JPH07289273A - Naphthoquinone derivative - Google Patents

Abstract

PURPOSE:To obtain a new naphthoquinone derivative, having a high ability to grow bifidus bacteria and useful for foods, medicines, measurement, of the bifidus bacteria, etc., by using a microorganism such as the genus Propionibacterium capable of producing 2-amino-3-carboxy-1,4-naphthoquinone. CONSTITUTION:This new 2-amino-3-carboxy-1,4-naphthoquinone expressed by the formula is obtained by culturing a microorganism, belonging to the genera Propionibacterium, Enterobacter, Bacillus, etc., capable of producing 2-amino-3- carboxy-1,4-naphthoquinone (e.g. Propionibacterium freudenreichii ATCC6207 strain) in a culture medium, producing two kinds of naphthoquinones in the culture and collecting the resultant naphthoquinones. The obtained derivative has promoting actions on the growth of bifidus bacteria at an ultralow concentration and can be utilized as foods or medicines and further for improving enteral floras or an assay system, etc., for measuring the bifidus bacteria.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel naphthoquinone derivative having an activity of promoting the growth of bifidobacteria at an extremely low concentration. This novel compound improves the intestinal flora by addition to various foods and drinks. It is also used as a selective medium for measuring the number of Bifidobacterium.

[0002] Previous comparative studies of intestinal flora in breast-fed infants and artificial infants have suggested that bifidobacteria are useful for human health. At present, it is effective in suppressing the carcinogenesis, suppressing intestinal rot, and preventing infectious diseases that the bifidobacteria significantly decrease with various digestive tract diseases and aging, and that the growth of intestinal bifidobacteria is promoted. It has been confirmed. Therefore, it can be said that selective growth of Bifidobacterium in the intestine is extremely important from the viewpoint of health maintenance and prevention / treatment of various adult diseases.

From the above viewpoints, the number of foods containing fermented bifidobacteria (fermented milk, yogurt, etc.) has been increasing in recent years. From the viewpoint of production and quality control of these foods, the number of bifidobacteria in those foods is increased. It is necessary to develop a selective medium that can measure the specificity.

[0004]

2. Description of the Related Art Regarding substances useful for promoting the growth of useful bifidobacteria, so-called bifidus factors, several substances have been studied and reported so far. For example, N-acetylglucosamine (Proc.
Soc. Exp. Biol. Med. , 90 , 219
(1955)), peptide-related substances (Am. J. Cli
n. Nutr. , 32 , 1428 (1974); Agr.
ic. Biol. Chem. , 48 , 2159 (198
4)), carrot extract (Nippon Kaika, 55 , 499 (198)
1); Chem. Pharm. Bull. , (Tokyo
o) 14 , 1191 (1966)), sugar-related substances (Tohoku Welfare Bulletin, 10 , 313 (1986)), etc. However, preparation of these growth-promoting substances for Bifidobacterium is complicated, and there is a point that the effect of selectively growing only Bifidobacterium is not always sufficient.

[0005]

DISCLOSURE OF THE INVENTION The present invention is directed to the preparation of a novel substance capable of selectively and rapidly growing only bifidobacteria, a composition for promoting the growth of bifidobacteria, and an analysis system for measuring the number of bifidobacteria using the substance. The purpose is to provide.

[0006]

Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and the present inventors have conducted extensive studies on metabolites of microorganisms that promote the growth of bifidobacteria. As a result, Propionibacterium (Pr
Bifidobacterium (Bifidobacterium)
longum, B.I. breve, B.I. adolescence
Ntis, B.N. bifidum, B. infanti
s, B. animalis, B .; pseudolong
It has been found to produce highly active growth promoting substances for um etc.). The present invention is based on this new finding.

Furthermore, when the physicochemical properties of this substance were studied in detail, it was confirmed that it was a novel substance which was unknown in the past, and its structure was also determined successfully, and an industrial production method was established. The invention was completed.

The bifidobacteria growth-promoting substance according to the present invention (hereinafter, also referred to as bifidus factor) is a novel substance having the physicochemical properties shown in the following 1 and 2 below.

[0009]

[Outer 1]

[0010]

[Outside 2]

The bifidus factor according to the present invention shows the properties of a low-molecular-weight quinone compound in view of the above-mentioned physicochemical properties. As a result of its structural determination, the bifidous factor succeeded and the formula (I ) Was obtained, and the compound was identified as a previously unknown novel compound, 2-amino-3-carboxy-1,4-naphthoquinone.

[0012]

[Chemical 2]

The novel substance 2-amino-3-carboxy-1,4-naphthoquinone according to the present invention is 2-amino-3-
It is obtained by culturing a strain having the ability to produce carboxy-1,4-naphthoquinone and collecting 2-amino-3-carboxy-1,4-naphthoquinone from the culture. It can also be manufactured by a chemical synthesis method.

2-Amino-3-carboxy-1,4-naphthoquinone (hereinafter sometimes abbreviated as the substance of the present invention)
As an example of a genus of fungi that produces a naphthoquinone derivative, there are Propionibacterium (Propionib).
enterium), Enterobacter (Entero)
Bacter), Bacillus and the like. For example, Propionibacterium fr.
eudenreichii) ATCC 6207 strain,
It is suitable as the substance-producing bacterium of the present invention.

In order to produce 2-amino-3-carboxy-1,4-naphthoquinone according to the present invention, a strain having the ability to produce the substance of the present invention is first added to a nutrient source capable of growing ordinary microorganisms. Culture aerobically or anaerobically in the medium. As the nutrient source, known ones which have been conventionally used for culturing microorganisms can be used. In particular, a medium composed of trypticase, phyton, yeast extract and glucose is preferably used.

As the culturing method, various known aerobic and anaerobic culturing methods can be used, but the aerobic or anaerobic culturing method using a liquid medium is most preferable from the viewpoint of mass production.
The culture temperature is about 20 to 40 ° C., and the pH of the medium is neutral to slightly acidic. In liquid culture, about 1 after the start of culture
After about 3 days, the substance of the present invention accumulates in the medium and cells. The culture is stopped, the culture supernatant is separated from the cells, and the cells are subjected to separation and purification of the substance of the present invention.

The method for separating and purifying the substance of the present invention is described below. First, bacterial cells are separated from the culture solution by a usual centrifugation method. Chloroform: methanol =
A 2: 1 solution is added and the active substance is extracted with stirring. Then
The extract fraction is concentrated under reduced pressure and then applied to a silica gel column. After that, when elution was carried out while increasing the concentration of ethyl acetate in n-hexane, the growth-promoting activity of Bifidobacterium was observed in the elution fraction in which the ethyl acetate concentration in n-hexane was 45 to 60%. After concentrating the eluted fraction under reduced pressure, S
Subject to gel filtration chromatography on an ephadex LH-20 column. When eluted with methanol, the growth promoting activity of Bifidobacterium is recognized at the elution position on the relatively low molecular side.

The eluate fraction is concentrated under reduced pressure and then subjected to high performance liquid chromatography using a reverse phase column.
Acetonitrile: methanol: water: acetic acid = 250: 10
When eluted with 0: 900: 0.6 (pH 5.6), the growth promoting activity of Bifidobacterium is eluted as a single peak. When the eluted fraction is concentrated under reduced pressure, a yellow powdery substance of the present invention can be obtained.

The substance of the present invention separated and purified as described above exhibits the physicochemical properties as described above. The structure was also determined, and as a result, the novel substance 2 represented by the formula (I) was obtained.
-Amino-3-carboxy-1,4-naphthoquinone.

Further, the substance of the present invention broadly includes not only the naphthoquinone derivative represented by the formula (I) but also a salt thereof. Examples of the salt of the substance of the present invention with a base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and salts with acid include hydrochloride and sulfate. Acid addition salts such as

Since the substance of the present invention has a very strong bifidobacteria growth-promoting action, as will be apparent from the following description, a composition containing the substance of the present invention is a bifidobacteria growth-promoting composition of a pharmaceutical type. , Food and drink type,
And / or can be used in the form of each of the analytical agent types, for example, by direct administration as a medicine, or by direct administration or ingestion as a nutritional food or functional food such as a food for specified health use, or In addition, by adding it to various foods (fermented milk, yogurt, etc.) and ingesting it, the intestinal flora can be improved. Further, the composition of the present invention can be used in an assay system such as Bifidobacteria count measurement.

When the composition of the present invention is used as a pharmaceutical composition, the substance of the present invention is administered in various forms.
Examples of the dosage form include oral administration such as tablets, capsules, granules, powders and syrups. These various preparations are usually used in the technical field of pharmaceutical preparation such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It is possible to formulate with known auxiliary agents.

When this preparation is applied to humans, oral administration is preferable. The therapeutically effective amount of the active ingredient varies depending on the age and condition of each patient to be treated, but in general, the active ingredient is administered in an amount of 0.01 to 1.
Oral administration of 0 mg.

Since the substance of the present invention can achieve the intended purpose by oral administration, the composition of the present invention can be used as a food and drink type. To that end, the substance of the present invention, using various auxiliary agents and other food and drink, a drink,
Various methods can be used, such as making into tablets and other various food and drink types, and adding directly to food and drink. Since the composition of the present invention in the form of food and drink as described above can be ingested over a long period of time, in addition to ordinary food and drink, it is put on the market as a food for specified health use, a nutritional supplement, a health food, etc. be able to.

The substance of the present invention is characterized by not only very high bifidobacteria growth-promoting ability, but also extremely high selectivity and specificity. Moreover, according to the present invention, the growth ability of bifidobacteria whose growth is suppressed by the presence of an antibiotic or the like is restored by adding the growth promoting substance (the substance of the present invention). Therefore, in addition to the usage of increasing the number of Bifidobacterium in the digestive tract by directly administering the substance of the present invention to humans or animals, it is added to a selective medium for Bifidobacterium analysis in foods, large intestine contents, fecal samples, etc. It is possible.

That is, as one of the systems for measuring the number of Bifidobacteria in a Bifidobacteria-containing sample, an agar medium containing a selective agent that suppresses the growth of contaminants other than Bifidobacterium was used, and the sample was placed in the medium. A method is known in which only bifidobacteria are proliferated specifically or selectively by inoculating and incubating with, and the presence or absence of bifidobacteria and the viable cell count are measured. However,
When a particularly high concentration of the selective agent is used in the selective medium to which the selective agent is added, not only the growth of contaminants other than Bifidobacterium is suppressed, but the growth of Bifidobacterium itself may also be suppressed, It was difficult to know the exact number of Bifidobacteria.

On the other hand, when the bifidobacteria growth-promoting substance of the present invention (the substance of the present invention) is added to the selective medium, the growth of bifidobacteria can be specifically promoted to form colonies, and the number of bifidobacteria can be increased. Accurate measurement is possible. Moreover, this effect can be obtained by using antibiotics such as paromomycin sulfate and neomycin sulfate as well as propionate, lithium chloride and any other known selective agent as a selective agent. The selected selective medium for Bifidobacterium assay can be effectively used for a wide range of assay applications.

[0028]

EXAMPLES Examples of the present invention will be described below.

[0029]

Example 1 Growth-promoting action of bifidobacteria: The substance of the present invention obtained in the examples described below was added to T at a concentration of 10 ng / ml.
PYG medium (8 g trypticase (BBL), 3 g phytonpeptone (BBL), 5 g yeast extract, 0.5 g L-cysteine hydrochloride, 20 g glucose, K ₂ HPO ₄
2g, KH ₂ PO ₄ 3g, MgCl ₂ · 6H ₂ O 0.5
_{g, FeSO 4 · 7H 2 O} 10mg, H 2 O 1000
ml, pH 6.5), and subsequently inoculated with various intestinal bacteria such as bifidobacteria described below, and anaerobically cultured at 37 ° C. for 20 hours by the gas pack method.

(A) Bifidobacterium longum ATCC 15707 (B) Bif. Breve ATCC 15700 (C) Bif. Adolescentis ATCC 15703 (D) Bif. Infantis ATCC 15697 (E) Bif. Bifidum ATCC 11146 (F) Clostridium perfringens ATCC 3626 ( G) Cl. Butyricum ATCC 860 (H) Cl. Ramosum ATCC 13937 (I) Enterobacter cloacae ATCC 961 (J) Escherichia coli ATCC 26 (K) Fusobacterium varium ATCC 8501 (L) Bacteroides fragilis ATCC 23745 (M) Eubacterium aerofaciens ATCC 25986 (N) Enterococcus faecalis IFO 3971 (O) Staphylococcus aureus ATCC 4012

After completion of the culture, OD _{580 of} each culture solution
(Absorbance at a wavelength of 580 nm) was measured, and Table 1 below was used.
Got the result. As a control, a TPYG medium to which the substance of the present invention was not added was inoculated with each test strain.

[0032]

[Table 1]

As shown in Table 1 above, it was confirmed that the addition of the substance of the present invention markedly promoted the growth of various bifidobacteria. On the other hand, with respect to other intestinal bacteria, the growth promoting action was extremely weak or none, and the selectivity or specificity of the substance of the present invention was demonstrated.

[0034]

Example 2: Degree of activity 1 to 0.01n of the substance of the present invention
It was added to the TPYG medium so as to be g / ml, and subsequently, Bifidobacterium (Bifidobacterium) was added.
longum ATCC 15707, Bif. bre
ve ATCC 15700, Bif. bifidum
ATCC 11146, Bif. adolescent
tis ATCC 15703, Bif. infant
is ATCC 15697) was inoculated and anaerobically cultured at 37 ° C. for 20 hours by the gas pack method, and then the OD ₅₈₀ (absorbance at a wavelength of 580 nm) of each culture solution was measured. The results are shown in Table 2 below. As a control,
The TPYG medium to which the substance of the present invention was not added was used by inoculating each of the test bacteria.

[0035]

[Table 2]

As shown in Table 2, the substance of the present invention is
It was confirmed that a very low concentration of 01 to 0.1 ng / ml exhibits a growth promoting action on various bifidobacteria.

[0037]

Example 3: Production of substance of the present invention 10 L of seed culture medium having the following composition was dispensed into 3 Erlenmeyer flasks having a volume of 5 L, and autoclave sterilization was performed at 121 ° C. for 15 minutes.
In addition to this, Propionibacterium freudenreich (P
ropionibacterium freudenr
eichii) Activation culture solution of ATCC 6207 strain 1
A seed culture was prepared by inoculating 00 ml and incubating at 30 ° C. for 3 days.

Seed medium composition (TPYG medium) Trypticase (BBL) 8 (g) Phyton peptone (BBL) 3 Yeast extract 5 L-Cysteine hydrochloride 0.5 Glucose 20 K ₂ HPO ₄ 2 KH ₂ PO ₄ 3 MgCl _{2 · 6H 2 O 0.5 FeSO 4} · 7H 2 O 0.01 purified water 1000m
l pH 6.5

In the main culture, 10 L of the seed culture prepared above was inoculated into 500 L of the above-mentioned TPYG medium sterilized at 135 ° C. for 5 seconds, and pressurized with nitrogen gas (0.5
The cells were statically cultivated at 30 ° C. for 96 hours under kg / cm ² ).
The culture broth thus obtained was subjected to continuous centrifugation to separate the culture supernatant from the cells. The obtained cells were freeze-dried to obtain 1.738 g of freeze-dried cells.

Chloroform: methanol = 2: 1 solution (15 L) was added to the dried cells obtained above, and the mixture was allowed to stand at room temperature for 30 minutes.
The substance of the present invention was extracted by stirring for a minute. The obtained extract was suction filtered, and the filtrate was concentrated under reduced pressure to obtain 22.8 g of an extract. This was applied to a silica gel column (column length 70 cm, column diameter 60 cm; Wako Gel C-300 manufactured by Wako Pure Chemical Industries, Ltd.), and elution was carried out while increasing the ethyl acetate concentration in n-hexane, and ethyl acetate in n-hexane was obtained. Bifidobacteria growth-promoting activity was observed in the eluted fractions with a concentration of 45-60%.

The elution fraction was concentrated to dryness under reduced pressure,
Dissolve in 8 ml of methanol, then Sephadex L
H-20 column (column length 70 cm, column diameter 2.6 c
m; manufactured by Pharmacia) and subjected to gel filtration chromatography. Methanol as the eluent, flow rate 36 ml
When eluted at / hr, eluate volume 497-539 ml
The bifidobacteria growth-promoting activity was observed in the fractions. This elution fraction was concentrated under reduced pressure.

Next, a reversed phase column (column length 250 mm,
Column diameter 20 mm; Shiseido Co., Ltd. CAPCELL PAK
High performance liquid chromatography using C ₁₈ ) was performed. Acetonitrile: methanol: water: acetic acid = 250:
The eluent is 100: 900: 0.6 (pH 5.6),
Elution at a flow rate of 10 ml / min gave a retention time of 38
Bifidobacteria growth-promoting activity was observed in the eluted fractions before and after the minute. The eluted fraction was concentrated under reduced pressure to obtain 7.1 mg of the present substance as a yellow powder. Its physicochemical properties are as described above, and its chemical structure was determined as in formula (I).

[0043]

Example 4 50 g of granulated sugar, 100 g of an equal mixture of corn starch and lactose, the substance 1 of the present invention obtained in Example 3
00 mg was added and mixed thoroughly. The mixture was divided into 100 equal parts and packed in a bag, and 100 bags of 1.5 g of stick-shaped Bifidus factor nutritional health food were manufactured.

[0044]

Example 5 The following formulations (1) to (4) were prepared. (1) 1 g of the substance of the present invention obtained in Example 3, (2) lactose 140 g, (3) corn starch 29 g, (4) magnesium stearate 1 g.

First, (1), (2), (3) (however, 17
g) were mixed and granulated with the paste prepared from (3) (but 7 g). In the obtained granules, (3) (however, 5
g) and (4) were added and mixed well, and this mixture was compressed by a compression tablet machine to produce 1000 tablets each containing 1 mg of the substance of the present invention.

[0046]

EFFECTS OF THE INVENTION The bifidos factor consisting of the naphthoquinone compound according to the present invention is excellent in the effect of promoting the growth of bifidobacterium. It is extremely effective in increasing the amount of Bifidobacterium.

Furthermore, the bifidous factor according to the present invention is
Not only is it excellent in the growth-promoting effect of bifidobacteria, but it has strong selectivity or specificity and no growth effect on other contaminants.Therefore, when this bifidus factor is added to the bifidobacterial selection medium, A colony consisting of only bacteria is rapidly formed, which can be advantageously used for measuring the viable cell count of Bifidobacterium and various other assays.

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of 2-amino-3-carboxy-1,4-naphthoquinone dissolved in methanol to a concentration of 10 μg / ml.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // A61K 35/74 A 7431-4C (C12P 13/04 C12R 1:01) (72) Inventor Takeo Nao 540 Narita, Odawara, Kanagawa, Meiji Dairy Co., Ltd.Health Science Laboratories (72) Inventor Yoshiro Sato, 540, Narita, Odawara, Kanagawa Meiji Dairy Co., Ltd., Health Science Laboratories

Claims (9)

[Claims] 1. A compound having the formula (I) represented by the following chemical formula 1.
-Amino-3-carboxy-1,4-naphthoquinone. [Chemical 1] 2. 2-amino-3-represented by formula (I)
A bifidobacteria growth-promoting composition comprising carboxy-1,4-naphthoquinone as an active ingredient. 3. The composition according to claim 2, wherein the bifidobacteria growth-promoting composition is a bifidobacteria growth-promoting food or drink. 4. The composition according to claim 2, wherein the bifidobacteria growth-promoting composition is a bifidobacteria growth-promoting agent. 5. The composition according to claim 2, wherein the bifidobacteria growth promoting composition is a bifidobacterial selective agent. 6. 2-amino-3-carboxy-1,4-
Production of 2-amino-3-carboxy-1,4-naphthoquinone represented by formula (I), which comprises culturing a naphthoquinone-producing microorganism, producing the naphthoquinone in the culture, and collecting the naphthoquinone. Law. 7. A propionibacterium (Propionibacterium) as the naphthoquinone-producing bacterium.
m), Enterobacter
The method according to claim 6, wherein a microorganism selected from the group consisting of the genus Bacillus and the genus Bacillus is used. 8. A Propionibacterium freuich (Propionibacterium freu)
The method according to claim 7, characterized in that denreichii) is used. 9. A selective medium for analysis of bifidobacteria, comprising the composition according to claim 5 added.