Glutathione production promoter

Abstract

To provide a novel glutathione production promoter.SOLUTION: Provided is a glutathione production promoter that has one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, striped bamboo, Polygonatum falcatum, yam, Moringa, devil's claw, Houttuynia cordata, and Gymnema sylvestre as an active ingredient.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention provides a glutathione production promoter. [Background technology]

[0002] Glutathione is an antioxidant component that is constantly present in cells at levels on the order of several mM, and is thought to exert a whitening effect together with cysteine ​​as a sulfide (SH)-containing molecule (Non-Patent Documents 1 and 2).

[0003] To date, known glutathione production promoters include N-acetylcysteine, 4-vinylguaiacol, a water extract of echinacea cultivated using a specific method, methoxychalcones, a group consisting of piceatannol and skirpsin B, and triterpenoid-containing plants (Patent Documents 1 to 6). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4598740 [Patent Document 2] Japanese Patent Application Publication No. 2019-65046 [Patent Document 3] Japanese Patent Application Publication No. 2019-6722 [Patent Document 4] Patent No. 6112920 [Patent Document 5] Patent No. 6294579 [Patent Document 6] Japanese Patent Application Laid-Open No. 2010-105937 [Non-patent literature]

[0005] [Non-Patent Document 1] J Cosmet Dermatol. 2019;18:728-737 [Non-patent document 2] Cellular and Molecular Biology 1999, Vol.45 No.7, 981-990 [Non-patent document 3] PLoS ONE 14(5): e0217394.https: / / doi.org / 10.1371 / journal.pone.0217394, May 28, 2019 [Non-patent document 4] Wako Pure Chemical News Vol.87 No.2 (June 2019 issue), p.9-11 [Non-patent document 5] Yoshinori Katakura, Searching for Anti-Brain-Aging Foods and Elucidating the Molecular Basis of Their Functionality, Asahi Beer Foundation, April 2010 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a glutathione production promoter. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, Kumazasa, Narcissus lily, Yamaimo, Moringa, Devil's claw, Houttuynia cordata, and Gymnema function as glutathione production promoters. Furthermore, the present inventors have found that these substances act on skin cells via intestinal cells. Based on these findings, the following inventions have been completed: (1) A glutathione production promoter containing, as an active ingredient, one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, Japanese bamboo grass, narcissus lily, mountain yam, moringa, devil's claw, Houttuynia cordata, and gymnema. (2) The glutathione production promoter according to (1), which promotes glutathione production in skin cells via intestinal cells. (3) The glutathione production promoter according to (1) or (2), which promotes glutathione production in skin cells when taken orally. (4) A composition containing the glutathione production promoter according to any one of (1) to (3). [Effects of the Invention]

[0008] Glutathione production can be promoted by administering the glutathione production promoter of the present invention. According to the present invention, a composition or an oral preparation containing the glutathione production promoter can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of the method used in Experiment 3 to evaluate interactions between intestinal epithelial cells and epidermal keratinocytes. [Figure 2] FIG. 2 shows the amount of glutathione in HaCat cells when each sample was added in Experiment 3, expressed as a relative value, with the amount when the negative control (dH 2 O or DMSO only) was added being set at 100. DETAILED DESCRIPTION OF THE INVENTION

[0010] Administration of the glutathione production enhancer of the present invention can promote glutathione production. Glutathione (Glutathione, GSH, Glutathione-SH) is a tripeptide composed of three amino acids (glutamic acid, cysteine, and glycine). It exists in both oxidized and reduced forms; however, the present specification refers to the reduced form (CAS number: 70-18-8). Reduced glutathione has been suggested to be useful for skin whitening, suppressing UV damage, erythema, pigmentation, oxidative stress, radical scavenging, regulating cellular function through redox, eliminating xenobiotics, and as an SH donor for various enzymes. It also has been suggested to have a whitening effect by promoting the dominance of skin-colored melanin (pheomelanin) over dark melanin (eumelanin) (Non-Patent Documents 1 and 2). The glutathione production enhancer of the present invention is expected to contribute to skin whitening and other skin beautification by promoting glutathione production in skin cells via the intestinal tract, particularly when taken orally.

[0011] In the present invention, beautiful skin refers to improvement of skin condition through promotion of glutathione production in skin cells, such as skin whitening, suppression of UV damage, suppression of erythema, suppression of spots, suppression of oxidative stress, and scavenging of radicals.

[0012] "Promotion of glutathione production" can mean, for example, that the amount of glutathione produced when a glutathione production promoter is administered is increased compared to when nothing is administered (control). For example, it can mean that the increase is statistically significant (e.g., Student's t-test) at a significance level of 5%. Alternatively, "promotion of glutathione production" in the present invention can mean that the amount of glutathione produced when a glutathione production promoter is administered is increased by, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 400% or more, or 500% or more compared to when nothing is administered (control).

[0013] The glutathione production enhancer of the present invention enhances glutathione production in skin cells via intestinal cells. "Enhancing glutathione production in skin cells via intestinal cells" refers to ultimately promoting glutathione production in skin cells via direct or indirect pathways, such as administering an active ingredient of the present invention to intestinal cells and absorbing it, resulting in the active ingredient directly reaching skin cells, or the active ingredient being absorbed by intestinal cells and reaching skin cells in a decomposed or modified state, or the active ingredient being taken up by intestinal cells and releasing a different component, which then acts on skin cells. An example of "enhancing glutathione production in skin cells via intestinal cells" is promoting glutathione production in skin cells by oral administration. "Enhancing glutathione production in skin cells by oral administration" refers to orally ingesting the glutathione production enhancer of the present invention, resulting in the active ingredient being absorbed via the intestinal tract, and the substance taken up from the intestine acting directly or indirectly to ultimately promote glutathione production in skin cells.

[0014] For example, even if a certain component is taken orally from the intestine, the component does not directly reach the target cells, but rather another component secreted by intestinal cells may reach the target cells and act indirectly. For example, as described in Non-Patent Documents 3-5, it has been reported that carnosine taken up from the intestine does not directly affect the brain, but rather activates CREB in intestinal cells, thereby enhancing BDNF production, which in turn activates neurons and improves brain function. Furthermore, it has been suggested that this activation of the brain-gut correlation is mediated by exosomes. In other words, even if a component is administered to the intestine, it is unclear what effect the component will have on the target cells. Therefore, it is necessary to administer the test substance to intestinal cells and then confirm what effect it has on the target cells.

[0015] Such intestinal cell-mediated effects can be measured by various methods, including in vivo, in vitro, and ex vivo. For example, they can be determined by administering a test substance to an intestinal cell layer and measuring glutathione production in skin cells via the intestinal cell layer. For example, as in the Examples herein, this can be determined by a method for evaluating intestinal epithelial cell-epidermal keratinocyte interactions, in which a test substance is added to one side of a layer of intestinal cells such as Caco-2, and the test substance passes through the layer to measure the amount of glutathione in skin cells such as HaCaT present on the opposite side of the layer. Methods for evaluating intestinal epithelial cell-epidermal keratinocyte interactions are known in the art or can be easily performed by those skilled in the art. However, the measurement method is not limited to the above methods, and any other method may also be employed. For example, an in vitro method may be employed, in which a test substance is administered to an intestinal cell culture medium and the resulting supernatant is added to skin cells, or an in vivo method may be employed, in which the test substance is orally administered to an animal such as a human and the amount of glutathione in the skin may be measured.

[0016] The amount of glutathione may be measured using a probe whose fluorescence intensity ratio of wavelengths changes depending on the increase or decrease in reduced glutathione concentration, as described in the Examples, or by any other known technique.

[0017] The present invention provides a glutathione production promoter containing, as an active ingredient, one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, Kuma bamboo, Narcissus lily, Yamaimo, Moringa, Devil's claw, Houttuynia cordata, and Gymnema.

[0018] The present invention provides a glutathione production enhancer containing, as an active ingredient, one or more herbal medicines selected from the group consisting of Mallotus japonicus, radish, Kuma bamboo, narcissus, mountain yam, moringa, devil's claw, Houttuynia cordata, and gymnema. The present invention also provides a composition containing, as an active ingredient, one or more herbal medicines selected from the group consisting of Mallotus japonicus, radish, Kuma bamboo, narcissus, mountain yam, moringa, devil's claw, Houttuynia cordata, and gymnema. The glutathione production enhancer and composition of the present invention may be in the form of an oral or enteral preparation, and may be for skin beautification.

[0019] The Mallotus japonicus (scientific name: Mallotus japonicus, Mueller Argoviensis, also known as Cymbolle, Misomoliba, and Gosaiba) used in the present invention is a deciduous tree of the Euphorbiaceae family. It is preferable to use the bark of Mallotus japonicus. The bark contains bergenin, rutin, and tannin, the leaves contain geraniin, rutin, maloprenol, and its linolenic acid ester, and the seeds contain colotoxigenin, malogenin, and cologlaucigenin. It is said to be effective against eczema, rashes, and itching.

[0020] The radish used in the present invention (scientific name: Raphanus sativus var. longipinnatus, also known as Japanese radish or Mooli) is a biennial plant of the Brassicaceae family, genus Raphanus. It is preferable to use radish roots. Radish is widely cultivated as a vegetable, and its leaves are rich in vitamin A and are used as an ingredient in green juice. Its roots are rich in vitamin C and diastase.

[0021] The Kumazasa (scientific name: Sasa veitchii, also known as Bamboo grass or Tall groundcover) used in the present invention is an evergreen bamboo of the Poaceae family, the genus Sasa. It is preferable to use Kumazasa leaves. Kumazasa leaves contain chlorophyll, polysaccharides, lignans, various vitamins and minerals, and dried leaves are said to be effective against high blood pressure, diabetes, and antibacterial properties.

[0022] The narcissus used in the present invention (scientific names: Polygonatum falcatum A. Gray, Polygonatum sibiricum Redout?, Polygonatum kingianum Collett et Hemsley, Polygonatum cyrtonema Hua) is a perennial herb belonging to the Polygonatum genus of the Liliaceae family. It is preferable to use the rhizome. It is said to be effective as a nourishing tonic.

[0023] The yam used in the present invention (scientific name: Dioscorea japonica Thunberg, Dioscorea batatas Decaisne, Dioscorea batatas, also known as Dioscorea japonica or Dioscorea batatas) is a climbing perennial plant of the Dioscoreaceae family, Dioscorea genus. It is preferable to use the rhizomes of Dioscorea japonica. It is edible and is said to have nutritional and strengthening effects.

[0024] The moringa used in the present invention (scientific name: Moringa oleifera LAM. (M. pterygosperma), also known as Moringa) is a plant of the Moringaceae family, Moringa genus, and it is preferable to use moringa leaves. Moringa leaves are used for food and medicine and are said to be effective in providing nutrition. They contain various vitamins and minerals and are known as a superfood.

[0025] Devil's claw (scientific name: Harpagophytum procumbens DC., also known as Lion's Claw) used in the present invention is a perennial plant of the Pedaliaceae family. It is preferable to use devil's claw tubers. They contain harpagoside, phenols, flavonoids, luteolin glycoside, etc., and are said to have anti-inflammatory and pain-reducing effects.

[0026] The Houttuynia cordata (scientific name: Houttuynia cordata Thunberg, also known as Jyaku, Shifuki, Isakoroshi, and Jigokusoba) used in the present invention is a perennial plant of the Houttuynia genus in the family Saururaceae. It is preferable to use the above-ground parts of Houttuynia cordata. It is used internally and as a tea, and is said to be effective for gastrointestinal disorders, detoxification, food poisoning, diarrhea, constipation, and diuresis.

[0027] Gymnema sylvestre (Retz.) Schult, also known as Gymnema sylvestre, is a climbing plant of the genus Gymnema in the family Apocynaceae. It is preferable to use the leaves. The leaves contain gymnemic acid and glucuronic acid, which have been reported to have appetite suppressing effects and to prevent a sudden rise in blood sugar levels.

[0028] The above-mentioned herbal medicines are known substances, can be easily dried, purified, extracted, etc. by known methods, and are readily available as commercial products. They can be used either raw or dried, but from the viewpoint of usability, formulation, etc., they can also be used as extracts, dried products, dried powders, powdered raw materials, squeezed juices, etc. The form to be used can be appropriately selected depending on the raw material, and treatments such as sterilization may be performed as necessary.

[0029] When used as an extract, the extract can be extracted, for example, by solvent extraction. In solvent extraction, the whole plant or various parts (e.g., leaves, flowers, roots) of the plant are dried as needed, and further shredded or crushed as needed. The extract is then extracted using an aqueous extractant, water (e.g., cold water, warm water, or hot water at or below boiling point), or a hydrous organic solvent, or an organic solvent (e.g., ethanol, methanol, ether, 1,3-butylene glycol), selected appropriately depending on the properties of the raw materials and the intended use of the composition, at room temperature or with heating. However, the extraction method is not limited to solvent extraction and may be any conventional method known in the art. The extraction method and form of the extract used in the present invention are arbitrary as long as they do not impair the effects of the present invention. The extract may be in the form of a liquid extract itself, or may be diluted or concentrated as appropriate using conventional methods. It may also be in the form of a powder or lumpy solid obtained by drying the extract.

[0030] Examples of the aqueous organic solvent include aqueous lower alcohols such as aqueous ethanol, and in this case, the water content may be, for example, 0 to 10 v / v%, 10 to 40 v / v%, 20 to 30 v / v%, 30 to 50 v / v%, 50 to 80 v / v%, 80 to 99.5 v / v%, etc.

[0031] Methods for obtaining dry powder include shredding or crushing the whole plant or various parts (leaves, flowers, roots, etc.) and then drying, or drying the plant and then shredding or crushing it to obtain dry powder. Alternatively, methods such as shredding or crushing the plant, fermenting or enzymatically treating it, drying it, and then crushing it to a predetermined particle size as needed can also be used.

[0032] The glutathione production promoter of the present invention is preferably taken orally or enterally, but other administration routes such as transdermal administration are not excluded. When the glutathione production promoter of the present invention is administered by various administration routes, it is preferable to apply the active ingredient of the present invention in an amount that fully exhibits the glutathione production-promoting effect. The amount of the herbal medicine of the present invention to be added can be determined appropriately depending on the type, purpose, form, method of use, etc.

[0033] When the glutathione production enhancer of the present invention is used as an oral or enteral preparation, it is preferably prepared so that the dry weight of the plant body or its solvent extract is about 0.00001 to 5% by weight, more preferably about 0.0001 to 0.5% by weight, and even more preferably about 0.001 to 0.05% by weight, based on the total weight of the oral or enteral preparation. Alternatively, it may be prepared so that the concentration in the intestine is about 1 to 1000 μg / ml, more preferably about 5 to 500 μg / ml, and even more preferably about 10 to 100 μg / ml.

[0034] Furthermore, when the glutathione production promoter of the present invention is used as an oral or enteral preparation, the intake amount of Mallotus japonicus, Japanese radish, Kumazasa, Narcissus japonica, Yamaimo, Moringa, Devil's claw, Houttuynia cordata, and Gymnema or a solvent extract thereof per adult is preferably prepared so as to be, for example, about 0.01 mg to about 1,000 mg (dry weight equivalent), more preferably about 0.1 mg to about 100 mg, and even more preferably about 1.0 mg to about 10 mg (dry weight equivalent) per day. The frequency of intake is not limited, but may be once a day, or, for example, once every two weeks, once a week, once every three days, once every two days, once a day, twice a day, three times a day, or four times a day. The glutathione production promoter may be taken as needed, continuously, or intermittently, for example, at intervals of several months.

[0035] The glutathione production enhancer of the present invention can be contained in a composition for oral or enteral ingestion, such as a food composition. The glutathione production enhancer and composition of the present invention can be in any form, such as powder, liquid, solid such as tablet, granule, particle, paste, or gel.

[0036] The glutathione production enhancer and composition of the present invention can be used in combination with any additive selected as needed. Examples of additives include excipients. Any excipients commonly used in the desired dosage form may be used, including starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, and cyclodextrin; crystalline celluloses; sugars such as lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharides, and emulsified oligosaccharides; and sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol. These excipients can be used alone or in combination of two or more.

[0037] Other known colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. can be appropriately selected and used.

[0038] The present invention also provides a method for promoting glutathione production in the skin by administering, for example, orally or enterally, one or more herbal medicines selected from the group consisting of Mallotus japonicus, radish, bracken, narcissus, mountain yam, moringa, devil's claw, houttuynia cordata, and gymnema.The present invention also provides a method for suppressing oxidative stress and / or whitening the skin through promoting glutathione production in the skin by administering, for example, orally or enterally, one or more herbal medicines selected from the group consisting of Mallotus japonicus, radish, bracken, narcissus, mountain yam, moringa, devil's claw, houttuynia cordata, and gymnema.The method of the present invention is a cosmetic method and may not be performed by a doctor or medical professional.

[0039] Furthermore, the present invention provides use of one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, Kuma bamboo, Narco lily, Yamaimo, Moringa, Devil's claw, Houttuynia cordata, and Gymnema in the manufacture of a pharmaceutical, such as an oral or enteral preparation, for suppressing oxidative stress in the skin and / or promoting skin whitening.The present invention also provides one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, Kuma bamboo, Narco lily, Yamaimo, Moringa, Devil's claw, Houttuynia cordata, and Gymnema for use in a method for suppressing oxidative stress in the skin and / or promoting skin whitening by promoting glutathione production in the skin, for example, by oral or enteral administration. [Example]

[0040] The present invention will now be described in more detail with reference to examples, although the present invention is not limited thereto.

[0041] Experiment 1: Sample preparation As candidate samples for glutathione production promoters, Mallotus japonicus, Japanese radish, Japanese bamboo grass, narcissus lily, mountain yam, moringa, devil's claw, Houttuynia cordata, and gymnema were prepared as shown in the table below.

[0042] [Table 1]

[0043] A total of 140 candidate samples were prepared, including natural and synthetic ingredients such as animal and plant extracts. Samples were adjusted to 10 mg / ml using DMSO and then diluted appropriately with culture medium to prepare evaluation samples at 10 μg / ml. DMSO containing 10 μg / ml and 100 μg / ml NAC (N-acetylcysteine) served as positive controls, while DMSO containing 10 μg / ml glutathione, plain water, and DMSO served as negative controls.

[0044] Experiment 2: Construction of a screening system for components that promote glutathione production Experiment 2-1: Cultivation of human skin epidermis-derived cell line HaCaT cells In this experiment, HaCaT cells were used as a model cell for human skin. Cells were subcultured in DMEM medium (Dulbecco's Modified Eagle Medium; Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, CA, USA) at 37°C and 5% CO2 in cell culture dishes (Greiner Bio-One, Tokyo, Japan). The DMEM medium was prepared by dissolving 10.0 g of DMEM powder in 1 L of Milli-Q water, adding 0.1 g of streptomycin sulfate (Meiji, Tokyo, Japan), 100,000 U of penicillin G potassium (Meiji), 2.38 g of 1 M HEPES (DOJINDO, Kumamoto, Japan), and 2.0 g of 10% NaHCO3 (Wako, Osaka, Japan), and sterilizing the medium through a 0.22 μm filter (Toyo Roshi Kaisha, Tokyo, Japan).

[0045] Experiment 2-2: Cultivation of Caco-2 cells, a human colon cancer cell line In this study, human colon cancer-derived Caco-2 cells were used as a model of human intestinal epithelium. Caco-2 cells were subcultured in Dulbecco's Modified Eagle Medium (DMEM) (Nissui, Tokyo, Japan) containing 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, CA, USA) in cell culture dishes (Greiner Bio-One, Tokyo, Japan) at 37°C and 5% CO2. DMEM medium was prepared by dissolving 10.0 g of DMEM powder in 1 L of Milli-Q water, supplemented with 100 U / mL penicillin (Meiji), 0.1 mg / mL streptomycin (Meiji), 2.38 g of 1 M HEPES (DOJINDO, Kumamoto, Japan), and 2.0 g of 10% NaHCO3 (Wako, Osaka, Japan), and sterilized through a 0.22 μm filter (Tokyo Roshi Kaisha, Tokyo, Japan).

[0046] Experiment 3: Test substance screening setup Caco-2 cells shown in Experiment 2-2 were plated in a 24-well plate (Corning) at 1.0 × 10 5 After 24 hours, each sample prepared in Experiment 1 was diluted with medium and added to the Caco-2 cells at 10 μg / ml. 5 After 24 hours, the HaCaT medium was removed and 100 μL of Caco-2 supernatant was added.

[0047] After the incubation, reduced glutathione was quantified using QuicGSH3.0 (Goryo Chemical Co., Ltd.). After 48 hours, the black plate on which the culture had been carried out was removed, and 100 μl / well of 8% paraformaldehyde (final concentration: 4%) was added to the top of the culture medium, followed by leaving the plate at room temperature for 15 minutes. The culture medium and fixative were discarded, and the cells were washed twice with 200 μl / well of PBS. A 1 mM DMSO solution of QuicGSH3.0 was diluted with PBS to prepare a staining solution with a final concentration of 1 μM. Pluronic F-127 was added to the staining solution at a final concentration of 0.01%. 100 μl of this solution was added to the well and incubated at 37°C for 10 minutes. The staining solution was discarded, and the cells were washed twice with 100 μl / well of PBS. 100 μl / well of 500-fold diluted Hoechst33342 (Dojindo Laboratories) was added and the cells were left at room temperature for 20 minutes. The staining solution was discarded, and the cells were washed twice with 100 μl / well of PBS. 100 μl of PBS was added to the well and the cells were analyzed using an IN Cell Analyzer 2200.

[0048] QuicGSH3.0 is a fluorescent probe whose fluorescence intensity ratio at two wavelengths changes with increases or decreases in reduced glutathione concentration. In the absence of glutathione, the maximum wavelength is 625 nm, but upon reaction with glutathione, the fluorescence at 625 nm weakens and the fluorescence at 582 nm increases. In the analysis protocol, the area illuminated by either Cy3-Cy3 or Cy3-Cy5 is considered the GSH area, and the fluorescence intensity (Dens-Levels) and its ratio (550 nm / 616 nm) are calculated. Cell nuclei are also identified and counted from Hoechst images.

[0049] result: The results are shown in Figure 2. As shown in Figure 2, when the herbal medicine of the present invention was administered, the amount of intracellular glutathione increased significantly compared to the negative control (DMSO only and DMSO containing astaxanthin), and was equal to or greater than the positive control (NAC). Therefore, it can be seen that the herbal medicine of the present invention has an excellent effect of promoting glutathione production in skin cells.

Claims

1. A glutathione production promoter containing one or more herbal medicines selected from the group consisting of Mallotus japonicus, Japanese radish, Japanese bamboo grass, narcissus lily, mountain yam, moringa, devil's claw, Houttuynia cordata, and gymnema as active ingredients.

2. The glutathione production promoter according to claim 1, which promotes glutathione production in skin cells via intestinal cells.

3. The glutathione production promoter according to claim 1 or 2, which promotes glutathione production in skin cells when taken orally.

4. A composition comprising the glutathione production promoter according to any one of claims 1 to 3.

Images