Ceramide e, and synthesis method therefor and use thereof

Abstract

The present invention belongs to the technical field of biomedicine. Disclosed is a ceramide E having the following structural formula (1). Further disclosed in the present invention is a method for synthesizing the ceramide E. The ceramide E has at least one of the effects of skin barrier repair, tissue healing, inflammation prevention, soothing and acne removal, and can be used in cosmetics, health care products and medicines, especially in essential oils or waterless formula systems of cosmetics. A ceramide E compound having a new structure is designed in the present invention. The synthetic route has economical steps and is easy to operate, thereby avoiding a lengthy post-treatment process and the use of a hazardous reagent. The synthetic route is suitable for large-scale production.

Description

Ceramide E and its synthesis method and use Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to ceramide E and its synthesis method and use. Background Art

[0002] Ceramide (also known as molecular nail) is naturally present in the skin and is a crucial component of the skin barrier (stratum corneum), with a content of up to 40-50% by weight. Chemically, ceramide is a sphingolipid composed of a long-chain sphingosine base and fatty acids. The carbon chain length, degree of unsaturation, and number of hydroxyl groups in the sphingosine and fatty acid moieties can vary, so ceramide molecules are not unique; they refer to a class of compounds.

[0003] Ceramide analogs are synthetic ceramides that are much cheaper than natural ceramides. They can also enhance epidermal cell cohesion and promote epidermal hydration, improving the skin barrier and increasing the skin's water retention capacity. There are many types of ceramide analogs on the market, the most representative of which are Amorepacific's Hydroxypropyl Bispalmitamide MEA and Symrise's N-Palmitoyl Hydroxyproline Cetyl Ester. Compared to the high melting point, poor solubility in oils and water, and difficulty in formulating and applying natural ceramides, ceramide analogs have lower production costs and lower melting points, making them more convenient for use in skincare cosmetics. As a result, ceramide analogs have a certain market share in the skincare field.

[0004] Therefore, considering the widespread market demand for ceramide-like compounds, it is essential to develop cost-effective and efficient synthetic methods to achieve their industrial production. Furthermore, while previous reports on the efficacy of ceramide-like compounds have been limited to moisturizing, as their roles in a growing number of skincare applications are discovered, their potential applications are likely to expand further. Summary of the Invention

[0005] The purpose of the present invention is to provide a ceramide-like compound with a novel structure - ceramide E.

[0006] Another object of the present invention is to provide a method for synthesizing ceramide E.

[0007] Another object of the present invention is to provide uses of ceramide E.

[0008] Ceramide E, which has the following structure:

[0009] The synthesis method of ceramide E comprises the following steps:

[0010] S1, hexadecanol react with epichlorohydrin and ethanolamine in sequence to obtain intermediate A

[0011] S2, intermediate A and methyl palmitate to obtain ceramide E

[0012] Both S1 and S2 adopted continuous flow reaction.

[0013] Furthermore, an organic base is added to the S1, and the organic base is DBU, DBN, or DMAP; a quaternary ammonium salt is added to the S1, and the quaternary ammonium salt is TBAB, TBAC, or TBAI.

[0014] Furthermore, the S1 is: adding hexadecanol to toluene, then adding an organic base, reflux reacting for 2 to 6 hours, cooling to room temperature, and the resulting reaction liquid is used as material one; epichlorohydrin and quaternary ammonium salt are dispersed in toluene as material two; ethanolamine is dissolved in toluene as material three; material one and material two are pumped into a microreactor, mixed in a mixer, and reacted in module 1 at a reaction temperature of 50 to 60°C, material three is pumped in to mix with the effluent from module 1, and then reacted in module 2 at a reaction temperature of 50 to 60°C, the effluent is collected, and post-processed to obtain the product.

[0015] Furthermore, the S2 is: adding intermediate A to ethanol, then adding sodium ethoxide, and stirring for 20 to 40 minutes to obtain material one; dissolving methyl palmitate in ethanol to obtain material two, pumping material one and material two into a microreactor, the reaction temperature is 70 to 90° C., collecting the effluent, and post-processing to obtain the product.

[0016] Use of ceramide E in cosmetics, medicines, dietary products or health products.

[0017] Ceramide E has at least one of the following effects: skin barrier repair, tissue healing, anti-inflammatory, soothing, and anti-acne. It can be used in cosmetics, health products, and medicines, especially cosmetic essence oils or cosmetic anhydrous formula systems.

[0018] Specifically, ceramide E has a skin barrier repair effect; when the concentration of ceramide E is 7.8125 mg / L, the cell viability is above 105.48%; when the concentration of ceramide E is 15.625 mg / L, the cell viability is above 104.85%; when the concentration of ceramide E is 31.25 mg / L, the cell viability is above 109.03%; when the concentration of ceramide E is 62.5 mg / L, the cell viability is above 99.87%; when the concentration of ceramide E is 125 mg / L, the cell viability is above 114.55%; when the concentration of ceramide E is 250 mg / L, the cell viability is above 105.56%; when the concentration of ceramide E is 500 mg / L, the cell viability is above 102.45%; when the concentration of ceramide E is 1000 mg / L, the cell viability is above 95.14%.

[0019] Ceramide E has anti-inflammatory effects; when the concentration of ceramide E is 7.8125 mg / L, the IL-6 factor level is 0.506 times that of the LPS model group; when the concentration of ceramide E is 15.625 mg / L, the IL-6 factor level is 0.307 times that of the LPS model group; when the concentration of ceramide E is 31.25 mg / L, the IL-6 factor level is 0.242 times that of the LPS model group.

[0020] Ceramide E has a soothing effect; when the concentration of ceramide E is 7.8125 mg / L, the TRPV1 factor level is 0.862 times that of the capsaicin model group; when the concentration of ceramide E is 15.625 mg / L, the TRPV1 factor level is 0.781 times that of the capsaicin model group; when the concentration of ceramide E is 31.25 mg / L, the TRPV1 factor level is 0.706 times that of the capsaicin model group.

[0021] Propionibacterium acnes is a species of the genus Propionibacterium, a pathogen that causes acne. Ceramide E has the function of inhibiting Propionibacterium acnes and plays an acne-removing role.

[0022] A composition comprising ceramide E, its isomers, its pharmaceutically acceptable salts, its hydrates or solvates as an active ingredient, and the composition has skin barrier repair, tissue healing, anti-inflammatory, soothing and acne-removing effects.

[0023] As used herein, "pharmaceutically acceptable salt" means a salt of one aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with inorganic acids or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, hydroxysuccinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid; or (2) salts formed when the acidic proton present in the parent compound is substituted.

[0024] As used herein, "hydrate" refers to a compound that is bound to water. The binding between the compound and water includes non-covalent binding.

[0025] As used herein, "solvate" means a complex formed by solute molecules or ions and solvent molecules or ions.

[0026] As used herein, "isomer" means that the compound of the present invention or a salt thereof has the same chemical formula or molecular formula but different optical or steric properties.

[0027] Unless otherwise indicated, the term "compound of the present invention" or "ceramide" includes the compound itself, its pharmaceutically acceptable salt, its hydrate, its solvate, and its isomer.

[0028] The present invention has the following beneficial effects:

[0029] 1. The present invention designs a ceramide-like compound with a novel structure, ceramide E, which is different from the previously reported ceramides.

[0030] 2. The present invention uses hexadecanol, epichlorohydrin and ethanolamine as starting materials and synthesizes products through continuous flow reaction, which has the following advantages: (1) The microchannel reactor has a very large specific surface area, which can increase the mass transfer and heat transfer efficiency by orders of magnitude and reduce the reaction time; (2) The reaction materials are precisely controlled by the injection pump, avoiding the increase of side reactions caused by local stoichiometric inaccuracies and the resulting decrease in yield; (3) The microchannel reactor has good reaction heat removal and cooling capabilities, which can avoid harsh reaction conditions such as ultra-low temperatures; (4) The equipment occupies a small area and is simple to operate, which can reduce manual operations and reduce production costs; (5) The online reaction volume is small, ensuring process safety, and is suitable for high-risk processes.

[0031] 3. Ceramide E has shown good effects in skin barrier repair, tissue healing, anti-inflammation, and soothing. More importantly, the excellent performance of ceramide E in inhibiting Propionibacterium acnes was discovered for the first time, which will expand the application of ceramide E in acne treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a bar graph showing the results of the cell proliferation activity test in Example 5;

[0033] FIG2 is the cell migration ability test results of Example 6;

[0034] FIG3 is a bar graph showing the expression of IL-6 factor in the anti-inflammatory and repair efficacy test of Example 7;

[0035] FIG4 is a bar graph showing the mRNA expression of TRPV-1 factor in the soothing efficacy test of Example 8;

[0036] FIG5 is a diagram showing the inhibition zone of Propionibacterium acnes in the acne-removing efficacy test of Example 9. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to specific embodiments.

[0038] All reactions were carried out under a nitrogen atmosphere. Unless otherwise stated, chemicals such as hexadecanol, epichlorohydrin, ethanolamine, and methyl palmitate were purchased from commercial products and were not further purified. The toluene, ethanol, methanol, and dichloromethane used in the experiment were all anhydrous solvents. Thin layer chromatography (TLC) used 60F254 silica gel plates. Silica gel column chromatography used Qingdao Marine Silica Gel (particle size 0.040-0.063 mm). TLC color development used UV light (254 nm) or iodine. NMR spectra were characterized using a Bruker DPX 400 nuclear magnetic resonance instrument. 1 H NMR was measured at 400 MHz, the solvent was deuterated chloroform, and tetramethylsilane (TMS) was used as the internal standard. The units of chemical shifts are ppm, and the units of coupling constants are Hz. 1In H NMR, δ represents chemical shift, s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet. DBU refers to 1,8-diazacyclo[5,4,0]undecene-7, DBN refers to bicyclo[5.4.0]-1,8-diaza-7-nonane, DMAP refers to 4-dimethylaminopyridine, TBAB refers to 4-n-butylammonium bromide, TBAC refers to octyltrimethylammonium chloride, and TBAI refers to tetrabutylammonium iodide. The inner diameter of the tubing in the microreactor is 0.8–2.0 mm, and the tubing volume is approximately 50–100 mL. The tubing length can be adjusted based on the amount of solvent used in the reaction.

[0039] Example 1

[0040] Step 1: Under nitrogen protection, 1.0eq (50g) of hexadecanol was added to 150g of toluene, and then 2.0eq (62.9g) of DBU was added. The temperature was raised to reflux for 4h, and then the reaction system was cooled to room temperature. The resulting reaction solution was used as material 1; 1.1eq (21g) of epichlorohydrin and 0.03eq (2g) of TBAB were dispersed in 30g of toluene as material 2; 2.0eq (24.5g) of ethanolamine was dissolved in 30g of toluene as material 3; Material 1 and material 2 were simultaneously pumped into a micro- In the pipeline of the reactor, after mixing in the mixer, the reaction is carried out in module 1 at a reaction temperature of 55°C. Material three is pumped in and mixed with the effluent from module 1, and then the reaction is carried out in module 2 at a reaction temperature of 55°C. The effluent is collected, cooled to room temperature, washed with 200 mL of water, and the phases are separated. Toluene is distilled under reduced pressure below 80°C in the organic phase to obtain a crude product. The crude product is added to 75 g of anhydrous methanol, cooled for crystallization, filtered, and the filter cake is rinsed with anhydrous methanol to obtain a wet product. The wet product is dried under reduced pressure to obtain 50 g of intermediate A (yield 67%).

[0041] Characterization data: 1 H NMR(400MHz,Chloroform-d)δ3.91(dq,J=8.1,3.9Hz,1H),3.68(t,J=5.0Hz,2H),3.53–3.32(m,5H),2.79(t,J= 5.1Hz, 3H), 2.69 (qd, J = 12.1, 5.9Hz, 3H), 1.59 (t, J = 6.9Hz, 2H), 1.28 (d, J = 12.5Hz, 26H), 0.89 (t, J = 6.6Hz, 3H).

[0042] Step 2: Under nitrogen protection, 1.0eq (50g) of intermediate A was added to 100g of ethanol at room temperature, 1.2eq (11.4g) of sodium ethoxide was added, and stirred for 30min as material one; 1.2eq (45g) of methyl palmitate was dissolved in ethanol as material two; material one and material two were simultaneously pumped into the pipeline of the microreactor, reacted in the microreactor, the reaction temperature was 80°C, the effluent was collected, ethanol was distilled under reduced pressure, 200mL of dichloromethane was added, the organic phase was washed with 140mL of water, and 140mL of 2wt% dilute hydrochloric acid aqueous solution was washed, the dichloromethane organic phase obtained after separation was distilled under reduced pressure to obtain a crude product, the crude product was added with 100mL of anhydrous ethanol and heated to 50°C for slurrying, and the insoluble matter was removed by hot filtration. The filtrate was cooled to 10°C and stirred for crystallization for 1h, and then filtered to obtain a wet product, which was dried under reduced pressure at 55°C for 8h to obtain 58.2g of ceramide E (yield 70%).

[0043] Characterization data: 1 H NMR(400MHz,Chloroform-d)δ4.11(ddq,J=8.6,5.9,3.0Hz,0.6H),3.96(dt,J=8.9,4.3Hz,0.4H),3.90–3.70(m,2H),3.63(ddd,J=14.4,7.7,3.6Hz,2 H),3.57–3.32(m,7.4H),3.29(dd,J=14.2,8.1Hz,0.6H),2.46–2.28(m,2H ), 1.59 (dp, J=27.9, 7.2Hz, 4H), 1.33–1.26 (m, 50H), 0.88 (t, J=6.7Hz, 6H).

[0044] Example 2

[0045] Step 1: Under nitrogen protection, 1.0eq (50g) of hexadecanol was added to 150g of toluene, and then 2.5eq (76.8g) of DBN was added, and the temperature was raised to reflux for 3h, and then the reaction system was cooled to room temperature. The resulting reaction solution was used as material one; 1.3eq (24.8g) of epichlorohydrin and 0.04eq (3.05g) of TBAI were dispersed in 30g of toluene as material two; 1.8eq (22.7g) of ethanolamine was dissolved in 30g of toluene as material three; material one and material two were simultaneously The mixture was pumped into the pipeline of the microreactor, mixed in the mixer and reacted in module 1 at a reaction temperature of 52°C. Material three was pumped in and mixed with the effluent from module 1, and then reacted in module 2 at a reaction temperature of 56°C. The effluent was collected, cooled to room temperature, washed with 300 mL of water, and the phases were separated. Toluene was distilled under reduced pressure below 80°C from the organic phase to obtain a crude product. The crude product was added to 85 g of anhydrous methanol, cooled for crystallization, filtered, and the filter cake was rinsed with anhydrous methanol to obtain a wet product. The wet product was dried under reduced pressure to obtain 54 g of intermediate A (yield 73%).

[0046] Step 2: Under nitrogen protection, 1.0eq (54g) of intermediate A was added to 100g of ethanol at room temperature, 1.1eq (11.2g) of sodium ethoxide was added, and stirred for 40min as material one; 1.1eq (44.7g) of methyl palmitate was dissolved in ethanol as material two; material one and material two were simultaneously pumped into the pipeline of the microreactor, reacted in the microreactor, the reaction temperature was 70°C, the effluent was collected, ethanol was distilled under reduced pressure, 250mL of dichloromethane was added, the organic phase was washed with 140mL of water, and washed with 140mL of 2wt% dilute hydrochloric acid aqueous solution. The dichloromethane organic phase obtained after separation was distilled under reduced pressure to obtain a crude product, the crude product was added with 120mL of anhydrous ethanol and heated to 50°C for pulping, and the insoluble matter was removed by hot filtration. The filtrate was cooled to 10°C and stirred for crystallization for 1h, and then filtered to obtain a wet product, which was dried under reduced pressure at 55°C for 8h to obtain 60.6g of ceramide E (yield 67%).

[0047] Example 3

[0048] Step 1: Under nitrogen protection, 1.0eq (50g) of hexadecanol was added to 150g of toluene, and then 1.6eq (50g) of DBU was added. The temperature was raised to reflux for 5h, and then the reaction system was cooled to room temperature. The resulting reaction solution was used as material 1; 1.2eq (22.9g) of epichlorohydrin and 0.02eq (1.14g) of TBAC were dispersed in 30g of toluene as material 2; 1.5eq (18.9g) of ethanolamine was dissolved in 30g of toluene as material 3; Material 1 and material 2 were pumped simultaneously. The mixture was introduced into the pipeline of the microreactor, mixed in the mixer and reacted in module 1 at a reaction temperature of 60°C. Material three was pumped in and mixed with the effluent from module 1, and then reacted in module 2 at a reaction temperature of 54°C. The effluent was collected, cooled to room temperature, washed with 250 mL of water, and the phases were separated. Toluene was distilled under reduced pressure below 80°C in the organic phase to obtain a crude product. The crude product was added to 70 g of anhydrous methanol, cooled for crystallization, filtered, and the filter cake was rinsed with anhydrous methanol to obtain a wet product. The wet product was dried under reduced pressure to obtain 48 g of intermediate A (yield 65%).

[0049] Step 2: Under nitrogen protection, 1.0eq (48g) of intermediate A was added to 100g of ethanol at room temperature, 1.4eq (12.7g) of sodium ethoxide was added, and stirred for 30min as material one; 1.4eq (50.5g) of methyl palmitate was dissolved in ethanol as material two; material one and material two were simultaneously pumped into the pipeline of the microreactor and reacted in the microreactor at a reaction temperature of 90°C. The effluent was collected, ethanol was distilled under reduced pressure, 230mL of dichloromethane was added, 120mL of water was added to the organic phase, and 120mL of 2wt% dilute hydrochloric acid aqueous solution was washed. The dichloromethane organic phase obtained after separation was distilled under reduced pressure to obtain a crude product. The crude product was added with 100mL of anhydrous ethanol and heated to 50°C for slurrying. The insoluble matter was removed by hot filtration. The filtrate was cooled to 10°C and stirred for crystallization for 1h, then filtered to obtain a wet product, which was dried under reduced pressure at 55°C for 8h to obtain 59.8g of ceramide E (yield 75%).

[0050] Example 4

[0051] Step 1: Under nitrogen protection, 1.0eq (50g) of hexadecanol was added to 150g of toluene, and then 1.8eq (45.3g) of DMAP was added. The temperature was raised to reflux for 6h, and then the reaction system was cooled to room temperature. The resulting reaction solution was used as material 1; 1.1eq (21g) of epichlorohydrin and 0.03eq (2g) of TBAB were dispersed in 30g of toluene as material 2; 2.2eq (27.7g) of ethanolamine was dissolved in 30g of toluene as material 3; Material 1 and material 2 were simultaneously pumped into a micro- In the pipeline of the reactor, after mixing in the mixer, the reaction is carried out in module 1 at a reaction temperature of 58°C. Material three is pumped in and mixed with the effluent from module 1, and then the reaction is carried out in module 2 at a reaction temperature of 54°C. The effluent is collected, cooled to room temperature, washed with 200 mL of water, and the phases are separated. Toluene is distilled under reduced pressure below 80°C in the organic phase to obtain a crude product. The crude product is added to 75 g of anhydrous methanol, cooled for crystallization, filtered, and the filter cake is rinsed with anhydrous methanol to obtain a wet product. The wet product is dried under reduced pressure to obtain 53 g of intermediate A (yield 71.5%).

[0052] Step 2: Under nitrogen protection, 1.0eq (53g) of intermediate A was added to 100g of ethanol at room temperature, 1.2eq (12g) of sodium ethoxide was added, and stirred for 25min as material one; 1.2eq (47.8g) of methyl palmitate was dissolved in ethanol as material two; material one and material two were simultaneously pumped into the pipeline of the microreactor and reacted in the microreactor at a reaction temperature of 80°C. The effluent was collected, ethanol was distilled, 250mL of dichloromethane was added, and the organic phase was washed with 150mL of water and 150mL of 2wt% dilute hydrochloric acid solution. The dichloromethane organic phase obtained after separation was distilled under reduced pressure to obtain a crude product. The crude product was added with 120mL of anhydrous ethanol and heated to 50°C for pulping. The insoluble matter was removed by hot filtration. The filtrate was cooled to 10°C and stirred for crystallization for 1h, then filtered to obtain a wet product, which was dried under reduced pressure at 55°C for 8h to obtain 63.5g of ceramide E (yield 72%).

[0053] Example 5

[0054] MTT assay to detect the effects of compounds on cell proliferation

[0055] Human keratinocytes HaCaT or fibroblasts L929 were cultured at 1×10 4 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated overnight in an incubator. After 24 hours, the supernatant was discarded and 100 μL of culture medium containing samples of different concentrations (the product of Example 1 was dissolved in a DMSO-glycerol golden ratio) was added. After further incubation for 24 hours, 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 the cell survival rate was calculated as A. 给药孔 / A 空白孔 ×100%.

[0056] The results are shown in Figure 1. For fibroblasts, the cell viability at ceramide E concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 mg / L was 105.48%, 104.85%, 109.03%, 99.87%, 114.55%, 105.56%, 102.45%, and 95.14%, respectively. Ceramide E has a certain growth-promoting effect on fibroblasts.

[0057] Example 6

[0058] Cell migration to assess skin barrier repair

[0059] Principle: When cells grow to a fused monolayer state, a scratch tool is used to create a blank area on the fused monolayer of cells. The cells in the blank area are removed by mechanical force. After a period of culture, the migration of cells to the cell-free area is observed. The migration distance of the cells is measured to reflect the migration ability of the cells.

[0060] Steps:

[0061] 1. Mark the culture plate. First, use a marker pen and a ruler to evenly mark horizontal lines on the back of the 6-well plate, approximately every 0.5 to 1 cm, across the wells. Make at least 5 lines per well. Be careful not to make the lines too thick.

[0062] 2. Add about 5×10 cells to the well. 5 L929 fibroblasts were seeded when the confluence rate reached 100% overnight.

[0063] 3. Streak the cells. On the second day, use the pipette tip, perpendicular to the cell plane, to scratch the cell layer along the line drawn on the back of the plate on the first day (it is best to use the same pipette tip between different wells).

[0064] 4. Wash the cells. After the scratch is completed, wash the cells three times with sterile PBS to remove the non-adherent cells, that is, the cells that were scratched during the scratching process. The gaps left after the scratching are clearly visible, and then replace with fresh serum-free medium.

[0065] 5. Cell Culture and Observation: The sample (product of Example 1) was diluted with culture medium (concentration of the product of Example 1 was 62.5 mg / L) and added to a cell culture dish. The cells were cultured in a 37°C, 5 wt% CO2 incubator. After 24 hours, the cells were removed, observed under a microscope, and the width of the scratch was measured. The scratches were photographed, and the healing rate was calculated using Image J software.

[0066] The results, as shown in Figure 2, show that the scratch width in the experimental group was narrower than in the solvent control group, indicating that ceramide E has better tissue healing ability. Ceramide E enhances interactions between cells and the extracellular matrix, and between cells, thereby enhancing cell migration and motility, increasing the cell healing rate, and demonstrating excellent skin tissue repair activity.

[0067] Example 7

[0068] LPS-induced cell assay to detect anti-inflammatory and repair effects

[0069] RAW macrophages were cultured at a density of 1 × 10 4 Each well was seeded in a 96-well plate and placed in an incubator to adhere overnight. After 24 hours, the supernatant was discarded and 100 μL of samples (products of Example 1) diluted in DMEM medium at different concentrations were added. The negative control group was a DMEM medium without sample. Each group had 3 replicates and was incubated in a 5wt% CO2, 37°C environment. 2 hours after administration, 10 μg / mL LPS was added to the lipopolysaccharide model group and the experimental group and incubated together for 24 hours. After the reaction, cell RNA was extracted, reverse transcribed, and the IL-6 factor mRNA content was tested on a fluorescent quantitative PCR instrument. The data were processed and analyzed by the 2^-ΔCp method.

[0070] The results are shown in Figure 3. Under LPS stimulation at a working concentration of 10 μg / mL, IL-6 levels were 3.796 times the basal level. Under the action of ceramide E at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, IL-6 levels were significantly reduced, reaching 0.506, 0.307, and 0.242 times that of the LPS model group, respectively, in a dose-dependent manner. IL-6 expression is closely related to the occurrence of inflammation, demonstrating that ceramide E has a good anti-inflammatory effect and can promote the repair of inflammation-damaged skin.

[0071] Example 8

[0072] TRPV1 inhibition assay for soothing efficacy

[0073] HaCaT cells were cultured at a density of 1×10 4Cells were seeded in 96-well plates and placed in an incubator overnight. After 24 hours, the supernatant was discarded and 100 μL of samples (products of Example 1) diluted in DMEM medium at different concentrations were added. The negative control group was DMEM medium without sample, and the positive control group was 0.4% trans-tetra-tert-butylcyclohexanol. Three replicates were added to each group and incubated in a 5wt% CO2, 37°C environment. 2 hours after administration, 50 μmol / L capsaicin was added to the experimental group and incubated together for 24 hours. After the reaction, cell RNA was extracted, reverse transcribed, and the TRPV1 factor mRNA content was tested on a fluorescent quantitative PCR instrument. Data processing and analysis were performed by the 2^-ΔCp method.

[0074] The results are shown in Figure 4. Under stimulation with capsaicin at a working concentration of 50 μmol / L, TRPV1 levels were 5.759 times the basal level. The TRPV1 factor level in the positive control group, 0.4% trans-tetra-tert-butylcyclohexanol, was 0.341 times that of the capsaicin model group, demonstrating the reliability of the method. Under the action of ceramide E at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, TRPV1 factor levels were significantly reduced, reaching 0.862, 0.781, and 0.706 times that of the capsaicin model group, respectively, in a dose-dependent manner. TRPV1 factor expression is closely correlated with the occurrence of skin sensitivity and tingling, demonstrating that ceramide E has a good soothing effect and can soothe sensitive skin.

[0075] Example 9

[0076] Preparation of standard strain suspension of Propionibacterium acnes: Inoculate the Propionibacterium acnes strain on a blood agar plate, place it in an anaerobic incubator, and then place it in a constant temperature bacterial incubator for anaerobically incubation at 37°C for 48 hours. Then make a bacterial smear of the bacterial culture, perform Gram staining, and observe the basic morphology of the bacteria under a microscope.

[0077] Determination of inhibition zones: Samples of varying concentrations were added to the experimental groups. Using a pipette, 100 μL of a freshly prepared suspension of a standard strain of Propionibacterium acnes was added to each experimental group (the product of Example 1). The suspension was shaken evenly and the tubes were incubated in a 37°C anaerobic incubator for 48 hours. After completion of the incubation period, 1 mL of the suspension from each tube was plated onto blood agar plates of the same number and incubated in a 37°C anaerobic incubator for 48 hours. The inhibition zones were measured and the data recorded. 0.1 μg / mL penicillin was used as a positive control.

[0078] The results are shown in Figure 5 and the table below. The inhibition zone for penicillin in the positive control group was 15.60 mm, demonstrating the reliability of the method. At concentrations of 15 μg / mL, 30 μg / mL, and 30 μg / mL, the inhibition zone significantly increased to 9.28, 10.34, and 14.54 mm, respectively, in a dose-dependent manner. Propionibacterium acnes is closely associated with the development of acne, demonstrating that ceramide E has a potent inhibitory effect on acne and can be used to treat it.

[0079] 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 changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Ceramide E, which has the following structure:

2. Method for synthesizing ceramide E, comprising the following steps: S1. Cetyl alcohol reacts with epichlorohydrin and ethanolamine in sequence to obtain intermediate A S2, intermediate A and methyl palmitate are used to obtain ceramide E Both S1 and S2 adopt continuous flow reaction.

3. The synthesis method according to claim 2, characterized in that, An organic base is added in S1, and the organic base is DBU, DBN, or DMAP; a quaternary ammonium salt is added in S1, and the quaternary ammonium salt is TBAB, TBAC, or TBAI.

4. The synthesis method according to claim 3, characterized in that, S1 is as follows: Add hexadecanol into toluene, then add the organic base, and reflux for 2 - 6 h. Cool to room temperature, and use the obtained reaction solution as Material 1; Disperse epichlorohydrin and the quaternary ammonium salt in toluene as Material 2; Dissolve ethanolamine in toluene as Material 3; Pump Material 1 and Material 2 into a microreactor, mix them through a mixer, and then react in Module 1 at a reaction temperature of 50 - 60 °C. Pump in Material 3 to mix with the effluent from Module 1, and then react in Module 2 at a reaction temperature of 50 - 60 °C. Collect the effluent and perform post-treatment to obtain the product; S2 is as follows: Add intermediate A into ethanol, then add sodium ethoxide, and stir for 20 - 40 min as Material 1; Dissolve methyl palmitate in ethanol as Material 2. Pump Material 1 and Material 2 into a microreactor, and react at a reaction temperature of 70 - 90 °C. Collect the effluent and perform post-treatment to obtain the product.

5. Use of the ceramide E according to claim 1 in cosmetics, pharmaceuticals, dietary foods, or health products.

6. The use according to claim 5, characterized in that, The ceramide E has at least one of the effects of skin barrier repair, tissue healing, anti-inflammation, soothing, and acne treatment.

7. The use according to claim 5, characterized in that, The ceramide E has a skin barrier repair effect; when the concentration of the ceramide E is 7.8125 mg / L, the cell viability is above 105.48%; when the concentration of the ceramide E is 15.625 mg / L, the cell viability is above 104.85%; when the concentration of the ceramide E is 31.25 mg / L, the cell viability is above 109.03%; when the concentration of the ceramide E is 62.5 mg / L, the cell viability is above 99.87%; when the concentration of the ceramide E is 125 mg / L, the cell viability is above 114.55%; when the concentration of the ceramide E is 250 mg / L, the cell viability is above 105.56%; when the concentration of the ceramide E is 500 mg / L, the cell viability is above 102.45%; when the concentration of the ceramide E is 1000 mg / L, the cell viability is above 95.14%.

8. The use according to claim 5, characterized in that, The ceramide E has an anti-inflammatory effect; when the concentration of the ceramide E is 7.8125 mg / L, the level of IL-6 factor is 0.506 times that of the LPS model group; when the concentration of the ceramide E is 15.625 mg / L, the level of IL-6 factor is 0.307 times that of the LPS model group; when the concentration of the ceramide E is 31.25 mg / L, the level of IL-6 factor is 0.242 times that of the LPS model group.

9. The use according to claim 5, wherein The ceramide E has a soothing effect; when the concentration of the ceramide E is 7.8125 mg / L, the level of the TRPV1 factor is 0.862 times that of the capsaicin model group; when the concentration of the ceramide E is 15.625 mg / L, the level of the TRPV1 factor is 0.781 times that of the capsaicin model group; when the concentration of the ceramide E is 31.25 mg / L, the level of the TRPV1 factor is 0.706 times that of the capsaicin model group.

10. A composition comprising, as an active ingredient, the ceramide E of claim 1, its isomers, its pharmaceutically acceptable salts, its hydrates or its solvates.

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