A pharmaceutical composition containing olive leaf extract, medicine and application thereof

Abstract

The present invention provides a pharmaceutical composition comprising an olive leaf extract, a medicament, and its application, belonging to the field of biomedical technology. The present invention provides a pharmaceutical composition based on an olive leaf extract, mesenchymal stem cell exosomes, and T lymphocyte exosomes. The synergistic application of these components can effectively promote tissue repair and immune function recovery after tumor surgery. The present invention has developed a drug with postoperative tumor therapeutic effects that can effectively inhibit the proliferation of various tumor cells, improve postoperative immune status, and significantly promote tissue repair. The present invention provides a natural, safe, and innovative solution for adjuvant treatment of tumors after surgery, with flexible dosage form development potential, broad clinical application prospects, and market value.

Description

Technical Field

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

[0002] Postoperative tumor recurrence and metastasis are major challenges that urgently need to be addressed in cancer treatment. Although surgery, radiotherapy, and chemotherapy can eliminate the primary tumor, inadequate regulation of the postoperative microenvironment often leads to recurrence and even distant metastasis of residual tumor cells, significantly reducing patient survival and quality of life. Therefore, exploring novel adjuvant therapies that can effectively regulate the postoperative microenvironment and inhibit tumor recurrence and metastasis is of great scientific significance and clinical value.

[0003] Olive leaf extract is a natural product extracted from a traditional herb, rich in compounds such as oleuropein, olive glycosides, and luteolin. While oleuropein has been shown to be effective in cancer treatment, the efficacy of olive leaf extract alone is limited, and its strength and mechanism of action have not yet been fully explored. Therefore, there is a need to compound olive leaf extracts and develop drugs with specific therapeutic effects. Summary of the Invention

[0004] The present invention provides a pharmaceutical composition comprising an olive leaf extract, a medicine thereof, and an application thereof. The pharmaceutical composition or medicine can improve the patient's postoperative recovery quality and living standard 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 therapeutic effect after tumor surgery, 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 in a serum-free medium.

[0009] In a preferred embodiment of the present invention, 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.

[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 treating tumors after surgery.

[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 drug for postoperative tumor treatment 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 medicinal effects of olive leaf extract with the modern biological functions of the two exosomes to give full play to their synergistic effects in immunomodulation, inflammation suppression, and tissue repair, providing a new solution for postoperative tumor repair. The olive leaf extract of the present invention contains active ingredients such as oleuropein, oleuropein, and luteolin, which have antioxidant, anti-inflammatory, and immunomodulatory effects; mesenchymal stem cell exosomes accelerate postoperative tissue healing by promoting cell repair and regeneration; and 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 tumor tissue repair and immune function recovery through synergistic application.

[0016] The present invention utilizes the pharmaceutical composition to develop a drug with a postoperative tumor treatment effect. Cell-based experiments have demonstrated that the pharmaceutical composition or drug of the present invention has universal applicability to the postoperative repair of various tumor cells, effectively inhibiting their proliferation, improving postoperative immune status, and significantly promoting tissue repair. In animal experiments, the pharmaceutical composition also demonstrated excellent postoperative tumor repair effects, improving immune status, accelerating tissue repair, and functional recovery. This invention provides a natural, safe, and innovative solution for adjuvant treatment of postoperative tumors, with flexible dosage form development potential, 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 Figure 1 shows the results of exosome detection provided by the present invention, in which (A) is a transmission electron micrograph of MSCs exosomes; (B) is a transmission electron micrograph of T lymphocyte exosomes;

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

[0020] Figure 4 Figure 2 is a graph showing the immunoblotting 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;

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

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

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

[0024] The present 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). 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 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%. The solvent used in the extraction process includes an ethanol-water solution. In one embodiment, a 70-90% ethanol-water solution is selected, mixed at a material-liquid ratio of 1:5 to 1:15, and then moistened at room temperature for 20-60 minutes to ensure uniform penetration of the solvent into the leaf tissue. The present invention selects healthy, mold-free olive leaves, cleans and dries them, and then crushes them into 40-100 mesh particles before moistening. 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 to -0.08 MPa; a 70% to 90% ethanol solution preheated to 40 to 50° C. is injected at 60 to 80° C., and the solvent is rapidly vaporized to form an internal and external pressure difference, thereby promoting the rapid release of active ingredients in cells; the extraction is continuously stirred for 12 hours, and each batch is extracted 2 to 3 times; the extract is filtered and concentrated under reduced pressure to 1 / 3 to 1 / 4 of the original volume for concentration.

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

[0029] The present invention concentrates the above-mentioned eluate 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 the 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℃, gradually warmed to -10℃~20℃, and freeze-dried for 12~48 hours. The olive leaf extract freeze-dried powder prepared by the present invention is a light yellow or light green powder with no odor when detected by appearance. The content of oleuropein and oleuropein is detected by high performance liquid chromatography (HPLC), and it is found that the content of oleuropein is 45~65%, the content of oleuropein 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, oleuropein and luteolin, and has antioxidant, anti-inflammatory and immunomodulatory effects.

[0030] The preparation process of the mesenchymal stem cell exosomes described herein includes culturing mesenchymal stem cells, and after subculturing for 3-4 generations, culturing in a serum-free medium. For example, in an embodiment of the present invention, human umbilical cord mesenchymal stem cells are isolated from the umbilical cord of a healthy full-term fetus and tested for markers such as N-cadherin, CD44, RANKL, CD105, CD56, and / or CD90. The validated cells are cultured under conventional conditions for mesenchymal stem cell culture. After subculturing for 3-4 generations, the medium is replaced with a serum-free medium that is free of animal components and protein. The conventional medium for mesenchymal stem cells can be DMEM / F12 containing 5% hPL, 1% L-Glut, and 1% Pen / Strep, and cultured in a culture flask. In one embodiment, the serum-free medium described herein uses Rohto Pharmaceutical's AOF 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. At this stage, an inducer can be added 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 or a combination of two or more 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).

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

[0033] The present invention extracts mesenchymal stem cell exosomes from the supernatant after removing cell debris. The present invention is not particularly limited to 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 through a cell filtration membrane with a pore size of 0.22 μm; the filtered supernatant is added to an ultrafiltration device (Amicon Ultra-15 centrifugal filter) with a molecular weight cutoff of 100 kDa, and centrifuged 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, Wash and resuspend with a balanced salt solution, such as PBS. Dissolve the exosomes on the ultrafiltration membrane with 200-300 μL of PBS. Repeat the washing twice to obtain an exosome solution with an osmotic pressure between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. The MSC exosomes prepared by the present invention have a typical cup-shaped structure with a diameter distribution range of approximately 40 to 150 nm. They are positive for at least two of the markers CD9, CD63, CD81, and Hsp70.

[0035] The present invention dries the identified mesenchymal stem cell exosomes to prepare a 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 exosome samples containing a protective agent by placing them in a -80°C ultra-low temperature freezer (Thermo Fisher Forma) for 2-4 hours until completely frozen. Vacuum sublimation is then performed using a freeze dryer (Labconco FreeZone). During the sublimation phase, the condensation temperature is controlled at -40°C to -50°C, the vacuum pressure is 10-100 Pa, and the temperature is raised to -40°C to -20°C for 2-6 hours. During the desorption phase, the temperature is further raised to 25°C to 35°C for 4-8 hours, with the temperature increasing slowly throughout the process to avoid structural damage to the exosomes. After freeze-drying, the dried exosomes are sealed and stored in a light-proof and moisture-proof container. Short-term storage is at 2-8°C, and long-term storage is at -20°C to -80°C. The mesenchymal stem cell exosomes described in this invention accelerate postoperative tissue healing by promoting cell repair and regeneration.

[0036] The T lymphocyte exosomes of the present invention include the cultivation of T lymphocytes in the preparation process, and the T lymphocytes after subculture are cultured until the cell density reaches 70-80%, and then cultured 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 subculture until the cell density reaches 70-80%; wherein the inducer used can be any one of gamma-interferon, anti-CD3 antibody, anti-CD28 antibody and interleukin-2 (IL-2) or a combination of two or more; the T lymphocytes after subculture are cultured. Cultivate, and when the cell density reaches 70-80%, use a serum-free medium without animal components and protein for culture, 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 10 minutes, take the supernatant, then centrifuge at 2000g for 20 minutes, take the supernatant, then centrifuge at 10,000g for 30 minutes, take the supernatant, and finally centrifuge at 120,000g for 70 minutes, take the precipitate, resuspend, and obtain T lymphocyte exosomes; the above T lymphocyte exosomes are freeze-dried. The freeze-drying method is the same as the freeze-drying method for mesenchymal stem cell exosomes and 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 postoperative immune status by regulating the immune microenvironment.

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

[0038] The postoperative treatment of tumors referred to in this invention includes inhibiting tumor recurrence, promoting postoperative tissue repair, and modulating the immune microenvironment. The tumors described include lung cancer, breast cancer, liver cancer, cervical cancer, gastric cancer, prostate cancer, and colon cancer. The dosage forms of the postoperative treatment of tumors described in this invention include injections, oral preparations, or transdermal preparations.

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

[0040] The dosage forms of the drug of the present invention include injections, oral preparations or transdermal preparations, and corresponding excipients 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 injection solvent of the present invention includes physiological saline. Preparation of the injection comprises mixing the dry powders with physiological saline, adjusting the pH to 7.5-8, filtering, encapsulating, and sterilizing. The filtration process can be performed using a 0.22 μm ultrafiltration membrane to remove bacteria and impurities. The clarity is then checked and, if acceptable, encapsulated. The compound injection of the present invention exhibits stable quality and high bioavailability. The injection can be stored at either 4°C or -20°C to ensure the activity and stability of the exosomes.

[0043] To further illustrate the present invention, a pharmaceutical composition comprising an olive leaf extract, a medicine thereof, and applications thereof provided by the present invention 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, mold-free olive leaves, clean, dry, and crush into 70-mesh particles. Mix the olive leaf powder with 80% ethanol solution at a solid-liquid ratio of 1:10 and moisten at 25°C for 60 minutes to ensure that the solvent evenly penetrates the leaf tissue. The moistened olive leaf liquid was placed in a vacuum extraction device and the vacuum degree was adjusted to -0.05 MPa; at 80°C, an 80% ethanol solution preheated to 50°C was injected; the extraction was continued with stirring for 12 hours, and each batch was extracted 3 times; the extract was filtered and concentrated under reduced pressure to 1 / 3 of the original volume; purification was carried out using LSA type macroporous adsorption resin, eluted with deionized water, the sample flow rate was 10BV / h, and 30% ethanol aqueous solution was used for decomposition, and the decomposition flow rate was 5BV / h; the above eluate was concentrated to a viscous state; vacuum freeze-drying was carried out with a vacuum degree of 0.1-0.5mbar, pre-frozen to -40°C, gradually heated to 20°C, and freeze-drying continued for 48 hours; the content of oleuropein and oleuropein was detected by HPLC to ensure batch consistency; the appearance inspection showed that it was 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 fetus and assayed for the presence of markers such as N-cadherin, CD44, RANKL, CD105, CD56, and CD90. The validated cells were cultured in DMEM / F12 medium (supplemented with 5% hPL, 1% L-Glut, and 1% Pen / Strep) in culture flasks. After three passages, the medium was replaced with serum-free medium (Rohto Pharmaceutical AOF medium). When the cells reached 70% confluency, they were cultured for an additional 48 hours to promote exosome secretion. At this stage, inducers added 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 300g for 10 min at 4°C to remove residual intact cells. The supernatant was centrifuged again at 2000 g for 20 min to remove cell debris. The supernatant was first passed through a cell filter membrane with a pore size of 4 μm and then through a cell filter membrane with a pore size of 0.22 μm to further remove larger impurity particles. The exosomes were filtered using a 100 kDa ultrafiltration membrane, and the filtered supernatant was added to an ultrafiltration device (Amicon Ultra-15 centrifugal filter) and centrifuged at 3000 g for 1.5 h. The exosomes on the ultrafiltration membrane were dissolved with 200 μL of PBS and washed twice to obtain an exosome solution with an osmotic pressure of 280 mOsmol / 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 conventional lymphocyte separation fluid. The obtained peripheral blood mononuclear cells were sorted, and a T lymphocyte-enriched cell suspension was collected. The T lymphocyte-enriched cell suspension was cultured in RPMI 1640 medium with the addition of inducers including 20 ng / mL gamma 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). After ensuring good cell growth and reaching a cell confluence rate of 75%, the cells were switched to serum-free medium (Rohto Pharmaceutical AOF medium, Japan) 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 minutes at 4°C to remove the supernatant and remove intact cells; the supernatant was then centrifuged at 2000g for 20 minutes to further remove dead cells and impurities; the supernatant was then centrifuged at 10000g for 30 minutes to remove cell debris, and finally centrifuged at 120000g for 70 minutes, the supernatant was discarded, and 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 and frozen 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 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 exosomes, and T lymphocyte exosomes were dissolved in sterile saline for injection and stirred evenly. The weight ratio of olive leaf extract, mesenchymal stem cell exosomes, and T lymphocyte exosomes was 10:15:4, respectively. Glycerol was added as a stabilizer (final concentration of 5%), and the pH of the compound injection was adjusted to 7.5 using sodium hydroxide solution. The prepared compound injection was filtered through a 0.22μm filter membrane. The filtered injection solution was dispensed into sterile glass ampoules and stored sealed 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 the figure, under transmission electron microscopy, exosomes were observed to have a saucer-shaped double-layer capsule ultrastructure, and nanoparticle tracer 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 exosome markers (CD9, CD63, CD81 and Hsp70). At least two of these markers were positive, while the exosome negative marker (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] Based on 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. The rest of the technical solutions remained unchanged.

[0066] Comparative Example 2

[0067] Based on 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. The rest of the technical solutions remained unchanged.

[0068] Comparative Example 3

[0069] Based on 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] Based on 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] Based on 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] Based on 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 efficacy test was conducted on the injections prepared in Examples 1 to 4 and Comparative Examples 1 to 6.

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

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

[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 revived 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 reached about 80%, they were passaged, washed twice with PBS, added with 0.25% trypsin, and digested at 37°C for 90 seconds. The culture flask was gently shaken to remove 90% of the cells, and then added into 5 mL of mixed culture medium 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 seeded in 96-well plates with three replicate wells. After overnight adherence, 10 μL of the injection solutions prepared in Examples 1 to 4 and Comparative Examples 1 to 6 were added directly to the culture medium, gently shaken to mix, and then placed in a 37°C 5% CO2 incubator for continued culture. At the detection time point of 2 days (48 hours), the cells were removed, the cell supernatant discarded, and gently washed twice with PBS. 100 μL / well of the pre-prepared CCK-8 reagent was added to the cells. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a spectrophotometer. The absorbance of cells not treated with the injection solution was used as the 100% cell viability 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 against seven tumor cell lines in the logarithmic growth phase is shown in Table 1. The compound injections prepared in Examples 1 to 4 all exhibited significant inhibitory effects on tumor cell proliferation, with Example 3 showing the best effect, with inhibition rates of 78%±4% against lung cancer A549, 72%±5% against breast cancer MCF-7, 82%±5% against liver cancer HepG2, 74%±4% against cervical cancer Hela, 70%±6% against gastric cancer SNU-1, 76%±5% against prostate cancer PC3, and 79%±4% against colon cancer SW480.

[0082] Compared with Examples 1-4, the injections prepared in Comparative Examples 1-6 exhibited significantly lower inhibition rates, indicating that each component alone had a weak inhibitory effect on tumor cell proliferation and lacked a synergistic effect among the components. In particular, the inhibition rates in Comparative Examples 4 and 6 were significantly lower than those in the other groups. This suggests that the synergistic anti-tumor effect cannot be effectively exerted by a single component alone, and that the synergistic effect of olive leaf extract and exosomes is key to improving 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 immune cell function 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 a 37°C, 5% CO2 incubator.

[0089] The volume of the drug 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 enzyme marker, and the cytokine concentration in each group of samples was calculated according to the standard curve. The results are shown in Table 2. The compound injection of Example Groups (1 to 4) significantly improved the level of immune factors, especially in the concentrations 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 their ratio is 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 stimulating effect of these components alone on the immune system is relatively limited.

[0090] Table 2 Regulatory effects of Examples and Comparative Examples on immune factor secretion

[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 repair after surgery. Human non-small cell lung cancer A549 cells were inoculated subcutaneously on the back of the mice, with each mouse receiving an injection of 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 12 groups in each group, with 10 mice. 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 dose of the injection 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. Imaging examinations (ultrasound and CT scans) were used to monitor whether the mice in each group had tumor recurrence, and the recurrence rate was calculated. At the same time, the mice were weighed regularly, and their weight changes were evaluated as an indicator of overall health and postoperative recovery. After the treatment, 3 mice were randomly selected from each group, and the surgical resection site and surrounding tissues were taken, fixed in 4% paraformaldehyde solution, and paraffin sections were prepared conventionally. 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 statistically analyze 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 Example groups showed a significant inhibitory effect on tumor recurrence rate, especially in Example 4, which had the lowest tumor recurrence rate (10% ± 3%). The recurrence rate in the Example groups was generally lower than that in the control group, indicating that the compound injection has a good effect on tumor repair after surgery. Among the control groups, the negative control group had the highest recurrence rate, reaching 70% ± 8%, demonstrating that the group lacking therapeutic intervention had more severe tumor recurrence. The positive control group (interferon group) had a recurrence rate of 32% ± 5%, showing a certain effect, but still lower than that of the Example group.

[0096] The results are as follows Figure 6As shown, mice in the Example groups also showed a significant advantage in weight recovery, particularly in Example 4 (+10% ± 4%). In contrast, the negative control group experienced weight loss (-8% ± 4%). Changes in body weight reflect postoperative recovery. The weight gain of mice in the Example groups suggests that the compound injection has a positive effect on postoperative recovery. Control groups, particularly the comparative and positive control groups, experienced limited or decreased weight changes, indicating that the effects of these individual components are 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 negative control group (normal saline) had a lower VEGF positivity rate (30% ± 7%), indicating that the tissue repair effect of normal saline treatment alone was limited. The positive control group (interferon) had a VEGF positivity rate of 78% ± 5%, indicating that it had a certain angiogenesis-promoting effect, but the effect of the compound injection was more significant to a certain extent. The VEGF positivity rate of the comparative example groups 1 to 6 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 1-4) has a significant positive impact on postoperative tumor recovery, effectively reducing tumor recurrence rates and promoting weight recovery in mice. Furthermore, the high VEGF positivity rate in the Example group indicates that the compound injection has a positive effect in promoting angiogenesis, further supporting its potential to reduce tumor recurrence by improving angiogenesis and tissue repair, and highlighting its potential as a postoperative adjuvant therapy.

[0099] Table 3 Repair effects of Examples 1 to 4 and Comparative Examples 1 to 6 on non-small cell lung cancer in mice after surgery

[0100]

[0101] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. A pharmaceutical composition having a therapeutic effect after tumor surgery, characterized in that: It is composed of olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes; The mass ratio of the olive leaf extract, mesenchymal stem cell exosomes and T lymphocyte exosomes is (10-20): (15-30): (4-10), wherein the olive leaf extract is an ethanol extract of olive leaves; The mesenchymal stem cell exosomes are derived from the umbilical cord of a healthy full-term birth.

2. The pharmaceutical composition according to claim 1, characterized in that 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%.

3. The pharmaceutical composition according to claim 1, 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 in a serum-free medium.

4. The pharmaceutical composition according to claim 1, 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.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for treating tumors after surgery, characterized in that: The tumor is lung cancer.

6. The application according to claim 5, characterized in that The dosage form of the drug for postoperative tumor treatment includes injection, oral preparation or transdermal preparation.

7. A drug for treating tumors after surgery, characterized in that: The invention comprises the pharmaceutical composition according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

8. An injection for treating tumors after surgery, characterized in that: The injection comprises 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; The mesenchymal stem cell exosomes are derived from the umbilical cord of a healthy full-term birth.

Images