Preparation method of Japanese cayratia herb outer vesicles and application of Japanese cayratia herb outer vesicles in skin repair

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of Japanese cayratia herb outer vesicles and application of the Japanese cayratia herb outer vesicles in skin repair. The preparation method comprises the following steps: mixing cayratia japonica and a PBS solution, juicing, filtering, sequentially carrying out differential centrifugation on the filtrate, extracting by combining polyethylene glycol precipitation and a size exclusion method, and accurately controlling the raw material ratio, the molecular weight of a precipitation reagent polyethylene glycol, the centrifugation condition, the temperature, the size exclusion method and other parameter settings to obtain the cayratia japonica extract. The prepared cayratia japonica extracellular vesicles are clear in outline, complete in structure and high in content. The extracted cayratia japonica extracellular vesicles are further studied, and it is found that the cayratia japonica extracellular vesicles can successfully contribute to migration of human embryo skin fibroblasts (CCC-ESF) on the cellular level and can also have the effect of accelerating wound healing on animal skin tissue.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a preparation method of plant-derived Cayratia japonica extracellular vesicles and their application in anti-inflammatory repair of skin wounds. Background Art

[0002] Extracellular vesicles are a class of nanoscale membranous vesicles secreted by cells, widely present in animals, plants, and microorganisms. They can naturally carry various biomolecules, such as proteins, lipids, DNA, and RNA. In recent years, the application potential of extracellular vesicles in the medical and cosmetic fields has attracted extensive attention. Plant extracellular vesicles are a special type of vesicles secreted by plant cells, and their structure is similar to that of animal extracellular vesicles. Their natural origin endows them with excellent biocompatibility and safety.

[0003] Cayratia japonica, also known as five-leaf raspberry or Cayratia herb, belongs to the Vitaceae family. Relevant studies have shown that Cayratia japonica is rich in various bioactive components, and Cayratia japonica cream (an extract of Cayratia japonica) has certain wound repair functions. Currently, there is no report on the extracellular vesicles derived from Cayratia japonica, and there is a technical gap in the research and development of Cayratia japonica extracellular vesicles in the field of repair.

[0004] Apigenin is a flavonoid compound widely present in plants such as celery, chamomile, and citrus fruits, with multi-target biological activities. For wound healing, apigenin promotes epithelial cell migration, fibroblast proliferation, and orderly deposition of collagen by activating the PI3K / Akt and Wnt / β-catenin pathways, while inhibiting TGF-β / Smad signal transduction to reduce scar formation. Its unique dual regulatory mechanism can not only accelerate wound closure (by upregulating MMP-9 to promote tissue remodeling), but also inhibit excessive ECM accumulation (by downregulating TIMP-1), thus achieving high-quality tissue repair (https: / / doi.org / 10.1016 / j.jafr.2025.101816.).

[0005] As an ω-6 essential fatty acid, linoleic acid (LA) promotes wound healing through a multi-stage dynamic regulation mechanism: in the early stage, it accelerates the initiation of inflammation by enhancing neutrophil migration (such as H2O2 release and chemotaxis mediated by chemokine CINC-2αβ), and at the same time upregulates L-selectin to promote leukocyte-endothelial cell adhesion; subsequently, it reduces the expression of pro-inflammatory factors such as IL-1β and IL-6 by inhibiting the NF-κB pathway to prevent excessive inflammation, and promotes the proliferation of fibroblasts and keratinocytes by activating the AP-1 transcription factor. In the proliferation phase, LA induces angiogenesis by upregulating VEGF and ANGPT-2, enhances the expression of vinculin to promote fibroblast migration and collagen synthesis, and forms functional granulation tissue; its antioxidant effect relieves oxidative damage by neutralizing ROS and protects the newly formed tissue. In addition, LA regulates cell migration through the signal pathway mediated by the GPR40 receptor (such as the mTOR / Akt / PKCζ cascade), and metabolizes to generate lipid mediators (such as PGE2) to coordinate inflammation resolution and tissue remodeling, and finally optimizes extracellular matrix remodeling by balancing the activities of MMPs / TIMPs, accelerating wound closure and functional recovery.

[0006] Extracellular vesicles carry various biomolecules, such as proteins, lipids, DNA, and RNA, etc. Whether they can be sufficiently enriched with small molecule compounds is doubtful and technically difficult. The present invention finds that the Cayratia japonica extract (Cayratia japonica paste) lacks linoleic acid (LA), and the content of apigenin is also relatively low; its wound repair function is also relatively limited.

[0007] In addition, in the relevant research on the extraction of extracellular vesicles, their separation and purification are still the key steps restricting their research and application. The present invention finds that the PEG precipitation method makes extracellular vesicles aggregate and precipitate but with low purity. Considering using the SEC method to improve the purity, however, at present, for extracellular vesicles derived from animal cells, the SEC (size exclusion protocol) is mainly used for purification. However, plant extracellular vesicles often have higher sugar modifications, more cellulose, and may contain contaminants such as pectin that are different from animal cell supernatants. Moreover, the contents of these components in different plants vary greatly. Therefore, it is unknown whether the SEC chromatographic column can be used for effective purification during the extraction of Cayratia japonica extracellular vesicles, and generally the purification effects of different chromatographic columns are not significantly different. The present invention uses CaptoCore700, Sanarose6B, and G-25 chromatographic columns for purification, and the effect is not ideal. Summary of the Invention

[0008] The object of the present invention is to provide a method for preparing Cayratia japonica extracellular vesicles and their application in skin repair, verify that Cayratia japonica extracellular vesicles have the function of anti-inflammatory repair of skin wounds, and their application in the preparation of skin repair products from plant-derived materials. The Cayratia japonica extracellular vesicles in this article are the Cayratia japonica extracellular vesicles.

[0009] To achieve the above object, in the first aspect of the present invention, the following technical solution is adopted: A method for preparing Cayratia japonica extracellular vesicles, the method for preparing Cayratia japonica extracellular vesicles includes the following steps:

[0010] Step 1: Take Cayratia japonica, soak it in PBS, juice it with a blender, collect the supernatant after differential centrifugation to separate the solid and liquid, and obtain Cayratia japonica juice;

[0011] Step 2: The Cayratia japonica juice is pretreated, centrifuged, filtered, and precipitated with PEG, then the supernatant is discarded, and the precipitate is crude extracellular vesicles. After resuspending the precipitate with PBS, a resuspended crude extract is obtained; where the molecular weight of PEG is 9000 - 10000;

[0012] Step 3: Treat the resuspended crude extract with an SEC chromatographic column to purify and obtain Cayratia japonica extracellular vesicles.

[0013] By adopting the above technical solution, when preparing Cayratia japonica extracellular vesicles, after Cayratia japonica is soaked and broken, the effective substances inside the cell wall are released, and then the juice containing extracellular vesicles is obtained by differential centrifugation. After the Cayratia japonica juice is treated in Step 2, the obtained Cayratia japonica extracellular vesicles contain more impurities. After further treatment in Step 3, purified Cayratia japonica extracellular vesicles are obtained to improve their repair effect on skin wounds.

[0014] Step 1 includes the following steps: Take Cayratia japonica and soak it in a PBS solution according to a material-liquid ratio of 1 g: 15 - 25 mL, juice it with a blender, conduct preliminary filtration, and centrifuge the filtrate under the condition of 0 - 4 °C by differential centrifugation, filter, and collect the supernatant; the specific steps of the differential centrifugation are as follows: Centrifuge at 500 g - 1500 g for 10 min - 30 min; Centrifuge at 2000 g - 6000 g for 20 min - 30 min; Centrifuge at 8000 g - 12000 g for 30 min - 60 min, take the supernatant, and this supernatant is Cayratia japonica juice; where the Cayratia japonica is fresh Cayratia japonica stems and leaves or leaves, fresh Cayratia japonica whole herb; dried Cayratia japonica whole herb, stems and leaves or leaves.

[0015] By adopting the above technical solution, after centrifuging the filtrate at 500 g to 1500 g for 10 min to 30 min, macromolecular precipitation occurs, and the Cayratia japonica extracellular vesicles remain in the supernatant; by further increasing the centrifugation speed and centrifuging at 2000 g to 6000 g for 20 min to 30 min, impurities with slightly smaller particle sizes precipitate, and after filtration, the Cayratia japonica extracellular vesicles still remain in the supernatant; then, by centrifuging at 8000 g to 12000 g for 30 min to 60 min, large particle precipitation is further removed at this time, and Cayratia japonica extracellular vesicles with smaller molecular weights are left in the supernatant, and the supernatant is collected again.

[0016] Optionally, different molecular weight PEG precipitation reagents are used in step two, including: low molecular weight PEG200 - PEG600, medium molecular weight PEG1000 - PEG4000, high molecular weight PEG6000 - PEG12000. In the implementation example, the selected PEG molecular weights are PEG600, PEG4000, and PEG10000. In the present invention, it is found that for the preparation of Cayratia japonica extracellular vesicles, the size of the PEG molecular weight has a great influence on the particle size distribution and concentration of the finally prepared extracellular vesicles, which should be related to the characteristics and composition of Cayratia japonica and Cayratia japonica extracellular vesicles themselves.

[0017] The preparation of the PEG precipitation reagent includes using pure water as the buffer initial solution for the precipitation reagent, taking a certain amount of NaCl to prepare 0.5 M, 1 M, and 2 M NaCl, and taking a certain amount of polyethylene glycol to prepare 10%, 20%, and 30% polyethylene glycol.

[0018] In step two, the Cayratia japonica juice is filtered to obtain the plant cell supernatant; take 50 ml to 1000 ml of the plant cell supernatant and centrifuge it at 1000 g to 3000 g for 10 min to 30 min under the condition of 0 - 4 °C to obtain the centrifuged supernatant; take 50 ml to 1000 ml of the centrifuged supernatant and mix it with the PEG precipitation reagent according to a volume ratio of 1:1 to 1:3, and precipitate it for 12 - 24 h under the condition of 0 - 4 °C, and then centrifuge it at 9000 g to 11000 g for 0.5 - 1 h.

[0019] In step three, take the resuspended crude extract, filter it, add it to the SEC chromatographic column, elute it with PBS, collect several tubes of eluate, and retain the fractions of some tubes to obtain the purified Cayratia japonica extracellular vesicles.

[0020] Optionally, the SEC chromatographic columns of different packing types include: CaptoCore700 packing column, Sanarose6B packing column, G - 25 packing column, Smartarose6FF packing column.

[0021] The contents of active ingredients in Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes and Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li paste obtained by different purification systems were detected and compared by mass spectrometry, including flavonoid apigenin and ω-6 essential fatty acid linoleic acid.

[0022] In the second aspect of the present invention, Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes prepared by the method for preparing Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes described above are provided.

[0023] In the third aspect of the present invention, a composition containing Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes is disclosed.

[0024] In the fourth aspect of the present invention, the use of Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes in the preparation of skin wound repair products is provided.

[0025] Preferably, Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes are used for skin repair and wound healing. The types of repair include, but are not limited to, regeneration of various tissues, granulation tissue hyperplasia, and repair of scar tissue. The products include, but are not limited to, foods, skin care products, health products or drugs. The dosage forms of the products include, but are not limited to, emulsions, capsules, sprays, ointments or injections; the excipients of the products include, but are not limited to, excipients, solubilizers, sustained release agents or binders.

[0026] The verification method for the use of Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li extracellular vesicles for skin repair in the present invention is as follows:

[0027] Through the extraction of Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li exosomes, verification was carried out from two aspects: animal experiments and the cellular level.

[0028] In animal experiments, the treatment concentration of Cayratia japonica (Thunb.) Gagnep. ex W. T. Wang & C. L. Li extracellular vesicles was 1*10

[0025] ,

[0024] , 2 , ,

[0023] , 10 , , 8 ,

[0022] , , ,

[0028] , , ,

[0027] , ,

[0026] particles / mL. Three days before drug administration, 0.1 ml of a Staphylococcus aureus solution with an injection concentration of 10 8 CFU / mL was applied to the surgical wound site on the back of SD rats (the wound area of each group of SD rats was 4 cm 2) An infectious trauma model was induced in SD rats to cause skin wound infections. On the premise of successful model establishment, except for the non-infected control group and the model group, different drugs were applied to the wounds. The results showed that the wound infections of SD rats in the Cayratia japonica Thunb. paste group improved; while the wound infections of SD rats in the Cayratia japonica Thunb. extracellular vesicles group improved significantly, the wound gradually shrank, and the effect was most prominent on the 7th and 12th days, with the best healing effect. In contrast, it could be seen with the naked eye that there were still large areas of infected wounds in the non-infected control group and the model group, and there was a large amount of purulent tissue in the model group (the model group induced wound infections but did not apply any drugs with therapeutic effects). All of the above indicate that the extracellular vesicles of Cayratia japonica Thunb. of the present invention have the use for treating skin scars and repair. Compared with the traditional Cayratia japonica Thunb. paste drug, the extracellular vesicles of Cayratia japonica Thunb. of the present invention have the advantages of faster wound healing effect, inhibition of scar formation, natural raw materials, good tissue compatibility, and low side effects.

[0029] Through cell scratching, the present invention analyzed the migration of cells in the same position under the microscope at different times for each group. After adding 1×10 10 particles / mL extracellular vesicles group, the cell migration speed was faster, the healing area was larger, and the scratch area became smaller after 24 hours, indicating that compared with the control group, the Cayratia japonica Thunb. extracellular vesicles group had a greater tendency to promote cell migration and regeneration, and also had the functions of promoting wound healing and skin repair.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1) During the preparation of Cayratia japonica Thunb. extracellular vesicles, the present invention found that the PEG precipitation method makes extracellular vesicles aggregate and precipitate by changing the solution osmotic pressure, but its purity is low, while SEC uses porous fillers to screen particles of different sizes to achieve the separation of extracellular vesicles, but its yield is low. By exploring the SEC chromatographic column and optimizing the molecular weight of PEG used, the present invention found that the effects of Smartarose6FF chromatographic column and PEG10000 were significantly better than the combinations of other chromatographic columns and PEG molecular weights, which not only solved the PEG purity problem but also solved the SEC yield problem, further improving the extraction efficiency of extracellular vesicles and providing high-quality samples for the functional research and clinical application of extracellular vesicles;

[0032] The present invention extracts by mixing Cayratia japonica Thunb. with PBS solution, juicing, filtering, and sequentially subjecting the filtrate to differential centrifugation, combined polyethylene glycol precipitation, and size exclusion method. By precisely controlling parameters such as the raw material ratio, the molecular weight of the precipitation reagent polyethylene glycol, the centrifugation conditions, the temperature, and the size exclusion method, the prepared Cayratia japonica Thunb. extracellular vesicles have clear outlines, complete structures, and a high content of Cayratia japonica Thunb. extracellular vesicle nanoparticles.

[0033] 2) The present invention provides the therapeutic use of Cayratia japonica extracellular vesicles in anti-inflammatory repair of skin wounds. Experiments have shown that Cayratia japonica extracellular vesicles have obvious improvement effects on skin regeneration and are a natural drug raw material, which solves the side effect problems of existing related drugs to a certain extent; and the nano-scale size of extracellular vesicles makes them easy to be absorbed and the curative effect is better.

[0034] 3) When preparing Cayratia japonica extracellular vesicles in the present invention, Cayratia japonica stems, leaves, and blades are used as raw materials. First, differential centrifugation is used to release Cayratia japonica extracellular vesicles outside the cells, and then the PEG precipitation method and the SEC method are used to finally obtain Cayratia japonica extracellular vesicles with excellent effects and high purity; the selected PEG precipitation combined with the SEC method has the highest content of active ingredients (apigenin and linoleic acid) in the obtained EVs. Brief Description of the Drawings

[0035] Figure 1 is the electron micrograph of Cayratia japonica extracellular vesicles obtained in the present invention;

[0036] Figure 2 is the particle size distribution diagram of Cayratia japonica extracellular vesicles obtained in Example 1 and the comparative example of the present invention;

[0037] Figure 3 is the effect diagram of the migration of CCC-ESF cells after scratching after applying Cayratia japonica extracellular vesicles of the present invention;

[0038] Figure 4 is the schematic diagram of the cell migration rate of CCC-ESF cells after scratching after applying Cayratia japonica extracellular vesicles of the present invention;

[0039] Figure 5 is the photo of the skin lesions and recovery on the back of rats after applying Cayratia japonica extracellular vesicles of the present application;

[0040] Figure 6 is the analysis of the wound healing conditions of each group of SD rats at different healing stages;

[0041] Figure 7 is the H&E section diagram of the skin of rats after applying Cayratia japonica extracellular vesicles of the present application. Detailed Description of the Invention

[0042] The present invention will be specifically introduced below in conjunction with the drawings and specific embodiments.

[0043] In the following examples, the stems and leaves of Cayratia japonica are collected from a planting base in Bozhou, Anhui.

[0044] In the following examples, the Cayratia japonica paste comes from the in-hospital preparation of Nanjing Traditional Chinese Medicine Hospital.

[0045] In the following examples, the raw materials not described in detail are directly purchased from the market.

[0046] Example 1

[0047] A method for preparing Cayratia japonica extracellular vesicles specifically comprises the following steps:

[0048] Step 1: Select 10 g of dry Cayratia japonica stems and leaves, wash them clean with water, soak them in 20 times the amount of 1×PBS at 4°C for 12 hours (the dosage ratio of Cayratia japonica stems and leaves to 1×PBS is 1 g:20 mL), juice them with a blender for 5 min each time, repeat 6 times, preliminarily filter impurities with sterile gauze, collect the preliminary filtrate, and obtain the crude extract filtrate. The crude extract filtrate is subjected to differential centrifugation at 4°C. The differential centrifugation means centrifugation at 1000 g for 20 min; centrifugation at 3000 g for 30 min; centrifugation at 10000 g for 90 min (removing excess cells and impurities during differential centrifugation), take the supernatant, collect the supernatant (which is Cayratia japonica juice), and store it at -80°C.

[0049] Step 2: Filter the supernatant obtained in Step 1 through a filter screen with a pore size of 40 mesh (this is a pretreatment), and collect the plant cell supernatant again (i.e., the filtrate obtained after filtration through the filter screen). Use pure water as the precipitation reagent buffer initial solution, take 29.2 g of NaCl and add 500 ml of pure water to prepare a 1 M NaCl buffer solution, and take 100 g of polyethylene glycol 10000 (PEG10000) plus 500 ml of 1 M NaCl buffer solution to prepare a PEG precipitation reagent. Take 50 ml of the pretreated plant cell supernatant and centrifuge it at 3000 g for 30 min at 4°C to filter out cell debris and large apoptotic bodies. Then, mix 50 ml of the supernatant after centrifugation with the PEG precipitation reagent in an equal volume ratio of 1:1 and precipitate overnight at 4°C. Further, centrifuge the product after overnight treatment at 10000 g for 1 h, discard the supernatant, and the precipitate is the crude extract of plant extracellular vesicles. Add 2 ml of 1×PBS to the precipitate to resuspend it, and obtain the resuspended crude extract.

[0050] Step 3: Subject the obtained resuspended crude extract to size exclusion treatment. The specific steps of the size exclusion enrichment method include filtering the resuspended crude extract using a 0.45 μm filter membrane; after filtration, take out the Smartarose6FF SEC chromatographic column (Changzhou Tiandi Renhe Company) from the refrigerator, fix it vertically, and place an empty beaker below; empty the storage solution in the tube, add 15 mL of 1×PBS to the column until it all flows out, and then load the sample. Sample loading process: Take 8 EP tubes, label them with numbers 1 - 8, add 1 mL of the filtered resuspended crude extract sample along the wall from the top of the SEC chromatographic column. After the sample completely enters the packing and there is no liquid flowing out from the bottom outlet, add 0.5 mL of 1×PBS, and use EP tube 1 to collect the eluate with a collection volume of 0.5 mL; after the previous 0.5 mL of 1×PBS completely enters the exclusion column, add another 0.5 mL of 1×PBS, and use EP tube 2 to collect the eluate with a collection volume of 0.5 mL; repeat the operation 6 more times, and collect a total of 8 tubes of eluate. Collect extracellular vesicles with high recovery rate: Retain the fractions (i.e., eluate) of EP tubes 3, 4, 5, 6, and 7, totaling 2.5 mL; collect them into one collection tube. Finally, obtain the Cayratia japonica extracellular vesicle sample.

[0051] After the collection is completed, add 10 mL of cleaning solution to the SEC chromatographic column, then rinse with 15 mL of 1×PBS, then rinse with 10 mL of 1× storage solution, fill the SEC chromatographic column with 1× storage solution, and store it at 4°C.

[0052] Comparative Example

[0053] Compared with Example 1, change the SEC chromatographic column, replace the SEC chromatographic column with CaptoCore700 chromatographic column (cytiva company), Sanarose6B (Xiamen Sanji Technology) chromatographic column or G-25 chromatographic column (Thermo Fisher Scientific); change the molecular weight of PEG, replace PEG10000 with PEG600, PEG4000. The rest are the same as in Example 1.

[0054] Specifically, it includes CaptoCore700 - PEG600 treatment, CaptoCore700 - PEG4000 treatment, CaptoCore700 - PEG10000 treatment, Sanarose6B - PEG600 treatment, Sanarose6B - PEG4000 treatment, Sanarose6B - PEG10000 treatment, G-25 - PEG600 treatment, G-25 - PEG4000 treatment, G-25 - PEG10000 treatment, Smartarose6FF - PEG600 treatment, Smartarose6FF - PEG4000 treatment, Smartarose6FF - PEG10000 treatment.

[0055] The extracellular vesicles prepared in Example 1 and the comparative example were characterized and analyzed, and the results are as follows:

[0056] 1. Transmission electron microscopy analysis

[0057] The Cayratia japonica Thunb. excludosomes were fixed and examined by transmission electron microscopy using conventional procedures, and observed with a Hitachi TEM transmission electron microscope. The specific steps were as follows: The sample was taken out from -80 °C and melted at 4 °C, vortexed thoroughly, and diluted to a certain multiple with 20 mM Tris-HCl according to the sample concentration for standby. The copper grid was picked out with forceps and placed on a weighing paper. 10 - 20 μL of the sample was taken and vertically suspended and dropped on the copper grid, covering the copper grid. It was left standing for 5 min, and the edge of the copper grid was blotted dry with filter paper. The copper grid was transferred to the filter paper, and the copper grid was placed on the filter paper, and 4% paraformaldehyde solution was vertically suspended and dropped appropriately, and waited for 3 - 5 min. It was observed under a transmission electron microscope to observe the size and shape of the sample.

[0058] The Cayratia japonica Thunb. excludosomes obtained in Example 1 were as shown in the Figure 1 transmission electron microscope field of view. A disc-shaped or cup-shaped nanostructure with a depression on one side was observed. Through the scale bar, the particle size of this structure was found to be about 60 - 200 nm.

[0059] 2. Particle size analysis

[0060] The particle size of the Cayratia japonica Thunb. excludosomes was measured using a Nanocoulter nanoparticle tracking analyzer (NTA) manufactured by Ruixin Smart Manufacturing. The specific steps were as follows: The frozen Cayratia japonica Thunb. excludosome sample was taken out from -80 °C, naturally thawed in a 4 °C refrigerator, vortexed to mix evenly, and 10 μl - 20 μl was aliquoted into a 0.2 ml centrifuge tube and mixed again; the Cayratia japonica Thunb. excludosome sample was diluted 150 times with PBS and directly used for NTA to detect the concentration and particle size of the Cayratia japonica Thunb. excludosomes. In this article, μl is microliter.

[0061] Among them, the particle size distribution and concentration of the Cayratia japonica Thunb. excludosomes obtained in Example 1 and the comparative example after dilution with PBS were as shown in the Figure 2 figure. Most of the particle sizes of the Cayratia japonica Thunb. excludosome particles were between 50 - 200 nm. The particle count of the excludosomes obtained according to the particle size analysis was shown in Table 1. The combined results of the Smartarose 6FF chromatographic column and different molecular weight PEGs were the best, and the yield of PEG10000 and the Smartarose 6FF chromatographic column was the highest. Followed by the CaptoCore700 chromatographic column, the Sanarose6B chromatographic column, and the G-25 chromatographic column in turn.

[0062] Table 1 Total particle count of Cayratia japonica Thunb. excludosomes obtained using SEC chromatographic columns with different molecular weight PEGs and different packing types

[0063]

[0064] Furthermore, the protein concentration of the extracellular vesicles purified by different chromatographic columns was detected, and the purity of the extracted extracellular vesicles was evaluated by the number of particles / protein amount, that is, the higher the ratio, the less the contaminating proteins and the higher the purity. The results are shown in Table 2. The highest purity was obtained with the Smartarose6FF combined with PEG10000 protocol.

[0065] Table 2 Purity of Cayratia japonica extracellular vesicles obtained using SEC columns with different molecular weight PEGs and different packing types (expressed as number of particles / protein amount)

[0066]

[0067] 3. Active ingredient analysis

[0068] The Cayratia japonica paste was purchased from Nanjing Hospital of Traditional Chinese Medicine and is an in-hospital preparation of Nanjing Hospital of Traditional Chinese Medicine. The Cayratia japonica paste is made by mixing Cayratia japonica powder with vaseline. Each 1 g of the paste is equivalent to 0.15 g of the crude drug (Cayratia japonica). To the Cayratia japonica extracellular vesicles and the Cayratia japonica paste corresponding to an equal amount of Cayratia japonica medicinal materials (i.e., comparing the Cayratia japonica extracellular vesicles prepared from a weight of Cayratia japonica with the Cayratia japonica paste containing a weight of the crude drug), 400 μL of extraction solution (methanol:acetonitrile = 3:1, pre-cooled at -40 °C) was added, vortexed for 5 min, sonicated for 15 min, and allowed to stand at 4 °C for 1 h. The sample was taken out and centrifuged for 15 min (12000 rpm, 4 °C), an equal amount was taken, and vacuum concentrated to dryness. 50 μL of 50% methanol aqueous solution (methanol:water = 1:1, v / v) was added for reconstitution, vortexed for 3 min (4 °C, 2000 rpm), centrifuged for 15 min (12000 rpm, 4 °C), and the supernatant was taken for injection analysis. The instrument used was an Oribtrap HF-X mass spectrometer. The metabolites in the Cayratia japonica paste and the Cayratia japonica extracellular vesicles were analyzed, especially the active ingredients apigenin and linoleic acid. The mass spectrometry identification results showed that the ion intensities of apigenin and linoleic acid obtained by the SEC-4 protocol (Example 1) were the highest, indicating that the contents of apigenin and linoleic acid in the extracellular vesicles obtained by this method were the highest. At the same time, for the analysis of the active ingredients in the Cayratia japonica paste, no linoleic acid content was detected in the Cayratia japonica paste, and the detected amount of apigenin was significantly lower than that in the extracellular vesicles purified by the SEC-4 protocol, as shown in Table 3.

[0069] Table 3 Contents of active ingredients in the inclusions of Cayratia japonica extracellular vesicles and Cayratia japonica paste obtained by different extraction methods identified by mass spectrometry

[0070]

[0071] To further verify the repair effects of the Cayratia japonica Thunb. extracellular vesicles prepared in Example 1 on cells and animals, the following tests were also conducted:

[0072] 1. Cell scratch verification of the efficacy of Cayratia japonica Thunb. exosomes

[0073] In the present invention, the promoting effect of Cayratia japonica Thunb. exosomes on cell migration was verified by cell scratch.

[0074] Cell scratch experiment: Collect CCC-ESF cells in the logarithmic growth phase, adjust the cell density to 300,000 / ml, inoculate them in a 6-well plate, with 2 ml of culture medium in each well, and culture them under the conditions of 37 °C, 5%, and CO2. When the CCC-ESF cells in each well grow to 90%, after washing twice with PBS, use the tip of a sterile yellow pipette tip to scratch the confluent cell monolayer, and change the culture medium to DMEM complete culture medium without extracellular vesicles. Add 1×10 10 particles / mL extracellular vesicles to the culture medium of the Cayratia japonica Thunb. exosome group, which is the group added with Cayratia japonica Thunb. exosomes. Specifically, it is divided into a blank group, a control group, a group added with Cayratia japonica Thunb. paste, and a group added with Cayratia japonica Thunb. exosomes, with 3 replicates in each group. Among them, the blank group only contains cells and culture medium, the control group has cells and culture medium, has a scratch operation and does not add any therapeutic substances, the group added with Cayratia japonica Thunb. paste has cells and culture medium, has a scratch operation and adds Cayratia japonica Thunb. paste; the mass of the crude drug in the applied Cayratia japonica Thunb. paste is b, and the amount of the applied Cayratia japonica Thunb. exosomes is also obtained from Cayratia japonica Thunb. with a mass of b through Example 1, and b is obtained through conversion. Observe the migration rate of CCC-ESF cells at 0 h and 24 h after culture, and perform quantitative analysis using the formula. Among them, the migration rate = (A0 - An) / A0 × 100%, where A0 is the initial scratch area and An is the remaining area of the scratch measured after 24 h.

[0075] Among them, the results of the cell experiment are as Figure 3 and Figure 4 shown: The effect of Cayratia japonica Thunb. extracellular vesicles on the migration of CCC-ESF cells was detected through an in vitro cell migration experiment. The migration of cells in each group at the same position under the microscope at different times (0 h, 24 h) was analyzed by area comparison. Among them, the migration rates of the control group, the Cayratia japonica Thunb. paste group, and the Cayratia japonica Thunb. extracellular vesicle group after 24 h were 35.6%, 46.6%, and 66.3% respectively. Figure 3 and Figure 4 In, 0 h represents 0 h after scratching in the control group, 24 h control group represents 24 h after scratching in the control group, 24 h Cayratia japonica Thunb. extracellular vesicle group represents 24 h after scratching in the group added with Cayratia japonica Thunb. exosomes, and 24 h Cayratia japonica Thunb. paste group represents 24 h after scratching in the group added with Cayratia japonica Thunb. paste.

[0076] The results showed that compared with the control group at 0 h, it was visible to the naked eye that the cell migration of the control group at 24 h was not obvious and the cell migration ability was slow; compared with the control group at 0 h and the Cayratia japonica extracellular vesicle group at 24 h, the migration of the Cayratia japonica extracellular vesicle group at 24 h was obvious, which could significantly promote the migration of CCC-ESF cells and significantly improve the cell migration ability with a significant difference (P < 0.01); compared with the 24 h control group and the 24 h group with added Cayratia japonica paste respectively and the 24 h Cayratia japonica extracellular vesicle group, the extracellular vesicle group had a more obvious trend of promoting cell migration. It was shown that the Cayratia japonica extracellular vesicle group had the function of promoting fibroblast migration, wound healing and skin repair (P < 0.01).

[0077] 2. Animal experiment on wound healing of Cayratia japonica extracellular vesicles

[0078] In the animal experiment of the present invention, the method was as follows: 40 SD rats weighing about 200 g were prepared, the hair was wiped with sterile normal saline, and the hair on the back of the SD rats was shaved off. At the same time, the remaining hair on the back of the rats was thoroughly removed with 75% alcohol. Wounds with a diameter of 2 cm * 2 cm were cut on both sides of the rat spine. Three days before drug administration, a model of Staphylococcus aureus suspension was established (injecting 0.1 ml of Staphylococcus aureus solution with a concentration of 10 8 CFU / mL to the surgical wound site on the back of the SD rats, and the surgical wound site was the aforementioned 2 cm * 2 cm wound surface). The experiment was divided into 4 groups: the first group was the non-infected control group with only the wound surface and no Staphylococcus aureus infection; the second group was the model group, which was the group with the wound surface infected by Staphylococcus aureus without treatment; the third group was the Cayratia japonica paste treatment group, and an appropriate amount of paste was taken and applied to cover the infected wound surface; the fourth group was the Cayratia japonica extracellular vesicle treatment group, and the treatment dose of the Cayratia japonica extracellular vesicles was 1 * 10 10 particles (the concentration was 5 * 10 10 particles / mL and the volume was 200 ul), where particles referred to the number of Cayratia japonica extracellular vesicle nanoparticles, and particles / mL referred to the number of Cayratia japonica extracellular vesicle nanoparticles contained in each mL of the Cayratia japonica extracellular vesicle sample obtained in Example 1; the mass of the crude drug in the applied Cayratia japonica paste was c, and the amount of the applied Cayratia japonica extracellular vesicles was also obtained from Cayratia japonica with a mass of c through Example 1, and c was obtained through conversion.

[0079] After the operation of the rat wounds, 150 ul of normal saline or bacterial solution was dropped onto the wound surfaces of the corresponding groups according to the grouping (150 ul of normal saline corresponded to the non-infected control group, and the Staphylococcus aureus solution corresponded to the model group, the Cayratia japonica extracellular vesicle treatment group, and the Cayratia japonica paste treatment group. Sterile gauze of the corresponding size was attached to the wound surface, and at the same time, medical adhesive tape was used to fix it on the surface of the dressing film.

[0080] After the operation, each rat was raised in an independent ventilation cage. An obvious infection focus could form at the wound site 72 hours after the operation, successfully establishing a rat wound infection model while ensuring the normal survival of the rats.

[0081] On the 3rd, 7th, and 12th days, the infection conditions on the wound surfaces of each group were observed, and the changes in the wound surface size were visually estimated. After rinsing the wounds with normal saline every day, the same dose of Cayratia japonica Thunb. cream and Cayratia japonica Thunb. extracellular vesicles as on the 0th day were applied to the corresponding treatment groups. Moreover, 3 rats in each group were sacrificed every time to obtain wound tissues.

[0082] Among them, the results of the animal experiment are as Figure 5 and Figure 6 shown: The intuitive wound healing conditions of the non-infected control group monitoring the wound surface, the non-treated model group with an infected wound surface, the Cayratia japonica Thunb. cream treatment group, and the Cayratia japonica Thunb. extracellular vesicle treatment group were observed on the 0th, 3rd, 7th, and 12th days. The wound surface area of the Cayratia japonica Thunb. extracellular vesicle treatment group was the largest at 0d, but the smallest at 12d. The results showed that among the 4 groups, the wound healing state of the model group was the worst, and the other groups all had a certain healing effect. The wound healing effects were in the order of the Cayratia japonica Thunb. extracellular vesicle group being better than the Cayratia japonica Thunb. cream treatment group being better than the non-treated model group with an infected wound surface, and the effect was the most significant on the 7th and 12th days. And the Cayratia japonica Thunb. extracellular vesicles could significantly promote wound healing and accelerate skin repair. Further, the ImageJ software was used to analyze the wound healing conditions of the SD rats in each group at different healing stages, and the wound healing areas of each group were calculated. The results showed that in different healing cycles, compared with any other group, the wound healing area of the model group at different stages was significantly the lowest, especially prominent on the 7th day, and the lowest compared with the Cayratia japonica Thunb. extracellular vesicle group, with a highly significant difference (P < 0.0001). At the same time, comparing the wound healing areas of the Cayratia japonica Thunb. cream group and the Cayratia japonica Thunb. extracellular vesicle group at different stages, the healing area of the Cayratia japonica Thunb. cream group from the 3rd to the 12th day was lower than that of the Cayratia japonica Thunb. extracellular vesicle group on each day. The Cayratia japonica Thunb. extracellular vesicle group could significantly promote wound healing from the 3rd to the 12th day (P < 0.01), and the wound healing on the 12th day in the Cayratia japonica Thunb. extracellular vesicle group was extremely significantly increased (P < 0.001). Through statistics, on the 12th day, the wound area of the Cayratia japonica Thunb. extracellular vesicle group decreased by 81% compared with the model group, while the Cayratia japonica Thunb. cream group only decreased by 58% compared with the model group. The Cayratia japonica Thunb. extracellular vesicle group had the best wound healing effect in the wound healing areas at different stages.

[0083] 3. Tissue HE staining experiment

[0084] The HE staining method is one of the most commonly used staining methods in paraffin sections, applicable to the staining of any animal tissue. After staining, the cell nuclei, calcium salts, mucus, etc. are blue, and the dye eosin makes the various components of the cytoplasm show different shades of pink, which can be used to observe the basic morphology and pathological lesions of animal tissues.

[0085] Take the skin tissue specimens of the back wounds of the experimental rats on the 7th day in the animal experiment of treating infectious wounds with Cayratia japonica extracellular vesicles, fix them with 4% paraformaldehyde, prepare paraffin sections to be stained, and perform the following specific experimental steps:

[0086] Deparaffinize the sections routinely to water. The steps are as follows: sequentially place the sections in xylene I for 10 minutes, xylene II for 10 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and wash with distilled water.

[0087] Immerse in hematoxylin aqueous solution for 3 minutes and wash with water, differentiate with hydrochloric acid alcohol for 2 seconds, rinse with running water for 1 hour, then place in distilled water for a moment, dry after washing. Stain with alcoholic eosin staining solution for 6 seconds, wash with water for 3 seconds, and dehydrate with 100% ethanol for 2 seconds.

[0088] Dehydrate and mount the sections. Sequentially place the sections in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes for dehydration and clearing. Take out the sections from xylene, air-dry or dry them slightly, and mount them with neutral balsam. Observe the sections under an optical microscope and obtain images.

[0089] Among them, the results of H&E staining are as Figure 7 shown. Compare the HE staining of tissue sections in each group (×400 and ×1000 represent magnification factors). In the H&E staining sections of the back skin of the rats in the non-infected control group, the structures of each layer of the skin are relatively clear, and the structures of the epidermis and dermis are normal. However, there are obvious inflammatory cell infiltrations between cells in the model group, indicating that the modeling is relatively successful at the cellular level. Compared with the non-infected control group, visible new granulation tissue is generated in the back skin lesions of the rats in the Cayratia japonica paste group. Then, compare the Cayratia japonica paste group with the Cayratia japonica extracellular vesicle group. Compared with the Cayratia japonica paste treatment group, it is obvious that the Cayratia japonica extracellular vesicle group shows more angiogenesis and new granulation tissue (the pink part is new blood vessels and granulation tissue), indicating that the Cayratia japonica extracellular vesicle group has a better effect on skin improvement.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A preparation method of Cayratia japonica exosomes, characterized in that, The method for preparing the Cayratia japonica exosomes comprises the following steps: Step 1: Take Cayratia japonica, soak it in PBS, juice it with a blender, and collect the supernatant after differential centrifugation to separate the solid and liquid, obtaining Cayratia japonica juice; Step 2: The Cayratia japonica juice is pretreated, centrifuged, filtered, and precipitated with PEG, and then the supernatant is discarded. The precipitate is the crude extracellular vesicles. The precipitate is resuspended with PBS to obtain a resuspended crude extract; wherein the molecular weight of PEG is 9000 - 10000; Step 3: Treat the resuspended crude extract with an SEC chromatographic column to purify the Cayratia japonica extracellular vesicles.

2. The preparation method of Cayratia japonica outer vesicles according to claim 1, wherein, Step 1 includes the following steps: Take Cayratia japonica and soak it in a PBS solution according to a material-liquid ratio of 1 g: 15 - 25 mL, juice it with a blender, and perform preliminary filtration. The filtrate is subjected to differential centrifugation at 0 - 4 °C, filtered, and the supernatant is collected; the specific steps of the differential centrifugation are as follows: Centrifuge at 500 g - 1500 g for 10 min - 30 min; centrifuge at 2000 g - 6000 g for 20 min - 30 min; centrifuge at 8000 g - 12000 g for 30 min - 60 min, and take the supernatant, which is the Cayratia japonica juice; the Cayratia japonica is fresh Cayratia japonica stems and leaves or leaves, fresh Cayratia japonica whole herb; dried Cayratia japonica whole herb, stems and leaves or leaves.

3. The method for preparing the extracellular vesicles of Raspberry according to claim 1, characterized in that: In Step 2, the Cayratia japonica juice is filtered to obtain a plant cell supernatant; take 50 ml - 1000 ml of the plant cell supernatant and centrifuge it at 1000 g - 3000 g for 10 min - 30 min at 0 - 4 °C to obtain the centrifuged supernatant; take 50 ml - 1000 ml of the centrifuged supernatant and mix it with a PEG precipitation reagent according to a volume ratio of 1:1 - 1:3, and precipitate it at 0 - 4 °C for 12 - 24 h, and then centrifuge it at 9000 g - 11000 g for 0.5 - 1 h.

4. The preparation method of Cayratia japonica outer vesicles according to claim 1, characterized in that In Step 3, take the resuspended crude extract, filter it, add it to an SEC chromatographic column, elute it with PBS, collect several tubes of eluate, and retain the fractions of some tubes to obtain the purified Cayratia japonica extracellular vesicles; the material of the SEC chromatographic column is Smartarose6FF.

5. The Cayratia japonica exosomes prepared by the method for preparing Cayratia japonica exosomes according to any one of claims 1 - 4.

6. A composition comprising the Cayratia japonica exosomes according to claim 5.

7. The application of the Cayratia japonica exosomes according to claim 5 in the preparation of a skin repair product.

8. Use of the Cayratia japonica exosomes according to claim 7 in the preparation of a skin repair product, characterized in that, The Cayratia japonica exosomes are used for skin repair and wound healing.

9. Use of the Cayratia japonica exosomes according to claim 7 in the preparation of a skin repair product, characterized in that, The product is a food, skin care product, health product or drug.

10. Use of the Cayratia japonica exosomes according to claim 7 in the preparation of a skin repair product, characterized in that, The dosage form of the product is an emulsion, capsule, spray, ointment or injection; the excipients of the product include excipients, cosolvents, sustained release agents or binders.

Images