Codonopsis tangshen exosome, extraction method thereof and application of codonopsis tangshen exosome in tumor resistance

Abstract

The invention belongs to the technical field of preparation of anti-tumor drugs, and particularly relates to a codonopsis pilosula exosome, an extraction method thereof and application of the codonopsis pilosula exosome in tumor resistance. The codonopsis tangshen exosome is extracted from codonopsis tangshen, the size of the codonopsis tangshen exosome is 95-105 nm, and the shape of the codonopsis tangshen exosome is a vesicle with a lipid bilayer structure. The codonopsis tangshen exosome has an excellent anti-tumor effect and can significantly inhibit tumor growth, and the tumor growth inhibition effect is significantly superior to that of a common extract lobetyolin in codonopsis tangshen.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of anti-tumor drugs, and particularly relates to Codonopsis tangshen exosomes, an extraction method thereof, and uses thereof in anti-tumor treatment. Background Art

[0002] Exosomes are nanoscale vesicles (30 nm to 200 nm in diameter) secreted by cells, which have a phospholipid bilayer structure and carry bioactive substances such as nucleic acids, proteins, and lipids inside. They can mediate intercellular information transmission and regulate physiological functions. Currently, the research on animal exosomes in the fields of disease diagnosis, drug delivery, etc. is relatively mature, but there are problems such as the risk of immunogenicity and high cost of large-scale preparation, which limit their clinical application. In contrast, plant exosomes have gradually become a research hotspot for new drug carriers due to their wide sources and excellent biocompatibility.

[0003] Existing research shows that plant exosomes (EVs) are highly similar to animal exosomes in terms of morphology (40 nm to 150 nm in diameter), structure (cup-shaped / tray-shaped), and inclusions (miRNA, lipids, functional proteins), and at the same time have the advantages of low toxicity and natural stability. However, the secretion mechanism of plant exosomes has not been fully elucidated, and there are significant differences in the composition of exosomes among different plant varieties, resulting in the lag in the development of function verification and standardized extraction technology.

[0004] Moreover, current research on plant exosomes mostly focuses on model plants (such as broccoli, grapes, etc.), and there is still a systematic deficiency in the exploration of active ingredients and function research of exosomes from traditional medicinal plants. Summary of the Invention

[0005] The present invention extracts Codonopsis tangshen exosomes from Codonopsis tangshen Oliv., and verification shows that the Codonopsis tangshen exosomes have excellent anti-tumor activity, especially against breast cancer, and can be used in the preparation of anti-tumor drugs.

[0006] To achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a kind of Codonopsis tangshen exosomes, which are obtained by extracting from Codonopsis tangshen. The particle size of the Codonopsis tangshen exosomes is 95 nm - 105 nm, and the shape is a vesicle with a lipid bilayer structure.

[0008] On the other hand, the present invention provides an extraction method of the Codonopsis tangshen exosomes in the present invention, which includes: (1) crushing Codonopsis tangshen to obtain Codonopsis tangshen juice; (2) centrifuging the Codonopsis tangshen juice to obtain a precipitate; (3) resuspending the precipitate to obtain Codonopsis tangshen exosomes.

[0009] Preferably, the above extraction method satisfies one or more of the following conditions: (i) In step (2), the centrifugation step includes: first centrifuging the Codonopsis tangshen juice for the first time, taking the supernatant after the first centrifugation for the second centrifugation, taking the supernatant after the second centrifugation for the third centrifugation, and removing the supernatant after the third centrifugation to obtain a precipitate; wherein, the first centrifugation includes: centrifuging successively at 2600×g to 3400×g and 3600×g to 4400×g; the centrifugal force of the second centrifugation is 8000×g to 12000×g; the centrifugal force of the second centrifugation is 8000×g to 12000×g;

[0010] (ii) In step (3), after the precipitate is resuspended, it is filtered successively through a filter with a pore size of 0.6 μm to 0.8 μm and a pore size of 0.4 μm to 0.5 μm to obtain Codonopsis tangshen exosomes.

[0011] On the other hand, the present invention provides a Codonopsis tangshen exosome product, which is in different forms containing the effective active ingredients of the Codonopsis tangshen exosomes in the present invention. The Codonopsis tangshen exosome product includes a suspension, a lyophilized powder, a gel, a cream, a microcapsule or a liposome.

[0012] On the other hand, the present invention provides an anti-tumor composition, which includes the Codonopsis tangshen exosomes in the present invention or the Codonopsis tangshen exosome product in the present invention.

[0013] On the other hand, the present invention provides an anti-tumor drug, which includes the Codonopsis tangshen exosomes in the present invention, the Codonopsis tangshen exosome product in the present invention or the anti-tumor composition in the present invention.

[0014] Preferably, the dosage form of the above anti-tumor drug includes oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

[0015] On the other hand, the present invention provides a use of the Codonopsis tangshen exosomes in the present invention or the Codonopsis tangshen exosome product in the present invention, and the uses include any one of the following uses: (a) the use in the preparation of anti-tumor drugs; (b) the use in inhibiting the proliferation of tumor cells.

[0016] Preferably, in the above use, the tumor is breast cancer.

[0017] Preferably, in the above use, the dosage form of the anti-tumor drug is oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

[0018] The beneficial effects of the present invention include:

[0019] (1) The Chuan Codonopsis pilosula exosomes provided by the present invention have excellent anti-tumor effects, can significantly inhibit tumor growth, and the effect of inhibiting tumor proliferation is significantly better than that of Lobetyolin (LOB for short), a common extract in Chuan Codonopsis pilosula. For example, in the experiments of the present invention, under the same conditions, the tumor weight of the tumor model mice of 4T1 (mouse breast cancer cells) treated with Chuan Codonopsis pilosula exosomes was reduced by about 57.1% compared with that of the 4T1 tumor model mice treated with Lobetyolin, and was reduced by about 76.9% compared with that of the model mice treated with PBS solution.

[0020] (2) The extraction method of the Chuan Codonopsis pilosula exosomes provided by the present invention is simple, stable and efficient, suitable for large-scale production; and the extracted Chuan Codonopsis pilosula exosomes have high purity, complete morphology and good biological activity. Description of the Drawings

[0021] Figure 1 is the extraction flow chart of the Chuan Codonopsis pilosula exosomes extracted by the present invention;

[0022] Figure 2 is the transmission electron micrograph of the Chuan Codonopsis pilosula exosomes extracted by the present invention;

[0023] Figure 3 is the particle size distribution diagram of the Chuan Codonopsis pilosula exosomes extracted by the present invention;

[0024] Figure 4 is the Zeta potential distribution diagram of the Chuan Codonopsis pilosula exosomes extracted by the present invention;

[0025] Figure 5 is the flat-plate cloning experiment to detect the inhibitory effect of Chuan Codonopsis pilosula exosomes on the proliferation of 4T1 tumor cells; among them, A is the colony situation of 4T1 tumor cells treated with different concentrations of Chuan Codonopsis pilosula exosomes; B is the bar chart of the colony numbers of 4T1 tumor cells treated with different concentrations of Chuan Codonopsis pilosula exosomes;

[0026] Figure 6 is the change trend diagram of the body weights of different groups of mice at different time points after tumor inoculation;

[0027] Figure 7 is the result diagram of the inhibition of tumor growth by the Chuan Codonopsis pilosula exosomes extracted by the present invention; among them, A is the tumor physical objects of different groups of mice after the drug administration; B is the change situation of the tumor volumes of different groups of mice with the drug administration time; B is the tumor weight situation of different groups of mice after the drug administration. Detailed Embodiments

[0028] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation schemes according to the above-mentioned invention content still fall within the protection scope of the present invention.

[0029] The terms used in this disclosure are only for describing specific embodiments and are not intended to limit the present disclosure. Unless clearly different in context, singular expressions include plural expressions. As used herein, it should be understood that terms such as "including", "having", "containing", etc. are intended to indicate the existence of features, numbers, operations, components, parts, elements, materials, or combinations. Terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations may exist or may be added. As used herein, depending on the context, " / " may be interpreted as "and" or "or".

[0030] An embodiment of the present invention provides a Codonopsis tangshen exosome, which is obtained by extracting from Codonopsis tangshen. The particle size of the Codonopsis tangshen exosome is 95 nm to 105 nm, and the shape is a vesicle with a lipid bilayer structure.

[0031] It should be noted that Codonopsis tangshen Oliv. is a plant commonly used in traditional Chinese medicine, and has effects such as invigorating qi and nourishing blood, enhancing immunity, etc.; the present invention extracts Codonopsis tangshen exosomes from Codonopsis tangshen Oliv., and the particle size is mainly concentrated around 100 nm through a nanoparticle tracking analyzer. In addition, transmission electron microscopy images show that the Codonopsis tangshen exosome is a vesicle with a lipid bilayer structure, and the size is relatively uniform and the morphology is complete.

[0032] An embodiment of the present invention also provides a method for extracting the Codonopsis tangshen exosome in the present invention, which includes: (1) crushing Codonopsis tangshen to obtain Codonopsis tangshen juice; (2) centrifuging the Codonopsis tangshen juice to obtain a precipitate; (3) resuspending the precipitate to obtain Codonopsis tangshen exosomes.

[0033] It should be noted that "crushing", "centrifuging", and "resuspending" in the present invention are all conventional experimental operations in the present invention and have no specific meaning; in addition, the specific manner of the operation can be with or without equipment, and can be selected according to specific experimental conditions. In addition, when using equipment, the model of the equipment can also be selected according to specific experimental conditions.

[0034] In some specific examples, the above extraction method satisfies one or more of the following conditions:

[0035] (i) In step (2), the centrifugation step includes: first centrifuging the Codonopsis tangshen juice, taking the supernatant after the first centrifugation for the second centrifugation, taking the supernatant after the second centrifugation for the third centrifugation, and removing the supernatant after the third centrifugation to obtain a precipitate; wherein, the first centrifugation includes: centrifuging successively at 2600×g - 3400×g and 3600×g - 4400×g; the centrifugal force for the second centrifugation is 8000×g - 12000×g; the centrifugal force for the second centrifugation is 8000×g - 12000×g;

[0036] (ii) In step (3), after the precipitate is resuspended, it is filtered successively using pores with diameters of 0.6μm - 0.8μm and 0.4μm - 0.5μm to obtain Codonopsis tangshen exosomes.

[0037] In some specific examples, in step (2), the centrifugation step may include: first centrifuging the Codonopsis tangshen juice, taking the supernatant after the first centrifugation for the second centrifugation at 10000×g for 60 min, taking the supernatant after the second centrifugation for the third centrifugation at 100000×g for 120 min, and removing the supernatant after the third centrifugation to obtain a precipitate; wherein, the first centrifugation includes: centrifuging successively at 3000×g and 4000×g for 30 min.

[0038] In some specific examples, in step (3), after the precipitate is resuspended, it is filtered successively using pores with diameters of 0.7μm and 0.45μm to obtain Codonopsis tangshen exosomes.

[0039] It should be noted that the extraction method of Codonopsis tangshen exosomes in the present invention can preferably be any one of the above conditions, and more preferably satisfy both of the above conditions simultaneously. The purity of the extracted Codonopsis tangshen exosomes is higher and the anti-tumor activity is better.

[0040] The embodiment of the present invention also provides a Codonopsis tangshen exosome product, and the Codonopsis tangshen exosome product is in different forms containing the effective active ingredients of the Codonopsis tangshen exosomes in the present invention. The Codonopsis tangshen exosome product includes a suspension, a freeze-dried powder, a gel, a cream, a microcapsule or a liposome.

[0041] It should be noted that in order to better preserve and utilize the Codonopsis tangshen exosomes in the present invention, they can be prepared into different forms containing effective active ingredients, such as a suspension, a freeze-dried powder, a gel, a cream, a microcapsule or a liposome. In addition, the methods for preparing the above-mentioned different forms of related products from Codonopsis tangshen exosomes are well-known in the art and can be selected according to specific test conditions or specific usage scenarios.

[0042] The embodiment of the present invention also provides an anti-tumor composition, which includes the Codonopsis tangshen exosomes in the present invention or the Codonopsis tangshen exosome product in the present invention.

[0043] It should be noted that the Codonopsis tangshen exosomes or their related products in the present invention have anti-tumor activity, and they can be combined with other active ingredients to form an anti-tumor composition. The other active ingredients can be chemically synthesized anti-tumor ingredients, or naturally extracted anti-tumor active ingredients, or protein small molecules, and the specific selection can be made according to the type of tumor.

[0044] The embodiment of the present invention also provides an anti-tumor drug, which includes the Codonopsis tangshen exosomes in the present invention, or the Codonopsis tangshen exosome products in the present invention, or the anti-tumor composition in the present invention.

[0045] It should be noted that, as described above, the Codonopsis tangshen exosomes or their related products and the above anti-tumor composition in the present invention have anti-tumor activity. In order to better meet different clinical medication needs, the Codonopsis tangshen exosomes or their related products and the above anti-tumor composition can be added with pharmaceutically acceptable carriers to prepare different dosage forms, such as oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository; in addition, the pharmaceutically acceptable carriers are well-known in the art and can be selected according to different drug dosage forms.

[0046] The embodiment of the present invention also provides a use of the Codonopsis tangshen exosomes in the present invention or the Codonopsis tangshen exosome products in the present invention, and the uses include any one of the following uses:

[0047] (a) Use in the preparation of anti-tumor drugs;

[0048] (b) Use in inhibiting the proliferation of tumor cells.

[0049] It should be noted that the Codonopsis tangshen exosomes or the Codonopsis tangshen exosome products in the present invention have anti-tumor activity, and can be used to prepare anti-tumor drugs in vivo; they can also be used in vitro to only inhibit the proliferation of tumor cells; it should be understood that inhibiting the proliferation of tumor cells can be used to study the changes in related protein expression in vitro.

[0050] In some specific examples, in the above uses, the tumor is breast cancer.

[0051] It should be noted that the present invention has verified that Codonopsis tangshen exosomes have excellent anti-tumor effects, can significantly inhibit tumor growth, and the effect of inhibiting tumor proliferation is significantly better than that of the common extract Lobetyolin (LOB) in Codonopsis tangshen. For example, in the present invention, under the same conditions, the tumor weight of 4T1 tumor model mice treated with Codonopsis tangshen exosomes was reduced by about 57.1% compared with that of 4T1 tumor model mice treated with Lobetyolin, and was reduced by about 76.9% compared with that of model mice treated with PBS.

[0052] In some specific examples, in the above uses, the dosage form of the anti-tumor drug is oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

[0053] It should be noted that as described above, the Codonopsis tangshen exosomes or their related products in the present invention and the above anti-tumor composition have anti-tumor activity. In order to better meet different clinical medication needs, the Codonopsis tangshen exosomes or their related products and the above anti-tumor composition can be added with pharmaceutically acceptable carriers to prepare different dosage forms, such as oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository; in addition, pharmaceutically acceptable carriers are well known in the art and can be selected according to different drug dosage forms.

[0054] To better understand the present invention, the content of the present invention will be further clarified below with specific examples, but the content of the present invention is not limited to the following examples.

[0055] I. Extraction of Codonopsis tangshen exosomes

[0056] Example 1

[0057] In the embodiment of the present invention, the extraction process of Codonopsis tangshen exosomes can refer to Figure 1 , and the specific steps are as follows:

[0058] (1) Take an appropriate amount of fresh Codonopsis tangshen, wash and dry it. Add pre-cooled PBS solution (8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate and 1 L deionized water, pH 7.2 - 7.4, the same below) to a juicer, and crush it with the juicer. Stop for 1 minute every 1 minute of crushing, repeat 5 times, then filter out the Codonopsis tangshen residue with a filter screen, and divide the collected Codonopsis tangshen juice into centrifuge tubes and place them on ice temporarily;

[0059] (2) Place the collected Codonopsis tangshen juice in a pre-cooled centrifuge for gradient centrifugation. At 4°C, first centrifuge at 3000×g for 30 minutes, collect the supernatant, and then centrifuge the supernatant at 4000×g for 30 minutes and collect the supernatant into a clean centrifuge tube;

[0060] (3) Centrifuge the supernatant obtained in step (2) in an ultracentrifuge at 4°C at 10000×g for 60 minutes, and take the supernatant;

[0061] (4) Centrifuge the supernatant obtained in step (3) in an ultracentrifuge at 4°C at 100000×g for 120 minutes, discard the supernatant, and resuspend the precipitate with 1 mL of sterile pre-cooled PBS solution to obtain a Codonopsis tangshen exosome suspension;

[0062] (5) Filter the suspension successively with 0.7μm and 0.45μm pore size filter membranes in the ultra-clean bench to obtain the purified Codonopsis tangshen exosome suspension (hereinafter also referred to as the purified Codonopsis tangshen exosome suspension).

[0063] II. Characterization of Codonopsis tangshen exosomes

[0064] (I) Observation of morphology by transmission electron microscopy

[0065] Place the purified Codonopsis tangshen exosome suspension prepared in Example 1 above under a transmission electron microscope to observe the morphology. The steps are as follows:

[0066] (1) Adsorption: Pipette 10μL of the purified Codonopsis tangshen exosome suspension and drop it onto a disposable sealing film. Invert the copper mesh face down and let it adsorb naturally for 15 minutes;

[0067] (2) Fixation: Pipette 10μL of 2% paraformaldehyde fixative and drop it on the front of the copper mesh, and place it for 20 minutes;

[0068] (3) Staining: Pipette 10μL of 2% phosphotungstic acid solution and drop it on the front of the copper mesh, and let it stand for 90 seconds;

[0069] (4) Drying: Absorb the excess liquid droplets with a filter paper strip and air dry in the dark at room temperature for half an hour;

[0070] (5) Observation and photography: Observe under a transmission electron microscope.

[0071] The detection results are as Figure 2 shown. The results show that the Codonopsis tangshen exosomes extracted by the present invention are vesicles with a lipid bilayer structure, with relatively uniform sizes and complete morphologies.

[0072] (II) Detection of particle size under a nanoparticle tracking analyzer

[0073] Place the purified Codonopsis tangshen exosome suspension prepared in Example 1 above under a nanoparticle tracking analyzer (NTA, Nanoparticle Tracking Analysis; Malvern NanoSight) to detect the particle size. The steps are as follows:

[0074] (1) Cleaning: Wash the sample cell with 20μL of PBS solution through a syringe, and turn on the device switch and software for self-check;

[0075] (2) Standard product detection and instrument pre-calibration: Pipette 1 μL of the exosome size detection standard product (the standard product refers to an exosome particle sample with a known and certified particle size) into 2 mL of PBS solution, mix well, and check whether the measured particle size range is within 100 nm - 200 nm to verify the accuracy of the instrument and ensure the reliability of the experimental results. That is, by comparing the difference between the particle size value measured by the instrument and the standard product particle size value, the instrument can be calibrated and its settings adjusted;

[0076] (3) Sample detection: Pipette 200 μL of the purified Codonopsis tangshen exosome suspension, then dilute it to 2 mL with pre-cooled PBS solution to obtain the test sample. Then use a 1 mL syringe to inject the test sample into the sample cell and start the detection;

[0077] (4) Report generation: Export a PDF report after the detection is completed.

[0078] The detection results are as Figure 3 shown. The size of Codonopsis tangshen exosomes is mainly concentrated around 100 nm, which belongs to the size range of exosomes (30 nm - 200 nm).

[0079] (3) Detecting the potential of particles under a Zeta potential instrument

[0080] Place the purified Codonopsis tangshen exosome suspension prepared in Example 1 above under a Zeta potential instrument (MalvernPanalytical) to detect the potential of particles. The specific steps are as follows:

[0081] (1) Dilute the exosome sample: Take 20 μL of the purified Codonopsis tangshen exosome suspension prepared in Example 1 above, and dilute the exosomes with PBS solution to 10 μg / mL to obtain the diluted sample (usually, measurements need to be carried out under low concentrations (such as 10 μg / mL - 100 μg / mL protein concentration or 1×10 9 -1×10 11 particle / mL) conditions to avoid the influence of high ionic concentration on the Zeta potential);

[0082] (2) Filter to remove large particle impurities: Filter the diluted sample through a 0.22 μm filter to remove cell debris and large particles to obtain the sample to be measured;

[0083] (3) Preparation of Zeta potential instrument: Ensure that the Zeta potential measuring instrument is in normal working condition and perform calibration; Select the measurement mode: Use Laser Doppler Electrophoresis for Zeta potential measurement; Select an appropriate measurement cell: a disposable folded capillary cell; Sample loading: Using a bubble-free pipetting method, slowly inject 20 μL of the sample to be measured in step (2) into the measurement cell.

[0084] (4) Clean the measurement cell and rinse it with Milli-Q water to remove residual charge interference.

[0085] (5) Set the measurement parameters: Temperature: Set at 25 °C (to avoid temperature fluctuations affecting the measurement); Refractive Index: 1.330; Dielectric constant: 80.4; Viscosity: 0.8872 cP; Measurement voltage: Set to 120 V; Data acquisition time: Set for 10 repeated measurements to obtain stable data.

[0086] (6) Sample loading: Use a pipette to aspirate an appropriate amount (0.7 mL - 1 mL) of the diluted exosome sample and slowly inject it into the Zeta potential measurement cell, avoiding the generation of bubbles; Ensure that the electrodes are correctly inserted into the sample cell to prevent measurement errors.

[0087] (7) Start the measurement: Start the instrument to apply an electric field and measure the electrophoretic mobility of exosomes in the solution.

[0088] The detection results are as Figure 4 shown. The results show that the zeta potential of Codonopsis tangshen exosomes mainly concentrates around -25 mV, indicating that the Codonopsis tangshen exosomes extracted by the present invention are relatively stable and well-dispersed.

[0089] III. In vitro anti-tumor application of Codonopsis tangshen exosomes

[0090] (I) Experimental steps for cell resuscitation

[0091] (1) Take out the 4T1 tumor cells (mouse breast cancer cells) stored in liquid nitrogen and quickly place them in a 37 °C water bath to melt.

[0092] (2) Operate in a laminar flow hood, gently pipette and mix the cells in the cryopreservation tube, and centrifuge at 1000 rpm for 5 min.

[0093] (3) Discard the supernatant, resuspend the cell pellet with DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS), and transfer it to a cell culture flask.

[0094] (4) Place the cell culture flask in an incubator and culture it at 37°C with 5% carbon dioxide.

[0095] (II) Cell passage

[0096] (1) Operate in a laminar flow hood. Aspirate the culture medium (DMEM high-glucose medium containing 10% FBS) in the culture flask and add sterile PBS solution to wash the cells.

[0097] (2) Add a certain volume of trypsin solution to ensure that the trypsin covers the entire cell monolayer. Shake gently to allow the trypsin to come into full contact with the cells. Place it in an incubator for 1 minute and then observe under a microscope until the cell-cell connections disappear.

[0098] (3) Add DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS) to terminate the digestion. Suspend the cells in the solution and transfer the cells to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes.

[0099] (4) Discard the supernatant, resuspend the cells with DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS), and subculture them into culture flasks at a volume ratio of 1:4.

[0100] (5) Add complete culture medium for culture.

[0101] (III) Cell cryopreservation

[0102] (1) Place the cryo-box at 4°C and change the fresh culture medium one day before cryopreservation.

[0103] (2) Digest the cells with trypsin, terminate the digestion with complete culture medium, and then transfer the cells to a centrifuge tube.

[0104] (3) Centrifuge at 1000 rpm for 5 minutes.

[0105] (4) After discarding the supernatant, add the prepared commercial cell cryopreservation solution (Xinsaimi), gently pipette to mix evenly, and aliquot 1 mL into each cryotube.

[0106] (5) After labeling the cells with information, place the cryotubes in the cryo-box and transfer them to an -80°C refrigerator.

[0107] (6) Transfer the cells in the cryo-box to liquid nitrogen for storage.

[0108] (IV) Cell counting

[0109] (1) Preparation of single cell suspension from digested cells: Aspirate and discard the culture medium, gently wash the cells twice with 2 mL of pre-cooled sterile PBS solution, add an appropriate amount of 0.25% trypsin-EDTA (Gibco) to cover the cells, incubate at 37 °C for 2 min, and observe the digestion degree under a microscope; when the cells become round, partially detached but still have a small amount of cells adhering, add an equal volume of high-glucose DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) to terminate the digestion; gently pipette the cells to make them completely detached, collect the cell suspension, centrifuge at 1200 rpm for 3 min, discard the supernatant; resuspend the cell pellet with PBS solution, aspirate 10 μL of the cell suspension and drop it on the edge of the coverslip, and pay attention to avoid generating bubbles;

[0110] (2) After standing for 3 min, count under an inverted microscope; Counting principle: "Count the cells on the upper side and left side, not the lower side and right side".

[0111] (3) Count the total number of cells in four large squares, and the number of the cell suspension is recorded as: (N / 4)×10 4 cells / mL (N is the total number of cells in four large squares).

[0112] (V) In vitro anti-tumor effect of vesicles from Codonopsis tangshen Oliv.

[0113] In the examples of the present invention, the colony formation assay:

[0114] (1) Take out 4T1 cells from the cell culture incubator, wash them 3 times with 3 mL of PBS solution, add 1 mL of trypsin and digest for 5 min, then use an equal volume of high-glucose DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) to terminate the digestion;

[0115] (2) Set the rotation speed at 1000 rpm, centrifuge for 5 min, discard the supernatant, and resuspend the cells with an equal volume of high-glucose DMEM medium (Gibco) containing 10% fetal bovine serum (FBS); add the mixture to the cell counting chamber for counting;

[0116] (3) Inoculate 4T1 cells into a 6-well plate, about 500 cells per well, then add 2 mL of high-glucose DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) to the 6-well plate, and then culture for 24 hours to allow the cells to adhere to the wall;

[0117] (4) After culturing for 24 hours, add exosome solutions of different concentrations of Codonopsis tangshen Oliv. prepared with complete medium (such as 0, 4 μg / mL, 16 μg / mL, and 32 μg / mL);

[0118] (5) After culturing in the incubator for 10 days, discard the culture medium, wash 3 times with PBS, and then add paraformaldehyde to fix for 30 min;

[0119] (6) After fixation, add 1 mL of crystal violet solution and stain for 30 min. After staining, wash twice with PBS for 5 min each time.

[0120] (7) Take pictures using an EVOS M 7000 fully automatic live cell fluorescence imaging system and perform counting statistics using ImageJ.

[0121] The results are as Figure 5 shown ( Figure 5 in B, the ordinate shows the number of colony formations, unit (pieces)). The results show that after co - culturing 4T1 tumor cells with Codonopsis tangshen exosomes at different concentrations (0, 4 μg / mL, 16 μg / mL, and 32 μg / mL), the colony formation of 4T1 cells was inhibited; meanwhile, the effect of Codonopsis tangshen exosomes on the colony formation of 4T1 cells was dose - dependent.

[0122] IV. Antitumor effect of Codonopsis tangshen vesicles in vivo

[0123] (1) Construction of tumor - bearing mouse model: Trypsinize and count 4T1 tumor cells in the logarithmic growth phase; use a sterile syringe to inject 100 μL of cell suspension (i.e., inoculate about 2×10 5 tumor cells) into the tail vein of mice (female C57 mice, 4 - 6 weeks old, 20 g ± 2 g); after inoculation, observe the status of mice daily and start drug treatment 3 days later.

[0124] (2) Grouping and treatment: Randomly divide the tumor - bearing mice into the following groups, with 8 mice in each group; the groups are: Control group, Lobetyolin group (abbreviated as LOB, MCE, product number: HY - N0327), and Codonopsis tangshen exosome group (CDNPs); among them, each mouse in the control group is injected with 200 μL of PBS solution, each mouse in the Lobetyolin group is intraperitoneally injected with 200 μL of Lobetyolin solution (Lobetyolin is dissolved in PBS solution, and the concentration of Lobetyolin is 50 mg / mL), and each mouse in the Codonopsis tangshen exosome group is intraperitoneally injected with 200 μL of Codonopsis tangshen exosome solution (the Codonopsis tangshen exosome suspension prepared in Example 1 is dispersed in PBS solution, and the concentration of Codonopsis tangshen exosomes is 50 mg / mL (i.e., 1 mL of PBS solution contains 50 mg of Codonopsis tangshen exosome suspension));

[0125] (3) Drug administration plan: Inject each group of mice once every two days, measure the body weight and tumor size of the mice, and record the data;

[0126] (4) Mouse disposal and sample collection: After the drug administration is completed (a total of 8 times of drug administration), decapitate the mice, dissect the tumor tissue, and measure the weight of the mouse tumor, unit (g).

[0127] The weight changes of each group of mice are shown as Figure 6 follows. The results show that there are no significant differences in the weight changes of mice in the Codonopsis tangshen exosome group (CDNPs) and the lobetyolin group (LOB) compared with the control group (Control), indicating that neither Codonopsis tangshen exosomes nor lobetyolin have obvious toxic and side effects on mice, and lobetyolin and Codonopsis tangshen exosomes do not significantly affect the overall energy metabolism, food intake or consumption of mice at the doses used.

[0128] In addition, the tumor conditions of each group of mice are shown as Figure 7 follows. The results show that the tumor volume and tumor weight in the Codonopsis tangshen exosome group (CDNPs) and the lobetyolin group (LOB) are both smaller than those in the control group (Control); among them, the average tumor weight of mice in the control group is 0.65 ± 0.05 g, the average tumor weight of mice in the lobetyolin group is 0.35 ± 0.04 g, and the average tumor weight of mice in the Codonopsis tangshen exosome group is 0.15 ± 0.03 g. That is, statistical analysis shows that the tumor weight in the CDNPs group is significantly lower than that in the LOB group (p < 0.05), indicating that Codonopsis tangshen exosomes can effectively inhibit tumor growth, and the therapeutic effect is better than that of lobetyolin (One-way ANOVA, Mean ± SEM, p < 0.05 is considered to have statistical significance).

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Exosomes of Codonopsis pilosula, characterized in that The exosomes of Codonopsis pilosula are extracted from Codonopsis pilosula. The particle size of the exosomes of Codonopsis pilosula is 95nm~105nm, and the shape is a vesicle with a lipid bilayer structure.

2. The method for extracting exosomes from Codonopsis pilosula according to claim 1, characterized in that: include: (1) crushing Codonopsis pilosula to obtain Codonopsis pilosula juice; (2) centrifuging the Codonopsis pilosula juice to obtain a precipitate; (3) The precipitate was resuspended to obtain exosomes from Codonopsis pilosula.

3. The extraction method according to claim 2, characterized in that The extraction method meets one or more of the following conditions: (i) In step (2), the centrifugation step comprises: firstly subjecting the Codonopsis pilosula juice to a first centrifugation, taking the supernatant after the first centrifugation to a second centrifugation, taking the supernatant after the second centrifugation to a third centrifugation, and removing the supernatant to obtain a precipitate after the third centrifugation; wherein the first centrifugation comprises: centrifugation at 2600×g to 3400×g and 3600×g to 4400×g in sequence; the centrifugal force of the second centrifugation is 8000×g to 12000×g; the centrifugal force of the second centrifugation is 8000×g to 12000×g; (ii) In step (3), after the precipitate is resuspended, it is filtered using 0.6 μm to 0.8 μm pore size and 0.4 μm to 0.5 μm pore size to obtain Codonopsis pilosula exosomes.

4. A Codonopsis pilosula exosome product, characterized in that: The exosome preparation of Codonopsis pilosula is a different form of the effective active ingredients of the exosomes of Codonopsis pilosula described in claim 1, and the exosome preparation of Codonopsis pilosula includes a suspension, a lyophilized powder, a gel, a cream, a microcapsule or a liposome.

5. An antitumor composition, characterized in that: Including the Codonopsis pilosula exosomes described in claim 1 or the Codonopsis pilosula exosome product described in claim 4.

6. An anti-tumor drug, characterized in that: It includes the Codonopsis pilosula exosomes described in claim 1, the Codonopsis pilosula exosome product described in claim 4, or the anti-tumor composition described in claim 5.

7. The anti-tumor drug according to claim 6, characterized in that: The dosage forms of anti-tumor drugs include oral solution, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

8. Use of the Codonopsis pilosula exosomes according to claim 1 or the Codonopsis pilosula exosome product according to claim 4, including any one of the following uses: (a) Use in the preparation of anti-tumor drugs; (b) Use in inhibiting tumor cell proliferation.

9. The use according to claim 8, characterized in that The tumor is breast cancer.

10. The use according to claim 8 or 9, characterized in that The dosage forms of anti-tumor drugs are oral solution, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

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