysaccharide crude extract (OG) in oyster acidic polysaccharose finished product and the inventive method in liquid, it can be seen that the inventive method is made It is 121.08% to the proliferation rate of mouse spleen lymphocyte during standby 250 μ g/mL of oyster neutral polysaccharide, can effectively improves small Mouse spleen lymphocyte proliferation rate.
Embodiment 5：The Pacific oyster neutral polysaccharide prepared using the inventive method is released mouse spleen lymphocyte (MSL) Put IL-2 influence
(1) sample is prepared：With 1 × 106Individual/mL density is inoculated with MSL to 96 orifice plates, per the μ L of hole 100, adds many containing oyster Sugar, (final concentration for making Con A is 5.0 μ g/mL to the Con A μ L of RPMI-1640 nutrient solutions 100, is prepared using the inventive method Pacific oyster neutral polysaccharide final concentration of 250 μ g/mL), every group is all provided with control group (only plus RPMI-1640 nutrient solutions), Each sample sets 4 multiple holes, is placed in 5%CO2, cultivate 24h in 37 DEG C of incubator, draw nutrient solution, 1500rpm centrifugations 5min, collects supernatant.
(2) standard curve is drawn：The hole of gauge orifice 10 (5 concentration are all provided with 2 multiple holes) is set on enzyme mark coating plate, is passed through Dilution makes standard concentration be respectively 2400,1600,800,400,200pg/mL.
(3) it is loaded：This bottom outlet (being not added with sample and enzyme marking reagent), oyster polysaccharide hole, Positive control wells, blank are set respectively Hole, respectively sets 3 multiple holes.First add the μ L of sample diluting liquid 40 on enzyme mark coating plate, the μ L of testing sample 10 are then added again, gently Rock and shake up.
(4) incubate：With the rearmounted 37 DEG C of incubations 30min of shrouding film shrouding.
(5) wash：Concentrated cleaning solution is diluted, it is standby.Take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, Discarded after standing 30s.It is repeated 5 times, pats dry.
(6) it is enzyme-added：Every hole adds except 50 μ L enzyme marking reagents, this bottom outlet.
(7) incubate, washing.
(8) develop the color：Add the μ L of developer 50 per hole, then add developer B50 μ L, concussion is mixed, 37 DEG C of lucifuges colour developing 15min.
(9) terminate：Add the μ L of terminate liquid 50 per hole.
(10) determine：Determined adding within terminate liquid 15min.Returned to zero with this bottom outlet, absorbance is determined under 450nm.
Obtained according to standard curve after the corresponding IL-2 concentration of each experimental group, stimulus index is calculated using below equation：
Stimulus index SI (%)=(wExperiment-wBlank)/wBlank× 100% (5).
W in formulaExperiment、wBlankRespectively experimental group and naive mice splenic lymphocytes IL-2 concentration.
Figure 10 is that the Pacific oyster neutral polysaccharide prepared using the inventive method is released the IL-2 of mouse spleen lymphocyte Measure high-volume, it can be seen that the μ g/mL of Pacific oyster neutral polysaccharide 250 prepared using the inventive method are to mice spleen lymph Cell IL-2 stimulus index reaches 124.81%, can effectively stimulate mouse spleen lymphocyte immunocompetence.
Embodiment 6：The external suppression to Hepg-2 cells of Pacific oyster neutral polysaccharide prepared using the inventive method Rate is determined
(1) take the logarithm the tumour cell in growth period, DMEM nutrient solutions (DMEM culture mediums are used after Trypsin Induced：Hyclone： The 89 of penicillin/streptomycin:10:1 proportions) cell is adjusted to 4 × 104/mL.Add by every μ L of hole 100 (4000 cells) Enter in 96 well culture plates.
(2) each 100 μ of Pacific oyster neutral polysaccharide prepared by use the inventive method of different diluted concentrations is added after 4h L, control group adds isometric DMEM nutrient solutions, and each concentration is all provided with 4 multiple holes, puts 37 DEG C of volume fractions for 5%CO2Incubator In continuous culture 48h.Each holes of 4h add MTT (5g/L) 20 μ L before culture terminates, and place and continue to be incubated 4h in above-mentioned incubator, abandon Clearly, the μ L of dimethyl sulfoxide (DMSO) 150 are added per hole, fully vibration is mixed surveys absorption photometric value (A) at ELIASA 570nm, calculates Inhibiting rate, calculation formula is as follows：
Inhibiting rate (%)=(AControl group-AMedicine group)/AControl group× 100% (6).
Figure 11 is suppression of the Pacific oyster neutral polysaccharide prepared using the inventive method to human liver cancer cell Hepg-2 Rate, it can be seen that to human liver cancer cell Hepg-2 during the 200 μ g/mL of Pacific oyster neutral polysaccharide prepared using the inventive method Inhibiting rate be 55.81%, can effectively suppress the activity of liver cancer cell growth.
Embodiment 7：Annexin V-FITC Apoptosis is detected
(1) take the logarithm the tumour cell in growth period, cell is adjusted to 4 × 10 with DMEM complete culture solutions after Trypsin Induced4/ mL.Added by every μ L of hole 100 (4000 cells) in 96 well culture plates.
(2) each 100 μ of Pacific oyster neutral polysaccharide prepared by use the inventive method of different diluted concentrations is added after 4h L, control group adds isometric DMEM nutrient solutions, and each concentration is all provided with 4 multiple holes, puts 37 DEG C of volume fractions for 5%CO2In incubator Continuous culture 48h.
(3) cell culture fluid is absorbed after apoptosis induction terminates, PBS is added and washed once, add 195 μ LAnnexin V- FITC combination liquid, adds 5 μ L Annexin V-FITC, gently mixes.
(4) room temperature (20-25 DEG C) lucifuge is incubated 10min, and lucifuge is carried out using aluminium foil.Solution removed by aspiration, adds 190 μ L Annexin V-FITC combination liquid.
(5) 10 μ L propidium iodide stain liquid are added, are gently mixed, ice bath avoid light place.
(6) immediately in fluorescence microscopy Microscopic observation, Annexin V-FITC are green fluorescence, and PI is red fluorescence.
Figure 12 is double detections of the dye method to human liver cancer cell Hepg-2 Apoptosis of Annexin V-FITC, wherein (a), (b), (c), the corresponding blank group of (d) difference, positive group (the μ g/mL of 5-Fluorouracil 50), oyster neutral polysaccharide (200 μ g/mL), The neutral polysaccharide (200 μ g/mL) that traditional extraction process is obtained, shown dead color is the downright bad tumour cell with apoptosis late period, Light tone is the tumour cell of apoptosis early stage.
Although above having made to retouch in detail to the present invention with general explanation, embodiment and experiment State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art 's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed Scope.

Claims (7)

1. a kind of preparation method of Pacific oyster neutral polysaccharide, comprises the following steps：
（1）By Pacific oyster dry powder by acetone degreasing, degreasing ostreae testa pulverata is obtained after drying；
（2）Degreasing ostreae testa pulverata is soluble in water, handled with ultrasonic-microwave extraction instrument, by the solution centrifugal after processing, supernatant steams Hair concentration, obtains oyster polysaccharide crude extract；
（3）Oyster polysaccharide crude extract is added into ethanol-(NH4)2SO4Double-aqueous phase system carries out extract and separate, and the lower phase after extraction is Oyster neutral polysaccharide crude extract；
（4）Oyster neutral polysaccharide crude extract is dialysed, oyster polysaccharide refining liquid is obtained, oyster is can obtain after concentrate drying Neutral polysaccharide.
2. preparation method as claimed in claim 1, wherein step（2）Middle ultrasonic-microwave extraction instrument processing time is 20-30 points Clock, treatment temperature is 80-90 DEG C.
3. preparation method as claimed in claim 1, wherein step（3）Middle ethanol-(NH4)2SO4Ethanol in double-aqueous phase system, (NH4)2SO4Weight ratio with PBS is 0.5:0.32:1, and the ratio of double-aqueous phase system and oyster polysaccharide crude extract is：10：1, Crude extract concentration is about 9mg/mL.
4. the Pacific oyster neutral polysaccharide prepared by method described in a kind of use claim any one of 1-3.
5. application of the Pacific oyster neutral polysaccharide as claimed in claim 4 in Ia medicine is prepared.
6. application of the Pacific oyster neutral polysaccharide as claimed in claim 4 in antineoplastic is prepared.
7. application as claimed in claim 6, wherein the tumour is liver cancer.
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