External preparation for moisturizing skin

Abstract

To provide a skin care preparation for external use for moisture retention using an immature mandarin orange as a functional material for cosmetics.SOLUTION: The external preparation for moisturizing the skin contains hesperidin which is an extract of flesh and pericarp of unripe mandarin orange as an active ingredient. The unripe mandarin orange is preferably CitrusunshiuMarcow. (Qingdao species and Otsu species) or Shonan Gold.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a moisturizing skin preparation for external use that enhances the skin's moisture-retaining ability and thereby enhances the skin's moisturizing effect. [Background technology]

[0002] The skin acts as a physical barrier between the external environment, which includes temperature and humidity changes, ultraviolet rays, and dryness, and the internal environment, protecting and maintaining the latter. In particular, the skin regulates the evaporation of moisture from the body. The skin is divided into three layers, from the outside in: the epidermis, the dermis, and the subcutaneous tissue. The epidermis contains keratinocytes, which produce keratin fibers; melanocytes, which produce melanin; and Langerhans cells, which process foreign substances, and plays a role in preventing moisture evaporation from the body. The dermis is composed of collagen fibers and elastic fibers, as well as fibroblasts, which produce these fibers, and a matrix containing hyaluronic acid, and plays a role in moisturizing and receiving sensations such as touch. The subcutaneous tissue is mainly composed of fat cells, and acts as a shock absorber for external stimuli and regulates body temperature.

[0003] Hyaluronic acid is known to exist in the intercellular spaces of the epidermis and in the connective tissue of the dermis. Hyaluronic acid is a linear chain of N-acetylglucosamine and D-glucuronic acid (GlcNAcβ1-4GlcAβ1-3). It has an extremely high molecular weight, estimated to be between 800,000 and 1.2 million, with the potential to reach a maximum of 2 million. Hyaluronic acid can retain a large amount of water, but this decreases with age, causing a decrease in the skin's ability to retain moisture and thought to be a cause of dry skin.

[0004] JP 2024-524621 A discloses a skin moisturizing cosmetic composition containing 0.01% to 10% phosphatidylcholine, a mixture of propylene glycol and glycerol, a polymer moisturizer consisting essentially of sodium hyaluronate, Tremella fuciformis polysaccharide, and polyethylene glycol (PEG)-400, and purified water. Japanese Patent Publication No. 5016212 discloses a skin moisturizing cosmetic composition containing, as active ingredients, 0.001 to 10 wt. % of subtilisin protease, 0.001 to 20 wt. % of a specific jujube extract, and 0.000001 to 10 wt. % of hyaluronic acid, based on the total weight of the composition, and further containing 0.001 to 20 wt. % of a specific Astragalus chinensis extract or a specific Scutellaria baicalensis extract. Japanese Patent No. 4024189 discloses a topical skin preparation that is a moisturizing cosmetic, containing polyethylene glycol having an average molecular weight of 1000 or more, a polyoxyethylene-added nonionic surfactant having an average number of ethylene oxide moles of 30 or more, dimethicone copolyol, phospholipid, hyaluronic acid, and at least one selected from hyaluronic acid salts, 1) one or more polymers having a basic amino acid residue selected from polyarginine and polymethacryloyl lysine in their structure, and 2) xanthan gum. These techniques provide moisturizing effects by externally replenishing hyaluronic acid to the skin, but there are problems in terms of penetration when increasing hyaluronic acid in the dermis.

[0005] JP 2024-502775 A discloses a cosmetic composition for moisturizing skin, which contains cedrol or ferulic acid and at least one selected from the group consisting of beta-sitosterol and linoleic acid. The document teaches that the cosmetic composition for moisturizing skin has the effects of scavenging free radicals, reducing melanin, promoting collagen synthesis, and promoting hyaluronic acid synthesis. While this technology is excellent in promoting hyaluronic acid synthesis in keratinocytes of the human epidermis, it is unclear whether it can also promote hyaluronic acid synthesis in the human dermis.

[0006] Unshu mandarins are seedless, sweet citrus fruits widely consumed in East Asia, including Japan. However, their production has declined. To restore citrus consumption, various varieties of citrus fruits have been cultivated through selective breeding. The Aoshima mandarin (Unshu mandarin) of Mikkabi Town is Japan's most famous mandarin. Other varieties are cultivated in other regions. Shonan Gold (C. flaviculpus) was developed in 1988 from seedlings obtained by crossing Imamura Satsuma (C. unshiu (Swingle) Marcow) with Ogonkan (C. flaviculpus Hort. ex Tanaka) and was registered as a citrus variety (Manago et al. 2004). Shonan Gold contains the same amount of sugar as Unshu mandarins, but has approximately twice the acidity and a unique aroma (Sato et al. 2020). Approximately 113 tons of Shonan Gold were produced in 2016, and production is increasing every year. As production increases, the number of unripe fruits that are discarded also increases. Therefore, it is important to make effective use of unripe fruits.

[0007] Consumption of Aoshima mandarins has been reported to benefit bone health and lower blood pressure in hypertensive patients (Yamaguchi et al. 2012). Representative studies on the health benefits of agricultural products include the main components, β-cryptoxanthin and GABA (gamma-aminobutyric acid), which are known to promote bone health and lower blood pressure. Fruit thinning is necessary to produce mandarins rich in these components. Therefore, developing applications of unripe mandarins through picking is important. Dietary intake of unripe Aoshima mandarins has been shown to suppress basement membrane degradation and mitigate UVB-induced photoaging in hairless mice (Tamaru et al. 2020). The main components of the unripe mandarin peel are narirutin and hesperidin, and their consumption has been reported to improve blood flow and exert anti-inflammatory effects due to their strong antioxidant properties (Matsuda et al. 1991, Kubo et al. 2004). Oral ingestion of unripe Satsuma mandarin extract has been reported to improve skin moisture in hairless mice (Choi et al. 2017) and in women aged 40 to 60 years (Ham et al. 2022). Kim et al. demonstrated that Bacillus subtilis fermentation products of aqueous and ethanolic extracts of dried Satsuma mandarin peel enhanced type I procollagen production by inhibiting collagenase activity in dermal fibroblasts (Kim et al. 2018). However, Kim et al. demonstrated that aqueous and ethanolic extracts of dried Satsuma mandarin peel themselves did not have this effect. Thus, Kim et al.'s results suggest that naringenin, rutin, hesperetin, tangeretin, and nobiletin, specifically contained in the Bacillus subtilis fermentation product, are the active ingredients responsible for inhibiting collagenase activity in dermal fibroblasts.

[0008] Therefore, the purpose of this study was to clarify the characteristics of unripe mandarin oranges from different growing regions and varieties in Japan.To confirm their potential use as functional ingredients in cosmetics, we examined the hyaluronic acid production ability of human skin cells cultured with unripe fruit extracts. [Prior art documents] [Patent documents]

[0009] [License 1] Special Announcement No. 2024-524621 [License 2] Special Announcement No. 2024-502775 [License 3] Patent No. 5016212 [License 4] Patent No. 4024189 [Non-licensed literature]

[0010] [Non-licensed Document 1] Abatangelo G, Vindigni V, Avruscio G, Pandis L, Brun P. Hyaluronic acid: redefining its role. Cells. 2020;9(7):1743. doi: 10.3390 / cells9071743. [Non-licensed Document 2] Bae JT, Ko HJ, Kim GB, Pyo HB, Lee GS. Protective effects of fermented citrus unshiu peel extract against ultraviolet-a-induced photoageing in human dermal fibrobolasts. Phytother Res. 2012;26:1851-1856. doi: 10.1002 / ptr.4670. [Non-licensed Document 3] Chiang HM, Lin JW, Hsiao PL, Tsai SY, Wen KC. Hydrolysates of citrus plants stimulate melanogenesis protecting against UV-induced dermal damage. Phytother Res. 2011;25(4):569-576. doi: 10.1002 / ptr.3302. [Non-licensed Document 4] Choi SH, Choi SI, Jung TD, Cho BY, Lee JH, Kim SH, Yoon SA, Ham YM, Yoon WJ, Cho JH, Lee OH. Anti-photoaging effect of Jeju putgyul (Unripe Citrus) extracts on human dermal fibroblasts and ultraviolet B-induced hairless mouse skin. Int J Mol Sci. 2017;18(10):2052. doi: 10.3390 / ijms18102052.

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[0011] The problem to be solved by the present invention is to provide an external skin preparation that promotes hyaluronic acid synthesis not only in the human epidermis but also in the dermis, thereby improving the skin's moisture retention, while effectively utilizing unripe mandarin orange fruits that would otherwise be discarded. [Means for solving the problem]

[0012] The most important feature of the present invention is that the external moisturizing agent for skin contains hesperidin as an active ingredient. [Effects of the Invention]

[0013] By applying the topical skin moisturizing agent of the present invention to the skin, the production of hyaluronic acid in the epidermis and dermis is promoted, thereby providing the advantage of moisturizing the skin. [Brief explanation of the drawings]

[0014] [Figure 1] Reversed-phase HPLC patterns of methanol extracts (June 29th) of unripe Aoshima, Otsu, and Shonan Gold. HPLC analysis was performed using a Tosoh HPLC system (Tosoh, Tokyo, Japan) equipped with a DP-8020 pump and a UV-8020 detector. The chromatography column used was an ODS-80 Ts QA (4.6 × 150 mm). The mobile phase was acetonitrile:acetic acid:water (18:1:81). The flow rate was 1.0 mL / min, the detection wavelength was 285 nm, and the column temperature was 40°C. Narirutin and hesperidin were used as standards. Samples were collected on June 29th. [Figure 2] The contents of narirutin and hesperidin in extracts of unripe Aoshima, Otsu, and Shonan Gold were analyzed by HPLC. Data are presented as mean ± standard deviation. One-way analysis of variance was performed. Statistical analysis was performed using the Tukey-Kramer test in Statcel version 4.0. Statistical significance was set at p < 0.05. [Figure 3]Quantification of HAS-2 and HAS-3 mRNA expression in skin cells treated with methanol extracts of immature Aoshima, Otsu, and Shonan Gold. Human dermal fibroblasts (HFB) (A) and keratinocytes (HaCat) (B) were treated with methanol extracts of immature Aoshima, Otsu, and Shonan Gold (6 / 29). After 3 hours, cells were harvested and HAS-2 and -3 were extracted. Gene expression levels were measured by qRT-PCR. Statistical analysis was performed using the Tukey-Kramer test. Statistical significance was determined at p<0.05. [Figure 4] Hyaluronan content in skin cells treated with methanol extracts of immature Aoshima, Otsu, and Shonan Gold (6 / 29). HFB (A) and HaCat (B) were treated with methanol extracts of immature Aoshima, Otsu, and Shonan Gold (6 / 29). After 48 hours, the supernatants were collected and hyaluronan levels were detected by ELISA. Statistical analysis was performed using the Tukey-Kramer test. Statistical significance was determined at p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention has the following configuration. [Aspect 1] A topical moisturizing agent for the skin containing hesperidin as an active ingredient. [Aspect 2] The method of claim 1, wherein the skin is the dermis. [Aspect 3] The agent according to aspect 1 or 2, wherein the moisturizing is achieved by enhancing hyaluronic acid production. [Aspect 4] The agent according to any one of Aspects 1 to 3, wherein the agent is a cosmetic product. [Aspect 5] The agent according to Aspect 4, wherein the cosmetic product is in the form of a liquid, spray, mist, gel, cream, lotion, skin lotion, essence, compress, pack, powder, foundation, balm, ointment, bath additive, or cleanser. [Aspect 6] The agent according to any one of Aspects 1 to 5, wherein the hesperidin is an extract of the pulp and peel of an unripe mandarin orange. [Aspect 7] The agent according to Aspect 6, wherein the unripe mandarin orange is Citrus unshiu Markow. (Aoshima variety and Otsu variety) or Citrus flaviculpus (Shonan Gold).

[0016] In one embodiment, the agent of the present invention does not contain naringenin, rutin, hesperetin, tangeretin, nobiletin, naringin, apigenin, luteolin, quercetin, kaempferol, limonin, nomilin, and / or coumarin.

[0017] Any commercially available hesperidin can be used as the hesperidin used in the present invention. Furthermore, any known production method can be used to produce hesperidin. For example, one method for obtaining hesperidin from citrus fruits has been disclosed in which immature citrus fruits are extracted with an aqueous ethanol solution having an ethanol concentration of 40 to 60% to obtain an extract containing hesperidin (Japanese Patent Laid-Open Publication No. 2005-132791). Another method has been disclosed in which an alkaline earth metal compound and a pH adjuster are added to the citrus pomace (peel) to adjust the pH to 11.5 to 12.5, followed by stirring and mixing, squeezing the stirred mixture, and centrifuging the resulting squeezed liquid to obtain hesperidin (Japanese Patent Laid-Open Publication No. 8-188593). Furthermore, a method for producing hesperidin has been disclosed, which comprises an extraction step in which citrus fruits are brought into contact with a solvent consisting of either water or an organic solvent, or both, and subjected to extraction treatment while irradiating with microwaves to obtain an extract, and a hesperidin extraction step in which solid components contained in the extract are removed to obtain a clear extract containing hesperidin (JP 2011-105646 A).

[0018] The agent of the present invention may contain ingredients such as oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, coating agents, sweeteners, and acidulants, which are used in cosmetics, quasi-drugs, pharmaceuticals, foods, etc., within the range that does not impair the effects of hesperidin.

[0019] The content of hesperidin used in the agent of the present invention is preferably 0.5 to 2000 μg / mL, more preferably 2 to 500 μg / mL, and most preferably 10 to 100 μg / mL. [Example]

[0020] Immature Shonan Gold and Otsu varieties (C. unshuu varieties) were obtained from a fruit grower in Odawara City, Kanagawa Prefecture, and immature Aoshima varieties (C. unshuu varieties) were provided by a fruit grower in Mikkabi Town, Hamamatsu City, Shizuoka Prefecture. The immature mandarin oranges were collected on June 29, July 15, July 31, August 13, August 31, September 14, and September 29, 2020, and stored at -30°C until further analysis. All three varieties were collected as immature fruits. The frozen immature mandarin oranges were chopped in a blender and freeze-dried.

[0021] Freeze-dried samples of unripe mandarin oranges were extracted with 100% methanol, dried in an evaporator, and analyzed on the whole fruit, including the peel and pulp. The hesperidin and narirutin contents of unripe mandarin oranges were analyzed using high-performance liquid chromatography (HPLC) according to a previously described method (2016). Data are presented as mean ± standard deviation. One-way analysis of variance was performed. Statistical analysis was performed using the Tukey-Krame test in Statcel version 4.0. Statistical significance was set at p < 0.05.

[0022] The peaks detected by HPLC were narirutin and hesperidin in the methanol extract (Figure 1). Figure 2 shows the hesperidin and narirutin contents per weight of unripe mandarins harvested at different times. The hesperidin content in unripe mandarins did not vary seasonally. In contrast, the narirutin content was high in unripe Shonan Gold mandarins. The average weights of unripe mandarins harvested on September 29 were 66 g for unripe Aoshima mandarins, 79 g for unripe Otsu mandarins, and 42 g for unripe Shonan Gold mandarins. Because the weight of unripe mandarins increases seasonally, the narirutin content of unripe Satsuma mandarins at the early harvest stage was 0.2 mg and hesperidin content was 1.1 mg. On the final harvest day, the narirutin content was 0.2 mg and hesperidin content was 0.9 mg. The amounts of hesperidin and narirutin produced during the early stages of fruiting were maintained. A similar trend was observed in unripe Shonan Gold fruits, where the narirutin content was 0.4 mg and hesperidin content was 1.0 mg at the beginning of harvest, and 0.6 mg and 0.8 mg at the end of harvest. This trend was similar to that reported by Tamaru et al. (2020) for Satsuma mandarins (Aoshima).

[0023] Considering the raw food, tangerines are rich in flavonoids such as hesperidin and narirutin, which may function as a biological defense against predators. This is also suggested by the fact that high concentrations of flavonoids inhibit cell proliferation in cell addition tests. Methanol extracts of unripe tangerines were added to skin cells to examine their ability to produce hyaluronic acid.

[0024] Human dermal fibroblasts (HFB), a model system of human dermis, and human epidermal keratinocytes (HaCat), a model system of human epidermis, were purchased from the RIKEN BioResource Center (Tsukuba, Japan). Hesperidin, narirutin, and other reagents were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dulbecco's modified Eagle's medium (low glucose and high glucose) and fetal bovine serum were purchased from Sigma (St. Louis, MO, USA). 1% penicillin-streptomycin-neomycin antibiotic mixture (Gibco, Grand Island, NY, USA) was used for the skin cells.

[0025] Methanol extracts from unripe mandarin oranges (Aoshima, Otsu, and Shonan Gold) harvested on June 29 were dissolved in 0.1% dimethyl sulfoxide (DMS0), filtered through a 0.22 μm filter, and stored at -20°C until analysis. Each sample was adjusted to a final concentration of 50 μg / mL in medium and filtered through a 0.20 μm cellulose acetate membrane filter (ADVANTEC, Taipei, Taiwan).

[0026] Human dermal fibroblasts were grown in Dulbecco's modified Eagle's medium (low glucose) containing 10% fetal bovine serum and 1% penicillin-streptomycin-neomycin antibiotic mixture. Keratinocytes (HaCat) were cultured in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin, streptomycin, and neomycin antibiotic mixture. Cells were cultured in an incubator (MCO-17AIC, Dai-Sankyo, Tokyo, Japan) at 37°C with 5% CO2.

[0027] Human fibroblasts and keratinocytes (HaCat) were cultured to 80% confluence, and a methanol extract of unripe mandarin orange was added to the plate at 50 μg / mL and incubated at 37°C for 24 hours. 0.1% DMS0 solvent was added as a control. Cell addition experiments were repeated three times to ensure reproducibility.

[0028] Gene expression analysis in cultured cells was performed using quantitative real-time RT PCR. Human fibroblasts were cultured in 24-well plates at 1.2 × 10 4 Keratinocytes (HaCat) were seeded at 1.8 × 10 cells / well in a 24-well plate. 4 Cells / well were seeded and cultured for 48 hours, washed once with PBS, and then replaced with supplemented medium containing the test substance.

[0029] Three hours after sample addition, cells were lysed by adding 500 μl of cell lysis solution (TRIzol Reagent: Invitrogen) and RNA was collected. cDNA synthesis was performed using a Prime Script RT-PCR kit (Takara Bio Inc.). Reverse transcription was performed using a thermal cycler (GeneAmp PCR System 9700: PE Applied Biosystems). The reaction program was set as follows: 37°C for 15 minutes, 85°C for 5 minutes, and 4°C.

[0030] After the reaction was completed, the reaction mixture was diluted 2-fold with Easy Dilution and stored at -80°C until use. Real-time PCR was performed using the intercalator method with TB Green I. PCR reactions were performed using a Thermal Cycler Dice Real-Time System TP950 (Takara Bio Inc.). The reaction conditions were: (denaturation) 95°C, 5 seconds, 45 cycles; (annealing) 60°C, 30 seconds.

[0031] After the reaction was completed, the relative gene expression levels were analyzed using dedicated analysis software (Thermal Cycle Dice Real Time System TP950 Software Ver. 6.01D). Relative expression levels were determined using the standard curve method, and the gene expression levels of each sample were normalized to the co-amplified GAPDH. Primers were purchased from Eurofins Genomics Inc. (Tokyo, Japan).

[0032] The primer sequences were as follows: HAS-2 primer sequence (F) 5'-AGTCATGTACACAGCCTTCAGAGCA-3' (SEQ ID NO: 1) (R) 3'-CACCTCCAACCATGGGATCTTC-5' (SEQ ID NO: 2) HAS-3 primer sequence (F) 5'-TCGGCGATTCGGTGGACTA-3' (SEQ ID NO: 3) (R) 3'-CCTCCAGGACTCGAAGCATCTCTC-5' (SEQ ID NO: 4) GAPDH primer sequences (F) 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 5) (R) 3'-TGGTGAAGACGCCAGTGGA-5' (SEQ ID NO: 6)

[0033] Hyaluronan levels in the culture medium from human fibroblasts and keratinocytes were measured according to the method of Haserodt et al. (2011). Hyaluronan-binding protein (HABP) was coated onto multiwell plates blocked with Block Ace (Megmilk Snow Brand, Tokyo, Japan). Cell culture medium and methanol extract of unripe mandarin orange were added, and anti-HABP antibody (Funakoshi, Tokyo, Japan) was reacted. The antigen-antibody reaction was stopped by adding 1N sulfuric acid, and the absorbance at 450 / 630 nm was immediately measured. A hyaluronan calibration curve was prepared using hyaluronan standards (250, 62.5, 15.6, 3.9, and 0 ng / mL, Kewpie, Tokyo, Japan). All measurements were performed in duplicate, and the hyaluronan concentration in the culture medium was calculated using the standard curve.

[0034] The effects of the hyaluronan synthase genes HAS-2 and HAS-3 were investigated. Unripe mandarin extract was added to human fibroblasts and keratinocytes, and the amounts of HAS-2 and HAS-3 mRNA produced were compared (Figure 3). A sample containing DMSO without the test substance was used as a control. Addition of unripe Otsu mandarin extract to fibroblasts resulted in an approximately two-fold increase in HAS-2 mRNA gene expression. Addition of unripe Aoshima and Shonan Gold mandarin extracts slightly increased HAS-2 mRNA levels. However, the addition of hesperidin or narirutin had no effect. In keratinocytes, the addition of narirutin increased HAS-2 mRNA levels. The addition of unripe mandarin increased HAS-3 mRNA levels. The addition of hesperidin or narirutin to keratinocytes did not affect HAS-3 mRNA levels. Addition of unripe mandarin to keratinocytes did not affect the expression of genes encoding other moisturizing factors, such as filaggrin, aquaporin, and involucrin (data not shown).

[0035] The amount of hyaluronic acid produced in the HFB shown in Figure 4A was higher in immature Otsu, Shonan Gold, and Aoshima varieties than in the control. In keratinocytes, the values ​​were higher in immature Otsu, Shonan Gold, and Aoshima varieties compared to the control (Figure 4B). Comparing the effects of adding hesperidin and narirutin to skin cells, hesperidin induced higher hyaluronic acid production than narirutin.

[0036] Hyaluronic acid is known to have different roles in the epidermis and dermis. Increasing the amount of hyaluronic acid in the dermis improves skin moisture retention. Oral administration of unripe mandarin oranges improved skin moisture in a photoaging model mouse (Tamaru et al. 2020). Tamaru et al. suggested that orally administered flavonoids act by converting them to aglycones (i.e., naringenin and hesperetin) in the body. It has also been reported that the addition of Schizophyllum commune fermentation extract, a Satsuma mandarin peel extract containing naringenin and hesperetin, to skin fibroblasts protected against damage caused by UVA exposure (Bae et al. 2012). Furthermore, Jung et al. showed that the addition of ethyl acetate, hexane, and butanol fractions of a 50% ethanol extract of yuzu peel, which contains limonoids (i.e., nomilin, etc.) and coumarins, to keratinocytes (HaCat) restored hyaluronic acid levels reduced by UVB irradiation (Jung et al. 2022).

[0037] One of the clear functions of hyaluronan is to maintain the extracellular space between cells underlying the epidermal layer structure, facilitating the diffusion of nutrients to overlying cells and the removal of waste products (Abatangelo et al. 2020). Enhanced production of hyaluronan in the epithelium is thought to activate the metabolism of epithelial cells.

[0038] It has been shown that unripe mandarin orange extract promotes skin metabolism by promoting the production of HAS-3 mRNA and hyaluronic acid in epidermal cells. It is clear that unripe mandarin orange extract promotes the synthesis of hyaluronic acid in skin cells, and it is thought that it could be used as an ingredient in functional foods and cosmetics. Hyaluronic acid is a biopolymer involved in the moisture retention of skin.

[0039] Additionally, 3,5,6,7,8,3',4'-heptamethoxyflavone, a flavonoid derived from Satsuma mandarin peel, has been shown to increase type I collagen production by inhibiting MMP-1 activity (Kim et al. 2018). Fermented Satsuma mandarin peel exhibits anti-inflammatory effects in LPS-stimulated RAW264.7 cells, increases hyaluronic acid production in HaCat cells, and increases the amount of the moisturizing factor filaggrin (Bae et al. 2012). Nobiletin, a component of Satsuma mandarin, has been reported to suppress skin inflammation, oxidative stress, and carcinogenesis in mice (Chiang et al. 2011). [Industrial Applicability]

[0040] Hesperidin is found in large amounts in unripe mandarins (Figure 2). This study revealed that hesperidin is the component that increases hyaluronic acid production in fibroblasts when immature mandarin extract is added. Shonan Gold is highly rare due to its limited production area, and its added value lies in its ability to be harvested even when unripe. Shonan Gold is a cross between Ogonkan and Imamura Unshu, and is therefore thought to contain the beneficial components of Unshu. Unripe mandarins are highly valuable because they can be harvested before pesticide spraying, considering the harvest time. The content of hesperidin and narirutin varies depending on the mandarin variety, and their effects on skin cells may differ. Furthermore, their high antioxidant potential warrants consideration for applications beyond beauty products. The results of this characterization may expand the potential uses of unripe mandarins. [Explanation of symbols]

[0041] [Figures 1-4] Intensty: Intensity Retention Time: Retention time Narirutin: Narirutin Hesperidin: Hesperidin Aoshima: Aoshima species Otsu: Otsu species Shonan gold: Shonan Gold variety [Figures 3 and 4] Control: Control Hes: Hesperidin Nari: Narirutin [Figure 4] HA content: HA content

Claims

1. A topical moisturizing agent for the skin containing hesperidin as an active ingredient.

2. The agent according to claim 1, wherein the skin is the dermis.

3. The agent according to claim 2, wherein the moisturizing effect is achieved by enhancing hyaluronic acid production.

4. The agent according to claim 1, wherein the agent is a cosmetic product.

5. 5. The agent according to claim 4, wherein the cosmetic product is in the form of a liquid, spray, mist, gel, cream, lotion, skin lotion, essence, compress, pack, powder, foundation, balm, ointment, bath additive, or cleanser.

6. 2. The agent according to claim 1, wherein the hesperidin is an extract of the pulp and peel of unripe mandarin oranges.

7. The agent according to claim 6, wherein the unripe mandarin orange is Citrus unshiu Markow. (Aoshima variety and Otsu variety) or Citrus flaviculpus (Shonan Gold).

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