JP6872680B2 - Calcium absorption promoter - Google Patents

Description

The present invention relates to a calcium absorption-promoting agent containing a processed product of Hyuga Natsudaidai, and a calcium absorption-promoting food or pharmaceutical product containing the calcium absorption-promoting agent.

Osteoporosis is a systemic disease in which a decrease in bone mineral content causes a breakdown of bone microstructure, resulting in a decrease in bone strength and an increased risk of fracture. As we enter an aging society in the future, it is expected that prevention and treatment for this disease will become even more important.

Therapeutic agents for osteoporosis are roughly classified into three types: agents that suppress the bone resorption system, agents that stimulate the bone formation system, and agents that promote calcium absorption from the intestinal tract. The present inventors have previously reported that an extract of Citrus natsudaira can be an effective therapeutic agent for osteoporosis by suppressing the bone resorption system and stimulating the bone formation system (Patent Document 1). ). In addition to this, if a drug that promotes calcium absorption can be found, it is expected that osteoporosis can be treated more effectively based on a different mechanism of action.

JP 2006-008625

An object of the present invention is to provide a novel calcium absorption promoter.

The present inventor has found that a processed product of Hinata Natsudaidai has a calcium absorption promoting effect, and has completed the present invention.

Therefore, the present invention includes the following aspects.
(1) A calcium absorption promoter containing a processed product of Hinata Natsudaidai.
(2) The calcium absorption promoter according to (1), wherein the processed product of Hinata Natsudaidai is an extract of Hinata Natsudaidai.
(3) The calcium absorption promoter according to (2), wherein the extract of Hyuga Natsudaidai contains arabinogalactan having a molecular weight of 50,000 to 100,000.
(4) Arabinogalactan
It has β1,3-bonded galactose as the main chain, has a side chain branched from the 6-position of the main chain galactose, and contains β-galactose with 1,6-bonded side chains and α-arabinose with 1,5-bonded α-arabinose.
The calcium absorption promoter according to (3).
(5) A food composition or pharmaceutical product for promoting calcium absorption, which comprises the calcium absorption promoter according to any one of (1) to (4).
(6) A method for producing a calcium absorption-promoting food composition or a pharmaceutical product, which comprises adding the calcium absorption-promoting agent according to any one of (1) to (4).

INDUSTRIAL APPLICABILITY The present invention provides new agents, foods, pharmaceuticals and the like having a calcium absorption promoting effect.

FIG. 1 shows a chromatogram when the Hyuganatsu treated product 3 prepared in the example was subjected to HPLC. FIG. 2 shows a chromatogram when the active fraction 1 prepared in the example was subjected to HPLC. FIG. 3 shows the Ca absorption promoting effect of the Hyuganatsu treated product 2 (“treated product” in the figure) or the active fraction 1 (“active fraction” in the figure) prepared in the examples in the rat inversion small intestine. The value of Ca concentration shows the average value (n = 5, 6) of the inverted small intestinal fluid, and the boxplot shows the standard error. * Indicates that there is a significant difference at P <0.05 with respect to the control.

1. 1. Calcium Absorption Promoter In one aspect, the present invention relates to a calcium absorption enhancer, which comprises a processed product of Hyuga Natsudaidai. Hyuganatsu oranges (also referred to as "Hyuganatsu" in this specification) are citrus fruits specially produced in Miyazaki prefecture, and are extremely unique fruits that eat not only the flesh but also a part of the skin and the flesh.

In the present specification, the "processed product" of Hyuga Natsudaidai includes all those derived from any part of the plant body of Hyuga Natsudaidai such as roots, stems, leaves, flowers, and fruits, but it is preferable. Is derived from the fruit part. Fruits are mainly divided into pericarp and flesh, both of which are preferably used. For example, the squeezed residue after squeezing Hinata Natsudaidai is preferably used. Each part of these plants can also be used raw. The raw state is also included in the "processed product" in the present invention. In addition, each part of the above-mentioned plant body can be further semi-dried or dried before use. Semi-drying or drying can be carried out by a usual method, for example, a method of drying in the sun or a method of drying with a dryer. In addition, each part of the plant body or its semi-dried product or dried product may be used as it is, but is preferably used in a state of being crushed to an appropriate size.

As the processed product of the present invention, an extract of Hinata Natsudaidai is also preferably used. The extract includes an extract obtained by extracting the above-mentioned processed product with an extraction solvent, a diluted extract, a concentrated extract, a dried extract, or a crudely purified extract thereof. ..

The solvent used for extraction may be water, a hydrophilic organic solvent, a hydrophobic organic solvent, or a mixture of two or more of these, preferably water. The type of water is not particularly limited, and may be pure water, tap water, or the like. In addition, water may be subjected to usual treatments such as purification, sterilization, sterilization, filtration, osmotic pressure adjustment, and buffering. For example, a buffer solution such as physiological saline or phosphate buffer solution is also used as an extraction solvent. It is possible. Hydrophilic organic solvents include alcohols such as methyl alcohol, ethyl alcohol, butyl alcohol, glycerin, propylene glycol, 1,3-butylene glycol, acetone, tetrahydrofuran, acetonitrile, 1,4-dioxane, pyridine, dimethyl sulfoxide, N, Known hydrophilic organic solvents such as N-dimethylformamide and acetic acid can be mentioned. Examples of the hydrophobic organic solvent include known hydrophobic organic solvents such as hexane, cyclohexane, carbon tetrachloride, chloroform, dichloromethane, 1,2-dichloroethane, diethyl ether, ethyl acetate, benzene, toluene, n-hexane, and isooctane. Can be mentioned. Extraction using a supercritical fluid is also possible.

The extraction operation is not particularly limited and may be performed according to a conventional method. In order to improve the extraction efficiency, heating, stirring, shaking and the like can also be used in combination. In addition, for the purpose of improving the extraction efficiency, the Hinata Natsudaidai sample can be appropriately treated before extraction. Examples of such treatment include pulverization and degreasing. During or after extraction, the extract can be treated with an enzyme such as pectinase, protease, lipase, amylase, etc. to remove components other than the target substance.

In one embodiment, the processed product of Hinata Natsudaidai of the present invention contains arabinogalactan. Arabinogalactan may be the active ingredient in the processed product of Hinata Natsudaidai of the present invention. As used herein, the term "arabinogalactan" refers to a polysaccharide containing arabinose and galactose as its main components. The arabinogalactan of the present invention has β1,3 bound galactose as the main chain and has a side chain branched from the 6-position of the main chain galactose. As used herein, the "main chain" refers to the relatively longest chain in a sugar chain molecule, and the "side chain" refers to a relatively short chain branched from the main chain.

The arabinogalactan of the present invention may preferably contain 1,6 bound β-galactose and 1,5-bound α-arabinose as side chains. More preferably, the 1,6 bond in the side chain is a β1,6 bond, and the 1,5 bond is an α1,5 bond.

In the present specification, the inclusion of β-galactose in which the side chain is 1,6 bound means that the side chain contains a structure of 6) -βGal- (1 →, and this structure is a monosaccharide unit in the side chain. It may be present in disaccharide units or more. The side chain 6) -βGal- (1 → is directly bound to the 6th position of the main chain galactose by β1,6 bond. It may be bound to the main chain via other sugars such as arabinose, and galactose may be bound to other sugars in the side chain, for example, 6) -βGal- (6) -βGal- (. 1 → is in the side chain via the β1 → 6 bond with galactose (βGal- (1 → 6) -Gal) or with arabinose via the β1 → 5 bond (βGal-(1 → 5) -Ara). Alternatively, it may be bound via the α1 → 6 bond (αAra- (1 → 6) -Gal), preferably 6) -βGal- (1 → is in the side chain via the β1 → 6 bond. Combined with other galactose.

Similarly, in the present specification, the inclusion of α-arabinose in which the side chain is 1,5 bound means that the side chain contains the structure of → 5) -αAra- (1 →, and this structure is contained in the side chain. May be present in monosaccharide units or in disaccharide units or more. → 5) -αAra- (1 → is directly bound to the 6-position of galactose in the main chain by α1,6 binding. It may be bound to the main chain via other sugars such as galactose, and arabinose may be bound to other sugars in the side chain. For example, → 5)-. αAra- (1 → is in the side chain via β1 → 5 bond with galactose (βGal- (1 → 5) -Ara) or via α1 → 6 bond (αAra- (1 → 6) -Gal) , Or may be bound to arabinose via an α1 → 5 bond (αAra-(1 → 5) -Ara), preferably 5) -αAra- (1 → is side via an α1 → 5 bond. Combines with other arabinose in the chain.

Further, the arabinogalactan of the present invention may preferably have βGal (1 → and / or αAra (1 →) at the end of its side chain.

The arabinogalactan of the present invention preferably does not contain 1,3 bonded galactose in the side chain, that is, 3) -Gal- (1 →. Also, the arabinogalactan of the present invention is preferably contained in the side chain. Does not contain 1,3 bound arabinose, ie 3) -Ara- (1 →.

In the arabinogalactan of the present invention, the number of sugar units contained in each side chain is not limited, and may be a monosaccharide unit or a disaccharide unit or more. In addition, each side chain may consist of either a galactose unit or an arabinose unit, or these sugar units may be mixed in one side chain. Preferably, all galactose in the side chain is β-galactose and all arabinose is α-arabinose.

The ratio of galactose to arabinose in arabinogalactan of the present invention is not particularly limited. For example, when the mole ratio of galactose is 1, the molar ratio of arabinose is 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.45 or more. Or it may be 0.5 or more, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.55 or less.

The side chain of arabinogalactan of the present invention may contain a small amount of sugars other than galactose and arabinose, such as glucose, rhamnose, fucose, and glucuronic acid, in the side chain, for example. In this case, when the mole of galactose is 1, the molar ratio of sugars other than galactose and arabinose is 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, preferably 0.15 or less, 0.1 or less, 0.05 or less, or 0.01 or less. You can.

The molecular weight of arabinogalactan of the present invention may be, for example, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, preferably 65,000 or more, 66,000 or more, 67,000 or more, or 68,000 or more, and 120,000 or less, 110,000 or more. Below, it may be 100,000 or less, 90,000 or less, 80,000 or less, preferably 75,000 or less, 74,000 or less, 73,000 or less, or 72,000 or less.

Analysis of the structure of polysaccharides such as arabinogalactan can be performed by methods known to those skilled in the art, such as methylation analysis and / or NMR. Similarly, analysis of polysaccharide constituents and molecular weights can also be performed by methods known to those of skill in the art, such as HPLC and size exclusion chromatography, respectively.

The processed product or calcium absorption promoter of the present invention has an action of promoting the absorption of calcium from the intestinal tract into the body. The present inventor has previously found that a processed product of Hyuga Natsudaidai has an effect of improving bone metabolism. However, it has not been suggested that the treated product of Hyuga Natsudaidai has a calcium absorption promoting effect in addition to a bone metabolism improving effect. By utilizing the calcium absorption promoting action of the treated product of Hyuga Natsudaidai of the present invention, it becomes possible to prevent and / or treat osteoporosis based on an action mechanism different from the action mechanism for improving bone metabolism. In addition to the prevention and / or treatment of osteoporosis, the calcium absorption promoting action can also be used to improve the nutritional value of, for example, calcium-containing foods. Whether or not a substance has an action of promoting calcium absorption can be determined by a method known to those skilled in the art, for example, creating an inverted small intestine, adding the substance to an external solution containing calcium, and calcium from the external solution to the inside of the inverted small intestine. It can be easily confirmed by confirming whether or not the uptake of calcium increases.

The calcium absorption promoter of the present invention may consist of a processed product of the Hinata Natsudaidai of the present invention, or other components other than the treated product of the Hinata Natsudaidai of the present invention, such as excipients, binders, disintegrants, and surfactants. It may contain activators, lubricants, fluidity promoters, flavoring agents, colorants, fragrances and the like.

The calcium absorption promoter of the present invention can be formulated by a conventional method. The dosage form is not particularly limited and is appropriately selected as needed, but is generally oral such as tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, and elixirs. It can be administered as a parenteral agent such as an agent, an injection, an infusion, a suppository, an inhalant, a transdermal absorbent, a transmucosal absorbent, a patch, and an ointment.

Oral preparations are prepared according to conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethyl cellulose, cornstarch, and inorganic salts.

In addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances and the like can be appropriately used in this type of preparation. ..

Specific examples of the binder include crystalline cellulose, crystalline cellulose carmellose sodium, methyl cellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, and carmellose sodium. , Ethyl cellulose, carboxymethyl ethyl cellulose, hydroxyethyl cellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, purulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl Examples thereof include methacrylate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, polyvinyl alcohol, gum arabic, rubber arabic powder, agar, gelatin, white cellac, tragant, purified sucrose, and macrogol.

Specific examples of the disintegrant include crystalline cellulose, methyl cellulose, low-substituted hydroxypropyl cellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partial pregelatinization. Included are starch, hydroxypropyl starch, sodium carboxymethyl starch, and tragant.

Specific examples of surfactants include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, and sorbitan monostearate. , Polysorbate monopalmitate, sorbitan monolaurate, polysorbate, glycerin monostearate, sodium lauryl sulfate, and lauromacrogol.

Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc, etc. Examples thereof include magnesium aluminometasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, braces, hydrogenated vegetable oil, and polyethylene glycol.

Specific examples of the fluidity accelerator include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

In addition, the calcium absorption promoter of the present invention may contain a flavoring agent and / or a coloring agent when administered as a liquid agent, a syrup agent, a suspension agent, an emulsion, or an elixir agent.

In one aspect, the present invention relates to a method for preventing and / or treating osteoporosis in a subject, which comprises administering the calcium absorption promoter of the present invention to the subject.

Also, in one aspect, the present invention relates to the use of the calcium absorption enhancer of the present invention in the prevention and / or treatment of osteoporosis.

2. Calcium Absorption Promoting Food Compositions or Pharmaceuticals In one aspect, the present invention relates to calcium absorption promoting food compositions or pharmaceuticals containing the calcium absorption promoting agents of the present invention. In another aspect, the present invention relates to a calcium absorption promoting food composition or pharmaceutical product containing the calcium absorption promoting agent of the present invention.

The food composition of the present invention (also simply referred to as "food") can be prepared by a conventional method. For example, the form of such food is not limited to ordinary "food", for example, the form of tablets containing candy, troches and the like, the form of sugar-coated tablets, the form of granules, the form of powdered beverages, powdered soup and the like. Any form of block confectionery such as biscuits, capsules, jellies, etc., paste forms such as jam, gum forms such as chewing gum, soft drinks containing tea, beverage forms such as alcoholic beverages, etc. It may be a food in the form, and can also be a food for specified health use (for example, a food for preventing osteoporosis). The food containing the calcium absorption enhancer of the present invention may contain various other ingredients usually used as food raw materials as long as the desired effects of the present invention are not impaired. Other ingredients include, for example, water, alcohols, sweeteners, acidulants, colorants, preservatives, flavors, excipients, stabilizers, pH regulators, sugars, various vitamins, minerals, antioxidants, etc. Examples thereof include solubilizers, binders, lubricants, suspending agents, wetting agents, film-forming substances, flavoring agents, odorants, surfactants, and fluidity promoters. These ingredients can be used alone or in combination. Similarly, the pharmaceutical product of the present invention can be prepared by a conventional method, for example, according to the method described for the calcium absorption enhancer in 1 above.

The content of the calcium absorption-promoting agent in the foods and pharmaceuticals of the present invention is not particularly limited as long as the desired action is exhibited, and those skilled in the art can use the sex, age, body weight, height, etc. of the subject. And it can be determined as appropriate in consideration of the degree of symptoms and the like.

3. 3. Method for Producing Food Composition or Pharmaceutical In one aspect, the present invention relates to a method for producing a food composition or pharmaceutical, which comprises adding the calcium absorption promoter of the present invention. The food composition or pharmaceutical product may be for promoting calcium absorption.

The method for producing a food or pharmaceutical product of the present invention can be carried out by a method known to those skilled in the art, except that the calcium absorption promoter of the present invention is added. The addition of the calcium absorption enhancer of the present invention can be carried out at any time during the manufacturing process of the food or drug as long as the obtained food or drug produces the desired effect. For example, the calcium absorption enhancer of the present invention may be mixed with other raw materials in the early or middle stages of production, or may be added to the semi-finished product in the final stage of production.

Further, in one aspect, the present invention relates to the use of the calcium absorption promoter of the present invention in the production of a food composition or a pharmaceutical product.

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

<Example 1: Preparation of Hyuganatsu processed product>
1) Preparation of Hyuganatsu processed product 1 and Hyuganatsu processed product 2 From the Hyuganatsu squeezed residue, Hyuganatsu processed product 1 and Hyuganatsu processed product 2 were prepared as follows.

Hyuganatsu was squeezed with an in-line juice machine (John Bean Technology), and the juice residue was collected in a cardboard box and stored frozen. Subsequently, the frozen Hyuganatsu squeezed residue is thawed, twice the amount of warm water at 60 ° C is added to the thawed Hyuganatsu squeezed residue, and water is extracted by immersing while stirring for 15 minutes. Was done. Subsequently, the water-extracted Hyuganatsu juice residue was crushed with a chopper pulper, and then foreign substances were removed by sieving at 5 mm, 1 mm, and 0.5 mm and by magnet wrap. Next, the crushed Hyuganatsu juice residue from which foreign substances have been removed is squeezed (rotation speed 3,900 rpm, 3,000 g) with a decanter juice machine (Mitsubishi Kakoki) and centrifuged (flow rate 4 to 5 m ³ / h, discharged). After 1 time / 15 minutes), the mixture was sterilized at 95 ± 1 ° C for about 8 to 9 seconds and cooled to 45 to 50 ° C. Next, 0.05% w / w of pectinase derived from Aspergillus niger (Yakult Pharmaceutical Industry Co., Ltd.) was added to the heat-sterilized solution, subjected to enzymatic treatment at 45 to 50 ° C. for 1 hour, primary filtration using diatomaceous earth, and then 95. The enzyme was inactivated by heating to ± 1.5 ° C. for about 8 to 9 seconds, and cooled to 30 ° C. or lower. Subsequently, after secondary filtration using diatomaceous earth, foreign substances were removed using a 3μ filter. Subsequently, after concentration under reduced pressure, the mixture was cooled to 25 ° C. or lower, Bx was adjusted to 35.1 to 35.5 by adding water, and foreign substances were removed with 80 mesh. Subsequently, it was sterilized at 120 ± 1.5 ° C. for about 15 seconds and cooled to 30 ° C. or lower. Subsequently, foreign substances were removed with an 80 mesh and a magnet trap, filled in a fully open 18 L can, and stored frozen.

Subsequently, the obtained Hyuganatsu processed product 1 is dried and mixed with an equal amount of dextrin (Matsutani Chemical Industry Co., Ltd., Hyogo Prefecture) in order to reduce the hygroscopicity of the obtained powder, and an extract powder (pomace) is used. : PO) (Hyuganatsu processed product 2) was prepared.

2) Preparation of active fraction 1 The Hyuganatsu treated product 1 obtained in 1) above was treated with activated carbon as follows.
First, the above Hyuganatsu treated product 1 was thawed, diluted to about Bx6.8 to 7.2, and then activated carbon (Osaka Gas Chemical) was added at 1.0% w / w and treated for 1 hour. Subsequently, after primary filtration using diatomaceous earth, the mixture was heated to 95 ± 1.5 ° C. for about 8 to 9 seconds and then cooled to 25 ° C. or lower. Subsequently, after secondary filtration using diatomaceous earth, foreign substances were removed using a 3μ filter. Subsequently, after concentration under reduced pressure, the mixture was cooled to 25 ° C. or lower, Bx was adjusted to 35.1 to 35.5 by adding water, and foreign substances were removed with 80 mesh. Subsequently, foreign substances were removed with an 80 mesh and a magnet trap, filled in a fully open 18 L can, and stored frozen. It has been confirmed that there is no difference in the peaks in the Hyuganatsu treated product 1 before and after the activated carbon treatment (data not shown).

Subsequently, the activated carbon-treated Hyuganatsu treated product 1 (hereinafter, “Hyuganatsu treated product 3”) was subjected to ultrafiltration as follows, and a fraction (active fraction 1) (pomace) having a single peak on HPLC was used. extract: PE) was prepared.

First, the Hyuganatsu treated product 3 was subjected to HPLC under the conditions shown in Table 1 below, and the chromatogram of FIG. 1 was obtained.

In order to obtain the peak of about 16 minutes RT seen in the chromatogram, particles having a particle size of 1 μm or more were first removed by prefiltration with a Tosell cartridge filter (ADVANTEC). Next, ultrafiltration was performed using Pellicon 2 Cassette Biomax 100 kDa (Millipore), and the permeated solution was recovered. Subsequently, the recovered solution was subjected to ultrafiltration using Pellicon 2 Cassette Biomax 30 kDa (Millipore). When the solution in the tank was reduced to less than half, the holding solution was hydrated to the same amount as the original solution and ultrafiltered using Pellicon 2 Cassette Biomax 30 kDa (Millipore). The solution held in a 100 kDa membrane was purified using a microza MF lab module (Asahi Kasei Chemicals) with a nominal pore size of 0.2 μm. In the same manner as above, when the amount of the solution in the beaker was reduced to less than half, the holding solution was hydrated to the same amount as the original solution, and purification was repeated. The obtained permeate was combined with a solution that had permeated a 100 kDa membrane, and ultrafiltration was performed using a 30 kDa membrane. The holding solution was analyzed by HPLC according to the above conditions, watering and ultrafiltration were repeated until a single peak was obtained, and finally the holding solution was recovered to prepare an active fraction 1. Figure 2 shows the results when the active fraction 1 was subjected to HPLC under the above conditions.

Moreover, when the molecular weight of the HPLC retention time was measured using pullulan (Shodex) or dextran as a standard sample, it was shown that the molecular weight of the active fraction 1 was about 68,000 to 72,000 (data not shown).

<Example 2: Promotion of Ca absorption in the inverted small intestine of the Hyuganatsu treated product>
An 8-week-old male Sprague Dawley rat (purchased from Kudo Co., Ltd.) was anesthetized with Nembutal and the small intestine was removed. Pieces of the small intestine were cut to a length of 12 cm from the upper, middle and lower parts of the small intestine, inverted, and roped about 1 cm from both ends to create an inverted small intestine. In the inverted small intestine, 1 mL of 0.9% NaCl was added as an internal solution.

Subsequently, inversion small intestine was added to an external solution (pH 6.6) consisting of 16.9 mM CaCl ₂ , 5.8 mM KH ₂ PO ₄ , 0.9% NaCl, or a solution prepared by adding 0.02% of PO or PE prepared in Example 1 to the external solution. After soaking and incubating for 40 minutes, the internal solution of the inverted small intestine was collected and stored frozen. The internal solution was thawed before use, 10 μL was mixed with 180 μL of R-1 solution of Aqua Autokinos Ca Reagent (Kainos Co., Ltd., Japan), and Ca concentration was measured based on the absorbance at 660 nm according to the manufacturer's instructions. ..

The results are shown in Figure 3. FIG. 3 shows that the Hyuganatsu treated product 1 and the active fraction 1 containing the peak considered to be the active ingredient thereof promote the absorption of calcium in the rat inversion small intestine.

<Example 3: Identification of components contained in the active fraction>
1) Preparation of active fraction 2 The active fraction 2 of the Hyuganatsu treated product 1 obtained in Example 1 was prepared as follows without being treated with activated carbon and subjected to the following analysis.

First, the Hyuganatsu treated product 1 obtained in Example 1 was prefiltered with NITROCELLULOSE MEMBRANE 0.22 μm (Millipore) to remove particles having a large particle size. Subsequently, the pre-filtered solution was ultrafiltered using Ultrafiltration Discs Ultracel 30 kDa (Millipore) to prepare a fraction to be retained (hereinafter referred to as "active fraction 2").

The active fraction 2 showed the same peak as the active fraction 1 in the HPLC analysis (data not shown). In addition, when the molecular weight of the HPLC retention time was measured using pullulan (Shodex) or dextran as a standard sample, it was shown that the molecular weight of the components contained in the active fraction 2 was about 68,000 to 72,000 (data not shown). ).

2) Analysis of acid hydrolyzate of active fraction 2 by HPLC The active fraction 2 prepared by the above method was acid hydrolyzed by heating with 1N sulfuric acid at 105 ° C. for 3 hours. As a result of analyzing this acid hydrolyzate by HPLC under the conditions shown in Table 2 below, it was confirmed that galactose and arabinose were contained as constituent sugars (data not shown). The molar ratio of galactose to arabinose was about 1: 0.53 (data not shown).

Subsequently, methylation analysis was performed. Specifically, 2 ml of NaOH / DMSO solution and 0.3 ml of methyl iodide were added to a polysaccharide sample obtained by freeze-drying the active fraction 2, and the mixture was treated at 30 ° C. for 20 minutes. After 20 minutes, 0.7 ml of methyl iodide was further added, and the treatment was carried out at 30 ° C. for 40 minutes. After adding 2 ml of 0.88% KCl to the methylation reaction solution, 2 ml of chloroform was added to carry out liquid-liquid partitioning. After removing the upper layer (aqueous layer), 3 ml of 0.88% KCl was newly added to perform liquid-liquid distribution, and the upper layer was removed in the same manner. Then, 0.5 ml of toluene was added to the chloroform layer, and the mixture was dried under reduced pressure. The methylated polysaccharide was dissolved in 2 ml of 2 M trifluoroacetic acid (TFA) and hydrolyzed at 110 ° C. for 4 hours. Then, 0.5 ml of toluene was added, then 3 ml of anhydrous methanol was added, and the mixture was dried under reduced pressure. Sodium borohydride (NaBH ₄ ) 40 mg is dissolved in 2 ml of purified water, _{1.2 ml of NaBH 4} solution prepared by adding 0.4 ml of 1 M ammonia water is added to the sample and reacted for 3 minutes, and then the remaining NaBH ₄ solutions were added and left at 25 ° C. for 12 hours. It was neutralized with acetic acid (0.6 ml), 0.5 ml of toluene was added and dried under reduced pressure, and then 4 ml of anhydrous methanol was added and dried under reduced pressure again. 0.5 ml each of acetic anhydride and pyridine was added and acetylated at 100 ° C. for 1 hour. Then, 3 ml of toluene was added and the mixture was dried under reduced pressure. Next, 2 ml of distilled water and 1.5 ml of chloroform were added to perform liquid-liquid distribution, and after removing the aqueous layer, 2 ml of distilled water was newly added to remove the upper layer in the same manner. Then, 0.5 ml of toluene was added to the chloroform layer, and the mixture was dried under reduced pressure. Next, 0.5 ml of chloroform was added to extract the reaction product, and GC / MS analysis was performed under the following conditions.
Mass spectrometer: Shimadzu GCMS-QP2010SE,
Capillary column: InertCap RTX 5MS,
Carrier gas: helium,
Analysis temperature: 180 ℃ 5 min, 180-250 ℃ 2 ℃ / min, 250 ℃ 5 min,
Interface temperature: 250 ° C.

As a result of methylation analysis, the polysaccharides contained in the sample are → 3) -Gal- (1 → is the main chain (peaks 4 and 6), → 6) -Gal- (1 → and → 5) -Ara- (1). It was clarified that the side chain containing → (peaks 2 and 5) is an arabinogalactan branched at the 6-position (Table 3). In addition, this arabinogalactan has a terminal Gal and a terminal Ara. Was also clarified (Table 3, Peaks 1 and 3).

Subsequently, two-dimensional NMR (HSQC) analysis was performed. Specifically, the polysaccharide sample obtained by freeze-drying the active fraction 2 was dissolved in heavy water, placed in a sample tube (178 x 4 mmφ), ¹ H-NMR and ¹³ C-NMR were measured, and HSQC was performed. (Hetero-nuclear Single Quantum Coherence) measurement was performed. 1,4-dioxane was used as a standard substance. A BRUKER AV400M was used as the nuclear magnetic resonance apparatus.

As a result, it was clarified that the binding modes of galactose and arabinose are β-bond and α-bond, respectively.

From these results, the active ingredient derived from Hinata Natsu Mikan has β1,3-bound galactose as the main chain, a side chain branched from the 6-position of the main chain galactose, and β-galactose with 1,6 side chains. , And 1,5-bound α-arabinose were suggested to be arabinogalactan.

Claims (5)

A calcium absorption enhancer containing a processed product of Hyuga Natsudaidai containing arabinogalactan with a molecular weight of 50,000 to 100,000. The calcium absorption promoter according to claim 1, wherein the processed product of Hyuga Natsudaidai is an extract of Hyuga Natsudaidai. Arabinogalactan,
It has β1,3-bonded galactose as the main chain, has a side chain branched from the 6-position of the main chain galactose, and contains β-galactose with 1,6-bonded side chains and α-arabinose with 1,5-bonded α-arabinose.
The calcium absorption promoter according to claim 1 or 2. A food composition or pharmaceutical product for promoting calcium absorption, which comprises the calcium absorption promoter according to any one of claims 1 to 3. A method for producing a calcium absorption-promoting food composition or a pharmaceutical product, which comprises adding the calcium absorption-promoting agent according to any one of claims 1 to 3.

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