Olive oil ceramide and its synthesis method and use

Abstract

This invention belongs to the field of biomedicine and discloses olive oil ceramide, which is obtained by reacting olive oil fatty acids with sphingosine compounds selected from sphingosine, phytosphingosine, and dihydrosphingosine. This invention also discloses a synthesis method and uses of olive oil ceramide. Olive oil ceramide exhibits excellent properties in repairing the skin's natural barrier, anti-inflammatory, tissue healing, and anti-aging, and has broad application prospects in cosmetics, health products, biopharmaceuticals, and other fields.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to olive oil ceramide and a synthesis method and use thereof. Background Art

[0002] Ceramide (also known as molecular nail) is naturally present in the skin and is a crucial component of the skin barrier (stratum corneum), comprising up to 40-50% by weight. Ceramides are a class of lipids composed of a long-chain sphingosine base and fatty acids. The carbon chain length, degree of unsaturation, and number of hydroxyl groups in the sphingosine and fatty acid moieties can vary. Ceramides represent a class of compounds that exhibit excellent properties in regulating skin barrier function, restoring skin moisture, and enhancing adhesion between keratinocytes.

[0003] Due to the importance of ceramides, many cosmetics and pharmaceutical companies are researching and developing related products. Natural plant-derived ceramides, due to their more sustainable and environmentally friendly raw material sources and similar composition to skin ceramides, can form an effective skin barrier to prevent moisture loss and protect against external damage. They may become the next generation of environmentally friendly, safe and reliable ceramide products.

[0004] Olive oil, generally referring to olive oil, is cold-pressed directly from fresh olive fruits, untreated by heat or chemical treatment, preserving its natural nutrients. It is known in the West as "liquid gold," "the queen of vegetable oils," and "nectar of the Mediterranean." Rich in the monounsaturated fatty acid oleic acid, as well as vitamins A, B, D, E, K, and antioxidants, olive oil is considered the most beneficial oil ever discovered for human health. Data indicates that olive oil can reduce catecholamine synthesis while inhibiting the production of reactive oxygen species, lipid peroxidation, and protein carbonylation in the skin. It offers excellent anti-aging, antioxidant, whitening, and pore-minimizing benefits. Its rich content of seborrheic vitamins is beneficial for the skin, making it a safe and reliable beauty product, earning it the nickname "edible cosmetics." Furthermore, research indicates that olive oil has medicinal benefits, including promoting blood circulation, reducing cardiovascular disease, promoting bone and nervous system development, promoting weight loss, healing wounds, and slowing neuronal degeneration in the elderly. Olive oil can also be used as a sedative and as an adjunct treatment for jaundice and gallstones. Summary of the Invention

[0005] The present invention aims to provide a ceramide synthesized from olive oil fatty acids of plant origin.

[0006] Another object of the present invention is to provide a method for synthesizing olive oil ceramide, which utilizes olive oil or olive oil fatty acid, which is readily available from natural plant sources, as a raw material.

[0007] Another object of the present invention is to provide uses of olive oil ceramide.

[0008] In order to achieve one of the above purposes, the present invention adopts the following technical solutions:

[0009] In a first aspect of the present invention, olive oil ceramide is obtained by reacting olive oil fatty acid with a sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine, and dihydrosphingosine.

[0010] The reaction can be a chemical synthesis reaction (as described in detail below) or a microbial fermentation method, that is, using Pichia sherwoodii or Saccharomyces cerevisiae to ferment under certain conditions to obtain sphingosine compounds, and then adding fatty acids to finally obtain ceramide; or using olive oil as the raw material, selecting a suitable strain to ferment to obtain olive oil ceramide.

[0011] 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.

[0012] Furthermore, the olive oil fatty acid is obtained by hydrolyzing olive oil.

[0013] Furthermore, the olive oil fatty acid contains 20 to 83 wt% oleic acid, preferably 55 to 83 wt% oleic acid.

[0014] Furthermore, the olive oil fatty acid contains 7.5-20 wt% palmitic acid.

[0015] Furthermore, the olive oil fatty acid contains 3.5 to 70 wt% linoleic acid, preferably 3.5 to 21 wt% linoleic acid.

[0016] Furthermore, the olive oil fatty acid contains 0.5 to 5 wt% of stearic acid.

[0017] Furthermore, the olive oil fatty acid contains 0.1 to 1.0 wt% of linolenic acid.

[0018] In addition, olive oil fatty acids also contain 0-3.5wt% palmitic acid, 0-0.6wt% arachidic acid, 0-0.4wt% eicosenoic acid, 0-0.3wt% heptadecanoic acid, 0-0.3wt% heptadecanoic acid, 0-0.2wt% behenic acid, 0-0.2wt% lignoceric acid, and 0-0.05wt% myristic acid.

[0019] The fatty acid composition of olive oil is: 20-83wt% oleic acid, 7.5-20wt% palmitic acid, 3.5-70wt% linoleic acid, 0.5-5wt% stearic acid, 0.1-1.0wt% linolenic acid, 0-3.5wt% palmitoleic acid, 0-0.6wt% arachidic acid, 0-0.4wt% eicosenoic acid, 0-0.3wt% heptadecanoic acid, 0-0.3wt% heptadecanoic acid, 0-0.2wt% behenic acid, 0-0.2wt% tetracosanoic acid, and 0-0.05wt% myristic acid.

[0020] The main component of olive oil fatty acids is oleic acid. Other fatty acids include linoleic acid, palmitic acid, stearic acid, and linolenic acid, which are necessary components. The content of each component will vary depending on the tree species, soil, climate, origin, picking season, and extraction process. Palmitoleic acid, arachidic acid, eicosenoic acid, heptadecanoic acid, heptadecanedetonanoic acid, behenic acid, tetracosanoic acid, and myristic acid are not necessarily present and are optional or non-essential components.

[0021] Olive oil ceramide, comprising: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, and linolenic acid ceramide. Since all fatty acids participate in the same reaction, the mass proportion of ceramide after the reaction does not change much. Therefore, the composition of olive oil fatty acids can be used as a reference. The composition of olive oil ceramide is: 20-83 wt% oleic acid ceramide, 7.5-20 wt% palmitic acid ceramide, 3.5-70 wt% linoleic acid ceramide, 0.5-5 wt% stearic acid ceramide, and 0.1-1.0 wt% linolenic acid ceramide. A preferred composition is: 55-83 wt% oleic acid ceramide, 7.5-20 wt% palmitic acid ceramide, 3.5-21 wt% linoleic acid ceramide, 0.5-5 wt% stearic acid ceramide, and 0.1-1.0 wt% linolenic acid ceramide. The content of each component varies depending on the content of each fatty acid in olive oil fatty acids or oils. In addition, olive oil ceramide also includes ceramide obtained by reacting one or more of palmitic acid, arachidic acid, eicosenoic acid, heptadecanoic acid, heptadecanedioic acid, behenic acid, tetradecanoic acid, and myristic acid with sphingosine compounds, that is, it also includes 0-3.5wt% palmitic acid ceramide, 0-0.6wt% arachidic acid ceramide, 0-0.4wt% eicosenoic acid ceramide, 0-0.3wt% heptadecanoic acid ceramide, 0-0.3wt% heptadecanedioic acid ceramide, 0-0.2wt% behenic acid ceramide, 0-0.2wt% tetradecanoic acid ceramide, and 0-0.05wt% myristic acid ceramide.

[0022] Olive oil ceramide, which comprises: oleic acid ceramide, palmitic acid ceramide, and linoleic acid ceramide; oleic acid ceramide accounts for 20 to 83 wt%, preferably 55 to 83 wt%, palmitic acid ceramide accounts for 7.5 to 20 wt%, and linoleic acid ceramide accounts for 3.5 to 70 wt%, preferably 3.5 to 21 wt%.

[0023] Furthermore, the olive oil ceramide includes stearic acid ceramide, and the stearic acid ceramide accounts for 0.5 to 5 wt%.

[0024] Furthermore, the olive oil ceramide includes linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.1 to 1.0 wt%.

[0025] Oleic acid ceramide is obtained by the condensation reaction of oleic acid and sphingosine compounds, including oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide, and oleic acid dihydrosphingosine ceramide; linoleic acid ceramide is obtained by the condensation reaction of linoleic acid and sphingosine compounds, including linoleic acid phytosphingosine ceramide, linoleic acid sphingosine ceramide, and linoleic acid dihydrosphingosine ceramide; palmitic acid ceramide is obtained by the condensation reaction of palmitic acid and sphingosine compounds, including palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide, and palmitic acid sphingosine ceramide. Phytosphingosine ceramide, palmitic acid dihydrosphingosine ceramide; stearic acid ceramide is obtained by the condensation reaction of stearic acid and sphingosine compounds, including stearic acid phytosphingosine ceramide, stearic acid sphingosine ceramide, stearic acid dihydrosphingosine ceramide; linolenic acid ceramide is obtained by the condensation reaction of linolenic acid and sphingosine compounds, including linolenic acid phytosphingosine ceramide, linolenic acid sphingosine ceramide, linolenic acid dihydrosphingosine ceramide; arachidic acid ceramide, eicosenoic acid ceramide, etc. and so on.

[0026] The second aspect of the present invention is a method for synthesizing olive oil ceramide, comprising the following steps:

[0027] Olive oil fatty acid reacts with sphingosine compounds under condensation agent conditions, and the condensation agent is EDCI and Et3N.

[0028] Furthermore, the molar ratio of the olive oil fatty acid, sphingosine compound, EDCI, and Et3N is 1:(1-1.5):(1-2):(1-2), and the solvent of the reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate, and acetonitrile.

[0029] Olive oil purchased on the market is generally in the form of oil and needs to be hydrolyzed into olive oil fatty acids, so the following steps are also included:

[0030] Olive oil is hydrolyzed through saponification to produce olive oil fatty acids.

[0031] Furthermore, the saponification reaction is the hydrolysis of olive oil in a potassium hydroxide solution.

[0032] Furthermore, the mass ratio of the olive oil fat to potassium hydroxide is 1:(1-2).

[0033] The third aspect of the present invention is the use of olive oil ceramide in cosmetics and medicines.

[0034] Furthermore, the olive oil ceramide has at least one of the following effects: skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, promotion of collagen synthesis, increase of elastin, and whitening.

[0035] A composition comprises olive oil ceramide, and the composition has at least one of the following effects: skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidation, promotion of collagen synthesis, increase of elastin, and whitening.

[0036] The composition contains acceptable excipients, including one or more of solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, moisturizers, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, flavors, and pigments; the composition is in the form of a cream, emulsion, solution, film, aerosol, or spray.

[0037] The present invention has the following beneficial effects:

[0038] Olive oil fatty acids are naturally occurring fatty acids. The main component is monounsaturated fatty acids - oleic acid or linoleic acid. In addition, it also contains palmitic acid, stearic acid, linolenic acid, etc. It reacts with sphingosine compounds naturally present in the skin to produce olive oil ceramide through a mild reaction. It shows excellent performance in repairing the skin's natural barrier, anti-oxidation, and anti-aging, and has broad application prospects in cosmetics, biomedicine and other fields.

[0039] 1. Better results than single ceramides. Different ceramides have different effects due to their structural differences, and a single ceramide generally cannot achieve comprehensive effects. This solution is based on a biomimetic approach, using naturally derived olive oil or fatty acids as raw materials to synthesize composite ceramides to compensate for the differences in the effects of different ceramides. The trace fatty acids in the olive source can form trace ceramides, which play a complementary role in efficacy.

[0040] 2. It has better effects than compound ceramides. In addition to oleic acid (or oil), olive oil also contains vitamins, polyphenols and other ingredients. These nutrients have the effects of moisturizing the skin and enhancing cell vitality. Ceramide synthesized from olive oil has a synergistic effect with other active ingredients contained in olive oil. Compared with ceramides compounded in similar proportions, it has better effects.

[0041] 3. Lower cost. The method of the present invention quickly produces a composite of multiple ceramides. Olive oil or its fatty acids, which are plant-derived, are widely available, easily commercially available, and relatively low in cost, making them more environmentally friendly and economical. This is different from the idea of mixing and compounding different single ceramides. Single-ingredient fatty acids not only have high raw material prices but also require the separate production of different ceramides and then compounding, increasing preparation costs.

[0042] 4. Simple synthesis method: The method of the present invention can be prepared by chemical synthesis to prepare multiple ceramides in one step, or by microbial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 、 2 This is a bar graph of the cell proliferation activity test results in Example 4;

[0044] Figure 3 The results of cell migration ability test in Example 5 are as follows;

[0045] Figure 4 This is a bar graph of the elastase inhibition rate in Example 6;

[0046] Figure 5 This is a bar graph showing the anti-inflammatory and repair efficacy test results of Example 7;

[0047] Figure 6 This is a bar graph showing the expression level of MMP1 in Example 8;

[0048] Figure 7 、 8 This is a bar chart of the antioxidant test results of Example 9;

[0049] Figure 9 This is a bar chart of the whitening activity test results of Example 10. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to specific embodiments.

[0051] EDCI refers to 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and Et3N refers to triethylamine. Silica gel column chromatography used Qingdao Marine Silica Gel (particle size 0.040-0.063 mm). Thin layer chromatography (TLC) used 60F254 silica gel plates, and TLC color development used UV light (254 nm) or iodine. NMR spectra were characterized using a Bruker DPX 400 nuclear magnetic resonance instrument. 1 H NMR is performed at 400 MHz, with deuterated methanol, deuterated DMSO or deuterated tetrahydrofuran as the solvent, and tetramethylsilane (TMS) as the internal standard. The unit of chemical shift is ppm, and the unit of coupling constant is Hz. 1 In H NMR, δ represents chemical shift, s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet.

[0052] Example 1

[0053] Synthesis of ceramide from olive oil fatty acids and phytosphingosine

[0054] Step 1: Dissolve 50 g of olive oil in 60 mL of tetrahydrofuran, cool in an ice bath, and add 100 mL of potassium hydroxide (25 wt%) solution dropwise. After the addition is complete, warm the mixture to room temperature and react until the reaction is complete as determined by TLC.

[0055] Post-treatment: dilute hydrochloric acid (3N) was added to adjust the pH value of the reaction system to 3, 150 mL of ethyl acetate was added to extract the aqueous phase, and 100 mL of saturated brine was added to wash once. The organic phase was added with anhydrous Na2SO4 to dry, filtered and concentrated in vacuo to obtain 40 g of olive oil fatty acid.

[0056] Step 2: Add 250 mL of olive oil fatty acid (50 mmol, molecular weight based on oleic acid), EDCI (60 mmol), and Et3N (60 mmol) into a round-bottom flask, then add 100 mL of dichloromethane, and stir at room temperature for 1 hour. Then, add phytosphingosine (60 mmol) to the reaction system and stir at room temperature until the reaction is completed as detected by TLC.

[0057] Post-treatment: water was added to quench the reaction, the organic layer was separated, dried, filtered, concentrated in vacuo, and then washed with a solvent to obtain olive oil ceramide. The product was analyzed by HPLC. HPLC chromatographic conditions: using a Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), using an InnovaLDS-2 4.6*250mm, 5μm column, column temperature: 30°C, injection volume: 10μL, flow rate: 1.0mL / min, evaporation temperature: 40°C, carrier gas flow rate: 2.5L / min, mobile phase: 100% methanol.

[0058] The HPLC retention times of each component are: oleic acid-phytosphingosine ceramide 11.3 min, linoleic acid-phytosphingosine ceramide 9.5 min, palmitic acid-phytosphingosine ceramide 10.7 min, stearic acid-phytosphingosine ceramide 13.9 min, and linolenic acid-phytosphingosine ceramide 8.2 min.

[0059] The resulting product was separated 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 (the nuclear magnetic hydrogen spectrum was consistent with the standard spectrum), with mass ratios of 21.3%, 10.5%, 63.2%, 2.4%, and 0.4%, respectively. The rest were other components with low contents. This embodiment uses domestic olive oil fatty acid, the main component of which is linoleic acid, and the resulting product is mainly linoleic acid ceramide.

[0060] Example 2

[0061] Synthesis of ceramide from olive oil fatty acids and sphingosine

[0062] Step 1: Dissolve 50 g of olive oil in 60 mL of tetrahydrofuran, cool in an ice bath, and add 100 mL of potassium hydroxide (25 wt%) solution dropwise. After the addition is complete, warm the mixture to room temperature and react until the reaction is complete as determined by TLC.

[0063] Post-treatment: dilute hydrochloric acid (3N) was added to adjust the pH value of the reaction system to 3, 150 mL of ethyl acetate was added to extract the aqueous phase, and 100 mL of saturated brine was added to wash once. The organic phase was added with anhydrous Na2SO4 to dry, filtered and concentrated in vacuo to obtain 40 g of olive oil fatty acid.

[0064] Step 2: Add 250 mL of olive oil fatty acid (50 mmol, molecular weight based on oleic acid), EDCI (75 mmol), and Et3N (75 mmol) into a round-bottom flask, then add 100 mL of dichloromethane, and stir at room temperature for 1 hour. Then, add sphingosine (50 mmol) to the reaction system and stir at room temperature until the reaction is complete as detected by TLC.

[0065] Post-treatment: water was added to quench the reaction, the organic layer was separated, dried, filtered, concentrated in vacuo, and then washed with a solvent to obtain olive oil ceramide. The product was analyzed by HPLC. HPLC chromatographic conditions: using a Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), using an Innova! ODS-2 4.6*250 mm, 5 μm column, column temperature: 30 ° C, injection volume: 10 μL, flow rate: 1.0 mL / min, evaporation temperature: 40 ° C, carrier gas flow rate: 2.5 L / min, mobile phase: (2 wt% water + 98 wt% methanol) both containing 0.1 wt% formic acid.

[0066] The HPLC retention time of each component is: oleic acid-sphingosine ceramide 20.7 min, linoleic acid-sphingosine ceramide 16.6 min.

[0067] The resulting product was separated 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 (the nuclear magnetic resonance 1H spectrum was consistent with the standard spectrum), with mass ratios of 61.8%, 12.6%, 19.3%, 3.4%, and 0.6%, respectively. The remainder was other components with a small content. This embodiment uses imported olive oil fatty acid, the main component of which is oleic acid, and the resulting product is mainly oleic acid ceramide.

[0068] Oleic acid-sphingosine ceramide hydrogen spectrum: 1 H NMR(400MHz,Methanol-d4)δ7.70(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(dd d,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, 6.0Hz, 6H), 1.64-1.51 (m, 2H), 1.30 (d, J = 14.5Hz, 41H), 0.90 (t, J = 6.7Hz, 6H). Linoleic acid-sphingosine ceramide hydrogen spectrum: 1H 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),2.1 3-1.94(m,6H),1.67-1.49(m,2H),1.31(d,J=19.6Hz,40H),0.90(td,J=6.8,3.8Hz,6H).

[0069] Example 3

[0070] Synthesis of ceramide from olive oil fatty acids and dihydrosphingosine

[0071] Step 1: Dissolve 50 g of olive oil in 60 mL of tetrahydrofuran, cool in an ice bath, and add 100 mL of potassium hydroxide (25 wt%) solution dropwise. After the addition is complete, warm the mixture to room temperature and react until the reaction is complete as determined by TLC.

[0072] Post-treatment: dilute hydrochloric acid (3N) was added to adjust the pH value of the reaction system to 3, 150 mL of ethyl acetate was added to extract the aqueous phase, and 100 mL of saturated brine was added to wash once. The organic phase was added with anhydrous Na2SO4 to dry, filtered and concentrated in vacuo to obtain 40 g of olive oil fatty acid.

[0073] Step 2: Add 250 mL of olive oil fatty acid (50 mmol, molecular weight based on oleic acid), EDCI (100 mmol), and Et3N (100 mmol) into a round-bottom flask, then add 100 mL of dichloromethane, and stir at room temperature for 1 hour. Then, add dihydrosphingosine (75 mmol) to the reaction system and stir at room temperature until the reaction is completed as detected by TLC.

[0074] Post-treatment: water was added to quench the reaction, the organic layer was separated, dried, filtered, concentrated in vacuo, and then washed with a solvent to obtain olive oil ceramide. The product was analyzed by HPLC. HPLC chromatographic conditions: using a Shimadzu high performance liquid chromatograph (LC-2030C 3DPlus), using an Innova! ODS-2 4.6*250 mm, 5 μm column, column temperature: 30 ° C, injection volume: 10 μL, flow rate: 1.0 mL / min, evaporation temperature: 40 ° C, carrier gas flow rate: 2.5 L / min, mobile phase: (2 wt% water + 98 wt% methanol) both containing 0.1 wt% formic acid.

[0075] The HPLC retention time of each component is: oleic acid-dihydrosphingosine ceramide 23.6 minutes, linoleic acid-dihydrosphingosine ceramide 19.1 minutes.

[0076] The resulting product was separated by preparative high-performance liquid chromatography to yield oleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, linoleic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, and linolenic acid-dihydrosphingosine ceramide (H NMR spectra were consistent with the standard spectrum), with mass ratios of 29.9%, 18.2%, 45.1%, 2.3%, and 0.9%, respectively. The remainder consisted of other components, present in relatively small amounts.

[0077] Oleic acid-dihydrosphingosine ceramide hydrogen spectrum: 1 H NMR (400 MHz, Methanol-d4) δ 5.34 (t, J = 4.9 Hz, 2H), 3.81 (q, J = 5.6 Hz, 1H), 3.75-3.65 (m, 2H), 3.65-3.51 (m, 1H), 2.22 (dd, J = 9.2, 5.4 Hz, 2H), 2.03 (q, J = 6.3 Hz, 4H), 1.70-1.49 (m, 5H), 1.48-1.05 (m, 45H), 0.90 (t, J = 6.6 Hz, 6H). Linoleic acid-dihydrosphingosine 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),2.2 2(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).

[0078] Example 4

[0079] MTT assay to detect the effects of compounds on cell proliferation

[0080] HaCaT cells were cultured at a rate of 1 × 10 4 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated overnight in an incubator. After 24 hours, the supernatant was discarded and 100 μL of culture medium containing the product of Example 1 at different concentrations was added. After further incubation for 24 hours, the culture medium was removed and 100 μL of MTT was added to each well. The absorbance at 450 nm was measured and the cell survival rate was calculated as A. 给药孔 / A空白孔 ×100%.

[0081] The results are as follows Figure 1 As shown, olive oil ceramide has a promoting effect on cell viability. The cell survival rates at concentrations of 70, 150, 250, and 300 mg / L are 102.7%, 121.2%, 145.2%, and 165.2%, respectively, showing a significant effect of promoting cell proliferation and having good tissue repair ability.

[0082] The same method was used to test the effect of ceramide 3B (i.e., oleic acid ceramide) on cell proliferation activity. The results are as follows: Figure 2 As shown in the data, the cell survival rates at concentrations of 70, 150, 250, and 300 mg / L were 101.7%, 111.2%, 126.8%, and 141.9%, respectively. Its effect in promoting cell proliferation and tissue repair potential was not as good as that of olive oil ceramide.

[0083] Example 5

[0084] Cell migration to assess skin barrier repair

[0085] Principle: When cells grow to a fused monolayer state, a scratch tool is used to create a blank area on the fused monolayer of cells. The cells in the blank area are removed by mechanical force. After a period of culture, the migration of cells to the cell-free area is observed. The migration distance of the cells is measured to reflect the migration ability of the cells.

[0086] Steps:

[0087] 1. Mark the culture plate. First, use a marker pen and a ruler to evenly mark horizontal lines on the back of the 6-well plate, approximately every 0.5 to 1 cm, across the wells. Make at least 5 lines per well. Be careful not to make the lines too thick.

[0088] 2. Add about 5×10 cells to the well. 5 cells (the number of different cells varies and is adjusted according to the growth rate of the cells). The inoculation principle is that the fusion rate reaches 100% after overnight.

[0089] 3. Streak the cells. On the second day, use the pipette tip, perpendicular to the cell plane, to scratch the cell layer along the line drawn on the back of the plate the day before (it is best to use the same pipette tip between different wells).

[0090] 4. Wash the cells. After the scratch is completed, wash the cells three times with sterile PBS to remove the non-adherent cells, that is, the cells that were scratched during the scratching process. The gaps left after the scratching are clearly visible, and then replace with fresh serum-free medium.

[0091] 5. Cell Culture and Observation: The sample (product from Example 1, ceramide 3B) was diluted with culture medium (concentration of 0.08 mM) and added to a cell culture dish. The cells were cultured in a 37°C, 5 wt% CO2 incubator. After 24 hours, the cells were removed, and the scratch width was measured and photographed under a microscope. The healing rate was calculated using Image J software.

[0092] The results are as follows Figure 3 As shown, the scratch width in the experimental group was narrower than that in the solvent control group, demonstrating that olive oil ceramide possesses superior tissue healing capabilities. The healing rate after 24 hours was 31.25% for the solvent control group, 74.48% for olive oil ceramide, and 59.32% for ceramide 3B. The compound of the present invention significantly enhanced the cell healing rate, exhibiting excellent skin tissue repair activity, and exhibited superior efficacy compared to ceramide 3B.

[0093] Example 6

[0094] Elastase inhibition assay to test anti-aging effects

[0095] Elastase inhibition method: 2 mL of 2 mg / mL elastase solution was added to different concentrations of the product of Example 1, and the mixture was thoroughly vortexed and shaken at 37°C and 400 rpm for 20 min. 5 mL of 0.5 mol / L phosphate buffer (pH 6.0) was immediately added and vortexed. An appropriate amount of the mixture was transferred to a 2 mL centrifuge tube and centrifuged at 9,391 × g for 10 min. 200 μL of the supernatant was accurately pipetted into a 96-well plate and the absorbance was measured using a microplate reader at a wavelength of 495 nm, while simultaneously performing a spectral scan from 400 to 800 nm.

[0096] The substrate plus enzyme solution served as the blank control group, the substrate plus enzyme and sample solution served as the enzyme inhibition group, and the substrate plus sample without enzyme solution served as background subtraction. Each group had three replicate wells. Inhibition rate (%) = [1-(An-An') / (A0-A0')] × 100%, where A0 is the absorbance with enzyme but no sample, A0' is the absorbance with substrate alone without sample and enzyme, An is the absorbance with sample solution alone, and An' is the absorbance with sample but no enzyme. If An'>An, it indicates a promoting effect, and the promotion rate (%) = [1-(An'-An) / (A0-A0')] × 100%.

[0097] The results are as follows Figure 4As shown in the results, olive oil ceramide has a good inhibitory effect on elastase at different concentrations. Specifically, the inhibition rate of elastase is 11.13% at a concentration of 250 mg / L, 21.67% at a concentration of 500 mg / L, 30.26% at a concentration of 1000 mg / L, and 19.81% at a concentration of 2000 mg / L.

[0098] The inhibitory activity of ceramide 3B on elastase was tested using the same method. The elastase inhibition rate was 9.76% at a concentration of 582 mg / L (ie, 1 mmol / L), which was inferior to that of olive oil ceramide at a similar concentration.

[0099] Example 7

[0100] LPS-induced cell assay to detect anti-inflammatory and repair effects

[0101] B16 mouse melanoma cells were cultured at a density of 1×10 4 Cells were seeded in 96-well plates and placed in an incubator to adhere overnight. After 24 hours, the supernatant was discarded and 100 μL of the product of Example 1 diluted with DMEM medium at different concentrations was added. The negative control group was a DMEM medium without sample. Each group had 3 replicates and was incubated in a 5wt% CO2, 37°C environment. 2 hours after administration, 10 μg / mL LPS was added to the lipopolysaccharide model group and the experimental group and incubated together for 24 hours. After the reaction was completed, 50 μL of the cell supernatant was taken and the intracellular IL-6 gene expression was detected using an IL-6 ELISA kit.

[0102] The results are as follows Figure 5 As shown in the results, under the stimulation of LPS at a working concentration of 10 μg / mL, the IL-6 level was 12.51 times the basal level. Under the action of olive oil ceramide at concentrations of 50 mg / L, 100 mg / L, 200 mg / L, and 400 mg / L, the IL-6 factor level was significantly reduced to 11.13, 10.26, 8.31, and 6.10 times the basal level, respectively, in a dose-dependent manner. This proves that olive oil ceramide has a good anti-inflammatory effect and can promote the repair of inflammatory damaged skin.

[0103] Example 8

[0104] MMP1, also known as interstitial collagenase or matrix metalloproteinase, belongs to the matrix metalloproteinase family. Its primary substrate is fibrillar collagen, degrading collagen fibers and gelatin within the extracellular matrix and altering the cellular microenvironment. MMP1 plays a crucial role in elastin production. Inhibiting MMP1 can enhance collagen and elastin synthesis in fibroblasts, while reducing MMP activity can increase the rate of collagen synthesis.

[0105] HaCaT cells were cultured at a rate of 1 × 10 5 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated overnight in an incubator. After 24 hours, the supernatant was discarded and 100 μL of culture medium containing different concentrations of the product of Example 1 (no drug was added to the model group). The negative control group was treated with DMEM culture medium without drug. Each group had 3 replicates. After incubation for 2 hours in an environment of 5% CO2 and 37°C, UVA was irradiated. The distance between the UV radiation source and the cells was 15 cm, and the UVA intensity was 200 mJ / cm 2 The irradiation time was 1.5 hours. After the irradiation, the cells were incubated in the incubator for 12 hours. The expression of MMP-1 gene in the cells was detected using an MMP-1 ELISA kit.

[0106] The results are as follows Figure 6 As shown in the figure, the MMP1 expression level of the negative control group was set to 1, the expression level of the model group was 1.67, the expression level of MMP1 was 1.50 when the concentration of olive oil ceramide was 50 mg / L, the expression level of MMP1 was 1.39 when the concentration was 100 mg / L, the expression level of MMP1 was 1.32 when the concentration was 150 mg / L, and the expression level of MMP1 was 1.34 when the concentration was 200 mg / L. The higher the concentration, the more significant the inhibitory effect on MMP1.

[0107] After UVA radiation, keratinocytes promote the increase in MMP1 expression in fibroblasts, which causes the degradation of the skin's extracellular matrix and skin collagen, leading to skin photoaging. These results indicate that olive oil ceramide can inhibit the production of MMP1 in fibroblasts caused by UV radiation, and has a certain effect on preventing skin photoaging.

[0108] Example 9

[0109] DPPH free radical scavenging assay for antioxidant activity

[0110] DPPH is 1,1-diphenyl-2-trinitrophenylhydrazine, which can be used in antioxidant experiments.

[0111] Samples of corresponding concentrations (50, 100, 200, 400, and 800 mg / L) were mixed with 0.1 mol / L DPPH and anhydrous ethanol solutions at a 1:1 volume ratio. DPPH and anhydrous ethanol were then mixed in equal volumes at a 1:1 ratio. The mixture was allowed to react at room temperature in the dark for 30 minutes, and the absorbance was measured at 517 nm. The absorbance of the sample-DPPH reaction solution was designated as A1, the absorbance of the sample-anhydrous ethanol reaction solution as A2, and the absorbance of the DPPH-anhydrous ethanol reaction solution as A3. The DPPH scavenging efficiency of the sample = [1-(A1-A2) / A3] × 100%.

[0112] The results are as follows Figure 7 As shown in Figure 2, the DPPH free radical scavenging rates at concentrations of 50, 100, 200, 400, and 800 mg / L were 29.42%, 39.49%, 46.77%, 55.26%, and 67.01%, respectively, demonstrating excellent antioxidant effects. The antioxidant effects of ceramide 3B (i.e., oleic acid ceramide) were tested using the same method, and the results were as follows: Figure 8 As shown in the results, the DPPH free radical scavenging rates at concentrations of 50, 100, 200, 400, and 800 mg / L were 7.76%, 12.82%, 24.10%, 29.60%, and 33.16%, respectively. Olive oil ceramide has a higher DPPH scavenging rate than ceramide 3B (oleic acid ceramide), demonstrating a superior antioxidant effect.

[0113] Example 10

[0114] Whitening activity test

[0115] B16 cells in the exponential growth phase were obtained and digested with 0.25% trypsin-EDTA and pipetted evenly. The cells were plated at 3×10 5 Cells were seeded into 12-well plates at a density of 100 cells / well. Cultured overnight at 37°C and 5% CO2. The supernatant was discarded and culture medium containing samples of different mass concentrations was added. The blank group was incubated with RPMI-1640 medium without drugs, and the model group was incubated with DMEM medium. Each group had 3 replicates and incubated for 24 hours in a 5% CO2, 37°C environment. The culture medium in the well plate was discarded, and after washing once or twice with phosphate buffered saline (PBS), 1 mL of NaOH solution (1 mol / L) containing 10% DMSO was added. The plate was kept constant at 80°C or 100°C for 2 hours until the cells were completely dissolved. The plate was placed in a microplate reader and the absorbance was measured at 405 nm. The melanin inhibition rate was calculated as (OD value of each well / OD value of the blank group) × 100%.

[0116] The results are as follows Figure 9 As shown, the melanin expression of the modeling group was 157.36%. At concentrations of 10, 20, 40, 80, and 100 mg / L, the melanin inhibition rates of olive oil ceramide were 147.77%, 131.84%, 120.56%, 108.82%, and 96.28%, respectively. Olive oil ceramide showed a good whitening effect.

[0117] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Olive oil ceramide, obtained by reacting olive oil fatty acids with sphingosine compounds, wherein the sphingosine compounds are selected from sphingosine, dihydrosphingosine, and phytosphingosine, characterized in that: The olive oil ceramide composition comprises: 21.3-61.8% oleic acid ceramide, 10.5-18.2% palmitic acid ceramide, 19.3-63.2% linoleic acid ceramide, 2.3-3.4% stearic acid ceramide, and 0.4-0.9% linolenic acid ceramide; The olive oil ceramide is synthesized according to the following steps: Olive oil fatty acid reacts with sphingosine compounds under condensation conditions, wherein the condensation agent is EDCI and Et3N; The molar ratio of the olive oil fatty acid, sphingosine compound, EDCI, and Et3N is 1:(1-1.5):(1-2):(1-2), and the solvent of the reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate, and acetonitrile.

2. The olive oil ceramide according to claim 1, characterized in that The olive oil fatty acid is obtained by hydrolyzing olive oil.

3. The olive oil ceramide according to claim 1, characterized in that The olive oil ceramide composition also includes: 0-3.5wt% palmitoleic acid ceramide, 0-0.6wt% arachidic acid ceramide, 0-0.4wt% eicosenoic acid ceramide, 0-0.3wt% heptadecanoic acid ceramide, 0-0.3wt% heptadecanoic acid ceramide, 0-0.2wt% behenic acid ceramide, 0-0.2wt% lignoceramide, and 0-0.05wt% myristic acid ceramide.

4. The method for synthesizing olive oil ceramide according to any one of claims 1 to 3, comprising the following steps: Step 1: Dissolve 50 g of olive oil in 60 mL of tetrahydrofuran, cool in an ice bath, and dropwise add 100 mL of a 25 wt% potassium hydroxide solution. After the addition is complete, warm the mixture to room temperature and react until the reaction is complete as determined by TLC. Post-treatment: Add 3N dilute hydrochloric acid to adjust the pH value of the reaction system to 3, add 150 mL of ethyl acetate to extract the aqueous phase, add 100 mL of saturated brine to wash once, add anhydrous Na2SO4 to dry the organic phase, filter and concentrate in vacuo to obtain 40 g of olive oil fatty acid; Step 2: Add 50 mmol of olive oil fatty acid (molecular weight based on oleic acid), 60 mmol of EDCI, and 60 mmol of Et3N to 250 mL of a round-bottom flask, then add 100 mL of dichloromethane, and stir at room temperature for 1 hour. Then, add 60 mmol of phytosphingosine to the reaction system and stir at room temperature until the reaction is complete as detected by TLC. Post-treatment: add water to quench the reaction, separate the organic layer, dry, filter and concentrate in vacuo, and then wash with solvent to obtain olive oil ceramide; or: 50 mmol of olive oil fatty acid (molecular weight based on oleic acid), 75 mmol of EDCI, and 75 mmol of Et3N were added to a 250 mL round-bottom flask, followed by 100 mL of dichloromethane, and then stirred at room temperature for 1 hour. 50 mmol of sphingosine was then added to the reaction system, and stirred at room temperature until the reaction was complete as detected by TLC. Post-treatment: add water to quench the reaction, separate the organic layer, dry, filter and concentrate in vacuo, and then wash with solvent to obtain olive oil ceramide; or: 50 mmol of olive oil fatty acid (molecular weight based on oleic acid), 100 mmol of EDCI, and 100 mmol of Et3N were added to a 250 mL round-bottom flask, followed by 100 mL of dichloromethane, and stirred at room temperature for 1 hour. Subsequently, 75 mmol of dihydrosphingosine was added to the reaction system and stirred at room temperature until the reaction was complete as detected by TLC. Post-treatment: water was added to quench the reaction, the organic layer was separated, dried, filtered, concentrated in vacuo, and then washed with a solvent to obtain olive oil ceramide.

5. Use of the olive oil ceramide according to any one of claims 1 to 3 in the preparation of cosmetics and medicines, wherein the olive oil ceramide has anti-inflammatory effects.

6. A composition comprising the olive oil ceramide according to any one of claims 1 to 3, wherein the composition has anti-inflammatory effect.

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