CN116023289A

CN116023289A - Avocado oil ceramide and synthesis method and application thereof

Info

Publication number: CN116023289A
Application number: CN202310051748.3A
Authority: CN
Inventors: 杨超文; 叶柳
Original assignee: Shenzhen Dikeman Biotechnology Co ltd
Current assignee: Shenzhen Dikeman Biotechnology Co ltd
Priority date: 2023-02-02
Filing date: 2023-02-02
Publication date: 2023-04-28

Abstract

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

Avocado oil ceramide and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to avocado 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.

Avocado oil, also known as avocado oil, obtained from the flesh of avocados, is one of a few non-seed-made vegetable oils. The avocado oil has rich nutritive value, contains abundant healthy fat (unsaturated fatty acid reaches about 80 wt%), and also contains nutrients required by human body: vitamin a, vitamin D, vitamin E, vitamin K, carotenoids, plant sterols, squalene, etc. The avocado oil can be used for supplementing nutrition, reducing blood pressure, reducing weight, improving blood lipid, preventing cardiovascular and cerebrovascular diseases, and promoting health of diabetes. The avocado oil has wide application in the field of skin care and beauty, is particularly suitable for dry skin and aged skin, has the function of filtering ultraviolet rays, and has good sun protection effect. The avocado oil can be used alone or in combination; can be used for external or oral administration, or used as cooking oil for heating. The fatty acids of avocado oils are mainly oleic acid, linoleic acid and less common palmitoleic acid. The combination of the three unsaturated fatty acids ensures that avocado oil is easily absorbed on the skin to achieve the best care effect and ensure skin moistening. The high levels of phytosterols, such as beta-sitosterol, in avocado oil form a natural sun protection factor which helps to protect the skin from sunlight and also relieves skin discomfort due to sunburn. In addition, the components such as multiple vitamins, carotenoid, squalene and the like are excellent blocking agents, inhibitors and antioxidants, and can protect skin from being influenced by environmental factors such as ultraviolet rays, sunlight and the like under the combined action of the blocking agents, the inhibitors and the antioxidants, so that the effect of preventing skin cancer is achieved.

Disclosure of Invention

The invention aims to provide ceramide synthesized by using avocado oil fatty acid of plant origin.

It is another object of the present invention to provide a method for synthesizing avocado oil ceramide which utilizes avocado oil or avocado fatty acid which are naturally derived and readily available from plants as raw materials.

It is another object of the present invention to provide the use of avocado 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, avocado oil ceramide is obtained by reacting avocado oil fatty acids with a sphingoid 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 using avocado oil as raw material, selecting proper strain, and fermenting to obtain avocado 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 avocado oil fatty acid is obtained by hydrolyzing avocado oil.

Further, the avocado oil fatty acid contains 50 to 80wt% oleic acid.

Further, the avocado oil fatty acid contains 5 to 25wt% palmitoleic acid.

Further, the avocado oil fatty acid contains 5 to 20wt% linoleic acid.

Further, the avocado oil fatty acid contains 6 to 18wt% palmitic acid.

Further, the avocado oil fatty acid contains 0.1 to 2wt% stearic acid.

Further, the avocado oil fatty acid contains 0.1 to 1wt% linolenic acid.

In addition, avocado oil fatty acids also contain 0 to 1wt% of eicosapentaenoic acid (i.e. arachidonic acid) 0 to 0.5 wt%.

The avocado oil fatty acid comprises the following components: 50 to 80 weight percent of oleic acid, 5 to 25 weight percent of palmitoleic acid, 5 to 20 weight percent of linoleic acid, 6 to 18 weight percent of palmitic acid, 0.1 to 2 weight percent of stearic acid, 0.1 to 1 weight percent of linolenic acid, 0 to 1 weight percent of arachic acid and 0 to 0.5 weight percent of eicosapentaenoic acid.

The main component of avocado oil fatty acid is oleic acid, other fatty acids including palmitoleic acid, linoleic acid, palmitic acid, stearic acid and linolenic acid are essential components, and are influenced by tree species, soil, climate, production place, picking season and extraction process, the content of each component is different, and eicosanoic acid are not necessarily contained, and are optional components or unnecessary components.

Avocado oil ceramide, which comprises the following components: oleic acid ceramide, palmitoleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic 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 avocado oil ceramide is similar to that of avocado oil fatty acid: 50 to 80 weight percent of oleic acid ceramide, 5 to 25 weight percent of palmitoleic acid ceramide, 5 to 20 weight percent of linoleic acid ceramide, 6 to 18 weight percent of palmitic acid ceramide, 0.1 to 2 weight percent of stearic acid ceramide and 0.1 to 1 weight percent of linolenic acid ceramide. The content of each component is different due to the different content of each fatty acid in avocado oil fatty acid or grease. In addition, the avocado oil ceramide also comprises ceramide obtained by the reaction of one or more of eicosanoids and eicosanoids with sphingosine compounds, namely 0 to 1 weight percent of eicosanoids ceramide and 0 to 0.5 weight percent of eicosanoids ceramide. Avocado oil ceramide also includes compounds that are present in avocado oil fatty acids but do not react with sphingosine compounds, such as vitamin a, vitamin D, vitamin E, vitamin K, carotenoids, phytosterols, squalene, and the like.

Avocado oil ceramide, which comprises the following components: oleic acid ceramide, palmitoleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide; 50-80 wt% of oleic acid ceramide, 5-25 wt% of palmitoleic acid ceramide, 5-20 wt% of linoleic acid ceramide and 6-18 wt% of palmitoleic acid ceramide.

Further, the avocado oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 0.1-2 wt%.

Further, the avocado oil ceramide comprises linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.1 to 1 weight percent.

Further, avocado oil ceramides include not more than 1% by weight of eicosapentaenoic acid ceramides and not more than 0.5% by weight of eicosapentaenoic acid ceramides, and in particular, 0.1 to 1% by weight of eicosapentaenoic acid ceramides and 0.1 to 0.5% by weight of eicosapentaenoic acid ceramides.

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; palmitoleic acid ceramide is obtained by condensation reaction of palmitoleic acid and sphingosine compounds, and comprises palmitoleic acid phytosphingosine ceramide, palmitoleic acid sphingosine ceramide and palmitoleic acid dihydrosphingosine 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; 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, eicosapentaenoic acid ceramide, and the like.

In a second aspect of the invention, a method for synthesizing avocado oil ceramide comprises the following steps:

under the conditions of condensing agent and organic base, the avocado oil fatty acid reacts with sphingosine compound, the condensing agent is EDCI, and the organic base is Et ₃ N。

Further, the avocado oil fatty acid, sphingosine, EDCI, et ₃ The molar ratio of N 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.

Avocado oils purchased on the market are generally in the form of oils and fats, which need to be hydrolysed to fatty acids of the avocado oil, and therefore comprise the following steps:

the avocado oil fat is hydrolyzed by saponification reaction to obtain avocado oil fatty acid.

Further, the saponification reaction is hydrolysis of avocado oil in potassium hydroxide solution.

Further, the mass ratio of the avocado oil and fat to the potassium hydroxide is 1: (1-2).

In a third aspect of the invention, the use of avocado oil ceramide in cosmetics, pharmaceutical products, dietary or health care products.

Further, the avocado 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 avocado oil ceramide, the composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, promoting collagen synthesis, maintaining elastin viability, 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 avocado oil fatty acid belongs to naturally-formed fatty acid, the main component is monounsaturated fatty acid-oleic acid, and the avocado oil fatty acid also contains palmitoleic acid, linoleic acid, palmitic acid and the like, and the avocado oil ceramide prepared by mild reaction with a sphingosine compound naturally existing in skin has excellent performance in the aspects of repairing a natural skin barrier, resisting oxidation, resisting aging and the like, and has wide application prospect in the fields of cosmetics, health-care products, biological medicines and the like.

1. 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 avocado 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 avocado oil can form trace ceramide to play a role in efficacy supplement.

2. Compared with the compounded ceramide, the effect is better. Besides fatty acid (or grease), the avocado oil also contains nutrients required by human body, such as vitamin A, vitamin D, vitamin E, vitamin K, carotenoid, plant sterol, squalene and the like, and the nutrients have the effects of moistening skin, filtering ultraviolet rays, providing barrier for skin and the like, and the ceramide synthesized by the avocado oil has a synergistic effect with other active ingredients contained in the avocado 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, and the vegetable-derived avocado oil and fat or fatty acid thereof has wide sources, easy commercial acquisition, lower cost, environmental protection and economy, is different from the idea of mixing and compounding different single ceramides, and the fatty acid with a single component has high raw material price (such as palmitoleic acid), needs to respectively produce different ceramides, and then is compounded, thereby 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;

FIG. 6 is a bar graph of DPPH radical scavenging for oxidation resistance test of example 8;

fig. 7 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, et ₃ N refers to triethylamine. 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 avocado oil fatty acid and phytosphingosine

The first step: 50g of avocado oil and fat are dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 110mL 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 120mL of ethyl acetate to extract a water phase, adding 80mL 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 39g of avocado oil fatty acid.

And a second step of: avocado oil fatty acid (50 mmol based on main component fatty acid), EDCI (60 mmol), et ₃ N (60 mmol) was added to a 250mL round bottom flask, followed by 80mL of dichloromethane, followed by stirring at room temperature for 1 hour, followed by phytosphingosine (60 mmol) added to the reaction system, and 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 avocado 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 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: palmitoleic acid-phytosphingosine ceramide 8.0min, linolenic acid-phytosphingosine ceramide 8.3min, linoleic acid-phytosphingosine ceramide 9.5min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.3min, eicosapentaenoic acid-phytosphingosine ceramide 12.9min, stearic acid-phytosphingosine ceramide 13.9min.

The obtained products are analyzed by high performance liquid chromatography, and the content ratio of oleic acid-phytosphingosine ceramide, palmitoleic acid-phytosphingosine ceramide, linoleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide and linolenic acid-phytosphingosine ceramide is 70%, 10%, 7%, 8%, 1%, 0.5% and 0.5% in sequence, and the rest is other components, and the content is less.

Example 2

Synthesis of ceramide from avocado oil fatty acid and sphingosine

Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 120mL of ethyl acetate to extract a water phase, adding 80mL 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 39.3g of avocado oil fatty acid.

And a second step of: avocado oil fatty acid (50 mmol based on main component fatty acid), EDCI (65 mmol), et ₃ N (60 mmol) was added to a 250mL round bottom flask, followed by 100mL of dichloromethane, followed by stirring at room temperature for 1 hour, followed by sphingosine (55 mmol) added to the reaction system, and stirring at room temperature until TLC detection was complete.

The retention time of each component HPLC was: linolenic acid-sphingosine ceramide 7.9min, palmitoleic acid-sphingosine ceramide 8.2min, linoleic acid-sphingosine ceramide 8.6min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.5min, stearic acid-sphingosine ceramide 13.5min, arachidic acid-sphingosine ceramide 17.8min.

The obtained products are analyzed by high performance liquid chromatography, and the contents of oleic acid-sphingosine ceramide, palmitoleic acid-sphingosine ceramide, linoleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, linolenic acid-sphingosine ceramide and arachidic acid-sphingosine ceramide are 52%, 8%, 19%, 16%, 2%, 0.5% and 1% in sequence, and the rest are other components, with smaller contents.

Example 3

Synthesis of ceramide from avocado oil fatty acid and sphinganine

Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 120mL of ethyl acetate to extract a water phase, adding 80mL 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 39.2g of avocado oil fatty acid.

And a second step of: avocado oil fatty acid (50 mmol based on main component fatty acid), EDCI (70 mmol), et ₃ N (65 mmol) was added to a 250mL round bottom flask, followed by 100mL of dichloromethane, followed by stirring at room temperature for 1 hour, followed by addition of sphinganine (65 mmol) to the reaction system, stirring at room temperature, and detection by TLC was complete.

The retention time of each component HPLC was: linolenic acid-dihydrosphingosine ceramide 8.3min, palmitoleic acid-dihydrosphingosine ceramide 9.0min, linoleic acid-dihydrosphingosine ceramide 9.5min, palmitic acid-dihydrosphingosine ceramide 10.6min, oleic acid-dihydrosphingosine ceramide 11.2min, stearic acid-dihydrosphingosine ceramide 13.5min, eicosapentaenoic acid-dihydrosphingosine ceramide 14.0min, arachidic acid-dihydrosphingosine ceramide 16.0min.

The obtained products are analyzed by high performance liquid chromatography, the content of oleic acid-dihydrosphingosine ceramide, palmitoleic acid-dihydrosphingosine ceramide, linoleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, linolenic acid-dihydrosphingosine ceramide, arachidic acid-dihydrosphingosine ceramide and eicosapentaenoic acid-dihydrosphingosine ceramide is 61%, 20%, 6%, 7%, 1%, 0.5% and 0.5% in sequence, and the rest is other components, and the content is 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. Sample (example 1 product)After dilution of the product, ceramide 3B, with the medium (product concentration of example 1: 50mg/L, ceramide 3B concentration: 100 mg/L) was added to 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 figure 1, which shows that the scratch width of the experimental group is narrower than that of the solvent control group, thus indicating that avocado oil ceramide has better tissue healing capacity. The solvent control group had a rate of 35.23% after 24 hours, avocado oleoyl ceramide had a rate of 62.71% 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, avocado oil ceramide has a good inhibitory effect on elastase at various concentrations, specifically, an inhibitory rate of 11.47% on elastase at a concentration of 0.25g/L, an inhibitory rate of 18.33% on elastase at a concentration of 0.5g/L, an inhibitory rate of 25.27% on elastase at a concentration of 1.0g/L, and an inhibitory rate of 25.33% on elastase at a concentration of 2.0 g/L.

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 10.16 times the basal levels at a working concentration of 10. Mu.g/mL of LPS stimulation. Under the action of avocado 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 1.07, 0.77, 0.80 and 0.67 times of that of an LPS model group respectively, and the avocado oil ceramide is dose-dependent, which proves that the avocado 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 24 hours, the supernatant was discarded and samples containing different concentrations were added (example 1 productSubstance) of the culture medium 100 μl, no sample was added to the model group, the negative control group was DMEM medium without sample, 3 wells per group, and the mass fraction of CO was 5% 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 17%, 32%, 45% at the concentrations of 125, 250, 400mg/L of avocado 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 was 34%, 44% and 55% relative to the model group at the concentrations of 125, 250 and 400mg/L of avocado 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 avocado oil ceramide can inhibit the fibroblast from producing MMP1 caused by ultraviolet radiation, and has a certain effect on 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 7.09%, 12.40%, 18.73%, 23.77% and 27.44%, respectively, and excellent antioxidant effects were exhibited.

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. 7, 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 avocado oil ceramide were 4.58%, 10.70%, 15.95%, 19.30% and 20.98% at concentrations of 10, 20, 40, 80 and 100mg/L, respectively, and a good whitening effect was 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

1. Avocado oil ceramide, which is obtained by reacting avocado oil fatty acids with a sphingoid compound selected from the group consisting of sphingosine, phytosphingosine, dihydrosphingosine.

2. The avocado oil ceramide of claim 1, wherein the avocado oil fatty acid is obtained by hydrolysis of avocado oil.

3. Avocado oil ceramide according to claim 1 or 2, characterized in that the avocado oil fatty acid contains 50-80 wt.% oleic acid, 5-25 wt.% palmitoleic acid, 5-20 wt.% linoleic acid, 6-18 wt.% palmitic acid, 0.1-2 wt.% stearic acid, 0.1-1 wt.% linolenic acid.

4. Avocado oil ceramide, which comprises the following components: oleic acid ceramide, palmitoleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide.

5. The avocado oil ceramide of claim 4, comprising the composition of: 50 to 80 weight percent of oleic acid ceramide, 5 to 25 weight percent of palmitoleic acid ceramide, 5 to 20 weight percent of linoleic acid ceramide, 6 to 18 weight percent of palmitic acid ceramide, 0.1 to 2 weight percent of stearic acid ceramide and 0.1 to 1 weight percent of linolenic acid ceramide.

6. The avocado oil ceramide of claim 4 or 5, further comprising: 0 to 1 weight percent of arachidic acid ceramide and 0 to 0.5 weight percent of eicosapentaenoic acid ceramide.

7. The method for synthesizing avocado oil ceramide according to any one of claims 1 to 6, comprising the steps of:

under the conditions of condensing agent and organic base, the avocado oil fatty acid reacts with sphingosine compound, the condensing agent is EDCI, and the organic base is Et ₃ N；

The avocado oil fatty acid, sphingosine compound, EDCI, et ₃ The molar ratio of N 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 avocado oil ceramide as defined in any one of claims 1 to 6 in cosmetics, pharmaceuticals, dietary or health products.

9. The use according to claim 8, wherein the avocado 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 avocado oil ceramide of any one of claims 1-6, having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, promoting collagen synthesis, maintaining elastin viability, whitening efficacy.