CN115894279A

CN115894279A - Olive oil ceramide and synthesis method and application thereof

Info

Publication number: CN115894279A
Application number: CN202211493023.1A
Authority: CN
Inventors: 杨超文; 叶柳
Original assignee: Shenzhen Dikeman Biotechnology Co ltd
Current assignee: Shenzhen Dikeman Biotechnology Co ltd
Priority date: 2022-11-25
Filing date: 2022-11-25
Publication date: 2023-04-04

Abstract

The invention belongs to the technical field of biological medicines, and discloses olive oil ceramide which is obtained by reacting olive oil fatty acid with sphingoid compounds, wherein the sphingoid compounds are selected from sphingosine, phytosphingosine and dihydrosphingosine. The olive oil ceramide has excellent performances in the aspects of repairing natural barriers of skin, resisting inflammation, healing tissues, resisting aging and the like, and has wide application prospects in the fields of cosmetics, health products, biological medicines and the like.

Description

Olive oil ceramide and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to olive oil ceramide, a synthetic method and application thereof.

Background

Ceramide (Ceramide, also called molecular nail) naturally exists in skin, is a very important component of skin barrier (stratum corneum) and is up to 40-50 wt%, and is a sphingolipid consisting of sphingoid long-chain bases and fatty acids, wherein the carbon chain length, the unsaturation degree and the hydroxyl number of the sphingoid part and the fatty acid part can be changed, and the Ceramide represents a compound. Ceramides exhibit excellent properties in regulating skin barrier function, restoring skin moisture, and enhancing adhesion between skin keratinocytes, etc.

Due to the importance of ceramides, many cosmetic and pharmaceutical companies are researching and developing corresponding products. The natural plant-derived ceramide can form an effective skin barrier to prevent water loss and resist external damage due to a more sustainable and more environment-friendly raw material source and a similar component to skin ceramide, and can possibly become a next-generation environment-friendly, safe and reliable ceramide product.

The olive oil is generally olive oil grease, is prepared by directly cold pressing fresh olive fruits, does not undergo heating and chemical treatment, retains natural nutritional ingredients, and is known as 'liquid gold', 'vegetable oil queen', 'Mediterranean nectar' in the west. Olive oil is rich in oleic acid, which is a monounsaturated fatty acid, and also vitamin a, vitamin B, vitamin D, vitamin E, vitamin K, antioxidants, and the like, and is considered to be the most suitable oil for the human body among the oils found so far. The data show that the olive oil can reduce the synthesis of catecholamine, inhibit the increase of active oxygen generation, lipid peroxidation and protein carbonylation of the skin, and has good effects of resisting aging and oxidation, whitening and beautifying, shrinking pores and the like. Because it contains abundant seborrheic vitamin, it is very beneficial to skin, and is a safe and reliable good cosmetic product, so that it is known as "edible cosmetic product". In addition, research shows that the olive oil has the effects of promoting blood circulation, reducing cardiovascular diseases, promoting the development of bones and nervous systems, losing weight, treating injuries, slowing down the functional degeneration of nerve cells of the old and the like in the aspect of medicine, and the olive oil can also be used as a sedative and an auxiliary treatment means for treating jaundice and gallstones.

Disclosure of Invention

The invention aims to provide ceramide synthesized by olive oil fatty acid from plants.

Another object of the present invention is to provide a method for synthesizing olive oil ceramide using olive oil or olive oil fatty acid, which are naturally derived from plants and easily available, as a raw material.

It is another object of the present invention to provide the use of olive oil ceramide.

In order to achieve one of the purposes, the invention adopts the following technical scheme:

in a first aspect of the invention, an olive oil ceramide, obtained by reacting an olive oil fatty acid with a sphingoid compound selected from sphingosine, phytosphingosine, dihydrosphingosine.

The reaction can be a chemical synthesis reaction (as detailed below), or a microbial fermentation method, namely, pichia pastoris or saccharomyces cerevisiae is used for fermentation under certain environment to obtain sphingoid compounds, and then fatty acid is added to finally obtain ceramide; or fermenting olive oil as raw material with proper strain to obtain olive 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 olive oil fatty acid is obtained by hydrolysis of olive oil.

Further, the olive oil fatty acid contains 20 to 83wt% oleic acid, preferably 55 to 83wt% oleic acid.

Further, the olive oil fatty acid contains 7.5 to 20wt% of palmitic acid.

Further, the olive oil fatty acid contains 3.5 to 70wt% linoleic acid, preferably 3.5 to 21wt% linoleic acid.

Further, the olive oil fatty acid contains 0.5 to 5wt% of stearic acid.

Further, the olive oil fatty acid contains 0.1-1.0 wt% of linolenic acid.

In addition, the olive oil fatty acid also contains 0 to 3.5 weight percent of palmitoleic acid, 0 to 0.6 weight percent of arachidic acid, 0 to 0.4 weight percent of eicosenoic acid, 0 to 0.3 weight percent of heptadecanoic acid, 0 to 0.3 weight percent of heptadecenoic acid, 0 to 0.2 weight percent of behenic acid, 0 to 0.2 weight percent of tetracosanoic acid and 0 to 0.05 weight percent of myristic acid.

The olive oil fatty acid has the following components: 20 to 83 weight percent of oleic acid, 7.5 to 20 weight percent of palmitic acid, 3.5 to 70 weight percent of linoleic acid, 0.5 to 5 weight percent of stearic acid, 0.1 to 1.0 weight percent of linolenic acid, 0 to 3.5 weight percent of palmitoleic acid, 0 to 0.6 weight percent of arachidic acid, 0 to 0.4 weight percent of eicosenoic acid, 0 to 0.3 weight percent of heptadecanoic acid, 0 to 0.3 weight percent of heptadecenoic acid, 0 to 0.2 weight percent of behenic acid, 0 to 0.2 weight percent of tetracosanic acid and 0 to 0.05 weight percent of myristic acid.

The main component of olive oil fatty acid is oleic acid, other fatty acids include linoleic acid, palmitic acid, stearic acid and linolenic acid, which are essential components, and the content of each component is different under the influence of tree species, soil, climate, producing area, picking season and extraction process, while palmitoleic acid, arachidic acid, eicosenoic acid, heptadecanoic acid, heptadecenoic acid, behenic acid, lignoceric acid and myristic acid are optional components or unnecessary components.

Olive oil ceramide, the composition of which comprises: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide; since fatty acids all participate in the same reaction, and the mass ratio of ceramide after the reaction does not change much, the composition of olive oil fatty acid can be referred to, and the composition of olive oil ceramide is: 20 to 83 weight percent of oleic acid ceramide, 7.5 to 20 weight percent of palmitic acid ceramide, 3.5 to 70 weight percent of linoleic acid ceramide, 0.5 to 5 weight percent of stearic acid ceramide and 0.1 to 1.0 weight percent of linolenic acid ceramide; the preferred composition is: 55 to 83 weight percent of oleic acid ceramide, 7.5 to 20 weight percent of palmitic acid ceramide, 3.5 to 21 weight percent of linoleic acid ceramide, 0.5 to 5 weight percent of stearic acid ceramide, and 0.1 to 1.0 weight percent of linolenic acid ceramide. The content of each component is different due to different contents of olive oil fatty acid or fatty acid in the oil. In addition, the olive oil ceramide also comprises ceramide obtained by reacting one or more of palmitoleic acid, arachidic acid, eicosenoic acid, heptadecylic acid, heptadecenoic acid, behenic acid, lignoceric acid and myristic acid with sphingoid compounds, namely, 0-3.5 wt% of palmitoleic acid ceramide, 0-0.6 wt% of arachidic acid ceramide, 0-0.4 wt% of eicosenoic acid ceramide, 0-0.3 wt% of heptadecenoic acid ceramide, 0-0.2 wt% of behenic acid ceramide, 0-0.2 wt% of lignoceric acid ceramide and 0-0.05 wt% of myristic acid ceramide.

Olive oil ceramide, the composition of which comprises: oleic acid ceramide, palmitic acid ceramide, linoleic acid ceramide; oleic acid ceramide 20-83 wt%, preferably 55-83 wt%, palmitic acid ceramide 7.5-20 wt%, linoleic acid ceramide 3.5-70 wt%, preferably 3.5-21 wt%.

Further, the olive oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 0.5-5 wt%.

Furthermore, the olive oil ceramide comprises linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.1-1.0 wt%.

The oleic acid ceramide is obtained by condensation reaction of oleic acid and sphingoid compounds, and comprises oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide and oleic acid dihydrosphingosine ceramide; the linoleic acid ceramide is obtained by condensation reaction of linoleic acid and sphingoid compounds, and comprises linoleic acid phytosphingosine ceramide, linoleic acid sphingosine ceramide, and linoleic acid dihydrosphingosine ceramide; the palmitic acid ceramide is obtained by condensation reaction of palmitic acid and sphingoid compounds, and comprises palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide, and palmitic acid dihydrosphingosine ceramide; the stearic acid ceramide is obtained by condensation reaction of stearic acid and sphingoid compounds, and comprises stearic acid phytosphingosine ceramide, stearic acid sphingosine ceramide and stearic acid dihydrosphingosine ceramide; linolenic acid ceramide is obtained by condensation reaction of linolenic acid and sphingoid compounds, and comprises linolenic acid phytosphingosine ceramide, linolenic acid sphingosine ceramide, and linolenic acid dihydrosphingosine ceramide; arachidic acid ceramide, eicosenoic acid ceramide, and the like.

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

reacting olive oil fatty acid with sphingoid compound under the condition of condensing agent, wherein the condensing agent is EDCI and Et ₃ N。

Further, the olive oil fatty acid, sphingoid compound, EDCI, et ₃ The molar ratio of N is 1: (1-1.5): (1-2): (1-2), wherein the solvent for reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.

Olive oil, which is generally available on the market in the form of fat and needs to be hydrolysed to olive oil fatty acids, therefore it also comprises the following steps:

the olive oil fatty acid is obtained by hydrolyzing olive oil through saponification reaction.

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

Further, the mass ratio of the olive oil grease to the potassium hydroxide is 1: (1-2).

In a third aspect of the present invention, the use of olive oil ceramide in cosmetics, pharmaceuticals, dietary foods or health products.

Further, the olive oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidation, collagen synthesis promotion, elastin enhancement, and whitening effects.

A composition comprising olive 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 increasing, and whitening effects.

The composition contains acceptable adjuvants including one or more of solubilizer, antiseptic, antioxidant, pH regulator, penetration enhancer, liposome, humectant, thickener, chelating agent, skin feeling regulator, surfactant, emulsifier, essence and pigment; the composition is in the form of cream, emulsion, solution, pellicle, aerosol or spray.

The invention has the following beneficial effects:

the olive oil fatty acid belongs to naturally formed fatty acid, the main component is monounsaturated fatty acid-oleic acid or linoleic acid, and in addition, palmitic acid, stearic acid, linolenic acid and the like are also contained, and the olive oil ceramide is prepared by mild reaction with sphingoid compounds naturally existing in skin, shows excellent performance in the aspects of repairing, antioxidation, anti-aging and the like of natural barriers of the skin, and has wide application prospect in the fields of cosmetics, health care products, biological medicines and the like.

1. It is more effective than ceramide alone. Different ceramides have different effects due to their structural differences, and ceramides with a single structure generally have poor comprehensive effects. The scheme is based on a bionic idea, olive oil grease or fatty acid from natural sources is used as a raw material to synthesize the composite ceramide so as to make up for the difference of the efficacies of different ceramides, and trace fatty acid in olive sources can form trace ceramide, so that the function supplement effect is achieved.

2. Compared with compounded ceramide, the effect is better. Except oleic acid (or grease), the olive oil also contains components such as vitamins and polyphenol, and the like, the nutrient substances have the effects of moistening skin, enhancing cell activity and the like, and the ceramide synthesized by the olive oil has the synergistic effect with other active components contained in the olive 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 quickly obtain the composition compounded by various ceramides, the olive oil grease of plant source or the fatty acid thereof has wide source, easy commercial acquisition, lower cost, more environmental protection and economy, and is different from the idea of mixing and compounding different single ceramides, the fatty acid of single component has high raw material price, and the different ceramides are respectively produced and then compounded, thereby increasing the preparation cost.

4. The synthetic method is simple. The method can adopt chemical synthesis to realize one-step preparation of various ceramides, and can also use a microbial fermentation method.

Drawings

FIGS. 1 and 2 are bar graphs of the cell proliferation activity test results of example 4;

FIG. 3 shows the results of the measurement of cell migration ability in example 5;

FIG. 4 is a bar graph of the elastase inhibition of example 6;

FIG. 5 is a bar graph of the anti-inflammatory repair efficacy assays of example 7;

FIG. 6 is a bar graph showing the expression level of MMP1 in example 8;

FIGS. 7 and 8 are bar graphs of the oxidation resistance test results of example 9;

fig. 9 is a bar graph of the results of the whitening activity test of example 10.

Detailed Description

The present invention will be further described with reference to the following specific examples.

EDCI means 1-ethyl- (3-dimethylaminopropyl) carbodiimide, et ₃ N refers to triethylamine. The silica gel column chromatography uses Qingdao marine silica gel (particle size 0.040-0.063 mm). Thin Layer Chromatography (TLC) was performed using a 60F254 silica gel plate, and TLC developed using UV light (254 nm) or iodine. NMR spectra were characterized using a Bruker DPX 400 nuclear magnetic resonance apparatus, ¹ h NMR is 400MHz, solvent is deuterated methanol, deuterated DMSO or deuterated tetrahydrofuran, and Tetramethylsilane (TMS) is used as internal standard. Chemical shifts are in ppm and coupling constants are in Hz. In that ¹ In H NMR, δ represents chemical shift, s represents singlet, d represents doublet, t represents triplet, q represents quartet, and m represents multiplet.

Example 1

Synthesis of ceramide from olive oil fatty acid and phytosphingosine

The first step is as follows: 50g of olive oil grease is dissolved in 60mL of tetrahydrofuran, cooled in ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is heated to room temperature after the dropwise addition is finished to react until the TLC detection reaction is finished.

And (3) post-treatment: adding dilute hydrochloric acid (3N) to adjust pH to 3, adding 150mL ethyl acetate to extract water phase, adding 100mL saturated salt water to wash once, adding anhydrous Na to organic phase ₂ SO ₄ Drying, filtering and vacuum concentrating to obtain 40g olive oil fatty acid.

The second step is that: mixing olive oil fatty acid (50 mmol, molecular weight is calculated as oleic acid), EDCI (60 mmol), et ₃ N (60 mmol) was added to 250mL in a round-bottom flask, followed by addition of 100mL of dichloromethane, 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 completion of the TLC detection reaction.

And (3) post-treatment: adding water to quench and react, separating an organic layer, drying, filtering, concentrating in vacuum, washing by a solvent to obtain the olive oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the column temperature was adjusted by Innoval ODS-2.6 × 250mm,5 μm column: 30 ℃, injection volume: 10 μ L, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.

The HPLC retention time of each component was: oleic acid-phytosphingosine ceramide 11.3min, linoleic acid-phytosphingosine ceramide 9.5min, palmitic acid-phytosphingosine ceramide 10.7min, stearic acid-phytosphingosine ceramide 13.9min, linolenic acid-phytosphingosine ceramide 8.2min.

Separating the obtained product by preparative high performance liquid chromatography to obtain oleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, linoleic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide, and linolenic acid-phytosphingosine ceramide (nuclear magnetic hydrogen spectrum is consistent with standard spectrum), with mass ratio of 21.3%, 10.5%, 63.2%, 2.4%, 0.4%, and the balance of other components with less content. In this example, a domestic olive oil fatty acid was used, the main component of which was linoleic acid, and the resulting product was mainly linoleic acid ceramide.

Example 2

Synthesis of ceramide from olive oil fatty acid and sphingosine

And (3) post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 150mL ethyl acetate to extract the aqueous phase, adding 100mL saturated saline water to wash once, and adding anhydrous Na into the organic phase ₂ SO ₄ Drying, filtering and vacuum concentrating to obtain 40g olive oil fatty acid.

The second step is that: mixing olive oil fatty acid (50 mmol, molecular weight is calculated as oleic acid), EDCI (75 mmol), et ₃ Adding N (75 mmol) into 250mL of the mixture, adding 100mL of dichloromethane into the mixture, stirring the mixture at room temperature for 1 hour, adding sphingosine (50 mmol) into the reaction system, stirring the mixture at room temperature until TLC detectionAnd (4) finishing the reaction.

And (3) post-treatment: adding water to quench and react, separating an organic layer, drying, filtering, concentrating in vacuum, washing by a solvent to obtain the olive oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the mixture was purified by Innoval ODS-2.6 × 250mm,5 μm column, column temperature: 30 ℃, injection volume: 10 μ L, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: (2 wt% water +98wt% methanol) each contained 0.1wt% formic acid.

The HPLC retention time of each component was: oleic acid-sphingosine ceramide 20.7min, linoleic acid-sphingosine ceramide 16.6min.

Separating the obtained product by preparative high performance liquid chromatography to obtain oleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, linoleic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, and linolenic acid-sphingosine ceramide (nuclear magnetic hydrogen spectrum is consistent with standard spectrum), with mass ratio of 61.8%, 12.6%, 19.3%, 3.4%, and 0.6%, and other components with less content. This example uses imported olive oil fatty acid, the main component of which is oleic acid, the resulting product being predominantly oleic ceramide.

Oleic acid-sphingosine ceramide hydrogen profile: ¹ h NMR (400mhz, methanol-d 4) δ 7.70 (d, J =8.9hz, 1h), 5.69 (dt, J =15.3,6.7hz, 1h), 5.45 (dd, J =15.4,7.5hz, 1h), 5.34 (t, J =4.9hz, 2h), 4.03 (t, J =7.5hz, 1h), 3.85 (ddd, J =9.5,4.6,2.4hz, 1h), 3.68 (d, J =5.0hz, 2h), 2.19 (t, J =7.6hz, 2h), 2.03 (q, J =6.9, 6.4hz, 6h), 1.64-1.51 (m, 2H), 1.30 (d, J =14.5hz, 41h), 0.90 (t, J = 6.0h, 6.7h). Linoleic acid-sphingosine ceramide hydrogen spectrum: ¹ H NMR(400MHz,Methanol-d4)δ7.69(d,J＝8.9Hz,1H),5.69(dt,J＝15.3,6.7Hz,1H),5.46(dd,J＝15.3,7.4Hz,1H),5.41–5.25(m,4H),4.03(t,J＝7.5Hz,1H),3.85(tdd,J＝7.5,4.7,2.2Hz,1H),3.68(d,J＝5.0Hz,2H),2.77(t,J＝6.4Hz,2H),2.20(q,J＝7.6,6.9Hz,2H),2.13–1.94(m,6H),1.67–1.49(m,2H),1.31(d,J＝19.6Hz,40H),0.90(td,J＝6.8,3.8Hz,6H)。

example 3

Synthesis of ceramide from olive oil fatty acid and dihydrosphingosine

And (3) post-treatment: adding dilute hydrochloric acid (3N) to adjust pH to 3, adding 150mL ethyl acetate to extract water phase, adding 100mL saturated salt water to wash once, adding anhydrous Na to organic phase ₂ SO ₄ Drying, filtration and vacuum concentration gave 40g of olive oil fatty acid.

The second step is that: mixing olive oil fatty acid (50 mmol, molecular weight is calculated as oleic acid), EDCI (100 mmol), et ₃ N (100 mmol) was added to 250mL in a round-bottom flask, followed by addition of 100mL of methylene chloride, followed by stirring at room temperature for 1 hour, followed by addition of sphinganine (75 mmol) to the reaction system, followed by stirring at room temperature until completion of the TLC detection reaction.

And (3) post-treatment: adding water to quench and react, separating an organic layer, drying, filtering, concentrating in vacuum, washing by a solvent to obtain the olive oil ceramide, analyzing a product by HPLC, and carrying out HPLC chromatographic conditions: using Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), the column temperature was adjusted by Innoval ODS-2.6 × 250mm,5 μm column: 30 ℃, injection volume: 10 μ L, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: (2 wt% water +98wt% methanol) each contained 0.1wt% formic acid.

The HPLC retention time of each component was: oleic acid-sphinganine ceramide 23.6min, linoleic acid-sphinganine ceramide 19.1min.

Separating the obtained product by preparative high performance liquid chromatography to obtain oleic acid-sphinganine ceramide, palmitic acid-sphinganine ceramide, linoleic acid-sphinganine ceramide, stearic acid-sphinganine ceramide, and linolenic acid-sphinganine ceramide (nuclear magnetic hydrogen spectrum is consistent with standard spectrum), with mass ratio of 29.9%, 18.2%, 45.1%, 2.3%, and 0.9%. The balance of other components with less content.

Oleic acid-dihydrosphingosine ceramide hydrogen profile: ¹ h NMR (400mhz, methanol-d 4) δ 5.34 (t, J =4.9hz, 2h), 3.81 (q, J =5.6hz, 1h), 3.75-3.65 (m, 2H), 3.65-3.51 (m, 1H), 2.22 (dd, J =9.2,5.4hz, 2h), 2.03 (q, J =6.3hz, 4h), 1.70-1.49 (m, 5H), 1.48-1.05 (m, 45H), 0.90 (t, J =6.6hz, 6h). Linoleic acid-sphinganine ceramide: ¹ H NMR(400MHz,Methanol-d4)δ5.42–5.24(m,4H),3.89–3.75(m,1H),3.75–3.64(m,2H),3.64–3.53(m,1H),2.77(t,J＝6.4Hz,2H),2.22(t,J＝7.4Hz,2H),2.06(q,J＝6.8Hz,4H),1.57(ddt,J＝33.9,13.9,5.8Hz,4H),1.31(d,J＝21.5Hz,40H),0.90(td,J＝6.8,3.7Hz,6H)。

example 4

MTT method for detecting cell proliferation activity of compound

HaCaT cells were cultured at 1X 10 ⁴ The density of cells/well was plated in 96-well plates overnight in an incubator. After 24h the supernatant was discarded, 100. Mu.L of medium containing different concentrations of the product of example 1 was added, incubation was continued for 24h and the medium was removed, 100. Mu.L of thiazole blue (MTT) was added to each well, absorbance at 450nm was measured and cell viability = A was calculated _{Medicine feeding hole} /A _{Blank hole} ×100％。

As shown in fig. 1, the results show that the olive oil ceramide has a promoting effect on cell viability, and the cell viability is 102.7%, 121.2%, 145.2% and 165.2% respectively at the concentrations of 70, 150, 250 and 300mg/L, showing a significant cell proliferation promoting effect and having good tissue repair ability.

The proliferation activity of ceramide 3B (i.e., oleic acid ceramide) on cells was tested in the same manner, and as a result, as shown in fig. 2, the cell viability was 101.7%, 111.2%, 126.8%, 141.9% at concentrations of 70, 150, 250, 300mg/L, respectively, which was less effective in promoting cell proliferation and tissue repair than olive oil ceramide.

Example 5

Evaluation of skin Barrier repair by cell migration

The principle is as follows: when the cells grow to be fused into a monolayer, a blank area is made on the fused monolayer by a scratching tool, the cells in the blank area are removed by mechanical force, the migration of the cells to a cell-free area is observed through a period of culture, and the migration capability of the cells is reflected by measuring the migration distance of the cells.

The method comprises the following operation steps:

1. the plates were streaked. Firstly, a Marker pen is used on the back of a 6-hole plate, a straight ruler is used for uniformly drawing transverse lines which are about every 0.5-1 cm and cross through holes, each hole at least penetrates through 5 lines, and the lines are not too thick when drawing lines.

2. And (5) laying cells. About 5X 10 additions to the wells ⁵ And (3) inoculating each cell (the number of different cells is different and is adjusted according to the growth speed of the cells), wherein the inoculation principle is that the fusion rate reaches 100 percent after the overnight inoculation.

3. And (4) cell lineation. The next day, the cell layer was scored with a tip perpendicular to the cell plane along the line drawn on the back of the plate on the first day (preferably the same tip is used between different wells).

4. The cells were washed. After the scoring was completed, cells were washed 3 times with sterile PBS, non-adherent cells were washed away, i.e., cells streaked during streaking, leaving a clear gap after streaking, and then fresh serum-free medium was replaced.

5. And (5) culturing and observing cells. Diluting the sample (example 1 product, ceramide 3B) with the culture medium (concentration of 0.08 mM), adding to a cell culture dish, placing the cells at 37 deg.C, 5wt% ₂ The cells were incubated in an incubator and taken out at 24h, microscopically observed and the width of the scratch was measured and photographed, and the healing rate was calculated using Image J software.

The results are shown in fig. 3, and the scratch width of the experimental group is narrower than that of the solvent control group, indicating that the olive oil ceramide has better tissue healing ability. The healing rate of the solvent control group after 24h was 31.25%, the healing rate of olive oil ceramide after 24h was 74.48%, and the healing rate of ceramide 3B after 24h was 59.32%. The compound provided by the invention obviously improves the cell healing rate, has good skin tissue repair activity, and has a better effect than ceramide 3B.

Example 6

Elastase inhibition experiments to test anti-aging effect

Elastase inhibition method: 2mL of 2mg/mL elastase solution is taken, added with the products of the embodiment 1 with different concentrations, fully and uniformly mixed by vortex, shaken in a shaking table at 37 ℃ and 400r/min for 20min, immediately added with 5mL of 0.5mol/L phosphate buffer solution with pH6.0, uniformly mixed by vortex, taken a proper amount of the uniformly mixed solution to a 2mL centrifugal tube, centrifuged for 10min at 9 x 391 g, the supernatant liquid is precisely absorbed into a 96-well plate, the absorbance is measured by an enzyme-labeling instrument at the wavelength of 495nm, and simultaneously spectral scanning of 400-800 nm is carried out.

And taking a substrate and enzyme solution as a blank control group, taking a substrate and enzyme solution and a sample solution as an enzyme inhibition group, and taking the substrate and sample without the enzyme solution as background. Each group is provided with 3 holes. Inhibition (%) = [1- (An-An ')/(A0-A0') ]. Times.100%, wherein A0 is the absorbance of a sample without adding An enzyme, A0 'is the absorbance of a sample without adding only a substrate and An enzyme, an is the absorbance of a solution with only a sample, and An' is the absorbance of a sample without adding An enzyme. If An ' > An, a promoting effect is exhibited, and the promoting rate (%) = [1- (An ' -An)/(A0-A0 ') ] × 100%.

As shown in FIG. 4, the olive oil ceramide has a good inhibitory effect on elastase at different concentrations, specifically, the inhibitory rate on elastase is 11.13% at a concentration of 250mg/L, 21.67% at a concentration of 500mg/L, 30.26% at a concentration of 1000mg/L, and 19.81% at a concentration of 2000 mg/L.

The inhibitory activity of ceramide 3B against elastase was tested in the same manner, and the elastase inhibitory effect at a concentration of 582mg/L (i.e., 1 mmol/L) was 9.76%, which was not as good as that of olive oil ceramide at a similar concentration.

Example 7

LPS induced cell method for detecting anti-inflammatory repair effect

B16 mouse melanoma cells at a density of 1X 10 ⁴ One/well is planted in a 96-well plate, the 96-well plate is placed in an incubator and is adhered to the wall overnight, supernatant is discarded after 24 hours, 100 mu L of the product of the example 1 with different concentrations diluted by DMEM medium is added, a negative control group is DMEM medium without samples, 3 wells are added in each group, and the weight of the negative control group is 5wt% of the DMEM medium without samples％CO ₂ And incubating at 37 ℃. After 2h administration, the LPS model group and the experimental group were added with 10. Mu.g/mL LPS and incubated together for 24h. After the reaction, 50. Mu.L of cell supernatant was collected and the expression of IL-6 gene in the cells was detected by using IL-6ELISA kit.

The results are shown in FIG. 5, where IL-6 levels were 12.51-fold higher than basal levels when stimulated with LPS at a working concentration of 10. Mu.g/mL. Under the action of the olive oil ceramide with the concentrations of 50mg/L, 100mg/L, 200mg/L and 400mg/L respectively, the IL-6 factor level is obviously reduced and is 11.13 times and 10.26,8.31,6.10 times of the basic level respectively, and the dose dependence is formed, so that the olive oil ceramide has a good anti-inflammatory effect and can promote the repair of inflammatory damaged skin.

Example 8

MMP1 is also called interstitial collagenase and matrix metalloproteinase, belongs to the family of matrix metalloproteinase, and has the main function of a substrate, namely fibrous collagen, degrading collagen fibers and gelatin in extracellular matrix and changing microenvironment of cells. MMP1 plays an important role in elastin, inhibition of MMP1 can improve synthesis of collagen and elastin of fibroblasts, and reduction of MMP activity can increase collagen synthesis speed.

HaCaT cells were cultured at 1X 10 ⁵ The density of cells/well was plated in 96-well plates overnight in an incubator. After 24h, the supernatant was discarded, 100. Mu.L of the medium containing the product of example 1 at various concentrations (no drug was added to the model group), and the negative control group was DMEM medium containing no drug, 3 multiple wells each, and the CO was reduced at a mass fraction of 5% ₂ After incubation at 37 ℃ for 2h, UVA ultraviolet radiation was applied. The distance between the ultraviolet radiation source and the cell was 15cm, and the UVA intensity was 200mJ/cm ² The radiation time is 1.5h, and after the radiation is finished, the incubation is continued in the incubator for 12h. MMP-1 gene expression in cells was detected using an MMP-1ELISA kit.

As shown in FIG. 6, the MMP1 expression level of the negative control group was 1, the MMP1 expression level of the model group was 1.67, the concentration of olive oil ceramide was 50mg/L, the MMP1 expression level was 1.50, the MMP1 expression level was 1.39 at a concentration of 100mg/L, the MMP1 expression level was 1.32 at a concentration of 150mg/L, and the MMP1 expression level was 1.34 at a concentration of 200mg/L, and the inhibitory effect on MMP1 was more significant at higher concentrations.

After UVA ultraviolet radiation, keratinocytes promote increased fibroblast MMP1 expression, thereby causing degradation of skin extracellular matrix and skin collagen, resulting in skin photoaging. The results show that the olive oil ceramide can inhibit fibroblast cells caused by ultraviolet radiation from generating MMP1, and has certain effect on preventing skin photoaging.

Example 9

DPPH free radical scavenging and detecting antioxidant performance

DPPH is 1,1-diphenyl-2-trinitrophenylhydrazine, and can be used for antioxidant experiments.

Samples of 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 mixing DPPH and absolute ethyl alcohol in a volume ratio of 1:1, mixing the mixture in equal volume, reacting the mixture for 30min in a dark place at room temperature, and measuring the light absorption value at 517 nm. The absorbance of the sample and the DPPH reaction solution was denoted as A1, the absorbance of the sample and the absolute ethanol reaction solution was denoted as A2, the absorbance of the DPPH and the absolute ethanol reaction solution was denoted as A3, and the DPPH clearance of the sample = [1- (A1-A2)/A3 ] × 100%.

As shown in FIG. 7, the DPPH radical scavenging rates at concentrations of 50, 100, 200, 400, and 800mg/L were 29.42%, 39.49%, 46.77%, 55.26%, and 67.01, respectively, and excellent antioxidant effect was exhibited. Ceramide 3B (i.e., oleic ceramide) was tested for antioxidant effect in the same manner, and as shown in FIG. 8, DPPH radical scavenging rates at concentrations of 50, 100, 200, 400, 800mg/L were 7.76%, 12.82%, 24.10%, 29.60%, 33.16%. The clearance rate of olive oil ceramide on DPPH is higher than that of ceramide 3B (oleic acid ceramide), and the olive oil ceramide has better antioxidant effect.

Example 10

Whitening Activity test

Taking B16 cells in exponential growth phase, digesting with trypsin-EDTA with the mass fraction of 0.25%, blowing uniformly, and mixing the cells according to the proportion of 3 multiplied by 10 ⁵ The density of each well was seeded in 12-well plates. At 37 ℃ and 5% by mass of CO ₂ Culturing in the environmentOvernight. Discarding supernatant, adding culture solution containing samples with different mass concentrations, incubating with RPMI-1640 medium without drug as blank group, incubating with DMEM medium as molding group, each group having 3 multiple wells, and making CO content of 5% by mass ₂ And incubating for 24 hours at 37 ℃. After the medium in the well plate was discarded and washed once to twice with Phosphate Buffered Saline (PBS), 1mL of a NaOH solution (1 mol/L) containing 10% by mass of DMSO was added and the mixture was incubated at 80 ℃ or 100 ℃ for 2 hours until the cells were completely lysed. The sample was placed in a microplate reader and absorbance was measured at 405 nm. Melanin production inhibition rate = (each well OD value/blank group OD value) × 100% was calculated.

As a result, as shown in fig. 9, the melanin of the model group was 157.36%, and the melanin inhibition ratios of olive oil ceramide were 147.77%, 131.84%, 120.56%, 108.82%, and 96.28% at concentrations of 10, 20, 40, 80, and 100mg/L, respectively, and olive oil ceramide showed excellent whitening effects.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. An olive oil ceramide, which is obtained by reacting an olive oil fatty acid with a sphingoid compound selected from sphingosine, phytosphingosine, dihydrosphingosine.

2. The olive oil ceramide according to claim 1, wherein the olive oil fatty acid is obtained from hydrolysis of olive oil.

3. The olive oil ceramide according to claim 1 or 2, wherein the olive oil fatty acid contains 20 to 83wt% oleic acid and 3.5 to 70wt% linoleic acid.

4. Olive oil ceramide, the composition of which comprises: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide.

5. The olive oil ceramide according to claim 4, whose composition comprises: 20 to 83 weight percent of oleic acid ceramide, 7.5 to 20 weight percent of palmitic acid ceramide, 3.5 to 70 weight percent of linoleic acid ceramide, 0.5 to 5 weight percent of stearic acid ceramide and 0.1 to 1.0 weight percent of linolenic acid ceramide.

6. The olive oil ceramide according to claim 4 or 5, the composition of which further comprises: 0 to 3.5 weight percent of palmitoleic acid ceramide, 0 to 0.6 weight percent of arachidic acid ceramide, 0 to 0.4 weight percent of eicosenoic acid ceramide, 0 to 0.3 weight percent of heptadecanoic acid ceramide, 0 to 0.3 weight percent of heptadecenoic acid ceramide, 0 to 0.2 weight percent of behenic acid ceramide, 0 to 0.2 weight percent of tetracosanoic acid ceramide, and 0 to 0.05 weight percent of myristic acid ceramide.

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

reacting olive oil fatty acid with sphingoid compound under the condition of condensing agent, wherein the condensing agent is EDCI and Et ₃ N；

The olive oil fatty acid, sphingoid compound, EDCI, et ₃ The molar ratio of N is 1: (1-1.5): (1-2): (1-2), wherein the solvent for reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.

8. Use of the olive oil ceramide as claimed in any one of claims 1 to 6 in cosmetics, pharmaceuticals, dietary foods or health products.

9. The use according to claim 8, wherein said olive oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promotion, elastin enhancement, and whitening efficacy.

10. A composition comprising the olive oil ceramide of any one of claims 1 to 6, having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, collagen synthesis promoting, elastin enhancement, and whitening effects.