JP2022169354A - Arginase production promoter - Google Patents

Abstract

To provide a new substance that promotes the production of arginase and use thereof.SOLUTION: An arginase production promoter contains lactic acid bacteria as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to an arginase production promoter.

Arginase is an enzyme that catalyzes the urea-producing reaction. Arginase is contained in the liver, kidney, etc. of humans, mammals, and amphibians, and in the urea cycle, it decomposes arginine to produce ornithine and urea. Arginase is also activated by divalent metal ions such as iron, cobalt and manganese, and inactivated by mercury ions, silver ions, citric acid and the like.

In general, blood ammonia, which increases after exercise, is detoxified (metabolized) and excreted by urea synthesis in the liver. Ammonia is metabolized into urea through the urea cycle in hepatocytes, which is then excreted in the urine by the kidneys. Therefore, it is said that the blood ammonia level becomes high when the urea cycle activity is lowered and the production of ammonia in the intestine is increased due to the deterioration of liver function. Therefore, congenital urea cycle enzyme deficiency and liver dysfunction often develop hyperammonemia.

As mentioned above, the urea cycle functions primarily in the liver, performing a detoxifying function of converting ammonia to urea. It is known that when this function declines, liver cells are damaged by ammonia, resulting in various liver diseases and serious symptoms such as hyperammonemia. Patients with hyperammonemia develop symptoms such as nausea, vomiting, disturbance of consciousness, lethargy, convulsions, and respiratory distress, and delay in treatment can be life threatening.

Ammonia produced in the body moves in the blood, enters the urea cycle in the liver, passes through carbamoyl phosphate, is converted to citrulline, and finally urea is produced from arginine for detoxification. Arginase, one of the enzymes in the urea cycle, is an enzyme that controls the reaction that produces ornithine from arginine. Arginine plays an important role in ammonia detoxification.

As substances having arginase activating action, extracts of herbal medicine tree (Mokutsu) (Patent Document 1), Bakumondou extract (Patent Document 2), apricot juice (Patent Document 3), plant extracts of the family Asclepiadaceae (Patent Document 3) Plant extracts such as those disclosed in Patent Document 4) are known. Lactoferrin (Patent Document 5) and stigmasterol (Patent Document 6), which are milk-derived proteins, are also known to have urea cycle activating action and hyperammonemia improving action by utilizing arginase activating action. there is Furthermore, an invention has been proposed in which these extracts are used as skin quality improving agents.

In recent years, the use of microorganisms and their cultured metabolites has attracted attention as functional materials. In particular, white koji mold fungus extract (Patent Document 7) is known to exhibit arginase activating action, and also has other effects such as blood pressure elevation and blood sugar elevation, activation of genes related to tight junction formation, collagenase inhibitory action, Hyaluronidase inhibitory action is exemplified. In addition, lactic acid bacteria are known to have various functions such as immunity enhancement, anti-inflammatory, lipid metabolism, and skin quality improvement.

Arginase exists in multiple isotypes. For example, an antibody capable of distinguishing between arginase in red blood cells and arginase derived from liver is known (Patent Document 8). Arginase in the liver has been named Arginase 1 and is responsible for most of the arginase activity in the liver. Decreased activity of arginase 1 in the liver slows detoxification of ammonia and causes hyperammonemia. Therefore, by promoting the activity of arginase 1, it becomes possible to prevent or treat disorders such as hyperammonemia.

In addition, Patent Document 9 discloses recombinant human arginase I (also referred to as arginase 1 or ARG-1). It has been revealed that when arginase 1 is expressed at a high concentration, immunocompetent cells consume arginine, thereby suppressing the function of T cells and suppressing allergic reactions.

Furthermore, in Patent Document 10, it was found that arginase 1 exists in the skin and contributes to the suppression of erythema of the skin that occurs due to ultraviolet irradiation, and it can be used as an indicator of resistance to ultraviolet rays. We propose an evaluation method for sunburn as an index.

Japanese Patent No. 3823373 JP 2003-277285 A Japanese Patent Application Laid-Open No. 2006-143608 JP 2008-255078 A Japanese Patent Application Laid-Open No. 2012-223100 Japanese Patent No. 6605794 Japanese Patent No. 6783962 WO96/28732 Japanese Patent Publication No. 2010-512168 Japanese Patent No. 4446010

An object of the present invention is to provide a new substance that promotes the production of arginase and its use.

That is, the present invention is as follows.
[1]
An arginase production promoter containing lactic acid bacteria as an active ingredient.
[2]
promoting the production of said arginase in skin tissue;
The production promoter according to [1].
[3]
The lactic acid bacterium includes the genus Lactococcus,
The production promoter according to [1] or [2].
[4]
Using the lactic acid bacteria, which are dead bacteria, as an active ingredient,
[1] The production promoter according to any one of [3].
[5]
A skin softening agent containing lactic acid bacteria as an active ingredient.
[6]
An agent for promoting the production of arginase in liver tissue, containing lactic acid bacteria as an active ingredient.
[7]
A urea cycle activator containing lactic acid bacteria as an active ingredient.
[8]
A hyperammonemia-improving agent containing lactic acid bacteria as an active ingredient.

ADVANTAGE OF THE INVENTION According to this invention, the new substance which promotes production of arginase, and its utilization can be provided.

Hereinafter, an embodiment of the present invention (hereinafter referred to as "this embodiment") will be described in detail, but the present invention is not limited to this, and various modifications are possible without departing from the gist thereof. is.

The production promoter of the present embodiment is an arginase production promoter containing lactic acid bacteria as an active ingredient. The present embodiment also relates to the use of the arginase production promoter. Specifically, it is as follows.

By using the production-enhancing agent of the present embodiment, arginase activity is promoted, activation of the urea cycle, and thus ammonia metabolism are promoted. Therefore, the production promoter of the present embodiment is useful for suppressing hyperammonemia (alleviating symptoms).

In addition, by using the production promoter of the present embodiment, the production of arginase enzyme in skin tissue is promoted, so that the urea content in skin tissue increases, resulting in softening of the skin, and Tissue water retention is improved. Furthermore, by using the production promoter of the present embodiment, the activity of arginase is promoted, thereby suppressing melanin production in skin tissue. Therefore, the production promoter of the present embodiment is also useful as a skin whitening agent. Agents for such uses are also collectively referred to as skin function improving agents.

Furthermore, the production promoter of the present embodiment can be used as a drug, quasi-drug, food (functional food, supplement, drink, etc.) that activates arginase in the liver, and for promoting arginase production in the skin. It can be suitably used as cosmetics, pharmaceuticals, quasi-drugs, foods (functional foods, supplements, drinks, etc.) and the like. Agents for such uses are also referred to as liver function-enhancing agents.

In the present embodiment, arginase is an enzyme that degrades arginine to produce ornithine and urea, and includes arginase 1, an isotype present mainly in the liver.

Lactic acid bacteria used in the present embodiment are Gram-positive, rod-shaped or coccus-producing bacteria that produce 50% or more of lactic acid relative to sugar consumed. Examples thereof include lactic acid bacteria of the genera Lactococcus, Leuconostoc, Streptococcus, and Pediococcus. Preferably, it belongs to the genus Lactococcus. Since these lactic acid bacteria are lactic acid bacteria commonly present in food, lactic acid bacteria used in food production, or lactic acid bacteria isolated from the feces of healthy individuals, there is no risk of side effects. In addition, since it can be easily obtained in large amounts by culturing, the use of microbial cells obtained by culturing is economical with low production costs. In recent years, it has various uses as food, medicine, or cosmetics.

As a culture method for growing the lactic acid bacteria by culturing, conventionally known methods for culturing lactic acid bacteria can be used, and there is no particular limitation. For example, using a medium for growing lactic acid bacteria, culturing at 37° C. for 5 to 120 hours, preferably 16 to 28 hours while maintaining the pH near neutrality, the number of viable bacteria is about 10 ⁷ to 10 ¹⁰ /ml, preferably about 10 10 /ml. is a method of obtaining a culture solution of about 10 ⁸ to 10 ¹⁰ /ml.

The lactic acid bacteria used in the present embodiment may be dead cells, a cell suspension, or an extract obtained by extracting a cell suspension with hot water. Preferably, it contains only lactic acid bacteria. As dead cells, the cells of lactic acid bacteria cultured and collected by a conventional method are washed, centrifuged and dehydrated, and if necessary, after repeated washing and dehydration, suspended in distilled water, physiological saline, etc. A dead cell suspension obtained by heating the suspension at, for example, 80 to 115° C. for 30 minutes to 3 seconds, or a dried product thereof. As a method for drying the dead cell suspension, a known drying method can be used, and there is no particular limitation, but methods such as spray drying and freeze drying can be used. If necessary, before and after the sterilization treatment by heating or the like, or before and after the drying treatment, an enzyme treatment, a surfactant treatment, and a grinding/pulverization treatment can be performed.

Since the arginase production-enhancing agent of the present embodiment has the ability to promote arginase production in liver cells, the urea cycle is activated, and thus the ammonia metabolism is promoted. ). In addition, since it has the ability to promote the production of arginase in skin cells, the urea content in skin tissue increases, resulting in softening of the skin and improvement in water retention of the skin tissue. In addition, since the produced arginase suppresses melanin production, it is also useful as a whitening agent.

The arginase production promoter of the present embodiment can be used as an external composition or an oral composition. Specifically, pharmaceuticals, quasi-drugs, topical or systemic skin cosmetics, various external skin preparations including medicinal or cosmetic preparations applied to the scalp and hair, bath preparations, and orally ingested pharmaceuticals.・It can be used in various forms such as internal use compositions of quasi-drugs, general food and drink, supplements, drinks, and food compositions such as functional foods.

The amount of lactic acid bacteria contained in the arginase production promoter of the present embodiment may be appropriately adjusted according to each form, dosage form, and application, and is not particularly limited. The suspension is adjusted to contain 10 ⁶ or more, preferably 1×10 ⁷ or more, more preferably 1×10 ⁸ or more, and preferably 1×10 ⁹ or more. More preferred.

A suspension of the lactic acid bacteria is preferably used as the lactic acid bacteria used in the present embodiment. The amount of lactic acid bacteria in the suspension is not particularly limited, but it is usually 1×10 ⁹ or more per gram, preferably 1×10 ¹⁰ or more per gram. 1×10 ¹¹ or more is more preferable, and 1×10 ¹² or more per gram is even more preferable. The amount of lactic acid bacteria suspension blended with the arginase production promoter of the present embodiment is usually 0.00001% to 20% by weight, preferably 0.00005% to 10% by weight, and 0.0001% by weight. % to 5% by weight, more preferably 0.0005% to 2% by weight, and particularly preferably 0.001% to 1% by weight.

The invention related to the arginase production promoter of the present embodiment may contain other ingredients in addition to the lactic acid bacteria within a range that does not impair the effects of the present embodiment. For example, it can be used in combination with a drug or the like that is commonly used for treatment of materials for external use or internal use, and a combination in which the effect of the present embodiment is more likely to be exhibited by the combined drug is particularly preferred. Other ingredients include oily ingredients, lubricants, excipients, binders, disintegrants and the like. In addition, additives such as sweeteners, sour agents, flavoring agents, coloring agents, pigments, etc. may be contained in appropriate amounts. In addition, in the various compositions and various agents of the present embodiment, each of these components may be used alone, or two or more thereof may be used in combination.

Examples of oily components include various fatty acid esters, hydrocarbons, higher fatty acids, higher alcohols, and the like. Lubricants include magnesium stearate, stearic acid, sodium stearyl fumarate, sucrose fatty acid esters, polyethylene glycol (macrogol), talc and the like.

Binders include hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, gelatin, starch, dextrin, gum arabic, sodium alginate, tragacanth, refined gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol (partially saponified product), ethyl cellulose, Pullulan, polyethylene glycol (macrogol) and the like can be exemplified.

Disintegrants include carboxymethylcellulose calcium (carmellose calcium), carboxymethylcellulose sodium (croscarmellose sodium), low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylstarch sodium, cross-linked carboxymethylcellulose sodium (croscarmellose sodium). , powdered cellulose, cellulose or derivatives thereof, crosslinked polyvinylpyrrolidone (crospovidone), starch, carboxymethyl starch, hydroxypropyl starch, agar and the like.

Excipients include polysaccharides such as crystalline cellulose, sodium alginate, xanthan gum, starches such as pregelatinized starch, hydroxypropyl starch, corn starch, potato starch and derivatives thereof, sucrose, glucose, xylitol, erythritol, sorbitol, lactitol, Trehalose, palatinose, palatinite (reduced palatinose), mannitol, maltitol, lactitol, lactose, fructose, sugars and sugar alcohols such as powdered hydrogenated maltose starch syrup, powdered cellulose, partially pregelatinized starch, cellulose and its derivatives such as ethyl cellulose, light Silicic anhydride, titanium oxide, aluminum hydroxide gel, synthetic aluminum silicate, aluminum trisilicate, silicon dioxide, kaolin, cocoa butter, citric acid or its salts, stearic acid or its salts, calcium hydrogen phosphate, hydrogen phosphate Sodium etc. can be illustrated.

Examples of sweeteners include aspartame, acesulfame potassium, saccharin sodium, sucralose, stevia, thaumatin and the like. Examples of acidulants include citric acid, succinic acid, tartaric acid, malic acid, fumaric acid and the like. Examples of flavoring agents include monoterpenes such as menthol, camphor, borneol, and limonene, and various fragrances.

The dosage form of various formulations related to the arginase production-enhancing agent of the present embodiment is not particularly limited, and can be appropriately selected according to the mode of administration. For oral administration, tablets, capsules, granules, powders, fine granules, syrups, sustained-release tablets, tablets, chewable tablets, drops and the like can be used.

When the arginase production promoter of the present embodiment is used as an external preparation, an oily component, a moisturizing component, or the like can be appropriately blended depending on the application site.

When used as an external preparation for skin, the dosage form is not particularly limited. Formulations include, for example, creams, emulsions, oils, lotions, packs, aqueous gels, oil gels and ointments. From the viewpoint of percutaneous absorbability, creams, milky lotions, oils and the like can be said to be more preferable dosage forms. These external preparations for skin can be produced in the same manner as ordinary external preparations for skin, except that the triterpenoid compound is appropriately selected and blended according to the dosage form.

In addition to lactic acid bacteria, external skin preparations include liquid paraffin, vaseline, hydrocarbons such as squalane, isopropyl myristate (IPM), synthetic gairou, jojoba oil, carnauba wax, and the like, which are usually applied to external skin preparations. animal and vegetable oils such as olive oil and beef tallow; higher alcohols such as cetanol and stearyl alcohol; higher fatty acids such as stearic acid and oleic acid; anionic surfactants such as sodium lauryl sulfate and alkyl sulfosuccinate; Cationic surfactants such as alkylamine salts, nonionic surfactants such as fatty acid monoglycerides and polyoxyethylene hydrogenated castor oil, surfactants such as amphoteric surfactants such as alkylbetaine, polyhydric alcohols such as glycerin and propylene glycol lower alcohols such as ethanol and propanol; preservatives such as parabens and chlorhexidine gluconate; UV absorbers such as para-aminobenzoic acid derivatives and benzophenone derivatives; antioxidants such as vitamin E and butylhydroxytoluene; , thickeners such as carboxyvinyl polymer, moisturizing agents such as polyethylene glycol, pH adjusters such as citrates and acetates, powders such as titanium oxide, silica gel, talc, fragrances, pigments, etc., hyaluronic acid, placenta Medicinal ingredients such as extracts, ginseng extracts, vitamins, and sterol glycosides can be appropriately selected and blended according to various purposes.

Since the enzyme activator of the present embodiment can activate the urea cycle, it can also be used as a urea cycle activator. In the present embodiment, activation of the urea cycle means that the expression of each enzyme in the urea cycle is increased, the degree of action of each enzyme that constitutes the urea cycle in the living body is increased more than usual, and metabolism It means that the whole is promoted, etc.

The arginase production promoter of the present embodiment promotes the metabolism (detoxification) of ammonia by activating the arginase enzyme. It is also useful as a symptom improving agent.

When the arginase production promoter of the present embodiment is administered as an oral preparation, the concentration of lactic acid bacteria, the dosage form, the age, weight, and sex of the subject of administration, and the exercise load when taken before exercise. It can be determined as appropriate depending on the conditions. For example, when the dosage form is a tablet, it is preferable to take with water or the like. The administration interval can be determined appropriately, and may be, for example, before meals, after meals, between meals, before exercise, or after exercise.

The oral dose of the arginase production-enhancing agent of the present embodiment is usually 1 mg to 5,000 mg/day, preferably 50 mg to 1,000 mg/day, in terms of human intake of lactic acid bacteria per day. Preferably 100 mg to 500 mg/day.

When the arginase production promoter of the present embodiment is administered as an external preparation, it can be appropriately determined according to conditions such as the concentration of lactic acid bacteria, dosage form, age, weight, sex, and application site of the subject. For example, when the topical agent is a cream, it is applied to the skin 1 to 3 times a day.

When the agent for promoting arginase production of this embodiment is administered as an external preparation, it is blended in the external preparation at a concentration of 0.01 to 100 μg/ml. It is preferably 0.05 to 50 μg/ml, particularly preferably 0.1 to 10 μg/ml.

Specific examples will be described below, but the present invention is not limited to the following examples.

Example 1 (Preparation of lactic acid bacteria suspension)
Lactococcus lactis strain LLA-34 was statically cultured at 37° C. for 24 hours in an MRS liquid medium prepared from a commercially available MRS powder medium (manufactured by Difuco & BBD). The resulting preculture was inoculated into a new MRS liquid medium and statically cultured at 37° C. for 24 hours. After culturing, the cells were separated and collected by centrifugation at 7000 rpm. This operation was repeated several times, and the cells were washed with distilled water. Next, the washed cells were resuspended in distilled water to an optical density (OD) of 40 and sterilized at 121°C for 20 minutes to obtain a suspension of lactic acid bacteria.

<Test 1: Evaluation test of ability of lactic acid bacteria to promote arginase production (human keratinocytes)>
1. Test method Human keratinocytes (PHK16-0b) were seeded in a 6-well plate at 2.0×10 ⁵ cells/well, and EpiLife (Gibco) was added with Human KG growth additive (Gibco) and 100 ug/ml penicillin-streptomycin. Cultured for 3 days in supplemented medium. As a sample (Example 1), a suspension of lactic acid bacteria was added to the medium so that OD=0.08 (concentration 1) and OD=0.4 (concentration 2), and cultured for 4 days. After washing the cells with PBS(-), the cells were lysed with 100 μl of Passive Lysis Buffer (manufactured by Gibco). This cell lysate was centrifuged, and the arginase activity of the obtained supernatant was measured using QuantiChrom Arginase Assay Kit (manufactured by Bioassay Systems) according to the enclosed procedure. The amount of urea was calculated from the absorption at 430 nm using a plate reader (Perkin Elmer, ARVO X3). Separately, the total protein amount in the cell lysate was measured with a protein assay kit (manufactured by Takara Bio Inc.), and the amount of urea per unit cell protein was converted into intracellular arginase activity. The intracellular arginase activity at the time of addition of the test sample was calculated with the value of no test sample added (Comparative Example 1: Control) set to 100%.

2. Results The measurement results are shown in Table 1 below.

As shown in Table 1, it was confirmed that the Lactococcus lactis strain LLA-34 increased the arginase activity of human keratinocytes. Keratinocytes are present in human skin, and enhanced arginase activity in skin was observed. This indicates moisturization of the skin and improved metabolism.

<Test 2: Evaluation test of ability of lactic acid bacteria to promote arginase production (human liver cancer cells)>
1. Test method Human liver cancer cells (HepG2) were seeded in a 6-well plate at 2.0×10 ⁵ cells/well, and HepG2 medium (manufactured by CET) supplemented with 10% fetal bovine serum and 100 ug/ml penicillin-streptomycin. was cultured for 1 day. As a sample (Example 1), a suspension of lactic acid bacteria was added to the medium at OD=0.08 (concentration 1) and OD=0.4 (concentration 2), and cultured for 1 day. Preparation of cell lysate and measurement of intracellular arginase activity were performed in the same manner as in Test 1. The intracellular arginase activity at the time of addition of the test sample was calculated, taking the value of no test sample added (Comparative Example 1) as 100%.

2. Results The measurement results are shown in Table 2 below.

As shown in Table 2, it was confirmed that the Lactococcus lactis strain LLA-34 increased the arginase activity of human liver cancer cells. These strains are effective in arginase activation in liver as well as in skin.

<Test 3: Arginase production promoting ability evaluation test>
In order to verify the effect of Example 1, the arginase production promoting ability evaluation test of Comparative Example 2 was carried out.
Comparative Example 2: Okesa persimmon skin (freeze-dried powder): Commercially available okesa persimmon skin freeze-dried and then pulverized

1. Test method Human keratinocytes (PHK16-0b) were seeded in a 6-well plate at 2.0×10 ⁵ cells/well, and EpiLife (Gibco) was added with Human KG growth additive (Gibco) and 100 ug/ml penicillin-streptomycin. Cultured for 3 days in supplemented medium. Comparative Example 2 was added as a specimen to a final concentration of 100 ug/ml in the medium and cultured for 4 days. Preparation of cell lysate and measurement of intracellular arginase activity were performed in the same manner as in Test 1. The intracellular arginase activity at the time of addition of the test sample was calculated, taking the value of no test sample added (Comparative Example 1) as 100%.

2. Results The measurement results are shown in Table 3 below together with the results of Test 1 (Example 1).

Comparative Example 2 is known to have an effect of improving skin quality and liver function, but it was found that the lactic acid bacterium of Example 1 has a higher effect in promoting arginase production.

The present application has industrial applicability as an arginase production promoter.

Claims (8)

An arginase production promoter containing lactic acid bacteria as an active ingredient. promoting the production of said arginase in skin tissue;
The production promoter according to claim 1. The lactic acid bacterium includes the genus Lactococcus,
The production promoter according to claim 1 or 2. Using the lactic acid bacteria, which are dead bacteria, as an active ingredient,
The production promoter according to any one of claims 1 to 3. A skin softening agent containing lactic acid bacteria as an active ingredient. An agent for promoting the production of arginase in liver tissue, containing lactic acid bacteria as an active ingredient. A urea cycle activator containing lactic acid bacteria as an active ingredient. A hyperammonemia-improving agent containing lactic acid bacteria as an active ingredient.