JP7438816B2 - Intestinal equol-producing bacteria increase agent, equol production promoter, and blood equol concentration increase agent - Google Patents

Description

The present invention relates to an oral composition suitably used to increase equol-producing bacteria present in the intestines. The present invention also relates to an oral composition suitably used to promote equol production in the body. Furthermore, the present invention relates to an oral composition suitably used for increasing blood equol concentration.

Equol is a compound with various characteristics such as higher estrogen-like activity compared to O-desmethylangolensin (O-DMA), a metabolite of daidzein, a typical isoflavone, and has traditionally been used to treat osteoporosis. Therapeutic and preventive effects on breast cancer, etc. have been reported (e.g., Patent Document 1), alleviation of menopausal symptoms, prevention of metabolic syndrome, improvement of skin wrinkles and sagging, and preventive effects on breast cancer, etc. (e.g., Non-Patent Document 1). 1st prize). Equol has also been reported to have other antioxidant effects (improving age spots, whitening) and anti-androgenic effects (preventing prostate cancer, etc., improving hair loss).

Equol is known to be produced by isoflavones being metabolized in the body. Figure 1 shows the metabolic pathway of isoflavones in the body.
(1) When isoflavone glycosides (e.g., daidzein) are orally ingested, they are converted to aglycones (e.g., daidzein) by the action of saliva, enzymes in the small intestinal mucosa, or β-glucosidase derived from intestinal bacteria. Ru.
(2) Daidzein is further metabolized by intestinal bacteria to dihydrodaidzein (DHD), an equol precursor, and then to equol by equol-producing bacteria. Furthermore, it is known that many of the equol-producing bacteria present in the intestines can directly produce equol from daidzein (Non-Patent Document 2).
(3) O-desmethylangorensin (O-DMA) is produced from daidzein and dihydrodaidzein (DHD) by the action of intestinal bacteria other than equol-producing bacteria (eg, Eubacterium genus, Clostrium genus, etc.).
(4) After aglycones such as daidzein and their metabolites are absorbed into the intestinal tract, unconjugated forms are distributed to target organs and tissues, and conjugated forms are excreted in urine and feces.

Thus, equol is a metabolite produced when daidzein is metabolized by intestinal bacteria in the intestine. Therefore, the amount of equol produced in the body, that is, the amount of equol in the body, varies from person to person due to differences in the equol-producing bacteria and intestinal flora present in the intestines, and also due to differences between men and women and racial differences. It is known that there are differences.

In recent years, attempts have been made to increase the production efficiency of equol by influencing the intestinal flora. Oligosaccharides such as fructooligosaccharides (Patent Document 2), lactobionic acid (Patent Document 3), oligosaccharides such as cellooligosaccharides (Patent Document 4), bittern (Mg, Ca) components (Patent Document 5), and resistant starch (Patent Document 5) An increase in equol production and an increase in equol-producing bacteria have been reported in Non-Patent Document 3).

Japanese Patent Application Publication No. 2005-232074 International Publication No. 1999/007392 Japanese Patent Application Publication No. 2008-001608 Japanese Patent Application Publication No. 2014-125431 Japanese Patent Application Publication No. 2019-162060

"Chemistry and Biology", published by the Japanese Society of Agricultural Chemistry, Vol.44, No.3, p.151-153 (2006) Kenneth et al., Equol: History, Chemistry, and Formation. J Nutr., 140(7), 1355S-1362S, 2010. Tousen et al., The combined effects of soya isoflavones and resistant starch on equol production and trabecular bone loss in ovariectomised mice. Br J Nutr., 116(2), 247-257, 2016.

In view of the above-mentioned circumstances, it is an object of the present invention to provide a composition that has the effect of increasing equol-producing bacteria present in the intestine. Another object of the present invention is to provide a composition that has the effect of promoting equol production in the body. Furthermore, it is an object of the present invention to provide a composition that has the effect of increasing the amount of equol in the blood.

The present inventors have conducted extensive studies to solve the above problems, and have discovered that when black soybean seed coat extract is ingested, equol-producing bacteria among intestinal bacteria increases. From this, it is expected that by ingesting black soybean seed coat extract, the ability of intestinal bacteria to produce equol will be enhanced, thereby increasing the amount of equol produced in the body. In addition, when black soybean seed coat extract was ingested together with soybean isoflavones contained in soybeans, blood concentrations of dihydrodaidzein (DHD) and equol, which are equol precursors among isoflavone metabolites, significantly increased; Among the metabolites of isoflavones, it was confirmed that O-desmethylangorensin (O-DMA) did not increase. Based on this, intake of black soybean seed coat extract affects the intestinal flora, increasing the number of equol-producing bacteria, and in the metabolism of soy isoflavones, converting them to O-desmethylangolensin (O-DMA). It is thought that it acts to favor conversion to equol rather than equol. In other words, it is thought that by ingesting black soybean seed coat extract, the intestinal flora is improved so that equol production in the intestines becomes dominant.
The present invention was completed based on these findings, and includes the following embodiments.

(I) Agent for increasing intestinal equol-producing bacteria (I-1) An agent for increasing intestinal equol-producing bacteria, which contains black soybean seed coat extract as an active ingredient.
(I-2) The agent for increasing equol-producing bacteria according to (I-1), wherein the equol-producing bacteria are bacteria of the genus Adlercreutzia.

(II) Equol production promoter (II-1) An equol production promoter containing black soybean seed coat extract as an active ingredient.
(II-2) Equol production described in (II-1), which acts to favor the conversion of soy isoflavones to equol over the conversion of soy isoflavones to O-desmethylangorensin in the metabolism of soy isoflavones in the body. Accelerator.

(III) Blood equol concentration increasing agent A blood equol concentration increasing agent containing black soybean seed coat extract and soybean isoflavones.

(IV) Method for using black soybean seed coat extract (IV-1) Adding black soybean seed coat extract to an oral composition to impart an effect of enhancing equol production ability by intestinal bacteria to the oral composition. How to use black soybean seed coat extract.
(IV-2) A method of using a black soybean hull extract for enhancing the effect of increasing blood equol concentration of the oral composition by incorporating the black soybean hull extract into an oral composition, the method comprising:
The method, wherein the oral composition contains soy isoflavones.

By orally ingesting a composition containing the black soybean seed coat extract of the present invention, the number of equol-producing bacteria present in the intestines can be increased. Furthermore, by orally ingesting a composition containing the black soybean seed coat extract of the present invention, the intestinal flora can be improved so that equol production becomes predominant. As a result, the ability of intestinal bacteria, including equol-producing bacteria, to produce equol is enhanced, which promotes the metabolism of equol precursors such as daidzein and dihydrodaidzein (DHD) to equol, thereby increasing the production of equol. can be promoted. In addition, by ingesting the composition containing the black soybean seed coat extract of the present invention together with soybean isoflavones, as mentioned above, the metabolism to equol is promoted, and as a result, the equol concentration in the blood can be efficiently reduced. It is possible to rise.

As described above, according to the present invention, it is possible to efficiently metabolize soybean isoflavones ingested as food or feed into equol regardless of the presence or absence of circumstances such as a small number of equol-producing bacteria existing in the intestine. As a result, it becomes possible to efficiently absorb and ingest equol into the body. As a result, it is possible to maintain and improve health based on the effects of equol.

FIG. 2 is a diagram showing the metabolic pathway of isoflavones in the body. The results of Experimental Example 1 are shown. Specifically, the abundance ratio (%) of equol-producing bacteria (genus Adlercreutzia) in the cecal contents of mice ingested with black soybean seed coat extract or soybean isoflavones is shown. The results of Experimental Example 2 are shown. Specifically, (A) contains four measured compounds (Daidzein, dihydrodaidzein (DHD), Equol, and O-desmethylangolensin (O-DMA)) contained in 1 L of serum. (B) is a diagram comparing the proportions of four measured compounds contained in 1 L of serum for each compound.

(I) Agent for increasing intestinal equol-producing bacteria
(II) Equol production promoter The agent for increasing intestinal equol-producing bacteria of the present invention (hereinafter also simply referred to as "the present bacteria increasing agent") and the equol production promoter of the present invention (hereinafter also simply referred to as "the present promoter") ) are all characterized by containing an extract of black soybean seed coat, preferably an edible extract, as an active ingredient. Hereinafter, the present invention, including the agent for increasing intestinal equol-producing bacteria and the agent for promoting equol production, will be collectively referred to as the "present invention".

The black soybean used in the present invention is the black seed (grain) (black soybean) of a short-day annual plant belonging to the family Fabaceae, genus Glycine max (L.) Merrill. There are varieties of black soybeans, such as Nakasei Kokuro, Tokachikuro, Iwaikuro, Tamadaikoku, Tambaguro, Shinanoguro, and Gankui, but seeds of any black soybean variety may be used.

Black soybeans can be separated into seed coats and embryos (cotyledons and hypocotyls) by, for example, using a sorting machine. In the present invention, the seed coat of black soybean obtained by the separation can be used as a processing raw material. During processing, black soybean seed coats may be separated and left as they are (raw or dried), or they may be crushed or ground (crushed, pulverized, or powdered). ).

As a method for extraction from black soybean seed coat, commonly used methods can be used. Examples include, but are not limited to, a method of immersing raw or dried black soybean seed coat (as is, or in coarse, finely chopped, crushed, or pulverized form) in a water-soluble solvent; a method of extraction with stirring as necessary; ; or a percolation method. The temperature conditions used for extraction are not particularly limited, and may be any of low temperature, room temperature, and heating conditions (including high temperatures), but preferably heating conditions (including high temperatures). More specifically, in the case of extraction with a water-containing lower alcohol described below, the temperature is 30°C or higher, preferably in the range of 40°C to 60°C, and although not limited, extraction at such temperature conditions is carried out for 60 minutes or more, preferably 90°C. Do this for about 120 minutes. In addition, when extracting with an acidic aqueous solution, the temperature is 50° C. or higher, preferably in the range of 50 to 80° C., and although not limited, extraction is carried out at such temperature conditions for 10 minutes or more, preferably about 20 minutes to 120 minutes.

The water-soluble solvent used for extraction includes, but is not particularly limited to, water, lower alcohols, or mixtures thereof. Examples of the lower alcohol include lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropyl alcohol, and butanol. Ethanol can be preferably mentioned as the lower alcohol. The water-soluble solvent is preferably water or a water-containing lower alcohol (especially water-containing ethanol), and more preferably water. In addition, when using a water-containing lower alcohol as a solvent, it is preferable that the amount of lower alcohol contained therein is 80% by volume or less.

The water-soluble solvent used for extraction is preferably adjusted to be acidic. Although not particularly limited, the pH range of the water-soluble solvent is preferably about 1 to 4, particularly preferably about 1 to 2. In order to adjust the pH of the water-soluble solvent to fall within this range, an appropriate acidic substance such as an organic acid or an inorganic acid can usually be used.

Examples of acidic substances include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid; as well as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, and fluorosulfonic acid. (hereinafter referred to as sulfonic acid), formic acid, acetic acid, citric acid, and oxalic acid (hereinafter referred to as carboxylic acid). Acids having a sulfo group are preferred, and specific examples include sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, and fluorosulfonic acid. Among them, sulfuric acid is preferred. Note that the normality of the acid in the water-soluble solvent is not particularly limited as long as the pH of the water-soluble solvent is within the above pH range, but is preferably in the range of 0.01 to 0.5N, more preferably 0.03N. ~0.5N.

Within the scope of achieving the effects of the present invention, the obtained extract may be further subjected to purification treatments such as removal of solids by filtration, co-precipitation or centrifugation, extraction treatment, adsorption treatment, etc., as necessary. Good too.

The black soybean seed coat extract prepared in this manner may be further sterilized by a known method such as UHT sterilization or retort sterilization, if necessary, as long as it exhibits the effects of the present invention.

The form of the present bacteria increasing agent and the present promoter is not particularly limited as long as it is an oral administration form. In addition, as long as it has an oral administration (oral ingestion) form, it can be classified according to its use (drugs, quasi-drugs, food and drink [foods with health functions such as foods for specified health uses, foods with functional claims, and foods with nutritional functions)]. and supplements]) are not particularly limited. Preferably it is a food or drink, and more preferably a food for specified health uses or a food with functional claims that can claim its action or effect. Further, the present bacteria increasing agent and the present promoter may be applied to feed or pet food for animals other than humans (including livestock, poultry, and pets). Furthermore, the present bacteria increasing agent and the present promoter may be additives added to pharmaceuticals, quasi-drugs, foods and drinks, feed, or pet food.

Specifically, the oral dosage form includes the extract prepared by the above extraction method in the form of a liquid (including extract form and syrup) or jelly; Powders, fine granules, or granules formulated into capsules; capsules (hard capsules, soft capsules) filled with liquids, powders, or granules; or powders or granules that are further compressed to form tablets. (solid preparations).

The present bacteria increasing agent and present promoter are prepared by combining the above black soybean hull extract with conventionally known edible carriers, excipients, etc. that are pharmaceutically acceptable for food or feed, and are prepared in various dosage forms (oral administration It can also be prepared in the following form.

When the present bacteria increasing agent and present promoter are in the form of liquid preparations, they may be stored frozen, or may be stored after removing moisture by freeze-drying or the like. Freeze-dried preparations, dry syrups, etc. are redissolved by adding sterile water or the like before use.

When the present bacteria increasing agent and the present promoter are in the form of a solid dosage form, for example, in the case of a tablet, a wide variety of carriers conventionally known in the art can be used. Such carriers include, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, and silicic acid; water, ethanol, propanol, simple syrup, glucose solution, starch solution, and gelatin. Solution, binder such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, crystalline cellulose, hydroxypropylcellulose, hypromellose, sodium alginate; dried starch, agar powder, laminaran powder, sodium bicarbonate, polyoxyethylene sorbitan fatty acid Disintegrants such as esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, crospovidone, povidone, and low-substituted hydroxypropylcellulose; disintegration inhibitors such as stearin, cocoa butter, and hydrogenated oil; quaternary ammonium salts, lauryl Absorption enhancers such as sodium sulfate; humectants such as glycerin; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid; lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol, etc. can be used. Furthermore, the tablets may be coated with a conventional coating if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets. Furthermore, the composition containing the active ingredient can be filled into conventionally known capsules made from gelatin, pullulan, starch, gum arabic, hydroxypropyl methylcellulose (HPMC), etc., to form capsules.

In addition, when it is in the form of a pill, a wide variety of carriers conventionally known in the art can be used as carriers. Examples include excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc, binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol, and disintegrants such as laminaran and agar. can be used.

In addition to the above, additives such as surfactants, absorption enhancers, adsorbents, fillers, preservatives, stabilizers, emulsifiers, and solubilizers may be selected and used as appropriate depending on the form of the formulation. can.

All of these forms can be prepared using methods commonly used in the field; for example, tablets can be prepared by adding the above active ingredient and other excipients necessary to obtain tablets, mixing and dispersing them thoroughly, and then It can be obtained by compressing into tablets. Further, the powder can be obtained by appropriately adding the above-mentioned active ingredient and other excipients necessary for obtaining the powder, and mixing and pulverizing the mixture by a suitable method.

In addition to the above-mentioned formulation form, the present bacteria increasing agent and the present promoter may be in the form of a normal food or drink. The food and drink can be produced by adding an additive containing the black soybean seed coat extract described above or the black soybean seed coat extract described below to various foods and drinks. The food and drink may be in any form that can be orally ingested, such as a solution, suspension, emulsion, jelly (gel), sol, powder, or solid molded product, and is not particularly limited. Specifically, instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups/miso soups, and freeze-dried foods; soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, and tea. Beverages such as beverages, powdered drinks, concentrated drinks, nutritional drinks, and alcoholic drinks; Flour products such as bread, pasta, noodles, cake mixes, fried chicken powder, and bread crumbs; Candy, caramel, chewing gum, chocolate, cookies, biscuits, and cakes Confectionery such as pies, snacks, crackers, Japanese sweets, and desserts; Seasonings such as sauces, processed tomato seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, and curry/stew ingredients; Fats and oils such as processed oils, butter, margarine, and mayonnaise; Dairy products such as milk drinks, yogurts, cheese, fermented milk, lactic acid bacteria drinks, ice creams, and creams; Processed egg products such as puddings and mayonnaise; Fish, meat, ham, Processed seafood products such as sausages and fish paste products; Processed livestock products such as meat hams and sausages; Processed agricultural products such as canned agricultural products, jams and marmalades, pickles, boiled beans, and cereals; Frozen foods, nutritional foods, etc. . Although not limited, acidic foods are preferred from the viewpoint of suppressing precipitation of polyphenols derived from black soybean seed coat extract and deterioration of color.

Moreover, the present bacteria increasing agent and the present promoter may be in the form of ordinary feed or pet food. These feeds and pet foods can also be produced by adding the above-mentioned black soybean husk extract to various feeds and pet foods.

The content of black soybean seed coat extract in this bacteria increaser and this promoter is set at 100% by mass as the upper limit, depending on the type of form and the type of use (medicines, foods, feed/pet food, etc.) mentioned above. It can be set as appropriate. The dosage (intake amount) of the present bacteria increasing agent and the present promoter may be changed as appropriate depending on the type of human or animal, the sex and age of the subject, and the condition and severity of symptoms of the subject. Although not limited, for example, the daily dose (intake amount) for one adult human (body weight 50 kg) is converted to the amount of black soybean seed coat extract (dry amount) contained in this bacteria increasing agent and this promoter. Usually, the amount is about 10 to 500 mg. It is usually administered orally (ingested) once a day or in two to three divided doses. The time of administration is not particularly limited, and can be exemplified, for example, during one or more of the morning, noon, and evening meal times. Although not limited, it may be taken together with a meal, or within 30 minutes before or after a meal.

The subject of the present bacteria increasing agent and the present promoting agent may be any person who needs to increase the amount of equol in the body or has such a desire, and is not particularly limited as long as this is the case. These people include middle-aged and elderly people, preferably women, whose estrogen activity in the body has decreased or tends to decrease. Increased bone density, treatment and prevention of osteoporosis, alleviation of menopausal symptoms, lowering of cholesterol, prevention and improvement of metabolic syndrome, improvement of skin wrinkles, sagging or age spots, prevention of breast cancer, prostate cancer, etc. This includes those who request it. In addition, it is known that consuming high-fat foods causes dysbiosis of the intestinal flora. Those who are present can also be suitably targeted. By improving the intestinal flora, the number of equol-producing bacteria increases, and the equol-producing ability of the intestinal bacteria can be enhanced.

The intestinal equol-producing bacteria targeted by the present invention are resident intestinal bacteria that are involved in the metabolism of soybean isoflavone aglycones (e.g., daidzein, etc.) and their metabolites (e.g., dihydrodaidzein (DHD), etc.) to equol. . Such equol-producing bacteria include, in addition to the well-known Adlercreutzia equolifaciens, Asaccharobacter celatus AHU1763 (Ref.1), Asaccharobacter celatus gen. nov., sp nov. strain do03T (Ref.2), and Bacteroides ovatus (Ref. 3), Bifidobacterium animalis (Ref.4), Coriobacteriaceae sp MT1B9 (Ref.5), Eggerthella sp (YY7918 (Ref.6), Julong732 (Ref.7), MT4s-5 (Ref.8)), Enterococcus faecium ( Ref.9), Eubacterium sp D1 and D2 (Ref.10), Finegoldia magna (Ref.11), Lactobacillus mucosae (Ref.11), Lactobacillus sp Niu-O16 (Ref.12), Lactococcus garvieae (Lc 20-92) ) (Ref.13), Ruminococcus productus (Ref.14), Slackia sp (HE8 (Ref.15), FJK1 (Ref.16), TM-30 (Ref.17), NATTS (Ref.18)), Slackia Examples include Strain DZE (Ref. 19), Streptococcus intermedius (Ref. 14), and Veillonella sp (Ref. 20). Note that the document name corresponding to each Ref. number is described at the end of this specification. Preferably, it is a resident intestinal bacteria belonging to the genus Adlercreutzia, such as Adlercreutzia equolifaciens.

By orally taking (administering, ingesting) this bacteria increasing agent, the number of equol-producing bacteria in the intestine can be increased. Whether or not the number of equol-producing bacteria in the intestine increases by taking (administering or ingesting) this bacteria-increasing agent can be evaluated and confirmed by measuring the number of equol-producing bacteria in feces.
Furthermore, by orally taking (administering, ingesting) this promoter, production of equol can be promoted. Here, the cause of "promotion of equol production" is not particularly limited, but oral administration of this promoter increases the number of equol-producing bacteria as described above, as well as As shown in Experimental Example 2, in the metabolism of soybean isoflavones, the conversion of equol proceeds more predominately than the conversion to O-desmethylangolensine (O-DMA). Whether or not equol production is promoted by taking (administering or ingesting) this promoter can be evaluated and confirmed by measuring the equol concentration in the blood (equol blood amount). Furthermore, since equol is produced by the metabolism of soy isoflavone aglycones and their metabolites into equol in the intestine, when evaluating the promotion of equol production, soy isoflavones should be taken (administered, administered, Ingestion) is preferred.

Equol concentration in blood can be measured using a liquid chromatograph mass spectrometer (LC/MASS) (for example, Stephen Barnes etc., HPLC-Mass Spectrometry Analysis of Isoflavones, P.S.E.B.M., Vol.217: pp254- 262, 1998, etc.). Specifically, it can be measured by the method (LC-MS/MS analysis) described in the experimental examples described later.

(III) Blood equol concentration-increasing agent The blood equol concentration-increasing agent of the present invention (hereinafter also simply referred to as "this increasing agent") comprises an extract of black soybean seed coat, preferably an edible extract, and a soybean isoflavone. It is characterized by containing.

Soybean isoflavones are a type of flavonoid, and are a general term for compounds based on the aglycone skeleton of daidzein and its analogues glycitein and genistein. In addition to aglycones, isoflavones include glycosides and their acetylated and malonylated forms. Specifically, daidzein, glycitein, and genistein are soybean isoflavone aglycones, daidzin, glycitin, and genistein are soybean isoflavone glycosides, acetyldaidzine, acetylglycitin, and acetylgenistein are soybean isoflavone glycosides, and soybean isoflavone glycosides are daidzein, glycitein, and genistein. Examples of malonylated glycosides include malonyldaidzine, malonylglycitin, and malonylgenistein. These may be used alone or in any combination of two or more. Further, the present elevating agent may contain soybean isoflavone itself, but is not limited thereto, and may also contain edible materials containing soybean isoflavone. Such edible materials include, for example, isoflavone-containing parts (soybeans, soybean hypocotyls, etc.) of plants containing isoflavones (for example, leguminous plants such as soybeans), processed products thereof (such as dried products and extracts), Processed foods (for example, tofu, soy milk, fermented soy milk, natto, etc.) can be mentioned. In particular, soybean hypocotyl extract and purified products thereof can be preferably exemplified, although not limited thereto, since they have a high content of soybean isoflavones and can be ingested efficiently.

Like the above-mentioned bacteria increasing agent and promoter, this increasing agent can be used in any oral administration form, such as pharmaceuticals, quasi-drugs, food and drinks (foods for specified health uses, functional foods, etc.). It can be prepared as labeled foods, health functional foods such as nutritional functional foods, and supplements), feed, or pet food. The details are as described above regarding the present bacterial increase agent and the present promoter.

Although the proportion of soybean isoflavones in this elevating agent is not limited, it can be adjusted as appropriate based on the daily intake of soybean isoflavones. For example, the daily intake of soy isoflavones for one adult (weight 50 kg) is usually 10% in terms of the soy isoflavones (dry amount) contained in this elevating agent, although this may be changed as appropriate depending on the condition and severity of the subject. ~150mg. It is usually administered orally once or in two to three divided doses a day. The time of administration is not particularly limited, and can be exemplified, for example, during one or more of the morning, noon, and evening meal times. Also, although not limited, it is preferable to take it within 30 minutes either with a meal, before a meal, or after a meal since it affects the absorption of lipids contained in food. On the other hand, the amount of black soybean hull extract blended is not particularly limited.

By orally taking (administering, ingesting) this elevating agent, it becomes possible to increase the equol concentration in the blood. Whether or not the equol concentration in the blood increases by taking (administering, ingesting) this increasing agent can be evaluated and confirmed by measuring the equol concentration in the blood (equol blood amount). The measurement method is as described above.

By orally taking (administering, ingesting) this elevating agent, the effects based on equol can be enjoyed. In addition, effects based on equol include, but are not limited to, alleviation of symptoms of menopause (hot flashes, headaches, dizziness, symptoms of autonomic nervous system imbalance, tachycardia, blood pressure fluctuations, etc.); and reduction of lipid peroxide production. Suppression; suppression of excessive secretion of sebum (prevention or improvement of acne and pimples); prevention of cancer (breast cancer, endometrial cancer, prostate cancer, stomach cancer, etc.); type II diabetes (fasting blood sugar level, insulin resistance) Prevention or improvement of lifestyle-related diseases (diabetes, hyperlipidemia, arteriosclerosis, high cholesterol, etc.); Prevention or improvement of osteoporosis, increase in bone density, increase in bone mineral concentration; skin aging ( Preventing or improving skin firmness and elasticity (decrease in skin firmness and elasticity, age spots, wrinkles, sagging); Improving blood flow (preventing or improving sensitivity to cold, stiff shoulders, tension headaches, dullness and dryness of the skin, etc.); Preventing or improving male pattern hair loss At least one effect selected from improvements etc. can be expected.

(IV) Method of using black soybean seed coat extract The present invention also provides a method of using black soybean seed coat extract.
One method is to use a black soybean seed coat extract to impart an effect of enhancing the ability of intestinal bacteria to produce equol to an oral composition. The method can be carried out by incorporating the black soybean seed coat extract into the target oral composition. In addition, instead of the black soybean seed coat extract, the above-mentioned present bacteria increasing agent or present promoter containing black soybean seed coat extract as an active ingredient can also be used.

Targeted oral compositions include compositions that are orally administered to humans or compositions that are ingested by humans, specifically oral pharmaceuticals, oral quasi-drugs, and foods and drinks. Preferably it is food and drink. Furthermore, when targeting non-human animals, feed or pet food can be used as the oral composition.

The type of black soybean used as a raw material for the black soybean seed coat extract used in the method of the present invention, the method for obtaining black soybean seed coat, the black soybean seed coat extract, especially the black soybean seed coat acidic extract that is a preferred embodiment of the extract. The preparation method is as explained in (I) above, and can be cited in this section. The amount of the black soybean seed coat extract added to the oral composition is not particularly limited as long as it can provide the oral composition with an effect of enhancing the ability of intestinal bacteria to produce equol. The evaluation was conducted when subjects continuously ingested an oral composition containing black soybean seed coat extract (target composition) and when subjects took an oral composition containing no black soybean coat extract (comparison composition). This can be done by comparing the degree of enhancement of the ability of intestinal bacteria to produce equol by continuously ingesting equol. The degree of increase in equol production ability (difference before and after ingestion) when the target composition is continuously ingested is the same as the degree of increase in equol production ability (difference before and after ingestion) when the comparative composition is continuously ingested. ), it can be determined that the oral composition is given the effect of enhancing the ability of intestinal bacteria to produce equol by incorporating the black soybean hull extract into the oral composition. .

The enhancement of equol-producing ability by intestinal bacteria can be determined from the abundance ratio of equol-producing bacteria in the intestines. If the number of equol-producing bacteria present in the intestine increases to a greater extent by ingesting the target composition than the comparative composition, it can be said that the effect of the present invention is being exerted. It can also be determined from the amount of intestinal equol produced when soybean isoflavones are ingested. If the amount of equol present in the intestine increases to a greater extent by ingesting the target composition than the comparative composition, it can be said that the effect of the present invention is being exerted. These evaluations can be performed on mammals other than humans instead of humans, as shown in the experimental examples described below.
In addition, although the enhancement of equol production ability by intestinal bacteria can be indirectly determined, it can also be determined from the blood or urine equol concentration obtained when soybean isoflavones are ingested. If the degree of increase in blood or urine equol concentration is greater by ingesting the target composition than the comparative composition, it can be said that the effect of the present invention is being exerted. The evaluation can also be performed on mammals other than humans instead of humans, as shown in the experimental examples described below.

Another method of the present invention is the use of a black soybean seed coat extract in an oral composition containing soybean isoflavones to enhance the effect of the composition on increasing blood equol concentration. The method can be carried out by blending the black soybean seed coat extract into the target soybean isoflavone-containing oral composition. In addition, instead of the black soybean seed coat extract, the above-mentioned present bacteria increasing agent or present promoter containing black soybean seed coat extract as an active ingredient can also be used.

Targeted soy isoflavone-containing oral compositions include compositions that are orally administered to humans or compositions that are ingested by humans, specifically oral pharmaceuticals, oral quasi-drugs, and foods and drinks. . Preferably it is food or drink. Furthermore, when targeting animals, feed or pet food can be used as the oral composition. Soybean isoflavones are as described above.

The type of black soybean used as a raw material for the black soybean seed coat extract used in the method of the present invention, the method for obtaining black soybean seed coat, the black soybean seed coat extract, especially the black soybean seed coat acidic extract that is a preferred embodiment of the extract. The preparation method is as explained in (I) above, and can be cited in this section. The amount of black soybean hull extract added to the soybean isoflavone-containing oral composition is not particularly limited as long as it can enhance the effect of increasing the blood or urine equol concentration of the oral composition. The evaluation was based on continuous intake of a soybean isoflavone-containing oral composition containing black soybean hull extract and continuous intake of a soybean isoflavone-containing oral composition without black soybean hull extract. This can be done by comparing the degree of increase in equol concentration in blood or urine. If the degree of increase in blood or urinary equol concentration in the former is higher than in the latter, the method of the present invention is carried out by blending black soybean seed coat extract with the oral composition containing soybean isoflavones. It can be said that.

As mentioned above, in this specification, the terms "comprising" and "containing" include the meanings of "consisting of" and "consisting essentially of."

Hereinafter, the present invention will be explained using experimental examples in order to help understand the structure and effects of the present invention. However, the present invention is not limited in any way by these experimental examples. The following experiments were conducted at room temperature (25±5° C.) and atmospheric pressure conditions unless otherwise noted. In addition, unless otherwise mentioned, "%" described below means "mass %" and "part" means "mass part."

Experimental example 1 Black soybean seed coat extract intake test (effect on intestinal equol-producing bacteria)
Test animals (mice) were made to ingest black soybean seed coat extract, and their body weight, food intake, and amount of cecal content were measured. Genomic DNA was extracted from the cecal content, and intestinal flora analysis was performed. The abundance ratio (%) of producing bacteria (genus Adlercreutzia) in the cecum was measured.

(1) Test animal: Male C57BL/6J mouse, 8 weeks old (purchased from Japan SLC)
Breeding environment: room temperature 25°C, humidity 55%, indoor fluorescent lights were turned on on a 12-hour cycle from 7 a.m. to 7 p.m.
Breeding period: After the animals were brought in and after a two-week acclimatization period using solid food samples, they were divided into the following test sections (a) to (d) so that the average weight of each group was equal (each group n=6-7). During the breeding period, each feed and drinking water were available ad libitum.

Test area:
(a) Control group: intake of feed (normal food: solid sample D12450J: Research Diet, the same applies hereinafter) + drinking water (distilled water, the same hereinafter) (b) Control group A: feed (normal food) + 0. Ingestion of drinking water supplemented with 3% cholic acid (c) Black soybean coat extract group: Intake of feed (normal food + 1.7% black soybean coat extract) + drinking water supplemented with 0.3% cholic acid (d) Isoflavone group: Ingested feed (regular food + 0.8% isoflavones) + drinking water added with 0.3% cholic acid.

Chronocare (manufactured by Fujico Co., Ltd.) was used as the "black soybean seed coat extract". Chronocare is an acidic extract of black soybean hulls containing 58% by mass or more of black soybean hull polyphenols, and contains excipients at a rate of approximately 15% by mass. Black soybean seed coat polyphenols include, for example, Procyanidin B2, Procyanidin C1, Procyanidin A2, Procyanidin B5, epicatechin, and cyanidin-3-glucoside. In addition, "1.7%" of the above-mentioned "1.7% black soybean seed coat extract" is the amount converted to the black soybean seed coat extract content contained in Chronocare.
Further, as the "isoflavone", Fujiflavone P40 (manufactured by Fujico Co., Ltd.) was used. Fujiflavone P40 contains more than 37% by mass of soybean isoflavones. Among soybean isoflavones, the total amount of glycosides of three isoflavones (aglycones), genistein, daidzein, and glycitein, is about 85% by mass or more, and aglycones are included at a ratio of about 15% by mass. Note that "0.8%" in the above "0.8% isoflavone" is the amount converted to the isoflavone content contained in Fujiflavone P40.

(2) Test method and results 1. Body weight, food intake, and cecal content The test animals in each test group were allowed to freely consume each feed and drinking water for 2 weeks. ) was measured. The results are shown in Table 1 as average values for each group.

As shown in Table 1 above, compared to both the (a) control group and (b) control A group, the amount of cecal content was lower in the (c) black soybean hull extract group and (d) isoflavone group. A significant increasing trend was observed. On the other hand, no major differences in food intake were observed between the groups.

2. Analysis of intestinal flora in cecal contents DNA was extracted from the cecal contents collected from test animals in each test group after 2 weeks of ad libitum ingestion of each feed and drinking water. We amplified the V3-V4 region of the 16S rRNA gene and performed meta-16S bacterial flora analysis using Illumina MiSeq. A total of 1,195,799 reads (average 51,991 reads) from the 23 samples obtained were analyzed using QIIME (Quantitave Insights Into Microbial Ecology).

Figure 2 shows the abundance ratio (%) of Adlercreutzia, an equol-producing bacterium, in the cecum from bacterial flora analysis. As shown in Figure 2, compared to (a) the control group and (b) control group A, the number of equol-producing bacteria in the intestine was significantly increased in the (c) black soybean coat extract group. was confirmed. This effect of increasing intestinal equol-producing bacteria was only slightly observed in the (d) isoflavone group compared to the (c) black soybean seed coat extract group, so there was no benefit from ingesting black soybean seed coat extract. This is considered to be a unique effect.
It is known that continued intake of high-fat foods disrupts the intestinal flora. In this experiment, instead of ingesting a high-fat diet, test animals ((b) control A group, (c) black soybean hull extract group, (d) isoflavone group) were given drinking water containing primary bile acid (cholic acid). Experiments were conducted with the intestinal flora disrupted or disturbed by ingesting . As shown in FIG. 2, it was confirmed that even in this state, the number of equol-producing bacteria in the intestine was significantly increased by ingesting black soybean seed coat extract. Based on these facts, orally ingesting black soybean seed coat extract changes the bacterial composition ratio of the intestinal flora, such as increasing the number of equol-producing bacteria in the intestine, and as a result, equol in the intestinal bacteria changes. It was suggested that the production ability was enhanced and the amount of equol in the body could be increased.

Experimental example 2 Black soybean seed coat extract intake test (effect on equol production in the body)
Based on the results of Experimental Example 1, test animals (mice) were allowed to ingest black soybean seed coat extract together with soybean isoflavones for a certain period of time, and then their body weight, cecal content, and daily intake (g/ day/mice) was measured. Furthermore, in order to evaluate the amount of equol produced in the body, blood equol concentration was measured.

(1) Test animal: Male C57BL/6J mouse, 8 weeks old (purchased from Japan SLC)
Breeding environment: room temperature 25°C, humidity 55%, indoor fluorescent lights were turned on on a 12-hour cycle from 7 a.m. to 7 p.m.
Breeding period: After the animals were brought in and after a two-week acclimatization period using solid food samples, they were divided into test groups (a) and (b) below so that the average weight of each group was equal ( n=6 for each group). During the breeding period, each feed and drinking water (distilled water) were freely available.

Test area:
(a) Isoflavone group (Iso): Feed (normal + 0.2% isoflavones) + drinking water intake (b) Isoflavones + black soybean husk extract group (Iso + Chrono): Feed (normal + 0.2% isoflavones + 0.425%) Intake of black soybean seed coat extract) + drinking water

Chronocare (manufactured by Fujico Co., Ltd.) was used as the "black soybean seed coat extract". In addition, "0.425%" of the above-mentioned "0.425% black soybean hull extract" is the amount converted to the black soybean hull extract content contained in Chronocare. Further, as the "isoflavone", Fujiflavone P40 (manufactured by Fujico Co., Ltd.) was used. Its composition is as described above. Note that "0.2%" in the above "0.2% isoflavone" is the amount converted to the isoflavone content contained in Fujiflavone P40.

(2) Test method and results 1. Body weight, food intake, and cecal content The test animals in each test group were allowed to freely consume each feed and drinking water for 2 weeks. ) was measured. The results are shown in Table 2 as average values for each group.

As shown in Table 2 above, compared to (a) the group that ingested isoflavones (Iso), (b) the group that ingested black soybean hull extract in addition to isoflavones (Iso + Chrono), the amount of cecal content decreased. An increasing trend was observed. No major differences were observed in body weight and food intake.

2. Measurement of blood equol amount (1) Serum deconjugation treatment
After the test animals in each test group were allowed to freely consume each feed and drinking water for 2 weeks, blood was collected, and the serum fraction was frozen. This frozen serum sample was subjected to the following enzyme treatment to deconjugate it, and then subjected to column treatment.

(1-1) Enzyme treatment
Enzyme treatment was performed by the following method.
1. Add 10 μL of 100 ppm naligenin to 200 μL of serum.
2. Add 200 μL of 50 U/mL sulfatase solution and incubate at 37°C for 2 hours.
3. Add 200 μL of 200 U/mL β-glucuronidase solution and incubate at 37°C for 2 hours.

(1-2) Column processing
Column treatment was carried out by the following method.
1. After passing the entire serum sample enzyme-treated in (1-1) through Sep-Pak (Waters Corporation), wash the Sep-Pak three times with 1 mL of 15% by volume aqueous methanol.
2. Pour 1 mL of 80% by volume water-containing methanol through the Sep-Pak and collect the eluted sample.

(2) LC-MS/MS analysis Using the serum that has undergone the deconjugation treatment (enzyme treatment and column treatment) as a sample, daidzein, dihydrodaidzein (DHD), equol, and O-dessin contained in the serum sample are The amount of methylangolensin (O-DMA) was measured by LC-MS/MS analysis.
LC-MS/MS analysis was performed using a Prominence UFLC system (Shimazhu) coupled to a triple quadrupole mass spectrometer (API2000 LC/MS/MS system, Applied Biosystems) using an ESI probe as an ion source. Ta. The substance to be measured was ionized in positive mode and analyzed in multiple reaction monitoring (MRM) mode.
[LC conditions]
Column: InertSustain C18 column (5μm, 2.1x 50 mm, GL Science)
Mobile phase: 0.1% formic acid + 50% aqueous methanol Separation method: Isocratic separation Flow rate: 0.2 mL/min
Injection volume: 5μL
Analysis time: 10 minutes.
[MC conditions]
Curtain gas: 30 psi
Collitin gas: 3 psi
Ionspray voltage: 5.5kV
Interface temperature：500℃
Ion source gas 1 (nebulizing gas): 60 psi
Ion source gas 2 (turbo gas): 60 psi

The m/z values are as follows.
Daidzein: 255.2 > 199.1, 91.0, 65.0,
Dihydrodidzein (DHD): 257.2 > 122.9, 77.0, 95.0,
Equol: 243.3 > 123.0, 133.1, 106.9,
O-desmethylangolensine (O-DMA): 259.2 > 149.1, 121.1, 77.1,
Naringenin (internal standard): 273.2 > 153.1, 147.1, 90.9.
Analyst 1.5 software (Applied Biosystems) was used as the analysis software.

(3) Results The amounts of daidzein, dihydrodaidzein (DHD), equol, and O-desmethylangolensin (O-DMA) contained in the serum collected from test animals in each test group were measured. The results are shown in Figure 3. Figure 3 (A) compares the total amount and proportion of these four measured compounds contained in 1 L of serum for test animals in each test group ((a) Iso, (b) Iso + Chrono). 3B is a diagram comparing the proportions of each compound.
As shown in Figure 3, blood equol concentration tended to increase more significantly when subjects ingested black soybean hull extract in addition to soybean isoflavones than when ingesting soybean isoflavones alone. In addition, blood levels of dihydrodidzein (DHD), a precursor of equol, tended to increase significantly when subjects ingested black soybean hull extract in addition to soybean isoflavones. In contrast, no difference was observed in the blood concentration of O-desmethylangolensin (O-DMA), a daidzein metabolite, between intake of soy isoflavone alone and intake of black soybean hull extract. Ta.

As shown in the above experiments, it was confirmed that equol production in the body increased by ingesting black soybean seed coat extract in addition to soybean isoflavones. An increase in dihydrodaidzein (DHD), a precursor of equol, was also observed. On the other hand, the amount of O-DMA, a daidzein metabolite, did not change. From this, it is thought that black soybean seed coat extract does not affect the intestinal bacteria involved in O-desmethylangolensin (O-DMA) production, but selectively affects the intestinal bacteria involved in equol production. In other words, by ingesting black soybean seed coat extract, as shown in Experimental Example 1, equol-producing bacteria and intestinal bacteria involved in equol production were increased, while O-desmethylangolensin (O It is thought that it can improve the intestinal flora to one suitable for efficient production of equol, as it does not affect the intestinal bacteria involved in the production of equol (-DMA).

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Claims (6)

An agent for increasing intestinal equol-producing bacteria, which contains black soybean seed coat extract as an active ingredient. The agent for increasing equol-producing bacteria according to claim 1, wherein the equol-producing bacteria are bacteria of the genus Adlercreutzia. An equol production promoter containing black soybean seed coat extract as an active ingredient. A blood equol concentration increasing agent containing black soybean seed coat extract and soybean isoflavones. A method of using a black soybean seed coat extract, which comprises blending the black soybean seed coat extract into an oral composition to impart an effect of enhancing equol production ability by intestinal bacteria to the oral composition. A method of using a black soybean seed coat extract for enhancing the blood equol concentration increasing effect of the oral composition by incorporating the black soybean seed coat extract into an oral composition, the method comprising:
The method, wherein the oral composition contains soy isoflavones.