A composition with the effect of repairing and resisting skin cell aging and application thereof

Abstract

This invention discloses a composition with repairing and anti-aging effects on skin cells and its application, belonging to the field of cosmetics and skin care technology. Firstly, this invention provides a composite PDRN, composed of PDRN-1 derived from male salmon testes, PDRN-2 derived from *Lactobacillus plantarum*, and PDRN-3 derived from *Lactobacillus bulgaricus* in a mass ratio of 100:(0.5-1.5):(0.5-1.5). These three components synergistically act on multiple skin aging-related pathways. Further, it provides *Lactobacillus plantarum* microbial vesicles loaded with the above composite PDRN. The preparation method involves mixing a vesicle suspension with a protein content of 0.05-0.15 mg / mL with a composite PDRN solution of 1 wt%-2 wt% at a volume ratio of 1:(1-3), sonicating at 40 kHz for 30 min in an ice bath, followed by centrifugation purification and lyophilization. This invention achieves synergistic effects of carrier-load by scientifically compounding PDRN from multiple sources and optimizing the microbial vesicle delivery process, significantly enhancing anti-inflammatory, barrier repair, and anti-skin cell aging efficacy, while exhibiting good biocompatibility. It can be widely applied to the preparation of various repair and anti-skin cell aging cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetics and skin care technology, specifically to a composition with repairing and anti-aging effects on skin cells and its application. Background Technology

[0002] Skin aging is a complex physiological process involving the combined effects of endogenous factors (such as genes and hormones) and exogenous factors (such as ultraviolet radiation and pollution). Its core characteristics include impaired skin barrier function, degradation of collagen and elastin, chronically elevated inflammation levels, and decreased self-repair capabilities. Developing active ingredients with both highly effective repair and anti-aging functions to address skin aging has become a research hotspot in the cosmetics and skincare fields.

[0003] Polydeoxyribonucleotides (PDRNs), as bioactive polymers composed of deoxyribonucleotides, have shown potential in skin repair and anti-aging applications due to their properties such as promoting cell proliferation, tissue repair, and anti-inflammation. Currently, commercially available and research-used PDRNs are mainly derived from animal tissues (such as salmon testes), and their bioactivity has been confirmed to some extent. However, PDRNs from single sources have relatively limited targets and pathways of action, and their efficacy is limited when addressing the complex multifactorial and multi-pathway problem of skin aging, making it difficult to comprehensively and systematically address the multiple challenges of inflammation, matrix degradation, and barrier repair. In recent years, microbial-derived PDRNs have also gradually attracted attention, as they may possess immunomodulatory properties different from animal-derived PDRNs. However, how to scientifically combine PDRNs from different sources with different characteristics to produce synergistic effects, rather than simply adding them together, remains a technical challenge that has not yet been solved in the current technology.

[0004] On the other hand, the efficacy of active ingredients largely depends on their delivery efficiency. Many active ingredients, due to issues with molecular weight, hydrophilicity, or stability, struggle to effectively penetrate the skin's stratum corneum barrier to reach their target cells. While delivery systems such as liposomes and nanoparticles have been extensively studied, their application is limited by problems such as complex preparation, high cost, unstable encapsulation efficiency, or poor biocompatibility. Microbial vesicles, especially those derived from probiotics (such as Lactobacillus), offer a novel biological delivery vehicle with good biocompatibility, low immunogenicity, and a naturally amphiphilic membrane structure, theoretically capable of encapsulating hydrophilic active ingredients and promoting their cellular uptake. However, systematic research and optimization are currently lacking in how to efficiently and stably load specific combinations of active ingredients into microbial vesicles and optimize preparation process parameters (such as the ratio of vesicles to active ingredients, mixing methods, and particle size control) to maximize encapsulation efficiency, loading rate, and final bioefficacy. Furthermore, whether microbial vesicles themselves, as delivery carriers, merely function as "carriers" or can produce unexpected synergistic biological effects with specific loads, thus transcending simple physical mixing, remains a blank area to be explored in this field.

[0005] Therefore, developing a novel composition that utilizes a scientific blend of PDRNs from different sources and employs microbial vesicles for efficient delivery to produce significant synergistic repair and anti-skin cell aging effects is of great significance for improving the efficacy of cosmetics and skin care products. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition with repairing and anti-aging effects on skin cells and its applications. This composition enhances multiple effects, including soothing and anti-inflammatory properties, by combining animal-derived and microbial-derived PDRN in a specific ratio and utilizing *Lactobacillus plantarum* vesicles as a delivery carrier.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a compound PDRN with repair and anti-skin cell aging effects, characterized in that the compound PDRN includes PDRN-1, PDRN-2, and PDRN-3; wherein PDRN-1 is extracted from the testes of male salmon; PDRN-2 is extracted from Lactobacillus plantarum; and PDRN-3 is extracted from Lactobacillus bulgaricus.

[0008] Preferably, the PDRN-2 is extracted from Lactobacillus plantarum, with accession number CGMCC NO.25187; the PDRN-3 is extracted from Lactobacillus bulgaricus, with accession number SHBCC D24344.

[0009] Preferably, the mass ratio of PDRN-1, PDRN-2, and PDRN-3 in the composite PDRN is 100:(0.5-1.5):(0.5-1.5).

[0010] More preferably, the mass ratio of PDRN-1, PDRN-2, and PDRN-3 in the composite PDRN is 100:1.2:0.8.

[0011] Secondly, the present invention provides a microbial vesicle with repair and anti-skin cell aging effects, wherein the microbial vesicle is loaded with the composite PDRN described in the first aspect.

[0012] Preferably, the vesicle extract is derived from Lactobacillus plantarum; More preferably, the vesicle extract is derived from Lactobacillus plantarum, with accession number CGMCCNO.25187, and was purchased from Biostime Biotechnology Co., Ltd.

[0013] Thirdly, the present invention provides a method for preparing the microbial vesicles with repairing and anti-skin cell aging effects described in the second aspect, comprising the following preparation steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve the composite PDRN in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and then sonicate them to obtain mixture C; S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0014] Preferably, in the preparation step, the protein content in the mixture A in step S1 is 0.05-0.15 mg / mL; the particle concentration is ≥1.0 × 10⁻⁶. 8 particles / mL; the content of composite PDRN in the mixture B in step S2 is 1wt%-2wt%; the volume ratio of mixture A to mixture B in step S3 is 1:(1-3); the ultrasonic treatment is carried out under ice bath conditions, and the ultrasonic conditions are: ultrasonic frequency 40kHz; ultrasonic time 30min; the centrifugation speed in step S4 is 15000g, time 20min, and temperature 4℃.

[0015] Fourthly, the present invention provides the application of the PDRN described in the first aspect, or the microbial vesicles described in the second aspect, or the microbial vesicles prepared by the preparation method described in the third aspect, in the preparation of cosmetics with repairing and anti-skin cell aging effects.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Synergistic Effect of PDRNs from Multiple Sources: This invention creatively combines animal-derived (salmon testes) PDRNs with specific microbial-derived (Lactobacillus plantarum, Lactobacillus bulgaricus) PDRNs, rather than simply combining substances. Through specific mass ratios, these three PDRN molecules, from different sources and potentially differing in structure or activity profile, can act on multiple signaling pathways and cellular processes related to skin aging. The mechanism of this synergistic effect can be explained as follows: animal-derived PDRNs provide abundant nucleotide raw materials, promoting cellular energy metabolism and DNA repair; Lactobacillus plantarum-derived PDRNs effectively regulate skin microecology and innate immunity, inhibiting excessive inflammation; and Lactobacillus bulgaricus-derived PDRNs assist in regulating other skin repair pathways. The three work together to achieve synergistic effects. The synergistic effect of these three can more comprehensively regulate inflammatory responses and inhibit the activity of matrix-degrading enzymes such as elastase, addressing skin barrier damage, inflammation, and aging from multiple targets and levels, achieving a synergistic enhancement effect of "1+1+1>3".

[0017] 2. A "carrier-load" synergistic delivery and efficacy amplification system was constructed: This invention does not simply physically mix composite PDRN with microbial vesicles, but rather uses an optimized process to efficiently load composite PDRN into the interior of *Lactobacillus plantarum* vesicles. This microbial vesicle carrier not only solves the problems of transdermal absorption and easy degradation of biomacromolecules such as PDRN, but also promotes skin penetration and cellular uptake through its nanoscale size and biomembrane structure. More importantly, the carrier and load generate a synergistic biological effect that goes beyond simple physical addition. The membrane components of *Lactobacillus plantarum* vesicles themselves (such as lipids and proteins) may have mild immunomodulatory activity. When loaded with a specific ratio of composite PDRN, the two may activate complementary or mutually reinforcing signaling networks within the cell. While delivering PDRN, the vesicles' own components may interact with cells, creating a more favorable microenvironment for the efficacy of PDRN, or acting together at upstream regulatory nodes, thereby significantly amplifying the final anti-inflammatory, repair, and anti-skin cell aging responses.

[0018] 3. Optimized preparation process ensures maximum synergistic effect: The preparation method of this invention precisely controls the vesicle source, concentration, mixing ratio with active ingredients, ultrasonic treatment conditions, and particle size range of the final product. These optimized process parameters are not set in isolation, but rather to ensure that the composite PDRN can be loaded into vesicles with the highest efficiency, forming a final product with uniform size, good stability, and a particle size within the optimal range for skin delivery (50-220 nm). Optimal encapsulation and loading are the foundation of the aforementioned "carrier-load" synergistic effect, while optimal particle size is crucial for the effective delivery of this synergistic system to the site of action. Therefore, the preparation process and active ingredient formulation design of this invention complement each other, together forming a complete technical system that maximizes the synergistic repair and anti-skin cell aging effects, ensuring that the synergistic effects discovered in the laboratory can be realized and translated into the final product. Detailed Implementation

[0019] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0020] The sources of some of the bacterial strains in this invention are as follows: PDRN-1 was purchased from Baihong Synthetic Biotechnology (Yantai) Co., Ltd., product number: HYPDRN-ZH; The preparation method of PDRN-2 was based on the preparation method of Sample B in Example 1 of Patent Publication No. CN120330277B. The strain used was Lactobacillus plantarum (Latin name: Lactobacillus plantarum), with accession number CGMCCNO.25187, which was deposited on June 27, 2022 at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and was purchased from Shenghe Biotechnology Co., Ltd.

[0021] The preparation method of PDRN-3 refers to the preparation method of sample B in Example 1 of patent publication number CN120330277B; the bacterial strain used is Lactobacillus delbrueckii subsp. bulgaricus, which was purchased from Shanghai Biotechnology Center, accession number SHBCC D24344.

[0022] All other raw materials, reagents, and equipment are commercially available products.

[0023] Preparation of composite PDRN Composite PDRN-1: Composed of PDRN-1, PDRN-2, and PDRN-3 in a mass ratio of 100:1.2:0.8; Preparation method: Simply mix PDRN-1, PDRN-2, and PDRN-3 lyophilized powders in a specific mass ratio until homogeneous.

[0024] Composite PDRN-2: The only difference from composite PDRN-1 is that the mass ratio of PDRN-1, PDRN-2, and PDRN-3 is 100:0.5:0.5.

[0025] The only difference between composite PDRN-3 and composite PDRN-1 is that the mass ratio of PDRN-1, PDRN-2, and PDRN-3 is 100:1.5:1.5.

[0026] Composite PDRN-A: Composed of PDRN-1 and PDRN-3 in a mass ratio of 100:0.8; Preparation method: Simply mix PDRN-1 and PDRN-3 lyophilized powders in a specific mass ratio until homogeneous.

[0027] Composite PDRN-B: Composed of PDRN-1 and PDRN-2 in a mass ratio of 100:1.2; Preparation method: Simply mix PDRN-1 and PDRN-2 lyophilized powders in a specific mass ratio until homogeneous.

[0028] Composite PDRN-C: Composed of PDRN-2 and PDRN-3 in a mass ratio of 1.2:0.8; Preparation method: Simply mix PDRN-2 and PDRN-3 lyophilized powders in a specific mass ratio until homogeneous.

[0029] Composite PDRN-D: Composed of PDRN-1, PDRN-2, and PDRN-3 in a mass ratio of 5:1:2; Preparation method: Simply mix PDRN-1, PDRN-2, and PDRN-3 lyophilized powders in a specific mass ratio until homogeneous.

[0030] Preparation of Lactobacillus plantarum vesicles (1) Take 40g of wet Lactobacillus plantarum mud and resuspend it in 400mL PBS buffer. Centrifuge and wash twice at 15000g at 4℃ for 10min each time. Finally, resuspend it in 200mL PBS buffer and freeze at -80℃ for 20 hours. (2) After the resuspended liquid is completely thawed at room temperature, it is placed at -80℃ for 2 hours and then thawed at room temperature for use.

[0031] (3) The thawed resuspension was ultrasonically broken up in an ice bath using an ultrasonic cell disruptor to obtain the disrupted liquid. The disruption conditions were: working power 1000W, working time 2s, interval 3s, and total processing time 60min.

[0032] (4) Centrifuge the above-mentioned broken liquid at 4℃ and 15000g for 20min and collect the supernatant.

[0033] (5) The supernatant was filtered using a 0.22μm filter. The filtrate was rich in Lactobacillus plantarum vesicles. The Lactobacillus plantarum vesicles were frozen at -20℃ for later use.

[0034] Preparation of Lactobacillus bulgaricus vesicles The only difference from the preparation of Lactobacillus plantarum vesicles is that Lactobacillus bulgaricus is used to replace Lactobacillus plantarum with a similar amount.

[0035] Preparation of microbial vesicles: Microbial Vesicle-1 Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0036] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 9 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:1.7; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0037] Microbial Vesicle-2 Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve the composite PDRN-2 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0038] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 9 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:1.7; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0039] Microbial Vesicle-3 Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve the composite PDRN-3 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0040] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 9 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:1.7; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0041] Microbial Vesicle-4 Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0042] In step S1, the protein content in mixture A is 0.15 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 11 particles / mL; In step S2, the content of composite PDRN in mixture B is 2 wt%; In step S3, the volume ratio of mixture A to mixture B is 1:1; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0043] Microbial Vesicle-5 Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0044] In step S1, the protein content in mixture A is 0.05 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 8 particles / mL; In step S2, the content of composite PDRN in mixture B is 1 wt%; In step S3, the volume ratio of mixture A to mixture B is 1:3; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0045] Microbial vesicles-① Includes the following steps: S1: Mix Lactobacillus bulgaricus vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0046] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 10 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:1.7; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0047] Microbial vesicles-② Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0048] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 10 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:4; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0049] Microbial vesicles - ③ Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0050] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 10 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:0.5; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0051] Microbial vesicles-④ Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0052] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 9 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:1.7; The ultrasound conditions were: ultrasound frequency 20kHz; ultrasound time 30min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0053] Microbial vesicles-⑤ Includes the following steps: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve composite PDRN-1 in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and perform ultrasonic treatment in an ice bath to obtain mixture C. Take 10 mL to test the encapsulation rate and loading rate. S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

[0054] In step S1, the protein content in mixture A is 0.1 mg / mL; the particle concentration is 1.0 × 10⁻⁶. 9 particles / mL; In step S2, the content of composite PDRN in mixture B is 1.02 wt%. In step S3, the volume ratio of mixture A to mixture B is 1:1.7; The ultrasound conditions were: ultrasound frequency 40kHz; ultrasound time 15min. The centrifugation speed in step S4 is 15000g, the time is 20min, and the temperature is 4℃.

[0055] Efficacy testing Test Example 1 examines the effect of compound PDRN on inhibiting the secretion of TNF-α, IL-1β, and IL-6. Samples: Composite PDRN1-3, Composite PDRNA-D; Sample preparation: The above samples were mixed with ultrapure water to obtain test samples of different concentrations, as shown in Table 1.

[0056] Experimental method: An LPS-induced inflammation model of mouse monocytes and macrophages RAW264.7 was used.

[0057] Experimental Procedure: RAW264.7 mouse mononuclear macrophages were seeded into culture plates, and 20 μL of the test sample was added to each group. A blank control group and an LPS model group were set up. Both the blank control group and the LPS model group were treated with 20 μL of ultrapure water. After pretreatment for 2 hours, except for the blank control group, all other groups were stimulated with LPS (1 μg / mL) for 24 hours. Cell supernatants were collected, and the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 were detected using an ELISA kit. The inhibition rate of each group relative to the model group was calculated.

[0058] Inhibition rate / % = (LPS model group detection value - Test sample group detection value) / (LPS model group detection value - Blank control group detection value) × 100% Table 1. Determination of anti-inflammatory efficacy of the test samples

[0059] Test Example 2 tested the inhibition of elastase by the Example and Comparative Examples. Samples: Composite PDRN1-3, Composite PDRNA-D; Sample preparation: The above samples were mixed with ultrapure water to obtain test samples of different concentrations, as shown in Table 2.

[0060] Experimental method: Elastase inhibition assay was used.

[0061] Experimental Method: 100 μL of 0.2 mol / L tris-HCl buffer, 25 μL of 10 mmol / L N-methoxysuccinyl-alanine-proline-valine-4-nitroaniline, and 20 μL of the test sample were mixed and incubated at 25 °C for 10 min. Then, 25 μL of 0.3 U / mL elastase was added and the mixture was incubated for another 10 min. Finally, the OD value was measured at 410 nm. 20 μL of deionized water was used as a blank control instead of the test sample. The results are shown in Table 2 below.

[0062] Elastase inhibition rate (%) = (OD value of blank control group - OD value of test sample group) / OD value of blank control group × 100%.

[0063] Table 2. Effects of test substances on elastase activity Test sample concentration elastase inhibition rate / % Composite PDRN-1 0.3wt% 76.3 Composite PDRN-1 1wt% 88.5 Composite PDRN-1 0.05wt% 34.2 Composite PDRN-2 0.3wt% 68.9 Composite PDRN-3 0.3wt% 71.4 Composite PDRN-A 0.3wt% 52.6 Composite PDRN-B 0.3wt% 57.9 Composite PDRN-C 0.3wt% 44.7 Composite PDRN-D 0.3wt% 28.9 This test case used an LPS-induced RAW264.7 cell inflammation model and an elastase inhibition assay to verify the anti-inflammatory and anti-skin cell aging effects of different ratios of compound PDRN. Data showed that the compound PDRN-1 with the optimized mass ratio (PDRN-1:PDRN-2:PDRN-3=100:1.2:0.8) exhibited the best overall activity at a concentration of 0.3 wt%, with inhibition rates of 68.4%, 72.1%, and 65.8% against pro-inflammatory factors TNF-α, IL-1β, and IL-6, respectively, and an inhibition rate of 76.3% against elastase. This excellent effect confirms the synergistic effect between animal-derived PDRN-1 and microbial-derived PDRN-2 and PDRN-3 at the specified ratio. The possible mechanism is that PDRN molecules from different sources can jointly regulate inflammatory signaling pathways (such as NF-κB), and this specific compound formulation has a stronger affinity for the active site of elastase. In contrast, samples using suboptimal ratios (such as 100:0.5:0.5 for compound PDRN-2 and 100:1.5:1.5 for compound PDRN-3) showed observable decreases in all efficacy indicators, indicating that deviations from the ratio weaken the synergistic effect. The efficacy of comparative ratios lacking any component (compounds PDRN-A, B, and C) was significantly reduced, especially compound PDRN-C lacking the core component PDRN-1, which showed the weakest effect. Furthermore, the efficacy of compound PDRN-D (5:1:2), with its severely imbalanced ratio, was even lower than that of samples lacking some components. This, conversely, confirms that the ternary components and their specific mass ratios are necessary conditions for producing the aforementioned synergistic effect. Concentration gradient experiments further showed that the efficacy of compound PDRN-1 was concentration-dependent, reaching a peak at 1 wt% and significantly decreasing at 0.05 wt%, clarifying its effective range.

[0064] Test Example 3: Determination of Encapsulation Efficiency and Loading Rate of Microbial Vesicles The test samples are shown in Table 3.

[0065] Take 10 mL of liquid C of mixture, centrifuge at 15000 g for 20 min, discard the supernatant, then resuspend the drug-loaded Lactobacillus plantarum vesicles at the bottom of the centrifuge tube with 10 mL of pure water, centrifuge at 15000 g for 20 min, discard the supernatant, freeze-dry the resuspended liquid to obtain the total mass of drug-loaded microbial vesicles.

[0066] The lyophilized vesicles were resuspended in 10 mL of ultrapure water and then placed in an ultrasonic cell disruptor for ultrasonic disruption in an ice bath (ultrasonic frequency 40 kHz, ultrasonic time 2 s, interval 3 s, total ultrasonic time 10 min). After disruption, the cells were centrifuged at 15000 g for 10 min, and the supernatant was collected for PDRN content determination to obtain the PDRN content (mg / mL) encapsulated inside the vesicles.

[0067] The encapsulation ratio and load factor are calculated using the following formulas: Encapsulation efficiency EE% = W / W total × 100% W: The quality of PDRN encapsulated within the vesicle; W: Total investment in PDRN quality; Load factor DL% = Q / Qtotal × 100% Q: The quality of PDRN encapsulated within the vesicles Q: Total mass of freeze-dried drug-loaded microbial vesicles The results are shown in Table 3.

[0068] Table 3 Encapsulation efficiency and load factor Test sample Load rate DL% Encapsulation efficiency (EE%) Microbial Vesicle-1 8.72 34.26 Microbial Vesicle-2 7.89* 28.21* Microbial Vesicle-3 8.13* 29.64* Microbial Vesicle-4 5.87* 15.42* Microbial Vesicle-5 4.95* 8.76* Microbial vesicles-① 8.64 33.71 Microbial vesicles-② 6.23* 22.15* Microbial vesicles - ③ 7.05* 25.38* Microbial vesicles-④ 5.12* 11.89* Microbial vesicles-⑤ 3.42* 18.47* Note: "*" indicates p < 0.05 compared to microbial vesicle-1.

[0069] This test case determined the effects of different preparation processes on the encapsulation efficiency and loading rate of microbial vesicles, providing a basis for the selection of core process parameters. Microbial vesicle-1, prepared using center-point process parameters (vesicle protein concentration 0.1 mg / mL, composite PDRN-1 concentration 1.02 wt%, volume ratio 1:1.7, 40 kHz ultrasound), achieved the highest encapsulation efficiency (34.26%) and loading rate (8.72%). This indicates that these parameters most effectively drive the loading of composite PDRN across the vesicle membrane. The mechanism may involve the optimal matching between the cavitation effect generated by a specific ultrasound frequency (40 kHz) and the vesicle membrane permeability, as well as the optimal collision frequency between vesicles and solute molecules at the aforementioned mixed volume ratio. Microbial vesicle-2 (loaded with PDRN-2) and microbial vesicle-3 (loaded with PDRN-3), using the same process but loaded with different ratios of composite PDRN, showed statistically significant decreases in both encapsulation efficiency and loading rate (p<0.05), demonstrating that the ratio and structure of the active ingredient (composite PDRN) itself also affect its interaction efficiency with the vesicle carrier. Samples with altered key process parameters, such as microbial vesicle-4 (vesicle to PDRN concentration too high, volume ratio 1:1) and microbial vesicle-5 (vesicle concentration too low, volume ratio 1:3), showed a significant decrease in encapsulation and loading efficiency, indicating that an imbalance in reactant concentration and ratio disrupts loading equilibrium. While microbial vesicle-① (Lactobacillus bulgaricus vesicles) using vesicles from different sources exhibited similar loading capacities, deviations from the preferred volume ratios (1:4 for microbial vesicle-②, 1:0.5 for microbial vesicle-③) or ultrasonic frequencies (20kHz for microbial vesicle-④) all resulted in a significant decrease in efficiency. This collectively confirms that the vesicle source, mixing volume ratio (1:1-3), and ultrasonic frequency (40kHz) defined in this invention are key process characteristics for achieving efficient encapsulation.

[0070] Test Example 4: Detection of the anti-inflammatory and elastase-inhibiting effects of microbial vesicles Test samples: microbial vesicles-1, Lactobacillus plantarum vesicles, compound PDRN-1, microbial vesicles-①, Lactobacillus bulgaricus vesicles.

[0071] Test sample concentration: Microbial vesicle-1 or microbial vesicle-① was prepared into a test sample with a mass concentration of 0.3 wt% using ultrapure water; the concentrations of Lactobacillus plantarum vesicles, Lactobacillus bulgaricus vesicles, and composite PDRN-1 in the microbial vesicles were calculated using the loading rate determined in Test Example 3, and test samples of corresponding concentrations were prepared. The solvent was ultrapure water, and the test samples were obtained.

[0072] Experimental methods: The inhibition of TNF-α, IL-1β and IL-6 expression is described in Test Example 1; the detection method for elastase inhibition rate is described in Test Example 2, and the results are shown in Table 4.

[0073] Table 4. Detection of the anti-inflammatory and elastase-inhibiting effects of microbial vesicles.

[0074] This test case aims to demonstrate the synergistic effect of "microbial vesicles" as a delivery system, rather than simply the sum of the individual components. Data shows that microbial vesicle-1 (0.3 wt%) loaded with compound PDRN-1 significantly outperformed its physical components (0.27 wt% Lactobacillus plantarum vesicles and 0.03 wt% compound PDRN-1) in all indicators, including anti-inflammation and elastase inhibition, compared to the combined efficacy of the physical components alone. For example, the TNF-α inhibition rate of microbial vesicle-1 was 84.6%, while the two individual components were only 15.8% and 9.2%, respectively. This result clearly confirms that encapsulating compound PDRN into Lactobacillus plantarum vesicles using an ultrasonic loading process produces a significant synergistic effect, with efficacy significantly higher than the sum of the individual component efficacies. The mechanism may lie in the fact that the vesicle carrier not only enhances the intracellular delivery efficiency of the active ingredient, but its membrane components may also act as biological signaling molecules, working synergistically with the loaded compound PDRN to activate a stronger or more durable repair and anti-inflammatory network within the cell. Microbial vesicles-① prepared using Lactobacillus bulgaricus vesicles showed better efficacy than individual components, but slightly lower efficacy than microbial vesicles-1. At the same time, Lactobacillus bulgaricus vesicles alone were less effective than Lactobacillus plantarum vesicles. This together indicates that vesicles derived from Lactobacillus plantarum are more advantageous in synergistic effects with the specific PDRN complex.

[0075] Test Example 5: The Effect of Particle Size on Microbial Vesicles Test samples: Microbial vesicle-1 solution (solvent: ultrapure water) was subjected to pressure-based fractional filtration using polycarbonate microporous membranes with diameters of 0.05 μm, 0.22 μm, 0.45 μm, and 1.0 μm, respectively. Four particle size fractions were collected: <50 nm, 50-220 nm, 220-450 nm, and 450-1000 nm. The four particle sizes were freeze-dried to obtain four sample sizes, which were named Microbial Vesicle-1A, Microbial Vesicle-1B, Microbial Vesicle-1C, and Microbial Vesicle-1D, respectively. The four sample sizes were then prepared into a 0.3 wt% test sample using ultrapure water for analysis.

[0076] Experimental methods: The methods for detecting the inhibition of TNF-α, IL-1β, and IL-6 are the same as those in Test Example 1; the method for detecting the inhibition rate of elastase is the same as those in Test Example 2. The results are shown in Table 5.

[0077] Table 5. Effect of particle size on microbial vesicles

[0078] Note: The data for microbial vesicle-1 in Table 5 are referenced from Table 4.

[0079] This test case, through particle size analysis, clarified the optimal particle size range of the loaded microbial vesicles, providing crucial evidence for product quality control and application. After sieving, microbial vesicle-1B with a particle size range of 50-220 nm showed the best performance in all efficacy evaluations (e.g., TNF-α inhibition rate of 89.7%, elastase inhibition rate of 90.2%), even outperforming the unscreened, broad-distribution stock solution microbial vesicle-1. This demonstrates that this particle size range is the golden range for achieving optimal biological efficacy. The mechanism lies in the fact that a particle size of 50-220 nm is most conducive to particles passing through the intercellular lipid channels of the skin epidermis and being efficiently taken up by fibroblasts and immune cells in the dermis through endocytosis, thereby maximizing the bioavailability of the active ingredients. Microbial vesicles with excessively small particle size -1A (<50nm) may have reduced efficacy due to limited drug loading and different behavior in vivo; while microbial vesicles with excessively large particle size -1C (220-450nm) and microbial vesicles -1D (450-1000nm) have significantly reduced efficacy due to increased transdermal resistance and a sharp drop in cellular uptake efficiency.

[0080] Test Example 6 Security Test Test Example 6-1 Mouse Skin Stimulation Test Samples: See Table 7 below Sample preparation: The sample was mixed with ultrapure water to obtain a sample with a concentration of 0.3 wt%.

[0081] Skin irritation tests were conducted on the test samples. The test subjects were nude BALB / c-nu mice, with 10 mice per sample (5 males and 5 females randomly selected). The test sample was applied to the back of the mice (0.5cm × 0.5cm) twice daily (9:00 AM and 6:00 PM) for 4 consecutive weeks. Mouse behavior and skin condition were observed daily. Scoring was performed according to the criteria in Table 6 below. Table 6 Scoring Criteria Mouse skin condition Rating / points Normal skin 5 No irritation, normal skin color 4 Mild irritation (redness, etc.) 3 Rash and swelling 2 Ulcers, blisters, poor mobility, death 0 The experimental results are shown in Table 7 below: Table 7 Security Test Results

[0082] After a 4-week trial, Table 7 above shows that the test samples have good biocompatibility and did not cause skin irritation in mice.

[0083] Test Example 6-2 Human Safety Evaluation Safety evaluation through human trial: Human skin trial was conducted, and the specific test methods were in accordance with the "Cosmetic Safety Technical Specifications" (2015 edition).

[0084] Samples: 1-5 microbial vesicles; Preparation of test sample: The above sample was mixed with ultrapure water to obtain a test sample with a concentration of 0.3 wt%.

[0085] The specific method is as follows: Volunteers aged 25-45 years with no history of allergies were randomly divided into groups of 10, with each group corresponding to one test sample. The test substance was placed in a patch applicator at a dosage of 0.020-0.025g. The patch applicator containing the test substance was covered with non-irritating cloth-based adhesive tape and applied to the inner forearm of the subject. The patch was then gently pressed with the palm of the hand to ensure even adhesion to the skin surface. Each group of volunteers was assigned one test substance, and the treatment lasted for 24 hours. Thirty minutes after removing the patch applicator, the skin reaction was observed after the pressure mark disappeared. If the result was negative, the results were repeated at 24 hours and 48 hours after the patch test.

[0086] Evaluation criteria: Grade 0: Negative reaction; Grade 1: Suspicious reaction, with only slight erythema; Grade 2: Weak positive reaction, erythema, infiltration, edema, and possible papules; Grade 3: Strong positive reaction, erythema, infiltration, edema, papules may be present, and the reaction may extend beyond the test area; Grade 4: Extremely positive reaction, with obvious erythema, severe infiltration, edema, confluent herpes, and reaction extending beyond the test area.

[0087] Test results: All subjects had negative skin reactions, indicating that the test samples provided by this invention are safe and non-irritating.

[0088] Test Example 6-3 examines the effects of the test examples and comparative examples on skin elasticity and skin barrier repair.

[0089] Samples: 1-5 microbial vesicles; Preparation of test sample: The above sample was mixed with ultrapure water to obtain a test sample with a concentration of 0.3 wt%.

[0090] The anti-aging test of the essence prepared in this invention was carried out for 8 weeks in accordance with the method described in the literature (Zhu Liping et al. Clinical efficacy test and analysis of ginseng anti-aging mask [J], Chinese Journal of Aesthetic Medicine, 2016, 25(02):33-36).

[0091] Ten participants were selected for each test sample. Inclusion criteria: ① No gender restriction, age 30-65 years; some degree of facial skin laxity or fine wrinkles; ② Good health, serious attitude, good communication skills, and able to truthfully reflect the user's experience; ③ Voluntary participation, signing of informed consent, strict adherence to the study protocol, use of the product as required, and completion of follow-up. Exclusion criteria: ① Pregnant or breastfeeding women; ② Sensitive skin; ③ Damaged facial skin or skin diseases affecting facial observation; ④ Facial treatments, cosmetic procedures, or other tests that may affect the results; ⑤ Sunburn within the past 3 months or use of hormonal drugs or immunosuppressants; ⑥ Participation in other facial clinical studies or treatment by a dermatologist within the past 3 months. Termination and exclusion criteria: ① Participant requests to discontinue the trial; ② Poor compliance; ③ Inability to continue the trial due to adverse reactions or special physiological or pathological changes; ④ Use of other skincare products that may affect the experiment, or excessive exposure to ultraviolet radiation during the test.

[0092] Product usage instructions: After cleansing each evening, subjects should apply 0.5–0.6 mL of the test sample evenly to the face, massage until absorbed, and then continue for 8 weeks without further cleansing or application of any other cosmetics. Data should be measured on the same area on day 0 (the day before the trial) and day 57 (the day after the trial).

[0093] Instrument measurement method: The Tewameter skin moisture loss meter and the MPA580 skin elasticity tester from CK (Germany) were used for testing. Specifically: ① Measurement of transepidermal water loss (TEWL value) of facial skin: Using a skin moisture loss meter, the transepidermal water loss of the left and right sides of the face was measured, and the average value was taken from two measurements. ② Facial skin elasticity measurement: Use a skin elasticity tester to measure the skin elasticity at the outer corners of the left and right eyes and under the eyes, and calculate the average value of R2, R2=Ua / Uf; Ua is the skin recovery value from the time the negative pressure is removed to the time the negative pressure is applied again in the next continuous test; Uf is the maximum stretch of the skin when there is negative pressure.

[0094] TEWL change rate (%) = (TEWL value on day 0 - TEWL value on day 57) / TEWL value on day 0 × 100% Skin elasticity change rate (%) = (R² value on day 57 - R² value on day 0) / R² value on day 0 × 100% Average rate of change = Sum of rates of change within the group / Number of people in the group Table 8. Human efficacy evaluation of the test samples Group TEWL change rate average / % Average percentage of skin elasticity change Microbial Vesicle-1 26.45 19.83 Microbial Vesicle-2 21.73 16.54 Microbial Vesicle-3 23.89 17.92 Microbial Vesicle-4 11.24 9.67 Microbial Vesicle-5 9.89 6.45 This test case, through human clinical trials, ultimately verified the actual efficacy of different microbial vesicle samples in improving skin barrier function and enhancing skin elasticity. Microbial vesicle-1, prepared using all optimized conditions (compound PDRN-1 ratio and center-point process), achieved the most significant clinical effect (TEWL reduction of 26.45% and skin elasticity improvement of 19.83%), which confirms the ultimate effectiveness of the aforementioned optimized ratio and process from a human application perspective. Microbial vesicle-2 and microbial vesicle-3, using a less optimized PDRN ratio, showed correspondingly smaller clinical improvement, consistent with the results in test cases 1 and 2 where the compound PDRN activity was slightly weaker, and test case 3 where the encapsulation rate was lower, indicating that optimizing the active ingredient ratio is fundamental to the final efficacy. Microbial vesicles-4 and-5, prepared using a suboptimal process, despite using the same active ingredient (compound PDRN-1), showed significantly lower clinical improvement (TEWL change rates of 11.24% and 9.89%, respectively) than microbial vesicles-1. This is directly attributed to the extremely low encapsulation and loading efficiency revealed in Test Example 3. This result strongly demonstrates that the specific preparation process described in this invention is crucial for achieving and maintaining the synergistic effect of the compound PDRN; without this process, the expected superior efficacy cannot be obtained in the final product.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A compound PDRN with repairing and anti-skin cell aging effects, characterized in that, The composite PDRN includes PDRN-1, PDRN-2, and PDRN-3; wherein PDRN-1 is extracted from the testes of male salmon; PDRN-2 is extracted from Lactobacillus plantarum; and PDRN-3 is extracted from Lactobacillus bulgaricus.

2. The composite PDRN as described in claim 1, characterized in that, The mass ratio of PDRN-1, PDRN-2, and PDRN-3 in the composite PDRN is 100:(0.5-1.5):(0.5-1.5).

3. The composite PDRN as described in claim 2, characterized in that, The mass ratio of PDRN-1, PDRN-2, and PDRN-3 in the composite PDRN is 100:1.2:0.

8.

4. The composite PDRN as described in any one of claims 1-3, characterized in that, The PDRN-2 was extracted from Lactobacillus plantarum, with accession number CGMCCNO.25187; the PDRN-3 was extracted from Lactobacillus bulgaricus, with accession number SHBCCD24344.

5. A type of microbial vesicle with repairing and anti-aging effects on skin cells, characterized in that, The microbial vesicles are loaded with the composite PDRN as described in any one of claims 1-4.

6. The microbial vesicles as described in claim 5, characterized in that, The vesicles were extracted from Lactobacillus plantarum.

7. The microbial vesicles as described in claim 6, characterized in that, The vesicle extract was derived from Lactobacillus plantarum, with the preservation number CGMCCNO.25187.

8. A method for preparing microbial vesicles as described in any one of claims 5-7, characterized in that, The preparation steps include the following: S1: Mix Lactobacillus plantarum vesicles with ultrapure water to obtain mixture A; S2: Dissolve the composite PDRN in ultrapure water to obtain mixture B; S3: Mix mixture A and mixture B, and then sonicate them to obtain mixture C; S4: Centrifuge the mixture C, discard the supernatant, then add an equal volume of ultrapure water to resuspend the mixture, centrifuge again, discard the supernatant, and freeze-dry to obtain microbial vesicles.

9. The preparation method according to claim 8, characterized in that, In step S1, the protein content in mixture A is 0.05–0.15 mg / mL; the particle concentration is ≥1.0 × 10⁻⁶. 8 particles / mL; the content of composite PDRN in the mixture B in step S2 is 1wt%-2wt%; the volume ratio of mixture A to mixture B in step S3 is 1:(1-3); the ultrasonic treatment is carried out under ice bath conditions, and the ultrasonic conditions are: ultrasonic frequency 40kHz; ultrasonic time 30min; the centrifugation speed in step S4 is 15000g, time 20min, and temperature 4℃.

10. The use of the composite PDRN as described in any one of claims 1-4, or the microbial vesicles as described in any one of claims 5-7, or the microbial vesicles prepared by the preparation method described in claim 8 or 9, in the preparation of cosmetics with repairing and anti-skin cell aging effects.

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