Skin beautifying composition containing botryocene as active ingredient

Abstract

The purpose of the present invention is to provide a composition for cosmetic treatment of skin, which contains botryocene as an active ingredient, and which is for preventing or alleviating undesirable cosmetic changes in skin. Botryocene has been demonstrated by a number of tests to inhibit the production of ROS and its downstream action that results in cosmetically undesired changes in the skin, and thus it was found that botryocene can utilize such unknown likelihood in the prevention or mitigation of cosmetically undesired changes in the skin.

Description

TECHNICAL FIELD

[0001] The present application relates to a skin cosmetic composition containing botenylen as an effective ingredient for preventing or alleviating cosmetically undesirable changes in the skin. BACKGROUND

[0002] The skin is constantly exposed to stress caused by various exogenous and endogenous factors. As such stress factors, there can be mentioned exposure to sunlight (particularly UVB contained in sunlight; photoaging), wind, heat, dryness, moisture, contact with chemical substances such as household surfactants, contact with substances which give mechanical stimulation to the skin such as abrasives, smoking, alcohol, side effects of drugs, physical or psychological stress, biochemical changes in the skin caused by aging and the like, hormonal abnormalities, fatigue, acne, malnutrition, diseases, biological clock disorders and the like.

[0003] Stress caused by these factors brings about cosmetically undesirable decreases in the macroscopic appearance, clinical and physical properties, and physiological and histological functions of the skin. The skin is fatigued, the biological and mechanical functions of the skin tissue are weakened with time, and symptoms of aging of the skin are produced.

[0004] As remarkable cosmetically undesirable changes, there are production of dryness and fine lines and / or wrinkles, decrease in elasticity, relaxation of the skin, water-losing skin, decrease in hardness, thinning of the skin, decrease in uniformity of the blood color of the face, facial unevenness, excess pigmentation, spots, age spots, erythema, rough surface texture and mottled complexion and the like. Dull damaged hair, hair loss and uneven scalp are also common symptoms.

[0005] Further, upon aging of the skin or chronic exposure to mild environmental stress, although not as remarkable as the direct stress described above, measurable cosmetically undesirable changes also occur. As such changes, there can be mentioned general decrease in the activity of tissues and cells, slowing of cell replication, decrease in protein synthesis, increase in protein decomposition, decrease in blood circulation of the skin or dilation of blood vessels caused by stasis, infiltration of blood, decrease in water content, erroneous accumulation of the structure and function of proteins, undesirable changes in the skin barrier, adhesion to connective tissues and decrease in the ability of the skin to reconstruct or repair and the like.

[0006] As a main process of the cosmetic undesirable changes of the skin which occur remarkably or can be measured by the various stresses, aging and the like described above, these factors are known to promote the generation of reactive oxygen species (ROS) as oxidative stress, and to cause inflammatory responses, lipid, protein or DNA damage and the accompanying cell function reduction or cell death, tissue destruction and the like by the increase in the concentration of ROS. The skin is often exposed to some oxidative stress, and in the case where the intensity of the oxidative stress exceeds the allowable amount or the oxidative stress handling mechanism in the body is reduced, some cosmetic undesirable changes occur.

[0007] Therefore, it can be considered that the prevention or reduction of the cosmetic undesirable changes of the skin is effective by preventing the generation of ROS in the skin and removing it.

[0008] As a mechanism of eliminating ROS in the living body, a representative ROS eliminating enzyme, superoxide dismutase (SOD) catalyzes the reaction of converting superoxide, which is a reactive oxygen species, to oxygen and hydrogen peroxide. The formed hydrogen peroxide is also harmful, but it is further eliminated by catalase and glutathione peroxidase (GPx).

[0009] Various low molecules such as vitamins, polyphenols, catechins, glutathione, ascorbate, tocopherol, ubiquinone, bilirubin, uric acid and the like can function as natural antioxidants in cooperation with the antioxidant mechanism in the living body or independently. For example, vitamin B2 functions as a coenzyme of GPx, and polyphenols have SOD-like activity. Carotenoids also function as antioxidants and can exert a defense effect against oxidative stress and related chronic diseases. For example, Non-Patent Literature 1 summarizes reports on the relationship between various chronic diseases such as coronary heart disease, cataract and cancer and carotenoids.

[0010] Therefore, a cosmetic preparation having a material with such an antioxidant activity as an effective ingredient can be utilized in the prevention or reduction of the cosmetic undesirable changes of the skin associated with the action of ROS. In fact, skin cosmetic compositions using various antioxidant materials as effective ingredients are reported (Patent Literature 1, Patent Literature 2, Patent Literature 3).

[0011] A new compound having a good antioxidant activity, and the discovery of a good antioxidant activity possessed by an existing compound are highly expected as a material which is an alternative to an antioxidant in high demand in the fields of medicine, food and cosmetics.

[0012] For example, Patent Literature 4 discloses a compound, a method for producing the same, and an antioxidant agent containing the compound as an effective ingredient, as a flavonoid compound exhibiting excellent antioxidant action, which is synthesized as gambogin 6-C-arabinoside. Further, Patent Literature 5 discloses that epigallocatechin dimer and trimer have superoxide anion radical scavenging activity. Alternatively, Patent Literature 6 discloses a novel nitrogen-containing heterocyclic compound or a salt thereof, and a therapeutic agent for oxidative stress-related diseases containing the same.

[0013] Prior Art Documents

[0014] Patent Literature

[0015] Patent Literature 1: Japanese Patent Application Laid-Open No. 01 / 026670

[0016] Patent Literature 2: Japanese Patent Application Laid-Open No. 2021-095370

[0017] Patent Literature 3: Japanese Patent Application Laid-Open No. 2017-537091

[0018] Patent Literature 4: Japanese Patent Application Laid-Open No. 2006-265249

[0019] Patent Literature 5: Japanese Patent Application Laid-Open No. 2010-280716

[0020] Patent Literature 6: Japanese Patent Application Laid-Open No. 2007-016011

[0021] Patent Literature 7: Japanese Patent Application Laid-Open No. Hei 7-265059

[0022] Patent Literature 8: Japanese Patent Application Laid-Open No. 2013-504332

[0023] Patent Literature 9: Japanese Patent Application Laid-Open No. 2013-035791

[0024] Patent Literature 10: Japanese Patent Application Laid-Open No. 2012 / 077801

[0025] Non-Patent Literature

[0026] Non-Patent Literature 1: Canfield, et al., (1992) Proc. Soc. Exp. Biol. Med. 200:260

[0027] Non-Patent Literature 2: Kaya, K., Microbiol. Cult. Coll. 26 (1): 1-10, 2010 SUMMARY

[0028] PROBLEMS TO BE SOLVED BY THE INVENTION

[0029] The present application has an object to provide a skin cosmetic composition containing botryococcene as an effective ingredient for preventing or alleviating cosmetically undesirable changes in the skin.

[0030] Means for solving the problem

[0031] Algae belonging to the genus Botryococcus are microalgae that fix carbon dioxide using light energy to produce hydrocarbons. In recent years, with concern about depletion of fossil energy, carbon dioxide fixation using algae of the genus Botryococcus is considered to be an effective energy acquisition method. Furthermore, global warming caused by an increase in the amount of carbon dioxide accompanying combustion of fossil energy and the like is also a problem, and the use of algae of the genus Botryococcus, which have the ability to convert carbon dioxide into hydrocarbons, is expected not only from the aspect of the use of light energy, but also from the aspect of reduction in the amount of carbon dioxide (Patent Literature 7).

[0032] Botryococcus braunii (B. braunii), which is one of the colonial algae of the genus Botryococcus, is classified into race-A, race-B, and race-L based on the structural characteristics of the produced hydrocarbons. Among these, race-B produces C n H 2n-10 (n = 30 to 37) represents botryococcene, which is a branched triterpenoid hydrocarbon (Non-Patent Literature 2). Triterpenoid hydrocarbons can be used as a raw material for hydrocracking in petroleum refining for the production of, for example, octane (gasoline, petrol), kerosene, and diesel. Botryococcene has the potential to be converted into biofuels with higher octane numbers (Patent Literature 8).

[0033] On the other hand, the use as a cosmetically effective ingredient of botryococcene has also been studied (Patent Literature 9). The present applicant is one of the co-applicants of this document. In this document, it is reported that a combination of botryococcene and a hydrogen-modified botryococcene can be utilized as a moisturizer with superior effects compared to squalane. The cosmetic use of botryococcene can become a powerful driving force for the development of botryococcene production technology using cultivation of B. braunii, since it can add value far higher than the biofuel use.

[0034] The present inventors conducted further studies on the cosmetic use of botryococcene, and as a result, it was confirmed through a plurality of tests that botryococcene suppresses the production of ROS, which leads to cosmetically undesirable changes in the skin, and the action downstream thereof, and it was found that botryococcene can utilize such unknown possibilities in the prevention or alleviation of cosmetically undesirable changes in the skin.

[0035] The patent document 9 described above shows the cosmetic use of squalen, like the present application, but the moisturizing function of the skin shown in the document is a different use that is not related to the prevention or reduction of the cosmetically undesirable changes of the skin related to ROS shown in the present application.

[0036] Therefore, the present application provides the following invention.

[0037] 1. A cosmetic composition for preventing or reducing cosmetically undesirable changes of the skin, comprising squalen as an effective ingredient.

[0038] 2. The cosmetic composition according to item 1, wherein the cosmetically undesirable changes of the skin are one or more of dryness and the generation of fine lines and / or wrinkles, a decrease in elasticity, a loosening of the skin, a water- depleted skin, a decrease in hardness, a thinning of the skin, a decrease in the uniformity of the blood color of the face, a facial uneven color, a pigment excess, age spots, erythema, a coarse surface texture, a mottled facial color, a general decrease in the activity of tissues and cells, a decrease in cell replication, a decrease in protein synthesis, an increase in protein decomposition, a decrease in the blood circulation of the skin or a vascular dilation caused by congestion, a blood infiltration, a decrease in the water content, an erroneous accumulation of the structure and function of proteins, an undesirable change in the skin barrier, an adhesion to connective tissue and a decrease in the reconstruction or repair capacity of the skin.

[0039] 3. The cosmetic composition according to item 1 or 2, wherein the cosmetically undesirable changes of the skin are the formation of wrinkles or spots of the skin.

[0040] 4. The cosmetic composition according to any one of items 1 to 3, wherein the prevention or reduction of the cosmetically undesirable changes of the skin is achieved by any one or more of the inhibition of ROS generation in the skin tissue by the squalen, the inhibition of the generation of inflammatory cytokines caused by ROS, the inhibition of the phagocytosis of the melanosome by pigment cells caused by inflammatory cytokines, or the inhibition of the decrease in collagen production by fibroblasts caused by inflammatory cytokines or the inhibition of the enhancement of the production of MMP-1.

[0041] 5. The cosmetic composition according to item 4, wherein the ROS in the skin tissue is generated by one or more of the sunlight exposure of the skin, wind, heat, dryness, humidity, contact with chemicals, contact with substances that bring mechanical stimulation, smoking, alcohol, side effects of drugs, physical or psychological stress, biochemical changes in the skin caused by aging, hormone abnormalities, fatigue, acne, malnutrition, diseases, a biological clock disorder.

[0042] 6. The cosmetic composition according to item 4, wherein the ROS in the skin tissue is generated by the sunlight exposure of the skin.

[0043] 7. The cosmetic composition according to any one of items 1 to 6, comprising further one or more effective ingredients for preventing or reducing cosmetically undesirable changes in the skin.

[0044] 8. The cosmetic composition according to item 7, wherein the further one or more effective ingredients is an antioxidant.

[0045] 9. The cosmetic composition according to item 8, wherein the antioxidant is selected from the group consisting of vitamin A / retinol, vitamin C / ascorbic acid, vitamin E / alpha-tocopherol, coenzyme Q10, N-acetylcysteine (NAC), S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine persulfide, cysteine trisulfide, uric acid, allicin, alliin, allyl disulfide, cyanidin chloride, cyanidin-3,5-diglucoside, anthocyanin, isoflavone, quercetin, catechin, theaflavin, sesamin, sesamol, resveratrol, curcumin, chlorogenic acid, ferulic acid, 3,4-dihydroxy cinnamic acid, beta-carotene, lycopene, astaxanthin, zeaxanthin, L-cysteine methyl ester hydrochloride (LCM), L-cysteine ethyl ester hydrochloride (LCE), D-cysteine hydrochloride monohydrate (DCH), D-cysteine methyl ester hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, L-methionine methyl ester hydrochloride (LMM).

[0046] 10. The cosmetic composition according to any one of items 1 to 9, which is in the form of a cosmetic water, cream, emulsion, gel, aerosol, pack, cleanser, bath, foundation, powder, lipstick, ointment, cataplasm, paste, plaster, essence for skin application. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Optical microscope images of Brown Botryococcus sp. showing secreted botryococcus oil droplets and the structure of botryococcus.

[0048] Figure 2 Cell toxicity evaluation caused by botryococcus treatment.

[0049] Figure 3 Cellular ROS scavenging caused by botryococcus treatment Figure 3 A) and mitigation of cell viability reduction caused by H2O2 addition Figure 3 B) by botryococcus treatment.

[0050] Figure 4 Inflammatory cytokines IL-1a Figure 4 A) and PGE2 Figure 4B) production inhibition.

[0051] Figure 5 The study showed inhibition of fluorescent bead phagocytosis in fibroblasts induced by glucocorticoid treatment.

[0052] Figure 6 It showed inhibition of decreased collagen production in epidermal cells caused by phycocyanin treatment. Figure 6 A) and MMP-1 produce inhibition ( Figure 6 B).

[0053] Figure 7 A schematic diagram illustrating the process by which UVB causes cosmetically undesirable changes to the skin. Figures 3 to 6 The position of the effect produced by glucosamine treatment in this process was observed. Detailed Implementation

[0054] Botrycocele is a type of polycystic alginate produced by a portion of Botryococcus braunii using C... n H 2n-10 (n = 30-37) represents a branched triterpenoid hydrocarbon. The *Botrytis cinerea* race-B, which produces this phycocyanin, is useful for the large-scale production of phycocyanin because it has the property of producing large quantities of phycocyanin with high purity, secreting more than 90% of it extracellularly and accumulating it within the colony (Patent Document 10).

[0055] Botryne itself can be directly used as diesel fuel in ships and agricultural vehicles, but it can be further converted into light oil, naphtha, kerosene, and gasoline through existing catalytic cracking processes. Furthermore, due to its excellent moisturizing properties, botryne has also been found to be useful as a moisturizing ingredient in cosmetics (Patent Document 9). Thus, botryne holds promise for various industrial applications, and the skin cosmetic applications proposed in this invention further enhance its value as a promising additional use.

[0056] In this invention, a cosmetic composition containing phycocyanin as an active ingredient is prepared, which can prevent or reduce undesirable cosmetic changes in the skin.

[0057] Such preventive or mitigating effects of undesirable cosmetic changes in the skin caused by glucophagene, as described later in the examples, can be reasonably inferred from experimental results such as glucophagene's inhibition of various events that cause undesirable cosmetic changes in the skin, including the production of inflammatory cytokines caused by increased ROS concentration in the skin, melanosome phagocytosis by pigment cells caused by inflammatory cytokines, or decreased collagen production or MMP-1 production by fibroblasts caused by inflammatory cytokines.

[0058] Increased ROS concentration in the skin, mediated by various stress factors, leads to various undesirable cosmetic changes. These stress factors, whether directly or indirectly causing increased ROS concentration, are not limited to a few, but can include: sun exposure (UVB; photoaging), wind, heat, dryness, humidity, contact with chemicals such as household surfactants, contact with substances that mechanically irritate the skin such as abrasives, smoking, alcohol, drug side effects, physical or psychological stress, biochemical changes in the skin due to aging, hormonal abnormalities, fatigue, acne, malnutrition, disease, and circadian rhythm disruption.

[0059] In this invention, sunlight exposure is a particularly important stress factor. Undesirable cosmetic changes in the skin caused by such sunlight exposure are generally termed photoaging. Ultraviolet (UV) radiation, a component of sunlight, especially medium-wave UV (UVB, wavelength 290-320 nm), primarily causes photoaging. The UVB radiation dose required to cause photoaging is currently unknown, but repeated exposure to UVB levels that cause transient erythema and sunburn is usually closely associated with photoaging. Clinically, photoaging can be identified as rough skin, wrinkle formation, pigmentation, yellowing, sagging, telangiectasia, nevus formation, purpura, skin fragility, atrophy, the formation of fibrotic hypopigmented areas, and the development of precancerous and malignant tumors. Photoaging occurs on skin habitually exposed to sunlight, such as the face, ears, head, neck, and hands.

[0060] Undesirable cosmetic changes that can be prevented or mitigated by the skin cosmetic composition of the present invention are not limited to those mentioned above, but include dryness and the formation of fine lines and / or wrinkles, decreased elasticity, skin laxity, loss of moisture, decreased firmness, thinning of the skin, decreased evenness of facial color, uneven complexion, excess pigmentation, age spots, erythema, rough surface texture, and mottled complexion. Furthermore, as less significant but measurable undesirable cosmetic changes, generalized reductions in tissue and cell activity, slowed cell replication, decreased protein synthesis, increased protein breakdown, reduced skin blood circulation or vasodilation due to congestion, blood infiltration, decreased moisture content, accumulation of errors in protein structure and function, undesirable changes in the skin barrier, adhesion to connective tissue, and reduced skin reconstruction or repair capabilities are also expected to be prevented or improved by the skin cosmetic composition of the present invention.

[0061] The undesirable cosmetic changes that are particularly to be prevented or mitigated in the skin cosmetic composition of the present invention are the formation of wrinkles or blemishes. Increased ROS concentration in the skin primarily enhances the production of inflammatory cytokines IL-1α and PGE2, which, through their action, promote the phagocytosis of melanosomes by epidermal cells, leading to blemishes. Furthermore, collagen production by dermal fibroblasts is inhibited, or MMP-1 production is promoted, resulting in wrinkles. In the embodiments of this application, the skin cosmetic composition of the present invention has been shown to exhibit inhibitory effects at various points along the pathway from increased ROS concentration to the formation of these blemishes or wrinkles. Figure 7 ).

[0062] The skin-beautifying compositions of the present invention can be prepared in any suitable dosage form intended for application to the skin to prevent or reduce undesirable cosmetic changes. Therefore, although not limited, examples of dosage forms include lotions, creams, lotions, gels, aerosols, masks, cleansers, bath products, foundations, powders, lipsticks, ointments, poultices, pastes, ointments, serums, etc. Determining the dosage form in which the skin-beautifying compositions of the present invention are prepared, and designing the component composition suitable for constituting the desired dosage form, is generally within the scope of formulation condition optimization or condition studies for those skilled in the art.

[0063] The appropriate amount of phycocyanin in the skin-cosmetic composition of the present invention to achieve the desired effect can be determined by considering factors such as the dosage form of the composition, usage, dosage, desired cosmetic effect, and the types or degrees of undesirable cosmetic changes. Furthermore, it can be appropriately determined through preliminary experiments typically conducted by those skilled in the art in the cosmetic field. In one embodiment, the skin-cosmetic composition of the present invention contains phycocyanin at a concentration of 0.001 to 20% by weight, more preferably 0.001 to 15% by weight, even more preferably 0.001 to 10% by weight, even more preferably 0.01 to 3% by weight, and even more preferably 0.01 to 2% by weight. It should be noted that the phycocyanin concentrations in the embodiments of the present application refer to concentrations used for in vitro cultured cells, and do not specify a range for the amount of phycocyanin in the skin-cosmetic composition of the present invention.

[0064] In addition to glucophage, the skin-beautifying composition of the present invention may also contain one or more additional active ingredients. Such additional active ingredients can assist, additively, or synergistically enhance the effects of the skin-beautifying composition of the present invention in preventing or mitigating undesirable cosmetic changes. For example, antioxidants may be incorporated into the skin-beautifying composition of the present invention as one or more additional active ingredients.

[0065] Antioxidants that are added to the skin-beautifying composition of the present invention are not limited, and examples include vitamin A / retinol, vitamin C / ascorbic acid, vitamin E / α-tocopherol, coenzyme Q10, N-acetylcysteine ​​(NAC), S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine ​​persulfate, cysteine ​​trisulfide, uric acid, allicin, alliin, allyl disulfide, cyanidin chloride, cyanidin-3,5-diglucoside, anthocyanins, isoflavones, and quercetin. The ingredients include: catechins, theaflavins, sesamin, sesamol, resveratrol, curcumin, chlorogenic acid, ferulic acid, 3,4-dihydroxycinnamic acid, β-carotene, lycopene, astaxanthin, zeaxanthin, L-cysteine ​​methyl hydrochloride (LCM), L-cysteine ​​ethyl hydrochloride (LCE), D-cysteine ​​hydrochloride monohydrate (DCH), D-cysteine ​​methyl hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, and L-methionine methyl hydrochloride (LMM).

[0066] Example

[0067] The following examples demonstrate the effects of phycocyanin, incorporated into the skin cosmetic compositions of the present invention, in in vitro models of various molecular biological events leading to undesirable changes in skin cosmetics.

[0068] Cell culture

[0069] Normal human epidermal keratinocytes (NHEKs) were cultured in HuMedia-KG2 (KG2) medium supplemented with 10% fetal bovine serum, 1% antibiotics, and 1% human epidermal keratinocyte proliferation supplement at 37°C in a 5% CO2 incubator.

[0070] Normal human skin fibroblasts (NHDFs) were cultured in DMEM supplemented with 10% fetal bovine serum, 1% antibiotics, and 1% human epidermal keratinocyte proliferation supplement in a 37°C, 5% CO2 incubator.

[0071] Assay for cell damage caused by phycocyanin treatment

[0072] NHEKs cells were cultured in KG2 medium at a rate of 2.5 × 10⁻⁶. 4Cells were seeded at 96-well plates at various densities and incubated for 24 hours. The supernatant was aspirated, and the medium was replaced with HuMedia-KB2 (KB2) medium supplemented with 0, 12.5, 25, 50, 100, 200, and 400 μM glucophage, and incubated for another 24 hours. After incubation, cells were washed with PBS and then replaced with KB2 medium containing 0.003% neutral red for 2 hours. Neutral red was extracted from the cells using 1M HCl in 30% MeOH. The absorbance at 550 nm was measured using a microplate reader.

[0073] The results of this experiment are presented as follows: Figure 2 Cell viability remained unchanged at concentrations of 0–200 μM for alginate, but a significant decrease was observed at 400 μM. Based on these results, the maximum lginate concentration used in the following experiments was 200 μM.

[0074] Example 1. Effect of glucosamine on intracellular reactive oxygen species removal

[0075] NHEKs cells were cultured in KG2 medium at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 96-well plates and incubated for 24 hours. The supernatant was removed, and the medium was replaced with KB2 medium supplemented with 0, 25, 50, 100, or 200 μM glucosamine, and incubated for another 24 hours. After incubation, cells were washed with PBS, replaced with HBSS buffer containing 200 μM H2O2, and incubated for 1 hour. The supernatant was removed, and the cells were incubated with HBSS buffer containing 20 μM DCFH-DA for 30 minutes. Fluorescence intensity was measured using a microplate reader (Emission = 485 nm, Extation = 530 nm). Cells were then lysed with 50 μL of PBS containing 0.5% Triton X-100, protein levels were quantified, and fluorescence intensity per unit of protein was normalized.

[0076] The results of the above experiments are shown in Figure 3 In A, intracellular ROS concentration increased after treatment with 200 μM H2O2. Both the increased intracellular ROS concentration after H2O2 treatment and the untreated intracellular ROS concentration showed a concentration-dependent decrease in ROS concentration upon the addition of glucosamine.

[0077] Example 2. Effect of glucosamine on cell damage induced by H2O2 addition.

[0078] NHEKs cells were cultured in KG2 medium at a rate of 2.5 × 10⁻⁶. 4Cells were seeded at a density of 0.5 g / well in 96-well plates and incubated for 24 hours. The supernatant was aspirated, and the medium was replaced with KB2 medium supplemented with 0, 25, 50, 100, and 200 μM glucophage, and incubated for 24 hours. After incubation, cells were washed with HBSS containing PBS and 300 μM H2O2 for 1 hour, and then incubated with KB2 medium for 24 hours. After incubation, cells were washed with PBS, replaced with KB2 medium containing 0.003% neutral red, and incubated for 2 hours. After incubation, cells were washed with PBS. Neutral red in the cells was extracted with 1 M HCl in 30% MeOH. The absorbance at 550 nm was measured using a microplate reader.

[0079] The results of the above experiments are shown in Figure 3 In B, it was presumed that the decrease in cell viability after treatment with 300 μM H2O2 was due to the increased intracellular ROS concentration. The decrease in cell viability caused by H2O2 treatment was restored in a concentration-dependent manner by the addition of glucosamine.

[0080] Example 3. The promoting effect of glucophagene on IL-1α and PGE2 induced by UVB irradiation.

[0081] NHEKs cells were cultured in KG2 medium at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 0.5 μM / well in 96-well plates and incubated for 24 hours. The supernatant was aspirated, and the cells were replaced with KB2 medium containing 0, 25, 50, 100, and 200 μM phycocyanin and cultured for 24 hours. The cells were then washed with PBS and replaced with HBSS. The cells were then irradiated with UVB (280–320 nm, 20 mJ / cm²). 2 Cells were cultured in fresh KB2 medium for 24 hours. The expression levels of IL-1α and PGE2 in the culture supernatant were determined using an ELISA kit. Cells were then lysed with 50 μL of PBS containing 0.5% Triton X-100 to quantify protein levels and calculate the amount of IL-1α and PGE2 produced per unit of protein.

[0082] The results of the above experiments are shown in Figure 4The concentrations of inflammatory cytokines IL-1α and PGE2 were significantly increased by UVB irradiation, reproducing the skin's inflammatory response to UVB irradiation, which induces the production of inflammatory cytokines due to oxidative stress in cells. The induction of IL-1α and PGE2 production induced by UVB irradiation was inhibited in a concentration-dependent manner by the addition of fucoidan up to a concentration of 100 μM. At a fucoidan concentration of 200 μM, the induction of IL-1α and PGE2 production was not inhibited. The production of IL-1α and PGE2 under UVB non-irradiation conditions also showed that fucoidan concentration-dependent inhibition of IL-1α up to 100 μM and PGE2 production up to 50 μM were confirmed for fucoidan up to 50 μM.

[0083] Example 4. Effect of glucophagene on promoting the phagocytosis of fluorescent beads induced by UVB irradiation

[0084] NHEKs cells were cultured in KG2 medium at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 0.5 μM / well in 96-well plates and incubated for 24 hours. The supernatant was aspirated, and the medium was replaced with KB2 medium containing 0, 25, 50, 100, and 200 μM phycocyanin and cultured for 24 hours. Cells were then washed with PBS and replaced with HBSS. The cells were then irradiated with UVB (280–320 nm, 20 mJ / cm²). 2 Cells were cultured in fresh KB2 medium for 24 hours. After replacing the medium with KB2 medium containing FluoSpheres (trademark) and incubating for 4 hours, the cells were lysed with 50 μL PBS containing 0.5% Triton X-100. The fluorescence of the cell lysate was measured using a microplate reader. The fluorescence of the cell lysate was detected using a microplate reader at excitation / emission wavelengths of 535 nm / 590 nm. Then, the cells were lysed with 50 μL PBS containing 0.5% Triton X-100 to quantify the protein content, and the fluorescence intensity per unit of protein was normalized.

[0085] The results of the above experiments are shown in Figure 5 In vitro, phagocytosis of FluoSpheres (trademark) induced by NHEK cells recreated the system by which pigment cells stimulated by UVB irradiation (via ROS and inflammatory cytokines) accumulate melanosomes intracellularly, leading to the formation of skin spots. Phagocytosis of FluoSpheres induced by UVB irradiation was concentration-dependently inhibited by the addition of phycocyanin up to a concentration of 100 μM. At a phycocyanin concentration of 200 μM, the inhibitory effect of FluoSpheres phagocytosis was less pronounced compared to 50 μM and 100 μM.

[0086] Example 5. Effects of phycocyanin on the inhibition of type I collagen production and the promotion of MMP-1 production induced by UVB irradiation.

[0087] NHEKs cells were cultured in KG2 medium at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 0.5 μM / well in 96-well plates and incubated for 24 hours. The supernatant was aspirated, and the medium was replaced with KB2 medium containing 0, 25, 50, 100, and 200 μM phycocyanin and cultured for 24 hours. Cells were then washed with PBS and replaced with HBSS. The cells were then irradiated with UVB (280–320 nm, 20 mJ / cm²). 2 The cells were cultured in fresh KB2 medium for 24 hours. After culture, the supernatant (K-CM) was recovered. NHDFs cells were then cultured in DMEM medium at 2.0 × 10⁻⁶. 4 Cells were seeded at a density of 96-well plates and incubated for 24 hours. Following incubation, K-CM was added, and the cells were cultured for an additional 24 hours. The supernatant was then used for analysis of type I collagen and MMP-1 production using ELIZA. Cells were then lysed with 50 μL of PBS containing 0.5% Triton X-100, and protein levels were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific) to calculate the production of type I collagen and MMP-1 per unit of protein.

[0088] The results of the above experiments are shown in Figure 6 This experiment recreated in vitro the system by which UVB-stimulated epidermal cells produce inflammatory cytokines, which in turn inhibit collagen production in fibroblasts or promote MMP-1 production, leading to wrinkle formation. The culture supernatant of UVB-irradiated NHEK cells exhibited strong inhibitory effects on collagen production and promoting effects on MMP-1 production, presumably due to the release of inflammatory cytokines from UVB-irradiated NHEK cells in the culture medium. Decreased collagen production and promoted MMP-1 production were inhibited in a concentration-dependent manner by the addition of alginate up to a concentration of 100 μM. At a glucose concentration of 200 μM, the inhibitory effect was less pronounced compared to 50 μM and 100 μM.

Claims

1. A cosmetic composition for preventing or reducing unwanted cosmetic changes in the skin, and containing glucophage as an active ingredient.

2. The cosmetic composition according to claim 1, wherein the undesirable cosmetic changes to the skin are one or more of the following: dryness and the formation of fine lines and / or wrinkles; decreased elasticity; skin laxity; dehydrated skin; decreased firmness; thinning of the skin; decreased evenness of facial color; poor complexion; excess pigmentation; age spots; erythema; rough surface texture; mottled complexion; general decrease in tissue and cell activity; slowed cell replication; decreased protein synthesis; increased protein breakdown; decreased blood circulation in the skin or vasodilation caused by congestion; blood infiltration; decreased moisture content; accumulation of errors in protein structure and function; undesirable changes to the skin barrier; adhesion to connective tissue; and decreased ability of the skin to rebuild or repair.

3. The cosmetic composition according to claim 1, wherein the undesirable cosmetic change to the skin is the formation of wrinkles or blemishes.

4. The cosmetic composition according to claim 1, wherein the prevention or reduction of undesirable cosmetic changes to the skin is achieved by the action of the glucosamine on: Inhibition of reactive oxygen species (ROS) production in skin tissue; inhibition of ROS-induced inflammatory cytokine production; inhibition of melanosome phagocytosis via pigment cells induced by inflammatory cytokines; inhibition of decreased collagen production via fibroblasts induced by inflammatory cytokines; or inhibition of enhanced MMP-1 production. It can be achieved using any one or more of the methods listed.

5. The cosmetic composition according to claim 4, wherein the ROS in the skin tissue is generated by one or more of the following: sun exposure, wind, heat, dryness, moisture, contact with chemicals, contact with substances that cause mechanical stimulation, smoking, alcohol, side effects of drugs, physical or psychological stress, biochemical changes in the skin due to aging, hormonal abnormalities, fatigue, acne, malnutrition, disease, and circadian rhythm disorder.

6. The cosmetic composition according to claim 4, wherein the ROS in the skin tissue is generated by sun exposure of the skin.

7. The cosmetic composition according to claim 1, further comprising one or more active ingredients for preventing or reducing undesirable cosmetic changes to the skin.

8. The cosmetic composition according to claim 7, wherein the further one or more active ingredients is an antioxidant.

9. The cosmetic composition according to claim 8, wherein the antioxidant is selected from vitamin A / retinol, vitamin C / ascorbic acid, vitamin E / α-tocopherol, coenzyme Q10, N-acetylcysteine ​​(NAC), S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine ​​persulfate, cysteine ​​trisulfide, uric acid, allicin, alliin, allyl disulfide, cyanidin chloride, cyanidin-3,5-diglucoside, anthocyanins, isoflavones, quercetin, and pediatric acid. Theaflavin, theaflavins, sesamin, sesamol, resveratrol, curcumin, chlorogenic acid, ferulic acid, 3,4-dihydroxycinnamic acid, β-carotene, lycopene, astaxanthin, zeaxanthin, L-cysteine ​​methyl hydrochloride (LCM), L-cysteine ​​ethyl hydrochloride (LCE), D-cysteine ​​hydrochloride monohydrate (DCH), D-cysteine ​​methyl hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, L-methionine methyl hydrochloride (LMM).

10. The cosmetic composition according to claim 1 is in the form of a lotion, cream, lotion, gel, aerosol, mask, cleanser, bath product, foundation, powder, lipstick, ointment, poultice, paste, plaster, or serum for skin application.

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