CN106986948B - 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 PDFInfo
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Abstract
The invention discloses a kind of preparation methods of Pacific oyster neutral polysaccharide, comprising the following steps: (1) Pacific oyster dry powder is obtained degreasing ostreae testa pulverata after dry by acetone degreasing；(2) degreasing ostreae testa pulverata is soluble in water, it is handled with ultrasonic-microwave extraction instrument, by treated, solution is centrifuged, and supernatant is concentrated by evaporation, and obtains oyster polysaccharide crude extract；(3) ethyl alcohol-(NH is added in oyster polysaccharide crude extract4)2SO4Double-aqueous phase system carries out extraction and separation, and lower phase extracted is oyster neutral polysaccharide crude extract；(4) oyster neutral polysaccharide crude extract is dialysed, obtains oyster polysaccharide refining liquid, oyster neutral polysaccharide can be obtained after concentrate drying.Extraction process of the present invention is simple, at low cost; suitable for intermittent and large-scale production processing high-purity, high yield, high bioactivity oyster neutral polysaccharide finished product; and obtained oyster neutral polysaccharide can be long time stored, with high purity; it can be improved mouse spleen lymphocyte immunocompetence, inhibit Hepg-2 liver cancer cells activity.
Description
Technical field
The present invention relates to one kind to be related to natural products field of deep, and in particular to a kind of Pacific oyster neutral polysaccharide and
Its extracting method and application,
Background technique
Oyster is that the first big cultivated shellfish and China important marine shellfish, category Ostreidae, Bivalve software are dynamic in the world
Object, is distributed in each ocean stretch of coastal water in temperate zone and the torrid zone, 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 adductor muscle etc..Left housing is recessed, big and thick, can be used to
Adhere to his object, there is more radial rib on shell surface, clear denumerable；Right shell is smaller and flat, and there is the cricoid scale of multilayer concentric 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, except rich in meat
Protein is outer there is also the polysaccharide of a large amount of glycogen structures, and the content of polysaccharide accounts for the 20%-40% or so of oyster dry weight.Oyster is thick
Polysaccharide mainly has neutrality and two kinds of acidic polysaccharose.As other polysaccharide molecules, the structure of oyster glycogen be divided into primary structure and
Higher structure (including second level, three-level, quaternary structure).
Extraction for polysaccharide, for same materials, the extracting method taken is different, and obtained polysaccharide structures are not yet
Together, the polysaccharide of the different parts of same materials also has different structures, therefore processing step is also not quite similar.Therefore, it selects
Suitable method is most important, on the basis of destroying glycogen original structure as few as possible, obtains higher recovery rate.
Currently, the extracting method of oyster polysaccharide mainly has water extraction method, alkali extraction method and enzymatic extraction method.
Water extraction method has many advantages, such as equipment and simple to operate, widely applicable, but the operating time is long, low efficiency,
And it needs to operate repeatedly, therefore energy consumption is higher.Water extraction method process is substantially are as follows: after fresh oyster meat accurately weighs, after cleaning
Triangular flask equipped with oyster slurry, is then put into boiling water bath and boils 1h by homogenate, and alcohol precipitation is concentrated in cold filtration.
Alkali extracting method can more completely extract polysaccharide from tissue, this be based on animal body in, sugar mostly with albumen
In conjunction with glycoprotein is formed, it is unstable that covalent bond meets alkali, to achieve the purpose that discharge polysaccharide；Generally with alkali solubles such as NaOH, KOH
For liquid as extractant, recovery rate is higher, but polysaccharide molecule is possible to be degraded, and the structure and activity to polysaccharide have certain shadow
It rings.Alkali extraction method process is substantially are as follows: is homogenized oyster meat, 30% potassium hydroxide, 100 DEG C of heating 1h, after being cooled to room temperature are added
95% ethyl alcohol is added, filters to obtain precipitating, is i.e. oyster Thick many candies enzymatic extraction method extracts active polysaccharide using biological enzyme, because of enzyme
Specificity and selectivity it is stronger, so method has mild condition, do not destroy active polysaccharide, it is excellent that the purity of polysaccharide of extraction is higher etc.
Point, but production cost can be improved, extraction conditions are required also higher.Enzyme formulation enzyme is generally pepsin and trypsase, enzyme
Oyster Thick many candies after solution are further purified using ion-exchange chromatography, available different component.
Also contain pigment, protein, the impurity such as oligosaccharide in Thick many candies.Frequently with active carbon decoloring or H2O2Decoloration,
The problems such as this will cause neutral polysaccharide and is adsorbed, oxidation.Column chromatography is the method for removing these impurity more universal at present.
In short, the above is extracted and goes deimpurity method there are operating procedures more, product yield is low, the operation cycle
The problems such as long, introducing impurity, activity reduce.
Summary of the invention
It is an object of the present invention to overcome the deficiencies of existing technologies, a kind of new Pacific oyster neutral polysaccharide is provided
Preparation method, comprising the following steps:
(1) Pacific oyster dry powder is obtained into degreasing ostreae testa pulverata after dry by acetone degreasing；
(2) degreasing ostreae testa pulverata is soluble in water, it is handled with ultrasonic-microwave extraction instrument, by treated, solution is centrifuged, supernatant
Liquid is concentrated by evaporation, and obtains oyster polysaccharide crude extract；
(3) ethyl alcohol-(NH is added in oyster polysaccharide crude extract4)2SO4Double-aqueous phase system carries out extraction and separation, under extracted
It is mutually oyster neutral polysaccharide crude extract；
(4) oyster neutral polysaccharide crude extract is dialysed, obtains oyster polysaccharide refining liquid, can be obtained after concentrate drying
Oyster neutral polysaccharide.
As further improvement to above-mentioned technical proposal, wherein the ultrasonic-microwave extraction instrument processing time is in step (2)
20-30 minutes, treatment temperature was 80-90 DEG C.
As further improvement to above-mentioned technical proposal, wherein ethyl alcohol-(NH in step (3)4)2SO4In double-aqueous phase system
Ethyl alcohol, (NH4)2SO4Weight ratio with PBS is 0.5:0.32:1, and the ratio of double-aqueous phase system and oyster polysaccharide crude extract are as follows:
10:1, crude extract concentration are about 9mg/mL.
Another object of the present invention is to provide a kind of Pacific oyster neutral polysaccharides prepared using the above method.
Yet another object of the invention be to provide a kind of above-mentioned Pacific oyster neutral polysaccharide prepare it is immune-related
Application in drug.
Yet another object of the invention is to provide a kind of Pacific oyster neutral polysaccharide in the preparation of antitumor drugs
Using.Preferably, wherein the tumour is liver cancer.
The invention has the following advantages that
(1) present invention is using ultrasonic-microwave extraction, ethyl alcohol-(NH4)2SO4What double-aqueous phase system extraction and dialysis combined
Mode extracts oyster neutral polysaccharide, avoids low (< 5% oyster neutral polysaccharide/male of the at high cost of traditional chromatographic technique, yield
Oyster powder), the drawbacks such as time-consuming, extraction process is simple, at low cost, is suitable for intermittent and large-scale production processing high-purity, high receives
The oyster neutral polysaccharide finished product of rate.
(2) oyster polysaccharide that the method for the prior art is extracted is usually the mixture of neutral polysaccharide and acidic polysaccharose, and this
The extracted oyster polysaccharide of the method for invention is pure neutral polysaccharide.The present inventor is the study found that oyster neutral polysaccharide compares acid
Property polysaccharide can preferably improve the activity of mice spleen lymphocyte immune cell, there is stronger inhibition to human liver cancer cell Hepg-2
Proliferation, apoptosis-induced effect.
(3) present invention prepared by Pacific oyster neutral polysaccharide can be stored at 4 DEG C for a long time, activity will not reduce or
It loses.
(4) the Pacific oyster neutral polysaccharide prepared by the present invention can promote the immunocompetence of mouse spleen lymphocyte,
Has the function of Inhibit proliferaton, apoptosis-induced to human liver cancer cell Hepg-2.
Detailed description of the invention
Fig. 1 is Pacific oyster neutral polysaccharide extraction process flow chart of the invention.
Fig. 2 is traditional oyster polysaccharide extraction process flow chart.
Fig. 3 is using ethyl alcohol-(NH4)2SO4The result that double-aqueous phase system is enriched with oyster polysaccharide crude extract.
Fig. 4 is using ethyl alcohol-(NH of the invention4)2SO4The Pacific oyster Thick many candies that double-aqueous phase system method is extracted
Finished product.
Fig. 5 is in comparative example using the finished product of the Pacific oyster Thick many candies of traditional method for extracting.
Fig. 6 is the purity that oyster neutral polysaccharide prepared by the method for the present invention of photodetector measurement is evaporated using HPLC-
Map.
Fig. 7 (a) (b) is respectively the scanning of the ultraviolet specrophotometer of oyster polysaccharide crude extract and oyster neutral polysaccharide finished product
Map.
Fig. 8 is that oyster neutral polysaccharide monosaccharide prepared by the method for the present invention of HPLC-ELSD instrument detection measures map, with Portugal
Grape saccharide map coincide.
Fig. 9 is oyster neutral polysaccharide (OGN2) prepared by traditional oyster neutral polysaccharide (OGN1), the method for the present invention, passes
The oyster acidic polysaccharose (OGA2) and the present invention in liquid are abandoned after system acidic polysaccharose (OGA1), the method for the present invention aqueous two-phase extraction
Polysaccharide crude extract (OG) measures the proliferation rate of mouse spleen lymphocyte in method.
Figure 10 is survey of the oyster neutral polysaccharide to the IL-2 burst size of mouse spleen lymphocyte prepared by the method for the present invention
It is fixed.
Figure 11 is inhibiting rate of the oyster neutral polysaccharide prepared by the method for the present invention to human liver cancer cell Hepg-2.
Figure 12 is detection of the bis- dye methods of Annexin V-FITC to human liver cancer cell Hepg-2 Apoptosis, wherein (a),
(b), (c) corresponding blank group, positive group (50 μ g/mL of 5-Fluorouracil), oyster is neutral prepared by the method for the present invention
Polysaccharide (200 μ g/mL).
Specific embodiment
Below in conjunction with specific embodiment, invention is further explained.The embodiment of the present invention is intended to illustrate this hair
Bright technical solution, is not construed as limiting technical solution of the present invention.
Embodiment 1: the Pacific oyster neutral polysaccharide finished product prepared using the method for the present invention
It is prepared by the technique of Pacific oyster neutral polysaccharide finished product as shown in Figure 1, the specific steps are as follows:
(1) prepare degreasing ostreae testa pulverata: oyster dry powder after crushed, crosses 40 meshes, in an oven 60 DEG C of dryings to constant weight.Claim
It takes the oyster powder of 6g to be added in the deionized water of 60ml, the acetone washing of 3 times of volumes is added, 6000rpm is centrifuged at 4 DEG C
20min, low temperature drying obtain degreasing ostreae testa pulverata.
(2) it prepares oyster polysaccharide crude extract: the glass that the obtained degreasing ostreae testa pulverata of step (1) is put into 1000mL is extracted
In extracting container, addition 600mL deionized water, which is placed in 500W ultrasonic-microwave extraction instrument, to be handled, and 86 DEG C of temperature, time 26min,
It is cooling after having extracted.Extracting solution is centrifuged (4000rpm, 10min), merges supernatant, concentrated by rotary evaporation obtains oyster polysaccharide and slightly mentions
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: ethyl alcohol-(NH is prepared4)2SO4Double-aqueous phase system solution phase composition are as follows:
17.7wt% (NH4)2SO4, 27.29wt% ethyl alcohol, 55.01wt%PBS.3.54g is sequentially added in 10mL centrifuge tube
40wt% (NH4)2SO4, 0.8g Thick many candies solution (concentration 9mg/mL), 1.4768g 0.01M PBS (pH=7.2), 2.1832g
Ethyl alcohol mixes 30min, stands 10min.As shown in figure 3, using ethyl alcohol-(NH4)2SO4Double-aqueous phase system slightly mentions oyster polysaccharide
After liquid is extracted, pigment impurity etc. is enriched in phase on ethyl alcohol, the acidic polysaccharose and protein-enriched of macromolecule in middle layer,
(NH4)2SO4Lower phase is mainly enriched oyster neutral polysaccharide.
Phase is discarded, removes and oyster neutral polysaccharide crude extract is mutually made.
(4) it prepares oyster neutral polysaccharide finished product: oyster neutral polysaccharide refining liquid prepared by step (3) being taken to be placed in 2000KD
Bag filter in flowing water dialysis for 24 hours, then for 24 hours in deionized water dialysis, dialyzate rotary evaporation concentration, freeze-drying be made
White powder oyster neutral polysaccharide finished product (OGN2) (as shown in Figure 4).
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.During obtained product is
Property polysaccharide and acidic polysaccharose mixture, be yellow floccule (as shown in Figure 5).
Polysaccharide in different extraction steps is measured by phend-sulphuric acid and BCA method in embodiment 1 and comparative example 1
The content of polysaccharide and protein in solution.
Phend-sulphuric acid: precision is weighed and is dried at 105 DEG C to the glucose 20mg of constant weight, is set in 500mL measuring bottle, 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,
1.0,1.2,1.4,1.6,1.8mL, are respectively placed in 10mL plug test tube, are respectively mended with water to 2.0mL, be then respectively adding
5% phenol solution 1mL, shakes up, and is rapidly added concentrated sulfuric acid 5mL, and shaking is placed at room temperature for 5min, is put into 15min in boiling water, taking-up is set
Cooling 30 min, finally add water to scale, shake up in cold water, using the distilled water of 2mL by same color operation as blank, with purple
Outer spectrophotometer measures absorbance at 490nm.Standard curve is drawn to concentration of glucose using A value as ordinate, with standard
Curve blank is control, dilutes the polysaccharide solution of certain multiple, finds sample 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 method: taking 50 parts of reagent As to be uniformly mixed with 1 part of reagent B, and accurate 25 μ L sample solution of drawing add in enzyme mark hole
Enter 200 μ L of BCA reagent, jog is control, the 562nm in microplate reader with blank after being cooled to room temperature in 37 DEG C of heat preservation 30min
Locate colorimetric, using bovine serum albumin content as abscissa, using light absorption value as ordinate, draws standard curve.With standard curve sky
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.
It is for statistical analysis to the yield of oyster neutral polysaccharide in different extraction steps simultaneously, and observe different extraction steps
The appearance of middle oyster neutral polysaccharide.
By detection learn, using the method for the present invention extract oyster neutral polysaccharide yield compare conventional method from
2.86% is increased 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) be respectively oyster polysaccharide crude extract and neutral polysaccharide ultraviolet specrophotometer scanning spectra, in
Property polysaccharide at 280nm without obvious absorption peaks, illustrate to achieve the effect that remove removing protein well.
The molecular weight and purity for the Pacific oyster neutral polysaccharide that embodiment 2:HPLC detection is prepared using the method for the present invention
Condition: splitter: TSKgel G4000SW (300mm × 7.5mm)；Mobile phase: ammonium acetate solution (0.05mol/
L)；Sample concentration: detailed data below；Sample volume: 20 μ L；Flow velocity: 0.5mL/min, drift tube temperature: 55 DEG C, flow rate of carrier gas:
Ram: 2L/min is opened.It as a result as indicated with 6, is the relatively narrow symmetrical peak type of peak width.
Embodiment 3: detection is using monosaccharide composition measuring (1) in the Pacific oyster neutral polysaccharide of the method for the present invention preparation
The Pacific oyster neutral polysaccharide 10mg using the method for the present invention preparation is weighed, is placed in 10mL plug test tube, adds 1mol/
The H of L2SO42mL, sealing.4h is hydrolyzed in 100 DEG C of constant temperature, with saturation Ba (OH) after hydrolyzate is cooling2It is neutralized to neutrality, 3000rpm
It is centrifuged 10min and removes BaSO4Precipitating.Supernatant is fully transferred in 10mL measuring bottle, is diluted to scale, solution warp with distilled water
Oyster neutral polysaccharide hydrolyzate is obtained after 0.45 μm of filtering with microporous membrane.
(2) HPLC-ELSD instrument detects: 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 volume: 20mL, flow velocity:
1mL/min, drift tube temperature: 83 DEG C, flow rate of carrier gas 2.1L/min.
Fig. 8 is that monosaccharide is surveyed in the Pacific oyster neutral polysaccharide using the method for the present invention preparation of HPLC-ELSD instrument detection
Determine map, coincide with dextrose standard sample map, illustrates that monosaccharide composition is glucose.
Embodiment 4: using the Pacific oyster neutral polysaccharide of the method for the present invention preparation to the proliferation of mouse spleen lymphocyte
Measurement
1. the preparation of mouse spleen lymphocyte suspension
(1) mouse is moved back in super-clean bench with 75% ethyl alcohol soaking disinfection 3min, takes out mouse and is placed in sterile plastics
On platform, left abdomen upward, cuts off osculum in the middle part of abdomen, exposure spleen is lifted spleen with tweezers, following knot is separated with eye scissors
It forms tissue, takes out spleen, it is online to be placed in 70 μm of cell filtration, is placed below on 50mL centrifuge tube, with appropriate (about 15mL) nothing
Bacterium PBS liquid (pH7.2-7.4) slowly rinses, and on one side grinds spleen with grinding rod, individual cells is made to be flushed to centrifuge tube by PBS
In, lapping liquid 1000rpm is centrifuged 10min later, discards supernatant liquor.
(2) erythrocyte cracked liquid 6mL is added in precipitating, piping and druming mixes, and static 5min is centrifuged 10min (1000rpm), abandons
Supernatant is removed, plasma composition, cell fragment and some red blood cells are removed.Appropriate RPMI1640 complete medium (RPMI- is added
1640 culture mediums: fetal calf serum: the 89:10:1 ratio of penicillin/streptomycin is prepared) suspension splenocyte, it is blown and beaten with liquid-transfering gun equal
It is even, it is placed in 5%CO2, cultivate 2h in 37 DEG C of incubator after, RPMI1640 complete medium suspension splenocyte is replaced and is cultivated
Bottle, to remove the heteroproteose cell of adherent growth, is placed in 5%CO again later2, cultivate for 24 hours in 37 DEG C of incubator.Use cell counting board
The number for counting cell, adding suitable culture medium adjustment cell concentration is 1 × 106A/mL.
2. using the proliferation of mtt assay measurement mouse spleen lymphocyte
(1) by the cell suspension inoculation prepared to 96 orifice plates, every 100 μ L of hole is added containing oyster polysaccharide, sword bean egg
White A (ConA), 100 μ L of RPMI-1640 culture solution (final concentration of Con A is 5.0 μ g/m L) make using the method for the present invention
Final concentration of 10,50,250 μ g/mL of the Pacific oyster neutral polysaccharide of preparation), every group is all provided with control group and (only adds RPMI-
1640 culture medium), each sample sets 4 multiple holes, is placed in 5%CO2, cultivate 48h in 37 DEG C of incubator.
(2) 20 hole μ L/ of MTT solution is added after cultivating, continues to cultivate 4h, three liquid (10g dodecyl sulphur is added
Sour sodium, 5mL isobutanol, 0.1mL 12mol/L HCI are dissolved in distilled water, are settled to 100mL) 80 holes μ L/, it was placed in 37 DEG C
After night, the absorbance A of wavelength 570nm is surveyed with microplate reader570nmValue judges lymphocyte using proliferation rate as index for experimental result
Transforming degree:
Fig. 9 is that oyster polysaccharide measures the proliferation rate of mouse spleen lymphocyte, OGN1, OGN2, OGA1, OGA2, OG difference
To be abandoned after oyster neutral polysaccharide prepared by traditional oyster neutral polysaccharide, the method for the present invention, convention acidic polysaccharide, aqueous two-phase extraction
Polysaccharide crude extract (OG) in oyster acidic polysaccharose finished product and the method for the present invention in liquid, it can be seen that the method for the present invention is made
It is 121.08% to the proliferation rate of mouse spleen lymphocyte when standby 250 μ g/mL of oyster neutral polysaccharide, can effectively improves small
Mouse spleen lymphocyte proliferation rate.
Embodiment 5: using the Pacific oyster neutral polysaccharide of the method for the present invention preparation to mouse spleen lymphocyte (MSL)
Discharge the influence of IL-2
(1) sample is prepared: with 1 × 106To 96 orifice plates, every 100 μ L of hole is added containing oyster the density inoculation MSL of a/mL
Polysaccharide, (final concentration for making Con A is 5.0 μ g/mL to the 100 μ L of RPMI-1640 culture solution of Con A, using the method for the present invention
The final concentration of 250 μ g/mL of the Pacific oyster neutral polysaccharide of preparation), every group is all provided with control group and (only adds RPMI-1640 culture
Liquid), each sample sets 4 multiple holes, is placed in 5%CO2, cultivate for 24 hours in 37 DEG C of incubator, draw culture solution, 1500rpm centrifugation
5min collects supernatant.
(2) it draws standard curve: setting 10 hole of gauge orifice (5 concentration are all provided with 2 multiple holes) on enzyme mark coating plate, pass through
Dilution makes standard concentration be respectively 2400,1600,800,400,200pg/mL.
(3) it is loaded: setting this bottom outlet (sample and enzyme marking reagent is not added), oyster polysaccharide hole, Positive control wells, blank respectively
Hole sets 3 multiple holes respectively.First add 40 μ L of sample diluting liquid on enzyme mark coating plate, then adds 10 μ L of sample to be tested again, gently
Shaking shakes up.
(4) it incubates: using 37 DEG C of incubation 30min of sealing plate film sealing plate postposition.
(5) it washs: dilution concentrated cleaning solution, it is spare.It takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up in every hole,
It is discarded after standing 30s.It is repeated 5 times, pats dry.
(6) enzyme: every hole adds 50 μ L enzyme marking reagents, except this bottom outlet.
(7) it incubates, washing.
(8) develop the color: every hole adds 50 μ L of color developing agent, then plus color developing agent B50 μ L, concussion mix, 37 DEG C are protected from light colour developing 15min.
(9) terminate: every hole adds 50 μ L of terminate liquid.
(10) it measures: being measured being added within terminate liquid 15min.With the zeroing of this bottom outlet, absorbance is measured under 450nm.
After obtaining the corresponding IL-2 concentration of each experimental group according to standard curve, it is calculated using the following equation stimulus index:
Stimulus index SI (%)=(wExperiment-wBlank)/wBlank× 100% (5).
W in formulaExperiment、wBlankThe respectively concentration of experimental group and naive mice splenic lymphocytes IL-2.
Figure 10 is that the Pacific oyster neutral polysaccharide prepared using the method for the present invention releases the IL-2 of mouse spleen lymphocyte
Measurement high-volume, it can be seen that mice spleen is drenched using the 250 μ g/mL of Pacific oyster neutral polysaccharide of the method for the present invention preparation
The stimulus index of bar cell IL-2 reaches 124.81%, can effectively stimulate mouse spleen lymphocyte immunocompetence.
Embodiment 6: the Pacific oyster neutral polysaccharide inhibition to Hepg-2 cell in vitro prepared using the method for the present invention
Rate measurement
(1) tumour cell of logarithmic growth phase uses DMEM culture solution (DMEM culture medium: tire ox after trypsin digestion
Serum: the 89:10:1 ratio of penicillin/streptomycin is prepared) cell is adjusted to 4 × 104/mL.It is (4000 thin by every 100 μ L of hole
Born of the same parents) it is added in 96 well culture plates.
(2) each 100 μ of Pacific oyster neutral polysaccharide of different diluted concentrations prepared using the method for the present invention is added after 4h
L, control group add isometric DMEM culture solution, and each concentration is all provided with 4 multiple holes, and setting 37 DEG C of volume fractions is 5%CO2Incubator
In continuously cultivate 48h.MTT (5g/L) 20 μ L is added in each hole 4h before culture terminates, and places and continues to be incubated for 4h in above-mentioned incubator, abandons
150 μ L of dimethyl sulfoxide is added in supernatant, every hole, and sufficiently oscillation surveys absorption photometric value (A) at microplate reader 570nm after mixing, meter
Inhibiting rate is calculated, calculation formula is as follows:
Inhibiting rate (%)=(AControl group-AMedicine group)/AControl group× 100% (6).
Figure 11 is the inhibition of the Pacific oyster neutral polysaccharide that is prepared using the method for the present invention to human liver cancer cell Hepg-2
Rate, it can be seen that human liver cancer cell Hepg-2 when the 200 μ g/mL of Pacific oyster neutral polysaccharide prepared using the method for the present invention
Inhibiting rate be 55.81%, can effectively inhibit the activity of liver cancer cell growth.
The detection of embodiment 7:Annexin V-FITC Apoptosis
(1) tumour cell of logarithmic growth phase, after trypsin digestion with DMEM complete culture solution adjust cell to 4 ×
10 4/mL.It is added in 96 well culture plates by every 100 μ L of hole (4000 cells).
(2) each 100 μ of Pacific oyster neutral polysaccharide of different diluted concentrations prepared using the method for the present invention is added after 4h
L, control group add isometric DMEM culture solution, and each concentration is all provided with 4 multiple holes, and setting 37 DEG C of volume fractions is 5%CO2Incubator
In continuously cultivate 48h.
(3) cell culture fluid is absorbed after apoptosis induction, PBS, which is added, washed once, and 195 μ LAnnexin are added
V-FITC combination liquid is added 5 μ L Annexin V-FITC, mixes gently.
(4) room temperature (20-25 DEG C), which is protected from light, is incubated for 10min, is protected from light using aluminium foil.190 μ L are added in solution removed by aspiration
Annexin V-FITC combination liquid.
(5) 10 μ L propidium iodide stain liquid are added, mix gently, ice bath avoid light place.
(6) immediately in fluorescence microscopy microscopic observation, Annexin V-FITC is green fluorescence, and PI is red fluorescence.
Figure 12 is detection of the bis- dye methods of Annexin V-FITC to human liver cancer cell Hepg-2 Apoptosis, wherein (a),
(b), (c), (d) corresponding blank group, positive group (50 μ g/mL of 5-Fluorouracil), oyster neutral polysaccharide (200 μ g/mL),
The neutral polysaccharide (200 μ g/mL) that traditional extraction process obtains, shown dead color are the tumour cell in necrosis and apoptosis advanced stage,
Light tone is the tumour cell of apoptosis early stage.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, 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, belong to claimed
Range.
Claims (6)
1. a kind of preparation method of Pacific oyster neutral polysaccharide, comprising the following steps:
(1) Pacific oyster dry powder is obtained into degreasing ostreae testa pulverata after dry by acetone degreasing；
(2) degreasing ostreae testa pulverata is soluble in water, it is handled with ultrasonic-microwave extraction instrument, by treated, solution is centrifuged, and supernatant steams
Hair concentration, obtains oyster polysaccharide crude extract；
(3) ethyl alcohol-(NH is added in oyster polysaccharide crude extract4)2SO4Double-aqueous phase system carries out extraction and separation, and lower phase extracted is
Oyster neutral polysaccharide crude extract；
(4) oyster neutral polysaccharide crude extract is dialysed, obtains oyster polysaccharide refining liquid, oyster can be obtained after concentrate drying
Neutral polysaccharide；
Wherein ethyl alcohol-(NH in step (3)4)2SO4Ethyl alcohol, (NH in double-aqueous phase system4)2SO4Weight ratio with PBS is 0.5:
0.32:1, and the ratio of double-aqueous phase system and oyster polysaccharide crude extract are as follows: 10:1, crude extract concentration are about 9mg/mL.
2. preparation method as described in claim 1, wherein the ultrasonic-microwave extraction instrument processing time is 20-30 points in step (2)
Clock, treatment temperature are 80-90 DEG C.
3. a kind of using Pacific oyster neutral polysaccharide prepared by the described in any item methods of claim 1-2.
4. Pacific oyster neutral polysaccharide as claimed in claim 3 is preparing the application in immune-related drug.
5. Pacific oyster neutral polysaccharide application in preparation of anti-tumor drugs as claimed in claim 3.
6. application as claimed in claim 5, wherein the tumour is liver cancer.
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| CN108013471A (en) * | 2017-11-02 | 2018-05-11 | 金华市飞凌生物科技有限公司 | The preparation method of oyster polysaccharide compound |
| CN107857824A (en) * | 2017-11-02 | 2018-03-30 | 金华市飞凌生物科技有限公司 | A kind of extracting method of oyster polysaccharide |
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