A composition containing double pdrn with skin cell anti-wrinkle repair and whitening effect and its application

Abstract

This invention discloses a composition containing dual PDRNs with anti-wrinkle, repairing, and whitening effects on skin cells, and its application. The active ingredients of this composition consist of 0.005-10% red camellia PDRN and 0.01%-4% salmon PDRN, accounting for a weight percentage of the total composition. The dual PDRN composition provided by this invention can significantly increase the EDU positivity rate and reduce the content of γ-H2AX protein and melanin, thereby exerting anti-wrinkle, firming, repairing, and whitening effects, and further contributing to skin rejuvenation.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic materials and their application technology, and relates to a composition containing dual PDRN with anti-wrinkle repair and whitening effects on skin cells and its application, especially a composition containing dual PDRN derived from red camellia with anti-wrinkle repair and whitening effects and its application. Background Technology

[0002] PDRN (polydeoxyribonucleotide), a tissue regeneration active substance, is a polymer with a molecular weight of less than 1500 kDa made from DNA that promotes the self-regeneration of damaged cells and tissues. Unlike ordinary DNA, PDRN does not transmit genetic information and exhibits pharmacological activity. It stimulates A2 receptors, which act as signal transducers for skin regeneration, and has the functions of promoting the secretion of various growth factors, generating capillaries through vascular endothelial growth factor (VEGF), improving blood circulation, anti-inflammatory effects, and preventing capillary leakage.

[0003] Currently, commercially available PDRNs are mainly derived from animal tissues (such as salmon testes). These animal-derived PDRNs have the advantages of high bioactivity and mature technology, but their potential limitations are also receiving increasing attention, such as the limitations of raw material supply due to species reproductive cycles, potential marine pollution risks, and high costs.

[0004] In recent years, breakthroughs have been achieved in the production of plant-derived PDRN (or "plant DNA fragments") obtained through microbial fermentation or plant cell culture techniques. These ingredients perfectly align with the trends of pure beauty and sustainable development, offering advantages such as controllable sourcing, ethical compliance, and extremely low allergenicity.

[0005] Currently, the application of PDRN in the cosmetics field is limited to a single source (either animal or plant). Simply mixing the two may only be seen as a substitute or supplement for an ingredient, without exploring its inherent synergistic mechanism and application value. How to break through the limitation of existing technologies and create a new PDRN composite system that combines the advantages of both and can produce a synergistic effect of "1+1>2" has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a dual PDRN composition with anti-wrinkle, repairing, and whitening effects, and its application. The active ingredients of this composition consist of 0.005-10% camellia PDRN and 0.01%-4% salmon PDRN, accounting for a weight percentage of the total composition. This composition can significantly promote EDU expression and reduce the content of γ-H2AX protein and melanin, thereby achieving anti-wrinkle, firming, repairing, and whitening effects.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a dual PDRN composition, wherein the active ingredients of the composition consist of 0.005-10% by weight of camellia PDRN and 0.01%-4% by weight of salmon PDRN.

[0008] As one embodiment, the molecular weight of the camellia PDRN is less than 100 bp. Camellia PDRN with a molecular weight of less than 100 bp derived from camellia, including camellia flower PDRN with a molecular weight of less than 100 bp, are all suitable for the system of the present invention.

[0009] As one implementation, the salmon PDRN has an average molecular weight of ≤1000 KDa and a nucleic acid content of ≥90.0%.

[0010] As one embodiment, the mass ratio of red camellia PDRN to salmon PDRN in the composition is 0.01 to 0.5:1.

[0011] In some embodiments, the composition comprises: 0.005%–10% red camellia PDRN, 0.01%–4% salmon PDRN, 0–30% preservative, and the balance being water, based on the total weight of the composition. The preservative may be butylene glycol, 1,3-propanediol, or glycerin, etc.

[0012] As one implementation, the red camellia PDRN is prepared by a method comprising the following steps: S1. After drying the red camellia flowers, grind them into powder, add water, cell wall lysin and protease, and carry out enzymatic hydrolysis to obtain the enzymatic hydrolysis system; S2. Centrifuge and filter the enzymatically hydrolyzed system; add anhydrous ethanol to the supernatant to precipitate the DNA, centrifuge and collect the precipitate, dry it, reconstitute it and filter it to obtain the red camellia PDRN.

[0013] As one implementation scheme, in step S1, the ratio of camellia pollen to water is 1:10; the amount of cell wall lysin added is 0.2-1.0% of the total weight of camellia pollen and water; and the amount of protease added is 0.2-1.0% of the total weight of camellia pollen and water.

[0014] As one implementation scheme, in step S1, the enzymatic hydrolysis temperature is 55-65℃ and the enzymatic hydrolysis time is 0.5-1.5 h.

[0015] As one implementation scheme, in step S1, red camellia pollen is homogenized with water to obtain a homogenate, which is then filtered and collected to obtain supernatant A; cell wall lysin and protease are added to the supernatant A to carry out enzymatic hydrolysis.

[0016] Add 5-15 mmol / L Tris-HCl, 0.5-2 mol / L NaCl, 1-3% CTAB and 0.1-1% EDTA to the enzymatically hydrolyzed solution obtained from the above enzymatic reaction to obtain a mixture; incubate the mixture at 55-65℃ for 20 min, centrifuge for 1 h, and collect the supernatant described in S2.

[0017] Secondly, the present invention relates to the use of the aforementioned dual PDRN composition as a cosmetic ingredient.

[0018] As one embodiment, the cosmetic includes anti-wrinkle, firming, repairing, and / or whitening functional cosmetics. The amount of the dual PDRN composition added to the cosmetic is 0.001% to 10%.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The composition of the present invention, which is mainly composed of red camellia PDRN and salmon PDRN, can significantly promote the expression of EDU and enhance the skin's repair ability; (2) The composition of the present invention, which is mainly composed of red camellia PDRN and salmon PDRN, can reduce the content of γ-H2AX protein, repair HSF cell DNA damage, maintain normal cell function, and avoid the decline of collagen synthesis capacity, thereby playing a role in skin anti-wrinkle and firming. (3) The composition of the present invention, which is mainly composed of red camellia PDRN and salmon PDRN, can significantly reduce the content of melanin and has a significant whitening effect. Attached Figure Description

[0020] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is an agarose gel electrophoresis image of PDRN derived from red camellia flowers according to the present invention; Figure 2 This is a comparative graph showing the effects of the PDRN compositions of the examples and control / comparative examples on HSF cell proliferation; Figure 3 This is a comparative graph showing the effects of the PDRN compositions of the examples and control / comparative examples on γ-H2AX protein in cells; Figure 4 This is a comparative graph showing the effects of the PDRN compositions of the examples and control / comparative examples on the inhibition of melanin production in zebrafish. Detailed Implementation

[0021] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0022] In the following embodiments, Salmon PDRN has an average molecular weight of ≤1000 KDa and a nucleic acid content of ≥90.0%.

[0023] The molecular weight of PDRN in red camellia is less than 100 bp.

[0024] The preparation of PDRN from red camellia flowers includes the following steps: S1. Grind the dried red camellia flowers, then take 100 g of the pulverized material and homogenize it with 1000 mL of pure water to obtain a homogenate. Filter and collect the supernatant A. S2. Add 0.5% cell wall lysin and 0.5% protease to the above supernatant A, and perform enzymatic hydrolysis at 60°C for 1 h to obtain the enzymatically hydrolyzed solution; S3. Add 10 mmol / L Tris-HCl, 1 mol / L NaCl, 2% CTAB and 0.5% EDTA to the enzymatically hydrolyzed solution to obtain a mixture; incubate the mixture at 60℃ for 20 min, centrifuge for 1 h, filter and collect the supernatant B; S4. Add anhydrous ethanol to the supernatant B to precipitate the DNA, centrifuge for 10 min, and collect the precipitate A. S5. The precipitate A was dried, reconstituted, and filtered to obtain a red camellia PDRN solution, which was then subjected to agarose gel electrophoresis to obtain… Figure 1 The result.

[0025] Example 1 This embodiment relates to a dual PDRN composition and its application; the composition and dosage of the dual PDRN composition are shown in Table 1, wherein the mass ratio of red camellia-derived PDRN and salmon PDRN is 0.05.

[0026] Salmon PDRN and red camellia PDRN alone were used as control 1 and control group 2, respectively. The amount used is shown in Table 1, so that the nucleic acid content of red camellia double PDRN, salmon PDRN and red camellia PDRN all meet the requirement of 0.2%.

[0027] Examples 2-7 Examples 2-7 relate to dual PDRN compositions and their applications; the composition and dosage of the corresponding dual PDRN compositions are shown in Table 1.

[0028] Table 1

[0029] Test Example 1: Effect of Camellia japonica double PDRN on HSF cell proliferation; Principle: Human skin fibroblasts (HSF) are the main functional cells of the loose connective tissue in the dermis and are a classic model for in vitro studies of skin development, homeostasis, aging, and damage repair. EdU (5-ethynyl-2'-deoxyuridine) is a thymine nucleoside analog that can replace thymine during cellular DNA synthesis (S phase) and be specifically incorporated into newly synthesized DNA strands. By specifically labeling the DNA-incorporated EdU with a fluorescent group, cells in the proliferating state can be identified by fluorescence detection. The proliferative activity of HSF cells can be quantitatively evaluated by statistically analyzing the proportion of EdU-positive cells to the total number of cells (EdU positivity rate).

[0030] Specific experimental steps: 1) Cell Culture and Seeding: Frozen HSF cells were revived and seeded into cell culture flasks at an appropriate density. DMEM medium was added, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2. When the HSF cells were in the logarithmic growth phase, they were removed, washed twice with PBS, digested with trypsin, and the digestion was terminated with DMEM medium. After centrifugation, the supernatant was discarded, and DMEM medium was added to prepare a cell suspension. The suspension was then seeded into six-well plates and cultured in a cell culture incubator.

[0031] 2) Sample addition treatment: 2 mL of DMEM medium was added to the blank control group, 2 mL of DMEM medium containing 0.5 μM ergothioneine was added to the positive control group, and 2 mL of DMEM medium containing different concentrations of camellia PDRN and salmon PDRN was added to the sample groups respectively (the contents of camellia PDRN and / or salmon PDRN in the DMEM medium of the control examples 1-2 and the corresponding sample groups of examples 1-7 are as shown in Table 1).

[0032] 3) EdU cell proliferation detection: After 24 h of treatment with the corresponding samples, cells in each group were added to the prepared EdU working solution and incubated for another 4 h in an incubator. After incubation, the culture medium was discarded, and the cells were fixed at room temperature with 4% paraformaldehyde. Then, they were permeabilized with an immunostaining and permeabilization solution containing Triton X-100. After treatment, Click reaction working solution was added, and the cells were incubated at room temperature in the dark for 30 min. The reaction solution was discarded, and the cell nuclei were stained with DAPI staining solution. Finally, fluorescence images of the EdU and DAPI channels were acquired under a fluorescence microscope. The EdU positivity rate of each group was calculated by analyzing the number of EdU-positive cells and the total number of cells to reflect the cell proliferation level.

[0033] 4) Data statistics: Excel and GraphPad Prism 8.0 software were used for data processing and statistical analysis. The experimental results are expressed as mean ± SEM. The t-test or one-way ANOVA was used for comparison between groups. P < 0.05 was considered statistically significant.

[0034] Experimental results showed that under the experimental conditions, red camellia double PDRN, salmon PDRN, and red camellia PDRN could all repair and promote EDU production, enhancing the skin's repair ability; and under the same concentration conditions set in this experiment, the effect of red camellia double PDRN was better than that of salmon PDRN and red camellia PDRN (e.g., Figure 2 The data for each comparative example and embodiment are shown in Table 2.

[0035] Test Example 2: Effect of Camellia japonica double PDRN on γ-H2AX protein in cells; Principle: Phosphorylated histone H2AX (γ-H2AX) is widely recognized as a biomarker for assessing the degree of cellular DNA damage and repair efficiency due to its extremely high sensitivity and specificity to DNA double-strand breaks (DSBs). This experiment uses immunofluorescence staining to label γH2AX protein in the cell nucleus with specific antibodies. By observing and quantifying changes in the intensity and number of fluorescent focal points (Foci) formed by γH2AX, the accumulation or clearance of intracellular DNA damage after sample treatment can be directly reflected, thereby assessing the regulatory effect of the sample on the DNA damage repair capacity of HSF cells and clarifying its potential in maintaining skin cell stability and photoprotection.

[0036] Specific experimental steps: 1) Sample addition treatment HSF cells resuscitated using Test Example 1 were used. Except for the blank control group, the model control group, positive control group, and sample concentration groups were all irradiated under a UVA lamp. After irradiation, the old culture medium of each group was discarded, and the cells were rinsed with sterile PBS solution. After discarding the PBS, samples were added. 2 mL of DMEM medium was added to the blank control group and the model control group; 2 mL of DMEM medium containing ergothioneine was added to the positive control group; and 2 mL of DMEM medium containing different concentrations of camellia PDRN and salmon PDRN was added to the sample groups (same as Test Example 1). After sample addition, the cells in each group were cultured for 24 h in a 37°C, 5% CO2 cell culture incubator.

[0037] 2) Detection of γ-H2AX protein expression by immunofluorescence method After incubation, each cell group was fixed with 4% paraformaldehyde at room temperature for 20 min, and then permeabilized with 1% Triton X-100 at room temperature for 15 min. After washing with PBS, the cells were blocked at room temperature for 1 h, and the blocking solution was discarded. The cells were incubated with primary antibody overnight at 4°C, and then with fluorescent secondary antibody at 37°C in the dark for 1 h. After washing with PBS, the cells were incubated with DAPI at room temperature in the dark for 5 min. The slides were then removed, fixed with mounting solution overnight, and images were taken under a fluorescence microscope for data analysis.

[0038] Experimental results showed that under the conditions of this experiment, salmon PDRN had no significant effect, while both camellia PDRN and camellia PDRN could repair HSF cell DNA damage, maintain normal cell function, and prevent the decline in collagen synthesis capacity, providing potential biological support for skin anti-wrinkle and firming. Furthermore, under the same concentration conditions set in this experiment, the effect of camellia PDRN was better than that of salmon PDRN and camellia PDRN (e.g., Figure 3 The data for each comparative example and embodiment are shown in Table 2.

[0039] Test Example 3: Effect of Camellia oleifera dual PDRN on the inhibition of melanin production in zebrafish; Principle: Zebrafish share 87% genetic homology with humans and possess genes related to melanin formation. Zebrafish have distinctive zebra-like stripes on their bodies and primarily consist of three types of cells: melanocytes, xanthocyanin cells, and argyrocytes. Zebrafish pigment cells originate from the neural crest, and early pigment formation occurs at 24 hpf, appearing as dots throughout the skin and retina. Therefore, the skin-whitening effect of samples is evaluated by detecting the formation and distribution of melanin in zebrafish.

[0040] Specific experimental steps: The experiment included a blank control group, a positive control group, and sample groups with different concentrations. Wild-type AB zebrafish embryos with normal development (6-8 hpf) were selected under a stereomicroscope and placed in 6-well cell culture plates, with 30 embryos per well and 3 replicates per group. 5 mL of the corresponding solution was added to each well. Standard dilution water was added to the blank control group, 2% arbutin solution was added to the positive control group, and the corresponding concentration of sample solution was added to the sample groups (same as in Test Example 1). The zebrafish embryos were then incubated in an incubator until 72 hpf. After incubation at 72 hpf, the pigment distribution on the zebrafish body surface was photographed and recorded. The zebrafish embryos were then transferred to EP tubes, lysis buffer (5 mg / mL sodium deoxycholate) was added, homogenized, centrifuged, and the precipitate was collected. 1 M sodium hydroxide solution was added, and the sample was analyzed using a microplate reader. The formula for calculating the melanin production inhibition rate is as follows: .

[0041] Experimental results showed that 0.2% red camellia double PDRN, salmon PDRN, and red camellia PDRN all had whitening effects under the experimental conditions. Furthermore, under the same concentration conditions set in this experiment, the effect of red camellia double PDRN was better than that of salmon PDRN and red camellia PDRN (e.g., Figure 4 The data for each comparative example and embodiment are shown in Table 2.

[0042] Table 2

[0043] As shown in Table 2, comparing the efficacy data of Examples 1-3 with those of Control Examples 1 and 2, the composition of the present invention, mainly composed of 0.005-10% red camellia PDRN and 0.01%-4% salmon PDRN, significantly promotes EDU expression and enhances skin repair ability compared to using red camellia PDRN or salmon PDRN alone; it reduces γ-H2AX protein content, repairs HSF cell DNA damage, maintains normal cell function, and prevents a decline in collagen synthesis capacity, thereby achieving anti-wrinkle and firming effects on the skin; at the same time, it can also significantly reduce melanin content, exhibiting significant whitening effects. Comparing the efficacy data of Examples 1-3 and Examples 4 and 5, it is shown that when the mass ratio of red camellia PDRN to salmon PDRN in the composition is controlled at 0.01-0.5:1, it exhibits optimal skin repair, anti-wrinkle and firming, and whitening effects. As can be seen from the efficacy data of Examples 6 and 7, the composition can exert anti-wrinkle, firming, repairing and whitening effects even at low concentrations, and the higher the concentration of the composition, the more significant the effect.

[0044] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A dual PDRN composition, characterized in that, The active ingredients of the composition consist of 0.005-10% red camellia PDRN and 0.01%-4% salmon PDRN, accounting for a weight percentage of the total weight of the composition.

2. The dual PDRN composition according to claim 1, characterized in that, The molecular weight of the PDRN in the red camellia is less than 100 bp.

3. The dual PDRN composition according to claim 1, characterized in that, The average molecular weight of the salmon PDRN is ≤1000 KDa, and the nucleic acid content is ≥90.0%.

4. The dual PDRN composition according to claim 1, characterized in that, The mass ratio of red camellia PDRN to salmon PDRN in the composition is 0.01 to 0.5:

1.

5. The dual PDRN composition according to claim 1, characterized in that, The red camellia PDRN is prepared by a method including the following steps: S1. After drying the red camellia flowers, grind them into powder, add water, cell wall lysin and protease, and carry out enzymatic hydrolysis to obtain the enzymatic hydrolysis system; S2. Centrifuge and filter the enzymatically hydrolyzed system, add anhydrous ethanol to the supernatant to precipitate the DNA, centrifuge and collect the precipitate, dry it, reconstitute it and filter it to obtain the red camellia PDRN.

6. The dual PDRN composition according to claim 5, characterized in that, In step S1, the ratio of camellia pollen to water is 1:10; the amount of cell wall lysin added is 0.2-1.0% of the total weight of camellia pollen and water; the amount of protease added is 0.2-1.0% of the total weight of camellia pollen and water.

7. The dual PDRN composition according to claim 5, characterized in that, In step S1, the enzymatic hydrolysis temperature is 55-65℃ and the enzymatic hydrolysis time is 0.5-1.5 h.

8. The dual PDRN composition according to claim 5, characterized in that, In step S1, red camellia pollen is homogenized with water to obtain a homogenate; the supernatant A is collected by filtration; cell wall lysin and protease are added to the supernatant A to carry out enzymatic hydrolysis.

9. The use of a dual PDRN composition according to any one of claims 1 to 8 as a cosmetic ingredient.

10. The application according to claim 9, characterized in that, The cosmetics include anti-wrinkle, firming, repairing, and / or whitening functional cosmetics.

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