CN116003287A - Sunflower seed oil ceramide and synthesis method and application thereof - Google Patents
Sunflower seed oil ceramide and synthesis method and application thereof Download PDFInfo
- Publication number
- CN116003287A CN116003287A CN202310051732.2A CN202310051732A CN116003287A CN 116003287 A CN116003287 A CN 116003287A CN 202310051732 A CN202310051732 A CN 202310051732A CN 116003287 A CN116003287 A CN 116003287A
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- CN
- China
- Prior art keywords
- ceramide
- seed oil
- sunflower seed
- acid
- fatty acid
- 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.)
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- 239000008158 vegetable oil Substances 0.000 description 1
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Abstract
The invention belongs to the technical field of biological medicines, and discloses sunflower seed oil ceramide which is obtained by reacting sunflower seed oil fatty acid with a sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. The sunflower seed oil ceramide has excellent performances in the aspects of repairing natural skin barriers, anti-inflammatory, tissue healing, anti-aging and the like, and has wide application prospects in the fields of cosmetics, health-care products, biological medicines and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to sunflower seed oil ceramide and a synthesis method and application thereof.
Background
Ceramides (ceramides, also known as molecular nails) naturally occur in the skin and are very important components of the skin barrier (stratum corneum), in amounts of up to 40-50 wt.%, ceramides are a class of sphingolipids consisting of long-chain bases of sphingosine and fatty acids, in which the carbon chain length, unsaturation and number of hydroxyl groups of the sphingosine moiety, fatty acid moiety are all variable, and ceramides represent a class of compounds. Ceramide has excellent properties in regulating skin barrier function, recovering skin moisture, enhancing adhesion between skin keratinocytes, and the like.
Because of 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 more sustainable and more environment-friendly raw material source and the characteristics similar to the skin ceramide components, and can become a next-generation environment-friendly, safe and reliable ceramide product.
Sunflower seed oil is a healthy vegetable oil extracted from sunflower seeds and known for its high content of linoleic acid. Sunflower seed oil is rich in various fatty acids essential to human body, mainly comprising: linoleic acid, oleic acid, stearic acid, palmitic acid. In addition, it also contains vitamin E, plant sterol, phospholipid, carotene, etc. The refined sunflower seed oil is clear faint yellow or green yellow, has fragrant smell, pure taste and high nutritive value, and has good effects of resisting oxidation, preserving moisture and delaying cell aging. In the health care field, the sunflower seed oil also has the effects of reducing cholesterol, preventing arteriosclerosis, preventing coronary heart disease, regulating metabolism, maintaining blood pressure balance and the like.
Disclosure of Invention
The invention aims to provide ceramide synthesized by sunflower seed oil fatty acid of plant origin.
Another object of the present invention is to provide a method for synthesizing sunflower seed oil ceramide, which uses sunflower seed oil or sunflower seed oil fatty acid which is natural plant source and is easy to obtain as raw material.
It is another object of the present invention to provide the use of sunflower seed oil ceramide.
In order to achieve one of the above purposes, the present invention adopts the following technical scheme:
in a first aspect of the invention, sunflower seed oil ceramide is obtained by reacting sunflower seed oil fatty acid with a sphingosine compound selected from the group consisting of sphingosine, phytosphingosine and sphinganine.
The reaction can be chemical synthesis reaction (as detailed below), or microbial fermentation method, i.e. using Pichia pastoris or Saccharomyces cerevisiae, fermenting under certain environment to obtain sphingosine compound, and adding fatty acid to obtain ceramide; or using sunflower seed oil as raw material, selecting proper strain, and making fermentation so as to obtain the invented sunflower seed 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 sunflower seed oil fatty acid is obtained by hydrolyzing sunflower seed oil grease.
Further, the sunflower seed oil fatty acid contains 55-75wt% linoleic acid.
Further, the sunflower seed oil fatty acid contains 20-40 wt% of oleic acid.
Further, the sunflower seed oil fatty acid contains 0.5 to 7 weight percent of stearic acid.
Further, the sunflower seed oil fatty acid contains 1 to 8wt% of palmitic acid.
In addition, the sunflower seed oil fatty acid also contains 0 to 2 weight percent of behenic acid and 0 to 1 weight percent of arachidic acid.
The composition of sunflower seed oil fatty acid is: 55 to 75 weight percent of linoleic acid, 20 to 40 weight percent of oleic acid, 0.5 to 7 weight percent of stearic acid, 1 to 8 weight percent of palmitic acid, 0 to 2 weight percent of behenic acid and 0 to 1 weight percent of arachidic acid.
The main component of the sunflower seed oil fatty acid is linoleic acid, other fatty acids comprise oleic acid, stearic acid and palmitic acid, which are essential components, and are influenced by plant varieties, soil, climate, production places, picking seasons and extraction processes, the content of each component is different, and the behenic acid and the arachidic acid are not necessarily contained, and are optional components or unnecessary components.
Sunflower seed oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, stearic acid ceramide, palmitic acid ceramide; because fatty acids all participate in the same reaction, the mass ratio of ceramide after the reaction is not changed greatly, so the composition of the ceramide of sunflower seed oil is similar to that of sunflower seed oil fatty acid, and the composition of the ceramide of sunflower seed oil is as follows: 55 to 75 weight percent of linoleic acid ceramide, 20 to 40 weight percent of oleic acid ceramide, 0.5 to 7 weight percent of stearic acid ceramide and 1 to 8 weight percent of palmitic acid ceramide. The content of each component is different due to the different content of each fatty acid in sunflower seed oil fatty acid or grease. In addition, the sunflower seed oil ceramide also comprises ceramide obtained by reacting one or more of behenic acid and arachidic acid with sphingosine compounds, namely, 0-2wt% of behenic acid ceramide and 0-1wt% of arachidic acid ceramide. Sunflower seed oil ceramides also include compounds that are present in sunflower seed oil fatty acids but that are not reactive with sphingosine compounds, vitamin E, phytosterols, phospholipids, carotenes, and the like.
Sunflower seed oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide; the linoleic acid ceramide accounts for 55 to 75 weight percent, and the oleic acid ceramide accounts for 20 to 40 weight percent.
Further, the sunflower seed oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 0.5-7wt%.
Further, the sunflower seed oil ceramide comprises palmitic acid ceramide, and the palmitic acid ceramide accounts for 1-8wt%.
Further, the sunflower seed oil ceramide comprises not more than 2wt% of behenic acid ceramide and not more than 1wt% of arachidic acid ceramide, especially 0.1 to 2wt% of behenic acid ceramide and 0.1 to 1% of arachidic acid ceramide.
The linoleic acid ceramide is obtained by condensation reaction of linoleic acid and sphingosine compounds, and comprises linoleic acid phytosphingosine ceramide, linoleic acid sphingosine ceramide and linoleic acid dihydrosphingosine ceramide; oleic acid ceramide is obtained by condensation reaction of oleic acid and sphingosine compounds, and comprises oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide and oleic acid dihydrosphingosine ceramide; stearic acid ceramide is obtained by condensation reaction of stearic acid and sphingosine compound, and comprises stearic acid phytosphingosine ceramide, stearic acid sphingosine ceramide and stearic acid dihydrosphingosine ceramide; the palmitic acid ceramide is obtained by condensation reaction of palmitic acid and sphingosine compounds, and comprises palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide and palmitic acid dihydrosphingosine ceramide; behenic acid ceramide, arachidic acid ceramide, and the like.
In a second aspect of the invention, the method for synthesizing sunflower seed oil ceramide comprises the following steps:
under the conditions of condensing agent and organic base, sunflower seed oil fatty acid reacts with sphingosine compound, the condensing agent is EDCI, and the organic base is DIPEA.
Further, the molar ratio of the sunflower seed oil fatty acid to the sphingosine compound to the EDCI to the DIPEA 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.
Commercially available sunflower seed oil is generally in the form of oil and requires hydrolysis to sunflower seed oil fatty acids, and therefore comprises the following steps:
the sunflower seed oil fat is hydrolyzed by saponification reaction to obtain sunflower seed oil fatty acid.
Further, the saponification reaction is hydrolysis of sunflower seed oil fat in potassium hydroxide solution.
Further, the mass ratio of the sunflower seed oil grease to the potassium hydroxide is 1: (1-2).
In a third aspect of the invention, the use of sunflower seed oil ceramide in cosmetics, pharmaceutical products, dietary or health care products.
Further, the sunflower seed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, collagen synthesis promoting, elastin activity maintaining, and whitening effects.
A composition comprising sunflower seed 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, whitening efficacy.
The composition contains acceptable auxiliary materials, including one or more of solubilizer, antiseptic, antioxidant, pH regulator, penetration enhancer, liposome, humectant, thickener, chelating agent, skin feel regulator, surfactant, emulsifier, essence and pigment; the composition is in the form of cream, emulsion, solution, film, aerosol or spray.
The invention has the following beneficial effects:
the sunflower seed oil fatty acid belongs to naturally-formed fatty acid, the main component is unsaturated fatty acid-linoleic acid, and in addition, oleic acid, saturated fatty acid such as stearic acid, palmitic acid and the like are contained, and the sunflower seed oil ceramide is prepared by mild reaction with a sphingosine compound naturally existing in skin, so that the sunflower seed oil ceramide has excellent performance in the aspects of repairing a natural skin barrier, resisting oxidation, resisting aging and the like, and has wide application prospects in the fields of cosmetics, health-care products, biological medicines and the like.
1. Better results compared to ceramide alone. Different ceramides have different effects due to the structural differences, and ceramides with a single structure generally have difficulty in having comprehensive effects. The scheme is based on a bionic thought, and the natural sunflower seed oil or fatty acid is used as a raw material to synthesize the compound ceramide so as to make up the difference of different ceramide effects, and trace fatty acid in the sunflower seed oil can form trace ceramide to play a role in efficacy supplementation.
2. Compared with the compounded ceramide, the effect is better. Besides fatty acid (or oil), sunflower seed oil also contains nutrient components such as vitamin E, plant sterol, phospholipid, carotene and the like, and the nutrient components have good effects of resisting oxidation, preserving moisture and delaying cell aging. The ceramide synthesized by sunflower seed oil has a synergistic effect with other active ingredients contained in the sunflower seed oil, and has better effect compared with ceramide compounded according to similar proportion.
3. The cost is lower. The method of the invention can rapidly obtain the composition compounded by various ceramides, and the plant-derived sunflower seed oil or the fatty acid thereof has wide sources, is easy to obtain commercially, has lower cost, is more environment-friendly and economical, is different from the idea of mixing and compounding different single ceramides, has high raw material price, needs to separately produce different ceramides and then compound, and increases the preparation cost.
4. The synthesis 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 showing the results of the cell proliferation activity test of example 4;
FIG. 3 is the result of the cell migration ability test of example 5;
FIGS. 4 and 5 are bar graphs of elastase inhibition ratios of example 6;
FIG. 6 is a bar graph showing the detection of IL-6 factor expression level in anti-inflammatory repair efficacy in example 7;
FIGS. 7 and 8 are bar charts of MMP1 expression levels in the photo-aging test of example 8;
FIGS. 9 and 10 are bar graphs of DPPH radical scavenging for oxidation resistance test of example 9;
FIG. 11 is a bar graph showing the whitening activity test melanin content of example 10.
Detailed Description
The invention will be further illustrated with reference to specific examples.
EDCI refers to 1-ethyl- (3-dimethylaminopropyl) carbodiimide and DIPEA refers to N, N-diisopropylethylamine. The silica gel column chromatography uses Qingdao ocean silica gel (particle size 0.040-0.063 mm). Thin Layer Chromatography (TLC) using 60F254 silica gel plates was performed using UV light (254 nm) or iodine.
Example 1
Synthesis of ceramide from sunflower seed oil fatty acid and phytosphingosine
The first step: 50g of sunflower seed oil is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is cooled to room temperature for reaction after the dropwise addition until TLC detection is completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 150mL of ethyl acetate to extract a water phase, adding 100mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Dried, filtered and concentrated in vacuo to yield 40.5g of sunflower oil fatty acid.
And a second step of: sunflower seed oil fatty acid (50 mmol, based on the main component fatty acid), EDCI (60 mmol), DIPEA (60 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and phytosphingosine (60 mmol) was added to the reaction system, followed by stirring at room temperature until TLC detection was complete.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain sunflower seed oil ceramide, and analyzing a product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an shimadzu high performance liquid chromatograph (LC-2030 c3d Plus), column temperature with Innoval ODS-2.6x250 mm,5 μm column: 30 ℃, sample injection volume: 10 μl, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The retention time of each component HPLC was: linoleic acid-phytosphingosine ceramide 9.4min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.3min, stearic acid-phytosphingosine ceramide 13.9min, arachidic acid-phytosphingosine ceramide 16.5min, and behenic acid-phytosphingosine ceramide 18.3min.
The obtained product is analyzed by high performance liquid chromatography, and the contents of linoleic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, behenic acid-phytosphingosine ceramide and arachidic acid-phytosphingosine ceramide are 58%, 27%, 4%, 5%, 2%, 1% and the rest are other components, and the content is less.
Example 2
Synthesis of ceramide from sunflower seed oil fatty acid and sphingosine
The first step: 50g of sunflower seed oil is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is cooled to room temperature for reaction after the dropwise addition until TLC detection is completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 150mL of ethyl acetate to extract a water phase, adding 100mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Drying, filtration and concentration in vacuo gave 41g of sunflower oil fatty acid.
And a second step of: sunflower seed oil fatty acid (50 mmol, based on the main component fatty acid), EDCI (70 mmol), DIPEA (70 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and then sphingosine (65 mmol) was added to the reaction system, followed by stirring at room temperature until TLC detection was complete.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain sunflower seed oil ceramide, and analyzing a product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an shimadzu high performance liquid chromatograph (LC-2030 c3d Plus), column temperature with Innoval ODS-2.6x250 mm,5 μm column: 30 ℃, sample injection volume: 10 μl, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The retention time of each component HPLC was: linoleic acid-sphingosine ceramide 8.6min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.4min, stearic acid-sphingosine ceramide 13.5min, arachidic acid-sphingosine ceramide 17.8min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-sphingosine ceramide, oleic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide and arachidic acid-sphingosine ceramide are 65%, 22%, 6%, 3%, 1% in sequence, and the rest are other components, and the content is low.
Example 3
Synthesis of ceramide from sunflower seed oil fatty acid and sphinganine
The first step: 50g of sunflower seed oil is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is cooled to room temperature for reaction after the dropwise addition until TLC detection is completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 150mL of ethyl acetate to extract a water phase, adding 100mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Dried, filtered and concentrated in vacuo to yield 42g sunflower oil fatty acid.
And a second step of: sunflower seed oil fatty acid (50 mmol, based on the main component fatty acid), EDCI (80 mmol), DIPEA (65 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, then sphinganine (55 mmol) was added to the reaction system, and stirring at room temperature was performed until TLC detection was complete.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain sunflower seed oil ceramide, and analyzing a product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an shimadzu high performance liquid chromatograph (LC-2030 c3d Plus), column temperature with Innoval ODS-2.6x250 mm,5 μm column: 30 ℃, sample injection volume: 10 μl, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The retention time of each component HPLC was: linoleic acid-sphinganine ceramide 9.4min, palmitic acid-sphinganine ceramide 10.6min, oleic acid-sphinganine ceramide 11.2min, stearic acid-sphinganine ceramide 13.5min, and behenic acid-sphinganine ceramide 21.9min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-dihydrosphingosine ceramide, oleic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide and behenic acid-dihydrosphingosine ceramide are 55%, 32%, 3%, 7%, 1% in sequence, and the rest are other components, and the content is less.
Example 4
MTT method for detecting proliferation activity of compound on cell
HaCaT cells were grown at 1X 10 4 The density of individuals/wells was seeded in 96-well plates and the incubator was overnight. 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, 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 Drug delivery hole /A Blank hole ×100％。
As shown in FIG. 1, sunflower seed oil ceramide has a promoting effect on cell viability, and cell viability rates at concentrations of 1.95313, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, and 250mg/L are 91.25%, 83.90%, 88.78%, 98.94%, 97.12%, 102.17%, 100.88%, and 107.99%, respectively. The composition has the effect of promoting cell proliferation at a higher concentration, and has a certain tissue repair capability.
The proliferation activity of ceramide 2 on cells was measured in the same manner, and as a result, as shown in FIG. 2, cell viability was 61.49%,60.03%,55.41%,54.64%,53.37%,46.95%,44.05%,40.35%,39.42% at concentrations of 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000mg/L, respectively, which had an inhibitory effect on cell proliferation and a tissue repair potential inferior to that of sunflower seed oil ceramide.
Example 5
Assessment of skin barrier repair by cell migration
Principle of: when the cells grow to be fused into a single-layer state, a scratch tool is manufactured on the fused single-layer cells, the cells in the blank area are removed by mechanical force, the migration condition of the cells to the cell-free area is observed through a period of culture, and the migration capability of the cells is reflected by measuring the migration distance of the cells.
The operation steps are as follows:
1. the culture plate is streaked. Firstly, a Marker pen is used for uniformly scribing transverse lines by comparing with a straight ruler, and the transverse lines are crossed through the through holes at intervals of about 0.5 cm to 1cm, and each hole at least passes through 5 lines, so that attention lines are not too thick when scribing.
2. And (5) paving cells. About 5X 10 is added to the well 5 Individual cells (the number of different cells is different, and the cell growth speed is regulated), and the inoculation principle is that the fusion rate reaches 100% after overnight.
3. Cell streaking. The next day the tip is used to scratch the cell layer along the line marked on the back of the plate on the first day, perpendicular to the cell plane (the same tip is preferably used between the different wells).
4. Washing cells. After the streaking was completed, cells were washed 3 times with sterile PBS, cells that did not adhere to the wall, i.e., streaked cells at streaking, and the gap left after streaking was clearly visible, followed by replacement of fresh serum-free medium.
5. And (5) culturing and observing the cells. After the sample (product of example 1, ceramide 3B) was diluted with the medium (product of example 1, concentration of ceramide 3B was 50mg/L, concentration of ceramide 3B was 100 mg/L), the cells were placed in a cell culture dish, and the cells were placed in 5wt% CO at 37 ℃C 2 Incubator culture, after 24 hours, cells were removed, observed with a microscope and the width of scratches was measured, and photographed, and the healing rate was calculated using 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 sunflower seed oil ceramide has better tissue healing capacity. The solvent control group had a healing rate of 12.57% after 24 hours, sunflower seed oil ceramide had a healing rate of 83.52% after 24 hours, and ceramide 3B had a healing rate of 59.32% after 24 hours. The compound obviously improves the cell healing rate, has good skin tissue repair activity and has better effect than ceramide 3B.
Example 6
Elastase inhibition experiment tests anti-aging effect
Elastase inhibition method: 2mg/mL elastase solution (product of example 1) is taken, samples with different concentrations (2 mL) are added, vortex mixing is carried out fully, shaking is carried out for 20min at 37 ℃ by a 400r/min shaking table, 5mL of 0.5mol/L phosphate buffer solution with pH of 6.0 is added immediately, vortex mixing is carried out, a proper amount of mixed solution is taken into a 2mL centrifuge tube, centrifugation is carried out for 10min at 9 391×g, 200 mu L of supernatant is sucked into a 96-well plate precisely, absorbance is measured by an enzyme-labeled instrument at a wavelength of 495nm, and spectrum scanning at 400-800 nm is carried out simultaneously.
The substrate enzyme adding solution is used as a blank control group, the substrate enzyme adding and sample solution is used as an enzyme inhibition group, and the substrate enzyme adding and sample solution is used as a background. Each group is provided with 3 multiple holes. Inhibition ratio (%) = [1- (An-An ')/(A0-A0') ] ×100%, where A0 is absorbance with no enzyme added to the sample, A0 'is absorbance with no enzyme added to the substrate and no sample added to the enzyme, an is absorbance with only sample solution, an' is absorbance with no enzyme added to the sample. When An ' > An, the effect is expressed as acceleration, and the acceleration rate (%) = [1- (An ' -An)/(A0-A0 ') ] ×100%.
As shown in FIG. 4, sunflower seed oil ceramide has a good effect of inhibiting elastase at various concentrations, specifically, an inhibition rate of 17.47% for elastase at a concentration of 0.25g/L, an inhibition rate of 21.67% for elastase at a concentration of 0.5g/L, an inhibition rate of 23.60% for elastase at a concentration of 1.0g/L, and an inhibition rate of 33.33% for elastase at a concentration of 2.0 g/L.
The inhibitory activity of ceramide 2 against elastase was measured in the same manner, and as a result, as shown in FIG. 5, the elastase inhibition rates at concentrations of 0.25, 0.5, 1.0 and 2.0g/L were 10.12%, 18.06%, 28.84% and 19.78%, respectively, which were not as good as those of sunflower seed oil ceramide at the same concentrations.
Example 7
LPS induced cell method for detecting anti-inflammatory repair efficacy
B16 mouse melanoma cells were grown at a density of 1X 10 4 The cells/wells were seeded in 96-well plates, placed in an incubator overnight, the supernatant was discarded after 24 hours, 100. Mu.L of samples of different concentrations diluted with DMEM medium (product of example 1) were added, the negative control group was DMEM medium without samples, 3 wells per group, and mixed with CO at 5 wt.% 2 Incubate at 37 ℃. Lipopolysaccharide model group and experimental group were added with 10 μg/mL LPS and incubated together for 24h 2h after dosing. After the reaction, 50. Mu.L of the cell supernatant was collected, and the intracellular IL-6 gene expression was detected using an IL-6ELISA kit.
The results are shown in FIG. 6, where IL-6 levels were 11.51 times the basal levels at a working concentration of 10. Mu.g/mL of LPS stimulation. Under the action of sunflower seed oil ceramide with the concentration of 50mg/L, 100mg/L, 200mg/L and 400mg/L respectively, the IL-6 factor level is obviously reduced and is 0.84 times, 0.68 times, 0.52 times and 0.31 times of that of an LPS model group respectively, and the sunflower seed oil ceramide is dose-dependent, so that the sunflower seed 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 matrix metalloproteinase family, and its main acting substrate is fibrous collagen, which can degrade collagen fiber and gelatin in extracellular matrix and change microenvironment of cells. MMP1 plays an important role in elastin, inhibiting MMP1 can improve the synthesis of fibroblast collagen and elastin, and reducing MMP activity can increase the collagen synthesis speed.
HaCaT cells were grown at 1X 10 5 The density of individuals/wells was seeded in 96-well plates and the incubator was overnight. After 24h, the supernatant was discarded, 100. Mu.L of medium containing samples of different concentrations (product of example 1) was added, no samples were added to the model group, the negative control group was DMEM medium without samples, 3 wells per group, and the mass fraction was 5% CO 2 After incubation for 2h at 37℃either UVA or UVB ultraviolet radiation is irradiated. Purple (purple)The distance between the source of the external radiation and the cells was 15cm and the UVA intensity was 200mJ/cm 2 The irradiation time was 2 hours, and the UVB intensity was 50mJ/cm 2 The irradiation time was 1h. After the end of irradiation, incubation was continued for 12h in the incubator. Intracellular MMP-1 gene expression was detected using an MMP-1ELISA kit. Inhibition = 1- (experimental group MMP1 expression level/model group MMP1 expression level) ×100%.
As shown in fig. 7 and 8, 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 ratio of MMP1 expression in the sunflower seed oil ceramide was 16%, 30%, 42% relative to the model group at the concentrations of 125, 250, and 400 mg/L; the expression level of MMP1 in the negative control group was 1, the expression level in the model group was 2.33, and the inhibition rate of MMP1 expression in the sunflower seed oil ceramide was 30%, 41% and 54% relative to the model group at concentrations of 125, 250 and 400 mg/L.
After UVA uv radiation, keratinocytes promote elevated expression of MMP1 by fibroblasts, thereby causing degradation of the extracellular matrix of the skin and collagen of the skin, leading to photoaging of the skin. The results show that the sunflower seed oil ceramide can inhibit the fibroblast from producing MMP1 caused by ultraviolet radiation, and has a certain effect of preventing skin photoaging.
Example 9
DPPH free radical scavenging detection of antioxidant performance
DPPH is 1, 1-diphenyl-2-trinitrophenylhydrazine, and can be used for antioxidant experiments.
Samples (product of example 1) at 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 DPPH and absolute ethyl alcohol 1:1, and the absorbance at 517 nm. The absorbance of the sample and the reaction solution of DPPH was designated as A1, the absorbance of the sample and the reaction solution of absolute ethyl alcohol was designated as A2, the absorbance of the reaction solution of DPPH and absolute ethyl alcohol was designated as A3, and the clearance rate of DPPH of the sample was = [1- (A1-A2)/A3 ]. Times.100%.
As a result, as shown in FIG. 9, the DPPH radical scavengers at concentrations of 50, 100, 200, 400 and 800mg/L were 9.43%, 23.91%, 35.82%, 42.16% and 52.24%, respectively, and excellent antioxidant effects were exhibited. The antioxidant effect of ceramide 3B (i.e., oleic acid ceramide) was measured in the same manner, and the result was shown in FIG. 10, in which DPPH radical scavenging rate was 7.76%, 12.82%, 24.10%, 29.60% and 33.16% at concentrations of 50, 100, 200, 400 and 800 mg/L. The clearance rate of the sunflower seed oil ceramide to DPPH is higher than that of ceramide 3B, and the sunflower seed oil ceramide has better antioxidation effect.
Example 10
Whitening Activity test
Taking B16 cells in exponential growth phase, digesting with trypsin-EDTA with mass fraction of 0.25%, blowing uniformly, and mixing the cells according to 3×10 5 Density of individual/well was seeded in 12-well plates. At 37 ℃, the mass fraction of CO is 5 percent 2 Incubated overnight in the environment. Removing supernatant, adding culture solution containing samples with different mass concentrations (product of example 1), incubating with RPMI-1640 culture medium without sample as blank group, incubating with DMEM culture medium as mould group, and incubating with 3 compound holes in each group at mass fraction of 5% CO 2 Incubation was carried out for 24h at 37 ℃. The culture medium in the well plate is discarded, and after washing with Phosphate Buffer (PBS) for one to two times, 1mL of NaOH solution (1 mol/L) containing 10% DMSO by mass fraction is added to lyse the cells, and the cells are kept at a constant temperature of 80℃or 100℃for 2 hours until the cells are completely lysed. The absorbance was measured at 405nm in a microplate reader. The melanin inhibition rate=1- (OD value per well/OD value of model group) ×100% was calculated.
As a result, as shown in FIG. 11, the melanin content of the blank group was 1, the melanin expression of the model group was 1.51, and the melanin inhibition rates of sunflower seed oil ceramide were 2.38%, 10.70%, 11.54%, 14.90% and 25.38% at concentrations of 10, 20, 40, 80 and 100mg/L, respectively, and excellent whitening effects were exhibited.
The foregoing is merely illustrative embodiments of the present invention, and the present invention is not limited thereto, and any changes or substitutions that may be easily contemplated by those skilled in the art within the scope of the present invention should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (10)
1. Sunflower seed oil ceramide, which is obtained by reacting sunflower seed oil fatty acid with a sphingosine compound selected from the group consisting of sphingosine, phytosphingosine and dihydrosphingosine.
2. Sunflower seed oil ceramide of claim 1, wherein the sunflower seed oil fatty acid is obtained by hydrolysis of sunflower seed oil.
3. Sunflower seed oil ceramide according to claim 1 or 2, wherein the sunflower seed oil fatty acid comprises 55-75 wt% linoleic acid, 20-40 wt% oleic acid, 0.5-7 wt% stearic acid, 1-8 wt% palmitic acid.
4. Sunflower seed oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, stearic acid ceramide, palmitic acid ceramide.
5. The sunflower seed oil ceramide of claim 4, comprising the composition of: 55 to 75 weight percent of linoleic acid ceramide, 20 to 40 weight percent of oleic acid ceramide, 0.5 to 7 weight percent of stearic acid ceramide and 1 to 8 weight percent of palmitic acid ceramide.
6. The sunflower seed oil ceramide of claim 4 or 5, further comprising: 0 to 2 weight percent of behenic acid ceramide and 0 to 1 weight percent of arachidic acid ceramide.
7. The method for synthesizing sunflower seed oil ceramide according to any one of claims 1 to 6, comprising the following steps:
under the conditions of condensing agent and organic base, sunflower seed oil fatty acid reacts with sphingosine compound, wherein the condensing agent is EDCI, and the organic base is DIPEA;
the molar ratio of the sunflower seed oil fatty acid to the sphingosine compound to EDCI to DIPEA 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.
8. Use of the sunflower seed oil ceramide of any one of claims 1-6 in cosmetics, pharmaceuticals, dietary or health products.
9. The use according to claim 8, wherein the sunflower seed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, promoting collagen synthesis, maintaining elastin viability, whitening efficacy.
10. A composition comprising the sunflower seed oil ceramide of any one of claims 1-6, the composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, promoting collagen synthesis, maintaining elastin viability, whitening efficacy.
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