Celastrus orbiculatus rhizome exosome-like vesicles as well as extraction method and application thereof

Abstract

The invention discloses celastrus orbiculatus rootstock exosome-like vesicles and an extraction method and application thereof. The celastrus orbiculatus rootstock exosome-like vesicles are obtained by cleaning and grinding celastrus orbiculatus rootstocks, then performing centrifugal separation, then performing resuspension and then performing collection by using a sucrose density gradient centrifugation method. The obtained exosome-like vesicle is stable in property, low in toxicity, high in biocompatibility and good in targeting property. The celastrus orbiculatus rhizome exosome-like vesicles can inhibit gastric cancer cell activity, inhibit gastric cancer cell migration, promote gastric cancer cell apoptosis and reduce gastric cancer cell proliferation ability, and the vesicles can be enriched in the stomach in a targeted manner after oral administration. The celastrus orbiculatus rootstock exosome-like vesicles are clear in anti-gastric cancer effect and reliable in curative effect, and have clinical transformation potential.

Description

TECHNICAL FIELD

[0001] The present application relates to a kudzu vine rhizome exosome-like vesicle and its extraction method and its anti-gastric cancer application, belonging to the technical field of plant exosomes. BACKGROUND

[0002] Gastric cancer is a common malignant tumor of the digestive tract. Due to its strong invasiveness, most patients are in the middle and advanced stages when diagnosed, and about 60% have distant metastasis. At present, the therapeutic effect of targeted drugs and chemotherapeutic drugs is still not ideal, and the survival period of patients is limited, so the development of new candidate drugs has become an urgent need.

[0003] Kudzu vine is a plant of the family Celastraceae, with bitter, pungent and slightly warm properties. It has the effects of expelling wind and dampness, unblocking channels and relieving pain, promoting blood circulation and detoxification. Its stems, roots, fruits and leaves can be used as medicine. Kudzu vine mainly contains active ingredients such as sesquiterpenes, triterpenes, steroids, organic acids, flavonoids and tannins, which have certain effects on anti-inflammatory, antiviral, antibacterial and antioxidant.

[0004] The homogenate of kudzu vine plant tissue contains a large amount of starch, pectin, pigments (such as chlorophyll), tannins, polysaccharides and secondary metabolites (such as alkaloids in kudzu vine itself), which can seriously interfere with subsequent separation and purification, and may mask or destroy the target vesicles. A set of standard methods are needed to prove that the extract is indeed kudzu vine rhizome exosome-like vesicles, not other membrane fragments or nanoparticles.

[0005] However, traditional Chinese medicine preparations often face problems such as low solubility, poor dissolution rate, limited bioavailability and insufficient stability, which affect the full play of their therapeutic effect. Plant exosome-like vesicles, as a natural source of nanoscale carriers, have good biocompatibility, multifunctionality, stability and low toxicity, and are easy to control, showing broad prospects in the fields of drug delivery, disease diagnosis and treatment. SUMMARY

[0006] The purpose of the present application is to provide a kudzu vine rhizome exosome-like vesicle, another purpose of the present application is to provide an extraction method of the kudzu vine rhizome exosome-like vesicle, and the last purpose of the present application is to provide the application of the kudzu vine rhizome exosome-like vesicle in preparing a drug for treating and / or preventing cancer.

[0007] Technical scheme: the extraction method of the kudzu vine rhizome exosome-like vesicle provided by the present application comprises the following steps:

[0008] (1) washing fresh kudzu vine rhizomes with deionized water, grinding, filtering with gauze, and obtaining juice;

[0009] (2) centrifuging the juice, collecting the precipitate, and obtaining a crude extract of the A. henryi rhizome exosome-like vesicle, and resuspending the crude extract to obtain a resuspension;

[0010] (3) collecting the A. henryi rhizome exosome-like vesicle from the resuspension by sucrose density gradient centrifugation, and filtering to obtain the A. henryi rhizome exosome-like vesicle.

[0011] Further, in step (2), the centrifugal separation is performed by differential centrifugation and ultracentrifugation. The differential centrifugation includes the following steps:

[0012] centrifuging at 500xg-1500xg for 10-20 minutes at 4°C, collecting the supernatant, centrifuging at 3000xg for 30 minutes, collecting the supernatant, centrifuging at 10000xg-13000xg for 60-70 minutes at 4°C, and collecting the supernatant.

[0013] The ultracentrifugation includes the following steps:

[0014] centrifuging the collected supernatant at 120000xg-150000xg for 90-120 minutes at 4°C to obtain a precipitate.

[0015] Further, in step (3), the sucrose density gradient centrifugation includes the following steps:

[0016] transferring the resuspension to sucrose solutions with different density gradients, ultracentrifuging at 120000xg-150000xg for 2 hours at 4°C, collecting the A. henryi rhizome exosome-like vesicle from the interface between 30% and 45% sucrose, wherein the different density gradients are 8%, 15%, 30%, 45%, and 60% (w / v), resuspending the collected A. henryi rhizome exosome-like vesicle in pre-cooled 1xPBS buffer, filtering with a 0.22μm filter membrane, and storing at -80°C.

[0017] The A. henryi rhizome exosome-like vesicle obtained by the extraction method.

[0018] The A. henryi rhizome exosome-like vesicle can be used for preparing a drug for treating and / or preventing cancer, wherein the cancer is gastric cancer and / or intestinal cancer.

[0019] The A. henryi rhizome exosome-like vesicle successfully extracted by the present application contains 653 compounds, including 393 lipid components, and traditional anti-tumor active components of A. henryi such as triterpenes and sesquiterpenes. Experiments have proved that the A. henryi rhizome exosome-like vesicle can be effectively internalized by gastric cancer cells, and further exert an inhibitory effect through various mechanisms such as inhibiting cell proliferation, inducing cell apoptosis, and inhibiting cell migration and invasion.

[0020] Advantages: Compared with the prior art, the present application has the following advantages:

[0021] (1) The extracted kudzu vine rhizome exosome-like vesicle has simple and controllable preparation process, stable property, low toxicity, high biocompatibility, good targeting property and good application prospect.

[0022] (2) The kudzu vine rhizome exosome-like vesicle system contains rich lipids and organic heterocyclic compounds and other active substances on the basis of prolonging the original effective anti-tumor components (such as triterpenes and sesquiterpenes) of kudzu vine, which indicates that it may play an anti-gastric cancer role through multiple pathways. This provides a material basis for its potential synergistic anti-gastric cancer effect, and makes it have more comprehensive potential and value in the application of anti-tumor.

[0023] (3) The kudzu vine rhizome exosome-like vesicle can inhibit the activity of gastric cancer cells, inhibit the migration of gastric cancer cells, promote the apoptosis of gastric cancer cells, reduce the proliferation ability of gastric cancer cells, and the vesicle can be targeted to accumulate in the stomach after oral administration. The anti-gastric cancer effect of the kudzu vine rhizome exosome-like vesicle is clear, the curative effect is reliable, and it has clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a morphological diagram of kudzu vine rhizome exosome-like vesicle for transmission electron microscope detection;

[0025] Figure 2 It is a particle size analysis result diagram of kudzu vine rhizome exosome-like vesicle;

[0026] Figure 3 It is a zeta potential analysis result diagram of kudzu vine rhizome exosome-like vesicle;

[0027] Figure 4 It is a protein BCA quantitative result diagram of kudzu vine rhizome exosome-like vesicle;

[0028] Figure 5 It is a qualitative and quantitative result diagram of chemical components of kudzu vine rhizome exosome-like vesicle;

[0029] Figure 6 It is a qualitative and quantitative result diagram of lipid components of kudzu vine rhizome exosome-like vesicle;

[0030] Figure 7 It is a schematic diagram of cell uptake of exosome of kudzu vine rhizome exosome-like vesicle;

[0031] Figure 8 It is a kudzu vine rhizome exosome-like vesicle enrichment diagram in mice after gavage;

[0032] Figure 9 It is a kudzu vine rhizome exosome-like vesicle treatment gastric cancer cell AGS, HGC-27 CCK-8 experiment result diagram;

[0033] Figure 10 Figure for AGS, HGC-27 tranwell experiment results of treating gastric cancer cells with Radix Acanthopanacis Senticosi exosome-like vesicles;

[0034] Figure 11 Figure for AGS, HGC-27 scratch experiment results of treating gastric cancer cells with Radix Acanthopanacis Senticosi exosome-like vesicles;

[0035] Figure 12 Figure for AGS, HGC-27 apoptosis flow results of treating gastric cancer cells with Radix Acanthopanacis Senticosi exosome-like vesicles;

[0036] Figure 13 Figure for AGS, HGC-27 EdU proliferation experiment results of treating gastric cancer cells with Radix Acanthopanacis Senticosi exosome-like vesicles. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be further described below in combination with the drawings and examples.

[0038] Cell strain: human gastric cancer cell lines AGS, HGC-27, both purchased from Shanghai Cell Library of Chinese Academy of Sciences.

[0039] Reagents: RMPI-1640 medium (HYCLONE company, SH30809.01), phosphate (PBS) buffer (Hyclone company, SH30256.01), 0.25% trypsin digestion solution (Hyclone company, SH30256.01), fetal bovine serum (FBS) (Gibco 10270-106), penicillin-streptomycin mixture (Solebo, P1400), paraformaldehyde (China National Pharmaceutical, 80096618), DIO staining solution (MCE, D0969), DAPI staining solution (Bi Yun Tian, C1006), Actin-Tracker Red-555 (Bi Yun Tian, C2203S), immunostaining permeation liquid (Triton X-100) (Bi Yun Tian, P0096), immunofluorescence staining secondary antibody diluent (Bi Yun Tian, P0108), Cell Counting Kit-8 (CCK-8) (Solebo, CA1210), DIR dye (MCE, HY-D1048), Annexin V-FITC / PI apoptosis detection kit (Solebo, C1062S), deionized water (ddH2O), EdU cell proliferation detection kit (Abbkine, KTA2031).

[0040] Instruments and equipment: carbon dioxide incubator (Thermo Scientific), clean bench (Thermo Scientific), upright fluorescence microscope (Olympus), sterile six-well plate (Corning), TC treated cell climbing sheet (Solaybao), full-automatic enzyme-labeled instrument (Perkin Elmer), sterile 96-well plate (Corning), small animal live imaging system (Perkin Elmer), sterile 24-well plate Transwell chamber and 8.0 μm pore size polycarbonate membrane (Corning), flow cytometer (BD FACSVERSE).

[0041] Experimental animals: 12 C57BL / 6 mice aged 5-6 weeks were selected and purchased from the Comparative Medicine Center of Yangzhou University, and the weight was about 20 g.

[0042] Example 1 Extraction of Kadsura longipedunculata rhizome exosome-like vesicles

[0043] (1) Wash 200 g of fresh Kadsura longipedunculata rhizome with deionized water, grind, filter with gauze, and obtain juice;

[0044] (2) Centrifuge the above juice at 500xg for 10 minutes at 4°C, collect the supernatant; centrifuge at 3000xg for 30 minutes, collect the supernatant; centrifuge at 10000xg for 60 minutes at 4°C, collect the supernatant; centrifuge at 150000xg for 90 minutes at 4°C, obtain the precipitate, and obtain Kadsura longipedunculata rhizome exosome-like vesicle crude extract;

[0045] (3) Collect the precipitate and resuspend with 1xPBS;

[0046] (4) Spread the resuspension on the top of five sucrose solutions with w / v of 8%, 15%, 30%, 45%, and 60% (in centrifuge tubes, from low concentration to high concentration in order), and centrifuge at 150,000xg at 4°C for 2 hours. After centrifugation, an obvious band will appear at the junction between the 30% and 45% sucrose solutions, and the sample is collected from the band, which is Kadsura longipedunculata rhizome-derived exosome-like vesicles. Resuspend the collected Kadsura longipedunculata rhizome exosome-like vesicles in pre-cooled 1xPBS buffer, filter with a 0.22 μm filter membrane, and store at -80°C.

[0047] Comparative Example 1

[0048] (1) Wash 200 g of fresh Kadsura longipedunculata rhizome with deionized water, grind, add 1 L of deionized water and boil for 2 h, filter with gauze, and obtain concentrated juice;

[0049] (2) The juice was centrifuged at 1500×g for 20 minutes at 4℃ and the supernatant was collected; centrifuged at 3000×g for 30 minutes and the supernatant was collected; centrifuged at 16000×g for 60 minutes at 4℃ and the supernatant was collected; centrifuged at 150000×g for 120 minutes at 4℃ to obtain the precipitate, which was resuspended in 1×PBS. Electron microscopy results showed that no exosome-like vesicles were obtained from the rhizomes of Celastrus orbiculatus.

[0050] Comparative Example 2:

[0051] (1) Wash 200g of fresh Celastrus orbiculatus rhizomes with deionized water, grind them, filter them with gauze, and obtain the juice;

[0052] (2) The juice was centrifuged at 1500×g for 20 minutes at 4℃ and the supernatant was collected; centrifuged at 3000×g for 30 minutes and the supernatant was collected; centrifuged at 16000×g for 60 minutes at 4℃ and the supernatant was collected; centrifuged at 150000×g for 120 minutes at 4℃ to obtain the precipitate, which was resuspended in 1×PBS. Electron microscopy results showed that no pure Celastrus orbiculatus rhizome exosome-like vesicles were obtained.

[0053] Physicochemical characteristics of exosome-like vesicles from the rhizome of Celastrus orbiculatus:

[0054] 1. Morphological characterization

[0055] Morphological characterization of exosome-like vesicles from the rhizome of Celastrus orbiculatus under transmission electron microscopy, such as... Figure 1 As shown, the exosome-like vesicles of Celastrus orbiculatus rhizome exhibit a typical spherical cup-shaped nano-exosome bilayer membrane structure.

[0056] 2. Particle size determination

[0057] Nanoparticle tracking analysis (NTA) determined the average particle size of exosome-like vesicles from the rhizome of *Celastrus orbiculatus* to be 179.2 nm, with a median particle size of 190.4 nm. The results are as follows: Figure 2 As shown, the size of the exosome-like vesicles in the rhizome of Celastrus orbiculatus is approximately 120-250 nm.

[0058] 3. Potential Measurement

[0059] The zeta potential of exosome-like vesicles from the rhizomes of Celastrus orbiculatus was measured using a laser particle size analyzer (DLS). The results are as follows: Figure 3 As shown, the results indicate that the zeta potential of exosome-like vesicles in the rhizome of Celastrus orbiculatus is -16.85±1.25mV.

[0060] 4. Protein concentration determination

[0061] like Figure 4 As shown, BCA detected a protein concentration of 29.35325 mg / ml in the exosome-like vesicles of Celastrus orbiculatus rhizome.

[0062] 5. Chemical composition identification:

[0063] 5.1 Sample Processing

[0064] Take 100 μL of Celastrus orbiculatus rhizome exosome vesicle sample into a 2 mL centrifuge tube; add 400 μL of pre-cooled methanol:acetonitrile (1:1, v / v), vortex for 30 s; freeze at -20℃ for 30 min; centrifuge at 12,000 rpm, 4℃ for 10 min, take 400 μL of the supernatant and concentrate to dryness under vacuum; add 150 μL of 50% methanol (containing 5 ppm 2-chlorophenylalanine) to reconstitute, vortex for 30 s; centrifuge at 12,000 rpm, 4℃ for 10 min, take the supernatant and filter through a 0.22 μm filter membrane, add the filtrate to the test bottle; take 10-20 μL of each sample filtrate and mix them to form a QC sample for evaluating instrument stability and data reliability.

[0065] 5.2 Chromatographic Methods

[0066] An ACQUITY UPLCHSS T3 column (100 Å, 1.8 µm, 2.1 mm × 100 mm) was used at a flow rate of 0.4 mL / min, a column temperature of 40 °C, and an autosampler temperature of 8 °C. The injection volume was 2 μL. The mobile phases for both positive and negative modes were: mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile (containing 0.1% formic acid). The elution gradients are shown in Table 1 below.

[0067] Table 1. Elution gradients for positive and negative modes

[0068] Time (min) B% 0 5% 1 5% 7 95% 8 95% 8.1 5% 12 5%

[0069] 5.3 Mass Spectrometry Methods

[0070] A Thermo Orbitrap Exploris 120 mass spectrometer was used to acquire DDA mass spectrometry data in both positive and negative ion modes under the control of Xcalibur software (version 4.7, Thermo). An HESI source was used with a spray voltage of 3.5 kV / -3.0 kV, a sheath gas temperature of 40 alb, an auxiliary gas temperature of 15 alb, a capillary temperature of 325 °C, an auxiliary gas temperature of 300 °C, a first-order resolution of 60,000, a scan range of 100-1000 m / z, AGC Target Standard, and a Max IT time of 100 ms. The top four ions in the response were selected for secondary fragmentation, with a dynamic exclusion time of 8 s. The second-order resolution was 15,000, the HCD collision energy was 30%, and the AGC Target Standard and Max IT Auto settings were used.

[0071] Data Analysis: The raw data was imported into CompoundDiscoverer™ 3.3 (version 3.3.2.31, Thermo, Waltham, USA). Based on the software's new peak detection and peak quality scoring algorithms, peak extraction, alignment, and correction were performed, and the total peak area (Sum) was normalized. Metabolite identification was based on a self-built library, the mzCloud online library (https: / / www.mzcloud.org / ), LIPIDMAPS (https: / / www.lipidmaps.org / ), HMDB (https: / / hmdb.ca / ), MoNA (https: / / mona.fiehnlab.ucdavis.edu / ), and the NIST_2020_MSMS spectral library. Results are as follows: Figure 5 As shown. The results are as follows. Figure 5 Identification analysis revealed that the most abundant category of chemical components in the exosome-like vesicles of *Celastrus orbiculatus* rhizome was organic heterocyclic compounds, followed by lipids and lipid-like molecules. Other significant categories included organic acids and derivatives, benzeneoids, organic oxygen compounds, phenylpropanoids and polyketides, and organic nitrogen compounds. Less abundant categories included alkaloids and derivatives, organic sulfur compounds, organic metallic compounds, organic halogen compounds, nucleosides, nucleotides, and analogues. Among these, triterpenes and sesquiterpenes, the main antitumor active components of *Celastrus orbiculatus* rhizome, were detected in the exosome-like vesicles.

[0072] 6. Identification of lipid composition

[0073] 6.1 Sample processing:

[0074] Take an appropriate amount of exosome vesicle sample from *Celastrus orbiculatus* rhizome into a 2.0 mL centrifuge tube, add 750 μL of chloroform:methanol (2:1, v / v), vortex for 60 s; place on ice and let stand for 40 min; add 200 µL of water, vortex for 30 s, sonicate at 40 Hz for 10 min, and let stand at -20℃ for 20 min; centrifuge at 12,000 rpm, 4℃ for 10 min; take 300 μL of the lower organic phase and transfer to a new 2 mL centrifuge tube; add 500 µL of chloroform:methanol (2:1) solution to the upper residue, vortex for 30 s; centrifuge at 12,000 rpm for 10 min, take 400 µL of the lower organic layer solution and combine with a 2 mL centrifuge tube, concentrate under vacuum to dryness; add 150 μL of isopropanol (containing 5 ppm 2-chlorophenylalanine) to reconstitute, vortex for 60 s; centrifuge at 12,000 rpm, 4℃ for 10 min. min; take the supernatant and filter it through a 0.22μm filter membrane, then add the filtrate to the sample vial.

[0075] 6.2 LC-MS / MS Analysis:

[0076] Lipid molecules were separated using a Thermo Vanquish Flex chromatography system (Thermo Fisher Scientific, Waltham, MA, USA), and lipid molecules in positive and negative ion modes were identified using a Thermo Q Exactive HF-X mass spectrometer with an ESI source (Thermo Fisher Scientific, Waltham, MA, USA). A Waters BEH C18 column (1.7 µm, 2.1 mm × 100 mm) was used at a flow rate of 0.25 mL / min, a column temperature of 50 °C, an autosampler temperature of 8 °C, and an injection volume of 2 μL. Mobile phase A was acetonitrile:water (6:4) containing 0.1% formic acid + 5 mM ammonium formate, and mobile phase B was isopropanol:acetonitrile (9:1) containing 0.1% formic acid + 5 mM ammonium formate. The mobile phase gradient was as follows: 0-2 min, 30% B-50% B; 2-12 min, 50% B-75% B; 12-12.1 min, 75% B; 12.1-18 min, 75% B-95% B; 18 min-21 min, 95% B; 21-21.1 min, 95% B-30% B; 21.1-24 min, 30% B.

[0077] The mass spectrometry parameters were as follows: fog voltage 3.5 kV / -2.5 kV, sheath gas 40 arb, auxiliary gas 10 arb, capillary temperature 320℃, auxiliary gas temperature 300℃, primary resolution 60,000, scan range 150-2000 m / z, AGC Target Standard, Max IT 100 ms, screening the top 8 ions for secondary fragmentation, dynamic exclusion time 4 s, secondary resolution 15,000, HCD collision energy 20% and 30%, AGC Target Standard, Max IT 25 ms.

[0078] 6.3 Data Analysis

[0079] The data extracted by LipidSearch underwent preprocessing including filtering, imputation, correction, and normalization, followed by quality assessment. Only after passing this assessment were data analyzed. The data analysis included statistical identification of lipid samples, lipid composition analysis, and lipid differential analysis. Lipid composition analysis included lipid subclass composition and lipid content distribution analysis; lipid differential analysis included lipid content, chain length, and chain saturation analysis; and lipid content variation analysis involved multiple dimensions, including overall, subclass, and molecular analysis. The LC-MS / MS analysis results are shown below. Figure 6 The results showed that the main lipid substances in the exosome-like vesicles of Celastrus orbiculatus rhizome were triglycerides (TG), ceramides (Cer), phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), diglycerides (DAG), phosphatidylinositol (PI), and phosphatidylglycerol (PG).

[0080] Example 2

[0081] 1. Uptake of exosome-like vesicles from Celastrus orbiculatus rhizomes

[0082] 200 μL of Celastrus orbiculatus root exosome vesicles were added to 10 μL of 1 mM DIO dye, vortexed for 1 min, and incubated at 37 °C for 30 min in a Thermo Scientific CO2 incubator to allow the dye to fully bind to the Celastrus orbiculatus root exosome vesicles. The solution was then filtered through a 0.22 μm filter to remove free dye, resulting in DIO-labeled Celastrus orbiculatus root exosome vesicles. These vesicles were then diluted with PBS buffer to obtain 2 mg / mL DIO-labeled Celastrus orbiculatus root exosome vesicles.

[0083] TC-treated cell slides were placed in sterile six-well plates (Corning). Human gastric cancer cell lines AGS and HGC-27 with good growth were selected and prepared into 0.3×10⁻⁶ cells using 1×PBS. 6Cell suspension of 10 cells / ml was evenly seeded into each well of a plate. After the cells were in stable growth, 2 mg / ml of Celastrus orbiculatus root exosome-like vesicles labeled with LDIO were added, and the plates were incubated at 37°C for 6 hours in a CO2 incubator.

[0084] Cells were washed twice with PBS buffer, and 1 mL of paraformaldehyde was added to each well for fixation at room temperature for 30 min. After removing the fixative, cells were washed twice with PBS buffer for 3 min each time. TC-treated cell slides were carefully transferred to glass slides, and immunostaining permeabilization buffer (0.1% Triton X-100) was added for cell permeabilization at room temperature for 5 min. After removing the permeabilization buffer, cells were washed twice with PBS buffer for 3 min each time. Then, Actin-Tracker Red-555 (1:50) diluted with immunofluorescence staining secondary antibody was added and incubated at room temperature in the dark for 60 min. Cells were washed thoroughly three times with PBS, counterstained with DAPI staining solution in the dark for 5 min, blotted with absorbent paper, dried in the dark, and finally mounted with neutral resin.

[0085] Observation and photography were performed using an upright fluorescence microscope (Olympus). Figure 7 As shown, exosomes stained with DIO appear green, the cytoskeleton stained with Actin-Tracker Red-555 appears red, and cell nuclei stained with DAPI appear blue. Based on fluorescence colocalization, it can be determined that exosome-like vesicles from Celastrus orbiculatus rhizomes are taken up and internalized by AGS and HGC-27 cells.

[0086] 2. Enrichment of exosome-like vesicles from Celastrus orbiculatus rhizomes in normal mice

[0087] Take 500 μL of Celastrus orbiculatus rhizome exosome vesicles and add 10 μL of 5 mM DIR dye. Vortex mix for 1 min and incubate at 37°C for 30 min in a CO2 incubator to allow the dye to fully bind with the Celastrus orbiculatus rhizome exosome vesicles, thus obtaining DIR dye-labeled Celastrus orbiculatus rhizome exosome vesicles. Dilute with PBS buffer to obtain 1 mg / ml DIR-labeled Celastrus orbiculatus rhizome exosome vesicles.

[0088] C57BL / 6 mice were randomly divided into four groups of three mice each. After fasting for 24 hours, the mice were administered 6 mg / kg of their body weight via gavage using a curved gavage needle. The groups were divided into four subgroups: 4 h post-gavage (6 mg / kg), 8 h post-gavage (6 mg / kg), 12 h post-gavage (6 mg / kg), and 24 h post-gavage (6 mg / kg). Organs were dissected at different time points for in vivo imaging. Results are as follows: Figure 8As shown, 4 hours after drug administration, fluorescence accumulation first appeared in the stomach, intestines, and liver. 8 hours later, fluorescence accumulation appeared in the lungs and kidneys, with the liver showing the strongest fluorescence. 12 hours later, kidney fluorescence increased, while liver fluorescence gradually decreased. 24 hours later, fluorescence in the liver, kidneys, and lungs decreased, while gastrointestinal fluorescence remained stable. No significant fluorescence was observed in the heart and spleen. This indicates that after 24 hours, the drug mainly accumulates in the stomach and intestines.

[0089] Experimental Example 3: Inhibitory Effect of Celastrus orbiculatus Rhizome Exosome-like Vesicles on Gastric Cancer Cells in Vitro

[0090] Drug preparation: Take the exosome-like vesicles from the rhizome of Celastrus orbiculatus and dilute them with RMPI-1640 complete medium containing 10% FBS to concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL and 300 μg / mL respectively.

[0091] Cell culture: Human gastric cancer cell lines AGS and HGC-27 were cultured in a carbon dioxide constant temperature incubator at 37°C, saturated humidity, and 5% carbon dioxide.

[0092] CCK-8 Experiment:

[0093] AGS and HGC-27 cells in the exponential growth phase were collected, and an appropriate amount of 0.25% trypsin digestion solution was added. After pipetting, the adherent cells were detached. After cell counting, a single-cell suspension was prepared in RPMI-1640 medium containing 10% fetal bovine serum. 3000 cells per well were seeded into 96-well culture plates, with 3 replicates per group. The volume of complete culture medium in each well was 100 μL. 200 μL of sterile PBS buffer was added to the surrounding blank wells to prevent liquid evaporation. After adhesion, the culture medium was removed, and 100 μL of different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL) of Celastrus orbiculatus rhizome exosome-like vesicles were added. The cells were cultured in a CO2 incubator at 37°C, 5% CO2, and saturated humidity for 24 h. After culture, 10 μL of LCK-8 solution was added to each well, and the plate was incubated in a CO2 incubator for 3 hours. The 96-well plate was then removed from the CO2 incubator and placed in a multi-mode microplate reader, where the absorbance was measured at 460 nm. Cell growth inhibition rate = (OD value of blank wells - OD value of experimental wells) / (OD value of blank wells - OD value of control wells) * 100%.

[0094] Data Processing: The obtained data were analyzed using Graphpad Prism 10.1.2 statistical software. Data are expressed as mean ± standard deviation (s), and one-way ANOVA was used for statistical analysis. A p-value < 0.05 was considered statistically significant. Results are as follows: Figure 9As shown, exosome-like vesicles from the rhizome of *Celastrus orbiculatus* significantly inhibited the gastric cancer cells used in this experiment, exhibiting a clear dose-dependent effect. Calculations revealed that the IC50 of *Celastrus orbiculatus* rhizome exosome-like vesicles against AGS gastric cancer cells was... 50 The value was 137.3 μg / mL, and the IC50 value for HGC-27 was... 50 The value was 191.1 μg / mL.

[0095] Experiment Example 4: In vitro inhibitory effect of exosome-like vesicles from Celastrus orbiculatus rhizome on gastric cancer cell migration.

[0096] Drug preparation: Take the exosome-like vesicles extracted from the rhizomes of Celastrus orbiculatus in Example 1 and dilute them with RMPI-1640 complete medium containing 10% FBS to concentrations of 100 μg / mL, 200 μg / mL and 300 μg / mL respectively.

[0097] Cell culture: Same as in Example 3.

[0098] 1. Transwell experiment:

[0099] AGS and HGC-27 cells in the exponential growth phase were digested, centrifuged, and resuspended. Using sterile 24-well Transwell chambers and 8.0 μm pore size polycarbonate membranes, 600 μL of RMPI-1640 complete medium containing different concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL) of Celastrus orbiculatus root exosome-like vesicles was added to the lower chamber. The control group received 600 μL of RMPI-1640 complete medium containing 10% FBS without vesicles. 250 μL of empty RMPI-1640 medium containing 20,000 cells was added to the upper chamber. The chambers were incubated in a CO2 incubator at 37°C, saturated humidity, and 5% CO2 for 24 h. Afterward, the chambers were removed, fixed with paraformaldehyde for 15 min, stained with crystal violet for 15 min, washed with PBS, and air-dried.

[0100] The images were observed and photographed using an inverted fluorescence microscope, and the results were statistically analyzed and counted. The results are as follows: Figure 10 As shown in the Transwell assay, the number of cells penetrating the membrane was significantly reduced in the vesicle-treated group compared to the control group. Furthermore, after treatment with *Celastrus orbiculatus* root exosome-like vesicles at concentrations of 100 μg / mL, 200 μg / mL, and 300 μg / mL, the number of cells penetrating the membrane decreased progressively with increasing concentration. This indicates that *Celastrus orbiculatus* root exosome-like vesicles significantly inhibited the migration ability of both AGS and HGC-27 gastric cancer cells, exhibiting a clear concentration-dependent effect.

[0101] 2. Cell scratch assay:

[0102] AGS and HGC-27 cells in the exponential growth phase were digested, centrifuged, and resuspended. Cell counting was performed. Two × 10⁶ cells were seeded in each well of a six-well plate. When the cells reached approximately 90% confluence as a monolayer, a vertical line was drawn on the confluent cells using a 200 μL pipette tip to create a linear scratch. Exfoliated cells were washed away with PBS. Each well was then divided into three regions using a marker. After removing the liquid, 2 mL of different concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL) of Celastrus orbiculatus root exosome-like vesicles were added and incubated for 24 h. The supernatant was discarded, and the cells were washed three times with PBS.

[0103] Images were taken at 0h and 24h using an inverted fluorescence microscope, and the mobility was calculated.

[0104] Data Processing: The obtained data were analyzed using Graphpad Prism 10.1.2 statistical software. Data are expressed as mean ± standard deviation (s), and one-way ANOVA was used for statistical analysis. A p-value < 0.05 was considered statistically significant. Results are as follows: Figure 11 As shown, exosome-like vesicles from the rhizome of *Celastrus orbiculatus* significantly inhibited the migration of two gastric cancer cell lines, AGS and HGC-27, exhibiting a clear concentration-dependent effect: In AGS cells, the migration rate of the control group was approximately 0.72, decreasing to approximately 0.42, 0.20, and 0.12 after treatment with 100, 200, and 300 μg / ml vesicles, respectively, with highly significant differences between each treatment group and the control group (P < 0.0001). In HGC-27 cells, the migration rate of the control group was approximately 0.74, decreasing to approximately 0.55, 0.41, and 0.23 after treatment with 100, 200, and 300 μg / ml vesicles, respectively, with statistically significant or highly significant differences between each treatment group and the control group (P < 0.001 for the 100 μg / ml group, P < 0.001 for the 200 μg / ml group, and P < 0.001 for the 300 μg / ml group). (P<0.0001) Overall, this indicates that the exosome-like vesicles of Celastrus orbiculatus rhizome can effectively inhibit the migration of gastric cancer cells, and the inhibitory effect increases with increasing vesicle concentration, with the inhibitory effect on AGS cells being more prominent.

[0105] Experimental Example 5: The effect of Celastrus orbiculatus rhizome exosome-like vesicles on promoting apoptosis in gastric cancer cells in vitro.

[0106] Drug preparation and cell culture: Same as in Example 4.

[0107] Flow cytometry apoptosis experiment:

[0108] AGS and HGC-27 cells in the exponential growth phase were digested, centrifuged, and resuspended. The cells were seeded into six-well plates, and after attachment, 2 ml of RMPI-1640 complete medium containing different concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL) of Celastrus orbiculatus root exosome-like vesicles was added to each well. The control group was added to RMPI-1640 complete medium containing 10% FBS without vesicles. The plates were incubated in a CO2 incubator at 37°C, saturated humidity, and 5% CO2 for 24 h.

[0109] Remove the 6-well plate, collect the supernatant, and transfer it to a 1.5 ml centrifuge tube. Wash the bottom cells with PBS buffer. Digest the cells with 500 ml of 0.25% trypsin digestion solution, and stop digestion with an equal volume of RMPI-1640 complete culture medium containing 10% FBS. Transfer the cell suspension to a centrifuge tube, mix well, and centrifuge at 1200 rpm for 5 min. Remove the supernatant and collect the cell pellet. Resuspend the cells in PBS buffer and centrifuge again at 1200 rpm for 5 min. Remove the supernatant and collect the cell pellet. Dilute 3 ml of Binding Buffer (10×) to 1× with 27 ml of ddH2O. Resuspend the cells in 1 ml of Binding Buffer (1×) and centrifuge at 300 x g for 10 min. Resuspend the cells in 1 ml of Binding Buffer (1×) to achieve a cell density of 1×10⁻⁶ cells / mL. 6 per mL.

[0110] Take an appropriate number of centrifuge tubes, add 100 μL of cell suspension to each tube, add 5 μL of Annexin V-FITC staining solution, mix well, and incubate at room temperature in the dark for 10 min. Add 5 μL of PI staining solution to each tube, mix well, and incubate in the dark for 5 min. Add 400 μL of PBS buffer and mix well. Detect apoptosis data using flow cytometry within 1 h. Annexin V-FITC shows green fluorescence, and PI shows red fluorescence.

[0111] Data processing: The apoptosis rate of the data obtained after the experiment was analyzed using FlowJo_v10.8.1 software, and the statistically processed data were analyzed using Graphpad Prism 10.1.2 statistical software. Data are expressed as mean ± standard deviation (s), and one-way ANOVA was used for statistical analysis. A p-value < 0.05 was considered statistically significant. Results are as follows: Figure 12As shown, exosome-like vesicles from the rhizome of *Celastrus orbiculatus* exhibited significant pro-apoptotic effects on both AGS and HGC-27 gastric cancer cells, showing a clear concentration-dependent effect. In AGS cells, the apoptosis rate in the control group was approximately 10%. After treatment with vesicles at concentrations of 100, 200, and 300 μg / ml, the apoptosis rate increased sequentially, reaching approximately 24% in the 300 μg / ml group. The difference between the 100 μg / ml group and the control group was significant (P<0.01), while the difference between the 300 μg / ml group and the control group was extremely significant (P<0.001). In HGC-27 cells, the apoptosis rate in the control group was approximately 9%. After treatment with vesicles at concentrations of 100, 200, and 300 μg / ml, the apoptosis rate showed a more pronounced upward trend, reaching approximately 40% in the 300 μg / ml group. The difference between the 100 μg / ml group and the control group was significant (P<0.05), while the difference between the 200 μg / ml group and the control group was extremely significant (P<0.05). The differences between the group and the control group were extremely significant (P<0.001), and the differences between the 300 μg / ml group and the control group were extremely significant (P<0.0001). Overall, this indicates that the exosome-like vesicles of Celastrus orbiculatus rhizome can effectively induce apoptosis in gastric cancer cells, and the pro-apoptotic effect on HGC-27 cells is more prominent.

[0112] Experimental Example 6: In vitro inhibitory effect of exosome-like vesicles from Celastrus orbiculatus rhizome on the proliferation of gastric cancer cells.

[0113] The drug preparation and cell culture were the same as in Example 4.

[0114] EdU proliferation experiment:

[0115] AGS and HGC-27 cells in the exponential growth phase were digested, centrifuged, resuspended, and evenly seeded into 24-well plates. When the cells reached 50% confluence, different concentrations (100 μg / mL, 200 μg / mL, and 300 μg / mL) of Celastrus orbiculatus rhizomes were added, and the cells were cultured for 24 h in a CO2 incubator at 37°C, saturated humidity, and 5% CO2.

[0116] According to the instructions of the Abbkine EdU cell proliferation assay kit, RMPI-1640 complete medium containing 10% FBS was diluted with the EdU solution in the kit at a ratio of 1000:1. 300 μL of medium containing EdU was added to each well and then incubated in a CO2 incubator at 37°C, saturated humidity, and 5% CO2 for 2 hours. The liquid was then aspirated and the cells were washed with PBS buffer solution.

[0117] Add 300 μL of freshly prepared 1× Click-IT staining solution to each well (refer to Table 2 for Click-IT staining solution preparation), incubate in the dark at room temperature for 30 min, then remove the reaction solution and wash the cells in the wells with BSA Wash Solution (1×) for 5 min, repeating twice. Dilute Hoechst 33342 (1000×) staining solution to 1× with PBS, add 300 μL to each well, and incubate in the dark at room temperature for 30 min. Aspirate the liquid and wash three times with BSA Wash Solution (1×).

[0118] Observation and recording were performed using an inverted fluorescence microscope.

[0119] Table 2 Preparation of Click-IT staining reaction solution

[0120] Order of Preparation Click-IT Stain Reaction 500 μL 1 mL 1 Deionized Water 379 758 2 Reaction Buffer (10x) 50 100 3 Copper Reagent 20 40 4 AbFluor 545 Azide 1 2 5 Reducing Agent (10x) 50 100

[0121] Data Processing: The obtained data were analyzed using Graphpad Prism 10.1.2 statistical software. Data are expressed as mean ± standard deviation (s), and one-way ANOVA was used for statistical analysis. A p-value < 0.05 was considered statistically significant. Results are as follows: Figure 13 As shown, exosome-like vesicles from the rhizome of Celastrus orbiculatus significantly inhibited the proliferation of two gastric cancer cells, AGS and HGC-27, in a concentration-dependent manner: In AGS cells, the proliferation rate of the control group was approximately 42%; after treatment with vesicles at concentrations of 100, 200, and 300 μg / ml, the proliferation rates decreased to approximately 29%, 18%, and 9%, respectively. The differences between each treatment group and the control group were statistically significant or extremely significant (P<0.0001 for the 100 μg / ml group, P<0.01 for the 200 μg / ml group, and P<0.01 for the 300 μg / ml group), and the differences between the groups were also extremely significant (P<0.0001). In HGC-27 cells, the cell proliferation rate of the control group was approximately 45%. After treatment with 100, 200, and 300 μg / ml vesicles, the proliferation rate decreased to approximately 36%, 28%, and 20%, respectively. The differences between each treatment group and the control group were statistically significant or highly statistically significant (P<0.0001 for the 100 μg / ml group, P<0.01 for the 200 μg / ml group, and P<0.01 for the 300 μg / ml group), and the differences between the groups were also highly statistically significant (P<0.0001). Overall, exosome-like vesicles from the rhizome of *Celastrus orbiculatus* effectively inhibited the proliferation of gastric cancer cells, and this inhibitory effect increased with increasing vesicle concentration, with a more pronounced inhibitory effect on AGS cells.

Claims

1. A method for extracting an exosome-like vesicle of a rootstock of Celastrus orbiculatus, characterized by, The method comprises the following steps: (1) washing fresh Celastrus angulatus rhizome with deionized water, grinding, filtering with gauze to obtain juice; (2) centrifuging the juice, collecting the precipitate to obtain a Celastrus angulatus rhizome exosome-like vesicle crude extract, and resuspending to obtain a resuspension; (3) collecting the Celastrus angulatus rhizome exosome-like vesicle by sucrose density gradient centrifugation, and filtering to obtain the Celastrus angulatus rhizome exosome-like vesicle.

2. The extraction method according to claim 1, characterized in that, In step (2), the centrifugal separation is performed by differential centrifugation and ultracentrifugation.

3. The extraction method according to claim 2, characterized in that, The differential centrifugal separation comprises the following steps: centrifuging at 500xg-1500xg at 4℃ for 10-20 minutes to collect supernatant; centrifuging at 3000xg for 30 minutes to collect supernatant; centrifuging at 10000xg-13000xg at 4℃ for 60-70 minutes to collect supernatant.

4. The extraction method of claim 2, wherein, The ultracentrifugation comprises the following steps: centrifuging the collected supernatant at 120000xg-150000xg at 4℃ for 90-120 minutes to obtain a precipitate.

5. The extraction method of claim 1, wherein, In step (3), the sucrose density gradient centrifugation comprises the following steps: transferring the resuspension to sucrose solutions with different density gradients, and centrifuging at 120000xg-150000xg at 4℃ for more than 2 hours to collect the Celastrus angulatus rhizome exosome-like vesicle from the interface of 30%-45% sucrose.

6. The extraction method according to claim 5, characterized in that, The different density gradients are 8%, 15%, 30%, 45% and 60% (w / v).

7. The extraction method of claim 1, wherein, In step (3), the collected Celastrus angulatus rhizome exosome-like vesicle is resuspended in pre-cooled 1xPBS buffer, filtered with a 0.22μm filter membrane, and stored at -80℃.

8. The Celastrus angulatus rhizome exosome-like vesicle obtained by the extraction method of any one of claims 1-7.

9. The use of the Celastrus angulatus rhizome exosome-like vesicle of claim 8 in the preparation of a medicament for treating and / or preventing cancer.

10. Use according to claim 9, characterized in that, The cancer is gastric cancer and / or intestinal cancer.

Images