A nanoemulsion preparation containing multi-source PDRN and a preparation method and application thereof

Abstract

The present application relates to the technical field of daily chemicals, and particularly relates to a nanoemulsion preparation containing multi-source PDRN and a preparation method and application thereof. The preparation raw materials of the nanoemulsion preparation containing multi-source PDRN include rose PDRN, salmon PDRN, probiotic fermented PDRN, polyglycerol fatty acid ester non-ionic emulsifier, microbial extracellular polysaccharide and the like. The present application can maintain the structural integrity of the nano-sized emulsion droplets in the nanoemulsion preparation by compounding the above components, significantly improve the centrifugal stability, physical stability and long-term stability of the nanoemulsion preparation, and improve the anti-aging, repair and anti-inflammatory effects of the nanoemulsion preparation. The nanoemulsion preparation can withstand the application environment of high rotation speed, high temperature, light and low temperature, and is convenient for storage and transportation. The nanoemulsion preparation can up-regulate the expression of longevity genes, improve the level of mitochondrial NAD+ and increase the content of anti-inflammatory cytokines, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of daily chemical products technology, and in particular to a nanoemulsion formulation containing multi-source PDRN, its preparation method and application. Background Technology

[0002] Skin aging is a gradual physiological process, influenced by various factors such as environment, lifestyle, and disease. With advancements in technology and improved living standards, the demand for functional skincare products is increasing, especially for products with anti-aging properties.

[0003] Polydeoxyribonucleic acid (PDRN) is widely used in functional skincare products due to its anti-inflammatory, soothing, energy metabolism regulating, and tissue repair effects. Currently, PDRN sources are becoming increasingly diversified, each with its own unique biological advantages: plant-derived rose PDRN extracted from rose petals focuses on repairing effects; animal-derived salmon PDRN extracted from salmon sperm cells focuses on anti-aging effects; and microbial-derived probiotic fermented PDRN extracted from probiotic strains excels in regulating skin microecological balance and reducing inflammation. Theoretically, if these three different PDRN sources can be effectively combined, they could have a more comprehensive application value compared to PDRN from a single source. However, in actual production applications, the preparation of skin care formulations using these three multi-source PDRNs still faces many technical bottlenecks, which seriously limit the industrial application and market promotion of compound PDRN efficacy products: There are very few systematic research studies on the compounding of plant-derived, animal-derived, and microbial-derived PDRNs in the existing technology, and there is a lack of mature formulation system guidance. At the same time, PDRN itself has poor stability and is easily affected by environmental factors such as temperature and light. During storage, molecular degradation and inactivation of biological activity are prone to occur, which not only shortens the product shelf life, but also fails to guarantee the stable performance of its biological efficacy. In addition, the preparation of traditional skin care formulations on the market mostly relies on high-temperature emulsification processes. High-temperature environments will destroy the biological activity of PDRN, resulting in a large amount of PDRN inactivation during the preparation process, which further significantly reduces the skin care efficacy of the finished product.

[0004] Therefore, developing a proprietary compound system adapted to multi-source PDRN and preparing highly stable composite PDRN skincare products are technical problems that urgently need to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a nanoemulsion preparation containing multi-source PDRN, its preparation method and application, which can achieve mitochondrial function repair, longevity gene activation and anti-inflammatory and soothing effects. The nanoemulsion preparation exhibits significant anti-aging, repair and anti-inflammatory effects.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The first aspect of the present invention provides a nanoemulsion formulation containing multi-source PDRN, wherein, by mass percentage, the raw materials for preparing the nanoemulsion formulation containing multi-source PDRN include the following components: 3% to 15% of the active ingredient, 7% to 14% of the polyglycerol fatty acid ester nonionic emulsifier, 15% to 30% of the emollient, 6% to 8% of the moisturizer, 0.1% to 0.3% of the microbial extracellular polysaccharide, and the balance being water; The active ingredient consists of rose PDRN, salmon PDRN, and probiotic fermented PDRN.

[0007] In the above-mentioned nanoemulsion formulation containing multi-source PDRN, the HLB value of the polyglycerol fatty acid ester nonionic emulsifier is 6-17. The polyglycerol fatty acid ester nonionic emulsifier is one of two polyglycerol-10 laurate or polyglycerol-6 stearate.

[0008] The nanoemulsion formulation containing multi-source PDRN as described above, wherein the microbial extracellular polysaccharide includes xanthan gum.

[0009] In the nanoemulsion formulation containing multi-source PDRN as described above, the mass ratio of the rose PDRN, the salmon PDRN, and the probiotic fermented PDRN is (2-4):(0.1-0.5):(2-6).

[0010] The nanoemulsion formulation containing multi-source PDRN as described above, wherein the humectant is at least one of glycerin, butylene glycol, dipropylene glycol, and glyceryl polyether-26; And / or, the emollient is at least one of C15-19 alkyl, caprylic / capric triglyceride, meadowfoam seed oil, and isododecane.

[0011] A second aspect of the present invention provides a method for preparing the nanoemulsion formulation containing multi-source PDRN, comprising the following steps: S1. Mix polyglycerol fatty acid ester nonionic emulsifier and emollient, heat to a first temperature under stirring, and keep at the first temperature to obtain oil phase; S2. Mix the humectant, extracellular polysaccharide and water, and heat to a second temperature under stirring to obtain the aqueous phase; S3. Add the aqueous phase dropwise to the oil phase, stir, and adjust the pH of the system to 5.5–6.5 to obtain a nanoemulsion mixture; S4. Rose PDRN, salmon PDRN and probiotic fermented PDRN are mixed and added to the nanoemulsion mixture. After stirring, a nanoemulsion preparation containing multi-source PDRN is obtained.

[0012] In the preparation method of the nanoemulsion formulation containing multi-source PDRN as described above, in step S1, the stirring speed is 700-1200 rpm, the first temperature is 40-50℃, and the holding time is 30-40 minutes. And / or, in step S2, the second temperature is 50–65°C.

[0013] In the preparation method of the nanoemulsion formulation containing multi-source PDRN as described above, in step S3, the stirring temperature is 35-45°C and the stirring time is 30-40 minutes. And / or, in step S4, the stirring process is carried out at a speed of 700-1200 rpm for 30-40 minutes; the stirring process is carried out at room temperature.

[0014] A third aspect of the present invention provides the application of a nanoemulsion formulation containing multiple PDRN in the preparation of products with anti-aging, repair and anti-inflammatory effects, wherein the nanoemulsion formulation containing multiple PDRN is the above-mentioned nanoemulsion formulation containing multiple PDRN, or the nanoemulsion formulation containing multiple PDRN is a nanoemulsion formulation containing multiple PDRN prepared by the above-mentioned preparation method.

[0015] The above-mentioned application of nanoemulsion formulations containing multi-source PDRN in the preparation of products with anti-aging, repair and anti-inflammatory effects, wherein the nanoemulsion formulations containing multi-source PDRN can upregulate the expression of longevity genes SIRT1, SIRT3 and CAT. And / or, the nanoemulsion formulation containing multi-source PDRN can increase mitochondrial NAD+ levels; And / or, the nanoemulsion formulation containing multi-source PDRN can increase the content of the anti-inflammatory cytokine IL-10.

[0016] The solution of the present invention has at least the following beneficial effects: The present invention provides a nanoemulsion formulation containing multi-source PDRN, the raw materials of which include the following components: an active ingredient composed of rose PDRN, salmon PDRN, and probiotic-fermented PDRN; a polyglycerol fatty acid ester nonionic emulsifier; a skin emulsifier; a moisturizer; and microbial extracellular polysaccharides. The present invention uses rose PDRN, salmon PDRN, and probiotic-fermented PDRN as active ingredients, polyglycerol-10 laurate and polyglycerol-6 stearate as polyglycerol fatty acid ester nonionic emulsifiers, and xanthan gum as microbial extracellular polysaccharides. By compounding the active ingredients (rose PDRN, salmon PDRN, and probiotic-fermented PDRN) with polyglycerol-10 laurate, polyglycerol-6 stearate, xanthan gum, etc., the structural integrity of the nano-sized droplets in the nanoemulsion formulation can be maintained, significantly improving the centrifugal stability, physical stability, and long-term stability of the nanoemulsion formulation, and improving the anti-aging, repair, and anti-inflammatory effects of the nanoemulsion formulation. This nanoemulsion formulation contains densely distributed core-shell droplets (also known as nanoscale droplets) with a particle size of 10-200 nm. These nanoscale droplets have a uniform particle size distribution and excellent dispersibility, which is beneficial to improving the bioavailability of the active ingredients (rose PDRN, salmon PDRN, and probiotic fermented PDRN) in the formulation. This nanoemulsion formulation exhibits excellent centrifugal stability, physical stability, and long-term stability. It can withstand high-speed (e.g., 3000-5000 rpm, 30 min), high temperature (e.g., 45-50℃), light (e.g., 4500 lx illuminance), and low temperature (e.g., -18-4℃) application environments. After being stored at room temperature for 3 months, no oil or precipitation was observed after high-speed centrifugation for 30 min, demonstrating excellent stability and facilitating storage and transportation. This nanoemulsion formulation can upregulate the expression of longevity genes (e.g., SIRT1, SIRT3, CAT), increase mitochondrial NAD+ levels, and increase the content of anti-inflammatory cytokines (e.g., IL-10), exhibiting significant anti-aging, repair, and anti-inflammatory effects.

[0017] The present invention provides a method for preparing nanoemulsion formulations containing multi-source PDRN, based on PIC (phase inversion) emulsification technology. Its core principle is to induce a phase inversion in the system by changing the proportions of its components at a constant temperature (after heat preservation of the oil phase), thereby preparing a nanoemulsion. By slowly and controllably altering the system composition (water content), the system completes the transition from a W / O phase to an O / W phase. The entire reaction is mild, requiring no high-intensity homogeneous shearing, and relies on spontaneous recombination at the phase interface to form uniform and stable nano-sized droplets. These nano-sized droplets are then used to encapsulate and anchor rose PDRN, salmon PDRN, and probiotic fermentation PDRN at the interface, avoiding problems such as raw material degradation and component incompatibility at the process level, maximizing the preservation of the bioactivity of multi-source PDRN, and enhancing system stability through the size advantage of the nano-sized droplets. The nanoemulsion formulations containing multi-source PDRN obtained using the method of the present invention exhibit excellent centrifugal stability, physical stability, and long-term stability, and demonstrate significant anti-aging, repair, and anti-inflammatory effects, showing broad application prospects.

[0018] This invention uses plant-derived polyglycerol fatty acid ester nonionic emulsifiers, specifically a combination of polyglycerol-10 laurate and polyglycerol-6 stearate. These emulsifiers are inherently green, biodegradable, mild, and low-irritant, making them suitable for all skin types, including sensitive skin. By precisely controlling the ratio of the two emulsifiers, a nanoemulsion formulation with a nanoscale droplet structure was successfully formed, exhibiting advantages such as mildness and high safety. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a TEM image of the nanoemulsion formulation (composition A) in Example 1 of the present invention, with a scale bar of 200 nm; Figure 2 The image shows a TEM image of the nanoemulsion formulation (composition E) in Comparative Example 1 of this invention, with a scale bar of 500 nm. Figure 3 The image shows a TEM image of the nanoemulsion formulation (composition F) in Comparative Example 2 of this invention, with a scale bar of 200 nm. Figure 4 The image shows a TEM image of the nanoemulsion formulation (composition G) in Comparative Example 3 of this invention, with a scale bar of 500 nm. Figure 5The image shows a TEM image of the nanoemulsion formulation (composition H) in Comparative Example 4 of this invention, with a scale bar of 1.0 μm. Figure 6 This is a microstructure diagram of the nanoemulsion formulation (composition A) in Example 1 of the present invention under an optical microscope, with a scale bar of 10 μm; Figure 7 The image shows the microstructure of the nanoemulsion formulation (composition E) in Comparative Example 1 of the present invention under an optical microscope, with a scale bar of 20 μm. Figure 8 The image shows the microstructure of the nanoemulsion formulation (composition F) in Comparative Example 2 of the present invention under an optical microscope, with a scale bar of 20 μm. Figure 9 The image shows the microstructure of the nanoemulsion formulation (composition G) in Comparative Example 3 of the present invention under an optical microscope, with a scale bar of 20 μm. Figure 10 The image shows the microstructure of the nanoemulsion formulation (composition H) in Comparative Example 4 of this invention under an optical microscope, with a scale bar of 20 μm. Figure 11 This is a DLS particle size distribution diagram of the nanoemulsion formulation (composition A) in Example 1 of the present invention; Figure 12 This is a Zeta potential diagram of the nanoemulsion formulation (composition A) in Example 1 of the present invention; Figure 13 This is the result of detecting the expression level of the SIRT1 gene in Test Example 3 of the present invention; Figure 14 This is the result of detecting the expression level of the SIRT3 gene in Test Example 3 of the present invention; Figure 15 The results of CAT gene expression level detection in test example 3 of this invention; Figure 16 The results of NAD+ concentration detection in Test Example 3 of this invention; Figure 17 The results of IL-10 content detection in Test Example 3 of this invention; Figures 13-17 In the table, # indicates a significant difference between the negative control group or model group and the control group, where # indicates P-value < 0.05, ## indicates P-value < 0.01, and ### indicates P-value < 0.001; * indicates a significant difference between the sample group and the negative control group or model group, where * indicates P-value < 0.05, ** indicates P-value < 0.01, and *** indicates P-value < 0.001. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0022] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0023] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0024] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, the technical / scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] The first aspect of the present invention provides a nanoemulsion formulation (also known as a nanoemulsion formulation) containing multi-source PDRN. The preparation raw materials of the nanoemulsion formulation containing multi-source PDRN, by mass percentage, include the following components: 3%–15% active ingredient, 7%–14% polyglycerol fatty acid ester nonionic emulsifier, 15%–30% emollient, 6%–8% moisturizer, 0.1%–0.3% microbial extracellular polysaccharide, and the balance being water. The active ingredient consists of rose PDRN, salmon PDRN, and probiotic fermented PDRN.

[0027] This invention combines the active ingredients (rose PDRN, salmon PDRN, and probiotic fermented PDRN) with nonionic emulsifiers of polyglycerol fatty acid esters (polyglycerol-10 laurate and polyglycerol-6 stearate in this embodiment) and microbial extracellular polysaccharides (xanthan gum in this embodiment). This combination maintains the structural integrity of nano-sized droplets in nanoemulsion formulations, significantly improves the centrifugal stability, physical stability, and long-term stability of nanoemulsion formulations, and enhances their anti-aging, repair, and anti-inflammatory effects. This nanoemulsion formulation contains densely distributed core-shell droplets (also known as nanoscale droplets) with a particle size of 10-200 nm. These nanoscale droplets have a uniform particle size distribution and excellent dispersibility, which is beneficial to improving the bioavailability of the active ingredients (rose PDRN, salmon PDRN, and probiotic fermented PDRN) in the formulation. This nanoemulsion formulation exhibits excellent centrifugal stability, physical stability, and long-term stability. It can withstand high-speed (e.g., 3000-5000 rpm, 30 min), high temperature (e.g., 45-50℃), light (e.g., 4500 lx illuminance), and low temperature (e.g., -18-4℃) application environments. After being stored at room temperature for 3 months, no oil or precipitation was observed after high-speed centrifugation for 30 min, demonstrating excellent stability and facilitating storage and transportation. This nanoemulsion formulation can upregulate the expression of longevity genes (e.g., SIRT1, SIRT3, CAT), increase mitochondrial NAD+ levels, and increase the content of anti-inflammatory cytokines (e.g., IL-10), exhibiting significant anti-aging, repair, and anti-inflammatory effects.

[0028] In some embodiments, the HLB value of the polyglycerol fatty acid ester nonionic emulsifier is 6 to 17.

[0029] In some embodiments, the polyglycerol fatty acid ester nonionic emulsifier is two of polyglycerol-10 laurate and polyglycerol-6 stearate. This invention uses a combination of polyglycerol-10 laurate and polyglycerol-6 stearate. These emulsifiers are inherently green, biodegradable, mild, and low-irritant, making them suitable for all skin types, including sensitive skin. By precisely controlling the ratio of the two emulsifiers, a nanoemulsion formulation with a nanoscale droplet structure is successfully formed, effectively improving the stability of the nanoemulsion formulation and offering advantages such as mildness and high safety. Preferably, the HLB value of the polyglycerol-10 laurate is 15-17, and the HLB value of the polyglycerol-6 stearate is 7-9.

[0030] Further, by mass percentage, the raw materials used in the preparation comprise 5%–8% polyglycerol-10 laurate and 2%–6% polyglycerol-6 stearate. Specifically, by mass percentage, the raw materials for preparing the nanoemulsion formulation containing multi-source PDRN include the following components: 3%–15% active ingredient, 5%–8% polyglycerol-10 laurate, 2%–6% polyglycerol-6 stearate, 15%–30% emollient, 6%–8% moisturizer, 0.1%–0.3% microbial extracellular polysaccharide, and the balance being water.

[0031] In some embodiments, the microbial extracellular polysaccharide includes xanthan gum. The use of xanthan gum in this invention helps maintain the structural integrity of nano-sized droplets in nanoemulsion formulations, improves the centrifugal stability, physical stability, and long-term stability of nanoemulsion formulations, and enhances their anti-aging, repair, and anti-inflammatory effects.

[0032] In some embodiments, the mass ratio of the rose PDRN, the salmon PDRN, and the probiotic fermented PDRN is (2-4):(0.1-0.5):(2-6), for example, 2.5:0.5:5, 2:0.2:3, 3:0.15:5, 4:0.3:2.5, etc.

[0033] In some embodiments, the humectant is at least one of glycerin, butylene glycol, dipropylene glycol, and glyceryl polyether-26.

[0034] In some embodiments, the emollient is at least one of C15-19 alkyl, caprylic / capric triglyceride, meadowfoam seed oil, and isododecane.

[0035] A second aspect of the present invention provides a method for preparing the nanoemulsion formulation containing multi-source PDRN, comprising the following steps: S1. Mix polyglycerol fatty acid ester nonionic emulsifier and emollient, heat to a first temperature under stirring, and keep at the first temperature to obtain oil phase; S2. Mix the humectant, extracellular polysaccharide and water, and heat to a second temperature under stirring to obtain the aqueous phase; S3. Add the aqueous phase dropwise to the oil phase, stir, and adjust the pH of the system to 5.5–6.5 to obtain a nanoemulsion mixture; S4. Rose PDRN, salmon PDRN and probiotic fermented PDRN are mixed and added to the nanoemulsion mixture. After stirring, a nanoemulsion preparation containing multi-source PDRN is obtained.

[0036] In this invention, the purpose of heating to a first temperature under stirring conditions in step S1 is to fully dissolve the polyglycerol fatty acid ester nonionic emulsifier and the emollient.

[0037] In step S2, the purpose of heating to the second temperature under stirring conditions is to ensure that the aqueous phase is dissolved uniformly and free of particles.

[0038] In step S3, the purpose of adding the aqueous phase dropwise to the oil phase is to facilitate the transformation of the system from W / O phase to O / W phase by slowly and controllably changing the system composition (water content) at a constant temperature.

[0039] In step S4, the purpose of stirring is to encapsulate PDRN from different sources (rose PDRN, salmon PDRN, and probiotic fermented PDRN) in the nanoemulsion mixture to form a nanoemulsion formulation.

[0040] In some embodiments, in step S1, the stirring speed is 700-1200 rpm, the first temperature is 40-50°C, and the heat preservation time is 30-40 minutes.

[0041] In some implementations, in step S2, the second temperature is 50–65°C.

[0042] In some embodiments, in step S3, the stirring temperature is 35-45°C and the stirring time is 30-40 minutes.

[0043] In some embodiments, in step S4, the stirring process is carried out at a speed of 700-1200 rpm for 30-40 minutes; the stirring process is carried out at room temperature.

[0044] A third aspect of the present invention provides the application of a nanoemulsion formulation containing multiple PDRN in the preparation of products with anti-aging, repair and anti-inflammatory effects, wherein the nanoemulsion formulation containing multiple PDRN is the above-mentioned nanoemulsion formulation containing multiple PDRN, or the nanoemulsion formulation containing multiple PDRN is a nanoemulsion formulation containing multiple PDRN prepared by the above-mentioned preparation method.

[0045] In some embodiments, the nanoemulsion formulation containing multi-source PDRN can upregulate the expression of longevity genes SIRT1, SIRT3 and CAT; the nanoemulsion formulation containing multi-source PDRN can increase the level of mitochondrial NAD+; and the nanoemulsion formulation containing multi-source PDRN can increase the content of the anti-inflammatory cytokine IL-10.

[0046] The nanoemulsion formulation containing multi-source PDRN of the present invention contains densely distributed core-shell droplets (also known as nano-sized droplets) with a particle size of 10-200 nm. These nano-sized droplets have a uniform particle size distribution and excellent dispersibility, which is beneficial for promoting the transdermal absorption of active ingredients (rose PDRN, salmon PDRN, and probiotic fermented PDRN) and improving the bioavailability of active ingredients. The nanoemulsion formulation exhibits excellent centrifugal stability, physical stability, and long-term stability, and can withstand high rotational speeds (e.g., 3000-5000 rpm). The nanoemulsion formulation exhibits excellent stability and is suitable for various application environments, including high temperature (e.g., 45–50°C), light (e.g., 4500 lx illuminance), and low temperature (e.g., -18–4°C). After being stored at room temperature for 3 months, it showed no oil release or precipitation after high-speed centrifugation for 30 minutes, making it easy to store and transport. The nanoemulsion formulation can upregulate the expression of longevity genes (e.g., SIRT1, SIRT3, CAT), increase mitochondrial NAD+ levels, and increase the content of anti-inflammatory cytokines (e.g., IL-10), demonstrating significant anti-aging, repair, and anti-inflammatory effects.

[0047] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0048] In the following examples: Rose PDRN: Trade name: HYPDRNZ(ROsa)-PF, Manufacturer: Microsec Biotechnology (Yantai) Co., Ltd. Salmon PDRN: Trade name: HYPDRN-ZH (Sodium DNA, salmon-derived PDRN), Manufacturer: Baihong Synthetic Biotechnology (Yantai) Co., Ltd. Probiotic fermented PDRN: Trade name: HYPDRNZ(LAB), Manufacturer: Microsec Biotechnology (Yantai) Co., Ltd. NIKKOL DECAGLYN 1-L (Polyglycerol-10 Laurate): CAS No. 34406-66-1. NIKKOL Hexaglyn 1-SV (Polyglycerol-6 Stearate): CAS No. 95461-65-7. EMOGREEN L19 (C15-19 Alkane): Purchased from SEPPIC. Caprylic / Capric Triglyceride: Purchased from Evonik. Glycerin: CAS No. 56-81-5. Xanthan gum (Keltrol® AP CG-T): Purchased from CP Kelco, CAS No. 11138-66-2. Lakelandia alba (Limenthanes Alba) seed oil: Purchased from Haimings (Shanghai) New Materials Co., Ltd. Butylene glycol: CAS No. 107-88-0. Isododecane: CAS No. 31807-55-3.

[0049] Example 1 This embodiment provides a nanoemulsion formulation and its preparation method.

[0050] To prepare the nanoemulsion formulation, the following raw materials were prepared by weight percentage: 2.5% rose PDRN, 0.5% salmon PDRN, 5% probiotic fermented PDRN, 6% polyglycerol-10 laurate (HLB value=15.5), 4% polyglycerol-6 stearate (HLB value=8), 12% C15-19 alkyl, 8% caprylic / capric triglyceride, 8% glycerol, 0.2% xanthan gum, and the balance being deionized water.

[0051] Specific preparation steps: (1) Weigh each raw material according to the above proportions.

[0052] (2) Mix polyglycerol-10 laurate, polyglycerol-6 stearate, C15-19 alkyl and octanoic acid / decanoic acid triglyceride evenly, heat to 40°C under stirring (stirring speed is 800 rpm), stir until all raw materials are completely dissolved, and keep at 40°C for 35 minutes to obtain the oil phase; (3) Mix glycerol, xanthan gum and deionized water, heat to 60°C under stirring (stirring speed is 300 rpm), and stir until all raw materials are completely dissolved to obtain an aqueous phase; (4) Add the aqueous phase obtained in step (3) dropwise to the oil phase obtained in step (2), stir for 40 minutes in a 40°C water bath, and adjust the pH of the system to 5.8 with citric acid (this pH value is in the stable activity range of PDRN (pH 5.5-6.5), which can protect the DNA fragment structure of PDRN from degradation and maintain its biological activity), to obtain a nanoemulsion mixture; (5) After mixing rose PDRN, salmon PDRN and probiotic fermented PDRN evenly, add them to the nanoemulsion mixture obtained in step (4), stir at room temperature, the stirring speed is 800 rpm, and the stirring time is 35 minutes to obtain the nanoemulsion preparation (referred to as composition A).

[0053] Example 2 This embodiment provides a nanoemulsion formulation and its preparation method.

[0054] To prepare the nanoemulsion formulation, the following raw materials were prepared by weight percentage: 2% rose PDRN, 0.2% salmon PDRN, 5.2% probiotic fermented PDRN, 7% polyglycerol-10 laurate (HLB value = 15.5), 5% polyglycerol-6 stearate (HLB value = 8), 10% C15-19 alkyl, 6% caprylic / capric triglyceride, 2% meadowfoam seed oil, 6% glycerol, 0.2% xanthan gum, and the balance being deionized water.

[0055] The specific preparation steps of the nanoemulsion formulation in this embodiment are basically the same as those in Example 1, except that in step (1), each raw material is weighed according to the proportions described above in this embodiment.

[0056] The nanoemulsion preparation obtained in this embodiment is designated as composition B.

[0057] Example 3 This embodiment provides a nanoemulsion formulation and its preparation method.

[0058] To prepare the nanoemulsion formulation, the following raw materials were prepared by weight percentage: 3% rose PDRN, 0.15% salmon PDRN, 5% probiotic fermented PDRN, 7% polyglycerol-10 laurate (HLB value = 15.5), 4% polyglycerol-6 stearate (HLB value = 8), 10% isododecane, 6% caprylic / capric triglyceride, 6% butylene glycol, 0.2% xanthan gum, and the balance being deionized water.

[0059] The specific preparation steps of the nanoemulsion formulation in this embodiment are basically the same as those in Example 1, except that in step (1), each raw material is weighed according to the proportions described above in this embodiment.

[0060] The nanoemulsion preparation obtained in this embodiment is designated as composition C.

[0061] Example 4 This embodiment provides a nanoemulsion formulation and its preparation method.

[0062] To prepare the nanoemulsion formulation, the following raw materials were prepared by weight percentage: 4% rose PDRN, 0.3% salmon PDRN, 2.5% probiotic fermented PDRN, 6% polyglycerol-10 laurate (HLB value = 15.5), 5% polyglycerol-6 stearate (HLB value = 8), 10% C15-19 alkyl, 7% caprylic / capric triglyceride, 7% glycerol, 0.2% xanthan gum, and the balance being deionized water.

[0063] The specific preparation steps of the nanoemulsion formulation in this embodiment are basically the same as those in Example 1, except that in step (1), each raw material is weighed according to the proportions described above in this embodiment.

[0064] The nanoemulsion preparation obtained in this embodiment is denoted as composition D.

[0065] Comparative Example 1 (without added rose PDRN, salmon PDRN, and probiotic fermented PDRN) This comparative example provides a nanoemulsion formulation and its preparation method.

[0066] The raw materials used in this comparative example for preparing the nanoemulsion formulation are basically the same as those in Example 1, except that rose PDRN, salmon PDRN, and probiotic fermentation PDRN were not added. Specifically, the nanoemulsion formulation was prepared by weight percentage as follows: 6% polyglycerol-10 laurate (HLB value = 15.5), 4% polyglycerol-6 stearate (HLB value = 8), 12% C15-19 alkyl, 8% caprylic / capric triglyceride, 8% glycerol, 0.2% xanthan gum, and the balance being deionized water.

[0067] The specific preparation steps of the nanoemulsion formulation in this comparative example are basically the same as those in Example 1, except that step (5) is not performed. Specifically, the specific preparation steps of the nanoemulsion formulation in this comparative example are as follows: (1) Weigh each raw material according to the above proportions.

[0068] (2) Mix polyglycerol-10 laurate, polyglycerol-6 stearate, C15-19 alkyl and octanoic acid / decanoic acid triglyceride evenly, heat to 40°C under stirring (stirring speed is 800 rpm), stir until all raw materials are completely dissolved, and keep at 40°C for 35 minutes to obtain the oil phase; (3) Mix glycerol, xanthan gum and deionized water, heat to 60°C under stirring (stirring speed is 300 rpm), and stir until all raw materials are completely dissolved to obtain an aqueous phase; (4) The aqueous phase obtained in step (3) is added dropwise to the oil phase obtained in step (2), and stirred for 40 minutes in a water bath at 40°C. The pH of the system is adjusted to 5.8 with citric acid (this pH value is in the stable activity range of PDRN (pH 5.5–6.5), which can protect the DNA fragment structure of PDRN from degradation and maintain its biological activity), to obtain the nanoemulsion preparation (denoted as composition E).

[0069] Comparative Example 2 (without xanthan gum) This comparative example provides a nanoemulsion formulation and its preparation method.

[0070] The raw materials used in this comparative example for preparing the nanoemulsion formulation are basically the same as those in Example 1, except that xanthan gum was not added. Specifically, the nanoemulsion formulation was prepared by weight percentage as follows: rose PDRN 2.5%, salmon PDRN 0.5%, probiotic fermented PDRN 5%, polyglycerol-10 laurate (HLB value = 15.5) 6%, polyglycerol-6 stearate (HLB value = 8) 4%, C15-19 alkyl 12%, caprylic / capric triglyceride 8%, glycerol 8%, and the balance being deionized water.

[0071] The specific preparation steps of the nanoemulsion formulation in this comparative example are basically the same as those in Example 1, except that in step (3), "mixing glycerol, xanthan gum and deionized water" is replaced with "mixing glycerol and deionized water".

[0072] The nanoemulsion preparation obtained in this comparative example is denoted as composition F.

[0073] Comparative Example 3 (without polyglycerol-10 laurate) This comparative example provides a nanoemulsion formulation and its preparation method.

[0074] The raw materials used in this comparative example for preparing the nanoemulsion formulation are basically the same as those in Example 1, except that polyglycerol-10 laurate is not added. Specifically, the nanoemulsion formulation is prepared by weight percentage as follows: rose PDRN 2.5%, salmon PDRN 0.5%, probiotic fermented PDRN 5%, polyglycerol-6 stearate (HLB value=8) 4%, C15-19 alkyl 12%, caprylic / capric triglyceride 8%, glycerol 8%, xanthan gum 0.2%, and the balance being deionized water.

[0075] The specific preparation steps of the nanoemulsion formulation in this comparative example are basically the same as those in Example 1, except that in step (2), “polyglycerol-10 laurate, polyglycerol-6 stearate, C15-19 alkyl and octanoic acid / decanoic acid triglyceride are mixed evenly” is replaced with “polyglycerol-6 stearate, C15-19 alkyl and octanoic acid / decanoic acid triglyceride are mixed evenly”.

[0076] The nanoemulsion preparation obtained in this comparative example is denoted as composition G.

[0077] Comparative Example 4 (without polyglycerol-6 stearate) This comparative example provides a nanoemulsion formulation and its preparation method.

[0078] The raw materials used in this comparative example for preparing the nanoemulsion formulation are basically the same as those in Example 1, except that polyglycerol-6 stearate is not added. Specifically, the nanoemulsion formulation is prepared by weight percentage as follows: rose PDRN 2.5%, salmon PDRN 0.5%, probiotic fermented PDRN 5%, polyglycerol-10 laurate (HLB value = 15.5) 6%, C15-19 alkyl 12%, caprylic / capric triglyceride 8%, glycerol 8%, xanthan gum 0.2%, and the balance being deionized water.

[0079] The specific preparation steps of the nanoemulsion formulation in this comparative example are basically the same as those in Example 1, except that in step (2), “polyglycerol-10 laurate, polyglycerol-6 stearate, C15-19 alkyl and octanoic acid / decanoic acid triglyceride are mixed evenly” is replaced with “polyglycerol-10 laurate, C15-19 alkyl and octanoic acid / decanoic acid triglyceride are mixed evenly”.

[0080] The nanoemulsion formulation prepared in this comparative example is denoted as composition H.

[0081] Test Example 1 This test case provides an experimental study for structural testing of nanoemulsion formulations.

[0082] I. Compositions A, E, F, G, and H of this invention were imaged using a transmission electron microscope (TEM) with negative staining technique. The results are as follows: Figures 1-5 As shown. Figure 1 This is a TEM image of the nanoemulsion formulation (composition A) in Example 1 of the present invention, with a scale bar of 200 nm; Figure 2 The image shows a TEM image of the nanoemulsion formulation (composition E) in Comparative Example 1 of this invention, with a scale bar of 500 nm. Figure 3 The image shows a TEM image of the nanoemulsion formulation (composition F) in Comparative Example 2 of this invention, with a scale bar of 200 nm. Figure 4 The image shows a TEM image of the nanoemulsion formulation (composition G) in Comparative Example 3 of this invention, with a scale bar of 500 nm. Figure 5 The image shown is a TEM image of the nanoemulsion formulation (composition H) in Comparative Example 4 of this invention. The scale bar is 1.0 μm.

[0083] Depend on Figures 1-5 It can be concluded that composition A exhibits densely packed, spherical core-shell droplets with a particle size distribution of 10-200 nm. Composition E also shows spherical core-shell droplets with a particle size distribution of 100-200 nm and a relatively dense arrangement. Composition F retains the spherical core-shell structure with a particle size distribution of 100-200 nm, but the arrangement is relatively loose. Compositions G and H show varying degrees of droplet damage and structural incompleteness, with particle sizes increasing to 200-500 nm, and significant droplet aggregation and adhesion. Compared to composition A, the droplet size in compositions G and H is significantly increased, and the structural stability is significantly reduced, failing to maintain the integrity of the spherical core-shell structure.

[0084] II. Compositions A, E, F, G, and H of this invention were photographed under an optical microscope, and the results are as follows: Figures 6-10 As shown. Figure 6This is a microscopic image of the nanoemulsion formulation (composition A) in Example 1 of the present invention under an optical microscope, with a scale bar of 10 μm; Figure 7 This is a microscopic image of the nanoemulsion formulation (composition E) in Comparative Example 1 of the present invention under an optical microscope. The scale bar is 20 μm. Figure 8 This is a microscopic image of the nanoemulsion formulation (composition F) in Comparative Example 2 of the present invention under an optical microscope. The scale bar is 20 μm. Figure 9 This is a microscopic image of the nanoemulsion formulation (composition G) in Comparative Example 3 of the present invention under an optical microscope. The scale bar is 20 μm. Figure 10 The image shows the microstructure of the nanoemulsion formulation (composition H) in Comparative Example 4 of this invention under an optical microscope. The scale bar is 20 μm.

[0085] Depend on Figures 6-10 It can be concluded that the droplets in composition A are small in size and uniformly distributed under microscopic conditions, with no significant aggregation or adhesion. Compared to composition A, the droplet size in compositions F (without xanthan gum), G (without polyglycerol-10 laurate), and H (without polyglycerol-6 stearate) is significantly larger, and the uniformity is poor. Furthermore, due to... Figure 9 It can be seen that a large number of dispersed droplets in composition G are enriched at the oil-water interface. The spherical outline of the droplets and the integrity of the interfacial film are destroyed, making it impossible to form a continuous and stable nanoscale dispersion system. This easily induces droplet aggregation, merging and phase separation behavior, ultimately leading to a decrease in the stability of the nanoemulsion formulation.

[0086] III. Dynamic light scattering (DLS) and zeta potential tests were performed on the nanoemulsion formulation (composition A) of this invention, and the results are as follows: Figures 11-12 As shown. Figure 11 This is a DLS particle size distribution diagram of the nanoemulsion formulation (composition A) in Example 1 of the present invention; Figure 12 This is a Zeta potential diagram of the nanoemulsion formulation (composition A) in Example 1 of the present invention.

[0087] Depend on Figure 11 It is known that the average particle size (Z-Average) of composition A is 112.9 nm, the polydispersity index (PDI) is 0.233, the particle size distribution curve is unimodal with the main peak particle size at 146.8 nm, and there are no secondary peaks or abnormally large particle size components. The overall particle size is concentrated in the range of 10-200 nm, with a narrow particle size distribution and excellent uniformity. This confirms that composition A forms a nanoemulsion system with uniform particle size distribution and excellent dispersibility. This provides core structural support for the physical and long-term stability of nanoemulsion formulations and can also improve the bioavailability of active ingredients (rose PDRN, salmon PDRN and probiotic fermented PDRN) by taking advantage of the transdermal effect of nano-sized particles.

[0088] Depend on Figure 12 It is known that the zeta potential of composition A is -30.4 mV, the zeta deviation is 7.36 mV, the potential distribution curve is single-peaked with no secondary peaks or abnormal distribution, and the charge distribution is uniform, indicating that the surface charge distribution of the droplets in the system is excellent. The zeta potential is in the stable range of >-30 mV, and the electrostatic repulsion between droplets can effectively inhibit aggregation and delamination. At the same time, composition A has a low conductivity of 0.0102 mS / cm, indicating that the overall ionic strength of the system is low and the double electric layer structure on the surface of the droplets is stable, which further supports the long-term stability of the system. Moreover, the mild environment of low ionic strength can better preserve the bioactivity of the active ingredients (rose PDRN, salmon PDRN and probiotic fermented PDRN).

[0089] Test Example 2 This test example provides a stability test experiment for nanoemulsion formulations.

[0090] 1. Using compositions A through H of this invention as samples, the pH value changes and appearance of the samples under different experimental conditions (influence factor experiment, accelerated experiment, and long-term experiment) were tested (observing whether the samples showed stratification and recording the results; if no stratification was observed, the samples were considered to be without abnormalities) to evaluate the physical stability of each composition. Influencing factor experiment: 100g samples were placed in high temperature (50℃), light (illuminance: 4500lx), and cold resistance (-18℃) environments, and the pH value and appearance of the samples were tested on the 10th day (10D), 30th day (1M), 60th day (2M), or 90th day (3M). Accelerated testing: Place 100g of sample in an environment of 45°C and test the pH value and appearance of the sample on day 10 (10D), day 30 (1M), day 60 (2M), or day 90 (3M). Long-term experiment: 100g samples were placed in environments at room temperature and refrigerated (4℃), and the pH value and appearance of the samples were tested on day 10 (10D), day 30 (1M), day 60 (2M), or day 90 (3M). Specific details and test results are shown in Table 1.

[0091] Table 1. Physical stability test results of compositions A to H under different experimental conditions. In Table 1, / indicates that it was not tested.

[0092] As shown in Table 1, compositions A through E showed no stratification and had minimal pH changes, demonstrating excellent physical stability and the ability to withstand extreme environments under different conditions, making them easy to store and transport. In contrast, compositions F through H exhibited poor physical stability and stratified under extreme environments under different conditions.

[0093] II. Compositions A through H of this invention were used as samples for centrifugal stability testing. The specific testing method is as follows: 10g of sample was incubated at 40℃ for 1 hour, and then centrifuged at 3000rpm, 4000rpm, and 5000rpm for 30 minutes respectively. After centrifugation, the sample was observed and recorded for oil release or stratification. If no oil release or stratification was observed after centrifugation at the corresponding speed, the sample was considered to be in normal condition, indicating that the sample could withstand the speed conditions and had excellent centrifugal stability. The centrifugal stability test results of compositions A through H are shown in Table 2.

[0094] Table 2. Centrifugal stability test results of compositions A to H As shown in Table 2, compositions A through E showed no oil exudation or stratification, and could withstand high speeds of 3000–5000 rpm, exhibiting excellent centrifugal stability. In contrast, compositions F through H showed oil exudation or stratification, were not resistant to high speeds of 3000–5000 rpm, and had poor centrifugal stability.

[0095] III. Compositions A through H of this invention were used as samples for long-term stability testing. The specific testing method is as follows: 100g of the sample was stored at room temperature for three months, then centrifuged for 30 minutes at different speeds (2000 rpm, 3000 rpm, 4000 rpm). After centrifugation, the sample was observed and recorded for any oil release or precipitation. If no oil release or precipitation occurred, the sample was considered to be in normal condition, indicating excellent long-term stability. Compositions A through H of this invention were used as samples for testing, and the results are shown in Table 3.

[0096] Table 3. Results of long-term stability tests for compositions A through H As shown in Table 3, compositions A through E showed no oil exudation or precipitation and could withstand high speeds of 2000–4000 rpm after being stored at room temperature for 3 months, demonstrating excellent long-term stability. In contrast, compositions F through H exhibited oil exudation and precipitation, indicating poorer long-term stability.

[0097] Based on the analysis in Tables 1-3, a comparison of Composition A and Composition F shows that the addition of xanthan gum can effectively improve the centrifugal stability, physical stability, and long-term stability of nanoemulsion formulations. The reason for this may be that xanthan gum and polyglycerol fatty acid ester nonionic emulsifiers (polyglycerol-10 laurate and polyglycerol-6 stearate) have a synergistic effect. Together, they construct a three-dimensional network structure with pseudoplastic characteristics, which strengthens the steric hindrance and electrostatic repulsion between emulsion droplets, while anchoring emulsifier molecules and reinforcing the stability of the interfacial film. The stability of the nanoemulsion formulation is significantly improved, thereby effectively inhibiting the phenomena of droplet aggregation and stratification under high-speed centrifugation. By comparing composition A with compositions G and H, it can be seen that using polyglycerol-10 laurate and polyglycerol-6 stearate as a compound emulsifier can effectively improve the centrifugal stability of nanoemulsion formulations. The reason may be that polyglycerol-10 laurate, with its high HLB value and polyhydroxy hydrophilic long-chain structure, can form a dense and elastic hydration interfacial film at the oil-water interface. Through steric hindrance and electrostatic repulsion, it can effectively reduce interfacial tension, inhibit emulsion droplet aggregation, and prevent stratification. The combination of polyglycerol-10 laurate and polyglycerol-6 stearate has excellent formulation compatibility and significantly improves the stability of nanoemulsion formulations.

[0098] Test Example 3 This test case provides an experiment to test the efficacy of nanoemulsion formulations.

[0099] I. Longevity gene expression level test To evaluate the anti-aging and repair effects of the nanoemulsion formulation of the present invention, composition A and composition E were used as samples, and relevant tests were carried out based on the UVB-induced photodamage model of human skin fibroblasts.

[0100] 1. Test principle: This embodiment uses a photodamage model induced by UVB irradiation of human skin fibroblasts to detect changes in SIRT1, SIRT3, and CAT genes after sample treatment, and evaluates the anti-aging and repair efficacy of the sample based on UVB-induced photodamage to human skin fibroblasts.

[0101] 2. Experimental cells: Human skin fibroblasts (HFB), purchased from Shaanxi Boxi Biotechnology Co., Ltd., cell passage number P6.

[0102] 3. Main reagents and equipment: DMEM medium (Gibco), fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.), PBS (Solvebao), trypsin (GIBCO), RNA EASY FAST total RNA extraction kit (Tiangen, DP452), PrimeScript TMRT Master Mix (TAKARA, RR06A), TBGreenpremixExTaqTMII (TAKARA, RR820A); CO2 incubator (Thermo, 150i), clean bench (Suzhou Antai, SW-CJ-1F), inverted microscope (Leica Microsystems Shanghai Co., Ltd., DMI1), real-time quantitative PCR instrument (Quantstudio3), MiniCentrifuge (JoAnLAB), spinometer (Scientificindustries), micro-volume nucleic acid and protein analyzer (Shanghai Jinpeng Analytical Instruments Co., Ltd.), UVB lamp (Philips).

[0103] 4. All PCR primers used in this invention were synthesized by Qingke Biotechnology Co., Ltd.

[0104] 5. Experimental steps: (1) Cell resuscitation and culture: Human skin fibroblasts (HFB cells) of passage P6 were taken out from the liquid nitrogen tank, resuscitated and seeded into culture flasks, and cultured in an incubator at 37℃ and 5% CO2; after the cell confluence reached 70%-80%, they were digested and counted to obtain cultured cells. (2) Cell seeding: 4×10 4 / The seeding density of the wells is to seed the cultured cells into 12-well plates with a seeding volume of 1 mL per well. Then, the seeded 12-well plates are placed in a cell culture incubator and cultured for 24 h at 37°C and 5% CO2. (3) Experimental drug administration and grouping: control group (no UVB irradiation required), model group and sample group are set up. The drug administration volume is 1 mL per well. Each experimental group is set up with 3 replicate wells. See Table 4 for specific experimental details.

[0105] Table 4 Experimental Scheme (4) Extraction of total RNA from cells: Follow the instructions of the RNA EASY FAST Total RNA Extraction Kit.

[0106] (5) cDNA acquisition: Using extracted total cellular RNA as a template, reverse transcription was performed to obtain cDNA. The reverse transcription reaction system was PrimeScript. TM Add 4 μL of RT Master Mix and bring the RNase-Free dH2O to a final volume of 20 μL. Reaction conditions: 37 °C for 15 min, 85 °C for 5 s, and 4 °C.

[0107] (6) Real-time quantitative PCR: Using cDNA as a template, real-time quantitative PCR was performed to detect three longevity-related genes, SIRT1, SIRT3, and CAT; GADPH was used as an internal reference gene. The primer sequences used in the real-time quantitative PCR detection are shown in Table 5. The real-time quantitative PCR reaction system was as follows: TBGreen premix ExTaq™ II 5 μL, forward and reverse primers (10 μM) 0.4 μL each, ROX Reference Dyeor DyeII 0.2 μL, cDNA template 1 μL, and sterile water to a final volume of 10 μL; the reaction conditions were: 195℃ pre-denaturation for 30 s; 95℃ amplification for 5 s, 55℃ amplification for 30 s, for 30 cycles; and finally, a dissolution reaction was performed: 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s. Each experimental group had 3 replicate wells, and the arithmetic mean of the test results was taken as the final data.

[0108] Table 5 Primer sequences used in quantitative real-time PCR detection This invention uses composition A and composition E as samples, respectively, and performs longevity-related gene detection (SIRT1, SIRT3, and CAT) according to the above experimental steps, obtaining the following results: Figures 13-15 The results are shown.

[0109] Figure 13 This is the result of detecting the expression level of the SIRT1 gene in Test Example 3 of the present invention; Figure 14 This is the result of detecting the expression level of the SIRT3 gene in Test Example 3 of the present invention; Figure 15 This is the result of detecting the expression level of the CAT gene in Test Example 3 of the present invention.

[0110] Depend on Figures 13-15 It can be seen that, compared with the control group, the expression levels of SIRT1, SIRT3 and CAT genes in the model group were significantly reduced, indicating that the UVB-induced photodamage model of human skin fibroblasts was successfully established.

[0111] Depend on Figure 13 It was found that, compared with the model group, the expression level of SIRT1 gene in composition A increased significantly, with an increase rate of 182.03%. Compared with the model group, the expression level of SIRT1 gene in composition E increased, with an increase rate of 60.25%. t-test statistical analysis showed that composition A significantly enhanced the expression of the SIRT1 gene, indicating that composition A has a more significant anti-aging and repair effect on mitochondria.

[0112] Depend on Figure 14It was found that, compared with the model group, the expression level of SIRT3 gene in composition A increased significantly, with an increase rate of 230.78%. Compared with the model group, the expression level of SIRT3 gene in composition E increased, with an increase rate of 58.67%. t-test statistical analysis showed that composition A significantly enhanced the expression of SIRT3 gene, indicating that composition A has a more significant anti-aging and repair effect on mitochondria.

[0113] Depend on Figure 15 It was found that, compared with the model group, the expression level of the CAT gene in composition A increased significantly, with an increase rate of 58.91%. Compared with the model group, the expression level of the CAT gene in composition E increased, with an increase rate of 40.56%. t-test statistical analysis showed that composition A significantly enhanced the expression of the CAT gene, indicating that composition A has a more significant anti-aging and repair effect on mitochondria.

[0114] II. Mitochondrial NAD+ Concentration Test To further evaluate the anti-aging and repair effects of the nanoemulsion formulation of the present invention, compositions A and E were used as samples, and relevant tests were carried out based on the UVA-induced photoaging model of human skin fibroblasts.

[0115] 1. Test Principle: NADP (Nicotinamide adenine dinucleotide phosphate) is a coenzyme for many redox reactions, including both NADP+ (oxidized form) and NADPH (reduced form). NADPH is a phosphorylated derivative of the ribonucleotide ring linked to adenine in NAD+ (nicotinamide adenine dinucleotide), participating in various anabolic reactions such as the synthesis of lipids, fatty acids, and nucleotides.

[0116] 2. Testing Basis: Referring to "Detection Method of Mitochondrial NAD+ Concentration Based on Fibroblasts", a UVA-induced photoaging model of human skin fibroblasts was used. The NAD+ level was detected by colorimetric method to evaluate the anti-aging and repair efficacy of the test samples.

[0117] 3. Experimental cells: Human skin fibroblasts (HFB), purchased from Shaanxi Boxi Biotechnology Co., Ltd., cell passage number P6.

[0118] 4. Main reagents and equipment: DMEM culture medium (Gibco), newborn calf serum (NBS, Lanzhou Rongye), PBS (Solepro), quercetin (Aladdin), NAD+ detection kit (Beyotime); CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-2F), UVA irradiator (Philips), fluorescence microscope (Olympus, BX43), super-resolution microscope (NanoInsights-TechCo., Multi-SIM).

[0119] 5. Testing Method: (1) Cell seeding: Human skin fibroblasts (HFB cells) with a replacement number of P6 were thawed and seeded into culture flasks. They were cultured at 37°C and 5% CO2. When the cell confluence reached 60%, they were digested with trypsin and counted to obtain cultured cells. 5 The seeding density of the cells after culture was determined by seeding them into 6-well plates and culturing them at 37°C and 5% CO2 for 24 hours.

[0120] (2) Experimental grouping and administration: Blank control group (BC), negative control group (NC), positive control group (PC) and sample group were set up. The administration volume per well was 2 mL, and each experimental group was set up with 3 replicate wells. The experimental scheme is shown in Table 1 below. After administration, the 6-well plate was placed in an incubator at 37℃ and 5% CO2 for 24 h.

[0121] (3) Colorimetric detection: After incubation, cells were collected and NAD+ detection was performed according to the instructions of the NAD+ detection kit.

[0122] Table 6 Experimental Scheme 6. Calculation of Improvement Rate: The improvement rate is calculated using the following formula: .

[0123] 7. Statistical Analysis of Results: GraphPadPrism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, P < 0.01 was considered significantly significant, and P < 0.001 was considered highly significant. Each experimental group had three replicates, and the arithmetic mean of the test results was used as the final data.

[0124] This invention uses composition A and composition E as samples, and performs NAD+ concentration tests according to the above experimental steps to obtain the following results. Figure 16 The results are shown. Figure 16The results show the NAD+ concentration detection in Test Example 3 of this invention.

[0125] Depend on Figure 16 It was found that, compared with the blank control group (BC), the concentration of NAD+ per unit protein in the negative control group (NC) was significantly decreased, indicating that the stimulation conditions in this test were effective. Compared with the negative control group (NC), the concentration of NAD+ per unit protein in the positive control group (PC) was significantly increased, indicating that the positive control was effective in this test. Compared with the negative control group (NC), the concentration of NAD+ per unit protein in sample group - composition A was significantly increased, with an increase rate of 89.54%. Compared with the negative control group (NC), the concentration of NAD+ per unit protein in sample group - composition E was increased, with an increase rate of 50.58%. t-test statistical analysis showed that composition A had a significant effect on increasing NAD+ levels, indicating that composition A can effectively increase NAD+ levels in human skin fibroblasts, showing significant potential for mitochondrial function repair and improvement of cell aging.

[0126] III. Anti-inflammatory cytokine content test To evaluate the anti-inflammatory efficacy of the nanoemulsion formulation of the present invention, compositions A and E were used as samples, and relevant tests were carried out based on the lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation model.

[0127] 1. Test basis: Referring to the "Evaluation Methods for Anti-inflammatory Efficacy of Cosmetics (Cell Model Method)", "Technical Specifications for Construction of In Vitro Cell Inflammation Models" and industry-standard evaluation criteria, an LPS-induced RAW264.7 cell inflammation model was used. The secretion level of the anti-inflammatory factor IL-10 was detected by ELISA to evaluate the anti-inflammatory activity of the test samples.

[0128] 2. Test principle: Lipopolysaccharide (LPS) can specifically activate the inflammatory signaling pathway in RAW264.7 macrophages, inducing cellular inflammatory responses. IL-10 is an endogenous anti-inflammatory cytokine, and its secretion level is positively correlated with cellular anti-inflammatory activity. Pretreatment of cells with the test sample can regulate the secretion of the anti-inflammatory cytokine IL-10 by modulating the inflammatory pathway. The ELISA method, utilizing antigen-antibody specific binding and enzymatic colorimetric reaction, was used to quantitatively detect the content of the anti-inflammatory cytokine IL-10 in the cell supernatant, thereby assessing the anti-inflammatory efficacy of the sample.

[0129] 3. Main materials: RAW264.7 macrophages; lipopolysaccharide (LPS); Samples: Composition A, Composition E; double-distilled water (ddH2O); mouse interleukin-10 (IL-10) ELISA kit.

[0130] 4. Testing Method: (1) Cell seeding: Raw264.7 cells in the logarithmic growth phase were revived and seeded into culture plates and cultured at 37°C and 5% CO2 until adherence.

[0131] (2) Sample pretreatment: A control group, a model group, and a sample group were set up. The control group and the model group were added with culture medium containing ddH2O, and the sample group was added with culture medium containing an equal volume of sample solution. The samples were pre-incubated at 37℃ and 5% CO2 for 24h. The sample solution was obtained by mixing the sample with double-distilled water (ddH2O) evenly, and the concentration of the sample in the sample solution was 20mg / mL. The amount of ddH2O in the culture medium containing ddH2O was the same as the amount of sample solution in the culture medium containing the sample solution.

[0132] (3) Inflammation modeling: After the pre-incubation, except for the control group, each group was given LPS with a final concentration of 1 μg / mL and cultured for 24 h.

[0133] (4) Sample collection: After the stimulation culture is completed, the cell supernatant of each group is collected, and after centrifugation to remove impurities, IL-10 is detected.

[0134] (5) IL-10 detection: The mouse interleukin-10 (IL-10) ELISA kit was tested according to the instructions. The absorbance value was measured by an ELISA reader, and the IL-10 content of each group was calculated according to the standard curve. Each group was set up with 3 parallel replicates, and the arithmetic mean of the test results was taken as the final data.

[0135] This invention uses composition A and composition E as samples, and tests the content of the anti-inflammatory cytokine IL-10 according to the above experimental steps, obtaining the following results. Figure 17 The results are shown. Figure 17 The results show the detection of IL-10 content in Test Example 3 of this invention.

[0136] Depend on Figure 17 The results showed that the IL-10 content in the model group was significantly increased compared with the control group, indicating that the LPS-induced RAW264.7 cell inflammation model was successfully established. Compared with the model group, the IL-10 content in both sample group-composition A and sample group-composition E increased, with increases of 32.22% and 20.97%, respectively. The effect of sample group-composition A on increasing IL-10 content was significantly greater than that of composition E, indicating that composition A has a more significant anti-inflammatory effect.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoemulsion formulation containing multi-source PDRN, characterized in that, The raw materials for preparing the nanoemulsion formulation containing multi-source PDRN, by mass percentage, include the following components: 3%–15% active ingredient, 7%–14% polyglycerol fatty acid ester nonionic emulsifier, 15%–30% emollient, 6%–8% moisturizer, 0.1%–0.3% microbial extracellular polysaccharide, and the balance being water; The active ingredient consists of rose PDRN, salmon PDRN, and probiotic fermented PDRN.

2. The nanoemulsion formulation containing multi-source PDRN according to claim 1, characterized in that, The HLB value of the polyglycerol fatty acid ester nonionic emulsifier is 6 to 17; The polyglycerol fatty acid ester nonionic emulsifier is one of two polyglycerol-10 laurate or polyglycerol-6 stearate.

3. The nanoemulsion formulation containing multi-source PDRN according to claim 1, characterized in that, The microbial extracellular polysaccharides include xanthan gum.

4. The nanoemulsion formulation containing multi-source PDRN according to claim 1, characterized in that, The mass ratio of the rose PDRN, the salmon PDRN, and the probiotic fermented PDRN is (2-4):(0.1-0.5):(2-6).

5. The nanoemulsion formulation containing multi-source PDRN according to claim 1, characterized in that, The moisturizer is at least one of glycerin, butylene glycol, dipropylene glycol, and glyceryl polyether-26; And / or, the emollient is at least one of C15-19 alkyl, caprylic / capric triglyceride, meadowfoam seed oil, and isododecane.

6. A method for preparing a nanoemulsion formulation containing multi-source PDRN as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Mix polyglycerol fatty acid ester nonionic emulsifier and emollient, heat to a first temperature under stirring, and keep at the first temperature to obtain oil phase; S2. Mix the humectant, extracellular polysaccharide and water, and heat to a second temperature under stirring to obtain the aqueous phase; S3. Add the aqueous phase dropwise to the oil phase, stir, and adjust the pH of the system to 5.5–6.5 to obtain a nanoemulsion mixture; S4. Rose PDRN, salmon PDRN and probiotic fermented PDRN are mixed and added to the nanoemulsion mixture. After stirring, a nanoemulsion preparation containing multi-source PDRN is obtained.

7. The method for preparing a nanoemulsion formulation containing multi-source PDRN according to claim 6, characterized in that, In step S1, the stirring speed is 700-1200 rpm, the first temperature is 40-50℃, and the heat preservation time is 30-40 minutes. And / or, in step S2, the second temperature is 50–65°C.

8. The method for preparing a nanoemulsion formulation containing multi-source PDRN according to claim 5, characterized in that, In step S3, the stirring temperature is 35-45°C and the stirring time is 30-40 minutes; And / or, in step S4, the stirring process is carried out at a speed of 700-1200 rpm for 30-40 minutes; the stirring process is carried out at room temperature.

9. The application of a nanoemulsion formulation containing multi-source PDRN in the preparation of products with anti-aging, repairing, and anti-inflammatory effects, characterized in that, The nanoemulsion formulation containing multi-source PDRN is the nanoemulsion formulation containing multi-source PDRN as described in any one of claims 1 to 5, or the nanoemulsion formulation containing multi-source PDRN is a nanoemulsion formulation containing multi-source PDRN prepared by the preparation method described in any one of claims 6 to 8.

10. The application of the nanoemulsion formulation containing multi-source PDRN according to claim 9 in the preparation of products with anti-aging, repairing, and anti-inflammatory effects, characterized in that, The nanoemulsion formulation containing multi-source PDRN can upregulate the expression of longevity genes SIRT1, SIRT3 and CAT. And / or, the nanoemulsion formulation containing multi-source PDRN can increase mitochondrial NAD+ levels; And / or, the nanoemulsion formulation containing multi-source PDRN can increase the content of the anti-inflammatory cytokine IL-10.

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