CN116003289A - Watermelon seed oil ceramide and synthesis method and application thereof - Google Patents
Watermelon seed oil ceramide and synthesis method and application thereof Download PDFInfo
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- CN116003289A CN116003289A CN202310051767.6A CN202310051767A CN116003289A CN 116003289 A CN116003289 A CN 116003289A CN 202310051767 A CN202310051767 A CN 202310051767A CN 116003289 A CN116003289 A CN 116003289A
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- CN
- China
- Prior art keywords
- ceramide
- seed oil
- watermelon seed
- acid
- fatty acid
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- 229940106189 ceramide Drugs 0.000 title claims abstract description 138
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 title claims abstract description 128
- 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 128
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 title claims abstract description 128
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Abstract
The invention belongs to the technical field of biological medicines, and discloses watermelon seed oil ceramide which is obtained by reacting watermelon seed oil fatty acid with a sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine and dihydrosphingosine. The watermelon seed oil ceramide has excellent performances in the aspects of repairing natural skin barriers, anti-inflammatory, 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 watermelon 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 watermelon seed oil is prepared by cold pressing watermelon seeds, is light yellow liquid, and contains various fatty acids required by human body, including linoleic acid, oleic acid, palmitic acid, stearic acid and the like. Wherein the linoleic acid not only can soften cutin, but also has effects of promoting hair growth and providing nutrition for hair, and can maintain skin barrier function in hot and severe environment. The watermelon seed oil is light, fresh and not greasy, has extremely strong penetrability in skin care, can be absorbed rapidly, is an excellent humectant for skin, is particularly suitable for baby care, and is good baby skin softening oil. Unsaturated fatty acid in watermelon seed oil is helpful for reconstructing natural barrier of skin, deeply nourishing and delaying damage of free radical to cells, thereby preventing skin aging. In addition, the watermelon seed oil is rich in vitamins, trace elements and the like, can remove free radicals, and has the effects of resisting oxidation, resisting aging and the like.
Disclosure of Invention
The invention aims to provide ceramide synthesized by utilizing watermelon seed oil fatty acid of plant origin.
Another object of the present invention is to provide a method for synthesizing watermelon seed oil ceramide, which uses natural plant-derived and readily available watermelon seed oil fat or watermelon seed oil fatty acid as a raw material.
Another object of the invention is to provide the use of watermelon 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, a watermelon seed oil ceramide is obtained by reacting a watermelon 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 watermelon seed oil is used as a raw material, and proper bacterial strain is selected for fermentation to obtain the watermelon 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 watermelon seed oil fatty acid is obtained by hydrolyzing watermelon seed oil grease.
Further, the watermelon seed oil fatty acid contains 60-80 wt% linoleic acid.
Further, the watermelon seed oil fatty acid contains 3-15 wt% of palmitic acid.
Further, the watermelon seed oil fatty acid contains 10-30wt% oleic acid.
Further, the watermelon seed oil fatty acid contains 1-5 wt% stearic acid.
The watermelon seed oil fatty acid comprises the following components: 60-80 wt% of linoleic acid, 3-15 wt% of palmitic acid, 10-30 wt% of oleic acid and 1-5 wt% of stearic acid.
The main component of the watermelon seed oil fatty acid is linoleic acid, other fatty acids comprise palmitic acid, oleic acid and stearic acid, are essential components, are influenced by plant varieties, soil, climate, production places, picking seasons and extraction processes, and have different contents.
Watermelon seed oil ceramide, its composition includes: linoleic acid ceramide, palmitic acid ceramide, oleic acid ceramide, stearic acid ceramide; because the fatty acid participates in the same reaction, the mass ratio of the ceramide after the reaction is not changed greatly, so the composition of the ceramide is similar to that of the watermelon seed oil fatty acid, and the composition of the ceramide of the watermelon seed oil is as follows: 60 to 80 weight percent of linoleic acid ceramide, 3 to 15 weight percent of palmitic acid ceramide, 10 to 30 weight percent of oleic acid ceramide and 1 to 5 weight percent of stearic acid ceramide. The content of each component is different due to the different content of each fatty acid in the watermelon seed oil fatty acid or grease.
Watermelon seed oil ceramide, its composition includes: linoleic acid ceramide, palmitic acid ceramide, oleic acid ceramide; the linoleic acid ceramide accounts for 60 to 80 weight percent, the palmitic acid ceramide accounts for 3 to 15 weight percent, and the oleic acid ceramide accounts for 10 to 30 weight percent.
Further, the watermelon seed oil ceramide comprises stearic acid ceramide, and the stearic acid ceramide accounts for 1-5 wt%.
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; 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; 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.
In a second aspect of the invention, a method for synthesizing watermelon seed oil ceramide comprises the following steps:
under the conditions of condensing agent and organic alkali, the watermelon seed oil fatty acid reacts with a sphingosine compound, the condensing agent is EDCI, and the organic alkali is NMM.
Further, the mol ratio of the watermelon seed oil fatty acid to the sphingosine compound to the EDCI to the NMM 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 watermelon seed oil purchased in the market is generally in the form of oil and needs to be hydrolyzed into watermelon seed oil fatty acid, and therefore the method further comprises the following steps:
the watermelon seed oil fat is hydrolyzed by saponification reaction to obtain the watermelon seed oil fatty acid.
Further, the saponification reaction is hydrolysis of watermelon seed oil grease in potassium hydroxide solution.
Further, the mass ratio of the watermelon seed oil grease to the potassium hydroxide is 1: (1-2).
In a third aspect of the invention, the use of watermelon seed oil ceramide in cosmetics, pharmaceutical products, dietary or health care products.
Further, the watermelon seed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, collagen synthesis promoting, elastin activity maintaining, and whitening effects.
A composition comprising watermelon seed oil ceramide, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, anti-oxidant, 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 watermelon seed oil fatty acid belongs to naturally-formed fatty acid, the main component is unsaturated fatty acid-linoleic acid, and in addition, oleic acid and saturated fatty acid such as palmitic acid and stearic acid are contained, and the watermelon seed oil ceramide is prepared by mild reaction with a sphingosine compound naturally existing in skin, has excellent performance in the aspects of repairing a natural skin barrier, resisting oxidation, resisting aging and the like, and has wide application prospect 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 watermelon 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 watermelon 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 watermelon seed oil is rich in vitamins, trace elements, proteins and the like, and the active ingredients can remove free radicals and have the effects of resisting oxidation, resisting aging and the like. The ceramide synthesized by the watermelon seed oil has a synergistic effect with other active ingredients contained in the watermelon 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 a plurality of ceramide compound compositions, the plant source watermelon seed oil grease or the fatty acid source thereof has wide sources, is easy to obtain commercially, has lower cost, is more environment-friendly and economical, is different from the idea of mixing and compounding different single ceramides, 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
FIGS. 1 and 2 are bar graphs showing the results of the cell proliferation activity test of example 4;
FIG. 3 is the result of the cell migration ability test of example 5;
FIGS. 4 and 5 are bar graphs of elastase inhibition ratios of example 6;
FIG. 6 is a bar graph showing the detection of IL-6 factor expression level in anti-inflammatory repair efficacy in example 7;
FIGS. 7 and 8 are bar charts of MMP1 expression levels in the photo-aging test of example 8;
FIGS. 9 and 10 are bar graphs of DPPH radical scavenging for oxidation resistance test of example 9;
FIG. 11 is a bar graph showing the whitening activity test melanin content of example 10.
Detailed Description
The invention will be further illustrated with reference to specific examples.
EDCI refers to 1-ethyl- (3-dimethylaminopropyl) carbodiimide and NMM refers to N-methylmorpholine. 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 watermelon seed oil fatty acid and phytosphingosine
The first step: 50g of watermelon seed oil is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the reaction is carried out after the dropwise addition is completed, and the temperature is raised to room temperature until the TLC detection reaction is completed.
Post-treatment: adding dilute hydrochloric acid (3N) to regulate the reaction systemThe pH value is 3, 150mL of ethyl acetate is added to extract the water phase, 100mL of saturated saline is added to wash once, and the organic phase is added with anhydrous Na 2 SO 4 Drying, filtering and concentrating in vacuum to obtain 40.2g of watermelon seed oil fatty acid.
And a second step of: watermelon seed oil fatty acid (50 mmol, calculated as main component fatty acid), EDCI (60 mmol), NMM (60 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, then phytosphingosine (60 mmol) was added to the reaction system, and stirring at room temperature was carried out until TLC detection was completed.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain the watermelon 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: linoleic acid-phytosphingosine ceramide 9.6min, palmitic acid-phytosphingosine ceramide 10.7min, oleic acid-phytosphingosine ceramide 11.4min, stearic acid-phytosphingosine ceramide 13.9min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-phytosphingosine ceramide, palmitic acid-phytosphingosine ceramide, oleic acid-phytosphingosine ceramide and stearic acid-phytosphingosine ceramide are 63%, 12%, 18%, 4% in sequence, and the rest is other components, and the content is low.
Example 2
Synthesis of ceramide from fatty acid and sphingosine of watermelon seed oil
The first step: 50g of watermelon seed oil is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the reaction is carried out after the dropwise addition is completed, and the temperature is raised to room temperature until the TLC detection reaction is completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, and adding 150mL of ethyl acetateThe aqueous phase was extracted, 100mL of saturated brine was added and washed once, and the organic phase was added anhydrous Na 2 SO 4 Drying, filtering and concentrating in vacuo gave 39.8g of watermelon seed oil fatty acid.
And a second step of: watermelon seed oil fatty acid (50 mmol, based on main component fatty acid), EDCI (70 mmol), NMM (70 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, then sphingosine (50 mmol) was added to the reaction system, and stirring at room temperature was carried out until TLC detection was completed.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain the watermelon 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: linoleic acid-sphingosine ceramide 8.7min, oleic acid-sphingosine ceramide 10.1min, palmitic acid-sphingosine ceramide 10.4min, stearic acid-sphingosine ceramide 13.5min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-sphingosine ceramide, palmitic acid-sphingosine ceramide, oleic acid-sphingosine ceramide and stearic acid-sphingosine ceramide are 76%, 5%, 13% and 2% in sequence, and the rest is other components, so that the content is low.
Example 3
Synthesis of ceramide from watermelon seed oil fatty acid and sphinganine
The first step: 50g of watermelon seed oil is dissolved in 60mL of tetrahydrofuran, cooled in an ice bath, 100mL of potassium hydroxide (25 wt%) solution is added dropwise, and the reaction is carried out after the dropwise addition is completed, and the temperature is raised to room temperature until the TLC detection reaction is completed.
Post-treatment: adding dilute hydrochloric acid (3N) to adjust the pH value of the reaction system to 3, adding 150mL 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 watermelon seed oil fatty acid.
And a second step of: watermelon seed oil fatty acid (50 mmol, calculated as main component fatty acid), EDCI (65 mmol), NMM (65 mmol) were added to a 250mL round bottom flask, 100mL dichloromethane was added, followed by stirring at room temperature for 1 hour, then sphinganine (60 mmol) was added to the reaction system, and stirring at room temperature was carried out until TLC detection was completed.
Post-treatment: adding water for quenching reaction, separating an organic layer, drying, filtering and concentrating in vacuum, washing by a solvent to obtain the watermelon 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: linoleic acid-dihydrosphingosine ceramide 9.5min, palmitic acid-dihydrosphingosine ceramide 10.5min, oleic acid-dihydrosphingosine ceramide 11.1min, stearic acid-dihydrosphingosine ceramide 13.5min.
The obtained product is analyzed by high performance liquid chromatography, the contents of linoleic acid-dihydrosphingosine ceramide, palmitic acid-dihydrosphingosine ceramide, oleic acid-dihydrosphingosine ceramide and stearic acid-dihydrosphingosine ceramide are 66%, 8%, 23%, 1% and the rest are other components, and the content is low.
Example 4
MTT method for detecting proliferation activity of compound on cell
HaCaT cells were grown at 1X 10 4 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, incubation was continued for 24h, medium was removed, 100. Mu.L of thiazole blue (MTT) was added to each well, absorbance at 450nm was measured, and cell viability = A was calculated Drug delivery hole /A Blank hole ×100％。
As shown in FIG. 1, the watermelon seed oil ceramide has a promoting effect on cell viability, and the cell viability rates at the concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000mg/L are 113.93%, 120.33%, 113.79%, 100.42%, 94.15%, 89.97% and 69.22%, respectively. The effective concentration is as low as 8mg/L, the safe concentration is 62.5mg/L, the concentration gradient is relatively stable, the obvious effect of promoting cell proliferation is shown, and the tissue repair capability is good.
The proliferation activity of ceramide 2 on cells was measured in the same manner, and as a result, as shown in FIG. 2, cell viability was 61.49%, 60.03%, 55.41%, 54.64%, 53.37%, 46.95%, 44.05%, 40.35%, 39.42% at concentrations of 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000mg/L, respectively, which had an inhibitory effect on cell proliferation and a tissue repair potential inferior to that of watermelon seed oil ceramide.
Example 5
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, concentration of ceramide 3B was 20mg/L, concentration of ceramide 3B was 100 mg/L), the cells were placed in 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. 3, and the scratch width of the experimental group is narrower than that of the solvent control group, which indicates that the watermelon seed oil ceramide has better tissue healing capacity. The solvent control group had a healing rate of 35.21% after 24 hours, the watermelon seed oil ceramide had a healing rate of 88.53% after 24 hours, and ceramide 3B had a healing 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 6
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. 4, the watermelon seed oil ceramide has a good effect of inhibiting elastase at various concentrations, specifically, the inhibition rate of elastase at a concentration of 0.25g/L is 11.47%, the inhibition rate of elastase at a concentration of 0.5g/L is 23.93%, the inhibition rate of elastase at a concentration of 1.0g/L is 35.00%, and the inhibition rate of elastase at a concentration of 2.0g/L is 48.00%.
The inhibitory activity of ceramide 2 against elastase was measured in the same manner, and as a result, as shown in FIG. 5, 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 were not as good as those of the same concentration of watermelon seed oil ceramide.
Example 7
LPS induced cell method for detecting anti-inflammatory repair efficacy
B16 mouse melanoma cells were grown at a density of 1X 10 4 The cells/wells were seeded in 96-well plates, placed in an incubator overnight, the supernatant was discarded after 24 hours, 100. Mu.L of samples of different concentrations diluted with DMEM medium (product of example 1) were added, the negative control group was DMEM medium without samples, 3 wells per group, and mixed with CO at 5 wt.% 2 Incubate at 37 ℃. Lipopolysaccharide model group and experimental group were added with 10 μg/mL LPS and incubated together for 24h 2h after dosing. After the reaction, 50. Mu.L of the cell supernatant was collected, and the intracellular IL-6 gene expression was detected using an IL-6ELISA kit.
The results are shown in FIG. 6, where IL-6 levels were 10.97 times the basal levels at a working concentration of 10. Mu.g/mL of LPS stimulation. Under the action of the watermelon seed oil ceramide with the concentration of 50mg/L, 100mg/L, 200mg/L and 400mg/L respectively, the IL-6 factor level is obviously reduced and is 0.85, 0.66, 0.61 and 0.45 times of that of an LPS model group respectively, and the watermelon seed oil ceramide is dose-dependent, so that the watermelon seed oil ceramide has good anti-inflammatory effect and can promote the repair of inflammatory damaged skin.
Example 8
Photo aging resistance test
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. 7 and 8, 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 34%, 44%, 64% at the concentrations of 125, 250, 400mg/L for watermelon seed oil 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 the watermelon seed oil ceramide at 125, 250 and 400mg/L was 36%, 45% and 59% relative to the MMP1 expression level of the model group.
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 watermelon seed oil ceramide can inhibit the fibroblast from producing MMP1 caused by ultraviolet radiation, and has a certain effect of preventing skin photoaging.
Example 9
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. 9, the DPPH radical scavengers at concentrations of 50, 100, 200, 400 and 800mg/L were 24.09%, 32.67%, 39.00%, 44.60% and 48.13%, 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 result was shown in FIG. 10, in which DPPH radical scavenging rate was 7.76%, 12.82%, 24.10%, 29.60% and 33.16% at concentrations of 50, 100, 200, 400 and 800 mg/L. The clearance rate of the ceramide of the watermelon seed oil to DPPH is higher than that of ceramide 3B, and the watermelon seed oil has better antioxidation effect.
Example 10
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. 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 ℃. Discarding culture medium in the well plate, washing with Phosphate Buffer (PBS) for one to two times, adding 1mL NaOH solution (1 mol/L) containing 10% DMSO by mass fraction, lysing cells, and standing at 80deg.C or 100deg.C for 2 hr until 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. 11, the melanin content of the blank group was 1, the melanin expression of the model group was 1.54, and the melanin inhibition rates of the watermelon seed oil ceramide were 17.48%, 21.12%, 21.08%, 27.84%, and 29.71% 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 (9)
1. Watermelon seed oil ceramide is prepared by reacting watermelon seed oil fatty acid with sphingosine compound, wherein the sphingosine compound is selected from sphingosine, phytosphingosine and dihydrosphingosine.
2. The watermelon seed oil ceramide of claim 1, wherein the watermelon seed oil fatty acid is obtained by hydrolysis of watermelon seed oil.
3. The watermelon seed oil ceramide of claim 1 or 2, wherein the watermelon seed oil fatty acid comprises 60-80 wt% linoleic acid, 3-15 wt% palmitic acid, 10-30 wt% oleic acid, 1-5 wt% stearic acid.
4. Watermelon seed oil ceramide, its composition includes: linoleic acid ceramide, palmitic acid ceramide, oleic acid ceramide, stearic acid ceramide.
5. The watermelon seed oil ceramide of claim 4, comprising the composition of: 60 to 80 weight percent of linoleic acid ceramide, 3 to 15 weight percent of palmitic acid ceramide, 10 to 30 weight percent of oleic acid ceramide and 1 to 5 weight percent of stearic acid ceramide.
6. The method for synthesizing the watermelon seed oil ceramide according to any one of claims 1-5, comprising the following steps:
under the conditions of condensing agent and organic alkali, the watermelon seed oil fatty acid reacts with a sphingosine compound, wherein the condensing agent is EDCI, and the organic alkali is NMM;
the mol ratio of the watermelon seed oil fatty acid to the sphingosine compound to the EDCI to the NMM 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.
7. Use of the watermelon seed oil ceramide of any one of claims 1-5 in cosmetics, pharmaceuticals, dietary or health products.
8. The use according to claim 7, wherein the watermelon seed oil ceramide has at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, promoting collagen synthesis, maintaining elastin viability, whitening efficacy.
9. A composition comprising the watermelon seed oil ceramide of any one of claims 1-5, said composition having at least one of skin barrier repair, tissue healing, anti-aging, anti-inflammatory, anti-photoaging, antioxidant, promoting collagen synthesis, maintaining elastin viability, whitening efficacy.
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| CN202310051767.6A CN116003289A (en) | 2023-02-02 | 2023-02-02 | Watermelon seed oil ceramide and synthesis method and application thereof |
| 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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| 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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