CN102676613B - Preparation method for disaccharide, tetrasccharide and hexaose of chondroitin sulfuric acid - Google Patents
Preparation method for disaccharide, tetrasccharide and hexaose of chondroitin sulfuric acid Download PDFInfo
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- CN102676613B CN102676613B CN 201210159448 CN201210159448A CN102676613B CN 102676613 B CN102676613 B CN 102676613B CN 201210159448 CN201210159448 CN 201210159448 CN 201210159448 A CN201210159448 A CN 201210159448A CN 102676613 B CN102676613 B CN 102676613B
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Abstract
The invention discloses a preparation method for disaccharide, tetrasccharide and hexaose of chondroitin sulfuric acid, and belongs to the technical field of food. The method comprises the following steps that: the chondroitin sulfuric acid adopted as a raw material is degraded by hyaluronidase, so as to obtain chondroitin sulfuric acid oligosaccharide; through filtering separation by G-25 gel, desalination by G-10, DEAE cellulose 52 anion exchange chromatographic column separation, natural preparation of gel electrophoretic separation, and the like, the disaccharide, tetrasccharide and hexaose of the chondroitin sulfuric acid can be prepared. The invention relates to the preparation method for the disaccharide, the tetrasccharide and the hexaose of the chondroitin sulfuric acid. According to the invention, simultaneous preparation of three types of chondroitin sulfuric acid oligosaccharide can be firstly achieved, and a technical base for industrial preparation for single degree of polymerization chondroitin sulfuric acid oligosaccharide can be provided.
Description
Technical field
The preparation method of a kind of chondroitin sulfate disaccharides, tetrose, six sugar belongs to food industrial technical field.
Background technology
Chondroitin sulfate (CS), a kind of as glycosaminoglycan is one of three kinds of most important glycosaminoglycan.It is made up of D-glucuronic acid and N-acetyl-D-amino galactosonic acid sulfuric ester, is the class acidic mucopolysaccharide in the cartilaginous tissues such as the larynx bone that extensively is present in the human and animal, nasal septum, tracheae.The CS of high molecule mass shows high biological activity, as anticancer disease, study of anti-atherogenic effect, anti-inflammatory, immunomodulatory, anti-oxidant etc., not only in bioprocesss such as cell transfer, differentiation, propagation, identification and tissue formation, play an important role, and be used for medicine and Clinical Application.But because high molecular, high apparent viscosity, low water solubility and complicated structure, most of macromolecule polysaccharide is difficult to by organizing the receptors bind of barrier and cell interior.And because the factors such as selection perviousness of cytolemma, most of glycosaminoglycan faces the lower subject matter of bioavailability in the clinical application.Recent years, the functional performance of low molecular mass CS more and more is subject to people's attention.With other glycosaminoglycan, as hyaluronic acid, heparitin sulfate is compared, and the research of lower molecular weight CS is backward relatively.At present domestic the research of CS is mainly concentrated on the aspects such as extraction, physiologically active and derivative of CS, the research of CS oligosaccharides is not appeared in the newspapers, also few to the separation of the CS oligosaccharides report of purifying in the world, the research of single oligosaccharides is less.The preparation method of a kind of chondroitin sulfate disaccharides that the present invention relates to, tetrose, six sugar, be raw material with the chondroitin sulfate, adopt the hyaluronic acid enzyme liberating to prepare the chondroitin sulfate oligosaccharides, through G-25 gel-filtration separation, G-10 desalination, the separation of DEAE Mierocrystalline cellulose 52 anion-exchange columns, natural preparative gel electrophoresis separation etc., prepare chondroitin sulfate disaccharides, tetrose and six sugar, preparation when having realized three kinds of chondroitin sulfate oligosaccharides first is for the industrial preparation of single polymerization degree chondroitin sulfate oligosaccharides provides technical basis; For the foundation in the sugared storehouse of saccharide compound and the screening of carbohydrate medicine provide important separation means; The production that can be clinical medicine provides certain foundation.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of chondroitin sulfate disaccharides, tetrose, six sugar.
Technical scheme of the present invention: the preparation method of a kind of chondroitin sulfate disaccharides, tetrose, six sugar, be raw material with the chondroitin sulfate, adopt the hyaluronic acid enzyme liberating to prepare the chondroitin sulfate oligosaccharides, through G-25 gel-filtration separation, G-10 desalination, the separation of DEAE Mierocrystalline cellulose 52 anion-exchange columns, natural preparative gel electrophoresis separation etc., prepare chondroitin sulfate disaccharides, tetrose and six sugar.Step is:
(1) enzymic degradation of chondroitin sulfate
0.2 g chondroitin sulfate is dissolved in the phosphate buffered saline buffer of 10 mL pH 5.9, places 37 ℃ of insulation 10 min, make itself and temperature of reaction reach consistent, the back adds Unidasa HAase in substrate solution, and making its enzyme work is 1.4 * 10
5U/L is with 150 rev/mins hunting speed oscillatory reaction 22h-24h; After reaction finished, heated and boiled 20min made enzyme deactivation, and the centrifugal 15min of 4000r/min gets supernatant liquor and adds 3 times of volume 95% ethanol sedimentations, left standstill, and with 10mL dehydrated alcohol dehydration 3 times, vacuum-drying gets the chondroitin sulfate oligosaccharides;
(2) gel-filtration of chondroitin sulfate oligosaccharides separates
The Sephadex G-25 that handles well is dressed up the glass chromatography column of 1.6 * 150 cm, the chondroitin sulfate oligosaccharides is mixed with the solution of 40 mg/mL with deionized water, applied sample amount 1 mL, with the ultrapure water of 5 times of column volumes with 4 mL/min flow velocity wash-outs, collect with distribution collector Redfrac95 timing automatic, the 4mL/ pipe detects glucuronic acid content to determine the peak position that of sugar with carbazole reaction;
(3) G-10 desalination: 40 ℃ of rotary evaporations of the sample of collecting concentrate by Sephadex G-10,1.6 * 150 cm, after desalination separates, concentrating sample is to about 50mL, freeze-drying again, serve as to detect index to catch the free radical ability, filter out the highest chondroitin sulfate oligosaccharides of anti-oxidant activity, as next step separate object;
(4) anion exchange separation of chondroitin sulfate oligosaccharides
Dress post: with soaked DEAE Mierocrystalline cellulose 52, slowly join 2cm * 20cm in the chromatography column along glass stick, avoid (if the generation bubble of bubble generation in the glue, the useable glass rod stirs gently, bubble is overflowed), make slowly sedimentation of gel according to action of gravity, and keep surfacing;
Pre-equilibration: after chromatography column connects constant flow pump, with ultrapure water balance pillar, steady to baseline with 3-5 column volume of speed wash-out of 2mL/min;
Sample preparation: the chondroitin sulfate oligosaccharides that step (3) is filtered out is dissolved to 10 mL with ultrapure water, and last sample is to DEAE Mierocrystalline cellulose 52 anion-exchange columns;
Wash-out: go up sample 1mL, with 0-1mol/L NaCl gradient elution 75min, elution rate is 2mL/min, the 210nm ultraviolet detection; Collect each elution peak;
(5) the natural preparative gel electrophoresis of chondroitin sulfate disaccharides, tetrose, six sugar separates
The solution preparation:
A liquid: boric acid 0.1mol/L, Tris 0.1mol/L, ethylenediamine tetraacetic oxalic acid 0.01 mol/L, being dissolved in water makes pH8.3.
The concentrated glue of mass concentration 5%: per 100 mL concentrate glue and contain acrylamide 4.75 g, methylene diacrylamide 0.25 g, and transfer pH 6.3 with hydrochloric acid; Every 3mL concentrates glue and adds 10% ammonium persulfate aqueous solution, 100 μ L, TEMED10 μ L.
The separation gel of mass concentration 35%: per 100 mL separation gels contain acrylamide 31.82 g, methylene diacrylamide 3.18 g and sucrose 15 g.Every 10mL separation gel adds 10% ammonium persulfate aqueous solution, 200 μ L, TEMED 20 μ L.
Separation gel and concentrated glue are all prepared with A liquid.
Electrode buffer: glycine 1.25 mol/L, Tris 0.2 mol/L, being dissolved in water makes pH 8.3.
Last sample: will be on the elution peak that the DEAE-52 anion-exchange chromatography separates sample.
Deposition condition: room temperature, 400V, 4-5h.
Dyeing: 0.5% toluidine blue is dissolved in 2% acetic acid solution, dyeing 10min.
Decolouring: 2% acetic acid decolouring.
Sample collection: after the sample decolouring, respectively the maximum components of content in the electrophoretic band are cut, smashed to pieces, place 1mL PBS solution to shake.Centrifugal behind the 12h, get supernatant liquor, place 4 ℃ of preservations.Precipitation continues to place 1mL PBS solution to shake.Behind the triplicate, three gained supernatant liquors are crossed the desalination of AKTA superdex peptide post, lyophilize namely gets chondroitin sulfate disaccharides, tetrose, six sugar.
Beneficial effect of the present invention: the inventive method has realized the preparation of chondroitin sulfate disaccharides, tetrose, three kinds of chondroitin sulfate oligosaccharides of six sugar first, preparation when having realized three kinds of chondroitin sulfate oligosaccharides first is for the industrial preparation of single polymerization degree chondroitin sulfate oligosaccharides provides technical basis.
Description of drawings
Fig. 1: DEAE Mierocrystalline cellulose 52 tomographic maps of chondroitin sulfate oligosaccharides.
Fig. 2: the preparation PAGE electrophorogram of chondroitin sulfate oligosaccharides.A is the PAGE electrophorogram at A peak among Fig. 1, and B is the PAGE electrophorogram at B peak among Fig. 1, and C is the PAGE electrophorogram at C peak among Fig. 1; Part draw a circle among the figure for providing next step to carry out component a, b, the c of molecular weight identification through the gel filtration chromatography post.
Fig. 3: chondroitin sulfate disaccharides, tetrose, six glycan molecule flow measurement figure.A is component a(chondroitin sulfate disaccharides among Fig. 2) molecular weight determination figure, b be amount of component b (chondroitin sulfate six sugar) molecular weight determination figure among Fig. 2 for components b (chondroitin sulfate tetrose) molecular weight determination figure, c among Fig. 2.
Embodiment
Embodiment 1
1, enzymic degradation prepares the chondroitin sulfate oligosaccharides
0.2 g chondroitin sulfate is dissolved in the phosphate buffered saline buffer of 10 mL pH 5.9, place 37 ℃ of insulation 10 min, make itself and temperature of reaction reach consistent, the back adds Unidasa (HAase in substrate solution, from bull testis), making its enzyme work is 1.4 * 10
5U/L, 150 rev/mins of oscillatory reaction 22h.After reaction finished, heated and boiled 20min made enzyme deactivation, the centrifugal 15min of 4000r/min, get supernatant liquor and add 3 times of volume 95% ethanol sedimentations, leave standstill, with 10mL dehydrated alcohol dehydration 3 times, vacuum-drying gets chondroitin sulfate oligose A, and measures the total antioxidant activity (TOA) of gained degraded product.
2, the mensuration of total antioxidant activity
Measuring the test kit specification sheets according to Total antioxidant capacity (TOA) measures.
Measuring principle: many antioxidant are arranged in the body, can make Fe
3+Be reduced into Fe
2+, the latter can form firm complex compound with luxuriant and rich with fragrance quinoline class material, can measure the height of its resistance of oxidation by colorimetric.
In the time of 37 ℃, the every ml soln of per minute makes the every increase by 0.01 of absorbancy (OD) value of reaction system, is a Total antioxidant capacity unit.
3, the gel-filtration of chondroitin sulfate oligosaccharides separates
The Sephadex G-25 that handles well is dressed up the glass chromatography column of 1.6 * 150 cm, o-CS is mixed with the solution of 40 mg/mL with deionized water, applied sample amount 1 mL, with the ultrapure water of 5 times of column volumes with 4 mL/min flow velocity wash-outs, collect with distribution collector (Redfrac95) timing automatic, the 4mL/ pipe detects glucuronic acid content to determine the peak position that of sugar with carbazole reaction.40 ℃ of rotary evaporations of the sample of collecting concentrate by Sephadex G-10(1.6 * 150 cm) desalination.Concentrating sample is to certain volume freeze-drying again after separating.Serve as to detect index screening to go out the highest lower molecular weight CS of anti-oxidant activity as next step separate object with total antioxidant activity (TOA).
4, carbazole reaction detects glucuronic acid content
The preparation of carbazole test solution: carbazole 0.0625g, add dehydrated alcohol 50mL, concussion makes dissolving, namely.
Borax sulfuric acid liquid: sodium tetraborate 2.385g is dissolved in vitriol oil 500mL, namely.
The making of typical curve:
CS standard substance 500mg is dissolved in the 10mL water.Precision is measured CS reference liquid 0.0mL, 0.1mL, and 0.2mL, 0.3mL, 0.4mL, 0.5mL places tool plug test tube, respectively adds water to 1mL and shakes up, and is cooled to about 4 ℃.Slowly add borax sulfuric acid liquid 5mL under the back jolting, place boiling water bath to heat 10min in test tube, put and be cooled to room temperature in the normal-temperature water.The accurate carbazole test solution 0.2mL that adds, mixing is put reheat 15min in the boiling water bath, is cooled to room temperature.Measure absorbance A at wavelength 530nm place, with absorbance A to concentration C mapping drawing standard curve.
5, the anion exchange separation of chondroitin sulfate oligosaccharides
Dress post: with soaked DEAE Mierocrystalline cellulose 52, slowly join (2cm * 20cm), avoid bubble generation (if produce bubble, the useable glass rod stirs gently, and bubble is overflowed) in the glue in the chromatography column along glass stick.Make slowly sedimentation of gel according to action of gravity, and keep surfacing.
Pre-equilibration: after chromatography column connects constant flow pump, with ultrapure water balance pillar, steady to baseline with 3-5 column volume of speed wash-out of 2mL/min.
Sample preparation: the component lyophilized powder that the anti-oxidant activity that separates through gel-filtration is the highest is dissolved to 10mL with ultrapure water, and last DEAE Mierocrystalline cellulose 52 anion-exchange columns further separate.
Wash-out: go up sample 1mL, with 0-1mol/L NaCl gradient elution 75min, elution rate is 2mL/min, the 210nm ultraviolet detection; Collect each elution peak (Fig. 1).
6, the natural preparation electrophoretic separation of chondroitin sulfate disaccharides, tetrose, six sugar
The solution preparation:
A liquid: boric acid 0.1mol/L, Tris 0.1mol/L, ethylenediamine tetraacetic oxalic acid 0.01 mol/L, being dissolved in water makes pH8.3.
5% concentrates glue: per 100 mL concentrate glue and contain acrylamide 4.75 g, methylene diacrylamide 0.25 g, and transfer pH 6.3 with hydrochloric acid.Every 3mL concentrates glue and adds 10% ammonium persulfate aqueous solution, 100 μ L, TEMED10 μ L.
35% separation gel: per 100 mL separation gels contain acrylamide 31.82 g, methylene diacrylamide 3.18 g and sucrose 15 g.Every 10mL separation gel adds 10% ammonium persulfate aqueous solution, 200 μ L, TEMED20 μ L.
Separation gel and concentrated glue are all prepared with A liquid.
Electrode buffer: glycine 1.25 mol/L, Tris 0.2 mol/L, being dissolved in water makes pH 8.3.
Last sample: will be on the elution peak that the DEAE-52 anion-exchange chromatography separates sample.
Deposition condition: room temperature, 400V, 4-5h.
Dyeing: 0.5% toluidine blue is dissolved in 2% acetic acid solution, dyeing 10min.
Decolouring: 2% acetic acid decolouring.
Sample collection: after the sample decolouring, respectively the maximum components of content in the electrophoretic band are cut, smashed to pieces, place 1ml PBS solution to shake.Centrifugal behind the 12h, get supernatant liquor, place 4 ℃ of preservations.Precipitation continues to place 1ml PBS solution to shake.Behind the triplicate, three gained supernatant liquors are crossed the desalination of AKTA superdex peptide post, lyophilize namely gets chondroitin sulfate disaccharides, tetrose, six sugar (Fig. 2).
7, the mensuration of the molecular weight of chondroitin sulfate disaccharides, tetrose, six sugar
Adopt high-efficient gel filtration chromatography (HPGPC) method to measure separating obtained chondroitin sulfate disaccharides, tetrose, six sugar (Fig. 3).Chromatographic column: Ultrahydrogel Linear 300mm * 7.8mmid * 2; Moving phase: 0.1mol/L NaNO
3Flow velocity: 0.9 mL/min; Column temperature: 45 ℃; Detector: 2410 differential refraction detectors.
Claims (1)
1. the preparation method of a chondroitin sulfate disaccharides, tetrose, six sugar, it is characterized in that, be raw material with the chondroitin sulfate, adopt the hyaluronic acid enzyme liberating to prepare the chondroitin sulfate oligosaccharides, separate through G-25 gel-filtration separation, G-10 desalination, the separation of DEAE Mierocrystalline cellulose 52 anion-exchange columns, natural preparative gel electrophoresis, prepare chondroitin sulfate disaccharides, tetrose and six sugar; Concrete steps are:
(1) enzymic degradation of chondroitin sulfate
0.2 g chondroitin sulfate is dissolved in the phosphate buffered saline buffer of 10 mL pH 5.9, places 37 ℃ of insulation 10 min, make itself and temperature of reaction reach consistent, the back adds Unidasa HAase in substrate solution, and making its enzyme work is 1.4 * 10
5U/L is with 150 rev/mins hunting speed oscillatory reaction 22h-24h; After reaction finished, heated and boiled 20min made enzyme deactivation, and the centrifugal 15min of 4000r/min gets supernatant liquor and adds 3 times of volume 95% ethanol sedimentations, left standstill, and with 10mL dehydrated alcohol dehydration 3 times, vacuum-drying gets the chondroitin sulfate oligosaccharides;
(2) gel-filtration of chondroitin sulfate oligosaccharides separates
The Sephadex G-25 that handles well is dressed up the glass chromatography column of 1.6 * 150 cm, the chondroitin sulfate oligosaccharides is mixed with the solution of 40 mg/mL with deionized water, applied sample amount 1 mL, with the ultrapure water of 5 times of column volumes with 4 mL/min flow velocity wash-outs, collect with distribution collector Redfrac 95 timing automatic, the 4mL/ pipe detects glucuronic acid content to determine the peak position that of sugar with carbazole reaction;
(3) G-10 desalination: after separating through 1.6 * 150 cm chromatography column desalinations of being dressed up by Sephadex G-10 after 40 ℃ of rotary evaporations of the sample of collecting concentrate, concentrating sample is to 50mL, freeze-drying again, serve as to detect index to catch the free radical ability, filter out the highest chondroitin sulfate oligosaccharides of anti-oxidant activity, as next step separate object;
(4) anion exchange separation of chondroitin sulfate oligosaccharides
Dress post: with soaked DEAE Mierocrystalline cellulose 52, slowly join 2cm * 20cm in the chromatography column along glass stick, avoid bubble generation in the glue, if the generation bubble stirs gently with glass stick, bubble is overflowed, make slowly sedimentation of gel according to action of gravity, and keep surfacing;
Pre-equilibration: after chromatography column connects constant flow pump, with ultrapure water balance pillar, steady to baseline with 3-5 column volume of speed wash-out of 2mL/min;
Sample preparation: the chondroitin sulfate oligosaccharides that step (3) is filtered out is dissolved to 10 mL with ultrapure water, and last sample is to DEAE Mierocrystalline cellulose 52 anion-exchange columns;
Wash-out: go up sample 1mL, with 0-1mol/L NaCl gradient elution 75min, elution rate is 2mL/min, the 210nm ultraviolet detection; Collect each elution peak;
(5) the natural preparative gel electrophoresis of chondroitin sulfate disaccharides, tetrose, six sugar separates
The solution preparation:
A liquid: boric acid 0.1mol/L, Tris 0.1mol/L, ethylenediamine tetraacetic oxalic acid 0.01 mol/L, being dissolved in water makes pH8.3;
The concentrated glue of mass concentration 5%: per 100 mL concentrate glue and contain acrylamide 4.75 g, methylene diacrylamide 0.25 g, and transfer pH 6.3 with hydrochloric acid; Every 3mL concentrates glue and adds 10% ammonium persulfate aqueous solution, 100 μ L, TEMED10 μ L;
The separation gel of mass concentration 35%: per 100 mL separation gels contain acrylamide 31.82 g, methylene diacrylamide 3.18 g and sucrose 15 g; Every 10mL separation gel adds 10% ammonium persulfate aqueous solution, 200 μ L, TEMED 20 μ L;
Separation gel and concentrated glue are all prepared with A liquid;
Electrode buffer: glycine 1.25 mol/L, Tris 0.2 mol/L, being dissolved in water makes pH 8.3;
Last sample: will be on the elution peak that the DEAE-52 anion-exchange chromatography separates sample;
Deposition condition: room temperature, 400V, 4-5h;
Dyeing: 0.5% toluidine blue is dissolved in 2% acetic acid solution, dyeing 10min;
Decolouring: 2% acetic acid decolouring;
Sample collection: after the sample decolouring, respectively the maximum components of content in the electrophoretic band are cut, smash to pieces, place 1mL PBS solution to shake, centrifugal behind the 12h, get supernatant liquor, place 4 ℃ of preservations, precipitation continues to place 1mL PBS solution to shake, behind the triplicate, three gained supernatant liquors are crossed the desalination of AKTA superdex peptide post, and lyophilize namely gets chondroitin sulfate disaccharides, tetrose, six sugar.
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| CN103583943A (en) * | 2013-10-10 | 2014-02-19 | 青岛贝尔特生物科技有限公司 | Formula of healthcare product for increasing bone mineral density |
| CN104610467B (en) * | 2015-01-27 | 2017-02-22 | 江南大学 | Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide |
| CN106467563B (en) * | 2015-08-21 | 2019-08-06 | 北京大学 | The synthetic method and its midbody compound of chondroitin sulfate tetrose |
| CN105851200A (en) * | 2016-04-22 | 2016-08-17 | 浙江大学宁波理工学院 | Natural biological preserving agent for aquatic products and preparation method and application thereof |
| CN107727762A (en) * | 2017-09-28 | 2018-02-23 | 山东大学 | A kind of method of nitrous acid degradation analysis LMWHs disaccharides structure |
| CN110041383A (en) * | 2018-01-15 | 2019-07-23 | 中国医学科学院药物研究所 | Chondroitin sulfate oligosaccharides, preparation method and application |
| CN109613132A (en) * | 2018-12-06 | 2019-04-12 | 上海景峰制药有限公司 | A kind of detection method of heterozygosis chondroitin sulfate |
| WO2021081999A1 (en) * | 2019-11-01 | 2021-05-06 | 南京汉欣医药科技有限公司 | Low-molecular-weight chondroitin sulfate and preparation method therefor |
| JP2023500294A (en) | 2019-11-01 | 2023-01-05 | ナンチン ハンシン ファーマシューティカル テクノロジー カンパニー，リミティド | Low molecular weight chondroitin sulfate, composition, method of preparation and use thereof |
| CN111575329B (en) * | 2020-05-26 | 2021-12-07 | 山东大学 | Preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property |
| CN113603732B (en) * | 2021-08-25 | 2024-02-13 | 江南大学 | Non-animal chondroitin sulfate oligosaccharide and preparation method thereof |
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