JP2002131233A - Method for labeling and analysis of uronic acid- containing polysaccharides - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the labeling and analysis of uronic acid- containing polysaccharides, in which the uronic acid-containing polysaccharides can be measured with high sensitivity, specifically, precisely, with satisfactory quatitation and reproducibility, and in a simple manner. SOLUTION: A substance, which comprises a primary amino group, a hydrazino group or a hydrazide group and which is fluorescent or ultrasonic absorbent, is bonded to the uronic acid-containing polysaccharides so as to be labeled. The labeled polysaccharrides are isolated by means of liquid chromatography, so as to be analyzed and determibed quantitatively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting and quantifying polysaccharides, which had been difficult to detect, by introducing a fluorescent or ultraviolet-absorbing substance into uronic acid-containing polysaccharides. And how to.

[0002]

2. Description of the Related Art Hitherto, as a fluorescent labeling method for monosaccharides and oligosaccharides, a method has been used in which a fluorescent reagent having an amino group is introduced into the reducing terminal of a sugar via a Schiff base. Although these methods are effective means for analyzing monosaccharides and oligosaccharides, polysaccharides have problems in sensitivity and separation because the ratio of the reducing terminal in the molecule is low. For this reason, a colorimetric method, a method of covalently binding a protein to glycosaminoglycan, and the like have been proposed (JP-A-2000-2000).
-97940). However, these methods have low specificity and complicated operation. In particular, when the uronic acid-containing polysaccharide having a wide molecular weight distribution, for example, when using a natural product containing the polysaccharide, or when targeting a mixture containing other substances such as a beverage containing the polysaccharide, when performing labeling and quantification, Problems such as a quantitative method and a resolving power were remarkable.

Further, as a method for detecting uronic acid, a method of labeling in a non-aqueous solvent and a method of labeling a carboxyl group of uronic acid in an aqueous system have been proposed. Especially DMEQ
Method using (6,7-dimethoxy-1-methyl-2 (1H) -quinoxalinone-3-propionylcarboxylic acid) hydrazide (Journal Chromatogr)
aphy B, 654 (1994), 171-176.
Page) is an excellent labeling method that can be reacted in aqueous systems, but only the labeling of monosaccharides is described here, and there is no application to other sugars (oligosaccharides and polysaccharides). However, its applicability to polysaccharides has not been suggested. When this method is applied to a polysaccharide, similarly to the method of introducing a fluorescent reagent having an amino group as described above, when the ratio of a carboxyl group in a molecule is low, the sensitivity to a quantitative label in terms of sensitivity and separation is high. It was considered difficult to apply.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to quantitatively label a uronic acid-containing polysaccharide (ie, a polysaccharide having a carboxyl group) in an aqueous solvent, and to detect a highly sensitive polysaccharide. , And to provide a method that allows quantification.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have found that many polysaccharides having physiological significance are those containing uronic acid, and these polysaccharides are dissolved in non-aqueous solvents. Therefore, the present inventors have found a method of labeling and analyzing the carboxyl group of uronic acid using an aqueous solvent, and have completed the present invention.

[0006] The present invention relates to a method for preparing a uronic acid-containing polysaccharide by using a water-soluble polysaccharide.
And an amino group (—NH_Two), Hydrazi
Group (-NHNH_Two) Or a hydrazide group (-CON
HNH _Two) And has fluorescence or ultraviolet absorption
Characterized by labeling with urethane,
It is intended to provide a method for labeling an acid-containing polysaccharide.

Further, the present invention provides a method for separating and analyzing a uronic acid-containing polysaccharide, which comprises separating and analyzing a labeled substance obtained by the above method by liquid chromatography.

Further, the present invention provides a method for quantifying a uronic acid-containing polysaccharide, which comprises measuring the fluorescence intensity or the ultraviolet absorption intensity of the label obtained by the above method.

[0009]

The above-mentioned uronic acid-containing polysaccharide is a polysaccharide containing a carboxyl group derived from seaweed, microorganism, plant or animal, for example, fucoidan obtained from Okinawa mozuku, alginic acid of brown algae; pectin Mucopolysaccharides (glycosaminoglycans) such as heparin, chondroitin sulfate, hyaluronic acid, and teicuronic acid. Here, the polysaccharide means a monosaccharide polymerized by about 10 or more.

Examples of the substance having an amino group, a hydrazino or hydrazide group and having a fluorescent property include secondary amino acids such as 2-aminopyridine, 2-aminoethylpyridine and alkyl 2-aminobenzoate (for example, methyl or ethyl). Examples include hydrazines and hydrazides such as 1 amine, lucifer yellow (manufactured by Molecular Probes), hydrazinopyridine, pyridinehydrazide, dansylhydrazide, and DMEQ-hydrazide. Preferred fluorescent substance reagents are DMEQ-hydrazide, hydrazinopyridine, aminobenzoic acid ethyl ester, and lucifer yellow, which have good reaction efficiency and quantitative performance at the time of labeling,
Particularly, DMEQ-hydrazide.

Substances having an amino group, a hydrazino group or a hydrazide group and having an ultraviolet absorbing property include:
Primary amines such as -aminoethylpyridine, 2-aminopyridine, alkyl 2-aminobenzoate (eg, methyl or ethyl) ester; Lucifer Yellow (manufactured by Molecular Probes), hydrazinopyridine, pyridinehydrazide, dansylhydrazide, DMEQ Hydrazides, hydrazines such as DNP (2,4-dinitrophenol) -hydrazide and hydrazides.

The carboxyl group of the uronic acid-containing polysaccharide is
Water-soluble condensing agent (a substance having a dehydrating action), for example, 1-
Activated by a water-soluble carbodiimide such as ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide, the above-mentioned fluorescent or ultraviolet absorbing substance (reagent) Binds to an amino group, a hydrazino group or a hydrazide group. This reaction can be carried out in an aqueous system. The reagent is used in an amount such that the carboxyl group of the uronic acid-containing polysaccharide sufficiently reacts. For example, the molar ratio of the uronic acid-containing polysaccharide to the reagent is 1: 1 to 1:20, preferably 1: 5.
Use in an amount such that it becomes １ ： 1: 10. When the amount of the reagent is less than equimolar, the reaction efficiency is reduced, and when the amount is more than 20 times, the workability and the ease of analysis are reduced. Excess reagents can be removed by a pre-treatment column such as a cation exchange column such as Bond Elute or a lipophilic resin column to quantify uronic acid-containing polysaccharides, i.e., to stabilize the baseline and eliminate interference with excessive addition. And excess reagent may be removed by dialysis, a desalting column or the like. The removal means may be appropriately determined according to the type of reagent used and the like. For example, when DMEQ-hydrazide is used, a cation exchange column is preferred from the viewpoint of removal efficiency. In this way, the uronic acid-containing polysaccharide is labeled with a fluorescent or ultraviolet absorbing substance. The reaction temperature and the reaction time are appropriately adjusted depending on the properties of each reagent.
° C, preferably about 0 to 60 ° C, particularly preferably 0 to 40 ° C.
° C. This is because if the temperature is high, the decomposition reaction or excess reaction (reaction other than the carboxyl group) of the polysaccharide bonded to the reagent is promoted, and the quantitative property is reduced. The reaction time is usually about 10 to 90 minutes.

The amount of the water-soluble carbodiimide is usually preferably 5 to 10 times the amount of the carboxyl group from the viewpoint of reaction efficiency. Further, the water-soluble carbodiimide with the addition of pyridine, and the like, it is preferable for the reaction is allowed to proceed after the pH was adjusted <br/>. Further, in order to stabilize the water-soluble condensing agent and improve the reaction efficiency, a reaction aid such as N-hydroxysuccinimide and sulfo N-hydroxysuccinimide may be used.

The labeled product of the uronic acid-containing polysaccharide derivative obtained by the above operation is separated and analyzed by liquid chromatography, particularly high performance liquid chromatography (HPLC), using the lipophilicity obtained by labeling. can do. Furthermore, the uronic acid-containing polysaccharide can be quantified by measuring the fluorescence intensity or ultraviolet absorption intensity of the label using a fluorescence detector or an ultraviolet detector.

Columns and solvents used for liquid chromatography may be selected from reverse-phase, normal-phase, gel filtration, and ion-exchange columns according to the properties of the reagent used for derivatization (labeling) of the uronic acid-containing polysaccharide. It is appropriately selected and used, but a reversed-phase system is preferable in terms of quantitativeness, separation ability, and the like.

The method of the present invention can be used for detecting and quantifying uronic acid-containing polysaccharides in foods and drinks and cosmetics, or for quantifying glucosaminoglycan as a marker for liver disease, rheumatism or bladder cancer. . It can be applied to any sample for detection, quantification, etc. from a sample containing uronic acid-containing polysaccharide, but it is a target such as a mixture containing components other than uronic acid-containing polysaccharide or polysaccharide having a wide molecular weight distribution. The effect when applied to a sample in which separation and quantification of a polysaccharide is difficult is particularly remarkable. Examples of such specimens include foods and drinks containing uronic acid-containing polysaccharides, urine and blood of patients with the above diseases.

[0017]

EXAMPLES The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[Preparation of uronic acid-containing polysaccharide derivative] Example 1: Preparation of 2-hydrazinopyridine derivative 1 ml of an aqueous solution of fucoidan (1 mg / mL) derived from Mozuku
In addition, as a water-soluble carbodiimide, 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide (hereinafter WS)
C) (solution in methanol at a ratio of 1.92 μg / 10 mL), 400 μL of 2-hydrazinopyridine solution (dissolved in methanol to 15 mM), and 20% pyridine methanol solution 200 μL was added, reacted at 45 ° C. for 45 minutes, and desalted with a NAP10 column (manufactured by Amersham Pharmacia) to obtain a 2-hydrazinopyridine derivative of fucoidan.

Example 2 Preparation of 2-Hydrazinopyridine Derivative In the same manner as in Example 1, 2M was added to a fucoidan sample (2 mL).
Of WSC (1 mL), 1M 2-hydrazinopyridine (1 mL), and 20% pyridinemethanol (1 mL) were added and reacted at 45 ° C. for 3 hours. Then NaCl 3
0 mg was added and dialyzed (10000 cuts). The target fucoidan 2-hydrazinopyridine derivative was obtained in the dialysate.

Example 3 Preparation of 2-Aminoethylpyridine Derivative A fucoidan solution sample (1 mL) similar to that of Example 1 was added to
Add 10 μL of aminoethylpyridine stock solution and add
15 μL of N hydrochloric acid was added. 400 μm of 1M WSC solution
L and 200 μL of a 100 mg / mL sulfo N-hydroxysuccinimide solution were added, and the mixture was reacted at room temperature for 20 hours. By desalting with a NAP10 column, a fucoidan fluorescent derivative (2-aminoethylpyridine derivative) was obtained.

Example 4 Preparation of Ethyl Derivative of 2-Aminobenzoic Acid Derivative of Mozuku-derived fucoidan solution (1 mg / mL) 100
1 μL of 1N hydrochloric acid was added to μL. 1M W in this solution
SC (50 μL), N-hydroxysuccinimide (NH
S) (50 μL) was added and reacted at room temperature for 1 hour. W
SC and NHS were added by 50 μL each, and the mixture was further reacted for 1 hour. Add 300 μL ethanol and add 20 mM
50 μL of 2-aminobenzoic acid ethyl ester was added. Add 20 μL of 0.5 M NaHCO ₃ and add
Allowed to react for hours. 50 μL of WSC was added, and the mixture was further reacted for 1 hour. The salt was desalted with a NAP5 column to obtain ethyl 2-aminobenzoate derivative of fucoidan.

Example 5: Preparation of Lucifer Yellow Derivative Mozuku fucoidan solution sample (1 mg / mL) 100 μL
, 30 μL of 20% pyridine / methanol was added to
50 μL of 0 mg / mL Lucifer Yellow was added.
Further, 120 μL of a 1M WSC solution was added and reacted at 37 ° C. for 1 hour to prepare a lucifer yellow fluorescent derivative of fucoidan.

Example 6: Preparation of DMEQ-hydrazide derivative 100 µL of an aqueous solution of Mozukufucoidan (1 mg / mL) was added with 100 µL of an ethanol solution containing 4% pyridine. 50 mM 6,7-dimethoxy-1-methyl-2
100 μL of (1H) -quinoxalinone-3-propionylcarboxylic acid (DMEQ) -hydrazide solution (dissolved in dimethylformamide) was added, and 10 μL of a 0.2 M WSC solution was further added and mixed well. 6 at room temperature, protected from light
After reacting for 0 minutes, a Bond Elut SCX column (100 mg / piece) pre-conditioned with 200 μL of the reaction solution
A cation exchange column manufactured by Varian, Bond Elut SCX (100 mg / column) was washed with 1 mL of methanol, then passed through 1 mL of water and conditioned, and then applied to adsorb excess DMEQ-hydrazide. Removed. The filtrate was collected to obtain a DMEQ-hydrazide derivative of fucoidan.

[Quantitative Analysis by HPLC] Example 7: A 10 mM acetate buffer (pH 3.10) was used for the DMEQ-hydrazide derivative of fucoidan of Example 6.
A solution obtained by adding methanol to a concentration of 20% to 6) (acetate buffer: methanol = 8: 2) was used as a mobile phase.
Analysis was performed by high performance liquid chromatography (HPLC) using a polymer-based C-18 column (YMC-Pack polymer C-18, 4.6 × 150 nm, manufactured by YMC). The chromatography was carried out using a mobile phase flow rate of 0.
The operation was performed at 5 mL / min, an excitation wavelength (Ex) of 367 nm, and an emission wavelength (Em) of 445 nm. By analyzing with a fluorometer, fucoidan could be detected at around 1.9 minutes. The result is shown in FIG. 1B together with the case where water was used as a blank (A).

Since Mozuku fucoidan was quantitatively labeled by the above-mentioned method, and sharp separation and detection were possible with a reversed-phase column, its quantitative nature was examined. FIG. 2 is a graph plotting the amount of fucoidan and the peak area of the fluorescence intensity. As shown in FIG. 2, in the method of the present invention, linearity was observed at an injection of up to 1 mg / mL. The variation of the measured value is 5-6%
It was below.

Examples 8 to 12 Under the same conditions as in Example 6, instead of Mozukufucoidan, heparin (Example 8), hyaluronic acid (Example 9),
Each was derivatized with DMEQ-hydrazide and labeled with chondroitin sulfate (Example 10), alginic acid (Example 11) or pectin (Example 12). All were quantitatively labeled.

[Separation and Detection Using Gel Filtration Column] Example 13 The hyaluronic acid derivative and the heparin derivative labeled in Examples 8 and 9 were subjected to gel filtration column (Sho).
(dex GS620HQ) and separation detection was performed by HPLC using water as a mobile phase. The results are shown in FIGS.

As shown in FIGS. 3 and 4, not only Mozukufucoidan but also uronic acid-containing polysaccharides such as hyaluronic acid and heparin can be labeled and detected by the method of the present invention. The conditions for derivatization for labeling may be other than those described in the above examples, and optimal conditions can be set according to the properties of the polysaccharide. As the column to be used, an ion exchange column or the like is effective according to the properties of the polysaccharide in addition to the above-mentioned reversed phase column system and gel filtration column.

Example 14 9 to 50 g of mixed tea (hub tea, dokudami tea, fennel)
1 kg of ion-exchanged water at 0 ° C was added, and the mixture was extracted with stirring for 10 minutes. After roughly filtering the tea material using a 100-mesh stainless steel filter, the extract was cooled to 30 ° C. or less,
The mixture was treated at 0 rpm for 5 minutes to obtain a clear liquid. After dilution 3 times, 50% sodium ascorbate is used to stabilize the quality.
After adding mg, sodium bicarbonate was added to adjust the pH to 6.0. Mozuku fucoidan was added to the tea beverage thus obtained at a ratio of 0.4 mg / mL to 100 μL of the sample,
Add 100 μL of ethanol solution containing% pyridine.
Add 100 μL of a 50 mM DMEQ-hydrazide solution, and further add 10 μL of a 0.2 M WSC solution and mix well. After reaction at room temperature for 60 minutes, a cation exchange column manufactured by Varian (Bond Elut SCX column, 100 mg / tube) in which 200 μL of the reaction solution was previously conditioned.
To remove excess DMEQ-hydrazide by adsorption. Collect the permeate and use as a sample for HPLC. Analysis conditions <Examples of high-performance liquid chromatography operation conditions> Column: YMC-PackPolymerC18 (4.6 mm inner diameter, 150 mm length) Flow rate: 0.5 mL / min Column temperature: 30 ° C. Detector: Fluorometer (measurement wavelength: Ex367 nm, Em
445 nm) Injection volume: 10 μL Mobile phase: eluent for HPLC analysis (10 mM acetate buffer [pH 3.6]: methanol = 8: 2 (V / V))

Under these conditions, the fucoidan in the tea is
, And was separated and identified from other tea components as well. The area reflected the amount of fucoidan.

[0031]

As described above, the method of the present invention has made it possible to efficiently label and detect the uronic acid-containing polysaccharide. The detection method according to the present invention is a very practical method because it can measure uronic acid-containing polysaccharides with high sensitivity, specifically and accurately, with good quantitativeness and reproducibility, and easily.
In addition, it is suitable for analyzing trace components such as biological samples. In particular, when the derivatization method using DMEQ-hydrazide and the separation method using a reversed-phase column are combined, broadening of the peak due to the influence of the molecular weight distribution of the polysaccharide and the like can be avoided, so that the quantitative property is particularly excellent. Since the number of constituent reagents is small, it can be used at low cost. This has high utility as a diagnostic method for mucopolysaccharide-related diseases or as a method for measuring uronic acid-containing polysaccharides in pharmaceuticals and foods.

[Brief description of the drawings]

1 is a high performance liquid chromatogram of water and B is a high performance liquid chromatogram of fucoidan DMEQ-hydrazide derivative.

FIG. 2 is a graph plotting the amount of fucoidan and the peak area of fucoidan fluorescence intensity.

FIG. 3 is a liquid chromatogram of a hyaluronic acid derivative using a gel filtration column.

FIG. 4 is a liquid chromatogram of a heparin derivative using a gel filtration column.

FIG. 5 is a high performance liquid chromatogram of fucoidan added to tea.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) G01N 30/88 G01N 30/88 N (72) Inventor Hideyuki Shibata 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo No. Yakult Honsha (72) Inventor Ritsuo Aiyama 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo (72) Inventor Shusuke Hashimoto 1-1-1, Higashi-Shimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha (72) Inventor Sadao Ueyama 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo F-term (Reference) 2G054 EA03 GB02 4C090 AA10 BA50 BA66 BA67 BA68 BA72 BB18 BB20 BB21 BB22 BB23 BB35 BB36 BB52 BB53 BB54 BB55 BD14 DA12

Claims (5)

[Claims] 1. A method for labeling a uronic acid-containing polysaccharide using a water-soluble condensation activating reagent and a substance having an amino group, a hydrazino group or a hydrazide group and having a fluorescent or ultraviolet absorbing property. A method for labeling a uronic acid-containing polysaccharide, which is characterized in that: 2. The method according to claim 1, wherein the water-soluble condensation activating reagent is a water-soluble carbodiimide. 3. The method according to claim 1, wherein the uronic acid-containing polysaccharide is selected from the group consisting of fucoidan, alginic acid, pectin, heparin, chondroitin sulfate, hyaluronic acid, glucosaminoglycan and teicuronic acid. 4. A method for separating and analyzing uronic acid-containing polysaccharides, wherein the uronic acid-containing polysaccharides labeled by the method according to claim 1 are separated and analyzed by liquid chromatography.
Analysis method. 5. A method for quantifying a uronic acid-containing polysaccharide, comprising separating a uronic acid-containing polysaccharide labeled by the method of claim 1 by liquid chromatography and measuring a fluorescence intensity or an ultraviolet absorption intensity. Law.