JP2023010837A - Secondary bile acid generation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel use of soybean isoflavone as a food material.

SOLUTION: The present invention provides, as a secondary bile acid generation inhibitor, a composition containing soybean isoflavone as an active ingredient.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention relates to a new use of soy isoflavone as a food material.

The idea that abnormal intestinal flora (dysbiosis) is an important factor in the pathogenesis of obesity, type 2 diabetes, colon cancer, inflammatory bowel disease, and the like is spreading. For example, one of the causes of obesity is a change in the composition of the intestinal flora. Specifically, obese humans tend to have a high abundance of Firmicutes bacteria and a low abundance of Bacteroidetes bacteria. On the other hand, in the case of normal or lean humans, the tendency is opposite to that of the obese type, and the abundance of Bacteroidetes bacteria tends to be high and the abundance of Firmicutes bacteria tends to be low (Non-Patent Document 1). . For this reason, it has been proposed to change the composition of the intestinal flora from obese to normal or lean by ingesting health foods. It has been reported that the abundance of Bacteroidetes is increased and the abundance of Firmicutes is decreased (Patent Document 1). In many patients with ulcerative colitis, the abundance of butyric acid-producing bacteria is reduced compared to healthy subjects. One of the reasons is that the intestinal butyric acid concentration is reduced due to the reduction of intestinal butyric acid-producing bacteria (Non-Patent Document 2).

In addition, bile acid has been reported as a regulator of the intestinal flora, and it has been reported that the increase in bile acid caused by ingestion of a high-fat diet affects changes in the intestinal flora. Bile acids are the main components of bile involved in lipid absorption, and their main role is to promote the formation of micelles in the gastrointestinal tract when fat is ingested, making fat easier to absorb. Bile acids consist of primary and secondary bile acids. Primary bile acids are synthesized in the liver from cholesterol as conjugated bile acids conjugated with glycine or taurine and secreted into the intestine. Conjugated bile acids that have finished their role in the intestine are reabsorbed in the small intestine and returned to the liver. However, some conjugated bile acids go to the large intestine, where glycine or taurine is released (deconjugated) by intestinal bacteria, and deconjugated primary bile acids (such as cholic acid and chenodeoxycholic acid) are further converted into deoxycholic acid and Converted to secondary bile acids such as lithocholic acid. When converted to secondary bile acids, the hydrophobicity of the molecules increases and they exhibit strong bactericidal activity. For example, deoxycholic acid (DCA), which is a secondary bile acid, is known to exhibit ten times the bactericidal activity of cholic acid (CA), which is a primary bile acid (see Non-Patent Document 3).

Based on these findings, as a mechanism for changes in the intestinal microflora, ingestion of a high-fat diet results in excess bile acid secretion, resulting in an increase in the concentration of deoxycholic acid (DCA) by intestinal bacteria. A "bile acid hypothesis" has been proposed, which is a selective pressure (see Non-Patent Document 4). In addition, deoxycholic acid (DCA) is known to have colon cancer-promoting effects (Non-Patent Document 5). Furthermore, according to the description of Patent Document 2, the reason for the recent increase in colorectal cancer in Japan is the relationship between the increase in lipid intake due to the westernization of the diet and the accompanying increase in the amount of intestinal bile acids. Some literature points out. Furthermore, in Non-Patent Document 6, intestinal bacteria (deoxycholic acid-producing bacteria) that convert primary bile acids (cholic acid, etc.) that help digestion and absorption of lipids into secondary bile acids (deoxycholic acid, etc.) when obesity occurs ), the increased secondary bile acids in the body cause cellular senescence in hepatic stellate cells and secrete inflammatory cytokines, promoting carcinogenesis in the surrounding hepatocytes. It has been described that a similar mechanism is involved in the development of liver cancer associated with obesity in humans, and that suppressing the growth of deoxycholic acid-producing bacteria may lead to the prevention of liver cancer development. Suggested.

Thus, conventionally, various secondary bile acid-lowering agents (two bile acid production inhibitors) have been proposed. For example, secondary bile acid lowering agents containing plant-derived polyphenols such as curcumin as active ingredients (Patent Document 3), secondary bile acid production inhibitors containing α-linked galacose-containing oligosaccharides as active ingredients (Patent Document 4) A secondary bile acid inhibitor (Patent Document 5) containing a reishi mushroom extract as an active ingredient, and a deoxycholic acid-reducing agent containing as an active ingredient a lactic acid bacterium belonging to Lactobacillus gasseri and/or its culture. agent (Patent Document 2) and the like have been proposed. In particular, according to the lactic acid bacteria, by suppressing the change from primary bile acids to secondary bile acids, the production of secondary bile acids can be suppressed without reducing the amount of useful primary bile acids, that is, selective It is said to be excellent in that it can reduce the amount of secondary bile acids.

JP 2015-127340 A JP 2017-66086 A JP 2009-227609 A JP 2004-244365 A JP 2018-43955 A

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., 589:47-65, 2005. Japan Science and Technology Agency website "Cancer Research Foundation, Japan Science and Technology Agency (JST) June 27, 2013" Joint announcement: Changes in intestinal bacteria associated with obesity promotes the onset of cancer” (http://www.jst.go.jp/pr/announce/20130627-2/)

An object of the present invention is to provide a new use of soy isoflavone as a food material. Specifically, based on the useful bioregulatory function (tertiary function) exerted on the living body when soy isoflavone is ingested, the object is to provide a new functional use as a food material.

The present inventors have conducted intensive studies in order to solve the above problems. Changes in the composition of the internal flora) are significantly improved by ingesting soy isoflavones at the same time, and equol-producing bacteria, butyric acid bacteria and other short-chain fatty acid-producing bacteria, which are useful intestinal bacteria, or / and that the decrease in lactic acid bacteria was suppressed and further increased.

In addition, we found that soy isoflavone intake significantly reduced the increase in the production of secondary bile acids caused by ingestion of a diet supplemented with primary bile acids. It was confirmed that the amount did not decrease, but rather increased.

The present invention has been completed based on such findings, and includes the following embodiments.
(I) Improving agent for intestinal flora
(I-1) An agent for improving intestinal flora containing soy isoflavone as an active ingredient.
(I-2) The intestinal flora-improving agent according to (I-1), which exhibits at least one action selected from the group consisting of the following (a) to (e):
(a) an increase in the Shanon index;
(b) Decrease in the "Firmicutes/Bacteroidetes" ratio in intestinal bacteria, (c) 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 abundance of lactic acid bacteria (Lactobacillus) in intestinal bacteria.

(II) Secondary bile acid production inhibitor (II-1) Secondary bile acid production inhibitor containing soy isoflavone as an active ingredient.
(II-2) The secondary bile acid production inhibitor according to (II-1), which increases the amount of primary bile acid production.

(III) Additive for intestinal microflora improving agent, secondary bile acid production inhibitor additive (III-1) Intestinal microflora improving agent containing soy isoflavone as an active ingredient or for suppressing secondary bile acid production Additives for agents.

(IV) Method of using soy isoflavone (IV-1) Soy isoflavone or the additive described in (III-1) is blended into an oral composition, and the oral composition has an intestinal flora improving effect, secondary A method of using soy isoflavone for imparting at least one action selected from the group consisting of a bile acid production inhibitory action and a primary bile acid production increasing action.

According to the present invention, it is possible to provide an intestinal microflora-improving agent containing soy isoflavone as an active ingredient. According to the intestinal flora-improving agent of the present invention, (a) an increase in the Shanon index, (b) a decrease in the ratio of "Firmicutes/Bacteroidetes" in intestinal bacteria, (c) intestinal bacteria (d) an increase in the abundance of short-chain fatty acid-producing bacteria in intestinal bacteria, and (e) an increase in the abundance of lactic acid bacteria (Lactobacillus) in intestinal bacteria. At least one effect selected from the group consisting of increasing can be exerted. From the effect of (a), according to the intestinal microflora-improving agent of the present invention, it is possible to improve the diversity of the intestinal microflora that has decreased due to the ingestion of a high-fat diet. Moreover, according to the effect of (b), it is possible to change the constitution from an obese type to a normal type or lean type by changing the composition of the intestinal flora. According to the function and effect of (c), equol, which has an estrogenic effect, is produced in the body, making it possible to compensate for female hormones that tend to be deficient with aging. According to the effect of (d), it becomes possible to prevent or improve diseases and conditions caused by reduction of short-chain fatty acids in the intestine, such as inflammatory bowel disease. In addition, according to the effect of (e), it is possible to prevent or improve autoimmune diseases and colds caused by lactic acid bacteria, and to improve intestinal regulation, diarrhea, bowel movements, etc. by increasing the number of good bacteria and suppressing the bad bacteria. .

Further, according to the present invention, it is possible to provide a secondary bile acid production inhibitor containing soy isoflavone as an active ingredient. The secondary bile acid production inhibitor of the present invention is excellent in reducing the amount of secondary bile acids in the intestine. Therefore, it can be effectively used to prevent or improve the onset of diseases and pathological conditions (for example, colon diseases, liver diseases, etc.) caused by secondary bile acids. In addition, according to the secondary bile acid production inhibitor of the present invention, by suppressing the production of secondary bile acids from primary bile acids in the intestine, the amount of primary bile acids that are important for lipid absorption can be reduced. Secondary bile acid production can be selectively reduced by increasing rather than decreasing.

Furthermore, since the secondary bile acid production inhibitor of the present invention contains soybean isoflavone, which has been confirmed to be food safe, as an active ingredient, there is no concern about side effects, and it can be ingested without difficulty by incorporating it into daily diet. and the effects described above can be enjoyed.

Furthermore, according to the additive for improving the intestinal flora of the present invention, the additive for suppressing the production of secondary bile acid, or the method of using soy isoflavone of the present invention, the food composition etc. , the intestinal flora-improving agent and/or secondary bile acid production inhibitor of the present invention can be easily prepared and provided.

The Shanon index (mean ± SEM (n = 6), Tukey-HSD test) according to the "intestinal microflora analysis in the contents of the cecum" in the experimental example was divided into the control group, the CA group (cholic acid administration group), and the CA + 3% raffinose. The results of comparison between groups, CA + 0.5% curcumin group, and CA + 0.8% isoflavone group are shown. Different letters in each graph indicate significant differences between groups (p<0.05). Among the "intestinal microbiota analysis in the contents of the cecum" in the experimental example, (A) the composition of the intestinal microbiota at the phylum level (n = 6) was divided into the control group, the CA group (cholic acid-administered group), and the CA + 3 The results of comparison among the % raffinose group, the CA+0.5% curcumin group, and the CA+0.8% isoflavone group are shown. (B) shows the results of comparing the ratio of the phylum Firmicutes to the phylum Bacteroidetes (Firmicutes/Bacteroidetes ratio) in each group. FIG. 2 is a diagram comparing the abundance ratio (%) of equol-producing bacteria (Adlercreutzia genus) in cecal contents for each group among the results obtained in the "intestinal microflora analysis in cecal contents" in Experimental Examples. . 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). FIG. 2 is a diagram comparing abundance ratios (%) of lactic acid bacteria (genus Lactobacillus) in cecal contents for each group among the results obtained in the "intestinal microflora analysis in cecal contents" in Experimental Examples. Fig. 2 shows a comparison of the abundance ratio (%) of various short-chain fatty acid-producing bacteria present in the cecal contents for each group among the results obtained in the "intestinal microflora analysis of the cecal contents" in the experimental example. is. A is a diagram comparing the abundance ratio (%) of butyric acid-producing bacteria (Coprococcus genus) in the cecal contents for each group; B is butyric acid, propionic acid, and isovaleric acid-producing bacteria in the cecal contents for each group. A diagram comparing the abundance ratio (%) of (Prevotella genus); C compares the abundance ratio (%) of succinic acid-producing bacteria (Parabacteroides genus) in the contents of the cecum for each group; Fig. 10 is a diagram comparing abundance ratios (%) of acetic acid/lactic acid-producing bacteria (genus Blautia) in cecal contents. FIG. 2 is a diagram comparing the abundance ratio (%) of bacteria belonging to the phylum Firmicutes present in the cecal contents for each group among the results obtained in the "intestinal microflora analysis in the cecal contents" in the experimental example. . A indicates the abundance ratio (%) of bacteria in the genus Turicibacter and B in the genus Dorea. In Fig. A, different characters in each graph indicate significant differences between groups (Tukey-HSD test, p<0.05). * in Figure B indicates that there is a significant difference compared with the CA group by Welch test (p<0.05). Among the results obtained in the "intestinal microflora analysis of cecal contents" in the experimental example, this is a diagram comparing the abundance ratio (%) of bacteria of the phylum Proteobacteria (family Desulfovibrionaceae) in the contents of the cecum for each group. be. 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 bacterial flora-improving agent") is characterized by containing soy isoflavone as an active ingredient.

Soybean isoflavone is a kind of flavonoid that is abundantly contained mainly in the germ of soybeans, and is a general term for compounds having a basic skeleton of the compound represented by the following formula:

Soy isoflavones are known to include compounds having the above-mentioned basic skeleton (soy isoflavone aglycones), glycosides (soy isoflavone glycosides) in which sugars are bound thereto, and acetylated and malonylated glycosides of the glycosides. ing. The term "soybean isoflavone" in the present invention includes these soybean isoflavone aglycones, soybean isoflavone glycosides, and their acetylated and malonylated forms without discrimination. Specifically, soy isoflavone aglycones include genistein, daidzein, and glycitein. Soy isoflavone glycosides include genistin, daidzin, and glycitin. Acetylated soybean isoflavone glycosides include acetylgenistin, acetyldaidzin, and acetylglycitin, and malonylated soybean isoflavone glycosides include malonylgenistin, malonyldaidzin, and malonylglycitin. These soy isoflavones may be used singly or in combination of two or more. In the present invention, soy isoflavones may be those refined from soybeans, but they may also be crudely refined products, such as those extracted from soybeans (extracts), pulverized or shredded soybeans, and the like. can be used. Methods for purifying or extracting isoflavones from soybeans are known, and in the present invention, purified products or extracts of soybean isoflavones obtained according to known methods can be used. Commercially available soy isoflavones can also be used.

The form of the present bacterial flora improving agent is not particularly limited as long as it is an oral administration form. In addition, as long as it has an oral administration (orally ingested) form, it is classified according to its use (pharmaceuticals, quasi-drugs, foods and drinks [foods with specified health uses, foods with function claims, foods with nutritional functions, etc.) and supplements]) is not particularly limited. It is preferably a food or drink, and more preferably a food for specified health use or a food with function claims, which can claim its actions and effects.

Oral administration forms, specifically those prepared in the form of liquids (including extract forms and syrups) or jelly formulations; Capsules filled with liquid, powder or granules (hard capsules, soft capsules); or powders or granules further tableted into tablets (solid formulations).

The agent for improving bacterial flora can also be prepared into various dosage forms (oral administration forms) by combining the above soy isoflavone with conventionally known edible carriers, excipients, etc. that are pharmaceutically or food acceptable.

When the present bacterial flora improving agent is in the form of a liquid preparation, it can be stored frozen, or it may be stored after removing moisture by freeze-drying or the like. Freeze-dried preparations, dry syrups, etc. are used by adding sterilized water or the like to dissolve them again.

When the present bacterial flora-improving agent is in the form of a solid formulation, for example, in the case of tablets, conventionally known carriers in the art can be widely used. Examples of such carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, silicic acid; water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin. solution, binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, crystalline cellulose, hydroxypropylcellulose, hypromellose, sodium alginate; dry starch, agar powder, laminaran powder, sodium bicarbonate, polyoxyethylene sorbitan fatty acid Disintegrants such as esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, crospovidone, povidone, low-substituted hydroxypropyl cellulose; disintegration inhibitors such as stearin, cocoa butter, hydrogenated oil; quaternary ammonium salts, lauryl Absorption promoters such as sodium sulfate; moisturizing agents such as glycerin; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid; lubricants such as refined talc, stearate, boric acid powder, polyethylene glycol, etc. can be used. Further, the tablets may be tablets coated with conventional coatings, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multi-layer tablets, if necessary. In addition, the composition containing the active ingredient can be filled into conventionally known capsules made from gelatin, pullulan, starch, gum arabic, hydroxypropylmethylcellulose (HPMC), etc., to form capsules.

In addition, when the formulation is in the form of pills, conventionally known carriers in the art can be widely used as carriers. Examples thereof include excipients such as glucose, lactose, starch, cacao 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 promoters, adsorbents, fillers, preservatives, stabilizers, emulsifiers, solubilizers, etc. can be appropriately selected and used according to the form of the formulation. can.

Any of these forms can be prepared using a method commonly used in the art. For example, tablets can be obtained by appropriately adding the above-mentioned active ingredients and other excipients necessary for obtaining tablets, and mixing and dispersing them well. It can be obtained by tableting. Moreover, the powder can be obtained by appropriately adding the above active ingredient and other excipients necessary for obtaining the powder, and mixing and pulverizing them by a suitable method.

The bacterial flora-improving agent may be in the form of a usual food or drink, in addition to the above-described formulation form. The food and drink can be produced by adding the soybean isoflavone described above or an additive containing the soybean isoflavone described below to various food and drink. Food and drink are not particularly limited as long as they are orally ingestible forms such as solutions, suspensions, emulsions, jelly (gels), sols, powders and solid moldings. Specifically, instant foods such as instant noodles, retort pouch foods, canned foods, microwave oven foods, instant soups/miso soups, freeze-dried foods; soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea Beverages such as beverages, powdered drinks, concentrated drinks, nutritional drinks and alcoholic beverages; Flour products such as bread, pasta, noodles, cake mixes, fried chicken powder and bread crumbs; candy, caramel, chewing gum, chocolate, cookies, biscuits and cakes , pies, snacks, crackers, Japanese sweets, dessert confections; Oils and fats such as processed oils and fats, butter, margarine and mayonnaise; dairy products such as milk drinks, yogurts, cheese, fermented milk, lactic acid beverages, ice creams and creams; processed egg products such as pudding and mayonnaise; Processed marine 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, marmalades, pickles, boiled beans and cereals; .

(Action of intestinal microflora-improving agent)
The subject of the microflora-improving agent is a human or non-human animal. Humans are preferred. Non-human animals include pets (companions), laboratory animals, and animals kept in zoos and aquariums. By administering (ingesting) the present bacterial flora-improving agent to a human or non-human animal, it is possible to adjust the composition of the intestinal flora of the human or non-human animal, especially when a high-fat diet is ingested. It is possible to ameliorate the changes in the composition of the intestinal microflora caused by the high-fat diet and return it to a state close to or better than before the intake of the high-fat diet. For example, ingestion of the present bacterial flora-improving agent induces the following changes in the intestinal flora.

(A) Increase in Shannon index Ingestion of this microflora-improving agent by humans or non-human animals can increase the Shanon index, which has decreased due to ingestion of a high-fat diet (increase in Shanon index). The Shanon index is an index obtained by adding the frequency of each bacterial species to the number of bacterial species, and is used as an index for judging the diversity of the intestinal microflora. In other words, ingestion of this bacterial flora-improving agent by humans or non-human animals improves the diversity of the intestinal flora, which has decreased due to ingestion of a high-fat diet, and normal It becomes possible to return to a state (intestinal microbiota diversity) or a state close to it.

(B) Changes in the composition of the intestinal flora at the phylum level Firmicutes against Bacteroidetes bacteria present in the intestine by ingestion of the present bacterial flora-improving agent by humans or non-human animals The proportion of phylum bacteria ("Firmicutes/Bacteroidetes" ratio) is reduced. This decrease in abundance ratio may be caused by an increase in the proportion of Bacteroidetes bacteria relative to the proportion of Firmicutes bacteria, or by an increase in the proportion of Firmicutes bacteria relative to the proportion of Bacteroidetes bacteria. or may be caused by a decrease in the proportion of Firmicutes bacteria and an increase in the proportion of Bacteroidetes bacteria. As described in Non-Patent Document 1, the "Firmicutes/Bacteroidetes" ratio tends to be high in obese or obese-prone subjects and low in normal or lean subjects. As shown in the experimental examples described later, ingestion of this bacterial flora-improving agent can change the composition of the intestinal flora from an obese type to a normal or lean type (non-obesity type). Crocodile-improving agents may reduce obesity, prevent obesity, or maintain a non-obesity state. In other words, the present bacterial flora-improving agent can be expected to have a dieting effect such as suppression of weight gain and weight loss.

(C) Increase in the abundance of bacteria beneficial to the body (1) Increase in the abundance of equol-producing bacteria (Adlercreutzia) in intestinal bacteria increase the proportion of Adlercreutzia, an equol-producing bacterium present in the This bacterium has the effect of converting isoflavones ingested into the body into equol, which has a higher estrogen-like activity. In other words, when taken into the body, this bacterial flora-improving agent, which contains soy isoflavone as an active ingredient, increases the proportion of equol-producing bacteria (Adlercreutzia) present in the intestine, thereby voluntarily metabolizing and activating isoflavones. to produce equol by promoting Therefore, by improving the intestinal flora of humans (adjusting the composition of the intestinal flora), this microbial flora-improving agent enhances the production of equol from isoflavones and exerts an action and effect based on equol. becomes possible. Actions and effects based on equol include, but are not limited to, alleviation of menopausal symptoms (hot flashes, headaches, dizziness, autonomic imbalance-like symptoms, tachycardia, blood pressure fluctuations, etc.); 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; prevention or improvement of lifestyle-related diseases (diabetes, hyperlipidemia, arteriosclerosis, high cholesterol, etc.); prevention or improvement of osteoporosis, increased bone density, increased bone mineral concentration; skin aging ( prevention or improvement of skin firmness and elasticity, blemishes, wrinkles, sagging); improvement of blood flow (prevention or improvement of sensitivity to cold, stiff shoulders, tension headaches, dullness and dryness of the skin); prevention or improvement of male pattern baldness At least one effect selected from improvement and the like can be expected.

(2) Increase in the ratio of short-chain fatty acid-producing bacteria in intestinal bacteria Ingestion of this microflora-improving agent by humans or non-human animals increases the ratio of short-chain fatty acid-producing bacteria in the intestine. be able to. Specifically, the short-chain fatty acid-producing bacteria include bacteria of the genus Coprococccus 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 acetic acid-producing bacteria. Mention may be made of bacteria of the genus Blautia that produce lactic acid. These short-chain fatty acid-producing bacteria can increase the abundance in the intestine of short-chain fatty acids that exert useful functions in the intestine.
The role of short-chain fatty acids in the intestine and their relation to health are explained below.

a. Strengthening the barrier function against harmful substances It is said that acetic acid works to enhance the barrier function of the large intestine. Butyric acid is also said to have the effect of activating the MUC2 gene in intestinal cells, promoting the secretion of mucin, which is a mucosal substance, and protecting the large intestine.

b. Prevention of carcinogenesis Short-chain fatty acids are said to help prevent colon cancer by making the intestines weakly acidic, making it difficult for harmful secondary bile acids to be produced. In addition, butyric acid is said to suppress the development of colon cancer by suppressing abnormal proliferation of colon cells, promoting apoptosis, suppressing lesions of colon cells, and the like. There is a research report that propionic acid acts on short-chain fatty acid receptors on liver cancer cells to suppress the growth of liver cancer cells.

c. Prevention of obesity Short-chain fatty acids act on short-chain fatty acid receptors in adipocytes to suppress the uptake of energy into adipocytes and prevent adipocyte enlargement. It also acts on short-chain fatty acid receptors in nerve cells and promotes energy consumption via the sympathetic nervous system, thereby regulating energy balance.

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

e. Suppression of appetite Butyrate and propionate secrete GLP-1 and other intestinal hormones such as PYY from intestinal L cells. GLP-1 and PYY act on the brain to suppress appetite, and are known to maintain satiety and prevent overeating.

f. Regulation of immune function Approximately 60% of the immune cells of the whole body are concentrated in the intestine, and it is said that the collapse of the immune balance (especially excessive immune reaction) in the intestine affects the whole body. Butyric acid has the effect of increasing immune cells called Treg cells that suppress excessive immune reactions, and it is known that this is related to the function of butyric acid to promote the acetylation of histones in colon epithelial cells. It is also said that butyric acid is useful for inflammatory bowel disease, which is an intestinal immune disease.

(3) Increase in the proportion of lactic acid bacteria present in intestinal bacteria By ingestion of this bacterial flora-improving agent by humans or non-human animals, the proportion of lactic acid bacteria present in the intestine can be increased. An increase in lactic acid bacteria can be expected to prevent or improve autoimmune diseases and colds caused by lactic acid bacteria, and improve intestinal regulation, diarrhea, bowel movements, etc. by increasing good bacteria and suppressing bad bacteria.

Subjects to whom the present bacterial flora improving agent is applied may be any person who needs to enjoy the above effects, and is not particularly limited in this respect, but is preferably a factor that changes the composition of the intestinal flora. It can broadly target people who take a fatty diet. A person who likes to take a high-fat diet is preferred, and a person who is obese or prone to obesity is more preferred. A fatty diet is a diet containing a large amount of fat, and although it is not strictly defined, it includes a diet in which the ratio of fat to total energy intake (fat energy ratio) is about 30 to 40% or more. be able to. For example, meat such as beef ribs, beef loin, ground beef, corned beef (cow), pork ribs, pork loin, bacon (pig), sausage (pork); Eggs and dairy products such as egg yolk, fresh cream (milk fat), and Camembert cheese; Grains such as croissants, Danish pastries, corn snacks, and potato chips; Macadamia nuts, peanuts, almonds, cashew nuts, etc. Nuts; meals containing a lot of fat-containing ingredients such as fried tofu, soybean flour, mayonnaise, French dressing, olive oil, sesame oil, salted butter, and margarine tend to be high-fat meals. Obesity is judged based on BMI (Body Mass Index: weight [kg]/height [m] squared). can be done.

Whether or not administration (administration, ingestion) of the present bacterial flora-improving agent improves the intestinal flora can be evaluated and confirmed by analyzing the intestinal bacteria contained in feces.

In addition, soy isoflavone, which is the active ingredient of this bacterial flora improving agent, can be obtained by changing the composition of the intestinal flora and/or increasing the abundance of useful intestinal bacteria, as shown in the experimental examples described later. . It is thought to inhibit the production of secondary bile acids from primary bile acids in the intestine. As a result, according to this bacterial flora-improving agent, the production of primary bile acids, which are useful for lipid absorption, is increased rather than reduced, while secondary bile acids, which cause colorectal cancer and liver cancer, are produced. Its production can be suppressed significantly better and its abundance in the intestine can be reduced.

The dosage (ingestion) of this bacterial flora improving agent can be appropriately changed depending on the condition and severity of symptoms of the subject, but the daily dosage (ingestion) for an adult (50 kg body weight) is The amount of soybean isoflavone (dry amount) contained in the improving agent is usually about 10 to 150 mg. It is usually used in the form of oral administration once or divided into 2 to 3 times a day. The dosing time is not particularly limited, and can be exemplified by one or more times during meals in the morning, noon, and evening. In addition, although not limited, it is preferable to take it within 30 minutes together with a meal or before or after a meal because it 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 simply referred to as "this production inhibitor") is characterized by containing soybean isoflavone as an active ingredient.

As with the present bacterial flora-improving agent described above, the present production-suppressing agent is not particularly limited as long as it is in the form of oral administration. In addition, as long as it has an oral administration (orally ingested) form, it is classified according to its use (pharmaceuticals, quasi-drugs, foods and drinks [foods with specified health uses, foods with function claims, foods with nutritional functions, etc.) and supplements]) is not particularly limited. It is preferably a food or drink, and more preferably a food for specified health use or a food with function claims, which can claim its actions and effects.

The present production inhibitor may be in the form of a usual food or drink in addition to various formulation forms. The food and drink can be produced by adding the soybean isoflavone described above or an additive containing the soybean isoflavone described below to various food and drink.

The dosage (intake) of this production inhibitor can be appropriately changed depending on the condition and severity of the symptoms of the subject. It is usually about 10 to 150 mg in terms of the amount of soy isoflavone (dry amount) contained in the soybean. It is usually used in the form of oral administration once or divided into 2 to 3 times a day. The dosing time is not particularly limited, and can be exemplified by one or more times during meals in the morning, noon, and evening. In addition, although not limited, it is preferable to take it within 30 minutes together with a meal or before or after a meal because it affects the absorption of lipids contained in food.

Subjects for this production inhibitor are not particularly limited as long as they need to reduce the amount of secondary bile acid production. It can broadly target people who take a fatty diet. A person who likes to take a high-fat diet is preferred, and a person who is obese or prone to obesity is more preferred.

Whether or not administration (administration, ingestion) of this production inhibitor suppresses the production of secondary bile acids and reduces the production amount will be evaluated and confirmed by analyzing and measuring the amount of bile acids in feces. be able to. 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 later, this production inhibitor does not decrease, but rather increases, the production of primary bile acids useful for lipid absorption, while preventing colorectal cancer and liver cancer. It can significantly suppress the production of secondary bile acids, which are also the cause, and reduce their abundance in the intestine.

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

The additive of the present invention is an additive used for imparting an intestinal microflora-improving effect to a target oral composition based on the intestinal microflora-improving effect of soy isoflavone, which is an active ingredient thereof. is. The additive of the present invention can also be used to further enhance the action of an oral composition having an action of improving intestinal flora.

The additive of the present invention is used to impart a secondary bile acid production inhibitory effect to a target oral composition based on the secondary bile acid production inhibitory effect of soy isoflavone, which is an active ingredient of the additive. It can also be an additive that is used. The additive of the present invention can also be used to further enhance the action of an oral composition that inhibits secondary bile acid production. In addition, the additive of the present invention increases the amount of primary bile acid produced and decreases the amount of secondary bile acid produced in the target oral composition based on the action of soy isoflavone, which is the active ingredient thereof. It is also an additive used to impart the effect of The additive of the present invention can also be used for oral compositions having these effects to further enhance those effects.

Here, the oral compositions targeted by the present invention include compositions administered orally to humans or compositions ingested by humans, specifically oral pharmaceuticals, oral quasi-drugs, and food and drink. is included. Food and drink are preferred.

The type of soy isoflavone used as a raw material for the additive of the present invention, its preparation method, etc. are as described in (I) above, and can be used in this section (III).

The additive of the present invention may be soy isoflavone itself, or may be prepared by combining soy isoflavone with conventionally known edible carriers, excipients, etc. that are pharmaceutically or food acceptable. There may be. The additive of the present invention is added to the above-mentioned oral pharmaceuticals, oral quasi-drugs, and/or food and drink, and used to prepare the aforementioned secondary bile acid production inhibitor. For this reason, regardless of its form, it may be in the form of a liquid (including extracts and syrups) or jelly, and the liquid may be formulated into powder or granules by a conventional method. powders, fine granules, granules; capsules (hard capsules, soft capsules) filled with liquids, powders, or granules; (solid formulation).

Oral pharmaceuticals, oral quasi-drugs, and / or the amount of the additive of the present invention used by adding and blending in food and drink, the intestinal flora improving agent prepared by blending the additive of the present invention or The daily administration (ingestion) of the secondary bile acid production inhibitor can be adjusted so that the daily administration (ingestion) is usually about 10 to 150 mg in terms of soybean isoflavone (dry amount).

In addition, the additive of the present invention is used as one of raw materials together with other raw materials in the process of preparing the oral composition (oral drug, oral quasi-drug, and/or food and drink), or When taking (administering or ingesting) the composition, it can be used by adding it to the oral composition at the time of use.

(IV) Method of using soy isoflavone The present invention also provides a method of using soy isoflavone. In the method, the oral composition has an action of improving the intestinal flora, an action of suppressing the production of secondary bile acids, and/or an action of increasing the production of primary bile acids and reducing the production of secondary bile acids. A method of using soy isoflavones for delivery, the method can be carried out by incorporating the soy isoflavones into a subject oral composition. Instead of soy isoflavone, the above-described additive containing soy isoflavone as an active ingredient can also be used.

The details of the target oral composition and the method of using soy isoflavones are as described above.

Hereinafter, the terms "including" and "containing" include the meanings of "consisting of" and "consisting essentially of".

Hereinafter, the present invention will be described using experimental examples in order to facilitate understanding of the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were performed at room temperature (25±5° C.) and atmospheric pressure conditions.

Experimental example Bile acid intake test Test animals (mice) were given soybean isoflavone in addition to primary bile acid (cholic acid), and body weight, food intake, cecal content weight, and bile acid content in the cecal content ( Primary bile acid content, secondary bile acid content, and total bile acid content) were measured. In addition, genomic DNA was extracted from the contents of the cecum, and the intestinal flora was analyzed.

(1) Test animal Animal: male C57BL/6J mouse 8 weeks old (purchased from Japan SLC)
Breeding period: After bringing in the animals, after a two-week acclimation period with solid samples of normal food, the animals were divided into the following test groups so that the average body weight of each group was equal.
Breeding environment: room temperature 25°C, humidity 55%, indoor fluorescent lights were turned on in a 12-hour cycle from 7:00 am to 7:00 pm.

Test zone:
After the acclimation period, the test animals were divided into the following groups (n = 6 or 7 in each group), and 0.3% (w/v) call was administered to the bile acid administration groups (b) to (e). The rats were allowed to drink an acid (CA) aqueous solution, and were allowed to freely ingest a diet in which each material was mixed with a control diet for two weeks.
(a) Control group: ingested control diet (normal diet solid sample D12450J: Research Diet) + drinking water (distilled water; hereinafter the same) (b) Bile acid administration group (CA group): control diet + 0.3% Drinking water supplemented with cholate (c) 3% raffinose + bile acid administration group (CA + 3% raffinose group): Control diet + 3% raffinose + 0.3% drinking water supplemented with cholate (d) 0.5% curcumin + bile acid Administration group (CA + 0.5% curcumin group): Control diet + 0.5% curcumin + 0.3% cholic acid added drinking water (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 As the soy isoflavone, Isoflavone P40 (manufactured by Fujicco Co., Ltd.) was used. Isoflavone P40 contains 37% by mass or more of isoflavone. Among all isoflavones, the ratio of glycosides of three kinds of isoflavones, genistein, daidzein, and glycitein, is about 85% by mass or more, and aglycones are contained at a ratio of about 15% by mass. The above "0.8%" is the amount in terms of isoflavone content contained in Isoflavone P40.

(2) Test method and results 1. Body weight, amount of food intake, amount of food in the cecum For the test animals in each test group, the body weight, the amount of food in the cecum, and the amount of food consumed per day (g/day/mice) were measured after the administration period of 2 weeks. The results are shown in Table 1 as mean + standard error (n=6-7) for each group.

As shown in Table 1 above, a significant decrease in body weight was observed in the (e) CA + 0.8% isoflavone group compared to the control group and the CA group, although the amount of food intake did not change significantly. rice field. In contrast, no significant weight loss was observed in (c) CA + 3% raffinose group and (d) CA + 0.5% curcumin group. In addition, compared to the control group and the CA group, (c) CA + 3% raffinose group and (e) CA + 0.8% isoflavone group showed a tendency to increase cecal contents, especially in (e) CA + 0.8% isoflavone group. showed a significant increase.

3. Amount of Bile Acids in Cecal Contents The amount of bile acids in the cecal contents was quantified. Hagio, M., M. Matsumoto, and S. Ishizuka. 2011. Bile acid analysis in various biological samples using ultra performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI- MS). Methods Mol. Biol. 708: 119-129.), samples for LC-MS were prepared from cecal contents, and bile acid components were analyzed and quantified using LC-MS.

The results of comparing the amount of each primary bile acid, secondary bile acid, and total bile acid in the control group, CA group, CA + 3% raffinose group, CA + 0.5% curcumin group, and CA + 0.8% isoflavone group are shown. 2. Table 2 also shows the results of the Tukey HSD test. The upper right superscript letter of each numerical value indicates that different letters are significantly different between groups (p<0.05), and there is no significant difference when the same letter is included.

As shown in Table 2, the amount of primary bile acids in the CA group, which received cholic acid, decreased compared to the control group, and the amount of secondary bile acids, including highly toxic deoxycholic acid (DCA), significantly increased. was increasing. On the other hand, in the CA + 0.8% isoflavone group, in which soy isoflavone was ingested in addition to cholic acid, primary bile acid levels increased significantly compared to both the control group and the CA group. The amount of deoxycholic acid (DCA) was also markedly decreased along with the amount of both secondary bile acids compared to the CA group in which the amounts of cholic acid (DCA) and secondary bile acids were increased.

The above-mentioned bile acid administration test showed that soy isoflavone has the effect of suppressing the increase in secondary bile acids caused by intake of CA, in other words, the intake of high-fat diet, that is, the effect of reducing secondary bile acids (secondary bile acid generation suppression effect) was confirmed. 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, suggesting that soy isoflavones have the effect of primary bile acids (e.g. , promotion of emulsification of fat, excretion of cholesterol, etc.) without impairing (or rather enhancing) the production and increase of secondary bile acids, and have the effect of reducing the amount of secondary bile acids produced. confirmed.

As shown in Table 1, the amount of cecal content increased in the CA + 0.8% isoflavone group, and it is known that the increase in cecal content is related to the increase in short-chain fatty acids that have beneficial effects on the host. It is From this, in the CA + 0.8% isoflavone group, the metabolites of short-chain fatty acid-producing bacteria, which are specific beneficial bacteria, increased, the intestinal pH decreased, and/or the diversity of the intestinal flora improved. As a result, an increase in primary bile acid production and a decrease in secondary bile acid production are thought to occur. In addition, 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 flora caused by soy isoflavone intake.

Recently, it has been reported that an antioxidant called Tempole suppresses obesity caused by high-fat diet intake, decreases the Firmicutes phylum that increases with a high-fat diet, and increases the Bacteroidets phylum that 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)). Administration of Tempole increases a conjugated bile acid called tauro-β-muricholic acid (TβMCA), which acts as an antagonist to farnesoid X receptor (FXR), a nuclear receptor in the gastrointestinal tract, and inhibits FXR activation. It has been suggested that suppression of obesity occurs in Although the mechanism of anti-obesity by isoflavone intake is not clear, this study showed a significant increase in tauro-β-muricholic acid (TβMCA), a primary bile acid, suggesting that improvement of the intestinal flora may reduce primary bile acid production. Obesity suppression may occur through increased production.

3. Intestinal microflora composition in cecal contents For test animals in each test group, DNA was extracted according to a standard method from the cecal contents collected after the administration period of 2 weeks, and V3 of the 16S rRNA gene was requested to Biotechnology Research Institute Co., Ltd. -V4 region was amplified and subjected to meta16S flora analysis by Illumina MiSeq. A total of 1,511,039 reads (average of 50,368 reads) of the 30 samples obtained were subjected to bacterial flora analysis using QIIME (Quantitave Insights Into Microbial Ecology).

From the results of the microbial flora analysis, the Shanon index (an index obtained by adding the frequency of each species to the number of bacterial species), phylum-level microbial flora composition, and family/genus-level microbial flora composition were determined. The Shanon index is used as an index for judging bacterial flora diversity.

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

[Phylum-level microflora composition]
The phylum-level gut microbiota composition (n=6) in the cecal contents is shown in FIG. 2A. As shown in Figure 2A, CA administration increased the Proteobacteria phylum and decreased the Bacteroidetes phylum. The ratio of the phylum Firmicutes to the phylum Bacteroidetes (Firmicutes/Bacteroidetes ratio) is shown in FIG. 2B. As shown in FIGS. 2A and B, 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, it is known that the intestinal flora of obese humans has a low proportion of bacteria belonging to the phylum Bacteroidetes and a high proportion of bacteria belonging to the phylum Firmicutes. On the other hand, as the body weight decreases, that is, as the body becomes normal or lean, the proportion of bacteria belonging to the phylum Bacteroidetes increases and the proportion belonging to the phylum Firmicutes decreases. The results in Fig. 2B were correlated with the weight loss in the CA + 0.8% Iso group shown in Table 1, suggesting that intake of soy isoflavones changes the composition of the intestinal flora, resulting in a lean constitution. was confirmed.

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

4. Discussion In general, high-fat diet intake is known to cause an increase in secondary bile acids, an increase in the phylum Firmicutes, and a decrease in the phylum Bacteroidetes, leading to the development of obesity and metabolic syndrome. In addition, it has been pointed out that an increase in secondary bile acids due to intake of a high-fat diet is associated with colon diseases and liver diseases (Non-Patent Document 2). In this study, it was confirmed that soy isoflavone administration reduced the secondary bile acids increased by CA administration. Furthermore, the diversity of the intestinal flora decreased by CA administration was ameliorated by administration of soy isoflavone (genus Turicibacter, genus Dorea) and Proteobacteria (family Desulfovibrionaceae) are both reduced by soy isoflavone administration, and the ratio of Firmicutes to Bacteroidetes (Firmicutes/Bacteroidetes ratio) is reduced). It was confirmed that administration increased various short-chain fatty acid-producing bacteria such as butyric acid-producing bacteria (genus Coprococcus), useful bacteria such as equol-producing bacteria (genus Adlercreutzia), and lactic acid bacteria (genus Lactobacillus) in the intestine.

Although the details of the relationship between these intestinal bacteria and bile acids are unknown, administration of soy isoflavones improved the diversity of the intestinal flora, which was reduced by CA administration. and an increase in primary bile acids. In this way, obesity, metabolic rhodome, colon diseases, It is thought that it exerts an action to improve or prevent other diseases and conditions such as liver disease.

Claims (4)

A secondary bile acid production inhibitor containing soy isoflavone as an active ingredient. 2. The inhibitor of secondary bile acid production according to claim 1, which exhibits the effect of increasing the production of primary bile acids and reducing the production of secondary bile acids. An additive for a secondary bile acid production inhibitor containing soybean isoflavone as an active ingredient. Soy isoflavone or the additive according to claim 3 is blended into an oral composition, and the oral composition has at least an effect of suppressing secondary bile acid production and an effect of increasing primary bile acid production. A method of using soy isoflavones to impart one action.

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