JP7758787B2 - Primary bile acid synthesis promoter - Google Patents

Description

This invention relates to a new use of soy isoflavones as a food ingredient.

Abnormalities in the intestinal microbiota (dysbiosis) are increasingly considered to be an important factor in the development of conditions such as obesity, type 2 diabetes, colon cancer, and inflammatory bowel disease. For example, changes in the composition of the intestinal microbiota have been cited as one of the causes of obesity. Specifically, obese individuals tend to have a high proportion of Firmicutes bacteria and a low proportion of Bacteroidetes bacteria. On the other hand, normal or lean individuals tend to exhibit the opposite trend to obese individuals, with a high proportion of Bacteroidetes bacteria and a low proportion of Firmicutes bacteria (Non-Patent Document 1). For this reason, it has been proposed that the intake of health foods can change the composition of the intestinal microbiota from obese individuals to normal or lean individuals. For example, it has been reported that the use of an extract from a plant in the genus Salacia as an active ingredient increases the proportion of Bacteroidetes and decreases the proportion of Firmicutes (Patent Document 1). Furthermore, because the proportion of butyrate-producing bacteria present is reduced in many ulcerative colitis patients compared to healthy individuals, it has been suggested that one of the causes of inflammatory bowel disease (ulcerative colitis, Crohn's disease) is a decrease in intestinal butyrate concentration due to a decrease in butyrate-producing bacteria in the intestine (Non-Patent Document 2).

Bile acids have also been reported as a factor controlling the intestinal microbiota, and it has been reported that bile acids, which increase with the intake of a high-fat diet, affect changes in the intestinal microbiota. Bile acids are the main component of bile, which is involved in lipid absorption. Their main role is to promote the formation of micelles in the digestive tract when fat is ingested, making fat more easily absorbed. Bile acids consist of primary and secondary bile acids. Primary bile acids are synthesized in the liver as conjugated bile acids by conjugating cholesterol with glycine or taurine and secreted into the intestine. After completing their role in the intestine, conjugated bile acids are reabsorbed in the small intestine and returned to the liver. However, some conjugated bile acids reach the large intestine, where intestinal bacteria remove the glycine or taurine (deconjugation). The deconjugated primary bile acids (cholic acid, chenodeoxycholic acid, etc.) are further converted to secondary bile acids such as deoxycholic acid and lithocholic acid. Once converted to a secondary bile acid, the molecule's hydrophobicity increases and it exhibits strong bactericidal activity. For example, the secondary bile acid deoxycholic acid (DCA) is known to exhibit 10 times the bactericidal activity of the primary bile acid cholic acid (CA) (see Non-Patent Document 3).

Based on these findings, the "bile acid hypothesis" has been proposed as a mechanism by which changes in the intestinal flora occur, stating that the intake of a high-fat diet results in excess secretion of bile acids, which in turn results in increased deoxycholic acid (DCA) concentrations by intestinal bacteria, acting as selective pressure (see Non-Patent Document 4). Deoxycholic acid (DCA) is also known to have a colon cancer-promoting effect (Non-Patent Document 5). Furthermore, according to Patent Document 2, there are also documents pointing out that the recent increase in colon cancer in Japan is due to an increase in lipid intake due to the Westernization of dietary habits, and the resulting increase in the amount of bile acids in the intestine. Furthermore, Non-Patent Document 6 describes that when one becomes obese, there is a significant increase in intestinal bacteria (deoxycholic acid-producing bacteria) that convert primary bile acids (such as cholic acid) that aid in the digestion and absorption of lipids into secondary bile acids (such as deoxycholic acid), and that this increase in secondary bile acids in the body causes hepatic stellate cells in the liver to undergo cellular senescence, leading to the secretion of inflammatory cytokines, which promotes carcinogenesis in surrounding liver cells. It suggests that a similar mechanism is involved in the development of liver cancer associated with human obesity, and that inhibiting the growth of deoxycholic acid-producing bacteria may lead to the prevention of liver cancer.

As such, various secondary bile acid lowering agents (secondary bile acid production inhibitors) have been proposed as methods for improving or preventing diseases caused by secondary bile acids (colon disease, liver disease) and their symptoms. Examples include a secondary bile acid lowering agent containing a plant-derived polyphenol such as curcumin as an active ingredient (Patent Document 3), a secondary bile acid production inhibitor containing an α-linked galactose-containing oligosaccharide as an active ingredient (Patent Document 4), a secondary bile acid inhibitor containing an extract of a Ganoderma lucidum component as an active ingredient (Patent Document 5), and a deoxycholic acid reducer containing cells of lactic acid bacteria belonging to Lactobacillus gasseri and/or a culture thereof as an active ingredient (Patent Document 2). These lactic acid bacteria are particularly advantageous in that they inhibit the conversion of primary bile acids to secondary bile acids, thereby suppressing the production of secondary bile acids without reducing the amount of useful primary bile acids, i.e., selectively reducing the amount of secondary bile acids.

JP 2015-127340 A JP 2017-66086 A JP 2009-227609 A Japanese Patent Application Laid-Open No. 2004-244365 Japanese Patent Application Laid-Open No. 2018-43955

Ley R.E., et al., Nature, 2006, Vol.444, No.7122. pp.1022-1023 Machiels K., et al., Gut, 2014, Vol.63, No.8, pp.1275-1283 Kurdi et al., J Bacteriol., 188(5), 1979-1986 (2006) Yokota et al., Gut Microbes., 3(5):455-459 (2012) H. Bernstein et al., Mutat. Res., Vol. 589, pp. 47-65, 2005 Japan Science and Technology Agency website: "Joint announcement: Obesity-related changes in intestinal bacteria promote the development of liver cancer" (JST, Japan Foundation for Cancer Research, June 27, 2013) (http://www.jst.go.jp/pr/announce/20130627-2/)

The objective of the present invention is to provide new uses for soy isoflavones as food ingredients. Specifically, the objective is to provide new functional uses for soy isoflavones as food ingredients based on the useful bioregulatory function (tertiary function) that they have on the body when ingested.

The inventors of the present invention conducted extensive research to solve the above-mentioned problems and discovered that the deterioration of the intestinal flora (changes in the composition of the intestinal flora) that occurs in mice when they are fed a diet simulating a high-fat diet (a diet supplemented with primary bile acids) is significantly improved by simultaneously feeding them soy isoflavones; furthermore, the decline of equol-producing bacteria, short-chain fatty acid-producing bacteria such as butyric acid bacteria, and/or lactic acid bacteria, which are considered to be beneficial intestinal bacteria, is inhibited and even increased.

The researchers also found that ingestion of soy isoflavones significantly reduced the increase in secondary bile acid production that occurs when animals are fed a diet supplemented with primary bile acids, while confirming that the amount of primary bile acids, which are useful for fat absorption, did not decrease but rather increased.

The present invention was completed based on this finding and includes the following embodiments.
(I) Agent for improving intestinal flora
(I-1) An agent for improving intestinal flora, containing soy isoflavone as an active ingredient.
(I-2) The agent for improving intestinal flora according to (I-1), which exhibits at least one effect selected from the group consisting of the following (a) to (e):
(a) an increase in the Shannon index,
(b) a decrease in the ratio of “Firmicutes bacteria/Bacteroidetes bacteria” in intestinal bacteria;
(c) an increase in the proportion of equol-producing bacteria (Adlercreutzia) in intestinal bacteria;
(d) an increase in the proportion of short-chain fatty acid-producing bacteria in intestinal bacteria;
(e) Increase in the proportion of lactic acid bacteria (Lactobacillus) in intestinal bacteria.

(II) Secondary bile acid production inhibitor, primary bile acid production promoter (II-1) A secondary bile acid production inhibitor containing soy isoflavone as an active ingredient.
(II-2) A secondary bile acid production inhibitor according to (II-1), characterized by increasing the amount of primary bile acid produced.
(II-3) A primary bile acid production promoter containing soy isoflavone as an active ingredient.
(II-4) The primary bile acid production promoter according to (II-3), wherein the primary bile acid is at least one selected from the group consisting of cholic acid and taurocholic acid.

(III) Additive for intestinal flora improver, additive for secondary bile acid production inhibitor, additive for primary bile acid production promoter (III-1) Additive for intestinal flora improver or secondary bile acid production inhibitor containing soy isoflavone as an active ingredient.
(III-2) An additive for a primary bile acid production promoter containing soybean isoflavone as an active ingredient.

(IV) Method of using soy isoflavones (IV-1) A method of using soy isoflavones by incorporating soy isoflavones or the additive described in (III-1) into an oral composition to impart to the oral composition at least one effect selected from the group consisting of an effect of improving the intestinal flora, an effect of inhibiting secondary bile acid production, and an effect of increasing the amount of primary bile acid produced.

The present invention provides an intestinal microbiota improver containing soy isoflavones as an active ingredient. The intestinal microbiota improver of the present invention can exert at least one effect selected from the group consisting of (a) an increase in the Shanon index, (b) a decrease in the "Firmicutes/Bacteroidetes" ratio in intestinal bacteria, (c) an increase in the proportion of equol-producing bacteria (Adlercreutzia) in intestinal bacteria, (d) an increase in the proportion of short-chain fatty acid-producing bacteria in intestinal bacteria, and (e) an increase in the proportion of lactic acid bacteria (Lactobacillus) in intestinal bacteria. Based on the effect of (a), the intestinal microbiota improver of the present invention can improve the diversity of intestinal microbiota that has been reduced by the intake of a high-fat diet. Furthermore, based on the effect of (b), it is possible to change the constitution of an individual from an obese type to a normal type or a lean type by changing the composition of the intestinal microbiota. The effect of (c) is that equol, which has estrogen-like effects, is produced in the body, making it possible to supplement the female hormones that tend to become deficient with age. The effect of (d) is that it is possible to prevent or improve diseases and conditions caused by a decrease in short-chain fatty acids in the intestines, such as inflammatory bowel disease. Furthermore, the effect of (e) is that lactic acid bacteria can prevent or improve autoimmune diseases and colds, and can improve intestinal regulation, diarrhea, bowel movements, etc. by increasing good bacteria and suppressing bad bacteria.

The present invention also provides a secondary bile acid production inhibitor containing soy isoflavones as an active ingredient. The secondary bile acid production inhibitor of the present invention is excellent at reducing the amount of secondary bile acids in the intestine. Therefore, it can be useful in preventing or ameliorating diseases and conditions caused by secondary bile acids (e.g., colon disease, liver disease, etc.). Furthermore, the secondary bile acid production inhibitor of the present invention inhibits the production of secondary bile acids from primary bile acids in the intestine, thereby increasing rather than reducing the production of primary bile acids, which are important for lipid absorption, and selectively reducing the production of secondary bile acids.

Furthermore, the secondary bile acid production inhibitor of the present invention uses soy isoflavones, which have been confirmed to be safe for consumption, as its active ingredient, so there is no need to worry about side effects. It can be easily ingested by incorporating it into your daily diet, and you can enjoy the aforementioned effects.

Furthermore, according to the method of using the additive for an intestinal flora improver, the additive for an inhibitor of secondary bile acid production, or the soy isoflavone of the present invention, by adding it to a target food composition or the like, the intestinal flora improver and/or secondary bile acid production inhibitor of the present invention can be easily prepared and provided.

This shows the results of a comparison of the Shanon index (mean ± SEM (n = 6), Tukey-HSD test) from the "analysis of intestinal bacterial flora in cecal contents" in an experimental example between the control group, CA group (cholic acid administration group), CA + 3% raffinose group, CA + 0.5% curcumin group, and CA + 0.8% isoflavone group. Different letters in each graph indicate significant differences between groups (p < 0.05). From the "analysis of intestinal bacterial flora in cecal contents" in the experimental example, (A) the results of comparing the intestinal bacterial flora composition at the phylum level (n=6) among the control group, CA group (cholic acid administration group), CA + 3% raffinose group, CA + 0.5% curcumin group, and CA + 0.8% isoflavone group are shown. (B) The results of comparing the ratio of the Firmicutes phylum to the Bacteroidetes phylum (Firmicutes/Bacteroidetes ratio) among each group are shown. This figure compares the abundance (%) of equol-producing bacteria (genus Adlercreutzia) in the cecal contents of each group from the results of the "analysis of intestinal bacterial flora in cecal contents" in the experimental example. Mean ± standard error (n=6) (the same applies to Figures 4 to 7 below). Different letters in each graph indicate significant differences between groups (Tukey-HSD test, p<0.05). This is a graph comparing the abundance ratio (%) of lactic acid bacteria (genus Lactobacillus) in the cecal contents of each group, based on the results obtained from the ``Analysis of intestinal bacterial flora in cecal contents'' in the experimental example. The results of the "analysis of intestinal bacterial flora in cecal contents" in the experimental example show a comparison of the abundance ratio (%) of various short-chain fatty acid-producing bacteria present in the cecal contents of each group. A shows a comparison of the abundance ratio (%) of butyric acid-producing bacteria (Coprococcus genus) in the cecal contents of each group; B shows a comparison of the abundance ratio (%) of butyric acid, propionic acid, and isovaleric acid-producing bacteria (Prevotella genus) in the cecal contents of each group; C shows a comparison of the abundance ratio (%) of succinic acid-producing bacteria (Parabacteroides genus) in the cecal contents of each group; and D shows a comparison of the abundance ratio (%) of acetic acid and lactic acid-producing bacteria (Blautia genus) in the cecal contents of each group. This figure compares the percentage of bacteria belonging to the phylum Firmicutes in the cecal contents of each group, based on the results of the "intestinal microbiota analysis in cecal contents" in the experimental example. A indicates the percentage of bacteria belonging to the genus Turicibacter, and B indicates the percentage of bacteria belonging to the genus Dorea. In Figure A, different letters in each graph indicate significant differences between groups (Tukey-HSD test, p<0.05). In Figure B, * indicates a significant difference compared to the CA group, based on the Welch test (p<0.05). This figure compares the abundance (%) of Proteobacteria (Desulfovibrionaceae) bacteria in the cecal contents of each group from the results of "analysis of intestinal bacterial flora in cecal contents" in an experimental example. Different letters in each graph indicate significant differences between groups (Tukey-HSD test, p<0.05).

(I) Intestinal Flora Improving Agent The intestinal flora improving agent of the present invention (hereinafter also simply referred to as "the present flora improving agent") is characterized by containing soy isoflavone as an active ingredient.

Soy isoflavones are a type of flavonoid found in large amounts mainly in the germ of soybeans, and are a general term for compounds with the basic structure shown in the following formula:

Known soy isoflavones include compounds with the above basic structure (soy isoflavone aglycones), glycosides with sugars attached (soy isoflavone glycosides), and acetylated and malonylated versions of these glycosides. In this specification, the term "soy isoflavones" encompasses these soy isoflavone aglycones, soy isoflavone glycosides, and their acetylated and malonylated versions. Specifically, soy isoflavone aglycones include genistein, daidzein, and glycitein. Soy isoflavone glycosides include genistin, daidzin, and glycitin. Acetylated versions of soy isoflavone glycosides include acetylgenistin, acetyldaidzin, and acetylglycitin, and malonylated versions include malonylgenistin, malonyldaidzin, and malonylglycitin. These soy isoflavones may be used alone or in combinations of two or more. Furthermore, in the present invention, soy isoflavones may be those purified from soybeans, or they may be crudely purified products, such as those extracted from soybeans (extracts), or crushed or shredded soybeans. Methods for purifying or extracting isoflavones from soybeans are known, and in the present invention, purified products or extracts of soy isoflavones obtained according to known methods can be used. Commercially available soy isoflavones can also be used.

The present microbiota improver can take any form as long as it is administered orally. Furthermore, as long as it is administered orally (orally ingested), there are no particular limitations on its intended use (drugs, quasi-drugs, food and drink [including health functional foods such as foods for specified health uses, foods with functional claims, and nutritional functional foods, as well as supplements]). Food and drink are preferred, and foods for specified health uses or foods with functional claims that can claim to have certain actions or effects.

Specific oral dosage forms include those prepared in the form of liquids (including extracts and syrups) or jellies; powders, fine granules, or granules formulated into powder or granules using conventional methods; capsules (hard capsules, soft capsules) in which liquids, powders, or granules are filled; and powders or granules further compressed into tablets (solid formulations).

This bacterial flora improver can also be prepared in various dosage forms (oral administration forms) by combining the above-mentioned soy isoflavones with conventionally known edible carriers, excipients, etc. that are pharmaceutically or food-acceptable.

When the present bacterial flora improver is in the form of a liquid preparation, it can be stored frozen, or it can be stored after removing water by freeze-drying or other methods. Freeze-dried preparations and dry syrups can be reconstituted by adding sterilized water or other suitable solution at the time of use.

When the present bacterial flora-improving agent is in the form of a solid preparation, for example, a tablet, a wide variety of carriers conventionally known in the art can be used. Examples of such carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, and silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, crystalline cellulose, hydroxypropylcellulose, hypromellose, and sodium alginate; and binders such as dry starch, powdered agar, powdered laminarin, sodium bicarbonate, and polio. Disintegrants such as ethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, crospovidone, povidone, and low-substituted hydroxypropyl cellulose can be used. Disintegration inhibitors such as stearin, cocoa butter, and hydrogenated oils can be used. Absorption promoters such as quaternary ammonium salts and sodium lauryl sulfate can be used. Moisturizing agents such as glycerin can be used. Adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid can be used. Lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol can also be used. Furthermore, tablets can be coated with conventional coatings, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, or double-layered or multi-layered tablets, as needed. Furthermore, compositions containing the active ingredient can be filled into conventional capsules made from materials such as gelatin, pullulan, starch, gum arabic, and hydroxypropylmethylcellulose (HPMC) to form capsules.

When formulated in pill form, a wide variety of carriers conventionally known in the art can be used. Examples include excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, and talc; binders such as powdered gum arabic, powdered tragacanth, gelatin, and ethanol; and disintegrants such as laminaran and agar.

Additives other than those mentioned above, such as surfactants, absorption enhancers, adsorbents, fillers, preservatives, stabilizers, emulsifiers, solubilizers, etc., can be selected and used appropriately depending on the form of the formulation.

All of these forms can be prepared using methods commonly used in the art. For example, tablets can be obtained by adding the above-mentioned active ingredient and other excipients necessary to obtain tablets, mixing and dispersing them thoroughly, and then compressing them into tablets. Powders can be obtained by adding the above-mentioned active ingredient and other excipients necessary to obtain powders, mixing and pulverizing them using a suitable method.

In addition to the formulations described above, the present microbiota-improving agent may also be in the form of ordinary food or beverage. Such food or beverage can be produced by adding the soy isoflavones described above or an additive containing soy isoflavones described below to various foods or beverages. The food or beverage may be in any form that can be orally ingested, such as a solution, suspension, emulsion, jelly (gel), sol, powder, or solid molding, and is not particularly limited. Specific examples include instant foods such as instant noodles, retort pouch foods, canned foods, microwave foods, instant soups/miso soups, and freeze-dried foods; beverages such as soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, nutritional drinks, and alcoholic beverages; wheat flour products such as bread, pasta, noodles, cake mix, fried chicken flour, and breadcrumbs; sweets such as candy, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, and dessert sweets; sauces, processed tomato seasonings, and flavors. Examples include condiments such as seasonings, cooking mixes, sauces, dressings, soups, curry and stew bases; processed oils and fats such as butter, margarine and mayonnaise; dairy products such as milk drinks, yogurt, cheese, fermented milk, lactic acid bacteria drinks, ice cream and cream; processed egg products such as pudding and mayonnaise; processed seafood products such as fish ham and sausage and fish paste products; processed livestock products such as meat ham and sausage; processed agricultural products such as canned agricultural products, jams and marmalades, pickles, boiled beans and cereals; frozen foods, nutritional foods, etc.

(Effect of intestinal flora improver)
The target of this microbiota improver is a human or a non-human animal. Preferably, it is a human. Examples of non-human animals include pets (companion animals), laboratory animals, and animals kept in zoos and aquariums. By administering (ingesting) this microbiota improver to a human or a non-human animal, it is possible to adjust the composition of the intestinal microbiota of the human or non-human animal, and in particular, to improve changes in the composition of the intestinal microbiota caused by ingestion of a high-fat diet, and to return it to a state before or close to the state before ingestion of the high-fat diet, or even to an even better state. For example, ingestion of this microbiota improver induces the following changes in the intestinal microbiota:

(A) Increase in Shanon Index: Ingestion of this microbiota improver by humans or non-human animals can increase the Shanon index (increase in Shanon index) that has decreased due to ingestion of a high-fat diet. The Shanon index is an index that takes into account the number of bacterial species and the frequency of each species, and is used as an index to determine the diversity of the intestinal microbiota. In other words, ingestion of this microbiota improver by humans or non-human animals can improve the diversity of the intestinal microbiota that has decreased due to ingestion of a high-fat diet, and can restore the normal state (intestinal microbiota diversity) to or close to the normal state before ingestion of the high-fat diet.

(B) Changes in the composition of intestinal microbiota at the phylum level. When humans or non-human animals ingest this microbiota-improving agent, the ratio of Firmicutes bacteria to Bacteroidetes bacteria present in the intestine (the "Firmicutes bacteria/Bacteroidetes bacteria" ratio) decreases. This decrease in abundance ratio may be caused by an increase in the ratio of Bacteroidetes bacteria to Firmicutes bacteria, or by an increase in the ratio of Firmicutes bacteria to Bacteroidetes bacteria, or by a decrease in the ratio of Firmicutes bacteria and an increase in the ratio of Bacteroidetes bacteria. As described in Non-Patent Document 1, the "Firmicutes bacteria/Bacteroidetes bacteria" ratio tends to be high in obese or obese-prone subjects and low in normal or lean subjects. As will be shown in the experimental examples described below, ingestion of this microbiota improver can change the composition of the intestinal microbiota from obese to normal or lean (non-obese), and therefore it is believed that this microbiota improver can reduce or prevent obesity, or maintain a non-obese state. In other words, this microbiota improver can be expected to have diet effects such as suppressing weight gain and weight loss.

(C) Increasing the Prevalence of Beneficial Bacteria (1) Increasing the Prevalence of Equol-Producing Bacteria (Adlercreutzia) in Intestinal Bacteria. Ingestion of this microbiota improver by humans or non-human animals increases the proportion of Adlercreutzia, an equol-producing bacterium, in the intestine. This bacterium converts ingested isoflavones into equol, which has higher estrogenic activity. In other words, when ingested, this microbiota improver, which contains soy isoflavones as its active ingredient, increases the proportion of equol-producing bacteria (Adlercreutzia) in the intestine, thereby promoting the metabolism and activation of isoflavones and producing equol. Therefore, by improving the human intestinal microbiota (by adjusting the composition of the intestinal microbiota), this microbiota improver enhances the production of equol from isoflavones, enabling it to exert its equol-based effects. The effects of equol can be expected to include, but are not limited to, at least one effect selected from the following: alleviation of menopausal symptoms (hot flashes, headache, dizziness, autonomic nervous system disorder-like symptoms, tachycardia, blood pressure fluctuations, etc.); suppression of lipid peroxide production; suppression of excessive sebum secretion (prevention or improvement of acne and pimples); prevention of cancer (breast cancer, uterine cancer, prostate cancer, stomach cancer, etc.); prevention or improvement of type II diabetes (fasting blood glucose 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; prevention or improvement of skin aging (loss of skin firmness and elasticity, age spots, wrinkles, sagging); improvement of blood flow (prevention or improvement of cold sensitivity, stiff shoulders, tension headaches, dull and dry skin, etc.); and prevention or improvement of male pattern baldness.

(2) Increase in the proportion of short-chain fatty acid-producing bacteria in intestinal bacteria. By ingesting this microbiota improver in humans or non-human animals, the proportion of short-chain fatty acid-producing bacteria in the intestine can be increased. Specifically, short-chain fatty acid-producing bacteria include bacteria of the genus Coprococcccus that produce butyric acid, bacteria of the genus Parabacteroides that produce succinic acid, bacteria of the genus Prevotella that produce butyric acid, propionic acid, and isovaleric acid, and bacteria of the genus Blautia that produce acetic acid and lactic acid. These short-chain fatty acid-producing bacteria can increase the amount of short-chain fatty acids present in the intestine, which have useful functions in the intestine.
The role of short-chain fatty acids in the intestine and their relationship to health is explained below.

Strengthening the barrier function against harmful substances Acetic acid is said to enhance the barrier function of the large intestine. In addition, butyric acid is said to activate the MUC2 gene in intestinal cells, promoting the secretion of mucin, a mucosal substance, and protecting the large intestine.

b. Cancer prevention: Short-chain fatty acids are said to prevent colon cancer by making the intestines slightly acidic, making it harder for harmful secondary bile acids to form. Furthermore, butyric acid is said to suppress the onset of colon cancer by suppressing abnormal colon cell proliferation, promoting apoptosis, and suppressing colon cell lesions. Research has shown that propionic acid acts on short-chain fatty acid receptors on liver cancer cells, thereby suppressing their proliferation.

c. Prevention of obesity Short-chain fatty acids act on the short-chain fatty acid receptors in fat cells to suppress the uptake of energy into fat cells and prevent fat cells from enlarging. They also act on the short-chain fatty acid receptors in nerve cells, promoting energy consumption via the sympathetic nervous system and thus regulating energy balance.

d. Prevention of diabetes Butyric acid acts on L cells in the intestinal tract and promotes the secretion of the intestinal hormone GLP-1. GLP-1 has the effect of preventing and improving diabetes, suppressing the decrease in the number of pancreatic beta cells that secrete insulin, and promoting insulin secretion.

e. Suppression of appetite Butyrate and propionate induce the secretion of intestinal hormones such as GLP-1 and PYY from the L cells of the intestinal tract. GLP-1 and PYY act on the brain to suppress appetite, prolonging the feeling of fullness and preventing overeating.

f. Regulation of immune function: Approximately 60% of the body's immune cells are concentrated in the intestines, and it is said that an imbalance in the immune system (especially an excessive immune response) in the intestines affects the entire body. Butyrate has the effect of increasing Treg cells, immune cells that suppress excessive immune responses, and it is known that this is related to butyrate's ability to promote histone acetylation in colonic epithelial cells. Butyrate is also said to be useful in inflammatory bowel disease, an intestinal immune disorder.

(3) Increase in the proportion of lactic acid bacteria in intestinal bacteria. By ingesting this microflora improver in humans or non-human animals, the proportion of lactic acid bacteria present in the intestines can be increased. This increase in lactic acid bacteria can be expected to prevent or improve autoimmune diseases and colds caused by lactic acid bacteria, and to improve intestinal conditions, diarrhea, bowel movements, etc. by increasing good bacteria and suppressing bad bacteria.

The target population for this microbiota-improving agent is not particularly limited as long as it is a person who needs to enjoy the above-mentioned effects, but preferably it can broadly include those who consume a high-fat diet, which is a factor that alters the composition of the intestinal microbiota. Preferably, it is a person who prefers to consume a high-fat diet, and more preferably, a person who is obese or has a tendency toward obesity. A fatty diet is a diet that contains a lot of fat, and although it is not strictly defined, it can include a diet in which the proportion of fat in the total energy intake (fat energy ratio) is approximately 30-40% or more. For example, meats such as beef belly, beef loin, ground beef, corned beef (beef), pork belly, pork loin, bacon (pork), and sausage (pork); seafood such as monkfish kimo (sweet bean kimo), fatty tuna, grilled eel, saury, and yellowtail; eggs and dairy products such as egg yolk, cream (milk fat), and Camembert cheese; grains such as croissants, Danish pastries, corn snacks, and potato chips; nuts such as macadamia nuts, peanuts, almonds, and cashews; and diets high in fat-containing ingredients such as fried tofu, soybean flour, mayonnaise, French dressing, olive oil, sesame oil, salted butter, and margarine are likely to be high-fat. Obesity is assessed based on BMI (Body Mass Index: weight [kg] / height [m] squared), with a BMI of 30 or higher being considered obese and a BMI of 25 to 30 being considered slightly obese.

Whether or not taking (administering, ingesting) this microbiota-improving agent improves the intestinal microbiota can be evaluated and confirmed by analyzing the intestinal bacteria contained in feces.

Furthermore, as shown in the experimental examples below, soy isoflavones, the active ingredient in this microbiota improver, are thought to suppress the conversion of primary bile acids to secondary bile acids in the intestine by changing the composition of the intestinal microbiota and/or increasing the abundance of beneficial intestinal bacteria. As a result, this microbiota improver does not decrease the production of primary bile acids, which are useful for lipid absorption, but rather increases them, while significantly suppressing the production of secondary bile acids, which can cause colon cancer and liver cancer, and reducing their abundance in the intestine.

The dosage (intake) of this microbiota improver can be adjusted as appropriate depending on the condition and severity of symptoms of the subject, but the daily dosage (intake) for an adult (body weight 50 kg) is typically around 10-150 mg, converted into the amount of soy isoflavones (dry weight) contained in this microbiota improver. It is usually administered orally once a day or in two to three divided doses. There are no particular restrictions on the time of administration, and examples include one or more of the following: breakfast, lunch, and dinner. Furthermore, although not limited, it is preferable to take it with a meal, or within 30 minutes either before or after a meal, as this affects the absorption of lipids contained in food.

(II) Secondary Bile Acid Production Inhibitor The secondary bile acid production inhibitor of the present invention (hereinafter also referred to simply as "the production inhibitor") is characterized by containing soybean isoflavone as an active ingredient.

Like the aforementioned bacterial flora improver, the present production inhibitor can take any form as long as it is administered orally. Furthermore, as long as it is administered orally (orally ingested), there are no particular limitations on its intended use (drugs, quasi-drugs, food and beverages [including health functional foods such as foods for specified health uses, foods with functional claims, and nutritional functional foods, as well as supplements]). Food and beverages are preferred, and foods for specified health uses or foods with functional claims that can claim to have certain actions or effects.

The production inhibitor may be in various formulations or may be in the form of a regular food or beverage. Such food or beverage can be produced by adding the soy isoflavones described above or an additive containing soy isoflavones described below to various foods or beverages.

The dosage (intake) of this production inhibitor can be adjusted as appropriate depending on the subject's condition and the severity of symptoms, but the daily dosage (intake) for an adult (body weight 50 kg) is typically around 10-150 mg, converted into the amount of soy isoflavones (dry amount) contained in this production inhibitor. It is usually administered orally once a day or in two to three divided doses. There are no particular restrictions on the time of administration, and examples include one or more of the following: breakfast, lunch, and dinner. Furthermore, although not limited, it is preferable to take it with a meal, or within 30 minutes either before or after a meal, as it affects the absorption of lipids contained in food.

The target population for this production inhibitor is anyone who needs to reduce the amount of secondary bile acid production, and while there are no particular limitations in this regard, it can broadly include anyone who consumes a high-fat diet, which is a factor that increases the amount of secondary bile acid production. Preferably, this is someone who prefers to consume a high-fat diet, and more preferably, someone who is obese or has a tendency toward obesity.

Whether or not taking (administering, ingesting) this production inhibitor inhibits the production of secondary bile acids and reduces their production levels can be evaluated and confirmed by analyzing and measuring the amount of bile acids in feces. This production inhibitor is thought to inhibit the production of secondary bile acids from primary bile acids in the intestine. As a result, as shown in the experimental examples described below, this production inhibitor does not decrease, but rather increases, the production of primary bile acids that are useful for lipid absorption, while significantly suppressing the production of secondary bile acids that can cause colon cancer and liver cancer and reducing their presence in the intestine.

(III) Intestinal flora improving agent-like additive or additive for suppressing secondary bile acid production The additive of the present invention is characterized by containing soybean isoflavone, preferably edible soybean isoflavone, as an active ingredient.

The additive of the present invention is an additive used to impart an intestinal flora-improving effect to a target oral composition based on the intestinal flora-improving effect of its active ingredient, soy isoflavone. The additive of the present invention can also be used to further enhance the effect of oral compositions that have an intestinal flora-improving effect.

The additive of the present invention can also be an additive used to impart the effect of inhibiting the production of secondary bile acids to a target oral composition, based on the effect of soy isoflavone, its active ingredient, on inhibiting the production of secondary bile acids. The additive of the present invention can also be used to further enhance the effect of oral compositions that have the effect of inhibiting the production of secondary bile acids. The additive of the present invention can also be an additive used to impart the effect of increasing the production of primary bile acids and decreasing the production of secondary bile acids to a target oral composition, based on the effect of soy isoflavone, its active ingredient. The additive of the present invention can also be used to further enhance the effect of oral compositions that have these effects.

The oral compositions covered by the present invention include compositions that are orally administered to or ingested by humans, specifically oral pharmaceuticals, oral quasi-drugs, and food and beverages. Food and beverages are preferred.

The types of soy isoflavones used as raw materials for the additive of the present invention, their preparation methods, etc. are as explained in (I) above, and can be used by reference in this section (III).

The additive of the present invention may be soy isoflavone itself, or it may be prepared by combining soy isoflavone with conventionally known edible carriers, excipients, etc. that are pharmaceutically and food-acceptable. The additive of the present invention is used to prepare the aforementioned secondary bile acid production inhibitor by adding it to the above-mentioned oral pharmaceuticals, oral quasi-drugs, and/or foods and beverages. Therefore, to that extent, its form is not particularly limited, and it may be in the form of a liquid (including extracts and syrups) or jelly, or the liquid may be formulated into a powder or granule form using conventional methods to form a powder, fine granules, or granules; a capsule (hard capsule, soft capsule) in which the liquid, powder, or granules is filled into a capsule, or the powder or granules may be further compressed into a tablet form (solid formulation).

The amount of the additive of the present invention to be added to and incorporated into oral pharmaceuticals, oral quasi-drugs, and/or foods and beverages can be increased so that the daily dose (ingestion) of the intestinal flora improver or secondary bile acid production inhibitor prepared by incorporating the additive of the present invention is typically approximately 10 to 150 mg, converted into the amount of soy isoflavones (dry weight).

The additive of the present invention can be used as one of the ingredients together with other ingredients in the process of preparing the oral composition (oral pharmaceuticals, oral quasi-drugs, and/or food and beverages), or can be added to the oral composition immediately when the oral composition is taken (administered or ingested).

(IV) Method of Using Soy IsoflavonesThe present invention also provides a method of using soy isoflavones. This method is a method of using soy isoflavones to impart to an oral composition the effects of improving the intestinal flora, inhibiting the production of secondary bile acids, and/or increasing the production of primary bile acids and decreasing the production of secondary bile acids, and this method can be carried out by incorporating soy isoflavones into the oral composition. Note that instead of soy isoflavones, the aforementioned additives containing soy isoflavones as an active ingredient can also be used.

Details of the subject oral composition and method of using soy isoflavones are as described above.

Hereinafter, in this specification, the terms "comprise" and "contain" include the meanings of "consist of" and "consist essentially of."

The following describes the present invention using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited in any way by these experimental examples. Unless otherwise specified, the following experiments were conducted at room temperature (25±5°C) and atmospheric pressure.

Experimental Example: Bile Acid Intake Test Test animals (mice) were given soy isoflavones in addition to primary bile acids (cholic acid), and body weight, food intake, cecal content weight, and the amount of bile acids in the cecal content (primary bile acids, secondary bile acids, total bile acids) were measured. Genomic DNA was also extracted from the cecal content, and intestinal bacterial flora analysis was performed.

(1) Test animals: Male C57BL/6J mice, 8 weeks old (purchased from SLC Japan)
Breeding period: After the animals were brought in, they were allowed to acclimate for two weeks on normal solid food, after which they were divided into the following test groups so that the average weight of each group was equal.
Rearing environment: Room temperature 25°C, humidity 55%, indoor fluorescent lights were lit on a 12-hour cycle from 7:00 AM to 7:00 PM.

Test area:
After the acclimatization period, the test animals were divided into the following test groups (n=6 or 7 per group). The bile acid administration groups (b) to (e) were given 0.3% (w/v) cholic acid (CA) aqueous solution as drinking water, and were also given ad libitum access to a control diet containing each material for two weeks.
(a) Control group: Control diet (regular solid diet sample D12450J: Research Diet) + drinking water (distilled water; the same applies below).
(b) Bile acid administration group (CA group): Control diet + drinking water containing 0.3% cholic acid.
(c) 3% raffinose + bile acid administration group (CA + 3% raffinose group): Received control diet + 3% raffinose + 0.3% cholic acid-added drinking water.
(d) 0.5% curcumin + bile acid administration group (CA + 0.5% curcumin group): They consumed a control diet plus drinking water containing 0.5% curcumin and 0.3% cholic acid.
(e) 0.8% soy isoflavone + bile acid administration group (CA + 0.8% isoflavone group): Intake of control diet + 0.8% soy isoflavone + 0.3% cholic acid-added drinking water.
The soy isoflavone used was Isoflavone P40 (manufactured by Fujicco Co., Ltd.). Isoflavone P40 contains 37% or more by mass of isoflavones. Of the total isoflavones, the glycosides of the three isoflavones genistein, daidzein, and glycitein account for approximately 85% by mass or more, and the aglycones account for approximately 15% by mass. The "0.8%" above is the amount converted into the isoflavone content contained in Isoflavone P40.

(2) Test Method and Results 1. Body Weight, Food Intake, and Cecal Content After the 2-week administration period, the body weight, cecal content, and daily food intake (g/day/mice) of the test animals in each test group were measured. The results are shown in Table 1 as the mean value + standard error (n = 6-7) for each group.

As shown in Table 1 above, although food intake did not change significantly in either group, a significant decrease in body weight was observed in the (e) CA + 0.8% isoflavone group compared to the control group and CA group. In contrast, no significant decrease in body weight was observed in either the (c) CA + 3% raffinose group or the (d) CA + 0.5% curcumin group. Furthermore, compared to the control group and CA group, a tendency for an increase in cecal contents was observed in the (c) CA + 3% raffinose group and the (e) CA + 0.8% isoflavone group, with a particularly significant increase observed in the (e) CA + 0.8% isoflavone group.

2. Bile acid content in cecal contents. Bile acid content in cecal contents was quantified. LC-MS samples were prepared from cecal contents according to the method described by Hagio et al. (Hagio, M., M. Matsumoto, and S. Ishizuka. 2011. Bile acid analysis in various biological samples using ultraperformance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS). Methods Mol. Biol. 708: 119-129.) and bile acid components were analyzed and quantified using LC-MS.

Table 2 shows the results of a comparison of the amounts of each primary bile acid, secondary bile acid, and total bile acid for the control group, CA group, CA + 3% raffinose group, CA + 0.5% curcumin group, and CA + 0.8% isoflavone group. Table 2 also shows the results of the Tukey HSD test. The letters in the upper right corner of each value indicate that different letters indicate a significant difference between groups (p<0.05), while the same letter indicates no significant difference.

As shown in Table 2, the CA group that ingested cholic acid had a reduced amount of primary bile acids and a significant increase in the amount of secondary bile acids, including the highly toxic deoxycholic acid (DCA), compared to the control group. In contrast, the CA + 0.8% isoflavone group, which ingested soy isoflavones in addition to cholic acid, had a significantly increased amount of primary bile acids compared to both the control and CA groups. Furthermore, compared to the CA group, which had increased amounts of deoxycholic acid (DCA) and secondary bile acids, the amount of deoxycholic acid (DCA) and the amount of secondary bile acids were significantly reduced.

The above bile acid administration test confirmed that soy isoflavones have the effect of suppressing the increase in secondary bile acids caused by CA intake, in other words, by consuming a high-fat diet, in other words, a secondary bile acid-reducing effect (secondary bile acid production-inhibiting effect). On the other hand, in the CA + 0.8% isoflavone group, the production of primary bile acids such as cholic acid (CA) and taurocholic acid (TCA) increased, confirming that soy isoflavones have the effect of suppressing the production and increase of secondary bile acids and reducing the production of secondary bile acids without impairing (in fact, enhancing) the effects of primary bile acids (e.g., promoting fat emulsification, cholesterol excretion, etc.).

As shown in Table 1, the CA + 0.8% isoflavone group had an increased cecal content volume, which is known to be associated with an increase in short-chain fatty acids that have beneficial effects on the host. This suggests that the CA + 0.8% isoflavone group had an increased level of metabolites from specific beneficial bacteria that produce short-chain fatty acids, resulting in a decrease in intestinal pH and/or an improved diversity of the intestinal microflora, resulting in an increase in primary bile acid production and a decrease in secondary bile acid production. Furthermore, as shown in Table 1, weight loss was observed in the CA + 2% isoflavone group, suggesting that the anti-obesity effect is related to changes in the intestinal microflora caused by soy isoflavone intake.

Recently, it has been reported that an antioxidant called Tempole suppresses obesity caused by a high-fat diet, reducing the Firmicutes phylum, which increases with a high-fat diet, and increasing the Bacteroidets phylum, which decreases with a high-fat diet (Li et al., Microbiome remodeling leads to inhibition of intestinal farnesoid X receptor signaling and decreased obesity. Nat Commun., 4:2384. doi: 10.1038/ncomms3384 (2013)). It has been suggested that administration of Tempole increases the conjugated bile acid called tauro-β-muricholic acid (TβMCA), which acts as an antagonist of the nuclear receptor farnesoid X receptor (FXR) in the gastrointestinal tract, thereby inhibiting FXR activation and suppressing obesity. Although the mechanism by which isoflavone intake has an anti-obesity effect is unclear, the significant increase in the primary bile acid tauro-β-muricholic acid (TβMCA) in this study suggests that obesity may be suppressed through an increase in the production of primary bile acids due to improvements in the intestinal flora.

3. Composition of the intestinal microbiota in the cecal contents DNA was extracted from the cecal contents of the test animals in each test group after the two-week administration period according to standard methods, and the V3-V4 region of the 16S rRNA gene was amplified by Seibu Giken Co., Ltd., followed by meta-16S microbiota analysis using Illumina MiSeq. A total of 1,511,039 reads (average 50,368 reads) from the 30 samples obtained were used for microbiota analysis using QIIME (Quantitave Insights Into Microbial Ecology).

From the results of the bacterial flora analysis, the Shanon index (an index that takes into account the number of bacterial species and the frequency of each species), the bacterial flora composition at the phylum level, and the bacterial flora composition at the family and genus level were calculated. The Shanon index is used as an index to assess bacterial flora diversity.

[Shanon index]
The results of the Shanon index (mean ± SEM (n = 6), Tukey-HSD test) are shown in Figure 1. As shown in Figure 1, a significant decrease in the Shanon index (decreased microbiota diversity) was observed in the CA group compared to the control group, but a significant improvement (improved microbiota diversity) was observed in the CA + 0.8% isoflavone group.

[Phylum-level bacterial flora composition]
Figure 2A shows the phylum-level composition of the intestinal microbiota in the cecal contents (n = 6). As shown in Figure 2A, CA administration increased the Proteobacteria phylum and decreased the Bacteroidetes phylum. Figure 2B shows the ratio of the Firmicutes phylum to the Bacteroidetes phylum (Firmicutes/Bacteroidetes ratio). As shown in Figures 2A and 2B, particularly in the CA + 0.8% Iso group, the Firmicutes/Bacteroidetes ratio tended to decrease as the Bacteroidetes phylum increased. As described in Non-Patent Document 1, the intestinal microbiota of obese individuals is known to have a low proportion of bacteria belonging to the Bacteroidetes phylum and a high proportion of bacteria belonging to the Firmicutes phylum. On the other hand, with weight loss, i.e., as individuals become normal or lean, the proportion of bacteria belonging to the Bacteroidetes phylum increases and the proportion of bacteria belonging to the Firmicutes phylum decreases. The results in Figure 2B correlated with the weight loss in the CA + 0.8% Iso group shown in Table 1, confirming that intake of soy isoflavones changes the composition of the intestinal flora and leads to a leaner constitution.

[Family and genus level bacterial flora composition]
The family and genus-level intestinal microbiota composition (n=6) in cecal contents is shown in Figures 3, 4, 5A-D, and 6-8. As shown in Figure 3, the equol-producing Adlercreutzia genus, which was decreased by CA administration, was significantly increased by isoflavone administration. As shown in Figure 4, the lactic acid bacteria Lactobacillus genus, which was decreased by CA administration, was significantly increased by isoflavone administration. Furthermore, as shown in Figures 5A-D, the short-chain fatty acid-producing Coprococcus genus (butyric acid-producing bacteria) (Figure 5A) and Prevotella genus (butyric acid, propionic acid, and isovaleric acid-producing bacteria) (Figure 5B), which were decreased by CA administration, were significantly increased by isoflavone administration. In particular, the Prevotella genus is known to be a bile acid-sensitive bacterium within the Bacteroidetes phylum. Furthermore, as shown in Figures 5C and 5D, it was confirmed that the short-chain fatty acid-producing bacteria Parabacteroides (succinic acid-producing bacteria) and Blautia (acetic acid- and lactic acid-producing bacteria) also increased with isoflavone administration. Furthermore, as shown in Figures 6 and 7, it was confirmed that the Firmicutes phylum (Turicibacter, Dorea) (Figures 6A and 6B) and Proteobacteria phylum (Desulfovibrionaceae) (Figure 7), which increased with CA administration, both decreased with soy isoflavone administration.

4. Discussion It is generally known that a high-fat diet increases secondary bile acids, as well as increases the Firmicutes phylum and decreases the Bacteroidetes phylum, leading to the development of obesity and metabolic syndrome. It has also been suggested that an increase in secondary bile acids due to a high-fat diet is associated with colon and liver disease (Non-Patent Document 2). This study confirmed that the increase in secondary bile acids due to CA administration was reduced by the administration of soy isoflavones. Furthermore, it was confirmed that the diversity of the intestinal flora, which was reduced by CA administration, was improved by the administration of soy isoflavones (for example, the Bacteroidetes phylum, which was reduced by CA administration, increased by soy isoflavone administration; the Firmicutes phylum (Turicibacter genera, Dorea genera) and Proteobacteria phylum (Desulfovibrionaceae family), which were increased by CA administration, both decreased by soy isoflavone administration; and the ratio of Firmicutes to Bacteroidetes phylum (Firmicutes/Bacteroidetes ratio) decreased).It was also confirmed that the administration of soy isoflavones increased beneficial bacteria in the intestines, such as butyric acid-producing bacteria (Coprococcus genus), various short-chain fatty acid-producing bacteria, equol-producing bacteria (Adlercreutzia genus), and lactic acid bacteria (Lactobacillus genus).

Although the details of the relationship between these intestinal bacteria and bile acids are unknown, it is thought that the administration of soy isoflavones improves the diversity of the intestinal flora, which was reduced by CA administration, resulting in the suppression of secondary bile acid production and an increase in primary bile acids. In this way, it is thought that the effects of consuming soy isoflavones, such as improving the intestinal flora and/or reducing secondary bile acids (inhibiting secondary bile acid production), may improve and prevent other diseases and conditions, including obesity, metabolic syndrome, colon disease, and liver disease.

Claims (2)

A primary bile acid production promoter containing soy isoflavone as an active ingredient,
The primary bile acid production promoter is an orally administered agent for increasing primary bile acid production in the intestine,
The primary bile acid is at least one selected from the group consisting of cholic acid, glycocholic acid, α-muricholic acid, and tauro-β-muricholic acid.
The primary bile acid production promoter . An additive for a primary bile acid production promoter containing soy isoflavone as an active ingredient ,
the primary bile acid production promoter is an orally administered agent for increasing primary bile acid production in the intestine,
The primary bile acid is at least one selected from the group consisting of cholic acid, glycocholic acid, α-muricholic acid, and tauro-β-muricholic acid.
An additive for the primary bile acid production promoter .