WO2005105852A1 - Novel arabinogalactan, substance with antidiabetic activity and method of use thereof - Google Patents

Abstract

A highly effective safely ingestible antidiabetic medicine or functional food with antidiabetic activity, provided as a result of finding a novel ingredient with antidiabetic activity contained in food. In particular, there is provided an arabinogalactan extracted from the skin of white sweet potato with a water base solvent and purified, comprising a skeleton of β1,6-bonded Gal sugar chain highly branched by β1,3-bonds so that (1) αAraf is α1,3-bonded or (2) αAraf(1→5)αAraf is α1,3-bonded, as side chains, to about 65 to 75% of Gal’s constituting the skeleton and that (3) αRha(1→4)βGlcUA is β1,6-bonded to some or all of terminal Gal’s of the skeleton, and having a molecular weight of 10×104 to 20×104, wherein the constitutional ratio of rhamnose to glucuronic acid is approximately 1:1, the constitutional ratio of arabinose to galactose approximately 1:2 and the constitutional ratio of rhamnose to arabinose approximately 1 : 5 to 6. This arabinogalactan is used as a medicine with antidiabetic activity.

Description

 Specification

 Novel arabinogalatatan, antidiabetic substance and method of use thereof

 The present invention relates to a novel arabinogalatatan, an antidiabetic substance and a method for using the same

More specifically, the present invention relates to a novel arabinogalatatan having an antidiabetic action. Background art

 [0002] Diabetes is the most serious problem in lifestyle-related diseases. According to the Ministry of Health, Labor and Welfare's 2002 Survey on Diabetes Mellitus, the total number of diabetic patients and diabetic reserves is estimated to be about 16.2 million, and this number is steadily increasing. It is said that the increase in lifestyle-related diseases, including diabetes mellitus, is mainly caused by changes in dietary habits. In particular, increase in fat intake and excessive intake of salt have been pointed out as major causes. Above all, it is thought that the shortage of dietary fiber is deeply involved. Adequate dietary fiber intake is important for the prevention and treatment of lifestyle-related diseases, and a daily intake of 20-30g is recommended.

[0003] Dietary fiber includes cellulose, hemicellulose, lignin, chitin and other insoluble dietary fibers and konnyakumannan (darcomannan), guar gum (galatatomannan), arabinoxylan, arabinogalatatan, and the like. There is pure water-soluble dietary fiber. So far, both have been regarded as substances that are not degraded by human digestive enzymes, have no nutritional value, and hinder the efficiency of use of nutrients, and have been treated as unnecessary foods. However, their usefulness, such as improving the intestinal flora, activating gastrointestinal motility, and reducing digestion and absorption of carbohydrates and lipids, has been gradually clarified and attracted attention. The mechanism has not been elucidated yet, but short-chain fatty acids, etc., produced during fermentation by enteric bacteria regulate hormonal secretion in the body, thereby affecting cholesterol synthesis and sugar metabolism in the liver. (J Am Diet Ass, 103 (l), 86-96, 2003, Diabetes care., 23 (l), 9-142000, Am J Gastroenterol., 85, 549-553, 1990 ). In addition, it has been revealed that water-soluble dietary fiber has many physiological functions such as immunological activity and antitumor activity (Carbohydr Polym., 25, 269-276, 1994, (Phytochemistry, 27 (8), 251-2517, 1988) ₀ The essence of dietary fiber is polysaccharides, and it is said that among polysaccharides, insoluble polysaccharides require high intake. In recent years, attention has been focused on water-soluble polysaccharides that can achieve an effect with a lower volume.

[0004] Galatatmannan (Nutr.

 Res., 20 (9), 1301-1307, 2000) perarabinoxylan (Clin.

 Exper. Pharm. Phys., 27 (l-2), 41-45, 2000) has been shown to have antidiabetic activity. However, almost all of the water-soluble polysaccharides, arabinogalatatan, have been reported to exhibit immunostimulatory effects, and galactoses with galactose (Gal) bound to | In the main chain, the oligosaccharide in which / 3 arabinopyranose (Amp) and arabinofuranose (Araf) are α 1,3 linked at the 6-position of galactan ゃ Araf and Gal are j8 1,3 linked Oligosaccharides, arabinogalactan (AGII) with oligosaccharides linked to Gal and Gal as side chains, have been reported to lower blood glucose levels in hyperlipidemic patients V ヽ(2001 IFT Annual Meeting).

[0005] Arabinogalatatan is subdivided! /, But is roughly classified into two types. That is, it is classified into pectinic arabinogalatatan (hereinafter type I-AG) and arabinose 3,6-galactan (hereinafter type II-AG). The former type I—AG has galactose (Gal) with β 1,4 linked | 8 1,4 galactan in the main chain and two α-arabinos at position 3 of the constituent Gal residue. Oligosaccharide (α Araf (1 → 5) a Araf (1 →)) in which furanose (α Araf) is linked to α 1,5 is linked. On the other hand, type III—AG is mainly Liquids and other powers are also obtained, and many have an arabino 3,6-galactan structure in the main chain and side chains consisting of Araf, Gal, etc. are bonded to the main chain by | 81,3. The composition ratio of Ga 1 varies greatly depending on the plant species. Generally, Gal is high, and arabinogalatatan contains fucose (Fuc) in addition to the main constituent sugars Ara and Gal. , Rhamnose (Rha), xylose (Xyl), glucuronic acid (GlcUA), etc., and their structures are very diverse. AGIIa has j8 1,6-galactan with galactose linked to | 8 1,6 in the main chain, and a Araf and two α Arafs are α ΐ, 5-linked oligosaccharide Araf (1 → 5) α Araf (1 →) or an oligosaccharide in which Gal or a Araf and Gal are α 1,3 linked (α Araf (1 → 3) α Gal (1 →) And It has a structure.

 [0006] Under these circumstances, diet treatment is the least burdensome in treating diabetes, since long-term self-management is required. Further, in consideration of side effects and ease of ingestion, it is desired to develop an antidiabetic drug which is preferably derived from food or is close to a food form.

 [0007] Thus, the present applicant has searched for antidiabetic substances from various foods, and found that an extract from white sweet potatoes has an antidiabetic effect on humans. (Patent Document 1: Japanese Patent Application Laid-Open No. 10-120584). This extract has a molecular weight of about 20,000 and its main component is said to be protein. The inventors of the present application have investigated an anti-diabetic substance that is more effective, and found that arabinogalatatan having a completely different structure from the above-mentioned conventional arabinogalatatan has an antidiabetic effect. And completed the invention of the present application.

 Patent Document 1: JP-A-10-120584

 Disclosure of the invention

 Problems to be solved by the invention

[0008] An object of the present invention is to find a new antidiabetic component existing in food and provide a more effective and safe ingestible antidiabetic drug or a functional food having an antidiabetic effect. .

 Means for solving the problem

The present invention relates to arabinogalatatan having a skeleton in which a β 1,6-bonded Gal sugar chain is highly branched by β 1,3 bonds, wherein Gal constituting the skeleton has a side chain of ( 1) a Ar af is α ΐ, 3 bond or (2) a Araf (l → 5) α Araf (1 → is α 1,3 bond and (3) _a Rha (l → 4) | 8 GlcUA is Arabinogalatatan with an average molecular weight of 100,000 to 200,000 bonded to part or all of the terminal Gal of the backbone of the skeleton, of which the constituent ratio of rhamnose: dalcuronic acid is approximately 1: 1, and arabinose: The present invention relates to a novel substance called arabinogalatatan having a galactose composition ratio of about 1: 2 and a rhamnose: arabinose composition ratio of 1: 5 to 6. The present invention relates to the substance. Focusing on the anti-diabetic effect of the drug.

[0010] Further, the antidiabetic substance of the present invention can be used in an aqueous system for the skin portion or tuber portion of white sweet potato. A lower alcohol or a lower ketone was added to the concentrated dialysis solution of the extract, and the supernatant obtained after removing the precipitate was fractionated by gel filtration chromatography and Z or ion exchange chromatography.

 (1) Average molecular weight 100,000-200,000

 (2) the force of color development by phenolic sulfuric acid hardly shows absorption around 280 nm,

(3) After adsorption on a weakly basic ion exchange resin, it is eluted with an ionic strength eluent of about 100 to 400 mM aqueous sodium chloride solution.

 Characterized as being an extract! /

[0011] The present invention provides an antidiabetic drug or a health food containing the above substance as an active ingredient, a method of using the substance as an antidiabetic agent, and a method of adding a blood sugar level so that the blood sugar level can be maintained in a normal range. Food production methods.

 The invention's effect

 The structure of arabinogalatatan of the present invention is completely different from the structure of conventional arabinogalatatan, and can be used as an antidiabetic substance. This is mainly contained in white sweet potatoes and is considered to be excellent in safety. In addition, arabinogalatatan in the present invention is a kind of water-soluble dietary fiber, like glycomannan arabinoxylan, and is therefore not only an antidiabetic substance but also the same as other dietary fibers such as promoting defecation. Action can be expected at the same time. Since it is water-soluble, it can be taken and has a small amount of rinsing power.Even with a small amount, it is effective in various forms of food, such as drinks and jelly-like foods, and powdered forms such as sprinkles. It is often offered as a food product.

 Brief Description of Drawings

FIG. 1 is a view showing a basic skeleton of arabinogalatatan according to the present invention.

 FIG. 2 is a diagram exemplifying a mode of bonding a side chain to a basic skeleton of arabinogalatatan according to the present invention.

 FIG. 3 is a view schematically showing a purification step of arabinogalatatan according to the present invention.

FIG. 4 is a view showing an elution pattern of the crude extract in FIG. 3 by gel filtration chromatography. ―Country は means absorption at 490nm by phenol sulfuric acid method, ― ♦ ― means 280nm Shows the absorption at. In the figure, the fractionation fractions are indicated by F1 to F4.

 FIG. 5 is a view showing an elution pattern of ion exchange chromatography of an F1 fraction in filtration gel chromatography. -■-indicates absorption at 490 nm by the phenol sulfuric acid method, and ♦ indicates absorption at 280 nm. In the figure, the fractionation fractions are indicated by “200 mM NaCl” and “400 mM NaCl”.

 FIG. 6 is an analysis chart by MALLS-SEC of a 200 mM NaCl fraction in ion exchange chromatography. The dotted line shown in the figure indicates the molecular weight distribution of arabinogalatatan.

 FIG. 7 is an analysis chart by HP AEC-PAD of a 200 mM NaCl fraction in ion exchange chromatography.

 FIG. 8 is an analysis chart by GS-MS of a 200 mM NaCl fraction in ion exchange chromatography.

 FIG. 9 is an HMBC analysis diagram of arabinogalatatan according to the present invention.

 FIG. 10 is a graph showing a hypoglycemic effect in a continuous oral administration test.

 FIG. 11 is a graph showing a time-dependent change in a blood glucose level during a glucose load.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0014] The arabinogalatatan, which is the active ingredient of the present invention, is obtained mainly by extracting and separating the skin power of white sweet potatoes. White sweet potatoes have a scientific name of Ipomoea Batatas SD, a type of sweet potato (Chimoto iDomoea Batatas Poiret), and are sometimes called "Simon potato", "Kiapo potato", or "White satsimo". . In the present invention, tubers of white sweet potato, particularly those obtained from the skin thereof, are preferably used. However, as long as it has the following structure, it is not limited to white sweet potatoes and their related plant power.

[0015] The arabinogalatatan has a basic skeleton shown in Fig. 1 in which galactose is / 31,6 linked to a sugar chain, and is highly branched by 1,3 bonds. In other words, the main chain having galactose j8 1,6 bonds has a large number of oligosaccharides having galactose / 31,6 bonds or galactatodalican has a large number of j8 1,3 bonds with side chains. The sugar chain with j8 1,6 linked to galactose is complicated by the 131,3 bond, as in the case of oligosaccharide or galactoglycan with j8 1,6 linked to galactose having a | 81,3 linked side chain. Branched It has a skeleton. As a result, it has a relatively bulky structure, and in this point, it has a feature that is significantly different from conventional arabinogalatatan having a linear sugar chain as a main chain of galactose.

[0016] The arabinogalatatan of the present invention has three types of side chains (1) (2) with respect to the basic skeleton shown in Fig. 1 as shown in Figs. 2 (A) and (B). (3) is combined. Side chain (1) is terminal a Amf, Side chain (2) is oligosaccharide ( _a Araf (l → 5) a Ar a (l →), side chain of two arabinofuranoses linked by a1,5) (3) a 1, 4 linked oligosaccharides in rhamnose glucuronic acid _{(a Rha (l → 4)} | 8 G1 a _C UA (1 →), the side chain (1) (2) respectively the skeleton The side chain (3) has j81,6 bonds to galactose, which constitutes the above skeleton, in the side chain (3).

 [0017] Although the bonding position of the side chains (1), (2), and (3) and the branching position of the extra-galactose sugar chain are unknown, about 65 to 75% of the side chain (1 ) Or the side chain (2) is bound, and the side chain (3) is bound to 5 to 10% of the galactose constituting the skeleton has an antidiabetic action. It should be noted that the side chain (1), (2) and (3) binding ratios to Gal may be out of this range. In other words, the bond ratio is only a guide, and even if the obtained bond ratio of arabinogalatatan is out of this range, the basic skeleton and the side chains (1) (2) (3) and the average As long as it has a molecular weight, it can be said that it is the same substance as arabinogalatatan of the present invention.

 As described above, in the novel arabinogalatatan according to the present invention, as shown in FIG. 1, galactose has β 1,6 linked sugar chains, and galactose has β 1,6 bonds. As shown in Fig. 2 (A) and (II), the side chain (1) or (2) is attached to the galactose sugar chain linked to ΐ, 6 while the sugar chain is branched by j8 1,3 bond everywhere. Thus, the structure in which the side chain (3) is linked to the galactose of the sugar chain by β 1, 3 and the side chain (3) is | 81,6 linked to a part or all of the sugar chain terminal of the galactose as shown in FIG. Have.

The average molecular weight of arabinogalatatan in the present invention is about 100,000 to 200,000, and as shown in FIG. 6, its molecular weight distribution is extremely narrow as compared with other polysaccharides. Is also its characteristic. Here, the average molecular weight means a value obtained by the method described in Examples, and the arabinogalatatan in the present invention has a value between 100,000 and 200,000. Therefore, when the molecular weight is measured by the following MALLS-SEC method, Some peaks with molecular weights outside the range of 100,000 to 200,000 may be mixed.

 [0020] According to actual measurement, the constituent sugar ratio of this arabinogalatatan is rhamnose: arabinose: galatatose: glucuronic acid = 1.2: 6.7: 14.1: 1.0, and approximately rhamnose: glucuronic acid = 1: 1, arabinose: galactose = 1: 2, rhamnose: arabinose = 1: 5-6.

 [0021] The arabinogalatatan of the present invention can be obtained, for example, by the method shown in FIG. First, a dried product of white sweet potato, so-called caiapoimo skin, is crushed or powdered, and water is added. Since arabinogalatatan is considered to be mainly present in the tuber skin in large amounts, the viewpoint of yield is preferably used mainly as the tuber skin. Of course, the whole potato can be used as a raw material, and above ground stems and whole plants can be used. Similarly, when extracting from other plants and the like, a portion where a high yield is expected is preferably used.

 [0022] Since arabinogalatatan is a type of water-soluble polysaccharide, an aqueous extraction solvent is used first as in the case of extraction of other water-soluble polysaccharides. Water is most preferred as the extraction solvent. A lower alcohol (C1 to C4) such as ethanol, a hydrophilic solvent such as acetone, or a mixture of water and such a hydrophilic solvent may be used. Further, at the time of extraction, heating and heating may be appropriately performed. The extraction method is not particularly limited. For example, the juice is crushed and squeezed by using a juicer or a mixer, and then the insoluble matter is removed by a centrifugal separation method or filtration. Alternatively, the extraction solvent may be added again to the residue once extracted, and then added to the previous extract. These are common methods for extracting polysaccharides.

Next, the obtained extract is dialyzed against deionized water. By dialysis against deionized water, low molecular substances such as salts and monosaccharides in the extract, which are impurities, are removed. The dialysis membrane to be used preferably has a molecular weight rejection of about 12000 to 15000 Da or more. A smaller rejection (limit) molecular weight can achieve the object sufficiently, but is disadvantageous because the analysis time becomes longer. In addition, dialysis membranes with a high efficiency of analysis, that is, dialysis membranes with a large number of dialysis holes per membrane, are commercially available as dialysis membranes of about 12000 to 15000 Da (for example, Spectra Biotech Membrane Z manufactured by Funakoshi). Pore 2.1), since efficiency is desired, those in the above range are preferred. In addition, dialysis produces precipitates. The precipitate may be removed by centrifugation or the like as appropriate. Thus, impurities having a low molecular weight of about 100 OODa or less are removed. This step is not an essential step, but by performing this step, the working efficiency in the following step, gel filtration chromatography, can be significantly increased. Then, purification is further performed using the obtained dialysis solution. At this time, the concentration in the dialysis solution cannot be said to be sufficiently high, so concentration should be performed as necessary. The concentration is performed under reduced pressure using an evaporator or the like, preferably at a low temperature of about 40 ° C.

 Then, a lower alcohol or a lower ketone is added to the obtained internal dialysis solution (or a solution obtained by concentrating the same), and the mixture is allowed to stand. Further, impurities other than the target substance are precipitated and removed. Ethanol or methanol is preferred as the lower alcohol, and acetone is preferred as the lower ketone. This operation is preferably repeated. When repeating this operation, it is preferable to gradually increase the concentration of alcohol or the like (ethanol, methanol, acetone).

. That is, first, an alcohol or the like is added at a low concentration to form a precipitate, which is removed. Then, alcohol and the like are further added to the liquid from which the precipitate has been removed to increase the concentration of the alcohol and the like, and further a precipitate is generated. The process is repeated until no precipitation occurs when alcohol or the like is added. However, care must be taken that the alcohol concentration does not exceed about 60 v / v%. If the alcohol concentration exceeds about 60 v / v%, the target substance may precipitate, so add it while paying attention to the alcohol concentration. In addition, it is preferable that the concentration of alcohol be as high as at least 20 v / v% or more to generate a precipitate. The supernatant separated by centrifugation or the like is concentrated to obtain a crude extract. Since the arabinogalatatan of the present invention contains rhamnose as a constituent sugar, precipitation hardly occurs even when the concentration of alcohol or the like is increased to about 60 v / v%. Even when the concentration is low, the target substance may precipitate (for example, at a liquid temperature of 4 ° C, it precipitates with 60 v / v% ethanol). Therefore, sufficient attention must be paid to the operating environment. In addition, if the object is left for a long time, for example, about 48 hours, the target substance may precipitate. Therefore, it is preferable that the object is kept still for about 24 hours. This purification technique is a general technique for purifying polysaccharides. Also, in view of the high rhamnose content, the concentration of arabinogalatatan of the present invention should be as high as not to precipitate. Alcohol and the like are added all at once to remove most of the impurities by precipitation.Also, alcohol and the like are added to a high concentration (at least 60 v / v% or more) to precipitate the desired product to obtain a crude extract. A little monster.

 [0025] Then, the crude extract is further fractionated to obtain the desired arabinogalatatan.

 The fractionation is performed by a combination of gel filtration chromatography and ion exchange chromatography.

 [0026] In the fractionation by gel filtration chromatography, as a filler, trade name Toyopearl H W-65S (700 to 1.3 million limiting molecular weight dextran, Tosoh Corporation) or trade name

 Sephacryl S-400HR (Limited molecular weight dextran: 1 to 2,000,000, globular protein: 2 to 8,000,000, Amersham Bioscience Co., Ltd.) is exemplified. The eluent varies depending on the filler used, but is preferably water. Then, the absorbance of the eluate at 280 nm and 490 nm after color development with phenolic sulfuric acid was measured, and there was no absorption in the ultraviolet region of 280 ηm, showing absorption at 490 nm, corresponding to a molecular weight of about 100,000 to 200,000 Da. Sample peaks.

 Next, the fractionated substance is further fractionated by ion-exchange resin to obtain a target substantially single substance, arabinogalatatan. The ion exchange resin used at this time is, for example, a weakly basic ion exchange resin represented by DEAE-Sephadex A50 (manufactured by Amersham Bioscience Co., Ltd.) and Amberlite IRA96SB, IRA67. Then, the antidiabetic substance captured by the ion exchange resin is eluted with the strong ionic electrolyte. An aqueous sodium chloride solution is preferably used for the strong ionic electrolyte, and the ion strength at that time is preferably equivalent to the ion strength of an aqueous sodium chloride solution of 100 to 400 mM, preferably about 200 mM. . If the concentration exceeds 400 mM, other than the target substance, for example, a basic substance such as protein may be eluted together. Also in this fractionation, the absorbance at a wavelength of 280 nm of the eluate and a wavelength of 490 ηm after coloring with phenol sulfuric acid are used as indices, and a fraction showing a substantially single peak is collected.

[0028] Then, the fractionated eluate is, for example, concentrated under reduced pressure, and then lyophilized to obtain powdery arabinogalatatan as a substantially single substance. The obtained arabinogalata The specific rotation of the tin is [α] ²⁰ = —37 ° ± 1 ° (c = 0.08, water)

 D

 A remarkable spectrum showing j8-1,6-galactan and a Rha (l →

4) A very specific peak is shown for the arabinogalatatan of the present invention, which indicates the presence of β GlcUA (l →.) The composition ratio of this saccharide is as described above. For practical use as an antidiabetic agent, both fractionation steps by gel filtration chromatography and ion exchange chromatography are not essential, and at least one of gel filtration chromatography and ion exchange chromatography is more preferable. It is possible to use an extract, which is fractionated by gel filtration chromatography and almost all proteins are almost polysaccharide components, and of course, it is highly purified by both fractionation steps. Is obtained.

 [0029] As described below, this arabinogalatatan has an action of lowering the blood sugar level or maintaining the blood sugar level within a certain range by oral administration or intravenous administration. It is powdery at room temperature and easily soluble in water. Therefore, excipients such as starch and lactose can be added to this arabinogalatatan to form solid preparations such as powders, tablets, capsules, etc., or it can be dissolved in physiological saline and provided as injections. it can. Further, it may be dissolved in water and provided as an oral liquid preparation.

 [0030] In addition, the present invention is not limited to pharmaceuticals, and is provided as a so-called health food in the form of tablets, capsules, and the like, which is added to soft drinks and low-calorie foods in all forms of food. Is done.

 [0031] As described above, the arabinogalatatan of the present invention is intended to maintain and reduce the blood sugar level regardless of the form thereof, not only when it is used as an antidiabetic substance in human or animal drugs. For this purpose, it is provided as so-called health food or food for specified health use (also referred to as functional food), and as a general food or animal feed to which it is added.

 [0032] The dose may be appropriately adjusted depending on the administration subject, symptoms, etc., but is generally about 0.1 to: LOOmgZ body weight kg per day, and the above substance is added in an amount capable of maintaining a blood glucose level in a normal range. Is done.

Example 1 Hereinafter, the present invention is more specifically described based on the following examples. The present invention is not limited to the following examples.

 [0034] Arabinogalatatan was obtained through a purification process according to FIG. First, 10 L of water was added to 1 kg of dried skin of white sweet potato (crushed skin) and extracted with stirring at room temperature for 4 hours. The obtained mixture was centrifuged (7500 rpm, 30 minutes), and the residue was separated by filtration. Next, the obtained extract was dialyzed against deionized water at room temperature for 3 days. For dialysis, put the extract into a dialysis tube of the product name Dialysis Membrane Size 36 (pore size 24A, limiting molecular weight 12000-14000 Da, manufactured by Wako Pure Chemical Industries, Ltd.), and 5-6 L of deionized water per 1 L of extract Was performed by exchanging deionized water three times a day. Then, the insoluble portion generated in the internal solution for the separation was removed by centrifugation (7500 rpm, 30 minutes).

 [0035] The dialysate from which the precipitate has been removed is heated and concentrated at about 40 ° C using an evaporator, and then an appropriate amount of ethanol is added so as to have a concentration of about 20 v / v%, and allowed to stand at 4 ° C. did. The resulting precipitate was removed by centrifugation (7500 rpm, 30 minutes). Furthermore, ethanol was added to the supernatant, and the alcohol concentration was adjusted to about 40 v / v%. Then, ethanol was added again to adjust the alcohol concentration to about 60 v / v%, and the mixture was allowed to stand for one day and night (under 4 ° C) .The supernatant was collected, concentrated, and lyophilized to freeze the crude polysaccharide. An extract (3.8 g) was obtained.

[0036] The crude polysaccharide extract was subjected to gel filtration chromatography and ion exchange chromatography, and further purified. An aqueous lwZv% solution (lgZlOO mL) of the crude polysaccharide extract was prepared, and centrifuged (15000 rpm for 20 minutes) to collect a water-soluble portion. This was fractionated into four fractions (hereinafter referred to as F1 to F4) using gel filtration chromatography. The fractionation was performed on a 15 mL Zl tube using Toyopearl HW-65S (manufactured by Tosoichi Co., Ltd .: 880 mL packed in a 4.5 × 700 cm column) as a carrier and water at a flow rate of 0.5 mL Zmin as an eluent. Detection by the phenol-sulfuric acid method (wavelength 490 nm) and UV absorption (280 nm) was performed for each tube and divided into four fractions F1 to F4. Figure 4 shows the results. The obtained F1 to F4 fractions were F1 fraction: tube No. 19 24Z concentrated mass 246.5mg, F2 fraction: tube No. 25 30Z concentrated mass 167.5mg, F3 fraction: tube No31 36Z concentrated mass 150.5mg, F4 fraction : The tube No. 37-43Z had a concentrated mass of 146.5 mg.

 [0037] Next, as a preliminary test, the extracts of the obtained fractions F1 to F4 were examined for hypoglycemic action. Blood glucose was measured after oral administration of lOOmgZ kg of each of the extract concentrates F1 to F4 to each group of 5 male 7-week-old dbZdb mice for 5 weeks, and blood glucose levels were measured. Was seen. In addition, since the extract of F1 was more effective than the extract of F2, the extract of the fraction F1 was used in the following analysis.

 Next, the extract of the above-mentioned fraction F1 was subjected to ion exchange chromatography and further purified. After loading the above extract on DEAESephadexA-50 (manufactured by Amersham Biosciences) as an ion exchange resin, deionized water and 200 mM and 400 mM aqueous sodium chloride solutions were eluted stepwise. Elution was performed at a flow rate of 0.5 mL, and the eluate was collected in a 15 mL / l tube. The first 10 tubes were eluted with deionized water, the next 10 were eluted with a 200 mM aqueous sodium chloride solution, and the rest were eluted with a 400 mM aqueous sodium chloride solution. The eluate was detected for each tube by the phenol-sulfuric acid method (wavelength 490 nm) and UV absorption (280 nm). Figure 5 shows the results. As a result, the water fraction did not contain any substance considered to be an active ingredient, and the 400 mM fraction showed the presence of polysaccharides and proteins. On the other hand, in the 200 mM fraction, only the polysaccharide with almost no protein was present. From these results and the results obtained by gel filtration chromatography, it was considered that the fraction eluted with the 200 mM aqueous sodium chloride solution was a component having a hypoglycemic effect. The yield of the extract obtained with a 200 mM fraction was about 0.09% based on the dried white sweet potato skin.

Next, the molecular weight and molecular size of the above components were determined using a multi-angle light scattering detector (MALLS) and size exclusion chromatography (SEC). The measurement conditions are as follows. The columns used were OH-pak SB-806MHQ (manufactured by Showa Denko KK), the detectors were a multi-angle light scattering detector DAWN · E (manufactured by Wyatt) and a differential refractive index detector RI 8020 (manufactured by Tosoh Corporation), The column temperature is room temperature (25 ° C), the mobile phase is a 200 mM NaCl aqueous solution, the sample concentration is lmgZmL (dissolved in the mobile phase), the flow rate is 0.5 mLZmin, and the injection volume is 100 L. In addition, use the above 200 mM eluted fraction for the sample, attach a syringe filter (pore size 0.2 μm) to the syringe at the time of injection, and inject while filtering. It was. Figure 6 shows the results. As can be seen from FIG. 6, the retention time of this material was about 19 minutes, almost a single peak. The molecular weight of this product was about 100,000 in small places and about 200,000 in large places, and the average molecular weight was about 130,000. Its molecular weight distribution is high purity and relatively homogeneous, showing a nearly single peak with a narrow width of about 100,000. Here, the average molecular weight refers to a weight average molecular weight in a single peak indicating the substance obtained by measurement using the above measuring device.

 [0040] The structure of arabinogalatatan of the present invention was determined as follows. First, the composition ratio of the constituent sugars was examined. The fraction eluted at 200 mM by ion exchange chromatography was completely hydrolyzed with formic acid and trifluoroacetic acid, and the sugar composition was determined by the HPAEC-PAD method. The measurement conditions are as follows. The column used was CarboPac PA-1 (4 X 250 mm: manufactured by Nippon Dionetas Co., Ltd.), elution was performed with 0.2M NaOH (0 → 5 minutes) followed by a linear gradient 0 → 0.45M sodium acetate (in0.2N NaOHZ5 → 35 minutes), The flow rate is l.OmL Zmin and the detector is a pulsed envelometry detector. The results are shown in FIG. As a result, it was found that the arabinogalatatan was composed of Rha, Ara, Gal, and GlcUA at a ratio of 1.2: 6.7: 14.1: 1.0.

 [Table 1] Detection peaks between Nadera (min) Area Height Content ratio

Rha 2.77667 9.77629 80.74749 1.2

Ara 3.23000 54.67402 389.90249 6.7

Gal 3.85333 116.06262 716.32060 14.1

GlcUA 19.3233 8.20344 45.36891 1.0

Next, the fraction eluted with a 200 mM sodium chloride aqueous solution was subjected to partial methylation analysis. The analysis was performed using a gas mass analyzer (GC-17A, GCMS-QP5000: Shimadzu Corporation), DB-225GC column (J & W Scientific: 0.25m X 30m X 0.25m), 170 ° C for 5 minutes, 170 → 210 The procedure was carried out under a column heating program of ° C (heating rate 2 ° CZmin) at a vaporization chamber temperature of 230 ° C and a detector temperature of 230 ° C. The results are shown in Figure 8 and Table 2.

[Table 2] Peak binding mode Area Content ratio Fragment

1 Araf-(1 → 16444834 18.9 117,101,129,87,71,161,59,145

 Rha- (1 →

 2 → 5) -Araf- (l → 7004480 8.0 117,87, 129, 101,71,58, 189, 161

3 Gal-(l- → 872049 1.0 101,117,145,129,161,87,71,205,59

4 → 3,5) -Araf- (1 → 875469 1.0 117,85,58,127,99,159,71

 5 → 3) -Gal- (l → 5121040 5.9 117,129,101,71,161,87,58,233,173

6 → 4) -GlcUA- (l → 1465712 1.7 117, 113,99,87, 101,71,233, 129, 173

7 → 6) -Gal- (l → 2576058 3.0 101,99, 117,87, 129,71,59, 189,233

8 → 3,6) -Gal- (l → 16207472 18.6 117, 129,87,58, 101,74, 189,159, 139

As can be understood from FIG. 8 and Table 2, the arabinogalatatan of the present invention has a terminal a Araf or terminal Rha (peak 1) and terminal Gal (peak 3), 1,5 bond. A with Araf (peak 2), Gal with 1,3 bond (peak 5), Gal with 1,6 bond (peak 7), Gal with 1,3,6 bond (peak 8) and 1,3 It has been revealed that Gal (peak 4) has eight types of bonding formulas having five, five bonds. Although no Rha-derived peak was detected, it is probable that the retention time with the terminal Araf was very close, and the two were not sufficiently separated and were detected at the same position.

Next, to clarify the linkage of each sugar, various NMR ^ vectors (, ¹³ C-NMR, ¾

-'HCOSY, HOHAHA, HMQC, HMBC) were analyzed. First, we clarified the chemical shift of each sugar. The results are shown in Table 3. Furthermore, based on the results, HMBC analysis (see FIG. 9) was performed to clarify the partial structure and sugar chain linkage shown in FIG. In other words, Gal has a galactose chain with j8 1,6 bonds and a galactose chain branched by 1,3 bonds, and 65-75% of the glassose has (1) terminal a Araf or (2) two α-arabinoffs. Ranosu is shed 1, 5 linked oligosaccharide (2 monosaccharide) is 3 1, 3 bound, and (3) rhamnose 1 _alpha to glucuronic acid, 4 linked oligosaccharide (2 monosaccharide) whose 5-10 Β 1,6 bonds to% galactose.

[Table 3] Position

 1 2 3 4 5 6

 aAraf (1 → δΗ πι) 5.08 3.96 3.76 3.93 3.63 3.54

 5C¾) pm) 110.7 83.1 78.5 85.8 63.3

 5H¾) pm) 4.91 3.87 3.76 3.91 3.63 3.54

 5C¾) pm) 1092 82.8 78.5 85.8 63.3

 → 5) aArai l → SH ^ ipm) 5.08 3.96 3.84 3.98 3.70 3.62

 5C (ppm) 110.7 83.1 78.7 84.0 68.8

 aRha (l → δΗήρρηχ) 4.60 3.74 3.58 325 3.84 1.07

 5C (pin) 102.4 75.4 72.1 73.9 70.7 18.5

 → 4) p (MJA (l → 4.33 S20 3.41 3.45 3.56

 105.1 752 76.3 81.0 n.a. 176.7

[Confirmation of hypoglycemic action]

 A hypoglycemic effect test was performed using 5-week-old dbZdb mice (male). The breeding environment consisted of a light-dark cycle of 7:00 to 19:00 with a 12-hour light-on cycle, a temperature of 23 to 24 ° C, and a humidity of 60 to 70% RH. The values were divided into two groups (six in each group) so that there was no significant difference between the groups. The administration groups were the non-administration group (physiological saline) and the arabinogalatatan administration group (20 mgZkg) obtained in the above Example. The administration was oral once daily using a sonde for 5 consecutive weeks. During the test period, both feed and drinking water were freely available. Blood was collected from the mouse tail vein before administration and every week after the start of administration, and the obtained serum was used to confirm the hypoglycemic effect. The measurement results are shown in Table 4 and FIG.

 [Table 4]

 Blood: Mean Sat SD 0.05, ** υ <0.01 vs control group

[Confirmation of glucose tolerance improving action]

After the test for confirming the hypoglycemic effect, glucose was orally administered to each group at 2 gZkg, and blood was collected from the mouse tail vein at 0 (before glucose loading), 60 minutes and 120 minutes later, and the blood glucose level was measured over time. The changes were examined. The results are shown in Table 5 and FIG. This test was performed after a 12-hour fast after the end of the hypoglycemic effect test. [0050] [Table 5]

 Blood fiber: Mean Sat SD * p <0.05, ** p <0.01 vs control group Example 2

 Next, using the eluted fraction (concentrated, lyophilized product) of a 200 mM aqueous sodium chloride solution in liquid chromatoda fluffy, the health food of the present invention was prepared in a conventional manner according to the following formulation (per tablet). A tablet (1000 tablets) was prepared.

 140 mg of arabinogalatatan according to the present invention

 4mg sucrose fatty acid ester

 4mg calcium carbonate

 Reduced maltose syrup lOmg

 Lactic anhydride 22mg

 Crystalline senorelose lOmg

 Milk calcium lOmg

 [0052] The arabinogaratatan (AG) is a kind of Apiaceae tortoise, Angelica acutiloba KITAGAWA (Carbohydr

 Res, 193, 173-192, 1989), Viscum album L.

 Phytochemistry, 27 (8), 2511-2517, 1988), Leguminosae gum Acacia Senegal gum (Carbohydr Res.2.104.1966), Obaco family Oobako Plantago major L. (Carbohvdr Res, 35, 145-153, 1998), Asteraceae Echinacea purpurea (Carbohvdr

Res., 327, 497-504, 2000), and there are many AGs having a partial structure (side chain) of Am found in arabinogalactan derived from white sweet potato. However, the arabinogalatatan of the present invention differs from those in the basic skeleton or the side chain, and is composed of → 6) | 8 G _a1 (l → force). There is no report of a polysaccharide having a structure in which a Rha (l → 4) j8 GlcUA (l → It was concluded that the white sweet potato-derived arabinogalatatan according to the present invention was completely novel. Butane has a hypoglycemic effect and is very useful as an antidiabetic substance.

Claims

The scope of the claims  [1] A galapinogalactan having a skeleton in which β 1,6-linked Gal sugar chains are highly branched by β 1,3 bonds,  (1) a Araf has a << 1, 3 bond or (2) a Ara f (l → 5) a Araf has an α ΐ, 3 bond as a side chain to Gal constituting the skeleton, and (3) a Rha (l → 4) arabinogalatatan with an average molecular weight of 100,000 to 200,000 in which j8 GlcUA is | 81,6-bonded to part or all of the terminal Gal of the skeleton.  [2] The ara of claim 1, wherein the ratio of rhamnose: glucuronic acid is approximately 1: 1, the ratio of arabinose: galactose is approximately 1: 2, and the ratio of rhamnose: arabinose is 1: 5-6. Pinogalactan. [3] The β 1,6-linked Gal sugar chain has a skeleton highly branched by the β 1,3 bond, and (1) a Araf has << 1, 3 bond or (2) a Araf (l → 5) a Araf binds << 1, 3 bond and (3) _a Rha (l → 4) GlcUA binds to part or all of terminal Gal of the skeleton | 8 1,6 bond Average molecular weight 100,000-200,000, rhamnose: glucuronic acid composition ratio is about 1: 1, arabinose: galactose composition ratio is about 1: 2, rhamnose: arabinose composition ratio is 1: 5-6 Nogaratatan A powerful antidiabetic substance.  [4] Lower alcohol or lower ketone is added to the dialysis solution of the aqueous extract of the skin or tuber of the white sweet potato, and the supernatant obtained after removing the precipitate is subjected to gel filtration chromatography and Z or ion exchange chromatography. Fractionated by graphy  (1) Average molecular weight 100,000-200,000  (2) the force of color development by phenolic sulfuric acid hardly shows absorption around 280 nm, (3) After adsorption on a weakly basic ion exchange resin, it is eluted with an ionic strength eluent of about 100 to 400 mM aqueous sodium chloride solution.  An antidiabetic substance, which is an extract. [5] An antidiabetic drug, characterized in that the antidiabetic substance according to any one of claims 3 and 4 is used as an active ingredient. [6] The antidiabetic substance according to any one of claims 3 and 4 as an active ingredient. A health food characterized by that:  [7] This is an alara pinogalactan having a skeleton in which a β 1,6-linked Gal sugar chain is highly branched by β 1,3 bonds, and (1) a Araf is a side chain of Gal constituting the skeleton. << 1, 3 bond or (2) a Araf (l → 5) a Araf force Sal, 3 bond and (3) a Rha (l → 4) iS GlcUA is part or all of terminal Gal of the skeleton | 8 1,6-linked average molecular weight 100,000-200,000, rhamnose: glucuronic acid composition ratio is about 1: 1, arabinose: galactatose composition ratio is about 1: 2, rhamnose: arabinose composition ratio is 1 : Add lower alcohol or lower ketone to the dialysate of aqueous extract of arabinogalatatan or white sweet potato skin or tuber root which is 5-6, and remove supernatant by gel filtration chromatography and Z or Fractionated by ion exchange chromatography (1) Average molecular weight 100,000 to 200,000 (2) Force to show color by phenolic sulfuric acid Almost no absorption around 280 nm, (3) About 100 to 40 to 40 to 40 after adsorption to weakly basic ion-exchange resin A use method characterized in that an extract eluted with an ionic strength eluate of an aqueous OmM salted sodium salt solution is used as an antidiabetic substance.  [8] A method for producing a food, comprising adding an antidiabetic substance according to any one of claims 3 and 4 in an amount capable of maintaining a blood glucose level in a normal range.