Use of a portulaca oleracea extracellular vesicle in anti-acne and regulating acne microecological flora

Abstract

The application discloses application of a Portulaca oleracea extracellular vesicle in resisting acne and regulating acne microecological flora. The Portulaca oleracea extracellular vesicle PDEVs provided by the application can significantly inhibit growth of Propionibacterium acnes, and has obvious bactericidal effect under a lower MIC condition, has less influence on skin beneficial bacteria, is helpful to maintaining or regulating skin microecological balance, and has the advantage of selectively regulating skin flora. Meanwhile, the PDEVs can significantly reduce mRNA expression levels of inflammatory factors IL-6, IL-8, IL-1beta and TNF-alpha of HaCaT cells under stimulation of inactivated Propionibacterium acnes and intracellular reactive oxygen species ROS levels, thereby reducing inflammatory reaction and oxidative stress reaction; the PDEVs have a synergistic effect in regulating skin microecology, inhibiting inflammatory reaction and relieving oxidative stress, have good biocompatibility and safety, and provide more choices for developing natural source active ingredients for preventing or improving acne.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of extracellular vesicles derived from purslane in anti-acne treatment and regulation of acne microbiota. Background Technology

[0002] Acne is a common skin disease characterized by chronic inflammation of the pilosebaceous unit. Its occurrence is closely related to multiple factors, including abnormal sebum secretion, abnormal follicular keratinization, skin microecological imbalance, and abnormal inflammatory response. Among these factors, Propionibacterium acnes (…) Cutibacterium acnes *Propionibacterium acnes* is considered a key microbial factor inducing inflammatory responses in acne. After proliferating in the hair follicle environment, this bacterium stimulates keratinocytes to produce various inflammatory factors and induces elevated levels of intracellular reactive oxygen species (ROS), thereby exacerbating local inflammation and affecting skin barrier function. Therefore, interventions targeting *Propionibacterium acnes*-related inflammatory pathways, particularly by inhibiting bacterial growth while regulating inflammatory factor expression and reducing oxidative stress levels, are of great significance for the prevention and improvement of acne.

[0003] Currently, antibiotics, retinoids, salicylic acid, and benzoyl peroxide are commonly used in clinical and skin care treatments for acne. While these ingredients have some efficacy, long-term use may lead to increased bacterial resistance, skin irritation, or impaired skin barrier function. Existing anti-acne drugs may also affect the skin's microecology. Some anti-acne ingredients primarily work through broad-spectrum antibacterial activity; while inhibiting Propionibacterium acnes, they can also affect beneficial skin flora, thus disrupting the skin's microecological balance. Therefore, developing naturally derived, highly safe active substances with multi-faceted regulatory potential has become an important research direction in this field.

[0004] purslane( Portulaca oleraceaPurslane (L.) is a common plant used for both medicinal and edible purposes, containing various active ingredients such as polysaccharides, flavonoids, unsaturated fatty acids, and alkaloids. Existing literature reports that purslane extract possesses antibacterial, anti-inflammatory, and antioxidant activities and has been applied in the field of skin care. However, traditional plant extracts often suffer from problems such as complex composition, limited batch stability, and low bioavailability of active ingredients. Currently, purslane extract is commonly obtained through water extraction or alcohol extraction and added as a functional ingredient to skin care products. Existing studies show that different purslane extracts have varying antibacterial effects against Propionibacterium acnes, with MICs ranging from 2 to 4 mg / mL. It can inhibit skin inflammation caused by ultraviolet radiation, chemical irritation, or allergens (such as contact dermatitis), but there are no reports of it simultaneously regulating acne-induced keratinocyte inflammatory responses and oxidative stress. Meanwhile, existing studies report that purslane extract has a significant inhibitory effect on the skin microbiota Staphylococcus epidermidis, with a minimum inhibitory concentration reaching 50 μg / mL; long-term use may affect skin barrier function.

[0005] Although existing purslane extracts possess antibacterial or anti-inflammatory activities, they also affect the beneficial flora of the skin. Furthermore, existing purslane extracts have not been systematically validated for use in acne-induced inflammatory responses and oxidative stress, nor have comprehensive regulatory solutions been developed. Therefore, it is necessary to develop more effective anti-acne products that do not affect the beneficial flora of the skin and better maintain the skin barrier. This invention application is thus filed. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies, such as the impact of purslane extract on skin microecology, inhibition of antibacterial flora, and the need for improved antibacterial effects, as well as its ability to better regulate inflammatory responses and oxidative stress caused by acne. The purpose of this invention is to provide an application of purslane extracellular vesicles in anti-acne treatment and regulation of the acne microecological flora.

[0007] Specifically, this invention aims to: (1) provide a naturally derived extracellular vesicle from purslane with a well-defined structure as the active ingredient; (2) verify its antibacterial effect in a Propionibacterium acnes-related model and evaluate its impact on inflammatory response levels and oxidative stress; (3) while inhibiting Propionibacterium acnes, have minimal impact on the growth of beneficial skin bacteria such as Staphylococcus epidermidis, thereby achieving a relatively balanced regulation of the skin's microecological environment; and (4) provide a comprehensive application scheme suitable for acne prevention or improvement. Therefore, the purpose of this invention is to provide a new application of purslane extracellular vesicles in anti-acne treatment and regulation of the acne microecological flora.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: The purslane-derived extracellular vesicles (PDEVs) provided by this invention exist in the form of natural nanovesicles with a particle size of approximately 118.2 nm and a typical lipid bilayer structure. Compared with traditional plant extracts, these nanovesicles can more effectively encapsulate and deliver plant-derived bioactive components, thereby improving the stability of active substances and cellular uptake efficiency. Experimental results show that PDEVs can significantly inhibit the growth of Propionibacterium acnes at a concentration of 128 μg / mL and exhibit significant bactericidal activity at 8×MIC. Meanwhile, no significant inhibitory effect was observed on the growth of Staphylococcus epidermidis, a commensal bacterium of the skin, within the same concentration range. This indicates that purslane-derived extracellular vesicles can inhibit acne-related pathogens while having minimal impact on beneficial skin bacteria, thus helping to maintain or regulate the skin's microecological balance. Compared with existing broad-spectrum antibacterial or simple bacteriostatic technologies, this invention has the advantage of selectively regulating the skin flora.

[0009] Furthermore, compared to existing technologies that utilize plant-derived vesicles with only single anti-inflammatory or antioxidant mechanisms, this invention establishes an in vitro cell model based on the inflammatory process induced by *Propionibacterium acnes*, and systematically evaluates the multiple biological functions of PDEVs. Experimental results show that, within a concentration range of 25-100 μg / mL, PDEVs significantly reduce the mRNA expression levels of inflammatory factors IL-6, IL-8, IL-1β, and TNF-α in HaCaT cells stimulated by inactivated *Propionibacterium acnes*. Simultaneously, flow cytometry analysis revealed that PDEVs significantly reduced intracellular reactive oxygen species (ROS) levels at concentrations of 25 μg / mL and 50 μg / mL, thereby alleviating oxidative stress. In addition, CCK-8 assays showed that HaCaT cell survival rates were above 80% within a concentration range ≤100 μg / mL, indicating good cell compatibility and safety. This indicates that extracellular vesicles derived from purslane can exert synergistic effects through multiple pathways, such as inhibiting the growth of Propionibacterium acnes, regulating the expression of inflammatory factors, and reducing oxidative stress levels, thus exhibiting more significant technical effects in the prevention and improvement of acne.

[0010] Therefore, the purslane-derived extracellular vesicles provided by this invention not only have good biocompatibility and safety, but also exhibit synergistic effects in regulating skin microecology, inhibiting inflammatory responses and alleviating oxidative stress, providing a new technical solution for developing natural-derived active ingredients for the prevention or improvement of acne.

[0011] This invention provides the application of purslane extracellular vesicles in the preparation of products that regulate the skin microecology of acne-prone skin.

[0012] Preferably, the regulation of acne skin microecology refers to the regulation of acne skin flora.

[0013] More preferably, the regulation of acne-causing skin flora is selectively regulated by Staphylococcus epidermidis, a beneficial bacterium of the skin.

[0014] This invention provides the application of purslane extracellular vesicles in the preparation of products that inhibit Propionibacterium acnes.

[0015] This invention provides the application of purslane extracellular vesicles in the preparation of anti-acne drugs.

[0016] This invention provides the use of purslane extracellular vesicles in the preparation of products that alleviate and reduce inflammatory responses and oxidative stress levels caused by acne.

[0017] Preferably, the product is a cosmetic or a pharmaceutical.

[0018] Furthermore, the product can reduce the mRNA expression levels of inflammatory factors IL-6, IL-8, IL-1β and TNF-α.

[0019] Furthermore, the product can reduce intracellular reactive oxygen species levels.

[0020] Preferably, the purslane extracellular vesicles are plant-derived extracellular vesicles with nanoscale particle size and membrane structure characteristics obtained by extraction and purification from fresh purslane.

[0021] More preferably, the extracellular vesicles of purslane have a particle size of 100-200 nm and a lipid bilayer structure.

[0022] Specifically, in this embodiment of the invention, fresh purslane plant material is used as the source of extracellular vesicles. In other embodiments, different tissues or parts of purslane, such as leaves, stems, whole herb extracts, or homogenized plant material, can also be used for vesicle isolation. As long as extracellular vesicles derived from purslane can be obtained and have similar biological activity, they can be used to achieve the purpose of this invention.

[0023] Preferably, the extraction method employs differential centrifugation, density gradient centrifugation, ultrafiltration concentration, size exclusion chromatography, or a combination of ultracentrifugation and ultrafiltration.

[0024] More preferably, the differential centrifugation conditions are as follows: first, centrifuge at 800-1200 g for 8-15 min, discard the precipitate, and collect the supernatant; then, centrifuge at 2000-4000 g for 10-30 min, discard the precipitate, and collect the supernatant; continue centrifuging at 10000-20000 g for 50-60 min, discard the precipitate, collect the supernatant, and remove cell debris and large particulate impurities.

[0025] Furthermore, the concentration of purslane extracellular vesicles in the product is 25-100 μg / mL. In other embodiments, the concentration or dosage of extracellular vesicles may be appropriately adjusted according to the specific application scenario or formulation, as long as the technical effect of inhibiting acne-related inflammatory response or regulating skin microecology can be achieved, it falls within the protection scope of this invention.

[0026] Preferably, the drug further contains a pharmaceutically acceptable carrier or excipient.

[0027] In this invention, extracellular vesicles derived from purslane can be used as an active ingredient in products for treating acne or improving skin inflammation. In other embodiments, these extracellular vesicles can be further prepared into different types of topical skin preparations, such as solutions, gels, lotions, serums, masks, or sprays, or used in combination with other skin care ingredients. As long as the core active ingredient is extracellular vesicles derived from purslane and can achieve the anti-acne or anti-inflammatory effects described in this invention, they fall within the scope of protection of this invention.

[0028] The present invention has the following beneficial effects: This invention discloses a novel application of purslane extracellular vesicles (PDEVs) in anti-acne treatment and regulation of the acne microbiota. The PDEVs provided by this invention significantly inhibit the growth of *Propionibacterium acnes*, with a lower MIC than existing purslane extracts, and have minimal impact on beneficial skin bacteria, helping to maintain or regulate the skin microecological balance and exhibiting the advantage of selectively regulating the skin microbiota. Simultaneously, PDEVs significantly reduce the mRNA expression levels of inflammatory factors IL-6, IL-8, IL-1β, and TNF-α in HaCaT cells stimulated by inactivated *Propionibacterium acnes*, as well as the intracellular reactive oxygen species (ROS) levels, thereby alleviating inflammatory and oxidative stress responses. The purslane extracellular vesicles provided by this invention demonstrate a synergistic effect in regulating the skin microecology, inhibiting inflammatory responses, and alleviating oxidative stress, exhibiting good biocompatibility and safety, and providing more options for developing naturally derived active ingredients for acne prevention or improvement. Attached Figure Description

[0029] Figure 1 The particle size, zeta potential and morphological characterization of PDEVs are shown in the figure (a is the particle size distribution of PDEVs; b is the zeta potential of PDEVs; c is the morphology of PDEVs under transmission electron microscopy (200 nm); d is the morphology of PDEVs under transmission electron microscopy (100 nm)).

[0030] Figure 2The selective antibacterial effects of PDEVs against Propionibacterium acnes and Staphylococcus epidermidis are shown in the figure (a in the figure shows the antibacterial effect of different concentrations of PDEVs against Propionibacterium acnes; b shows the antibacterial effect of different concentrations of PDEVs against Staphylococcus epidermidis).

[0031] Figure 3 For PDEVs against Propionibacterium acnes ( Cutibacterium acnes Evaluation of bactericidal activity.

[0032] Figure 4 To evaluate the cytotoxicity of PDEVs on HacaT cells.

[0033] Figure 5 The effects of PDEVs on the expression of inflammatory factors in HaCaT cells induced by inactivated Propionibacterium acnes (Figure a shows the effect of different concentrations of PDEVs on IL-1β expression; b shows the effect of different concentrations of PDEVs on IL-6 expression; c shows the effect of different concentrations of PDEVs on TNF-α expression; d shows the effect of different concentrations of PDEVs on IL-8 expression).

[0034] Figure 6 The effect of PDEVs on ROS levels in HaCaT cells (Figure a shows the fluorescence intensity distribution of ROS levels in each group detected by flow cytometry; b shows the quantitative statistical analysis of relative ROS levels in each group). Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] The Propionibacterium acnes used in the examples ( Cutibacterium acnes ATCC 6919: From Guangdong Provincial Microbial Culture Collection Center; Staphylococcus epidermidis ( Staphylococcus epidermidisBNCC 330867, purchased from BeiNa Biotechnology Co., Ltd.; Brain Heart Infusion Broth (BHI): purchased from Beijing Bairddi Biotechnology Co., Ltd., brand: OXOID (catalog number 3555371); Columbia Blood AgarBase: purchased from Beijing Bairddi Biotechnology Co., Ltd., brand: OXOID (catalog number 3534315); LB lysogeny broth: purchased from Kaiji Biotechnology, brand: Kaiji Biotechnology (catalog number KGH2105-5.0L); Human immortalized keratinocytes (HacaT cells), from the China Center for Type Culture Collection; BCA protein concentration assay kit: purchased from Shenzhen High-Tech Technology Co., Ltd., brand: Beyotime (catalog number P0012); High-glucose Dulbecco's Modified Eagle Medium (DMEM): purchased from Shenzhen Hikvision Technology Co., Ltd., brand: GIBCO (catalog number 8121263); Fetal bovine serum: purchased from Shenzhen Hikvision Technology Co., Ltd., brand: Pronosai (catalog number 164210); CCK8 reagent: purchased from Shenzhen Hikvision Technology Co., Ltd., brand: Dongren (catalog number PH632); ROS reactive oxygen species detection kit: purchased from Shenzhen Hikvision Technology Co., Ltd., brand: Beyotime (catalog number S0033S).

[0038] Example 1: Extraction of extracellular vesicles from purslane Take fresh purslane ( Portulaca oleraceaThe aerial parts of the plant tissue were thoroughly rinsed with deionized water to remove surface mud and impurities, then rinsed twice with phosphate-buffered saline (PBS, pH 7.4), blotted dry, and cut into small segments. Pre-cooled PBS buffer was added at a mass-to-volume ratio of 1:3, and the tissue was mechanically homogenized for 3–5 min using a juicer to obtain a plant tissue homogenate. The homogenate was filtered through multiple layers of sterile gauze to remove larger tissue fragments and incompletely broken plant residues, yielding a preliminary filtrate. This filtrate was then subjected to differential centrifugation at 4°C: first, centrifuged at 1000 g for 10 min, discarding the precipitate and collecting the supernatant; then centrifuged at 3000 g for 20 min, discarding the precipitate and collecting the supernatant; finally, centrifuged at 10000 g for 60 min, discarding the precipitate and collecting the supernatant, to further remove cell debris and large particulate impurities. The resulting supernatant was filtered through a 0.45 μm microporous membrane and transferred to an ultracentrifuge tube, then ultracentrifuged at 150000 g for 90 min at 4°C. Discard the supernatant and collect the precipitate, which is the crude extract of plant-derived extracellular vesicles (PDEVs) from purslane. Then, slowly resuspend the precipitate in an appropriate amount of pre-cooled PBS to obtain a purslane plant exosome suspension. The obtained sample can be stored at 4℃ for a short period, or placed in a container... Store at 80℃ for extended periods, avoiding repeated freeze-thaw cycles.

[0039] Example 2: Morphological detection of extracellular vesicles derived from purslane (1) Transmission electron microscopy to detect the morphology of extracellular vesicles: 20 μL of extracellular vesicles from Portulaca oleracea prepared in Example 1 were placed on a 300-mesh copper grid, dried naturally, and then 20 μL of phosphotungstic acid was added for negative staining. After drying, the vesicles were then examined by the instrument.

[0040] (2) Particle size and potential of extracellular vesicles were detected by particle size analyzer: The particle size distribution of extracellular vesicles from Portulaca oleracea was detected by dynamic light scattering method using Malvern particle size analyzer, and their Zeta potential was determined by electrophoretic light scattering method.

[0041] The particle size and zeta potential of extracellular vesicles derived from purslane were detected using a particle size analyzer. The results are as follows: Figure 1 As shown, the average particle size of PDEVs is approximately 118.2 nm, and the Zeta potential is -13.9 mV, indicating that they possess typical nanoscale particle characteristics. Figure 1 ab). Further observation of its morphology and structure using transmission electron microscopy revealed that the vesicle exhibited a cup-shaped or spherical structure, and a membrane bilayer structure was also observed. Figure 1 (cd), which conforms to the typical morphological characteristics of extracellular vesicles from plant sources.

[0042] Example 3: Evaluation of the in vitro antibacterial activity of extracellular vesicles derived from purslane 1. Source of strain and culture conditions Under aseptic conditions, the lyophilized Propionibacterium acnes standard strain (ATCC 6919) was reconstituted with sterile phosphate-buffered saline (PBS) in a laminar flow hood. After gentle mixing, an appropriate amount of bacterial suspension was taken with a sterile inoculation loop and streaked onto the surface of Brain Heart Infusion agar (BHI agar). The inoculated plates were placed in an anaerobic incubator at 37°C for 48 h to obtain single colonies. After incubation, morphologically typical single colonies were picked and inoculated into BHI liquid medium, and cultured under anaerobic conditions at 37°C until the logarithmic growth phase. Before the experiment, the bacterial suspension was appropriately diluted with sterile PBS, and the concentration of the bacterial suspension was adjusted to approximately 1 × 10⁻⁶ using a McFarland turbidity meter. 8 CFU / mL was used as a standard inoculum for subsequent antibacterial experiments.

[0043] Under aseptic conditions, in a laminar flow hood, the lyophilized Staphylococcus epidermidis standard strain (BNCC 330867) was reconstituted with sterile PBS. After gentle pipetting and mixing, an appropriate amount of bacterial suspension was taken with a sterile inoculation loop and streaked onto the surface of LB solid medium (Luria-Bertani agar). The inoculated plates were placed in a 37°C incubator for 24 h to obtain single colonies. After incubation, morphologically typical single colonies were picked and inoculated into LB liquid medium (Luria-Bertani broth), and cultured with shaking at 37°C until the logarithmic growth phase. Before the experiment, the bacterial suspension was appropriately diluted with sterile PBS, and the concentration of the bacterial suspension was adjusted to approximately 1 × 10⁻⁶ using a McFarland turbidimeter. 8 CFU / mL was used as a standard inoculum for subsequent antibacterial experiments.

[0044] 2. Assay for the growth inhibitory activity of Propionibacterium acnes A suspension of Propionibacterium acnes in the logarithmic growth phase was diluted to 1×10⁻⁶ with BHI liquid medium. 6 CFU / mL. In a sterile 96-well plate, add 100 μL of bacterial suspension to each well, followed by 100 μL of working solution of extracellular vesicles derived from purslane at different concentrations, and mix gently. By mixing in equal volumes, the final concentrations of extracellular vesicles in the reaction system were achieved to be 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL.

[0045] A blank control group (BHI medium + PDEVs) and a negative control group (bacterial culture + BHI medium) were set up. The 96-well plates were incubated anaerobically at 37°C for 72 h. After incubation, the absorbance (OD) of each well was measured at 600 nm using a microplate reader. 600 To evaluate the inhibitory activity of purslane-derived extracellular vesicles on the growth of Propionibacterium acnes.

[0046] The effect of PDEVs on the growth of skin-associated microbiota was evaluated using a two-fold serial dilution method. The results are as follows: Figure 2 As shown, PDEVs can significantly inhibit the growth of Propionibacterium acnes at a concentration of 128 μg / mL.

[0047] 3. Assay for the bactericidal activity of Propionibacterium acnes Suspensions of Propionibacterium acnes at various concentrations after treatment with the minimum inhibitory concentration (MIC) were selected, and 100 μL of bacterial suspension from each of the MIC, 2MIC, 4MIC, and 8MIC treatment groups were inoculated onto Columbia blood agar plates. The bacterial suspension was evenly spread on the plate surface using a sterile spreader to ensure full distribution. The inoculated plates were then incubated at 37°C under anaerobic conditions for 72 h.

[0048] After incubation, observe the colony growth. The lowest drug concentration at which no visible colonies grow on the plate is determined as the minimum bactericidal concentration (MBC).

[0049] MBC results are as follows Figure 3 As shown, under 8×MIC conditions, PDEVs have a significant bactericidal effect on Propionibacterium acnes. After the treated bacterial solution was spread on Columbia blood agar plates and cultured anaerobically, no Propionibacterium acnes colony formation was observed.

[0050] 4. Assay for Staphylococcus epidermidis growth inhibitory activity A suspension of Staphylococcus epidermidis in the logarithmic growth phase was diluted to 1×10⁻⁶ with LB liquid medium. 6 CFU / mL. In a sterile 96-well plate, add 100 μL of bacterial suspension to each well, followed by 100 μL of working solution of extracellular vesicles derived from purslane at different concentrations, and mix gently. By mixing in equal volumes, the final concentrations of extracellular vesicles in the reaction system were achieved to be 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL.

[0051] A blank control group (LB medium + PDEVs) and a negative control group (bacterial culture + LB medium) were set up. The 96-well plates were incubated at 37°C under aerobic conditions for 24 h. After incubation, the absorbance (OD) of each well was measured at 600 nm using a microplate reader. 600 To evaluate the inhibitory activity of purslane-derived extracellular vesicles on the growth of Staphylococcus epidermidis.

[0052] Results of inhibition of Staphylococcus epidermidis growth as follows Figure 2 As shown, PDEVs did not show a significant inhibitory effect on the growth of Staphylococcus epidermidis at a concentration of 128 μg / mL.

[0053] In summary, the results show that PDEVs have different effects on different skin-associated microbiota, inhibiting the growth of Propionibacterium acnes while having a smaller effect on the growth of Staphylococcus epidermidis.

[0054] Example 4 Cytotoxicity evaluation of extracellular vesicles derived from purslane (1) HacaT in the logarithmic growth phase is fed according to 1×10 4 HaCaT cells were seeded at a density of 1000 cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator until the cells adhered. Then, different concentrations of purslane-derived extracellular vesicles were added for treatment. A blank control group (containing only culture medium, no cells) and a negative control group (cells + culture medium, without PLEVs) were also included. Each treatment group was divided into 6 replicates and cultured for another 24 h.

[0055] (2) After the treatment was completed, 100 μL of basic culture medium working solution containing 10% CCK-8 (Cell Counting Kit-8) reagent was added to each well and incubated at 37°C in the dark for 1 h. Then, the absorbance value of each well was measured at 450 nm using an ELISA reader.

[0056] Cell proliferation rate % = (OD) 450 Experimental group - OD 450 Blank control group) / (OD 450 Negative control group - OD 450 (blank control group) × 100%.

[0057] The cytotoxicity of extracellular vesicles derived from purslane to HaCaT cells was evaluated using the CCK-8 assay. The results are as follows: Figure 4 As shown, the relative survival rate of HaCaT cells was above 80% within the concentration range of ≤100 μg / mL. This indicates that PDEVs did not exhibit significant cytotoxicity to HaCaT cells within this concentration range, demonstrating good cell compatibility and safety.

[0058] Example 5: Evaluation of the in vitro anti-inflammatory activity of extracellular vesicles derived from purslane 1. HacaT cell culture Human immortalized keratinocytes (HaCaT) were obtained from the China Center for Type Culture Collection. Cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0059] Cells were cultured in a 37°C, 5% CO2 incubator, with the culture medium changed every 1-2 days. When cell confluence reached 80%–90%, the culture medium was discarded, and the cells were washed with sterile PBS buffer, then digested with 0.25% EDTA for 7–10 min. Complete culture medium was added to terminate the digestion, and the cells were pipetted to prepare a single-cell suspension. Cells were seeded at an appropriate density into 6-well or 96-well plates according to experimental requirements and cultured at 37°C, 5% CO2 until cell adhesion and approximately 70%–80% confluence were achieved. These cells were then used for subsequent inflammation model construction and drug intervention experiments.

[0060] 2. Preparation of inactivated Propionibacterium acnes and establishment of an inflammation model A suspension of *Propionibacterium acnes* in the logarithmic growth phase was centrifuged at 6000 rpm for 10 min at 4°C. The supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were resuspended in sterile PBS and washed twice. The concentration of the bacterial suspension was then adjusted to approximately 1 × 10⁻⁶. 9 CFU / mL. The obtained bacterial suspension was inactivated by heating in an 80℃ water bath for 30 min. The inactivated suspension was then set aside. After inactivation, an appropriate amount of the bacterial suspension was inoculated onto Columbia blood agar plates and anaerobically cultured at 37℃ for 72 h to confirm the absence of colony growth, thus verifying complete inactivation. 3 × 10⁻⁶ cells were then inoculated into 6-well plates. 5 HaCaT cells were cultured at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 60%, different concentrations of purslane-derived extracellular vesicles were added for pretreatment for 12 hours. After pretreatment, the drug-containing culture medium was discarded. The inactivated Propionibacterium acnes suspension was diluted to 1×10⁻⁶ using complete culture medium. 8 CFU / mL was added to each well and incubated with cells for 12 h to construct an inflammatory stimulation model.

[0061] 3. Total RNA extraction Discard the culture medium and gently wash the cells 1–2 times with pre-cooled PBS buffer. Add 1 mL of TRIzol lysis buffer to each well, repeatedly pipetting to fully lyse the cells, and transfer the lysis buffer to a 1.5 mL RNase-free centrifuge tube. Add 200 μL of chloroform to the lysis buffer, tighten the cap, and vortex vigorously to mix. Incubate at room temperature for 5–10 min. Then centrifuge at 12000 g for 15 min at 4°C to separate the solutions. Carefully aspirate the upper aqueous phase to a new RNase-free centrifuge tube. Add 500 μL of isopropanol to the aqueous phase, mix gently, incubate at room temperature for 5–10 min, and centrifuge at 12000 g for 10 min at 4°C. Discard the supernatant and retain the RNA precipitate. Add 1 mL of 75% ethanol to the precipitate and wash, centrifuging at 7500 g for 5 min at 4°C. Repeat the washing 1–2 times. After discarding the ethanol, briefly dry the precipitate at room temperature to evaporate any residual ethanol. Finally, an appropriate amount of RNase-free water (DEPC-treated water) was added to dissolve the RNA. The concentration and purity of total RNA were determined using a NanoDrop micro-spectrophotometer, and RNA quality was evaluated using the A260 / A280 ratio.

[0062] 4. Reverse transcription and real-time quantitative PCR detection RNA was reverse transcribed into cDNA using a full-gold reverse transcription kit. The specific steps are as follows: (1) Prepare the reaction solution according to a total reaction volume of 20 μL, with a total RNA content not exceeding 1000 ng. The entire preparation process should be carried out on ice. The specific preparation of the reaction solution is as follows: Table 1. Preparation of reverse transcription cDNA reaction solution

[0063] (2) After gently mixing, place the mixture in a PCR instrument for reverse transcription. The reaction program is as follows: react at 42℃ for 15 min, then heat at 85℃ for 5 s to terminate the reaction. The obtained cDNA can be used for subsequent real-time quantitative PCR (qPCR) detection. Prepare a 10 μL qPCR reaction system. The reaction solution is shown in Table 2 below, and the amplification primers are shown in Table 3. After preparation, perform amplification according to the reaction system in Table 4.

[0064] Table 2 qPCR reaction system

[0065] Table 3 Primer upstream and downstream sequences

[0066] Table 4 Amplification Procedure

[0067] (4) Using GAPDH as an internal reference gene, 2 -△△Ct The relative expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were calculated using a method.

[0068] The mRNA expression levels of inflammatory factors IL-6, IL-8, IL-1β, and TNF-α in HaCaT cells stimulated with inactivated Propionibacterium acnes were detected by real-time quantitative PCR. The results are as follows: Figure 5 As shown, compared with the model group, PDEVs significantly reduced the expression levels of the above-mentioned inflammatory factors at concentrations of 25, 50 and 100 μg / mL, suggesting that PDEVs can effectively inhibit the inflammatory response of HaCaT cells induced by Propionibacterium acnes.

[0069] Example 6 Evaluation of the antioxidant activity of extracellular vesicles derived from purslane (1) HaCaT cells were prepared at a ratio of 3 × 10⁻⁶ 5 Cells were seeded at a density of cells / well in 6-well plates and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached about 60%, different concentrations of Portulaca oleracea-derived extracellular vesicles (PDEVs) were added for pretreatment for 12 h.

[0070] (2) Discard the drug-containing culture medium after pretreatment. Dilute the inactivated Propionibacterium acnes suspension with complete culture medium to 1×10⁻⁶. 8 CFU / mL was added to each well and incubated with cells for 12 h to induce oxidative stress.

[0071] (3) After the treatment, discard the culture medium and gently wash the cells twice with sterile PBS pre-warmed at 37°C to remove residual treatment solution and culture medium. Then add 2 mL of the prepared 10 μM DCFH-DA serum-free working solution to each well and incubate in the dark for 20-30 min, shaking the culture plate every 5 min to ensure that the probe is in full contact with the cells.

[0072] (4) After incubation, immediately aspirate the working solution from each well and gently wash the cells twice with pre-warmed 37°C sterile PBS. Then add 0.25% EDTA-free trypsin for digestion. After the cells become round and detach from the cell wall, add serum-containing culture medium to stop digestion and gently pipette until a single-cell suspension is formed.

[0073] (5) The cell suspension was then centrifuged at 700 g for 5 min, the supernatant was discarded, and the cells were resuspended in pre-warmed PBS and washed twice. Finally, 500 μL of PBS was added to resuspend the cells, and the intracellular reactive oxygen species level was detected by flow cytometry. The degree of oxidative stress was evaluated by fluorescence intensity.

[0074] Flow cytometry was used to detect the effect of extracellular vesicles derived from purslane on the oxidative stress response induced by inactivated Propionibacterium acnes in HaCaT cells. The results are as follows: Figure 6 As shown, compared with the model group, PDEVs significantly reduced intracellular reactive oxygen species (ROS) levels at concentrations of 25 μg / mL and 50 μg / mL. These results indicate that PDEVs can effectively alleviate oxidative stress induced by inactivated Propionibacterium acnes in HaCaT cells.

[0075] In summary, the extracellular vesicles derived from purslane provided by this invention are obtained through plant tissue homogenization, differential centrifugation, and ultracentrifugation, resulting in plant-derived extracellular vesicles with nanoscale particle size and membrane structure characteristics. Purslane-derived extracellular vesicles exhibit significant activity in inhibiting the growth of *Propionibacterium acnes*, while having minimal impact on the growth of *Staphylococcus epidermidis* within the same concentration range, thus achieving differential regulation of skin-related microbiota. Simultaneously, purslane-derived extracellular vesicles can inhibit the expression of inflammatory factors induced by *Propionibacterium acnes*, including IL-6, IL-8, IL-1β, and TNF-α, thereby exerting an anti-inflammatory effect; they can also reduce intracellular reactive oxygen species levels, alleviating oxidative stress induced by *Propionibacterium acnes*, and can be used as an anti-acne active ingredient in pharmaceuticals, skincare products, or skin care compositions, with an effective concentration range of 25-100 μg / mL.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of purslane extracellular vesicles in the preparation of products that regulate the skin microecology of acne-prone skin.

2. Application of purslane extracellular vesicles in the preparation of products that inhibit Propionibacterium acnes.

3. Application of purslane extracellular vesicles in the preparation of anti-acne drugs.

4. Application of purslane extracellular vesicles in the preparation of products that alleviate and reduce inflammatory responses and oxidative stress levels caused by acne.

5. The application according to claim 4, characterized in that, The product can reduce the mRNA expression levels of inflammatory factors IL-6, IL-8, IL-1β, and TNF-α, as well as the level of intracellular reactive oxygen species.

6. The application according to any one of claims 1 to 4, characterized in that, The purslane extracellular vesicles are plant-derived extracellular vesicles with nanoscale particle size and membrane structure characteristics, obtained by extraction and purification from fresh purslane.

7. The application according to claim 6, characterized in that, The extraction is performed using differential centrifugation, density gradient centrifugation, ultrafiltration concentration, size exclusion chromatography, or a combination of ultracentrifugation and ultrafiltration.

8. The application according to claim 7, characterized in that, The differential centrifugation conditions are as follows: first centrifuge at 800~1200 g for 8~15 min, discard the precipitate and collect the supernatant; then centrifuge at 2000~4000 g for 10~30 min, discard the precipitate and collect the supernatant; continue centrifuging at 10000~20000 g for 50~60 min, discard the precipitate and collect the supernatant to remove cell debris and large particulate impurities.

9. The application according to claims 1-3, characterized in that, The concentration of purslane extracellular vesicles in the product or drug is 25-100 μg / mL.

10. The application according to claim 2, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

Images