CN115974716A - Cottonseed oil ceramide and synthesis method and application thereof - Google Patents
Cottonseed oil ceramide and synthesis method and application thereof Download PDFInfo
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- CN115974716A CN115974716A CN202310051764.2A CN202310051764A CN115974716A CN 115974716 A CN115974716 A CN 115974716A CN 202310051764 A CN202310051764 A CN 202310051764A CN 115974716 A CN115974716 A CN 115974716A
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- ceramide
- cottonseed oil
- acid
- fatty acid
- oil fatty
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- 229940106189 ceramide Drugs 0.000 title claims abstract description 170
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 title claims abstract description 161
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 title claims abstract description 161
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 title claims abstract description 161
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 title claims abstract description 161
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- 238000001308 synthesis method Methods 0.000 title description 3
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- AERBNCYCJBRYDG-UHFFFAOYSA-N D-ribo-phytosphingosine Natural products CCCCCCCCCCCCCCC(O)C(O)C(N)CO AERBNCYCJBRYDG-UHFFFAOYSA-N 0.000 claims abstract description 11
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Abstract
The invention belongs to the technical field of biological medicines, and discloses cottonseed oil ceramide, which is obtained by reacting cottonseed oil fatty acid with a sphingoid compound, wherein the sphingoid compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. The cottonseed oil ceramide has excellent performances in the aspects of repairing natural barriers of skin, resisting inflammation, healing tissues, resisting aging and the like, and has wide application prospects in the fields of cosmetics, health products, biological medicines and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to cottonseed oil ceramide, a synthesis method and application thereof.
Background
Ceramide (also called molecular nail) naturally exists in skin, is a very important component of skin barrier (stratum corneum) and is up to 40-50 wt%, and is a sphingolipid consisting of sphingoid long-chain bases and fatty acids, wherein the carbon chain length, unsaturation degree and hydroxyl number of the sphingosine part and the fatty acid part can be changed, and the Ceramide represents a compound. Ceramides exhibit excellent properties in regulating skin barrier function, restoring skin moisture, and enhancing adhesion between skin keratinocytes, etc.
Due to the importance of ceramides, many cosmetic and pharmaceutical companies are researching and developing corresponding products. The natural plant-derived ceramide can form an effective skin barrier to prevent water loss and resist external damage due to the characteristics of more sustainable and more environment-friendly raw material sources and the similar components of the ceramide and the ceramide of the skin, and can become a next-generation environment-friendly, safe and reliable ceramide product.
The cottonseed oil is oil obtained by squeezing cotton seeds as a raw material, contains a large amount of fatty acids necessary for a human body, and comprises the following components: linoleic acid, oleic acid, palmitic acid. The refined cottonseed oil is orange yellow or brown, has high nutritive value, is easy to be absorbed by human body, and has absorption rate up to 98%. The cottonseed oil has effects of promoting metabolism, resisting oxidation and aging. In addition, since the linoleic acid content in cottonseed oil is particularly high, it has a good effect of suppressing the increase in blood cholesterol. Meanwhile, the health food contains a large amount of grease, and can play a role in clearing intestines, detoxifying and relieving constipation.
Disclosure of Invention
The invention aims to provide ceramide synthesized by utilizing cottonseed oil fatty acid of plant origin.
Another object of the present invention is to provide a method for synthesizing cottonseed oil ceramide, which uses cottonseed oil fat or cottonseed oil fatty acid, which are easily available from natural plants, as a raw material.
Another object of the present invention is to provide the use of ceramide in cottonseed oil.
In order to achieve one of the purposes, the invention adopts the following technical scheme:
in a first aspect of the invention, a cottonseed oil ceramide, which is derived from the reaction of cottonseed oil fatty acids with a sphingoid compound selected from the group consisting of sphingosine, phytosphingosine, dihydrosphingosine.
The reaction can be a chemical synthesis reaction (as detailed below), or a microbial fermentation method, namely, pichia pastoris or saccharomyces cerevisiae is used for fermentation under certain environment to obtain sphingoid compounds, and then fatty acid is added to finally obtain ceramide; or the cottonseed oil is used as a raw material, and a proper strain is selected for fermentation to obtain the cottonseed oil ceramide.
Sphingosine refers to 2-amino-4-octadecene-1, 3-diol, phytosphingosine refers to 2-amino-octadecane-1, 3, 4-triol, and dihydrosphingosine refers to 2-amino-octadecane-1, 3-diol.
Further, the cottonseed oil fatty acid is obtained by hydrolyzing cottonseed oil fat.
Furthermore, the cottonseed oil fatty acid contains 55-80 wt% of linoleic acid.
Further, the cottonseed oil fatty acid contains 10 to 25wt% palmitic acid.
Further, the cottonseed oil fatty acid contains 6-18 wt% of oleic acid.
Further, the cottonseed oil fatty acid contains 1-3 wt% of stearic acid.
Furthermore, the cottonseed oil fatty acid contains 0.1-1 wt% of arachidic acid.
Further, the cottonseed oil fatty acid contains 0.02 to 1wt% of palmitoleic acid.
Furthermore, the cottonseed oil fatty acid contains 0.01-1 wt% of linolenic acid.
The composition of the cottonseed oil fatty acid is as follows: 55-80 wt% of linoleic acid, 10-25 wt% of palmitic acid, 6-18 wt% of oleic acid, 1-3 wt% of stearic acid, 0.1-1 wt% of arachidic acid, 0.02-1 wt% of palmitoleic acid and 0.01-1 wt% of linolenic acid.
The main components of the cottonseed oil fatty acid are linoleic acid, palmitic acid and oleic acid, other fatty acids comprise stearic acid, arachidic acid, palmitoleic acid and linolenic acid, which are necessary components, and the content of each component is different under the influence of plant varieties, soil, climate, production area, picking season and extraction process.
Cottonseed oil ceramide, the composition of which comprises: linoleic acid ceramide, palmitic acid ceramide, oleic acid ceramide, stearic acid ceramide, arachidic acid ceramide, palmitoleic acid ceramide, linolenic acid ceramide; because fatty acids all participate in the same reaction, and the mass ratio of the ceramide after the reaction is not changed greatly, the composition of the cotton seed oil ceramide is similar to that of cotton seed oil fatty acid: 55-80 wt% of linoleic acid ceramide, 10-25 wt% of palmitic acid ceramide, 6-18 wt% of oleic acid ceramide, 1-3 wt% of stearic acid ceramide, 0.1-1 wt% of arachidic acid ceramide, 0.02-1 wt% of palmitoleic acid ceramide and 0.01-1 wt% of linolenic acid ceramide. The content of each component is different due to different contents of fatty acid in cottonseed oil or oil.
Cottonseed oil ceramide, the composition of which comprises: linoleic acid ceramide, palmitic acid ceramide, oleic acid ceramide; linoleic acid ceramide 55-80 wt%, palmitic acid ceramide 10-25 wt%, and oleic acid ceramide 6-18 wt%.
Further, the cottonseed oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 1-3 wt%.
Furthermore, the cottonseed oil ceramide comprises 0.1-1 wt% of arachidic ceramide.
Further, the cottonseed oil ceramide comprises palmitoleic acid ceramide, and the palmitoleic acid ceramide accounts for 0.02-1 wt%.
Further, the cottonseed oil ceramide comprises linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.01-1 wt%.
Linoleic acid ceramide is obtained by condensation reaction of linoleic acid and sphingoid compounds, and comprises linoleic acid phytosphingosine ceramide, linoleic acid sphingosine ceramide, and linoleic acid dihydrosphingosine ceramide; the palmitic acid ceramide is obtained by condensation reaction of palmitic acid and sphingoid compounds, and comprises palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide, and palmitic acid dihydrosphingosine ceramide; the oleic acid ceramide is obtained by condensation reaction of oleic acid and sphingoid compounds, and comprises oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide and oleic acid dihydrosphingosine ceramide; stearic acid ceramide, arachidic acid ceramide, palmitoleic acid ceramide, linolenic acid ceramide, and the like.
In a second aspect of the present invention, a method for synthesizing ceramide in cottonseed oil, comprising the steps of:
the cottonseed oil fatty acid reacts with the sphingoid compound under the conditions of a condensing agent and an organic base, wherein the condensing agent is EDCI, and the organic base is NMM.
Further, the mole ratio of the cottonseed oil fatty acid to the sphingoid compound to EDCI to NMM is 1: (1-1.5): (1-2): (1-2), wherein the solvent for reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.
Cottonseed oil is generally commercially available in the form of oil and requires hydrolysis to cottonseed oil fatty acids, thus further comprising the steps of:
and hydrolyzing the cottonseed oil grease through saponification reaction to obtain cottonseed oil fatty acid.
Further, the saponification reaction is hydrolysis of the cottonseed oil grease in a potassium hydroxide solution.
Further, the mass ratio of the cottonseed oil grease to the potassium hydroxide is 1: (1-2).
In a third aspect of the invention, the use of cottonseed oil ceramide in cosmetics, pharmaceuticals, dietary foods or health products.
Further, the cottonseed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promotion, and elastin vitality maintenance effects.
A composition comprising cottonseed oil ceramide, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promoting, elastin viability maintaining efficacy.
The composition contains acceptable adjuvants including one or more of solubilizer, antiseptic, antioxidant, pH regulator, penetration enhancer, liposome, humectant, thickener, chelating agent, skin feeling regulator, surfactant, emulsifier, essence and pigment; the composition is in the form of cream, emulsion, solution, pellicle, aerosol or spray.
The invention has the following beneficial effects:
the cottonseed oil fatty acid belongs to naturally formed fatty acid, the main component is unsaturated fatty acid-linoleic acid, and in addition, the cottonseed oil fatty acid also contains oleic acid and saturated fatty acid such as palmitic acid, and the cottonseed oil ceramide is prepared by mild reaction with sphingoid compounds naturally existing in the skin, shows excellent performance in the aspects of repairing, oxidation resistance, aging resistance and the like of the natural barrier of the skin, and has wide application prospect in the fields of cosmetics, health products, biological medicines and the like.
1. It is more effective than ceramide alone. Different ceramides have different effects due to their structural differences, and ceramides with a single structure generally have poor comprehensive effects. The scheme is based on a bionic idea, cottonseed oil grease or fatty acid from a natural source is used as a raw material to synthesize the composite ceramide so as to make up for the difference of efficacies of different ceramides, and trace fatty acid in the cottonseed oil can form trace ceramide, so that the function supplement effect is achieved.
2. Compared with compounded ceramide, the effect is better. Besides fatty acid (or grease), cottonseed oil is rich in other nutritional ingredients, and the ingredients have the effects of resisting oxidation, resisting aging and the like. The ceramide synthesized by the cottonseed oil has a synergistic effect with other active ingredients contained in the cottonseed oil, and has a better effect compared with the ceramide compounded according to a similar proportion.
3. The cost is lower. The method of the invention can quickly obtain the composition compounded by various ceramides, the plant-derived cottonseed oil grease or fatty acid thereof has wide sources, is easy to be obtained commercially, has lower cost, is more environment-friendly and economic, and is different from the idea of mixing and compounding different single ceramides, the fatty acid with a single component has high raw material price, and different ceramides are required to be produced respectively and then compounded, so the preparation cost is increased.
4. The synthetic method is simple. The method can adopt chemical synthesis to realize one-step preparation of various ceramides, and can also use a microbial fermentation method.
Drawings
FIGS. 1 and 2 are bar graphs of the cell proliferation activity test results of example 4;
FIG. 3 shows the results of the cell migration ability test in example 5;
FIG. 4 is a bar graph of the elastase inhibition of example 6;
FIG. 5 is a bar graph showing the IL-6 factor expression levels measured for anti-inflammatory repair efficacy in example 7;
FIGS. 6 and 7 are graphs showing MMP1 expression levels in the anti-photoaging test of example 8;
FIGS. 8 and 9 are bar graphs of DPPH radical clearance for oxidation resistance testing of example 9.
Detailed Description
The present invention will be further described with reference to the following specific examples.
EDCI means 1-ethyl- (3-dimethylaminopropyl) carbodiimide and NMM means N-methylmorpholine. The silica gel column chromatography uses Qingdao marine silica gel (particle size 0.040-0.063 mm). Thin Layer Chromatography (TLC) was performed using 60F254 silica gel plates, and TLC developed using UV light (254 nm) or iodine.
Example 1
Synthesis of ceramide from fatty acid of cottonseed oil and phytosphingosine
The first step is as follows: dissolving 50g of cottonseed oil fat in 60mL of tetrahydrofuran, cooling in ice bath, dropwise adding 100mL of potassium hydroxide (25 wt%) solution, heating to room temperature after dropwise adding, and reacting until the TLC detection reaction is finished.
And (3) post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 150mL ethyl acetate to extract the aqueous phase, adding 100mL saturated saline water to wash once, and adding anhydrous Na into the organic phase 2 SO 4 Drying, filtration and concentration in vacuo gave 40.5g of cottonseed oil fatty acid.
The second step is that: cottonseed oil fatty acid (50 mmol in terms of main component fatty acid), EDCI (65 mmol) and NMM (65 mmol) were added to a 250mL round-bottomed flask, 100mL of dichloromethane was added, followed by stirring at room temperature for 1 hour, followed by adding phytosphingosine (55 mmol) to the reaction system, followed by stirring at room temperature until the TLC detection reaction was completed.
And (3) post-treatment: adding water to quench and react, separating an organic layer, drying, filtering, concentrating in vacuum, washing by a solvent to obtain cottonseed oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the column temperature was adjusted by Innoval ODS-2.6 × 250mm,5 μm column: 30 ℃, injection volume: 10 μ L, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The HPLC retention time of each component was: palmitoleic acid-phytosphingosine ceramide for 8.1min, linolenic acid-phytosphingosine ceramide for 8.3min, linoleic acid-phytosphingosine ceramide for 9.5min, palmitic acid-phytosphingosine ceramide for 10.7min, oleic acid-phytosphingosine ceramide for 11.3min, stearic acid-phytosphingosine ceramide for 13.9min, and arachidic acid-phytosphingosine ceramide for 16.5min.
The obtained product is analyzed by high performance liquid chromatography, and the contents of linoleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide, arachidic acid-phytosphingosine ceramide, palmitoleic acid-phytosphingosine ceramide and linolenic acid-phytosphingosine ceramide are 74%, 12%, 7%, 2%, 1% and 1% in sequence, and the rest is other components with less content.
Example 2
Synthesis of ceramide from fatty acid of cottonseed oil and sphingosine
The first step is as follows: dissolving 50g of cottonseed oil fat in 60mL of tetrahydrofuran, cooling in ice bath, dropwise adding 100mL of potassium hydroxide (25 wt%) solution, heating to room temperature after dropwise adding, and reacting until the reaction is detected by TLC.
And (3) post-treatment: adding dilute hydrochloric acid (3N) to adjust pH to 3, adding 150mL ethyl acetate to extract water phase, adding 100mL saturated salt water to wash once, adding anhydrous Na to organic phase 2 SO 4 Dried, filtered and concentrated in vacuo to give 38g of cottonseed oil fatty acid.
The second step: cottonseed oil fatty acid (50 mmol in terms of main component fatty acid), EDCI (70 mmol) and NMM (70 mmol) were added to a 250mL round-bottom flask, 100mL of dichloromethane was added, followed by stirring at room temperature for 1 hour, and then sphingosine (60 mmol) was added to the reaction system, followed by stirring at room temperature, until the TLC detection reaction was completed.
And (3) post-treatment: adding water to quench and react, separating an organic layer, drying, filtering, concentrating in vacuum, washing by a solvent to obtain cottonseed oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the column temperature was adjusted by Innoval ODS-2.6 × 250mm,5 μm column: 30 ℃, injection volume: 10 μ L, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The HPLC retention times for the components were: linolenic acid-sphingosine ceramide 7.9min, palmitoleic acid-sphingosine ceramide 8.2min, linoleic acid-sphingosine ceramide 8.7min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.4min, stearic acid-sphingosine ceramide 13.6min, arachidic acid-sphingosine ceramide 17.8min.
The obtained product is analyzed by high performance liquid chromatography, and the contents of linoleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, oleic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, arachidic acid-sphingosine ceramide, palmitoleic acid-sphingosine ceramide and linolenic acid-sphingosine ceramide are 59%, 16%, 17%, 3%, 1%, 0.5% and 0.5% in sequence, and the rest is other components with less content.
Example 3
Synthesis of ceramide from fatty acid of cottonseed oil and dihydrosphingosine
The first step is as follows: dissolving 50g of cottonseed oil fat in 60mL of tetrahydrofuran, cooling in ice bath, dropwise adding 100mL of potassium hydroxide (25 wt%) solution, heating to room temperature after dropwise adding, and reacting until the reaction is detected by TLC.
And (3) post-treatment: adding dilute hydrochloric acid (3N) to adjust pH to 3, adding 150mL ethyl acetate to extract water phase, adding 100mL saturated salt water to wash once, adding anhydrous Na to organic phase 2 SO 4 Drying, filtration and concentration in vacuo gave 40g of cottonseed oil fatty acid.
The second step is that: cottonseed oil fatty acid (50 mmol, as fatty acid as the main component), EDCI (80 mmol) and NMM (80 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was further added, followed by stirring at room temperature for 1 hour, followed by adding dihydrosphingosine (65 mmol) to the reaction system, followed by stirring at room temperature until the TLC detection reaction was completed.
And (3) post-treatment: adding water to quench and react, separating an organic layer, drying, filtering, concentrating in vacuum, washing by a solvent to obtain cottonseed oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the column temperature was adjusted by Innoval ODS-2.6 × 250mm,5 μm column: 30 ℃, injection volume: 10 μ L, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The HPLC retention time of each component was: linolenic acid-sphinganine ceramide 8.3min, palmitoleic acid-sphinganine ceramide 8.9min, linoleic acid-sphinganine ceramide 9.5min, palmitic acid-sphinganine ceramide 10.6min, oleic acid-sphinganine ceramide 11.1min, stearic acid-sphinganine ceramide 13.5min, arachidic acid-sphinganine ceramide 16.0min.
According to the analysis of the obtained product by high performance liquid chromatography, the contents of linoleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, oleic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, arachidic acid-dihydrosphingosine ceramide, palmitoleic acid-dihydrosphingosine ceramide and linolenic acid-dihydrosphingosine ceramide are respectively 62%, 22%, 11%, 1%, 0.5% and 1% and the rest is other components with less content.
Example 4
MTT method for detecting cell proliferation activity of compound
HaCaT cells were cultured at 1X 10 4 The density of cells/well was plated in 96-well plates overnight in an incubator. After 24h the supernatant was discarded, 100. Mu.L of medium containing samples of different concentrations (product of example 1) was added, incubation was continued for 24h and the medium was removed, 100. Mu.L of thiazole blue (MTT) was added to each well, absorbance at 450nm was measured and cell viability = A was calculated Medicine feeding hole /A Blank hole ×100％。
The results are shown in fig. 1, and the cottonseed oil ceramide has a promoting effect on cell viability, and the cell viability is 122.63%, 108.38%, 94.33%, 100.86%, 119.66%, 113.59%, 100.40%, 115.47% and 124.87% at the concentrations of 0.97657, 1.95313, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125 and 250mg/L, respectively. The concentration of the effective components is as low as 1mg/L, the concentration gradient is stable, the obvious effect of promoting cell proliferation is shown, and the tissue repair capability is good.
The proliferation activity of ceramide 2 on cells was tested in the same manner, and the results are shown in fig. 2, and the cell survival rates at concentrations of 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000mg/L were 61.49%, 60.03%, 55.41%, 54.64%, 53.37%, 46.95%, 44.05%, 40.35% and 39.42%, respectively, which had inhibitory effects on cell proliferation and had tissue repair potentials inferior to that of cottonseed oil ceramide.
Example 5
Evaluation of skin Barrier repair by cell migration
The principle is as follows: when the cells grow to be fused into a monolayer, a scratch tool is used for manufacturing a blank area on the fused monolayer, the cells in the blank area are removed by mechanical force, the migration of the cells to a cell-free area is observed through a period of culture, and the migration capacity of the cells is reflected by measuring the migration distance of the cells.
The method comprises the following operation steps:
1. the plates were streaked. Firstly, a Marker pen is used on the back of a 6-hole plate, a straight ruler is used for uniformly drawing transverse lines which are about every 0.5-1 cm and cross through holes, each hole at least penetrates through 5 lines, and the lines are not too thick when drawing lines.
2. And (5) laying cells. About 5X 10 additions to the wells 5 And (3) inoculating each cell (the number of different cells is different and is adjusted according to the growth speed of the cells), wherein the inoculation principle is that the fusion rate reaches 100 percent after the overnight inoculation.
3. And (4) scribing cells. The next day, the cell layer was scored using a tip, perpendicular to the cell plane, along the line drawn on the back of the plate on the first day (preferably the same tip is used between different wells).
4. The cells were washed. After the scoring was completed, cells were washed 3 times with sterile PBS, non-adherent cells were washed away, i.e., cells streaked during streaking, leaving a clear gap after streaking, and then fresh serum-free medium was replaced.
5. And (5) culturing and observing cells. Samples (example 1 product, ceramide 3B) were diluted with medium (example 1 product concentration 20mg/L, ceramide 3B concentration 100 mg/L) and added to a cell culture dish, and the cells were placed at 37 ℃ and 5wt% CO 2 The cells were cultured in an incubator, taken out after 24 hours, observed and measured for width of scratch by a microscope, photographed, and the healing rate was calculated with Image J software.
The results are shown in fig. 3, and the scratch width of the experimental group is narrower than that of the solvent control group, which indicates that the cotton seed oil ceramide has better tissue healing capability. The healing rate of the solvent control group after 24h was 38.22%, the healing rate of the cottonseed oil ceramide after 24h was 96.21%, and the healing rate of the ceramide 3B after 24h was 59.32%. The compound provided by the invention obviously improves the cell healing rate, has good skin tissue repair activity, and has a better effect than ceramide 3B.
Example 6
Elastase inhibition experiment for testing anti-aging effect
Elastase inhibition methods: 2mL of 2mg/mL elastase solution is taken, samples (products in example 1) with different concentrations are added, the mixture is fully and evenly mixed in a vortex mode, the mixture is shaken in a shaking table with 400r/min at 37 ℃ for 20min, 5mL of 0.5mol/L phosphate buffer solution with pH6.0 is immediately added, the mixture is evenly mixed in a vortex mode, a proper amount of evenly mixed solution is taken to be put into a 2mL centrifugal tube, the centrifugal tube is centrifuged for 10min at 9 391 Xg, 200 mu L of supernatant is precisely absorbed into a 96-well plate, absorbance is measured by an enzyme-labeling instrument at the wave length of 495nm, and spectrum scanning of 400-800 nm is carried out at the same time.
And taking a substrate and enzyme solution as a blank control group, taking the substrate and enzyme solution and a sample solution as an enzyme inhibition group, and taking the substrate and sample without the enzyme solution as a background. Each group is provided with 3 holes. Inhibition (%) = [1- (An-An ')/(A0-A0') ] × 100%, where A0 is the absorbance of a sample with no enzyme, A0 'is the absorbance of a sample with no substrate and no enzyme, an is the absorbance of a solution with only a sample, and An' is the absorbance of a sample with no enzyme. If An ' > An, a promoting effect is exhibited, and the promoting rate (%) = [1- (An ' -An)/(A0-A0 ') ] × 100%.
As shown in FIG. 4, the cottonseed oil ceramide has a good inhibitory effect on elastase at various concentrations, specifically, the inhibitory rate on elastase is 11.80% at a concentration of 0.25g/L, 18.33% at a concentration of 0.5g/L, 23.60% at a concentration of 1.0g/L, and 35.00% at a concentration of 2.0 g/L.
Example 7
Detection of anti-inflammatory repair effect by LPS induced cell method
B16 mouse melanoma cells at a density of 1X 10 4 Planting in 96-well plate, placing in incubator, standing overnight, discarding supernatant after 24 hr, and adding 100 μL samples of different concentrations diluted in DMEM medium (product of example 1), negative control group was DMEM medium without sample, 3 duplicate wells per group, at 5wt% CO 2 And incubating at 37 ℃. After 2h administration, the LPS model group and the experimental group were added with 10. Mu.g/mL LPS and incubated together for 24h. After the reaction, 50. Mu.L of cell supernatant was collected and the expression of IL-6 gene in the cells was detected by using IL-6ELISA kit.
As a result, as shown in FIG. 5, the IL-6 level was 10.16 times the basal level in LPS stimulation at a working concentration of 10. Mu.g/mL. Under the action of cottonseed oil ceramide with the concentrations of 50mg/L, 100mg/L, 200mg/L and 400mg/L respectively, the IL-6 factor level is obviously reduced and is 0.99, 0.87, 0.72 and 0.57 times of those of an LPS model group respectively, and the dosage dependence is presented, so that the cottonseed oil ceramide has a good anti-inflammatory effect and can promote the repair of inflammatory damaged skin.
Example 8
Light aging resistance test
MMP1 is also called interstitial collagenase and matrix metalloproteinase, belongs to a family of matrix metalloproteinase, and has the main action substrates of fibrous collagen, can degrade collagen fibers and gelatin in extracellular matrix and change the microenvironment of cells. MMP1 plays an important role in elastin, inhibition of MMP1 can improve synthesis of collagen and elastin of fibroblasts, and reduction of MMP activity can increase collagen synthesis speed.
HaCaT cells were cultured at 1X 10 5 The density of cells/well was plated in 96-well plates overnight in an incubator. After 24h, the supernatant was discarded, 100. Mu.L of medium containing samples (product of example 1) at different concentrations was added, no sample was added to the model group, the negative control group was DMEM medium without sample, 3 wells each, and CO was added at a mass fraction of 5% 2 After incubation at 37 ℃ for 2h, UVA or UVB ultraviolet radiation was applied. The distance between the ultraviolet radiation light source and the cell was 15cm, and the UVA intensity was 200mJ/cm 2 The irradiation time is 2h, the UVB intensity is 50mJ/cm 2 The irradiation time is 1h. After the irradiation was finished, incubation was continued in the incubator for 12h. MMP-1 gene expression in cells was detected using an MMP-1ELISA kit. Inhibition =1- (experimental group MMP1 expression amount/model group MMP1 tableAmount) x 100%.
As a result, as shown in fig. 6 and 7, the expression level of MMP1 in the negative control group was 1, the expression level in the model group was 1.90, and the inhibition rates of MMP1 expression in the model group were 27%, 48%, and 62% for cottonseed oil at concentrations of 125, 250, and 400 mg/L; in UVB, the MMP1 expression level of the negative control group is 1, the expression level of the model group is 2.33, and the inhibition rates of cottonseed oil at concentrations of 125, 250 and 400mg/L relative to the MMP1 expression of the model group are 35%, 49% and 56%.
After UVA ultraviolet radiation, keratinocytes promote increased fibroblast MMP1 expression, thereby causing degradation of skin extracellular matrix and skin collagen, resulting in skin photoaging. The results show that the cottonseed oil ceramide can inhibit the fibroblast cells caused by ultraviolet radiation from generating MMP1, and has a certain effect on preventing skin photoaging.
Example 9
DPPH free radical scavenging and detecting antioxidant performance
DPPH is 1, 1-diphenyl-2-trinitrophenylhydrazine, and can be used for antioxidant experiments. Samples (product of example 1) at the corresponding concentrations (50, 100, 200, 400, 800 mg/L) were mixed with 0.1mol/L DPPH, absolute ethanol solution at a ratio of 1:1, mixing the mixture evenly, mixing DPPH and absolute ethyl alcohol 1:1, mixing the mixture in equal volume, reacting the mixture for 30min in a dark place at room temperature, and measuring the light absorption value at 517 nm. The absorbance of the sample and the DPPH reaction solution was denoted as A1, the absorbance of the sample and the absolute ethanol reaction solution was denoted as A2, the absorbance of the DPPH and the absolute ethanol reaction solution was denoted as A3, and the DPPH clearance of the sample = [1- (A1-A2)/A3 ] × 100%.
As shown in FIG. 8, the DPPH radical clearance rates at concentrations of 50, 100, 200, 400, and 800mg/L were 9.43%, 23.91%, 33.61%, 41.93%, and 49.19%, respectively, and the excellent antioxidant effect was exhibited. Ceramide 3B (i.e., oleic ceramide) was tested for antioxidant effect in the same manner, and as shown in FIG. 9, DPPH radical scavenging rates at concentrations of 50, 100, 200, 400, 800mg/L were 7.76%, 12.82%, 24.10%, 29.60%, 33.16%. The clearance rate of the cottonseed oil ceramide to DPPH is higher than that of ceramide 3B, and the cottonseed oil ceramide has a better antioxidant effect.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. Cottonseed oil ceramide, which is obtained by reacting cottonseed oil fatty acid with a sphingoid compound, wherein the sphingoid compound is selected from sphingosine, phytosphingosine and dihydrosphingosine.
2. The cottonseed oil ceramide of claim 1, wherein the cottonseed oil fatty acid is obtained from hydrolysis of cottonseed oil.
3. The cottonseed oil ceramide of claim 1 or claim 2, wherein the cottonseed oil fatty acid comprises 55 to 80wt% linoleic acid, 10 to 25wt% palmitic acid, 6 to 18wt% oleic acid, 1 to 3wt% stearic acid.
4. Cottonseed oil ceramide, the composition of which comprises: linoleic acid ceramide, palmitic acid ceramide, oleic acid ceramide, stearic acid ceramide, arachidic acid ceramide, palmitoleic acid ceramide, linolenic acid ceramide.
5. The cottonseed oil ceramide according to claim 4, whose composition comprises: 55-80 wt% of linoleic acid ceramide, 10-25 wt% of palmitic acid ceramide, 6-18 wt% of oleic acid ceramide, 1-3 wt% of stearic acid ceramide, 0.1-1 wt% of arachidic acid ceramide, 0.02-1 wt% of palmitoleic acid ceramide, and 0.01-1 wt% of linolenic acid ceramide.
6. The method for synthesizing the ceramide of any one of claims 1 to 5, comprising the steps of:
reacting cottonseed oil fatty acid with a sphingoid compound under the conditions of a condensing agent and an organic base, wherein the condensing agent is EDCI, and the organic base is NMM;
the molar ratio of the cottonseed oil fatty acid to the sphingoid compound to the EDCI to the NMM is 1: (1-1.5): (1-2): (1-2), wherein the solvent for reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.
7. Use of the cottonseed oil ceramide of any one of claims 1 to 5 in cosmetics, pharmaceuticals, dietary foods or health products.
8. The use according to claim 7, wherein the cottonseed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promoting, elastin viability maintaining efficacy.
9. A composition comprising the cottonseed oil ceramide of any one of claims 1 to 5, the composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promoting, elastin viability maintaining efficacy.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN202310051764.2A CN115974716A (en) | 2023-02-02 | 2023-02-02 | Cottonseed oil ceramide and synthesis method and application thereof |
| PCT/CN2023/133524 WO2024109867A1 (en) | 2022-11-25 | 2023-11-23 | Vegetable oil ceramides, synthesis method therefor, and use thereof |
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| WO2024109867A1 (en) * | 2022-11-25 | 2024-05-30 | 深圳市迪克曼生物科技有限公司 | Vegetable oil ceramides, synthesis method therefor, and use thereof |
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| WO2024109867A1 (en) * | 2022-11-25 | 2024-05-30 | 深圳市迪克曼生物科技有限公司 | Vegetable oil ceramides, synthesis method therefor, and use thereof |
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