A natural extracellular vesicle derived from a jack-in-the-pulpit, a preparation method thereof and application thereof in preparing a medicine for preventing or treating a neurodegenerative disease

Abstract

The application provides a natural extracellular vesicle derived from arisaema, a preparation method of the extracellular vesicle and application of the extracellular vesicle in preparation of a medicine for preventing or treating a neurodegenerative disease. The extracellular vesicle is extracted from fresh tissues of arisaema, has a membrane structure composed of a lipid bilayer, and has a particle size distribution of 50 nm to 700 nm; the extracellular vesicle encapsulates an active metabolite group derived from arisaema, the active metabolite group at least contains apigenin, curcumin, sappanin, ginkgolide B, hypericin and naringenin; and the extracellular vesicle does not contain calcium oxalate needle crystals and agglutinins in raw arisaema which cause mucous membrane irritation and neurotoxicity, or contains a small amount of calcium oxalate needle crystals and agglutinins which do not cause mucous membrane irritation and neurotoxicity. The application realizes the unity of toxicity separation and drug efficacy concentration, resolves the long-term controversy on the nasal brain delivery anatomical pathway in the field, can reverse an inflammatory storm induced by A beta and repair synaptic plasticity.

Description

Technical Field

[0001] This invention relates to the fields of nanomedicine and modernization of traditional Chinese medicine, specifically to a plant-derived extracellular vesicle (PDEV) extracted from Arisaema heterophyllum and its preparation method, as well as its development into a nasal delivery formulation for the prevention or treatment of neurodegenerative diseases related to neuroimmunity, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Background Technology

[0002] The clinical treatment of Alzheimer's disease (AD) has long faced two major bottlenecks: first, the absolute impermeability of the blood-brain barrier (BBB) ​​prevents more than 98% of small molecule drugs and almost 100% of large molecule drugs from entering the brain; second, single-target drugs (such as single Aβ monoclonal antibodies) targeting complex neurodegenerative networks have extremely limited efficacy.

[0003] Arisaema heterophyllum Blume (AHB) has a long history in traditional medicine for treating neurological disorders such as stroke and epilepsy. However, raw Arisaema heterophyllum contains calcium oxalate needle crystals and lectin proteins, which have strong mucosal irritation and neurotoxicity, thus its modern clinical application is strictly limited. Traditional Chinese medicine often uses bile fermentation (processed into "Bile Arisaema") to reduce toxicity and enhance efficacy, but this involves complex chemical reactions and is difficult to standardize.

[0004] Currently, most nanocarriers used for brain-targeted delivery are synthetic liposomes or exosomes derived from mammalian cells. However, synthetic liposomes lack intrinsic biological activity, while mammalian exosomes suffer from low yield and high risk of cross-species immunogenicity. Therefore, there is an urgent need for a natural nanodelivery system that can bypass the brain-body block (BBB), achieve multi-target therapy, and is highly safe. Summary of the Invention

[0005] Technical issues This invention aims to provide a novel strategy of "detoxification-by-nanotechnology" to directly unlock the potential of raw Arisaema heterophyllum in treating central nervous system diseases. Through specific physical extraction techniques, avoiding traditional processing methods, its inherently toxic macromolecules are removed, while its multi-functional active ingredients are enriched to prepare a natural nano-therapeutic entity.

[0006] Technical solution In a first aspect, the present invention provides extracellular vesicles from Arisaema heterophyllum Blume (AHBEVs), wherein the extracellular vesicles are extracted from fresh Arisaema heterophyllum Blume tissue and have a membrane structure composed of a lipid bilayer with a particle size distribution between 50 nm and 700 nm; the extracellular vesicles encapsulate an active metabolite group from Arisaema heterophyllum, the active metabolite group including at least apigenin, curcumin, shampoosin, ginkgolide B, hypericin, naringenin, etc.; and the extracellular vesicles do not contain calcium oxalate needle crystals and lectins that cause mucosal irritation and neurotoxicity in raw Arisaema heterophyllum, or contain a small amount of calcium oxalate needle crystals and lectins that do not cause mucosal irritation and neurotoxicity.

[0007] In some embodiments, the fresh tissue of the Arisaema includes tubers (rhizomes).

[0008] In a second aspect, the present invention provides a method for preparing extracellular vesicles derived from Araceae (as described in the first aspect), the method comprising the following steps: (1) Homogenization and coarse filtration: Fresh Arisaema tubers are thoroughly mixed with pre-cooled extraction buffer and homogenized. The homogenate is collected, filtered to remove plant residues, and crude extract is obtained. (2) Differential centrifugation to remove cell residue: Centrifuge the crude extract obtained in step (1) at 2,000-5,000 ×g, especially 4,000 ×g, for 30-60 minutes, especially 40-45 minutes, and take the supernatant; then centrifuge the supernatant at 10,000-50,000 ×g, especially 10,000 ×g, for 90-120 minutes to remove cell residue by physical precipitation. (3) Filtration and purification: The supernatant obtained by removing cell residue in step (2) is filtered through a microporous membrane and centrifuged at 100,000-150,000×g, especially 100,000×g, for 2-3 hours at 0-4 ℃, and the precipitate is collected. (4) Gradient centrifugation to remove macromolecular toxic proteins and crystal structures: The precipitate obtained in step (3) is resuspended in pre-cooled extraction buffer, and the sample is spread on the upper layer of a sucrose density gradient solution with a concentration gradient of 8%, 30%, 45%, and 60%, and then centrifuged again at 100,000-150,000×g, especially at 100,000×g for 1-3 hours. (5) Collection: Take the fraction between the 8% and 30% sucrose layers, dilute it with pre-cooled extraction buffer, and centrifuge it at 100,000-150,000×g, especially at 100,000×g, for 1-3 hours at 0-4 ℃ to obtain extracellular vesicles (AHBEVs) derived from Arisaema.

[0009] In some embodiments, the extraction buffer in steps (1), (4), and (5) can refer to a buffer system used in biochemical, cell culture, and other experiments to maintain the acid-base balance of the system and adapt to the physiological environment. Examples include, but are not limited to, phosphate-buffered saline (PBS), physiological saline, and HEPES buffer. In a preferred embodiment, the extraction buffer is phosphate-buffered saline (PBS).

[0010] In some implementations, in steps (1), (4), and (5), the precooling can be 0-8°C, preferably 4°C.

[0011] In some embodiments, in step (1), the filtration is performed using multiple layers of gauze or a filter screen.

[0012] In some embodiments, in step (2), the crude extract obtained in step (1) is centrifuged at 4,000 × g for 40-45 minutes and the supernatant is collected; then the supernatant is centrifuged at 10,000 × g for 90 minutes to remove cell residue.

[0013] In some embodiments, in step (3), a microporous filter membrane with a diameter of 0.2-0.5 μm, preferably 0.22 μm to 0.45 μm, is used for the filtration.

[0014] In some embodiments, in step (4), the resuspension is performed using sterile phosphate-buffered saline (PBS).

[0015] In some embodiments, in step (5), the dilution is performed using sterile phosphate buffer (PBS).

[0016] In a third aspect, the present invention provides extracellular vesicles (AHBEVs) derived from Arisaema heterophyllum prepared by the preparation method of the second aspect.

[0017] In a fourth aspect, the present invention provides a pharmaceutical composition comprising extracellular vesicles (AHBEVs) derived from Arisaema heterophyllum in the first or third aspect, and pharmaceutically acceptable excipients.

[0018] In some embodiments, the dosage form of the pharmaceutical composition may include nasal delivery formulations, injectable formulations, oral administration formulations, rectal administration formulations, particularly nasal delivery formulations, including but not limited to nasal drops, nasal sprays, nasal powders, or in-situ gels; preferably, the excipients include penetration enhancers, pH adjusters, or adhesives suitable for absorption by the nasal mucosa.

[0019] In a fourth aspect, the present invention provides the use of extracellular vesicles derived from Arisaema in the first or third aspect, or pharmaceutical compositions in the fourth aspect, in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases.

[0020] In this invention, the neurodegenerative disease can refer to a chronic disease whose onset is closely related to age and is accompanied by neuroimmune imbalance. Examples include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS).

[0021] In some embodiments, the extracellular vesicles in the drug include at least one of the following effects: (1) As a biological sieve, it enriches phytochemicals that resist Aβ toxicity and improve chemotactic perception defects; (2) Bypassing the blood-brain barrier (BBB) ​​via the intraaxonal transport pathway of olfactory sensory nerves (OSNs), it directly targets the central nervous system; (3) Inhibit the NF-κB signaling cascade, downregulate M1 microglia markers (CD86, IL-1β, TNF-α), upregulate M2 markers (CD206, Arg1, IL-10), and reshape the polarization state of microglia; (4) Restore neuroimmune homeostasis, lipid metabolism, antioxidant defense and synaptic plasticity.

[0022] In a fifth aspect, the present invention provides a method for preventing or treating neurodegenerative diseases, the method comprising administering to a subject in need a therapeutically effective amount of an extracellular vesicle derived from Arisaema in the first or third aspect, or a pharmaceutical composition in the fourth aspect.

[0023] Beneficial effects A groundbreaking "All-in-one" Entity: This invention demonstrates that AHBEVs are not merely inert drug carriers. Their unique extraction process acts as a "biological sieve," physically eliminating large molecules that cause mucosal irritation and toxicity (completely resolving the application pain points of raw Arisaema heterophyllum), while seamlessly enriching their lipid bilayer with abundant synergistic bioactive metabolites (such as apigenin and curcumin). This perfectly achieves the unity of toxicity separation and drug efficacy concentration.

[0024] Unraveling the precise intracerebral delivery pathway: This invention, through the establishment of a zinc sulfate (ZnSO4)-induced olfactory nerve ablation model, provides the first conclusive in vivo demonstration that the essential pathway for AHBEVs to enter the central nervous system is "intra-axonal transport via olfactory sensory nerves (OSNs)." This discovery quells the long-standing controversy in the field regarding the anatomical pathway of nasobrain delivery.

[0025] Microglia targeting: It promotes the transformation of microglia from the pro-inflammatory M1 type to the M2 type with phagocytic and repair functions, completely reversing the Aβ-induced inflammatory storm and repairing synaptic plasticity. Attached Figure Description

[0026] Figure 1 The isolation, physicochemical characterization, and metabolomics analysis results of Arisaema extracellular vesicles (AHBEVs) in Example 1 are shown. A: Representative transmission electron microscopy (TEM) image: AHBEVs exhibit complete, typical spherical or characteristic cup-shaped vesicle structures with a clearly visible lipid bilayer (scale bar = 200 nm); B: Dynamic light scattering (DLS) analysis of the hydrodynamic particle size distribution of purified AHBEVs; C: Nanoparticle tracking analysis (NTA) spectrum, showing uniform particle size distribution and abundance of AHBEVs. D: Zeta potential detection results, reflecting the surface charge and colloidal dispersion stability of AHBEVs; E: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein map, Coomassie brilliant blue staining; Compared with Arisaema heterophyllum extract, AHBEVs specifically enriched multiple protein components; FH: Dynamic monitoring of AHBEV stability after freezing at -80 ℃ for 7, 14, 30 and 90 days, evaluating vesicle structure integrity, particle size distribution and particle concentration changes (transmission electron microscopy icon bar = 200 nm).

[0027] Figure 2The results of stability analysis of Arisaema extracellular vesicles (AHBEVs) cryopreserved for 450 days in Example 1 are shown. A: Representative transmission electron microscopy (TEM) image: AHBEVs exhibit intact, typical spherical or characteristic cup-shaped vesicle structures with a clearly visible lipid bilayer (scale bar = 100 nm); B: Nanoparticle tracking analysis (NTA) spectrum, showing uniform particle size distribution and abundance of AHBEVs.

[0028] Figure 3 The results of stability analysis of Arisaema extracellular vesicles (AHBEVs) after repeated freeze-thaw cycles in Example 1 are shown. A: Hydrodynamic particle size distribution after 1, 3, and 5 freeze-thaw cycles, as detected by dynamic light scattering (DLS); B: Particle size distribution and abundance after 1, 3, and 5 freeze-thaw cycles, as determined by nanoparticle tracking analysis (NTA).

[0029] Figure 4The results of AHBEV protein quantification in Example 2 are shown. Wherein: A: Actual caliper measurement of AHB tubers with a diameter greater than 30 mm; B: Standard curve for BCA protein quantification; C: Statistical comparison of BCA-protein concentration between AHB extract and AHBEVs samples; D: Protein content (per 100g AHB tuber) in AHB extract and AHBEVs samples; E: Pie chart showing the classification and proportion distribution of metabolites obtained from non-targeted metabolomics identification of AHBEVs in Example 2, including organic acids and derivatives, lipids and lipid-like molecules, organic heterocyclic compounds, organic oxygen compounds, benzeneoids, nucleosides, phenylpropanoids and polyketides, and organic nitrogen compounds. Compounds, alkaloids and their derivatives, lignans, etc.; F: The heatmap shows the relative abundance of representative secondary metabolites in three independently prepared batches of Arisaema tuber-derived extracellular vesicles (AHBEV-1, AHBEV-2, AHBEV-3), with data obtained based on metabolomics analysis. The color bar represents the relative signal intensity: blue indicates low abundance, and red indicates high abundance.The main components include naringenin, schaftoside, ginkgolide B, convallatoxin, hypericin, albiflorin, vitamin B6 (pyridoxine), curcumin, apigenin, salannin, dihydromysticin, eleutheroside E, phenylethyl primeveroside, catalposide, genipin gentiobioside, neoruscogenin, and luminol (dimethylisophorazine). M501T208 (mass spectrometry number), 4- Thiazolidine-4-carboxylic acid and Aceroside-VIII.

[0030] Figure 5The results of scanning electron microscopy observation of calcium oxalate crystals and quantitative detection of calcium oxalate content in different AHB samples in Example 2 are shown. Wherein: A: Oxalic acid content in AHB extract and AHBEVs. B: Scanning electron microscope (SEM) image of fresh AHB tuber slices (AHB Slice), with yellow triangles indicating calcium oxalate needle crystals, scale bar 25 μm; Scanning electron microscope (SEM) image of AHB tuber powder (AHB Bowders), with yellow triangles indicating calcium oxalate needle crystals, scale bar 25 μm; C: High-magnification scanning electron microscope (SEM) image of AHB extract (AHB Extract), showing numerous calcium oxalate needle crystals (yellow triangles), scale bar 2 μm; D: Scanning electron microscope (SEM) image of AHBEVs, with no visible calcium oxalate crystals, scale bar 25 μm; E: Quantitative bar chart of calcium oxalate content in three groups: raw extract, AHB extract, and AHBEVs (unit: mg / 100 g raw material); P < 0.0001 indicates extremely significant differences between groups, and a single scatter plot represents an independent replicate sample; F: Results of semi-quantitative SDS-PAGE detection of endogenous lectins in AHBEVs. AHB extract and total AHBEVs protein SDS-PAGE electrophoresis images stained with Coomassie Brilliant Blue. The box indicates the band of endogenous AHB lectin protein near 20 kDa. G: Semi-quantitative statistical bar chart of relative AHB lectin expression. P < 0.0001 indicates highly significant difference between the two groups; a single scatter plot represents an independent technical replication.

[0031] Figure 6 This image shows representative HE-stained images of mouse nasal tissue after nasal administration of AHB Extract or AHBEVs in Example 3, as well as the levels of IL-6, IL-1β, and TNF-α in nasal mucosal homogenates. In the figures, A: Overall view of the olfactory epithelium and nasal lamina propria; B: Integrity of olfactory cilia; C: Structure of the ethmoidal concha (scale bar 50 μm); DF: ELISA results of pro-inflammatory cytokine levels in the nasal mucosa, with quantitative comparisons of IL-6, IL-1β, and TNF-α pro-inflammatory proteins in the nasal mucosa of the blank group, AHB Extract group, and AHBEVs group. P < 0.0001 indicates a highly significant difference between the two groups. Individual scatter plots in the figure represent independent biological samples.

[0032] Figure 7The results of the in vitro hemolysis experiment of rat erythrocytes treated with gradient concentrations of AHBEVs in Example 3 are shown. A: Visual chromatogram of hemolysis in a 96-well plate: Blank group was the negative control, and 0.1% Triton X-100 was the positive control for complete hemolysis; rat erythrocytes were co-incubated with gradient concentrations of AHB extract (1, 10, 50, 250, and 500 μg / mL); B: Quantitative statistics of hemolysis rate (%) for different concentrations of AHB extract; C: Quantitative statistics of hemolysis rate for different concentrations of AHBEVs. Extremely significant differences between groups were marked as P < 0.0001, and each scatter plot represents one independent technical replicate.

[0033] Figure 8 The results of serum biochemical marker detection after 28 days of AHBEV intervention in Example 3 are shown. These include a quantitative statistical bar chart of serum albumin (ALB), urea (UREA), uric acid (UA), blood glucose (GLU), alanine aminotransferase (ALT), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TRIG), total cholesterol (CHOL), and aspartate aminotransferase (AST). ns represents no statistically significant difference between the two groups, and P=0.0490 represents a statistically significant difference between the groups. Each scatter point in the chart represents one independent biological replicate sample.

[0034] Figure 9 This document illustrates how Arisaema extracellular vesicles (AHBEVs) promote microglia polarization in Example 4. A: Western blot images of representative proteins of p-P65 / P65, M1 marker (CD86), and M2 marker (CD206) after Aβ stimulation of BV2 microglia; BD: Quantitative analysis results of the representative proteins of p-P65 / P65, M1 marker (CD86), and M2 marker (CD206) from the Western blot images; EG: Quantitative detection of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory cytokines (IL-10) in the supernatant of BV2 cells using ELISA, confirming the cells' shift towards functional M2 polarization.

[0035] Figure 10The results of Arisaema extracellular vesicles (AHBEVs) in Example 5 are shown to alleviate Aβ-induced neurotoxicity in Caenorhabditis elegans. Among them: A: Blank group, Aβ-induced Caenorhabditis elegans model, low-dose AHBEVs (10 μg / mL), and high-dose AHBEVs (50 μg / mL); the progressive paralysis curve of the CL2006 nematode strain with muscle-specific Aβ expression was observed at 72 h. Among them, high-dose AHBEVs (50 μg / mL) significantly delayed the onset of paralysis (P = 0.0472); BD: Blank group, Aβ-induced Caenorhabditis elegans model, low-dose AHBEVs (10 μg / mL), and high-dose AHBEVs (50 μg / mL); behavioral test results of CL2355 nematode with whole-neuron Aβ expression. Among them, AHBEVs intervention significantly restored the nematode chemotactic index (CI), 1 h (P = 0.025) and 3 h (P = 0.025). < 0.001) all showed improvement (B and C), and significantly alleviated 5-hydroxytryptamine (5-HT) hypersensitivity reaction (P = 0.010) (D).

[0036] Figure 11This demonstrates that intranasal administration of Arisaema extracellular vesicles (AHBEVs) in Example 6 improved cognitive impairment, cleared Aβ pathological deposits, and inhibited glial cell overactivation in APP / PS1 mice. The study included a wild-type control group (WT), an APP / PS1 transgenic model group (Model), and an AHBEVs-treated group. The Morris water maze (MWM) test was used to evaluate the spatial learning and reference memory abilities of the mice. A: Escape latency curve during the 6-day orientation navigation phase; from day 3, the spatial learning ability of the AHBEVs intervention group mice was significantly improved (P < 0.05); B: Time spent in the target quadrant during the spatial exploration experiment (P = 0.0524); C: Number of times the mice crossed the original platform location (P = 0.0055); D: Novel object recognition (NOR) test to evaluate long-term recognition memory; the recognition index (RI) results showed that the preference for novel objects in the AHBEVs group mice was significantly restored (P = 0.0358); E: Burying food test (BFT) quantitatively detected the olfactory sensitivity of the mice. Intranasal delivery of AHBEVs significantly shortened the abnormally prolonged latency of mice searching for buried food particles (P < 0.0308); F: Representative immunofluorescence panorama of the cerebral cortex and hippocampus. Brain tissue sections were stained multiple times to label Aβ plaques (6E10 antibody), microglia (IBA-1), and astrocytes (GFAP), respectively, to visually present neurotoxicity and the inflammatory microenvironment; GL: Statistical analysis of fluorescence signals of Aβ plaques (6E10 antibody), astrocytes (GFAP), microglia (IBA-1) in the hippocampus, and Aβ plaques (6E10 antibody), astrocytes (GFAP), and microglia (IBA-1) in the cortex. Detailed Implementation

[0037] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1: Extraction and physicochemical characterization of AHBEVs.

[0041] Extracellular vesicle extraction: Arisaema tubers were thoroughly washed and homogenized in pre-cooled phosphate-buffered saline (PBS) at 4°C. The homogenate was collected and filtered through multiple layers of gauze or a filter to remove plant residues, yielding a crude extract (raw extract). The crude extract was then centrifuged at differential speed (4000 g for 45 min, 10000 g for 90 min) to remove cell residues. The supernatant was filtered through a 0.45 μm filter and ultracentrifuged at 4°C and 100000 g for 2 h. The crude precipitate was collected and resuspended in PBS. The sample was then layered on top of a sucrose density gradient solution with concentrations of 8%, 30%, 45%, and 60%, and ultracentrifuged again at 100000 g for 2 h. The fraction between the 8% and 30% sucrose layers was collected, diluted with PBS, and ultracentrifuged again for 2 hours to obtain purified Arisaema-derived extracellular vesicles (AHBEVs).

[0042] Preparation of Arisaema heterophyllum extract: Arisaema heterophyllum tubers were mixed with phosphate-buffered saline (PBS) pre-cooled to 4°C and mechanically homogenized. The entire homogenate was then pre-centrifuged at low speed (2000 g, 10 min). Due to significant differences in component density, the denser calcium oxalate needle crystals, unbroken cells, and cell wall debris settled rapidly, resulting in a coarse precipitate (toxic components); while soluble active ingredients, proteins, and low-density nanoscale vesicles remained in the supernatant, which is the Arisaema heterophyllum extract (AHB Extract).

[0043] Physicochemical characterization of extracellular vesicles: The AHBEV stock solution obtained by sucrose density gradient separation was serially diluted to an appropriate concentration with pre-cooled PBS, and gently pipetted to avoid vesicle aggregation. The microscopic morphology of AHBEVs was observed using a Hitachi HT7800 transmission electron microscope (TEM); the vesicle size distribution and zeta potential were determined using a Zetasizer Advance instrument through dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA).

[0044] DLS detection: 1 mL of diluted sample was placed in a disposable particle size cuvette and equilibrated at 25 °C for 5 min. Detection was performed using 173° backscatter mode. Five sets of measurements were repeated for each sample, with 12 scans per set, to obtain the vesicle hydration average particle size (Z-average) and polydispersity index (PDI). Samples from the same dilution system were injected into a capillary cell for Zeta potential measurement, with three parallel measurements for each sample. Before NTA detection, the instrument flow path and recognition parameters were calibrated using 100 nm polystyrene microspheres, and the sample was diluted to 10... 6 -10 7Particle / mL range; five 30-second imaging videos were continuously acquired for each sample, and the Brownian motion trajectory of individual vesicles was tracked by software to statistically analyze the actual particle size distribution and particle concentration. All experiments were set up in three independent biological replicates.

[0045] To separate the therapeutic activity of Arisaema heterophyllum from its macromolecular toxic substances, this study successfully extracted AHBEVs using a standardized and complete stepwise preparation process combining differential centrifugation and sucrose density gradient ultracentrifugation. The extracted AHBEVs were then subjected to physicochemical characterization and metabolomics analysis. Transmission electron microscopy (TEM) analysis showed that AHBEVs exhibited intact, typical spherical or characteristic cup-shaped vesicle structures, with a clearly visible lipid bilayer (scale bar = 200 nm). Figure 1 (A). Dynamic light scattering (DLS) detection results showed that the AHBEVs had a uniform hydrodynamic particle size distribution, with an average particle size of 162.30 ± 63.88 nm. Figure 1 (B). Nanoparticle tracking analysis (NTA) revealed that AHBEVs had a uniform particle size distribution, with an average particle size of 136.60 ± 61.60 nm. The total particle concentration (particle abundance) was also measured, and the vesicle yield reached 1.3 × 10¹¹ vesicles / mL. Figure 1 (C). The zeta potential is -7.45 ± 0.34 mV ( Figure 1 (D) The negative charge on the surface can generate sufficient electrostatic repulsion, ensuring the stability of the colloidal dispersion system.

[0046] To further verify the purity of the isolated nanovesicles and distinguish the vesicle components from the impurities in the Arisaema heterophyllum extract, this study conducted protein mapping analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic band results showed significant differences in protein composition: the protein bands of the Arisaema heterophyllum extract were disordered and diffuse, with numerous protein aggregates; while AHBEVs selectively encapsulated characteristic proteins, with protein bands mainly concentrated in the low to medium molecular weight range (10–100 kDa), and a significantly enriched characteristic band appearing near 20 kDa. Figure 1 (E). This differential banding not only demonstrates the presence of specific molecular carriers in AHBEVs, but also confirms the high separation efficiency of the density gradient separation process used in this study, which can effectively remove soluble impurities and plant macromolecules without vesicle structures.

[0047] Excellent colloidal stability is an indispensable prerequisite for the reproducible clinical translation of nanomedicines. Therefore, this study systematically investigated the long-term cryopreservation stability and handling stability of AHBEVs. Continuous monitoring results showed that after being cryopreserved at -80 °C for 7, 14, 30, and 90 days, the vesicle structure integrity, narrow particle size distribution, and particle concentration of AHBEVs remained largely unchanged. Figure 1(FH). Furthermore, AHBEVs can be cryopreserved at -80 °C for up to 450 days with intact vesicle structure (FH). Figure 2 The particle size distribution and particle concentration in the middle (A) region showed no significant changes. Figure 2 (B)

[0048] To simulate storage disturbances in actual clinical practice, rigorous repeated freeze-thaw experiments were conducted on the vesicles. DLS and NTA test results showed that AHBEVs were well-tolerated, maintaining their original physicochemical properties even after three consecutive freeze-thaw cycles. Figure 3 (AB); However, after 5 freeze-thaw cycles, the vesicle system showed significant instability, manifested as a sharp increase in particle size (>300 nm), aggregation of a large number of particles, and damage to the vesicle membrane structure (AB). Figure 3 (AB).

[0049] The above-mentioned complete set of stability evaluation results confirms that AHBEVs are stable in physicochemical properties under standard low-temperature storage conditions. At the same time, a strict "single-use, single-fill" quality control standard has been established to ensure the integrity of vesicle structure in all subsequent in vitro and in vivo experiments.

[0050] Example 2: Component identification of AHBEVs.

[0051] This experiment selected intact AHB tubers with a diameter greater than 30 mm as raw materials. Figure 4 According to Example 1, crude AHB extract was collected from tuber homogenates, and AHBEVs were obtained, followed by protein separation and purification. Protein quantification was performed on both groups of samples using the BCA method, and a protein standard curve with good linear fit was plotted. Figure 4 The linear regression equation for AHB extract was (Y = -100.8 + 875X), with a coefficient of determination (R² = 0.9925), demonstrating the reliability of the linearity and accuracy of the quantitative system. Protein concentration statistics showed that the total protein content of the AHB extract was significantly higher than that of the purified AHBEVs (B). Figure 4 The difference between the two groups was statistically significant (P<0.0001), which is consistent with the experimental rule that a large amount of soluble proteins are removed during the separation and purification of AHBEVs.

[0052] Protein content detection: Protein concentration was determined using the BCA protein quantification kit (Lablead, B5001). A gradient of BSA standards was prepared according to the manufacturer's instructions for plotting a standard curve. Diluted protein samples and blank lysis buffer were added to 96-well plates, followed by freshly prepared BCA working solution and mixing. The plates were incubated at 37°C in the dark for 30 min. The absorbance of each well was measured using a microplate reader at 562 nm. The original protein concentration was calculated based on the standard curve, and the actual protein concentration was obtained by converting the dilution factor. Figure 4 The figure shows the protein content (per 100g AHB tuber) in the two groups of samples: AHB Extract and AHBEVs.

[0053] Metabolomics: Metabolites were extracted from purified Arisaema heterophyllum extracellular vesicles (AHBEVs) using a pre-cooled methanol-acetonitrile-water mixture. Samples were analyzed using a Vanquish ultra-high performance liquid chromatography system in tandem with an Orbitrap Exploris 480 mass spectrometer. This study employed non-targeted metabolomics techniques to comprehensively detect AHBEV metabolites and categorized and statistically analyzed all annotated metabolites according to chemical structure classification standards. Figure 4 (E). The pie chart visually presents the relative proportions of various metabolites: organic acids and their derivatives are the most abundant metabolic category, accounting for 30.04% of all annotated metabolites; followed by lipids and lipid molecules, accounting for 25.24%. These two categories together account for more than half of the total metabolites, indicating that organic acid metabolism and lipid metabolism are the core metabolic pathways in the sample. Undefined metabolites account for 10.76%, suggesting that there are still a large number of unknown small molecules with unclear structures that need further analysis. Other categories with relatively high proportions are, in descending order, organic heterocyclic compounds (8.44%), organic oxygen-containing compounds (6.98%), and benzene compounds (5.82%); while alkaloids, lignans, and other miscellaneous substances each account for less than 1%, belonging to the trace metabolic components in the sample. Figure 4The heatmap shows the relative abundance of representative secondary metabolites in three independently prepared Arisaema tuber-derived extracellular vesicles (AHBEV-1, AHBEV-2, and AHBEV-3), with data obtained based on metabolomics analysis. The color bar represents the relative signal intensity: blue indicates low abundance, and red indicates high abundance. The main components include naringenin, schaftoside, ginkgolide B, convallatoxin, hypericin, albiflorin, vitamin B6 (pyridoxine), curcumin, apigenin, salannin, dihydromysticin, eleutheroside E, phenylethyl primeveroside, catalposide, genipin gentiobioside, neoruscogenin, and luminol (dimethylisophorazine). M501T208 (mass spectrometry number), 4- Thiazolidine-4-carboxylic acid and Aceroside-VIII.

[0054] Scanning electron microscopy (SEM) observation of calcium oxalate needles: Sample preparation for SEM: AHB tuber slices, AHB powder (AHB tuber slices were dried at 40℃ with a forced air dryer until the moisture content was ≤8%; then pulverized to obtain AHB powder), AHB extract, and purified AHBEVs samples were taken, fixed, dehydrated, dried, and sputter-coated with gold. The morphology and distribution of calcium oxalate needles were then observed under different magnifications using a scanning electron microscope.

[0055] Calcium oxalate content determination by high performance liquid chromatography (HPLC): Chromatographic conditions Chromatographic separation was carried out using a high-performance liquid chromatography system equipped with an ultraviolet detector; the chromatographic column used was a Mairis C18 column (250 mm × 4.6 mm, particle size 5 μm).

[0056] The mobile phase was a 0.5% (v / v) aqueous phosphoric acid solution; isocratic elution mode was used, with a flow rate of 0.8 mL / min; the column temperature was kept constant at 25℃, the injection volume was 10 μL, and the detection wavelength was set to 220 nm.

[0057] Sample pretreatment and acid hydrolysis Calcium oxalate is poorly soluble in water, therefore it must be converted into soluble oxalic acid before HPLC detection. The specific procedure is as follows: Accurately weigh 0.5 g of dried AHB medicinal powder (or an equivalent mass of AHB extracellular vesicle sample), add 5 mL of ultrapure water, and soak at 60 ℃ for 20 min. Repeat the washing twice. After centrifugation, discard the supernatant to completely remove the original water-soluble free oxalic acid and other water-soluble impurities in the sample.

[0058] Add 5 mL of ultrapure water and 1 mL of hydrochloric acid solution (hydrochloric acid:water = 1:1, volume ratio) to the precipitate, and incubate at 70℃ for 30 min to ensure complete hydrolysis of calcium oxalate. After centrifugation, collect the supernatant, add ultrapure water to a final volume of 10 mL, filter through a 0.22 μm aqueous syringe filter, and then perform HPLC analysis on the filtrate.

[0059] Methodological validation and content calculation Accurately weigh oxalic acid reference standard, dissolve it in ultrapure water to prepare a reference standard stock solution with a concentration of 2.0 mg / mL; prepare a series of reference standard working solutions with a concentration range of 0.1~2.0 mg / mL by serial dilution for plotting standard curves.

[0060] In accordance with the relevant guidelines of the Pharmacopoeia of the People's Republic of China, the analytical methods were fully validated, and the evaluation indicators included linearity, intra-day precision, repeatability, sample stability after 72 h at room temperature, and recovery rate (accuracy).

[0061] The concentration of oxalic acid in the sample is calculated based on the standard curve, and then multiplied by the molar mass ratio of anhydrous calcium oxalate to oxalic acid to obtain the content of calcium oxalate in the sample.

[0062] Methodology Validation and Data Analysis The linearity, limit of detection (LOD), and limit of quantitation (LOQ) of the established HPLC method were investigated: a standard curve was plotted with oxalic acid concentration on the x-axis and peak area on the y-axis, and the linear correlation coefficient R was calculated. 2 >0.999; the limit of detection and limit of quantitation were calculated using signal-to-noise ratios S / N=3 and S / N=10, respectively.

[0063] Compared to the AHB extract, the oxalic acid peak area in the chromatogram of the AHB extracellular vesicle sample decreased significantly. This result quantitatively confirms that toxic calcium oxalate needle crystals were successfully removed by physical means during the vesicle preparation process.

[0064] In the test, equal amounts of crude extract of raw materials, AHB extract and AHBEVs were pretreated, and the calcium oxalate content corresponding to each 100 g of raw materials was determined and converted. Statistical analysis was performed by one-way ANOVA combined with multiple comparisons. P < 0.0001 was considered to be extremely significant between groups. Figure 5 Figure A shows the oxalic acid content in AHB extract and AHBEVs. Endogenous lectin detection: Equal amounts of AHB extract and total AHBEV protein were loaded and separated by SDS-PAGE electrophoresis. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 and imaged. Image analysis software was used to read the gray value of the AHB lectin target band at 20 kDa, and the relative protein expression level was calculated to complete a semi-quantitative comparison between the two groups of samples. Independent samples t-tests were used to analyze the data from both groups; P < 0.0001 was considered highly significant between the groups.

[0065] This study used scanning electron microscopy (SEM) to observe the morphology of calcium oxalate needle crystals in different AHB samples. SEM results showed that typical needle-like calcium oxalate crystals were clearly observed in fresh AHB tuber slices, AHB powder, and AHB extracts. Figure 5 In the middle BC, numerous slender needle-like crystals were observed in the extract under high magnification; however, no calcium oxalate crystal structure was observed in the purified AHBEVs sample. Figure 5 (D). The quantitative detection results of calcium oxalate content were consistent with the electron microscopy morphology results. The calcium oxalate content of the three groups of samples was in the order of crude extract > AHB extract > AHBEVs, and the differences between the groups were all extremely significant (P < 0.0001). Figure 5 (E). The above results demonstrate that the AHBEVs separation and purification process can effectively remove irritating calcium oxalate needle crystals from the raw materials. This is also the core reason why AHBEVs have significantly lower hemolytic toxicity and nasal mucosal pro-inflammatory effects compared to AHB extract.

[0066] SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining was used for semi-quantitative comparison of endogenous AHB lectins in AHB extract and purified AHBEVs. Electrophoresis results showed that both groups of samples exhibited characteristic AHB lectin bands at approximately 20 kDa, but the staining depth of the corresponding band in AHB extract was significantly higher than that in AHBEVs. Further semi-quantitative grayscale analysis confirmed that the relative expression level of AHB lectins in AHBEVs was much lower than that in AHB extract, with a highly significant difference between the two groups (P < 0.0001). This result indicates that the AHBEVs separation and purification process can effectively remove most of the endogenous AHB lectins from the crude extract. Experimental results are shown below. Figure 5 FG (Chinese character)

[0067] Example 3: Safety evaluation of AHBEVs.

[0068] Blood biochemical detection: Eight-week-old male C57BL / 6 wild-type mice (Beijing Sibeifu Biotechnology Co., Ltd., license number: SYXK (Jing) 2023-0011) were adaptively fed for 3 to 5 days in an SPF-grade experimental animal room, with an alternating cycle of 12 h light / 12 h darkness, and free access to food and water. The experiment was approved by the Medical and Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (approval number: BUCM20250630-007). For intranasal administration, the mice were gently fixed in a supine position without anesthesia. The AHBEVs group was given AHBEVs suspension every other day, with a single administration volume of 20 μL at a concentration of 5 μg / μL, and a total single administration dose of 100 μg per mouse. The continuous intervention lasted for 28 days, with a total of 14 administrations. The administration was performed by fractional dripping with a sterile micro pipette, with 2 to 3 μL each time, dripping alternately into the left and right nostrils. The liquid medicine was inhaled independently by the mice to avoid aspiration of liquid into the lungs. The control group was given an equal volume of sterile phosphate buffered saline (PBS) as the vehicle control.

[0069] After the animal intervention, peripheral blood was collected from each group, and serum was separated by standing at room temperature; a fully automatic biochemical analyzer and matching commercial detection kits were used to quantitatively detect the contents of serum albumin, urea, uric acid, glucose, alanine aminotransferase, high-density lipoprotein, low-density lipoprotein, triglyceride, total cholesterol and aspartate aminotransferase in strict accordance with the instructions. Independent sample t-test was used for statistical analysis of the data of the two groups. P < 0.05 was determined that the difference between groups was statistically significant, and P > 0.05 was marked as no significant difference (ns).

[0070] HE staining: To systematically evaluate the biocompatibility of local mucosa (to solve the problem of strong mucosal irritation caused by crude Arisaematis Rhizoma), and to observe the overall tissue morphology, this study carried out hematoxylin-eosin (HE) staining detection. After corresponding intervention treatment in each group, the target tissues (complete nasal tissue, brain tissue and main viscera) were collected and fixed in 4% paraformaldehyde; nasal tissue samples were decalcified with EDTA solution, followed by paraffin embedding. The slice thickness of tissue blocks was set to 5 μm, and staining was performed with standard HE staining reagents. Histopathological changes were observed and photographed under an optical microscope, with emphasis on evaluating the structural integrity of nasal epithelium and the morphology of neurons in the hippocampus. Figure 6 In the figure, A, B and C show representative HE staining images of nasal tissues of mice after intranasal administration of AHB extract (AHB Extract) or AHBEVs.

[0071] Detection of nasal mucosal inflammatory factors: Eight-week-old male C57BL / 6 wild-type mice (Beijing Splasher Biotechnology Co., Ltd., license number: SYXK (Jing) 2023-0011) were adaptively fed for 3 to 5 days in an SPF-grade experimental animal room, with a 12 h light / 12 h dark alternating cycle, and free access to food and water. The experiment was approved by the Medical and Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (approval number: BUCM20250630-007). For intranasal administration, the mice were gently fixed in a supine position without anesthesia. The AHBEVs group and AHB extract group were administered continuously for 3 days, with a single administration volume of 20 μL, a concentration of 5 μg / μL, and a total single administration dose of 100 μg per mouse. The administration was performed by fractional infusion using a sterile micropipette, with 2 to 3 μL each time, alternately instilled into the left and right nostrils. The liquid was inhaled spontaneously by the mice to avoid aspiration of the liquid into the lungs. The control group was given an equal volume of sterile phosphate buffered saline (PBS) as the vehicle control. After 3 consecutive days of corresponding intranasal administration to the experimental animals, the nasal mucosal tissues of each group were isolated, fully homogenized, and total tissue proteins were extracted; the BCA method was used to determine the tissue protein concentration for data correction. Commercial ELISA kits were used to quantitatively detect the contents of IL-6, IL-1β and TNF-α in nasal mucosal homogenates, and the results were finally expressed as cytokine content per unit of total protein (pg / mg protein). One-way analysis of variance combined with post-hoc multiple comparison was used for statistical testing, and P < 0.0001 was considered as extremely significant difference between groups.

[0072] The results showed that compared with the blank group, the contents of IL-6, IL-1β and TNF-α in the nasal mucosa of the AHB extract group were all significantly increased ( Figure 6 D-F in , P < 0.0001), indicating that AHB extract can induce obvious nasal mucosal inflammatory response; while the levels of the three inflammatory factors in the AHBEVs group were maintained at low levels similar to those in the blank group, and the difference was extremely significant compared with the AHB extract group ( Figure 6 D-F in , P < 0.0001). This result indicates that short-term intranasal administration of AHBEVs does not induce nasal mucosal inflammation, while AHB extract has strong nasal mucosal irritation and pro-inflammatory effect.

[0073] Hemolysis experiment: Male rats (250-350g) were collected from SPF-grade laboratory animal facilities and acclimatized for 3-5 days using a 12-hour light / 12-hour dark cycle, with free access to food and water. The experiment was approved by the Medical and Laboratory Animal Ethics Committee of Beijing University of Chinese Medicine (Approval No.: BUCM20250630-007). Fresh whole blood was centrifuged to separate red blood cells, which were then diluted to prepare a red blood cell suspension. A PBS negative control group and a 0.1% Triton X-100 complete hemolysis positive control group were simultaneously set up. Serially diluted AHB extract and AHBEVs (1, 10, 50, 250, 500 μg / mL) were added to the rat red blood cell suspension and incubated at 37°C. Incubate at a constant temperature of ℃; after incubation, centrifuge and collect the supernatant, measure absorbance, and calculate the hemolysis rate of each group. One-way ANOVA was used for statistical testing; P < 0.0001 was considered highly significant between groups.

[0074] This study used an in vitro hemolysis assay with rat erythrocytes to evaluate the blood compatibility of AHB extract and purified AHBEVs. Figure 7 The results of an in vitro hemolysis experiment on rat erythrocytes treated with gradient concentrations of AHBEVs are shown. Visual colorimetric results from a 96-well plate show that AHB extract induced significant hemolysis and reddening of the supernatant starting at 50 μg / mL, while AHBEVs at the highest concentration of 500 μg / mL only showed very weak hemolysis. Figure 7 (A). Quantitative statistical results of supernatant absorbance showed that the AHB extract had a concentration-dependent strong hemolytic effect; the hemolysis rate increased sharply at concentrations ≥50 μg / mL, and the difference was extremely significant compared with the blank group. Figure 7 (B, P<0.0001); the hemolysis rate of AHBEVs was less than 3% at all tested concentrations, with only a slight increase in hemolysis rate in the 500 μg / mL high concentration group, which was highly significant compared with the blank group. Figure 7 (C, P<0.0001). The above results indicate that the AHB extract has a significant erythrocyte damaging effect, while the isolated and purified AHBEVs have excellent blood compatibility and almost no risk of hemolysis within the experimental concentration range.

[0075] This study measured serum biochemical indicators to evaluate the effects of AHBEVs on liver and kidney function and glucose and lipid metabolism in vivo. After 28 days of AHBEV intervention, serum albumin (ALB) levels were significantly higher than those in the blank control group (P=0.0490); however, there were no statistically significant differences between the two groups in renal function indicators such as urea, uric acid, blood glucose, liver injury markers such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as all lipid indicators, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, and total cholesterol. Figure 8(All values ​​are in ns, P>0.05). The above results indicate that AHBEVs intervention can significantly increase serum albumin levels without causing abnormalities in liver, kidney, or glucose and lipid metabolism-related indicators, suggesting that AHBEVs have good in vivo biocompatibility.

[0076] Example 4: Arisaema extracellular vesicles (AHBEVs) promote microglia polarization.

[0077] Cell culture In vitro experiments were conducted using the immortalized mouse microglia cell line (BV2) (SNL-155, CCTCC). Cells were cultured in DMEM (Gibco) medium using standard methods; the medium was supplemented with a mixture of 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin). Cells were cultured in a sterile humidified incubator at 37°C with 5% carbon dioxide; the culture medium was changed every two days.

[0078] When the cell confluence reached 70%–80%, the cells were digested and passaged using 0.25% trypsin-EDTA digestion solution, and seeded in separate flasks at an appropriate ratio. Only cells with viability >95% and in the logarithmic growth phase were selected for subsequent co-incubation and drug intervention experiments.

[0079] Construction of an in vitro AD cell model and drug intervention To investigate the neuroprotective and immunomodulatory activities of AHB extracellular vesicles (AHBEVs) in vitro, this study used BV2 microglia to construct a β-amyloid (Aβ)-induced Alzheimer's disease cell damage model.

[0080] Cells were seeded at an appropriate cell density into 12-well plates and cultured overnight in an adherent manner.

[0081] Then pre-prepared Aβ was added to the culture medium. 1-42 Oligomers: BV2 cells were initially concentrated at 1 μM (to induce inflammatory polarization in microglia); after 12 h of incubation, biochemical indicators, cell morphology, and molecular level assays were performed.

[0082] The blank control group was treated with an equal volume of phosphate-buffered saline (PBS); the model group was given only Aβ. 1-42 Stimulation. The intervention group was pretreated with purified AHBEVs at final concentrations of 2 μg / mL and 10 μg / mL for 12 h.

[0083] Western blot (WB) protein immunoblotting assay Total protein was extracted from cell lysates, flash-frozen hippocampal and cortical brain tissue using pre-chilled RIPA lysis buffer (a mixture of protease inhibitors and phosphatase inhibitors). Samples were thoroughly homogenized on ice, briefly sonicated, and then centrifuged at 4°C and 12,000 rpm for 10 minutes. Protein concentration was accurately determined using a BCA protein quantification kit.

[0084] Equal amounts of total protein (50 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the molecular weight of the target protein, the samples were transferred to 0.22 μm or 0.45 μm polyvinylidene fluoride (PVDF) membranes (Bio-Rad). After transfer, the membranes were blocked with TBST buffer containing 5% skim milk at room temperature for 2 hours; then incubated overnight at 4°C with primary antibody.

[0085] After washing the membrane, horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated at room temperature for 2 hours. Protein bands were obtained by imaging using an ECL chemiluminescence imaging system. The gray values ​​of the bands were quantitatively analyzed using ImageJ software, and β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control protein to correct the loading amount.

[0086] Enzyme-linked immunosorbent assay (ELISA) BV2 cell culture supernatant was collected (centrifuged at 1000 rpm for 10 min to remove cell debris) for the detection of neuroinflammatory cytokine secretion levels. Following the kit instructions strictly, a commercially available ELISA kit (BioLegend) was used to detect the levels of pro-inflammatory cytokines (interleukin-1β, interleukin-6) and the anti-inflammatory cytokine interleukin-10.

[0087] The absorbance at 450 nm was measured using a microplate reader (SpectraMax iD3), and the background value was corrected at a wavelength of 630 nm. The cytokine content of the brain tissue sample needs to be standardized and corrected in conjunction with the total protein concentration of the corresponding tissue homogenate measured by the BCA method.

[0088] Experimental results are as follows Figure 9 As shown. Figure 9This paper illustrates how Arisaema extracellular vesicles (AHBEVs) promote microglia polarization in Example 4. A: Western blot images of representative proteins of p-P65 / P65, the pro-inflammatory M1 marker (CD86), and the anti-inflammatory M2 marker (CD206) after Aβ stimulation of BV2 microglia; BD: Quantitative analysis results of the representative proteins of p-P65 / P65, the M1 marker (CD86), and the M2 marker (CD206) from the Western blot images; EG: Quantitative detection of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory cytokines (IL-10) in the supernatant of BV2 cells using ELISA, confirming the cells' shift towards functional M2 polarization.

[0089] Example 5: Validation of the efficacy of Caenorhabditis elegans (AD) model (reversing Aβ-induced paralysis and chemotactic perception defects).

[0090] Wild-type Caenorhabditis elegans N2 (Bristol strain), transgenic lines CL2355 and CL2006 were all purchased from the Caenorhabditis elegans Genetic Resource Center (CGC).

[0091] Nematode paralysis model experiment: This study used the transgenic Caenorhabditis elegans CL2006 strain to evaluate the protective effect of AHBEVs against Aβ toxicity; this strain can continuously express human Aβ in body wall muscle cells under the regulation of a temperature-sensitive promoter. 1-42 Protein. N2 nematodes that had been synchronized to the L3 late instar were selected and transferred to NGM culture plates (1 L of standard NGM basic dry powder components: 3 g sodium chloride, 2.5 g peptone, 17 g agar; after autoclaving and cooling, potassium phosphate buffer, calcium chloride, magnesium sulfate, and ethanol were aseptically added to dissolve cholesterol, providing the nematodes with carbon and nitrogen nutrition, osmotic pressure, and cholesterol as a raw material for cell membrane synthesis; E. coli OP50 bacterial flora was laid as a food source for the nematodes); groups were set up as a blank solvent control group and AHBEVs treatment groups (10 μg / mL and 50 μg / mL). Wild-type N2 nematodes under the same culture conditions served as a blank control.

[0092] To induce large amounts of Aβ 1-42 To induce aggregation, the culture temperature of all culture plates was increased from 16 ℃ to 25 ℃, and this time point was recorded as 0 h. After 24 h of high-temperature induction, the paralysis status of nematodes was observed and counted every 12 h under a stereomicroscope. To avoid subjective observation bias, the group information was blinded for the observers. The criteria for paralysis were: only head movement, or inability to complete at least one complete, spontaneous sinusoidal body movement after light stimulation with a platinum wire. Nematodes that crawled out of the agar surface, burrowed into the culture medium, or exhibited internal egg hatching (bag-like death) were not included in the statistics.

[0093] Paralysis rate at each time point = number of paralyzed individuals among the effective nematodes on the plate / total number of effective nematodes. At least three independent biological replicates should be set up for each group, with approximately 50–100 nematodes per treatment condition.

[0094] Nematode chemotaxis experiment: CL2355 transgenic nematodes can express human Aβ in whole neurons. 1-42 Severe chemoreceptive impairment may occur. This experiment uses this strain to evaluate the neuroprotective and cognitive-improving effects of AHBEVs.

[0095] Synchronized L3 stage wild-type N2 nematodes and CL2355 transgenic nematodes were inoculated into NGM plates containing solvent and AHBEVs, respectively; the culture temperature was adjusted to 25 ℃ for 48 h to induce Aβ expression in neurons.

[0096] After induction, nematodes were collected and washed three times with sterile M9 buffer. Thorough washing completely removes E. coli OP50 food, briefly starving the nematodes and eliminating interference from food odors. Chemotaxis experiments were performed on uncoated 90 mm fresh NGM plates: samples were spotted at both ends of a single diameter line on the plate. 5 μL of chemotactic attractant (0.1% benzaldehyde in anhydrous ethanol solution) was added to one side, and 5 μL of anhydrous ethanol blank solvent was added to the other side. Simultaneously, 5 μL of 1 mol / L sodium azide (NaN3) was added to each spot to fix the nematodes in the affected area, and the plates were air-dried at room temperature for 10 min.

[0097] Approximately 100 washed nematodes were carefully dropped into the center of an agar plate and incubated at room temperature in the dark for 60 minutes to allow the nematodes to crawl freely and sense odor signals. After incubation, the number of nematodes (A) in the attracted area, (C) in the solvent control area, and (O) in the remaining areas were counted under a stereomicroscope.

[0098] The chemotaxis index is calculated as follows: CI = (A - C) / T, where T is the total number of effective nematodes in the plate (T = A + C + O). A CI closer to +1 indicates normal chemotaxis and strong chemosensory function; a CI close to 0 indicates random distribution of nematodes and severely impaired chemosensory function.

[0099] 5-HT Hypersensitivity Assay: CL2355 nematodes, which express Aβ throughout neurons, exhibit significant hypersensitivity to exogenous serotonin (5-HT) due to synaptic damage and neurotransmitter signaling disorders caused by Aβ. This experiment was used to evaluate the restorative effect of AHBEVs on the aforementioned functional deficits.

[0100] Synchronized nematodes were cultured at 16 ℃ to the L3 larval stage, then transferred to treatment plates containing solvent and AHBEVs, and cultured at 25 ℃ for 48 h to induce Aβ expression.

[0101] After induction, the nematodes were gently collected, washed with M9 buffer to remove food residue, and transferred to 96-well microplates. 200 μL of 1 mmol / L 5-HT hydrochloride solution was added to each well to begin the experiment. The nematodes were exposed to 5-HT for a precise 5-minute timer, and their movement was observed under a stereomicroscope: each nematode was strictly observed for 5 seconds; individuals that completely ceased spontaneous head shaking and body twisting during the observation period were considered paralyzed.

[0102] The severity of 5-HT hypersensitivity was quantified by paralysis rate: Paralysis rate = number of paralyzed nematodes in a single well / total number of nematodes detected in that well.

[0103] This study used a mature transgenic Caenorhabditis elegans model of Alzheimer's disease for experimental purposes. The CL2006 strain can continuously express human Aβ in muscle cells. 1-42 Protein, when heated, can induce large amounts of Aβ aggregation and cause progressive paralysis in nematodes. In the solvent-treated model group, paralysis occurred rapidly, with a paralysis rate of 73.0 ± 0.2% at 72 h. Figure 10 High-dose AHBEVs (50 μg / mL) intervention significantly delayed the paralysis progression (P = 0.0472). Figure 10 (A)

[0104] To further evaluate the protective effect of AHBEVs on neuronal circuits, this study used the CL2355 nematode strain, which expresses Aβ throughout neurons. AHBEV intervention significantly repaired the damaged benzaldehyde chemotactic index (CI): P = 0.025 at 1 h, and P < 0.001 at 3 h. Figure 10 (B and C), while significantly alleviating serotonin (5-HT) hypersensitivity reactions (P = 0.01, Figure 10 (D). The above results indicate that AHBEVs can significantly repair chemosensory-related cognitive functions and improve abnormal neurotransmitter signal transduction, confirming that they can effectively protect neural networks from Aβ protein toxicity.

[0105] Example 6: Cognitive function recovery and Aβ plaque clearance effect in APP / PS1 mouse model.

[0106] Experimental grouping and intervention: Six-month-old APP / PS1 double transgenic AD mice (Shanghai Southern Model Biotechnology Co., Ltd., license No.: SYXK (Hu) 2023-0005) were adaptively fed for 3-5 days in an SPF-grade experimental animal room, with a 12 h light / 12 h dark alternating cycle, and free access to food and water. The experiment was approved by the Medical and Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (approval No.: BUCM20250630-007). For intranasal administration, the mice were gently fixed in a supine position without anesthesia. The AHBEVs group was given AHBEVs suspension every other day, with a single administration volume of 20 μL and a concentration of 5 μg / μL, and the total single administration dose per mouse was 100 μg. The intervention lasted for 28 consecutive days, with a total of 14 administrations. Aseptic micro-pipette was used for drip infusion in divided doses, 2 to 3 μL each time, alternating between the left and right nostrils. The liquid medicine was inhaled independently by the mice to avoid liquid choking into the lungs. The control group was given an equal volume of sterile phosphate buffered saline (PBS) as the vehicle control.

[0107] Immunofluorescence staining: Coronal sections (40 μm thick) of brain tissue from the above APP / PS1 mice were taken for subsequent staining. The samples were permeabilized and blocked with PBS containing 0.5% Triton X-100 and 3% donkey serum at room temperature for 60 min. Then the corresponding primary antibodies were added to specifically label each target protein (IBA-1, GFAP, 6E10), followed by incubation overnight at 4 ℃. After sufficient washing with PBS, the matched Alexa Fluor fluorescent secondary antibodies (488, 594, 647 fluorescent channels) were added and incubated for 2 h in the dark; DAPI was used to counterstain the cell nuclei. Finally, the slides were mounted with an anti-fluorescence quenching mounting medium, and fluorescence images were collected with an Olympus FV3000 laser confocal microscope. ImageJ software was used to perform semi-quantitative analysis of fluorescence intensity and colocalization coefficient (Pearson correlation coefficient).

[0108] Morris water maze test (MWM): The Morris water maze test was used to evaluate the spatial learning ability and reference memory of mice. The experimental device is a circular pool with a diameter of 90 cm, which is virtually divided into four equal quadrants. Non-toxic titanium dioxide (TiO₂) was added into the water to make the water turbid, completely shielding the underwater escape platform, forcing the mice to locate only by spatial orientation clues and unable to directly see the platform. The platform was placed in the center of the target quadrant, 1 cm below the water surface. A light-shielding enclosure was arranged around the pool to avoid external visual interference; four off-site visual markers with high contrast and different shapes (circle, triangle, square, pentagram) were pasted on the inner wall of the pool as spatial reference markers. A video trajectory tracking system was equipped above the pool to record and analyze the swimming trajectory of the mice throughout the whole process.

[0109] The spatial learning phase (orientation and navigation phase) involved six consecutive days of training. Each day, mice were placed face-down on the pool wall and gently submerged from a randomly selected entry point in a non-target quadrant. The longest time a mouse could search for the hidden platform was 60 seconds; this time was recorded as the escape latency. If a mouse failed to find the platform within 60 seconds, it was gently guided to it and allowed to remain there for 10 seconds to consolidate spatial memory. The spatial exploration experiment (memory retention test) was conducted 24 hours after the final orientation and navigation training session (day 7). The mice were removed from the underwater platform, and their memory retention was assessed. They were then placed in the water from the quadrant opposite to the target quadrant and allowed to swim freely for 60 seconds. Spatial memory indicators were quantified using video tracking software: the percentage of time spent in the target quadrant, the number of times the mouse crossed the original platform location, and the latency to first reach the target area.

[0110] Novel Object Recognition (NOR) Experiment: This experiment evaluates recognition memory in mice. The principle is that rodents are naturally more inclined to explore new objects than familiar ones. The experimental setup is a matte, non-reflective, square open-field box (50 cm × 50 cm × 40 cm) to reduce visual interference from reflections. The experiment is conducted in a quiet environment with soft, uniform red light illumination to reduce stress on the mice. A high-definition camera is mounted above the box to record animal behavior, and an automated trajectory analysis system completes the data analysis. The experimental props are made of odorless, high-density plastic to prevent displacement by the mice: familiar objects A1 and A2 are blue cuboids (5 cm × 3 cm × 3 cm), and new object B is a red cylinder (base radius 1.5 cm, height 5 cm).

[0111] Adaptation Phase. 24 hours before formal training, place a single mouse facing the box wall into an empty box and allow it to explore freely for 5 minutes to eliminate its fear of the unfamiliar environment. After each mouse's test, thoroughly wipe the box with 75% ethanol to remove any residual odor interference.

[0112] Training phase (learning period). Two identical familiar objects, A1 and A2, are placed symmetrically inside the box, approximately 5 cm from the box wall. The mouse is placed in the box with its back to the objects and allowed to explore freely for 10 minutes.

[0113] The criteria for determining effective exploration are: the mouse's nose tip is ≤2 cm from the object, it sniffs the object or touches the object with its front paws; occasional rubbing, turning around, sitting still, or climbing the object are not counted as effective exploration time.

[0114] Testing phase (memory retention period). Testing was conducted 24 hours after the end of training to assess long-term memory. One familiar object A1 was replaced with a new object B, while the other familiar object A2 remained in its original position. The mouse was then allowed to explore freely in the box for another 10 minutes.

[0115] Data analysis. The total time (T) spent by mice exploring new objects was statistically analyzed.novel ) and total time to become familiar with an object (T) Familiar The recognition index (RI) is used to quantify recognition and memory ability. The calculation formula is: Recognition Index RI = Time spent exploring new objects ÷ (Time spent exploring new objects + Time spent exploring familiar objects) Buried Food Test (BFT): This study used the buried food test to quantitatively detect the olfactory sensitivity of mice and determine whether the olfactory perception pathway is damaged. The principle of this experiment is that rodents in a state of hunger rely on their innate instinct to search for buried food using olfactory signals.

[0116] Adaptation period. To avoid mice developing fear of unfamiliar food and to fully stimulate their foraging desire, the mice were allowed to become familiar with the target smell 3 days before the experiment. In addition to regular feed, standard feed pellets with peanut butter and good palatability were added to the cages.

[0117] Fasting period. Mice were completely fasted for 24 hours before the experiment to induce a strong feeding urge; during this period, mice were allowed free access to water to avoid dehydration.

[0118] Testing period. The experiment was conducted in a dedicated clean and transparent experimental cage (40 cm × 40 cm × 30 cm), with a 3 cm thick layer of brand new, odorless wood shavings bedding. One feed pellet coated with peanut butter was randomly buried about 1 cm below the surface of the bedding, completely obscuring it so that the mice could not detect it visually.

[0119] A single mouse was placed in the center of the cage, and the time from when it was placed in the cage until it first picked up a food pellet and grasped the food with its forepaws or teeth was recorded. This time was recorded as the olfactory latency. The longest observation period in the experiment was set at 600 s; if the mouse did not find food within 600 s, the latency was uniformly assigned to 600 s for statistical purposes.

[0120] This study investigated a 28-day intranasal drug administration intervention in APP / PS1 double transgenic Alzheimer's disease model mice and evaluated their cognitive function and brain pathological changes. During the intranasal drug administration procedure, mice were gently immobilized in a supine position without anesthesia. The AHBEVs group received AHBEVs suspension every other day, with a single administration volume of 20 μL and a concentration of 5 μg / μL, for a total single administration of 100 μg per mouse, for 28 consecutive days, totaling 14 administrations. Sterile micropipette was used to administer the drug in multiple doses of 2–3 μL, alternating between the left and right nostrils, relying on the mice to inhale the drug spontaneously to avoid aspiration into the lungs. The control group received an equal volume of sterile phosphate-buffered saline (PBS) as a solvent control.

[0121] First, the classic Morris water maze (MWM) test was used to assess the spatial learning and reference memory abilities of mice. The results of the 6-day orienteering phase showed that, compared with the wild-type (WT) control group, the escape latency of the APP / PS1 model group mice was significantly prolonged, indicating a severe impairment in their spatial learning ability. Intranasal administration of AHBEVs effectively reversed this cognitive deficit; from day 3 of the experiment, the escape latency of the AHBEV-treated group was significantly shorter than that of the model group (P < 0.05). Figure 11 In subsequent spatial exploration experiments after the platform was removed, the model mice showed a significant reduction in the time spent in the target quadrant and a significant decrease in the number of times they crossed the original platform site. In stark contrast, AHBEV intervention significantly increased the time mice spent in the target quadrant and the number of times they crossed the platform. Figure 11 (Chinese BC).

[0122] To further confirm the cognitive-improving effect of AHBEVs, this study used a novel object recognition (NOR) test to evaluate mouse recognition memory. During the 24-hour memory retention phase, the model group mice showed a significant loss of preference for exploring new objects, and their recognition index (RI) decreased significantly. Figure 11 (D); while AHBEVs administration can significantly improve the recognition index ( Figure 11 (D), restoring the natural novelty-seeking preference of mice. Furthermore, olfactory dysfunction is a typical early characteristic of Alzheimer's disease. The food-burying experiment (BFT) results showed that AHBEV intervention significantly shortened the abnormally prolonged food-seeking latency in model mice and improved olfactory impairment ( Figure 11 (E).

[0123] This study conducted comprehensive immunofluorescence detection on the core lesion areas of Alzheimer's disease—the cerebral cortex and hippocampus. Aβ plaque deposition is the initiating core of the Alzheimer's disease cascade pathological response. 6E10 antibody staining revealed a large number of densely aggregated Aβ-positive plaques widely distributed in the cerebral cortex and hippocampus of APP / PS1 model mice. Figure 11 In the middle F), the fluorescence area of ​​cortical patches was significantly increased ( Figure 11 In the middle J), ​​the hippocampal plaque burden also increased significantly ( Figure 11 (G). After AHBEVs intervention, the Aβ fluorescence intensity and total plaque area in the mouse cortex and hippocampus were significantly reduced (G). Figure 11 J, Figure 11 The study confirmed that AHBEVs can effectively inhibit amyloid pathological deposition and promote Aβ clearance in vivo.

[0124] Aβ aggregates persistently induce severe neuroinflammation. This study further examined the activation level of glial cells in the neurotoxic microenvironment. IBA-1 staining results showed that microglia in the model group were significantly overactivated: the cell morphology pathologically transformed into an amoeboid state, with enlarged cell bodies, short and thickened processes, and a significant increase in cell number. Consistent with the Aβ clearance results, AHBEVs significantly reversed this inflammatory phenotype. In the treatment group, microglia basically recovered to a multi-branched resting homeostatic morphology, and the IBA-1 fluorescence intensity in the whole brain was significantly reduced. Figure 11 L, Figure 11 Middle I).

[0125] GFAP staining was used to assess astrocyte activation (reactive astrocyte proliferation). The results showed that astrocytes in the brain tissue of model mice were significantly enlarged and their fibrous processes were thickened. AHBEV intervention effectively inhibited excessive astrocyte activation, and GFAP expression levels recovered to near the wild-control level. Figure 11 (Middle K, 11 Middle H).

[0126] In summary, the combined experimental evidence from behavioral and histopathological studies fully confirms that the cognitive protective effect of nasal delivery of AHBEVs stems from its dual mechanism of action—both clearing neurotoxic Aβ plaques and synergistically inhibiting overactivated inflammatory glial networks.

Claims

1. An extracellular vesicle derived from Araceae, wherein, The extracellular vesicles are extracted from fresh Arisaema heterophyllum tissue and have a membrane structure composed of a lipid bilayer with a particle size distribution between 50 nm and 700 nm. The extracellular vesicles encapsulate a group of active metabolites derived from Arisaema heterophyllum, which includes at least apigenin, curcumin, shampooside, ginkgolide B, hypericin, and naringenin. Furthermore, the extracellular vesicles do not contain calcium oxalate needle crystals and lectins that cause mucosal irritation and neurotoxicity in raw Arisaema heterophyllum, or contain a small amount of calcium oxalate needle crystals and lectins that do not cause mucosal irritation and neurotoxicity.

2. The extracellular vesicles derived from Arisaema according to claim 1, wherein, The fresh tissues of the arisaema include tubers (rhizomes).

3. A method for preparing extracellular vesicles derived from Arisaema heterophyllum, the method comprising the following steps: (1) Homogenization and coarse filtration: Fresh Arisaema tubers are thoroughly mixed with pre-cooled extraction buffer and homogenized. The homogenate is collected, filtered to remove plant residues, and crude extract is obtained. (2) Differential centrifugation to remove cell residue: Centrifuge the crude extract obtained in step (1) at 2,000-5,000 ×g, especially 4,000 ×g, for 30-60 minutes, especially 40-45 minutes, and take the supernatant; then centrifuge the supernatant at 10,000-50,000 ×g, especially 10,000 ×g, for 90-120 minutes to remove cell residue by physical precipitation. (3) Filtration and purification: The supernatant obtained by removing cell residue in step (2) is filtered through a microporous membrane and centrifuged at 100,000-150,000×g, especially 100,000×g, for 2-3 hours at 0-4 ℃, and the precipitate is collected. (4) Gradient centrifugation to remove macromolecular toxic proteins and crystal structures: The precipitate obtained in step (3) is resuspended in pre-cooled extraction buffer, and the sample is spread on the upper layer of a sucrose density gradient solution with a concentration gradient of 8%, 30%, 45%, and 60%, and then centrifuged again at 100,000-150,000×g, especially at 100,000×g for 1-3 hours. (5) Collection: Take the fraction between the 8% and 30% sucrose layers, dilute it with pre-cooled extraction buffer, and centrifuge it at 100,000-150,000×g, especially at 100,000×g, for 1-3 hours at 0-4 ℃ to obtain extracellular vesicles (AHBEVs) derived from Arisaema.

4. The method according to claim 3, wherein: In steps (1), (4), and (5), the extraction buffer is phosphate-buffered saline (PBS), physiological saline, HEPES buffer, or a combination thereof; and / or In steps (1), (4), and (5), precooling is performed at 0-8°C, preferably at 4°C; and / or The filtration is performed in step (1) using multiple layers of gauze or a filter screen.

5. The method according to claim 3 or 4, wherein, In step (2), the crude extract obtained in step (1) is centrifuged at 4,000×g for 40-45 minutes and the supernatant is collected; then the supernatant is centrifuged at 10,000×g for 90 minutes to remove cell residue.

6. The method according to any one of claims 3 to 5, wherein, In step (3), a microporous filter membrane with a diameter of 0.2-0.5 μm, preferably 0.22 μm to 0.45 μm, is used for the filtration.

7. An extracellular vesicle derived from Araceae, prepared by the method according to any one of claims 3 to 6.

8. A pharmaceutical composition, wherein, The pharmaceutical composition comprises extracellular vesicles derived from Arisaema as described in claim 1, 2 or 7, and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, wherein, The dosage forms of the pharmaceutical composition include nasal delivery formulations, injectable formulations, oral administration formulations, rectal administration formulations, and especially nasal delivery formulations, including nasal drops, nasal sprays, nasal powders, or in-situ gels; preferably, the excipients include penetration enhancers, pH adjusters, or adhesives suitable for absorption by the nasal mucosa.

10. The use of the extracellular vesicles derived from Arisaema according to claim 1, 2, or 7, or the pharmaceutical composition according to claim 8 or 9, in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases. Specifically, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In particular, the extracellular vesicles described herein have at least one of the following effects in the drug: (1) As a biological sieve, it enriches phytochemicals that resist Aβ toxicity and improve chemotactic perception defects; (2) Bypassing the blood-brain barrier (BBB) ​​via the intraaxonal transport pathway of olfactory sensory nerves (OSNs), it directly targets the central nervous system; (3) Inhibit the NF-κB signaling cascade, downregulate M1 microglia markers (CD86, IL-1β, TNF-α), upregulate M2 markers (CD206, Arg1, IL-10), and reshape the polarization state of microglia; (4) Restore neuroimmune homeostasis, lipid metabolism, antioxidant defense and synaptic plasticity.

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