Skin care composition of olive origin and use thereof

Abstract

This invention provides a skin care composition derived from olive oil and its application. It combines olive leaf extract with ceramide NP derived from olive oil to maintain intracellular calcium ion homeostasis, reduce inflammatory response, and reduce UV damage to the skin. Cosmetics using this composition can maintain the stability of the skin barrier, solve a series of skin problems, and have a high degree of effectiveness and significant application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to a skincare composition of Olea europaea origin. BACKGROUND

[0002] Olea europaea L. is a evergreen tree of Oleaceae, which is originally from the Mediterranean coast. It is a famous woody oil tree species in the world, and its cultivation history can be traced back to 4,000 years ago. Olive oil is extracted from the fruit of Olea europaea L. by cold pressing process. The main component is glycerol triester containing various unsaturated fatty acids. In addition, it also contains various active substances such as olive polyphenol, squalene, carotene, vitamin E, etc., which has special nutritional value and is known as "liquid gold". In addition to the application in diet, olive oil is also widely used in the fields of medicine and cosmetics. Research shows that olive oil has certain anti-cancer, cardiovascular system improvement, anti-aging, wound healing promotion and other effects.

[0003] In addition to fruit oil, the leaves of Olea europaea also contain rich active ingredients, including oleuropein, hydroxytyrosol, flavonoids and polyphenols, etc. Research shows that the extract of Olea europaea leaves has effects including antioxidant, anti-inflammatory, antibacterial, antiviral, etc., and has wide application prospects in the fields of medicine, health food and cosmetics.

[0004] Human stratum corneum (SC) is a "brick wall structure" formed by the stacking of dead corneocytes transformed from keratinocytes, filled with lipids and natural moisturizing factor between cells, which forms the skin barrier. The main components of lipids are ceramides, cholesterol and free fatty acids. The structure of ceramides consists of two parts, i.e. long-chain fatty acids connected to the sphingosine backbone through an amide bond. The highest content of ceramides in human stratum corneum is NP, N represents non-hydroxy fatty acid, and P represents phytosphingosine. In addition, there is a relatively wide distribution of fatty acid carbon chains in human stratum corneum ceramides, which can form a variety of lamellar structure crystalline domains in the stratum corneum, which is conducive to maintaining the function of the skin barrier. The ceramides derived from plant oils also have a relatively wide distribution of fatty acid carbon chains, and have better compatibility with human stratum corneum lipids (Oh, M.J. et al. Novel phytoceramides containing fatty acids of diverse chain lengths are better than a single C18-ceramide N-stearoyl phytosphingosine to improve the physiological properties of human stratum corneum. Clin Cosmet Investig Dermatol. 2017; 10: 363-371). Supplementing stratum corneum lipids helps to improve the structure of stratum corneum, increase the content of lipids in stratum corneum, repair and enhance the skin barrier, and reduce the transdermal water loss of the skin. SUMMARY

[0005] In order to comprehensively utilize various active substances derived from Olea europaea, it is unexpectedly found that the combination of two active substances derived from Olea europaea: olive leaf extract and ceramide NP derived from olive oil can maintain the stability of the skin barrier and has certain repair effect on ultraviolet-induced skin damage.

[0006] Olive leaf extract has a relatively wide application in cosmetics. For example, patent CN111789799A discloses the anti-inflammatory and soothing effects of olive leaf extract and its composition. Patent CN114469820A discloses that olive leaf extract can achieve whitening effect by inhibiting the activity of tyrosinase. Ceramide NP is also widely used in cosmetics for repairing skin barrier. For example, patent CN115006324A discloses the effect of a nourishing and repairing cream containing ceramide NP on repairing skin barrier. Patent CN112402282A discloses a ceramide composition which can reduce the penetration of irritants, increase the water content of stratum corneum and the water retention capacity of skin. The olive oil-derived ceramide has good soothing and repairing effects, but there is no related patent application. Both substances have multiple excellent effects on the skin, but there is no patent protecting the combination of the two.

[0007] In the prior art, the effects of olive oil-derived ceramide and olive leaf extract are studied respectively, and the combination of the two is not studied. Moreover, the soothing and repairing effects of olive oil-derived ceramide are not specifically described.

[0008] To solve the above problems, the present application provides an olive-derived skin care composition, which contains olive leaf extract and olive oil-derived ceramide NP.

[0009] Further, the content of oleuropein in the olive leaf extract is greater than 0.1%.

[0010] Further, the olive leaf extract in the composition can be extracted by extraction, ultrasonic-assisted extraction, microwave-assisted extraction, supercritical carbon dioxide extraction and the like, and the extraction method is not particularly limited. The extract can be a solution or a solid, and the form is not particularly limited. The content of oleuropein in the olive leaf used for extraction is greater than 0.1%, and the olive leaf is stored in a cool and ventilated place to avoid degradation of active substances.

[0011] Preferably, the olive leaf extract is purified by macroporous resin adsorption.

[0012] Further, the ceramide is olive oil-derived ceramide NP, and the structure is shown in formula (1). In formula (1), R represents a saturated or unsaturated alkyl group with 11-30 carbon atoms.

[0013]

[0014] Specifically, the fatty acid source of the olive oil-derived ceramide NP is olive oil.

[0015] Specifically, the olive oil-derived ceramide NP refers to a final product obtained by subjecting a phytosphingosine obtained by fermentation to an amine-ester exchange reaction with triglyceride in olive oil.

[0016] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0017] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0018] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0019] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0020] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0021] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0022] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0023] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0024] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0025] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0026] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0027] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0028] Preferably, the fatty acid donor olive oil in the olive oil-derived ceramide NP and the olive leaf come from the same origin.

[0029] The significant advantages of the present application are as follows:

[0030] 1. The present application provides an olive-derived skin care composition, comprising olive leaf extract and olive oil-derived ceramide NP.

[0031] 2. The present application provides an olive-derived skin care composition, the composition comprising olive leaf extract and olive oil-derived ceramide NP, which can maintain intracellular calcium homeostasis, reduce inflammation, reduce cell apoptosis, and thus reduce UV-induced skin damage and maintain skin barrier stability by promoting the expression of barrier genes.

[0032] 3. The present application provides an olive-derived skin care composition, the composition comprising olive leaf extract and olive oil-derived ceramide NP, which can be used in various skin care products, including but not limited to milk, cream, water, and essence. The skin care products applied with the composition have the effect of maintaining skin barrier and repairing UV-induced skin damage. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the description of the embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0034] Figure 1 HPLC spectrum of olive oil-derived ceramide NP from Gansu Longnan

[0035] Figure 2 Effect of olive oil-derived ceramide NP on the morphological changes of epidermal model tissues caused by SLS stimulation

[0036] Figure 3 Effect of olive oil-derived ceramide NP on the changes in filaggrin FLG content of epidermal model caused by SLS stimulation

[0037] Figure 4 Effect of olive oil-derived ceramide NP on the changes in transglutaminase TGM1 content of epidermal model caused by SLS stimulation

[0038] Figure 5 Structural formula of olive oil-derived ceramide NP

[0039] Figure 6 GO enrichment analysis of up-regulated genes in epidermal model after application of the composition compared with negative control

[0040] Figure 7 GO enrichment analysis of down-regulated genes in epidermal model after application of the composition compared with negative control

[0041] In the following examples, the commercially available ceramide NP was purchased from Wincat Speciality Chemicals Co. Ltd. and its characteristic is that the fatty acid part is stearic acid; the olive oil was from Longnan, Gansu, and was purchased from Gansu Longnan Xiangyu Olive Development Co. Ltd.; the fermented plant sphingosine was purchased from Solstice Biotech Co. Ltd. in Korea; the olive leaf was from Longnan, Gansu, and the olive leaf extract was purified by macroporous resin adsorption method. The extract was a powder, and the content of oleuropein was 32%. The preparation method of the olive leaf extract was as follows: after the dried olive leaves were crushed, 70% ethanol aqueous solution was added and heated to reflux for 3 times. The combined extract was filtered to obtain the concentrated extract. The extract was adsorbed by a macroporous resin column, and after the adsorption was completed, the resin column was washed with water to remove impurities, and then desorbed with 70% ethanol aqueous solution. The desorption liquid was collected, and after the addition of an appropriate amount of dextrin, it was freeze-dried to prepare the product, the olive leaf extract powder.

[0042] Example 1: Preparation of ceramide NP from olive oil

[0043] The conventional amine ester exchange method was used to prepare ceramide. 3 g of plant sphingosine was added to 10 g of olive oil from Longnan, Gansu, and stirred under nitrogen protection to 100℃, and refluxed for 10 hours until the reaction was complete. The heat was removed, and the temperature was cooled to room temperature under nitrogen atmosphere. Petroleum ether recrystallization was used to obtain the final product of ceramide, and the yield was 93%. HPLC showed that the purity was more than 92%, and the details were shown in the specification attached Figure 1 .

[0044] The above synthesis method is only one of the possibilities. The amine ester exchange reaction can use acid, base, catalyst or enzyme to improve the reaction rate or reduce the degree of side reaction. The reaction can be carried out under nitrogen, air or vacuum conditions, and the present application does not make specific limitations on the synthesis method.

[0045] Example 2: Carbon chain distribution of fatty acid chain in ceramide NP from olive oil and raw material olive oil

[0046] The ester exchange method is used to determine the ceramide NP from olive oil and the raw material olive oil. For the olive oil sample, about 0.3 g of the sample is precisely weighed, 5 mL of sodium hydroxide-methanol solution is added, and the mixture is shaken and reacted at room temperature for 40 min, 5 mL of petroleum ether is added, the mixture is shaken and placed, and then 5 mL of distilled water is added. The upper organic phase is taken into a centrifuge tube, dried with anhydrous sodium sulfate, centrifuged, filtered, diluted, and then analyzed by GC-MS. For the ceramide sample, about 0.2 g of the sample is precisely weighed, 5 mL of potassium hydroxide-methanol solution is added, and the mixture is shaken and reacted in a water bath at 80°C for 1 hour, 5 mL of BF3-methanol solution is added, and the mixture is shaken and reacted in a water bath at 80°C for 1 hour. 20 mL of chloroform and 10 mL of 0.9% sodium chloride solution are added, the mixture is shaken and centrifuged, and then the lower solution is taken and washed with 10 mL of methanol and 10 mL of 0.9% sodium chloride solution once, respectively. The mixture is dried with anhydrous sodium sulfate, and then detected by GC-MS and analyzed by database comparison and retrieval. The experimental results are shown in Table 1.

[0047] Table 1: Comparison of carbon chain distribution of fatty acid chains

[0048] Raw material olive oil (%) Olive oil-derived ceramide NP (%) C16:0 12.09 10.69 C16:1 1.59 1.63 C18:0 1.89 2.03 C18:1 74.69 75.37 C18:2 7.92 8.13 C18:3 0.79 0.80 C20:0 0.34 0.32 C20:1 0.30 0.29 Others 0.40 0.74

[0049] As shown in the data in Table 1, the contents of C18:0, C18:1 and C18:2 in the raw material olive oil are 1.89%, 74.69% and 7.92%, respectively, and the contents of the three in the ceramide NP from olive oil are 2.03%, 75.37% and 8.13%, respectively. For C16:0 and C16:1, the contents in the raw material olive oil are 12.09% and 1.59%, respectively, and the contents of the three in the ceramide NP from olive oil are 10.69% and 1.63%, respectively. That is, the ceramide NP from olive oil has a similar fatty acid chain distribution as the raw material olive oil, and the content of unsaturated fatty acid is high, which indicates that the fatty acid in the ceramide NP prepared by the method of the present application is derived from olive oil.

[0050] Case three: comparison of soothing effects of ceramide NP from olive oil, ceramide NP from other plant oils and commercially available ceramide NP

[0051] The effect of various ceramide NPs on the content of inflammatory factor IL-1α and inflammatory mediator PGE2 of keratinocytes stimulated by UVB was determined. Sample group one was the above-mentioned ceramide from Longnan olive oil, sample group two was ceramide from Mediterranean olive oil, sample group three was ceramide from shea butter, sample group four was ceramide from mixed vegetable oil (the vegetable oil included shea butter, white pool seed oil, mowrah seed oil and macadamia seed oil), and sample group five was commercially available ceramide from stearic acid. The ceramide NPs from other vegetable oils in the sample group were obtained by using different vegetable oils as raw materials and by adopting a similar synthesis and purification method as in the first embodiment. The test concentration of sample groups one to five was 0.003%, the concentration of the positive control dexamethasone was 0.01%, and the UVB stimulation was 300 mJ / cm2. The experimental results are shown in Table 2.

[0052] Table 2: Effect of ceramide NPs on the content change of inflammatory factor IL-1α and inflammatory mediator PGE2

[0053]

[0054] As can be seen from the data in Table 2, the ceramides in sample groups one to five all had an inhibitory effect on the content of inflammatory factors and inflammatory mediators of keratinocytes caused by UVB stimulation, among which the inhibitory effect of the ceramide from olive oil was the strongest, and the inhibitory effect of the commercially available ceramide from stearic acid was the weakest. By comparing sample groups one and two with sample groups three and four, it can be seen that the ceramide with a higher content of unsaturated fatty acids has a better soothing effect. By comparing sample group one with sample group two, it can be seen that the soothing effect of the ceramide from Longnan olive oil was significantly better than that of the ceramide from Mediterranean olive oil (p<0.01).

[0055] Embodiment four: comparison of anti-inflammatory effects of ceramide NPs from olive oil and commercially available ceramide NPs from stearic acid

[0056] The effect of ceramides on skin barrier damage stimulated by SLS (sodium lauryl sulfate) was studied by using a 3D skin model (purchased from Guangdong Boxi Biological Technology Co., Ltd.), and the efficacy of ceramide NPs from olive oil and commercially available ceramide NPs was compared.

[0057] The skin models were divided into blank control, negative control, positive control and sample groups, each group containing 3 parallel experiments. The blank control group was not treated, the negative control group was added with 25 μL of 0.2% SLS solution on the surface, the positive control group was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.01% dexamethasone on the surface, sample group one was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.05% olive oil-derived ceramide NP on the surface, sample group two was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.1% olive oil-derived ceramide NP on the surface, and sample group three was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.1% commercial ceramide NP on the surface

[0058] After the administration was completed, the skin models were incubated in an incubator for 24 hours, and after the incubation was completed, the surface of the model was washed with sterile PBS solution to remove the remaining test substances, and the remaining liquid inside and outside the model was wiped off with a sterile cotton swab. After the incubation was completed, the epidermal skin model culture solution was collected in a centrifuge tube, and the content of inflammatory factor IL-1α and inflammatory mediator PGE2 was determined by using an ELISA kit. The experimental results are shown in Table 3.

[0059] Table 3: Changes in the content of inflammatory factor IL-1α and inflammatory mediator PGE2

[0060]

[0061] As can be seen from the data in Table 3, compared with the negative control group and the blank control group, SLS stimulation led to an increase in the content of inflammatory factor IL-1α and inflammatory mediator PGE2. Compared with the negative control group, the olive oil-derived ceramide NP can significantly reduce the content of inflammatory factor IL-1α and inflammatory mediator PGE2, and the p value is less than 0.01. The inhibition rate of 0.05% olive oil-derived ceramide NP on IL-1α and PGE2 was 24.67% and 33.01%, respectively, and the inhibition rate of 0.1% olive oil-derived ceramide NP on IL-1α and PGE2 was 40.71% and 34.39%, respectively. Commercial ceramide NP has a significant inhibitory effect on inflammatory factor IL-1α, with an inhibition rate of 21.47%, but has no significant effect on inflammatory mediator PGE2. The inhibitory effect of olive oil-derived ceramide NP on inflammatory factor IL-1α and inflammatory mediator PGE2 is significantly enhanced compared with commercial ceramide NP. For inflammatory factor IL-1α, sample group two was compared with sample group three, and the p value was 0.045 < 0.05; for inflammatory mediator PGE2, sample group two was compared with sample group three, and the p value was 0.002 < 0.01.

[0062] Among the experimental data in this group, there was a significant difference in the inhibitory effect of olive oil-derived ceramide NP and commercially available ceramide NP on inflammatory factors IL-1α and inflammatory mediators PGE2.

[0063] Case five: Comparison of barrier repair effects of olive oil-derived ceramide NP and commercially available ceramide NP

[0064] The 3D skin model (purchased from Guangdong Boxi Biological Technology Co., Ltd.) was used to study the effect of ceramide on SLS (sodium lauryl sulfate) stimulated skin barrier damage, and the efficacy of olive oil-derived ceramide NP and commercially available ceramide NP was compared.

[0065] The skin model was divided into blank control, negative control, positive control and sample group, each group containing 3 parallel experiments. The blank control group was not treated, the negative control group was added with 25 μL of 0.2% SLS solution on the surface, the positive control group was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 50 μM PPAR agonist WY14643 on the surface, sample group one was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.05% olive oil-derived ceramide NP on the surface, sample group two was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.1% olive oil-derived ceramide NP on the surface, sample group three was added with 12.5 μL of 0.4% SLS solution and 12.5 μL of 0.1% commercially available ceramide NP on the surface.

[0066] Filaggrin (FLG) interacts with keratin fibers to form dense keratin fiber bundles, and is connected with Loricrin (LOR) and other proteins under the action of transglutaminase to form a water-insoluble stable cornified envelope (CE), which is the basis of skin barrier. In addition, filaggrin is gradually degraded into small molecule active substances such as natural moisturizing factor by proteases such as Caspase-14 at the end of keratinocyte differentiation, maintaining skin hydration. Many skin disorders such as ichthyosis vulgaris and atopic dermatitis may be related to skin barrier damage caused by abnormal function of filaggrin. Transglutaminase-1 (TGM1) is mainly expressed in epidermal cells and participates in the formation of ε-(γ-glutamyl) lysine isopeptide cross-linking in the process of cornified envelope formation. This cross-linking is very stable and is a key step for keratinocytes to terminally differentiate into cornified envelope. Therefore, the content of FLG and TGM1 enzyme is one of the key indicators for characterizing the function of skin barrier.

[0067] After drug administration, the skin model was incubated in an incubator for 24 hours. Afterward, the model surface was cleaned with sterile PBS solution to remove any remaining test material, and residual fluid inside and outside the model was wiped away with sterile cotton swabs. The model used for testing was fixed in 4% paraformaldehyde for 24 hours. The contents of filaggrin and transglutaminase I were determined by immunofluorescence, and tissue morphology changes were analyzed after H&E staining. Experimental results are shown in Table 4 and the appendix to the instruction manual. Figures 2-4 As shown.

[0068] Table 4: Changes in FLG and TGM1 enzyme content

[0069]

[0070] Depend on Figure 2 It was found that, compared with the blank control and negative control groups, SLS stimulation caused tissue morphological damage in the skin model, specifically manifested as a reduction in the number of viable cell layers and the appearance of vacuoles. Olive oil-derived ceramide NP showed a certain repair effect on tissue morphological damage; 0.05% olive oil-derived ceramide NP reduced the number and volume of vacuoles, and 0.1% olive oil-derived ceramide NP reduced vacuoles, resulting in a more compact arrangement of viable cell layers and a significant improvement in viable cell damage. However, commercially available ceramide NP had no effect on improving vacuoles or viable cell layers in the tissue.

[0071] From Table 4 and Figure 3 It was found that, compared with the blank control and negative control group, SLS stimulation led to a reduction in the area of ​​the green fluorescent region in the skin model (the upper half of the image, the lighter-colored part of the stratum corneum), indicating a decrease in filaggrin content. Both olive oil-derived ceramide NP and commercially available ceramide NP significantly increased filaggrin content in the skin model. The promoting effects of 0.05%, 0.1% olive oil-derived ceramide NP, and 0.1% commercially available ceramide NP on filaggrin content were 225.58%, 281.40%, and 262.79%, respectively. At the same concentration, olive oil-derived ceramide NP was significantly more effective than commercially available ceramide NP in increasing filaggrin content (p=0.041<0.05).

[0072] From Table 4 and Figure 4 It was found that SLS stimulation reduced the area of ​​the green fluorescent region in the skin model (upper half of the image, the lighter-colored part of the stratum corneum), indicating a decrease in TGM1 content. Both olive oil-derived ceramide NP and commercially available ceramide NP significantly increased TGM1 content in the skin model. The promoting effects of 0.05%, 0.1% olive oil-derived ceramide NP, and 0.1% commercially available ceramide NP on filaggrin content were 491.67%, 558.33%, and 366.67%, respectively. At the same concentration, olive oil-derived ceramide NP was significantly more effective than commercially available ceramide NP in increasing TGM1 content (p=0.012<0.05).

[0073] Among the experimental data in this group, the promoting effects of olive oil-derived ceramide NP and commercially available ceramide NP on filaggrin FLG and transglutaminase 1 (TGM1) showed significant differences.

[0074] The above experimental results show that olive oil-derived ceramide NP has a significant effect on the inflammatory response and skin barrier damage of the epidermal skin model caused by SLS stimulation. The reduction of the content of inflammatory factors and inflammatory mediators indicates that olive oil-derived ceramide NP has good soothing effect. The repair of skin tissue structure and the increase of the content of barrier-related proteins indicate that olive oil-derived ceramide NP has good skin barrier repair effect. Compared with commercially available ceramide NP, it can be found that olive oil-derived ceramide NP has more outstanding effects in soothing and repairing.

[0075] Case Six: An Olea europaea-derived skin care composition

[0076] The present application provides an Olea europaea-derived skin care composition, which uses Olea europaea leaves from Longnan, Gansu, and obtains an Olea europaea leaf extract in powder form through macroporous resin purification technology, wherein the content of oleuropein is 32%. Olea europaea oil from Longnan, Gansu, is used to prepare ceramide NP. The product, olive oil-derived ceramide NP, is obtained by mixing and heating the fermentation-derived phytosphingosine with olive oil to make the phytosphingosine undergo transesterification with triglyceride. The effects of the Olea europaea leaf extract, olive oil-derived ceramide NP, and the composition (Olea europaea leaf extract + olive oil-derived ceramide NP) on the UVB-irradiated skin model are tested by gene microarray sequencing method to verify the soothing and repairing effects of single components and the composition on the skin damage caused by UV.

[0077] Case Seven: GO enrichment analysis of gene expression of epidermal model after application of the composition

[0078] The full-thickness epidermal reconstruction model EpiDerm FT (purchased from MatTek Corporation, USA) is divided into 5 groups, namely blank control (no treatment), negative control (only UVB treatment), Olea europaea leaf extract (0.2%), olive oil-derived ceramide NP (0.05%), and the composition prepared in the present application (Olea europaea leaf extract 0.2% + olive oil-derived ceramide NP 0.05%), each group including 3 parallel experiments. The gene microarray sequencing method is used to treat the epidermal model with 200 μL of the corresponding test substance for 4 hours, UVB irradiation (280-340 nm, 200 mJ / cm 2) and then treated with the corresponding test substance for 24 hours. After incubation was complete, the model surface was washed with sterile PBS solution to remove any test substance residue, and the inside and outside of the model were wiped with sterile cotton swabs to remove any residual liquid. Total RNA was extracted for microarray analysis (Affymetrix Human Clariom S, a total of 21448 genes). Genes with a p-value less than 0.05 (significant change) in the test composition group compared to the negative control group were subjected to GO enrichment analysis, and the results are shown in Tables 1 and 2. Figure 6 、 7

[0079] The results show that, after administration of the composition of olive leaf extract and ceramide NP of olive oil origin, the upregulated genes are related to processes including copper ion detoxification, regulation of exogenous apoptotic signaling pathway, negative regulation of cysteine endopeptidase activity during apoptosis process, mitochondrial membrane, etc. After administration of the composition of olive leaf extract and ceramide NP of olive oil origin, the downregulated genes are related to processes including DNA damage site, double-stranded RNA response, cell-matrix adhesion, regulation of DNA damage stimulus response, etc.

[0080] Example VIII: Composition promotes expression of barrier-related genes in epidermal model

[0081] ​Calcium ions play an important role in maintaining the structure and stability of the skin barrier. There is a calcium ion concentration gradient in the normal epidermis, with the highest concentration in the stratum corneum and the lowest concentration in the basal layer. Lower calcium ion concentration promotes keratinocyte proliferation, and higher calcium ion concentration promotes keratinocyte differentiation and stratification. Intracellular and extracellular calcium ion concentrations influence each other and work together to maintain the barrier function. Increased ROS caused by UV irradiation can affect the concentration of calcium ions in the endoplasmic reticulum, causing endoplasmic reticulum stress, and thus affecting the skin barrier function. VAPB encodes a vesicle-associated membrane protein-binding protein B located on the surface of the endoplasmic reticulum, interacts with mitochondrial protein PTPIP51, and regulates calcium ion concentration. The absence of VAPB affects the process of calcium ions released by the endoplasmic reticulum being taken up by mitochondria (De Vos, K.J. et al. VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis. Hum. Mol. Genet 21, 1299-1311 (2012)). MICU1 regulates mitochondrial calcium transporter MCU (Antony, A.N. et al. MICU1 regulation of mitochondrial Ca(2+) uptake dictates survival and tissue regeneration. Nat. Commun 7, 10955 (2012)), affecting the uptake of calcium ions by mitochondria. The absence of MICU1 leads to excessive concentration of calcium ions taken up by mitochondria, inducing cell death processes and affecting wound healing. The expression levels of the above two genes affect the calcium ion homeostasis in cells, and may also affect the calcium ion concentration in the stratum corneum of the skin, leading to changes in the skin barrier function. CBR1 encodes an NADPH-dependent carbonyl reductase-1 that catalyzes the reduction of various carbonyl compounds including prostaglandins, which can reduce inflammation caused by oxidative stress. IFNA7 encodes interferon alpha 7, a low molecular weight glycoprotein produced by macrophages during the immune process, which is involved in the JAK / STAT signaling pathway and affects immune regulation.WIF1 encodes Wnt Inhibitor Factor 1, which inhibits the anti-inflammatory canonical Wnt signaling pathway and inhibits the proliferation and differentiation of keratinocytes (Schlüter, H. et al. WIF1 Is Expressed by Stem Cells of the Human Interfollicular Epidermis and Acts to Suppress Keratinocyte Proliferation. J Invest Dermatol 133, 1669-1673 (2013)). PMAIP1 encodes a pro-apoptotic protein belonging to the Bcl-2 protein family, also known as Noxa, which is involved in the p53-mediated apoptosis process.

[0082] The change rates of the expression of the relevant genes are shown in Table 6.

[0083] Table 6: Change rate of gene expression

[0084]

[0085] After UVB irradiation, the expression of VAPB, MICU1 and CBR1 decreased, while the expression of IFNA7, WIF1 and PMAIP1 increased, that is, the calcium homeostasis in the skin barrier was destroyed (VAPB, MICU1), the skin inflammation was aggravated (CBR1, IFNA7), and the cell differentiation and apoptosis process was affected (WIF1, PMAIP1). After the application of olive leaf extract, ceramide NP derived from olive oil and the combination of the two, the expression of VAPB, MICU1 and CBR1 increased, while the expression of IFNA7, WIF1 and PMAIP1 decreased, that is, the skin barrier damage, skin inflammation and cell differentiation and apoptosis were all relieved to some extent, compared with the negative control group treated only with UVB irradiation. And compared with the single raw material group, the gene expression of the composition group changed more significantly, with obvious synergistic effect. The above changes in gene expression show that the combination of olive leaf extract and ceramide NP derived from olive oil has good repair and soothing effect, and can reduce the damage of UVB irradiation to the skin tissue.

[0086] The above skin model microarray sequencing results show that the combination of olive leaf extract and ceramide NP from olive oil has a significant effect on maintaining skin barrier function. Promoting the expression of VAPB and MICU1 genes maintains the homeostasis of calcium ions in the skin barrier, promotes the expression of CBR1 gene to reduce the generation of inflammation. Inhibit the expression of IFNA7 gene, reduce the process of ultraviolet-induced inflammation; inhibit the expression of WIF1 gene, reduce the influence on the anti-inflammatory Wnt pathway, regulate the proliferation and differentiation process of cells; inhibit the expression of PMAIP1 gene, inhibit the process of ultraviolet-induced apoptosis. The composition plays a role in soothing repair and maintaining the skin barrier by regulating the expression of related genes, so the skin care product containing the composition also has the effects of soothing repair and maintaining the skin barrier.

[0087] The above embodiments illustrate the technical solutions of the present application, and further research on the process, formula and application will be carried out subsequently. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the present application. Any modification, supplement or similar substitution within the principle range of the present application should be included in the protection scope of the present application.

[0088] Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be limited by the scope defined in the claims.

Claims

1. A composition characterized in that, The composition consists of an Olea europaea leaf extract and an olive oil-derived ceramide NP, and the structure of the olive oil-derived ceramide NP in the composition can be described by formula (1), R is a saturated or unsaturated alkyl group with 11-30 carbon atoms: 。 2. The composition of claim 1, wherein The Olea europaea leaf extract contains 32% of oleuropein.

3. Use of a composition for the manufacture of a skin care preparation for reducing the damage of UV light to the skin, characterized in that, The composition consists of an Olea europaea leaf extract and an olive oil-derived ceramide NP.

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