CN117902996A

CN117902996A - Ceramide E and synthesis method and application thereof

Info

Publication number: CN117902996A
Application number: CN202410043732.2A
Authority: CN
Inventors: 杨超文; 叶柳; 宋家良
Original assignee: Shenzhen Dikeman Biotechnology Co ltd
Current assignee: Shenzhen Dikeman Biotechnology Co ltd
Priority date: 2024-01-11
Filing date: 2024-01-11
Publication date: 2024-04-19

Abstract

The invention belongs to the technical field of biological medicine, and discloses ceramide E, which has the following structure: The invention also discloses a synthesis method of the ceramide E, and the ceramide E has at least one of skin barrier repairing, tissue healing, anti-inflammatory, soothing and acne removing effects, and can be used for cosmetics, health products and medicines, in particular to a cosmetic essential oil or a cosmetic anhydrous formula system. The invention designs the ceramide E compound with a novel structure, which has the advantages of economic steps of a synthetic route, simple and convenient operation, avoids the use of lengthy post-treatment process and dangerous reagent, and is suitable for large-scale production.

Description

Ceramide E and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to ceramide E and a synthesis method and application thereof.

Background

Ceramides (also known as molecular nails), which naturally occur in the skin, are very important components of the skin barrier (stratum corneum), in amounts of up to 40 to 50% by weight. In terms of chemical structure, ceramide is a sphingolipid consisting of sphingosine long-chain bases and fatty acids, in which the sphingosine moiety, the carbon chain length of the fatty acid moiety, the degree of unsaturation and the number of hydroxyl groups are all variable, so that the ceramide molecule is not the only one, and refers to a class of compounds.

The ceramide analogue is artificially synthesized ceramide, has a much lower price than natural ceramide, but can also enhance the cohesion of epidermal cells and promote the hydration of epidermis, and has the effects of improving skin barrier and improving skin water retention capacity. The class of ceramide on the market is a wide variety, most typically that of jasmine, hydroxypropyl dipalmitin MEA and delphinidin, cetyl palmitate. Compared with the problems of high melting point, indissolvable oil and water, difficult formula application and the like of natural ceramide, the ceramide has lower production cost and lower melting point, and is more convenient to be applied to the formula of the skin care cosmetics. Therefore, the ceramide has a certain market in the field of skin care products.

Therefore, in view of the wide market demand for ceramide-like compounds, it is very necessary to realize the industrial production of ceramide-like compounds by developing an economical and efficient synthesis method. In addition, the efficacy report of the ceramide-like compound is limited to moisturizing effect, and as the effect of the ceramide-like compound in more and more skin care efficacy fields is explored, the potential application fields of the ceramide-like compound can be further expanded.

Disclosure of Invention

The invention aims to provide a ceramide compound-ceramide E with a novel structure.

It is another object of the present invention to provide a method for synthesizing ceramide E.

It is another object of the present invention to provide the use of ceramide E.

Ceramide E, having the following structure:

the synthesis method of the ceramide E comprises the following steps:

s1, cetyl alcohol reacts with epoxy chloropropane and ethanolamine in turn to obtain an intermediate A

S2, intermediate A and methyl palmitate to obtain ceramide E

S1 and S2 both use continuous flow reactions.

Further, adding organic base into the S1, wherein the organic base is DBU, DBN, DMAP; and quaternary ammonium salt is added into the S1, and the quaternary ammonium salt is TBAB, TBAC, TBAI.

Further, the S1 is: adding hexadecanol into toluene, then adding organic alkali, carrying out reflux reaction for 2-6 h, and cooling to room temperature, wherein the obtained reaction liquid is used as a material I; dispersing epichlorohydrin and quaternary ammonium salt into toluene to obtain a second material; dissolving ethanolamine in toluene as a material III; pumping the first material and the second material into a microreactor, mixing the materials by a mixer, then carrying out reaction in a module 1 at the reaction temperature of 50-60 ℃, pumping the third material into the reactor to mix with effluent of the module 1, then carrying out reaction in a module 2 at the reaction temperature of 50-60 ℃, collecting effluent, and carrying out post-treatment to obtain the product.

Further, the step S2 is: adding ethanol into the intermediate A, then adding sodium ethoxide, and stirring for 20-40 min to obtain a material I; dissolving methyl palmitate in ethanol to obtain a material II, pumping the material I and the material II into a microreactor, reacting at 70-90 ℃, collecting effluent, and post-treating to obtain the product.

The use of ceramide E in cosmetics, pharmaceutical products, dietary or health care products.

The ceramide E has at least one of skin barrier repairing, tissue healing, anti-inflammatory, soothing and acne removing effects, and can be used for cosmetics, health products and medicines, in particular cosmetic essential oil or a cosmetic anhydrous formula system.

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

Ceramide E has anti-inflammatory effect; at a concentration of 7.8125mg/L, the IL-6 factor level is 0.506 times that of the LPS model group; at a concentration of 15.625mg/L, the IL-6 factor level was 0.307-fold that of the LPS model group; at a concentration of 31.25mg/L, the IL-6 factor level was 0.242 fold that of the LPS model group.

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

Propionibacterium acnes is a species of Propionibacterium, causes pathogenic bacteria of acne, and ceramide E has the purpose of inhibiting Propionibacterium acnes and plays a role of acne removal.

A composition comprising ceramide E, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient, which has skin barrier repair, tissue healing, anti-inflammatory, soothing, acne-removing effects.

As used herein, "pharmaceutically acceptable salt" means a salt of an aspect of the invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: (1) Acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with organic acids such as acetic acid, propionic acid, caproic 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-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2, 2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid; or (2) a salt formed when an acidic proton present in the parent compound is substituted.

As used herein, "hydrate" means a compound that binds to water. Binding between the compound and water includes non-covalent binding.

As used herein, "solvate" means a complex formed by a solute molecule or ion and a solvent molecule or ion.

As used herein, "isomers" means that the compounds of the present invention or salts thereof have the same chemical formula or molecular formula but different optical or spatial properties.

The term "compound of the invention" or "ceramide" includes the compound itself, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, and an isomer thereof, unless otherwise specified.

The invention has the following beneficial effects:

1. The invention designs a ceramide compound-ceramide E with a novel structure, and the structure is different from the reported ceramide.

2. The invention uses hexadecanol, epichlorohydrin and ethanolamine as starting materials, synthesizes products through continuous flow reaction, and has the following advantages: (1) The microchannel reactor has extremely large specific surface area, can increase mass and heat transfer efficiency by orders of magnitude, and reduces reaction time; (2) The reaction materials are precisely controlled by a syringe pump, so that the yield is prevented from being reduced due to the increase of side reactions caused by inaccurate local stoichiometry; (3) The microchannel reactor has better capability of removing reaction heat and cooling capability, and can avoid harsh reaction conditions such as ultralow temperature and the like; (4) The equipment occupies small area, is simple to operate, can reduce manual operation and can reduce production cost; (5) The on-line reaction volume is small, the process safety is ensured, and the method is suitable for high-risk processes.

3. The ceramide E has good effects in the aspects of skin barrier repair, tissue healing, anti-inflammatory, relieving and the like, and more importantly, the excellent performance of the ceramide E in the aspect of inhibiting the acne propionibacteria is discovered for the first time, so that the application of the ceramide E in the aspect of acne removal is expanded.

Drawings

FIG. 1 is a bar graph showing the results of the cell proliferation activity test of example 5;

FIG. 2 is the result of the cell migration ability test of example 6;

FIG. 3 is a bar graph showing the detection of IL-6 factor expression level in anti-inflammatory repair efficacy in example 7;

FIG. 4 is a bar graph showing the TRPV-1 factor mRNA expression level of the soothing efficacy test of example 8;

fig. 5 is a chart showing the detection of acne removing efficacy by propionibacterium inhibition zone of example 9.

Detailed Description

The invention will be further illustrated with reference to specific examples.

All reactions were carried out under nitrogen atmosphere. Unless otherwise indicated, the chemicals cetyl alcohol, epichlorohydrin, ethanolamine, methyl palmitate, etc. were purchased from commercial products and were not further purified. Toluene, ethanol, methanol and methylene dichloride used in the experiment are all anhydrous solvents. Thin Layer Chromatography (TLC) was performed using 60F254 silica gel plates. The silica gel column chromatography uses Qingdao ocean silica gel (particle size 0.040-0.063 mm). TLC developed using UV light (254 nm) or iodine. The NMR spectra were characterized using a Bruker DPX 400 NMR apparatus, ¹ H NMR at 400MHz, solvent deuterated chloroform, tetramethylsilane TMS as internal standard. Chemical shifts are in ppm and coupling constants are in Hz. In ¹ H NMR, δ represents a chemical shift, s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet. DBU means 1, 8-diazabicyclo [5,4,0] undecene-7, DBN means bicyclo [5.4.0] -1, 8-diaza-7-non, DMAP means 4-dimethylaminopyridine, TBAB means 4-n-butylammonium bromide, TBAC means octyltrimethylammonium chloride, TBAI tetrabutylammonium iodide. The inner diameter of the pipeline of the micro-reaction device is selected to be 0.8-2.0 mm, the volume of the pipeline is about 50-100 mL, and the length of the pipeline can be correspondingly adjusted according to the amount of the solvent used in the reaction.

Example 1

The first step: under the protection of nitrogen, 1.0eq (50 g) of hexadecanol is added into 150g of toluene, then 2.0eq (62.9 g) of DBU is added, the temperature is raised and reflux is carried out for 4 hours, then the reaction system is cooled to room temperature, and the obtained reaction liquid is taken as a material I; 1.1eq (21 g) of epichlorohydrin and 0.03eq (2 g) of TBAB were dispersed in 30g of toluene as material two; 2.0eq (24.5 g) ethanolamine was dissolved in 30g toluene as material three; pumping the first material and the second material into a pipeline of a microreactor at the same time, mixing the materials by a mixer, then carrying out reaction in a module 1, pumping the third material into the reactor at a reaction temperature of 55 ℃ to be mixed with effluent of the module 1, then carrying out reaction in a module 2, collecting effluent, cooling to room temperature, adding 200mL of water for washing, separating phases, distilling toluene under reduced pressure below 80 ℃ of an organic phase to obtain a crude product, adding the crude product into 75g of absolute methanol, cooling for crystallization, filtering, rinsing a filter cake by the absolute methanol to obtain a wet product, and carrying out reduced pressure drying on the wet product to obtain 50g of intermediate A (yield 67%).

Characterization data ：¹H 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).

And a second step of: under the protection of nitrogen, 1.0eq (50 g) of intermediate A is added into 100g of ethanol at room temperature, 1.2eq (11.4 g) of sodium ethoxide is added, and stirring is carried out for 30min, thus obtaining a material I; 1.2eq (45 g) methyl palmitate was dissolved in ethanol as material two; pumping the first material and the second material into a pipeline of a microreactor at the same time, reacting in the microreactor at the reaction temperature of 80 ℃, collecting effluent liquid, distilling ethanol under reduced pressure, adding 200mL of dichloromethane, adding 140mL of water for washing an organic phase, 140mL of 2wt% dilute hydrochloric acid water solution for washing, distilling the dichloromethane organic phase obtained after liquid separation under reduced pressure to obtain a crude product, adding 100mL of absolute ethanol into the crude product, heating to 50 ℃ for pulping, filtering while the crude product is hot to remove insoluble substances, cooling the filtrate to 10 ℃, stirring and crystallizing for 1h, filtering to obtain a wet product, and drying under reduced pressure at 55 ℃ for 8h to obtain 58.2g of ceramide E (yield 70%).

Characterization data ：¹H 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,2H),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).

Example 2

The first step: under the protection of nitrogen, 1.0eq (50 g) of hexadecanol is added into 150g of toluene, then 2.5eq (76.8 g) of DBN is added, the temperature is raised and reflux is carried out for 3 hours, then the reaction system is cooled to room temperature, and the obtained reaction liquid is taken as a material I; 1.3eq (24.8 g) epichlorohydrin and 0.04eq (3.05 g) TBAI were dispersed in 30g toluene as feed two; 1.8eq (22.7 g) ethanolamine was dissolved in 30g toluene as material three; pumping the first material and the second material into a pipeline of a microreactor at the same time, mixing the materials by a mixer, then carrying out reaction in a module 1, pumping the third material into the reactor at the reaction temperature of 52 ℃ to be mixed with effluent of the module 1, then carrying out reaction in a module 2, collecting effluent at the reaction temperature of 56 ℃, cooling to room temperature, adding 300mL of water for washing, separating phases, distilling toluene under reduced pressure below 80 ℃ of an organic phase to obtain a crude product, adding the crude product into 85g of anhydrous methanol, cooling for crystallization, filtering, rinsing a filter cake by the anhydrous methanol to obtain a wet product, and drying the wet product under reduced pressure to obtain 54g of intermediate A (yield 73%).

And a second step of: under the protection of nitrogen, 1.0eq (54 g) of intermediate A is added into 100g of ethanol at room temperature, 1.1eq (11.2 g) of sodium ethoxide is added, and stirring is carried out for 40min, thus obtaining a material I; 1.1eq (44.7 g) methyl palmitate was dissolved in ethanol as material two; pumping the first material and the second material into a pipeline of a microreactor at the same time, reacting in the microreactor at the reaction temperature of 70 ℃, collecting effluent liquid, distilling ethanol under reduced pressure, adding 250mL of dichloromethane, adding 140mL of water for washing, 140mL of 2wt% diluted hydrochloric acid water solution for washing, distilling the dichloromethane organic phase obtained after liquid separation under reduced pressure to obtain a crude product, adding 120mL of absolute ethanol into the crude product, heating to 50 ℃, pulping, filtering while the crude product is hot to remove insoluble matters, cooling the filtrate to 10 ℃, stirring and crystallizing for 1h, filtering to obtain a wet product, and drying under reduced pressure at 55 ℃ for 8h to obtain 60.6g of ceramide E (yield 67%).

Example 3

The first step: under the protection of nitrogen, 1.0eq (50 g) of hexadecanol is added into 150g of toluene, then 1.6eq (50 g) of DBU is added, the temperature is raised and reflux is carried out for 5 hours, then the reaction system is cooled to room temperature, and the obtained reaction liquid is taken as a material I; 1.2eq (22.9 g) epichlorohydrin and 0.02eq (1.14 g) TBAC were dispersed in 30g toluene as feed two; 1.5eq (18.9 g) ethanolamine was dissolved in 30g toluene as material three; pumping the first material and the second material into a pipeline of a microreactor at the same time, mixing the materials by a mixer, then carrying out reaction in a module 1, pumping the third material into the reactor at a reaction temperature of 60 ℃ to be mixed with effluent of the module 1, then carrying out reaction in a module 2, collecting effluent, cooling to room temperature, adding 250mL of water for washing, separating phases, distilling toluene under reduced pressure below 80 ℃ of an organic phase to obtain a crude product, adding the crude product into 70g of absolute methanol, cooling for crystallization, filtering, rinsing a filter cake by the absolute methanol to obtain a wet product, and carrying out reduced pressure drying on the wet product to obtain 48g of intermediate A (yield 65%).

And a second step of: under the protection of nitrogen, 1.0eq (48 g) of intermediate A is added into 100g of ethanol at room temperature, 1.4eq (12.7 g) of sodium ethoxide is added, and stirring is carried out for 30min, thus obtaining a material I; 1.4eq (50.5 g) methyl palmitate was dissolved in ethanol as material two; pumping the first material and the second material into a pipeline of a microreactor at the same time, reacting in the microreactor at the reaction temperature of 90 ℃, collecting effluent liquid, distilling ethanol under reduced pressure, adding 230mL of dichloromethane, adding 120mL of water for washing an organic phase, 120mL of 2wt% diluted hydrochloric acid water solution for washing, performing reduced pressure distillation on the dichloromethane organic phase obtained after liquid separation to obtain a crude product, adding 100mL of absolute ethanol into the crude product, heating to 50 ℃, pulping, filtering while the crude product is hot to remove insoluble substances, cooling the filtrate to 10 ℃, stirring and crystallizing for 1h, filtering to obtain a wet product, and performing reduced pressure drying at 55 ℃ for 8h to obtain 59.8g of ceramide E (yield 75%).

Example 4

The first step: under the protection of nitrogen, 1.0eq (50 g) of hexadecanol is added into 150g of toluene, then 1.8eq (45.3 g) of DMAP is added, the temperature is raised and reflux is carried out for 6 hours, then the reaction system is cooled to room temperature, and the obtained reaction liquid is taken as a material I; 1.1eq (21 g) of epichlorohydrin and 0.03eq (2 g) of TBAB were dispersed in 30g of toluene as material two; 2.2eq (27.7 g) ethanolamine was dissolved in 30g toluene as material three; pumping the first material and the second material into a pipeline of a microreactor at the same time, mixing the materials by a mixer, then carrying out reaction in a module 1, pumping the third material into the reactor at 58 ℃ to carry out mixing with effluent of the module 1, then carrying out reaction in a module 2 at 54 ℃, collecting effluent, cooling to room temperature, adding 200mL of water for washing, separating phases, distilling toluene under reduced pressure below 80 ℃ of an organic phase to obtain a crude product, adding the crude product into 75g of absolute methanol, cooling for crystallization, filtering, rinsing a filter cake by the absolute methanol to obtain a wet product, and carrying out reduced pressure drying on the wet product to obtain 53g of intermediate A (yield 71.5%).

And a second step of: under the protection of nitrogen, 1.0eq (53 g) of intermediate A is added into 100g of ethanol at room temperature, 1.2eq (12 g) of sodium ethoxide is added, and stirring is carried out for 25min, thus obtaining a material I; 1.2eq (47.8 g) methyl palmitate was dissolved in ethanol as material two; pumping the first material and the second material into a pipeline of a microreactor at the same time, reacting in the microreactor at the reaction temperature of 80 ℃, collecting effluent liquid, distilling ethanol, adding 250mL of dichloromethane, adding 150mL of water for washing an organic phase, washing 150mL of 2wt% dilute hydrochloric acid aqueous solution, performing reduced pressure distillation on the dichloromethane organic phase obtained after liquid separation to obtain a crude product, adding 120mL of absolute ethanol into the crude product, heating to 50 ℃, pulping, filtering while the crude product is hot to remove insoluble substances, cooling the filtrate to 10 ℃, stirring and crystallizing for 1h, filtering to obtain a wet product, and performing reduced pressure drying at 55 ℃ for 8h to obtain 63.5g of ceramide E (yield 72%).

Example 5

MTT method for detecting proliferation activity of compound on cell

Human keratinocyte cells HaCaT or fibroblasts L929 were seeded at a density of 1×10 ⁴ cells/well in 96-well plates and in an incubator overnight. After 24h, the supernatant was discarded, 100. Mu.L of medium containing samples of different concentrations (the product of example 1 was dissolved in DMSO-glycerol golden ratio) was added, incubation was continued for 24h, 100. Mu.L of thiazole blue (MTT) was added to each well, absorbance at 450nm was measured, and cell viability = A _{Drug delivery hole}/A_{Blank hole} X100% was calculated.

As a result, as shown in FIG. 1, the cell viability of the fibroblasts was 105.48%, 104.85%, 109.03%, 99.87%, 114.55%, 105.56%, 102.45% and 95.14% at concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000mg/L, respectively. Ceramide E has a certain growth promoting effect on fibroblasts.

Example 6

Assessment of skin barrier repair by cell migration

Principle of: when the cells grow to be fused into a single-layer state, a scratch tool is manufactured on the fused single-layer cells, the cells in the blank area are removed by mechanical force, the migration condition of the cells to the cell-free area is observed through a period of culture, and the migration capability of the cells is reflected by measuring the migration distance of the cells.

The operation steps are as follows:

1. the culture plate is streaked. Firstly, a Marker pen is used for uniformly scribing transverse lines by comparing with a straight ruler, and the transverse lines are crossed through the through holes at intervals of about 0.5 cm to 1cm, and each hole at least passes through 5 lines, so that attention lines are not too thick when scribing.

2. And (5) paving cells. About 5X 10 ⁵ fibroblasts L929 were added to the wells and the seeding principle was that the fusion rate reached 100% after overnight.

3. Cell streaking. The next day the tip is used to scratch the cell layer along the line marked on the back of the plate on the first day, perpendicular to the cell plane (the same tip is preferably used between the different wells).

4. Washing cells. After the streaking was completed, cells were washed 3 times with sterile PBS, cells that did not adhere to the wall, i.e., streaked cells at streaking, and the gap left after streaking was clearly visible, followed by replacement of fresh serum-free medium.

5. And (5) culturing and observing the cells. After the sample (product of example 1) was diluted with the medium (product concentration of example 1: 62.5 mg/L), the cells were placed in a 37℃and 5wt% CO ₂ incubator for culturing, and after 24 hours, the cells were taken out, observed with a microscope and the width of the scratch was measured, and photographed, and the healing rate was calculated using Image J software.

The results are shown in fig. 2, which shows that the scratch width of the experimental group is narrower than that of the solvent control group, indicating that ceramide E has better tissue healing ability. Ceramide E enhances the interaction between cells and extracellular matrix and between cells, thereby enhancing the migration and movement capacity of cells, improving the healing rate of cells and having good skin tissue repair activity.

Example 7

LPS induced cell method for detecting anti-inflammatory repair efficacy

Macrophages RAW were seeded at a density of 1×10 ⁴ per well in 96-well plates, placed in an incubator overnight, after 24h the supernatant was discarded, 100 μl of samples of different concentrations diluted with DMEM medium (product of example 1), negative control group was DMEM medium without sample, 3 wells per group incubated in 5wt% co ₂, 37 ℃. Lipopolysaccharide model group and experimental group were added with 10 μg/mL LPS and incubated together for 24h 2h after dosing. After the reaction is finished, cell RNA is extracted, reverse transcription is carried out, the content of IL-6 factor mRNA is tested on a fluorescent quantitative PCR instrument, and data processing analysis is carried out by a 2-delta Cp method.

The results are shown in FIG. 3, where IL-6 levels were 3.796 times the basal levels at a working concentration of 10. Mu.g/mL of LPS stimulation. Under the action of ceramide E with the concentration of 7.8125mg/L, 15.625mg/L and 31.25mg/L, the IL-6 factor level is obviously reduced, which is 0.506 times, 0.307 times and 0.242 times of LPS model group respectively, and the IL-6 expression is closely related to inflammation, which proves that the ceramide E has good anti-inflammatory effect and can promote the repair of the skin with inflammation damage.

Example 8

TRPV1 inhibition method for detecting soothing efficacy

HaCaT cells were seeded at a density of 1×10 ⁴ per well in 96-well plates, placed in an incubator overnight, after 24h the supernatant was discarded, 100 μl of samples of different concentrations diluted with DMEM medium (product of example 1), negative control was DMEM medium without sample, positive control was 0.4% trans-tetra-t-butylcyclohexanol, 3 wells per group, incubated in 5wt% co ₂ at 37 ℃. The experimental group was added with 50. Mu. Mol/L capsaicin 2h after dosing and incubated together for 24h. After the reaction is finished, cell RNA is extracted, reverse transcription is carried out, the TRPV1 factor mRNA content is tested on a fluorescent quantitative PCR instrument, and a 2-delta Cp method is used for data processing analysis.

The results are shown in FIG. 4, where TRPV1 levels were 5.759 times the basal levels at a working concentration of 50. Mu. Mol/L capsaicin stimulation. The TRPV1 factor level of 0.4% trans-tetra-tert-butylcyclohexanol in the positive control group was 0.341 fold that in the capsaicin model group, demonstrating reliable method. Under the action of ceramide E with the concentration of 7.8125mg/L, 15.625mg/L and 31.25mg/L, the TRPV1 factor level is obviously reduced, and is 0.862 times, 0.781 times and 0.706 times of that of a capsaicin model group respectively, and the TRPV1 factor is dose-dependent. While TRPV1 factor expression is closely related to the occurrence of skin sensitive stinging, which proves that ceramide E has a good soothing effect and can soothe sensitive skin.

Example 9

Preparation of propionibacterium acnes standard strain suspension: the propionibacterium acnes strain is inoculated on a blood agar plate, placed in an anaerobic incubator, placed in a constant temperature bacteria incubator, subjected to anaerobic culture at 37 ℃ for 48 hours, taken out, then the bacteria culture is manufactured into a bacteria smear, subjected to a gram staining method, and observed under a microscope.

Determination of the zone of inhibition: respectively adding samples with different concentrations into the test groups, respectively absorbing 100uL of freshly prepared propionibacterium acnes standard strain suspension by a pipetting gun, respectively adding the samples into the test groups (products of the example 1), uniformly oscillating, placing test tubes into a constant-temperature anaerobic incubator at 37 ℃ for culturing for 48 hours, respectively absorbing 1ml of suspension in each group of test tubes by a flat pouring method after the culture of each group of test tubes is finished, inoculating the suspension onto blood agar flat plates with the same number, and placing the blood agar flat plates into the constant-temperature anaerobic incubator at 37 ℃ for culturing for 48 hours; the zone of inhibition was measured and the data recorded. Penicillin 0.1. Mu.g/mL was used as a positive control.

As shown in FIG. 5 and the following table, the inhibition zone of penicillin in the positive control group is 15.60mm, which indicates that the method is reliable. Under the action of ceramide E with the concentration of 15 mug/mL, 30 mug/mL and 30 mug/mL respectively, the inhibition zone is obviously increased and is 9.28 mm, 10.34 mm and 14.54mm respectively, and the inhibition zone is dose-dependent. The propionibacterium acnes is closely related to the occurrence of acne, and the ceramide E has good acne inhibition effect and can remove acnes.

The foregoing is merely illustrative embodiments of the present invention, and the present invention is not limited thereto, and any changes or substitutions that may be easily contemplated by those skilled in the art within the scope of the present invention should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Ceramide E, having the following structure:

2. The synthesis method of the ceramide E comprises the following steps:

S2, intermediate A and methyl palmitate to obtain ceramide E

S1 and S2 both use continuous flow reactions.

3. The method of synthesis according to claim 2, wherein S1 is added with an organic base, the organic base being DBU, DBN, DMAP; and quaternary ammonium salt is added into the S1, and the quaternary ammonium salt is TBAB, TBAC, TBAI.

4. A method of synthesis according to claim 3, wherein S1 is: adding hexadecanol into toluene, then adding organic alkali, carrying out reflux reaction for 2-6 h, and cooling to room temperature, wherein the obtained reaction liquid is used as a material I; dispersing epichlorohydrin and quaternary ammonium salt into toluene to obtain a second material; dissolving ethanolamine in toluene as a material III; pumping the first material and the second material into a microreactor, mixing the materials by a mixer, then carrying out reaction in a module 1 at the reaction temperature of 50-60 ℃, pumping the third material into the reactor to be mixed with effluent of the module 1, then carrying out reaction in a module 2at the reaction temperature of 50-60 ℃, collecting effluent, and carrying out post treatment to obtain a product; the S2 is as follows: adding ethanol into the intermediate A, then adding sodium ethoxide, and stirring for 20-40 min to obtain a material I; dissolving methyl palmitate in ethanol to obtain a material II, pumping the material I and the material II into a microreactor, reacting at 70-90 ℃, collecting effluent, and post-treating to obtain the product.

5. Use of ceramide E of claim 1 in cosmetics, pharmaceuticals, dietary or health care products.

6. The use according to claim 5, wherein said ceramide E has at least one of skin barrier repair, tissue healing, anti-inflammatory, soothing, acne-removing efficacy.

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

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

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

10. A composition comprising the ceramide E of claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof as an active ingredient.