A plant-based exovesicle preparation for treating liver cancer, its preparation method and application.

Abstract

This invention relates to the field of biomedical technology, specifically disclosing a plant-derived exovesicle preparation for treating liver cancer, its preparation method, and its application. The plant-derived exovesicle preparation is used to prepare drugs for treating liver cancer, and includes Uncaria rhynchophylla exovesicles and tea exovesicles. The plant-derived exovesicle preparation provided by this invention treats liver cancer by inhibiting liver cancer cell proliferation, promoting liver cancer cell apoptosis, and inhibiting oncogene expression.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a plant exosome preparation for treating liver cancer and a preparation method and application thereof. BACKGROUND

[0002] Primary liver cancer is a common fatal malignant tumor, and most patients are diagnosed with intermediate or advanced liver cancer, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). HCC is the main type of liver cancer, accounting for 75% of the total, and CCA accounts for 15%. CCA is divided into three subtypes: distal CCA (dCCA), perihilar CCA (pCCA) and intrahepatic CCA (iCCA). The incidence of CCA tends to increase with age. The proliferation and differentiation of cancer cells and the expression of oncogenes are key factors in the development of cancer, so inhibiting the proliferation and differentiation of cancer cells and inhibiting the expression of oncogenes is an important way to treat cancer. Therefore, it is of great significance to explore new ways to inhibit the proliferation and differentiation of liver cancer cells for the treatment of cancer. SUMMARY

[0003] In order to develop a new way to inhibit the proliferation and differentiation of liver cancer cells, the present application provides a plant exosome preparation for treating liver cancer and a preparation method and application thereof. The plant exosome preparation provided by the present application can inhibit the proliferation of liver cancer cells, promote the apoptosis of liver cancer cells, and inhibit the expression of oncogenes to treat liver cancer.

[0004] The present application provides the use of a plant exosome preparation in the preparation of a drug for treating liver cancer, wherein the plant exosome preparation comprises Gouteng exosome and tea leaf exosome.

[0005] The plant exosome preparation provided by the present application can inhibit the proliferation of liver cancer cells, promote the apoptosis of liver cancer cells, and inhibit the expression of oncogenes to treat liver cancer.

[0006] Further, the plant exosome preparation is prepared by mixing Gouteng exosome and tea leaf exosome in a particle number ratio of 1:1-10.

[0007] Further, the Gouteng exosome is extracted from Gouteng, and the tea leaf exosome is extracted from tea leaves.

[0008] Further, the drug is used to inhibit the proliferation of liver cancer cells.

[0009] Further, the drug is used to promote the apoptosis of liver cancer cells.

[0010] Further, the drug is used to inhibit the expression of oncogenes P13K 、 AKT1 and mTOR .

[0011] The application also provides a preparation method of the plant extracellular vesicle preparation, and the preparation of the plant extracellular vesicle preparation comprises the following steps:

[0012] Preparation of Gouteng extracellular vesicles: Gouteng is crushed to obtain juice to obtain Gouteng juice, and supernatant is collected by centrifugation, and then Gouteng extracellular vesicles are obtained by using a plant tissue exosome extraction kit for extraction;

[0013] Preparation of tea extracellular vesicles: tea is crushed to obtain juice to obtain tea juice, and supernatant is collected by centrifugation, and then tea extracellular vesicles are obtained by using a plant tissue exosome extraction kit for extraction;

[0014] The Gouteng extracellular vesicles and the tea extracellular vesicles are mixed to obtain the plant extracellular vesicle preparation.

[0015] Further, in the preparation process of the Gouteng extracellular vesicles, the extraction process using the plant tissue exosome extraction kit is as follows: after obtaining the Gouteng juice, supernatant is collected by centrifugation, the collected supernatant is mixed with Isolation Reagent A reagent in the plant tissue exosome extraction kit, and then extraction is carried out by standing for 10-15 minutes, and then supernatant is collected by centrifugation, the collected supernatant is mixed with Isolation Reagent B reagent in the plant tissue exosome extraction kit, and then extraction is carried out by standing for 50-70 minutes, and then the precipitate is collected by centrifugation, and the precipitate is resuspended in sterile normal saline and then centrifuged, and the supernatant is collected to obtain the Gouteng extracellular vesicles.

[0016] Further, in the preparation process of the tea extracellular vesicles, the extraction process using the plant tissue exosome extraction kit is as follows: after obtaining the tea juice, supernatant is collected by centrifugation, the collected supernatant is mixed with Isolation Reagent A reagent in the plant tissue exosome extraction kit, and then extraction is carried out by standing for 10-15 minutes, and then supernatant is collected by centrifugation, the collected supernatant is mixed with Isolation Reagent B reagent in the plant tissue exosome extraction kit, and then extraction is carried out by standing for 50-70 minutes, and then the precipitate is collected by centrifugation, and the precipitate is resuspended in sterile normal saline and then centrifuged, and the supernatant is collected to obtain the tea extracellular vesicles.

[0017] The application also provides the plant extracellular vesicle preparation obtained by the preparation method.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The plant exosome preparation is prepared by mixing the Gouteng exosome and the tea exosome, has obvious ability of inhibiting tumor growth, and has small side effect. The plant exosome preparation provided by the application can inhibit the growth of liver cancer cells by promoting cell apoptosis and inhibiting the expression of tumor growth genes, and provides a new technical idea for treating liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0021] Figure 1 The transmission electron microscope graph and the nanometer particle size graph of the Gouteng exosome and the tea exosome prepared by the application;

[0022] In the figure, A is the transmission electron microscope graph of the Gouteng exosome prepared by the application;

[0023] B is the transmission electron microscope graph of the tea exosome prepared by the application;

[0024] C is the nanometer particle size graph of the Gouteng exosome prepared by the application;

[0025] D is the nanometer particle size graph of the tea exosome prepared by the application.

[0026] Figure 2 The influence of the plant exosome preparation (Gouteng and tea exosome combined preparation) prepared by the application on the growth of HepG2 cells;

[0027] In the figure, A is the growth of HepG2 cells in the control group;

[0028] B is the growth of HepG2 cells in the positive group;

[0029] C is the influence of the EVs (Gouteng+tea) plant exosome preparation on the growth of HepG2 cells;

[0030] D is the influence of the EVs (ginseng+curcuma) plant exosome preparation on the growth of HepG2 cells.

[0031] Figure 3 The absorption of Gouteng exosome and tea exosome by HepG2 cells at different times.

[0032] Figure 4 Effects of plant exosome preparation (gouteng and tea exosome combined preparation) on apoptosis of HepG2 cells;

[0033] In the figure, A is the apoptosis of HepG2 cells in the control group;

[0034] B is the apoptosis of HepG2 cells in the positive group;

[0035] C is the effect of EVs (gouteng + tea) plant exosome preparation on the apoptosis of HepG2 cells;

[0036] D is the effect of EVs (ginseng + turmeric) plant exosome preparation on the apoptosis of HepG2 cells;

[0037] E is a statistical graph of the apoptosis of HepG2 cells in different groups.

[0038] Figure 5 qPCR detection of the effects of plant exosome preparation (gouteng and tea exosome combined preparation) on the expression of tumor-related genes in HepG2 cells. DETAILED DESCRIPTION

[0039] The specific embodiments of the present application will be described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0040] Example 1: A plant exosome preparation and its preparation method and identification.

[0041] 1. Preparation method of gouteng exosome

[0042] Take 1 kg of fresh gouteng and wash it clean in a sink with drinking water, then rinse it with ultrapure water.

[0043] Cut the washed gouteng into pieces, crush it in a juicer, filter it with a 150-mesh nylon screen, take the juice, and obtain gouteng original juice.

[0044] Centrifuge the original juice of Uncaria rhynchophylla at 10℃ and 6500 g for 10 min to obtain the first supernatant; centrifuge the first supernatant at 10℃ and 10000 g for 20 min to obtain the second supernatant; mix the second supernatant with extraction reagent A at a volume ratio of 2:1 and let it stand at 4℃ for 10 min. After standing, centrifuge at 10℃ and 12000 g for 10 min to obtain the third supernatant; mix the third supernatant with extraction reagent B at a volume ratio of 3:1 and let it stand at 4℃ for 1 h. After standing, centrifuge at 10℃ and 13500 g for 30 min, discard the supernatant and collect the precipitate.

[0045] The precipitate was resuspended in sterile saline and centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which is the outer vesicle of Uncaria rhynchophylla.

[0046] The extraction reagent A is Isolation Reagent A from the Plant Tissue Exosome Extraction Kit (Catalog No.: S309) purchased from Shenzhen Shifangjie Technology Co., Ltd. The extraction reagent B is Isolation Reagent B from the Plant Tissue Exosome Extraction Kit (Catalog No.: S309) sold by Shenzhen Shifangjie Technology Co., Ltd.

[0047] 2. Identification of Uncaria rhynchophylla exovesicles

[0048] The extracted outer vesicles of Uncaria rhynchophylla were examined by transmission electron microscopy, and the results are as follows: Figure 1 As shown, the results indicate that a distinct cup-shaped double-layered vesicle structure can be observed under a transmission electron microscope.

[0049] The particle size distribution and density of Uncaria rhynchophylla exovesicles were determined using a nanoparticle size tracer (NTA). The purity was initially assessed based on the peak distribution; fewer peaks indicate higher purity. The yield of exovesicles was determined by the number of exovesicles per milliliter; a higher number of exovesicles indicates a higher yield. Results are as follows: Figure 1 As shown, the NTA results indicate that the average particle size of the isolated Uncaria rhynchophylla exovesicles is 35.9 nm, and the concentration is 2.68 × 10⁻⁶. 10 The particle size per mL is within the range of 30 nm to 150 nm for external vesicles, and the purity and yield are high.

[0050] 3. Preparation of Tea Leaf Vesicles

[0051] Take 1 kg of fresh tea leaves, rinse them thoroughly with drinking water in a water tank, and then rinse them with ultrapure water. Place the rinsed tea leaves in a juicer and grind them. Filter the juice through a 150-mesh nylon screen to obtain the original tea juice. Centrifuge the original tea juice at 10℃, 6500 g for 10 min to obtain the first supernatant. Centrifuge the first supernatant at 10℃, 10000 g for 20 min to obtain the second supernatant. Mix the second supernatant with extraction reagent A at a volume ratio of 2:1 and let it stand at 4℃ for 10 min. After standing, centrifuge at 10℃, 12000 g for 10 min to obtain the third supernatant. Mix the third supernatant with extraction reagent B at a volume ratio of 3:1 and let it stand at 4℃ for 1 h. After standing, centrifuge at 10℃, 13500 g for 30 min, discard the supernatant, and collect the precipitate. The precipitate was resuspended in sterile physiological saline and centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which is the outer vesicle of the tea leaves.

[0052] 4. Identification of tea leaf vesicles

[0053] The extracted tea leaf vesicles were examined using transmission electron microscopy, and the results are as follows: Figure 1 As shown, a distinct cup-shaped double-layered vesicle structure can be observed under a transmission electron microscope.

[0054] The particle size distribution and density of tea leaf exovesicles were determined using a nanoparticle size analyzer (NTA). The purity was initially assessed based on the peak distribution; fewer peaks indicate higher purity. The yield of exovesicles was determined by the number of exovesicles per milliliter; a higher number of exovesicles indicates a higher yield. Results are as follows: Figure 1 The NTA results show that the average particle size of the separated tea leaf vesicles was 125.4 nm, and the concentration was 1.06 × 10⁻⁶. 10 The particle size per mL is within the range of 30 nm to 150 nm for external vesicles, and the purity and yield are high.

[0055] 5. Preparation of plant-derived exovesicle preparations

[0056] The prepared Uncaria rhynchophylla exovesicles and tea exovesicles were mixed at a particle ratio of 1:4 to form a plant exovesicle preparation, denoted as EVs (Uncaria rhynchophylla + tea).

[0057] The mixture was filtered sequentially using 0.45 μm and 0.22 μm syringe filters (Servicebio), and the protein concentration was determined using a BCA protein concentration assay kit (Beyotime Biotechnology Co., Ltd.).

[0058] Example 2: Application of plant-derived exovesicle preparations in inhibiting the proliferation of liver cancer cells and promoting apoptosis of liver cancer cells.

[0059] I. Effects of plant-derived exovesicle preparations on the growth of liver cancer cells

[0060] 1. Experimental Materials

[0061] Cells: HepG2 human liver cancer cells.

[0062] Complete cell culture medium: DMEM basal medium (Servicebio) containing a mixture of 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin (Servicebio).

[0063] 2. Experimental Methods

[0064] (1) Grouping

[0065] The subjects were divided into four groups: a control group, a positive control group (10 μM cisplatin), EVs (Uncaria rhynchophylla + tea), and EVs (ginseng + turmeric). The treatments for each group were as follows:

[0066] Control group: cells that grow normally and are not treated.

[0067] Positive group (10 μM cisplatin): Cisplatin was added to the cells and diluted with complete cell culture medium to a final concentration of 10 μM.

[0068] EVs (Uncaria rhynchophylla + tea): Add the extravesicle combination preparation prepared in Example 1 to each well according to the protein assay concentration, and dilute with complete cell culture medium to a final concentration of 100 μg / mL;

[0069] EVs (ginseng + turmeric): Add EVs (ginseng + turmeric) to each well according to the protein assay concentration, and dilute with complete cell culture medium to a final concentration of 100 μg / mL.

[0070] The preparation process of EVs (ginseng + turmeric) is as follows:

[0071] Take 1 kg of fresh ginseng, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water.

[0072] The washed ginseng was cut into pieces and placed in a juicer for blending and pulverization. The mixture was then filtered through a 150-mesh nylon screen to obtain the ginseng plant juice. The ginseng plant juice was centrifuged at 10℃ and 6500 g for 10 min to obtain the first supernatant. The first supernatant was centrifuged at 10℃ and 10000 g for 20 min to obtain the second supernatant. The second supernatant was mixed with extraction reagent A at a volume ratio of 2:1 and allowed to stand at 4℃ for 10 min. After standing, it was centrifuged at 10℃ and 12000 g for 10 min to obtain the third supernatant. The third supernatant was mixed with extraction reagent B at a volume ratio of 3:1 and allowed to stand at 4℃ for 1 h. After standing, it was centrifuged at 10℃ and 13500 g for 30 min. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in sterile physiological saline and centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which is the ginseng exovesicle.

[0073] Take 1 kg of fresh turmeric, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water.

[0074] Cut the washed turmeric into pieces, place them in a juicer and grind them. Filter the juice through a 150-mesh nylon screen to obtain the original turmeric juice. Centrifuge the original turmeric juice at 10℃, 6500 g for 10 min to obtain the first supernatant. Centrifuge the first supernatant at 10℃, 10000 g for 20 min to obtain the second supernatant. Mix the second supernatant with extraction reagent A at a volume ratio of 2:1 and let it stand at 4℃ for 10 min. After standing, centrifuge at 10℃, 12000 g for 10 min to obtain the third supernatant. Mix the third supernatant with extraction reagent B at a volume ratio of 3:1 and let it stand at 4℃ for 1 h. After standing, centrifuge at 10℃, 13500 g for 30 min, discard the supernatant, and collect the precipitate. Resuspend the precipitate in sterile physiological saline and centrifuge at 2000 g for 10 min to obtain the fourth supernatant, which is the turmeric exovesicle.

[0075] The prepared ginseng exovesicles and turmeric exovesicles were mixed at a particle ratio of 1:5 to obtain EVs (ginseng + turmeric).

[0076] (2) Experimental steps

[0077] Log-phase cells of human hepatocellular carcinoma HepG2 were collected and arranged at a ratio of 2 × 10⁻⁶ cells / cells. 5 The cells were seeded into 24-well plates. After cell adhesion, the corresponding substances were added according to the grouping and treatment methods described above. The cells were then incubated in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cell growth status was observed and photographed under a microscope.

[0078] (3) Experimental results

[0079] like Figure 2As shown, compared with the control group, the positive group cells were in significantly worse condition, lacking basic cell morphology, and almost all cells were dead. The EVs (Uncaria rhynchophylla + tea leaves) group had slightly more cells than the positive group, but the cell condition was similar to the positive group, lacking basic cell morphology and with most cells dead. Ginseng and turmeric, which are also combination preparations, had no significant effect on the state of HepG2 cells. Therefore, it can be seen that the plant exovesicle preparation prepared in this invention can specifically induce HepG2 cell death.

[0080] II. HepG2 cell uptake of Uncaria rhynchophylla and tea leaf vesicles

[0081] 1. Experimental Materials

[0082] External vesicles: Uncaria rhynchophylla external vesicles and tea external vesicles prepared in Example 1.

[0083] Cells: HepG2 human liver cancer cells.

[0084] Cell culture medium: DMEM basal medium (Servicebio) containing a mixture of 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin (Servicebio).

[0085] Reagents: DiO cell membrane green fluorescence staining kit and 1× sterile PBS, both purchased from Servicebio. The DiO cell membrane green fluorescence staining kit includes a DiO cell membrane green fluorescent probe and staining buffer.

[0086] 2. Experimental Methods

[0087] Log-phase cells of human hepatocellular carcinoma HepG2 were collected and arranged at a ratio of 2 × 10⁻⁶ cells / cells. 5 The cells were seeded into 24-well plates and placed in a cell culture incubator. The Dio cell membrane green fluorescent probe was diluted with staining buffer at a volume ratio of 1:250 to prepare the Dio staining working solution. 100 μL of each of the Uncaria rhynchophylla and Tea vesicles were centrifuged at 13500g for 30 min, and the precipitates were collected separately. Each precipitate was then resuspended in 100 μL of the Dio staining working solution, and the resuspended samples were incubated at 37℃ in the dark for 30 min, followed by centrifugation at 13500g for 30 min to remove excess dye. The Uncaria rhynchophylla and Tea vesicle precipitates were collected separately and resuspended in 1× sterile PBS to obtain Dio-labeled Uncaria rhynchophylla and Tea vesicles. The Dio-labeled Uncaria rhynchophylla and Tea vesicles were co-cultured with cells for 24 h and 48 h, respectively, and the absorption was observed and recorded using a fluorescence microscope.

[0088] 3. Experimental Results

[0089] like Figure 3As shown, when cells were co-incubated with DiO green fluorescent vesicles, both the Uncaria rhynchophylla and Tea vesicle groups showed green fluorescence at 24 h, with a greater number of green fluorescent cells at 48 h. This indicates that HepG2 cells can absorb Uncaria rhynchophylla and Tea vesicles, and the uptake rates of these two types of vesicles are similar, starting at 24 h and increasing at 48 h.

[0090] III. Effects of exovesicle combination formulation on HepG2 cell apoptosis

[0091] 1. Experimental Materials

[0092] External vesicles: Uncaria rhynchophylla external vesicles and tea external vesicles prepared in Example 1.

[0093] Cells: HepG2 human liver cancer cells.

[0094] Cell culture medium: DMEM basal medium (Servicebio) containing a mixture of 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin (Servicebio).

[0095] Reagents: Hoechst 33258 was purchased from Sangon Biotech Co., Ltd., 1× sterile PBS, and tissue fixative was purchased from Servicebio.

[0096] 2. Experimental Methods

[0097] (1) Grouping

[0098] The study group was divided into a control group, a positive group (final concentration of 10 μM cisplatin), EVs (Uncaria rhynchophylla + tea leaves), and EVs (ginseng + turmeric). The final concentration of EVs (Uncaria rhynchophylla + tea leaves) and EVs (ginseng + turmeric) was 100 μg / mL.

[0099] (2) Experimental steps

[0100] HepG2 liver cancer cells in the logarithmic phase were collected and arranged at a ratio of 2 × 10⁻⁶ cells / cells. 5 The cells were seeded into 24-well plates and incubated at 37°C with 5% CO2. After cell adhesion, different reagents were added according to the grouping, and the cells were incubated at 37°C with 5% CO2 for further incubation. The culture medium was aspirated, and 200 μL of tissue fixative was added to each well for fixation for 10 min. The fixative was removed, and the cells were washed once with 1× sterile PBS. After aspirating the liquid, 500 μL of Hoechst 33258 staining solution (10 μg / ml) was added, and staining was performed for 10 min. The staining solution was removed, and the cells were washed once with 1× sterile PBS. The liquid was aspirated. Cell apoptosis was observed and recorded under a fluorescence microscope.

[0101] 3. Experimental Results

[0102] like Figure 4 As shown, some cells in the control group were stained with a blue fluorescent probe, and the fluorescence intensity was relatively weak. The fluorescence intensity of EVs (ginseng + turmeric) was similar to or even weaker than that of the control group. However, the number and intensity of fluorescence in the positive group and the EVs (Uncaria + tea) group were stronger than those in the control group, and there was no significant difference between the positive group and the EVs (Uncaria + tea) group. This indicates that plant exovesicle preparations can specifically induce cell death in HepG2 cells by promoting apoptosis, thereby achieving an anti-cancer effect.

[0103] IV. Effects of plant-derived vesicle preparations on HepG2 cell tumor pathways

[0104] 1. Experimental Materials

[0105] Cells: HepG2 human liver cancer cells.

[0106] Cell culture medium: DMEM basal medium (Servicebio) containing a mixture of 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin (Servicebio).

[0107] 2. Experimental Methods

[0108] (1) Grouping

[0109] The study was divided into three groups: a blank control group, a positive control group (final concentration of 10 μM cisplatin), and EVs (plant exovesicle preparation with a final concentration of 100 μg / mL).

[0110] (2) Experimental steps

[0111] Log-phase cells of human hepatocellular carcinoma HepG2 were collected and arranged at a ratio of 4 × 10⁻⁶ cells / cells. 5 Cells were seeded into 12-well plates and placed in a cell culture incubator. After cell attachment, the corresponding reagents were added according to the cell groups, and the plates were incubated at 37°C with 5% CO2 for 48 hours. Cells were collected and analyzed according to the instructions of the RNA extraction kit, reverse transcription kit, and qPCR assay. P13K , AKT1 as well as mTOR The expression is shown in Table 1.

[0112] Table 1 Primer sequences

[0113]

[0114] (3) Data processing

[0115] Statistical analysis was performed using Prism 6.0 (GraphPad) software. Two-way ANOVA was used for comparisons among multiple groups. Results are expressed as mean ± standard deviation (x ± s).

[0116] 3. Experimental Results

[0117] like Figure 5 As shown, compared with the control group, both the positive group and EVs (Uncaria rhynchophylla + tea leaves) inhibited the expression of PI3K, AKT1, and mTOR at the mRNA level, and there was no significant difference between the two groups. This indicates that Uncaria rhynchophylla / tea leaf vesicles can achieve anti-cancer effects by inhibiting the expression of tumor-forming genes and promoting tumor cell apoptosis, with efficacy comparable to cisplatin.

[0118] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0119] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a plant exosome preparation for the manufacture of a medicament for treating liver cancer, characterized in that, The plant exosome preparation is prepared by mixing Gouteng exosome and tea exosome in a particle number ratio of 1:1-10.

2. Use of the plant exosome preparation according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The Gouteng exosome is extracted from Gouteng as raw material; and the tea exosome is extracted from tea as raw material.

3. Use of the plant exosome preparation according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The medicine is used for inhibiting proliferation of liver cancer cells.

4. Use of the plant exosome preparation according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The medicine is used for promoting apoptosis of liver cancer cells.

5. Use of the plant exosome preparation according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, The medicament is used for inhibiting expression of oncogenes P13K , AKT1 and mTOR .

6. A plant extracellular vesicle preparation for treating liver cancer, characterized by, The plant exosome preparation is prepared by mixing Gouteng exosome and tea exosome in a particle number ratio of 1:1-10.

7. The plant extracellular vesicle formulation for treating liver cancer according to claim 6, characterized in that, The average particle size of the Gouteng exosome in the plant exosome preparation is 35.9 nm, and the average particle size of the tea exosome is 125.4 nm.

8. A method for preparing the plant-derived vesicular preparation for treating liver cancer according to any one of claims 6 to 7, characterized by, The method comprises the following steps: Preparation of Gouteng exosome: Gouteng is crushed and filtered to obtain Gouteng juice, the supernatant is collected by centrifugation, and then Gouteng exosome is extracted by using a plant tissue exosome extraction kit; Preparation of tea exosome: tea is crushed and filtered to obtain tea juice, the supernatant is collected by centrifugation, and then tea exosome is extracted by using a plant tissue exosome extraction kit; Mixing of Gouteng exosome and tea exosome to obtain plant exosome preparation.

9. The method of claim 8, wherein the plant extracellular vesicle preparation is prepared by the steps of, During the preparation of Gouteng exosome, the extraction process by using the plant tissue exosome extraction kit is as follows: the collected supernatant is mixed with Isolation Reagent A reagent in the plant tissue exosome extraction kit, and then is left to extract for 10-15 min; the supernatant is collected by centrifugation, the collected supernatant is mixed with Isolation Reagent B reagent in the plant tissue exosome extraction kit, and then is left to extract for 50-70 min; the precipitate is collected by centrifugation, the precipitate is resuspended in sterile normal saline, and then is centrifuged; the supernatant is collected to obtain Gouteng exosome.

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