Application of exosome-like nano-particles from purple perilla in preparation of composition with anti-inflammatory effect or anti-oxidation effect

Abstract

The invention relates to the technical field of exosomes, in particular to application of exosome-like nano-particles sourced from perilla frutescens to preparation of a composition with an anti-inflammatory effect or an anti-oxidation effect. According to the application, the exosome-like nano-particles sourced from purple perilla are obtained through extraction, zebra fish is taken as an example, the anti-inflammatory effect and the anti-oxidation effect of the exosome-like nano-particles sourced from purple perilla are analyzed, and the result shows that the exosome-like nano-particles sourced from purple perilla have better anti-inflammatory effect and anti-oxidation effect.

Description

Technical Field

[0001] The present application relates to the technical field of exosomes, and in particular to the use of exosome-like nanoparticles derived from Perilla frutescens in the preparation of a composition with anti-inflammatory or antioxidant effects. Background Art

[0002] Exosomes are multivesicular bodies produced in cells. They are membrane vesicles with a diameter of about 30-150nm and a density of 1.13-1.19g / ml. They have a typical "cup-and-disc" shape. Almost all types of cells in the human body can produce exosomes, with an average of 1,000-10,000 per human cell. Usually, there are 1×10 12 Exosomes are heterogeneous, and even exosomes secreted by the same cell type may have very different functions.

[0003] The main methods for isolating exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, filtration, polymer precipitation, immunoseparation, isolation and screening, etc. However, the mainstream separation method in the literature on exosome research is still ultracentrifugation. Ultracentrifugation separation can accurately and repeatedly obtain exosomes while minimizing the co-purification of protein aggregates and other membrane particles.

[0004] Exosomes exist in almost all tissues, intercellular spaces, and body fluids, including blood, saliva, urine, and breast milk. Exosomes carry proteins, miRNAs, lncRNAs, circRNAs, mRNAs, and their degradation fragments involved in intracellular signal transduction, and participate in the important regulation of cell activities; they have made their mark in the treatment of difficult and complicated diseases such as tumor metastasis, immune regulation mechanisms, disease occurrence and development, Alzheimer's disease, and immune diseases, and are expected to become early diagnostic markers for a variety of diseases. Summary of the invention

[0005] The present application provides a use of exosome-like nanoparticles derived from Perilla frutescens in the preparation of a composition with anti-inflammatory or antioxidant effects.

[0006] Optionally, the perilla-derived exosome-like nanoparticles are capable of reducing the number of neutrophils at the site of inflammation.

[0007] Optionally, the perilla-derived exosome-like nanoparticles can reduce the expression of immune-related genes mpx and lcp1 after zebrafish injury.

[0008] Optionally, the perilla-derived exosome-like nanoparticles can reduce the expression level of inflammatory factors.

[0009] Optionally, the inflammatory factors include ctsla, ctss2.1, c4b, c8a, mpx, lcp1, lect2.1, cts12, vtna, c8b, cfh and ela2.

[0010] Optionally, the perilla-derived exosome-like nanoparticles are capable of reducing H2O2-induced ROS levels.

[0011] Optionally, the perilla-derived exosome-like nanoparticles can increase the SOD activity and GSH content after H2O2 induction.

[0012] In summary, the present application includes at least one of the following beneficial technical effects:

[0013] The separation effect of the perilla-derived exosome-like nanoparticles prepared in the present application is good, and they are typical exosomes, and the size is within the range of exosomes.

[0014] After experimental analysis, this application obtained exosome-like nanoparticles derived from Perilla by extraction, and took zebrafish as an example to analyze the anti-inflammatory and antioxidant effects of exosome-like nanoparticles derived from Perilla. The results showed that exosome-like nanoparticles derived from Perilla have good anti-inflammatory and antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Transmission electron microscopy image of exosome-like nanoparticles derived from Perilla frutescens.

[0016] Figure 2 Schematic diagram of the particle size and concentration of exosome-like nanoparticles derived from Perilla frutescens.

[0017] Figure 3 This is a standard curve created using standards.

[0018] Figure 4 Analysis of the anti-inflammatory efficacy of zebrafish - statistical results of neutrophils in the tail (Figure A is the statistical area of ​​the neutrophil count in the blank group; Figure B is the statistical area of ​​the neutrophil count in the model group; Figure C is the statistical area of ​​the neutrophil count in the sample group (6.25μg / mL); Figure D is the statistical area of ​​the neutrophil count in the sample group (12.5μg / mL); Figure E is the number of neutrophils in each group).

[0019] Figure 5 Analysis of the anti-inflammatory efficacy of zebrafish - transcriptomic analysis and qRT-PCR verification results (Figure A is KEGG enrichment analysis; Figure B is GO functional annotation analysis; Figure C is the volcano plot of differentially expressed genes in the immune system process and response to stimulus pathways in GO functional annotation analysis; Figure D is the qRT-PCR results).

[0020] Figure 6 Whole-mount in situ hybridization results of immune-related genes for zebrafish anti-inflammatory efficacy analysis.

[0021] Figure 7 These are the results of zebrafish anti-inflammatory efficacy analysis-safety evaluation (Figure A is the phenotype of the control group; Figure B is the phenotype of the model group; Figure C is the phenotype of the group treated with perilla-derived exosome nanoparticles; Figure D is the survival rate comparison result; Figure E is the normal rate comparison result; Figure F is the heart rate comparison result; Figure G is the body length comparison result).

[0022] Figure 8 This is the zebrafish antioxidant efficacy analysis - ROS fluorescence staining quantification results (Figure A is the fluorescence image of the blank group fluorescence quantification; Figure B is the fluorescence image of the model group fluorescence quantification; Figure C is the fluorescence image of the sample group fluorescence quantification; Figure D is the results of ROS fluorescence staining quantification in each group).

[0023] Fig. 9 This is an analysis of the antioxidant efficacy of zebrafish - oxidation index detection (Figure A is the detection result of SOD; Figure B is the detection result of CAT; Figure C is the detection result of GSH; Figure D is the detection result of MDA).

[0024] Fig.10 Comparative analysis results of the anti-inflammatory efficacy between exosome-like nanoparticles derived from perilla and exosomes derived from other plants (Figure A is the statistical area of ​​the number of neutrophils in the blank group; Figure B is the statistical area of ​​the number of neutrophils in the model group; Figure C is the statistical area of ​​the number of neutrophils in the perilla-derived exosome-like nanoparticle group (12.5μg / mL); Figure D is the statistical area of ​​the number of neutrophils in the loquat exosome group (12.5μg / mL); Figure E is the statistical area of ​​the number of neutrophils in the osmanthus fragrans exosome group (12.5μg / mL); Figure F is the statistical area of ​​the number of neutrophils in the scabra exosome group (12.5μg / mL); Figure G is the number of neutrophils in each group). DETAILED DESCRIPTION

[0025] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0027] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0028] In the present application, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application, but not excluding other contents.

[0029] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] In the following examples, some of the reagents used are as follows: PBS (E607008) was purchased from Sangon Biotech; 0.45 μm filter membrane (FPV403030) was purchased from Jie Te; uranyl acetate (GZ02625-5) was purchased from Henan Xinrui.

[0032] In the following examples, some of the instruments used are as follows: a transmission electron microscope (HT-7700) was purchased from Hitachi; and a multifunctional microplate reader (Varioskan LUX) was purchased from Thermo.

[0033] In the following examples, SPSS 26.0 statistical software was used for data analysis. The bar graphs are presented as mean ± standard deviation. The differences between the groups were determined by one-way ANOVA, and the least significant difference (LSD) test was performed, with a confidence interval of 95% significance level.

[0034] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.

[0035] Example

[0036] This embodiment provides a kind of exosome-like nanoparticles derived from Perilla frutescens. The sample is a Perilla frutescens plant.

[0037] The specific steps include:

[0038] (I) Sample pretreatment

[0039] (1) Cut the whole plant sample with a scalpel, wash it with distilled water, and absorb the surface moisture with filter paper.

[0040] (2) All sampling instruments were sterilized by high temperature and high pressure in an ultra-clean bench. The samples were chopped into small pieces (1 mm × 1 mm), added with an appropriate amount of 1× PBS, and blended in a juicer.

[0041] (3) Filter the residue with high-temperature sterilized emery cloth, centrifuge the filtrate at 10,000 rpm for 20 min, collect the supernatant after the residue settles, and filter with a 0.45 μm filter membrane to obtain the supernatant, which is the sample treatment solution. Store it at -80°C for future use.

[0042] (II) Extraction of exosomes

[0043] (1) Quickly thaw the sample treatment solution at 37°C.

[0044] (2) Transfer the sample to a new centrifuge tube and centrifuge at 2000 × g and 4°C for 30 min.

[0045] (3) Transfer the supernatant to a new centrifuge tube and centrifuge again at 10,000 × g and 4°C for 45 min to remove larger vesicles.

[0046] (4) Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate.

[0047] (5) Transfer the filtrate to a new centrifuge tube and centrifuge at 100,000 × g and 4°C for 70 min in an ultraspeed rotor.

[0048] (6) Remove the supernatant, resuspend with 10 mL of pre-cooled 1× PBS, select an ultraspeed rotor, and centrifuge again at 100,000 × g and 4°C for 70 min.

[0049] (7) Remove the supernatant and resuspend with 150 μL of pre-cooled 1× PBS to obtain perilla-derived exosome-like nanoparticles, which are then stored at -80°C for later use.

[0050] Test 1

[0051] The following tests were performed on the perilla-derived exosome-like nanoparticles of the example.

[0052] (1) Transmission electron microscopy observation

[0053] The method is as follows: take 10 μL of perilla-derived exosome-like nanoparticles, drop them on a copper grid and precipitate them for 1 min, and absorb the floating liquid with filter paper; continue to take 10 μL of uranyl acetate, drop them on a copper grid and precipitate them for 1 min, and absorb the floating liquid with filter paper; dry them at room temperature for several minutes, perform electron microscopy detection and imaging at 80 kV, and obtain transmission electron microscopy imaging results.

[0054] Test results such as Figure 1 shown.

[0055] Depend on Figure 1 It can be seen that the separation effect of exosome-like nanoparticles derived from Perilla is better, and they all show a spherical or cup-shaped structure with intact membranes, have clear lipid bilayer structural characteristics, and the size is consistent with the range of exosomes, which is highly consistent with the typical exosome morphology.

[0056] (2) Particle size analysis

[0057] The method is as follows: take 5 μL of perilla-derived exosome-like nanoparticles, dilute them 5 times, and use standard samples to perform instrument performance tests before loading exosome samples. Note that gradient dilution is required to avoid clogging of the injection needle by the sample. After completing the sample test, the particle size and concentration information of the exosomes can be obtained.

[0058] The analysis results are as follows Figure 2 shown.

[0059] Depend on Figure 2 It can be seen that the particle size range of exosome-like nanoparticles derived from Perilla is 50-130nm, the average particle size is 79.5nm, and the concentration is 9.83e+10Particles / mL.

[0060] (3) Protein extraction and concentration determination

[0061] The method is as follows: quickly melt the perilla-derived exosome-like nanoparticles at 37°C, and quickly add 5× RIPA lysis buffer, mix well, and lyse on ice for 30 minutes, mixing well during the period. Prepare the standard sample for BCA method protein concentration, and take 5μL of the diluted sample and add it to the BCA mixture, mix well. Incubate at 37°C for 30 minutes, and read the OD value on the microplate reader. 562nm Detect the absorbance value and record it. Calculate the protein concentration of the sample to be tested according to the standard curve.

[0062] The standard curve is as follows Figure 3 As shown.562nm Substitute them into the standard curve for calculation.

[0063] The calculation results show that the protein concentration of the perilla-derived exosome-like nanoparticles (after 4-fold dilution and addition of lysis buffer) is 0.2823 μg / μL, and the protein concentration (original concentration before dilution) is 2.823 μg / μL.

[0064] Test result 2

[0065] The anti-inflammatory efficacy of the exosome-like nanoparticles derived from perilla in the embodiment was analyzed. The detection object used was zebrafish. The details are as follows:

[0066] (1) Tail neutrophil staining and statistics

[0067] Sixty healthy zebrafish embryos 3 hours after fertilization were collected and cultured until 72 hours after fertilization. They were randomly divided into 4 groups, 15 in each group, namely blank group, model group and sample group, and 3 groups were set up in parallel.

[0068] Among them, the blank group was not subjected to tail amputation; the model group had the tail fin of the zebrafish fry cut off with a sterile scalpel; the sample group had the tail fin of the zebrafish fry cut off with a sterile scalpel, and at the same time, 6.25 μg / mL or 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the embodiment were added.

[0069] After 24 hours of incubation, 15 zebrafish larvae from each of the blank group, model group and two sample groups were placed in centrifuge tubes and fixed with 4% PFA overnight. The larvae were rinsed twice with PBST on the second day, 5 minutes each time. 50 μL of Sudan black staining solution was added to each centrifuge tube, and the tubes were stained on a shaker in the dark for 40 minutes, and then eluted with 70% ethanol until the signal was clearly visible. Observe once every 15 minutes during the elution period. After staining, the larvae were decolorized with a decolorizing solution to make the signal easier to observe. The decolorized larvae were fixed in 4% PFA for 20 minutes, and then glycerol was added to permeabilize the larvae overnight. The larvae were placed in glycerol on the second day, observed and photographed under a microscope, and then the neutrophils in the tail were counted in the fixed area.

[0070] The results are as follows Figure 4 As shown. Figure A is the statistical area of ​​the number of neutrophils in the blank group; Figure B is the statistical area of ​​the number of neutrophils in the model group; Figure C is the statistical area of ​​the number of neutrophils in the sample group (6.25μg / mL); Figure D is the statistical area of ​​the number of neutrophils in the sample group (12.5μg / mL); Figure E is the number of neutrophils in each group.

[0071] Depend on Figure 4It can be seen that the exosome-like nanoparticles derived from perilla provided in the present application can effectively reduce the number of neutrophils in zebrafish after tail fin amputation, indicating that the exosome-like nanoparticles derived from perilla provided in the present application have good anti-inflammatory effects.

[0072] (2) Transcriptomic analysis and qRT-PCR verification

[0073] Forty-five healthy zebrafish embryos 3 hours after fertilization were selected and cultured until 72 hours after fertilization. They were randomly divided into three groups, each with 15 animals, namely, a blank group, a model group, and a sample group. The three groups were set up in parallel.

[0074] Among them, the blank group was not subjected to tail amputation; the model group had the tail fin of the zebrafish fry cut off with a sterile scalpel; the sample group had the tail fin of the zebrafish fry cut off with a sterile scalpel, and at the same time, 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the example were added.

[0075] After incubation for 24 h, 15 zebrafish fry from each of the blank group, model group and sample group were placed in 2 mL RNase-free centrifuge tubes, quickly frozen in liquid nitrogen, and then stored at -80°C for transcriptome sequencing.

[0076] The qRT-PCR validation steps are as follows: RNA from zebrafish larvae was extracted using a kit and cDNA was synthesized immediately. qRT-PCR amplified gene fragments using the following parameters: 95°C pre-denaturation for 30 seconds; then 39 cycles, including 95°C denaturation for 10 seconds, 58°C annealing for 30 seconds, and 72°C extension for 30 seconds; and finally a final extension, including 95°C for 10 seconds, 65°C for 5 seconds, and 95°C for 1 minute.

[0077] The results are as follows Figure 5 Figure A is the KEGG enrichment analysis; Figure B is the GO functional annotation analysis; Figure C is the volcano map of differentially expressed genes in the immune system process and response to stimulus pathways in the GO functional annotation analysis; Figure D is the qRT-PCR results.

[0078] Depend on Figure 5It can be seen that KEGG enrichment analysis showed that the differentially expressed genes between the model group and the control group were mainly enriched in the complement and coagulation cascades, systemic lupus erythematosus, PPAR signaling, and antigen processing and presentation pathways. In contrast, the differentially expressed genes between the perilla-derived exosome-like nanoparticles-treated group and the model group showed enrichment in pathways related to fat digestion and absorption and glycerolipid metabolism. GO annotation analysis of differentially expressed genes revealed the enrichment of immune-related biological processes, including immune system processes and responses to stimuli, highlighting the potential regulation of perilla-derived exosome-like nanoparticles on inflammatory responses. qRT-PCR validation of ten immune-related genes, including ctsla, ctss2.1, c4b, c8a, mpx, lcp1, lect2.1, cts12, vtna, c8b, cfh, and ela2, further confirmed that these results were consistent with the transcriptome sequencing data (all of which were reduced in expression compared with the model group after treatment with perilla-derived exosome-like nanoparticles).

[0079] (3) Whole-mount in situ hybridization of immune-related genes

[0080] 90 healthy zebrafish embryos 3 hours after fertilization were taken and cultured until 72 hours after fertilization, and randomly divided into 6 groups, 15 in each group, namely blank group, model group and sample group, and 3 groups were set up in parallel. The immune-related genes were mpx gene and lcp1 gene.

[0081] Among them, the blank group was not subjected to tail amputation; the model group had the tail fin of the zebrafish fry cut off with a sterile scalpel; the sample group had the tail fin of the zebrafish fry cut off with a sterile scalpel, and at the same time, 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the example were added.

[0082] After 24 hours of incubation, 15 zebrafish larvae from each of the blank group, model group, and sample group were placed in centrifuge tubes and fixed with 4% PFA overnight. The next day, the larvae were rinsed twice with PBST, 5 minutes each time. 50 μL of Sudan black staining solution was added to each centrifuge tube, and the tubes were stained on a shaker in the dark for 40 minutes, and then eluted with 70% ethanol until the signal was clearly visible. Observe once every 15 minutes during the elution period. After staining, the larvae were decolorized with a decolorizing solution to make the signal easier to observe, and then stored in methanol.

[0083] The hybridization steps are as follows:

[0084] Day 1: The zebrafish larvae placed in methanol were washed with PBST (PBS + 1% Tween) gradients, digested with proteinase K (10 μg / mL) for 30 min, washed 4 times with PBST, added with 1 mL of pre-prepared HYB, and placed in a hybridization box at 60°C for 2-5 hours. Then, the prepared hybridization solution containing the probe was added to the centrifuge tube containing the zebrafish larvae, and then placed in a hybridization box at 65°C overnight.

[0085] On the second day, the hybridized fry were taken out of the hybridization box and the probe was aspirated. After a highly stringent gradient wash, they were placed in a blocking buffer and incubated at room temperature for 3-4 hours. Subsequently, anti-digoxigenin antibody was added to the blocking buffer at a dilution of 1:10000 and incubated overnight at -4°C in a shaker.

[0086] On the third day, after washing away the antibody-containing solution, color development was performed, followed by gradient decolorization with methanol until the signal was clearly visible, and 4% PFA was added for fixation for 20 minutes. After fixation, glycerol was added and shaken at 4°C overnight.

[0087] On the fourth day, after permeabilization was completed, the fry were observed under a microscope and photographs were taken to record the experimental results.

[0088] Test results such as Figure 6 As shown. Among them, PELNs are exosome-like nanoparticles derived from Perilla frutescens.

[0089] Depend on Figure 6 It can be seen that perilla-derived exosome-like nanoparticles can reduce the expression of immune-related genes mpx and lcp1 in zebrafish after injury.

[0090] (4) Safety evaluation

[0091] Forty-five healthy zebrafish embryos 3 hours after fertilization were selected and cultured until 72 hours after fertilization. They were randomly divided into three groups, each with 15 animals, namely, a blank group, a model group, and a sample group. The three groups were set up in parallel.

[0092] Among them, the blank group was not subjected to tail amputation; the model group had the tail fin of the zebrafish fry cut off with a sterile scalpel; the sample group had the tail fin of the zebrafish fry cut off with a sterile scalpel, and at the same time, 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the example were added.

[0093] After 24 hours of incubation, 15 zebrafish fry were taken from each of the blank group, model group and sample group, and the survival rate, normal rate, heart rate, and basic developmental indicators such as body length were recorded using a stereomicroscope, and photographs were taken to measure them.

[0094] The results are as follows Figure 7Figure A is the phenotype of the control group; Figure B is the phenotype of the model group; Figure C is the phenotype of the group treated with perilla-derived exosome-like nanoparticles; Figure D is the comparison result of survival rate; Figure E is the comparison result of normal rate; Figure F is the comparison result of heart rate; and Figure G is the comparison result of body length.

[0095] Depend on Figure 7 It can be seen that perilla-derived exosome-like nanoparticles did not change the zebrafish phenotype, survival rate, normal rate, heart rate and body length, and had no obvious toxicity.

[0096] Test result three

[0097] The antioxidant efficacy of the exosome-like nanoparticles derived from perilla in the embodiment was analyzed. The detection object used was zebrafish. The details are as follows:

[0098] (1) Quantification of ROS fluorescence staining

[0099] Forty-five healthy zebrafish embryos 3 hours after fertilization were randomly divided into three groups, each with 15 embryos, namely a blank group, a model group and a sample group, and the three groups were set up in parallel.

[0100] The blank group was not stimulated; the model group was stimulated with 30% hydrogen peroxide; the sample group was stimulated with 30% hydrogen peroxide, and 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the embodiment were added.

[0101] Incubate until 96 hours after fertilization, and change the incubation solution daily. Use DCFH-DA staining solution at 1:2000, incubate at 28°C for 20 minutes, wash, and observe the fluorescence intensity of zebrafish larvae under dark conditions using a stereo fluorescence microscope and take photos.

[0102] The results are as follows Figure 8 As shown. Figure A is the fluorescence image of the blank group fluorescence quantification; Figure B is the fluorescence image of the model group fluorescence quantification; Figure C is the fluorescence image of the sample group fluorescence quantification; Figure D is the results of ROS fluorescence staining quantification in each group.

[0103] Depend on Figure 8 It can be seen that exosome-like nanoparticles derived from Perilla can reduce the level of ROS induced by H2O2.

[0104] (2) Oxidation index detection

[0105] 150 healthy zebrafish embryos 3 hours after fertilization were randomly divided into 3 groups, 50 in each group, namely blank group, model group and sample group, and the 3 groups were set up in parallel.

[0106] The blank group was not stimulated; the model group was stimulated with 30% hydrogen peroxide; the sample group was stimulated with 30% hydrogen peroxide, and 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the embodiment were added.

[0107] Incubate until 96 hours after fertilization, and change the incubation solution daily. 30 fixed numbers of each group were placed in 2 mL RNase-free centrifuge tubes, quickly frozen in liquid nitrogen, and then pre-cooled 0.9% sodium chloride solution was added to the centrifuge tubes and homogenized. Centrifuge at 2500r / min at 4°C for 10 minutes, and the supernatant was collected and placed at 4°C for use. Use the BCA protein assay kit to make a standard curve and calculate the protein concentration of the sample. According to the instructions of the SOD, CAT, GSH, and MDA kits, perform corresponding experiments and calculations.

[0108] The results are as follows Fig. 9 As shown. Figure A is the detection result of SOD; Figure B is the detection result of CAT; Figure C is the detection result of GSH; Figure D is the detection result of MDA.

[0109] Depend on Fig. 9 It can be seen that exosome-like nanoparticles derived from Perilla can increase the SOD activity and GSH content after H2O2 induction.

[0110] Test result 4

[0111] The exosome-like nanoparticles derived from Perilla obtained in the present embodiment were compared with exosomes derived from other plants for anti-inflammatory efficacy analysis. The test object used was zebrafish. The details are as follows:

[0112] (1) Tail neutrophil staining and statistics

[0113] A total of 90 healthy zebrafish embryos at 3 hours after fertilization were collected and cultured until 72 hours after fertilization. They were randomly divided into 6 groups, with 15 in each group, namely, blank group, model group, sample group (PELNs), control group 1 (loquat exosomes), control group 2 (Daoshouxiang exosomes), control group 1 (Scutellaria baicalensis exosomes), and 3 groups were set up in parallel.

[0114] Among them, the blank group was not tail-cut; the model group used a sterile scalpel to cut off the tail fin of the zebrafish fry; the sample group used a sterile scalpel to cut off the tail fin of the zebrafish fry, and 12.5 μg / mL of the perilla-derived exosome-like nanoparticles of the embodiment were added; the control group 1 used a sterile scalpel to cut off the tail fin of the zebrafish fry, and 12.5 μg / mL of loquat exosomes were added; the control group 2 used a sterile scalpel to cut off the tail fin of the zebrafish fry, and 12.5 μg / mL of the hand-fragrant exosomes were added; the control group 3 used a sterile scalpel to cut off the tail fin of the zebrafish fry, and 12.5 μg / mL of the scattered bamboo root seven exosomes were added.

[0115] After 24 hours of incubation, 15 zebrafish larvae from each of the blank group, model group, sample group and three control groups were placed in centrifuge tubes and fixed with 4% PFA overnight. The larvae were rinsed with PBST twice the next day for 5 minutes each time. 50 μL of Sudan black staining solution was added to each centrifuge tube, and the tubes were stained on a shaker in the dark for 40 minutes, and then eluted with 70% ethanol until the signal was clearly visible. Observe once every 15 minutes during the elution period. After staining, the larvae were decolorized with a decolorizing solution to make the signal easier to observe. The decolorized larvae were fixed in 4% PFA for 20 minutes, and then glycerol was added to permeabilize the larvae overnight. The larvae were placed in glycerol the next day, observed and photographed under a microscope, and then the neutrophils in the tail were counted in the fixed area.

[0116] The results are as follows Fig.10 As shown. Among them, Figure A is the statistical area of ​​the number of neutrophils in the blank group; Figure B is the statistical area of ​​the number of neutrophils in the model group; Figure C is the statistical area of ​​the number of neutrophils in the perilla-derived exosome-like nanoparticles group (12.5μg / mL); Figure D is the statistical area of ​​the number of neutrophils in the loquat exosome group (12.5μg / mL); Figure E is the statistical area of ​​the number of neutrophils in the scutellaria exosome group (12.5μg / mL); Figure F is the statistical area of ​​the number of neutrophils in the scatted bamboo root seven exosome group (12.5μg / mL); Figure E is the number of neutrophils in each group.

[0117] Depend on Fig.10 It can be seen that, in comparison, the exosome-like nanoparticles derived from perilla provided in the present application can effectively reduce the number of neutrophils in zebrafish after tail fin amputation, while the exosomes from other plant sources in the control group can hardly reduce the number of neutrophils in zebrafish after tail fin amputation, indicating that the exosome-like nanoparticles derived from perilla provided in the present application have good anti-inflammatory effects.

[0118] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of exosome-like nanoparticles derived from Perilla frutescens in the preparation of a composition with anti-inflammatory or antioxidant effects.

2. The use according to claim 1, characterized in that: The perilla-derived exosome-like nanoparticles can reduce the number of neutrophils in inflammatory sites.

3. The use according to claim 1, characterized in that: The exosome-like nanoparticles derived from Perilla frutescens can reduce the expression of immune-related genes after zebrafish injury. mpx and lcp1 expression.

4. The use according to claim 1, characterized in that: The perilla-derived exosome-like nanoparticles can reduce the expression level of inflammatory factors.

5. The use according to claim 4, characterized in that: The inflammatory factors include ctsla , ctss2.1 , c4b , c8a , mpx , lcp1 , lect2.1 , cts12 , vtna , c8b , cfh and ela2 .

6. The use according to claim 1, characterized in that: The perilla-derived exosome-like nanoparticles can reduce the level of ROS induced by H2O2.

7. The use according to claim 1, characterized in that: The perilla-derived exosome-like nanoparticles can increase the SOD activity and GSH content after H2O2 induction.

Images