Pharmaceutical composition containing olive leaf extract as well as medicine and application thereof

Abstract

The invention provides a pharmaceutical composition containing an olive leaf extract as well as a medicine and application thereof, and belongs to the technical field of biomedicine. The invention provides a pharmaceutical composition based on an olive leaf extract, a mesenchymal stem cell exosome and a T lymphocyte exosome. The synergistic application of all the components can effectively promote postoperative tissue repair and immune function recovery of tumors. The medicine with the tumor postoperative treatment effect is developed, proliferation of various tumor cells can be effectively inhibited, the postoperative immune state is improved, and tissue repair is remarkably promoted. The invention provides a natural, safe and innovative solution for tumor postoperative adjuvant therapy, and has flexible dosage form development potential and wide clinical application prospect and market value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a pharmaceutical composition comprising an olive leaf extract, a medicine thereof and an application thereof. Background Art

[0002] Tumor recurrence and metastasis after surgery are major problems that need to be solved in cancer treatment. Although surgery, radiotherapy and chemotherapy can remove primary tumors, insufficient regulation of the postoperative microenvironment often leads to recurrence of residual tumor cells and even metastasis to distant sites, significantly reducing the patient's survival rate and quality of life. Therefore, exploring new adjuvant treatments that can effectively regulate the postoperative microenvironment and inhibit tumor recurrence and metastasis has important scientific significance and clinical value.

[0003] Olive leaf extract is a natural product extracted from traditional herbal plants, rich in compounds such as oleuropein, olive glycosides and luteolin. In tumor treatment, although oleuropein has been shown to have certain effects, the efficacy of single olive leaf extract is limited, and its intensity and mechanism of action have not been fully utilized. Therefore, it is necessary to compound olive leaf extract and prepare drugs with definite therapeutic effects. Summary of the invention

[0004] The present invention provides a pharmaceutical composition comprising an olive leaf extract, a medicine and an application thereof. The pharmaceutical composition or the medicine can improve the postoperative recovery quality and living standard of patients through comprehensive effects such as regulating the immune microenvironment, promoting tissue repair and inhibiting postoperative tumor recurrence.

[0005] The first object of the present invention is to provide a pharmaceutical composition having a postoperative therapeutic effect on tumors, comprising olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes.

[0006] In a preferred embodiment of the present invention, the mass ratio of the olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes is (10-20): (15-30): (4-10).

[0007] In a preferred embodiment of the present invention, the olive leaf extract comprises an ethanol extract of olive leaves, and the mass percentage of oleuropein in the dry powder of the olive leaf extract is not less than 45%, and the mass percentage of oleuropein is not less than 4%.

[0008] In a preferred embodiment of the present invention, the preparation process of the mesenchymal stem cell exosomes includes culturing the mesenchymal stem cells, and after subculturing to the third to fourth generations, culturing is performed in a serum-free medium.

[0009] In a preferred embodiment of the present invention, the T lymphocyte exosomes include culturing T lymphocytes during the preparation process, and culturing the subcultured T lymphocytes until the cell density reaches 70-80%, and then culturing them in a serum-free medium.

[0010] The second object of the present invention is to provide the use of the above-mentioned pharmaceutical composition in the preparation of drugs for postoperative treatment of tumors.

[0011] In a preferred embodiment of the present invention, the dosage form of the drug for postoperative treatment of tumors includes injection, oral preparation or transdermal preparation.

[0012] In a preferred embodiment of the present invention, the therapeutic spectrum of the postoperative tumor treatment drug includes: lung cancer, breast cancer, liver cancer, cervical cancer, gastric cancer, prostate cancer and colon cancer.

[0013] The third object of the present invention is to provide a drug for treating tumors after surgery, comprising the above-mentioned pharmaceutical composition and pharmaceutically acceptable excipients.

[0014] The fourth object of the present invention is to provide an injection for the postoperative treatment of tumors, comprising olive leaf extract dry powder, mesenchymal stem cell exosome dry powder, T lymphocyte exosome dry powder and an injection solvent; and the mass percentages of olive leaf extract dry powder, mesenchymal stem cell exosome dry powder and T lymphocyte exosome dry powder in the injection are 10-20%, 15-30% and 4-10%, respectively.

[0015] Beneficial effects: The present invention provides a pharmaceutical composition based on olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes. The pharmaceutical composition combines the traditional herbal efficacy of olive leaf extract with the modern biological functions of the two exosomes to give full play to their synergistic effects in immunoregulation, inflammation inhibition and tissue repair, providing a new solution for postoperative repair of tumors. The olive leaf extract of the present invention contains active ingredients such as oleuropein, oleuropein and luteolin, which have antioxidant, anti-inflammatory and immunoregulatory effects; mesenchymal stem cell exosomes accelerate postoperative tissue healing by promoting cell repair and regeneration; T lymphocyte exosomes improve postoperative immune status by regulating the immune microenvironment. The present invention creatively prepares the three functional components of olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes independently, and effectively promotes postoperative tissue repair and immune function recovery of tumors through synergistic application.

[0016] The present invention uses the pharmaceutical composition to develop a drug with a postoperative therapeutic effect on tumors. Cell experiments have verified that the pharmaceutical composition or drug of the present invention has universal applicability to the postoperative repair of various tumor cells, can effectively inhibit their proliferation, improve postoperative immune status, and significantly promote tissue repair. In animal experiments, the pharmaceutical composition also showed excellent postoperative tumor repair effects, improved immune status, accelerated tissue repair and functional recovery. The present invention provides a natural, safe, and innovative solution for postoperative adjuvant therapy of tumors, with flexible dosage form development potential and broad clinical application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 High performance liquid chromatography (HPLC) analysis of the effective components of the olive leaf extract provided by the present invention;

[0018] Figure 2 The exosome detection result diagram provided by the present invention, in which (A) is a transmission electron microscopy photo of MSCs exosomes; (B) is a transmission electron microscopy photo of T lymphocyte exosomes;

[0019] Figure 3 The particle size distribution diagram of exosomes provided by the present invention, wherein (A) is a particle size distribution diagram of mesenchymal stem cell exosomes; (B) is a particle size distribution diagram of T lymphocyte exosomes;

[0020] Figure 4 The immunoblot analysis results of mesenchymal stem cells (MSCs), T lymphocytes (T cells), mesenchymal stem cell exosomes (MSCs-Exo) and T lymphocyte exosomes (T cell-Exo) provided by the present invention are shown;

[0021] Figure 5 A bar graph showing the inhibitory effect of each test group provided by the present invention on the tumor recurrence rate of a mouse model after tumor surgery;

[0022] Figure 6 A bar graph of weight changes of the mouse model after tumor surgery in each test group provided by the present invention;

[0023] Figure 7 The present invention provides a bar graph of the positive rate of vascular endothelial growth factor (VEGF) in each test group of the mouse model after tumor surgery. DETAILED DESCRIPTION

[0024] The invention provides a pharmaceutical composition having a postoperative therapeutic effect on tumors, comprising an olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes.

[0025] In a preferred embodiment of the present invention, the mass ratio of the olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes is (10-20): (15-30): (4-10), and the mass ratio can be any ratio of the following values: (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20): (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30): (4, 5, 6, 7, 8, 9, 10). For example, in one embodiment, the mass ratio is 10:15:4, or 14:20:6, or 18:25:8, or 20:30:10.

[0026] The olive leaf extract of the present invention includes an ethanol extract of olive leaves, and the mass percentage of oleuropein in the dry powder of the olive leaf extract is not less than 45%, and the mass percentage of oleuropein is not less than 4%. When the present invention performs the extraction, the solvent used includes an ethanol aqueous solution, such as selecting a 70-90% ethanol aqueous solution in one embodiment, and mixing it according to a solid-liquid ratio of 1:5-1:15, and then wetting it at room temperature for 20-60 minutes to ensure that the solvent evenly penetrates into the leaf tissue. The present invention selects healthy and mildew-free olive leaves, washes and dries them, and then crushes them into 40-100 mesh particles, and then performs the wetting. The room temperature referred to in the present invention refers to 20-30°C.

[0027] The invention adopts a vaporization extraction method to extract from moistened olive leaf liquid. In a specific embodiment, the moistened olive leaf liquid is placed in a vacuum extraction device, and the vacuum degree is adjusted to -0.05--0.08MPa; at 60-80°C, a 70%-90% ethanol solution preheated to 40-50°C is injected, and the solvent is rapidly vaporized to form an internal and external pressure difference, thereby promoting the rapid release of active components in cells; the extraction is continuously stirred for 12 hours, and each batch is extracted 2-3 times; the extract is filtered and concentrated under reduced pressure to 1 / 3-1 / 4 of the original volume for concentration.

[0028] The present invention elutes the reduced pressure concentrate. In one embodiment, LSA type macroporous adsorption resin is used for purification, deionized water is used for elution, the sample flow rate is 5BV / h~15BV / h, and 20%~80% ethanol aqueous solution is used for analysis, and the analysis flow rate is 5BV / h~15BV / h.

[0029] The present invention concentrates the above-mentioned eluent to a viscous state, and then obtains olive leaf extract powder after drying. The drying method includes spray drying, vacuum freeze drying or microwave drying. In one embodiment, the drying is carried out by a vacuum freeze drying method to obtain olive leaf extract freeze-dried powder. The vacuum degree of the vacuum freeze drying of the present invention can be set to 0.1-0.5mbar, pre-frozen to -40°C, gradually warmed to -10°C-20°C, and freeze-dried for 12-48 hours. The olive leaf extract freeze-dried powder prepared by the present invention is light yellow or light green powder in appearance detection, without odor; the content of oleuropein and olive glycoside is detected by high performance liquid chromatography (HPLC), and it is found that the content of oleuropein is 45-65%, the content of olive glycoside is 4-8%, and the content of luteolin is 2%-8%. The olive leaf extract of the present invention contains active ingredients such as oleuropein, olive glycoside and luteolin, and has antioxidant, anti-inflammatory and immunomodulatory effects.

[0030] The mesenchymal stem cell exosomes described in the present invention include the culture of mesenchymal stem cells in the preparation process, and after subculturing to 3 to 4 generations, they are cultured in a serum-free medium. As in the embodiments of the present invention, human umbilical cord mesenchymal stem cells are isolated from the umbilical cord of a healthy full-term delivery, and the markers N-cadherin, CD44, RANKL, CD105, CD56 and / or CD90 are detected; the verified cells are cultured under any conventional conditions for mesenchymal stem cell culture; after subculturing to 3 to 4 generations, the serum-free medium without animal components and proteins is replaced; wherein the conventional culture medium for mesenchymal stem cells can be DMEM / F12 containing 5% hPL, 1% L-Glut and 1% Pen / Strep, and cultured in a culture bottle. The serum-free culture medium described in the present invention, in one embodiment, uses Rohto Pharmaceutical AOF culture medium.

[0031] The present invention cultured the mesenchymal stem cells after passage using a serum-free medium. When the cells reached 70-80% fusion, the serum-free medium was continued to be used for 40-48 hours to promote exosome secretion. Inducers can be added at this stage to increase the exosome yield and functional activity, and the culture supernatant is collected for exosome extraction; the inducer can be selected from any one of transforming growth factor (TGF-β1), fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and interleukin-6 (IL-6), or a combination of two or more thereof.

[0032] The present invention centrifuges the cell supernatant to remove residual intact cells. The centrifugal force used may be 300g, the temperature is 4°C, and the centrifugation time is 10min to remove residual intact cells. Then, the cell debris is removed by centrifugation. The centrifugal force at this time is 2000g, and the centrifugation time is 20min.

[0033] The present invention extracts mesenchymal stem cell exosomes from the supernatant after removing cell debris. The present invention does not specifically limit the extraction method, such as ultrafiltration, polymer precipitation, size exclusion chromatography or ultracentrifugation. In one embodiment, the mesenchymal stem cell exosomes used in the present invention are prepared by ultrafiltration.

[0034] The ultrafiltration method used in the embodiment of the present invention includes passing the supernatant through a cell filtration membrane with a pore size of 4 μm, and then passing through a cell filtration membrane with a pore size of 0.22 μm; adding the filtered supernatant to an ultrafiltration device (Amicon Ultra-15 centrifugal filter), the molecular weight cutoff value is 100 kDa, and centrifuging at a centrifugal force of 3000g for 1 to 2 hours to retain the exosomes on the ultrafiltration membrane; after the ultrafiltration is completed, phosphate buffered saline (PBS), lactated Ringer's solution, A balanced salt solution, such as PBS, is used for washing and resuspending, and 200 to 300 μL of PBS is used to dissolve the exosomes on the ultrafiltration membrane, and the exosomes are repeatedly washed twice to obtain an exosome solution with an osmotic pressure between 250 and 310 mOsmol / L and a pH value between 6.0 and 8.0. The MSCs exosomes prepared by the present invention have a typical cup-shaped structure with a diameter distribution range of about 40 to 150 nm; at least two of the markers CD9, CD63, CD81 and Hsp70 are positive.

[0035] The present invention dries the identified mesenchymal stem cell exosomes to prepare dry powder. For example, the freeze-dried powder is prepared by a freeze-drying method in the embodiment, and the mesenchymal stem cell exosomes are mixed with a protective agent to prevent damage to the exosomes during the freeze-drying process. The protective agent can be one or more of trehalose, sucrose and mannose, and the added amount of the protective agent can be 2-10% w / v, such as 5% w / v. The present invention pre-freezes the exosome sample with the protective agent added: the sample is placed in an ultra-low temperature refrigerator (Thermo FisherForma) at -80°C for 2 to 4 hours until the sample is completely frozen; a freeze dryer (Labconco FreeZone) is used for vacuum sublimation: in the sublimation stage, the condensation temperature is controlled at -40°C to -50°C, the vacuum pressure is 10 to 100 Pa, the temperature rises to -40°C to -20°C, and the time is 2 to 6 hours; in the analysis stage, the temperature is further increased to 25°C to 35°C, the time is 4 to 8 hours, and the temperature is slowly increased throughout the process to avoid damage to the exosome structure; after freeze drying, the dried exosomes are sealed and packaged and stored in a light-proof and moisture-proof container; short-term storage is placed at 2 to 8°C, and long-term storage is in an environment of -20°C to -80°C. The mesenchymal stem cell exosomes of the present invention accelerate the healing of postoperative tissues by promoting cell repair and regeneration.

[0036] The T lymphocyte exosomes of the present invention include culturing T lymphocytes in the preparation process, and culturing the subcultured T lymphocytes until the cell density reaches 70-80%, and then culturing them in a serum-free medium, and the subculture includes sorting allogeneic peripheral blood mononuclear cells, collecting a cell suspension rich in T lymphocytes, culturing the cell suspension rich in T lymphocytes, adding an inducer, and subculturing until the cell density reaches 70-80%; wherein the inducer used can be any one of γ-interferon, anti-CD3 antibody, anti-CD28 antibody and interleukin-2 (IL-2) or a combination of two or more; the subcultured T lymphocytes are subjected to Cultivate, and when the cell density reaches 70-80%, use a serum-free medium without animal components and proteins for cultivation, collect the conditioned medium, and extract exosomes by differential ultracentrifugation; the conditions for the differential ultracentrifugation extraction are: at 4°C, centrifuge at 300g for 10min, take the supernatant, then centrifuge at 2000g for 20min, take the supernatant, then centrifuge at 10000g for 30min, take the supernatant, and finally centrifuge at 120000g for 70min, take the precipitate, resuspend, and obtain T lymphocyte exosomes; the above T lymphocyte exosomes are freeze-dried, and the freeze-drying method is the same as the freeze-drying method of mesenchymal stem cell exosomes, which will not be repeated here. The serum-free medium used in the present invention is, in one embodiment, Rohto Pharmaceutical AOF medium. The T lymphocyte exosomes described in the present invention can improve the postoperative immune status by regulating the immune microenvironment.

[0037] The present invention also provides the use of the above-mentioned pharmaceutical composition in preparing a drug for treating tumors after surgery.

[0038] The postoperative treatment of tumors referred to in the present invention includes inhibiting tumor recurrence, promoting postoperative tissue repair and regulating the immune microenvironment, and the tumors include: lung cancer, breast cancer, liver cancer, cervical cancer, gastric cancer, prostate cancer and colon cancer. The dosage form of the postoperative treatment drug for tumors of the present invention includes injection, oral preparation or transdermal preparation.

[0039] The present invention also provides a drug for treating tumors after surgery, comprising the above-mentioned drug composition and pharmaceutically acceptable excipients.

[0040] The dosage form of the drug of the present invention includes injection, oral preparation or transdermal preparation, and the corresponding auxiliary materials can be selected according to the corresponding dosage form for conventional preparation.

[0041] The present invention also provides an injection for postoperative treatment of tumors, comprising olive leaf extract dry powder, mesenchymal stem cell exosome dry powder, T lymphocyte exosome dry powder and an injection solvent; and the mass percentages of olive leaf extract dry powder, mesenchymal stem cell exosome dry powder and T lymphocyte exosome dry powder in the injection are 10-20%, 15-30% and 4-10%, respectively.

[0042] The solvent for injection of the present invention includes physiological saline. When preparing the injection, the steps include mixing the dry powder with physiological saline, adjusting the pH value to 7.5 to 8, filtering, encapsulating and sterilizing. The filtration of the present invention can be performed using a 0.22 μm ultrafiltration membrane to remove bacteria and impurities, check the clarity, and encapsulate after passing the test. The compound injection of the present invention has stable quality and high bioavailability. The injection of the present invention can be stored at 4°C or -20°C to ensure the activity and stability of the exosomes.

[0043] In order to further illustrate the present invention, a pharmaceutical composition comprising olive leaf extract provided by the present invention, its medicine and application are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] 0.25% trypsin: 0.25% trypsin-EDTA digestion solution was purchased from Gibico, USA;

[0045] DMEM medium was purchased from Gibco, USA; AOF serum-free medium was purchased from Rohto Pharmaceutical, Japan.

[0046] Example 1

[0047] 1. Olive leaf extract is prepared by the following method:

[0048] Select healthy olive leaves without mildew, wash, dry and crush to 70 mesh particles. Mix olive leaf powder with 80% ethanol solution at a solid-liquid ratio of 1:10, moisten at 25℃ for 60 minutes, and ensure that the solvent evenly penetrates into the leaf tissue. The moistened olive leaf liquid is placed in a vacuum extraction device and the vacuum degree is adjusted to -0.05MPa; at 80°C, an 80% ethanol solution preheated to 50°C is injected; the extraction is continued with stirring for 12 hours, and each batch is extracted 3 times; the extract is filtered and concentrated to 1 / 3 of the original volume under reduced pressure; purification is performed using LSA type macroporous adsorption resin, eluted with deionized water, the sample loading flow rate is 10BV / h, and 30% ethanol aqueous solution is used for analysis, and the analysis flow rate is 5BV / h; the above eluate is concentrated to a viscous state; vacuum freeze-drying is performed with a vacuum degree of 0.1 to 0.5mbar, pre-frozen to -40°C, gradually heated to 20°C, and freeze-drying is continued for 48 hours; the content of oleuropein and oleuropein is detected by HPLC to ensure batch consistency; the appearance inspection is a light yellow powder with no odor.

[0049] 2. Mesenchymal stem cell exosomes were prepared by the following method:

[0050] Human umbilical cord mesenchymal stem cells were isolated from the umbilical cord of a healthy full-term delivery, and the markers N-cadherin, CD44, RANKL, CD105, CD56, and CD90 were detected; the verified cells were cultured in DMEM / F12 medium (supplemented with 5% hPL, 1% L-Glut, 1% Pen / Strep) in a culture flask. After subculture to the third generation, the serum-free medium (Japan Rohto Pharmaceutical AOF medium) was replaced. When the cells reached 70% fusion, they were cultured for 48 hours to promote exosome secretion. Inducers added at this stage included 5 ng / mL transforming growth factor (TGF-β1, Sigma-Aldrich, T7039), 20 ng / mL fibroblast growth factor (bFGF, Sigma-Aldrich, F0291), 10 ng / mL epidermal growth factor (EGF, Sigma-Aldrich, E9644), 20 ng / mL insulin-like growth factor (IGF-1, Sigma-Aldrich, I3769), 30 ng / mL vascular endothelial growth factor (VEGF, Sigma-Aldrich, V7259), 20 ng / mL hepatocyte growth factor (HGF, PeproTech, 100-39), and 20 ng / mL interleukin-6 (IL-6, PeproTech, 200-06); the culture supernatant was collected for exosome extraction; the collected culture supernatant was centrifuged at 300 g for 10 min at 4°C to remove residual intact cells. Centrifuge again at 2000g for 20min to remove cell debris in the supernatant; pass the supernatant through a cell filter membrane with a pore size of 4μm first, and then through a cell filter membrane with a pore size of 0.22μm to further remove larger impurity particles; use a 100kDa ultrafiltration membrane to filter the exosomes, add the filtered supernatant to an ultrafiltration device (Amicon Ultra-15 centrifugal filter), and centrifuge at 3000g for 1.5h; dissolve the exosomes on the ultrafiltration membrane with 200μL PBS and wash twice to obtain an exosome solution with an osmotic pressure of 280mOsmol / L and a pH of 7.5.

[0051] 3. T lymphocyte exosomes were prepared by the following method:

[0052] Peripheral blood mononuclear cells were isolated from the peripheral blood of allogeneic subjects using a lymphocyte separation solution by conventional methods; the obtained peripheral blood mononuclear cells were sorted to collect a cell suspension rich in T lymphocytes; the cell suspension rich in T lymphocytes was cultured in RPMI 1640 medium, and inducers including 20 ng / mL γ-interferon (ThermoFisher, 300-02-500UG), 12 μg / mL anti-CD3 antibody (Anti-CD3 antibody, Thermo Fisher, 16-0037-81), 15 μg / mL anti-CD28 antibody (Anti-CD28 antibody, Thermo Fisher, 16-0289-81) and 10 ng / mL IL-2 (interleukin-2, Thermo Fisher, PHC0026) were added to ensure that the cells were in good growth state. After the cell fusion rate reached 75%, serum-free medium (Japan Rohto Pharmaceutical AOF medium) was used for subculture. After 72 hours, the conditioned medium was collected and exosomes were extracted by differential ultracentrifugation: the conditioned medium was centrifuged at 300g for 10 min at 4°C to obtain the supernatant and remove intact cells; the conditioned medium was centrifuged at 2000g for 20 min to obtain the supernatant and further remove dead cells and impurities; the conditioned medium was centrifuged at 10000g for 30 min to obtain the supernatant and remove cell debris, and finally centrifuged at 120000g for 70 min, the supernatant was discarded, the precipitate was resuspended in 50L PBS, and filtered through a 0.22μm ultrafiltration membrane to obtain the T lymphocyte exosome solution.

[0053] 4. Freeze-dry the above mesenchymal stem cell exosomes and T lymphocyte exosomes:

[0054] 5% w / v trehalose was added to the exosome solution as a protective agent. The exosome samples with the protective agent were pre-frozen: the samples were placed in an ultra-low temperature freezer (Thermo FisherForma) at -80°C for 2 hours, and then vacuum sublimated using a freeze dryer (Labconco FreeZone). During the sublimation stage, the condensation temperature was controlled at -40°C, the vacuum pressure was set to 80Pa, and the temperature was raised to -20°C for 5 hours; during the analysis stage, the temperature was raised to 35°C for 5 hours, and the temperature was slowly raised throughout the process to avoid damage to the exosome structure. After freeze-drying, the dried exosomes were sealed and packaged, stored in a light-proof and moisture-proof container at -80°C.

[0055] 5. The preparation method of the compound injection comprises the following steps:

[0056] Under sterile conditions, olive leaf extract, mesenchymal stem cell exosome solution and T lymphocyte exosome were dissolved in sterile saline for injection and stirred evenly; the mass ratio of olive leaf extract, mesenchymal stem cell exosome and T lymphocyte exosome was 10:15:4 respectively. Glycerol was added as a stabilizer (final concentration was 5%), and the pH value of the compound injection was adjusted to 7.5 with sodium hydroxide solution; the prepared compound injection was filtered through a 0.22μm filter membrane; the filtered injection was dispensed into sterile glass ampoules and sealed and stored at 4°C.

[0057] The HPLC analysis results of the olive leaf extract obtained in Example 1 are as follows: Figure 1 As shown; the mesenchymal stem cell exosomes and T lymphocyte exosomes obtained in Example 1 were identified, as shown Figure 2 As shown in Figure 2, under transmission electron microscopy, exosomes were observed to have a saucer-shaped double-layer capsule ultrastructure, and nanoparticle tracing analysis (NAT) showed that the particle size was mainly distributed in the range of 40 to 150 nm ( Figure 3 ), which is consistent with the results observed under transmission electron microscopy (TEM). Western blot analysis was used to detect specific markers of exosomes (CD9, CD63, CD81 and Hsp70), and at least two of these markers were positive, while the negative marker of exosomes (Calnexin) was negative ( Figure 4 ).

[0058] Example 2

[0059] On the basis of Example 1, the difference from Example 1 is as follows: the ratio of the components of the compound injection: the mass proportions of olive leaf extract, mesenchymal stem cell exosomes, and T lymphocyte exosomes are 14:20:6 respectively.

[0060] Example 3

[0061] On the basis of Example 1, the difference from Example 1 is as follows: the ratio of the components of the compound injection: the mass proportions of olive leaf extract, mesenchymal stem cell exosomes, and T lymphocyte exosomes are 18:25:8 respectively.

[0062] Example 4

[0063] On the basis of Example 1, the difference from Example 1 is as follows: the ratio of the components of the compound injection: the mass proportions of olive leaf extract, mesenchymal stem cell exosomes, and T lymphocyte exosomes are 20:30:10 respectively.

[0064] Comparative Example 1

[0065] On the basis of Example 3, the main components of the compound injection were adjusted to include only olive leaf extract and mesenchymal stem cell exosomes, and no longer included T lymphocyte exosomes, and the rest of the technical solutions remained unchanged.

[0066] Comparative Example 2

[0067] On the basis of Example 1, the main components of the compound injection were adjusted to include only olive leaf extract and T lymphocyte exosomes, and no longer included mesenchymal stem cell exosomes, and the rest of the technical solutions remained unchanged.

[0068] Comparative Example 3

[0069] On the basis of Example 1, the main components of the compound injection were adjusted to include only mesenchymal stem cell exosomes and T lymphocyte exosomes, and no longer included olive leaf extract, and the rest of the technical solutions remained unchanged.

[0070] Comparative Example 4

[0071] On the basis of Example 1, the main components of the compound injection were adjusted to include only olive leaf extract, and no longer included mesenchymal stem cell exosomes and T lymphocyte exosomes, and the rest of the technical solutions remained unchanged.

[0072] Comparative Example 5

[0073] On the basis of Example 1, the main components of the compound injection were adjusted to include only T lymphocyte exosomes, and no longer included olive leaf extract and mesenchymal stem cell exosomes, and the rest of the technical solutions remained unchanged.

[0074] Comparative Example 6

[0075] On the basis of Example 1, the main components of the compound injection were adjusted to include only mesenchymal stem cell exosomes, and no longer included olive leaf extract and T lymphocyte exosomes, and the rest of the technical solutions remained unchanged.

[0076] The injections prepared in Examples 1 to 4 and Comparative Examples 1 to 6 were subjected to efficacy tests.

[0077] 1. Tumor cell inhibition CCK-8 method

[0078] The CCK-8 method was used to detect the proliferation inhibition effects of the injections prepared in Examples 1 to 4 and Comparative Examples 1 to 6 on lung cancer cells A549, breast cancer cells MCF-7, liver cancer cells HepG2, cervical cancer Hela, gastric cancer SNU-1, prostate cancer PC3, and colon cancer cells SW480. The above cell lines were provided by the JCRB Cell Collection Center.

[0079] Experimental cells Lung cancer cells A549, breast cancer cells MCF-7, liver cancer cells HepG2, cervical cancer cells Hela, gastric cancer cells SNU-1, prostate cancer cells PC3, and colon cancer cells SW480 were resuscitated in a 37°C water bath for 90 seconds and then centrifuged at 1000 rpm. The centrifuged cells were cultured in the prepared mixed culture medium; when the cell density grew to about 80%, the cells were passaged, the cells were rinsed twice with PBS, 0.25% trypsin was added, and digested at 37°C for 90 seconds. The culture flask was gently shaken to make 90% of the cells fall off, and then 5 mL of mixed culture medium was added to terminate the digestion. Finally, the cells were centrifuged at 1000 rpm for 5 minutes and divided into culture flasks at a ratio of 1:3 for continued culture.

[0080] Cells in the logarithmic growth phase were inoculated in a 96-well plate, and three duplicate wells were set; after overnight attachment, 10 μL of the injection solution prepared in Examples 1 to 4 and Comparative Examples 1 to 6 was directly added to the culture medium, gently shaken to mix, and placed in a 37°C 5% CO2 incubator for continued cultivation; at the detection time point of 2d (48h), the cells were taken out, the cell supernatant was discarded, and gently washed twice with PBS; the CCK-8 reagent prepared in advance was added to the cells, 100 μL / well; after incubation at 37°C for 2 hours, the absorbance value at 450nm was detected with a marker. The absorbance of cells not treated with the injection solution was used as the 100% cell activity control at each time point, and etoposide (VP16) was used as a positive control.

[0081] The cytotoxicity of the injections of Examples 1 to 4 and Comparative Examples 1 to 6 on seven tumor cell lines in the logarithmic growth phase is shown in Table 1: the compound injections prepared in Examples 1 to 4 all showed significant tumor cell proliferation inhibition, especially Example 3 had the best effect, with an inhibition rate of 78%±4% on lung cancer A549, 72%±5% on breast cancer MCF-7, 82%±5% on liver cancer HepG2, 74%±4% on cervical cancer Hela, 70%±6% on gastric cancer SNU-1, 76%±5% on prostate cancer PC3, and 79%±4% on colon cancer SW480.

[0082] Compared with Examples 1 to 4, the injections prepared in Comparative Examples 1 to 6 showed significantly lower inhibition rates, indicating that each individual component had a weak inhibitory effect on tumor cell proliferation and lacked a synergistic effect between the components. In particular, the inhibition rates of Comparative Examples 4 and 6 were significantly lower than those of the other groups. This indicates that when only a single component acts, it cannot effectively exert a synergistic anti-tumor effect, and the synergistic effect of olive leaf extract and exosomes is the key to improving the efficacy.

[0083] Table 1 Inhibitory activity of Examples 1 to 4 and Comparative Examples 1 to 6 on tumor cells

[0084]

[0085] 2. ELISA method for immune factor regulation

[0086] The effects of the injections prepared in Examples 1 to 4 and Comparative Examples 1 to 6 on the function of immune cells were detected by ELISA, and their regulatory effects on the secretion of immune factors interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4) and interleukin-10 (IL-10) were evaluated.

[0087] In this experiment, peripheral blood mononuclear cells, T lymphocytes and RAW 264.7 macrophages were selected, and the control groups included a negative control (PBS solution) and a positive control (immune activator: anti-CD3 antibody).

[0088] Cell culture conditions: PBMCs were isolated from the peripheral blood of healthy volunteers by Ficoll density gradient centrifugation and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. T lymphocytes and RAW 264.7 macrophages were cultured in RPMI-1640 and DMEM medium, respectively, also supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were cultured in an incubator at 37°C and 5% CO2.

[0089] The drug volume for each experimental treatment was 100 μL, and it was ensured that each experimental group was repeated at least three times. After cell treatment, the culture was continued at 37 ° C and 5% CO2 for 48 hours. The cell supernatant of each group was collected for cytokine detection, and each experimental group was repeated at least three times. The absorbance (OD value) was read at the corresponding wavelength by an ELISA instrument, and the cytokine concentration in each group of samples was calculated according to the standard curve. As shown in Table 2, the compound injection of the embodiment group (1 to 4) significantly improved the level of immune factors, especially in the concentration of IFN-γ and TNF-α, indicating that the compound injection can effectively activate the immune system and enhance the anti-tumor immune response. Example 3 showed the strongest immune activation effect, which may be due to the synergistic effect between olive leaf extract and T lymphocyte exosomes, and the ratio was appropriate. The comparative examples containing only olive leaf extract or mesenchymal stem cell exosomes showed a significantly weakened immune activation effect, especially in comparative examples 4 and 6, where the cytokine levels were low, indicating that the stimulation of the immune system by using these components alone was relatively limited.

[0090] Table 2 Regulatory effects of the examples and comparative examples on the secretion of immune factors

[0091] Group IFN-γ (pg / mL) TNF-α(pg / mL) IL-4 (pg / mL) IL-10 (pg / mL) Example 1 150±10 120±8 20±5 30±4 Example 2 160±8 140±7 22±4 28±5 Example 3 180±12 150±10 25±6 35±3 Example 4 170±10 130±9 23±5 32±4 Comparative Example 1 80±5 90±6 15±3 18±2 Comparative Example 2 85±7 95±7 16±4 20±3 Comparative Example 3 70±5 85±5 13±2 15±2 Comparative Example 4 50±3 70±4 12±3 10±2 Comparative Example 5 65±5 80±6 15±4 14±3 Comparative Example 6 60±4 75±5 14±3 12±3 Negative control 20±3 30±5 10±2 8±1 Positive Control 200±15 180±12 30±6 45±5

[0092] 3. Tumor recurrence inhibition effect

[0093] 120 healthy C57BL / 6 mice were selected as experimental animals to establish an animal model of tumor postoperative repair. Human non-small cell lung cancer cells A549 were inoculated subcutaneously on the back of mice, with each mouse injected with approximately 1×10 6 When the tumor volume grows to 50mm 3 When the tumor is removed, surgery is performed. All mice will be randomly assigned to each group to ensure that the mice in each group are balanced in terms of weight, tumor size and gender. There are 10 mice in each group, for a total of 12 groups. The groups include Example 1 to 4 groups, Comparative Example 1 to 6 groups, a negative control group (normal saline) and a positive control group (interferon). During the recovery period after surgery, the compound injection or the solution of different control groups is administered by intraperitoneal injection. The injection dose is 10 mg / kg body weight, and the injection is continued for 7 days.

[0094] The tumor recurrence and weight changes of mice were measured regularly to evaluate the effect of the compound injection. The mice in each group were monitored for tumor recurrence by imaging examination (ultrasound and CT scan), and the recurrence rate was calculated. At the same time, the mice were weighed regularly to evaluate their weight changes as an indicator of overall health and postoperative recovery. After the treatment, 3 mice in each group were randomly selected, and the surgical resection site and surrounding tissues were fixed in 4% paraformaldehyde solution, and paraffin sections were prepared routinely. The sections were stained with anti-VEGF antibodies to detect the expression levels of angiogenesis-related proteins. The distribution and number of VEGF-positive cells were observed by optical microscopy. ImageJ software was used to count the area and density of positive cells and compare the differences between the groups.

[0095] The results are shown in Table 3 and Figure 5 As shown, the mice in the embodiment group showed a significant tumor recurrence rate inhibition effect, especially in the embodiment 4 group, the tumor recurrence rate was the lowest (10% ± 3%). The recurrence rate of the embodiment group was generally lower than that of the control group, indicating that the compound injection had a good effect on postoperative tumor repair. Among the control groups, the recurrence rate of the negative control group was the highest, reaching 70% ± 8%, proving that the tumor recurrence in the group lacking therapeutic intervention was more serious. The recurrence rate of the positive control group (interferon group) was 32% ± 5%, showing a certain effect, but still not as good as the embodiment group.

[0096] The results are as follows Figure 6As shown, the mice in the Example groups also showed a significant advantage in weight recovery, especially in Example 4 group (+10% ± 4%), while the negative control group showed a weight loss (-8% ± 4%). The change in body weight reflects the postoperative recovery. The weight gain of the mice in the Example groups indicates that the compound injection has a positive effect on postoperative recovery. The mice in the control groups, especially the comparative group and the positive control group, had limited or decreased weight changes, indicating that the effects of these individual components were relatively weak.

[0097] The experimental data of VEGF positive rate are as follows Figure 7 As shown, the embodiment groups (groups 1-4) all showed a high VEGF positivity rate (72% to 85%), indicating that the compound injection may help postoperative tissue repair and reduce tumor recurrence by promoting angiogenesis. In contrast, the VEGF positivity rate of the negative control group (normal saline) was low (30% ± 7%), showing that the tissue repair effect of normal saline treatment alone was limited. The VEGF positivity rate of the positive control group (interferon) was 78% ± 5%, indicating that it has a certain angiogenesis-promoting effect, but the effect of the compound injection is more significant to a certain extent. The VEGF positivity rate of comparative examples 1 to 6 groups was lower than that of the embodiment group as a whole, further confirming the advantages of the compound injection.

[0098] In summary, the compound injection (Examples 1 to 4) has a significant positive effect on tumor postoperative recovery, can effectively reduce tumor recurrence rate and promote weight recovery of mice. In addition, the VEGF positive rate of the Example group is high, indicating that the compound injection has a good effect in promoting angiogenesis, further supporting its ability to reduce tumor recurrence by improving angiogenesis and tissue repair, and providing its potential as a postoperative adjuvant therapy.

[0099] Table 3 Effects of Examples 1 to 4 and Comparative Examples 1 to 6 on postoperative repair of non-small cell lung cancer in mice

[0100]

[0101] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition having a postoperative therapeutic effect on tumors, characterized in that: Including olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes.

2. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes is (10-20): (15-30): (4-10).

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The olive leaf extract comprises an ethanol extract of olive leaves, and the mass percentage of oleuropein in the dry powder of the olive leaf extract is not less than 45%, and the mass percentage of oleuropein is not less than 4%.

4. The pharmaceutical composition according to claim 1 or 2, characterized in that The preparation process of the mesenchymal stem cell exosomes includes culturing the mesenchymal stem cells, and after subculturing to the third to fourth generations, culturing is performed in a serum-free medium.

5. The pharmaceutical composition according to claim 1 or 2, characterized in that: The preparation process of the T lymphocyte exosomes includes culturing T lymphocytes, and culturing the subcultured T lymphocytes until the cell density reaches 70-80%, and then culturing them in a serum-free medium.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of drugs for postoperative treatment of tumors.

7. The use according to claim 6, characterized in that: The dosage form of the tumor postoperative therapeutic drug includes injection, oral preparation or transdermal preparation.

8. The use according to claim 6, characterized in that: The therapeutic spectrum of the tumor postoperative treatment drug includes: lung cancer, breast cancer, liver cancer, cervical cancer, gastric cancer, prostate cancer and colon cancer.

9. A drug for treating tumors after surgery, characterized in that: The invention comprises the pharmaceutical composition according to any one of claims 1 to 5 and pharmaceutically acceptable excipients.

10. An injection for treating tumors after surgery, characterized in that: It includes olive leaf extract dry powder, mesenchymal stem cell exosome dry powder, T lymphocyte exosome dry powder and injection solvent; and the mass percentages of olive leaf extract dry powder, mesenchymal stem cell exosome dry powder and T lymphocyte exosome dry powder in the injection are 10-20%, 15-30% and 4-10%, respectively.

Images