CN117304059A - Tomato seed oil ceramide and synthetic method and application thereof - Google Patents

Tomato seed oil ceramide and synthetic method and application thereof Download PDF

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CN117304059A
CN117304059A CN202310051741.1A CN202310051741A CN117304059A CN 117304059 A CN117304059 A CN 117304059A CN 202310051741 A CN202310051741 A CN 202310051741A CN 117304059 A CN117304059 A CN 117304059A
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ceramide
seed oil
tomato seed
acid
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 tomato seed oil ceramide which is obtained by reacting tomato seed oil fatty acid with a sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. The tomato 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

Tomato seed oil ceramide and synthetic method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to tomato 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.
The tomato seed oil is natural high-quality edible oil prepared by subcritical low-temperature extraction of tomato seeds. The tomato seed oil contains unsaturated fatty acid as high as 80wt% including linoleic acid, oleic acid, palmitic acid, etc. In addition, tomato seed oil is rich in lycopene, vitamin E and precursor carotene of vitamin A. Research shows that tomato seed oil has the functions of regulating physiological function and development of human body, preventing cell aging, enhancing skin elasticity, etc. Meanwhile, the lycopene and various oil-soluble vitamins are contained, so that the lycopene-containing health care tea has obvious prevention and inhibition effects on diseases such as prostate cancer, digestive tract cancer, cervical cancer, skin cancer and the like. Tomato seed oil is also unique to the treatment of skin diseases, and can improve skin allergy and eliminate skin dryness and itching caused by skin allergy. The tomato seed oil also has the effects of protecting cardiovascular system, delaying aging, enhancing immunity, resisting ultraviolet radiation, etc.
Disclosure of Invention
The invention aims to provide ceramide synthesized by tomato seed oil fatty acid of plant origin.
Another object of the present invention is to provide a method for synthesizing tomato seed oil ceramide, which uses tomato seed oil grease or tomato 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 tomato 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, tomato seed oil ceramide is obtained by reacting tomato seed oil fatty acid with a sphingosine compound selected from the group consisting of sphingosine, phytosphingosine, 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 tomato seed oil is used as a raw material, and proper strains are selected for fermentation to obtain the tomato 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 tomato seed oil fatty acid is obtained by hydrolyzing tomato seed oil grease.
Further, the tomato seed oil fatty acid contains 55-80 wt% linoleic acid.
Further, the tomato seed oil fatty acid contains 10 to 25wt% of oleic acid.
Further, the tomato seed oil fatty acid contains 3 to 20wt% of palmitic acid.
Further, the tomato seed oil fatty acid contains 1-8 wt% of stearic acid.
Further, the tomato seed oil fatty acid contains 0.2-2 wt% linolenic acid.
In addition, the tomato seed oil fatty acid also contains 0-1 wt% of arachidic acid.
The tomato seed oil fatty acid comprises the following components: 55 to 80 weight percent of linoleic acid, 10 to 25 weight percent of oleic acid, 3 to 20 weight percent of palmitic acid, 1 to 8 weight percent of stearic acid, 0.2 to 2 weight percent of linolenic acid and 0 to 1 weight percent of arachidic acid.
The main component of tomato seed oil fatty acid is linoleic acid, other fatty acids comprise oleic acid, palmitic acid, stearic acid and linolenic 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 arachidic acid is not necessarily contained, and is an optional component or an unnecessary component.
Tomato seed oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide; because fatty acids all participate in the same reaction, the mass ratio of ceramide after the reaction is not greatly changed, so the composition of the ceramide of tomato seed oil is similar to that of tomato seed oil fatty acid, and the composition of the ceramide of tomato seed oil is as follows: 55 to 80 weight percent of linoleic acid ceramide, 10 to 25 weight percent of oleic acid ceramide, 3 to 20 weight percent of palmitic acid ceramide, 1 to 8 weight percent of stearic acid ceramide and 0.2 to 2 weight percent of linolenic acid ceramide. The content of each component is different due to the different content of each fatty acid in tomato seed oil fatty acid or grease. In addition, the tomato seed oil ceramide also comprises ceramide obtained by reacting arachidic acid with sphingosine compounds, namely 0-1 wt% of arachidic acid ceramide. Tomato seed oil ceramides also include compounds that are present in tomato seed oil fatty acids but that are not reactive with sphingosine compounds, lycopene, vitamin E, carotene, etc.
Tomato seed oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide; the linoleic acid ceramide accounts for 55-80 wt%, the oleic acid ceramide accounts for 10-25 wt%, and the palmitic acid ceramide accounts for 3-20 wt%.
Further, the tomato seed oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 1-8wt%.
Further, the tomato seed oil ceramide comprises linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.2-2 wt%.
Further, tomato seed oil ceramide comprises no more than 1wt% arachidic acid ceramide, especially 0.1-1 wt% 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; 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; stearic acid ceramide, linolenic acid ceramide, arachidic acid ceramide, and the like, and so forth.
In a second aspect of the invention, a method for synthesizing tomato seed oil ceramide comprises the following steps:
under the conditions of condensing agent and organic alkali, the tomato seed oil fatty acid reacts with sphingosine compound, the condensing agent is EDCI, and the organic alkali is DIPEA.
Further, the molar ratio of the tomato 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.
The tomato seed oil purchased on the market is generally in the form of oil and fat, and needs to be hydrolyzed into tomato seed oil fatty acid, and therefore the method further comprises the following steps:
the tomato seed oil fat is hydrolyzed by saponification reaction to obtain tomato seed oil fatty acid.
Further, the saponification reaction is hydrolysis of tomato seed oil grease in potassium hydroxide solution.
Further, the mass ratio of the tomato seed oil grease to the potassium hydroxide is 1: (1-2).
In a third aspect of the invention, the use of tomato seed oil ceramide in cosmetics, pharmaceutical products, dietary or health care products.
Further, the tomato 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 tomato 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 tomato seed oil fatty acid belongs to naturally-formed fatty acid, the main component is unsaturated fatty acid-linoleic acid, oleic acid and palmitic acid are contained, and the tomato seed oil ceramide is prepared by mild reaction with a sphingosine compound naturally existing in skin, 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 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 tomato seed oil grease 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 tomato 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 grease), tomato seed oil is rich in lycopene, vitamin E and precursor carotene of vitamin A, and the nutrients can prevent cell aging, enhance skin elasticity and play roles in moisturizing and beautifying. The ceramide synthesized by the tomato seed oil has a synergistic effect with other active ingredients contained in the tomato seed oil, and has better effect compared with the ceramide compounded according to similar proportion.
3. The cost is lower. The method of the invention can rapidly obtain a plurality of ceramide compound compositions, the tomato seed oil grease of plant sources or the fatty acid sources thereof have wide sources, are easy to obtain commercially, have lower cost, are more environment-friendly and economical, and are different from the idea of mixing and compounding different single ceramides, the fatty acid of a single component has high raw material price, and different ceramides are required to be produced respectively and then compounded, thus increasing 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
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 of the whitening activity test melanin content of example 9.
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 tomato seed oil fatty acid and phytosphingosine
The first step: 50g of tomato seed oil grease is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the reaction is carried out after the dropwise addition is completed, and the temperature is raised to room temperature until the TLC detection reaction 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 40.1g of tomato seed oil fatty acid.
And a second step of: tomato 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 completed.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain tomato 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: linolenic acid-phytosphingosine ceramide 8.2min, linoleic acid-phytosphingosine ceramide 9.4min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.2min, stearic acid-phytosphingosine ceramide 13.9min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide and linolenic acid-phytosphingosine ceramide are 63%, 15%, 11%, 6%, 1% in sequence, and the rest are other components, and the content is low.
Example 2
Synthesis of ceramide from tomato seed oil fatty acid and sphingosine
The first step: 50g of tomato seed oil grease is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the reaction is carried out after the dropwise addition is completed, and the temperature is raised to room temperature until the TLC detection reaction 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 tomato seed oil fatty acid.
And a second step of: tomato seed oil fatty acid (50 mmol, based on the main component fatty acid), EDCI (70 mmol), 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 sphingosine (55 mmol) was added to the reaction system, followed by stirring at room temperature until TLC detection was completed.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain tomato 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: linolenic acid-sphingosine ceramide 7.9min, linoleic acid-sphingosine ceramide 8.7min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.4min, stearic acid-sphingosine ceramide 13.6min, arachidic acid-sphingosine ceramide 17.8min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-sphingosine ceramide, oleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, linolenic acid-sphingosine ceramide and arachidic acid-sphingosine ceramide are 74%, 13%, 5%, 2%, 1% and the balance of other components in sequence, and the contents are less.
Example 3
Synthesis of ceramide from tomato seed oil fatty acid and sphinganine
The first step: 50g of tomato seed oil grease is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the reaction is carried out after the dropwise addition is completed, and the temperature is raised to room temperature until the TLC detection reaction 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 39.8g of tomato seed oil fatty acid.
And a second step of: tomato seed oil fatty acid (50 mmol, based on the main component fatty acid), EDCI (80 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 sphinganine (65 mmol) was added to the reaction system, followed by stirring at room temperature until TLC detection was completed.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain tomato 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: linolenic acid-dihydrosphingosine ceramide 8.3min, linoleic acid-dihydrosphingosine ceramide 9.5min, palmitic acid-dihydrosphingosine ceramide 10.6min, oleic acid-dihydrosphingosine ceramide 11.1min, stearic acid-dihydrosphingosine ceramide 13.5min, arachidic acid-dihydrosphingosine ceramide 16.0min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-dihydrosphingosine ceramide, oleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, linolenic acid-dihydrosphingosine ceramide and arachidic acid-dihydrosphingosine ceramide are 55%, 20%, 17%, 4%, 1% and 1% in sequence, and the rest are other components, and the contents are less.
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 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. 1, and the scratch width of the experimental group is narrower than that of the solvent control group, which indicates that the tomato seed oil ceramide has better tissue healing capacity. The solvent control group had a rate of 21.62% after 24 hours, tomato seed oil ceramide had a rate of 81.23% after 24 hours, and ceramide 3B had a 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 5
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. 2, tomato seed oil ceramide has good inhibition effect on elastase at different concentrations, specifically, the inhibition rate of elastase at the concentration of 0.25g/L is 8.80%, the inhibition rate of elastase at the concentration of 0.5g/L is 16.93%, the inhibition rate of elastase at the concentration of 1.0g/L is 23.00%, and the inhibition rate of elastase at the concentration of 2.0g/L is 25.00%.
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 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. 3, where IL-6 levels were 10.70 times the basal levels at a working concentration of 10. Mu.g/mL of LPS stimulation. Under the action of tomato 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 0.97, 0.85, 0.73 and 0.46 times of that of an LPS model group respectively, and the tomato seed oil ceramide is dose-dependent, so that the tomato seed oil ceramide has good 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 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. The distance between the ultraviolet radiation source 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. 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 ratio of MMP1 expression in the model group was 42%, 62%, 81% at the concentrations of 125, 250, 400mg/L for tomato seed oil ceramide; for UVB, the MMP1 expression level of the negative control group was 1, the expression level of the model group was 2.33, and the inhibition rate of MMP1 expression of the model group was 39%, 49% and 69% at concentrations of 125, 250 and 400mg/L of tomato seed oil ceramide.
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 tomato seed oil ceramide can inhibit the fibroblast from producing MMP1 caused by ultraviolet radiation, and has a certain effect of preventing skin photoaging.
Example 8
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. 6, the DPPH radical scavengers at concentrations of 50, 100, 200, 400 and 800mg/L were 9.43%, 23.91%, 35.10%, 41.93% and 50.27%, 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 tomato seed oil ceramide to DPPH is higher than that of ceramide 3B, and the tomato seed oil ceramide has better antioxidation effect.
Example 9
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. 8, the melanin content of the blank group was 1, the melanin expression of the model group was 1.54, and the melanin inhibition rates of the tomato seed oil ceramide were 13.59%, 21.12%, 28.66%, 38.66% and 40.53% 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. Tomato seed oil ceramide, it is obtained from tomato seed oil fatty acid and sphingosine compound reaction, said sphingosine compound is selected from sphingosine, phytosphingosine, dihydrosphingosine.
2. Tomato seed oil ceramide of claim 1, wherein the tomato seed oil fatty acid is obtained by hydrolysis of tomato seed oil.
3. Tomato seed oil ceramide according to claim 1 or 2, wherein the tomato seed oil fatty acid comprises 55-80 wt% linoleic acid, 10-25 wt% oleic acid, 3-20 wt% palmitic acid, 1-8 wt% stearic acid, 0.2-2 wt% linolenic acid.
4. Tomato seed oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide.
5. Tomato seed oil ceramide of claim 4, comprising the composition of: 55 to 80 weight percent of linoleic acid ceramide, 10 to 25 weight percent of oleic acid ceramide, 3 to 20 weight percent of palmitic acid ceramide, 1 to 8 weight percent of stearic acid ceramide and 0.2 to 2 weight percent of linolenic acid ceramide.
6. Tomato seed oil ceramide of claim 4 or 5, further comprising: 0 to 1wt% of arachidic acid ceramide.
7. The method for synthesizing tomato seed oil ceramide according to any one of claims 1 to 6, comprising the following steps:
under the conditions of condensing agent and organic alkali, the tomato seed oil fatty acid reacts with sphingosine compound, the condensing agent is EDCI, and the organic alkali is DIPEA;
the molar ratio of the tomato 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 tomato 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 said tomato 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 tomato seed oil ceramide of any one of claims 1-6, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, promotion of collagen synthesis, maintenance of elastin viability, whitening efficacy.
CN202310051741.1A 2022-11-25 2023-02-02 Tomato seed oil ceramide and synthetic method and application thereof Pending CN117304059A (en)

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PCT/CN2023/133524 WO2024109867A1 (en) 2022-11-25 2023-11-23 Vegetable oil ceramides, synthesis method therefor, and use thereof

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