Composition for skin improvement comprising kestose

Abstract

The present invention relates to a skin improvement composition comprising kestose as an active ingredient, specifically for use in improving skin photoaging and for use in improving skin elasticity by increasing the expression of hyaluronic acid.

Description

Technology Field

[0001] The present invention relates to a composition comprising kestose as an active ingredient, and to the use of said composition for improving photoaging, for example, for the prevention, treatment, or improvement of skin damage caused by UV rays. Background Technology

[0002] Recently, probiotics have been attracting attention as health supplements following research results indicating their effectiveness in alleviating atopic symptoms and strengthening immunity due to their ability to increase beneficial bacteria in the gut and suppress harmful bacteria.

[0003] However, reports suggest that caution is required when taking probiotics due to concerns about side effects, such as worsening immunity in patients with intestinal damage or immunocompromised conditions.

[0004] Recently, in addition to the term probiotics, research on prebiotics—which refers to nutrients that serve as food for probiotics and can maximize their function by activating intestinal probiotics—is being actively conducted.

[0005] Most prebiotics take the form of oligosaccharides or polysaccharides, and in the past, dietary fibers such as inulin and fructooligosaccharides were mainly used as food for beneficial bacteria such as Bifidobacteria. The problem to be solved

[0006] The present invention relates to a composition for improving skin condition comprising kestose as an active ingredient, and more specifically, to a composition for preventing or improving skin condition deterioration caused by ultraviolet rays, wherein the composition has skin antioxidant, anti-inflammatory, moisturizing, skin wrinkle improvement, skin regenerative activity, and / or skin elasticity recovery properties.

[0007] One example of the present invention relates to a use for improving skin photoaging comprising kestose as an active ingredient, and more specifically, to a use for preventing, treating, or improving photoaging, and a composition for controlling inflammation caused by photoaging.

[0008] A further example of the present invention relates to the use of improving skin elasticity through increased hyaluronan biosynthesis comprising kestose as an active ingredient. means of solving the problem

[0009] The present invention will be described in more detail below.

[0010] One example of the present invention relates to a use for improving skin conditions comprising kestose as an active ingredient. The skin condition may be one or more conditions selected from the group consisting of skin cell damage caused by ultraviolet rays, generation of reactive oxygen species (ROS), skin oxidation, skin inflammation, skin photoaging, skin wrinkles, and reduced skin elasticity.

[0011] A specific embodiment of the present invention relates to a use for improving skin photoaging comprising kestose as an active ingredient, and more specifically, to a use for preventing, treating, or improving photoaging, and to a composition for controlling inflammation caused by photoaging.

[0012] Another specific embodiment relates to a composition for preventing or improving skin photoaging comprising kestose as an active ingredient, wherein the skin photoaging includes skin cell damage caused by ultraviolet rays, generation of reactive oxygen species (ROS), and / or skin oxidation.

[0013] An additional embodiment relates to a composition for controlling inflammation caused by skin photoaging, comprising kestose as an active ingredient.

[0014] Further embodiments relate to a composition for improving skin elasticity comprising kestose as an active ingredient, specifically, a composition for improving skin elasticity by increasing hyaluronic acid biosynthesis, a composition for moisturizing the skin, a composition for regenerating the skin, or a composition for improving skin wrinkles.

[0015] The skin condition improvement composition according to the present invention can be administered via various routes such as external application to the skin or oral intake, and can be applied as a pharmaceutical composition, a cosmetic composition, or a food composition.

[0017] In this specification, the improvement of skin condition may be due to one or more effects selected from the group consisting of the ability of Kestose to protect skin cells from ultraviolet rays, the ability to remove reactive oxygen species (ROS), the skin antioxidant ability, the skin anti-inflammatory ability, the skin anti-photoaging ability, the skin collagen synthesis ability, and the ability to increase skin hyaluronan biosynthesis.

[0018] Specifically, the reactive oxygen species scavenging ability of the kestose according to the present invention is an increase in the expression level of one or more genes selected from the group consisting of PI3K, AKT, Nrf2, and HO-1 as factors related to Nrf2 / HO-1 signaling activated by ROS, or

[0019] The above anti-inflammatory ability is an increase in the expression level of one or more genes selected from the group consisting of p50, p65, TNF-α, IL-6, and COX-2 as factors related to NF-κB signaling that regulates inflammatory responses, or

[0020] The above skin collagen synthesis ability is due to the regulation of the expression levels of one or more genes selected from the group consisting of p38, ERK, JNK, c-Jun, c-Fos, MMP-1, MMP-2, MMP-3, and MMP-9 genes as factors related to MAPK-ERK / JNK / p38 / AP-1 signaling that degrades and regulates collagen, or

[0021] The above-mentioned skin collagen synthesis ability is due to the regulation of the expression levels of one or more genes selected from the group consisting of TGF-β1, Smad2 / 3, COL1A1, and COL2A1 genes as factors related to TGF-β1 / Smad signaling that induces collagen synthesis, or

[0022] The above-mentioned ability to increase skin hyaluronan biosynthesis may be due to the regulation of the expression levels of one or more genes selected from the group consisting of HAS-1, HAS-2, and HAS-3 genes as factors related to hyaluronic acid synthesis.

[0023] Extrinsic aging of the skin is primarily due to photodamage caused by ultraviolet (UV) irradiation. UV irradiation is a major environmental factor in acute and chronic diseases of human skin. Chronic UV exposure is a major cause of premature skin aging, so-called photoaging, and clinically is characterized by thickening, roughness, coarse wrinkles, and mottled pigmentation. Histological changes in the skin caused by UV include collagen cross-linking, excessive deposition of abnormal elastic fibers, and an increase in glycosaminoglycans.

[0024] Ultraviolet radiation consists of UVA (320–400 nm), UVB (280–320 nm), and UVC (100–280 nm). UVB generates reactive oxygen species (ROS) that directly damage DNA and induce the formation of pyrimidine dimers. ROS also activate signals for DNA repair and antioxidant defenses, and induce the expression of matrix metalloproteinases (MMPs). MMPs belong to a family of matrix-degrading enzymes structurally associated with tissue degradation processes such as aging, e.g., skin aging and arthritis. MMPs also degrade collagen and other extracellular matrix proteins, and MMP-1 targets collagen types I, II, and III in the skin.

[0025] UVB is known to stimulate the production of reactive oxygen species (ROS), causing oxidative stress and skin damage. Furthermore, it has been revealed that the upregulation of transcription factors such as NF-kB (nuclear factor kappa B) and AP-1 (activator protein 1) is present in UVB-treated cells. The aforementioned effects lead to the degradation of the extracellular matrix (ECM) and the induction of inflammatory cytokine production. The dermal extracellular matrix consists of collagen, elastin, fibronectin, gelatin, and glycosaminoglycans. Collagen is a major structural protein of skin connective tissue; synthesized by dermal fibroblasts, it plays a role in providing strength and elasticity to the skin. Additionally, hyaluronan is a major glycosaminoglycan widely distributed in connective tissue; in the dermis, HA is responsible for skin moisture regulation and maintaining cellular structure using its high water retention capacity and viscosity, indicating that HA is an important substance for maintaining healthy skin.

[0026] The breakdown of collagen and elastin in UV-induced photoaged skin is primarily caused by the increased expression of their degrading enzymes, such as MMP proteins. Additionally, pro-inflammatory cytokines interfere with collagen synthesis and promote its breakdown. Type I collagen is the most abundant protein in the extracellular matrix of connective tissue. The extracellular matrix contains other types of proteins, such as Type III, V, and VII collagen, elastin, proteoglycans, and fibronectin.

[0027] In the present invention, the efficacy of Kestos in improving skin condition was specifically confirmed through cytotoxicity evaluation of Kestos and protective efficacy against cells damaged by UVB. Specifically, epidermal keratinocyte (HaCaT) cells were treated with Kestos at various concentrations and cultured, and then an MTT assay was performed to evaluate the effect on cells at non-toxic Kestos concentrations in HaCaT cells irradiated with UVB. It was confirmed that Kestos effectively protected against UVB-induced damage, showing a cell viability approximately 20% higher than the UVB control group.

[0028] In addition, the efficacy of Kestos in scavenging reactive oxygen species (ROS) was evaluated by confirming the inhibitory effect using a 2',7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. When HaCaT cells were reacted with DCFH-DA fluorescence and the effect on UVB-activated ROS was confirmed, it was found that treatment with Kestos reduced ROS by approximately 30% compared to the UVB control group.

[0029] The antioxidant efficacy of Kestos on UVB-damaged HaCaT cells was analyzed by examining the gene and protein expression levels of factors (PI3K, AKT, Nrf2, HO-1) associated with Nrf2 / HO-1 signaling, which is activated by ROS, through RT-PCR and Western blot. As a result of analyzing the expression levels of the factors associated with Nrf2 / HO-1 signaling, when HaCaT cells were treated only with UVB, the expression levels of AKT and PI3K increased while the expression levels of Nrf2 and HO-1 decreased; however, when Kestos was treated, the expression of AKT, PI3K, Nrf2, and HO-1 showed a pattern similar to that of the control group not treated with Kestos, confirming that it did not affect the expression of oxidative capacity.

[0030] In addition, to confirm the anti-inflammatory efficacy of Kestose on UVB-damaged HaCaT cells, the gene expression levels of factors (p50, p65, TNF-α, IL-6, COX-2) related to NF-κB signaling that regulates inflammatory responses were checked via RT-PCR. As a result, the gene expression of activated NF-κB related factors p50, p65, TNF-α, IL-6, and COX-2, which are used to confirm the regulation of inflammatory responses, was reduced by Kestose, and among them, it was confirmed that there was an excellent anti-inflammatory effect at 10 mM.

[0031] To evaluate the anti-aging efficacy of Kestose on UVB-damaged HaCaT cells, the protein expression levels of factors (p38, ERK, JNK, c-Jun, c-Fos, MMP-1, MMP-2, MMP-3, MMP-9) associated with MAPK-ERK / JNK / p38 / AP-1 signaling, which degrades and regulates collagen, were examined. In the control group (UVB), an increase in the expression of MAPK protein phosphorylation (p-ERK, p-JNK, p-p38) was observed, while phosphorylation expression was inhibited when treated with different concentrations of Kestose. Calculation of protein expression density also confirmed that the expression levels of p-ERK / ERK, p-JNK / JNK, and p-p38 / p38 significantly decreased as the concentration of Kestose increased. In addition, it was confirmed that UVB-induced ROS activates the MAPK signaling pathway through phosphorylated ERK, JNK, and p38 signals, and that ketose significantly reduces the expression of MAPK signaling.

[0032] To evaluate the skin-protective efficacy of Kestose on UVB-damaged HaCaT cells, the expression levels of factors (TGF-β1, Smad2 / 3, COL1A1, COL2A1) associated with TGF-β1 / Smad signaling, which induces collagen synthesis, were examined. UVB exposure reduced TGF-β1 levels by approximately 54% compared to normal cells. Furthermore, in the experimental group treated with both UVB exposure and Kestose, TGF-β1 production increased by approximately 178% compared to cells irradiated with UVB alone, which was significantly higher (Fig. 10). Upon examining Smad signaling induced by TGF-β1 expression, it was found that Kestose increased expression levels, indicating an increase in the level of transforming growth factor (TGF)-β1, a factor involved in collagen production. Specifically, p-Smad 2 / 3 / Smad 2 / 3 is a factor activated by TGF-β1, and UVB exposure reduced p-Smad 2 / 3 / Smad 2 / 3 by approximately 38% compared to normal cells. Additionally, in the experimental group treated with UVB exposure and kestose, p-Smad 2 / 3 / Smad 2 / 3 increased by approximately 50% compared to cells irradiated with UVB only (Fig. 11). These results demonstrated that the TGF-β1 / Smad signaling pathway transporter, which is inhibited by ultraviolet rays, is increased by kestose, confirming its efficacy in improving photoaging.

[0033] The expression levels of factors (HAS-1, HAS-2, HAS-3) related to hyaluronan biosynthesis in UVB-damaged HaCaT cells were analyzed. It was confirmed that the expression levels of HAS-1, HAS-2, and HAS-3, genes related to hyaluronan biosynthesis that help with skin hydration, significantly increased at a concentration of 10 mM of Kestose. Therefore, it was confirmed that Kestose promotes hyaluronan biosynthesis by increasing the expression of genes related to hyaluronan biosynthesis, thereby possessing efficacy in maintaining skin structure and function, hydrating the skin, regenerating the skin, and increasing skin elasticity.

[0035] The present invention comprises kestose or 1-kestose as an active ingredient in a composition for improving skin condition, wherein kestose may be used alone or as a sugar composition containing it. Kestose may be used in liquid or powder form, and the powder may be amorphous or crystalline. The kestose may be used by purchasing a commercially available product or by using a product manufactured using a specific raw material.

[0036] One example of the above sugar composition may be a kestose-containing fructooligosaccharide (FOS). FOS consists of a linear chain having 1 to 9 fructose residues connected to a sucrose molecule by a β2→1 linkage. The kestose-containing fructooligosaccharide is preferably a main component of 1-kestose, and may include, for example, a high content of 1-kestose (GF2) and one or more selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4).

[0037] The kestose or sugar composition containing kestose usable in the present invention is not particularly limited and can be prepared using sugar as a substrate by using an enzyme having kestose conversion activity or a microorganism that produces said enzyme.

[0038] The enzyme having the above-mentioned kestose-converting activity is an enzyme having the activity of converting a substrate containing sugar into a fructooligosaccharide containing kestose, and may be an enzyme derived from one or more selected from the group consisting of, for example, Aspergillus niger strain, Pichia farinose strain, Yarrowia lipolytica, Millerozyma farinose, and Aspergillus oryzae strain.

[0039] In the present invention, as the intake amount (administration amount) of kestose, for example, as the intake amount (administration amount) of kestose, for example, 0.01 to 0.34 g / kg body weight, preferably 0.01 to 0.30 g / kg body weight, more preferably 0.01 to 0.24 g / kg body weight, 0.03 to 0.34 g / kg body weight, 0.05 to 0.34 g / kg body weight, 0.06 to 0.34 g / kg body weight, 0.03 to 0.24 g / kg body weight, 0.05 to 0.24 g / kg body weight, 0.06 to 0.24 g / kg body weight, 0.03 to 0.30 g / kg body weight, 0.05 to 0.30 g / kg body weight, Examples include 0.06 to 0.30 g / kg body weight, 0.03 to 0.26 g / kg body weight, 0.05 to 0.26 g / kg body weight, or 0.06 to 0.26 g / kg body weight. These intake amounts may be consumed in multiple divided doses, rather than limited to once a day.

[0040] One method involves administering kestose orally to humans or animals, either as is or in the form of food or medicine.

[0041] The above kestose composition may be a composition containing a total of 1 g to 30 g, 1 g to 20 g, 1 g to 15 g, 2 g to 15 g, 1 g to 10 g, 2 g to 10 g, 1 g to 7 g, and 2 g to 7 g of kestose as an active ingredient per day, based on a 60 kg adult.

[0042] In addition, the above kestose composition may be a composition containing 400 mg / g or more, 500 mg / g or more, 800 mg / g or more, or 900 mg / g or more in the combined amount of kestose, nistose, and fructopuranosylnistose.

[0043] The kestose usable in the present invention may be in liquid or powder form and may be included in the composition according to the present invention in various amounts. The kestose may be used as a single component or as a mixed composition containing other sugars, and may be, for example, fructooligosaccharide (FOS).

[0044] The above liquid or powder form kestose may be a composition having a content of 50 (w / w) % or more, 60 (w / w) % or more, 70 (w / w) % or more, 80 (w / w) % or more, preferably 85 (w / w) % or more, based on total solid content. Additionally, the crystalline form kestose may be a composition having a content of 98% or more based on total solid content.

[0045] The formulations of pharmaceuticals, quasi-pharmaceuticals, and supplements containing kestose are not specifically limited, and a formulation suitable for the method of administration can be appropriately selected. For example, in the case of oral administration, they may be in solid or liquid formulations such as powder, tablet, sugar-coated preparation, capsule, granule, dry syrup, liquid, syrup, drop, or drink.

[0046] Kestose can be added to the normal manufacturing process of various foods, food additives, and animal feed. Since the sweetness of 1-kestose is 30 and its quality of taste, physical properties, and processability are similar to those of sucrose, it can be used in various foods, beverages, food additives, medicines, or feed by treating it like sugar, such as by replacing part or all of the sugar with 1-kestose during the manufacturing process of various foods.

[0047] Specific examples of the composition according to the present invention include, for instance, beverages, dairy products, granules provided for consumption, pastes, seasonings, retort foods, baby food, fermented foods, preserved foods, processed seafood products, processed meat products, processed grain products, processed foods, food additives, health foods, animal feed, etc. Effects of the invention

[0048] The present invention can improve skin immunity by improving photoaging in humans or animals simply and effectively, with minimal concern for side effects or safety. Brief explanation of the drawing

[0049] Figure 1 is a diagram showing the cytotoxicity evaluation of kestose and the efficacy of protecting cells damaged by UVB according to Example 1. Figure 2 is a diagram showing the ROS removal ability of kestose through the DCFH-DA assay according to Example 2. Figure 3 is a diagram showing the results of analyzing gene and protein expression levels of factors (PI3K, AKT, Nrf2, HO-1) related to Nrf2 / HO-1 signaling activated by ROS according to Example 3 through RT-PCR and Western blot. Figure 4 is a diagram showing the analysis of gene expression levels of factors related to NF-κB signaling that regulate inflammatory responses through RT-PCR as an anti-inflammatory effect of Kestose in UVB-damaged HaCaT cells according to Example 4. Figures 5 and 6 are figures showing the effect of kestose on the expression level of MAPK signaling factors as the anti-aging efficacy of kestose in UVB-damaged HaCaT cells according to Example 5. Figures 5 and 6 are figures showing the effect of kestose on the expression level of MAPK signaling factors as the anti-aging efficacy of kestose in UVB-damaged HaCaT cells according to Example 5. Figures 7 and 8 are figures showing the effect of kestose on the expression level of the activator protein (AP)-1 signaling factor as the anti-aging efficacy of kestose in UVB-damaged HaCaT cells according to Example 5. Figure 9 is a figure showing the ability of kestose to reduce MMP expression as a skin-protective effect of kestose in UVB-damaged HaCaT cells according to Example 6. Figure 10 is a figure showing the results of analyzing the expression level of the TGF-β1 / Smad signaling factor as the anti-aging efficacy of Kestose in UVB-damaged HaCaT cells according to Example 7. Figure 11 is a diagram showing the increase in the expression of collagen synthesis protein of Kestose in UVB-damaged HaCaT cells according to Example 7. Figure 12 is a figure showing the results of evaluating the hyaluronic acid biosynthesis-increasing ability of Kestose by analyzing the expression levels of factors (HAS-1, HAS-2, HAS-3) related to hyaluronic acid synthesis according to Example 8. Specific details for implementing the invention

[0050] The present invention will be explained in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples.

[0052] Example 1: Evaluation of UV-protective ability of Kestos

[0053] To evaluate the cytotoxicity of kestose and confirm its protective efficacy against UVB-damaged cells, HaCaT cells were simultaneously treated with UVB and kestose at different concentrations and cultured for 24 hours, after which the results were measured using an MTT assay.

[0054] Specifically, 5 × 10 HaCaT cells in 96-well plates 4 Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. The cultured cell medium was removed, 0.5 mg / ml of MTT solution was added, and the reaction was carried out for 2 hours. 200 μl / well of dimethyl sulfoxide (DMSO) was added to dissolve formazan at room temperature for 10 minutes, and the absorbance was measured at 590 nm to calculate cell activity (%). MTT was determined by comparing the activity of cells with added samples with that of cells without added samples.

[0055] As a result obtained from the above experiment, the cytotoxicity evaluation of kestose and the efficacy of protecting cells damaged by UVB are shown, specifically the toxicity evaluation according to kestose concentration and UVB irradiation.

[0056] The cytotoxicity of kestose at 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM was checked, and it was confirmed that there was no toxicity at concentrations of 4 mM, 6 mM, and 10 mM.

[0057] In addition, the effects of non-toxic kestose concentrations on HaCaT cells irradiated with UVB were evaluated, and it was confirmed that kestose effectively protected against UVB-induced damage, showing a cell viability approximately 20% higher than the UVB control group.

[0059] Example 2: Evaluation of ROS Removal Ability of Castose

[0060] To confirm the efficacy of Kestos in removing reactive oxygen species (ROS), the ability to remove ROS activity was confirmed through a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay.

[0061] Specifically, 5 × 10 HaCaT cells in 96-well plates 4 Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. The cultured cell medium was removed, and 20 μM of DCFH-DA diluted in DMEM (HaCaT culture medium) was added to each well and cultured for 30 minutes under conditions of 5% CO2 and 37°C. After washing the cells with PBS, the amount of ROS produced was measured using fluorescence absorbance at excitation 488 nm and emission 535 nm.

[0062] Figure 2 shows the results of the analysis of the ROS activity removal ability of Kestose through the DCFH-DA assay obtained from the above experiment. In Figure 2, the control group (-) represents the results of the analysis of the ROS activity removal ability in HaCaT cells that were not subjected to UVB irradiation, and the control group (+) represents the experimental group in which Kestose was not treated in HaCaT cells that were subjected to UVB irradiation in the same way as the above experimental group.

[0063] From the above experimental results, the effect on UVB-activated ROS was confirmed after reacting HaCaT cells with DCFH-DA fluorescence, and it was confirmed that ROS was reduced by about 30% when treated with Kestose compared to the UVB control group.

[0065] Example 3: Evaluation of the antioxidant capacity of kestose by UV light

[0066] (Increase in antioxidant capacity inhibited by ROS)

[0067] To confirm the antioxidant effect of kestose on UVB-damaged HaCaT cells, RT-PCR gene expression levels of factors (PI3K, AKT, Nrf2, HO-1) related to Nrf2 / HO-1 signaling activated by ROS were checked.

[0068] Specifically, 2 × 10⁶ HaCaT cells in 24-well plates 5 Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. After culture, the cells were washed with PBS. RNA was extracted using Trizol, and cDNA was synthesized using a cDNA synthesis kit. The gene expression levels were then determined by qRT-PCR using primers designed for the genes of factors related to Nrf2 / HO-1 signaling (PI3K, AKT, Nrf2, HO-1). The PCR conditions used were denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and elongation at 72°C for 1 minute.

[0069] Figure 3 shows the results of analyzing the gene and protein expression levels of factors (PI3K, AKT, Nrf2, HO-1) related to Nrf2 / HO-1 signaling activated by ROS using RT-PCR and Western blot, based on the results obtained from the above experiment. In Figure 3, the control group (-) represents the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (+) represents the experimental group in which HaCaT cells were subjected to UVB irradiation in the same manner as the above experimental group but were not treated with Kestose.

[0070] According to the experimental results above, Kestose reduced the gene expression of oxidation-related factors PI3K, AKT, Nrf2, and HO-1, confirming that Kestose has an excellent antioxidant effect. Specifically, when HaCaT cells were treated with only UVB to check their expression levels, the expression of AKT and PI3K increased, while the expression of Nrf2 and HO-1 decreased. When Kestose was treated, the expression of AKT, PI3K, Nrf2, and HO-1 showed a pattern similar to that of the control group not treated with Kestose, confirming that it did not affect the expression of oxidation-related factors (p < 0.001). Through these results, the antioxidant effect of Kestose was confirmed.

[0072] Example 4: Confirmation of the anti-inflammatory effect of Kestose on UVB-damaged HaCaT cells

[0073] The gene expression levels of factors (p50, p65, TNF-α, IL-6, COX-2) related to NF-κB signaling that regulates inflammatory responses were confirmed through RT-PCR.

[0074] Specifically, 2 × 10⁶ HaCaT cells in 24-well plates 5 Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. After culture, the cells were washed with PBS. RNA was extracted using Trizol, and cDNA was synthesized using a cDNA synthesis kit. The expression levels of inflammation-related factors (p50, p65, TNF-α, IL-6, COX-2) were determined by qRT-PCR using primers designed for these genes. The PCR conditions used involved denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and elongation at 72°C for 1 minute.

[0075] Figure 4 shows the results of analyzing gene expression levels via RT-PCR for factors related to NF-κB signaling, which regulates inflammatory responses, as an anti-inflammatory effect of Kestose in UVB-damaged HaCaT cells obtained from the above experiment. In Figure 4, the control group (-) represents the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (+) represents the experimental group in which HaCaT cells were subjected to UVB irradiation in the same manner as the above experimental group but were not treated with Kestose.

[0076] According to the experimental results above, to confirm the regulation of the inflammatory response, the gene expression of activated NF-κB related factors p50, p65, TNF-α, IL-6, and COX-2 was reduced by Kestose, and among them, it was confirmed that there was an excellent anti-inflammatory effect at 10 mM.

[0078] Example 5: Evaluation of the anti-aging efficacy of Kestos on UVB-damaged HaCaT cells

[0079] 5-1: Decrease in the expression ability of MAPK signaling factors

[0080] We aimed to determine the expression levels of MAPK signaling genes and proteins that regulate collagen degradation.

[0081] Specifically, 2 × 10⁶ HaCaT cells in 24-well plates 5Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. Proteins were extracted from HaCaT cells using RIPA lysis buffer containing a protease inhibitor. The extracted proteins were pretreated by incubation at 95°C for 5 minutes, followed by protein separation via SDS-PAGE electrophoresis. Subsequently, the proteins were transferred to a PVDF membrane and subjected to a blocking process in 5% (w / v) BSA solution for 1 hour. The primary antibodies for each factor were then diluted 1:1000 with 5% (w / v) BSA solution and incubated at 4°C for one day. The membrane reacted with the primary antibody was reacted with the secondary antibody in a tris-buffered saline solution containing 0.1% (v / v) Tween 20 at room temperature for 1 hour. Bands were detected using a LAS 4000 system, and expression density was calculated using image detection software (Image J software) to determine the expression level.

[0082] As a result obtained from the above experiment, the effect of Kestose on the expression level of MAPK signaling factors as an anti-aging efficacy of Kestose in UVB-damaged HaCaT cells is shown in Figures 5 and 6. Figures 5 and 6 show a comparison of the expression levels of the MAPKs phosphorylation signal pathway following UVB irradiation. In Figure 6, the control group refers to the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (UVB) refers to the experimental group in which Kestose was not treated in HaCaT cells that were subjected to UVB irradiation in the same way as the above experimental group.

[0083] According to the experimental results above, it was confirmed that the expression levels of MAPK protein phosphorylation (p-ERK, p-JNK, p-p38) increased in the control group (UVB), while phosphorylation expression was inhibited when treated with different concentrations of kestose. Calculation of protein expression density also confirmed that the expression levels of p-ERK / ERK, p-JNK / JNK, and p-p38 / p38 significantly decreased as the concentration of kestose increased (p < 0.0001; kestose 10 mM). Furthermore, it was confirmed that ROS induced by UVB activates the MAPK signaling pathway through phosphorylated ERK, JNK, and p38 signals, and that kestose significantly reduces the expression levels of MAPK signaling.

[0085] 5-2: Expression levels of activator protein (AP)-1 signaling factor

[0086] As MAPK signaling activates the activator protein (AP)-1 signaling pathway, the expression levels of the subunits c-Fos and c-Jun increased in the UVB control group, but kestose decreased the expression level of c-Fos, while the expression level of c-Jun was not different from that of the UVB control group.

[0087] Specifically, proteins were extracted from HaCaT cells using RIPA lysis buffer containing a protease inhibitor. The extracted proteins were pretreated by reacting at 95°C for 5 minutes, and then separated by SDS-PAGE electrophoresis. Subsequently, the proteins were transferred to a PVDF membrane and subjected to a blocking process in 5% (w / v) BSA solution for 1 hour. The primary antibodies for each factor were then diluted 1:1000 with 5% (w / v) BSA solution and reacted at 4°C for one day. The membrane reacted with the primary antibodies was then reacted with the secondary antibodies in a tris-buffered saline solution containing 0.1% (v / v) Tween 20 at room temperature for 1 hour. Bands were detected using a LAS 4000 system, and expression levels were calculated by determining expression density using image detection software (Image J software).

[0088] Figures 7 and 8 show the results of analyzing the effect of Kestose on the expression level of the activator protein (AP)-1 signaling factor as an anti-aging efficacy of Kestose in UVB-damaged HaCaT cells, as obtained from the above experiment. In Figure 8, the control group refers to the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (UVB) refers to the experimental group in which Kestose was not treated in HaCaT cells that were subjected to UVB irradiation in the same way as the above experimental group.

[0089] According to the experimental results above, in the control group (UVB), the expression of AP-1 signaling proteins c-Fos, c-Jun, and phosphorylated c-Fos and c-Jun increased, but treatment with kestose attenuated the phosphorylation of c-Fos.

[0090] Calculation of protein expression density also showed that kestose treatment reduced c-Fos / Fos expression (p < 0.0001). However, kestose treatment also showed a decrease in pc-Jun / c-Jun expression. This demonstrated that kestose treatment inhibits the AP-1 inflammatory response activated by MAPK signaling.

[0092] Example 6: Confirmation of the skin-protective effect of Kestos on UVB-damaged HaCaT cells

[0093] The efficacy of inhibiting skin photoaging was evaluated through the ability of Kestose to reduce MMP expression.

[0094] Specifically, 2 × 10⁶ HaCaT cells in 24-well plates 5 Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. After culture, the cells were washed with PBS. RNA was extracted using Trizol, and cDNA was synthesized using a cDNA synthesis kit. The expression levels of inflammation-related factors (p50, p65, TNF-α, IL-6, COX-2) were determined by qRT-PCR using primers designed for these genes. The PCR conditions used involved denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and elongation at 72°C for 1 minute.

[0095] As a result of the above experiment, the ability of Kestose to reduce MMP expression as a skin protective effect of Kestose in UVB-damaged HaCaT cells is shown in Fig. 9. In Fig. 9, the control group (-) represents the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (+) represents the experimental group in which Kestose was not treated in HaCaT cells that were subjected to UVB irradiation in the same way as the above experimental group.

[0096] According to the experimental results above, MMP expression levels were reduced by kestose, which inhibited skin aging caused by photoaging. Specifically, when examining the gene expression levels of MMP-1, MMP-2, MMP-3, and MMP-9, which play the most important roles in degrading type I collagen of the basement membrane and basement membrane cell destruction, it was found that the expression levels increased by UVB were reduced by treatment with kestose at different concentrations. At 10 mM, the gene expression of MMP-1 (p < 0.0001), MMP-2 (p < 0.05), MMP-3 (p < 0.001), and MMP-9 (p < 0.001) was significantly reduced. These results suggest that kestose may be effective against aging caused by ultraviolet radiation.

[0098] Example 7: Evaluation of the collagen synthesis ability of Kestose using UVB-damaged HaCaT cells

[0099] 7-1: Increase in protein expression of TGF-β1 / Smad signaling genes

[0100] The expression levels of the TGFβ1 / Smad signaling gene and protein that induce collagen synthesis were confirmed.

[0101] Specifically, proteins were extracted from HaCaT cells using RIPA lysis buffer containing a protease inhibitor. The extracted proteins were pretreated by reacting at 95°C for 5 minutes, and then separated by SDS-PAGE electrophoresis. Subsequently, the proteins were transferred to a PVDF membrane and subjected to a blocking process in 5% (w / v) BSA solution for 1 hour. The primary antibodies for each factor were then diluted 1:1000 with 5% (w / v) BSA solution and reacted at 4°C for one day. The membrane reacted with the primary antibodies was then reacted with the secondary antibodies in a tris-buffered saline solution containing 0.1% (v / v) Tween 20 at room temperature for 1 hour. Bands were detected using a LAS 4000 system, and expression levels were calculated by determining expression density using image detection software (Image J software).

[0102] Figure 10 shows the results of analyzing the expression level of the TGF-β1 / Smad signaling factor as the anti-aging efficacy of Kestose in UVB-damaged HaCaT cells as a result of the above experiment. In Figure 10, the control group refers to the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (UBV) refers to the experimental group in which HaCaT cells were subjected to UVB irradiation in the same way as the above experimental group but were not treated with Kestose.

[0103] According to the experimental results above, UVB exposure reduced TGF-β1 by approximately 54% compared to normal cells. In addition, in the experimental group treated with UVB exposure and kestose, TGF-β1 production increased by approximately 178% compared to cells irradiated with UVB only, which was significantly higher (Fig. 10).

[0104] As a result of examining Smad signaling induced by TGF-β1 expression, expression levels were increased by kestose, indicating an increase in the levels of transforming growth factor (TGF)-β1, a factor involved in collagen production. Specifically, p-Smad 2 / 3 / Smad 2 / 3 is a factor activated by TGF-β1, and UVB exposure reduced p-Smad 2 / 3 / Smad 2 / 3 by approximately 38% compared to normal cells. Furthermore, in the experimental group treated with UVB exposure and kestose, p-Smad 2 / 3 / Smad 2 / 3 increased by approximately 50% compared to cells irradiated with UVB alone (Fig. 11). These results demonstrated that the TGF-β1 / Smad signaling pathway transporter, which is inhibited by ultraviolet rays, is increased by kestose, confirming its efficacy in improving photoaging.

[0106] 7-2: Increase in the expression of collagen synthesis proteins in Kestose

[0107] As a result of checking the gene expression levels of collagen synthesis proteins COL1A1 and COL2A1 generated by TGFβ1 / Smad signaling, it was confirmed that the expression levels of all proteins increased at kestose levels of 6 mM and 10 mM.

[0108] As a result of the above experiment, the increase in the expression of collagen synthesis protein of Kestose in UVB-damaged HaCaT cells is shown in Fig. 11. In Fig. 11, the control group (-) represents the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (+) represents the experimental group in which HaCaT cells were subjected to UVB irradiation in the same way as the above experimental group but were not treated with Kestose.

[0110] Example 8: Evaluation of the ability of Kestose to increase hyaluronan biosynthesis

[0111] To investigate the effects of Kestose on factors related to hyaluronan biosynthesis, which plays a role in maintaining skin structure and function, the expression levels of Kestose HAS-1, HAS-2, and HAS-3 in UVB-damaged HaCaT cells were analyzed.

[0112] Specifically, 2 × 10⁶ HaCaT cells in 24-well plates 5 Cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Simultaneously with 40 mJ UVB, cells were treated with kestose at concentrations of 4 mM, 6 mM, 10 mM, 14 mM, and 18 mM, respectively, and cultured for 12 hours. After culture, the cells were washed with PBS. RNA was extracted using Trizol, and cDNA was synthesized using a cDNA synthesis kit. The expression levels of inflammation-related factors (p50, p65, TNF-α, IL-6, COX-2) were determined by qRT-PCR using primers designed for these genes. The PCR conditions used involved denaturation at 95°C for 15 seconds, annealing at 62°C for 30 seconds, and elongation at 72°C for 1 minute.

[0113] Figure 12 shows the results of evaluating the potential of Kestose to increase hyaluronan biosynthesis by analyzing the expression levels of factors (HAS-1, HAS-2, HAS-3) related to hyaluronan biosynthesis based on the above experimental results. In Figure 12, the control group (-) represents the analysis of experimental results in HaCaT cells that were not subjected to UVB irradiation, and the control group (+) represents the experimental group in which HaCaT cells were subjected to UVB irradiation in the same way as the above experimental group but were not treated with Kestose.

[0114] According to the experimental results above, it was confirmed that the expression levels of HAS-1, HAS-2, and HAS-3, genes related to hyaluronan biosynthesis that help moisturize the skin, significantly increased at a concentration of 10 mM of Kestose. Therefore, it was confirmed that Kestose promotes hyaluronan biosynthesis by increasing the expression of genes related to hyaluronan biosynthesis, and thereby possesses efficacy in maintaining skin structure and function, moisturizing the skin, regenerating the skin, and increasing skin elasticity.

Claims

Claim 1 A composition for skin moisturization or skin regeneration containing kestose as an active ingredient. Claim 2 A composition for improving skin condition comprising kestose as an active ingredient, wherein the skin condition is one or more conditions selected from the group consisting of skin cell damage caused by ultraviolet rays, generation of reactive oxygen species (ROS), and skin oxidation. Claim 3 A composition according to claim 1 or 2, wherein the composition is based on one or more efficacy selected from the group consisting of the ability of kestose to protect skin cells from ultraviolet rays, ability to remove reactive oxygen species (ROS), skin antioxidant ability, skin anti-inflammatory ability, skin anti-photoaging ability, skin collagen synthesis ability, and ability to increase skin hyaluronan biosynthesis. Claim 4 In paragraph 3, the reactive oxygen species scavenging ability is an increase in the expression level of one or more genes selected from the group consisting of PI3K, AKT, Nrf2, and HO-1 as factors related to Nrf2 / HO-1 signaling activated by ROS; the anti-inflammatory ability is an increase in the expression level of one or more genes selected from the group consisting of p50, p65, TNF-α, IL-6, and COX-2 as factors related to NF-κB signaling that regulates inflammatory responses; the skin collagen synthesis ability is due to the regulation of the expression level of one or more genes selected from the group consisting of p38, ERK, JNK, c-Jun, c-Fos, MMP-1, MMP-2, MMP-3, and MMP-9 genes as factors related to MAPK-ERK / JNK / p38 / AP-1 signaling that degrades and regulates collagen; or the skin collagen synthesis ability is related to TGF-β1 / Smad signaling that induces collagen synthesis A composition in which the increased ability of skin hyaluronan biosynthesis is due to the regulation of the expression levels of one or more genes selected from the group consisting of TGF-β1, Smad2 / 3, COL1A1, and COL2A1 genes as factors, or the increased ability of skin hyaluronan biosynthesis is due to the regulation of the expression levels of one or more genes selected from the group consisting of HAS-1, HAS-2, and HAS-3 genes as factors related to hyaluronic acid synthesis. Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A composition according to claim 1 or 2, wherein the kestose is included in the composition at a total daily dosage of 1 g to 30 g based on a 60 kg adult. Claim 9 A composition according to claim 1 or 2, wherein the kestose is provided as a syrup containing kestose.

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