Use of a notoginseng exosome-like nanovesicle in preparation of a medicament for treating and / or preventing neurological diseases

Abstract

This application discloses the use of Morinda officinalis exosome-like nanovesicles in the preparation of drugs for the treatment and / or prevention of neurological diseases. This application creatively tests the physiological efficacy of Morinda officinalis exosome-like nanovesicles, finding that they can be fully internalized and absorbed by nerve cells, promoting nerve cell proliferation and regeneration of atrophied processes, thereby restoring neural connections, and exhibiting in vivo brain targeting. Through mouse behavioral studies and Nissl staining experiments, it is confirmed that these vesicles can significantly improve learning and memory abilities and neuronal structural integrity in Alzheimer's disease model mice, providing a new strategy for the prevention and / or treatment of Alzheimer's disease.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to the application of Morinda officinalis exosome-like nanovesicles in the preparation of drugs for the treatment and / or prevention of neurological diseases. Background Technology

[0002] Alzheimer's disease is one of the most common neurodegenerative diseases, characterized by memory impairment and motor dysfunction. Currently used medications (such as donepezil and memantine) can only temporarily relieve or stabilize symptoms and cannot stop the long-term progression of the disease. While anti-amyloid antibody drugs approved in recent years have some disease-modifying effects, they also carry certain safety risks. Therefore, developing safe, effective, and multi-pathway synergistic interventions in the pathological process of Alzheimer's disease is of significant clinical importance.

[0003] Morinda officinalis How, a perennial vine belonging to the Rubiaceae family, is one of the "Four Great Southern Chinese Herbs." Multiple studies have shown that Morinda officinalis has a clear foundation and development potential in the treatment of Alzheimer's disease. However, traditional Chinese medicine extracts face serious challenges such as low bioavailability and the difficulty for active ingredients to cross the blood-brain barrier, which severely limits the full realization of Morinda officinalis's anti-Alzheimer's efficacy.

[0004] Therefore, how to develop a drug for treating Alzheimer's disease based on Morinda officinalis biomaterials, deliver its active ingredients to the central nervous system as a whole, give full play to the advantages of multi-component synergistic therapy, and make up for the technical defects of traditional extracts that are difficult to cross the blood-brain barrier and have low bioavailability, is a scientific problem that still needs to be solved and is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application aims to provide the application of Morinda officinalis exosome-like nanovesicles in the preparation of drugs for the treatment and / or prevention of neurological diseases. This application creatively tests the physiological efficacy of Morinda officinalis exosome-like nanovesicles, finding that they can be fully internalized and absorbed by nerve cells, promoting nerve cell proliferation and regeneration of atrophied processes, thereby restoring neural connections, and exhibiting in vivo brain targeting. Through mouse behavioral studies and Nissl staining experiments, it is confirmed that these vesicles can significantly improve learning and memory abilities and neuronal structural integrity in Alzheimer's disease model mice, providing a new strategy for the prevention and / or treatment of Alzheimer's disease.

[0006] To achieve this objective, the present application adopts the following technical solution: In one aspect, this application provides the use of Morinda officinalis exosome-like nanovesicles (MOEVLPs) in the preparation of drugs for the treatment and / or prevention of neurological diseases.

[0007] This application creatively tested the physiological efficacy of exosome-like nanovesicles from the traditional Chinese medicine Morinda officinalis, finding that they can be fully internalized and absorbed by nerve cells, promote nerve cell proliferation and regeneration of atrophied protrusions, thereby restoring neural connections, and have in vivo brain targeting. Through mouse behavioral and Nissl staining experiments, it was confirmed that these vesicles can significantly improve the learning and memory abilities and neuronal structural integrity of Alzheimer's disease model mice, providing a new strategy for the prevention and / or treatment of Alzheimer's disease.

[0008] In some embodiments, the neurological disease includes Alzheimer's disease, Parkinson's disease, stroke, or central nervous system infection, preferably Alzheimer's disease.

[0009] This application demonstrates through mouse model experiments that Morinda officinalis exosome-like nanovesicles can significantly improve the open field activity ability, new object recognition index and water maze learning and memory ability of model mice. At the same time, Nissl staining of brain tissue showed reduced neuronal damage and increased Nissl bodies, indicating that the vesicles have the effect of protecting neurons and improving cognitive function, and have no obvious toxic side effects.

[0010] In some embodiments, the preparation method of the Morinda officinalis exosome-like nanovesicles includes the following steps: The pulp of Morinda officinalis plant was extracted, juiced, filtered, and then centrifuged at differential speed. The supernatant was collected, followed by ultracentrifugation to collect the precipitate and purification, yielding the Morinda officinalis exosome-like nanovesicles.

[0011] In some implementations, the differential centrifugation includes four centrifugations.

[0012] In some embodiments, during differential centrifugation, the first centrifugation is performed at a speed of 200-500 g for 5-10 min; the second centrifugation is performed at a speed of 1000-4000 g for 10-20 min; the third centrifugation is performed at a speed of 5000-8000 g for 30-40 min; and the fourth centrifugation is performed at a speed of 9000-10000 g for 50-60 min.

[0013] The first centrifugation can be performed at speeds of 200 g, 250 g, 300 g, 350 g, 400 g, 450 g, or 500 g, for times of 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min. The second centrifugation can be performed at speeds of 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, 3500 g, or 4000 g, for times of 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min. The third centrifugation can be performed at speeds of 5000 g, 5500 g, 6000 g, 6500 g, 7000 g, 7500 g, or 8000 g, for times of 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, etc. The fourth centrifugation can be performed at speeds of 9000 g, 9100 g, 9200 g, 9300 g, 9400 g, 9500 g, 9600 g, 9700 g, 9800 g, 9900 g, or 10000 g, and for times of 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min, but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0014] In some embodiments, the ultracentrifugation speed is 100,000-110,000 g, and the time is 70-90 min. The speed can be 100,000 g, 101,000 g, 102,000 g, 103,000 g, 104,000 g, 105,000 g, 106,000 g, 107,000 g, 108,000 g, 109,000 g, or 110,000 g, and the time can be 70 min, 75 min, 80 min, 85 min, or 90 min, etc., but is not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0015] The specific centrifugation parameters mentioned above enable more effective extraction of Morinda officinalis exosome-like nanovesicles and effectively prevent the loss of excessive impurities or active ingredients, which could lead to a decrease in the concentration of Morinda officinalis exosome-like nanovesicles and thus affect the efficacy.

[0016] In some embodiments, the ultracentrifugation to collect the precipitate and the purification are repeated at least twice.

[0017] Secondly, this application provides an application of Morinda officinalis exosome-like nanovesicles in the preparation of a nerve cell proliferation promoter.

[0018] According to the test results of this application, Morinda officinalis exosome-like nanovesicles can promote the proliferation of nerve cells at the cellular level (in vitro level). That is, Morinda officinalis exosome-like nanovesicles can be made into a simple experimental preparation for exploring the physiological process of nerve cell proliferation. The nerve cell proliferation promoter claimed in this application is not for eliminating the cause or lesion, that is, it is an application in the preparation of nerve cell proliferation promoter for a non-therapeutic purpose.

[0019] Thirdly, this application provides an application of Morinda officinalis exosome-like nanovesicles in the preparation of a nerve cell atrophy protrusion regeneration promoter.

[0020] According to the test results of this application, Morinda officinalis exosome-like nanovesicles can effectively reverse the atrophy of nerve cell processes induced by β-amyloid protein, increase process length, increase the number of process branches, repair damaged neural connections, and improve nerve conduction function.

[0021] Fourthly, this application provides an application of Morinda officinalis exosome-like nanovesicles in the preparation of brain-targeting formulations.

[0022] This application also creatively discovers that Morinda officinalis exosome-like nanovesicles have brain-targeting properties in vivo, effectively crossing the blood-brain barrier and accumulating in brain lesions. This discovery can be further applied to the preparation of brain-targeting agents for the study of the pathogenesis of central nervous system diseases such as Alzheimer's disease and the treatment of related symptoms.

[0023] Compared with the prior art, this application has the following advantages: This application provides the application of Morinda officinalis exosome-like nanovesicles in the preparation of drugs for the treatment and / or prevention of neurological diseases. Through creative testing of the physiological efficacy of Morinda officinalis exosome-like nanovesicles, it was found that they can be fully internalized and absorbed by nerve cells, promote nerve cell proliferation and regeneration of atrophied protrusions, thereby restoring neural connections, and have in vivo brain targeting. Through mouse behavioral and Nissl staining experiments, it was confirmed that these vesicles can significantly improve the learning and memory abilities and neuronal structural integrity of Alzheimer's disease model mice, providing a new strategy for the prevention and / or treatment of Alzheimer's disease. Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope image of the Morinda officinalis exosome-like nanovesicles extracted in Example 1; Figure 2 This is an RNA detection image of the Morinda officinalis exosome-like nanovesicles extracted in Example 1; Figure 3 The figure shows the results of the MTT assay for detecting the effect of MOEVLPs extracted in Example 1 on the proliferation of PC12 cells. Figure 4 Figure 1 shows the internalization and uptake of MOEVLPs extracted in Example 1 by PC12 cells at different time points as detected by fluorescence microscopy. Figure 5 The image shows the fluorescence staining results of MOEVLPs extracted in Example 1 on the atrophic processes of nerve cells under a fluorescence microscope. Figure 6 The image shows the results of fluorescence microscopy analysis of the average synaptic length of nerve cell atrophy processes by MOEVLPs extracted in Example 1. Figure 7 This is a diagram showing the brain targeting experimental results of MOEVLP extracted in Example 1; Figure 8 The open field trajectory map of MOEVLP-enhanced behavior in SAMP8 mice, extracted in Example 1; Figure 9 The graph shows the results of MOEVLP improving the behavior of SAMP8 mice, as extracted in Example 1; Figure 10 This is a graph showing the total exploration distance of mice in SAMP8 mice that were improved by MOEVLP in Example 1; Figure 11 The image shows the experimental results of the new object recognition method for improving the behavior of SAMP8 mice, obtained from the MOEVLP extraction in Example 1. Figure 12 The water maze trajectory diagram of MOEVLP-enhanced behavior in SAMP8 mice obtained in Example 1; Figure 13 The graph shows the time of first arrival at the platform in SAMP8 mice after MOEVLP improved behavior, as extracted in Example 1. Figure 14 The graph shows the number of times mice crossed the platform, which was obtained from Example 1 and improved the behavior of SAMP8 mice using MOEVLP. Figure 15 Image showing the results of Nissl staining; Figure 16 This is a graph showing the statistical results of the number of Nissl bodies in the mouse cerebral cortex; Figure 17 This is a graph showing the statistical results of the number of Nissl bodies in the CA3 region of the mouse hippocampus. Detailed Implementation

[0025] The technical solution of this application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely to help understand this application and should not be regarded as specific limitations on this application.

[0026] Example 1: Preparation of Morinda officinalis exosome-like nanovesicles (1) Extract the pulp from the Morinda officinalis plant, extract the juice, and filter the juice.

[0027] (2) Centrifuge the filtrate at a differential speed and collect the supernatant: the first centrifugation speed is 500 g and the first centrifugation time is 10 min; the second centrifugation speed is 2000 g and the second centrifugation time is 20 min; the third centrifugation speed is 5000 g and the third centrifugation time is 30 min; the fourth centrifugation speed is 10000 g and the fourth centrifugation time is 60 min.

[0028] (3) The supernatant was collected by ultracentrifugation at a speed of 100,000 g for 70 min. The sample was purified by using a 0.22 μm filter and repeated 3 times to obtain Morinda officinalis exosome-like nanovesicles.

[0029] The extracted Morinda officinalis exosome-like nanovesicles were characterized and their morphology was as follows: Figure 1 As shown, the vesicles exhibit a typical cup-shaped morphology with a clear membrane structure and a clean background. RNA analysis of the extracted Morinda officinalis exosome-like nanovesicles yielded the following results: Figure 2 As shown, the bands are concentrated at the bottom, consistent with the characteristic that most exosome-like nanovesicles are miRNAs (microRNAs). These results indicate that the extracted Morinda officinalis exosome-like nanovesicles meet the specified requirements.

[0030] Unless otherwise specified, the following examples all use Morinda officinalis exosome-like nanovesicles extracted in Example 1.

[0031] Example 2: Effect of MOEVLPs on PC12 cell proliferation To verify the bioactivity of MOEVLPs, we first conducted in vitro cellular studies to explore the proliferative effect of MOEVLPs on PC12 (adrenal pheochromocytoma cells).

[0032] The effect of MOEVLPs on PC12 cell proliferation was detected by the MTT assay (thiazolyl blue colorimetric method): PC12 cells were inoculated at a concentration of 1*102 4PDL (poly-L-lysine)-coated 96-well plates were seeded at a density of 100 cells / well. After 2 days of incubation, MOEVLPs at different concentrations (0.005, 0.01, 0.015, 0.02 mg / mL) were added directly, and incubation continued for another day. Subsequently, MTT (thiazolyl blue) solution (5 mg / mL, 20 μL / well) was added, and the 96-well plates were incubated in a light-protected incubator for 4 h. After incubation, the culture medium was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the MTT crystals. The absorbance (OD) was measured at 570 nm using a microplate reader, and cell viability (%) was calculated.

[0033] Experimental results show that ( Figure 3 MOEVLPs can significantly promote the proliferation of PC12 neurons. Compared with the control group, the PC12 cells treated with different concentrations of MOEVLPs showed increased viability, demonstrating that MOEVLPs has good neuronal proliferation-promoting activity, providing experimental evidence for its further application in neuroprotection and Alzheimer's disease treatment.

[0034] Example 3: Internalization and absorption experiment of MOEVLPs The full internalization and absorption of MOEVLPs by PC12 is a prerequisite for the subsequent action of MOEVLPs on PC12. Therefore, it is necessary to verify whether MOEVLPs can be internalized and absorbed by PC12 before conducting subsequent experiments.

[0035] The experimental steps are as follows: (1) MOEVLPs fluorescent labeling: FITCs (fluorescein isothiocyanate, 1 mg / mL) were added to MOEVLPs and then incubated at 4°C in the dark with gentle shaking for 8 h.

[0036] (2) Cell incubation and fluorescent labeling: FITC-MOEVLPs (fluorescently labeled MOEVLPs) were diluted in the culture medium and then incubated with cells in an incubator for 24 h. The culture medium was discarded, and the cells were washed with PBS (phosphate buffered saline), and fixed with 4% PFA (paraformaldehyde) at room temperature (20°C) for 30 min. The cells were washed twice with 0.2% Triton-PBS (polyethylene glycol octylphenyl ether-phosphate buffer). 300 μL of DAPI (diamidindole) was added to each well, and the cells were incubated in the dark at room temperature (20°C) for 2 h. The DAPI was removed, and the cells were washed three times with Triton-PBS for 5 min each time. One drop of anti-fluorescence attenuation mounting medium was added, and the cells were stored in the dark.

[0037] The results are as follows Figure 4The image shows the internalization and uptake of FITC-labeled Morinda officinalis exosome-like nanovesicles (FITC-MOEVLPs) by PC12 cells at different time points as detected by fluorescence microscopy. FITC-MOEVLPs can be internalized and absorbed by PC12 cells, and the degree of internalization increases with the extension of co-incubation time. At 4 h, a green fluorescence signal is visible in the cells; by 8 h, the green fluorescence signal is significantly enhanced, and the internalized MOEVLPs are mainly distributed around the cell nucleus. No obvious fluorescence signal was observed in the control group. These results demonstrate that Morinda officinalis exosome-like nanovesicles can be fully internalized and absorbed by nerve cells, laying an experimental foundation for subsequent neuroprotective and anti-Alzheimer's disease efficacy studies.

[0038] Example 4: Experiment on the neuroprotective effect of MOEVLPs on nerve cells The protective effect of MOEVLPs on the neural cell injury model is a prerequisite for MOEVLPs to exert their anti-Alzheimer's drug efficacy.

[0039] The experimental steps are as follows: (1) Fixation: Remove the culture medium from the 24-well plate. Add 4% PFA and fix at room temperature for 25-30 min. Wash twice with 0.2% Triton-PBS and add slowly.

[0040] (2) Blocking and staining: Add primary antibody solution and incubate the plate at 4°C. Remove the primary antibody on the second day. Wash twice with 0.2% Triton-PBS. Wash a third time with PBS to reduce foaming. Add 300 μL of secondary antibody solution to each well and incubate at room temperature in the dark for 2 h. Remove the secondary antibody. Wash three times with Triton-PBS. Add 1 drop of anti-fluorescence attenuation mounting medium and store in the dark.

[0041] (3) Observation and quantification: Observe and photograph in the corresponding wavelength range of the fluorescence microscope, perform fluorescence quantification and image processing, and finally perform data processing and analysis.

[0042] Experimental results show that ( Figure 5-6 Morinda officinalis exosome-like nanovesicles can significantly promote the regeneration of processes in atrophied neurons, increase process length and branch number, and reconstruct the neural cell connection network. This provides direct cellular evidence for its ability to improve cognitive function in Alzheimer's disease.

[0043] Example 5: Brain-targeting experiment of MOEVLPs In vivo targeted studies are a prerequisite for subsequent studies on in vivo biological activity.

[0044] The experimental steps are as follows: (1) MOEVLPs fluorescent labeling: Same as in Example 3.

[0045] (2) FITC-PBS (control group) fluorescent labeling: FITCs (fluorescein isothiocyanate, 1 mg / mL) were added to PBS and then incubated at 4°C in the dark with gentle shaking for 8 h.

[0046] (3) Animal in vivo imaging: 150 μL of FITC-MOEVLPs (MOEVLPs group) and FITC-PBS (control group) were injected into the tail vein of C57BL / 6J mice. The fluorescence signal intensity of FITC was measured using an in vivo imaging system at 24 h and 48 h after injection.

[0047] The results are as follows Figure 7 As shown, no significant fluorescence signal was detected in the brain tissue of the control group mice at any time point, while specific fluorescence signals were detected in the brain tissue of the MOEVLPs group mice, and the intensity of the fluorescence signal showed dynamic changes over time: fluorescence signal was present at 24 h, and the fluorescence signal became stronger at 48 h. These results fully demonstrate that Morinda officinalis exosome-like nanovesicles have clear brain targeting properties, can effectively cross the blood-brain barrier and accumulate in brain tissue, and that the brain is the target site for MOEVLPs to exert their anti-Alzheimer's disease bioactivity in vivo.

[0048] Example 6: MOEVLPs improve learning and memory abilities and neuronal structure in diseased mice. 1. Laboratory animals: Male SPF-grade SAMP8 mice (rapid aging model) and SAMR1 mice (normal anti-aging control), aged 7-8 months, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All mice were acclimatized for one week after purchase, during which time they were weighed daily and their mental state and activity levels were observed.

[0049] 2. Animal grouping and administration: After acclimatization, SAMP8 mice were randomly divided into four groups (n=8 per group): model group (PBS), low-dose group (0.1 mg / mL), medium-dose group (0.2 mg / mL), and high-dose group (0.4 mg / mL). SAMR1 mice served as the normal control group (n=8, PBS). The positive control group (n=8) received rapamycin (0.2 mg / mL). Administration was via tail vein injection, once every two days, with each injection volume of 200 μL. The normal control group and model group received an equal volume of PBS. The administration period was from day 0 to day 24 (a total of 12 administrations).

[0050] 3. Behavioral tests: Open Field Test: Mice were placed in an open box, and a video tracking system was used to record the total distance and average speed of movement over 5 minutes. This was used to assess the mice's autonomous activity and anxiety-like behavior. New Object Recognition Test: Adaptation Period: Mice were placed in an empty test box and allowed to explore freely for 5 minutes. Familiarization Period: Two identical objects were placed in the box, and the mice were allowed to explore for 5 minutes, recording the exploration time for each object. Testing Period: One hour after the familiarization period, one of the objects was replaced with a new object, and the mice were allowed to explore for 5 minutes, recording the exploration time for the old object (A) and the new object (B). The Discrimination Index (DI) = (Time spent exploring the new object - Time spent exploring the old object) / Total exploration time × 100%, used to assess short-term cognitive memory. Water Maze Test: Mice were placed in the water facing the pool wall, and the time required for the mice to find the hidden platform was recorded (escape latency). Training was conducted 4 times a day, with 15-minute intervals between each session, for 5 consecutive days. If the platform is not found within 60 seconds, guide the mouse to the platform and stay there for 15 seconds; the escape latency period is recorded as 60 seconds. In the spatial exploration experiment, the platform is removed, and the mouse is placed into the water from the entry point. Record the number of times the mouse crosses the original platform position, the percentage of time spent in the target quadrant, and the swimming path within 60 seconds.

[0051] 4. Tissue sampling and Nissl staining: After behavioral testing, mice were anesthetized and perfused with pre-cooled saline via the heart, followed by brain harvesting. Brain tissue was fixed in 4% paraformaldehyde, routinely dehydrated, and embedded in paraffin. It was stained with 0.1% toluidine blue for 30 min, differentiated, dehydrated, cleared, and mounted. The morphology of neurons in the CA3 region of the hippocampus and the cortex was observed under a light microscope, the number of Nissl bodies was counted, and neuronal damage and survival were assessed.

[0052] Behavioral results ( Figure 8-14 The results showed that, compared with the model group, the mice in the Morinda officinalis exosome-like nanovesicle drug administration group had significantly increased autonomous exploration activities, significantly improved average speed and total exploration distance, and reduced anxiety-like behaviors; the new object recognition experiment showed that the recognition index of the drug administration group mice was significantly increased and short-term cognitive memory ability was restored; the water maze experiment showed that the escape latency of the drug administration group mice was significantly shortened, the number of times they crossed platforms increased, and spatial learning and memory ability was significantly improved.

[0053] Nissl staining results Figure 15-17 The results of Nissl staining of brain tissue showed that Morinda officinalis exosome-like nanovesicles could alleviate neuronal damage in the hippocampus of SAMP8 mice, increase the number of Nissl bodies, protect the structural integrity of neurons, and inhibit neuronal atrophy.

[0054] The applicant declares that this application illustrates the application of Morinda officinalis exosome-like nanovesicles in the preparation of drugs for the treatment and / or prevention of neurological diseases through the above embodiments. However, this application is not limited to the above embodiments, that is, it does not mean that this application must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of this application, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

[0055] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0056] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. The application of Morinda officinalis exosome-like nanovesicles in the preparation of drugs for the treatment and / or prevention of neurological diseases.

2. The application according to claim 1, characterized in that, The neurological diseases include Alzheimer's disease, Parkinson's disease, stroke, or central nervous system infection, with Alzheimer's disease being the preferred option.

3. The application according to claim 1, characterized in that, The preparation method of the Morinda officinalis exosome-like nanovesicles includes the following steps: The pulp of Morinda officinalis plant was extracted, juiced, filtered, and then centrifuged at differential speed. The supernatant was collected, followed by ultracentrifugation to collect the precipitate and purification, yielding the Morinda officinalis exosome-like nanovesicles.

4. The application according to claim 3, characterized in that, The differential centrifugation includes four centrifugations.

5. The application according to claim 4, characterized in that, In the differential centrifugation, the first centrifugation is performed at a speed of 200-500 g for 5-10 min; the second centrifugation is performed at a speed of 1000-4000 g for 10-20 min; the third centrifugation is performed at a speed of 5000-8000 g for 30-40 min; and the fourth centrifugation is performed at a speed of 9000-10000 g for 50-60 min.

6. The application according to any one of claims 3-5, characterized in that, The ultracentrifugation speed is 100,000-110,000 g, and the time is 70-90 min.

7. The application according to any one of claims 3-6, characterized in that, The ultracentrifugation to collect the precipitate and the purification were repeated at least twice.

8. Application of Morinda officinalis exosome-like nanovesicles in the preparation of nerve cell proliferation promoters.

9. Application of Morinda officinalis exosome-like nanovesicles in the preparation of a nerve cell atrophy protrusion regeneration promoter.

10. Application of Morinda officinalis exosome-like nanovesicles in the preparation of brain-targeting agents.

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