CN115947667A - Coconut oil ceramide and synthesis method and application thereof - Google Patents
Coconut oil ceramide and synthesis method and application thereof Download PDFInfo
- Publication number
- CN115947667A CN115947667A CN202310051777.XA CN202310051777A CN115947667A CN 115947667 A CN115947667 A CN 115947667A CN 202310051777 A CN202310051777 A CN 202310051777A CN 115947667 A CN115947667 A CN 115947667A
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- Prior art keywords
- ceramide
- acid
- coconut oil
- fatty acid
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 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 159
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and discloses coconut oil ceramide which is obtained by reacting coconut oil fatty acid with sphingoid compounds, wherein the sphingoid compounds are selected from sphingosine, phytosphingosine and dihydrosphingosine. The coconut 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 coconut oil ceramide, a synthetic 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 with skin ceramide, and can possibly become a next-generation environment-friendly, safe and reliable ceramide product.
Coconut oil is a plant saturated oil, the content of saturated fatty acid is up to 92wt%, the main component is medium-chain fatty acid, the highest content of lauric acid, and other medium-chain and short-chain saturated fatty acids are as follows: caproic acid, capric acid, and long-chain saturated fatty acids such as myristic acid, palmitic acid, and stearic acid. Medium chain fatty acids are different from long chain fatty acids and do not negatively affect cholesterol. Medium chain fatty acids help to reduce the risk of atherosclerosis and heart disease and help to prevent heart disease. Medium chain fatty acids are only used for energy production, do not enter human body fat, and do not cause obesity. To date, the only vegetable oils with medium-chain fatty acids are coconut oil and palm kernel oil. Coconut oil is basically saturated fatty acid, and does not lack hydrogen atoms and double bonds in carbon chains, so that the coconut oil is not easy to oxidize and generate free radicals. In addition, coconut oil has excellent anti-inflammatory, weight reducing, and insulin sensitivity enhancing effects.
Disclosure of Invention
The invention aims to provide ceramide synthesized by coconut oil fatty acid of plant origin.
Another object of the present invention is to provide a method for the synthesis of coconut oil ceramide, which uses coconut oil fatty acid, which is naturally derived from plants and is easily available, as a raw material.
It is another object of the present invention to provide the use of coconut oil ceramide.
In order to achieve one of the purposes, the invention adopts the following technical scheme:
in a first aspect of the invention, a coconut oil ceramide, obtained by reacting a coconut oil fatty acid 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 coconut oil is used as a raw material, and proper bacterial strains are selected for fermentation to obtain the coconut 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 coconut oil fatty acid contains 20 to 70wt% of lauric acid.
Further, the coconut oil fatty acid contains 10-50 wt% myristic acid.
Further, the coconut oil fatty acid contains 10-20 wt% of palmitic acid.
Further, the coconut oil fatty acid contains 2 to 10wt% of stearic acid.
Further, the coconut oil fatty acid contains 2 to 10wt% of oleic acid.
In addition, the coconut oil fatty acid also contains 0 to 3 weight percent of capric acid and 0 to 3 weight percent of caproic acid.
The coconut oil fatty acid comprises the following components: 20-70 wt% of lauric acid, 10-50 wt% of myristic acid, 10-20 wt% of palmitic acid, 2-10 wt% of stearic acid, 2-10 wt% of oleic acid, 0-3 wt% of capric acid and 0-3 wt% of caproic acid.
The main component of coconut oil fatty acid is lauric acid, other fatty acids comprise myristic acid, palmitic acid, stearic acid and oleic acid which are necessary components, the content of each component can be different under the influence of plant species, soil, climate, producing area, picking season and extraction process, and capric acid and caproic acid are not necessarily contained and are optional components or unnecessary components.
Coconut oil ceramide, the composition of which comprises: lauric acid ceramide, myristic acid ceramide, palmitic acid ceramide, stearic acid ceramide, oleic acid ceramide; because fatty acids all participate in the same reaction, the mass ratio of the ceramide after the reaction is not greatly changed, and the composition of the coconut oil ceramide is similar to that of coconut oil fatty acid: 20 to 70 weight percent of lauric acid ceramide, 10 to 50 weight percent of myristic acid ceramide, 10 to 20 weight percent of palmitic acid ceramide, 2 to 10 weight percent of stearic acid ceramide and 2 to 10 weight percent of oleic acid ceramide. The content of each component is different due to different contents of each fatty acid in coconut oil fatty acid. In addition, the coconut oil ceramide also comprises ceramide obtained by reacting one or more of capric acid and caproic acid with sphingoid compounds, namely 0-3 wt% of capric acid ceramide and 0-3 wt% of caproic acid ceramide.
Coconut oil ceramide, the composition of which comprises: lauric acid ceramide, myristic acid ceramide, palmitic acid ceramide; 20-70 wt% of lauric acid ceramide, 10-50 wt% of myristic acid ceramide and 10-20 wt% of palmitic acid ceramide.
Further, the coconut oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 2-10 wt%.
Further, the coconut oil ceramide comprises oleic acid ceramide, and the oleic acid ceramide accounts for 2-10 wt%.
Further, the coconut oil ceramide comprises no more than 3wt% of decanoic acid ceramide and no more than 3wt% of hexanoic acid ceramide, especially 0.1-3 wt% of decanoic acid ceramide and 0.1-3 wt% of hexanoic acid ceramide.
The lauric acid ceramide is obtained by condensation reaction of lauric acid and sphingoid compounds, and comprises lauric acid phytosphingosine ceramide, lauric acid sphingosine ceramide and lauric acid dihydrosphingosine ceramide; the myristic acid ceramide is prepared by condensation reaction of myristic acid and sphingoid compounds, and comprises myristic acid phytosphingosine ceramide, myristic acid sphingosine ceramide, and myristic 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 stearic acid ceramide is obtained by condensation reaction of stearic acid and sphingoid compounds, and comprises stearic acid phytosphingosine ceramide, stearic acid sphingosine ceramide and stearic 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; capric acid ceramide, caproic acid ceramide, and the like.
In a second aspect of the invention, a method for synthesizing coconut oil ceramide comprises the following steps:
reacting coconut oil fatty acid with sphingoid compound under the conditions of condensing agent and organic base, wherein the condensing agent is EDCI, and the organic base is Et 3 N。
Further, the coconut oil fatty acid, sphingoid compound, EDCI, et 3 The molar ratio of N 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.
In a third aspect of the invention, the use of coconut oil ceramide in cosmetics, pharmaceuticals, dietary foods or health products.
Further, the coconut oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promotion, elastin vitality maintenance, and whitening effects.
A composition comprising coconut oil ceramide, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promotion, elastin viability maintenance, and whitening 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:
coconut oil fatty acid is a naturally occurring fatty acid, and the main component is medium-chain fatty acid lauric acid, and further contains long-chain saturated fatty acids, such as myristic acid, palmitic acid, stearic acid, oleic acid, and the like. The invention utilizes the excellent characteristics of coconut oil which is sourced from organic sources, rich saturated fatty acid, especially special high-content medium-chain fatty acid, and sphingoid compounds which naturally exist in skin to prepare the coconut oil ceramide through mild reaction, shows excellent performances in the aspects of repairing natural barriers of skin, deep moisture preservation, oxidation resistance, aging resistance and the like, and has wide application prospect in the fields of cosmetics, health care products, biological medicines and the like
1. Compared with single ceramide, the effect is better. Different ceramides have different effects due to their structural differences, and ceramides with a single structure generally have poor comprehensive effects. The coconut oil fatty acid composition is based on a bionic thought, coconut oil fatty acid from a natural source is used as a raw material to synthesize composite ceramide, so that the difference of efficacies of different ceramides is made up, and trace fatty acid in the coconut oil can form trace ceramide, so that the function supplement effect is achieved.
2. Compared with compounded ceramide, the effect is better. Besides fatty acids, coconut oil also contains other nutrients, which have the effects of moisturizing skin, enhancing cell viability, etc. The ceramide synthesized by the coconut oil has synergistic effect with other active ingredients contained in the coconut oil, and has better effect compared with the ceramide compounded according to similar proportion.
3. The cost is lower. The method of the invention can quickly obtain the composition compounded by various ceramides, the coconut oil fatty acid of plant source has wide source, easy commercial acquisition, lower cost, more environmental protection and economy, and is different from the idea of mixing and compounding different single ceramides, the fatty acid of single component has high raw material price, and different ceramides are required to be respectively produced and then compounded, thereby increasing the preparation cost.
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 graphs of DPPH radical scavenging rate for oxidation resistance test of example 9;
fig. 10 is a bar graph of melanin content in the whitening activity test of example 10.
Detailed Description
The present invention will be further described with reference to the following specific examples.
EDCI refers to 1-ethyl- (3-dimethylaminopropyl) carbodiimide, et 3 N refers to triethylamine. The silica gel column chromatography uses Qingdao marine silica gel (particle size 0.040-0.063 mm). Thin Layer Chromatography (TLC) was performed using a 60F254 silica gel plate, and TLC developed using UV light (254 nm) or iodine.
Example 1
Synthesis of ceramide from coconut oil fatty acid and phytosphingosine
Mixing coconut oil fatty acid (50 mmol, calculated as main component fatty acid), EDCI (65 mmol), et 3 N (65 mmol) was added to a 250mL round-bottom flask, followed by addition of 100mL of methylene chloride, followed by stirring at room temperature for 1 hour, followed by addition of phytosphingosine (55 mmol) to the reaction system, followed by stirring at room temperature until completion of the TLC detection reaction.
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 coconut 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: caproic acid-phytosphingosine ceramide 5.7min, capric acid-phytosphingosine ceramide 6.3min, lauric acid-phytosphingosine ceramide 7.5min, myristic acid-phytosphingosine ceramide 9.4min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.3min, stearic acid-phytosphingosine ceramide 13.9min.
According to the analysis of the obtained product by high performance liquid chromatography, the contents of lauric acid-phytosphingosine ceramide, myristic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide, capric acid-phytosphingosine ceramide and caproic acid-phytosphingosine ceramide are 41%, 29%, 12%, 6%, 4%, 1% and 3% in sequence, and the rest is other components with less content.
Example 2
Synthesis of ceramide from coconut oil fatty acid and sphingosine
Mixing coconut oil fatty acid (50 mmol, calculated as main component fatty acid), EDCI (70 mmol), et 3 N (70 mmol) was added to a 250mL round-bottom flask, followed by addition of 100mL of methylene chloride, followed by stirring at room temperature for 1 hour, followed by addition of sphingosine (60 mmol) to the reaction system, followed by stirring at room temperature until completion of the TLC detection reaction.
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 coconut 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: caproic acid-sphingosine ceramide 5.4min, lauric acid-sphingosine ceramide 6.5min, myristic acid-sphingosine ceramide 8.3min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.5min, stearic acid-sphingosine ceramide 13.6min.
The obtained product is analyzed by high performance liquid chromatography, and the contents of lauric acid-sphingosine ceramide, myristic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, oleic acid-sphingosine ceramide and caproic acid-sphingosine ceramide are 60%, 13%, 15%, 3%, 5% and 2% in sequence, and the rest is other components with less content.
Example 3
Synthesis of ceramide from coconut oil fatty acid and dihydrosphingosine
Mixing coconut oil fatty acid (50 mmol, calculated as main component fatty acid), EDCI (75 mmol), et 3 Adding N (75 mmol) into a 250mL round-bottom flask, adding 100mL dichloromethane, stirring at room temperature for 1 hr, adding sphinganine (70 mmol) into the reaction system, stirring at room temperature, and detecting by TLCAnd (6) finishing.
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 coconut 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: capric acid-sphinganine ceramide 6.2min, lauric acid-sphinganine ceramide 7.0min, myristic acid-sphinganine ceramide 8.5min, palmitic acid-sphinganine ceramide 10.6min, oleic acid-sphinganine ceramide 11.1min, stearic acid-sphinganine ceramide 13.6min.
According to the analysis of the obtained product by high performance liquid chromatography, the contents of lauric acid-sphinganine ceramide, myristic acid-sphinganine ceramide, palmitic acid-sphinganine ceramide, stearic acid-sphinganine ceramide, oleic acid-sphinganine ceramide and capric acid-sphinganine ceramide are 37%, 34%, 17%, 8% and 3% in sequence, 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 Density of individual/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, the medium was removed after further incubation for 24h, 100. Mu.L of blue thiazole (MTT) was added to each well, absorbance at 450nm was measured, and cell survival = A was calculated Medicine feeding hole /A Blank hole ×100％。
As shown in figure 1, the coconut oil has the effect of promoting cell viability, the cell survival rates of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000mg/L are 113.93%, 120.33%, 108.79%, 100.42%, 94.15%, 99.15%, 89.97% and 89.22 respectively, the effective concentration is as low as 7.8mg/L, the safe concentration is 63mg/L, the concentration gradient is stable, the obvious cell proliferation promoting effect is shown, and the coconut oil has good tissue repair capacity.
The proliferation activity of ceramide 2 on cells is tested according to the same method, and the results are shown in figure 2, the cell survival rates at the concentrations of 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000mg/L are respectively 61.49%, 60.03%, 55.41%, 54.64%, 53.37%, 46.95%, 44.05%, 40.35% and 39.42%, and the cell proliferation is inhibited, and the tissue repair potential is not as good as that of coconut 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 operation steps are as follows:
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) cell spreading. 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 with 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, the cells were washed 3 times with sterile PBS, the nonadherent cells, i.e., the scored cells during the scoring, were washed away, the gap left after scoring was clearly visible, 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 incubated in an incubator and taken out after 24h, microscopically observed and the width of the scratch was measured, photographed, and the healing rate was calculated using Image J software.
The results are shown in fig. 3, where the scratch width of the experimental group is narrower than that of the solvent control group, indicating that the coconut oil ceramide has better tissue healing ability. The healing rate of the solvent control group after 24h was 29.58%, the healing rate of coconut oil ceramide after 24h was 82.31%, and the healing rate of 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 a substrate and enzyme solution and a sample solution as an enzyme inhibition group, and taking the substrate and sample without the enzyme solution as background. Each group is provided with 3 times of 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, coconut oil showed good inhibitory effect on elastase at different concentrations, specifically, the inhibitory rate on elastase was 9.47% at a concentration of 0.25g/L, 20.60% at a concentration of 0.5g/L, 25.33% at a concentration of 1.0g/L, and 28.00% 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 One/well of the strain was placed in a 96-well plate, and the plate was placed in an incubator overnight, after 24 hours the supernatant was discarded, 100. Mu.L of samples (product of example 1) diluted in DMEM medium at various concentrations were added, the negative control group was sample-free DMEM medium, 3 duplicate wells were added, and the CO was calculated at 5wt% in each group 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.
The results are shown in FIG. 5, where IL-6 levels were 11.24-fold higher than the basal levels when stimulated with LPS at a working concentration of 10. Mu.g/mL. Under the action of coconut 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 respectively 0.85, 0.74, 0.71 and 0.75 times of LPS model group, and is dose-dependent, which proves that the coconut oil ceramide has good anti-inflammatory effect and can promote the repair of inflammatory damaged skin.
Example 8
MMP1 is also called interstitial collagenase and matrix metalloproteinase, belongs to the family of matrix metalloproteinase, and has the main function of a substrate, namely fibrous collagen, degrading collagen fibers and gelatin in extracellular matrix and changing 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 and samples containing different concentrations (product of example 1) were addedSubstance) of the test samples, the model group without the samples, the negative control group with the DMEM medium without the samples, each group with 3 multiple wells with 5% CO by mass 2 After incubation at 37 ℃ for 2h, UVA or UVB ultraviolet radiation was applied. The distance between the ultraviolet radiation 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. The inhibition rate =1- (MMP 1 expression amount in experimental group/MMP 1 expression amount in model group) × 100%.
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 coconut oil ceramide at concentrations of 125, 250, and 400mg/L with respect to MMP1 expression in the model group were 31%, 49%, and 67%; in UVB, the MMP1 expression level of the negative control group is set to be 1, the expression level of the model group is 2.33, and the inhibition rates of coconut oil ceramide at the concentrations of 125, 250 and 400mg/L relative to the MMP1 expression of the model group are 38%, 58% and 74%.
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 result shows that the coconut oil ceramide can inhibit the fibroblast caused by ultraviolet radiation from generating MMP1, and has 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, and mixing DPPH and absolute ethyl alcohol in a volume ratio of 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 29.09%, 32.72%, 35.10%, 41.93%, and 49.67%, respectively, and excellent antioxidant effects were 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 coconut oil ceramide has higher DPPH clearance rate than ceramide 3B, and has better antioxidant effect.
Example 10
Whitening Activity test
Taking B16 cells in exponential growth phase, digesting with trypsin-EDTA with the mass fraction of 0.25%, blowing uniformly, and mixing the cells according to the proportion of 3 multiplied by 10 5 The density of each well was seeded in 12-well plates. At 37 deg.C, mass fraction of 5% CO 2 Incubate overnight in the environment. Discarding the supernatant, adding culture solution containing samples (products of example 1) with different mass concentrations, incubating with RPMI-1640 medium without sample as a blank group, incubating with DMEM medium as a molding group, and re-incubating with 3 re-wells per group at a mass fraction of 5% CO 2 And incubating for 24 hours at 37 ℃. After the medium in the well plate was discarded and washed once to twice with Phosphate Buffered Saline (PBS), 1mL of a NaOH solution (1 mol/L) containing 10% by mass of DMSO was added to lyse the cells, and the cells were incubated at 80 ℃ or 100 ℃ for 2 hours until they were completely lysed. The sample was placed in a microplate reader and absorbance was measured at 405 nm. Melanin inhibition =1- (each well OD value/model group OD value) × 100% was calculated.
As shown in fig. 10, the melanin content of the blank control group was 1, the melanin expression of the building block was 1.54, and the inhibitory rates of the melanin of coconut oil ceramide were 11.42%, 18.95%, 20.00%, 20.27%, and 23.43%, respectively, at concentrations of 10, 20, 40, 80, and 100mg/L, thereby exhibiting excellent whitening effects.
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. Coconut oil ceramides obtained by reacting coconut oil fatty acids with sphingoid compounds selected from sphingosine, phytosphingosine, dihydrosphingosine.
2. The coconut oil ceramide according to claim 1, characterized in that the coconut oil fatty acid contains 20-70 wt% lauric acid, 10-50 wt% myristic acid, 10-20 wt% palmitic acid, 2-10 wt% stearic acid, 2-10 wt% oleic acid.
3. Coconut oil ceramide, the composition of which comprises: lauric acid ceramide, myristic acid ceramide, palmitic acid ceramide, stearic acid ceramide, oleic acid ceramide.
4. The coconut oil ceramide according to claim 3, having a composition comprising: 20-70 wt% of lauric acid ceramide, 10-50 wt% of myristic acid ceramide, 10-20 wt% of palmitic acid ceramide, 2-10 wt% of stearic acid ceramide and 2-10 wt% of oleic acid ceramide.
5. The coconut oil ceramide according to claim 3 or 4, further comprising in composition: 0 to 3 weight percent of decanoic acid ceramide and 0 to 3 weight percent of hexanoic acid ceramide.
6. A method of synthesizing the coconut oil ceramide as recited in any one of claims 1-5, comprising the steps of:
reacting coconut oil fatty acid with sphingoid compound under the conditions of condensing agent and organic base, wherein the condensing agent is EDCI, and the organic base is Et 3 N；
The coconut oil fatty acid, sphingoid compound, EDCI and Et 3 Of NThe molar ratio is 1: (1-1.5): (1-2): (1-2), wherein the solvent for the reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.
7. Use of the coconut 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 coconut oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promotion, elastin viability maintenance, whitening efficacy.
9. A composition comprising the coconut oil ceramide of any one of claims 1-5, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promotion, elastin viability maintenance, and whitening efficacy.
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| PCT/CN2023/133524 WO2024109867A1 (en) | 2022-11-25 | 2023-11-23 | Vegetable oil ceramides, synthesis method therefor, and use thereof |
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