Composition for promoting silk polyprotein gene expression and preparation method thereof

Abstract

The invention relates to the technical field of functional compositions, in particular to a composition for promoting sericin gene expression and a preparation method thereof.The composition at least comprises the following components: acetylchitosamine, capparis spinosa fruit extract or citrus fruit extract and xanthan gum or tremella polysaccharide. The composition of xanthan gum or tremella polysaccharide can significantly improve the mRNA expression quantity of cell filagarin (FLG) under the low-concentration dosage of 1.25% (m / V), has the effect of promoting proliferation of keratinocytes, has the effect of promoting the mRNA expression of filagarin, has the effect of improving the content of filagarin, and can be used as an effective component for maintaining skin barriers and preserving moisture.

Description

Technical Field

[0001] The present invention relates to the technical field of efficacy compositions, and particularly to a composition for promoting the expression of filaggrin gene and a preparation method thereof. Background Art

[0002] The skin has many functions, and the most important of which is its protective and barrier functions. The stratum corneum of the skin forms a protective barrier between the body and the environment, which can isolate pollutants, irritants and allergens in the environment.

[0003] Filaggrin is derived from filaggrin precursor protein and is processed from profilaggrin. It is a key protein that promotes the terminal differentiation of the epidermis and the formation of the skin barrier. Profilaggrin is a large inactive polypeptide precursor and is the main component in the granular cell layer of the epidermis. The profilaggrin gene is located on chromosome 1q21 and consists of three exons. Exon 3 encodes the main polypeptide of profilaggrin. Profilaggrin is a large polypeptide with a molecular weight greater than 400 kDa (dalton) and is highly phosphorylated. At the end stage of keratinization and differentiation, profilaggrin is dephosphorylated and decomposed into 10 to 12 substantially identical filaggrins with a molecular weight of 37 kDa. Filaggrin efficiently aggregates keratin to form an important component of the skin barrier against external stimuli. In addition, filaggrin is subsequently decomposed by proteases into natural moisturizing factors and amino acids, which firmly lock the moisture in the skin. On the other hand, filaggrin plays a key role in the keratinocyte apoptosis system. It can initiate keratinocyte apoptosis, causing keratinocytes to transform from granular layer cells with nuclei to corneocyte cells without nuclei. The decrease in the content of filaggrin will not only reduce the natural moisturizing factors and lead to a decrease in the water content of the skin, but also cause the loss of the skin barrier function, resulting in a decrease in the skin's ability to resist external stimuli, and in severe cases, it will cause related skin diseases such as ichthyosis, atopic dermatitis and psoriasis. Thus, it can be seen that filaggrin plays an important role in participating in biological activity functions such as skin barrier composition and moisturization. Based on the biological functions of filaggrin, the research on filaggrin gene expression promoters is of great significance.

[0004] The existing patent research on filaggrin gene expression promoters focuses on single compounds or certain substances, lacking the exploration of compositions. For example, Chinese Patent (authorized publication number: CN104519894B) discloses a filaggrin gene expression promoter, and discloses that the combination of zanthoxylum extract and 3-(1'-piperidine)-propionic acid is preferably used as a filaggrin gene expression promoter. However, the effect of promoting filaggrin gene expression needs to be further improved, and due to the fact that some components of zanthoxylum extract may cause irritation or allergic reactions to the skin of some people, its practical application is subject to certain limitations. Summary of the Invention

[0005] To solve the above problems, the present invention provides a composition for promoting the expression of filaggrin gene and a preparation method thereof, which has a proliferation-promoting effect on keratinocytes, can promote the expression of filaggrin mRNA (messenger ribonucleic acid), increase the content of filaggrin, and can be used as an effective ingredient for maintaining skin barrier and moisturizing.

[0006] On the one hand, the present invention provides a composition for promoting the expression of filaggrin gene, which at least comprises the following components: chitooligosaccharide, Capparis spinosa fruit extract or Citrus fruit extract, xanthan gum or Tremella polysaccharide.

[0007] In one embodiment, the composition for promoting the expression of filaggrin gene at least comprises the following components: chitooligosaccharide, Capparis spinosa fruit extract, xanthan gum or Tremella polysaccharide.

[0008] In one embodiment, the composition for promoting the expression of filaggrin gene at least comprises the following components: chitooligosaccharide, Capparis spinosa fruit extract, xanthan gum.

[0009] In one embodiment, the composition for promoting the expression of filaggrin gene, by weight, at least comprises the following raw materials: 1-10 parts of chitooligosaccharide solution, 0.1-5 parts of Capparis spinosa fruit extract solution, 0.1-5 parts of xanthan gum solution.

[0010] In one embodiment, the composition for promoting the expression of filaggrin gene, by weight, at least comprises the following raw materials: 2-7 parts of chitooligosaccharide solution, 0.5-3 parts of Capparis spinosa fruit extract solution, 0.5-3 parts of xanthan gum solution.

[0011] In one embodiment, the composition for promoting the expression of filaggrin gene, by weight, at least comprises the following raw materials: 4-6 parts of chitooligosaccharide solution, 1-2 parts of Capparis spinosa fruit extract solution, 1 part of xanthan gum solution.

[0012] In one embodiment, the chitooligosaccharide solution is an aqueous chitooligosaccharide solution with a mass concentration of 5-15%.

[0013] In one embodiment, the chitooligosaccharide solution is an aqueous chitooligosaccharide solution with a mass concentration of 5%.

[0014] In one embodiment, the mass proportion of Capparis spinosa fruit extract in the Capparis spinosa fruit extract solution is 5-50%.

[0015] In one embodiment, the mass proportion of Capparis spinosa fruit extract in the Capparis spinosa fruit extract solution is 30%.

[0016] In one embodiment, the solvent in the Capparis spinosa fruit extract solution is deionized water.

[0017] The capparis spinosa fruit extract is obtained commercially or prepared by ethanol extraction. The chitosan and xanthan gum are obtained commercially.

[0018] In one embodiment, the method for preparing the capparis spinosa fruit extract includes at least the following steps:

[0019] (1) Wash, chop, dry, and pulverize the capparis spinosa fruit to make a powder;

[0020] (2) Add an ethanol aqueous solution for hydrothermal extraction to obtain an extract;

[0021] (3) Centrifuge the extract and take the supernatant. The supernatant is rotary evaporated and concentrated to an extract-like state to obtain the capparis spinosa fruit extract.

[0022] In one embodiment, the mass ratio of the powder to the ethanol aqueous solution is 1:(10 - 20).

[0023] In one embodiment, the mass ratio of the powder to the ethanol aqueous solution is 1:10.

[0024] In one embodiment, the volume concentration of the ethanol aqueous solution is 85 - 95%.

[0025] In one embodiment, the volume concentration of the ethanol aqueous solution is 90%.

[0026] In one embodiment, the conditions for centrifugation are: the centrifugation speed is 4500 - 5000 rpm, and the centrifugation time is 10 - 15 min.

[0027] In one embodiment, the conditions for centrifugation are: the centrifugation speed is 5000 rpm, and the centrifugation time is 15 min.

[0028] In one embodiment, the xanthan gum solution is an aqueous xanthan gum solution with a mass concentration of 0.05 - 0.5%.

[0029] In one embodiment, the xanthan gum solution is an aqueous xanthan gum solution with a mass concentration of 0.5%.

[0030] Existing substances for promoting filaggrin gene expression include, but are not limited to, Selaginella tamariscina extract, Zanthoxylum bungeanum extract, rose oil, cedarwood oil, white pine oil, geranium oil, 2-ketoglutaric acid, 3-(1'-piperidine)-propionic acid, pepper oil, basil oil, methyl o-toluate, methyl anthranilate, and methyl o-methylaminobenzoate, etc. However, the actual effective concentration is usually very high, and the dosage of some substances needs to be controlled during actual application. Therefore, the actual effect exerted is not significant. During the exploration process of the present invention, it was unexpectedly found that a composition containing chitosan oligosaccharide, Capparis spinosa fruit extract or citrus fruit extract, xanthan gum or tremella polysaccharide can significantly increase the mRNA expression level of cellular filaggrin (FLG) at a low dosage of 1.25% (m / V), and the improvement rate increased from 61.02% to 71.19%. Further, by optimizing the composition to a combination of chitosan oligosaccharide, Capparis spinosa fruit extract, xanthan gum or tremella polysaccharide, the effect of promoting filaggrin gene expression was further improved, and the improvement rate increased from 71.19% to 89.83%. Especially, when chitosan oligosaccharide, Capparis spinosa fruit extract, and xanthan gum are combined and prepared in the form of a solution to obtain the composition, while ensuring the stability of the composition, the composition can achieve an improvement rate of 110.17 - 227.12% at a low dosage of 1.25% (m / V), and can be added to the cosmetic system in a relatively low amount to fully exert the effects of maintaining the skin barrier and moisturizing, and the resulting economic benefits are huge.

[0031] On the other hand, the present invention provides a preparation method of a composition for promoting filaggrin gene expression, which at least includes the following steps: by weight, mixing a chitosan oligosaccharide solution, a Capparis spinosa fruit extract solution, and a xanthan gum solution to obtain the composition for promoting filaggrin gene expression.

[0032] The composition for promoting filaggrin gene expression provided by the present invention has good compatibility and can be added to cosmetics based on actual needs to achieve a repair effect, which has great practical significance and application prospects.

[0033] Beneficial effects

[0034] 1. The present invention provides a composition for promoting filaggrin gene expression and a preparation method thereof, which have a proliferation-promoting effect on keratinocytes, can promote the expression of filaggrin mRNA and increase the content of filaggrin, and can be used as an effective ingredient for maintaining the skin barrier and moisturizing.

[0035] 2. By using a composition containing chitosan oligosaccharide, Capparis spinosa fruit extract or citrus fruit extract, xanthan gum or tremella polysaccharide, the present invention can significantly increase the mRNA expression level of cellular filaggrin (FLG) at a low dosage of 1.25% (m / V), and the improvement rate increases from 61.02% to 71.19%.

[0036] 3. By optimizing the composition to be a combination of chitosan oligosaccharide, Capparis spinosa fruit extract, xanthan gum or tremella polysaccharide, the present invention further improves the effect of promoting the expression of filaggrin gene, and the improvement rate is increased from 71.19% to 89.83%.

[0037] 4. The present invention prepares the composition by combining chitosan oligosaccharide, Capparis spinosa fruit extract, and xanthan gum in the form of solutions respectively. While ensuring the stability of the composition, the composition can achieve an improvement rate of 110.17 - 227.12% at a low dosage of 1.25% (m / V), and can be added to the cosmetic system in a relatively low amount to fully exert the effects of maintaining the skin barrier and moisturizing. The resulting economic benefits are huge.

[0038] 5. The composition for promoting the expression of filaggrin gene provided by the present invention has good compatibility, can be added to cosmetics based on actual needs to achieve the repair effect, and has great practical significance and application prospects. Description of the Drawings

[0039] Figure 1 It is a bar chart of the expression level of the FLG gene in the detection of the expression of barrier-related genes in keratinocytes stimulated by UVB. Detailed Embodiments

[0040] Examples 1 - 2, Comparative Examples 1 - 3

[0041] Examples 1 - 2 and Comparative Examples 1 - 3 of the present invention provide a composition for promoting the expression of filaggrin gene, and the formula is shown in Table 1.

[0042] Table 1

[0043] Raw materials (parts by weight) Example 1 Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 5 wt% acetylchitooligosamine aqueous solution 4 6 0 4 4 5 wt% chitooligosamine aqueous solution 0 0 4 0 0 Capparis spinosa fruit extract solution 1 2 1 0 1 Citrus fruit extract solution 0 0 0 1 0 0.5 wt% xanthan gum aqueous solution 1 1 1 1 0 0.5 wt% tremella polysaccharide aqueous solution 0 0 0 0 1

[0044] Among them: the brand of chitosan oligosaccharide in the 5wt% aqueous solution of chitosan oligosaccharide is chitosan oligosaccharide, which is sourced from Jiangsu Aoxin Bioengineering Co., Ltd. The brand of chitosan in the 5wt% aqueous solution of chitosan is chitosan, which is sourced from Huzhou Pure Biotechnology Co., Ltd. The brand of xanthan gum in the 0.5wt% aqueous solution of xanthan gum is CG-T XANTHAN GUM, which is sourced from CP Kelco U.S., Inc. The brand of tremella polysaccharide in the 0.5wt% aqueous solution of tremella polysaccharide is Tremella heteropolysaccharide (WSK), which is sourced from Shanghai Huiwen Biotechnology Co., Ltd.

[0045] The mass ratio of the Capparis spinosa fruit extract in the Capparis spinosa fruit extract solution is 30%, and the solvent in the Capparis spinosa fruit extract solution is deionized water.

[0046] The preparation method of the capparis spinosa fruit extract comprises the following steps:

[0047] (1) Wash, chop, dry, and pulverize the capparis spinosa fruit to form a powder;

[0048] (2) Add an ethanol aqueous solution for hydrothermal extraction to obtain an extract;

[0049] (3) Centrifuge the extract and take the supernatant. The supernatant is rotary evaporated and concentrated to an extract paste to obtain the capparis spinosa fruit extract.

[0050] The mass ratio of the powder to the ethanol aqueous solution is 1:10. The volume concentration of the ethanol aqueous solution is 90%. The conditions for centrifugation are: the centrifugation speed is 5000 rpm, and the centrifugation time is 15 min.

[0051] The mass proportion of the citrus fruit extract in the citrus fruit extract solution is 30%, and the solvent in the citrus fruit extract solution is deionized water.

[0052] The preparation method of the citrus fruit extract is the same as that of the capparis spinosa fruit extract, except that the capparis spinosa fruit in the preparation method of the capparis spinosa fruit extract is replaced by the citrus fruit.

[0053] On the other hand, in Examples 1-2 and Comparative Examples 1-3 of the present invention, a preparation method of a composition for promoting the expression of the filaggrin gene is provided. By weight, after mixing each raw material, a composition for promoting the expression of the filaggrin gene is obtained.

[0054] Performance test

[0055] For the compositions for promoting the expression of the filaggrin gene provided in Examples 1-2 and Comparative Examples 1-3 of the present invention, a repair efficacy test - detection of the expression of genes related to the keratinocyte barrier stimulated by UVB is carried out. The detection is based on the Q / WP-SHACDDTW-WIF-1027 test specification for the expression of genes related to keratinocytes stimulated by UVB. The detection methods and results are as follows, where: capparis spinosa fruit extract: xanthan gum: chitosan amine = 1:1:4 (Comparative Example 1), N-acetylchitosan amine: xanthan gum: citrus fruit extract = 4:1:1 (Comparative Example 2), N-acetylchitosan amine: capparis spinosa fruit extract: xanthan gum = 4:1:1 (Example 1), N-acetylchitosan amine: capparis spinosa fruit extract: tremella polysaccharide = 4:1:1 (Comparative Example 3), N-acetylchitosan amine: capparis spinosa fruit extract: xanthan gum = 6:2:1 (Example 2).

[0056] 1. Test Purpose: Filaggrin (FLG) is a key protein for maintaining skin barrier function and plays an important role in the construction of epidermal mechanical and permeability barriers. Therefore, detecting the expression level of the filaggrin (FLG) gene can be used as an important indicator for evaluating the skin barrier repair efficacy of the test sample. This test uses human immortalized keratinocytes (HaCaT) as the research object, and evaluates the repair efficacy of the sample by detecting the changes in the relative expression levels of the barrier-related gene FLG before and after treatment with the test sample.

[0057] 2. Test Items: Repair Efficacy Test - Detection of the Expression of Barrier-Related Genes in UVB-Stimulated Keratinocytes

[0058] 3 Test Materials

[0059] 3.1 Test System: Human immortalized keratinocytes (HaCaT).

[0060] 3.2 Main Reagents: High-glucose DMEM culture medium (VivaCell), fetal bovine serum (Gibco), PBS (Gibco), trypsin (Gibco), MTT (sigma), DMSO (Sinopharm), HiScript II Q RT SuperMix for qPCR (+gDNAwiper) (Vazyme), PowerUp TM SYBR TM Green Master Mix (Applied Biosystems TM ) and cell lysate (Vazyme).

[0061] 3.3 Main Equipment: CO2 incubator (Thermo, 160i), ultraviolet phototherapy instrument (Sigma, SS-03), biological safety cabinet (Suzhou Jing'an Antai, BSC-1604ⅡA2), microplate reader (Tecan, Spark), real-time fluorescence quantitative PCR instrument (ABI, 7500).

[0062] 3.4 Sample Information: The sample information is shown in Table 1 below.

[0063] Table 1

[0064]

[0065] 4. Test Methods

[0066] 1) Cell Seeding: Collect cells in the logarithmic growth phase and seed them into a 24-well plate at a cell density of 2×10 5 cells / well. Culture them in an incubator (37°C, 5% CO2) for 24 h.

[0067] 2) Administration: According to the cytotoxicity results, experiments were conducted according to Table 2, with untreated cells as the control, and 3 replicate wells were set in each group. Incubate in an incubator (37 °C, 5% CO2) and continue to culture for 24 h.

[0068] Table 2

[0069]

[0070] 3) Detection: Add 0.5 mL of lysis buffer to each well, pipette to lyse. Extract RNA, reverse transcribe to cDNA, and then perform fluorescence quantitative PCR detection. The 2-ΔΔCT method was used for result calculation.

[0071] 5. Test results: After the sample acted on keratinocytes, the FLG gene expression results are shown in Table 3 and Figure 1 as follows.

[0072] Table 3

[0073]

[0074] Note: When comparing the NC group with the BC group, the significance is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##; when comparing the PC group and the sample group with the NC group, the significance is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0075] Compared with the BC group, the expression level of the FLG gene in the NC group decreased significantly (p < 0.01), indicating that the UVB stimulation model was successfully established in this experiment. Compared with the NC group, at the administration concentration of 100 μg / mL VC + 7 μg / mL VE in the PC group, the expression level of the FLG gene increased significantly (p < 0.01), indicating that the positive control in this experiment was effective. Compared with the NC group, for the sample Capparis spinosa fruit extract: xanthan gum: chitosan amine = 1:1:4, N-acetylchitosan amine: xanthan gum: citrus fruit extract = 4:1:1, N-acetylchitosan amine: Capparis spinosa fruit extract: tremella polysaccharide = 4:1:1 at a concentration of 1.25% (m / V), the expression level of the FLG gene increased significantly (p < 0.05); compared with the NC group, for the samples N-acetylchitosan amine: Capparis spinosa fruit extract: xanthan gum = 4:1:1, N-acetylchitosan amine: Capparis spinosa fruit extract: xanthan gum = 6:2:1 at a concentration of 1.25% (m / V), the expression level of the FLG gene increased significantly (p < 0.01).

[0076] 6. Conclusion

[0077] Based on the human immortalized keratinocyte (HaCaT) model stimulated by UVB, samples of Capparis spinosa fruit extract: xanthan gum: chitosan = 1:1:4, N-acetylchitosan: xanthan gum: citrus fruit extract = 4:1:1, N-acetylchitosan: Capparis spinosa fruit extract: tremella polysaccharide = 4:1:1 at a concentration of 1.25% (m / V) can increase the mRNA expression level of filaggrin (FLG) in cells, and the improvement rates are 61.02%, 71.19%, and 89.83% respectively, and there are statistical differences compared with the NC group (p < 0.05); samples of N-acetylchitosan: Capparis spinosa fruit extract: xanthan gum = 4:1:1, N-acetylchitosan: Capparis spinosa fruit extract: xanthan gum = 6:2:1 at a concentration of 1.25% (m / V) can increase the mRNA expression level of filaggrin (FLG) in cells, and the improvement rates are 110.17% and 227.12% respectively, and there are statistical differences compared with the NC group (p < 0.01).

[0078] It shows that samples of Capparis spinosa fruit extract: xanthan gum: chitosan = 1:1:4, N-acetylchitosan: xanthan gum: citrus fruit extract = 4:1:1, N-acetylchitosan: Capparis spinosa fruit extract: tremella polysaccharide = 4:1:1, N-acetylchitosan: Capparis spinosa fruit extract: xanthan gum = 4:1:1, N-acetylchitosan: Capparis spinosa fruit extract: xanthan gum = 6:2:1 have the ability to promote the expression of FLG in keratinocytes and achieve the repair effect by increasing the mRNA expression of cell FLG.

Claims

1. A composition for promoting the expression of the filaggrin gene, characterized in that, It comprises at least the following components: chitosan oligosaccharide, Capparis spinosa fruit extract or citrus fruit extract, xanthan gum or tremella polysaccharide.

2. The composition for promoting the expression of the filaggrin gene according to claim 1, wherein The composition for promoting the expression of filaggrin gene comprises at least the following components: chitosan oligosaccharide, Capparis spinosa fruit extract, xanthan gum or tremella polysaccharide.

3. The composition for promoting the expression of the filaggrin gene according to claim 2, wherein The composition for promoting the expression of filaggrin gene comprises at least the following components: chitosan oligosaccharide, Capparis spinosa fruit extract, xanthan gum.

4. The composition for promoting the expression of the filaggrin gene according to claim 3, wherein, The composition for promoting the expression of filaggrin gene comprises at least the following raw materials by weight: 1 - 10 parts of chitosan oligosaccharide solution, 0.1 - 5 parts of Capparis spinosa fruit extract solution, 0.1 - 5 parts of xanthan gum solution.

5. The composition for promoting the expression of the filaggrin gene according to claim 4, wherein The chitosan oligosaccharide solution is an aqueous solution of chitosan oligosaccharide with a mass concentration of 5 - 15%.

6. The composition for promoting the expression of the filaggrin gene according to claim 4, wherein The xanthan gum solution is an aqueous solution of xanthan gum with a mass concentration of 0.05 - 0.5%.

7. The composition for promoting the expression of the filaggrin gene according to claim 4, wherein The mass proportion of Capparis spinosa fruit extract in the Capparis spinosa fruit extract solution is 5 - 50%.

8. The composition for promoting filaggrin gene expression according to claim 7, wherein The preparation method of the Capparis spinosa fruit extract comprises at least the following steps: (1) Wash, cut, dry and crush the Capparis spinosa fruit to make powder. (2) Add an ethanol aqueous solution for hydrothermal extraction to obtain an extract. (3) Centrifuge the extract and take the supernatant, and rotary evaporate and concentrate the supernatant to an extract-like state to obtain the Capparis spinosa fruit extract.

9. The composition for promoting the expression of filaggrin gene according to claim 8, wherein The volume concentration of the ethanol aqueous solution is 85 - 95%.

10. A method for preparing a composition for promoting the expression of the filaggrin gene according to any one of claims 4-9, characterized in that, It comprises at least the following steps: Mix the chitosan oligosaccharide solution, Capparis spinosa fruit extract solution and xanthan gum solution by weight to obtain the composition for promoting the expression of filaggrin gene.

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