JP6879498B2 - Akkermansia bacterium growth promoter and its use - Google Patents

Description

The present invention relates to an Akkermansia bacterium growth promoter, a pharmaceutical composition for improving the intestinal flora, foods and drinks, and a method for promoting the growth of Akkermansia bacterium.

Procyanidins are one of the major polyphenols, catechins and their polymers (see FIG. 1). It is known that procyanidins are widely contained in fruits such as apples and grapes, spices, cereals, teas, cacao and the like. In recent years, procyanidins have a strong antioxidant effect, fat accumulation suppression, obesity prevention effect, triglyceride absorption suppression effect, carcinogenesis suppression effect, peripheral circulation improvement effect, blood fluidity improvement effect, liver function improvement effect, and platelet aggregation suppression effect. Various effects such as are reported. In order for procyanidins to exert the above-mentioned effects, it is necessary to take an appropriate amount and absorb it into the body. In general, it has been clarified that polyphenols are absorbed into the body from the intestinal tract after ingestion and act on the liver and adipose tissue, and bioavailability is considered to be important. Among procyanidins, it has been reported that tetrameric low-molecular-weight procyanidins are absorbed into the body. In addition, it has been reported that decomposition products of low-molecular-weight procyanidins by intestinal bacteria are absorbed from the large intestine (see, for example, Non-Patent Document 1). However, the role of high molecular weight procyanidins of pentamers or more in functionality is unknown because they cannot pass through the intestinal tract and are not absorbed into the body due to their large molecular weight.

Recent studies have reported that changes in gut microbiota composition affect host energy regulation, nutritional intake, immune function, etc., and are closely related to metabolic disorders such as obesity and diabetes (for example). , See Non-Patent Document 2.). Comparing the intestinal flora of normal mice and obese mice by 16S rRNA analysis, obese mice had fewer bacteria belonging to the phylum Bacteroides and a higher proportion of bacteria belonging to the phylum Firmicutes than normal mice. In addition, when the intestinal flora of obese mice and normal mice was transplanted to sterile mice, the body fat was significantly higher in the mice transplanted with the intestinal bacteria of obese mice than in the mice transplanted with the intestinal bacteria of normal mice. It has been reported that the amount increases (Non-Patent Document 2).

As a mechanism by which intestinal bacteria affect pathological conditions such as obesity of the host and metabolic disorders such as diabetes, a pathway by metabolites in the intestinal tract such as short-chain fatty acids and bile acids is known. Short-chain fatty acids such as acetic acid, butyric acid, and propionic acid, which are metabolites of dietary fiber and polysaccharides from the intestinal flora, not only play an important role as an energy source, but also act as signal molecules to produce intestinal hormones and consume energy. It also affects appetite and is involved in the pathophysiology of obesity. For this reason, preventive and therapeutic methods via intestinal bacteria are being studied as methods for improving and treating lifestyle diseases such as obesity and diabetes, such as Lactobacillus (lactic acid bacteria) and Bifidobacterium. Probiotics that bring about beneficial effects such as improvement of the host's intestinal flora by ingesting bacteria of the genus Bifidobacterium, and dietary fibers and oligos that have a beneficial effect on the maintenance of the intestinal flora. Prebiotics that ingest sugar and the like are carried out.

On the other hand, it has been reported that in obesity, mild chronic inflammation in adipose tissue occurs, causing insulin resistance and impaired glucose tolerance. Obese mice fed a high-fat diet showed decreased intestinal barrier function, and it is thought that chronic inflammation is caused by an increase in endotoxin in the blood. Recently, the identification of bacteria and the search for drugs and food components for suppressing the increase in blood concentration of endotoxin such as LPS by enhancing the intestinal barrier function and improving chronic inflammation have been carried out. As a result, it has been reported that Akkermansia bacteria maintain the mucin mucosal layer of the intestinal tract and enhance the intestinal barrier function (see, for example, Non-Patent Document 3). In addition, as a technique for treating or preventing lifestyle-related diseases such as obesity and diabetes by enhancing the intestinal barrier function by Akkermansia bacteria, for example, cranberries and grape procyanidins improve the composition of the intestinal flora. It has been reported to do so and to grow Akkermansia bacterium (see, for example, Non-Patent Documents 4 and 5).

Shoji, T., et al., “Apple Procyanidin Oligomers Absorption in Rats after Oral Administration: Analysis of Procyanidins in Plasma Using the Porter Method and High-Performance Liquid Chromatography / Tandem Mass Spectrometry”, J. Agric. Food Chem., Vol. . 54, p884-892, 2006. Backhed, F., et al., “The gut microbiota as an environmental factor that regulates fat storage”, PNAS, vol.101, no.44, p15718-15723. Everard, A., et al., “Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity”, PNAS, vol.110, no.22, p9066-9071, 2013. Anhe, FF, et al., “A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. Population in the gut microbiota of mice”, Gut microbiota, vol.0, p1-12, 2014. Roopchand, E.D., et al., “Dietary Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat Diet-Induced Metabolic Syndrome”, Diabetes, vol.64, p2847-2858, 2015.

Non-Patent Documents 4 and 5 disclose that procyanidins of cranberries and grapes improve the composition of the intestinal flora, but the degree of polymerization and chemical structure of procyanidins are not specified.

The present invention has been made in view of the above circumstances, and is a novel Akkermansia bacterium growth-promoting agent containing procyanidins having a specified degree of polymerization and chemical structure and having an excellent effect of enhancing the intestinal barrier function. provide.

In order to solve the above problems, the present inventors conducted various experiments on plant-derived proanthocyanidins, and as a result of intensive research from a biochemical and medical point of view, proanthocyanidins of plant-derived pentamers or more. They have found that species improve the gut microbiota and have completed the present invention.

That is, the present invention includes the following aspects.
[1] An Akkermansia bacterium growth promoter containing a plant-derived pentamer or more of proanthocyanidins or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] At least one of the plants selected from apples, pears, peaches, grapes, lychees, blueberries, cassis, avocados, barley, guava, hops, red beans, walnuts, chestnuts, cacao, pine bark, black tea, green tea, and wine. The Ackermancia genus bacterial growth promoter according to the species [1].
[3] The Akkermansia bacterium growth promoter according to [1] or [2], wherein the proanthocyanidins are procyanidins.
[4] The intestine is characterized by containing at least one of the Akkermansia bacterium growth promoter according to any one of [1] to [3], and a pharmaceutically acceptable carrier and diluent. A pharmaceutical composition for improving flora.
[5] A food or drink containing the Akkermansia genus bacterial growth promoter according to any one of [1] to [3].
[6] A method for promoting the growth of Akkermansia bacteria, which comprises administering a plant-derived pentamer or more of proanthocyanidins.

According to the present invention, it is possible to provide an Akkermansia bacterium growth promoter containing procyanidins having a specified degree of polymerization and chemical structure and having an excellent effect of enhancing the intestinal barrier function.

It is a structural formula showing procyanidins which are multimers of dimer or more and 15 or less. It is a graph which shows the change of the body weight in the mouse of each group in Test Example 1 during the test period and after breeding for 20 weeks. It is a graph which shows the feed amount during the test period in the mouse of each group in Test Example 1. It is a graph which shows the amount of water drinking during the test period in the mouse of each group in Test Example 1. It is a graph which shows the liver weight after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the visceral fat weight after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the subcutaneous fat weight after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the blood glucose amount after 20 weeks breeding in the mouse of each group in Test Example 1. 6 is a graph showing the amount of triglyceride in blood after 20 weeks of rearing of mice in each group in Test Example 1. It is a graph which shows the total cholesterol after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the blood LPS amount after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the blood TNF-α amount after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the blood IL-6 amount after 20 weeks breeding in the mouse of each group in Test Example 1. It is a graph which shows the result of the main coordinate analysis of the intestinal flora after 20 weeks breeding in the mouse of each group by 16S rRNA analysis in Test Example 1. It is a graph which shows the profile at the phylum level of the intestinal flora after 20 weeks breeding in the mouse of each group by 16S rRNA analysis in Test Example 1. It is a graph which shows the abundance ratio (Firmicultes / Bacteroidetes) of the phylum Bacteroidetes to the phylum Bacteroidetes among the intestinal bacteria after 20 weeks breeding in the mouse of each group by 16S rRNA analysis in Test Example 1. It is a graph which shows the abundance ratio of the bacterium of the genus Akkermansia to the total intestinal bacterium among the intestinal bacterium after 20 weeks breeding in each group of mice by 16S rRNA analysis in Test Example 1.

<< Akkermansia Bacterial Growth Promoter >>
In one embodiment, the present invention provides an Akkermansia bacterium growth promoter containing a plant-derived pentamer or more of proanthocyanidins or a pharmaceutically acceptable salt thereof as an active ingredient.

The Akkermansia genus bacterial growth promoter of the present embodiment contains pentamers or more of procyanidins and has an excellent effect of enhancing the intestinal barrier function.
Conventionally, procyanidins having a pentamer or more have a large molecular weight, so that they cannot pass through the intestinal tract and are not absorbed into the body, and their functions have been unknown. On the other hand, the present inventors have found that pentamers or more procyanidins have an excellent Akkermansia bacterial growth promoting effect, and by ingesting pentamers or more procyanidins, the intestinal barrier function is enhanced. It was clarified that it can be enhanced.
In addition, in this specification, "intestinal barrier function" means to prevent harmful substances produced by bad bacteria among intestinal bacteria, or viruses and bacteria invading from the mouth from being absorbed into the body through the intestinal wall. It means a defensive function.

In general, Akkermansia bacterium is a useful intestinal bacterium present in the intestinal mucosal layer of humans from infants to the elderly, and Akkermansia bacterium is found in non-obese humans and rodents. It is known to be present in large quantities and only in small amounts in inflamed and obese humans and rodents. Furthermore, it has been clarified in experiments using obesity and type 2 (adult-onset) diabetes model mice that it is beneficial for improving metabolic deficiency associated with obesity (see, for example, Non-Patent Document 3).

<Proanthocyanidins>
In the present specification, "proanthocyanidins" are condensed tannins existing in plants, that is, flavan-3-ols as a constituent unit, and are condensed or condensed in a 4-8 bond type or a 4-6 bond type. It means a mixture of compounds bonded by polymerization, and these proanthocyanidins produce anthocyanidins such as cyanidins, delphinidins, and pelargonidins by acid treatment.
Examples of proanthocyanidins in the present embodiment include procyanidins, prodelphinidins, properargonidins and the like. Among them, the proanthocyanidins in the present embodiment are preferably procyanidins.
The proanthocyanidins contained in the Akkermansia genus bacterial growth promoter of the present embodiment are pentamers or more, and preferably pentamers or more and 15 or less.

In addition, the Akkermansia genus bacterial growth promoter of the present embodiment may contain a pharmaceutically acceptable salt of proanthocyanidins.

As used herein, the term "pharmaceutically acceptable" generally means a degree that does not cause side effects when properly administered to a test animal.

As the salt, a pharmaceutically acceptable acid addition salt or basic salt is preferable.
Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid; acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, and malic acid. , Salts with organic acids such as benzoic acid, methanesulfonic acid, benzenesulfonic acid and the like.
Examples of the basic salt include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and magnesium hydroxide; and salts with organic bases such as caffeine, piperidine, trimethylamine and pyridine. ..

The Akkermansia bacterium growth promoter of the present embodiment may contain, for example, a buffer solution such as PBS and Tris-HCl, and an additive such as sodium azide and glycerol as other components.

The Akkermansia genus bacterial growth promoter of the present embodiment can be used to provide a therapeutic method for improving the intestinal flora.
The treatment target is not particularly limited, and examples thereof include humans and mammals other than humans (for example, monkeys, mice, rats, rabbits, pigs, dogs, horses, cows, etc.), and among them, humans are preferable.

<Manufacturing method of proanthocyanidins>
The proanthocyanidins used in the Akkermansia bacterium growth promoter of the present embodiment may be commercially available, may be directly extracted and separated from a plant, or may be chemically synthesized.

Plants from which proanthocyanidins are derived include, for example, apples, pears, peaches, grapes, lychees, blueberries, cassis, avocados, barley, guava, hops, red beans, walnuts, chestnuts, cacao, pine bark, black tea, green tea, etc. Examples include, but are not limited to, wine. The proanthocyanidins obtained from these plants may be derived from one type of plant, or may be used in combination of those derived from a plurality of types of plants.

As a method for directly extracting and separating proanthocyanidins from plants, for example, a method for extracting and purifying a proanthocyanidin component from apple fruit (References: JP-A-7-285876, JP-A-2000-16951, And Japanese Patent Application Laid-Open No. 2002-87978). The apple used as a raw material may be an immature apple fruit (Reference: Japanese Patent Application Laid-Open No. 7-285876) or a wild apple species (Crab Apple) (Reference: Nan Li, et al. , “Profile and Antioxidant Activity of Phenolic Extracts from 10 Crabapples (Malus Wild Species)”, J. Agric. Food Chem., Vol. 62, p574-581, 2014.).

More specific examples of the method for directly extracting and separating proanthocyanidins from apples include the following extraction methods (Reference: Japanese Patent Application Laid-Open No. 7-285876).
First, the apple fruit is washed and crushed and squeezed to obtain fruit juice as it is or while adding sulfite. Then, it is centrifuged, and a clear juice is prepared by filtration or the like. The obtained clear juice may be appropriately concentrated by a known method.
As another extraction method, for example, fruits are mixed with alcohols, crushed, immersed as they are, and proanthocyanidins are extracted by pressing or heating under reflux. Then, after distilling off the alcohol, centrifugation and filtration, or distribution and filtration with an organic solvent such as hexane and chloroform are performed to obtain a clarified extract.

Next, as a method for purifying the crude apple polyphenol component from the obtained clear juice or the clear extract, for example, the following purification method (Reference: Japanese Patent Application Laid-Open No. 2000-16951) and the like are used. Can be mentioned.
First, by passing the above-mentioned clarified fruit juice or clarified extract through a column packed with an adsorbent capable of selectively adsorbing polyphenols (for example, styrenedivinylbenzene-based synthetic adsorbent resin, anion exchange resin, etc.), Adsorbs polyphenol components. Then, after washing the column with distilled water, the polyphenol component is eluted and recovered by passing an alcohol solution of 20 to 100%, preferably 30 to 60% through the column. When alcohol is distilled off from the obtained alcohol solution fraction, a crude apple polyphenol component is obtained. The crude apple polyphenol component contains procyanidins which are multimers of dimers or more and 15 or less as shown in FIG.

Next, as a method for obtaining proanthocyanidins from the obtained crude apple polyphenol component, for example, the method shown below (reference: JP-A-2002-87978) and the like can be mentioned.
First, the above-mentioned crude apple polyphenol component is separated and purified into a procyanidin 2-tetramer fraction and a pentamer or more fraction by solid-liquid extraction using methyl acetate as a liquid phase. A fraction of procyanidin pentamer or more that is not extracted into methyl acetate can be obtained by distilling methyl acetate by a known method. On the other hand, the procyanidin 2 to tetramer fraction extracted to methyl acetate is concentrated in an extraction solution by a known method and then dissolved in distilled water, and the procyanidin 2 to 5 dimer fraction is further subjected to normal phase chromatography. By separating and purifying according to the degree of polymerization (by molecular weight), a procyanidin oligomer having a uniform degree of polymerization can be obtained.

Further, proanthocyanidins are synthesized, for example, by an intermolecular condensation reaction in which an electrophile (E-unit) and a nucleofile (N-unit) represented by the following formula (1) are condensed between molecules (reference: [Saito,] A., et al., Biosci. Bitotech. Biochem., 66, p1764, 2002.], [Saito, A., et al., Tetrahedron Lett., 44, p5449, 2002.], [Saito, A., et al., Heterocycles, 62, p479, 2004.], [Saito, A., et al., Synlett, p2040, 2004.], [Saito, A., et al., Bioorganic Med. Chem., 12, p4783, 2004.], [Saito, A., et al., Tetrahedron, 60, p12043, 2004.], and [Saito, A., et al., Bioorganic Med. Chem., 13, p2759, 2005.] ) And other known methods may be used for chemical synthesis.

<Use>
The Akkermansia genus bacterial growth promoter of the present embodiment can be used, for example, in a pharmaceutical composition for improving the intestinal flora, food and drink, and the like, as described later.
In addition, the Akkermansia genus bacterial growth promoter of the present embodiment can be used, for example, in a medium, a culture preparation, or the like. By adding a culture preparation containing the Akkermansia bacterium growth promoter of the present embodiment to the medium, or by culturing the Akkermansia bacterium using the medium containing the Akkermansia bacterium growth promoter of the present embodiment. It can effectively promote the growth of Akkermansia bacterium.

<< Pharmaceutical composition for improving intestinal flora >>
In one embodiment, the present invention provides a pharmaceutical composition for improving gut flora, which comprises the above-mentioned Akkermansia bacterium growth promoter and at least one of a pharmaceutically acceptable carrier and diluent.

According to the pharmaceutical composition for improving the intestinal flora of the present embodiment, the intestinal barrier function can be effectively enhanced. In addition, it is safe and effective to ingest plant-derived pentamers or more of proanthocyanidins because they have previous eating experience from the viewpoint of safety and it is considered that there are few problems in ingesting them every day. A pharmaceutical composition for improving the intestinal flora can be provided.

In general, the "intestinal flora" is also called the intestinal flora, and a wide variety of intestinal bacteria from the ileum to the large intestine form a group for each type and inhabit the intestinal wall surface. Means. Gut microbiota are bacteria that have a beneficial effect on the body of the host (particularly human) (good bacteria), and bacteria that promote intestinal rot and produce toxic substances (for example, ammonia, phenol, indol, etc.) (bad bacteria). ), And it does not apply to good bacteria or bad bacteria, and is composed of three types of bacteria.
Examples of good bacteria include bacteria of the genus Lactobacillus (lactic acid bacteria), bacteria of the genus Bifidobacterium (Bifidobacterium), and bacteria of the genus Akkermansia.
Bad bacteria include, for example, Welsh, staphylococcus, and toxic strains of Escherichia coli (eg, intestinal pathogenic Escherichia coli, intestinal invasive Escherichia coli, toxinogenic Escherichia coli, intestinal hemorrhagic Escherichia coli (eg, O1, O18, O26, O111) , O128, O157, etc.) Intestinal pathogenic Escherichia coli, etc.) and the like.
Examples of opportunistic bacteria include Bacteroides, non-toxic strain Escherichia coli, streptococcus and the like.
Further, in the present specification, "improvement of intestinal flora" means a certain balance of intestinal bacteria in a form in which good bacteria suppress bad bacteria (for example, good bacteria: bad bacteria: opportunistic bacteria = 2: 1: 1: 7 etc.) means to improve so that it is maintained.

<Dose>
The pharmaceutical composition of the present embodiment takes into consideration the age, sex, body weight, symptoms, treatment method, administration method, treatment time, etc. of the test animal (various mammals including humans or non-human animals, preferably humans). It is adjusted as appropriate.
The dose of proanthocyanidins contained in the pharmaceutical composition of the present embodiment varies depending on the symptoms, but in the case of oral administration, generally in adults (assuming a body weight of 60 kg), 100 mg or more and 1500 mg or less per day, It is considered that it is preferably 300 mg or more and 500 mg or less.

As for the number of administrations, it is preferable to administer once to several times per week on average.
Examples of the administration form include methods known to those skilled in the art enterally or orally, and oral administration is preferable.
Injections can also be prepared as non-aqueous diluents (eg, vegetable oils such as polyethylene glycol and olive oil, alcohols such as ethanol), suspensions, or emulsions. Such sterilization of the injection can be performed by filtration sterilization with a filter, blending of a bactericidal agent, or the like. The injectable preparation can be produced as a form of preparation for errands. That is, a sterile solid composition can be prepared by a freeze-drying method or the like, and the composition can be dissolved in distilled water for injection or another solvent before use.

<Composition component>
The pharmaceutical composition of this embodiment comprises a therapeutically effective amount of the Akkermansia bacterium growth promoter described above, as well as a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents are excipients, diluters, bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavors. Examples include agents, solubilizers, additives and the like. By using one or more of these carriers, pharmaceutical compositions in the form of injections, liquids, capsules, suspensions, emulsions, syrups and the like can be prepared.

A colloidal dispersion system can also be used as the carrier. The colloidal dispersion system has the effect of enhancing the in vivo stability of the above-mentioned Akkermansia bacterium growth promoter and the effect of enhancing the transferability of the above-mentioned Akkermansia genus bacterium growth promoter to a specific organ, tissue or cell. Be expected. Examples of the colloidal dispersion system include polyethylene glycol, polymer composites, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. , Liposomal or artificial membrane vesicles, which have the effect of efficiently transporting the above-mentioned Ackermancia bacterial growth promoter to a specific organ, tissue, or cell, are preferable.

Examples of the formulation of the pharmaceutical composition of the present embodiment include those used orally as sugar-coated tablets, capsules, elixirs, and microcapsules as needed.
Alternatively, aseptic solutions with water or other pharmaceutically acceptable liquids, or those used parenterally in the form of injections of suspensions can be mentioned. Furthermore, pharmacologically acceptable carriers or diluents, specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, etc. Examples thereof include those formulated by appropriately combining with preservatives, binders and the like and mixing them in a generally accepted unit dose form required for pharmaceutical practice.

Additives that can be mixed with tablets and capsules include, for example, binders such as gelatin, cornstarch, traganth gum, gum arabic, excipients such as crystalline cellulose, swelling such as cornstarch, gelatin and alginic acid. Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, reddish oil or cherry are used. When the dispensing unit form is a capsule, the above-mentioned material can further contain a liquid carrier such as fat or oil. The sterile composition for injection can be formulated according to the usual pharmaceutical practice using a vehicle such as distilled water for injection.

When the pharmaceutical composition of the present embodiment is an injection, the sterile composition can be formulated according to the usual formulation practices using a vehicle such as distilled water for injection. Examples of the aqueous solution for injection include physiological saline, isotonic solutions containing glucose and other auxiliary agents, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like, which are appropriately dissolved. With auxiliary agents (eg, alcohol (specifically, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol, etc.)), or nonionic surfactants (eg, polysorbate 80 (TM), HCO-50, etc.) It may be used together.

Examples of the oily liquid include sesame oil and soybean oil, and examples of the solubilizing agent may be used in combination with, for example, benzyl benzoate, benzyl alcohol and the like. Further, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (for example, procaine hydrochloride, etc.), a stabilizer (for example, benzyl alcohol, phenol, etc.), or an antioxidant is further added. You may. The prepared injection solution is usually filled in a suitable ampoule.

Injectables can also be prepared as non-aqueous diluents (eg, vegetable oils such as polyethylene glycol and olive oil, alcohols such as ethanol), suspensions, or emulsions. Such sterilization of the injection can be performed by filtration sterilization with a filter, blending of a bactericidal agent, or the like. Injectables can be produced in the form of errand preparation. That is, a sterile solid composition can be prepared by a freeze-drying method or the like, and the composition can be dissolved in distilled water for injection or another solvent before use.

The pharmaceutical composition of the present embodiment may be used alone or in combination with other pharmaceutical compositions for improving intestinal flora. Other pharmaceutical compositions for improving intestinal flora include, for example, an intestinal regulator containing good bacteria such as bifidus bacterium preparation, butyric acid bacterium preparation, lactomin preparation, and resistant lactomin preparation as an active ingredient; pepsin, pancreatin and the like as active ingredients. Examples thereof include an intestinal regulator containing a digestive enzyme agent such as an animal digestive enzyme agent and a plant digestive enzyme agent containing diastase as an active ingredient.

<Treatment method>
One aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned Akkermansia bacterium growth promoter for improving the intestinal flora.
Also, one aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned Akkermansia bacterium growth promoter, as well as a pharmaceutically acceptable carrier or diluent.
In addition, one aspect of the present invention provides an Akkermansia bacterium growth promoter containing the pharmaceutical composition.
In addition, one aspect of the present invention provides the use of an Akkermansia bacterium growth promoter for producing a therapeutic agent for improving the intestinal flora.
In addition, one aspect of the present invention provides a therapeutic method for improving the intestinal flora, which comprises administering an effective amount of the above-mentioned Akkermansia bacterium growth promoter to a patient in need of treatment.

<< Food and drink >>
In one embodiment, the present invention provides a food or drink containing the above-mentioned Akkermansia bacterium growth promoter.

According to the food and drink of the present embodiment, the intestinal barrier function can be effectively enhanced. In addition, it is safe and effective to ingest plant-derived pentamers or more of proanthocyanidins because they have previous eating experience from the viewpoint of safety and it is considered that there are few problems in ingesting them every day. Food and drink can be provided.

In the present specification, "food and drink" is a combination of food and beverage, and mainly means processed food. In addition, the food and drink of the present embodiment includes health foods (including foods for specified health use), functional foods, health drinks, and functional drinks.

The form of the food or drink containing the above-mentioned Akkermansia bacterium growth promoter may be solid or liquid, and the above-mentioned Akkermansia bacterium growth promoter is widely added as a food additive in foods in general. Can be used. Specific examples of food and drink types include soft drinks (for example, mineral water, carbonated drinks, nutritional drinks, sports drinks, cocoa drinks, fruit drinks, dairy drinks, coffee drinks, tea-based drinks, soy milk drinks, and vegetable drinks. , Alcoholic beverages (eg, non-alcoholic beer, non-alcoholic wine, etc.), alcoholic beverages (eg, beer, sparkling liquor, cocktails, chewy, shochu, Japanese liquor, whiskey, brandy, wine, etc.) (these beverages) Concentrated stock solution and adjustment powder); Cold confectionery such as ice cream, ice sherbet, shaved ice; Soba, udon, spaghetti, Harusame, Gyoza skin, Shumai skin, Chinese noodles, Instant noodles and other noodles; Candy, chewing gum , Candy, gummy, gum, caramel, chocolate, tablet confectionery, snack confectionery, baked confectionery such as biscuits, confectionery such as jelly, jam, cream; marine or livestock processed food such as kamaboko, hamburger, ham, sausage; processed milk, Dairy products such as fermented milk, yogurt, butter, and cheese; fats and oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings and processed fats and oils; seasonings such as sauces and sauces; soups, stews, curries, Examples include, but are not limited to, breads, jams, salads, side dishes, pickles, etc.

The food and drink of the present embodiment may appropriately contain additives that are usually used depending on the type of food and drink. Examples of additives include sweeteners such as sugar, fructose, isomerized liquid sugar, glucose, aspartame and stevia, acidulants such as citric acid, malic acid and tartaric acid, excipients such as dextrin and starch, and binders. Diluting agents, fragrances, buffers, thickeners, gelling agents, coloring agents, stabilizers, emulsifiers, dispersants, suspending agents, preservatives and the like can be mentioned.

The blending amount of proanthocyanidins in the food and drink of the present embodiment may be an amount capable of exerting its physiological and pharmacological actions, and is administered orally in the above-mentioned << pharmaceutical composition for improving intestinal flora >>. In consideration of the dose and the general intake of the target food or drink, the daily intake for an adult may be usually 100 mg or more and 1500 mg or less, preferably 300 mg or more and 500 mg or less. For example, in the case of a solid food, it may be 10 to 50% by weight, and in the case of a liquid food such as a beverage, it may be 0.1 to 10% by weight.

<< How to grow Akkermansia bacteria >>
In one embodiment, the present invention provides a method for promoting the growth of Akkermansia bacteria, which administers plant-derived pentamers or more of proanthocyanidins.

According to the method for promoting the growth of Akkermansia bacterium of the present embodiment, the growth of Akkermansia bacterium can be effectively promoted.

As shown in Examples described later, administration of plant-derived pentamers or more of proanthocyanidins to dietary obesity model mice was able to promote the growth of Akkermansia bacterium, and thus Akkermansia genus. It was revealed that plant-derived pentamers or more proanthocyanidins are effective for bacterial growth.

When proanthocyanidins are administered to humans or non-human animals, the dose is the same as the dose in the above-mentioned << pharmaceutical composition for improving intestinal flora >> orally or parenterally. Can be mentioned.

As for the number of administrations and the administration form, the same ones as those exemplified in the above-mentioned << Pharmaceutical composition for improving intestinal flora >> can be mentioned.

In addition, proanthocyanidins may be added to a medium or the like to promote the growth of Akkermansia bacteria. As a result, the resulting Akkermansia bacterium is suitably used for prebiotic applications such as direct administration by humans or non-human animals.
In general, "prebiotics" means, for example, substances such as edible substances, which may not be digested by humans, but may be used by bacteria in the intestinal microflora and are probiotics in the intestine. It is believed to promote bacterial growth.
In addition, "probiotics" are preparations of microbial cells (for example, living microbial cells, etc.) or components of microbial cells, and when administered in an effective amount, the health or health condition (well) of the subject. -Being) means something that can provide a beneficial effect. As used herein, "probiotics" refers to non-pathogenic ones. Health benefits of probiotics include improving the balance of human or non-human animal microbiota in the gastrointestinal tract and / or restoring normal microbiota.

The dose (concentration contained in the medium) when proanthocyanidins are added to the medium is preferably 0.01 mg / mL or more and 10 mg / mL or less, and 0.1 mg / mL or more and 10 mg / mL or less. Is more preferable.
When the concentration of proanthocyanidins contained in the medium is within the above range, the growth of Akkermansia bacteria can be efficiently promoted.

The medium used for the growth of Akkermansia bacterium may have a composition suitable for culturing Akkermansia bacterium, and may contain a carbon source, a nitrogen source, vitamins, inorganic salts and the like that can be used as a nutrient source. All you need is. Examples of the medium include mucin-containing basal medium (reference material: Muriel D., et al., “Akkermansia muciniphila gen. Nov., Sp. Nov., A human intestinal mucin-degrading bacterium”, Int J Syst Evoll Microbiol. , Vol.54, p1469-1476, 2004.), etc., but not limited to this.
Further, the culture conditions for Akkermansia bacterium are preferably anaerobic conditions, and for example, the culture may be carried out by static culture, shaking culture, stirring culture or the like.
The culture temperature may be, for example, 20 ° C. or higher and 40 ° C. or lower.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Extraction and purification of proanthocyanidins (1) Extraction of crude apple polyphenol fraction Unripe fruit of apple "Fuji" collected in June in Hirosaki City, Japan (average weight of individual fruits is about 10 g) ) A total of 3 kg was homogenized in the presence of potassium pyrosulfite (0.1% w / w), and the ground product was kept at 4 ° C. for 24 hours. Then, the obtained pulverized product was squeezed to collect fruit juice, clarified by centrifugation (3500 g × 20 minutes), and filtered through a glass filter. Next, the obtained filtrate (1.8 L) was applied to a column (inner diameter 25 mm × 285 mm) packed with styrene-divinylbenzene synthetic adsorption resin Sepabeads SP = 850 (manufactured by Mitsubishi Kasei). Then, it was washed with distilled water (300 mL) to remove the water-soluble component, and then eluted with 80% ethanol. Then, ethanol was removed from the obtained 80% ethanol fraction (200 mL) and freeze-dried to obtain a crude apple polyphenol fraction. When this crude apple polyphenol fraction was analyzed by reverse phase high performance liquid chromatography, chlorogenic acids (about 20%), phloretin glycosides (about 5%), flavonols (about 15%), proanthocyanidins It was confirmed that it consisted of (about 50%) and other browning substances (about 10%). Furthermore, as a result of analysis by MALDI-TOF / MS, these proanthocyanidins were confirmed to be dimers to 15-mer composed of catechins and epicatechins, which are flavan-3-ols, and were high molecular weight polyphenols. ..

(2) Purification of Proanthocyanidins Next, the obtained crude apple polyphenol fraction was dissolved in distilled water and adjusted to pH 6.5 with 5N sodium hydroxide. Then, the solution of the crude apple polyphenol fraction was prepared and applied to a column packed with styrene-divinylbenzene synthetic adsorption resin Diaion HP-20ss (manufactured by Mitsubishi Kasei). Then, the column was washed with distilled water and then eluted with 25% ethanol. Then, the obtained 25% ethanol fraction was concentrated and lyophilized to obtain a proanthocyanidin fraction. In addition, references [Shoji, T., et al., “Apple (Malus pumila) procyanidins fractionated according to the degree of polymerization using normal-phase chromatography and characterized by HPLC-ESI-MS and MALDI-TOF-MS”, J. Chromatogr., Vol.1102, no.1, p206-213, 2006.] was used to separate apple procyanidins according to the degree of polymerization, and the tetramer was low from the monomer. It was separated into a molecular procyanidin fraction (hereinafter, may be referred to as “OP”) and a high molecular weight procyanidin fraction of pentamers or more (hereinafter, may be referred to as PP). The obtained apple-derived proanthocyanidin fraction was used as a sample for evaluating the effect on the intestinal flora in the dietary obesity model described later.
As a result of analysis by HPLC normal phase chromatography, the content of pentameric proanthocyanidins in the pentameric or higher polymer procyanidin fraction (PP) was 27.1%, and hexameric proanthocyanidins were found. The content of anthocyanidins is 23.2%, the content of 7-mer proanthocyanidins is 17.5%, the content of octameric proanthocyanidins is 8.8%, and 9 The content of proanthocyanidins above the dimer was 9.5%.

[Test Example 1]
The apple-derived proanthocyanidin fractions (OP and PP) obtained in Example 1 were used in the test by the method shown below. In order to investigate the possibility of acting on obesity, the apple-derived high molecular weight proanthocyanidin fractions (OP and PP) were freely orally ingested by dietary obese mice, and their effects were examined.

(1) Ingestion and weight measurement of apple-derived proanthocyanidin fractions (OP and PP) to dietary obese mice That is, 10-week-old C57BL / 6J mice were ingested in a control diet (normal diet) (hereinafter, "ND group"). ”), High-fat, high-sucrose diet intake group (hereinafter, may be referred to as“ HFHS group ”), high-fat, high-sucrose diet + low-molecular-weight procyanidin intake group (hereinafter,“ OP group ”). ”) And a high-fat, high-sucrose diet + high-molecular-weight procyanidin intake group (hereinafter, may be referred to as“ PP group ”), and the diet was continuously ingested for 20 weeks.
In the procyanidin intake group, OP and PP were each dissolved in tap water at 0.5% (w / w) and ingested by free drinking water. During the breeding period, body weight, food intake, and water intake were measured over time. After 20 weeks of breeding, autopsy was performed to determine the weight of organs (liver, spleen, pancreas, kidney), and the weight of peri-testis, peri-renal, mesentery, and subcutaneous fat. The results are shown in FIGS. 2-7. FIG. 2 is a graph showing changes in body weight during the test period and after 20 weeks of breeding in the mice of each group. 3 and 4 are graphs showing the amount of food and water consumed during the test period in the mice of each group, respectively. Further, FIGS. 5 to 7 are graphs showing liver weight, visceral fat weight, and subcutaneous fat weight after 20 weeks of rearing in each group of mice.

(2) Biochemical analysis Next, for each group of mice after 20 weeks of rearing, blood biochemical general component analysis (blood glucose level (blood glucose level), triglyceride, total cholesterol, LPS, and inflammatory cytokines) (TNF-α, IL-6)) was performed. The results are shown in FIGS. 8 to 13. FIGS. 8 to 11 show blood glucose level, triglyceride level, total cholesterol level, blood LPS level, blood TNF-α level, and blood IL- after 20 weeks of rearing in each group of mice. It is a graph which shows 6 quantities.
In addition, "LPS (Lipopolysaccharide; lipopolysaccharide)" is endotoxin produced from bad bacteria.

(3) Gut microbiota analysis by 16S rRNA Next, for mice in each group after 20 weeks of breeding, the 16S rDNA gene region (V3-V4 region) was amplified by PCR from the DNA extracted from the cecal contents. For PCR, TakaRa Ex Taq HS DNA polymerase (manufactured by TakaRa Bio) was used, and as a primer, a universal primer (314f / 785r) having an adapter sequence for Roche FLX analysis added to the 5'end side shown in Table 1 below was used. There was. The PCR reaction was carried out at 94 ° C. for 1 minute for 1 cycle, 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds for 20 cycles, and 72 ° C. for 5 minutes for 1 cycle.

Then, after confirming the band of the obtained PCR product by agarose gel electrophoresis, it was matched with the Illumina sequencing adapter and the dual index bar code sequence using the Nextera DNA Library Preparation Kit (manufactured by Illumina), and AMPure XP Beads. Purified using Kit (manufactured by Beckman Coulter). Next, analysis was performed by a next-generation sequencer, and the obtained data were analyzed using Quantitative Into Microbial Ecology (QIIME) pipeline (version 1.8.0.), And the classification of intestinal bacteria was based on the Green Genes database. I used it. The results are shown in FIGS. 14-17. FIG. 14 is a graph showing the results of main coordinate analysis of the intestinal flora after 20 weeks of rearing in mice of each group by 16S rRNA analysis. In addition, FIG. 15 is a graph showing the profile of the intestinal flora at the phylum level after 20 weeks of rearing in mice of each group by 16S rRNA analysis. In addition, FIG. 16 is a graph showing the abundance ratio of Firmicutes phylum bacteria to Bacteroidetes phylum bacteria (Firmicutes / Bacteroidetes) among the intestinal bacteria after 20 weeks of rearing in mice of each group by 16S rRNA analysis. In addition, FIG. 17 is a graph showing the abundance ratio of Akkermansia bacteria to total intestinal bacteria among the intestinal bacteria after 20 weeks of rearing in mice of each group by 16S rRNA analysis.

(4) Results and Discussion According to the methods described in (1) to (3) above in Test Example 1, the obesity of oral free intake of apple-derived high molecular weight proanthocyanidin fractions (OP and PP) in dietary obese mice The effect on abnormal sugar and lipid metabolism was investigated.
First, although there was no difference in the amount of food intake and water intake in terms of body weight, the weight gain was significantly suppressed in the procyanidin intake group (OP group, PP group) as compared with the HFHS group. (P <0.05) (see FIGS. 2, 3 and 4).
In addition, the increase in liver weight and visceral fat weight after 20 weeks of breeding was significantly suppressed in both the procyanidin intake group (OP group and PP group) as compared with the HFHS group (p <0.05). (See FIGS. 5, 6 and 7).

In addition, in the blood biochemical general component analysis, the increase in blood glucose level, triglyceride and total cholesterol was significantly suppressed in the procyanidin intake group (OP group, PP group) as compared with the HFHS group. (P <0.05) (see FIGS. 8, 9 and 10).
However, the increase in blood LPS level was significantly suppressed only in the PP group (p <0.05) (see FIG. 11). Similarly, the increase in inflammatory cytokines TNF-α and IL-6 was significantly suppressed only in the PP group (p <0.05) (see FIGS. 12 and 13).

Next, when the main coordinate analysis of the intestinal flora in the cecal contents by 16S rRNA analysis was performed, the similarity of the flora structure of each group was clearly separated at the phylum level (see FIG. 14). Furthermore, at the phylum level, Firmicutes was the most predominant bacterium, accounting for 60-80%, followed by Bacteroidetes, which accounted for more than 10-20% (see FIG. 15).
In addition, it has been reported that the Firmicutes / Bacteroidetes ratio increases due to deterioration of the intestinal environment caused by obesity and lifestyle-related diseases, but the Firmicutes / Bacteroidetes ratio is significantly suppressed in the PP group as compared with the HFHS group. (P <0.05) (see FIG. 16).
In addition, at the genus level, Akkermansia bacteria, which are known to enhance intestinal barrier function, were significantly increased in the PP group compared to the HFHS group (p <0.05) (p <0.05). (See FIG. 17).

It is considered that the cause of lifestyle-related diseases such as obesity and diabetes is that a chronic inflammatory state is caused by the influx of LPS into the body due to deterioration of the intestinal environment. Bacteria of the genus Akkermansia have been reported to enhance the intestinal barrier function and prevent LPS influx and chronic inflammation (see, for example, Non-Patent Document 3). Although an increase in Akkermansia bacteria due to procyanidin intake has been reported, there are no reports of differences due to chemical structure, and this was first clarified by utilizing a technique for separating procyanidins with high purity.

In recent years, improvement of the intestinal environment by intestinal bacteria has been studied as one of the preventive and therapeutic methods for metabolic diseases such as obesity and diabetes. A method of ingesting Akkermansia bacterium as a probiotic has been investigated, but a method for culturing Akkermansia bacterium that can be carried out at the human test level has not yet been established. On the other hand, a method of effectively growing Akkermansia bacterium by administering a food component such as dietary fiber or oligosaccharide as a prebiotic has been studied, but according to the present invention, a pentamer or more high molecular weight procyanidin is used. It was thought that it could be realized by utilizing it.

[Prescription example]
(1) Tablets Each part by weight shown in Table 2 below was uniformly mixed to prepare tablets according to a conventional method.

(2) Capsules Each part by weight shown in Table 3 below was uniformly mixed to prepare capsules according to a conventional method.

(3) Powders and granules Each part by weight shown in Table 4 below was uniformly mixed to prepare powders and granules according to a conventional method.

(4) Candy Each component of each weight part shown in Table 5 below was used to make a candy according to a conventional method.

(5) Juice Using each component of each part by weight shown in Table 6 below, juice was prepared according to a conventional method.

(6) Barley tea First, the barley tea components were extracted according to a conventional method using each component of each weight part shown in Table 7 below.

Next, each component of each part by weight shown in Table 8 below was used to prepare barley tea according to a conventional method.

According to the Akkermansia genus bacterial growth promoter of the present invention, the intestinal barrier can be effectively enhanced. Furthermore, ingestion of plant-derived pentamers or more proanthocyanidins contained in the Akkermansia genus bacterial growth promoter of the present invention has been eaten from the viewpoint of safety, and should be ingested daily. It is thought that there are few problems. Therefore, by using the Akkermansia genus bacterial growth promoter of the present invention, it is possible to provide a safe and effective pharmaceutical composition for improving the intestinal flora, or a food or drink.

Claims (6)

Ackermann, which comprises a plant-derived pentamer or more of proanthocyanidins or a pharmaceutically acceptable salt thereof as an active ingredient , wherein the proanthocyanidins are procyanidins, prodelphinidins or properalgonidins. Anthocyanidin growth promoter. The plant is at least one selected from apples, pears, peaches, grapes, lychees, blueberries, cassis, avocados, barley, guava, hops, red beans, walnuts, chestnuts, cacao, pine bark, black tea, green tea, and wine. The Ackermancia genus bacterial growth promoter according to claim 1. The Akkermansia bacterium growth promoter according to claim 1 or 2, wherein the proanthocyanidins are procyanidins. Propagation of Akkermansia bacterium, which comprises at least one of the Akkermansia bacterium growth promoter according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier and diluent. A pharmaceutical composition for improving the intestinal flora by promoting. A food or drink composition for promoting the growth of Akkermansia bacteria, which comprises the agent for promoting the growth of Akkermansia bacteria according to any one of claims 1 to 3. A method for promoting the growth of Akkermansia bacteria, which comprises administering a plant-derived pentamer or more of proanthocyanidins , wherein the proanthocyanidins are procyanidins, prodelphinidins or properalgonidins (medical practice for humans). except for.).

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