JP7008324B2 - Bile acid adsorbent and adiponectin secretagogue - Google Patents

Description

The present invention relates to a bile acid adsorbent and an adiponectin secretagogue.

Bile acids are synthesized from cholesterol in the liver, assisting in the digestion and absorption of lipids in the intestine, and then about 95% are reabsorbed from the lower ileum of the intestine and returned to the liver via the portal vein and re-absorbed. It's being used. By adsorbing bile acid and suppressing its reabsorption, the intestinal circulation of bile acid is inhibited and the new synthesis of bile acid from cholesterol is promoted, so that bile acid reduces the cholesterol level in the liver and blood. It is well known.

However, recent studies have shown that in addition to the digestive and absorptive functions of lipids and cholesterol, bile acids only regulate the synthetic absorption of bile acids themselves by binding to bile acid-specific receptors. Instead, it has become clear that it controls sugar / lipid metabolism and energy metabolism. If it becomes possible to increase energy consumption by this ability to enhance energy metabolism, it is expected that treatment of obesity and diabetes will be advanced with few side effects.

Dietary fiber is a typical food of an ingredient that effectively excretes bile acids. Dietary fiber is classified into water-soluble and insoluble, and in particular, water-soluble dietary fiber has an action of binding to bile acid and excreting bile acid in the same manner as a bile acid adsorbent. In particular, seaweed is rich in dietary fiber. Alginic acid, which is a typical water-soluble dietary fiber contained in wakame seaweed and kelp, promotes the excretion of bile acids, further suppresses the rise of blood glucose level, suppresses cholesterol and obesity cells, and is expected to improve diabetes. It has been reported (Non-Patent Document 1).

Non-Patent Document 2 shows the results of examining the glycine- or taurine-conjugated adsorption action of cholic acid, chenodeoxycholic acid, and deoxycholic acid by water-soluble and insoluble dietary fibers of wakame seaweed, hijiki, kelp, and neopyropia yezoensis. In Non-Patent Document 3, seaweeds such as wakame, kombui, hijiki, and seaweed were used as samples to investigate their binding to bile acids. However, it is stated that the adsorption of all bile acids was generally higher than that of insoluble dietary fiber.

Adiponectin is a kind of cytokine (adipocytokine) secreted from adipocytes, and the amount of adiponectin secreted decreases with the enlargement of adipocytes. The resulting hypoadiponectinemia can be deeply involved in the onset or progression of obesity, insulin resistance, metabolic syndrome and the like. Examples of foodstuffs that increase adiponectin secretion include seaweeds containing dietary fiber.

For example, Porphyran is a water-soluble dietary fiber derived from the red alga Neopyropia yezoensis and is also a type of sulfated polysaccharide. In Non-Patent Document 4, when porphyran is fed to KK-Ay mice of type II diabetes model, the blood insulin concentration and insulin resistance index are significantly decreased as compared with the control group fed with a diet containing cellulose. And a significant increase in adiponectin secretion was shown. In this paper, the decrease in insulin resistance index and the increase in porphyran secretion are discussed in relation to changes in the type and amount of intestinal bacteria in mice.

By the way, it has been reported that dietary fiber contained in seaweed is a polysaccharide, and that the physiological action of the polysaccharide has a correlation with the molecular weight (Non-Patent Documents 5 and 6).

Patent Document 1 describes a method for producing a seaweed high-temperature extraction composition by heating seaweeds to a high temperature under pressure in an aqueous medium in which carbon dioxide coexists, and the method is used to obtain seaweeds. It has been confirmed that the contained polysaccharides such as alginic acid have a low molecular weight, and that the product obtained by such a method (low molecular weight polysaccharide) has a low molecular weight, and its physiological activity is to lower blood pressure. It is further stated that. In Patent Document 2, chondroitin sulfate is hydrolyzed by heating it to a high temperature under pressure in an aqueous medium in which carbon dioxide coexists, and chondroitin sulfate whose molecular weight is reduced by hydrolysis exhibits α-glucosidase inhibitory activity. It is stated to show. Patent Document 3 states that alginic acid is preferably obtained from seaweed (brown algae), that alginic acid is hydrolyzed by heating it to a high temperature under pressure in an aqueous medium in which carbon dioxide coexists, and as such. It is described that the anti-diabetic effect was confirmed with hydrolyzed alginic acid.

A search for a better material for bile acid adsorption has been made. In addition, a search for a better material for adiponectin secretion promoting action has been made.

Japanese Unexamined Patent Publication No. 2006-320320 Japanese Unexamined Patent Publication No. 2012-12430 JP-A-2015-333772

New Diet Therapy, 30 (4), pp. 45-51, 2015 Journal of Japanese Society of Fisheries Science, Vol. 68 (4), pp. 579-581, 2002 Effect of seaweed dietary fiber and bile acid binding and lipid digestion and absorption (https://kaken.nii.ac.jp/d/p/08660247.ja.html) J. Nutr. Sci. Votaminol, Vol. 58, pp. 14-19, 2012 International Journal of Biological Macromolecules, Vol. 45, pp. 42-47, 2009 Carbohydrate Polymers, Vol. 87, pp. 1206-1210, 2012

An object of the present invention is to provide a material exhibiting a bile acid adsorbing action that can be used for foods and a food containing the material.

It is also an object of the present invention to provide a material having an adiponectin secretagogue action that can be used in foods and a food containing the material.

The present invention provides a bile acid adsorbent containing a hydrolyzate of a polysaccharide derived from seaweed.

Regarding the bile acid adsorbent, in one embodiment, the polysaccharide derived from the seaweed contains a sulfated polysaccharide.

Regarding the bile acid adsorbent, in one embodiment, the hydrolyzate is
A hydrolyzate derived from arame and having a weight average molecular weight in the range of 10,000 Da to 306,000 Da;
A hydrolyzate derived from Gagome and having a weight average molecular weight in the range of 10,000 Da to 860000 Da;
A hydrolyzate derived from hijiki seaweed and having a weight average molecular weight in the range of 10,000 Da to 268,000 Da;
A hydrolyzate derived from Sargassum and having a weight average molecular weight in the range of 10000 Da to 332000 Da;
Hydrolyzate derived from kelp and having a weight average molecular weight in the range of 10000 Da to 713000 Da;
A hydrolyzate derived from wakame seaweed and having a weight average molecular weight in the range of 10000 Da to 564000 Da;
Hydrolyzate derived from Sargassum horneri and having a weight average molecular weight in the range of 10000 Da to 272000 Da;
Hydrolysates derived from turmo and having a weight average molecular weight in the range of 10000 Da to 112000 Da;
A hydrolyzate derived from coontail and having a weight average molecular weight in the range of 10,000 Da to 180,000 Da;
Hydrolysates derived from mekabu and having a weight average molecular weight in the range of 10,000 Da to 501,000 Da;
Hydrolysates derived from Cladosiphon okalui and having a weight average molecular weight in the range of 10000 Da to 192000 Da;
A hydrolyzate derived from giraffe and having a weight average molecular weight in the range of 10,000 Da to 276,000 Da;
A hydrolyzate derived from black rose paste and having a weight average molecular weight in the range of 10,000 Da to 291,000 Da;
A hydrolyzate derived from Meristotheca papulosa and having a weight average molecular weight in the range of 10000 Da to 196000 Da;
A hydrolyzate derived from Gracilaria and having a weight average molecular weight in the range of 10,000 Da to 328,000 Da;
A hydrolyzate derived from Monostroma nitidum and having a weight average molecular weight in the range of 10,000 Da to 280000 Da; or a combination of two or more of these.

Regarding the bile acid adsorbent, in one embodiment, the seaweed is hijiki seaweed, and the weight average molecular weight of the hydrolyzate is in the range of 10,000 Da to 268,000 Da.

The present invention is a method for producing a bile acid adsorbent, which is derived from the seaweed by pressurizing and heating a seaweed or an aqueous medium containing a polysaccharide derived from the seaweed in the presence of carbon dioxide gas. Provided is a method comprising the step of obtaining a polysaccharide hydrolyzate.

The present invention is also a method for producing a bile acid adsorbent, in which a seaweed or a polysaccharide derived from the seaweed is heated, pressurized and heated in a vinegar medium to hydrolyze the polysaccharide derived from the seaweed. Provided is a method including a step of obtaining a product.

The present invention further provides an adiponectin secretagogue containing a hydrolyzate of a polysaccharide derived from seaweed.

Regarding the adiponectin secretagogue, in one embodiment, the polysaccharide derived from the seaweed contains a sulfated polysaccharide.

Regarding the adiponectin secretagogue, in one embodiment, the seaweed is hijiki and the weight average molecular weight of the hydrolyzate is in the range of 10,000 Da to 268,000 Da.

The present invention is a method for producing an adiponectin secretion-promoting agent, which is derived from the seaweed by pressurizing and heating a seaweed or an aqueous medium containing a polysaccharide derived from the seaweed in the presence of carbon dioxide gas. Provided is a method comprising the step of obtaining a hydrolyzate of a polysaccharide.

The present invention is also a method for producing an adiponectin secretion-promoting agent, wherein a seaweed or a polysaccharide derived from the seaweed is heated, pressurized and heated in a vinegar medium to hydrolyze the polysaccharide derived from the seaweed. Provided is a method including a step of obtaining a product.

The present invention further provides a food containing the above-mentioned bile acid adsorbent.

The present invention further provides a food containing the above-mentioned adiponectin secretagogue.

The present invention further provides a diabetes ameliorating agent, which comprises a hydrolyzate of a polysaccharide derived from hijiki and whose weight average molecular weight of the hydrolyzate is in the range of 10,000 Da to 268,000 Da.

The present invention further provides a constipation ameliorating agent, which comprises a hydrolyzate of a polysaccharide derived from hijiki and whose weight average molecular weight of the hydrolyzate is in the range of 10,000 Da to 268,000 Da.

The present invention also provides a food for preventing or treating diabetes, which comprises the above-mentioned diabetes improving agent.

The present invention also provides a constipation-improving food containing the above-mentioned constipation-improving agent.

INDUSTRIAL APPLICABILITY According to the present invention, a material exhibiting a bile acid adsorption effect that can be used in foods is provided. Further, according to the present invention, there is provided a material showing an adiponectin secretagogue effect that can be used in foods. Further, according to the present invention, there is provided a material showing a bile acid adsorption effect, an adiponectin secretion promoting effect, a diabetes improving effect and a constipation improving effect.

It is a graph which showed the adsorption rate of various seaweed powder samples in a bile adsorption test as a relative value when the adsorption rate of cholestyramine is 100%. It is a graph which showed the adsorption rate of various seaweed powder samples in the sodium (Na cholic acid) adsorption test as a relative value when the adsorption rate of cholestyramine is 100%. It is a graph which shows the dry weight of the feces excreted 24 hours after administration for the low-molecular-weight Hijiki group and the untreated Hijiki group. It is a graph which shows the amount of bile acid in the dry feces of the feces excreted 24 hours after administration about the low molecular weight hijiki group and the untreated hijiki group. It is a graph which shows the adiponectin level at the 3rd week of administration for the small molecule Hijiki group and the untreated Hijiki group. It is a graph which shows the hemoglobin A1c value at the 3rd week of administration for the small molecule Hijiki group and the untreated Hijiki group. It is a graph which shows the blood glucose level at the end of administration for the low-molecular-weight Hijiki group and the untreated Hijiki group. It is a graph which shows the insulin level at the end of administration for the low-molecular-weight Hijiki group and the untreated Hijiki group. It is a graph which shows the insulin resistance index calculated from the blood glucose level and the insulin level at the end of administration for a small molecule Hijiki group and an untreated Hijiki group.

Hereinafter, the present invention will be described in detail. In addition, in this specification, "Da" shown as a unit of a molecular weight means "Dalton".

As used herein, the term "polysaccharide derived from seaweed" refers to any polysaccharide contained in seaweed and its salt, for example, alginic acid, fucoidan, agarose, carrageenan, porphyran, and ramnan sulfate, and salts thereof. Can be mentioned. The "polysaccharide derived from seaweed" may be a single polysaccharide or a mixture of different polysaccharides.

As used herein, "alginic acid" is an acidic polysaccharide having a carboxyl group, and is a linear polymer in which β-D-mannuronic acid and α-L-gluuronic acid are 1,4 glycosidic bonded and a salt thereof. If it is such a thing, it is not particularly limited. At least a part of β-D-mannuronic acid may be a C-5 epimer.

As used herein, the term "fucoidan" is a sulfated polysaccharide containing L-fucose (6-deoxy-L-galactose) as a main constituent sugar. Fucoidan mainly contains 1,2 glycoside bonds of L-fucose 4 sulfuric acid, and may contain 1,3 bonds and / or 1,4 bonds. The composition of fucoidan's constituent sugars depends on the type of seaweed from which they are derived, which may also include uronic acid and galactose, as well as small amounts of xylose, mannose, rhamnose and the like.

Regarding the structure of fucoidan, the molar ratio of constituent sugar and sulfuric acid content is, for example, in the sugar composition of fucoidan derived from mozuku (for example, mozuku fucoidan from Okinawa, manufactured by Tanglewood: AH fucoidan 85), L-fucose: D-galactose: D-glucose: D-mannose: D-xylose, D-acetylated glucose: sulfuric acid = 5: 1: 1: 0.5: 0.5: 2: 7. The sulfated polysaccharide contained in "Hijiki from Miura, Kanagawa Prefecture" shown as an example of Hijiki in the present specification is a kind of fucoidan. Fucoidan contained in such hijiki can be referred to as hijiki fucoidan.

In the present specification, "agarose" is contained in gelidiaceae and the like of red algae, and its chemical structure is 1,3 bond β-D-galactose and 1,4 bond 3,6-anhydro-α-L-. It is a series of galactose repeatedly. Agarose is generally a neutral polysaccharide, but may have a small amount of ester sulfuric acid.

As used herein, the term "carrageenan" refers to a series of D-galactose in which α-1,3 bonds or β-1,4 bonds are alternately repeated. Carrageenan is a sulfated polysaccharide and most galactose units on the carrageenan molecule can have ester sulfate.

As used herein, "porphyran" is a sulfated polysaccharide consisting of a derivative of galactose. That is, L-galactose-6-sulfate, the methylated sugar of the hydroxyl group at the 6-position, 3,6-anhydro-L-galactose and D-galactose were alternately bound at α-1,3 and β-1,4. It has been partially replaced with D-galactose-6-O-methylgalactose. Porphyran is a sulfated polysaccharide, similar in structure to agarose, the major polysaccharide in agar.

As used herein, "ramnan sulfuric acid" is a polysaccharide containing L-rhamnose as a main constituent sugar, and has ester sulfuric acid as a part of its structure. The molar ratio of the constituent sugars of ramnan sulfuric acid to the sulfate group is, for example, L-rhamnose: D-acetylated glucose: D-xylose: D-glucose: D-galactose: sulfuric acid = 7: 1: 0.5: 0.1. : 0.5: 5.

As used herein, the term "sulfated polysaccharide" is a general term for sugars in which at least a part of the hydroxyl groups of the sugar is modified with ester sulfuric acid. For example, when the main skeleton of sugar is fucose, it is generally called fucoidan.

The salt of "polysaccharide derived from seaweed" is one or more of the groups in which hydrogen ions (H ⁺ ) such as carboxy group (-COOH) and hydroxyl group (-OH) can be dissociated. Is substituted with other cations to form a salt, and the number of salt forming sites may be only one or two or more, and all the groups in which hydrogen ions can be dissociated are salts. May be formed. Examples of the salt of the polysaccharide include sodium salt, potassium salt, ammonium salt, calcium salt, magnesium salt, aluminum salt and the like. When there are a plurality of sites for forming salts of the polysaccharide, the plurality of salts may be the same, all may be different, or only some of them may be different. For example, both the carboxy group and the hydroxyl group in the polysaccharide may form a salt. The cation forming the salt may be only one kind, two or more kinds, and when there are two or more kinds, the combination and the ratio thereof are not particularly limited. For example, among a plurality of carboxy groups existing in the polysaccharide molecule, those forming a sodium salt and those forming a potassium salt may coexist.

As used herein, the term "seaweed" is a general term for algae in the marine species group, and examples thereof include algae that can be easily discriminated with the naked eye. Seaweeds include brown algae, red algae and green algae. Seaweeds are not limited to their place of harvest or time of harvest. The "seaweed" in the present specification includes, for example, brown algae such as Arame, Gagomekonbu (also referred to as "Gagome" in the present specification), Hijiki, Hondawara, Macombu, Wakame, Akamoku, Tsurumo, Matsumo, Mekabu, and Mozuku. Examples of red algae include giraffe, black-and-white seaweed (also known as "Susabinori"), Tosakanori, Ogonori, etc.; and examples of green algae include human exa (also known as "sea lettuce"). In one embodiment, the seaweed is a seaweed selected from the group consisting of arame, gagome, hijiki, sargassum, kelp, wakame, akamoku, tsurumo, matsumo, mekabu, mozuku, giraffe, kurobaranori, tosakanori, ogonori and human sardine. .. In one embodiment, the seaweed is Hijiki. In the examples in the present specification, "Hijiki from Miura, Kanagawa Prefecture" is used as an example, but the production area of Hijiki is not limited to this.

A "hydrolyzed product" of a polysaccharide derived from seaweed (also referred to herein as a "hydrolyzed product") can be obtained by any method that causes hydrolysis. The hydrolysis method is not particularly limited. Hydrolysates of seaweed-derived polysaccharides can be produced by heating, stirring, etc. in the drying, crushing, and extraction operations of the raw material seaweed. In certain embodiments, the hydrolysis methods include, for example, (1) a method of pressurizing and heating a seaweed or an aqueous medium containing a polysaccharide derived from the seaweed in the presence of carbon dioxide gas, and (2). ) A method of heating, pressurizing and heating seaweed or a polysaccharide derived from the seaweed in a vinegar medium can be mentioned.

The "seaweed" in the above methods (1) and (2) is preferably a powder, and the average particle size of the powder is, for example, 1 μm to 10 mm, preferably 10 μm to 1 mm. Such powders can be prepared by a grinding technique commonly used by those skilled in the art (eg, the method described in Preparation Example 1 below). In the above methods (1) and (2), as the "polysaccharide derived from seaweed", a polysaccharide fraction previously extracted from seaweed, an isolated polysaccharide, or an isolated and purified polysaccharide can be used. .. Such a polysaccharide fraction or polysaccharide can be obtained by hot water extraction of seaweed after ethanol precipitation (eg, the method described in Preparation Example 2 below). The polysaccharide fraction or polysaccharide can be pulverized by drying or the like, if necessary.

In addition, in this specification, the term "seaweed or polysaccharide derived from the seaweed" means "seaweed" and "the seaweed" in addition to the case of either "seaweed" or "polysaccharide derived from the seaweed". It is also intended to include cases where it is combined with "polysaccharides derived from seaweed".

In the method (1) above, "in the presence of carbon dioxide gas" means that carbon dioxide gas is blown into an aqueous medium (for example, water) before adding seaweed or a polysaccharide derived from the seaweed (for example). It can be done by bubbling). "In the presence of carbon dioxide gas" is preferably in a state where the carbon dioxide gas is saturated in the aqueous medium.

In the method (1) above, the seaweed or the polysaccharide derived from the seaweed is derived from the ratio of the weight of the polysaccharide to the total of the weight of the aqueous medium and the weight of the polysaccharide (that is, derived from the seaweed) with respect to the aqueous medium. The concentration of the polysaccharide) can be added so as to be preferably 0.05 to 20% by weight, more preferably 0.1 to 10% by weight. When the concentration of the polysaccharide derived from seaweed is 0.05% by weight or more, the yield of the hydrolyzate is improved, and when the concentration of the polysaccharide derived from seaweed is 20% by weight or less, it is an aqueous medium. Polysaccharides derived from seaweed can be more uniformly dispersed or dissolved therein, whereby the hydrolysis reaction proceeds more efficiently and the molecular weight can be controlled more precisely.

In the method (2) above, the "vinegar medium" refers to a medium in which an aqueous medium (for example, water) and vinegar are mixed, for example, before adding a polysaccharide derived from seaweed to the aqueous medium (for example, water). Can be prepared by adding vinegar to an aqueous medium. Vinegar refers to vinegar to be edible, and refers to vinegar that falls under either the Japanese Agricultural Standards (JAS) or the vinegar quality labeling standards announced by the Consumer Affairs Agency. Vinegar includes vinegar and synthetic vinegar, and these vinegar and synthetic vinegar are also defined in the vinegar quality labeling standards. Examples of the brewed vinegar include grain vinegar (for example, rice vinegar, rice black vinegar, barley black vinegar, etc.) and fruit vinegar (for example, apple vinegar, grape vinegar, etc.). The acidity of vinegar is preferably in the range of 4-5%, and one example is an acidity of 4.2% as found in grain vinegar. For example, in the case of vinegar having an acidity of 4.2%, the concentration of vinegar in the aqueous medium is preferably 0.1 to 30% by volume, more preferably 2 to 20% by volume. Such acidity and concentration can improve the yield of the hydrolyzate and the precision of molecular weight control by the hydrolysis reaction.

In the method (2) above, the seaweed or the polysaccharide derived from the seaweed is derived from the ratio of the weight of the polysaccharide to the total of the weight of the vinegar medium and the weight of the polysaccharide (that is, derived from the seaweed) with respect to the vinegar medium. The concentration of the polysaccharide) can be added so as to be preferably 0.05 to 20% by weight, more preferably 0.1 to 10% by weight. When the concentration of the polysaccharide derived from seaweed is 0.05% by weight or more, the yield of the hydrolyzate is improved, and when the concentration of the polysaccharide derived from seaweed is 20% by weight or less, it is an aqueous medium. Polysaccharides derived from seaweed can be more uniformly dispersed or dissolved therein, whereby the hydrolysis reaction proceeds more efficiently and the molecular weight can be controlled more precisely.

In the above methods (1) and (2), the temperature during heating in the hydrolysis reaction is preferably 80 ° C. or higher, more preferably 80 ° C. to 130 ° C., and 90 ° C. to 120 ° C. It is more preferable to have. In the method (2) above, the temperature during heating in the hydrolysis reaction may depend on the heating time, but the vinegar medium is not pressurized, for example, at a temperature of 80 ° C. or higher and lower than 100 ° C. It may be imparted, or it may be imparted with a temperature of 100 ° C. or higher (for example, 100 ° C. to 130 ° C.) under pressure. In the method (1) above, it is preferable to apply a temperature of 100 ° C. or higher (for example, 100 ° C. to 130 ° C.) to the aqueous medium under pressure as the temperature during heating in the hydrolysis reaction. When the heating temperature is 80 ° C. or higher, the rate of hydrolysis reaction is improved, and when the heating temperature is 130 ° C. or lower, the amount of by-products is suppressed and the yield of decomposition products is suppressed. Can be improved.

In the above methods (1) and (2), for example, when the heating temperature is 105 ° C to 130 ° C, more preferably 110 ° C to 120 ° C, the pressure at pressurization is 0.05 MPa or more. It is preferably 0.12 MPa to 0.27 MPa, more preferably 0.14 MPa to 0.2 MPa. When the pressure at the time of pressurization is 0.05 MPa or more, the rate of hydrolysis reaction is improved, and when the pressure at the time of pressurization is 0.27 MPa or less, the amount of by-products is suppressed and decomposition is performed. The yield of goods is improved.

The heating time may be appropriately set in consideration of conditions such as the temperature at the time of heating. When the temperature at the time of heating is within the above range, it is preferably 5 minutes to 6 hours, more preferably 10 minutes to 4 hours, and particularly preferably 15 minutes to 2 hours. More specifically, the heating time can be determined depending on the type of seaweed and the molecular weight of the decomposition product obtained by the hydrolysis method.

The rate of hydrolysis reaction of polysaccharides derived from seaweed is particularly susceptible to the temperature at the time of heating and the initial pressure (initial pressure) after the start of hydrolysis, so these conditions should be adjusted as appropriate. Therefore, the molecular weight of the decomposition product can be easily adjusted.

The pH of the reaction solution at the end of hydrolysis is preferably 4.0 to 6.8, more preferably 4.5 to 5.5.

The hydrolysis reaction may be carried out by either a batch method or a continuous method.

In one embodiment of the present invention, the hydrolyzate may be treated with de-alginic acid (alginic acid removal). The de-alginic acid treatment may be performed either before or after the hydrolysis reaction of the polysaccharide derived from seaweed. The de-alginic acid treatment can be performed, for example, according to the procedure described in Preparation Example 2 below.

Hydrolysates of polysaccharides derived from seaweed can be used, for example, by depigmenting and extracting water-soluble polysaccharides from powdered seaweed samples; removing alginic acid under hydrochloric acid acidity; and bleaching, ethanol precipitation and dialysis prior to the hydrolysis reaction. It can be produced by subjecting a hydrolysis reaction after obtaining a purified polysaccharide by purification according to the above. Hydrolysates of polysaccharides derived from seaweed can also be obtained by subjecting to a hydrolysis reaction followed by alginic acid removal under hydrochloric acid acidity; and decolorization, ethanol precipitation and purification by dialysis. Each of the above procedures may follow, for example, as described in Preparation Example 2 below.

When de-alginic acid treatment is applied, the hydrolyzate of the polysaccharide derived from seaweed can be a hydrolyzate of a polysaccharide other than alginic acid, for example, a sulfated polysaccharide (eg, fucoidan (eg, hijiki fucoidan)). ..

The molecular weight of the polysaccharide derived from seaweed depends on the type of seaweed from which it is derived, the time of collection, the place of origin, etc., but the hydrolyzate of the polysaccharide derived from seaweed has, for example, the weight average molecular weight shown in Table 1 below. Have. Table 1 shows the weight average molecular weights of high molecular weight, medium molecular weight and low molecular weight dealginate polysaccharides derived from seaweed. The "polymer" shown in Table 1 is the weight average molecular weight of the dealginate polysaccharide (for example, prepared according to Preparation Example 2) obtained by heating, stirring, etc. in the drying, crushing, and extraction operations of seaweed. Is shown. "Medium molecular weight" and "low molecular weight" are classified into a medium molecular weight area having a weight average molecular weight of 100,000 Da or less and a range of up to 50,000 Da, and a low molecular weight area having a weight average molecular weight of 50,000 Da or less, respectively, as a guideline for adjusting the molecular weight. It can be obtained based on the weight average molecular weight of a sample further hydrolyzed from the above-mentioned "high molecular weight" dearginate polysaccharide obtained by adjusting the reaction temperature and reaction time for each seaweed (medium molecular weight region polysaccharide is simply "". "Medium molecular weight" and low molecular weight area polysaccharides are also simply referred to as "small molecules").

In one embodiment, a hydrolyzate of a polysaccharide derived from seaweed has a weight average molecular weight of about the weight average molecular weight of its unhydrolyzed product (ie, a polysaccharide that can naturally be present in seaweed). The value is 80% or less. Further, the lower limit of the weight average molecular weight of the hydrolyzate of the polysaccharide derived from seaweed may be 10,000 Da. When the polysaccharide derived from seaweed contains a sulfated polysaccharide, the hydrolyzate is preferably 10,000 Da or more from the viewpoint of controlling the hydrolysis reaction.

In one embodiment, the hydrolysates of polysaccharides derived from seaweed are:
A hydrolyzate derived from arame and having a weight average molecular weight in the range of 10,000 Da to 306,000 Da;
A hydrolyzate derived from Gagome and having a weight average molecular weight in the range of 10,000 Da to 860000 Da;
A hydrolyzate derived from hijiki seaweed and having a weight average molecular weight in the range of 10,000 Da to 268,000 Da;
A hydrolyzate derived from Sargassum and having a weight average molecular weight in the range of 10000 Da to 332000 Da;
Hydrolyzate derived from kelp and having a weight average molecular weight in the range of 10000 Da to 713000 Da;
A hydrolyzate derived from wakame seaweed and having a weight average molecular weight in the range of 10000 Da to 564000 Da;
Hydrolyzate derived from Sargassum horneri and having a weight average molecular weight in the range of 10000 Da to 272000 Da;
Hydrolysates derived from turmo and having a weight average molecular weight in the range of 10000 Da to 112000 Da;
A hydrolyzate derived from coontail and having a weight average molecular weight in the range of 10,000 Da to 180,000 Da;
Hydrolysates derived from mekabu and having a weight average molecular weight in the range of 10,000 Da to 501,000 Da;
Hydrolysates derived from Cladosiphon okalui and having a weight average molecular weight in the range of 10000 Da to 192000 Da;
A hydrolyzate derived from giraffe and having a weight average molecular weight in the range of 10,000 Da to 276,000 Da;
A hydrolyzate derived from black rose paste and having a weight average molecular weight in the range of 10,000 Da to 291,000 Da;
A hydrolyzate derived from Meristotheca papulosa and having a weight average molecular weight in the range of 10000 Da to 196000 Da;
A hydrolyzate derived from Gracilaria and having a weight average molecular weight in the range of 10,000 Da to 328,000 Da;
Hydrolysates derived from Monostroma nitidum and having a weight average molecular weight in the range of 10,000 Da to 280000 Da; or a combination of two or more of these. In one embodiment, the seaweed is hijiki and the weight average molecular weight of the hydrolyzate is in the range of 10,000 Da to 268,000 Da.

In one embodiment, the seaweed-derived polysaccharide hydrolyzate is a hydrolyzate derived from hijiki seaweed and having a weight average molecular weight in the range of 10,000 Da to 268,000 Da.

The bile acid adsorbent, adiponectin secretagogue, diabetes-improving agent or constipation-improving agent of the present invention contains a hydrolyzate of a polysaccharide derived from seaweed. "Containing a hydrolyzate of a polysaccharide derived from seaweed" is not limited in its mode as long as it contains the hydrolyzate. For example, the liquid itself of the product produced by the hydrolysis method, or a powder obtained by removing water from the liquid (for example, a lyophilized product or a powder obtained by evaporating water by heating), or the said product. It may contain a liquid or a hydrolyzate of a seaweed-derived polysaccharide extracted from the powder. By "containing a polysaccharide hydrolyzate derived from seaweed", the seaweed can be subjected to treatments such as de-alginic acid (alginic acid removal), decolorization, and purification as described above, in addition to the hydrolysis reaction, if necessary. It is also included when it includes those that have been applied.

The bile acid adsorbent of the present invention is, for example, a substance or material that exhibits an adsorption effect in the bile adsorption test and the sodium cholic acid adsorption test described in Study Example 1 below, or a test substantially equivalent thereto. The bile acid adsorbent of the present invention has a bile acid adsorbing effect of, for example, 70% or more, preferably 80% or more, more preferably 85% or more, with respect to the effect of cholestyramine known to have a high bile acid adsorbing effect. Even more preferably, it has a bile acid adsorption effect of 90% or more. The bile acid adsorbent of the present invention can also exert a bile acid excretion effect. With respect to the bile acid adsorbent, seaweeds, which are hydrolyzates of seaweed-derived polysaccharides, are preferably arame, gagome, hijiki, sargassum, kelp, wakame, akamoku, tsurumo, matsumo, mekabu, mozuku, giraffe, black rose, tosakanori, ogonori. And the seaweed. In one embodiment, the hydrolysates of polysaccharides derived from seaweed are:
A hydrolyzate derived from arame and having a weight average molecular weight in the range of 10,000 Da to 306,000 Da;
A hydrolyzate derived from Gagome and having a weight average molecular weight in the range of 10,000 Da to 860000 Da;
A hydrolyzate derived from hijiki seaweed and having a weight average molecular weight in the range of 10,000 Da to 268,000 Da;
A hydrolyzate derived from Sargassum and having a weight average molecular weight in the range of 10000 Da to 332000 Da;
Hydrolyzate derived from kelp and having a weight average molecular weight in the range of 10000 Da to 713000 Da;
A hydrolyzate derived from wakame seaweed and having a weight average molecular weight in the range of 10000 Da to 564000 Da;
Hydrolyzate derived from Sargassum horneri and having a weight average molecular weight in the range of 10000 Da to 272000 Da;
Hydrolysates derived from turmo and having a weight average molecular weight in the range of 10000 Da to 112000 Da;
A hydrolyzate derived from coontail and having a weight average molecular weight in the range of 10,000 Da to 180,000 Da;
Hydrolysates derived from mekabu and having a weight average molecular weight in the range of 10,000 Da to 501,000 Da;
Hydrolysates derived from Cladosiphon okalui and having a weight average molecular weight in the range of 10000 Da to 192000 Da;
A hydrolyzate derived from giraffe and having a weight average molecular weight in the range of 10,000 Da to 276,000 Da;
A hydrolyzate derived from black rose paste and having a weight average molecular weight in the range of 10,000 Da to 291,000 Da;
A hydrolyzate derived from Meristotheca papulosa and having a weight average molecular weight in the range of 10000 Da to 196000 Da;
A hydrolyzate derived from Gracilaria and having a weight average molecular weight in the range of 10,000 Da to 328,000 Da;
Hydrolysates derived from Monostroma nitidum and having a weight average molecular weight in the range of 10,000 Da to 280000 Da; or a combination of two or more of these. Further, in one embodiment, the hydrolyzate is a hydrolyzate having a weight average molecular weight shown in the medium molecule or small molecule of Table 1 above, and may be a mixture of two or more thereof. In one embodiment, the seaweed is hijiki, the hydrolyzate is derived from hijiki, and the weight average molecular weight is, for example, in the range of 10,000 Da to 268,000 Da, preferably in the range of 11,000 Da to 174,000 Da. More preferably, it is in the range of 12000 Da to 112000 Da.

The bile acid adsorbent of the present invention can be used as a food additive based on its bile acid adsorption effect. The present invention also provides foods containing bile acid adsorbents. The food "containing a bile acid adsorbent" may be either a food containing the bile acid adsorbent of the present invention in a food or a food containing the bile acid adsorbent itself as a food. The "food containing a bile acid adsorbent" of the present invention may be a food for reducing cholesterol levels in the liver and blood. Further, the "food containing a bile acid adsorbent" of the present invention may be a food for preventing or improving metabolic syndrome, a food for preventing or improving diabetes, and the like. The bile acid adsorbent of the present invention may be contained in a pharmaceutical product. Examples of such a drug include a drug for lowering cholesterol levels, a drug for preventing or ameliorating lifestyle-related diseases such as diabetes, and the like.

The adiponectin secretagogue of the present invention is a substance or a material that exerts an effect of promoting adiponectin secretion into blood. The adiponectin secretagogue effect can be measured by conventional methods. With respect to the adiponectin secretagogue, the seaweed derived from the hydrolyzate of the seaweed-derived polysaccharide is preferably hijiki. When the seaweed is hijiki, the weight average molecular weight of the hydrolyzate is, for example, in the range of 10000 Da to 268000 Da, preferably in the range of 11000 Da to 174000 Da, and more preferably in the range of 12000 Da to 112000 Da. be.

The adiponectin secretagogue of the present invention can be used as a food additive based on its adiponectin secretagogue effect. The present invention further provides a food containing an adiponectin secretagogue. The food "containing the adiponectin secretagogue" may be either a food containing the adiponectin secretagogue of the present invention in the food or a food containing the adiponectin secretagogue itself. The "food containing an adiponectin secretagogue" of the present invention may be a food for improving diabetes. The "food containing an adiponectin secretagogue" of the present invention may be a food for reducing cholesterol levels in the liver and blood. Further, the "food containing an adiponectin secretion promoter" of the present invention is a food for preventing or improving obesity, a food for preventing or improving metabolic syndrome, a food for preventing or improving hyperlipidemia, and a food for preventing or improving hypertension. , Foods for preventing or improving arteriosclerosis, foods for preventing cancer, and the like. The adiponectin secretagogue of the present invention may be contained in a pharmaceutical product. Examples of such drugs include drugs for preventing or ameliorating obesity, hyperlipidemia, hypertension, diabetes, arteriosclerosis, cancer and the like.

Furthermore, the present invention also provides a diabetes ameliorating agent, which comprises a hydrolyzate of a polysaccharide derived from hijiki and whose weight average molecular weight of this hydrolyzate is in the range of 10,000 Da to 268,000 Da. The weight average molecular weight of this hydrolyzate is preferably in the range of 11000 Da to 174000 Da, more preferably in the range of 12000 Da to 112000 Da. The diabetes improving agent of the present invention can be used as a food additive based on its diabetes improving effect. The present invention also provides a food for preventing or treating diabetes, which comprises a diabetes improving agent. The food "containing the diabetes improving agent" may be either a food containing the diabetes improving agent of the present invention in the food or a food containing the diabetes improving agent itself as a food. The diabetes improving agent of the present invention may be contained in a pharmaceutical product. Examples of such a drug include a drug for preventing or ameliorating diabetes. As described above, the diabetes improving agent of the present invention may be provided in any form such as a food additive, a food and a pharmaceutical product for the prevention or treatment of diabetes by itself, or the improving agent may be a food additive. , Can be contained as an ingredient in foods and pharmaceuticals.

Furthermore, the present invention also provides a constipation ameliorating agent containing a hydrolyzate of a polysaccharide derived from hijiki, and the weight average molecular weight of the hydrolyzate is in the range of 10,000 Da to 268,000 Da. The weight average molecular weight of this hydrolyzate is preferably in the range of 11000 Da to 174000 Da, more preferably in the range of 12000 Da to 112000 Da. The constipation improving agent of the present invention can be used as a food additive based on its constipation improving effect. The present invention also provides a constipation-improving food containing a constipation-improving agent. The food "containing the constipation improving agent" may be either a food containing the constipation improving agent of the present invention in the food or a food containing the constipation improving agent itself. The constipation improving agent of the present invention may be contained in a pharmaceutical product. Examples of such a drug include a drug for improving constipation. As described above, the constipation improving agent of the present invention may be provided in any form of a food additive, a food or a pharmaceutical product for the purpose of preventing or improving constipation, or the improving agent may be a food additive, a food product or the like. And can be contained as an ingredient in pharmaceutical products.

The seaweed is hijiki, and the weight average molecular weight of the hydrolyzate is, for example, in the range of 10000 Da to 268000 Da, preferably in the range of 11000 Da to 174000 Da, and more preferably in the range of 12000 Da to 112000 Da. In the embodiment, a bile acid adsorption effect, an adiponectin secretion promoting effect, a diabetes improving effect and a constipation improving effect can be exhibited. In this embodiment, food additives, foods and pharmaceuticals for applications based on each of the above effects can be obtained.

When the food additives, foods and pharmaceuticals as described above are formulated, the form of the formulation is not particularly limited. For example, depending on the purpose, it can be an oral preparation such as a tablet, a powder, a granule, a capsule, a fine granule, a liquid (liquid medicine, etc.), and all of these can be formulated by a known method. can. In addition, various additives usually used in the production of the pharmaceutical product may be blended in an amount appropriately selectable by those skilled in the art. Examples of such pharmaceutical additives include excipients, lubricants, plasticizers, surfactants, binders, disintegrants, wetting agents, stabilizers, flavoring agents, colorants, fragrances and the like. As the pharmaceutical additive, one type may be used alone, or two or more types may be used in combination. When two or more kinds are used in combination, the combination and the ratio may be appropriately selected according to the purpose.

The intake amount of the bile acid adsorbent, the adiponectin secretagogue, the diabetes improving agent or the constipation improving agent of the present invention can be appropriately adjusted depending on the age, condition and the like. For example, the intake of a bile acid adsorbent, an adiponectin secretagogue, a diabetes improving agent or a constipation improving agent is a mouse dose of Hijiki seaweed after a hydrolysis reaction (for example, Hijiki-derived polysaccharide hydrolysis having a weight average molecular weight of 18,000 Da). It is preferable that it is 7.6% by weight based on the dietary amount of 4.5 g of the mouse per day in terms of (which may contain substances). Conversion of intake to humans can be made by methods commonly used by those skilled in the art.

As described above, the bile acid adsorbent, adiponectin secretagogue, diabetes improving agent or constipation improving agent of the present invention can be used not only in pharmaceutical products but also in foods, and are therefore more versatile, regardless of age or gender. , Many people can easily take it.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

(Preparation Example 1: Preparation of powdered seaweed sample)
Seaweed was crushed using a mixer and sieved (mesh size (#) = 250 μm) to obtain a powdered seaweed sample having a particle size of 250 μm or less.

(Examination example 1: Bile acid adsorption test)
<Sample used>
In this study example, a total of 16 types of seaweeds, including arame, gagome, hijiki, sargassum, kelp, wakame, akamoku, matsumo, tsurumo, mekabu, mozuku, giraffe, kurobaranori, tosakanori, ogonori, and monostroma nitidum, were used as adsorbents. The action of was investigated. As the adsorbent, bile and sodium cholic acid, which is a typical bile acid in the living body, were used. Cholestyramine, a bile acid adsorbent, was used as a comparative sample of the adsorbent. Cholestyramine is a strong basic anion exchange resin. The biochemical reagents sodium cholic acid and cholestyramine were stored in a vacuum desiccator and used.

<Purification of powdered seaweed sample>
8 g of the powdered seaweed sample obtained in Preparation Example 1 was mixed with 400 mL of pure water to obtain a powdered seaweed mixed solution. This mixed solution is placed in four 40 mm × 30 cm dialysis cellulose tubes (manufactured by EIDIA Co., Ltd., product name: UC30-32, molecular weight cut off: MWCO 12,000 to 16,000: the same tube is used unless otherwise specified). , 3000 mL of pure water was put outside the membrane, and dialysis was performed with stirring for 24 hours. At that time, the pure water outside the membrane was replaced 2 hours and 4 hours after the start of dialysis. The mixed solution in the membrane was recovered and freeze-dried to obtain purified seaweed powder.

<Bile powder refining operation>
2 g of bile powder (manufactured by Wako Pure Chemical Industries, Ltd.) and 200 mL of pure water were mixed to obtain a bile powder mixed solution. This mixed solution was placed in two 40 mm × 30 cm dialysis cellulose tubes, 1000 mL of pure water was placed outside the membrane, and dialysis was performed with stirring for 24 hours. At that time, the pure water outside the membrane was replaced 2 hours and 4 hours after the start of dialysis. The bile powder solution that had come out of the membrane was collected and freeze-dried to obtain purified bile powder.

<Cholestyramine purification operation>
5 g of cholestyramine (manufactured by SIGMA-ALDRICH) was mixed with 250 mL of pure water to obtain a cholestyramine mixed solution. This mixed solution was placed in a 40 mm × 40 cm dialysis cellulose tube, 800 mL of pure water was placed outside the membrane, and dialysis was performed with stirring for 24 hours. At that time, the pure water outside the membrane was replaced 2 hours and 4 hours after the start of dialysis. The mixed solution in the membrane was recovered and lyophilized to obtain purified cholestyramine.

<Bile adsorption test>
The pure water used in the adsorption test was preheated in the incubator until it reached 37 ° C., and this warmed pure water was used for the subsequent tests. 1 g of purified seaweed powder and 0.2 g of purified bile powder were placed in 200 mL of pure water and shaken at 37.0 ° C. in a constant temperature bath with a shaker at 80 rpm for 2 hours. Next, this solution was placed in three 40 mm × 35 cm dialysis cellulose tubes, 350 mL of pure water was placed outside the membrane, and the mixture was shaken at 65 rpm for 2 hours in a constant temperature bath equipped with a shaker at 37.0 ° C. .. The in-membrane and extra-membrane solutions were freeze-dried and the yield was measured.

Weigh the amount of bile adsorbed on seaweed from the solution inside the membrane and the amount of bile not adsorbed on seaweed from the solution outside the membrane, and use the following formula (“starting bile amount” in the formula is 0.2 g). Based on this, the adsorption rate was calculated from the amount of bile that was not adsorbed.

<Sodium cholic acid adsorption test>
1 g of purified seaweed powder and 0.2 g of sodium cholic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were placed in 200 mL of pure water and shaken at 37.0 ° C. in a constant temperature bath with a shaker at 80 rpm for 2 hours. The adsorption rate was determined by the same procedure as in the adsorption test using purified bile powder, except that the amount of sodium cholic acid was measured instead of the amount of bile.

<Bile adsorption test and sodium cholic acid adsorption test with purified cholestyramine>
The adsorption rate was determined by the same procedure as in each of the above adsorption tests except that 1 g of purified cholestyramine was used.

<Results of bile acid adsorption test>
The results are shown in Table 2 below. Table 2 shows the adsorption rate of various powdered seaweed samples as a relative value (%) when the adsorption rate of cholestyramine is 100%.

Further, FIGS. 1 and 2 show relatives of the adsorption rates of various seaweed powder samples in the bile adsorption test (FIG. 1) and the sodium cholic acid adsorption test (FIG. 2), respectively, when the adsorption rate of cholestyramine is 100%. It is a graph shown by a value. In both FIGS. 1 and 2, the vertical axis shows a relative value (%) when the adsorption rate of cholestyramine is 100%.

From the results of this study example, in the bile adsorption test and the sodium cholic acid adsorption test, the ratio of the adsorption rate of the seaweed sample to the adsorption rate of cholestyramine was about 70% or higher in all the seaweeds used, which was high. The adsorption rate was confirmed. In the bile adsorption test and the sodium coliate adsorption test, for example, the seaweeds that showed high adsorption rates of 80% or more are as follows: Arame, Gagome, Hijiki, Kombu, Wakame, Akamoku, Tsurumo, Matsumo, Kirinsai. , Black seaweed, Meristotheca papulosa, Gracilaria, Hijiki seaweed (Table 2 and FIGS. 1 and 2). In addition, since the bile used in this study example is derived from bovine bile, it is expected that the same effect will be observed in vivo.

From the results of this study example, there were seaweeds showing a bile acid adsorption effect similar to that of cholestyramine, whether it was a bile acid alone or a bile mixture derived from a living body. In particular, seaweeds showing a high adsorption rate of about 80% or more with respect to the bile acid adsorption rate of cholestyramine are expected to have higher functionality as a bile acid adsorbent.

(Preparation Example 2: Preparation of hydrolyzate of polysaccharide derived from seaweed)
In the α-glucosidase activity inhibition test of Study Example 2 below, the same 16 types of seaweed as in the bile acid adsorption test of Study Example 1 above were adopted, and alginic acid was removed from the polysaccharides contained in the seaweeds. Polysaccharides were used. Dyes, alginic acid, etc., which are substances having enzyme inhibitory activity, were removed from the seaweed sample and purified by dialysis to obtain a dearginic acid polysaccharide, which was used as a material for an α-glucosidase activity inhibition test.

<Dye removal from powdered seaweed sample and extraction of water-soluble polysaccharides>
To the powdered seaweed sample prepared in Preparation Example 1, 3 times the dry weight of the seaweed was added with ethanol, and the mixture was stirred at room temperature for 24 hours to extract and remove the seaweed-containing pigments. It was separated from the containing ethanol solution. The precipitate after separation was subjected to an evaporator to remove ethanol, and then freeze-dried. Ten times the amount of pure water was added to the freeze-dried seaweed sample, and hot water extraction was performed at 80 ° C. for 4 hours. Then, this hot water extract was centrifuged and solid-liquid separated. The separated liquid was freeze-dried to obtain a polysaccharide powder which is a water-soluble dietary fiber.

<Removal of alginic acid under hydrochloric acid acidity>
The polysaccharide powder obtained by the above extraction operation may consist of alginic acid, a sulfated polysaccharide containing a mixture of fucoidan, carrageenan and the like, agarose and the like. The removal of alginic acid was carried out by a pH adjustment method using the acid dissociation constant of alginic acid. The acid dissociation constant of glucuronic acid, which is a constituent sugar of alginic acid, is 3.38, and that of mannuronic acid is 3.65. When the hydrogen ion concentration of the solution in which alginic acid is dissolved is set to pH = 1.0 or less, high-molecular-weight alginic acid cannot be dissociated as ions and precipitates due to the acid dissociation constant of alginic acid. However, since the sulfate group of a sulfated polysaccharide such as fucoidan is an ester bond, it does not depend on the hydrogen ion concentration in the solution and remains dissolved, so that alginic acid and the sulfated polysaccharide can be separated.

The polysaccharide powder obtained by the above extraction operation is dissolved in a small amount of pure water, hydrochloric acid is added dropwise while stirring the solution, the pH of the solution is adjusted to 1.0 or less, and then the solution is allowed to stand in a refrigerator for 24 hours. Alginic acid was precipitated. The precipitated alginic acid was separated into solid and liquid by a centrifuge, and the precipitate was freeze-dried to obtain alginic acid powder. The solution after centrifugation was neutralized with a 20 w / v% sodium carbonate aqueous solution and freeze-dried to obtain a mixed powder of the salts produced by the neutralization and the dealginate polysaccharide.

For Gracilaria and Monostroma nitidum, the addition of hydrochloric acid only clouded and did not produce a clear precipitate. In addition, after the Tosakanori was allowed to stand in the refrigerator for 24 hours, the entire solution was gelled, so that it was somewhat difficult to completely separate alginic acid. However, by performing the same operation as above, an alginic acid powder and a mixed powder of salts and a dearginic acid polysaccharide were obtained.

The results of alginic acid content for each seaweed are shown in Table 3. The alginic acid content was determined based on the following formula. In the table, "sample" indicates a polysaccharide powder obtained by hot water extraction (before removal of alginic acid), and "alginic acid" indicates an alginic acid powder recovered by precipitating by a pH adjustment method.

<Decolorization, ethanol precipitation and purification by dialysis>
Dissolve a mixed powder of salts and dealginate polysaccharide in a small amount of pure water, and stir in a hot water bath at 70 ° C. under acetic acid acid conditions until the sample solution turns pale yellow, 5 w / v% chlorite. The sodium solution was added dropwise. After the decolorization was completed, a 20 w / v% sodium carbonate solution was added, and the hydrogen ion concentration of the solution was set to about pH = 9.5. After stirring for a while, ethanol was added dropwise until the ethanol concentration reached 80 v / v%, and the mixture was allowed to stand in a refrigerator for 24 hours. The ethanol solution after standing was separated into solid and liquid by a centrifuge. The separated precipitate was subjected to an evaporator to remove ethanol, and then freeze-dried to obtain a decolorized mixed powder of salts and dealginate polysaccharides produced by neutralization.

The obtained mixed powder is dissolved in a small amount of pure water, and this solution is used as a dialysis membrane (manufactured by Funakoshi Co., Ltd., product name: Spectra / Por 3, fractional molecular weight: 3500; hereinafter, the same dialysis membrane is used unless otherwise specified). And dialyzed with stirring for 24 hours. At that time, the pure water outside the membrane was exchanged 2 hours and 4 hours after the start of dialysis, and the solution inside the membrane was recovered 24 hours later. The recovered solution was freeze-dried to obtain a de-alginate polysaccharide powder. The purified dealginate polysaccharide obtained here was designated as a "polymer" in this preparation example.

<Molecular weight adjustment of dealginate polysaccharide by pressurization and heating under carbon dioxide gas>
250 mL of pure water was put into a pressure resistant reaction vessel, and the temperature was raised to 30 ° C. When the temperature reached 30 ° C., carbon dioxide gas was blown into the mixture, and bubbling was performed for 15 minutes while stirring at 200 rpm. After adding 5 g of the de-alginate polysaccharide powder, the entire reaction vessel was sealed, the device pressure was adjusted to 0.30 MPa with carbon dioxide gas, and the temperature was raised to a predetermined temperature at a rotation speed of 200 rpm. The reaction start time was defined as the time when the temperature reached a predetermined temperature, and the reaction was carried out for a predetermined time. Immediately after the reaction was completed, the reaction vessel was cooled with ice water. After the sample solution was cooled, the pH was measured and lyophilized.

For various seaweeds, as a guideline for adjusting the molecular weight, the weight average is in the range of 100,000 Da or less and up to 50,000 Da as the medium molecular weight area (also referred to as "medium molecule"), and the weight average as the low molecular weight area (also referred to as "low molecule"). The de-alginate polysaccharide powder derived from each seaweed was hydrolyzed by setting the temperature, pressure and time for the reaction so that the molecular weight was 50,000 Da or less. Table 4 shows the temperature, pressure and time for the hydrolysis reaction set for the medium and low molecular weight regions of the de-alginate polysaccharide powders from various seaweeds, respectively.

However, the pressure is shown as a gauge pressure with an atmospheric pressure of 0.0 MPa. In the above hydrolysis reaction experiment, the initial pressure was 0.30 MPa at 30 ° C. in the presence of carbon dioxide, so the reaction pressure was water vapor pressure + 0.30 MPa, but due to the solubility of carbon dioxide, the gauge at the time of the experiment. The pressure display showed 0.53 MPa at 110 ° C.

The macromolecules, medium molecules, and small molecules obtained for various seaweeds were subjected to analysis by high performance liquid chromatography (HPLC), and the weight average molecular weight (Mw [Da]) was determined by GPC calculation software. However, since the upper limit of the exclusion limit of the analytical column is 400,000 Da and the lower limit is 10,000 Da, the weight average molecular weight outside these ranges is shown by extrapolation value. The software names of HPLC analyzer, analytical column, analytical conditions and weight average molecular weight calculation are shown below.
(HPLC device and analysis conditions)
Agilent 1100 Binary Pump Agilent 1100 Degassa RI Detector: JASCO Differential Refractometer 2031 plus
Column: KS-804 made by SHODEX (exclusion limit: 400,000),
KS-802 made by SHODEX (exclusion limit: 10000),
KS-G (Gad Column) made by SHODEX
Sample loop: PHEOMYNE 500 μL loop Eluent: 0.1 mol / L NaCl
Flow rate: 0.700 mL / min Column temperature: 40.0 ° C
Weight average molecular weight calculation software: Chromato-PRO-GPC (manufactured by Runtime Instruments)

Table 5 shows the weight average molecular weights of the macromolecules, medium molecules, and small molecules obtained for various seaweeds by GPC analysis.

The molecular weight-adjusted dealginate polysaccharide was dissolved in a small amount of pure water, this solution was placed in a dialysis membrane, and dialysis was performed with stirring for 24 hours. At that time, the pure water outside the membrane was exchanged 2 hours and 4 hours after the start of dialysis, and the solution inside the membrane was recovered 24 hours later. The recovered solution was freeze-dried to obtain a purified dealginate polysaccharide. This purified dealginate polysaccharide was subjected to the following Study Example 2 (α-glucosidase inhibitory activity test).

(Study Example 2: α-glucosidase inhibitory activity test)
As the standard substance in this study example, Trizma Base (manufactured by Wako Pure Chemical Industries, Ltd .: Trizma Base) known as a general α-glucosidase activity inhibitor was used.

14.75 mg of purified dearginite polysaccharide (per high molecular weight, medium molecular weight, and low molecular weight) powder in a phosphate buffer (Wako Pure Chemical Industries, Ltd .: neutral phosphorus pH standard solution, pH = 6.8) solution. The mixture was added, the concentration was adjusted to 0.5 w / v%, and the mixture was completely dissolved. Based on this dearginic acid polysaccharide solution, the concentration was readjusted to various concentrations, and 0.100 mL of GSH (SIGMA: 3 mM L-Glutathione, reduced) and α-glucosidase enzyme solution (5.1 mg /), which are reaction accelerators, were adjusted. After adding 0.100 mL of 100 mL α-Glucosidase from Saccharomyces cerevisiae (SIGMA) and stirring with a vortex mixer, the mixture was preheated at 37 ° C. for 10 minutes.

After preheating, 0.250 mL of 10 mM p-nitrophenyl α-D-glucoside (manufactured by SIGMA) was added as a substrate, and the mixture was stirred with a vortex mixer, and the mixture was incubated at 37 ° C. for 20 minutes. After incubation, 8.00 mL of 100 mM sodium carbonate (solution manufactured by Kishida Chemical Co., Ltd.) was added to stop the reaction, and the absorbance was measured at 400 nm. The one measured without adding the de-alginate polysaccharide was used as a control (activity 100%). Further, the value measured by adding a buffer instead of the enzyme solution was used as a blank value. Based on the following formula, the inhibition rate (Activity Inhibition: (%)) of the enzyme activity of α-glucosidase of the dealginate polysaccharide was determined.

Table 6 shows the inhibition rate [%] of high molecular weight, medium molecule and small molecule α-glucosidase activity for each seaweed together with the concentration [mg / mL] indicating the inhibition rate. However, for kelp and coontail medium and small molecules, the inhibition rate data could not be obtained because the crystal structure of the dealginate polysaccharide changed and the solution became cloudy or the sample did not dissolve.

As shown in Table 6, it was arame, hijiki and sargassum that showed an inhibition rate of 50% or more in any of high molecular weight, medium molecule and small molecule. Arame and Hijiki showed an inhibition rate of about 80% or more, and these showed a numerical value of 95% or more.

For Arame, Hijiki and Sargassum, which showed a high inhibition rate of 50% or more regardless of the concentration, the 50% inhibition concentration ( _IC50 value: [mg / mL]) in the α-glucosidase inhibitory activity test is shown in Table 7. ..

From the _IC50 values in Table 7, the _IC50 value [mg / mL] of Hijiki showed a low value of 0.0831 for small molecules. Others with an IC ₅₀ value of 1.00 [mg / mL] or less were 0.524, which is a polymer of arame, 0.210, which is a medium molecule of hijiki, and 0.944, which is a small molecule of sargassum. .. The dealginate polysaccharide in the molecular region of these seaweeds is expected to have high functionality as an α-glucosidase inhibitor.

As described above, in both the bile acid adsorption test of Study Example 1 and the α-glucosidase inhibitory activity test of Study Example 2, arame and hijiki showed good results. In addition, Sargassum showed a sufficiently high adsorption rate of about 70% in the bile acid adsorption test result of Study Example 1, although it did not reach 80%, which was one of the criteria in the study example. In the α-glucosidase inhibitory activity test, the results were good. From this, it was judged that Sargassum also conformed to the good results of Arame and Hijiki.

(Study Example 3: Elemental analysis of de-alginate polysaccharide)
Regarding the dearginic acid polysaccharides obtained in Preparation Example 2, elemental analysis (manufactured by Elementer, fully automatic elemental analyzer vario EL III) was performed for the four types of polysaccharides, human exa, hijiki, gagomekonbu, and mozuku, whose main components are sulfated polysaccharides. ) Performed elemental analysis on carbon, hydrogen, oxygen, nitrogen, and sulfur. Sulfanilic acid (manufactured by Merck Millipore) was used as an internal standard substance, and Okinawan mozuku fucoidan (manufactured by Tanglewood: AH fucoidan 85) was used as a comparative substance. This commercially available AH fucoidan was purified by the ethanol precipitation method as a pretreatment and subjected to elemental analysis. The degree of sulfation (SO ₃ Na [%]) of the elemental analysis sample (de-alginate polysaccharide) was determined from the elemental analysis values according to the following formula.

The results of elemental analysis are shown in Table 8.

By this elemental analysis, the average degree of sulfation of the dearginate polysaccharide of mozuku prepared in Preparation Example 2 was 16.3%, whereas the average value of the degree of sulfation of mozuku fucoidan from Okinawa was almost the same. It showed about 16.6%. From this elemental analysis data, it was judged that the purification step from the raw algae used in Preparation Example 2 to the purified dealginate sulfated polysaccharide was generally performed correctly. Since the extraction / purification process from seaweed was performed almost correctly, the sulfated degree analysis value suggested that the purified dealginate polysaccharide derived from Hijiki was hijiki fucoidan.

<Elimination limit of ester sulfuric acid due to lower molecular weight of sulfated polysaccharide>
The ester-sulfuric acid bond of the sulfated polysaccharide is an ester bond, which is weaker than the glucosidic bond, which is a bond between sugars and sugars, in terms of binding force. Therefore, the cleavage of the ester bond may proceed slightly more predominantly than the cleavage of the glucoside bond and the progress of the low molecular weight reduction.

The desorption limit of ester sulfuric acid was confirmed by conducting a long-term hydrolysis experiment using AH fucoidan 85 as a sample. Except for using 5 g of AH fucoidan and 500 mL of pure water, the hydrolysis reaction was carried out according to the molecular weight adjustment with carbon dioxide. The reaction temperature and reaction time are as shown in Tables 9 and 10. Table 9 shows the results of the weight average molecular weight for each reaction time and reaction temperature, and Table 10 shows the pH value of the solution.

From Tables 9 and 10, it was shown that when the weight average molecular weight was less than 10000 Da, the pH value of the corresponding solution was lower than 4.0. The change in the pH value of the reaction solution means that the ester bond of the sulfated polysaccharide with the ester sulfuric acid is broken, the sulfate group is desorbed, and the sulfate group reacts with water in the solution to generate sulfuric acid. As the production of sulfuric acid progresses, the hydrolysis reaction proceeds rapidly, inducing further elimination of sulfuric acid groups, and it becomes difficult to control the reaction. From these facts, it was estimated that the limit of reducing the molecular weight of the sulfated polysaccharide was approximately 10,000 Da.

(Preparation Example 3)
Dried hijiki (produced off Miura, Kanagawa Prefecture: Miura Seaweed Co., Ltd.) was crushed according to Preparation Example 1 to obtain a hijiki powder sample (also in Preparation Examples 1 and 2, dried hijiki produced off Miura, Kanagawa Prefecture). Was used).

<Preparation of untreated hijiki powder sample>
From the obtained Hijiki powder sample, <Dye removal and water-soluble polysaccharide extraction from powdered seaweed sample>, <Alginic acid removal under hydrochloric acid acidity>, and <Decolorization, ethanol precipitation, and purification by dialysis> in Preparation Example 2. Purified de-alginate polysaccharide was obtained according to the procedure of. This purified dealginate polysaccharide was referred to as "untreated hijiki" in a mouse test (Example 1 below).

<Preparation of low molecular weight hijiki powder sample>
About 40 g of the hijiki powder sample obtained by pulverizing according to Preparation Example 1 above was added to 1.5 L of pure water and heated at 80 ° C. to 90 ° C. for 2 hours to obtain an extract. The whole extract was subjected to a centrifuge for solid-liquid separation to obtain a supernatant (which may contain polysaccharides such as alginic acid and fucoidan), which was lyophilized to obtain a polysaccharide derived from hijiki seaweed. In the preliminary experiment, these procedures were performed twice. In that case, the yield of the polysaccharide was 23% by weight.

10 g of the obtained hijiki-derived polysaccharide and 250 mL of a vinegar solution having a vinegar concentration shown in Table 11 (prepared from pure water and vinegar (rice vinegar: acidity 4.2%)) were placed in a high-pressure reactor and shown in Table 11. The hydrolysis reaction was carried out under the predetermined conditions shown. The obtained hydrolyzate was neutralized with a 0.5 w / v% sodium carbonate aqueous solution so that the pH was in the range of 6.6 to 6.8, and the hydrolyzate powder of the hijiki-derived polysaccharide was lyophilized by freeze-drying. Obtained.

The obtained hydrolyzate was purified and treated with alginic acid according to the procedures of <Removal of alginic acid under hydrochloric acid acidity> and <Decolorization, ethanol precipitation and purification by dialysis> of Preparation Example 2.

The obtained "untreated Hijiki" and a polysaccharide hydrolyzed in a vinegar solution to be purified and de-alginic acid-treated (purified de-alginic acid polysaccharide hydrolyzate derived from Hijiki) were subjected to molecular weight analysis by GPC. The GPC analysis method was measured according to the GPC analysis method of Preparation Example 2. The molecular weight of the purified de-alginate polysaccharide hydrolyzate derived from Hijiki by GPC analysis is also shown in Table 11 together with the molecular weight of the purified de-alginate polysaccharide (“untreated Hijiki”).

In the α-glucosidase inhibitory activity test of Study Example 2, when the seaweed was hijiki, remarkable results were observed in a small molecule having a weight average molecular weight of 12000 Da. In hydrolysis with vinegar, the vinegar concentration is 10% by volume, the reaction temperature and the reaction time are 120 ° C., and the molecular weight at 2 hours is 11000 Da, which is close to this molecular weight. As described above, as the hydrolysis reaction proceeds, the pH (hydrogen ion concentration) of the reaction solution decreases, and the sulfate ester group tends to be gradually eliminated. Further, the amount of the raw material that requires hydrolysis for preparing the feed for the mouse administration test is large compared to the amount for the α-glucosidase inhibitory activity test as in Study Example 2. From these facts, there is a concern about the elimination of ester sulfuric acid in the hydrolysis reaction in the mouse test. Therefore, a hydrolyzed sample of 18,000 Da obtained at a reaction temperature of 120 ° C. with a smaller amount of vinegar used as a catalyst of 5% by volume was provided as a mouse feed (in Example 1 below, this purified dearginite polysaccharide derived from hijiki seaweed). The hydrolyzate was referred to as "low molecular weight hijiki").

(Example 1: Continuous administration in type II diabetes model mice)
Using the low-molecular-weight sardine powder sample and the untreated sardine powder sample prepared in Preparation Example 3, the fecal weight and the amount of bile acid excreted in feces were examined as bile acid adsorption action by administration to type II diabetes model mice. Furthermore, the action on adiponectin was also investigated.

<Experimental animals, feed and breeding conditions>
KK-Ay / Ta Jcl male mice (Japan Claire: 4 weeks old), which are type II diabetes model mice, were preliminarily bred with a commercially available solid feed (CE-2, Japan Claire) for 1 week, and the low molecular weight Hijiki group (8 animals) and untreated Hijiki group (9 animals) were grouped. It is known that KK- ^Ay mice induce hyperinsulinemia and decreased insulin sensitivity (insulin resistance) by ingesting a high-fat diet, which further exacerbates the pathological condition.

After 1 week of pre-breeding, the low-molecular-weight Hijiki group was fed a high-fat, high-sucrose diet (F2HFHSD, manufactured by Oriental Yeast Co., Ltd.) with a 7.6% low-molecular-weight Hijiki powder sample once a day. It was ingested at 4.5 g for 3 weeks. In the untreated hijiki group, a high-fat, high-sucrose diet (F2HFHSD, manufactured by Oriental Yeast Co., Ltd.) mixed with 7.6% of untreated hijiki powder sample was ingested once a day at 4.5 g for 3 weeks. I let you. The feed was given daily at 17:00 and was ingested until 9:00 the next day, and the amount of food consumed was weighed. As for the beverage, tap water was allowed to be drunk freely. During this period, the mice were individually placed in cages under an environment of room temperature 23 ± 2 ° C., humidity 55 ± 5%, and a 12-hour light-dark cycle (light period 7:00 to 19:00, dark period 19:00 to 7:00). Raised in. Mice were weighed at the beginning and end of ingestion.

The experimental animals of the low-molecular-weight Hijiki group and the untreated Hijiki group were examined for the following items. The experimental results are shown by the average value of each group, and the t-test was performed on the untreated Hijiki group, and the significant difference test was performed.

<Increase / decrease in feed intake and body weight>
As shown in Table 12 below, there was a particularly large difference between the low molecular weight Hijiki group and the untreated Hijiki group in the feed intake of the mice from the start to the end of the experiment and the body weight of the mice at the end. I couldn't.

<Weight of feces and amount of bile acid excreted in feces>
The excreted feces were collected every 24 hours and weighed dry. Further, after crushing the dry feces, 0.1 g of the dry powder feces was weighed in a screw cap test tube. To that, 2 mL of 99.5 v / v% ethanol was added, mixed well with a touch mixer, heated at 80 ° C. for 1 hour, and then only the supernatant was collected in a separate test tube. This operation was performed twice, and about 6 mL of the supernatant was collected. After evaporating at 85 ° C. to 1 mL or less, the volume was adjusted to 1 mL with ethanol to obtain an extract. The total bile acid concentration of this extract was measured using a total bile acid test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).

The dry weight of the feces excreted every 24 hours and the amount of bile acid in the dried feces are shown in FIGS. 3 and 4.

The fecal weight [g / 24 hours] showed an average value of 0.662 in the low-molecular-weight Hijiki group, which was higher than the average value of 0.613 in the untreated Hijiki group (FIG. 3).

Furthermore, the amount of bile acid excreted in feces [μmol / 24 hours] showed an average value of 0.827 in the low-molecular-weight Hijiki group, which was higher than the average value of 0.671 in the untreated Hijiki group (. FIG. 4).

<Measurement of adiponectin>
Blood was collected from the tail vein in the final week of administration (3rd week), and the adiponectin level in the blood was measured using the conventional method.

The adiponectin level [ng / mL] at 3 weeks after administration is shown in FIG. As for the adiponectin value, the average value of the low molecular weight Hijiki group was 3182, which was higher than the average value of 2328 of the untreated Hijiki group.

<Measurement of hemoglobin A1c (HbA1c) value>
Blood was collected from the tail vein in the final week of administration (3rd week), and the hemoglobin A1c level was measured. Measurements of the hemoglobin A1c value were performed using a small rapid hemoglobin A1c, a Malb / C analyzer (DCA2000 system, manufactured by Bayer Medical) and a cartridge (manufactured by Siemens).

The hemoglobin A1c value [%] at 3 weeks after administration is shown in FIG. As for the hemoglobin A1c value, the average value of the low molecular weight Hijiki group was 7.19, which was lower than the average value of 7.24 of the untreated Hijiki group.

<Measurement of blood glucose and insulin levels at the end>
At the end of administration, 2 to 4 hours after fasting, all blood was collected from the abdominal aorta under isoflurane suction anesthesia and euthanized. The collected blood is centrifuged (3000 rpm, 10 minutes), and the glucose concentration in the obtained serum is measured by a biochemical automatic analyzer (Fuji Drychem 4000, manufactured by Fuji Film Medical Co., Ltd.) and a sample slide (Fuji Film Medical Co., Ltd.). Measured using (manufactured by the company). The serum insulin concentration was measured using a commercially available measurement kit (Levis insulin mouse H type, manufactured by Shiba Goat Co., Ltd.).

The blood glucose level [mg / dL] at the end of administration is shown in FIG. In the end-of-life blood glucose level, the average value of the low-molecular-weight Hijiki group was 432, which was lower than the average value of 505 of the untreated Hijiki group.

The insulin value [μU / mL] at the end of administration is shown in FIG. In the end insulin value, the mean value of the low molecular weight hijiki group was 0.156, which was lower than the mean value of the untreated hijiki group of 0.369.

In addition, the insulin resistance index (HOMA-R) was calculated using blood glucose and insulin levels. The insulin resistance index (HOMA-R) is a homeostasis model assessment ratio and can be calculated by the following formula:

The insulin resistance index calculated from the blood glucose level and the insulin level is shown in FIG. In the insulin resistance index, the average value of the low molecular weight Hijiki group was 0.180, which was lower than the average value of 0.470 of the untreated Hijiki group.

According to this example, the low molecular weight Hijiki group increased by 8.1% and 10% in dry feces weight and 23% in bile acid excretion as compared with the untreated Hijiki group. And 10%, and adiponectin levels increased by 37% and 5%, respectively. In addition, a 0.79% reduction in hemoglobin A1c, a 14% reduction in blood glucose level and a significance of 5%, a 58% reduction in insulin level and a significance of 20%, and an insulin resistance index. A 62% reduction and a 20% significance were shown in the low molecular weight insulin group, respectively.

Table 13 shows the fecal weight, fecal excretion bile acid amount, adiponectin level, blood glucose level, insulin level and measured values (mean value) and standard deviation of the insulin resistance index in mouse experiments, and the untreated elbow group is used as the denominator. Table 14 shows the rate of increase / decrease [%] between groups, the determination of the rate of increase / decrease, the p-value in the significance test, and the significance level derived from the p-value.

As described above, the type II diabetes model mouse had a tendency to promote biliary acid excretion and 10% by ingesting the low molecular weight hijiki sample (purified dearginate polysaccharide (hijikifucoidan) hydrolyzed powder derived from hijiki) of Preparation Example 3. It was shown that there was a significant increase in the amount of adiponectin secreted and a significance of 5%.

Furthermore, in this example, ingestion of a low molecular weight Hijiki sample of type II diabetes model mice showed a tendency to significantly reduce the low insulin resistance index, and also showed an effect of suppressing deterioration of insulin resistance. ..

The present invention is useful, for example, in the field of manufacturing for food additives, foods and materials thereof, as well as pharmaceuticals and materials thereof.

Claims (4)

A bile acid adsorbent containing a hydrolyzate of a polysaccharide derived from seaweed, wherein the hydrolyzate is a purified de-alginic acid polysaccharide hydrolyzate derived from Hijiki having a weight average molecular weight of 18,000 Da. .. A method for producing a bile acid adsorbent.
Including the step of pressurizing and heating a seaweed or an aqueous medium containing a polysaccharide derived from the seaweed in the presence of carbon dioxide gas to obtain a hydrolyzate of the polysaccharide derived from the seaweed.
The hydrolyzate is a purified dealginate polysaccharide hydrolyzate derived from hijiki seaweed having a weight average molecular weight of 18,000 Da.
Method. A method for producing a bile acid adsorbent.
It comprises a step of heating, pressurizing and heating a seaweed or a polysaccharide derived from the seaweed in a vinegar medium to obtain a hydrolyzate of the polysaccharide derived from the seaweed.
The hydrolyzate is a purified dealginate polysaccharide hydrolyzate derived from hijiki seaweed having a weight average molecular weight of 18,000 Da.
Method. A food containing the bile acid adsorbent according to claim 1.

Images