Preparation method of forsythia suspensa leaf exosome and application thereof

Abstract

The application provides a preparation method of forsythia suspensa leaf exosome and application thereof, and the application can effectively remove cell debris and macromolecular impurities, and precisely separate target exosome bands through discontinuous sucrose gradient, so that high-purity forsythia suspensa leaf exosome is obtained; the application is based on forsythia suspensa leaves which are geographical indication agricultural products in Shanxi Province, exosomes with anti-aging potential are extracted from the forsythia suspensa leaves, and structural characteristics of the exosomes are identified, biological activity of the exosomes is verified, a theoretical basis is provided for mining new candidate drugs with multiple biological activities from the forsythia suspensa leaves, and research value and development potential of the forsythia suspensa leaves are improved, a theoretical basis is provided for development and utilization of the forsythia suspensa leaves in the direction of human health, and the application has important significance for deep development of forsythia suspensa leaf resources and improvement of medicinal value.

Description

Technical Field

[0001] This invention relates to the field of plant natural exosome extraction technology, and in particular to a method for preparing exosomes from Forsythia suspensa leaves and their application. Background Technology

[0002] In the long course of evolution in nature, aging is an unavoidable physiological process for all organisms. According to the World Health Organization, by 2050, the global population aged 60 and over is projected to exceed 2.1 billion. With aging, the incidence of many age-related diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, and diabetes, gradually increases. Extending healthy lifespan and mitigating the negative impacts of aging have become key focuses of anti-aging research in recent years. Numerous studies have demonstrated that delaying aging and extending healthy lifespan are feasible in commonly used animal models such as mice, fruit flies, and *Caenorhabditis elegans*. Therefore, research into the principles, pathways, and mechanisms of anti-aging has significant scientific and social value.

[0003] Exosomes are nanoscale vesicle-like bodies encapsulated in a lipid bilayer. They are highly stable in bodily fluids such as peripheral blood, urine, pleural effusion, cell supernatant, and cerebrospinal fluid. They are not only products of normal cellular metabolism, but exosome secretion is also a common cellular function. Exosomes possess various biological functions, including regulating the body's immune response, antigen presentation, and tumor invasion. Among these, their anti-inflammatory and anti-aging effects are particularly noteworthy. Although aging is an inevitable physiological process, senescent cells still possess significant plasticity, and exosomes can slow down or even reverse some aging phenotypes through multiple pathways. Furthermore, exosomes offer advantages such as reusability, low immunogenicity, and lack of ethical restrictions, making them promising candidates for both basic research and clinical applications.

[0004] Forsythia, a plant belonging to the genus Forsythia in the family Oleaceae, is a traditional Chinese herbal medicine with various effects such as clearing heat and detoxifying, reducing swelling and dissipating nodules, and dispelling wind-heat. It has no special requirements for its growing soil environment and is widely cultivated in Shanxi, Henan, Hebei, and other regions of my country. Forsythia leaves are the leaflets of the Forsythia plant. The *Chinese Materia Medica* records that "Forsythia stems and leaves are cold in nature and mainly treat accumulated heat in the heart and lungs." Modern pharmacological studies have shown that Forsythia leaves contain various chemical components such as forsythoside A, forsythoside B, and forsythoside C. The content of forsythoside A and forsythoside B is much higher than that in the Forsythia fruit, which is commonly used as a medicinal raw material. These compounds have strong antioxidant and antiviral biological activities. However, current applications of Forsythia are mostly concentrated on the traditional extraction of Forsythia fruit, with insufficient development and utilization of other parts such as the leaves.

[0005] Currently, the mainstream extraction methods for plant exosomes include differential centrifugation, ultrafiltration, polymer precipitation, and size exclusion chromatography. Differential centrifugation removes impurities step-by-step through gradient centrifugation, but suffers from long operation times and damage to exosome membrane structures caused by high-speed centrifugation. Ultrafiltration relies on membrane pore size separation, which can shorten the time, but membrane clogging and low recovery rates limit its application. Polymer precipitation can improve yield, but the introduced chemical reagents are difficult to remove completely, affecting exosome purity. Commercial reagent kits are expensive and have poor compatibility with plant samples. Ultracentrifugation is one of the commonly used extraction methods, but it requires expensive ultracentrifuges, is complex and time-consuming, and can also lead to exosome aggregation and damage, affecting its purity and integrity. Density gradient centrifugation can improve exosome purity, but it requires density gradient media, which may cause non-specific adsorption of exosomes, leading to loss or contamination. Furthermore, plant cells release a large amount of intracellular substances, such as proteins, nucleic acids, and polysaccharides, during the disruption and extraction process. These impurities are similar in size and physical properties to exosomes, making them difficult to remove completely. Plant tissues may also contain various secondary metabolites, such as polyphenols and flavonoids, which may interact with exosomes and affect the extraction results.

[0006] Currently, there are no reports on the extraction of exosomes from Forsythia suspensa leaves and their anti-aging effects. Therefore, there is an urgent need in this field to develop a method for efficiently extracting high-purity exosomes from Forsythia suspensa leaves and to verify their application potential in the field of anti-aging. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing exosomes from Forsythia suspensa leaves and their applications, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a method for preparing exosomes from Forsythia suspensa leaves, comprising the following steps:

[0010] S1. Take dried forsythia leaves, wash them, add PBS buffer to a blender and homogenize, filter to remove residue, and collect the juice.

[0011] S2. Centrifuge the juice obtained in S1 multiple times at 4°C, discard the precipitate, and collect the supernatant.

[0012] S3. Centrifuge the supernatant obtained in S2 at 4°C, discard the supernatant, retain the precipitate and resuspend it with PBS buffer.

[0013] S4. Transfer the resuspension obtained in S3 to discontinuous sucrose media with different mass fractions and perform ultracentrifugation to collect the bands between the 30%-45% sucrose gradient layers.

[0014] S5. The bands obtained in S4 were diluted with Tris-HCl and then subjected to ultracentrifugation to wash away excess sucrose solution. The final precipitate was resuspended in pre-cooled PBS buffer, which is the exosome of Forsythia suspensa.

[0015] Preferably, the multiple centrifugation processes in step S2 are as follows: centrifugation at 1000g for 15 minutes, centrifugation at 3000g for 30 minutes, and centrifugation at 10000g for 40 minutes.

[0016] Preferably, the ultracentrifugation conditions in step S3 are 150,000g centrifugation for 1.5h.

[0017] Preferably, the mass fractions of the discontinuous sucrose medium in step S4 are 8%, 30%, 45%, and 60%, respectively, and the conditions for ultracentrifugation are 150,000g for 2 hours.

[0018] Preferably, the ultracentrifugation conditions in step S5 are 100,000g centrifugation for 1 hour, and the concentration of Tris-HCl is 20 mmol / L and the pH is 7.4.

[0019] Preferably, step S5 further includes filtering the resuspended Forsythia leaf exosomes through a 0.22 μm filter membrane for sterilization, determining the protein concentration using the BCA method, and then storing them at -80°C.

[0020] Preferably, in step S1, the ratio of dried forsythia leaves to PBS buffer is 5-10 mL of PBS buffer per 1 g of dried forsythia leaves.

[0021] The present invention also provides a method for preparing forsythia leaf exosomes.

[0022] Preferably, the average particle size of the forsythia leaf exosomes is 100-200 nm, and the zeta potential is negative.

[0023] This invention also provides the application of Forsythia leaf exosomes in the preparation of anti-aging functional foods, special dietary foods, health products or pharmaceuticals.

[0024] The present invention achieves the following beneficial technical effects compared to the prior art:

[0025] This invention provides a method for preparing exosomes from Forsythia suspensa leaves and their applications. This invention utilizes a combination of multiple differential centrifugation and sucrose density gradient centrifugation to effectively remove cell debris and macromolecular impurities while precisely separating target exosome bands using a discontinuous sucrose gradient, thereby obtaining high-purity Forsythia suspensa leaf exosomes. Based on Forsythia suspensa leaves, a geographical indication agricultural product of Shanxi Province, this invention extracts exosomes with anti-aging potential, identifies their structural characteristics, and verifies their biological activity. This provides a theoretical basis for exploring novel candidate drugs with multiple biological activities from Forsythia suspensa leaves and enhances the research value and development potential of Forsythia suspensa leaves. This invention evaluates the anti-aging effects of Forsythia suspensa leaves at the cellular level and using Caenorhabditis elegans as a model organism. The results show that Forsythia suspensa leaf exosomes have certain anti-aging activities, effectively inhibiting D-gal-induced senescence of NIH-3T3 cells, promoting the cell cycle transition from G1 to S phase, and regulating the expression of p53 and p21 genes. At the same time, they can significantly prolong the lifespan of nematodes, improve their motility, reduce lipofuscin accumulation, enhance antioxidant enzyme activity, and improve stress resistance. This provides a theoretical basis for the development and utilization of Forsythia suspensa leaves in the field of human health and is of great significance for the in-depth development of Forsythia suspensa leaf resources and the enhancement of their medicinal value. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Characterization results of exosomes from Forsythia suspensa leaves, including: A: Transmission electron micrograph of FELNs; B: Particle size map of FELNs; C: Zeta potential of FELNs.

[0028] Figure 2 The anti-aging effect of Forsythia leaf exosomes on NIH-3T3 cells, including: A: Schematic diagram of NIH-3T3 uptake in mouse embryonic fibroblasts; B: Statistical chart of β-galactosidase staining and content in NIH-3T3 cells; C: Statistical chart of cell cycle changes and the proportion of each part of the cell cycle in NIH-3T3 cells.

[0029] Figure 3 The anti-aging effects of Forsythia suspensa leaf exosomes on Caenorhabditis elegans, including: A: Effect of different concentrations of FELNs on the lifespan of N2 nematodes; B: Measurement of the motility of N2 nematodes; C: Measurement of lipofuscin accumulation in N2 nematodes; D: Measurement of SOD, CAT, GSH, and MDA content in N2 nematodes; E: Effect of FELNs on the stress resistance of N2 nematodes. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The purpose of this invention is to provide a method for preparing exosomes from Forsythia suspensa leaves and their applications, in order to solve the problems existing in the prior art.

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Method for extracting exosomes from Forsythia leaves

[0034] The method for extracting exosomes from Forsythia suspensa leaves includes the following steps:

[0035] Extraction of crude exosome extract from Forsythia suspensa leaves: After washing dried Forsythia suspensa leaves, add an appropriate amount of PBS buffer and homogenize in a high-speed blender. Filter to remove residue, and centrifuge the Forsythia suspensa leaf juice sequentially at 4℃: centrifuge at 1000g for 15min, discard the precipitate and retain the supernatant; centrifuge at 3000g for 30min, discard the precipitate and retain the supernatant; centrifuge at 10000g for 40min, discard the precipitate and retain the supernatant.

[0036] Purification of Forsythia suspensa leaf exosomes: Prepare 8%, 30%, 45%, and 60% sucrose solutions, adding them sequentially to ultracentrifuge tubes in ascending order of concentration, with each sucrose gradient solution added in 2 mL volumes. Transfer the crude extract suspension of Forsythia suspensa leaf exosomes to the prepared discontinuous sucrose gradient, centrifuge at 100,000 g, 4°C for 2 h, and allow the solutions to separate into layers. Collect the bands between 30% and 45% concentrations, centrifuge at 100,000 g, 4°C for 1 h, wash away excess sucrose, discard the supernatant, and resuspend the precipitate to obtain the Forsythia suspensa leaf exosomes.

[0037] The exosomal suspension was filtered through a 0.22 μm filter, and the concentration of exosomal proteins was determined using a BCA kit. A standard curve was plotted based on the absorbance values ​​of different concentrations of bovine serum albumin standards at 570 nm. The concentration of exosomal proteins was calculated based on the standard curve, and the solutions were aliquoted into EP tubes and stored at -80 °C.

[0038] Example 2: Characterization analysis of Forsythia leaf exosomes

[0039] Transmission electron microscopy analysis: 20 μL of the obtained Forsythia leaf exosome suspension was pipetted onto a carbon-supported copper grid and left to stand for 3-5 minutes. Excess liquid was absorbed with filter paper. 2% phosphotungstic acid was then dropped onto the carbon-supported copper grid and left to stand for 1-2 minutes. Excess liquid was absorbed with filter paper, and the mixture was allowed to dry at room temperature. The results were observed under a transmission electron microscope. Figure 1 As shown, bilayered vesicle particles with translucent edges and a size between 100-200 nm can be observed under a transmission electron microscope.

[0040] Particle size and zeta potential analysis: After rinsing the sample cell of the nanoparticle size analyzer with ultrapure water, the instrument was calibrated with polystyrene microspheres (100 nm). The obtained high-purity Forsythia suspensa leaf exosome suspension was diluted to a suitable concentration range with PBS buffer and analyzed by the instrument, repeated three times. The results are as follows: Figure 1 As shown, nanoparticle tracking analysis and Zeta potential results indicate that the average particle size of Forsythia leaf exosomes is approximately 175.5 nm, the particle surface is negatively charged, and the Zeta potential is approximately -39.73 mV.

[0041] Example 3: Anti-aging effect of Forsythia leaf exosomes on NIH-3T3 cells

[0042] Uptake of NIH-3T3 cells from mouse embryonic fibroblasts: Add an appropriate amount of DIO solution to Forsythia suspensa exosomes and mix thoroughly. Incubate at 37°C in the dark for 30 min, then centrifuge at 150,000 g for 90 min at 4°C. Discard the supernatant and resuspend the bottom-labeled Forsythia suspensa exosome pellet in an appropriate amount of PBS buffer for later use. NIH-3T3 cells in the logarithmic growth phase were then... 5 Cells were seeded at a density of 12-well plates and incubated in a cell culture incubator. When the cells reached 60% confluence, they were replaced with serum-free medium and starved overnight. Then, a suspension of stained Forsythia leaf exosomes was added and incubated overnight at 37°C. The 12-well plates were removed from the cell culture incubator, the medium was discarded, and the plates were washed three times with PBS. 500 μL of 4% paraformaldehyde solution was added to each well for fixation at 37°C for 15 min. After fixation, the plates were washed three times with PBS, and 500 μL of 0.2% Triton X-100 was added to each well for permeabilization for 10 min. After permeabilization, the plates were washed three times with PBS in the dark, and then incubated at 37°C in the dark for 30 min with prepared Rhodamine-labeled pirarulide dye. After incubation, the plates were washed three times with PBS in the dark. DAPI was dropped onto a glass slide, the slide was inverted, and the slide was mounted with nail polish. The uptake of exosomes by NIH-3T3 cells was observed using a fluorescence inverted microscope.

[0043] Effects of Forsythia leaf exosomes on β-galactosidase in NIH-3T3 cells: Cells in good condition during the logarithmic growth phase were cultured in 12-well plates at a cell density of 5 × 10⁶ cells / well.4 Cells were added to each well (except for the blank well) with a final concentration of 50 mg / mL of D-gal to establish a senescence model. After 24 hours, the treatment groups were successively treated with Forsythia suspensa exosomes at final concentrations of 30 and 60 μg / mL, with three replicates for each concentration gradient. After 24 hours of treatment, the culture supernatant was discarded, and the cells were washed three times with PBS. β-galactosidase staining was then performed according to the kit instructions, and the cells were incubated overnight in a cell culture incubator. The staining was observed the next day using a fluorescence inverted microscope. Results showed that senescent cells exhibited a distinct blue staining characteristic. Compared with the normal control group, β-galactosidase expression was significantly upregulated in the model group, as evidenced by a significant increase in the number of blue-stained cells. The proportion of β-galactosidase-positive cells decreased dose-dependently with increasing Forsythia suspensa exosome treatment concentration. These findings indicate that Forsythia suspensa exosomes can effectively inhibit D-gal-induced senescence of NIH-3T3 cells and have a significant protective effect on the cells.

[0044] Effects of Forsythia leaf exosomes on the NIH-3T3 cell cycle: Cells in good condition during the logarithmic growth phase were cultured in 12-well plates at a cell density of 5 × 10⁶ cells / well. 4 Cells / well. After cell adhesion, except for the blank wells, D-gal was added to each well at a final concentration of 50 mg / mL to establish a senescence model. After 24 h, forsythia leaf exosomes at final concentrations of 30 μg / mL and 60 μg / mL were added sequentially to the drug-treated groups, with three replicates for each concentration gradient. After 24 h of treatment, the culture supernatant was discarded, and the cells were washed with PBS and fixed and stained according to the kit instructions. Finally, light scattering was detected by flow cytometry, and cell DNA content and light scattering were analyzed using NovoExpress software. The results showed that D-gal treatment caused cell cycle arrest in the G1 phase and inhibited cell proliferation. However, forsythia leaf exosomes could promote the transition of G1 phase cells to S phase, effectively alleviating D-gal-induced cell cycle arrest, thereby exerting a protective effect against cell senescence.

[0045] Effects of Forsythia suspensa exosomes on p53 and p21 gene expression in NIH-3T3 cells were detected by RT-qPCR: Cells in good condition during the logarithmic growth phase were adjusted in number and passaged in four small dishes. After cell attachment, except for the control group, each group was treated with D-galactose at a final concentration of 50 mg / mL to establish a senescence model. After 24 h, the treated groups were treated with Forsythia suspensa exosomes at final concentrations of 30 and 60 μg / mL, respectively. After 24 h of treatment, the culture supernatant was discarded, and cells were scraped from the cells with PBS into 1.5 mL centrifuge tubes for total RNA extraction.

[0046] Extraction of total RNA from cells: After washing the scraped cells three times with PBS, centrifuge them in 1.5 mL enzyme-free centrifuge tubes (4℃, 1100 rpm) for 5 min; then add 500 μL of Trizol lysis buffer to each tube and let stand for 5 min; add 100 μL of chloroform to each tube, shake vigorously for 15 s, let stand for 5 min, and then centrifuge (4℃, 13000 rpm) for 15 min; transfer 200 μL of the colorless aqueous phase to a new 1.5 mL enzyme-free centrifuge tube, add an equal volume of isopropanol, and slowly invert to mix, let stand for 10 min, and then centrifuge (4℃, 13000 rpm) for 10 min; carefully aspirate the supernatant, then wash each tube with 500 μL of 75% ethanol (prepared with DEPC water) and centrifuge (4℃, 13000 rpm) for 5 min, discard the supernatant, repeat the washing 2-3 times, and then place in a fume hood to air dry until transparent. After drying, 10 μL of DEPC water was added to each tube and the tube was placed in a 57°C metal bath for 10 min to aid dissolution, and then the RNA concentration was measured.

[0047] cDNA synthesis: Prepare the reverse transcription system in an enzyme-free sterile tube according to the reverse transcription instructions. After mixing and centrifuging, perform reverse transcription on a PCR instrument under the following conditions: 42℃, 25 min, 85℃, 5 s. After reverse transcription, add 180 μL of ddH2O to each tube, mix well, and store at -20℃ for later use.

[0048] The reverse transcription system is shown in Table 1:

[0049] Table 1 Reverse transcription system

[0050] template RNA 500 ng 5×Master Mix 4 μL <![CDATA[RNase free H2O]]> To 20 μL

[0051] qRT-PCR detection of gene expression levels: cDNA synthesized by reverse transcription can be directly used for qRT-PCR. The primer sequences for real-time quantitative PCR in this experiment are shown in Table 2.

[0052] Table 2 q-PCR primer sequences

[0053]

[0054] The results showed that different concentrations of Forsythia leaf exosomes could reduce the expression levels of p53 and p21 in D-gal-induced NIH-3T3 cells, indicating that Forsythia leaf exosomes can protect cells and delay the senescence of NIH-3T3 cells by regulating the expression of p53 and p21.

[0055] Example 4: Anti-aging effect of Forsythia leaf exosomes on Caenorhabditis elegans

[0056] Effects of Forsythia suspensa leaf exosomes on the lifespan of N2 nematodes: Synchronized nematodes were cultured on NGM plates to the L4 stage. NGM plates containing 0.1 mg / mL 5-fluorouracil were prepared. One day before nematode transfer, E. coli OP50 and Forsythia suspensa leaf exosomes at final concentrations of 0.4 and 0.8 mg / mL were spread onto FuDR plates and cultured. Fifty L4-stage nematodes were transferred from each plate to the spread plates and cultured at 20°C. Nematode survival status was monitored daily, with survival, death, and removal of individuals recorded. The culture medium was changed every 24 hours. The criteria for determining death were: nematodes were considered dead when they completely ceased feeding and showed no reaction to light touch with platinum wire. Individuals excluded included those that died from dehydration due to crawling out of the culture area, those whose eggs had hatched and formed a pouch-like morphology, and those that burrowed deep into the agar medium. The experimental period was calculated from the L4 larval stage until the individual was confirmed dead; this was the lifespan of the nematode. Results showed that, compared to the control group, treatment of N2 nematodes with different concentrations of Forsythia suspensa exosomes significantly prolonged their lifespan. At a Forsythia suspensa exosome concentration of 0.8 mg / mL, the lifespan extension rate reached 18.2%.

[0057] N2 nematode motility assay: N2 nematodes synchronized to the L4 stage were transferred to FuDR medium containing different concentrations of Forsythia suspensa leaf exosomes and cultured. Motility was evaluated on days 6, 10, 14, and 18. Nematodes that could move freely without platinum wire were categorized as "Normal," those that could move after being touched by platinum wire were categorized as "Sluggish," and those that did not move after being touched by platinum wire were categorized as "Immobile." One hundred nematodes were counted in each group. Results showed that on day 10, 55% of the nematodes in the control group exhibited "Normal" motility, while the proportions in the treatment groups were 65% and 75%, respectively. On day 18, 10% of the nematodes in the control group exhibited "Normal" motility, while the proportions in the treatment groups were 20% and 30%, respectively. This indicates that Forsythia suspensa leaf exosomes can enhance nematode motility in a concentration-dependent manner.

[0058] Assay for lipofuscin accumulation in N2 nematodes: N2 nematodes synchronized to the L4 stage were transferred to fresh culture medium containing different concentrations of Forsythia suspensa leaf exosomes. The nematodes were transferred to a new culture medium every other day. On day 7, the accumulation of lipofuscin in the nematodes was observed using a fluorescence inverted microscope. Thirty nematodes were counted in each group. The results showed that compared with the control group, the lipofuscin content in the treated nematodes was reduced, indicating that Forsythia suspensa leaf exosomes reduced the accumulation of lipofuscin in the nematodes, thereby improving their health.

[0059] Determination of SOD, CAT, GSH, and MDA content in N2 nematodes: N2 nematodes synchronized to the L4 stage were transferred to FuDR medium containing different concentrations of Forsythia suspensa leaf exosomes and cultured for 7 days. Every other day, the nematodes were transferred to fresh medium. On the 7th day, all nematodes were washed off the plates with M9, allowed to stand for 10 minutes, centrifuged, and the supernatant was discarded. The nematodes were washed again with M9, centrifuged, and the supernatant was discarded. An appropriate amount of M9 was added to each tube, and the nematodes were lysed using a cryogenic homogenizer. After lysation, the supernatant was collected by centrifugation, and the contents of SOD, CAT, GSH, and MDA were measured according to the manufacturer's instructions. Each group contained at least 1000 nematodes. The results showed that treatment with Forsythia suspensa leaf exosomes significantly enhanced the enzyme activity of SOD and CAT in nematodes, while increasing GSH content and decreasing MDA levels, and these effects showed a clear dose-dependent relationship. This indicates that Forsythia suspensa leaf exosomes provide effective protection to the nematode under oxidative and heat stress conditions by activating the nematode's antioxidant enzyme system.

[0060] Effects of Forsythia leaf exosomes on the stress resistance of N2 nematodes:

[0061] Oxidative stress: N2 strain *Caenorhabditis elegans* nematodes at the L4 stage were synchronized and then inoculated into fresh NGM medium containing different concentrations of *Forsythia suspensa* leaf exosomes. They were cultured at a constant temperature of 20℃ for 5 days, with the medium changed every 24 hours. Then, they were transferred to NGM plates containing hydrogen peroxide for further culture. Nematode survival was recorded every 30 minutes. Fifty nematodes were observed in each group. Experimental data were analyzed using statistical software, survival curves were plotted, and statistical processing was performed.

[0062] Heat stress: N2 strain *Caenorhabditis elegans* nematodes developed to the L4 stage were synchronized and then inoculated into fresh NGM medium containing different concentrations of *Forsythia suspensa* leaf exosomes. They were cultured at a constant temperature of 20℃ for 5 days, with the medium changed every 24 hours. Then, they were transferred to fresh NGM plates and incubated at 37℃. Nematode survival was recorded every hour, with 50 nematodes observed in each group. Experimental data were analyzed using statistical software, survival curves were plotted, and statistical processing was performed.

[0063] UV stress: N2 strain *Caenorhabditis elegans* nematodes at stage L4 were synchronized and then inoculated into fresh NGM medium containing different concentrations of *Forsythia suspensa* leaf exosomes. The medium was incubated at 20°C for 5 days, with the medium changed every 24 hours. The nematodes were then transferred to fresh NGM plates and irradiated with UV light for 1 hour in a clean bench. Survival was then recorded every hour under dark conditions. Fifty nematodes were observed in each group. Experimental data were analyzed using statistical software, survival curves were plotted, and statistical processing was performed.

[0064] The experimental results showed that under oxidative stress, the survival time of nematodes treated with Forsythia leaf exosomes was significantly increased compared to the control group, with increases of 22.22% and 33.33% in the two treatment groups, respectively. In the heat stress experiment, the survival time of nematodes in the treatment groups was also extended by 25.0% and 37.5% compared to the control group, respectively. In the ultraviolet stress experiment, the survival time of nematodes in the treatment groups was also extended by 8.33% and 33.33% compared to the control group, respectively. These data fully demonstrate that Forsythia leaf exosomes can effectively enhance the stress tolerance of nematodes.

[0065] Comparative Example 1

[0066] Unlike Example 1, in step S4, the mass fractions of discontinuous sucrose media are 10%, 25%, 40%, and 55%, respectively, while the remaining steps and conditions are the same as in Example 1.

[0067] Comparative Example 2

[0068] Unlike Example 1, in step S2, the multiple centrifugation processes are as follows: centrifugation at 800g for 10 min, centrifugation at 2500g for 20 min, and centrifugation at 8000g for 30 min. The remaining steps and conditions are the same as in Example 1.

[0069] Comparative Example 3

[0070] Unlike Example 1, in step S3, the ultracentrifugation conditions are 100,000g for 1 hour, while the remaining steps and conditions are the same as in Example 1.

[0071] Comparative Example 4

[0072] Unlike Example 1, the sucrose density gradient centrifugation purification step S4 was omitted, and the precipitate obtained in step S3 was directly resuspended in PBS buffer as Forsythia leaf exosomes. The remaining steps and conditions were the same as in Example 1.

[0073] Comparative Example 5

[0074] Unlike Example 1, distilled water was used instead of Tris-HCl for dilution in step S5, while the remaining steps and conditions were the same as in Example 1.

[0075] Performance testing

[0076] 1. Nanoparticle tracking analysis method for detecting exosome concentration and particle size distribution.

[0077] The particle concentration and average particle size of the Forsythia leaf exosomes obtained in each example and comparative example were detected using a Malvern NanoSight NS300 nanoparticle tracking analyzer. Samples were diluted to an appropriate concentration with sterile PBS before being analyzed. Each sample was analyzed three times consecutively, with five images acquired each time. The instrument automatically calculated the average particle concentration and standard deviation. The results are shown in Table 1.

[0078] 2. Exosome purity detection.

[0079] The protein impurity content of Forsythia leaf exosomes obtained in each example and comparative example was detected by SDS-PAGE electrophoresis. 20 μL of exosome sample was taken, 5 μL of 5×SDS loading buffer was added, and the sample was boiled for 5 min to denature it. After cooling at 4℃, the supernatant was collected by centrifugation. A 12% separating gel and a 5% stacking gel were prepared, and 15 μL of the treated sample was loaded into each well. Electrophoresis was performed at a constant voltage of 80V until bromophenol blue entered the separating gel. The voltage was then adjusted to 120V, and electrophoresis continued until bromophenol blue reached the bottom of the gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 for 2 h, and then destained with destaining solution until the background was clear. The gel was scanned using a gel imaging system, and the gray values ​​of the bands were analyzed using ImageJ software. The proportion of the total gray value of the impurity protein band to the total gray value of all bands was calculated, which represents the impurity protein content. The detection results are shown in Table 1.

[0080] Table 3 shows the particle size, concentration, and purity of Forsythia leaf exosomes obtained from each example and comparative example.

[0081] Example 1 175.5 <![CDATA[8.5×10 11 ]]> 2.6 Example 2 173.2 <![CDATA[8.2×10 11 ]]> 2.8 Example 3 176.8 <![CDATA[7.9×10 11 ]]> 2.9 Comparative Example 1 182.3 <![CDATA[5.2×10 11 ]]> 5.8 Comparative Example 2 180.5 <![CDATA[4.8×10 11 ]]> 6.2 Comparative Example 3 178.9 <![CDATA[4.5×10 11 ]]> 6.5 Comparative Example 4 185.7 <![CDATA[6.8×10 11 ]]> 12.3 Comparative Example 5 177.2 <![CDATA[6.5×10 11 ]]> 5.6

[0082] As shown in Table 3, the exosomes from Forsythia suspensa leaves prepared using the methods of Examples 1-3 of this invention have an average particle size in the range of 173-177 nm, and a particle concentration of 7.9 × 10⁻⁶. 11 The particle count was above 100 particles / mL, and the proportion of impurities in the total protein was less than 3.0%, indicating that the method of this invention can obtain high-purity and high-concentration Forsythia leaf exosomes. In contrast, Comparative Examples 1-3 changed the centrifugation parameters or sucrose gradient concentration, resulting in a significant decrease in particle concentration and a significant increase in impurities in protein content; Comparative Example 4 omitted the sucrose density gradient centrifugation step, and although the particle concentration was still acceptable, the impurities in protein content was as high as 12.3%, and the purity was significantly reduced; Comparative Example 5 used distilled water instead of Tris-HCl for dilution, and the impurities in protein content also increased. The above results show that this invention, through optimized differential centrifugation parameters, sucrose density gradient centrifugation conditions, and appropriate dilution solution selection, can synergistically achieve efficient extraction and high-purity purification of Forsythia leaf exosomes.

[0083] Anti-aging efficacy verification

[0084] The anti-aging efficacy of the Forsythia suspensa leaf exosomes obtained in Example 1 was verified at the cellular and nematode levels according to the methods in Examples 3 and 4. Cellular experiments showed that the Forsythia suspensa leaf exosomes could be effectively taken up by NIH-3T3 cells, dose-dependently inhibiting D-gal-induced β-galactosidase-positive expression in NIH-3T3 cells, promoting the transition of G1 phase cells to S phase, and downregulating the expression levels of p53 and p21 genes. Nematode experiments showed that the Forsythia suspensa leaf exosomes at a concentration of 0.8 mg / mL prolonged the lifespan of N2 nematodes by 18.2%, significantly improved nematode motility, reduced lipofuscin accumulation, enhanced SOD and CAT enzyme activity, increased GSH content, and reduced MDA levels, while significantly enhancing the survival ability of nematodes under oxidative stress, heat stress, and UV stress. These results indicate that the Forsythia suspensa leaf exosomes prepared in this invention possess excellent anti-aging biological activity.

[0085] This invention extracts exosome nanoparticles from Forsythia suspensa leaves through the above embodiments, and studies and verifies their anti-aging effects at the cellular level and in a Caenorhabditis elegans aging model. This provides a theoretical basis for the development and utilization of Forsythia suspensa leaves in the field of human health, and is of great significance for the in-depth development of Forsythia suspensa leaf resources and the enhancement of their medicinal value.

[0086] This invention has illustrated its principles and implementation methods using specific examples. The descriptions of these embodiments are merely illustrative of the method and its core ideas; furthermore, those skilled in the art will recognize that modifications may be made to the specific implementation methods and application scope based on the principles of this invention. Therefore, the content of this specification should not be construed as limiting the invention.

Claims

1. A method for preparing exosomes from Forsythia suspensa leaves, characterized in that, Includes the following steps: S1. Take dried forsythia leaves, wash them, add PBS buffer to a blender and homogenize, filter to remove residue, and collect the juice. S2. Centrifuge the juice obtained in S1 multiple times at 4°C, discard the precipitate, and collect the supernatant. S3. Centrifuge the supernatant obtained in S2 at 4°C, discard the supernatant, retain the precipitate and resuspend it with PBS buffer. S4. Transfer the resuspension obtained in S3 to discontinuous sucrose media with different mass fractions and perform ultracentrifugation to collect the bands between the 30%-45% sucrose gradient layers. S5. The bands obtained in S4 were diluted with Tris-HCl and then subjected to ultracentrifugation to wash away excess sucrose solution. The final precipitate was resuspended in pre-cooled PBS buffer, which is the exosome of Forsythia suspensa.

2. The method for preparing Forsythia leaf exosomes according to claim 1, characterized in that, The multiple centrifugation processes in step S2 are as follows: centrifugation at 1000g for 15 minutes, centrifugation at 3000g for 30 minutes, and centrifugation at 10000g for 40 minutes.

3. The method for preparing Forsythia leaf exosomes according to claim 1, characterized in that, The conditions for ultracentrifugation in step S3 are 150,000g for 1.5h.

4. The method for preparing Forsythia leaf exosomes according to claim 1, characterized in that, The mass fractions of the discontinuous sucrose medium in step S4 are 8%, 30%, 45%, and 60%, respectively, and the conditions for ultracentrifugation are 150,000g for 2 hours.

5. The method for preparing Forsythia leaf exosomes according to claim 1, characterized in that, The ultracentrifugation conditions in step S5 are: centrifugation at 100,000g for 1 hour, and the concentration of Tris-HCl is 20 mmol / L with a pH of 7.

4.

6. The method for preparing Forsythia leaf exosomes according to claim 1, characterized in that, Step S5 also includes filtration of the resuspended Forsythia leaf exosomes through a 0.22 μm filter membrane for sterilization, determination of protein concentration using the BCA method, and storage at -80°C.

7. The method for preparing Forsythia leaf exosomes according to claim 1, characterized in that, In step S1, the ratio of dried forsythia leaves to PBS buffer is 5-10 mL of PBS buffer per 1 g of dried forsythia leaves.

8. Forsythia leaf exosomes prepared by the method for preparing Forsythia leaf exosomes according to any one of claims 1 to 7.

9. The forsythia leaf exosomes according to claim 8, characterized in that, The average particle size of the forsythia leaf exosomes is 100-200 nm, and the zeta potential is negative.

10. The use of Forsythia leaf exosomes prepared by any one of claims 1 to 7 in the preparation of anti-aging functional foods, special dietary foods, health products or pharmaceuticals.

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