CN117304057A - Sea buckthorn oil ceramide and its synthesis method and use - Google Patents

Abstract

The invention belongs to the technical field of biomedicine and discloses sea buckthorn oil ceramide, which is obtained by reacting sea buckthorn oil fatty acids with sphingosine compounds. The sphingosine compounds are selected from the group consisting of sphingosine, phytosphingosine, and dihydrosphingosine. Amino alcohol, this plan also discloses the synthesis method and use of sea buckthorn oil ceramide. Sea buckthorn oil ceramide has excellent properties in repairing the skin's natural barrier, tissue healing, and anti-aging, and has broad application prospects in cosmetics, health products, biomedicine and other fields.

Description

Sea buckthorn oil ceramide and its synthesis method and use

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to sea buckthorn oil ceramide and its synthesis method and use.

Background technique

Ceramide (Ceramide, also known as molecular nail) naturally exists in the skin and is a very important component of the skin barrier (stratum corneum). The content is as high as 40-50wt%. Ceramide is a type of long-chain base composed of sphingosine. Sphingosine lipids composed of fatty acids, in which the sphingosine part and the carbon chain length of the fatty acid part, the degree of unsaturation and the number of hydroxyl groups can all be changed, ceramide represents a class of compounds. Ceramides have excellent properties in regulating skin barrier function, restoring skin moisture, and enhancing adhesion between skin keratinocytes.

Due to the importance of ceramides, many cosmetics and pharmaceutical companies are researching and developing corresponding products. Natural plant-derived ceramides are likely to become the next generation of environmentally friendly products due to their more sustainable and environmentally friendly raw material sources and similar properties to skin ceramide components. They can form an effective skin barrier to prevent moisture loss and resist external damage. , safe and reliable ceramide products.

Sea buckthorn oil is a brown-yellow to brown-red transparent oily liquid obtained from sea buckthorn seeds or sea buckthorn fruits through supercritical extraction or subcritical biotechnology low-temperature extraction. It is a highly concentrated product that integrates the active ingredients of sea buckthorn and contains flavonoids, organic acids, biological More than 140 biologically active ingredients including bases, sterols, triterpenes and various vitamins. Sea buckthorn oil contains a large amount of unsaturated fatty acids, such as linoleic acid, oleic acid, linolenic acid, etc. and a small amount of saturated fatty acids, such as palmitic acid, stearic acid, myristic acid, etc., and is considered to be a very effective vegetable oil in the treatment of eczema. In addition, it also contains palmoleoleic acid, which has a composition similar to human sebum, so it can strengthen skin regeneration and repair damaged mucous membranes. As a health food raw material, sea buckthorn oil has been widely used in antioxidant, anti-fatigue, liver protection, and lowering blood lipids. As a medicinal raw material, sea buckthorn oil has obvious biological effects. It has strong anti-infection and promotes fast healing. It is widely used to treat burns, scalds, frostbite, knife wounds, etc. Sea buckthorn oil has good and stable effects on tonsillitis, stomatitis, conjunctivitis, keratitis, gynecological cervicitis, etc. Sea buckthorn oil is a complex of multiple vitamins and bioactive substances. It can nourish the skin, promote metabolism, resist allergies, sterilize and reduce inflammation, promote epithelial cell regeneration, repair the skin, maintain the acidic environment of the skin, and has strong penetration. Sex, so it is also an important raw material for beauty and skin care. The total flavonoids in seabuckthorn can directly capture superoxide free radicals and hydroxyl free radicals. VE and VC superoxide dismutase (SOD) have antioxidant and eliminate free radicals on cell membranes, effectively delaying human aging; VC in seabuckthorn Its content ranks first among fruits and vegetables, and it is known as the "King of VC". VC is a natural whitening agent in the body, which can effectively inhibit the deposition of abnormal pigments on the skin and the activity of tyrosinase, and help many Reduction of melanin (the intermediate in which tyrosine is converted into melanin), thereby reducing the formation of melanin and effectively whitening the skin.

Contents of the invention

The object of the present invention is to provide a ceramide synthesized from plant-derived sea buckthorn oil fatty acids.

Another object of the present invention is to provide a method for synthesizing sea buckthorn oil ceramide, which uses natural plant-derived and easily available sea buckthorn oil or sea buckthorn oil fatty acid as raw material.

Another object of the present invention is to provide the use of sea buckthorn oil ceramide.

In order to achieve one of the above objectives, the present invention adopts the following technical solutions:

The first aspect of the present invention is sea buckthorn oil ceramide, which is obtained by reacting sea buckthorn oil fatty acids with sphingosine compounds, and the sphingosine compounds are selected from the group consisting of sphingosine, phytosphingosine, and dihydrosphingosine.

The reaction can be a chemical synthesis reaction (as detailed below) or a microbial fermentation method, that is, using Pichia pastoris or Saccharomyces cerevisiae to ferment under a certain environment to obtain sphingosine compounds, and then adding fatty acids to finally obtain Ceramide; or use sea buckthorn oil as raw material, select a suitable strain, and ferment it to obtain sea buckthorn 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 sea buckthorn oil fatty acid is obtained by hydrolyzing sea buckthorn seed oil or sea buckthorn fruit oil.

Further, the sea buckthorn oil fatty acid contains 20-35wt% oleic acid.

Further, the sea buckthorn oil fatty acid contains 8 to 30 wt% palmitic acid.

Further, the sea buckthorn oil fatty acid contains 4 to 40 wt% linoleic acid.

Further, the sea buckthorn oil fatty acid contains 2 to 30 wt% linolenic acid.

Further, the sea buckthorn oil fatty acid contains 0.5 to 35 wt% palmitic acid.

Further, the sea buckthorn oil fatty acid contains 0.5-3wt% stearic acid.

In addition, sea buckthorn oil fatty acid also contains 0 to 1 wt% myristic acid.

The fatty acid composition of sea buckthorn oil is: 20~35wt% oleic acid, 8~30wt% palmitic acid, 4~40wt% linoleic acid, 2~30wt% linolenic acid, 0.5~35wt% palmitic acid, 0.5~3wt% hard Fatty acid, 0~1wt% myristic acid.

The main fatty acid component of sea buckthorn oil is oleic acid. Other fatty acids include palmitic acid, linoleic acid, linolenic acid, palmitic acid, and stearic acid. These are essential components and are affected by plant species, soil, climate, origin, and picking. The content of each component will be different due to the influence of season and extraction process. However, myristic acid may not be included and is an optional or non-essential component.

Sea buckthorn oil ceramide, its composition includes: oleic acid ceramide, palmitic acid ceramide, linoleic acid ceramide, linolenic acid ceramide, palmitic acid ceramide, stearic acid ceramide; because fatty acids all participate in the same reaction , the mass proportion of ceramide does not change much after the reaction, so it is similar to the composition of sea buckthorn oil fatty acids. The composition of sea buckthorn oil ceramide is: 20~35wt% oleic acid ceramide, 8~30wt% palmitic acid ceramide, 4~ 40wt% linoleic acid ceramide, 2-30wt% linolenic acid ceramide, 0.5-35wt% palmitic acid ceramide, 0.5-3wt% stearic acid ceramide. The content of each component varies depending on the content of fatty acids in sea buckthorn oil or oil. In addition, sea buckthorn oil ceramide also includes ceramide obtained by reacting myristic acid and sphingosine compounds, that is, it also includes 0 to 1 wt% myristic acid ceramide. Sea buckthorn oil ceramides also include flavonoids, alkaloids, sterols, triterpenes, vitamins and other compounds that are present in the fatty acids of sea buckthorn oil but do not react with sphingosine compounds.

Sea buckthorn oil ceramide, its composition includes: oleic acid ceramide, palmitic acid ceramide, linoleic acid ceramide, linolenic acid ceramide; oleic acid ceramide accounts for 20 to 35wt%, and palmitic acid ceramide accounts for 8 to 30wt %, linoleic acid ceramide accounts for 4 to 40 wt%, and linolenic acid ceramide accounts for 2 to 30 wt%.

Further, the sea buckthorn oil ceramide includes palmitic acid ceramide, and the palmitic acid ceramide accounts for 0.5 to 35wt%.

Further, the sea buckthorn oil ceramide includes stearic acid ceramide, and stearic acid ceramide accounts for 0.5 to 3 wt%.

Further, the sea buckthorn oil ceramide includes no more than 1 wt% myristate ceramide, especially 0.1 to 1 wt% myristate ceramide.

Oleic acid ceramide is obtained by the condensation reaction of oleic acid and sphingosine compounds, including oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide, oleic acid dihydrosphingosine ceramide; palmitic acid ceramide is It is obtained by the condensation reaction of palmitic acid and sphingosine compounds, including palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide, palmitic dihydrosphingosine ceramide; linoleic acid ceramide is obtained from linoleic acid and sphingosine ceramide. Obtained from the condensation reaction of sphingosine compounds, including linoleic acid phytosphingosine ceramide, linoleic acid sphingosine ceramide, linoleic acid dihydrosphingosine ceramide; linolenic acid ceramide is a combination of linolenic acid and sphingosine Obtained from the condensation reaction of alcohol compounds, including linolenic acid phytosphingosine ceramide, linolenic acid sphingosine ceramide, linolenic acid dihydrosphingosine ceramide; stearic acid ceramide is a compound of stearic acid and sphingosine Obtained by condensation reaction, including stearic acid phytosphingosine ceramide, stearic acid sphingosine ceramide, stearic dihydrosphingosine ceramide; palmitic acid ceramide, myristic acid ceramide, etc. analogy.

The second aspect of the present invention, the synthesis method of sea buckthorn oil ceramide, includes the following steps:

Sea buckthorn oil fatty acids react with sphingosine compounds under the conditions of a condensing agent and an organic base. The condensing agent is EDCI and the organic base is Et ₃ N.

Further, the molar ratio of the sea buckthorn oil fatty acid, sphingosine compounds, EDCI, and Et ₃ N is 1: (1～1.5): (1～2): (1～2), and the solvent of the reaction is At least one of methylene chloride, tetrahydrofuran, ethyl acetate, and acetonitrile.

Seabuckthorn oil purchased on the market is generally in the form of oil and needs to be hydrolyzed into seabuckthorn oil fatty acids, so the following steps are also included:

Sea buckthorn oil and fat are hydrolyzed through saponification reaction to obtain sea buckthorn oil fatty acids.

Further, the saponification reaction is hydrolysis of sea buckthorn oil in a potassium hydroxide solution.

Further, the mass ratio of the sea buckthorn oil and potassium hydroxide is 1: (1-2).

The third aspect of the present invention is the use of sea buckthorn oil ceramide in cosmetics, medicines, dietary products or health care products.

Furthermore, the sea buckthorn oil ceramide has at least one of the effects of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, promoting collagen synthesis, maintaining elastin vitality, and whitening.

A composition, including sea buckthorn oil ceramide, has at least one of the effects of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, promoting collagen synthesis, maintaining elastin vitality, and whitening.

The composition contains acceptable excipients, including solubilizers, preservatives, antioxidants, pH adjusters, penetration enhancers, liposomes, moisturizers, thickeners, chelating agents, skin texture modifiers, and surfactants. , emulsifier, flavor and pigment; the composition is in the form of cream, emulsion, solution, film, aerosol or spray.

The invention has the following beneficial effects:

Sea buckthorn oil fatty acids are naturally occurring fatty acids. The main components are oleic acid, palmitic acid and linoleic acid. In addition, they also contain linolenic acid, palmitic acid, stearic acid, etc., which are closely related to sphingosine naturally present in the skin. Alcohol compounds, sea buckthorn oil ceramide prepared through mild reaction, show excellent performance in the repair of skin's natural barrier, antioxidant, anti-aging, etc., and have broad application prospects in cosmetics, health products, biomedicine and other fields. .

1. Better effect than single ceramide. Different ceramides have different effects due to their structural differences. Ceramides with a single structure are generally difficult to have a comprehensive effect. This solution is based on the idea of bionics. It uses sea buckthorn oil or fatty acids from natural sources as raw materials to synthesize complex ceramides to make up for the differences in the efficacy of different ceramides. The trace fatty acids in sea buckthorn oil can form trace amounts of ceramides to supplement the efficacy. role.

2. It has better effect than compound ceramide. In addition to fatty acids, sea buckthorn oil also contains more than 140 biologically active ingredients such as flavonoids, organic acids, alkaloids, sterols, triterpenes and various vitamins. These nutrients have the effects of moisturizing the skin and enhancing cell vitality. Ceramides synthesized from oil have a synergistic effect with other active ingredients contained in sea buckthorn oil, and have better effects than ceramides compounded in similar proportions.

3. Lower cost. The method of the present invention quickly obtains a composite composition of multiple ceramides. Plant-derived sea buckthorn oil or its fatty acids have a wide range of sources, are easy to obtain commercially, have lower costs, are more environmentally friendly and economical, and are different from combining different single ceramides. According to the idea of mixing and compounding, single-component fatty acids not only have high raw material prices, but also need to produce different ceramides separately and then compound them, which increases the preparation cost.

4. The synthesis method is simple. The method of the present invention can adopt chemical synthesis to prepare multiple ceramides in one step, or can also use microbial fermentation.

Description of the drawings

Figure 1 is the test result of cell migration ability in Example 4;

Figures 2 and 3 are bar graphs of the elastase inhibition rate in Example 5;

Figures 4 and 5 are bar graphs of MMP1 expression in the photoaging resistance test of Example 6;

Figures 6 and 7 are bar graphs of DPPH free radical scavenging rate in the antioxidant test of Example 7;

Figure 8 is a bar graph of melanin content in the whitening activity test of Example 8.

Detailed ways

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

EDCI refers to 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and Et ₃ N refers to triethylamine. Silica gel column chromatography uses Qingdao marine silica gel (particle size 0.040-0.063mm). Thin layer chromatography (TLC) uses 60F254 silica gel plate, and TLC color development uses UV light (254nm) or iodine.

Example 1

Sea buckthorn oil fatty acid and phytosphingosine synthesize ceramide

Step 1: Dissolve 50g of sea buckthorn seed oil in 60mL of tetrahydrofuran, cool it in an ice bath, add dropwise 100mL of potassium hydroxide (25wt%) solution, and after completion of the dropwise addition, rise to room temperature for reaction until TLC detects that the reaction is complete.

Post-processing: Add dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, add 150 mL ethyl acetate to extract the aqueous phase, add 100 mL saturated brine and wash once, add anhydrous Na ₂ SO ₄ to the organic phase to dry, filter and concentrate in vacuum. 39.6g of sea buckthorn oil fatty acids were obtained.

Step 2: Add sea buckthorn oil fatty acid (50mmol, calculated as the main component fatty acid), EDCI (60mmol), and Et ₃ N (60mmol) into a 250mL round-bottomed flask, then add 100mL dichloromethane, and then stir at room temperature for 1 hours, then phytosphingosine (60 mmol) was added to the reaction system, and stirred at room temperature until the reaction was completed as detected by TLC.

Post-processing: add water to quench the reaction, separate the organic layer, dry, filter and concentrate in vacuum, then wash with solvent to obtain sea buckthorn oil ceramide. The product is analyzed by HPLC. HPLC chromatography conditions: use Shimadzu high-performance liquid chromatography (LC-2030C3DPlus) ), using Innoval ODS-2 4.6*250mm, 5μm column, column temperature: 30℃, injection volume: 10μL, flow rate: 1.0mL/min, evaporation temperature: 40℃, carrier gas flow rate: 2.5L/min, flowing Phase: 100% methanol.

The HPLC retention times of each component are: palmitic acid-phytosphingosine ceramide 8.1 min, linolenic acid-phytosphingosine ceramide 8.6 min, linoleic acid-phytosphingosine ceramide 9.5 min, palmitic acid- Phytosphingosine ceramide 10.7 minutes, oleic acid-phytosphingosine ceramide 11.3 minutes, stearic acid-phytosphingosine ceramide 13.9 minutes.

The obtained products were analyzed by high performance liquid chromatography and found that oleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, linoleic acid-phytosphingosine ceramide, linolenic acid-phytosphingosine ceramide, The contents of palmitic acid-phytosphingosine ceramide and stearic acid-phytosphingosine ceramide are 31%, 22%, 14%, 17%, 11% and 2% respectively, and the rest are other ingredients. less.

Example 2

Sea buckthorn oil fatty acids and sphingosine synthesize ceramide

Step 1: Dissolve 50g of sea buckthorn fruit oil in 60mL of tetrahydrofuran, cool it in an ice bath, add dropwise 100mL of potassium hydroxide (25wt%) solution, and after completion of the dropwise addition, rise to room temperature for reaction until TLC detects that the reaction is complete.

Post-processing: Add dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, add 150 mL ethyl acetate to extract the aqueous phase, add 100 mL saturated brine and wash once, add anhydrous Na ₂ SO ₄ to the organic phase to dry, filter and concentrate in vacuum. 39.6g of sea buckthorn oil fatty acids were obtained.

Step 2: Add sea buckthorn oil fatty acid (50mmol, based on the main component fatty acid), EDCI (65mmol), and Et ₃ N (65mmol) into a 250mL round-bottomed flask, then add 100mL dichloromethane, and then stir at room temperature for 1 hours, then sphingosine (55 mmol) was added to the reaction system, and stirred at room temperature until the reaction was completed as detected by TLC.

Post-processing: add water to quench the reaction, separate the organic layer, dry, filter and concentrate in vacuum, then wash with solvent to obtain sea buckthorn oil ceramide. The product is analyzed by HPLC. HPLC chromatography conditions: use Shimadzu high-performance liquid chromatography (LC-2030C3DPlus) ), using Innoval ODS-2 4.6*250mm, 5μm column, column temperature: 30℃, injection volume: 10μL, flow rate: 1.0mL/min, evaporation temperature: 40℃, carrier gas flow rate: 2.5L/min, flowing Phase: 100% methanol.

The HPLC retention times of each component are: linolenic acid-sphingosine ceramide 7.8 min, palmitic acid-sphingosine ceramide 8.1 min, myristic acid-sphingosine ceramide 8.3 min, linoleic acid-sphingosine ceramide Alcohol ceramide 8.7 minutes, oleic acid-sphingosine ceramide 10.1 minutes, palmitic acid-sphingosine ceramide 10.5 minutes, stearic acid-sphingosine ceramide 13.5 minutes.

The obtained product was analyzed by high performance liquid chromatography, and it was found that oleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, linoleic acid-sphingosine ceramide, linolenic acid-sphingosine ceramide, palmitic acid The contents of -sphingosine ceramide, stearic acid-sphingosine ceramide, and myristic acid-sphingosine ceramide are 22%, 13%, 35%, 6%, 16%, 3%, and 1%, respectively. The rest are other ingredients in smaller amounts.

Example 3

Sea buckthorn oil fatty acid and dihydrosphingosine synthesize ceramide

Step 1: Dissolve 50g of sea buckthorn seed oil in 60mL of tetrahydrofuran, cool it in an ice bath, add dropwise 100mL of potassium hydroxide (25wt%) solution, and after completion of the dropwise addition, rise to room temperature for reaction until TLC detects that the reaction is complete.

Post-processing: Add dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, add 150 mL ethyl acetate to extract the aqueous phase, add 100 mL saturated brine and wash once, add anhydrous Na ₂ SO ₄ to the organic phase to dry, filter and concentrate in vacuum. 39.6g of sea buckthorn oil fatty acids were obtained.

Step 2: Add sea buckthorn oil fatty acid (50mmol, based on the main component fatty acid), EDCI (80mmol), and Et ₃ N (80mmol) into a 250mL round-bottomed flask, then add 100mL dichloromethane, and then stir at room temperature for 1 hour, then dihydrosphingosine (60 mmol) was added to the reaction system, and stirred at room temperature until the reaction was completed as detected by TLC.

Post-processing: add water to quench the reaction, separate the organic layer, dry, filter and concentrate in vacuum, then wash with solvent to obtain sea buckthorn oil ceramide. The product is analyzed by HPLC. HPLC chromatography conditions: use Shimadzu high-performance liquid chromatography (LC-2030C3DPlus) ), using Innoval ODS-2 4.6*250mm, 5μm column, column temperature: 30℃, injection volume: 10μL, flow rate: 1.0mL/min, evaporation temperature: 40℃, carrier gas flow rate: 2.5L/min, flowing Phase: 100% methanol.

The HPLC retention times of each component are: linolenic acid-sphingosine ceramide 8.3 min, palmitic acid-sphingosine ceramide 9.0 min, linoleic acid-sphingosine ceramide 9.6 min, Palmitic acid-sphingosine ceramide 10.6 minutes, oleic acid-sphingosine ceramide 11.1 minutes, stearic acid-sphingosine ceramide 13.6 minutes.

The obtained products were analyzed by high performance liquid chromatography and found that oleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, linoleic acid-sphingosine ceramide, and linolenic acid-sphingosine ceramide The contents of alcohol ceramide, palmitic acid-sphingosine ceramide, and stearic acid-sphingosine ceramide are 28%, 27%, 6%, 25%, 7%, and 1%, respectively. The rest are other ingredients in smaller amounts.

Example 4

Cell migration to assess skin barrier repair

Principle: When the cells grow to fuse into a single layer, a scratch tool is used to create a blank area on the fused single layer of cells. The cells in the blank area are removed by mechanical force. After a period of culture, the cells are observed to become cell-free. Regional migration reflects the migration ability of cells by measuring the migration distance of cells.

Steps:

1. Line the culture plate. First, use a Marker pen on the back of the 6-well plate, use a ruler to compare, and draw horizontal lines evenly, about every 0.5 to 1cm, across the holes. Each hole should pass through at least 5 lines. When drawing, be careful not to draw lines. Too thick.

2. Spread the cells. Add about 5 × 10 ⁵ cells into the well (the number of cells varies, and is adjusted according to the growth rate of the cells). The seeding principle is to reach 100% confluence rate after overnight.

3. Cell line drawing. On the second day, use a pipette tip perpendicular to the cell plane to scratch the cell layer along the line drawn on the back of the plate on the first day (it is best to use the same pipette tip between different wells).

4. Wash the cells. After scratching is completed, wash the cells 3 times with sterile PBS to remove non-adherent cells, that is, cells that were crossed during scratching. The gap left after scratching is clearly visible, and then replace with fresh serum-free medium.

5. Cell culture and observation. After the sample (Example 1 product, Ceramide 3B) is diluted with culture medium (Example 1 product concentration is 100 mg/L, Ceramide 3B concentration is 100 mg/L), add it to the cell culture dish, and place the cells in 37°C, 5wt% Incubate in a CO ₂ incubator, take out the cells after 24 hours, observe with a microscope, measure the width of the scratch, and take photos, and use Image J software to calculate the healing rate.

The results are shown in Figure 1. Compared with the solvent control group, the scratch width of the experimental group was narrower, indicating that sea buckthorn oil ceramide has better tissue healing ability. The healing rate of the solvent control group after 24 hours was 21.35%, and the healing rate of sea buckthorn oil ceramide after 24 hours was 59.48%. The compound of the present invention significantly improves the cell healing rate and has good skin tissue repair activity.

Example 5

Elastase inhibition assay to test anti-aging effects

Elastase inhibition method: Take 2 mL of 2 mg/mL elastase solution, add samples of different concentrations (product of Example 1), vortex and mix thoroughly, shake on a shaker at 37°C, 400 r/min for 20 min, and immediately add 0.5 pH 6.0 5 mL of mol/L phosphate buffer, vortex to mix, take an appropriate amount of the mixed solution into a 2 mL centrifuge tube, centrifuge at 9 391 × g for 10 min, accurately pipette 200 μL of the supernatant into a 96-well plate, and enzyme at a wavelength of 495 nm Measure the absorbance with a standard instrument and perform a spectral scan from 400 to 800 nm at the same time.

The substrate plus enzyme solution was used as the blank control group, the substrate plus enzyme and sample solution was the enzyme inhibition group, and the substrate plus sample without enzyme solution was used to subtract the background. Each group is equipped with 3 multiple holes. Inhibition rate (%) = [1–(An–An′)/(A0–A0′)]×100%, in the formula, A0 is the absorbance of adding enzyme without adding sample, A0′ is adding only substrate without adding sample and the absorbance of the enzyme, An is the absorbance of the sample solution only, and An′ is the absorbance of the sample without enzyme. If An′>An, it shows a promotion effect, promotion rate (%) = [1–(An′–An)/(A0–A0′)]×100%.

The results are shown in Figure 2. Sea buckthorn oil ceramide has a good inhibitory effect on elastase at different concentrations. Specifically, the inhibition rate of elastase at a concentration of 0.25g/L is 11.47%, and at a concentration of 0.5 The inhibition rate of elastase at the concentration of g/L is 23.93%, the inhibition rate of elastase at the concentration of 1.0g/L is 35.00%, and the inhibition rate of elastase at the concentration of 2.0g/L is 50.00 %.

The inhibitory activity of ceramide 2 on elastase was tested according to the same method. The results are shown in Figure 3. The elastase inhibition rates at concentrations of 0.25, 0.5, 1.0, and 2.0g/L were 10.12%, 18.06%, and 28.84 respectively. % and 19.78%, its inhibitory effect is not as good as the same concentration of sea buckthorn oil ceramide.

Example 6

MMP1, also known as interstitial collagenase and matrix metalloproteinase, belongs to the matrix metalloproteinase family. Its main substrate is fibrillar 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 collagen and elastin synthesis in fibroblasts, and reducing MMP activity can increase the rate of collagen synthesis.

HaCaT cells were seeded in a 96-well plate at a density of 1×10 ⁵ cells/well and kept in an incubator overnight. After 24 hours, the supernatant was discarded, and 100 μL of culture medium containing samples (products of Example 1) of different concentrations was added. No samples were added to the model group. The negative control group was DMEM culture medium without samples. Each group had 3 duplicate wells. After incubating for 2 hours in a mass fraction of 5% CO ₂ and 37°C environment, UVA or UVB ultraviolet rays were irradiated. The distance between the ultraviolet radiation source and the cells is 15cm, the UVA intensity is 200mJ/cm ² and the radiation time is 2h, the UVB intensity is 50mJ/cm ² and the radiation time is 1h. After irradiation, continue incubation in the incubator for 12 hours. MMP-1 ELISA kit was used to detect intracellular MMP-1 gene expression. Inhibition rate = 1-(Expression amount of MMP1 in the experimental group/Expression amount of MMP1 in the model group) × 100%.

The results are shown in Figures 4 and 5. For UVA, the expression level of MMP1 in the negative control group was set to 1, and the expression level in the model group was 1.90. When sea buckthorn oil ceramide was at concentrations of 125, 250, and 400 mg/L, relative to the model group The inhibition rates of MMP1 expression were 31%, 46%, and 56%; for UVB, the MMP1 expression level in the negative control group was set to 1, the expression level in the model group was 2.33, and sea buckthorn oil ceramide was at 125, 250, and 400 mg/L. At different concentrations, the inhibition rates of MMP1 expression relative to the model group were 35%, 51%, and 69%.

After UVA ultraviolet radiation, keratinocytes promote the increase of MMP1 expression in fibroblasts, which causes the degradation of skin extracellular matrix and skin collagen, leading to skin photoaging. The above results show that sea buckthorn oil ceramide can inhibit the production of MMP1 by fibroblasts caused by ultraviolet radiation, and has a certain effect on preventing skin photoaging.

Example 7

DPPH free radical scavenging test for antioxidant properties

DPPH is 1,1-diphenyl-2-trinitophenylhydrazine, which can be used in antioxidant experiments.

Mix the corresponding concentration (50, 100, 200, 400, 800mg/L) samples (product of Example 1) with 0.1mol/LDPPH and absolute ethanol solutions in a volume ratio of 1:1. DPPH and absolute ethanol are 1:1. : 1 Mix equal volumes, react at room temperature for 30 minutes in the dark, and measure the absorbance value at 517nm. The absorbance of the reaction solution between the sample and DPPH is recorded as A1, the absorbance of the reaction solution between the sample and absolute ethanol is recorded as A2, the absorbance of the reaction solution between DPPH and absolute ethanol is recorded as A3, and the DPPH clearance rate of the sample = [1-(A1-A2 )/A3]×100%.

The results are shown in Figure 6. The DPPH free radical scavenging rates at concentrations of 50, 100, 200, 400, and 800 mg/L were 26.76%, 34.38%, 40.77%, 51.72%, and 54.44% respectively, showing excellent anti- Oxidation effect. The antioxidant effect of ceramide 3B (oleic acid ceramide) was tested according to the same method. The results are shown in Figure 7. The DPPH free radical scavenging rate at concentrations of 50, 100, 200, 400 and 800 mg/L was 7.76. %, 12.82%, 24.10%, 29.60%, 33.16%. Sea buckthorn oil ceramide has a higher clearance rate for DPPH than ceramide 3B and has a better antioxidant effect.

Example 8

Whitening Activity Test

Take B16 cells in the exponential growth phase, digest them with 0.25% trypsin-EDTA and pipet evenly, and seed the cells in a 12-well plate at a density of 3×10 ⁵ cells/well. Cultivate overnight at 37°C and 5% _CO2 . Discard the supernatant, add culture medium containing samples of different mass concentrations (products of Example 1), incubate with RPMI-1640 culture medium without adding samples as the blank group, add DMEM culture medium and incubate as the modeling group, each group has 3 Multiple wells were incubated for 24 h in a mass fraction of 5% CO ₂ and 37°C environment. Discard the culture medium in the well plate, wash it once or twice with phosphate buffer saline (PBS), add 1 mL of NaOH solution (1 mol/L) containing 10% DMSO to lyse the cells, and place it at 80°C or 100°C. Keep at constant temperature for 2 hours until the cells are completely dissolved. Place it in a microplate reader and measure the absorbance value at 405nm. Calculate the melanin inhibition rate=1-(OD value of each well/OD value of model group)×100%.

The results are shown in Figure 8. The melanin content of the blank control group was set to 1, and the melanin expression of the model group was 1.54. When the concentrations were 10, 20, 40, 80, and 100 mg/L, the melanin inhibition rate of sea buckthorn oil ceramide was They are 15.10%, 21.55%, 24.98%, 35.20%, and 46.59% respectively, showing a good whitening effect.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. All are 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 (10)

1. Sea buckthorn oil ceramide is obtained by reacting sea buckthorn oil fatty acids with sphingosine compounds, and the sphingosine compounds are selected from the group consisting of sphingosine, phytosphingosine, and dihydrosphingosine. 2. Seabuckthorn oil ceramide according to claim 1, characterized in that the seabuckthorn oil fatty acid is obtained by hydrolysis of seabuckthorn seed oil or seabuckthorn fruit oil. 3. The sea buckthorn oil ceramide according to claim 1 or 2, characterized in that the sea buckthorn oil fatty acid contains 20 to 35 wt% oleic acid, 8 to 30 wt% palmitic acid, 4 to 40 wt% linoleic acid, 2 to 30wt% linolenic acid, 0.5-35wt% palmitic acid, 0.5-3wt% stearic acid. 4. Sea buckthorn oil ceramide, its composition includes: oleic acid ceramide, palmitic acid ceramide, linoleic acid ceramide, linolenic acid ceramide, palmitic acid ceramide, stearic acid ceramide. 5. Seabuckthorn oil ceramide according to claim 4, its composition includes: 20-35wt% oleic acid ceramide, 8-30wt% palmitic acid ceramide, 4-40wt% linoleic acid ceramide, 2-30wt% Linolenic acid ceramide, 0.5~35wt% palmitoneic acid ceramide, 0.5~3wt% stearic acid ceramide. 6. The sea buckthorn oil ceramide according to claim 4 or 5, further comprising: 0 to 1 wt% myristate ceramide. 7. The synthesis method of sea buckthorn oil ceramide according to any one of claims 1 to 6, comprising the following steps: Under the conditions of a condensing agent and an organic base, sea buckthorn oil fatty acids react with sphingosine compounds, the condensing agent is EDCI, and the organic base is Et ₃ N; The molar ratio of the sea buckthorn oil fatty acid, sphingosine compounds, EDCI, and Et ₃ N is 1: (1～1.5): (1～2): (1～2), and the solvent of the reaction is dichloromethane , at least one of tetrahydrofuran, ethyl acetate, and acetonitrile. 8. Use of the sea buckthorn oil ceramide according to any one of claims 1 to 6 in cosmetics, medicines, dietary products or health care products. 9. The use according to claim 8, characterized in that the sea buckthorn oil ceramide has skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, promoting collagen synthesis, maintaining elastin vitality, and whitening effects. at least one of them. 10. A composition, comprising the sea buckthorn oil ceramide according to any one of claims 1 to 6, said composition having skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, promoting collagen synthesis, Maintain at least one of elastin vitality and whitening effects.