JPH04266896A - Purification of alginic acid oligosaccharide and sulfuric acid group-containing oligosaccharide - Google Patents

Abstract

PURPOSE:To extremely readily fractionate an oligosaccharide by the once employment of a chromatography comprising a HPLC using an anion exchange resin column. CONSTITUTION:A method for purifying an oligosaccharide containing an alginic acid oligosaccharide or a sulfuric acid group-containing polysaccharide is characterized by making the alginic acid oligosaccharide prepared from alginic acid or a polysaccharide containing sulfuric acid groups in the structure of the alginic acid oligosaccharide adsorbed onto an anion exchange resin column and then applying to a high performance liquid chormatography to fractionate the oligosaccharide by a concentration gradient elution method using water and sodium chloride.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a method for purifying alginate oligosaccharides and sulfate group-containing oligosaccharides by high performance liquid chromatography (hereinafter abbreviated as HPLC).

000
2]

BACKGROUND OF THE INVENTION Examples of acidic polysaccharides having sulfate groups, carboxyl groups, etc. in their structures include heparin, heparan sulfate, chondroitin sulfate, and alginic acid, which have been commercially available and used as pharmaceuticals and foods. However, oligosaccharides, which are decomposition products of these polysaccharides, are not commercially available, or even if they are rarely available, they are very expensive, and at present, research on how to use them has not made much progress. In the case of heparin, it has been widely used as an anticoagulant until now, but heparin fragments, which are lower molecular weight heparin, are used as anticoagulants [L
inhardt et al. , (1982) J.
Biol. Chem. 257, 7310-73
13] or antitumor agents [Folkman et al.
, (1983) Science 221, 719-
725] has also been reported to have the effect of

[0003]Currently, as a method for purifying oligosaccharides of these polysaccharides, a multidimensional high performance liquid chromatography method using an anion exchange resin [Vella et al. , (198
9) J. Liquid Chromatograph
hy 12(8), 1333-1346]), also a method using an open column of anion exchange resin [Method for purifying succinic acid-containing oligosaccharides TOYOPEARL NE
WS No. 45], a combination of electrodialysis and an anion exchange resin open column method [Purification method of sialic acid-containing oligosaccharides, Japanese Patent Publication No. 63-28428],
Gel filtration method under acidic conditions [purification method of alginic acid hydrolyzate Anzai et al. , (1990) Nipp.
on Suisan Gakkaisi56(1),
73-81], but these purification methods using gel filtration and ion exchange methods require two to several purification steps to obtain purified oligosaccharides, making it difficult to use them on an industrial scale. was difficult.

0004

[Problems to be Solved by the Invention] Therefore, the present inventors conducted extensive research to develop a method for easily and quickly preparing highly pure alginate oligosaccharides and sulfate group-containing oligosaccharides. They focused on the fact that sulfate group-containing oligosaccharides have a negative charge, and found that the above development objective could be achieved by combining an anion exchange resin and HPLC, leading to the completion of the present invention.

[0005]

[Means for Solving the Problems] That is, the present invention adsorbs alginic acid oligosaccharide prepared from alginic acid onto an anion exchange resin column, subjects it to high performance liquid chromatography, performs concentration gradient elution with water and sodium chloride, and fractionates it. This is a method for purifying alginate oligosaccharides.

Furthermore, the present invention involves adsorbing a sulfate group-containing oligosaccharide prepared from a polysaccharide containing a sulfate group in its structure onto an anion exchange resin column, subjecting it to high performance liquid chromatography, and eluting with a concentration gradient using water and sodium chloride. This is a method for purifying sulfate group-containing oligosaccharides, which is characterized by fractionating the oligosaccharides. In the above purification method, concentration gradient elution with water and sodium chloride increases the concentration of sodium chloride from 0 to 0.25.
After increasing linearly to M in 40 minutes, increasing to 2M by 5
This is done by holding for 15 minutes and then flushing only water for 15 minutes.

[0007] As the anion exchange resin column for HPLC used in the present invention, a commercially available one can be used, and an example thereof is Protein Pak G-DEAE (
Waters), Protein Pak G-QA
(manufactured by Waters), COSMOGEL DEAE
GLASS (manufactured by Nacalai Tesque), COSMOGEL
QA GLASS (manufactured by Nacalai Tesque), Mono
Q (manufactured by Pharmacia), TSKgel D
EAE (manufactured by Tosoh), TSKgel QAE (manufactured by Tosoh)
(manufactured by).

Raw materials containing alginate oligosaccharides to be subjected to the purification method of the present invention include sodium alginate and potassium alginate decomposed with alginate lyase, and alginic acid from brown algae (for example, wakame, kelp, hijiki, etc.) A product obtained by decomposing a substance containing alginic acid with alginic acid lyase, or a product obtained by hydrolyzing a substance containing alginic acid with an acid such as sulfuric acid is used. As raw materials containing sulfate group-containing oligosaccharides, heparin decomposed with heparinase, heparan sulfate decomposed with heparitinase, chondroitin sulfate decomposed with chondroitinase, etc. are used.

Next, the operating procedure of the purification method of the present invention will be explained. First, a column filled with an anion exchange resin is connected to HPLC, and water is used as a solvent for equilibration. An alginic acid oligosaccharide or sulfate group-containing oligosaccharide solution from which insoluble matter has been removed by centrifugation or filtration is injected into the column and adsorbed onto the resin. At this time, there is no need to decolorize the sample. Next, increase the concentration of sodium chloride from 0 to 0.
．． After ascending linearly to 25M in 40 minutes, 2M
5 minutes, then run only water for 15 minutes. Purification of the alginic acid oligosaccharide or the sulfate group-containing oligosaccharide is completed by fractionating the oligosaccharide using a fraction collector and collecting each fraction of the oligosaccharide. In this series of operations, injection and elution can be repeated in units of 60 minutes. After this, desalting and concentration are performed as necessary.

0010

[Examples] The present invention will be specifically explained below using examples. However, the technical scope of the present invention is not limited to these Examples.

[0011]

[Example 1] Sodium alginate (manufactured by Wako Pure Chemical Industries)
Alteromonas sp. Microtechnology Research Institute No. 11
No. 685 (Patent Application No. 2-294897) A purification method using a column for DEAE analysis of a decomposed product (hereinafter referred to as alginate oligosaccharide) decomposed by alginate lyase derived from DEAE GLASS is an analytical column using ethyl (DEAE) type weak anion exchange resin.
Made by Nacalai Tesque, 8mm x 75mm, 10μm)
and a DEAE GLASS guard column (manufactured by Nacalai Tesque, 8 mm x 10 mm, 30 μm).
This was equilibrated with distilled water. 10% (w/
v) Add the alginate oligosaccharide solution to 10,000 rpm. p. m
．． 2 μl of the supernatant obtained by centrifugation for 10 minutes was injected. Next, using distilled water and a 2M sodium chloride aqueous solution, the concentration of sodium chloride was linearly increased from 0 to 0.25M in 40 minutes, and then the sodium chloride concentration was immediately raised to 2M and kept at that concentration for 5 minutes. , and then eluted by running distilled water for 15 minutes. During this time, measure the absorption at 230 nm.
(Since lyase creates double bonds in decomposed products through an elimination reaction, we used 230 nm, which is the maximum absorption wavelength of double bonds.) When the elution state was monitored by this method, the elution time was 15.465 minutes. 8 until 005 minutes
Several peaks were observed (see Figure 1). At the same time, when we fractionated each peak using peak mode with a fraction collector, the results were 15.465 minutes and 16.942 minutes.
19.742 minutes, 21.942 minutes and 23.275 minutes, 2
It was fractionated into six fractions: 5.369 minutes, 27.977 minutes, and 31.005 minutes. As a result of measuring the amount of reducing sugar in these six fractions using the 3,6-dinitrophthalic acid method,
The peak observed by absorption at 230 nm was found to be a sugar peak (see Figure 2). Flow rate is 0
．． The flow rate was 6 ml/min.

0012

[Example 2] Purification method of alginate oligosaccharide using a QA analysis column. Quaternary ammonium (QA) type strong anion exchange resin analytical column QA GLASS (manufactured by Nacalai Tesque, 8
mm x 75mm, 10μm) and QA GLASS
Guard column (manufactured by Nacalai Tesque, 8mm x 10mm
, 30 μm) were equilibrated in the same manner as in Example 1, and 2 μl of alginate oligosaccharide supernatant was injected for adsorption. Elution was carried out in the same manner as in Example 1 using a concentration gradient of distilled water and a 2M aqueous sodium chloride solution. When the elution state was monitored by absorption at 230 nm, the elution time was 28.50.
Although it was possible to fractionate and purify 8 fractions from 7 minutes to 43.792 minutes, the column retention time was longer than that of DEAE.
(See Figure 3).

[0013]

[Example 3] Purification method of alginate oligosaccharide using a preparative column HPLC (ALC/GPC 609G, Water
A preparative DEAE GLASS (manufactured by Nacalai Tesque, 20 mm x 100 mm, 30 μm) and a DEAE GLASS guard column were connected to a DEAE GLASS (manufactured by Nacalai Tesque) and equilibrated with distilled water. In the same manner as in Example 1, 200 μl of alginate oligosaccharide supernatant was injected. Flow rate is 5ml/min
The elution was carried out in the same manner as in Example 1. When the elution state was monitored by absorption at 230 nm, the results were as follows: 20.84 minutes, 22.39 minutes,
26.43 minutes, 28.50 minutes, 29.56 minutes, 31
．． Eight peaks at 51 minutes, 33.27 minutes, and 35.24 minutes were observed, and each peak was fractionated into 8 peaks and collected using a fraction collector (see Figure 4). Then, as a result of calculating the total sugar content using D-mannofuranurono-6,3-lactone as a standard using the phenol/sulfuric acid method, the yields of the 8 fractions were 0.9 mg and 4 mg, respectively.
．． 5mg, 6.2mg, 1.8mg, 4.4mg
, 2.0 mg, 1.8 mg, and 4.6 mg.

[0014]

[Example 4] Purification method using an analytical column for a decomposition product (hereinafter referred to as kelp oligosaccharide) obtained by decomposing kelp powder (Kombu kelp powder), which is a typical product containing alginic acid as a main component, using alginate lyase. Similarly, DEAE GLA for HPLC analysis
The SS column and DEAE GLASS guard column were connected and equilibrated with distilled water. A 10% (w/v) kelp oligosaccharide solution was added at 10,000 rpm. p. m. 10 μl of the supernatant obtained by centrifugation for 10 minutes was injected. Flow rate is 0.6ml
/min, and elution and fractionation were performed in the same manner as in Example 1. As in the case of alginate oligosaccharides, fractionation and purification into eight components was possible from elution time of 27.522 minutes to 44.544 minutes (see Figure 5).

[0015]

[Example 5] Chondroitin sulfate C (manufactured by Seikagaku Corporation), a sulfate group-containing polysaccharide, was converted into chondroitinase ABC.
(manufactured by Seikagaku Corporation) (hereinafter referred to as ChS-
Purification method using an analytical column for (referred to as C oligosaccharide) As in Example 1, an analytical DEAE GLASS column and a DEAE GLASS guard column were connected and equilibrated with distilled water. 10mg/ml ChS-C oligosaccharide solution
0 μl was injected. The flow rate was 0.6 ml/min, and elution was carried out in the same manner as in Example 1. As a result, a disaccharide fraction, which is the final degradation product of chondroitin sulfate C by chondroitinase ABC, was obtained at 31.114 minutes (see FIG. 6).

[0016]

[Example 6] Chondroitin sulfate A (manufactured by Nacalai Tesque), a sulfate group-containing polysaccharide, was added to chondroitinase AB.
Purification method using an analytical column for a decomposed product (hereinafter referred to as ChS-A oligosaccharide) decomposed with C As in Example 1, an analytical DEAE GLASS column and a DEAE GLASS guard column were connected and equilibrated with distilled water. 50 μl of 25 mg/ml ChS-A oligosaccharide solution was injected. As a result, 28.718 minutes
(See Figure 7) A disaccharide fraction, which is the final degradation product of chondroitin sulfate A by chondroitinase ABC, was obtained.

[0017]

Example 7 shows the chromatography of alginate oligosaccharides at different concentration profiles. HPLC as in Example 1
DEAE GLASS and DEAE GLA for analysis
The SS guard column was connected and equilibrated. 2 μl of alginate oligosaccharide supernatant was injected and adsorbed. Only distilled water was allowed to flow for the first 5 minutes, then the concentration of sodium chloride was increased from 0 to 0.5M over 20 minutes, and then only distilled water was allowed to flow immediately. The time required for elution was halved (30 minutes) compared to when the concentration ratio of the present invention was used, but the separation was worse. (See Figure 8)

[0018]

[Example 8] NOVA PAK C which is a C18 column
18 (4μSpherical, 3.9 × 150
, Waters). H
NOVA PAK C18 was connected to a PLC, equilibrated with acetonitrile:distilled water (80:20) mixture, 5 μl of alginate oligosaccharide supernatant was injected, and elution was performed with the same mixture. A flow rate of 1 ml/min was used. (See Figure 9) We tried varying the ratio and flow rate of acetonitrile and distilled water, but the separation under the above conditions (see Figure 9) was the best and no further improvement could be made.

[0019]

Effects of the Invention The present invention was able to very easily fractionate these oligosaccharides by using HPLC and an anion exchange resin column. That is, in the present invention, the product can be purified by a single chromatography, and since the only solvents used for the purification are distilled water and sodium chloride, it is extremely easy to desalt afterward. Furthermore, in the case of the present invention, one chromatography is assembled in 60 minutes, including column washing, so automation is possible by using an autoinjector and a fraction collector together. Additionally, anion exchange resin columns are easier to handle, have better reproducibility, and are more durable than other HPLC columns (e.g., ODS columns, gel filtration columns, NH2 columns, etc.).
It is also easy to transfer to large-scale preparation on an industrial scale.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the elution results of alginic acid oligosaccharides using DEAE GLASS for analysis in Example 1.

FIG. 2 is a diagram showing the results of measuring the amount of reducing sugar in each fraction of Example 1 using the 3,6-dinitrophthalic acid method. The vertical axis shows the amount of reducing sugar in each fraction calculated using glucose as a standard, and the horizontal axis shows each fraction in the order of elution.

FIG. 3 is a diagram showing the elution results of alginate oligosaccharide using analytical QA GLASS in Example 2.

FIG. 4 is a diagram showing the elution results of alginate oligosaccharides in preparative DEAE GLASS in Example 3.

FIG. 5 is a diagram showing the elution results of kelp oligosaccharide in DEAE GLASS for analysis in Example 4.

FIG. 6 is a diagram showing the elution results of ChS-C oligosaccharides in DEAE GLASS for analysis in Example 5.

FIG. 7 is a diagram showing the elution results of ChS-A oligosaccharides in DEAE GLASS for analysis in Example 6.

FIG. 8 is a diagram showing the elution curves of alginate oligosaccharides at different concentrations in Example 7.

FIG. 9 is a diagram showing the results of elution with acetonitrile and distilled water in Example 8.

Claims (4)

[Claims] Claim 1: Purification of alginic acid oligosaccharides, which is characterized by adsorbing alginic acid oligosaccharides prepared from alginic acid onto an anion exchange resin column and subjecting them to high performance liquid chromatography, followed by concentration gradient elution and fractionation with water and sodium chloride. Law. Claim 2: Perform concentration gradient elution with water and sodium chloride, increasing the concentration of sodium chloride from 0 to 0.25M.
After raising it linearly for 0 minutes, keep it at 2M for 5 minutes,
2. The method for purifying alginate oligosaccharide according to claim 1, which is then carried out by flowing only water for 15 minutes. Claim 3: A sulfate group-containing oligosaccharide prepared from a polysaccharide containing a sulfate group in its structure is adsorbed onto an anion exchange resin column, subjected to high performance liquid chromatography, and fractionated by concentration gradient elution with water and sodium chloride. A method for purifying sulfate group-containing oligosaccharides. Claim 4: Concentration gradient elution with water and sodium chloride is performed with the concentration of sodium chloride ranging from 0 to 0.25M.
After raising it linearly for 0 minutes, keep it at 2M for 5 minutes,
4. The method for purifying a sulfate group-containing oligosaccharide according to claim 3, which is then carried out by flowing only water for 15 minutes.