Application of linolenic acid derived ceramide

Abstract

The invention belongs to the technical field of biological medicines, and discloses linolenic acid-derived ceramide which has a structure of a general formula I or an isomer of the general formula I: # imgabs0, R1 is selected from # imgabs1, and R2 is selected from one of the following structures: # imgabs2. The invention also discloses a preparation method and application of the linolenic acid-derived ceramide. Long-chain linolenic acid reacts with sphingosine and phytosphingosine to obtain a ceramide compound with a novel structure, and the ceramide compound has an obvious inhibition effect on tyrosinase and can effectively prevent generation of melanin, so that the ceramide compound has a good skin whitening effect and can be applied to the fields of cosmetics and the like.

Description

[0001] This application is a divisional application of the application with the application number 202211358116.3, the application date of November 1, 2022, and the invention title of "Linolenic Acid-Derived Ceramide and Its Preparation Method and Application". Technical Field

[0002] The present invention belongs to the field of biomedical technologies, and particularly relates to linolenic acid-derived ceramide and its preparation method and application. Background Art

[0003] Ceramide molecules are compounds composed of a sphingosine molecule and a fatty acid molecule. The carbon chain length, degree of unsaturation, and number of hydroxyl groups in the sphingosine part and fatty acid part can all vary. Ceramide is involved in various physiological functions, including apoptosis, cell growth arrest, differentiation, cell senescence, cell migration, and adhesion. The roles of ceramide and its downstream metabolites have also been mentioned in some pathological conditions, including cancer, neurodegenerative diseases, diabetes, microbial lesions, obesity, and inflammation.

[0004] Ceramide is the main component of the lipids in the extracellular matrix of the epidermal layer of human skin. Together with cholesterol and saturated fatty acids, ceramide forms a water-impermeable protective structure to prevent excessive water evaporation and also block the entry of microorganisms. Currently, 15 types of ceramides have been detected in the skin, which can generally be divided into three categories: short-chain ceramides, long-chain ceramides, and ceramides bonded to keratinocytes.

[0005] Due to the importance of ceramide, there is a wide demand for functional ceramide in the market. However, the extraction and purification of natural ceramide have economic problems. Therefore, it is very necessary to rapidly construct new ceramides through chemical synthesis pathways and explore the efficacy uses and efficacy differences of these ceramides. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of ceramides with novel structures, namely linolenic acid-derived ceramides.

[0007] Another purpose of the present invention is to provide a synthesis method of linolenic acid-derived ceramide.

[0008] Another purpose of the present invention is to provide the uses of linolenic acid-derived ceramide.

[0009] To achieve one of the above purposes, the present invention adopts the following technical solutions;

[0010] In the first aspect of the present invention, linolenic acid-derived ceramide has a structure of general formula I or an isomer of general formula I:

[0011]

[0012] Among them, R 1 is selected from R 2 is selected from one of the following structures:

[0013]

[0014] Furthermore, R 2 is selected from one of the following structures: Isomers include enantiomers, diastereomers, cis-trans isomers.

[0015] Furthermore, the ceramides derived from linolenic acid are selected from α-linolenic acid sphingosine

[0016] α-linolenic acid phytosphingosine

[0017] γ-linolenic acid sphingosine

[0018] γ-linolenic acid phytosphingosine.

[0019] Furthermore, the ceramide derived from linolenic acid is γ-linolenic acid sphingosine.

[0020] In the second aspect of the present invention, a method for preparing ceramides derived from linolenic acid includes the following steps:

[0021]

[0022] Compound S1 reacts with p-nitrobenzenesulfonyl chloride and an organic base to obtain compound S2;

[0023] Compound S2 reacts with sphingosine base to obtain compound I.

[0024] Furthermore, the molar ratio of the compound S1, p-nitrobenzenesulfonyl chloride, organic base, and sphingosine base is (1-2):(1-2):(2-6):1.

[0025] Furthermore, the organic base is triethylamine.

[0026] Furthermore, the solvent for the reaction is ethyl acetate.

[0027] In the third aspect of the present invention, the use of α-linolenic acid sphingosine, α-linolenic acid phytosphingosine, γ-linolenic acid sphingosine or γ-linolenic acid phytosphingosine in cosmetics, health products, and pharmaceuticals.

[0028] Specifically, α-linolenic acid sphingosine, α-linolenic acid phytosphingosine, γ-linolenic acid sphingosine or γ-linolenic acid phytosphingosine has a whitening effect.

[0029] Specifically, sphingosine α-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is more than 5% at a concentration of 1 mM, and / or more than 8% at a concentration of 2 mM, and / or more than 10% at a concentration of 3 mM, and / or more than 13% at a concentration of 4 mM, and / or more than 16% at a concentration of 5 mM, and / or more than 18% at a concentration of 6 mM.

[0030] Specifically, phytosphingosine α-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is more than 7% at a concentration of 1 mM, and / or more than 9% at a concentration of 2 mM, and / or more than 9% at a concentration of 3 mM, and / or more than 12% at a concentration of 4 mM, and / or more than 11% at a concentration of 5 mM, and / or more than 14% at a concentration of 6 mM.

[0031] Specifically, sphingosine γ-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is more than 27% at a concentration of 1 mM, and / or more than 33% at a concentration of 2 mM, and / or more than 40% at a concentration of 3 mM, and / or more than 35% at a concentration of 4 mM, and / or more than 33% at a concentration of 5 mM, and / or more than 33% at a concentration of 6 mM.

[0032] Specifically, phytosphingosine γ-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is more than 6% at a concentration of 1 mM, and / or more than 12% at a concentration of 2 mM, and / or more than 18% at a concentration of 3 mM, and / or more than 24% at a concentration of 4 mM, and / or more than 31% at a concentration of 5 mM, and / or more than 35% at a concentration of 6 mM.

[0033] Specifically, sphingosine α-linolenate, phytosphingosine α-linolenate, sphingosine γ-linolenate or phytosphingosine γ-linolenate has an anti-aging effect.

[0034] Specifically, sphingosine α-linolenate inhibits elastase, and has an inhibition rate of tyrosinase of more than 10% at a concentration of 3 mg / L, and / or an inhibition rate of tyrosinase of more than 15% at a concentration of 6 mg / L, and / or an inhibition rate of tyrosinase of more than 16% at a concentration of 9 mg / L, and / or an inhibition rate of tyrosinase of more than 23% at a concentration of 15 mg / L, and / or an inhibition rate of tyrosinase of more than 24% at a concentration of 18 mg / L.

[0035] Specifically, phytosphingosine α-linolenate inhibits elastase, and has an inhibition rate of tyrosinase of more than 7% at a concentration of 3 mg / L, and / or an inhibition rate of tyrosinase of more than 10% at a concentration of 9 mg / L, and / or an inhibition rate of tyrosinase of more than 11% at a concentration of 12 mg / L, and / or an inhibition rate of tyrosinase of more than 13% at a concentration of 18 mg / L.

[0036] Specifically, sphingosine γ-linolenate inhibits elastase, and has an inhibition rate of tyrosinase of more than 24% at a concentration of 3 mg / L, and / or an inhibition rate of tyrosinase of more than 16% at a concentration of 6 mg / L, and / or an inhibition rate of tyrosinase of more than 16% at a concentration of 9 mg / L, and / or an inhibition rate of tyrosinase of more than 12% at a concentration of 15 mg / L, and / or an inhibition rate of tyrosinase of more than 23% at a concentration of 18 mg / L.

[0037] Specifically, phytosphingosine γ-linolenate inhibits elastase, and has an inhibition rate of tyrosinase of more than 8% at a concentration of 3 mg / L, and / or an inhibition rate of tyrosinase of more than 9% at a concentration of 6 mg / L, and / or an inhibition rate of tyrosinase of more than 13% at a concentration of 9 mg / L, and / or an inhibition rate of tyrosinase of more than 14% at a concentration of 12 mg / L, and / or an inhibition rate of tyrosinase of more than 17% at a concentration of 18 mg / L.

[0038] Sphingosine α-linolenate, phytosphingosine α-linolenate, sphingosine γ-linolenate or phytosphingosine γ-linolenate has at least one of the following effects: tissue repair, anti-inflammation, tissue healing.

[0039] Specifically, sphingosine α-linolenate, phytosphingosine α-linolenate, sphingosine γ-linolenate or phytosphingosine γ-linolenate inhibits the production of interstitial collagenase MMP1 and has an anti-photoaging effect.

[0040] A composition comprising at least one of sphingosine α-linolenate, phytosphingosine α-linolenate, sphingosine γ-linolenate or phytosphingosine γ-linolenate, and the composition has at least one of the effects of whitening, anti-aging, anti-photoaging, barrier, repair and anti-inflammation.

[0041] The composition contains acceptable excipients, including one or more of solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, fragrances and pigments; the composition is in the form of a cream, emulsion, solution, film, aerosol or spray.

[0042] α-Linolenic acid is one of the essential nutrients for people, and has the functions of enhancing intelligence, improving memory, protecting eyesight and improving sleep. α-Linolenic acid has stronger and safer effects than DHA, etc. α-Linolenic acid can be converted into DHA, DPA, EPA, etc. in the body, while supplementing DHA, etc. only plays a partial role. α-Linolenic acid as an energy source for growth, cell metabolism and muscle movement is only part of its function, and it is more used as a structural substance and a metabolic regulatory substance to play a structural function and a regulatory function. γ-Linolenic acid is one of the essential polyunsaturated fatty acids for the human body and is a precursor substance for synthesizing human prostaglandins, and can be applied to fields such as medicine and food.

[0043] The present invention has the following beneficial effects:

[0044] The present invention reacts long-chain linolenic acid with sphingosine and phytosphingosine to obtain a novel class of ceramide compounds. This class of compounds has an obvious inhibitory effect on tyrosinase and can effectively prevent the formation of melanin, thus having a good skin whitening effect. The prior art has not systematically studied the whitening effect of linolenic acid-derived ceramides. The present invention discovers and verifies through experiments that linolenic acid-derived ceramides have a high inhibitory rate on tyrosinase, can reduce melanin production, prevent melanin deposition, and have a whitening effect; the present invention also discovers that linolenic acid-derived ceramides have an inhibitory effect on elastase, can reduce the degradation of elastin, and promote the skin to be more firm and elastic, and have a more excellent anti-aging effect, and can be used in fields such as cosmetics, health products and drugs. In addition, linolenic acid-derived ceramides also have tissue repair, anti-inflammatory and tissue healing effects. Description of the Drawings

[0045] Figures 1 to 4 It is a bar chart of the tyrosinase inhibitory rate of the ceramide in Example 2;

[0046] Figures 5 to 6 It is a bar chart of the detection results of cell proliferation viability in Example 3;

[0047] Figures 7 to 8Column chart of the anti-inflammatory repair efficacy test results in Example 4;

[0048] Figure 9 Results of the healing ability test in Example 5;

[0049] Figures 10 to 13 Column chart of the elastase inhibition rate in Example 6;

[0050] Figure 14 Column chart of the MMP1 expression level in Example 7. Detailed implementation mode

[0051] The present invention will be further described below in conjunction with specific embodiments.

[0052] Unless otherwise specified, chemicals are purchased from commercial products and are not further purified. Ethyl acetate used in the experiments is an anhydrous solvent. Thin layer chromatography (TLC) uses 60F254 silica gel plates. Silica gel column chromatography uses Qingdao Marine silica gel (particle size 0.040 - 0.063 mm). TLC color development is carried out using UV light (254 nm) or iodine. NMR spectra are characterized using a Bruker DPX 400 nuclear magnetic resonance instrument, 1 The 1H NMR is at 400 MHz, the solvents are deuterated methanol, deuterated DMSO or deuterated tetrahydrofuran, and tetramethylsilane (TMS) is used as the internal standard. The unit of chemical shift is ppm, and the unit of coupling constant is Hz. In 1 1H NMR, δ represents the chemical shift, s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet.

[0053] Example 1

[0054] Synthesis of linolenic acid-derived ceramides

[0055]

[0056] Dissolve 1.2 eq (60 mmol) of p-nitrobenzenesulfonyl chloride in 70 ml of ethyl acetate, dissolve 1.32 eq (66 mmol) of compound S1 (α-linolenic acid or γ-linolenic acid) and 3 eq (150 mmol) of triethylamine in 30 ml of ethyl acetate, and drop it into the ethyl acetate solution of p-nitrobenzenesulfonyl chloride. React at 40 °C for 12 h, and detect by TCL until the p-nitrobenzenesulfonyl chloride reaction is completed. Add 2 eq (100 mmol) of triethylamine and 1 eq (50 mmol) of sphingosine or phytosphingosine, and react overnight at 40 °C. Detect by TLC until the sphingosine base reaction is complete.

[0057] Workup: Add 100 mL of water, adjust the pH to 5 - 6 with 2N dilute hydrochloric acid, extract with 100 mL of saturated brine twice, dry over anhydrous sodium sulfate, filter and concentrate in vacuo. The residue thus obtained was purified by silica gel column chromatography to give the product (50 - 70% yield).

[0058]

[0059] α-Linolenic acid phytosphingosine

[0060] 1 H NMR (400 MHz, Methanol-d 4 ) δ 5.42–5.22 (m, 6H), 4.07 (td, J = 5.8, 4.3 Hz, 1H), 3.79–3.65 (m, 3H), 3.58 (t, J = 5.8 Hz, 1H), 3.52 (ddd, J = 9.6, 5.6, 2.4 Hz, 1H), 2.80 (t, J = 6.0 Hz, 4H), 2.22 (t, J = 7.5 Hz, 2H), 2.12–2.04 (m, 4H), 1.91–1.80 (m, 1H), 1.67–1.52 (m, 4H), 1.35–1.28 (d, J = 22.7 Hz, 31H), 0.97 (t, J = 7.6 Hz, 3H), 0.89 (t, J = 7.6 Hz, 3H).

[0061]

[0062] α-Linolenic acid sphingosine

[0063] 1 H NMR (400 MHz, Methanol-d 4 ) δ 5.74–5.63 (m, 1H), 5.49–5.42 (m, 1H), 5.41–5.24 (m, 6H), 4.04 (t, J = 7.4 Hz, 1H), 3.85 (dt, J = 7.5, 5.0 Hz, 1H), 3.68 (d, J = 5.0 Hz, 2H), 2.80 (t, J = 6.0 Hz, 4H), 2.24–2.14 (m, 2H), 2.14–1.97 (m, 6H), 1.59 (dt, J = 8.0, 3.9 Hz, 2H), 1.41–1.22 (m, 30H), 0.97 (t, J = 7.6 Hz, 3H), 0.89 (t, J = 7.6 Hz, 3H).

[0064]

[0065] γ-Linolenic acid phytosphingosine

[0066] 11H NMR (400 MHz, Methanol-d 4 ) δ 5.52–5.33 (m, 6H), 4.09–4.02 (m, 1H), 3.72–3.68 (m, 3H), 3.53 (t, J = 5.8 Hz, 1H), 3.50–3.44 (m, 1H), 2.76 (t, J = 6.2 Hz, 4H), 2.18 (t, J = 7.6 Hz, 2H), 2.10–2.02 (m, 4H), 1.94–1.82 (m, 1H), 1.69–1.54 (m, 4H), 1.32–1.25 (d, J = 22.4 Hz, 30H), 0.92 (t, J = 7.6 Hz, 3H), 0.83 (t, J = 7.6 Hz, 3H).

[0067]

[0068] γ-Linolenoyl Sphingosine

[0069] 1 1H NMR (400 MHz, Methanol-d 4 ) δ 5.76–5.66 (m, 1H), 5.49 (ddt, J = 15.3, 7.4, 1.5 Hz, 1H), 5.45–5.30 (m, 6H), 4.07 (t, J = 7.4 Hz, 1H), 3.88 (dt, J = 7.5, 5.0 Hz, 1H), 3.71 (dd, J = 5.0, 1.2 Hz, 2H), 2.84 (td, J = 5.7, 3.3 Hz, 4H), 2.23 (t, J = 7.5 Hz, 2H), 2.18–1.99 (m, 6H), 1.64 (tt, J = 9.2, 6.9 Hz, 2H), 1.46–1.24 (m, 30H), 0.93 (t, J = 7.6 Hz, 3H), 0.90 (t, J = 7.6 Hz, 3H).

[0070] Example 2

[0071] Comparing the Whitening Efficacy of the Compound of Example 1 by Intracellular Tyrosinase Inhibition Method

[0072] Detecting Whitening Efficacy by Intracellular Tyrosinase Method: Seed B16 cells in a 96-well plate (density 1×10 4(cells / well), after incubating in an incubator for 24 h, discard the supernatant, add 100 μL of compounds at different concentrations diluted with DMEM medium. The DMEM medium without drugs is used as the negative control group. After continuing to incubate for 24 h, discard the culture medium containing drugs, wash twice with PBS, then add 100 μL of 1 wt% Triton X-100, freeze at -80 °C for 1 h to form ice, and then thaw at room temperature. After thawing, add 20 μL of 0.5 wt% L-DOPA to each well and react at room temperature with horizontal oscillation for 4 h. After the reaction, measure the absorbance at a wavelength of 490 nm, and calculate the cell tyrosinase inhibition rate = A 给药孔 / A 空白孔 × 100%.

[0073] The results are as Figures 1 to 4 shown in Figure 1 Table 1 and are the results of α-linolenic acid phytosphingosine, Figure 2 are the results of γ-linolenic acid phytosphingosine, Figure 3 are the results of α-linolenic acid sphingosine, Figure 4 are the results of γ-linolenic acid sphingosine. α-Linolenic acid sphingosine, α-linolenic acid phytosphingosine, γ-linolenic acid sphingosine or γ-linolenic acid phytosphingosine all have good inhibitory effects on tyrosinase at different concentrations. In particular, γ-linolenic acid sphingosine still has a high inhibition rate at a very low concentration (1 mM), while γ-linolenic acid phytosphingosine requires a higher concentration (4 mM) to have a high inhibition rate.

[0074] Table 1

[0075] Concentration (mM) 1 2 3 4 5 6 Sphingosine α-linolenate (%) 5.1 8.2 10.4 13.5 16.1 18.6 Phytosphingosine α-linolenate (%) 7.1 9.3 9.1 12.6 11.6 14.3 Sphingosine γ-linolenate (%) 27.7 33.6 40.4 35.2 33.8 33.4 Phytosphingosine γ-linolenate (%) 6.8 12.6 18.2 24.0 31.6 35.6

[0076] Example 3

[0077] MTT assay for detecting the proliferative viability of compounds on cells

[0078] Seed HaCaT cells at a density of 1 × 10 4 (cells / well) in a 96-well plate and incubate overnight in an incubator. After 24 h, discard the supernatant, add 100 μL of medium containing different concentrations of compounds (α-linolenic acid phytosphingosine, γ-linolenic acid phytosphingosine). After continuing to incubate for 24 h, remove the medium, add 100 μL of thiazolyl blue (MTT) to each well, measure the absorbance at 450 nm, and calculate the cell survival rate = A 给药孔 / A 空白孔 × 100%.

[0079] The results are as Figure 5 , 6 shown, Figure 5 are the results of α-linolenic acid phytosphingosine, Figure 6The result is phytosphingosine of γ-linolenic acid. Phytosphingosine of α-linolenic acid and phytosphingosine of γ-linolenic acid have a promoting effect on cell viability at lower concentrations (0.0625 mmol / L, 0.125 mmol / L), showing an obvious effect of promoting cell proliferation and having good tissue repair ability. Even at a very high concentration (2 mmol / L), the cell viability is still above 50%, indicating that the cytotoxicity of the compound is controllable and showing good biosafety.

[0080] Example 4

[0081] Detecting anti-inflammatory and repair efficacy by LPS-induced cell method

[0082] Seed B16 mouse melanoma cells at a density of 1×10 4 cells / well in a 96-well plate, place it in an incubator to adhere overnight. After 24 h, discard the supernatant, add 100 μL of different concentrations of compounds (phytosphingosine of α-linolenic acid, phytosphingosine of γ-linolenic acid) diluted with DMEM medium. The model group is cells without adding drugs, and the negative control group is DMEM medium without drugs. Each group has 3 replicate wells. Incubate in an environment of 5% CO 2 2, 37 °C. After 2 h of drug administration, add 10 μg / mL LPS to the lipopolysaccharide model group and the experimental group and incubate together until 24 h. After the reaction ends, take 50 μL of cell supernatant, and use an IL-6 ELISA kit to detect the expression of IL-6 gene in the cells.

[0083] The results are as Figure 7 、 8 shown. Figure 7 The result is phytosphingosine of α-linolenic acid. Figure 8 The result is phytosphingosine of γ-linolenic acid. Both phytosphingosine of α-linolenic acid and phytosphingosine of γ-linolenic acid have excellent effects on inhibiting the expression of IL-6 cytokine. Under the stimulation of LPS with a working concentration of 10 μg / mL, the IL-6 level is 40.7 times the basal level. Under the action of phytosphingosine of α-linolenic acid at concentrations of 0.0625, 0.125, 0.25, 0.5 mM respectively, the levels of IL-6 factor are significantly reduced, which are 29.6, 24.8, 19.5, 14.7 times the basal level respectively, showing a dose-dependent relationship; under the action of phytosphingosine of γ-linolenic acid at concentrations of 0.0625, 0.125, 0.25, 0.5 mM respectively, the levels of IL-6 factor are 30.2, 26.1, 18.2, 17.2, 18.8 times the basal level respectively. Therefore, these two compounds have good anti-inflammatory effects and can promote the repair of inflamed and damaged skin.

[0084] Example 5

[0085] Scratch method for detecting tissue healing ability

[0086] Principle: When cells grow to a confluent monolayer state, a blank area is created on the confluent monolayer cells using a scratching tool. The cells in the blank area are removed by mechanical force. After a period of culture, the migration of cells into the cell-free area is observed, and the migration ability of cells is reflected by measuring the migration distance of cells.

[0087] Operation steps:

[0088] 1. Mark lines on the culture plate. First, use a Marker pen to draw horizontal lines evenly on the back of the 6-well plate with the help of a ruler. Draw lines approximately every 0.5 - 1 cm, crossing the wells. Each well should have at least 5 lines crossed. Pay attention not to make the lines too thick when drawing.

[0089] 2. Seed cells. Add about 5×10 5 cells (the number of different cells varies, adjusted according to the growth rate of the cells) into the wells. The seeding principle is that the confluence rate reaches 100% after overnight culture.

[0090] 3. Scratch the cells. The next day, use a pipette tip, perpendicular to the cell plane, to scratch the cell layer along the lines drawn on the back of the plate the day before (it is best to use the same pipette tip for different wells).

[0091] 4. Wash the cells. After scratching, wash the cells 3 times with sterile PBS to remove non-adherent cells, that is, the cells scratched during the line drawing. The gaps left after scratching are clearly visible, and then replace with fresh serum-free medium.

[0092] 5. Cell culture and observation. Dilute the compounds (phytosphingosine α-linolenate, phytosphingosine γ-linolenate) with the medium (concentration is 0.08 mM) and add them to the cell culture dish. Place the cells in an incubator at 37°C and 5 wt% CO 2 for culture. Take out the cells at 24 h and 48 h, observe and measure the width of the scratch under a microscope, and take pictures.

[0093] The results are as Figure 9 shown. Compared with the solvent control group, the scratch width of the experimental group is narrower, and phytosphingosine α-linolenate and phytosphingosine γ-linolenate have better tissue healing ability.

[0094] Example 6

[0095] Elastase inhibition experiment to test the anti-aging effect

[0096] Elastase inhibition method: Take 2 mL of 2 mg / mL elastase solution, add compounds with different concentrations, vortex thoroughly to mix evenly, shake in a shaker at 37 °C and 400 r / min for 20 min, immediately add 5 mL of 0.5 mol / L phosphate buffer solution with pH 6.0, vortex to mix evenly, take an appropriate amount of the mixed solution into a 2 mL centrifuge tube, centrifuge at 9 391×g for 10 min, precisely pipette 200 μL of the supernatant into a 96-well plate, measure the absorbance with an enzyme-labeling instrument at a wavelength of 495 nm, and simultaneously perform a spectral scan from 400 to 800 nm.

[0097] Use the substrate plus enzyme solution as the blank control group, the substrate plus enzyme and sample solution as the enzyme inhibition group, and the substrate plus sample without enzyme solution for background subtraction. Each group is set with 3 replicate wells. Inhibition rate (%) = [1 – (An – An′) / (A0 – A0′)] × 100%, where A0 is the absorbance of adding enzyme without sample, A0′ is the absorbance of adding only substrate without sample and enzyme, An is the absorbance of adding only the sample solution, and An′ is the absorbance of adding sample without enzyme. If An′ > An, it shows a promoting effect, and the promotion rate (%) = [1 – (An′ – An) / (A0 – A0′)] × 100%.

[0098] The results are as Figures 10 to 13 shown in Figure 10 and Table 2, Figure 11 which are the results of α-linolenic acid phytosphingosine, Figure 12 which are the results of γ-linolenic acid phytosphingosine, Figure 13 which are the results of α-linolenic acid sphingosine,

[0099] Table 2

[0100] Concentration (mg / L) 3 6 9 12 15 18 Sphingosine α-linolenate (%) 10.6 15.8 16.7 — 23.7 24.5 Phytosphingosine α-linolenate (%) 7.1 — 10.3 11.9 — 13.2 Sphingosine γ-linolenate (%) 24.6 16.1 16.7 — 12.3 23.7 Phytosphingosine γ-linolenate (%) 8.2 9.3 13.7 14.4 — 17

[0101] Elastin is a component of elastic fibers in connective tissue, which can make the tissue elastic and also jointly maintain the structural stability of the tissue with other extracellular matrices such as collagen. Elastase can degrade elastin under conditions such as inflammation or long-term ultraviolet irradiation, thus causing the aging of skin tissue. The compounds of the present invention have good anti-aging ability by inhibiting the activity improvement of elastase.

[0102] Example 7

[0103] MMP1, also known as interstitial collagenase and matrix metalloproteinase, belongs to the matrix metalloproteinase family. Its main substrate is fibrous collagen, which can degrade collagen fibers and gelatin in the extracellular matrix and change the microenvironment of cells. MMP1 plays an important role in elastin. Inhibiting MMP1 can increase the synthesis of collagen and elastin by fibroblasts, and a decrease in MMP activity can increase the rate of collagen synthesis.

[0104] Seed HaCaT cells in a 96-well plate at a density of 1×10 5 cells / well and incubate overnight in an incubator. After 24 hours, discard the supernatant and add 100 μL of medium containing different concentrations of phytosphingosine α-linolenate (the model group does not add the drug). The negative control group is DMEM medium without the drug. There are 3 replicate wells in each group. Incubate for 2 hours in an environment of 5% CO 2 2, 37 °C, and then irradiate with UVA ultraviolet light. The distance between the ultraviolet light radiation source and the cells is 15 cm, the UVA intensity is 200 mJ / cm 2 2, and the radiation time is 1.5 hours. After the radiation ends, continue to incubate in the incubator for 12 hours. Use the MMP-1 ELISA kit to detect the expression of the MMP-1 gene in the cells.

[0105] The results are as Figure 14 shown. The expression level of MMP1 in the negative control group is set as 1, the expression level in the model group is 1.64. When the concentration of phytosphingosine α-linolenate is 50 mg / L, the expression level of MMP1 is 1.41; when the concentration is 100 mg / L, the expression level of MMP1 is 1.23; when the concentration is 150 mg / L, the expression level of MMP1 is 1.26; when the concentration is 200 mg / L, the expression level of MMP1 is 1.13, showing a concentration-dependent manner. The higher the concentration, the more significant the inhibitory effect on MMP1.

[0106] After UVA ultraviolet radiation, keratinocytes promote the increased expression of MMP1 in fibroblasts, thereby causing the degradation of the skin extracellular matrix and skin collagen, leading to skin photoaging. The above results indicate that phytosphingosine α-linolenate can inhibit the production of MMP1 by fibroblasts caused by ultraviolet radiation and has a certain effect on preventing skin photoaging.

[0107] As described above, this is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection 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. Use of linolenic acid-derived ceramides, the use of sphingosine α-linolenate, phytosphingosine α-linolenate or phytosphingosine γ-linolenate in the preparation of cosmetics, health products or pharmaceuticals.

2. The use according to claim 1, wherein, the sphingosine α-linolenate, phytosphingosine α-linolenate or phytosphingosine γ-linolenate has a whitening effect.

3. The use according to claim 1 or 2, wherein, sphingosine α-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is above 5% at a concentration of 1 mM, and / or the inhibition rate of tyrosinase is above 8% at a concentration of 2 mM, and / or the inhibition rate of tyrosinase is above 10% at a concentration of 3 mM, and / or the inhibition rate of tyrosinase is above 13% at a concentration of 4 mM, and / or the inhibition rate of tyrosinase is above 16% at a concentration of 5 mM, and / or the inhibition rate of tyrosinase is above 18% at a concentration of 6 mM.

4. The use according to claim 1 or 2, wherein, phytosphingosine α-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is above 7% at a concentration of 1 mM, and / or the inhibition rate of tyrosinase is above 9% at a concentration of 2 mM, and / or the inhibition rate of tyrosinase is above 9% at a concentration of 3 mM, and / or the inhibition rate of tyrosinase is above 12% at a concentration of 4 mM, and / or the inhibition rate of tyrosinase is above 11% at a concentration of 5 mM, and / or the inhibition rate of tyrosinase is above 14% at a concentration of 6 mM.

5. The use according to claim 1 or 2, wherein, phytosphingosine γ-linolenate inhibits tyrosinase, and the inhibition rate of tyrosinase is above 6% at a concentration of 1 mM, and / or the inhibition rate of tyrosinase is above 12% at a concentration of 2 mM, and / or the inhibition rate of tyrosinase is above 18% at a concentration of 3 mM, and / or the inhibition rate of tyrosinase is above 24% at a concentration of 4 mM, and / or the inhibition rate of tyrosinase is above 31% at a concentration of 5 mM, and / or the inhibition rate of tyrosinase is above 35% at a concentration of 6 mM.

6. The use according to claim 1, wherein, the sphingosine α-linolenate, phytosphingosine α-linolenate or phytosphingosine γ-linolenate has an anti-aging effect.

7. The use according to claim 1, wherein, the sphingosine α-linolenate, phytosphingosine α-linolenate or phytosphingosine γ-linolenate has an anti-photoaging effect.

8. The use according to claim 1, wherein, the sphingosine α-linolenate, phytosphingosine α-linolenate or phytosphingosine γ-linolenate has at least one of the following effects: tissue repair, anti-inflammation, tissue healing.

9. A composition comprising at least one of sphingosine α-linolenate, phytosphingosine α-linolenate or phytosphingosine γ-linolenate, and the composition has at least one of the effects of whitening, anti-aging, anti-photoaging, barrier, repair, and anti-inflammation.

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