CN109897117B - Phosphorylated TP2 polysaccharide, and preparation method and application thereof - Google Patents
Phosphorylated TP2 polysaccharide, and preparation method and application thereof Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 92
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 92
- -1 Phosphorylated TP2 polysaccharide Chemical class 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims abstract description 20
- 238000004108 freeze drying Methods 0.000 claims abstract description 20
- 150000004676 glycans Chemical class 0.000 claims abstract description 20
- 230000026731 phosphorylation Effects 0.000 claims abstract description 20
- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 20
- 241000606124 Bacteroides fragilis Species 0.000 claims abstract description 16
- 238000000502 dialysis Methods 0.000 claims abstract description 9
- UGTZMIPZNRIWHX-UHFFFAOYSA-K sodium trimetaphosphate Chemical compound [Na+].[Na+].[Na+].[O-]P1(=O)OP([O-])(=O)OP([O-])(=O)O1 UGTZMIPZNRIWHX-UHFFFAOYSA-K 0.000 claims abstract description 8
- 235000019832 sodium triphosphate Nutrition 0.000 claims abstract description 8
- 239000002244 precipitate Substances 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 14
- 239000010452 phosphate Substances 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- 238000010612 desalination reaction Methods 0.000 claims description 4
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- 230000000865 phosphorylative effect Effects 0.000 claims description 4
- 239000010802 sludge Substances 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 229960000583 acetic acid Drugs 0.000 claims description 2
- 238000009835 boiling Methods 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 239000012362 glacial acetic acid Substances 0.000 claims description 2
- 238000002386 leaching Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 2
- 150000003839 salts Chemical class 0.000 claims 2
- 239000004480 active ingredient Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 5
- 230000009266 disease activity Effects 0.000 abstract description 4
- 238000001556 precipitation Methods 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 230000004071 biological effect Effects 0.000 description 7
- 210000001072 colon Anatomy 0.000 description 6
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 5
- 206010009887 colitis Diseases 0.000 description 5
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- 241001465754 Metazoa Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 206010022714 Intestinal ulcer Diseases 0.000 description 2
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- 210000000936 intestine Anatomy 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000000779 smoke Substances 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- UETNIIAIRMUTSM-UHFFFAOYSA-N Jacareubin Natural products CC1(C)OC2=CC3Oc4c(O)c(O)ccc4C(=O)C3C(=C2C=C1)O UETNIIAIRMUTSM-UHFFFAOYSA-N 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
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- 230000006978 adaptation Effects 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
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- 229940010552 ammonium molybdate Drugs 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
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- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
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- 230000024949 interleukin-17 production Effects 0.000 description 1
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- 230000002147 killing effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
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- 238000005556 structure-activity relationship Methods 0.000 description 1
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- NCEXYHBECQHGNR-QZQOTICOSA-N sulfasalazine Chemical compound C1=C(O)C(C(=O)O)=CC(\N=N\C=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-QZQOTICOSA-N 0.000 description 1
- 229960001940 sulfasalazine Drugs 0.000 description 1
- NCEXYHBECQHGNR-UHFFFAOYSA-N sulfasalazine Natural products C1=C(O)C(C(=O)O)=CC(N=NC=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a preparation method and application of phosphorylated TP2 polysaccharide with anti-inflammatory activity, wherein the TP2 polysaccharide is derived from bacteroides fragilis ZY-312, a phosphorylation reagent is prepared by mixing sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio, the usage amount of the phosphorylation reagent is 0.1-0.15 g/mL, the concentration of the polysaccharide is 0.01-0.02 g/mL, the polysaccharide solution and the phosphorylation reagent are uniformly mixed, the pH value of the solution is adjusted to 8.0-9.0, the reaction is carried out, ethanol solution is subjected to alcohol precipitation, precipitates are collected, and the phosphorylated TP2 polysaccharide is obtained through freeze drying, redissolution, dialysis and freeze drying. The phosphorylated TP2 polysaccharide had higher anti-inflammatory bowel disease activity than the TP2 polysaccharide.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to the technical field of application of bacteroides fragilis, and particularly relates to phosphorylated TP2 polysaccharide and a preparation method and application thereof.
Background
Inflammatory Bowel Disease (IBD) is a group of chronic Inflammatory diseases of intestinal tract with uncertain etiology, and the etiology and pathogenesis of IBD are complex and related to factors such as immunity, heredity, environment, microbial infection and the like. In recent years, it has been found that immune factors play an important role in the onset and change of inflammatory bowel disease, including helper T cells, regulatory T cells, cytokines, autoantibodies, and the like.
Studies have shown that Bacteroides fragilis capsular polysaccharide can reduce intestinal inflammation in animals by inhibiting IL-17 production; there have also been studies showing that bacteroides fragilis capsular polysaccharide can treat colitis.
The polysaccharide is a macromolecular compound with a complex structure, has various biological activities such as immunoregulation, anti-tumor, anti-oxidation and the like, exerts the biological activity closely related to the specific structure of the polysaccharide, has limited biological activity of the extracted natural polysaccharide, and can achieve the purpose of enhancing the biological activity through chemical modification. At present, phosphorylated polysaccharides are important derivatives of polysaccharides, and the main research is that the phosphorylated polysaccharides have various biological activities such as immunoregulation, anti-tumor and anti-virus, so that phosphorylation modification of polysaccharides and investigation of changes of biological activities before and after modification are important contents of the research on structure-activity relationship of polysaccharides at present. However, no report about phosphorylation modification of Bacteroides fragilis capsular polysaccharide is found so far.
At present, domestic and foreign application research on Bacteroides fragilis capsular polysaccharide mainly focuses on preventing and treating inflammation, enhancing immunity, multiple sclerosis, asthma, abscess and the like. There are few reports on studies on chemical modification of bacteroides fragilis capsular polysaccharide (TP2 polysaccharide) to enhance its biological activity.
Disclosure of Invention
The invention aims to provide a preparation method of phosphorylated TP2 polysaccharide for preventing and treating inflammatory bowel diseases so as to develop a novel anti-inflammatory drug.
A method for preparing phosphorylated TP2 polysaccharide having anti-inflammatory bowel disease activity, comprising the steps of:
(1) preparation of phosphorylating reagent: mixing sodium tripolyphosphate and sodium trimetaphosphate, dissolving with water, and preparing a phosphorylation reagent, wherein the concentration of the phosphorylation reagent is 0.1-0.15 g/mL;
(2) preparation of TP2 polysaccharide: carrying out hot water leaching, ultrafiltration concentration, acidolysis, ultrafiltration desalination, column chromatography and freeze drying on bacterial sludge of Bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 to obtain TP2 polysaccharide;
(3) adding freeze-dried TP2 polysaccharide into a phosphorylation reagent, and dissolving to obtain a solution, wherein the concentration of the TP2 polysaccharide in the solution is 0.01-0.02 g/mL;
(4) adjusting the pH value of the solution to 8.0-9.0, reacting, taking out, and cooling to room temperature;
(5) slowly adding 3-4 times of 95% ethanol solution into the solution, and standing; centrifuging, taking the precipitate, placing the precipitate in an environment with the temperature of 50 +/-3 ℃ for 2-3 hours to remove residual ethanol, and freeze-drying; adding ultrapure water into the polysaccharide obtained after freeze drying, redissolving, dialyzing, and freeze drying to obtain the phosphorylated TP2 polysaccharide.
In one embodiment, the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is 5-6: 1.
In one embodiment, the Bacteroides fragilis is existing Bacteroides fragilis ZY-312 with the preservation number of CGMCC No. 10685.
In one embodiment, the reaction conditions are: and (3) reacting for 5-6 h at the temperature of 80-85 ℃.
In one embodiment, in the step (5), the phosphate TP2 polysaccharide is obtained by re-dissolving at 50-60 ℃, transferring the phosphate TP2 polysaccharide into a dialysis bag with the molecular weight cutoff of 10000-14000D for dialysis for 48 +/-2 h, and freeze-drying.
Another objective of the invention is to provide a phosphorylated TP2 polysaccharide, namely, a phosphorylated TP2 polysaccharide obtained by the above preparation method.
It is another object of the present invention to provide the use of said phosphorylated TP2 polysaccharide.
The phosphorylated TP2 polysaccharide obtained by the preparation method is applied to preparing medicaments for preventing and treating Inflammatory Bowel Diseases (IBD).
A medicine for preventing and treating Inflammatory Bowel Disease (IBD) contains the phosphorylated TP2 polysaccharide as active component.
The invention has the following beneficial effects:
according to the invention, the phosphorylated TP2 polysaccharide is obtained by carrying out phosphorylation modification on the Bacteroides fragilis TP2 polysaccharide, and the phosphate radical grafting amount of the phosphorylated TP2 polysaccharide is controlled to be 4-6%. The phosphorylated polysaccharide obtained by the invention has the activity of resisting inflammatory bowel diseases and has the potential of developing new anti-inflammatory drugs.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The reagents and starting materials used in the following examples were all commercially available unless otherwise specified.
The steps in the following preparation method can be replaced by the order of steps, for example, the steps (1) and (2) can be different in sequence according to the common knowledge of the skilled person, but the preparation result is not changed.
Example 1
The preparation method of phosphorylated TP2 polysaccharide with the activity of preventing and treating inflammatory bowel diseases, which is described in the embodiment, comprises the following steps:
(1) preparation of phosphorylating reagent: the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is 5:1, the ultrapure water is dissolved, and the concentration of a phosphorylation reagent is 0.1 g/mL;
(2) TP2 polysaccharide is derived from Bacteroides fragilis ZY-312 with preservation number of CGMCC No.10685, 20g of bacterial mud, 100mL of ultrapure water is added, the mixture is uniformly mixed, stirred and extracted for 2h at 90 ℃ and 15000g, the mixture is centrifuged at normal temperature for 20min, supernatant is taken, glacial acetic acid with volume concentration of 10% is added, the mixture reacts for 3h under a boiling state, the mixture is cooled to room temperature, the pH of reaction liquid is adjusted to be neutral by using a sodium hydroxide solution with mass concentration of 10%, ultrafiltration and desalination are carried out by using a 10KD ultrafiltration membrane, DEAE Sepharose Fast Flow column chromatography (20cm multiplied by 1.6cm), the pH is 8.5, elution is carried out by using a 20mM Tris-HCl solution, the ultraviolet detection wavelength is 206nm, eluent corresponding to an absorption peak is collected, ultrafiltration and desalination are carried out by using the 10KD ultrafiltration membrane, and freeze drying is carried out, so as to obtain TP2 polysaccharide;
(3) adding freeze-dried TP2 polysaccharide into phosphorylation reagent, and dissolving to make TP2 polysaccharide concentration 0.02 g/mL;
(4) adjusting the pH value of the solution to 9.0; the solution is placed in an environment with the temperature of 80 ℃ for reaction for 5 hours, taken out and cooled to the room temperature;
(5) slowly adding 4 times of 95% ethanol solution into the solution, and standing for 24 h; centrifuging, collecting precipitate, standing at 50 deg.C for 3 hr to remove residual ethanol, and freeze drying; adding ultrapure water into the polysaccharide obtained after freeze drying, redissolving at 55 ℃, transferring into a dialysis bag with the molecular weight cutoff of 10000D for dialysis for 48h, and freeze drying to obtain the phosphorylated TP2 polysaccharide.
Drawing of phosphate radical standard curve (molybdenum blue colorimetric method)
Respectively placing potassium dihydrogen phosphate as standard substance in 25mL colorimetric tubes, sequentially adding 2.0mL of 5% ammonium molybdate solution, shaking, standing for several seconds, respectively adding 1.0mL of 2% sodium sulfite solution and 1.0mL of 0.5% hydroquinone solution, shaking, adding water to scale, standing for 30min, measuring absorbance at 700nm wavelength, taking phosphate radical (in P) mass concentration as abscissa and corresponding absorbance as ordinate as standard curve, and obtaining standard curve equation as standard curve equation
y＝0.952x-0.894，R2＝0.9991
The phosphate grafting amount of the phosphorylated TP2 polysaccharide of the present invention was determined as follows:
taking a proper amount of sample, adding 1mL of concentrated sulfuric acid and concentrated nitric acid into a beaker respectively, heating the beaker on an electric furnace until smoke is produced, then cooling the beaker, adding 1mL of 30% hydrogen peroxide solution, slowly heating the beaker, and repeating the steps until no smoke is produced in the flask, wherein the solution is colorless and transparent or light yellow. After cooling, 1mL of 6mol/L hydrochloric acid was added, and the mixture was heated on an electric furnace to completely decompose the acid, and transferred to a 50mL volumetric flask to fix the volume. And (3) taking 0.5mL to measure the absorbance according to a standard curve operation method, and calculating the content of phosphate (in terms of P) according to a standard curve equation.
In the formula: a is the absorbance of the sample at a wavelength of 700nm, and m is the mass of the sample/mg.
The phosphorylated TP2 polysaccharide prepared in this example had a phosphate graft of 4.71%.
Example 2
The preparation method of phosphorylated TP2 polysaccharide having anti-inflammatory bowel disease activity described in this example comprises the following steps:
(1) preparation of phosphorylating reagent: the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is 6:1, the ultrapure water is dissolved, and the concentration of a phosphorylation reagent is 0.1 g/mL;
(2) TP2 polysaccharide was prepared as in example 1;
(3) adding freeze-dried TP2 polysaccharide into phosphorylation reagent, and dissolving to make TP2 polysaccharide concentration 0.015 g/mL;
(4) adjusting the pH value of the solution to 8.0, placing the solution in an environment of 85 ℃, reacting for 5 hours, taking out, and cooling to room temperature;
(5) slowly adding 3 times of 95% ethanol solution into the solution, and standing for 24 h; centrifuging, collecting precipitate, standing at 50 deg.C for 2.5h to remove residual ethanol, and freeze drying; adding ultrapure water into the polysaccharide obtained after freeze drying, redissolving at 60 ℃, transferring into a 14000D dialysis bag with molecular weight cutoff for dialysis for 48h, and freeze drying to obtain the phosphorylated TP2 polysaccharide.
According to the method for measuring the phosphate grafting amount of phosphorylated TP2 polysaccharide described in example 1, the phosphate grafting amount of phosphorylated TP2 polysaccharide prepared according to the present invention was 5.68%.
Comparative example 1
The preparation method of phosphorylated TP2 polysaccharide is the same as that of example 1, but the mass ratio of sodium tripolyphosphate to sodium trimetaphosphate is 8:1, and the concentration of the phosphorylation reagent is 0.3 g/mL; adding freeze-dried TP2 polysaccharide into the phosphorylation reagent in the step (3), and dissolving to ensure that the concentration of TP2 polysaccharide is 0.4 g/mL; the other steps are the same as in example 1. The obtained acidified TP2 polysaccharide was prepared with a phosphate grafting of 0.413%.
Comparative example 2
The preparation method of phosphorylated TP2 polysaccharide is the same as that of example 1, but the mass ratio of sodium tripolyphosphate to sodium trimetaphosphate is 4:1, and the concentration of the phosphorylation reagent is 0.4 g/mL; adding freeze-dried TP2 polysaccharide into the phosphorylation reagent in the step (3), and dissolving to ensure that the concentration of TP2 polysaccharide is 0.05 g/mL; the other steps are the same as in example 1. The acidified TP2 polysaccharide was prepared with a phosphate graft of 7.53%.
Example 3
Anti-inflammatory bowel disease activity of phosphorylated TP2 polysaccharide:
the research establishes a Balb/c mouse colitis model induced by trinitrobenzenesulfonic acid (TNBS) as an animal model for researching inflammatory bowel disease, and discusses the treatment effect of TP2 polysaccharide and phosphorylated TP2 polysaccharide on inflammatory bowel disease. The Balb/c mice are 16-18 g (70 mice without spare animals) and are 6-8 weeks old, and are fed with common feed in an animal laboratory, so that the modeling is carried out after one week adaptation.
The grouping and dosing regimen was as follows:
table 1 grouping and dosing regimens
The day of modeling was designated day0, except for the negative control group, the enteritis of other groups was induced by perfusing 50. mu.L TNBS perfusate (50% ethanol solution containing 2mg TNBS) into the rectum of the mice and perfusing 100. mu.L TNBS perfusate into the colon. Mice were weighed every 24h and recorded; the activity and mortality of the mice were observed and recorded. Killing the mouse by using a cervical dislocation method at day7, fixing the mouse in a supine position, opening the abdominal cavity of the mouse, intercepting colon tissues of the mouse, carrying out general observation, intercepting part of the colon tissues, then placing the colon tissues in a formaldehyde solution for fixing, dehydrating, embedding, slicing and staining, observing pathological changes of the colon tissues under a light microscope, and carrying out TNBS-colitis histological scoring.
TNBS-colitis histological scoring method
Results
(1) After modeling, TNBS-colitis is characterized in that the weight of mice is obviously reduced, the mice die after 4 days, the death rate of the mice reaches 30 percent on the 5 th day, colon is shortened, intestinal wall is thickened, inflammatory exudation exists, part of intestinal segments are adhered to surrounding tissues, and the histological score is 4 grade.
(2) The positive drug sulfasalazine gradually recovers the weight of the mouse, and reduces the weight of the intestines and the area of the intestinal ulcer.
(3) The TP2 polysaccharide and the phosphorylated TP2 polysaccharide in the embodiment 1 of the invention have similar effects with positive drugs, and can reduce the weight of intestines and the area of intestinal ulcer and relieve the inflammatory infiltration degree. The histological score of the phosphorylated TP2 polysaccharide described in example 1 of the present invention was superior to that of the TP2 polysaccharide. In general, the phosphorylated TP2 polysaccharide described in example 1 of the present invention was superior to the TP2 polysaccharide and also superior to the group of TP2 polysaccharides described in comparative examples 1 and 2 in treating inflammatory bowel disease.
TABLE 3 mouse mortality, intestinal length, weight, ulcer area and histological score
The technical features of the embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the description in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. A preparation method of phosphorylated TP2 polysaccharide is characterized by comprising the following steps:
(1) preparation of phosphorylating reagent: mixing sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of 5:1, dissolving with water, and preparing a phosphorylation reagent, wherein the concentration of the phosphorylation reagent is 0.1 g/mL;
(2) preparation of TP2 polysaccharide: carrying out hot water leaching, ultrafiltration concentration, acidolysis, ultrafiltration desalination, column chromatography and freeze drying on bacterial sludge of bacteroides fragilis to obtain TP2 polysaccharide;
(3) adding freeze-dried TP2 polysaccharide into a phosphorylation reagent, and dissolving to obtain a solution, wherein the concentration of the TP2 polysaccharide in the solution is 0.01-0.02 g/mL;
(4) adjusting the pH value of the solution to 8.0-9.0, reacting, taking out, and cooling to room temperature;
(5) slowly adding 3-4 times of 95% ethanol solution into the solution, and standing; centrifuging, taking the precipitate, placing the precipitate in an environment with the temperature of 50 +/-3 ℃ for 2-3 hours to remove residual ethanol, and freeze-drying; adding ultrapure water into the polysaccharide obtained after freeze drying, redissolving, dialyzing, and freeze drying to obtain the phosphorylated TP2 polysaccharide.
2. The method according to claim 1, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a accession number of CGMCC No. 10685.
3. The process according to claim 1 or 2, wherein the reaction conditions are: and (3) reacting for 5-6 h at the temperature of 80-85 ℃.
4. The preparation method according to claim 1 or 2, wherein in the step (5), the phosphate TP2 polysaccharide is obtained by re-dissolving at 50-60 ℃, transferring the re-dissolved solution into a dialysis bag with the molecular weight cutoff of 10000-14000D for dialysis for 48 +/-2 h, and freeze-drying.
5. The preparation method according to claim 2, wherein in the step (2), the bacterial sludge of Bacteroides fragilis ZY-312 with CGMCC number 10685 is taken, ultrapure water is added, the mixture is uniformly mixed, stirred and extracted, centrifuged at normal temperature, supernatant is taken, glacial acetic acid is added, the mixture reacts for 2 to 4 hours in a boiling state, the mixture is cooled to room temperature, the pH of the reaction solution is adjusted to be neutral, ultrafiltration is performed by an ultrafiltration membrane to remove salt, DEAE Sepharose Fast Flow column chromatography is performed, pH is 8.5, and 20mM Tris-HCl solution is eluted, eluent corresponding to an absorption peak is collected, ultrafiltration is performed by an ultrafiltration membrane to remove salt, and freeze drying is performed to obtain the TP2 polysaccharide.
6. Phosphorylated TP2 polysaccharide obtainable by the process according to any one of claims 1 to 5.
7. The phosphorylated TP2 polysaccharide of claim 6, wherein the phosphorylated TP2 polysaccharide has a phosphate grafting amount of 4 to 6%.
8. Use of a phosphorylated TP2 polysaccharide of claim 6 or 7 in the manufacture of a medicament for the prevention or treatment of inflammatory bowel disease.
9. A medicament for the prevention and treatment of inflammatory bowel disease, characterized in that the active ingredient comprises phosphorylated TP2 polysaccharide according to claim 6 or 7.
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| CN103319619A (en) * | 2013-06-25 | 2013-09-25 | 合肥工业大学 | Application of lachnum extracellular polysaccharide phosphorylated derivative and application thereof in preparation of antitumor drugs |
| CN105560352A (en) * | 2016-02-01 | 2016-05-11 | 南京农业大学 | Duck viral hepatitis resisting astraglus polysaccharide phosphorylated molecular modification method |
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