Plant external vesicle combined preparation for treating non-small cell lung cancer as well as preparation method and application of plant external vesicle combined preparation

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a plant exovesicle combined preparation for treating non-small cell lung cancer and a preparation method and application thereof, and the plant exovesicle combined preparation comprises ginseng exovesicles and turmeric exovesicles. The plant external vesicle combined preparation provided by the invention is used for inhibiting non-small cell lung cancer cells and promoting apoptosis of the non-small cell lung cancer cells, and can be used for preparing medicines for treating the non-small cell lung cancer.

Description

Technical Field

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

[0002] Lung cancer originates from the trachea, bronchial mucosa, or glands and is a common primary lung malignancy. It can be divided into two major categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for over 80% to 85% of lung cancer cases, of which approximately 40% are adenocarcinomas, 25% to 30% are squamous cell carcinomas, 10% to 15% are large cell carcinomas, and 20% are other types. Cancer has a high mortality rate and is difficult to detect in its early stages, often diagnosed in the late stages. The overall five-year survival rate for patients with advanced lung cancer is around 20%. Existing cancer treatments mostly rely on chemotherapy and radiotherapy, but these have significant side effects, severely impacting patients' quality of life during treatment. Therefore, there is an urgent need to explore new approaches to treat lung cancer. Summary of the Invention

[0003] To develop a new approach for treating lung cancer, the present invention provides a plant exosome combination formulation for treating non-small cell lung cancer, as well as its preparation method and application. The plant exosome combination formulation provided by the present invention is used to inhibit non-small cell lung cancer cells and promote their apoptosis, and can be used to prepare a drug for treating non-small cell lung cancer.

[0004] The present invention provides an application of a plant exosome combination preparation in preparing a drug for treating non-small cell lung cancer. The plant exosome combination preparation comprises ginseng exosomes and turmeric exosomes.

[0005] The plant extracellular vesicle combination preparation provided by the present invention is used for inhibiting non-small cell lung cancer cells and promoting their apoptosis, and further for preparing a drug for treating non-small cell lung cancer.

[0006] Furthermore, the plant exosome combination preparation is prepared by mixing ginseng exosomes and turmeric exosomes in a particle number ratio of 1:1-10 to 1:1-10.

[0007] Furthermore, the ginseng exosomes are extracted from ginseng using a plant tissue exosome extraction kit; and the turmeric exosomes are extracted from turmeric using a plant tissue exosome extraction kit.

[0008] Furthermore, the drug is used to inhibit the proliferation of human non-small cell lung cancer cells.

[0009] Furthermore, the drug is used to promote apoptosis of human non-small cell lung cancer cells.

[0010] Furthermore, the drug is used to inhibit oncogenes P13K 、 AKT1as well as mTOR expression.

[0011] The present invention also provides a method for preparing a plant extracellular vesicle combination preparation, wherein the preparation of the plant extracellular vesicle combination preparation comprises the following steps: Preparation of ginseng exosomes: Ginseng exosomes were extracted from ginseng using a plant tissue exosome extraction kit; Preparation of turmeric exosomes: Extracting turmeric exosomes from turmeric using a plant tissue exosome extraction kit; The plant exovesicle combination preparation is obtained by mixing ginseng exovesicles and turmeric exovesicles.

[0012] Furthermore, the ratio of the number of mixed particles of ginseng exosomes and turmeric exosomes is 1:1-10.

[0013] Furthermore, the ginseng exosome extraction process is as follows: after crushing ginseng, the ginseng plant juice is collected, the supernatant is collected by centrifugation, and the Isolation Reagent A reagent and the Isolation Reagent B reagent in the plant tissue exosome extraction kit are used for extraction according to the instructions to collect the ginseng exosomes.

[0014] Furthermore, the turmeric exosome extraction process is as follows: after crushing turmeric, the turmeric plant juice is collected, the supernatant is collected by centrifugation, and the Isolation Reagent A reagent and the Isolation Reagent B reagent in the plant tissue exosome extraction kit are used for extraction according to the instructions to collect the turmeric exosomes.

[0015] The present invention also provides a plant exosome combination preparation prepared by the preparation method, wherein the average particle size of the ginseng exosomes in the plant exosome combination preparation is 182.7 nm, and the average particle size of the turmeric exosomes is 127.8 nm.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention achieves the purpose of treating non-small cell lung cancer by inhibiting the growth of non-small cell lung cancer cells, promoting the apoptosis of non-small cell lung cancer cells, and inhibiting the growth of non-small cell lung tumors through ginseng exosomes and turmeric exosomes.

[0017] The plant exosome combination preparation prepared by the present invention includes ginseng exosomes and turmeric exosomes, which can provide a stable source of exosomes for the treatment of non-small cell lung cancer. Ginseng exosomes and turmeric exosomes carry multiple bioactive molecules, transmit signals and participate in anti-cancer.

[0018] The traditional Chinese medicine raw materials ginseng and turmeric are selected as plant-derived medicines in the present invention. The extracted exosomes show obvious tumor inhibition ability with small side effects, providing a new treatment method for non-small cell lung cancer treatment and cancer prevention, and are of great significance to the scientific, rational and sustainable development and utilization of ginseng and turmeric resources.

[0019] This invention, based on an in vitro model of non-small cell lung cancer (NSCLC) cells, examined the effects of a plant exovesicle combination formulation containing ginseng exovesicles and turmeric exovesicles on A549 cell apoptosis. The results showed that the plant exovesicle combination formulation promoted A549 cell apoptosis and inhibited tumor gene expression, providing a new technical approach for the treatment of NSCLC. The extraction method for the plant exovesicle combination formulation of the invention offers the advantages of high efficiency and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 The nanoparticle size diagram and transmission electron microscopy diagram of the combined preparation of ginseng and turmeric exosomes of the present invention; In the figure, A is a transmission electron micrograph of ginseng extracellular vesicles; B is a transmission electron micrograph of turmeric extracellular vesicles; C is the nanoparticle size diagram of ginseng extracellular vesicles; D is the nanoparticle size diagram of turmeric extracellular vesicles.

[0022] Figure 2 The effect of the combined preparation of ginseng and turmeric extracellular vesicles of the present invention on the growth of A549 cells; In the figure, A shows the growth of A549 cells in the control group; B is the effect of ginseng group on the growth of A549 cells; C shows the effect of turmeric group on the growth of A549 cells; D shows the growth of A549 cells in the positive group (10 μM cisplatin); E is the effect of EVs (ginseng + turmeric) on the growth of A549 cells; F shows the effect of EVs (Uncaria rhynchophylla + tea leaves) on the growth of A549 cells.

[0023] Figure 3 The present invention detects the absorption of ginseng extracellular vesicles and turmeric extracellular vesicles by A549 cells.

[0024] Figure 4 The present invention detects the effect of the combined preparation of ginseng and turmeric extracellular vesicles on the apoptosis of A549 cells; In the figure, A shows the apoptosis of A549 cells in the control group; B is the effect of ginseng group on apoptosis of A549 cells; C shows the effect of turmeric group on apoptosis of A549 cells; D shows the apoptosis of A549 cells in the positive group (10 μM cisplatin); E is the effect of EVs (ginseng + turmeric) on apoptosis of A549 cells; F shows the effect of EVs (Uncaria rhynchophylla + tea leaves) on the apoptosis of A549 cells.

[0025] Figure 5 The present invention uses qPCR to detect the expression of tumor-related genes in A549 cells by the combined preparation of ginseng and turmeric extracellular vesicles. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1: Preparation and identification of a plant extracellular vesicle combination preparation for treating non-small cell lung cancer.

[0028] 1. Preparation of Ginseng Exovesicles, Turmeric Exovesicles, and Plant Exovesicle Combination Preparations 1. Preparation steps of ginseng extracellular vesicles Take 1kg of fresh ginseng, put it in a sink, rinse it with drinking water, and then rinse it with ultrapure water.

[0029] The washed ginseng was cut into pieces, placed in a juicer for mixing and crushing, filtered through a 150-mesh nylon mesh, and the juice was taken to obtain ginseng plant juice.

[0030] Centrifuge the ginseng plant 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 2:1 volume ratio, 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 3:1 volume ratio, 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.

[0031] The precipitate was resuspended with sterile physiological saline and centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which was the ginseng exocyst.

[0032] The extraction reagent A is the Isolation Reagent A in the Plant Tissue Exosome Extraction Kit (Cat. No. S309) sold by Shenzhen Shifangjie Technology Co., Ltd. The extraction reagent B is the Isolation Reagent B in the Plant Tissue Exosome Extraction Kit (Cat. No. S309) sold by Shenzhen Shifangjie Technology Co., Ltd.

[0033] 2. Preparation steps of turmeric extracellular vesicles Take 1 kg of fresh turmeric, put it in the sink and rinse it with drinking water, then rinse it with ultrapure water.

[0034] Cut the washed turmeric into pieces, place in a juicer for mixing and crushing, filter through a 150-mesh nylon mesh, and extract the juice to obtain turmeric plant juice.

[0035] Centrifuge the turmeric plant 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 2:1 volume ratio, 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 3:1 volume ratio, 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.

[0036] The precipitate was resuspended with sterile saline and centrifuged at 2000 g for 10 min to obtain the fourth supernatant, which was the turmeric exosomes.

[0037] The extraction reagent A is the Isolation Reagent A in the Plant Tissue Exosome Extraction Kit (Cat. No. S309) sold by Shenzhen Shifangjie Technology Co., Ltd. The extraction reagent B is the Isolation Reagent B in the Plant Tissue Exosome Extraction Kit (Cat. No. S309) sold by Shenzhen Shifangjie Technology Co., Ltd.

[0038] 3. Plant extracellular vesicle combination preparations The prepared ginseng extracellular vesicles and turmeric extracellular vesicles were mixed in a particle ratio of 1:5 to prepare a plant extracellular vesicle combination preparation, which was recorded as EVs (ginseng + turmeric).

[0039] 2. Identification of Ginseng and Turmeric Extracellular Vesicles 1. Identification of ginseng extracellular vesicles The extracted ginseng extracellular vesicles 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.

[0040] The particle size distribution and density of ginseng 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 As shown in the figure, the NTA results showed that the average particle size of the isolated ginseng extracellular vesicles was 182.7 nm and the concentration was 1.82 × 10 10 particles / mL. This is consistent with the particle size range of extracellular vesicles and offers high purity and yield.

[0041] 2. Identification of Curcuma Extracellular Vesicles The extracted turmeric extracellular vesicles were examined by transmission electron microscopy. Figure 1 As shown, the results show that a clear cup-shaped double-layer vesicle structure can be observed under a transmission electron microscope. The particle size distribution and density of the turmeric exosome particles were measured by a 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. The results are shown in Figure 2. Figure 1 As shown in the NTA results, the average particle size of the isolated turmeric extracellular vesicles was 127.8 nm and the concentration was 2.09×10 10 particles / mL.

[0042] The extracellular vesicle combination preparation was filtered using 0.45 μm and 0.22 μm syringe filters (Servicebio), and the protein concentration of the extracellular vesicle combination preparation was determined using a BCA protein concentration assay kit (Beyotime Biotechnology Co., Ltd.).

[0043] Example 2: Use of the plant extracellular vesicle combination preparation prepared in Example 1 in the treatment of non-small cell lung cancer.

[0044] 1. Effects of plant extracellular vesicle combination preparations on A549 cell growth 1. Experimental Materials The combined preparation of ginseng exosomes, turmeric exosomes and plant exosomes prepared in Example 1.

[0045] Cells: Human non-small cell lung cancer A549 cells.

[0046] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0047] 2. Experimental methods (1) Experimental groups Six groups were divided into the control group, the ginseng group (ginseng extracellular vesicles), the turmeric group (turmeric extracellular vesicles), the positive group (10 μM cisplatin), the EVs (ginseng + turmeric), and the EVs (Uncaria rhynchophylla + tea leaves). The reagents and dosages used in each group are as follows:

[0048] Control group: only normally growing cells without treatment; Ginseng group: Ginseng extracellular vesicles, according to the protein concentration determination, ginseng extracellular vesicles prepared in Example 1 were added to each well and diluted with cell culture medium to a final concentration of 100 μg / mL; Turmeric group: Turmeric exosomes, according to the protein determination concentration, were added to each well of the turmeric exosomes prepared in Example 1 and diluted with cell culture medium to a final concentration of 100 μg / mL; Positive group: 10 μM cisplatin, cisplatin was added to the cells and diluted with cell culture medium to a final concentration of 10 μM; EVs (ginseng + turmeric) group: add the extracellular vesicle combination preparation prepared in Example 1 to each well according to the protein determination concentration and dilute with cell culture medium to a final concentration of 100 μg / mL; EVs (Uncaria rhynchophylla + tea leaves) group: EVs (Uncaria rhynchophylla + tea leaves) were added to each well according to the protein concentration and diluted with cell culture medium to a final concentration of 100 μg / mL.

[0049] The preparation process of EVs (Uncaria rhynchophylla + tea leaves) is as follows: Take 1 kg of fresh Uncaria rhynchophylla, put it in the sink, rinse it with drinking water, and then rinse it with ultrapure water.

[0050] Washed Uncaria rhynchophylla was cut into pieces, crushed in a juicer, and filtered through a 150-mesh nylon mesh to obtain the original Uncaria rhynchophylla juice. The original Uncaria rhynchophylla 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, incubated at 4°C for 10 minutes, and then 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, incubated at 4°C for 1 hour, and then 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 Uncaria rhynchophylla exovesicles.

[0051] 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.

[0052] EVs (Uncaria rhynchophylla + tea leaves) were obtained by mixing Uncaria rhynchophylla extracellular vesicles and tea leaves extracellular vesicles in a particle ratio of 1:4.

[0053] (2) Experimental steps A549 cells were collected at the logarithmic phase and plated at 2×10 5 After the cells adhered to the wall, the corresponding reagents were added according to the experimental groups mentioned above and 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.

[0054] 0. Experimental Results like Figure 2As shown, the results showed that compared with the control group, cells in all other groups exhibited varying degrees of changes. The EVs (Uncaria rhynchophylla + tea leaves) and ginseng groups were less affected. The EVs (Uncaria rhynchophylla + tea leaves) group and the control group displayed similar cell states, indicating that EVs (Uncaria rhynchophylla + tea leaves) had no significant effect on A549 cells. Cells in the ginseng group became elongated and began to deteriorate, while cells in the turmeric group further elongated and rounded. Cells in the EVs (ginseng + turmeric) group and the positive group almost lost their morphology and displayed a high level of cell debris, indicating a high rate of A549 cell death. These results indicate that treatment with turmeric, ginseng, and a combination of plant EVs all affected lung cancer cell growth, and the combined plant EVs were more effective than either turmeric or ginseng alone. The combination of Uncaria rhynchophylla and tea leaves had virtually no effect on A549 cells, demonstrating that the combined ginseng and turmeric plant EVs formulations are specifically targeted to A549 cells.

[0055] 2. Uptake of Ginseng and Turmeric Extracellular Vesicles by A549 Cells 1. Experimental Materials Ginseng exosomes and turmeric exosomes prepared in Example 1.

[0056] Cells: Human non-small cell lung cancer A549 cells.

[0057] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0058] 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.

[0059] 2. Experimental methods A549 cells were collected at the logarithmic phase and plated at 2×10 5Each well was plated into a 24-well plate and placed in a cell culture incubator. Dio cell membrane green fluorescent probe was diluted with staining buffer at a volume ratio of 1:250 to prepare Dio staining working solution. 100 μL of ginseng and 100 μL of turmeric exovesicles were centrifuged at 13,500 g for 30 minutes to obtain ginseng and turmeric exovesicle pellets, respectively. The pellets were resuspended in 100 μL of Dio staining working solution, incubated at 37°C in the dark for 30 minutes, and centrifuged at 13,500 g for 30 minutes to remove excess dye. The exovesicle pellets were collected and resuspended in 1× sterile PBS to obtain Dio-labeled ginseng and turmeric exovesicle pellets. The Dio-labeled ginseng and turmeric exovesicle pellets were co-cultured with A549 cells for 24 and 48 hours, respectively, and the uptake was observed and recorded using a fluorescence microscope.

[0060] 1. Experimental results like Figure 3 As shown, after 24 hours of co-incubation, all groups began to show fluorescence, indicating that A549 cells began to absorb the exovesicles. After 48 hours of co-incubation, all groups showed a large amount of fluorescence, indicating that the ginseng exovesicles and turmeric exovesicles were successfully absorbed by the recipient A549 cells and most of them were present in the cytoplasm.

[0061] Effects of extracellular vesicle combination preparations on apoptosis of A549 cells 1. Experimental Materials The combined preparation of ginseng exosomes, turmeric exosomes and plant exosomes prepared in Example 1.

[0062] Cells: Human non-small cell lung cancer A549 cells.

[0063] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0064] Reagents: Hoechst 33258 was purchased from Shenggong Bioengineering Co., Ltd., 1× sterile PBS, and tissue fixative were purchased from Servicebio.

[0065] 2. Experimental methods (1) Experimental groups Six groups were divided into treatment groups: control group, ginseng group (ginseng extracellular vesicles), turmeric group (turmeric extracellular vesicles), positive group (10 μM cisplatin), EVs (ginseng + turmeric), and EVs (Uncaria rhynchophylla + tea leaves). The reagents and dosages used in each treatment group are as follows:

[0066] Control group: only normally growing cells without treatment; Ginseng group: Ginseng extracellular vesicles, according to the protein concentration determination, ginseng extracellular vesicles prepared in Example 1 were added to each well and diluted with cell culture medium to a final concentration of 100 μg / mL; Turmeric group: Turmeric exosomes, according to the protein assay concentration, were added to each well and diluted with cell culture medium to a final concentration of 100 μg / mL; Positive group: 10 μM cisplatin, cisplatin was added to the cells and diluted with cell culture medium to make the final concentration 10 μM; EVs (ginseng + turmeric) group: add the extracellular vesicle combination preparation prepared in Example 1 to each well according to the protein determination concentration and dilute with cell culture medium to a final concentration of 100 μg / mL; EVs (Uncaria rhynchophylla + tea leaves) group: EVs (Uncaria rhynchophylla + tea leaves) were added to each well according to the protein concentration and diluted with cell culture medium to a final concentration of 100 μg / mL.

[0067] (2) Experimental steps The cells in the logarithmic phase were collected and plated at 2×10 5 Plate 100 μL of cells / well into a 24-well plate and place in a cell culture incubator. After the cells adhere, add the corresponding reagents according to the experimental groupings described above and continue incubation in a 37°C, 5% CO2 cell culture incubator. Aspirate the culture medium and add 200 μL of tissue fixative to each well for 10 minutes. Remove the fixative, rinse 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.

[0068] 3. Experimental results like Figure 4 As shown, under a fluorescence microscope, the blue fluorescence intensity of each group was ranked as follows: positive group > EVs (ginseng + turmeric) > turmeric group > ginseng group = EVs (Uncaria rhynchophylla + tea) = control group, indicating that cisplatin, plant extracellular vesicle combination preparations and turmeric extracellular vesicles all significantly promoted the apoptosis of A549 cells, and the pro-apoptotic effect of the plant extracellular vesicle combination preparation was stronger than the effect of the extracellular vesicle alone, and the Uncaria rhynchophylla and tea extracellular vesicles, which were also combination preparations, had no obvious effect on the apoptosis of A549, indicating that the combination preparation of ginseng and turmeric extracellular vesicles can specifically regulate the apoptosis of A549 cells.

[0069] Effects of extracellular vesicle combination preparations on tumor pathways in A549 cells 1. Experimental Materials Example 1 Preparation of plant extracellular vesicle combination preparations.

[0070] Cells: Human non-small cell lung cancer A549 cells.

[0071] Cell culture medium: DMEM basal medium (Servicebio) containing 10% fetal bovine serum (Sunview) and 1% penicillin-streptomycin mixture (Servicebio).

[0072] Reagents: RNA extraction kit and reverse transcription kit were purchased from Servicebio, and qPCR reagents were purchased from Sunview.

[0073] 2. Experimental methods (1) Experimental groups The cells were divided into three groups: control group, positive group (10 μM cisplatin) and EVs (ginseng + turmeric).

[0074] The reagents and dosages used in each group are as follows: Control group: Normally grown cells without treatment.

[0075] Positive group: cisplatin was added to the cells and diluted with cell culture medium to a final concentration of 10 μM; EVs (ginseng + turmeric) group: The extracellular vesicle combination preparation prepared in Example 1 was added to each well according to the protein determination concentration and diluted with cell culture medium to a final concentration of 100 μg / mL.

[0076] (2) Experimental steps A549 cells were collected at the logarithmic phase and plated at 4×10 5 After A549 cells adhered to the wall, the corresponding reagents were added according to the grouping and incubated in the incubator for 48 hours. The cells were collected and tested according to the instructions of RNA extraction kit, reverse transcription kit and qPCR detection. P13K 、 AKT1 as well as mTOR The primer sequences are shown in Table 1.

[0077] 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).

[0078] 3. Experimental results like Figure 5 As shown, both the positive group and the EVs (ginseng + turmeric) group significantly inhibited tumor-related pathways P13K 、 AKT1 and mTORIt can be seen that the EVs (ginseng + turmeric) group and cisplatin achieve anti-tumor effects by inhibiting the growth and proliferation of tumor cells.

[0079] The present invention prepares ginseng and turmeric exovesicles and explores their anti-tumor effects, providing not only a method for preparing plant exovesicles but also a new approach to anti-cancer research. The findings of this invention will facilitate the scientific, comprehensive, and sustainable development and utilization of ginseng and turmeric resources, and also provide support and assistance for the subsequent development of new drugs for the treatment of non-small cell lung cancer, such as new arthritis drugs.

[0080] 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.

[0081] 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 combination preparation in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The plant exosome combination preparation includes ginseng exosomes and turmeric exosomes.

2. Use of the plant extracellular vesicle combination preparation according to claim 1 in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The plant exosome combination preparation is prepared by mixing ginseng exosomes and turmeric exosomes in a particle number ratio of 1:1 to 10.

3. Use of the plant extracellular vesicle combination preparation according to claim 2 in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The ginseng exosomes are extracted from ginseng using a plant tissue exosome extraction kit; the turmeric exosomes are extracted from turmeric using a plant tissue exosome extraction kit.

4. Use of the plant extracellular vesicle combination preparation according to claim 1 in preparing a drug for treating non-small cell lung cancer, characterized in that: The drug is used for inhibiting the proliferation of human non-small cell lung cancer cells.

5. Use of the plant extracellular vesicle combination preparation according to claim 1 in preparing a drug for treating non-small cell lung cancer, characterized in that: The drug is used for promoting apoptosis of human non-small cell lung cancer cells.

6. Use of the plant extracellular vesicle combination preparation according to claim 1 in preparing a drug for treating non-small cell lung 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 combination preparation, characterized in that: The plant extracellular vesicle combination preparation is the plant extracellular vesicle combination preparation according to claim 1; The preparation of the plant extracellular vesicle combination preparation comprises the following steps: Preparation of ginseng exosomes: Ginseng exosomes were extracted from ginseng using a plant tissue exosome extraction kit; Preparation of turmeric exosomes: Extracting turmeric exosomes from turmeric using a plant tissue exosome extraction kit; The plant exovesicle combination preparation is obtained by mixing ginseng exovesicles and turmeric exovesicles.

8. The method for preparing the plant extracellular vesicle combination preparation according to claim 7, characterized in that: The extraction process of ginseng exosomes is as follows: after crushing ginseng, collect the ginseng plant juice, collect the supernatant by centrifugation, and use the Isolation Reagent A reagent and Isolation Reagent B reagent in the plant tissue exosome extraction kit to extract according to the instructions to collect ginseng exosomes.

9. A plant extracellular vesicle combination preparation obtained by the preparation method according to any one of claims 7 to 8, characterized in that: The average particle size of the ginseng exosomes in the plant exosome combination preparation is 182.7 nm, and the average particle size of the turmeric exosomes is 127.8 nm.

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