CN117285434A - Monkey bread seed oil ceramide and synthesis method and application thereof - Google Patents

Monkey bread seed oil ceramide and synthesis method and application thereof Download PDF

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CN117285434A
CN117285434A CN202310186326.7A CN202310186326A CN117285434A CN 117285434 A CN117285434 A CN 117285434A CN 202310186326 A CN202310186326 A CN 202310186326A CN 117285434 A CN117285434 A CN 117285434A
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ceramide
monkey
acid
seed oil
sphingosine
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杨超文
叶柳
李青
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Shenzhen Dikeman Biotechnology Co ltd
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Shenzhen Dikeman Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of biological medicines, and discloses monkey-bread-seed-oil ceramide which is obtained by reacting monkey-bread-seed-oil fatty acid with a sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. The monkey seed oil ceramide has excellent performances in the aspects of repairing natural skin barriers, tissue healing, anti-aging and the like, and has wide application prospects in the fields of cosmetics, health-care products, biological medicines and the like.

Description

Monkey bread seed oil ceramide and synthesis method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to monkey seed oil ceramide and a synthesis method and application thereof.
Background
Ceramides (ceramides, also known as molecular nails) naturally occur in the skin and are very important components of the skin barrier (stratum corneum), in amounts of up to 40-50 wt.%, ceramides are a class of sphingolipids consisting of long-chain bases of sphingosine and fatty acids, in which the carbon chain length, unsaturation and number of hydroxyl groups of the sphingosine moiety, fatty acid moiety are all variable, and ceramides represent a class of compounds. Ceramide has excellent properties in regulating skin barrier function, recovering skin moisture, enhancing adhesion between skin keratinocytes, and the like.
Because of the importance of ceramides, many cosmetic and pharmaceutical companies are researching and developing corresponding products. The natural plant-derived ceramide can form an effective skin barrier to prevent water loss and resist external damage due to the more sustainable and more environment-friendly raw material source and the characteristics similar to the skin ceramide components, and can become a next-generation environment-friendly, safe and reliable ceramide product.
The monkey seed oil is golden yellow, has nut fragrance, contains a plurality of fatty acids which are not easy to synthesize by human body and multivitamins with excellent cosmetic effect, and contains 33wt% of saturated fatty acid, 36wt% of unsaturated fatty acid unit, 31wt% of polyunsaturated fatty acid and A, D, E, K of vitamins. Wherein, oleic acid has extremely high moisture retention capability, and can resist dry skin and aging; linoleic acid can promote skin activation; palmitic acid has anti-aging effect and can prevent fine lines, wrinkles, etc.; stearic acid has extremely strong antioxidant power; vitamin A enhances the activation of skin, has strong antioxidant power and enhances the skin elasticity; vitamin C can prevent wrinkles, spots and black spots; vitamin D can activate skin immunity, improve skin water locking performance, and prevent skin dryness; the vitamin E can resist ultraviolet damage to skin and has extremely strong oxidation resistance; vitamin K can promote skin metabolism and repair striae gravidarum. The monkey seed oil has the effects of moistening skin and softening horny layer, and has the effects of increasing skin elasticity and promoting skin cell tissue reproduction without blocking pores, so that the skin breathes normally. Monkey seed oil is often used, and skin color can be effectively lightened. The monkey seed oil has extremely strong regeneration capability, can help repair the stratum corneum, and is very suitable for people with thin stratum corneum.
Disclosure of Invention
The invention aims to provide ceramide synthesized by utilizing plant-derived monkey seed oil fatty acid.
Another object of the present invention is to provide a method for synthesizing monkey seed oil ceramide, which uses a monkey seed oil fat or a monkey seed oil fatty acid which is derived from natural plants and is easily available as a raw material.
It is another object of the present invention to provide the use of monkey seed oil ceramide.
In order to achieve one of the above purposes, the present invention adopts the following technical scheme:
in a first aspect of the invention, monkey seed oil ceramide is obtained by reacting a monkey seed oil fatty acid with a sphingosine compound selected from the group consisting of sphingosine, phytosphingosine, sphinganine.
The reaction can be chemical synthesis reaction (as detailed below), or microbial fermentation method, i.e. using Pichia pastoris or Saccharomyces cerevisiae, fermenting under certain environment to obtain sphingosine compound, and adding fatty acid to obtain ceramide; or using monkey seed oil as raw material, selecting proper strain, and fermenting to obtain monkey seed 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 monkey seed oil fatty acid is obtained by hydrolyzing monkey seed oil.
Further, the monkey seed oil fatty acid contains 30 to 50 weight percent of oleic acid.
Further, the monkey seed oil fatty acid contains 15 to 35 weight percent linoleic acid.
Further, the monkey seed oil fatty acid contains 13 to 30wt% palmitic acid.
Further, the monkey seed oil fatty acid contains 2 to 8wt% of stearic acid.
Further, the monkey seed oil fatty acid contains 0.5 to 5 weight percent of linolenic acid.
Further, the monkey seed oil fatty acid contains 0.2 to 2 weight percent of arachidic acid.
In addition, the monkey seed oil fatty acid also contains 0 to 1 weight percent of palmitoleic acid and 0 to 0.5 weight percent of myristic acid.
The composition of the monkey seed oil fatty acid is as follows: 30 to 50 weight percent of oleic acid, 15 to 35 weight percent of linoleic acid, 13 to 30 weight percent of palmitic acid, 2 to 8 weight percent of stearic acid, 0.5 to 5 weight percent of linolenic acid, 0.2 to 2 weight percent of arachic acid, 0 to 1 weight percent of palmitoleic acid and 0 to 0.5 weight percent of myristic acid.
The main components of the monkey seed oil fatty acid are oleic acid and linoleic acid, other fatty acids comprise palmitic acid, stearic acid, linolenic acid and arachidic acid, which are essential components, are influenced by tree species, soil, climate, production place, picking season and extraction process, the content of each component is different, and palmitoleic acid and myristic acid are not necessarily contained, and are optional components or unnecessary components.
Monkey seed oil ceramide, its composition includes: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide, arachidic acid ceramide; because fatty acids all participate in the same reaction, the mass ratio of ceramide after the reaction is not greatly changed, so the composition of the ceramide of the monkey bread seed oil is similar to that of the fatty acid of the monkey bread seed oil, and the composition of the ceramide of the monkey bread seed oil is as follows: 30 to 50 weight percent of oleic acid ceramide, 15 to 35 weight percent of linoleic acid ceramide, 13 to 30 weight percent of palmitic acid ceramide, 2 to 8 weight percent of stearic acid ceramide, 0.5 to 5 weight percent of linolenic acid ceramide and 0.2 to 2 weight percent of arachidic acid ceramide. The content of each component is different due to the different content of each fatty acid in the monkey seed oil fatty acid or the grease. In addition, the monkey seed oil ceramide also comprises ceramide obtained by reacting one or more of palmitoleic acid and myristic acid with sphingosine compounds, namely 0-1 wt% palmitoleic acid ceramide and 0-0.5 wt% myristic acid ceramide. Monkey seed oil ceramide also includes compounds such as vitamin A, D, E, K which are present in monkey seed oil fatty acids but which do not react with sphingosine compounds.
Monkey seed oil ceramide, its composition includes: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide; 30-50 wt% of oleic acid ceramide, 15-35 wt% of linoleic acid ceramide, 13-30 wt% of palmitic acid ceramide and 2-8 wt% of stearic acid ceramide.
Further, the monkey seed oil ceramide comprises linolenic acid ceramide, and the linolenic acid ceramide accounts for 0.5-5 wt%.
Further, the monkey seed oil ceramide comprises arachidic acid ceramide, and the arachidic acid ceramide accounts for 0.2-2 wt%.
Further, the monkey seed oil ceramide comprises not more than 1wt% palmitoleic acid ceramide and not more than 0.5wt% myristic acid ceramide, particularly 0.1 to 1wt% palmitoleic acid ceramide and 0.1 to 0.5wt% myristic acid ceramide.
Oleic acid ceramide is obtained by condensation reaction of oleic acid and sphingosine compounds, and comprises oleic acid phytosphingosine ceramide, oleic acid sphingosine ceramide and oleic acid dihydrosphingosine ceramide; the linoleic acid ceramide is obtained by condensation reaction of linoleic acid and sphingosine compounds, and comprises linoleic acid phytosphingosine ceramide, linoleic acid sphingosine ceramide and linoleic acid dihydrosphingosine ceramide; the palmitic acid ceramide is obtained by condensation reaction of palmitic acid and sphingosine compounds, and comprises palmitic acid phytosphingosine ceramide, palmitic acid sphingosine ceramide and palmitic acid dihydrosphingosine ceramide; stearic acid ceramide, linolenic acid ceramide, arachidic acid ceramide, palmitoleic acid ceramide, myristic acid ceramide, and the like.
In a second aspect of the invention, a method for synthesizing monkey seed oil ceramide comprises the following steps:
under the conditions of condensing agent and organic alkali, the monkey bread seed oil fatty acid reacts with the sphingosine compound, wherein the condensing agent is EDCI, and the organic alkali is Et 3 N。
Further, the monkey seed oil fatty acid, sphingosine compound, EDCI, et 3 The molar ratio of N is 1: (1-1.5): (1-2): (1-2), wherein the solvent for the reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.
The commercially available monkey seed oil is generally in the form of an oil and fat, and needs to be hydrolyzed into a monkey seed oil fatty acid, and therefore further comprises the following steps:
the monkey bread seed oil fat is hydrolyzed by saponification reaction to obtain the monkey bread seed oil fatty acid.
Further, the saponification reaction is hydrolysis of the monkey seed oil fat in potassium hydroxide solution.
Further, the mass ratio of the monkey seed oil grease to the potassium hydroxide is 1: (1-2).
In a third aspect of the invention, the use of monkey seed oil ceramide in cosmetics, pharmaceuticals, dietary or health care products.
Further, the monkey seed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, antioxidant, collagen synthesis promoting, elastin activity maintaining, and whitening effects.
A composition comprising monkey seed oil ceramide, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, antioxidant, collagen synthesis promoting, elastin viability maintaining, whitening efficacy.
The composition contains acceptable auxiliary materials, including one or more of solubilizer, antiseptic, antioxidant, pH regulator, penetration enhancer, liposome, humectant, thickener, chelating agent, skin feel regulator, surfactant, emulsifier, essence and pigment; the composition is in the form of cream, emulsion, solution, film, aerosol or spray.
The invention has the following beneficial effects:
the monkey seed oil fatty acid belongs to naturally-formed fatty acid, the main components are unsaturated fatty acid-oleic acid and linoleic acid, in addition, palmitic acid, stearic acid and the like, and the monkey seed oil ceramide is prepared by mild reaction with sphingosine compounds naturally existing in skin, has excellent performance in the aspects of repairing natural skin barriers, resisting oxidation, resisting aging and the like, and has wide application prospects in the fields of cosmetics, health-care products, biological medicines and the like.
1. Better results compared to ceramide alone. Different ceramides have different effects due to the structural differences, and ceramides with a single structure generally have difficulty in having comprehensive effects. The scheme is based on a bionic thought, and the natural monkey seed oil grease or fatty acid is used as a raw material to synthesize the compound ceramide so as to make up the difference of different ceramide effects, and trace fatty acid in the monkey seed oil can form trace ceramide to play a role in efficacy supplement.
2. Compared with the compounded ceramide, the effect is better. Besides fatty acid (or grease), the monkey seed oil also contains some rare fatty acid and multiple vitamins, and the nutrient substances have the effects of moistening skin, filtering ultraviolet rays, providing a barrier for skin and the like, and the ceramide synthesized by the monkey seed oil has a synergistic effect with other active ingredients contained in the monkey seed oil, and has better effect compared with the ceramide compounded according to similar proportion.
3. The cost is lower. The method of the invention can rapidly obtain the composition compounded by various ceramides, and the vegetable-source monkey seed oil grease or the fatty acid thereof has wide sources, is easy to obtain commercially, is more environment-friendly and economical, is different from the idea of mixing and compounding different single ceramides, has high raw material price, needs to separately produce different ceramides, and then is compounded, thereby increasing the preparation cost.
4. The synthesis method is simple. The method can adopt chemical synthesis to realize one-step preparation of various ceramides, and can also use a microbial fermentation method.
Drawings
FIG. 1 is a graph showing the results of the cell migration ability test of example 4;
FIGS. 2 and 3 are bar graphs of elastase inhibition ratios of example 5;
FIGS. 4 and 5 are bar charts of MMP1 expression levels in the anti-photoaging test of example 6;
FIGS. 6 and 7 are bar graphs of DPPH radical scavenging for oxidation resistance test of example 7;
FIG. 8 is a bar graph showing the whitening activity test melanin content of example 8.
Detailed Description
The invention will be further illustrated with reference to specific examples.
EDCI refers to 1-ethyl- (3-dimethylaminopropyl) carbodiimide, et 3 N refers to triethylamine. The silica gel column chromatography uses Qingdao ocean silica gel (particle size 0.040-0.063 mm). Thin Layer Chromatography (TLC) using 60F254 silica gel plates was performed using UV light (254 nm) or iodine.
Example 1
Synthesis of ceramide from monkey seed oil fatty acid and phytosphingosine
The first step: 50g of monkey seed oil was dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 110mL of potassium hydroxide (25 wt%) solution was added dropwise, and after the addition was completed, the reaction was allowed to stand at room temperature until TLC detection was completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 120mL of ethyl acetate to extract a water phase, adding 80mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Drying, filtration and concentration in vacuo gave 39g of monkey seed oil fatty acid.
And a second step of: mixing monkey seed oil fatty acid (50 mmol based on main component fatty acid), EDCI (65 mmol), et 3 N (70 mmol) was added to a 250mL round bottom flask, followed by 80mL of dichloromethane, followed by stirring at room temperature for 1 hour, followed by phytosphingosine (60 mmol) added to the reaction system, and stirring at room temperature until TLC detection was complete.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain monkey seed oil ceramide, and analyzing a product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an shimadzu high performance liquid chromatograph (LC-2030 c3d Plus), column temperature with Innoval ODS-2.6x250 mm,5 μm column: 30 ℃, sample injection volume: 10 μl, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The retention time of each component HPLC was: palmitoleic acid-phytosphingosine ceramide 8.0min, linolenic acid-phytosphingosine ceramide 8.4min, myristic acid-phytosphingosine ceramide 9.3min, linoleic acid-phytosphingosine ceramide 9.6min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.2min, stearic acid-phytosphingosine ceramide 13.9min, arachidic acid-phytosphingosine ceramide 16.4min.
The obtained products are analyzed by high performance liquid chromatography, and the content ratio of oleic acid-phytosphingosine ceramide, linoleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide, linolenic acid-phytosphingosine ceramide, arachidic acid-phytosphingosine ceramide, palmitoleic acid-phytosphingosine ceramide and myristic acid-phytosphingosine ceramide is 34%, 32%, 21%, 4%, 1.5%, 0.5% and 0.5% in sequence, and the rest is other components, and the content is less.
Example 2
Synthesis of ceramide from monkey seed oil fatty acid and sphingosine
The first step: 50g of monkey seed oil was dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 110mL of potassium hydroxide (25 wt%) solution was added dropwise, and after the addition was completed, the reaction was allowed to stand at room temperature until TLC detection was completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 120mL of ethyl acetate to extract a water phase, adding 80mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Dried, filtered and concentrated in vacuo to yield 38.8g of monkey seed oil fatty acid.
And a second step of: mixing monkey seed oil fatty acid (50 mmol based on main component fatty acid), EDCI (80 mmol), et 3 N (80 mmol) was added to a 250mL round bottom flask, followed by 100mL of dichloromethane, followed by stirring at room temperature for 1 hour, followed by sphingosine (70 mmol) added to the reaction system, and stirring at room temperature until TLC detection was complete.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain monkey seed oil ceramide, and analyzing a product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an shimadzu high performance liquid chromatograph (LC-2030 c3d Plus), column temperature with Innoval ODS-2.6x250 mm,5 μm column: 30 ℃, sample injection volume: 10 μl, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The retention time of each component HPLC was: linolenic acid-sphingosine ceramide 7.9min, palmitoleic acid-sphingosine ceramide 8.2min, myristic acid-sphingosine ceramide 8.4min, linoleic acid-sphingosine ceramide 8.6min, oleic acid-sphingosine ceramide 10.1min, palmitic acid-sphingosine ceramide 10.5min, stearic acid-sphingosine ceramide 13.5min, arachidic acid-sphingosine ceramide 17.8min.
The obtained products are analyzed by high performance liquid chromatography, and the content of oleic acid-sphingosine ceramide, linoleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, linolenic acid-sphingosine ceramide, arachidic acid-sphingosine ceramide, palmitoleic acid ceramide and myristic acid-sphingosine ceramide is 41%, 26%, 15%, 8%, 2%, 1% and 0.5% in sequence, and the rest is other components, and the content is less.
Example 3
Synthesis of ceramide from monkey seed oil fatty acid and sphinganine
The first step: 50g of monkey seed oil was dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 110mL of potassium hydroxide (25 wt%) solution was added dropwise, and after the addition was completed, the reaction was allowed to stand at room temperature until TLC detection was completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 120mL of ethyl acetate to extract a water phase, adding 80mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Drying, filtration and concentration in vacuo gave 40.4g of monkey seed oil fatty acid.
And a second step of: mixing monkey seed oil fatty acid (50 mmol based on main component fatty acid), EDCI (70 mmol), et 3 N (75 mmol) was added to a 250mL round bottom flask, followed by 100mL of dichloromethane, followed by stirring at room temperature for 1 hour, followed by addition of sphinganine (65 mmol) to the reaction system, stirring at room temperature, and detection by TLC was complete.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain monkey seed oil ceramide, and analyzing a product by HPLC (high performance liquid chromatography) under the condition of HPLC chromatography: using an shimadzu high performance liquid chromatograph (LC-2030 c3d Plus), column temperature with Innoval ODS-2.6x250 mm,5 μm column: 30 ℃, sample injection volume: 10 μl, flow rate: 1.0mL/min, evaporation temperature: 40 ℃, carrier gas flow rate: 2.5L/min, mobile phase: 100% methanol.
The retention time of each component HPLC was: linolenic acid-sphinganine ceramide 8.2min, myristic acid-sphinganine ceramide 8.5min, palmitoleic acid-sphinganine ceramide 8.9min, linoleic acid-sphinganine ceramide 9.5min, palmitic acid-sphinganine ceramide 10.7min, oleic acid-sphinganine ceramide 11.1min, stearic acid-sphinganine ceramide 13.5min, arachidic acid-sphinganine ceramide 16.0min.
The obtained products are analyzed by high performance liquid chromatography, and the content of oleic acid-dihydrosphingosine ceramide, linoleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide, linolenic acid-dihydrosphingosine ceramide, arachidic acid-dihydrosphingosine ceramide, palmitoleic acid-dihydrosphingosine ceramide and myristic acid-dihydrosphingosine ceramide is 46%, 17%, 26%, 5%, 2%, 1%, 0.5% and 0.5% in sequence, and the rest is other components, and the content is less.
Example 4
Assessment of skin barrier repair by cell migration
Principle of: when the cells grow to be fused into a single-layer state, a scratch tool is manufactured on the fused single-layer cells, the cells in the blank area are removed by mechanical force, the migration condition of the cells to the cell-free area is observed through a period of culture, and the migration capability of the cells is reflected by measuring the migration distance of the cells.
The operation steps are as follows:
1. the culture plate is streaked. Firstly, a Marker pen is used for uniformly scribing transverse lines by comparing with a straight ruler, and the transverse lines are crossed through the through holes at intervals of about 0.5 cm to 1cm, and each hole at least passes through 5 lines, so that attention lines are not too thick when scribing.
2. And (5) paving cells. About 5X 10 is added to the well 5 Individual cells (the number of different cells is different, and the cell growth speed is regulated), and the inoculation principle is that the fusion rate reaches 100% after overnight.
3. Cell streaking. The next day the tip is used to scratch the cell layer along the line marked on the back of the plate on the first day, perpendicular to the cell plane (the same tip is preferably used between the different wells).
4. Washing cells. After the streaking was completed, cells were washed 3 times with sterile PBS, cells that did not adhere to the wall, i.e., streaked cells at streaking, and the gap left after streaking was clearly visible, followed by replacement of fresh serum-free medium.
5. And (5) culturing and observing the cells. After the sample (product of example 1, ceramide 3B) was diluted with the medium (product concentration of example 1 was 100mg/L,ceramide 3B concentration of 100 mg/L) was added to a cell culture dish, and the cells were placed in 5wt% CO at 37℃C 2 Incubator culture, after 24 hours, cells were removed, observed with a microscope and the width of scratches was measured, and photographed, and the healing rate was calculated using Image J software.
The results are shown in fig. 1, and the scratch width of the experimental group is narrower than that of the solvent control group, which indicates that the monkey seed oil ceramide has better tissue healing capacity. The solvent control group had a rate of 21.34% after 24 hours, monkey seed oil ceramide had a rate of 93.28% after 24 hours, and ceramide 3B had a rate of 59.32% after 24 hours. The compound obviously improves the cell healing rate, has good skin tissue repair activity and has better effect than ceramide 3B.
Example 5
Elastase inhibition experiment tests anti-aging effect
Elastase inhibition method: 2mg/mL elastase solution (product of example 1) is taken, samples with different concentrations (2 mL) are added, vortex mixing is carried out fully, shaking is carried out for 20min at 37 ℃ by a 400r/min shaking table, 5mL of 0.5mol/L phosphate buffer solution with pH of 6.0 is added immediately, vortex mixing is carried out, a proper amount of mixed solution is taken into a 2mL centrifuge tube, centrifugation is carried out for 10min at 9 391×g, 200 mu L of supernatant is sucked into a 96-well plate precisely, absorbance is measured by an enzyme-labeled instrument at a wavelength of 495nm, and spectrum scanning at 400-800 nm is carried out simultaneously.
The substrate enzyme adding solution is used as a blank control group, the substrate enzyme adding and sample solution is used as an enzyme inhibition group, and the substrate enzyme adding and sample solution is used as a background. Each group is provided with 3 multiple holes. Inhibition ratio (%) = [1- (An-An ')/(A0-A0') ] ×100%, where A0 is absorbance with no enzyme added to the sample, A0 'is absorbance with no enzyme added to the substrate and no sample added to the enzyme, an is absorbance with only sample solution, an' is absorbance with no enzyme added to the sample. When An ' > An, the effect is expressed as acceleration, and the acceleration rate (%) = [1- (An ' -An)/(A0-A0 ') ] ×100%.
As shown in FIG. 2, the monkey seed oil ceramide has a good inhibitory effect on elastase at various concentrations, specifically, the inhibition rate of elastase at a concentration of 0.25g/L was 9.47%, the inhibition rate of elastase at a concentration of 0.5g/L was 20.33%, the inhibition rate of elastase at a concentration of 1.0g/L was 32.93%, and the inhibition rate of elastase at a concentration of 2.0g/L was 26.67%.
The inhibitory activity of ceramide 2 against elastase was measured in the same manner, and as a result, as shown in FIG. 3, the elastase inhibition rates at concentrations of 0.25, 0.5, 1.0 and 2.0g/L were 10.12%, 18.06%, 28.84% and 19.78%, respectively, which are generally inferior to that of monkey seed oil ceramide at the same concentrations.
Example 6
MMP1 is also called interstitial collagenase and matrix metalloproteinase, belongs to matrix metalloproteinase family, and its main acting substrate is fibrous collagen, which can degrade collagen fiber and gelatin in extracellular matrix and change microenvironment of cells. MMP1 plays an important role in elastin, inhibiting MMP1 can improve the synthesis of fibroblast collagen and elastin, and reducing MMP activity can increase the collagen synthesis speed.
HaCaT cells were grown at 1X 10 5 The density of individuals/wells was seeded in 96-well plates and the incubator was overnight. After 24h, the supernatant was discarded, 100. Mu.L of medium containing samples of different concentrations (product of example 1) was added, no samples were added to the model group, the negative control group was DMEM medium without samples, 3 wells per group, and the mass fraction was 5% CO 2 After incubation for 2h at 37℃either UVA or UVB ultraviolet radiation is irradiated. The distance between the ultraviolet radiation source and the cells was 15cm, and the UVA intensity was 200mJ/cm 2 The irradiation time was 2 hours, and the UVB intensity was 50mJ/cm 2 The irradiation time was 1h. After the end of irradiation, incubation was continued for 12h in the incubator. Intracellular MMP-1 gene expression was detected using an MMP-1ELISA kit. Inhibition = 1- (experimental group MMP1 expression level/model group MMP1 expression level) ×100%.
As shown in fig. 4 and 5, the expression level of MMP1 in the negative control group was 1, the expression level in the model group was 1.90, and the inhibition ratio of MMP1 expression in the model group was 24%, 34%, 54% at concentrations of 125, 250, 400mg/L for monkey seed oil ceramide; the expression level of MMP1 in the negative control group was 1, the expression level in the model group was 2.33, and the inhibition rate of MMP1 expression in the model group was 25%, 48% and 60% at concentrations of 125, 250 and 400mg/L for monkey seed oil ceramide.
After UVA uv radiation, keratinocytes promote elevated expression of MMP1 by fibroblasts, thereby causing degradation of the extracellular matrix of the skin and collagen of the skin, leading to photoaging of the skin. The results show that the monkey seed oil ceramide can inhibit the fibroblast from producing MMP1 caused by ultraviolet radiation, and has a certain effect on preventing skin photoaging.
Example 7
DPPH free radical scavenging detection of antioxidant performance
DPPH is 1, 1-diphenyl-2-trinitrophenylhydrazine, and can be used for antioxidant experiments.
Samples (product of example 1) at corresponding concentrations (50, 100, 200, 400, 800 mg/L) were mixed with 0.1mol/L DPPH, absolute ethanol solution at a ratio of 1:1, and DPPH and absolute ethyl alcohol 1:1, and the absorbance at 517 nm. The absorbance of the sample and the reaction solution of DPPH was designated as A1, the absorbance of the sample and the reaction solution of absolute ethyl alcohol was designated as A2, the absorbance of the reaction solution of DPPH and absolute ethyl alcohol was designated as A3, and the clearance rate of DPPH of the sample was = [1- (A1-A2)/A3 ]. Times.100%.
As a result, as shown in FIG. 6, the DPPH radical scavengers at concentrations of 50, 100, 200, 400 and 800mg/L were 9.43%, 17.38%, 23.10%, 33.93% and 48.37%, respectively, and excellent antioxidant effects were exhibited. The antioxidant effect of ceramide 3B (i.e., oleic acid ceramide) was measured in the same manner, and the DPPH radical scavenging rate at 50, 100, 200, 400, 800mg/L was 7.76%, 12.82%, 24.10%, 29.60%, 33.16%, as shown in FIG. 7. The clearance rate of the monkey seed oil ceramide to DPPH is higher than that of ceramide 3B, and the monkey seed oil ceramide has better antioxidation effect.
Example 8
Whitening Activity test
Taking B16 cells in exponential growth phase, digesting and blowing uniformly by using trypsin-EDTA with mass fraction of 0.25%, and pressing the cells by 3×10 5 Density of individual/well was seeded in 12-well plates. At 37 ℃, the mass fraction of CO is 5 percent 2 Incubated overnight in the environment. Removing supernatant, adding culture solution containing samples with different mass concentrations (product of example 1), incubating with RPMI-1640 culture medium without sample as blank group, incubating with DMEM culture medium as mould group, and incubating with 3 compound holes in each group at mass fraction of 5% CO 2 Incubation was carried out for 24h at 37 ℃. The culture medium in the well plate is discarded, and after washing with Phosphate Buffer (PBS) for one to two times, 1mL of NaOH solution (1 mol/L) containing 10% DMSO by mass fraction is added to lyse the cells, and the cells are kept at a constant temperature of 80℃or 100℃for 2 hours until the cells are completely lysed. The absorbance was measured at 405nm in a microplate reader. The melanin inhibition rate=1- (OD value per well/OD value of model group) ×100% was calculated.
As shown in FIG. 8, the melanin content of the blank group was 1, the melanin expression of the model group was 1.51, and the melanin inhibition rates of the monkey seed oil ceramide were 7.25%, 12.52%, 16.90%, 22.48% and 20.63% at concentrations of 10, 20, 40, 80 and 100mg/L, respectively, and excellent whitening effect was exhibited.
The foregoing is merely illustrative embodiments of the present invention, and the present invention is not limited thereto, and any changes or substitutions that may be easily contemplated by those skilled in the art within the scope of the present invention should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (10)

1. Monkey seed oil ceramide, it is got from monkey seed oil fatty acid and sphingosine compound reaction, the said sphingosine compound is selected from sphingosine, phytosphingosine, dihydrosphingosine.
2. The monkey seed oil ceramide of claim 1, wherein the monkey seed oil fatty acid is hydrolyzed from monkey seed oil.
3. The monkey seed oil ceramide of claim 1 or 2, wherein the monkey seed oil fatty acid comprises 30 to 50wt% oleic acid, 15 to 35wt% linoleic acid, 13 to 30wt% palmitic acid, 2 to 8wt% stearic acid, 0.5 to 5wt% linolenic acid, 0.2 to 2wt% arachidic acid.
4. Monkey seed oil ceramide, its composition includes: oleic acid ceramide, linoleic acid ceramide, palmitic acid ceramide, stearic acid ceramide, linolenic acid ceramide, arachidic acid ceramide.
5. The monkey seed oil ceramide of claim 4, comprising: 30 to 50 weight percent of oleic acid ceramide, 15 to 35 weight percent of linoleic acid ceramide, 13 to 30 weight percent of palmitic acid ceramide, 2 to 8 weight percent of stearic acid ceramide, 0.5 to 5 weight percent of linolenic acid ceramide and 0.2 to 2 weight percent of arachidic acid ceramide.
6. The monkey-bread seed oil ceramide of claim 4 or 5, further comprising: 0 to 1wt% of palmitoleic acid ceramide and 0 to 0.5wt% of myristic acid ceramide.
7. The method for synthesizing monkey-bread seed oil ceramide according to any one of claims 1 to 6, comprising the steps of:
under the conditions of condensing agent and organic alkali, the monkey bread seed oil fatty acid reacts with the sphingosine compound, wherein the condensing agent is EDCI, and the organic alkali is Et 3 N；
The monkey seed oil fatty acid, sphingosine compound, EDCI, et 3 The molar ratio of N is 1: (1-1.5): (1-2): (1-2), wherein the solvent for the reaction is at least one of dichloromethane, tetrahydrofuran, ethyl acetate and acetonitrile.
8. Use of the monkey-bread seed oil ceramide of any one of claims 1 to 6 in cosmetics, pharmaceuticals, dietary foods or health care products.
9. The use according to claim 8, wherein the monkey seed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, antioxidant, promotion of collagen synthesis, maintenance of elastin activity, whitening efficacy.
10. A composition comprising the monkey seed oil ceramide of any one of claims 1-6, the composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, promotion of collagen synthesis, maintenance of elastin viability, whitening efficacy.
CN202310186326.7A 2022-11-25 2023-03-01 Monkey bread seed oil ceramide and synthesis method and application thereof Pending CN117285434A (en)

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PCT/CN2023/133524 WO2024109867A1 (en) 2022-11-25 2023-11-23 Vegetable oil ceramides, synthesis method therefor, and use thereof

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