A method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation

Abstract

This invention provides a method for extracting exosome-like nanoparticles from tea leaves using differential centrifugation, relating to the field of extracellular vesicle technology in tea. The method includes homogenization and filtration, differential centrifugation, ultracentrifugation, and resuspension. By sequentially combining tea homogenization, filtration, multi-stage differential centrifugation, and ultracentrifugation, the method achieves the stepwise separation of tissue fragments, larger particles, and other non-target components in the tea homogenate, and enriches the exosome-like nanoparticles. The resulting method has a clear process and well-defined operating conditions, and can be applied to teas processed using different methods, such as sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea. It shows promising application prospects in the study of functional components in tea, the evaluation of tea-derived nanoparticles, and the development of related products.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle technology in tea, and more particularly to a method for extracting exosome-like nanoparticles from tea based on differential centrifugation. Background Technology

[0002] Tea is a natural plant product made from the fresh leaves of the tea plant through various processing techniques. Based on processing methods and the degree of fermentation, tea can generally be classified into types such as green tea, white tea, yellow tea, oolong tea, black tea, and dark tea. Yunnan Province is rich in tea resources, boasting distinctive tea varieties including sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea. Due to differences in raw material varieties, processing temperatures, fermentation levels, and the involvement of microorganisms, the composition of polyphenols, flavonoids, phenolic acids, amino acids, alkaloids, proteins, and lipids varies among different tea types, potentially affecting the composition and form of their functional components.

[0003] Plant exosome-like nanoparticles are a class of nanoscale vesicles released from plant cells or derived from plant tissues. They typically possess a phospholipid bilayer structure and can carry bioactive components such as lipids, proteins, metabolites, and RNA. In recent years, plant exosome-like nanoparticles have attracted attention due to their natural origin, membrane structure characteristics, and ability to load various endogenous substances. Existing studies have isolated exosome-like nanoparticles from some plant tissues, fruits and vegetables, and tea leaves or tea flowers, and investigated their morphology, particle size, and some biological activities.

[0004] However, after processing such as rolling, drying, fermentation, and post-fermentation, tea leaves have a complex tissue structure and chemical composition. Tea homogenates typically contain plant tissue fragments, insoluble particles, macromolecular aggregates, and membrane structures of varying sizes, which can easily interfere with the separation and characterization of exosome-like nanoparticles. Existing research on exosome-like nanoparticles derived from tea leaves is mostly focused on single tea types or specific research objectives. Different research institutes employ varying raw material processing methods, centrifugation conditions, and resuspension methods, lacking a well-defined extraction method with relatively uniform parameters applicable to teas with different processing techniques. This hinders cross-sectional comparisons of particles from different tea sources.

[0005] Furthermore, existing technologies lack systematic research on the differences in particle concentration, protein content, lipid composition, metabolite composition, and miRNA expression of exosome-like nanoparticles derived from tea at different fermentation levels. There is also a lack of unified evaluation of the stability of the obtained particles under heat treatment, simulated gastrointestinal environments, and different storage temperatures. In particular, for Yunnan specialty teas such as sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea, it is necessary to establish technical methods encompassing sample pretreatment, step-by-step impurity removal, particle enrichment, and structural and physicochemical property verification to obtain tea exosome-like nanoparticles suitable for subsequent compositional analysis, stability studies, and functional evaluation.

[0006] Therefore, it is necessary to provide a method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation. By performing stepwise centrifugation to remove impurities and ultracentrifugation to enrich the tea leaf homogenate, the interference of impurities of different particle sizes on the target particles can be reduced, and the extraction of tea leaves from different processing processes can be completed under relatively uniform conditions. This provides a technical basis for the characterization, comparison, and further development and utilization of exosome-like nanoparticles from tea leaves. Summary of the Invention

[0007] In view of this, the present invention provides a method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation. This invention combines tea homogenization, filtration, multi-stage differential centrifugation, and ultracentrifugation sequentially to achieve the stepwise separation of tissue fragments, larger particles, and other non-target components in the tea homogenate, and to enrich the exosome-like nanoparticles from the tea leaves. The resulting method has a clear process flow and well-defined operating conditions, and can be applied to teas processed using different methods, such as sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea. It has promising applications in the study of functional components in tea, the evaluation of tea-derived nanoparticles, and the development of related products.

[0008] This invention relates to a method for extracting exosome-like nanoparticles from tea leaves using differential centrifugation, comprising the following steps: S1. Homogenization and Filtration: Mix dried tea leaves with pure water in a certain proportion, homogenize, filter and collect the filtrate; S2. Differential centrifugation: The filtrate is centrifuged sequentially at 800-1000 r / min for 5-10 min, 1000-3000 r / min for 15-20 min, 3000-5000 r / min for 25-30 min, and 8000-10000 r / min for 25-30 min at 4℃. The supernatant is retained after each centrifugation. S3. Ultracentrifugation: Centrifuge the supernatant obtained in step S2 at 4℃ and 100,000×g (corresponding to 31,100 r / min for a Type 70 Ti rotor) for 1.5-2 h, and discard the supernatant. S4. Resuspension: The precipitate obtained in step S3 was resuspended in pure water to obtain a suspension of tea exosome-like nanoparticles (T-ELNs).

[0009] Preferably, in step S1, the tea is at least one of sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu'er ripe tea made from fresh leaves of Yunnan large-leaf tea trees; In step S1, the tea leaves and pure water are mixed at a ratio of 1 g: 4 mL and homogenized. The homogenization speed is 10000 r / min and the homogenization time is 150 s. Then, the mixture is filtered through a filter material with an equivalent pore size of 400-600 μm. The pure water is pre-cooled at 4℃ to avoid vesicle degradation. More preferably, the filtration uses 4 layers of general-purpose coarse filter gauze with an equivalent pore size of 400-600 μm.

[0010] Preferably, in step S4, the amount of pure water used is 4 mL, the precipitate is resuspended by ultracentrifugation, the precipitate is first dispersed by blowing and then vortexed to mix, and then allowed to stand at low temperature to ensure uniform dispersion of nanoparticles, which meets the standard operation for exosome preparation.

[0011] Preferably, the obtained tea exosome-like nanoparticles are cup-shaped or spherical and have a double-layer membrane structure under a transmission electron microscope. The average hydrated particle size measured by dynamic light scattering is 229.00-279.43 nm, the polydispersity index is less than 0.5, and the Zeta potential is -25.97 to -19.10 mV.

[0012] The present invention also provides uses for T-ELNs extracted by the above method, wherein the T-ELNs can be used as oral delivery carriers, or used to prepare functional foods and health products with anti-inflammatory, antioxidant and / or intestinal immune-modulating effects, or used to prepare biological agents that regulate cell function across species.

[0013] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention employs differential centrifugation, progressing from low to high speeds. This process gradually removes larger tissue fragments and particulate impurities from the tea homogenate based on differences in particle size and sedimentation characteristics. Subsequent ultracentrifugation allows for the sedimentation and enrichment of nanoscale membrane particles. The method described in this invention can process teas processed using different methods under a unified technical process, improving the comparability of extraction results across different tea types and facilitating repeated implementation.

[0014] The tea exosome-like nanoparticles obtained by the method described in this invention exhibit a cup-shaped or spherical shape under a transmission electron microscope and possess a bilayer film structure. Their average hydrated particle size under dynamic light scattering is at the nanoscale, with a polydispersity index less than 0.5 and a negative Zeta potential, indicating that the obtained particles have a relatively concentrated particle size distribution and certain aqueous dispersion stability. In the examples, the average hydrated particle size of the four types of tea exosome-like nanoparticles obtained was measured by dynamic light scattering, ranging from 229.00 to 279.43 nm, and the Zeta potential ranged from -25.97 to -19.10 mV, which meets the sample requirements for subsequent structural characterization, composition detection, and stability evaluation.

[0015] This invention enables the acquisition of corresponding exosome-like nanoparticles from tea processed using different techniques, thus providing a unified sample preparation basis for comparing the concentration, protein content, lipid composition, metabolite composition, and miRNA expression characteristics of particles from different tea sources. By employing the same extraction process, experimental biases caused by differences in pretreatment and separation conditions can be reduced, which is beneficial for reflecting the relationship between tea processing and fermentation processes and the differences in the composition of the obtained particles.

[0016] The tea exosome-like nanoparticles obtained in this invention can also be used to evaluate thermal stability, simulated gastric juice stability, simulated intestinal juice stability, and storage stability. The relevant experimental results can provide a basis for the processing tolerance, digestive environment stability, and storage condition selection of particles from different tea sources, and lay a technical foundation for subsequent functional research, quality control, and development and utilization of tea exosome-like nanoparticles. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings.

[0018] Figure 1 The particle concentration, morphology, particle size, surface potential, and protein content of exosome-like nanoparticles extracted from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea using the method described in this invention are characterized. Among them, (a) to (d) are the particle concentration and particle size distribution of exosome-like nanoparticles from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea, respectively, measured by a nanoparticle tracking analyzer, and atomic force microscopy images; (e) to (h) are the dynamic light scattering particle size and transmission electron microscopy images of the above four types of tea exosome-like nanoparticles, respectively. Figure 1 (i) shows the zeta potential detection results of the above four types of tea exosome-like nanoparticles, and (j) shows the protein concentration detection results of the above four types of tea-derived nanoparticles. Figure 2 The results of lipidomics analysis of exosome-like nanoparticles from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu'er ripe tea are shown. Among them, (a) to (d) are rose diagrams of lipid composition of exosome-like nanoparticles from the above four types of tea; (e) is the principal component analysis score plot of lipidomics data; (f) is the orthogonal partial least squares discriminant analysis score plot of lipidomics data; (g) is the permutation test plot of the orthogonal partial least squares discriminant analysis model; and (h) is the hierarchical clustering heatmap of the top 20 differentially expressed lipids in exosome-like nanoparticles from the four types of tea. Figure 3The results of non-targeted metabolomics analysis of exosome-like nanoparticles from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu'er ripe tea are shown below. (a) is a distribution map of the identified metabolites; (b) is a principal component analysis score map of the non-targeted metabolomics data; (c) is a score map of orthogonal partial least squares discriminant analysis of the non-targeted metabolomics data; (d) is a permutation test map of the orthogonal partial least squares discriminant analysis model; (e) to (j) are volcano plots of differential metabolites obtained from pairwise comparisons of exosome-like nanoparticles from different teas; (k) is a hierarchical clustering heatmap of differential metabolites; and (l) is a KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification results of differential metabolites. Figure 4 The sequencing and composition analysis results of miRNAs contained in tea exosome-like nanoparticles are shown below. (a) is the expression profile of miRNAs in Pu-erh ripe tea exosome-like nanoparticles; (b) is the principal component analysis score plot of miRNA expression data; (c) is the alignment result of sequencing reads with the reference genome sequence; (d) is the length distribution plot of sRNA sequences in different tea exosome-like nanoparticles; (e) is the statistical result of the number of miRNAs identified in different tea exosome-like nanoparticles; (f) is the Venn diagram of the common and specific miRNAs identified in SDT-ELNs, YGT-ELNs and YBT-ELNs; (g) is the expression of the top 30 miRNAs in each sample; (h) to (j) are volcano diagrams of differentially expressed miRNAs among different tea exosome-like nanoparticles. Figure 5 The results of functional enrichment analysis of target genes corresponding to differentially expressed miRNAs among different tea exosome-like nanoparticles are shown. Among them, (a) to (c) are the gene ontology functional enrichment analysis results of target genes of differentially expressed miRNAs in different groups; (d) to (f) are the pathway enrichment analysis results of target genes of differentially expressed miRNAs in different groups. Figure 6 The results of the thermal stability and simulated gastrointestinal fluid stability evaluation of exosome-like nanoparticles from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea are shown. Among them, (a) to (d) show the changes in average hydrated particle size and Zeta potential of the above four types of tea exosome-like nanoparticles after heat treatment at 80℃ for different times, respectively; (e) shows the changes in average hydrated particle size and Zeta potential of the above four types of tea exosome-like nanoparticles after incubation in simulated gastric fluid for different times; (f) shows the changes in average hydrated particle size and Zeta potential of the above four types of tea exosome-like nanoparticles after incubation in simulated intestinal fluid for different times. Figure 7The results of stability evaluation of exosome-like nanoparticles of sun-dried green tea, Yunnan green tea, Yunnan black tea and Pu-erh ripe tea during storage under different temperature conditions are shown. Among them, (a) to (d) show the changes in average hydrated particle size of exosome-like nanoparticles of sun-dried green tea, Yunnan green tea, Yunnan black tea and Pu-erh ripe tea after storage at 25℃, 4℃ and -20℃ for different times. Below each sub-figure is the transmission electron microscope image of the corresponding sample after 28 days of storage. Figure 8 The nanoparticle tracking analysis results of exosome-like nanoparticles from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea obtained under initial differential centrifugation conditions during the parameter screening stage are shown. Among them, (a) to (d) are the particle size distribution curves (particle size-particle concentration) of exosome-like nanoparticles from sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea, respectively, measured by NTA (nanoparticle tracking analyzer). Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] In the embodiments, “SDT-ELNs”, “YGT-ELNs”, “YBT-ELNs” and “RPT-ELNs” refer to exosome-like nanoparticles derived from sun-dried green tea, Yunnan green tea, Yunnan black tea and Pu’er ripe tea, respectively.

[0021] The Yunnan green tea was Yunnan Dianlv (batch number: 2025200228, product standard number: GB / T 14456.2), and the Yunnan black tea was Fengpai Dianhong (batch number: 2024200647, product standard number: GB / T 13738.2), purchased from Yunnan Dianhong Group Co., Ltd.; the Pu'er ripe tea and sun-dried green tea (batch numbers: 250904 and 250903 respectively, product standard number: GB / T22111) were purchased from Menghai Bafang Tea Garden Tea Industry Co., Ltd.

[0022] Unless otherwise specified, all experiments were repeated three times. All research data were statistically processed using IBM SPSS Statistics 27 software and plotted using Origin 2022. A p-value < 0.05 was considered statistically significant.

[0023] Example 1: Extraction and Characterization of Tea Leaf Exosome-like Nanoparticles During parameter screening, the filtrate was centrifuged sequentially at 800 r / min for 5 min, 1000 r / min for 15 min, 3000 r / min for 25 min, and 8000 r / min for 25 min at 4℃, retaining the supernatant after each centrifugation. The supernatant was then centrifuged at 100000×g (corresponding to a Type 70 Ti rotor at 31100 r / min) for 1.5 h at 4℃, the supernatant was discarded, and the precipitate was resuspended in pure water. The particle size distribution of the obtained samples was determined using a nanoparticle tracking analyzer. The results showed ( Figure 8 The presence of numerous impurity peaks in the particle size distribution indicates insufficient impurity removal under these conditions. Based on these results, the differential centrifugation times for each stage were further extended, and the centrifugation speeds were increased to achieve the centrifugation conditions described in Example 1.

[0024] Sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu-erh ripe tea were weighed separately and mixed with pure water at a ratio of 1 g: 4 mL. A high-speed juicer was used to homogenize the mixture at a speed of 10,000 r / min. Pure water that had been stored at 4℃ for 24 hours in advance was used to avoid vesicle degradation. The homogenization time was 150 s. The mixture was filtered through four layers of general-purpose coarse filter gauze with an equivalent pore size of 400 μm, and the filtrate was collected.

[0025] The filtrate was centrifuged sequentially at 1000 r / min for 10 min, 3000 r / min for 20 min, 5000 r / min for 30 min, and 10000 r / min for 30 min at 4℃; the supernatant was collected after each centrifugation. The filtrate was then centrifuged at 100000 g (corresponding to a Type 70 Ti rotor at 31100 r / min) for 2 h at 4℃. The supernatant was discarded, and the precipitate was resuspended in 4 mL of pure water to obtain suspensions of SDT-ELNs, YGT-ELNs, YBT-ELNs, and RPT-ELNs, respectively.

[0026] The particle size distribution and concentration of T-ELNs were determined using a NanoSight NS300 nanoparticle tracking analyzer (Malvin Panaco Ltd., UK). Samples were diluted 100-fold to an appropriate concentration using sterile PBS buffer (composition: NaCl, KCl, Na₂HPO₄, KH₂PO₄, pH=7.4), and 500 μL was injected into the sample cell. The syringe pump flow rate was set to 40, the video frame rate to 25.0 fps, and the total number of frames acquired per sample was 1498. After acquiring videos of particle Brownian motion, the video data was analyzed using NTA 3.4 software (Malvin Panaco Ltd., UK) to obtain the particle size distribution and concentration of T-ELNs. Particle size and potential were measured using a Zetasizer NanoZS90 dynamic light scattering instrument (Malvin Panaco Ltd., UK). Take 200 μL of sample, bring the volume to 1 mL with sterile PBS buffer, and transfer the sample to a square glass particle size cuvette. Use the H2O-SIZE-sop program to measure particle size and the H2O-ZETA-sop program to measure potential. Use the matching Zetasizer Nano analysis software (Malvin Panaco Ltd., UK) to obtain the average hydrated particle size and polydispersity index (PDI) of the sample. After the Zeta potential is detected, use the same software to output the Zeta potential value.

[0027] T-ELNs suspension was dropped onto a copper grid and allowed to stand for 3 minutes. Excess liquid was absorbed with filter paper, and 10 μL of 1% phosphotungstic acid staining solution was added. After staining for 2 minutes, the staining solution was absorbed, and the mixture was allowed to air dry at room temperature. Observation was performed using a Tecnai G2 Spirit transmission electron microscope (FEI, USA). T-ELNs suspension was also dropped onto a mica sheet and dried completely at 40°C. Its morphology was observed using a DimensionIcon atomic force microscope (Bruck, Germany).

[0028] Follow the instructions for the BCA kit. Mix reagent A and reagent B at a volume ratio of 50:1 to prepare the BCA working solution. Add 20 μL of BSA standard solution at concentrations of (0, 125, 250, 500, 750, 1000, 1500, 2000 μg / mL) to each well of a 96-well plate. Dilute the sample 10-fold with 1×PBS, and add 20 μL of the diluted solution to each well. Add 200 μL of BCA working solution to each well, mix well, and incubate at 37°C for 30 min. Measure the absorbance at 562 nm using an ELX808 microplate reader (Porton Microplate, USA). Use the absorbance without BSA as a blank control. Plot a standard curve with absorbance on the x-axis and BSA concentration on the y-axis, and calculate the protein concentration of the sample.

[0029] Nanoparticle tracking analysis showed that the concentrations of SDT-ELNs, YGT-ELNs, YBT-ELNs, and RPT-ELNs were 1.78 × 10⁻⁶. 12 1.97×10 12 2.26×10 11 2.83×10 11 The particles / mL; the AFM of these particles all showed discrete and independent spikes, indicating good particle dispersibility. DLS and TEM results further showed that the average hydrated particle size of the four T-ELNs was 229.00-279.43 nm, and the polydispersity index (PDI) was less than 0.5. All four T-ELNs exhibited the typical cup-shaped morphology and bilayer membrane structure of exosomes, with no significant morphological differences. The Zeta potentials of the T-ELNs were all negative. The protein concentrations of the four T-ELNs, measured using a BCA kit, were 21.81±0.18, 16.27±0.55, 2.07±0.20, and 2.31±0.11 mg / mL, respectively. (See attached table). Figure 1 .

[0030] Example 2: Omics Analysis of Four Types of Tea Exosome-like Nanoparticles (1) Lipidomics detection Lipid composition was determined by liquid chromatography-mass spectrometry (LC-MS). Take 200 μL of T-ELNs into 2 mL centrifuge tubes, add 750 μL of methyl tert-butyl ether-methanol (10:3, v / v) solution, vortex for 30 s; place on ice for 40 min, add 190 μL of H2O, mix and vortex for 30 s using a BE-2600 mixer (Haimen Qilinbei Instrument Manufacturing Co., Ltd., Jiangsu, China), and let stand on ice for 10 min; centrifuge at 12000 r / min at room temperature for 5 min using an H1650-W centrifuge (Xiangyi Laboratory Instrument Development Co., Ltd., Hunan, China), take 300 μL of the organic layer and transfer it to a new 2 mL centrifuge tube; add 500 μL of methyl tert-butyl ether-methanol (10:3, v / v), vortex for 30 s; centrifuge at 12000 r / min at room temperature for 5 min, take 400 μL of the organic layer and transfer it to the same 2 mL centrifuge tube, concentrate the sample using a 5305 vacuum concentrator (Eppendorf, Germany); and concentrate using 200... The sample was dissolved in μL of isopropanol and filtered through a 0.22 μm PES microporous membrane to obtain the test sample, ready for LC-MS analysis. Separation was then performed using a Vanquish high-performance liquid chromatograph (Thermo Fisher Scientific, USA). Chromatographic conditions: ACQUITY UPLC was used. ®A BEH C18 column (1.7 µm, 2.1 × 100 mm) was used. The autosampler temperature was set to 8 °C, the column temperature to 50 °C, the flow rate to 0.25 mL / min, and the injection volume to 2 μL. Mobile phase A2 consisted of acetonitrile-water (60:40, v:v) containing 0.1% formic acid and 10 mM ammonium formate; mobile phase B2 consisted of isopropanol-acetonitrile (90:10, v:v) containing 0.1% formic acid and 10 mM ammonium formate. The gradient elution program was as follows: 0–2 min, 60–55% A2; 2–2.1 min, 55%–45% A2; 2.1–10 min, 45%–30% A2; 10–10.1 min, 30% A2; 10.1–15 min, 30%–1% A2; 15–17 min, 1% A2; 17–17.1 min, 1%–60% A2; 17.1–20 min, 60% A2. The charge-to-mass ratio of lipid molecules was determined using a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA). Mass spectrometry conditions: electrospray ionization (ESI) was used, with detection in both positive and negative ion modes. The spray voltage was 3.50 kV for positive ion mode and 2.50 kV for negative ion mode. The sheath gas pressure was 30 arb, and the auxiliary gas pressure was 10 arb. The capillary temperature was 325℃, and a full scan was performed at a resolution of 35,000, with a mass-to-charge ratio (m / z) of 150-2000. High-energy collisional dissociation (HCD) was used for secondary mass spectrometry analysis at a collision voltage of 30 eV. Dynamic exclusion was employed to remove unnecessary MS / MS information. Based on the results, lipid characteristic peaks were first identified using MS-DIAL 4.70 software (RIKEN Center for Comprehensive Medical Sciences, Japan) combined with the Lipid Map database (www.lipidmaps.org) and retention times and mass spectrometry information of standard compounds. Peak extraction and alignment were then performed. Finally, relative quantification of lipids was completed using the total peak area normalization method.

[0031] The results showed that triglycerides (TG) were the dominant lipid component in all four T-ELNs, with relative contents ranging from 25.19% (YGT-ELNs) to 31.53% (RPT-ELNs). Despite the dominance of TG, the different T-ELNs exhibited significant differences in minor lipid categories. YGT-ELNs showed a phospholipid-rich pattern of "high PC, low TG," with phosphatidylcholine (PC) accounting for 12.98%. Principal component analysis and the top 20 differential lipid heatmaps showed that YBT-ELNs and RPT-ELNs clustered similarly, while SDT-ELNs and YGT-ELNs formed another cluster. (See attached results). Figure 2 .

[0032] (2) Non-targeted metabolomics detection Take 50 μL of T-ELNs, add homogenizing beads, add 100 μL of extraction buffer (methanol-acetonitrile-water = 2:2:1, v:v:v), the extraction buffer contains isotope-labeled internal standard, vortex mix for 30 s, place in a JXFSTPRP-24 homogenizer (Jingxin Technology Co., Ltd., Shanghai, China) at 35 Hz, homogenize for 4 min, then place the sample in an ice-water bath and sonicate for 5 min using a PS-60AL sonicator (Redbond Electronics Co., Ltd., Shenzhen, China). Repeat this step 3 times. Freeze in liquid nitrogen for 1 min, remove and thaw, vortex mix for 30 s, repeat the step 3 times, sonicate in an ice-water bath for 10 min; after standing at -40℃ for 1 h, centrifuge at 4℃ and 12000 r / min for 15 min using a CenLee 16K benchtop high-speed centrifuge (Hunan Xiangli Scientific Instruments Co., Ltd., China), and take the supernatant for 0. The sample was filtered through a .22 μm PES microporous membrane, and the filtrate was transferred to a sample vial for analysis. Equal volumes of supernatant from each sample were mixed to form a QC sample, which was then analyzed using the same method. Chromatographic separation of the target compounds was performed using a Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific, USA) with a Phenomenex Kinetex C18 column (2.1 × 50 mm, 2.6 μm). Mobile phase A: water (containing 0.01% acetic acid), mobile phase B: isopropanol-acetonitrile (1:1, v / v), sample pan temperature 4 °C, injection volume 2 μL. Mass spectrometry was performed using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, USA), with data acquisition controlled by Xcalibur software (version 4.4, Thermo). Mass spectrometry parameters: sheath gas pressure 50 Arb, auxiliary gas pressure 15 Arb, capillary temperature 320℃, primary scan resolution 60000, secondary mass spectrometry resolution 15000, nebulization voltage 3.8 kV (positive) or -3.4 kV (negative).

[0033] Untargeted metabolomics identified 2444 metabolites from four T-ELNs, with phenylpropanoids accounting for the highest proportion (31.3%, 766 metabolites). These metabolites were also rich in terpenes, lipids, alkaloids, polyketides, amino acids, and their derivatives. PCA and OPLS-DA showed that SDT-ELNs and YGT-ELNs had similar metabolographic profiles, while YBT-ELNs and RPT-ELNs were significantly separated. Differential metabolite analysis using P<0.05, VIP>1, FC>2, or FC<0.5 as screening criteria revealed the highest number of differentially expressed metabolites between SDT-ELNs and RPT-ELNs (706 upregulated and 726 downregulated). RPT-ELNs were significantly enriched in medium-chain fatty acids (such as hexanoic acid) and phenolic acids, while SDT-ELNs were enriched in flavanols (such as (+)-gallic acid and epigallic acid) and flavonols (such as quercetin). KEGG enrichment analysis not only covers the global metabolic map but also significantly enriches the "amino acid biosynthesis" pathway. Results are shown in [link to results]. Figure 3 .

[0034] (3) miRNA sequencing and analysis First, total RNA was extracted from T-ELNs using TRIzol reagent, and then NanoDrop was used. TM RNA concentration and purity were detected using a 2000 micro-volume UV-Vis spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity was assessed by agarose gel electrophoresis, and RNA quality number (RQN) was determined using a Fragment Analyzer 5300 automated fragment analyzer (Agilent Technologies, Shanghai, China). Single-sample library preparation criteria were: total RNA ≥ 1 μg, concentration ≥ 50 ng / μL, RQN > 7, OD260 / 280 ratio 1.8-2.2. Specific adapters were ligated to the 3' and 5' ends of the qualified RNA, respectively. Using the adapter-ligated RNA as a template, one-stranded cDNA was synthesized using random primers and reverse transcriptase catalysis, followed by double-stranded synthesis to obtain stable double-stranded DNA. Then, 11-12 cycles of PCR amplification were performed using sequencing primers to enrich the library fragments. Based on miRNA length characteristics, the target fragments were separated, purified, and recovered using a 6% Novex TBE polyacrylamide gel (1.0 mm, 10 wells). Purified library via Invitrogen Qubit TM 4. After quantitative PCR using a fluorescence quantification instrument (Thermo Fisher Scientific, USA), the mixed libraries were prepared according to the sequencing data requirements. The mixed libraries were then amplified by bridge PCR using a cBot automated sequencing cluster generator (Inmena Inc., USA) to generate sequencing clusters. Finally, high-throughput sequencing was performed on the NovaSeq X Plus high-throughput gene sequencing system (Inmena Inc., USA). The above library construction and sequencing work was completed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0035] Only eight miRNAs were detected in RPT-ELNs and their abundance was extremely low. Therefore, subsequent differential analyses focused on SDT-ELNs, YGT-ELNs, and YBT-ELNs.

[0036] The results showed that almost no miRNA expression was detected in RPT-ELNs (only 8 types, and their abundance was extremely low). Therefore, subsequent analyses focused on SDT-ELNs, YGT-ELNs, and YBT-ELNs. The three T-ELNs exhibited good intra-group repetition and inter-group differences in expression profiles. YBT-ELNs had the highest number of sRNAs that could be aligned to the reference sequence (approximately 3.57 million, accounting for 26.19% of the total sRNAs), followed by SDT-ELNs and YGT-ELNs with approximately 790,000 and 590,000 sRNAs, respectively. sRNA length distribution showed that SDT-ELNs and YGT-ELNs were mainly concentrated in the 18-21 nt range, while YBT-ELNs were concentrated in the 23-24 nt range. miRNA identification results showed that YBT-ELNs had the highest number of identified known miRNAs (2499), followed by SDT-ELNs (2130) and YGT-ELNs (1327). The Venn diagram shows that 1875 miRNAs are co-expressed by the three T-ELNs, while SDT-ELNs-specific miRNAs number 484, far exceeding the number of miRNAs specific to YGT-ELNs (30) and YBT-ELNs (411). Analysis of the top 30 most abundant miRNAs reveals that YGT-ELNs and YBT-ELNs are primarily enriched in the miR159 family, while YBT-ELNs are also enriched in the miR166 and miR168 families. Figure 4 g). Differential miRNA expression analysis (P<0.05, VIP>1, FC>2) showed that the number of differentially expressed miRNAs was highest between SDT-ELNs and YBT-ELNs (422 upregulated, 763 downregulated), while the difference was lowest between SDT-ELNs and YGT-ELNs (115 upregulated, 40 downregulated). Notably, all differentially expressed miRNAs with an upregulation fold greater than 18 between SDT-ELNs and YGT-ELNs were members of the miR828 family (such as osa-miR5083, ssl-miR828, etc.); gma-miR4995 was upregulated more than 100-fold. (See attached results). Figure 4 .

[0037] GO and KEGG enrichment analyses were performed on the target genes of differentially expressed miRNAs. GO analysis showed that the target genes of SDT-ELNs vs YGT-ELNs were significantly enriched in the P450 electron transport chain, heterochromatin formation, and epigenetic regulation. The target genes of SDT-ELNs vs YBT-ELNs were highly enriched in immune-related pathways such as defense responses, stress responses, and ADP binding. The target genes of YGT-ELNs vs YBT-ELNs were also enriched in defense responses and enzyme activity regulation. KEGG pathway enrichment analysis showed that SDT-ELNs vs YGT-ELNs were mainly enriched in monotypic acid biosynthesis, branched-chain amino acid synthesis, and base excision repair. Both SDT-ELNs vs YBT-ELNs and YGT-ELNs vs YBT-ELNs were enriched in pathways such as pyruvate metabolism, plant-pathogen interactions, and photosynthetic carbon fixation. Results are shown below. Figure 5 .

[0038] Example 3: Stability evaluation of tea exosome-like nanoparticles Take 2 mL of T-ELNs and incubate them in a water bath at 80℃ for 20 min, 40 min, and 60 min, then immediately cool them rapidly in an ice bath. The particle size and zeta potential are determined using a dynamic light scattering method.

[0039] The particle size of SDT-ELNs showed a trend of first significantly increasing, then slightly decreasing, and then increasing again after 60 min. After 60 min, the particle size increased by approximately 138.874% compared to the initial value (the particle size after 60 min was 2.39 times the initial particle size). The particle size of YGT-ELNs generally showed an initial increase followed by a gradual decrease, and the absolute values ​​of the Zeta potentials of both decreased with the progress of the reaction. The particle size of YBT-ELNs generally showed a slow upward trend over time, while the particle size of RPT-ELNs initially decreased slightly and remained at a low level, before a sharp increase occurred after 60 min. Specifically, after 60 min, the particle size increased by 18.10% and 130.41% respectively compared to the initial value (the particle size after 60 min was 2.30 times the initial particle size). The results are shown in [Figure number missing]. Figure 6 (ad).

[0040] Example 4: Stability evaluation of tea exosome-like nanoparticles in simulated gastrointestinal fluid T-ELNs were added to simulated gastric fluid (enzyme-free, sterile, containing hydrochloric acid, pH 2.5±0.5) or simulated intestinal fluid (sterile, containing 0.464 g / L trypsin and phosphate, pH 6.8) at a gastric / intestinal fluid volume ratio of 1:20 (v:v). After thorough mixing, the mixture was incubated at 37°C and 120 r / min in a shaker. Samples were taken from each solution at 2 h and 4 h to determine their particle size and stability in the simulated gastrointestinal environment.

[0041] In simulated gastric fluid, both SDT-ELNs and YGT-ELNs showed an overall increase in particle size. After 4 hours, SDT-ELNs increased by approximately 11.30%, while the Zeta potential of YGT-ELNs changed from -25.56 mV to -18.00 mV. YBT-ELNs and RPT-ELNs maintained particle sizes close to their initial values. (See attached table for details.) Figure 6 (e).

[0042] In simulated intestinal fluid, the particle size of the four T-ELNs decreased slowly after 2 h, with RPT-ELNs showing the largest decrease (approximately 24.33%) and YGT-ELNs the smallest decrease (approximately 3.25%). At 4 h, the particle size of the four T-ELNs tended to stabilize, and the Zeta potential remained stable between -18.17 and -25.5 mV. The overall stability of the four T-ELNs in SGF was better than that in SIF. Overall, SDT-ELNs, YGT-ELNs, and RPT-ELNs showed better stability in simulated digestion than YBT-ELNs, the latter exhibiting greater particle size fluctuations in gastrointestinal fluid. Nevertheless, all particles maintained a nanoscale size (approximately 213.40–256.10 nm) after the simulated digestion was completed, demonstrating good stability in simulated gastrointestinal fluid. The results are shown in [Figure number missing]. Figure 6 (f).

[0043] Example 5: Evaluation of the storage stability of tea exosome-like nanoparticles T-ELNs were stored at 25℃, 4℃, and -20℃, and the particle size changes were measured at different storage times (1 d, 7 d, 14 d, and 28 d). The morphology was observed by transmission electron microscopy on the 28th day.

[0044] At room temperature (25℃), except for YBT-ELNs, the particle size of all groups decreased significantly within 14 days, with a decrease of 9.06%–18.38%, and slightly rebounded at 28 days. At 4℃, the particle size changes of SDT-ELNs and YGT-ELNs were relatively small within 28 days, while those of YBT-ELNs and RPT-ELNs were larger. TEM showed that the bilayer film remained intact, exhibiting good stability. At -20℃, the particle size changes of SDT-ELNs and YGT-ELNs were minimal, while the decrease in RPT-ELNs was still significant. Even with changes in particle size, transmission electron microscopy observation of all T-ELNs at 28 days showed that the bilayer film structure remained intact, as shown in the results below. Figure 7 (ad).

[0045] Example 6: Comparison of the composition of tea-derived particles at different fermentation levels Comparing the protein concentrations of four tea exosome-like nanoparticles, the protein concentrations decreased from 21.81±0.18 mg / mL and 16.27±0.55 mg / mL for SDT-ELNs and YGT-ELNs to 2.07±0.20 mg / mL and 2.31±0.11 mg / mL for YBT-ELNs and RPT-ELNs. Principal component analysis and the top 20 differential lipid heatmaps showed that YBT-ELNs and RPT-ELNs clustered similarly, while SDT-ELNs and YGT-ELNs formed another cluster. PCA and OPLS-DA showed that SDT-ELNs and YGT-ELNs had similar metabolic profiles, while YBT-ELNs and RPT-ELNs were significantly separated. RPT-ELNs were significantly enriched in medium-chain fatty acids (such as hexanoic acid) and phenolic acids, while SDT-ELNs were enriched in flavanols (such as (+)-gallocatechin and epigallocatechin) and flavonols (such as quercetin). Only eight miRNAs were detected in RPT-ELNs, and their abundance was extremely low. This comparison indicates that the fermentation process is related to the differences in the composition of the resulting particles.

[0046] Example 7: Stability comparison in simulated gastric and intestinal fluids The four T-ELNs exhibited better overall stability in simulated gastric fluid than in simulated intestinal fluid. Overall, SDT-ELNs, YGT-ELNs, and RPT-ELNs showed better stability in simulated digestion than YBT-ELNs, the latter exhibiting greater particle size fluctuations in gastrointestinal fluid. Nevertheless, all particles maintained their nanoscale size (approximately 213.40–256.10 nm) after simulated digestion, demonstrating good structural stability in simulated gastrointestinal fluid.

[0047] Example 8: Effect of different storage temperatures on particle stability Storage conditions at 25℃, 4℃, and -20℃ were compared. At 25℃, particle size varied significantly; low temperatures (4℃ or -20℃) effectively maintained the structural stability of T-ELNs. SDT-ELNs and YGT-ELNs exhibited the best storage tolerance, while RPT-ELNs were the most sensitive to temperature changes. Even with variations in particle size, TEM images of all T-ELNs at day 28 showed a complete bilayer membrane structure, and the core vesicle morphology was preserved throughout the experimental period.

[0048] The particle size of SDT-ELNs and YGT-ELNs can be well maintained at 4℃ and -20℃. The particle size of RPT-ELNs still decreased by 39.08% at -20℃.

[0049] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation, characterized in that, Includes the following steps: S1. Homogenization and Filtration: Mix dried tea leaves with pure water in a certain proportion, homogenize, filter and collect the filtrate; S2. Differential centrifugation: The filtrate is centrifuged sequentially at 800-1000 r / min for 5-10 min, 1000-3000 r / min for 15-20 min, 3000-5000 r / min for 25-30 min, and 8000-10000 r / min for 25-30 min at 4℃. The supernatant is retained after each centrifugation. S3. Ultracentrifugation: Centrifuge the supernatant obtained in step S2 at 4℃ and 100,000 g for 1.5-2 h, and discard the supernatant. S4. Resuspension: The precipitate obtained in step S3 is resuspended in pure water to obtain a suspension of tea exosome-like nanoparticles.

2. The method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation according to claim 1, characterized in that, In step S1, the ratio of dried tea leaves to pure water is 1 g: 4 mL.

3. The method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation according to claim 1, characterized in that, In step S1, the tea is at least one of sun-dried green tea, Yunnan green tea, Yunnan black tea, and Pu'er ripe tea, made from fresh leaves of Yunnan large-leaf tea trees.

4. The method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation according to claim 3, characterized in that, In step S1, tea leaves and pure water are mixed at a ratio of 1 g: 4 mL and homogenized. The homogenization speed is 10000 r / min and the homogenization time is 150 s. Then, the mixture is filtered through four layers of gauze with an equivalent pore size of 400-600 μm. The pure water is pre-cooled at 4℃.

5. The method for extracting exosome-like nanoparticles from tea leaves based on differential centrifugation according to claim 1, characterized in that, In step S4, the amount of pure water used is 4 mL. The precipitate is resuspended by ultracentrifugation, the precipitate is first dispersed by blowing and then vortexed to mix, and then allowed to stand at low temperature to ensure uniform dispersion of nanoparticles, which meets the standard operation for exosome preparation.

6. The tea exosome-like nanoparticles prepared by the method according to any one of claims 1-5, characterized in that, The tea exosome-like nanoparticles, under transmission electron microscopy, appear as cup-shaped or spherical structures with a double-layer membrane structure. The average hydrated particle size measured by dynamic light scattering is 229.00-279.43 nm, the polydispersity index is less than 0.5, and the zeta potential is -25.97 to -19.10 mV.

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