Application of hydrolyzed hyaluronic acid or salt thereof as 11beta-HSD1 expression inhibitor

Abstract

The invention provides application of hydrolyzed hyaluronic acid or a salt thereof as an 11beta-HSD1 expression inhibitor. The application finds that hydrolyzed hyaluronic acid or a salt thereof can be used as a 11 [beta]-HSD1 expression inhibitor, and thus can be used for inhibiting excessive conversion of cortisol into cortisol, such as cortisol production. Further, the hydrolyzed hyaluronic acid or the salt thereof can prevent and / or alleviate skin problems caused by cortisol imbalance, such as skin aging such as reduced skin collagen and reduced skin microcosin, skin barrier injury, skin inflammation, slowed skin self-repairing speed, skin atrophy, and the like.

Description

Technical Field

[0001] The present application belongs to the technical field of personal care products, and specifically relates to the use of hydrolyzed hyaluronic acid or a salt thereof as an inhibitor of 11β-HSD1 expression. Background Art

[0002] Human skin is closely related to the nervous system. In recent years, the beauty industry has paid more and more attention to the interaction between nerve signaling molecules and skin cells and is committed to finding neurobeauty ingredients to solve various skin problems and improve skin health.

[0003] Studies have shown that psychological stress activates the hypothalamic-pituitary-adrenal axis (HPA axis), which stimulates the production of endogenous glucocorticoids (GC) and activates 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), converting cortisone (inactive GC) into hydrocortisone (also known as cortisol, active GC), resulting in increased cortisol levels in serum and skin (the pattern is as follows Figure 1 shown [1] ). Under normal circumstances, cortisol levels rise rapidly after stress and return to normal after a few hours. However, long-term stress can lead to continuous activation of the HPA axis, resulting in an imbalance in cortisol levels and a series of skin problems. For example, it inhibits the proliferation and differentiation of skin cells, reduces the speed of skin self-repair, increases transepidermal water loss (TEWL), damages skin barrier function, decreases collagen content and the resulting skin atrophy, releases pro-inflammatory cytokines, and increases reactive oxygen species (ROS) levels, causing oxidative stress. [1][2][3][4][6] .

[0004] Blocking 11β-HSD1 activity can help the skin balance cortisol levels, increase dermal collagen synthesis, enhance barrier function, restore homeostasis and resist skin aging. [5] Previous studies have shown that topical application of 11β-HSD1 inhibitors can accelerate the healing of full-thickness back wounds in mice. [2] Finding active ingredients that can reduce the excessive conversion of cortisone to cortisol in the skin and alleviate skin problems remains a challenge.

[0005] [1]Terao M, Katayama I.Local cortisol / corticosterone activation inskin physiology and pathology[J]. Journal of Dermatological Science, 2016:11-16.

[0006] [2]TIGANESCU A, TAHRANI AA, MORGAN SA, et al. 11β-Hydroxysteroiddehydrogenase blockade prevents age-induced skin structure and functiondefects[J]. The Journal of Clinical Investigation, 2013, 123(7): 3051-3060.

[0007] [3]Sung-Jay C, Dong-Hye K, Eun-Jung K, et al. Psychological StressDeteriorates Skin Barrier Function by Activating 11β-HydroxysteroidDehydrogenase 1and the HPAAxis[J]. Scientific Reports, 2017.

[0008] [4]Terao M, Tani M, Itoi S, et al.11β-Hydroxysteroid Dehydrogenase1Specific Inhibitor Increased Dermal Collagen Content and Promotes FibroblastProliferation[J].Plos One,2014,9.

[0009] [5] Xin Yuchen et al., Principles and research progress of neurocosmetology[J], China Food and Drug Administration, No. 1, 2025 (Total No. 252).

[0010] [6]TOLLENAERE MD,MEUNIER M,SCANDOLERAA,et al.Well-aging: a new strategy for skin homeostasis under multi-stressed conditions[J].J CosmetDermatol,2020,19(2):444-455. Summary of the Invention

[0011] In response to the problems existing in the prior art, the inventors of the present application conducted in-depth research and found that hydrolyzed hyaluronic acid or its salts can inhibit the expression of 11β-HSD1, thereby weakening the conversion of cortisone to cortisol, and preventing and / or alleviating skin problems caused by cortisol imbalance.

[0012] Specifically, this application involves the following aspects:

[0013] 1. Use of hydrolyzed hyaluronic acid or its salt as an inhibitor of 11β-HSD1 expression.

[0014] 2. The use according to item 1, wherein the hydrolyzed hyaluronic acid or a salt thereof is used to reduce the production of cortisol.

[0015] 3. The use according to item 1, wherein the hydrolyzed hyaluronic acid or a salt thereof is used to reduce the conversion of cortisone to cortisol in the skin.

[0016] 4. The use according to item 1, wherein the hydrolyzed hyaluronic acid or its salt is used to prepare cosmetics for emotional skin care.

[0017] 5. Use of hydrolyzed hyaluronic acid or a salt thereof as an 11β-HSD1 expression inhibitor for preventing and / or alleviating skin problems caused by cortisol.

[0018] 6. Use of hydrolyzed hyaluronic acid or a salt thereof as an 11β-HSD1 expression inhibitor for preventing and / or alleviating skin problems caused by stress.

[0019] 7. The use according to item 5 or 6, wherein the skin problems include one or more of skin aging, damaged skin barrier, skin inflammation, slowed skin self-repair, and skin atrophy.

[0020] The present application discovered that hydrolyzed hyaluronic acid or its salt can serve as an inhibitor of 11β-HSD1 expression, thereby being used to inhibit the excessive conversion of cortisone to cortisol.

[0021] Furthermore, hydrolyzed hyaluronic acid or its salt can prevent and / or alleviate skin problems caused by cortisol imbalance, such as skin aging, skin barrier damage, skin inflammation, slowed skin self-repair, skin atrophy, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of Reference 1;

[0023] Figure 2 Fluorescence results showing the expression level of 11β-HSD1 in experimental group 1;

[0024] Figure 3 Fluorescence results showing the expression level of 11β-HSD1 in experimental group 2;

[0025] Figure 4 Fluorescence results showing the expression level of collagen I in experimental group 3;

[0026] Figure 5 Fluorescence results showing the expression level of collagen I in experimental group 4;

[0027] Figure 6 Fluorescence results showing the expression level of collagen III in experimental group 3;

[0028] Figure 7 Fluorescence results showing the expression level of loricrin in experimental group 5. DETAILED DESCRIPTION

[0029] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0030] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0031] As used herein, the term "expression inhibitor" refers to any agent capable of reducing or downregulating the expression of a protein of a corresponding gene. Those skilled in the art will appreciate that such reduction or downregulation can be determined using conventional techniques in the art. An 11β-HSD1 expression inhibitor refers to an agent capable of reducing or downregulating 11β-HSD1 expression.

[0032] As used herein, the term "Cortisol" refers to a naturally occurring glucocorticoid produced by the zona fasciculata of the adrenal glands, also known as hydrocortisone.

[0033] The present application provides the use of hydrolyzed hyaluronic acid or a salt thereof as an inhibitor of 11β-HSD1 expression.

[0034] Wherein, described hydrolyzed hyaluronic acid or its salt can be any hydrolyzed hyaluronic acid or its salt known in the art.In some embodiments, the molecular weight of described hydrolyzed hyaluronic acid or its salt is below 10kDa, for example, can be 10kDa, 9kDa, 8kDa, 7kDa, 6kDa, 5kDa, 4kDa, 3kDa, 2kDa, 1kDa, 950Da, 900Da, 850Da, 800Da, 750Da, 700Da, 650Da, 600Da, 550Da, 500Da, 400Da, 379Da etc., and the arbitrary numerical value between these numerical values.In some embodiments, the molecular weight of described hydrolyzed hyaluronic acid or its salt is 379-1000Da.

[0035] In the application, the hydrolyzed hyaluronic acid or its salt acts as an inhibitor of 11β-HSD1 expression and can reduce the production of cortisol.

[0036] The present application also provides use of the hydrolyzed hyaluronic acid or its salt in reducing the production of cortisol.

[0037] In some embodiments, the hydrolyzed hyaluronic acid or a salt thereof is used to reduce the conversion of cortisone to cortisol in the skin, thereby reducing the production of cortisol.

[0038] Psychological stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which stimulates the production of endogenous glucocorticoids (GCs) and activates 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), converting cortisone (an inactive GC) into hydrocortisone (also known as cortisol, an active GC). This leads to elevated cortisol levels in serum and skin. Chronic stress can lead to sustained activation of the HPA axis, resulting in an imbalance in cortisol levels and a series of skin problems. Therefore, cortisol plays a key role in stress and mood regulation. Hydrolyzed hyaluronic acid or its salts can be used to create cosmetics for emotional skincare. Emotional skincare generally refers to a skincare concept that focuses on the relationship between emotional well-being and skin health. Chivalrous emotional skincare can be understood as skincare products that directly heal and soothe emotions through smell, sight, and touch. Examples include adding soothing aromas like essential oils and fragrances, packaging with a sense of ritual or playfulness, and materials with a smooth, moisturizing feel. Emotional skin care in a broad sense also includes influencing the user's emotions and feelings through physiological effects on the skin, relying on the two-way communication mechanism between the skin and emotions in the human body to achieve the purpose of improving skin condition or psychological state.

[0039] The present application also provides use of hydrolyzed hyaluronic acid or a salt thereof as an 11β-HSD1 expression inhibitor in preventing and / or alleviating skin problems caused by cortisol.

[0040] The present application also provides use of hydrolyzed hyaluronic acid or a salt thereof in preventing and / or alleviating skin problems caused by cortisol.

[0041] The present application also provides use of hydrolyzed hyaluronic acid or a salt thereof as an 11β-HSD1 expression inhibitor in preventing and / or alleviating skin problems caused by stress.

[0042] The present application also provides use of hydrolyzed hyaluronic acid or a salt thereof in preventing and / or alleviating skin problems caused by stress.

[0043] In the use for preventing and / or alleviating skin problems caused by cortisol or preventing and / or alleviating skin problems caused by stress, the molecular weight of the hydrolyzed hyaluronic acid or its salt is as described above.

[0044] Skin problems caused by cortisol are known in the art and may include, for example, one or more of skin aging, damaged skin barrier, skin inflammation, slowed skin self-repair, and skin atrophy.

[0045] The present application has found that hydrolyzed hyaluronic acid or its salt can suppress the expression of 11β-HSD1, thereby can prevent and / or alleviate the skin problem caused by the imbalance of cortisol. In addition, hydrolyzed hyaluronic acid or its salt can improve the collagen reduction caused by cortisol, thereby can alleviate the skin aging caused by cortisol. Hydrolyzed sodium hyaluronate can also significantly promote the generation of loricrin of epidermal keratinocytes after cortisone treatment, and plays an important role in maintaining the skin barrier function caused by cortisol. Therefore, hydrolyzed hyaluronic acid or its salt has good prevention and / or alleviation effect on the skin problems such as skin aging caused by cortisol, damaged skin barrier, skin inflammation, slowing down of skin self-repair speed, skin atrophy.

[0046] Example

[0047] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0048] Example 1. Expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)

[0049] This test uses fluorescence microscopy to detect and analyze whether the test sample can inhibit the overexpression of 11β-HSD1 in the human epidermal immortalized keratinocyte (HaCaT) cell line caused by UV irradiation, and evaluates whether the test sample has the efficacy of an 11β-HSD1 expression inhibitor based on the significant difference.

[0050] 1. Cell Model and Experimental Materials

[0051] Table 1

[0052]

[0053]

[0054] 2. Main equipment

[0055] CO2 incubator (Thermo, 160i), biological safety cabinet (Thermo), fluorescence inverted microscope (Leica), solar simulator

[0056] 3. Methods

[0057] 3.1 Experimental Grouping

[0058] Table 2 Experimental groups

[0059]

[0060] Among them, experimental sample solution 1: hydrolyzed sodium hyaluronate powder was diluted with DMEM culture medium to prepare a solution with 0.05 wt% of hyaluronic acid.

[0061] Experimental sample solution 2: dilute thioneine powder with DMEM medium to prepare a solution containing 5 μM thioneine.

[0062] 3.2 Experimental methods

[0063] Human immortalized keratinocytes were plated at a density of 30,000 cells / well in a 24-well plate containing a cell slide and cultured in an incubator for 72 hours until the cell confluence reached 70-80%. The cells were divided into three groups: untreated group, experimental group, and UVB irradiation group.

[0064] Untreated group: The cell culture medium was aspirated and replaced with PBS solution, and then the liquid in the cell wells was replaced with DMEM medium and cultured for another 24 h.

[0065] Experimental Groups 1 and 2: The cell culture medium was aspirated and replaced with PBS solution. The well plates were then placed under a solar simulator for irradiation at a dose of UVB = 15 mJ. After irradiation, the liquid in the cell wells was replaced with experimental sample solutions 1 and 2, respectively, and the cells were cultured for another 24 h.

[0066] UVB irradiation group: the cell culture medium was aspirated and replaced with PBS solution, and the well plate was placed under a solar simulator for irradiation with an irradiation dose of UVB = 15 mJ. After irradiation, the liquid in the cell wells was replaced with DMEM medium and cultured for another 24 h.

[0067] Cell slides were collected and fixed with ice-cold methanol for 15 minutes. Anti-HSD11B1 antibody was added and incubated overnight at 4°C. The supernatant was discarded, and Goat anti-mouse IgG secondary antibody was added and incubated for 1.5 hours at room temperature. The slides were mounted with DAPI mounting medium. Fluorescence images were obtained under a Leica inverted fluorescence microscope, and fluorescence intensity was quantified using Image J software. Results for experimental groups 1 and 2 and their respective UVB-irradiated groups were normalized to the respective untreated group as 1.

[0068] Statistical analysis was performed using the t-test to test the significance of 11β-HSD1 expression levels among samples. P<0.05 indicated a statistical difference, P<0.01 indicated a significant statistical difference, P<0.001 indicated a very significant statistical difference, and P>0.05 indicated no statistical difference.

[0069] 4. Experimental Results

[0070] The results of 11β-HSD1 expression levels are shown in Tables 3, 4, Figure 2 、 3 shown.

[0071] Table 3

[0072]

[0073] Table 4

[0074]

[0075]

[0076] The results showed that after UVB irradiation, the expression of 11β-HSD1 in HaCaT cells was significantly upregulated, indicating that the model was successfully established.

[0077] After treatment with hydrolyzed hyaluronic acid, its expression level decreased significantly, indicating that hydrolyzed hyaluronic acid can significantly inhibit the expression of 11β-HSD1 on keratinocytes after UVB irradiation, and plays an important role in reducing the increase in cortisol concentration in the skin under stress.

[0078] After treatment with ergothioneine, its expression level did not change significantly, indicating that ergothioneine could not inhibit the expression of 11β-HSD1 in keratinocytes after UVB irradiation.

[0079] Example 2: Determination of collagen content

[0080] 1. Cell Model and Experimental Materials

[0081] Table 5 Cell models and experimental materials

[0082] Cell models and experimental materials Supplier Human dermal fibroblast cell line ATCC DMEM medium Gibco Hydrolyzed sodium hyaluronate <![CDATA[Hybloom TM Micro-True TM (800-1000Da), Bloomage Biotechnology Ergothioneine Bloomage Bio FBS Gibco PBS Gibco

[0083] 2. Main equipment

[0084] Carbon dioxide incubator (Thermo, 160i), biological safety cabinet (Thermo), fluorescence inverted microscope (Leica)

[0085] 3. Methods

[0086] 3.1 Experimental Grouping

[0087] Table 6 Experimental groups

[0088]

[0089]

[0090] Cortisone solution: a certain amount of cortisone powder was weighed and diluted with DMEM culture medium to prepare a cortisone solution with a concentration of 100 μM.

[0091] 3.2 Experimental methods

[0092] Human dermal fibroblasts were plated at a density of 15,000 per well in a 24-well plate containing a cell slide and cultured in an incubator for 24 hours.

[0093] Untreated group: The cell culture medium was aspirated and replaced with fresh DMEM medium, and culture was continued for 72 h.

[0094] Experimental group 3: The cell culture medium was aspirated and replaced with cortisone solution and experimental sample solution 1, and culture was continued for 72 h.

[0095] Experimental group 4: The cell culture medium was aspirated and replaced with cortisone solution and experimental sample solution 2, and culture was continued for 72 h.

[0096] Raw material control group: aspirate the above cell culture medium and replace it with experimental sample solution 2, and continue culturing for 72 hours.

[0097] Pressure treatment group: the cell culture medium was aspirated and replaced with cortisone solution, and the cells were cultured for another 72 h.

[0098] Cell slides were harvested and fixed with ice-cold methanol for 15 minutes. Anti-collagen I or anti-collagen III antibodies were added and incubated overnight at 4°C. The supernatant was discarded, and the slides were incubated with Goat anti-rabbit IgG secondary antibodies for 1.5 hours at room temperature. The slides were mounted with DAPI mounting medium. Fluorescence images were captured under a Leica inverted fluorescence microscope. "Collagen I" represents collagen type 1, "Collagen III" represents collagen type 3, "Nuclear" represents nuclei, and "Merge" represents a fusion of the two. Fluorescence intensities of type I and type III collagen were quantified using Image J software. Results for experimental groups 3 and 4, as well as the respective stress-treated groups, were normalized to the respective untreated group as 1.

[0099] Statistical analysis was performed using the t-test to test the significance of collagen expression levels between samples. P < 0.05 indicated a statistical difference, P < 0.01 indicated a significant statistical difference, P < 0.001 indicated an extremely significant statistical difference, and P > 0.05 indicated no statistical difference.

[0100] 4. Experimental Results

[0101] The results of collagen I expression levels are shown in Tables 7, 8, Figure 4 、 Figure 5 The results of collagen III expression levels are shown in Table 9. Figure 6 shown.

[0102] Table 7

[0103]

[0104] Table 8

[0105]

[0106] Table 9

[0107]

[0108]

[0109] The results showed that the production of type I and type III collagen in the stress-treated group was significantly lower than that in the untreated group, indicating that cortisone was converted into cortisol, which in turn affected the expression of type I and type III collagen. That is, stress would reduce the production of type I and type III collagen in dermal fibroblasts, and the model was successfully established.

[0110] Compared with the stress-treated group, the production of type I and type III collagen in experimental group 3 was significantly increased, indicating that hydrolyzed sodium hyaluronate can improve the reduction of collagen caused by cortisol, that is, hydrolyzed sodium hyaluronate can alleviate skin aging caused by cortisol.

[0111] Example 3: Determination of loricrin content

[0112] 1. Cell Model and Experimental Materials

[0113] Table 10 Cell models and experimental materials

[0114]

[0115] 2. Main equipment

[0116] Carbon dioxide incubator (Thermo, 160i), biological safety cabinet (Thermo), fluorescence inverted microscope (Leica)

[0117] 3. Methods

[0118] 3.1 Experimental Grouping

[0119] Table 11 Experimental groups

[0120]

[0121] 4. Experimental Methods

[0122] Human immortalized keratinocytes were plated at a density of 30,000 per well in a 24-well plate containing a cell slide and cultured in an incubator for 24 hours.

[0123] Untreated group: The cell culture medium was aspirated and replaced with fresh DMEM medium, and culture was continued for 72 h.

[0124] Experimental group 5: The above cell culture medium was aspirated and replaced with experimental sample solution 1, and culture was continued for 72 h.

[0125] Pressure treatment group: the cell culture medium was aspirated and replaced with cortisone solution, and the cells were cultured for another 72 h.

[0126] Cell slides were collected and fixed with ice-cold methanol for 15 minutes. Anti-loricrin antibody was added and incubated overnight at 4°C. The supernatant was discarded, and Goat anti-rabbit IgG secondary antibody was added and incubated at room temperature for 1.5 hours. The slides were mounted with DAPI mounting medium. Fluorescence images were observed and captured under a Leica inverted fluorescence microscope, and fluorescence intensity was quantified using Image J software. Results were normalized to the untreated group as 1.

[0127] Statistical analysis was performed using the t-test to test the significance of the expression levels of loricrin between samples. P < 0.05 indicated a statistical difference, P < 0.01 indicated a significant statistical difference, P < 0.001 indicated an extremely significant statistical difference, and P > 0.05 indicated no statistical difference.

[0128] 4. Experimental Results

[0129] The results of the expression level of loricrin are shown in Table 12. Figure 7 shown.

[0130] Table 12 Experimental data

[0131]

[0132]

[0133] After cortisone treatment (stress treatment group), the cell loricrin was significantly lost.

[0134] Compared with the treatment with cortisone solution alone, experimental group 5 restored the production of loricrin, indicating that hydrolyzed sodium hyaluronate can significantly promote the production of loricrin in epidermal keratinocytes after cortisone treatment, and plays an important role in maintaining the damaged skin barrier function caused by cortisol.

[0135] From the above experiments, it can be seen that hydrolyzed sodium hyaluronate has the effect of inhibiting the expression of 11β-HSD1, and can thus regulate many skin problems caused by cortisol imbalance, such as skin aging, skin barrier damage, skin inflammation, slower skin repair, skin atrophy, etc. caused by cortisol imbalance.

Claims

1. Use of hydrolyzed hyaluronic acid or its salt as an inhibitor of 11β-HSD1 expression.

2. The use according to claim 1, wherein the hydrolyzed hyaluronic acid or its salt is used to reduce the production of cortisol.

3. The use according to claim 1, wherein the hydrolyzed hyaluronic acid or a salt thereof is used to reduce the conversion of cortisone to cortisol in the skin.

4. The use according to claim 1, wherein the hydrolyzed hyaluronic acid or its salt is used to prepare cosmetics for emotional skin care.

5. Use of hydrolyzed hyaluronic acid or a salt thereof as an 11β-HSD1 expression inhibitor for preventing and / or alleviating skin problems caused by cortisol.

6. Use of hydrolyzed hyaluronic acid or a salt thereof as an 11β-HSD1 expression inhibitor for preventing and / or alleviating skin problems caused by stress.

7. The use according to claim 5 or 6, wherein the skin problems include one or more of skin aging, damaged skin barrier, skin inflammation, slowed skin self-repair, and skin atrophy.

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