Plant external vesicle preparation for treating liver cancer as well as preparation method and application of plant external vesicle preparation

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a plant external vesicle preparation for treating liver cancer and a preparation method and application thereof, the plant external vesicle preparation is used for preparing a medicine for treating liver cancer, and the plant external vesicle preparation comprises uncaria external vesicles and tea external vesicles. The plant external vesicle preparation provided by the invention is used for treating liver cancer by inhibiting proliferation of liver cancer cells, promoting apoptosis of the liver cancer cells and inhibiting expression of oncogenes.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a plant extracellular vesicle preparation for treating liver cancer, and a preparation method and application thereof. Background Art

[0002] Primary liver cancer is a common and fatal malignancy. Patients are often diagnosed at intermediate or advanced stages of the disease, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). HCC is the predominant type of liver cancer, accounting for 75% of cases, while CCA accounts for 15%. CCA is divided into three subtypes: distal CCA (dCCA), perihilar CCA (pCCA), and intrahepatic CCA (iCCA). The incidence of CCA increases with age. Cancer cell proliferation and differentiation, as well as oncogene expression, are key factors in the development and progression of cancer. Therefore, inhibiting cancer cell proliferation and differentiation, as well as suppressing oncogene expression, is an important approach for cancer treatment. Therefore, identifying new pathways to inhibit the proliferation and differentiation of liver cancer cells is of great significance for cancer treatment. Summary of the Invention

[0003] To develop a new approach to inhibit the proliferation and differentiation of liver cancer cells, the present invention provides a plant exosome preparation for treating liver cancer, as well as its preparation method and application. The plant exosome preparation provided by the present invention treats liver cancer by inhibiting liver cancer cell proliferation, promoting liver cancer cell apoptosis, and suppressing oncogene expression.

[0004] The present invention provides an application of a plant exovesicle preparation in preparing a drug for treating liver cancer. The plant exovesicle preparation comprises Uncaria rhynchophylla exovesicles and tea exovesicles.

[0005] The plant extracellular vesicle preparation provided by the present invention treats liver cancer by inhibiting the proliferation of liver cancer cells, promoting the apoptosis of liver cancer cells, and inhibiting the expression of oncogenes.

[0006] Furthermore, the plant exovesicle preparation is prepared by mixing Uncaria rhynchophylla exovesicles and tea exovesicles in a particle number ratio of 1:1 to 10.

[0007] Furthermore, the Uncaria rhynchophylla exovesicles are obtained by extracting Uncaria rhynchophylla as raw material; and the Tea leaves exovesicles are obtained by extracting Tea leaves as raw material.

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

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

[0010] Furthermore, the drug is used to inhibit oncogenes P13K 、 AKT1 as well as mTOR expression.

[0011] The present invention also provides a method for preparing a plant extracellular vesicle preparation, which comprises the following steps: Preparation of Uncaria rhynchophylla exosomes: Grinding Uncaria rhynchophylla and filtering to obtain the juice to obtain Uncaria rhynchophylla original juice, centrifuging to collect the supernatant, and then extracting the Uncaria rhynchophylla exosomes using a plant tissue exosome extraction kit to obtain Uncaria rhynchophylla exosomes; Preparation of tea exosomes: The tea leaves are crushed and filtered to obtain tea juice, the supernatant is collected by centrifugation, and then the tea exosomes are extracted using a plant tissue exosome extraction kit to obtain tea exosomes; The plant exovesicle preparation is obtained by mixing the exovesicles of Uncaria rhynchophylla and the exovesicles of tea.

[0012] Furthermore, in the preparation process of Uncaria rhynchophylla exosomes, the extraction process using a plant tissue exosome extraction kit is as follows: after obtaining the Uncaria rhynchophylla juice, the supernatant is collected by centrifugation, the collected supernatant is mixed with the Isolation Reagent A reagent in the plant tissue exosome extraction kit, and the mixture is allowed to stand for 10 minutes to 15 minutes, and then the supernatant is collected by centrifugation, the collected supernatant is mixed with the Isolation Reagent B reagent in the plant tissue exosome extraction kit, and the mixture is allowed to stand for 50 minutes to 70 minutes, centrifuged and the precipitate is collected, the precipitate is resuspended with sterile saline and centrifuged, and the supernatant is collected to obtain Uncaria rhynchophylla exosomes.

[0013] Furthermore, in the preparation process of tea exosomes, the extraction process using a plant tissue exosome extraction kit is as follows: after obtaining the tea juice, the supernatant is collected by centrifugation, the collected supernatant is mixed with the Isolation Reagent A reagent in the plant tissue exosome extraction kit, and then allowed to stand for extraction for 10 minutes to 15 minutes, and then centrifuged to collect the supernatant, the collected supernatant is mixed with the Isolation Reagent B reagent in the plant tissue exosome extraction kit, and then allowed to stand for extraction for 50 minutes to 70 minutes, centrifuged and collected the precipitate, resuspended the precipitate with sterile saline and centrifuged, and the supernatant is collected to obtain tea exosomes.

[0014] The present invention also provides a plant exovesicle preparation prepared by the above-mentioned preparation method. In the plant exovesicle preparation, the average particle size of the Uncaria rhynchophylla exovesicles is 35.9 nm, and the average particle size of the tea exovesicles is 125.4 nm.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention extracts and obtains exovesicles from Uncaria rhynchophylla and tea leaves, and mixes them to prepare a plant exovesicle preparation. The plant exovesicle preparation prepared by the present invention has a significant ability to inhibit tumor growth with minimal side effects. The present invention tested the effect of the plant exovesicle preparation on HepG2 cells in an in vitro model of human liver cancer. The results showed that the plant exovesicle preparation can inhibit the growth of liver cancer cells by promoting cell apoptosis and inhibiting the expression of tumor growth genes, providing a new technical approach for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 Transmission electron microscopy images and nanoparticle size images of the Uncaria rhynchophylla exovesicles and tea exovesicles prepared in the present invention; In the figure, A is a transmission electron micrograph of the Uncaria rhynchophylla exovesicles prepared by the present invention; B is a transmission electron micrograph of the tea exocysts prepared in the present invention; C is a graph showing the nanoparticle size of the Uncaria rhynchophylla exovesicles prepared in the present invention; D is a diagram of the nanoparticle size of the tea exosomes prepared by the present invention.

[0018] Figure 2 Effects of the plant extracellular vesicle preparation prepared by the present invention (combination preparation of Uncaria rhynchophylla and tea extracellular vesicles) on the growth of HepG2 cells; In the figure, A shows the growth of HepG2 cells in the control group; B shows the growth of HepG2 cells in the positive group; C shows the effect of EVs (Uncaria rhynchophylla + tea leaves) plant extracellular vesicle preparation on the growth of HepG2 cells; D shows the effect of EVs (ginseng + turmeric) plant extracellular vesicle preparation on the growth of HepG2 cells.

[0019] Figure 3 The results show that HepG2 cells absorb Uncaria rhynchophylla extracellular vesicles and tea extracellular vesicles at different times.

[0020] Figure 4 Effects of plant extracellular vesicle preparations (combination preparations of Uncaria rhynchophylla and tea leaf extracellular vesicles) on apoptosis of HepG2 cells; In the figure, A shows the apoptosis of HepG2 cells in the control group; B shows the apoptosis of HepG2 cells in the positive group; C shows the effect of EVs (Uncaria rhynchophylla + tea leaves) plant extracellular vesicle preparation on HepG2 cell apoptosis; D shows the effect of EVs (ginseng + turmeric) plant extracellular vesicle preparation on HepG2 cell apoptosis; E is the statistical graph of HepG2 cell apoptosis in different groups.

[0021] Figure 5 The qPCR method was used to detect the effect of plant extracellular vesicle preparations (combination preparations of Uncaria rhynchophylla and tea extracellular vesicles) on the expression of tumor-related genes in HepG2 cells. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0023] Example 1: A plant extracellular vesicle preparation, its preparation method and identification.

[0024] 1. Preparation method of Uncaria rhynchophylla extracellular vesicles Take 1 kg of fresh Uncaria rhynchophylla, put it in a sink, rinse it with drinking water, and then rinse it with ultrapure water.

[0025] Cut the washed Uncaria rhynchophylla into pieces, crush them in a juicer, filter them through a 150-mesh nylon mesh, and obtain the juice to obtain the Uncaria rhynchophylla original juice.

[0026] The Uncaria rhynchophylla juice was centrifuged at 6500 g for 10 min at 10°C to obtain the first supernatant; the first supernatant was centrifuged at 10000 g for 20 min at 10°C to obtain the second supernatant; the second supernatant was mixed with extraction reagent A at a volume ratio of 2:1, and then allowed to stand at 4°C for 10 min. After standing, the mixture was centrifuged at 12000 g for 10 min at 10°C to obtain the third supernatant; the third supernatant was mixed with extraction reagent B at a volume ratio of 3:1, and then allowed to stand at 4°C for 1 h. After standing, the mixture was centrifuged at 13500 g for 30 min at 10°C, and the supernatant was discarded to collect the precipitate.

[0027] Sterile physiological saline was added to resuspend the precipitate, and the mixture was centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which was the Uncaria rhynchophylla exovesicles.

[0028] The extraction reagent A is Isolation Reagent A in the Plant Tissue Exosome Extraction Kit (Cat. No. S309) purchased from Shenzhen Shifangjie Technology Co., Ltd. The extraction reagent B is Isolation Reagent B in the Plant Tissue Exosome Extraction Kit (Cat. No. S309) sold by Shenzhen Shifangjie Technology Co., Ltd.

[0029] 2. Identification of Uncaria rhynchophylla extracellular vesicles The extracted extracellular vesicles of Uncaria rhynchophylla were examined by transmission electron microscopy. Figure 1 As shown, the results showed that an obvious cup-shaped double-layer vesicle structure could be observed under transmission electron microscopy.

[0030] The particle size distribution and density of the Uncaria rhynchophylla exosomes were measured by nanoparticle tracking analyzer (NTA). The purity was preliminarily determined based on the peak distribution. The fewer the peaks, the higher the purity. The yield of exosomes was determined by the number of exosomes per milliliter. The greater the number, the higher the yield. Figure 1 As shown in the NTA results, the average particle size of the isolated Uncaria rhynchophylla extracellular vesicles was 35.9 nm and the concentration was 2.68×10 10 particles / mL. This is consistent with the typical particle size range of 30nm to 150nm for extracellular vesicles, and offers high purity and yield.

[0031] 3. Preparation of tea vesicles Take 1 kg of fresh tea leaves, rinse them in a sink with drinking water, and then rinse with ultrapure water. Crush the washed tea leaves in a juicer and filter through a 150-mesh nylon mesh to obtain the tea juice. Centrifuge the tea juice at 6500 g for 10 minutes at 10°C to obtain the first supernatant. Centrifuge the first supernatant at 10000 g for 20 minutes at 10°C to obtain the second supernatant. Mix the second supernatant with extraction reagent A at a volume ratio of 2:1, incubate at 4°C for 10 minutes, and then centrifuge at 12000 g for 10 minutes at 10°C to obtain the third supernatant. Mix the third supernatant with extraction reagent B at a volume ratio of 3:1, incubate at 4°C for 1 hour, and then centrifuge at 13500 g for 30 minutes at 10°C. Discard the supernatant and collect the precipitate. Sterile physiological saline was added to resuspend the precipitate, and the mixture was centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which was the tea leaf exocyst.

[0032] 4. Identification of extracellular vesicles of tea leaves The extracted tea vesicles were examined by transmission electron microscopy. Figure 1 As shown, a clear cup-shaped double-layer vesicle structure can be observed under a transmission electron microscope.

[0033] The particle size distribution and density of tea exosome particles were measured by nanoparticle tracking analyzer (NTA). The purity was preliminarily determined based on the peak distribution. The fewer the peaks, the higher the purity. The yield of exosomes was determined by the number of exosomes per milliliter. The greater the number, the higher the yield. Figure 1 The NTA results showed that the average particle size of the isolated tea extracellular vesicles was 125.4 nm and the concentration was 1.06×10 10 particles / mL. This is consistent with the typical particle size range of 30nm to 150nm for extracellular vesicles, and offers high purity and yield.

[0034] 5. Preparation of plant extracellular vesicle preparations The prepared Uncaria rhynchophylla exosomes and tea leaves exosomes were mixed in a particle ratio of 1:4 to prepare a plant exosome preparation, which was recorded as EVs (Uncaria rhynchophylla + tea leaves).

[0035] The mixture was filtered using 0.45 μm and 0.22 μm syringe filters (Servicebio) in sequence, and the protein concentration of the mixed preparation was determined using a BCA protein concentration assay kit (Beyotime Biotechnology Co., Ltd.).

[0036] Example 2: Application of plant extracellular vesicle preparations in inhibiting the proliferation of liver cancer cells and promoting the apoptosis of liver cancer cells.

[0037] 1. Effects of plant extracellular vesicle preparations on the growth of liver cancer cells 1. Experimental Materials Cells: Human hepatocellular carcinoma cell HepG2.

[0038] Complete cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0039] 2. Experimental methods (1) Grouping The animals were divided into four groups: control group, positive group (10 μM cisplatin), EVs (Uncaria rhynchophylla + tea leaves), and EVs (Ginseng + Curcuma longa). The treatments for each group were as follows:

[0040] Control group: Normally grown cells without treatment.

[0041] 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.

[0042] EVs (Uncaria rhynchophylla + tea leaves): Add the extracellular vesicle combination preparation prepared in Example 1 to each well according to the protein determination concentration and dilute with complete cell culture medium to a final concentration of 100 μg / mL; EVs (ginseng + turmeric): Add EVs (ginseng + turmeric) to each well according to the protein concentration determined and dilute with complete cell culture medium to a final concentration of 100 μg / mL.

[0043] The preparation process of EVs (ginseng + turmeric) is as follows: Take 1kg of fresh ginseng, put it in a sink, rinse it with drinking water, and then rinse it with ultrapure water.

[0044] Washed ginseng was cut into pieces, blended and crushed in a juicer, and filtered through a 150-mesh nylon mesh to obtain the ginseng plant juice. The ginseng plant juice was centrifuged at 6500 g for 10 minutes at 10°C to obtain the first supernatant. The first supernatant was centrifuged at 10000 g for 20 minutes at 10°C to obtain the second supernatant. The second supernatant was mixed with extraction reagent A at a volume ratio of 2:1, then allowed to stand at 4°C for 10 minutes. After the standstill, the mixture was centrifuged at 12000 g for 10 minutes at 10°C to obtain the third supernatant. The third supernatant was mixed with extraction reagent B at a volume ratio of 3:1, allowed to stand at 4°C for 1 hour. After the standstill, the mixture was centrifuged at 13500 g for 30 minutes at 10°C. The supernatant was discarded and the precipitate was collected. The precipitate was resuspended in sterile saline and centrifuged at 2000 g for 10 minutes to obtain the fourth supernatant, which is the ginseng exovesicles.

[0045] Take 1 kg of fresh turmeric, put it in the sink and rinse it with drinking water, then rinse it with ultrapure water.

[0046] Wash and chop turmeric, place it in a juicer, blend and crush it, and filter it through a 150-mesh nylon mesh to obtain the turmeric plant juice. Centrifuge the turmeric plant juice at 6500 g for 10 minutes at 10°C to obtain the first supernatant. Centrifuge the first supernatant at 10,000 g for 20 minutes at 10°C to obtain the second supernatant. Mix the second supernatant with extraction reagent A at a volume ratio of 2:1, let it stand at 4°C for 10 minutes, and then centrifuge at 12,000 g for 10 minutes at 10°C to obtain the third supernatant. Mix the third supernatant with extraction reagent B at a volume ratio of 3:1, let it stand at 4°C for 1 hour, and then centrifuge at 13,500 g for 30 minutes at 10°C. Discard the supernatant and collect the precipitate. Resuspend the precipitate in sterile saline and centrifuge at 2000 g for 10 minutes to obtain the fourth supernatant, which is the turmeric exovesicles.

[0047] The prepared ginseng extracellular vesicles and turmeric extracellular vesicles were mixed in a particle ratio of 1:5 to obtain EVs (ginseng + turmeric).

[0048] (2) Experimental steps Human hepatoma cell line HepG2 was collected at the logarithmic phase and the cells were plated at 2×10 5 After the cells adhered to the wall, the corresponding substances were added according to the above grouping treatment methods. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cell growth status was observed under a microscope and photographed.

[0049] (3) Experimental results like Figure 2 As shown, compared with the control group, the cells in the positive group showed a significant deterioration in state, with no basic cell morphology and almost all cell death. The EVs (Uncaria rhynchophylla + tea) group had slightly more cells than the positive group, but the cell state was similar to that of the positive group, with no basic cell morphology and most cells dead. Ginseng and turmeric, also combined preparations, had no significant effect on the state of HepG2 cells. This shows that the plant extracellular vesicle preparation prepared by the present invention can specifically cause HepG2 cell death.

[0050] 2. Uptake of Uncaria rhynchophylla and tea leaf extracellular vesicles by HepG2 cells 1. Experimental Materials Exovesicles: Uncaria rhynchophylla exovesicles and tea exovesicles prepared in Example 1.

[0051] Cells: Human hepatocellular carcinoma cell HepG2.

[0052] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0053] Reagents: DiO Cell Membrane Green Fluorescent Staining Kit and 1× sterile PBS were purchased from Servicebio. The DiO Cell Membrane Green Fluorescent Staining Kit includes Dio Cell Membrane Green Fluorescent Probe and staining buffer.

[0054] 2. Experimental methods Human hepatoma cell line HepG2 was collected at the logarithmic phase and the cells were plated at 2×10 5100 μL of each of the Uncaria rhynchophylla and tea leaf extravesicles were centrifuged at 13,500 g for 30 minutes, and the precipitates were collected and resuspended in 100 μL of the Dio working solution. The resuspended samples were incubated at 37°C in the dark for 30 minutes and centrifuged at 13,500 g for 30 minutes to remove excess dye. The precipitates of the Uncaria rhynchophylla and tea leaf extravesicles were collected and resuspended in 1× sterile PBS to obtain Dio-labeled Uncaria rhynchophylla and tea leaf extravesicles. The Dio-labeled Uncaria rhynchophylla and tea leaf extravesicles were co-cultured with cells for 24 and 48 hours, respectively, and the absorption was observed and recorded using a fluorescence microscope.

[0055] 3. Experimental results like Figure 3 As shown, when DiO green fluorescent-labeled exovesicles were co-incubated with cells, both the Uncaria rhynchophylla and tea leaves exovesicle groups showed green fluorescence at 24 hours, with an increased number of green fluorescent cells at 48 hours. This indicates that HepG2 cells can absorb both Uncaria rhynchophylla and tea leaves exovesicles, and that the uptake rates of both exovesicles are similar, starting at 24 hours and increasing at 48 hours.

[0056] 3. Effect of extracellular vesicle combination preparation on apoptosis of HepG2 cells 1. Experimental Materials Exovesicles: Uncaria rhynchophylla exovesicles and tea exovesicles prepared in Example 1.

[0057] Cells: Human hepatocellular carcinoma cell HepG2.

[0058] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0059] Reagents: Hoechst 33258 was purchased from Shenggong Bioengineering Co., Ltd., 1× sterile PBS, and tissue fixative were purchased from Servicebio.

[0060] 2. Experimental methods (1) Grouping The cells were divided into a control group, a positive group (final concentration of 10 μM cisplatin), EVs (Uncaria rhynchophylla + tea leaves), and EVs (Ginseng + Curcuma longa). The final concentrations of EVs (Uncaria rhynchophylla + tea leaves) and EVs (Ginseng + Curcuma longa) were both 100 μg / mL.

[0061] (2) Experimental steps HepG2 hepatocellular carcinoma cells were collected and plated at 2×105 Plate 100 μL of cells / well in a 24-well plate and place in a cell culture incubator at 37°C, 5% CO2. After the cells adhere, add different reagents according to grouping and continue incubation in a cell culture incubator at 37°C, 5% CO2. Aspirate the culture medium and add 200 μL of tissue fixative to each well for 10 minutes. Remove the fixative, wash once with 1x sterile PBS, aspirate the liquid, and add 500 μL of Hoechst 33258 staining solution (10 μg / mL) for 10 minutes. Remove the staining solution, wash once with 1x sterile PBS, and aspirate the liquid. Observe and record cell apoptosis under a fluorescence microscope.

[0062] 3. Experimental results like Figure 4 As shown, some cells in the control group were stained with a blue fluorescent probe, with relatively weak fluorescence intensity. The fluorescence intensity of the 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 rhynchophylla + tea) group were both stronger than those in the control group, with no significant difference between the positive group and the EVs (Uncaria rhynchophylla + tea) group. This suggests that plant extracellular vesicles can specifically promote apoptosis in HepG2 cells, leading to cell death and achieving anti-cancer effects.

[0063] Effects of plant extracellular vesicle preparations on tumor pathways in HepG2 cells 1. Experimental Materials Cells: Human hepatocellular carcinoma cell HepG2.

[0064] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0065] 2. Experimental methods (1) Grouping The cells were divided into three groups: blank group, positive control group (final concentration of 10 μM cisplatin) and EVs (plant extracellular vesicle preparation with a final concentration of 100 μg / mL).

[0066] (2) Experimental steps HepG2 human hepatoma cells were collected at the logarithmic phase and the cells were plated at 4 × 10 5 The cells were plated into 12-well plates and placed in a cell culture incubator. After the cells adhered to the wall, the corresponding reagents were added according to the grouping and continued to be incubated in a cell culture incubator at 37°C and 5% CO2 for 48 hours. The cells were collected and tested according to the instructions of the RNA extraction kit, reverse transcription kit and qPCR test. P13K 、 AKT1 as well as mTOR The primer sequences are shown in Table 1.

[0067] Table 1 Primer sequences (3) Data processing Statistical analysis was performed using Prism 6.0 (GraphPad) software. Two-way ANOVA was used to compare multiple groups. The results were expressed as mean ± standard deviation (x ± s).

[0068] 3. Experimental results like Figure 5 As shown in the results, compared to the control group, both the positive group and EVs (Uncaria rhynchophylla + tea leaves) inhibited the expression of P13K, AKT1, and mTOR at the mRNA level, with no significant differences between the two groups. This suggests that Uncaria rhynchophylla / tea leaves EVs can achieve anti-cancer effects by inhibiting the expression of tumorigenic genes and promoting tumor cell apoptosis, with efficacy comparable to that of cisplatin.

[0069] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0070] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. Application of a plant extracellular vesicle preparation in the preparation of a drug for treating liver cancer, characterized in that: The plant extracellular vesicle preparation includes Uncaria rhynchophylla extracellular vesicles and tea leaf extracellular vesicles.

2. The use of the plant extracellular vesicle preparation according to claim 1 in preparing a drug for treating liver cancer, characterized in that: The plant exovesicle preparation is prepared by mixing Uncaria rhynchophylla exovesicles and tea exovesicles in a particle number ratio of 1:1-10.

3. The use of the plant extracellular vesicle preparation according to claim 2 in the preparation of a drug for treating liver cancer, characterized in that: The Uncaria rhynchophylla exovesicles are obtained by extracting Uncaria rhynchophylla as raw material; and the Tea leaves exovesicles are obtained by extracting tea leaves as raw material.

4. Use of the plant extracellular vesicle preparation according to claim 1 in preparing a drug for treating liver cancer, characterized in that: The drug is used for inhibiting the proliferation of liver cancer cells.

5. Use of the plant extracellular vesicle preparation according to claim 1 in preparing a drug for treating liver cancer, characterized in that: The medicine is used for promoting apoptosis of liver cancer cells.

6. Use of the plant extracellular vesicle preparation according to claim 1 in preparing a drug for treating liver cancer, characterized in that: The drug is used to inhibit oncogenes P13K 、 AKT1 as well as mTOR expression.

7. A method for preparing a plant extracellular vesicle preparation, characterized in that: The plant extracellular vesicle preparation is the plant extracellular vesicle preparation according to claim 1, and the preparation of the plant extracellular vesicle preparation comprises the following steps: Preparation of Uncaria rhynchophylla exosomes: Grinding Uncaria rhynchophylla and filtering to obtain the juice to obtain Uncaria rhynchophylla original juice, centrifuging to collect the supernatant, and then extracting the Uncaria rhynchophylla exosomes using a plant tissue exosome extraction kit to obtain Uncaria rhynchophylla exosomes; Preparation of tea exosomes: The tea leaves are crushed and filtered to obtain tea juice, the supernatant is collected by centrifugation, and then the tea exosomes are extracted using a plant tissue exosome extraction kit to obtain tea exosomes; The plant exovesicle preparation is obtained by mixing the exovesicles of Uncaria rhynchophylla and the exovesicles of tea.

8. The method for preparing the plant extracellular vesicle preparation according to claim 7, characterized in that: In the preparation process of Uncaria rhynchophylla exosomes, the extraction process using the plant tissue exosome extraction kit is as follows: the collected supernatant is mixed with the Isolation Reagent A reagent in the plant tissue exosome extraction kit and then allowed to stand for extraction for 10 minutes to 15 minutes, and then centrifuged to collect the supernatant, and the collected supernatant is mixed with the Isolation Reagent B reagent in the plant tissue exosome extraction kit and then allowed to stand for extraction for 50 minutes to 70 minutes, centrifuged and collected the precipitate, resuspended the precipitate with sterile saline and centrifuged, and the supernatant is collected to obtain Uncaria rhynchophylla exosomes.

9. A plant extracellular vesicle preparation obtained by the preparation method according to any one of claims 7 to 8, characterized in that: The average particle size of Uncaria rhynchophylla exosomes in the plant exosome preparation is 35.9 nm, and the average particle size of tea exosomes is 125.4 nm.

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