Application of gardenia-derived extracellular vesicles in preparation of drugs for preventing and treating neurodegenerative diseases

Abstract

The invention discloses application of gardenia-derived extracellular vesicles in preparation of drugs for preventing and treating neurodegenerative diseases, and relates to the field of biological medicines. According to the invention, the gardenia-derived extracellular vesicles are applied to oxidation resistance and neuroprotection, and after being taken, the gardenia-derived extracellular vesicles can significantly improve the activity reduction of rotenone-induced PC12 cells, and effectively reduce the generation of reactive oxygen species (ROS) and superoxide anions (O2 <->) in the rotenone-induced PC12 cells, thereby relieving oxidative stress; in addition, the treatment of the extracellular vesicles derived from gardenia fruits can also significantly reduce DNA breakage and cell membrane damage in nerve cells caused by rotenone treatment, and has an obvious protection effect on nuclei and cell membranes of the nerve cells. Therefore, the extracellular vesicles derived from the gardenia fruits have remarkable oxidation resistance and nerve cell protection potential.

Description

Technical Field

[0001] The invention relates to biomedicine, and in particular to application of Gardenia jasminoides-derived extracellular vesicles in preparing drugs for preventing and treating neurodegenerative diseases. Background Art

[0002] Extracellular vesicles (EVs) are a type of nano-sized vesicles that are formed inside cells and released into the extracellular environment after fusion with the plasma membrane. EVs mainly include exosomes, microvesicles, and apoptotic bodies, among which exosomes are the smallest type, with a diameter usually between 30-150nm, while microvesicles (MVs) have a wide range of sizes, with a diameter of 100-1,000nm. EVs carry a variety of biologically active molecules, such as nucleic acids, proteins, and metabolites, which play an important role in intercellular communication, signal transduction, and material transport. Almost all types of living cells, including animal and plant cells, can secrete EVs.

[0003] In recent years, plant-derived extracellular vesicles have received widespread attention. Plant EVs have high biocompatibility and low immunogenicity, and can carry rich bioactive molecules, which have potential application value in disease treatment. After entering the body, these plant EVs can participate in the regulation of cell signaling pathways and exhibit a variety of biological activities such as antioxidant and anti-inflammatory. For example, EVs from plants such as honeysuckle, astragalus, and licorice have outstanding antioxidant and anti-inflammatory properties. In the field of neuroprotection, plant-derived EVs may be rich in neuroprotective factors and antioxidants, which can effectively reduce oxidative stress, reduce neurotoxicity, and promote the survival and regeneration of neurons. Studies have found that EVs from plants such as ginseng, ginger, and dendrobium can improve neural function and behavioral performance by regulating cell signaling pathways, reducing neurotoxicity, and protecting the integrity of nerve cell membranes.

[0004] Gardenia jasminoides J.Ellis is a traditional Chinese medicinal material, also known as yellow gardenia, gardenia, white toad, belonging to the genus Gardenia of the Rubiaceae family. The fruit of the plant Gardenia is also called "gardenia", which is a traditional Chinese medicine and the first batch of Chinese medicinal materials with the same origin of medicine and food. Its fruit is widely used in traditional Chinese medicine. Gardenia is rich in bioactive ingredients such as iridoids, flavonoids and triterpenes, which give it significant pharmacological effects such as antioxidant, antidepressant, antianxiety and neuroprotective effects. Although there have been many studies on the chemical components and direct pharmacological effects of gardenia, domestic and foreign researchers have isolated chemical components such as iridoids, flavonoids, diterpenes, triterpenes, phenolic acids and organic acid esters from gardenia flowers. In addition, the extraction process of total flavonoids from gardenia flowers, the extraction and purification process of gardenia oil, the extraction process of gardenia yellow pigment and the analysis and determination of amino acid components have also been studied. Patent applications including publication number CN1 16855438A disclose a method for preparing plant nanovesicles derived from Gardenia jasminoides, which has the advantages of simple preparation method, low cost, and high yield. Plant-derived exosome-like nanoparticles can carry out interspecies communication by inducing a variety of cytokines. They have the advantages of high positioning and targeting ability, non-toxicity, high thermal stability, easy metabolic degradation by the human body, and low immunogenicity. However, the biological activity of extracellular vesicles (EVs) derived from Gardenia jasminoides has not been deeply explored. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies in existing research, the present invention proposes an extracellular vesicle derived from Gardenia, which can improve oxidative stress and nerve cell damage, and has antioxidant and neuroprotective effects. It has good prospects in promoting the use of Gardenia resources, which are both medicinal and edible, for the prevention and treatment of neurodegenerative diseases.

[0006] Compared with other plant EVs, Gardenia EVs (GDEVs) themselves may be rich in bioactive ingredients such as flavonoids and terpenes, which can improve neurological function and alleviate symptoms of mental illness through multiple mechanisms such as promoting nerve cell repair, promoting synaptic plasticity, protecting nerve cell membranes, and regulating neurotransmitter release. The present invention provides a new direction for the in-depth utilization of Gardenia in the treatment and prevention of degenerative neurological diseases, which can not only promote the further development of Gardenia as a dual-purpose medicinal and edible resource, but also lay the foundation for its application in related fields in the future.

[0007] The present invention provides application of Gardenia jasminoides-derived extracellular vesicles in preparing medicines for preventing and treating neurodegenerative diseases.

[0008] The present invention utilizes rotenone (Rot) to induce a PC12 neuronal cell injury model, and uses Gardenia jasminoides extracellular vesicles to reduce nuclear damage, DNA breakage and cell apoptosis (externalization of phosphatidylserine on the cell membrane) of PC12 cells, evaluates the neuroprotective effect of the Gardenia jasminoides extracellular vesicles, and explores their use in the treatment of Alzheimer's disease and other related neurodegenerative diseases.

[0009] The Gardenia jasmine extracellular vesicles protect nerve cells by reducing damage to the nerve cell nuclei, reducing DNA breakage in the nuclei and reducing cell membrane damage, thereby preventing neurodegenerative diseases caused by nerve cell damage.

[0010] The present invention also provides the use of the Gardenia-derived extracellular vesicles in the preparation of antioxidant drugs.

[0011] The present invention uses the rotenone-induced PC12 cell oxidative stress model as an experimental model and finds that Gardenia jasminoides extracellular vesicles can be taken up by PC12 cells after being co-cultured with PC12 cells, thereby improving cell viability and reducing reactive oxygen species (ROS) and superoxide anion (O2 - ) levels, indicating that the Gardenia jasminoides extracellular vesicles have antioxidant potential.

[0012] Preferably, the neurodegenerative disease is Alzheimer's disease, ischemic stroke, hemorrhagic stroke, brain trauma or Parkinson's disease.

[0013] Preferably, when used, that is, when exerting an antioxidant effect or a neuroprotective effect, the protein concentration of the Gardenia jasminoides-derived extracellular vesicles is 6.25-50 μg / mL. The intervention treatment time is at least 24 hours.

[0014] More preferably, the protein concentration of the Gardenia jasminoides-derived extracellular vesicles is 20-50 μg / mL.

[0015] The extracellular vesicles described in the present invention can be any as long as they are from Gardenia and have a protein concentration of 6.25-50 μg / mL. In the embodiments of the present invention, as an example, the preparation method of the Gardenia-derived extracellular vesicles includes the following steps:

[0016] (1) adding a buffer solution to the raw material of Gardenia jasminoides and crushing the raw material to obtain a puree; centrifuging the puree for multiple times at the same speed, collecting the supernatant after each centrifugation for the next centrifugation, and collecting the supernatant after the last centrifugation;

[0017] (2) subjecting the supernatant collected after the last centrifugation in step (1) to ultracentrifugation to collect the precipitate;

[0018] (3) resuspending the precipitate obtained in step (2) with a buffer solution and subjecting the precipitate to density gradient ultracentrifugation to collect the suspended matter between each density layer;

[0019] (4) resuspending the suspension collected in step (3) with a buffer solution and then ultracentrifuging again to collect the precipitate;

[0020] (5) The precipitate collected in step (4) is resuspended in a buffer solution and then filtered through a membrane to obtain the gardenia-derived plant nanovesicles, wherein the pore size of the filter membrane is 0.1 to 0.5 μm.

[0021] Preferably, in step (1), the gardenia is gardenia fruit and / or gardenia flower;

[0022] Use a juicer for crushing, the speed of the juicer should be 1000-15000 rpm, and the crushing time should be 30-120 seconds; avoid crushing too finely or for too long, which may cause gelation of the original pulp.

[0023] The multiple centrifugation is divided into two centrifugations, the centrifugal force of the two centrifugations is 20000×g, and the centrifugation time is 20 minutes. The purpose is to initially separate the pomace from the puree.

[0024] Specifically, in step (2), the supernatant collected after the last centrifugation in step (1) is first filtered to collect the filtrate; the collected filtrate is then subjected to ultracentrifugation; wherein the pore size of the filter paper used for the filtration is 0.45 μm or 0.22 μm, so as to further improve the collection efficiency of the precipitate when passing through the membrane in steps (2) and (4).

[0025] In steps (2) and (4), the centrifugal force of ultracentrifugation is 100,000 to 170,000×g, and the centrifugation time is 60 to 180 minutes. A relatively low rotation speed can improve production safety, and a relatively short centrifugation time is convenient for improving the output rate per unit time.

[0026] In step (3), the density gradient ultracentrifugation uses sucrose, cesium chloride, rubidium chloride or cesium bromide as the gradient material; the mass concentrations of the density gradients are 8%, 30%, 45% and 60% respectively; preferably, sucrose is used as the gradient material to effectively improve biosafety.

[0027] In step (5), when filtering the membrane, first pass it through a filter membrane with a pore size of 0.45 μm, and then pass it through a filter membrane with a pore size of 0.22 μm. Passing through two membranes can reduce the risk of the membrane being directly blocked and improve the utilization rate.

[0028] The pH range of the buffer in each step is 7.0-7.6; the buffer in each step is at least one of the following: phosphate buffer, Tris buffer, Tris-HCl buffer, TBS buffer, TBST buffer, TE buffer and Good's buffer.

[0029] All centrifugation-related operations were performed at 4-8°C to reduce the breakage of nanovesicles caused by the heat generated by high-speed centrifugation.

[0030] Beneficial effects of the present invention:

[0031] 1. The present invention applies Gardenia jasminoides-derived extracellular vesicles to anti-oxidation and neuroprotection. After ingestion, Gardenia jasminoides-derived extracellular vesicles can significantly improve the decreased viability of PC12 cells induced by rotenone, and effectively reduce the levels of reactive oxygen species (ROS) and superoxide anions (O2 - ) to alleviate oxidative stress; in addition, the treatment of extracellular vesicles derived from gardenia fruit can also significantly reduce the DNA breakage and cell membrane damage of nerve cells caused by rotenone treatment, and has a significant protective effect on the nucleus and cell membrane of nerve cells. Therefore, the extracellular vesicles derived from gardenia fruit of the present invention have significant anti-oxidation and nerve cell protection potential.

[0032] 2. The present invention can promote the use of Gardenia resources for both medicinal and edible purposes, and can be used for the treatment and prevention of neurodegenerative diseases, thereby expanding new application areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a morphological picture of the extracellular vesicles derived from gardenia fruit extracted and prepared in Example 1 observed by transmission electron microscopy.

[0034] Figure 2 This is the particle size distribution diagram of extracellular vesicles derived from gardenia fruit obtained by Malvern laser particle size analyzer detection.

[0035] Figure 3 This is the uptake of extracellular vesicles derived from Gardenia jasminoides by PC12 cells after intervention for 1h, 8h and 24h.

[0036] Figure 4 This is the effect of 24h intervention with extracellular vesicles derived from Gardenia jasminoides on the viability of PC12 cells.

[0037] Figure 5 The study shows that 24h intervention with extracellular vesicles derived from Gardenia jasminoides improves the decreased viability of PC12 neurons induced by rotenone.

[0038] Figure 6 This is the effect of 24h intervention of Gardenia fruit-derived extracellular vesicles on the generation of reactive oxygen species (ROS) in rotenone-induced PC12 neurons and the quantitative statistical graph, where the scale bar is 50μm.

[0039] Figure 7 The intervention of Gardenia jasminoides-derived extracellular vesicles for 24 h can enhance the expression of superoxide anion (O2 -) and quantitative statistical diagrams generated, where the scale bar is 50 μm.

[0040] Figure 8 This is the effect of 24h intervention with extracellular vesicles derived from Gardenia jasminoides on rotenone-induced nuclear damage in PC12 neurons. The scale bar is 50μm.

[0041] Fig. 9 This is the effect of 24h intervention of extracellular vesicles derived from Gardenia jasminoides on rotenone-induced DNA fragmentation in PC12 neurons. The scale bar is 50μm.

[0042] Fig.10 This is the effect of 24h intervention with extracellular vesicles derived from Gardenia jasminoides on rotenone-induced cell membrane damage (phosphatidylserine externalization) in PC12 neurons.

[0043] Figure 5-10 Middle: Ctrl group, representing the blank control group without any treatment; Rot group, representing the group treated with 1 μM rotenone; Rot+GDEVs group (20 μg / mL and 50 μg / mL), representing the group co-treated with 1 μM rotenone and extracellular vesicles derived from Gardenia jasminoides with different protein concentrations. DETAILED DESCRIPTION

[0044] The method for preparing the Gardenia jasminoides-derived extracellular vesicles of the present invention comprises the following steps:

[0045] (1) adding a buffer solution to the raw material of Gardenia jasminoides and crushing the raw material to obtain a puree; centrifuging the puree for multiple times at the same speed, collecting the supernatant after each centrifugation for the next centrifugation, and collecting the supernatant after the last centrifugation;

[0046] (2) subjecting the supernatant collected after the last centrifugation in step (1) to ultracentrifugation to collect the precipitate;

[0047] (3) resuspending the precipitate obtained in step (2) with a buffer solution and subjecting the precipitate to density gradient ultracentrifugation to collect the suspended matter between each density layer;

[0048] (4) resuspending the suspension collected in step (3) with a buffer solution and then ultracentrifuging again to collect the precipitate;

[0049] (5) The precipitate collected in step (4) is resuspended in a buffer solution and then filtered through a membrane to obtain the gardenia-derived plant nanovesicles, wherein the pore size of the filter membrane is 0.1 to 0.5 μm.

[0050] Preferably, in step (1), the gardenia is gardenia fruit and / or gardenia flower;

[0051] Use a juicer for crushing, the speed of the juicer should be 1000-15000 rpm, and the crushing time should be 30-120 seconds; avoid crushing too finely or for too long, which may cause gelation of the original pulp.

[0052] The multiple centrifugation is divided into two centrifugations, the centrifugal force of the two centrifugations is 20000×g, and the centrifugation time is 20 minutes. The purpose is to initially separate the pomace from the puree.

[0053] Specifically, in step (2), the supernatant collected after the last centrifugation in step (1) is first filtered to collect the filtrate; the collected filtrate is then subjected to ultracentrifugation; wherein the pore size of the filter paper used for the filtration is 0.45 μm or 0.22 μm, so as to further improve the collection efficiency of the precipitate when passing through the membrane in steps (2) and (4).

[0054] In steps (2) and (4), the centrifugal force of ultracentrifugation is 100,000 to 170,000×g, and the centrifugation time is 60 to 180 minutes. A relatively low rotation speed can improve production safety, and a relatively short centrifugation time is convenient for improving the output rate per unit time.

[0055] In step (3), the density gradient ultracentrifugation uses sucrose, cesium chloride, rubidium chloride or cesium bromide as the gradient material; the mass concentrations of the density gradients are 8%, 30%, 45% and 60% respectively; preferably, sucrose is used as the gradient material to effectively improve biosafety.

[0056] In step (5), when filtering the membrane, first pass it through a filter membrane with a pore size of 0.45 μm, and then pass it through a filter membrane with a pore size of 0.22 μm. Passing through two membranes can reduce the risk of the membrane being directly blocked and improve the utilization rate.

[0057] The pH range of the buffer in each step is 7.0-7.6; the buffer in each step is at least one of the following: phosphate buffer, Tris buffer, Tris-HCl buffer, TBS buffer, TBST buffer, TE buffer and Good's buffer.

[0058] All centrifugation-related operations were performed at 4-8°C to reduce the breakage of nanovesicles caused by the heat generated by high-speed centrifugation.

[0059] The present invention is described below by means of specific embodiments.

[0060] Example 1

[0061] In this example, extracellular vesicles derived from gardenia fruit were prepared and their morphology was observed. The specific method was consistent with the method for preparing extracellular vesicles derived from gardenia in Example 1 of patent application with publication number CN116855438A.

[0062] The prepared Gardenia fruit-derived extracellular vesicles were negatively stained with uranyl acetate, and then their morphology was observed under an electron microscope. Figure 1 The extracellular vesicles derived from gardenia fruit showed membrane-structured nanoparticles under transmission electron microscopy, with a particle size range of 50-200nm. This confirmed that the nanoscale vesicles derived from gardenia fruit were extracted, that is, the extracellular vesicles were successfully extracted.

[0063] Example 2

[0064] In this example, the particle size of the extracellular vesicles derived from Gardenia jasminoides extracted in Example 1 was detected.

[0065] NTA measurements were performed using a Nanosight LM10 system, using a 488 nm blue laser. Freshly prepared Gardenia-derived EVs stock solution was diluted with deionized water to adjust the number of vesicles per field of view to allow for optimal counting. Three 30-second videos were captured and analyzed using the batch analysis tool of the Nanosight NTA 3.2 software. Particle concentration was determined by averaging the area under the histogram of the three measurements. Results are shown in Figure 2 The particle concentration of Gardenia-derived extracellular vesicles was 3.62±0.22×1011 particles / mL.

[0066] Example 3

[0067] This example observes the uptake of extracellular vesicles derived from gardenia fruit extracted in Example 1 by PC12 cells after intervention for different time periods.

[0068] DiD Perchlorate (DiIC18 (5)) was mixed with extracellular vesicles derived from Gardenia jasminoides, incubated for 30 minutes, and then loaded into a 100 kDa ultrafiltration tube (Millipore, USA). DiD Perchlorate-labeled GDEVs (GDEVs-DiD) were recovered by elution with PBS. After GDEVs-DiD was co-cultured with PC12 cells for 1 h, 8 h, and 24 h, the cells were fixed with 2.5% glutaraldehyde for 30 minutes, and then the cell nuclei were stained with DAPI dye for 15 minutes. The cells were washed three times with PBS to remove excess DAPI dye, and the GDEVs-DiD with red fluorescence and the cell nuclei with blue fluorescence were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 3 As shown, obvious red fluorescence of DiD-GDEVs was observed near the nucleus and between the cell membranes, indicating that Gardenia-derived extracellular vesicles could be taken up by PC12 cells after 24 h of treatment.

[0069] Example 4

[0070] This example verifies the effect of 24h intervention of the Gardenia fruit-derived extracellular vesicles extracted in Example 1 on improving the decreased viability of PC12 neurons induced by rotenone.

[0071] PC12 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin at 37°C and 5% CO2. First, GDEVs (1.5625, 3.125, 6.25, 12.5, 25 and 50 μg / mL) were treated for 24 h to determine that they would not significantly affect PC12 cell viability. Then, it was studied whether these protein concentrations of GDEVs co-treated for 24 h could reduce the reduction in PC12 cell viability induced by 1 μM rotenone (Rot). Cell viability was evaluated by methylthiazole tetrazolium (MTT) assay. PC12 cells treated with trypsin were cultured in an incubator and attached to the wall, then treated with the corresponding concentrations of Rot or GDEVs for 24 h, and then cultured with 0.5 mg / mL of MTT at 37°C for 4 h. After removing the MTT solution, 150 μL of dimethyl sulfite was added and the absorbance at 570 nm was measured on a multifunctional microplate reader. Figure 4 and Figure 5 The results showed that treatment with Gardenia jasminoides with different protein concentrations alone would not affect the viability of PC12 cells, but intervention with Gardenia jasminoides with a protein concentration higher than 6.25 μg / mL for 24 hours could improve the rotenone-induced decrease in PC12 neuronal cell viability.

[0072] Example 5

[0073] This example verifies the effect of the Gardenia fruit-derived extracellular vesicles extracted in Example 1 on reducing the generation of reactive oxygen species in PC12 neurons induced by rotenone after 24 hours of intervention.

[0074] The experiment was divided into a control group (blank group), a rotenone group (1 μM Rot), and a rotenone and Gardenia-derived extracellular vesicles co-treated group (Rot+GDEVs, 20 μg / mL and 50 μg / mL). After treating PC12 cells for 24 h, the cells were fixed with 2.5% glutaraldehyde for 30 minutes. Subsequently, the cells were washed three times with PBS, and the generation of intracellular reactive oxygen species (ROS) was evaluated using a DCFH-DA probe (10 μM, 45 minutes). After two light washes with PBS, the fluorescence intensity in the cells was observed under a fluorescence microscope using consistent exposure settings. Figure 6 The fluorescence results and quantitative statistics showed that the ROS generation in PC2 cells increased significantly after treatment with 1 μM rotenone alone. However, 20 μg / mL and 50 μg / mL of extracellular vesicles derived from gardenia fruit could significantly reduce the generation of ROS, indicating that extracellular vesicles derived from gardenia fruit can alleviate the oxidative stress level in PC12 cells.

[0075] Example 6

[0076] This example verifies the effect of the Gardenia fruit-derived extracellular vesicles extracted in Example 1 on reducing the generation of superoxide anions in PC12 neurons induced by rotenone after 24 hours of intervention.

[0077] The experiment was divided into a control group (blank group), a rotenone group (1 μM Rot), and a group treated with rotenone and Gardenia-derived extracellular vesicles (Rot+GDEVs, 20 μg / mL and 50 μg / mL). After treating PC12 cells for 24 h, the cells were fixed with 2.5% glutaraldehyde for 30 min. Subsequently, the cells were washed three times with PBS, and the superoxide anion (O2 - After two gentle washes with PBS, the fluorescence intensity in the cells was observed under a fluorescence microscope using consistent exposure conditions. Figure 7 The fluorescence results and quantitative statistics showed that after treatment with 1 μM rotenone alone, the intracellular O - Relatively speaking, 20μg / mL and 50μg / mL of Gardenia fruit derived extracellular vesicles can significantly reduce O2 - The production of EVs indicated that Gardenia fruit-derived EVs could alleviate the oxidative stress level in PC12 cells.

[0078] Example 7

[0079] This example verifies the effect of the Gardenia fruit-derived extracellular vesicles extracted in Example 1 on reducing rotenone-induced nuclear damage in PC12 neurons after 24 hours of intervention.

[0080] The experiment was divided into a control group (blank group), a rotenone group (1 μM Rot), and a group treated with rotenone and Gardenia-derived extracellular vesicles (Rot+GDEVs, 20 μg / mL and 50 μg / mL). After treating PC12 cells for 24 h, the cells were fixed with 2.5% glutaraldehyde for 30 minutes. Subsequently, the cells were washed three times with PBS and the nuclei were stained with 2 μg / mL DAPI for 5 minutes at room temperature. After washing twice with PBS, the fluorescence intensity in the cells was observed under a fluorescence microscope using consistent exposure settings. Figure 8 Quantitative statistics showed that after treatment with 1 μM rotenone alone, the bright spots formed by nuclear shrinkage in PC12 cells increased significantly. Both 20 μg / mL and 50 μg / mL of extracellular vesicles derived from gardenia fruit could significantly reduce nuclear damage, indicating that extracellular vesicles derived from gardenia fruit have a protective effect on the nuclei of PC12 cells.

[0081] Example 8

[0082] This example verifies the effect of the Gardenia fruit-derived extracellular vesicles extracted in Example 1 on reducing rotenone-induced DNA breakage in PC12 neurons after 24 hours of intervention.

[0083] The experiment was divided into a control group (blank group), a rotenone group (1 μM Rot), and a rotenone and Gardenia jasminoides-derived extracellular vesicles co-treated group (Rot+GDEVs, 20 μg / mL and 50 μg / mL). TM 488-12-dUTP will be incorporated into the 3'-hydroxyl end of the broken DNA, thus detecting DNA breakage and cell damage through fluorescence changes. First, PC12 cells were treated with 20μg / mL proteinase K at room temperature for 5 minutes and then washed twice with PBS. Next, 50μL of YSFluor TM The cells were incubated with 488-12-dUTP and terminal deoxyribonucleotidyl transferase (TdT) incubation buffer at 37°C for 60 min and then washed twice with PBS. Then, the nuclei were stained with 2 μg / mL DAPI for 5 min at room temperature. Finally, YSFluor was observed by fluorescence microscopy. TM Green fluorescence intensity of 488-12-dUTP. Fig. 9 The results showed that compared with the control group, YSFluor TM The fluorescence intensity of 488-12-dUTP increased significantly, indicating that DNA fragmentation increased; and the intervention of extracellular vesicles derived from Gardenia fruit could significantly reduce the expression of YSFluor TM The fluorescence intensity of 488-12-dUTP indicates that DNA fragmentation in cells is reduced.

[0084] Example 9

[0085] This example verifies the effect of the Gardenia fruit-derived extracellular vesicles extracted in Example 1 on reducing rotenone-induced phosphatidylserine externalization on the cell membrane of PC12 neurons after 24 hours of intervention.

[0086] The experiment was divided into a control group (blank group), a rotenone group (1 μM Rot), and a group treated with rotenone and Gardenia jasminoides (Rot+GDEVs, 20 μg / mL and 50 μg / mL). Annexin V-FITC / PI apoptosis detection kit identifies apoptotic cells by FITC-labeled Annexin V. After treatment, the cells were washed three times with PBS and then digested with EDTA-free trypsin. Subsequently, the cells were collected by centrifugation at 300 × g for 5 minutes at 4 °C, and after rinsing twice with pre-cooled PBS, the cells were resuspended in 100 μL of 1×Binding Buffer. 5 μL Annexin V-FITC and 10 μL PI staining solution were added, mixed thoroughly, and incubated in the dark at room temperature for 10-15 minutes. Then, 400 μL 1×Binding Buffer was added and the cells were filtered using a 70 μm filter. Finally, the samples were analyzed by flow cytometry, and late apoptotic cells showed both green and red fluorescence, appearing in the Q2 area. Fig.10 The results showed that after Rot treatment, cell membrane damage occurred, and the proportion of cells with phosphatidylserine externalization on the cell membrane increased from 0.17% to 13.4%, while 50μg / mL of Gardenia jasminoides extracellular vesicles could effectively reduce its proportion to 0.42%. This indicates that Gardenia jasminoides extracellular vesicles can reduce cell membrane damage and have a protective effect on the cell membrane of nerve cells.

[0087] The above results show that the extracellular vesicles derived from gardenia fruit obtained by ultracentrifugation extraction from gardenia fruit and purification by sucrose gradient centrifugation and membrane filtration in the present invention are morphologically membrane-structured nanoparticles with a diameter of 50-200 nm and a concentration of 3.62±0.22×1011 particles / ml; the extracellular vesicles derived from gardenia fruit can be successfully taken up by PC2 cells after treatment for 24 hours, and 20 μg / mL and 50 μg / mL of gardenia extracellular vesicles can significantly improve the decreased viability of PC12 cells induced by 1 μM rotenone after being taken up, and effectively reduce the reactive oxygen species (ROS) and superoxide anion (O2 - ) to alleviate oxidative stress; in addition, the treatment of extracellular vesicles derived from gardenia fruit can also significantly reduce the DNA breakage and cell membrane damage of nerve cells caused by rotenone treatment, and has a significant protective effect on the nucleus and cell nucleus of nerve cells. Therefore, the extracellular vesicles derived from gardenia fruit of the present invention have significant antioxidant and nerve cell protection potential.

Claims

1. Application of Gardenia-derived extracellular vesicles in the preparation of drugs for the prevention and treatment of neurodegenerative diseases.

2. Application of Gardenia-derived extracellular vesicles in the preparation of antioxidant drugs.

3. The use according to claim 1, characterized in that: The neurodegenerative disease is Alzheimer's disease, ischemic stroke, hemorrhagic stroke, brain trauma or Parkinson's disease.

4. The use according to claim 1 or 2, characterized in that: When used, the protein concentration of the Gardenia-derived extracellular vesicles is 6.25-50 μg / mL.

5. The use according to claim 4, characterized in that: The intervention time was at least 24 hours.

6. The use according to claim 1 or 2, characterized in that: The method for preparing the Gardenia jasminoides-derived extracellular vesicles comprises the following steps: (1) adding a buffer solution to the raw material of Gardenia jasminoides and crushing the raw material to obtain a puree; centrifuging the puree for multiple times at the same speed, collecting the supernatant after each centrifugation for the next centrifugation, and collecting the supernatant after the last centrifugation; (2) subjecting the supernatant collected after the last centrifugation in step (1) to ultracentrifugation to collect the precipitate; (3) resuspending the precipitate obtained in step (2) with a buffer solution and subjecting the precipitate to density gradient ultracentrifugation to collect the suspended matter between each density layer; (4) resuspending the suspension collected in step (3) with a buffer solution and then ultracentrifuging again to collect the precipitate; (5) The precipitate collected in step (4) is resuspended in a buffer solution and then filtered through a membrane to obtain the gardenia-derived plant nanovesicles, wherein the pore size of the filter membrane is 0.1 to 0.5 μm.

7. The use according to claim 6, characterized in that: In step (1), the gardenia is gardenia fruit and / or gardenia flower; A juicer is used for crushing, the speed of the juicer is 1000-15000 rpm, and the crushing time is 30-120 seconds; The multiple centrifugations were divided into two centrifugations, the centrifugal force of both centrifugations was 20,000×g, and the centrifugation time was 20 minutes.

8. The use according to claim 6, characterized in that: In step (2), the supernatant collected after the last centrifugation in step (1) is first filtered to collect the filtrate; and the collected filtrate is then subjected to ultracentrifugation; In steps (2) and (4), the centrifugal force of ultracentrifugation is 100,000 to 170,000×g, and the centrifugation time is 60 to 180 minutes.

9. The use according to claim 6, characterized in that: In step (3), the density gradient ultracentrifugation uses sucrose, cesium chloride, rubidium chloride or cesium bromide as the gradient material; the mass concentrations of the density gradients are 8%, 30%, 45% and 60% respectively; In step (5), when filtering the membrane, first pass it through a filter membrane with a pore size of 0.45 μm, and then pass it through a filter membrane with a pore size of 0.22 μm.

10. The use according to claim 6, characterized in that: The pH range of the buffer in each step was 7.0 to 7.6; The buffer in each step is at least one of the following: phosphate buffer, Tris buffer, Tris-HCl buffer, TBS buffer, TBST buffer, TE buffer and Good's buffer.

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