Immature bitter orange extracellular vesicle-like particle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses an immature bitter orange-derived extracellular vesicle-like nanoparticle, which is characterized in that the immature bitter orange-derived extracellular vesicle-like nanoparticle is prepared by the steps of crushing immature bitter orange, filtering, centrifuging and resuspending, the average particle size is 60-120 nm, and the particle size of the immature bitter orange-derived extracellular vesicle-like nanoparticle is 20-30 nm. The extracellular vesicle-like nanoparticles are used for entrapment of at least one of naringin, neohesperidin, naringenin and hesperidin. The extracellular vesicle-like nanoparticles derived from immature bitter oranges are simple in preparation method, have liver targeting and good stability, and can be used for preparing medicines for preventing and / or treating liver injury, depression and breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to extracellular vesicle-like particles of Fructus Aurantii Immaturus, and a preparation method and application thereof. Background Art

[0002] In modern society, chronic diseases related to metabolic disorders and mental stress, such as non-alcoholic fatty liver disease (NAFLD), depression, and certain types of tumors, have become major global public health challenges. More and more clinical and basic research shows that there are profound internal connections among these seemingly independent diseases in pathophysiology. Among them, the liver, as the metabolic center and important endocrine organ of the body, plays a core role in maintaining whole-body homeostasis, regulating emotions, and resisting disease progression. Therefore, starting from the overall concept of "soothing the liver and relieving depression" in traditional Chinese medicine theory, finding strategies that can target and regulate liver function to synergistically intervene in multiple related diseases has become a frontier direction for the research and development of innovative drugs.

[0003] Plant-derived extracellular vesicle-like nanoparticles are nanoscale particles naturally secreted by plant cells and wrapped by a phospholipid bilayer. They encapsulate various bioactive substances from the source plant, including unique proteins, lipids, small molecule compounds, and small RNAs. As a novel natural nanocarrier and bioactive entity, plant vesicles have shown great application potential in the biomedical field due to their good biocompatibility, low toxicity, high safety, and ability to protect the activity of the encapsulated substances. Combining traditional Chinese herbs with modern vesicle technology provides a new perspective for exerting the overall efficacy of traditional Chinese medicine with multiple components and multiple targets.

[0004] As a traditional Chinese medicine, Fructus Aurantii Immaturus has the effects of soothing the liver and relieving depression, and breaking qi and dissipating accumulation. Based on the effects of Fructus Aurantii Immaturus, the naturally isolated extracellular vesicle-like particles from Fructus Aurantii Immaturus may have protective and therapeutic effects on liver-related diseases, which can be a research direction with great scientific value and transformation potential. However, there are no reports in the existing technology on systematically isolating and purifying extracellular vesicle-like particles of Fructus Aurantii Immaturus, confirming their tissue targeting in vivo, and comprehensively evaluating their biological activities and application potential in multiple liver-related diseases and mental stress-related diseases. Therefore, a preparation method for extracellular vesicle-like nanoparticles derived from Fructus Aurantii Immaturus with stable properties needs to be developed. Summary of the Invention

[0005] The purpose of the present invention is to develop a preparation method for extracellular vesicle-like nanoparticles derived from Fructus Aurantii Immaturus. Through this method, nanoparticles with stable properties can be prepared, and various liver-related diseases and mental stress-related diseases can be treated, so as to solve the technical problems that there are no extracellular vesicle-like nanoparticles derived from Fructus Aurantii Immaturus, their preparation methods and applications in the existing technology.

[0006] Citrus aurantium L., a plant of the Rutaceae family, is the dried young fruit of Citrus aurantium L. and its cultivated varieties or Citrus sinensis Osbeck. It has a bitter, pungent, and sour taste, and is slightly cold in nature. It has the effects of soothing the liver and relieving depression, breaking up qi stagnation and eliminating stagnation.

[0007] According to a first aspect of the present invention, extracellular vesicle-like nanoparticles derived from Citrus aurantium are provided, which are prepared by crushing, filtering, centrifuging and resuspending Citrus aurantium. The average particle size of the extracellular vesicle-like nanoparticles derived from Citrus aurantium is 60-120 nm.

[0008] In some embodiments, the potential of the extracellular vesicle-like nanoparticles derived from Citrus aurantium is -13 to -15 mV.

[0009] In some embodiments, extracellular vesicle-like nanoparticles derived from Citrus aurantium encapsulate at least one of naringin, neohesperidin, naringenin, and hesperidin.

[0010] In some embodiments, when the extracellular vesicle-like nanoparticles derived from Citrus aurantium are resuspended in 400 μL of buffer solution, the resulting suspension contains 250–300 μg / mL of naringin, 300–320 μg / mL of neohesperidin, 20–25 μg / mL of naringenin, and 11–18 μg / mL of hesperidin. The buffer solution is PBS buffer.

[0011] In some embodiments, extracellular vesicle-like nanoparticles derived from Citrus aurantium have liver-targeting properties.

[0012] According to a second aspect of the present invention, a method for preparing extracellular vesicle-like nanoparticles derived from Citrus aurantium is provided, comprising the following steps: (1) After mixing the immature bitter orange with PBS buffer, the juice was filtered, and the filtrate was collected and then subjected to gradient centrifugation at 4℃ and 2500~13000×g. The supernatant was collected. (2) Centrifuge the supernatant at 4℃ and 120,000~150,000×g, retain the precipitate, and resuspend the precipitate in PBS buffer to obtain a suspension; (3) Centrifuge the suspension at 4℃ and 120,000~150,000×g, retain the precipitate, resuspend the precipitate with PBS buffer, filter, and collect the filtrate to obtain the product.

[0013] In some implementations, in step (1), the volume ratio of Citrus aurantium to PBS buffer is 1:(1.2~2.0).

[0014] In some implementations, the parameters used for gradient centrifugation in step (1) are: S1. First centrifugation: Centrifuge at 4℃ and 2500~3500×g for 8~12 minutes, and take the supernatant; S2. Second centrifugation: Centrifuge at 4℃ and 11000~13000×g for 18~22 minutes, and discard the precipitate and the uppermost oil layer; S3. Third centrifugation: Centrifuge at 4℃ and 11000~13000×g for 18~22 minutes, and take the supernatant.

[0015] In some embodiments, the oil is the volatile oil of Citrus aurantium. The volatile oil mainly originates from the oil chambers of Citrus aurantium.

[0016] In gradient centrifugation, the first centrifugation aims to remove larger impurities (intact cells, large cell wall fragments, cell nuclei, and large polymers formed after plant tissue disruption). The second centrifugation aims to remove medium-sized and dense organelles and debris. Due to the abundance of oil cells in *Citrus aurantium*, the volatile oil components are larger in volume and lower in density, floating on the surface of the supernatant. To remove the volatile oil components and cell debris from *Citrus aurantium*, centrifugation conditions of 11000–13000 × g are selected. After centrifugation, the uppermost layer of volatile oil and the bottommost precipitate are removed, retaining the intermediate layer. The third centrifugation aims to remove polysaccharides, pectin, and proteoglycans that may re-aggregate in the plant homogenate after the second centrifugation. Centrifugation at 11000–13000 × g significantly reduces the probability of these re-aggregates co-precipitating with subsequent ultraprecipitation, improving the purity and stability of the supernatant. Selecting these parameters ensures maximum removal of interfering impurities while minimizing the loss of target vesicles.

[0017] In some embodiments, in step (1), the filtration method used is filtration with gauze. The purpose is to remove large particulate impurities from the immature bitter orange.

[0018] In some implementations, in steps (2) and (3), centrifugation is performed at 4°C and 140,000 × g for 60 to 80 minutes.

[0019] According to a third aspect of the present invention, the use of the above-mentioned extracellular vesicle-like nanoparticles derived from Citrus aurantium in the preparation of medicaments for the prevention and / or treatment of acute liver injury or non-alcoholic fatty liver disease is provided.

[0020] In some implementations, acute liver injury can be drug-induced liver injury, and non-alcoholic fatty liver disease can be high-fat diet-induced non-alcoholic fatty liver disease. High-fat diet-induced non-alcoholic fatty liver disease is a type of chronic liver injury.

[0021] According to a fourth aspect of the present invention, the use of the above-mentioned extracellular vesicle-like nanoparticles derived from Citrus aurantium in the preparation of medicaments for the prevention and / or treatment of mental stress disorders is provided.

[0022] In some implementations, mental stress disorders are defined as depression and mental disorders. Specifically, depression and mental disorders include depressive disorder or depressive-like behavior.

[0023] In some implementations, the type of depression can be mild to moderate depressive disorder or persistent depressive disorder.

[0024] In some implementations, depressive-like behaviors include hopelessness-like behaviors, anhedonia, reduced mobility, anxiety, and insomnia.

[0025] According to a fifth aspect of the present invention, the use of the above-mentioned extracellular vesicle-like nanoparticles derived from Citrus aurantium in the preparation of a medicament for treating breast cancer is provided.

[0026] In some implementations, breast cancer is defined as breast cancer mediated by chronic psychological stress.

[0027] The beneficial effects of this invention are as follows: (1) This invention provides a stable and efficient method for preparing extracellular vesicle-like nanoparticles derived from Citrus aurantium, which can obtain high-purity, uniformly physicochemically and medicinally suitable extracellular vesicle-like particles from the natural medicinal material Citrus aurantium, providing a new technical approach for the modernization of traditional Chinese medicine.

[0028] (2) This invention prepares extracellular vesicle-like particles with a typical saucer-like structure, an average particle size of 60-120 nm, and a negatively charged surface through the steps of crushing, filtering, centrifugation, and resuspension. It also reveals for the first time that the vesicles of Citrus aurantium have significant natural liver-targeting properties. This key discovery not only provides modern biological evidence for the traditional efficacy of Citrus aurantium in "soothing the liver and relieving depression," but also lays the material and theoretical foundation for the therapeutic effect of Citrus aurantium vesicles as a novel active substance form of Citrus aurantium.

[0029] (3) The extracellular vesicle-like nanoparticles derived from Citrus aurantium of the present invention have good stability and can show clear therapeutic potential in a variety of diseases such as acute liver injury, non-alcoholic fatty liver, depression and breast cancer mediated by chronic psychological stress. They provide a novel and effective therapeutic entity for clinical intervention of these diseases and have broad application prospects in the preparation of drugs for the prevention or treatment of the above-mentioned diseases. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the preparation process of the Citrus aurantium vesicles of the present invention. Figure 2 This is a transmission electron microscope image of the Citrus aurantium vesicles of the present invention; Figure 3 The particle size distribution and nanoflow cytometry of the Citrus aurantium vesicles of this invention are shown below. Figure 4 This is a potential distribution diagram of the Citrus aurantium vesicles of the present invention; Figure 5 The purity test results of the Citrus aurantium vesicles of the present invention; Figure 6 This is a protein band diagram of the contents of the Citrus aurantium vesicles of the present invention; Figure 7 This is a diagram showing the small RNA bands within the contents of the Citrus aurantium vesicles of the present invention. Figure 8 This is a thin-layer chromatogram of lipids from the Citrus aurantium vesicles of the present invention; Figure 9 This is a particle size distribution diagram for the stability testing of Citrus aurantium vesicles according to the present invention. Figure 10 White light spectrum for stability testing of Citrus aurantium vesicles according to the present invention; Figure 11 This is a high-performance liquid chromatogram of the Citrus aurantium vesicles of the present invention; Figure 12 This is a liquid chromatography-mass spectrometry (LC-MS) spectral image of the Citrus aurantium vesicles of the present invention; Figure 13 (A) is a live fluorescence image of a mouse; Figure 13 (B) is a fluorescence image of an isolated mouse organ; Figure 14 (A) H&E staining images of mice in each group during the acetaminophen-induced acute liver injury experiment; Figure 14 (B) represents the levels of alanine aminotransferase and aspartate aminotransferase in each group of mice during the acetaminophen-induced acute liver injury experiment. Figure 15 (A) H&E staining images of mice in each group during the high-fat diet-induced non-alcoholic fatty liver experiment; Figure 15 (B) shows Oil Red O staining images of mice in each group during the high-fat diet-induced non-alcoholic fatty liver experiment; Figure 15 (C) shows the general physiological indicators and serum biochemical test results of mice in each group in the experiment of non-alcoholic fatty liver induced by high-fat diet; Figure 16 (A) Results of open field tests for mice in each group during the chronic unpredictable mild stress depressive-like behavior experiment; Figure 16 (B) represents the sugar water preference rate of mice in each group during the chronic unpredictable mild stress depressive-like behavior experiment; Figure 16 (C) Serum corticosterone levels in each group of mice during the chronic unpredictable mild stress mouse depressive-like behavior experiment; Figure 17 (A) Tumor images of mice in each group during the chronic psychological stress breast cancer mouse experiment; Figure 17 (B) represents the volume of mammary tumors in each group of mice in the chronic psychological stress breast cancer mouse experiment; Figure 17 (C) represents the body weight of mice in each group during the chronic psychological stress breast cancer mouse experiment; Figure 18 (A) is a live fluorescence image of the lungs of mice in each group in the experiment of chronic psychological stress-mediated lung metastasis of breast tumors in mice according to the present invention; Figure 18 (B) shows a gross specimen of representative isolated lung tissue from each group of mice; Figure 18 (C) shows H&E staining pathological images of representative lung tissues from each group of mice; Figure 18 (D) Quantitative statistical analysis of fluorescence intensity, number of metastatic lesions and area of ​​lung metastases in mice of each group; Figure 19 The expression levels of Ki67, a tumor proliferation marker for breast cancer in mice, are shown in the figures for each group of mice in this invention. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0032] The bitter orange used in this invention is the bitter orange of the Citrus aurantium L. variety from Xingan County, Jiangxi Province.

[0033] Example 1 This embodiment provides a method for preparing extracellular vesicle-like nanoparticles derived from Citrus aurantium, including the following steps: (1) Take 200g of immature bitter orange, wash it three times with pure water for 1-3 minutes each time, and then let it air dry. (2) Add the immature bitter orange and PBS buffer pre-cooled at 4℃ to a blender at a volume ratio of 1:1.5. First, blend for 10 seconds, then continue blending for 1-2 minutes to obtain immature bitter orange juice. Filter the obtained immature bitter orange juice with gauze to remove large particles of impurities. After filtration, centrifuge the immature bitter orange juice at a gradient of 4℃, 3000×g, 10 min; 12000×g, 20 min; 12000×g, 20 min. After centrifugation, take the supernatant and discard the precipitate. (3) Centrifuge the supernatant at 4°C, 140,000×g for 70 min in a refrigerated ultracentrifuge (Beckman Optima L-100XP). After centrifugation, discard the supernatant, take the precipitate, suspend it in sterile PBS buffer pre-cooled at 4°C, and bring the volume up to the maximum volume of the ultracentrifuge tube. (4) Centrifuge the suspension again at 4°C, 140,000×g for 70 min in an ultra-high speed refrigerated centrifuge. Discard the supernatant after centrifugation and take the precipitate. Resuspend the precipitate in 400 μL of sterile PBS buffer pre-cooled at 4°C. Let the suspension stand at 4°C for 1-2 hours. Take the supernatant and filter it through a 0.45 μm filter membrane. Collect the filtrate to obtain a solution containing extracellular vesicle-like nanoparticles (AFI-EVs) derived from Citrus aurantium.

[0034] The preparation route of extracellular vesicle-like nanoparticles derived from Citrus aurantium (hereinafter referred to as "Citrus aurantium vesicles") is as follows: Figure 1 As shown. Extracellular vesicle-like nanoparticles derived from Citrus aurantium were aliquoted into 0.5 mL EP tubes, 50-100 μL per tube, avoiding repeated freeze-thaw cycles, and stored at -80℃.

[0035] The following tests were performed on the Citrus aurantium vesicles prepared in Example 1: (1) Transmission electron microscopy test (2) Particle size detection The instrument used was a nanoBrook 90 Plus PALS (Brookhaven Instruments, USA). The specific procedure was to dilute the Citrus aurantium vesicles with phosphate buffer at a volume ratio of 1:10, mix well, and add 2 mL of the mixture to a dedicated cuvette for detection.

[0036] (3) Nanoflow cytometry detection The instrument used was a Flow NanoFCM NanoAnalyzer U30E. The specific steps were as follows: Pre-start preparation: Prepare 150 μL of washing solution, 150 μL of ultrapure water, and 100 μL of control beads (250 nm, 200X). Check the sheath fluid, waste fluid, and washing solution, ensuring the height difference between the sheath fluid and waste fluid is 20-30 mL, and the washing solution level is >10 mL. Start-up preparation: First, turn on the instrument, then start the computer and software, and perform system initialization, liquid path initialization, and air bubble removal. Routine quality control: Load one tube of control beads (control beads concentration 2.04 × 10⁻⁶). 10(Particles / mL, Catalog No.: 2211181), click Sample-Boosting, and adjust the bright spot in the camera window to its brightest point using 10µm increments. Click Sample-Sampling, and simultaneously enable SPCM; the real-time signal waveform will then be visible in the graphics area. Using the waveform and other information, adjust the fluorescence and scattered light channels along the X and Y axes to ensure the signal is strongest and most uniform. After ensuring quality control passes, you can begin acquiring control spheres. Particle size standard sphere acquisition: The particle size standard spheres range from 68-155 nm, divided into four groups: group 1 (68 nm), group 2 (91 nm), group 3 (113 nm), and group 4 (155 nm). Sample detection (the method is the same as for acquiring control spheres and particle size standard spheres).

[0037] (4) Potential detection (5) Purity testing Considering the membrane structure characteristics of vesicles, we used the surfactant Triton X-100 to disrupt the membrane structure to identify the purity of the obtained Citrus aurantium vesicles. The specific steps were as follows: six different concentrations of Triton X-100 were prepared (0%, 0.05%, 0.1%, 0.5%, 1%, and 2%). The Triton X-100 solution was mixed with the AFI-EVs solution at a volume ratio of 1:1 and incubated at room temperature for 5 min. After incubation, the proportion of membrane-bound particles at different Triton X-100 concentrations was detected by nanoflow cytometry.

[0038] (6) Contents detection Proteins in *Citrus aurantium* vesicles were identified by polyacrylamide gel electrophoresis. The protein concentration in *Citrus aurantium* vesicles was 2.86 mg / mL using a BCA kit. Electrophoresis was performed using 12% SDS-PAGE. 1 / 4 volume of 5× protein loading buffer was added to the protein sample, and the sample was heated in a boiling water bath for 10 min to denature the protein. The protein loading amount was 20 μg. The electrophoresis conditions were: first run at 80 V for 30 min, then at 120 V for 70 min. After the electrophoresis, the sample was stained with Coomassie brilliant blue for 90 min, and then destained with destaining solution. The solution was changed every 1 h, and after two changes, the sample was destained overnight. The sample was then observed using a Bio-Rad gel imaging system.

[0039] Small RNAs in *Citrus aurantium* vesicles were identified by agarose gel electrophoresis: RNA was extracted from *Citrus aurantium* vesicles using a nucleic acid purification column (RNA-specific) kit (Solarbio, Beijing, China), and RNA concentration was detected using a NanoDrop (Thermo Scientific, USA) instrument. Electrophoresis was performed on a 2% formaldehyde-denaturing agarose gel. Half a volume of 2×RNA loading buffer was added to the RNA sample, and the gel was heated at 65°C for 10 min to denature it. The loading volume was 2.5 μg, and the electrophoresis conditions were 110V for 45 min. After electrophoresis, the gel was observed using a Bio-Rad gel imaging system.

[0040] Lipids in Citrus aurantium vesicles were identified by thin-layer chromatography. Citrus aurantium vesicle samples were dissolved in a chloroform / methanol mixture (1:2:1, v / v) and centrifuged at 10000×g for 10 min. The samples separated into three layers (upper layer water, intermediate protein, and lower layer lipids). The lower layer was collected and dried under nitrogen. The dried sample was reconstituted in 50 µL of chloroform and spotted capillarily onto a silica gel GF254 thin-layer chromatography plate (silica gel 60 high-performance thin-layer plate, glass plate, containing fluorescent indicator F254). The plate was placed in a developing container with a chloroform / methanol / acetic acid mixture (190:9:1, v / v) as the developing solvent and imaged using ultraviolet fluorescence.

[0041] (7) Stability test The Citrus aurantium vesicle solution was stored at -80℃ and observed for 28 days. The particle size changes of the Citrus aurantium vesicle solution were observed on days 1, 7, 14, and 28.

[0042] (8) High performance liquid chromatography (HPLC) detection Take 200 μL of Citrus aurantium vesicle solution from a -80℃ freezer, enrich it with nitrogen until the PBS is completely evaporated, add 400 μL of chromatographic methanol, vortex thoroughly for 5 min, sonicate for 15 min, centrifuge at 8000×g for 20 min at room temperature, take the supernatant and filter it through a 0.22 μm filter membrane for analysis.

[0043] (9) Detection by liquid chromatography-mass spectrometry (LC-MS) Take 500 μL of Citrus aurantium vesicle solution from a -80℃ freezer, enrich it with nitrogen until the PBS is completely evaporated, add 200 μL of chromatographic methanol, vortex thoroughly for 5 min, sonicate for 15 min, centrifuge at 8000×g for 20 min at room temperature, take the supernatant and filter it through a 0.22 μm filter membrane for instrumental analysis.

[0044] Transmission electron microscopy results of Citrus aurantium vesicles are as follows: Figure 2 As shown. From Figure 2It can be seen that the Citrus aurantium vesicles obtained by the above preparation method are tea saucer-like membrane vesicle structures with clear membranes.

[0045] The particle size distribution and nano-flow cytometry results of Citrus aurantium vesicles are as follows: Figure 3 As shown, the results indicate that the particle size of the *Citrus aurantium* vesicles is 79.74 nm ± 13.86 nm, which falls within the typical particle size range (30-300 nm) for traditional Chinese medicine vesicles. The average concentration measured three times by nanoflow cytometry was 2.78 × 10⁻⁶. 11 Particles / mL. Potential detection results are as follows: Figure 4 As shown, the potential of the Citrus aurantium vesicles is -13.75 mV, indicating that the surface of the Citrus aurantium vesicles carries a negative charge, which helps the Citrus aurantium vesicles maintain structural stability and integrity.

[0046] The purity test results of Citrus aurantium vesicles are as follows: Figure 5 As shown, Triton X-100 can specifically disrupt the membrane structure of vesicles. For example... Figure 5 As shown, the number of Citrus aurantium vesicle particles decreased significantly with increasing Triton X-100 concentration. Compared with the untreated control group, treatment with 2% Triton X-100 reduced the particle number to 5.51%, indicating that approximately 95% of the particles in the sample were vesicles with intact membrane structures.

[0047] In the content detection, the protein band diagram of the contents of Citrus aurantium vesicles (AFI-EVs) is as follows: Figure 6 As shown, the vesicles of Citrus aurantium contain a relatively high abundance of small proteins, with molecular weights concentrated around 25 kDa. The small RNA banding diagram of the contents (EVs) is shown below. Figure 7 As shown, the RNA concentration in the vesicles of Citrus aurantium was 559.4 ng / μL, indicating that the vesicles contained abundant small RNAs with a molecular weight of approximately 100 bp. The lipid thin-layer chromatography chromatogram is shown below. Figure 8 As shown, by Figure 8 It is known that the vesicles of Citrus aurantium contain different forms of lipids.

[0048] In the stability test, the particle size distribution of Citrus aurantium vesicles on days 1, 7, 14, and 28 is as follows: Figure 9 As shown, the images on days 1, 7, 14, and 28 are as follows. Figure 10 As shown, from 9 and Figure 10 It can be seen that the Citrus aurantium vesicles obtained in Example 1 can maintain their properties after being stored at 4°C for 28 days.

[0049] The HPLC detection results of Citrus aurantium vesicles are as follows: Figure 11 As shown. From Figure 11It can be seen that the vesicles of Citrus aurantium contain flavonoid active compounds unique to Citrus aurantium, such as naringin, neohesperidin, naringenin, and hesperidin. In the vesicles, the content of naringin is approximately 273.9 μg / mL, the content of neohesperidin is approximately 302.4 μg / mL, while the contents of naringenin and hesperidin are relatively lower, at 23.0 μg / mL and 14.7 μg / mL, respectively.

[0050] The LC-MS detection results of Citrus aurantium vesicles are as follows: Figure 12 As shown, the vesicles of Citrus aurantium contain 2,972 small molecule compounds, including organic acids (39.57%), flavonoids (3.20%), alkaloids (13.50%), volatile oils (4.80%), and other substances.

[0051] The following experiments were conducted on the Citrus aurantium vesicles prepared in Example 1.

[0052] I. In vivo tissue distribution and liver targeting of Citrus aurantium vesicles Thirty 6-week-old BALB / c mice were acclimatized for one week. Citrus aurantium vesicles were labeled with the lipophilic fluorescent probe DiR, and the free DiR dye was removed by ultracentrifugation to obtain DiR-labeled Citrus aurantium vesicles (EVs-DiR). The experimental animals were randomly divided into two groups: the DiR probe group (injected with free DiR probe as a control) and the Citrus aurantium vesicle group (injected with EVs-DiR).

[0053] Equal doses of DiR probes or EVs-DiR were administered to mice via intraperitoneal injection. At 3, 6, 12, 24, and 48 hours post-injection, the dynamic distribution of fluorescence signals within the mice was observed and recorded using an in vivo small animal imaging system (IVIS Spectrum). Simultaneously, at each time point, three mice from each group were sacrificed, and their major organs, including the heart, liver, spleen, lungs, and kidneys, were completely dissected for immediate in vitro fluorescence imaging to accurately quantify and compare the fluorescence signal intensity in each tissue and organ.

[0054] Mouse in vivo imaging results are shown in Figure 13 (A) It can be observed that the fluorescence signal of the Citrus aurantium vesicle group is significantly concentrated in the mouse liver region. The results of isolated mouse organ imaging are shown in [Figure A]. Figure 13 (B) It can be observed that compared with the DiR probe group, the Citrus aurantium vesicle group at different time points showed extremely strong fluorescence signal enrichment in the liver tissue, and its cumulative fluorescence intensity was much higher than that of other organs and the liver of the DiR probe group, further confirming that Citrus aurantium vesicles have significant liver targeting in mice.

[0055] II. Protective effect of Citrus aurantium vesicles on acetaminophen (APAP)-induced acute liver injury in mice. Thirty-two 8-week-old C57BL / 6J mice were used and acclimatized for one week. Before modeling, all mice were fasted for 12 hours to reduce individual differences. The mice were randomly divided into four groups: control group, model group (APAP + Vehicle), and low-dose Citrus aurantium vesicle group (APAP + EV). low ) and the high-dose group of Citrus aurantium vesicles (APAP + EV) high ).

[0056] Except for the control group, which received intraperitoneal injection of saline, the other three groups of mice were injected intraperitoneally with acetaminophen (APAP) at a dose of 300 mg / kg to establish an acute liver injury model. The drug administration intervention regimen was as follows: mice in the low- and high-dose groups of Citrus aurantium vesicles received intraperitoneal injections of 3.75 mg / kg and 7.5 mg / kg Citrus aurantium vesicles, respectively, 24 hours and 2 hours before APAP injection; a second therapeutic injection was administered 1 hour after APAP injection. The control and model groups received an equal volume of saline at the corresponding time points.

[0057] Twenty-four hours after APAP injection, blood was collected from the orbital venous plexus of all mice, and serum was used for biochemical assays. The mice were then sacrificed, and liver tissue was collected and fixed in 4% paraformaldehyde for subsequent histopathological analysis.

[0058] H&E staining was performed on mice in each group, and the results are as follows: Figure 14 As shown in (A). Observation of the staining results revealed that the hepatocytes of the control group mice were neatly arranged. The livers of the model group mice showed extensive hepatocyte necrosis, accompanied by a large amount of inflammatory cell infiltration. In contrast, the liver tissue damage in both the low- and high-dose Citrus aurantium vesicle intervention groups was significantly improved, the area of ​​hepatocyte necrosis was significantly reduced, and the degree of inflammatory infiltration was also greatly reduced.

[0059] Serological tests were performed on mice in each group, and the results are shown in the table below. Figure 14 (B). As shown in the figure, compared with the control group, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group mice were significantly increased, indicating that APAP successfully induced acute hepatocyte necrosis. Compared with the model group, the serum ALT and AST levels in mice were significantly reduced after low- and high-dose intervention with Citrus aurantium vesicles, and the protective effect of the high-dose group was more significant, showing a dose-dependent effect.

[0060] Figure 14 The results confirmed that the vesicles could significantly alleviate acute liver injury induced by APAP, and this finding indicates that the Citrus aurantium vesicles described in this invention have important application potential in the prevention and / or treatment of acute liver diseases such as drug-induced liver injury.

[0061] III. The intervention effect of Citrus aurantium vesicles on high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD). Thirty-two C57BL / 6J mice (6 weeks old) were randomly divided into four groups after one week of acclimatization: control group, model group (HFD+Vehicle), and low-dose Citrus aurantium vesicle treatment group (HFD+EV). low ) and the high-dose treatment group of Citrus aurantium vesicles (HFD+EV) high ).

[0062] Except for the control group fed a normal diet, the other three groups of mice were fed a high-fat diet (HFD) for 8 weeks to establish a non-alcoholic fatty liver disease (NAFLD) model. During the modeling period, the mice underwent drug intervention. The low- and high-dose treatment groups received intraperitoneal injections of 3.75 mg / kg and 7.5 mg / kg of *Citrus aurantium* vesicles, respectively, three times a week. The control and model groups received injections of the same volume of physiological saline at corresponding time points.

[0063] Eight weeks after intervention, the final body weight of mice in each group was recorded. After euthanasia, the liver was immediately dissected and weighed, and the liver index was calculated (liver index % = liver weight / body weight × 100%). Blood was collected from the orbital venous plexus to collect serum for subsequent biochemical assays. A portion of liver tissue was fixed with 4% paraformaldehyde or frozen sections were prepared for histopathological analysis.

[0064] H&E staining and Oil Red O staining were performed on mice in each group. The results are shown in the figure. Figure 15 (A) and Figure 15 (B) Histopathological examination of liver tissue showed that in the control group, H&E-stained liver sections showed neatly arranged hepatocytes without obvious fatty degeneration, and Oil Red O staining was negative. In contrast, H&E-stained liver sections of the model group mice showed extensive and severe hepatocyte fatty degeneration (manifested as vacuolar degeneration) and inflammatory cell infiltration. Oil Red O staining revealed numerous, diffusely distributed orange-red lipid droplets within the liver tissue. Compared with the model group, liver tissue damage was significantly improved in both the low- and high-dose *Citrus aurantium* vesicle treatment groups. H&E staining showed a significant reduction in the number and volume of hepatocyte vacuolar degeneration, reduced inflammatory infiltration, and Oil Red O staining also confirmed a significant reduction in intrahepatic lipid droplet deposition.

[0065] General physiological indicators and serum biochemical test results (see) Figure 15(C) shows that, compared with the control group, the liver index, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC) levels in the model group mice were significantly increased, indicating that HFD successfully induced hepatic steatosis, liver injury, and dyslipidemia. Compared with the model group, all the above indicators were significantly improved in mice with non-alcoholic fatty liver disease after low- and high-dose intervention with Citrus aurantium vesicles, and the treatment effect of the high-dose group was more significant.

[0066] In summary, histopathological analysis confirmed that *Citrus aurantium* vesicles can significantly improve non-alcoholic fatty liver disease induced by a high-fat diet. Its effects are manifested in multiple aspects, including reducing hepatomegaly, restoring liver function, regulating blood lipid levels, and inhibiting excessive fat deposition in the liver. This finding indicates that the *Citrus aurantium* vesicles described in this invention have significant application potential for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0067] IV. The effect of Citrus aurantium vesicles on improving depressive-like behavior in mice under chronic unpredictable mild stress Thirty-two male C57BL / 6J mice (8 weeks old) were used as experimental animals. After one week of acclimatization, they were randomly divided into four groups (n=8 / group): control group, model group (CUMS+Vehicle), and low-dose Citrus aurantium vesicle group (CUMS+EV). low ) and the high-dose group of Citrus aurantium vesicles (CUMS+EV) high Except for the normally fed control group, the other three groups of mice underwent 5 weeks of Chronic Unpredictable Mild Stress (CUMS) to induce depressive-like behavior. Stress protocols included diurnal reversal, damp bedding, 45° cage tilt, restraint, fasting, water deprivation, forced swimming in ice water, and tail clamping; one of these protocols was randomly selected daily. From the second week of modeling, mice in each group were given drug intervention, administered intraperitoneally three times a week for four weeks. The control and model groups received the same volume of saline, while the low-dose and high-dose groups of Citrus aurantium vesicles received 3.75 mg / kg and 7.5 mg / kg of Citrus aurantium vesicles, respectively.

[0068] At the end of the 5-week intervention, the following behavioral tests were conducted: (1) Open Field Test (OFT): The mouse is placed in the center of the open field box and the total distance of movement of the mouse is recorded by a camera system within 5 minutes to assess the mouse’s autonomous activity ability and depressive-like behavior.

[0069] (2) Sucrose Preference Test (SPT): The sucrose preference test was used to assess the degree of anhedonia in mice. Mice were allowed to freely choose between drinking a 1% sucrose solution or pure water within 24 hours. The consumption of each liquid was recorded, and the sucrose preference rate was calculated. The formula for calculating the sucrose preference rate is: Sucrose preference rate = Sucrose consumption ÷ (Sucrose consumption + Pure water consumption) × 100%.

[0070] The results of the open field experiment are shown below. Figure 16 (A). In the open field experiment, the total movement distance of the model group mice was significantly shorter than that of the control group, suggesting that CUMS induced significant depressive-like behavior. After intervention with Citrus aurantium vesicles, the total movement distance of mice in the open field in the low- and high-dose Citrus aurantium vesicle groups was significantly increased compared with that in the model group.

[0071] The results of the sugar water preference experiment are shown below Figure 16 (B). In the sucrose preference experiment, the sucrose preference rate of mice in the model group was significantly lower than that in the control group, indicating that CUMS successfully induced anhedonia behavior in mice. Compared with the model group, the sucrose preference rate of mice was significantly improved after low-dose and high-dose intervention with Citrus aurantium vesicles, with the sucrose preference rate in the high-dose group recovering to near that of the control group.

[0072] At the experimental endpoint, blood was collected from the eyes of mice, and the supernatant was collected by centrifugation after standing. The concentration of corticosterone in the serum of mice in each group was detected using an ELISA kit to assess the functional status of the hypothalamus-pituitary-adrenal (HPA) axis. Results are as follows: Figure 16 As shown in (C), compared with the control group, the serum corticosterone (CORT) level in the model group mice was significantly increased, indicating that the CUMS model caused hyperfunction of the HPA axis in mice. Compared with the model group, both the low- and high-dose groups of Citrus aurantium vesicles significantly reduced the serum CORT content, and the CORT level in the high-dose group had returned to a normal range similar to that in the control group.

[0073] The above experimental results confirm that *Citrus aurantium* vesicles can significantly improve CUMS-mediated reduction in spontaneous activity and anhedonia, as well as core depressive-like behaviors, in mice, and can significantly correct HPA axis dysfunction under stress. This finding demonstrates the potential of *Citrus aurantium* vesicles described in this invention for the prevention and / or treatment of depression-related diseases.

[0074] V. Fructus Aurantii Immaturus vesicles inhibit the growth and metastasis of breast tumors mediated by chronic psychological stress. First, a chronic psychological stress-induced breast cancer mouse model was constructed, and control, emotional stress model group (CUMS), and low-dose Citrus aurantium vesicle group (CUMS+EV) were set up. low) and the high-dose group of Citrus aurantium vesicles (CUMS+EV) high Except for the control group, the other three groups of mice were randomly subjected to two mild stimuli daily (including restraint, overnight light exposure, cage tilt at 45 degrees, fasting and water restriction, moist bedding, forced swimming in ice water, and tail clamping). After one week of stress, 4T1-Luc cells were in situ seeded into the mammary fat pads of Babl / c mice. The successful model construction was verified 28 days after the stress period using behavioral tests such as the open field test and sucrose preference test. After verifying the successful model construction, mice in the low-dose and high-dose groups of Citrus aurantium vesicles were intraperitoneally injected with Citrus aurantium vesicles at a dose of 3.75 mg / kg and 7.5 mg / kg, respectively, every two days. Mouse body weight and tumor size were observed every 3 days during the intervention period for a total of 36 days.

[0075] Before the above experiment reached its endpoint, 15 mg / mL of D-luciferin was prepared and injected intraperitoneally at a volume of 10 μL / g based on the mouse's body weight. Small animal in vivo imaging was then used to detect fluorescence in the mouse lungs and assess lung metastasis.

[0076] At the end of the experiment, tumor tissue from the mammary fat pads of mice was collected for immunohistochemical staining to detect the expression level of the tumor proliferation marker Ki-67. The specific method for immunohistochemical staining was as follows: the tumor tissue was dehydrated, embedded, sectioned, baked at 60°C for 2 h, dewaxed, and antigen retrieval solution (sodium citrate buffer) was prepared for antigen retrieval. Peroxidase incubation for 10 min, followed by 3 washes with PBS (3 min each time); 5% BSA blocking for 20 min, followed by 3 washes with PBS (3 min each time); primary antibody incubation overnight at 4°C, followed by 3 washes with PBS (3 min each time); reaction enhancement solution incubation for 20 min at room temperature, followed by 3 washes with PBS (3 min each time); enzyme-labeled goat anti-mouse / rabbit IgG polymer incubation for 20 min at room temperature, followed by 3 washes with PBS (3 min each time); DAB chromogenic solution incubation for 3-5 min, followed by microscopic observation; hematoxylin staining for 30 s, followed by microscopic observation, followed by rinsing with tap water to achieve blue reflection; dehydration, followed by 2 mins of 95% ethanol, 2 mins of anhydrous ethanol, and 5 mins of xylene twice; mounting with neutral resin; photographing and statistical analysis.

[0077] Tumor size of mice in each group at each time point is as follows: Figure 17 As shown in (A)~(C). From Figure 17 It can be seen that Citrus aurantium vesicles can significantly inhibit the growth of breast tumors, and there is no significant difference in body weight between mice treated with Citrus aurantium vesicles and the model group. More importantly, in mice mediated by chronic psychological stress, the breast tumor volume after low-dose Citrus aurantium vesicle administration was basically the same as that of the control group, while the growth rate of breast tumors after high-dose Citrus aurantium vesicle administration was lower than that of the control group.

[0078] In vivo bioluminescence imaging of mice in each group is shown below. Figure 18 (A). Compared with the control group, the biofluorescence signal intensity in the lungs of CUMS model mice was significantly enhanced, indicating more severe lung metastasis of tumors. The fluorescence signal intensity in the low-dose and high-dose Citrus aurantium vesicle groups was significantly weakened. Low-dose and high-dose Citrus aurantium vesicle intervention significantly reduced the fluorescence signal intensity in the lungs of CUMS model mice. To validate the in vivo imaging results at the tissue level, ex vivo lung tissues from each group of mice were then examined. Gross specimens (see...) Figure 18 (B) and H&E stained pathological images (see Figure 18 Consistent with the results of (C)), chronic unpredictable mild stress (CUMS) intervention significantly increased the number and size of lung metastases, while *Citrus aurantium* vesicles effectively reversed this process, significantly reducing the number and size of lung metastases under CUMS. Finally, quantitative statistical analysis of lung metastasis burden ( Figure 18 (D) showed that the fluorescence intensity, number, and total area of ​​metastatic lesions in the CUMS model group were significantly higher than those in the control group (P<0.01). Importantly, compared with the CUMS model group, high-dose Citrus aurantium vesicles significantly reduced the above three indicators in the CUMS model group (P<0.01), while low-dose Citrus aurantium vesicles also significantly reduced the fluorescence intensity and total area of ​​metastatic lesions (P<0.01). In conclusion, from the overall level to the histopathological level, it is shown that Citrus aurantium vesicles can effectively inhibit lung metastasis of breast cancer mediated by chronic psychological stress. Immunohistochemical staining results of mice in each group are shown in [Figure 1]. Figure 19 .from Figure 19 It can be seen that, compared with the model group, low-dose and high-dose Citrus aurantium vesicles can reduce the expression of the tumor proliferation marker Ki67.

[0079] The above experimental results confirm that the Citrus aurantium vesicles prepared in Example 1 can significantly inhibit the growth and metastasis of breast tumors promoted by chronic psychological stress. This finding indicates that the Citrus aurantium vesicles described in this invention have important application value in the prevention and / or treatment of breast cancer related to emotional stress.

[0080] This invention has discovered that *Citrus aurantium* vesicles possess unique liver-targeting properties and have preventive and / or therapeutic effects on a variety of diseases: (1) Protective effect in acute liver injury. In an acetaminophen (APAP)-induced acute liver injury model in mice, the Citrus aurantium vesicles prepared in this invention can significantly reduce serum transaminase levels and effectively alleviate liver tissue necrosis and inflammatory infiltration, showing significant hepatoprotective activity.

[0081] (2) Therapeutic effect in non-alcoholic fatty liver disease (NAFLD). In a high-fat diet-induced NAFLD model, the Citrus aurantium vesicles prepared in this invention can significantly reduce liver index, improve blood lipid disorders, restore liver function, and effectively inhibit excessive deposition of fat in the liver.

[0082] (3) Improvement effect in depression. In a mouse model of depression induced by chronic unpredictable mild stress (CUMS), the Citrus aurantium vesicles prepared in this invention can effectively improve core depressive-like behaviors such as anhedonia and decreased voluntary activity, and can significantly correct stress-induced HPA axis dysfunction.

[0083] (4) Inhibitory effect in stress-related tumors: In a breast cancer model promoted by chronic psychological stress, the Citrus aurantium vesicles prepared in this invention can effectively inhibit the growth of in situ tumors and metastasis to the lungs, and reduce the proliferative activity of tumor cells.

[0084] In summary, Citrus aurantium vesicles have potential applications in treating various diseases related to liver function and mental stress, and can be used to prepare drugs for the prevention and / or treatment of liver damage, depression, and breast cancer. These drugs exert their effects in the body by targeted delivery of Citrus aurantium vesicles to the liver.

[0085] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. Extracellular vesicle-like nanoparticles derived from Citrus aurantium, characterized in that, The extracellular vesicle-like nanoparticles derived from Citrus aurantium are prepared by crushing, filtering, centrifuging, and resuspending Citrus aurantium. They have liver-targeting properties and an average particle size of 60-120 nm.

2. The extracellular vesicle-like nanoparticles derived from Citrus aurantium according to claim 1, characterized in that, The potential of the extracellular vesicle-like nanoparticles derived from Citrus aurantium is -13 to -15 mV.

3. The extracellular vesicle-like nanoparticles derived from Citrus aurantium according to claim 1 or 2, characterized in that, The extracellular vesicle-like nanoparticles derived from Citrus aurantium encapsulate at least one of naringin, neohesperidin, naringenin, and hesperidin.

4. A method for preparing extracellular vesicle-like nanoparticles derived from Citrus aurantium as described in any one of claims 1 to 3, comprising the following steps: (1) After mixing the immature bitter orange with PBS buffer, the juice was filtered, and the filtrate was collected and then subjected to gradient centrifugation at 4℃ and 2500~13000×g. The supernatant was collected. (2) Centrifuge the supernatant at 4℃ and 120,000~150,000×g, retain the precipitate, and resuspend the precipitate in PBS buffer to obtain a suspension; (3) Centrifuge the suspension at 4℃ and 140000×g, retain the precipitate, resuspend the precipitate with PBS buffer, filter, and collect the filtrate to obtain the product.

5. The preparation method according to claim 4, characterized in that, In step (1), the volume ratio of Citrus aurantium to PBS buffer is 1:(1.2~2.0).

6. The preparation method according to claim 4, characterized in that, In step (1), the parameters used for gradient centrifugation are: S1. First centrifugation: Centrifuge at 4℃ and 2500~3500×g for 8~12 minutes; S2. Second centrifugation: Centrifuge at 4℃ and 11000~13000×g for 18~22 minutes; S3. Third centrifugation: Centrifuge at 4℃ and 11000~13000×g for 18~22 minutes.

7. The preparation method according to claim 4, characterized in that, In steps (2) and (3), centrifuge at 4℃ and 140000×g for 60~80 minutes.

8. The use of the extracellular vesicle-like nanoparticles derived from Citrus aurantium as described in any one of claims 1 to 3 in the preparation of a medicament for the prevention and / or treatment of acute liver injury or non-alcoholic fatty liver disease.

9. The use of the extracellular vesicle-like nanoparticles derived from Citrus aurantium as described in any one of claims 1 to 3 in the preparation of medicaments for the prevention and / or treatment of mental stress disorders.

10. The use of the extracellular vesicle-like nanoparticles derived from Citrus aurantium as described in any one of claims 1 to 3 in the preparation of a medicament for treating breast cancer.

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