CN101220101A - Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof - Google Patents
Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof Download PDFInfo
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- CN101220101A CN101220101A CNA2008100140478A CN200810014047A CN101220101A CN 101220101 A CN101220101 A CN 101220101A CN A2008100140478 A CNA2008100140478 A CN A2008100140478A CN 200810014047 A CN200810014047 A CN 200810014047A CN 101220101 A CN101220101 A CN 101220101A
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Abstract
The invention discloses a polysaccharide from Trichoderma pseudokoningii, which is characterized in that: (1) the high performance gel filtration chromatography test shows that the polysaccharide has a symmetrical peak with 18325 molecular weight; (2) an improved sulfuric acid-phenol method and a meta-phenylphenol method test show that the neutral polysaccharide content is 65.2 percent, while the uronic acid content is 32.6 percent; (3) the GC analysis of the alditol acetate shows that the monosaccharide components are rhamnose, glucose and galactose, the molar ratio is that Rha:Glc:Gal equals to 5.6:2.7:1.0, while when the polysaccharide is completely reduced, the molar ratio is that Rha:Glc:Gal equals to 14.5:9.3: 1.0, having 26.6 molar percent glucuronic acid. The preparation method of the polysaccharide from Trichoderma pseudokoningii consists of the crude polysaccharide preparation and the polysaccharide purification. The polysaccharide from the Trichoderma pseudokoningii has broad applications in preparing the drugs or functional food for improving the mammal immunocompetence and for treating or adjuvant treating the tumors.
Description
Technical field
The invention belongs to microorganism field, relate to a kind of polysaccharide that has enhancing immunity, suppresses tumor growth, the immunocompetent exocellular polysaccharide that has that is particularly related to that trichoderma pseudokiningii CGMCC No.1443 (Trichoderma pseudokoningii) produced (is called for short wooden mould polysaccharide, EPS) and its production and use.
Background technology
The mould traditional classification system of pressing of wood is under the jurisdiction of Deuteromycotina (Deuteromycotina), hyphomycetes (Hyphomycetes), hyphomycetales (Hyphomycetales), silk spore section (Hyphomycetaceae).Wood is mould to be the widely distributed fungi of a class, except that indivedual kinds be the weak pathogenic bacterium, most have antagonistic action to phytopathogen, is research and use a kind of at most in the biocontrol fungi, it all has restraining effect to multiple pathogenic bacteria, is that a class ideal is prevented and treated the biocontrol microorganisms of soil-borne disease.
Fungus polysaccharide is isolated from fungus sporophore, mycelium, fermented liquid, can control the cell fission differentiation, regulates the old and feeble class active polysaccharide of cell growth.The compound of polysaccharide of fungi has very strong biological activity, mainly concentrates on immunomodulatory, antitumor, antiviral, anti-mutation, anti-ageing, reducing blood-fat, antiulcer agent, aspect such as anti-oxidant.Strong immunity and anti-tumor activity are the polysaccharide important biological.Studies show that, the secretion that polysaccharide can be by enhancing immunity cytoactive, the activating cells factor, induce effect enhancing body immunologic functions such as generating antibody and activating complement system.The antitumor approach of polysaccharide is mainly the synthetic of the growth, protein and the nucleic acid that suppress tumour cell, the growth characteristics of inducing apoptosis of tumour cell, the expression that influence oncogene and change tumor cell membrane.Trichoderma pseudokiningii CGMCC No.1443 is a kind of the wooden mould of Johnson ﹠ Johnson's growing way very that have, wood is mould grow and envrionment conditions (nutrition, temperature, pH value, illumination etc.) that the generation of spore needs all fairly simple, it is very fast to nourish and grow.Trichoderma pseudokiningii CGMCC No.1443 secretes a large amount of active exocellular polysaccharides, and its exocellular polysaccharide secretion condition is simple, the content height, and in every liter of nutrient solution, the content of Crude polysaccharides can reach 700mg, and its separation and purification mode is simple, has stronger using value.CN1748511 (200510044527.5) discloses the biotechnological formulation that contains trichoderma pseudokiningii CGMCC No.1443, is used to prevent and treat the plant multiple diseases.Also do not have immunocompetent report and patent disclosure at present about trichoderma pseudokiningii CGMCCNo.1443 exocellular polysaccharide, the polysaccharide of our separation and purification from the trichoderma pseudokiningii fermented liquid, has stronger immuno-stimulating ability, can significantly induce the apoptosis of fibroma cell, prolong S180 ascitic tumor mouse life, the knurl that alleviates S180 solid tumor mouse is heavy, increases its spleen index.
Summary of the invention
At the deficiencies in the prior art, one of purpose of the present invention aims to provide a kind of trichoderma pseudokoningii exocellular polysaccharide.
Two of purpose of the present invention aims to provide a kind of preparation method of trichoderma pseudokoningii exocellular polysaccharide.
Three of purpose of the present invention aims to provide the important use of trichoderma pseudokoningii exocellular polysaccharide.
Trichoderma pseudokoningii exocellular polysaccharide of the present invention is characterized in that,
(1) detect by the efficient gel filtration chromatography, it has single symmetrical peak, and molecular weight is 18325;
(2) sulfuric acid-phynol method and the phenylphenol method by improvement detects, and its neutral polysaccharide content is 65.2%, and glucuronic acid content is 32.6%;
(3) analyze by the gas-chromatography (GC) of alditol acetate, its monose consists of rhamnosyl (Rha), glucose (Glc) and semi-lactosi (Gal), and mol ratio is Rha: Glc: Gal=5.6: 2.7: 1.0.
After this polysaccharide reduced fully, the mol ratio of rhamnosyl, glucose and semi-lactosi was Rha: Glc: Gal=14.5: 9.3: 1.0, and its to contain molar content be 26.6% glucuronic acid.This trichoderma pseudokoningii exocellular polysaccharide is with trichoderma pseudokiningii fermented liquid decolouring, concentrate, behind alcohol precipitation and the Deproteinization, the homogeneous polysaccharide that obtains by the gel chromatography column purification.
The preparation method of trichoderma pseudokoningii exocellular polysaccharide of the present invention comprises the preparation of step (1) Crude polysaccharides and the purifying of step (2) polysaccharide, it is characterized in that the preparation of described step (1) Crude polysaccharides comprises:
A, trichoderma pseudokiningii inserted liquid nutrient medium after, 28 ℃, 130 rpms (rpm) shaking culture 6 days (d);
B, centrifugal mycelia and the spore removed of filtration are crossed the decolouring of 3520 resin columns, collect elutriant, are evaporated to 1/4 of original volume;
C, concentrated solution Sevag method deproteinated;
D, adding ethanol are in 4 ℃ of standing over night, and centrifugal collecting precipitation washs successively with ether, acetone, 95% ethanol, and precipitation is dissolved in distilled water, and dialysing was concentrated to original volume 1/4 after (h) in 48 hours, got the crude extracellular polysaccharide sample through lyophilize,
The purifying of described step (2) polysaccharide comprises:
A, employing dextrane gel (Sephadex) G-75 column chromatography accurately take by weighing wooden mould crude extracellular polysaccharide 1g, be dissolved in the water of 10ml, and upper prop, water carries out wash-out under the room temperature, control flow velocity 0.4ml/min;
B, with branch's automatic receptor per 15 minutes one pipes (min/ pipe), the phenolsulfuric acid development process is followed the tracks of detection by pipe, in 490nm place mensuration absorbance value;
C, light absorption value is mapped, merge the polysaccharide soln in single peak district according to elution volume, concentrating under reduced pressure, lyophilize obtains trichoderma pseudokoningii exocellular polysaccharide preliminary purification component;
With aforementioned gained preliminary purification component S ephadex G-100 sephadex chromatography purifying, get the purpose product.
This trichoderma pseudokoningii exocellular polysaccharide has widely in preparing raising mammalian immune active medicine or functional foodstuff and in the medicine for preparing treatment or adjuvant therapy of tumors, functional foodstuff to be used.Polysaccharide of the present invention all has promoter action to specificity and non-specific immunity, humoral immunization or cellular immunization, and rat fibroma cell is had stronger apoptosis-induced effect, and S180 ascitic tumor and the solid tumor of mouse all had restraining effect.
Trichoderma pseudokoningii exocellular polysaccharide disclosed by the invention is by the trichoderma pseudokiningii liquid fermentation liquid, through decolouring, concentrate, after methods such as Deproteinization, alcohol precipitation obtain Crude polysaccharides, the further homogeneous component (EPS) that obtains by gel chromatography column.Polysaccharide of the present invention detects by the efficient gel filtration chromatography, finds that this component is single symmetrical peak, and molecular weight is 18325; By a sulfuric acid-phynol method and a phenylphenol method of improvement, find that neutral polysaccharide content is 65.2% in this polysaccharide, glucuronic acid content is 32.6%.Show that through sugared compositional analysis the TLC of complete hydrolysis product analyzes demonstration and contains a certain amount of uronic acid; The GC analysis revealed of alditol acetate contains rhamnosyl, glucose and a spot of semi-lactosi, and mol ratio is Rha: Glc: Gal=5.6: 2.7: 1.0.EPS carries out carboxyl reduction with the Conrad method, reduces to obtain EPS-0-R after 3 times, and it is carried out sugared compositional analysis, and TLC analyzes and do not contain uronic acid, shows that reduction is complete.TPP-0-R GC analysis revealed, contain rhamnosyl, glucose and semi-lactosi in it, mol ratio is Rha: Glc: Gal=14.5: 9.3: 1.0, illustrate that this polysaccharide contains glucuronic acid (GlcA), its molar content is 26.6%, the quality percentage composition is about 31.9%, and this is consistent with the result that a phenylphenol method is measured.
The large-scale producing method of trichoderma pseudokoningii exocellular polysaccharide of the present invention may further comprise the steps:
(1) preparation of Crude polysaccharides:
Trichoderma pseudokiningii CGMCC No.1443 is inoculated in potato dextrose agar (PDA) activation, inserts in the liquid nutrient medium 28 ℃, 100rpm shaking culture 6d then by flat board.Remove mycelia and spore with four layers of filtered through gauze, the centrifugal 15min of 4000rpm, supernatant cross the decolouring of 3520 resin columns, collect elutriant, and 48 ℃ are evaporated to 1/4 of original volume.Concentrated solution Sevag method (chloroform: propyl carbinol=4: 1, V/V) deproteinated.95% ethanol that adds 3 times of volumes is in 4 ℃ of standing over night, the centrifugal 15min of 5000rpm, collecting precipitation, ether, acetone, 95% ethanol wash successively, precipitation is dissolved in distilled water, is concentrated to original volume 1/4 behind the dialysis 48h, gets crude extracellular polysaccharide sample (EPS) through lyophilize.
(2) purifying of polysaccharide:
Adopt Sephadex G-75 dextrane gel column chromatography, accurately take by weighing wooden mould crude extracellular polysaccharide 1g, be dissolved in the water of 10ml, upper prop, water carries out wash-out under the room temperature, and flow velocity 0.4ml/min is with the every 15min/ pipe of branch's automatic receptor.Follow the tracks of detection with the phenolsulfuric acid development process by pipe, measure absorbance value in the 490nm place.According to elution volume light absorption value is mapped, merge the polysaccharide soln in single peak district, concentrating under reduced pressure, lyophilize obtains trichoderma pseudokoningii exocellular polysaccharide component EPS.Use Sephadex G-100 sephadex chromatography to be further purified two kinds of components that obtain.
Polysaccharide of the present invention improves active functional food of mammalian immune and medicine in preparation, and the medicine aspect of treatment or auxiliary for treating cancer has widely to be used.Just activating splenocyte propagation, strengthening the macrophage phagocytic activity, having greater activity aspect the output that stimulates scavenger cell NO, activity of acid phosphatase when the cell in vitro experimental result shows polysaccharide EPS low concentration of the present invention; Rat fibroma cell there is stronger apoptosis-induced effect, and has dose-dependent effect; Simultaneously, S 180 ascitic tumors and the solid tumor to mouse all has significant retarding effect.Toxicological experiment shows that this sugar does not have toxicity at the following pair cell of 30mg/ml concentration.The polysaccharide that shows this invention can be used as activeconstituents preparation raising active functional food of mammalian immune and medicine, the medicine of treatment or auxiliary for treating cancer.
Description of drawings
Fig. 1: trichoderma pseudokoningii exocellular polysaccharide EPS is to the influence of splenocyte propagation.
Fig. 2: the collaborative ConA of trichoderma pseudokoningii exocellular polysaccharide EPS is to the influence of splenocyte propagation.
Fig. 3: the collaborative LPS of trichoderma pseudokoningii exocellular polysaccharide EPS engulfs the influence of toluylene red ability to lymphocyte.
Fig. 4: the collaborative LPS of trichoderma pseudokoningii exocellular polysaccharide EPS produces NO to lymphocyte influence.
Fig. 5: the collaborative LPS of trichoderma pseudokoningii exocellular polysaccharide EPS is to the influence of lymphocyte activity of acid phosphatase.
Fig. 6: trichoderma pseudokoningii exocellular polysaccharide EPS is to the influence of rat fibroma cell.
Embodiment
Further specify the present invention below in conjunction with embodiment and experimental example, but be not limited thereto.
The preparation of embodiment 1 trichoderma pseudokoningii exocellular polysaccharide
(1) preparation of Crude polysaccharides: trichoderma pseudokiningii CGMCC No.1443 is inoculated on the PDA substratum, behind 28 ℃ of activation 2d, inserts in the liquid nutrient medium 28 ℃, 130rpm shaking culture 6d by flat board.Remove mycelia and spore with four layers of filtered through gauze, the centrifugal 15min of 4000rpm, supernatant cross the decolouring of 3520 resin columns, collect elutriant, and 48 ℃ are evaporated to 1/4 of original volume.Concentrated solution Sevag method (chloroform: propyl carbinol=4: 1, V/V) deproteinated.95% ethanol that adds 3 times of volumes is in 4 ℃ of standing over night, the centrifugal 15min of 5000rpm, collecting precipitation, ether, acetone, 95% ethanol wash successively, precipitation is dissolved in distilled water, is concentrated to original volume 1/4 behind the dialysis 48h, gets crude extracellular polysaccharide sample (EPS) through lyophilize.
(2) purifying of polysaccharide: adopt Sephadex G-75 dextrane gel column chromatography.Accurately take by weighing wooden mould crude extracellular polysaccharide 1g, be dissolved in the water of 10ml, upper prop, water carries out wash-out under the room temperature, and flow velocity 0.4ml/min collects the 15min/ pipe with branch's automatic receptor.Follow the tracks of detection with the phenolsulfuric acid development process by pipe, measure absorbance value in the 490nm place.According to elution volume light absorption value is mapped, merge the polysaccharide soln in single peak district, concentrating under reduced pressure, lyophilize obtains trichoderma pseudokoningii exocellular polysaccharide component EPS.Use Sephadex G-100 sephadex chromatography to be further purified two kinds of components that obtain.
Large scale fermentation is produced the medium optimization of exocellular polysaccharide: with biomass and exopolysaccharides is index, adopts single factor and orthogonal test that the liquid state fermentation condition of the trichoderma pseudokiningii of shake-flask culture is optimized.Trichoderma pseudokiningii fermentation controlled variable after the optimization is: the optimal medium prescription is wheat bran 30g/l, Semen Maydis powder 30g/l, glucose 7.5g/l, potassium primary phosphate 1g/l; Fermented incubation time is 6d; The initial pH value of substratum is 5.0～6.0; Leavening temperature is 30 ± 1 ℃.The output of mycelia dry weight and exocellular polysaccharide difference 5.272g/l, 0.622g/l under the optimal culture condition.
The purifying of embodiment 2 trichoderma pseudokoningii exocellular polysaccharides
Adopt Sephadex G-75 dextrane gel column chromatography.Take by weighing 20g Sephadex G-75 and be suspended in the water of large volume, allow its natural subsidence 4h, remove the fine particle of crossing of suspension with decantation, repeatedly several times after, fill up water and be warming up to gradually and boil insulation 3h.Cooling vacuumizes, and (in 2.5 * 80cm), the water by 2-3 times of column volume is stablized column cap gently to stir down the chromatography column of packing into continuously.Accurately take by weighing wooden mould crude extracellular polysaccharide 1.0g, be dissolved in the water of 5ml, upper prop carries out wash-out with distilled water under the room temperature, flow velocity 0.4ml/min, 15min/ pipe.Follow the tracks of detection with the phenolsulfuric acid development process by pipe, measure absorbance value in the 490nm place.According to elution volume light absorption value is mapped, merge the polysaccharide soln in single peak district, concentrating under reduced pressure, lyophilize obtains trichoderma pseudokoningii exocellular polysaccharide component EPS.Use Sephadex G-100 sephadex chromatography to be further purified the component that obtains.
The purity of embodiment 3 trichoderma pseudokoningii exocellular polysaccharides is identified
The purity of polysaccharide sample and molecular weight are measured with efficient gel filtration method (HPGPC), relation according to the molecular weight and the elution volume of polysaccharide on gel column, earlier with poly-(Dextran) production standard curve of warding off in the following series standard Portugal of different molecular weight, then elution volume per sample (because flow velocity is certain, thus in fact usefulness be retention time) with the typical curve contrast.Peak shape is per sample judged its purity, and the molecular weight of sample is tried to achieve by typical curve.
Used HPLC chromatographic instrument is by water generation (Waters) 515 HPLC pumps, and Waters 2410 differential detectors constitute; Chromatographic column is the strong hydroxyl gel column of water generation (Waters Ultrahydrogel) 2000 and 500 liang of post series connection of Ultrahydrogel.Moving phase is 0.003mol/l sodium-acetate (NaAc), and flow velocity 0.5ml/min, sample are dissolved in the moving phase, and concentration is 1% (w/v), and sample size is 20 μ l.The known dextran of molecular weight (Dextran) T-series standard dextran is T-2000, T-500, and T-110, T-70, T-40 and T-20, the drafting of typical curve and data processing are all carried out with the software of water generation company.
Detect through the efficient gel filtration chromatography, find that this component is single symmetrical peak, its molecular weight is 18325; By a sulfuric acid-phynol method and a phenylphenol method of improvement, find that neutral polysaccharide content is 65.2% in this polysaccharide, glucuronic acid content is 32.6%.
The evaluation of the structure of embodiment 4 trichoderma pseudokoningii exocellular polysaccharides
(1) glycosyl compositional analysis
The reduction of uronic acid is carried out with the Conrad method in the polysaccharide: polysaccharide sample (20mg) is dissolved in the 10ml water, add N-cyclohexyl-N '-(2-N-methyl beautiful jade is for ethyl)-carbodiimide-tosilate N-Cyclohexyl-N '-(2-morpholinoethyl)-carbodiimide methyl-p-toluenesulfonate then, abbreviate CMC as) 300mg, drip 0.01mol/l HCl under the magnetic agitation, make pH remain 4.75 with automatical potentiometric titrimeter, keep 2h.Drip 2mol/l sodium borohydride solution (NaBH
4) 20ml, this moment, the pH value sharply rose, and pH is 7.00 with the control of 4mol/l hydrochloric acid, continues to stir NaBH
4Solution adds in 45min.If generate a large amount of foams, can add several primary isoamyl alcohol froth breakings in the reaction.NaBH
4After adding, at room temperature continue reaction 1h, it is 6.80 that the dropping glacial acetic acid makes pH.Reaction solution is to distill water dialysis (1l * 3), and dialyzed solution is evaporated to 10ml, lyophilize.
EPS is carried out sugared compositional analysis, and the TLC of complete hydrolysis product analyzes demonstration and contains a certain amount of uronic acid; The GC analysis revealed of sugar alcohol acetate vinegar contains that sandlwood is warded off, glucose and a spot of semi-lactosi, and mol ratio is Rha: Glc: Gal=5.6: 2.7: 1.0.EPS carries out carboxyl reduction with the Conrad method, reduces to obtain EPS-R after 3 times, and it is carried out sugared compositional analysis, and TLC analyzes and do not contain uronic acid, shows that reduction is complete.EPS-R GC analysis revealed, contain in it that sandlwood is warded off, grape is warded off and gala is warded off, mol ratio is Rha: Glc: Gal=14.5: 9.3: 1.0, illustrating that this wards off more contains glucuronic acid (GlcA), its molar content is 26.6, the quality percentage composition is about 31.9, and this is consistent with the result that a phenylphenol method is measured.
(2) structure elucidation
Infrared spectroscopy: take by weighing 2.0mg polysaccharide sample (pressing potassium bromide troche), at 40000～400cm
-1Carry out infrared scan in the scope.IR spectrum result shows that EPS has the charateristic avsorption band of polysaccharide.3403.8cm
-1The strong absorption peak at place is that the stretching vibration of O-H key in the glycan molecule absorbs 2933.2cm
-1The peak at place is that the stretching vibration of c h bond in the glycan molecule absorbs 1604.5cm
-1The strong absorption peak at place is the absorption of combination water in the glycan molecule.1297.9cm
-1And 1417.4cm
-1The peak be the angle absorption of vibrations of c h bond in the glycan molecule.1076.1cm
-1And 1033.7cm
-1The peak be that the stretching vibration of C-O key in the glycan molecule absorbs.
13C NMR spectrum: after getting polysaccharide 40-60mg vacuum-drying, be dissolved in 0.5ml heavy water (D
2O) in, place φ 5mm nuclear-magnetism pipe, add 1 μ l acetone as interior mark (δ 30.50ppm), in room temperature measuring;
1H NMR spectrum: get exsiccant polysaccharide sample (15-20mg), use D
2O exchanges once, and then is dissolved in 0.5ml D
2Among the O, place φ 5mm nuclear-magnetism pipe, with HOD peak calibration (δ 4.80ppm), in room temperature measuring.Chemical shift is all with tetramethylsilane (Me
4Si) be external standard, measure and all on Bruker AM-400 nuclear magnetic resonance analyser, carry out (table 1).
The part of table l TPP-0
13The ownership of C NMR signal
| C1 | C2 | C3 | C4 | C5 | C6 | |
| Rha GlcA | 102.86 98.99 | 77.06 71.42 | 75.03 73.13 | 74.01 70.52 | 72.85 74.32 | 16.61 176.77 |
The cellular immunization activity of experimental example 1 trichoderma pseudokoningii exocellular polysaccharide
(1) splenocyte proliferation experiment
1.1 the preparation of splenocyte: kunming mice, male and female half and half, 4-6 week, body weight 18～20g.The eyeball of mouse bloodletting causes death, 75% alcohol-pickled 1min, and mouse spleen is taken out in aseptic technique, place 100 order stainless (steel) wires to grind, make single splenocyte see through screen cloth and fall into the culture dish that fills D-Hanks liquid, be transferred to centrifuge tube, the centrifugal 8min of 1200rpm, supernatant discarded adds the 2ml sterilized water, adds 1640 liquid after 20 seconds, centrifugal supernatant discarded adds 1640 complete culture solutions, piping and druming makes cell suspension repeatedly, draws splenocyte to another centrifuge tube, and it is 1 * 10 that blood counting is adjusted cell concn
7Individual/ml.
1.2 splenocyte propagation: obtained cell suspension adds in the 96 porocyte culture plates, every hole 100 μ l.Experiment is divided into control group (not adding medicine and ConA), medicine (final concentration is respectively 25,50,100,500,1000 μ g/ml) stimulating group, the independent stimulating group of ConA (final concentration is 5 μ g/ml) and collaborative ConA (final concentration the is 5 μ g/ml) stimulating group of medicine (final concentration is respectively 25,50,100,500,1000 μ g/mL) separately, and every group of every concentration is established three multiple holes.In 37 ℃, 5%CO
2Place CO under the condition
2Cultivate 72h in the incubator.The every hole of 4h adds MTT solution 10 μ l before cultivating end, continues to cultivate.Take out, the careful suction removed supernatant 100 μ l, and every hole adds DMSO 150 μ l dissolving first a ceremonial jade-ladle, used in libation, reads every hole OD value at microplate reader 570nm wavelength place.The result shows: EPS can utmost point significant stimulation splenocyte propagation.EPS, multiplication effect has utmost point significant difference (P＜0.01) compared with the control when concentration 25,50,100,500 and 1000 μ g/ml, amplification is respectively 48.4%, 69.8%, 72.6%, 70.2%, 62.3%, has dose-dependently (Fig. 1) during less than 250 μ g/ml in concentration.Show that with the synergy experiment of ConA the mouse lymphocyte propagation of EPS (25-1000 μ g/ml) thorn regular menstruation during early pregnancy ConA effect in the finite concentration scope has certain synergy; EPS concentration has significant difference (P＜0.05) when being 100,500 μ g/ml, amplification is 11.1%, 8.6% (Fig. 2) of contrast.
(2) the immunocompetent mensuration of mouse macrophage
2.1 the preparation of Turnover of Mouse Peritoneal Macrophages: mouse peritoneal is injected 3% thioglycollate medium 2ml, and the cervical vertebra dislocation is put to death after 3 days, opens belly, and in abdominal injection PBS solution 5ml, the massage stomach wall extracts the belly transudate, and transudate is collected in flushing repeatedly.Washed twice, centrifugal (200g * 6min).Put into glass dish in 37 ℃, 5%CO
2Place CO under the condition
2Cultivate 2h in the incubator, take out,, remove non-adherent cell, scrape gently with the rubber rod and get, be the Turnover of Mouse Peritoneal Macrophages of purifying with RPMI-1640 flushing 3 times.Counting makes 1 * 10 with the RPMI-1640 that contains 10% foetal calf serum
7The cell suspension of/ml.
2.2 mouse macrophage activate the phagocytic capacity: obtained cell suspension adds in the 96 porocyte culture plates, and every hole 100 μ l also add the LPS that final concentration is 1 μ g/ml.Experiment is divided into control group (concentration is the LPS of 1 μ g/ml) and medicine (final concentration is respectively 25,50,100,500,1000 μ g/ml) stimulating group, and every group of every concentration is established three multiple holes.In 37 ℃, 5%CO
2Place CO under the condition
2Cultivate 24h in the incubator.Take out, remove supernatant, every hole adds 1% toluylene red normal saline solution, in 37 ℃ of placement 20min, takes out, and removes supernatant, uses PBS solution washing 3 times, removes the toluylene red dyestuff of not engulfing.Add dehydrated alcohol: Glacial acetic acid (1: 1) mixed solution 100 μ l, room temperature reads every hole OD value at microplate reader 490nm wavelength place after placing 20min.The result shows: EPS all has the effect (P＜0.01) of the activate the phagocytic capacity that extremely significantly promotes scavenger cell under 5 gradient concentrations that experiment is set, amplification compared with the control is respectively 108.2%, 97.6%, 90.6%, 62.4%, 55.3% and 35.3%, minimal effective concentration is 25 μ g/ml, along with its promoter action of rising decline (as Fig. 3) to some extent on the contrary of concentration.
2.3.NO Determination on content
Obtained cell suspension adds in the 96 porocyte culture plates, and every hole 100 μ l also add the LPS that final concentration is 1 μ g/ml.Experiment is divided into control group (concentration is the LPS of 1 μ g/ml) and medicine (final concentration is respectively 25,50,100,500,1000 μ g/ml) stimulating group, and every group of every concentration is established three multiple holes.In 37 ℃, 5%CO
2Place CO under the condition
2Cultivate 24h in the incubator.Take out, every hole is carefully drawn supernatant 50 μ l and is placed another 96 porocyte culture plate, and every hole adds Griess reagent 50 μ l, mixing.Room temperature is placed 10min, reads every hole OD value at microplate reader 490nm wavelength place.Use NaNO
2Make typical curve and with NO
2Concentration conversion become the content of NO.The result shows: EPS can produce NO by the significant stimulation scavenger cell.As Fig. 4, EPS has utmost point significant stimulation and produces NO effect (P＜0.01) when concentration is 25,50,100 and 500 μ g/ml, amplification is respectively 126.30%, 133.0%, 135.9%, 151.5%, 149.6% of contrast, when being 1000 μ g/ml, concentration has significant stimulation effect (P＜0.05), amplification is 171.5% of contrast, show as more weak dose-dependently, best induced concentration is 25 μ g/ml.
2.4 the mensuration of activity of acid phosphatase
Obtained cell suspension adds in the 96 porocyte culture plates, and every hole 100 μ l also add the LPS that final concentration is 1 μ g/ml.Experiment is divided into control group (concentration is the LPS of 1 μ g/ml) and medicine (final concentration is respectively 25,50,100,500,1000 μ g/ml) stimulating group, and every group of every concentration is established three multiple holes.In 37 ℃, 5%CO
2Place CO under the condition
2Cultivate 24h in the incubator.Take out, remove supernatant, every hole adds pNPP solution 100 μ l, and mixing in 37 ℃ of placement 30min, takes out, and adds 1.0mol/lNaOH solution 10 μ l termination reactions immediately.Read every hole OD value at microplate reader 405nm wavelength place.The result shows: EPS has the effect of activating macrophage activity of acid phosphatase, when concentration 50,100 and 250 μ g/ml, has the extremely significantly effect (P＜0.01) of activating macrophage activity of acid phosphatase, amplification is respectively 44.5%, 42.1% and 43.6% of contrast, have remarkable activation effect (P＜0.05) when 25 and 1000 μ g/ml, amplification is respectively 29.3%, 16.6% (see figure 5).
The external inducing action of experimental example 2 trichoderma pseudokoningii exocellular polysaccharides to rat fibroma natural death of cerebral cells
Get the rat fibroma cell suspension that goes down to posterity repeatedly and add in the 96 porocyte culture plates, every hole 100 μ l.Experiment is divided into control group (not adding medicine), medicine (final concentration is respectively 250,500,750,1000,1250,1500 μ g/ml) effect group, and every group of every concentration is established three multiple holes.In 37 ℃, 5%CO
2Place CO under the condition
2Cultivate 72h in the incubator.The every hole of 4h adds MTT solution 10 μ l before cultivating end, continues to cultivate.Take out, the careful suction removed supernatant 100 μ l, and every hole adds DMSO 150 μ l dissolving first a ceremonial jade-ladle, used in libation, reads every hole OD value at microplate reader 570nm wavelength place.Its result shows: die as the accent that Fig. 6 trichoderma pseudokoningii exocellular polysaccharide (250-1500 μ g/ml) effect can both inducing tumor cell, each group all has positive effect, best results when concentration is 1250 μ g/ml has been compared utmost point significant difference (P＜0.01) with control group.Compare with the normal fiber oncocyte, the fibroma cell shrinkage after the effect of trichoderma pseudokiningii bacterium exopolysaccharide is agglomerating to present the tangible accent symptom of dying.Above result shows that trichoderma pseudokoningii exocellular polysaccharide has the effect of inducing the fibroma natural death of cerebral cells in the finite concentration scope.
Experimental example 3 trichoderma pseudokoningii exocellular polysaccharide body innerlich anwendens are to the inhibition experiment of mouse S180 ascitic tumor
Select ascitic type go down to posterity 7-10d and the good mouse of tumor growth, dislocation causes death.Extract the dense thick ascites of intraperitoneal white, be prepared into 1 * 10
7The cell suspension of individual/ml, trypan blue chromoscopy tumor cell activity after more than 95%, under aseptic condition, mouse peritoneal injection 0.2ml tumor cell suspension.
Mouse is divided into 5 groups at random, every group 12: respectively by 0,25,50,75, the dosage of 100mg/kg.d is dissolved in the physiological saline of 0.2ml, set time every day section is expelled to mouse peritoneal, every 5d weighing is the mouse body weight once, successive administration 30d, the survival time after the record mouse inoculation tumour.The result shows: behind the inoculation ascitic tumor, the weight increase of mouse is slow, and its weight increase speed has only about 20% of healthy mice.After the injection of 25-100mg/kg.d dosage EPS belly, can both increase the body weight of ascitic tumor mouse, wherein the EPS effect of 75mg/kg.d is best, and advancing the speed of mouse is lower by about 18% than healthy mice, is the EPS of 100mg/kg.d secondly.Plan Kang Shi exocellular polysaccharide the results are shown in Table 2 to the influence of S180 ascites carcinoma mouse body weight (g).
Behind the inoculation ascitic tumor, on average there is 16.4d in the life-span of mouse, behind the EPS of injection various dose, has prolonged the life-span of ascitic tumor mouse.Wherein, even the life-span of the dosage ascitic tumor mouse of 25mg/kg.d has prolonged 17.7%, the best 75mg/kg.d of effect has prolonged ascitic tumor mouse life 38.4%.Plan Kang Shi exocellular polysaccharide the results are shown in Table 3 to the influence of S180 ascites carcinoma mouse survival time.
Table 2: intend the Kang Shi exocellular polysaccharide to the influence of S180 ascites carcinoma mouse body weight (g) (n=12, x ± s)
| Handle (mg/kg.d) | 0d (before the administration) | 5d | 10d | 15d | 20d |
| Blank 0 (contrast) 25 50 75 100 | 18.5±1.1 18.3±1.2 18.2±1.0 18.2±1.0 18.3±1.1 18.2±1.1 | 23.0±1.2 20.0±1.1 20.1±1.1 21.9±1.3 22.7±1.1 21.1±1.2 | 28.1±1.1 21.7±1.2 22.5±1.2 23.8±1.2 25.7±1.3 24.0±1.2 | 32.2±1.1 23.4±1.1 24.4±1.3 26.2±1.2 28.7±1.4 26.4±1.3 | 36.5±1.1 24.6±1.1 26.5±1.3 28.8±1.3 33.1±1.4 29.2±1.1 |
Table 3: intend the Kang Shi exocellular polysaccharide to the influence of mouse survival time of S180 ascites carcinoma
| Handle (mg/kg.d) | Average survival fate (d) | Increase in life span (%) |
| 0 (contrast) 25 50 75 100 | 16.4±2.1 19.3±2.6 20.5±2.4 22.7±2.8 21.1±2.2 | - 17.7 * 25.0 * 38.4 ** 28.7 * |
Experimental example 4 trichoderma pseudokoningii exocellular polysaccharide body innerlich anwendens are to the inhibition experiment of mouse S180 solid tumor
Get above-mentioned prepare 1 * 10
7The S180 ascitic type tumor cell suspension of individual/ml in mouse right upper extremity oxter subcutaneous vaccination tumour cell suspension 0.2ml, is set up S180 mice with tumor model.
Inoculation back administration group: behind the mouse inoculation 24h, random packet, is divided into control group and treatment group (giving EPS 25,50,75 and 100mg/kg.day) by 12 every group.The subcutaneous injection administration, once a day, continuous 10 days.Treatment group injection 0.2ml contains the physiological saline of various dose EPS, the physiological saline of control group injection 0.2ml.
Pre-administration group: 7d random packet before the mouse inoculation, is divided into control group and treatment group (giving EPS 25,50,75 and 100mg/kg.d) by 12 every group.The mouse stomach administration, once a day, continuous 7d.Treatment group gives the EPS aqueous solution of various dose, and control group gives the distilled water of equal volume.Set up the S180 tumor model on the 8th day, continue administration 10d.
Behind last administration 24h, S180 solid tumor mouse is plucked the eyeball sacrificed by exsanguination, gets the about 1ml of blood, strips subcutaneous solid tumor knurl piece, removes fatty fibrous tissue, weighs, and calculates tumour inhibiting rate (%) as follows.The experiment triplicate.
Tumour inhibiting rate (%)=(the average knurl of the average knurl weight-treatment group of control group is heavy)/average knurl of control group heavy * 100%
Winning spleen weighs and calculates spleen index: spleen index=solid tumor mice spleen weight/mouse body weight
The result shows: administration and pre-administration all have an obvious suppression effect (seeing Table 4 and 5) to mouse S180 sarcoma behind the inoculation ascitic tumor cell.Inoculation back administration, the tumour inhibiting rate of 25mg/kg.d is 10.5%, compares with control group, reaches significant difference, the 75mg/kg.d tumour inhibiting rate is still best, reaches 37.7%; 50-100mg/kg.d after the pre-administration, inoculate tumour cell, knurl representation work reduces, and reaches significant difference compared with the control; It is best that wherein the dosage of 75mg/kg.d suppresses effect, inhibitory rate to 41.1%.Spleen index also has similar effect, and the EPS of 25-100mg/kg.d can both significantly improve the spleen index of S180 solid tumor mouse, increases the resistance of mouse, and wherein the effect of 75mg/kg.d is best, and the 100mg/kg.d effect slightly descends.Simultaneously, the treatment group of same dose, the effect of pre-administration group all are better than inoculation back administration group, show that pre-administration can effectively suppress the generation of tumour.
Table 4: injection EPS is to the restraining effect of mouse S180 sarcoma (n=12, x ± s) behind the inoculation ascitic tumor cell
| Handle (mg/kg.d) | Solid tumor | Spleen | ||
| Knurl heavy (g) | Tumour inhibiting rate (%) | Spleen heavy (g) | Spleen index | |
| 0 (contrast) 25 50 75 100 | 1.62±0.51 1.45±0.41 * 1.21±0.32 ** 1.01±0.46 ** 1.17±0.35 ** | 10.5 25.3 37.7 27.8 | 0.187±0.054 0.231±0.053 0.267±0.061 0.285±0.075 0.278±0.052 | 0.0076±0.0027 0.093±0.0037 * 0.0102±0.0041 ** 0.0114±0.0036 ** O.0105±0.0044 ** |
Table 5: give behind the EPS 7d restraining effect to mouse S180 sarcoma (n=12, x ± s) in advance
| Handle (mg/kg.d) | Solid tumor | Spleen | ||
| Knurl heavy (g) | Tumour inhibiting rate (%) | Spleen heavy (g) | Spleen index | |
| 0 (contrast) 25 50 75 100 | 1.92±0.57 1.54±0.53 ** 1.37±0.32 ** 1.13±0.46 ** 1.29±0.35 ** | - 19.8 28.6 41.1 32.8 | 0.181±0.04 0.243±0.07 0.272±0.06 0.298±0.06 0.282±0.05 | 0.0078±0.0021 0.0102±0.0037 ** 0.0110±0.0045 ** 0.0117±0.0041 ** 0.0113±0.0039 ** |
Claims (6)
1. a trichoderma pseudokoningii exocellular polysaccharide is characterized in that,
(1) detect by the efficient gel filtration chromatography, it has single symmetrical peak, and molecular weight is 18325;
(2) sulfuric acid-phynol method and the phenylphenol method by improvement detects, and its neutral polysaccharide content is 65.2%, and glucuronic acid content is 32.6%;
(3) analyze by the gas-chromatography (GC) of alditol acetate, its monose consists of rhamnosyl, glucose and semi-lactosi, and mol ratio is Rha: Glc: Gal=5.6: 2.7: 1.0.
2. a trichoderma pseudokoningii exocellular polysaccharide is characterized in that, after this polysaccharide reduced fully, the mol ratio of rhamnosyl, glucose and semi-lactosi was Rha: Glc: Gal=14.5: 9.3: 1.0, and its to contain molar content be 26.6% glucuronic acid.
3. a trichoderma pseudokoningii exocellular polysaccharide is characterized in that, it is with trichoderma pseudokiningii fermented liquid decolouring, concentrate, behind alcohol precipitation and the Deproteinization, the homogeneous polysaccharide that obtains by the gel chromatography column purification.
4. the preparation method of the arbitrary described trichoderma pseudokoningii exocellular polysaccharide of claim 1-3 comprises the preparation of step (1) Crude polysaccharides and the purifying of step (2) polysaccharide, it is characterized in that,
The preparation of described step (1) Crude polysaccharides comprises:
A, trichoderma pseudokiningii inserted liquid nutrient medium after, 28 ℃, 130 rpms shaking culture 6 days;
B, centrifugal mycelia and the spore removed of filtration are crossed the decolouring of 3520 resin columns, collect elutriant, are evaporated to 1/4 of original volume;
C, concentrated solution Sevag method deproteinated;
D, adding ethanol are in 4 ℃ of standing over night, and centrifugal collecting precipitation washs successively with ether, acetone, 95% ethanol, and precipitation is dissolved in distilled water, are concentrated to original volume 1/4 behind the dialysis 48h, get the crude extracellular polysaccharide sample through lyophilize,
The purifying of described step (2) polysaccharide comprises:
A, employing sephadex G-75 dextrane gel column chromatography take by weighing wooden mould crude extracellular polysaccharide 1g, be dissolved in the water of 10ml, and upper prop, water carries out wash-out under the room temperature, control flow velocity 0.4ml/min;
B, with branch's automatic receptor per 15 minutes one pipes, the phenolsulfuric acid development process is followed the tracks of by pipe and is detected, in 490nm place mensuration absorbance value;
C, light absorption value is mapped, merge the polysaccharide soln in single peak district according to elution volume, concentrating under reduced pressure, lyophilize obtains trichoderma pseudokoningii exocellular polysaccharide preliminary purification component,
With aforementioned gained preliminary purification component S ephadex G-100 sephadex chromatography purifying, get the purpose product.
5. the application of the arbitrary described trichoderma pseudokoningii exocellular polysaccharide of claim 1-3 in preparation raising mammalian immune active medicine or functional foodstuff.
6. the arbitrary described trichoderma pseudokoningii exocellular polysaccharide of claim 1-3 is in preparation treatment or the medicine of adjuvant therapy of tumors and the application in the functional foodstuff.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102406680A (en) * | 2010-09-20 | 2012-04-11 | 陕西嘉诺生物科技企业集团有限公司 | Complex polysaccharide immune preparation and preparation method thereof |
| CN102657674A (en) * | 2012-04-11 | 2012-09-12 | 山东大学 | Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities |
| CN103027924A (en) * | 2012-11-26 | 2013-04-10 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide as medicine for treating gastric cancer |
| CN103816370A (en) * | 2014-03-17 | 2014-05-28 | 鞠芳 | Chinese medicament group for assisting chemotherapy |
| CN105412155A (en) * | 2015-12-02 | 2016-03-23 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide in preventing and treating colon cancer and application of chemotherapeutic drug combined with trichoderma pseudokoningii exopolysaccharide in treating colon cancer |
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2008
- 2008-01-23 CN CNA2008100140478A patent/CN101220101A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102406680A (en) * | 2010-09-20 | 2012-04-11 | 陕西嘉诺生物科技企业集团有限公司 | Complex polysaccharide immune preparation and preparation method thereof |
| CN102657674A (en) * | 2012-04-11 | 2012-09-12 | 山东大学 | Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities |
| CN103027924A (en) * | 2012-11-26 | 2013-04-10 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide as medicine for treating gastric cancer |
| CN103027924B (en) * | 2012-11-26 | 2015-02-25 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide as medicine for treating gastric cancer |
| CN103816370A (en) * | 2014-03-17 | 2014-05-28 | 鞠芳 | Chinese medicament group for assisting chemotherapy |
| CN105412155A (en) * | 2015-12-02 | 2016-03-23 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide in preventing and treating colon cancer and application of chemotherapeutic drug combined with trichoderma pseudokoningii exopolysaccharide in treating colon cancer |
| CN105412155B (en) * | 2015-12-02 | 2019-03-12 | 皖南医学院 | Trichoderma pseudokoningii exocellular polysaccharide treats and prevents the application of colon cancer and its is combined the application of chemotherapeutic drug therapy colon cancer |
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