Composition for promoting gene expression of skin cells in moisturizing and repairing aspects

Abstract

The invention relates to a composition for promoting gene expression of skin cells in the aspects of moisturizing and repairing. The composition is composed of ceramide, composite hyaluronic acid and cosmetic acceptable auxiliary materials, ceramide is selected from ceramide 1 to ceramide 11, and the composite hyaluronic acid is a compound of hyaluronic acid and two sodium hyaluronates with different molecular weights. According to the composition of the composite hyaluronic acid and the ceramide, disclosed by the invention, the effects of moisturizing and repairing the skin can be synergistically exerted. And compared with single use of the two components, the effect is better.

Description

Technical Field

[0001] The present application relates to the fields of medicine and cosmetics, and in particular to a composition for promoting gene expression in skin cells in terms of moisturizing and repairing. Background Art

[0002] Ceramide exists in the skin and is an important type of natural skin lipid. Together with cholesterol and free fatty acids, it constitutes the skin's lipid barrier.

[0003] There are two naming systems for ceramides: one is based on the order of chromatographic migration, namely ceramides 1-8, and then named according to the time of discovery, namely ceramides 9-11. The other is named according to the molecular structure, with the last letter representing the sphingosine group, including S (sphingosine), dS (dihydrosphingosine), P (phytosphingosine), and H (6-hydroxysphingosine); the first letter represents the acyl chain, including N (no hydroxyl), A (α-hydroxy), and EO (linoleated ω-hydroxy). Different subtypes of ceramides are present in different proportions in the stratum corneum of the skin, with NP (ceramide 3) accounting for 22.1%, NH (ceramide 8) accounting for 14.5%, and AH (ceramide 7) accounting for 10.8%.

[0004] Abnormal ceramide metabolism is associated with a variety of skin diseases, including psoriasis, atopic dermatitis, acne, autosomal recessive congenital ichthyosis, etc.

[0005] Hyaluronic acid (HA), another natural moisturizer found in human skin, is also known by another name: hyaluronic acid. Hyaluronic acid is a high-molecular-weight linear polysaccharide that is widely present in human skin and other tissues and is a key component of the extracellular matrix. It is composed of repeating alternating units of glucuronic acid and N-acetylglucosamine. This structure makes it highly hygroscopic. Hydrated hyaluronic acid can hold up to 1,000 times its weight in water, giving it a powerful moisturizing and hydrating ability. Hyaluronic acid has different molecular weights and can be classified as large, medium, small, and ultra-low molecules.

[0006] Despite its simple structure, hyaluronic acid possesses numerous benefits. Found in high concentrations in human joints and the eye, it plays a role in lubricating joints, reducing friction between the eye and surrounding tissues, and bonding gel-like connective tissue. Therefore, it is widely used in the medical field as a filler or viscosupplement for joint surgery, cartilage regeneration, and vitreous supplements for ophthalmic surgery. Furthermore, because hyaluronic acid is a naturally occurring component of the human body and boasts a high safety profile, it has been widely used in the medical aesthetics industry in recent decades for applications such as facial fillers, plastic surgery, and derma-stimulating treatments. In the skincare industry, hyaluronic acid, as a non-invasive ingredient, is also widely used. Hyaluronic acid's primary benefit is moisturizing, but it also offers anti-aging and anti-wrinkle properties, barrier repair, and film-forming properties. It is a star ingredient in skincare products.

[0007] CN107854367A discloses a plant extract composition and an essence containing the plant extract composition. The essence contains the aforementioned plant extract composition, ceramide, sodium hyaluronate, polyglutamic acid, trehalose, and allantoin. The essence contains numerous ingredients, including ceramide and sodium hyaluronate, as well as other ingredients. These ingredients, when used together, can repair damaged skin barriers in sensitive skin.

[0008] CN106511141A discloses a moisturizing cream comprising a mixture of ingredients including meadowfoam seed oil, light liquid paraffin, polydimethylsiloxane, cetearyl alcohol, glycerin, ceramide, sodium hyaluronate, carbomer, aminomethyl propanol, fragrance, kaolin, and water. In addition to ceramide and sodium hyaluronate, the cream also contains numerous other water-soluble and oil-soluble ingredients. Together, these ingredients moisturize and repair the skin.

[0009] CN118340929A discloses a medical collagen scar repair silicone gel dressing, comprising a composite hydrogel, silicone gel, medical collagen, ceramide, sodium hyaluronate, biocompatible additives, silicone omentum, diclofenac sodium, excipients, and a polyurethane nonwoven backing. This dressing also promotes cell regeneration and scar repair through the combination of multiple raw materials.

[0010] CN116747153A proposes a composition for skin barrier repair and soothing, as well as its preparation method and application. The composition comprises the following ingredients, by weight: 10-15 parts ceramide, 8-12 parts hyaluronic acid, 5-9 parts Centella asiatica extract, 12-18 parts oligopeptide, 50-70 parts water, and 1-5 parts butylene glycol. In addition to the ceramide and hyaluronic acid, the composition also contains Centella asiatica extract, oligopeptide, water, and butylene glycol, which synergistically provide excellent repair and moisturizing properties. Summary of the Invention

[0011] Technical Solution

[0012] The inventors of this application, through a large number of experiments, unexpectedly discovered that the combination of ceramide and hyaluronic acid in a certain ratio can better moisturize, replenish water and repair the barrier than when they are used alone.

[0013] The present invention involves only two types of ingredients: ceramide and composite hyaluronic acid. The combination of ceramide and composite hyaluronic acid in a certain ratio has better hydration, moisturizing, and barrier repairing functions than when used alone.

[0014] According to one aspect of the present invention, a composition is provided, which is composed of ceramide, composite hyaluronic acid, and cosmetic or pharmaceutical acceptable excipients, wherein

[0015] The ceramide is selected from ceramide 1 to ceramide 11, preferably selected from ceramide 3, ceramide 7 and ceramide 8, more preferably ceramide 3,

[0016] The composite hyaluronic acid is preferably a complex of hyaluronic acid and two sodium hyaluronates with different molecular weights, wherein the molecular weight of the hyaluronic acid is preferably 4000-8000, more preferably 6000, wherein the molecular weight of one sodium hyaluronate is preferably 1.2 million to 1.8 million, more preferably 1.5 million, and the molecular weight of the other sodium hyaluronate is preferably 1.9 million to 2.3 million, preferably 2.14 million. Preferably, the mass ratio of the hyaluronic acid with a molecular weight of 4000-8000, the sodium hyaluronate with a molecular weight of 1.2 million to 1.8 million, and the sodium hyaluronate with a molecular weight of 1.9 million to 2.3 million is 1:(0.5 to 2):(0.5 to 2), preferably 1:1:1.

[0017] According to one embodiment of the present invention, the total amount of the composite hyaluronic acid and the ceramide is 0.01% to 1% based on the mass percentage of the raw materials.

[0018] According to one embodiment of the present invention, the mass ratio of the composite hyaluronic acid: the ceramide is 5:1 to 30:1, preferably 30:4.

[0019] According to one embodiment of the present invention, the mass ratio of the composite hyaluronic acid: the ceramide is 50:1 to 300:1, preferably 300:4.

[0020] According to one embodiment of the present invention, the mass ratio of the composite hyaluronic acid: the ceramide is 500:1 to 3000:1, preferably 3000:4.

[0021] According to another aspect of the present invention, there is provided a use of the composition as a moisturizing agent and / or repairing agent in cosmetics, pharmaceutical external application preparations, or medical devices.

[0022] Beneficial effects

[0023] The composite composition of hyaluronic acid and ceramide according to the present invention can synergistically exert the effects of moisturizing and repairing the skin, and the effects are better than when the two components are used alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a trend diagram of the changes in the expression levels of different genes in different embodiments according to the experimental examples of the present invention.

[0025] Figure 2 It is a bar graph of different gene expression enhancement rates for different embodiments according to the experimental examples of the present invention. DETAILED DESCRIPTION

[0026] To facilitate understanding of the features and effects of the present invention by persons having ordinary skill in the art, the following provides a general description and definition of terms and expressions used in this specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art in connection with the present invention. In the event of any conflict, the definitions in this specification shall prevail.

[0027] Throughout this document, all features or conditions defined as numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered to encompass and specifically disclose all possible subranges and individual values ​​within those ranges, particularly integer values. For example, a description of a range "1 to 8" should be considered to specifically disclose all possible subranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, and so forth, particularly those defined by all integer values, and should be considered to specifically disclose individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, 7, and 8. Unless otherwise indicated, the foregoing interpretation applies to all of the present disclosure, regardless of whether the ranges are comprehensive or not.

[0028] In this document, numerical values ​​should be understood to have the accuracy of the number of significant digits of the numerical value, provided that the purpose of the invention can be achieved. For example, the number 40.0 should be understood to cover the range from 39.50 to 40.49.

[0029] The following detailed description is merely illustrative in nature and is not intended to limit the present invention and its uses. In addition, this document is not bound by any theory described in the foregoing prior art or summary of the invention or in the following detailed description or examples.

[0030] Unless otherwise specified, the materials used in the following examples include the following composite hyaluronic acid: hyaluronic acid (molecular weight 6000, purity 100%, purchased from Shandong Yinhe), sodium hyaluronate (molecular weight 1.5 million, purity 100%, purchased from Shandong Yinhe), sodium hyaluronate (molecular weight 2.14 million, purity 100%, purchased from Shandong Yinhe), ceramide (Ceramide LCS, wherein the solid content of ceramide 3 is 4%, and the rest are excipients, purchased from LCS Biotech Co., Ltd. Eco), and the instrument was purchased from IKA.

[0031] Example 1

[0032] Step 1: Preparation of composite hyaluronic acid solution: Weigh 0.5% hyaluronic acid (molecular weight 6000), 0.5% sodium hyaluronate (molecular weight 1.5 million), and 0.5% sodium hyaluronate (molecular weight 2.14 million) by mass, add 98.5% deionized water, and stir at room temperature until clear and transparent. This yields a composite hyaluronic acid solution.

[0033] Step 2: Add 2% of the composite hyaluronic acid solution prepared in Step 1, 0.01% Ceramide LCS, and 97.99% deionized water, by mass percentage, at room temperature and stir thoroughly. This yields a pure composite hyaluronic acid and ceramide mixture, S3, at a ratio of 300:4.

[0034] Example 2

[0035] Step 1: Preparation of composite hyaluronic acid solution: Weigh 0.5% hyaluronic acid (molecular weight 6000), 0.5% sodium hyaluronate (molecular weight 1.5 million), and 0.5% sodium hyaluronate (molecular weight 2.14 million) by mass, add 98.5% deionized water, and stir at room temperature until clear and transparent. This yields a composite hyaluronic acid solution.

[0036] Step 2: Add 2% composite hyaluronic acid solution and 0.001% Ceramide LCS, based on mass percentage, to 97.999% deionized water and stir at room temperature to obtain a pure hyaluronic acid and ceramide mixture S4 sample with a mass ratio of 3000:4.

[0037] Example 3

[0038] Step 1: Preparation of composite hyaluronic acid solution: Weigh 0.5% hyaluronic acid (molecular weight 6000), 0.5% sodium hyaluronate (molecular weight 1.5 million), and 0.5% sodium hyaluronate (molecular weight 2.14 million) by mass, add 98.5% deionized water, and stir at room temperature until clear and transparent. This yields a composite hyaluronic acid solution.

[0039] Step 2: Take 2% composite hyaluronic acid solution and 0.1% Ceramide LCS, add 97.9% deionized water, and stir at room temperature to obtain a pure mixture S5 of composite hyaluronic acid and ceramide with a mass ratio of 30:4.

[0040] Comparative Example 1

[0041] Step 1: Preparation of composite hyaluronic acid solution: Weigh 0.5% hyaluronic acid (molecular weight 6000), 0.5% sodium hyaluronate (molecular weight 1.5 million), and 0.5% sodium hyaluronate (molecular weight 2.14 million) by mass, add 98.5% deionized water, and stir at room temperature until clear and transparent. This yields a composite hyaluronic acid solution.

[0042] Step 2: Take 2% of the composite hyaluronic acid solution by mass percentage, add 98% of deionized water, and stir evenly at room temperature to obtain a composite hyaluronic acid solution S1 sample.

[0043] Comparative Example 2

[0044] Step 1: Take 0.01% Ceramide LCS by mass, add 99.99% deionized water, and stir evenly at room temperature to obtain a ceramide solution S2 sample.

[0045] Experimental Example 1

[0046] The cells used were keratinocytes, batch number: Ep210707, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0047] Main reagents

[0048] KC2500 culture medium (Boxi Biotechnology), PBS (Boster), fetal bovine serum (FBS, Lanzhou Rongye), MTT (Sigma), DMSO (Sigma), RNAiso Plus (Takara), reverse transcription kit (Takara), fluorescent dye (Takara), and CaCl2 (Sigma).

[0049] Main equipment

[0050] CO2 incubator (Thermo, 150i), clean bench (Suzhou Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), fluorescence quantitative PCR instrument (BioRad, CFX-96).

[0051] Sample Information

[0052] Table 1

[0053]

[0054] Based on P7 generation keratinocyte gene testing

[0055] Test Method

[0056] 1) Inoculation: 2×10 5 The cells were seeded into 6-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0057] 2) Solution preparation: Prepare the test substance working solution according to the test plan (Table 2).

[0058] Table 2 Test plan

[0059]

[0060] Calcium chloride was used to verify the validity of the model. The concentration of the positive control sample was much higher than that of the sample group.

[0061] 3) Drug administration: According to the test scheme in Table 2, when the cell plating rate in the 6-well plate reaches 40% to 60%, group drug administration is carried out, with a dosage of 2 mL per well, 3 replicate wells per group, and incubation in an incubator (37°C, 5% CO2) for 24 h.

[0062] 4) Sample collection: After 24 hours of incubation, discard the supernatant, wash twice with 1 mL / well PBS, add 1 mL of RNAiso Plus to each well, lyse the cells by pipetting, and collect the samples.

[0063] 5) Detection: RNA was extracted and reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed. -△△CT Method to calculate the results.

[0064] 6) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Comparisons between groups were analyzed using the t-test. All statistical analyses were two-tailed. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference.

[0065] Based on the test method, keratinocytes were treated with RNAiso Plus and then collected. RNA extraction, reverse transcription, and fluorescence quantitative PCR were performed according to the kit instructions. The test results are shown in Table 3 below. The gene change trends are as follows: Figure 1 As shown, the gene expression improvement rate is shown in Table 4, and the gene expression improvement rate bar graph is shown in Figure 2 shown.

[0066] Among them, filaggrin (FLG) is an important molecule that connects keratin fibers in the stratum corneum of human skin. With the help of FLG monomers, keratin fibers are regularly aggregated, forming a solid physical barrier in the outermost layer of the epidermis, which can prevent the loss of epidermal moisture and the invasion of external allergens, helping to repair and moisturize the skin.

[0067] Loricrin (LOR) is a major component of the keratinocyte seal, accounting for approximately 80% of the total keratinocyte protein content. LOR is primarily found in the stratum granulosum of the epidermis and plays a crucial role in the epidermal barrier function, earning it the nickname "cellular armor." LOR strengthens the keratinocyte seal and enhances the permeability barrier. LOR interacts with intermediate filaments, increasing the flexibility of the keratinocyte seal structure and aiding in repair and moisturizing.

[0068] Table 3 Summary of FLG and LOR gene detection results

[0069]

[0070] Note: Use 2 -△△CT Methods: Results were calculated and statistically analyzed using the t-test. Significance compared to the BC group is indicated by *, P value < 0.05 is indicated by *, and P value < 0.01 is indicated by **. A larger average value indicates better repair / moisturizing results.

[0071] Table 4 Summary of gene expression enhancement rates

[0072]

[0073] From the above Table 3-4 and Figure 1-2 It can be seen that compared with the blank control group, samples S3 and S4 at a concentration of 0.0781% had a significant effect on increasing the expression of the gene FLG (P<0.05), with the increase rates being 26.09% and 23.47% respectively;

[0074] Samples S2, S3 and S4 had a significant effect on increasing the expression of gene LOR at a concentration of 0.0781% (P<0.05), with the increase rates being 42.88%, 119.58% and 28.98% respectively.

[0075] Compared with the control group, the P7 generation keratinocytes

[0076] Sample S1 (complexed hyaluronic acid) at a concentration of 0.0781% had no significant effect on increasing the expression levels of genes FLG and LOR.

[0077] At a concentration of 0.0781%, sample S2 (ceramide) increased the gene expression of LOR by 42.88%, but had no significant effect on the expression of FLG.

[0078] At a concentration of 0.0781%, sample S3 (composite hyaluronic acid:ceramide=300:4) increased the gene expression of FLG by 26.09% and the gene expression of LOR by 119.58%.

[0079] At a concentration of 0.0781%, sample S4 (composite hyaluronic acid:ceramide=3000:4) increased the gene expression of FLG by 23.47% and the gene expression of LOR by 28.98%.

[0080] Sample S5 (composite hyaluronic acid:ceramide=30:4) had no significant effect on increasing the expression levels of genes FLG and LOR at a concentration of 0.0781%.

[0081] When the ratio of hyaluronic acid to ceramide is 300:4, it can enhance the gene expression of FLG and LOR, and has a repair and moisturizing effect. The repair and moisturizing effect is better than using hyaluronic acid or ceramide alone.

[0082] When the ratio of hyaluronic acid to ceramide is 3000:4, it can increase the gene expression of FLG and LOR, providing repair and moisturizing effects. This is even more effective than using hyaluronic acid alone. Compared to using ceramide alone, it also increases the expression of FLG, providing even better moisturizing and repair effects.

[0083] When the ratio of hyaluronic acid to ceramide is 300:4 or 3000:4, it has a repairing effect and can increase the gene expression of FLG. The repair effect is better than when using hyaluronic acid and ceramide alone.

[0084] When the ratio of hyaluronic acid to ceramide is 300:4 or 3000:4, it has a repairing effect and can increase LOR gene expression. The repair effect is even greater than when using hyaluronic acid alone. In particular, when the ratio of hyaluronic acid to ceramide is 300:4, the repair effect is even greater than when using ceramide alone.

Claims

1. A composition comprising ceramide, composite hyaluronic acid, and cosmetic or pharmaceutically acceptable excipients, wherein: The ceramide is selected from ceramide 1 to ceramide 11, The composite hyaluronic acid is a complex of hyaluronic acid and two sodium hyaluronates with different molecular weights.

2. The composition according to claim 1, wherein The ceramide is selected from ceramide 3, ceramide 7 and ceramide 8, The molecular weight of the hyaluronic acid is 4000-8000, the molecular weight of one type of sodium hyaluronate is 1.2 million to 1.8 million, and the molecular weight of another type of sodium hyaluronate is 1.9 million to 2.3 million.

3. The composition according to claim 1, wherein The ceramide is ceramide 3, The mass ratio of the hyaluronic acid with a molecular weight of 4000-8000, the sodium hyaluronate with a molecular weight of 1.2 million to 1.8 million, and the sodium hyaluronate with a molecular weight of 1.9 million to 2.3 million is 1: (0.5 to 2): (0.5 to 2).

4. The composition according to claim 3, wherein The ceramide is ceramide 3, The mass ratio of the hyaluronic acid with a molecular weight of 4000-8000, the sodium hyaluronate with a molecular weight of 1.2 million to 1.8 million, and the sodium hyaluronate with a molecular weight of 1.9 million to 2.3 million is 1:1:

1.

5. The composition according to any one of claims 1 to 4, wherein Calculated by raw material mass percentage, the total content of the composite hyaluronic acid and the ceramide is 0.01% to 1%.

6. The composition according to any one of claims 1 to 4, wherein The mass ratio of the composite hyaluronic acid to the ceramide is 5:1 to 30:1, preferably 30:

4.

7. The composition according to any one of claims 1 to 4, wherein The mass ratio of the composite hyaluronic acid to the ceramide is 50:1 to 300:

1.

8. The composition according to any one of claims 1 to 4, wherein The mass ratio of the composite hyaluronic acid to the ceramide is 300:

4.

9. The composition according to any one of claims 1 to 4, wherein The mass ratio of the composite hyaluronic acid to the ceramide is 500:1 to 3000:1, preferably 3000:

4.

10. Use of the composition according to any one of claims 1 to 9 as a moisturizing agent and / or repairing agent in cosmetics, pharmaceutical external application preparations, or medical devices.

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