Use of ectoin ceramide in skin care

Abstract

Use of ectoin ceramide in skin care. The ectoin ceramide has an excellent effect in the aspects of repairing the skin barrier, promoting skin tissue healing, resisting photoaging, moisturizing, resisting inflammation and soothing, and the like. Especially in the aspects of cell viability proliferation and resisting inflammation and soothing, the ectoin ceramide shows superior effects than ectoin, and has good application value in the fields of preparing cosmetics, drugs, and health care products with corresponding efficacy or indications.

Description

Uses of ectoin ceramide in skin care Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of ectoin ceramide in skin care. Background Technology

[0002] Ceramides, as an important physiological lipid in the skin, have attracted special attention from researchers due to their unique skin-care effects. Ceramides are present in all eukaryotic cells and play a crucial regulatory role in cell differentiation, proliferation, apoptosis, aging, and other vital processes. As a major component of the intercellular lipids of the stratum corneum, ceramides not only act as second messenger molecules in the sphingomyelin pathway but also play a vital role in the formation of the epidermal stratum corneum, thus possessing functions such as maintaining the skin barrier, soothing, moisturizing, anti-aging, whitening, and disease treatment.

[0003] Ectoin is a cyclic amino acid derivative and an amphoteric organic molecule with excellent hydrophilicity. It can bind to surrounding water molecules and amino acid derivatives to form a stable hydration shell, maintaining osmotic pressure balance and reducing excessive water evaporation from internal cells. Scientific research has found that ectoin not only has strong anti-stress and excellent repairing abilities but also effectively repairs the skin barrier.

[0004] Ceramides are a class of sphingosine lipids composed of long-chain sphingosine bases and fatty acids. The carbon chain length, degree of unsaturation, and number of hydroxyl groups in both the sphingosine and fatty acid moieties can vary. Ceramides represent a class of compounds. If the long-chain fatty acid moieties are replaced with hydrophilic ectoine, they dock with the long-chain sphingosine bases to form a new type of ceramide product, namely ectoine ceramide. Currently, research on the efficacy of this type of compound is lacking, and there is no specific experimental data to support its efficacy. Therefore, studying the skin care efficacy of ectoine ceramide at the cellular level, exploring its specific biological activities, and determining its suitability for use in cosmetic formulations is of significant research importance and practical value. Summary of the Invention

[0005] The purpose of this invention is to provide the use of ectoin ceramide in skin care, especially for skin barrier repair, tissue healing, anti-photoaging, moisturizing, anti-inflammatory and soothing.

[0006] The ectoine ceramide referred to in this invention is a product obtained by condensation of ectoine with sphingosine, dihydrosphingosine, and phytosphingosine, and has the following structure:

[0007] This invention demonstrates through experiments that ectoin ceramide has effects such as skin barrier repair, tissue healing, anti-photoaging, moisturizing, anti-inflammatory and soothing, and can be used in cosmetics, pharmaceuticals or health products.

[0008] On the other hand, ectoine ceramides are used in the preparation of cosmetics, pharmaceuticals, or health products for skin barrier repair.

[0009] On the other hand, ectoin ceramides are used in the preparation of cosmetics, pharmaceuticals, or health products for tissue healing.

[0010] On the other hand, ectoin ceramides are used in the preparation of cosmetics, pharmaceuticals, or health products for anti-photoaging purposes.

[0011] On the other hand, ectoine ceramides are used in the preparation of cosmetics, pharmaceuticals, or health products for moisturizing.

[0012] On the other hand, ectoine ceramides are used in the preparation of cosmetics, pharmaceuticals, or health products for anti-inflammatory and soothing purposes.

[0013] On the other hand, a composition comprising ectoin ceramide, said composition having at least one of the following effects: skin barrier repair, tissue healing, anti-photoaging, moisturizing, and anti-inflammatory soothing.

[0014] The present invention has the following beneficial effects:

[0015] This invention reacts water-soluble ectoine with sphingosine derivatives to derive ectoine ceramide. The chemical structure incorporates N and NH atoms, enabling hydrogen bonding and resulting in better hydrophilicity than traditional ceramides. Ectoine ceramide exhibits excellent effects in repairing the skin barrier, promoting skin tissue healing, combating photoaging, moisturizing, and soothing inflammation. Particularly in cell vitality proliferation and anti-inflammatory effects, ectoine ceramide demonstrates superior efficacy compared to ectoine, making it highly valuable for the preparation of cosmetics, pharmaceuticals, and health products with corresponding efficacy or indications. Attached Figure Description

[0016] Figures 1 and 2 are bar charts showing the cell proliferation activity detection results of Example 2;

[0017] Figure 3 shows the results of the cell scratch experiment in Example 3;

[0018] Figure 4 is a bar chart showing the anti-inflammatory and repair efficacy test results of Example 4;

[0019] Figure 5 is a bar chart showing the results of the moisturizing efficacy test in Example 5;

[0020] Figure 6 is a bar chart showing the anti-photoaging test results of Example 6. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments.

[0022] Example 1

[0023] Preparation of ectoine phytosphingosine ceramide

[0024] Ectoin (50 mmol), EDCI (60 mmol), and HOBT (60 mmol) were placed in a 250 mL round-bottom flask, followed by the addition of 100 mL of dichloromethane. The mixture was stirred at room temperature for 1 hour. Then, phytosphingosine (48 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 36 hours until TLC showed complete disappearance of the phytosphingosine. Post-treatment: Water was added to quench the reaction mixture. The organic layer was separated, dried, filtered, and concentrated under vacuum. The residue obtained was purified by silica gel column chromatography to yield ectoin phytosphingosine product (10.6 g, yield 50%). High performance liquid chromatography (LC-2030C 3D Plus) was used. The column was Innoval ODS-2 (4.6 mm × 250 mm, 5 μm). Mobile phase A was 0.05% TFA-H2O, mobile phase B was acetonitrile, flow rate was 1.0 mL / min, detector was ELSD, evaporation temperature was 40 °C, carrier gas flow rate was 2.5 L / min, and the product elution time was 4.95 min.

[0025] 1 H NMR (400MHz, Methanol-d4) δ4.18(dt,J=6.6,4.5Hz,1H),3.82(dd,J=11.3,4.2Hz,1H),3.71(dd,J=11.3,6.6Hz,1H),3.60–3.44(m,3H), 1.95(s,4H),1.79(dt,J=14.0,6.9Hz,1H),1.68(t,J=10.6Hz,1H),1.54(d,J=11.7Hz,1H),1.29(d,J=8.8Hz,26H),0.89(t,J=6.8Hz,3H).

[0026] Preparation of ectoin sphingosine ceramide

[0027] Ectoin (50 mmol), EDCI (60 mmol), and HOBT (60 mmol) were placed in a 250 mL round-bottom flask, followed by the addition of 100 mL of dichloromethane. The mixture was stirred at room temperature for 1 hour. Then, sphingosine (50 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 32 hours until TLC showed complete disappearance of phytosphingosine. Post-treatment: Water was added for quenching. The organic layer was separated, dried, filtered, and concentrated under vacuum. The residue obtained was purified by silica gel column chromatography to give the ectoin sphingosine ceramide product (9.54 g, yield 45%).

[0028] 1 H NMR (400MHz, Methanol-d4) δ5.75 (dt, J=14.1, 6.7Hz, 1H), 5.49 (dd, J=15.4, 6.4Hz, 1H), 4 .22(dt,J=6.8,4.4Hz,1H),3.85(dd,J=11.2,4.4Hz,1H),3.74(dd,J=11.2,6.8Hz,1H),3.6 6–3.51(m,2H),3.29–3.16(m,1H),2.02(dq,J=12.2,6.8Hz,2H),2.01–1.95(m,1H),1.92(s ,3H),1.79–1.69(m,1H),1.54–1.46(m,1H),1.29(d,J=8.8Hz,23H),0.92(t,J=6.8Hz,3H).

[0029] Preparation of ectoin dihydrosphingosine ceramide

[0030] Ectoin (50 mmol), EDCI (60 mmol), and HOBT (60 mmol) were placed in a 250 mL round-bottom flask, followed by the addition of 100 mL of dichloromethane. The mixture was stirred at room temperature for 1 hour. Then, dihydrosphingosine (52 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 28 hours until TLC showed complete disappearance of phytosphingosine. Post-treatment: Water was added for quenching. The organic layer was separated, dried, filtered, and concentrated under vacuum. The residue obtained was purified by silica gel column chromatography to give the ectoin dihydrosphingosine ceramide product (8.94 g, yield 42%).

[0031] 1H NMR(400MHz, Methanol-d4)δ4.2(dt,J=6.4,4.6Hz,1H),3.80(dd,J=11.2,4.1Hz,1H),3.70–3.65(m,1H),3.60–3.44(m,2H),3.32–3.19(m,1 H), 2.01–1.94(m,1H),1.93(s,3H),1.79–1.73(m,1H),1.64(t,J=10.6Hz,1H),1.54–1.51(m,1H),1.29–1.22(m,28H),0.86(t,J=6.8Hz,3H).

[0032] Example 2

[0033] MTT assay for the effect of ectoin on cell proliferation activity

[0034] Human keratinocytes (HaCaT cells) and mouse mononuclear macrophages (RAW cells) were used at a ratio of 1×10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded, and 100 μL of culture medium containing different concentrations of the sample (ectoine phytosphingosine, ectoine) or blank was added (three parallel sets for each concentration). Incubation continued for another 24 hours, after which the culture medium was removed, and 100 μL of thiazolyl blue (MTT) was added to each well. The absorbance at 450 nm was measured, and cell viability was calculated as A. 给药孔 / A 空白孔 ×100%.

[0035] Table 1. Results of Ectoin Phytosphingosine Effect on Cell Proliferation Viability (HaCaT Cells)

[0036] Table 2 Results of Ectoin's effect on cell proliferation (HaCaT cells)

[0037] Table 3 Results of Ectoin Phytosphingosine Effect on Cell Proliferation Viability (RAW Cells)

[0038] Table 4. Results of Ectoin's effect on cell proliferation (RAW cells)

[0039] The results of the effects of ectoine phytosphingosine and ectoine on cell proliferation activity are shown in Tables 1-4 and Figures 1 and 2. For HaCaT cells, the cell viability of ectoine phytosphingosine at concentrations of 3.90625 mg / L, 7.8125 mg / L, and 15.625 mg / L (0.0088 mmol / L, 0.0177 mmol / L, and 0.0354 mmol / L) were 146.83%, 137.05%, and 98.35%, respectively. The cell viability of ectoine at concentrations of 7.8125 mg / L and 15.625 mg / L (0.0550 mmol / L and 0.1099 mmol / L) were 122.14% and 128.61%, respectively. Comparing the molar concentrations, it was found that ectoine phytosphingosine showed stronger cell proliferation ability than ectoine at lower concentrations. For RAW cells, the cell viability of ectoine phytosphingosine at concentrations of 3.90625 mg / L, 7.8125 mg / L, and 15.625 mg / L (0.0088 mmol / L, 0.0177 mmol / L, and 0.0354 mmol / L) was 126.99%, 119.22%, and 93.00%, respectively. The cell viability of ectoine at concentrations of 3.90625 mg / L, 7.8125 mg / L, and 15.625 mg / L (0.0275 mmol / L, 0.0550 mmol / L, and 0.1099 mmol / L) was 131.36%, 126.49%, and 131.36%, respectively. Comparing molar concentrations, low concentrations of ectoine phytosphingosine showed excellent cell proliferation ability.

[0040] Example 3

[0041] The effect of ectoin on cell migration was detected using the cell scratch assay.

[0042] The scratch assay is an in vitro experimental method for studying cell migration. When keratinocytes grow to a fused monolayer, an artificially created blank area (scratch) is made on the fused monolayer. Cells at the edge of the scratch gradually move into the blank area, causing the "scratch" to heal. This simulates the process of epidermal keratinocyte migration to some extent. By observing the state of cells in the scratch area at different stages, the migration ability of cells can be judged. It is an important method in in vitro experiments for studying skin wound healing and repair.

[0043] Operating steps:

[0044] 1. Marking the culture plate. First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate with a ruler, about every 0.5 to 1 cm, passing through the wells. Each well should have at least 5 lines.

[0045] 2. Seeding cells. Add approximately 5 × 10⁻⁶ cells to the wells. 5One fibroblast L929 cell was seeded, with the principle being that the confluence rate reached 100% after overnight inoculation.

[0046] 3. Cell streaking. After 24 hours, use a micropipette tip perpendicular to the cell plane to streak along the lines previously streaked on the back of the plate on the monolayer of adherent cells (use the same tip between different wells) to create a scar model.

[0047] 4. Washing cells. After scratching, wash the cells three times with sterile PBS to remove non-adherent cells, i.e., the cells that were scratched off during the streaking process. Then replace with fresh serum-free culture medium.

[0048] 5. Cell Culture and Observation. The sample (ectoin sphingosine) was diluted with DMEM medium (concentration 62.5 mg / L) and added to cell culture dishes. The cells were incubated at 37°C in a 5 wt% CO2 incubator. After 24 hours, the cells were removed, observed under a microscope, and the width of the scratches was measured and photographed. The control group consisted of DMEM medium without the sample.

[0049] As shown in Figure 3, compared to the control group, the scratch width in the experimental group was narrower, indicating that ectoin sphingosine has a stronger ability to promote the healing of keratinocytes (skin tissue) and has a significant repair effect on cell damage. This is because ectoin sphingosine enhances the interaction between cells and the extracellular matrix, as well as between cells, thereby enhancing cell migration and movement.

[0050] Example 4

[0051] The anti-inflammatory and restorative effects of ectoin phytosphingosine were detected using the LPS-induced cell method.

[0052] Interleukin 6 (IL-6) is a cytokine most typically associated with inflammation. It plays a crucial role in host defense by regulating immune and inflammatory responses. Inflammation affects the skin barrier, increasing epidermal moisture loss and hindering keratinocyte growth, making barrier repair difficult once damaged. It also breaks down the extracellular matrix, causing skin collapse and inhibiting collagen synthesis, leading to loose skin and wrinkles. Therefore, effectively reducing IL-6 production in keratinocytes and fibroblasts induced by external damage and UV radiation, and lowering the inflammatory response, is essential for restoring the skin barrier and maintaining skin elasticity and stability.

[0053] Macrophages were RAW at a density of 1×10⁻⁶. 4Cells were seeded per well in 96-well plates and incubated overnight in an incubator. After 24 hours, the supernatant was discarded, and 100 μL of different concentrations of samples (ectoin phytosphingosine and ectoin) diluted in DMEM medium were added. The blank control group contained no sample in DMEM medium. Each group was divided into three replicates, and incubated at 37°C with 5 wt% CO2. Two hours after drug administration, 10 μg / mL LPS was added to both the lipopolysaccharide model group and the experimental group, and they were co-incubated for 24 hours. After the reaction, 50 μL of cell supernatant was collected, and the expression of intracellular IL-6 factor was detected using an IL-6 ELISA kit.

[0054] Table 5 Results of the ectoin phytosphingosine anti-inflammatory test

[0055] Table 6 Results of Ectocin Anti-inflammatory Test

[0056] The results are shown in Tables 5-6 and Figure 4. The IL-6 expression level in the blank control group was set as 100. Under stimulation with LPS at a working concentration of 10 μg / mL, the IL-6 level was 3.93 times the baseline level. Under the action of ectoine phytosphingosine at concentrations of 3.90625 mg / L, 7.8125 mg / L, and 15.625 mg / L, the relative mRNA expression levels of IL-6 were 156.55, 122.89, and 52.26, respectively. Under the action of ectoine at concentrations of 3.90625 mg / L, 7.8125 mg / L, and 15.625 mg / L, the relative mRNA expression levels of IL-6 were 184.70, 145.04, and 144.06, respectively.

[0057] Co-incubation of ectoine phytosphingosine with cells significantly reduced the expression of intracellular IL-6 inflammatory factor in a dose-dependent manner. Unexpectedly, the inhibitory effect of ectoine phytosphingosine on IL-6 was far superior to that of ectoine, demonstrating excellent anti-inflammatory and soothing effects and promoting the repair of inflamed and damaged skin. This was an unexpected discovery by the inventors.

[0058] Example 5

[0059] AQP3 cell assay for the moisturizing effect of ectoin dihydrosphingosine

[0060] Aquaporin 3 (AQP3) is a transporter protein factor on the cell membrane responsible for the transport of substances such as water, glycerol, and urea. It is mainly expressed in keratinocytes and skin fibroblasts. AQP3 not only participates in skin moisturizing and barrier function, but also plays an important role in skin damage and repair, healing, and anti-aging, and is an important guarantee for maintaining normal skin morphology and function.

[0061] Human keratinocytes HaCat cells were arranged at a density of 1×10⁻⁶.4 Cells were seeded per well in 96-well plates and incubated overnight in an incubator. After 24 hours, the supernatant was discarded, and 100 μL of different concentrations of ectoin dihydrosphingosine diluted in DMEM medium was added. The blank control group was prepared with DMEM medium without the drug. Each group had 3 replicates. The experimental groups were dried in a clean bench at a fan speed of 0.4 m / s for 20 min, and then incubated in a 5% CO2 environment at 37°C for 24 h. After the reaction, 50 μL of cell supernatant was collected, and the expression of AQP3 cytokines was detected using an AQP3 kit.

[0062] Table 7 Results of Ectoin Dihydrosphingosine Moisturizing Efficacy Test

[0063] The results are shown in Table 7 and Figure 5. Ectoycin dihydrosphingosine effectively increased the expression level of AQP3 aquaporin in a concentration-dependent manner. At concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, the expression levels were 1.15, 1.26, and 1.31 times that of the blank control group, respectively.

[0064] Example 6

[0065] Anti-photoaging test of ectoin phytosphingosine

[0066] MMP1, also known as interstitial collagenase or matrix metalloproteinase, belongs to the matrix metalloproteinase family. Its main substrate is fibrous collagen, which can degrade collagen fibers and gelatin in the extracellular matrix and alter the cellular microenvironment. MMP1 plays an important role in elastin; inhibiting MMP1 can increase collagen and elastin synthesis in fibroblasts, and reducing MMP activity can increase the rate of collagen synthesis.

[0067] HFF-1 cells were fed at a rate of 1×10 5 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded, and 100 μL of medium containing different concentrations of ectoine phytosphingosine (no drug added to the model group) was added. The negative control group received drug-free DMEM medium. Each group was divided into 3 replicates. After incubation at 37°C with 5% CO2 for 4 hours, both experimental and model groups were exposed to UVA or UVB ultraviolet radiation. The distance between the UV radiation source and the cells was 15 cm, and the UVA or UVB intensity was 200 mJ / cm². 2 The irradiation time was 1.0 h, and after irradiation, the cells were incubated in an incubator for another 24 h. Intracellular MMP-1 gene expression was detected using an MMP-1 ELISA kit.

[0068] Table 8. Results of MMP-1 expression level detection by ectoine phytosphingosine against UVA-induced resistance.

[0069] Table 9. Results of MMP-1 expression level detection for UVB-induced resistance by ectoine phytosphingosine.

[0070] The results of MMP-1 overexpression inhibition caused by UVA are shown in Table 8 and Figure 6. The MMP1 expression level in the blank control group was set to 1, and the expression level in the model group was 2.06. At the concentrations of ectoin phytosphingosine of 5 mg / L, 10 mg / L, and 20 mg / L, the MMP1 expression levels were 1.41, 1.21, and 0.79, respectively.

[0071] The results of UVB-induced MMP-1 overexpression inhibition are shown in Table 9 and Figure 6. The MMP1 expression level in the blank control group was set to 1, and the expression level in the model group was 2.32. At concentrations of ectoin phytosphingosine of 5 mg / L, 10 mg / L, and 20 mg / L, the MMP1 expression levels were 1.50, 1.20, and 0.52, respectively.

[0072] Following ultraviolet radiation, keratinocytes promote increased MMP1 expression in fibroblasts, leading to degradation of the skin's extracellular matrix and collagen, resulting in photoaging. These results indicate that ectoine phytosphingosine can inhibit MMP1 production in fibroblasts induced by ultraviolet radiation, thus playing a role in preventing photoaging.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of ectoin ceramide in cosmetics, pharmaceuticals or health products.

2. The use according to claim 1, characterized in that, The ectoine ceramide is ectoine sphingosine, ectoine dihydrosphingosine, or ectoine phytosphingosine.

3. The use according to claim 1 or 2, characterized in that, The ectoine ceramide has at least one of the following effects: skin barrier repair, tissue healing, anti-photoaging, moisturizing, and anti-inflammatory soothing.

4. Use of ectoin ceramide in the preparation of cosmetics, pharmaceuticals or health products for skin barrier repair.

5. Use of ectoin ceramide in the preparation of cosmetics, pharmaceuticals or health products for tissue healing.

6. Use of ectoin ceramide in the preparation of cosmetics, pharmaceuticals or health products for anti-photoaging.

7. Use of ectoin ceramide in the preparation of cosmetics, pharmaceuticals or health products for moisturizing purposes.

8. Use of ectoin ceramide in the preparation of cosmetics, pharmaceuticals or health products for anti-inflammatory and soothing purposes.

9. A composition comprising ectoin ceramide, said composition having at least one of the following effects: skin barrier repair, tissue healing, anti-photoaging, moisturizing, and anti-inflammatory soothing.

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