JP2017147960A - Proliferation stimulant of intestinal blautia coccoides, anxiety-ameliorating agent, and therapeutic agent for inflammation of intestine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a proliferation stimulant of intestinal Blautia coccoides, and a composition for stabilizing mood and ameliorating anxiety, or suppressing and treating inflammation of the intestine by stimulating proliferation of intestinal Blautia coccoides.SOLUTION: The invention relates to a sphingoglycolipid-containing composition comprising a sphingoglycolipid as an active ingredient. More particularly, the invention relates to a proliferation stimulant of intestinal Blautia coccoides comprising a sphingoglycolipid, or an anxiety-ameliorating agent comprising a sphingoglycolipid, or a therapeutic agent for inflammation of the intestine comprising a sphingoglycolipid.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a growth stimulator for intestinal Brautia coccoides containing a glycosphingolipid.

  Many foods that are orally ingested are known to be directly digested and absorbed as nutrients such as carbohydrates, lipids, proteins, vitamins, minerals, etc., and as ingredients that exhibit pharmacological effects and functions. Yes. Although it is involved in digestion and absorption, there exist intestinal bacteria that are resident in the human intestine, and these enteric bacteria are known to have an important relationship with human health.

  There are a wide variety of enterobacteria, and the whole is called the intestinal flora, and its balance is considered important. This balance is known to vary greatly depending on factors such as the individual, age, physical condition, nutritional status, and stress. In general, useful microorganisms in the human digestive tract are also called good bacteria, and bifidobacteria and lactic acid bacteria are known as good bacteria.

  The regulation of intestinal microbiota, especially the growth of good bacteria, is influenced by food, and it is known that yogurt and other foods containing lactic acid bacteria, which are also good bacteria in the first place, have a function of regulating intestinal microflora. It has been. However, even the foods that are said to be widely healthy, such as Japanese foods, have not been fully studied about the effects of their diet on the gut microbiota.

  In recent years, in addition to the bacteria generally known as good bacteria, the influence of Clostridium, which has been confirmed to exist as an intestinal bacterium, has begun to attract attention. For example, it is disclosed that direct ingestion of this bacterium has an effect of improving anxiety and hyperactivity (Patent Document 1). In addition, a technique relating to the proliferation of regulatory T cells by a bacterial composition containing bacteria belonging to the genus Clostridium is disclosed, and thereby a technique for treating or preventing autoimmune diseases, inflammatory diseases, infectious diseases, etc. is disclosed. (Patent Document 2).

  On the other hand, glycosylceramide, a kind of glycosphingolipid, is extracted from plants and moss and is widely used as a cosmetic application for dry skin, and has been reported to be effective in improving skin function ( Patent Document 3). Furthermore, a method for improving the composition of the intestinal flora by administering a food containing excess sphingolipid to a bird or mammal has been disclosed (Patent Document 4).

JP 2013-119546 A Special table 2015-500792 gazette Japanese Patent Laid-Open No. 06-145039 JP-T-2006-516278

  Traditionally, Japanese food has been said to be good for health, but how it has changed the gut microbiota has not been fully studied. On the other hand, Patent Document 4 discloses a method for improving intestinal bacterial flora by ingesting sphingolipid, but here, many types of sphingolipid are not desired in the intestine. It is based on the discovery that bacteria can be killed selectively while other bacteria remain virtually undisturbed.

  In other words, sphingolipids are considered to have only their growth-inhibiting effects on specific bacteria, and only the bacteria corresponding to so-called bad bacteria are excluded, and other good bacteria are maintained relatively. It is intended to increase the composition and improve the gut microbiota. It can be used as an alternative to antibiotics, or it can suppress various symptoms caused by harmful bacteria (diarrhea, gastrointestinal tract infection, liver damage, intestinal cancer).

  Here, as disclosed in Patent Documents 1 and 2, it is expected that Blautia coccoides, which is a kind of intestinal bacteria, obtain various functions by ingestion directly. However, there are no known techniques for selectively growing these bacteria despite their presence in the gut microbiota.

  Under such circumstances, an object of the present invention is to provide a new regulator of intestinal bacterial flora. Here, an object of the present invention is to provide a growth stimulating agent for intestinal Blautia coccoides whose importance is beginning to attract attention. In addition, by stimulating the proliferation of intestinal Blautia coccoides, effects of stabilizing the mind and suppressing enteritis are also expected.

  The present inventors selectively concentrated glycosphingolipids, particularly glycosylceramides contained in sputum etc., and orally ingested so that intestinal bacterium coccoides have not been known in the intestinal microflora. It was found to grow selectively.

  As a result of intensive studies to solve the above problems, the present inventor has found that the following inventions meet the above object, and have reached the present invention.

That is, the present invention relates to the following inventions.
<1> A growth stimulant for intestinal Blaucia coccoides containing glycosphingolipid.
<2> An anxiety-improving agent containing a glycosphingolipid.
<3> A therapeutic agent for enteritis containing glycosphingolipid.
<4> The intestinal Blautia coccoides growth stimulator according to <1>, wherein the glycosphingolipid is glycosylceramide.
<5> The anxiety-improving agent according to <2>, wherein the glycosphingolipid is glycosylceramide.
<6> The therapeutic agent for enteritis according to <3>, wherein the glycosphingolipid is glycosylceramide.

  According to the present invention, there is provided a growth stimulating agent that stimulates the growth of intestinal Brautia coccoides. In addition, an anxiety-improving agent and an enteritis-inhibiting agent are provided by proliferating intestinal Brautia coccoides.

It is a figure which shows the abundance ratio of Brautia coccoides in the intestinal microflora of the mouse | mouth which fed the Brautia coccoides growth stimulating agent which concerns on this invention. It is the graph which confirmed the growth stimulating effect of Blaucia coccoides by the Blaucia coccoides proliferation stimulating agent which concerns on this invention.

  DESCRIPTION OF EMBODIMENTS Embodiments of the present invention will be described in detail below. However, the description of constituent elements described below is an example (representative example) of an embodiment of the present invention, and the present invention is described below unless the gist thereof is changed. It is not limited to the contents.

  The present invention relates to a composition having various functions containing a glycosphingolipid. Here, the present invention relates to a growth stimulant for Blaucia coccoides, an anxiety ameliorating agent, and a therapeutic agent for enteritis, all of which relate to a composition containing a glycosphingolipid. It may be referred to as a “sphingoglycolipid-containing composition”.

[Glycosphingolipid]
The glycosphingolipid-containing composition of the present invention contains glycosphingolipid as an active ingredient. Glycosphingolipid is a lipid having a sphingoid base, which is a long-chain amino alcohol. As glycosphingolipids, galactosylceramide in which ceramide is bound to galactose, glucosylceramide in which ceramide is bound to glucose, etc. are known, and what is generally called glycosylceramide can be used.

  As described above, the glycosphingolipid-containing composition of the present invention comprises a glycosphingolipid. As the glycosphingolipid, in addition to those derived from Aspergillus, those derived from animals such as bovine brain, plants, and microorganisms can be used. A plurality of glycosphingolipids derived from these may be used as a mixture, or may be used alone or in combination with other components as necessary. Examples of the plant-derived glycosphingolipid include glycosphingolipids derived from rice, corn, konjac, maitake, tamogitake, soybean, beet, and wheat.

[Lipids derived from Aspergillus oryzae]
The glycosphingolipid-containing composition of the present invention can contain a glycosphingolipid derived from Aspergillus as a glycosphingolipid. Aspergillus oryzae-derived lipids are mainly lipid components obtained by extracting lipid components from Aspergillus or removing components other than lipids such as water from Aspergillus oryzae. Specifically, the koji mold is mixed with an organic solvent such as alcohol to extract the lipid component of koji mold into the organic solvent, or the koji mold cell membrane is crushed with a homogenizer and centrifuged to separate the components, The lipid component of Aspergillus is obtained by drying to remove water or combining these steps. The lipid derived from Aspergillus oryzae contains glycosphingolipids such as glycosylceramide. This gonococcal lipid is a lipid and, like gonococcus and cocoon itself, does not need to be maintained alive or by freeze-drying, etc. Are better. Moreover, the lipid derived from Aspergillus can be obtained from a culture of Aspergillus oryzae and has high production efficiency.

[Koji mold]
Aspergillus is a fungus used for sputum. Rice bran is a product obtained by breeding bacteria, such as Aspergillus spp. Described later, effective in food fermentation on grains such as rice, wheat, and soybean. This koji is used when producing fermented foods such as sake, miso, vinegar, pickles, soy sauce, shochu, and awamori. Here, the koji mold of the present invention is not particularly limited as long as it has a glycosphingolipid as an active ingredient. For example, koji mold belonging to the genus Aspergillus can be used. In the present invention, shochu treated as industrial waste containing these koji molds can be used, and industrial waste containing this koji mold can be used as a raw material for the glycosphingolipid-containing composition of the present invention. It is also useful as a processing method.

  More specifically, Aspergillus oryzae, Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachii, Aspergillus saitoi, Aspergillus saitoi, Aspergillus saitoi, Aspergillus saitoi, Aspergillus saitoi, Aspergillus saitoi, Aspergillus saitoi (Aspergillus awamori), Aspergillus nakazawai, Aspergillus usamii, Aspergillus luchensis, Aspergillus niger and the like.

  In addition, the koji mold that can be used in the present invention is not limited to the koji mold belonging to the genus Aspergillus, and other genus, for example, the bacteria belonging to the genus Rhizopus, the bacteria belonging to the genus Monuscus and the bacteria belonging to the genus Penicillium are also used. be able to. Examples of bacteria belonging to these genera are given below.

  Fungi belonging to the genus Rhizopus: Rhizopus oryzae, Rhizopus oligosporus, Rhizopusniveus, Rhizopus microspores, Ronzo pus , Monuscus genus: Monuscus anka, Monuscus purpureus, Monuscusruber, Monuscus pilosus, Monuscus aurantiacus , Such as Monuscus kaoliang, belonging to the genus Penicillium: Penicillium camemberti, Penicillium roqueforti, Penicillium grauca (Penicillium glaucum), such as Penicillium casei column (Penicillium caseicolum), in the present invention may be used in combination of Aspergillus singly or in combination. Among them, it is preferable to include Aspergillus kawachi which is widely used for fermentation of alcoholic beverages such as shochu.

[Brautia coccoides growth-improving agent]
Brautia coccoides is a kind of bacteria present in the intestinal flora. By using the glycosphingolipid-containing composition of the present invention, it is possible to proliferate the intestinal Brautia coccoides. Therefore, it is possible to prepare an intestinal bacterial flora that focuses on this bacterium.

[Anxiety improver]
The glycosphingolipid-containing composition of the present invention can be used as an anxiety-improving agent and exhibit an effect of improving anxiety or hyperactivity. The effect of this anxiety-improving agent is due to the growth of intestinal Brautia coccoides. Developmental disorders called attention deficit / hyperactivity disorder are known for anxiety improvement and hyperactivity improvement. More specifically, this results in conditions such as maintenance of attention, abnormal time sensation, decreased information processing, and symptoms that interfere with daily life due to overactivity and impulsivity. . Many of these symptoms are seen especially in children, but some symptoms may be seen even in adults. Conventionally, a specific treatment method for this symptom has not yet been established, but according to the present invention, an anxiolytic agent that suppresses these developmental disorders by ingesting the glycosphingolipid-containing composition of the present invention. Is provided.

[Therapeutic agent for enteritis]
The glycosphingolipid-containing composition of the present invention can be used as a therapeutic agent for enterocolitis and exhibit enterocolitis treatment effects. This enterocolitis treatment effect is due to the proliferation of Blautia coccoides in the intestine. Regulatory T cells (Treg cells) are a kind of CD4 positive helper T cells and are said to have an immunosuppressive function and the like. Proliferating and accumulating Treg cells is said to have a function of suppressing excessive inflammation by secreting anti-inflammatory cytokines such as IL-10 and TGFβ in addition to suppressing immune responses against self. Treg cells can be proliferated and accumulated by ingesting Blaucia coccoides, but if the sphingoglycolipid-containing composition of the present invention is orally ingested, Treg cells can be proliferated and selectively proliferated by intestinal Bleucia coccoides. Accumulation can be achieved in the intestine. Thereby, inflammation in digestive organs, such as an intestine, can be improved / suppressed.

  Therefore, the present invention can also be achieved as the use of a glycosphingolipid (preferably glycosylceramide) for the production of any one of the growth stimulating agent of Blaucia coccoides, an anxiety-improving agent and a therapeutic agent for enteritis.

  The glycosphingolipid-containing composition of the present invention preferably contains 70 to 5,000 mg of glycosphingolipid as an active ingredient per day for a human weighing 60 kg. In addition, humans can ingest 1.2 to 80 mg / kg body weight per day. When the intake is less than this, effects such as intestinal blastia coccoides growth stimulation, anxiety improvement, and enterocolitis treatment may not be sufficiently achieved. On the other hand, the glycosphingolipid of the present invention has low toxicity and little adverse effects due to overdose, but the effect is saturated even if it is excessively consumed, so it is not necessary to ingest more than the upper limit. The daily intake of a 60 kg body weight human (adult) is more preferably 200 mg or more, and even more preferably 600 mg or more.

  The content of the glycosphingolipid in the composition of the glycosphingolipid-containing composition of the present invention is appropriately changed depending on the dosage form and the like, and is not particularly limited, but the content assumed as a dosage form for oral consumption As 0.1 to 50% by weight.

  The glycosphingolipid-containing composition of the present invention contains a glycosphingolipid and is in the form of a solid formulation such as a soft capsule, a hard capsule, a capsule tablet such as a tableting tablet, or the like so as to be pharmaceutically acceptable. Can be appropriately formulated into a dosage form of a pharmaceutical preparation suitable for oral administration such as liquid or liquid. And it is orally administered as the formulation, The oral dose can be controlled. Therefore, this intake is an amount taken from what was prepared as a composition, and can be set as an amount taken separately from a normal meal, for example, as a tablet or a capsule. In addition, glycosphingolipids are also contained in general foods, but the amount consumed by a general meal may not be sufficient as an amount that exerts the effect of the present invention, and the intake amount is defined as the active ingredient concentration. In the management, it is considered that there are a certain number of layers with insufficient intake from the normal intake derived from these foods. Therefore, it is preferable to manage the intake amount as a sufficient amount by the glycosphingolipid-containing composition of the present invention.

  The glycosphingolipid-containing composition of the present invention can also be achieved as a functional food related to intestinal blastia coccoides growth stimulation, anxiety improvement, and enterocolitis treatment. The glycosphingolipid content in the composition of the glycosphingolipid-containing composition as a food is appropriately changed depending on the food, but can be about 0.1 to 50% by weight in each food. Any kind of food may be used, for example, mainly flour such as bread and noodles and starch, confectionery such as chocolate, jelly and gummy, dairy products such as yogurt and cheese, fermented food, Examples include beverages and seasonings. As a method of blending the glycosphingolipid, it may be blended appropriately at the stage of the production process according to various foods.

  Moreover, the glycosphingolipid as an active ingredient of the present invention includes foods that have been eaten from the past, and has low toxicity. Also, in the case of food forms containing glycosphingolipids, it is preferable to adjust the concentration to be higher than those generally contained so that an effective amount can be ingested efficiently. In addition, since glycosphingolipid is excellent in palatability, it is easy to use as various foods suitable for continuous intake.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to a following example, unless the summary is changed.

[Example 1] Abundance ratio of Blaucia coccoides in gut microbiota By ingesting a glycosphingolipid-containing composition orally, changes in the abundance ratio of Blacia coccoides in gut microbiota were evaluated.

[Evaluation item]
[Intestinal flora analysis]
The mouse intestinal bacterial flora was analyzed using the next-generation sequencer NGS “Miseq” (manufactured by Illumina).

[Glycosphingolipid (glycosylceramide)]
<Extraction of coconut lipid components>
(1) Lipid components are extracted with chloroform / methanol (1: 1) from dried rice bran (grown Aspergillus oryzae, 70% polished rice), roughly purified by Bligh & Dyer method, dried, and dried lipid components Got. When this sputum lipid component is analyzed, at least “N-2′-hydroxy-3′-octadecanoyl-1-O-β-D-”, which is a glycosylceramide (a kind of glycosphingolipid) specific to bacilli. It was confirmed that glycosylceramides such as “glucopyranosyl-9-methyl-4,8 sphingadianin” were contained. This coconut lipid component is a lipid containing about 10% of glycosylceramide.

[Glycosphingolipid-containing composition]
Based on the above coconut lipid component (containing 10% glycosylceramide) based on a diet for mice “AIN-76” (standard purified diet for nutritional studies using mice and rats by the National Institute of Nutrition (AIN)) Then, it was mixed with sucrose to prepare a sphingosaccharide-containing composition having a jaundice lipid component concentration of 1 wt%. This bait is hereinafter referred to as “GlcCer bait” (Kgc). Moreover, the jaundice lipid component of the GlcCer bait was replaced with sucrose, and the bait without the jaundice lipid component was used as a “standard bait” (Nor) for comparison. These more specific component ratios (% by weight) are shown in Table 1.

^* Prepared based on AIN-76 composition.

[Glycosylceramide donation to mice]
"Experimental system"
The experimental mice (C57BL / 6N) were divided into 2 groups of 3 mice, the group fed with the “GlcCer diet (Kgc)” (“Kgc group”), and the general mouse diet (“standard diet” in Table 1). (Nor) ") was fed (" Nor group "). Each group is fed with each bait for one week, the genome is extracted from its feces, the 16S rRNA gene V1-V2 variable region of all bacteria is amplified by PCR, and the next-generation genome analyzer (" Sequence analysis was performed using “Miseq” (manufactured by Illumina), and microbiota data was obtained using the MiSeq Reporter 16S metadataomics system attached to the apparatus. For the cluster analysis, EMBL's R environment intestinal bacteria analysis software was used (see “http://enterotype.embl.de/enterotypes.html”).

  FIG. 1 is an appearance frequency (relative value) relating to the abundance ratio of Blue coccoides in the feces of mice fed with the growth stimulating agent of Blue coccoides according to the present invention (* p <0.05 (Student's t-test)).

  As a result of the intestinal microbiota analysis, it has been confirmed that the gram-positive bacterial phylum is most present. As shown in FIG. 1, the median value in the Kgc group was about 4.2%, and a significant increase in Blaucia coccoides was observed compared to the median value in the Nor group of about 2.1%.

[Example 2] Growth of Blaucia coccoides in medium containing coconut lipid component The influence of the growth of Blaucia coccoides by the presence or absence of the cocoon lipid component was evaluated.

[Evaluation item]
[Turbidity (OD600)]
By measuring the turbidity of the fermentation test solution using a turbidimeter (Shimadzu Corporation "UV-1800": test wavelength 600 nm, quartz cell used, optical path length 1 cm), The index to be evaluated. In addition, distilled water was used as a blank in which the turbidity (OD600) was zero.

[Basic medium]
A mixture of 1.5 mL of sodium cholate (1.5% w / v) in 1.5 mL of YPD medium was used as the basic medium.

[Medium (A): Glycosylceramide-containing medium]
Furthermore, 7.5 μL of glycosylceramide-containing ethanol (glycosylceramide concentration 4 μg / μL) was mixed with the basic medium to obtain a medium (A) (glycosylceramide-containing medium). As the glycosylceramide, the dried coconut lipid component described in Example 1 was used.

[Medium (B): Control medium]
Moreover, instead of the glycosylceramide-containing ethanol in the medium (A), the same amount of ethanol was mixed in the basic medium as a medium (B) for the control test.

[Culture test]
The medium (A) or the medium (B) was infused with Brautia coccoides so that OD600 = 0.1, and anaerobically cultured at 37 ° C. under anaerobic conditions (Aneropack) for 24 hours. The culture result is shown in FIG. 2 (proliferation stimulation of Blaucia coccoides by addition of glycosylceramide).

  As shown in FIG. 2, in the group cultured with the medium (A) containing glycosylceramide, OD600 increased, so that Blaucia coccoides grew.

  By using the glycosphingolipid-containing composition according to the present invention, since a significant increase in B. coccoides in the intestinal flora was confirmed, a more stable effect of B. coccoides can be obtained than direct ingestion of B. cocoides. Expected to improve anxiety and treat enteritis.

  According to the present invention, the intestinal flora can be prepared. Specifically, it is used as an intestinal Blaucia coccoides growth stimulating agent that selectively proliferates intestinal Blaucia coccoides. Moreover, anxiety improvement and enterocolitis treatment can be performed by selectively proliferating intestinal Brautia coccoides.

Claims (6)

  An intestinal Blaucia coccoides growth stimulant containing glycosphingolipid.   An anxiety ameliorating agent containing a sphingoglycolipid.   A therapeutic agent for enteritis containing glycosphingolipid.   The intestinal Blautia coccides growth stimulator according to claim 1, wherein the glycosphingolipid is glycosylceramide.   The anxiety ameliorating agent according to claim 2, wherein the glycosphingolipid is glycosylceramide.   The therapeutic agent for enteritis according to claim 3, wherein the glycosphingolipid is glycosylceramide.