CN115947666A - Rice bran oil ceramide and synthesis method and application thereof - Google Patents
Rice bran oil ceramide and synthesis method and application thereof Download PDFInfo
- Publication number
- CN115947666A CN115947666A CN202310051756.8A CN202310051756A CN115947666A CN 115947666 A CN115947666 A CN 115947666A CN 202310051756 A CN202310051756 A CN 202310051756A CN 115947666 A CN115947666 A CN 115947666A
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- CN
- China
- Prior art keywords
- ceramide
- acid
- rice bran
- bran oil
- fatty acid
- Prior art date
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- 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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Abstract
The invention belongs to the technical field of biological medicines, and discloses rice bran oil ceramide, which is obtained by reacting rice bran oil fatty acid with sphingoid compounds, wherein the sphingoid compounds are selected from sphingosine, phytosphingosine and dihydrosphingosine. The rice bran 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 rice bran 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 rice bran oil is prepared from rice bran which is a byproduct obtained in the rice processing process by a squeezing method or a leaching method. The oil content in the rice bran is 14-24 wt%, belongs to functional oil, and is high-quality vegetable oil. Rice bran oil is composed mainly of three fatty acids: oleic acid, linoleic acid and palmitic acid, and further stearic acid and linolenic acid, in a total amount of about 92% of the total fatty acids. Rice bran oil has the advantages of containing the most balanced fatty acid ratio, namely saturated fatty acid: monounsaturated fatty acids: polyunsaturated fatty acids approach 1:1:1, known as "heart oil"; secondly, the rice bran oil contains a unique substance of oryzanol which is an oxidant and can improve sleep, relieve fatigue and the like; thirdly, the rice bran oil contains rich vitamin E and has strong antioxidation. In addition, the fatty acid composition, vitamin E, sterol, oryzanol and the like of the rice bran oil are beneficial to absorption of a human body, and have the beneficial effects of removing cholesterol in blood, reducing blood fat, promoting growth and development of the human body and the like, so the rice bran oil is recognized as nutritional and healthy oil at home and abroad.
Disclosure of Invention
The invention aims to provide ceramide synthesized by rice bran oil fatty acid from plant sources.
Another object of the present invention is to provide a method for synthesizing rice bran oil ceramide, which uses rice bran oil fatty acid which is naturally derived from plants and is easily available as a raw material.
Another object of the present invention is to provide the use of rice bran 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 rice bran oil ceramide, derived from the reaction of a rice bran 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 rice bran oil is used as a raw material, and proper strains are selected for fermentation to obtain the rice bran 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 rice bran oil fatty acid contains 40-60 wt% of oleic acid.
Further, the rice bran oil fatty acid contains 20-45 wt% of linoleic acid.
Further, the rice bran oil fatty acid contains 5 to 20wt% of palmitic acid.
Further, the rice bran oil fatty acid contains 0.5 to 5wt% of stearic acid.
Further, the rice bran oil fatty acid contains 0.1-2 wt% of linolenic acid.
In addition, the rice bran oil fatty acid also contains 0-2 wt% of arachidic acid.
The rice bran oil fatty acid comprises the following components: 40 to 60 weight percent of oleic acid, 20 to 45 weight percent of linoleic acid, 5 to 20 weight percent of palmitic acid, 0.5 to 5 weight percent of stearic acid, 0.1 to 2 weight percent of linolenic acid and 0 to 2 weight percent of arachidic acid.
The main component of the fatty acid of the rice bran oil is linoleic acid, and other fatty acids comprise oleic acid, palmitic acid, linolenic acid and stearic acid which are necessary components, the content of each component can be different under the influence of plant varieties, soil, climate, producing area, picking season and extraction process, and the content of the arachidic acid is not necessary and is an optional component or an unnecessary component.
Rice bran oil ceramide, its composition includes: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide; because fatty acids all participate in the same reaction, and the mass ratio of ceramide after the reaction is not changed greatly, the composition of the rice bran oil ceramide is similar to that of rice bran oil fatty acid: 40 to 60 weight percent of oleic acid ceramide, 20 to 45 weight percent of linoleic acid ceramide, 5 to 20 weight percent of palmitic acid ceramide, 0.5 to 5 weight percent of stearic acid ceramide, and 0.1 to 2 weight percent of linolenic acid ceramide. The content of each component is different due to different content of each fatty acid in the rice bran oil fatty acid. In addition, the rice bran oil ceramide also comprises ceramide obtained by reacting arachidic acid with sphingoid compounds, namely 0-2 wt% of arachidic acid ceramide.
Rice bran oil ceramide, its composition includes: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide; oleic acid ceramide accounts for 40-60 wt%, linoleic acid ceramide accounts for 20-45 wt%, and palmitic acid ceramide accounts for 5-20 wt%.
Further, the rice bran oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 0.5-5 wt%.
Furthermore, the rice bran oil ceramide comprises linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.1-2 wt%.
Further, the rice bran oil ceramide comprises not more than 2wt% of arachidic acid ceramide, and especially 0.1-2 wt% of arachidic acid 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; 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; stearic acid ceramide, linolenic acid ceramide, arachidic acid ceramide, and the like.
In a second aspect of the invention, a method for synthesizing rice bran oil ceramide comprises the following steps:
under the conditions of a condensing agent and an organic base, rice bran oil fatty acid reacts with a sphingoid compound, wherein the condensing agent is EDCI, and the organic base is DIPEA.
Further, the molar ratio of the rice bran oil fatty acid to the sphingoid compound to EDCI to DIPEA 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 rice bran oil ceramide in cosmetics, pharmaceuticals, dietary foods or health products.
Further, the rice bran oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidation, collagen synthesis promotion, elastin activity maintenance, and whitening effects.
A composition comprising rice bran 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:
the rice bran oil fatty acid belongs to naturally formed fatty acid, the main component is unsaturated fatty acid-oleic acid, in addition, linoleic acid, linolenic acid and saturated fatty acid such as palmitic acid and stearic acid are also contained, and the rice bran oil ceramide is prepared by mild reaction with sphingoid compounds naturally existing in the skin, has excellent performance in the aspects of repairing natural barriers of the skin, resisting oxidation, resisting aging 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 different structures, and ceramides with a single structure generally have poor comprehensive effects. The scheme is based on a bionic idea, rice bran oil fatty acid from natural sources is used as a raw material to synthesize the composite ceramide so as to make up for the difference of the efficacies of different ceramides, and trace fatty acid in the rice bran 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, the rice bran oil also contains active ingredients such as vitamin E, sterol and oryzanol, and the nutrient substances have the effects of moistening skin, enhancing cell activity and the like. The ceramide synthesized by the rice bran oil has a synergistic effect with other active ingredients contained in the rice bran 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 rice bran 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
FIG. 1 shows the results of the cell migration ability test in example 4;
FIG. 2 is a bar graph of the elastase inhibition of example 5;
FIG. 3 is a bar graph of IL-6 factor expression levels measured for anti-inflammatory repair efficacy in example 6;
FIGS. 4 and 5 are bar graphs showing MMP1 expression levels in the anti-photoaging test of example 7;
FIG. 6 is a bar graph of DPPH radical scavenging for oxidation resistance test in example 8;
fig. 7 is a bar graph of melanin content in the whitening activity test of example 9.
Detailed Description
The present invention will be further described with reference to the following specific examples.
EDCI refers to 1-ethyl- (3-dimethylaminopropyl) carbodiimide and DIPEA refers to N, N-diisopropylethylamine. 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 rice bran oil fatty acid and phytosphingosine
Adding rice bran oil fatty acid (50 mmol, calculated as main component fatty acid), EDCI (75 mmol) and DIPEA (70 mmol) into a 250mL round-bottom flask, adding 100mL dichloromethane, stirring at room temperature for 1 hour, adding phytosphingosine (70 mmol) into the reaction system, and stirring at room temperature until TLC detection reaction is finished.
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 the rice bran 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-phytosphingosine ceramide 8.3min, linoleic acid-phytosphingosine ceramide 9.4min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.2min, stearic acid-phytosphingosine ceramide 13.8min, arachidic acid-phytosphingosine ceramide 16.5min.
The obtained product is analyzed by high performance liquid chromatography, and the contents of oleic acid-phytosphingosine ceramide, linoleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide, linolenic acid-phytosphingosine ceramide and arachidic acid-phytosphingosine ceramide are 51%, 30%, 12%, 2%, 1% and 1% in sequence, and the rest is other components with less content.
Example 2
Synthesis of ceramide from rice bran oil fatty acid and sphingosine
Adding rice bran oil fatty acid (50 mmol based on main component fatty acid), EDCI (65 mmol) and DIPEA (60 mmol) into a 250mL round-bottom flask, adding 100mL dichloromethane, stirring at room temperature for 1 hour, adding sphingosine (60 mmol) into the reaction system, and stirring at room temperature until TLC detection reaction is finished.
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 rice bran oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the mixture was purified by Innoval ODS-2.6 × 250mm,5 μm column, column temperature: 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-sphingosine ceramide 7.9min, linoleic acid-sphingosine ceramide 8.6min, oleic acid-sphingosine ceramide 10.1min, palmitic acid-sphingosine ceramide 10.4min, and stearic acid-sphingosine ceramide 13.6min.
The obtained product has high performance liquid chromatography analysis, and contains 43%, 40%, 7%, 5%, 2% of oleic acid-sphingosine ceramide, linoleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, and linolenic acid-sphingosine ceramide, and the rest is other components with low content.
Example 3
Synthesis of ceramide from rice bran oil fatty acid and dihydrosphingosine
Rice bran oil fatty acid (50 mmol, based on the main component fatty acid), EDCI (55 mmol) and DIPEA (55 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and then dihydrosphingosine (55 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 rice bran 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, linoleic acid-sphinganine ceramide 9.5min, palmitic acid-sphinganine ceramide 10.5min, oleic acid-sphinganine ceramide 11.1min, stearic acid-sphinganine ceramide 13.5min, and arachidic acid-sphinganine ceramide 16.1min.
According to the analysis of the obtained product by high performance liquid chromatography, the contents of oleic acid-dihydrosphingosine ceramide, linoleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, linolenic acid-dihydrosphingosine ceramide and arachidic acid-dihydrosphingosine ceramide are 54%, 21%, 17%, 3%, 1% and 2% in sequence, and the balance is other components with less content.
Example 4
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) 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) cell lineation. 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, 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 Cultured in an incubator atAfter 24h, the cells were removed, 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. 1, and the scratch width of the experimental group is narrower than that of the solvent control group, which indicates that the rice bran oil ceramide has better tissue healing capability. The healing rate of the solvent control group after 24h was 32.15%, the healing rate of the rice bran oil ceramide after 24h was 74.78%, 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 5
Elastase inhibition experiments to test 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 uniformly mixed in a vortex mode, the shaking table is shaken for 20min at 37 ℃ and 400r/min, 5mL of 0.5mol/L phosphate buffer solution with the pH value of 6.0 is immediately added, the mixture is uniformly mixed in a vortex mode, a proper amount of mixed solution is taken into a 2mL centrifugal tube, the centrifugal tube is centrifuged for 10min at the speed of 9 391 Xg, 200 mu L of supernatant liquid is precisely absorbed into a 96-well plate, the absorbance is measured by an enzyme-labeling instrument at the position of the wavelength of 495nm, and meanwhile, spectrum scanning of 400-800 nm is carried out.
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 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. When An ' > An exhibits a promoting effect, the promoting rate (%) = [1- (An ' -An)/(A0-A0 ') ] × 100%.
As shown in FIG. 2, the rice bran oil showed a good inhibitory effect on elastase at various concentrations, specifically, 8.47% at a concentration of 0.25g/L, 19.60% at a concentration of 0.5g/L, 22.33% at a concentration of 1.0g/L, and 25.67% at a concentration of 2.0 g/L.
Example 6
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. 3, where IL-6 levels were 10.43 times the basal levels when stimulated with LPS at a working concentration of 10. Mu.g/mL. Under the action of the rice bran 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 0.97, 0.85, 0.91 and 0.81 times of that of an LPS model group, and the factor level is dose-dependent, so that the rice bran oil ceramide is proved to have a certain anti-inflammatory effect and can promote the repair of inflammatory damaged skin.
Example 7
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, 100. Mu.L of medium containing samples (products of example 1) of different concentrations were added, no sample was added to the model group, DMEM medium without sample was used as the negative control group, 3 wells were added to each group, and CO was added at a mass fraction of 5% 2 After incubation at 37 ℃ for 2h, UVA or UVB ultraviolet radiation was applied. Purple pigmentThe distance between the external 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. The inhibition rate =1- (MMP 1 expression amount in experimental group/MMP 1 expression amount in model group) × 100%.
As shown in fig. 4 and 5, 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 rice bran oil ceramide at concentrations of 125, 250, and 400mg/L with respect to MMP1 expression in the model group were 29%, 41%, and 56%; in UVB, the MMP1 expression level of a negative control group is set as 1, the expression level of a model group is set as 2.33, and the inhibition rates of rice bran oil ceramide at concentrations of 125, 250 and 400mg/L relative to the MMP1 expression of the model group are 37%, 52% and 70%.
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 rice bran oil ceramide can inhibit the fibroblast caused by ultraviolet radiation from generating MMP1, and has certain effect on preventing skin photoaging.
Example 8
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. 6, the DPPH radical scavenging rates at concentrations of 50, 100, 200, 400, and 800mg/L were 8.43%, 11.91%, 16.02%, 20.60%, and 25.63%, respectively, and a certain antioxidant effect was exhibited.
Example 9
Whitening Activity test
Taking B16 cells in exponential growth phase, digesting with trypsin-EDTA (0.25 mass percent) and blowing uniformly, and dividing the cells into 3 multiplied by 10 5 The density of each well was seeded in 12-well plates. At 37 ℃ and 5% by mass of 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 blank group, incubating with DMEM medium as make module group, each group having 3 duplicate wells, and determining the concentration of CO at mass fraction of 5% 2 And incubating for 24 hours at 37 ℃. After discarding the medium from the well plate, washing once to twice with Phosphate Buffered Saline (PBS), 1mL of NaOH solution containing 10% by mass of DMSO (1 mol/L) was added to lyse the cells, and the cells were left to stand 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. 7, the melanin content of the blank control group was 1, the melanin expression of the building block was 1.51, and the melanin inhibition rates of the rice bran oil ceramide were 11.68%, 18.27%, 24.42%, 25.58% and 31.25% at concentrations of 10, 20, 40, 80 and 100mg/L, respectively, 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 should be subject to the protection scope of the claims.
Claims (9)
1. A rice bran oil ceramide obtained by reacting a rice bran oil fatty acid with a sphingoid compound selected from the group consisting of sphingosine, phytosphingosine, dihydrosphingosine.
2. The rice bran oil ceramide of claim 1, wherein the rice bran oil fatty acid comprises 40 to 60wt% oleic acid, 20 to 45wt% linoleic acid, 5 to 20wt% palmitic acid, 0.5 to 5wt% stearic acid, and 0.1 to 2wt% linolenic acid.
3. Rice bran oil ceramide, the composition of which comprises: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide.
4. The rice oil ceramide of claim 3, having a composition comprising: 40 to 60 weight percent of oleic acid ceramide, 20 to 45 weight percent of linoleic acid ceramide, 5 to 20 weight percent of palmitic acid ceramide, 0.5 to 5 weight percent of stearic acid ceramide, and 0.1 to 2 weight percent of linolenic acid ceramide.
5. The rice oil ceramide according to claim 3 or 4, further comprising in composition: 0-2 wt% of arachidic acid ceramide.
6. A method of synthesizing rice oil ceramide as claimed in any one of claims 1 to 5, comprising the steps of:
under the conditions of a condensing agent and organic base, rice bran oil fatty acid reacts with a sphingoid compound, wherein the condensing agent is EDCI, and the organic base is DIPEA;
the molar ratio of the rice bran oil fatty acid to the sphingoid compound to the EDCI to the DIPEA 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 rice bran 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 rice bran 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, and whitening efficacy.
9. A composition comprising the rice bran 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 promotion, elastin viability maintenance, and whitening efficacy.
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| CN202310051756.8A CN115947666A (en) | 2023-02-02 | 2023-02-02 | Rice bran 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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| CN202310051756.8A CN115947666A (en) | 2023-02-02 | 2023-02-02 | Rice bran oil ceramide and synthesis method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116173151A (en) * | 2023-04-21 | 2023-05-30 | 广东海洋大学 | Application of ceramide compound in antioxidation stress |
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