CN116003286A - Prinsepia utilis royle oil ceramide and synthesis method and application thereof - Google Patents
Prinsepia utilis royle oil ceramide and synthesis method and application thereof Download PDFInfo
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- CN116003286A CN116003286A CN202310051539.9A CN202310051539A CN116003286A CN 116003286 A CN116003286 A CN 116003286A CN 202310051539 A CN202310051539 A CN 202310051539A CN 116003286 A CN116003286 A CN 116003286A
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- China
- Prior art keywords
- ceramide
- prinsepia utilis
- acid
- utilis royle
- royle oil
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- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 title claims abstract description 135
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 title claims abstract description 135
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Abstract
The invention belongs to the technical field of biological medicines, and discloses prinsepia utilis royle oil ceramide which is obtained by reacting prinsepia royle oil fatty acid with a sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. Prinsepia utilis royle 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
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to prinsepia utilis royle 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.
Prinsepia utilis royle oil is vegetable oil extracted from Prinsepia utilis royle, which is the fruit of Prinsepia utilis royle of Rosaceae, and is mainly distributed in mountain areas with altitude of 1800-3200 m in Yunnan and other places in China. The fatty acid composition of Prinsepia utilis royle oil is mainly unsaturated fatty acid-oleic acid and linoleic acid, and also contains palmitic acid and stearic acid, and contains multiple vitamins such as vitamin A, vitamin D, vitamin E, vitamin K, etc. Prinsepia utilis royle oil is rich in monounsaturated fatty acids compared to other vegetable oils. Studies have shown that monounsaturated fatty acids can lower cholesterol in the blood and low density lipoproteins that are detrimental to cardiovascular health, but not lower beneficial high density lipoproteins. The food rich in monounsaturated fatty acid has effects of relieving hypertension and resisting atherosclerosis, and the monounsaturated fatty acid has the characteristic of being not easy to oxidize. Prinsepia utilis royle oil has good safety: no matter the oral administration or the external application, the Chinese medicinal composition has no toxic or side effect on human body and no stimulation on human skin; the skin has strong permeability, has a fatty acid structure which is the most suitable for the human body constitution fat proportion mode, has good skin permeability, can improve the degradation of cutin, and has the effects of moisturizing, preventing cracking and supplementing nutrition to the skin; obvious anti-inflammatory and antibacterial effects; ultraviolet injury resistance; improving circulation and promoting metabolism.
Disclosure of Invention
The invention aims to provide ceramide synthesized by using Prinsepia utilis oil fatty acid of plant origin.
Another object of the present invention is to provide a method for synthesizing prinsepia utilis royle oil ceramide, which uses prinsepia utilis royle oil grease or prinsepia royle oil fatty acid which is a natural plant source and is easily available as a raw material.
It is another object of the present invention to provide the use of prinsepia utilis royle oil ceramides.
In order to achieve one of the above purposes, the present invention adopts the following technical scheme:
in a first aspect of the invention, prinsepia utilis royle oil ceramide is obtained by reacting prinsepia utilis royle oil fatty acids 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 taking Prinsepia utilis royle oil as raw material, selecting proper strain, and fermenting to obtain Prinsepia utilis royle 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 prinsepia utilis royle oil fatty acid is obtained by hydrolyzing prinsepia utilis royle oil grease.
Further, the prinsepia utilis royle oil fatty acid contains 30-50 wt% linoleic acid.
Further, the Prinsepia utilis royle oil fatty acid contains 25-40 wt% oleic acid.
Further, the prinsepia utilis royle oil fatty acid contains 10-25 wt% of palmitic acid.
Further, the prinsepia utilis royle oil fatty acid contains 3-10wt% of stearic acid.
In addition, prinsepia utilis royle oil fatty acid contains 0-1 wt% palmitoleic acid and 0-1 wt% arachidic acid.
The components of the prinsepia utilis royle oil fatty acid are as follows: 30 to 50 weight percent of linoleic acid, 25 to 40 weight percent of oleic acid, 10 to 25 weight percent of palmitic acid, 3 to 10 weight percent of stearic acid, 0 to 1 weight percent of palmitoleic acid and 0 to 1 weight percent of arachidic acid.
The main components of the prinsepia utilis royle oil fatty acid are oleic acid and linoleic acid, other fatty acids comprise palmitic acid and stearic acid, the essential components are affected by plant varieties, soil, climate, production places, picking seasons and extraction processes, the content of each component is different, and the palmitoleic acid and the arachidic acid are not necessarily contained and are optional components or unnecessary components.
Prinsepia utilis royle oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, stearic acid ceramide; because fatty acids all participate in the same reaction, the mass ratio of ceramide after the reaction is not changed greatly, so the composition of the prinsepia utilis royle ceramide is similar to that of prinsepia utilis royle oil acid, and is as follows: 30 to 50 weight percent of linoleic acid ceramide, 25 to 40 weight percent of oleic acid ceramide, 10 to 25 weight percent of palmitic acid ceramide and 3 to 10 weight percent of stearic acid ceramide. The content of each component is different due to the different content of the prinsepia utilis royle oil fatty acid or the fatty acid in the oil. In addition, the prinsepia utilis royle oil ceramide also comprises ceramide obtained by reacting one or more of palmitoleic acid and arachidic acid with sphingoid compounds, namely 0-1 wt% of palmitoleic acid ceramide and 0-1 wt% of arachidic acid ceramide. Prinsepia utilis royle oil ceramide also comprises compounds which exist in Prinsepia utilis royle oil fatty acid but do not react with sphingosine compounds, such as vitamin A, vitamin D, vitamin E, vitamin K and the like.
Prinsepia utilis royle oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide; 30-50 wt% of linoleic acid ceramide, 25-40 wt% of oleic acid ceramide and 10-25 wt% of palmitic acid ceramide.
Further, the prinsepia utilis royle oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 3-10wt%.
Further, the prinsepia utilis royle oil ceramides comprise no more than 1wt% of palmitoleic acid ceramides and no more than 1wt% of arachidic acid ceramides, in particular 0.1 to 1wt% of palmitoleic acid ceramides and 0.1 to 1wt% of arachidic acid ceramides.
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; 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 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 is obtained by condensation reaction of stearic acid and sphingosine compound, and comprises stearic acid phytosphingosine ceramide, stearic acid sphingosine ceramide and stearic acid dihydrosphingosine ceramide; palmitoleic acid ceramide, arachidic acid ceramide, and the like, and so forth.
In a second aspect of the invention, a method for synthesizing prinsepia utilis royle ceramide comprises the following steps:
under the conditions of condensing agent and organic alkali, the prinsepia utilis royle oil acid reacts with the sphingosine compound, the condensing agent is EDCI, and the organic alkali is DMAP.
Further, the molar ratio of the prinsepia utilis royle oil fatty acid to the sphingosine compound to the EDCI to the DMAP 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.
Prinsepia utilis royle oil purchased on the market is generally in the form of oil and fat, and needs to be hydrolysed to prinsepia utilis royle oil acid, and therefore comprises the following steps:
the prinsepia utilis royle oil is hydrolyzed by saponification reaction to obtain prinsepia utilis royle oil acid.
Further, the saponification reaction is hydrolysis of prinsepia utilis royle oil grease in potassium hydroxide solution.
Further, the mass ratio of the prinsepia utilis royle oil to the potassium hydroxide is 1: (1-2).
In a third aspect of the invention, the use of prinsepia utilis royle oil ceramide in cosmetics, pharmaceutical products, dietary or health care products.
Further, the prinsepia utilis royle oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, collagen synthesis promotion, elastin activity maintenance, and whitening effects.
A composition comprising prinsepia utilis royle oil ceramide, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidant, promotion of collagen synthesis, maintenance of elastin viability, 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:
prinsepia utilis royle oil fatty acid belongs to naturally-formed fatty acid, the main components are oleic acid and linoleic acid, in addition, palmitic acid and stearic acid are contained, and the Prinsepia utilis royle 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 prinsepia utilis royle oil 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 prinsepia utilis royle 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), prinsepia utilis royle oil also contains various fat-soluble vitamins and microelements which are easy to be absorbed by human body and a large number of amino acids which are necessary for human body, and these active substances have the effects of keeping skin nutrition, moisture and elasticity, improving degradation of skin horny layer, repairing rough skin and the like. The ceramide synthesized by the prinsepia utilis royle oil has a synergistic effect with other active ingredients contained in the prinsepia utilis royle 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 plant-derived prinsepia utilis royle oil or the fatty acid thereof has the advantages of wide sources, easy commercial acquisition, lower cost, environmental protection and economy, and the method is different from the idea of mixing and compounding different single ceramides, and the fatty acid with a single component has high raw material price, and needs to produce different ceramides respectively and then compound, 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;
FIG. 2 is a bar graph of elastase inhibition of example 5;
FIGS. 3 and 4 are bar charts of MMP1 expression levels in the photo-aging test of example 6;
FIG. 5 is a bar graph of DPPH radical scavenging for oxidation resistance test of example 7;
FIG. 6 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 and DMAP refers to N, N-diisopropylethylamine. 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
Prinsepia utilis royle oil fatty acid and phytosphingosine to synthesize ceramide
The first step: 50g of prinsepia utilis royle oil and fat is dissolved in 80mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is cooled to room temperature for reaction after the dripping is finished until TLC detection is finished.
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 100mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Drying, filtering and concentrating in vacuum to obtain 40.6g of Prinsepia utilis oil fatty acid.
And a second step of: prinsepia utilis oil fatty acid (50 mmol, calculated as fatty acid as main ingredient), EDCI (55 mmol) and DMAP (55 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and phytosphingosine (55 mmol) was added to the reaction system, followed by 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 prinsepia utilis royle 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: linoleic acid-phytosphingosine ceramide 9.5min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.3min, stearic acid-phytosphingosine ceramide 13.9min, arachidic acid-phytosphingosine ceramide 16.5min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, stearic acid-phytosphingosine ceramide and arachidic acid-phytosphingosine ceramide are 39%, 36%, 12%, 9%, 1% in sequence, and the rest are other components, and the content is less.
Example 2
Prinsepia utilis royle oil fatty acid and sphingosine synthesis of ceramide
The first step: 50g of prinsepia utilis royle oil and fat is dissolved in 80mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is cooled to room temperature for reaction after the dripping is finished until TLC detection is finished.
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 100mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Drying, filtering and concentrating in vacuum to obtain 40.4g of Prinsepia utilis oil fatty acid.
And a second step of: prinsepia utilis oil fatty acid (50 mmol, calculated as fatty acid as main ingredient), EDCI (65 mmol) and DMAP (65 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and then sphingosine (65 mmol) was added to the reaction system, followed by 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 prinsepia utilis royle 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-sphingosine ceramide 8.2min, linoleic acid-sphingosine ceramide 8.7min, oleic acid-sphingosine ceramide 10.2min, palmitic acid-sphingosine ceramide 10.5min, stearic acid-sphingosine ceramide 13.6min, arachidic acid-sphingosine ceramide 17.8min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-sphingosine ceramide, oleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, stearic acid-sphingosine ceramide, palmitoleic acid-sphingosine ceramide and arachidic acid-sphingosine ceramide are 34%, 32%, 22%, 6%, 1%, 0.5% in sequence, and the balance is other components, and the contents are less.
Example 3
Prinsepia utilis royle oil fatty acid and sphinganine for synthesizing ceramide
The first step: 50g of prinsepia utilis royle oil and fat is dissolved in 80mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the mixture is cooled to room temperature for reaction after the dripping is finished until TLC detection is finished.
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 100mL of saturated saline water for washing once, and adding anhydrous Na into an organic phase 2 SO 4 Drying, filtering and concentrating under vacuum to obtain 40.5g Prinsepia utilis oil fatty acid.
And a second step of: prinsepia utilis oil fatty acid (50 mmol, calculated as fatty acid as main ingredient), EDCI (75 mmol), DMAP (75 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, and then sphinganine (75 mmol) was added to the reaction system, 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 prinsepia utilis royle 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-sphinganine ceramide 8.9min, linoleic acid-sphinganine ceramide 9.4min, palmitic acid-sphinganine ceramide 10.6min, oleic acid-sphinganine ceramide 11.1min, stearic acid-sphinganine ceramide 13.5min.
The obtained products are analyzed by high performance liquid chromatography, the contents of linoleic acid-dihydrosphingosine ceramide, oleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, stearic acid-dihydrosphingosine ceramide and palmitoleic acid-dihydrosphingosine ceramide are 48%, 27%, 16%, 4%, 0.5% in sequence, and the balance is other components, and the contents are 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 of example 1 at 100mg/L, ceramide 3B at 100 mg/L), the cells were placed in a cell culture dish at 37℃and 5wt% CO 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 prinsepia utilis royle ceramide has better tissue healing capacity. The healing rate of the solvent control group after 24 hours is 39.12%, the healing rate of the prinsepia utilis royle ceramide after 24 hours is 92.38%, and the healing rate of the ceramide 3B after 24 hours is 59.32%. 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 results show that the Prinsepia utilis oil ceramide has excellent inhibitory effect on elastase at various concentrations, specifically, the inhibition rate of elastase at a concentration of 0.25g/L is 9.84%, the inhibition rate of elastase at a concentration of 0.5g/L is 17.26%, the inhibition rate of elastase at a concentration of 1.0g/L is 28.00%, and the inhibition rate of elastase at a concentration of 2.0g/L is 27.33%.
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. 3 and 4, 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 13%, 24%, 43% at the concentrations of 125, 250, 400mg/L of prinsepia utilis ceramide; for UVB, the MMP1 expression level of the negative control group was 1, the expression level of the model group was 2.33, and the inhibition rate of MMP1 expression was 28%, 46% and 58% relative to the model group at concentrations of 125, 250 and 400 mg/L.
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 prinsepia utilis royle 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. 5, the DPPH radical scavengers at concentrations of 50, 100, 200, 400 and 800mg/L were 9.43%, 19.26%, 21.77%, 30.60% and 32.24%, respectively, and excellent antioxidant effects were exhibited.
Example 8
Whitening Activity test
Taking B16 cells in exponential growth phase, digesting with trypsin-EDTA with mass fraction of 0.25%, blowing uniformly, and mixing the cells according to 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. The supernatant was discarded and cultures containing samples of different mass concentrations (product of example 1) were addedIncubating the solution with RPMI-1640 culture medium without sample as blank group, adding DMEM culture medium as mould group, and incubating each group with 3 multiple holes 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 a result, as shown in FIG. 6, 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 prinsepia utilis royle ceramide were 8.14%, 12.96%, 16.90%, 22.48% and 21.52% at concentrations of 10, 20, 40, 80 and 100mg/L, respectively, and excellent whitening effects were 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. Prinsepia utilis royle oil ceramide is obtained by reacting Prinsepia utilis royle oil fatty acid with sphingosine compound selected from sphingosine, phytosphingosine and dihydrosphingosine.
2. The prinsepia utilis royle oil ceramide of claim 1, wherein the prinsepia utilis royle oil fatty acid is obtained by hydrolysis of prinsepia utilis royle oil.
3. The prinsepia utilis royle oil ceramide of claim 1 or 2, wherein the prinsepia utilis royle oil ceramide comprises 30-50 wt% linoleic acid, 25-40 wt% oleic acid, 10-25 wt% palmitic acid, 3-10 wt% stearic acid.
4. Prinsepia utilis royle oil ceramide, its composition includes: linoleic acid ceramide, oleic acid ceramide, palmitic acid ceramide, stearic acid ceramide.
5. The prinsepia utilis royle oil ceramide of claim 4, which comprises: 30 to 50 weight percent of linoleic acid ceramide, 25 to 40 weight percent of oleic acid ceramide, 10 to 25 weight percent of palmitic acid ceramide and 3 to 10 weight percent of stearic acid ceramide.
6. The prinsepia utilis royle oil ceramide of claim 4 or 5, further comprising: 0 to 1 weight percent of palmitoleic acid ceramide and 0 to 1 weight percent of arachidic acid ceramide.
7. The synthetic method of prinsepia utilis royle oil ceramide according to any one of claims 1 to 6, comprising the steps of:
under the conditions of a condensing agent and organic alkali, the prinsepia utilis royle oil acid reacts with a sphingosine compound, wherein the condensing agent is EDCI, and the organic alkali is DMAP;
the molar ratio of the prinsepia utilis royle oil fatty acid to the sphingosine compound to the EDCI to the DMAP 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 a prinsepia utilis royle oil ceramide according to any one of claims 1 to 6 in cosmetics, pharmaceutical products, dietary products or health products.
9. The use according to claim 8, wherein the prinsepia utilis royle ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidation, promotion of collagen synthesis, maintenance of elastin activity, whitening efficacy.
10. A composition comprising the prinsepia utilis royle oil ceramide of any one of claims 1 to 6, the composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-photoaging, anti-oxidant, promotion of collagen synthesis, maintenance of elastin activity, whitening efficacy.
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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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| WO2024109867A1 (en) * | 2022-11-25 | 2024-05-30 | 深圳市迪克曼生物科技有限公司 | Vegetable oil ceramides, synthesis method therefor, and use thereof |
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