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

Abstract

The present invention discloses the use of Gardenia jasminoides-derived extracellular vesicles in the preparation of drugs for preventing and treating neurodegenerative diseases, and relates to the field of biomedicine. The present invention applies Gardenia jasminoides-derived extracellular vesicles to anti-oxidation and neuroprotection. After being ingested, 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 ‑ ) production, thereby alleviating oxidative stress. Furthermore, treatment with gardenia fruit-derived extracellular vesicles significantly reduced DNA breakage and cell membrane damage in neurons caused by rotenone treatment, demonstrating a significant protective effect on the nuclei and cell membranes of neurons. Therefore, the gardenia fruit-derived extracellular vesicles described in the present invention possess significant antioxidant and neuroprotective potential.

Description

Technical Field

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

[0002] Extracellular vesicles (EVs) are a type of nanoscale vesicle that forms inside cells, fuses with the plasma membrane, and is then released into the extracellular environment. EVs primarily include exosomes, microvesicles, and apoptotic bodies. Exosomes are the smallest, typically ranging in diameter from 30 to 150 nm, while microvesicles (MVs) have a wide range of sizes, ranging from 100 to 1,000 nm in diameter. EVs carry a variety of bioactive molecules, such as nucleic acids, proteins, and metabolites, which play an important role in intercellular communication, signaling, and transport of substances. EVs can be secreted by almost all types of living cells, including animal and plant cells.

[0003] In recent years, plant-derived extracellular vesicles have received widespread attention. Plant EVs have high biocompatibility and low immunogenicity, and can carry a wealth of 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 multiple biological activities such as antioxidant and anti-inflammatory activities. For example, EVs from plants such as honeysuckle, astragalus, and licorice have outstanding performance in antioxidant and anti-inflammatory aspects. In the field of neuroprotection, plant-derived EVs may be rich in neuroprotective factors and antioxidants, which can effectively reduce oxidative stress, alleviate neurotoxicity, and promote the survival and regeneration of nerve cells. 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 (Gardenia jasminoides J. Ellis), also known as yellow gardenia, mountain gardenia, and white toad, belongs to the genus Gardenia in the Rubiaceae family. The fruit of the plant, also known as "gardenia," is a traditional Chinese medicine and one of the first Chinese medicinal herbs to be used as both a medicine and a food. Its fruit is widely used in Traditional Chinese Medicine. Gardenia is rich in bioactive compounds such as iridoids, flavonoids, and triterpenes, which impart significant antioxidant, antidepressant, anxiolytic, and neuroprotective properties. While the chemical composition and direct pharmacological effects of gardenia have been extensively studied, researchers both domestically and internationally have isolated chemical components such as iridoids, flavonoids, diterpenes, triterpenes, phenolic acids, and organic acid esters from gardenia flowers. Furthermore, research has been conducted on the extraction of total flavonoids from gardenia flowers, the extraction and purification of gardenia oil, the extraction of gardenia yellow pigment, and the analysis and determination of its amino acid composition. Patent applications, including one with publication number CN1 16855438A, disclose a method for preparing plant nanovesicles derived from Gardenia jasminoides. This patent application boasts advantages such as simplicity, low cost, and high yield. Plant-derived exosome-like nanoparticles can promote interspecies communication by inducing multiple cytokines. They possess advantages such as high targeting capabilities, non-toxicity, high thermal stability, easy metabolic degradation by the human body, and low immunogenicity. However, the biological activity of Gardenia jasminoides-derived extracellular vesicles (EVs) has not been thoroughly explored. Summary of the Invention

[0005] To overcome the above-mentioned deficiencies in existing research, the present invention proposes an extracellular vesicle derived from Gardenia jasminoides, 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 jasminoides, a dual-purpose medicinal and edible resource, for the prevention and treatment of neurodegenerative diseases.

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

[0007] The present invention provides the use of Gardenia jasminoides-derived extracellular vesicles in the preparation of drugs for preventing and treating neurodegenerative diseases.

[0008] The present invention utilizes a rotenone (Rot)-induced PC12 neuronal cell injury model, where Gardenia jasminoides extracellular vesicles reduce nuclear damage, DNA breakage, and apoptosis (externalization of phosphatidylserine on the cell membrane) in PC12 cells. This is to evaluate the neuroprotective effects of Gardenia jasminoides extracellular vesicles and explore their potential use in the treatment of Alzheimer's disease and other related neurodegenerative diseases.

[0009] The Gardenia jasminoides 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 Gardenia jasminoides-derived extracellular vesicles in the preparation of antioxidant drugs.

[0011] The present invention used the rotenone-induced oxidative stress model of PC12 cells as an experimental model and found that Gardenia jasminoides extracellular vesicles could 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 anions (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, ie, 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 of Gardenia jasminoides and have a protein concentration of 6.25-50 μg / mL. As an example, the preparation method of the Gardenia jasminoides-derived extracellular vesicles in the present invention 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 pulp; centrifuging the raw material for multiple times at the same speed, collecting the supernatant after each centrifugation for the next centrifugation, and collecting the supernatant after the final 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 performing 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 the pulp to gel.

[0023] The multiple centrifugations are divided into two centrifugations, both of which are performed at a centrifugal force of 20,000 × g and for 20 minutes, in order to preliminarily 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 the 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 can help increase 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), the membrane is first filtered through a membrane with a pore size of 0.45 μm, and then through a membrane with a pore size of 0.22 μm. Passing through two membranes can reduce the risk of direct clogging of the membrane and improve utilization.

[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 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 antioxidant 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 - ) production, thereby alleviating oxidative stress. Furthermore, treatment with gardenia fruit-derived extracellular vesicles significantly reduced DNA breakage and cell membrane damage in neurons caused by rotenone treatment, demonstrating a significant protective effect on the nuclei and cell membranes of neurons. Therefore, the gardenia fruit-derived extracellular vesicles described in the present invention possess significant antioxidant and neuroprotective potential.

[0032] 2. The present invention can promote the use of Gardenia jasminoides 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 image of the extracellular vesicles derived from Gardenia jasminoides 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 jasminoides obtained by Malvern laser particle size analyzer.

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

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

[0037] Figure 5 This is the improvement effect of Gardenia jasminoides-derived extracellular vesicles intervention for 24 hours on the decreased viability of PC12 neurons induced by rotenone.

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

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

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

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

[0042] Figure 10 This study investigates the effects 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 from Gardenia jasminoides at different protein concentrations. DETAILED DESCRIPTION

[0044] The method for preparing 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 pulp; centrifuging the raw material for multiple times at the same speed, collecting the supernatant after each centrifugation for the next centrifugation, and collecting the supernatant after the final 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 performing 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 the pulp to gel.

[0052] The multiple centrifugations are divided into two centrifugations, both of which are performed at a centrifugal force of 20,000 × g and for 20 minutes, in order to preliminarily 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 the 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 can help increase 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), the membrane is first filtered through a membrane with a pore size of 0.45 μm, and then through a membrane with a pore size of 0.22 μm. Passing through two membranes can reduce the risk of direct clogging of the membrane and improve utilization.

[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 heat generated by high-speed centrifugation.

[0059] The present invention is described below through specific embodiments.

[0060] Example 1

[0061] In this example, extracellular vesicles derived from Gardenia jasminoides were prepared and their morphology was observed. The specific method was consistent with the method for preparing extracellular vesicles derived from Gardenia jasminoides in Example 1 of patent application 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 Under a transmission electron microscope, the extracellular vesicles derived from gardenia fruit appeared as membrane-structured nanoparticles with a particle size range of 50-200 nm. This confirmed the successful extraction of nanoscale vesicles derived from gardenia fruit.

[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, employing a 488 nm blue laser. Freshly prepared Gardenia jasminoides-derived extracellular vesicle 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 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. Figure 2 The particle concentration of Gardenia jasminoides-derived extracellular vesicles was 3.62±0.22×1011 particles / mL.

[0066] Example 3

[0067] This example observed the uptake of extracellular vesicles derived from Gardenia jasminoides 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 hour, 8 hours, and 24 hours, 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 the excess DAPI dye, and the GDEVs-DiD showing red fluorescence and the cell nuclei showing blue fluorescence were observed under a fluorescence microscope. The results are shown in FIG. Figure 3 As shown, obvious red fluorescence of DiD-GDEVs was observed near the cell nucleus and between the cell membranes, indicating that Gardenia jasminoides-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 the Gardenia jasminoides-derived extracellular vesicles extracted in Example 1 on improving the decreased viability of PC12 neurons induced by rotenone after 24 hours of intervention.

[0071] PC12 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO₂. First, treatment with GDEVs at varying protein concentrations (1.5625, 3.125, 6.25, 12.5, 25, and 50 μg / mL) for 24 hours confirmed that they did not significantly affect PC12 cell viability. Next, we investigated whether co-treatment with GDEVs at these protein concentrations for 24 hours could attenuate the decrease in PC12 cell viability induced by 1 μM rotenone (Rot). Cell viability was assessed using the methylthiazolium tetrazolium (MTT) assay. Trypsinized PC12 cells were cultured in an incubator, allowed to adhere, and then treated with the corresponding concentrations of Rot or GDEVs for 24 hours. The cells were then incubated with 0.5 mg / mL MTT for 4 hours at 37°C. After removing the MTT solution, 150 μL of dimethyl sulfite was added, and the absorbance at 570 nm was measured using a multifunctional microplate reader. Figure 4 and Figure 5 The results showed that treatment with Gardenia jasminoides-derived extracellular vesicles with different protein concentrations alone did not affect the viability of PC12 cells, but intervention with Gardenia jasminoides-derived extracellular vesicles with a protein concentration higher than 6.25 μg / mL for 24 hours could improve the rotenone-induced decrease in PC12 neuronal viability.

[0072] Example 5

[0073] This example verifies the effect of the Gardenia jasminoides-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 group treated with rotenone and Gardenia jasminoides-derived extracellular vesicles (Rot+GDEVs, 20 μg / mL and 50 μg / mL). After treating PC12 cells for 24 hours, 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 assessed 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 Fluorescence results and quantitative statistics showed that treatment with 1 μM rotenone alone significantly increased ROS production in PC12 cells. However, 20 μg / mL and 50 μg / mL of Gardenia jasminoides-derived extracellular vesicles significantly reduced ROS production, indicating that Gardenia jasminoides-derived extracellular vesicles can alleviate oxidative stress in PC12 cells.

[0075] Example 6

[0076] This example verifies the effect of the Gardenia jasminoides-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 jasminoides-derived extracellular vesicles (Rot+GDEVs, 20 μg / mL and 50 μg / mL). After treating PC12 cells for 24 hours, the cells were fixed with 2.5% glutaraldehyde for 30 minutes. Subsequently, the cells were washed three times with PBS and the superoxide anion (O2 - After washing twice 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 O2 - Relatively speaking, 20μg / mL and 50μg / mL of Gardenia jasminoides-derived extracellular vesicles can significantly reduce O2 - The results showed that extracellular vesicles derived from Gardenia jasminoides could alleviate the oxidative stress level in PC12 cells.

[0078] Example 7

[0079] This example verifies the effect of the Gardenia jasminoides-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 jasminoides-derived extracellular vesicles (Rot+GDEVs, 20 μg / mL and 50 μg / mL). After treating PC12 cells for 24 hours, 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 two light washes with PBS, the fluorescence intensity in the cells was observed under a fluorescence microscope using consistent exposure settings. Figure 8 Quantitative statistics showed that treatment with 1 μM rotenone alone significantly increased the number of bright spots formed by nuclear shrinkage in PC12 cells. However, 20 μg / mL and 50 μg / mL of Gardenia jasminoides-derived extracellular vesicles significantly reduced nuclear damage, indicating that Gardenia jasminoides-derived extracellular vesicles have a protective effect on the nuclei of PC12 cells.

[0081] Example 8

[0082] This example verifies the effect of the Gardenia jasminoides-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 control group (blank group), rotenone group (1μM Rot) and rotenone and Gardenia jasminoides-derived extracellular vesicles co-treated group (Rot+GDEVs, 20μg / mL and 50μg / mL). TM 488-12-dUTP will be incorporated into the 3'-hydroxyl end of DNA fragments, thus detecting DNA fragmentation and cell damage by fluorescence changes. First, PC12 cells were treated with 20μg / mL proteinase K at room temperature for 5 minutes, and then washed twice with PBS. Then, 50μL of YSFluor TM The cells were incubated with 488-12-dUTP and recombinant TdT enzyme in terminal deoxyribonucleotidyl transferase (TdT) incubation buffer at 37°C for 60 minutes and then washed twice with PBS. Then, the nuclei were stained with 2 μg / mL DAPI for 5 minutes at room temperature. Finally, the YSFluor was observed by fluorescence microscopy. TM Green fluorescence intensity of 488-12-dUTP. Figure 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 jasminoides could significantly reduce the expression of YSFluor TM The fluorescence intensity of 488-12-dUTP decreases the DNA fragmentation in cells.

[0084] Example 9

[0085] This example verifies the effect of the Gardenia jasminoides-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-derived extracellular vesicles (Rot+GDEVs, 20 μg / mL and 50 μg / mL). The Annexin V-FITC / PI apoptosis detection kit identifies apoptotic cells using FITC-labeled Annexin V. After treatment, cells were washed three times with PBS and then digested with EDTA-free trypsin. Subsequently, cells were harvested by centrifugation at 300 × g for 5 minutes at 4°C, rinsed twice with pre-cooled PBS, and resuspended in 100 μL of 1× Binding Buffer. 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added, mixed thoroughly, and incubated in the dark at room temperature for 10-15 minutes. Then, 400 μL of 1× Binding Buffer was added, and the cells were filtered through a 70 μm filter. Finally, the samples were analyzed by flow cytometry. Late apoptotic cells exhibit both green and red fluorescence, appearing in the Q2 zone. Figure 10 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%. However, 50 μg / mL of Gardenia jasminoides EVs effectively reduced this proportion to 0.42%, indicating that Gardenia jasminoides EVs can reduce cell membrane damage and have a protective effect on the cell membrane of neurons.

[0087] The above results show that the extracellular vesicles derived from gardenia fruit obtained by the present invention are extracted from gardenia fruit by ultracentrifugation and purified by sucrose gradient centrifugation and membrane filtration. Morphologically, the cell vesicles present 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. Moreover, 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 - ) production, thereby alleviating oxidative stress; in addition, treatment with extracellular vesicles derived from gardenia fruit can significantly reduce DNA breakage and cell membrane damage in neurons caused by rotenone treatment, and has a significant protective effect on the nuclei and cell nuclei of neurons. Therefore, the extracellular vesicles derived from gardenia fruit described in the present invention have significant antioxidant and neuronal cell protection potential.

Claims

1. Application of Gardenia jasminoides-derived extracellular vesicles in the preparation of neuronal cell protective and / or antioxidant drugs; The method for preparing Gardenia jasminoides-derived extracellular vesicles comprises the following steps: (1) Adding buffer solution to the raw material of Gardenia jasminoides and crushing it to obtain the raw pulp; The slurry is centrifuged multiple times at the same speed, and the supernatant is collected after each centrifugation for the next centrifugation, and the supernatant is collected after the last centrifugation; The gardenia is gardenia fruit and / or gardenia flower; When crushing, use a juicer with a speed of 1000 to 15000 rpm and a crushing time of 30 to 120 seconds; Multiple centrifugation was divided into two centrifugations, the centrifugal force of both centrifugations was 20,000 × g, and the centrifugation time was 20 minutes; (2) First, the supernatant collected after the last centrifugation in step (1) is filtered to collect the filtrate; then the collected filtrate is subjected to ultracentrifugation to collect the precipitate; The centrifugal force of ultracentrifugation is 100,000 to 170,000 × g, and the centrifugation time is 60 to 180 minutes; (3) resuspending the precipitate obtained in step (2) with a buffer solution and performing density gradient ultracentrifugation to collect the suspended matter between each density layer; Density gradient ultracentrifugation uses sucrose, cesium chloride, rubidium chloride or cesium bromide as gradient materials; the density gradient mass concentrations are 8%, 30%, 45% and 60% respectively; (4) resuspending the suspension collected in step (3) with a buffer solution and then ultracentrifuging again to collect the precipitate; The centrifugal force of ultracentrifugation is 100,000 to 170,000 × g, and the centrifugation time is 60 to 180 minutes; (5) resuspending the precipitate collected in step (4) with a buffer solution and filtering 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; 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.

2. The use according to claim 1, characterized in that When used, the protein concentration of the Gardenia jasminoides-derived extracellular vesicles is 6.25-50 μg / mL, and the intervention treatment time is at least 24 hours.

3. The use according to claim 1, characterized in that The pH range of the buffer in each step was 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.

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