New application of schizonepeta fordii vesicles

Abstract

The invention discloses a new application of schizonepeta fordii vesicles, which is realized through in-vitro cells, and proves that the schizonepeta fordii vesicles can obviously reduce the expression of TNF-alpha and NF-kappa B inflammatory factors of macrophages, reduce the expression of TRPV1 of keratinocytes, increase the expression of LOR and FLG of the keratinocytes and reduce the expression of SREBP-1 and ACC of sebaceous gland cells. The schizonepeta tenuifolia vesicle has the advantages that the schizonepeta tenuifolia vesicle has obvious soothing, repairing, oil control and tightening effects and can be applied to a skin care product with at least one of soothing, repairing, oil control and tightening effects, and accordingly the schizonepeta tenuifolia vesicle can be applied to skin care products with at least one of soothing, repairing, oil control and tightening effects by the aid of the schizonepeta tenuifolia vesicle.

Description

Technical Field

[0001] This invention relates to a vesicle of Nepeta cataria, and more particularly to a new application of a Nepeta cataria vesicle. Background Technology

[0002] The skin is the largest organ in the human body, playing a vital protective role and serving as the body's first line of defense. In daily life, various factors such as climate change, work stress, lifestyle, and natural aging can cause skin damage or aging.

[0003] Currently, the main ingredients used in skin care cosmetics or topical medications are plant extracts, fermentation products, and monomers. However, these ingredients still face challenges in application, such as poor permeability, low transdermal penetration, and delivery difficulties, making it difficult to effectively exert their efficacy.

[0004] Plant-derived vesicles (PDVLs) refer to particles obtained from plants and plant tissues that possess a lipid bilayer structure and cannot self-replicate. These particles can be uniformly dispersed in water. In terms of composition, unlike plant essential oils and traditional plant extracts which contain only secondary metabolites, PDVLs encapsulate a variety of active ingredients, including proteins, lipids, miRNAs, and other secondary metabolites. This multi-component synergistic mechanism, composed of abundant functional molecules, has greater application potential than single-component formulations. In terms of applications, the lipid bilayer structure provides a natural advantage in biocompatibility compared to traditional plant extracts. Compared to animal-derived vesicles, plant-derived vesicles have higher production efficiency, shorter extraction cycles, lower immunogenicity, and are free from ethical controversies. Currently, literature reports that plant-derived vesicles have good application value in anti-tumor, anti-inflammatory, intestinal homeostasis, wound regeneration, cell differentiation, and tissue repair. Plant-derived vesicles also have broad application prospects in skin care cosmetics and topical medications.

[0005] *Schizonepeta tenuifolia* is a plant belonging to the genus *Schizonepeta* in the family Lamiaceae. According to the *Flora of China*, the whole plant and flower spikes are commonly used in traditional Chinese medicine to treat symptoms such as colds, headaches, sore throats, and itchy skin. The whole plant is also rich in aromatic oils, which can be extracted from it.

[0006] Existing patent CN118338892A discloses a new composition containing effluxes derived from Nepeta cataria as an active ingredient, revealing that effluxes obtained from fresh Nepeta cataria or callus cell culture medium possess activities that improve skin elasticity, reduce wrinkles, and promote wound healing. However, the Lamiaceae family is one of the most widely distributed families globally, comprising approximately 220 genera and over 3,500 species worldwide. China alone has 99 genera and over 800 species, and objective differences exist between different genera. Nepeta cataria and Nepeta dissecta belong to different genera, with clear intergeneric differences. Currently, there is no research on Nepeta dissecta vesicles, and their application in skin care remains unclear. Summary of the Invention

[0007] The purpose of this invention is to provide a novel application for Nepeta cataria vesicles. This invention discovers that Nepeta cataria vesicles can be used as a soothing, repairing, oil-controlling, and firming active ingredient in skin care cosmetics or topical pharmaceutical products.

[0008] The technical solution of this invention:

[0009] Application of Nepeta cataria vesicles in the preparation of skin care products with at least one of the following effects: soothing, repairing, oil control, and firming.

[0010] In the aforementioned applications, the product is a cosmetic or a drug.

[0011] In the aforementioned applications, the method for extracting the vesicles of *Nepeta cataria* includes the following steps:

[0012] S1. Pulping: Add buffer solution to Nepeta cataria and pulverize to obtain slurry;

[0013] S2. Enzymatic hydrolysis: Add a hydrolysis-specific enzyme to the slurry, stir and filter to obtain the filtrate;

[0014] S3. Separation: The filtrate is centrifuged sequentially at low speed, medium speed, and high speed at low temperature to obtain the supernatant;

[0015] S4. Purification: Add a high molecular weight precipitant to the supernatant, refrigerate, centrifuge, and resuspend in buffer to obtain Nepeta cataria vesicles.

[0016] In the aforementioned application, in step S1, the buffer solution is a phosphate buffer solution with a concentration of 0.1~0.2 mol / L and a material-to-liquid ratio of (1:8)~(1:12) (W / V).

[0017] In the aforementioned application, in step S2, the amount of hydrolysis-specific enzyme added is 0.1~0.5% of the raw material amount (W / W). The hydrolysis-specific enzyme is composed of at least one of cellulase, pectinase or hemicellulase, and the enzyme activity of the hydrolysis-specific enzyme is ≥300,000 U / g.

[0018] In the aforementioned application, in step S3, the low temperature is 2~6℃, the low speed centrifugation is 800~1200×g, the medium speed centrifugation is 2500~3500×g, the high speed centrifugation is 9000~11000×g, and the centrifugation time for each centrifugation is 10~30min.

[0019] In the aforementioned application, in step S4, the centrifugation conditions are 9000~12000×g for 10~20min.

[0020] In the aforementioned application, in step S4, the polymeric precipitant is polyethylene glycol (PEG-6000), and the amount of polyethylene glycol added is 8~12% (w / v) of the supernatant.

[0021] In step S4, the buffer solution is a phosphate buffer solution, and the amount of phosphate buffer solution added is 0.25 to 1 times (V / W) of the raw material.

[0022] A cosmetic product with soothing, repairing, oil-controlling, or firming effects, containing Nepeta cataria vesicles.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a method for extracting *Nepeta cataria* vesicles from *Nepeta cataria*. The extracted *Nepeta cataria* vesicles were observed to have a distinct cup-shaped structure under transmission electron microscopy, proving that the extraction was successful.

[0025] This invention utilizes in vitro cell experiments on *Nepeta cataria* vesicles to demonstrate that *Nepeta cataria* vesicles can significantly reduce the expression of inflammatory factors such as tumor necrosis factor-α (TNF-α) and NF-κB in macrophages, decrease transient receptor potential vanillic acid subtype 1 (TRPV1) in keratinocytes and increase the expression of lobe rhinol (LOR) and filaggrin (FLG), decrease the expression of sterol regulatory element-binding protein-1 (SREBP-1) and acetyl-CoA carboxylase (ACC) in sebaceous gland cells, increase the expression of type III collagen in fibroblasts, repair damaged skin barrier, control lipid synthesis, and increase collagen production.

[0026] Therefore, Nepeta cataria vesicles have significant soothing, repairing, oil-controlling and firming effects. Nepeta cataria vesicles can be used as an active ingredient with soothing, repairing, oil-controlling and firming effects in skin care cosmetics or topical drugs, effectively expanding the application scope of Nepeta cataria vesicles. Attached Figure Description

[0027] Figure 1 This is a flowchart of the extraction method of the present invention.

[0028] Figure 2 These are typical transmission electron microscope images of *Nepeta cataria* vesicles from different embodiments of the present invention.

[0029] Figure 3 This is a typical particle size distribution diagram of the Nepeta cataria vesicles in Embodiment 2 of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0031] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, a hinged connection, a rotatable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0032] Example 1:

[0033] like Figure 1 As shown, a method for extracting vesicles from Nepeta cataria includes the following steps:

[0034] S1. Pulping: Take 100g of dried Nepeta cataria and pre-pulverize it. Add 0.2mol / L PBS buffer at a ratio of 1:10 (W / V) of plant to PBS buffer and pulverize to obtain a slurry.

[0035] S2. Enzymatic hydrolysis: Add 0.1% (w / w) of cellulase to the slurry, stir at room temperature, filter, and obtain filtrate.

[0036] S3. Separation: The filtrate is centrifuged sequentially at 6°C at low speed (800×g) for 10 min, medium speed (3000×g) for 20 min, and high speed (9000×g) for 30 min to remove large particles and cell debris, and the supernatant is obtained.

[0037] S4. Purification: Add PEG-6000 to the supernatant at a concentration of 8% (W / V) of the supernatant. Refrigerate overnight to precipitate the vesicles. Centrifuge at 12000×g for 10 min to obtain the precipitate. Resuspend the precipitate in 0.25 times the amount of the raw material in PBS buffer (V / W) to obtain the Nepeta cataria vesicle sample.

[0038] Example 2:

[0039] The method for extracting vesicles from Nepeta cataria includes the following steps:

[0040] S1. Pulping: Take 500g of dried Nepeta cataria and pre-pulverize it. Add 0.15mol / L PBS buffer at a ratio of 1:12 (W / V) of plant to PBS buffer and pulverize to obtain a slurry.

[0041] S2. Enzymatic hydrolysis: Add 0.5% (w / w) of pectinase to the slurry, stir at room temperature, filter, and obtain filtrate.

[0042] S3. Separation: The filtrate is centrifuged sequentially at 4°C at low speed (1000×g) for 10 min, medium speed (3500×g) for 20 min, and high speed (10000×g) for 30 min to remove large particles and cell debris, and the supernatant is obtained.

[0043] S4. Purification: PEG-6000 was added to the supernatant at a concentration of 12% (W / V). The mixture was refrigerated overnight to precipitate the vesicles. The precipitate was obtained by centrifugation at 9000×g for 15 min. The precipitate was resuspended in 0.5 times the amount of the raw material in PBS buffer (V / W) to obtain the Nepeta cataria vesicle sample.

[0044] Example 3:

[0045] The method for extracting vesicles from Nepeta cataria includes the following steps:

[0046] S1. Pulping: Take 1000g of dried Nepeta cataria and pre-pulverize it. Add 0.1mol / L PBS buffer at a ratio of 1:8 (W / V) of plant to PBS buffer and pulverize to obtain a slurry.

[0047] S2. Enzymatic hydrolysis: Add 0.3% (w / w) of hemicellulase to the slurry, stir at room temperature, filter, and obtain filtrate.

[0048] S3. Separation: The filtrate is centrifuged sequentially at 2℃ at low speed (1200×g) for 10 min, medium speed (2500×g) for 20 min, and high speed (11000×g) for 30 min to remove large particles and cell debris, and the supernatant is obtained.

[0049] S4. Purification: PEG-6000 was added to the supernatant at a concentration of 10% (W / V). The mixture was refrigerated overnight to precipitate the vesicles. The precipitate was obtained by centrifugation at 10000×g for 20 min. The precipitate was resuspended in PBS buffer (V / W) at a concentration equal to the amount of the raw material to obtain the Nepeta cataria vesicle sample.

[0050] Test Example 1: Morphological characterization of vesicles in Nepeta cataria.

[0051] (1) Experimental method: Transmission Electron Microscope (TEM) analysis.

[0052] (2) Experimental steps:

[0053] a. Dilute the Nepeta cataria vesicle sample with 1×PBS buffer, drop it onto the carbon membrane of the copper grid, let it precipitate for 1 min, and then absorb the floating liquid with filter paper.

[0054] b. Apply phosphotungstic acid staining agent or uranyl acetate staining agent to the copper mesh, allow it to precipitate, absorb off any excess dye, and let the copper mesh dry naturally in a dry environment.

[0055] c. Perform electron microscopy imaging and record images and data.

[0056] (3) Experimental results:

[0057] Typical transmission electron micrographs of Nepeta cataria vesicles obtained in Examples 1-3 are shown below. Figure 2 As shown in the figure, the vesicles of *Nepeta cataria* in the three embodiments all have a cup-shaped structure, and the structure is regularly circular. The above results prove that the extraction method of the present invention achieves successful extraction of *Nepeta cataria* vesicles.

[0058] Test Example 2: Characterization experiment of particle size and concentration of Nepeta cataria var. spp. vesicles.

[0059] (1) Experimental method: Nanoparticle Tracking Analysis (NTA).

[0060] (2) Experimental steps: Dilute the Nepeta cataria vesicle sample with 1×PBS buffer, put it into the sample cell of the nanoparticle tracker, and record data and images such as the average particle size and concentration of the vesicles.

[0061] (3) Experimental results:

[0062] The average particle size and concentration of *Nepeta cataria* vesicles in the three embodiments are shown in Table 1. The average particle size of *Nepeta cataria* vesicles ranged from 121.57 to 126.13 nm, and the concentration was 9.57 × 10⁻⁶. 10~4.83×10 11 Particles / mL. Figure 3 Typical particle size distribution of Nepeta cataria vesicles obtained in Example 2.

[0063] Table 1. Mean particle size, concentration, zeta potential, and menthol content of *Nepeta cataria* vesicles

[0064]

[0065] Test Example 3: Measurement of Zeta potential of Nepeta cataria var. spp. vesicles.

[0066] (1) Experimental method: Zeta potential analyzer.

[0067] (2) Experimental procedure: The Zeta potential of the vesicle dispersion of Nepeta cataria was measured according to the operating procedure of the Zeta potential analyzer.

[0068] (3) Experimental results: The Zeta potentials of the vesicles of Nepeta cataria are shown in Table 1.

[0069] Test Example 4: Characteristic component analysis of Nepeta cataria vesicles.

[0070] (1) Experimental method: High performance liquid chromatography

[0071] (2) Experimental steps:

[0072] a. Preparation of standard solution: Dissolve an appropriate amount of menthol standard in methanol in a 10 mL volumetric flask, dilute to volume, and mix well to obtain the standard stock solution. Dilute the stock solution and filter it through a 0.22 μm microporous membrane to obtain the standard solution for later use.

[0073] b. Preparation of test solution: Take the test sample, filter it through a 0.22μm microporous membrane to prepare the test solution for later use.

[0074] c. Analysis: The prepared standard and test solutions were analyzed by high-performance liquid chromatography (HPLC). The mobile phase was methanol-0.1% formic acid in water, and the detection wavelength was 250 nm.

[0075] (3) Experimental results:

[0076] Catnipae lanceolata has a distinctive, refreshing aroma, primarily derived from components such as menthol. Therefore, menthol can serve as an indicator component for identifying Catnipae lanceolata. The menthol content in the Catnipae lanceolata vesicle samples obtained in Examples 1-3 was analyzed, and the results are shown in Table 1. Menthol was detected in all Catnipae lanceolata vesicle samples obtained in Examples 1-3, indicating that the plant vesicles obtained in this invention are derived from Catnipae lanceolata.

[0077] The physicochemical characterization of the Nepeta cataria vesicle samples obtained in Examples 1-3 was basically stable, and the sample in Example 2 was selected for efficacy verification.

[0078] Test Example 5: Test on the soothing effect of Nepeta cataria vesicles.

[0079] 5.1 Cytotoxicity assay:

[0080] (1) Experimental method: MTT method.

[0081] (2) Experimental cells: RAW 264.7 macrophages and HaCaT keratinocytes.

[0082] (3) Experimental steps:

[0083] a. Cells were seeded in 96-well plates and cultured overnight at 37°C in 5% CO2. When the cell seeding rate reached 60%, the cells were injected with the appropriate concentration of culture medium containing the sample from Example 2. The solvent control group was replaced with fresh culture medium, the positive control group was given culture medium containing 10% DMSO, and the zeroing group was given blank culture medium without cell seeding. The cells were incubated at 37°C in 5% CO2 for 24 hours.

[0084] b. After incubation, discard the supernatant, add MTT solution to each well, incubate in the dark for 4 hours, then discard the culture medium, add DMSO solution, shake to mix, and measure the absorbance value at 490 nm (OD). 490 ).

[0085] c. Cell viability (%) = (OD 490样品组 -OD 490调零组 ) / (OD 490溶剂对照组 -OD 490调零组 ) × 100%.

[0086] (4) Experimental results:

[0087] Table 2. Effects of Nepeta cataria vesicles on the cell viability of RAW 264.7 macrophages and HaCaT keratinocytes.

[0088]

[0089] Note: Cell viability ≥90% indicates no cytotoxicity.

[0090] The experimental results are shown in Table 2, indicating that the Nepeta cataria vesicle sample of Example 2 did not show significant cytotoxicity to RAW264.7 macrophages in the 2.5% concentration range, and did not show significant cytotoxicity to HaCaT keratinocytes in the 5.0% concentration range.

[0091] Therefore, in subsequent experiments, 0.1% and 2.5% were selected as the drug concentrations for the LPS-stimulated macrophage experiment and the capsaicin (CAP)-stimulated keratinocyte experiment.

[0092] 5.2 Effects of Nepeta cataria vesicles on TNF-α and NF-κB in macrophages:

[0093] (1) Experimental steps:

[0094] a. Cell grouping, modeling, and drug administration: Macrophages were seeded into 6-well plates and divided into a blank control group, a model group, a positive control group, and experimental groups with different concentrations. Cells were cultured overnight at 37°C in 5% CO2. When the cell deposition rate in the 6-well plates reached 60%, drugs were administered to the groups. Each experimental group was added with culture medium containing different concentrations of the sample from Example 2. The positive control group was added with culture medium containing dexamethasone, and the blank control group and model group were added with blank culture medium. After incubation for 2 hours, all groups except the blank control group were treated with working solution containing LPS and incubated for 22 hours.

[0095] b. Indicator detection: After incubation, RNA was extracted from each group, cDNA was reverse transcribed, and the gene expression of TNF-α and NF-κB was detected by real-time PCR.

[0096] c. Calculation of downregulation rate: Downregulation rate (%) = (model group - experimental group) / model group * 100%.

[0097] (2) Experimental results:

[0098] Table 3. Effects of Nepeta cataria vesicles on the relative expression levels of TNF-α and NF-κB in macrophages.

[0099]

[0100] Note: When performing statistical analysis using the t-test method, compared with the blank control group... # This indicates that the p-value is less than 0.05. ## This indicates a P-value < 0.01. Compared to the model group, * indicates a P-value < 0.05, and ** indicates a P-value < 0.01.

[0101] In addition to dryness, peeling, redness, and itching, skin barrier damage is often accompanied by inflammation. The LPS-stimulated macrophage inflammation model is a classic in vitro model for screening anti-inflammatory components. The experimental results of the effects of *Nepeta cataria* vesicles on TNF-α and NF-κB in macrophages are shown in Table 3. In this experiment, 0.1% and 2.5% of the Example 2 samples significantly reduced the gene expression of TNF-α and NF-κB. Compared with the model group, the 0.1% Example 2 sample downregulated the relative expression of TNF-α by 32.18% and the relative expression of NF-κB by 7.69%. The 2.5% Example 2 sample downregulated the expression of the two inflammatory factors by 33.62% and 8.46%, respectively. These results indicate that *Nepeta cataria* vesicles have a significant inhibitory effect on the expression of inflammatory factors, which can alleviate redness and swelling caused by skin inflammation and can be applied to skin care cosmetics or topical medications with soothing effects.

[0102] 5.3 Effects of Nepeta cataria vesicles on TRPV1:

[0103] (1) Experimental steps:

[0104] a. Cell grouping, modeling, and drug administration: Keratinocytes were seeded into 6-well plates and divided into a blank control group, a model group, a positive control group, and experimental groups with different concentrations. Cells were cultured at 37°C and 5% CO2 for 24 hours. When the cell deposition rate in each well reached 60%, culture medium containing CAP and different concentrations of the sample from Example 2 was added to each experimental group. The positive control group was added to culture medium containing CAP and trans-4-tert-butylcyclohexanol, the model group was added to culture medium containing CAP, and the blank control group was added to blank culture medium. After drug administration, the cells were incubated for 24 hours.

[0105] b. Indicator detection: After incubation, RNA was extracted from each group, cDNA was reverse transcribed, and TRPV1 gene expression was detected by real-time PCR.

[0106] c. Calculation of downregulation rate: Downregulation rate (%) = (model group - experimental group) / model group * 100%.

[0107] (2) Experimental results:

[0108] Table 4. Effects of Nepeta cataria vesicles on relative TRPV1 expression in keratinocytes

[0109]

[0110] Note: When performing statistical analysis using the t-test method, compared with the blank control group... # This indicates that the p-value is less than 0.05. ##This indicates a P-value < 0.01. Compared to the model group, * indicates a P-value < 0.05, and ** indicates a P-value < 0.01.

[0111] TRPV1 is a core molecular switch for pain and temperature sensation in human skin. When the skin is stimulated by external factors, TRPV1 triggers rapid neuronal transmission of pain signals, which is the main physiological source of stinging and burning sensations in individuals with impaired skin barriers or sensitive skin. The effects of *Nepeta cataria* vesicles on TRPV1 are shown in Table 4. Compared to the model group, the addition of 0.1% of the Example 2 sample downregulated TRPV1 expression by 45.84%, and the addition of 2.5% of the Example 2 sample downregulated TRPV1 expression by 74.63%. These results indicate that *Nepeta cataria* vesicles significantly block the transmission of pain signals, relieving stinging sensations caused by skin damage, and can be applied to skin care cosmetics or topical medications with soothing effects.

[0112] Test Example 6: Repair Efficacy Test of Nepeta cataria vesicles.

[0113] 6.1 Cytotoxicity assay:

[0114] (1) Experimental method: MTT method.

[0115] (2) Experimental cells: HaCaT keratinocytes.

[0116] (3) The experimental procedure is the same as the experimental procedure in 5.1 of test example 5.

[0117] (4) Experimental results: Same as Table 2, 0.1% and 2.5% were selected as the drug concentrations for this repair efficacy test.

[0118] 6.2 Effects of Nepeta cataria vesicles on LOR and FLG:

[0119] (1) Experimental steps:

[0120] a. Cell grouping and drug administration: Keratinocytes were seeded in 6-well plates and divided into a blank control group, a positive control group, and experimental groups with different concentrations. The cells were cultured overnight at 37°C in 5% CO2. When the plating rate reached 40%–60%, culture medium containing different concentrations of the sample from Example 2 was added to each experimental group. Culture medium containing WY14643 was added to the positive control group, and blank culture medium was added to the blank control group. The cells were incubated for 24 hours.

[0121] b. Indicator detection: After incubation, RNA was extracted from each group, cDNA was reverse transcribed, and the gene expression of LOR and FLG was detected by real-time PCR.

[0122] c. Calculation of improvement rate: Improvement rate (%) = (Experimental group - Blank control group) / Blank control group * 100%.

[0123] (2) Experimental results:

[0124] Table 5. Effects of Nepeta cataria vesicles on the relative expression levels of LOR and FLG in keratinocytes.

[0125]

[0126] Note: When performing statistical analysis using the t-test method, compared with the blank control group, * indicates P-value < 0.05, and ** indicates P-value < 0.01.

[0127] Lobelin (LOR) and filaggrin (FLG) are important components of the skin barrier. LOR participates in the formation of a dense keratinized capsule, while FLG aggregates keratin fibers, jointly maintaining the integrity of the skin barrier. Simultaneously, FLG can degrade into natural moisturizing factor (NMF), maintaining skin hydration and preventing dryness and cracking. The experimental results of the effects of *Nepeta cataria* vesicles on LOR and FLG are shown in Table 5. In Example 2, the samples at concentrations of 0.1% and 2.5% significantly increased the expression levels of LOR and FLG genes, with increases of 433.00% and 506.00% and 238.00% and 558.00%, respectively. This indicates that *Nepeta cataria* vesicles can improve damaged skin barriers by upregulating the expression of LOR and FLG, and can be applied to skin care cosmetics or topical pharmaceutical products with repairing effects.

[0128] Test Example 7: Oil-controlling efficacy test of Nepeta cataria vesicles.

[0129] 7.1 Cytotoxicity assay:

[0130] (1) Experimental method: MTT method.

[0131] (2) Experimental cells: sebaceous gland cells.

[0132] (3) The experimental procedure is the same as the experimental procedure in 5.1 of test example 5.

[0133] (4) Experimental results:

[0134] Table 6. Effects of Nepeta cataria vesicles on sebaceous gland cell viability

[0135]

[0136] Note: Cell viability ≥90% indicates no cytotoxicity.

[0137] According to the MTT test results shown in Table 6, the vesicle samples of Nepeta cataria in Example 2 did not show significant cytotoxicity to sebaceous gland cells in the concentration range of 2.5%. Therefore, 0.1% and 2.5% were selected as the drug concentrations for subsequent oil-control efficacy testing.

[0138] 7.2 Effects of Nepeta cataria vesicles on SREBP-1 and ACC:

[0139] (1) Experimental steps:

[0140] a. Cell grouping, modeling, and drug administration: Sebaceous gland cells were seeded in 6-well plates and divided into a blank control group, a model group, a positive control group, and experimental groups with different concentrations. Cells were cultured overnight at 37°C in 5% CO2. When the cell deposition rate reached 40%–60%, culture medium containing different concentrations of the sample from Example 2 and DHT was added to each experimental group. The positive control group was added to culture medium containing isotretinoin and DHT, the model group was added to culture medium containing DHT, and the blank control group was added to blank culture medium. Cells were incubated for 24 hours.

[0141] b. Indicator detection: RNA was extracted from each group, cDNA was reverse transcribed, and the gene expression of SREBP-1 and ACC was detected by real-time PCR.

[0142] c. Calculation of downregulation rate: Downregulation rate (%) = (model group - experimental group) / model group * 100%.

[0143] (2) Experimental results:

[0144] Table 7. Effects of Nepeta cataria vesicles on the relative expression levels of SREBP-1 and ACC in sebaceous gland cells.

[0145]

[0146] Note: When performing statistical analysis using the t-test method, compared with the blank control group... # This indicates that the p-value is less than 0.05. ## This indicates a P-value < 0.01. Compared to the model group, * indicates a P-value < 0.05, and ** indicates a P-value < 0.01.

[0147] SREBP-1 is a core transcription factor regulating fatty acid synthesis genes, directly controlling the biosynthesis of fatty acids and cholesterol in sebaceous gland cells. ACC catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, and is a key rate-limiting enzyme in fatty acid synthesis and a crucial metabolic enzyme connecting upstream signals and downstream lipid synthesis. Driven by SREBP-1c, the expression level of downstream ACC increases, leading to the synthesis of large amounts of fatty acids. The experimental results of the effects of Nepeta cataria vesicles on SREBP-1 and ACC are shown in Table 7. In Example 2 of this invention, the samples at concentrations of 0.1% and 2.5% significantly reduced the expression levels of SREBP-1 and ACC. These results indicate that Nepeta cataria vesicles have an inhibitory effect on lipid synthesis and can be applied to skin care cosmetics or topical medications with oil-controlling effects.

[0148] Test Example 8: Test on the firming effect of Nepeta cataria vesicles.

[0149] 8.1 Cytotoxicity assay:

[0150] (1) Experimental method: MTT method.

[0151] (2) Experimental cells: fibroblasts.

[0152] (3) The experimental procedure is the same as the experimental procedure in 5.1 of test example 5.

[0153] (4) Experimental results:

[0154] Table 8. Effects of Nepeta cataria vesicles on fibroblast viability

[0155]

[0156] Note: Cell viability ≥90% indicates no cytotoxicity.

[0157] Based on the MTT assay results shown in Table 8, the sample from Example 2 did not exhibit significant cytotoxicity against fibroblasts within the 5% concentration range. Therefore, 0.1% and 2.5% were selected as the dosage concentrations for subsequent firming efficacy testing.

[0158] 8.2 Effects of Nepeta cataria vesicles on Collagen III:

[0159] (1) Experimental steps:

[0160] a. Cell grouping, modeling, and drug administration: Fibroblasts were seeded in 6-well plates and divided into a blank control group, a model group, a positive control group, and experimental groups with different concentrations. Cells were cultured overnight at 37°C in 5% CO2. When the cell deposition rate reached 30-50%, culture medium containing different concentrations of the sample from Example 2 was added to each experimental group. Culture medium containing TGF-β1 was added to the positive control group, while blank culture medium was added to the blank control group and the model group. Incubation was carried out for 24 hours. After incubation, except for the blank control group, all other groups were irradiated with UVA and cultured for another 24 hours after irradiation.

[0161] b. Indicator detection: After incubation, RNA was extracted from each group, cDNA was reverse transcribed, and the gene expression of Collagen III was detected by quantitative real-time PCR.

[0162] c. Calculation of improvement rate: Improvement rate (%) = (Experimental group - Model group) / Model group * 100%.

[0163] (2) Experimental results:

[0164] Table 9. Effects of Nepeta cataria vesicles on the relative expression level of Collagen III in fibroblasts.

[0165]

[0166] Note: When performing statistical analysis using the t-test method, compared with the blank control group... # This indicates that the p-value is less than 0.05. ## This indicates a P-value < 0.01. Compared to the model group, * indicates a P-value < 0.05, and ** indicates a P-value < 0.01.

[0167] Collagen III is a major component of fibrous collagen, accounting for 5% to 20% of all collagen in the human body. This collagen often assembles with Collagen I to form collagen fiber structures and is abundant in elastic tissue, making the skin appear plump and full. However, when the skin experiences increased free radicals due to natural aging or external stimuli, pathways such as MAPK and NF-κB are activated, and the expression of matrix metalloproteinases (MMPs) is upregulated, accelerating collagen degradation and leading to skin atrophy and sagging. The experimental results of the effect of Nepeta cataria vesicles on Collagen III are shown in Table 9. Treatment with 0.1% and 2.5% of the Sample 2 from Example 2 significantly increased the expression level of Collagen III, indicating that Nepeta cataria vesicles have a collagen-promoting effect and can be applied to skin care cosmetics or topical medications with firming effects.

[0168] In summary, *Nepeta cataria* vesicles significantly reduce the expression of inflammatory factors such as TNF-α and NF-κB in macrophages, decrease TRPV1 in keratinocytes while increasing LOR and FLG in keratinocytes, decrease the expression of SREBP-1 and ACC in sebaceous gland cells, and increase the expression of Collagen III in fibroblasts. These effects can alleviate redness, swelling, and stinging caused by skin inflammation, repair damaged skin barriers, control sebum secretion, and promote collagen production. This suggests that *Nepeta cataria* vesicles can be used as an active ingredient with soothing, repairing, oil-controlling, and firming properties in skin care cosmetics or topical medications.

[0169] The parts of this invention not described in detail are prior art and therefore will not be specifically described here.

Claims

1. Application of Nepeta cataria vesicles in the preparation of skin care products with at least one of the following effects: soothing, repairing, oil control, and firming.

2. The application according to claim 1, characterized in that: The product in question is either a cosmetic or a pharmaceutical.

3. The application according to claim 1, characterized in that: The method for extracting vesicles from *Nepeta cataria* includes the following steps: S1. Pulping: Add buffer solution to Nepeta cataria and pulverize to obtain slurry; S2. Enzymatic hydrolysis: Add a hydrolysis-specific enzyme to the slurry, stir and filter to obtain the filtrate; S3. Separation: The filtrate is centrifuged sequentially at low speed, medium speed, and high speed at low temperature to obtain the supernatant; S4. Purification: Add a high molecular weight precipitant to the supernatant, refrigerate, centrifuge, and resuspend in buffer to obtain Nepeta cataria vesicles.

4. The application according to claim 3, characterized in that: In step S1, the buffer solution is a phosphate buffer solution with a concentration of 0.1~0.2 mol / L and a material-to-liquid ratio of (1:8)~(1:12) (W / V).

5. The application according to claim 3, characterized in that: In step S2, the amount of hydrolysis-specific enzyme added is 0.1~0.5% of the raw material amount (W / W). The hydrolysis-specific enzyme is composed of at least one of cellulase, pectinase or hemicellulase, and the enzyme activity of the hydrolysis-specific enzyme is ≥300,000 U / g.

6. The application according to claim 3, characterized in that: In step S3, the low temperature is 2~6℃, the low speed centrifugation is 800~1200×g, the medium speed centrifugation is 2500~3500×g, the high speed centrifugation is 9000~11000×g, and the centrifugation time for each centrifugation is 10~30min.

7. The application according to claim 3, characterized in that: In step S4, the centrifugation conditions are 9000~12000×g for 10~20min.

8. The application according to claim 3, characterized in that: In step S4, polyethylene glycol is used as the polymeric precipitant, and the amount of polyethylene glycol added is 8-12% (w / v) of the supernatant.

9. The application according to claim 3, characterized in that: In step S4, the buffer solution is a phosphate buffer solution, and the amount of phosphate buffer solution added is 0.25 to 1 times (V / W) of the raw material.

10. A cosmetic product having soothing, repairing, oil-controlling, or firming effects, characterized in that: It contains Nepeta cataria vesicles.

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