Pogostemon cablin extracellular vesicle as well as extraction and separation method and application thereof

Abstract

The invention provides pogostemon cablin extracellular vesicles and an extraction and separation method and application thereof. Pogostemon cablin tissue juice is subjected to differential centrifugation, PEG precipitation and ultrafiltration, and the pogostemon cablin extracellular vesicles are extracted. The method is simple and convenient, equipment and materials are easy to obtain, the obtained extracellular vesicle crude extract is purified by combining an ultrafiltration method, and the obtained purified solution is clear and transparent and high in concentration and purity. The compound has no toxicity to HaCaT human immortalized epidermal cells and zebrafish, has good biocompatibility, shows good anti-photoaging, anti-oxidation and anti-inflammatory effects in in-vitro and in-vivo experiments, and is expected to be prepared into an anti-photoaging natural plant drug.

Description

Technical Field

[0001] This application belongs to the field of plant extracellular vesicles and biological agents, and particularly relates to a patchouli extracellular vesicle, its extraction and separation method, and its application. Background Technology

[0002] Prolonged sun exposure is a significant environmental factor accelerating skin aging, characterized by elevated collagenase levels, particularly MMP1 and 2, fragmentation of collagen and elastin fibers, and inflammatory responses. Simultaneously, repeated UVB exposure leads to the degradation of reactive oxygen species, transcription factors, and elastin, ultimately resulting in skin wrinkling. Although phytochemicals are currently available for anti-photoaging purposes, their side effects, such as cytotoxicity, low bioavailability, and the skin allergies or inflammatory reactions they may cause, limit their application. Therefore, there is an urgent need to develop anti-photoaging drugs with low or no toxic side effects and good biocompatibility.

[0003] Extracellular vesicles (EVs) are nanovesicles secreted by plant cells. They consist of a lipid bilayer and contain bioactive components such as nucleic acids, proteins, lipids, and small active molecules, playing a role in intercellular communication and substance transport. In recent years, with the deepening of research on plant EVs, their cross-species regulatory functions have been demonstrated. Because plant EVs contain bioactive small molecules, they can serve as novel drugs with various pharmacological activities such as antioxidant, antibacterial, antitumor, and anti-osteoporosis effects. They can also act as drug delivery carriers, binding to bioactive small molecules to exhibit dual activities, while maintaining stability, safety, and biocompatibility. This brings innovative therapeutic strategies and broad application prospects to the biomedical field.

[0004] Patchouli ( Pogostemon cablin *Blanco* Benth. is a plant belonging to the genus *Blanco* in the Lamiaceae family. The medicinal part is the dried aerial portion. It possesses aromatic and dampness-resolving, appetite-stimulating and nausea-relieving, and exterior-releasing and summer-heat-relieving effects. Clinically, it is mainly used to treat summer-dampness syndrome or the initial stage of damp-heat syndrome. Patchouli is used in various therapeutic forms, mostly in the form of volatile oil and water extracts. Its main active components are patchouli alcohol, patchouli ketone, and patchoulene. Modern pharmacological studies show that patchouli has anti-inflammatory, analgesic, antioxidant, antitumor, antibacterial, and antiviral pharmacological effects. Regarding antioxidant activity, under oxidative stress, excessive ROS can damage cellular proteins, lipids, and DNA, leading to fatal cellular damage and consequently affecting various pathologies such as aging, neurodegenerative diseases, cancer, and cardiovascular diseases. Studies show that patchouli ketone has antioxidant activity and can resist UV-induced photoaging.

[0005] There are currently no reports on patchouli extracellular vesicles (PL-EVs), therefore, research on how to effectively and easily extract patchouli extracellular vesicles and their applications is of great value. Summary of the Invention

[0006] This application provides a method for extracting and separating patchouli extracellular vesicles, along with their applications, to fill the gap in the extraction and separation of patchouli extracellular vesicles. This application overcomes the shortcomings and deficiencies of existing technologies, providing a method for preparing plant extracellular vesicles that are naturally derived, safe, low-cost, and have high extraction purity. Furthermore, this patchouli extracellular vesicle, as a natural plant extract, can be developed into a drug and used in the preparation of anti-photoaging drugs, exhibiting safety and good biocompatibility. The technical solution is as follows: In a first aspect, embodiments of this application provide a method for extracting and separating extracellular vesicles from patchouli, including: Extracellular vesicles of patchouli were extracted from the tissue juice by differential centrifugation, PEG precipitation, and ultrafiltration.

[0007] In one embodiment, the patchouli tissue fluid is at least one sap from fresh patchouli leaves, stems, or roots.

[0008] In one embodiment, the patchouli tissue fluid is the juice from fresh patchouli leaves.

[0009] In one embodiment, the method for preparing patchouli tissue fluid is as follows: Fresh patchouli tissue was washed with pure water and dried; then mixed with a phosphate buffer solution, juiced, and the juice was collected and filtered to obtain the patchouli tissue liquid.

[0010] In one embodiment, the weight-to-volume ratio of fresh patchouli tissue to phosphate-buffered saline solution is 1 g: (1-5) mL.

[0011] In one embodiment, the juice is extracted 2-6 times, with each extraction lasting 10-120 seconds and an interval of 30-180 seconds between extractions.

[0012] In one implementation, the differential centrifugation process is as follows: The patchouli tissue fluid was centrifuged at 500~1000×g for 10~20min, and the first supernatant was collected. The first supernatant was centrifuged at 3000~6000g×g for 25~40min, and the second supernatant was collected. The second supernatant was centrifuged at 10,000–13,000 × g for 50–70 min; the third supernatant was then collected.

[0013] In one implementation, the centrifugation temperature is 3~8°C.

[0014] In one embodiment, PEG is added to the third supernatant and precipitated overnight at 3-8°C.

[0015] In one embodiment, the molecular weight of the PEG is 3000-6000.

[0016] In one embodiment, PEG is added to a mass concentration of 5%-12%.

[0017] In one embodiment, after PEG precipitation, the liquid phase is collected and centrifuged at 10000~13000×g for 30~60min; the precipitate is collected; and the crude extract is obtained by resuspending in PBS.

[0018] In one embodiment, the crude extract is ultrafiltered using an ultrafiltration tube to obtain a dispersion of the patchouli extracellular vesicles.

[0019] In one embodiment, a 3-100 kDa ultrafiltration tube is used to centrifuge at 1000-3000×g for 10-30 min, and the fourth supernatant is collected to obtain a dispersion of the extracellular vesicles of the patchouli.

[0020] In one embodiment, a dispersion of patchouli extracellular vesicles is filtered through a sterile filter to obtain patchouli extracellular vesicles.

[0021] In one embodiment, the extracellular vesicles of patchouli are stored at -80 to -60°C. In one embodiment, before adding PEG to the third supernatant, the third supernatant is filtered through a 0.22~1.0um microporous membrane, and PEG is added to the obtained filtrate for precipitation.

[0022] Secondly, this application provides a patchouli extracellular vesicle, which is prepared by the above-described method for extracting and separating patchouli extracellular vesicles.

[0023] Thirdly, embodiments of this application provide an application of the aforementioned patchouli extracellular vesicles in the preparation of anti-skin photoaging products.

[0024] In one embodiment, the anti-photoaging product is a pharmaceutical product, health product, or beauty product.

[0025] In one embodiment, the dosage form of the medicine is any one of oral liquid, capsule, ointment, gel patch, plaster, and traditional Chinese medicine.

[0026] The advantages or beneficial effects of the above technical solutions include at least the following: The method for extracting and separating extracellular vesicles of patchouli in this application uses patchouli as raw material. Extracellular vesicles of patchouli are obtained from its juice through differential centrifugation, PEG precipitation and ultrafiltration. The method is simple, the equipment and materials are readily available, and the obtained extracellular vesicle dispersion is clear and transparent with high concentration and purity. The extracellular vesicles have intact morphology, good membrane stability, uniform particle size and are rich in lipids and proteins.

[0027] Furthermore, the outer vesicles are non-toxic to HaCaT human immortalized epidermal cells and zebrafish, exhibiting good biocompatibility. They demonstrate good anti-photoaging, antioxidant, and anti-inflammatory effects in both in vitro and in vivo experiments, and are expected to be prepared as a natural plant drug for anti-photoaging.

[0028] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0029] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0030] Figure 1 This is a transmission electron microscope image of extracellular vesicles from patchouli cells; Figure 2 This is a particle size diagram of extracellular vesicles in patchouli. Figure 3 This is an extracellular vesicle potential diagram of patchouli cells; Figure 4 This is a thin-layer chromatogram of lipids in extracellular vesicles of patchouli. Figure 5 This is an SDS-PAGE image of proteins from extracellular vesicles of patchouli. Figure 6 A diagram showing the differences between extracellular vesicles and components of patchouli; Figure 7 A stacked bar chart showing the classification of extracellular vesicles and metabolic components of patchouli. Figure 8 Venn diagram of extracellular vesicles and metabolites of patchouli; Figure 9 Volcano diagram showing the differences in components between extracellular vesicles and patchouli. Figure 10 This is a graph showing the cytotoxicity of extracellular vesicles of patchouli against HaCaT cells; Figure 11This is a diagram illustrating the role of patchouli extracellular vesicles in preventing UVB-induced damage to HaCaT cells. Figure 12 This is a diagram illustrating the effect of patchouli extracellular vesicles on reducing oxidative damage of H2O2 to HaCaT cells. Figure 13 The values ​​are: (A) Typical ROS fluorescence intensity of zebrafish after treatment with patchouli extracellular vesicles for 24 h; (B) ROS fluorescence intensity of zebrafish after treatment with patchouli extracellular vesicles; Statistical differences were analyzed using independent t-tests: *** represents p<0.001; Figure 14 The images show the caudal fin area of ​​zebrafish after treatment with patchouli extracellular vesicles for 24 hours; (A) a typical image of zebrafish caudal fin after treatment with patchouli extracellular vesicles (the red dashed box represents the caudal fin area); (B) the caudal fin area of ​​zebrafish after treatment with patchouli extracellular vesicles; statistical differences were analyzed using independent t-tests: * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. Figure 15 Zebrafish treated with patchouli extracellular vesicles for 24 hours cola1a1a Relative gene expression levels; statistical differences were analyzed using independent t-tests: * represents p < 0.05, *** represents p < 0.001; Figure 16 The total distance traveled by zebrafish after treatment with patchouli extracellular vesicles for 24 hours is shown in Figure (A). (A) Typical movement trajectory of zebrafish after treatment with patchouli extracellular vesicles (black line: slow movement, green line: medium movement, red line: fast movement); (B) Total distance traveled by zebrafish after treatment with patchouli extracellular vesicles (mm). Statistical differences were analyzed using independent t-tests: ** represents... p <0.01, *** represents p <0.001; Figure 17 The results show the number of neutrophils in zebrafish skin after 18 hours of treatment with patchouli extracellular vesicles; (A) Typical image of neutrophils in zebrafish skin after treatment with patchouli extracellular vesicles (the red dashed box indicates the zebrafish skin counting area); (B) Number of neutrophils in zebrafish skin after treatment with patchouli extracellular vesicles; Statistical differences were analyzed using independent t-tests: *** represents p <0.001. Detailed Implementation

[0031] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0032] To fill the gap in the isolation and extraction of extracellular vesicles from patchouli, this application optimizes the extraction and purification method of extracellular vesicles from patchouli, a valuable traditional Chinese medicine resource, and establishes a simple preparation method for isolating and purifying extracellular vesicles from patchouli. This provides a methodological reference for subsequent researchers to isolate plant-derived extracellular vesicles and offers relevant reference and guidance for their future in-depth research and application in medical aesthetics and disease treatment.

[0033] Example 1: Preparation, characterization and identification of extracellular vesicles from patchouli. 1.1 Preparation of extracellular vesicles from patchouli This embodiment presents a method for preparing extracellular vesicles of patchouli that is simple to operate, time-saving, low in equipment cost, and high in extraction purity. The method comprises the following steps: Step (1) Preparation of patchouli leaf homogenate: Wash the patchouli leaves three times with pure water. Take 60g of the washed patchouli leaves and put them into a juicer and add 1×PBS buffer solution (patchouli leaves:PBS mass-volume ratio = 1:2). Juice 4 times, 30s each time, with a 60s interval between each juice extraction to obtain patchouli leaf homogenate. The homogenate of patchouli leaves was filtered through gauze. The filtrate was centrifuged at 4°C, 1000×g for 10 min, and the supernatant was collected. The supernatant was then centrifuged at 4°C, 3000×g for 30 min, and the supernatant was collected. Finally, the supernatant was centrifuged at 4°C, 10000×g for 60 min. After centrifugation, plant fibers, dead cells and large particulate impurities were removed, and the supernatant was collected. The collected supernatant was filtered through a 0.45 μm microporous membrane, and the resulting clear solution was used to further separate the extracellular vesicles of patchouli.

[0034] Step (2) Crude extraction of extracellular vesicles from patchouli: Add an appropriate amount of PEG6000 solution to the collected supernatant to make the final concentration of PEG6000 5.75%, mix well, and let stand at 4°C for 12 hours. After standing, the solution was centrifuged at 10000×g for 60 minutes at 4°C. The supernatant was discarded, and the centrifuge tube was inverted on paper for 5 minutes to remove excess supernatant. The precipitate was resuspended in a small amount of 1×PBS solution. The resuspended crude extract was turbid and contained a lot of impurities, requiring further purification.

[0035] Step (3) Purification of patchouli extracellular vesicles: The resuspended crude extract was sonicated at 100W for 10 seconds, and the solution was collected into a 100kD ultrafiltration tube. The ultrafiltration tube was centrifuged at 4°C and 3500 rpm for 30 minutes, and the supernatant was collected. The supernatant was sonicated at 100W for 10 seconds and then filtered through a 0.45µm filter. The resulting patchouli leaf extracellular vesicles were stored at -80°C.

[0036] 1.2 Characterization and Identification of Extracellular Vesicles in Patchouli 1. The extracellular vesicles of patchouli extracted in Example 1 were observed by transmission electron microscopy (TEM). The TEM image is shown below. Figure 1 As shown; the particle size distribution was analyzed using a single-particle nanobioanalyzer, and the particle size distribution diagram is shown below. Figure 2 As shown.

[0037] from Figure 1 The TEM images show that the extracted patchouli extracellular vesicles are mostly spherical or bowl-shaped nanoparticles with a distinct double membrane structure. Figure 2 The particle size distribution results were similar to those obtained by transmission electron microscopy, showing uniform particle size concentrated in the range of 50–150 nm, with an average diameter of 86.3 nm. The vesicle yield was high, with a concentration reaching 8.1 × 10⁻⁶. 10 Particles / ml.

[0038] 2. Dynamic light scattering measurements were performed on the outer vesicles, and the vesicle potential was measured as follows: Figure 3 As shown.

[0039] Figure 3 The study showed that the vesicles exhibited negative potential, with an average potential of -11.3 mV, indicating good membrane stability.

[0040] 3. Lipid extraction and thin-layer chromatography experiments (1) Extraction of lipids: Take 300uL of the extracellular vesicles of patchouli extracted in Example 1, vortex mix them in the ratio of chloroform:methanol:EVs (8:4:3) for 5min, centrifuge at 10000×g for 10min, take out the centrifuged EP tube, at this time the sample in the centrifuge tube is divided into three layers, the upper layer is the water and methanol phase, the middle layer is the protein precipitation phase, and the lower layer is the organic phase. Take the lower organic phase and place it in another EP tube, dry it at 100℃ to obtain non-polar lipids, and add 100uL of chloroform to resuspend.

[0041] (2) Take the dried and activated silica gel G plate, and lightly draw a line 1 cm away from the bottom of the plate with a pencil as the spotting line. Use a capillary tube to spot the lipid sample on the spotting line. Spot the sample multiple times, and the spotting diameter should not be greater than 3 mm. Add about 1 cm of developing solvent (dichloromethane:methanol:acetic acid = 190:9:1) to the developing tank. Place the silica gel G plate into the developing tank to separate the lipids. When the developing solvent is about 1 cm away from the front edge of the silica gel G plate, take out the thin-layer plate and dry it with hot air.

[0042] (3) Remove the silica gel G plate, spray it with a color developer (spraying 10% CuSO4 and 8% phosphoric acid solution successively), and carbonize the silica gel G plate at 105℃ to develop color. The results are as follows: Figure 4 As shown.

[0043] from Figure 4 As can be seen from the present invention, the extracellular vesicles of patchouli extracted and purified by the present invention are rich in lipids.

[0044] 4. Protein extraction and SDS-PAGE identification The protein components of PL-EVs were analyzed using SDS-PAGE with Coomassie Brilliant Blue staining. First, the protein concentration of the PL-EVs sample was determined using a BCA protein quantification kit. 20 μL of PL-EVs sample was taken and lysed with Ripa lysis buffer containing 1% protease inhibitor at a 1:1 volume ratio. The mixture was incubated on ice for 30 min, vortexing for 5 s every 10 min. Subsequently, it was centrifuged at 10000×g for 10 min at 4°C. The supernatant was collected, and 5×SDS-PAGE protein loading buffer was added at a 4:1 volume ratio. After vortexing, the sample was heated at 95°C for 10 min to denature the protein and then rapidly cooled on ice. The cooled sample was then used for SDS-PAGE electrophoresis to separate the protein components. Finally, Coomassie Brilliant Blue staining was used for color development and detection. The results are as follows: Figure 5 As shown.

[0045] Figure 5 The study showed that the isolated patchouli extracellular vesicles contained protein components, mainly distributed in the range of 10-35 kDa.

[0046] 5. Analysis of the components of extracellular vesicles in patchouli UPLC-MS / MS was used to perform extensive targeted analysis of the component metabolomics of patchouli and its extracellular vesicles; such as... Figure 6 As shown, A is a PCA diagram of the components of patchouli and patchouli extracellular vesicles; B is a stacked bar chart of the classification of the components of patchouli and patchouli extracellular vesicles; C is a Venn diagram of the components of patchouli and patchouli extracellular vesicles; and D is a volcano diagram of the differential components of patchouli and patchouli extracellular vesicles.

[0047] PCA analysis showed that the first principal component could explain 84% of the features in the original dataset, and the patchouli leaf group was clearly separated from the PL-EVs group, indicating that their component spectra were quite different. Figure 6PL-EVs are rich in metabolites, exhibiting similar metabolite types to those found in patchouli leaf tissue. The main metabolites include 21% terpenes, followed by 18.3% flavonoids and 11% lipids. In addition, amino acids, phenolic acids, nucleotides, quinones, and organic acids were also detected. Among these, 1973 metabolites were identical to those found in patchouli leaves. The abundance of lipids, nucleotides, amino acids, and their derivatives was higher in PL-EVs than in patchouli leaf tissue. Figure 7 and Figure 8 Differential metabolic analysis of patchouli leaves and PL-EVs, using Fold Change (FC) ≥2 or ≤0.5 and VIP ≥1 as criteria, showed that 376 metabolites were upregulated and 741 metabolites were downregulated. Figure 9 ).

[0048] Example 2: Anti-photoaging and antioxidant effects of patchouli extracellular vesicles on HaCaT 2.1 MTT assay to determine the cytotoxicity of patchouli extracellular vesicles to HaCaT cells.

[0049] HaCaT immortalized human epidermal cells in good growth condition were evenly seeded into 96-well plates at a density of 10,000 cells / well and cultured for 18 hours until the cells adhered. Then, 0 and 1.0 × 10⁻⁶ cells were added to each well. 8 2.5×10 8 5.0×10 8 1.0×10 9 100 μL of DMEM medium (particles / mL) was added to each well. After culturing the cells for 24 h, the medium was discarded, and 10 μL of LMTT (5 mg / mL) was added to each well. The cells were incubated for 4 h, the medium was discarded, and 100 μL of LDMSO solution was added to each well. The cells were shaken for 10 min on a shaker, and the absorbance was measured at 490 nm using a microplate reader. The results are as follows. Figure 10 As shown.

[0050] Figure 10 Data from the study showed that patchouli extracellular vesicles at 1×10 9 and 5×10 8 Under particle / mL conditions, it showed no cytotoxicity to HaCaT human immortalized epidermal cells and even promoted cell viability.

[0051] 2.2 The role of patchouli extracellular vesicles in preventing UVB-induced damage to HaCaT cells.

[0052] HaCaT immortalized human epidermal cells in good growth condition were evenly seeded into 96-well plates at a density of 10,000 cells / well and cultured for 18 hours until the cells adhered. Then, 0 and 1.0 × 10⁻⁶ cells were added to each well. 8 2.5×10 8 5.0×108 1.0×10 9 100 μL of DMEM medium (particles / mL) was added to each well. After culturing the cells for 24 h, the medium was discarded, and the cells were washed twice with 1×PBS. 50 μL of 1×PBS was added to each well, and the cells were incubated for 5 s before UVB irradiation at a dose of 210 mJ / cm². 2 After irradiation, 100 u LDM-MEM medium was added to each well, and the mixture was incubated for 24 h. Then, 10 u LMTT solution (5 mg / mL) was added to each well, and the mixture was incubated for another 4 h. After 4 h, the medium was discarded, and 100 u LDMSO solution was added to each well. The mixture was shaken for 10 min on a shaker, and the absorbance was measured at 490 nm using a microplate reader. The results are as follows: Figure 11 As shown.

[0053] Figure 11 The data showed that, compared with the control group, the UVB irradiation model group significantly inhibited the survival rate of HaCaT cells (P<0.001). Compared with the model group, the HaCaT cells in the experimental group (1×10⁶ cells) showed a significantly higher survival rate. 9 2.5×10 8 and 1×10 8 After co-incubation with PL-EVs per mL, the survival rate of HaCaT cells increased significantly, suggesting that PL-EVs have a role in preventing photoaging damage to HaCaT cells.

[0054] 2.3 Patchouli extracellular vesicles reduce the oxidative damage of H2O2 to HaCaT cells.

[0055] Healthy HaCaT immortalized human epidermal cells were evenly seeded into 96-well plates at a density of 10,000 cells / well. After the cells adhered to the wells the following day, 0 and 1.0 × 10⁻⁶ cells were added to each well. 7 1.0×10 8 5.0×10 8 1.0×10 9 100 μL of DMEM medium (particles / mL) was added to each well. After culturing cells for 24 h, the medium was discarded. Cells were washed twice with 1×PBS, and H2O2 solution was added to bring the final DMEM medium concentration to 800 μM. After culturing for 4 h, the medium was discarded. 100 μL of DMEM medium and 10 μL of LMTT (5 mg / mL) were added to each well, and the cells were incubated for 4 h. The medium was then discarded, and 100 μL of LDMSO solution was added to each well. The cells were shaken on a shaker for 10 min, and the absorbance was measured at 570 nm using a microplate reader. The results are as follows: Figure 12 As shown.

[0056] Figure 12The data showed that, compared with the control group, the model group induced oxidative damage to HaCaT cells with 800 uMH2O2, resulting in a significant decrease in cell survival (P<0.001). In contrast to the model group, co-incubation of HaCaT cells with different concentrations of PL-EVs in the experimental group showed that PL-EVs effectively alleviated H2O2-induced oxidative damage to HaCaT cells.

[0057] Example 3: Anti-photoaging effect of patchouli extracellular vesicles on zebrafish.

[0058] 3.1 MTC Detection Experimental groups: normal control group, model control group (UVB 45min), and sample group (samples were set with 4 concentration gradients: 1.74E+9 particles / mL, 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0059] Methods: Zebrafish 2 days post-fertilization (2 dpf) were randomly selected and subjected to the experiment in 6-well plates (3 mL / well), with 30 fish per well. The normal control group underwent routine culture, while the model control group and the sample group received 45 min of UVB irradiation (without sample addition) to establish a photoaging model. After irradiation, different concentrations of patchouli extracellular vesicles were added to the sample group. After 24 h of treatment, the maximum detectable concentration of the sample in the model zebrafish was determined. The results are shown in Table 1.

[0060] Table 1 Results of the experiment to explore the concentration of extracellular vesicles in patchouli (n=30)

[0061] In Table 1, after 24 hours of exposure to zebrafish, the phenotypes of patchouli extracellular vesicles at all four treatment concentrations were similar to those of the model control group. Therefore, the MTC of patchouli extracellular vesicles in zebrafish can be determined to be 1.39E+10 particles / mL.

[0062] 3.2 ROS Detection Experimental groups: normal control group, model control group (UVB 45min), positive control group (resveratrol 20μg / mL), and sample group (samples were set with 3 concentration gradients: 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0063] Treatment method: Zebrafish 2 days post-fertilization (2 dpf) were randomly selected and subjected to the experiment in 6-well plates (3 mL / well), with 30 fish per well. Except for the normal control group, which underwent routine culture, the model control group, positive control group, and sample group were all subjected to 45 min of UVB irradiation (without sample) to establish a photoaging model. After irradiation, the positive control group was treated with 20 μg / mL resveratrol, while the sample group was treated with different concentrations of patchouli extracellular vesicles. 24 h after treatment, 10 zebrafish from each group were randomly selected and imaged under a microscope. The anti-photoaging efficacy of the samples was evaluated by statistically analyzing the ROS fluorescence intensity of the zebrafish. Results are as follows: Figure 13 As shown.

[0064] Figure 13 According to the data, after 24 hours of treatment, compared with the model control group, the extracellular vesicles of patchouli showed a significant reduction in the ROS fluorescence intensity of zebrafish. At concentrations of 3.47E+9, 6.94E+9, and 1.39E+10 particles / mL, their anti-photoaging efficacy was 44%, 66%, and 76%, respectively.

[0065] 3.3 Caudal fin area measurement Experimental groups: normal control group, model control group (UVB 45min), positive control group (resveratrol 20μg / mL), and sample group (samples were set with 3 concentration gradients: 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0066] Treatment method: Zebrafish 2 days post-fertilization (2 dpf) were randomly selected and subjected to the experiment in 6-well plates (3 mL / well), with 30 fish per well. In addition to the normal control group undergoing routine culture, the model control group, positive control group, and sample group were all subjected to 45 min of UVB irradiation (without sample) to establish a photoaging model. After irradiation, the positive control group was given 20 μg / mL of resveratrol, and the sample group was given different concentrations of patchouli extracellular vesicles. 24 h after treatment, 10 zebrafish from each group were randomly selected and placed under a microscope. Results are as follows: Figure 14 As shown.

[0067] Figure 14 According to the data, after 24 hours of treatment, compared with the model control group, the extracellular vesicles of patchouli showed a significant increase in the caudal fin area of ​​zebrafish. At concentrations of 3.47E+9, 6.94E+9, and 1.39E+10 particles / mL, their anti-photoaging efficacy was 23%, 25%, and 37%, respectively.

[0068] 3.4 Elastin gene detection Experimental groups: normal control group, model control group (UVB 45min), positive control group (resveratrol 20μg / mL), and sample group (samples were set with 3 concentration gradients: 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0069] Treatment method: Zebrafish 2 days post-fertilization (2 dpf) were randomly selected and subjected to experiments in 6-well plates (3 mL / well), with 30 fish per well. The normal control group underwent routine culture, while the model control group, positive control group, and sample group were irradiated with UVB for 45 min (without sample) to establish a photoaging model. The positive control group was treated with 20 μg / mL resveratrol, and the sample groups were treated with different concentrations of patchouli extracellular vesicles. After 24 h of treatment, total RNA was extracted, and β-actin was used as an internal reference gene. The RNA was detected using real-time quantitative PCR. cola1a1a The relative gene expression level was used to assess the anti-photoaging efficacy of the samples. Results were as follows: Figure 15 As shown.

[0070] Figure 15 According to the data, after 24 hours of treatment, compared with the model control group, the extracellular vesicles of patchouli had an effect on the elastin gene in zebrafish. cola1a1a The relative expression level of the substance showed a significant increase. According to the formula for calculating the anti-photoaging efficacy, the anti-photoaging efficacy was 10%, 19%, and 73% at concentrations of 3.47E+9, 6.94E+9, and 1.39E+10 particles / mL, respectively.

[0071] 3.5 Total Movement Distance Detection Experimental groups: normal control group, model control group (UVB 45min), positive control group (resveratrol 20μg / mL), and sample group (samples were set with 3 concentration gradients: 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0072] Methods: Zebrafish 2 days post-fertilization (2 dpf) were randomly selected and subjected to experiments in 6-well plates (3 mL / well), with 30 fish per well. The normal control group underwent routine culture, while the model control group, positive control group, and sample group were irradiated with UVB for 45 min (without sample) to establish a photoaging model. The positive control group was treated with 20 μg / mL resveratrol, and the sample groups were treated with different concentrations of patchouli extracellular vesicles. After 24 h of treatment, 10 zebrafish from each experimental group were randomly transferred to 96-well plates (1 fish / well) and then placed under a zebrafish 2D behavior analyzer for 1 h of behavioral measurement. The anti-photoaging efficacy of the samples was evaluated by counting the total movement distance of the zebrafish. Results are as follows: Figure 16 As shown.

[0073] Figure 16According to the data, after 24 hours of treatment, compared with the model control group, the extracellular vesicles of patchouli significantly increased the total movement distance of zebrafish. At concentrations of 3.47E+9, 6.94E+9, and 1.39E+10 particles / mL, their anti-photoaging efficacy was 22%, 48%, and 58%, respectively. Among them, at the lowest concentration (3.47E+9 particles / mL), there was no significant difference compared with the model control group (p>0.05).

[0074] Example 4: Anti-inflammatory effect of patchouli extracellular vesicles on zebrafish.

[0075] 4.1 MTC Detection Experimental groups: normal control group, model control group (SLS 60 μg / mL), and sample group (samples were set with 4 concentration gradients: 1.74E+9 particles / mL, 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0076] Treatment method: Zebrafish 3 days post-fertilization (3dpf) were randomly selected and subjected to experiments in 6-well plates (3 mL / well), with 30 fish per well. The normal control group was cultured normally, while the model control group and sample group were induced with sodium dodecyl sulfate (SLS).

[0077] In a zebrafish anti-inflammatory model, different concentrations of patchouli extracellular vesicles were simultaneously administered to the sample groups for 18 hours. The maximum detectable concentration of the sample in the model zebrafish was then determined. The results are shown in Table 2.

[0078] Table 2 Results of the experiment to explore the concentration of extracellular vesicles in patchouli (n=30)

[0079] After 18 hours of exposure to zebrafish, the phenotypes of the extracellular vesicles of patchouli in the sample were similar to those of the model control group at all four treatment concentrations. Therefore, the MTC of the extracellular vesicles of patchouli in zebrafish can be determined to be 1.39E+10 particles / mL.

[0080] 4.2 Neutrophil Detection Experimental groups: normal control group, model control group (SLS 60 μg / mL), positive control group (dipotassium glycyrrhizate 0.031%), and sample group (samples were set with 3 concentration gradients: 3.47E+9 particles / mL, 6.94E+9 particles / mL, and 1.39E+10 particles / mL).

[0081] Methods: Zebrafish 3 days post-fertilization (3 dpf) were randomly selected and subjected to experiments in 6-well plates (3 mL / well), with 30 fish per well. The normal control group was cultured normally. The model control group, positive control group, and sample group were all treated with sodium dodecyl sulfate (SLS) to induce an anti-inflammatory model in zebrafish. The positive control group was treated with 0.031% dipotassium glycyrrhizate, and the sample group was treated with different concentrations of patchouli extracellular vesicles. Treatment lasted 18 hours. Ten zebrafish from each group were randomly selected and imaged under a microscope. The anti-inflammatory efficacy of the samples was evaluated by counting the number of neutrophils in the zebrafish skin. Results are as follows: Figure 17 As shown.

[0082] Figure 17 In the study, after 18 hours of treatment, compared with the model control group, the extracellular vesicles of patchouli showed a significant reduction in the number of neutrophils in zebrafish skin. At concentrations of 3.47E+9, 6.94E+9, and 1.39E+10 particles / mL, the anti-inflammatory efficacy was 45%, 74%, and 78%, respectively.

[0083] Therefore, the patchouli extracellular vesicles prepared by the technical solution provided by the present invention are non-toxic to HaCaT human immortalized epidermal cells and zebrafish, have good biocompatibility, and exhibit good anti-photoaging, antioxidant and anti-inflammatory effects in both in vitro and in vivo experiments, and are expected to be prepared as a natural plant drug for anti-photoaging.

[0084] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0085] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0086] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for extracting and separating P. heterophyllum extracellular vesicles, characterized in that, P. heterophyllum tissue juice is extracted and separated by differential centrifugation, PEG precipitation and ultrafiltration to obtain P. heterophyllum extracellular vesicles. 2.The method for extracting and separating P. heterophyllum extracellular vesicles according to claim 1, characterized in that, the P. heterophyllum tissue juice is at least one of juice of fresh leaves, stems or root systems of P. heterophyllum; preferably, the P. heterophyllum tissue juice is juice of fresh leaves of P. heterophyllum. 3.The method for extracting and separating P. heterophyllum extracellular vesicles according to claim 2, characterized in that, the preparation method of the P. heterophyllum tissue juice is as follows: the fresh P. heterophyllum tissue is washed with pure water and then dried; a phosphate buffered saline solution is added and mixed uniformly, and then the P. heterophyllum tissue is squeezed to collect juice, which is filtered to obtain the P. heterophyllum tissue juice; preferably, the weight / volume ratio of the fresh P. heterophyllum tissue to the phosphate buffered saline solution is 1 g: (1-5) mL; preferably, the juice is squeezed for 2-6 times, and each time the juice is squeezed for 10-120 s, and each time the juice is squeezed with an interval of 30-180 s. 4.The method for extracting and separating P. heterophyllum extracellular vesicles according to claim 1, characterized in that, the differential centrifugation process is as follows: the P. heterophyllum tissue juice is sequentially centrifuged at 500-1000 × g for 10-20 min to collect a first supernatant; the first supernatant is centrifuged at 3000-6000 × g for 25-40 min to collect a second supernatant; the second supernatant is centrifuged at 10000-13000 × g for 50-70 min to collect a third supernatant; preferably, the centrifugation temperature is 3-8 ℃. 5.The method for extracting and separating P. heterophyllum extracellular vesicles according to claim 1, characterized in that, PEG is added to the third supernatant, and the third supernatant is precipitated overnight at 3-8 ℃; preferably, the molecular weight of the PEG is 3000-6000; and preferably, the PEG is added to a mass concentration of 5%-12%. 6.The method for extracting and separating P. heterophyllum extracellular vesicles according to claim 1, characterized in that, after the PEG precipitation, a liquid phase is collected, and the liquid phase is centrifuged at 10000-13000 × g for 30-60 min to collect a precipitate; the precipitate is resuspended with PBS to obtain a crude extract; the crude extract is ultrafiltered by using an ultrafiltration tube to obtain a dispersion of the P. heterophyllum extracellular vesicles; preferably, a 3-100 kd ultrafiltration tube is used to centrifuge at 1000-3000 × g for 10-30 min to collect a fourth supernatant; the dispersion of the P. heterophyllum extracellular vesicles is obtained. 7.The method for extracting and separating P. heterophyllum extracellular vesicles according to claim 5, characterized in that, before the PEG is added to the third supernatant, the third supernatant is filtered through a 0.22-1.0 um microporous filter membrane, and the PEG is added to the obtained filtrate for precipitation.

8. A P. peltatum extracellular vesicle, characterized in that, The P. heterophyllum extracellular vesicles are prepared by the method for extracting and separating P. heterophyllum extracellular vesicles according to any one of claims 1-7. 9.The use of the P. heterophyllum extracellular vesicles according to claim 8 in the preparation of an anti-skin photoaging product.

10. Use according to claim 9, characterized in that, The anti-skin photoaging product is a drug, a health care product or a cosmetic product. Preferably, the dosage form of the medicine is any one of oral liquid, capsule, ointment, gel patch, patch and traditional Chinese medicine.

Images