CN105483187A - Anti-tumor effect of sulfated polysaccharide - Google Patents
Anti-tumor effect of sulfated polysaccharide Download PDFInfo
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- CN105483187A CN105483187A CN201510916155.4A CN201510916155A CN105483187A CN 105483187 A CN105483187 A CN 105483187A CN 201510916155 A CN201510916155 A CN 201510916155A CN 105483187 A CN105483187 A CN 105483187A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention provides uses of a sulfated polysaccharide in anti-tumor drugs. A polysaccharide (about 10-20 kDa in molecular weight) obtained by Escherichia coli K5 fermenting is subjected to sulfating bio-modification (namely enzymatic controllable modification) by using 3-sulfotransferase (3-OST-1) and aryl sulfotransferase (AST-IV); after sulfating modification, a sulfated polysaccharide product is obtained by separation and purification. The sulfated polysaccharide of the invention has high inhibitory action on the proliferation and growth of various tumor cells, such as human liver cancer cells (Hepg2), colon cancer cells (HCT116) and lung cancer cells (A549), has no bad influence on normal liver cells LO2 and is applicable to the development of anti-cancer drugs.
Description
Technical field
The present invention relates to medical art.The particularly new medical use of sulfated polysaccharides.
Background technology
Cancer, also known as malignant tumour, for by controlling the not normal caused disease of cell proliferation machinery.Along with the deterioration etc. of environment, cancer has become one of serious disease of harm humans health, and capture cancer is the research topic attracted attention in the world always.Non-equilibrium coupling and the transfer diffusion thereof of Cell apoptosis and proliferation are the bases that malignant tumour occurs.The regulation and control of apoptotic intervention and Cell apoptosis and proliferation are important channel and the means of tumor disease therapeutic.Under normal physiological condition, in good time, the apoptosis regulation of appropriateness removes old and feeble and the proemial cell of tool to organism, the growth maintaining genomic stability and normal individual is very important, deposits inducing apoptosis of tumour cell in case can reach the object of oncotherapy in tumour.
Polysaccharide (Polysaccharide) is also known as saccharan, in the cytolemma being distributed in higher animal plant or microorganism wall, it is the natural polymer polymer that a class is formed by connecting by sugar former times key by aldose or ketose, it is the abundantest biological ployose of nature content, form one of large base substance of vital movement four, not only for biology provides skeleton structure and energy derive, also take part in the various biological phenomena of cell and the adjustment of physiological process widely.Along with the chemical research of molecular biology, phylactic agent and the searching of development and novel drugs resource and exploitation, the research of polysaccharide comes into one's own more and more widely.Can carry out structure of modification by methods such as chemistry, physics and biology to polysaccharide, the biologic activity of polysaccharide is obviously increased, and much research shows polysaccharide to be carried out anti-tumor activity and the immunoregulatory activity that sulphation modification can improve polysaccharide.
EscherichiacoliK5 fermentation using bacteria gained polysaccharide, its structure is the repetition disaccharides structure that glucuronic acid is linked by Isosorbide-5-Nitrae glycosidic link with n acetylglucosamine n.The present invention adopts three sulfate transferring enzymes (3-OST-1) and aromatic sulfuric acid based transferase (AST-IV) to 3 sulfation bio-modifications of n acetylglucosamine n in this polysaccharide disaccharides structure, controlled by reaction conditions, to obtaining serial highly active polyose modification product.
Summary of the invention
Originally the object of the invention is to the novelty teabag that sulfated polysaccharides is provided.
Particularly, the invention provides sulfated polysaccharides and prepare the effect in antitumor medicine.
Wherein, the separation and purification of EscherichiacoliK5 fermentation using bacteria obtains the single capsular polysaccharide of the higher structure of purity, to be glucuronic acid (GlcUA) to be alternately connected in molar ratio with acetamido glucose (GlcNAc) at 1: 1 for the structure of polysaccharide, its structural formula is [-GlcUA-β-(1,4)-GlcNAc-α-(1,4)-] n, the content recording polysaccharide through nuclear-magnetism marker method and HPGPC method is 95.27% be about 10 ~ 20kDa with molecular size range, and n is about 240 ~ 480.
Described sulfated polysaccharides, for bacterial eapsular polysaccharide obtains product through three sulfate transferring enzymes (3-OST-1) and aromatic sulfuric acid based transferase (AST-IV) enzymatic modification, products therefrom be by glucosamine 3 sulfations in polysaccharide disaccharides structure after sulfated polysaccharides.
Measure in the specific embodiment of the invention and show, in product, sulfated polysaccharides content is 94 ± 2%w/w, this sulfating product purity is higher, biological activity test is carried out with the sulfating product under this purity, its result can reflect the biological activity of sulfated polysaccharides really, efficiently avoid the interference of other compositions to sulfated polysaccharides determination of activity.
The method of bacterial eapsular polysaccharide deacetylation is: 100mgheparosan is dissolved in 25mL, the NaOH of 2mol/L, is placed in 60 DEG C of water-baths and reacts 5h.After reaction terminates, sample is cooled to room temperature, adjusts pH to 7.0 with HCl.Use 3500Da dialysis tubing, pure water is dialysed, freeze-drying.
Further, after bacterial eapsular polysaccharide deacetylation, deacetylation polysaccharide concentration 1mg/L; Substrate 40 μm of ol/LPAP and 1mmol/LPNPS, 3-OST-1 are 0.88U/L, AST-IV is 1.688UU/L, pH7.0 (50mmol/LMES, 1%Triton, 1mmol/LMgCl
2, 1mmol/LMnCl
2damping fluid); Temperature 30 DEG C; Time 24h.
Further, after reaction terminates, solution is heated to 100 DEG C, insulation 10min, then centrifugal (4000rpm) removes zymoprotein, supernatant liquor is put into dialysis tubing (molecular weight cut-off is 3.5kDa) dialysis (4 DEG C, 24h), by centrifugal for sample (4000rpm) after dialysis terminates, get supernatant liquor lyophilize, white powder sulfated polysaccharides modified outcome can be obtained.
MTT experiment proves, sulfated polysaccharides all has stronger restraining effect to being in three kinds of cell human liver cancer cells (Hepg2) of logarithmic phase, the propagation of colon cancer cell (HCT116) and lung carcinoma cell (A549) and growth, normal cell human liver cell LO2 growth is had no adverse effects, for research sulfated polysaccharides derivative PTS provides the foundation.
Accompanying drawing explanation
Fig. 1 EscherichiacoliK5 fermentation using bacteria gained polysaccharide
1h-NMR collection of illustrative plates
Fig. 2 EscherichiacoliK5 fermentation using bacteria gained polysaccharide HPGPC collection of illustrative plates
Fig. 3 sulfated polysaccharides
1h-NMR collection of illustrative plates
Embodiment
The present invention will be further explained for the following examples, but the present invention is not limited only to these embodiments, the scope that these embodiments do not limit the present invention in any way.Those skilled in the art within the scope of the claims made some changes and adjustment also should be thought and belongs to the scope of the invention
Embodiment 1
By bacterial eapsular polysaccharide deacetylation, method is dissolved in 25mL, the NaOH of 2mol/L for getting 100mg polysaccharide, is placed in 60 DEG C of water-baths and reacts 5h.After reaction terminates, sample is cooled to room temperature, adjusts pH to 7.0 with HCl.Use 3500Da dialysis tubing, pure water is dialysed, freeze-drying.
Embodiment 2
Adopting three sulfate transferring enzymes (3-OST-1) and aromatic sulfuric acid based transferase (AST-IV)) modification method carries out bio-modification (i.e. the controlled modification of enzyme process) to bacterial eapsular polysaccharide, after bacterial eapsular polysaccharide deacetylation, deacetylation polysaccharide concentration 1mg/L; Substrate 40 μm of ol/LPAP and 1mmol/LPNPS, 3-OST-1 are 0.88U/L, AST-IV is 1.688UU/L, pH7.0 (50mmol/LMES, 1%Triton, 1mmol/LMgCl
2, 1mmol/LMnCl
2damping fluid); Temperature 30 DEG C; Time 24h.
Embodiment 3
Remove zymoprotein: solution is heated to 100 DEG C, insulation 10min, then centrifugal (4000rpm) removes zymoprotein, supernatant liquor is put into dialysis tubing (molecular weight cut-off is 3.5kDa) dialysis (4 DEG C, 24h), by centrifugal for sample (4000rpm) after dialysis terminates, get supernatant liquor lyophilize, white powder 3 sulfated polysaccharides modified outcomes can be obtained.
Embodiment 4
The anti-tumor activity of sulfated polysaccharides:
Mtt assay is adopted to detect sulfated polysaccharides product to the anti-tumor activity of human liver cancer cell (Hepg2), colon cancer cell (HCT116) and lung carcinoma cell (A549).Arrange drug concentration gradient be 200,100,80,40,20,10,5,0mg/ml, cultivate with RPMI-1640+10%FBS substratum or DMEM high glucose medium, get growth logarithmic phase cell dissociation bed board, cell number is by 6000/hole bed board, 180 μ L/ holes, in 37 DEG C, cultivate in 5%CO2 incubator.After cell attachment, add sulfated polysaccharides and the positive drug taxol 20 μ L/ hole of different concns, each concentration all establishes three parallel holes, and after administration is hatched and cultivated 48h, every hole adds MTT20 μ l, continues to cultivate 4h.Microplate reader detects OD
570take drug level as transverse axis, inhibiting rate is the longitudinal axis, does broken line graph, by SPSS software calculate medicine to the half inhibiting rate IC of different cell
50value.
Result shows that sulfated polysaccharides product has good anti-tumor activity, it all has obvious suppression relation to the propagation of human liver cancer cell (Hepg2), colon cancer cell (HCT116) and lung carcinoma cell (A549) and growth, the IC50 value of its correspondence is 53.456 μ g/ml, 33.328 μ g/ml and 22.344 μ g/ml respectively, has no adverse effects to normal cell human liver cell LO2 growth.
Claims (5)
1. the present invention adopts the separation and purification of EscherichiacoliK5 fermentation using bacteria to obtain the single capsular polysaccharide of the higher structure of purity, to be glucuronic acid (GlcUA) to be alternately connected in molar ratio with acetamido glucose (GlcNAc) at 1: 1 for the structure of polysaccharide, its structural formula is [-GlcUA-β-(1,4)-GlcNAc-α-(1,4)-] n, the content recording polysaccharide through nuclear-magnetism marker method and HPGPC method is 95.27% be about 10 ~ 20kDa with molecular size range, and n is about 240 ~ 480.
2. polysaccharide according to claim 1, by the method for its deacetylation is: 100mgheparosan is dissolved in 25mL, the NaOH of 2mol/L, is placed in 60 DEG C of water-baths and reacts 5h.After reaction terminates, sample is cooled to room temperature, adjusts pH to 7.0 with HCl.Use 3500Da dialysis tubing, pure water is dialysed, freeze-drying.
3. lyophilized products according to claim 2, adopts three sulfate transferring enzymes (3-OST-1) and aromatic sulfuric acid based transferase (AST-IV) to carry out sulfation bio-modification (i.e. the controlled modification of enzyme process) to it.Through separation and purification after sulphation modification, obtain 3 sulfated polysaccharides products, its content is 94% ± 2%w/w.。
4. application according to claim 3, is characterized in that the action condition of sulfation transferring enzyme: after polysaccharide deacetylation, deacetylation polysaccharide concentration 1mg/L; Substrate 40 μm of ol/LPAP and 1mmol/LPNPS, 3-OST-1 are 0.88U/L, AST-IV is 1.688UU/L, pH7.0 (50mmol/LMES, 1%Triton, 1mmol/LMgCl
2, 1mmol/LMnCl
2damping fluid); Temperature 30 DEG C; Time 24h.
5. 3 sulfated polysaccharides products according to claim 3, it all shows obvious restraining effect to the growth of human liver cancer cell (Hepg2), colon cancer cell (HCT116) and lung carcinoma cell (A549) to it is characterized in that adopting mtt assay inspection side, its corresponding IC50 value is, has no adverse effects to normal cell human liver cell LO2 growth.
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Cited By (1)
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CN112791186A (en) * | 2021-04-14 | 2021-05-14 | 江苏艾洛特医药研究院有限公司 | Polysaccharide nanocomposite and preparation and application thereof |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006124801A2 (en) * | 2005-05-12 | 2006-11-23 | The University Of North Carolina At Chapel Hill | Enzymatic synthesis of sulfated polysaccharides |
CN1916030A (en) * | 2001-02-27 | 2007-02-21 | 乔治·佐派蒂 | Highly sulfated derivatives of k5 polysaccharide and their preparation |
CN101531723A (en) * | 2009-02-27 | 2009-09-16 | 江南大学 | Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure |
CN102660610A (en) * | 2012-05-31 | 2012-09-12 | 江南大学 | Method for preparing high-activity and low-molecular-weight heparin by enzymic method |
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2015
- 2015-12-09 CN CN201510916155.4A patent/CN105483187A/en active Pending
Patent Citations (4)
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CN1916030A (en) * | 2001-02-27 | 2007-02-21 | 乔治·佐派蒂 | Highly sulfated derivatives of k5 polysaccharide and their preparation |
WO2006124801A2 (en) * | 2005-05-12 | 2006-11-23 | The University Of North Carolina At Chapel Hill | Enzymatic synthesis of sulfated polysaccharides |
CN101531723A (en) * | 2009-02-27 | 2009-09-16 | 江南大学 | Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure |
CN102660610A (en) * | 2012-05-31 | 2012-09-12 | 江南大学 | Method for preparing high-activity and low-molecular-weight heparin by enzymic method |
Non-Patent Citations (1)
Title |
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Cited By (1)
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CN112791186A (en) * | 2021-04-14 | 2021-05-14 | 江苏艾洛特医药研究院有限公司 | Polysaccharide nanocomposite and preparation and application thereof |
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Application publication date: 20160413 |