Preparation method of bird's nest extracellular vesicles and application thereof in treatment of central nervous system diseases

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of bird's nest extracellular vesicles and application thereof in treatment of central nervous system diseases. The application provides a bird's nest extracellular vesicle extracted by a mild non-chemical or stewing method. Through research and identification, it is found that the bird's nest extracellular vesicle contains special oligopeptide compositions. The oligopeptide compositions have small molecular weight, high activity and multiple effects of improving central nervous system diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation method of extracellular vesicles from bird's nest and their application in the treatment of central nervous system diseases. Background Technology

[0002] Neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease) and mental illnesses (such as anxiety disorders and depression) constitute a significant group of central nervous system diseases that seriously threaten human health. Currently, first-line clinical treatments primarily rely on small-molecule compounds to antagonize or regulate single neurotransmitter receptors (such as monoaminergic antidepressants, dopamine replacement therapy, or cholinesterase inhibitors). These traditional single-target drugs generally suffer from poor therapeutic efficacy, high relapse rates, and significant systemic side effects. Therefore, there is an urgent clinical need to develop drugs with high safety and minimal side effects for treating central nervous system diseases.

[0003] Extracellular vesicles (EVs) are a class of nanoscale membrane-bound vesicles secreted by cells. Because they carry various bioactive molecules such as proteins, lipids, and nucleic acids, they play unique roles in intercellular communication, immune regulation, tissue repair, and blood-brain barrier penetration. Recent studies have shown that plant extracellular vesicles have potential in the field of neuroprotection. For example, patent CN120939071A discloses that exosome-like nanovesicles derived from Hangzhou white chrysanthemum have the effect of protecting nerve cells and improving anxiety behavior and cognitive impairment in mice. Another example is patent CN120860146A, which discloses that exosomes from Lilium lancifolium can penetrate biological membranes and the blood-brain barrier, act on the central nervous system, and treat depression.

[0004] As a traditional medicinal and edible tonic, bird's nest is gaining increasing attention for its potential value in the prevention and treatment of central nervous system diseases. However, current research remains unclear regarding the effects of bird's nest on neurodegenerative diseases (such as Parkinson's disease) and mental illnesses (such as depression and anxiety). Its active ingredients, targets, and in vivo behavior are far from being elucidated. Currently, research on the effective components of bird's nest mainly focuses on macromolecular components such as sialic acid and glycoproteins. Even though some researchers have proposed targeted hydrolysis of glycoproteins in bird's nest through enzymatic hydrolysis to obtain active sialylated glycopeptides or bird's nest polysaccharides, existing research has two main limitations: firstly, current methods often employ relatively drastic extraction methods such as heating, proteolytic hydrolysis, and organic solvent precipitation, which can cause significant loss or structural damage of trace active components; secondly, the neuroprotective effect of bird's nest is currently mainly attributed to anti-neuroinflammatory responses or relies on the indirect pathway of the gut-brain axis. However, no research has yet provided clear evidence regarding whether the active ingredients in bird's nest can effectively cross the blood-brain barrier and directly act on the central nervous system, or their neuroprotective targets. These limitations severely restrict the high-value development and clinical application of bird's nest in this field. Therefore, it is necessary to propose a product derived from bird's nest with potential therapeutic value for central nervous system diseases to supplement the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a new application for extracellular vesicles in bird's nest, partially solving or alleviating the above-mentioned deficiencies in the prior art. The technical solution of this invention includes the following.

[0006] The first aspect of this invention relates to the use of extracellular vesicles from bird's nest in the preparation of a medicament for treating mental illnesses, wherein the active ingredient of the extracellular vesicles from bird's nest includes an oligopeptide composition, wherein the oligopeptide composition is a combination of dipeptides and tripeptides; wherein the oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

[0007] Preferably, the molecular weight of the oligopeptide composition is in the range of 150 Da to 380 Da.

[0008] As a preferred option, the extracellular vesicles of the bird's nest are extracted using a mild, non-chemical or stewing method.

[0009] Furthermore, the mental illness mentioned is anxiety disorder.

[0010] Furthermore, the mental illness mentioned is depression.

[0011] Furthermore, the dosage form of the drug includes dry powder, capsules, or solution; or, the route of administration of the drug includes oral administration, nasal administration, or intravenous administration.

[0012] Furthermore, the average particle size of the extracellular vesicles in the bird's nest ranges from 100 to 200 nm, and the extracellular vesicles in the bird's nest exhibit a typical saucer-shaped or cup-shaped structure.

[0013] A second aspect of the present invention relates to the application of bird's nest extracellular vesicles in the preparation of products for regulating mood, said products including health products or functional foods; the active ingredient of the bird's nest extracellular vesicles includes an oligopeptide composition, said oligopeptide composition being a combination of dipeptides and tripeptides; said oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

[0014] Furthermore, the regulation of emotions refers to the regulation of anxiety or depression.

[0015] A third aspect of this invention relates to the application of bird's nest extracellular vesicles in the preparation of products that improve oxidative stress damage to nerve cells, said products including pharmaceuticals, health products, or functional foods; the active ingredient of the bird's nest extracellular vesicles includes an oligopeptide composition, said oligopeptide composition being a combination of dipeptides and tripeptides; said oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

[0016] A fourth aspect of this invention relates to the application of bird's nest extracellular vesicles in the preparation of products that improve the energy metabolism of nerve cells, said products including pharmaceuticals, health products, or functional foods; the active ingredient of the bird's nest extracellular vesicles includes an oligopeptide composition, said oligopeptide composition being a combination of dipeptides and tripeptides; said oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

[0017] A fifth aspect of the present invention relates to the application of bird's nest extracellular vesicles in the preparation of products that improve bile acid metabolism, said products including pharmaceuticals, health products, or functional foods; the active ingredient of said bird's nest extracellular vesicles includes an oligopeptide composition, said oligopeptide composition being a combination of dipeptides and tripeptides; said oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

[0018] The final aspect of this invention also provides a method for preparing the above-mentioned extracellular vesicles of bird's nest, comprising the following steps: S01: Soak the dried bird's nest thoroughly at 10-37℃; S02: After soaking the bird's nest thoroughly, homogenize and filter it. Centrifuge the filtered material at 12000 g for 30-90 min at low temperature; the low temperature is set to 2-6℃. Take the supernatant and filter it through a 0.22μm filter membrane to collect the filtrate. S03: The filtrate was purified and extracted by ultrasonic nanofiltration for 50-120 min, and then resuspended in PBS to obtain the extracellular vesicle solution of bird's nest.

[0019] Beneficial technical effects: This invention proposes a method for extracting extracellular vesicles from bird's nest using a mild, non-chemical or non-stewing method. Research and identification of its components revealed that these extracellular vesicles contain a special oligopeptide composition, a combination of dipeptides and tripeptides, including L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine. These oligopeptide compositions have small molecular weights, high activity, and various effects on improving central nervous system diseases. Verification has shown that the extracellular vesicles from bird's nest provided by this invention: (1) It has antidepressant and anti-anxiety effects.

[0020] (2) It can directly cross the blood-brain barrier and enter the brain region.

[0021] (3) It has multiple target effects, including anti-oxidative stress, improvement of mitochondrial energy metabolism and improvement of bile acid metabolism. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0023] Figure 1 The following are characterization results of extracellular vesicles of bird's nest in one embodiment of the present invention (A is a TEM image of extracellular vesicles of bird's nest, B is the particle size / concentration result of extracellular vesicles of bird's nest, and C is the Zeta potential result of extracellular vesicles of bird's nest). Figure 2 This is a diagram of the proteomics and bioinformatics analysis of extracellular vesicles in bird's nest in one embodiment of the present invention (A is the statistical analysis of protein components identified by LC-MS / MS mass spectrometry in extracellular vesicles of bird's nest; B is the result of KEGG pathway enrichment analysis of the obtained proteins). Figure 3 This invention provides an embodiment for analyzing the metabolite profile and pathway enrichment results of extracellular vesicles in bird's nest. Figure 4 This is an example of the LC-MS / MS mass spectrometry detection results of six core oligopeptides in extracellular vesicles of bird's nest according to one embodiment of the present invention; Figure 5 This is a brain tracer image of extracellular vesicles from bird's nest cells in one embodiment of the present invention; Figure 6 This is the result of the protective effect of bird's nest extracellular vesicle treatment on hydrogen peroxide-induced HT22 cell damage in one embodiment of the present invention; Figure 7 This is an example of the protective effect of bird's nest extracellular vesicles on nerve cell energy metabolism in one embodiment of the present invention (A is the intracellular ATP content of each experimental group model group, B is the protein blotting result of each experimental group, and C is the gray-scale quantitative result of each experimental group). Figure 8 This invention illustrates the effect of treating extracellular vesicles from bird's nest on improving anxiety-depression-like behavior induced by chronic restraint stress in mice (A represents the results of an open field experiment in a depressed mouse model, and B represents the results of a forced swimming experiment in a depressed mouse model). Figure 9 In one embodiment of the present invention, treatment with extracellular vesicles of bird's nest can significantly increase the concentration of bile acid metabolites (cholic acid, lithocholic acid, tauride-deoxycholic acid) in the hippocampus of mice induced by chronic restraint stress. Figure 10 This is a representation of the extracellular vesicles of bird's nest after stewing in one embodiment of the present invention, showing their no effect on nerve cells (A is a TEM image of extracellular vesicles of bird's nest after stewing, B is the particle size / concentration result of extracellular vesicles of bird's nest after stewing, C is the Zeta potential result of extracellular vesicles of bird's nest after stewing, and D is the effect of extracellular vesicles of bird's nest on nerve cells after stewing). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0026] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0027] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0028] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0029] Example 1 Preparation and characterization of extracellular vesicles from bird's nest 1. Preparation of extracellular vesicles from bird's nest Preparation of extracellular vesicles from bird's nest: Bird's nest was soaked, homogenized, filtered, purified, and resuspended to obtain extracellular vesicles. A certain amount of dried bird's nest was weighed and added to purified water at a material-to-liquid ratio of 1:20-1:40 for thorough soaking at 10-37℃ for 4-6 hours. The soaked bird's nest was homogenized for 3-5 minutes and filtered. The filtrate was centrifuged at 12000 g for 30-90 minutes at low temperature (preferably 2-6℃). The supernatant was filtered through a 0.22 μm filter membrane to obtain the filtrate. A certain volume (30-40 mL) of the filtrate was purified and extracted using ultrasonic nanofiltration for 50-120 minutes. Finally, the filtrate was resuspended in a certain volume (200-400 μL) of PBS to obtain the purified extracellular vesicle solution from bird's nest.

[0030] The obtained bird's nest extracellular vesicles showed a protein removal rate of >99.2%, a particle recovery rate of >30%, and a final product particle concentration of >1.2×10⁻⁶. 10 particles / mL, and the particle number / protein ratio > 5.1 × 10⁻⁶. 8particles / μg. This method is a mild, non-chemical or non-cooking process that yields high-purity, high-recovery, and highly enriched extracellular vesicles from bird's nest.

[0031] 2. Characterization of extracellular vesicles in bird's nest: (1) Observation by transmission electron microscopy Take 10 μL of extracellular vesicle solution and drop it onto a pre-prepared Parafilm sealing film (with the back of the Parafilm sealing film adhering to the table). Place the copper mesh of the film face down and allow it to naturally absorb the suspension droplet for 15 min. Then, use filter paper to absorb the excess droplet and let the copper mesh dry slightly. Next, take 10 μL of 2% phosphotungstic acid solution (w / v) as the staining solution and drop it onto the Parafilm sealing film. Place the copper mesh face down with the staining solution and invert it to stand for 5 min. Use filter paper to absorb the excess droplet, let the copper mesh dry under an incandescent lamp, and observe and photograph it under a transmission electron microscope.

[0032] (2) Particle size and concentration The particle size distribution and concentration of extracellular vesicles were determined using a nanoflow cytometer (NanoFCM, Flow NanoAnalyzer U30E).

[0033] (3) Zeta potential The zeta potentials of extracellular vesicles were determined using a nanoparticle tracking analyzer (ParticleMetrix, ZetaView version 8.05.14SP7).

[0034] Experimental results: Transmission electron microscopy scanning results as follows Figure 1 As shown in Figure A, a typical saucer-shaped exosome-like vesicle structure is visible under the microscope. The vesicle boundaries are clear, the membrane structure is intact, and there are no obvious damages, fragments, or aggregates of impurities. The morphology fully meets the classic morphological criteria for animal-derived extracellular vesicles. Figure 1 Particle size and concentration analysis results for B showed that the particle concentration of extracellular vesicles in bird's nest was 1.2 × 10⁻⁶. 10 The particles / mL and the average particle size of the extracellular vesicles were 176.4 nm, indicating that extracellular vesicles with intact morphology and concentrated particle size distribution were successfully prepared. Figure 1 The Zeta potential detection results of C showed that the Zeta potential was -35.52±0.96 mV, indicating that the extracellular vesicles of bird's nest cells do not easily aggregate in solution and have good stability.

[0035] Unless otherwise specified, all experiments conducted in the following embodiments of the present invention used the extracellular vesicles of bird's nest prepared in this embodiment.

[0036] Example 2 Identification and functional analysis of extracellular vesicle protein components in bird's nest based on DIA proteomics 1. Extraction of extracellular vesicle proteins The extracellular vesicle solution extracted from bird's nest in Example 1 was frozen. A portion of the extracellular vesicle sample was then removed from the frozen state and transferred to an MP shaking tube. An appropriate amount of plant protein extract (BPP solution) was added. The tube was shaken three times using a high-throughput tissue homogenizer, 40 seconds each time. The supernatant was collected after centrifugation at 12000g for 20 minutes at 4°C. An equal volume of Tris-saturated phenol was then added to the supernatant, and the tube was shaken in a vortex at 4°C for 10 minutes. The phenol phase was collected after centrifugation at 12000g for 20 minutes at 4°C, and an equal volume of BPP solution was added. The tube was shaken in a vortex at 12000g for 20 minutes at 4°C. The phenol phase was collected after centrifugation at 12000g for 20 minutes at 4°C, and 5 times the volume of pre-cooled ammonium acetate methanol solution was added. The tube was then incubated overnight at -20°C to precipitate the protein. The following day, the protein was centrifuged at 12000g for 20 min at 4°C, and the supernatant was discarded. 90% pre-cooled acetone was added to the precipitate, and after mixing, the mixture was centrifuged again, and the supernatant was discarded. This process was repeated twice. The precipitate was dissolved in protein lysis buffer (8M urea + 1% SDS, containing a protease inhibitor cocktail). The mixture was sonicated on ice for 2 min, and then centrifuged at 12000g for 20 min at 4°C. The protein supernatant was collected. Protein content was then determined using the BCA method, strictly following the BCA reagent instructions. After protein quantification, SDS-PAGE electrophoresis was performed.

[0037] 2. Proteolytic enzyme digestion Take 100 μg of the protein sample from the previous step, add lysis buffer, and add 100 mM triethylammonium bicarbonate buffer (TEAB); add 10 mM tris(2-carboxyethyl)phosphine (TCEP) and react at 37℃ for 60 min; add 40 mM iodoacetamide and react at room temperature in the dark for 40 min; add pre-cooled acetone (acetone:sample v:v = 6:1) to each tube and precipitate at -20℃ for 4 h; centrifuge at 10000 g for 20 min and collect the precipitate; dissolve the sample thoroughly with 100 µL of 100 mM TEAB; add Trypsin at a mass ratio of 1:50 (enzyme:protein) and incubate overnight at 37℃.

[0038] 3. Peptide desalting and quantification The product from the previous step was digested with trypsin to obtain peptides, which were then dried using a vacuum pump. The dried peptides were then reconstituted with 0.1% trifluoroacetic acid (TFA); desalted using HLB; and dried using a vacuum concentrator. Finally, peptide quantification was performed using UV spectrophotometry on a Nano Drop One (ThermoScientific) instrument.

[0039] 4. DIA mass spectrometry detection Peptide separation was performed using a VanquishNeo (Thermo) chromatograph. The analytical column was a uPAC HighThroughptu column (75 μm × 5.5 cm, Thermo, USA). Mobile phase A consisted of a mixed aqueous solution of 2% acetonitrile and 0.1% formic acid, and mobile phase B consisted of a mixed aqueous solution of 80% acetonitrile and 0.1% formic acid. Data acquisition software was ThermoXcalibur 4.7 (Thermo, USA). The nano-level high-performance liquid chromatography (HPLC) separated samples were analyzed by mass spectrometry using an Orbitrap Astral (Thermo) mass spectrometer. The Orbitrap Astral (Thermo) mass spectrometer was operated in DIA mode with positive ion detection and an ion source voltage set to 1.5 kV. The primary scan range was 380–980 m / z, and the secondary scan range was 150–2000 m / z.

[0040] 5. DIA Data Analysis Import raw DIA data into Spectronaut TM 18. Software system was used for database search and analysis. The databases used in this study were Aerodramus fuciphagus (species) and Apus apus (species). Six peptides and three daughter ions of each peptide were selected for quantitative analysis. The parameters were as follows: Protein FDR ≤ 0.01, Peptide FDR ≤ 0.01, Peptide Confidence ≥ 99%, XIC width ≤ 75ppm. Shared peptides and modified peptides were excluded, and the peak areas were calculated and summed to obtain the quantitative results.

[0041] 6. Bioinformatics Analysis The KEGG pathway database was used to analyze the metabolic pathways involved in the detected proteins.

[0042] Experimental results: The protein components of the extracted bird's nest extracellular vesicles were identified by LC-MS / MS mass spectrometry. Figure 2 A). A total of 2025 peptides were identified in extracellular vesicles derived from bird's nest. After library search analysis, 568 proteins belonging to the genera *Aerodramus fuciphagus* and *Apus apus* were identified. Further KEGG pathway enrichment analysis was performed on the identified proteins. Figure 2(B) The results showed that the proteins are widely involved in the regulation of multiple biological pathways, including the immune system, endocrine system, sensory system, nervous system, and development and regeneration. Notably, KEGG enrichment analysis indicated that these proteins are closely related to neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. These results suggest that extracellular vesicles from bird's nest may function by regulating signaling pathways related to neurodegenerative diseases, indicating their potential application value as adjunctive intervention agents for neurodegenerative diseases.

[0043] Example 3 Identification and Functional Analysis of Extracellular Vesicle Metabolites in Bird's Nest Based on Metabolomics 1. Extraction of extracellular vesicle metabolites from bird's nest (1) Take out the bird's nest extracellular vesicle sample extracted in Example 1 from the -80℃ freezer and place it on ice to thaw until there are no ice cubes in the sample (all subsequent operations are performed on ice); (2) After the sample is thawed, vortex for 10 s to mix, and transfer 50 μL of the sample into the corresponding numbered centrifuge tube; (3) Add 150 μL of 20% acetonitrile methanol internal standard extraction solution, vortex for 3 min, and centrifuge at 12000 r / min for 10 min at 4℃; (4) After centrifugation, transfer 150 μL of the supernatant to another centrifuge tube with the corresponding number, and let it stand in a -20℃ refrigerator for 30 min; (5) Centrifuge at 12000 r / min for 3 min at 4℃, and transfer 120 μL of the supernatant into the inner liner of the corresponding sample bottle for analysis.

[0044] 2. Chromatographic and mass spectrometric acquisition conditions 2.1 T3 Chromatographic Conditions (1) Chromatographic column: Waters ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 mm * 100 mm (2) Mobile phase A: 0.1% formic acid / water; Mobile phase B: 0.1% formic acid / acetonitrile (3) Column temperature: 40 ℃; flow rate: 0.4 mL / min; injection volume: 3 μL Table 1. Mobile phase gradient conditions for T3 column 2.2 Mass Spectrometry Conditions Table 2 Exactive HF-X Mass Spectrometry Conditions 2.3 Data Preprocessing Raw mass spectrometry data were converted to mzML format using ProteoWizard. Peak extraction, alignment, and retention time correction were performed using XCMS. Peaks with a missing rate >50% in each sample group were filtered, and blank values ​​were filled using KNN with 1 / 5 minimum value (1 / 5 minimum value for blank values ​​>50%, KNN for blank values ​​<50%). Peak area was corrected using the SVR method. Metabolite identification was performed on the corrected and filtered peaks by searching the laboratory's self-built database, integrating public libraries, and prediction libraries. Finally, substances with a comprehensive score of 0.5 or higher and a QC sample CV value of less than 0.3 were extracted and identified. Positive and negative patterns were then merged (if duplicate substances were found, the substance with the highest qualitative grade and highest score was retained), resulting in the all_sample_data.xlsx file.

[0045] 3. Qualitative and quantitative analysis of metabolites The results are summarized based on the metabolite numbers, integral values, and corresponding metabolite names detected in the experiment.

[0046] Experimental results: Based on non-targeted metabolomics data, a chemical classification and statistical analysis of metabolites in the extracellular vesicles of bird's nest was completed (Figure 3), identifying a total of 3046 metabolites. Among them, amino acids, peptides and their analogues were the largest dominant components, totaling 398, accounting for 13.07% of the total metabolites; fatty acids and their conjugates were the second largest, accounting for 6.14%; carbonyl compounds, organic heterocyclic compounds, carbohydrates and their conjugates, and other clearly defined small molecule components accounted for 8.84%; unclassified metabolites accounted for 52.43%, indicating that a large number of unidentified trace active substances still exist in the vesicles. Further targeted mass spectrometry was used to qualitatively detect the characteristic oligopeptides (Figure 4), successfully identifying six oligopeptides: dipeptides Phe-Pro (L-phenylalanine-L-proline, molecular weight 262.1 Da), Leu-Gly (L-leucine-L-glycine, molecular weight 189.1 Da), Trp-Pro (tryptophan-proline, molecular weight 302.2 Da), and tripeptides Leu-Gly-Leu (leucine-glycine-leucine, molecular weight 301.2 Da), Thr-Val-Leu (threonine-valine-leucine, molecular weight 331.2 Da), and Ile-Gln-Ile (isoleucine-glutamine-isoleucine, molecular weight 372.2 Da). The m / z of the molecular ion peaks of each oligopeptide perfectly matched the theoretical molecular weight, suggesting that the extracellular vesicles of bird's nest are rich in small molecule active oligopeptides, which is consistent with the component classification and statistical results.

[0047] Example 4 Fluorescent tracing of extracellular vesicles in bird's nest 1. PKH26 fluorescent dye labeling of extracellular vesicles in bird's nest cells (1) Sample preparation: Take 50 μg of extracellular vesicles extracted from bird's nest in Example 1, with a volume ≤ 50 μL. (2) Preparation of staining working solution: Based on the volume of extracellular vesicles of bird's nest prepared in the previous step, dilute the fluorescent dye with buffer at a ratio of 1:25 to prepare an equal volume of 2× staining working solution; taking 50μL of extracellular vesicles of bird's nest to be stained as an example, add 2μL of fluorescent dye to 48μL of buffer to obtain 50μL of 2× staining working solution. (3) Fluorescent labeling of extracellular vesicles in bird's nest: Add the suspension of extracellular vesicles in bird's nest to the staining working solution at a volume ratio of 1:1, mix quickly by pipetting, and then incubate at room temperature in the dark for 5 minutes to obtain fluorescently labeled extracellular vesicles in bird's nest.

[0048] 2. Purification of fluorescently labeled extracellular vesicles from bird's nest cells (1) Fluorescent adsorption column buffer replacement ① Invert the fluorescent adsorption column to expel air bubbles. Break off and discard the tip of the adsorption column, then place it into a 2mL buffer collection tube. Open the protective cap on the top of the adsorption column to allow the buffer to drip out naturally. If the buffer does not drip out, replace the protective cap and open it again. ② Discard the buffer solution in the collection tube, then put the adsorption column back into the collection tube and centrifuge at 1000g for 2 minutes at room temperature. ③ Discard the buffer solution in the collection tube, then put the adsorption column back into the collection tube, slowly add 500 μL of 1×PBS to the top of the adsorption column packing, and centrifuge at 1000g for 1 min at room temperature. ④ Repeat step ③ twice. ⑤ Place the adsorption column into a new 1.5 mL collection tube and proceed to the next step immediately.

[0049] (2) Removal of free fluorescence. After the fluorescent labeling of bird's nest extracellular vesicles is completed, the reaction solution (≤ 100 μL) is slowly added to the top of the fluorescent adsorption column packing and centrifuged at 1000 g for 4 min at room temperature. The collected liquid is the purified fluorescently labeled bird's nest extracellular vesicles.

[0050] 3. Gavage tracing of fluorescently labeled extracellular vesicles from bird's nest C57BL / 6 mice were pretreated with environmental acclimatization and antibiotics before being administered PKH26-labeled extracellular vesicles from bird's nest cells via gavage, with physiological saline serving as a control. Brain tissue was harvested and frozen sections were prepared 24 hours after gavage, and the uptake of bird's nest extracellular vesicles by the tissues was observed using fluorescence microscopy.

[0051] Experimental results: The results are as follows Figure 5As shown, after mice were administered fluorescently labeled extracellular vesicles from bird's nest via gavage, their brain tissue sections were observed using a fluorescence microscope. The results showed that a clear red fluorescent signal was detected in the mouse brain tissue, and the fluorescently labeled extracellular vesicles were specifically distributed primarily in the hippocampus and cortical regions. These results indicate that the extracellular vesicles extracted from bird's nest using this invention can penetrate the blood-brain barrier and accumulate in target areas of the central nervous system, laying an anatomical foundation for their subsequent central nervous system regulatory activity.

[0052] Example 5 The protective effect of extracellular vesicles in bird's nest against oxidative stress damage in nerve cells Experimental methods: 1. Place the frozen HT22 cell tubes in a 37°C water bath and shake rapidly until they are the size of soybeans. Transfer them to 15 mL centrifuge tubes, centrifuge at 800 rpm for 4 min, collect the pellet, resuspend it in complete culture (DMEM + 10% FBS + 1% penicillin-streptomycin, v / v), add it to a culture dish, and incubate at 37°C and 5% CO2 for 36 h.

[0053] 2. When the cell aggregation reaches 75%, discard the culture medium, rinse twice with PBS buffer, add 1 mL of 0.25% trypsin solution (v / v) and digest at 37℃ for 40 s, add complete culture medium to stop digestion, pipette and centrifuge at 800 rpm for 4 min, resuspend the precipitate and add fresh complete culture medium to continue culturing.

[0054] 3. Seed 100 μL of HT22 cell suspension into each well of a 96-well plate. After complete cell adhesion, proceed with the corresponding treatments according to the following groups: (1) Blank group: Replace 100 μL of complete culture medium in each well (this group has no cells and no drug treatment); (2) Control group: 100 μL of complete culture medium was replaced in each well to maintain normal culture of HT22 cells without drug intervention; (3) Hydrogen peroxide group (H2O2): Replace 100 μL of complete culture medium in each well, and add H2O2 to a final concentration of 500 μM after 2 h; (4) Bird's nest extracellular vesicle group: Discard the original culture medium and add 100 μL of bird's nest extracellular vesicle solution prepared in Example 1, diluted 1000 times with complete culture medium to each well; (5) Hydrogen peroxide + bird's nest extracellular vesicle treatment group: Discard the original culture medium, add 100 μL of bird's nest extracellular vesicle solution diluted 1000 times with complete culture medium to each well, and add hydrogen peroxide to the final concentration of 500 μM after 48 h.

[0055] 4. After culturing for another 12 hours, cell viability was detected using a CCK-8 assay kit. 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 30 minutes. The absorbance at 450 nm was then measured using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = [(Experimental wells - Blank wells) / (Control wells - Blank wells)] × 100%.

[0056] Experimental results: The results are as follows Figure 6 As shown, hydrogen peroxide (H2O2) treatment significantly reduced the survival rate of hippocampal neurons in HT22 mice (P<0.05), indicating that H2O2 successfully induced a neuronal injury model. Treatment with extracellular vesicles from bird's nest alone had no significant effect on cell survival, indicating that the extracellular vesicles from bird's nest prepared in this invention have no significant cytotoxicity and possess good biosafety.

[0057] Further research revealed that the hydrogen peroxide + bird's nest extracellular vesicle treatment group significantly reversed the hydrogen peroxide-induced decrease in cell viability (P<0.05). These results indicate that bird's nest extracellular vesicles have a significant protective and repairing effect on H2O2-induced nerve cell damage.

[0058] Previous studies have shown that H2O2, as a significant member of reactive oxygen species (ROS), can induce severe oxidative stress when it accumulates excessively in the nervous system, attacking biomolecules such as proteins, lipids, and nucleic acids, leading to neuronal structural damage and functional impairment. H2O2-mediated oxidative stress has been proven to be widely involved in the pathological processes of various central nervous system diseases, including neurodegenerative diseases (such as Alzheimer's and Parkinson's), ischemia-reperfusion injury, and depression. Therefore, effectively clearing or neutralizing H2O2 and alleviating oxidative stress damage has become one of the important treatment strategies for these diseases. Extracellular vesicles from bird's nest have a significant protective effect against oxidative stress caused by hydrogen peroxide-induced neuronal damage, thus possessing potential therapeutic value for central nervous system diseases.

[0059] Example 6 The protective effect of extracellular vesicles in bird's nest on mitochondrial energy metabolism in nerve cells 1. Place the frozen HT22 cell tubes in a 37°C water bath and shake rapidly until they are the size of soybeans. Transfer them to 15 mL centrifuge tubes, centrifuge at 800 rpm for 4 min, collect the pellet, resuspend it in complete culture medium (DMEM + 10% FBS + 1% penicillin-streptomycin, v / v), add it to a culture dish, and incubate at 37°C and 5% CO2 for 36 h.

[0060] 2. When the cell aggregation reaches 75%, discard the culture medium, rinse twice with PBS buffer, add 1 mL of 0.25% trypsin solution (v / v) and digest at 37℃ for 40 s, add complete culture medium to stop digestion, pipette and centrifuge at 800 rpm for 4 min, resuspend the precipitate and add fresh complete culture medium to continue culturing.

[0061] 3. Seed 2 mL of HT22 cell suspension into each well of a 6-well plate. After complete cell adhesion, add 200 μL of lysis buffer (equivalent to 1 / 10 of the 2 mL cell culture medium volume) to each well of the corresponding treatment plates to lyse the cells. After lysis, centrifuge at 12000 g for 5 minutes at 4ºC, and collect the supernatant for subsequent assays. The groups were: control group, hydrogen peroxide group (H2O2), bird's nest extracellular vesicle group, and hydrogen peroxide + bird's nest extracellular vesicle treatment group.

[0062] 4. Two methods for supernatant determination: ① One method involves preparing an appropriate amount of ATP detection working solution according to the ratio of 100 μL of ATP detection working solution required for each sample or standard. Thaw the reagent to be used on an ice bath. Take an appropriate amount of ATP detection reagent and dilute it with ATP detection reagent diluent at a ratio of 1:9. For example, add 100 μL of ATP detection reagent to 900 μL of ATP detection reagent diluent to prepare 1 mL of ATP detection working solution. The diluted ATP detection reagent is the ATP detection working solution used for subsequent experiments. The ATP detection working solution can be temporarily stored on an ice bath. Add 100 μL of ATP detection working solution to the detection well or detection tube. Incubate at room temperature for 3-5 minutes to allow all background ATP to be consumed, thereby reducing the background. 100 μL of ATP detection working solution can be added to 10-20 detection wells or detection tubes at once. Then add 20 μL of sample or standard to the detection well or tube, mix quickly with a pipette (micropipette), and after at least 2 seconds, measure the RLU value with a chemiluminescence analyzer.

[0063] ② A type of cellular protein extraction was performed. The experiment included three groups: a control group, a hydrogen peroxide group (H2O2), and a group treated with hydrogen peroxide and bird's nest extracellular vesicles. BCA protein quantification was performed on the supernatant: serially diluted BSA standard + protein sample, OD562 was measured using an ELISA reader, and the protein concentration of each group was calculated. Protein samples underwent denaturation, electrophoresis, electroporation, and chemiluminescence imaging. Images of p-ATF2, ATF2, and GAPDH protein bands were acquired for data analysis.

[0064] Experimental results: like Figure 7As shown in Figure A, compared with the blank control group, the ATP concentration of the group treated with bird's nest extracellular vesicles alone did not change significantly (P > 0.05). The ATP content in nerve cells treated with H2O2 was significantly reduced (P < 0.0001), indicating that H2O2 successfully induced cellular energy metabolism disorders. Compared with the H2O2-only model group, the ATP content in nerve cells treated with H2O2 + bird's nest extracellular vesicles significantly increased after 24 h of treatment (P < 0.001). These results indicate that bird's nest extracellular vesicles can reverse H2O2-induced mitochondrial energy metabolism disorders and maintain intracellular ATP homeostasis. It should be noted that there was no significant difference in ATP measurement results between the control group and the group treated with bird's nest extracellular vesicles alone.

[0065] Western blot (7B) and grayscale quantification (7C) results show that the p-ATF2 / total ATF2 ratio characterizes the ATF2 pathway activation level. Compared with the blank control group, the p-ATF2 / total ATF2 ratio in the H2O2 model group was significantly downregulated (P<0.0001), suggesting that H2O2-induced mitochondrial damage can significantly inhibit the phosphorylation activation of ATF2 protein. After intervention with bird's nest extracellular vesicles, the p-ATF2 / total ATF2 ratio in the H2O2 + bird's nest extracellular vesicle treatment group was significantly restored compared with the H2O2 model group alone (P<0.01), indicating that bird's nest extracellular vesicles can effectively restore the phosphorylation activation level of the ATF2 pathway in oxidatively damaged cells.

[0066] The above experimental results demonstrate that extracellular vesicles from bird's nest can significantly reverse H2O2-induced ATP depletion and restore ATF2 phosphorylation activation levels. The extracellular vesicles from bird's nest prepared in this invention may exert potential therapeutic effects on central nervous system diseases by improving mitochondrial energy metabolism and regulating ATF2 signaling pathway activity, and have clear application value in anti-mitochondrial damage and cell protection.

[0067] Example 7 The effect of extracellular vesicles from bird's nest on improving depressive and anxious behavior in mice. 1. Laboratory animals and grouping After 1 week of adaptive feeding, 5-6 week old male C57BL / 6J mice were randomly divided into a control group, a chronic restraint stress + PBS group, and a CRS + bird's nest extracellular vesicle group, with no fewer than 9 mice in each group.

[0068] 2. Construction of a depression and anxiety model and intervention program Chronic Restrictive Stress (CRS) Modeling: CRS is a classic method for constructing a depression model. Mice in the CRS + PBS group and the CRS + bird's nest extracellular vesicle group were subjected to 6 hours of restriction stimulation daily in a restriction tube for 14 days. During restriction, the mice only experienced limited movement; the restriction tube did not compress the body or tail. All mice were deprived of food and water during the restriction period. After the restriction ended each day, the mice were provided with normal water and food. After the CRS modeling was completed, the CRS + bird's nest extracellular vesicle group was administered bird's nest extracellular vesicles (0.5 mg / kg, prepared in Example 1) by gavage, while the control group and the CRS + PBS group were administered an equal volume of PBS by gavage. Forced swimming and open field tests were performed after the CRS modeling and drug administration to assess the mice's depressive-anxiety-like behaviors.

[0069] 3. Behavioral experiments (1) Open field experiment Evaluation of anxiety-like behavior. During the experiment, mice were removed from their cages and placed in the center of a 50 cm × 50 cm × 30 cm white open area, facing away from the experimenter, and allowed to move freely for 5 minutes. VisuTrack animal behavior analysis software was used to monitor the mice's movement in real time. After each mouse's experiment, any remaining feces and urine were cleaned up, and 75% alcohol was sprayed into the open area to completely remove any odor left by the mice. The next round of the experiment was conducted after the alcohol had completely evaporated. The time spent in the central area and the total distance traveled within 5 minutes were recorded.

[0070] (2) Forced swimming To evaluate depressive behavior, a transparent cylindrical container approximately 10 cm in diameter and 25 cm in height was used. The water depth was approximately 10–15 cm, and the water temperature was maintained at 23–25°C. Mice were forced to swim for 6 minutes, and the immobility time within the last 4 minutes was recorded as the core endpoint (immobility was defined as the mouse ceasing to struggle, adopting a floating posture, and only making slight limb movements to keep its head above water). The first 2 minutes were considered an adaptation period and were not included in the analysis.

[0071] Experimental results: In the open field experiment, the central region activity time of mice in each group was as follows: Figure 8 As shown in Figure A (the vertical axis represents the activity time in the central region, in minutes), compared with the control group, the activity time in the central region of mice in the CRS+PBS group was significantly shortened (P<0.05); compared with the CRS+PBS group, the central activity time of mice in the CRS+bird's nest extracellular vesicle group was significantly increased (P<0.05), and similar to that of the control group, suggesting that the anxiety state of mice was improved after intervention with bird's nest extracellular vesicles. Figure 8As shown in B, in the forced swimming experiment, compared with the CRS+PBS group, the immobility time of the mice in the CRS+bird's nest extracellular vesicle group was significantly reduced (P<0.05), suggesting that the depressive state of the mice was improved after the intervention of bird's nest extracellular vesicles.

[0072] The above results suggest that chronic restraint stimulation can induce a depression-anxiety-like phenotype in mice, and treatment with extracellular vesicles from bird's nest can effectively alleviate this phenotype, indicating that extracellular vesicles from bird's nest have an antidepressant and anti-anxiety effect.

[0073] Example 8 Extracellular vesicles from bird's nest increased the content of bile acid metabolites in the hippocampus of depressed and anxious mice. 1. Extraction of mouse hippocampal tissue samples (1) Take out the hippocampal tissue samples of mice in the chronic restraint stress CRS+PBS group and CRS+bird's nest extracellular vesicle group in Example 7 from the -80℃ freezer and thaw them on ice until there are no ice cubes in the samples (all subsequent operations are performed on ice). (2) After the sample is thawed, vortex for 10 s to mix, and transfer 50 μL of the sample into the corresponding numbered centrifuge tube; (3) Add 150 μL of 20% acetonitrile methanol internal standard extraction solution, vortex for 3 min, and centrifuge at 12000 r / min for 10 min at 4℃; (4) After centrifugation, transfer 150 μL of the supernatant to another centrifuge tube with the corresponding number, and let it stand in a -20℃ refrigerator for 30 min; (5) Centrifuge at 12000 r / min for 3 min at 4℃, and transfer 120 μL of the supernatant into the inner liner of the corresponding sample vial for analysis.

[0074] 2. Chromatographic and mass spectrometric acquisition conditions 2.1 T3 Chromatographic Conditions (1) Chromatographic column: Waters ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 mm * 100 mm (2) Mobile phase A: 0.1% formic acid / water; Mobile phase B: 0.1% formic acid / acetonitrile (3) Column temperature: 40℃; flow rate: 0.4 mL / min; injection volume: 3 μL Table 3. Mobile phase gradient conditions for T3 column 2.2 Mass Spectrometry Conditions Table 4 Exactive HF-X Mass Spectrometry Conditions 2.3 Data Preprocessing Raw mass spectrometry data were converted to mzML format using ProteoWizard. Peak extraction, alignment, and retention time correction were performed using XCMS. Peaks with a missing rate >50% in each sample group were filtered, and blank values ​​were filled using KNN with 1 / 5 minimum value (1 / 5 minimum value for blank values ​​>50%, KNN for blank values ​​<50%). Peak area was corrected using the SVR method. Metabolite identification was performed on the corrected and filtered peaks by searching the laboratory's self-built database, integrating public libraries, and prediction libraries. Finally, substances with a comprehensive score of 0.5 or higher and a QC sample CV value of less than 0.3 were extracted and identified. Positive and negative patterns were then merged (if duplicate substances were found, the substance with the highest qualitative grade and highest score was retained), resulting in the all_sample_data.xlsx file.

[0075] 3. Data Results Analysis Before performing differential analysis, principal component analysis was first conducted on the grouped samples for comparison to observe the magnitude of variability between and within different groups. Log2 transformation and unit variance scaling (UV) were applied to the data, and the OPLSR.Anal function in the MetaboAnalyst package of R software was used for analysis. Based on the Variable Importance in Projection (VIP) obtained from the OPLS-DA model (biological replicates ≥ 3), metabolites showing differential differences between different groups were initially screened. Simultaneously, the p-value / FDR (biological replicates ≥ 2) from univariate analysis could be used to further screen differentially expressed metabolites. Metabolites with p < 0.05 (t-test) were selected. A statistically significant difference in metabolites between different groups was considered statistically significant.

[0076] Experimental results: Quantitative and qualitative analysis of metabolites in mouse hippocampus tissue was performed based on non-targeted metabolomics data, such as... Figure 9 Statistical analysis showed that, compared with the CRS+PBS group, treatment with extracellular vesicles from bird's nest significantly increased the concentrations of tauride-deoxycholic acid, lithocholic acid, and cholic acid in the hippocampus of mice. Previous studies have found that tauride-deoxycholic acid, lithocholic acid, and cholic acid have anti-apoptotic and neuroinflammation-reducing effects. The above results suggest that extracellular vesicles from bird's nest may exert neuroprotective effects by improving bile acid metabolism in mice.

[0077] Example 9 This embodiment provides a control experiment example. 1. Preparation of extracellular vesicles from bird's nest after stewing Preparation of extracellular vesicles from stewed bird's nest: Bird's nest was soaked, stewed, homogenized, filtered, purified, and resuspended to obtain extracellular vesicles. The specific method was as follows: A certain amount of dried bird's nest was weighed and added to purified water at a material-to-liquid ratio of 1:20-1:40. The mixture was soaked at 10-37℃ for 4-6 hours, then stewed in boiling water for 1-3 hours. The bird's nest was homogenized for 3-5 minutes and filtered. The filtrate was centrifuged at 12000 g for 30-90 minutes at low temperature (preferably 2-6℃). The supernatant was filtered through a 0.22 μm filter membrane to obtain the filtrate. A certain volume (30-40 mL) of the filtrate was purified and extracted using ultrasonic nanofiltration (this step is the same as in Example 1), with a purification time of 50-120 minutes. Finally, the filtrate was resuspended in a certain volume (200-400 μL) of PBS to obtain the purified extracellular vesicle solution from the stewed bird's nest.

[0078] 2. Characterization of extracellular vesicles in bird's nest after stewing: (1) Observation by transmission electron microscopy Take 10 μL of extracellular vesicle solution and drop it onto a pre-prepared Parafilm sealing film (with the back of the Parafilm sealing film adhering to the table). Place the copper mesh of the film face down and allow it to naturally absorb the suspension droplet for 15 min. Then, use filter paper to absorb the excess droplet and let the copper mesh dry slightly. Next, take 10 μL of 2% phosphotungstic acid solution (w / v) as the staining solution and drop it onto the Parafilm sealing film. Place the copper mesh face down with the staining solution and invert it to stand for 5 min. Use filter paper to absorb the excess droplet, let the copper mesh dry under an incandescent lamp, and observe and photograph it under a transmission electron microscope.

[0079] (2) Particle size and concentration The particle size distribution and concentration of extracellular vesicles were determined using a nanoflow cytometer (NanoFCM, Flow NanoAnalyzer U30E).

[0080] (3) Zeta potential The zeta potentials of extracellular vesicles were determined using a nanoparticle tracking analyzer (ParticleMetrix, ZetaView version 8.05.14SP7).

[0081] 3. Detection of the effect of extracellular vesicles of bird's nest on nerve cell activity after stewing The experimental method is the same as in Example 5, but the grouping is set as follows: (1) Blank: Replace 100 μL of complete culture medium in each well (this group has no cells and no drug treatment). The data in this group are only needed for activity analysis and are not included in the statistical comparison. (2) Control: 100 μL of complete culture medium was replaced in each well to maintain normal culture of HT22 cells without drug intervention; (3) Hydrogen peroxide group (H2O2): Replace 100 μL of complete culture medium in each well, and add H2O2 to a final concentration of 500 μM after 2 h; (4) Hydrogen peroxide + stewed bird's nest extracellular vesicle group: the original culture medium was discarded, and 100 μL of extracellular vesicle solution (prepared in Example 1) diluted 1000 times with complete culture medium was added to each well. After 48 h, hydrogen peroxide was added to the final concentration of 500 μM.

[0082] (5) Hydrogen peroxide + bird's nest extracellular vesicle group: Discard the original culture medium, add 100 μL of extracellular vesicle solution (prepared in Example 1) diluted 1000 times with complete culture medium to each well, and add hydrogen peroxide to the final concentration of 500 μM after 48 h.

[0083] After culturing for another 12 hours, cell viability was detected using a CCK-8 assay kit. 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 30 minutes. The absorbance at 450 nm was then measured using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = [(Experimental wells - Blank wells) / (Control wells - Blank wells)] × 100%.

[0084] Experimental results: This study compared the morphology, particle size concentration, zeta potential, and neuroprotective activity of extracellular vesicles from uncooked and high-temperature cooked bird's nests. The results are as follows: Figure 10 As shown: The extracellular vesicles derived from bird's nest exhibit a typical saucer-like double-membrane structure, with an intact membrane structure consistent with the morphological characteristics of animal-derived extracellular vesicles. This indicates that stewing does not significantly damage the basal membrane morphology of the vesicles. Figure 10 A).

[0085] Nanoparticle tracking analysis ( Figure 10 (B and particle size data in Tables 5 and 6) Comparing the two groups of samples: The median extracellular vesicle size of the uncooked bird's nest was 176.4 nm, and the particle size concentration was 1.2 × 10⁻⁶. 10 The median particle size of the extracellular vesicles in the bird's nest after stewing was 194.9 nm, and the particle size concentration was 8.0 × 10⁻⁶. 10 The particle size / mL also showed an increase in the proportion of large-diameter aggregated particles and the appearance of new secondary particle size peaks, proving that high-temperature stewing may change the original particle size and distribution characteristics.

[0086] Zeta potential detection ( Figure 10As shown in C), the Zeta potential of extracellular vesicles in bird's nest after stewing was -41.07±1.47 mV. The higher absolute value of the negative potential indicates that the colloidal stability of the vesicle aqueous solution improved after stewing, making it less prone to aggregation and sedimentation between particles. The CCK-8 experimental results show that ( Figure 10 (D) Regarding nerve cells damaged by oxidative stress, the protective effect of stewed bird's nest extracellular vesicles in improving nerve cell survival rate was significantly weaker than that of unstewed bird's nest extracellular vesicles. Although stewing improved the colloidal dispersion stability of the vesicles, cell function experiments showed that stewing severely weakened the neuroprotective efficacy of bird's nest extracellular vesicles.

[0087] Table 5. Extracellular vesicle size and concentration in bird's nest Table 6. Extracellular vesicle size and concentration of bird's nest after stewing A comprehensive comparison shows that while high-temperature stewing can improve the dispersion stability of extracellular vesicles in solution, it can also cause vesicle aggregation, increase particle size, and significantly reduce their biological activity against neuro-oxidative damage. The neuroprotective effect of extracellular vesicles in stewed bird's nest is significantly worse, which is not conducive to preserving their neuro-repair-related effects.

[0088] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0089] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. The application of extracellular vesicles from bird's nest in the preparation of drugs for treating mental illnesses, characterized in that... The active ingredient of the extracellular vesicles of the bird's nest includes an oligopeptide composition, which is a combination of dipeptides and tripeptides; the oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

2. The application as described in claim 1, characterized in that, The molecular weight range of the oligopeptide composition is 150 Da to 380 Da.

3. The application as described in claim 1, characterized in that, The mental illness mentioned is anxiety disorder.

4. The application as described in claim 1, characterized in that, The mental illness mentioned is depression.

5. The application as described in claim 1, characterized in that, The dosage form of the drug includes dry powder, capsules, or solution; or, the route of administration of the drug includes oral administration, nasal administration, or intravenous administration.

6. The application as described in claim 1, characterized in that, The average particle size of the extracellular vesicles in the bird's nest ranges from 100 to 200 nm, and the extracellular vesicles in the bird's nest exhibit a typical saucer-shaped or cup-shaped structure.

7. The application of extracellular vesicles from bird's nest in the preparation of products for regulating mood, characterized in that... The product includes health supplements or functional foods; the active ingredient of the extracellular vesicles of the bird's nest includes an oligopeptide composition, which is a combination of dipeptides and tripeptides; the oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

8. The application of extracellular vesicles from bird's nest in the preparation of products that improve oxidative stress damage in nerve cells, characterized in that... The product includes pharmaceuticals, health products, or functional foods; the active ingredient of the extracellular vesicles of the bird's nest includes an oligopeptide composition, which is a combination of dipeptides and tripeptides; the oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

9. The application of extracellular vesicles from bird's nest in the preparation of products that improve the energy metabolism of nerve cells, characterized in that... The product includes pharmaceuticals, health products, or functional foods; the active ingredient of the extracellular vesicles of the bird's nest includes an oligopeptide composition, which is a combination of dipeptides and tripeptides; the oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

10. The application of extracellular vesicles from bird's nest in the preparation of products that improve bile acid metabolism, characterized in that... The product includes pharmaceuticals, health products, or functional foods; the active ingredient of the extracellular vesicles of the bird's nest includes an oligopeptide composition, which is a combination of dipeptides and tripeptides; the oligopeptide composition includes L-phenylalanine-L-proline, leucine-glycine-leucine, L-leucyl-L-glycine, tryptophan-proline, threonine-valine-leucine, and isoleucine-glutamine-isoleucine.

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