A peony multi-function active composition targeting the TRPV1 / SIRT1 pathway, a preparation method and applications thereof

Abstract

The application discloses a peony multi-function active composition targeting TRPV1 / SIRT1 pathway, a preparation method and application thereof, and belongs to the technical field of cosmetics.The peony multi-function active composition comprises, in percentage by weight, 40-60% of glycerol, 5-15% of peony flower water, 5-15% of peony seed oil, 5-15% of peony extract, 5-15% of lactobacillus fermentation lysate, 0.5-6% of hydrogenated lecithin, 0.01-5% of peony root bark extract, 0.05-6% of polyglyceryl-10 laurate, 0.1-1% of p-hydroxyacetophenone, 0.1-1% of 1,2-hexanediol, 0.01-1% of pseudomonas fermentate and 0.0001-0.05% of paeonol, and the balance is deionized water.The peony multi-function active composition integrates active ingredients of multiple parts of peony, compounding two kinds of fermentation products, and constructs a multi-component synergistic activity system, can target the regulation of skin TRPV1 sensitive pathway, MITF melanin synthesis pathway and SIRT1 aging pathway, simultaneously improves skin sensitive stinging, pigment deposition, oxidative aging and barrier damage and other problems, and has multiple skin care functions of soothing sensitive skin, brightening skin color, anti-aging and firming and barrier repair.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway, its preparation method, and its application. Background Technology

[0002] Peony is a classic Chinese medicinal and edible natural active ingredient, rich in bioactive components such as polyphenols, flavonoids, phenolic glycosides, unsaturated fatty acids and polysaccharides. It has excellent antioxidant, anti-inflammatory, soothing and skin repair potential, and has good application value in the field of functional skin care.

[0003] Currently, skincare formulas containing peony active ingredients on the market are relatively simple in their application. Most products simply combine basic peony extracts, achieving only basic skincare effects such as basic moisturizing and superficial anti-oxidation, without targeting the intrinsic molecular regulatory mechanisms of skin damage. Skin sensitivity and stinging, abnormal melanin deposition, skin oxidation and aging, and barrier damage correspond to core regulatory pathways such as the TRPV1 sensitivity pathway, the MITF melanin synthesis pathway, and the SIRT1 aging pathway, respectively. These pathways collectively determine the overall condition of the skin. Existing conventional peony formulas only utilize the basic activity of the raw materials and do not perform multi-target synergistic regulation of the above key pathways, resulting in a lack of precision and targeting in skincare effects.

[0004] Therefore, most existing peony extract skincare formulas only utilize a single peony ingredient and rarely construct multi-component synergistic systems. They are difficult to simultaneously regulate multiple pathways such as TRPV1, SIRT1, and MITF, resulting in limited synergistic effects. They cannot simultaneously regulate multiple core skin pathways and cannot fundamentally improve multiple skin problems such as sensitivity and stinging, pigmentation, oxidative aging, and barrier damage. Overall, their skincare benefits are limited, and their comprehensive repair capabilities are weak, making it difficult to meet the current market's high-end demand for multi-pathway, complex, and fundamental skincare products. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a multi-functional active composition of peony targeting the TRPV1 / SIRT1 pathway, its preparation method, and its applications. The present invention employs the following technical solution: This invention provides a peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway, which comprises the following components by weight percentage: Glycerin 40-60%, peony flower water 5-15%, peony seed oil 5-15%, peony extract 5-15%, Lactobacillus fermentation lysate 5-15%, hydrogenated lecithin 0.5-6%, peony root bark extract 0.01-5%, polyglycerol-10 laurate 0.05-6%, p-hydroxyacetophenone 0.1-1%, 1,2-hexanediol 0.1-1%, Pseudomonas aeruginosa fermentation product 0.01-1%, paeonol 0.0001-0.05%, deionized water to 100%.

[0006] Furthermore, the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway comprises, by weight percentage: 50% glycerin, 10% peony flower water, 10% peony seed oil, 10% peony extract, 10% Lactobacillus fermentation lysate, 4% hydrogenated lecithin, 3% peony root bark extract, 3% polyglycerol-10 laurate, 0.5% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, 0.5% Pseudomonas aeruginosa fermentation product, 0.025% paeonol, and deionized water to bring the total to 100%.

[0007] This invention provides a method for preparing the aforementioned peony multifunctional active composition, comprising the following steps: S1. Mix peony seed oil, hydrogenated lecithin, polyglycerol-10 laurate, and paeonol, heat to 45-55℃, and stir until uniformly mixed to obtain an oil phase mixture. S2. Mix glycerol, deionized water, peony flower water, peony extract, peony root bark extract, Lactobacillus fermentation lysate, and Pseudomonas aeruginosa fermentation product, heat to 45-55℃, stir evenly, and obtain an aqueous phase mixture. S3. Slowly add the oil phase mixture to the water phase mixture, stir at 45~55℃ for 3~10 min, and homogenize under high pressure 1~5 times at a pressure of 15000-30000 PSI; S4. After homogenization, cool to 25~35℃, add p-hydroxyacetophenone and 1,2-hexanediol, stir evenly to obtain the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway.

[0008] This invention provides the application of the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway in the preparation of skin care products.

[0009] Furthermore, the percentage by weight of the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway added to the skin care product is no more than 10%.

[0010] Furthermore, the skincare products include any one of serums, toners, lotions, creams, and masks.

[0011] The beneficial effects of this invention are: The composition of this invention uses the active ingredients of the whole peony plant, including peony flowers, peony root bark, and peony seeds, and is combined with fermentation lysate of Lactobacillus and fermentation product of Pseudomonas aeruginosa to construct a synergistic active system. This system can simultaneously target the skin's TRPV1 sensitive pathway, MITF melanin synthesis pathway, and SIRT1 aging pathway, thereby improving multiple skin problems such as skin sensitivity and stinging, abnormal melanin deposition, skin oxidative aging, and skin barrier damage. It achieves the core skin care effects of soothing sensitivity, brightening skin tone, anti-aging and firming, and barrier repair. Attached Figure Description

[0012] Figure 1 A bar chart of TRPV1 content in cells after modeling for a soothing efficacy test; Figure 2 A bar chart showing the content of type I collagen in cells after UVA damage in an anti-wrinkle efficacy test. Detailed Implementation

[0013] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods.

[0014] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0015] Unless otherwise specified, all materials, reagents, raw materials, etc. used in the following examples are commercially available.

[0016] The sources of the raw materials used in this invention are shown in Table 1 below.

[0017] Table 1. Sources of raw materials Peony Flower Water Peony Flower Water Guangzhou Ruiran Biotechnology Co., Ltd. Peony extract Peony extract Guangzhou Ruiran Biotechnology Co., Ltd. Peony root bark extract YH51002gp Peony Root Bark Extract Quzhou Zhanhong Biotechnology Co., Ltd. Peony seed oil Peony seed oil Guangzhou Viang Biotechnology Co., Ltd. glycerin Refined Glycerin Guangzhou Bino Biotechnology Co., Ltd. Lactobacillus fermentation lysate LAVA-DERMABIOTICS HDB1234GN Guangzhou Baihaobo Co., Ltd. fermentation products of Pseudomonas aeruginosa ANTARCTICINE® marine ingredient C Guangzhou Rongdaxing Chemical Co., Ltd. Hydrogenated lecithin Phoslip PD10 Hydrogenated Lecithin Jinjiuqi (Fushun) Pharmaceutical Co., Ltd. Polyglycerol-10 lauryl ester UN-110 Polyglycerol-10 Laurate Foshan Yinmei United Technology Co., Ltd. Paeonol Paeonol Jiangxi Cedar Natural Medicinal Oil Co., Ltd. 1,2-Hexanediol Asumcare HDO800 Guangzhou Huixianggu Chemical Co., Ltd. p-Hydroxyacetophenone Ecosave™ HAP Pro Jiangsu Xinhan New Materials Co., Ltd. Example Example 1 A multifunctional peony active composition targeting the TRPV1 / SIRT1 pathway, with the following formulation (by weight percentage): 40% glycerol, 5% peony flower water, 5% peony seed oil, 5% peony extract, 5% Lactobacillus fermentation lysate, 0.5% hydrogenated lecithin, 0.01% peony root bark extract, 0.05% polyglycerol-10 laurate, 0.1% p-hydroxyacetophenone, 0.1% 1,2-hexanediol, 0.01% Pseudomonas aeruginosa fermentation product, 0.0001% paeonol, deionized water to 100%.

[0018] The preparation method of the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway is carried out according to the following steps: S1. Mix peony seed oil, hydrogenated lecithin, polyglycerol-10 laurate, and paeonol, heat to 50°C, and stir until uniformly mixed to obtain an oil phase mixture; S2. Mix glycerol, deionized water, peony flower water, peony extract, peony root bark extract, Lactobacillus fermentation lysate, and Pseudomonas aeruginosa fermentation product, heat to 48°C, and stir until homogeneous to obtain an aqueous mixture. S3. Slowly add the oil phase mixture to the water phase mixture, stir at 55°C for 5 minutes, and homogenize under high pressure twice at a pressure of 20000 PSI. S4. After homogenization, cool to 30°C, add p-hydroxyacetophenone and 1,2-hexanediol, stir until homogeneous, and obtain the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway.

[0019] Example 2 A multifunctional peony active composition targeting the TRPV1 / SIRT1 pathway, with the following formulation (by weight percentage): 50% glycerin, 10% peony flower water, 10% peony seed oil, 10% peony extract, 10% Lactobacillus fermentation lysate, 4% hydrogenated lecithin, 3% peony root bark extract, 3% polyglycerol-10 laurate, 0.5% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, 0.5% Pseudomonas aeruginosa fermentation product, 0.025% paeonol, and deionized water to bring the total to 100%.

[0020] The composition was prepared using the same method as in Example 1.

[0021] Example 3 A multifunctional peony active composition targeting the TRPV1 / SIRT1 pathway, with the following formulation (by weight percentage): Glycerin 60%, Peony flower water 15%, Peony seed oil 15%, Peony extract 15%, Lactobacillus fermentation lysate 15%, Hydrogenated lecithin 6%, Peony root bark extract 5%, Polyglycerol-10 lauryl ester 6%, p-hydroxyacetophenone 1%, 1,2-hexanediol 1%, Pseudomonas aeruginosa fermentation product 1%, Paeonol 0.05%, deionized water to 100%.

[0022] The composition was prepared using the same method as in Example 1.

[0023] Comparative Example Comparative Example 1 In this comparative example, the composition formulation does not include peony flower water, and the reduced mass is made up by deionized water. The amounts of the remaining components are completely consistent with the preparation process and Example 2.

[0024] Comparative Example 2 In this comparative example, peony extract was not added to the formulation of the composition, and the reduced mass was made up by deionized water. The amounts of the remaining components were completely consistent with the preparation process and Example 2.

[0025] Comparative Example 3 In this comparative example, no peony root bark extract was added to the formulation of the composition, and the reduced mass was made up by deionized water. The amounts of the remaining components were completely consistent with the preparation process and Example 2.

[0026] Comparative Example 4 In this comparative example, no paeonol was added to the formulation of the composition, and the reduced mass was made up by deionized water. The amounts of the remaining components were completely consistent with the preparation process and Example 2.

[0027] Comparative Example 5 In this comparative example, peony seed oil was not added to the formulation of the composition, and the reduced mass was made up by deionized water. The amounts of the remaining components were completely consistent with the preparation process and Example 2.

[0028] Comparative Example 6 In this comparative example, hydrogenated lecithin was not added to the formulation of the composition, polyglycerol-10 laurate was increased to 7%, and the amounts of the remaining components were completely consistent with the preparation process and Example 2.

[0029] Comparative Example 7 In this comparative example, the fermentation product of *Pseudomonas aeruginosa* in the composition formulation was replaced with *Lactobacillus* fermentation lysate, i.e., *Pseudomonas aeruginosa* fermentation product was not added. The amount of *Lactobacillus* fermentation lysate added was 10.5%, and the amounts of the remaining components were completely consistent with the preparation process and Example 2.

[0030] Comparative Example 8 The formulation and dosage are completely consistent with those in Example 2; The homogenization and emulsification steps were omitted in the preparation process, and all components were stirred at 500 rpm for 30 min at room temperature.

[0031] Effect verification test This experiment is divided into two parts: cellular activity verification uses direct testing of the raw materials of the composition to verify the composition's biological activity and pathway regulation; human efficacy verification uses testing of finished skincare products containing the composition to simulate skincare effects in real-world usage scenarios.

[0032] I. In vitro cell experiments 1. Skin brightening and skin repair test 1.1 Experimental Grouping This experiment used mouse melanoma cells (B16) and human skin fibroblasts (HFF-1) to verify their in vitro viability. Both cell lines were purchased from Wuhan Pronosei Biotechnology Co., Ltd. B16 cells were cultured in DMEM high-glucose basal medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin to form a complete culture medium. HFF-1 cells were cultured in MEM basal medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 100 U / mL penicillin, and 100 μg / mL streptomycin to form a complete culture medium. The pH of all culture media was adjusted to 7.2–7.4, and the cells were routinely cultured in a constant temperature incubator at 37℃ and 5% CO2 saturated humidity.

[0033] The experiment included a blank control group, a model control group, Examples 1-3, and Comparative Examples 1-8, with 6 replicates per group to ensure repeatability. All samples were added using a standardized concentration system: a 10% (v / v) stock solution of sterile serum-free basal medium for the corresponding cells was prepared; the total volume of each cell culture well was 1000 μL, with 10 μL of the stock solution added to achieve a final concentration of 0.1% (v / v) in the culture system. The blank control group and model control group received an equal volume of sterile serum-free basal medium to eliminate interference from the solvent system and ensure consistent experimental conditions across groups. Specific grouping and processing methods are as follows: Blank control group: Both experimental cells were routinely cultured in the corresponding complete culture medium without any stimuli or sample solutions added. They were cultured normally for the corresponding experimental duration and served as a reference for normal cells.

[0034] Model control group: The two cell types were subjected to corresponding damage modeling treatments. B16 cells were induced to develop melanin using α-melanocyte-stimulating hormone (α-MSH), while HFF-1 human skin fibroblasts were induced to develop oxidative damage using hydrogen peroxide (H2O2). No composite samples were added during the modeling and culture process, only an equal volume of sterile serum-free basal culture medium was added to construct a stable cell damage model control.

[0035] Each experimental group (Examples 1-3, Comparative Examples 1-8): Cells in each group underwent the same modeling stimulation treatment as the control group, and were simultaneously added with the corresponding sample solution of the Example and Comparative Example compositions at a final concentration of 0.1% (v / v). Cells were cultured simultaneously under the same culture conditions. The activity of different formulation compositions on melanin synthesis inhibition and cell barrier repair was evaluated by assessing the differences in cell indicators among the groups.

[0036] 1.2 Detection Indicators, Methods and Calculation Formulas (1) Tyrosinase activity inhibition rate: MITF is a core transcription factor in melanin synthesis and can directly bind to the promoter of tyrosinase genes, upregulating the expression and activity of tyrosinase. Tyrosinase is a key rate-limiting enzyme in melanin synthesis, and its activity directly determines the amount of melanin produced. Inhibition of tyrosinase activity can directly reflect the regulatory effect of the composition on the MITF pathway, corresponding to the strength of the skin brightening effect.

[0037] Experimental methods: B16 mouse melanoma cells in the logarithmic growth phase were used, and 1×10⁻⁶ cells were injected with the solution. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours until 70% confluence was achieved. Except for the blank control group, all other groups were replaced with complete culture medium containing 100 nmol / L α-melanocyte-stimulating hormone (α-MSH) to induce melanin synthesis. Simultaneously, each treatment group was treated with the corresponding composition sample at a final concentration of 0.1% (screened by a CCK-8 cytotoxicity pre-test, at which relative cell viability ≥90%), while the blank control group received an equal volume of complete culture medium. After 48 hours of further culture, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. A lysis buffer containing 1% Triton X-100 was added to the PBS, and the cells were subjected to two freeze-thaw cycles to achieve complete lysis. The cells were centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. An equal volume of the supernatant was added to each well of a 96-well plate, and 100 μL of 0.1% L-DOPA solution was added to each well. The plates were incubated at 37°C in the dark for 30 minutes, and the absorbance was measured at 475 nm using a microplate reader. Tyrosinase activity was corrected for the total protein concentration in each well. The inhibition rate of tyrosinase activity is calculated according to the following formula:

[0038] The higher the inhibition rate, the stronger the inhibition of tyrosinase activity, the better the regulation of the MITF pathway, and the more significant the skin brightening effect.

[0039] (2) Cell scratch healing rate HFF-1 human skin fibroblasts in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours until complete confluence and the formation of a monolayer. Uniform scratches were made perpendicular to the bottom of the wells using a 200 μL sterile pipette tip, and the cells were washed three times with PBS to remove detached cells. Except for the blank control group, all other groups were treated with serum-free medium containing 200 μmol / L H2O2 to induce cell damage; simultaneously, each treatment group was treated with the corresponding sample at a final concentration of 0.1% (v / v), while the blank control group received an equal volume of serum-free medium. The initial scratch width was recorded by photographing under an inverted microscope; after another 24 hours of culture, the same location was photographed again, and the scratch width was measured using ImageJ software. Calculation formula:

[0040] The higher the scratch healing rate, the stronger the cell migration and repair ability, and the better the skin repair effect of the composition.

[0041] Pre-test results of modeling effectiveness: Basal tyrosinase activity: The relative tyrosinase activity in the blank control group (B16 cells) was 1.00±0.05, while the relative tyrosinase activity in the α-MSH-induced model control group was 1.76±0.08. The model group showed a highly significant increase compared to the blank group (p<0.01), indicating that the model of excessive melanin synthesis was effective and could be used to evaluate the melanin-inhibiting activity of the composition. The effectiveness of the scratch injury model can be directly verified by the scratch healing index data in Table 2 below.

[0042] 1.3 The results of the cell experiments are shown in Table 2.

[0043] Table 2 Results of cell experiments Blank control group - - 34.1±2.3 - Model control group - - 18.6±1.7 ## Example 1 30.2±1.9 && 40.5±1.3 ** Example 2 43.1±2.5 - 49.3±2.8 ** Example 3 39.6±2.1 & 45.7±1.5 **& Comparative Example 1 25.4±1.6 && 30.1±1.2 *&& Comparative Example 2 27.8±1.5 && 31.5±0.5 *&& Comparative Example 3 26.4±1.8 && 30.4±2.0 *&& Comparative Example 4 26.7±1.3 && 27.2±1.9 *&& Comparative Example 5 27.7±1.7 && 28.8±2.5 *&& Comparative Example 6 29.2±1.0 && 29.8±0.7 *&& Comparative Example 7 17.7±1.4 && 22.8±1.4 && Comparative Example 8 17.9±1.2 && 22.6±1.7 && Note: Compared with the blank control group, ##p<0.01, #p<0.05; compared with the model control group, **p<0.01, *p<0.05; compared with the Example 2 group, &&p<0.01, &p<0.05.

[0044] The scratch healing rate and tyrosinase inhibition rate were calculated from the original test data. No tyrosinase inhibition rate was calculated for the blank control group and the model group; the corresponding cells in the table are marked with "-". For details of the original tyrosinase modeling data, please refer to section "1.2" above for the effectiveness of the modeling.

[0045] In Table 2, the scratch healing rate of cells in the model control group was significantly lower than that in the blank control group (##p<0.01), indicating that after the modeling intervention, the cells showed obvious barrier damage and decreased repair ability. The skin barrier damage model in this experiment was successfully established, the experimental system is reliable, and it can be used to accurately evaluate the repair activity of each sample.

[0046] Based on the scratch barrier repair indicators, it can be seen that after intervention with the compositions of Examples 1, 2, and 3 of this invention, the level of inhibition of tyrosinase activity and the cell scratch healing ability of damaged cells were significantly improved compared with the model control group. This proves that the peony multifunctional active composition defined in this invention can effectively inhibit abnormal melanin synthesis, enhance the cell's self-repair ability, and has clear skin brightening and barrier repair activities.

[0047] Comparative Examples 1, 2, 3, 4, and 5 were found to lack peony flower water, peony extract, peony root bark extract, paeonol, and peony seed oil, respectively, which are the core active components of peony. Although the indicators of the above groups were significantly improved compared with the model control group, the efficacy values ​​were all lower than those of Examples 1-3 due to the lack of key active ingredients. Moreover, the skin care efficacy was significantly weakened compared with the optimal Example 2. This indicates that the active components of the peony series are the core functional raw materials for the composition to exert the effects of inhibiting melanin and repairing skin. The absence of any one of the peony active substances will directly weaken the overall activity of the formula.

[0048] Comparative Example 6 used a single emulsification system, and all indicators were significantly lower than those of Examples 1-3. The results show that the compound emulsification system defined in this invention can ensure the stable dispersion and penetration absorption of active components, which is necessary for the efficacy of the formulation.

[0049] In Comparative Example 7, after adjusting the fermentation products using a single fermentation system, there were no significant differences in various indicators compared to the model control group (p≥0.05), indicating a near loss of skin brightening and barrier repair capabilities. Furthermore, there was a highly significant difference compared to Example 2 (p<0.01). This demonstrates that a single fermentation product cannot achieve the synergistic effect of a dual fermentation system. The specific ratio of dual fermentation products in this invention is not a simple additive of raw materials, but rather a key functional system highly compatible with the multiple active components of peony. Arbitrarily replacing the fermentation functional raw materials will directly lead to the near-complete failure of the multi-pathway skincare efficacy of the composition, meaning it cannot effectively improve abnormal melanin synthesis and damaged cell barriers.

[0050] Comparative Example 8 retained the optimal formulation components and dosages of Example 2, but did not undergo homogenization and emulsification; it was prepared using only conventional low-speed stirring. The two efficacy indicators in this group showed a significant decrease, demonstrating that the high-temperature phase separation and high-pressure homogenization emulsification preparation process specified in this invention can effectively improve the bioavailability of the active components in the system. This is a crucial condition for ensuring the full efficacy of the formulation; process simplification directly leads to a complete loss of the skin-care activity of the composition.

[0051] 2. The soothing efficacy of the composition from Example 2 was tested (TRPV1 sensitive pathway verification). 2.1 Experimental Grouping This experiment used immortalized human epidermal cells (HaCaT) to verify the soothing efficacy, and constructed a skin sensitivity injury model by activating the TRPV1 pathway of cells with capsaicin (CAP). HaCaT cells were cultured in DMEM medium containing 10% FBS as the complete culture medium, with the pH of the medium adjusted to 7.2-7.4. The cells were routinely cultured in a constant temperature incubator at 37℃ and 5% CO2 saturated humidity.

[0052] The experiment included a blank control group, a model control group, a positive control group, and Example 2 group, with 6 replicates in each group to ensure the repeatability of the experiment.

[0053] Blank control group: cells were cultured in complete culture medium under normal conditions without capsaicin stimulation or the addition of any sample solution, and were cultured for the normal experimental duration as a reference for normal cells.

[0054] Model control group: Cells were stimulated with capsaicin-containing culture medium to construct a sensitive damage model. No composition samples were added during the modeling and culture process, only an equal volume of basal culture medium (serum-free) was added to construct a cell sensitive damage model control.

[0055] Positive control group: After modeling, 4-tert-butylcyclohexanol (optimal working concentration) was added to the model as a positive control for soothing efficacy.

[0056] In Example 2, cells were treated with capsaicin in the same manner as the control group, and simultaneously added to the sample of the composition from Example 2 (selected as the experimental concentration of 1.00% (v / v) with relative cell viability ≥90% after screening by CCK-8 cytotoxicity pre-test). Cells were cultured simultaneously under the same conditions. The regulatory effects and soothing efficacy of different formulations on the TRPV1-sensitive pathway were evaluated by the difference in TRPV1 protein content in the cells.

[0057] 2.2 Detection Indicators, Methods and Calculation Formulas (1) TRPV-1 protein content: TRPV1 is a key ion channel for skin to sense stinging, burning and redness. TRPV1 is overactivated under sensitive conditions. The intracellular TRPV-1 protein content was detected to evaluate the inhibitory effect of the composition on the TRPV1 sensitive pathway and the corresponding soothing effect.

[0058] Experimental method: HaCaT immortalized human epidermal cells in logarithmic growth phase were used, and 1×10⁻⁶ cells were extracted. 4 Cells were seeded per well in 96-well plates and cultured for 18-24 h. Except for the blank control group, all other groups were stimulated with capsaicin-containing medium for 4 h to induce high TRPV-1 expression. The original medium was discarded, and the cells were gently washed with PBS to remove residual capsaicin. The positive control group and the Example 2 group were incubated with maintenance medium containing the positive control substance and the Example 2 composition, respectively, for 24 h. The blank control group and the model control group were incubated with an equal volume of maintenance medium. After incubation, the medium was discarded, and cells were lysed on ice with cell lysis buffer. The TRPV-1 protein content was detected according to the TRPV-1 ELISA kit instructions. The TRPV-1 downregulation rate was calculated using the following formula:

[0059] In the formula, T 样品 The average TRPV-1 protein content in the test group; T 空白对照represents the average value of TRPV-1 protein content in the blank control group; T 裸细胞 represents the baseline TRPV-1 protein content of nude cells; a down-regulation rate >0 indicates that the test substance can inhibit the capsaicin-induced up-regulation of TRPV1 protein; a higher value indicates a stronger ability to inhibit the TRPV1 pathway and better soothing activity.

[0060] Pre-test results for modeling effectiveness: after capsaicin induction modeling, the TRPV-1 down-regulation rate of the positive control group is a positive number with extremely significant difference (P<0.001), which proves that capsaicin stimulation can stably activate the TRPV-1 pathway of HaCaT cells, the sensitive cell model is successfully constructed, and can be used to evaluate the soothing activity of the composition.

[0061] Judgment indicators: the content of the test sample is lower than that of the blank control group and the statistical difference P value < 0.05, the test sample has a soothing effect.

[0062] 2.3 The cell test results are shown in Table 3 and Figure 1 as shown.

[0063] Table 3 Cell test results of soothing efficacy Blank control 6.69±0.22 - - Positive control group 4.57±0.39 31.69 <0.001 Example 2 Group (1% concentration sample) 5.17±0.46 22.72 <0.001 Note: P>0.05 means there is no statistical difference between the two groups of data; 0.01<P<0.05 means statistical significance; 0.001<P<0.01 means significant difference; P<0.001 means extremely significant difference.

[0064] The TRPV-1 down-regulation rate is a ratio index converted from the original test data; the blank control group has no calculation result of down-regulation rate, and the corresponding cell in the table is marked with "-"; the effectiveness of capsaicin modeling is detailed in the pre-test content of modeling effectiveness in 2.2 above.

[0065] In Table 3, the TRPV-1 down-regulation rate of the positive control group is a positive value, which is extremely significantly different from that of the blank control group (P<0.001), indicating that the capsaicin-HaCaT sensitive injury model is successfully modeled, and the test system is reliable and can be used to evaluate the soothing activity of the sample.

[0066] Figure 1 shows the TRPV-1 content results; combined with Table 3, it can be seen from the TRPV-1 down-regulation data that after the intervention of the composition of Example 2, the cellular TRPV-1 protein content is significantly lower than that of the blank control group, and the TRPV-1 down-regulation rate reaches 22.75%, with an extremely significant difference (P<0.001); it is proved that the composition of the present invention can effectively inhibit the excessive activation of the TRPV-1 pathway and has definite soothing sensitive activity.

[0067] 3. Anti-wrinkle efficacy test (indirect verification of SIRT1 aging pathway) 3.1 Experimental Grouping This experiment used normal human skin fibroblasts (HSF) to verify their in vitro anti-wrinkle activity. The cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd. HSF cells were cultured in DMEM high-glucose basal medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin to prepare a complete culture medium. The pH of the medium was adjusted to 7.2-7.4, and the cells were routinely cultured in a constant temperature incubator at 37℃ and 5% CO2 saturated humidity.

[0068] The experiment included a blank control group, a model control group, a positive control group, and Example 2 group, with 6 replicates per group to ensure reproducibility. All samples used a standardized concentration system: test samples were prepared using sterile serum-free DMEM basal medium; based on the preliminary cell viability test results, the final concentration of the composition for the formal test was determined to be 0.50% (v / v). An equal volume of sterile serum-free basal medium was added to the blank control group and the model control group to eliminate interference from the solvent system and ensure completely consistent experimental conditions across groups.

[0069] The specific grouping and treatment methods are as follows: Blank control group: HSF cells are routinely cultured in the corresponding complete culture medium without UVA light damage modeling stimulation or the addition of any sample solution. They are cultured normally for the corresponding experimental time and serve as a normal cell reference.

[0070] Model control group: Cells were subjected to UVA irradiation for photodamage modeling. The modeling protocol was matched with the cell aging damage model corresponding to the type I collagen detection index. No composite sample was added during the modeling and culture process. Only an equal volume of sterile serum-free basal culture medium was added to construct a stable cell photoaging damage model control.

[0071] Positive control group: Cells were simultaneously treated with UVA stimulation identical to those in the model control group. After modeling, a final concentration of 0.1% vitamin C was added, and cells were cultured synchronously in the same culture environment to verify the effectiveness of the model.

[0072] Example 2 group: Cells were simultaneously treated with UVA damage stimulation identical to the model control group. After modeling, the sample solution of the Example 2 composition at a final concentration of 0.50% (v / v) was added, and the cells were cultured synchronously under the same culture conditions. The difference in type I collagen content among the groups was used to evaluate the promoting effect of the composition on collagen synthesis in fibroblasts, indirectly reflecting the composition's regulation of the SIRT1 aging pathway and its anti-wrinkle activity.

[0073] 3.2 Detection Indicators, Methods and Calculation Formulas (1) Type I collagen upregulation rate: SIRT1 is a key deacetylase that regulates cell aging. Activation of the SIRT1 pathway can promote the synthesis of type I collagen in fibroblasts. Type I collagen is the main structural protein in the dermis, and its increased content indicates enhanced anti-wrinkle activity. The content of type I collagen in cell supernatant after UVA damage and the upregulation rate can be calculated to indirectly evaluate the anti-wrinkle efficacy of the composition.

[0074] Experimental Methods: Normal human skin fibroblasts (HSF) in logarithmic growth phase were seeded at 5 × 10³ / well in 96-well plates and cultured for 18-24 h until adherence. The supernatant was discarded, and the cells were covered with PBS buffer. Except for the blank control group, all other groups were irradiated with UVA lamps. The irradiation intensity was calibrated using a UV radiometer, with a cumulative dose of 15 J / cm² (determined in a preliminary experiment). The temperature was controlled to ≤37℃ during irradiation. After irradiation, the positive control group and the Example 2 group were respectively given maintenance culture medium containing the positive control substance and the composition of Example 2; the blank control group and the model control group were given an equal volume of maintenance culture medium. Cells were incubated according to the experimental sequence. After incubation, the cell supernatant was collected, and the type I collagen content was measured according to the type I collagen instructions.

[0075] The upregulation rate of type I collagen is calculated using the following formula:

[0076] In the formula: T is the average type I collagen content of the test group; C is the average type I collagen content of the blank control group.

[0077] The higher the upregulation rate, the stronger the effect of promoting type I collagen synthesis, the more significant the anti-wrinkle effect, and the better the activation effect of the SIRT1 pathway.

[0078] Preliminary test results of modeling effectiveness: After UVA photodamage modeling, the content of type I collagen in the positive control group was significantly higher than that in the blank control group and the difference was extremely significant (P<0.001), which proved that the UVA-induced fibroblast photoaging model was successfully constructed and can be used to evaluate the anti-wrinkle activity of the composition.

[0079] Judgment criteria: The content of the test sample is higher than that of the blank control group and the difference is statistically significant. P If the value is <0.05, the test sample has anti-wrinkle effect.

[0080] 3.3 The results of the cell experiments are shown in Table 4 and... Figure 2 As shown.

[0081] Table 4. Cellular Experiment Results of Anti-wrinkle Efficacy Blank control 3.78±0.46 - - Positive control group 5.11±0.25 35.19 <0.001 Example 2 (0.5% concentration sample) 5.37±0.57 42.06 <0.001 Note: P>0.05 indicates no statistically significant difference between the two groups of data; 0.01<P<0.05 indicates statistical significance; 0.001<P<0.01 indicates a significant difference; P<0.001 indicates an extremely significant difference.

[0082] The type I collagen up-regulation rate is a ratio index converted from the original test data; no up-regulation rate calculation results are obtained for the blank control group and the model control group, and the corresponding cells in the table are marked with "-"; the effectiveness of UVA photodamage modeling is detailed in the pre-test of modeling effectiveness in Section 3.2 above.

[0083] In Table 4, the up-regulation rate of type I collagen in the positive control group is a positive value and is extremely significantly different from that in the blank control group (P<0.001), which indicates that the UVA-HSF photoaging damage model is successfully established, the test system is reliable, and can be used to evaluate the anti-wrinkle activity of the sample.

[0084] Figure 2 The figure shows the type I collagen content in cells after UVA damage in the anti-wrinkle efficacy test; combining with the type I collagen up-regulation index in Table 4, it can be seen that after the intervention of the composition of Example 2, the type I collagen content in UVA-damaged cells is significantly increased compared with the blank control group, the up-regulation rate of type I collagen reaches 42.24%, with an extremely significant difference (P<0.001). This effect is related to the composition activating the SIRT1 aging pathway and improving collagen metabolism in photodamaged fibroblasts, which proves that the composition of the present invention has definite anti-wrinkle and firming activity.

[0085] II. Human Efficacy Verification 1. Preparation of Test Samples The test dosage form is essence, and the basic essence matrix formula is: 87.7% deionized water, 5% glycerin, 4% butanediol, 0.1% carbomer, 0.2% triethanolamine, 0.1% sodium hyaluronate, 0.5% p-hydroxyacetophenone, 0.3% 1,2-hexanediol, and 0.05% disodium EDTA.

[0086] Blank matrix group: The above basic essence matrix is used directly.

[0087] Example 2 finished product group: 5% of the composition of Example 2 is added to the basic essence matrix, replacing an equal amount of deionized water.

[0088] Comparative Example 3 finished product group: 5% of the composition of Comparative Example 3 is added to the basic essence matrix, replacing an equal amount of deionized water.

[0089] Comparative Example 7 finished product group: 5% of the composition of Comparative Example 7 is added to the basic essence matrix, replacing an equal amount of deionized water.

[0090] Comparative Example 8 finished product group: 5% of the composition of Comparative Example 8 is added to the basic essence matrix, replacing an equal amount of deionized water.

[0091] 2. Subjects and Trial Design Seventy-five healthy participants, aged 25-45, with sensitive skin, dull complexion, and mild fine lines, and no history of skin diseases, were recruited. Participants were randomly divided into five groups of 15 each. After cleansing in the morning and evening, the test product was evenly applied to the face and marked areas on the inner forearms of both hands for 28 consecutive days. Performance indicators were measured on day 0 and day 28. The testing environment was 22±2℃ with a relative humidity of 50±5%. Participants underwent environmental acclimatization for 30 minutes before the test.

[0092] 3. Detection Indicators and Methods (1) Soothing effect: On the test day of day 0 and day 28, the test site was cleaned first. The subject sat quietly in the constant temperature and humidity room for 30 minutes to adapt. No test sample or skin care product was applied to the test site within 4 hours before the test. The lactic acid stinging test was performed directly to test the skin's tolerance to lactic acid stimulation under baseline conditions. Specifically, 50 μL of 10% lactic acid solution was applied to the subject's nasolabial fold on one side. The subject scored the stinging sensation on a scale of 0 to 10 at 2.5 min and 5.0 min after application. 0 points was no stinging and 10 points was severe stinging. The average score at the two time points was taken as the final result. The higher the score, the stronger the stinging.

[0093] (2) Skin brightening effect: The melanin index of the subject's inner forearm was measured using a skin melanin analyzer. Each area was tested three times in parallel and the average value was taken. The lower the melanin index value, the less melanin content in the skin, and the better the skin brightening effect of the test sample.

[0094] (3) Anti-aging efficacy: The skin elasticity R2 value of the fixed marked area at the corner of the eye was measured using a skin elasticity tester. This value is the ratio of elastic recovery to total stretching. Each area was tested in parallel three times, and the average value was taken. The absolute value of R2 ranged from 0 to 1. The closer the value is to 1, the better the skin elasticity. In this experiment, the change in R2 was used to evaluate the anti-aging effect of the test sample. The change was the difference between the test value on day 28 and the initial value on day 0. A positive R2 change and a larger value indicate a more significant improvement in skin elasticity after using the test sample, corresponding to a better anti-aging efficacy of the test sample.

[0095] (4) Repair efficacy: The TEWL value of the fixed marked area on the inner forearm of the subject was tested using a transdermal water loss tester. Each area was tested in parallel for 3 times and the average value was taken. The lower the TEWL value, the stronger the skin barrier water retention capacity and the better the barrier repair efficacy of the test sample.

[0096] 4. The test results (28-day test values) are shown in Table 5.

[0097] Table 5 Results of Human Efficacy Verification Blank matrix group 6.7±0.4 -1.1±0.5 +0.02±0.01 -2.0±0.3 Example 2 Finished Product 2.4±0.6** -8.8±1.2** +0.19±0.04** -8.0±0.5** Comparative Example 3 Finished Product 4.2±0.8& -5.2±1.0& +0.12±0.04& -4.9±0.2& Comparative Example 7 Finished Product 5.8±0.5&& -2.6±0.8&& +0.07±0.07&& -3.2±0.4&& Comparative Example 8 Finished Products 6.0±0.7&& -2.2±0.9&& +0.05±0.05&& -3.1±0.3&& Note: Compared with the blank control group, **p<0.01, *p<0.05; compared with the Example 2 group, &&p<0.01, &p<0.05.

[0098] 2.4 Results Analysis: After 28 days of continuous use of each component, the actual skin care efficacy of different product compositions was evaluated using four human skin characterization indicators: skin irritation score, melanin index (MI), skin elasticity R2 value, and transepidermal water loss (TEWL). The changes in each indicator were consistent with the results of previous cell experiments.

[0099] In Example 2, after 28 days of use, all four test indicators showed highly significant differences compared to the blank matrix group (**p<0.01). Specifically, the skin stinging sensation score decreased from 6.7±0.7 in the blank group to 2.4±0.5, significantly reducing skin sensitivity and stinging reactions, effectively improving skin tolerance, and demonstrating excellent skin soothing effects. The skin melanin index (MI) changed by -8.8±1.2, showing a significant improvement in melanin metabolism compared to the blank group, confirming that the composition can effectively inhibit abnormal melanin deposition and achieve skin brightening and dullness improvement by regulating the MITF pathway. The skin elasticity R2 increased by +0.19±0.02, significantly improving skin laxity and firmness, corresponding to the anti-oxidative and anti-aging mechanism activated by the SIRT1 pathway. Simultaneously, the TEWL value significantly decreased to -8.0±1.5, indicating that the product can effectively reduce transepidermal water loss, strengthen the skin barrier structure, and achieve highly efficient barrier repair effects, fully verifying that the optimal formulation of this invention possesses comprehensive skincare advantages of soothing, brightening, anti-aging, and repairing.

[0100] Comparative Example 3, lacking the addition of peony root bark extract to its formula, showed significantly weaker improvements in all four human efficacy indicators compared to the product group in Example 2 (p < 0.05). Compared to Example 2, this group exhibited a significant decrease in pain relief and melanin metabolism improvement, fully validating that peony root bark extract is the core active ingredient responsible for the composition's soothing, anti-allergic, and skin-brightening effects. Simultaneously, the improvement in skin elasticity and skin barrier repair capacity decreased in this group, confirming that peony root bark extract is not a single-function ingredient but can synergistically enhance the effects of fermentation products and other peony active components within the system, jointly participating in skin anti-aging and barrier repair regulation.

[0101] Compared with the finished product of Example 7, after replacing the fermentation product of *Pseudomonas alterniflora* with *Lactobacillus* fermentation lysate, the overall skin care efficacy was significantly reduced. The four indicators showed extremely significant differences compared with the finished product of Example 2. The skin stinging improvement effect and TEWL barrier repair effect were the most significantly weakened in this group. This indicates that the combination of *Lactobacillus* fermentation lysate and *Pseudomonas alterniflora* fermentation product in this formula is the key functional component for barrier repair and skin tolerance improvement in the system of this invention. The two work together to strengthen the skin physical barrier and reduce skin sensitivity. When combined with the active ingredients of the peony series, they form a multi-dimensional synergistic active system for barrier repair, soothing and anti-sensitivity, and anti-aging and brightening.

[0102] Comparative Example 8 retained the complete optimal formula components, but omitted the homogenization and emulsification fine preparation process and only used conventional stirring preparation. Its four measured indicators showed the lowest improvement among all test groups, and there was a highly significant difference from the product of Example 2. This result fully demonstrates that the high-pressure homogenization and emulsification process defined in this invention can effectively improve the transdermal absorption efficiency of active ingredients and maximize the release of peony active components and fermentation products for skin care efficacy. It is an indispensable key preparation process to ensure the final application effect of the finished product.

[0103] In summary, the peony multi-functional active composition of the present invention, when added to the finished skin care product, can simultaneously achieve multiple skin care effects such as soothing, brightening, anti-aging, and repairing. The combination of all components and the process together constitute a complete efficacy system. Changing the composition formula or preparation process will lead to a significant decrease in the overall efficacy of the finished product.

[0104] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A multifunctional active composition of peony targeting the TRPV1 / SIRT1 pathway, characterized in that, The peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway comprises, by weight percentage, the following components: Glycerin 40-60%, peony flower water 5-15%, peony seed oil 5-15%, peony extract 5-15%, Lactobacillus fermentation lysate 5-15%, hydrogenated lecithin 0.5-6%, peony root bark extract 0.01-5%, polyglycerol-10 laurate 0.05-6%, p-hydroxyacetophenone 0.1-1%, 1,2-hexanediol 0.1-1%, Pseudomonas aeruginosa fermentation product 0.01-1%, paeonol 0.0001-0.05%, deionized water to 100%.

2. The peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway according to claim 1, characterized in that, The peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway comprises, by weight percentage: 50% glycerin, 10% peony flower water, 10% peony seed oil, 10% peony extract, 10% Lactobacillus fermentation lysate, 4% hydrogenated lecithin, 3% peony root bark extract, 3% polyglycerol-10 laurate, 0.5% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, 0.5% Pseudomonas aeruginosa fermentation product, 0.025% paeonol, and deionized water to 100%.

3. A method for preparing a peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway as described in any one of claims 1-2, characterized in that, Includes the following steps: S1. Mix peony seed oil, hydrogenated lecithin, polyglycerol-10 laurate, and paeonol, heat to 45-55℃, and stir until uniformly mixed to obtain an oil phase mixture. S2. Mix glycerol, deionized water, peony flower water, peony extract, peony root bark extract, Lactobacillus fermentation lysate, and Pseudomonas aeruginosa fermentation product, heat to 45-55℃, stir evenly, and obtain an aqueous phase mixture. S3. Slowly add the oil phase mixture to the water phase mixture, stir at 45~55℃ for 3~10 min, and homogenize under high pressure 1~5 times at a pressure of 15000-30000 PSI; S4. After homogenization, cool to 25~35℃, add p-hydroxyacetophenone and 1,2-hexanediol, stir evenly to obtain the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway.

4. The use of the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway according to any one of claims 1-3 in the preparation of skin care products.

5. The application according to claim 4, characterized in that, The percentage by weight of the peony multifunctional active composition targeting the TRPV1 / SIRT1 pathway added to the skin care product is no more than 10%.

6. The application according to claim 4, characterized in that, The skincare products include any one of the following: serum, toner, lotion, cream, and mask.

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