JP2008037797A - Adipocyte differentiation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an adipocyte differentiation inhibitor which excels in adipocyte differentiation inhibition and has high safety and stability. <P>SOLUTION: The adipocyte differentiation inhibitor has an acidic xylooligosaccharide having a uronic acid residue in the xylooligosaccharide molecule as an active ingredient, and the acidic xylooligosaccharide is a mixed composition of oligosaccharides having a different degree of polymerization of xylose and the average degree of polymerization is preferably 2.0-15.0. A preferred acidic xylooligosaccharide is the one obtained by enzymatically and/or physicochemically treating a lignocellulose material to obtain a composite of a xylooligosaccharide component and a lignin component, then subjecting the composite to acid hydrolysis treatment to obtain a xylooligosaccharide mixture, and separating a xylooligosaccharide having at least one uronic acid residue in the molecule as a side chain from the obtained xylooligosaccharide mixture. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an adipocyte differentiation inhibitor, and more particularly, to an adipocyte differentiation inhibitor that has an excellent adipocyte differentiation inhibitory action and is highly safe for humans and animals.

In recent years, with the Westernization of eating habits, diseases such as diabetes and arteriosclerosis are increasing due to excessive intake of fat and sugar. Preadipocytes present in the body are differentiated into fat cells by excessive intake of fat and sugar, and fat is accumulated in the fat cells. As fat accumulates in fat cells, fat cells become enlarged (hypertrophic fat cells). Fat is normally decomposed in fat cells, but in fat cells, fat accumulated in the cells is abnormally decomposed, causing diseases such as diabetes and arteriosclerosis.
Therefore, if differentiation from preadipocytes to adipocytes can be suppressed, it is considered possible to prevent diabetes, arteriosclerosis and the like.

  To date, cyclic peptides and gramineous plant extract components have been reported as components involved in the suppression of adipocyte differentiation (see Patent Literature 1 and Patent Literature 2). When ingested by humans, there are concerns such as side effects such as allergies and difficulty in ingestion due to odor and taste. Accordingly, there is a need for an adipocyte differentiation inhibitor that is highly safe to the human body and tasteless and odorless.

The present applicants have reported a method for producing acidic xylo-oligosaccharides and physiological actions of acidic xylo-oligosaccharides such as an intestinal environment-improving agent and an atopic dermatitis-improving agent (Patent Document 3, Patent Document 4 and (See Patent Document 5). However, there is no report on the action of acidic xylo-oligosaccharides to suppress adipocyte differentiation.
Japanese Patent Laid-Open No. 2005-220074 JP 2005-247695 A JP 2003-183303 A JP 2004-182609 A Japanese Patent Laid-Open No. 2004-210666

  The subject of this invention is providing the adipocyte differentiation inhibitor which is excellent in the adipocyte differentiation inhibitory effect, and has high safety | security and stability.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that an acidic xylo-oligosaccharide composition to which a uronic acid residue is added has an excellent adipocyte differentiation inhibitory action.
In order to solve the above problems, the following configuration is adopted.
That is, the first of the present invention is an “adipocyte differentiation inhibitor characterized by comprising an acidic xylo-oligosaccharide having a uronic acid residue in the xylo-oligosaccharide molecule as an active ingredient”.
According to a second aspect of the present invention, in the first aspect, the acidic xylo-oligosaccharide is a mixed composition of oligosaccharides having different degrees of xylose polymerization, and the average degree of polymerization is 2.0 to 15.0. An adipocyte differentiation inhibitor ”.
A third aspect of the present invention is the composite of the xylooligosaccharide component and the lignin component obtained by subjecting the lignocellulose material to enzymatic and / or physicochemical treatment in the first or second invention. Then, the complex is subjected to an acid hydrolysis treatment to obtain a xylo-oligosaccharide mixture, and from the resulting xylo-oligosaccharide mixture, xylo-oligosaccharide having at least one uronic acid residue as a side chain in one molecule is decomposed. An adipocyte differentiation inhibitor characterized by being obtained in the above ".
A fourth aspect of the present invention is the “adipocyte differentiation inhibitor characterized in that uronic acid is glucuronic acid or 4-O-methyl-glucuronic acid” in the first to third aspects of the present invention.

  According to the present invention, an adipocyte differentiation inhibitor having an excellent adipocyte differentiation inhibitory action and high safety and stability is provided.

  Hereinafter, although the structure of this invention is explained in full detail, this invention is not limited by this. The xylooligosaccharide refers to a xylose polymer that is a dimer of xylose, a xylotriose that is a trimer, or a tetramer to a 20-mer polymer of xylose. The acidic xylo-oligosaccharide used in the present invention means one having at least one uronic acid residue in one molecule of xylo-oligosaccharide.

  Moreover, the mixed composition of the oligosaccharide from which the polymerization degree of xylose differs may be sufficient. Generally, it is often obtained as such a composition in order to produce it from a natural product. Hereinafter, an acidic xylo-oligosaccharide composition will be mainly described.

  In the composition, the numerical value represented by the average degree of polymerization is an average value of the xylose chain length of the acidic xylooligosaccharide having a normal distribution, preferably 2.0 to 15.0, and more preferably 2.0 to 11.0. The difference between the upper limit and the lower limit of the xylose chain length is preferably 20 or less, and more preferably 10 or less. Uronic acid is known in nature as a component of a polysaccharide having various physiological activities such as pectin, pectinic acid, alginic acid, hyaluronic acid, heparin, chondroitin sulfate, and deltaman sulfate. The uronic acid in the present invention is not particularly limited, but glucuronic acid or 4-O-methyl-glucuronic acid is preferable.

If the acidic xylo-oligosaccharide composition as described above can be obtained, its production method is not particularly limited. (1) A method of extracting xylan from wood and enzymatically decomposing it (cellulase research group published, Cellulase Research) Newsletter Volume 16, issued on June 14, 2001, p17-26), (2) Lignocellulose material is treated enzymatically and / or physicochemically to obtain a complex of xylooligosaccharide component and lignin component, Then, the complex is subjected to an acid hydrolysis treatment to obtain a xylooligosaccharide mixture, and a method for separating xylooligosaccharide having at least one uronic acid residue as a side chain in one molecule from the obtained xylooligosaccharide mixture is mentioned. It is done.
In particular, the method (2) is preferable because it can produce a large amount of a polymer having a relatively high degree of polymerization, such as a 5-10 mer, at a low cost.

  The acidic oligosaccharide composition can be obtained through a hydrolysis process, a concentration process, a dilute acid treatment process, and a purification process using a lignocellulosic material derived from chemical pulp as a raw material. In the hydrolysis process, xylan in lignocellulose is selectively hydrolyzed with dilute acid treatment, high-temperature and high-pressure steam (cooking / explosion) treatment, or hemicellulase, and a high molecular weight complex composed of xylooligosaccharide and lignin is intermediated. Get as a body. In the concentration step, the xylooligosaccharide-lignin-like substance complex is concentrated by a reverse osmosis membrane or the like, and oligosaccharides having a low polymerization degree, low-molecular impurities, and the like can be removed. In the concentration step, a reverse osmosis membrane is preferably used, but ultrafiltration membrane, salting out, dialysis and the like are also possible. A lignin-like substance is released from the complex by the diluted acid treatment step of the obtained concentrated liquid, and a diluted acid-treated liquid containing acidic xylo-oligosaccharides and neutral xylo-oligosaccharides can be obtained. At this time, the lignin-like substance separated from the complex condenses and precipitates under acidic conditions, and can be removed by filtration using a ceramic filter or filter paper. In the dilute acid treatment step, acid hydrolysis is preferably used, but enzymatic degradation using lignin degrading enzyme or the like is also possible.

  The purification process includes an ultrafiltration process, a decolorization process, and an adsorption process. Some lignin-like substances remain in the solution as soluble polymers, but are removed by an ultrafiltration process, and most of impurities such as coloring substances are removed by a decolorization process using activated carbon. In the ultrafiltration step, an ultrafiltration membrane is preferably used, but reverse osmosis membrane, salting out, dialysis and the like are also possible. Acid xylo-oligosaccharides and neutral xylo-oligosaccharides are dissolved in the sugar solution thus obtained. Only an acidic xylo-oligosaccharide can be extracted from this sugar solution by an adsorption process using an ion exchange resin. First, the sugar solution is treated with a strong cation exchange resin to remove metal ions in the sugar solution. Next, sulfate ions and the like in the sugar solution are removed using a strong anion exchange resin. In this step, simultaneously with the removal of sulfate ions, a part of the organic acid, which is a weak acid, and the colored component are simultaneously removed. The sugar solution treated with the strong anion exchange resin is treated again with the strong cation exchange resin to further remove metal ions. Finally, it is treated with a weak anion exchange resin to adsorb acidic xylo-oligosaccharides to the resin.

By eluting the acidic xylo-oligosaccharide adsorbed on the resin with a low-concentration salt (NaCl, CaCl ₂ , KCl, MgCl _2, etc.), an acidic xylo-oligosaccharide solution free from impurities can be obtained. From this solution, for example, a powder of a white acidic xylo-oligosaccharide composition can be obtained by spray drying or freeze-drying treatment.

  The merit of the above-mentioned production method of acidic xylooligosaccharide composition using chemical pulp-derived lignocellulose as a raw material and high molecular weight complex consisting of xylooligosaccharide and lignin as an intermediate is economical and acidic with high average polymerization degree of xylose. The xylo-oligosaccharide composition is easily obtained. The average degree of polymerization can be changed, for example, by adjusting dilute acid treatment conditions or treating with hemicellulase again. In addition, by changing the salt concentration of the eluate used for elution of the weak anion exchange resin, acidic xylo-oligosaccharide compositions having different numbers of uronic acid residues bound per molecule can be obtained. Furthermore, it is also possible to obtain an acidic xylo-oligosaccharide composition in which the uronic acid binding site is limited to the terminal by acting an appropriate xylanase or hemicellulase.

  Although the intake form of the adipocyte differentiation inhibitor containing the acidic xylo-oligosaccharide of the present invention may be taken directly, it can be added to beverages or added to foods. When ingested directly, it may be pulverized or tableted by tableting.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by this. First, an outline of each measurement method and Preparation Examples 1 to 3 of acidic xylo-oligosaccharides (UX10, UX5, UX2) contained as active ingredients in the present invention are shown.
<Outline of measurement method>
(1) Quantification of total sugar amount The total sugar amount is prepared using D-xylose (manufactured by Wako Pure Chemical Industries, Ltd.) with a calibration curve, and the phenol-sulfuric acid method (quantitative method for reducing sugar, published by Academic Publishing Center). And quantified.
(2) Quantification of reducing sugar amount The reducing sugar amount was prepared by using D-xylose (manufactured by Wako Pure Chemical Industries, Ltd.) as a calibration curve, and the Sommoji-Nelson method (quantitative method for reducing sugar, published by Academic Publishing Center). And quantified.
(3) Determination of the amount of uronic acid The uronic acid was prepared using a calibration curve using D-glucuronic acid (manufactured by Wako Pure Chemical Industries, Ltd.), with the carbazole sulfate method (quantitative method for reducing sugar, published by the Academic Publishing Center). Quantified.
(4) Determining the average degree of polymerization Keep the sample sugar solution at 50 ° C., centrifuge at 15,000 rpm for 15 minutes to remove insoluble matter, and use the total sugar amount in the supernatant solution as the reducing sugar amount (both converted to xylose) The average degree of polymerization was determined by dividing by.
(5) Method for analysis of acidic xylooligosaccharides:
The oligosaccharide chain distribution was analyzed using an ion chromatograph (Dionex, analytical column: Carbo Pac PA-10). A 100 mM NaOH solution is used as a separation solvent, and sodium acetate is added to the above-mentioned separation solvent so as to have a concentration of 500 mM as an elution solvent, so that the separation ratio: elution solvent = 10: 0 to 4: 6 in the solution ratio. These linear gradients were combined and separated. From the obtained chromatogram, the difference between the upper limit and the lower limit of the xylose chain length was determined.
(6) Determination of the number of uronic acid residues per molecule of oligosaccharide Keep the sample sugar solution at 50 ° C., centrifuge at 15,000 rpm for 15 minutes to remove insoluble matter, and the amount of uronic acid in the supernatant ( The number of uronic acid residues per oligosaccharide molecule was determined by dividing (D-glucuronic acid equivalent) by the reducing sugar amount (xylose equivalent).
(7) Definition of enzyme titer Kaxylan (manufactured by Sigma) was used for measuring the activity of xylanase used as an enzyme. Enzyme titer is defined by measuring the reducing power of reducing sugar obtained by xylanase degrading xylan using the DNS method (quantitative method for reducing sugar, published by Academic Publishing Center). The amount of enzyme that generates a reducing power corresponding to xylose was 1 unit.

<Preparation Example: Preparation Example of Acidic Xylooligosaccharide>
<Preparation Example 1>
Oxygen delignified pulp slurry (kappa number 9.6, pulp viscosity 25.1 cps) was obtained from mixed hardwood chips (domestic hardwood 70%, eucalyptus 30%) as a raw material through kraft cooking and oxygen delignification processes. After the pulp was filtered and washed from the slurry, the following enzyme treatment with xylanase was performed using a pulp slurry adjusted to a pulp concentration of 10% and pH 8.
After adding xylanase produced by Bacillus sp. S-2113 strain (National Institute of Advanced Industrial Science and Technology, Patent Microorganism Deposit Center, Deposited Strain FERM BP-5264) to 1 unit / g of pulp, 120 at 60 ° C. Treated for minutes. Thereafter, the pulp residue was removed by filtration to obtain 1050 L of an enzyme treatment liquid.
Next, the obtained enzyme treatment solution was subjected to a concentration step, a dilute acid treatment step, and a purification step in this order.
In the concentration step, a concentrated solution (40-fold concentration) was prepared using a reverse osmosis membrane (Nonto Denko Corporation, RONTR-7410). In the dilute acid treatment step, the pH of the obtained concentrated solution was adjusted to 3.5 and then heat-treated at 121 ° C. for 60 minutes to form precipitates of polymer contaminants such as lignin. Further, the precipitate was removed by ceramic filter filtration to obtain a diluted acid treatment solution.
In the purification process, the ultrafiltration / decolorization process and the adsorption process were performed in this order. In the ultrafiltration / decolorization step, after passing the dilute acid treatment solution through an ultrafiltration membrane (Osmonics, molecular weight cut off 8000), addition of 770 g of activated carbon (Wako Pure Chemical Industries, Ltd.) and ceramic filter filtration To obtain a decolorization treatment solution. In the adsorption process, the decolorization treatment liquid is a strong cation exchange resin (PK218 manufactured by Mitsubishi Chemical Corporation), a strong anion exchange resin (PA408 manufactured by Mitsubishi Chemical Corporation), and a strong cation exchange resin (manufactured by Mitsubishi Chemical Corporation). PK218) Each was sequentially passed through a column packed with 100 kg, and then applied to a column packed with 100 kg of a weak anion exchange resin (WA30 manufactured by Mitsubishi Chemical Corporation). The solution eluted from the weak anion exchange resin-packed column with a 75 mM NaCl solution was spray-dried to obtain acidic xylooligosaccharide powder (total sugar amount 353 g, recovery rate 13.1%). Hereinafter, this acidic xylo-oligosaccharide is referred to as UX10. According to the measurement method described above, UX10 was a sugar composition compound having an average degree of polymerization of 10.3, a difference between the upper limit and the lower limit of the xylose chain length of 10, and one uronic acid residue per molecule of acidic xylooligosaccharide.
<Preparation Example 2>
To 1160 ml of the diluted acid treatment solution obtained in the same manner as in Preparation Example 1, 28 mg of Sumiteam X (Xylanase manufactured by Shin Nippon Chemical Industry Co., Ltd.) was added and reacted at 40 ° C. for 20 hours. After inactivating the enzyme by heat treatment (70 ° C., 1 hour), the Sumiteam X treatment solution was subjected to the same purification step as in Preparation Example 1 to produce acid xylo-oligosaccharide powder (total sugar amount 21.3 g, recovery rate 22. 2%). Hereinafter, this acidic xylo-oligosaccharide is referred to as UX5. According to the measurement method described above, UX5 was a sugar composition compound having an average degree of polymerization of 4.8, a difference between the upper limit and the lower limit of the xylose chain length of 9, and one uronic acid residue per molecule of acidic xylooligosaccharide.
<Preparation Example 3>
To 100 ml of 10% aqueous solution of UX10 obtained from Preparation Example 1, 50 mg of Sumiteam X (Xylanase manufactured by Shin Nippon Chemical Industry Co., Ltd.) was added, reacted at 60 ° C. for 20 hours, and then 10 g of weak anion exchange resin (WA30). To a column packed with After the column was washed with water, the solution eluted with 75 mM NaCl solution was freeze-dried to obtain acidic xylooligosaccharide powder (total sugar amount 2.1 g, recovery rate 21%). Hereinafter, this acidic xylo-oligosaccharide is referred to as UX2. According to the measurement method described above, UX2 was a saccharide composition compound having an average degree of polymerization of 2.3, a difference between the upper limit and the lower limit of the xylose chain length of 2, and one uronic acid residue per molecule of acidic xylooligosaccharide.

<Example 1>
An aqueous solution containing three types of acidic xylo-oligosaccharides (UX2, UX5, UX10) having different average polymerization degrees obtained by the above-described adjustment examples was prepared. Using this acidic xylooligosaccharide aqueous solution, the adipocyte differentiation inhibitory action was measured by the following method.
Subcultured mouse-derived preadipocytes (3T3-L1) were suspended in DME medium (containing 10% calf serum) (cell density 4 × 10 ⁴ / ml), and 200 μl was added to each well of a 96-well plate. ° C., and cultured under the conditions of 5% CO _2. Two days after the start of the culture, the medium was replaced with a DME medium (containing 10% calf serum) containing dexamethasone (0.5 mM), methylisobutylxanthine (1 μM) and insulin (10 μg / ml), and cultured under the same conditions as described above. Four days after the start of the culture, the medium was replaced with a DME medium (containing 10% calf serum) containing insulin (10 μg / ml) and the medium was changed every two days under the same conditions as described above. Twelve days after the start of the culture, the medium was removed, oil red was added to the cells, and neutral fat was stained. The stained dye was extracted from the cells and the absorbance (520 nm) was measured. In addition, it added to the culture medium so that the final concentration of acidic sugar (UX2, UX5, UX10) might be 1%, 0.1%, and 0.01% from 2 days after the start of the culture until the end of the culture. Distilled water was added to the medium as a control. In addition, it was confirmed in advance using an MTT assay kit (R & D SYSTEM) that acidic sugars (UX2, UX5, UX10) were not cytotoxic to mouse-derived preadipocytes (3T3-L1). The results are shown in Table 1.

In the test group to which acidic xylo-oligosaccharide (UX2, UX5, UX10) was added, the intracellular triglyceride content was lower than that in the control.

<Example 2>
<Safety test>
As a safety test for acidic xylooligosaccharides, a skin irritation test and an acute oral toxicity test were conducted.

<Skin irritation test>
100 μl of 2% by weight acidic xylo-oligosaccharide (UX2, UX5, UX10) aqueous solution was applied to the back skin of C3H mice (male, 6 weeks old, manufactured by Charles River Japan) after hair removal for about 1 month. , Was applied continuously (once / day, 10 animals per group). Abnormalities such as erythema, edema, and inflammation were not particularly observed in the mouse back skin during the application period and 2 weeks after the application was completed. In addition, compared with the blank (water application group), no significant difference (P <0.05) was observed in the body weight transition.

<Acute oral toxicity test>
A 60% by mass acidic xylo-oligosaccharide (UX2, UX5, UX10) aqueous solution was orally administered to each ICR mouse (male, 6 weeks old, manufactured by Charles River Japan Co., Ltd.) using a stomach tube (dosage amount). : 5 g / mouse body weight 1 kg, 10 in each group). There were no deaths until 2 weeks after administration. In addition, there was no significant difference (P <0.05) in weight transition compared to the blank (water administration group).

<Example 3>
<Stability test>
A 60% by mass aqueous acid xylo-oligosaccharide (UX2, UX5, UX10) aqueous solution was prepared and stored at room temperature. The acid xylooligosaccharide aqueous solution immediately after preparation and after storage for 1 month was analyzed by ion chromatogram. The chromatogram pattern of the sample after storage for 1 month did not change compared to the sample immediately after preparation. Moreover, the difference in the area of each peak in the chromatogram was less than 5% between the sample after storage for 1 month and the sample immediately after preparation.

The adipocyte differentiation inhibitor of the present invention can be mixed with enteral nutrients or pharmaceuticals and used as medical foods. It can also be provided as a quasi-drug or pharmaceutical by mixing with ingredients generally used in quasi-drugs or pharmaceuticals. Note that food, medical foods and pharmaceuticals can be used not only as humans but also as foods for dogs and cats and functional foods.

Claims (4)

An adipocyte differentiation inhibitor comprising, as an active ingredient, an acidic xylo-oligosaccharide having a uronic acid residue in the xylo-oligosaccharide molecule. The adipocyte differentiation inhibitor according to claim 1, wherein the acidic xylo-oligosaccharide is a mixed composition of oligosaccharides having different degrees of polymerization of xylose and having an average degree of polymerization of 2.0 to 15.0. . The acidic xylo-oligosaccharide is “a lignocellulosic material is enzymatically and / or physicochemically treated to obtain a complex of xylo-oligosaccharide component and lignin component, and then the complex is subjected to an acid hydrolysis treatment to form a xylooligosaccharide mixture. The obtained and obtained xylo-oligosaccharide mixture is obtained by decomposing xylo-oligosaccharide having at least one uronic acid residue as a side chain in one molecule ". 2. The adipocyte differentiation inhibitor according to 2. The adipocyte differentiation inhibitor according to any one of claims 1 to 3, wherein the uronic acid is glucuronic acid or 4-O-methyl-glucuronic acid.