Cosmetic composition containing enzymatic hydrolysate of Jeju dangyuja for strengthening skin barrier or relieving skin itching

Abstract

The present invention relates to a cosmetic composition for alleviating skin itching and strengthening the skin barrier, comprising an enzyme extract prepared by treating Jeju Citrus grandis Osbeck with a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides'. Compared to existing hot water extracts or ethanol extracts, the Citrus grandis enzyme extract of the present invention effectively promotes the expression of skin barrier-related factors such as filaggrin, aquaporin-3, and CD44, and has the effect of inhibiting the expression of itching-related factors such as TRPV1 and PAR-2.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition for strengthening the skin barrier or alleviating skin itching, and specifically, to a cosmetic composition for strengthening the skin barrier or alleviating skin itching comprising an enzyme hydrolysate of Jeju citrus fruit as an active ingredient. Background Technology

[0003] Skin itching (pruritus) is defined as an unpleasant sensation that triggers a desire to scratch or rub the skin. However, despite being a common symptom in both skin and systemic diseases, its characteristics are not fully understood. Skin itching is caused by various internal and external factors and causes significant discomfort, particularly for patients with atopic dermatitis. Nevertheless, existing treatments have limitations in that long-term use is difficult due to concerns regarding side effects.

[0004] The skin barrier plays a vital role in blocking the intrusion of harmful external substances and preventing internal moisture loss. Skin conditions such as atopic dermatitis and xerosis are closely linked to damage to the skin barrier. A damaged skin barrier accelerates moisture loss, causing skin dryness and allowing external allergens or irritants to easily penetrate. The penetration of these irritants triggers an immune response, leading to inflammation and ultimately forming a vicious cycle that causes chronic itching. Therefore, to fundamentally alleviate itching, it is crucial to go beyond merely suppressing symptoms and restore the damaged skin barrier function.

[0005] The cosmetics industry is developing various cosmetic formulations and related ingredients to strengthen the skin barrier and alleviate itching. However, existing ingredients have several limitations.

[0006] First, regarding chemically synthesized raw materials, despite their excellent efficacy, they frequently cause side effects such as skin allergies or contact dermatitis in some users, leading to ongoing concerns regarding safety.

[0007] Second, while animal-derived ingredients were widely used in the past for components such as collagen and elastin, their use is rapidly declining due to the recent rise in consumer awareness of animal ethics, a decrease in preference, and safety issues such as the Bovine Spongiform Encephalopathy (BSE) outbreak.

[0008] Third, while microbial-derived raw materials can yield useful components through the fermentation process, there are limitations to their application to sensitive skin due to the potential presence of skin-irritating and toxic substances, such as lipopolysaccharides (LPS) or mycotoxins, derived from bacteria or fungi used in the manufacturing process.

[0009] Due to these issues, plant-based ingredients that have few side effects, are safe, and can be supplied sustainably are currently the most sought-after in the cosmetics industry. Prior art literature

[0011] Korean Registered Patent No. 10-2015-0139688 (December 14, 2015) describes a whitening cosmetic composition using a citronella extract, focusing on the efficacy of inhibiting melanin production. The problem to be solved

[0012] In the present invention, as a plant-based raw material, Jeju citron (scientific name: Citrus grandis We intend to develop and provide cosmetics effective for strengthening the skin barrier and alleviating itching using Osbeck. means of solving the problem

[0014] The present invention relates to a citron ( Citrus grandis OsbeckA cosmetic composition is provided containing a sugar-based enzyme hydrolysate obtained by treating the whole fruit with a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides'.

[0015] In the cosmetic composition of the present invention, the 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides' may preferably be Viscozyme L.

[0016] In the cosmetic composition of the present invention, the above-mentioned sugar citrus enzyme hydrolysate may preferably be the product obtained by treating with the above-mentioned 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides' and then adding ethanol to inactivate the enzyme.

[0017] In the cosmetic composition of the present invention, the cosmetic composition may preferably be for strengthening the skin barrier or for improving skin itching.

[0018] In the cosmetic composition of the present invention, the enzyme hydrolysate of the sugar citrus fruit may preferably contain naringenin, which is a non-glycosidic flavonoid.

[0019] In the cosmetic composition of the present invention, the cosmetic composition may be any one selected from basic cosmetic formulations or makeup formulations, such as, for example, lotion, gel, cream, essence, makeup base, foundation. Effects of the invention

[0021] In the cosmetic composition of the present invention, flavonoids are converted into low molecular weight non-glycosidic forms through enzymatic treatment. As a result, the skin permeability is significantly increased, and consequently, while causing no skin irritation, the expression of skin barrier factors such as filaggrin, aquaporin-3, and CD44 is increased, and the expression of itching-related factors such as TRPV1 and PAR-2 ​​is suppressed, thereby maximizing the efficacy of strengthening (or improving) the skin barrier and improving (or alleviating) skin itching compared to conventional hot water or ethanol extracts. Brief explanation of the drawing

[0023] Figure 1 shows the difference between the citronella enzyme extract (Example 1) and the citronella hot water extract (Comparative Example 1) and ethanol extract (Comparative Example 2). ex-vivo This is an immunofluorescence staining image and a graph of quantitative results showing a significant increase in the expression of filaggrin, CD44, and aquaporin-3 in skin tissue. Figure 2 shows the difference between the citronella enzyme extract (Example 1) and the citronella hot water extract (Comparative Example 1) and ethanol extract (Comparative Example 2). ex-vivo This is an immunofluorescence staining image and a graph of quantitative results showing that TRPV-1 and PAR-2 ​​expression was significantly inhibited in skin tissue. Figure 3 is a graph of a photograph and quantitative results confirming that naringenin, a flavonoid non-glycoside of the citronella enzyme extract (Example 1), has increased skin permeability in artificial skin compared to naringin, a flavonoid glycoside of the citronella extract. Figure 4 shows the results of evaluating skin irritation in a human skin model (Reconstructed Human Epidermis) to confirm the skin safety of the citron enzyme extract. Specific details for implementing the invention

[0024] Jeju Dangyuja (scientific name: Citrus grandis Osbeck Yuja is a native Jeju citrus fruit that has been used for medicinal and culinary purposes in folk medicine since ancient times. It has about twice the free sugar content compared to yuja from other regions and contains more than four times the amount of Vitamin C found in tangerines or lemons. Additionally, it has been revealed that the peel of Dangyuja contains large amounts of flavonoids such as hesperidin, naringin, and neohesperidin. However, existing research and patents related to Jeju Dangyuja and similar citrus fruits have mainly focused on antioxidant, whitening, and anti-aging effects.

[0025] These existing studies aimed to achieve general skin improvement effects, such as antioxidant, whitening, and anti-aging effects, by utilizing the natural components of citronella extract, and mainly used hot water extraction or ethanol extraction methods. However, these extraction methods had the problem of low extraction efficiency of active ingredients and limited skin absorption rates due to the large number of high-molecular-weight components.

[0026] The present invention focuses on resolving specific skin problems, namely the improvement of skin itching and the strengthening of the skin barrier. There have been no confirmed reports of maximizing specific active ingredients (e.g., water-soluble dietary fiber, specific phenolic acids, etc.) of *Tangyuja* or extracting them in new forms by applying Viscozyme L enzyme treatment technology. By introducing an enzymatic hydrolysis process utilizing a "carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides," such as Viscozyme L, the present invention achieves an optimized composition of ingredients and efficacy that is difficult to obtain through general extraction methods. Experiments of the present invention have confirmed that this can create a much more effective synergy for strengthening the skin barrier and improving itching compared to existing simple extracts. Therefore, the significance and differentiation of the present invention lie in solving the difficult problem of improving skin itching and strengthening the skin barrier by applying a "carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides," specifically Viscozyme L enzyme treatment technology.

[0027] Furthermore, conventional citrus fruit extracts were not suitable for use in cosmetics because their high-viscosity water-soluble polysaccharide components exhibited significant viscosity changes due to salts and pH, and they also tended to gel when in contact with alcohols. In addition, due to the hydrophilic polymeric structure of the polysaccharides, transdermal penetration was difficult, presenting a disadvantage in that various physiological activities were hard to manifest through transdermal application, which is the typical application method for cosmetics. However, in this invention, by treating the beneficial flavonoid component with a "carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides" to convert it into a non-glycosidic form, a Jeju citrus fruit enzyme extract with enhanced skin absorption was developed. Consequently, it became possible to effectively improve or alleviate skin itching and strengthen or improve damaged skin barrier function.

[0028] Based on this, the present invention provides a cosmetic composition for strengthening the skin barrier and improving skin itching, characterized by containing as an active ingredient an enzyme extract of *Tangyuja* obtained by treating *Tangyuja* with a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides' and then inhibiting the enzyme activity to obtain an extract.

[0029] In the cosmetic composition of the present invention, the extract is characterized by being extracted from *Tangyuja* using a Viscozyme L enzyme having carbohydrate-degrading enzyme activity. For example, by effectively degrading the complex carbohydrate structure of the *Tangyuja* cell wall through treatment with the Viscozyme L enzyme, the release of active ingredients is promoted and reduced in molecular weight. Through this, the present invention was able to overcome the disadvantage that notable physiological activity does not appear upon transdermal application due to the characteristics of hydrophilic polymers, and furthermore, it was confirmed that the content of non-glycosidic flavonoids in the enzyme-degraded *Tangyuja* extract was increased.

[0030] In addition, in this invention, an increase in the content of active ingredients was confirmed in a cultured citron enzyme extract using a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides,' and it was confirmed that this citron enzyme extract effectively alleviates skin itching and safely improves damaged skin barrier function as a cosmetic composition without causing skin irritation.

[0031] Based on this, the present invention ( Citrus grandis Osbeck A cosmetic composition is provided containing a sugar-based enzyme hydrolysate obtained by treating the whole fruit with a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides'.

[0032] The enzyme hydrolysate of the citronella of the present invention can be prepared, for example, through the following processes ((a) to (d)).

[0033] <Step (a): Step of mixing sugar citron and distilled water>

[0034] In this step (a), it is preferable to use citrons from Jeju Island, for example. Additionally, the part of the citron used should preferably be the whole fruit. In this step (a), it is preferable to cut the citron before use. It is preferable to add an equal amount of distilled water to the cut whole citron and proceed to the following step (b).

[0036] <Step (b): A step of adding a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides' to the mixture of the above-mentioned whole citron and distilled water to induce a reaction at 43–45°C for 12–36 hours>

[0037] In step (b), hydrolysis is performed by adding a 'carbohydrate hydrolyzing enzyme that breaks down plant cell wall polysaccharides,' and preferably, Viscozyme L is used. Viscozyme L is a commercial enzyme sold by Novozymes A / S. At this time, it is preferable to add Viscozyme L to the mixture of the whole fruit of the citrus fruit and distilled water in an amount of 2 to 10% (v / v) relative to the volume of the mixture. By effectively breaking down the complex carbohydrate structure of the citrus fruit cell wall through Viscozyme L enzyme treatment, the release of active ingredients is promoted and they are broken down into low molecular weights. This overcomes the disadvantage that notable physiological activity is not observed through transdermal application due to the characteristics of hydrophilic polymers. Furthermore, the content of non-glycosidic flavonoids in the enzyme-hydrolyzed citrus fruit extract could be further increased.

[0039] <Step (c): Step of adding ethanol to inhibit and inactivate enzyme activity>

[0040] In this step (c), ethanol is added to inactivate the enzyme; preferably, the amount of ethanol added is equal to the amount of the 'mixture of the sugar-coated fruit and distilled water'. The ethanol inhibits enzyme activity and removes the separated sugars.

[0042] <Step (d): A step of obtaining an extract by filtration, concentration, and drying>

[0043] Step (d) is a step of obtaining an extract in the form of a filter, concentration, and drying after enzyme inactivation. After the enzyme inactivation, preferably, the supernatant is obtained by centrifugation, followed by primary filtration using a mesh, and secondary microfiltration using a 0.2 μm filter. Afterward, the extract is concentrated using a concentrator and then freeze-dried to obtain an extract in the form of a powder (hereinafter also referred to as 'Tangyuja enzyme hydrolysate'). The Tangyuja enzyme hydrolysate obtained in this way preferably contains naringenin, a non-glycosidic flavonoid.

[0044] The hot water extract of the citronella obtained in the present invention can be dissolved in one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol, if necessary, preferably, and more preferably 1-30% by weight in one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol.

[0046] Meanwhile, the cosmetic composition of the present invention may preferably be for strengthening the skin barrier or for improving skin itching. The skin barrier strengthening of the present invention may preferably be achieved by increasing the expression of skin barrier-related factors such as filaggrin, aquaporin-2, and / or CD44. In addition, the skin itching relief of the present invention may preferably be achieved by suppressing the expression of itching factors such as TRPV1 and / or PAR-2.

[0047] Meanwhile, the cosmetic composition of the present invention is not necessarily limited to a specific formulation, but may be any one selected from basic cosmetic formulations or makeup formulations, such as a lotion, gel, cream, essence, makeup base, or foundation.

[0049] The contents of the present invention will be explained in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0051] [Example 1: Preparation of Jeju Dangyuja Enzyme Hydrolysate (Viscozyme L Treatment)]

[0052] (1) 1 kg of whole Jeju Island citron was washed and then cut into pieces approximately 0.5 cm to 1 cm in size.

[0053] (2) An equal amount (1L) of distilled water was added to the cut citron fruit.

[0054] (3) Viscozyme L (Novozymes A / S product), a carbohydrate-degrading enzyme complex, was added to the above solution at a ratio of 5% (v / v) relative to the 'mixture of whole citron and distilled water'.

[0055] (4) Enzyme hydrolysis was carried out for 24 hours in a shaking incubator at 45°C.

[0056] (5) After the reaction was finished, an equal amount of EtOH was added to inactivate the enzyme, and the sugar was removed through precipitation.

[0057] (6) The supernatant was recovered by centrifugation. Afterwards, the recovered supernatant was filtered first (mesh) and then filtered second (0.2 μm filter).

[0058] (7) After concentrating the filtered extract using a rotary concentrator, freeze-dry the resulting product to obtain a final powder-form Jeju citron enzyme hydrolysate (hereinafter referred to as "the extract of the present invention").

[0060] [Comparative Example 1: Preparation of Jeju Tangyuja Hot Water Extract]

[0061] (1) 1 kg of whole Jeju Island citron fruit was prepared by cutting it in the same way as in Example 1.

[0062] (2) 10 times (10L) of distilled water was added to the cut citron.

[0063] (3) Hot water extraction was performed at 90°C for 4 hours without adding enzymes.

[0064] (4) The extract was filtered, concentrated, and freeze-dried in the same way as in Example 1 to obtain a hot water extract (hereinafter referred to as "Comparative Example 1 extract").

[0066] [Comparative Example 2: Preparation of Jeju Tangyuja Ethanol Extract]

[0067] (1) 1 kg of whole Jeju Island citron fruit was prepared by cutting it in the same way as in Example 1.

[0068] (2) 10 times (10L) of 70% (v / v) ethanol was added to the cut citron.

[0069] (3) Extraction was carried out by immersion at room temperature for 24 hours without adding enzymes.

[0070] (4) The extract was filtered, concentrated, and freeze-dried in the same way as in Example 1 to obtain an ethanol extract (hereinafter referred to as "Comparative Example 2 extract").

[0072] [Experimental Example 1: Analysis of Flavonoid Content in Extracts]

[0073] This experiment was conducted to confirm the effect of enhancing the content of non-glycoside flavonoids (hesperidin, naringenin) of the enzyme-treated extract of *Tangyuja* (Example 1). The content of non-glycoside flavonoids was analyzed using high-performance liquid chromatography (HPLC).

[0074] 1) Analysis Equipment and Conditions

[0075] The measurement conditions for liquid chromatography are as follows.

[0076] Detector: PDA detector (288 nm)

[0077] Column: Shim-pack GIS C18 (4.6 mm × 250 mm, 5 μm)

[0078] Mobile phase: Gradient conditions using acetonitrile and 0.1% trifluoroacetic acid aqueous solution

[0079] Mobile phase flow rate: 1.0 mL / min

[0080] Injection volume: 10 μL

[0081] Oven temperature: 40 ℃

[0083] 2) Reagents and Solutions

[0084] The main reagents and solutions used in the analysis are as follows:

[0085] Standards: Naringenin (for HPLC analysis), Hesperetin (for HPLC analysis)

[0086] Solvents: Acetonitrile, distilled water, methanol, trifluoroacetic acid (p. 4)

[0087] Analysis Sample: Citron Enzyme Extract

[0089] 3) Preparation of experimental solution

[0090] (1) Preparation of standard solution

[0091] ① Preparation of individual standard stock solutions

[0092] 0.1 g of Naringenin standard was accurately weighed, dissolved in methanol, and diluted to 100 mL to prepare a standard stock solution.

[0093] A specific amount of each Hesperetin standard was also accurately weighed and dissolved in methanol to prepare a highly concentrated stock solution (e.g., 1 mg / mL).

[0094] ② Preparation of mixed standard solution for calibration curve

[0095] Mixed standard solutions of various concentrations, ranging from 10 to 200 μg / mL, were prepared by appropriately diluting two standard stock solutions with 80% methanol (p. 4) or a methanol / water / acetic acid mixture. Calibration curves for each component were constructed using these mixed standard solutions.

[0097] (2) Preparation of test solution

[0098] 20 mg of the analysis sample (Tangyuja enzyme extract) was accurately weighed and 1 mL of 80% methanol was added. After dissolving for 30 minutes using an ultrasonic extractor, the solution was filtered through a PTFE filter (0.45 μm) and used as the test solution.

[0100] 4) Test Procedure and Quantification

[0101] Under the above conditions, the test solution and standard solution were injected into the liquid chromatograph, and the content of Naringenin and Hesperetin in the sample was calculated using the obtained peak area and the calibration curve of each standard substance.

[0102] Flavonoid content based on extract powder Naringin (mg / kg) Naringenin (mg / kg) Neohesperidin (mg / kg) Hesperetin (mg / kg) Example 1 40.12 51620.55 50.26 47300.9 Comparative Example 1 30220.45 49.59 48400.41 31.41 Comparative Example 2 40110.23 58.45 56790.12 38.79

[0104] Flavonoids in the enzyme extract of *Tangyuja* were converted from glycoside form to aglycone form, and through this, changes in the efficacy and permeability of the extract could be confirmed.

[0106] [Experimental Example 2: of the enzyme extract of the citrus fruit of the present invention ex-vivo evaluation]

[0107] In this experimental example, the citron enzyme extract (Example 1) prepared in Example 1 above ex-vivo An evaluation was performed. To confirm the skin barrier strengthening and itching relief effects, the effects on the expression of filaggrin, aquaporin-3, CD44, TRPV-1, and PAR-2 ​​were examined using human-derived skin tissue.

[0108] 1) Sample processing and tissue fixation

[0109] Research human skin was cultured in an Insert / Transwell system at 37°C in a 5% CO2 incubator for 24 hours. After UV irradiation, the above-mentioned citrus fruit enzyme extract (Example 1), citrus fruit hot water extract (Comparative Example 1), and citrus fruit ethanol extract (Comparative Example 2) were each diluted to a concentration of 1% and treated, and cultured in a 37°C incubator at 5% CO2 for 5 days. The tissues treated with the samples were fixed in a 10% neutral formalin solution for 24 hours. Subsequently, they were embedded in an OCT Compound, sliced ​​to a thickness of 10 µm, attached to gelatin-coated slides, dried and stored, and used for staining.

[0110] 2) Immunofluorescence staining

[0111] Tissue sections prepared on slides were hydrated in distilled water. They were placed in 0.01 M citrate solution at pH 6.0, heated in a microwave for 15 minutes, and then incubated at room temperature for 20 minutes. The sections were blocked with anti-goat serum and incubated overnight at 4°C with primary antibodies filaggrin, aquaporin-3, CD44, TRPV-1, and PAR-2. The sections were washed with PBS solution, mounted in mounting medium with DAPI, and observed under a fluorescence microscope.

[0112] The results were objectively analyzed by quantifying the positive staining area using the Image J program on images taken with a fluorescence microscope at 200x magnification (Equation 1).

[0113] [Mathematical Formula 1]

[0114]

[0116] 3) Confirmation of the effect of promoting the expression of filaggrin, aquaporin-3, and CD44, factors involved in the skin barrier.

[0117] As a result of measuring the effect of increasing the expression of filaggrin, aquaporin-3, and CD44, which are factors involved in the skin barrier, the enzyme extract of *Tangyuja* (Example 1) showed a higher effect of increasing filaggrin, aquaporin-3, and CD44 expression compared to the hot water extract of *Tangyuja* (Comparative Example 1) and the ethanol extract of *Tangyuja* (Comparative Example 2), as shown in Figure 1. The enzyme extract of *Tangyuja* (Example 1) showed high expression increase rates of 41.79% for filaggrin, 70.57% for aquaporin-3, and 54.89% for CD44 compared to the UV irradiation group (Figure 1).

[0118] 4) Confirmation of the inhibitory effect on the expression of TRPV-1 and PAR-2, factors involved in itching As a result of measuring the inhibitory effect on the expression of TRPV-1 and PAR-2, factors involved in itching, the enzyme extract of *Trichosanthes kirilowii* (Example 1) showed a higher TRPV-1 and PAR-2 ​​expression inhibition rate compared to the hot water extract of *Trichosanthes kirilowii* (Comparative Example 1) and the ethanol extract of *Trichosanthes kirilowii* (Comparative Example 2), as shown in Figure 2. The enzyme extract of *Trichosanthes kirilowii* (Example 1) showed a high expression inhibition rate of 92.32% for TRPV-1 and 60.22% for PAR-2 ​​compared to the UV irradiation group (Figure 2).

[0120] [Experimental Example 3: Evaluation of skin permeability of Naringenin, an active ingredient of the citronella enzyme extract of the present invention}

[0121] 1) Conjugation of narigenin and fluorescent substance (Example 1 active ingredient conjugate)

[0122] 10 mg of naringenin (active substance of Example 1) was weighed into a 1.5 ml microtube, and then 0.5 mL of dimethylformamide anhydrous (DMF) was added to completely dissolve the naringenin. After that, Flamma ® 5 mg of Fluors 552 Dichlorotriazine (BioActs) was added. 50 μl of pyridine anhydrous, which acts as a reaction catalyst and base, was added. The reaction vessel was sealed, and the reaction was carried out by stirring at room temperature for 4–12 hours while blocking light. The mixture produced after the labeling reaction was PD MidiTrap ™ Naringenin-Flamma® Fluors 552 conjugates were prepared by removing unreacted free dyes using a G-10 column.

[0123] 2) Naringin and fluorescent substance combination (Effective substance combinations of Comparative Examples 1 and 2)

[0124] 5 mg of naringin (active substance of Comparative Examples 1 and 2) was weighed into a 1.5 ml microtube, and then 0.4 mL of dimethylformamide anhydrous (DMF) was added to completely dissolve the naringin. After that, Flamma ®5 mg of Fluors 552 Dichlorotriazine (BioActs) was added. To maintain the pH of the reaction solution above 9.0, a high concentration of bicarbonate buffer was added, the reaction vessel was sealed, and the reaction was carried out by stirring at room temperature for 4–12 hours under light protection. The mixture produced after the labeling reaction, PD MidiTrap ™ Naringin-Flamma® Fluors 552 conjugates were prepared by removing unreacted free dyes using a G-10 column.

[0125] 3) Tissue sample processing

[0126] The artificial skin used for the skin penetration test utilized a custom-made Coseed Skin containing a dermal layer. 2.5 ml of artificial skin culture medium was added, and the skin was equilibrated for 16 hours in a 37°C, 5% CO2 incubator before being used for the skin penetration experiment. Flamma ® 50 μL of Fluors 552 was applied to artificial skin, including one control sample labeled with Fluors 552 (naringenin, naringin). After application, the permeation reaction was carried out for 24 hours in a 32°C, 5% CO2 incubator. After 24 hours, the surface of the artificial skin was washed three times with PBS to ensure no residual sample remained, and the skin tissue was separated and placed in an embedding cassette. The embedding cassette containing the artificial skin tissue was fixed in a 10% formalin solution for 24 hours, followed by sucrose infiltration, after which frozen sections were prepared. The degree of permeation of the two samples (naringenin, naringin) – Fluors 552, including one control sample, on the artificial skin was observed and photographed using a THUNDER Imager 3D Tissue microscope (Leica Microsystems, Germany).

[0127] Permeability analysis experiments were repeated three times for each experiment, and the statistical significance of the data according to the experimental group was verified using Student's t-test. p If the value was 0.05 or less, it was determined that there was a statistically significant difference.

[0128] As shown in Figure 3, which is a photograph taken with a microscope, naringenin (active substance of Experimental Example 1) showed a stronger fluorescence intensity than naringin (active substances of Comparative Examples 1 and 2). Based on the above results, it was confirmed that a larger amount of naringenin, which has a smaller molecular weight, penetrated the epidermis and was delivered to the dermis.

[0130] [Experimental Example 4: Evaluation of Skin Irritation from Tangyuja Extract]

[0131] For the enzyme extract of *Tangyuja* (Example 1), hot water extract of *Tangyuja* (Comparative Example 1), and ethanol extract of *Tangyuja* (Comparative Example 2), a human skin model (Keraskin) established in accordance with the OECD TG439 guidelines ™ A skin irritation evaluation test was conducted using ).

[0132] 50 μL each of the enzyme extract of *Tangyuja* (Example 1), the hot water extract of *Tangyuja* (Comparative Example 1), and the ethanol extract of *Tangyuja* (Comparative Example 2) were applied to the surface of an artificial skin model. A 5% SDS (Sodium, Dodecyl Sulfate) solution was used as a positive control, and phosphate-buffered saline was used as a negative control. After treatment in a 37°C, 5% CO2 incubator for 30 minutes, residual samples were removed by washing thoroughly with PBS. Subsequently, the samples were cultured in fresh medium for an additional 42 hours. After the culture was completed, each artificial skin model was treated with the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide) and reacted for a certain period of time. MTT is reduced by mitochondrial dehydrogenase in living cells to produce insoluble purple formazan crystals. The generated formazan was extracted, and its absorbance was measured at a wavelength of 570 nm using a microplate reader. The cell viability of each sample treatment group was calculated based on the absorbance value of the negative control as the 100% viability (Equation 2).

[0133] [Mathematical Formula 2]

[0134]

[0135] As a result of the experiment, the enzyme extract of *Tangyuja* (Example 1), the hot water extract of *Tangyuja* (Comparative Example 1), and the ethanol extract of *Tangyuja* were all calculated to be greater than the judgment criterion of 50%, and were therefore judged to be non-irritating substances. The enzyme extract of *Tangyuja* showed a high survival rate of 90.84%. On the other hand, the hot water extract of *Tangyuja* (Comparative Example 1) and the ethanol extract of *Tangyuja* (Comparative Example 2) showed relatively low cell survival rates of 75.12% and 68.55%, respectively. Therefore, the enzyme extract of *Tangyuja* of the present invention was evaluated to have excellent safety when applied to the skin and to be suitable for use as an active ingredient in cosmetic compositions.

Claims

Claim 1 Dangyuja ( Citrus grandis Osbeck A cosmetic composition for strengthening the skin barrier, comprising a sugar citrus enzyme hydrolysate obtained by treating the whole fruit with Viscozyme L, a carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides, wherein the sugar citrus enzyme hydrolysate is characterized by having an enhanced content of naringenin, a non-glycosidic flavonoid. Claim 2 Dangyuja ( Citrus grandis Osbeck A cosmetic composition for improving skin itching, comprising a sugar citrus enzyme hydrolysate obtained by treating the whole fruit with Viscozyme L, a carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides, wherein the sugar citrus enzyme hydrolysate is characterized by having an enhanced content of naringenin, a non-glycosidic flavonoid. Claim 3 A cosmetic composition according to claim 1 or 2, characterized in that the enzyme hydrolysate of the sugar citrus fruit is obtained by treating it with Viscozyme L, which is a 'carbohydrate hydrolyzing enzyme that decomposes plant cell wall polysaccharides,' and then adding ethanol to inactivate the enzyme. Claim 4 delete Claim 5 delete Claim 6 A cosmetic composition according to claim 1 or 2, characterized in that the cosmetic composition is selected from any one of a basic cosmetic formulation or a makeup formulation including a lotion, gel, cream, essence, makeup base, and foundation.

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