CN102659955A - Novel polysaccharide sulfating process based on physical-chemical combination - Google Patents
Novel polysaccharide sulfating process based on physical-chemical combination Download PDFInfo
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- CN102659955A CN102659955A CN2012101052521A CN201210105252A CN102659955A CN 102659955 A CN102659955 A CN 102659955A CN 2012101052521 A CN2012101052521 A CN 2012101052521A CN 201210105252 A CN201210105252 A CN 201210105252A CN 102659955 A CN102659955 A CN 102659955A
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Abstract
The invention aims to provide a polysaccharide sulfating process through adopting a chemical-physical combination means. The process comprises the following steps: 1, purifying to obtain uniform pure polysaccharides; 2, preparing a sulfating reagent; 3, applying a microwave auxiliary method; 4, carrying out infrared detection; 5, carrying out SD detection; and 6, detecting the in-vitro antioxidant activity. The process of the invention has the advantages of reaching of the sulfating degree of traditional processes, maintenance of a high biological activity, and substantial shortening of the sulfating time.
Description
Technical field
The present invention relates to the technology of polysaccharide chemistry modification aspect, be based on the novel polysaccharide process of sulfating that physical chemistry combines specifically.
Technical background
The polysaccharide that from medicinal fungi, extracts has multiple biological activity, particularly immunostimulation and antineoplastic function, makes its focus that becomes current research, is widely used in protective foods and field of medicaments.This biomacromolecule can not produce direct toxic action by pair cell, but plays a role through acting on defence and immunity system.
The biological activity that four factor affecting polysaccharide are arranged usually: be respectively water-soluble, molecular weight, molecular chain structure and substituted radical.Thereby the means through chemical modification can make the new BA of polysaccharide acquisition develop new medicinal field.
Polysaccharide is the polymer that is linked to each other through glycosidic link by monosaccharide residue, and with respect to other biomacromolecule, it has the ability of the more bioinformations of carrying.The activity of polysaccharide receives the influence of its molecular structure directly or indirectly, takes certain method that the polysaccharide molecule structure is modified, and can improve or give active polysaccharide, reduce its toxic side effect.The common methods of at present polysaccharide being modified has sulfation, phosphorylation, acetylize, alkylation, sulfonylation, carboxymethylation etc.These method of modifying have not only changed the physico-chemical property of polysaccharide, can also change its physiologically active.
Up to nineteen seventies, the sulfation of polysaccharide is active just to be familiar with, yet now, sulfation is that change polysaccharide BA is main, also is the most effective means.There are some researches show that abroad Sulfated lentinan has restraining effect to HIV virus.The increase slowly of sulfate in the polysaccharide, immunocompetence diminishes gradually, and when the amount of sulfate was increased to a certain value, the immunocompetence completely dissolve changed antiviral activity into.
Usually the sulfation method of modifying is to use some sulfur acidizing reagents, through chemical reaction, on the unit of polysaccharide hydroxyl, connects sulfate group.Sulfation system commonly used has: chlorsulfonic acid---pyridine, sulphur trioxide---triethylamine, the vitriol oil.And reaction is to accomplish Sulfated process through direct-fired means.Some researchs show that temperature is high more, and Sulfated substitution value is high more, yet too high temperature can influence the higher structure of molecule, cause the forfeiture of BA.Therefore can only under the temperature of gentleness, through prolonging the time of reaction, reach the purpose of sulfation high substitution value, general sulfating reaction all will pass through 8-10 hour reaction times now, just can reach the ideal degree.
Microwave can be to the instantaneous heat temperature raising of dielectric material, and energy consumption is also very low.On the other hand, the output rating of microwave is adjustable at any time, but there is not the residual activity phenomenon in the change thereupon of medium temperature rise inertialess, the needs that be highly advantageous to control automatically and serialization are produced.
Summary of the invention
The object of the present invention is to provide a kind of technology that adopts chemical physics bonded means to carry out polysaccharide sulfated production; This technology can reach the degree of traditional technology; And kept higher BA, shortened the Sulfated time greatly simultaneously.
Technical scheme of the present invention is: carry out according to the following steps:
1, with lentinan as the substrate of participating in reaction, extract purifying and the pure article polysaccharide of homogeneous that obtains in the fermented liquid that the mushroom exocellular polysaccharide produces through airlift fermentation.
2, the preparation of sulfur acidizing reagent: adopt the higher sulfation system of replacement rate---chlorsulfonic acid-pyridine system: chlorsulfonic acid slowly is added drop-wise in the container that pyridine is housed; The dropping process should be carried out in cryosel is bathed; And use magnetic stirrer, controlled temperature at 0 degree centigrade to 4 degrees centigrade in case overheated.
3, microwave assisting method
Pure article polysaccharide is dissolved in the methane amide, and sulfur acidizing reagent slowly drips, and stirs.Utilize microwave oven to react.After reaction finished, mixed solution was adjusted to pH neutrality, and uses alcohol precipitation, and is centrifugal multiple water-soluble, concentrates dialysis, and freeze-drying obtains the sulfation product.
4, infrared detection
KBr pressed disc method Fourier infrared spectrograph detects, 400-4000cm
-1Observe the substituted particular peaks of sulfuric acid.
5, SD detects
The phenol sulfuric acid process is adopted in the detection of carbohydrate content, and bariumchloride gelatin method is adopted in the detection of sulfate radical content.
6, the detection of antioxidation activity in vitro.Ultra oxygen root radical scavenging activity:
Simulation xanthine and XOD reaction system produce ultra-oxygen anion free radical, add electron transport substance and griess developer, in the 550nm colorimetric.Add the sample of different concns in the testing tube, the superoxide anion of calculation sample is removed ability.
The hydroxide radical radical scavenging activity: utilize fenton reaction to produce hydroxy radical qiao, give with electron acceptor(EA) after, with the griess colour developing, the 550nm colorimetric adds the sample of different concns in the testing tube, the hydroxyl negatively charged ion of calculation sample is removed ability.
The invention has the advantages that: microwave assisting method is with respect to traditional method, and is consuming time shorter.Traditional method needs about 10 hours, and novel process only needs just completion reaction in short 6 minutes.Utilize the characteristic of microwave, promptly reach the purpose of heating, adopt the means of batch process to prevent the inactivation that superheated causes.Because microwave makes molecule in solvent, fully unfold, better contact simultaneously with sulfur acidizing reagent.Through detecting, the sample that novel process obtains has reached Sulfated result equally, and its replacement rate is high, does not lose biological activity simultaneously.
Description of drawings
Fig. 1 is that KBr pressed disc method Fourier infrared spectrograph detects comparison diagram.
Embodiment:
The present invention combines concrete example to carry out through following steps:
1, with lentinan as the substrate of participating in reaction, extract purifying and the pure article polysaccharide of homogeneous that obtains in the fermented liquid that the mushroom exocellular polysaccharide produces through airlift fermentation.
2, the preparation of sulfur acidizing reagent: adopt the higher sulfation system of replacement rate---chlorsulfonic acid-pyridine system: the chlorsulfonic acid of 5ml slowly is added drop-wise in the three-necked flask that the 25ml pyridine is housed; The dropping process should be carried out in cryosel is bathed; And use magnetic stirrer, controlled temperature at 0 degree centigrade to 4 degrees centigrade in case overheated.
3, microwave assisting method
The pure article polysaccharide of 100mg is dissolved in the 20ml methane amide, and sulfur acidizing reagent slowly drips, and stirs.Utilize common microwave stove to react.With power modulation gentle (about 100W), the reaction system heating stopped after 2 minutes, took out cooling.Repeat twice again, finish sulfating reaction.After reaction finished, mixed solution was adjusted to pH neutrality, and uses alcohol precipitation, and is centrifugal multiple water-soluble, concentrates dialysis, and freeze-drying obtains sulfation sample sLNT-2.
4, the physico-chemical property of product check
In the following stated: sLNT-1 is that traditional method gained sulfation sample, sLNT-2 are the inventive method gained sulfation sample.
1) infrared detection, KBr pressed disc method Fourier infrared spectrograph detects, 400-4000cm
-1Observe the substituted particular peaks of sulfuric acid.As shown in Figure 1, for the comparison of traditional method product: 977cm
-1Be the β glycosidic link, 1650and 1415cm
-1Be the stretching vibration of carbonyl and carboxyl, 1132 and 1060cm
-1Be (C-O-C) and stretching vibration (C-OH), 3361cm
-1(OH) and 2933cm
-1(C-H) be the characteristic peak of sugar ring.On sulfating product, 1246/1225cm
-1The S=O stretching vibration and the 855/854cm at place
-1The C-O-S stretching vibration at place shows that sulfation forms sulfuric ester.
2) SD detects, and the phenol sulfuric acid process is adopted in the detection of carbohydrate content, and bariumchloride gelatin method is adopted in the detection of sulfate radical content.It below is comparison with the traditional method product.
| Sample | Carbohydrate content (%) | SD |
| LNT | - | |
| sLNT-1 | 58.8％ | 1.45 |
| sLNT-2 | 49.6％ | 1.47 |
3) detection of antioxidation activity in vitro
Ultra oxygen root radical scavenging activity, simulation xanthine and XOD reaction system produce ultra-oxygen anion free radical, add electron transport substance and griess developer, in the 550nm colorimetric.Add the sample of different concns in the testing tube, the superoxide anion of calculation sample is removed ability.It below is comparison with the traditional method product.
The hydroxide radical radical scavenging activity:
Utilize fenton reaction to produce hydroxy radical qiao, give with electron acceptor(EA) after, with griess colour developing, 550nm colorimetric.Add the sample of different concns in the testing tube, the hydroxyl negatively charged ion of calculation sample is removed ability.It below is comparison with the traditional method product.
Claims (1)
1. the novel polysaccharide process of sulfating that combines based on physical chemistry is characterized in that carrying out according to the following steps:
1) with lentinan as the substrate of participating in reaction, extract purifying and the pure article polysaccharide of homogeneous that obtains in the fermented liquid that the mushroom exocellular polysaccharide produces through airlift fermentation;
2) preparation of sulfur acidizing reagent: adopt the higher sulfation system of replacement rate---chlorsulfonic acid-pyridine system: chlorsulfonic acid slowly is added drop-wise in the container that pyridine is housed; The dropping process should be carried out in cryosel is bathed; And use magnetic stirrer, controlled temperature at 0 degree centigrade to 4 degrees centigrade in case overheated;
3) microwave assisting method: pure article polysaccharide is dissolved in the methane amide, and sulfur acidizing reagent slowly drips, and stirs, and utilizes microwave oven to react; After reaction finished, mixed solution was adjusted to pH neutrality, and uses alcohol precipitation, and is centrifugal multiple water-soluble; Concentrate dialysis, freeze-drying obtains the sulfation product;
4) infrared detection: KBr pressed disc method Fourier infrared spectrograph detects, 400-4000cm
-1Observe the substituted particular peaks of sulfuric acid;
5) SD detects: the phenol sulfuric acid process is adopted in the detection of carbohydrate content, and bariumchloride gelatin method is adopted in the detection of sulfate radical content;
6) detection of antioxidation activity in vitro
Ultra oxygen root radical scavenging activity: simulation xanthine and XOD reaction system produce ultra-oxygen anion free radical; Add electron transport substance and griess developer; In the 550nm colorimetric, add the sample of different concns in the testing tube, the superoxide anion of calculation sample is removed ability;
The hydroxide radical radical scavenging activity: utilize fenton reaction to produce hydroxy radical qiao, give with electron acceptor(EA) after, with the griess colour developing, the 550nm colorimetric adds the sample of different concns in the testing tube, the hydroxyl negatively charged ion of calculation sample is removed ability.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104592319A (en) * | 2015-01-16 | 2015-05-06 | 中国科学院上海有机化学研究所 | Simultaneous N,O-sulfating method of glycosaminoglycan class compound and reaction intermediate thereof |
| CN106109487A (en) * | 2016-07-20 | 2016-11-16 | 安徽大学 | A kind of novel antioxidant having antitumor action concurrently and preparation method thereof |
| CN112245647A (en) * | 2020-10-26 | 2021-01-22 | 杭州电子科技大学 | Preparation method of sponge wound dressing |
Citations (1)
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| CN1970782A (en) * | 2006-11-02 | 2007-05-30 | 浙江大学 | Method for fermentation preparation of Ganoderma lucidum polysaccharide |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970782A (en) * | 2006-11-02 | 2007-05-30 | 浙江大学 | Method for fermentation preparation of Ganoderma lucidum polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| 易建平等: ""枸杞多糖硫酸酯的微波制备"", 《第十二届全国微波能应用学术会议论文集》 * |
| 燕航等: ""硫酸酯化香菇多糖衍生物制备的研究"", 《化学与生物工程》 * |
| 贺亮等: ""香菇多糖的化学修饰及结构表征"", 《第二届全国食用菌中青年专家学术交流会论文集》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104592319A (en) * | 2015-01-16 | 2015-05-06 | 中国科学院上海有机化学研究所 | Simultaneous N,O-sulfating method of glycosaminoglycan class compound and reaction intermediate thereof |
| CN104592319B (en) * | 2015-01-16 | 2017-12-08 | 中国科学院上海有机化学研究所 | The method and its reaction intermediate of a kind of glycosaminoglycan compound N, O while sulphation |
| CN106109487A (en) * | 2016-07-20 | 2016-11-16 | 安徽大学 | A kind of novel antioxidant having antitumor action concurrently and preparation method thereof |
| CN106109487B (en) * | 2016-07-20 | 2018-12-28 | 安徽大学 | A kind of antioxidant and preparation method thereof having both antitumor action |
| CN112245647A (en) * | 2020-10-26 | 2021-01-22 | 杭州电子科技大学 | Preparation method of sponge wound dressing |
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Application publication date: 20120912 |