CN103613678A - Sipunculus nudus polysaccharide sulfation modification method - Google Patents
Sipunculus nudus polysaccharide sulfation modification method Download PDFInfo
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Abstract
The invention discloses a Sipunculus nudus polysaccharide sulfation modification method. The Sipunculus nudus polysaccharide has the functions of resisting microbes, resisting viruses, resisting radiation, enhancing the immunity of organisms and the like. The activity of the polysaccharide is directly or indirectly influenced by the molecular structure, and proper modification on the molecular structure of the polysaccharide can change the activity of the polysaccharide and enhance the effect. A chlorosulfonic acid-pyridine method is utilized to perform sulfation modification on the Sipunculus nudus polysaccharide; the purification of the Sipunculus nudus polysaccharide, the sulfation modification of the Sipunculus nudus polysaccharide and other specific technical measures are taken to obtain the Sipunculus nudus sulfated polysaccharide; and a disc diffusion test is utilized to perform antibacterial activity determination on the Sipunculus nudus polysaccharide and sulfated polysaccharide under optimal modification conditions by using 6 bacteria as indication bacteria. The antimicrobial test result indicates that the Sipunculus nudus sulfation molecular modification product prepared by the method can further enhance the antibacterial activity of the Sipunculus nudus polysaccharide.
Description
Technical field
What the present invention relates to is the polysaccharide sulfated modifying method of a kind of Sipunculus nudus, belongs to the structure of modification technical field of polysaccharide.
Background technology
Polysaccharide is isolating protein and the outer important biomacromolecule material of another class of nucleic acid in organism, is extensively present in animal cell membrane, plant and microorganism wall, has biological activity widely.Polysaccharose substance has immunomodulatory, antitumor, radioprotective, antibacterial, antifatigue and the anti-ageing effect of waiting for a long time.The biological activity of polysaccharide and its structure have close relationship, take certain method to carry out suitable modification to polysaccharide molecule structure and can change active polysaccharide, improve its effect.Sulphation modification is one of method common in polyose modification, because the introducing of sulfate has had polyanion characteristic, and polyanion characteristic is the principal element that its biologic activity changes, some natural polysaccharides have obtained purposes polysaccharide derivates more widely after sulphation modification.
Sipunculus nudus; have the title of " ocean Cordyceps sinensis "; a kind of dietotherapy excellent herbal cuisine of holding concurrently, have significantly delay senility, the effect such as anti-oxidant, antifatigue, hypoxia tolerance, high temperature resistant, lactagogue and nourishing Yin and falling fire, removing heat from the lung and dissipating phlegm, cardiovascular systems is also had to significant protective effect.Polysaccharide is as the main effective constituent of Sipunculus nudus, its bioactive research is day by day increased, according to bibliographical information and the applicant's previous work, prove, Sipunculus nudus polysaccharide has anti-microbial activity, but this anti-microbial activity is slightly inadequate, therefore, find a kind of method that improves Sipunculus nudus polysaccharide anti-microbial activity, be more conducive to improve the utility value of Sipunculus nudus.Sipunculus nudus polysaccharide is carried out to structural modification, is one of approach improving Sipunculus nudus polysaccharide anti-microbial activity, does not find that there is in this respect the disclosure of correlation technique through retrieval.
Summary of the invention
The object of the present invention is to provide the polysaccharide sulfated modifying method of a kind of Sipunculus nudus, for the low problem of Sipunculus nudus polysaccharide anti-microbial activity, Sipunculus nudus polysaccharide structures is carried out to sulphation modification, thereby improve the anti-microbial activity of Sipunculus nudus polysaccharide.
For achieving the above object, the present invention has taked following technical proposals, the polysaccharide sulfated modifying method of a kind of Sipunculus nudus, and concrete technological step is:
(1) purifying of Sipunculus nudus polysaccharide
1) cellulose chromatography: get Sipunculus nudus Crude polysaccharides and carry out cellulose chromatography, column chromatography working method routinely, get Sipunculus nudus Crude polysaccharides 0.5g and be dissolved in 10mL ultrapure water loading, with 0.3-0.5moL/L NaCL wash-out, flow velocity is 1mL/min, obtains elutriant, with glassine paper dialysis, remove freshen, being evaporated to 30 ℃ of relative densities of temperature is 0.9829 g/mL, carries out lyophilize, and the Sipunculus nudus polysaccharide dried frozen aquatic products that obtains water content and be 1-5% is standby;
2) gel filtration chromatography: get Sipunculus nudus polysaccharide dried frozen aquatic products and dissolve with ultrapure water, 0.45 μ m membrane filtration, carry out according to a conventional method gel filtration chromatography, with ultrapure water with 0.2 mL/min flow velocity wash-out, obtain elutriant, being evaporated to and recording relative density when temperature is 28 ℃ is 0.9856-0.9918 g/mL, carries out lyophilize, and the Sipunculus nudus polysaccharide of purifying that obtains water content and be 1-5% is standby;
(2) sulphation modification of Sipunculus nudus polysaccharide
1) sulfur acidizing reagent preparation: the three-necked flask with condensing works and magnetic agitation is placed in to cryosel and bathes, the pyridine that adds 10-15 mL, comparatively violent stirring 5-10 min, it by chlorsulfonic acid and pyridine, is the volume ratio of 1:6-10, in 40min, dropwise add while stirring chlorsulfonic acid to pyridine, to occurring a large amount of faint yellow solids, reaction finishes, and obtains esterifying reagent standby;
2) sulphation modification operation: the Sipunculus nudus polysaccharide that accurately takes 0.500g purifying, miscible in 35mLDMF, with magnetic stirrer 30min, blend together uniform suspension, pour in esterifying reagent and be stirred in and in water-bath, make its reaction, bath temperature is 45-70 ℃, reaction times is 2-4 hour, after finishing, reaction is cooled to room temperature, reaction solution is poured in the 100mL frozen water that is chilled in advance 0-4 ℃, with 15% NaOH, be neutralized to pH7.0-7.2, add the long-pending dehydrated alcohol of triploid, standing 24h, centrifugal, hot water dissolving's precipitation, flowing water dialysis 48h, lyophilize to water content is 1-2%, obtain Sipunculus nudus controlling sulfate polyose (sulfated SPS, sSPS).
Take the present invention of above-mentioned measure, wherein in controlling sulfate polyose, sugared assay adopts phenolsulfuric acid method; The mensuration of sulfate content adopts BaCl
2-gelatin turbidimetry, measures absorbancy according to standard curve making method, by regression equation calculation sulfate concentration, is calculated as follows substitution value DS:DS=(1.62 * S%)/(32-1.02 * S%).Gained Sipunculus nudus controlling sulfate polyose of the present invention is carried out to anti-microbial activity comparison, adopt disk diffusion method, with 6 kinds of bacterium (intestinal bacteria
escherichia coli, subtilis
bacillus subtilis, streptococcus aureus
staphylococcus aures, Sarcina lutea
sarcina lutea, Vibrio parahaemolyticus
vibrio parahemolyticusand Vibrio anguillarum
vibrio anguillarum) be indicator, Sipunculus nudus polysaccharide and optimum modification condition gained controlling sulfate polyose sSPS5 have been carried out to Determination of Antibacterial Activity.Result is as shown in the table:
Sipunculus nudus polysaccharide and controlling sulfate polyose are to the restraining effect of bacterium (mm)
| Bacterial species | Sipunculus nudus polysaccharide | Sipunculus nudus controlling sulfate polyose |
| Intestinal bacteria | 7.3±0.2 | 7.7±0.1 |
| Subtilis | 9.8±0.2 | 11.0±0.2 |
| Streptococcus aureus | 7.7±0.3 | 8.1±0.2 |
| Vibrio parahaemolyticus | 8.0±0.2 | 8.9±0.2 |
| Vibrio anguillarum | 7.1±0.1 | 7.2±0.2 |
| Sarcina lutea | 10.1±0.2 | 12.2±0.4 |
From above-mentioned test, the Sipunculus nudus sulfating numerator modification product of the inventive method gained, can further improve the anti-microbial activity of Sipunculus nudus polysaccharide.
Accompanying drawing explanation
Fig. 1 sulfate typical curve of the present invention.
Embodiment
Embodiment 1
1. the extraction of Sipunculus nudus polysaccharide
Sipunculus nudus body wall body wall meat pulp adds 6 times of volume 6%NaOH, and 4 h are extracted in 50 ℃ of water-baths, adjust pH 7.0, centrifugal, supernatant liquor trichloroacetic acid method Deproteinization 2～3 times, being concentrated into volume is 1/4 original left and right, adding ethanol to final concentration is that 75%, 4 ℃ of precipitation is spent the night.Centrifugal, precipitation with dehydrated alcohol, washing with acetone repeatedly, is put ventilation and is dried rear ultrapure water and dissolve, and with holding back relative molecular weight, is 10000 glassine paper flowing water, 48 h that dialyse, ethanol redeposition, and centrifugal, lyophilize obtains Sipunculus nudus Crude polysaccharides.
2. the purifying of Sipunculus nudus polysaccharide
(1) DEAE-52 cellulose chromatography: repeatedly clean after column packing wet method dress DEAE-52 cellulose column, ultrapure water equilibrate overnight with 1mol/L NaOH, ultrapure water, 1mol/L HCL, ultrapure water.Get Sipunculus nudus Crude polysaccharides 0.5g and be dissolved in 10mL ultrapure water loading, with 0.5moL/L NaCL wash-out, flow velocity is 1mL/min, automatic collector is collected every pipe 5mL, draws elution curve, merges peak elutriant, glassine paper dialysis is except freshen, concentrating under reduced pressure, lyophilize.
(2) Sephadex G-100 gel filtration chromatography: the Sipunculus nudus polysaccharide dried frozen aquatic products through the separation of DEAE-52 cellulose column dissolves with ultrapure water, 0.45 μ m membrane filtration, upper Sephadex G-100 post, uses ultrapure water with 0.2 mL/min flow velocity wash-out, every pipe 5mL, draws elution curve.Merge absorption peak elutriant, concentrating under reduced pressure, lyophilize, obtains Sipunculus nudus polysaccharide Sephadex G-100 gel column isolate.
3. the sulphation modification of Sipunculus nudus polysaccharide
(1) sulfating reaction orthogonal design: choose the factor (volume ratio of chlorsulfonic acid and pyridine, reaction times and temperature of reaction) of three major effect esterification techniques by the orthogonal test L of 3 factor 3 levels
9(3
4) design modification condition (in Table 1), the degree of substitution of take is determined optimal conditions as investigating index.
Table 1 sulfation orthogonal test level of factor table
(2) sulfur acidizing reagent preparation: the three-necked flask with condensing works and stirring (magnetic agitation) is placed in to cryosel and bathes, the pyridine that adds 10-15 mL, comparatively violent stirring 5-10 min, it by chlorsulfonic acid and pyridine, is the volume ratio of 1:6-10, in 40min, dropwise add while stirring chlorsulfonic acid to pyridine, to occurring a large amount of faint yellow solids, reaction finishes, and obtains esterifying reagent standby;
(3) sulphation modification operation: the Sipunculus nudus polysaccharide that accurately takes 0.500g purifying, miscible in 35mLDMF, with magnetic stirrer 30min, blend together uniform suspension, pour in esterifying reagent and stir, in water-bath, make its reaction, bath temperature is 45-70 ℃, reaction times is 2-4 hour, after finishing, reaction is cooled to room temperature, reaction solution is poured in the 100mL frozen water that is chilled in advance 0-4 ℃, with 15% NaOH, be neutralized to pH7.0-7.2, add the long-pending dehydrated alcohol of triploid, standing 24h, centrifugal, hot water dissolving's precipitation, flowing water dialysis 48h, lyophilize to water content is 1-2%, obtain 9 kinds of Sipunculus nudus esterificated polysaccharide (sulfated SPS, sSPS) sSPS1, sSPS2, sSPS3, sSPS4, sSPS5, sSPS6, sSPS7, sSPS8 and sSPS9.In controlling sulfate polyose, sugared assay adopts phenolsulfuric acid method; The mensuration of sulfate content adopts BaCl2-gelatin turbidimetry, according to standard curve making method, measure absorbancy, by regression equation sulfate typical curve as shown in Figure 1, calculate sulfate concentration, be calculated as follows substitution value DS:DS=(1.62 * S%)/(32-1.02 * S%).Take degree of substitution and sugared content is index, adopts L
9(3
4) orthogonal design carries out preferably modification conditions such as reagent proportioning, reaction times and temperature of reaction.Result demonstration, the R value of temperature of reaction is maximum, is respectively 0.125 and 20.29, all maximum on the impact of DS and sugared content; The R value of chlorsulfonic acid and pyridine proportioning is taken second place, and is respectively 0.075 and 12.97; Reaction times is minimum, be respectively 0.064 and 9.59(table 2).Comprehensive evaluation determines that its best modification condition is: chlorsulfonic acid-pyridine volume ratio 1:8, reaction times 3h, 75 ℃ of temperature of reaction.
Table 2 sulfation orthogonal test and statistics
4.
5. the anti-microbial activity comparison of sulfation Sipunculus nudus polysaccharide
Adopt disk diffusion method, with 6 kinds of bacterium (intestinal bacteria
escherichia coli, subtilis
bacillus subtilis, streptococcus aureus
staphylococcus aures, Sarcina lutea
sarcina lutea, Vibrio parahaemolyticus
vibrio parahemolyticusand Vibrio anguillarum
vibrio anguillarum) be indicator, Sipunculus nudus polysaccharide and optimum modification condition gained controlling sulfate polyose sSPS5 have been carried out to Determination of Antibacterial Activity.Scraping is for trying bacterium lawn in 5 mL sterilized waters, concussion shakes up to obtain bacteria suspension, pour into through autoclaving and be cooled in the substratum of 40～45 ℃, after shaking up, be sub-packed in culture dish, treat that it solidifies, the circular paper (diameter D=6 mm) of getting sterilizing injects respectively polysaccharide and controlling sulfate polyose (concentration 6.25 mg/mL) 20 μ L, after drying up, is affixed on culture medium flat plate, does blank simultaneously.After 37 ℃ of constant temperature culture 24 h, measure inhibition zone size.Repeat to test 3 times, antibacterial circle diameter is three mean values of measuring.
By antibacterial experiment result known (table 3), Sipunculus nudus controlling sulfate polyose is all better than Sipunculus nudus polysaccharide to the restraining effect of 6 kinds of bacteriums, and in prompting the present invention, sulfating numerator modification can further improve the anti-microbial activity of Sipunculus nudus polysaccharide.
Table 3 Sipunculus nudus polysaccharide and controlling sulfate polyose are to the restraining effect of bacterium (mm)
| Bacterial species | Sipunculus nudus polysaccharide | Sipunculus nudus controlling sulfate polyose |
| Intestinal bacteria | 7.3±0.2 | 7.7±0.1 |
| Subtilis | 9.8±0.2 | 11.0±0.2 |
| Streptococcus aureus | 7.7±0.3 | 8.1±0.2 |
| Vibrio parahaemolyticus | 8.0±0.2 | 8.9±0.2 |
| Vibrio anguillarum | 7.1±0.1 | 7.2±0.2 |
| Sarcina lutea | 10.1±0.2 | 12.2±0.4 |
Claims (1)
1. the polysaccharide sulfated modifying method of Sipunculus nudus, concrete technological step is:
(1) purifying of Sipunculus nudus polysaccharide
1) cellulose chromatography is got Sipunculus nudus Crude polysaccharides and is carried out cellulose chromatography, column chromatography working method routinely, get Sipunculus nudus Crude polysaccharides 0.5g and be dissolved in 10mL ultrapure water loading, with 0.3-0.5moL/L NaCL wash-out, flow velocity is 1mL/min, obtains elutriant, with glassine paper dialysis, remove freshen, being evaporated to 30 ℃ of relative densities of temperature is 0.9829 g/mL, carries out lyophilize, and the Sipunculus nudus polysaccharide dried frozen aquatic products that obtains water content and be 1-5% is standby;
2) gel filtration chromatography is got Sipunculus nudus polysaccharide dried frozen aquatic products and is dissolved with ultrapure water, 0.45 μ m membrane filtration, carry out according to a conventional method gel filtration chromatography, with ultrapure water with 0.2 mL/min flow velocity wash-out, obtain elutriant, being evaporated to and recording relative density when temperature is 28 ℃ is 0.9856-0.9918 g/mL, carries out lyophilize, and the Sipunculus nudus polysaccharide of purifying that obtains water content and be 1-5% is standby;
(2) sulphation modification of Sipunculus nudus polysaccharide
1) sulfur acidizing reagent preparation is placed in cryosel bath by the three-necked flask with condensing works and magnetic agitation, the pyridine that adds 10-15 mL, comparatively violent stirring 5-10 min, it by chlorsulfonic acid and pyridine, is the volume ratio of 1:6-10, in 40min, dropwise add while stirring chlorsulfonic acid to pyridine, to occurring a large amount of faint yellow solids, reaction finishes, and obtains esterifying reagent standby;
2) sulphation modification operation accurately takes the Sipunculus nudus polysaccharide of 0.500g purifying, miscible in 35mLDMF, with magnetic stirrer 30min, blend together uniform suspension, pour in esterifying reagent and be stirred in and in water-bath, make its reaction, bath temperature is 45-70 ℃, reaction times is 2-4 hour, after finishing, reaction is cooled to room temperature, reaction solution is poured in the 100mL frozen water that is chilled in advance 0-4 ℃, with 15% NaOH, be neutralized to pH7.0-7.2, add the long-pending dehydrated alcohol of triploid, standing 24h, centrifugal, hot water dissolving's precipitation, flowing water dialysis 48h, lyophilize to water content is 1-2%, obtain Sipunculus nudus controlling sulfate polyose (sulfated SPS, sSPS).
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| CN1544477A (en) * | 2003-11-25 | 2004-11-10 | 武汉大学 | Urushi polysaccharide sulfation and its preparation method and uses |
| US20080207895A1 (en) * | 2002-11-27 | 2008-08-28 | Rosenberg Robert D | Methods for synthesizing polysaccharides |
| CN102153666A (en) * | 2011-02-22 | 2011-08-17 | 中国人民解放军海军医学研究所 | Sipunculidae polysaccharide and preparation method thereof and application thereof to anti-fatigue function |
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Patent Citations (3)
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| US20080207895A1 (en) * | 2002-11-27 | 2008-08-28 | Rosenberg Robert D | Methods for synthesizing polysaccharides |
| CN1544477A (en) * | 2003-11-25 | 2004-11-10 | 武汉大学 | Urushi polysaccharide sulfation and its preparation method and uses |
| CN102153666A (en) * | 2011-02-22 | 2011-08-17 | 中国人民解放军海军医学研究所 | Sipunculidae polysaccharide and preparation method thereof and application thereof to anti-fatigue function |
Non-Patent Citations (3)
| Title |
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| CHEN-XIAO ZHANG,等: "Immunomodulatory activities on macrophage of a polysaccharide from Sipunculus nudus L.", 《FOOD AND CHEMICAL TOXICOLOGY》 * |
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| 黄小燕，等: "多糖硫酸化修饰和多糖硫酸酯的研究进展", 《天然产物研究与开发》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112409504A (en) * | 2020-12-09 | 2021-02-26 | 北部湾大学 | Preparation method and application of Sipunculus nudus polysaccharide |
| CN112409504B (en) * | 2020-12-09 | 2021-12-14 | 北部湾大学 | Preparation method and application of Sipunculus nudus polysaccharide |
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