Separation method of extracellular vesicles derived from non-medicinal parts of paris polyphylla and application of extracellular vesicles in anti-inflammatory and whitening

Abstract

The invention discloses a separation method of extracellular vesicles derived from non-medicinal parts of paris polyphylla and application of the extracellular vesicles in anti-inflammatory and whitening. According to the method, the extracellular vesicles are separated from stems, leaves and root hairs of non-medicinal parts of paris polyphylla by adopting double isoelectric precipitation combined with electrophoresis dialysis, and biological activity research is systematically performed on the extracellular vesicles sourced from the stems, leaves and root hairs. The cell and zebra fish model comprehensive evaluation proves that the extracellular vesicles from the two sources have obvious anti-inflammatory effects. However, in the melanin inhibition activity, the extracellular vesicles of the stems and leaves of the paris polyphylla are good in performance, and the inhibition effect of the extracellular vesicles on melanin generation in a zebra fish in-vivo model and the inhibition capacity of the extracellular vesicles on tyrosinase in vitro are obviously superior to those of a classic whitening agent arbutin. The limitation that only paris polyphylla tubers are utilized traditionally is broken through, development and efficacy verification of the paris polyphylla stem leaf and root hair extracellular vesicles are achieved for the first time, and the application prospect in the aspect of developing anti-inflammatory drugs and whitening cosmetics is wide.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and functional cosmetics, specifically to a method for isolating extracellular vesicles from non-medicinal parts of Paris polyphylla and their application in anti-inflammatory and whitening effects. This invention is the first to isolate extracellular vesicles from the non-medicinal parts of the traditional Chinese medicinal herb Paris polyphylla—the stems and leaves—and reveals for the first time their outstanding bioactivity and novel uses in anti-inflammatory and whitening effects. Background Technology

[0002] The dried rhizome of Paris polyphylla, a traditional Chinese medicine, has been clinically proven to have effects such as clearing heat and detoxifying, reducing swelling and relieving pain. Current research and industrial development almost entirely focus on the active ingredients (such as various saponins) in its rhizome, and related products have been developed. However, during the harvesting and processing of Paris polyphylla, a large amount of non-medicinal parts (such as roots, stems, and leaves) are discarded as agricultural waste, resulting in significant resource waste. How to achieve high-value utilization of these non-medicinal parts is one of the key issues in improving the comprehensive utilization efficiency of Chinese medicinal resources.

[0003] Extracellular vesicles are nanoscale vesicles actively secreted by cells, carrying important functional molecules such as proteins, lipids, and nucleic acids, and mediating intercellular communication. In recent years, plant-derived extracellular vesicles have become a cutting-edge research hotspot in the development of novel drug delivery systems and functional cosmetic ingredients due to their good biocompatibility, low immunogenicity, and potential cross-species regulatory activity.

[0004] Currently, the isolation of plant extracellular vesicles mainly relies on ultracentrifugation and its improved techniques. However, this method has significant limitations when applied to plant tissues: First, the sedimentation coefficients of soluble proteins and polysaccharides, which are abundant in plant extracts, may partially overlap with those of extracellular vesicles, making it difficult to completely remove them using centrifugation alone, thus affecting product purity. Second, the strong mechanical stress generated by ultracentrifugation may damage the integrity of the extracellular vesicle structure, potentially affecting its biological activity. Therefore, developing a highly efficient purification method based on novel separation principles that can remove impurities more gently and selectively has become an important technological need in this field. Isoelectric point precipitation, based on the surface charge characteristics of biomolecules, offers a potentially novel technological path to meet this need.

[0005] Although research on the tubers of Paris polyphylla is relatively in-depth, to date, there have been no publicly available reports on the successful isolation of extracellular vesicles from non-medicinal parts of Paris polyphylla (especially stems and leaves) and a systematic evaluation of their anti-inflammatory and skin-whitening activities. This invention aims to fill this gap and turn waste into treasure. Summary of the Invention

[0006] The purpose of this invention is to break through the traditional limitation of focusing only on the medicinal parts (rhizome) of Paris polyphylla, and to disclose for the first time a method for isolating extracellular vesicles (SL-EXO) from the non-medicinal parts of Paris polyphylla, namely stems and leaves. Furthermore, this invention systematically reveals for the first time the bioactivity of these extracellular vesicles from stems and leaves, as well as extracellular vesicles (RP-EXO) from another non-medicinal part of Paris polyphylla, namely roots, particularly their clear efficacy in anti-inflammatory and skin-whitening effects. This provides a new material basis and application approach for the whole-plant and high-value-added development of Paris polyphylla resources.

[0007] Meanwhile, this invention utilizes the difference in solubility between extracellular vesicles and impurity proteins / particles at a specific pH for separation. This is a mild physicochemical method that can maximize the preservation of the biological activity of extracellular vesicles, and is cost-controllable and easy to scale up.

[0008] Traditional ultracentrifugation methods primarily rely on differences in particle size and density, making it difficult to efficiently remove soluble proteins and polymeric impurities with similar particle size and density to the target extracellular vesicles. This invention innovatively introduces a two-step isoelectric point precipitation strategy:

[0009] 1. Ambient IEP Precipitation: By adjusting the pH of the extract to the isoelectric point range (pH 4-5) of a large number of impurities in the plant juice, most of the soluble impurity proteins are denatured and precipitated, while extracellular vesicles remain dispersed under these conditions due to their stable surface charge. This allows for the efficient removal of major impurities during the crude extraction stage.

[0010] 2. Target IEP Focusing: After obtaining relatively pure crude extracellular vesicles, the pH of the suspension is further precisely adjusted to near the isoelectric point of the target extracellular vesicles (pH 6.0-7.8), causing the extracellular vesicles to gently aggregate and precipitate, thus separating them from the small amount of residual impurities (such as certain polysaccharides and nucleic acids) that remain soluble at this pH. Finally, residual small molecule impurities are further removed by electrophoretic dialysis to improve product purity. Protein concentration is quantified by BCA method.

[0011] To achieve the above objectives, the present invention specifically adopts the following technical solution: In a first aspect, the present invention provides a method for isolating extracellular vesicles from non-medicinal parts of Paris polyphylla, comprising the following steps:

[0012] (1) Environmental isoelectric point precipitation to remove impurities: The pH of the crude extract of Paris polyphylla roots or stems and leaves is adjusted to the isoelectric point range of impurity proteins in the plant juice. After standing, centrifugation is performed to remove impurity protein denaturation precipitation. Extracellular vesicles remain dispersed due to stable surface charge. The supernatant rich in extracellular vesicles is collected.

[0013] (2) Target isoelectric point focusing purification: Adjust the pH of the supernatant obtained in step (1) to the isoelectric point range of the target extracellular vesicle itself, let it stand to allow the extracellular vesicles to aggregate and precipitate, remove residual impurities, and collect the extracellular vesicle precipitate by centrifugation.

[0014] (3) Dissolution and deaggregation: The extracellular vesicle precipitate obtained in step (2) was resuspended with neutral buffer and the extracellular vesicle components were collected by electrophoresis dialysis.

[0015] Furthermore, in step (1), the pH of the crude extract is adjusted to 4.0-5.0, and in step (2), the pH of the supernatant is adjusted to 6.0-7.8.

[0016] Further, the pH is adjusted to 4.5 in step (1). The pH is adjusted to 7.2 in step (2).

[0017] Furthermore, steps (1) and (2) are both carried out at a low temperature of 2-8°C.

[0018] Secondly, the present invention also provides the application of extracellular vesicles derived from non-medicinal parts of Paris polyphylla in the preparation of anti-inflammatory products.

[0019] Furthermore, the extracellular vesicles are extracellular vesicles of Paris polyphylla stems, leaves, or roots.

[0020] Furthermore, the anti-inflammatory product is a pharmaceutical product.

[0021] Thirdly, the present invention also provides a composition with anti-inflammatory effects, comprising an effective amount of extracellular vesicles of Paris polyphylla roots or extracellular vesicles of Paris polyphylla stems and leaves or a combination thereof, and a pharmaceutically acceptable carrier.

[0022] Fourthly, the present invention also provides the application of extracellular vesicles from stems and leaves of Paris polyphylla (a non-medicinal part) in the preparation of products for skin whitening.

[0023] Furthermore, the whitening product is a cosmetic.

[0024] Fifthly, the present invention also provides a composition having whitening effects, comprising an effective amount of extracellular vesicles of Paris polyphylla stems and leaves, and a cosmetically acceptable carrier.

[0025] Furthermore, the method for preparing the composition includes separation and purification from the extract of Paris polyphylla roots or stems and leaves by a three-stage combination of differential centrifugation, polymer precipitation and electrophoretic dialysis, differential centrifugation combined with ultracentrifugation, sucrose density gradient centrifugation combined with ultracentrifugation, and differential centrifugation combined with size exclusion chromatography.

[0026] The core findings of this invention have been confirmed through a series of in vitro and in vivo experiments:

[0027] 1. Anti-inflammatory activity:

[0028] In the transgenic neutrophil fluorescent zebrafish (Tg(lyz:eGFP)) in vivo model, both the extracellular vesicles of Paris polyphylla stem and leaf cells (SL-EXO) and the extracellular vesicles of root cells (RP-EXO) significantly inhibited the enhancement of inflammation-induced fluorescence signals, demonstrating a clear anti-inflammatory effect.

[0029] 2. Whitening activity:

[0030] In vitro tyrosinase inhibition: In cellular enzyme activity assays, extracellular vesicles (SL-EXO) from the stems and leaves of Paris polyphylla exhibited strong tyrosinase inhibitory activity, with the inhibitory effect showing a concentration-dependent relationship. However, extracellular vesicles (RP-EXO) from the roots did not show significant inhibitory activity in this model.

[0031] In vivo melanin inhibition: In a zebrafish embryonic melanin production model, extracellular vesicles (SL-EXO) from Paris polyphylla stem and leaf cells significantly reduced melanin deposition in vivo in a dose-dependent manner, with the same effect as arbutin at the same concentration, further verifying its excellent whitening efficacy.

[0032] Compared with the prior art, the present invention has the following significant advantages:

[0033] 1. Waste utilization: This invention is the first to focus the research on the traditionally discarded stems and leaves of Paris polyphylla and successfully isolate extracellular vesicles with clear activity from them, realizing the transformation from "waste" to "high-value active raw material", which is a major breakthrough in the understanding and utilization of Paris polyphylla resources.

[0034] 2. Efficacy proof: Through cell and zebrafish models, it was confirmed that the extracellular vesicles of the two non-medicinal parts have anti-inflammatory activity. Furthermore, it was discovered and confirmed for the first time that the extracellular vesicles of Paris polyphylla stem and leaf (SL-EXO) have a whitening effect superior to the classic whitening agent arbutin.

[0035] 3. Application Orientation: This invention provides a novel and highly promising core ingredient for the development of new, naturally derived anti-inflammatory drugs for the skin (such as for adjunctive treatment of dermatitis and eczema), soothing and anti-allergic products, as well as high-end cosmetics or functional skin care products with high whitening and brightening effects. It has broad application prospects.

[0036] 4. In line with the concept of sustainable development: The implementation of this invention can greatly improve the comprehensive utilization rate and economic value of Paris polyphylla resources, reduce resource waste, and conform to the green and circular industrial development direction.

[0037] 5. Innovative Preparation Method: The combined separation method provided by this invention innovatively employs a dual isoelectric point precipitation strategy to achieve highly selective purification based on charge differences. This method can efficiently remove plant-derived soluble protein impurities under mild conditions, significantly improve the purity of extracellular vesicle products, and better maintain their natural biological activity, providing a better raw material guarantee for subsequent functional applications. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 The images shown are TEM images and NTA results of plant extracellular vesicles prepared according to the present invention. Among them, A and C are TEM and NTA images of extracellular vesicles in Paris polyphylla root cells; B and D are TEM and NTA images of extracellular vesicles in Paris polyphylla stems and leaves.

[0040] Figure 2 Zeta potential (A), ABTS free radical scavenging rate (B), and tyrosinase inhibition rate (C) of extracellular vesicles in the roots and leaves of Paris polyphylla prepared in this invention.

[0041] Figure 3 Distribution of neutrophils in zebrafish embryos (A) and quantitative analysis of neutrophils (B).

[0042] Figure 4 The analysis included the distribution of melanin in the head and abdomen of zebrafish embryos (A), quantitative analysis of melanin (B), quantitative analysis of melanin content in vivo (C), and quantitative analysis of tyrosinase activity in vivo (D).

[0043] Figure 5 The 24-h and 48-h SL-EXO and Arbutin cytotoxicity were measured.

[0044] Figure 6 The values ​​represent the relative tyrosinase activity (A) and relative melanin content (B) within the cell. Detailed Implementation

[0045] The following examples are used to further illustrate the technical solutions of the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the experimental methods are all implemented in accordance with national or international standards, and the reagents and equipment are all commercially available products in the art.

[0046] Example 1: Isolation and characterization of extracellular vesicles from roots and stems of Paris polyphylla

[0047] Materials: Fresh non-medicinal parts of Paris polyphylla (roots, stems and leaves), phosphate-buffered saline (PBS).

[0048] Separation method:

[0049] 1) Extraction:

[0050] 1. Tissue pretreatment and crude extraction: Wash and chop the roots or stems and leaves of Paris polyphylla, add pre-cooled extraction buffer (0.1M PBS) and homogenize. Filter through a 200-mesh filter to obtain the crude extract.

[0051] Step 1 (Isoelectric point precipitation to remove large amounts of impurities): Place the crude extract at 4 °C and adjust the pH to 4.5 precisely with dilute hydrochloric acid while stirring slowly. Let it stand at 4 °C for 1-2 hours to allow a large amount of plant impurities to precipitate near this isoelectric point. Then, centrifuge at 10000 × g for 30 minutes at 4 °C and carefully collect the supernatant. At this point, most of the soluble impurities have been removed.

[0052] Step 2 (Ultrafiltration Concentration and Buffer Replacement): The supernatant was concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa.

[0053] Step 3 (Target Isoelectric Point Focusing for Fine Purification of Extracellular Vesicles): At 4°C with slow stirring, precisely adjust the pH of the concentrated extracellular vesicle suspension to 7.2 using dilute hydrochloric acid. Incubate overnight (12-16 hours) at 4°C to allow the extracellular vesicles to slowly and gently aggregate and precipitate. Centrifuge at 12,000 × g for 40 minutes at 4°C, collect the precipitate, and resuspend in PBS.

[0054] 2) Purification of extracellular vesicles:

[0055] a. The obtained dispersion was first filtered through a 0.45 μm filter and then through a 0.22 μm filter for sterilization.

[0056] b. Use a tangential flow filtration system to repeatedly ultrafilter the sample to 10-15 ml at 3500-5000g and 4℃.

[0057] c. Perform electrophoretic dialysis on the above concentrate at 300-350 mA for 30 min, and repeat the above operation 4-6 times.

[0058] d. Then, repeatedly ultrafilter the dialyzed liquid at 3500g and 4℃ to obtain 4-5 ml. Aliquot and store at -80℃.

[0059] e. The protein concentration of the purified extracellular vesicles was determined using a BCA assay kit.

[0060] Characterization and testing:

[0061] Transmission electron microscopy observation:

[0062] Take 20 μL of freshly prepared Paris polyphylla root and stem / leaf extracellular vesicle samples and drop them onto a carbon membrane copper grid. Let stand for 15 minutes and blot away excess liquid with filter paper. Add 20 g / L phosphotungstic acid to negatively stain the extracellular vesicles on the carbon membrane copper grid for 90 seconds and blot away excess liquid with filter paper. After drying at room temperature, place the sample under a transmission microscope for observation and image acquisition.

[0063] Particle size analysis and potential analysis:

[0064] (1) Particle size: The particle size and concentration of extracellular vesicles were tested using NanoSight NS300. The sample pool was cleaned with PBS using a syringe, the device was turned on and the software was self-tested. 1 μL of standard was taken into 2 ml of PBS and mixed to check if the particle size was in compliance. An appropriate volume of extracellular vesicles was taken, diluted with PBS to 2 ml, and injected into the sample pool through a 1 ml syringe to start the test.

[0065] (2) Potential analysis: After diluting the sample with PBS, the potentiometer was inserted into the sample cell of the Zetasizer Nano and the potential value was measured. The measurement was repeated three times.

[0066] Characterization results:

[0067] a. Transmission electron microscopy (TEM) observation: such as Figure 1 A (RP-EXO) and Figure 1 As shown in B (SL-EXO), both types of extracellular vesicles exhibit typical bilayer membrane vesicle structures under transmission electron microscopy, with intact morphology, clear outlines, and round or near-round morphological characteristics. The images show continuous and intact membrane structures, with visible low-electron-density contents, further confirming their vesicular nature. The particle size distribution is relatively uniform, roughly within the range of 30–150 nm, conforming to the general morphological standards of extracellular vesicles. The samples underwent negative staining during preparation, resulting in clear contrast of the membrane structure and demonstrating typical ultrastructural characteristics of extracellular vesicles, indicating that the extracted samples possess good structural integrity and are suitable for subsequent analysis.

[0068] b. Nanoparticle tracking analysis (NTA) is a technique for visualizing and analyzing the particle size of nanoparticles in liquid suspensions through light scattering and Brownian motion properties. For example... Figure 1 C (RP-EXO) and Figure 1As shown in D(SL-EXO), the particle size distribution of the two extracellular vesicles is mainly concentrated in the range of 50 to 150 nm, with the main peak of RP-EXO located at 151 nm and the main peak of SL-EXO located at 135 nm. This particle size distribution is consistent with the physical characteristics of typical extracellular vesicles, further confirming that the extracted particles do indeed belong to the category of extracellular vesicles. The overall distribution shows a single-peak morphology, indicating that the sample particle size is relatively uniform, with the vast majority of particles falling within the common extracellular vesicle size range, and no obvious aggregates or debris interference were observed. c. Zeta potential test: The surface charge of RP-EXO and SL-EXO was measured using a Zeta potential analyzer, as shown in... Figure 2 As shown in Figure A, the average potentials of the two particles are -6.14 mV and -7.1 mV, respectively. These negative values ​​indicate that the particle surface carries a negative charge, consistent with the electrical characteristics of typical plant extracellular vesicles, suggesting good colloidal stability and biocompatibility. This further supports the conclusion that both are plant-derived extracellular vesicles from an electrochemical perspective.

[0069] Example 2: ABTS free radical scavenging experiment

[0070] Experimental materials: ABTS, K2S2O8, microplate reader, extracellular vesicles prepared in Example 1, 0.01M PBS solution;

[0071] Experimental methods:

[0072] Preparation of ABTS test solutions: ABTS stock solution (7.4 mmol / L, 0.4 mL): 0.0045 g ABTS, 1.1025 mL distilled water (MW=548.7); K2S2O8 stock solution (2.6 mmol / L, 1.43 mL): Take 0.0025 g K2S2O8, add 3.575 mL distilled water (MW=270.32), mix the two solutions, let stand in the dark at room temperature for 12 hours, then dilute with PBS to an absorbance of approximately 0.7 (734 nm). A0 value detection: Take 0.2 mL of this ABTS solution and 50 μL of PBS solution, mix thoroughly, and measure the absorbance at 734 nm. A value detection: Take 50 mL of ABTS test solution, mix thoroughly with 50 μL of extracellular vesicles of varying concentrations, and measure the absorbance at 734 nm. The experiment was independently repeated three times. The formula for calculating ABTS clearance rate is as follows:

[0073]

[0074] Experimental results:

[0075] like Figure 2As shown in Figure B, both RP-EXO and SL-EXO exhibited significant concentration-dependent ABTS radical scavenging capabilities. As the sample concentration gradually increased from 0.05 mg / mL to 0.5 mg / mL, the clearance rates of both extracellular vesicles continuously increased, showing an overall positive correlation trend. However, there were significant differences in their clearance efficiency and kinetic characteristics.

[0076] SL-EXO exhibits extremely strong antioxidant activity, with significant scavenging ability even at low concentrations. At a low concentration of 0.05 mg / mL, the scavenging rate reached 19%; when the concentration was increased to 0.1 mg / mL, the scavenging rate rose sharply to 57.3%, showing strong initial reactivity; with further increases in concentration, the rate of increase in scavenging rate gradually slowed, reaching 78.7% at 0.3 mg / mL, and finally reaching a maximum of 83.3% at 0.5 mg / mL. This change indicates that SL-EXO approaches its scavenging capacity saturation at low concentrations, demonstrating its highly efficient and rapidly responding antioxidant properties.

[0077] In comparison, RP-EXO exhibited weaker overall ABTS radical scavenging ability, with a relatively gradual and linear growth pattern. Within the concentration range of 0.05–0.2 mg / mL, the scavenging rate only slowly increased from 12% to 31.3%; when the concentration was further increased to 0.5 mg / mL, the scavenging rate peaked at only 56.7%, significantly lower than that of SL-EXO at the same concentration. These results indicate that RP-EXO did not show a significant scavenging saturation phenomenon within the experimental concentration range, and its antioxidant capacity steadily but to a limited extent increased with increasing concentration.

[0078] Example 3: In vitro tyrosinase inhibition experiment

[0079] Experimental materials: L-tyrosine, extracellular vesicles prepared in Example 1, arbutin, 0.01M PBS solution;

[0080] Experimental methods:

[0081] A. Treatment of the test substance:

[0082] Positive controls α-arbutin, SL-EXO, and RP-EXO were diluted with PBS buffer to prepare concentration gradient solutions of 0.06 mg / mL, 0.08 mg / mL, 0.12 mg / mL, 0.15 mg / mL, 0.2 mg / mL, and 0.4 mg / mL, respectively.

[0083] B. Experimental Groups:

[0084] In a 96-well microplate, three groups were set up: solvent background wells (Ta), solvent reaction wells (Tb), sample background wells (TC), and sample reaction wells (Td). Specifically, Ta wells contained neither L-tyrosine substrate solution nor sample solution; Tb wells contained L-tyrosine substrate solution but no sample solution; TC wells contained neither L-tyrosine substrate solution nor sample solution; and Td wells contained both L-tyrosine substrate solution and sample solution. Each group was run in triplicate.

[0085] C. Experimental steps:

[0086] Referring to the reagent dosage in Table 1, add L-tyrosine solution and PBS buffer to each well in sequence, mix thoroughly, incubate at 37℃ for 10 min, then add 20 μL of tyrosine solution in sequence, mix at 37℃ for 5 min ± 5 s, and immediately place in a microplate reader for measurement at a wavelength of 475 nm.

[0087] Table 1. Sample loading table for tyrosinase activity inhibition test

[0088]

[0089] Note: The time from adding tyrosine solution to measuring absorbance was consistent for each well (5±5s).

[0090] Experimental results: such as Figure 2 As shown in Figure C, the inhibitory activities of samples RP-EXO and SL-EXO at different concentrations were evaluated in an in vitro tyrosinase inhibition experiment, with the classic inhibitor arbutin used as a positive control. Experimental data showed that both arbutin and SL-EXO exhibited clear concentration-dependent inhibitory effects. As the concentration increased from 0.06 mg / mL to 0.40 mg / mL, the inhibition rate of arbutin increased from 5.50% to 49.78%, while SL-EXO showed a more significant inhibitory ability, with its inhibition rate increasing sharply from 6.51% to 92.66%. In contrast, sample RP-EXO did not show significant inhibitory activity at any of the tested concentrations, with its inhibition rate consistently below 2% and showing no concentration-dependent trend. Specifically, the inhibitory efficacy of SL-EXO far exceeded that of the positive control. At a concentration of 0.15 mg / mL, the average inhibition rate of SL-EXO reached 58.08%, exceeding the inhibitory effect of arbutin at a concentration of 0.40 mg / mL, indicating its half-maximal inhibitory concentration (IC50). 50The activity level was significantly lower than that of arbutin. The activity trend of the positive control arbutin was consistent with known literature reports, verifying the reliability of the experimental system. All active samples showed good consistency in three parallel experiments at each concentration, with small standard deviations, indicating high repeatability and reliable data. In conclusion, SL-EXO is a highly efficient tyrosinase inhibitor with significantly stronger in vitro activity than arbutin; while RP-EXO did not show inhibitory activity under the conditions of this experiment.

[0091] Example 4: Validation of anti-inflammatory activity (zebrafish model)

[0092] Experimental materials: zebrafish embryos, LPS, extracellular vesicles prepared in Example 1, ibuprofen.

[0093] Experimental Methods: Zebrafish embryos of the neutrophil fluorescent strain Tg(lyz:eGFP) were selected and divided into 10 groups. Unfertilized / abnormal embryos were removed at 12 hpf in E3 medium at 28.5 ℃. Ten embryos from each group were transferred to 6-well plates, with 3 replicates per experimental group, for a total of 30 wells. 5 mL of the corresponding concentration of drug solution was added to each well. The 10 experimental groups were: RP-EXO: 0.0001 mg / mL, 0.0005 mg / mL, 0.001 mg / mL; SL-EXO: GX: 0.0001 mg / mL, 0.0005 mg / mL, 0.001 mg / mL; Ibuprofen: 0.0001 mg / mL, 0.0005 mg / mL, 0.001 mg / mL. A blank control group without added drugs was also included.

[0094] The cells were cultured in a 28.5 ℃ incubator in the dark for 96 hours, with 50% of the drug solution replaced daily. Images were acquired using a fluorescence microscope 108 hours after fertilization (hpf). Neutrophils were labeled with green fluorescent dye, and 15 samples were randomly selected from each group for imaging. Fluorescence intensity was positively correlated with neutrophil activation; fluorescence signal increased in inflammatory states, while the degree of fluorescence intensity reduction was positively correlated with the anti-inflammatory effect of the drug. To enhance the inflammatory phenotype, lipopolysaccharide (LPS) was added to each well at a final concentration of 0.5 μg / mL. Copper sulfate (CuSO4) was not used because LPS specifically activates macrophages / neutrophils through the Toll-like receptor 4 (TLR4) pathway, and the induced inflammatory response is highly similar to that of human bacterial infections; while CuSO4 modeling is more suitable for heavy metal poisoning models and easily leads to non-specific tissue damage.

[0095] After image acquisition, fluorescence intensity quantification and statistical analysis were performed using ImageJ software (Version 1.53). The specific operation procedure is as follows:

[0096] 1. Image - Color - 16-bit

[0097] 2. Image - Adjust-Threshold (set to 15)

[0098] 3. Image - Adjust - Auto Threshold (default parameter)

[0099] 4. Analyze - Set Measurements (check Area, Mean gray value, Standarddeviation, Integrated density, Area fraction, Limit to threshold)

[0100] 5. Analyze - Measure (Record Area values)

[0101] The obtained data were used to create charts and perform statistical significance analysis using GraphPad Prism software (Version 9.0).

[0102] Experimental results: such as Figure 3As shown, an anti-inflammatory effect evaluation experiment was conducted using a transgenic neutrophil fluorescent zebrafish model. The effects of samples SL-EXO and RP-EXO at different concentrations were tested, with the nonsteroidal anti-inflammatory drug ibuprofen used as a positive control. The experiment quantified the inflammation level by measuring the fluorescence intensity in zebrafish after inflammation induction; fluorescence intensity was positively correlated with the degree of inflammation. Statistical analysis showed that, compared with the blank control group, the positive control ibuprofen significantly reduced fluorescence intensity at concentrations of 0.0005 and 0.001 mg / mL, confirming the effectiveness of this model in evaluating the anti-inflammatory effect of drugs. Sample SL-EXO exhibited significant anti-inflammatory activity at all tested concentrations, with a clear concentration-dependent effect. At the lowest concentration of 0.0001 mg / mL, SL-EXO significantly inhibited the fluorescence signal; when the concentration increased to 0.001 mg / mL, its fluorescence intensity decreased to the lowest level, with an inhibitory effect comparable to that of ibuprofen at the same concentration, indicating that SL-EXO has strong in vivo anti-inflammatory potential. In contrast, sample RP-EXO also showed a certain trend of fluorescence inhibition within the tested concentration range, but its effect was relatively weak and did not exhibit the clear concentration-dependent relationship like SL-EXO. At a concentration of 0.0005 mg / mL, RP-EXO reached its peak inhibitory effect, but its activity was still inferior to that of SL-EXO and ibuprofen at the same concentration. In summary, the results of this experiment indicate that sample SL-EXO has significant anti-inflammatory efficacy in a zebrafish in vivo inflammation model, and its effect increases with increasing concentration within a certain range, being superior to the positive control ibuprofen. While sample RP-EXO also showed some activity, its effect was weaker. These results provide important in vivo experimental evidence for SL-EXO as a potential anti-inflammatory lead compound.

[0103] Based on the aforementioned in vitro and in vivo experimental results, SL-EXO demonstrates superior potential compared to RP-EXO in several key bioactivity indicators. SL-EXO may possess stronger bioactivity in anti-oxidative stress, inhibition of inflammatory factor release, and regulation of melanin synthesis pathways due to its unique morphological structure or active ingredient composition. Therefore, compared to RP-EXO, SL-EXO shows greater potential for comprehensive skincare applications, particularly in anti-oxidation, anti-inflammation, and whitening functions. Based on these preliminary conclusions, future research will focus on conducting more in-depth and systematic verification of the whitening mechanism of SL-EXO in in vitro cell models and in vivo zebrafish models, including quantitative assessments of its inhibition of tyrosinase activity and melanin content.

[0104] Example 5: Whitening Activity Verification (Zebrafish Model)

[0105] 1. Apparent whitening experiment in zebrafish

[0106] Experimental materials:

[0107] Wild-type AB strain zebrafish embryos, stem and leaf extracellular vesicles prepared in Example 1, arbutin, microscope.

[0108] Experimental methods:

[0109] AB zebrafish embryos were selected and divided into 7 groups, placed in 12-well plates, with 3 replicates per experimental group and 10 embryos per well. Three groups each received drugs JY and XG, and a blank control group was included. Drug-containing culture medium was added at 12 hpf, and the embryos were cultured for another 48 hours. Embryo morphology was photographed under a stereomicroscope, with 15 embryos photographed per group. A parallel experiment was also set up, where embryos were collected but not photographed, and all experimental conditions were identical.

[0110] Experimental results: such as Figure 4 A and Figure 4 As shown in Figure B, in an in vivo melanin production inhibition experiment using AB strain zebrafish embryos, this invention evaluated the depigmentation effect of sample SL-EXO, using the classic skin-whitening ingredient arbutin as a positive control. The experiment involved treating embryos 12 hours after fertilization with the drug for 48 hours, followed by quantitative analysis of their in vivo melanin content.

[0111] The experimental results showed that, compared with the blank control group, the positive control arbutin significantly reduced the melanin content of zebrafish embryos at concentrations of 0.0005 and 0.001 mg / mL, validating the applicability of this in vivo model. Sample SL-EXO exhibited extremely strong melanin production inhibition at all tested concentrations, with the effect showing a significant dose-dependent relationship. Notably, at the highest tested concentration (0.001 mg / mL), the melanin content in the SL-EXO-treated group decreased to extremely low levels, and its inhibitory effect numerically far exceeded that of arbutin at the same concentration, indicating that SL-EXO possesses excellent depigmentation activity in the in vivo environment. Even at a lower concentration of 0.0005 mg / mL, the inhibitory effect of SL-EXO was already very significant, suggesting its high in vivo activity.

[0112] In contrast, while arbutin also showed a dose-dependent inhibitory trend, its reduction in melanin content at the same concentration was far less than that of SL-EXO. For example, at 0.001 mg / mL, the residual melanin in the arbutin group was significantly higher than that in the SL-EXO group.

[0113] In summary, the in vivo experimental results demonstrate that SL-EXO effectively inhibits melanin production in zebrafish embryos, exhibiting significantly stronger depigmentation activity than the standard control arbutin. This provides crucial in vivo efficacy evidence for SL-EXO as a promising novel candidate compound for skin whitening or treating hyperpigmentation.

[0114] 2. Experiment on melanin content and tyrosinase activity in zebrafish

[0115] Experimental materials:

[0116] Wild-type AB strain zebrafish embryos, stem and leaf extracellular vesicles prepared in Example 1, arbutin, L-tyrosine, NaOH, DMSO, RIPA Lysis Buffer, microscope.

[0117] Experimental methods:

[0118] Zebrafish embryos of the Tg(lyz:eGFP) neutrophil fluorescent strain were selected and divided into 7 groups. At 12 hpf, the corresponding drug was added. After 48 h of culture, 10 juvenile fish were selected from each group. After abstaining from water, 200 μL of lysis buffer containing 1% v / v protease inhibitor was added. The embryos were homogenized and centrifuged at 4000 rpm for 10 minutes. The precipitate and supernatant were used to determine melanin content and tyrosinase activity, respectively. Each group was repeated 3 times.

[0119] Add 250 μL of NaOH solution (1M, containing 10% v / v DMSO) to the precipitate and incubate at 85 °C for 2 hours. Pour 100 μL of the reaction solution into a 96-well plate and measure the absorbance at 405 nm. Take 20 μL of the supernatant, add 180 μL of L-Dopa solution (1 mg / mL), and incubate at 37 °C in the dark for 2 hours. Pour 100 μL of the reaction solution into a 96-well plate and measure the absorbance at 475 nm.

[0120] Experimental results: such as Figure 4 C and Figure 4 As shown in D;

[0121] In an in vivo mechanism study of Tg(lyz:eGFP) zebrafish embryos, this invention simultaneously determined the effects of sample SL-EXO and the positive control arbutin on melanin content and the activity of tyrosinase, a key enzyme in melanin synthesis, using biochemical methods. Experimental data showed that both compounds effectively intervened in the melanin production process in zebrafish, but they exhibited differences in their modes of action.

[0122] In terms of melanin content determination (precipitation data), both SL-EXO and arbutin showed significant concentration-dependent inhibitory effects. As the concentration increased from 0.0001 mg / mL to 0.001 mg / mL, the melanin deposition in both groups of zebrafish embryos gradually decreased. Notably, at the highest concentration (0.001 mg / mL), the melanin residue in the SL-EXO-treated group decreased to a level comparable to that of the arbutin group. At lower concentrations (0.0001 mg / mL), the inhibitory effect of SL-EXO was numerically more pronounced, indicating its stronger depigmentation efficacy at this concentration.

[0123] Both compounds showed significant effects in inhibiting tyrosinase activity (supernatant data). Arbutin exhibited extremely strong inhibitory activity at all tested concentrations, especially at a low concentration of 0.0001 mg / mL, where its inhibitory effect was already very significant, suggesting a high direct action on tyrosinase. SL-EXO also showed a clear dose-response effect in inhibiting tyrosinase activity, inhibiting enzyme activity to extremely low levels at 0.001 mg / mL, but its initial inhibitory strength at low concentrations was weaker than that of arbutin.

[0124] Based on the combined analysis of the two indicators, the results of this experiment show that both SL-EXO and the positive control drug arbutin can effectively reduce the synthesis and deposition of melanin in zebrafish by inhibiting tyrosinase activity, a key pathway. Arbutin showed excellent performance in directly inhibiting enzyme activity, while SL-EXO, especially within a specific concentration range, demonstrated potential that was no less effective than, and perhaps even superior to, SL-EXO in reducing overall melanin production. The difference in their modes of action suggests that, in addition to directly inhibiting tyrosinase, SL-EXO may have other accessory pathways involved in its potent depigmentation effect. This provides an important experimental direction for further elucidating the whitening mechanism of SL-EXO.

[0125] Example 6: Validation of skin whitening activity (cell model)

[0126] Experimental materials: B16F10 cells, CCK8, extracellular vesicles prepared in Example 1, arbutin.

[0127] Experimental methods:

[0128] 1. Evaluation of the sample's effect on cell growth and toxicity - CCK-8 assay;

[0129] (1) Cell resuscitation: Take B16F10 cells stored in liquid nitrogen, thaw them rapidly at 37 ℃, transfer the cells in the cryopreservation tube to a centrifuge tube containing 5 ml PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet in complete culture medium after resuscitation, and place the culture flask at 37 ℃ and 5% v / v CO2 for culture.

[0130] (2) Cell passage: Once the cells have grown into a monolayer, discard the culture medium, wash once with PBS, digest with trypsin and collect the cell suspension, centrifuge at 1000 rpm for 5 min, remove the supernatant, add an appropriate amount of culture medium to make it a single cell suspension; passage at a ratio of 1 to 3.

[0131] (3) Cell plating: When the passaged cells reach 80-90% of the culture flask volume, discard the culture medium and add 1 ml of trypsin for digestion. Observe under a microscope. After the cells become rounded, add 1 ml of PBS and resuspend them in water. Transfer the suspension to a 15 ml centrifuge tube, centrifuge, discard the supernatant, resuspend the cells in 4 ml of culture medium, mix by refluxing, and take 10 μL for counting. Adjust the cell density to 1×10⁻⁶ cells based on the counting results. 5 / ml, 100μL / well for 96-well plate.

[0132] (4) Sample and sample culture medium preparation: Take the sample immediately before use and prepare arbutin at 0.04, 0.08, 0.2, 0.4, 0.8 mg / mL using complete culture medium, and prepare arbutin at 10, 50, 100, 250, 500 μM.

[0133] (5) Intervention of cells with sample culture medium: Discard the cell culture supernatant in the 96-well plate, add sample culture medium, three wells for each concentration, add an equal volume of culture medium to the normal control, and incubate the 96-well plate at 37 ℃ under 5% CO2 conditions.

[0134] (6) CCK-8 detection: After the samples were incubated for 24 h and 48 h, the culture supernatant was discarded, and culture medium containing 10% v / v CCK-8 was added to each well. The samples were then incubated at 37 ℃ and 5% v / v CO2 for another 2 h. The absorbance of each well was measured at 450 nm using an ELISA reader.

[0135] 2. Detection of intracellular tyrosinase activity and protein concentration;

[0136] Following the above grouping, except for the blank control group, all other groups used freshly prepared α-MSH (200 nM DMEM + 0.1% v / v DMSO) and were cultured at 37 ℃ and 5% CO2 for 1 hour. After 1 hour of pretreatment, each group was added with sample-containing medium. After 48 hours, the samples were washed 2–3 times with pre-cooled PBS (4 ℃) to thoroughly remove residual medium. 50–100 μL of PBS containing 1% Triton X-100 (pH 7.4) was added to each well (adjusted according to the well area), and the samples were lysed on ice for 30 minutes. After centrifugation (12,000 rpm, 10 min, 4 ℃), the supernatant was collected. The supernatant was mixed with an equal volume of L-DOPA (100 μL) solution and incubated at 37 ℃ in the dark for 2 hours. The absorbance was measured at 475 nm using a microplate reader. Protein concentration was determined using a partial lysate of the supernatant according to the BCA kit.

[0137] 3. Detection of intracellular melanase activity

[0138] Following the above grouping, except for the blank control group, all other groups were cultured with freshly prepared α-MSH (200 nM + DMEM + 0.1% DMSO) at 37 ℃ and 5% v / v CO2 for 1 h. After 1 h of pretreatment, each group was added with sample-containing culture medium. After 48 h, 250 μl of 1M NaOH (containing 10% DMSO) solution was added, and the cells were heated in an 80 ℃ water bath for 1 h to fully lyse the cells and dissolve the melanin. After cooling to room temperature, the cells were centrifuged (12,000 rpm, 10 min) to remove insoluble debris, and the absorbance was measured at 405 nm using a microplate reader.

[0139] Experimental results:

[0140] 1. Cytotoxicity;

[0141] like Figure 5 As shown, SL-EXO at concentrations ranging from 0.04 to 0.80 mg / mL, and arbutin at concentrations ranging from 50 to 100 μM, did not exhibit significant cytotoxicity against B16F10 melanoma cells after 24 and 48 hours of treatment. Cell viability remained at a high level, indicating that the selected concentrations can be used for subsequent functional experiments.

[0142] 2. Intracellular tyrosinase activity;

[0143] like Figure 6As shown in Figure A, in the α-MSH-induced melanin synthesis model, SL-EXO at concentrations of 0.2, 0.4, and 0.8 mg / mL significantly inhibited intracellular tyrosinase activity, with the inhibitory effect increasing with increasing concentration, exhibiting a clear dose-dependent trend. The positive control, arbutin, also showed a significant inhibitory effect, further confirming the reliability and effectiveness of the experimental system. This provides a foundation for studying the melanin production mechanism and highlights the potential role of SL-EXO in regulating enzyme activity.

[0144] 3. Intracellular melanin content;

[0145] like Figure 6 As shown in Figure B, in the α-MSH-induced melanin synthesis experimental model, compared with the model group, the melanin content of each SL-EXO treatment group was significantly reduced, showing a certain concentration-dependent trend. This result indicates that SL-EXO can effectively inhibit the α-MSH-induced melanin production process, and its mechanism of action may be related to regulating tyrosinase activity or affecting the melanin synthesis signaling pathway. Therefore, SL-EXO exhibits potential whitening or pigmentation regulation biological functions, providing preliminary experimental evidence for its application in cosmetics or dermatology.

[0146] In this experimental example, the total protein concentration of the plant extracellular vesicle-like nanoparticles in the above-described embodiments was determined using the BCA method. The specific procedure is as follows:

[0147] The total protein concentration was determined using the BCA Protein Assay Kit (Enhanced Version) (Beyotime). Following the kit's instructions, a protein standard curve was obtained. The prepared plant extracellular vesicle-like nanoparticles were diluted a certain factor, and 20 μl was added to a 96-well plate according to the instructions. Then, 200 μl of working solution was added, and the plate was wrapped in aluminum foil and incubated at 37 °C for 30 min in the dark. The absorbance was then measured at 562 nm. The total protein concentration of the sample was calculated based on the standard curve and the dilution factor.

[0148] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for isolating extracellular vesicles from non-medicinal parts of Paris polyphylla, characterized in that, Includes the following steps: (1) Environmental isoelectric point precipitation to remove impurities: The pH of the crude extract of Paris polyphylla roots or stems and leaves is adjusted to the isoelectric point range of impurity proteins in the plant juice. After standing, centrifugation is performed to remove impurity protein denaturation precipitation. Extracellular vesicles remain dispersed due to stable surface charge. The supernatant rich in extracellular vesicles is collected. (2) Target isoelectric point focusing purification: Adjust the pH of the supernatant obtained in step (1) to the isoelectric point range of the target extracellular vesicle itself, let it stand to allow the extracellular vesicles to aggregate and precipitate, remove residual impurities, and collect the extracellular vesicle precipitate by centrifugation. (3) Dissolution and deaggregation: The extracellular vesicle precipitate obtained in step (2) was resuspended with neutral buffer and the extracellular vesicle components were collected by electrophoresis dialysis.

2. The application of extracellular vesicles derived from non-medicinal parts of Paris polyphylla obtained by the method described in claim 1 in the preparation of anti-inflammatory products.

3. The application according to claim 2, characterized in that, The extracellular vesicles are extracellular vesicles of Paris polyphylla stems, leaves, or roots.

4. The application according to claim 2, characterized in that, The anti-inflammatory product is a pharmaceutical product.

5. A composition having anti-inflammatory effects, characterized in that, It comprises an effective amount of Paris polyphylla root extracellular vesicles or Paris polyphylla stem and leaf extracellular vesicles or combinations thereof obtained by the separation method of claim 1, and a pharmaceutically acceptable carrier.

6. The application of extracellular vesicles from stems and leaves of Paris polyphylla obtained by the method described in claim 1 in the preparation of skin whitening products.

7. The application according to claim 6, characterized in that, The whitening product in question is a cosmetic product.

8. A composition having whitening effects, characterized in that, It contains an effective amount of Paris polyphylla stem and leaf extracellular vesicles obtained by the separation method of claim 1, and a cosmetically acceptable carrier.

9. The composition according to claim 5 or 8, characterized in that, The preparation method includes separation and purification from the extract of Paris polyphylla roots or stems and leaves by a three-stage combination of differential centrifugation, polymer precipitation and electrophoretic dialysis, differential centrifugation combined with ultracentrifugation, sucrose density gradient centrifugation combined with ultracentrifugation, and differential centrifugation combined with size exclusion chromatography.

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