CN103319619A - Application of lachnum extracellular polysaccharide phosphorylated derivative and application thereof in preparation of antitumor drugs - Google Patents
Application of lachnum extracellular polysaccharide phosphorylated derivative and application thereof in preparation of antitumor drugs Download PDFInfo
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Abstract
The invention discloses an application of lachnum extracellular polysaccharide phosphorylated derivative and an application thereof in preparation of antitumor drugs. The lachnum extracellular polysaccharide phosphorylated derivative is obtained by performing phosphorylated decoration on extracellular polysaccharide generated by lachnum with strain preservation number of CCTCC No: M2011196. As shown by an experiment, the extracellular polysaccharide phosphorylated derivative generated by lachnum with strain preservation number of CCTCC No: M2011196 not only has excellent antitumor function, but also has no toxicity or side effect, and the preparation method is simple and economic, so that the lachnum extracellular polysaccharide phosphorylated derivative can be used for preparing tablet or capsule antitumor drugs.
Description
Technical field
The present invention relates to a kind of Lachnum exocellular polysaccharide phosphorylated derivative and the application in the preparation antitumor drug thereof.
Background technology
Malignant tumour is one of common clinically disease, also is to cause one of human dead Etiological.Therefore at present, the methods such as Radiotherapy chemotherapy are mainly adopted in the treatment of malignant tumour, and normally used chemotherapeutics mostly is the chemosynthesis material but not natural product, in the kill tumor cell, normal cell are also had to a certain degree lethal effect; And chemotherapy can make the patient lose the appetite, and weight loss causes body weak, and causes immunosuppression to a certain degree, and toxic side effect is larger.Therefore, excavating and develop antitumor drug natural, that have no side effect has great importance to safeguarding human health.
Fungus polysaccharide is the secondary metabolite of fungi, and the biological activity of polysaccharide depends on its molecular structure, takes certain method that it is carried out molecular modification, can improve the active of polysaccharide or make polysaccharide produce new activity.It is a kind of covalent modification that the phosphorylated molecules of polysaccharide is modified, and is the process that the hydroxyl on the side chain is replaced by phosphate radical.
It is the saprophytic property of class fungi that Lachnum belongs to, and can produce various bioactivators under the liquid culture condition, such as polysaccharide, polyphenol, pigment etc.China's " mathematics medicine and pharmacology magazine " (2009 the 3rd phase of the 22nd volume the 337th～338 page) discloses the antitumor activity of lentinan, experimental result shows that mushroom has significant antitumor action, but the polysaccharide that relates in the document is lentinan, does not mention the anti-tumor function of Lachnum polysaccharide; Chinese patent application numbers 201010582408.6 discloses the application of a kind of Lachnum polysaccharide in the preparation anti-fatigue medicament, but does not relate to phosphorylation modification and the anti-tumor function thereof of Lachnum polysaccharide; " Carbohydrate Polymers " (the 83rd volume was the 737th～742 page in 2011) discloses phosphorylation modification and the herpesvirus resisting activity research thereof of David's-harp polysaccharide, the result shows behind phosphorylation modification, and the herpesvirus resisting activity of David's-harp polysaccharide obviously improves.But mention the phosphorylated derivative of Lachnum exocellular polysaccharide and the report that conduct prepares the application facet of antitumor drug thereof so far there are no open source literature.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of Lachnum exocellular polysaccharide phosphorylated derivative.
The another one technical problem that the present invention will solve provides the application of a kind of Lachnum exocellular polysaccharide phosphorylated derivative in the preparation antitumor drug.
For Lachnum exocellular polysaccharide phosphorylated derivative, the technical solution used in the present invention is: this derivative is prepared by following methods:
(1) the purification exocellular polysaccharide is extracted in the Lachnum fermentation that culture presevation is numbered CCTCC No:M2011196 afterwards, sodium sulfate with 90～110mL distilled water dissolving 9～11g phosphorylation agent and 4～6%, add the above-mentioned polysaccharide of 0.9～1.1g after the dissolving, use NaHCO
3After transferring pH=8～10, react 4～6h under 75～85 ℃ of conditions, sample liquid obtains the alcohol precipitation polysaccharide with 95% ethanol precipitation, the 20～28h of 2～3 times of volumes;
(2) with alcohol precipitation polysaccharide vacuum lyophilization, in 55～65 ℃ of water-baths, redissolve again, the solution after the redissolution in dialysis tubing take distilled water as dialyzate the dialysis 24～36h, lyophilize namely gets Lachnum polysaccharide phosphorylated derivative.
As preferably, phosphorylation agent is mixed for 6:1 in mass ratio by tripoly phosphate sodium STPP and Trisodium trimetaphosphate described in the step (1).
The application of Lachnum exocellular polysaccharide phosphorylated derivative in the preparation antitumor drug.
The invention has the beneficial effects as follows:
The exocellular polysaccharide that Lachnum produces that the present invention uses culture presevation to be numbered CCTCC No:M2011196 prepares phosphorylated derivative, and take above-mentioned exocellular polysaccharide phosphorylated derivative as raw material as the preparation antitumor drug application, prepared medicine has overcome existing chemotherapeutics in the kill tumor cell, normal cell also there is to a certain degree lethal effect, and chemotherapy can make the patient lose the appetite, weight loss, cause body weak, and cause to a certain degree immunosuppression, the shortcoming such as toxic side effect is larger.Confirm through experimental study of the present invention, above-mentioned Lachnum exocellular polysaccharide derivative not only has significant anti-tumor function, and have no side effect, its preparation method simple economy is therefore can be applied to above-mentioned Lachnum exocellular polysaccharide derivative to prepare the antitumor drug of tablet or capsule formulation.
Embodiment
Embodiment 1:
One, the preparation of Lachnum exocellular polysaccharide:
The Lachnum that culture presevation is numbered CCTCC No:M2011196 is inoculated in the 5L fermentor tank, culture medium prescription is: glucose 40g/L, yeast extract paste 12g/L, glycine 6.2mg/L, MgSO47H2O 1g/L, KH2PO4 1g/L, the coefficient volume ratio is 0.7, inoculum size is the 15%(volume ratio), culture temperature is 35 ℃, air flow is 1.0L/min, and mixing speed is 180r/min, and fermentation time is 10 days; With the fermented liquid suction filtration, concentrated after, the massfraction that adds its 3 times of volumes is that 95% ethanol is in 4 ℃ of alcohol precipitation 24h, alcohol is analysed liquid with the centrifugal 10min of 5000r/min, remove supernatant, precipitation dehydrated alcohol, washing with acetone 2～3 times, then use dissolved in distilled water, Sevag method deproteinated and concentration expressed in percentage by volume are after 30% H2O2 insulation (50 ℃) decolouring is processed, distilled water normal temperature dialysis 24h,-50 ℃ of lyophilizes namely get the Lachnum exocellular polysaccharide that culture presevation is numbered CCTCC No:M2011196.
Two, the preparation of Lachnum exocellular polysaccharide phosphorylated derivative
Sodium sulfate (the foaming phenomenon when reducing reaction) with 100mL distilled water dissolving 10g phosphorylation agent (8.57g tripoly phosphate sodium STPP+1.43g Trisodium trimetaphosphate) and 5%, add the above-mentioned polysaccharide of 1g after the dissolving, behind NaHCO3 accent pH=9, react 5h under 80 ℃ of conditions, sample liquid precipitates 24h with 95% long-pending ethanol of triploid.With alcohol precipitation polysaccharide vacuum lyophilization, in 60 ℃ of water-baths, redissolve again, the solution after the redissolution in dialysis tubing (relative molecular weight that dams is 13,000Da) in take distilled water as dialyzate the dialysis 24h, lyophilize namely gets Lachnum exocellular polysaccharide phosphorylated derivative.
Three, the detection of Lachnum polysaccharide derivates phosphorylation modification degree
The phosphate radical standard curve determination: take by weighing 3.6g Tutofusin tris and 120mg MgCl26H2O and be dissolved in the 300mL distilled water, the HCl of 1mol/L transfers pH=7, namely gets Tris buffered soln.The massfraction of getting respectively equal volume is that the H2SO4 solution of 20% the VC aqueous solution, 3mol/L and massfraction are 3% ammonium molybdate aqueous solution, mixes namely to get and decides phosphorus reagent.Accurately take by weighing 1g KH2PO4 and be dissolved in the 100mL water, with 100 times of gained solution dilutions, namely getting concentration is the phosphate radical reference liquid of 0.1mg/mL.Accurately draw above-mentioned phosphate radical reference liquid 0,0.5,1,1.5,2,2.5,3,3.5,4,4.5,5mL, move into respectively in the colorimetric cylinder, distilled water is supplemented to 5mL, adds Tris buffered soln 3mL, shake up rear adding 3mL and decide phosphorus reagent, 45 ℃ of water bath with thermostatic control 30min take out the rear light absorption value of measuring at the 580nm place, take phosphate concentration as X-coordinate, absorbancy is ordinate zou, the drawing standard curve.
Phosphate radical is measured: get the 0.1g sample in beaker, add the 1mL vitriol oil and 1mL concentrated nitric acid, be heated to and smolder, add the H of 1mL30% after the cooling
2O
2Solution, slowly heating repeats above step until no longer smolder in the beaker again, and solution is water white transparency or faint yellow.The hydrochloric acid that adds 1mL6mol/L after the cooling, heating thoroughly decompose acid, are transferred to constant volume in the 50mL volumetric flask.Get the 5mL mentioned solution, measure absorbancy according to the typical curve working method, according to typical curve regression equation calculation phosphate content.
Infrared measurement: get respectively above-mentioned Lachnum exocellular polysaccharide and derivative 2mg thereof, compressing tablet after grinding with KBr adopts Fourier transform infrared spectrometer (Nexus 670) to carry out the IR spectroscopic analysis, and sweep limit is 4000-400cm
-1
Nuclear magnetic resonance spectrometry: get respectively the above-mentioned Lachnum exocellular polysaccharide of 50mg and derivative thereof, be dissolved in 1.0mLD
2Among the O, adopt Bruker AV-500 nuclear magnetic resonance spectrometer to carry out 13C NMR and analyze.Get the above-mentioned Lachnum polysaccharide derivates of 50mg, be dissolved in 1.0mL D
2Among the O, adopt Bruker AV-500 nuclear magnetic resonance spectrometer to carry out
31P NMR analyzes.
Four, Lachnum exocellular polysaccharide and phosphorylated derivative thereof are to mouse antineoplastic action pharmacological evaluation:
(1) preparation of Lachnum exocellular polysaccharide and phosphorylated derivative normal saline solution thereof: be the aqueous solution that 0.9% physiological saline is configured to respectively 100mg/mL and 400mg/mL with above-mentioned Lachnum exocellular polysaccharide and phosphorylated derivative powder mass percentage concentration thereof, the administration volume of taking be 0.25mL/ only.
(2) animal: 80 of kunming mices, male and female half and half, body weight 22 ± 2g, random packet.
(3) test kit: superoxide-dismutase (SOD) testing cassete, TNF-α ELISA testing cassete and IL-2ELISA testing cassete, for building up the product that bio-engineering research is produced in Nanjing; Positive drug: endoxan is the product of Hualian Pharmaceutical Co., Ltd., Shanghai's production.
(4) method: get mouse sarcoma S180cell, behind the cultivation 3d, adjusting concentration is 2 * 10 under 37 ℃ of conditions
6Individual/mL.Get 20 of kunming mices, abdominal injection 0.5mL cell suspension/only carry out rising in value in the oncocyte body.Mouse adapts to a week under experimental situation, select the good mouse of growth conditions to get ascites, and the centrifugal 3min of 3000r/min gets supernatant, and it is 10 that stroke-physiological saline solution is diluted to cell concn
7Individual/mL, every above-mentioned cell suspension of right side of mice armpit subcutaneous vaccination 0.2mL, behind the conventional raising 4d, the mouse that the sarcoma diameter reaches about 0.5cm shows the modeling success.
Get 60 of the S180 sarcoma model mices of above-mentioned modeling success, be divided at random 6 groups (10 every group), be respectively model control group (0.9% physiological saline), positive controls (endoxan, 25mg/kg), polysaccharide low dose group (100mg/kg), polysaccharide high dose group (400mg/kg), polysaccharide derivates low dose group (100mg/kg), polysaccharide derivates high dose group (400mg/kg), get 10 healthy mices as the blank group, weigh respectively and record.Gastric infusion 0.2mL/, successive administration 10d measures food ration every day, calculating mean value.2d after the last administration, mouse is weighed, and takes off cervical vertebra and puts to death.
(4) index of correlation is measured:
Tumour inhibiting rate and Immune Organs Index are measured: separate the knurl piece, claim the knurl quality, calculate tumour inhibiting rate; Get spleen and thymus gland, claim quality, calculate organ index.
Tumour inhibiting rate=(model group knurl quality-administration group knurl quality)/model group knurl quality * 100%;
Organ index=Organ weight/weight * 100%
The mensuration of SOD enzyme activity, IL-2 and TNF-alpha levels: eyeball of mouse is got blood, the centrifugal 3min of 5000r/min, get serum and be put in-20 ℃ for subsequent use.Return to before use room temperature, operate according to test kit specification sheets step, measure the level of SOD enzyme activity, IL-2 and TNF-α.
(5) experimental result:
Table 1 has provided above-mentioned Lachnum exocellular polysaccharide and phosphorylated derivative thereof to the impact of S180 sarcoma mouse food ration, body weight gain rate, tumour inhibiting rate and organ index:
Table 1 polysaccharide and derivative thereof are on the impact of S180 sarcoma mouse food ration, body weight gain rate, tumour inhibiting rate and organ index
Annotate: compare a P ﹤ 0.05, b P ﹤ 0.01 with model control group.
The index parameter of respectively organizing in the his-and-hers watches 1 compares analysis, can find out that Lachnum exocellular polysaccharide and phosphorylated derivative thereof are on the impact of each treatment group mouse index of correlation.
Compare with model control group, the food ration of Lachnum exocellular polysaccharide group and phosphorylated derivative group mouse all has increase (table 1) in various degree, wherein the food ration of phosphorylated derivative high dose group mouse has increased by 12.19% than model control group, have significant difference (P ﹤ 0.05), and the food ration of positive controls mouse has significantly reduced 24.37%(P ﹤ 0.01).The body weight gain rate of Lachnum exocellular polysaccharide group and phosphorylated derivative group mouse all significantly is lower than the model control group mouse, but with the body weight gain rate of normal group mouse without significant difference, show that Lachnum exocellular polysaccharide and polysaccharide derivates all can significantly suppress the growth of mouse tumor piece, and to the growth unrestraint effect of this heavy sensation of the whole body of mouse; The positive controls mouse has reduced by 64.91% than the body weight gain rate of model control group mouse, reduced by 29.67% than normal group mouse, show that endoxan (chemotherapeutics) has significantly suppressed the growth of mouse tumor piece, also suppressed the growth of this heavy sensation of the whole body of mouse simultaneously.Compare with the polysaccharide group, positive controls mouse One's spirits are drooping, fur is matt, perpendicular hair phenomenon is obvious, movable obviously minimizing, the serious toxic side effect of this phenomenon and chemotherapeutics is relevant, show Lachnum exocellular polysaccharide group and phosphorylated derivative thereof to S180 sarcoma mouse all without obvious toxic side effect.
Each dosage group polysaccharide has all shown significant tumor-inhibiting action (table 1), wherein concentration is that the tumour inhibiting rate of the phosphorylated derivative group mouse of 100mg/kg has improved 24.52% than exocellular polysaccharide group, concentration is that the tumour inhibiting rate of polysaccharide derivates group mouse of 400mg/kg is higher by 13.05% than exocellular polysaccharide group, polysaccharide derivates is remarkable than exocellular polysaccharide to the tumor-inhibiting action of S180 sarcoma mouse, shows that the anti-tumor activity of phosphorylated derivative is more remarkable than polysaccharide.
Compare with Normal group, the thymus index of model control group mouse obviously descends, and the thymus index of each dosage polysaccharide group mouse all has rising in various degree, and the thymus index of polysaccharide derivates group mouse is high than the exocellular polysaccharide group.Compare with the model control group mouse, the index and spleen index of Lachnum exocellular polysaccharide group and polysaccharide derivates group mouse is without significant difference.Positive drug (endoxan) has reduced the Thymus and spleen index of mouse, illustrates that endoxan may cause the immunosuppression of mouse, has shown serious toxic side effect.
Table 2 provided above-mentioned Lachnum exocellular polysaccharide and phosphorylated derivative thereof on S180 sarcoma SOD in Mice enzyme live, the impact of IL-2 and TNF-α:
Table 2 polysaccharide and derivative thereof are on the impact of the work of S180 sarcoma SOD in Mice enzyme, IL-2 and TNF-α
| Grouping | SOD enzyme (U/mL) alive | IL-2（pg/mL） | TNF-α（pg/mL） |
| Normal group | 119.04±1.99 | 10.57±1.90 | 47.13±1.44 |
| Model control group (physiological saline) | 106.56±4.84 | 5.05±1.01 | 44.63±1.81 |
| The polysaccharide low dose group | 122.08±3.53 a | 5.67±1.33 | 46.36±2.09 |
| The polysaccharide derivates low dose group | 114.23±2.86 | 7.56±1.06 a | 46.99±1.31 |
| The polysaccharide high dose group | 115.27±3.16 | 7.93±1.47 a | 48.04±1.77 |
| The polysaccharide derivates high dose group | 117.21±2.36 | 8.68±1.41 a | 50.49±1.89 b |
| Positive controls (endoxan) | 121.87±4.15 a | 10.62±1.82 b | 49.94±1.78 a |
Annotate: compare with model control group,
aP ﹤ 0.05,
bP ﹤ 0.01.
Compare with Normal group, the level of the work of SOD enzyme, IL-2 and TNF-α obviously reduces (table 2) in the model control group mice serum.Compare with model control group, the work of SOD enzyme, IL-2 and the TNF-α of Lachnum exocellular polysaccharide group, phosphorylated derivative group and positive controls mouse all has increase in various degree, and all near normal group mouse level.The polysaccharide derivates of same concentrations is remarkable than exocellular polysaccharide to the restraining effect of S180 sarcoma mouse tumor, shows that phosphorylation modification has improved the anti-tumor activity of this polysaccharide.
The most representative index of mouse tumor illness has been measured in above-mentioned pharmacological experiment analysis, as can be seen from the above results, when dosage is 100-400mg/kg, the Lachnum product exocellular polysaccharide that culture presevation is numbered CCTCC No:M 2011196 has significant antitumor action, can significantly significantly improve food ration, body weight, tumour inhibiting rate and the thymus index of S180 sarcoma mouse, effectively increase the level of the work of SOD enzyme, IL-2 and TNF-α, have obvious anti-tumor activity.The anti-tumor activity of same concentrations phosphorylated derivative obviously strengthens.Positive drug also has significant anti-tumor activity, but food ration, body weight, thymus index and the spleen index of positive drug group mouse all obviously reduce, show that positive drug has affected the immunologic function of mouse to a certain extent, have toxic side effect, and above-mentioned Lachnum product exocellular polysaccharide and polysaccharide derivates thereof are all without obvious toxic side effect.
Embodiment 2:
Prepare the Lachnum exocellular polysaccharide by following condition and mode first: the Lachnum that culture presevation is numbered CCTCC No:M2011196 is inoculated in the 4L fermentor tank, culture medium prescription is: glucose 44g/L, yeast extract paste 12g/L, glycine 6.5mg/L, MgSO47H2O 1.2g/L, KH2PO4 1.2g/L, the coefficient volume ratio is 0.6, inoculum size is the 14%(volume ratio), culture temperature is 30 ℃, air flow is 1.1L/min, and mixing speed is 175r/min, and fermentation time is 8 days; With the fermented liquid suction filtration, concentrated after, the massfraction that adds its 2 times of volumes is that 95% ethanol is in 4 ℃ of alcohol precipitation 30h, alcohol is analysed liquid with the centrifugal 8min of 5000r/min, remove supernatant, then precipitation dehydrated alcohol, washing with acetone 3 times use dissolved in distilled water, Sevag method deproteinated and concentration expressed in percentage by volume are after 30% H2O2 insulation (60 ℃) decolouring is processed, distilled water normal temperature dialysis 30h ,-49 ℃ of lyophilizes namely get the Lachnum product exocellular polysaccharide that culture presevation is numbered CCTCC No:M2011196.
Prepare Lachnum polysaccharide phosphorylated derivative with following condition and mode again: with the sodium sulfate (the foaming phenomenon when reducing reaction) of 105mL distilled water dissolving 10.5g phosphorylation agent (9g tripoly phosphate sodium STPP+1.5g Trisodium trimetaphosphate) and 5.5%, add the above-mentioned polysaccharide of 1.05g after the dissolving, behind NaHCO3 accent pH=8, react 4h under 85 ℃ of conditions, sample liquid precipitates 26h with 95% ethanol of 2 times of volumes.With alcohol precipitation polysaccharide vacuum lyophilization, in 55 ℃ of water-baths, redissolve again, the solution after the redissolution in dialysis tubing (relative molecular weight that dams is 13,000Da) in take distilled water as dialyzate the dialysis 30h, lyophilize namely gets Lachnum polysaccharide phosphorylated derivative.
Then be the aqueous solution that 0.9% physiological saline is configured to 100mg/mL, 400mg/mL with above-mentioned Lachnum exocellular polysaccharide and phosphorylated derivative powder mass percentage concentration thereof, the administration volume be 0.25mL/ only.
Lachnum exocellular polysaccharide and the phosphorylated derivative thereof of above-mentioned each dosage are carried out mouse antineoplastic action pharmacological evaluation, the mode step that adopts and other conditions all with embodiment 1 in identical, also all obtained with embodiment 1 in similar result.
The description of test of above-described embodiment, Lachnum exocellular polysaccharide and phosphorylated derivative thereof that culture presevation is numbered CCTCC No:M2011196 not only have significant anti-tumor function, and have no side effect, the its preparation method simple economy is therefore can be numbered culture presevation the Lachnum exocellular polysaccharide of CCTCC No:M2011196 and the antitumor drug that polysaccharide derivates is applied to prepare tablet or capsule formulation.
Embodiment 3: the processing of Lachnum exocellular polysaccharide phosphorylated derivative preparation
The present invention produces the exocellular polysaccharide phosphorylated derivative as the application of preparation antitumor drug with the Lachnum that culture presevation is numbered CCTCC No:M2011196, and the mode that specifically can be processed as following formulation is used:
1, tablet processing
Behind the Lachnum exocellular polysaccharide phosphorylated derivative of 300-600g and pharmaceutical excipient thinner 200-250g, glidant 30-60g mixing, mix with 250-600mL ethanol and to make softwood, softwood sieves and obtains wet granular, whole grain after dry, add at last compressing tablet behind the lubricant 3-10g mixing, make 1000 tablets of tablets.Described pharmaceutical excipient thinner is selected dextrin or dry starch; Described glidant adopts talcum powder or micropowder silica gel or Microcrystalline Cellulose; Described lubricant adopts Magnesium Stearate or Zinic stearas.
The preference of the tablet processing that the present embodiment is recommended is: the above-mentioned Lachnum exocellular polysaccharide phosphorylated polysaccharide 500g, thinner dextrin 240g, the glidant talcum powder 50g that got the 80-100 mesh sieve, adding 400mL ethanol after mixing mixes and makes softwood, softwood sieves and obtains wet granular, whole grain after dry, add at last compressing tablet behind the magnesium stearate lubricant 10g mixing, make 1000, the heavy 0.8g of sheet, polysaccharide content 0.5g/ sheet.
2, capsule processing
To mix with the above-mentioned Lachnum exocellular polysaccharide phosphorylated polysaccharide of 300-600g behind pharmaceutical excipient thinner 200-270g, glidant 40-70g, the wetting agent 3-6g mixing, dress 1# Capsules makes 1000 of capsules.Described pharmaceutical excipient thinner is selected dextrin or dry starch; Described glidant adopts talcum powder or micropowder silica gel or Microcrystalline Cellulose; Described wetting agent adopts sodium lauryl sulphate or dioctyl sulphosuccinate.
The capsule processing preference that the present embodiment is recommended is: get thinner dry starch 250g, glidant talcum powder 45g, wetting agent sodium lauryl sulphate 5g mixes with the above-mentioned Lachnum exocellular polysaccharide of 500g phosphorylated derivative and mixes 1000 of rear dress 1# Capsuleses, polysaccharide content 0.5g/ grain.
The exocellular polysaccharide that Lachnum produces that the present invention uses culture presevation to be numbered CCTCC No:M2011196 prepares the phosphorylated polysaccharide derivative, and take above-mentioned exocellular polysaccharide phosphorylated derivative as raw material as the preparation antitumor drug application, prepared medicine has overcome existing chemotherapeutics in the kill tumor cell, normal cell also there is to a certain degree lethal effect, and chemotherapy can make the patient lose the appetite, weight loss, cause body weak, and cause to a certain degree immunosuppression, the shortcomings such as toxic side effect is larger, and preparation method's simple economy.
Above-described embodiment of the present invention does not consist of the restriction to protection domain of the present invention.Any modification of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., all should be included within the claim protection domain of the present invention.
Claims (3)
1. Lachnum exocellular polysaccharide phosphorylated derivative is characterized in that, this derivative is prepared by following methods:
(1) the purification exocellular polysaccharide is extracted in the Lachnum fermentation that culture presevation is numbered CCTCC No:M2011196 afterwards, sodium sulfate with 90～110mL distilled water dissolving 9～11g phosphorylation agent and 4～6%, add the above-mentioned polysaccharide of 0.9～1.1g after the dissolving, use NaHCO
3After transferring pH=8～10, react 4～6h under 75～85 ℃ of conditions, sample liquid obtains the alcohol precipitation polysaccharide with 95% ethanol precipitation, the 20～28h of 2～3 times of volumes;
(2) with alcohol precipitation polysaccharide vacuum lyophilization, in 55～65 ℃ of water-baths, redissolve again, the solution after the redissolution in dialysis tubing take distilled water as dialyzate the dialysis 24～36h, lyophilize namely gets Lachnum exocellular polysaccharide phosphorylated derivative.
2. Lachnum exocellular polysaccharide derivative claimed in claim 1 is characterized in that, phosphorylation agent described in the step (1) by tripoly phosphate sodium STPP and Trisodium trimetaphosphate in mass ratio 6:1 mix.
3. the as claimed in claim 1 application of Lachnum exocellular polysaccharide derivative in the preparation antitumor drug.
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