JP2001086999A - Composition containing xylooligosaccharide and carcinoma cell apoptosis inducer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an enzyme and algae, capable of producing β-1,3- xylooligosaccharide having high bioactivity when producing the β-1,3- xylooligosaccharide from the algae, and further to effectively utilize the bioactivity of the xylooligosaccharide. SOLUTION: This xylooligosaccharide-containing composition is obtained by treating polysaccharides derived from cell walls of the algae with β-1,3- xylanase produced by the strain AX-4 of the genus Bibrio, or treating the polysaccharides derived from cell walls of Caulerpa racemosa verlaete-virens of the algae belonging to the genus Caulerpa of green algae, and has <=5 average polymerization degree (total amount of sugar/amount of reducing sugar), and a protein content below the limit of detection. The carcinoma cell apoptosis inducer consists essentially of the xylooligosaccharide obtained by treating the β-1,3-xylan obtained from the cell walls of algae with the β-1,3-xylanase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel oligosaccharide composition derived from a natural product, and more particularly to a β-1,3-derived from seaweed.
The present invention relates to a xylooligosaccharide-containing composition obtained by treating xylan with β-1,3-xylanase.
The present invention also relates to a cancer cell apoptosis inducer containing xylo-oligosaccharide as a main component.

[0002]

2. Description of the Related Art β-1,3-xylan is a homopolysaccharide having D-xylose as a constituent sugar, similarly to β-1,4-xylan constituting a hemicellulose main chain of land plants. β-
1,3-xylan has one hydroxyl group in the constituent sugar xylose.
There is no high crystallinity unlike β-1,4-xylan due to β-bonding at the 3rd and 3rd positions. In general, β-1, as a polysaccharide,
3-xylan is known as a constituent polysaccharide of the cell wall of seaweed,
It has been extracted from the seaweeds of the Iridaceae and Iridaceae. According to Nishizawa et al., Who has been studying the cell wall of seaweed, the cell wall of seaweed is different from the cell wall of land plants.
Many of them are said to be polysaccharides without crystallinity. It is speculated that the lack of crystallinity may be due to the fact that the living space of the algae was in water where there was little influence of gravity and the cells themselves were not required to be as robust as land plants.

On the other hand, it is a well-known fact that the cell walls of algae are variously modified in their sugar chains. It is known that sugar chains including modified polysaccharides and oligosaccharides have various physiological activities, particularly to agar-derived oligosaccharides and alginic acid-derived oligosaccharides, and more recently to fucoidan extracted from brown algae. There are reports on oligosaccharides and polysaccharides derived from them.

[0004] β-1,3-glucan whose structure is very similar to that of β-1,3-xylan of interest in the present invention is well known as a cell wall constituent polysaccharide of terrestrial fungi.
1,3-glucan-based anticancer drugs have been very well studied. On the other hand, β-1,3-xylan is present in a large amount in the cell wall of marine algae, and has not been studied so far. At present, enzymes that degrade homopolysaccharides having β-1,3-xylan as a main chain are mainly obtained from marine bacteria. β-1,3-xylanase screened using β-1,3-xylan as a substrate is used for producing protoplasts of marine algae such as Aosa and Amanori, which are used in industrially useful algal breeding. It is used. Araki et al. Report from Mie University
From bacteria cultured using 1,3-xylan as a substrate, β-
Although 1,3-xylanase has been prepared, the use of this enzyme is described as preparation of protoplasts of Amanori. (JP-A-10-295372) However, β-1,3-xylanase produced by these marine bacteria is β-
Β-1,3- formed when 1,3-xylan is decomposed
There are no reports that studied the biological activity of xylo-oligosaccharide compositions.

[0005]

Since β-1,3-xylan makes it difficult to obtain a purified polysaccharide due to its specialty as a raw material, β-1,3-xylanase degrades β-1,3-xylan. However, there has been a problem that research and development on the biological activity of the β-1,3-xylo-oligosaccharide composition produced in this method do not proceed. An object of the present invention is to obtain a xylo-oligosaccharide having an effective physiological activity.

[0006]

In order to solve the above problems, the present invention employs the following configuration. That is, the first invention of the present invention relates to the following (1) and (1) obtained by treating a polysaccharide derived from the cell wall of algae with β-1,3-xylanase produced by a strain AX-4 of the genus Vibrio. Xylooligosaccharide-containing composition having the property of (2) (1) Average degree of polymerization (total sugar content / reducing sugar content): 5 or less (2) Protein content: below the detection limit.

The second invention of the present invention relates to the following (1) and (2), which are obtained by treating a polysaccharide derived from the cell wall of the green alga Sricopodaceae with β-1,3-xylanase. (1) Average degree of polymerization (total sugar content / reducing sugar content): 5 or less. (2) Protein content: below the detection limit.)

[0008] The third invention of the present invention is based on the principle that "xylooligosaccharides obtained by treating β-1,3-xylan obtained from a cell wall of algae with β-1,3-xylanase are used as a main component. Cancer cell apoptosis inducer ".

[0009] The present inventors have converted β-1,3-xylan derived from Srikkogita, an algae of the family Ivy, to β-1,3-xylan.
The β-1,3-xylo-oligosaccharide composition obtained by treatment with xylanase (derived from Vibrio sp.) Was used to assay the apoptosis-inducing ability of human leukemia cancer cell line HL60. It has been found that the sugar composition induces apoptosis of human leukemia cancer cell line HL60 very effectively, and the present invention has been completed.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As a raw material for obtaining the β-1,3-xylo-oligosaccharide composition of the present invention, algae such as green algae, brown algae and red algae are used. Examples of such algae include algae belonging to the Iridaceae family of the order Milliaceae, and algae such as Aosa, Amanori and Darus. Above all, algae of the family Iraceaceae containing a large amount of β-1,3-xylan are preferred.

[0011] The Iridaceae of the order Miridae are algae widely distributed from the tropics to the temperate zone. Some species of the algae of the order Mirida have customarily used their hot-water extract as a pesticide for human parasites. There is a familiar algae such as there. Scrophulariaceae, a member of the family Ivy, thrives on the sea surface south of Honshu and is available in relatively large quantities. Sliced Potter ( Caulerpa
racemosa ver. laete-virens ) can be easily collected from the coastal waters of Kagoshima Prefecture, and dried and powdered to extract β-1,3-xylan from the cell wall.

Next, an extraction method will be described. In the extraction, algae are dried and extracted with an aqueous alkaline solution. Generally, it is relatively easy to recover β-1,3-xylan by dissolving and extracting cell walls using an aqueous solution having a high alkali concentration, but at the same time, other polysaccharides such as β
-1,4-glucan, β-1,3-glucan, and even β
Since -1,4 xylan and the like are also extracted, it is preferable to use an alkaline aqueous solution having a relatively low concentration.

As such a method, the method of Iriki et al. (N
187, 82-83 (1960)), and this method is most recommended in the present invention. A detailed extraction method will be described in an embodiment described later. In the extracted xylan-containing composition, coloring components such as those derived from chlorophyll are simultaneously recovered.After extraction, the polysaccharide is bleached using bleaching powder, and further fractionated using a solvent such as ethanol or acetone. Purify and recover only the polysaccharide component. The obtained dried and purified powder, β-1,3-xylan, is in the form of a white powder. Since the fractionated and purified product thus obtained contains almost no other saccharides such as glucose and galactose, it is suitable for producing β-1,3-xylo-oligosaccharide.

Next, the production of xylo-oligosaccharide will be described. As a general method for producing β-1,3-xylo-oligosaccharide, physicochemical prescription such as explosion or enzymatic treatment is known, but in the present invention, the enzymatic method is used. Enzymes that can be used in the present invention include generally known β-1,
Any 3-xylanase can be used.

However, in the present invention, purified β-1,
A strain AX- of the genus Vibrio isolated from seawater by screening in a minimal medium using 3-xylan as the sole carbon source
4 (T.Aoki, T.Araki, andM.Kitamikado: Purification a
nd characterization of endo-β-1,3-xylanase from V
ibrio sp.Nippon Suisan Gakkaishi, 54, 277-281 (1988)
) Was cultured using the purified β-1,3-xylan as a main carbon source, and the β-
Most preferably, 1,3-xylanase is used.
The AX-4 strain was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (Tsukuba City, Ibaraki Prefecture), and had accession number FE.
RM P-17557 is provided.

Hereinafter, specifically, β-1,3-
The production of xylo-oligosaccharide will be described. Purified β-1,
3-Xylan is suspended in distilled water containing 2 mM calcium chloride and adjusted to a pH in the range of 7 to 5.5 using an acetate buffer. Although any buffer may be used for this adjustment, the optimum pH is 6.0. Then β-1,3
-Xylanase is added in an amount of 0.5 to 1 unit (1 unit is an enzyme activity that generates a reducing power equivalent to 1 μmol xylose per minute when the enzyme protein acts on the substrate β-1,3-xylan), The enzymatic reaction is performed in the range of 27 ° C to 45 ° C. An enzyme reaction time of at least 60 minutes is sufficient. After completion of the enzymatic reaction, the mixture is heated at 100 ° C. for 5 minutes to completely stop the enzymatic reaction and at the same time insolubilize the added enzyme protein.

Ethanol is added to this reaction solution in an amount of 1 to 3 times. The xylo-oligosaccharide having a relatively large degree of polymerization after addition of ethanol becomes cloudy and precipitates.
About 5 oligosaccharides hardly precipitate. After the addition of ethanol, the mixture is centrifuged using a centrifuge to separate into a precipitate and a supernatant, and the supernatant is concentrated to dryness by an evaporator, and β-1,3-xylo-oligosaccharide having an average degree of polymerization of 5 or less is obtained. A composition is present.

When determining the physicochemical properties of an oligosaccharide, the average degree of polymerization is important. Β-1,3 obtained by the present invention
-The average degree of polymerization of the xylooligosaccharide composition was determined by converting the total amount of sugar to xylose, and then determining the amount of reducing sugar of the same sample by conversion to xylose. The average degree of polymerization is defined as: total sugar amount (xylose conversion) / reducing sugar amount (xylose conversion). Incidentally, the total sugar content was determined by measuring the amount of reducing sugar by the phenol sulfate method and the amount of reducing sugar by the Somogyi Nelson method.

Since the β-1,3-xylo-oligosaccharide composition thus obtained contains some salts, the β-1,3-xylo-oligosaccharide composition is desalted and purified for measuring the physiological activity. Need to be done. The desalting method from the oligosaccharide composition may be any known method,
Desalination may be performed using a combination of a cation exchange resin and an anion exchange resin, or an amphoteric simple resin may be used. It is also possible to desalinate using a gel filtration method. The recovery of the thus-desalted β-1,3-xylo-oligosaccharide composition is about 5% by weight based on the initial purified β-1,3-xylan.

A protein assay kit I (manufactured by Bio-Rad) was used for protein measurement. A calibration curve used for protein quantification was prepared using bovine serum albumin as a standard substance. As a result, the β-1,3-xylo-oligosaccharide composition after desalting did not contain any protein.

The thus obtained β-1,
Since the average degree of polymerization of the 3-xylo-oligosaccharide composition is 5 or less, the protein is removed to below the detection limit by fractionation with ethanol, and the protein is highly purified as a sugar chain molecule. Test reproducibility is extremely high. Assay of apoptosis-inducing ability using these xylo-oligosaccharide-containing compositions was performed using a cancer cell (HL6) in a culture system to which a β-1,3-xylo-oligosaccharide-containing composition was added.
It is possible to examine the change in proliferation and morphology in 0).

The proliferation of HL60 in a culture system to which a β-1,3-xylo-oligosaccharide-containing composition has been added can be easily measured by examining the incorporation of labeled thymidine into cells when the cells proliferate. it can. The concentration of the β-1,3-xylo-oligosaccharide-containing composition in the culture system is about 0.5 mg / ml.
It has been found that in the culture system adjusted to ~ 1.0 mg / ml, the proliferation of HL60 is clearly inferior to the system without the β-1,3-xylo-oligosaccharide-containing composition.

Microscopic observation revealed that the decrease in the proliferation of HL60 in the culture system containing the β-1,3-xylo-oligosaccharide-containing composition was apparently due to the cell death of HL60. This HL60
Cell death is necrosis (cell death due to external environmental changes)
But not apoptosis (programmed cell death).

One of the indicators of cell apoptosis is intracellular chromatin aggregation. In the culture system used for the above-mentioned apoptosis assay, aggregation of chromatin at the time of cell death was observed only in the culture system containing the β-1,3-xylo-oligosaccharide-containing composition. DNA fragmentation occurs in the cells after the aggregation of chromatin, and the cells themselves shrink and disappear.

It has been found that DNA fragmentation in apoptosis is derived from a DNA endonuclease (DNase) specific to apoptosis. At present, more than 10 DNases have been reported in mammals. Since the fragmentation of DNA induced by β-1,3-xylo-oligosaccharide is completely prevented by adding 125 to 500 μM of zinc chloride to the medium and conducting the same experiment, the DNA fragment of HL60 in this test was used. It is suggested that the DNase involved in the formation may be related to a Zn ²⁺ -dependent endonuclease.

It has been found that the apoptotic signal transduction system is deeply involved in a protease cascade called the caspase family. Therefore, it was examined whether protease CPP32 belonging to the caspase family was activated by β-1,3-xylo-oligosaccharide.
As a result, surprisingly, about 1 mg / ml of β-1,3-
The addition of xylo-oligosaccharides to the culture medium resulted in
The activity was increased, and it was found that DNA fragmentation occurred accordingly.

ZVAD-fmk, a cell membrane permeability inhibitor of the caspase family, was added to HL60 at a concentration of 50 μM.
When β-1,3-xylo-oligosaccharide was added to the same culture system at the same time as adding to the cell culture system, HL60
And the fragmentation of DNA derived from β-1,3-xylo-oligosaccharide was also suppressed. From these results, it was found that apoptosis caused by β-1,3-xylo-oligosaccharide ultimately causes DNA fragmentation via activation of a protease CPP32 belonging to the caspase family.

[0028]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments, but it goes without saying that the present invention is not limited to the scope of the following embodiments. <Preparation of β-1,3-xylan from Sri Lanka ivy> The final green algae ( Caulerpa racemosa) from the coastal waters of Kagoshima Prefecture
ver. laete-virens ) was dried, pulverized with a pulverizer, and thoroughly washed with deionized water. After washing, the solution was filtered using a cloth bag, and the obtained residue was poured into a 1% hydrochloric acid solution and stirred at room temperature for 45 minutes. After washing with water, the obtained residue was filtered with a cloth bag. It was poured into 1.23% sodium hydroxide and stirred at 100 ° C. for 1 hour while blowing nitrogen gas. After washing, the obtained residue was filtered with a cloth bag and then decolorized with sodium chlorite. After the washing, the crude fiber of Sri Lanka irrigation was charged into a 10% sodium hydroxide solution, and stirred for 5 hours while blowing nitrogen gas to extract β-1,3-xylan and centrifuged. 2 in the resulting supernatant
Β-1,3-xylan was precipitated by adding ethanol twice as much. The precipitate is washed with ethanol containing 1% hydrochloric acid,
Drying under reduced pressure gave β-1,3-xylan as a white powder. The collected amount was 13% of the dried scotch ivy.

In order to examine the constituent sugars of the obtained β-1,3-xylan, 0.2 g of the white powder was placed in a test tube with a screw hole, mixed with 2 ml of trifluoroacetic acid, and hydrolyzed for 3 hours. Was. The solution was dried under reduced pressure after filtration. The obtained decomposition product was analyzed by thin-layer chromatography (solvent, n-butanol: acetic acid: water = 10: 5: 1 (V / V), color former, diphenylamine-aniline phosphate reagent). As a result, it was found that most was xylose.

<Preparation of β-1,3-xylanase> Vibr
AX-4 strain belonging to io sp. was cultured in a peptone slant medium (peptone 0.5%, yeast extract 0.1%, sodium chloride 3
%, Β-1,3-xylan 0.3% and agar 1.5%
After culturing for 2 days at 25 ° C. in a medium having a pH of 7.8 to 8.2 consisting of 0.5% peptone, 0.1% yeast extract, 3% sodium chloride, 0.3% β-1,3-xylan. 10% of pH 7.8-8.2 medium containing 3%)
And cultured with shaking at 25 ° C. for 2 days. On the second day of the liquid culture, 10 ml of the liquid medium was inoculated into 1000 ml of the medium in a 3000 ml Erlenmeyer flask, and shaking culture was performed at 25 ° C. for 5 days. Solid ammonium sulfate was added to the supernatant from which the cells were removed by centrifugation until 75% saturation, and the mixture was allowed to stand at 5 ° C. overnight. The precipitate obtained is washed with 50 mM containing 2 mM calcium chloride.
mM acetate buffer, pH 6.0. The recovery of the enzyme in the culture supernatant was 60%. The AX-4 strain was deposited with the National Institute of Advanced Industrial Science and Technology (Tsukuba, Ibaraki) and has a deposit number of FERM P-17557.
Is given.

<Β-1,3-xylan to β-1,3-xylan
Preparation of Xylooligosaccharide> β-1,3-xylan 200m
was suspended in 10 ml of water containing 2 mM calcium chloride, and 0.44 unit of β-1,3-xylanase dissolved in 3 ml of 50 mM acetate buffer (pH 6.0) was added, followed by reaction at 37 ° C. for 60 minutes. . The reaction was stopped by heating at 100 ° C. for 5 minutes, and twice the amount of ethanol was added. The supernatant was concentrated by centrifugation. Concentrate is Dowex-50X
8 (H ⁺ ) and Dowex-1X (CO ₃ ^2- ) were desalted by passing successively through two columns. The effluent and the washing were concentrated and fixed, and suspended in chloroform: methanol = 1: 1. Chloroform: methanol = 1: 1
(Phenyl boron) equilibrated in
ate agarose gel) column, washed with the same solvent, and eluted with methanol: water = 1: 1. The eluate was concentrated and freeze-dried. The yield was 5%.

The average degree of polymerization in the β-1,3-xylo-oligosaccharide composition obtained as described above was 4.2.
In addition, no protein was detected, and no other saccharides were detected.

<Test of apoptosis-inducing ability> β-1,3
-Human leukemia cell line HL60 was used to measure the apoptosis-inducing ability of xylo-oligosaccharide. HL60 is isolated from peripheral blood of patients with acute promyelocytic leukemia and is also known to differentiate into neutrophils and granulocytes. This cell line is available from any conventional cell bank (eg, the National Institutes of Health cell bank). HL60 can usually be cultured in an RPMI medium containing fetal calf serum (10%) or a serum-free medium containing insulin and transferrin. In the test of this example, the oligosaccharide concentration was 0.1% in the medium.
Oligosaccharides were added to the medium at a concentration of 5 mg / ml to 1.0 mg / ml, and the apoptosis-inducing ability was measured. Further, as a control experiment, similar tests were carried out for various oligosaccharides (described in Table 1) other than β-1,3-xylo-oligosaccharide.

As a first step in the assay for apoptosis-inducing ability, the incorporation of labeled tritiated thymidine into living cells during HL60 proliferation was measured. The growth inhibition of various oligosaccharides on HL60 was assayed by utilizing the fact that when the growth rate of HL60 decreases, the amount of tritiated thymidine incorporated into cells decreases. As a result, in the medium to which β-1,3-xylo-oligosaccharide was added, the ratio of incorporation of tritiated thymidine into HL60 was much lower than in the case where other various oligosaccharides were added. Was found to be inhibited. Table 1 shows the results.
Shown in

[0035]

[Table 1]

In addition, the inhibition of the synthesis of DNA was β-1,3.
-In order to examine whether or not it depends on the xylo-oligosaccharide concentration, the concentration of β-1,3-xylo-oligosaccharide added to the medium was changed and the amount of synthesized DNA was examined. There was a change in the synthesis amount of. Table 2 shows the results.

[0037]

[Table 2]

H provided by β-1,3-xylo-oligosaccharide
In order to examine whether the growth inhibition of L60 was caused by cell death due to apoptosis, the state of intracellular DNA in the β-1,3-xylo-oligosaccharide-added group was observed. Molecularization was observed. Chromatin condensation and chromosomal DNA fragmentation are both indicators of apoptosis.

0.5 mg of β-1,3-xylo-oligosaccharide
Photograph 1 in FIG. 1 shows the results of extracting DNA from HL60 cells supplemented with / ml and 1.0 mg / ml and performing agarose electrophoresis. <Explanation of the photograph> The lane is from the left. (1) φ × 174 / HincII (2) control (3) β-1,3-xylo-oligosaccharide addition section (0.5 mg)
(4) β-1,3-xylo-oligosaccharide addition group (1.0 mg
In the above, DNA fragmentation has occurred in both (3) and (4).

Further, the chromatin DNA of HL60 cells was stained with a fluorescent dye and observed with a fluorescent microscope. Photograph 2 shown in FIG. 2 is a control. Photograph 3 shown in FIG.
1,3-xylo-oligosaccharide-added group (1.0 mg / ml), where it was observed that chromatin condensation occurred.

[0041]

Industrial Applicability According to the present invention, when producing β-1,3-xylo-oligosaccharide from algae, β-β-3-xylo-oligosaccharide having high physiological activity is used.
An enzyme and an algae for producing 1,3-xylo-oligosaccharide are provided, and a cancer cell apoptosis-inducing agent containing xylo-oligosaccharide as a main component is provided.

[Brief description of the drawings]

FIG. 1 is a photograph showing the results of agarose electrophoresis.

FIG. 2: Observation of HL60 cells by fluorescence microscope (no addition = control)

FIG. 3: Observation of HL60 cells with a fluorescence microscope (β-
1,3-xylo-oligosaccharide added section)

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 AF04 CA02 CA08 CA21 CC03 CD19 DA05 4C057 BB01 BB04 4C086 AA01 AA02 AA03 EA01 MA04 NA14 ZB26

Claims (3)

[Claims] The present invention has the following properties (1) and (2) obtained by treating a polysaccharide derived from the cell wall of algae with β-1,3-xylanase produced by a strain AX-4 of the genus Vibrio. A xylo-oligosaccharide-containing composition. (1) Average polymerization degree (total sugar content / reducing sugar content): 5 or less. (2) Protein content: below the detection limit. 2. A xylo-oligosaccharide having the following properties (1) and (2), which is obtained by treating a polysaccharide derived from the cell wall of a green alga, Sricopodaceae, Srikkogita with β-1,3-xylanase. Composition. (1) Average polymerization degree (total sugar content / reducing sugar content): 5 or less. (2) Protein content: below the detection limit. 3. A β-1,3-obtained from an algal cell wall.
A cancer cell apoptosis-inducing agent containing xylooligosaccharide as a main component, which is obtained by treating xylan with β-1,3-xylanase.