JP6039891B2 - Epithelial sodium channel activator - Google Patents

Description

  The present invention relates to an epithelial sodium channel activator.

The sense of taste is stimulated by taste cells present in the taste buds present on the surface of the tongue, such as the encased papillae, foliate nipples, and rod-shaped nipples, and the stimulation is transmitted to the brain via the chorda tympani, glossopharyngeal nerve, etc. I feel for the first time here. Taste affects the physiological state, psychological state, and taste quality (deliciousness of food), and is one of the five important senses of animals such as humans.
There are five basic tastes (sweet, salty, sour, umami, and bitter) known as taste senses. Among these, salty taste is extremely important as a flavor of food and drink because it has not only a preference but also an appetite enhancing effect that draws the deliciousness of the food and drink.

  Sodium chloride is usually used to impart a salty taste to food and drink. The overdose of sodium, its main component, is a risk factor for many health diseases, including hypertension. Therefore, suppression of sodium chloride intake is recommended. So far, various low-salt foods and drinks with reduced sodium chloride content have been developed and marketed. For example, potassium chloride and magnesium sulfate are known as substitutes for sodium chloride. However, all of them have a weak salty taste compared to sodium chloride, and have an unpleasant taste such as a bitter taste and astringency in addition to the salty taste, and it is difficult to achieve both salt reduction and taste quality.

About 50 to 100 taste cells present in one taste bud, epithelial Na + channel consisting of α-subunit, β-subunit, and γ-subunit (hereinafter referred to as “ENaC” in the present specification). (Also called). ENaC is a kind of salty taste receptor that mediates the influx of sodium through the apical membrane of miso, and the activity of ENaC is controlled by temperature, osmotic pressure, and the like. To date, amiloride, an inhibitor of ENaC function, is known to attenuate the taste response to sodium chloride in mammals such as humans. Furthermore, a substance that promotes sodium influx mediated by ENaC functions as a general taste tasting synergist and enhances human taste sensation (see, for example, Patent Document 1).
Furthermore, the relationship between the salty taste threshold and the prevalence of hypertension is also known, and the prevalence of hypertension increases when it is insensitive to saltiness (see, for example, Non-Patent Document 1). For example, as a result of examining the salty taste perception threshold and the prevalence of hypertension in Japanese women, the prevalence of hypertension is 55.6% (55/99) in humans with a saltiness threshold of 1% or more, while It has been reported that the prevalence of hypertension is 32.9% (out of 127/380) in humans whose threshold is less than 1%, and that the prevalence of hypertension is significantly high when the saltiness threshold is high.
From the above viewpoint, when the ENaC function is inhibited, the salty taste threshold increases and the possibility of suffering from hypertension increases. Therefore, an ENaC activator is required from the viewpoint of prevention and treatment of hypertension.

International Publication No. 02/087306

T. Michikawa et al., Hypertens. Res., Vol. 32, No. 5, p. 399-403, 2009

  An object of the present invention is to provide an epithelial sodium channel activator having an epithelial sodium channel activity effect. Furthermore, this invention makes it a subject to provide the salty taste enhancer which has the effect of enhancing the salty taste of food and drink, without impairing the original flavor.

In view of the above problems, the present inventors have conducted intensive studies. As a result, it was found that when the epithelial sodium channel is activated, the chorda tympani nerve response is enhanced and the salty taste threshold is lowered (sensitive to salty taste). In addition, cabbage ( Brassica oleracea ) extract, ferulic acid, tocopherol, chrysanthemum ( Chrysanthemum morifolium ) extract, spinach ( Spinacia oleracea ) extract, Yaeyama Aoki ( Morinda citrifolia ) extract and dimethyl octenone activated epithelial sodium channel It has been found that it has an effect, enhances the chorda tympani response, and enhances the salty taste. The present invention has been completed based on these findings.

The present invention is selected from the group consisting of cabbage ( Brassica oleracea ) extract, ferulic acid, tocopherol, chrysanthemum ( Chrysanthemum morifolium ) extract, spinach ( Spinacia oleracea ) extract, Morinda citrifolia extract and dimethyl octenon The present invention relates to an epithelial sodium channel activator containing at least one active ingredient.
The present invention also relates to a salty taste enhancer containing as an active ingredient at least one selected from the group consisting of cabbage extract, ferulic acid, tocopherol, chrysanthemum extract, spinach extract, Yaeyama Aoki extract and dimethyloctenone.

  The epithelial sodium channel activator of the present invention has an excellent epithelial sodium channel activity effect. Moreover, the salty taste enhancer of the present invention has an excellent effect of enhancing the salty taste of foods and beverages.

  The ENaC activator and the salty taste enhancer of the present invention include at least one selected from the group consisting of cabbage extract, ferulic acid, tocopherol, chrysanthemum extract, spinach extract, Yaeyama Aoki extract and dimethyloctenone as an active ingredient. To do.

A plant extract is demonstrated among the active ingredients used for this invention.
The cabbage in the present invention is a perennial plant belonging to the genus Brassica in the Brassicaaceae family and is widely used as a vegetable.
Chrysanthemum in the present invention is a perennial herb belonging to the genus Chrysanthemum belonging to the Asteraceae family and is used for ornamental purposes.
The spinach in the present invention is a plant belonging to the genus Spinacia belonging to the family Chenopodiaceae and is widely used as a vegetable.
The term “Yaeyama Aoki” in the present invention refers to an evergreen small tree belonging to the genus Morinda belonging to the Rubiaceae family, and is used as an edible, pharmaceutical and dyeing material.

Any arbitrary part of the plant can be used as the plant in the present invention. For example, any part of the above-mentioned plant whole tree, whole grass, root, rhizome, stem, branch, stem, leaf, bark, sap, resin, flower, fruit, seed, pericarp, bud, bud, spikelet, heartwood, etc. And any one or more of these combinations can be used.
In order to obtain a cabbage extract in the present invention, it is preferable to extract cabbage leaves. In order to obtain a chrysanthemum extract in the present invention, it is preferable to extract chrysanthemum flowers. In order to obtain a spinach extract in the present invention, it is preferable to extract spinach leaves. In the present invention, in order to obtain a Yaeyama Aoki extract, it is preferable to extract a Yaeyama Aoki fruit.
Or in this invention, you may use the extract marketed as a foodstuff, a drink, etc. as an extract of the said plant. For example, noni juice marketed as a beverage may be used as the Yaeyama Aoki extract.

There are no particular limitations on the method for producing the cabbage, chrysanthemum, spinach and yaeyama aoki extract used in the present invention, and the extract can be obtained by extracting the above-mentioned plant by a conventional method. Specifically, dried products obtained by drying the plant, squeezed juice obtained by squeezing and extracting the pulverized product, steam distillates, crude extracts using various extraction solvents, distributing crude extracts, column chromatography, etc. Extract fractions obtained by purification by various chromatographic methods can be used as the extract in the present invention.
The above-mentioned plant can be subjected to extraction as it is, but it is also preferable to add steps such as drying, shredding, and pulverization in order to further increase the extraction efficiency. Moreover, in this invention, you may use any of the said extract, steam distillate, a pressing thing, etc. individually or in combination of 2 or more types. Especially, as a plant extract of this invention, it is more preferable to use the extract obtained using the extraction solvent from the dried material which dried the said plant, or its ground material.

  As the extraction solvent, either a polar solvent or a nonpolar solvent can be used, and these can also be mixed and used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran Linear and cyclic ethers such as diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; benzene and toluene Aromatic hydrocarbons such as; pyridines; supercritical carbon dioxide; fats and oils, waxes, and other oils. Or the mixture which combined 2 or more types of the said solvent can be used as an extraction solvent. Of these, water, alcohols, and a water-alcohol mixed solution are preferably used, and an aqueous ethanol solution is more preferably used.

  The extraction conditions for obtaining the extract used in the present invention are not particularly limited, depending on the solvent used, and normal conditions can be applied. For example, what is necessary is just to immerse the said plant at 0-100 degreeC for 1 hour-30 days, or to heat-reflux. Moreover, in order to improve extraction efficiency, you may stir together or may perform a homogenization process in a solvent.

  The extract obtained by extraction with the above-mentioned solvent may be used as it is. However, a fraction having high activity can be fractionated by an appropriate separation means such as gel filtration, chromatography, precision distillation, activated carbon treatment, etc. It can also be used. In the present invention, the plant extract includes the various extracts thus obtained, a diluted solution thereof, a concentrated solution thereof, a purified product thereof or a dry powder thereof.

Next, compounds other than the plant extract among the active ingredients used in the present invention will be described.
Ferulic acid (4-hydroxy-3-methoxycinnamic acid) used in the present invention is a derivative of cinnamic acid represented by the following formula.

It is known that ferulic acid can improve water solubility and increase physiological effectiveness by making it into a salt form. In the ENaC activator and salty taste enhancer of the present invention, ferulic acid may be used in the form of a salt.
The salt of ferulic acid is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples of such basic substances for salt formation include hydroxides of alkali metals such as lithium hydroxide, sodium hydroxide and potassium hydroxide; hydroxides of alkaline earth metals such as magnesium hydroxide and calcium hydroxide. Substances: inorganic bases such as ammonium hydroxide, basic amino acids such as arginine, lysine, histidine and ornithine; organic bases such as monoethanolamine, diethanolamine and triethanolamine. Of these, alkali metal or alkaline earth metal hydroxides are preferred.

  There is no restriction | limiting in particular in the manufacturing method of the said ferulic acid, What was chemically synthesized may be used, and what was extracted or refine | purified from the material derived from a natural product may be used. Moreover, what is marketed as a reagent can also be used as ferulic acid.

  Tocopherol used in the present invention is also called vitamin E and is a kind of fat-soluble vitamin. In addition, tocopherol does not have a double bond in the side chain, and there are four types of isomers (α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol) as shown below.

  There is no restriction | limiting in particular in the manufacturing method of the said tocopherol, What was chemically synthesized may be used and what was extracted or refine | purified from the material derived from a natural product may be used. Moreover, what is marketed as a reagent can also be used as tocopherol. Furthermore, as tocopherol, one of the isomers may be used, or two or more of the isomers may be used in combination.

  Dimethyloctenone (4,7-dimethyl-6-octen-3-one) used in the present invention is a compound represented by the following formula.

  There is no restriction | limiting in particular in the manufacturing method of the said dimethyl octenone, What was chemically synthesized may be used and what was extracted or refine | purified from the material derived from a natural product may be used. Moreover, what is marketed as a reagent can also be used as a dimethyl octenone.

  In the ENaC activator and salty taste enhancer of the present invention, any one of the above active ingredients may be used, or two or more may be used in combination.

  Activation of ENaC enhances the chorda tympani response and lowers the saltiness threshold. Furthermore, cabbage extract, ferulic acid, tocopherol, chrysanthemum extract, spinach extract, Yaeyama Aoki extract and dimethyl octenone exert ENaC activation action, enhance chorda tympani nerve response, and reduce salty taste threshold. Therefore, ENaC activator and salty taste enhancement by using as an active ingredient at least one selected from the group consisting of cabbage extract, ferulic acid, tocopherol, chrysanthemum extract, spinach extract, Yaeyama Aoki extract and dimethyloctenone An agent can be obtained. Furthermore, according to the present invention, by activating ENaC, it is possible to provide a salty taste enhancing technique that allows a sufficient salty taste to be felt even with a small amount of salt. It is a knowledge newly obtained by the present inventors that the active ingredient exerts an ENaC activating action, enhances the chorda tympani response, and lowers the saltiness threshold.

  In this specification, “ENaC activation” refers to the function of ENaC by increasing ion flux such as sodium ion, increasing membrane potential, increasing current amplitude, increasing potential opening, and promoting ENaC expression. Refers to strengthening. In the present invention, “ENaC activation” particularly refers to increasing the sodium ion flux and enhancing the function of ENaC.

Although there is no restriction | limiting in particular about the evaluation method of ENaC activation, For example, the method as described in Unexamined-Japanese-Patent No. 2007-528712 can be referred.
A specific example of an evaluation method for ENaC activation will be described. Evaluation of ENaC activation by contacting a test agent with a strain that stably and functionally expresses human ENaC (hereinafter also referred to as ENaC stable expression strain) and measuring the change in ion flux Is possible. The method for measuring changes in ion flux is not particularly limited, and examples thereof include patch clamp technique, measurement of whole cell current, radiolabeled ion flux assay, fluorescence assay using a potential sensitive dye or ion sensitive dye, and the like.

  According to the ENaC activator of the present invention, a tissue disorder or pathological condition in which ENaC activation is effective for treatment is prevented or improved. As tissues in which ENaC is expressed, taste bud, tubular membrane, colon, bladder, lung, cornea, respiratory tract, skin, salivary gland, sweat gland, mammary gland and the like are known. The tissue damaged by the reduced function of ENaC can be any tissue in the body, for example, tongue, eye, ear, skin, epithelium, blood, lymph, chorda tympani, esophagus, gastrointestinal tract, stomach, blood vessel , Skin, lung, heart, kidney, bladder, small intestine, large intestine and the like. Examples of tissue disorders and conditions include taste disorders, diarrhea, hyponatremia, pulmonary edema, hyperkalemia, skin barrier disorders, and the like (see, for example, the document S. Zeissig et al., Gastroenterology, vol. 134, No. 5, p.436-447, 2008; T. Maruo et al., J. Invest. Dermatol., Vol.118, No.4, p.589-594, 2002, N. Randrianarison et al. , Am. J. Physiol. Lung Cell Mol. Physiol., Vol. 294, No. 3, p.409-416, 2008, F. J. McDonald et al., Proc. Natl. Acad. Sci. A., vol. 96, No. 4, p. 1727-1731, 1999, etc.). Therefore, activation of ENaC makes it possible to prevent or improve these tissue disorders and pathological conditions.

The tissue disorder or pathological condition in which ENaC activation is effective for treatment will be described with specific examples.
There is a correlation between ENaC activity and the salty taste threshold, and substances that promote ENaC-mediated sodium influx expressed in taste cells present in taste buds function as general taste tasting synergists. The salty taste threshold is lowered (becomes sensitive to salty taste) (see, for example, WO 02/087306). Therefore, the ENaC activator of the present invention can be applied to a salty taste enhancer that makes a sufficient salty taste even with a small amount of salt, a preventive / ameliorating agent for hypertension that is one of the causes caused by excessive intake of salt. is there.
ENaC is also expressed in the tubule membrane that constitutes the human renal cortical collecting duct (distal renal tubule). ENaC expressed in the tubular membrane is known to precisely control the amount of sodium in the body by reabsorbing sodium, and plays a very important role in regulating body fluid volume, plasma osmotic pressure, blood pressure, etc. Have
Crohn's disease patients have chronic intestinal inflammation and have symptoms such as diarrhea. Analysis of intestinal ENaC function in patients with Crohn's disease revealed a decrease in Na permeability through ENaC and a decrease in γ subunit gene expression. This indicates that activation of ENaC can alleviate symptoms such as diarrhea (see S. Zeissig et al., Gastroenterology, vol. 134, No. 5, p. 436-447, 2008, etc.).
In mice lacking the αa subunit of ENaC, it has been reported that epidermal thickening and abnormal lipid secretion occur compared to wild-type mice, and that psoriatic-like shapes are observed when histological sections are observed. . These indicate that ENaC is involved in keratinocyte differentiation, etc., and suggesting that a decrease in ENaC function causes skin barrier disorders (T. Maruo et al., J. Invest. Dermatol., Vol. 118, No. 4, p. 589-594, 2002, etc.).
ENaC expressed in the lungs plays a role in preventing lung edema by permeating Na to remove lung water. It has been reported that in mice lacking the ENaC β-subunit, clearance (water removal capacity) is reduced by 32%. This indicates that pulmonary edema can be prevented by activating ENaC (N. Randrianarison et al., Am. J. Physiol. Lung Cell Mol. Physiol., Vol. 294, No. 3, p.409-416, 2008).
Deletion of βa subunit of ENaC has been reported to decrease blood sodium concentration and increase potassium concentration. This indicates that activation of ENaC can prevent hyponatremia and hyperkalemia (FJ McDonald et al., Proc. Natl. Acad. Sci. U. S. A). , Vol.96, No.4, p.1727-1731, 1999, etc.).

According to the salty taste enhancer of the present invention, the salty taste of a food or drink can be enhanced with a small amount of addition without impairing the original flavor of the food or drink. In addition, tissue disorders or pathological conditions for which salty taste enhancement is effective for treatment are prevented or improved.
The tissue that is damaged by an increase in the saltiness threshold (in which saltiness becomes insensitive) can be any tissue in the living body, and includes, for example, the tongue, kidney, heart, blood vessel, and the like. Examples of tissue disorders and pathological conditions include taste disorders, hypertension, renal disorders, myocardial infarction, stroke and the like (for example, Takeda et al., History of Medicine, Vol. 231, No. 8, pp. 805-808, 2009). See year). Therefore, the enhancement of salty taste can prevent or improve these tissue disorders and pathological conditions.

  The ENaC activator and salty taste enhancer of the present invention can be applied to uses such as pharmaceuticals, quasi drugs, foods and beverages. Furthermore, the ENaC activator and salty taste enhancer of the present invention are applied to humans and animals in liquid, solid, emulsion, paste, gel, powder (powder), granule, pellet, stick, etc. Various dosage forms can be taken.

  When applying the ENaC activator and salty taste enhancer of the present invention to pharmaceuticals and quasi-drugs, various additives are blended as necessary, and appropriate amounts of the above-mentioned active ingredients are contained, and various dosage forms of pharmaceuticals or quasi-drugs are used. It can be prepared as a product. For example, as an oral medicine such as tablets, coated tablets, capsules, granules, powders, syrups, extracts, or ointments, eye ointments, lotions, creams, patches, suppositories, eye drops, nasal drops, injections It can be prepared as a parenteral medicine such as an agent. These medicaments may be produced according to conventional methods using various additives. There is no restriction | limiting in particular in the additive to be used, The normally used thing can be used. Examples thereof include solid carriers such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, distilled water, physiological saline, aqueous glucose solution, alcohols such as ethanol, liquid carriers such as propylene glycol and polyethylene glycol, Various animal and vegetable oils, oily carriers such as white petrolatum, paraffin, waxes, stabilizers, wetting agents, emulsifiers, binders, tonicity agents, disintegrating agents, lubricants, diluents, osmotic pressure adjusting agents, pH Examples include regulators, preservatives, antioxidants, colorants, ultraviolet absorbers, humectants, thickeners, brighteners, buffers, flavoring agents, and flavoring agents.

  When the ENaC activator and salty taste enhancer of the present invention are applied to foods and drinks, pet foods, etc., provided in a form suitable for food or beverage, for example, granular, granular, tablet, capsule, paste, etc. can do. Furthermore, in addition to general foods and drinks, the foods and drinks develop as a result of a decrease in ENaC function or an increase in the saltiness threshold, and the concept is the prevention or improvement of symptoms in which ENaC function enhancement or salty taste enhancement is effective for treatment or prevention. If necessary, it can be in the form of a functional food or drink such as a beauty food, a food for a sick person, a nutritional functional food, or a food for specified health use.

There is no particular limitation on the form of the food and drink, for example, beverages such as fruit juice beverages, milk beverages, tea-based beverages, candy, drops, jelly, cookies, chocolate, cakes, yogurt, gum and other confectionery, seasonings, Examples include cooking oil, dairy products, breads, noodles, and processed rice. Moreover, it is good also as beauty foods, health food-drinks, etc., such as a tablet (tablet), a capsule, a granule, and a syrup.
These foods and drinks use, for example, an appropriate combination of food ingredients such as sweeteners, colorants, antioxidants, vitamins, fragrances, minerals, etc., proteins, lipids, carbohydrates, carbohydrates, dietary fibers, etc. It can be prepared by incorporating this and the ENaC activator and salty taste enhancer of the present invention into various foods and beverages according to conventional methods.

  The ENaC activator and salty taste enhancer of the present invention may be used as they are as pharmaceuticals, quasi drugs, foods, beverages, etc., or as additives or compounding agents for pharmaceuticals, quasi drugs, foods, beverages, etc. It may be used.

  The compounding amount of the active ingredient in the ENaC activator and salty taste enhancer of the present invention varies depending on the form of use, but pharmaceuticals such as oral solid preparations such as tablets, coated tablets, granules, powders and capsules, In the case of oral liquid preparations such as oral liquids and syrups, 0.00005 to 10% by mass is preferable, 0.0002 to 2% by mass is more preferable, and 0.005 to 0.1% by mass is even more preferable. When mix | blending with food-drinks, pet food, etc., 0.00005-10 mass% is preferable, 0.0002-2 mass% is more preferable, 0.005-0.1 mass% is further more preferable.

  The administration or intake of the ENaC activator and salty taste enhancer of the present invention may vary according to the individual's condition, weight, sex, age, or other factors. The daily dose or intake of an adult (body weight 60 kg) is preferably 0.005 to 2000 mg, more preferably 0.02 to 200 mg, and even more preferably 0.5 to 20 mg. The ENaC activator and salty taste enhancer of the present invention are 3 times a day, 2 times a day, 1 time per day, 1 time per 2 days, 1 time per 3 days, 1 time per week, or any Can be administered or taken at different periods and intervals.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Production Example 1 Preparation of cabbage extract To 10 g of cabbage powder (extraction site: leaves, obtained from Kodama Foods Co., Ltd.) was added 100 mL of a 50% aqueous ethanol solution, and the mixture was allowed to stand at room temperature for 7 days. Thereafter, filtration was performed to obtain a cabbage extract. The solid content concentration of the extract was 4.39% (w / v).

Production Example 2 Preparation of ferulic acid Trans-4-hydroxy-3-methoxycinnamic acid (obtained from Aldrich) was dissolved in 99.5% ethanol to a concentration of 1% (w / v) to prepare ferulic acid. .

Production Example 3 Preparation of Tocopherol Tocopherol was prepared by dissolving Emix 35L (trade name, manufactured by Eisai Food Chemical Co., Ltd.) in 99.5% ethanol to a concentration of 1% (w / v).

Production Example 4 Preparation of Chrysanthemum Extract Chrysanthemum extract powder MF (trade name, extraction site: Hana, manufactured by Maruzen Pharmaceutical Co., Ltd.) was dissolved in 20% ethanol to a concentration of 1% (w / v). Prepared.

Production Example 5 Preparation of Spinach Extract To 10 g of spinach powder (extraction site: obtained from leaf, kodama food) was added 100 mL of 50% aqueous ethanol solution, and the mixture was allowed to stand at room temperature for 7 days. Thereafter, filtration was performed to obtain a spinach extract. The solid content concentration of the extract was 3.64% (w / v).

Production Example 6 Preparation of Yaeyama Aoki Extract Noni juice (obtained from Air Green) was diluted with 100% ethanol to a concentration of 1% (v / v) to prepare a Yaeyama Aoki extract.

Production Example 7 Preparation of dimethyl octenone Dimethyl octenone (obtained from Dibodan) was diluted with 100% ethanol to a concentration of 1% (v / v) to prepare dimethyl octenone.

Test Example 1 ENaC Activation Test A human ENaC activation test using the samples prepared in Production Examples 1 to 7 was performed as follows according to the method described in JP-T-2007-528712.
Pyrobest (trade name) DNA polymerase (manufactured by Takara Bio Inc.) and primers for amplification of the following α subunit gene, using as a template cDNA (manufactured by Clontech) that has cloned DNA encoding the αa subunit of ENaC expressed in human kidney cells PCR (Polymerase chain reaction) was used to amplify a cDNA fragment encoding the α subunit of ENaC (α subunit PCR amplified fragment).
<Primer for α subunit gene amplification>
5 'primer: 5'-CACCATGGAGGGGAACAAGC-3' (SEQ ID NO: 1)
3 ′ primer: 5′-GGGCCCCCCCAGAGGACA-3 ′ (SEQ ID NO: 2)

Using cDNA (manufactured by Clontech) as a template for cloning DNA encoding the βa subunit or γ subunit of ENaC expressed in human kidney cells, Advantage 2 polymerase (manufactured by Clontech), and primers for amplifying the following β subunit gene Alternatively, PCR is performed using primers for amplifying the γ subunit gene, and a cDNA fragment encoding the β subunit of ENaC (β subunit PCR amplified fragment) and a cDNA fragment encoding the γ subunit (γ subunit PCR amplified fragment) Each was amplified.
<Primer for β subunit gene amplification>
5 'primer: 5'-ATGCGGTACCATGCACGTGAAGAAGTACCT-3' (SEQ ID NO: 3)
3 'primer: 5'-GCATCTCGAGTAGATGGCATCACCCTCACT-3' (SEQ ID NO: 4)
<Gamma subunit gene amplification primer>
5'-side primer: 5'-ATGCAAGCTTATGGCACCCGGAGAGAAGAT-3 '(SEQ ID NO: 5)
3'-side primer: 5'-GCATGAATTCCAGAGCTCATCCAGCATCTG-3 '(SEQ ID NO: 6)

  The α subunit PCR amplified fragment is used as a human mammal expression vector pcDNA3.1 (containing a neomycin resistance gene, manufactured by Invitrogen), and the β subunit PCR amplified fragment is used as a human mammal expression vector pcDNA4 (Zeocin). (Product name: Resistance gene-containing, manufactured by Invitrogen), and the γ subunit PCR amplified fragment was cloned into human mammalian expression vector pcDNA6 (Blasticidin resistance gene-containing, manufactured by Invitrogen), pcDNA3. 1 / αENaC, pcDNA4 / βENaC and pcDNA6 / γENaC were prepared.

HEK293 (human embryonic kidney cells) was seeded on a 24-well plate so that the number of cells was 1 × 10 ⁵ cells / well, and cultured at 37 ° C. under 5% CO _{2 for} 24 hours. Serum reduction medium (trade name: Opti-MEM, manufactured by Invitrogen), 100 μL, pcDNA3.1 / αENaC 0.3 μg, pcDNA4 / βENaC 0.3 μg, pcDNA6 / γENaC 0.3 μg, and cationic lipid for gene transfer (Product name: lipofectamine 2000, manufactured by Invitrogen) 1 μL was added and incubated at room temperature for 20 minutes. After incubation, the mixed solution was added to the 24-well plate and cultured at 37 ° C. under 5% CO _{2 for} 24 hours. D containing aminoglycoside antibiotic G418 (manufactured by Nacalai Tesque) 800 μg / mL, bleomycin antibiotic zeocin (trade name, manufactured by Invitrogen) 150 μg / mL, and nucleoside antibiotic blasticidin (manufactured by Invitrogen) 6 μg / mL -MEM medium (Dulbecco's Modified Eagle Medium, manufactured by Gibco Co., Ltd.) was changed, and after 3 days of culture, the same medium was washed and further cultured. Repeatedly, the grown cells were obtained as ENaC stable expression strains. It was.

The ENaC stable expression strain and HEK293 were seeded in 96-well plates (Becton Dickinson) at a cell count of 2.0 × 10 ⁴ cells / well, respectively, at 37 ° C. and 5% CO ₂ for 48 hours. Culture was performed. After removing the medium and washing twice with a sodium free ringer solution (Na free Ringer solution; 150 mM NMDG-Cl, 5 mM KCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM glucose, 10 mM HEPES), a sodium free Ringer solution was prepared. 10 μM of a fluorescent sodium ion fluorescent indicator (trade name: CoroNa Green, manufactured by Invitrogen) was added and incubated at 37 ° C. in a 5% CO ₂ environment for 45 minutes.
After washing twice with a sodium free ringer solution, the sample prepared in Production Examples 1 to 7 is added to the 96 well so that the final concentration is 0.005% by volume, and incubated at 37 ° C. in a 5% CO ₂ environment for 20 minutes. Went. Thereafter, NaCl is added to the culture solution to a final concentration of 150 mM, and the fluorescence intensity of sodium ions taken into the cells is measured using FDSS (Functional Drug Screening System, manufactured by Hamamatsu Photonics), and the ion flux is measured. Was analyzed. In addition, the fluorescence intensity was calculated as a relative value when the fluorescence intensity measured every second after NaCl addition was set to 1.0 when the fluorescence intensity before NaCl addition was 1.0, and was expressed as an average value measured for 5 minutes.

From the measured fluorescence intensity, ENaC activity was measured by the method shown below.
The value obtained by subtracting the relative fluorescence intensity (C) when each sample and NaCl are added to HEK293 from the relative fluorescence intensity (A) when each sample and NaCl are added to the ENaC stable expression strain is obtained as NaCl for the ENaC stable expression strain. The ENaC activity was obtained by dividing the relative fluorescence intensity (B) when only Na was added by the value obtained by subtracting the relative fluorescence intensity (D) when only NaCl was added to HEK293 (see the following formula (1)). When the numerical value obtained from the following formula is larger than 1, it indicates that the ion flux of sodium ions is increased, that is, the function of ENaC is activated. The results are shown in Table 1.

As is clear from the results in Table 1, the fluorescence intensity increased and ENaC was activated by adding each of the samples prepared in Production Examples 1-7.
In addition, when the dependency of ENaC activity in the ENaC stable expression strain on NaCl concentration was examined, the ENaC activity of 200 mM NaCl was calculated by comparing the relative fluorescence intensity when B and D were added only with 150 mM NaCl in formula (1), A and C were expressed by relative fluorescence intensity when only 200 mM NaCl was added, and corresponded to 2.0. From the results in Table 1, when cabbage extract, ferulic acid, tocopherol, chrysanthemum extract, spinach extract, Yaeyama Aoki extract and dimethyloctenone were added, ENaC activity was 2.0 or more. Therefore, these components have an ENaC activity effect equivalent to or better than 200 mM NaCl.

Test Example 2 Sensory evaluation of salty taste enhancer Addition of ferulic acid, Yaeyama Aoki extract, tocopherol and chrysanthemum extract prepared in the above production example to 75 mM saline (purified salt, manufactured by Japan Salt Co., Ltd.) to 0.005% by volume, respectively. A prepared taste solution was prepared.
Three men and women in their 30s irrigated 10 mL of distilled water for 10 seconds, and then included 10 mL of the taste solution in their mouths for 10 seconds and discharged them. The salty strength score of 75 mM saline was set to 5, the salty strength score of 150 mM saline was set to 10, and the salty strength of the taste solution was evaluated in 10 stages. Moreover, it was evaluated whether the flavor of the said taste solution had a change with respect to the salt solution. The results are shown in Table 2.

  As apparent from the results in Table 2, the salty taste enhancer of the present invention enhances the salty taste without impairing the original flavor of the food or drink.

Test example 3 Rat chorda tympani nerve response test
Wistar rats (female, approximately 15 weeks old retired rats, weight of about 250g, obtained from Japan SLC), pentobarbital (Dainippon Sumitomo Pharma) 65mg / kg and urethane (Wako) 75mg / kg Anesthesia was induced by intraperitoneal administration. Thereafter, a cannula was inserted into the trachea to ensure respiration, and then the muscle was cut by incising the cheek. After the arcuate bone and mandible were crushed and separated from the connective tissue, the chorda tympani nerve under the bone was exposed and the nerve was fixed to the electrode.

  A solution of 0 to 0.01% by volume ferulic acid (manufactured by Sigma) in 20 mM saline was dropped onto the entire tongue using a perfusion system, and signals from the chorda tympani nerve were recorded. The dropping of the ferulic acid solution was performed in a cycle of dropping the ferulic acid solution for 1 minute, washing with water for 3 minutes, and standing for 2 minutes. For data, the peak value of the response every 12 seconds was measured for 1 minute, the average value was calculated, and it was shown as a relative value to the response value when 0.3 M ammonium chloride was dropped. The results are shown in Table 3.

  As is clear from the results in Table 3, the ENaC activator of the present invention having ENaC activity as shown in Test Example 1 has an effect of enhancing the chorda tympani response. Furthermore, the neural response is enhanced in a concentration-dependent manner.

  The results of Test Examples 1 to 3 show that the ENaC activator of the present invention activates ENaC, and accordingly, the chorda tympani nerve response is enhanced, and the salty taste is enhanced without impairing the original flavor of food and drink. It is.

Claims (3)

The active ingredient of the alcohol mixture extract - cabbage (Brassica oleracea), chrysanthemum (Chrysanthemum morifolium), spinach (Spinacia oleracea)及 beauty Indian mulberry at least one selected from (Morinda citrifolia) or Ranaru group, alcohols or water An epithelial sodium channel activator.   2. The epithelial sodium channel activator according to claim 1, which increases sodium ion flux and enhances epithelial sodium channel function. Cabbage (Brassica oleracea), key click (Chrysanthemum morifolium), spinach (Spinacia oleracea)及 beauty Indian mulberry at least one selected from (Morinda citrifolia) or Ranaru group, alcohols or water - active ingredient alcohol mixture extract A salty taste enhancer.