CN117304060A - Soybean oil ceramide and synthesis method and application thereof - Google Patents

Soybean oil ceramide and synthesis method and application thereof Download PDF

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CN117304060A
CN117304060A CN202310051760.4A CN202310051760A CN117304060A CN 117304060 A CN117304060 A CN 117304060A CN 202310051760 A CN202310051760 A CN 202310051760A CN 117304060 A CN117304060 A CN 117304060A
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ceramide
acid
soybean oil
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fatty acid
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杨超文
叶柳
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Shenzhen Dikeman Biotechnology Co ltd
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Shenzhen Dikeman Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of biological medicines, and discloses soybean oil ceramide which is obtained by reacting soybean oil fatty acid with a coleopinol compound, wherein the coleopinol compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. The soybean 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

Soybean oil ceramide and synthesis method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to soybean oil ceramide and a synthesis method and application thereof.
Background
Ceramides (ceramides, also known as molecular nails) are naturally present in the skin and are very important components of the skin barrier (stratum corneum), in amounts up to 40-50 wt.%, and are a class of sphingolipids consisting of coleoplastics long-chain bases and fatty acids, in which the carbon chain length, unsaturation and hydroxyl number of the sphingosine moiety, fatty acid moiety are all variable, and ceramides represent a class of compounds. Ceramide exhibits excellent properties in regulating skin barrier function, recovering skin moisture, and enhancing adhesion between skin keratinocytes.
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 environment-friendly raw material source and the characteristics similar to the skin ceramide components, and is likely to become a next-generation environment-friendly, safe and reliable ceramide product.
Soybean oil is one of the most commonly used cooking oils, and contains a large amount of linoleic acid, which is a vegetable oil based on linoleic acid. Linoleic acid is a fatty acid essential to the human body and has important physiological functions. The infants lack linoleic acid, the skin becomes dry, scales thicken and the development and growth are slow; the old people lack linoleic acid, which can cause white internal disorder and cardiovascular and cerebrovascular diseases. In addition, soybean oil also contains oleic acid, again palmitic acid and linolenic acid, and the ratio of other fatty acids is not large.
Disclosure of Invention
The invention aims to provide ceramide synthesized by soybean oil fatty acid of plant origin.
It is another object of the present invention to provide a method for synthesizing soybean oil ceramide, which uses soybean oil fatty acid which is natural plant source and is easily available as raw material.
It is another object of the present invention to provide the use of soybean 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, a soybean oil ceramide is obtained by reacting a soybean oil fatty acid with a coleopinol compound selected from the group consisting of sphingosine, phytosphingosine, dihydrosphingosine.
The reaction can be chemical synthesis reaction (as detailed below), or microorganism fermentation method, i.e. Pichia pastoris or Saccharomyces cerevisiae is used to ferment under certain environment to obtain coleoplastics compound, and then fatty acid is added to obtain ceramide; or soybean oil is used as a raw material, and proper strains are selected for fermentation to obtain the soybean 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 soybean oil fatty acid contains 50 to 75wt% linoleic acid.
Further, the soybean oil fatty acid contains 15 to 30wt% oleic acid.
Further, the soybean oil fatty acid contains 2 to 12wt% palmitic acid.
Further, the soybean oil fatty acid contains 1 to 10wt% of linolenic acid.
Further, the soybean oil fatty acid contains 1 to 5wt% of stearic acid.
In addition, the soybean oil fatty acid also contains 0-2 wt% of peanut acid and 0-2 wt% of behenic acid.
The composition of the soybean oil fatty acid is as follows: 50 to 75 weight percent of linoleic acid, 15 to 30 weight percent of oleic acid, 2 to 12 weight percent of palmitic acid, 1 to 10 weight percent of linolenic acid, 1 to 5 weight percent of stearic acid, 0 to 2 weight percent of peanut acid and 0 to 2 weight percent of behenic acid.
The main component of the soybean oil fatty acid is linoleic acid, the other fatty acids comprise oleic acid, palmitic acid, linolenic acid and stearic 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 can be different, and the content of the peanut acid and the behenic acid is not necessarily contained, so that the soybean oil fatty acid is an optional component or an unnecessary component.
Soybean oil ceramide, the composition of which comprises: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, linolenic acid ceramide, stearic acid ceramide; since fatty acids all participate in the same reaction, the mass ratio of ceramide after the reaction does not change much, so the composition of the soybean oil ceramide is similar to that of soybean oil fatty acid, and the composition of the soybean oil ceramide is as follows: 50 to 75 weight percent of linoleic acid ceramide, 15 to 30 weight percent of oleic acid ceramide, 2 to 12 weight percent of palmitic acid ceramide, 1 to 10 weight percent of linolenic acid ceramide and 1 to 5 weight percent of stearic acid ceramide. The content of each component is different due to the different content of each fatty acid in the soybean oil fatty acid. In addition, the soybean oil ceramide also comprises ceramide obtained by the reaction of one or more of the peanut acid and the behenic acid with coleoplastics, namely 0 to 2 weight percent of the peanut acid ceramide and 0 to 2 weight percent of the behenic acid ceramide.
Soybean oil ceramide, the composition of which comprises: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, linolenic acid ceramide; 50-75wt% of linoleic acid ceramide, 15-30wt% of oleic acid ceramide, 2-12wt% of palmitic acid ceramide and 1-10wt% of linolenic acid ceramide.
Further, the soybean oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 1-5 wt%.
Further, the soybean oil ceramide comprises not more than 2wt% of the peanut acid ceramide and not more than 2wt% of the behenic acid ceramide, particularly 0.1 to 2wt% of the peanut acid ceramide and 0.1 to 2wt% of the behenic acid ceramide.
The linoleic acid ceramide is obtained by condensation reaction of linoleic acid and coleopinol 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 coleopinol compounds, and comprises oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide and oleic acid dihydrosphingosine ceramide; the palmitic acid ceramide is obtained by condensation reaction of palmitic acid and coleoplastics, and comprises palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide and palmitic acid dihydro sphingosine ceramide; linolenic acid ceramide, stearic acid ceramide, arachic acid ceramide, behenic acid ceramide, and the like.
In a second aspect of the invention, a method for synthesizing soybean oil ceramide comprises the following steps:
under the conditions of condensing agent and organic base, the soybean oil fatty acid reacts with coleopinol compound, wherein the condensing agent is EDCI, and the organic base is DIPEA.
Further, the molar ratio of the soybean oil fatty acid to the coleophagin 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.
In a third aspect of the invention, the use of soybean oil ceramide in cosmetics, pharmaceutical products, dietary or health care products.
Further, the soybean 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 soy 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 adjuvants including one or more of solubilizer, preservative, antioxidant, pH regulator, penetration enhancer, liposome, humectant, thickener, chelating agent, skin feel regulator, surfactant, emulsifying agent, 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 soybean oil fatty acid belongs to naturally-formed fatty acid, the main component is unsaturated fatty acid-linoleic acid, and in addition, oleic acid, linolenic acid and saturated fatty acid such as palmitic acid and stearic acid are contained, and the soybean oil fatty acid and coleophaga compounds naturally existing in skin are subjected to mild reaction to prepare the soybean oil ceramide, so that the soybean oil ceramide has excellent performance in the aspects of repairing natural skin barriers, 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 difference of structures, and ceramides with a single structure generally have difficulty in having comprehensive effects. The scheme is based on the bionic thought, and the soybean oil fatty acid from natural sources 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 soybean oil can form trace ceramide to play a role in efficacy supplement.
2. The effect is better compared with the compound ceramide. In addition to fatty acids, soybean oil also contains other nutrients which have effects of moisturizing skin, enhancing cell viability, etc. The ceramide synthesized by the soybean oil has a synergistic effect with other active ingredients contained in the soybean oil, and has better effect compared with the ceramide compounded according to similar proportion.
3. The cost is lower. The method can quickly obtain the composition compounded by various ceramides, and the botanical soybean oil fatty acid 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 is compounded, thereby increasing the preparation cost.
4. The synthesis method is simple. The method of the invention can adopt chemical synthesis to realize one-step preparation of various ceramides, and can also adopt a microbial fermentation method.
Drawings
FIG. 1 is a graph showing the results of the cell migration ability test of example 4;
FIG. 2 is a bar graph of elastase inhibition of example 5;
FIG. 3 is a bar graph showing the detection of IL-6 factor expression level in anti-inflammatory repair efficacy of example 6;
FIGS. 4 and 5 are bar charts of MMP1 expression levels in the photo-aging test of example 7;
FIGS. 6 and 7 are bar graphs of DPPH radical scavenging for oxidation resistance test of example 8;
FIG. 8 is a bar graph showing the whitening activity test melanin content of example 9.
Detailed Description
The invention will be further described 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 adopts Qingdao ocean silica gel (particle size 0.040-0.063 mm). Thin Layer Chromatography (TLC) using 60F254 silica gel plates and TLC developed using UV light (254 nm) or iodine.
Example 1
Synthesis of ceramide from soybean oil fatty acid and phytosphingosine
Soybean oil fatty acid (50 mmol based on 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 (55 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 organic layer, drying, filtering and vacuum concentrating, washing with solvent to obtain soybean oil ceramide, and analyzing the product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an island liquid high performance liquid chromatograph (LC-2030 c 3 dplus) with Innoval ODS-2.6x250 mm,5 μm column temperature: 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: linolenic acid-phytosphingosine ceramide 8.3min, linoleic acid-phytosphingosine ceramide 9.5min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.3min, stearic acid-phytosphingosine ceramide 13.9min, peanut acid-phytosphingosine ceramide 16.6min, and behenic acid-phytosphingosine ceramide 18.3min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, linolenic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide and behenic acid-phytosphingosine ceramide are 67%, 16%, 4%, 3%, 2%, 1% and the balance of other components in sequence, and the contents are less.
Example 2
Synthesis of ceramide from soybean oil fatty acid and sphingosine
Soybean oil fatty acid (50 mmol based on main component fatty acid), EDCI (65 mmol), DIPEA (65 mmol) were added to a 250mL round bottom flask, and 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and then sphingosine (60 mmol) was added to the reaction system, followed by stirring at room temperature until TLC detection was complete.
Post-treatment: adding water for quenching reaction, separating organic layer, drying, filtering and vacuum concentrating, washing with solvent to obtain soybean oil ceramide, and analyzing the product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an island liquid high performance liquid chromatograph (LC-2030 c 3 dplus) with Innoval ODS-2.6x250 mm,5 μm column temperature: 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: linolenic acid-sphingosine ceramide 7.9min, linoleic acid-sphingosine ceramide 8.7min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.5min, stearic acid-sphingosine ceramide 13.5min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-sphingosine ceramide, oleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, linolenic acid-sphingosine ceramide and stearic acid-sphingosine ceramide are 52%, 26%, 11%, 7%, 2% and the balance of other components in sequence, and the contents are small.
Example 3
Synthesis of ceramide from soybean oil fatty acid and dihydrosphingosine
Soybean oil fatty acid (50 mmol based on 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 dihydrosphingosine (70 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 organic layer, drying, filtering and vacuum concentrating, washing with solvent to obtain soybean oil ceramide, and analyzing the product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an island liquid high performance liquid chromatograph (LC-2030 c 3 dplus) with Innoval ODS-2.6x250 mm,5 μm column temperature: 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: linolenic acid-dihydrosphingoceramide 8.3min, linoleic acid-dihydrosphingoceramide 9.5min, palmitic acid-dihydrosphingoceramide 10.6min, oleic acid-dihydrosphingoceramide 11.2min, stearic acid-dihydrosphingoceramide 13.5min, peanut acid-dihydrosphingoceramide 16.0min, and behenic acid-dihydrosphingoceramide 21.8min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-dihydrosphingoceramide, oleic acid-dihydrosphingoceramide, palmitic acid-dihydrosphingoceramide, linolenic acid-dihydrosphingoceramide, stearic acid-dihydrosphingoceramide, peanut acid-dihydrosphingoceramide and behenic acid-dihydrosphingoceramide are 59%, 20%, 6%, 5%, 4%, 1% and 1% in sequence, and the rest are other components, and the content is low.
Example 4
Assessment of skin barrier repair by cell migration
Principle of: when the cells grow to be fused into a single-layer state, a scratching tool is manufactured on the fused single-layer cells, the cells in the blank area are mechanically removed, the migration of the cells to the 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 culture plate is streaked. Firstly, a Marker pen is arranged behind a 6-hole plate, straight lines are used for comparing, and uniformly scribing lines, approximately every 0.5 cm to 1cm is used for crossing holes, each hole at least penetrates 5 lines, and 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 is regulated according to the growth speed of the cells), and the inoculation principle is that the fusion rate reaches 100% after overnight.
3. Cell streaking. The next day was scored on the cell layer with a gun head perpendicular to the cell plane along the line of the first day that scored on the back of the plate (the same gun head is preferably used between the different wells).
4. Washing cells. After the streaking is completed, the cells are washed 3 times with sterile PBS, the cells which are not adherent are washed away, i.e. the streaked cells are streaked during streaking, the gap left after streaking is clearly visible, and then the fresh serum-free medium is replaced.
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 20mg/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 The incubator cultures, cells were removed after 24h, the width of the scratch was observed and measured with a microscope, and photographs were taken and the healing rate was calculated with 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 soybean oil ceramide has better tissue healing capacity. The solvent control had a healing rate of 34.18% after 24 hours, soybean oil ceramide had a healing rate of 74.37% after 24 hours, and ceramide 3B had a healing rate of 59.32% after 24 hours. The compound of the invention obviously improves the cell healing rate, has good skin tissue repair activity and better effect than ceramide 3B.
Example 5
Test of anti-aging effect by elastase inhibition experiment
The elastase inhibition method comprises the following steps: 2mg/mL elastase solution (2 mL) is taken, samples with different concentrations (the product of the example 1) 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 the mixed solution is taken into a 2mL centrifuge tube, centrifugation is carried out for 10min at 9 391 Xg, 200 mu L of supernatant is sucked into a 96-well plate precisely, absorbance is measured by an enzyme marker at the 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 complex holes. Inhibition ratio (%) = [1- (An-An ')/(A0-A0') ] ×100%, where A0 is absorbance with no sample added with enzyme, A0 'is absorbance with no sample added with substrate and enzyme only, an is absorbance with sample solution only, an' is absorbance with sample without enzyme. When An ' > An, the effect is expressed as acceleration, and the acceleration rate (%) = [1- (An ' -An)/(A0-A0 ') ] ×100%.
As shown in FIG. 2, soybean oil has a good effect of inhibiting elastase at various concentrations, specifically, the rate of inhibition of elastase at 0.25g/L is 7.13%, the rate of inhibition of elastase at 0.5g/L is 16.27%, the rate of inhibition of elastase at 1.0g/L is 21.67%, and the rate of inhibition of elastase at 2.0g/L is 22.67%.
Example 6
LPS induced cell method for detecting anti-inflammatory repair efficacy
B16 mouse melanoma cells were grown at a density of 1X 10 4 The seed/hole is planted in a 96-well plate and placedAdhering overnight in incubator, discarding supernatant after 24h, adding 100 μl of samples (product of example 1) diluted with DMEM medium at different concentrations, negative control group containing DMEM medium without sample, 3 wells per group, and mixing with 5wt% CO 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 completion of the reaction, 50. Mu.L of the cell supernatant was taken, and the intracellular IL-6 gene expression was detected using an IL-6ELISA kit.
The results are shown in FIG. 3, where IL-6 levels were 10.43 times the basal levels at a working concentration of 10. Mu.g/mL of LPS stimulation. Under the action of soybean 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 0.94 times, 0.88 times, 0.96 times and 0.89 times of that of an LPS model group respectively, and the soybean oil ceramide is dose-dependent, so that the soybean oil ceramide has a certain anti-inflammatory effect and can promote the repair of inflammatory damaged skin.
Example 7
MMP1 is also called as interstitial collagen enzyme and matrix metalloproteinase, belongs to matrix metalloproteinase family, and has main action substrate of 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 synthesis of collagen and elastin of fibroblasts, and reducing MMP activity can increase 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. The distance between the source of ultraviolet 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. The MMP-1ELISA kit is used for detecting the intracellular MMP-1 gene expression. Inhibition =1- (experimental group MMP1 expression level/model group MM)P1 expression amount). Times.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 rate of MMP1 expression in the model group was 26%, 46% and 54% at concentrations of 125, 250 and 400mg/L for the soybean oil ceramide; for UVB, the MMP1 expression level of the negative control group is set as 1, the expression level of the model group is 2.33, and the inhibition rate of the soybean oil ceramide to the MMP1 expression of the model group is 40%, 48% and 60% at the concentration 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 soybean oil ceramide can inhibit the generation of MMP1 by fibroblasts caused by ultraviolet radiation, and has a certain effect on preventing skin photoaging.
Example 8
DPPH free radical scavenging detection of antioxidant property
DPPH is 1,1-diphenyl-2-trinitrophenylhydrazine, and can be used for an antioxidant experiment.
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, mixing the mixture in equal volume, reacting for 30min at room temperature in a dark place, and measuring 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. 6, DPPH radical scavengers at concentrations of 50, 100, 200, 400 and 800mg/L were 13.43%, 27.91%, 35.24%, 38.26% and 43.50%, 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 DPPH radical scavenging rate at 50, 100, 200, 400, 800mg/L was 7.76%, 12.82%, 24.10%, 29.60%, 33.16%, as shown in FIG. 7. The clearance rate of the soybean oil ceramide to DPPH is higher than that of ceramide 3B, and the soybean oil ceramide has better antioxidation effect.
Example 9
Whitening Activity test
Taking B16 cells in exponential growth phase, digesting with 0.25% by mass of trypsin-EDTA, blowing uniformly, and mixing the cells according to a ratio of 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, each group having 3 multiple holes, and adding CO with mass fraction of 5% 2 Incubation was carried out for 24h at 37 ℃. The culture medium in the well plate is discarded, and after washing with Phosphate Buffer Solution (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 sample was placed in an enzyme marker and absorbance was measured at 405 nm. Melanin inhibition =1- (OD per well/model group OD) ×100% was calculated.
As a result, as shown in FIG. 8, the melanin content of the blank group was set to 1, the melanin expression of the model group was 1.51, and the melanin inhibition rates of soybean oil ceramide were 12.12%, 19.59%, 25.53%, 27.79% and 32.35% at concentrations of 10, 20, 40, 80 and 100mg/L, respectively, and excellent whitening effect was exhibited.
The foregoing is merely specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are easily conceivable by those skilled in the art within the technical scope of the present invention should be covered 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 soybean oil ceramide obtained by reacting a soybean oil fatty acid with a coleopinol compound selected from the group consisting of sphingosine, phytosphingosine and dihydrosphingosine.
2. The soybean oil ceramide of claim 1, wherein the soybean oil fatty acid comprises 50-75 wt% linoleic acid, 15-30 wt% oleic acid, 2-12 wt% palmitic acid, 1-10 wt% linolenic acid, 1-5 wt% stearic acid.
3. Soybean oil ceramide, the composition of which comprises: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, linolenic acid ceramide, stearic acid ceramide.
4. A soybean oil ceramide of claim 3, comprising the composition of: 50 to 75 weight percent of linoleic acid ceramide, 15 to 30 weight percent of oleic acid ceramide, 2 to 12 weight percent of palmitic acid ceramide, 1 to 10 weight percent of linolenic acid ceramide and 1 to 5 weight percent of stearic acid ceramide.
5. The soybean oil ceramide of claim 3 or 4, further comprising: 0 to 2 weight percent of peanut acid ceramide and 0 to 2 weight percent of behenic acid ceramide.
6. The method for synthesizing soybean oil ceramide of any one of claims 1 to 5, comprising the steps of:
under the conditions of condensing agent and organic base, soybean oil fatty acid reacts with coleopinol compound, wherein the condensing agent is EDCI, and the organic base is DIPEA;
the mol ratio of the soybean oil fatty acid to the coleophagin 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.
7. Use of the soybean oil ceramide of any one of claims 1 to 5 in cosmetics, pharmaceuticals, dietary or health products.
8. The use according to claim 7, wherein the soybean oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidation, promotion of collagen synthesis, maintenance of elastin activity, whitening efficacy.
9. A composition comprising the soybean 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-oxidation, promotion of collagen synthesis, maintenance of elastin viability, whitening efficacy.
CN202310051760.4A 2022-11-25 2023-02-02 Soybean oil ceramide and synthesis method and application thereof Pending CN117304060A (en)

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CN202310051760.4A CN117304060A (en) 2023-02-02 2023-02-02 Soybean 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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