JP2022108114A - Autophagy derivative - Google Patents

Abstract

To provide a skin-whitening agent, autophagy derivative, and epidermis hyperplasia inhibitor.SOLUTION: Provided are a humus extract containing fulvic acid and mineral, and/or a skin-whitening agent including uncoupler other than fulvic acid, an autophagy derivative including uncoupler in keratinocyte, and an epidermis hyperplasia inhibitor including uncoupler.SELECTED DRAWING: None

Description

There is an application for application of Article 30, Paragraph 2 of the Patent Law Date January 20, 2020 to January 22, 2020 Meeting name 10th Cosmetic Development Exhibition Venue Makuhari Messe (2-1 Nakase, Mihama-ku, Chiba-shi, Chiba)

TECHNICAL FIELD The present invention relates to a whitening agent, an autophagy inducer, and an epidermal hyperplasia inhibitor.

Autophagy is a general term for systems that degrade parts of the cytoplasm, such as cytoplasmic components including proteins and intracellular organelles such as mitochondria. ing. In autophagy, the isolating membrane generated from the intracellular rough endoplasmic reticulum/mitochondrial contact site takes in substrates, forms a double-membrane structure called autophagosome, fuses with lysosomes, and forms a single-membrane automaton. It is a series of processes in which phagolysosomes are formed, degrading enzymes are supplied, and internal substrates are decomposed. Autophagy is classified into macroautophagy, microautophagy and chaperone-mediated autophagy, and autophagy usually refers to macroautophagy. Autophagy is reported to be involved in skin color determination by regulating melanosome digestion in keratinocytes (Non-Patent Document 1).

Spots, freckles, and post-sunburn skin pigmentation occur as a result of increased melanin production due to activation of melanocytes present in the skin, and are one of the skin concerns of middle-aged and elderly people. In general, melanin is formed by the action of tyrosinase, an enzyme that is biosynthesized in pigment cells, and various whitening ingredients that inhibit tyrosinase activity and suppress melanin production are available. Proposed.

Humus is a polymer formed mainly by decomposition of carbohydrates and proteins in plant residues, microbial remains, etc. in soil over a long period of time by microorganisms and condensation of the decomposition products. Humus contains amorphous polymers such as fulvic acid and humic acid. Humic acid is a reddish-brown or black-brown amorphous polymeric organic acid that is soluble in alkali and insoluble in acid. On the other hand, fulvic acid is an amorphous macromolecular organic acid that is soluble in alkalis and acids. Humus is known to contain fatty acids, organic acids, amino acids, proteins, minerals, etc., in addition to fulvic acid and humic acid.

In this way, fulvic acid (FA) is an organic acid that exists in humus, and humus containing fulvic acid is recognized in some parts of the United States and Saga Prefecture in Japan, and industrial standardization of fulvic acid is recommended. It is

In addition, in Patent Document 1, the present inventors reported that fulvic acid has a marked effect on lifestyle-related diseases, that liver function is improved, and that fulvic acid acts as an uncoupling agent. there is

Regarding humic acid, for example, Non-Patent Document 2 reports its anti-inflammatory action.

WO2019/146790

Journal of Investigative Dermatology (2013) 133, 2416-2424 Phytother Res. 2015 Jun;29(6):791-5

The present invention provides a whitening agent, an autophagy inducer, and an epidermal hyperplasia inhibitor.

As a result of intensive studies aimed at achieving the above object, the present inventors have found that fulvic acid has the effect of activating autophagy in keratinocytes. Furthermore, based on the activation of autophagy by such fulvic acid, the inventors found that the digestive activity of melanosomes is activated in keratinocytes, and that fulvic acid containing minerals in particular has excellent activity. They also found that by combining fulvic acid with an uncoupling agent other than fulvic acid, the digestive activity of melanosomes can be further activated in keratinocytes.

The present invention was completed through further studies based on these findings, and provides the following whitening agents, autophagy inducers, and epidermal hyperplasia inhibitors.

Section 1. A whitening agent comprising a humic extract containing fulvic acid and minerals and/or an uncoupling agent other than fulvic acid.
Section 2. Item 2. The whitening agent according to item 1, wherein the uncoupling agent is metformin.
Item 3. Autophagy inducers in keratinocytes containing uncouplers.
Section 4. Item 4. The autophagy inducer according to Item 3, wherein the uncoupling agent is a humic extract containing fulvic acid and/or metformin.
Item 5. An epidermal thickening inhibitor containing an uncoupling agent.
Item 6. Item 6. The epidermal hyperplasia inhibitor according to Item 5, wherein the uncoupling agent is a humic extract containing fulvic acid and/or metformin.

The uncoupling agent has a remarkably excellent autophagy activation action in keratinocytes, and a whitening action and an epidermal hyperplasia suppression action based on the autophagy activation action. It is useful as an active ingredient in inhibitors.

In addition, humus extracts containing fulvic acid and minerals are particularly useful as an active ingredient of skin whitening agents because of their excellent whitening effect. Furthermore, by combining fulvic acid with an uncoupling agent other than fulvic acid, the whitening action can be further improved.

1 is a graph showing the effect of autophagy inhibitors on the digestive activity of melanosomes taken up by keratinocytes. n=8. Data represent the mean ± s.e.m. after subtracting the absorbance in wells to which no melanosomes were added (Blank). One-way ANOVA. ***: P<0.005 vs CRL, ###: P<0.005 vs Hu. FIG. 10 is a graph showing the additive effect of the uncoupler metformin (MET) and Humicle on the digestive activity of melanosomes taken up by keratinocytes. n=6. Data represent the mean ± s.e.m. after subtracting the absorbance in wells to which no melanosomes were added (Blank). One-way ANOVA. ***: P<0.005 vs CRL. Fig. 10 is a graph showing the synergistic effect of fulvic acid and minerals on digestive activity of melanosomes incorporated into keratinocytes. n=6. Data represent the mean ± s.e.m. after subtracting the absorbance in wells to which no melanosomes were added (Blank). One-way ANOVA. ***: P<0.005 vs CRL.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail.

As used herein, the term "comprise" includes both the meaning of "essentially consisting of" and the meaning of "consisting only of."

The whitening agent of the present invention is characterized by containing an uncoupling agent (in particular, a humus extract containing fulvic acid and minerals, and/or an uncoupling agent other than fulvic acid). Further, the autophagy inducer and the epidermal hyperplasia inhibitor of the present invention are characterized by containing an uncoupling agent.

The uncoupling agent is a compound that inhibits the coupling of the electron transport chain and the ATP synthesis reaction in the oxidative phosphorylation of organisms, and the uncoupling agent in the present invention is not particularly limited to known uncoupling agents. can be used for Such uncoupling agents include, for example, humus extracts containing fulvic acid, metformin, buformin, phenformin, and the like. In the present invention, the uncoupling agent can be used singly or in combination of two or more.

Fulvic acid is an amorphous macromolecular organic acid that is soluble in alkalis and acids among substances contained in humus. Any humus can be used as the humus as a raw material of fulvic acid without any particular restrictions on the place of production, condition, and the like.

Such humus includes, for example, humic shale obtained from ancient plant deposits (shales) in Emery County, Utah, USA. Humic shale is the nutrient-rich fertile soil from about 70 to 127 million years ago that has survived as organic matter without being transformed into oil and coal. Plants that lived in the above period were buried underground during the Ice Age, changed to peat moss by microorganisms such as bacteria, and new organic matter piled up on top of it, and was again decomposed by bacteria, etc., resulting in sedimentary layers over time. is formed. The humus contained in the formed sedimentary layer is mainly composed of organic macromolecular polymers such as fulvic acid and humic acid, and contains trace amounts of more than 70 kinds of minerals.

A method for extracting fulvic acid from humus is not particularly limited, and various known methods can be used. Extraction means include, for example, solid-liquid extraction, liquid-liquid extraction, immersion, leaching, decoction, reflux extraction, supercritical fluid extraction, microwave extraction, mixing and stirring, and the like. The extraction means can be carried out singly or in combination of two or more.

When extracting fulvic acid by mixing humus and an extraction solvent, the extraction solvent includes, for example, water (e.g., distilled water, ion-exchanged water, reverse osmosis water, etc.), alcohol (e.g., methanol, ethanol , propanol, etc.), mixtures thereof, and the like. The extraction time, extraction temperature, pH of the extraction solvent, and the like in the extraction operation can be appropriately set in consideration of extraction efficiency and the like.

The humus extract may be further filtered for removal of debris and sterilization. Filtration means are not particularly limited, and can be carried out by conventional methods.

The humus extract can be further processed to isolate or purify fulvic acid. Examples of such isolation or purification treatment include concentration, concentration under reduced pressure, distillation, fractional distillation, redissolution, solvent extraction, crystallization, recrystallization, chromatography and the like. These methods can be carried out singly or in combination of two or more.

The humus extract used in the present invention is not particularly limited as long as it is extracted from humus (humus, humus) and contains fulvic acid, preferably fulvic acid and minerals. In addition, the humus extract used in the present invention can be produced by the method described above.

The pH of the humus extract is in the acidic range, eg pH 1-6, preferably pH 2-5, especially pH 3. The pH of the humus extract may be appropriately adjusted using a pH adjuster.

As the humic extract, both those in which fulvic acid is not isolated or purified (such as crude extracts) and those in which fulvic acid is isolated and purified can be used.

It is known that humus extracts contain fatty acids, organic acids, amino acids, proteins, minerals, etc. in addition to fulvic acid and humic acid. In that case, the humus extract in the present invention may contain fatty acids, organic acids, amino acids, proteins, minerals, and the like, especially minerals, in addition to fulvic acid. Since fulvic acid can chelate minerals, when minerals are contained in the humus extract, fulvic acid may be in a state of chelating minerals. Thus, the inclusion of minerals in addition to fulvic acid in the humus extract improves the whitening effect.

Also, fulvic acid can be used in a free state or in a salt state. Examples of salts of fulvic acid include inorganic salts such as sodium, magnesium, potassium, calcium and aluminum; salts with organic bases such as methylamine, ethylamine and ethanolamine; salts with basic amino acids such as lysine, ornithine and arginine. salts and ammonium salts. The salt may be an acid addition salt, and specific examples of such salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, organic acids such as propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, ethanesulfonic acid; acid amino acids such as aspartic acid and glutamic acid; Acid addition salts are included. Fulvic acid also includes hydrates, solvates, and the like.

The form of the humus extract is not particularly limited, and examples thereof include powder, granule, solid, solution, suspension, emulsion, liquid concentrate, and slurry.

Both homemade and commercially available humus extracts can be used. Commercially available humus extracts include, for example, “Humicle (registered trademark) HC fulvic acid powder” sold by Style and Value Japan. The fulvic acid contained in the humicle has infrared absorption peaks at wave numbers 3362, 2875, 1675, 1559, 1360, 1200, 1047 and 835 cm ^-1 in the infrared absorption spectrum in the FT-IR method (see Japanese Patent No. 6120342 ). In addition, the average molecular weight of fulvic acid contained in the humicle is usually less than 4,000, preferably 3,500 or less, particularly preferably 3,000 or less, and still more preferably 1,000 to 3,000. The humicle contains 90% by mass or more of minerals, and the content of fulvic acid is less than 10% by mass.

The whitening agent of the present invention can further contain an uncoupling agent other than fulvic acid, and such a combination of fulvic acid and an uncoupling agent other than fulvic acid can further improve the whitening effect.

The ratio of the uncoupling agent contained in the whitening agent, autophagy inducer, and epidermal thickening inhibitor of the present invention includes, for example, 0.0001 to 99% by mass, 0.001 to 80% by mass, and 0.01 to 70% by mass. .

The whitening agent, autophagy inducer, and epidermal thickening inhibitor of the present invention can be used in cosmetics, food and drink (particularly food and drink intended for health, health maintenance, promotion, etc. (e.g., health food, functional food, nutritional composition, etc.). It can be used as a product (nutritional composition), dietary supplement, supplement, health food, food for specified health uses, food with nutrient function claims, or food with function claims)), quasi-drugs, pharmaceuticals, and the like. In addition, the whitening agent, autophagy inducer, and epidermal hyperplasia inhibitor of the present invention also include additives that impart a whitening effect, an autophagy inducer, and an epidermal hyperplasia inhibitory effect.

The uncoupling agent can be used as it is in the above food and drink, and if necessary, it can be used as an excipient, vitamins, minerals, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents. Agents, binders, sweeteners, disintegrants, lubricants, coloring agents, flavors, stabilizers, preservatives, sustained-release modifiers, surfactants, brighteners, solubilizers, wetting agents, etc. can.

Food and drink include all food and drink that can be ingested by mammals (including humans), such as dairy products; fermented foods (yogurt, cheese, etc.); beverages (coffee, juice, cocoa, tea drinks, sports drinks, Soft drinks such as nutritional drinks, milk drinks, lactic acid drinks, drinks containing lactic acid bacteria, yoghurt drinks, carbonated drinks, Japanese sake, western liquor, alcoholic beverages such as fruit wine, etc.); spreads (custard cream, etc.); pastes (fruit pastes) etc.); Confectionery (chocolate, donut, pie, cream puff, gum, gummy, jelly, candy, cookie, cake, pudding, biscuit, etc.); Beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, ham, sausage, bacon, etc.); seasonings (dressing, furikake, umami seasoning, soup base, miso, soy sauce, sauce, ketchup, oyster sources, etc.).

The method for producing the food and drink is not particularly limited, either, and any known method can be followed as appropriate.

The dosage unit form when used as a supplement is not particularly limited and can be appropriately selected, and examples thereof include tablets, capsules, granules, liquids, powders and the like.

The intake amount of food and drink can be appropriately set according to various conditions such as the body weight, age, sex, and symptoms of the person who consumes the food.

The uncoupling agent alone can be used in the above medicines, or it can be used in combination with other pharmaceutical ingredients such as vitamins and herbal medicines described in the Japanese Pharmacopoeia.

When the whitening agent, autophagy inducer, and epidermal hyperplasia inhibitor of the present invention are prepared as pharmaceuticals, an uncoupling agent is added to a tablet (raw material) together with a pharmaceutically acceptable non-toxic carrier, diluent or excipient. tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders (powders), fine granules, granules, liquids, suspensions, emulsions, Syrups, pastes, injections (including cases where they are mixed with distilled water or infusions such as amino acid or electrolytes at the time of use) can be prepared in the form of pharmaceutical preparations. is.

The method of administration of the drug is not particularly limited, and for example, transdermal administration, oral administration, intraarterial administration, intravenous administration, intraoral administration, rectal administration, enteral administration, and the like can be performed.

The dosage of the drug can be appropriately determined according to various conditions such as patient's body weight, age, sex and symptoms.

In addition to the uncoupling agent, the above-mentioned cosmetics contain ingredients that are commonly used in cosmetics, such as whitening agents, moisturizing agents, antioxidants, oily ingredients, ultraviolet absorbers, surfactants, thickeners, alcohols, and powders. Ingredients, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as needed.

Cosmetics include all cosmetics applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, etc. of animals (including humans).

There are a wide range of formulations for cosmetics, including aqueous solutions, solubilized systems, emulsified systems, powder systems, oil systems, gel systems, ointment systems, aerosol systems, water-oil two-layer systems, and water-oil-powder three-layer systems. Can take dosage form.

The use of cosmetics is also arbitrary, and for example, basic cosmetics include face wash, lotion, milky lotion, cream, gel, essence, serum, pack, mask, etc., and makeup cosmetics include foundation, Lipstick, blush, eye shadow, eyeliner, mascara, etc., nail cosmetics, nail polish, base coat, top coat, nail polish remover, etc.; , mouthwash, massage agent, cleansing agent, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling agent, hair tonic, hair restorer, antiperspirant, bath agent, etc. is mentioned.

The whitening agent, autophagy inducer, and epidermal hyperplasia inhibitor of the present invention are applicable to mammals including humans (preferably humans).

In addition, quasi-drugs are also included in the medicines and cosmetics in the present invention.

As shown in the test examples below, the present inventors found that the uncoupling agent fulvic acid activates autophagy in keratinocytes. Furthermore, it was observed that fulvic acid activates the digestive activity of melanosomes in keratinocytes based on the activating action of autophagy, and whitening action can be expected. As described above, fulvic acid has a whitening effect such as pigmentation after sunburn, lightening of spots, freckles, erythema, etc., and making dullness inconspicuous, and can maintain healthy skin. , skin care cosmetics, etc.

Autophagy-related gene deficiency is known to exhibit a phenotype characterized by thickening of the epidermal layer (Biochem Biophys Res Commun. 2013 Jan 11;430(2):689-94., J Dermatol Sci. 2013 Jul;71(1):67-75., Exp Dermatol. 2018 Oct;27(10):1142-1151.). Since it is known that the epidermis thickens when autophagy does not occur, fulvic acid has an autophagy-activating effect in keratinocytes, and is therefore expected to have an effect of suppressing epidermal hyperplasia.

Therefore, the uncoupling agent has a remarkably excellent autophagy activating action, and a whitening action and an epidermal hyperplasia inhibitory action based on the autophagy activating action. It can be suitably used as an active ingredient of an inhibitor. A humus extract containing fulvic acid and minerals has a more excellent skin-whitening effect and can be particularly suitably used as an active ingredient of a skin-whitening agent. Further, by combining fulvic acid with an uncoupling agent other than fulvic acid, the whitening effect can be further improved.

In addition, since fulvic acid is a naturally-derived component, it is highly safe.

Examples are given below to describe the present invention in more detail. However, the present invention is by no means limited to these examples.

Test example 1
pMRX-IP-GFP-LC3-RFP (addgene, #84573) was subcloned into the pMXs-puro retroviral vector. Using this, HaCaT stably expressing GFP-LC3-RFP was established by drug selection with 4 μg/ml puromycin, and autophagy was measured using this. In the experiment, MEM essential amino acid solution (50 ×) (WAKO) was added to an amino acid-free autophagy-specific medium (WAKO, Osaka, Japan) supplemented with 20% fetal bovine serum, 10 ng/ml hrEGF and antibiotics. A final concentration of × (1×AA) was added (hereinafter referred to as an autophagy-inducing medium), and Humicle was added at the same time. After culturing the cells in the medium, the cells were collected and the fluorescence intensities of GFP and RFP were measured with a flow cytometer.

From the results of this experiment, it was confirmed that the addition of Humicle enhances autophagy in a concentration-dependent manner.

Test example 2
The B16 melanoma cell line was purchased from the National Institute of Biomedical Innovation (JCRB0202, Osaka, Japan) and cultured in Dulbecco's Eagle's minimum essential medium supplemented with 10% fetal bovine serum. After the cells seeded in the 96-well plate became confluent, they were treated with Humicle. After 72 hours, the cells were solubilized with DMSO and the absorbance at 490 nm was measured using a microplate reader.

The results of this experiment confirmed that humicles do not affect melanin synthesis in melanoma.

Test example 3
Mouse melanoma B16 cells were crushed by adding 10 ml of cell lysate (0.1 M Tris, 1% NP-40, 0.01% SDS, pH 7.5) to 1×10 ⁸ cells, and mixing by pipetting. It was left on ice for 30 minutes. The cell lysate was centrifuged (1,000 g, 10 minutes), and the supernatant was recovered and centrifuged again (20,000 g, 10 minutes). The pellet was washed with PBS(-), centrifuged (20,000 g, 10 minutes), and suspended in 1 ml of autophagy-inducing medium to prepare a melanosome-extracted pellet. 6 μl (equivalent to 7.5×10 ⁵ cells) of melanosomes were added to each well.

HaCaT was seeded in 96-well plates. When the cells were confluent, melanosomes prepared from the B16 melanoma cell line were added with daily medium changes. Four days later, the cells were treated with Humicle using an autophagy-inducing medium. After 72 hours, the cells were solubilized with DMSO and the absorbance at 490 nm was measured using a microplate reader.

From the results of this experiment, it was confirmed that the addition of Humicle improved the digestive activity of melanosomes in keratinocytes in a concentration-dependent manner.

Test example 4
The effect of 3-methyladenine (3MA), an autophagy inhibitor, on melanin-digesting activity of keratinocytes was investigated. The procedure was carried out in the same manner as in Test Example 3, except that 3MA was added.

The results are shown in FIG. 5 mM 3MA reversed the melan-removing effect of humicule (Hu) on melanosome-loaded HaCaT. This indicates that melanin digestion in keratinocytes was caused by autophagy.

Test example 5
The effect of metformin (MET), an uncoupling agent, on the melanin-digesting activity of keratinocytes was investigated. The test was performed in the same manner as in Test Example 3, except that MET was added.

The results are shown in FIG. 500 μM MET and 0.0001% Hu each alone did not show significant melanosome-digesting activity, but both showed significant effects when treated simultaneously.

Test example 6
The effects of fulvic acid and minerals on the melanin-digesting activity of keratinocytes were investigated. The procedure was the same as in Test Example 3, except that fulvic acid and minerals were added. The mineral was obtained by subjecting Humicle to intense heat treatment (600° C., 12 hours×2 times) in an electric furnace, and repeating this process until the amount of the product reached 200 g.

The results are shown in FIG. No melanosome digestive activity was observed when exposed to 0.0001% standard fulvic acid (FA) from Dando purchased from the Japan Society of Humic Substances or to 0.01% minerals obtained by burning organic matter from Humicle. Simultaneous treatment showed a significant effect.

Claims (5)

A whitening agent comprising a humic extract containing fulvic acid and minerals and/or an uncoupling agent other than fulvic acid. 2. A whitening agent according to claim 1, wherein said uncoupling agent is metformin. Autophagy inducers in keratinocytes containing uncouplers. 4. The autophagy inducer according to claim 3, wherein the uncoupling agent is a humic extract containing fulvic acid and/or metformin. An epidermal thickening inhibitor containing an uncoupling agent.

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