Ligusticum wallichii extracellular vesicles and preparation method thereof, pharmaceutical composition and medicine for treating cerebral apoplexy, and application of ligusticum wallichii extracellular vesicles in preparation of medicine for treating cerebral apoplexy

Abstract

The invention discloses a preparation method of ligusticum wallichii extracellular vesicles, fresh ligusticum wallichii tissues are used as raw materials, and the ligusticum wallichii extracellular vesicles are obtained through the steps of cleaning, cutting, homogenizing, differential centrifugation, ultracentrifugation, gradient sucrose density separation and the like, are of a typical lipid double-layer structure, are uniform in particle size and have good dispersity and biocompatibility, and are used for preparing the ligusticum wallichii extracellular vesicles. Endogenous active ingredients of ligusticum wallichii can be carried; the ligusticum wallichii extracellular vesicles can span a blood brain barrier, are enriched in a cerebral ischemia area, and play a remarkable neuroprotective role in manners of regulating and controlling neuroinflammation, relieving oxidative stress, promoting neural restoration and the like; the preparation method and application scheme of the ligusticum wallichii extracellular vesicles have the advantages of being natural, safe, efficient, high in targeting performance and the like, and a new technical path and application prospects are provided for development of the plant-derived extracellular vesicles in cerebral apoplexy treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of ischemic stroke drug preparation, in particular to a ligusticum chuanxiong extracellular vesicle, a preparation method thereof, a drug composition for treating ischemic stroke, a drug and an application in the preparation of a drug for treating ischemic stroke. BACKGROUND

[0002] Stroke is a cerebrovascular disease with high mortality and disability rate worldwide, mainly including ischemic and hemorrhagic types. Among them, ischemic stroke accounts for about 70-80% of all strokes, and its pathological mechanism is complex, including reduced cerebral blood perfusion, energy metabolism disorder, activated inflammatory response, blood-brain barrier damage and neuronal death. The current clinical treatment methods mainly include thrombolysis, interventional thrombectomy and basic symptomatic treatment, but all have limitations such as short treatment time window, large side effects, limited applicable population, etc., and there is an urgent need to develop safe and effective new treatment strategies.

[0003] As a traditional Chinese medicinal material, ligusticum chuanxiong has the effects of promoting blood circulation to remove blood stasis, dilating blood vessels, improving cerebral circulation, anti-inflammation and anti-oxidation, etc. Its main active ingredients include ligustrazine, ferulic acid, ligustilide and the like. However, the active ingredients in ligusticum chuanxiong generally have poor water solubility, low bioavailability and difficulty in crossing the blood-brain barrier, which limits its application in central nervous system diseases.

[0004] In recent years, plant-derived extracellular vesicles have become a potential natural nanoscale drug delivery system due to their naturalness, good biocompatibility, low toxicity, ability to load endogenous active substances and easy crossing of biological barriers. Studies have shown that extracellular vesicles from many plants have anti-inflammatory, antioxidant, immunomodulatory and tissue repair-promoting effects. However, there is no public literature reporting the extraction process, structural characteristics of ligusticum chuanxiong-derived extracellular vesicles and their application in the treatment of stroke. As a traditional medicine with clear effects of promoting blood circulation to remove blood stasis and neuroprotection, ligusticum chuanxiong-derived extracellular vesicles may have better transmembrane delivery characteristics and targeting, so it is urgent to establish a stable and repeatable preparation system of ligusticum chuanxiong extracellular vesicles and verify its improvement effect on cerebral ischemic injury. SUMMARY

[0005] The present application designs and develops a ligusticum chuanxiong extracellular vesicle, and the purpose of the present application is to provide a high-purity extracellular vesicle rich in endogenous active ingredients of ligusticum chuanxiong.

[0006] The present application designs and develops a preparation method of ligusticum chuanxiong extracellular vesicle, and the purpose of the present application is to obtain a high-purity extracellular vesicle rich in endogenous active ingredients of ligusticum chuanxiong by constructing a standardized extraction process.

[0007] The application designs and develops an application of Ligusticum chuanxiong extracellular vesicles in preparation of a drug for treating ischemic stroke. The application aims to verify the neuroprotective effect through an ischemic cerebral infarction animal model, so as to provide a new technical path for carrierization of active ingredients of traditional Chinese medicines and treatment of stroke.

[0008] The application also designs and develops a drug for treating ischemic stroke.

[0009] The application also designs and develops a drug composition for treating ischemic stroke.

[0010] The application provides a technical scheme.

[0011] A preparation method of Ligusticum chuanxiong extracellular vesicles comprises the following steps:

[0012] Step one, after pretreatment, Ligusticum chuanxiong is processed by a juicer to filter residues, the filtrate is subjected to first centrifugal treatment, after removing the precipitate, second centrifugal treatment is performed, and after removing the precipitate, third centrifugal treatment is performed;

[0013] Step two, the filtered precipitate is transferred to a sucrose density gradient solution and subjected to fourth centrifugal treatment, the solution between the layers with concentrations of 30% and 45% is collected, and fifth centrifugal treatment is performed, the sample after discarding the supernatant is resuspended by adding a phosphate buffered saline solution, and the Ligusticum chuanxiong extracellular vesicles are obtained by filtering the sample through a filter after being sucked through a needle tube.

[0014] Preferably, in the step one, the operation process of pretreatment comprises the following steps: after fresh Ligusticum chuanxiong is washed with purified water for three times, the Ligusticum chuanxiong is cut and soaked in a phosphate buffered saline solution for 30 minutes.

[0015] The volume of the phosphate buffered saline solution is 2 times the mass of the cut Ligusticum chuanxiong.

[0016] Preferably, in the step one, the first centrifugal treatment is performed at 2000xg for 20 minutes and at a temperature of 4 DEG C.

[0017] The second centrifugal treatment is performed at 12000xg for 60 minutes and at a temperature of 4 DEG C.

[0018] The third centrifugal treatment is performed at 100000xg for 90 minutes and at a temperature of 4 DEG C.

[0019] Preferably, in the step two, the fourth centrifugal treatment and the fifth centrifugal treatment are both performed at 150000xg for 90 minutes and at a temperature of 4 DEG C.

[0020] Preferably, in the step two, the concentrations of the sucrose density gradient solution are 60%, 45%, 30% and 8% in sequence.

[0021] Preferably, in the step two, the Ligusticum Chuanxiong extracellular vesicles are obtained by filtering through a 0.45 μm filter at the needle after being sucked through a 1 mL needle tube.

[0022] A Ligusticum Chuanxiong extracellular vesicle prepared by using the preparation method of the Ligusticum Chuanxiong extracellular vesicle.

[0023] A pharmaceutical composition for treating ischemic stroke, comprising the Ligusticum Chuanxiong extracellular vesicle.

[0024] A medicine for treating ischemic stroke, comprising the Ligusticum Chuanxiong extracellular vesicle.

[0025] Use of a Ligusticum Chuanxiong extracellular vesicle in preparation of a medicine for treating ischemic stroke, using the Ligusticum Chuanxiong extracellular vesicle.

[0026] The beneficial effects of the present application are as follows:

[0027] 1. The present application provides a preparation method of Ligusticum Chuanxiong extracellular vesicles and the obtained product, and constructs a new type of natural drug delivery system which is efficient and stable; the vesicles are safe in source, complete in structure, have good biocompatibility and targeting, can encapsulate various active ingredients in Ligusticum Chuanxiong and maintain the biological activity, thereby overcoming the problems of dispersion of ingredients, low absorption rate and difficulty in crossing the blood-brain barrier in traditional Chinese medicine preparations, and providing a new way for the conversion and application of plant-derived exosomes in the treatment of brain diseases.

[0028] 2. The preparation process of the Ligusticum Chuanxiong extracellular vesicles is simple, has strong repeatability, high purity, uniform vesicle particle size distribution, and stable surface potential, and can be stored for a long time without aggregation or denaturation, thereby ensuring the controllability and batch stability in biological and pharmaceutical preparations; the preparation system can obtain vesicles with complete structure without using organic solvents or additional chemical modification, thereby significantly improving the extraction efficiency and product safety, and being suitable for large-scale preparation and industrialization.

[0029] 3. The present application first applies Ligusticum Chuanxiong extracellular vesicles to the research and development and treatment of ischemic stroke drugs, finds that the Ligusticum Chuanxiong extracellular vesicles can cross the blood-brain barrier in vivo, are enriched in ischemic lesion areas, can effectively reduce brain tissue damage, reduce infarction area and improve neurological dysfunction; compared with conventional therapeutic drugs, the Ligusticum Chuanxiong extracellular vesicles have better bioavailability and tissue targeting, can significantly improve the therapeutic efficiency of active ingredients of Ligusticum Chuanxiong, and exhibit superior neuroprotective effect and clinical conversion potential. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A preparation process flowchart of the Ligusticum Chuanxiong extracellular vesicle described in the present application is shown in the figure.

[0031] Figure 2This is a morphological structure diagram of the extracellular vesicles of Ligusticum chuanxiong after extraction and purification under a transmission electron microscope.

[0032] Figure 3 This is a diagram showing the particle size distribution of extracellular vesicles in Ligusticum chuanxiong as described in this invention.

[0033] Figure 4 This is a graph showing the Zeta potential detection results of extracellular vesicles of Ligusticum chuanxiong as described in this invention;

[0034] Figure 5 This is a Coomassie brilliant blue result of the extracellular vesicle protein components of Ligusticum chuanxiong described in this invention;

[0035] Figure 6 This is an LC-MS mass spectrometry and component identification diagram of the small molecule components of the extracellular vesicles of Ligusticum chuanxiong described in this invention;

[0036] Figure 7 This is a standard curve of the BCA protein quantification detection results described in this invention;

[0037] Figure 8 This is a graph showing the neurological function scores of mice in each group during the animal experiments described in this invention.

[0038] Figure 9 These are brain CT scan results of mice from different groups as described in this invention;

[0039] Figure 10 A graph showing the total offset distance analysis of brain edema in mice of different groups;

[0040] Figure 11 The images show the TTC staining results of each experimental group described in this invention.

[0041] Figure 12 This is a graph showing the measurement and analysis of cerebral infarction area in each experimental group described in this invention. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0043] like Figure 1 As shown, this invention provides a method for preparing extracellular vesicles from Ligusticum chuanxiong, comprising the following steps:

[0044] Step 1: First, wash off the dirt on the surface of the Ligusticum striatum, weigh it, and then wash the fresh Ligusticum striatum three times with ultrapure water; then, cut it into pieces and soak it in an appropriate amount of PBS (Ligusticum striatum mass:PBS volume = 1:2 (w:v)) for 30 minutes;

[0045] Step 2: Use a juicer to pulverize the chuanxiong (Ligusticum striatum) at 35,000 rpm for 3 minutes and 30 seconds each time, twice in total. After the first pulverization, let it stand in a 4°C refrigerator for 2 hours. After the second pulverization, filter out the residue and squeeze out as much water as possible. Divide the filtrate into six 50mL centrifuge tubes, about 40mL per tube.

[0046] Step 3: Centrifuge the filtrate at 4°C at 2000×g for 20 minutes to remove the precipitate. Divide the supernatant into 6 new 50mL centrifuge tubes. Centrifuge the supernatant at 4°C at 12000×g for 60 minutes.

[0047] Step 4: Then transfer the supernatant to six 38.5mL centrifuge tubes, weigh and balance them, and centrifuge at 100,000×g at 4°C for 90 minutes to collect the precipitate.

[0048] Step 5: Collect the precipitates from the above 6 tubes separately, and suspend each tube in 2 mL of PBS for later use; slowly add 60%, 45%, 30%, and 8% sucrose solutions sequentially to the wall of a 13.8 mL centrifuge tube, and finally slowly add 2 mL of the suspensions from the above 6 tubes to the top of the 6 prepared gradient sucrose solutions. Centrifuge at 150,000 × g for 90 minutes at 4 °C, and collect the bands between the 30% and 45% sucrose solutions from the 6 tubes into 6 new 13.8 mL centrifuge tubes.

[0049] Step 6: Weigh and balance the liquid obtained from the centrifugation of the above 6 tubes (the band between the 30% and 45% sucrose solutions) with PBS buffer, and then ultracentrifuge at 150,000 × g for 90 minutes at 4°C. After centrifugation, remove the supernatant, resuspend each tube in 300 μL of PBS, and filter the vesicles through a 0.45 μm filter into two 1.5 mL EP tubes using a 1 mL syringe to obtain the extracellular vesicles of Ligusticum chuanxiong. Store at 4°C.

[0050] This invention also provides extracellular vesicles of Ligusticum chuanxiong. The prepared extracellular vesicles of Ligusticum chuanxiong are characterized by transmission electron microscopy, particle size and concentration, and potential. The components of the extracellular vesicles of Ligusticum chuanxiong are identified, specifically including protein composition analysis and LC-MS component identification. At the same time, BCA protein quantification analysis of the extracellular vesicles of Ligusticum chuanxiong is performed.

[0051] In another embodiment, the transmission electron microscopy (TEM) characterization process includes: to further confirm the morphological characteristics of the extracellular vesicles of *Ligusticum chuanxiong* obtained in this invention, the vesicle samples obtained by gradient sucrose separation were lightly diluted with PBS for TEM observation. An appropriate amount of vesicle suspension was dropped onto a pre-dried carbon film copper grid, allowing the sample to spread naturally on the membrane surface and stand for several minutes to complete adsorption. Subsequently, while the droplet was not completely dry, a small amount of paraformaldehyde working solution was added for preliminary fixation, ensuring the vesicle surface structure remained stable under fixed conditions. After lightly drying, negative staining with uranium acetate was performed to ensure sufficient contrast between the vesicle outline and the membrane structure under electron beam irradiation. After the sample dried naturally, it was placed in a TEM and imaged under appropriate accelerating voltage conditions.

[0052] In another embodiment, the particle size and concentration characterization and potential characterization process includes: To further evaluate the colloidal stability and surface physicochemical properties of the extracellular vesicles of *Ligusticum chuanxiong*, their surface potential was measured. The purified vesicle sample was diluted with sterile PBS at an appropriate ratio to ensure its concentration was within the optimal detection range of the nanoparticle size analyzer, avoiding signal overlap or loss. Subsequently, the diluted suspension was gently mixed and slowly injected into a dedicated sample cell using a pipette, taking care to avoid introducing air bubbles to prevent interference with laser scattering and particle trajectory imaging. After sample loading, the laser source was turned on, and the optical path was adjusted so that the laser beam penetrated the sample cell perpendicularly, forming a clear scattered light field in the microscope field of view. The Brownian motion trajectory of the vesicles in the liquid phase was captured by a real-time video acquisition system, and the scattered light points of the particles in each frame were automatically identified and tracked. The system continuously recorded the displacement of particles within different particle size ranges, and calculated the hydrodynamic diameter of the particles from their Brownian motion velocity according to the Stokes-Einstein equation. Finally, the instrument statistically analyzed the motion data of thousands of particles to generate a particle size distribution spectrum, particle concentration data, and zeta potential values.

[0053] In another embodiment, the protein composition analysis (Coomassie Brilliant Blue) process includes: When analyzing the protein composition of extracellular vesicles of *Ligusticum chuanxiong*, the gradient-purified vesicle suspension is first ultracentrifuged at 150,000 × g for 90 minutes at 4°C to obtain a precipitate. After centrifugation, the supernatant is discarded, and an appropriate amount of RIPA lysis buffer (containing 1 μL PMSF per 50 μL) is added to thoroughly mix the precipitate. After standing at 4°C for approximately 30 minutes, the vesicles are completely lysed by continuous pipetting. Subsequently, following the protein loading buffer operation requirements, the appropriate proportion of loading buffer is added to 50 μL of the lysed vesicle sample and an equal volume of PBS, respectively. The mixture is then treated in a 95°C metal bath for 30 minutes and stored at –80°C for later use. Before loading, the treated vesicle proteins and the control PBS sample are reheated in a 100°C metal bath for 5–10 minutes to fully denature the system. After cooling to room temperature, the system is ready for loading. SDS-PAGE gel preparation: Select an appropriate separating gel concentration based on the desired protein separation range. After checking for leaks in the glass plates beforehand, begin preparing the separating gel. Mix the separating gel solution, buffer, and ammonium persulfate, then add the mixture between the glass slides. Cover the surface with pure water to maintain a smooth interface. After approximately 15 minutes, discard the pure water. Prepare the stacking gel in the same manner and insert the comb, allowing it to stand until the gel is completely cured. After removing the comb, slowly add 10 μL of the denatured vesicle protein sample to the corresponding sample well. Simultaneously, add a protein marker to another well. Then, place the gel in the electrophoresis tank for electrophoresis. First, run the stacking gel at a constant voltage of 80V for approximately 30 minutes to concentrate the proteins. Then, increase the voltage to 120V and continue running the separating gel for approximately 1 hour, until the bromophenol blue indicator approaches the bottom of the gel. Turn off the power and remove the glass slides. After peeling the gel, place it directly into a staining dish containing Coomassie Brilliant Blue staining solution, ensuring the staining solution completely covers the gel. Shake on a shaker at approximately 45 rpm for about 1 hour to ensure uniform staining of the protein bands. After staining, place the gel in destaining solution and continue shaking at the same speed for about 1 hour until the background color is mostly removed and the bands are clearly visible. Finally, place the destaining gel in a transparent protective film to remove air bubbles and then place it in an imaging system for photography.

[0054] In another embodiment, the LC-MS component identification process includes: to clarify the composition of small molecules carried by the extracellular vesicles of *Ligusticum chuanxiong* obtained in this invention, the vesicle samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). After purification, the vesicle suspension was lyophilized to obtain a dry powder, and a mixture of methanol, acetonitrile, and water was used as the extraction solvent. Sufficient shaking and short-term sonication were used to completely release the small molecules from the vesicle interior and membrane surface into the solvent. The resulting mixture was then centrifuged at low temperature to remove precipitates and impurities. The supernatant was then filtered through a 0.22 μm filter membrane and entered into the LC-MS system for detection. In the liquid chromatography system, the sample was separated by a C18 column. Different components flowed into the mass spectrometer in different time sequences according to their polarity and retention characteristics. The mass-to-charge ratio information of each ion peak was acquired in high-resolution mode and matched with a plant drug metabolism database.

[0055] In another embodiment, the BCA protein quantification process includes the following steps:

[0056] Step 1, Microplate Method: Prepare AB solution: Prepare centrifuge tubes and calculate the required AB solution volume [(number of standard wells × 3 + number of sample wells × 3) × 200 μl]. Prepare AB solution: (A:B = 50:1, mix thoroughly, and protect from light). BCA working solution is stable at room temperature for 24 hours; protein quantification must be completed within 24 hours.

[0057] Step 2, Prepare standard solutions: Prepare standards of corresponding concentrations using BSA and PBS according to Table 1. Take 8 EP tubes, prepare the solutions as shown in Table 1, and then vortex to mix. Pipette 8 × 3 20 μl AH standard solutions into the well plate, add them slowly, avoiding the formation of air bubbles, and add them along the well wall.

[0058] Table 1. Liquid Dosage for Pipe No. AH

[0059] Tube number 1 x PBS (μl) BSA (μl) BSA final concentration (μg / ml) A 320 80 1000 B 50 150A1 750 C 100 100A1 500 D 100 100B2 375 E 100 100C3 250 F 100 100E5 125 G 80 20F6 25 H 100 0 0

[0060] Step 3, Sample solution test: Dilute the sample to 2.5 times the original concentration;

[0061] 2x: 40 μl of test solution plus 40 μl of PBS;

[0062] 5x: 20 μl of detection solution plus 80 μl of PBS;

[0063] Step 4: Add working solution: Add 200 μL of working solution to each well, shake for 30 seconds to mix thoroughly, cover the microplate, wrap the well plate with aluminum foil, and try not to touch the bottom of the well during the process. Place it in an incubator and incubate at 37°C for 30 minutes.

[0064] Step 5, Detection by microplate reader: Cool to room temperature, detect the absorbance at a wavelength of 562 nm on the microplate reader, and calculate the sample concentration through the standard curve prepared with the standard product.

[0065] The present invention also provides an application of Ligusticum chuanxiong extracellular vesicles in the preparation of drugs for treating ischemic stroke.

[0066] The present invention determines the application of Ligusticum chuanxiong extracellular vesicles in the preparation of drugs for treating ischemic stroke through their neuroprotective effects in an animal model of ischemic stroke.

[0067] The specific process for preparing the animal model of ischemic stroke includes:

[0068] 1. Experimental animals and drugs

[0069] The animals selected in this experiment were 6-week-old SPF-grade male ICR mice, weighing 20 - 25 g. All mice were provided by Beijing Huafukang Biotechnology Co., Ltd. (Animal license number: SCXK Jing 2024 - 0003), and were housed in the experimental center of Tianjin University of Traditional Chinese Medicine, with 5 mice per cage, at room temperature (23 ± 3°C) and humidity (40 ± 5%). The ethical license number for experimental animals is (TCM-LAEC2025019Z0928). After 1 week of adaptive feeding, they were reserved for use. The administration concentration of edaravone injection was 7.5 mg / kg, and the administration volume was 200 μL.

[0070] All mice were randomly divided into 6 groups: sham operation group (Sham), model group (Model), low-dose group (RLCEVLP) 3 mg / kg, medium-dose group (RLCEVLP) 6 mg / kg, high-dose group (RLCEVLP) 12 mg / kg, and edaravone positive drug group (Edaravone) 7.5 mg / kg.

[0071] 2. Main reagents

[0072] BCA protein concentration assay kit (Solarbio, PC0020), iohexol (Chenxin Pharmaceutical Co., Ltd.), tribromoethanol (Aladdin, T161626), TTC staining solution (2%) (Solarbio, G3005), Coomassie Brilliant Blue staining solution (Beyotime, P0017B), edaravone injection (China National Pharmaceutical Corporation Guorui Pharmaceutical Co., Ltd.).

[0073] 3. Main instruments

[0074] MCAO model wire embolization (Shenzhen Rewod Life Science and Technology Co., Ltd.); incubator (Hunan Xiangyi Experimental Instrument Development Co., Ltd.); multifunctional microplate reader (Thermo Fisher); CT small animal imager (PerkinElmer, QuantumFX, USA), fluorescence and chemiluminescence imaging system (Shanghai Qinxiang Scientific Instruments Co., Ltd.).

[0075] 4. Experimental Methods

[0076] Laboratory animals were anesthetized with tribromoethanol. The mouse skull was exposed, and cerebral blood flow was monitored using a laser speckle flow imaging system (moorFLPI-2Pro, Moor Instruments, UK). The left common carotid artery (LCCA), internal carotid artery (ICA), and external carotid artery (ECA) were exposed. A silicone-coated 4-0 nylon monofilament was inserted into the ECA, gently passed through the ICA, and until its tip blocked the middle cerebral artery (MCA), causing localized ischemia in the left cerebral cortex. Cerebral blood flow was then monitored using the flow imaging system. After 60 minutes of ischemia, cerebral blood flow was measured again, and the internal carotid artery sutures were removed. Finally, cerebral blood flow was measured again to ensure recanalization, and the mouse wound was sutured to allow infection. To eliminate the influence of differences in physiological parameters among the mouse groups on subsequent experimental results, a small animal vital signs monitor (ZR-05, Puxin Instruments, China) and a non-invasive blood pressure monitoring system (CODA, Kent Scientific, USA) were used to continuously measure the blood pressure, blood oxygen saturation, and heart rate of the mice from 15 minutes before ischemia to 15 minutes after reperfusion. During the operation, a heating pad was used to maintain the mouse body temperature at 37.0±0.5℃. Subsequent experiments were conducted 24 hours after reperfusion. Six experimental groups were set up, including a sham surgery group (vascular exposure without suture insertion), a model group (successful modeling but no treatment), three treatment groups (low-dose, medium-dose, and high-dose Chuanxiong extracellular vesicle intervention), and a commercially available clinically used edaravone as a positive control group. After successful modeling, the extracted extracellular vesicles of Ligusticum chuanxiong were administered via tail vein injection. Three doses of RLCEVLP were used: low, medium, and high doses: 3 mg / kg, 6 mg / kg, and 12 mg / kg, 200 μL per mouse. Tail vein injection was administered for three consecutive days. As shown in Table 2, the mouse's motor coordination, sensory response, and balance were quantitatively evaluated daily using a mouse neurological function scoring system (1 point was awarded for failure to complete the task or lack of a tested reflex; 13-18 points indicated severe injury; 7-12 points indicated moderate injury; 1-6 points indicated mild injury; higher scores indicated more severe neurological functional impairment). This reflected the severity of ischemic brain injury and the recovery status after treatment intervention. To further observe the extent of brain tissue structural damage and infarction, mice underwent in vivo CT scans for cerebral edema on day 3. Mice were anesthetized and injected with a contrast agent (iohexol, 15 mL / kg) via the tail vein. Brain imaging was performed using a μCT small animal imaging system (PerkinElmer, Quantum FX, USA) with the following parameters: voltage 90 kV, current 180 μA, field of view 20 mm, standard scan time 4.5 min, and 360° field of view. The offset distance of the coronal midline of the brain was calculated using ImageJ analysis software (National Institutes of Health, Bethesda, Maryland). This distance was directly proportional to the severity of cerebral edema. After CT imaging, mice were anesthetized and euthanized by cervical dislocation, and brain tissue was rapidly collected for TTC staining.

[0077] Table 2 Mouse Neurological Function Scoring System

[0078]

[0079]

[0080] Example

[0081] This invention provides a method for preparing extracellular vesicles from Ligusticum chuanxiong, comprising the following steps:

[0082] Step 1: First, wash off the dirt on the surface of the Ligusticum striatum, weigh it, and then wash the fresh Ligusticum striatum three times with ultrapure water; then, cut it into pieces and soak it in an appropriate amount of PBS (Ligusticum striatum weight:PBS volume = 1:2 (w:v)) for 30 minutes;

[0083] Step 2: Use a juicer to grind the chuanxiong at a speed of 35,000 rpm for 3 minutes and 30 seconds each time, for a total of two times. After the first time, let it stand in the refrigerator at 4℃ for 2 hours. After the second time, filter the residue, squeeze out as much water as possible from the residue, and divide the filtrate into 6 centrifuge tubes of 50mL each, about 40mL in each tube.

[0084] Step 3: Centrifuge the filtrate at 4°C at 2000×g for 20 minutes to remove the precipitate. Divide the supernatant into 6 new 50mL centrifuge tubes. Centrifuge the supernatant at 4°C at 12000×g for 60 minutes.

[0085] Step 4: Then transfer the supernatant to six 38.5mL centrifuge tubes, weigh and balance them, and centrifuge at 100,000×g at 4°C for 90 minutes to collect the precipitate.

[0086] Step 5: Collect the precipitates from the above 6 tubes separately, and suspend each tube in 2 mL of PBS for later use; slowly add 60%, 45%, 30%, and 8% sucrose solutions sequentially to the wall of a 13.8 mL centrifuge tube, and finally slowly add 2 mL of the suspensions from the above 6 tubes to the top of the 6 prepared gradient sucrose solutions. Centrifuge at 150,000 × g for 90 minutes at 4 °C, and collect the bands between the 30% and 45% sucrose solutions from the 6 tubes into 6 new 13.8 mL centrifuge tubes.

[0087] Step 6: Weigh and balance the liquid obtained from the above 6 centrifuged tubes (the band between the 30% and 45% sucrose solutions) with PBS buffer, and then ultracentrifuge at 150,000 × g for 90 minutes at 4°C. After centrifugation, remove the supernatant, resuspend each tube in 300 μL of PBS, and filter the solution through a 0.45 μm filter into two 1.5 mL EP tubes using a 1 mL syringe to obtain extracellular vesicles of Ligusticum chuanxiong. Store at 4°C.

[0088] In this embodiment, the precipitate obtained by centrifugation was weighed and the yield was calculated. Approximately 8 mg of extracellular vesicles of Ligusticum chuanxiong could be separated from every 150 g of Ligusticum chuanxiong.

[0089] Test case

[0090] like Figure 2 As shown in the transmission electron microscope, the extracellular vesicles of Ligusticum chuanxiong exhibit a regular overall morphology, mostly nearly spherical or slightly flattened, with a continuous and clear double-membrane structure visible at the periphery. The surface is smooth, without obvious rupture, folding, or aggregation, indicating that the vesicles maintained good integrity during extraction and purification. The images show that the vesicle sizes are relatively concentrated, mostly typical cup-shaped EVs of 108.2 nm, consistent with the range obtained from particle size analysis, and there are few impurities in the background. This indicates that the extraction process of this invention can effectively separate high-purity Ligusticum chuanxiong-derived nanovesicles. Overall observation results confirm that the vesicles obtained by this invention possess the typical ultrastructural characteristics of plant-derived exosomes, providing reliable morphological evidence for subsequent functional studies.

[0091] like Figure 3 , 4 As shown, the extracellular vesicles of Ligusticum chuanxiong exhibit stable negative charge in the PBS system, with their zeta potential values ​​falling within the common negative charge range for biological vesicles. This indicates that the outer surface of the vesicles carries a certain number of anionic groups, a property consistent with its bilayer membrane composition and the natural structure of plant-derived vesicles. The particles are concentrated at approximately 108.2 nm, with a concentration of 5.3 × 10⁻⁶. 6 The particle size distribution was unimodal (particles / mL). The detection potential of extracellular vesicles of Ligusticum chuanxiong was around -19 mV. The stable negative potential not only reflects the low spontaneous aggregation tendency of vesicles in solution, but also suggests that they have good dispersibility and system stability during subsequent storage and application, providing the necessary physicochemical basis for their use as drug delivery carriers or in vivo experiments.

[0092] like Figure 5 As shown, continuous and clear protein bands were observed in the extracellular vesicle samples of Ligusticum chuanxiong, which showed a wide range of molecular weights. The more concentrated bands were located in the region of about 35-70 kDa, which is consistent with the protein composition characteristics of plant-derived vesicles. This indicates that the vesicles obtained by this invention not only have a complete structure, but their protein composition also has the characteristics of typical plant extracellular vesicles.

[0093] like Figure 6As shown in Table 3, the analysis results revealed that multiple characteristic compounds derived from Ligusticum chuanxiong were detected in the extracellular vesicles, including ferulic acid derivatives, ligustilide components, and some volatile or semi-volatile small molecule metabolites. These substances are believed to have anti-inflammatory, blood flow-improving, and neuroprotective effects in Ligusticum chuanxiong. The vesicles prepared in this invention not only maintain the structural characteristics of plant-derived exosomes but also carry the active components of Ligusticum chuanxiong itself, providing a compositional basis for its mechanism of action in cerebral ischemia-related diseases.

[0094] Table 3 Analysis Results

[0095]

[0096]

[0097] like Figure 7 As shown in Tables 4 and 5, the sample concentration was calculated using a standard curve prepared from the standard samples.

[0098] Table 4 Absorbance Data

[0099]

[0100]

[0101] Table 5 Sample Concentration Values

[0102]

[0103] like Figure 8 As shown, significant differences in the effects of different treatments were observed in the behavioral and histological evaluations of mice after reperfusion. Neurological function scores revealed that the model group mice exhibited significant motor impairment, sluggish sensory responses, and decreased balance after ischemia, with overall scores remaining at a high level, indicating severe neurological damage. Mice receiving extracellular vesicle intervention from Ligusticum chuanxiong, especially the medium- and high-dose groups, showed a gradual reduction in neurological deficits. The high-dose group showed the most significant improvement, characterized by enhanced voluntary activity, reduced hemiplegia, and accelerated recovery of limb coordination, with a degree of functional improvement similar to that of the edaravone group.

[0104] like Figure 9 , 10 As shown, the CT scan images and their analysis further validated the above behavioral results. Significant midline shift was observed in the brain regions of the model group, indicating severe cerebral edema on the affected side. In contrast, the midline shift distance was reduced to varying degrees in mice treated with extracellular vesicles of Ligusticum chuanxiong at all dose groups, especially at medium and high doses, suggesting a significant reduction in cerebral edema. The edaravone group also showed an effect in improving cerebral edema.

[0105] like Figure 11 , Figure 12 As shown, in the TTC staining observation model group, large infarct areas appeared distinctly white, while normal tissue remained bright red, forming a sharp contrast. In the Chuanxiong vesicle intervention group, the area of ​​white infarct areas was significantly reduced, and the degree was positively correlated with the dose. The staining of the high-dose group was closer to that of the sham-operated group, indicating that brain tissue activity was well preserved.

[0106] In summary, the results demonstrate that the method for preparing extracellular vesicles of Ligusticum chuanxiong established in this invention can stably obtain nanovesicles with intact structures, stable surface charges, and protein spectra and small molecule components characteristic of Ligusticum chuanxiong. These nanovesicles exhibit uniform morphology and well-defined physicochemical properties, meeting the requirements for subsequent biological applications. Animal experiments further confirmed that these vesicles can significantly improve neurological function, cerebral edema, and cerebral infarction area in mice with ischemic brain injury, exhibiting a good neuroprotective effect. These results collectively indicate that extracellular vesicles of Ligusticum chuanxiong possess both stable physicochemical properties and significant in vivo biological activity, making them a potential plant-derived nano-preparation for the treatment of stroke.

[0107] This invention utilizes fresh Ligusticum chuanxiong tissue as raw material. Through steps including washing, chopping, homogenization, differential centrifugation, ultracentrifugation, and gradient sucrose density separation, a reproducible, stable, and highly pure method for preparing Ligusticum chuanxiong extracellular vesicles was constructed. The obtained extracellular vesicles exhibit a typical lipid bilayer structure, uniform particle size, good dispersibility and biocompatibility, and can carry endogenous active components of Ligusticum chuanxiong. Furthermore, it was discovered that Ligusticum chuanxiong extracellular vesicles can cross the blood-brain barrier, accumulate in ischemic brain regions, and exert significant neuroprotective effects by regulating neuroinflammation, reducing oxidative stress, and promoting nerve repair. In a mouse model of ischemic stroke, administration of Ligusticum chuanxiong extracellular vesicles significantly reduced infarct volume, improved neurological deficits, and decreased tissue damage.

[0108] The method and application scheme for preparing extracellular vesicles from Ligusticum chuanxiong proposed in this invention have the advantages of being natural, safe, efficient and highly targeted, providing a new technical path and application prospect for the development of plant-derived extracellular vesicles in the treatment of stroke.

[0109] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing an extracellular vesicle of Chuanxiong, characterized by, Comprising the following steps: Step one, pretreated Chuanxiong is processed by a juicer to filter the residue, the filtrate is subjected to a first centrifugal treatment, the precipitate is removed, and then subjected to a second centrifugal treatment, the precipitate is removed again, and then subjected to a third centrifugal treatment; Step two, the filtered precipitate is transferred to a sucrose density gradient solution and subjected to a fourth centrifugal treatment, the solution between the 30% and 45% layers is collected, subjected to a fifth centrifugal treatment, the sample with the supernatant discarded is resuspended by adding a phosphate buffered saline solution, and then filtered by a filter through a needle tube to obtain the Chuanxiong extracellular vesicle.

2. The method of preparing the Chuanxiong extracellular vesicles according to claim 1, wherein, In the step one, the operation process of pretreatment includes: washing fresh Chuanxiong with purified water for three times, cutting and soaking in a phosphate buffered saline solution for 30 minutes. The volume of the phosphate buffered saline solution is 2 times the mass of the cut Chuanxiong.

3. The method of preparing the Chuanxiong extracellular vesicles according to claim 1 or 2, wherein, In the step one, the first centrifugal treatment is at 2000xg for 20 minutes at 4℃; The second centrifugal treatment is at 12000xg for 60 minutes at 4℃; The third centrifugal treatment is at 100000xg for 90 minutes at 4℃.

4. The method of preparing the Chuanxiong extracellular vesicles according to claim 3, wherein, In the step two, the conditions of the fourth and fifth centrifugal treatments are both at 150000xg for 90 minutes at 4℃.

5. The method of preparing the Chuanxiong extracellular vesicles according to claim 4, wherein, In the step two, the concentrations of the sucrose density gradient solution are 60%, 45%, 30% and 8% in sequence.

6. The method of preparing the Chuanxiong extracellular vesicles according to claim 4, wherein, In the step two, the Chuanxiong extracellular vesicle is obtained by filtering through a 0.45μm filter at the needle head through a 1mL needle tube.

7. A Ligusticum cell extracellular vesicle, characterized in that, Prepared by using the preparation method of the Chuanxiong extracellular vesicle as claimed in any one of claims 1-6.

8. A pharmaceutical composition for treating ischemic stroke, characterized by comprising the compound of claim 1. Comprising the Chuanxiong extracellular vesicle as claimed in claim 7.

9. A medicament for treating ischemic stroke, characterized by comprising the compound of claim 1. Comprising the Chuanxiong extracellular vesicle as claimed in claim 7.

10. Use of Ligusticum chuanxiong extracellular vesicles in the preparation of a medicament for treating ischemic stroke. Using the Chuanxiong extracellular vesicle as claimed in claim 7.

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