CN105669874A

CN105669874A - Peach gum polysaccharide degradation product PGP-2 as well as preparation method and application thereof

Info

Publication number: CN105669874A
Application number: CN201610072050.XA
Authority: CN
Inventors: 冉艳红; 王伟; 李弘剑; 周天鸿; 杨晓萍; 王雅秋
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2016-02-02
Filing date: 2016-02-02
Publication date: 2016-06-15
Anticipated expiration: 2036-02-02
Also published as: CN105669874B

Abstract

The invention provides a peach gum polysaccharide degradation product PGP-2. The PGP-2 is obtained by obtaining a fermentation broth by inoculating a peach gum culture medium with microbacterium A5 and performing separation and purification; the microbacterium A5 is preserved, with the preservation number of CCTCC NO:M209174 in the CCTCC (China Center for Type Culture Collection) on August 7, 2009; anion exchange is adopted for separation and purification, and a NaCl solution is adopted for gradient elution. The PGP-2 is simple to prepare, can significantly promote growth of Bifidobacterium while inhibiting growth of Enterococcus faecium, has higher scavenging activity for hydroxyl free radicals and DPPH free radicals and can significantly inhibit growth and proliferation of Hela cells, Galectin-3 mediated cell agglutination experiments indicate that the PGP-2 can significantly inhibit agglutination of Galectin-3 mediated red blood cells, Hela cells and MCF-7 cells.

Description

A kind of peach gum polysaccharide catabolite PGP-2 and its preparation method and application

Technical field

The invention belongs to peach gum polysaccharide preparing technical field, more particularly, to a kind of peach gum polysaccharide catabolite PGP-2 and its preparation method and application.

Background technology

Resina persicae is the resin being secreted in rosaceous plant Fructus Persicae or Prunus davidiana bark. About the medical value of Resina persicae, Chinese medical book is extensively on the books. Resina persicae is put down, and sweet-bitter flavor is good at promoting blood circulation and detumescence, treating stranguria pain relieving, is quenched the thirst, quenches one's thirst. The diseases such as stranguria with blood, stranguria caused by urinary stone, diabetes are all had good effect by clinical application.

China's Resina persicae aboundresources, is the natural resources of Chinese medicinal materials of a kind of high-quality. All being related to the report of peach gum polysaccharide composition both at home and abroad, but the enzyme hydrolysis of natural polysaccharide is all global problem all the time, the screening of efficient polysaccharide hydrolase bacterial strain and correlational study enjoy the concern of people always. Due to the steric hindrance of polysaccharide space conformation, single enzyme is difficult to complete for polysaccharide depolymerization. Steric hindrance produced by the protection of the short helical structure of Resina persicae itself and side chain so that general arabinogalactan hydrolytic enzyme such as tilactase, arabinosidase and galacturonic acid enzyme etc. are all difficult to be degraded. This seminar adopts the achievement of early-stage Study, the microbacterium A5 with decomposition Resina persicae ability is adopted to induce the method producing enzyme to be hydrolyzed peach gum polysaccharide in Resina persicae culture medium, and it is easily separated purification, expect to obtain a kind of product after enzymolysis, higher biologic activity can be represented, thus extracting further and application offer technical support for peach gum polysaccharide.

Summary of the invention

The present invention is according to the deficiency in current peach gum polysaccharide extractive technique, it is provided that a kind of peach gum polysaccharide catabolite PGP-2 and its preparation method and application.

The technical purpose of the present invention is achieved through the following technical solutions:

The invention provides a kind of peach gum polysaccharide catabolite PGP-2, described PGP-2 to be seeded in Resina persicae culture medium by microbacterium A5 and obtain fermentation liquid, separate purification and obtain;

Described microbacterium A5 is stored in China typical culture collection center on August 7th, 2009, and its deposit number is CCTCCNO:M209174;

Described separation purification adopts anion exchange to carry out, and eluting carries out gradient elution for adopting NaCl solution.

Preferably, gradient elution concentration respectively 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L, 1.0mol/L of described NaCl solution;

Described PGP-2 is the product after 0.2mol/LNaCl eluant solution.

Preferably, the preparation of described PGP-2 specifically includes following steps:

S1. by actication of culture, seed liquor is obtained;

S2. gained seed liquor in S1 being seeded to Resina persicae culture medium, carry out fermentation culture, in S2, fermentation temperature is 20 ~ 40 DEG C, initial Resina persicae concentration 4 ~ 8%, and medium pH is 4 ~ 6, and shaking speed is 160 ~ 200rpm, obtains fermentation liquid;

S3. gained fermentation liquid in S2 is easily separated purification, obtains described PGP-2;

Described S3 comprises the steps:

S31. gained fermentation liquid in S2 is centrifuged, obtains peach gum polysaccharide liquid, then filter, successively by filtration product through deproteinization, precipitate with ethanol, dried, obtain desciccate;

S32. by gained desciccate in S31 through anion exchange, concentration, dialysis, remove impurity, namely obtain described PGP-2 after drying.

The applicant adopts the achievement of early-stage Study, under specific enzymatic hydrolysis condition, adopt the microbacterium A5 with decomposition Resina persicae ability to induce the method producing enzyme to be hydrolyzed peach gum polysaccharide in Resina persicae culture medium, and in conjunction with separation purifying technique, extract and obtain peach gum polysaccharide PGP-2.

The polysaccharide product obtained after thalline is degraded has the impurity such as inorganic salt and the insoluble little molecule of alcohol in culture medium source, and the albumen of thalline and thalline secretion and other metabolite. Can by molecular size and shape classification (such as fractional precipitation, ultrafiltration, molecular sieve, chromatography etc.), it is possible to by the character classification (as by the electrophoresis of charge property classification, ion-exchange chromatography etc.) with group of the molecule institute and then polysaccharide catabolite is easily separated extraction.

Simultaneously, the present invention finds, PGP-2 can obtain through the mode of DEAE-Cellulose52 anion-exchange column, but can not be come by the Resina persicae crude polysaccharides product separation after by fermentation through the separate mode of SephadexG200 column chromatography and organic solvent eluting.

Preferably, in described S2, fermentation temperature is 30 DEG C, initial Resina persicae concentration 8%, and medium pH is 4, and shaking speed is 160rpm, and inoculum concentration is 3%.

Preferably, in described S1, actication of culture is that at 4 DEG C, described strain is preserved slant strains, and nutrient agar panel is rule, and is inverted for 37 DEG C and cultivates 24h, chooses monoclonal from flat board and be transferred to LB Tube propagation base, and in 200rpm rotating speed, at 37 DEG C, 12h is cultivated in concussion.

Preferably, in described S31, deproteinization adopts Sevage method deproteinization, and in described S31, precipitate with ethanol is the ethanol of employing 75%;

In described S32, concentration is for adopting PEG 8000 to carry out concentration, and described dialysis is the bag filter adopting molecular cut off 200.

In the present invention, the purification procedures of fermentation liquid is as follows:

。

The present invention is by carrying out relevant biological activity research to PGP-2, find that it is to suppressing Hela Growth of Cells and propagation, and inhibitory action increases with concentration and increases, and the coagulation of the chicken red blood cells that can Gal-3 be induced, Hela cell, MCF-7 Breast Cancer Cell produces inhibitory action. The growth of bacillus bifidus and bacillus subtilis can also be promoted, it is suppressed that the growth of enterococcus faecalis. Additionally, hydroxyl radical free radical and DPPH are had stronger Scavenging activity by PGP-2, the practical application for natural polysaccharide product provides technical support, possesses the prospect of being widely applied.

Compared with prior art, the method have the advantages that

The PGP-2 preparation of the offer of the present invention is simple, and the growth of bacillus bifidus can be remarkably promoted, suppress the growth of enterococcus faecalis, and possess the stronger Scavenging activity to hydroxyl radical free radical and DPPH free radical, the cell aggregation Inhibition test of Galectin-3 mediation is shown, it can significantly inhibit the coagulation of the chicken red blood cells of Galectin-3 mediation, Hela cell and MCF-7 cell.

Accompanying drawing illustrates:

Fig. 1 be in embodiment 2 Resina persicae crude polysaccharides through DEAE-52 anion-exchange column NaCl gradient elution curve.

Fig. 2 is the infrared spectrogram of PGP-2.

Fig. 3 is the MALDI-TOF-MS figure of PGP-2.

Fig. 4 is PGP-2 and the PGP-2 impact that several intestinal are grown.

Fig. 5 is the antioxidant activity impact of PGP-2.

Fig. 6 is that PGP-2 is to Hela cytotoxicity and cell growth inhibition.

Fig. 7 is the PGP-2 impact on Hela cell proliferation.

Detailed description of the invention

By the examples below the present invention is specifically described; be necessary it is pointed out here that be that the present embodiment is served only for the present invention is further described; but it is not intended that limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.

Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.

Embodiment 1: fermentation liquid prepared by microbacterium degraded Resina persicae:

1, actication of culture

4 DEG C preserve slant strains, and nutrient agar panel is rule, and are inverted for 37 DEG C and cultivate 24h, choose monoclonal from flat board and be transferred to LB Tube propagation base, and 6ml/ props up, 200rpm, 37 DEG C, and 12h is cultivated in concussion.

The LB fluid medium of 1.8%: 3.6g compound LB, deionized water is settled to 200ml, PH=4,121 DEG C, and sterilizing in 20 minutes is standby.

Strain is microbacterium A5, is stored in China typical culture collection center on August 7th, 2009, and its deposit number is CCTCCNO:M209174;

2, shake-flask culture

Being seeded in Resina persicae Shake flask medium by the seed liquor after activation, 500ml triangular flask, liquid amount is 200ml, and initial Resina persicae concentration is 8%, initial pH=4, and inoculum concentration is 3%, and cultivation temperature is 30 DEG C, 24h is cultivated in 160rpm concussion. Fermentation liquid after cultivation is for the separation of follow-up peach gum polysaccharide catabolite.

The preparation of peach gum polysaccharide culture medium: accurately weigh peach gum polysaccharide, deionized water constant volume is to 200ml, PH=4. 121 DEG C, sterilizing in 15 minutes is standby.

Embodiment 2: embodiment 1 gained fermentation liquid is easily separated purification:

The fermentation liquid first embodiment 1 prepared and Sevage reagent (chloroform: amylalcohol or n-butyl alcohol (mixing with 4: 1 or 5: 1 ratios) mix by a certain percentage, it is then centrifuged for removing the denatured protein between extracting solution Yu Sevage reagent intersection, then adopt the ethanol of 75% to precipitate above-mentioned substance, obtain Resina persicae crude polysaccharides.

DEAE-Cellulose52 anion exchange is selected to be further purified Resina persicae crude polysaccharides, specific as follows:

Take 10gDEAE-52 and add 500ml water, soak 24h, make cellulose grain fully expand, remove the fine grained suspended. Transfer thereafter to sucking filtration in buchner funnel (nylon wire of interior pad 200 order), 4h is soaked by 200ml0.5mol/LNaOH NaCl solution, transfer to sucking filtration in buchner funnel (nylon wire of interior pad 200 order), it is washed till neutrality with distilled water, transfer to after draining in beaker, soak 4h with 200ml0.5mol/LHCl. Being then transferred in buchner funnel sucking filtration, be washed till neutrality with distilled water, be transferred in beaker after draining, such soda acid is repeatedly taken turns and is washed, till eluate achromaticity and clarification. Finally soak 20min with the phosphate buffer of 150ml0.02mol/L, PH=6.5, after degassed, fill post, standby.

The preparation of DEAE-Cellulose52 anion-exchange resin column: first by good for the chromatographic column vertical rack of 1.8 × 40cm, with appropriate aqueous solution elder generation rinse leak detection, bottom is kept to have little water solution, then the DEAE-52 anticipated is stirred evenly, it is poured into continuously in post with by this suspension, when the 1/4-1/3 that its natural subsidence to post is high, open the outlet of post lower end, allow solvent ooze, make post upper end suspension slowly drop to the height of needs.Adsorbent surface is smooth, it should be made always to be immersed in solvent, be strictly on guard against that bubble produces. Control its flow velocity, allow 200m1's (about 5 times of column volumes) to flow through fixing phase by the phosphate buffer balance of 0.02mol/L, pH=6.5 so that it is reaching balance, fixing phase constant height is 30cm.

Sample is through the centrifugal 10min of 5000rpm. Take supernatant and cross 0.22um microporous filter membrane, loading. The loading volume of peach gum polysaccharide is 5ml, and sample concentration is 10g/L.

The eluting of DEAE-Cellulose52 anion-exchange resin column adopts the NaCl solution of variable concentrations to carry out gradient elution, eluting concentration respectively 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L, 1.0mol/L, collect eluted product respectively.

First carrying out eluting with after 200ml distilled water eluting with the NaCl that salinity is 0～1.0mol/L, each concentration elution volume is 100ml, and flow speed control is at 1ml/min, and fraction collector controls speed and manages at 5min/.

Through DEAE-Cellulose52 anion-exchange chromatography separation purification result as it is shown in figure 1, from figure 1 it appears that distilled water elution fraction does not have absworption peak appearance, can illustrate not contain neutral polysaccharide in the produced polysaccharide solution of A5 microbacterium degraded.

The present invention this stage be respectively adopted simultaneously SephadexG200 gel filtration chromatography and 75% ethanol be easily separated purification, peach gum polysaccharide component is not separated further after eluting, illustrate that the catabolite after by fermentation can not carry out the extraction of natural product by which.

Eluted product is adopted PEG 8000 to carry out concentration after completing by eluting. Step, for eluent is loaded bag filter, is placed in saturated PEG 8000 solution, and 4 DEG C overnight, and namely bag internal solvent is absorbed by Polyethylene Glycol, reaches the purpose of sample concentration.

Then the solution after concentration is put into flowing water dialysis 48h in bag filter (molecular cut off 200), 0.2MNaCl elution fraction obtains described PGP2.

The qualification of embodiment 3:PGP-2 and analysis

1, the IR spectrum analysis of PGP-2

The PGP2 weighing 5mg carries out KBr tabletting, at 400～4000cm^-1Between scanning.

As in figure 2 it is shown, PGP-2 is at 3430cm^-1The sugared ring stretching vibration absworption peak of O-H is only small, 2888cm^-1Place is for C-H(mainly includes CH, CH₂、CH₃) stretching vibration absworption peak, 1500～1200cm^-1Several absworption peak (1243cm that place exists^-1、1359cm^-1、1468cm^-1) it is the vibration of C-H angle, 1110cm^-1There is the angle vibration of C-O-C, 961cm in place^-1The peak at place is likely the vibration of D-Glucose to be caused, 843cm^-1Place is the vibration of pyranose β type C-H angle.

2, the MALDI-TOF-MS of PGP-2 analyzes:

Taking PGP-2 polysaccharide sample 1mg to be dissolved in the TFA of 10 μ l, carry out MALDI-TOF-MS analysis, result is as shown in Figure 3.

Being drawn by Fig. 3, peak below is in 877.5～1493.9 scopes, and differing molecular weight between each peak is 44, infers that PGP-1 is mainly made up of acidic polysaccharose, wherein contains substantial amounts of alduronic acid. according to monosaccharide composition analysis result calculate each degree of polymerisation Saccharide form 877.5 places corresponding be by 6 Arabic monosaccharide dehydrating condensations and the oxidized six arabinose aldehydic acid molecular weight formed, 921.6 what place was corresponding is six arabinose dialdehyde acid molecule amounts of 6 Arabic monosaccharide formation, 965.6 what place was corresponding is six xylose three aldehydic acid of 6 xylose monosaccharide formation, 1009.6 what place was corresponding is six xylose four aldehydic acid, what 1053 places were corresponding is six arabinose five aldehydic acid, 1097.7 what place was corresponding is six xylose six aldehydic acid, 1141.7 what place was corresponding is by three xyloses and four arabinose six aldehydic acid, 1229.8 what place was corresponding is three xylose four arabinose seven aldehydic acid, 1273.8 what place was corresponding is tri-glucose four mannose three aldehydic acid, 1317, what 3 places were corresponding is three lactose four glucose four aldehydic acid, the acidic polysaccharose that PGP-2 is made up of the alduronic acid that the degree of polymerization is different in summary.

Embodiment 4: compliance test result:

1, the impact that several intestinal are grown by PGP-2

(1) medicine Medilac-Vita one is wrapped (1g/ bag) and golden bifid a piece of (0.5g/ sheet) dissolves with aseptic 0.9%NaCl respectively, be made into the bacterium solution of 0.01g/ml, and dilute 10^-4Making sample bacterium solution again, by the method for mixing slat chain conveyor, each flat board bacterium solution 1ml, the PGP-2 adding 100mg, 200mg, 500mg and 1000mg respectively does three repetitions, to be not added with any sugar-like for blank.

(2) being cultivated by KF based in constant incubator 37 DEG C of cultivation 24h, selectivity cultivates enterococcus faecalis; TPY culture medium is placed in CO₂37 DEG C of Anaerobic culturel 24h in incubator, selectivity cultivates bacillus bifidus; Nutrient agar is 37 DEG C of cultivation 24h in constant incubator, and selectivity cultivates bacillus subtilis.

(3) after cultivating 24h, each culture medium being taken out, assessing the PGP-2 of the variable concentrations impact on three kinds of bacteria growings by the thalline quantity of experimental group Yu matched group being done contrast.

As shown in Figure 4, wherein a, b and c represent the impact on enterococcus faecalis, bacillus bifidus and bacillus subtilis to result respectively, and as can be known from Fig. 4, PGP-2 can remarkably promote the growth of above-mentioned several intestinal.

2,1) PGP-2 is to hydroxyl radical free radical elimination effect:

Principle is for utilizing H₂O₂With Fe²⁺Mixing produces hydroxy radical, it may be assumed that H₂O₂+Fe²⁺→·OH+H₂O₂+Fe²⁺. Adding salicylic acid again in system catch hydroxy radical and produce coloring matter, this material has absorption maximum at 510nm place. The content of hydroxy radical is represented with this light absorption value.

Specifically comprise the following steps that in reaction system containing 1ml8.8mmol/LH₂O₂,1ml9mmol/LFeSO₄, 1ml9mmol/L salicylic acid-ethanol, 1ml solution to be measured, wherein H₂O₂It is eventually adding and starts whole reaction. 37 DEG C of centrifugal 6min of reaction 0.5h, 12000r/min, then make reference with distilled water, measure absorbance under 510nm. Consider that the absorbance of solution to be measured itself is different, with 1.0ml9mmol/LFeSO₄Solution, 1.0ml9mmol/L salicylic acid-alcoholic solution, the sample solution of 1.0ml variable concentrations, 1.0ml distilled water are as the background absorption value of sample. Blank group distilled water replaces sample solution, and matched group distilled water replaces hydrogenperoxide steam generator.

In formula: A₀-for the absorbance of blank liquid;

A₁-for the absorbance after addition sample solution;

A₂-for being not added with H₂O₂The absorbance of sample solution background.

2), the PGP-2 removing to DPPH free radical

Take the sample solution of 2.0ml variable concentrations respectively in test tube, add 2.0ml1.0 × 10^-4The DPPH solution of mmol/L, mix homogeneously, after lucifuge stands 30min, returns to zero as blank using equal-volume distilled water and 50% alcohol mixeding liquid, measures absorbance A in 517nm place₁, measure the absorbance A 2 of 2.0ml sample solution and 2.0ml50% alcohol mixeding liquid and the absorbance A of 2.0ml50% alcohol mixeding liquid simultaneously₀。

;

Result is as it is shown in figure 5, PGP-2 possesses the significant Scavenging activity to hydroxyl radical free radical and DPPH free radical.

3, to Hela cytotoxicity experiment

(1) inoculating cell: the monolayer Hela adherent with 0.25% tryptic digestive juice digestion cultivates cell, it is configured to individual cells suspension with culture fluid in the DMEM complete medium containing 10% hyclone and dual anti-(penicillin 100U/ml and 100 μ g/ml streptomycins), adjusting the concentration of cell suspension inner cell, making concentration of cell suspension is 1.0 × 10⁴Individual/ml, then by cell suspension inoculation cell in 96 well culture plates, once 2 culture plates of inoculation, every piece of every hole of culture plate adds above-mentioned cell suspension 180 μ l.

(2) cell is cultivated: the culture plate inoculated is inserted saturated humidity, 37 DEG C, 5%CO₂Incubator is cultivated 24h, after cell attachment, with the supernatant in liquid-transfering gun gentle aspiration culture plate, discards.

(3) experiment packet: matched group (equivalent PBS), experimental group PGP-2 group, sets 6 concentration groups: 5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, 500 μ g/ml by PGP-2 group. Each group intervenes cervical cancer cell Hela cell 48,72h respectively, and in above experiment packet, each group arranges 3 parallel holes, repeats 3 times.

(4) colour generation: cultivate after 48h, 72h in incubator respectively, add 20 μ l5mg L-1MTT solution in the 96 every holes of orifice plate, continue to be placed in 37 DEG C, 5%CO₂, saturated humidity incubator is cultivated 4 hours, then terminate cultivating, carefully absorb supernatant culture fluid in every hole with liquid-transfering gun, then add DMSO150 μ l toward every hole, on shaking table, then shake 10min, after first is fully dissolved, use enzyme-linked immunosorbent assay instrument colorimetric.

(5) colorimetric: measure the optical density value (OD value) in each hole on 96 orifice plates at the 570nm wavelength place of enzyme-linked immunosorbent assay instrument, adds distilled water in blank well, is used for returning to zero.

(6) computing formula: inhibitory rate of cell growth (IR)=(1-adds PGP-2 group mean OD value/Normal group mean OD value) × 100%.

4, to Hela cell growth inhibition test

(1) inoculating cell: with the single-layer culturing cell that 0.25% tryptic digestive juice digestion is adherent, it is configured to individual cells suspension with containing 10% newborn calf serum RPMI-1640 culture fluid, adjusting the concentration of cell suspension inner cell, making concentration of cell suspension is 5.0 × 10⁴Individual/ml, then by cell suspension inoculation cell in 96 well culture plates, once 3 culture plates of inoculation, every piece of every hole of culture plate adds above-mentioned cell suspension 180 μ l.

(3) experiment packet: matched group (equivalent PBS), experimental group PGP-2 group, sets 3 concentration groups: 25 μ g/ml, 50 μ g/ml, 100 μ g/ml by PGP-2 group. Each group intervene respectively cervical cancer cell Hela cell 24,48,72h, in above experiment packet, each group arranges 3 parallel holes, repeats 3 times.

(4) in incubator, cultivate 24 respectively, after 48h, 72h, take out at room temperature or 37 DEG C, digest attached cell with trypsinization liquid, make cell suspension. Take each one piece of coverslip, counting slide, first clean with PBS, again with 75% ethanol wiping counting slide and coverslip repeatedly, then air-dry, covered, draw cell suspension 5 μ l with 10 μ l liquid-transfering guns to be instilled slowly in counting slide by coverslip side, make uniform full cell suspension body between microscope slide and coverslip.

(5) again with, under 10 × object lens microscope, observing counting lattice interior celliferous total number X in corner on counting slide. Counting principle: during cell pressure center line, under not several on number, the not number right side, a number left side, cell mass adds up to a cell. Counting gained total cellular score is substituted into formula, draws cell density=X/4 × 10⁴Individual/ml(cell number/milliliter stock solution).

Result as shown in Figures 6 and 7, draws from Fig. 6, and when PGP-2 concentration is more than 25 μ g/ml, growth over time, the growth of Hela cell is suppressed. As PGP-2 concentration 500 μ g/ml, 48h cell survival rate reduces about 10% compared with matched group, and 72h cell survival rate reduces about 25%.

Learning from Fig. 7, compared with the control, 0-3 days, along with the increase of PGP-2 concentration, the propagation of cell was subject to obvious suppression.

5, PGP-2 suppresses Gal-3 activity:

(1) the separation purification of chicken red blood cell: gather fresh Sanguis Gallus domesticus, pour in the beaker filling Alsever liquid and shake up, it is prevented that blood coagulation. Adding 5 times of volume 0.15MNaCl to be washed by Sanguis Gallus domesticus five times, 2500rpm is centrifuged 10min; Abandoning supernatant, add 0.02MPBS, hanged by cell, make the suspension of 4%, then washed 4 times by cell with 5 times of volume 0.15MNaCl in precipitation, 2500rpm is centrifuged 10min, abandons supernatant; Having hanged precipitation with 0.02MPBS, washed cell 2 times with PBS, finally hanged by cell PBS, 4 DEG C of conditions preserve.

(2) cultivation of Hela cell: Hela cell takes out recovery from liquid nitrogen container, is incubated in the DMEM complete medium containing 10% hyclone and dual anti-(penicillin 100U/ml and 100 μ g/ml streptomycins), is positioned over 37 DEG C, 5%CO₂Incubator is cultivated, carries out going down to posterity or conservation when cell fusion degree reaches about 90%.

(3) cultivation of MCF-7 Breast Cancer Cell: with (2).

(4) process of Hela cell: take out Hela cell from CO2 incubator, with the single-layer culturing cell that 0.25% tryptic digestive juice digestion is adherent under super-clean bench, then the centrifugal 10min of 2500rpm, abandons supernatant; Having hanged precipitation with 0.02MPBS, washed cell 2 times with PBS, finally hanged by cell PBS, 4 DEG C of conditions preserve.

(5) process of MCF-7 Breast Cancer Cell: with (4)

(6) the Galectin-3 concentration screening that cell agglutination is the suitableeest: with the PBS Galectin-3 solution preparing variable concentrations, simultaneously the erythrocyte PBS prepared is diluted to 4% suspension. Being separately added into the Galectin-3 of variable concentrations (25 μ g/ml, 50 μ g/ml, 100 μ g/ml) in V-type 96 orifice plate, then be sequentially added into 25 μ l normal saline, 25 μ l1%BSA and 25 μ l4% chicken red blood cells in every hole, cumulative volume is 100 μ l. Room temperature stands 30min, observes red cell agglutination situation. The method screening the suitableeest Galectin-3 concentration during with Hela cell with breast cancer cell line MCF-7 is identical with chicken red blood cells.

(7) PGP-2 is to Galectin-3 activity inhibition:

Preparation of samples: the PGP-2 taking variable concentrations (25 μ g/ml, 50 μ g/ml, 75 μ g/ml) is that 25 μ l mix with Galectin-3 respectively, room temperature stands 10min.

The mixture of application of sample: experimental port is sequentially added into: 1%BSA, sugar-like and Galectin-3, adds the erythrocyte of 4% after mixing. Positive control wells is sequentially added into: 1%BSA, Galactose and Galectin-3 mixture, adds the erythrocyte of 4% after mixing. Negative control hole is sequentially added into: 1%BSA, 0.15MNaCl and Galectin-3 mixture, adds the erythrocyte 25 μ l of 4% after mixing.

Observe: room temperature observes erythrocytic coagulation degree after standing 90min, the inhibitory activity of sugar-like is represented with the Cmin of sugar-like needed for suppressing red cell agglutination completely, namely needed for suppressing red cell agglutination completely, sugar-like concentration is more little, represent that the rejection ability of this sugar-like is strong, more strong with the binding ability of Galectin-3.

Hela cell is identical with the coagulation experiment of the coagulation experimental procedure of MCF-7 Breast Cancer Cell Yu chicken red blood cells.

Obtaining from above experiment, when PGP-2 concentration is 50 μ g/ml, haemagglutination phenomenon disappears, and the minimal inhibitory concentration to Galectin-3 (MIC) of PGP-2 is 50 μ g/ml.

Galectin-3 being mediated in the coagulation of Hela cell, when PGP-2 concentration is 200 μ g/ml, the cell agglutination of Galectin-3 mediation disappears, and the minimal inhibitory concentration (MIC) of PGP-2 is 200 μ g/ml.

Obtaining in the coagulation suppression of MCF-7 cell, when PGP-2 concentration is 100 μ g/ml, the cell agglutination of Galectin-3 mediation disappears, and the minimal inhibitory concentration (MIC) of PGP-2 is 100 μ g/ml.

Claims

1. a peach gum polysaccharide catabolite PGP-2, it is characterised in that described PGP-2 is seeded in Resina persicae culture medium by microbacterium A5 and obtains fermentation liquid, separates purification and obtains;

2. peach gum polysaccharide catabolite PGP-2 according to claim 1, it is characterized in that, gradient elution concentration respectively 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L, 0.8mol/L, 0.9mol/L, 1.0mol/L of described NaCl solution;

Described PGP-2 is the product after 0.2mol/LNaCl eluant solution.

3. peach gum polysaccharide catabolite PGP-2 according to claim 1, it is characterised in that the preparation of described PGP-2 specifically includes following steps:

S1. by actication of culture, seed liquor is obtained;

Described S3 comprises the steps:

4. preparation method according to claim 3, it is characterised in that in described S2, fermentation temperature is 30 DEG C, initial Resina persicae concentration 8%, medium pH is 4, and shaking speed is 160rpm, and inoculum concentration is 3%.

5. preparation method according to claim 3, it is characterized in that, in described S1, actication of culture is that at 4 DEG C, described strain is preserved slant strains, nutrient agar panel is rule, it is inverted for 37 DEG C and cultivates 24h, choosing monoclonal from flat board and be transferred to LB Tube propagation base, in 200rpm rotating speed, at 37 DEG C, 12h is cultivated in concussion.

6. preparation method according to claim 3, it is characterised in that in described S31, deproteinization adopts Sevage method deproteinization, in described S31, precipitate with ethanol is the ethanol of employing 75%.

7. preparation method according to claim 3, it is characterised in that in described S32, concentration is for adopting PEG 8000 to carry out concentration, and described dialysis is the bag filter adopting molecular cut off 200.

8. the peach gum polysaccharide catabolite PGP-2 described in claim 1 promotes the application in beneficial bacteria of intestinal tract and natural polysaccharide antioxidant and antitumor product in preparation.

9. the peach gum polysaccharide catabolite PGP-2 described in claim 1 suppresses the application in erythrocyte, Hela cell, MCF-7 Breast Cancer Cell aggregation products in preparation.

10. a Gal-3 inhibitor, it is characterised in that containing the peach gum polysaccharide catabolite PGP-2 described in claim 1;Described PGP-2 suppresses the minimal inhibitory concentration of the erythrocyte aggregation of Gal-3 mediation to be 50 μ g/ml; Described PGP-2 suppresses the minimal inhibitory concentration of the Hela cell aggregation of Gal-3 mediation to be 200 μ g/ml; Described PGP-2 suppresses the minimal inhibitory concentration of the MCF-7 cell aggregation of Gal-3 mediation to be 100 μ g/m.