Topical skin preparation

Abstract

To provide, knowing that glutamine metabolism is involved in the survival of senescent cells, a topical skin preparation for ameliorating or preventing skin spots, wrinkles, sagging and the like by removing or decreasing senescent cells, where the formulation contains a plant extract having glutaminase inhibitory activity while being excellent in safety, stability and efficacy.SOLUTION: This invention has found that an extract of Coptis exhibits glutaminase inhibitory activity. The present invention enables the use of the extract as an active ingredient in a glutaminase inhibitor.SELECTED DRAWING: None

Description

Detailed Description of the Invention [Technical field]

[0001] The present invention relates to an external skin preparation containing a glutaminase inhibitor. [Background technology]

[0002] Aging at the cellular level, or cellular senescence, is a state in which cells enter an irreversible state of cell division arrest. Cells in this state are called senescent cells, and they secrete inflammatory cytokines such as IL-1α, IL-6, and IL-8, as well as other extracellular matrix degrading enzymes, and exhibit the Senescence Associated Secretory Phenomenon (SASP). Senescent cells can be naturally removed by the immune system or apoptosis, but as we age, their ability to do so decreases, and they accumulate in the skin and organs without being removed. As a result, surrounding normal cells are also exposed to chronic inflammation, which can lead to problems with skin aging such as age-related spots, wrinkles, and sagging skin, as well as age-related diseases such as arteriosclerosis and Alzheimer's disease, so research into the regulation of aging and senescent cells is active.

[0003] Patent Document 1 reports an inhibitor of the enzyme glutaminase (GLS1), which produces glutamic acid and ammonia from glutamine, as a senescent cell removal agent that removes senescent cells in the body. In addition, Non-Patent Document 1 reports that expression of KGA, an isoform of GLS1, is increased in senescent cells, that there is a positive correlation between age and KGA expression in human skin, and that glutamic acid metabolism is involved in the survival of senescent cells and is essential for the survival of senescent cells. This is thought to be because there is a mechanism that maintains survival by neutralizing the intracellular pH by producing ammonia through the induction of GLS1 when the intracellular pH of senescent cells decreases due to abnormalities in intracellular organelles. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 095971

[0005] [Non-Patent Document 1] Y. Johmura et al., Science., 371(6526), ​​265-270(2021)Problem that the invention aims to solve

[0006] In view of the above-mentioned prior art, there is a demand for a technology to inhibit glutaminase activity and expression. The present invention aims to provide an external skin preparation that contains a plant-derived substance that is highly safe for the living body and inhibits glutaminase activity and expression in order to remove accumulated senescent cells. Means for solving the problem

[0007] As a result of intensive research aimed at solving the above problems, it has been found that an extract of Coptis japonica (Thunb.) Makino (Ranunculaceae) is an effective agent for inhibiting glutaminase activity.

[0008] The topical skin preparation of the present invention is expected to maintain healthy, youthful skin by removing senescent cells and improving and preventing age spots, wrinkles, and sagging caused by senescent cells that accumulate with age, since the Coptis japonica extract is excellent at inhibiting glutaminase activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Plant parts The part of the plant used in the present invention is not particularly limited, and the whole plant, above-ground parts, leaves, flowers, stems, rhizomes, seeds, roots, fruits, etc. may be used as appropriate, but in the case of Coptis japonica of the Ranunculaceae family, the rhizomes are preferably used.

[0010] Extraction Solvent In the present invention, as the extraction solvent, water, a lower alcohol having 1 to 5 carbon atoms and having a straight or branched chain, a lower ester having 1 to 5 carbon atoms and having a straight or branched chain, a lower acetone having 1 to 5 carbon atoms and having a straight or branched chain, and a mixture of two or more of these are used. Specific examples of the lower alcohol having 1 to 5 carbon atoms and having a straight or branched chain are preferably methanol, ethanol, propanol, butanol, 1,3-butylene glycol, propylene glycol, glycerin, etc., and more preferably methanol and ethanol. Specific examples of the lower ester having 1 to 5 carbon atoms and having a straight or branched chain are preferably methyl acetate, ethyl acetate, etc., and more preferably ethyl acetate. Examples of the lower acetone having 1 to 5 carbon atoms and having a straight or branched chain are preferably methyl ethyl ketone, acetone, etc., and more preferably acetone. In the present invention, when the solvent extract extracted with a solvent is used as it is, it is preferable to select and use a solvent that is also used as a food from among these solvents, taking into consideration the viewpoint of safety, and more preferably water, ethanol, and a mixture thereof.

[0011] The amount of extraction solvent to be added can be appropriately determined according to the amount of plant raw material, extraction temperature, etc. Solvent extraction may be performed at room temperature and normal pressure, and the extraction temperature can be appropriately determined taking into account the boiling point of each solvent, etc.

[0012] In the present invention, when solvent extraction is performed, the weight ratio of the raw material to the solvent is 1:20 to 1:1, preferably 1:15 to 1:5, and more preferably 1:10. The temperature when heating during extraction is about 30°C to 100°C, preferably the lower limit is 45°C or more and the upper limit is 90°C or less.

[0013] In the present invention, it is not necessary to carry out solvent extraction, and the extract may be a liquid or oil obtained by squeezing a natural material.

[0014] concentrated According to another aspect of the present invention, an extract can be obtained by removing the solvent from the solvent extract. Generally, the method of removing the solvent can be vacuum concentration, heat concentration, ventilation concentration, freeze concentration, spray concentration, other concentration methods, or a mixture of these. The pressure, temperature, wind power, spray, etc. during concentration can be appropriately set according to the amount of the extract to be obtained and the intended use. In the present invention, vacuum concentration is preferably used. When vacuum concentration is performed, the pressure and temperature can be appropriately set according to the amount of the concentrate to be obtained and the intended use.

[0015] The extract used in the present invention may be used as it is, or may be filtered or concentrated as necessary. The extract may also be fractionated and purified by column chromatography, countercurrent distribution, or the like before use.

[0016] Furthermore, the above-mentioned substances can be dried under reduced pressure or freeze-dried, and then prepared into a powder or paste form, which can then be appropriately formulated for use.

[0017] Dry / dried matter According to another embodiment of the present invention, the extract or the extract from which the solvent has been removed can be dried to obtain a dried product. In the present invention, the drying method used includes sun drying, (hot) air drying, vacuum drying, ventilation drying, fluidized bed drying, spray drying, freeze drying, reduced pressure drying, infrared drying, high frequency drying, and other drying methods, or a combination of these. Drying may be performed simultaneously with the above-mentioned concentration.

[0018] Skin preparations The extract composition according to the present invention is suitable for incorporation into a skin topical agent because it is excellent in usability and safety when applied to the skin. Specifically, the skin topical agent is an external agent comprising a composition containing a plant extract. The type of skin topical agent may be any that can be applied to the skin, and examples thereof include creams, skin lotions, mists, milky lotions, lotions, gels, packs, ointments, essences, hair tonics, hair liquids, liniments, hair rinses, hair shampoos, hair treatments, hair conditioners, aerosols, mousses, and the like. The amount of the plant extract added is about 0.0001% by weight or more and 20% by weight or less based on the total weight of the skin composition (cosmetics). In addition, when the extract is a liquid juice, oil, fermented product, etc., the amount is about 0.0001% by weight or more and 100% by weight or less (the extract or fermented product is used as is) based on the total weight of the skin topical agent, taking into consideration safety depending on the characteristics and properties of the extract itself. When the extraction composition according to the present invention is used as it is, the concentration of the extract is not particularly limited as long as the content of the dry solid matter, which is the solute, is within the above range.

[0019] The skin external preparation of the present invention can be prepared by blending with various types of bases known as normal skin external preparations according to the usual method, and the form of the external preparation is not particularly limited, and any dosage form can be selected, for example, a milky lotion, cream, lotion, pack, etc. In addition, it is not limited to general cosmetics, and includes medicines, quasi-drugs, and medicinal cosmetics.

[0020] In the topical skin preparation of the present invention, components that can be used together with the extract of Coptis japonica rhizome of the Ranunculaceae family include, for example, moisturizers, UV absorbers, complex lipids, anti-inflammatory agents, vitamins and their derivatives, oily components, surfactants, preservatives, powder components, purified water, polymer compounds, gelling agents, antioxidants, cholesterols, plant sterols, lipoproteins, microbial-derived components, algae extracts, blood circulation promoters, antiseborrheic agents, thickeners, colorants, and cosmetic components, and can be appropriately selected and used within a range that does not impair the effects of the present invention.

[0021] Glutaminase Glutaminase is an enzyme that produces glutamic acid and ammonia from glutamine. Two types of human glutaminase are known: kidney type (GLS1) and liver type (GLS2), and splice variants of GLS1 are known: KGA, GAC, and GAM. The glutaminase activity / expression inhibitor of the present invention is not particularly limited, but is preferably a GLS1 inhibitor. The method of inhibition is also not particularly limited, but possible methods include inhibition of GLS1 gene expression or protein expression. EXAMPLES

[0022] The present invention will now be described in more detail with reference to examples and comparative examples, but the present invention is in no way limited thereto. Furthermore, the extraction method for the extract of the drug used is also in no way limited.

[0023] <Extract Manufacturing Example 1> Manufacturing method of plant extract (Coptis japonica extract) As for the Coptis japonica, any plant with the scientific name Coptis japonica (Thunb.) Makino (Ranunculaceae) or the same genus may be used. 600g of 50v / v% ethanol (20 times the amount) was added to 30g of Coptis rhizome, extracted at 50℃ for 5 hours, filtered and concentrated, then freeze-dried and powdered to obtain an extract (Coptis japonica extract 1). As another method, 500g of 1,3-butylene glycol (10 times the amount) was added to 50g of Coptis rhizome, extracted at room temperature for 10 days, filtered, and obtained an extract (Coptis japonica extract 2).

[0024] Regarding cells, although there is no particular limitation, normal cells, senescent cells, cancer cells, etc. from various animal species and sites can be used. When using senescent cells, aging induction can be achieved by replication (cell division), exposure to hydrogen peroxide, UVA, UVB, gamma rays, and treatment with drugs that damage DNA. Examples include Nutlin-3a, an Mdm2 inhibitor, and doxorubicin, an anti-cancer drug. EXAMPLES

[0025] (Creation of senescent cells) In order to create stable senescent cells, replicative senescent cells were used for senescence induction. Normal human fibroblasts, which are also used in aging research and prior art literature, were also used. Normal human skin fibroblasts (Kurabo) were cultured at 10 cm 2 The cells were seeded in a petri dish and cultured until subconfluent, then transferred to another 10 cm 2 The cells were repeatedly passaged by seeding a small amount on a petri dish. The number of passages was not particularly limited, but was approximately 30 to 49 times. In addition, exposure to hydrogen peroxide was also performed as another method for inducing aging. Normal human skin fibroblasts (Kurabo) were seeded on a 10 cm 2 The cells were seeded in petri dishes and cultured until they became subconfluent. After washing with Hanks' buffer solution, 400 μM hydrogen peroxide was added and exposed, the medium was replaced with DMEM medium containing 5% fetal bovine serum, and cultured overnight at 37°C under 5% carbon dioxide. After repeated exposure to hydrogen peroxide, the cells exposed to hydrogen peroxide were significantly flattened compared to unexposed cells, and showed a senescent morphology. To determine whether senescence had occurred, SA-β-Gal activity, increased reactive oxygen species (ROS), p16INK4a expression, and other commonly known cellular senescence markers were checked, and it was determined that senescence had been induced comprehensively.

[0026] (Test Example 1) Measurement of glutaminase activity inhibitory effect The inhibitory effect of glutaminase activity was measured for the extract shown in Production Example 1 (Coptis Rhizome Extract 1). Replicative senescent cells (passage number: 36) and senescent cells exposed to hydrogen peroxide were seeded on a 6-well plate and cultured in DMEM medium containing 5% fetal bovine serum under 5% carbon dioxide gas for 24 hours. After 24 hours of culture, the test sample was dissolved in purified water, and a filter-sterilized solution was added, followed by culture under the same conditions in the above medium. After a certain period of culture, the cells were washed with PBS(-), PBS(-) containing 0.5% TritonX-100 was added, and the cells were peeled off with a scraper and left to stand at 4°C. The supernatant after centrifugation was used as a crude enzyme solution. A predetermined amount of 5 mM L-glutamine-containing PBS (-) and the crude enzyme solution were mixed and reacted at 37 ° C for 15 minutes. After 15 minutes, 0.5 M HCL aqueous solution was added and mixed to stop the reaction. 25 μL of the solution after the reaction was stopped or L-glutamic acid solution and 75 μL of a coloring solution (β-NAD +: Oriental Yeast, WST-1: Dojindo Laboratories, 1-Methoxy PMS: Dojindo Laboratories, Glutamate Dehydrogenase: Sigma-Aldrich-containing 0.1 M Tris-HCL buffer) were added, and the absorbance at 450 nm was measured after 10 minutes at 37 ° C. The glutaminase activity was evaluated by calculating the amount of L-glutamic acid in the glutaminase reaction solution from the calibration curve of L-glutamic acid and dividing it by the protein amount of the cell lysate measured separately by the BCA method (mean ± SD). Each glutaminase activity value is shown in Table 1.

[0027] [Table 1]

[0028] As shown in Table 1, it was confirmed that Coptis japonica extract has the effect of inhibiting glutaminase activity in senescent cells.

[0029] (Test Example 2) Measurement of KGA / GAC expression The KGA / GAC expression was measured for the extract (Coptis extract 1) shown in Production Example 1. Replicative senescent cells (passage number: 42) and cells exposed to hydrogen peroxide were seeded on a 6-well plate and cultured in DMEM medium containing 5% fetal bovine serum under 5% carbon dioxide gas for 24 hours. After 24 hours of culture, the test sample was dissolved in purified water, and the filter-sterilized solution was added, and the cells were cultured under the same conditions in the above medium. After a certain period of culture, the cells were washed with PBS(-), RIPA buffer (Santa Cruz Biotechnology) was added, and the cells were peeled off with a scraper and left to stand at 4°C. The supernatant after centrifugation was used as a cell lysate. The protein content of the cell lysate was measured by the BCA method, and the protein content of each cell lysate was uniformly adjusted with RIPA buffer, and then the cell lysate was diluted with sample buffer (Fujifilm Wako Pure Chemical) and boiled at 100°C for 5 minutes. Each cell lysate was subjected to SDS-PAGE to separate the cells, and after transferring to a PVDF membrane, KGA and GAC expression was evaluated by Western blotting using anti-KGA / GAC antibodies (Proteintech) (Figure 1). [Brief description of the drawings]

[0030] [Figure 1] The results of Western blotting of cell lysates from hydrogen peroxide-exposed senescent cells and replicative senescent cells cultured in a medium without Coptis japonica extract (0 ppm) and in a medium with Coptis japonica extract (25 ppm) using KGA and GAC antibodies are shown. Bands were observed at approximately 65 kDa for KGA and approximately 58 kDa for GAC.

[0031] As shown in Figure 1, it was found that the expression of KGA and GAC was significantly suppressed by the Coptis japonica extract compared to the control (0 ppm) senescent cells.

[0032] Examples of blending plant extracts into topical skin preparations containing the glutaminase inhibitor of the present invention are shown below, but the blending examples do not limit the present invention.

[0033] (Formulation example) Cream The following ingredients (1) to (10), and separately the following ingredients (11) to (17) and (19), were heated to 75°C to dissolve and emulsify, and the mixture was cooled to 50°C with stirring, and ingredient (18) was added to prepare a cream. (Ingredients) (Weight%) (1) Jojoba oil 3.0% (2) Squalane 2.0% (3) Methylpolysiloxane 0.5% (4) Stearyl alcohol 0.5% (5) Cetyl alcohol 0.5% (6) Caprylic / capric triglyceride 12.5% (7) Glyceryl monostearate 5.0% (8) Diglyceryl monostearate 1.5% (9) Decaglyceryl monostearate 3.0% (10) Propyl paraoxybenzoate 0.1% (11) Xanthan gum 0.1% (12) Coptis Rhizome Extract 2 0.1% (13) Phellodendron Amurense Bark Extract 0.1% (14) Ginger Rhizome Extract 0.1% (15) Melilot extract 0.8% (16) 1,3-Butylene glycol 2.5% (17) Methyl parahydroxybenzoate 0.2% (18)Fragrance 0.1% (19) Remaining purified water [Industrial Applicability]

[0034] Coptis japonica extract inhibits the expression of glutaminase, which is essential for the survival of aging cells, and is therefore expected to be used in cosmetics, quasi-drugs, and pharmaceuticals that prevent and improve skin aging.

Claims

1. A glutaminase inhibitor comprising an extract of the Coptis japonica plant.

2. A skin external preparation comprising the glutaminase inhibitor according to claim 1.

Images