Coptis root exosome as well as extraction method and anti-tumor application thereof

Abstract

The invention belongs to the technical field of preparation of anti-tumor drugs, and particularly relates to a coptis root exosome, an extraction method thereof and application of the coptis root exosome in tumor resistance. The coptis root exosome is extracted from coptis roots, the size of the coptis root exosome is 125nm-135nm, and the coptis root exosome is shaped like a vesicle with a lipid bilayer structure. The coptis root exosome has an excellent anti-tumor effect and can remarkably inhibit tumor growth, and the tumor growth inhibition effect is remarkably superior to that of a common extract berberine in coptis roots.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of anti-tumor drugs, and specifically relates to exosomes derived from the roots of Coptis chinensis, a method for extracting the same, and its use in anti-tumor treatment. Background Art

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

[0003] Existing studies have shown that plant exosomes (EVs) are highly similar to animal exosomes in terms of morphology (diameter 40 nm to 150 nm), 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 techniques.

[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 functional research of exosomes from traditional medicinal plants. Summary of the Invention

[0005] The present invention extracts exosome-like nanovesicles derived from the roots of Coptis chinensis (CENs) from the roots of Coptis chinensis (Coptis chinensis or Coptis chinensis Franch.), and verification shows that the exosome-like nanovesicles derived from the roots of Coptis chinensis 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 an exosome-like nanovesicle derived from the roots of Coptis chinensis, which is obtained by extracting from the roots of Coptis chinensis, and the exosome-like nanovesicle derived from the roots of Coptis chinensis has a particle size of 125 nm to 135 nm and a shape of a vesicle with a lipid bilayer structure.

[0008] On the other hand, the present invention provides a method for extracting exosomes from Coptis chinensis roots, which includes: (1) crushing Coptis chinensis roots to obtain Coptis chinensis root juice; (2) centrifuging the Coptis chinensis root juice to obtain a precipitate; (3) resuspending the precipitate to obtain exosomes from Coptis chinensis roots.

[0009] Preferably, the extraction method satisfies one or more of the following conditions: (i) in step (2), the centrifugation step includes: first centrifuging the Coptis chinensis root juice, taking the supernatant after the first centrifugation for a second centrifugation, taking the supernatant after the second centrifugation for a 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 third centrifugation is 8000×g - 12000×g; (ii) in step (3), after resuspending the precipitate, filtering with a pore size of 0.2μm - 0.3m to obtain exosomes from Coptis chinensis roots.

[0010] On yet another aspect, the present invention provides an exosome preparation from Coptis chinensis roots, which contains the exosomes from Coptis chinensis roots in the present invention, and the exosome preparation from Coptis chinensis roots includes a suspension, freeze-dried powder, gel, cream, microcapsule or liposome.

[0011] On yet another aspect, the present invention provides an anti-tumor composition, which includes the exosomes from Coptis chinensis roots in the present invention or the exosome preparation from Coptis chinensis roots in the present invention.

[0012] On yet another aspect, the present invention provides an anti-tumor drug, which includes the exosomes from Coptis chinensis roots in the present invention or the exosome preparation from Coptis chinensis roots in the present invention or the anti-tumor composition in the present invention.

[0013] Preferably, the dosage form of the above anti-tumor drug includes oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

[0014] On yet another aspect, the present invention provides a use of the exosomes from Coptis chinensis roots in the present invention or the exosome preparation from Coptis chinensis roots in the present invention, and the uses include any one of the following uses: (a) use in the preparation of anti-tumor drugs; (b) use in inhibiting the proliferation of tumor cells; (c) use in promoting the apoptosis of tumor cells.

[0015] Preferably, in the above uses, the tumor is breast cancer.

[0016] Preferably, in the above uses, the dosage form of the anti-tumor drug is oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

[0017] The beneficial effects of the present invention include:

[0018] (1) The exosomes from Coptis chinensis roots 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 berberine (ART), a common extract in Coptis chinensis roots. For example, in the present invention, under the same conditions, the tumor weight of the 4T1 (mouse breast cancer cells) tumor model mice treated with the exosomes from Coptis chinensis roots was reduced by about 19.1% compared with that of the 4T1 tumor model mice treated with berberine, and was reduced by about 54.1% compared with that of the model mice treated with PBS.

[0019] (2) The extraction method of the exosomes from Coptis chinensis roots provided by the present invention is simple, stable, and efficient, suitable for large-scale production; and the extracted exosomes from Coptis chinensis roots have high purity, complete morphology, and good biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the extraction flow chart of the exosomes from Coptis chinensis roots extracted by the present invention;

[0021] Figure 2 is the transmission electron micrograph of the exosomes from Coptis chinensis roots extracted by the present invention;

[0022] Figure 3 is the particle size distribution chart of the exosomes from Coptis chinensis roots extracted by the present invention;

[0023] Figure 4 shows the inhibitory effect of the exosomes from Coptis chinensis on the viability of tumor cell lines detected by the CCK-8 method;

[0024] Figure 5 shows the effect of the exosomes from Coptis chinensis on the apoptosis of tumor cell lines detected by the AnnexinV-FITC / PI double staining method;

[0025] Figure 6 shows the change of tumor volume of each group of mice during the drug administration process;

[0026] Figure 7 is the physical picture of the tumors of each group of mice after the drug administration;

[0027] Figure 8 is the tumor weight of each group of mice after the drug administration. DETAILED DESCRIPTION OF THE INVENTION

[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 document are only for describing specific embodiments and are not intended to limit the present disclosure. Unless there is a clearly different meaning in the context, the expressions in the singular form include those in the plural form. As used herein, it should be understood that terms such as "comprising", "having", "including" are intended to indicate the existence of features, numbers, operations, components, parts, elements, materials or combinations. The 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 their combinations may exist or can be added. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

[0030] An embodiment of the present invention provides an exosome from the root of Coptis chinensis. The exosome from the root of Coptis chinensis is obtained by extracting from the root of Coptis chinensis. The particle size of the exosome from the root of Coptis chinensis (Coptis chinensis or Coptis chinensis Franch.) is 125 nm to 135 nm, and the shape is a vesicle with a lipid bilayer structure.

[0031] It should be noted that Coptis chinensis (Coptis chinensis or Coptis chinensis Franch.) is a plant commonly used in traditional Chinese medicine, which has the effects of supplementing qi and nourishing blood, enhancing immunity, etc. The exosome from the root of Coptis chinensis is extracted from the root of Coptis chinensis. After being detected by a nanoparticle tracking analyzer, the particle size is mainly concentrated around 130 nm, that is, 125 nm to 135 nm. In addition, the transmission electron microscopy image shows that the exosome from the root of Coptis chinensis 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 exosome from the root of Coptis chinensis in the present invention, which includes: (1) crushing the root of Coptis chinensis to obtain the juice of the root of Coptis chinensis; (2) centrifuging the juice of the root of Coptis chinensis to obtain a precipitate; (3) resuspending the precipitate to obtain the exosome from the root of Coptis chinensis.

[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 Coptis root 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 third centrifugation is 8000×g - 12000×g;

[0036] (ii) In step (3), after the precipitate is resuspended, it is filtered through a filter with a pore size of 0.2μm - 0.3m to obtain Coptis root exosomes.

[0037] In some specific examples, in step (2) of the above extraction method, the centrifugation step includes: first centrifuging the Coptis root juice, taking the supernatant after the first centrifugation and centrifuging at 10000×g for 60 min for the second centrifugation, taking the supernatant after the second centrifugation and centrifuging at 100000×g for 120 min for the third centrifugation, 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) of the above extraction method, after the precipitate is resuspended, it is filtered through a filter with a pore size of 0.22μm to obtain Coptis root exosomes.

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

[0040] The embodiment of the present invention also provides a Coptis root exosome product, which contains the Coptis root exosomes in the present invention, and the Coptis root exosome product includes a suspension, a lyophilized powder, a gel, a cream, a microcapsule or a liposome.

[0041] It should be noted that in order to better preserve and utilize the Coptis root exosomes in the present invention, they can be prepared into different forms containing effective active ingredients, such as a suspension, a lyophilized powder, a gel, a cream, a microcapsule or a liposome. In addition, the methods for preparing the above different forms of products from Coptis root 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 Coptis root exosomes in the present invention or the Coptis root exosome product in the present invention.

[0043] It should be noted that the exosomes from Coptis chinensis roots or their 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 components, 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 embodiments of the present invention also provide an anti-tumor drug, which includes the exosomes from Coptis chinensis roots or the products of the exosomes from Coptis chinensis roots or the anti-tumor composition in the present invention.

[0045] It should be noted that as described above, the exosomes from Coptis chinensis roots or their 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 exosomes from Coptis chinensis roots or their 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.

[0046] The embodiments of the present invention also provide a use of the exosomes from Coptis chinensis roots or the products of the exosomes from Coptis chinensis roots 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] (c) Use in promoting the apoptosis of tumor cells.

[0050] It should be noted that the exosomes from Coptis chinensis roots or the products of the exosomes from Coptis chinensis roots in the present invention have anti-tumor activity, and can be used to prepare anti-tumor drugs in vivo; it can also be used to study the changes in the expression of certain anti-tumor proteins in vitro.

[0051] In some specific examples, in the above uses, the tumor is breast cancer.

[0052] It should be noted that the present invention has verified that the exosomes from Coptis chinensis roots have excellent anti-tumor effects, can significantly inhibit tumor growth, and the effect of inhibiting tumor proliferation is significantly better than that of berberine, a common extract in Coptis chinensis roots. For example, in the present invention, it was tested that under the same conditions, the tumor weight of the 4T1 (mouse breast cancer cells) tumor model mice treated with the exosomes from Coptis chinensis roots was reduced by about 19.1% compared with that of the 4T1 tumor model mice treated with berberine, and was reduced by about 54.1% compared with that of the model mice treated with PBS.

[0053] 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.

[0054] It should be noted that, as described above, the exosomes from Coptis chinensis roots or their 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 exosomes from Coptis chinensis roots or their 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.

[0055] 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 only.

[0056] I. Extraction of exosomes from Coptis chinensis roots

[0057] Example 1

[0058] In the embodiment of the present invention, the extraction process of exosomes from Coptis chinensis roots can refer to Figure 1 , and the specific steps are as follows:

[0059] (1) Take 100 g of fresh Coptis chinensis roots, wash and air-dry them. Add 300 ml of pre-cooled PBS solution (137 mM sodium chloride, 2.7 mM potassium chloride, 10.0 mM disodium hydrogen phosphate, 2.0 mM potassium dihydrogen phosphate, pH 7.4, the same below) to a juicer, and crush the roots in the juicer. Crush for 1 min and stop for 1 min, repeat 5 times. Then filter out the residues of Coptis chinensis roots with a filter screen, and aliquot the collected juice of Coptis chinensis roots into centrifuge tubes, and temporarily place them on ice.

[0060] (2) Place the collected juice of Coptis chinensis roots in a pre-cooled centrifuge for gradient centrifugation. At 4 °C, first centrifuge at 3000 × g for 30 min, collect the supernatant, and then centrifuge the supernatant at 4000 × g for 30 min, and collect the supernatant into a clean centrifuge tube.

[0061] (3) Centrifuge the supernatant obtained in step (2) in an ultracentrifuge at 4 °C at 10000 × g for 60 min, and take the supernatant.

[0062] (4) Centrifuge the supernatant obtained in step (3) in an ultracentrifuge at 4 °C at 100000 × g for 120 min, discard the supernatant, and resuspend the precipitate with 1 mL of sterile pre-cooled PBS solution to obtain a suspension of exosomes from Coptis chinensis roots.

[0063] (5) Filter the suspension with a 0.22 μm pore size filter membrane in a laminar flow hood in sequence to obtain the purified suspension of exosomes from Coptis chinensis roots (hereinafter also referred to as the purified suspension of exosomes from Coptis chinensis roots).

[0064] II. Characterization of Exosomes from Coptis chinensis Roots

[0065] (I) Observation of Morphology by Transmission Electron Microscope

[0066] Place the purified suspension of exosomes from Coptis chinensis roots prepared in Example 1 above under a transmission electron microscope to observe the morphology. The steps are as follows:

[0067] (1) Adsorption: Pipette 10 μL of the purified suspension of exosomes from Coptis chinensis roots and drop it onto a disposable sealing film. Invert the copper mesh face down and let it adsorb naturally for 15 min;

[0068] (2) Fixation: Pipette 10 μL of a 2% paraformaldehyde fixative and drop it onto the front of the copper mesh, and place it for 20 min;

[0069] (3) Staining: Pipette 10 μL of a 2% phosphotungstic acid solution and drop it onto the front of the copper mesh, and let it stand for 90 s;

[0070] (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;

[0071] (5) Observation and Photography: Observe under a transmission electron microscope.

[0072] The detection results are as Figure 2 shown. The results show that the exosomes from Coptis chinensis roots extracted by the present invention are vesicles with a lipid bilayer structure, with relatively uniform sizes and complete morphologies.

[0073] (II) Detection of Particle Size by Nanoparticle Tracking Analyzer

[0074] Place the purified suspension of exosomes from Coptis chinensis roots 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:

[0075] (1) Cleaning: Use 20 μL of PBS solution to clean the sample cell through a syringe, and turn on the device switch and software for self-check;

[0076] (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 and mix well to detect whether the particle size range is within 100 nm - 200 nm, so as 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 can be adjusted;

[0077] (3) Sample detection: Pipette 200 μL of the purified exosome suspension from Coptis chinensis roots, then dilute it to 2 mL with pre - cooled PBS solution to obtain the detection sample. Then use a 1 mL syringe to inject the detection sample into the sample cell to start the detection;

[0078] (4) Report generation: After the detection is completed, export the PDF report.

[0079] The detection results are as Figure 3 shown. The size of exosomes from Coptis chinensis roots is mainly concentrated around 130 nm, which belongs to the size range of exosomes (30 nm - 200 nm).

[0080] III. Antitumor application of exosomes from Coptis chinensis roots

[0081] (I) Experimental steps for cell resuscitation

[0082] (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;

[0083] (2) Operate in a laminar flow hood. Gently pipette and mix the cells in the cryopreservation tube with a pipette, and centrifuge at 1000 rpm for 5 min;

[0084] (3) Discard the supernatant, resuspend the cell pellet with 1640 medium (Gibco) containing 10% fetal bovine serum (FBS), and then transfer it to a cell culture flask;

[0085] (4) Place the cell culture flask in a cell culture incubator for cultivation under the conditions of 37 °C and 5% carbon dioxide.

[0086] (II) Cell passage

[0087] (1) Operate in a laminar flow hood. Aspirate the medium in the culture flask (1640 medium containing 10% FBS), and add sterile PBS solution to wash the cells;

[0088] (2) Add a certain volume of trypsin solution to ensure that the trypsin covers the entire cell bottom layer. Shake gently to make the trypsin fully contact with the cells, place it in the cell culture incubator for 2 min, and then observe under a microscope until the cell - to - cell connections disappear;

[0089] (3) Add 1640 medium (Gibco) (containing 10% fetal bovine serum (FBS)) to terminate digestion. Suspend the cells in the solution and transfer the cells to a centrifuge tube. Centrifuge at 1000 rpm for 5 min.

[0090] (4) Discard the supernatant, resuspend the cells with 1640 medium (Gibco) (containing 10% fetal bovine serum (FBS)), and aliquot into culture flasks at a volume ratio of 1:4 (the volume ratio of the precipitate to the MEM high-glucose medium).

[0091] (5) Add complete culture medium for culturing.

[0092] (III) Cell Cryopreservation

[0093] (1) Place the cryopreservation box at 4°C and replace the fresh medium one day before cryopreservation.

[0094] (2) Digest the cells with trypsin and transfer the cells to a centrifuge tube after terminating digestion with complete medium.

[0095] (3) Centrifuge at 1000 rpm for 5 min.

[0096] (4) After discarding the supernatant, add the prepared commercial cell cryopreservation solution (Xinsaimi), gently pipette to mix evenly, and aliquot 1 mL into each cryopreservation tube.

[0097] (5) After labeling the cells, place the cryopreservation tubes in the cryopreservation box and transfer them to an -80°C refrigerator.

[0098] (6) Transfer the cells in the cryopreservation box to liquid nitrogen for storage.

[0099] (IV) Cell Counting

[0100] (1) Digest the cells to prepare a single-cell suspension: Aspirate and discard the 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 adherent, add an equal volume of 1640 medium (Gibco) containing 10% fetal bovine serum (FBS) to terminate digestion. Gently pipette the cells to make them completely detached, collect the cell suspension, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cell pellet with PBS solution, aspirate 10 μL of the cell suspension and add it dropwise to the edge of the coverslip, taking care to avoid generating bubbles.

[0101] (2) After standing for 3 min, count the cells under an inverted microscope. Counting principle: "Count the cells in the upper and left areas, not the lower and right areas."

[0102] (3) Count the total number of cells in four large squares, and record the number of cell suspensions as: (N / 4)×10 4 cells / mL (N is the total number of cells in four large squares).

[0103] (V) In vitro anti-tumor effect of vesicles from Coptis chinensis roots

[0104] (1) Detect cell viability by CCK-8 method

[0105] 1) Cell seeding: Seed tumor cells in the logarithmic growth phase into a 96-well plate at a density of 2e 4 cells / mL, add 100 μL of cell suspension to each well, and culture in an incubator at 37 °C and 5% CO2 for 24 hours to allow the cells to adhere to the wall;

[0106] 2) Treat the cells: Divide the cells cultured in the 96-well plate into a control group (Vehicle) and an experimental group (Coptischinensis). Add 100 μL of PBS solution to the control group; add 100 μL of exosome solution from Coptis chinensis roots at a concentration of 30 μg / mL to the experimental group; set 3 replicates for each group;

[0107] 3) Continue to incubate the 96-well plate in the incubator for 24 h, 48 h, and 72 h;

[0108] 4) At each time point, add 10 μL of CCK8 solution to each well, shake gently, and then continue to incubate the 96-well plate in an incubator at 37 °C and 5% CO2 for 1 - 2 hours (adjust the specific time according to the cell situation until the solution color changes significantly to orange-yellow);

[0109] 5) Measure the absorbance: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm, and record the OD values of all groups;

[0110] 6) Calculate the cell proliferation inhibition rate: Calculate the cell proliferation inhibition rate according to the following formula: (1 - OD value of the experimental group / OD value of the control group)×100%.

[0111] The OD450nm values at different incubation times in different steps 3) are as Figure 4 shown. The results show that exosomes from Coptis chinensis do not affect the proliferation of tumor cells in vitro.

[0112] (2) Cell apoptosis detection assay

[0113] 1) Take out the 4T1 tumor cells from the cell culture incubator, wash them 3 times with 2 mL of PBS solution, add 1 mL of trypsin and digest for 2 min, and then terminate the digestion with complete medium (1640 medium (Gibco) containing 10% fetal bovine serum (FBS), the same below);

[0114] 2) Set the rotation speed to 1000 rpm. After centrifugation for 5 min, discard the supernatant, and resuspend the cells with an appropriate amount of complete medium; Take 10 μL of the cell suspension and add it to a hemocytometer for counting; Adjust the number of viable cells to 3e 5 cells / mL;

[0115] 3) Use a pipette to aspirate 1 mL of the adjusted cell suspension into a 6-well plate, then add 1 mL of complete medium to the 6-well plate. After shaking well, place it in a cell culture incubator for continued culture;

[0116] 4) After culturing for 24 hours, add the Coptis chinensis exosome solution (concentration 30 μg / mL) prepared with complete medium, and add the same volume of PBS solution as a control;

[0117] 5) After culturing for 48 hours, collect the original medium in the 6-well plate, add PBS to wash the adherent cells, and add 1 mL of trypsin for digestion;

[0118] 6) After digestion with trypsin for 2 min, add the original medium to terminate the digestion;

[0119] 7) Gently pipette the cells down, then transfer the liquid in the 6-well plate to a centrifuge tube, set the rotation speed to 1000 rpm, and set the centrifugation time to 5 min.

[0120] 8) Discard the supernatant, add 1 mL of PBS solution to resuspend the cells, centrifuge at a rotation speed of 1000 rpm for 5 min, discard the supernatant, add 500 μL of Annexin V-FITC binding solution, resuspend the cells, and then add 5 μL of Annexin V-FITC and 5 μL of propidium iodide staining solution respectively.

[0121] 9) After incubating in the dark at room temperature for 20 min, perform flow cytometry (the instrument is the FACSCanto flow cytometer from BD, USA) for detection.

[0122] The apoptosis index of cells in different groups was obtained through flow cytometry detection, and the results are as Figure 5 shown. Coptis chinensis exosomes do not affect the apoptosis of tumor cells in vitro.

[0123] (VI) Anti-tumor effect of Coptis chinensis root vesicles in vivo

[0124] (1) Construction of a tumor-bearing mouse model: Digest and count 4T1 tumor cells in the logarithmic growth phase; Use a sterile syringe to subcutaneously inject 100 μL of cell suspension (i.e., inoculate about 5×10 5 tumor cells) into the abdomen of mice (6-8-week-old female C57 mice, weighing about 20 g ± 2 g); After inoculation, observe the status of the mice daily. Wait until the tumor volume reaches about 50 mm 3 -100 mm3 (About 7 days later), drug treatment was started;

[0125] (2) Grouping and treatment: The tumor-bearing mice were randomly divided into the following groups, with 5 mice in each group; the groups were: control group (Vehicle), berberine group (Berberine) (MCE, product number HY-N0716), and Coptis chinensis exosome group (Coptischinensis); among them, each mouse in the control group was injected with 100 μL of PBS solution, each mouse in the berberine group was intraperitoneally injected with 100 μL of berberine solution (100 μL of berberine solution contains 0.05 mg of berberine) (dissolve 5 mg of berberine in 1 mL of mixed solution (the mixed solution contains 4% DMSO, 30% PEG300, and 66% ddH2O (all are volume fractions)), and the concentration of berberine in the mixed solution is 5 mg / mL), and each mouse in the Coptis chinensis exosome group was intraperitoneally injected with 100 μL of Coptis chinensis exosome solution (dilute the Coptis chinensis exosome suspension prepared in Example 1 with PBS solution to obtain a 5 mg / mL exosome solution, that is, 1 mL of PBS solution contains 5 mg of Coptis chinensis exosome suspension).

[0126] (3) Drug administration plan: Each group of mice was intraperitoneally injected once every two days, and the drug was administered 9 times. The tumor volume was measured every 3 days and the data was recorded;

[0127] (4) Tumor volume measurement: Use a vernier caliper to measure the long diameter (L) and short diameter (W) of the mouse tumor, and calculate the tumor volume according to the following formula: Tumor volume (mm 3 ) = [long diameter × (short diameter) 2 / 2. The average value of each group was taken to draw a tumor volume change curve; after the drug administration was completed, the mice were sacrificed by cervical dislocation, and the tumor tissue was dissected to measure the weight of the mouse tumor, in units of (g).

[0128] During the drug administration process, the tumor volume change curves of each group of mice were as Figure 6 shown. The results showed that the tumor volume growth rates of the Coptis chinensis exosome group (Coptis chinensis) and the berberine group (Berberine) were significantly lower than those of the control group (Vehicle), and the tumor volume growth rate of the Coptis chinensis exosome group (Coptis chinensis) was lower than that of the berberine group (Berberine).

[0129] In addition, after the drug administration was completed, the tumor size and tumor weight of each group of mice were as Figure 7 and Figure 8As shown, the results showed that the tumor volume and tumor weight of the exosome group from the roots of Coptis chinensis and the Berberine group were smaller than those of the control group (Vehicle); among them, the average tumor weight of the mice in the control group was 0.37 g, the average tumor weight of the mice in the Berberine group was 0.21 g, and the average tumor weight of the mice in the exosome group from the roots of Coptis chinensis was 0.17 g. That is, according to statistical analysis, the tumor weight of the exosome group from the roots of Coptis chinensis was significantly lower than that of the control group (p < 0.05), indicating that the exosomes from the roots of Coptis chinensis could effectively inhibit tumor growth, and the therapeutic effect was better than that of Berberine (One-way ANOVA, Mean ± SEM, p < 0.05 was considered to have statistical significance).

[0130] From the above, it can be known that although the exosomes from the roots of Coptis chinensis do not have an obvious effect of inhibiting the proliferation of tumor cells and promoting the apoptosis of tumor cells in vitro, they have a significant effect of inhibiting tumor growth in vivo, indicating that the exosomes from the roots of Coptis chinensis can be prepared into anti-tumor drugs for use.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not 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 from the roots of Coptis chinensis Franch., characterized in that, The exosomes from the roots of Coptis chinensis Franch are obtained by extracting from the roots of Coptis chinensis Franch. The particle size of the exosomes from the roots of Coptis chinensis Franch is 125 nm to 135 nm, and the shape is vesicles with a lipid bilayer structure.

2. The extraction method of the exosomes from the roots of Coptis chinensis described in claim 1, characterized in that, Including: (1) The roots of Coptis chinensis Franch are broken to obtain the juice of the roots of Coptis chinensis Franch; (2) The juice of the roots of Coptis chinensis Franch is centrifuged to obtain a precipitate; (3) The precipitate is resuspended to obtain the exosomes from the roots of Coptis chinensis Franch.

3. The extraction method according to claim 2, wherein The extraction method satisfies one or more of the following conditions: (i) In step (2), the centrifugation step includes: first centrifuging the juice of the roots of Coptis chinensis Franch, 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; among them, the first centrifugation includes: centrifuging successively at 2600×g to 3400×g and 3600×g to 4400×g; the centrifugal force for the second centrifugation is 8000×g to 12000×g; the centrifugal force for the third centrifugation is 8000×g to 12000×g; (ii) In step (3), after the precipitate is resuspended, it is filtered through a filter with a pore size of 0.2 μm to 0.3 m to obtain the exosomes from the roots of Coptis chinensis Franch.

4. The exosome product of Coptis chinensis roots is characterized in that, The exosome product from the roots of Coptis chinensis Franch contains the exosomes from the roots of Coptis chinensis Franch described in claim 1. The exosome product from the roots of Coptis chinensis Franch includes a suspension, freeze-dried powder, gel, cream, microcapsule or liposome.

5. An anti-tumor composition, characterized in that, (It) includes the exosomes from the roots of Coptis chinensis Franch described in claim 1 or the exosome product from the roots of Coptis chinensis Franch described in claim 4.

6. An anti-tumor drug, characterized in that, (It) includes the exosomes from the roots of Coptis chinensis Franch described in claim 1 or the exosome product from the roots of Coptis chinensis Franch described in claim 4 or the anti-tumor composition described in claim 5.

7. The anti-tumor drug according to claim 6, wherein, The dosage forms of the anti-tumor drug include oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

8. The use of the exosomes from the roots of Coptis chinensis Franch described in claim 1 or the exosome product from the roots of Coptis chinensis Franch described in claim 4, and the use includes any one of the following uses: (a) The use in the preparation of an anti-tumor drug; (b) The use in inhibiting the proliferation of tumor cells; (c) The use in promoting the apoptosis of tumor cells.

9. The use according to claim 8, wherein The tumor is breast cancer.

10. The use according to claim 8 or 9, characterized in that, The dosage form of the anti-tumor drug is oral liquid, injection, tablet, capsule, powder, patch, spray, ointment or suppository.

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