Application of Cacumen biotae-derived extracellular vesicles in preparation of drugs for treating anxiety, depression and insomnia

Abstract

The invention discloses application of Cacumen biotae-derived extracellular vesicles in preparation of drugs for treating anxiety, depression or insomnia. The EVs are separated and purified from cacumen biotae through a specific differential ultracentrifugation method, the EVs have a typical double-layer membrane structure, the average particle size is about 100 nm, about 1012 EVs can be extracted from each gram of cacumen biotae, and 62 volatile components are contained. Cacumen biotae EVs can be taken by PC12 cells, and corticosterone-induced neuron damage can be relieved; for chronic constraint stress model mice, anxiety behaviors and spatial learning and memory disorders can be improved, and central nervous inflammation can be inhibited; for chronic unpredictable mild stress model mice, the weight, the sucrose preference rate and the cognitive ability can be recovered, and the levels of 5-hydroxytryptamine and gamma-aminobutyric acid are increased; and for 4-chloro-DL-phenylalanine induced insomnia model mice, the sleep condition can be improved, the neurotransmitter level can be recovered, and the effect is equivalent to that of positive drugs such as citalopram, fluoxetine, diazepam and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of extracellular vesicles derived from Platycladus Orientalis Leaf (EVs) in the preparation of drugs for treating anxiety, depression and insomnia-related diseases, particularly suitable for acting on the central nervous system via intranasal administration to improve stress-induced mental illnesses and sleep disorders. Background Technology

[0002] Anxiety, depression, and insomnia are prevalent mental illnesses and sleep disorders worldwide. Their pathogenesis is complex, often involving neurotransmitter imbalances (such as abnormal levels of serotonin 5-HT and γ-aminobutyric acid GABA), circadian rhythm disturbances, and neuroinflammatory responses. These three often occur simultaneously, severely impacting patients' quality of life. Current clinical treatments have significant limitations: 1) The blood-brain barrier (BBB) ​​hinders drug delivery: The central nervous system has a strict blood-brain barrier; over 98% of potential therapeutic drugs cannot penetrate this barrier to reach their target sites, significantly reducing the efficacy of drugs for central nervous system disorders. 2) Existing drugs have significant side effects: Selective serotonin reuptake inhibitors (SSRIs) for anxiety and depression (such as citalopram and fluoxetine) have slow onset of action and are prone to causing gastrointestinal reactions and sexual dysfunction; benzodiazepines for insomnia... Drugs like diazepam carry risks such as dependence and drowsiness the following day. 3) The disease mechanism is multidimensional, and single-target drugs have limited effects: The pathological processes of anxiety, depression and insomnia involve abnormal regulation of multiple systems such as neurotransmitters, inflammatory responses and circadian rhythms. Traditional drugs with a single mechanism of action are difficult to achieve multidimensional intervention.

[0003] Extracellular vesicles (EVs) are a class of membrane-bound nanostructures secreted by cells. They can mediate intercellular communication by carrying lipids, proteins, RNA, and small molecule active substances, demonstrating the advantages of "natural nanocarriers" in disease treatment. Compared to animal-derived EVs, plant-derived EVs (Plant-EVs) are characterized by low immunogenicity, wide availability, rich in natural active ingredients (such as flavonoids and terpenoids), and high stability, making them suitable as drug delivery carriers or directly as therapeutic agents.

[0004] Chinese arborvitae (Platycladus orientalis (L.) Franco), a traditional East Asian medicinal plant, has its leaves used in traditional medicine to treat inflammation, infection, and other diseases. Modern research has confirmed that its terpenoids, flavonoids, and other components have antioxidant and neuroprotective effects. However, there are currently no reports on the isolation and purification methods of EVs derived from Chinese arborvitae leaves, nor on their application in treating anxiety, depression, and insomnia. Furthermore, the technical challenge of how to efficiently penetrate the blood-brain barrier and target the central nervous system with the active ingredients from Chinese arborvitae leaves remains unresolved. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing the application of extracellular vesicles derived from Platycladus orientalis leaves in the preparation of medicaments for treating anxiety, depression, or insomnia-related diseases.

[0006] Another object of the present invention is to provide a pharmaceutical composition for treating anxiety, depression or insomnia-related diseases and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] Application of extracellular vesicles derived from Platycladus orientalis leaves in the preparation of drugs for treating anxiety, depression or insomnia-related diseases.

[0009] Preferably, the extracellular vesicles derived from Platycladus orientalis leaves are isolated and purified by the following method:

[0010] (1) Material preparation: Select fresh arborvitae leaves, wash them and remove the petioles, add pre-cooled 0.9% physiological saline, and homogenize them at 4℃ using a tissue homogenizer to obtain arborvitae leaf homogenate.

[0011] (2) Differential ultracentrifugation: First, the homogenate was centrifuged at 4℃ and 3000×g for 30 min and the supernatant was collected. Second, the supernatant was filtered through a 0.45μm microporous membrane and centrifuged at 4℃ and 10000×g for 30 min and the supernatant was collected. Third, the supernatant was transferred to an ultracentrifuge tube and centrifuged at 4℃ and 120000×g for 60 min. The supernatant was discarded and the precipitate was retained. Fourth, the precipitate was resuspended in a wetting buffer (20mM MES hydrate, 2mM CaCl2, 0.1M NaCl, pH 6.0) and centrifuged again at 4℃ and 120000×g for 60 min. Finally, the precipitate was resuspended in 1mL of wetting buffer to obtain an extracellular vesicle suspension from Platycladus orientalis leaves.

[0012] Preferably, the extracellular vesicles derived from Platycladus orientalis leaves have a typical double-membrane structure, an average particle size of about 100 nm, and about 10 vesicles can be extracted from each gram of Platycladus orientalis leaves. 12 One extracellular vesicle.

[0013] Preferably, the extracellular vesicles of the Platycladus orientalis leaves contain 62 volatile components, mainly including 15 monoterpenes (70.77%), 11 oxygenated sesquiterpenes (16.22%), 20 sesquiterpenes (9.50%), 2 oxygenated diterpenes (1.97%), 12 oxygenated monoterpenes (1.81%), and 2 isovalerates (0.11%). Among them, α-pinene, β-pinene, d-limonene, and elemol are the main active ingredients, and α-pinene accounts for more than 40% of the total volatile components.

[0014] Preferably, the drug acts on the central nervous system via intranasal administration, and the drug dosage form is nasal drops.

[0015] Preferably, the nasal drops are prepared by taking a suspension of extracellular vesicles from Platycladus orientalis leaves, adding mannitol as a stabilizer, adding benzalkonium chloride as a preservative, adjusting the pH to 6.5-7.0 with PBS, filtering through a 0.22μm filter membrane for sterilization, and then dispensing.

[0016] 5. The application according to claim 6, characterized in that the concentration of the extracellular vesicle suspension of Platycladus orientalis leaves is 30 μg / μL-60 μg / μL, the final concentration of mannitol is 3%-5%, and the final concentration of benzalkonium chloride is 0.005%-0.01%.

[0017] A pharmaceutical composition for treating anxiety, depression or insomnia-related diseases, characterized in that it contains extracellular vesicles derived from Platycladus orientalis leaves, wherein the extracellular vesicles derived from Platycladus orientalis leaves are obtained by the following method: (1) Material preparation: Select fresh Platycladus orientalis leaves, wash them and remove the petioles, add pre-cooled 0.9% physiological saline, and homogenize them at 4°C using a tissue homogenizer to obtain Platycladus orientalis leaf homogenate.

[0018] (2) Differential ultracentrifugation: First, the homogenate was centrifuged at 4℃ and 3000×g for 30 min and the supernatant was collected. Second, the supernatant was filtered through a 0.45μm microporous membrane and centrifuged at 4℃ and 10000×g for 30 min and the supernatant was collected. Third, the supernatant was transferred to an ultracentrifuge tube and centrifuged at 4℃ and 120000×g for 60 min. The supernatant was discarded and the precipitate was retained. Fourth, the precipitate was resuspended in a wetting buffer (20mM MES hydrate, 2mM CaCl2, 0.1M NaCl, pH 6.0) and centrifuged again at 4℃ and 120000×g for 60 min. Finally, the precipitate was resuspended in 1mL of wetting buffer to obtain an extracellular vesicle suspension from Platycladus orientalis leaves.

[0019] Preferably, it also includes other pharmaceutical excipients for preparing the pharmaceutical composition into nasal drops.

[0020] The use of the pharmaceutical composition in the preparation of a medicament for treating anxiety, depression or insomnia-related disorders. Attached Figure Description

[0021] Figure 1 Images show the separation and characterization of EVs from Platycladus orientalis leaves. (A) Morphological characteristics of EVs from Platycladus orientalis leaves under transmission electron microscopy (TEM), showing their typical bilayer membrane structure, scale bar 100 nm; (B) Quantitative results of particle size distribution and concentration of EVs from Platycladus orientalis leaves obtained by nanoparticle tracking analysis (NTA), showing an average particle size of approximately 100 nm, and approximately 10 EVs can be extracted from each gram of Platycladus orientalis leaves. 12 EVs.

[0022] Figure 2 This is a chromatogram showing the volatile components in *Platycladus orientalis* leaf EVs. Gas chromatography-mass spectrometry (GC-MS) revealed 62 volatile components in *Platycladus orientalis* leaf EVs, mainly including monoterpenes (15 types, accounting for 70.77%) and oxygenated sesquiterpenes (11 types, accounting for 16.22%). Among these, α-pinene, β-pinene, d-limonene, and elemol were the main active ingredients, with α-pinene accounting for over 40% of the total volatile components.

[0023] Figure 3 This is a GO and KEGG enrichment analysis of the volatile components of Platycladus orientalis leaf EVs. GO analysis shows that the target functions of the volatile components are mainly enriched in biological processes directly related to anxiety, depression, and insomnia, such as "circadian rhythm regulation," "sleep-wake cycle regulation," and "GABAergic synaptic transmission." KEGG analysis shows that its pathways of action are concentrated in "mammalian circadian rhythm," "neuroactive ligand-receptor interaction," "GABAergic synapses," and "glutamatergic synapses," confirming that Platycladus orientalis leaf EVs can exert therapeutic effects by regulating key central signaling pathways.

[0024] Figure 4 The image shows the effect of arborvitae leaf EVs being taken up by PC12 cells and protecting against corticosterone-induced neuronal damage. (A) Laser confocal microscopy observation results: DiI-labeled arborvitae leaf EVs (red) can enter PC12 cells (cell nuclei are stained blue by DAPI), scale bar 10 μm; (B) Cell viability assay (CCK-8 assay) results: Compared with the corticosterone-only treatment group, the viability of PC12 cells treated with 5 μg / mL and 10 μg / mL arborvitae leaf EVs was significantly increased (p<0.05, p<0.01); (C) Lactate dehydrogenase (LDH) release assay results: Arborvitae leaf EVs can dose-dependently reduce corticosterone-induced LDH release in PC12 cells (p<0.05, p<0.01), indicating that it can alleviate neuronal damage.

[0025] Figure 5 The diagram shows the anti-anxiety effect of Platycladus orientalis leaf EVs on mice with chronic restraint stress (CRS). (A) Changes in body weight of mice in each group: The body weight of the CRS model group was significantly lower than that of the normal control group. After treatment with Platycladus orientalis leaf EVs (10 μg / kg), the body weight recovered to near normal levels (p<0.01); (B) Social interaction test (SI) results: The social interaction index of mice in the EVs-treated group was significantly higher than that of the model group (p<0.01), which was comparable to that of citalopram (10 mg / kg); (C) Novel object inhibition feeding test results: The feeding latency of mice in the EVs-treated group was significantly shortened (p<0.01); (DG) Morris water maze (MWM) test results: The escape latency of mice in the EVs-treated group was shortened and the time spent in the target quadrant was prolonged (p<0.01), indicating that it can improve CRS-induced spatial learning and memory impairment.

[0026] Figure 6 This image shows the regulatory effect of Platycladus orientalis leaf EVs on the mRNA expression of inflammatory factors in the hippocampus and cortex of CRS model mice. (AD) Compared with the CRS model group, the mRNA expression of pro-inflammatory factors TNF-α and IL-6 in the hippocampus and cortex of mice treated with Platycladus orientalis leaf EVs was significantly reduced (p<0.01), while the mRNA expression of anti-inflammatory factor TGF-β and phagocytic marker CD206 was significantly increased (p<0.01), confirming that it can inhibit central nervous system inflammation.

[0027] Figure 7 The image shows the antidepressant effect of Platycladus orientalis leaf EVs on a mouse model of chronic unpredictable mild stress (CUMS). (A) Changes in body weight of mice in each group: the body weight of the EVs-treated group was significantly higher than that of the CUMS model group (p<0.01); (B) Sucrose preference test (SPT) results: the sucrose preference rate of the EVs-treated group was significantly higher than that of the model group (p<0.01), improving anhedonia symptoms; (CF) MWM test results: the escape latency was shortened and the time spent in the target quadrant was prolonged in the EVs-treated group (p<0.01), improving cognitive impairment; (GJ) Detection of serum and hippocampal 5-HT and GABA concentrations (ELISA method): the levels of both neurotransmitters in the EVs-treated group were significantly higher than those in the model group (p<0.01), comparable to the effect of fluoxetine.

[0028] Figure 8The image shows the sleep-promoting effect of Platycladus orientalis leaf EVs on a PCPA-induced insomnia model mouse. (A) Changes in body weight of mice in each group. The body weight of the EVs-treated group was significantly higher than that of the PCPA model group (p<0.01); (BD) Results of sodium pentobarbital-induced sleep test. The EVs (5μg / kg, 10μg / kg) treatment group showed increased sleep onset rate, shortened sleep latency, and prolonged sleep duration (p<0.05, p<0.01), comparable to the effect of diazepam (1.2mg / kg); (EH) Results of serum and hippocampal 5-HT and GABA concentration detection. The levels of the two neurotransmitters in the EVs-treated group were significantly higher than those in the model group (p<0.01), confirming that it improves insomnia by restoring neurotransmitter balance. Detailed Implementation

[0029] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0030] Example 1: Isolation, purification and characterization of EVs from Platycladus orientalis leaves

[0031] 1) Material preparation

[0032] Fresh arborvitae leaves from the Lushan region (identified as Platycladus orientalis (L.) Franco by Lushan Botanical Garden and the Chinese Academy of Sciences) were selected, washed, and the petioles were removed, retaining the leaf tissue. The fresh arborvitae leaves were added to pre-cooled 0.9% physiological saline at a ratio of 1g:5mL, and homogenized using a tissue homogenizer at 4℃ for 5 minutes to obtain a arborvitae leaf homogenate.

[0033] 2) Differential overspeed centrifugal separation EVs

[0034] Step 1: Centrifugation: Centrifuge the homogenate at 4℃ and 3000×g for 30 min to remove large cell debris and collect the supernatant.

[0035] Filtration: The supernatant is filtered through a 0.45μm microporous membrane to further remove impurities;

[0036] Step 2: Centrifugation: Centrifuge the filtered supernatant at 4℃ and 10000×g for 30 min and collect the supernatant.

[0037] Step 3: Centrifugation: Transfer the supernatant to an ultracentrifuge tube and centrifuge at 4°C and 120,000 × g for 60 min. Discard the supernatant and retain the precipitate.

[0038] Washing and resuspension: The precipitate was resuspended in 10 mL of wetting buffer (20 mM MES hydrate, 2 mM CaCl2, 0.1 M NaCl, pH 6.0), centrifuged again at 4 °C and 120,000 × g for 60 min, and finally resuspended in 1 mL of wetting buffer to obtain the Platycladus orientalis leaf EV suspension.

[0039] 3) Characterization of EVs

[0040] Transmission electron microscopy (TEM) observation: 10 μL of EV suspension was dropped onto the surface of a copper mesh, fixed with 2.5% glutaraldehyde for 15 min, and then post-fixed with 2% osmium tetroxide for 10 min. After dehydration with graded acetone, the EVs were embedded in epoxy resin to prepare 80-90 nm ultrathin sections. The sections were stained with 5% uranium acetate for 15 min and 0.1% lead citrate for 5 min. Finally, the morphology was observed using a Hitachi 7500 transmission electron microscope.

[0041] Nanoparticle tracking analysis (NTA): The EV suspension was diluted to a suitable concentration with wetting buffer and detected using a NanoSight NS300 system (488nm laser) at 25°C, camera level 12, acquisition time 30s, and detection threshold 7.

[0042] Results: Extracellular vesicles (EVs) were isolated using differential ultracentrifugation and characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The results showed that the EVs possessed a typical bilayer membrane structure with an average diameter of approximately 100 nm. Figure 1 (AB). Approximately 10 mg of arborvitae leaves can be extracted per gram. 12 EVs.

[0043] Example 2: Volatile Component Analysis of Platycladus orientalis Leaves (EVs) (GC-MS)

[0044] Take 1 mL of EVs suspension, add 2 mL of HPLC-grade n-hexane, seal and soak at room temperature for 24 h (shaking once every 6 h during this period), add internal standard ethyl decanoate (final concentration 10 μg / mL), filter through a 0.22 μm nylon filter membrane, and analyze using an Agilent 7890A-5975C GC-MS system (HP-5MS capillary column, 30 m × 0.25 mm × 0.25 μm). Chromatographic conditions: injection port temperature 250℃, split ratio 10:1, carrier gas helium (1.0 mL / min), column temperature program: 50℃ for 2 min → 5℃ / min to 180℃ → 20℃ / min to 270℃ for 5 min; mass spectrometry conditions: electron ionization source (EI, 70 eV), ion source temperature 230℃, scan range 40-500 amu.

[0045] Results: The volatile components of extracellular vesicles (EVs) derived from Platycladus orientalis leaves were analyzed by gas chromatography-mass spectrometry (GC-MS), and a total of 62 compounds were identified. The main components of these EVs were monoterpenes (15 species, accounting for 70.77%), followed by oxygenated sesquiterpenes (11 species, accounting for 16.22%), sesquiterpenes (20 species, accounting for 9.50%), oxygenated diterpenes (2 species, accounting for 1.97%), oxygenated monoterpenes (12 species, accounting for 1.81%), and isovalerates (2 species, accounting for 0.11%). Figure 2 A. Table 1). Among these components, α-pinene, β-pinene, d-limonene, and elemol were present in the highest amounts, with α-pinene accounting for over 40% of the total volatile components. Figure 2 B. Table 1). The targets of action of volatile components in extracellular vesicles (EVs) derived from Platycladus orientalis leaves were screened and identified using the Swiss Target Prediction, PubChem, TCMSP, and PharmMapper databases. Metascape was used to perform pathway enrichment analysis on these targets, with screening criteria set at p < 0.01 and enrichment score > 20. Gene Ontology (GO) analysis showed that the functions of these targets mainly involve circadian rhythm regulation, circadian sleep-wake cycle regulation, sleep, and circadian rhythm-related processes. Figure 3 Kyoto Encyclopedia of Genetics and Genomes (KEGG) analysis identified pathways associated with mammalian circadian rhythms, circadian rhythm synchronization, neuroactive ligand-receptor interactions, GABAergic synapses, and glutamatergic synapses. Figure 3 This indicates that the main functions of volatile components in extracellular vesicles derived from Platycladus orientalis leaves are associated with anxiety, depression, and insomnia. These findings further confirm the potential of Platycladus orientalis leaf-derived EVs in treating anxiety, depression, and insomnia.

[0046] Example 3: Verification of cellular uptake and neuroprotective effects of Platycladus orientalis leaf EVs

[0047] DiI-labeled Platycladus orientalis leaf EVs: Take 100 μL of Platycladus orientalis leaf EV suspension (concentration approximately 10). 11 Add 1 μL of DiI staining solution (1 mg / mL) to the sample and incubate at room temperature in the dark for 30 min (gently invert and mix every 5 min). Then, remove free DiI by following the steps of "differential ultracentrifugation to separate EVs" in Example 1 (centrifugation at 3000×g for 30 min → filtration at 0.45 μm → centrifugation at 10000×g for 30 min → centrifugation at 120000×g for 60 min). Finally, resuspend the sample in 100 μL of DMEM medium to obtain DiI-labeled EVs.

[0048] Cell uptake assay: PC12 cells (rat adrenal medullary pheochromocytoma cell line) were seeded in confocal culture dishes (2×10⁻⁶ cells / year). 4 After culturing for 24 hours, DiI-labeled EVs (final concentration 10⁸ EVs / mL) were added, and the culture was continued for another 12 hours. The culture medium was discarded, and the EVs were washed three times with PBS. DAPI staining solution (1 μg / mL) was added and incubated in the dark for 10 minutes. After washing again, the EVs were observed using an Olympus FV3000 confocal microscope.

[0049] Corticosterone-induced neuronal injury protection experiment: PC12 cells were seeded in 96-well plates (1×10⁶ cells / wells). 5 The samples were divided into four groups: blank control group: DMEM medium only; model group: corticosteroids at a final concentration of 500 μM; low-dose EVs group: 500 μM corticosteroids + 5 μg / mL Platycladus orientalis EVs (approximately 10 samples / well). 9 EVs high-dose group: 500 μM corticosterone + 10 μg / mL Platycladus orientalis EVs (approximately 2 × 10⁻⁶ cells / mL); EVs high-dose group: 500 μM corticosterone + 10 μg / mL Platycladus orientalis EVs (approximately 2 × 10⁻⁶ cells / mL). 9 Cell viability was assessed using the CCK-8 assay after 24 h of culture (10 μL CCK-8 reagent was added, and absorbance was measured at 450 nm after 2 h of incubation). Cytotoxicity was assessed using the LDH release assay kit (supernatant was collected, and absorbance was measured at 490 nm according to the kit instructions). Results: DiI (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole carbocyanine perchlorate)-labeled extracellular vesicles (EVs) derived from Platycladus orientalis leaves were co-incubated with the rat adrenal medullary pheochromocytoma cell line PC12. After incubation, the cells were washed three times with phosphate-buffered saline (PBS) and then stained with 4',6-diamidinyl-2-phenylindole (DAPI). Microscopic observation revealed a large number of small granular DiI-labeled EVs (appearing blue) within PC12 cells (appearing red). Figure 4 A) This indicates that EVs derived from Platycladus orientalis leaves can be internalized in mammalian cells. Furthermore, the study found that these EVs could alleviate corticosterone-induced neuronal damage in PC12 cells and reduce the release of lactate dehydrogenase (LDH). Figure 4 BC).

[0050] Example 4: Preparation of Platycladus orientalis leaf EVs pharmaceutical formulation

[0051] Nasal drops: Take a suspension of EVs from Platycladus orientalis leaves (concentration 50 μg / μL), add an appropriate amount of mannitol (final concentration 5%) as a stabilizer, add 0.01% benzalkonium chloride as a preservative, adjust the pH to 6.5-7.0 with PBS, filter through a 0.22 μm filter membrane for sterilization, dispense into 100 μL light-protected dropper bottles, each bottle containing 5 mg of EVs, for intranasal administration (1-2 μL per nasal cavity each time).

[0052] Example 5: Anti-anxiety effect of Platycladus orientalis leaf EVs on CRS model mice

[0053] Animal model establishment and grouping: Eight-week-old male C57BL / 6 mice (purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.) were selected and, after acclimatization for 3 days, were divided into 4 groups (n=10 / group): Normal control group: No stress treatment was given, and an equal volume of PBS was administered intranasally daily; CRS model group: Mice were placed alone in a ventilated plastic cylinder (3 cm in diameter, length adjusted according to mouse weight) for 10 hours from 9:00 to 19:00 daily for 14 consecutive days, and an equal volume of PBS was administered intranasally daily; Citalopram positive control group: CRS treatment was the same as the model group, and 10 mg / kg citalopram was administered by gavage daily; EVs treatment group: CRS treatment was the same as the model group, and 10 μg / kg Platycladus orientalis EVs (diluted with PBS to a concentration of 50 μg / μL, with the mouse supine and head tilted at 45°, and administered intranasally using a micropipette) was administered intranasally daily.

[0054] Behavioral testing

[0055] Social Interaction Test (SI): The experiment consisted of two phases, each lasting 150 seconds. In the first phase, mice were placed in an open field box (45×45×45cm) containing an empty metal cage (10×6×45cm), and the time spent in the interaction area was recorded. In the second phase, an unfamiliar CD1 male mouse was placed in the metal cage, and the time spent in the interaction area was recorded again. The SI index was calculated as (time spent in the interaction area with a target / time spent in the interaction area without a target) × 100.

[0056] Novel object inhibition feeding test: After fasting for 24 hours, mice were placed in an open field box containing novel objects (such as plastic toys) and food particles, and the latency period from placement to the start of feeding was recorded.

[0057] Morris Water Maze (MWM) Test: A circular pool (160cm in diameter, water temperature 24-26℃) was divided into 4 quadrants. A platform was placed 2cm underwater in the target quadrant (Quadrant A). Training Phase: Twice daily, mice were placed in the water from different quadrants, and the escape latency to the platform within 90 seconds was recorded (if the latency exceeded 90 seconds, the mice were guided to the platform, and the latency was recorded as 90 seconds). This training was conducted for 3 consecutive days. Testing Phase: The platform was removed, and the time the mice spent in the target quadrant within 300 seconds was recorded.

[0058] Detection of mRNA expression of inflammatory factors: After the behavioral test, the mice were anesthetized with 350 mg / kg tribromoethanol in the peritoneum, euthanized by cervical dislocation, and the hippocampus and cortical tissue were separated. Total RNA was extracted with TRIzol reagent, reverse transcribed into cDNA, and then subjected to RT-qPCR (primer sequences are shown in Table 2) to detect the mRNA expression levels of TNF-α, IL-6, TGF-β, and CD206.

[0059] Results: The chronic restraint stress (CRS) model induced anxiety-like behaviors in mice, including social withdrawal and weight loss. Experimental results showed that mice in the CRS model group exhibited weight loss, reduced social interaction, and prolonged feeding latency. Figure 5 AC). Treatment with *Platycladus orientalis*-derived extracellular vesicles (EVs) was comparable to that with etanercept (10 mg / kg), significantly increasing mouse body weight, improving social interaction, and shortening feeding latency. Figure 5 AC). Assessment using the Morris water maze (MWM) test revealed impaired spatial learning and memory abilities in the CRS model group mice, specifically manifested as prolonged escape latency, longer swimming paths, decreased accuracy, and slower average speed. Figure 5 DG); while treatment with EVs derived from Platycladus orientalis leaves can reverse these functional defects, with effects comparable to epoxetine (DG). Figure 5 DG). Furthermore, treatment with EVs derived from Platycladus orientalis leaves restored the mRNA expression levels of CD206, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and interleukin-6 (IL-6) in the hippocampus and cortex of mice to normal levels. Figure 6 (AD), which suggests that these EVs can reduce neuroinflammatory responses.

[0060] Example 6: Antidepressant effect of Platycladus orientalis leaf EVs on CUMS model mice

[0061] Animal model establishment and grouping: Eight-week-old male C57BL / 6 mice were acclimatized to the environment for 3 days and then divided into 4 groups (n=10 / group): Normal control group: No stress treatment was given, and an equal volume of PBS was administered intranasally daily; CUMS model group: Three stressors were randomly selected daily (a total of 8: 24h fasting, 24h water deprivation, 2h restraint in 50mL centrifuge tubes, 10min tail clipping, 24h wet cage, 5min swimming in 4℃ cold water, 2h exposure to new odors, 10min cage shaking), and the treatment was continued for 28 days, with an equal volume of PBS administered intranasally daily; Fluoxetine positive control group: CUMS treatment was the same as the model group, and 10mg / kg fluoxetine was administered by gavage daily; EVs treatment group: CUMS treatment was the same as the model group, and 10μg / kg Platycladus orientalis EVs were administered intranasally daily.

[0062] Behavioral testing

[0063] Weight monitoring: Weigh the mice weekly and record weight changes.

[0064] Sucrose Preference Test (SPT): Mice were housed alone and first adapted to 1% sucrose water for 2 days. Then, they were fasted and deprived of water for 24 hours. They were given one bottle of 1% sucrose water and one bottle of pure water. After 12 hours, the bottles were exchanged. After 24 hours, the weight of the two bottles was measured. The sucrose preference rate was calculated as (sucrose water intake / total fluid intake) × 100%.

[0065] Morris water maze test: A circular pool (160cm in diameter, water temperature 24-26℃) is divided into 4 quadrants. A platform is placed 2cm underwater in the target quadrant (Quadrant A). Training phase: Twice daily, mice are placed in the water from different quadrants, and the escape latency to the platform within 90 seconds is recorded (if the latency exceeds 90 seconds, the mouse is guided to the platform, and the latency is recorded as 90 seconds). This training is continued for 3 days. Testing phase: The platform is removed, and the time the mouse spends in the target quadrant within 300 seconds is recorded.

[0066] Neurotransmitter detection: Serum and hippocampal tissue were collected after mouse sacrifice. 5-HT and GABA concentrations were detected using an ELISA kit (Wuhan Maike Biotechnology Co., Ltd.) (sensitivity 6.25 pg / mL). Results: The chronic unpredictable mild stress (CUMS) model induced a depressive-like phenotype in mice, including anhedonia and cognitive impairment. Experimental results showed that mice in the CUMS model group exhibited weight loss, decreased sucrose preference, impaired Morris water maze (MWM) test performance, and decreased serum and hippocampal levels of serotonin (5-HT) and gamma-aminobutyric acid (GABA). Figure 7 AF). Similar to the effects of fluoxetine (a selective serotonin reuptake inhibitor primarily used to treat major depressive disorder), intranasal administration of Platycladus orientalis-derived extracellular vesicles (EVs) restored body weight, sucrose intake, and spatial learning and memory abilities in mice. Figure 7 AF), and increased serum and hippocampal levels of 5-HT and GABA. Figure 7 (GJ). Notably, the Chronic Restraint Stress (CRS) model and the CUMS model share some overlap in behavioral deficits (reduced activity, cognitive impairment) and neurochemical disturbances (decreased 5-HT and GABA levels); however, their focuses differ. The CRS model primarily simulates anxiety-like behaviors and social dysfunction, while the CUMS model primarily simulates depressive-like behaviors (anhedonia, decreased sugar preference). Extracellular energy (EVs) derived from Platycladus orientalis leaves showed improvement in both anxiety-related and depression-related indicators, highlighting their potential for multimodal treatment across different stress-induced mental illness phenotypes.

[0067] Example 7: Hypnotic effect of Platycladus orientalis leaf EVs on 4-chloro-DL-phenylalanine (PCPA)-induced insomnia model mice. Animal model establishment and grouping: After 7 days of environmental adaptation, 50 mice with similar open field test (OFT) scores, body weight, and sucrose intake were selected and divided into 5 groups (n=10 / group): Normal control group: No modeling was performed, and the mice were given an equal volume of distilled water by gavage daily; PCPA model group: 0.4 g / kg was injected intraperitoneally at 8:00 am for 2 consecutive days. PCPA suspension (successful modeling criteria: disappearance of diurnal rhythm and increased daytime activity) was administered, followed by daily gavage with an equal volume of distilled water for 21 days; Diazepam positive control group: PCPA modeling was the same as the model group, followed by daily gavage with 1.2 mg / kg diazepam for 21 days; Low-dose EVs group: PCPA modeling was the same as the model group, followed by daily intranasal administration of 5 μg / kg Platycladus orientalis EVs for 21 days; High-dose EVs group: PCPA modeling was the same as the model group, followed by daily intranasal administration of 10 μg / kg Platycladus orientalis EVs for 21 days.

[0068] Sleep-related assays: Sodium pentobarbital-induced sleep test: 30 minutes after the last administration, mice were intraperitoneally injected with 40 mg / kg sodium pentobarbital. The time from the disappearance to the recovery of the righting reflex (sleep duration) and the time from injection to the disappearance of the righting reflex (sleep latency) were recorded. The sleep onset rate was calculated as (number of mice falling asleep / total number of mice × 100%). Body weight and neurotransmitter assays: Body weight was measured weekly; serum and hippocampal 5-HT and GABA concentrations were measured after mouse sacrifice.

[0069] Results: Parachlorophenylalanine (PCPA) is often used to treat carcinoid syndrome because it can reduce serotonin (5-HT) levels in mammals. Therefore, in the research field, PCPA is widely used to construct animal models of insomnia, especially suitable for research related to sleep disorders. Experimental results showed that PCPA-induced insomnia model mice exhibited weight loss, shortened total sleep time, and decreased levels of serotonin (5-HT) and γ-aminobutyric acid (GABA) in the hippocampus and serum. Figure 8 AH). With effects comparable to diazepam, treatment with extracellular vesicles (EVs) derived from Platycladus orientalis leaves significantly improved sleep onset, prolonged total sleep time, and restored neurotransmitter (5-HT and GABA) levels in mice. Figure 8 AH).

[0070] Table 1. Volatile components of EVs from Platycladus orientalis leaves.

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[0076] Table 2 Primer sequences

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Claims

1. Application of extracellular vesicles derived from Platycladus orientalis leaves in the preparation of drugs for treating anxiety, depression or insomnia-related diseases.

2. The application according to claim 1, characterized in that, The extracellular vesicles derived from Platycladus orientalis leaves were isolated and purified using the following method: (1) Material preparation: Select fresh arborvitae leaves, wash them and remove the petioles, add pre-cooled 0.9% physiological saline, and homogenize them at 4℃ using a tissue homogenizer to obtain arborvitae leaf homogenate. (2) Differential speed ultracentrifugation: First step, centrifuge the homogenate at 4℃ and 3000×g for 30min and collect the supernatant. The second step involves filtering the supernatant through a 0.45μm microporous membrane and centrifuging it at 4℃ and 10000×g for 30 min to collect the supernatant. The third step is to transfer the supernatant to an ultracentrifuge tube and centrifuge at 4°C and 120,000 × g for 60 min, discarding the supernatant and retaining the precipitate. Fourth, the precipitate was resuspended in wetting buffer (20 mM MES hydrate, 2 mM CaCl2, 0.1 M NaCl, pH 6.0), and centrifuged again at 4 °C and 120,000 × g for 60 min. Finally, the precipitate was resuspended in 1 mL of wetting buffer to obtain an extracellular vesicle suspension derived from Platycladus orientalis leaves.

3. The application according to claim 1, characterized in that, The extracellular vesicles derived from the leaves of Platycladus orientalis have a typical double-membrane structure, with an average particle size of approximately 100 nm. Approximately 10 vesicles can be extracted from each gram of Platycladus orientalis leaves. 12 One extracellular vesicle.

4. The application according to claim 1, characterized in that, The extracellular vesicles of the arborvitae leaves contain 62 volatile components, mainly including 15 monoterpenes (70.77%), 11 oxygenated sesquiterpenes (16.22%), 20 sesquiterpenes (9.50%), 2 oxygenated diterpenes (1.97%), 12 oxygenated monoterpenes (1.81%), and 2 isovalerates (0.11%). Among them, α-pinene, β-pinene, d-limonene, and elemol are the main active ingredients, and α-pinene accounts for more than 40% of the total volatile components.

5. The application according to claim 1, characterized in that, The drug acts on the central nervous system via intranasal administration, and the drug dosage form is nasal drops.

6. The application according to claim 5, characterized in that, The nasal drops are prepared as follows: take the extracellular vesicle suspension of Platycladus orientalis leaves, add mannitol as a stabilizer, add benzalkonium chloride as a preservative, adjust the pH to 6.5-7.0 with PBS, filter through a 0.22μm filter membrane for sterilization, and dispense into individual containers.

7. The application according to claim 6, characterized in that, The concentration of the extracellular vesicle suspension of Platycladus orientalis leaves was 30 μg / μL-60 μg / μL, the final concentration of mannitol was 3%-5%, and the final concentration of benzalkonium chloride was 0.005%-0.01%.

8. A pharmaceutical composition for treating anxiety, depression, or insomnia-related disorders, characterized in that, It contains extracellular vesicles derived from Platycladus orientalis leaves, which are obtained by isolation and purification using the following method: (1) Material preparation: Select fresh arborvitae leaves, wash them and remove the petioles, add pre-cooled 0.9% physiological saline, and homogenize them at 4℃ using a tissue homogenizer to obtain arborvitae leaf homogenate. (2) Differential speed ultracentrifugation: First step, centrifuge the homogenate at 4℃ and 3000×g for 30min and collect the supernatant. The second step involves filtering the supernatant through a 0.45μm microporous membrane and centrifuging it at 4℃ and 10000×g for 30 min to collect the supernatant. The third step is to transfer the supernatant to an ultracentrifuge tube and centrifuge at 4°C and 120,000 × g for 60 min, discarding the supernatant and retaining the precipitate. Fourth, the precipitate was resuspended in wetting buffer (20 mM MES hydrate, 2 mM CaCl2, 0.1 M NaCl, pH 6.0), and centrifuged again at 4 °C and 120,000 × g for 60 min. Finally, the precipitate was resuspended in 1 mL of wetting buffer to obtain an extracellular vesicle suspension derived from Platycladus orientalis leaves.

9. The pharmaceutical composition according to claim 8, characterized in that, It also includes other pharmaceutical excipients for preparing the pharmaceutical composition into nasal drops.

10. Use of the pharmaceutical composition of claim 8 or 9 in the preparation of a medicament for treating anxiety, depression or insomnia-related disorders.

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