JP6605794B2 - Arginase 1 production promoter - Google Patents

Description

  The present invention relates to an arginase 1 production promoter.

Arginase is an enzyme that catalyzes a urea production reaction. It is contained in humans, mammals, amphibian livers, kidneys, etc., and acts as a member of the urea cycle, breaking down arginine to produce ornithine and urea. In the case of humans, they are also included in teleosts, plants, yeasts and molds in addition to these animals that excrete urea as a final product of nitrogen metabolism. It is 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 that increases after exercise or the like is detoxified (metabolized) and excreted by urea synthesis in the liver. The metabolic process of ammonia becomes urea by the urea cycle in hepatocytes and is excreted in the urine from the kidney. Therefore, it is said that when the activity of the urea cycle is reduced due to a decrease in liver function and the production of ammonia in the intestine is increased, the blood ammonia concentration becomes a high value. Therefore, hyperammonemia often develops in the case of congenital urea cycle enzyme deficiency and liver function abnormality.

  As described above, the urea circuit functions mainly in the liver and performs a detoxification function that converts ammonia into urea. It is known that when this function is reduced, liver cells are damaged by ammonia, causing various liver diseases and causing serious symptoms such as hyperammonemia. In patients with hyperammonemia, symptoms such as nausea, vomiting, disturbance of consciousness, lethargy, convulsions, and breathing problems may appear and life delays may occur if treatment is delayed.

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

  Herbal medicines extract that has an arginase activating action (Mokutsu) extract (Patent Document 1), Bakumondou extract (Patent Document 2), apricot juice (Patent Document 3), plant extract of the genus Gullaceae Plant extracts such as Patent Document 4) are known. In addition to arginase activation, lactoferrin, a milk-derived protein, is known to activate ammonia-related enzymes such as carbamoyl phosphate synthetase I and argininosuccinate synthetase I to exert ammonia detoxification. (Patent Document 5). An invention has been proposed in which any of these extracts is used as a skin external preparation.

Arginase has multiple isotypes. For example, an antibody capable of discriminating between arginase in erythrocytes and arginase derived from liver is known (Patent Document 6). Arginase in the liver is named and classified as arginase 1 and is responsible for most of the arginase activity in the liver. A decrease in the activity of arginase 1 in the liver delays ammonia detoxification and causes hyperammonemia. Therefore, by promoting the activity of arginase 1, it becomes possible to prevent or treat disorders such as hyperammonemia.
Patent Document 7 discloses recombinant human arginase I (also referred to as arginase 1 or ARG-1). When arginase 1 is expressed at a high concentration, it has been clarified that in immunocompetent cells, arginine is consumed to suppress the function of T cells and suppress allergic reactions.
The present applicant has found that arginase 1 is present in the skin and contributes to the suppression of erythema in the skin caused by ultraviolet irradiation, and can be used as an index of resistance to ultraviolet rays. A sunburn evaluation method is proposed (Patent Document 8).

Japanese Patent No. 3823373 JP 2003-277285 A JP 2006-143608 A JP 2008-255078 A JP 2012-223100 A WO96 / 28732 publication Special table 2010-512168 gazette Japanese Patent No. 4444610

  This invention makes it a subject to provide the novel substance which accelerates | stimulates production of arginase 1, and its utilization.

The present invention has the following configuration.
(1) production promoter of arginase 1 containing 5.7~250μg / mL oleanolic acid as an active ingredient.
(2) the urea cycle activator comprising 5.7~250μg / mL and an active ingredient oleanolic acid.
(3) A hyperammonemia-improving agent comprising oleanolic acid as an active ingredient.
(4) production promoter of arginase 1 skin tissue containing 62.5~250μg / mL and an active ingredient oleanolic acid.

According to the present invention, the activity of arginase 1 is promoted, the urea cycle is activated, and consequently the metabolism of ammonia is promoted, which is useful for the suppression of hyperammonemia (reduction of symptoms). Moreover, since the production of the arginase 1 enzyme in the skin tissue is promoted, the urea content of the skin tissue is increased. As a result, the skin is softened and the water retention of the skin tissue is improved.
Furthermore, stigmasterol and / or oleanolic acid is useful as a skin whitening agent because it suppresses melanin production in the skin tissue.

It is a graph which shows the result of having measured the arginase 1 activation effect in a human keratinocyte cell suspension about stigmasterol and oleanolic acid. It is a graph which shows the result of having measured the production promotion effect of arginase 1 produced in the three-dimensional human epidermis model tissue constructed | assembled by the human normal epidermal keratinocyte and the melanocyte about stigmasterol and oleanolic acid. The measurement result of the production inhibitory effect of the amount of melanin produced in the three-dimensional human epidermis model tissue constructed by human normal epidermal keratinocytes and melanocytes is shown using stigmasterol and kojic acid as a positive control. It is a graph.

This invention is the invention which concerns on the production promoter of arginase 1 which uses stigmasterol and / or oleanolic acid as an active ingredient, and its utilization. Moreover, this invention is invention of the melanin production inhibitor which uses stigmasterol as an active ingredient.
In the present invention, arginase 1 is an enzyme that decomposes arginine to produce ornithine and urea, and is an isotype mainly present in the liver, and is also referred to as arginase-I.

Stigmasterol is a compound classified into a group of sterols (steroid alcohols) called phytosterols or plant sterols, and is a kind of phytochemical contained in plants. It is a white solid with a characteristic odor and is insoluble in water but soluble in alcohol. It has various uses as food additives, pharmaceuticals, and cosmetics.
Stigmasterol has the structure of the following chemical formula 1.

  The stigmasterol used in the present invention may be isolated and purified or may contain other sterols such as campesterol and brassicasterol known as phytosterols extracted from plants. Preferably, only stigmasterol is included. As stigmasterol or phytosterol, a purified product extracted from a plant or a mixture of phytosterols is commercially available, and such commercially available products can be used in the practice of the present invention.

Oleanolic acid used in the present invention is a substance generally known as white powder found on the surface of grape skin called olives and the surface of olive leaves, which prevents infection by pathogenic bacteria and at the same time maintains its freshness. It is the main component of a certain waxy component. It is known that oleanolic acid has physiological activities such as anticancer action, anti-inflammatory action, antioxidant action, and antihyperlipidemic effect.
Oleanolic acid is used as a triterpene compound in applications such as health foods, utilizing functions such as dental caries prevention and anti-aging.
Oleanolic acid can be represented by the structural formula shown by the following chemical formula 2.

  Oleanolic acid can be extracted from grape skins and olive leaves. JP 2013-91604 A discloses a method of extracting a mixture of maslinic acid and oleanolic acid, which are triterpene compounds, using a lower alcohol. In the present invention, such an extract may be used. Moreover, you may use the oleanolic acid marketed as a cosmetic raw material.

  In addition to stigmasterol and / or oleanolic acid, a pharmaceutically acceptable carrier can be added to the invention relating to the arginase 1 production promoter of the present invention. Examples of the pharmaceutically acceptable carrier include oily components, lubricants, excipients, binders, disintegrants and the like. Further, an appropriate amount of additives such as a sweetening agent, a sour agent, a flavoring agent, a coloring agent, and a pigment may be appropriately contained.

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

  As binders, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, gelatin, starch, dextrin, gum arabic, sodium alginate, tragacanth, purified gelatin, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, polyvinyl alcohol (partially saponified product), ethylcellulose, Examples include pullulan and polyethylene glycol (macrogol).

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

  As excipients, polysaccharides such as crystalline cellulose, sodium alginate, xanthan gum, pregelatinized starch, hydroxypropyl starch, corn starch, potato starch and other derivatives, sucrose, glucose, xylitol, erythritol, sorbitol, lactitol, Sugars and sugar alcohols such as trehalose, palatinose, palatinose (reduced palatinose), mannitol, maltitol, lactitol, lactose, fructose, powdered reduced maltose varicella, cellulose such as powdered cellulose, partially pregelatinized starch, ethylcellulose and derivatives thereof, light Silicic anhydride, titanium oxide, aluminum hydroxide gel, synthetic aluminum silicate, aluminum trisilicate, silicon dioxide, kaolin, cocoa butter, citric acid or its salt, stearic acid Or the salt, calcium hydrogenphosphate, sodium hydrogenphosphate, etc. can be illustrated.

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

  The dosage form of various preparations relating to the arginase 1 production promoter of the present invention is not particularly limited, and can be appropriately selected depending on the administration form. In the case of oral administration, tablets, capsules, granules, powders, fine granules, syrups, sustained-release tablets, tablets, chewable tablets, drop agents and the like can be mentioned.

When the arginase 1 production promoter of the present invention is used as an external preparation, an oily component, a moisturizing component and the like can be appropriately blended depending on the application site.
When used for an external preparation for skin, the dosage form is not particularly limited. Examples of the preparation include creams, emulsions, oils, lotions, packs, aqueous gels, oil gels and ointments. From the viewpoint of transdermal absorbability, creams, emulsions, oils and the like are more preferable dosage forms. These external preparations for skin can be produced by the same method as that for normal external preparations for skin, except that the triterpenoid compound is appropriately selected and blended depending on the dosage form.

  In addition to the stigmasterol and / or oleanolic acid, the topical skin preparation usually includes hydrocarbons such as liquid paraffin, petrolatum, squalane, isopropyl myristate (IPM), synthetic gallow, Esters such as jojoba oil and carnauba wax, 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, sodium lauryl sulfate, alkylsulfosuccinate Surfactants such as anionic surfactants, cation surfactants such as quaternary alkylamine salts, fatty acid monoglycerides, nonionic surfactants such as poxyethylene hydrogenated castor oil, amphoteric surfactants such as alkylbetaines, glycerin and Polyhydric polymers such as propylene glycol Cole, lower alcohols such as ethanol and propanol, preservatives such as parabens and chlorhexidine gluconate, UV absorbers such as paraaminobenzoic acid derivatives and benzophenone derivatives, antioxidants such as vitamin E and butylhydroxytoluene, Arabic Thickeners such as rubber and carboxyvinyl polymer, moisturizers such as polyethylene glycol, pH adjusters such as citrate and acetate, powders such as titanium oxide, silica gel and talc, fragrances, pigments, hyaluronic acid, Medicinal components such as placenta extract, ginseng extract, vitamins, sterol glycosides and the like can be appropriately selected and blended.

  Since the enzyme activator of the present invention can activate the urea cycle, it can also be used as a urea cycle activator. In the present invention, activation of the urea cycle means that the expression of individual enzymes in the urea cycle increases, the degree of action of individual enzymes constituting the urea cycle in the living body increases more than usual, and the entire metabolism is increased. It means that is promoted.

  The arginase 1 activator promoter of the present invention promotes the metabolism (detoxification) of ammonia by activating the arginase 1 enzyme. It is also useful as an ammoniaemia improving agent.

  When the arginase 1 activation promoter of the present invention is administered as an oral preparation, the concentration of stigmasterol and / or oleanolic acid, the dosage form, the age, weight, sex, and pre-exercise of the subject Can be appropriately determined depending on conditions such as exercise load. For example, when the dosage form is a tablet, it is preferably taken with water or the like. The administration interval can be determined as appropriate, and may be any of before meal, after meal, between meals, before exercise, and after exercise, for example.

  The oral dosage of the arginase 1 activity promoter of the present invention is usually 1 mg to 5,000 mg / day, preferably 50 mg to 1,000 mg as the daily stigmasterol and / or oleanolic acid intake per day. / Day, more preferably 100 mg to 500 mg / day.

  When administering the arginase 1 activation promoter of the present invention as an external preparation, it depends on the conditions such as the concentration of stigmasterol and / or oleanolic acid, the dosage form, the age, weight, sex, and application site of the subject. It can be determined as appropriate. For example, when the external preparation is a cream, it is applied to the skin 1 to 3 times a day.

When administering the arginase 1 activity promoter of this invention as an external preparation, it mix | blends so that it may become a 0.01-100 microgram / ml density | concentration in an external preparation. Preferably it is 0.05-50 microgram / ml, Most preferably, it is 0.1-10 microgram / ml.
Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.

<Test 1: Arginase activation effect test of stigmasterol and oleanolic acid>
1. Test method Human keratinocytes were seeded at 2.0 × 10 ⁴ cells / cm ² in a φ35 mm dish and cultured for 3 days in a medium in which HuLife KG growth additive set (manufactured by Kurashiki Spinning Co., Ltd.) was added to EpiLife (manufactured by Life Technologies). . The medium was replaced with a medium containing the test concentrations shown in Table 1 below and cultured for 4 days. The cells were washed with PBS (−) and then homogenized in 200 μl of 25 mM Tris-HCl pH7.5. After 50 μl of this cell suspension was incubated at 50 ° C. for 10 minutes, 40 μl of 0.1 M arginine aqueous solution was added, and incubated at 37 ° C. for 3 hours. After adding 10 μl of 60% perchloric acid, the amount of urea in the supernatant obtained by centrifugation at 10,000 g for 10 minutes was measured using QuantiChrom Urea Assay Kit (manufactured by Bioassay Systems) according to the enclosed operation method. Separately, the amount of Urea per unit cell protein was calculated using the results of measuring the protein mass in the suspension with a protein assay kit (manufactured by Bio-Rad), and converted as the amount of intracellular arginase activity.
The addition of the test sample was taken as 100%, and the intracellular arginase activity when the test sample was added was calculated.

2. Results The measurement results are shown in Table 1 and FIG.

  As shown in Table 1 and FIG. 1, it was confirmed that both stigmasterol and oleanolic acid increase the arginase activity of keratinocytes derived from human skin.

<Test 2: arginase production promoting effect test of human stigmasterol and oleanolic acid using 3D human epidermis model>
1. Test method Melanonoderm (manufactured by MatTek) (black donor type), a 3D human epidermis model constructed from normal human keratinocytes and melanocytes, is opened within 24 hours after delivery, and EPI-100LLMM maintenance medium according to the standard method Culture was started with (MatTek). After 24 hours, an evaluation sample was prepared with DPBS (−), and 100 μl was administered onto the skin model cup. The medium and sample were changed every 48 hours.
After culturing for 8 days from the start of sample administration, the tissue is dissolved in Cell Lysis Buffer, and 1 amount of arginase in this tissue lysate is used using “Arginase I, Human, ELISA kit” (Hycult Biotech). It was measured.
Further, the amount of protein in the tissue suspension was measured, and the amount of arginase per unit protein was calculated.
Arginase 1 production in the tissue without the sample was taken as 100%, and the arginase 1 production rate when the test sample was added was calculated.

2. Results The measurement results are shown in Table 2 and FIG.

  As shown in Table 2 and FIG. 2, an increase in the amount of arginase 1 in the three-dimensional human epidermis model tissue was confirmed for both stigmasterol and oleanolic acid.

From the above two tests, it was revealed that stigmasterol and oleanolic acid increase the activity of arginase 1 in the cells, and further promote the production of arginase 1 in the epidermal tissue from a three-dimensional human epidermis model test. .
Moreover, since the production amount of arginase 1 in the tissue increased, it was expected that the urea cycle was activated and the detoxification effect of ammonia was enhanced. Furthermore, it was expected that the amount of urea in the hiff tissue would increase. Therefore, it was expected to contribute to softening and water retention of the hiff.

<Test 3: Whitening effect test of stigmasterol>
In the course of the above test, a phenomenon in which the pigment of melanocytes became thin was observed, so a test for confirming the whitening effect was performed.
1. Test method Melanonoderm (manufactured by MatTek) (black donor type), a 3D human epidermis model constructed from normal human keratinocytes and melanocytes, is opened within 24 hours after delivery, and EPI-100LLMM maintenance medium according to the standard method Culture was started with (MatTek). After 24 hours, an evaluation sample was prepared with DPBS (−), and 100 μl was administered onto the skin model cup. Stigmasterol was selected as an evaluation sample. In addition, kojic acid, which is known to have a melanin production inhibitory effect, was selected as a positive control. The medium and sample were changed every 48 hours.
The cells were cultured for 8 days from the start of sample administration. After completion of the culture, the tissue was cut with a 3.5 mm biopsy punch, and the two sections were placed in 150 μl of 1N sodium hydroxide aqueous solution, heated and dissolved at 100 ° C. for 1 hour, and the absorbance at a wavelength of 475 nm was measured to measure the amount of melanin. did. Then, the amount of melanin per unit protein was calculated. The melanin production rate when the sample was added was calculated with the amount of melanin when the test sample was not added as 100%.

2. Results The test results are shown in Table 3 and FIG.

2. Results As shown in Table 3 and FIG. 3, stigmasterol is useful as a whitening agent because it suppresses melanin production in the Hiff tissue at a concentration of 1/70 or less of kojic acid known as a whitening component. I was able to confirm.

Claims (4)

Production promoter arginase 1 containing 5.7~250μg / mL and an active ingredient oleanolic acid. Urea cycle activator comprising 5.7~250μg / mL with an active ingredient oleanolic acid.   A hyperammonemia-improving agent containing oleanolic acid as an active ingredient. Production promoter of arginase 1 skin tissue containing 62.5~250μg / mL oleanolic acid as an active ingredient.