JP2016020339A - 1,4-dihydroxy-2-naphthoic acid derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stable novel compound having bifidobacteria growth action.SOLUTION: The present invention relates to a compound represented by formula (I), where Rand Rindependently represent a hydrogen atom or a group which can be hydrolysed by an enzyme in vivo; and at least one of Rand Ris not a hydrogen atom, or salt thereof.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a 1,4-dihydroxy-2-naphthoic acid derivative having a bifidobacteria proliferating action effective in improving intestinal flora. More specifically, the present invention relates to a stabilized 1,4-dihydroxy-2-naphthoic acid (DHNA) derivative. More specifically, 1) a sulfate ester of a DHNA hydroxyl group, a glucuronidation compound and a glucosylated compound of a DHNA hydroxyl group, 2 ) It relates to pharmaceuticals and foods containing these compounds.

1,4-dihydroxy-2-naphthoic acid (1,4-dihydroxy-2-naphthoic acid: DHNA) has been conventionally used as an industrial material such as dyes, pigments and photosensitive materials. In recent years, DHNA was discovered from propionic acid bacteria cultures and reported to be bifidobacterial growth factors and vitamin K precursors (Patent Documents 1 and 2, Non-Patent Document 1), and purified and concentrated from propionic acid bacteria cultures. The obtained DHNA fraction is effective for improvement of intestinal flora, prevention of metabolic bone disease, etc. (Patent Document 3), besides, IBS (inflammatory bowel disease), Helicobacter pylori eradication, allergy, osteoporosis It is considered to be effective in the field of health food and medicine.
However, since DHNA is easily decomposed by oxidation and is unstable, in order to prevent its decomposition, (1) foods and drinks to which DHNA fraction is added (beverages including dairy products, lactic acid bacteria beverages, vegetable juices, fruit juices, nutritional beverages, etc. ), Gelled food (yogurt, etc.)) and the like have been required to completely remove dissolved oxygen by nitrogen substitution or the like, or (2) coexisting an antioxidant (Patent Document 4).
In addition, since conventional DHNA is easily degraded particularly at pH 7 or higher, it is easily degraded during the movement of the gastrointestinal tract from the duodenum to the small intestine and from the small intestine to the large intestine. there were.
Thus, in order to stabilize DHNA, it does not require a complicated operation of removing oxygen during processing of food and drink, and is not accompanied by addition of an antioxidant that impairs the taste of food and drink. There is no known DHNA that can be reached without being broken down.

JP-A-8-98677 WO 01/28547 WO 03/016544 WO 2004/085364

Isawa K, et al, Isolation and identification of a new bifidogenic growth stimulator produced by propionibacterium freudenreichii ET-3, Biosci. Biotechnol. Biochem., 66, 679-681 (2002)

  An object of the present invention is to provide a stable compound having a bifidobacterial proliferating action, and a medicine and food containing them, which are useful for improving intestinal flora.

  In order to solve the above problems, the present inventors have conducted intensive research,

  Formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo; at least one of R ¹ and R ² is not a hydrogen atom. ]
Has been found to have an excellent bifidobacteria-proliferating action, is useful for improving intestinal flora, and is stable.

That is, the present invention is as follows.
[1] Formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo; at least one of R ¹ and R ² is not a hydrogen atom. ]
Or a salt thereof.
[2] The compound or salt thereof according to [1], wherein the group capable of being hydrolyzed by an enzyme in vivo is a glycosyl group or an oxo acid group.
[3] A group that can be hydrolyzed by an enzyme in vivo,

[In the formula, * represents a binding site. ]
The compound or a salt thereof according to [1], which is a group selected from the group consisting of:
[4] A group capable of being hydrolyzed by an enzyme in vivo is

[In the formula, * represents a binding site. ]
The compound or a salt thereof according to [1], which is a group selected from the group consisting of:
[5] A medicament comprising the compound or salt thereof according to any one of [1] to [4].
[6] The medicament according to [5], which is a bifidobacteria growth agent.
[7] The medicament according to [5], which is an intestinal flora improving agent.
[8] A food comprising the compound or salt thereof according to any one of [1] to [4].
[9] 1,4-dihydroxy-2-naphthoic acid (DHNA) is added to a plant tissue culture medium and cultured. Formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo; at least one of R ¹ and R ² is not a hydrogen atom. ]
Or a salt thereof.
[10] The production method according to [9], wherein the plant expresses a glycosyltransferase.
[11] The production method according to [9] or [10], wherein DHNA is derived from a culture solution obtained by culturing a microorganism that produces DHNA.

According to the present invention, the compound of formula (I) has an excellent bifidobacterial growth action, and therefore improves intestinal flora, and is effective in preventing and improving constipation and diarrhea, and preventing colorectal cancer.
The compounds of the present application have the effect of increasing bifidobacteria and activating and adjusting immunity, so they are effective in preventing and improving hay fever, improving atopic dermatitis, preventing cancer, lowering cholesterol, and improving high blood pressure. .
Furthermore, according to the present invention, it is effective in preventing or improving osteoporosis and the like.
Further, according to the present invention, when producing food and drink, etc., it can be easily added without going through complicated steps, and an antioxidant or the like that affects the flavor and taste of the food or drink is added. It can be manufactured without.
According to the present invention, since it reaches the large intestine without being decomposed, it is possible to administer DHNA at a small dose, and it is easy to take and safely administer to children and the elderly.

FIG. 1 shows UV spectra of DHNAS (compound represented by the following (a1)). Na, DHNAG (compound represented by the following formula (c1)) and DHNAg (compound represented by the following (d1)). . FIG. 2 shows a comparison of the room temperature stability of DHNA (1,4-dihydroxy-2-naphthoic acid), DHNAS, DHNAG and NHNAg at pH 7.4, pH 3.0 phosphate buffer, and water. Show. The vertical axis represents each concentration when the initial concentration is 100, and the horizontal axis represents time (hour or day). FIG. 3 shows the results of HPLC analysis of metabolites in the urine of mice administered with DHNA. FIG. 4 shows the results of HPLC analysis of metabolites in the urine of mice administered with DHNAS. FIG. 5 shows the results of HPLC analysis of metabolites in the urine of mice administered with DHNAg.

Hereinafter, the present invention will be described in detail.
The present invention provides a compound of formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo; at least one of R ¹ and R ² is not a hydrogen atom. ]
Or a salt thereof (hereinafter also referred to as the compound of the present invention or compound (I)).

In the present specification, R ¹ and R ² each independently represent a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo (hereinafter sometimes abbreviated as a specific group), and R ¹ and R ² At least one is not a hydrogen atom.
The group capable of being hydrolyzed by an enzyme in vivo is not particularly limited as long as it is hydrolyzed by an enzyme in vivo when Compound (I) is administered.

[In the formula, * represents a binding site. ]
Glycosyl group (monosaccharide glycoside, disaccharide glycoside, oligosaccharide glycoside), or the like

[In the formula, * represents a binding site. ]
Oxo acid groups such as

[In the formula, * represents a binding site. ]
Is preferred,

[In the formula, * represents a binding site. ]
Is more preferable.

  Examples of enzymes in the living body include enzymes present in the intestine. For example, a sulfatase that hydrolyzes a sulfated compound (DHNAS), a β-glucuronidase that hydrolyzes a β-glucuronidated compound (DHNAG), and also a phosphated compound (DHNAP) Examples thereof include phosphatase, β-glucosidase that hydrolyzes β-glucosylated DHNA, and β-galactosidase that hydrolyzes β-galactosylated DHNA. Of these, sulfatase, β-glucuronidase and phosphatase are preferable, and sulfatase and β-glucuronidase are more preferable.

  The enzyme may be derived from bacteria, fungi, plants, or animals, but is preferably an enzyme in the body of an animal such as a human.

In compound (I), both R ¹ and R ² may be specific groups, and R ¹ and R ² may be the same or different.
Further, either R ¹ or R ² may be a specific group. In this case, although not particularly limited, R ² is a specific group from the viewpoint of easiness of synthesis because —OH at the 1-position is inactive due to a hydrogen bond with —COOH at the 2-position. It is preferable.

  As preferable specific examples of the compound represented by the formula (I),

The compound etc. which are represented by these are mentioned,
More preferably,

And more preferably

It is.

  Examples of the salt of the compound represented by the formula (I) include pharmacologically acceptable salts such as metal salts with sodium, potassium, magnesium, calcium and the like; trimethylamine, triethylamine, pyridine, picoline, Salts with organic bases such as N-methylpyrrolidine, N-methylpiperidine, N-methylmorpholine; trifluoroacetic acid, acetic acid, lactic acid, succinic acid, maleic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, benzoic acid, Examples include methanesulfonic acid, p-toluenesulfonic acid, cinnamic acid, fumaric acid, phosphonic acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, acid addition salts with acids such as sulfuric acid, and the like. .

The compound represented by the formula (I) may be a crystal.
The crystal of the compound represented by the formula (I) can be produced by crystallization by applying a crystallization method known per se to the compound represented by the formula (I). When the compound represented by the formula (I) is a crystal, it is included in the compound represented by the formula (I) whether the crystal form is single or a crystal form mixture.

The compound represented by the formula (I) may be labeled with an isotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, etc.) and the like.
Furthermore, the compound represented by the formula (I) may be a hydrate, a non-hydrate, a non-solvate or a solvate.
Furthermore, a deuterium converter in which ¹ H is converted to ² H (D) is also included in the compound represented by formula (I).

  As the compound represented by the formula (I) or a salt thereof, any of chemical synthesis methods, natural products derived from animals and plants, those obtained by fermentation methods or gene recombination methods may be used.

  Compound (I) can be produced using a known chemical synthesis method.

For example, the compound (I) in which R ¹ and / or R ² is an oxo acid group includes 1,4-dihydroxy-2-naphthoic acid and a known oxo acid esterifying agent such as a sulfating agent or a phosphorylating agent. It can be obtained by reacting.
Examples of the sulfating agent include trimethylamine sulfur trioxide complex, triethylamine sulfur trioxide complex, dimethylformamide sulfur trioxide complex, pyridine sulfur trioxide complex, and chlorosulfonic acid.
As the phosphorylating agent, for example, phosphoric anhydride, orthophosphoric acid, polyphosphoric acid, phosphorus pentoxide and the like are used.
The amount of a known oxo acid esterifying agent such as a sulfating agent or phosphorylating agent is usually 1 to 10 molar equivalents relative to 1,4-dihydroxy-2-naphthoic acid.
The above reaction is usually performed in a solvent. Examples of the solvent include dichloromethane, chloroform, diethyl ether, diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran, dioxane, dimethoxyethane, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, and the like. Usually, it is 0 degreeC-100 degreeC, and reaction time is 0.1 to 24 hours normally.

On the other hand, for example, the compound (I) in which R ¹ and / or R ² is a β-glycosyl group has a hydroxy group at the 1-position as a leaving group with respect to a known sugar molecule, and other active groups (hydroxy groups). Carboxy group) is reacted with a carboxylic acid protector of 1,4-dihydroxy-2-naphthoic acid in the presence of an activator such as a Lewis acid or a metal salt. The product obtained can be obtained by subjecting it to a deprotection by a known method.
Examples of the leaving group include a halogen atom, an acetyloxy group, an imidate group, an alkoxy group, an alkylsulfanyl group, a phenylsulfanyl group, and a pyridylsulfanyl group.
Examples of the hydroxy-protecting group include acyl groups such as acetyl group and benzoyl group. Examples of the protecting group for the carboxy group include alkyl groups such as a tert-butyl group and a methyl group.
As the protected sugar fragment, a commercially available product may be used, or it may be obtained by a known method.
The amount of the protected sugar fragment used is usually from 0.1 to 10 molar equivalents relative to the 1,4-dihydroxy-2-naphthoic acid protected carboxylic acid.
Examples of the protected carboxylic acid of 1,4-dihydroxy-2-naphthoic acid include those protected with the protecting group of the carboxy group shown above, specifically, 1,4-dihydroxy-2 -Tert-butyl naphthoate, methyl 1,4-dihydroxy-2-naphthoate and the like. Moreover, as the carboxylic acid protector of 1,4-dihydroxy-2-naphthoic acid, one protecting one of the hydroxy groups may be used as necessary.
Examples of the activator include silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver perchlorate, silver chloride, silver bromide, silver silver iodide, silver silicate, and boron tetrafluoride. Silver acid, silver zeolite, stannous chloride, stannous bromide, stannous iodide, stannic chloride, stannic bromide, stannic iodide, tetraethylammonium bromide, tetraethylammonium chloride, trimethylsilyl Examples thereof include trifluoromethanesulfonate, etheric boron trifluoride, silicon tetrafluoride and the like.
The usage-amount of an activator is 0.01 mol equivalent to 100 mol equivalent normally with respect to the carboxylic acid protector of 1, 4- dihydroxy-2- naphthoic acid.
The glycosylation reaction is usually performed in a solvent. Examples of the solvent include dichloromethane, 1,2-dichloroethane, diethyl ether, benzene, toluene, acetonitrile, and the like. The reaction temperature is usually 0 ° C. to 100 ° C., and the reaction time is usually 0.1%. ~ 24 hours.
The ratio of α-form and β-form of the product obtained by glycosylation varies depending on the reagent used and reaction conditions.
When the protective group as exemplified above is used, the deprotection reaction of the product obtained by the glycosylation is performed by treating with a base such as potassium hydroxide, sodium hydroxide or sodium methoxide. It can be done easily. The amount of the base used is usually 0.01 molar equivalent to an excess amount with respect to the product obtained by glycosylation.
The deprotection reaction is usually performed in a solvent. Examples of the solvent include water, methanol, ethanol, hexane, heptane, dichloromethane, chloroform, tetrahydrofuran, dioxane and the like. The reaction temperature is usually 0 ° C. to 100 ° C., and the reaction time is usually 0.00. 1 to 24 hours.

  The compound obtained by the above method is isolated and purified from the reaction solution by a known separation and purification means such as concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, phase transfer, chromatography, etc. You may use for reaction.

  As the starting material for the production of the compound represented by the formula (I) or a salt thereof, 1,4-dihydroxy-2-naphthoic acid or a protected form thereof may be a commercially available one, or a known method Can also be obtained by

The compound represented by the formula (I) or a salt thereof includes those derived from a culture solution obtained by culturing microorganisms or plant tissues. DHNAS, DHNAG, and the like can be isolated from the culture medium of cultured microorganisms that express sulfotransferase (ST), UDP-glucuronosyltransferase (UGT), and the like. In addition, DHNAS, DHNAG, and the like can be obtained in high yield by adding DHNA to the medium. Further, when DHNA is added to a tissue culture medium of a plant expressing a glycosyltransferase (an enzyme involved in glucosylation, galactosylation, glucuronidation, etc.), glycosyl-DHNA is obtained.
Examples of bacteria that produce DHNA include Propionibacterium, Enterobacter, Sporolactobacillus, Bacillus, and the like. By introducing an ST or UGT expression vector, DHNAS, DHNAG and the like can be obtained in high yield. Among them, it is preferable to use a Propionibacterium genus. Furthermore, the above-mentioned compound can also be obtained by mixed culture of a DHNA-producing strain and an ST, UGT expression strain or glycosyltransferase expression cell strain.

Plant tissue culture is performed according to a conventional method.
The plant tissue is not limited as long as it is a plant expressing a glycosyltransferase, and examples thereof include tissues such as tobacco, Arabidopsis thaliana, eucalyptus, pokeweed and periwinkle.
As a medium used for plant tissue culture, a medium known in this field is usually used.
As components contained in the medium, glucose, sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid, etc. that are usually used for cultivation of plant tissues are used as carbon sources, and urea, ammonia, ammonium sulfate are used as nitrogen sources. , Ammonium chloride, nitrate, etc. are used. As the phosphoric acid and potassium source, potassium phosphate, ammonium phosphate, potassium chloride and the like are used. As other trace metals, inorganic salts such as Mg, Ca, Fe, Na, K, Mn, Co, Cu, and Zn are effective. Further, if necessary, organic substances such as corn steep liquor, casein, yeast extract and peptone may be added.
For example, examples of the medium include MS medium, Kano medium, and B5 medium.
Moreover, the culture medium which modified | changed the said culture medium may be sufficient.

The amount of DHNA added to the plant tissue culture medium is, for example, added to the medium so that the final concentration is 100 to 400 μM.
The DHNA to be added may be obtained by culturing the DHNA-producing bacterium by a conventional method, causing the bacterium to produce DHNA, and adding the culture solution as it is to the plant tissue culture medium. Culture conditions etc. are described in Furuichi K, et al., Enhancement of 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3 fed-batch culture, Appl Environ Microbiol, 73, 3137-3143 (2007). Follow.

  The pH of the medium may be neutral, weakly basic, or weakly acidic. Usually, the pH is 4.5 to 6.5.

  The culture temperature is usually 6 to 35 ° C, preferably 20 to 35 ° C, more preferably 25 to 32 ° C. Under temperature conditions of 6 to 35 ° C., it is desirable for tissue culture of plants.

  Pre-culture is preferably performed prior to the culture.

  The culture time of the main culture may vary depending on the cell concentration of the preculture medium and the amount of the preculture liquid relative to the amount of the medium of the main culture, but after entering the logarithmic growth phase, a new medium and DHNA are added for about 15 to 24 hours. It is. The culture is carried out by a batch culture method, a semi-batch culture method or a continuous culture method with standing, shaking, stirring, or aeration.

After completion of the culture, the compound (I) or a salt thereof is obtained by extraction and purification from a plant tissue or culture solution by a conventional method.
In addition, a method for obtaining DHNAS, DHNAG, and the like from the urine of a DHNA-administered animal is possible.

A medicament comprising the compound (I) of the present invention or a salt thereof is also encompassed by the present invention (hereinafter also referred to as the medicament of the present invention).
The medicament of the present invention can be administered orally or parenterally with the compound (I) of the present invention or a salt thereof as it is or in combination with a pharmacologically acceptable carrier.

  The pharmaceutical preparation of the present invention can be administered orally, for example, as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules and microcapsules), and syrups. Examples of dosage forms for parenteral administration include injections, infusions, drops, suppositories, and the like. It is also effective to make a sustained-release preparation by combining with an appropriate base.

  As a method for producing the pharmaceutical of the present invention into the above-mentioned dosage form, a known production method generally used in the art can be applied. In addition, when manufacturing into the above-mentioned dosage forms, excipients, binders, disintegrants, lubricants, sweeteners, interfaces commonly used in the pharmaceutical field when manufacturing into the dosage forms, if necessary. An activator, a suspending agent, an emulsifier and the like can be produced by appropriately containing appropriate amounts.

  For example, when the medicament of the present invention is produced into tablets, it can be produced by containing excipients, binders, disintegrants, lubricants, etc., and when produced into pills and granules. , Excipients, binders, disintegrants and the like. In addition, excipients and the like are used for powders and capsules, sweeteners and the like are used for syrups, and suspending agents and surfactants are used for emulsions and suspensions. It can be produced by adding an emulsifier and the like.

  Examples of excipients include lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, licorice powder, mannitol, sodium bicarbonate, calcium phosphate, calcium sulfate and the like.

  Examples of the binder include 5 to 10% by weight starch paste, 10 to 20% by weight gum arabic solution or gelatin solution, 1 to 5% by weight tragacanth solution, carboxymethyl cellulose solution, sodium alginate solution, glycerin and the like.

  Examples of disintegrants include starch and calcium carbonate.

  Examples of the lubricant include magnesium stearate, stearic acid, calcium stearate, purified talc and the like.

  Examples of sweeteners include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, simple syrup and the like.

  Examples of the surfactant include sodium lauryl sulfate, polysorbate 80, sorbitan monofatty acid ester, polyoxyl 40 stearate and the like.

  Examples of the suspending agent include gum arabic, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, bentonite and the like.

  Examples of emulsifiers include gum arabic, tragacanth, gelatin, polysorbate 80 and the like.

  Furthermore, when the medicament of the present invention is produced in the above-mentioned dosage form, an appropriate amount of a coloring agent, a preservative, a fragrance, a flavoring agent, a stabilizer, a thickener, etc., which are usually used in the pharmaceutical field, are added as desired. can do.

  The dose for oral administration of the medicament of the present invention varies depending on the sex, symptom, age, and administration method of the patient to be administered. Usually, the daily dose of the compound of the present invention per kg of adult weight is 0 0.003 μg to 3 μg, preferably 0.03 μg to 3 μg, and more preferably 0.1 μg to 1 μg. The daily dose can be administered at once or in several divided doses. Before meals, after meals, and between meals are not considered, but before meals are preferred. The administration period is not particularly limited.

When the pharmaceutical of the present invention is administered parenterally, it is usually administered in the form of a liquid (for example, an injection). The single dose varies depending on the administration subject, symptom, administration method, and the like. For example, in the form of an injection, an appropriate amount can be administered by intravenous injection in consideration of the oral dose.
In addition to intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous infusions, and the like are included as injections, and sustained-release preparations include iontophoretic transdermal agents and the like. Such an injection is prepared by a method known per se, that is, by dissolving, suspending or emulsifying the compound of the present invention in a sterile aqueous or oily liquid.
Aqueous solutions for injection include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, and suitable solubilizers such as alcohol (For example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant (for example, polysorbate 80, HCO-50) and the like may be used in combination.
Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate, benzyl alcohol or the like as a solubilizer.
Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (For example, benzyl alcohol, phenol, etc.) may be blended.

When the medicament of the present invention is administered (applied) as a transdermally absorbable external preparation, known additives and the like are mixed as necessary, and creams, solutions, lotions, emulsions, tinctures are prepared in a conventional manner. And ointments, aqueous gels, oily gels, aerosols, powders, shampoos, soaps, and other external preparations.
The compound of the present invention can be made into a quasi drug or the like as it is or by adding an appropriate additive.

  In addition to the above components, the above external preparation includes a water-soluble component, an oily component, a powder component, a surfactant, a polymer component, a thickener, a tackifier, a film forming agent, a pH adjuster, an antioxidant, Preservatives, preservatives, excipients, moisturizers, skin protectants, refreshing agents, fragrances, coloring agents, chelating agents, lubricants, antipruritic agents, blood circulation promoters, astringents, tissue repair promoters, antiperspirants In addition, additives necessary for plant extract ingredients, animal extract ingredients, cosmetics, quasi drugs, and the like can be blended as necessary.

  The medicament of the present invention is useful for mammals (eg, humans, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc.) as bifidobacteria proliferating agents or intestinal flora improving agents. .

The bifidobacteria proliferating agent of the present invention can increase the amount of intestinal bifidobacteria, thereby being effective in the prevention and improvement of constipation and diarrhea, and prevention of colorectal cancer, and preferably applied to the prevention and improvement of constipation Is done.
The intestinal flora improving agent of the present invention improves intestinal flora, activates and regulates immunity, prevents and improves hay fever, improves atopic dermatitis, cancer prevention, lowers cholesterol, improves hypertension It is effective for.
Intestinal flora refers to this microbial community, which is a complex microbial ecosystem composed of various and diverse bacteria that are constantly growing in the intestinal tract of mammals such as humans. “Improvement of flora” means that bacteria that act harmful to humans (bad bacteria) are kept inferior and bacteria that work useful to humans (good bacteria) become dominant.

  The compound of the present invention is combined with drugs such as an intestinal regulating agent, digestive agent, antidiarrheal agent, antiallergic agent, anti-inflammatory agent, antihistamine and the like for the purpose of enhancing the action of the compound or reducing the dose of the compound. Can be used. At this time, the administration timing of the compound of the present invention and the concomitant drug is not limited, and these may be administered simultaneously to the administration subject or may be administered with a time difference. Furthermore, the compound of the present invention and the concomitant drug may be administered as two types of preparations containing each active ingredient, or may be administered as a single preparation containing both active ingredients.

  A food containing the compound of the present invention or a salt thereof is also included in the present invention (hereinafter also referred to as the food of the present invention). The food of the present invention may be a food consisting only of a culture solution obtained by culturing bacteria or cells producing DHNAS, DHNAG, glucosyl-DHNA (DHNAg) and the like.

  The food of the present invention may be any general food form containing the compound of the present invention. For example, it can be used as a drink agent, for example, a soft drink or a powdered drink, by itself or by adding an appropriate flavor thereto. Specifically, it can be mixed with juice, milk, confectionery, jelly, etc. to eat and drink. It is also possible to provide such food as a health functional food. This health functional food includes foods and drinks that are labeled for use in the present invention such as bifidobacteria growth and intestinal flora improvement, particularly foods for specified health use, nutritional functional foods, and the like.

  Furthermore, the food of the present invention can be used as a food supplement. When used as a food supplement, it can be prepared in the form of tablets, capsules, powders, granules, suspensions, chewables, syrups and the like. The food supplement in the present invention refers to those taken for the purpose of supplementing nutrition in addition to those taken as food, and includes nutritional supplements, dietary supplements, and the like.

When the compound of the present invention is ingested as a food, the daily intake of the compound of the present invention per kg of adult weight is 0.003 μg to 3 μg, preferably 0.03 μg to 3 μg, more preferably 0.1 μg. ~ 1 μg.
It is preferable to take the above amount once a day or several times a day before meals. In this case, the daily intake or the intake per time can be made into one unit packaging.

  A method for producing a compound represented by the above formula (I), wherein DHNA is added to a plant tissue culture medium and cultured, is also encompassed in the present invention. The plant tissue preferably expresses glycosyltransferase. Various definitions are as described above.

  A compound represented by the above formula (I) or a salt thereof obtained by adding 1,4-dihydroxy-2-naphthoic acid (DHNA) to a plant tissue culture medium and culturing is also encompassed in the present invention. Various definitions are as described above.

  EXAMPLES Hereinafter, although a manufacture example and a test example are given and this invention is demonstrated in more detail, a test example etc. is described for description of this invention, and does not limit this invention.

(Production Example 1) Synthesis method of DHNAS · Na (Na salt of the compound represented by the above formula (a1)) 1,4-dihydroxy-2-naphthoic acid (200 mL of four-necked Kolben under argon atmosphere) To a DMF solution (50 mL) of Tokyo Kasei) (10.35 g, 50.69 mmol), a pyridine-sulfur trioxide complex (Wako Pure Chemical Industries) (10.72 g, 67.35 mmol) was added, and the internal temperature was 25 to 28 ° C. For 4 hours. The reaction solution was directly concentrated under reduced pressure at a bath temperature of 45 ° C., and AcOEt / MeOH (5/1 v / v) (100 mL) was added to the resulting residue to obtain a uniform solution. The solution was ice-cooled, and the precipitated solid was collected by filtration, washed with cold AcOEt / MeOH (5/1 v / v) (20 mL, x2), and dried under reduced pressure to obtain a crude DHNAS. This crude product was suspended in purified water (50 mL), and saturated aqueous sodium hydrogen carbonate was added to the homogeneous solution with stirring. Activated carbon (2 g) was added, and the mixture was stirred for 30 minutes, and then filtered off. The filtrate was eluted with water using a strongly acidic cation exchange resin Dowex 50Wx8 (Na) (500 mL) prepared in advance. Fractions containing the desired product were collected and concentrated under reduced pressure at a bath temperature of 45 ° C. After adding EtOH-toluene to the obtained residue, the operation of concentrating under reduced pressure was performed twice. Acetone (50 mL) was added to the residue, and the mixture was heated and suspended at 40 ° C. for 30 minutes, followed by ice cooling. The precipitated solid was collected by filtration, washed with acetone (10 mL, x2), and then dried under reduced pressure at 50 ° C. to isolate 13.54 g (yield 73%) of DHNAS · Na in acetone as a white solid as a white solid. did. The acetone adduct (13.53 g) was dissolved in water (300 mL) and concentrated under reduced pressure. The obtained aqueous solution was freeze-dried to obtain 11.61 g (yield: 73%) of DHNAS · Na as a light brown solid (assay by HPLC, purity> 99.9%).
The UV spectrum of the obtained DHNAS · Na is shown in FIG. The UV spectrum was measured using an aqueous solution containing methanol (12%), acetonitrile (20%), and ammonium formate (8 mM) as a solvent.
The NMR data of the obtained DHNAS · Na are shown in Tables 1 and 2. Signal assignment was performed by two-dimensional NMR methods such as correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC). As for the binding position (C-1 or C-4) of the sulfate ester, the 13C chemical shift of C-4 was calculated with simulation software. Since it was closer to the measured value (137.8ppm) than the attached value (149.7ppm), it was judged that it was bound to C-4.
Mass spectrum of the obtained DHNAS · Na; MH peak was observed at 305 [DHNAS · Na (MW = 306) -1 = 305] by FAB Mass (negative mode).

(Production Example 2) Method for producing DHNAG (compound represented by the above formula (c1))
One female mouse (7 weeks old) of ICR strain was orally administered 60 mg / 200 μL (2335 mg / Kg (1% tween 80 aqueous solution)) of DHNA once, then transferred to a metabolic cage for 3 days every 24 hours after administration. Urine and feces were collected to the eyes to obtain metabolic excreta. Metabolic excreta was analyzed, and M1 was fractionated to obtain DHNAG as shown in FIG.
The UV spectrum of the obtained DHNAG is shown in FIG. The UV spectrum was measured using an aqueous solution containing methanol (12%), acetonitrile (20%), and ammonium formate (8 mM) as a solvent.
The NMR data of the obtained DHNAG are shown in Tables 1 and 2.
Signal assignment was performed by a two-dimensional NMR method such as correlation spectroscopy (COSY) or heteromultiple bond correlation (HMBC). In HMBC, since H-1 ′ has a correlation with C-4, the binding position of the glucuronyl group was determined to be C-4.

(Production Example 3) Production method of DHNAg (compound represented by the above formula (d1)) Tobacco BY-2 cells (obtained from the RIKEN BioResource Center; culture is performed according to the BY-2 culture method designated by the center except for pH 5.5) 1 ml of the suspension was inoculated into 95 ml of MS medium ^* using a 300 ml Erlenmeyer flask and aerobically cultured at 26.5 ° C. in the dark for 3 days (shaking 100 times per minute). It was. When normal growth was confirmed, it was further diluted twice with MS medium and cultured for 18 hours. Then, 25 ml was transferred to a new 200 ml flask, 25 ml of MS medium was added, and cultured for 6 hours under the same conditions. 2.04 mg (chemical conversion) was added (100 μl of DHNA solution in DMSO (20.4 mg / ml), final concentration 200 μM). After culturing for 18 hours, a part of the culture solution was collected and centrifuged (150,000 rpm), and 20 μl of the supernatant was injected into HPLC ^** to analyze glucosyl-DHNA (retention time 16.5 minutes) (FIG. 5). As a result, it was found that β-glucosyl-DHNA (DHNAg) was produced from DHNA with a yield of 10-17% (molecular weight was confirmed to be 366 by FAB mass; it was hydrolyzed by β-glucosidase. Make sure). The UV spectrum and NMR data of DHNAg are shown in FIG. In HMBC of NMR, since H-1 ′ has a correlation with C-4, the binding position of the glucosyl group was determined to be C-4.
^* MS medium: Mixed salt for Murashige and Skog (WAKO), sucrose (30 g / l), KH ₂ PO ₄ (200 μg / ml), vitamin [Myo-inositol (100 μg / ml), Thiamin-HCl (1 μg / ml) )] And 2,4-dichlorophenoxyacetic acid (200 μg / l), adjusted to pH 5.5.
^** HPLC conditions: column, Shiseido CAPCELL PAK C18, 4.6 × 250 mm, 5 μm); elution, A: 15% MeOH, 10 mM ammonium formate, B: acetonitrile, A → 30% B (20 min) gradient; flow rate, 1 ml / min Temperature, 40 ° C

(Production Example 4) Production method by culturing glycosylated DHNA
Furuichi K, et al., Enhancement of 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3 fed-batch culture, Appl Environ Microbiol, 73, 3137-3143 (2007) Amberlite XAD-2 (Aldrich Chemical Company, Inc,) is a culture solution of Propionibacterium, Enterobacter, Sporolactobacillus, and Bacillus. USA) Introduce into a column to adsorb low molecular weight compounds in the culture. After thoroughly washing the column with deionized water, the low molecular weight compound is eluted with ethanol. The obtained ethanol solution is concentrated to dryness, the residue is dissolved in DMSO, and a certain amount thereof is added to a medium of plant culture cells such as tobacco, Arabidopsis thaliana, eucalyptus, pokeweed and periwinkle. After culturing for 15 to 24 hours, the culture solution is introduced into an Amberlite XAD-2 column to adsorb the produced glycosylated DHNA. The column is thoroughly washed with deionized water, and then glycosylated DHNA is eluted with an aqueous solution containing 10 → 60% ethanol by gradient (or stepwise increase in ethanol concentration). The β-glucosyl-DHNA compound is eluted at 40-60% ethanol concentration.

(Test Example 1)
Comparison of stability of DHNA, DHNAS, DHNAG and DHNAg DHNA, DHNAS obtained in Production Example 1, DHNAG obtained in Production Example 2 and DHNAg obtained in Production Example 3 were mixed with 0.1 M phosphate buffer (pH 7). .4) Each solution was prepared by adding the solution, 0.1 M phosphate buffer (pH 3.0) solution and water to a final concentration of 25 μg / ml. Each solution was allowed to stand at room temperature (23 ° C.), and the DHNA concentration was measured by HPLC over time to examine the decomposition of each compound.
DHNA rapidly degraded within 24 hours under each condition (FIG. 2). In particular, the degradation at pH 7.4 was remarkable.
For DHNAS, DHNAG, and DHNAg, almost no degradation was observed in 6 days of observation under any condition. The rapid degradation of DHNA and the stability of DHNAS, DHNAG and DHNAg were also confirmed by TLC.

HPLC separation conditions / column: Inert Sustain (registered trademark) C18 (4.6 × 250 mm, 3 μ), manufactured by GL Sciences Inc., temperature: 40 ° C.
Eluent: 12% methanol, 20% acetonitrile, 8 mM ammonium formate Flow rate: 0.65 ml / min
HPLC apparatus; Hewlett Packard 1100
TLC separation conditions and plate; Merck silica gel TLC plate (5 × 10 cm)
Developing solvent; 1-butanol: acetic acid: water (4: 1: 2)
Rf value: DHNA (0.87), DHNAS (0.56), DHNAG (0.43), DHNAg (0.71)
・ Detection: Detected as a blue fluorescent spot by UV lamp irradiation

(Test Example 2)
DHNAS Toxicity Test ICR strain female mice (7 weeks old, n = 6) were orally administered a single oral dose of 2 mg of DHNAS (2143 mg / kg). Six mice in the control group were given a single oral dose of 200 μL of water. One mouse and one control mouse each administered with DHNAS were transferred to a metabolic cage, and urine and feces were collected every 24 hours after the administration until the third day for analysis of metabolic excreta. In other mice, observations of body weight and condition were performed up to day 28 after administration. No toxicity was observed during the observation period. In the DHNAS administration group, fecal softening was observed.

Findings concerning toxicity of DHNA Regarding the acute toxicity of DHNA, a study was conducted using a DHNA-containing extract in an amount of 78 mg / Kg (1 mg / Kg as DHNA) administered to five 5-week-old ICR mice for 5 days, and a body weight It was reported that there was no abnormality in behavior and organ anatomy [Kitasato Institute Toxicity Test Report (1994)].

(Test Example 3)
DHNA administration experiment 60 mg / 200 μL (2335 mg / Kg (1% tween 80 aqueous solution)) of DHNA was orally administered to one ICR strain female mouse (7 weeks old), then transferred to a metabolic cage every 24 hours after administration. On day 3, urine and feces were collected and analyzed for metabolic excretion.

(Identification of DHNA and DHNAS metabolites)
Two metabolites M1 and M2 were detected when urine was collected 24 hours after administration of DHNA to mice and analyzed by HPLC (FIG. 3).

HPLC separation conditions / column; Inert Sustain (registered trademark) C18 (4.6 × 250 mm, 3 μ), manufactured by GL Sciences Inc .; eluent; linear gradient (0-15 minutes), solution A (12.5 mM citric acid, 25 mM acetic acid Sodium, 10 mM acetic acid, 30 mM sodium hydroxide) → A solution containing 20% acetonitrile; (15-35 minutes) A solution / flow rate containing 20% acetonitrile; 0.65 ml / min

As for the fractionated M1, from the mass analysis by FAB Mass (negative mode) (JEOL JMS-SX102), the MH peak was observed at 379, the molecular weight was 380, and it was estimated to be glucuronidated DHNA, and M1 was β-glucuronidase. Since it was hydrolyzed by (β-glucuronidase (Type H-1, SIGMA)) treatment to produce DHNA, it was identified as β-glucuronidated DHNA (DHNAG). The structure was confirmed from NMR.
M2 was observed as MH peak at 283 by FAB Mass (negative mode), so the molecular weight was estimated to be 284, DHNA sulfate (DHNAS), and the retention time and UV spectrum in the synthesized product and HPLC were consistent. Therefore, the structure was confirmed.
On the other hand, M1 (DHNAG) was also detected from the urine 24-hour urine of DHNAS-administered mice (FIG. 4). From this result, it was proved that the DHNAS → DHNA → DHNAG pathway was present in the mouse and DHNAS → DHNA conversion occurred in vivo. It was done.

Claims (11)

Formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo; at least one of R ¹ and R ² is not a hydrogen atom. ]
Or a salt thereof.   The compound or a salt thereof according to claim 1, wherein the group capable of being hydrolyzed by an enzyme in vivo is a glycosyl group or an oxo acid group. Groups that can be hydrolyzed by enzymes in vivo

[In the formula, * represents a binding site. ]
The compound or a salt thereof according to claim 1, which is a group selected from the group consisting of: Groups that can be hydrolyzed by enzymes in vivo

[In the formula, * represents a binding site. ]
The compound or a salt thereof according to claim 1, which is a group selected from the group consisting of:   The pharmaceutical containing the compound or its salt of any one of Claims 1-4.   The medicament according to claim 5, which is a bifidobacteria growth agent.   The medicament according to claim 5, which is an intestinal flora improving agent.   The foodstuff containing the compound or its salt of any one of Claims 1-4. 1,4-dihydroxy-2-naphthoic acid (DHNA) is added to a plant tissue culture medium and cultured. Formula (I):

[Wherein, R ¹ and R ² are each independently a hydrogen atom or a group that can be hydrolyzed by an enzyme in vivo; at least one of R ¹ and R ² is not a hydrogen atom. ]
Or a salt thereof.   The production method according to claim 9, wherein the plant is a plant expressing a glycosyltransferase.   The production method according to claim 9 or 10, wherein the DHNA is derived from a culture solution obtained by culturing a microorganism that produces DHNA.