Skin cosmetic composition containing botryococcene as active ingredient

Abstract

The purpose of the present invention is to provide a skin cosmetic composition containing botryococcene as an active ingredient, the skin cosmetic composition being used for preventing or reducing cosmetically undesirable changes of the skin. Multiple studies have proven that botryococcene inhibits the production of ROS and the downstream effects thereof that result in cosmetically undesirable changes of the skin, leading to the discovery of the unknown possibility that that botryococcene can be used to prevent or reduce cosmetically undesirable changes of the skin.

Description

Skin cosmetic composition containing botryococcene as an active ingredient

[0001] The present invention relates to a skin cosmetic composition containing botryococcene as an active ingredient for preventing or alleviating cosmetically undesirable changes in the skin.

[0002] Skin is constantly exposed to stress from a variety of exogenous and endogenous factors, including sun exposure (especially UVB contained in sunlight; 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, side effects of drugs, physical or psychological stress, biochemical changes in the skin due to aging or other reasons, hormonal abnormalities, fatigue, acne, malnutrition, disease, and malfunction of the biological clock.

[0003] Stress from these factors leads to cosmetically undesirable deterioration of the skin's visible appearance, clinical and physical properties, and physiological and organizational functions: the skin becomes fatigued, and the biological and mechanical functions of the skin tissue weaken over time, resulting in the signs of skin aging.

[0004] Significant cosmetically undesirable changes include dryness and the development of fine lines and wrinkles, loss of elasticity, sagging skin, dehydrated skin, decreased firmness, thinning skin, uneven facial complexion, pale complexion, hyperpigmentation, age spots, age spots, red spots, rough texture, and uneven complexion. Dull, damaged hair, hair loss, and scalp imbalances are also common symptoms.

[0005] Additionally, as skin ages or is chronically exposed to mild environmental stresses, measurable cosmetically undesirable changes occur that are less pronounced than those caused by the direct stresses described above, including a general decline in tissue and cell activity, slowed cell replication, decreased protein synthesis, increased protein degradation, decreased or congested cutaneous blood circulation leading to vasodilation, blood infiltration, decreased water content, accumulation of errors in protein structure and function, undesirable changes in the skin barrier, connective tissue adhesion, and a decrease in the skin's ability to remodel or repair itself.

[0006] It is known that the main process by which the various stresses and aging described above cause noticeable or measurable cosmetically undesirable changes in the skin is that these factors promote the generation of reactive oxygen species (ROS) as oxidative stress, and an increase in ROS concentration causes inflammatory responses, lipid, protein, or DNA damage, and accompanying damage such as impaired cellular function, cell death, and tissue destruction. The skin is constantly exposed to some kind of oxidative stress, and if the intensity of this oxidative stress exceeds the tolerable level or if the body's oxidative stress processing mechanism is weakened, some kind of cosmetically undesirable change will occur.

[0007] Therefore, preventing the generation of ROS in the skin and eliminating them is believed to be effective in preventing or reducing cosmetically undesirable changes in the skin.

[0008] Superoxide dismutase (SOD), a typical ROS-scavenging enzyme, catalyzes the conversion of reactive oxygen species, superoxide, into oxygen and hydrogen peroxide. The resulting hydrogen peroxide, which is also harmful, is further eliminated by catalase and glutathione peroxidase (GPx).

[0009] Various small molecules, such as various vitamins, polyphenols, catechins, glutathione, ascorbate, tocopherol, ubiquinone, bilirubin, and uric acid, can function as natural antioxidants, either in cooperation with the body's antioxidant mechanisms or independently. For example, vitamin B2 acts as a coenzyme for GPx, and polyphenols have SOD-like activity. Carotenoids also function as antioxidants and may exert protective effects against oxidative stress and related chronic diseases. For example, Non-Patent Document 1 summarizes reports on the relationship between carotenoids and various chronic diseases, such as coronary heart disease, cataracts, and cancer.

[0010] Therefore, cosmetic products containing such substances with antioxidant activity as active ingredients can be used to prevent or reduce undesirable cosmetic changes in the skin associated with the action of ROS. In fact, various cosmetic skin compositions using antioxidants as active ingredients have been reported (Patent Documents 1, 2, and 3).

[0011] The discovery of new compounds with good antioxidant activity, or the discovery of existing compounds with good antioxidant activity, is greatly welcomed as it will broaden the options for antioxidants, which are in high demand in the fields of medicine, food, and cosmetics.

[0012] For example, Patent Document 4 discloses the synthesis of luteolin 6-C-arabinoside as a flavonoid compound exhibiting excellent antioxidant activity, and discloses the compound, a method for producing the compound, and an antioxidant containing the compound as an active ingredient. Patent Document 5 discloses that epigallocatechin dimers and trimers have the activity of scavenging superoxide anion radicals. Patent Document 6 discloses novel nitrogen-containing heterocyclic compounds or salts thereof, and therapeutic agents for oxidative stress-related diseases containing them.

[0013] : Re-listed 01 / 026670: JP 2021-095370: JP 2017-537091: JP 2006-265249: JP 2010-280716: JP 2007-016011: JP 7-265059: JP 2013-504332: JP 2013-035791: Re-listed 2012 / 077801

[0014] : Canfield, et al. , (1992) Proc. Soc. Exp. Biol. Med. 200:260: Kaya, K. , Microbiol. Cult. Coll. 26 (1): 1-10, 2010

[0015] The present invention aims to provide a skin cosmetic composition containing botryococcene as an active ingredient for preventing or alleviating cosmetically undesirable changes in the skin.

[0016] Algae belonging to the genus Botryococcus are microalgae that use light energy to fix carbon dioxide and produce hydrocarbons. In recent years, concerns have arisen about the depletion of fossil fuels, and carbon dioxide fixation by algae of the genus Botryococcus is considered to be an effective method of energy acquisition. Furthermore, global warming caused by the increase in carbon dioxide levels associated with the combustion of fossil fuels has become a problem. The use of algae of the genus Botryococcus, which have the ability to convert carbon dioxide into hydrocarbons, is expected to be useful not only for utilizing light energy but also for reducing the amount of carbon dioxide (Patent Document 7).

[0017] Botryococcus braunii (B. braunii), a species of colonial algae belonging to the genus Botryococcus, is divided into Race-A, Race-B, and Race-L based on the structural characteristics of the hydrocarbons it produces. ｎ H ２ｎ－１０ The plant produces botryococcene, a branched triterpenoid hydrocarbon with the formula (n=30-37) (Non-Patent Document 2). Triterpenoid hydrocarbons can be used as a feedstock for hydrocracking in petroleum refineries to produce, for example, octane (gasoline, petrol), kerosene, and diesel. Botryococcene can potentially be converted into biofuels with higher octane numbers (Patent Document 8).

[0018] Meanwhile, the use of botryococcene as an active ingredient in cosmetics is also being investigated (Patent Document 9). The applicant of the present invention is one of the co-applicants of this document. This document reports that a combination of botryococcene and hydrogenated modified botryococcene can be used as a moisturizing agent with superior effectiveness to squalane. Since the cosmetic use of botryococcene can be valued far more highly than its use as a biofuel, this could provide a strong incentive to develop technology for producing botryococcene using Botryococcus braunii culture.

[0019] The inventors of the present invention conducted further research into the cosmetic applications of botryococcene and have demonstrated through multiple tests that botryococcene inhibits the production of ROS and its downstream effects that cause cosmetically undesirable changes in the skin, leading to the discovery of the hitherto unknown possibility that botryococcene may be used to prevent or alleviate cosmetically undesirable changes in the skin.

[0020] The above-mentioned Patent Document 9 discloses the cosmetic use of botryococcene, just like the present invention. However, the skin moisturizing function disclosed in the document and the prevention or reduction of cosmetically undesirable changes in the skin associated with ROS disclosed in the present invention are separate uses that are not related in any way.

[0021] Therefore, the present application provides the following inventions.

[0022] 1. A cosmetic composition containing botryococcene as an active ingredient for preventing or alleviating cosmetically undesirable changes in the skin. 2. The cosmetic composition according to item 1, wherein the cosmetically undesirable changes in the skin are one or more of the following: dryness and the appearance of fine lines and wrinkles, loss of elasticity, sagging skin, dehydrated skin, decreased firmness, thinning skin, uneven facial complexion, pale complexion, hyperpigmentation, age spots, red spots, rough surface texture, mottled complexion, a general decrease in tissue and cell activity, slowed cell replication, decreased protein synthesis, increased protein breakdown, dilated blood vessels due to reduced or congested blood circulation in the skin, blood infiltration, decreased water content, accumulation of errors in protein structure and function, undesirable changes in the skin barrier, connective tissue adhesion, and a decrease in the skin's ability to rebuild or repair itself. 3. The cosmetic composition according to either item 1 or 2, wherein the cosmetically undesirable changes in the skin are the formation of wrinkles or age spots. 4. 4. The cosmetic composition according to any one of items 1 to 3, wherein the prevention or reduction of cosmetically undesirable changes in the skin is achieved by the botryococcene acting on one or more of the following: suppressing the generation of ROS in skin tissue, suppressing the production of inflammatory cytokines caused by ROS, suppressing melanosome phagocytosis by pigment cells caused by inflammatory cytokines, or suppressing the decrease in collagen production by fibroblasts or the increase in MMP-1 production caused by inflammatory cytokines. 5. The cosmetic composition according to item 4, wherein the ROS in the skin tissue are generated by one or more of the following: exposure of the skin to sunlight, wind, heat, dryness, moisture, contact with chemicals, contact with a mechanically irritating substance, 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 malfunction of the biological clock. 6. The cosmetic composition according to item 4, wherein the ROS in the skin tissue are generated by exposure of the skin to sunlight. 7. 7. The cosmetic composition according to any one of items 1 to 6, further comprising one or more active ingredients that prevent or alleviate cosmetically undesirable changes to the skin. 8. The cosmetic composition according to item 7, wherein the one or more additional active ingredients are antioxidants.9. The antioxidant is selected from the group consisting of vitamin A / retinol, vitamin C / ascorbic acid, vitamin E / α-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, sesaminol, resveratrol, and cucurbits. Item 8. The cosmetic composition according to item 8, wherein the active ingredient is selected from the group consisting of cumin, chlorogenic acid, ferulic acid, 3,4-dihydroxycinnamic acid, β-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, and L-methionine methyl ester hydrochloride (LMM). 10. The cosmetic composition according to any one of items 1 to 9, which is in the form of a skin lotion, cream, emulsion, gel, aerosol, pack, cleanser, bath additive, foundation, powder, lipstick, ointment, cataplasm, paste, plaster, or essence.

[0023] FIG. 1 shows an optical microscope image of Botryococcus braunii secreting botryococcene oil droplets and the structural formula of botryococcene.

[0024] FIG. 2 shows the cytotoxicity evaluation by botryococcene treatment.

[0025] Figure 3 shows the effects of botryococcene treatment on intracellular ROS scavenging (Figure 3A) and H ２ O ２ The decrease in cell viability due to the addition of ATP was alleviated by botryococcene treatment (FIG. 3B).

[0026] Figure 4 shows the effects of botryococcene treatment on the inflammatory cytokine IL-1α (Figure 4A) and PGE ２ (Figure 4B) shows the suppression of production.

[0027] FIG. 5 shows the inhibition of fluorescent bead phagocytosis by fibroblasts by botryococcene treatment.

[0028] FIG. 6 shows the suppression of the decrease in collagen production in epidermal cells (FIG. 6A) and the suppression of MMP-1 production (FIG. 6B) by botryococcene treatment.

[0029] FIG. 7 shows a schematic diagram of the process by which UVB causes cosmetically undesirable changes in the skin and the points in the process where the effects of botryococcene treatment were observed in FIGS.

[0030] Botryococcene is a C produced by some strains of Botryococcus braunii. ｎ H ２ｎ－１０ (n=30 to 37) Botryococcus braunii race-B, which produces botryococcene, is useful for mass production of botryococcene because it produces botryococcene in high purity and in large quantities, secreting 90% or more of the produced botryococcene extracellularly and accumulating it within the colony (Patent Document 10).

[0031] Botryococcene itself can be used directly as diesel oil for ships and agricultural vehicles, but it can also be converted into diesel, naphtha, kerosene, and gasoline using conventional catalytic cracking. Furthermore, because botryococcene has excellent moisturizing properties, it has also been found to be used as a moisturizing ingredient in cosmetics (Patent Document 9). While botryococcene is thus expected to be used in a variety of industrial applications, the skin cosmetic application proposed in this invention represents a further promising use of botryococcene, further enhancing its value.

[0032] In the present invention, a cosmetic composition containing botryococcene as an active ingredient is prepared, and such a cosmetic composition can prevent or reduce cosmetically undesirable changes in the skin.

[0033] The preventive or alleviating effect of botryococcene on cosmetically undesirable changes in the skin can be reasonably estimated from test results, as will be described later in the Examples, showing that botryococcene suppresses various events that cause cosmetically undesirable changes in the skin, such as the production of inflammatory cytokines caused by an increase in 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.

[0034] Increased ROS levels in the skin mediate the effects of various stress factors on the skin, resulting in various cosmetically undesirable changes in the skin. Such stress factors that indirectly or directly cause increased ROS levels include, but are not limited to, sun exposure (UVB contained in sunlight; 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 or other reasons, hormonal abnormalities, fatigue, acne, malnutrition, disease, and malfunction of the biological clock.

[0035] A stress factor of particular importance in the present invention is sun exposure. The cosmetically undesirable changes in skin caused by such sun exposure are generally referred to as photoaging. Ultraviolet (UV) radiation, a component of sunlight, particularly mid-UV (UVB, wavelengths 290-320 nm), is the primary cause of photoaging. While the amount of UVB exposure required to induce photoaging is currently unknown, repeated exposure to UVB at levels that cause transient erythema or sunburn typically leads to photoaging. Clinically, photoaging can be identified as rough skin, wrinkle formation, pigmented patches, rosy skin, sagging skin, telangiectasia, lentigo formation, purpura, skin fragility, atrophy, the development of fibrotic hypopigmented areas, and the development of premalignant and malignant tumors. Photoaging occurs in skin habitually exposed to sunlight, such as the face, ears, head, neck, and hands.

[0036] Cosmetically undesirable changes that may be prevented or alleviated by the skin cosmetic composition of the present invention include, but are not limited to, dryness and the appearance of fine lines and wrinkles, loss of elasticity, sagging skin, dehydrated skin, loss of firmness, thinning skin, uneven facial complexion, pale complexion, hyperpigmentation, age spots, red spots, rough surface texture, and mottled complexion, etc. Also expected to be prevented or improved by the skin cosmetic composition of the present invention are less pronounced but measurable cosmetically undesirable changes such as a general decrease in tissue and cell activity, slowed cell replication, decreased protein synthesis, increased protein breakdown, dilated blood vessels due to decreased or congested blood circulation in the skin, blood infiltration, decreased water content, accumulation of errors in protein structure and function, undesirable changes in the skin barrier, connective tissue adhesion, and a decreased ability of the skin to rebuild or repair itself.

[0037] The cosmetically undesirable changes that should be particularly prevented or alleviated by the skin cosmetic composition of the present invention are the formation of wrinkles or age spots on the skin. Increased ROS levels in the skin are mainly caused by the inflammatory cytokines IL-1α and PGE. ２ These actions promote melanosome phagocytosis in epidermal cells, resulting in skin blemishes, and also suppress collagen production in dermal fibroblasts or promote MMP-1 production, resulting in skin wrinkles. The examples of this application demonstrate that the skin cosmetic composition of the present invention exhibits inhibitory effects at various points in the pathway leading from increased ROS concentration to the formation of these blemishes or wrinkles (Figure 7).

[0038] The skin cosmetic composition of the present invention can be prepared in any suitable formulation intended for application to the skin where undesirable cosmetic changes are to be prevented or alleviated. Accordingly, formulations include, but are not limited to, lotions, creams, emulsions, gels, aerosols, packs, cleansers, bath additives, foundations, powders, lipsticks, ointments, poultices, pastes, plasters, essences, etc. Determining the formulation of the skin cosmetic composition of the present invention and designing an appropriate component composition to constitute the desired formulation are within the scope of routine optimization or consideration of formulation conditions for those skilled in the art.

[0039] The amount of botryococcene to be incorporated into the skin cosmetic composition of the present invention sufficient to achieve the desired effect can be appropriately determined by those skilled in the art through preliminary tests commonly performed in the cosmetic field, taking into consideration factors such as the formulation, method of use, dosage, desired cosmetic effect, and the type or degree of cosmetically undesirable changes to be targeted. In one embodiment, the skin cosmetic composition of the present invention contains botryococcene 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. The botryococcene concentrations in the examples of the present application are concentrations applied to in vitro cultured cells, and do not define the range of the amount of botryococcene to be blended in the skin cosmetic composition of the present invention.

[0040] The skin cosmetic composition of the present invention may contain one or more additional active ingredients in addition to botryococcene. Such additional active ingredients may supplement, or additively or synergistically enhance, the effect of the skin cosmetic composition of the present invention in preventing or alleviating undesirable cosmetic changes. For example, the skin cosmetic composition of the present invention may contain an antioxidant as one or more additional active ingredients.

[0041] Antioxidants that may be additionally incorporated into the skin cosmetic composition of the present invention include, but are not limited to, vitamin A / retinol, vitamin C / ascorbic acid, vitamin E / α-tocopherol, coenzyme Q10, N-acetylcysteine ​​(NAC), S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine ​​persulfide, cysteine ​​trisulfide, uric acid, allicin, allyl disulfide, cyanidin chloride, cyanidin-3,5-diglucoside, anthocyanins, isoflavones, quercetin, catechin, theaflavin, Examples of such antioxidants include hydroxybenzoates, ...

[0042] The following examples demonstrate the effect of botryococcene incorporated into the skin cosmetic composition of the present invention in in vitro models of various molecular biological events that lead to cosmetically undesirable changes in the skin.

[0043] Cell Culture 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 growth supplement at 37°C and 5% CO . ２ The cells were cultured in an incubator.

[0044] Normal human dermal fibroblasts (NHDFs) were cultured in DMEM supplemented with 10% fetal bovine serum, 1% antibiotics, and 1% human epidermal keratinocyte growth supplement at 37°C and 5% CO. ２ The cells were cultured in an incubator.

[0045] Cytotoxicity Measurement by Botryococcene Treatment NHEKs cells were cultured at 2.5 × 10 ４Cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. The supernatant was aspirated and replaced with HuMedia-KB2 (KB2) medium supplemented with 0, 12.5, 25, 50, 100, 200, or 400 μM botryococcene, followed by 24 hours of incubation. After incubation, the cells were washed with PBS, replaced with KB2 medium containing 0.003% neutral red, and incubated for 2 hours. Intracellular neutral red was extracted with 1 M HCl in 30% MeOH. Absorbance at 550 nm was measured using a microplate reader.

[0046] The results of this test are shown in Figure 2. There was no change in cell viability at botryococcene concentrations of 0 to 200 μM, but a significant decrease was observed at 400 μM. Based on this result, the maximum botryococcene treatment concentration in subsequent tests was set at 200 μM.

[0047] Example 1. Effect of botryococcene on scavenging intracellular reactive oxygen species NHEKs cells were cultured at 2.5 × 10 in KG2 medium. ４ The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. The supernatant was removed and replaced with KB2 medium supplemented with 0, 25, 50, 100, or 200 μM botryococcene, followed by incubation for 24 hours. After incubation, the cells were washed with PBS and then added to KB2 medium containing 200 μM H ２ O ２ The medium was replaced with HBSS buffer containing 20 μM DCFH-DA and the cells were cultured for 1 hour. The supernatant was removed, and the cells were cultured for 30 minutes in HBSS buffer containing 20 μM DCFH-DA. Fluorescence intensity was measured using a microplate reader (Emission = 485 nm, Extension = 530 nm). The cells were then lysed in 50 μL of PBS containing 0.5% Triton® X-100, the protein amount was quantified, and the fluorescence intensity per unit protein was normalized.

[0048] The results of the above test are shown in Figure 3A. ２ O ２ Treatment increases intracellular ROS levels. ２ O ２ The treatment increased intracellular ROS concentration and H ２ O ２In both cases, the ROS concentration in the untreated cells was reduced by the addition of botryococcene in a concentration-dependent manner.

[0049] Example 2. H ２ O ２ Effect of botryococcene on cytotoxicity caused by the addition of ４ The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. The supernatant was aspirated and replaced with KB2 medium supplemented with 0, 25, 50, 100, or 200 μM botryococcene, and incubated for 24 hours. After incubation, the cells were resuspended in PBS and 300 μM H ２ O ２ The cells were then washed with HBSS containing 0.003% neutral red for 1 hour, and then incubated in KB2 medium for 24 hours. After incubation, the cells were washed with PBS and replaced with KB2 medium containing 0.003% neutral red for 2 hours. After incubation, the cells were washed with PBS. Intracellular neutral red was extracted with 1 M HCl in 30% MeOH. The absorbance at 550 nm was measured using a microplate reader.

[0050] The results of the above test are shown in Figure 3B. ２ O ２ The decrease in cell viability due to treatment is presumably due to an increase in intracellular ROS concentration. ２ O ２ The decrease in cell viability due to treatment was restored by the addition of botryococcene in a concentration-dependent manner.

[0051] Example 3. IL-1α and PGE induced by UVB irradiation ２ Effect of botryococcene on production promotion NHEKs cells were cultured at 2.5 × 10 in KG2 medium. ４ The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. The supernatant was aspirated and replaced with KB2 medium containing 0, 25, 50, 100, or 200 μM botryococcene, and the medium was cultured for 24 hours. After that, the cells were washed with PBS and replaced with HBSS. Then, the cells were exposed to UVB (280-320 nm, 20 mJ / cm). ２) were irradiated and cultured in fresh KB2 medium for 24 hours. IL-1α and PGE in the culture supernatant ２ The expression levels were measured using an ELISA kit. Then, the cells were lysed with 50 μL of PBS containing 0.5% Triton (registered trademark) X-100, the amount of protein was quantified, and the amount of IL-1α and PGE2 produced per unit protein was calculated.

[0052] The results of the above test are shown in Figure 4. UVB irradiation increased the levels of inflammatory cytokines IL-1α and PGE ２ The levels of IL-1α and PGE2 were significantly increased, which reproduces the inflammatory response of skin to UVB irradiation, in which UVB irradiation causes oxidative stress in cells and induces the production of inflammatory cytokines. ２ The induction of both IL-1α and PGE production was concentration-dependently suppressed by the addition of botryococcene up to 100 μM. ２ The induction of IL-1α and PGE production was not suppressed under UVB non-irradiation conditions. ２ The production of IL-1α was up to 100 μM, and PGE ２ For the α-glucan derivative, a concentration-dependent inhibition was observed up to 50 μM of botryococcene.

[0053] Example 4. Effect of botryococcene on the promotion of fluorescent bead phagocytosis by UVB irradiation. NHEKs cells were cultured at 2.5 × 10 ４ The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. The supernatant was aspirated and replaced with KB2 medium containing 0, 25, 50, 100, or 200 μM botryococcene, and the medium was cultured for 24 hours. After that, the cells were washed with PBS and replaced with HBSS. Then, the cells were exposed to UVB (280-320 nm, 20 mJ / cm ２) and cultured in fresh KB2 medium for 24 hours. The medium was replaced with KB2 medium containing FluoSpheres™ and allowed to stand for 4 hours. Afterwards, the cells were lysed in 50 μL PBS 0.5% Triton® X-100, and the fluorescence of the cell lysate was measured using a microplate reader. The fluorescence of the cell lysate was detected using a fluorescence reader at excitation / emission wavelengths of 535 nm / 590 nm. The cells were then lysed in 50 μL PBS containing 0.5% Triton® X-100, and the protein amount was quantified, and the fluorescence intensity per unit protein was normalized.

[0054] The results of the above test are shown in Figure 5. The phagocytosis of FluoSpheres™ by NHEKs cells reproduces in vitro the system in which pigment cells stimulated by UVB irradiation (via ROS and inflammatory cytokines) phagocytose melanosomes, which accumulate intracellularly, resulting in the formation of skin blemishes. The phagocytosis of fluorescent beads induced by UVB irradiation was concentration-dependently inhibited by the addition of botryococcene up to 100 μM. At a botryococcene concentration of 200 μM, the inhibitory effect on fluorescent bead phagocytosis was smaller than at 50 μM and 100 μM.

[0055] Example 5. Effect of botryococcene on the inhibition of type I collagen production and the promotion of MMP-1 production by UVB irradiation. NHEKs cells were cultured at 2.5 × 10 ４ The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. The supernatant was aspirated and replaced with KB2 medium containing 0, 25, 50, 100, or 200 μM botryococcene, and the medium was cultured for 24 hours. After that, the cells were washed with PBS and replaced with HBSS. Then, the cells were exposed to UVB (280-320 nm, 20 mJ / cm ２ ) were irradiated and cultured in fresh KB2 medium for 24 hours. After the culture, the supernatant (K-CM) was collected. NHDFs cells were cultured at 2.0 × 10 ４The cells were seeded onto a 96-well plate at a density of 1000 cells / well and incubated for 24 hours. After incubation, K-CM was added and the cells were further incubated for 24 hours. The supernatant was analyzed for type I collagen and MMP-1 production by ELISA. The cells were then lysed in 50 μL of PBS containing 0.5% Triton® X-100, and the protein amount was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific), and the type I collagen and MMP-1 production per unit protein was calculated.

[0056] The results of the above test are shown in Figure 6. This test reproduced in vitro the system in which epidermal cells stimulated by UVB irradiation produce inflammatory cytokines, which in turn suppress collagen production in fibroblasts or promote MMP-1 production, resulting in the formation of skin wrinkles. The culture supernatant of UVB-irradiated NHEKs cells had a strong collagen production inhibitory effect and MMP-1 production promoter effect, which is presumed to be due to the inflammatory cytokines released into the medium by UVB-irradiated NHEKs cells. The decrease in collagen production and the promotion of MMP-1 production were concentration-dependently inhibited by the addition of botryococcene up to 100 μM. At a botryococcene concentration of 200 μM, the inhibitory effect was smaller than at 50 μM and 100 μM.

Claims

1. A cosmetic composition containing botryococcene as an active ingredient for preventing or reducing cosmetically undesirable changes to the skin.

2. The cosmetic composition of claim 1, wherein the undesirable cosmetic changes to the skin are one or more of the following: dryness and the appearance of fine lines and wrinkles, loss of elasticity, sagging skin, dehydrated skin, loss of firmness, thinning skin, less even facial complexion, pale complexion, hyperpigmentation, age spots, erythema, rough surface texture, mottled complexion, general decrease in tissue and cell activity, slowed cell replication, decreased protein synthesis, increased protein breakdown, dilated blood vessels due to reduced or congested blood circulation in the skin, infiltration of blood, decreased water 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 remodel or repair itself.

3. The cosmetic composition according to claim 1, wherein the cosmetically undesirable change in the skin is the formation of wrinkles or age spots on the skin.

4. The cosmetic composition according to claim 1, wherein the prevention or reduction of cosmetically undesirable changes in the skin is achieved by the botryococcene acting on one or more of the following: suppressing the generation of reactive oxygen species (ROS) in skin tissue, suppressing the production of inflammatory cytokines caused by ROS, suppressing melanosome phagocytosis by pigment cells caused by inflammatory cytokines, or suppressing the decrease in collagen production or the increase in MMP-1 production by fibroblasts caused by inflammatory cytokines.

5. The cosmetic composition according to claim 4, wherein the ROS in the skin tissue is generated by one or more of the following: exposure of the skin to sunlight, wind, heat, dryness, moisture, contact with chemicals, contact with a substance that causes mechanical irritation, 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 malfunction of the biological clock.

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

7. The cosmetic composition of claim 1 further comprising one or more active ingredients that prevent or reduce undesirable cosmetic changes to the skin.

8. The cosmetic composition of claim 7, wherein the one or more additional active ingredients are antioxidants.

9. The antioxidant is selected from the group consisting of vitamin A / retinol, vitamin C / ascorbic acid, vitamin E / α-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, sesaminol, resveratrol, and curcuma. The cosmetic composition according to claim 8, wherein the active ingredient is selected from the group consisting of cumin, chlorogenic acid, ferulic acid, 3,4-dihydroxycinnamic acid, β-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, and L-methionine methyl ester hydrochloride (LMM).

10. The cosmetic composition according to claim 1, which is in the form of a lotion, cream, milk, gel, aerosol, pack, cleanser, bath additive, foundation, powder, lipstick, ointment, cataplasm, paste, plaster, or essence for application to the skin.

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